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Toxicology of trace metals : metallothionein production and carcinogenesis Brown, David Arthur 1978

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TOXICOLOGY  OF TRACE METALS:  METALLOTHIONEIN  PRODUCTION AND CARCINOGENESIS  by DAVID ARTHUR BROWN B.Sc,  University of B r i t i s h  C o l u m b i a , 1970  A T H E S I S SUBMITTED IN PARTIAL FULFILLMENT THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF  PHILOSOPHY  in THE FACULTY OF GRADUATE (The  Department of  STUDIES  Zoology  and the  Institute of  We a c c e p t t h i s  Oceanography)  t h e s i s as conforming  to the r e q u i r e d standard  THE UNIVERSITY OF B R I T I S H COLUMBIA November,  ©  David Arthur  1978  Brown,  1978  OF  In  presenting  an  advanced  the I  Library  further  for  degree shall  agree  scholarly  by  his  of  this  written  thesis  in  at  University  the  make that  it  thesis  purposes  for  freely  may  for  University  financial  gain  Zoology  of  British  January 15,  1979  of  of  Columbia,  British  Columbia  for  extensive by  the  is understood  permission.  of  fulfilment  available  be g r a n t e d  It  2075 Wesbrook Place Vancouver, Canada V6T 1W5  Date  partial  permission  representatives.  Department  The  this  shall  Head  be  requirements  reference copying  that  not  the  of  agree  and  of my  I  this  or  a l 1 owed  without  that  study. thesis  Department  copying  for  or  publication my  11  Abstract Many  reports  polluted  areas  inferred  from  cance  of  these  study  reports  actual of  protein, toxic  a  changes  in  trace  of  passing and  protein Cd  exposed  are  bound  by  and  binding  the were  study  were  in  of  sampled  from  duck  metal 1othionein was in  sumably  since  with  for  Mussels  Zn  the Cd  was  Zn-binding were  an  in  of  area  trace  to  exert weight  metal  the  Cu  high  and  role  as Zn  of  unless  the  high this  weight  Cd,  molecular  they  case,  components  stored  high  protein able  gel.  weight  are  tissue,  on  cadmium  was  weight levels  pool, to  and  weight  G-75  molecular  effectively  polluted  centrifuged,  were  kidney  in  be  molecular  and  In  to  Pharmacia  liver  to  the  levels  molecular  known  to  levels,  of  more  upon  detoxifying  free  elucidate  with  the  high  sites  exposed  are  This  the  metal  high  according  molecular  then  trace  homogenized,  deficient. high  s i g n i f i -  based  measures  which  to  packed  Excesses  it  made  be  organism.  assay  the  can  cancer.  were  present  relatively  In  was  etiology  a. c o l u m n  in  order  fractionated  zinc  pool  in  Tissues  through  pool  those  enzymes a  the  l i t t l e  from  biological  meaningful  that  metals.  appeared  Zn  the  metals:  metallothionein. to  to  trace  metal 1oenzymes.  bound  levels  the  supernatants  protein  actual  carcinogenesis  Organisms  Copper  the  of  organisms  but  to  Further,  in  in  literature,  biochemically  by  metals  levels  as  metal 1 o t h i o n e i n ,  pool.  by  metal  the  data  metal  metals  protein  the  in  these  effects  trace  trace  exist  toxicology  those  with  of  of  pre-  compete  metal 1oenzymes. Cu  and  Zn  near  a  sewer  out-  i i i fall  and  in  the  laboratory.  until  metal 1othionein  was  rapid  a  protein  of  Growth  detectable and  levels  in  to  are  metals  with  Both  Hg  in Hg  in  did  not  occur  saturated,  high  ratio  in  and  molecular  Cd  Zn by  was DEN  ducks  there  weight  Cd  was  enzyme  binding  Zn  were  be  had  high in it  is  It  is  division  nonessential  weight  of  protein  Cd  and  pool.  Cd.was  bound  exposed  to  low  doses,  When  molecular able  to  suggested with  processes.  weight  weight  compete that  the  this  conclude  Cd,  In to  carcinogen  protein  molecular to  Cd  the  with  weight  reasonable  interfering  trace  of  high  high  zinc  pool  portion  molecular the  metal-  and  of  increases  administered  effectively  sites.  cell  higher  deficient  more  might in  the  exposure,  pools,  involved  in  effects  In  of  Copper  Hg  metal 1oenzymes.  humans  was  pool.  protein  of  metal.lothionein.  (DEN)  where  capacity  weight  molecular  mice  protein  surpassed.  Toxic  to  exposures  the  interference  and  high  on  when  level.  in  exposed  those  molecular  the  much  decreased  with  organisms,  fish  When  accumulated  accumulated  high  were  weight  apparently  metals  a  in  occurred  the  organisms,  fish  this  to  essential  and  reduced  exposure  diethylnitrosamine  pool  the  molecular  was  attributed  metal 1 o t h i o n e i n .  Since  in  metal  high  the  cancerous  Cd:Zn  control  Cd  were  the  bind  decreased  metals  most  metals  rates  increasing  the  apparently  zooplankton  zooplankton,  lothionein  with  survival  pool.  mercury.  fish  was  increase  Phytoplankton,  was  Reduced  Cd,  more  pool. protein that,  as  protein  for in  metalTocancerous  Zn-containing  enzymes  i v Exposure and  Zn  in  to  the  DEN  high  metal 1othionein. reversed  When  losses  of  Cu  creased  levels  protein  pool  death  due  Cu  Zn were  of  over to  low  Cu  in  molecular  administration  Zn  s l i g h t l y ,  of  Cd  were  were more and  control tumors.  evident. in  the  values,  and  and Very high  and  of  pools, of  Zn  Cd, and  with  pretumorous high  Cd  molecular  greatly  Cu on  with  increased  administered  increased,  Zn  decreases  weight  and  levels  and  changes  to  of  low  logical  and  resulted  Concurrent  losses  time.  alone  DEN survival  DEN, histo-  doses  in-  weight  increased  time  Table  of  Contents  Page  Abstract Table  i i  of  Contents  List  of  Tables  List  of  Figures  v v i i x  Acknowledgements  CHARTER  xiv  "  I  Introduction  II  The W i l d l i f e Community o f Iona I s l a n d Jetty, Vancouver, B . C . , and Heavy-metal Pollution Effects  24  R e l a t i o n s h i p Between S u r v i v a l of Mussels E x p o s e d t o C d , Cu a n d Zn a n d t h e C y t o plasmic D i s t r i b u t i o n of these Metals  42  Interactions Cytoplasm of  59  III  IV  V  VI  VII  VIII  1  Between Cadmium and Z i n c Duck L i v e r and Kidney  in  R e l a t i o n s h i p Between C y t o p l a s m i c Distrib u t i o n of Mlercury and T o x i c E f f e c t s to Zooplankton and . Chum..Salmon (Oncorhynchus keta). Exposed to Mercury in a C o n t r o l l e d Ecosystem  80  The E f f e c t o f M e r c u r y E x p o s u r e on t h e Cytoplasmic D i s t r i b u t i o n of Mercury, Copper, and Zinc i n S k e l e t o n e m a c o s t a t urn  97  I n c r e a s e s o f Cd a n d t h e C d : Z n R a t i o i n t h e High M o l e c u l a r Weight P r o t e i n Pool from A p p a r e n t l y Normal Liver of Tumor-Bearing Flounders (Parophrys vetu1us)  11  I n c r e a s e s o f Cd a n d t h e C d : Z n R a t i o i n t h e High M o l e c u l a r Weight P r o t e i n Pool from A p p a r e n t l y Normal Kidney of Terminal Human Cancer Patients  '  3  3  2  v i  CHAPTER  IX  X  Page  D e c r e a s e s of, Copper and Z i n c i n the High M o l e c u l a r Weight P r o t e i n Pool from Livers of M i c e Exposed to D i e t h y l n i t r o s a m i n e , With or W i t h o u t Cadmium o r Z i n c S u p p l e m e n t a t i o n . .  144  D e c r e a s e s of Copper and Z i n c i n Pretumorous and Posttumorous L i v e r s of Mice Exposed to D i e t h y l n i t r o s a m i n e , With and Without Cadmium o r Z i n c S u p p l e m e n t a t i o n  ''^  XI  The E f f e c t o f Cadmium E x p o s u r e , With or W i t h o u t D i e t h y l n i t r o s a m i n e , on t h e Cytop l a s m i c L e v e l s and D i s t r i b u t i o n of Cadmium, Copper and Z i n c  XII  Discussion  2  3  8  XIII  References  2  5  0  XIV  Appendix  I:  Methodology  Summary  263  v i i List  of  Tables  Table 1.  2.  3.  4.  5.  6.  7.  8.  9.  10.  Page L e v e l s o f C d , C u a n d Zn a d d e d t o bioassay water ( c o l l e c t e d f r o m S t a t i o n E) for the l a b o r a t o r y exposure of mussels from Station E, Iona I s l a n d , Vancouver, B.C  45  T h e d i s t r i b u t i o n o f C d , C u a n d Zn a m o n g cytop l a s m i c pools of m u s s e l s exposed to various l e v e l s of these t r a c e elements in the laboratory (Exposures 1-5) or near Iona Island, Vancouver, B.C. (Station B-E)  50  T h e d i s t r i b u t i o n o f C d , C u a n d Zn amongst c y t o p l a s m i c p o o l s f r o m l i v e r and k i d n e y of ducks  66  D i s t r i b u t i o n o f H g , C u a n d Zn a m o n g s t cytop l a s m i c p o o l s f r o m l i v e r o f chum s a l m o n exp o s e d t o v a r i o u s c o n c e n t r a t i o n s o f Hg  86  D i s t r i b u t i o n o f H g , C u a n d Zn a m o n g s t cytop l a s m i c p o o l s from t i s s u e of z o o p l a n k t o n exp o s e d t o v a r i o u s c o n c e n t r a t i o n s o f Hg  97  The D i s t r i b u t i o n o f Z n , C u a n d Hg amongst c y t o p l a s m i c pools from phytopi ankton exposed to mercury in batch c u l t u r e s  106  Parophrys vetulus. D i s t r i b u t i o n of Cd, a n d Zn a m o n g s t p r o t e i n p e a k s f r o m l i v e r plasm of nontumorand tumor-bearing flounders  121  Parophrys vetulus. Ratio p r o t e i n peaks from l i v e r tumorand t u m o r - b e a r i n g  Cu • cyto-  of Cd:Zn in various c y t o p l a s m of nonflounders  123  Parophrys vetulus. Percentage of t o t a l Cd, C u o r Zn o n e a c h o f p r o t e i n p e a k s from l i v e r c y t o p l a s m of nontumorand tumorbearing flounders  124  D i s t r i b u t i o n o f C d , C u a n d Zn a m o n g s t cytoplasmic pools from kidney t i s s u e of cancer and n o n c a n c e r p a t i e n t s  139  v i i i Table 11.  12.  13.  14.  15.  Page Percentage of total C d , Cu o r c y t o p l a s m i c pools from kidney cancer and noncancer p a t i e n t s  Zn o n v a r i o u s tissue of 140  Ratio of Cd:Zn in cytoplasmic pools from kidney t i s s u e of cancer and noncancer patients  142  T h e d i s t r i b u t i o n o f C d , C u a n d Zn amongst p r o t e i n peaks from l i v e r of mice exposed to DEN ( d i e t h y l n i t r o s a m i n e ) , w i t h and without C d o r Zn  150  P e r c e n t a g e d e c r e a s e s o f C u a n d Zn i n t o t a l t i s s u e homogenate supernatant and the high molecular weight p r o t e i n pool from l i v e r s of m i c e e x p o s e d t o DEN (diethylnitrosamine), with and without C d o r Zn  152  Changes i n h i s t o l o g y of l i v e r a f t e r 12.5 w e e k s e x p o s u r e t o DEN (diethylnitrosamine) w i t h and w i t h o u t Cd a n d Zn  1°4  16.  Levels o f C d , Cu a n d Zn i n l i v e r tissue homogenate supernatant of mice exposed to diethylnitrosamine (DEN), w i t h and w i t h o u t s u p p l e m e n t a t i o n w i t h Cd o r Zn  17.  P e r c e n t a g e c h a n g e s o f C d a n d Zn l e v e l s i n t o t a l t i s s u e homogenate supernatant from l i v e r s of mice exposed to diethylnitrosamine (DEN), w i t h and w i t h o u t C d o r Zn. s u p p l e men t a t i o n  18.  19.  20.  T h e d i s t r i b u t i o n o f C d , Cu a n d Zn a m o n g c y t o p l a s m i c pools from l i v e r s of mice e x posed to d i e t h y l n i t r o s a m i n e (DEN), w i t h and without s u p p l e m e n t a t i o n w i t h Cd o r Zn The c y t o p l a s m i c d i s t r i b u t i o n o f Cd w i t h p o s u r e t o a r a n g e o f Cd c o n c e n t r a t i o n s , or without diethylnitrosamine (DEN)  exwith  T h e c y t o p l a s m i c d i s t r i b u t i o n o f Cu w i t h p o s u r e t o a r a n g e o f Cd c o n c e n t r a t i o n s , or without diethylnitrosamine (DEN)  exwith  ' '  8  1  8  3  2  0  2  i x Table 21.  22.  23.  24.  25.  26  Page T h e c y t o p l a s m i c d i s t r i b u t i o n o f Zn w i t h p o s u r e t o a r a n g e o f Cd c o n c e n t r a t i o n s , or w i t h o u t diethylnitrosamine (DEN)  exwith . . . .  204  Percentage d i s t r i b u t i o n . o f total Cd o n e a c h c y t o p l a s m i c pool w i t h exposure to a range of Cd c o n c e n t r a t i o n s , w i t h o r w i t h o u t diethyl-;-* nitrosamine (DEN)  205  Percentage d i s t r i b u t i o n , of t o t a l Cu on e a c h c y t o p l a s m i c pool w i t h exposure to a range of Cd c o n c e n t r a t i o n s , w i t h o r w i t h o u t diethylnitrosamine (DEN)  206  P e r c e n t a g e d i s t r i b u t i o n of t o t a l Zn o n e a c h c y t o p l a s m i c pool with exposure to a range of Cd c o n c e n t r a t i o n s , w i t h o r w i t h o u t diethylnitrosamine (DEN)  207  Recommended p r o c e d u r e s t i s s u e types  for  266  The s p e c i f i c a t i o n s f o r P h a r m a c i a c o l u m n s when s e p h a d e x G-75 gel  various sizes packed with  various  of 268  X  List  of  Figures  F i gure 1.  Page Metallothionein i s a p r o t e i n of molecular weight of a p p r o x i m a t e l y 10,000. One third of i t s amino a c i d s are c y s t e i n e . There are 24 c y s t e i n e r e s i d u e s p e r m e t a l 1 o t h i o n e i n molecule. Each 3 c y s t e i n e r e s i d u e s b i n d a metal i o n so t h a t e a c h m o l e c u l e o f metallothionein binds 3 metal ions. Exposure to l o w d o s e s o f Cd r e s u l t s i n m e t a l 1 o t h i o n e i n p r o d u c t i o n w i t h Cd a n d Zn e a c h i n approximately h a l f of the b i n d i n g s i t e s . Subsequent exposure t o a h i g h Cd d o s e , results i n d i s p l a c e m e n t o f Zn f r o m m e t a l l o - k thionein by t h e C d . Zn i s m u c h l e s s toxic than over  Cd 70  s i n c e Zn i s a n a t u r a l metal 1oenzymes  component  of the Fraser River B r i t i s h Columbia  of  estuary,  g  2.  Sketch-map Vancouver,  near  3.  Diagram of a s i m p l i f i e d food-web of the wildl i f e community around the Iona I s l a n d j e t t y .  4.  Graph of (D) protein fractions (composing peak. I t o V) a n d c o n c e n t r a t i o n s of (A) copper, (B) z i n c , and (C) cadmium, from duck l i v e r in each of these f r a c t i o n s  5.  Total quantity of metals (Cd, Cu, and Zn) p l o t t e d a g a i n s t t h e r a t i o o f t h e same metals on t h e m e t a l 1 o t h i o n e i n to high molecular weight protein (enzyme-containing) fractions for different animals  6.  Gel e l u t i o n p r o f i l e , of m u s s e l s B, Iona I s l a n d , Vancouver, B.C  7.  The l e v e l s o f t o t a l Cd molecular weight (HMW) metallothionein  8.  Gel (A)  elution profiles and k i d n e y (B)  of  from  Station 4 8  + Cu + Zn i n protein pool  Greater  ,  the and  Scaup  high  liver  63  •" x i F igure 9.  10.  11.  12.  13.  14.  15.  16.  17.  18.  19.  Page T h e v a r i a t i o n o f C d , C u a n d Zn molecular weight protein pool, t h i o n e i n and the low m o l e c u l a r plasmic pool with i n c r e a s e s of in l i v e r and k i d n e y c y t o p l a s m  on t h e high metallor' weight cytothese metals 67  The v a r i a t i o n of the p o r t i o n of t o t a l cytop l a s m i c Cd on t h e h i g h m o l e c u l a r weight p r o t e i n pool (HMW) + the low molecular weight c y t o p l a s m i c pool (LMW) with inc r e a s e s o f Zn o n t h e h i g h m o l e c u l a r weight p r o t e i n pool + the low m o l e c u l a r weight c y t o p l a s m i c pool  ' '  The v a r i a t i o n o f t h e p o r t i o n o f t o t a l cytop l a s m i c Cd o n m e t a l l o t h i o n e i n with levels o f Zn o n t h e h i g h m o l e c u l a r w e i g h t protein pool (HMW) + the low m o l e c u l a r weight cytoplasmic pool (LMW)  7 3  Gel e l u t i o n p r o f i l e s from c o n t r o l chum s a l m o n e x p o s e d t o 1 >ig H g / L (B), and those e x p o s e d t o 5 jjg Hg/L (C) f o r 72 d a y s ,  88  Gel e l u t i o n p r o f i l e s from c o n t r o l zooplankton (A), zooplankton e x p o s e d t o 1 jjg Hg/L (B), and t h o s e e x p o s e d t o 5 jjg Hg/L f o r 72 d a y s In v i v o f l u o r e s c e n c e Skeletonema costatum hours  of batch c u l t u r e s e x p o s e d t o Hg f o r  90  of 116 101  Gel e l u t i o n p r o f i l e of e x p o s e d t o 0 . 5 jjg Hg/L  Skel etonema costatum... f o r 116 hours  Parophrys;vetulus. p r o f i l e s from l i v e r f 1 ounders  Composite of gel elution of 3 tumor-bearing  Parophrys vetulus. p r o f i l e s from l i v e r f 1ounders  Composite of gel elution of nontumor-bearing  1 °  11  Composite of k i d n e y s of 2  gel e l u t i o n p r o f i l e s from terminal noncancer patients  Composite of kidneys of 2  gel e l u t i o n p r o f i l e s from terminal cancer patients  3  7  11 ^  . . . .  l  3  ^  I3'  xii Figure 20.  21  22  23.  24,  25  26,  27,  28,  Page Composite gel e1ution , profi1es from nonc a n c e r o u s 1 i vers of c o n t r o l mice and mice e x p o s e d t o DEN w i t h a n d . w i t h o u t C d o r Zn f o r 12.5 weeks ,  153  T h e v a r i a t i o n o f Cu molecular weight pro cancerous l i v e r s of e x p o s e d t o DEN w i t h f o r 12.5 weeks  1 55  l e v e l s in the high t e i n pool from nonc o n t r o l mice and mice and w i t h o u t C d o r Zn  T h e v a r i a t i o n o f Zn l e v e l s i n t h e h i g h molecular weight p r o t e i n pool from nonc a n c e r o u s l i v e r s of c o n t r o l mice and mice e x p o s e d t o DEN w i t h a n d w i t h o u t C d o r Z n f o r 12.5 weeks  157  The p e r c e n t a g e d e c r e a s e s o f Cu l e v e l s i n the high m o l e c u l a r weight p r o t e i n pool from noncancerous l i v e r s of DEN-exposed mice  1 60  T h e p e r c e n t a g e d e c r e a s e s o f Zn l e v e l s i n t h e high m o l e c u l a r weight p r o t e i n pool from noncancerous l i v e r s of DEN-exposed mice ,  1 62  T h e v a r i a t i o n o f Cu l e v e l s i n l i v e r s o f m i c e e x p o s e d t o DEN w i t h o u t C d o r Zn  cytoplasm w i t h and  1 79  T h e v a r i a t i o n o f Zn l e v e l s i n l i v e r s o f m i c e e x p o s e d t o DEN o u t C d o r Zn  cytoplasm from w i t h and w i t h -  The t i m e exposure C d o r Zn  from ,  ,  to d e a t h a f t e r commencement of of mice to DEN, w i t h and w i t h o u t supplementation  ,  181  1 85  The i n c r e a s e i n t i m e t o d e a t h w i t h increases o f Cu a n d Zn i n l i v e r , a t t i m e o f d e a t h , of m i c e e x p o s e d to DEN, w i t h and w i t h o u t Cd o r Zn s u p p l e m e n t a t i o n  188  29,  Composite gel control mice  208  30,  Composite gel e l u t i o n p r o f i l e s from three drinking m i c e e x p o s e d t o 2 5 mg C d / L i n t h e i r water, and from t h r e e m i c e e x p o s e d t o 2 5 mg C d / L + 2 5 mg D E N / L ..  elution  profiles  from  three  210  x i i i  Figure 31.  32  33.  34,  35,  36,  37.  38,  39.  Page T h e v a r i a t i o n o f Cd l e v e l s i n t h e high m o l e c u l a r weight p r o t e i n pool from mice exposed to Cd, w i t h or w i t h o u t DEN The v a r i a t i o n t h i o n e i n from without DEN  o f Cd l e v e l s mice exposed  in to  metalloCd, with  ,  or ,  T h e v a r i a t i o n o f Cu l e v e l s i n t h e high molecular weight p r o t e i n pool from mice exposed to Cd, with or without DEN T h e v a r i a t i o n o f Cu l e v e l s . i n t h i o n e i n from mice exposed to or without DEN  metalloCd, with  T h e v a r i a t i o n o f Zn l e v e l s i n t h e high m o l e c u l a r weight p r o t e i n pool from mice exposed to Cd, w i t h or w i t h o u t DEN T h e v a r i a t i o n o f Zn l e v e l s t h i o n e i n from mice exposed or w i t h o u t DEN  in to  metalloCd, with  The v a r i a t i o n o f t o t a l tissue supernatant Cu + Zn l e v e l s i n to Cd, w i t h or w i t h o u t DEN  homogenate mice exposed  The v a r i a t i o n o f l i v e r w e i g h t to v a r i o u s l e v e l s of Cd, w i t h DEN  with exposure or without  A f l o w diagram of the s t e p s i n v o l v e d in the e x t r a c t i o n of m e t a l l o t h i o n e i n and the high and low m o l e c u l a r w e i g h t p o o l s from tissue.  ,  212  214  217  219  222  224  226  228  264  x i v Acknowledgements Studies Environment  in  Canada,  and  the  was  supported  National  Council  of  introducing  Tim  to  reviewed  me  those  Ken  and some  the  Council  of  from  of  discussions provided  absorption  Parsons  topic  Chatel,  of  the  on  Agnes  Knight.  by  grants  Science Canada.  the  from  Foundation, The  National  author Research  and  with Chan,  Don  in  the  topic  use  of  of  and  Perkin  the  absorption  of  studies;  Bawden,  Louise  B.C.  Research  thesis,  before  provided  Elmer  While use  for  Thanks  these  Carole  spectrophotometer,  atomic  Thompson  carcinogenesis.  their  provided  in  this  publication, of  me  McLeay  for  the  Jeff  metallothionein.  manuscripts  Institute  furnace  Tim  collaborated  submitted  Environment  to  Brian  ting  graphite  supported  National  scholarship  who  were  atomic  a  were  U.S.  Research  to  they  Research  the  thanks  Parsons,  Cloutier,  by  thesis  Canada.  Special  also,  this  the  their  stimulaB.  C.  flame Pacific Perkin  spectrophotometer.  Elmer  1 CHAPTER  I  Introduction  Metallothionein  and  Metal 1oenzymes  Metal!othionein protein high  high  levels  Vallee,  in of  1957; can  to  exposed  the  complexes  the  bind  are  a  low  sulfhydryl amino  Kagi  groups  is  and  heavy  groups acid  metals  due  The  presence  of  and  sulfhydryl  rendering  (Piscator,  stored  the  (10,000)  (Margoshes  1960).  thus  weight  to  cysteine  Vallee,  organism usually  molecular  them  1964).  nontoxic  Metal-thionein  solubilized  in  liver  and  kidney  cytoplasm. Heavy to  high  levels  metals  molecular of  necessary (Friedberg,  for  normal  stimulated  by  levels  a l . ,  of  production sequester a  of all  1 973 ;  a l . ,  1 973)  metals  metals,  1972a, Winge  b; et  into  to such  heavy cell  high as  of  and  Cu)  metal  Low  1oenzymes  there  is  exposure  1 975 ;  Sugawara  there and  is  then a  kidney  not  metal.  In  cytoplasm,  molecular  production  Cousins,  are  be  of  and  sequestered  very  high  tissue,  case  heavy  weight  Either  1974;  inthen  sufficient  this  an  bound  are  and  may  enzymes.  be  enzymes.  When  metals  liver  may  metal 1othionein  al . ,  if  as Zn  Squib  heavy  metal!othionein  in  (e.g.,  1971).  However  excess  such  functioning  Excess  metals  metal 1othionein  proteins  heavy  (Webb,  "spillover",  metal 1othionein et  of  1975).  heavy  to  Underwood,  metal 1othionein.  flux  be  the  1974,  is  Sugawara,  weight heavy  higher  et  bound  certain  to  Davies  not  to  there  metals  proteins excess  may from  (Winge of  heavy  2 metal the  or  an  enzyme  et  1971).  with  a l . ,  the  1973).  with  Enzymes  1974).  may be  levels  of  to  lethal  the  on  The  toxicity  Use  of  a  Molar  and a  but  competing tion  of  heavy  affinity  also for  binding  their to  This the  than  the  fact  no  that  on  the  these  competition  that  there  metals  each  number  relate  sublethal or  enzymes  provide  degree  of  the  binding data  will  be  binding  metal ions  sites. not  a heavy  areas.  available  which  will  of  f i t  Units  will  for  success of  at  after  1974).  would  metal!othionein rate  metals  longer  industrialized  Weight  by  heavy  metallothionein  of  (Winge  required  molecules  highly  limited  Competitive  relative  sites  of  suggests  different  thionein.  in  by  due  level.  1 974 ; coin-  changes  quantitatively to  B renin e r ,  assumptions of the " s p i l l o v e r " theory  metals This  is  render  spillage  (Friedberg,  assay  Rather  amount  duced.  the  bound  limited  heavy  tween  metals  problem  One o f t h e is  enzymes to  of  can  nonfunctional  from  substrate  meaningful  1 974 ;  about  different  "spillover"  enzyme  pathological  Nonfunction  the  an  rendered  possible  heavy  biochemically metal  of  brought  changes,  sites  on  appearance  are  changes  conformational  It  the  properties  (Friedberg,  binding  Thus,  metal  (Fried berg,  appearance  conformational metals  heavy  nonfunctional  Williams, cides  incorrect  i t  of  to  sites not  on  be-  metallo-  only  on  sulfhydryl  binding  each  type  Proper  metal  interpreta-  be p o s s i b l e  using  there  detoxify  c a n be i n -  competition  depend for  i s that  weight  3 units  as  lecular in  heavy  metals  weight.  molar  tend  to  Therefore  units  which  be  of  heavy  relate  to  a  wide  metals  the  range  will  number  of  be  of  mo-  expressed  metal  ions  present. When quantity  molar of  portional thionein  metal  to  the  has  3  Vallee,  1961;  Bremner  and  per  al . ,  amount  assay  =  y  Most  mole  are  Buhler  (Nordberg  all  the  measurement not  metal  and  et  metal  on  metal  ion  and  (Kagi Kagi,  24  1 974 ;  sulfhydryls 1 972 ;  1974;  Winge  metal  et  1othionein by  i . e . ,  mole metal 1 o t h i o n e i n 24 m o l e SH  metal  pro-  metal 1othionein  necessary,  total  Metallo-  a l . ,  on  the  present.  and  Kagi,  of  then  directly  approximately  Buhler  is  is  per  1 966 ;  and  if  used,  X  1othionein  meta11othionein on  units.  In  to  study,  molar  1  total  reports  this  a l . ,  1975;  the  X  groups  1975)  methods  3 m o l e SH mole m e t a l mole  et  Therefore, for,  metal  metal 1othionein  molecule  Davies,  accounted  x  of  sulfhydryl Pulido  1 975).  of  metal 1othionein  Davies,  and  protein  The  on  metal 1othionein  Bremner  is  quantities  m e t a l l o t h i o n e in  order  that  all  data  data  from  referred  and  heavy  other to  metals  studies  have  been  may  use be  weight related  converted  to  quantities.  Process As  of  f i r s t  Detoxification proposed  metal 1othione in  is  the  by  by  Metal  Piscator  1othionein  (1964)  detoxification  of  the heavy  function metals.  of  4 Rabbits  were  thionein  with  Previously kidney  bound  Piscator  of  bound  Here  i t  to  i t  was  (1 9 7 2 )  bound  to  only  taken high  to  was  After  days  few  thionein. before Cd  would  be  the  able  of  exert  This  most  of  the  to  that  is  exert  and  suggests effects  i t s  of  that  a  apparatus  single  cells  kidney  where  molecular was  Cd  is  toxic  high  decline  binding  metal 1othione i n .  but  to  to  in  free  proteins.  to  bind  Therefore,  effects.  However, Cd w a s  in  of  is  as  bound  weight  levels  enzymes  i n i t i a l l y  molecular  enzymes,  f i r s t  'metallo-  available  more  in  f i r s t  Cd w a s  weight  bound  was  later  Cadmium  of  and was  remaining i t  tissue,  particles.  Cd  injection  Cadmium  proteins,  to  kidney.  few minutes  occurred,  less  by  a  synthesized.  metal 1othionein  toxic  synthesis  blood  high  suggests  a  thereafter.  to  the  l i v e r ,  tubules.  in  and  to  in  f i l t r a t i o n  organs  and  the  tissue,  transported  weight  from  With  synthesized  after  bound  metal!othionein,  teins. to  This  was  that  liver  metallothionein  synthesis to  In  liver  l i v e r s .  only  1961).  in  glomular  red  organelles  supernatant a  to  molecular  cell  and  found  into  i t  (metallo-  their  isolated  1960,  renal  slowly  up  metal 1othione i n . bound  the  distributed  redistributed was  in  from  been  also  that  the  Cd-thionein  isolated had  metals  through  and  Vallee,  proposed  reabsorbed  blood  was  and  heavy  Nordberg. Cd,  Cd  metal 1othionein  passed  was  Cd)  (Kagi  (1964)  then  with  metal 1othionein  tissue  discovery  and  injected  Cd  based  proable upon  5 For of  up  to  112  Cd-thionein  in  constant,  liver  kidney,  kidneys.  This  Piscator's the  and  absorbed  from  0.01%  the  of  sudden  decreased  with  study  some is  transported  body  tubules. of  in  Cd  with  appearance  of  tubular  to  small  Nordberg  least  carrier  after  Cd-thionein tion  and  become  saying  the  subsequent  absorption  is  from  tubular  reaches  damaged  Cd  by  accordance  with  Cd-thionein  and  formed is  found  that  excreted  was  that  per  observed  has  day.  Cd  was  the  is  When  amounts  of  is  an at  Further,  glomular  reabsorption.  Cd-thionein  bound  urine,  occurred. by  A  meta11othionein.  into  cortex  reabout  metal 1othionein  tissues  in  concomitantly  This to  the  is  Nordberg was  from  from  it  accumulated  decreased  kidney  excretion  identical  kidney  in  where  damage  tubular  the  that  proteinuria.  by  concentration  kidney  excretion  size  of  renal  Cd  with  summarizes  important  the  the  redistribution  in  that  to  burden  protein  slow  concurrent  hypothesis  kidney  a  while  therefore  increase  a  injection,  suggesting  (1964)  liver  after  liver  remained to  days  f i l t r a -  the  Cd,  tubules  re-  excreted  in  the  urine. Nordberg Cd  into  alone  rats  as  resulted  binding  of  appeared damage  et  in  this the  whereas  al . free  in Cd  Cd to  (1 9 7 5 ) Cd  or  equivalent  Cd-thionein.  uptake  by  liver  free  Cd-thionein Cd  did  not.  amounts  Cadmium  tissue  metallothionein,  kidneys. the  injected  exposure  and  while  Further,  subsequent  Cd-thionein  resulted  in 5  of  renal  times  as  6 much  Cd w a s  required  as  opposed  was  attributed  to  renal  rapid  release  Death that  liver  stores  would  be  of  excretion  This  be  due  could  result  thionein be  is  in  (Kagi  available It  is  then  to  to  bind  in  kidney and  Metal 1othionein Development  of  is  increased most  occur  would  be  Cu  not  reabsorbed  suggest  of Zn  the  that  stored  is  then  proteins. 1othionein  slowly  Renal in  metallo-  would  metal  and  for  urine  from  structural  kidney)  is  f a t a l .  organism.  These  bloodstream^. is  pH  1961).  summary  complex  the  and  renal  (or  authors  to  low  Cd,  1960,  liver the  the  be  metal 1othionein  trans-  reabsorption  kidney  cortex.  excreted.  Metal1othionein-mediated  be  To!erance  which  to  and  Kagi,  exists ion  1 960 ,  1974;  be  metal  Cd  results  doses  of  bind  the  for  each  1 961 ;  Bremner  to  1othionein  and  i t  saturated three  Pulido  would  detoxify  However,  in  in  Cd.  expected  metal 1othionein  quickly  metal  and V a l l e e ,  excess  exposures.  always one  might  excess  to  to  challenge  this  of  This  subsequent  i .e. ,  preexposure  subsequent  available  on  (Kagi  Buhler  by  that  synthesis  metallothionein  residues  to  way  by  readily  metals  known  exposure.  organisms,  1966;  of  the  obvious  then  in  via  tolerance  i n i t i a l  that  the  commonly  upon  heavy  that  this  of  harmful  and damage from  The  to  Metals It  The  fact  dissociation  apparent  occurs  Heavy  the  and V a l l e e ,  synthesized  ferred  to  Cd-thionein  damage.  of  purposes may  to  et  and D a v i e s ,  is  known  state  cysteine a l . , 1975).  7 Therefore must  some  1.0  (1974)  and  challenge and  8.2  was  found  3.2  doses,  to  Zn.  lenged an  the  with  tolerance  Cd  vivo  by as  levels  resulting  development  (Figure  to  it  is  a  free  cellular  Zn  results  Cd  Cd  to  of  Zn Zn  injections  from that  is  both the  much  component far  less  of  6.0 to  challenge  but  that  contained were  Cd-  chal-  resulted and  Zn-  increased from  less  tolerance  metallo-  toxic  many  toxic  Zn  while  mice  displacement  Zn  in  Cd  s a c r i f i c e , .'th i s  Zn  Zn  natural  4.5,  increased  or  and  pretreated  1).  Cd  of  of  cadmium  preexposure  zinc  from  of  subsequent  dose  dosages  by  metal 1othionein  suggests  than  values  Zn  prior  Cd  later  from  3  results  hours  contained  or  This  cadmium  Likewise,  of  cadmium hours  with  LD-50  LD-50  displacement  cases  thionein  the  48  pretreatment  metal1othionein. both  in  metallothionein  When  J_n  resulting  found  metallothionein only  followed  increase  with  Cd-induced  in  of  rats  respectively.  Leber  linearly  preinjected  mg/Kg,  mg/Kg,  doses.  in  mechanism  exist. Leber  0,  other  to  enzymes  enzymes.  effects  Hence,  than  free  Cd. Similarily, synthesis  of  equimolar  levels  1 975). or  exposure  to  metal 1othionein of  Therefore,  Zn  and  should  result  other  metals  upon  in  Cu  or  Hg  containing the  exposure  Zn  Ag,  to  exposure any  increased  subsequent  one  results  approximately metal of  tolerance  exposure.  in  (Winge  Cd,  Hg,  Ag,  to  any  of  et Cu the  al . ,  8  Figure  1.  Metal!othionein weight  of  of  its  amino  24  cysteine  acids  so  binds  doses  Cd  mately sequent  Cd of  the  in  displacement  by  the  since  and  Zn  is  Zn a  of is  One  third  There  residues  molecule ions.  of  bind  Exposure  metal 1othionein  Zn  each  in  to  low  pro-  approxi-  sites.  high  Cd  Zn  from  metal  less  a  metallo-  in  much  are  1othionein  a  natural  metal 1oenzymes.  metal  binding  to  molecular  cysteine.  cysteine each  of  10,000.  per  8 metal  exposure  Cd.  are  results  with half  3  that  thionein  duction  protein  residues Each  ion  of  a  approximately  molecule. metal  is  Sub-  dose,  1othionein  toxic  component  results  of  than over  Cd 70  9'  Metallothionein  M.W. 1 0 , 0 0 0  I l I l Ll I I I I I I I I I I I I I I I I I I  f x  2 4 cysteine  residues  Low Chronic Cd Exposures I I I I I I M J I I I II f M J I I I I I I Cd  Zn C d Zn C d  Zn C d  Zn  3 c y s t e i n e r e s i d u e s / m e t a l ion Zn in a p p r o x i m a t e l y 5 0 % of binding  F o l l o w e d by H i g h A c u t e C d E x p o s u r e  LU LU LU LU LU UJ LU LL' Cd C d C d C d C d C d C d C d  L e s s T o x i c Zn D i s p l a c e d by C d  sites  10 The  Occurrence  Heavy  of  Metallothionein  cortex  by  ammonium  sulfate and  protein  in  not  was  Margoshes  Cadmium  an  and  f i r s t  isolated  Vallee  (1957)  renal  were  found  associated  1  0.42  molar  ratio.  Later  Kagi  :  determined.  technique, and  present  in  high  amounts  while more  (Kagi  found  a  Mg,  ten  Sr,  Cr,  1  Sr,  and :  Cr  A l ,  and  DEAE-cel1ulose Vallee,  0.64 and  Mg  occurred  1961)  :  0.07  :  Pb  were  not  (1960)  using  Cd  and  in  pre-  from and  Fe  in  some  Zn,  Fe  molar  found  horse Zn  lesser  preparations.  chromatography  Cd,  0.03  metals  fractional  found  Ca,  isolated  heavy  metallothionein  Ba,  Pb  the  Vallee  preparations  amounts;  precise  included in  in  isolated  ethanol-  with  sulfate  renal  technique.  Other  and  horse  an  zinc  cortex  a  by  precipitatron  a  cipitation  Ca,  Associated  from  fractional  ethanol-ch1oroform-ammoniurn  When  Its  Metals Metallothionein  were  and  and  step Cu  ratio.  were  Al ,  associated  was  Ba,  with  metallothionein. Piscator tissue  of  rabbits  identified Zn  and  1966 renal 0.07  Cu  (1964)  in were  Pulido  et  cortex. molar  Cd-thionein  isolated  exposed  human  liver  found  in  a  to  Cd.  by  Buhler  1  a l .  isolated  Cd,  Zn,  ratio. from  Cu  Rugstad cultured  metallothionein  :  72.1  Hg  and human  liver  Metallothionein and :  Kagi  1.1  were  in  Norseth skin  a  was  (1974).  molar  metallothionein  and  from  1  (1975)  Cd,  ratio.  from :  In  human  1.08 have  epithelial  f i r s t  :  0.13  isolated  cells  grown  :  11 in  a  Cd  rich  medium.  Wisniewska and  found  kidney.  these  kidney  Nordberg  to  Zn  Co,  (1972)  Webb or  found kidney  (1972a)  shown  intestines  swine et  fed  a l .  four  rats  to  various  metals  thionein  Rats have  followed  Cd t o this  concluded  that  given  found  rats  their  Cd,  in  in  Cd,  Zn,  Hg,  42 Cu,  with  Ni  bound  or to  water of  their  Cousins  et  renal  cortex  of  their  diets.  Hg,  the  metal 1othionein of  preexposed  the walls  Ag,  with  plasma,  drinking  1975).  mice,  Piscator,  were  and Marafante  injections  only  injected  from  associated  stimulate  rats  cations  with  Sabbioni  with  were  Cd  into  erythrocytes,  when  in  in  preinjected  Cd  and  Cd-thionein  of  rats  (Nordberg  were  Cd  their  of  of  a n d Hg  in  urine  in  these  quantities  fraction.  with  that  of  Cd  Hg-thionein  injection  Cd-thionein  injected  were  in  later  with  found  and Sugawara,  isolated  (1975)  (1970)  After  found  rats  metallothionein  and u r i n e  days  (Sugawara  have  to  Cd-thionein  low q u a n t i t i e s  been  (1973)  a l .  mercury.  meta11othionein. have  et  Cd a n d 2 - 4  that  bound  injected  and o c c a s i o n a l l y  testes,  1972).  (1970)  metals  radioactive  liver,  a l .  Jakubowski  liver, with  et  or  Winge  Zn.  liver  A l l  metallo-  (1975)  injected  production  different  metals.  Ag  could  a n d Sn  al .  and They bind  m e t a l 1 o t h i onei n . In  1 9 7 2 MacLean  fraction n idulans)  binding exposed  Cd to  et  al .  a n d Zn  found in  a m e t a l 1 o t h i o n e i n - 1 i ke  blue  radioactive  green  Cd.  algae  However  (Anacyati s in  a  try-  panosomid the  flagellate  same  conditions,  occurring found  the fur  Thompson of  Atlantic seal  caurinus  but  soybean  plants.  an  of  with  could  not  mussels  from  appreciable exposed  tissue  of  (Howard  the  Hg-thionein  levels eels,  in  but  Sea. l i v e r s ,  i n much et  above  currence  of  metallothionein  most  studies thionein  a l l of  c e l l s ,  animal  and  in  urine may  found  in  Zn  Cd  exposed from  both  a n d Cu  kidney  amounts  Cd  Noel-Lambot in  has been  suggests animal  found  tissue  in  in  of  muscle  in  kidney,  tissue  metalloThe  to  the some  There  is  a  the  plant  skin  p o s s i b i l i t y level  paucity kingdom;  algae  oc-  testes,  cultured  suggests  blue-green  that  kingdom.  l i v e r ,  exist  tissues.  metallothionein  has been  the  erythrocytes,  metallothionein or  in  have  1975).  summary  ubiquitous  in  vulgata)  Cd,  and  lesser  a l . ,  is  plasma,  gill  (Sebastodes  (1975)  1975).  