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Fish cell DNA repair and breakage assays for assessing aquatic genotoxicity Walton, Douglas G. 1985

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FISH CELL DNA REPAIR AND BREAKAGE ASSAYS FOR ASSESSING AQUATIC GENOTOXICITY by DOUGLAS G. WALTON B . S c , U n i v e r s i t y o f V i c t o r i a , 1976 M.Sc, Memorial U n i v e r s i t y of Newfoundland, 1978 A T h e s i s Submitted i n P a r t i a l F u l f i l l m e n t o f the Requirements f o r the Degree o f DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF ZOOLOGY We ac c e p t t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA October 1985 © Douglas George Walton, 1985 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6G/81) ABSTRACT I t i s claimed that many cancers i n man are causally r e l a t e d to chemical carcinogen exposure. S i m i l a r l y epidemiological studies of f i s h populations have associated an elevated tumor incidence with environments contaminated with chemicals, some of which have demonstrated carcinogenic a c t i v i t y . Sampling and pathologically screening large numbers of f i s h i s labour intensive, costly, time-consuming, and f i e l d studies provide only circumstantial evidence f o r an e t i o l o g i c a l agent. A v a r i e t y of quick and inexpensive assays have been used with mammalian c e l l s to detect probable carcinogenic a c t i v i t y by analyzing f o r DNA-damaging events. More recently f i s h c e l l s and metabolizing enzymes are being used i n these assays i n order to provide t e s t s with more relevance to the aquatic environment, v a l i d a t e mammalian t e s t r e s u l t s , determine f i s h c e l l s e n s i t i v i t y to genotoxic agents, and, ultimately, to develop techniques to d i r e c t l y assess the genotoxic:carcinogenic p o t e n t i a l of a p a r t i c u l a r environment. The present study i n i t i a l l y compared the DNA r e p a i r response i n f i s h and mammalian c e l l s . Although the measured re p a i r response of f i s h c e l l s was found to be very low, i n comparison to mammalian c e l l s , the assay procedure was a l t e r e d to increase the f i s h c e l l s e n s i t i v i t y . Despite being able to detect DNA re p a i r i n f i s h c e l l s treated with 3,4-benzopyrene the r e p a i r technique was f e l t to lack the s e n s i t i v i t y necessary f o r in vivo monitoring. However, the low amount of rep a i r measurable i n f i s h c e l l s suggested that t e s t s f o r DNA - i i -b r e a k a g e may b e m o r e s e n s i t i v e . S u b s e q u e n t c o m p a r a t i v e e x p e r i m e n t a t i o n w i t h t h e c h r o m o s o m e a b e r r a t i o n a n d m i c r o n u c l e u s t e s t s c o n f i r m e d t h i s . T r e a t m e n t o f f i s h c e l l s w i t h 3 , 4 - b e n z o p y r e n e r e s u l t e d i n c h r o m o s o m e a b e r r a t i o n s . P o l y c y c l i c a r o m a t i c h y d r o c a r b o n s l i k e 3 , 4 - b e n z o p y r e n e a r e i m p o r t a n t a q u a t i c c o n t a m i n a n t s a n d h a v e b e e n f o u n d i n e l e v a t e d c o n c e n t r a t i o n s i n s e d i m e n t s f r o m S t u r g e o n B a n k , a n a r e a n e a r V a n c o u v e r a t w h i c h a s e w a g e t r e a t m e n t p l a n t d i s c h a r g e s . F i e l d t e s t i n g t h e d e v e l o p e d g e n o t o x i c i t y t e c h n i q u e s i n d i c a t e d c y t o t o x i c i t y a n d c h r o m o s o m e a b e r r a t i o n s , b u t n o DNA r e p a i r , i n c u l t u r e d f i s h a n d m a m m a l i a n c e l l s e x p o s e d t o S t u r g e o n B a n k s e d i m e n t e x t r a c t s . H o w e v e r , i n a p a r a l l e l t e s t , a n i n v i v o i n c r e a s e i n m i c r o n u c l e i f r e q u e n c y i n s t a r r y f l o u n d e r c o u l d n o t b e d e m o n s t r a t e d . TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i i i LIST OF FIGURES i x ACKNOWLE DGEMENTS x i i INTRODUCTION 1 MATERIALS AND METHODS 5 1. Chemicals 5 2. C e l l C u l t u r e s 5 a) T i s s u e c u l t u r e c e l l l i n e s 5 b) C e l l c u l t u r e s 6 c) DNA q u a n t i t a t i o n 7 d) P r e p a r a t i o n of S9 7 e) DNA r e p a i r s y n t h e s i s 8 f) Chromosome a b e r r a t i o n s 10 g) M i c r o n u c l e i 10 3. Primary C e l l s 11 a) I s o l a t i o n o f the c e l l s 11 b) DNA r e p a i r s y n t h e s i s w i t h p r i m a r y c e l l s . . 12 4. T i s s u e S l i c e s 12 DNA r e p a i r s y n t h e s i s i n t i s s u e s l i c e s 12 5. Sediments 13 C o l l e c t i o n , p r e p a r a t i o n , and a s s a y i n g o f sediment samples 13 -iv-6. F l a t f i s h 16 C o l l e c t i o n of f l a t f i s h and examination f o r m i c r o n u c l e i 16 RESULTS 18 A. DNA Rep a i r S y n t h e s i s 18 1. Rep a i r i n C u l t u r e d C e l l s 18 a) C e l l l i n e comparison 18 b) I n v e s t i g a t i o n i n t o the low f i s h c e l l DNA r e p a i r response 18 c) I n c r e a s i n g the f i s h c e l l DNA r e p a i r response 27 d) E f f e c t o f DMSO on DNA r e p a i r s y n t h e s i s . . . 35 e) Use of rainbow t r o u t S9 i n the DNA r e p a i r assay 44 f) DNA r e p a i r s y n t h e s i s f o l l o w i n g treatment w i t h PAHs 44 g) R e p a i r i n h i b i t i o n assay 49 2. DNA R e p a i r S y n t h e s i s i n Primary C e l l s I s o l a t e d From Rainbow T r o u t 54 3. DNA R e p a i r i n T i s s u e S l i c e s 57 B. DNA Breakage Assays 60 1. DNA Breakage Assays with C u l t u r e d C e l l s 60 a) C e l l l i n e comparisons 60 b) Examination o f the e f f e c t o f p o s t -exposure d u r a t i o n and DMSO c o n c e n t r a t i o n on chromosome a b e r r a t i o n frequency 73 c) * Chromosome a b e r r a t i o n s f o l l o w i n g PAH treatment 78 C. F i e l d T e s t i n g the DNA Repair and Breakage Assays... 78 1. Sediment E x t r a c t s 78 a) DNA Repair 78 b) Chromosome a b e r r a t i o n s 86 -v-2. M i c r o n u c l e i i n F i s h C e l l s In V i v o 92 DISCUSSION 95 1. Why T e s t f o r Aq u a t i c G e n o t o x i c i t y ? 95 2. The DNA Repair Assay as a T e s t For A q u a t i c G e n o t o x i c i t y 98 a) The DNA r e p a i r response i n mammalian ve r s u s f i s h c e l l s 98 b) E x p l a n a t i o n s f o r the low DNA r e p a i r response of f i s h c e l l s 99 c) Enhancing the measured f i s h c e l l DNA r e p a i r response 101 d) U s i n g the DNA r e p a i r i n h i b i t i o n a sssay w i t h f i s h c e l l s 105 e) DMSO and S9 use i n the DNA r e p a i r assay.. 105 f) S c r e e n i n g PAHs f o r g e n o t o x i c i t y i n the DNA r e p a i r assay 106 g) DNA r e p a i r assays w i t h f i s h p r i m a r y c e l l s and t i s s u e s l i c e s 108 3. Assays f o r DNA Damage as T e s t s f o r A q u a t i c G e n o t o x i c i t y 110 a) Comparison o f chromosomal a b e r r a t i o n frequency i n mammalian and f i s h c e l l s . . . . 110 b) P r o c e d u r a l d e t a i l s o f the chromosome a b e r r a t i o n t e s t I l l c) Chromosome a b e r r a t i o n s f o l l o w i n g PAH exposure 112 d) Use o f the micronucleus t e s t w i t h f i s h and mammalian c e l l s 112 4. F i e l d T e s t i n g the G e n o t o x i c i t y Assays 114 a) Overview 114 b) In v i t r o g e n o t o x i c i t y t e s t i n g o f t h e contaminated area 115 c) JJQ v i v o g e n o t o x i c i t y assessment o f t h e contaminated area 116 -v i -SUMMARY 118 1. DNA Re p a i r Assay 118 2. T e s t s f o r DNA Damage 119 3. F i e l d T e s t i n g the Developed G e n o t o x i c i t y T e s t s 120 REFERENCES CITED 121 APPENDIX I - P u b l i c a t i o n s from the T h e s i s M a t e r i a l 133 APPENDIX I I - L i s t o f A b b r e v i a t i o n s 134 APPENDIX I I I - T h e o r e t i c a l Aspects and S t a t i s t i c a l A n a l y s i s of the DNA Repair and Breakage Assays 135 - v i i -LIST OF TABLES PAGE TABLE 1 25 DNA r e p a i r s y n t h e s i s i n HF, CHO, and RTG c e l l s exposed t o u l t r a v i o l e t l i g h t . TABLE 2 55 DNA r e p a i r i n RTG c e l l s i r r a d i a t e d with u l t r a v i o l e t l i g h t , then incubated w i t h HTdR i n l i g h t o r dark c o n d i t i o n s . TABLE 3 63 Post-4NQ0 exposure HTdR l a b e l l i n g of d i v i d i n g Umbra l i m i f i n and h e a r t c e l l s , Chinese hamster ovary c e l l s , and human f i b r o b l a s t c e l l s . TABLE 4 74 Frequency o f chromosome a b e r r a t i o n s i n Umbra l i m i h e a r t and Chinese hamster ovary c e l l s exposed t o 4NQ0 and examined a t i n t e r v a l s t h e r e a f t e r . TABLE 5 76 E f f e c t o f DMSO on chromosome a b e r r a t i o n frequency i n Umbra  l i m i h e a r t and Chinese hamster ovary c e l l s exposed t o rainbow t r o u t S9 a c t i v a t e d a f l a t o x i n B 1. TABLE 6 81 DNA r e p a i r s y n t h e s i s i n human f i b r o b l a s t and rainbow t r o u t gonad c e l l s t r e a t e d w i t h sediment e x t r a c t s from Spanish Bank and Sturgeon Bank. TABLE 7 87 Frequency o f chromosome a b e r r a t i o n s i n Chinese hamster ovary and Umbra l i m i h e a r t c e l l s t r e a t e d w i t h sediment e x t r a c t s from Spanish Bank and Sturgeon Bank. TABLE 8 93 Frequency o f m i c r o n u c l e i i n b u c c a l , l i v e r , and r e d b l o o d c e l l s from s t a r r y f l o u n d e r ( P l a t i c h t h y s s t e l l a t u s ) c o l l e c t e d from Spanish Bank and Sturgeon Bank. LIST OF FIGURES PAGE FIGURE 1 14 L o c a t i o n o f the f i s h and sediment c o l l e c t i o n s i t e s a t Sturgeon and Spanish Banks. FIGURE 2 19 DNA r e p a i r s y n t h e s i s measured i n c u l t u r e d HF, CHO, RTG, RTO, FHM, and CH c e l l s exposed t o (A) MNNG, (B) 4NQO, (C) NA2AAF, and (D) r a t S9 a c t i v a t e d AFB 1. FIGURE 3 21 The d a t a from FIGURE 2 f o r the DNA r e p a i r s y n t h e s i s i n c u l t u r e d RTG, RTO, FHM, and CH exposed t o (A) MNNG, (B) 4NQ0, (C) NA2AAF, and (D) AFB, r e p l o t t e d t o examine the d i f f e r e n c e i n r e p a i r magnitude between the f i s h c e l l l i n e s . FIGURE 4 23 DNA r e p a i r s y n t h e s i s measured i n c u l t u r e d HF, CHO, RTG, RTO, FHM, and CH c e l l s exposed t o n o n - a c t i v a t e d hFB^. FIGURE 5 28 D u r a t i o n o f DNA r e p a i r s y n t h e s i s i n c u l t u r e d HF, CHO, and RTG c e l l s exposed t o (A) MNNG o r (B) 4NQ0. FIGURE 6 30 E f f e c t o f i n c r e a s i n g 3HTdR treatment time on DNA r e p a i r i n c u l t u r e d RTG c e l l s f o l l o w i n g a 30 min MNNG treatment. FIGURE 7 30 RTG c e l l s exposed t o emulsion f o r v a r y i n g p e r i o d s o f time up t o 28 days p r i o r t o deve l o p i n g . FIGURE 8 33 E f f e c t o f assay temperature on DNA r e p a i r i n c u l t u r e d RTG c e l l s t r e a t e d s i m u l t a n e o u s l y w i t h HTdR and MNNG. FIGURE 9 33 In c r e a s e i n DNA r e p a i r i n RTG c e l l s a t 25 C w i t h i n c r e a s e d t r e a t m e n t time i n 5xl0" 4M 4NQ0. FIGURE 10 36 DNA r e p a i r i n c u l t u r e d RTG c e l l s kept i n 2.5% ADM f o r a p e r i o d v a r y i n g from i t o 7 days p r i o r t o a 6h simultaneous exposure t o 5 x l 0 - 4 M MNNG and HTdR. FIGURE 11 A 36 DNA r e p a i r i n c u l t u r e d RTG c e l l s g i v e n a 3h exposure t o HTdR and MNNG d i l u t e d w i t h ADM supplemented w i t h v a r y i n g c o n c e n t r a t i o n s o f f e t a l c a l f serum. FIGURE 12 , 38 E f f e c t o f i n c r e a s i n g 3HTdR c o n c e n t r a t i o n on DNA r e p a i r i n c u l t u r e d RTG c e l l s . - i x -FIGURE 13 E f f e c t of c u l t u r e d RTG c e l l l i n e passage on DNA r e p a i r . 