binding  North  occurrence  contained  and N i c k l e s s ,  and t h e  Cd-exposed  metal .loth ione in  pollution  been  and  fish  in  (Patel1 a  has  grypus)  and Yip  Cd-thionein  Naturally  the  rock  12  under  01afson  report  copper  limpets  thionein  that  also  by  metallothionein  The  epithelial  (Halichoerus  Casterline  industrial  (Bouquegneau  muscle,  Cd-thionein  u r s i nus )  detect  Common  severe  found  induced)  the  Cd.  grown  was f o u n d .  seal  of  presence  fasciculata)  fraction  authors  livers  Zn-thioneins  (1976)  Hg  in  the  oysters  grey  These  injected  demonstrated  and  such  (CalTorhinus  (1974).  Cd-thionein  area  no  (nonexperimentally  in  Pacific  (Crithidia  in  of metallo-  (McLean  et a l . ,  13 1972)  but  not  in  Naturally advanced found and  Cu  and on  mainly  Cd  Cherian,  et  small  a l . ,  1974).  occurring thionein et  a l . ,  Ag  (Winge  Sn  (Sabbioni  been  1966; et  are  Kagi a l . , and  on  organisms,  normally  found  Affinity  of  and Zn  by  bound  to  their  4.6  Sabbioni  and  in  whereas  exposed for  Vallee  (1957)  found  with  hot  determined  they 50%  higher  Metals  of  noted Cd  by  metallo-  Fe  that  these  and  hence  than  those  are  organisms.  at  Cd  was  pH  7  that  of  the  of  amount with Zn  removed  not  but  acid.  not  and  experimental  that  dialysis  50%  1972a),  1975)  trichloroacetic  that  (Pulido  Metallothionein  decreased  was  naturally  (Webb,  be  injected,  Heavy  metallothionein data  been  for  Co  in  Hg  1 974;  Marafante,  only  of  (Noel-Lambot,  contain  may  been  involved  induced  Ni,  It  quantities  metallothionein  (1960)  and  1975).  have  Hg  more  recent  Thompson,  to  1961),  has  protein  sufficient  addition,  environmentally  treatment  Vallee  From pH  from  be  a  1975).  by  amounts  and  and  far  Thus,  experimentally  Vallee,  they  Various  Margoshes  was  in  Cu  metallothionein  present  in  as  Zn,  may  Marafante,  found  it  Yip,  isolated  lesser Fe.  to  and  thus  of  Olfafson  in  1975;  with  Cd,  this  and  because  it  for  shown  conditions  removed  refer  metallothionein, has  Zn  amounts  1 975 ;  While  techniques,  and  specifically  (Casterline  metallothionein,  metallothionein  Hinge  metals  occurring  possibly  binding  1 976 ;  plants  chromatography  binding  works in  gel  soybean  Cd  that Kagi or  decreasing was  until  removed pH  3.5.  pH. at  14 Therefore of  Cd r e m o v a l  hydrogen  bound  to  that  ions  requires  and  therefore  metallothionein  the  number  of  a  than  Cd i s is  sulfhydryl  remains  Cd  be  are  Zn.  able  to  Kagi 5 X 10"  Thus  tt  displace  and  Zn d i d  against  Cd d e c r e a s e d  Therefore, of  Cd c a n  versa. cedure of  it  not  can  displace  Kagi  and  neglects  buffer  They with  conditions,  et  and that  that  with  after  both  Ag  or  removal  of  Ag a n d Hg  metallothionein  pH 7 . 1 .  the  They  the  found  Cd c o n t e n t  Zn c o n t e n t  assumed  that  of  at  that  but  Vallee  state  that  electrostatic  body  this  factors  against  dialysis  that  dialysis  metallothionein. p H , an  Zn f r o m m e t a l l o t h i o n e i n  al.  (1966)  Cu b u t Cd was  to  passed  p.l  M HC1..  cadmium,  titration  Hg-thionein  titratable  dialyzed  then  metallothionein that  found  (1960)  human m e t a l l o t h i o n e i n  eluted  they  but  infusion  not  vice  experimental  and  specific  proeffects  ions.  Pulido  HC1.  Further  metallothionein.  affect  be  tightly  Zn f r o m  Zn a t  against  more  unchanged  assumed  concentration  Cd a n d  Vallee  M Cd o r  3  can  higher  probably  Zn.  groups  p-chloromercuribenzoate and  10-fold  not  of  this  Hg.  a  by  They  found  and  3 X IO  the  through  Hg r e m a i n e d  Wisniewska  et  addition  that  under  were  in  acidic  of  0.1  removed  with  displaced  al.  (1970)  medium.  at  N  column  from  They  Ag  of  these  bound.  from m e t a l l o t h i o n e i n  stable  M solution  - 5  a Sephadex .G-25  copper  metallothionein  removed is  pH 2 . 0  zinc  while  adjusted  also  also  found Cd,  Zn  concluded  pH 2 b u t  that  15 Webb  (1972a)  metallothionein on  dialysis  whereas  Zn  against  0.2  than  of  the  was  was  when  Cd.  high  Cd,  low  for  Cd^>  of  a  et  Ag  only  order  of a  but  in  those  Cd  least  their toxic  decreas-  be  deduced:  exposure  f i r s t ,  much  can  of  Zn  this  more  had a l m o s t very  to  to  next,  high  the followed  prediction,  in Zn  rats than  equal  high  receiving Cd;  levels  levels  of  of Cd,  metallothionein.  1963). affinity ions  Cu  spillover  with  receiving  toxicity  of  pool  dose  The  and W o l f ,  dialysis  order  after  should  contained  high  twice  displaced  metallothionein,  on  of  Cu  accordance  Cd,  i t  equimolar  was  metallothionein  protein  that  dialyzed  sodium  similar  following  Therefore,  Cd w a s f o u n d order  a n d Zn  buffer  Further,  Ni  using  was  2 0 mM  Cd.  of  retained  acetate  against  binding  the  of  element,  In  in  to  vitro.  Cu.  weight  receiving  (McKee  20mM  replaced  Cd  summary  Zn^>  found  injections  in  binding  and Hg.  and Zn;  against  (1970),  Hg,  bound  was c o m p l e t e l y  was d i a l y z e d  a l .  firmly  metallothionein  Hg  and C d ,  that  trace  (1974)  those  the  Ni  above  molecular  Leber  Cd  the  Ag^>  levels  by  of  found  affinity  Hg^>  When  7.4),  metallothionein, From  ing  (pH  Evans  procedures, from  Cadmium  preparation  thionein  concentrations of  Zn.  Cd w a s m o r e  mM p - c h l o r o m e r c u r i b e n z o a t e  buffer  that  that  removed.  phosphate found  found  of  This for  will  be  Cd, is  Zn  a n d Hg  exactly  the  metallothionein; released  f i r s t  is  Hg^>  same  as  Cd^>  Zn  the  conveniently, in  a  "spillover"  16 situation.  Furthermore,  Zn,  is  component  many  a normal  Mercury,  cadmium  effects  by  and zinc  binding  sulfhydryl  groups  which  most  bind  of  which  those  1971).  the  bind  groups  enzymes,  i . e . , Hg^>  sarily same  ability  result  metals  in  to  also  have  was p r o b a b l y  of  sulfhydryl-based binding  most  strongly  an  Evolution The  of  f i r s t  time  the  higher,  lava  active  (heat  quantities Furthermore, atmosphere  evolutionary  those  It  of  follows  a b i l i t y  to  neces-  since  these  render Therefore,  that  detoxifying  the  system  which  containing  metallo-  that  not  a metal,  metals  on  metals  sulfhydryl  does  necessity  heavy  sulfhydryl  on  earth,  metal  today.  higher  the  in  those  nonfunctional.  detoxification  began  heavy  than  toxicity  increased  high  groups  Cd^> Zn.  metallothionein  to  to  1974).  developed,  are  most  enzymes.  Metallothionein  metal  life  tightly  enzymes  strongly  bound  sulfhydryl  f i r s t ,  toxic  Therefore,  metallothionein  a reduced  sulfhydryl-containing it  most  bind  (Friedberg,  particularily  probably  increased  released  their  (Williams, to  be  exert  thionein, of  enzymes  generally  enzymes,  tightly  should  output,  there  was  and oceans  the  from  substances  high  then  then as  now  as  years  oceans  probably  resulting  as  may h a v e  billion  in  was  generating)  6 times  3.6  levels  This  system  due  the in now  600 times (Strahler,  developed ago.  when  At  that  w e r e presumably in  part  presence the  to of  the radio-  atmosphere  (Strahler, a s much 1972).  C02  much  in  1972). in  the  Therefore,  the  oceans  would lava  would  have than  being  resulted  in  more  by  volcanic  activity.  in  a metal may  have  polymers  of  1974). Cu,  acids,  thionein  as  the  rich  medium,  been  the  acids  Thus,  l i f e  Fe,  that  in  evolved  was  acids  probably  clay  bonding  of  the  (Paechtmany  metals  systems  evolved for  the  lava  metal  enzyme  system  from  l i f e  u t i l i z i n g  probably  detoxifying  more  fact for  in  acidity  extracted  and n u c l e i c  Mg a n d M n )  This  Therefore  and  and f u r t h e r m o r e ,  a natural  fact  catalysts  amino  Zn,  acidic.  being  produced  nucleic  and  metallo-  excesses  of  metals. As  metals  postulated levels  by  Brown  dropped  photosynthesis  began  time  i t  that  is  proposed  These  are  trace  metals  decreased more,  insoluble in  of  atmospheric  creased metals  2.7 Fe  the  these  CC^  from  lava.  now t h a n metals  metal  developed  are  As  when  often  deficiencies in  a metal  and  f i r s t  in  oxides  less  of  other  the  evolved,  it  is  1971). i t  in  in  Furtherin  i n -  leaching  of  environment,  formed.  resulting  factor  that  resulting  content  limiting  At  oceans.  therefore  heavy  genera-  ago.  thus  the  that 0^  quantities  sharply  (Underwood, rich  when  years  flocculant,  the metal  the  l i k e l y  suddenly  vast  decreased  l i f e  is  a n d Mn h y d r o u s  metals  pH o f " t h e s e a w a t e r  i t  billion  and absorb  a n d on  levels  (1976)  relatively  ting  of  metal to  (especially  that  more  addition  Horowitz,  lower  much  in  templates  heavy  been  now,  evolved  f i r s t  have  oceans not  growth  Because would  be  of is  surprising because l i f e expected  1 8 that  present  exposure fact  day  to  that  metallic  much  experiments and  is  (e. g. ,  life  not  parts  of  carcinogenesis,  in  cell  division  polymerase,  taining  sponsible,  in  by  substances enzyme  that  are  adapted  is  to  in  survive  indicated  physiological high  Thompson,  by  the  levels  metal  of  exposure  1 974).  part,  and  Zn  for to  cancers.  inhibit  normal  enzymes  et  and  a l . ,  metabolism  Cd,  High  function  arene  Cu Cd  of  and and  interest  kinase  the  Cu  1969)  con-  re-  noncarcinogenic  oxides. to  RNA  This  is  noncarcinogenic  glutathione-S-epoxide Zn  levels  both  of  as  involved  thymidine  bioactivation  hydrase  of  particular  1976),  carcinogenic  metals  polymerase,  and  (Yamane  environment,  contain  Of  DNA  Dreosti,  the  rich  containing  e . g . ,  system  Levels  of  the  the  epoxide  development  enzymes  structure.  subsequent  the  systems.  many  element  transcriptase,  Duncan  their by  trace  processes,  substances  followed  and  a  their  reverse  1976;  in  monooxygenase  organic  normal  Olafson  well  This  synthesized  began  in  (Vallee,  than  be  surprising  essential  be  Carcinogenesis  Since it  can  would  pollutants.  higher  metallothionein  Metals  organisms  Cu-  are can and  changed alter  with  or  Zn-containing  metal 1oenzymes. Elevated  liver  found  in  many  cancer  Tietz  et  a l . ,  1957;  Sandberg  et  a l .  Cd,  Cu  and  patients Lewis  (1958)  et  Zn  and  (.Olson a l . ,  reported  kidney et  1969;  al.. ,  Cd  1 954,  Morgan,  increased  and  Zn  1 958;  1970,  hepatic  are  Cu  1971). in  19 some  cases  of  widespread l i v e r , while in  metastasis,  kidney Pories  a l l  Zn  c u l a r l y  to  be  increased  duced  tumors.  1969)  report  changes Olson  animals that of  a l .  levels  (1958)  in  occur  metal  increased and Smith  Zn  ho  in  Cu/Zn  (1970)  found  and Sasse  (1961)  rats  DMBA  with  in_ F u r s t of  a l .  tumor in  subcutaneous  and  tumors  (1957)  increase  levels  serum  parti-  Cd c o n t e n t  before  patients  diseases,  (1 9 5 6 ) :  et  with  increased  cancer  Arnold  Tietz  report  found  lung  al .  the  transferred  alterations  do n o t  occur  Haro,  that  formation Zn  tumors,  i n -  induced  report  liver  '.  while in  suggesting  as  a  result  carcinoma.  Koch patients  et  (1963),  a l .  the  drug  that  be e x p l a i n e d  The  present  as  study  of  elevated  could  pretumorous  tissue.  bound  results  a l l  be  of  trace  As  It in  has Hg  been  in  by  the by  chelating  can  of  leukemia  Furst  and a n t i c a n c e r  whether  found  occurring  in  suggested  metal  diethylnitrosamine distribution  Cu  reduced  carcinogens  investigate  and c y t o p l a s m i c  Hg  this  those  will  carcinogen  to  reported  6-mercaptopurine.  actions  can  organic  (1957)  and found  antileukemic  levels  et  in  association  malignant  and d e c r e a s e d  increases  metal  in  carcinoma.  Gorodiskii  bearing  the  Kalsted  carcinogens.  in  et  Cu  in  report  decreased  metastatic  nonmetal  (1973)  in  (1970)  Cd/Zn  patients.  found  by  a l .  especially  Morgan  and serum et  cancer  plasma  neoplasia,  or  not  agents. the  influence  Cd,  Cu a n d Zn  that  a methyl  the  high  drugs  the in group  molecular  weight et  protein  a l . ,  Hg,  as  1973). more  molecular can  pool  bind  Thus,  Hg  is  weight Cd,  rather  and  high  weight  interfer  with  regulation In gether  the  subunit specific and  i t  is  1968). can  be  is  and  a  of  it  subunit be of  this  an DNA  creasing  K+  (Lazarus  et  It  4-5  into become  of  DNA  artifact  toxic in  the  organic  their  Cd w e r e  high  carcinogens  cytoplasmic  increased  then  it  enzymes  to  Na+  would  in be  involved  intact  holds can  the able  in  300,000  enzyme  the  by  has m o l e c u l a r  feedback  subunits  Zn.  If  Hg  that  polymerase  A  the  weight  Hecht  the et  catalytic  et  et  et  al ,  a l . , DNA  19 6 9 ) . polymerase  a l . ,  1973),  procedure;  increases  extraction a l . ,  enzyme,  polymerase  (Jovin  extraction  1973).  to  48,000,  (White  (Hecht  and  exposed  the  so-called  B obtained in  and  DNA  by  transcarbamy-  catalytic  control  by  separated  than the i n t a c t  apparent  of  be  to-  with  separate  higher  subunits  aspartic  transcarbamylase,  used  a l . , 1 973 ;  Zn  together  times  polymerase  or  more  enzymes  subunits  of  joined  aspartic  has  bind  enzymes,  weight  subject  separated  may  amount  not  than  pool,  example,  released.  Like  Recently B  subunits  activity  much  if  These  a molecular  is  is  division.  For  subunits  (Chen  Zn-containing  1970).  agents.  regulatory Hg,  the  metallothionein  changing  protein  Zn-containing  (Ulmer,  has  thus  in  Perhaps  particular,  cell  most  various lase  of  to  pool.  Zn ,  In  to  Hg  available  distribution. molecular  methyl  protein  Cu  than  with  the i n -  procedure DNA  polymerase  21 A  has  a  molecular  polymerase activity subject of  DNA  that by A  occur,  in  there  release  of  free  split  into  of  DNA  establishment  many  respects  isolation of  of  Kitron,  A,  DNA  polymerase or  splitting DNA  and  B  in B  site  are  DNA  proliferation  similar  polymerase  vivo,  B.  If  the  could  of  obvious.  chemically  of  not  Krasny,  catalytic  polymerase  subunits  is  activity  1973;  freed  Hg  specific  the  the  as  metallothionein molecular  weight  the  of  relationships  studies from  be  into  DNA  lack  of  instrumental  indicative  of  precisely  measured,  but  protein  pool.  are  in  both  of  and  molecular  protein  are  pool,  this  the  also As  in  As  cancer.  result  these  pools,  and  on  the  high  in  of  to  metal  measuring  quantifiable  saturation  with  result  quantifies  of  levels  metals  a  response  a  of  thesis  procedures  study  levels  the  between  spillover  this  standardized  produced  of  established  metallothionein, weight  only  in  studies.  metallothionein,  Not  metals  described  previous  metallothionein  treatments.  level  45,000,  DNA  Studies  of  levels  while  decrease  polymerase  cellular  in  the  and such  metallothionein  the  and  between  agents  differ  various  (Lazarus  of  polymerase  which  a  unchecked  for  DNA  be  control  The  weight  could  was  Present  100,000-200,000  agents  parallels  with  feedback  the  of  transcarbamylase  polymerase  The  times  increases  the  of  molecular  control  The  subunits  in  a  polymerase  aspartic  Cd  has  4-5 to  1973).  When  B  weight  to  onset  the of  levels  of  high pathological  effects.  Thus,  cytoplasmic  this  study  distribution  correlates  of  metals  the  with  levels  and  pathological  changes. This Almost  all  measured to  study  previous  only  clearly  the  and  cytoplasmic  have  to  studies  in  the  of  changes  Zn  would  enzymes  paper,  either for  journals  in  authors chapter.  as  of  metals  in  each  metallothionein the  present  levels  of  Cu  of  and  study  Cd  Zn  stages.  have  study.  have  study and  is  Hg  able  affect  between  various  only  not  published, submission;  each The  they  are  interrelated.  As  followed this  date,  tumors  in  Cd,  Cu  through  study  distribution if  metallothionein,  of in  chapters, press,  the  of changes  these  cellular  also  in  or  topic  of may  the  discuss of  each  of  submitted,  status  appeared  the  after  Clearly,  on  to  processes  affected.  described  prefaces  change.  influence  be  consists  are  cytopl asmic  from  cancer  alterations  These  levels  in  occurring  Furthermore,  in the  l i t t l e  would  levels  considers  tissue.  were  which  element  alterations  their  thesis  paration  are  mainly  present  levels  This  high  trace  alterations  and  metal  since  of  posttumorous  Cu  on  Thus,  how  pretumorous  considers Cd,  metal.  three  pools.  The  Zn  studies  distribution  considered  begin. and  one  measures  establish  levels  The  also  or  these  is  in  one  pre-  papers,  appear,  preface how  which  of  various  and  the  each chapters  metallothionein  is  un-  23 familiar  to  most,  each  description  of  has  materials  its  own  sections. and  methods  all  papers  mental  some  Further,  has  the  literature.  and  there  description are  given  findings  discussion  of  introduction  at  of  the  at  all end  relevant methods, is  in the  a  this  of are  I.  the  cursory Each  paper  discussion  material  References thesis.  discussed  thesis.  a  and  detailed  Appendix  studies of  results  more  end  given  from  Some in  an  funda-  overall  24 CHAPTER  II  The  Wildlife  Vancouver,  Community  B.C.,  and  of  Iona  Island  Heavy-metal  Jetty,  Pollution  Effects Preface This  paper  1977,  Volume  C.  Bawden,  A.  approach in  this  that  4,  no  one  else  molecular to  work  the  quantify  these  proposed  in  Fraser  delta  which  banks  (Figure  some  is  two  out  River  the  metal  of  metals on  A. The  and,  is  other in  Brown, basic  described  workers,  both  the  for  Winge  levels our  levels.  et  on  The  and  al  (1973),  the  studies  in  high  metallothionein,  metal  pool,  high  are  the  "spillover"  paper.  2). to  Five three  available  for  ship  of  mixing  these  (a f e r r y  by  stone  miles  edges  causes  the  characterized  the  f a c i l i t y  that  and  enters  to  purpose  D.  R..Parsons.  Except  this  were  ion  The  action  pool  considered  Conservation,  metallothionein  of  levels.  protein  T.  from  levels  protein  Authors  and on  differs  these  has  Environmental  Chatel ,  our  It  in  213-216.  weight  is  Introduct  is  in  weight  quantifies  built  W.  considers  it  for  K.  taken  molecular  theory  pages  paper.  it  f i r s t  appeared  of  the  stone  the  Ocean  series  j e t t i e s , to  of  through  large  extending  4.8  km),  mid-banks,  have  where  where  turbulent  river  water  with  and  is coal  either port),  to or  mud-  been  deep  the  water  tidal sea.  service to  a  seawards  and  jetties  terminal  a  (3.2  docking of  Pacific  act  a as  The port  25  Figure  2.  Sketch-map  of  Vancouver,  British  by The  the  Iona  the  Island  approximate  dicated  by  faint  Fraser  River  Columbia. Jetty  edges  of  dotting.  Scale  which the  estuary,  is  near  indicated  c_a 4  mud-banks  km  long.  are  i n -  restraining Along trench city  walls the  edge  through  sewers  is  graded,  anoxic  an  able  abundance  (e.g.  of  M y t i 1 us  mag i s t e r ) . large the  to  feed  upon  Living  on a  are  two  scandiaca) these  Scaup the  of  zone  a  aquatic  in  the  bivalves  and  Cryptomya  community of  this  particular  the  (Cancer  jetty  have  The  birds,  (Circus  of  distichus),  norvegicus),  (Asio  de-  appreci-  edulis).  owl  Iona  including  most  f1ammeus  is  are  associated  observed  are  preyed  common  of  Nyctea  In  which  forage  been  and  cyaneus).  a  which  rats  herodias) ,  addition of  Greater  directly  with  environment.  crossing  the  animal  rocks  the  degradation  populations  jetties  it  of  are  to  Thus  area  a  Vancouver  v i c i n i t y  there  contrast  attract  crab  is  biologically  birds  In  not  of  a  inconspicua ,  raptorial  harrier  raptorial  (Aythya  and  there  from  immediate  this  (Rattus  (Myt i 1 u s  of  in  Fucus  the  currents.  constituting  life-forms,  Macoma  rats  species  and  to  (e.g.  among  so  of  jetties  effluent  the  commercial  mussels number  in  flow  five  resulted  higher  a  intertidal  by  which  to  and  population  in  flow,  has  edulis,  ca1ifornica),  to  seaweeds  the  primary  peripheral  of  lateral  of  environment  However,  stands  the  This  the  one  allowed  outfall.  is  of  which  Island  trench.  against  such  appears community  the  Island  mid-banks  abundant that  Iona  the  because  and Iona of  lack  Jetty,  the  sewage  outfalls  diverse Island  the  local  animal jetty  other  four and  do  communities.  supports  enrichment  of  a  large  the  28 marine in  Figure  Otte of  environment 3.  The  and L e v i n g s  mud-flat  was  by base  in  in  populations,  large  population on  the  the  exists with  showed  v i c i n i t y  has  that  of  been  the  the  indicated studied  sewage  than  that  of  other  bivalves  is  utilized  by  by  the  populations  well  as  matter  outfall  organic  as  and  mud-flat  crab  by  productivity  both  flourishing  in  spite  of  taken ppm,  Thus  near  compared  Cadmium  fall  al . ,  inverteareas.  the  rat  1 973).  by  the  mercury  Similar  above  of the  studies  Oysters maxima  up  The populain  than  to  2  7  by  (1974)  the  should  (Crassostrea by  the  Act; not  the  for  exceed  the  the  3.7  other  areas.  animals sewer  out(Parsons basis,  levels  human  Vancouver  gigas)  to  encountered  than  in  crabs  1.5  from  a l l  near  that  the  from  a market-weight  Greater  showed  permitted  ppm i n  higher Drug  contaminated  contained  crabs  ppm w e r e to  be  range  except  and  levels  to  is  above  3-5-years-old  ppm i n  converted  Food  cadmium  District  less  1  appreciably  Canadian  and  Drainage  When  are  than  to  of  shown  indicated  area  believed  mud-banks, of  the  content  was  less  the  values  values  levels  with  from  where  these  Island  community  that  are  mercury  was g e n e r a l l y  analysed  fact  which  the  Iona  w i l d l i f e  the  heavy-metals  sewage.  ted  food-web  being  sea. The  et  who  food-web  of  of  jetty  i t s  this  the  terms  brate  tion  of  (1975),  areas  higher,  sewage,  permit-  consumption,  0 . 5 ppm. Sewerage  cadmium  and  and zinc  from  Iona  Island  Canadian  Food  and  were  Drug  Act.  29  Figure  3.  Diagram life  of  a  simplified  community  around  food-web the  Iona  of  the  Island  wildjetty.  31 Seaweeds of  analysed  heavy-metals  sewage  were and  that  that  away  they  from  farther  to  down  been  exists,  in  account it  which  many  in  an  of is  The  is  protected  discussed  in  the  Materials  and  The  mechanism process  of  which  was  involves  weight  binds  the  the  (e.g.  was  Figure  2),  mud-flats  2),  located  accumulations  of  which  an, u n e x p e c t e d of to  as  how  w i l d l i f e be  effects  situation  are  contaminated  this of  has  w i l d l i f e  heavy-metals  is  paper.  that  protein.  . Subsequent  tolerate from  proposed  production  of  called  through  comprises  a l . ,  to  result  10,000)  heavy-metal  et  may  f i r s t  cysteine,  Piotrowski  B,  community  known  the  this  animals  pollution  cular  of  that  to  it  mud-flats.  forms  is  the  pollution  the  (Figure  higher  from  Methods  a b i l i t y  heavy-metal  from  D  the  apparent  that  across  levels  (Ibid.)  Station  w i l d l i f e  question  community  rest  this  of  different  area  heavy-metals.  areas  no  distance data  (e.g.  Station  showed  heavy-metal  and  showed  other  above,  flourishing by  than  the  given  jetty,  of  there  Thus  the  analysis  outfall  from  generally  with  levels  basic  jetty. the  heavy-metals From  the  time  sediment  highest  spread  the  same  decreased  From  the  closest  the  which  o u t f a l l .  evident  at  have  detoxification  Piscator protein  sulphydryl  25-30%  of  chronic  (1964).  of  low  metallothionein,  the  investigations  1973)  a  by a  low-level  tended  linking  to  molewhich  with  amino-acids  (e.g.  of  Nordberg,  confirm  The  that  the 1972;  32 metallothioneins from  chronic  protein  is  are  instrumental  cadmium  and mercury  synthesized  in  the  in  conferring  exposure;  l i v e r ,  protection  the  kidneys,  protective and  other  organs. The which  procedure  was  Shaikh  used  and  Thompson  in  by  The  supernatant  Liver  (Webb, to  a  by  2.5  x  lOmM  lected  on  fraction  was  250 and used  which 250 by  as  i t  at  was t h e n  100  at  a  allows  tissue  the  27,000 heated  x  at  Elmer  x  G-75  Aliquots  proteins  of  but  60  g_ f o r  of  Sephadex  minutes.  for  was  detection  403 a t o m i c  10  supernatant  and  zinc,  and  debris  70°C  27,000  of  were  g_ f o r  to  60 m l / h r .  Cadmium,  that  and O l a f s o n  spectrophotometer. for  to  of  l o w 2 8 0 nm a b s o r p t i o n  Perkin  similar  samples  final,  collector  metallothionein  tissue  cm c o l u m n  Nh^HCOg  nm a b s o r p t i o n . a  (1972),  other  The  of  was  centrif ugation  2 8 0 nm i n  has a  using  or  1972a).  with  at  Webb  and most  liquid  eluted  a  work  c e n t r i f ugation  followed  directly  isolation  present  homogenized,  removed  seconds,  the  (1971),  (1974).  was  minutes  the  Lucis  thoroughly  for  applied and  were  were This  10  col-  measured procedure  metallothionein a  and  relatively copper,  absorption  high  were  measured  spectro-  photometer. Results In total in  and  Figure  amount  five  Discussion  of  protein  4  the  graphs  copper,  A,  zinc,  fractions  B, and  isolated  and  C,  illustrate  cadmium, from  the  the  respectively, liver  of  a  33  Figure  4.  Graphs peaks  of I  copper, duck  to (B)  liver  (D) V)  protein and  zinc, in  each  fractions  concentrations and of  (C)  (compos-ing of  cadmium,  these  (A) from  fractions.  Relative  absorbance  Cd (ppm)  Greater  Scaup  fractions as I  duck,  into  those  which  shown  by  corresponds  to  to  V  Peak  I  such  weights  as  as  II).  weight  of  10,000,  D of  protein  increased  Olafson  and Thompson,  copper other  occur  mainly  have  been  also  Vancouver  (1974);  quantity  peak  peak  peak  and  II  peaks  IV  cytoplasmic  acids,  ATP,  proteins, will  with  a  the of  etc. including  have  molecular  to  haemo-  molecular  much  larger  metallothionein  is  very  small  The  size  of  compared  this  animals  peak  with  with  the  c a n be  cadmium  (cf.  1974). a  natural  environment,  is  to  identified  in  Figure As  systems,  4), zinc  their  loading  and on  while and  relatively However,  in  surveys  analytical  effluent  D i s t r i c t , than  in  1974) the  of  the  cadmium  copper  be e x p e c t e d .  and Drainage  sewage  the  on m e t a l l o t h i o n e i n  and C,  enzyme I  same  thus  corresponds  after  protein the  proteins,  these  which  injecting  B,  in  in  of  metallothionein.  Sewerage  abundant  4  similar  (A,  naturally in  more  is  on  abundance  by  from  and z i n c proteins  occurs  The  fractions.  samples  a l l  eluted  Figure  greatly  In  nucleic  Metallothionein, is  are  metallothionein,  65,000,  molecule.  in  to  almost  than  (peak  other  III  The  absorbed  1 ow-moT.ecul a r - w e i g h t  globin  indicated  are  high-molecular-weight  greater  haemoglobin  column.  and Thompson  amino-acids,  enzymes,  ca  Sephadex  metals  Olafson  to  contains  cellular  the  peak  correspond  material  a  high-molecular-weight  haemoglobin,  and  using  both high  both  metals  (Greater as  being  surrounding  36 environment. evidence  In  of  the  example  low-level  shown  pollution  metallothionein  protein  excess  particularly  metals  In  Figure  zinc,  and  (III)  as  a  that  the  area  of  of  as  that  partly  on  ratio  they  can  mussels.  of  0.70  higher  metal  dicate  a  a  these  greater  degree  metal-contaminated function  the  of  food-web From  the  on  species (Figure data  data D  be  (Figure the  2,  of  high  from  degree  ends  both  mussels  four  the  and  D.  might  thus  highest  must  differences  the  also and  be  on  metal  Thus  metallothionein  inter-  jetty  partly  anima1s).  pollution,  However,  are  the  trophic  be relatively loading  while  peak  relative to  had  metal  which  of  metal-  mussels  of  Station  it  an  lowest  position  a  for seen  the  at  animals  (I)  areas  and  the  may  copper, peak  polluted  showed  of  of  these  of  peak  it  had  values,  for  absorb  quantity  contamination  feed  +0.19  to  is  amount  from  mussels  two  small  pool  Station  Thus  of  there  metallothionein  pollution)  Ducks  loadings  the  indicates  levels  a  the  these  animals  with  up  enzyme  from  metal-contaminated  metal-free  of  clearly  between  in the  ratios.  compared  argued  (a  B  demonstrate  mediate  while  higher  Station  clams  which  4,  cadmium.  From  heavy-metal  ratio,  appreciably  in  Figure  induced  totalled  that  and  in  been  occurring  of  mussels  to-protein  have  animals.  l i t t l e  loading  we  ratio  groups  rats  5  cadmium,  five  from  -  has  in  some  inposition extent  position  in  3).  which  have  been  presented  it  remains  a  37  Figure  5.  Total  quantity  plotted on  the  weight for  against  of  metals  the  ratio  metallothionein protein  different  to  (Cd, of  Cu, the  high  same  Zn) metals  molecular-  (enzyme-containing) animals.  and  fractions  8£  39 question  as  occur  this  in  to  change  would  answer  to  how much wildlife  be  this  question of  the  and  As  i t  enzymes. toxic  effect  metal 1oenzyme Friedberg, will  only  induced  a to  the  (or  animal  some  protection,  peak onset  the  of  enzymes tional would normal  the  total if  cadmium  then  changes result enzyme  in  their  having  functions  metals  the  that, no  be  when  (Friedberg, after  longer  or  the  metallothionein  if  i t  et  c a n be  are  1974).  entry  of  into  of  heavy-  with  concurrent  a l . ,  1973). by  The  conforma-  heavy-metals. different  bound  changes, on  is  metallothionein  to  the  This  from  their  Non-function  sites  which  metallothionein  nonfunctional binding  1 974;  loading  the  peak,  normal  animal).  the  from  exert  metallothionein  "spillover"  they  can  Bremner,  of  properties  binding  further  amount)  rate  conformational f i t  a  heavy-metal  (Winge  by  partial  replacing  the  total  of  a  rendered about  that  overcome  changes  be  1971;  within  to  will  brought  required  molecules  in  load  A  by  and  enzymes  hi.gh-mol e c u l ar-we i ght  would  metals  argued  rate  from  could pathological  heavy-metals  and mercury)  pathological  fact  of  enzymes  load  the  there  animals.  that  produced the  marked  inferred  protect  sufficient  then  the  (Williams,  c a n be  overcome  is  [e.g.  to  known binding  (or  point,  heavy-metals  metals  by  i t  rate  sufficient  At  is  contamination  before  absorption  preferentially  at  among c a n be  complexes  1974),  metal  community  observed  consideration  their  more  is  due  substrate enzymes.  to  Thus  pathological  apparent  if  changes  heavy-metal  metallothionein In Island  area  it that,  associated  wildlife  in  this  in  other  areas  it  has  this  area  abundance animals  this  to  be  from  of  natural  (Cloud, in  one  1 974).,  of  not  The  at  that  of  It  In  life  pollution  is  not  surprising,  an  inherent  mechanism  from  low-level  heavy-metal  toxicity.  ling  Shakespeare's  from  Twelth  Night  beaut.ious.wa.il  results effort (Act  doth  as  1,  oft  are  best  an  Scene close  in  on  from  the  in  these  the it  is  this  to  therefore, which  in  planet  volcanic  due  Man to  find  protects  summarized  ecologist 1)  to  context,  pollution,  community  these  affect  evolved  (i.e.  than  occurs  part,  this  of  Further,  mechanism  in  has  unnatural  greater  apparently  least  Iona  totality  pollution  protective  the  channel  the is  of  mud-flats.  a  Perhaps  become  rate  anoxic  region  metallothionein.  to  the  o u t f a l l ,  River  does  due,  recall  emissions)  would  surveys  the  heavy-metal  to  age  from  in  the  Fraser  appropriate an  with  w i l d l i f e .  of  apparent except  that  that  appears  production  the  shown  of  exceeded  sewage-polluted  of  but  community  loading  is  immediately  been  the  production.  conclusion jetty  in  in  "...Nature  by  the  it  recallines  with  a  pollution."  Summary Marine particularly  and  terrestial  abundant  in  a  animals w i l d l i f e  have  been  community  shown  to  be  associated  with  a marine  These  same  animals  heavy-metals poisonous  sewer  but  metal of its  loading  pollution position  are  are  effects  metallothionein.  outfall  by The  appears and in  the  contaminated  apparently the  to  of  depend on  food-web.  the  City with  protected  production  amount  secondly the  from  of  a  of  Vancouver.  high  levels  from protein  metallothionein primarily species  on of  of  their known and  the animal  as  heavy-  degree and  42 CHAPTER  III  Relationship  Between  Cd,  Cu  Survival  Exposed  to  and  Zn,  plasmic  Distribution  of  be  submitted  shortly  and  Chatel  of  and  Mussels  the  these  Cyto-  metals.  Preface This Authors  paper  are  D.  will A.  study  correlates  cular  weight  unpolluted oratory,  and  polluted to  that  survival  of  exposed  tells  one  related what  to  the  via  the  and  a  of  chances  of  high  Zn  in  mussels  is  related  Cu  from  demonstrates of  approach  goes  levels  of an  it  an  labto  element is  possible  level  laboratory  the  metal-  beyond in  meaningful  toxicology of  trace  that  from  the  "spillover"  nonsurviving  metal  a  mole-  environmentally  comparing  survival  the  and  biochemically  actual  into  This  Cd,  mussels  total  (1978b).  Mussels  of  this  publication.  survival.  these  by  Thus,  measure  to  metals  possibilities  organisms  mussels.  of  in  study  surviving  traditional It  This  W.  with  exposed  survival  area.  predict  pool  "spillover"  metal-exposed to  are  and  K.  "spillover"  protein  area  "spillover"  Brown  for  the  organisms. assay  trace  organism  ( i . e . ,  elements), are.  Introduction In  many  studies,  metallothionein trace Leber, Zn  elements 1974;  ( i . e . ,  has  may  be  able  such  as  Hg  Brown  above  it  et  levels  a l . ,  been to  and  reported  bind Cd  (e.g.,  1977).  required  and  for  that  thus  the  protein  detoxify  Piscator,  Excesses  of  Cu  1964; and  metal 1oenzymes)  also  43 appear  to  be  1978a).  If  exceeded, cular  stored the  effects  Hg  (Winge  et  Parsons,  are  attributable Cd  When  in this so  sites  on  that  protein  to  to  metal  we  be  Smith,  the  lose  a l . ,  of  mole-  Brown  These and  effects  Zn  by  Bremner,  normal  no  is  high  1977;  Cu  1 974 ;  their  molecules  Chatel ,  pathological  1976).  (Friedberg,  have  of  suggested  high be  levels  due,  Hg  1  974).  conformational  longer  relative  to  the  f i t  near  in  the  laboratory,  in  the  same  sewer  in  binding  ratio  outfall.  as  of  Thus  Cd,  Cu  sewer  are  near  and  of  a the  metallothionein protein  trophic was Zn  level  suggested  were  bound  area. from  a  outfall levels  found  correlation  it  organisms  There  weight  increasing  increasing they  A  levels  mussels  same  elements  1977).  molecular  of  of  synthesis  a l . ,  polluted  study,  to  to  o u t f a l l .  this  the  et  with  levels  survival  trace  part,  the  high  metal  high  present  area  in  sewer  metallothionein the  to  bound  in  that  of  (Brown  increases  abnormally  polluted  the  et  displacement  enzymes  to  concurrent  Brown  and  the  may  enzyme)  In  with  and  metallothionein  "spillover"  1973;  metallothionein  nearness  that  to  of  (Brown  enzymes.  appeared  or  pool  substrate  sewer.outfal1,  (e.g.,  may  Irons  occurs,  presence  bound  Cd  a l . ,  1978;  Recently, the  capacity  metal 1oenzymes  shape  in  or  protein  and  or  metallothionein  binding  then  weight  on  is  in  of the  then  relatively are Cd,  non-  exposed, Cu  and  sediment  Zn  near  constructed  44 between weight then  survival protein  sampled  outfall levels  thionein cular in  at  the of  they  cease  high  molecular  Iona  Island,  Vancouver,  (see  Brown  farthest  near  et  B  is  ferred of  the  Station  District, At  the  to  the  and  a map o f outfall  of  was  at  to  Metal  and  high  metallo-  mole-  levels  their  survival.  B,  D and  tanks  Cu a n d in  Table  similar  (Greater  or  (Greater  E  1974).  loading  of  that  Vancouver were  after  D and  at  was  were  14  to  ratio  found  in  Sewerage changed days  were  obtained  added  The  elevated elevated  E  1.  to  Solutions  death  Zn  is  slightly  Station  water  E  greatly  from a  area).  Station  D i s t r i c t ,  mussels  bioassay  are  E,  analysed  study  while  very  C,  until  the  Stations  Drainage  bioassay  B  and f r o z e n  B,  shown  of  for  and normal  19.74).  time  B.C.,  Stations  Station  Cadmium,.  used  from  near  watter;  levels  elements near  C,  glass  E.  sewer  pool  metal  and t o  are  o u t f a l l .  related  levels  portions  bioassay  then  element  Sewerage  to  Station at  are  the  the  protein  Trace  sediments  Other  weight  molecular  Mussels  towards  near  mussels,  a l . , 1977,  away.  Vancouver  in  exist  collected  closest  Station  high  Methods were  Station  the  and m e t a l l o t h i o n e i n  Mussels  the  to  mussels  protein  and  in  distances  laboratory-exposed  Materials  in  levels  and m e t a l l o t h i o n e i n . .  various  these  weight  the  pool  until in  and. metal  1  g  mussels/L  from  these  of  trans-  tanks  these  the  sediments  and  Drainage  every  exposure,  three  days.  mussels  45  Table  1.  Levels water  of  are  Exposure 1  Cu  and  (collected  laboratory E,  Cd,  Iona in  mg  Cd 0.  from  exposure  Island,  Zn  of  added  to  bioassay  Station  E)  for  mussels  Vancouver,  B.C.  metal/L.  Cu  Zn  0  0  2  .0001  .009  .007  3  .001  .09  .07  4  .01  .9  .7  5  .1  9.0  7.0  from  the Station  Metal  levels  46 were  sampled  and  Mussels  were  parts of  of  these  0.9%  STIR-R  NaCl  frozen thawed  were  laboratory  were  water  bath.  Samples  £  10  to  Samples  were  10  a  ml/hour,  of  Two  eluted  the  ml  with  was  weight.protein  molecular  weight  using  and a  403 a  gether  atomic  for  of  each  of  give  tissue  (wet  the  all the  total  weight).  of  a  4.5 a  on  x  250  TRI-R homogenizer. minutes.  5 minutes  in  a  at  27,000  supernatant  packed  with  at  a  nm  to  x  were  G-75  flow  establish  pools,  gel.  rate  i . e . ,  the  the  metallothionein, (Brown in  et  of  arc  low  ).  spectrophoto-  using  a  Perkin equipped  background  Metal  levels  cytoplasmic  pools  were  in  the  1 977  analysis.  level  high  fraction  spectrophotometer  Deuterium  posi-1  and  a l . ,  each  absorption  determined  metal  70°C  fractions.  pool  atomic  ml  10  M NH4HC03 ml  soft  g_ f o r  resulting  2  the 3  teflon  recentrifuged  determined  were  furnace.  used  to  303  in  27,000  column  of  3 minutes  exactly  pool,  absorption  was  fraction  at  were  levels  graphite  rection  of  Elmer  Cadmium  Elmer  as  cytoplasmic  levels  Perkin  meter.  with  Zn  of  portions  with  cytoplasmic  molecular  Copper  for  at  0.01  read  various  gram  setting  then  K9/60  collected  Absorbance tions  for  were  Pharmacia  and  one  equipped  heated  minutes.  applied  a  centrifuged  Supernatants  for  at  motor  were  and  analyzed.  homogenized  solution,  Homogenates  until  each  cor-  in  each  added  pool  per  togram  Results Mussels Cu  and  hours  Zn in  1,  2  At  Iona  and  is  D  and  and  in  in  from Zn  metallothionein and  Zn  cytoplasmic pool  Copper  and  at  Zn  were  of  period  of  Cd,  48 Exposures 14  Stations  days.  C,  profile Cu  (I)  with  (II)  (III).  D  from  C  Zn  E,  in  the  low  moleStations  levels  protein B  of pool  (Table  molecular but.Cu  amounts  from  lesser  to the  lesser  and  Station  low  to  and  weight  from  appears  Mussels  contained  the  mussels  Cd,  pool  in  2).  weight  was  increased  2).  Station  E  exposed  decreased  with  increased  of  exposure  The  levels  of  Cd  pools  + and  Cu  in  Zn  in  Cd,  Cu  and  Cd  on  all  of  levels  all  (Table +  to  levels  increasing  levels  protein  This  those  creasing  weight  6.  Stations  had  pools  in  from  molecular  lower  (Table  laboratory,  cytoplasmic  than  were  from  profile,  pool  high  than  Mussels  at  to  B.  Island  the  pool  Mussels the  in  plentiful  protein  Iona  less  exposure  levels  cytoplasmic  and  this  were  exposed  for  1).  metallothionein  Cu  in  entire  high  Island,  (Table  Figure  weight  Cd,  Cadmium  5  Station  by  metals  E  Iona  survived  elution  shown  weight  the  at  gel  molecular  cular  and  mussels  sparse  B  these  4  survived  typical  E,  laboratory  characterized  high  C,  the  Island,  Station  of  3  but  A  be  in  Station  Exposures  and  E,  from  three  of  Zn  in  three  exposure.  pools  with  in-  2). the  high  metallothionein  molecular  increased  with  48  Figure  6.  Gel B,  elution  profile  Iona.Island, weight  thionein;  III:  pool .  mussels  Vancouver,  molecular  plasmic  of  protein  low  from  B.C. pool;  molecular,  I: II:  weight  Station high metallocyto-  i  1  1  10  15  Fraction  Number  r  20  T a b l e 2.  The d i s t r i b u t i o n trace Levels  elements  o f Cd, Cu and Zn among c y t o p l a s m i c  i n the l a b o r a t o r y  of Exposures  (Exposures  1-5 a r e g i v e n  Total  Exposure  Station  , High pool  MW  b  1-5) o r n e a r  i n T a b l e 1.  Cd Metallothionein  Low MW poo 1  pools  A l l metal  of mussels Iona  exposed  to v a r i o u s  I s l a n d , V a n c o u v e r , B. C.  levels  a r e i n jjmole m e t a l / g  Cu Total  l e v e l s of these ( S t a t i o n B-E).  tissue  (wet w e i g h t ) . 3  Zn High pool  MW  Metallothionein  Low MW pool  Total  High pool  MW  Metallothionein  Low MW pool  .016  .077  1  .0190  .0089  .0018  .0083  .301  .074  .043  .184  .225  2  .0205  .0067  . 0022  .0116  . 394  .079  .044  .271  .413  .151  .019  . .243  3  .0181  . 0057  .0017  .0107  .542  .128  . 063  . 351  .583  .215  .027  .341  4  .0147  .0049  .0014  .0084  .965  .211  .063  .691  1.058  .398  .034  .626  5  .0098  .0034  . 0012  .0052  3.416  .401  .223  2. 792  .769  .422  .032  .315  B  .0413  .0187  . 0052  .01 74  .757  .405  .188  .473  .286  .029  .158  C  .0169  .0071  .0031  .0067  .318  .089  .060  .169  .270  .153  .024  .093  D  .0187  . 0077  .0027  .0083  .633  .125  .108  .400  .349  .181  .021  .147  E  .01 75  . 0078  .0024  .0073  .631  .072  .050  . 509  .301  .164  .015  .122  .164  .132  Data i s compiled from p r o f i l e s such as those shown in Figure 6. 'Molecular weight.  O  exposure  level  in  Metallothionein metal  relative  Where  this  crease This  from  of  to  (Figure levels  and  7).  The graphs in  of  levels  pool  which  exposed  to  the  mussels  +  Cu  1  to.  3,  weight  +Zn  but  there  protein  a  weight  in  the  laboratory  4  of  further to  mussels  from  7.  Mussels  were  from  on  of  the  in  as  corresponded  to  3  to  4.  in-  point  pool. between  exposures  of  metallothionein  Island  animals  are  molecular  nonsurvival  from  Stations  high  molecular  levels in  in  laboratory the  C  indicated  metal  in  the  laboratory-  to  E  the  were  on  protein  the pool  Stations  B  highest  metal  Station  E  exposures  in  the  experiment.  Discussion It that  appears  evident  metallothionein  is  from  this  present  in  and  previous  mussels  B  protein  from  from  Station  weight  experiment.  lowest  the  also  weight  mussels  mussels  on  populated  of  those  the  Iona  sparsely  high  indicative  high  levels  the  the  Mussels  from  5.  laboratory-exposed  metal  increase  pool.  protein  nonsurvivors a  of  increase  dramatic  divisional  was  to  plateau  was  7).  portion  appeared  to  molecular  the  as  laboratory  Cd  small  the  Metallothionein  exposure  of  a  to  portion  D were  molecular  high  exposures  of  only  (Figure  corresponded  animals.  survival graph.  in  exposure  bind  occurred,  positions  Figure  had  levels  There  from  to  high  Exposures  metal  survivors  the  plateau  plateau  laboratory  appeared  Metallothionein slightly  the  and  studies, may  be  im-  52  Figure  7.  The  levels  molecular  of  weight  thionein. given sels B.C.  in from  have  thionein. metal  (HMW)  Table  1.  Stations have  1-5  at  in  weight All each  +  Cu  levels  B-E,  been  B-E  Iona  protein  and  high metallo-  those  represent Island, on  or  levels, are tissue  the  are  levels pool  in  pool  positions  metal  pool/g  Zn.  1-5  placed  those  metal  +  protein  Points  corresponding  molecular  Cd  Exposure  These  exposures  total  (wet  the  Vancouver, graphs  where in  mus-  the  they high  metalloin  jjmole weight).  of  Bioassay Survivors C9  D CO CO  +->  U) c N +  u  +  U  Bioassay Nonsurvivors  1.0  • 0.8 •  H M W Pool Metallothionein  0.6 0.4  <s o 0.2 E  —  >—  &C  ~i  2  3  Exposure  4  T "  5  Level CO  54 portant  as  Lambot,  1976;  portion  of  thionein crease This  The  to  of  effects  a l . , 1973;  found,  for  that  survival  of  organisms  Zn  mussels  for  Toxic  metal  sites  a l . ,  in  attributed of  metallo-  of  -The  increase  has  occurred, in  1977;  changes  malfunction  discussed,  of  (Winge  excesses  Conformational  "spillover" i t  et  pool  mussels.  c a n be  or  in  protein  studies  Brown  pool.  " s p i l l -  and appearance  nonessential ,  result  with  these  effects  binding  i n -  enzymes of  another  caused  metallo-  has  been  study  by  (1978c).  Cd was  exclusion and  for  laboratory  with  1976;  metallo-  protein  weight of  on  dramatic  weight  previous  metabolism.  after  Chatel  in  Smith,  1974).  reasons  the  appears  Cu  with  survival  (Noel-  laboratory  levels  was a  "spillover"  1978). of  there  molecular  noted  and  normal  levels  and In  of  Irons  interferences  and  to  been  (Friedberg,  thionein  due  has  metal  the  metallothionein,  high  between  substance  In  molecular  decreased  Parsons,  essential  of  with  metals,  these  Brown  the  interference  enzymes  high of  1978).  when  plateau, the  into  relationship  the  study,  saturation  metal  and  to in  detoxification  and Magee,  present  metals  essential  by  Talbot  the  concurrent  Brown  element  appeared  of  toxic et  trace  apparent  over" is  a  portion  probably as  i t  resulting entry  not  was  increased  of  the  a major  decreased  exposure from  sites  present  factor in  level.  competition  into  the  study,  the  influencing cytoplasm  This of  mussels.  i t  may  be  high  levels  At  Station  B,  Iona  it  is  Island,  greatly  Cd  pool  exposed  mussels  compared were  found  in  the  As  Cd  is  increased  in  the  ratio  than  in  Iona  Island  of  the  organic  Coombs  of  have  chelators  increase  of  mussels  tory-exposed  the  the  weight mussels,  would  be  any  ever,  is  present  those  from  indicate water  the  Station 3  the  at  rate  must  is  Island Cd  at  high  levels and  f i r s t  be  organic  accumulation  molecular  B are  as  laboratory  weight  high  than  of  high  levels  pool  were  a  would  mussels  be at  as  exposure;  less  48  and  the  of  unlikely Station  molecular  B  levels i . e . ,  protein  those  in  these  labora-  hours Cu  causative  high  4,  there  Cd.  high  and  Island.  decreased  George  the  at  of  effluent.  that  it  of  ratio  Iona  Iona  presence  of  the  that  uptake  and  survived  then  Exposures  may  occur,  Station  surviving  from  but  can  presence  in  Island,  Cd  the  in  Iona  sewer  of  Zn  at  ionic  protein  of  mussels  in  +  that  highest  death  in  the  both  mussels if  molecular  in  uptake  from  Cu  Laboratory-  outfall  the  by  suggested  levels  from  in  +  Alternatively,  concentration  mussels  Therefore,  Zn  Cd  as  weight  sewer  this  increased  before  pool  the  survival  molecular  stations.  mussels^at  +  influencing  high  other  near  Cd:Cu  chelators  Copper  the  administered  in  may.be  tissue  factor  exposure,  complexed  final  in  sediments.  (1977)  a  with  sediments  laboratory  higher  be  increased  protein  a  may  in  protein  intermediate  high in  there  Zinc,  weight  halfway  the  factor  that  B..  exposure.  howpool  between  between  levels  56 found  in  Station  survivors B  are  controlling The to  to  level  levels the  very  of  Cd  in  +  Cu  from  these  +  in  levels  mussels  may  level  as  they  metal  Therefore, of  trace  mussels  from  0.40 of  while  0.25.  increased creasing It study  a  both  from  with  trophic is  does  not  those  D  the  Nor  crease.  Rather,  number  excesses  of  pool. exposed  Thus  the  to  trace  occur  factor  it  as  in  the  et  B  to  found  sewer  of  high  2  exposed but  lower  to  5.  adapt  to  of  metallo-  metal  a  of  mean  that  ratio  this  outfall  or  1977). mussels  in  the  present  on  cytoplasmic  metal  levels  survivors high  may  not  the  level  of of  in-  when  weight  survival  the  metallo-  decreases  molecular  be  ratio i n -  metal  the  excesses Thus,  of  of  level  D  mussels.  ratio  have  to  equivalent  bound  5  a l . ,  influencing  elements  Stations  longer  mean  was  the  seem  metallothionein.  to  survival as  to  time  a  2  of  not  laboratory  protein  to  (Brown  increases,  metal  of  have  nearness  thionein  the  does  Exposures  had.more  study,  decrease  to  in  one  relatively  subject  weight  that  is  at  mussels.  from  have  Exposures  level  apparent  may  compared  to  previous  Zn  metallothionein  synthesis B  of  Mussels  been  molecular  those In  via  that  laboratory-exposed  have  Stations  thionein / high  as  be  mussels  metallothionein  on  the  than  could  elements  Zn  have  exposure they  on  stations  metallothionein  may  Since  survival  survival.  nonsurvivors  Mussels  it  the  metals  with  of  nonsurvivors.  sparse,  factors  correlate  had  and  protein organisms  metal  in  these  bound  organisms,  to  rather  metallothionein,  molecular the  but  weight  length  and  protein  level  of  how  much  but  instead  pool.  This  of  the  occurs may  metal  is  the  high  in  depend  upon  not  both  exposure.  Summary Mussels  were  sampled  Island  s e w e r , o u t f a l .1 ,  from  relatively  a  laboratory in  the  ments  for  ratio near  the  days,  sewer  to  slightly  in  exposures  plateaued, weight  to  there  protein  survive the  the  outfall Zn  in  the  thionein  dramatic  these  the  levels  outfall  in  were  laboratory-exposed  sampled  of  occurred  Cd in  in +  the  Cu  the  +  Zn  sedi-  the  laboratory-exposed  Cd  +  +  Cu  and  Zn  then  plateaued.  bound  increase these  Toxic on  increased  Cd in  +  Cu  high  mussels  effects  enzymes  Zn  molecular did  are  in  +  At  not  attributed  the  high  pool. at  a  These  molecular  in  Iona  In  Cu+'Zn;  metals  protein  sparse.  high  +  period.  populations  were  nonsurvival  sewer  exposure  weight  Mussel  a  the  exposed,  levels  metallothionein  was  of  bound  of  Mussels  were  metals  exposures  bounded  effects  molecular  lower  where  area  outfal1.  metallothionein  B.C.  various  these  mussels,  the  vicinity  Vancouver,  which  the  the  nonpolluted  .14  in  in  sampling mussels  weight  had  protein  laboratory-exposed mussels higher  from than  mussels.  area  levels pool  These  the of  for  higher  areas all  Cd  sewer +  Cu  indicative  mussels.  sampling those  near  +  of  Metallonear  but  the  the highest  metallothionein  levels  may  levels  near  be  due  the  to  sewer  a  longer outfall  exposure than  in  to the  high  metal  laboratory.  59 CHAPTER  IV  Interactions  Between  Cytoplasm  Duck  of  Cadmium  Liver  and  and  Zinc  in  Kidney  Preface This  paper  has  been  accepted  Chemico-Biological  Interactions,  1978.  are  The  This  authors  study  and  demonstrates  kidney  tissue,  importantly, high is  i t  molecular  saturated  Thus, have  the to  D.  binding  be  excesses that  protein are  of  before  appearing  W.  Chatel  Cu a n d  pool  of  in  before  (1978a). liver  Most  in  the  metallothionein  of  Zn  in  metallothionein  apparent  in  duck  Zn.  Cd c a n o c c u r  deficiencies  capacity  exceeded  a n d K.  by  be  metallothionein,  demonstrates  there  publication  and w i l l  Brown  that  stores  weight  if  A.  for  this  does  "spillover"  pool.  not  occurs.  Introduction Copper many  enzymes  protein  of  metals  such  a  the  function result  of  replace,  are  they  as  maintain If  Cd o r  or  then  they  and  or  may  to  their  when  interfer  conformational  longer  1974).  f i t  an  heavy  a b i l i t y  Dysfunction  produced  metals  to  competing  lose  1974)<  of  quaternary  are exposed  may be  excesses  with,  on m e t a l 1 o e n z y m e s  After  may no  functioning  tertiary  changes  toxic  metals  (Friedberg,  the  metal  (Friedberg,  1974).  molecules  both  heavy  conformational metals  for  metal 1oenzymes  Hg,  normally  essential  enzymes  necessary  functional  Bremner,  substrate the  of  essential  1974;  as  Zn  structure.  excess  to  and  or  (Friedberg changes,  binding  Alternatively,  sites some  on toxic  60 metals and  (e.g.,  Hg)  regulatory  activity  of  Brown,  1 965;  moved  of  the  accomplished can  zone  by  of  heavy  inactive  protein high  high  levels  Vallee,  can  the  is  bind  nontoxic  and  or to  the  of  liver  and  metallothionein  may  These  apparatus  and  are  the  tubules  0.01% per  of day  the via  This levels  of  body the  study Cd,  Cu  be  pass  excreted and  stored  burden  urine  of  in  Zn  the  them  bio-  (10,000)  presence  sulfhydryl rendering  in  glomular  metallothionein 1964;  into  of the  f i l t r a t i o n  reabsorbed Only is  in  about  excreted  Nordberg,  1972).  interrelationships  between  on  and  metallothionein  cyto-  amounts  liver  kidney.  1964).  the  small  or  of  and  (Piscator,  the  urine  the  is  thus  Very  from  the  (Piscator,  reports and  in  the  solubilized  through  This  re-  metallothionein,  The  organism  released  be  (Margoshes  metals  tissue.  must  weight  to  1960).  stored  1 968;  977).  due  heavy  and  functioning,  rendering  cysteine  exposed  kidney  bloodstream.  renal  groups  Vallee,  is  metals  activity.  1  of  a l . ,  cellular  molecular  acid  et  protein  al..,  catalytic  (Gerhart  White  thereby  low  chelate  Metallothionein plasm  a  amino  Kagi  the  et  sulfhydryl  of  1957;  groups them  in  of  (Brown  lost  toxic  biological  into  regulation  proper  or  metals,  Metallothionein  is  1 969 ;  ensure  metals  enzymes  normal  a l . ,  production  bind  logically  to  that  subunit  et  essential  from  which  Jovin  Thus,  certain  so  catalytic  1977).  excesses  split  subunits  the  Schachman,  can  on  the the  high  molecular et  weight  a l . , 1977)  Materials  in  and  Greater  were  Scaup  1977). of  and f r o z e n  9 ml  centrifuged  at  27,000  10  minutes.  Supernatants  for  10  (.14  ml)  minute  a l . , 1977).  minutes were  Sephadex  0.01  M NH4HC03  Coleman  Perkin  by  Elmer  were  were  Samples  to  fine  x  at  a  403 atomic  occidental is) o u t f a l l ,  known  to  be  (Brown  et  a l . ,  removed  tissue The  at  were  the  time  at  then  in  6 ml  27 , 0 0 0 x  1974;  to 1977;  supernatants  were  ml  packed  eluted  with  fractions.  2 5 0 a n d 2 8 0 nm o n a Cadmium on e a c h  g_ f o r  Brown,  K25/1 00 column  10.2  absorption  super-  recentrifuged  Filtrates  furnace  were  and heated  Cherian,  Pharmacia  in  and the  rehomogenized  Resulting  g e l .  thoroughly  homogenates  minutes  were  spectrophotometer. graphite  metals  were  (Mel an i t t a  sewage  is  combined  g_.  and c o l l e c t e d  was r e a d  124D  determined  G-75  10  1972a;  27,000  applied  with  Absorbance  at  area  centrif ugation  (Webb,  Scoter  This  each  Pellets by  Surf  Island  heavy  x g_ f o r  followed  et  tissue.  Iona  0.9% NaCl.  0 . 9 % NaCl  Brown  (Brown  analyzed.  of  of  1  of  until  of  collected.  for  pool  (Aechmophorus  samples  portions  in  70°C  the  levels  and kidney  gram  mari1 a) , Grebe  near  homogenized  natants  and kidney  Columbia.  high  Liver  Three  and Wester  British  with  capture  liver  (Aythya  collected  Vancouver, polluted  duck  (enzyme-containing)  Methods  perspicillata) ducks  protein  Perkin;  levels  fraction  Elmer  were with  spectrophotometer.  a  Cu a  and  Zn  levels  Perkin  Elmer  Deuterium  were  303  determined  atomic  background  by  the  absorption  correction  was  flame  method  with  spectrophometer. used  for  all  deter-  or  kidney  minations. Res u l t s Typical tissue  elution  profiles  are  presented  in  Figure  identified  according  to  its  peaks  found  in  previous  1 974;  Winge  et  a l . ,  Cd  peaks  liver  on  tissue  weight  and  described of  the  in  Table  Cu  Cd,  molecular  et  level  to  weight et in  has  The  protein  all  30,000  molecular  A  is  of  moieties  1 977).  peaks  dual  pool  Cd-binding  a l . ,  to  Thompson,  1 977).  the  been  position and  a l . ,  weight  (Prohaska  Zn  of  (Olafson  correspond  Cu  has  or been  linearly  on  9A-B).  appears  Zinc  appears  protein  to  Cu/g to  pool  9A-I).  It  high  with low  become tissue  increase at  low  peak the  the  The  each  against  (Figures  metallothionein  pool  in  plotted  metal  cytoplasmic  weight  to  metals  (Figures  ;umole  peak  Brown  of  each  and  plasmic  Each  summary  presented  3.  of  plasm  8.  similarity  level  increases  pool  liver  molecular  previously  metal  level  appear  duck  studies  1 973 ;  high  115,000  total  The each  the  from  (Table total is  of  molecular  (wet  weight),  tissue  levels  in  at  protein cyto-  cytoabout  (figure the  of  that  weight  weight  saturated  on  cytoplasmic  Cu  Cu  linearly  for  apparent  molecular  increases  3)  Zn  high  0.40 9C).  molecul  (Figure  9D)  63  Figure  8.  Gel  elution  (A)  and  protein  profiles  kidney pool;  molecular  (B). II:  weight  of i  :  Greater high  Scaup  molecular  metallothionein; cytoplasmic  pool.  III:  liver weight low  0.40.3-  Cu (ppm)  0.80.6-  Zn(ppm)  0.40.2-  Table  3.  The d i s t r i b u t i o n o f Cd, Cu and Zn amongst c y t o p l a s m i c c o m p i l a t i o n s . of m e t a l MW:  molecular  levels  weight.  ND:  from  profiles  Greater  Surf  s u c h as F i g u r e 4 i n ^ m o l e / g t i s s u e  are;  Metallothioein  Low MW pool  Total  Zn . High pool  MW  Metallothionein  Low MW pool  Total  High pool  MW  Metallothionein  Low MW pool  .00418  .00053  . 625  .252  . 265  .108  .234  .101  .098  .035  K i dney  .00845  .00187  .00632  .00026  . 342  .156  .096  .090  .067  .043  .006  .018  L i ver  .00641  .00142  .00391  .00108  . 408  . 1 32  .153  .123  .151  .114  .020  .017  .071  .089  .058  .041  .005  .012  Scaup L i v e r K i dney  W e s t e r n Grebe L i v e r Kidney Greater  Data  (wet w e i g h t ) .  Cu H i g h MW poo 1  of ducks.  .00107  Kidney Greater  and k i d n e y  .00578  Scaup L i v e r  Scoter  from l i v e r  not d e t e c t a b l e .  Cd Total  pools  Scaup L i v e r Kidney  .00294  .00089  .00107  .00098  . 262  .102  .01141  .00320  .00290  .00530  .230  .074  .088  .069  .119  .106  .010  .003  .01330  .00590  .00370  .00370  .225  .079  . 084  .062  .045  .040  .003  .002  .00164  .00065  .00040  .00059  .181  .081  . 041  .059  .078  .072  .002  .004  .00147  .00068  .00043  .00036  .170  .077  . 037  .056  .029.  .028  ND  .001  .01650  .00212  .00481  .00957  .230  .070  .106  . 054  .170  .123  .045  .002  .01571  .00408  .00788  .00375  .189  .062  .075  .052  .065  .053  .006  .006  67  Figure  9.  The  variation  molecular and  the  with  weight  low  of  cytoplasm, levels  Cd,  Cu  protein  molecular  increases  kidney Metal  of  are  in  Zn  pool,  weight  these o:  and  jjmole/g  the  high  metallothionein  cytoplasmic  metals  l i v e r ,  on  ©:  in  liver  pool and  kidney.  tissue  (wet  weight).  HIGH MOLECULAR WEIGHT PROTEIN POOL  METALLOTHIONEIN  LOW MOLECULAR WEIGHT CYTOPLASMIC POOL  Cu TOTAL 0 CYTOPLASMIC  ~20  To  AO~  •15i Zn  .10 .05  TOTAL 0 CYTOPLASMIC Zn  o  .10  .20  .006 Cd  .10  .008  H  o  .004  8  TOTAL 0• CYTOPLASMIC Cd  .20  • .008-  .004 .002  o  .004  #  .005  .010  .015  .005  .010  .015  .005  .010  .015  The be  kidney Zn  while  jjmole  saturation an  in  Low  of  (Figures  kidney to  of  be  pools pools  to  appears  liver  to  Cd  10). the  high  Zn  saturation  pools  in  liver  the  on  of  protein  Zn  levels  total  and  kidney  the  portion  pool  is  l i t t l e  high  and  levels  of  Zn  high  molecular  at  low Zn  levels  weight  9D)  does  of  high  molecular  tissue  appears  cytoplasmic  than  on cytoweight the  pools 25%  (Figure  of  on  the  protein  coincide  Cd  appear  11).  weight to  portion  where  protein  more  Cd  molecular  percent  and  generally  the  low  or  9F).  pools  Seventy-five  of  Zn  cytoplasmic  metallothionein  the  with  (Figure  clearly,  to  at  (Figure  shows  more  related  molecular  appear  occur  cytoplasmic  Only  (Figure  Thus,  of  be  levels.  saturated  Zn  However,  on  Zn  weight  increasing  on  to  to  Zn/g  Concurrent  cytoplasmic  present  appears  metallothionein  three  9G-I). Cd  lower  increase  the  with  appears  cytoplasmic  an  appears  on  cytoplasmic on  pool  cytoplasmic  tissue.  levels  weight  (Figure  and Zn  Zn  increase  cytoplasmic  at  high  Cd  levels  pools  saturation  molecular  the  molecular  Cd  jumole  of  to  plasmic  0.06  protein  Zn/g  appear  these  about  liver  relationship  weight  weight  cytoplasmic  Levels  of  at  acceleration  9E). no  molecular  saturated  tissue 0.14  high  with  metallo-  th ione i n. Discussion Results molecular  of  weight  this  study  protein  indicate  pool  is  Zn  that  once  saturated,  the  high  excess  Zn  70 is  stored  high  on  metallothionein.  molecular  plasm,  Zn  saturation at  may  a  be  liver.  of  of a  the  portion  these  of  must  is  level  the  been  more  weight  requirements  on  the  high  the  That  Zn  in  a  of  of  pro-  hence,  molecular  pool  higher  certain  and  Zn  kidney,  activity  center  meta11oenzymes,  that  protein  that  enzymes,  the  cyto-  met.  metabolic  metabolic  in  cell  indicate  than  higher  contain  being  have  of  molecular  Zn  the  may  occur  have  weight  pro-  poo 1 . The  appear plasmic Copper for  high  to  be  Cu  molecular Cu  have  levels  are  requirements  found  levels (Brown,  on  Cu the  1 977),  phytoplankton  may  and  weight  protein  Cu  in  levels high  Cu  profiles  Zn  profiles.  higher  to  be  pool  would  higher then  than  to  8-fold  weight  (Brown,  1978).  than  than  Zn  Zn  Perhaps become those  appear  to  Cu  requirements  higher  1978),  studies  than  pool  Zn  of  fish  1 978b). and  high  zooplankton molecular  saturated  reported be  cyto-  levels.  protein  the  not  Previous  and . C h a t e l ,  Brown,  does  although  organisms.  3-  and  pool  ducks  higher  marine  mussels  might  be  protein  these  molecular  Parsons,  levels  in  3-fold  (Cloutier  (Brown  weight  saturated  metal 1oenzymes  with  pool  high  higher  liver  it  Zn  this  liver  2-fold  the  of  fraction  metal 1oenzymes  organisms,  tein  of  reflection As  higher  protein  saturation  requirements  occurs  weight  As.enzymes  in  similar  in this  in  ducks study,  shape  to  71  Figure  10.  The  variation  plasmic  Cd  protein  pool  of  on  the  the  high  (BMW)  +  cytoplasmic  pool  on  molecular  the o:  the low  high  ^umole/g  ®:  tissue  (wet  low  total  cyto-  weight  molecular  weight  with  increases  of  Zn  weight  protein  pool  +  weight  kidney.  of  molecular  the  (LMW)  molecular  l i v e r ,  portion  cytoplasmic Zinc  weight).  levels  pool. are  in  72  MW1+MWH  NO  PO  %  73  Figure  11.  The  variation  plasmic of  Zn on  pool  the  pool  levels  weight).  the  portion  of  Cd on m e t a l l o t h i o n e i n high  (HMW) + t h e  plasmic Zinc  of  molecular low  (LMW). are  liver,  i n >imole/g  with  weight  molecular o:  total  levels protein  weight ®:  tissue  cyto-  cyto-  kidney. (wet  74  N I 3 N 0 I H 1 0 1 1 V 1 3 W N O PO %  The plasmic is  present Cd  higher  appearing when  Zn  appears  as  exists,  whereby  the  high  occupy appears  a  be  high  75%  of  Cd  pools  with  it  probable  the in  high  upon  fewer  appears Zn  that  tissue  sites  tissue.  This  may  enzyme  binding  sites  in  Competition in  tissue  exerts  is  toxic  (Horvath, reduce has  also  until and  the Cd  well  and  toxic  been  occurs  on  and  via that  effects  shown  binding  Zn  Cd  that  its  the  high  available  most  Cd  so  bind,  for  will  that  it  is  metallothionein.  and  liver  reaction  Cd  is  pool  Zn  lower  is  lower  not from  Zn  levels  that  there  can  compete  levels  weight  tissue,  displaced by  not  Cd  kidney  similar  It  is  effect  Cd  toxic  capacity  It  molecular  for  high of  pool.  of  are in  metallo-  kidney.  established.  effects  1976)  the  of  pool  low  and  reflect the  weight  Rather,  suggests Cd  cyto-  and  protein  which  kidney  high  of  equilibrium  are  levels and  That  l i v e r ,  for  Zn  equilibrium  weight with  Zn  pools.  both  type.  this  pools,  of,  the  in  this  compared  binding  on  levels  molecular  kidney  synthesis  on  of  weight  by  portion  dependent  levels  displaced  Since  dependent  concentration high  the  molecular  lower  these  induce  is  are  on  to  that  high  sites  available  low  the  molecular  binding  to  on  when  low  indicates  levels  though  and  Zn  study  of  known  on  levels  binding  Zn  of  (Flick effects  prevent  a l . ,  of  Cd  metallothionein  molecular  Cd  metal 1oenzymes  Zn  et  that  sites  weight  or  1971). do  not  is  protein  It occur  exceeded pool  (Winge  et  Ganther Cd  may  a l . ,  (1975)  have  become  may  Irons  30,000 be  the  when  Zn  been  suggested,  on  the  high  molecular  toxic et  is  a l . ,  appear  via  1977).  The high  when  the  binding  exceeded,  but  also  high  effects  of  weight  Cd  pool,  of  increase  Zn  of  it  it is  increase  Cd  on  the  of are  this  anhydrase. levels  may  indicates  of  to  exert (Brown  Cd  pool,  may  not  metallothionein  is  deficiencies  Zn  pool.  l i k e l y ,  of  Since  on  the  effects  of  Cd  on  toxic  high  therefore,  Cd  indicate  that  protein  appears  high  suggest  high  pool  of of  metal 1oenzymes  weight  toxic  protein  metallothionein,  protein  when  a  they  that  study  there  weight  occur  protein  ficiencies an  when  on  and  effects  carbonic  with  capacity  Chen  toxic  weight;  from  molecular  molecular  the  protein  present  1 976).  occurs  interference  the  the  weight free  on  only  it  therefore,  available,  effects  that  metal 1oenzyme  has  Cd  Smith,  molecular  It  that  and  suggested  apparent  approximately protein  1 973 ;  molecular  that by  depermitting  molecular  weight  protein  study  be  of  significance  interactions  in  carcino-  pool. Results for  mice  12.5 the  the  understanding  genesis. of  from  It to  weeks high  liver  was  the  trace  metal  reported  carcinogen  resulted  molecular  tissue  present  (Brown  in  a  a  previous  study  diethylnitrosamine 39%  weight and  in  may  reduction  protein  Chan,  pool  1978).  of in When  that (DEN)  Zn  levels  exposure for on  pretumorous Cd  was  adminis-  tered Cd  concurrently  accumulated  Cadmium  1978).  of  were  Results  molecular Thus,  cytoplasmic  decreasing Cadmium  Cd  levels  bearing  is  a  (Brown,  It  carcinogenicity requiring  1977).  with  1 963 ,  1 964;  1972).  Cd.. o r  molecular  thus In  Zn  if  Cd  is  prevent  cancer.  previous  studies  portion  of  pool  in  cell  cancer  carcinogen 1971;  pool,  has been  Brown  1977), pool  Cd e x e r t s  binding  from (Gunn  on  (Brown,  occurring et  when  present  then  Zn  when  al . ,  Ciapparelli  found  i t s  sites  division  of  both  tumor-  protein  that Zn  mentioned  since  in  1971;  weight  the occurrence  i t  cancer  with  carcinogenic  prevent  of  Morgan,  involved  protein  deficient.  and has been  suggested  prevents  high  protein  increased  molecular  and Cohen,  weight  might  are  1970;  an o r g a n i c  Poswillo  Thus,  carcinogen and  enzymes  Chan,  that  Zn  the  binding  pool.  interference  Furthermore,  given  high  high  is  weight  the etiology ratio  and  on t h e  pool  pool.  the  indicate  increase  carcinogen  has been  via  might  protein  Brown  appear  this  additional  exceeded  1977;  molecular  (Morgan,  on t h e  1977).  Zn  in  Cd:Zn  organisms  i f  this  weight  study  on t h i s  potent  and the  (Brown,  pool  high  levels  a l l of  have  Cd w i l l  carcinogen  involved  particulari1y  the  protein  Zn  low to  present  liver  Cd o n t h e  almost  molecular  too  the  75% o f  an o r g a n i c  possibly  high  far  from  weight  as  DEN,  metallothionein  approximately  by  on t h e  levels  capacity  with  et  al . ,  on t h e  given  with  a  Cd on t h i s  pool  that  Hg  Cd o r  78 did  not  exert  their  in  levels  high  of  metallothionein;  toxic  enough if  then  they  occurred  pool  with  concurrent  1973;  Brown  and  on  that  protein  before  is  exceeded  is  an  this  if  pool  other  and  high  studies  of . C d ,  but  the  to  the  on  binding  pool  is  and  measure its  the  not  suggest only  status  the  weight  Since of  Cd  there on  possibility future  tissue  the  has  metallothionein  of  environ-  concentrations  distribution,  of  a l . ,  molecular  of  that  the  cytoplasmic Zn  of  et  study  occurrence  because we  high  capacity  protein  saturated.  between  present  exceeded  (Winge  present  the  Zn  was  weight  capacity  not  were  binding  capacity  The  occur  effects,  also  they  molecular  1978).  may  cancer,  sublethal  relation  the  exceeded  binding  association  mental  in  the  unless  pathological.effects  Cd  this  apparent  have  Parsons,  demonstrated pool  to  effects  especially  organism.  Summary Ducks to  be  were  polluted  G-75  molecular Zn  collected with  determined  Sephadex  ly  were  for  gel.  weight  saturated;  kidney  plasmic  Zn/g  liver  protein  pool  was  unless  a  marine  metals.  Gel  both  liver  Zinc  increased pool  saturation  Zn/g  Cd  heavy  protein  plasmic  cytoplasmic  from  (wet (wet  levels  weight)  on  was  the Zn  elution  kidney  this were  and  0.14  0.06  was  /jmole  molecular  saturated.  If  high apparent-  >imole  Seventy-five high  using  on. t h e  pool  known  profiles  tissue  linearly  until  weight).  found it  and  environment  cyto-  cytopercent  of  weight  this  pool  was  79 Zn  saturated,  plasmic  Cd  discussed  then  appeared in  terms  binding  sites  on  the  both  thionein,  on 1  high  with  excesses on of  of  Zn  and  up  metallothionein. a  competition  of  Cd  Copper  molecular  protein  increases  of  Cu  in  75%  of  Results  metal 1 oenzymes. weight  to  tissue.  and  Zn  cytoare  for  increased pool  and  Zn linearly metallo-  CHAPTER  V  Relationship of  Mercury  and to  Chum  Between  and  Toxic  Salmon  Mercury  in  Cytoplasmic Effects  to  (Oncorhynchus  a  Distribution  Controlled  Zooplankton  keta)  Exposed  Ecosystem  Preface This Research The  paper Board  authors  appeared of  are  in  Canada,  D.  A.  Brown  demonstrates  that  effects  of  appear  do  metallothionein molecular  is  weight  theory  described  study,  like  actions  in 12  of  paper  wouldn't In  and  be  high 13  as  the  able  to  and  et  not  the  in  the  high  supported.  and  This inter-  essential  protein  considered  of  "spillover"  is  the  paper  capacity  demonstrates  weight  understand  This  appears  the  element,  in  880=884.  pathological  binding  (1978b),  editor  pages  apparent  a l . (197.7)  appear  Fisheries  Parsons.  Thus,  trace  the  35,  mercury  pool.  molecular  did  R.  until  Chatel  toxic  T.  readily  Brown  and  a  the  Figures this  by  of  Volume  and  exceeded  protein  Brown  between  elements  not.  Journal  1978,  clearly  Hg  the  pool.  published that  trace  version  most  readers  them.  troduction Heavy  organisms acquired  metals but  1974;  of  prior  tolerance  Hummerstone, result  such  1971;  synthesis  Piscator,  as  Hg,  exposure to  1964).  to  increasing  Bremner, of  Cd,  the  Cu  and  these  protein  are  metals  metal  1974).  Zn  can  toxic  to  result  levels  (Bryan  and  Tolerance  occurs  as  metallothionein  Metallothionein  is  a  low  in  a  (Leber, molecular  weight the  (.10,000)  presence  protein  of  high  (Margoshes  and  sulfhydryl  groups  and  Zn  thus  (Winge  rendering  (Piscator,  can  them  in  in of  1957; bind  a l . ,  1964).  solubilized  levels  Vallee,  et  high  the  Kagi or  1975;  less  sulfhydryl amino and  chelate  Sabbioni  toxic  to  and  kidney  acid  due  to  cysteine  Vallee,  1960).  The  Hg,  Ag,  Sn  and  Cd,  Cu,  Marafante,  1975)  the  exposed  organism  is  usually  stored  Metallothionein  liver  groups  cytoplasm  in  higher  organ-  isms . One  obvious  means  tolerance  to  heavy  synthesis  of  excesses  This  excess  able  to  thionein  always  i.e.  metal  and  Vallee,  Kagi,  1974;  Hg,  Winge than and  et Cd  Vallee,  exposure  Ag  to  Hg  and  or  Cu  the  is  Pul.ido  metals  known  that  al . ,  1974;  This  Zn  in  has  metallothionein Pulido  Cd  Hg,  et  these  1 966 ;  al . ,  in  in  approximately  binding 1 966).  elements  organisms,  and  it  has  response half  Davies,  lower  Upon  of 1975;  affinity  sites  replace  (Kagi  Buhler  Recently  much  sub-  metallo-  produced  a  avail-  residues  and  by  exposure.  upon  state  Bremner  be  readily  heavy  1975).  Zn  would i n i t i a l  cysteine  et  Davies,  (Leber,  be  saturated  three  contains  1 960; or  it  upon  then  metal 1othionein  1975). for  protein  would  each  1 961 ;  acquired  detoxify  in  for  that  sites  a l . , or  this  and  exists  Bremner  to  binding  of  metallothionein-mediated  might.be  However,  1 960,  apparent  its  bind  ion  become Cd,  metals  exposure.  