38 FIGURE 14 40 Comparison o f DNA r e p a i r i n c u l t u r e d RTG c e l l s exposed t o MNNG or 4NQO u t i l i z i n g (A) the o r i g i n a l e xperimental c o n d i t i o n s (B) the c o n d i t i o n s found t o enhance g r a i n p r o d u c t i o n . FIGURE 15 42 E f f e c t o f i n c r e a s e d d i m e t h y l s u l f o x i d e c o n c e n t r a t i o n on DNA r e p a i r s y n t h e s i s i n c u l t u r e d RTG c e l l s f o l l o w i n g u l t r a v i o l e t l i g h t exposure. FIGURE 16 45 DNA r e p a i r s y n t h e s i s i n c u l t u r e d RTG c e l l s exposed t o AFB^ a c t i v a t e d u s i n g rainbow t r o u t S9. FIGURE 17 . . . 45 DNA r e p a i r response i n c u l t u r e d RTG c e l l s exposed t o l x l 0 ~ 4 M AFBTL a c t i v a t e d w i t h i n c r e a s i n g amounts o f rainbow t r o u t S9. FIGURE 18 47 DNA r e p a i r response measured i n c u l t u r e d HF c e l l s f o l l o w i n g exposure t o AFB^ a c t i v a t e d with S9 from o i l e x t r a c t - i n j e c t e d rainbow t r o u t , A r o c l o r 1 2 5 4 - i n j e c t e d rainbow t r o u t , or A r o c l o r 1 2 5 4 - i n j e c t e d r a t s . FIGURE 19 47 DNA r e p a i r i n c u l t u r e d RTG c e l l s t r e a t e d w i t h AFB^ a c t i v a t e d w i t h S9 from o i l e x t r a c t - i n j e c t e d rainbow t r o u t , A r o c l o r 1254-i n j e c t e d rainbow t r o u t , o r A r o c l o r 1 2 5 4 - i n j e c t e d r a t s . FIGURE 20 50 DNA r e p a i r s y n t h e s i s i n c u l t u r e d RTG and HF c e l l s exposed t o (A) BP, (B.) DBA, (C) BA, and (D) PY a c t i v a t e d w i t h S9 from A r o c l o r 1 2 5 4 - i n j e c t e d rainbow t r o u t . FIGURE 21 52 DNA r e p a i r s y n t h e s i s i n c u l t u r e d RTG c e l l s f o l l o w i n g treatment w i t h (A) BP, (B) DBA, (C) BA, and (D) PY a c t i v a t e d w i t h f i s h S9. FIGURE 22 58 DNA r e p a i r s y n t h e s i s i n primary rainbow t r o u t l i v e r , stomach, and i n t e s t i n e c e l l s exposed t o (A) MNNG, (B) 4NQO, and (C) AFB-p FIGURE 23 61 DNA r e p a i r i n U l - F and Ul-H, CHO, and HF c e l l s exposed t o (A) MNNG and (B) 4NQO. FIGURE 24 66 Frequency o f chromosome a b e r r a t i o n s i n Ul-H, CHO, and HF c e l l s t r e a t e d f o r 3h with (A) 4NQO and (B) MNNG. -x-FIGURE 2 5 69 Frequency of m i c r o n u c l e i i n (A) Ul-F, (B) Ul-H, (C) CHO, and (D) HF c e l l s measured a t 16h, 48h, 80h, 112h , and 144h f o l l o w i n g 3h treatment with MNNG. FIGURE 26 71 Frequency of m i c r o n u c l e i i n (A) U l - F , (B) Ul-H, (C) CHO, and (D) HF c e l l s measured a t 16h, 48h, 80h, 112h, and 144h f o l l o w i n g 3h treatment w i t h 4NQ0. FIGURE 27 79 Frequency o f chromosome a b e r r a t i o n s i n Ul-H and CHO c e l l s t r e a t e d w i t h (A) BP, (B) DBA, (C) BA, and (D) PY. FIGURE 28 136 Assay p r e p a r a t i o n s showing (A) DNA r e p a i r i n RTG c e l l s , (B) chromosome a b e r r a t i o n s i n an Ul-H c e l l , and (C) a micronucleus i n an Ul-H c e l l . FIGURE 29 139 P l o t s o f the data from F i g u r e 22 B showing v a r i a t i o n s on the mean as r e p r e s e n t e d with (A) standard d e v i a t i o n s , (B) 99% c o n f i d e n c e i n t e r v a l s , and (C) standard e r r o r s . - x i -ACKNOWLEDGEMENTS S i n c e r e s t thanks are extended t o the s t a f f of the Environmental C a r c i n o g e n e s i s U n i t a t the B.C. Cancer Research Centre where t h i s r e s e a r c h was conducted. In p a r t i c u l a r , I would l i k e t o express my a p p r e c i a t i o n t o my s u p e r v i s o r s Dr. A. Acton and Dr. H. S t i c h , as w e l l as Drs. Dunn, San, and Rosen f o r t h e i r h e l p and encouragement. The t e c h n i c a l a s s i s t a n c e o f Mrs. G. Wood, Mr. G. De Jong, Mr. G. T r a x l e r (Dept. o f F i s h e r i e s and Oceans, Nanaimo), and Dr. S. Campana ( c u r r e n t l y , Dept. o f F i s h e r i e s and Oceans, H a l i f a x ) i s g r a t e f u l l y acknowledged. Mrs. R. P r i t c h a r d and Mrs. A. Walton were i n s t r u m e n t a l i n h e l p i n g t o produce the f i n a l t h e s i s copy. Use o f t h e B.C. C h i l d r e n ' s H o s p i t a l EEG Department word p r o c e s s o r was k i n d l y p r o v i d e d by Dr. P. Wong. F i n a l l y , I would l i k e t o extend my deepest a p p r e c i a t i o n t o my w i f e and f a m i l y f o r t h e i r y e ars o f p a t i e n c e and i n v a l u a b l e h e l p . F i n a n c i a l support was p r o v i d e d by t h e B.C. Foundation f o r Non-Animal Research. INTRODUCTION * In a r e c e n t P e r s p e c t i v e s a r t i c l e M. G i l b e r t s o n (1984) c h a l l e n g e d the adequacy o f approaching a q u a t i c t o x i c o l o g y by f o c u s i n g on l a b o r a t o r y experimentation. In h i s o p i n i o n , i t would be much more i n f o r m a t i v e t o f i r s t undertake o b s e r v a t i o n s on f i s h p o p u l a t i o n s t o see whether any i n t e r e s t i n g anomalies are found, and then t o form hypotheses which can be e x p l o r e d through l a b o r a t o r y e x p e r i m e n t a t i o n and the development o f f u r t h e r b i o c h e m i c a l and h i s t o l o g i c a l methods. E p i d e m i o l o g i c a l s t u d i e s o f f i s h from l o c a l waters undertaken i n the 1970's by Dr. S t i c h and co-workers a t the B.C. Cancer Research Center determined t h a t s k i n p a p i l l o m a s among f l a t f i s h p o p u l a t i o n s o c c u r r e d a t e l e v a t e d f r e q u e n c i e s i n a s s o c i a t i o n w i t h contaminated environments ( S t i c h e t a l . , 1976, 1977a, 1977b). C o l l e c t i o n s o f E n g l i s h s o l e (Parophrys  v e t u l u s ) from Sturgeon Bank near Vancouver, f o r example, were found t o have a 57% frequency o f epidermal p a p i l l o m a s . A n a l y s e s o f the Sturgeon Bank sediments, e s p e c i a l l y near t h e Iona I s l a n d sewage treatment p l a n t o u t f a l l , showed h i g h l e v e l s o f t h e c a r c i n o g e n i c p o l y c y c l i c a r omatic hydrocarbon 3,4-benzopyrene (Dunn and S t i c h , 1976). However, f u r t h e r surveys f a i l e d t o show an obvious c o r r e l a t i o n between t h e f l a t f i s h s k i n p a p i l l o m a p r e v a l e n c e s and 3,4-benzopyrene l e v e l s i n bottom sediments which may be expected as t h e a r r a y o f p o t e n t i a l l y c a r c i n o g e n i c c h e m i c a l s w i l l l i k e l y v a r y between d i f f e r e n t l o c a t i o n s ( S t i c h e t a l . , u n p u b l i s h e d r e s u l t s ) . * see APPENDIX I - P u b l i c a t i o n s from the T h e s i s M a t e r i a l - l -More recently i t has been proposed that the f l a t f i s h epidermal papillomas or X - c e l l tumors are parasite inf e c t i o n s (Alpers et a l . , 1977; Dawe, 1981) and not a r e s u l t of exposure to chemical carcinogens (Stich et a l . , 1977a) and/or oncogenic viruses (Peters et a l . , 1978; Peters and Watermann, 1979) . Further studies are required to better determine the etiology of these tumors. Other epidemiological studies have correlated abnormally high tumor incidences i n f i s h with chemically contaminated environments. Brown et a l . (1973) found 4.4% of the f i s h surveyed from the Fox River i n I l l i n o i s had tumors of the ski n , stomach, and l i v e r . They note that the r i v e r had elevated heavy metal, p o l y c y c l i c aromatic hydrocarbon, and chlorinated hydrocarbon concentrations. Pierce et a l . (1978) showed a 32% incidence of hepatomas i n English sole (Parophrys  vetulus) from the Duwamish River estuary near Se a t t l e , Washington, and suspected chemical contaminants such as polychlorinated biphenyls as the e t i o l o g i c a l agent. In 1980, Black et a l . reported dermal tumors i n brown bullheads (Ictalurus nebulosus), gonadal tumors i n g o l d f i s h x carp hybrids, and neurolemmomas i n sheepshead (Aplodinotes  grunniens) from an area contaminated with p o l y c y c l i c aromatic hydrocarbons. ' Black et a l . (1982) found elevated frequencies (up t o 100%) o f several tumor types i n saugers (Stizostedion  canadense) and walleye (Stizostedion vitreum) from Torch Lake, Michigan, which i s heavily contaminated with copper-mining wastes. Also a recent Puget Sound study (Malins et a l . , 1984) associated chemical contaminants (aromatic hydrocarbons, - 2 -c h l o r i n a t e d o r g a n i c compounds, and heavy metals) with up to 16% n e o p l a s i a i n c i d e n c e i n E n g l i s h s o l e . A r e l a t i o n s h i p i s t h e r e f o r e apparent between f i s h i n h a b i t i n g e n v i r o n m e n t a l l y degraded areas and an e l e v a t e d r i s k of n e o p l a s t i c d i s e a s e p u r p o r t e d l y as a r e s u l t of exposure t o chemical contaminants. Sampling and s c r e e n i n g l a r g e numbers o f f i s h f o r p o s s i b l e p a t h o l o g i e s i s a l a b o u r i n t e n s i v e , c o s t l y , and time-consuming p r o j e c t . Such e p i d e m i o l o g i c a l s t u d i e s a l s o p r o v i d e o n l y c i r c u m s t a n t i a l evidence f o r an e t i o l o g i c a l agent. In order t o b e t t e r l o c a t e and d e l i n e a t e a contaminated a r e a , and demonstrate p r o b a b l e c a r c i n o g e n i c a c t i v i t y t h rough DNA-damaging events, a v a r i e t y o f r e l a t i v e l y f a s t and inexpensive assays have been developed u s i n g b a c t e r i a l o r mammalian c e l l s and enzyme p r e p a r a t i o n s ( S t i c h and San, 1981). In r e c e n t y e a r s f i s h b i o l o g i s t s have adapted these assays, u s i n g f i s h i n v i v o o r c u l t u r e d f i s h c e l l s and f i s h enzyme p r e p a r a t i o n s t o study t h e e f f e c t s of chemical carcinogens, and t o generate r e s u l t s which are more r e l e v a n t t o the a q u a t i c ecosystem. The U.B.C. Cancer Research Center l a b o r a t o r y was a c t i v e i n t h e development o f the human f i b r o b l a s t DNA r e p a i r assay i n t h e e a r l y 1970's ( L a i s h e s , 1974; San, 1976) and l a t e r conducted surveys w i t h the Chinese hamster ovary chromosome a b e r r a t i o n t e s t ( S t i c h e t a l . , 1977c; Lo and S t i c h , 1978; S t i c h e t a l . , 1979). S i n c e the t a r g e t f o r many c a r c i n o g e n s i s DNA ( H e i d e l b e r g e r , 1975), these assays f o r DNA breakage and r e p a i r a r e o f both p r a c t i c a l and t h e o r e t i c a l importance. The r e s e a r c h d i s c u s s e d here adapted such assays t o be more r e l e v a n t t o the a q u a t i c environment by employing f i s h - 3 -c e l l s and a c t i v a t i o n enzymes, and undertook a comparative approach, c o n t r a s t i n g f i s h c e l l responses t o t h a t of the mammalian c e l l s normally used. In a d d i t i o n t o s t u d y i n g model c a r c i n o g e n s such as N-methyl-N'-nitro-N-nitrosoguanidine and 4 - n i t r o q u i n o l i n e 1-oxide the e f