one  which  metal 1othione in  quickly  sequent  by  a. Zn  (Kagi second on  metallo-  thionein than  so  Cd o r  compared If or  Hg,  sites  that Hg  (Bremner,  organism  is  exposed  then  there  may n o t  on m e t a l l o t h i o n e i n  than  the  rate  will  result  pool.  novo  in  This  et  three  "spillover"  and  Cu a n d  Zn  (Friedberg,  of  1 9 6 8 ; Brown When these  et  latter  Hg  metals  weight  protein  and  are  natural  (Friedberg,  1974),  Zn  Cd o r  Hg  a l l  of  the  of  Cd t o  with  the  1 973 ;  the  Cd  binding Hg  may  These  or  a l . ,  exert  or be  Cd. slower  conditions enzyme  appearance Brown,  of  1 977 ;  1976). effects  by  metal 1oenzymes.  This  changes  the  no  1965;  shape  longer  ' s p l i t s  f i t  Jovin  et  may  Brown,  displace  Cu o r  may a l s o  components they  (Bremner,  are  of  into  be  lost  re-  enzymes  sites  subunits so  (Ger-  that  (White  et a l . ,  1 977). Zn  from in  the  pool. many  relatively  1974).  these  a l . , 1969)  appear  (enzyme-containing)  of  binding  enzymes  activity  a l . , 197.7;  Cd o r  Hg  dose  toxic  or  enzyme  nontoxic  their  molecules  and Schachman,  high  metals.  conformational  1974),  regulation  with  in  of  et  rather  Zn-containing  and S m i t h ,  cadmium  dimensional  substrate  Irons  very  metallothionein  coincides  (Winge  cytoplasm  relatively  detoxify  these  a l . , 1977;  a  enough  influx  effects  Mercury placing  a  is  to  of  of  the  1974).  synthesis  "spillover"  pathological Brown  of  be to  into  Zinc  Hg  de  hart  released  Cd o r  Further,  so  is  ( L e b e r 1 9 7 4 ) .  with an  Zn  metallothionein, high  molecular  However  as  Cu  metal 1oenzymes nontoxic  compared  83 This  study  previously plankton  reports  reported  exposed  "spillover" Materials  of  and  The  growth  to  Hg  findings  Hg  metallothionein  (Controlled  consists  Victoria  (Menzel  Ecosystem  lateral  currents  column,  conducted Hg  ment  on a  Inlet  the  added  form  of  marine of  on  day  H.gClg)  produce  the  captured  were  final  was  the  into 10.  By  day  i n i t i a l  a  On  on  with  enzyme  pool.  rate  matter  of  due  were  concentrations  day  An  the  1  experi-  of  this  CEEs.  of  of  Hg  10,000  et  a l . ,  Uniform  a  diffuser and  to  day.  one-tenth  1 977 ) .  Windom, the This  settling  molecular  approximately  to  throughout  2% p e r  Saanich  the  CEEs  (Topping  decreased  impact  fry  (in  1 jug/L.  surface  than  the  use of  of  Chum  two  by  large  population  5 and  the  determine  into of  Inlet,  experiment  mercury  the  (Grice  of  10,  attachment  greater  levels  to  approximately  to  simulate  effects  three  was a i d e d  m below  Experiment)  Saanich  day  concentrations  occurred  Hg  1976  of  concentrations  Mercury  72,  each  the  pelagic  introduced  10  particulate  of  Also  to  decrease  1977)  the  in  recycling.  natural  ring  at  zoo-  controlled  These  for  ecosystem.  mercury  experiment  and  Pollution  suspended  1977).  summer  of  1977).  (CEEs)  except  distribution lowered  to  polyethylene  and sediment  in  portion was  fish  of  :  1350 m  and Case,  water  of  of  ecosystems  natural  was  of  and W a l l a c e ,  3  experimental  relationship  Methods  CEPEX  f a c i l i t y  the  reduction  (Koeller  from  on  weight. those  of  On mesh  day  net  as  plankton Fish  72,  described  were  were  drained  captured  similarly exposure  zooplankton  frozen were  were  by  Grice  of  excess  with  until  a  sampled  et  randomly  specially  selected  a  a l . , (1977).  water  analyzed.  using  The  and frozen  designed  Three for  2 5 jum  at  seine  fish  from  analysis  zoo-  in  -20°C. and  each  the  present  study. Fish genized  livers  in  was c o l l e c t e d ,  fuged  at  fugation  heated 27,000  g_.  Pharmacia  measured  at  photometer.  photometer  correction.  method  u t i l i z i n g  cal  100,  a  1  min  (Webb,  for  contents  for  Zn  tion,  N=15),  and f o r  was J  this  + +  Hg w a s  were  were and  final  column  collected  of  fractions  the  spectro-  absorption deuterium by a  (Pharmacia  UV  model  3 . 3 % (friean 3 . 5 % (mean  + +  arc  cold  Control 100).  standard +  were  spectro-  standard  6 . 0 % (mean  centricentri-  Elmer  with  Unit  The in 0.9%  Perkin  was m e a s u r e d  Optical  197.2a)  cm P h a r m a c i a  303 atomic  Cu w a s J  N=15),  N=15).  UV  of  Fractions  method  g_.  Supernatants  g e l .  Elmer  homo-  rehomogenized  1.6  3 0 cm c e l l  tion,  deviation,  G-75  x £.  thawed,  27 , 0 0 0 x  100 x  Mercury  Pharmacia  precision  a  at  supernatant  the flame  ground  model  for  C u a n d Zn  Perkin  using  later  pellet  27,000  The to  the  2 5 0 a n d 2 8 0 nm o n a  The  on a  at 70°C  was a p p l i e d  containing  measured  to x  were  and c e n t r i f u g e d  and c e n t r i f u g e d  combined,  and  zooplankton  0 . 9 % NaCl  supernatant NaCl  or  back-  vapor Unit Analytideviadevia-  standard  By those  found  Leber, Brown as  comparing in  1974; et  containing) weight  the  1977;  Brown,  to  the  pool,  these  total  determined  was  not  cytoplasmic  on  pool  were  of  one  composite  in  protein  or  low  the Zn  in  added  of  identified (enzyme-  in  fractions  together,  pool. each  fractions.  t h e n . ca.l c u l a t e d  1976;  molecular  levels  each  sample  individual  were  with  1975;  Marafante,  were  each.metal  Yip,  weight  Cu.and pools  profiles  and  fractions  metallothionein,  detectable  each  1977),  The  metal  1974;  molecular  pool.  and  (Casterline  Thompson,  high  level  was  in  studies  and  cytoplasmic  comprising give  previous  absorbance  Olafson  a l . ,  belonging  protein  Mercury  pool  Metal  per  gram  of  the  very  large  to  as  it  levels  tissue  ex-  tracted. Results The of  Hg  most  in  prominent  the  high  molecular  of  both  fish  ing)  pool  Hg/L  (Tables  4  and  production  there  l i t t l e  When  this  reached,  limit  of  excesses  in  appears  at  pool  (Figures  In  fish  liver  Hg  1 jug  and  cytoplasm,  in  the  a  to  5 jjg  limit  to  Hg/L  exposure,  from  1  high  is  to  Hgas  5 jug  Hg/L.  apparently  molecular  weight  13). there  are  and  Zn  with  increasing  Hg  exposure.  appear  to  be  reflection  of  decreases  a  be  production  appear  12  the  to  increase  (enzyme-contain-  exposed  Hg-thionein  Hg-thionein of  protein  zooplankton  There  increase  is  weight  reached  protein  Cu  and  5).  thionein is  change  decreases These of  both  of  total  decreases Cu  and  Zn  Table  4.  Distribution to  various  Hg exposure c o n c e n t r a t ion Hg/L)  Metal  (f9  Hg Control  of  Hg,  Cu  and  concentrations  Zn of  amongst Hg.  Cytoplasmic total (jimole metal/g tissue, wet weight)  0.61  X  10"  MW:  cytoplasmic Molecular  0.11  X  1 .832  0.592  Zn  0.  0.111  Hg  0.87  X  10  -3  0.23  X  Cu  1 . 558  0.402  Zn  0.  0.071  Hg  4.77  104  10"  Weight.  H i g h MW p r o t e i n pool (jimole m e t a l / g tissue, wet weight)  Cu  164  pools  4.04  X  10"  from ND:  liver Not  or  Meta1 l o t h i o n e i n (jimole m e t a l / g tissue, wet we i g h t )  0.29  x  10  0.35  x  10"  X  0.22  X  10"  0.29  X  10"  0.733  ND  0.38  L o w MW c y t o p l a s m i c p o o l (.umole m e t a l / g t i s s u e , w e t we i g h t ) .  0.053  0.424  10"  exposed  0.708  ND -3  salmon  Detectable  0.532  10  chum  • 0.033  10"  0.35  X  Cu  1 .445  0 . 353  0 . 347  0.745  Zn  0.114  0.075  ND  0.039  10"  Table  5.  Distribution to  Hg  exposure  various  Metal  Hg Con t r o 1  of  Hg,  Cu  and  concentrations  Zn of  amongst Hg.  Cytoplasmic total (pmole metal/g tissue, wet weight)  0.40  X  10  MW:  cytqplasmic Molecular  0.32  X  0. 359  0.041  Zn  0.299  0.012  0.96  X  10  -4  Weight.  H i g h MW p r o t e i n p o o l (jimole m e t a l / g tissue, wet weight)  Cu  Hg  pools  0.40  X  10  -4  from ND:  tissue Not  of  Meta1lothionein (.umole m e t a l / g tissue, wet weight)  0.04  X  10  -4  0.27  Low MW c y t o p l a s m i c pool (jimole m e t a l / g t i s s u e , wet weight)  0.04  X  10"  0.286  ND  -4  exposed  Detectable.  0.033  10  zooplankton  0.288  X  10"  0.28  X  10"  Cu  0.352  0.046  0.039  0.267  Zn  0.283  0.016  0.005  0.262  Hg  1 .93  X  10"  1.17  X  10  0.26  X  10  -4  0.50  X  10"  Cu  0 . 326  0.031  0.045  0.249  Zn  0.212  0.011  0.026  0.1  75  OO  88  Figure  12.  Gel (A), and  elution chum those Hg;  I: II:  high  profiles  salmon  exposed  exposed o  -o  molecular  to  pool.  5 jug  Cu;  control to  1  III:  jjg  Hg/L  protein low  chum Hg/L  (C) -©  e  weight  metallothionein;  cytoplasmic  from  for  salmon (B), 72  days.  Zn . pool;  molecular  weight  90  Figure  13.  Gel (A), and  elution  profiles  zooplankton those  exposed  exposed  —Hg;  o — weight  thionein;  III: pool.  to  -oCu;  molecular  plasmic  from  5 jug  to  1 jjg  Hg/L  ®  protein  low  control  Hg/L  for ©Zn.  pool;  molecular  zooplankton  72 I:  II:  weight  (B), days. high  metallocyto-  in  the  and  enzyme-containing  of  Zn  (Table  low  crease  to in  of  unchanged  in  the  cytoplasmic  by Zn  changed  the  in  Cu  in  Zn  weight  and  metallothionein  cytoplasmic  to  exposed  decrease  pool  pool. (Table  in  both  there  5, .Figure  is  a  mercury  appears while low  net  molecu-  relatively  At  the  low  are  Hg/L.  pool,  in-  decrease  1 jjg  in  pool,  Cu  net  levels  cytoplasm  Hg/L,  a  Copper  decreased  Thus,  5 jjg  the  5).  enzyme-containing and  to  in  resulting  zooplankton  metallothionein, cytoplasmic  in  enzyme-containing  pool,  (Table  exposure  exposures,  Cu  zooplankton  metallothionein,  cytoplasmic  plasmic  molecular  decrease  weight  of  12).  cytoplasm  appears  in  the  4,.Figure  In  lar  in  pool,  to  be  un-  increased  molecular  decrease  in  weight  in  cyto-  13).  P i s c u s s i on Results (1977) et  appear  a l . ,  that occur  of  1974  this  to ;  support  Brown  intracellular unless  containing  study  and  et  pool.  the  a l . ,  toxic  until These  and  capacity  of  metallothionein  of  will  enzyme-containing  appears  thionein  was  thionein  increased  that  surpassed from  the in  is  the  Cd  (Winge  theory  suggests  Hg  will  appear  in  the  spillover when  control  to  enzyme-  the of  1 jug  metallo-  binding present metallo-  exposure. the  not  from  the  In  capacity Hg/L  Wallace  theory  exceeded.  5 jjg  and  and  pool  binding  the  Koeller  This  metals  metals  the  it  1 977 ) .  these  to  of  "spillover"  effects  thionein  study,  those  MercuryHg/L  exposure, Rather,  but  at  5 jug H g / . L ,  c o n t a i n ling  CEEs. about  CEEs  12.5  were  of  of  Hg  fish  while  (1977)  grams.  counts  decrease  or  those  of  where  in  growth.  enzyme  functioning  Brown  et  a l . , 1977;  likely  metal 1oenzyme  1974).  Hg  in  of  Cu a n d Z n .  Cu  a n d Zn  result  toxic  tertiary  are displaced  Bremner,  enzyme-  b y Hg In  in  Since  of  could  occur  result  quaternary  in  these  the present  toxic  effects  of  Hg,  of  in  concentra-  similar  part, or  therefore, altera-  1974;  Bremner,  a change  structure  study,  Cu o r  (Friedberg,  1974;  with  there results pool,  increases  were  of  decreases  in a reduction Hg e x p o s u r e  Thus,  the other  enzymatic  to  in  when  effects.  due s i m p l y  increase  was a  from  enzymes  pool,  Hg e x p o s u r e  independent  that  exposed  1977).  or  deficiency  1 jug H g / L  (Friedberg,  Brown,  fish  t h e 5 jjg Hg/L  inhibition  the enzyme-containing  in metal  exposed  there  least  b y Hg  the enzyme-containing  at  zoo-  weighed  in  or  and  5 jjg Hg/L  was a d r a m a t i c pool,  effects  fish  same  Hg/L  counts  At  tion  Zn  5 jjg H g / L .  in the  5 ,ug H g / L  Thus,  there  may be a r e s u l t  Most  to  both  control  on d a y 7 2 .  growth  1974;  1  these  1 pg  Zooplankton  reduced of  in to  the enzyme-containing  dramatic  found  exposed  controls  Hg e x p o s u r e  in  from  Hg o c c u r r e d  be d e c r e a s e d  one-fourth  zooplankton tion  to  day 72,  6 grams  weighed  l i t t l e  excess  and Wallace  growth  On  very  pool .  Koeller plankton  increased  growth  decreases  of  of  might  decreases  inhibitory C u a n d Zn  (Underwood,  1971).  While partially clusion As  explains  of  uptake  explained  and  Webb  and  Zn  by  to  saturated  and  by  therefore  from  the  levels Cd o r  they  Hg  metals  of  Cd o r  (Fowler  et  explains  losses  leakage  by  Hg.  DeFilippis  possibility. and Hg,  Stonard Cd,  Cu  into  a  When  metallothionein  cannot  in  potassium.  caused  this  Hg  ex-  metallothionein.  bind the  Cu a n d  Zn,  bloodstream  1976).  a l . , 1975;  including  a  of  by  metals,  (1975)  and Webb,  results  as  also  absorbed  (Stonard  C u a n d Zn  these  uptake  Hg,  constituents the  is  It. then  not  exposure  of  mucosal  Hg.  are  of  and Sugawara  duodenal  workers  that  these  losses  gastrointestinal  intestine  Other found  cellular of  via  high  displacement  Sugawara  (1976),  occurs  exposed is  intracellular  Overnell,  loss  of  certain  Overnell  due,to  cellular  (1975)  membrane  and P a l l a g h y  1975)  (1976)  damage found  that  65 Chiorella conclude tion and  of to  able  that the  a  for  tolerance by  exposed  there This  Zn  cell  of  fish  the  the  Chiorel l a of  this  l i v e r ,  as  concurrent  consistent  in  uptake  sensitive  in  in  had decreased  decrease  reduction  was a is  this  Hg  temperature  development In  to  with  number  well. to  exclusion Hg  of  Leber's  of  due to of  exchange  as  Zn. an Zn  sites  attributed  elements  They  inhibiuptake avail-  increased  characterized  mechanism.  increased  decrease  is  component  They  trace  uptake  of  in Cu  (1974)  metallothionein, in  metallothionein.  theory  suggesting  that  metallothionein  results  by  displacement  less  a  thionein placed  by  Zn  appears  In  in  toxic  not  with  of  crepancies ferent  were  levels fish  at  at  of  dicating the  present It toxic  whether  or  to  Hg/L  exposure  5 jjg  Hg/L pool,  found  in  less  levels of  Hg  in  from  Cd  or  Whether  or  not  employed  to  Cu  and  do. n o t  spillover  metallothionein  Hg  metallo-  here.  in  metalloeven  at  Therefore, by  increasing  occurred  in  fish.  may  levels  at  be in  liver  due  85%  cytoplasmic  and of  5). Hg  In  is  zooplankton  previous occur  in  synthesis  fish  pool,  liver the only  i n Therefore,  metallothionein.  studies  will can  in  of  probably  unti 1  occurs  Hg  cytoplasm  would Zn  dif-  those  found  zooplankton.  and  to  exposure  one-twenty-fifth 4  Dis-  zooplankton.  5 jug H g / L  zooplankton  this Hg  in  study,  liver  enzyme-containing  challenge  evident  not  in  . s p i l l o v e r of  a  while  the  fish  dis-  displaced  Hg  (Tables  exposure,  Cd  increased  as  fish and  that  occurred.  levels  part,  metallo-  present  zooplankton  of  in  metallothionein,  not  lesser  Hg  Hg/L  of  or  and  exposure  effects  occurs.  were  1 jug  less is  fish  types  much  in  Zn  spillover Zn  in  methods  and  Hg  those  is  lesser  of  one-fifth  at  Hg  Cu  the  Zinc  by  from  found  In  metallothionein,  enzyme-containing 60%  and  cytoplasmic  5 jjg  cytoplasm  Cu  in  tissue  only  where  between  Zooplankton  both  increases  level  Kg  metals  Leber  Cu.  detectable  zooplankton,  levels  toxic  ones.  displaced  zooplankton,  highest  detoxification,  metallothionein.  have  was  thionein the  from  to  thionein  more  of  in  " s p i 11  that over"  depend keep  pace  upon  with a  metal  long  levels and  period  of  of  but  Hg,  hence  with  ing  the  effects.  as  which  levels  is  all  of  of  shorter Hg  Hg,  the  not  be  whether  its  these  with  tissue  rate  of  exposure  very to  the  should  and  levels  of  high  over  tissue  periods  might  most  of  an of  but  result the  in  Hg  appear-  concurrent  to  Hg  metallothionein  exposure  levels  on  This  in  with  critical  occur  low  exposure  but  factor  to  bound  enzyme-con t a i n i n g . p o o l  may  and  result  Much  metallothionein.  level  exposure  might  levels  Thus,  survival rather,  time  higher  tissue  in  Thus,  detoxified.  much  lower  or  inflow.  toxic  organism's Cd  or  Hg,  but  enzyme-containing depend  upon  pool  both  the  uptake.  Summary Fish trace,  1  and and  5 jug  pathological of  Hg/L.  effects  metallothionein  molecular dental and  zooplankton  with  Zn.  terms  weight  of  were In  both  appeared  and  to  of  Hg  in  tertiary  quaternary  metal 1oenzymes  resulting  from  Hg.  in  Cu  concentration  are Cu  tissue  discussed  placement  of  and  exclusion  processes.  Zn  by  of  Hg  Hg  of  Hg  with into  were  are  as  with  both  an  and  as  the  high  pool.  Coinci-  decreases  structural  Zn  to  saturation  explained  replacement  and  exposed  zooplankton,  coincide  tissue  effects  Decreases  and  (enzyme-containing)  Pathological and  fish  "spillover"  protein  increases  simultaneously  of  in  and  increasing  Zn  and  by  Hg  intracellular  duodenal  Cu  in  changes Cu  of  dis-  cellular  97 CHAPTER  VI  The  Effect  of  Mercury  Exposure  Cytoplasmic  Distribution  and  Skeletonema  Zinc  in  of  on  the  Mercury,  Copper  costatum  Preface This and  D.  is  A.  Journal  my v e r s i o n  Brown  of  of  (1978),  a  Marine  paper  again  demonstrates  occur  until  i t  appears  that in  Interactions  between  weight  protein  in  fish  Hg  a n d Cu  and  high  Hg  and  but  L.  Cloutier  publication  and  of  Hg  do  Zn  high  the  are  not protein  molecular  similar  than  the  This  weight  in  in  Ecology.  molecular  different  Parsons,  by  effects  phytopi ankton,  and z o o p l a n k t o n , (Brown  for  Biology toxic  the  pool.  in  authored  and a c c e p t e d  Experimental  pool  paper  to  those  those  between  1978).  Introduction It  is  elements  (De  they  inflow  thionein  not  and  occur  Toxic of  as  in  of  phytoplankton  tolerance  Pallaghy,  Cd  is  can bind  of  and D a v i e s , of  Cd  plays  and thus It  levels 1 974;  but a  metals  exceeds  synthesis  (Winge  et  are  the  is in  not this  weight nontoxic,  stores  Cu  for  and  Zn  metallo-  and Chatel , found  rate  a l . , , 1 973 ;  trace  i t  role  render  required Brown  a n d Hg  these  also  to  subsequent  low molecular  and Hg.  excess  effects  a  to  1976),  metallothionein  which  such  (Bremner  1978a). of  or  protein  metals  enzymes  increased  Metal 1othione in  (10,000)  when  in  exposure  Filippis  whether  process.  heavy  that  results  exposures known  known  of  Brown  when  the  rate  metalloand  Parsons,  1978).  At  this  weight  protein  Cu  Zn  and  in  Parsons,  point, pool  metal 1oenzymes  of  animals  Olafson  et  binding active to  a  Cd  and  Cd.  In of  Hg  exposure  of  Cu  a n d Zn  The obtained  Zn  were  start  X  of  10"5  ZnS04, X  1 977 ;  molecular  by  replacing  Brown  and  10"8  1974).  Kagi,  1974;  Piscator,  However, remains  green  algae  study,  the to  protein  is  in  to  order  induced.  cytoplasmic  and  fraction radio-  are to  exposed  see  Also,  levels  c l a r i f i e d .  like  exposed  land  presence  be  phytoplankton  concentrations  the  in  a metallothionein  present  the  if  a  effects  distributions  Methods pennate  the  diatom  Northeast  Oceanography, of  the to  8.83  X  IO  Culture  University  of  a r t i f i c i a l  and R y t h e r ' s X  IO  - 8  - 4  Ske1etomema  Pacific  experiment,  M K2H2P04,  2.2  and  found  examined.  Guillard  consisted  1.3  are  from  modified  M  on  transferred  3.68  high  ubiquitously  Buhler  blue  like  coastal  of  been  found  in  the  and  Institute the  the  effects  phytoplankton  mercury  of  Materials  in  (1 9 7 2 )  metallothionein  At  toxic (Brown,  and Thompson,  al .  range  has (e.g.,  metallothionein  Maclean,  on  1978).  marine  1964;  occur  and e x e r t  Metallothionein and  they  M CoCl  M Na2Mo04-4H20 ,  2  X  of  enriched  medium.  The  TO-8  « 6H20,.  X  10"5  5 . 8 3 J J M Fe  X and  with  M  Na2Si03,  3.83  IO  culture  enrichment  M CuS04, 4.55  Columbia.  stock  seawater  5.3  was  Collection,  British  portions  M NaNO^» 1.96  costatum  - 9  M  X  IO  - 5  MnCl2m  5 . 8 5 JJM NaEDTA.  Vitamin  was added  The l i t e r , foil The  cultures  flat  in  cultures  covered under  were  bottom  sheet,  were  addition  of  grown  in  0 . 5 jjg Hg/L.  these  subsets  fugation,  in  centrifuge, were  NaCl  At  70  0.1  with  6 minutes, and the  of  laboratory centrifuged Superspeed  at  pellet  27,000  RC2-B  116  test  set at x £  for  automatic  was f r a c t i o n a t e d 60 cm) w i t h  on  0.01  fluorometer.  culture.  Two  hours  before  cultures  10  by  in  of .  of  the  centri-  a 6°C  analyzed. 3 ml  model  of 0.9%  563C  variable  Homogenates  minutes  using  refrigerated  a Sephadex M NH4HC03  each  supernatants  until  4 . 5 .  of  Another  hours  in  were  duration  The  STIR-R  by  (few  tubes,  g_.  frozen  a TRI-R  homogenizer at  at  grown  clean  the  was h o m o g e n i z e d  using  were  Hg/L.  pg  for  650 x  pellets  70  1.5  harvested  teflon  one c u l t u r e  jug H g / L  polycarbonate  5 minutes  (0.9 x  hours,  0.5°C.  III  very  two o t h e r  50 ml for  a  6  aluminum  was m e a s u r e d  costatum  while  an +  a  Model  with  were  for  Supernatant  Turner  S_.  17.0  with  Growth  a  by  Cultures  approximately  HgC^)  to  at  rpm.  Cells  gram  column  for  (as  One  Sorval  with  was p e r t u r b e d  discarded  speed were  grown  was e x p o s e d  experiment.  120  l o g phase  were  culture  at  borosi1icate,  stoppered  stirred  was s t a r t e d  cultures  in  maintained  illumination.  unialgal  Hg  flasks,  continuously  experiment  strength.  batchwise  bath  fluorescence  bacteria)  full  grown  stirrer  continuous  The  25%  boiling  a water  magnetic  monitoring  at  G-75  buffer  a  centrifuge. (Pharmacia) as  eluent.  1 00 Two  ml  fractions  were  Ultraviolet with  a  meter  Perkin to  weight and  Elmer  as  1977).  model  ( 2 8 0 nm)  124D  in  of  beam  the position  (enzyme-containing)  of  peak,  Copper  levels  were  a  Perkin  Elmer  absorption  spectrophotometer.  cold  method  vapor  utilizing  a  on  Pharmacia  UV  Optical  Unit  in  UV  model  et  a l . ,  each  atomic  was m e a s u r e d  fractions  (Pharmacia  molecular  (Brown  303 flame  Mercury  the combined  3 0 cm c e l l  pool  determined  model  were  metallothionein,  cytoplasmic  using  done  Peaks  the high  weight  a n d Zn  were  spectrophoto-  peaks.  low molecular  fraction  100,  spectra  double  the position  being  protein  the  absorbance  establish  identified  collected.  of  Control  by a  each  peak,  Unit  model  100).  Results At  the beginning  exponentially in  the  presence  i n i t i a l l y 0.27  (Figure of  exposed  hours  maintained  The  cellular  and  by A  typical  to  Hg  by  relatively  weight  is  shown  14).  Growth  those  a growth  rate  in high  cytoplasmic  Figure  of  levels pool  by  of  This  much  rate to  + 0.17  0 . 5 jjg  from  Zn  cultures of  Hg  in but  is the  1.31  until  +  70  divisions/day. 24  hours,  Hg/L.  S_. c o s t a t u m  profile  C u a n d Zn  (III),  The  25% a f t e r to  grew  deaccelerated  14).  1.92  profile  15.  was  had growth  exposure  gel elution  cultures  not exposed  was r e d u c e d  46 h o u r s  rate  (Figure  0 . 5 jjg Hg/L  while  density  55% a f t e r  the experiment,  0 . 5 jjg Hg/L to  divisions/day  of  exposed  characterized low  lesser  molecular amounts  101  Figure  14.  I_n  vivo  fluorescence  Skeletonema 116  hours.  with 0.5  1.5 jjg  jjg  Hg/L  costatum  of  exposed  — 0 — c o n t r o l ; Hg/L;  perturbation  70  hours  after  with the  cultures to  Hg  jjg  with  Hg/L;  1.5  jjg  1.5  jjg  Hg/L  start  of  the  of  for  —®-—control  — A — 0 . 5  perturbed  of  batch  perturbed —  Hg/L.  ^  time  approximately experiment.  102  200  5ioo u  80  o 40 i_  o 20  -i  2  1  3 Time (Days)  1 03  Figure  15.  Gel  elution  exposed  to  profile 0.5  jug  molecular  weight  thionein;  III:  pool .  of  Hg/L  Skeletonema for  protein low  116  pool;  molecular  costatum  hours. II:  weight  I:  metallo-  high "  cytoplasmic  Absorbance(280nm) p  Ki "i  p  o  'J^  J3>  1  1  O  bo o 1  r  Zn (mg/L) P O p O O O O O O O ~i  hJ 1  (jj 1  1  oi  r~  Cu(mg/L) P O O O f\) ^ 0) 00 O  ~f  1  1  1  r  105 of  Cu  and  in  the  rather  high  small  correspond  to  Cu  the  previous  studies  profiles  from  Figure the  15,  weight  in  costaturn levels  Copper  Levels  of  creased  Cu  to  Mercury  in  exposed  detectable,  by  0.5  resulted  a  plankton  Hg/L  decrease  All  exposed  of to  Hg  in  to  each  of  not the 0.5  uptake  in  the  exposed  to  low  the molecular  were  in  in-  those  S_.  Total  Zn  to  Hg  peak  molecular (Table were  6).  in-  exposed  to  1.5  jjg  increased  in  those  Hg/L. methods  employed  in  this  exposed  to  1.5  |ig  Hg/L.  addition  of  1.5  jjg  Hg/L  of  Hg to  detectable Hg/L,  high  like  be  preexposed  jjg  in  exposures.  before  in  Zn  concentrations.  p h y t o p l an k t o n  jjg  phytoplankton Hg/L.  in  shape  levels  p h y t o p l an k t o n  jjg  the  fractions  Hg  1.5  to  in  like  appeared to  of  Zinc  increased  those  Preexposure  jjg  in  6).  cultures  levels  was  all  and  metallothionein  those  1.5  in  of  in  those  (II)  which  elution  metals  decreases  pool  higher  were  only  in  in  (Table  the  the  slightly  p h y t o p l an k t o n  Gel  of  (I),  fractions  similar  levels  protein  pool  pool  Cu  those  1 977).  were  metallothionein  in  Total  study,  weight  levels  protein  the  in  pool  metallothionein  al . ,  resulted  decreased  weight  Hg/L.  Hg  exposed  were  in  protein  6).  to  the  et  of  exposures  cytoplasmic  creased  weight peaks  position  varied  molecular  Zn  (Brown  (Table  Exposure high  and  other  but  peaks  molecular  and  compared Hg  Hg  in  then  before those 1.5  jug  with addition phytoHg/L,  of  Table  6.  The in  distribution batch  pmole/g  of  cultures. tissue  (wet  Zn ,  Cu  Data  and are  weight).  Hg  amongst  cytoplasmic  c o m p i 1 a t i o n s of meta1 MW:  molecular  Zn Total  Control  pools  levels  weight.  ND:  from  from not  phytoplankton  profiles  such  exposed as  MW  Metallothionein  L o w MW pool  Total  mercury  15,  in  detectable.  Cu High pool  Figure  to  Hg High pool  MW  Metallothionein  Low MW pool  Total  High pool  MW  .0083  ND  .0148  .270  .032  .035  .203  ND  ND  ND  ND  ND  .0071  .275  .044  .034  .197  ND  ND  ND  ND  .253  .041  .032  .180  ND  ND  ND  ND  pg  Hg/L  .0136  .0065  3.5  jjg  Hg/L  .0115  . 0035  .0009  .  1.5  pg  Hg/L  .0144  . 0046  .0011  .0087  . 328  .042  .040  .246  .0138  .0117  0.5  +  .0130  . 0035  . 001 7  .0078  . 326  .062  .041  .223  .0034  . 0034  fig  Hg/L  L o w MW pool  .0231  D. 1  1.5  Metallothionein  0071  . 0021  ND  ND  ND  107 appeared those Hg  in  the  molecular  phytopl ankton  appeared  but  high  some  1  the  high  appeared  position al . ,  in  of  exposed  in  weight  only  to  molecular  those  protein  1.5  weight  fractions  metallothionein  in  jjg  pool.  Hg/L,  In  most  protein  pool,  corresponding  previous  studies  to  the  et  (Brown  977).  Discussion Results  from  this  study  thionein  doesn't  of  phytoplankton.  Hg  high  in  molecular  fractions  a  weight  major  (Brown  containing  the  et  role  Most  to  a l . ,  highest  in  pool  the  usual  1977).  Only  Hg  levels,  was  than  in of  those  a  metallo-  appeared  position in  that  detoxification  rather  the  Hg  indicate  in  the  those metallo-  phytoplankton  Hg-thionein  like  detectable. The  lack  of  Hg-thionein  at  be  due  to  poor  detection  it  may  be  that  metallothionein  levels  too  that  metallothionein  the  to  detectable  protein  corresponding  thionein  peak  play  appear  a  low  higher  creased  in  position viously  Hg  that  be  limits  like as  fractions  appearing  in  (Winge  al . ,  and  was  Hg  with  1 975 ) .  Upon  the Hg  It  is  was  these  induced  exposures,  been  the  of  the  second  binding  exposure,  i . e . ,  at evidence with Zn  in-  usual  suggested  metallothionein  half a  some  to  may  study,  saturated  There  has  levels  present  corresponding  induce  approximately  exposure  fraction  metallothionein. Cd  in  determined.  exposures  those  of  et  to  most  with  preZn  sites the  108 nontoxic 1974;  Zn  is  Brown  and  toxified. is  displaced Parsons,  Zinc  relatively  is  be  thionein, In the  at  the  weight,  protein  effects  are  Zn  metal 1oenzymes  pool.  (Brown  thought  conformational  changes  In  study,  present  exposures  where  protein  pool.  exposed  fish  molecular In  the  resulted  Hg  exposures.  of  Hg  to  found  upon  where many Zn,  appears  to  metallo-  is  exceeded.  in  the  1978).  high  molecular  become  molecular  in  growth  (Brown  to  of  0 . 5 jjg  resulted to  Hg  1.5  protein  were  in  in  Hg  weight  found  in  the  and P a r s o n s ,  Hg-  high  1978).  phytoplankton  during Hg/L  to  1974).  molecular  of  and  due  occurred  appeared  preexposure  uptake  exposure  high  Hg  Cu  Friedberg,  reductions when  pool  study,  the  patho-  weight  nonfunctional  suppression  When  high  displacing  1974;  until  Pathological  in  the  i t  metallothionein  Hg  growth  jjg Hg/L  to  from  Preexposure 1.5  like  de-  exposures.  appear  (Bremner,  less  of  result  protein  in  are  corresponding  Parsons,  appeared  present  Hg  then  zooplankton  weight  Cu  (Leber,  simultaneouslyoccurring  then  Similar and  to  exposure  Hg  component  bound  Hg  Hg  cytoplasm  study,  Hg  or  these  metallothionein  and  to  Metal 1oenzymes  the  is  with  effects  a  fractions  of  pool,  is  Cd  that  the  present  of  logical  from  i t  Hg  excesses  so  into  highest  most  capacity  occurs,  as  those two  toxic  1978),  the  on  animals,  binding  this  In  increased  more  released  harmless  metalloenzymes. to  by  subsequent with  in  only  2.5% o f  >ig  Hg/L  without  subsequent the  uptake  pre-  109 exposure.  De  exposure the  of  wall.  in  They  trace  the  also  mediated  There molecular  Cu  followed  binding  weight  by  to  Zinc  was  high  pool  both  weight  cytoplasmic  viously  found  lower  on  lower  Hg  pool,  from  of  its be  but  pool  Zn  to  exposure was at  the toxic  high mode  reduced  of  Cu  Hg/L.  Cu on  on  this  to  Hg  might  the  high  Hg,.  This  to  protein of  the  Cu  0.5  jjg  phyto-  Cu  and  levels Hg  With  high  high  the on  molecular  the  to  uptake.  exposure  of  the  Perhaps  and  exposure. on  in  for no  molecular  higher.  similar  all  of  study  costatum  weight  favors  Hg  cell  Chiorel1 a  preexposure  jug  decreased  protein  part  1.5  increasing  of  a  the  present  competition  were  in  exposure  with  molecular that  and  development  S.  with  to  uptake  of  mechanism.  levels  weight  as  of  that  inhibition  sites  the  increase  evident  an Zn  by  from  pool  found  tolerance  Hg  with  of  exchange  and  at  pool  an  enzymes  levels  lowest  displaces  so  on  pools  Hg  be  exposure  weight  the  Zn  exclusion  most  Hg,  Cu  protein  of  protein  sites  preexposure,  an  in  component  tolerance  to  is  resulted  Results  by  increase  exposed  Hg  (1976)  characterized  that  appears  of  plankton  Cu  idea  weight  increase  when  as  Pallaghy  increased  mechanism.  the  Hg/L  number  attributed  support be  to  sensitive  elements  exclusion  and  Chi ore!1 a  temperature  reduction  to  Filippis  This  high the  molecular effect  was  molecular low  molecular  levels. weight  action. high  low  Zinc  Perhaps protein was  molecular  preweight  no protein  pool  1978).  As  Zn  exposed  low  molecular  at  all  Hg  weight  molecular for  fish  the  depleted  lecular  of  enzymes  in  Metallothionein molecule ducks,  for  when  containing) Cu  and  Zn  small  the  of  in  cytoplasmic  metals  in  the  phytoplankton  low  acids and in can et  or  some these  In  bind  pool.  a l . ,  and  1962).  Zn,  has  not  but  much  Elucidation  been  of  In  Chatel  Zn  appear  acids only low  of  , to  other  relatively molecular  high  than  levels pool of  of in  a  metallothionein.  iron-binding  sporadically 1962);  strongly  of  pool.  and  a  defined.  the  storage  and  and  presence  a l . ,  less  Zn  organisms.  Cu  the  the  et  low  pool.  cytoplasmic  other  of  of  major  cytoplasmic  identified (Zahner  a  (Brown  of  possible  consists  the  excesses  presence  substance  plants  forms  the  that  saturated,  in  weight  be  be  nucleic  found  mo-  (enzyme-  animals,  The  molecular  siderochromes,  Cu  are  high  protein  weight  blocks.  substance  l i f e  to  excesses  acids,  is  protein  higher  Zn  amino  indicates  higher  in  pool  reservoir  weight  weight  molecular  storage/detoxification the  a  metallothionein  metals  weight  is  apparently  low  building  portion  Perhaps  it  Parsons,  the  may  as  appear  molecular  was  contains  cellular  as  while  it  acts  not  phytop 1 ankton ,  in  pool  Zn  high  pool  In  stored  This  the  not,  molecular  does  and  occurred  1 978a). be  Cu  is  and  cytoplasmic  levels  pool  high  (Brown  weight  pool  protein  the  Hg  exposure  protein  weight  to  hydroxamic  in  algae  their  function  Siderochromes than  structure  and  Fe  (Zahner identity  Ill of  the  be  a  of  the  low  molecular  valuable  weight  metal-binding  contribution  metabolism  of  to  trace  developing  elements  in  moeity an  should  understanding  phytoplankton.  Summary Skeletonema  costatum  were  with  and  without  exposure  to  Hg.  were  0.1  and  jjg  for  116  70  hours  jjg  Hg/L  0.5  followed for  70  rates  were  levels  equal  to  the  gel.  plankton of  this  tein to  were  the  with  pool.  jjg  of  Hg  jjg  hours,  1.5  jjg  Hg/L  Hg/L,  no  Hg/L the  after high  to  only  25% jjg  appeared  in  the  high  is  discussed  a  Hg  metal  exposure weight  exclusion  resulted protein  in and  Hg  Sephadex  with  in  phyto-  preexposure. weight  Most pro-  corresponding studies.  followed Hg  with  In  by  accumulated no  molecular as  to  packed  the  Hg/L  hours.  and  previous  Hg/L of  46  centrifuged  molecular  jjg  0.5  exposed  in. f r a c t i o n s  0.5  and  for  Hg/L.  no  in  levels  mercury  for  accumulated  1.5  to  jjg  column  to  by  molecular  46  exposure  Tolerance  Mercury  for  metallothionein  preexposed  characterized  high  1.5  a  levels  amounts  compared  the  Hg  lesser  1.5  or  0.5  seawater,  exposure  hours,  homogenized,  with  to  of  to  than  in  exposure  by  through  highest  position  a r t i f i c i a l  Mercury  Hg/L  accumulated  p h y t o p l an k t o n  all  greater  harvested,  exposed  pool,  jjg  followed  passed  The  Hg  1.5  in  decreased ih.phytoplankton  and  supernatant  G-75  by  hours  Growth  Cells  Hg/L  grown  preexposure; weight  being  protein  possibly  mechanism. decreases the  low  of  Zn  in  molecular  the weight  cytoplasmic  pools,  but  small  fractions  corresponding  studies.  Growth  decreases  are  effects  resulting  from  to  toxic  Zn-containing in  the  low  s i b i l i t y  of  to  increases  a  weight  discussed  is  As  most  cytoplasmic  discussed.  in  in  as  Cu  those previous  possibly  displacement  storage/detoxification  metallothionein  Zn  metallothionein  metal 1oenzymes.  molecular  of  of  and  pool,  substance  Zn  Zn the  due from  occurred pos-  other  than  113 CHAPTER  VII  Increases High  of  Cd a n d t h e  Molecular  Apparently  Weight  Normal  Flounders  Cd:Zn  Ratio  Protein  Liver  (Parophrys  of  in  Pool  the  from  Tumor-Bearing  VetuTus)  Preface This pages  203-209,  examined in  paper  in  elements  addition  of  then  the  lecular could  they  c e l l .  containing  as the  in  and n o t  these  protein  that  by  D.  Cd:Zn  If the  high  involved  changes  the  the to  cell  in  study  1970,  of  were  these  only  weight  biological  occurred  44,  increased  (Morgan,  molecular  then  in  were  Volume This  distribution  influence  pool,  -1 9 7 7 ,  Brown.  humans  these  changes  A.  ratio  cancerous  Cd w a s a v a i l a b l e  enzymes  Biology,  cytoplasmic  wouldn't  If  weight  mean  Marine  and the  determined.  metallothionein, pool,  Cd  fish,  was  in  and was a u t h o r e d  whether  cancerous  1971);  appeared  the  protein a c t i v i t y  high  increased  on  mo-  Cd:Zn  interfer  with  the  Zn-  division  processes.  Introduction It are of  has been  altered  in  both  tumor-bearing  Elevated been  liver  found  in  1 958; Tietz 1971; Morgan  (.1 9 7 0 )  established  tumorous  animals  Cd, many  et  Wright  well  a l . ,  Cu  and  1921; kidney  Cd  patients  (Olson  1 957 ;  Sandberg  et  1972;  increased  metal normal  Olsen  cancer  and Dormandy, found  Zn  heavy  and a p p a r e n t l y  (White,  and  that  a l . ,  et  and et  in  Zn  19 5 4 ) .  have  a l . , 1954,  1958;  l i v e r ,  tissues  al . ,  Kew a n d M a l l e t t ,  Cd:Zn  levels  Morgan, 1974).  kidney  and  114 serum  of  lung  found  plasma  cancer Zn  to  cularly  metastatic  creased  Zn  Arnold  in  decrease  blood  (1961)  Halsted  in  carcinoma.  white  and Sasse  patients.  malignant Vallee  cells  of  (1954), human  1958)  cancer  liver  of  Furst  and H a r o ,  of  tumors  (1957) tumor  induced  causal  in  liver  a n d do  amongst  the  containing)  of  vetulus).  studies  The  as  of  the  et  metals  (Webb, are in  weight  a l . ,  used  were  1972a;  known  liver  to  the  occur  of  detoxify  Cd,  no  subcutaneous are  Cu a n d  metallothionein pool  for  of  (Parophrys  the  study  and Thompson, and s t o r e  (Piscator,  before  (enzyme-  flounders  specific  al . ,  carcinoma.  cytoplasmic  Olafson  tissue  et  levels  protein  197 7 ) ,  weight  content  transferred  distribution  jn_  reported  metal  the  uninvolved  Cd  levels  et  tissue  Tietz  in  result  Olson  (1 9 5 6 ;  the  a l . , (1958)  nontumor-bearing  methods  in  alterations a  in  tumor  a l . ,  in  in metal  bearing  molecular  (Brown  in  carcinogens.  et  rats  occur  tumor--and  Metal 1othioneins heavy  in  low molecular  metallothionein  of  Olson  that  high  pool  and t h e  livers  Zn  not  report  changes  Zn  rats.  Zn  et  increases  nonmetal  while  suggesting  This  pool  by  increased  Gorodiskii  noted  that  formation,  tumors,  Zn  1969)  Zn  de-  patients.  decreased of  parti-  reported  leukemia  tumors  (1970)  diseases,  (1976)  decreased  with  patients.  concluded  increase  reported  patients  these  of  described  dimethylaminoazobenzene-induced a l . ,  and Smith  1964;  of  1974).  excesses Leber,  1 15 1974).  As  excesses  carcinogenesis, somehow  be  Materials  and  of  Campus.  British These  Canada;  Fish  were  were ml  Each  sample  heated  to  collected and  as  Samples  and the  with  2.0  fractions.  ml  2 8 0 rnu o n  photometer. graphite  Cd  and  furnace  atomic  absorption  by  flame  the  a  on  Perkin Zn  then  levels  each  were  fraction  with  a  Perkin  (Webb,  to  g e l .  then on  a  7.5)  a  1972a;  for  10  was r e a d  124D  303  was at  spectro-  determined  Cu was  min  Pharmacia  Filtrate  Perkin  Elmer  (pH  Supernatants  min  Coleman  in  rehomogenized  recentrifuged  spectrophotometer.  method  1  G-75  Each  buffer  min.  Absorbance  Elmer  length.  g.  applied  Sephadex  Bellingham,  thoroughly  for  supernatant  f i l l e d  Columbia,  chloride  10  and  collected  and suspended  was  70°C  Columbia  nontumor-  mm i n  0.05  for  were  from  120  5 0 mM T r i s  and  column  might  Research  British  British  homogenized  of  of  C o l umbia ; ..and  80 t o  collected  K9/60  250  British.  x £  x £  during  Cancer  vetulus)  Beach,  27,000  27,000  liver  process.  the  removed  approximately  MgC12-  1974).  from  (Parophrys  at  Cherian, at  2.5  been  centrifuged  were  in  metallothionein  University  Crescent  samples  approximately  and  had  Landing,  USA.  3 mM o f  that  obtained  flounders  1iver .sample.weighed  with  occur  carcinogenic  Columbia,  livers  Gibsons  Liver  the  were  around  Washington,  thought  in  samples  waters  metal  Methods,  tumor-bearing from  heavy  was  involved  Liver Center  'it  of  Elmer  by 403  determined atomic  116 absorption rection  spectrophotometer.  was  used  minations. +  for  both  Analytical  standard  deviation,  standard  deviation,  graphite  precision  deviation,  standard  Deuterium  M=12),  N= 13)  furnace  for  for  background  Zn  and flame  Cd was Y + was X ±  deter-  12.6%  15.5%  C u w a s T+_  and f o r  cor-  (mean  (mean  +  3 . 3 % (mean  +  3 nontumor  or  N=15).  Results Composite tumor are  (skin  have  in  metals has to and  a l l  been  As is  protein  is  each  also  is  a  supernatant Zinc, most  of  of  like  weight  Cd on  of  on  Cd  increased  the  high  the  levels  7.  Each  1974;  1 975 ;  the  the  in  peak position Olafson  striking  molecular  3.3-fold  of  Marafante,  most  high  of  weight  increase  nontumor-bearing  tumor-bearing is  a  cytoplasmic  increase  Cd,  markedly  of  7,  metal  peaks  similarity  (Leber,  the  peak; to  Table  i t s  Table  main  of  and Y i p ,  Cd o n  relative  the  to  studies  from  increases  2-fold  summary  vetulus),  individual  three  in  of  (Parophrys The  the  presented  (enzyme-containing)  are  of  Casterline  seen  fish  and 17.  and a  previous  each  flounders  according  increase  1 ow. m o l e c u l a r  there  but  in  c a n be the  16  together  1 974;  tumor-bearing There  of  identified  Thompson,  change  Figures  peaks  peaks, found  profiles,  -bearing  tube  added  in  1976).  the  in  each  been  elution  tumor)  presented  levels  gel  fish.  metallothionein pool  tissue  peaks.  a l l  homogenate  molecular  three weight  and  Overall,  fish. on  in  peaks, protein  11 7  Figure  16.  Parophrys profiles flounders.  vetulus. from I:  liver  Composite of  3  IT:  weight  cytoplasmic  metallothionein; pool.  gel  elution  tumor-bearing  high, m o l e c u l a r  pool;  of  weight III:  low  protein molecular  118  0-5 0-4 0-3 =3  0-2 0-1 010 +  |  008  *  006 004 • 002 • 0010 +  'g 0 0 0 8 £  0006 +  2  0004 0002 + 20 1-5  2  CC O to  CQ  10 05  5 FRACTION  10  15 NUMBER  11 9  Figure  17.  Parophrys profiles flounders.  vetulus. from I:  liver high  pool;  II:  weight  cytoplasmic  Composite of  gel  elution  nontumor-bearing  molecular  metallothionein; pool.  of  weight III:  low  protein molecular  05Q.  0-4 0-3 0-2 0- 1 010 008 006 004 002 0010  CL  0-008 0006  CJ  0-004 0-002 20  Uj  1- 5  • •  • 250 nm • 280 nm  m  OQ QC  O  (/> OQ  5 FRACTION  10  15 NUMBER  20  Table  7.  Parophrys v e t u l u s .  Distribution  o f n o n t u m o r - and t u m o r - b e a r i n g 15,  i n /imole  g tissue"^  o f C d , Cu and Zn a m o n g s t p r o t e i n peaks f r o m l i v e r  flounders.  (wet w e i g h t ) .  MW:  Data a r e Molecular  Cd  flounders  .0098 (3)  Tumor-bearing flounders  a  (3)  High pool  .0022  a  b  .0198  **P  <0.001.  .0071**  (.0093) (.0011)  Mean.  Student's  Mw  (.0048) ( . 0 0 0 3 )  ''standard d e v i a t i o n . *P < 0 . 0 5 ;  from F i g u r e s  t test.  Metallothionein  . 0016 (.0007)  .0029 (.0016)  Low MW pool  . 0060  Total  1.8117  Zn High pool  MW  . 4750  (.0046) ( 0 . 7 0 0 7 ) (. 2221 )  . 0099  1.8486  14 and  weight.  Cu  Total  Nontumor  compi1 a t i o n s of v a l u e s  cytoplasm  . 5429  (.0067) (0. 7912) (.2682)  Metallothionein  . 3977 (.1901)  .4161 (. 1 826)  Low MW pool  . 9390 (.3019  .8896  Total  .1976  High pool  MW  .0509  (.0768) (.0229)  .2930  .1242*  (.3416) (.0379) (.0439)  Metallothionein  Low MW pool  .0298  .1168  (.0053)  (.0575)  .0328  .1360  (.0122)  (.0194;  122 peak.  Here  it  Overall  there  bearing  fish  It  is  is is  liver  of  total  by  the  to  that are  the  than  in  fish  Cd  in  liver  of  a l l  peaks  fish  relative  also  clear  proportion  molecular  only  small  relative  to  of  weight  nontumor-bearing  shows  than  fish.  on  is  greater  in  tumor-  i n c r e a s e d r a t i o - o f • C d : Zn  It  high  increases  of  as  tumor-bearing  much  nontumor-bearing  Zn  Zn,  higher  and  in  of  a  bearing  increase  fish.  those  fish  Cu  1.5-fold  fish  8).  pool  tumor-bearing  apparent  (Table  in  in  a  fish  are  2.4-fold  relative  tumor-bearing evidenced  increased  to  that  nontumor-bearing in  tumor-bearing  cytoplasmic protein  fish  Cd  and  Zn  (enzyme-containing)  (Table  inconsistent  in  9).  changes  nontumor-bearing  in  fish  tumor-  (Tables  7  9).  Discuss ion It liver  of  Morgan, known  has  shown  tumor-bearing 1970,  that  protein  been  1971).  this  peak  Cd  However,  7).  that  excess  peak  (e.g.  Cherian,  Yip,  1975;  Marafante,  circumstances the  high (1)  usually  Capacity  appears  weight the  high  that  Cd  (Tietz  the is on  high an  protein Organism  it  in  1957;  has  not  molecular  been  weight  finding  in  metallothionein  1974;  Casterline  are,  levels  elevated  a l . ,  unusual  the  There  is  et  previously  Leber,  1976).  which  of  on  This  1974;  under  molecular  organisms  appears  (Table Cd  previously  of  however, Cd  are  and several  found  on  peak: to  Synthesize  Metallo-  1 23  Table  8.  Parophrys protein and from  vetulus.  peaks  from  tumor-bearing Table  7.  MW:  Ratio liver  of  Cd:Zn  in  various  cytoplasm  of  nontumor-  flounders. Molecular  Ratios weight  Cd:Zn T o t a l H i g h pool  MW  Metallothionein  L o w MW pool  Nontumor f1ounders (3)  .050  .043  .054  .052  Tumor-' bear ing •fl ounders (.3)  .068  .060  .087  .073  calculated  Table  9.  Parophrys liver from  vetulus.  cytoplasm Table  7.  Percentage  of MW:  nontumor-  of  and  Molecular  total  Cu  tumor-bearing  or  Zn  on  flounder.  each  of  protein  Percentages  Metal l o th i o n e i n  from  calculated  Zn  Cu MW  peaks  weight.  Cd High pool  Cd,  L o w Mw pool  High pool  MW  Metallothionein  L o w MW pool  High pool  MW  Metallothionein  L o w MW pool  Nontumor f1ounders  (3)  22.1  16.4  61. 5  26.2  22.0  51 . 8  25.8  15.1  59.1  (3)  35. 7  14.4  49. 9  29. 4  22. 5  48. 1  42.4  11.2  46.4  Tumor-bearing fl  ounders  •1 2 5 thionein  is  Surpassed.  pathological of  metallothionein  molecular of  weight  Cd w e r e  weight), mally and Cd  only  Webb  wet  Cd  High  that  2 jjg  is  found  their same  of  of  as  et  a l . , 1966).  from  for  of  Also,  Cd  (Kagi  to  nontumor-bearing  possible  is  unmeasured thionein, et  a l . , (3)  heavy e.g.  is  less  increased that  the  high  levels (wet  - 1  were  maxiStonard  higher  study  levels  of  ( 8 5 j j g Cd g  total  Metal.  Cd w a s f o u n d  there  metal  in are  or  Ag  (Kagi  Presence  of  Cd on  is  is  and V a l l e e , of  l i k e l y fish  which  Hg  It  by  on  as  I960;  Cu  the  levels  Pulido  metallo-..  ratio  can d i s p l a c e and V a l l e e ,  of  fish of  or  than  tumor-bearing  tumor-bearing high  by  less  Cd f r o m  in  Presence  unlikely  metal 1othionein  displacement  compared  It  much  metallothionein  Cu o r  is  with  capacity  weight).  Metallothionein  from  by  metals  on  study  (wet  1  90% o f  Heavy  thionein  these  Zn  binding  their  present  that  peak.  Another  that  the  present  85 t o  affinity  the  However,  even  the  be d i s p l a c e d  or  the  stated  6 0 . 0 jug Cd g t i s s u e in  that  in  (1973)  a n d Cd a p p e a r s  peak.  Displaced  Levels  a l .  Cd g t i s s u e "  weight),  Cd w o u l d as  exceeded  levels  found  et  Cd a p p e a r . w h e n  range  metallothionein (2)  Zn,  is  the  about  those  of  protein  the  (1976)  than  the  in  while  tissue"1,  of  effects  Winge  to  (Table  some  Cd f r o m 1960;  Cd  fish  8).  other metalloPulido  1966). High  Molecular  Weight  Protein  126 Peak  is  Due  by  Irons  in  the  presence  weight  of  of  stance,  in  of  Cu  high  This does  found  with  increases  not  liver  of  in  unlikely appear  to  for  cancer, in  can  of  a  As  in  ag-  molecular  in  the  be  significantly  present  7).  patients  i n -  Similarly,  Cu w e r e  while  shown  aggregate  resulting  (Table  values  Cu  Cu,  excess  fish  most  Metallothionein.  metallothionein  is  metastatic in  of  levels  tumor-bearing  (1958)  patients  (1976),  metallothionein  since  a l .  Aggregation  60,000.  elevated  no  an  and Smith  gregates  et  to  unaltered  Morgan  with  Olson in  (.1 9 7 2 )  found  bronchogenic  carcinoma. (4) Chen  et  Cd al .  thionein bind on  to  the  Bound  (.1 9 7 3 ) ,  and hence  to  an  free  high  methyl  but  is  will  while  rather  weight Hg  Carcinogen.  Hg  detoxified,  molecular  that  Organic  ionic  metallothionein,  conclude not  is  to  metallothionein.  The  of  methyl  Hg  when  high  is  in  accordance  conclude these that by Hg,  that  appear Cd  in  found with  toxic in  the  peak,  but  rather  on  of  effects high  of  fish  high  not  found  to  metallo-  will  Chen  toxic,  since  increased molecular et  a l . do  molecular  al .  it  is  weight  (1973),  It  peak  who until may  alkylated,  Hence, on  et  toxicity  peak.  similarly  occur  mainly  not. occur  weight  the  weight  by  not  found  peak.  metals  is  Hg  be  carcinogens.  Cd w o u l d the  Winge  molecular  organic  alkylated  the  data  tumor-bearing  "bioactivated" this  on  will  more  bound  bound  methyl  protein  much  be  As  like  be  but methyl  metallothionein peak.  As  127 explained  by  Furst  (1963),  explained  as  heavy  metal  organic  carcinogens  chelators  increasing  can  be  heavy  metal  uptake. In  agreement  with  1954,  1958;  Morgan,  study  using  Parophrys  1970,  organisms  relative  In  organisms,  normal  metallothionein However,  in  molecular reasons  as  Of liver a  (Webb,  1 972a,  protein  pool, Cd.  significance  may  be  normal  Zn  in  of  in  liver  body  Cd  are  genic It  process.(Tietz  has  blood  been and  diski i 1957; in  a l . ,  of  et  a l . ,  kidney), as  1957;  1 960;  Arnold  Thus,  it  and  appears  found  in  the  of  the  the  same  increased As  Cd  when  is  in  a l . ,  l i v e r ,  that  1972). kidney, (Goro-  and  Haro,  1 9 6 9 ; T i e t z e t  the  other  hand,  et  in  a l . , 1961; the  is  1 954,  is  1 958;  in  Herring  1 970 ;  carcinogenic  a l . ,  increased  decreased  Morgan ,  to  carcino-  organisms  but  not  bound  the  et  1 973). high  of  increased  Sasse, that  al . ,  for  7).  the  et  Strain  organisms  (Olson  on  part  tumor-bearing  blood  found  (Table  probable  of  on  tumor-bearing  is  tissue  Zinc  present  it  is  1970).  in  (except  Cd  Furst  the  organisms.  tissues  al . ,  Davies  is  that  1956,j_n_  and  b;  source  increased  tumor-bearing  tissue  a l . ,  1976).  tumor  Morgan,  liver  tumor et  et  found  and  in  et  organisms  possibly  the  most  metallothionein of  Zn  usually  Zn  tumor-bearing  constituent  burdens  is  excess  (Olson  elevated  Zn  for  and  was  excess  discussed  Cd  liver  nontumor-bearing  instance  weight  studies  1971),  vetulus  to  peak  this  previous  Vallee,  process,  128 Cd  and  Zn  increase  so  that  is  decreased  Zn  is  the  Cd:Zn in  in  the  ratio  blood  transferred  to  l i v e r ,  is  and the  but  Cd  more  increased  in  the  tumor liver  carcinogenesis.  Concurrently,  and  so  tumor  expected  tissue in  tumor  Morgan in  lung  increased  47%  in  study,  Morgan  liver  of  Cd:Zn  ratio.  the in  lung  liver the  Winge (1976)  have  appears Since and  in  function able  to  metals the by  the  liver.  and  that  high  bound  in  are  The  and  Zn  with  a  25%  a  increase  40%  this  protein  effects weight  fraction to  and ratio a  was  subsequent  increase  in in  increase  of  this  the of  ratio  Irons  and  Cd  occur  when  of  protein (White  Smith it  fraction. et  with it  a l . ,  1968)  enzymatic is  those  reasonheavy  and  hence  present  protein  peak,  exert  toxic  increased  Cd:Zn  ratio  in  weight  be  Zn  and  part,  metallothionein  Cd  increased  1974),  in  would  36%  interfere  least  during  peak.  (1974)  Friedberg, at  a  that  ratios  In  Cd  be  Zn  blood  Cd:Zn  kidney.  As  in  renal  found  toxic  1974;  to  and  study  Leber  known  that,  in  both  molecular  occur  Cd  22%  weight  (1973),  molecular  may  and  present  ratio  the  patients.  patients,  (Bremner,  not  liver  cancer  metals  binding It  in  found  conclude  high  than  liver  found  enzymes  heavy  tissue  cancer  a l .  increased Cd:Zn  molecular et  is  Zn,  liver.  areas  higher  (1971)  Cd:Zn  high  Cd  these  than  might  even  found  The  from  it  that  (.1 9 7 0 )  increased  tissue,  so  in effects  enzymes. be  that  the  the  high  129 molecular  weight  protein  results  in  interference  enzymes  in  pretumor  it  is  probable  toxicity enzymes.  Vallee  essential  to  acid as  as  et  polymerases  Zn  a l .  (1961)  primary  inhibitor  action  of  many  nonfunctional binding from  with  their  due  to  substrate enzymes  by  fact  dysfunction  transcarbamylase  cell  inhibition  Friedberg, via  a  for  a  potent  brought  about  by  different  1974).  Nonfunction  binding 1974).  on  the  Alternatively,  of  enzymes  (1965)  found  coli  c a n be  Hg,  changes,  sites  subunits  by  fere  Zn  subunits  together  holds  is  rendered  and r e g u l a t o r y which  i t  metal,  are  catalytic with  which  properties  s p l i t t i n g  Escherichia  Cd  Schroeder  conformational f i t  tissue  that  enzymes  enzymes  (Friedberg,  longer  may be  essential  changes  and Schachman  from  an  possessing  enzymes  normal  division.  is  These  in  ribonucleic in  these  of  1974;  Gerhart  on  role  in  It  that  after  i t s  deoxyribonucleic  Cd  no  can r e s u l t  and  if  metals  that  exerting  has a  that  that,  molecules  Zn  viruses.  of  states  sulfhydryl-containing  sites  metals  required  Zn ,  acid-dependent tumor  flounders  Zn-requiring  polymerases  conformational  heavy  the  of  acid  enzymes.  (Bremner,  subunits.  is  with  that  control  suggest  in  (1976)  metabolism  binding  in  Zn  Horvath  says  acid  from  instrumental  its  is  (1976)  tumor-bearing  Cd w i t h  inactivation  nucleic  with  of  competes  ribonucleic  interferes are  Cd  and d e o x y r i b o n u c l e i c  well  acid  of  tissue.  that  through  pool  which  into  that split might  (Griffin  aspartic into interet a l . ,  1 30 1973). tory to  Once  the  subunit,  feedback  it  also  be  Flick They  split et  can  be  a l .  the  important  that  states  Zn  produces  and  Cd  inorganic  that  to  hamsters  is  by  Cd  when  Cohen,  Dreosti, effect  1971;  when  given  otherwise  have  (Bischoff  and  development mice 1 975 ,  and  as  a  or  studies fact  that  spread  Woster  be in  et  ratio  et  a l . ,  a l . , has  study  the  col i  a l . , the age  effects that  Kolonel  inhibitor Zinc  in  organic  has  rats, with  mice the  1964;  Poswillo  1 972 ;  Duncan  same  preventive  to  mice  and  which  mammary Zn  would  gland  inhibited  and  tumors the  transplanted  Duncan  of  carcinogen  1963,  tissue  1 974;  Cd.  into  Dreosti,  1 975).  been in  E^.  on  ratio.  animals.  a l . ,  Further, tumor  from  conclude  occurring  spontaneous  of  increased this  et  early  1939).  (Duncan  Cd:Zn to  developed  and  1 976 ;  an  in  the  et  that  whose  simultaneously  with  (Gunn  they  competitive  from  subject  found  literature  Cd:Zn  tumours  cancer  from  have  polymerase  Further,  acts  regula-  longer  carcinogen,  the  the  Hg.  potent  demonstrated  Long,  rats  The  It  no  a  Ciapparelli  1975).  by  be  Cd  is  (1969)  acid  may  dimethylaminoazobenzene and  a l .  administered  carcinogen  and  from  the  malignant  prevent  freed  reviewed  Zn.  variable  (1976)  shown  et  subunits  (1971)  is  activity  Jovin  into  counteracted  and  high  subunit  deoxyribonucleic  conclude  been  has  control.  Zn-contain ing can  catalytic  shown  in  this  tumor-bearing Cd:Zn  ratio  and  other  organisms. has  been  shown  The to  131 be  increased  bearing in  in  the  organisms  levels  involved of  Zn  molecular  suggests  carcinogenesis,  enzymes  high  Cd  in  is  from  increased  levels  cell  division  occurring of  Cd  there  interfering  administered  carcinoma  that  protein  with by  for  a  is  pool  a  carcinogen  Thus, might  on  high prevent  competing  sites  that  Zn-containing  processes.  binding  tumor-  p o s s i b i l i t y  with  successfully  of  with  these  enzymes.  Summary It levels  is  evident  from  are  changed  in  nontumor-bearing normal role  liver  of  liver  organisms,  tissue.  of  zinc  metallothionein  the  In  high  cussed  but  were  of  enzymes  involved  reasons  for  weight are  peak,  in  excess  protein  discussed.  peak  on  the  the  weight  acid  occurring  rather  Copper  of  than  levels  was  the or  possible  Zn  in  the  vetulus).  3.3-  and  weight  increased  on  the  2.4protein by  40%  on  peak.  Results  are  Cd  Zn  Zn-requiring  and  for  metabolism. the  high  on  the  metallothionein  not  dis-  Possible  on  did  to  apparently  significantly  ratio  competition  Cd  Cu  molecular  protein  nucleic  in  increased  high  Cd:Zn  Cd,  metal.-  relative  (Parophrys  increased  addition,  terms  binding  flounders  heavy  organisms  considered  not  molecular  in  study  in  that  particularly  were  respectively,  peak.  This  tumor-bearing and  studies  tumor-bearing  me t a l l o t h i o n e i n s  Cadmium  fold,  previous  molecular  change.  peak  1 32 CHAPTER  VIII  Increases High  of  Cd  Molecular  Apparently Cancer  and  the  Weight  Normal  Cd:Zn  Ratio  Protein  Kidney  of  in  Pool  the  from  Terminal  Human  Patients  Preface This  study  was  done  (1978).  Unfortunately,  controls  or  is  cancerous  attained,  this  The  purpose  of  the  cytoplasmic  cancerous  fish  to  same,  be  the  cancerous  D.  the  be  was  humans.  suggests  organisms,  and  a  is  of  and  fact  such  2  for  sample  for  size  publication. if  changes  Zn  were  similar  that  they  do  changes  therefore  Knight  only  investigate Cd  The  B.  larger  submitted  to  that  and  size  When  will  study  Brown  sample  distribution and  A.  humans.  paper  this  by  may  be  are  in in  prove  common  etiologic  to  in  cancer. Introduction Recently, the  Cd:Zn  Brown  ratio  of  tumor-bearing  Cd  in  this  of  Cd  are  in  liver  Yip,  the  and  creases  may  of  of  kidney  and  tissue  and  Cd:Zn  Cd  serum ratio  increases weight  Occurrences to  (Cherian,  by  1974;  Previously, and of in  Zn  and  lung the  of  carcinoma,  detoxified  1976).  both  noted  molecular  unique  bound  Marafante,  the  high  be  kidney  increases  l i v e r ,  the  has  flounders.  usually  1975;  found  pool  in  (1977)  the  cancer  high  of  Cd  protein high since  and pool  levels  excesses  matallothionein Casterline Morgan Cd:Zn  and  (1970) ratio  patients.  molecular  of  in In-  weight  133 protein  pool  in  pool.  this  carcinoma, acid  metabolism  thymidine Cadmium  to  1971;  their  conformational Friedberg,  of  kidney  from  cancer  patients  grams  minutes  in  a  TRI-R  STIR-R  a  teflon  x  £  natants  were of  4 . 5 ml  e . g . , DNA and  and D r e o s t i ,  1976).  in  function  is  inhibited  or  changed  Brown,  ratio common  changes  1977).  humans  were  victims  in  The  with the  (Flick  in  these  et  present  carcinoma, high  factor  al . ,  study  to  see  molecular  if  weight  carcinoma.  of  minutes  obtained General  from  Vancouver  Hospital.  and two from  The age o f  the patients metastasis  not  be  believed  to  were  homogenized  0 . 9 % (W/V)  NaCl  at  laboratory  Homogenates  were  in a Sorvall  collected  a  unknown.  at  death  for 5  at  centrifuge. were  of  equipped  centrifuged  and t h e p e l l e t s  dying  is  setting  motor  RC2-B  these,  affected.  tissue  S63C  Of  patients  had g e n e r a l i z e d  kidney  Model  pestle.  were  Duncan  nucleic  Zn  causes.  kidneys  10  in  replacing  and Richmond  cancer  Both  for  involved  by  samples  Hospital  unrelated  Two  of  Methods  from  the  Cd:Zn  study  action  from  may be a  and  Four  came  the  pool  Materials  General  tissue  the  occur  transcriptase,  or Quaternary  1974;  enzymes  processes;  reverse  1976;  in  enzymes  division  i t s toxic  since  interest  Zn-containing  (Vallee,  so t h a t  protein  but  particular  polymerase,  kinase  increases  two  RNA  examine  significance  and c e l l  exerts  enzymes,  will  Of  are the  polymerase,  due  may be o f  6 on with  27,000 Super-  rehomogenized  1 34 for  3 minutes  guged  at  in  27,000  with  previous  then  placed  cellular  in  a  debris  nm  (1.6  a  x  to  via  10  minutes  establish  protein  pool,  weight  cytoplasmic and  Zn  and  bath  for  x  £  for  10  were  packed  Protein 124D  was  position  were  determined  each  metal  background  peak  levels  were in  was  each  done  tube  of  by  Pharmacia eluted  250  and  on  each  high  low  flame  atomic  a l . ,  graphite  summation peak.  profiles  of  15  ml  1  977).  absorption  furnace  employed  Total  each  280  molecular  et  Both  with  molecular  Brown  by  correction.  determined  then  and  and. t h e  by  spectrophotometry.  for  Cd  clear  were  a  at  the  1 977 ;  atomic  arc  to  gel,  of  metallothionein (Brown,  were  to  These  read  while  deuterium  5 minutes  G-75  pool  combined  supernatants  spectrophotometer  the  centri-  supernatants  applied  with  were  minutes.  spectrophotometry absorption  These  precipitation.  buffer. Elmer  NaCl.  Combined  water  heat  cm)  weight  Copper  for  27,000  100  Perkin  fraction  0.9%  supernatants  M NH4HC03 on  £  of  70°C  at  Resulting  0.01  x  ml  supernatants.  recentrifuged  column  2.5  metal of  levels  the  individual  Results Composite patients  are  shown  similar  total  this  occurs  Cd  occurs  in  10  11).  and  the  gel  Cd  in  Figures  levels,  on high  Zinc  elution  in  18  and  molecular levels  19.  noncancer  metallothionein.  were  weight higher  noncancer While  patients  In  cancer  protein in  cancer  both  have  most  of  patients  pool  cancer  or  more  (Tables  patients  but  1 35  Figure  18.  Composite of  2  of  gel  terminal  noncancer  molecular  weight  thionein;  III:  plasmic  pool.  elution  protein low  profiles  from  patients. pool;  molecular  II: weight  I:  kidneys high  metallocyto-  137  Figure  19.  Composite of  2  of  gel  terminal  cancer  molecular  weight  thionein;  III:  plasmic  pool.  elution  patients.  protein low  profiles  pool;  molecular  from  I: II: weight  kidneys  high metallo'cyto-  138  ^  .15  D)  .10  n  E  ^ .05 U  3  0.4  o> 0.3 £ 0.2 n 0.1  3  0.8 o> 0.6 § 0.4 "D  (J  0.2  o£4.0 ^23-0  %o2D o  to  1.0 1—i—i—i—i—i—i—i—i—i—i  5 Fraction  10 Number  15  Table  10.  D i s t r i b u t i o n o f Cd, noncancer p a t i e n t s . g tissue"  1  (wet  Cu  and  Zn amongst c y t o p l a s m i c  D a t a are  weight).  MW:  molecular  .192  Cancer patients  (2)  .186  a  t i s s u e of cancer  18 and  19  High pool  a  MW  Metal l o t h i one i n  and  i n >imole  Cu  Total  (2)  from kidney  weight.  Cd  Noncancer patients  pools  compi1 a t i o n s of v a 1 u e s f r o m F i g u r e s  Low MW pool  Total  Zn High pool  MW  Metal l o th ione in  Low MW pool  Total  High pool  MW  Metallothionein  Low MW pool  .0.65  .126  .001  .149  .044  .050  .055  .115  .076  .032  .007  .096  .084  .006  .147  .046  .048  . 053  .176  .072  .014  .090  Mean.  co LO  Table  11.  Percentage  of  cancer  noncancer  MW:  and  total  molecular  Cd,  Cu  or  patients.  Zn  on  various  Percentages  cytoplasmic calculated  pools from  from  Table  kidney  tissue  of  10.  weight.  Cd High pool  Cu MW  Metallothionein  L o w MW pool  High pool  Zn MW  Metallothionein  L o w MW pool  High pool  MW  Metallothionein  L o w MW pool  Non c a n c e r patients  (2)  33. 9  65. 6  0.5  29. 5  33.6  36.9  66.1  (2)  51 . 6  45. 2  3.2  31. 3  32.7  36.0  40. 9  27.8  6.1  Cancer patients  8.0  51 . 5  141 most  of  this  cytoplasmic The  ratio  the  high  (Table tween  increase pool  of  (Figures  Cd:Zn  18  weight  Copper  noncancer  the  low molecular  and 19;  was i n c r e a s e d  molecular  12).  was i n  in  protein  Tables cancer  pool  levels  appeared  and c a n c e r  victims.  10  and  11).  patients  and  to  weight  in  both  metallothionein  be u n c h a n g e d  be-  P i s c u s s i on The of  increase  of  the  Cd:Zn  cancer patients  in  the  present  changes  found  In  instances  both  cytoplasmic  in  ratio  tumor-bearing there  the high  pool  Cadmium  bound  to  metallothionein  that  i t  i s not  in  via  displacing  the  high  the  excess  is  In the  the  cancer  of  Zn  1977).  was a  in This  study.  this  affect  the high did not  increase  there  was a  similar  (Brown, in  bound  to  kidneys to  1977).  the portion  of  weight  by  produce  metallothionein. biologicaly  toxic  effects  metal 1oenzymes.  pool  pool  the  contains  in  Since  enzymes,  carcinogenesis,  Zn-containing  enzymes  processes.  flounders  However,  3.2-fold  patients,  in  human  is essentially  protein  division  tumor-bearing  present  there  cellular  to  portion  available  weight  available in  the  in  molecular  Zn-containing  Cd o c c u r r i n g  levels  (Brown,  from  molecular  likely  involved  Zn  to  is  increase  protein  inert  relative  in  study,  flounders  was an  Cd o c c u r r i n g  found  there  molecular occur in of  in  were  increases  weight cancer  protein patients  tumor-bearing Cd  in  1.5-fold  this  in pool in  flounders pool,  increase.  while  in  Therefore,  142  Table  12.  Ratio  of  Cd:Zn  in  tissue  of  Ratios  calculated  and  in  19  cancer  molar  cytoplasmic and  noncancer  from  units.  values MW:  pools  from  patients. in  Figures  molecular  Cd:Zn Total  High pool  MW  Metallothionein  L o w MW pool  2.59  0.13  12.00  0.04  Noncancer  patients(2)  1.19  0.65  1.58  1.11  Cancer patients(2)  kidney  18  weight.  143 although there  Cd  was  a  (1.7-fold) In Cu  humans  elements  carcinoma,  was  volved  in  of  Cd  et  a l . ,  of  cell  and  Zn  1971),  portant  for  the  flounders  with  cancer,  to  Thus both  increase protein  d i v i s i o n , binding  increases  of  degenerative  and  Cd:Zn  Since  in  the  in  humans  in  in  upon  as a  with  the  level  such  C d : Zn  change  ratio  these  flounders,  flounders  the  depend  sites  ratio  overall  enzymes,  will  flounders,  tumor-bearing  the  pool.  in  (1.4-fold).  and  the  pool  Cd:Zn  humans  of  Zn-containing  for  this  with  common an  in  of  unchanged.  weight  dysfunction  more  increase  compared  were  molecular  increased  larger  both  levels  trace  was  high  of  those  in-  competition  enzymes  ratio  (Flick  may  changes  such  as  those  levels  were  similar  be  im-  found  in  care inogenes i s. Summa r y Total cancer was  a  high  victims 1.5-fold  molecular  Cd  was  Zn  levels  the  cytoplasmic  found  low  sites  weight  Copper  in  terms  to  levels a  Zn-requiring  processes.  the  in  cancer  portion pool.  In  metallothionein.  weight  of  of  but  protein  increased  molecular  in  controls  increase  bound  were  victims. cussed  and  Cd  as  a  result  cytoplasmic were  of  Cd  involved  there  this  in  Total  Cd  of  in  Zn cell  most  of  Zn  in  cancer  Results and  the  cytoplasmic  increases  pool  human  victims  controls  unchanged.  competition enzymes  of  of  in  for  are  dis-  binding  division  144 CHAPTER  IX  Decreases  of  Molecular  Weight  of  Mice  With  Copper  Exposed  and Zinc  Protein to  and W i t h o u t  in  Pool  the  High  from  Livers  Diethylnitrosamine,  Cadmium  or  Zinc  Supple-  mentation. Preface This Chan  (1978),  Cancer an  paper  ratio  1977)  and in  (after  humans  12.5  induced  weeks  in  DEN  Knight,  in  mice.  weight  histological  protein  changes.  decrease  levels  protein  mice  weeks for  pool  and  d i d advance  and  Zn  in  of the  the  the  (Brown, a l l  time to  increase  appear  if  developed  tumors  did  Cu  in  administered  effect  see  fish  was  DEN  main  National  However,  tumors  was no  Y.  was t o  with  The of  10  There  the  changes  1978)..  studies,  of  of  cancerous  before  since  other  livers  in  and A.  study  induce  and  Cd a d m i n i s t e r e d  molecular  this  found  sampled  Brown  Journal  could  exposure),  exposed  of  A.  those  were  pretumorous  However,  weight  to  D.  the  purpose  (Brown  study  by  to  (DEN)  as n e c e s s a r y  in  to  The  similar  this  reported  be  submitted  carcinogen  Cd:Zn  C d : Zn  and  Institute.,  organic  mice  was a u t h o r e d  in  be  of  DEN. the  high  pretumorous  DEN  appeared  high  to  molecular  pool.  " Introduction Previous such It  as  Cd,  appears  studies  Cu with,  and  Zn  have are  indicated altered  occurrences  of  in  that  trace  heavy  tumor-bearing  cancers  other  than  metals  organisms. cancer  of  the  l i v e r ,  (Olson 1957;  et  that  a l . , 1954,  Sandberg  Brown,  liver  et  1977).  Cd,  Notabley, an  although  i s  tumorous  liver  and kidney  (Morgan,  1970,  1971;  than  Zn . ( . V a i l e e , . 1 9 7 6 ;  Cu  (White,  et.,  1956: With  creased the  in  tumors  and  to  Zinc  1961;  1958)  been  levels  liver  and increased  carcinogens.  of  metal  increased  a l .,  levels  levels  1 957 ;  (Arnold 1974)  tumors et  surrounding and  Dormandy,  and S a s s e ,  Zn is  i n -  in  1961)  surrounding  consistently  re-  a l .,  1 954,  1958;  and Dormandy,  1972;  Kew a n d  to  levels et  (Olson  1961)  in  levels  and S a s s e , c a n be a l .  et  a l .,  liver in  be d e c r e a s e d  (Arnold  of  Gorodiskii  report  Wright  Copper  Gorodiskii  Tumorous  has decreased  1954, 1958;  are normal  both  in non-  1977).  tissue  (Olson  tumors.  are i n -  organisms  liver  and S a s s e ,  reported  Changes  normal  liver  1972;  ratio  some-studies  decreased  in  et a l . ,  1969).  l i v e r ,  Wright  decreased.(Arnold  have  et  Teitz  Cd a n d Zn  Brown,  increased  1971,  the Cd:Zn  but  (Kew and M a l l e t t ,  Copper  rounding  1 977)  a l .,  be d e c r e a s e d  Sasse,  1974).  et  report  tissue.  ported  the  1970,  usually  (Teitz  apparently  Others  a l . , 1975;  both  1971;  liver  L i n ,  Cu a r e  tumor-bearing  and Haro,  in  (Olson  unchanged  liver  Furst  in  of  Flick,  a n d Cd  tumors  Zn  1972). or  in  increase  from  of  1921)  et  a l . , 1958; Morgan,  there  other  and sometimes  1 9 5 8 ; Koch  creased,  tissue  Zn  (Olson  Mallett,  1 958) or  tissue  liver  sur-  tumors et  al . ,  1961).  induced  (1956;  Arnold  in  by  Furst  organic and  Haro,  1969)  report  increased  nonmetal  carcinogens,  liver  levels  mice  Zn  decrease  increase  of  Arnold  in  liver  fed  DMBA.  selves. tumors  livers  Cu  of  slight  to  in  contradictory.  in  the  decreased  the  Cd:Zn  ratio  The  present  DEN  in  of  to  are  that  of  tissue  a l .  Cu  and  the  body  tissue them-  surrounding in  in  apfound  pretumorous  Fare  (1964) rat  DMBA Cu  Zn  are  liver  and  the  data  Zn  data  appear  to  findings  that  burdens  and  exposure.  consistent  cancer  rats  (1969)  changes for  of  tumors  metals  by  are  liver  pretumorous  only  with  in  content  However,  the  tissue  tissue  et  DMBA  for  decreased  in  increased  and  be  the  However,  that  data  in  these  gross  no  to  liver  Zn  Cu  found  changes  Zn  damage,  before  decreased  and  DMBA.  fact,  tumor  found  Yamane  liver.  metal  by  liver  However,  be  Cu  of  just  incrased  ( 1 963a,b)  In  peak  decreases  documented  be  are  but  indications  clearly  of  to  decreases  pretumorous  induced  observe  period  surrounding  progressively  are  study  and  tissue.  exposed  be  the  (1961)  DMBA  liver  rats  there  not  that  tumors  (1954)  evident.  found  tumors  Woodhouse  levels  levels are  or  is  decreases  normal  and  Thus,  liver  a l .  during  and  was  in  (dimethylaminoazobenzene)-exposed  Sasse  tumors  indicate  change  Fare  and  These  parently no  liver  Copper  surrounding  DMBA  et  regeneration  the  given.  content  Olson  i n i t i a l l y  during  neoplasia  both  of  Cd  of  Zn Cd  levels and  increased.  study  (diethylnitrosamine)  reports on  Cd,  the Cu  effect  of  and  levels  Zn  the  carcinogen in  pretumor  147 liver in  tissue.  the  pool  high  In  molecular  (Brown  et  et  a l .  (1954)  as  necessary  Further, replace  a l . , have  for  other Cu  al . ,  As  Zn  has  1 975 ;  and  et  a l . ,  1 974;  Woster  et  a l . ,  Zn  in  the  Cd,  of  Cd:Zn  ratio  Brown,  mice  pointed  Brown,  reported  1971;  Cu  Zn  water. and  Cd  as  also  977).  et  is  1 963 , 1972;  relative both  gave  evidence  organisms  a l . ,  1 976 ;  measured  we  cancer  a l . ,  1 975 ,  addition,  1971;  prevent  administered  there  tumor-bearing  1  et  may  enzyme  a l . ,  Gunn  Dreosti,  mice In  1 939;  in  Zn  systems. Cd  et  Ciapparelli  study  in  to  and  that  resulting  1 976;  Cu  Olson  enzyme  out  Kolonel,  Long,  of  and  Flick  this  and  and  another  of  (Morgan  DEN  increased  1970,  1971;  Methods  Mice  of  were  separated  cage. DEN,  enzymes  1961;  and  elements  reported.  a l . ,  1 975), and  are  importance  have  repeatedly  these  1977).  Materials  old,  of  and  DEN  in  the  of  (enzyme-containing)  1977)  Duncan  drinking  group  et  Cohen,  Duncan  in  Brown,  metal 1oenzymes  (Bischoff  Poswillo  levels  protein  activity  in  been  occurring  the  recognized  (Schroeder  et  changes  1977;  the  Zn  Yoshida  1964;  weight  investigators  and  dysfunction  from  particular,  Mice  the  Swiss  ppm  DEN  +  5  ppm  Cd  (as  DEN  +  on,  the  into  received  20  strain,  250  CdC12)  concentration  cages,  either ppm  of  approximately  tap  Zn for DEN  (as  3 mice  of  water,  water  the  ZnSo4•7H20),  five was  three  40  weeks. ppm.  months  same with or  From  sex 20  20 6  Drinking  per  ppm ppm  weeks solutions  148 (100  ml)  were  painted  Diet  consisted  ofj Purina  Mice  sacrificed  portion  were  (5jjm)  liver  order  different from  DEN  was  and s t a i n e d  In  1  liver  +  This  NaCl  and homogenized  S63C  for  exactly  homogenizing  time.  4  homogenates  minutes decanted Pellets ml and  of  in  pyrex  Each  a Sorvall  were  other  combined  RC2-B pyrex  with of  4  between  DEN,  1  a  time,  DEN  grams  + Cd, of  liver  of 0.9%  (setting  4.5)  motor  model  experimentation extracted  was p o u r e d tube  at  a  and each  g_ f o r  11  10 were  tubes.  2 minutes x £  with  group  Supernatants  centrifuge  27,000  varied into  27 , 0 0 0 x  exactly  at  The  analyzed.  at  Two  speed  metal  Sorvall  sectioned  four  4 . 5 ml  centrifuge.  centrifuges previous  1  with  centrifuge  A  standard  analysis  Previous  for  a  until  laboratory  centrifuged  rehomogenized  0.9% NaCl,  of  homogenate  Sorvall  DEN.  CO,,.  in  -20°C  time.  tube  jars  hematoxylin).  control,  of  with  analyzed  speed  amount  was t h e n  into  Groups  the  at  a standard  3 minutes.  that  capacity  at  variable  demonstrated  acid  exposure  a homogenizing  STIR-R  ml  (1  of  was wax e m b e d d e d ,  were  type  water  libitum.  