f e c t s o f t h e c a r c i n o g e n i c mycotoxin a f l a t o x i n B l f t o which rainbow t r o u t a r e v e r y s e n s i t i v e (Sinnhuber e t a l . , 1977), were a l s o examined. F i e l d a p p l i c a t i o n s o f the assays r e l a t e d t o our l a b o r a t o r y ' s e a r l i e r f i s h tumor work. Experiments f o c u s s e d on the e f f e c t s o f , f i r s t l y , p o l y c y c l i c aromatic hydrocarbons and, secondly, the Sturgeon Bank-Iona I s l a n d sewage treatment p l a n t o u t f a l l area where t h e r e are e l e v a t e d c o n c e n t r a t i o n s o f p o l y c y c l i c aromatic hydrocarbons, n o t a b l y 3,4-benzopyrene. -4-MATERIALS AND METHODS 1. Chemicals * Chemicals were obtained from the following sources: N-methyl-N 1-nitro-N-nitrosoguanidine and 4-nitroquinoline 1-oxide from the A l d r i c h Chemical Company, Milwaukee, Wisconsin; a f l a t o x i n 3,4-benzopyrene, 1,2-benzanthracene, and 1,2,5,6-dibenzanthracene from the Sigma Chemical Company, St. Louis, Missouri; pyrene from the Kodak Chemical Company, Rochester, New York; Aroclor 1254 from Analabs Incorporated, North Haven, Connecticut; [methyl- 3H]-thymidine (25 Ci/mmol s p e c i f i c a c t i v i t y ) from Amersham Corporation, Amersham, England; and co l c h i c i n e from the J.T. Baker Chemical Company, P h i l l i p s b u r g , New Jersey. The N-acetoxy-2-acetylaminofluorene was a g i f t from the National Cancer I n s t i t u t e ' s Standard Chemical Carcinogen Repository i n Chicago, I l l i n o i s , while the crude o i l was provided by S h e l l Canada Ltd. Only N-methyl-N'-nitro-N-nitrosoguanidine dissolved d i r e c t l y into the culture media, a l l of the other t e s t chemicals were prepared by i n i t i a l l y d i s s o l v i n g i n dimethylsulfoxide p r i o r to d i l u t i o n . Any other chemicals or solvents were of reagent or higher grade. * see APPENDIX*II - L i s t of Abbreviations 2. C e l l Cultures a) Tissue culture c e l l l i n e s The human f i b r o b l a s t c e l l l i n e used was a stock culture o r i g i n a t e d from t i s s u e explants at the Cancer Research Center. The chum salmon heart, rainbow trout ovary, rainbow trout -5-gonad, and fathead minnow were e s t a b l i s h e d c e l l l i n e s obtained from Mr. G. T r a x l e r ( P a c i f i c B i o l o g i c a l S t a t i o n , Nanaimo, B r i t i s h Columbia). The Chinese hamster ovary l i n e was a s t o c k l a b c u l t u r e which i s a v a i l a b l e , as are the rainbow t r o u t gonad and f a t h e a d minnow c e l l l i n e s , from the American Type C u l t u r e C o l l e c t i o n ( R o c k v i l l e , Maryland). As t h e r e were no e s t a b l i s h e d c e n t r a l mudminnow (Umbra  l i m i ) c e l l l i n e s these were i n i t i a t e d u s i n g the t i s s u e e x p l a n t t e c h n i q u e (Wolf and Quimby, 1976; Wolf, 1979). S e v e r a l l i v e mudminnows were ob t a i n e d from Mr. M. Rosenseld (Department o f Zoology, U n i v e r s i t y o f B r i t i s h Columbia). The l i v e r , s p l e e n , f i n s , h e a r t , swim b l a d d e r , and kidney were removed under s t e r i l e c o n d i t i o n s and t r a n s f e r r e d t o a s a l i n e s o l u t i o n c o n t a i n i n g a n t i b i o t i c s (83 ug/ml polymyxin B, 800 ug/ml b a c i t r a c i n , 500 ug/ml neomycin), p r i o r t o p l a t i n g i n s e v e r a l 25cm 2 f l a s k s ( F a l c o n P l a s t i c s ) . A f t e r a 60 min attachment p e r i o d , MEM (Eagle's minimal e s s e n t i a l medium, Grand I s l a n d B i o l o g i c a l Company) supplemented w i t h 20% f e t a l c a l f serum was added and subsequently r e p l a c e d a t two week i n t e r v a l s . Though i n i t i a l l y slow, t h e i r growth r a t e g r a d u a l l y i n c r e a s e d and a f t e r 6-9 months f i n (Ul-F) and h e a r t (Ul-H) c e l l l i n e s were f i r m l y e s t a b l i s h e d , b) C e l l c u l t u r e s A l l c e l l l i n e s were grown i n 75cm 2 p l a s t i c f l a s k s ( F a l c o n P l a s t i c s ) u s i n g MEM, a n t i b i o t i c s (streptomycin s u l f a t e , 29.5 ug/ml; kanamycin, 100 ug/ml; fungizone, 2.5 ug/ml), and sodium b i c a r b o n a t e (5 ml/800 ml medium f o r f i s h c e l l s ; 13.5 ml/800 ml medium f o r mammalian c e l l s ) . F i s h s t o c k c u l t u r e s -6-were maintained i n sealed flasks at 18 C while mammalian stocks were grown at 37 C i n a water-saturated C0 2 incubator. For experiments, c e l l s were seeded onto coverslips (mammalian c e l l s , 22mm2 coverslips i n Falcon P l a s t i c 3.5cm diameter p l a s t i c dishes; f i s h c e l l s , 10mm x 35mm cover s l i p s i n stoppered Bellco Leighton tubes) and maintained i n MEM supplemented with 10% serum. c) DNA quantitation The r e l a t i v e DNA content per c e l l i n each of the c e l l l i n e s was measured using the fluorescent s t a i n propidium iodide and laser-based flow microfluorimetry (Krishan, 1975; Fr i e d et a l . , 1976). Each c e l l culture was tryps i n i z e d to obtain a s i n g l e - c e l l suspension which was fi x e d i n 50% methanol then resuspended i n ribonuclease (1 mg/ml i n 0.2 M PBS, pH 7.0; Type III-A from bovine pancreas, Sigma Chemical Co., St. Louis, Missouri). This was replaced with a propidium iodide-sodium c i t r a t e solution (0.05 mg P.I./ml i n 0.1% NaCit) and a f t e r a 20 min staining period the c e l l s were resuspended i n 0.2 M phosphate buffer (pH 7.0). Aliquots were drawn into a Coultar Epics V (Florida) flow microfluorimeter where the r e l a t i v e DNA content was measured and the data p l o t t e d by computer. d) Preparation of S9 The S9 mixture was prepared as described by Ames et a l . (1975). Fischer male rats (150-250 g; Charles River Breeding Laboratory, St. Constant, Quebec) and rainbow trout (150-250 g; Sun Va l l e y Trout Farm, Mission, B r i t i s h Columbia) were i n j e c t e d i n t r a p e r i t o n e a l l y with Aroclor 1254 (500 mg/kg). -7-Rainbow t r o u t were a l s o i n j e c t e d (5 mg/kg) wit h an e x t r a c t o f crude o i l (W. Penrose, Argonne N a t i o n a l Laboratory, Argonne, I l l i n o i s , p e r s o n a l communication). The e x t r a c t was prepared by v o r t e x i n g 10 ml of crude o i l w i t h an equal volume o f di m e t h y l s u l f o x i d e : w a t e r , which was removed and e x t r a c t e d w i t h hexane. The hexane was v o l a t i l i z e d i n a r o t a r y evaporator and the r e m a i n i n g t a r r y r e s i d u e used f o r the i n j e c t i o n s . The l i v e r s were e x c i s e d 5 days l a t e r , r i n s e d w i t h c o l d 0.15 M KC1, poole d , and homogenized f o r 30-60 seconds i n a p o l y t r o n t i s s u e homogenizer (Brinkman Instruments, Rexdale, Ontario) u s i n g l g l i v e r / 3 ml o f c o l d 0.15 M KC1. The homogenate was c e n t r i f u g e d i n a r e f r i g e r a t e d c e n t r i f u g e (1-3 C) f o r 15 min a t 9000 g. 5ml a l i q u o t s o f the supernatant S9 f r a c t i o n were p i p e t t e d i n t o p o l y p r o p y l e n e tubes (Falcon P l a s t i c s ) and s t o r e d a t -80 C. The S9 mixture f o r t h e DNA r e p a i r experiments c o n t a i n e d 200 u l / d i s h o f S9 and the enzyme c o f a c t o r s NADP (1.8 mg/dish), G6P (2.7 mg/dish), and NADPH (0.6 mg/dish) d i s s o l v e d i n 0.3 ml PBS. F o r t h e chromosome a b e r r a t i o n experiments the S9 mixture c o n t a i n e d 50 u l / d i s h o f S9 and t h e c o f a c t o r s NADP (1.4 mg/dish) and G6P (0.7 mg/dish) i n i t i a l l y d i s s o l v e d i n Hepes b u f f e r . e) DNA r e p a i r s y n t h e s i s F o r each experiment, a p p r o x i m a t e l y 20,000 HF c e l l s (passage 5-7), 60,000 CHO c e l l s ( c o n t i n u o u s l y c u l t u r e d ) , and 80,000 c e l l s f o r each f i s h c e l l l i n e (RTG, passage 70-74; RTO, passage 15-18; CH, passage 19-22; FHM, passage 88-91; Ul-H, passage 9; U l - F , passage 11) were seeded onto c o v e r s l i p s and kept i n MEM supplemented w i t h 10% serum u n t i l t he c e l l s -8-were about 50% c o n f l u e n t . C e l l d i v i s i o n was i n h i b i t e d by a r g i n i n e - d e p r i v a t i o n i n o r d e r t o p r e v e n t f a l s e p o s i t i v e s r e s u l t i n g from the i n c o r p o r a t i o n of 3HTdR i n t o DNA d u r i n g r e p l i c a t i o n . The c e l l s were t r a n s f e r r e d i n t o a r g i n i n e -d e f i c i e n t medium (ADM) supplemented w i t h 2.5% serum and i n c u b a t e d f o r a f u r t h e r 4-5 days. ADM w i t h 2.5% serum was used t o d i l u t e t h e t e s t c h e m i c a l s . 3HTdR was a l s o d i l u t e d w i t h ADM t o a t t a i n a working c o n c e n t r a t i o n o f 10 uCi/ml. In an experiment the c u l t u r e medium was removed and 1.0 ml o f 3HTdR and t e s t c h e m i c a l added f o r the treatment p e r i o d . Where enzyme a c t i v a t i o n was r e q u i r e d , a 0.5 ml a l i q u o t o f S9 mixture (S9 p l u s c o f a c t o r s ) was added t o 0.5 ml o f the t e s t chemical and 1.0 ml 3HTdR. Upon completion of the chemical treatment, the c e l l s were t r e a t e d w i t h sodium c i t r a t e o r potassium c h l o r i d e (mammalian c e l l s , 1% N a C i t ; Ul-H and U l - F , 0.4% K C l ; a l l o t h e r f i s h c e l l l i n e s , 0.5% NaCit) f o r 10 min, immediately f o l l o w e d by f i x a t i o n i n e t h a n o l / a c e t i c a c i d (3:1) f o r 10-20 min. A i r -d r i e d c o v e r s l i p s were mounted on s l i d e s u s i n g p a r a f f i n and a u t o r a d i o g r a p h i c a l l y processed u s i n g an 18 o r 30 day exposure t o Kodak N u c l e a r Track Emulsion (NTB-3; Eastman Kodak, Rochester, New Y o r k ) . A f t e r d e v e l o p i n g the c e l l s were s t a i n e d w i t h 2% a c e t o - o r c e i n (50:50, a c e t i c acid/water) and a second c o v e r s l i p mounted wi t h Permount. DNA r e p a i r s y n t h e s i s was measured as t h e mean number o f s i l v e r g r a i n s over n u c l e i . The background g r a i n count, which v a r i e d from approximately 0 t o 5 g r a i n s , was s u b t r a c t e d from the n u c l e a r g r a i n count. A t l e a s t -9-30 n u c l e i were scored f o r each data p o i n t which were p l o t t e d w i t h a standard d e v i a t i o n on the mean. f) Chromosome a b e r r a t i o n s 150,000 Ul-H, 40,000 CHO, or 80,000 HF c e l l s were seeded per c o v e r s l i p and grown t o 60-80% c o n f l u e n c y . For t e s t chemical treatments the c u l t u r e medium was removed and 1.0 ml of chemical i n MEM with 2.5% serum added. Enzyme a c t i v a t i o n was p r o v i d e d as r e q u i r e d by adding 0.5 ml of an S9 mixture (S9 p l u s c o f a c t o r s ) t o 0.5 ml o f the t e s t c h e m i c a l . F o l l o w i n g a 3h treatment p e r i o d the c e l l s were r i n s e d w i t h MEM then i n c u b a t e d i n 1.5 ml MEM w i t h 10% serum (20% f o r Ul-H) f o r 2Oh (Ul-H, CHO) o r 28h (HF) . 