preserved  consistency  same  A l l  asphyxiation  tissue  livers  the  placed  a TRI-R  by  ad  was s t o r e d  ensure  were  on  in  chow  (Phosphotungstic  exposure  from  rat  days.  photodegradation  immediately  exposures,  Zn)  alternate  prevent  portion  to  each  on  to  solution.  remaining  one  black  were  of  formal in  renewed  in  2.5  for  10  minutes  placed  in  a  supernatants.  supernatants  were  then  70°C  of  149 water  bath  debris tubes same  for  via be  heat  time  at  of  the  time  to  a  Supernatants  250 on  and  280  each  high the  nm  10.2  molecular low  Brown  a l . ,  Copper absorption furnace  of  a  100  fraction  high  at  that  weight  length  x  £  applied  for  to  G-75  gel  was  10  a and  read  at  spectrophotometer the  position  of  metallothionein  cytoplasmic  of  data).  Protein  pool,  the  molecular  with  124D  all  exactly  27,000  then  establish  protein  for  packed  Elmer  to  weight  important  (unpubl.  was  cm)  cellular  increasing  buffer.  Perkin  weight  and  zinc  were  pool  d e t e r m i n e d . by  spectrophotometry  atomic  metal  the  x  clear  (Brown,  the  and 1977;  1977).  determinations Total  of  with  supernatant  M NH^HCO^  molecular  et  increases  (1.6  on  ml  amount  recentrifuged  column  to  temperature  were  Pharmacia  0.01  is  temperature  Resulting  with  the  It  high  minutes.  eluted  same  as  precipitated  exposed  3 minutes  precipitation.  heated  period  protein  approximately  absorption employed  levels  individual  for  Cd  was  atomic  done  spectrophotometry.  deuterium each  metal  while  flame  peak  levels  in  by  graphite  Both  arc  background  correction.  were  determined  by  each  tube  of  summing  each  peak.  Results Liver of and  Cu Zn  and is  increase  tissue Zn v  upon  homogenate exposure  apparent throughout  at  to  3 weeks  the  supernatant DEN.  This  (Table  exposure  13)  period  of  mice  is  depletion and of  depleted of  continues  12.5  weeks  Cu to  Table  13.  The d i s t r i b u t i o n  o f Cd, Cu a n d Zn amongst p r o t e i n peaks f r o m l i v e r  with  and w i t h o u t  Cd o r Zn.  MW:  Molecular  weight.  ND:  Data a r e c o m p i l a t i o n s o f data  o f m i c e e x p o s e d t o DEN ( d i e t h y l n i t r o s a m i n e )  from F i g u r e  20 i n ^ m o l e / g t i s s u e  Cd  Cu  Total  High pool  MW  (wet w e i g h t ) .  Not d e t e c t a b l e .  Zn  Metal l o th ione i n  Low MW pool  Total  High pool  .00070  .00161  .0968  .0477  .0189  .0303  .0754  .0700  .0020  .0034 .0029  Mw  Metallothionein  Low MW pool  Total  High pool  MW  Metallothionein  Low MW pool  0 Weeks Control  (2)  00391  .00160  Control  (2)  00333  .00116  .00073  .00144  .0851  .0417  .0164  .0270  .0703  .0670  .0005  DEN  (2)  00322  .00063  .00098  .00161  .0783  .0386  .0167  .0230  .0577  .0536  ND  .0041  DEN  + Zn (2)  00281  .00085  .00054  .00142  .0809  .0410  .0160  .0239  .0567  .0558  ND  .0009  DEN  + Cd (2)  00292  .00095  .00061  .00136  .0692  .0314  .0160  .0218  .0535  .0500  ND  .0035  3 Weeks  6 Weeks Control  (2)  00320  .00081  .00068  .00171  .0740  .0446  ND  .0295  .0813  .0773  ND  .0040  DEN  (2)  00315  .00073  .00067  .00174  .0548  .0348  ND  .0200  .0646  .0615  ND  .0031  DEN  + Zn (2)  00307  .00090  .00061  .00157  .0595  .0313  .0202  .0716  .0640  ND  .0076  DEN  + Cd (2)  00322  .00117  .00067  .00138  .0426  .0226  .0200  .0589  .0547  ND  .0042  ND  .0080 ND  12 .5 Weeks Control  (3)  00376  3  (.00140)° DEN DEN DEN  a  (3) + Zn ( 3 ) + Cd (3)  .00139  .00086  .00151  .1447  .0698  .0340  .0409  .0873  .0822  (.00068)  (.00042)  (.00034)  (.0073)  (.0004)  (.0148)  (.0087)  (.0046)  (.0034)  .00375  .00126  .00097  .00152  .0993*  (.00140)  (.00072)  (.00057)  (.00030)  (.0149)  00362  .00110  .00080  .00172  .1148*  (.00119)  (.0053)  (.00042)  (.00042)  (.0110)  00685  .00389  .00092  (.00276)  (.00317)  (.00046)  .0401** (.0052) .0510** (.0014)  .0188  .0404  .0538*  .0501*  (.0050)  (.0101)  (.0068)  (.0098)  .0231  .0407  .0590*  .0555*  (.0005)  (.0122)  (.0073)  (.0095)  .00204*  .1004*  .0466*  .0193  .0345  (.00005)  (.0096)  (.0089)  (.0093)  (.0093)  .0542** (.0036)  .0525** (.0020)  .0051 (.0059)  ND  .0037 (.0034)  ND  .0034 (.0022)  ND  .0017 (.0029)  Mean. Standard d e v i a t i o n .  *P <^0.05; 2 - t a i l e d Student's t t e s t . **P <0.001.  i—• cn o  151 (Table  14).  It flect  is  evident  decreases  (Tables  13  and  appears  to  be  with  and  20). at  "3.  on  and  Cd  6 weeks  the  reverse by  DEN  22), 3  and  molecular  weight  was  not  apparent  and  14;  Figures  DEN  or  in  DEN+:-Zn. of  at  weeks  12.5  high  Cd  on  molecular 6 weeks  tissue  Cd  (Table  13).  on  high  the  in  the  that  Cu  13  and  Cd  with  Figure  metallothionein  in  this  study.  tends  to  otherwise 20,  from  of  Zn  slightly induced  21  carcinogen  appeared  there  the  molecular  high 13).  and  i n i t i a l l y  losses  of  liver.  high  This  to  DEN  protein  be  unchanged  increased this  DEN by  increase  weight  to  weight  effect  (Tables  were  be  13  of  also  Cd  (Table  DEN  Cd  of  protein  Cd pool  at  on  13). 12.5  accumulated (Table  de-  pool  evident  alone +  by  slight  protein  decreases  pool  to  to  appeared  These  exposed  molecular  13;  on  exposure  However,  of  exposure,  22).  levels  were  pool also  DEN  (Table  Figures  Zn  re-  protein  with  increased  and  Zn  administration  Zn  the  and  Copper  water  and 14;  in  weeks  12.5  mice  Most  of  at  weight  levels  weeks  drinking  Cu  (Table  22).  employed  protein  Cd  and  12.5  apparent  of  and  21  Cu  weight  detectable  resulted  21  tissue  creases  at  methods  (Tables  6 weeks)  Total  Zn  depletions  exposure  20,  of  metallothionein  is  Administration  (at  and  it  decreases  molecular  always  by  carcinogen tissue  on  or  not  Furthermore, with  high  Figures  reduced  was  these  the  14;  without  Copper  that  13,  the at  3 Total  weeks  Table  14.  Percentage molecular with  and  decreases weight  without  of  Cu  protein Cd  or  and  pool  Zn.  Zn from  in  total  livers  Calculated  tissue of  from  Cu  exposed  DEN  in  Table  to 13.  and  the  high  (diethylnitrosamine)  MW:  Molecular  weight.  High pool  MW  Total  -  High pool  MW  Weeks DEN DEN  +  DEN 6  data  supernatant  Zn  Total  3  mice  homogenate  +  Zn Cd  20. 0  (2)  8. 0  7. 4  17  9  (2)  4. 9  1. 7  19  3  16  9  25. 4  5  20  4  7  (2)  18  7  24. 7  23  (2)  25  9  22  0  20  (2)  19  6  29  8  11  9  17  2  6  29  2  Weeks DEN DEN DEN  12.5  + +  Zn Cd  DEN  42  4  49  3  (3)  31  4  42  6  38  4  39  1  (3)  20  7  26  9  32  4  32  5  33  2  37  9  36  1  Weeks  DEN DEN  (2)  27  + +  Zn Cd  (3)  30  6  ro  153  Figure  20.  Composite  gel  cancerous  livers  exposed for is I: II:  to  12.5 the  DEN  elution  high  control  with  weeks. average  of  and  Each of  molecular  cytoplasmic  from  mice  without  Cd  III:  pool.  protein low  mice  or  Zn  profile  individual  weight  non-  and  composite  three  metallothionein;  weight  profiles  profiles. pool; molecular  1 55  Figure  21.  The  variation  lar  weight  livers DEN Each  of  with  presents  Cu  levels  protein control  and  point  animals  of  pool mice  without  at  0,  3  while  at  12.5  3  animals.  and  from and  Cd  on  or  high  molecu-  noncancerous  mice Zn  6 weeks weeks  the  exposed  for  12.5  to weeks.  represents  each  point  re-  2  157  Figure  22.  The  variation  lar  weight  livers DEN Each  of  with  presents  Zn  protein control  and  point  animals  of  levels pool  mice  without  at  0,  3  while  at  12.5  3  animals.  and  from and  Cd  on  or  high  molecu-  noncancerous  mice Zn  6 weeks weeks  the  exposed  for  12.5  to weeks.  represents  each  point  re-  2  Figure  20).  It  is  % metal  probable  (Table  portrayal  of  eliminates times  for  14;  the  metal  group  DEN-exposed tumorous.  +  mice,  hepatic  Zn-exposed  had  liver  ment.  the  DEN  +  livers, the  of  (Table  Pi  scussion This  and  Zn  in  the  and  (Table  than  a more  with  in  accurate  time  variations  indicated  without  as  in  this  heating  Zn,  were  alone  or  Mice  exposed  towards  indicative was  visible  livers,  two  of  exposed  for  those  exposed  presents  clear  evidence  to to  Cd DEN  +  Cd  develop-  in  all  variable,  mice  DEN  bile  Although  +  than  of  l i v e r s ,  for  DEN  to  the  of pre-  tumor  Zn-exposed  15).  rating  or  livers  regeneration  advanced  mouse  that  DEN  and  15).  most  +  Cd  receiving  (Table  that  give  slight  dysplasia  DEN  decreases  samples.  mice  cell  duct three  of  DEN-exposed  and  none  the DEN  DEN  of  overall +  or  Cd DEN  +  15).  report  pretumor  organic  carcinogen.  creases  liver  liver  to  24)  as  occurring  p r o l i f e r a t i o n ,  change  greater  Zn  of  Cd-exposed  controls  was  and  cholangiofibrosis,  cell  one  histology  of  pathologies  epithelial  due  with  livers  Gross  23  examination  Livers  more  expressed  changes  differences each  data  Figures  Histological  had  that  damage  Zn but  tissue Olsen  during they  resulting  do  et  the not  a l .  period  from  (1954) of  present  of  decreased  exposure report  to  OMBA  Cu an de-  carcinogen-induced any  data.  Arnold  and  1 60  Figure  23.  The  percentage  high  molecular  cancerous 8  decreases  week  livers  point  which  mice  point  at  animals  weight  is  were  0,  3  protein  a  levels pool  to  6 weeks from  animals.  25  from  ppm  and  the non-  The  study  in  DEN.  Each  represents 8  on  mice.  subsequent  exposed  those  Cu  DEN-exposed  from  3 and  while  represents  of  of  12.5  2 weeks  162  Figure  24.  The  percentage  high  molecular  cancerous week  point  mice  were  at  decreases weight  livers is  of  from  exposed  a to  0,  3 and  6 weeks  while  those  from  3  an i m a 1 s .  8  of  Zn  protein  levels pool  mice.  subsequent  study  ppm  DEN.  represents and  12.5  Each 2  weeks  the  from  DEN-exposed  25  on  nonThe  in  8  which  point  animals represents  163  Table  15.  Changes  in histology  Hepatocytes Increased regeneration  Control  of l i v e r  Dysplasia  after  12.5 weeks e x p o s u r e  Reticular collapse  Hepatitis  t o DEN  ( d i e t h y l n i t r o s a m i n e ) w i t h and w i t h o u t Cd o r Zn.  Hepatocellular necrosis  P o r t a l duct areas Portal B i l e duct c e l l triaditis proliferation and d i s a r r a y  Overall histology change rating 3  #1 #2 #3  DEN  DEN  #1 #2  1  #3  2  + Zn #1  1  #2 #3 DEN  + Cd #1 #2 #3  From 0 t o 3, b a s e d on m i c r o s c o p i c e x a m i n a t i o n o f p h o s p h o t u n g s t i c a c i d ''From 0 t o 3, b a s e d on m a c r o s c o p i c c  Absent.  ''present.  examination  at time o f autopsy.  hemotoxylin  stained  tissue.  Gross cholangiofibrosis rating* 5  165 Sasse in  (1961)  indicate  nontumorous  liver  areas  tumors.  carcinogen  The  can  unequivocally is  of  liver  liver  present  decrease that  of  and  rats  study  liver  these  Cu  Zn  with  confirms  Zn  and  changes  are  Cu  occur  decreased  DMBA-induced that  and,  an  organic  demonstrates  before  tumor  growth  evident. Decreases  from  the  occur  high  in  the  high  the  indication  of  heavy  Davies, Thus, only  and  Zn  molecular  1 974;  become  and  elements 1 977 ;  that  1 975 ;  decreases these  function  of  give  et  Cu  al . ,  Zn  977).  of  and 1 975).  were  decreases  would  and  cellular  hence  1  al . ,  and  good  for  (Bremner et  cell  a  excesses  Winge of  enzymes  available  Brown  apparent  Gower,  metallothionein, enzyme  this  metallothionein  from  on  pool  Zn  (Brown,  carcinogen-induced  effect  in  these  Riordan  Since  fraction  of  on  mainly  protein  levels  stored  pool.  are  weight and  has  tissue  protein  Cu  if  l i t t l e  pretumor  of  it  are  in  weight  functioning  years,  metals  Cu  levels the  metal 1oenzyme recent  of  molecular  cytoplasm,  In  that  have pro-  cesses. The weight Many  slight  protein  decrease pool  researchers  ratio  in  bearing Haro, 1977).  tumors  report and  organisms  1969; The  of  Teitz  the  of  Cd  observed  increase's  1975;  et  high  mice of  apparently  a l . , Cd  the  DEN-exposed  (Gorodiskii et  on  Cd  and  unexpected. the  Cd:Zn  livers  1956;  Morgan,  decreases  was  normal  a l . ,  molecular  1970,  concurrent  in  of  tumor-  Furst 1971;  with  and Brown,  decline  166 of  Cu a n d Zn  in  pretumorous  Zn  to  just  levels  liver.  i n i t i a l l y before  creased  in  liver  1972).  decreased  in  liver  tissue  tissue. Cu  starts  in  The  of  i t  tumor  pool  of  mice  Cd on t h e h i g h  bearing  flounders  accumulates  that  induction  increased Morgan, cance sulted  in  1970,  that in  Cd on  (Brown, pool  weight  at  1971;  exposure  after  is  seems  normal of  the patterns  protein both  too  vacated  metallothionein  but  This  the  molecular  Perhaps  sites  Cu i s  tumor of  tumor  tissue.  In  levels  and  that  tumors  + Cd w a s s i m i l a r  1977).  tumor-bearing  liver  as or  high  1977).  binding  of  the  DEN  (Brown,  enzyme  just  surrounding  molecular  on t h i s  metallothionein occupying  increase  in  development  follow  de-  Wright  apparently  with  peak  only  i t s e l f .  in  liver  to  found  liver  changes  remains  1958;  (1961)  Cu  to  but  Zinc  al . ,  increases  given  tissue  tissue  metal  report  increased  et  decreases  Cd c o n t i n u e s  of  (1954)  surrounding  i n t h e tumor  and/or  increase  and i s  and Sasse  might  early  i s evident.  Arnold  DMBA  i f  a l .  (Olson  tissue  of  pretumor  tissue  but that.Cu  Thus,  and Zn,  tein  that  in  et  tissue  liver  increased  indicate  Olson  neoplasia  the tumor  Dormandy,  to  be t y p i c a l  decrease  gross  surrounding  greatly  might  does  weight to  pool  Cd by  increases of  studies  low to is  tumorCd  have  saturated  simply  Cu a n d Zn  not  pro-  occur.  so As  Cd i s  organisms  (Teitz  et  a l .,  Brown,  1977),  may be o f  some  s i g n i f i -  to  +  study  re-  DEN  pathologies  Cd most  i t  in  the present  advanced  towards  1 957 ;  tumor  167 development. earlier tumor  with  Cu  and  exposure these  to  as  Zn  the  ture  is  perhaps  with  the  onset  as  Zn  since  Zn  with  Thus,  could most  DEN  As  organic metal  of  pretumorous  accordance that  Zn  and as  Long,  1964),  1939),  or  Ciapparelli of  et  transplanted  Dreosti, though  1 975 ,  the  Zn  excesses  growth  of  tumors  and  Zn  (Bischoff  the  of  drinking bind  Zn,  in while  Furst  a  (1963),  can  be  by  Zn  in  reports  in  the  from  gland by  DMBA  1 972).  Zn  (Duncan  Woster on  Zn  et  in-  cancer Cd  (Gunn  also  et and  prevents  a l . ,  a l . ,  is  Long,  l i t e r a -  (Poswillo  et  1974,  1 975).  It  contradictory  deficiencies and  this  occurring;  by  tumors  literature  both  by  of  losses  result  and  induced  induced  1 976 ;  in  could  mammary  a l . ,  increases  otherwise  changes  cancer  cancer  evidence  weeks'  of  Cu  pre-  chelators.  cancer  occurring  earlier  carcinogens  previous  prevents  spontaneously and  with  occurred  some  Zn  explained  all  Zn  reduced  would  alone  reduction in  DEN  excretionof  not. or  which  of  12.5  Perhaps  heavy  spread  appears  cancer.  of  1971;  Duncan  on,DEN  at  of  suggestive.  sites  and  is  indicative  those  1963,  Cohen,  of  subsequent  of  as  (Bischoff a l . ,  is  liver.  stating  cluding  the  Cd,  Cu  there  increasing  +  and  explained  the  DEN  actions  study  levels  of  indicative  are  complex  The  perhaps  Zn  in  chelation DEN-Zn  Cd,  decreases  Furthermore,  occupies  and  +  administration  well  water  large  changes.  metals That  Cu  DEN  metal  that  Cu  Also,  can  1 939;  prevent Gunn  et  a l . ,  168 1 963,  1 964 ;  Poswillo DeWys  Dreosti,  Zn  shown  and  might  in to  Woster  have  Fare,  Howell,  summarized Cu  by  result  metabolizing  in  carcinogens  Yoshida  et  a l .  in  (1975)  inhibition  Dysfunction  may  of  activity 1969).  1 970;  Duncan This  and  may  be  activities  of  1976)  while  zinc,  et  al . ,  Zn  which  has  been  DMBA-induced and  Zn-  (Duncan  processes  on  Fare  of  It  has  (1976),  which  tumors  Woodhouse,  of  the  this Cu  been  in  is  deficiencies activity  essential  noncarcinogenic  found  detoxification  carcinogens. onset  in  reductions the  to  possibly  metal 1oenzymes  reduce  1 958;  al . , 19172;  division  like  reductions  center,  of  induced  the  the  effect  Becking  version  on  1 974;  Vallee,  Copper,  al . ,  1975).  cell  prevent  et  1 963a,  1964).  and/or  result  or  1976;  inhibitory  1 946;  a l . ,  for  et  al . ,  could.lessen  Dreosti,  an  et  et  essential  reverse  McQuitty  Ciapparelli  carcinogenesis.  (Sharpless,  As  1976;  1 967 ;  Duncan  deficiencies  Duncan  result  a l . ,  1 972 ;  enzymes  excesses  drug  1971;  Pories,  dependent  Zn  Cohen,  1975,  because  b;  et  and  and  1974;  Petering  be  that  high  hepatic a  result  enzyme and of  Zn  of  Cd  subsequent  proposed  that  administered  with  of  drug  metabolizing  center  Zn  administration  has  Cd  DMBA  a  for  the  con-  of  Cd  could  replacing  Cu  when  Perhaps  hepatic  levels  (Yamane similar  Cu  could to  prevents by  center.  carcinogen-  exposures  cancer the  the  Thus,  high  .  of  metabolizing  system. and  both  metabolities .  levels  drug  of  the  stimulating et  a l . ,  effect  since  1 69 Zn  deficiencies Losses  of  inhibit  of  Zn  Zn-containing  and  Dreosti,  this  from  the  enzymes  1976;  Cd c o u l d  s i m i l a r i l y  Zn  on t h e s e  enzymes  (Brown,  Zn  carcinogen  preventing  ing  enzymes  involved  Chvapil portant  in  et  in  a l .  This  1977).  cell  in  that  the  losses  biomembranes.  Recent  sponsible. for  1977).  f o r moving  maintaining  and K r e s s ,  prevent  lipid  1977;  modifies  structure  fatty  organelle  prevents  These  membranes.  cancer  by  Zn-requir-  1977).  thus  cell  agents  weakening  the  have  is  that  are re-: and  membranes can such  also as C C l ^ .  conformational  lipoprotein  suggested  immune  or  division  Zinc  dimensional  Others  increasing  by  state  microtubules  cellular  induced  of  evidence  into  1977).  of  e t . a l . , (972)  are part  during  of  the three  acids,  a  proteolytic  microtubules  M i l l e r ,  peroxidation  with  breakdown  Chvapil  tubulin  chromosomes  Peroxidation  and  of  the continuity  (Gaskin  of  a n d RNA,.  of  related  and K r e s s ,  been  lysosomal  which  (Gaskin  has  of  suggests  the assembly  i t  via stabilization  macromolecules  stimulates  replacing  is im-  stabilizes  Zn  levels  Zn  that  to  by  Cd w i t h  suggested  which  closely  (Duncan  High  (Brown,  enzymes Zn  DNA  dysfunction  administered of  1976).  that  results  damage  1977).  Therefore,  when  have  in  division  division  division  cancer  result  Brown,  cell  (Becking,  cell  interference  theory  membrances  in  cancer  (1972)  preventing  membrances. lysosomal  prevents  system  might  1976;  disturb  proposed.that by  liver  involved  Vallee,  of  enzyme  that  response  cell Zn  (Woster  1 70 et  a l . ,  the of  1975;  fact Zn  Nieper,  that  lymphocytes  ; (Chvapil Both  reduced  Cu  by  both  have  down  the  et  and  al . ,  Zn  organic  been  shown  carcinogen.  of  Cu  been  responsible  for  in  Zn  the  it in  in  due  in  in  of  of  these  result  this  part  the  to  presence  to  naturally  are  an  Hence,  and slow  organic  the  in  re-  some  way  alleviation  occurring  a  be  or  with that  tissue  to  tissue,  prevent  l i k e l y  cancer.  in  study  pretumor  administered  pretumor  onset  may  be  in  appears  or  of  viable  studies  when  carcinogen-induced  prevention,  more  shown  other  of  elements  may  carcinogen  cancer  and  This  1 972).  Therefore,  of  are  have  an  onset  ductions  1977).  deficiencies  reduction,  or  even  .  cancer.  Summary Mice  were  given  drinking  water  Zn.  elution  Gel  genate All  molecular  Cu  at  3  and  Zn. 6  Cd  on  6  DEN  Cu  done and  +  alone  protein was  were 3,  Cd-  and  decreased  Zn  but the  not high  at  tissue  12.5  weeks  of  or  12.5  DEN  +  in  with  after  Zn  increased weeks,  molecular  39.1%  12.5 in  and  weight  or  homo-  Zn-exposed  increasing  and  Cd  exposure.  decreases  resulted  supplementation  with  liver  42.6%  pool  their  on  resulted  protein  in  supplementation  supplementation  weeks  accumulation  and  weight  Zinc  and  0,  DEN  Copper  exposure.. of  at  DEN-  weight  exposure.  without  profiles  pretumorous.  molecular  high  from  (diethylnitrosamine)  and  supernatant  livers  were  with  DEN  mice  of  high  time on  weeks  of the  of  lesser  depletions  Cu  Zn  and  resulted  protein  losses in  pool.  Cd  171 At  12.5  were in  to  higher  those  was 2  weeks, in  the  DEN  mice  exposed  evident  of  hepatic  in  mice  +  Zn,  Decreases animals  fed  of  cancer  to  prevent  benzene). is  due  to  mechanisms levels  of  cancer It the of Cu  is  Cu  are  both  +  3 of  none  of  DEN  as  DEN  exposed and  Cu  Gross  mice  of  the  controls.  Zn  in  proposed and  Zn  of  to  that  1  to  of  the  than  of  +  mice  Cd, exposed  of  in  the  etiology  in  other  studies  (Dimethylaminoazo-  preventative Cu  resulting  discussed.  Cd  livers  shown  DMBA  this  losses  +  DEN  relevant  been  with  inducement are  be  dysplasia  DEN  pretumorous  have  given  or  exposed  alone,  and  and  cholangiofibrosis  DEN  suggested  Zn  alone  to  when  cancer  DEN  Zn.  the  prevention  and  regeneration  receiving  to  a l l  cell  and  Zn.  from  effect Possible  reduced  1 72 CHAPTER  X  Decreases and  of  Copper  Posttumorous  and  Livers  Diethylnitrosamine, or  Zinc  Zinc of  With  in  Pretumorous  Mice  Exposed  and Without  to  Cadmium  Supplementation  Preface This will  be  this  study  pre-  to  of  Cu  in  the  were and  was  to  Chan, and  follow  Zn.  can  previously (Brown  (.1 9 7 8 ) ,  molecular cancer,  DEN  protein  higher  than  i t  As  was  weight  protein  Cu  and  control  in  pretumorous  Zn  Cd  and  Zn  had  and the pool;  fish  1978). in  from  decreases  exposures  that ratio  these  changes  maintained  1977)  Brown  decreases to  where  an  Cd:Zn  in  Time  of  liver  (Brown,  As  and  purpose  demonstrated  and Zn.  were  Cu  liver  protein  resulted  those  The  Cd,  cancerous  Cu  (1978),  in  not  increase  in  in  Brown  publication.  and K n i g h t ,  increased  A.  posttumorous  exposure  weight  weight  for  D.  changes  Thus,  high, m o l e c u l a r  humans  by  stages.  1978),  carcinogen  found  Chan  shortly  posttumorous and  organic  was w r i t t e n  submitted  (Brown Cd,  paper  and  of  high  death  high  closer  due  to  molecular to,  or  levels.  Introduction Recently 40  we  have  reported  mg d i e t h y l n i t r o s a m i n e  results liver 1978).  in  depletion  tissue  up  to  Furthermore,  of  12.5 we  that  (DEN)/L copper  in  and  administration the zinc  weeks' e x p o s u r e found  that  drinking in  of  25-  water  pretumorous  (Brown  and  administration  Chan, of  2 5 0 mg  173 Zn/L  with  DEN  resulted  When  5 trig C d / L  was  greater  depletions  but  at  not  high  (Brown,  in  as  weight the  flounders and  duck  of  Cd  pool and Zn  (Brown,  liver  might if  Chatel , in  that  the  in  simply  In  the  this  to  pool,  excesses  in  weight  protein  pool  a  human  study it  was  high  Therefore,  molecular occupies  trace  weight  Zn  as  DEN  binding  sites  of  Cd  (Brown interactions  that  this  excesses protein  pool  reduces  protein  metallo-  tumor-bearing  weight  in  the  molecu-  victims  determined  of  high  of  in  tissue  element  molecular  deficiencies  in  increases  cancer  of  exposure,  liver  the  Zn.  were  than  in  to  and  and  accumulated  Cd  Cd  Cu  weeks'  rather  of  of  six Cd  of  there  similar  the  1 978a).  Cd  of  DEN,  were  In  were  high  Most  kidney,  occur  there  up  increases  1977)  and  Zn  for  pool  1978).  and  protein  molecular  Knight,  in  Cu  These  protein  high  of  usual  1977).  depletions with  weight  is  lesser  administered  weeks.  molecular  thionein  lar  12.5  in  (Brown  levels  of  pool,  it  is  l i k e l y  vacated  by  Zn  in  this  pool .  tumors  present  develop.  tored  throughout  given  DEN  Zn  than  +  in  completely  study,  Copper, this  previous  reverse  Zn.  Another  mine  the  group  effect  of  zinc  the  to  exposed  cadmium  period.  determine  study  (.Brown  DEN  levels  +  100  of'Cd  to  DEN.until  levels  Another  if  higher  and  Chan,  carcinogen-induced  receives high  are  and  exposure  1 0 0 0 mg Z n / L the  mice  mg on  moni-  group  is  levels  of  1 978)  losses Cd/L  are  to  of  can Cu  deter-  carcinogenesis.  and  174 Materials  old,  of  were  separated  Mice  the Swiss  were  2 5 mg D E N / L  were  changed  black  to  Purina  from  type  were  study;  after  s i x of  analyzed  mice  visible Mice  exposure  tumors  type  + Cd)  genized  S63C  equipped  centrifuged  1  a  to  A l l mice  four  at  Control, ensure  using  until  Two g r a m s  speed  at  after  death.  in the  after  1  of  DEN of  +  present  s i x DENfor  presence  Zn,  and 1  were  homo-  tube  laboratory  with motor  4 . 5 ) and  Homogenates x £  each  analysis be-  liver  (setting  27,000  0,  exposure  one from  STIR-R  homogenizer.  at  asphyxia-  i n a homogenizing  a TRI-R  fed  analyzed.  a time, DEN,  painted  sampled  examined  consistency  standard  10 m i n u t e s  levels  were  1  were  d i e in each  sampled  frozen  3 minutes  a teflon  for  were  exposures.  set to  with  mice  Mice  via  )/L,  solutions  were  sampled  also  to  Q.9% NaCl  were  mice  ( i . e . ,  for exactly  ml o f  mice  water  jars  were  exposure  element  analyzed  different  of  tap  Drinking  DEN.  type  for trace  and then  in order  of  sex per  (as ZnS04•7H20  A l l water  exposure  these  had d i e d .  were  mg Z n  the start  Remaining  s i x control  exposed  +  1 000  months  t h e same  tap water,  photodegradation  each  of  two  either  days.  mice  CO,,.  3 mice  (as C d C l 2 ) / L .  Three  the f i r s t  model  1 0 0 mg C d  _ad_ 1 i b i t u m .  Only  4.5  2 5 mg D E N / L  chow  weeks  approximately  cages,  rat  with  tween  into  on a l t e r n a t e  tion  DEN  +  prevent  a n d 17  of  strain,  administered  2 5 mg D E N / L ,  With  8  Methods  Mice  cage.  or  and  were  in a Sorvall  RC2-B  175 centrifuge.  Supernatants  homogenized These and  for  were  the  Groups  exactly  then  4  for  exactly  heat  precipitation. x  £  for  Total were and  determined; Cd  These  Cu  and by  spectrophotometry.  weeks a  ml  was  column  fractions.  pool,  pool  levels  similar each  to  124D  (Brown were  total  elution  metal  and  in  higher  each  of  via  type ml  at  collected. levels by  flame,  absorption  homogenate.  from  0,  8 or  17  five  of  this  applied  100  cm), p a c k e d  with  G-75  read  and at  on  fraction  each  levels.  molecular  Brown each Total  determined each not  on  and  metal by  nm o n  weight  cyto-  Chan,  1978).  with  methods  levels summing  of  each  animals  to  protein  weight  fraction  fraction  done  15  280  low molecular  in  as  250 and  the  a l . , 1977;  to  g e l .  collected  and  were  water  and  was  in  70°C  debris  atomic  the  NaCl.  minutes  levels  high  levels  a  metal  Cd  furnace  M NH4HC03  were  10  supernatants  the  metal  0.9%  recentrifuged  supernatant  re-  supernatants.  cellular  then  the  of  for  placed  were  determined  profiles  x £  pellets of  of  et  pool  ml  then  spectrophotometer  position  cytoplasmic  individual Gel  0.01  metallothionein  plasmic Metal  the  with  x  Absorbance  Elmer  establish  (.1.6  2.5  previous  exposure  combined,  eluted  Perkin  each  ml  and  with  clear  and  Zn  in  27,000  graphite  Two  from  then  Pharmacia  This  a  were  to  homogenate  levels  supernatants  at  were  minutes  tissue  lower  minutes  5 minutes  10  collected  combined  supernatants  bath  27,000  2  centrifuged  supernatants of  were  for of  the  pool.  which  were  176 collected  after  death  therefore  it  likely  decreased  the  creased  the  providing  is  high  low  as  these  that  misleading  not  proteolytic  molecular  molecular  were  weight  weight  frozen  immediately;  enzymes  would  protein  pool,  cytoplasmic  have  and  in-  pool,  thus  levels  were  results.  'Results Total  tissue  decreased  by  decreases  ranged  of  death.  to  DEN  of  only  Zn  Figures  25  In Cu  and  mice  Cd  8  pool  weight  Cd,  was  thionein (Table  16  weeks  and to  reached  a  17).  in  to  or  DEN  at  54.0%  the  mice  maximum  Exposure  up  These  28.0%  unchanged  pool,  the  decreases  was  18). a  only and  +  to  time  exposed  decrease DEN  (Tables  Zn,  +  16  Cu  In  mice  but  17  slightly increased  in  of  Cd  and  17,  the  reductions high  exposed Cu  large  increases  After  these  of  decrease  pool ,  smaller  pool.  decreased  and  DEN  (Table  and  cytoplasmic  protein  were  Cu  protein  weight  weight  to  there  metallothionein,  Cu  8  Cu  26).  weeks,  molecular  at  and  of  reflected  protein  (.Tables  time o f d e a t h .  exposed  weight  DEN  weeks,  the  supernatant  21.8%  increases  levels  for  from  8  at  in  to  Cu.levels  for  8.1%  resulted  in  exposure  Total  +  homogenate  in  in  to  the  DEN  the  low  exposure  in  high  slightly  1ow  molecular  weight  levels  decreased  progressively  on  +  high  increases  weeks' the  molecular  in molecular to  molecular metallo-  cytoplasmic  pool  18).  Total  Zn  from  8  weeks'  Table  16.  L e v e l s o f C d , Cu a n d Zn I n l i v e r  tissue  with  with  and w i t h o u t s u p p l e m e n t a t i o n Cd  homogenate s u p e r n a t a n t o f m i c e e x p o s e d t o d 1 e t h y l n 1 t r o s a m 1 n e  Cd o r Zn. V a l u e s a r e e x p r e s s e d Cu  1n j i m o l e / g t i s s u e Zn  0 Weeks Control  (3)  0.00068"  0.0373  0.0739  (0.00017)°  (0.0033)  (0.0055)  8 Weeks Control  (3)  DEN DEN  (3) + Zn ( 3 )  DEN + Cd ( 3 )  0.00062  0.0344  0.0664  (0.0001 2)  (0.003Z)  (0.0062)  0.00029*  0.0269  0.0546*  (0.00013) 0.00043 (0.00020) 0; 1 2 6 6 * * (0,0116)  (0.0080) 0.0345 (0.0077) 0.0448 (0.0079)  (0.0038) 0.0583 (0.0080 0.0920 (0,0189)  17 Weeks Control  (3)  DEN  (3)  DEN + Zn ( 3 ) DEN  + Cd ( 3 )  0.00064  0.0291  0.0772  (0.00028)  (0.0010)  (0.0022)  0.00118 (0.00118) 0.00024* (0.00008)  0.0220** (0.0006) 0.0279 (0.0026)  0.0566* (0.0058) 0.0589** (0.0022)  0.1611** (0.0134)  0.0448** (0.0019)  0.0935* (0.0094)  Time o f D e a t h Control  (6)  0.00053 (0.00022)  0.0236 (0.0028)  0.0739 (0.0115)  DEN  (6)  0.00032* (0.00016)  0.0170* (0.0061)  0.0413** (0.0184)  DEN + Zn ( 6 )  0.00040 (0.00013)  0.0217 (0.0038)  0.0863 (0.0528)  DEN + Cd ( 6 )  0.2689** (0.0635)  0.0274 (0.0085)  0.1919* (0.0822)  "Mean. ''standard *P **P  < 0 . 0 5 ;  deviation. Student's  < 0 . 0 0 1 .  t  test.  (wet w e i g h t ) .  (DEN)  1 78  Table  17.  Percentage tissue mice and  8  homogenate  exposed without  to  of  C u a n d Zn  of  diethylnitrosamine  (DEN),  with  Zn  supplementation.  Cu  Zn  (.3)  -21 . 8  -17.8 -12.2  +  Zn  (3)  + 0.1  DEN  +  Cd  (3)  +30. 2  +38. 6  (3)  -24.4  -26. 7  weeks  DEN  +  Zn  (3)  -4.1  -23.7  DEN  +  Cd  (3)  + 54.0  + 21 . 1  (.6)  -28.0  ^44.1  Time  of  Death  DEN  total  livers  DEN  DEN  in  from  Cd o r  supernatant  levels  Weeks DEN  17  changes  DEN  +  Zn  (.6)  -8.1  DEN  +  Cd  (.6)  + 16.1  + 16.8 +159.7  179  Figure  25.  The  variation  livers Cd  or  of  of  mice  Zn.  Cu  levels  exposed  Each  point  represents  3 animals,  represents  6  the  mean  to  die  Cd  or  time  after Zn  animals to  at  DEN  cytoplasm with  and  from without  0,  8 and  17  weeks  while  those  at  30  each.  death  exposure  to  in  of to  supplementation.  Thirty the  DEN,  f i r s t with  weeks 6 and  weeks was  animals without  C9  ZD  to CO  0.05 h  D)  D E N + Ccl  | 0.04 E 1  ZL  ^ 0.03 U £ 0.02 DEN  CO  ai  o 0.01  u •+-» o h-  8  12  16  20 24 28 W e e k s of E x p o s u r e t o D E N  32  CO  o  1 81  Figure  26.  The  variation  livers Cd  or  of  of  mice  Zn.  Zn  levels  exposed  Each  point  represents  3 animals,  represents  6  the  mean  to  die  Cd  or  time  after Zn  animals to  at  DEN 0,  while each.  death  exposure  to  in  of to  supplementation.  8  cytoplasm with  and  without  and  17  weeks  those  at  30  Thirty the  DEN,  from  f i r s t with  weeks 6 and  weeks was  animals without  D  CO CO  +-> U) \  0.20  o E 0.15 3-  DEN+Cd  c N  u  0.10  Control  £  CO  _^o  q-^~~ D E N + Z n  (TJ  a 0.05 o  DEN  •i  u To •i o  —i  4  1  1  1  1  1  1—  8 12 16 20 24 28 W e e k s of E x p o s u r e t o D E N  32  Table  18.  The d i s t r i b u t i o n  o f Cd, Cu and Zn among c y t o p l a s m i c  diethylnitrosamine expressed  (DEN) w i t h  i n ^mole metal  and w i t h o u t  i n each  pool/g  from l i v e r s with,Cd  o f mice exposed t o  o r Zn.  Values are  t i s s u e (wet w e i g h t ) .  Cd Total  pools  supplementation  Cu High poo 1  HW  a  Zn  Metallothionein  Low MW pool  Total  High pool  MW  .00041  .00053  . 0520  . 0301  Metallothionein  Low MW pool  Total  .0070  .0149  .0726  High pool  MW  Metallothionein  Low MW pool  0 Weeks .0074  . 0652  ND  . 0644  .0626  ND  .0485  .0471  ND  .0014  .0137  . 0536  .0527  ND  .0009  .0243  .0199  . 0987  .0841  .0089  .0057  .0063  (3)  .00186  .00092  Control  (3)  .00168  .00080  .00039  .00049  .0481  .0292  .0062  .0127  DEN  (3)  .00153  .00061  .00047  .00045  . 0402  .0216  .0068  .0118  DEN + Zn (3)  .00159  .00075  .00042  .00042  .0488  .0294  .0057  DEN  .1084  .0212  .0860  .0012  .0630  .0188  Control  b  8 Weeks  + Cd (3)  .0018  17 Weeks Control  (3)  .00199  .00075  .00040  .00084  .0488  . 0281  .0077  .01 30  .0668  .0570  .0035  DEN  (3)  .00246  .00090  .00054  .00102  . 0374  . 01 93  .0082  .0099  .0487  .0435  .0008  .0044  DEN  + Zn (3)  .00177  .00063  .00036  .00078  .0385  .0198  .0069  .0118  .0478  .0415  .0005  .0058  DEN  + Cd (3)  .1927  .1241  .0174  .0512  .0547  .0249  .0085  .0213  . 0705  . 0553  .0033  .0119  a  Molecular  b  Not  weight.  detectable.  184 exposure the to  t i l l  time DEN  +  weeks,  resulted  in  lesser  increases  in  Zn  very  high  mice  (Figure  in  in  Zn  after  Zn  levels  in  the  fifth  up  to  (Table  apparent  that  Exposure  to  was  pool,  DEN  high,  exposure  to  Total  Cd  to the  DEN  at  time  sulted  in  8 of  8  high  only  Cd  and  weight  to  protein  low  than  in  increases  (Table to  the  17).  DEN  or  high  weeks' it  is  in  increased  protein  exposure molecular  reverse  to  metallothionein.  molecular  the  mice  less  exposure  in  surviving  four  17  of  to  pool  DEN  +  weight  losses  of  and  Cd,  cytoplasmic  Zn  from  pool,  otherwise  induced  to  decreased  by  the by  alone.  levels  weeks, death  8  weeks'  weeks'  longest  from  17  reflection  exposed  mainly  and  However,  death  resulted  seem  weight  of  weeks  17  in  did  17  a  resulted  time  after  8  death.  f i r s t  animals  Zn  at  is  at  Exposure  actually  Cd  decreased  for  After  DEN Cd  also  the  increased  molecular  pool  the  +  the  of  of  decrease  17).  Zn  sixth  were  of  of  death  in  DEN  at  After  is +  in  to  and  time  and  Zn,  levels  18).  Zn  although  Zn  protein  metallothionein. Zn  +  decreases  weight  Zn  DEN  159.7%  in  reflect  of  to  16  of  levels  Exposure  exposure  levels  Zinc  exposure  molecular  the  time  levels.  Zn  at  maximal  losses  the  27).  the  (Tables  at  ranging  +  44.1%  Zn  Reductions DEN  death;  Zn  increase  was  of  death  but  control  time  of  this  die  the  appeared  increased (Table  decreases  of  Cd  16). at  at  be 17  weeks,  Exposure all  sample  to  and DEN  times.  exposure  decreased +  Zn  re-  Exposure  at  1 85  Figure  27.  The  time  to  death  exposure  of  mice  Cd  supplementation.  or  Zn  after to  commencement  DEN,  with  and  of  without  186  J  o  oo CM  I I  o  co C\J  I I I I  o  C\J  o  CM  C\J  I I  o  o  c\j  l  <  o  00 v-  l  l  o  CD v-  187 to  DEN  +  Cd  exposure  gel  Changes  of  Cd  profiles  of  mice  the  (Table of  were  exposed  of  Cd  DEN  totals.  to  DEN  with  increases  of  apparent  on  metallothionein  most  Cd  was  found a  on  large  readily  or  DEN  Changes  +  on  was  as  equidistributed  Exposure  There  not  to  Cd  Cd at  the  +  in  over  Cd  all  resulted 8  decrease  three  on  tissue were,  pools  accumulation  However  molecular  of  as  levels  in  weeks.  high  Zn  at  weight  Cd-thionein  17 protein  from  8  to  weeks. All  mice  without time +  part,  18).  pool. 17  increases  levels  supernatant  most  most  weeks  in  time.  homogenate for  resulted  Cd  or  and  (Figure types  by  to a  be  appeared  to  +  longest mouse  Zn  readily  exposure visible  survival increase  levels  s t i l l  at  time with  the  mouse alive  by  by  with  the  of  to  three of  time  that  PEN  results  DEN  to  this  DEN +  Cd  exposure cyto-  (Figure  exposed  the  to  total  death  at  and  Survival  exposure  increases  one  DEN,  exposure  for  time  was  to  tumors.  slightly  larger, margin  surviving was  after  increased  much Mean  Cu  died  had  27).  plasmic  this  Zn,  appeared  Zn,  The  which  DEN  28). +  report  Zn;  was  written. Pi scuss ion This Cu  and  Zn  study in  confirms  pretumorous  decreases  are  veloped.  