4h p r i o r t o sampling 0.1 ml o f c o l c h i c i n e was added. The c o v e r s l i p s w i t h c e l l s were then p r o c e s s e d and f i x e d as noted i n the s e c t i o n on DNA r e p a i r methods. A i r - d r i e d c o v e r s l i p s were s t a i n e d w i t h 2% a c e t o -o r c e i n (50:50 a c e t i c a c i d : water), dehydrated, and mounted on s l i d e s w i t h Permount. In g e n e r a l , 100 metaphase p l a t e s p e r c o v e r s l i p were analyzed f o r chromosome breaks and exchanges, the most common forms o f a b e r r a t i o n s observed i n chromosome p r e p a r a t i o n s from these c e l l l i n e s . g) M i c r o n u c l e i 120,000 Ul-H, 120,000 U l - F , 14,000 CHO, o r 30,000 HF c e l l s were seeded onto c o v e r s l i p s and grown t o 20-30% c o n f l u e n c y i n MEM w i t h 10% serum. The c o v e r s l i p s o f c e l l s were then t r e a t e d w i t h a t e s t chemical i n MEM w i t h 2.5% serum f o r 3h f o l l o w i n g the chromosome a b e r r a t i o n assay methodology. A f t e r the treatment p e r i o d the chemical was r i n s e d from the c o v e r s l i p s w i t h MEM and the c e l l s were i n c u b a t e d i n MEM w i t h -10-10% serum u n t i l sampled a t 16, 48, 80, 112, and 144h. C o v e r s l i p s were processed and f i x e d as noted i n the s e c t i o n on DNA r e p a i r methods. A i r - d r i e d c o v e r s l i p s were s t a i n e d w i t h a c e t o - o r c e i n , dehydrated, and mounted on s l i d e s . In g e n e r a l , f o r each data p o i n t 1000 c e l l s were examined f o r the presence o f m i c r o n u c l e i . 3. Primary C e l l s a) I s o l a t i o n o f the c e l l s L i v e r , stomach, and i n t e s t i n e were removed from 150- t o 200- g rainbow t r o u t . U s i n g a s y r i n g e , t h e stomach and i n t e s t i n e were f l u s h e d w i t h c o o l , s t e r i l e b a l a n c e d s a l t s o l u t i o n (BSS) supplemented w i t h a n t i b i o t i c s (polymyxin B, 83 mg/1; b a c i t r a c i n , 800 mg/1; neomycin, 5 mg/1). The l i v e r was r i n s e d w i t h BSS, then minced w i t h 5 ml BSS i n a s t e r i l e p l a s t i c d i s h ( F a l c o n P l a s t i c s ) . The stomach and i n t e s t i n e were c u t l o n g i t u d i n a l l y t o expose the mucosa. Each t i s s u e was p l a c e d i n t o a s e a l e d s t e r i l e p o l ycarbonate Erlenmeyer f l a s k c o n t a i n i n g 10 ml BSS w i t h 0.5% pronase, and i n c u b a t e d a t 20 C f o r 45 t o 60 min w i t h o c c a s i o n a l a g i t a t i o n t o l o o s e n the c e l l s . F o l l o w i n g the i n c u b a t i o n p e r i o d , the s o l u t i o n was p i p e t t e d o f f and the remaining t i s s u e washed w i t h BSS. The enzyme s o l u t i o n and washings were p o o l e d and passed through a p i e c e o f s t e r i l e gauze t o remove l a r g e t i s s u e p i e c e s . The c e l l s u s p e n s i o n s were c e n t r i f u g e d a t 600 rpm f o r 5 min, resuspended i n 6 ml ADM w i t h 2.5% serum, and then c e n t r i f u g e d and resuspended i n f r e s h ADM w i t h 2.5% serum. C e l l v i a b i l i t y was t e s t e d a t 0 and 6 hours u s i n g the t r y p a n b l u e e x c l u s i o n t e c h n i q u e . -11-b) DNA r e p a i r s y n t h e s i s with primary c e l l s ADM w i t h 2.5% serum and 3HTdR ( f i n a l working c o n c e n t r a t i o n 10 uCi/ml) was used f o r making the t e s t chemical d i l u t i o n s . 1 ml a l i q u o t s o f c e l l s u s p e n s i o n and chemical: 3HTdR were added t o 3.5 cm p l a s t i c d i s h e s ( F a l c o n P l a s t i c s ) , which were p l a c e d i n a s e a l e d C 0 2 - f l u s h e d (5% CO z i n a i r ) c o n t a i n e r a t 18-20 C f o r 6h. At the end o f the treatment p e r i o d t h e c e l l s were p i p e t t e d i n t o t e s t tubes, c e n t r i f u g e d , and resuspended i n e t h a n o l : a c e t i c a c i d (3:1). A f t e r 10 min the c e l l s were a g a i n c e n t r i f u g e d and a l l but approximately 0.2 ml o f the f i x a t i v e removed. The remaining f i x a t i v e was used t o resuspend the c e l l s , which were dropped onto microscope s l i d e s . A i r - d r i e d s l i d e s were co a t e d w i t h Kodak Nuc l e a r Track Emulsion (NTB-3) and kept i n d e s i c c a t e d l i g h t - t i g h t boxes a t 4 C f o r 30 days. A f t e r d e v e l o p i n g , t h e c e l l s were s t a i n e d w i t h 2% a c e t o - o r c e i n and t h e amount o f DNA r e p a i r determined. The background g r a i n count was e s t i m a t e d from an e q u i v a l a n t area a d j a c e n t t o each n u c l e u s and s u b t r a c t e d from i t s count. DNA r e p a i r s y n t h e s i s was measured as t h e mean number o f s i l v e r g r a i n s o v e r a t l e a s t 30 n u c l e i f o r each d a t a p o i n t . These were p l o t t e d w i t h a st a n d a r d d e v i a t i o n on the mean. 4. T i s s u e S l i c e s DNA r e p a i r s y n t h e s i s i n t i s s u e s l i c e s S t a r r y f l o u n d e r ( P l a t l c h t h y s s t e l l a t u s ) w i t h epidermal p a p i l l o m a s were c o l l e c t e d by beach s e i n e from B e l l i n g h a m Bay (December, 1980) and kept i n an aquarium a t t h e Cancer -12-Research Center. F i s h s e l e c t e d f o r the experiments were k i l l e d by concussion and t h e i r p apillomas removed. S l i c e s of the p a p i l l o m a s were p l a c e d i n 3.5 cm p l a s t i c d i s h e s (Falcon P l a s t i c s ) w i t h 1.0 ml of t e s t chemical i n 2.5% ADM and 3HTdR (10 u C i / m l ) . The d i s h e s were p l a c e d i n a s e a l e d C 0 2 ~ f l u s h e d (5% C 0 2 i n a i r ) c o n t a i n e r a t 18-2 0 C f o r a 6h treatment p e r i o d . The t i s s u e s l i c e s were then removed, f i x e d i n f o r m a l i n , and h i s t o l o g i c a l l y processed. 2, 5, and 10 micron s e c t i o n s mounted on s l i d e s were coated w i t h Kodak Nuc l e a r T r a c k Emulsion (NTB-3) and h e l d i n l i g h t - t i g h t boxes a t 4 C f o r 30 days. A f t e r d e v e l o p i n g , t h e t i s s u e s e c t i o n s were s t a i n e d u s i n g hematoxylin and e o s i n , and the c e l l s examined f o r evidence of DNA r e p a i r . 5. Sediments C o l l e c t i o n , p r e p a r a t i o n , and a s s a y i n g of sediment samples Sediment samples were c o l l e c t e d from the Sturgeon Bank-Iona I s l a n d j e t t y area on October 31, 1983, and t h e Spanish Bank area on November 20, 1983 ( F i g u r e 1 ). A t each o f t h e 5 Sturgeon Bank and 3 Spanish Bank sampling l o c a t i o n s t h e top 5 cm o f sediment from t h r e e 30 cm 2 areas was removed and p l a c e d i n p l a s t i c bags. The sediment samples were f r o z e n a t -20 C u n t i l p r ocessed. A f t e r thawing the sediments f o r a p a r t i c u l a r l o c a t i o n , approximately equal p o r t i o n s o f t h e l o c a t i o n ' s t h r e e sediment samples were combined and f r e e z e - d r i e d . 500 g o f a f r e e z e - d r i e d uniform sample was then p l a c e d i n a c l e a n g l a s s b o t t l e , and e x t r a c t e d t h r e e times w i t h 500 ml p o r t i o n s o f methylene c h l o r i d e (HPLC grade, F i s h e r C h e m i c a l s ) . Suspended -13-FIGURE 1 L o c a t i o n o f t h e f i s h and sediment c o l l e c t i o n s i t e s a t Sturgeon and Spanish Banks (Greater Vancouver Sewerage and Drainage D i s t r i c t , 1983). -14-V A N C O U V E R 0 S km* I i Km ' C - S T U R G E O N BANK SAMPLING LOCATIONS VANCOUVER Area lono Island Sewage Treatment Plant Point Of Discharge Sediment Collection Locations Sturgeon Bank S E A ISLANO Scale km -15-p a r t i c l e s were removed from the combined s u p e r n a t a n t s by s e t t l i n g and f i l t r a t i o n (Whatman #1 f i l t e r ) . The methylene c h l o r i d e was removed with a r o t a r y e v a p o r a t o r and the r e s i d u e d i s s o l v e d i n d i m e t h y l s u l f o x i d e . The e x t r a c t s were s t o r e d a t -80 C u n t i l t e s t e d f o r genotoxic a c t i v i t y i n the DNA r e p a i r and chromosome a b e r r a t i o n assays, f o l l o w i n g the p r e v i o u s l y d i s c u s s e d methodology. 6. F l a t f i s h C o l l e c t i o n o f f l a t f i s h and examination f o r m i c r o n u c l e i F i s h were c o l l e c t e d by beach s e i n e from Sturgeon Bank on October 30, 1983, and Spanish Bank on November 19, 1983 ( F i g u r e 1 ) . S t a r r y f l o u n d e r ( P l a t i c h t h y s s t e l l a t u s ) , t h e predominant s p e c i e s found, were t r a n s p o r t e d t o t h e l a b o r a t o r y and h e l d i n a q u a r i a . W i t h i n 72h f o l l o w i n g t h e i r c o l l e c t i o n , 25 f i s h from Sturgeon Bank ( l e n g t h , 16.5 t o 28 cm) and 22 from Spanish Bank ( l e n g t h , 15.5 t o 25 cm) were k i l l e d and c e l l samples from the mouth, l i v e r , and b l o o d o b t a i n e d . The bottom o f t h e mouth was c u t back t o t h e operculum t o expose t h e b u c c a l e p i t h e l i u m . By drawing a s c a l p e l b l a d e a c r o s s t h e e p i t h e l i a l s u r f a c e , c e l l s were removed which were t h e n spread on s l i d e s . C e l l s were i s o l a t e d from the l i v e r f o l l o w i n g t h e procedure d e s c r i b e d i n the s e c t i o n on the i s o l a t i o n o f p r i m a r y c e l l s methods. Blood samples were o b t a i n e d by removing t h e t a i l and c o l l e c t i n g drops o f b l o o d on a s l i d e . L i v e r and b l o o d c e l l suspensions dropped onto s l i d e s were drawn i n t o a f a i r l y u n i f o r m c e l l l a y e r by p a s s i n g a second s l i d e a c r o s s i t s s u r f a c e . A i r - d r i e d s l i d e s were s t a i n e d by t h e p e r i o d i c a c i d -16-S c h i f f method and c o u n t e r s t a i n e d w i t h f a s t green. A c o v e r s l i p was a t t a c h e d w i t h Permount. 500-1000 b u c c a l e p i t h e l i a l , l i v e r , o r r e d b l o o d c e l l s were examined f o r m i c r o n u c l e i . -17-RESULTS A. DNA Repair S y n t h e s i s 1. Repair i n C u l t u r e d C e l l s a) C e l l l i n e comparison S i g n i f i c a n t l e v e l s o f DNA r e p a i r were observed f o l l o w i n g exposure o f a l l mammalian and f i s h c e l l l i n e s t o MNNG, 4NQ0, NA2AAF, and r a t S9 a c t i v a t e d AFB 1 ( F i g u r e 2 ) . Dose-dependent responses were observed w i t h a l l exposures, the l e v e l o f response v a r y i n g i n the o r d e r HF > CHO > a l l the f i s h c e l l l i n e s . DNA r e p a i r s y n t h e s i s was observed t o peak i n a l l o f the c e l l l i n e s a t approximately the same c o n c e n t r a t i o n o f a p a r t i c u l a r c hemical, and t h e r e p a i r magnitude was dependent on th e t e s t chemical used. C l o s e r examination o f the f i s h c e l l dose-response curves ( F i g u r e 3) i n d i c a t e s r e l a t i v e l y l i t t l e v a r i a t i o n between l i n e s w i t h r e g a r d t o s e n s i t i v i t y o r maximal res p o n s e . Only nominal DNA r e p a i r a c t i v i t y was d e t e c t e d f o l l o w i n g exposure t o n o n - a c t i v a t e d AFB^ ( F i g u r e 4 ) . b) I n v e s t i g a t i o n i n t o the low f i s h c e l l DNA r e p a i r response A number o f f a c t o r s which c o u l d account f o r t h e apparent low f i s h c e l l DNA r e p a i r response were examined. To ensure t h a t g r a i n count d i f f e r e n c e s were not s o l e l y due t o p o s s i b l e p o i k i l o t h e r m i c : homeothermic membrane d i f f e r e n c e s r e s u l t i n g i n v a r i a t i o n i n t e s t c h e m i c a l uptake, t h e amount o f r e p a i r f o l l o w i n g i r r a d i a t i o n w i t h u l t r a v i o l e t l i g h t was measured (Table 1) . These data confirmed t h e t e s t c h e m i c a l d a t a which showed th e magnitude o f t h e DNA r e p a i r response v a r i e d HF > CHO > RTG. - 1 8 -FIGURE 2 DNA r e p a i r s y n t h e s i s measured i n c u l t u r e d HF ( • ) , CHO ( v ) , RTG ( • ) , RTO ( T ) , FHM ( • ) , and CH ( • ) c e l l s exposed t o (A) MNNG, (B) 4NQO, (C) NA2AAF, and (D) r a t S9 a c t i v a t e d AFB 1. Assay c o n d i t i o n s : 37 C (mammalian c e l l s ) and 18 C ( f i s h c e l l s ) , 3h t e s t chemical treatment p e r i o d , emulsion-coated s l i d e s kept i n l i g h t - t i g h t boxes f o r 18 days p r i o r t o d e v e l o p i n g . Mean g r a i n counts p l o t t e d w i t h s t a n d a r d d e v i a t i o n s . -19-FIGURE 3 The d a t a from FIGURE 2 f o r the DNA r e p a i r s y n t h e s i s i n c u l t u r e d RTG ( • ) , RTO ( Y ) , FHM ( • ) , and CH ( A ) c e l l s exposed t o (A) MNNG, (B) 4NQO, (C) NA2AAF, and (D) AFB, r e p l o t t e d t o examine the d i f f e r e n c e i n r e p a i r magnitude between t h e f i s h c e l l l i n e s . -21-G R A I N S P E R N U C L E U S FIGURE 4 DNA r e p a i r s y n t h e s i s measured i n c u l t u r e d HF ( •) , CHO ( v ) , RTG ( • ) , RTO ( T ) , FHM ( • ), and CH ( A ) c e l l s exposed t o n o n - a c t i v a t e d AFB^. Assay c o n d i t i o n s : 18 C ( f i s h c e l l s ) and 37 C (mammalian c e l l s ) , 3h t e s t c h e m i c a l treatment, emulsion-coated s l i d e s kept i n l i g h t - t i g h t boxes f o r 18 days p r i o r t o d e v e l o p i n g . Mean g r a i n counts p l o t t e d w i t h the st a n d a r d d e v i a t i o n on the mean. -23-G R A I N S P E R N U C L E U S i I > n j i y O, -\—m~*-<D9—< l\ O J L . o, - i \\ M X X 1\N\ \\ / I / \ \ / \ \ M \ KJ O -I 1/ o,- 1 LO TABLE 1 DNA r e p a i r s y n t h e s i s i n HF, CHO, and RTG c e l l s exposed t o u l t r a v i o l e t l i g h t . Assay c o n d i t i o n s : i r r a d i a t e d c o v e r s l i p s i n c u b a t e d a t 18 C f o r 3 h, emulsion-coated s l i d e s kept i n l i g h t - t i g h t boxes f o r 18 days p r i o r t o d e v e l o p i n g . -25-Grains per NucLeus ± Standard Deviation uv uose 2 (ergs/mm ) RTG CHO HF 80 38 ± 7 Saturated Saturated 60 29 ± 6 86 ± 7 Saturated . 40 22 + 3 58 ± 8 192 ± 18 20 17 ± 3 23 ± 4 147 + 12 0 2 + 2 2 + 2 2 + 2 - 2 6 -As t he low amount o f f i s h c e l l DNA r e p a i r c o u l d be e x p l a i n e d by a de l a y e d o r prolonged r e p a i r response, t h e d u r a t i o n o f r e p a i r f o l l o w i n g t e s t c h e m i c a l t r e a t m e n t was i n v e s t i g a t e d . S i m i l a r time course responses were found a f t e r t r e a t i n g HF, CHO, and RTG c e l l s w i t h MNNG o r 4NQ0 ( F i g u r e 5 ) . The l e v e l o f r e p a i r peaked a t approximately 3h, then f e l l o f f r a p i d l y , a t t a i n i n g low, e s s e n t i a l l y background l e v e l s by 16-20h. The c e l l l i n e v a r i a t i o n i n r e p a i r magnitude c o u l d simply be t h e r e s u l t o f the amount o f DNA " t a r g e t " . However, the r e l a t i v e DNA q u a n t i t y per c e l l i n d i c a t e d o n l y 10 t o 15% l e s s DNA i n the f i s h than i n the mammalian c e l l l i n e s , c) I n c r e a s i n g the f i s h c e l l DNA r e p a i r response I t was d e c i d e d t h a t o n l y i f a f i s h c e l l r e p a i r response i n excess o f 50 g r a i n s per nucleus c o u l d be ach i e v e d , would the assay be deemed s u f f i c i e n t l y s e n s i t i v e t o be worth p u r s u i n g . S e v e r a l f a c t o r s were c o n s i d e r e d which c o u l d p o t e n t i a l l y improve the r e p a i r response. Extending the c e l l s ' i n c u b a t i o n p e r i o d i n 3HTdR ( F i g u r e 6) t o 6h f o l l o w i n g MNNG treatment r e s u l t e d i n h i g h e r g r a i n counts. Presumably a lo n g e r i n c u b a t i o n p e r i o d p e r m i t s more DNA t o be r e p a i r e d and hence a g r e a t e r i n c o r p o r a t i o n o f 3HTdR. No t o x i c e f f e c t s were observed w i t h t he l o n g e r 3HTdR i n c u b a t i o n time. G r a i n count i n c r e a s e s are not p r o p o r t i o n a l t o i n c r e a s e s i n 3HTdR i n c u b a t i o n time. T h i s i s l i k e l y a consequence o f the r a p i d l y f a l l i n g l e v e l o f r e p a i r f o l l o w i n g t e s t chemical treatment (Figure 5). Assay temperature was found t o i n f l u e n c e g r a i n p r o d u c t i o n -27-FIGURE 5 D u r a t i o n o f DNA r e p a i r s y n t h e s i s i n c u l t u r e d HF ( o ) , CHO ( v ) , and RTG ( • ) c e l l s exposed t o (A) MNNG o r (B) 4NQO. The c e l l s sampled a t 3h were s i m u l t a n e o u s l y t r e a t e d w i t h the t e s t chemical and HTdR f o r the i n i t i a l 3h segment, w h i l e the r e s t o f the c e l l s were t r e a t e d w i t h o n l y the ch e m i c a l f o r t h i s i n i t i a l p e r i o d , then l a t e r p u l s e d w i t h HTdR f o r 3h. Assay c o n d i t i o n s : 37 C (mammalian c e l l s ) and 18 C ( f i s h c e l l s ) , emulsion-coated s l i d e s kept i n l i g h t -t i g h t boxes f o r 18 days p r i o r t o d e v e l o p i n g . Mean g r a i n c o u n t s p l o t t e d w i t h a standard d e v i a t i o n on the mean. -28-FIGURE 6 E f f e c t o f i n c r e a s i n g HTdR treatment time (• , 30 min; • , 60 min; A , 90 min; • , 100 min; O , 360 min) on DNA r e p a i r i n c u l t u r e d RTG c e l l s f o l l o w i n g a 30 min MNNG treatment. Assay-c o n d i t i o n s : 18 C, emulsion-coated s l i d e s kept i n l i g h t - t i g h t boxes f o r 18 days p r i o r t o development. Mean g r a i n counts p l o t t e d w i t h a standard d e v i a t i o n on the mean. FIGURE 7 RTG c e l l s exposed t o emulsion f o r v a r y i n g p e r i o d s o f time up t o 28 days (7 da y s , * ; 14 d a y s , " ; 21 days,A ; 28 days, T ) p r i o r t o d e v e l o p i n g . Assay a t 18 C. Mean g r a i n counts p l o t t e d w i t h a s t a n d a r d d e v i a t i o n on t h e mean. -30-GRAINS PER NUCLEUS GRAINS PER NUCLEUS ( F i g u r e 8) . Conducting the assay over a temperature range from 4 t o 25 C i n d i c a t e d maximum r e p a i r s y n t h e s i s a t 25 C. C e l l death from the temperature change was not observed d u r i n g t h e experiment. A number of k a r y o r r h e x i c n u c l e i were observed i n RTG c e l l s maintained f o r s e v e r a l passages a t 25 C, p o s s i b l y i n d i c a t i n g t h a t the s t a b i l i t y o f t h e i r DNA may be i n f l u e n c e d by temperature. I n c r e a s i n g the d u r a t i o n of exposure t o t h e emulsion a l s o i n c r e a s e d n u c l e a r g r a i n counts ( F i g u r e 7) . A l o n g e r emulsion exposure p e r i o d p e rmits more 3HTdR t o decay and r e a c t w i t h the emulsion t o produce more g r a i n s over the n u c l e u s . As the h a l f - l i f e o f 3HTdR i s approximately 12 y e a r s (Rogers, 1979) t h e r a t e o f 3HTdR decay should be c o n s t a n t o v e r the 30 day exposure p e r i o d used here. The r e s u l t s , as expected, show a p r o p o r t i o n a l g r a i n count i n c r e a s e w i t h i n c r e a s e s i n emulsion exposure time. I n c r e a s e d g r a i n p r o d u c t i o n was found when the 4NQ0 treatment was lengthened t o approximately 30 min ( F i g u r e 9) . Only a s l i g h t g r a i n count i n c r e a s e i s r e a l i z e d between 30 t o 60 min o f chemical treatment and the s l i g h t d e c r e a s e beyond 60 min may i n d i c a t e a t o x i c e f f e c t . Other f a c t o r s were i n v e s t i g a t e d , none o f which enhanced g r a i n p r o d u c t i o n i n the RTG c e l l s . I t was not known i f the n u t r i t i o n a l r e q u i r e m e n t s o f RTG c e l l s , w i t h p o s s i b l e subsequent e f f e c t s on DNA r e p a i r c a p a b i l i t y , would be i m p a i r e d by p r o l o n g e d c e l l maintenance i n ADM p r i o r t o t h e assay, o r t h e 7.5% lower serum c o n c e n t r a t i o n i n t h e ADM. However, v a r y i n g t h e c e l l d i v i s i o n a r r e s t time from 1 t o 7 days i n 2.5% -32-FIGURE 8 E f f e c t of assay temperature (4 C, • ; 11 C, • ; 18 C, A ; 25 C, T ) on DNA r e p a i r i n c u l t u r e d RTG c e l l s t r e a t e d s i m u l t a n e o u s l y w i t h 3HTdR and MNNG. Assay c o n d i t i o n s : c e l l s seeded a t 18 C wit h a 48h a c c l i m a t i z a t i o n p e r i o d p r i o r t o the experiment, emulsion-coated s l i d e s kept i n l i g h t - t i g h t boxes f o r 18 days p r i o r t o development. Mean g r a i n counts p l o t t e d w i t h a standard d e v i a t i o n . FIGURE 9 Increas e i n DNA r e p a i r i n RTG c e l l s a t 25 C w i t h i n c r e a s e d treatment time i n 5x10 M 4NQ0 ( A , e x p e r i m e n t a l ; • , c o n t r o l ) . 4NQO treatment was f o l l o w e d by a 6h ^ HTdR treatment, and a 30 day emulsion exposure. Mean g r a i n counts a re p l o t t e d . -33-G R A I N S P E R N U C L E U S G R A I N S P E R N U C L E U S 5, ADM ( F i g u r e 10) p r i o r t o MNNG exposure had no e f f e c t on the l e v e l o f RTG DNA r e p a i r , w h i l e v a r y i n g the ADM serum c o n c e n t r a t i o n from 0 t o 10% r e s u l t e d i n a s l i g h t lowering o f g r a i n counts ( F i g u r e 11) . Another f a c t o r which c o u l d have l i m i t e d RTG c e l l r e p a i r was l a c k o f 3HTdR uptake. But the 10 uCi/ml o f 3HTdR appears not t o be l i m i t i n g as g r a i n p r o d u c t i o n was not enhanced beyond 2.5 uCi/ml ( F i g u r e 12). That 3HTdR i s adequately p e n e t r a t i n g the c e l l s i s f u r t h e r evidenced by a few h e a v i l y g r a i n e d ( i e . i n S-phase) n u c l e i observed i n c o n t r o l p r e p a r a t i o n s . L a s t l y RTG c e l l c u l t u r e s d i f f e r i n g by over 30 passages, when t r e a t e d w i t h MNNG and 3HTdR, f a i l e d t o demonstrate a l o s s o f r e p a i r c a p a b i l i t y w i t h time i n c u l t u r e ( F i g u r e 13). By c o n d u c t i n g the DNA r e p a i r assay a t 25 C i n s t e a d o f 18 C, i n c r e a s i n g t h e 3HTdR treatment p e r i o d from 3 t o 6 h, and ex t e n d i n g the emulsion exposure p e r i o d from 18 t o 30 days, RTG c e l l g r a i n counts were i n c r e a s e d 2 t o 3 - f o l d and mean counts i n e x cess o f 50 g r a i n s were achieved ( F i g u r e 14). Background g r a i n counts were a l s o observed t o i n c r e a s e s l i g h t l y but the e f f e c t was minimized by u s i n g a low c e l l d e n s i t y on each c o v e r s l i p , and by thorough r i n s i n g o f the c o v e r s l i p d u r i n g the f i x a t i o n procedure, t o remove n o n - i n c o r p o r a t e d 3HTdR. d) E f f e c t of'DMSO on DNA r e p a i r s y n t h e s i s As DMSO was used f o r i n i t i a l d i s s o l v i n g o f t e s t c hemicals i t s e f f e c t on DNA r e p a i r was examined ( F i g u r e 15) . U l t r a v i o l e t l i g h t i r r a d i a t e d RTG c e l l s e x h i b i t e d l e s s r e p a i r a t f i n a l DMSO c o n c e n t r a t i o n s i n excess o f 1%. C e l l l o s s from c o v e r s l i p s was observed a t 5% and 10%, i n d i c a t i n g t h a t t he -35-FIGURE 10 DNA r e p a i r i n c u l t u r e d RTG