Furthermore,  that  the  time  even  to  more  death  liver  tissue,  prevalent the  present  after  and  after study  exposure  in  to  decreases  shows  tumors  that  have  of these  de-  appears  to  PEN,  directly  is  suggest  188  Figure  28  The  increase  of  Cu  and  of  mice  Cd  or  Zn  Zn  in  time  in  l i v e r ,  exposed  to  to  DEN,  death at  time  with  supplementation.  with of  and  increases death, without  > cu 2 4 0 h  9 1 1  (0 Q  E c (fl  2  230 220 210 200 190 180 170 160  DEN i  1  0.1 0.2 T o t a l C y t o p l a s m i c C u + Z n (pmole/g t i s s u e ) a t t h e T i m e of D e a t h CO  to  1 90 related  to  liver  Cu  +  Zn  Cu  levels.  +  Zn ,  losses  of  and  these  levels  were  actually  increased  However,  this  increase  at  reflection so  that  of  increases  overall,  able  to  tion  increased  death  reduce  of  the  Zn  f i r s t  Cu  creases of  and of  have  of  been In  The  Cu  Zn  is  1970,  1971;  Brown,  more,  when  appeared  and  appear  to  be  of  able and  to  the  time  weeks of  death  die  (Figure  27).  large  was  a  with  High  large  of  in-  mechanism  by  which  in  carcinogenesis  result  Chan been  and  found  given  levels  that  in  DEN,  of  the  levels Cd  and  (Morgan,  bioactivation  for  organic  Further-  changes  present may  be  study a  administered  have  the  the  there  liver  studies,  Cd  Cd  1978).  pretumorous  high  re-  organisms  with  results  extension  (1978).  Knight,  previous  abnormally  .to  day  tumor-bearing  these  time  50  in  That  administra-  a  had  in  partially  increases  These  it  increase  1974)  mean  times.  and  were  Zn  the  in  might  Brown  deactivate  days)  coincided  Brown  1978).  Likewise,  resulted  Cd/L  study.  Daley,  to  levels  1977;  contradict  present  (Jerina  an  Chan,  reflection  to  be  (1 0  possible  by  5 mg  Zn.  exposure  increased  only  to  (Brown  the  Cd  previous•studies  ratio  and  mice  Zn  time.  Cd:Zn  in  +  discussed  of  17  only  all  and  the  time  of  and  was  Cu  two  at  8  adm i n i s t r a t i o n w"i t h DEN  of  Cu  the  at  animals,  six  at  total  survival  ductions  Zn  reduced  administered  six  of  only  slightly  Administration total  in  were  mice  the  losses only  Zn  those  DEN  death.  and  In  been  enzyme  carcinogens  shown system (Yoshida  1 91 et  a l . , 1975),  DEN  wouldn't If  then for  i . e . ,  be  losses  the the  of  Cu  and  reversal  of  these  of  the  study  when  a  there  were  increased  of  Cu  from  lower  (Brown  and  Zn  Cd  and  can  mucosal  be  a l . , 1970). Cd  mucosal by  Cd,  levels  to  that  Cd  Cu  were  mucosal  mucosal  outcompeted binding  in  exposure,  high  than  needed  more  available  Sugawara  and  drinking  water  in  the  Cd,  so  in  the so  levels  Zn  the  for  assumed, mucosal level  binding  Sugawara induced duodenal  is  were  exposure  binding  of  sites (.1 9 7 7 )  Cd for  of  Webb,  the  in  rats.  TOO and  i t  level  may  of  induction by  the  assumes  not  high  that  Cd  enough simply  metallothionein higher  were  uptake  Zn  1 976 ;  by  the  so  weeks'  aren't  study  rather  that  and  these  mucosal  metallothionein mucosa  Cu  were  exposure,  found  of  6  losses  argument  sites  the  changes,  increased  This  that  previous  3 and  so much  preexisting  a  increased  that  but  responsible In  present  increased  5 mg C d / L  carcinogenesis,  after  and  Cd,  bioactivated.  be  that  (..Stonard  metallothionein,  more  for  was  Zn  exclusion  enough  uptake  the  Also  many  by  of  pretumorous  These  as  for  process.  and  1978).  levels be  Cd may  metallothionein.  Cu a n d  sites.  Cu  However,  Zn  by  advanced  of  bloodstream  and  levels  induce  uptake  losses  high  necessary  losses  exposure  of  wouldn't  carcinogenic  metallothionein  Cd-induced that  the  i t  are  metallothionein  Evans  that  as  explained  into  be  presence  Zn  Chan,  absorbed et  the  carcinogenic  slowing  exposure  in  created  that of  Cu  there and  mg C d / L increased  were  Zn..  in Zn  the  192 In  the  creases i . e . ,  of  Cd  just  creased  in  1978).  fish  present in  Perhaps  tumors mice  fish  Cd  is  we  and  appears  contradict  to  Gorodiskii perhaps of  the  other  have  a l .  mode  this  perhaps  Cd  seems have  to  been  of  thionein  were Cd  most  pool  CBrown  molecular  were  (Brown  DEN,  does  not  have  given  weight  of  in  to  in  protein  by  DEN  the  and  Haro,  study  were  In  mice,  Cd  high  1 978c).. to  pool  if  Cd  however, (1 9 5 7 )  is  Cd  that  and  humans  weight  to  in  protein  which  these Third,  and  normal  when on  low  metallo-  given  weight  the  and Second,  than  fish  was  both  cancerous  study,  molecular  increase  In  changes.  Cd  Therefore  actual  in  accumulated  1 978c).; when  in  examined.  unnatural,  another  Knight,  1969).  carcinogens  in  was  molecular  these  was  the  al .  cancerous  caused  in-  liver.  different  high  that  et  DEN,  not  and  in  in-  to  it  This,  Tietz  is  both  the  potential  i . e . ,  in  First,  levels  tumors.  the  Chatel ,  Chatel,  the  Cd  Furst  exposed,  accumulated and  the  Since  this  (Brown  tissues  available.  and  humans  as  nontumorous  suggest  in  developed  tissue  in  action  levels  conditions  levels  levels  of  was  nontumorous  in  (1956;,  Cd  p o s s i b i l i t i e s .  findings  carcinogens.  organisms  DEN  decreased  increased  pool,  Cd  et  in  resulted exposure  However,  developed,  increased  DEN  weeks'  examined  humans,  organisms,  17  and  several have  to  tumors  1977)  tumors and  at  start.  after  suggests study  exposure  only  (Brown,  which  cancerous  study,  tissue  these  This  tissue  in  before  cancerous  the  present  with protein  carcinogen the  available.  high Perhaps,  1 93 if  Cd  had  study  in  mice  high  as  have  been  in  17  100  this  weeks  Cd/L)  then  is  creases  of  protein  pool  Cd  to  et  but Cd  weight  with  cancerous  organisms  haps  with  this  is  due  occurring  in  this  large weeks:, can  to  cause  High  high  pool  (Brown, study  drop  levels  metallothionein  to  accumulated  was  an  80%  of  usually Brown  this  levels  of  Cu  and  since  high  in  Cd  drop the  on  high  the  associated  and  in  aggregate  in-  Cd  Cd  like  al . ,  simply  of  Cd,  et  most  metallothionein of  report  weight  development  Alternatively, in  30  the  are  Levels  are  the  study  tumor  accumulated  1977;  levels  high  Cd  there  pool.  high the  +  weeks  protein  pool.  une*plainable perhaps  perhaps  humans  weeks  DEN  (Weisburger  molecular  17  present  very  17  at  protein  molecular  slowed  at  via  in  DEN  high  was  and  might  Others  resulting  as  least  to  Most  the  at  exposure  Cd-thionein.  in  tumors,  carcinogenesis.  in  but  to  high  fish  present  to  weeks'  weight  the  death  not  tumors  in  Fourth,  the  but  irrevelant  metallothionein,  1978),  in  cancerous  in  molecular  in  present  process  produce  as  the  Cd/L,  formation.  DEN  1 977).  Cd;Zn  and  due  tumor  19 ' w e e k s  al . ,  to  long;  to  5 mg  in  carcinogenic  time  for  15  of  the  death  exposure  and  coincidental, 8  for  levels,  (i^e.,  abnormally  of  and  higher  DEN  The  required  B.arbason  After  up.  required  weeks  formation 1 975 ;  to  mg  were  in  exposed  speeded  were  10  available  study  weeks  only  been  Knight, of  tumors  pool. Zn  also  there  from  Per-  is  a  8  to  17  levels  of  Cu,  (Irons  and  Smith,  1 94 1976). the be  high  are  Cd  only  human not  controls  Knight, Cd  this  Zn  is  not  and  Zn  (Brown  as  in of  reduced that  able  Zn  to  and  Zn  levels  patients  unlikely  probably weight  Cd  levels  are  In  high,  (Brown  that  but  and  high  levels  aggregation  by  spontaneously  and  binding  Zn;  to  and  Chan,  totally  however,  losses  simi-  of  both  of  Zn  with  DEN.  sites  on  DEN,  so  subsequently  reported  al . ,  that  Zn  carcinogen 1972),  tumors 1 976 ;  Cu  (Brown  remove  that  tissue  Cu  1978).  organic  1 975 ,  up  and  with  transplanted  failed  DEN  administration  ties  Chan,  et  with  DEN-induced  given  Ciapparelli  study  study,  bind,  an  previous  of  have  Dreosti,  is  Cd  in  would  exposed*mice.  losses  studies  1964),  Cd  since  be  therefore  molecular  1977) +  and  metallothionein  a  Other  1971;  high  cancer i t  for  previous  were  suggests  DEN  these  clumping  patients,  than  DEN-induced  DEN  with  study,  this  but  to  victims.  administration  to  This  (Brown,  l e v e l s . i n  responsible  In  and  and  Cd  fish  Therefore,  cancer  larly  when  in  pool,  appear  metallothionein,  increased  controls  in  counteract  This  in  of  for  Cd w o u l d  protein  and c a n c e r  1978).  were  1978),  Cu  of  of  functioning.  levels  10%  higher  only  aggregates enzyme  levels  weight  responsible  protein  of  to  affect not  high  molecular  bound  not is  Therefore,  Cd  (Duncan  Woster  occurring  et  prevents  (Pos'willo  (Gunn et  mammary  and  1975),  gland  Cohen,  et a . l . , 1 963.,  a l . , 1974;  a l . ,  cancer  or  cancer  Duncan to  mice  (Bischoff  1 95 and Zn  Long,  1939).  levels  period,  it  study, of  at  if  normal  is Zn  DEN  Zn  of  the +  longest  Zn  of  Zn  the  surviving  Perhaps  future  DEN  that  of  Cu Cd  (Morgan,  and  Zn and  to  to  is  seems  had  by  Zn  the  Zn  with  of  Cu  and  not  mice.  DEN,  at  then  it  can  to  the  study,  didn't  prevent  tumor  with 1977;  (Flick  the  this be  etiology  Cd  prevented  formation.  et  a l . ,  1971)  organisms  Brown  Knight,  and  study,  that  is  1978).  related  DEN-induced  development  DEN  means  cancerous  present  suggest  a  totally  If  relevant  carcinogen  was  Zn.  present  to  Also  to  the  the  highest  alternate  and  is  longest  compare  are  important  far  two  should  Zn  to  the  it  study  prevented,  in  present  present  not  Brown,  exposure  However,  exposed  is  a  the  the  tumors.  tissue  the  Cu  In  maintain  from  study,  +  in  associated  death,  are  DEN  losses  but  1971;  levels, Zn  Zn  be  1970,  time  and  i t s e l f  appears  That  both  tumors.  and  mice  studies  of  Cu  present  to  throughout  determine,  six  induced  induced  However, and  DEN  unable  levels  mouse  carcinogenesis  concluded  losses  the  f i r s t  administration  to  exposed  mouse.  point,  Cu  +  were  DEN-induced  in  exposed  counteract  of  d i f f i c u l t  that  surviving  we  control  prevents  interest,  levels  Since  of  to  Cu  losses  of  tumors.  Summary Mice with  or  sampled  were  exposed  without  1Q00  at  and  0,  8  mg 17  to  DEN  in  their mg  drinking  Zn/L  or  100  Cd/L.  weeks  of  exposure  and  water,  Mice at  the  were time  of  1 96 death. in  Cadmium,  tissue  copper  increasing  Cu  Cu  Zn  respectively,  time  revealed  of  and  that  exposed  mice  Zn  l i v e r s . and  Exposure  and  over  to  death  to  to  control 237  days. to  mean DEN  were  +  Cd  to  All  time  dead +  Zn  resulted and  and  28.0% at  17.8%  and 8  to  death had  to  17  at  the  weeks mo-  for  DEN  tumors  mean  in  of  least  increases  increased  at  high  losses  in  for  44.1%  and  the  reduced  in  resulted  from  mice  time  determined  exposure  21.8%  mainly  Mean  DEN  were  profiles  survival  values,  days.  weeks,  pool.  Exposure  extended  days. Zn  183  8  DEN from  elution  losses  protein  was  at  Gel  these  weight  and  Zn,  death.  lecular  their  levels  supernatant.  of  the  zinc  homogenate  losses and  and  Cu  198 of  Cu time  1 97 CHARTER  XI  The  Effect  of  Cadmium  Exposure,  Without  Diethylnitrosamine,  plasmic  Levels  Copper  and  on  With  or  the  and D i s t r i b u t i o n  Cyto-  of  Cadmium,  Zinc  Preface This  study  Chatel  (1978c),  tion.  The  whether pool it  or  was a u t h o r e d and w i l l  study not  Cd w h e n  but  DEN  protein  Cd w a s n o t  demonstrates but  high  was  demonstrated  the  drinking  water).  thionein  became  of  the  current  have  protein (Brown  the  Cd,  study  found  and K n i g h t ,  sults  from  this  over"  theory  weight  in  the  protein  occurs  on  however,  l o w Cd e x p o s u r e s  At  higher  DEN,  Cd e x p o s u r e s ,  study  metallomore  this  (<^100  in effect  mg C d / L  in  metallo-  was a  protein  mo-  This  accumulates  pool;  weight  study,  with  alone  and there  protein  high  control.  DEN  publica-  investigate  as a  with  W.  a previous  at  "spillover"  pool  with  con-  effects. demonstrates  potential  as  In  for  administered  alone  high•molecular  the  Cd.  when  Cd s a t u r a t e d  pathological  Thus, does  weight  only  to  molecular  Cd a d m i n i s t e r e d  molecular  Cd t o  pool  a n d K.  shortly  order  high  Cd a d m i n i s t e r e d  the  Brown  Cd a c c u m u l a t e d  administered  that  A.  in  with  that  D:  submitted  increased  administered  weight  thionein,  be  was c o n d u c t e d  was d e m o n s t r a t e d  lecular  by  in  to  study  suggested  increase  fish  1978), also by  that  i f  high  (Brown, Cd  is  strongly Brown  et  an  organic molecular  1977) readily support al  carcinogen  and  weight  humans  available. the  (1977),  " s p i l l -  and  Brown  Re-  1 98 and  Parsons  (1978).  Introduction Recently  i t  h a s become  organisms,  occurs  tein  in apparently  pool,  (Brown,  1977;  organisms, (Brown lar  bind  of  less many  (Brown than  has been  do n o t o c c u r  quantities (Winge and  in  et  sible  that  Brown  (1977)  from  Zn  until  1978).  Cd i s  involved  might in  polymerase, thymidine  et  in  RNA kinase  weight  in  polymerase, (Vallee,  in  1978;  with  cell  reverse 1976;  zinc components 1974).  of  the high  large pool  i t  molecular is  pos-  process.  Cd d i s p l a c e s Cd  t h e Zn  division;  in  Zn  cancerous  metal 1oenzymes e . g . , DNA  transcriptase,  Duncan  Cd o r  Cloutier  the carcinogenic  interferring of  and  protein  since  thus  available  Bremner,  organisms,  that  low molecu-  Cu a n d Z n ,  appear  in metal 1oenzymes,  the regulation  is a  effects  and P a r s o n s ,  cancerous  tissue  are natural  toxic  pro-  metallothionein  longer  1974;  Cd o c c u r s  weight  Copper  elements  molecular  important  be  Hg,  a r e no  that  cancerous  noncancerous  to  Cd,  (Friedbert,  Brown  sites  bound  Cd a s t h e y  these  of  In  a l . , 1977).  has hypothesized  binding  organisms  as t h e y  in  and kidney  1978).  can bind  Since  pool  liver  Cd,  Metallothionein  established  the high  protein  molecular  mainly  Hg o r  a l . , 1973;  Brown,  weight  which  metal 1oenzymes  It Hg  toxic  the high  and K n i g h t ,  nontoxic  enzymes  that  normal  1978).  protein them  in  occurs  and Knight,  weight  are  Brown  cadmium  rendering to  mainly  apparent  and D r e o s t i ,  and 1976).  1 99 The  present  carcinogen  distribution  increase  weight  bound  mg  Cd/L  to were  Chan,  stered have  to  pool  in  to of  Cd  old,  were  separated  were  administered  25  mg  Jars  are  the  of  particular high  decrease  previous  DEN,  most  that  the  In  does  the  this  Cd  was Cd  the  indeed  levels when  pool  5  not  (Brown admini-  would  and  of  Cd  otherwise  present  with  DEN  molecular  protein  study  the  that  study,  of  weight  that  DEN of  into  study,  without  influence  DEN,  the  Cd.  500  two  strain ,•approximately  cages, tap mg  water,  Cd  contained of  C02«  were  3 mice  (as  solutions DEN  the  sacrificed  with  Mice the  in  Swiss  Drinking  were  analyzed.  a  organic  influence  the  administered,  either or  which  asphyxiation  In  with  not  photodegradation Mice  each  of  200  DEN/L.  days. vent  100 ,  to  an  Methods  mice  50,  in  bound  demonstrate  or  Male  25,  can  DEN)  molecular  distribution  and  or  metallothionein.  whether  cytoplasmic  that  cadmium; Cd  However, to  confirm  concurrently  high  DEN  levels  Materials  of  and  the  bound  examine  of  administered  without  various  levels  1978).  been  to  metallothionein.  accumulated and  the  protein  Cd  aims  (diethylnitrosamine  cytoplasmic can  study  tap  water  CdCl2)/L, were  were  28  with  changed painted  days'  animals  analyzed  exposure  cage.  months  Mice containing or  on  without  alternate  black  to  pre-  DEN.  after  The  or  per  two  types  six  at  with  were a the  exposure, frozen  time, same  until  three Cd  via  from  concentra-  tion.  This  between were  was done  samples  removed,  was  in  laboratory and  for  ml  of  debris  then  minutes Two  tants  at  ml) o f  were  Pharmacia  each  centrifuged  column  15  fractions. fraction  establish  protein  pool,  cytoplasmic 1978).  the  a  Cadmium,  a  in  2 . 5 ml  for  10  minutes  the  heat  x  100  with  0.01  Perkin  position  of  (Brown  et  Cu a n d Zn  was a p p l i e d with  Elmer the  124D  high  and t h e  a l . , 1977; levels  at  were  for  collected.  this  was r e a d  a  centrifuged  homogenate  M NH4HC03  in  cellular  stable  packed  27,000  supernatants.  clear  were  pellets  at  heated  supernatants  of  cm)  to  were  of 0.9%  then  5 minutes,  4.5)  Sorvall  were  metallothionein,  pool  g_ i n  supernatants  and the  3  Homogenates  c o l l e c t e d and  These  liver  (setting  previous  Absorbance  using  exactly  with  a n d 5 ml  was e l u t e d  of  combined  of  (1.6  less  speed  x  were  2 minutes  x £  combined,  This  each  27,000  Livers  STIR-R  standard  precipitation.  gel. ml  at  exactly  27,000  analysis  a .TRI-R  homogenizer.  exactly  for  v i a heat  a  or for  using  Supernatants  were  bath  2 grams  0 . 9 % NaCl  to  of  Cd c o n c e n t r a t i o n .  tube,  minutes  6 combined  water  of  set  and the supernatants  70°C  to  same  a teflon  10  for  These  Groups  10  S63C  with  consistency  and then  4.5  centrifuge.  NaCl.  the  homogenizing  rehomogenized  £  in  motor  equipped  RC2-B  ensure  weighed,  a  centrifuged  x  with  homogenized  minutes,  to  supernato  Sephadex  a G-75  and c o l l e c t e d  as  2 5 0 a n d 2 8 0 nm o n spectrophotometer  molecular  weight  low molecular Brown  and  determined  weight  Chan, on  each  201 fraction atomic arc  using  the  flame  absorption  background  cytoplasmic metal  correction. were  in  on  a  spectrophotometer  pool  levels  method  Total  fraction  by  of  Elmer  equipped  metal  determined  each  Perkin  with  levels  summing  each  303 deuterium  in  of  each  the  individual  pool.  Results-'. Typical mg  Cd/L  in  Figures  bound  to  elution  exposure,  to  With  gel  29  with  or  30.  Two  and  metallothionein  Cd  administration  metallothionein  There  appeared  from  100  to  (Figure  large Cd/L  mice a  exposure,  Figure  32). increase  of  pool  at  Cd/L  alone,  a  increase 200 high  and  or  mg  Cd  1.4-fold mg  500  mg  Cd/L  molecular  shown  (Tables  or  Cd  31  DEN  the  and  with  at  resulted  Cd  from  Table  pool  without  a  500  mg  a  2.8-  protein  exposure  (calculated  was  was  22,  in  weight  and  Cd  the  and  Cd/L  more  molecu-  There  mg  protein  50  DEN  high  19  with  22).  plateau  32).  molecular  compared at  the  bound  production  this  (Tables Cd,  was  without  production  DEN  high  in  22).  (Table  Cd-thionein  of  (Figures  Cd  25  was  and  of  exposure  with  Cd  19  75%  increase  exposures,  weight  are  alone,  to  Cd/L  exposure,  Cd  increase  100  the  to  exposure  at  of  without  in  and  cytoplasmic  Cd/L  Cd-thionein  Exposure  fold  25  exposed  of  with  mg  controls  than  exposure,  pool  acceleration  more  plateau  large  protein  of  controls  100  Cd/L  DEN  thirds  alone,  a  from  without  in  to  be  mg  in  with  weight  up  to  200  32);  coincided lar  profiles  a  1.3-fold 19).  bound  to  At the  concurrent  the  202 Table  19.  The  cytoplasmic  posure  to  a  or  without  is  a  in  jumole/g  distribution  range  Cd  of  tissue  ND:  values  (wet  not  Cd  with  ex-  concentrations,  diethylnitrosamine  compilation  weight.  of  of  (DEN).  from  weight).  gel  with Data  profiles,  MW:  molecular  detectable.  Cd  Control 25  mg  25  mg  50  mg  50  mg  Cd/L Cd/L  DEN  +  Cd/L Cd/L  100  mg  Cd/L  100  mg  Cd/L  200  mg  Cd/L  200  mg  Cd/L  500  mg  Cd/L  500  mg  Cd/L  + DEN  +  +  +  DEN  DEN  DEN  Metal l o t h i one i n  L o w MW pool  .0004  . 0024  .0008  .0194  .0047  .0147  ND  .01  .0123  .0051  ND  .0765  . 0073  .0655  .0037  .0615  . 0103  . 0483  .0029  . 0880  .01 45  .0714  . 0021  .1188  .01  87  . 0985  .0016  . 1 593  .0822  . 0678  . 0093  .1408  .0303  . 1 081  . 0024  .  4125  . 0849  . 3276  ND  . 561 5  . 0464  . 51 51  ND  Total  High poo 1  . 0036  74  MW  203 Table  20.  The  cytoplasmic  exposure  to  a  range  with  or  without  Data  is  a  of  in  molecular  Cd  of  Cu  with  concentrations,  diethylnitrosamine  compilation  profiles, MW:  distribution  >imole/g weight.  of  values  tissue ND:  from  (wet not  (DEN). gel  weight). detectable  Cu  Control  25  mg  25  mg  50  mg  50  mg  Cd/L Cd/L  +  DEN  Cd/L Cd/L  100  mg  Cd/L  100  mg  Cd/L  200  mg  200  mg  500  mg  500  mg  +  DEN  . +  DEN  Cd/L Cd/L  +  DEN  Cd/L Cd/L  +  DEN  Metallothionein  L o w MW pool  .030  .018  . 024  .083  .044  . 028  .011  .053  .026  .014  .013  .092  . 029  .030  . 033  .089  . 022  . 032  . 035  .117  .032  . 039  .046  .096  .026  . 035  . 035  .101  . 036  . 027  . 038  . 084  .029  . 029  . 026  .165  . 037  . 040  .088  .161  . 044  . 049  . 068  Total  High pool  .072  MW  204 Table  21.  The  cytoplasmic  exposure  to  or  without  Data  is  a  MW:  2 5 mg  50  mg  50  mg  Cd/L Cd/L  DEN  +  Cd/L Cd/L  DEN  +  of  in  Cd  of  Zn  with  concentrations,  diethylnitrosamine  compilation  molecular  Con t r o l  mg  range  with  profiles,  25  a  distribution  jumole/g  of  values  tissue  weight.  ND:  from  (wet not  (DEN). gel  weight). detectable.  Metal l o th i o ne i n  L o w MW pool  . 080  .001  . 002  .120  . 1 01  .013  . 006  .093  .089  .002  .002  .126  . 098  . 020  .008  .085  . 073  . 009  .003  .102  .079  .021  . 002  .114  . 086  .025  . 002  . 1 37  . 126  . 007  . 004  .121  . 083  .034  .004  Total  High pool  . 083  MW  100  mg  Cd/L  100  mg  Cd/L  200  mg  Cd/L  200  mg  Cd/L +  500  mg  Cd/L  .175  .115  .047  .013  500  mg  Cd/L  . 1 98  .093  . 098  . 007  +  DEN  DEN  205 Table  22.  Percentage  distribution  cytoplasmic Cd  pool  with  concentrations,  diethylnitrosamine calculated weight.  ND:  from not  of  total  exposure  with  or  19.  to  a  MW:  molecular  detectable.  Metal l o th i o ne i n  L o w MW pool  Control  11.1  66. 7  22. 2  25  mg  25  mg  50  mg  50  mg  100  mg  100  mg  200  mg  200  mg  500  mg  500  mg  Cd/L  24. 2  75. 8  0,  Cd/L  +  DEN  70. 7  29.3  0  Cd/L  9.6  85. 6  4. 8  Cd/L  +  DEN  16.8  78.5  4. 7  Cd/L  1.6.5  81  .1  2. 4  Cd/L  +  DEN  15.8  82.9  : i . 3  Cd/L  51 . 6  42.6  5. 8  Cd/L  +  DEN  21 . 5  76.8  V. 7  Cd/L  20.6  79.4  0  Cd/L  +  DEN  91 . 7  0  8.3  MW  each  range  Percentage  Cd High pool  on  without  (DEN).  Table  Cd  of  Table  23.  Percentage each  distribution  cytoplasmic  range  of  Cd  pool  of  with  concentrations,  without  diethylnitrosamine  centage  calculated  MW:  molecular  from  weight.  total  Cu  on  exposure  to  with  ND:  or  (DEN).  Table not  Per-  20. detectable.  Cu Metallothionein  L o w MW pool  41 . 7  25.0  33. 3  53.0  33.7  1 3. 3  49. 1  26.4  24. 5  31 . 5  32. 6  35. 9  24. 7  36. 0  39. 3  33. 3  39. 3  34. 5  34. 5  31  35. 7  26.7  37.6  34. 5  34. 5  31 . 0  22.4  24.3  53.3  27. 3  30.4  42.3  High poo 1  Control  25>mg 25  mg  50  mg  50  mg  Cd/L Cd/L  Cd/L  mg  100  mg  2 00  mg  200  mg  500  DEN  Cd/L  100  500  +  mg mg  +  DEN  27.4  Cd/L Cd/L  +  DEN  Cd/L Cd/L  +  DEN  Cd/L Cd/L  +  DEN  MW  ;  a  .0  207 Table  24.  Percentage cytoplasmic Cd  distribution pool  with  concentrations,  nitrosamine from ND:  Table not  21.  total  exposure  with  (DEN).  of  or  to  without  Percentage  MW:  Zn  molecular  on  a  range  calculated weight.  Zn  Control  25  mg  Cd/L  25  mg  Cd/L  50  mg  Cd/L  50  mg  Cd/L  +  DEN  +  DEN  MW  Metallothionein  L o w MW pool  96. 4  1 . 2  2.4  84. 2  10.8  5.0  95.7  2.2  2.1  77.8  15.9  6.3  85. 9  10.6  3.5  77.5  20.6  1 . 9  75.4  22.8  1.8  1 00  mg  Cd/L  100  mg  Cd/L  200  mg  Cd/L  92.0  5.1  2.9  200  mg  Cd/L  68.6  28. 1  3.3  500  mg  Cd/L  65.7  26.9  7.4  500  mg  Cd/L  47.0  49. 5  3.5  +  +  DEN  DEN  of  diethyl-  detectable.  High pool  each  208  Figure  29.  Composite control  gel mice.  elution  profiles  from  three  Control ~  0.201  u CO  F r a c t i o n Number  210  Figure  30.  Composite mice  gel  exposed  water,  and  Cd/L  25  +  elution to  from mg  25  mg  three  DEN/L.  profiles  from  Cd/L  in  their  mice  exposed  three drinking to  25  mg  25ppm Celt DEN  2 5 p p m Cd 0.20 H  -020H  cn  E 0.10-  F r a c t i o n Number  F r a c t i o n Number  212  Figure  31.  The  variation  molecular exposed  to  of  weight Cd,  Cd  levels  protein  with  or  Each  point  represents  from  a  elution  gel  composite  sample  pool  the  a  value  three  high  from  without  profile  from  in  done  mice  DEN. calculated on  mice.  a  3  o.io-  1  —I  100  200 Cd  1  300  Exposure  1  400  1  500  Level ro co  t—»  214  Figure  32.  The  variation  thionein without value f i l e mice.  of  from DEN.  levels  in  metallo-  mice  exposed  to  Cd,  Each  point  calculated done  on  a  Cd  from  a  composite  with  represents gel  elution  sample  from  or  a prothree  M e t a l l o t h i o n e i n Cd (jjmole/g tissue)  913  exposure levels with Cd  to  DEN  were  (Table  higher  concurrent  at  levels  tein  pool  with  exposure  were  200  exposure  to  of  the  to  (Table  exposure,  there  molecular  weight  Cu  Cd 20,  31).  to  500  DEN,  Also, mg  Cd-thionein  Cd/L  compared  a  creasing  Cd  exposure  34).  of  exposures  with  the  point  exposure  200  mg  Cd/L  with  molecular general, three  Cu  tended  tended  to  exposure;  this  at  mg  the  25  The pool  were mg  to  25  increase  a  be  of  Zn by  compared  tended  dip  20, in  (Table  in  20,  of  to  23,  high  Cd/L Cd/L high  control with  in-  Figures of  in32  and  Cu-thionein 34);  this  of  metallothionein  to  the  31  and  Total  was  compared  the  Figure  increasing  pro-  parallel  equally  23).  (Table  exposure with  to  Cd  (Figures  however,  the  with  saturation  with  mg in  levels  distributed  exposure  Cu  Cd mg  increased  19,  (Table  increase,  of  compared  and  pool  increase  decreased  Cd/L,  the  "spillover"  pools  Cd/L  levels  At  apparent  protein  cytoplasmic  levels  33).  (Tables  concurrent  weight  500  exposure  with  +  the  pool,  be  DEN  at  Cu-thionein  to  to  weight  except  level,  appeared  coincided  Cd,  of  molecular  exposure  large  Cd-thionein  There  by  protein  levels  high  Figure  was  The  creases  in  alone,  values.  200  the  decreased  exposures  by  Figure  alone. The  at  19,  high  32).  In  among  the  cytoplasmic level  of  Cd  counteracted  by  Cu  DEN  20). molecular  to  exposure  DEN to  + Cd  Cd  weight  protein  at  50  alone  25,  (Table  and 21,  217  Figure  33.  The  variation  lecular to  Cd,  weight with  represents elution from  of  or a  levels  in  protein  pool  without  DEN.  value  profile  three  Cu  mice.  the  high  from, mice Each  on  a  exposed  point  c a l c u l a t e d from  done  mo-  composite  a  gel sample  Cu  o 0.40  U)  3" 0.35 to  +-»  aJ D U  °> 0.30 o E EL 0.2 5  • — • Cd 0 - - 0 Cd + DEN  o 0.20 cn I  100  200 300 400 Cd E x p o s u r e L e v e l  500 i—1 oo  219  Figure  34.  The  variation  from  mice  Each  point  a  gel  sample  of  exposed  levels  to  represents  elution from  Cu  profile  three  Cd, a  with value  done  mice.  in  on  metallothionein or  without  calculated a  composite  DEN. from  0.01 L (TJ  100  200 300 400 C d E x p o s u r e Level  500  Figure.35. 25  and  50  protein  mg  Zn  (Table with  This Cd/L.  Figure  increasing  and  of  36).  34  36).  had  the  high  molecular  with  (Table  creasing  most  Total  current  to  Cd,  at  100  at  mg  Cd/L  The  Cd  protein  remainder  level  mg  Cu  DEN,  except  Decreases with  exposure of  saturation  of  19,  weight  Figures  (Table  quarters  21)  of  protein  increased  with  increased  larger 500  31,  32  Cd/L  weight  in-  DEN to  by con-  ex-  were  ap-  exposure,  and  Cd  appeared  high  without  mg  metallothionein the  mice  metallothionein  were  liver  exposure  to  (Figures  of  weight  to  the  concurrent  with  liver  was  at of  in  21).  levels  increase  Cd/L  (Table  Zn  32  metallothionein  levels  (Table +  metallothionein  pool  Zn  dip  Zn  three  bound  this  37).  decrease  apparent from  to  to  molecular  cytoplasmic  but  exposure  parent  with  the  a  set  protein  bound  high  was  the  i n -  Figures  exposure  l e v e l ,  Zn  increased  24,  there  weight  level  parallel  and  Cd/L  at  weight  exposure  to  approximately  the  cytoplasmic  (Figure  38).  in  exposure  posure  200  of  Total  Cd  of  general, was  21  mg  exposure  percentage  Zn  24).  exposure  200  lowest  cytoplasmic pool,  the  Cd  In  Cd  tended  19,  each  24).  molecular  Cu-thionein, at  prevalent  Zn-thionein  and  In  lowest  (Table  of  (Tables  to  high  increasing  Levels  Zn-thionein  the  of  exposure,  Similarly  of  with  with  35).  Cd  particularly  level  Cd-thionein  levels and  was  The  increased  21,  creases  of  effect  alone to  (Figure  coincide  and  "spillover"  molecular  weight  and  Liver  38).  222  Figure  35.  The  variation  molecular exposed point gel  Zn  levels  weight.protein  to  Cd,  with  represents  elution  sample  of  from  a  done  mice.  pool  without  value  profile three  or  in  the  high  from DEN.  calculated on  a  mice Each from  composite  a  P o  H i g h M o l e c u l a r Weight Zn (jjmol/g tissue)  oo  o ^  o  o  rv>  O  o  o  Q.  rn x  TD  o c  2 Q  <  ro  o o  O  o  0 1  o o o o  zzz  I  o • o o a. +  (Jl  •  a m  GL  o _  ^  i  a  o  ^  —^  O)  oo  224  Figure  36.  The  variation  thionein without  from DEN.  calculated on  a  of  Zn  levels  in  metallo-  m i c e . e x p o seel  to  Cd ,  ; Ea'ch, p o i n t from  composite  a  gel  sample  with  represents  elution from  a  profile  three  mice.  or value done  0.1 O r  c N  C — U  -  C9 D CO  0O8  § $ 0.06 £ cn = "5 0.04 trJ f: •+-» t= 2  0.02  100  i  1  200  300  Cd E x p o s u r e  Leve  r 400  500 ro ro  226  Figure  37.  The  variation  of  total  supernatant  Cu  +  to  or  without  Cd,  with  represents elution from  a  value  profile  three  Zn  mice.  tissue  levels DEN.  in  homogenate mice Each  exposed point  c a l c u l a t e d from  done  on  a  composite  a  gel sample  Total Cu+Zn(|jmolc2/g tissue)  LZZ  228  Figure  38.  The to  variation various  Each mice.  f>'' M  L  point  of  levels  liver of  represents  weight  Cd, the  with mean  with or of  exposure  without three  DEN.  230 weights  were  exposure  to  less Cd  with  exposure  to  DEN  +  Cd  compared  with  at  Cd  exposures  alone.  Discussion This to  or  less  of  Cd  in  Cd  of  DEN  the  and  100  mg  increases  high  Chan  high  of  with  level  in  the  the  current DEN  became this  high  DEN +  Cd,  Cd  pool/g  tissue.  only  of  5 mg  of  (Brown,  as,  or  similar  mg  molecular  high  saturated  as  molecular  With  at  Cd  for  study  of  DEN,  protein  by  is  high  and of  Brown  since  it  the  the  possible molecular  humans  (Brown  carcinogens  there  pool  weight  about  the  that  weight  con-  with  exposure pool  jjmole  alone,  lower  less  protein  0.04  0.08  was  with  though,  about  effects  DEN.  exposure  until  explanation  to  protein  (plateaued)  tissue.  in  level  the  Also,  effects  appeared  con-  probable  exposures,  weight  It  the  Cd/L  of  a  1977)  same  500  exposure  is  in  Cd  the  An  it  Cd/L.  levels  fish  the  effects  to  and  become  the  and  metallothionein.  molecular  due  saturated  to  Thus,  equal  proportion  pool,  masking  was  that  appear  as  to  high  to  high  exposure.  pool/g  the  due  was  pool  200  Cd.  to  metallothionein,  of  the  due  the  protein  bound  magnitude  of  in  was  1978)  actions At  Cd in  occurrences  Knight,  weight  levels  in  levels  than  protein  DEN  presumably  (1978),  exposure  and  the  decreased  via  Cd/L,  molecular  decreases  increased,  weight  in  than  rather  that  to  that  occurrence  pool  Cd  demonstrated  effect  of  Cd  has  the  currently This  study  it  jjmole  Cd did  Cd  apparent  in  not this  231 saturation pool  levels  with  appears  to  ( i . e . ,  be  known  These  the  high  metallothionein (Webb,  1972b),  centrated might to  in  to  Results over"  theory  Parsons and  the  mg  appeared  posure  with  there  molecular and  lesser  Cd;  reasons  previous  as  by and  the.  et  Chatel  was  very  DEN large  protein  increases these  in  lesser At  that  mice  the  a l .  (1977),  Brown  200 It  of  of  mice  pool  in  and in mg  was Cd  on  exposed mice  were where  This exposed  confirm  100  point  con-  study.  (1 9 7 8 a , b ) ,  levels  enzymes  to  increases  this  since  of  present  exposure.  pool  in  destroyed.  to  from  sites  more  levels  appear  Brown  and  become  Cd-thionein  study  levels,  Also,  tissue  a l . , would  proteolytic  could  in  et  enzymes  binding  pool.  exposure  exposure,  (Chvapil  enzyme  by  liver  DEN  DEN  plateau  were  paragraph.  with  there  proteolytic  levels,  without  for  more  Cadmiurn-thionein  or  weight  DEN  proteolytic  degraded  this  to  with  that  protein  Cd/L-and  (1978).  study,  that  not  Brown  liver  metal 1oenzyme  discussed  (1 9 7 8 ) ,  Brown  of  higher  from  that  carcinogens of  protein  fact  cytoplasmic  liver  the  500  of  weight  the  lysosomes  weight  is  the  metallothionein  explain  100  from  reduce  molecular  of  levels  number  from  suggests  effect  certainly  therefore  This  higher  molecular  derives  necrosis  released  1972).  the  more  are a  high  exposure,  38).  is  almost  the  Figure  enzymes which  DEN  of  " s p i l l and  Cloutier the  present  Cd/L at  ex-  this  the to  point  high Cd  exposed discussed  alone,  to in  DEN the  metallothionein  +  appeared over a  to  to  become  the  high  simultaneous  reduced levels pool  at  and  appeared  lower  Cd  result  plasmic of  Zn  Cd  than  in in  from  account in  Zn  in  this  the  sudden  after  molecular  of  higher  Cd  to  protein  Cd  lower  found  that protein  in  of  induced  protein this  alone, Cd  proportions  weight of  i . e . ,  weight  weight  DEN  was  increased  at  have  mechanisms.  metallothionein coded  is  turned  of  Cd),  Cd  levels,  for on  but  to  induced of  is  Cd-thionein  cyto-  losses  pool  probably  pool,  rather  synthesis (1972a)  induced  at  metallothionein synthesize and  n e w mRNA  the  but  induced  has  the  Cousins  DMA  (1974),  coded  explaintwo  translational  exists  that  has  be by  occurring  dif-:  demonstrated  in  the  level  for  level  ( i . e . ,  cytoplasm  in  using  that  the  much  metallothionein  transcriptional  from  may  metallothionein  demonstrated at  is  production production  surprising,  Webb  is  Squib have  of  metallothionein  Cd-thionein  since  duction  mice,  occurred  Therefore,  increase  able  be  high  there  effects;  exposure  (1978a)  accumulation  plateaued,  also  pool,  molecular  with  also  molecular  apparently  mRNA  high  Chatel  pool.  high  exposed  than  the  protein  spilled  metallothionein.  5 0 0 mg C d / L ,  ferent  the  accumulation  the  for  The at  of  and  apparently  pathological  DEN  reductions  Brown  deficiencies  of  In in  a n d Cd  weight  exposures  weight  exposures.  pool  molecular  weights.  Cd  liver  saturated,  occurrence  liver of  Cd  and  presence higher can  ( i . e . ,  pro-  metallothionein).  233 As  Cd-thionein  but  synthesis  accelerates  that  synthesis  exposure this  is  again of  excess  in  scriptional  be  there  level.  with  is  DEN  creases  for  noted  in  (Brown  C u a n d Zn  is  in  found  accordance that  binding to  Cd  been  found  Zn  sites  (Suzuki found  by  by  of  to  Cd,  levels  at  the  which  an  peak  has  DEN  might  nucleus,  tran-. in  occurred.  exposures  that  A  then  second  higher  of  Cd  greatly be  i n -  very  similar  increase  apparent  plateau  spillover  had o c c u r r e d ,  Cu a n d Zn  in  in  the  increased,  previous a  studies portion  metallothionein  and Y o s h i k a w a , (1974)  so  metallothionein  a n d Cu o c c u p y  Leber  weight  the  of was  laboratory  1978b).  with  when  then  the  spillover  cancer.  and  l i m i t ,  into  the  after  When  saturated,  may be Cd,  production,  exposed  Cd-thionein  of  i t  penetration  and C h a t e l ,  As  with  Cd/L  molecular  penetrate  after  Cd a l o n e ,  production  mussels  i t s  exposure,  may be  level.  high  may e x p l a i n  i t  2 0 0 mg  metallothionein  development  metallothionein  to  was Cd  to  Cd-thionein  metallothionein  exposures, up  the  pool  of  2 0 0 mg C d / L  reached  to  available  This  with  nuclear  important  this  to  translational  production,  more  than  the  production  induction  metallothionein  in  at  After  Cd w o u l d  resulting  higher  spillover  pool.  up  metallothionein  induced  Cd w o u l d  protein  As  with  metallothionein  excess  plateaus  1972;  is  of  levels  fractions.  which  This  have  metallothionein  produced  Leber,  and Brown  did the  in  1974).  