c e l l s kept i n 2.5% ADM ( • , experimental;© , c o n t r o l ) f o r a p e r i o d v a r y i n g from 1 t o 7 days p r i o r t o a 6h simultaneous exposure t o 5 x l 0 _ 4 M MNNG and HTdR. Assay c o n d i t i o n s : 25 C, emulsion-coated s l i d e s kept i n l i g h t - t i g h t boxes f o r 30 days p r i o r t o dev e l o p i n g . Mean g r a i n counts are p l o t t e d . FIGURE 11 DNA r e p a i r i n c u l t u r e d RTG c e l l s g i v e n a 3h exposure t o HTdR and MNNG d i l u t e d w i t h ADM supplemented w i t h v a r y i n g c o n c e n t r a t i o n s o f f e t a l c a l f serum ( Y , 0% serum; •, 5% serum; A. , 7.5% serum; • , 10% serum). Assay c o n d i t i o n s : 18 C, emulsion-coated s l i d e s kept i n l i g h t - t i g h t boxes f o r 18 days. Mean g r a i n counts are p l o t t e d . -36-' GRAINS PER NUCLEUS GRAINS PER NUCLEUS —1 » * in uJj FIGURE 12 E f f e c t o f i n c r e a s i n g HTdR c o n c e n t r a t i o n (•, 2.5 uCi/ml;0 , 5 uC i / m l ; T , 10 uCi/ml; A , 15 uCi/ml; • , 20 uCi/ml) on DNA r e p a i r i n c u l t u r e d RTG c e l l s . Assay c o n d i t i o n s : simultaneous treatment w i t h HTdR and MNNG f o r 6h a t 25 C, emulsion-c o a t e d s l i d e s kept i n l i g h t - t i g h t boxes f o r 30 days. Mean g r a i n counts are p l o t t e d . FIGURE 13 E f f e c t o f c u l t u r e d RTG c e l l l i n e passage ( • , 63 P; • , 75 P; A , 82 P; Y , 96 P) on DNA r e p a i r . Assay c o n d i t i o n s : s i m ultaneous treatment w i t h HTdR and MNNG a t 25 C f o r 6h, emulsio n - c o a t e d s l i d e s kept i n l i g h t - t i g h t boxes f o r 30 days. Mean g r a i n counts are p l o t t e d . -38-GRAINS PER NUCLEUS FIGURE 14 Comparison o f DNA r e p a i r i n c u l t u r e d RTG c e l l s exposed t o MNNG ( • ) o r 4NQO ( • ) u t i l i z i n g (A) the o r i g i n a l e x p e r i m e n t a l c o n d i t i o n s (18 C assay temperature, 3h HTdR and t e s t chemical treatment, and 18 day emulsion exposure p e r i o d ) and (B) the c o n d i t i o n s found t o enhance g r a i n p r o d u c t i o n (25 C assay temperature, 6h HTdR and t e s t c h e m i c a l treatment, and 30 day emulsion exposure p e r i o d ) . Mean g r a i n counts p l o t t e d w i t h a standard d e v i a t i o n on t h e mean. -40-3 0 T 10H i UJ -I o 3 1 0 " 7 T 1 -\ 10"5 To"3 CONCENTRATION (M) S 1 0 0 - , CL E 8 0 -< o 6 0 H 404 20H 10"7 1ST • 1 B i d 5 i o 3 CONCENTRATION (M) -41-FIGURE 15 E f f e c t o f Increased d i m e t h y l s u l f o x i d e c o n c e n t r a t i o n on DNA r e p a i r s y n t h e s i s i n c u l t u r e d RTG c e l l s (O , experimental; • , c o n t r o l ! f o l l o w i n g u l t r a v i o l e t l i g h t exposure (100 ergs/mm 2). Assay c o n d i t i o n s : i r r a d i a t e d c e l l s i n c u b a t e d w i t h HTdR f o r 6h a t 25 C, emulsion-coated s l i d e s kept i n l i g h t -t i g h t boxes f o r 30 days. One standard d e v i a t i o n p l o t t e d on the mean g r a i n counts. -42-G R A I N S P E R N U C L E U S r e p a i r decrease may be the r e s u l t of t o x i c i t y . The f i n a l DMSO c o n c e n t r a t i o n i n the c u l t u r e medium f o r experiments t h e r e f o r e d i d not exceed 1%. e) Use o f rainbow t r o u t S9 i n the r e p a i r assay Because the m e t a b o l i c s p e c i f i c i t y o f f i s h S9 i s unknown, two types o f rainbow t r o u t h e p a t i c S9 were used t o a c t i v a t e AFB^ and e s t a b l i s h a dose-response curve ( F i g u r e 16) . Over the AFB^ c o n c e n t r a t i o n range t e s t e d , both S9 types ( i e . from A r o c l o r 1254 o r crude o i l e x t r a c t i n j e c t e d f i s h ) produced a p p r o x i m a t e l y the same l e v e l o f DNA r e p a i r . The q u a n t i t y of f i s h S9 r e q u i r e d t o a c t i v a t e AFB^ and generate DNA r e p a i r was t h e n o p t i m i z e d f o r the RTG c e l l s ( F i g u r e 17) . As the amount o f r e p a i r s y n t h e s i s was i n c r e a s e d v e r y l i t t l e beyond 200 u l S9/ml o f r e a c t i o n mixture t h i s S9 q u a n t i t y was then r o u t i n e l y used. Rat S9 i s a l s o r o u t i n e l y used a t t h i s c o n c e n t r a t i o n i n our l a b o r a t o r y f o r the DNA r e p a i r assay. Rat and f i s h S9 were compared f o r t h e i r a b i l i t y t o a c t i v a t e AFB^ (at 25 C) and cause DNA r e p a i r i n RTG and HF c e l l s ( F i g u r e 18, 19). The two f i s h S9s were found t o perform s l i g h t l y b e t t e r than the r a t S9 w i t h both c e l l l i n e s , w h i l e the amount o f r e p a i r w i t h a l l S9s was g r e a t e r i n t h e HF c e l l s . f ) DNA r e p a i r s y n t h e s i s f o l l o w i n g treatment w i t h PAHs P o l y c y c l i c aromatic hydrocarbons are an important c l a s s o f a q u a t i c contaminants o f which some, such as BP, DBA, and BA, a r e known c a r c i n o g e n s ( N a t i o n a l Academy of S c i e n c e s , 1972; IARC Monograph, 1973). DNA r e p a i r f o l l o w i n g treatment w i t h PAHs was s t u d i e d u s i n g S9 from rainbow t r o u t t o a c t i v a t e t h e t e s t c h e m i c a l s . -44-FIGURE 16 DNA r e p a i r s y n t h e s i s i n c u l t u r e d RTG c e l l s exposed t o AFB^ a c t i v a t e d u s i n g rainbow t r o u t S9 ( • , S9 from o i l e x t r a c t -i n j e c t e d f i s h ; S9 from A r o c l o r 1 2 5 4 - i n j e c t e d f i s h ) . Assay c o n d i t i o n s : 25 C, emulsion-coated s l i d e s kept i n l i g h t - t i g h t boxes f o r 30 days. Mean g r a i n counts p l o t t e d w i t h a s t a n d a r d d e v i a t i o n on the mean. FIGURE 17 DNA r e p a i r response i n c u l t u r e d RTG c e l l s exposed t o l x l O ~ 4 M AFB^ a c t i v a t e d w i t h i n c r e a s i n g amounts o f rainbow t r o u t S9 ( • , S9 from o i l e x t r a c t - i n j e c t e d f i s h ; • , S9 from A r o c l o r 1 2 5 4 - i n j e c t e d f i s h ) . Assay c o n d i t i o n s : 25 C, e m u l s i o n - c o a t e d s l i d e s kept i n l i g h t - t i g h t boxes f o r 30 days. Mean g r a i n counts p l o t t e d w i t h a standard d e v i a t i o n on t h e mean. -45-FIGURE 18 DNA r e p a i r response measured i n c u l t u r e d HF c e l l s f o l l o w i n g exposure t o AFB 1 a c t i v a t e d w i t h S9 from o i l e x t r a c t - i n j e c t e d rainbow t r o u t ( • ), A r o c l o r 1254-injected rainbow t r o u t ( • ) , o r A r o c l o r 1 2 5 4 - i n j e c t e d r a t s ( • ) . Assay c o n d i t i o n s : 30 min t e s t c h e m i c a l treatment a t 25 C was f o l l o w e d by a 3h i n c u b a t i o n w i t h HTdR a t 37 C,emulsion-c o a t e d s l i d e s kept i n l i g h t - t i g h t boxes f o r 18 days. Mean g r a i n counts p l o t t e d w i t h a standar d d e v i a t i o n on the mean. FIGURE 19 DNA r e p a i r i n c u l t u r e d RTG c e l l s t r e a t e d w i t h AFB^ a c t i v a t e d w i t h S9 from o i l e x t r a c t - i n j e c t e d rainbow t r o u t ( A ) , A r o c l o r 1 2 5 4 - i n j e c t e d rainbow t r o u t ( • )/or A r o c l o r 1254-i n j e c t e d r a t s ( • ). Assay c o n d i t i o n s : 30 min t e s t c h e m i c a l treatment, f o l l o w e d by 3h i n c u b a t i o n w i t h 3HTdR a t 25 C. Em u l s i o n - c o a t e d s l i d e s kept i n l i g h t - t i g h t boxes f o r 18 days. Mean g r a i n counts p l o t t e d w i t h a standar d d e v i a t i o n on t h e mean. -47-The r e p a i r assay was f i r s t conducted with both HF and RTG c e l l s u s i n g the o r i g i n a l assay c o n d i t i o n s i n o r d e r t o check the a c t i v a t i n g a b i l i t y o f t r o u t S9 produced from f i s h i n j e c t e d w i t h A r o c l o r 1254. HF c e l l s e x h i b i t e d the g r e a t e s t l e v e l s o f r e p a i r f o l l o w i n g treatment w i t h BP, o n l y a s l i g h t o r no response a f t e r DBA or BA treatment, and no response f o l l o w i n g PY treatment ( F i g u r e 20). Only BP generated a s m a l l amount o f r e p a i r i n the RTG c e l l s . As o n l y a v e r y l i m i t e d r e p a i r response was observed i n t h e RTG c e l l s , a second assay was conducted u s i n g the assay c o n d i t i o n s found t o enhance t h e r e p a i r response and both t r o u t S9s, thus r e - a s s e s s i n g t h e r e p a i r response and t e s t i n g the i n f l u e n c e o f the d i f f e r e n t S9s ( F i g u r e 21). The g r e a t e s t l e v e l o f RTG c e l l r e p a i r was observed f o l l o w i n g treatment w i t h BP a c t i v a t e d w i t h S9 from rainbow t r o u t i n j e c t e d w i t h A r o c l o r 1254. A lower l e v e l o f r e p a i r was noted i n RTG c e l l s t r e a t e d w i t h BP a c t i v a t e d w i t h S9 from the rainbow t r o u t i n j e c t e d w i t h t h e o i l e x t r a c t . Both t y p e s o f rainbow t r o u t S9 e l i c i t e d n e g l i g i b l e amounts o f r e p a i r i n t h e RTG c e l l s w i t h DBA and BA t r e a t m e n t s . As w i t h the HF c e l l s , no r e p a i r was observed a f t e r PY treatment, g) R e p a i r i n h i b i t i o n assay Although"DNA r e p a i r has been r e p o r t e d i n mammalian c e l l s f o l l o w i n g t r e a t m e n t w i t h a v a r i e t y o f n i t r o s a m i n e s , mycotoxins, p e s t i c i d e s , and p o l y c y c l i c aromatic hydrocarbons ( M i t c h e l l e t a l . , 1983), o t h e r c h e m i c a l s i n h i b i t r e p a i r ( P a i n t e r , 1981). When a n a l y z i n g complex mi x t u r e s b o t h DNA r e p a i r and i n h i b i t i o n assays can be conducted t o a c h i e v e a -49-FIGURE 20 DNA r e p a i r s y n t h e s i s i n c u l t u r e d RTG ( • , PAH+S9; O , PAH onl y ) and HF ( • , PAH+S9; • , PAH only) c e l l s exposed t o (A) BP, (B) DBA, (C) BA, and (D) PY a c t i v a t e d w i t h S9 from A r o c l o r 1 2 5 4 - i n j e c t e d rainbow t r o u t . Assay c o n d i t i o n s : 30 min a c t i v a t e d PAH treatment a t 25 C f o l l o w e d w i t h 3h HTdR i n c u b a t i o n a t 25 C (RTG) o r 37 C (HF), emulsion-coated s l i d e s k e p t i n l i g h t - t i g h t boxes f o r 18 days p r i o r t o d e v e l o p i n g . Mean g r a i n counts p l o t t e d w i t h a s t a n d a r d d e v i a t i o n on the mean. -50-GRAINS PER NUCLEUS FIGURE 21 DNA r e p a i r s y n t h e s i s i n c u l t u r e d RTG c e l l s f o l l o w i n g treatment w i t h (A) BP, (B) DBA, (C) BA, and (D) PY a c t i v a t e d w i t h f i s h S9 ( • , PAH on l y ; O , PAH+S9 from A r o c l o r 1254-i n j e c t e d rainbow t r o u t ; • , PAH+S9 from o i l e x t r a c t - i n j e c t e d rainbow t r o u t ) . Assay c o n d i t i o n s : 30 min a c t i v a t e d PAH treat m e n t and 3h HTdR i n c u b a t i o n a t 25 C, emulsion-coated s l i d e s k e p t i n l i g h t - t i g h t boxes f o r 30 days b e f o r e d e v e l o p i n g . Mean g r a i n counts p l o t t e d w i t h a s t a n d a r d d e v i a t i o n on t h e mean. -52-f u l l e r u n d e r s t a n d i n g o f the mixtures' e f f e c t s . To a v o i d p o s s i b l e c h e m i c a l i n t e r a c t i o n s , the i n i t i a l DNA damage i s u s u a l l y i n f l i c t e d by u l t r a v i o l e t l i g h t i r r a d i a t i o n . T h i s i s p o s s i b l e w i t h HF c e l l s , s i n c e they do not r e p a i r the thymidine dimers by p h o t o r e a c t i v a t i o n . But i n f i s h c e l l s t h i s DNA damage i s r e p a i r e d by p h o t o r e a c t i v a t i o n (Table 2) which l i m i t s t h i s assay's u s e f u l n e s s . 2. DNA R e p a i r S y n t h e s i s i n Primary C e l l s I s o l a t e d From Rainbow T r o u t The i n c o r p o r a t i o n o f S9 i n t o the jLn v i t r o DNA r e p a i r and o t h e r g e n o t o x i c i t y assays i s necessary as these c e l l u l a r t e s t systems u s u a l l y have a reduced c a p a b i l i t y t o m e t a b o l i z e t e s t c h e m i c a l s . How r e p r e s e n t a t i v e the i n v i t r o r e s u l t s a r e o f i n v i v o experiments has been q u e s t i o n e d due t o the p o s s i b i l i t y o f a r t e f a c t s b e i n g i n t r o d u c e d by the S9 a c t i v a t i o n . As f r e s h l y i s o l a t e d primary c e l l s r e t a i n t h e i r a b i l i t y t o m e t a b o l i z e t h e t e s t c h e m i c a l s , no S9 i s r e q u i r e d , and no assay a r t e f a c t s caused by the S9 a r e p o s s i b l e . I t was a l s o o f i n t e r e s t t o compare t h e amount o f primary c e l l and i n v i t r o RTG c e l l r e p a i r t o see i f t h e a b i l i t y t o r e p a i r DNA damage has been l o s t w i t h longterm c u l t u r i n g o f the RTG c e l l s . L a s t l y an u l t i m a t e g o a l was t o d i r e c t l y a s s e s s an a q u a t i c environment's g e n o t o x i c burden. One means o f doing t h i s would be t o c o l l e c t f i s h from a s u s p e c t area, remove t i s s u e s o f i n t e r e s t , i s o l a t e p r i m a r y c e l l s from them, and then determine t h e on-going amount o f DNA r e p a i r . -54-TABLE 2 DNA r e p a i r i n RTG c e l l s i r r a d i a t e d w i t h u l t r a v i o l e t l i g h t , t h e n i n c u b a t e d w i t h HTdR i n l i g h t (under l a b o r a t o r y f l u o r e s c e n t and incandescent l i g h t s ) o r dark (no l i g h t s ) c o n d i t i o n s f o r 3 h a t 18 C. And 18-day emulsion exposure p e r i o d was used. -55-- G r a i n s per Nucleus ± St a n d a r d D e v i a t i o n UV D o s e 2 — (ergs/mm ) L i g h t C o n d i t i o n s Dark C o n d i t i o n s 0 2 + 1.6 1 0.9 30 4 + 2.3 10 + 2.8 60 17 + 3.6 25 + 2.1 90 24 + 3.7 31 4.1 -56-The experiments conducted here measured DNA r e p a i r i n primar y c e l l s from rainbow t r o u t l i v e r , stomach, and i n t e s t i n e . C e l l v i a b i l i t y was estimated a t 2 and 6h u s i n g the t r y p a n b l u e e x c l u s i o n technique. Observed m o r t a l i t y o f the pri m a r y c e l l s was g e n e r a l l y l e s s than 5 t o 10% 6h a f t e r i s o l a t i o n . The l e v e l o f DNA r e p a i r was g r e a t e s t i n primary l i v e r c e l l s f o r a l l 3 o f the t e s t chemicals used ( F i g u r e 22) . The magnitude o f t h e i r response was g r e a t e s t w i t h 4NQO and l e a s t w i t h AFB^. The stomach c e l l s showed s m a l l amounts o f r e p a i r i n response t o MNNG and 4NQO but f a i l e d t o respond t o AFB^. I n t e s t i n a l c e l l s e x h i b i t e d low l e v e l s o f r e p a i r f o l l o w i n g 4NQ0 treatment o n l y . P r e l i m i n a r y primary c e l l DNA r e p a i r experiments f a i l e d t o d e t e c t any r e p a i r i n rainbow t r o u t r e d b l o o d c e l l s f o l l o w i n g MNNG o r 4NQO treatments. 3. DNA R e p a i r i n T i s s u e S l i c e s A n o t h e r t e c h n i q u e used which approximates i n v i v o t r eatments i s t o remove f i s h t i s s u e s , t r e a t s l i c e s o f them w i t h 3HTdR and t e s t c h e m i c a l s , and then t o examine c e l l s i n t i s s u e s e c t i o n s f o r evidence o f DNA r e p a i r . As t h e i n i t i a l DNA r e p a i r experiments i n d i c a t e d t h a t d i f f e r e n t c l a s s e s o f organisms may'possess d i f f e r e n t l e v e l s o f DNA r e p a i r i t was a n t i c i p a t e d t h a t t h e X - c e l l s , i f p a r a s i t e s , and e p i t h e l i a l c e l l s i n t h e f l a t f i s h e p i t h e l i a l p a p i l l o m a s c o u l d be d i s t i n g u i s h e d - (assuming t h e e p i t h e l i a l c e l l s demonstrated c o n s i d e r a b l e DNA r e p a i r and the X - c e l l s l i t t l e o r none). The o b s e r v a t i o n s made i n d i c a t e d no i n c r e a s e i n g r a i n counts above -57-FIGURE 22 DNA r e p a i r s y n t h e s i s i n primary rainbow t r o u t l i v e r ( • ) , stomach ( • ) , and i n t e s t i n e ( • ) c e l l s exposed t o (A) MNNG, (B) 4NQO, and (C) AFB,. Assay c o n d i t i o n s : 18 C, simultaneous treatment w i t h the t e s t chemical and HTdR f o r 6h, emulsion-coated s l i d e s kept i n l i g h t - t i g h t boxes f o r 30 days. A st a n d a r d d e v i a t i o n i s p l o t t e d on the mean g r a i n c o u n t s . -58-I On I 3,-J LT K GRAINS PER NUCLEUS II \ 1 JO z o o GRAINS PER NUCLEUS w \ - 'I v GRAINS PER NUCLEUS .2 2 s,- l l \ 1 1 \ z z z £> - 1 \ \ \ \ 1 o - i y b a s e l i n e c o n t r o l l e v e l s f o l l o w i n g MNNG o r 4NQ0 treatments. The X - c e l l s w i t h i n s t a r r y f l o u n d e r epidermal p a p i l l o m a s had few g r a i n s while some of the surrounding e p i t h e l i a l c e l l s were o c c a s i o n a l l y h e a v i l y g r a i n e d ( i e . i n S-phase). B. DNA Breakage Assays 1. DNA Breakage Assays w i t h C u l t u r e d C e l l s a) C e l l l i n e comparisons A DNA r e p a i r response s i m i l a r t o the e a r l i e r c e l l l i n e comparisons ( F i g u r e 2) was found w i t h the U l - F and Ul-H c e l l s ( F i g u r e 23). S i g n i f i c a n t l e v e l s o f r e p a i r were noted f o r t he c e l l l i n e s f o l l o w i n g treatment w i t h both 4NQ0 and MNNG. As p r e v i o u s l y found, dose-dependent responses peaked a t near the same c o n c e n t r a t i o n and t h e r e p a i r magnitude v a r i e d b o t h w i t h t h e c e l l l i n e (HF > CHO > Ul-H - Ul-F) and t h e t e s t chemical used (4NQ0 > MNNG). Examination o f p r e p a r a t i o n s f o r chromosome a b e r r a t i o n s i s c o m p l i c a t e d by a reduced number o f metaphase p l a t e s due t o a d e l a y i n m i t o t i c a c t i v i t y f o l l o w i n g both m a n i p u l a t i o n o f t h e c e l l s and, t o an even g r e a t e r e x t e n t , chemical treatment. The 3HTdR l a b e l l i n g technique was used w i t h d i v i d i n g c u l t u r e s t o determine t h e d u r a t i o n o f t h e m i t o t i c d e l a y a f t e r 4NQ0 treatment (Table 3 ) . A l l o f t h e c e l l l i n e s demonstrated a reduced d i v i s i o n r a t e i n c o n t r o l p r e p a r a t i o n s f o l l o w i n g t he i n t r o d u c t i o n o f j u s t f r e s h c u l t u r e medium i n t o t h e c u l t u r e d i s h . The post-treatment metaphase frequency was dependent on th e c e l l l i n e and the ch e m i c a l c o n c e n t r a t i o n s i n c e h i g h e r l e v e l s were more t o x i c . The l a r g e s t number o f metaphases were - 6 0 -FIGURE 23 DNA r e p a i r i n U l - F ( • ) and Ul-H ( T ) , CHO ( • ), and HF ( A ) c e l l s exposed t o (A) MNNG and (B) 4NQ0. Assay c o n d i t i o n s : t e s t c hemical and HTdR a p p l i e d s i m u l t a n e o u s l y f o r a 3h treatment p e r i o d a t 18 C ( f i s h c e l l s ) o r 37 C (mammalian c e l l s ) , emulsion-coated s l i d e s kept i n l i g h t - t i g h t boxes f o r 18 days. Mean g r a i n counts p l o t t e d w i t h a s t a n d a r d d e v i a t i o n on the mean. -61-GRAINS PER NUCLEUS GRAINS PER NUCLEUS TABLE 3 Post -4NQO exposure HTdR l a b e l l i n g o f d i v i d i n g Umbra l i m i f i n and heart: c e l l s , Chinese hamster ovary c e l l s , and human f i b r o b l a s t c e l l s . Assay conducted a t 18 C ( f i s h c e l l s ) o r 37 C (mammalian c e l l s ) w i t h an 18-day emulsion exposure p e r i o d . -63-a) Umbra 1 iral f i n c e l l s : number of heavily labelled nuclei per 1000 c e l l s Post-Exposure Time Period (Hours) 4NQO (M) 32-37 40-45 48-53 56-61 64-69 72-77 80-85 88-93 2.5x10 3 2 14 i e 24 - 15 5 1x10"° 2 7 12 17 25 8 12 11 Control 2 8 18 23 8 9 9 a b) Umbra limi heart c e l l s : number of heavily labelled nuclei per 1000 c e l l s Post-Exposure Time Period (Hours) 4NCO (M) 4-8 8-12 12-16 16-20 20-24 24-28 28-32 9 15 28 19 27 28 33 9 22 74 7S 44 22 25 S9 46 83 70 64 28 21 c) Chinese hamster ovary c e l l s : number of heavily labelled nuclei per 1000 c e l l s Post-Exposure Time Period (Hours) 4NQ0 (M) 4-8 8-12 12-16 16-20 20-24 24-28 28-32 5 x 1 0 - 6 7 7 17 36 34 64 37 1 x I O - 6 93 143 120 98 196 116 118 Control 113 121 118 167 201 85 145 d) Human fib r o b l a s t c e l l s : number of heavily labelled nuclei per 1000 c e l l s Post-Exposure Time Period (Hours) 4NQO (M) 16-20 20-24 24-28 30-34 34-38 38-42 42-46 1 x 10" 6 2 2 5 0 7 7 IS 5 x 1 0 - 7 2 6 3 13 4 15 28 Control 1 3 9 6 9 9 16 1 x 10" 6 1 x I O - 7 Control -64-observed with the CHO c e l l s . Waves o f r e p l i c a t i n g CHO c e l l s appeared by 12h wi t h 10~ 6 M 4NQO, but sampling a t 20 or 24h pr o v i d e d more metaphases over a broader c o n c e n t r a t i o n range. S i m i l a r l y , w i t h Ul-H c e l l s , by sampling between 16 t o 24h a f t e r 4NQO treatment t h e m i t o t i c d e l a y e f f e c t c o u l d be reduced. The U l - F c e l l s ' m i t o t i c d e l a y was extremely l o n g and o n l y by sampling between 50 t o 70h c o u l d a reasonable number o f metaphases be expected. Experience w i t h U l - F c e l l s i n c u l t u r e i n d i c a t e d t h a t t h e i r m i t o t i c r a t e was v e r y low. P r e v i o u s l y HF c e l l s were used i n our l a b o r a t o r y f o r s t u d y i n g chromosome a b e r r a t i o n s . D e s p i t e t h e low m i t o t i c r a t e noted h e r e and t h e 40 t o 45h d e l a y b e f o r e sampling, e a r l i e r experiments w i t h HF c e l l s had s u c c e s s f u l l y used a p o s t -treatment sampling time o f 30h. Having r o u g h l y e s t a b l i s h e d sampling times, i n i t i a l experiments were designed t o compare the frequency o f chromosome a b e r r a t i o n s i n CHO, Ul-H, and, d e s p i t e t h e i r apparent low m i t o t i c r a t e , HF c e l l s . S i g n i f i c a n t dose-dependent f r e q u e n c i e s o f chromosome a b e r r a t i o n s were observed i n a l l 3 c e l l l i n e s f o l l o w i n g treatment w i t h MNNG o r 4NQ0 ( F i g u r e 24). M i t o t i c i n h i b i t i o n a t h i g h e r chemical c o n c e n t r a t i o n s v a r i e d a c c o r d i n g t o t h e t e s t c h e m i c a l and the c e l l l i n e . M i t o t i c i n h i b i t i o n / t o x i c i t y ( i e . c e l l l o s s from c o v e r s l i p s ) was noted above 7.5 x 10~ 5 M MNNG f o r Ul-H, above 1 x 10~ 4 M MNNG f o r HF, and f o r b o t h o f th e s e c e l l l i n e s above 2.5 x 10~ 6 M 4NQ0. The peak a b e r r a t i o n f r e q u e n c y was dependent on t h e c e l l l i n e (CHO > Ul-H > HF), but t h e Ul-H c e l l s responded a t s l i g h t l y lower c o n c e n t r a t i o n s o f b o t h o f the t e s t c h e m i c a l s . -65-FIGURE 24 Frequency o f chromosome a b e r r a t i o n s i n Ul-H ( T ) , CHO (•), and HF ( A ) c e l l s t r e a t e d f o r 3h with (A) 4NQO and (B) MNNG. -66-% M E T A P H A S E S with C H R O M O S O M E ABERRATIONS % METAPHASES with CHROMOSOME ABERRATIONS In contrast to the