and Parsons  response It  has  (1978)  234 that  exposure  in  displacement  a  Thus, the  to  high  tolerance  presence  of  to  of  probable  decreased  on  of  cytoplasm.  Cu  Thus,  to  hypothesis  exposure,  so  that In  in Cu  liver.  Zn  interferes (Friedberg,  Zn  high  of  very  high  bind  in  into  the  with  Cu  1 974 ; of  this  a  and  Zn  Cu  and in  defense  to  1978) is  binding  Bremner,  the  Cd  than  Cu  high  is  and  Zn  that  in  mg  production, levels. 1978),  levels of  in  mucosal  transport Since  of Cd  metal 1oenzymes  l i k e l y  Zn  with  increasing  the  high  molecular toxic  500  (Brown,  induction  sites  Zn  according at  Cd  increased.  the  and  metallothionein  study Cu  the  enough  are  of  dis-  in  Presumably,  by  is  level  of  Cd,  by  were  through  more  so  1974),.it  against  Zn  metallothionein  increased  bloodstream  It  discussion,  previous  (Brown,  Zn.  and  induction  attributed  Cd  particularly  metal 1oenzymes.  in  metallothionein.  exposure  is  result  mediated  and  Cu  excess  of  is  levels  displaced  levels  levels,  a  level  Hg  Cd/L),  there  then  from  Cu  that mg  not  been by  or  both  (200  to  as  Cd  has  Cd  can  Zn  bound  at  where  were  study,  as  by  high  uptake  acts  of  Hg  metallothionein.  creased  pool ,  and  presented  and  This  and  Cu  transcriptional  metallothionein Cu  in  very  to  Zn  only  a  this  exposure  toxic  levels  induced  increased  resulted  that  and  levels  Cd/L  or  doses  occurred  metallothionein  the  acute  Cd  metallothionein  spillover  placement  less  of  metallothionein  therefore  where  levels  Cd  that  in-  exposure  weight actions  protein of  Cd  on  235 As in  in  previous  reduced  cularly and  levels  in  Chan,  protein shift weight  Zn  Zn  lecular  Brown  Cu  and  potential  been This  of  Cu by  has  study  and  shown, Zn  lower is  as  a  may i t  the  urine most  of  Brown  clearly  the  high  DEN  levels,  thus  allowing  exposure  which  so  that  levels.  aspects  of  the  (Brown  Zn  Chan  Cd  high to  What  is  actions  not of  metallo-  for  Zn.  bloodstream,  as  1978). being  possible  mechanistic  carcinogenesis  that  toxic of  Weight  molecular  effects clear,  DEN  are  effects  Cd  from  protein  spillover  pathological  accordance  complexing  "spillover"  molecular  reduces1  mo-  in  the  in  a  (1978).  demonstrated with  to  carcinogens  Cu*and  and  high  and Chan,  The  be  metallothionein  vehicle  through  to  that  due  weight  molecular  is  reported  chelators.  that  levels  high  This  been  (Brown  appeared  the  storage  have  simultaneously to  who  passes  described  has  to  increased.  is  exposure  there  parti-  molecular  of  as  by  high  percentage  reductions  metallothionein Also  When  and  pool  and t h e  element  of  protein  of  Zn  resulted  tissue  percentage  act  into  DEN,  the  a n d Zn  discussed  Cd a p p e a r  weight  (1978a)  to  DEN  trace  significance has  i . e . ,  to  liver  decreased,  protein  subsequently (1963)  in  metallothionein  appeared  a n d Zn  Furst  were  decreased  of  a n d Zn  1978).  and C h a t e l  Reductions  exposure  molecular  pool;  weight  thionein  of  from  was  Cu  Brown,  levels  protein  bound  of  high  1978;  Zn  of  with  the  studies,  weight at  Cu  lower  occur  at  pool .  this  important  at point, in  236 carcinogenesis.  This  study,  and  have  demonstrated  Brown  losses  of  strated and  (1978)  C u a n d Zn  that  Chan,  Cd  for  was n o t  but  and Chan,  1 978v  is  important  to  organisms Cd,  and  have  DEN  potential if  high  fore, the  to  been  shown  levels  does  relevance  of  of  seem  high  high  in  i t  this  Cd  are  to  be  cause  the  appears  study,  to  in  Brown  studies, DEN  that  Cd  cancerous  to  protein  have  the  protein  Cd  available.  continue of  demon-  appear  weight  weight  readily  etiology  have  by  However,  molecular  in  previous  molecular  of  Cd t o  Others  was d e c r e a s e d  1 978)  in  results  C u a n d Zn  Since,  rather  of  DEN  [1978),  (discussed  carcinogenesis.  increase  enough  there  losses  Brown,  increases  has  that  cancer  carcinogenesis.  increased,  a n d Chan  tissue.  prevent  Therefore  (Brown not  pretumor  C u a n d Zn  1978).  important  in  and Brown  to  Therestudy  cancer.  Summary Mice Cd/L, for  in  or  days.  DEN  the  100 At  with 28  that  were  + Cd  high  mg C d / L  exposed without  to DEN  G-75  resulted  in  molecular  was d i s c u s s e d  with  Cd e x p o s u r e  as  an  in  protein  more  with  2 0 0 a n d 5 0 0 mg  their  DEN  of  mice  due t o is  drinking  profiles  pool  at  25,  exposure  to in  exposed higher  known  to  of  Cd  50 a n d Cd  the to  water  revealed  accumulation  Cd a c c u m u l a t e d  pool  as  100,  increased  possibly  alone,  50,  elution  compared  protein  This  gel  weight  exposures,  weight  25,  exposure,  Sephadex  2 0 0 a n d 5 0 0 mg C d / L ,  molecular  0,  alone. high  Cd  alone.  enzyme  levels  damage  lysosomes,  thus  releasing  proteolytic  thionein  production  exposure  to  DEN  +  Metallothionein 200  mg  Cd/L  200  mg  Cd/L,  molecular of  Cd  there  weight  weight  weights.  Liver  +  Cd  thionein  sibly  and  a  and  or  to  500  pool.  pool  more  after  an  apparent  due  to  a  from  induction  of  metallothionein  alone,  then  with  from  of  Cd  of  Cd  the  in  mice  of  in  increased  to  high  high liver  exposed  of  Cd-  Cd-thionein  discussed  very  high  reduced  plateau  at  At  saturation  into  with  translational  to  the  Increases  are  100  in  apparent  alone.  these  exposures.  exposure.  reduced  Cd  reached;  metallo-  DEN  coincided  were  less  plateau  This  "spillover"  was  Cd/L  increase  to  switch  mg a  than  been  Cd  without  large  protein weights  There  reached  with  was  rather  had  200  protein  occurred  production  at  exposure,  metallothionein,  DEN  exposure  production  molecular  to  with  enzymes.  as  pos-  transcriptional  Cd  exposure  levels. Exposure Zn  in  the  weight  levels on  by  Cd and  exposure  increased  to  (200  mg  resulted in  pool.  on  metallothionein,  level due  l i v e r ,  protein  reduced  to  Cd/L)  displacement  particular,  These to  DEN  increases with  the  metallothionein but  declined  where by  in  Cd  the of  high  Cu  and  of  Zn  Copper  with  increases  somewhat  levels.  at  and  were  and  that  occurred;  Cu  molecular  Cd.  "spillover"  high  levels  zinc of  Cd  exposure presumably  238 CHAPTER  XII  Discussion  Metallothionein The  Studies  studies  presented  in  this  presence  of  metal  organisms  for  fractions  corresponding  thionein.  to  Phytoplankton  thionein and  the  peak,  but  low molecular  contained  Cu,  i t  was  not  elution  small peaks  Zn,  in  induced  metallothionein  synthesis  with  increasing  levels  increased  in  metallothionein, those  studies  produced  in  and  Zn,  the  though  have  response levels  C u a n d Zn  creasing  with  Winge  et  a l . , 1975;  of  on m e t a l l o t h i o n e i n  Zn  even  though  molecular  increasing  Zn  weight  peak  in  the  main  storage  area  for  the  of  Thus,  in nonthat  both  Zn  a n d Cu to  study.  also  (Leber,  1975).  binds  study  in  the  both is  i n -  increase  occurred  high  and low  a distinct in  Cu  1974;  The  present  in  in  metallothionein  level  there  Hg ,  detectable that  metallothionein  C u a n d Zn  peak  on m e t a l l o t h i o n e i n  and Webb,  decreased  pools.  position  in  This  levels  exposure,  exposure  Stonard  levels  that  Hg  high  phytoplankton  in  metallo-  the  corresponding  employed  Cd o r  of  evidence  was n o t  indicated to  of  Hg  elution  metallo-  with  level,  fractions  by m e t h o d s  Previous  in  a  15).  some  exposure  those  even  fractions  Hg  is  to,gel  have  detectable  There  since,  to  (Figure  phytoplankton.  examined  position  compared  exposed  have  bound  d i d appear  weight  but  the  thesis  Figure  phytoplankton  Zn 15.  The  appears  239 to  be  the  low m o l e c u l a r  Zooplankton in  those  did not  fractions  metallothionein (Cloutier of  and  Cu a n d Zn  these  like  5).  These  in  the  increases  for  plankton. Cu  and  weight  mussels  ings  in  in  has  Magee,  position  the  metallothionein increases  of  level  occurred  in  the  Hg of  of  in  level  weight  1othionein-  Hg  be  in  is  metallothionein main  pools.  induction  l e v e l ,  to  Hg  metal  elevated  the  ankton  concurrently  the  exposure  established  metallothionein The  gel  present  Cd,  Cu a n d  elution  (Figure  Cu a n d  exposure  were  of  of these  also  in  good  zoo-  storage  area  the  molecular  low  for  pool.  distinct  in  the  peaks  increases  exposure  in  for  The  phytopi  distinct  Hg  Zn  position  increases  Zn  appeared  clearly  1978).  since  1972a).  phytoplankton,  been  produce  and  evidence  zooplankton,  usual  low molecular  existence  cytoplasmic It  and  As  Zn  good  increasing  the  with  Cu a n d  like  were  occurred Cu  and  (Webb,  with  evidence  high  the  increasing  though  are  metallothionein fractions  fractions  increases  even  to  there  pool.  distinct  However,  1978),  these  These  decreasing  13).  and w i t h  fractions  very  corresponding  Brown, in  cytoplasmic  have  (Figure  fractions  (Table  weight  Zn  (Table high  other  study Zn  There  confirms  peaks  were  were  and  In  Talbot  these  find-  found  also  mussels,  low molecular  in  the  to  distinct  with  most  that  1976;  corresponding  on-metallothionein 2).  workers  (Noel-Lambot,  profile  6).  by  Cu  weight  increasing and  Zn  cytoplasmic  240 pool,  suggesting  plankton,  use  cytoplasmic The Cu  peak  pool  the  (Figure  12A).  (Figure  8B)  high  levels  very  high  of  had of  Cd-  29)  It  This  Cd, and  Mussels  had  high only  before  thionein  with  exposed in  Cd  Perhaps on  1.6-fold  pool in  from the  Tables  are  when in  in  a  to  amounts mice  Cd  (Figure  organisms  in  30).  can  portion  to  of  metallothionein. organisms  experiments.  levels  of  apparent  saturation  of  metal  the  high  Table  2).  a  containing  contained  lesser  laboratory  had  kidney  peaks  considering  from  distinct occurs  and  higher  to  Zn.  metallothionein  bound  increase  before  and a  an  of  metallo-  molecular Mice  6.1-fold  spillover  metallo-  increase  occurred  19-21).  differences  metallothionein  of  higher  since  laboratory,  levels  8A)  with  exposed  (Calculated  the  metallothionein  (calculated  levels  was  have  Laboratory  levels  levels  Cu  kidneys  19).  that  evident  there  and  levels  were  spillover  protein to  a  4  zoo-  weight  for  to  and  metallothionein  Human  and  therefore,  metal  area  metallothionein  metallothionein  thionein  weight  which  insignificant  metal  molecular  appeared  Zn.,  18  these  low  (Figures  and  (Figures  particularly to  salmon in  phytoplankton  storage  Cu-thionein  cytoplasmic  exposed  main  liver  Cu  unless  more  is  chum  1ike  the  distinctive  appears,  produce their  of  the  Duck  relatively  (Figure  as  of  fractions  Zn-thionein  had  mussels,  components  livers in  that  lower  between organisms  the  portion  ( i . e . ,  of  metal  phytoplankton  ,  241 zooplankton f i s h ,  and mussels)  ducks,  mice  and humans)  analyzed.  For  the  organism  entire  higher  can  other  tissues  levels trace  produce  elements  entire  were  to  higher  levels  that  The  for  organisms a  very  of  and  might  in  were  only  these  had  ,  very  other  in  the  of  the  analyzed,  i t  would  of  metal  equivalent  appeared  gland  molluscs  high  the  zooplankton,  have  tissues,  i f  portion  if  low  Therefore,  been  small  analyzed.  whereas  components  pool.  and d i g e s t i v e  metals  to  Perhaps  metal  In  metallothionein  metal 1oenzymes  pool,  mussels,  tissue  In  to  type  analysis.  kidney  of  phytoplankton  crop  heavy  and  for  ( i . e . ,  tissue  and  a l . , 1975).  only  from  the  zooplankton  cytoplasmic  higher  to  produce  bound  weight  due  organisms  organisms  protein  portion  thionein. areas  high  metallothionein.  analyzed  ,  liver  are  of  appeared  bound  a  body  is  only  et  higher  homogenized  higher  weight  molecular  have  in  was  (Bouquegneau  molecular low  phytoplankton  organisms,  These  versus  are  organs  or  mussels,  on  the  (Coughtrey  was  metallostorage  and  Martin,  1976). This  study,  vestigated cytoplasmic was  made  the  unlike  relative  pools  unique  these  various  to  point  established  trace  elements  binding  besides  truly  bound  of  almost  by  a l l  previous  of  metals  studies,  to  metallothionein.  other This  the  fact  that  pools  were  quantified.  by  these  do  not  studies,  occur  levels  was t h a t  unless,  and  i n -  of  study metals  The toxic  u n t i l ,  main effects these  trace  elements  pool.  This  plankton  Cd  to  were  exposed  Hg,  distribution  this  molecular  weight  Thus,  the  et  a l .  (1977),  do  not occur these  pool.  ference  of  with  metal  zooplankton exposed  extract  of  of in  pool  this  phyto-  and f i s h  were  Cu a n d Z n ,  the  cytoplasmic  cancerous  organisms,  thesis  toxic  elements to  have  fish  and  and  phyto-  in the occur  spillover  to  effects  metals,  or  sites  clearly  f i r s t  high  with  the  the  sub-  suggested  effects  metallothionein  binding  protein  metallothionein.  i . e . , that  Toxic  of  weight  in which  Cd,  was shown  theory"  toxic  to  from  trace  "spillover  metals  protein  studies  cancerous  protein  until  molecular  Cd, and, studies  appearance  studies  stantiated  were  the  saturation  by  Hg,  tissue  for  plankton,  and  to  in  Except  apparent  the high  to  mussels  exposed  humans.  in  was s u b s t a n t i a t e d  were  exposed mice  appear  by  Brown  of  trace  elements  becomes  metal  saturated  high  molecular  are attributed excesses  of  to  weight  the  required  i n metal 1oenzymes  inter-  metals,  (Brown  et  a l . ,  1977). In  phytoplankton  theory"  was n o t  in  most  cases,  or  Hg-thionein  However, in  gesting  confirmed too  that  low to  simply  reduced  the high  exposed  molecular  since,  Hg,  the entire  "spillover  either  Hg  levels  be d e t e c t a b l e  on  metallothionein,  wasn't  growth  to  produced  did correlate  protein  the occurrence  of  pool  in  toxic  in  these  with  were,  phytoplankton.  appearance  phytop 1 ankton, elements  in  the  of sughigh  Hg  molecular  weight  protein  fluencing  survival  in  pool  is  the  common  phytoplankton,  as  factor  it  is  in  inother  organisms. Carcinogenesis The that  Studies  studies  there  was  of  a  tumorous  possible  organisms,  i . e . ,  increases  of  and  molecular  weight  and  kidney  directed  tissue.  However,  it  was  creases  of  protein  pool  was  Cd, of  and  liver  demonstrated in  (DEN)  found Cu  the  that Zn  Cd  high  than  on  metallothionein,  with  the  the  it  appears  as  gel  elution  profiles  fish  and  bution,  of  humans. Cd  is  But,  it  necessary  tumorous  though  does for  an  not  To  an  in  add in  Cd  confusion, accumula-  was  DEN  +  fish  are  de-  weight  protein  appear  cancer.  was  molecular  to  liver  changes.  organic  which  high  resulted  weight  exposed  to  the  that  result  mice  Therefore, in  mice.  additional  similar  contain  normal  these  if  very  in  research  high  could  distributions  result  cause  molecular  fact,  in  apparently  of  DEN  ratio  exposure  from  that  present  demonstrate  DEN  tissue  of  In  to  suggested  organisms  further  could  tion  DEN.  of  Therefore,  attempting  carcinogen  these  Cd:Zn  pool  humans,  factor  that  the  protein  towards  organic  it  Cd  and  common  cancerous  both  fish  pool  rather  administered Cd  and  had  Cd  humans.  carcinogen  similar that  to  this  Therefore,  can tumorous d i s t r i -  high  244 levels may  of  Cd  simply  be  etiologic gens.  in  in  liver  (e.g.,  and  the  Zn  tumors  in  from  liver  so  be  Cu  and  kidney  ing  of  extended  the  that  stages,  there  carcino-  could  fish)  and  and  be  kidney tumorous  carcinogens  fish  and  increased of  DEN  various  some  other  the  exposed  necessary  remove  transfer  in  else,  in  at  death, Cd  the  causitive Cd  time  mice,  Since  the  investigate  cytoplasmic tissue  which  to  metals  possible  to  Cd,  them  liver.  to  pool by  finding Cu  and  either  death.  The  Cd  that  kidchanges  pools is  Zn  of  liver  develop-  Zn of  this  level  of  Cu  time  of  death.  That  Cd  increased  be  in  role  high in  conflict  molecular  weight  carcinogenesis  administered  with  (100  Zn  protein  Cd/L)  in  tumorous the  the  time  to  suggesting  pool  1977). in  that  extension  hypotheses  (Brown, mg  +  but  high  administration,  the  to  reduced  levels,  or  length  DEN  to  the  levels  losses  the  related  appears in  was  protein  these  directly  liver  interest  weight  reversal  the  are  and  certain  organisms,  somewhere  levels  tissue,  major  molecular  that  Zn  with  or  mice).  livers  studies  or  pool  carcinogens,  (e.g.,  bearing  are  to  of  protein  tumors. Of  was  the  transferred  in  liver  tumorous  these  in  Further  and  of  effect  possible  pretumorous  that  neys. Cd,  tumor  skin  develop  could  nontumorous from  weight  cancers  clearly  DEN-exposed  Perhaps  molecular  certain  is  in  human)  high  coincidental  it  differences  Cu  a  only  Also,  (e.g.,  the  has  a  However, study  in  245 which  Cd  probably the  extended  the  resulted  in  monooxygenase  (Yoshida would tion  et  of  the  levels  exposure could  of  have  total  of  and  Cd  Chan,  least  point  a  DEN  were  (5  the  was  not  the  Cd/L)  of  bioactivated then  An  DEN  administrato  12.5  increase weeks'  suggests  in  they  activity  case  after  This role  that  appeared  changes  1978).  high  of  state.  mg  catalytic  that  concentration indication  organism.  It  designed high  level  a  levels  be of  or  thionein  of  of  the  that  Cd  carcinogenesis.  no  a  metal  inflow  toxic  effects  into  fact  bound  by  to  would that  of  be  toxic be  level by  -  acute  be  able  in  caused a  by  In  A  this  one  of  same but  pace  doesn't  type  with  since  latter  the  there  similar  the  nondetoxified  measure  metallothionein,  keep  but  therefore  exposure,  to  resulting  organism,  effects.  obtained  organism.  without  an  that be  exposure  in  the  gives  of  could  metallothionein,  wouldn't the  metal  that  organism  experiment  metallothionein  might  high  an  is  survival  contamination  bound  synthesis  an  in for  chronic  evidence  after  demonstrated  metals  that  metal  contamination  be  potential  level  bound  to  toxic  l i k e l y  l i t t l e  metal  The  needs  of  low  metal  organism  l i t t l e  is  with  most  should  metal  this  precarcinogenic  at  key  l i t t l e  of  that  so  Studies  A  with  so  if  were  reduction  precarcinogenic  levels  (Brown  Future  in  a  death  large  1 975);  in  lower  a  to  system,  a l . ,  remain  time  metallo-  with organism,  metal.  portion  of  know  a  if  metal  is  exerting  immediate  application  monitoring. surveys But  The  of  their  levels  In  these  One  cannot  all  organisms,  the  rate  and  how  much  metal  much  assume  danger  a  organism.  metals  by  organisms shown  that  metabolism has  entire Such  and  been  lysosomes  no  in  that  of  should  readily  is  an  study  of  of  amenable  to  to  one  knows  trace  routine  and  how  toxic on  the  to  that  monitoring of areas.  detoxification  For  by  instance,  it  has  involved  in  the  metals. may an  produce  upon  m e t a l - p o l 1 uted  detoxification  attempt  depends  in  detoxification  metal  Therefore,  portion  organism.  comment  intimately of  to  detoxified  organism  metallothionein  metal  the  before  to  studied.  1974).  of  unless  ih i designated:  are  a b i l i t y  the  environmental  detoxification  shown  spectrum  be  within  present  within  mechanisms to  nothing  position  a  pollutants.  metallothionein,  be  future  other  organisms  And,  and  environmental  detoxified  by  is  lysosomes  (Porter,  studies  cedures  portion  must  with  portion  exposure.  metal 1 o t h i o n e i n  need  tell  same  incorporate  possible  the  direct  environmental  and  detoxified  pollutant'level  also  of  metal  has  of  metals  detoxified  one  should  Other  it  is  of  not,  replete  they  the  the  Therefore,  programs  been  since  occur, of  are  that  level  is  l i t t l e  words,  that  or  strategies  various  tell  nondetoxified  effects  to  of  other  metals  effects  literature  numbers  survive. of  toxic  be  Furthermore, stored  within  elucidation mechanisms  standardized  monitoring  of  is  the needed.  pro-  programs.  247 Since it  follows  able  for  sible each at  tolerance  most  to  the  pollutant,  been  can  surely of  done  detoxify  could  a b i l i t y  it  and  the  detoxify  metals  is  and  of  the  be  adipose  l i v e r ,  to  should  pos-  systems  levels  of  for  pollutant This  metallothionein -  e . g . ,  enzyme  readily  tissue,  store  be  avail-  surpassed.  toxicants  probably  pollutants,  systems  detoxification  work,  other  of  most  detoxification  monooxygenase-hydroxy1ase  the  reticulum  for  to  Therefore,  to  trace  done  PCB's  as  be  these  capacity for  be  the  may  developed  individual  how  their  has  well  there  be  pollutants.  study  which  ties  that  can  the  capaci-  systems  to  determined  and  these  and  as  endoplasmic  before  they  are  detoxified. The  study  of  carcinogenesis research. studies  As  causes of  with  that  This  losses  Unfortunately, DEN.  Future DEN,  counteract studies  of  both  Cu  utilized  should  Cu  and to  present  both  in  of  most  and  Zn  in  delay  that  the  onset  by  Cu  in  area are  of numerous  cancer  from  carcinogen tissue. of  as  (DEN)  Reversal  tumors.  administered  present  only  well  as  failed  to  Zn Zn  DEN. study  Zn  study  form  chemical  one  pretumor  carcinogen-induced the  there  prevent  administer  depleted  metals  promising  Zn  studies  the  trace  thesis,  shown  should are  a  this  has  appears  studies  the  be  in  thesis  the  since  Methods  to  discussed  decreases  these  involvement  appears  showing  occurring.  the  depletions. and  method  Future of  with  248 application  of  Cu  and  carcinogen-induced induced  losses  tion  with  will  be  of  of  the  Zn  that  metal  losses.  metal  can  carcinogen,  clear  most  be  and  readily If  these  prevented tumors  that  metal  losses  recent  study  by  counteracts carcinogen-  by  metal  prevented,  are  involved  administra-  then  in  it  the  etiology  cancer. A  that  very  the  organic  produces They Zn  Zn  and  furthermore  due  from  tumors  carcinogen  to  a  replete  deficiency  deficient to  MBM  diet.  than  They  et  a l .  (1978)  has  found  methyl benzylnitrosamine  demonstrated  Zn  exposure  Zn  Fong  in  that  diet,  the  rats  esophagus  bf  deficient-  in  developed  did  controls  conclude  (MBM)  tumors  given  rats.  faster  MBM  with  a  that:  "present studies with experimental animals i n d i c a t e t h a t z i n c i s somehow i n v o l v e d in the e t i o l o g y of esophageal c a n c e r . Since zinc usually interacts with other trace elements, our f i n d i n g s imply that the observed effects involve a trace element i m b a l a n c e on a b r o a d e r scale." An  attempt  thesis,  was  to  study  particularly in a  this  made  relatively  these  with  thesis,  common  fact  that  the  Cd  levels  in  in  carcinogenesis,  reported  imbalances to  elevation  the of  denominator  present  pretumor  molecular  studies  respect  the  The  high  in  while  weight  the  protein  a  this  ratio. ratio  present scale, As  derides increased of  that  the  indicated  appears  tumor-bearing  reports  pool  the  broader  Cd:Zn  of  study  tissue,  on  in  DEN  to  organisms. lowered  importance  Cd:Zn  be  ratio  tumor-bearing  of  in  Cd the  fish  and  humans,  and  the  high  low  Cd  the  molecular  doses,  genesis.  It  when  organic  such  as  may  present  studies simulate  and  the  in  use the  pool  Cd  levels  the  protein  that that  were  of  some  normally  report,  countered  be  portion  to  it  carcinogen  those  both  weight  points  carcinogenesis  Since  increased  is is  usual  levels  of  processes  a  necessary are  and  probably  environment,  lower  simply  in  Cd  far it  of  of  Cd  in  for  the  DEN  +  for  cancer  only  low,  i . e . ,  environment.  than  to  in  any  recommended order  in  carcino-  catalyst  higher  in  Cd  given  administered  is  both,  mice  relatively  encountered DEN  of  importance  levels  of  cytoplasmic  en-  that  more  carcinogenesis.  the  future  closely  250 CHAPTER  Arnold,  XIII  References  M . , a n d D. S a s s e . 1 9 6 1 . Q u a n t i t a t i v e and h i s t o c h e m i c a l a n a l y s i s o f C u , Zn a n d Fe i n s p o n t a n e o u s and i n d u c e d p r i m a r y t u m o r s o f r a t s . Cancer Res. 21: 761-766.  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M i c r o b i o l . 25: 708-736.  263  CHAPTER  XTV  Whereas of  this  based mary and  upon  methodology  high  and  in  cessed  the  in  (e.g.,  involved further  this  steps  presented  an  low  extraction  used  in  the  a  pestle.  For  £  for  size  levels  of is  (6-8) tissue in for  may  metal not  metal  fractions very  are  setting  on  be  done  exactly  laboratory  as  levels  zooplankton  eluted  with  collected samples  In  (15), would  and order  an  centrifugachloride  from  The  25. of  tissue. at  equipped done  or  that  in  a  dilution  Similar  liver  mussels,  a with  at  of  the  pellet  dilution  of  of  levels  metal  procedures  kidney  minimal  measureable  extraction  result  resultant  (17). of  a  minutes.  10  this  and  portion  are  (4,8&12)  pro-  l i t e r a -  3 minutes  motor  is  are  Table  the  extracted  a  pools  (2r4)  in  proteins for  sum-  procedures  sodium  given  A  current  after  increases  available.  are  done  small  are  of  procedures  (1)  These  obtained  Centrifugations  exactly  in  tissue  volumes  (1-9)  other  phytoplankton,  sample  and  process  (3&7)  speed  pellet  extractions  Homogenizations standard  the  tissue  1974). the  chapter  metallothionein  cytoplasmic  described  of  each  studies. of  kidney  Thompson,  sizes  and  x  as  i n i t i a l  Sample  or  in  recommended  these  weight  Liver  and  of  given  extraction  molecular  metallothionein  27,000  the  fashion  is  during  Olafson  extraction  teflon  in  Summary  describes  gained  39.  usual  extraction  solution  Methodology  section  used  Figure  (6-8).  double  detailed  experience  the  the  tion  I:  thesis,  of  ture  Appendix  tissue  are  followed  (Table  25).  264  Figure  39.  A flow  diagram  extraction and  low  See  text  of  of  steps  involved  metallothionein  molecular for  the  weight  details.  pools  and  the  from  in  the  high tissue.  265  0 )  Tissue  (2) (3)  I Add 0 . 9 % NaCl  i Homogenize  (4) (6)  Mussels, (5) ^ C o l l e c t  I Centrifuge  Supernatant-  —  Phytoplankton or Zooplankton  I  Add 0 . 9 % NaCl to P e l l e t (7)  1 Rehomogenize Pellet  (8)  1 Centrifuge  —  (9)  00) (ID  (12)  Discard  Pellet*  (13)  ^ Collect  SuDernatant  I Combine  Supernatants  Mussels Phytoplankton or Zooplankton  Heat to 7 0 ° C Centrifuge  1 Collect  Supernatant  U 4  Apply^ Supernatant t o Column  (15) Collect Fractions (16) Read A b s o r b a n c e a t 250 and 280 jjm (17) Read C u , Z n , C d o r Hg l e v e l s i n e a c h Fraction  Table 25.  Tissue  Recommended p r o c e d u r e s f o r v a r i o u s t i s s u e  Type  Sample S i z e f l ) (grams)  types.  F i r s t Homogen1zation(3) 0 . 9 % NaCl Volume ( m l )  Second Homogenization(7) 0 . 9 % NaCl Volume (ml)  Time t o H e a t ( m l n ) at 70°C(11)  Volume t o A p p l y ( m l ) t o Column(14)  Column Type Used  F r a c t i o n S1ze(ml) to C o l l e c t ( 1 5 ) a  Phytoplankton (whole tissue]  1  3  not  done  not  done  2  K.9/60  1.5  Zooplankton (whole t i s s u e )  1  3  not  done  not  done  2  K.9/60  1.5  Mussel (soft parts)  1  3  not  done  5  2  K.9/60  1.5  L i v e r or Kidney  0.1  1.5  not  done  5  1.0  K.9/60  0.8  0.5  2.0  not  done  5  1.5  K.9/60  1.0  1  2.5  1.5  5  2  K.9/60  1.5  2  4.5  2.5  5  5  KI.6/100  6  3  9  6  5  14  K2.5/100  15  a  To p r o d u c e a b o u t  30 f r a c t i o n s p e r p r o f i l e .  267 Supernatants tissue and  are  then  tract  at  cellular  and  cedure  the  is  utilized  was  not  many  not  the  necessary  after  to  one  gel  The  size  of  the  sample  is  used  preferable  to  (14)  for a  peaks. by  collecting  from  column.  an  was  ultracentrifuge  losses  of  heat  weight  is  protein  preferable;  this  molecular  weight  protein  denatured and  appear and  employed (Tables sample  column  as  Resolution  (free  of  a  with (14)  M  will  25  and  it  of  fraction  improves  sizes  down  to  with  narrower  longer  peaks  cellular  mainly  A  provides  super-  NH4HC03.  26). A  is  packed  depend  sizes.  It  (1-5).  column  0.01  are  pool,  heat.  zooplankton  clear  to  resolution  smaller  by  centrifugation  eluted  Better  procedure  a c t i v i t i e s  and  shorter  (13)  as  pro-  enzyme  applied  smaller  collected  supernatants  molecular  are  used  ex-  this  technique  these  column  is  tissue  if  extraction  size  obtained the  as  the  (11)  to  avoid  high  (10)  5 minutes  latter  phytop1 ankton  supernatants G-75  of  to  kidney  alternative  This  irreversibly  25)  clear  studies  the  high  to  Al  preferable  heat  Sephadex  lution  the  are  (Table  Resulting  column  from  for  supernatant  minutes.  present  and  bath  combined  However,  in  enzymes  natants.::  upon  the  particularly  measured  debris)  The  ultracentrifugation  is  as  60  liver  order  discarded.  for  in  (5),  water  in  debris.  enzymes  the  be  (12)  available.  coagulable  to  70°C  a  centrifuge  x £  not  method  in  pellet  to  105,000  pool,  mussel  centrifuged  of  (13)  heated  from  column  a  better  can  also  as 1-2%  resobe  eluant of  is  the  Table 26. The specifications for various sizes of Pharmacia columns when packed with Sephadex G-75 gel (from Pharmacia technical literature). Void Minimum/Maximum Maximum Bed Column Type (k) Volume F L o w Rate Sample Size Volume and size (cm) (ml) (ml/hr) (ml) (ml) (Kdiameter/length)  Time for First Macromolecules to Elute (hr)  0.4/10  17  13  0.8  200  2/50  44  70  1.6  485  5/120  114  168  1.5  K.9/60  38  Kl.6/100  K2.5/100  269 bed  volume.  give  less  diluted  The ing  Sample  position  absorbance  molecular whereas the  the  high  protein  pool  or  separate  if  applicable.  followed pool.  by  Each  be  The  Cu  enzymes  (Brown  levels  gel  et Hg  cular  weight  thionein  In  and  and  the to  the  low  due  Cd,  and  for  binds high  a  The  the  pool  /jm  bound,  the  to  of  tubes  26-45.  was  found the  1-15,  low In  next,  peak  of  pool metallothe  and  low of  shoulder  cytoplasmic  of  Cu  weight  Zn  molecular metals  phytoplankton  fractions,  the  a  binds  portion  tubes  to  hemoglobin,  protein  The  in  a  in and  that  in  high  mole-  a  metallo^  molecular other  to  may  appear  presence  it  of  jjm  due  due  with  weight  excesses  and  280  molecular  will  weight  portions  16-25,  comprised  280  peak,  peak  comprised  read-  low  at  presence  small  45  and  and  high  f i r s t  study,  by  250  Metallothionein also  resolution.  at  is  metal 1oenzymes.  present  tubes  Cd  correspond  to  comprising  protein  high  low  molecular  will  1977).  very  profile  pool  much  molecular  Zn  pools  but  comprised  cytoplasmic  as  less  Metallothionein  bonds.  due  high  required  organisms,  very  volume  i n i t i a l l y  absorbance  if  bed  with  The  acids.  /jm  peak  a l . ,  cytoplasmic  elution  250  peaked  contains  zooplankton.  amino  eluted  double  usually  above  high  the  but  (16).  metallothionein  The  metals,  jjm  usually  following  metals.  most  is  at  various  weight  have  absorbance  toxic  280  sulfhydryl-Cd  peak  a  is  and  aromatic  will  of  established  peaks  pools  of  25-40%  of  absorbance  presence  to  pools,  metallothionein of  up  cytoplasmic  250  weight  absence  have  at  sizes  gel  weight elution  270 profiles,  the  the  f i r s t  15/45  the  next  cular the  weight  molecular  (.33)  11/45  tubes  (.24)  of  tubes  cytoplasmic  weight  protein  the  pool  p r o f i l e ,  of  the  p r o f i l e ,  pool  the  last  comprised  metallothionein and  20/45  the  low  (.44)  mole-  tubes  of  profile. Metal  and  levels  expressed  plasmic in  high  a  as  pool  high  are a  per  determined  in  each  concentration  of  metal  gram  molecular  concentrations  of  correcting  the  for  OR  of  tissue  weight  Zn  in  pool  each  volume  {x, + x 9  (wet  each  (as  added,  each  weight). 10  cyto-  For  (e.g.,  mg  Zn  of  tissue  instance,  fractions,  mg Z n : / L )  fraction  + xrn)  +  in  comprising  fraction  of  fraction,  are 6  the added  ml):  X 6 ml  1000 ml  A correction (e.g.,  is applied for-the  2 g liver,  wet  wet w e i g h t  initially  homogenized  weight):  X 2 g liver(wet A  further  natant a  2  (13)  gram  water  correction  and  may  liver other  be  is  made  applied  sample  wt)  for  to  the  comprising  extractable  the  fact  column  that (14).  approximately  material  is  not  all  For 1.8  homogenized  super-  instance, ml  of i n i t i a l l y  271 in  4.5  ml  of  solution.  sodium Table  of  0.9% Of  discarded  and  this  chloride 25).  NaCl  8.8  ml  solution,  Therefore tissue  a  the  pellet  of  5 ml  extracted  extractable are  correction  2.5  material  applied factor  in  to  the  is  ml and  column  needed  for  extract:  X  8J3 5.0  A  correction  converted  is  into  be  evaluated  of  each  made /jmole  in  metal  (  X  '  so  terms  data  that  of  the  in  the  1 mmole Zn 6 5 . 4 mg Zn  competition relative  +  V -  +  X  ' °  )  r  o  9  Z  "  x e  m  x  x  l  numbers  *  metals  of  x  1 mmole Zn  (wet 1000  x  6 5 . 4 mg Zn  in  between  has  been can  molecules  1000 jwnole mmole  2 g liver x  Q.8  study  complete c a l u c u l a t i o n i s :  1000 ml  In  present  present. X  The  since  a  typical mg,  the  gel  there high  elution  would  be  molecular  profile  0.065 weight  M  wt)  x  5.0  jjmole  mmole where  >imole  x-j  Zn/g  protein  +  x^  liver  pool.  + (wet  X-JQ  =  weight)  PUBLICATIONS: McLeay, D. J . , and D. A. Brown. 1974. Growth stimulation and biochemical changes i n juvenile coho salmon (Oncorhynchus kisutch) exposed to bleached k r a f t pulpmill e f f l u e n t f o r 200 days. J . F i s h . Res. Board Can. 31« 1043-1049. McLeay, D. J , , and D. A. Brown. 1 9 7 5 . E f f e c t s of acute exposure to bleached k r a f t pulpmill e f f l u e n t on carbohydrate metabolism of juvenile coho salmon (Oncorhynchus kisutch) during r e s t and exercise. J . F i s h . Res. Board Can. 3 2 : 753-760. Brown, D. A., and D. J . McLeay. 1 9 7 5 . E f f e c t of bleached k r a f t m i l l e f f l u e n t on the s u r v i v a l of starved juvenile coho salmon (Oncorhynchus k i s u t c h ) . J . F i s h . Res. Board Can. 3 2 : 2 5 2 8 - 2 5 3 0 . Brown, D. A., and D. J . McLeay. 1 9 7 5 . E f f e c t of n i t r i t e on methemoglobin and t o t a l hemoglobin of juvenile rainbow t r o u t . Prog. F i s h - C u l t . 3 2 : 3 6 - 3 8 . Brown, D. A., C. A. Bawden, K. W. Chatel, and T. R. Parsons. 1977. The w i l d l i f e community of Iona Island j e t t y , Vancouver, B.C., and heavymetal p o l l u t i o n e f f e c t s . Environ. Conserv. 4: 213-216. Brown, D. A. 1977. Increases o f Cd and the CdiZn r a t i o i n the high molecular weight protein pool from apparently normal l i v e r of tumor-bearing flounders (Parophrys vetulus ). Mar. B i o l . 44: 203-209. Brown, D. A., and T. R Parsons. 1 9 7 8 . Relationship between cytoplasmic d i s t r i b u t i o n of mercury and toxic e f f e c t s to zooplankton and chum salmon (Oncorhynchus keta) exposed to mercury i n a controlled ecosystem. J . F i s h , Res. Board Can. 3 5 : 880-884. 0  Brown, D. A., and T. R. Parsons. 1978. The ubiquity and function of metallothionein i n marine organisms. (Presented a t the 41st annual meeting of the American Society of Limnology and Oceanography, V i c t o r i a , B.C.). Brown, D. A,, and K. W. Chatel. 1 9 7 8 . Interactions between cadmium and zinc i n cytoplasm of duck l i v e r and kidney. Biol.-Chem. Interactions 22: 2 7 1 - 2 7 9 . H a l l , J . B., and D. A. Brown. 1979. Plasma glucose and l a c t i c a c i d a l t e r a t i o n s i n response to a s t r e s s f u l exam. B i o l . Psych. (In press), Cloutier-Mantha, L., and D. A. Brown, 197<?. The e f f e c t of mercury exposure on the cytoplasmic d i s t r i b u t i o n of mercury, copper and zinc i n phytoplankton. J . Exp. Mar. B i o l , E c o l . (In press). Young, D. R,, C. K. Bertine, D. A. Brown, E . C r e c e l i u s , J . Martin, F. Morell, and G. Roesijadi. 1979, Trace metals, Ini;N0AA Recommendations to the U.S. Congress on Ocean P o l l u t i o n Research, Ed: E . Goldberg. (In press). Bayne, B. L., D. A. Brown, F. L . Harrison, and P. P. Yevich. 1979. Mussel health, In: The International Mussel Watch, Ed: E . Goldberg, ( i n press).  

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