chromosome aberration t e s t where c e l l s with a small number of large chromosomes are preferable, the micronucleus t e s t can be applied to most d i v i d i n g c e l l populations. In v i t r o comparisons were made with HF, CHO, U l -H, and Ul-F c e l l s following treatment with 4NQ0 or MNNG (Figures 25, 26). Since the appearance and frequency of micronuclei i s dependent, i n part, on the rate of c e l l d i v i s i o n , which varies according to the p a r t i c u l a r c e l l l i n e and may be delayed by mutagen exposure, the c e l l s were sampled at 32h in t e r v a l s up to 144h. A s i g n i f i c a n t increase i n micronuclei frequency was not noted i n any of the c e l l l i n e s by 16h a f t e r 4NQO treatment (Figure 26). Overall, the frequency increased only marginally (2 to 3-fold) i n Ul-F and HF c e l l s , but a s i g n i f i c a n t number of micronuclei were found with both CHO and Ul-H c e l l s , with the CHO c e l l s peaking at a s l i g h t l y higher frequency. Both CHO and Ul-H c e l l s peak at approximately the same 4NQ0 concentration, but the maximum number of micronuclei occurred at 48h f o r CHO and 144h f o r Ul-H c e l l s . Since a l l the c e l l l i n e s were exposed to a l l of the concentrations (5 x 10~ 8 to 5 x IO" 6 M), i t was noted that a m i t o t i c i n h i b i t i o n / t o x i c response often occurred above 1 - 2.5 x 10~ 6 M 4NQ0, and that t h i s e f f e c t became more apparent at the l a t e r sampling times. As with 4NQ0, the increase i n the frequency of micronuclei following exposure of Ul-F and HF c e l l s to MNNG was marginal (Figure 25), showing only a 2 to 3-fold increase over controls at the sampling times. Ul-H and CHO c e l l s again peak at approximately the same concentration (5 x 10~ 4 M -68-FIGURE 25 Frequency o f m i c r o n u c l e i i n (A) U l - F , (B) Ul-H, (C) CHO, and (0) HF c e l l s measured at 16h ( • ), 48h ( • ), 8Oh ( A ) , 112h ( r ) , and 144h ( o ) f o l l o w i n g 3h treatment w i t h MNNG. -69-% CELLS with MICRONUCLEI J FIGURE 26 F r e q u e n c y o f m i c r o n u c l e i i n (A) U l - F , (B) U l - H , (C) CHO, and (D) HF c e l l s measured a t 16h ( • ) , 48h ( • ) , 80h ( • ) , 112h ( T ) , and 144h ( O ) f o l l o w i n g 3h t rea tment w i t h 4NQO. -71-96 C E L L S w i t h M I C R O N U C L E I % C E L L S w i t h M I C R O N U C L E I MNNG), the magnitude of response being g r e a t e r i n the CHO c e l l s . The maximum m i c r o n u c l e i frequency f o l l o w i n g MNNG treatment o c c u r r e d a t 112h f o r both CHO and Ul-H. S i n c e a l l o f t he c e l l l i n e s were exposed t o the f u l l MNNG c o n c e n t r a t i o n range (5 x 1 ( T 8 t o 5 x 10~ 6 M), the m i t o t i c i n h i b i t i o n / t o x i c response noted a t the h i g h e r c o n c e n t r a t i o n s becomes more apparent a t the l a t e r sampling times. b) Examination o f the e f f e c t o f post-exposure d u r a t i o n and DMSO c o n c e n t r a t i o n on chromosome a b e r r a t i o n frequency P r i o r t o f u r t h e r chromosome a b e r r a t i o n experiments w i t h CHO and Ul-H c e l l s two assay d e t a i l s were examined. The a b e r r a t i o n frequency was examined a t i n t e r v a l s f o l l o w i n g exposure t o 4NQ0 (Table 4) . As p r e v i o u s l y noted, t h e Ul-H c e l l s respond a t a lower c o n c e n t r a t i o n , b u t t h e peak a b e r r a t i o n frequency was lower than i n CHO c e l l s . A b e r r a t i o n s can be d e t e c t e d a t s e v e r a l c o n c e n t r a t i o n s a f t e r 12h w i t h v a r i a t i o n i n frequency a t p a r t i c u l a r c o n c e n t r a t i o n s between 16 t o 24h. Beyond 24 t o 28h the a b e r r a t i o n frequency a t lower c h e m i c a l c o n c e n t r a t i o n s d e c r e a s e s , perhaps as a r e s u l t o f c o n t i n u i n g DNA r e p a i r . From the 4NQ0 model i t f o l l o w s t h a t sampling would be b e s t performed between 16 t o 24h a f t e r c h e m i c a l treatment. As i n the DNA r e p a i r experiments, DMSO was used t o d i s s o l v e t he t e s t c h e m i c a l s . The e f f e c t o f DMSO on a b e r r a t i o n frequency was examined i n a c t i v a t e d AFB 1 t r e a t e d CHO and Ul-H c e l l s (Table 5 ) . A t c o n c e n t r a t i o n s up t o 4% DMSO t h e r e was no e f f e c t on CHO chromosome a b e r r a t i o n s . M i t o t i c i n h i b i t i o n / t o x i c i t y w i t h Ul-H c e l l s was observed w i t h 4% DMSO -73-TABLE 4 Frequency o f chromosome a b e r r a t i o n s i n Umbra l i m i h e a r t and Chinese hamster ovary c e l l s exposed t o 4NQO and examined a t i n t e r v a l s t h e r e a f t e r (percent metaphase p l a t e s w i t h chromosome a b e r r a t i o n s ) . -74-Hours Post-Exposure 4NQO (M) 12 16 20 24 28 1 x 10 -5 Chinese hamster ovary c e l l s Umbra limi heart c e l l s 5 x 10 -6 2.5 x 10 -6 1 x 10 -6 1 x 10 Control -7 S x 10 -6 2.5 x 10 -6 1 x 10 -6 1 x 10 -7 S x 10 Control -8 MI/T« MI/T 25 12 2 1 MI/T MI/T 8 5 3 0 MI/T 51 22 3 2 1 MI/T 43 35 14 5 1 MI/T 55 13 3 0 1 MI/T 46 44 9 8 4 MI/T 65 23 2 2 0 MI/T 45 48 13 4 2 MI/T 55 9 0 0 0 MI/T MI/T 44 9 4 3 *MI/T - mitotic inhibition/toxicity. -75-TABLE 5 E f f e c t of DMSO on chromosome aberration frequency i n Umbra  l i m i heart and Chinese hamster ovary c e l l s exposed to rainbow trout S9 activated a f l a t o x i n B± (percent metaphase plates with chromosome aberrations) . -76-Percent DMSO AFB L (M) 0.1 1 2 4 Umbra l i m i heart c e l l s 5 x 10" 8 43 40 - MI/T* 1 x 10" 8 23 28 28 MI/T Control 0 3 0 MI/T Chinese hamster ovary 1 x I O - 5 83 88 83 80 c e l l s 5 x 10" 6 85 90 80 76 Control 2 1 0 2 *MI/T = m i t o t i c i n h i b i t i o n / t o x i c i t y . -77-o n l y . F u r t h e r experiments t h e r e f o r e used a f i n a l DMSO c o n c e n t r a t i o n o f not g r e a t e r than 1%. c) Chromosome a b e r r a t i o n s f o l l o w i n g PAH treatment Chromosome a b e r r a t i o n experiments p a r a l l e l t o the DNA r e p a i r assays w i t h PAHs were conducted. Up t o 40 and 50% chromosome a b e r r a t i o n s were observed i n Ul-H and CHO c e l l s r e s p e c t i v e l y , f o l l o w i n g treatment w i t h BP a c t i v a t e d by f i s h S9 ( F i g u r e 27) . T h i s p o s i t i v e response was d e t e c t e d a t or j u s t below the c o n c e n t r a t i o n found t o r e s u l t i n DNA r e p a i r . Few chromosome a b e r r a t i o n s were d e t e c t e d i n e i t h e r c e l l l i n e f o l l o w i n g DBA, BA, or PY treatment. C. F i e l d T e s t i n g the DNA R e p a i r and Breakage Assays 1. Sediment E x t r a c t s a) DNA r e p a i r R e p a i r assays were conducted w i t h RTG and HF c e l l s u s i n g A r o c l o r 1 2 5 4 - i n j e c t e d rainbow t r o u t S9 t o m e t a b o l i z e the e x t r a c t s (Table 6) . RTG and HF g r a i n counts f o r c u l t u r e s exposed t o e x t r a c t s from t h e Spanish Bank background a r e a were l i t t l e d i f f e r e n t from t h e counts f o r c e l l s t r e a t e d w i t h e x t r a c t s from the contaminated Sturgeon Bank a r e a . In the s e experiments no t o x i c e f f e c t s were noted f o l l o w i n g treatment w i t h t h e Spanish Bank sediment e x t r a c t s . T o x i c i t y was a l s o not e v i d e n t a f t e r treatment o f the HF c e l l s w i t h the Sturgeon Bank sediment e x t r a c t s . Loss o f RTG c e l l s from t h e c o v e r s l i p s , i n d i c a t i n g t o x i c i t y , was observed w i t h Sturgeon Bank sediment e x t r a c t treatment. T h i s t o x i c e f f e c t was -78-FIGURE 27 Frequency o f chromosome a b e r r a t i o n s i n Ul-H ( • , w i t h A r o c l o r 1 2 5 4 - i n j e c t e d rainbow t r o u t S9; O, no S9) and CHO ( • , w i t h A r o c l o r 1254-injected rainbow t r o u t S9; A , n o S9) c e l l s t r e a t e d with- (A) BP, (B) DBA, (C) BA, and (D) PY. 20 C 3h c h e m i c a l treatment f o l l o w e d by a 2 Oh recover y p e r i o d a t 18 C (Ul-H) o r 37 C (CHO) p r i o r t o h a r v e s t i n g . -79-% M e t a p h a s e s w i t h % M e t a p h a s e s w i t h C h r o m o s o m e A b e r r a t i o n s C h r o m o s o m e A b e r r a t i o n s TABLE 6 DNA r e p a i r s y n t h e s i s i n human f i b r o b l a s t and rainbow t r o u t gonad c e l l s t r e a t e d with sediment e x t r a c t s from Spanish Bank and Sturgeon Bank .(mean g r a i n count ± standa r d d e v i a t i o n ) . A c t i v a t i o n p r o v i d e d u s i n g A r o c l o r 1 2 5 4 - i n j e c t e d rainbow t r o u t S9. - 8 1 -A. UNA repair in human (fibroblast cells (assay temperature, 22*C; ) hr extract: 3HT<iR treatment; 18-day emulsion exposure period) I. Spanish Bank Spanish Bank II Spanish Bank 12 Spanish Bank 13 D i l u t i o n (-}S9 (*>S9 <-)S9 (*>S9 (-)S9 (»)S9 1- 0 • 10 2 1*1.1 1*1.0 111.4 111.0 l i l . S l t l . l 7.5 : 10 3 1*1.2 lt0.8 111.6 111.5 111.4 l t l . 2 5.0 : 10 3 lt0.9 1*0.8 111.4 l i l . O l l l . l l t l . l 2- 5 : IO 3 1*1.3 1*0.6 l t l . l 1*2.2 111.2 U0.8 1- ° • 10 3 1*1.4 1*1.9 2*2.4 1*1.1 1*1.5 1*1.4 7-S : IO 4 1*1.4 1*0.9 1*1.3 l t l . l 1*1.0 1*0.8 s - ° •• 10* 1*1.2 1*1.2 1*1.3 1*1.J 1*1.5 l t l . 2 2- s : 10 4 l t l . l 1*1.4 1*1.0 111.3 l i l . O l t l . l l - ° : 10 l t l . 2 l l l . O 1*1.J 111.3 1*1.2 111.6 S-° : 10 5 1*0.8 1*1.2 1*1.8 1*1.1 1*1.2 l l l . l l - ° •' 1 q S l l l . l l l l . O 1*1.5 l l l . l 212.4 111.4 Control 1*1.0 l t l . 2 l l l . l l t l . 3 HO.9 l i l . S -82-Table 6 (cont'd) 2. Sturgeon Bank #1 «2 13 #4 15 Dilution (-)S9 ( + )S9 (-)S9 (•)S9 C-)S9 (•)S9 (-)S9 ( + )S9 (-)S9 (•)S9 1.0 = i o 2 i t i . e ltl.3 2tl.9 ltl.3 111.6 111.2 211.8 111.2 211.6 111.2 7.5 : 103 111.3 itl.2 2±1.8 111.8 111.3 111.2 212.4 111.6 212.0 111.0 5.0 i o 3 1±1.2 lt0.9 l t l . 5 l t l . 6 111.2 111.2 211.7 111.5 211.7 ltl.O 2.5 i o 3 211.7 ltl.3 l t l . 2 ltl.3 211.7 111.3 211.7 112.0 211.7 111.2 1.0 i o 3 1±0.9 111.5 ltl . 5 ltl.O 211.6 211.6 211.6 111.7 211.8 lt l . 3 7.S 10« ltl . 3 l t l . l ltl.O ltl.5 211.7 ltl.3 ltl.3 111.5 111.4 110.8 5.0 : i o " l t i . 6 ltl.4 111.7 110.8 211.4 ltl.3 111.4 111.2 211.5 ltl.O 2.5 : i o 4 ltl.4 ltO.9 111.1 111.0 111.3 ltl.3 211.6 111.0 111.5 111.3 1.0 : i o 4 l t i . 2 l t l . l 111.8 111.9 111.6 211.7 211.3 111.6 111.6 111.4 5.0 : i o 5 ltl.4 lt0.8 110.8 111.0 111.5 ltl.3 211.3 111.3 112.0 111.4 1.0 : i o 5 l t l . 6 l t l . l 111.3 110.9 111.4 111.6 111.2 111.1 111.4 111.1 Control l t l . 3 lt0.7 111.0 111.6 111.3 110.8 212.0 111.9 211.8 212.0 - 8 3 -Table 6 (cont'd) B. DNA repair In rainbow trout gonad c e l l s (assay temperature, 22*C; 6 hr extract: 3HTdR treatment; 30-day emulsion exposure period) 1. Spanish Bank Spanish Bank «1 Spanish Bank #2 Spanish Bank 13 Dilu t i o n (-)S9 (•)S9 C-JS9 (•)S9 <-)S9 (•)S9 1.0 : 10 2 312.6 111.2 211.6 211.4 212.2 -7.5 : 10 3 2±1.6 111.4 212.1 211.7 212.0 211.7 5.0 : 10 3 111.4 111.3 211.6 211.8 211.6 -2.5 : 10 3 l t l . l 111.0 211.7 211.6 211.9 211.9 1.0 : 10 3 111.1 111.4 111.6 212.0 212.2 211.4 7.5 : 10 4 211.8 111.9 2t2.1 211.7 312.1 212.1 5.0 ; : 10 4 111.7 211.4 111.5 212.1 211.8 212.1 2.5 : : 10 4 211.4 - 111.3 - 2tl.7 311.9 1.0 : ; 10 4 111.3 110.9 211.8 111.4 . - 312.0 5.0 : i o 5 212.2 111.3 212.2 211.7 211.5 212.2 1.0 : i o 5 l t l . l 111.2 111.8 312.1 211.7 212.1 Control 211.8 111.4 212.3 312.3 _ -84-Table 6 (cont'd) 2. Sturg*eon Bank «1 12 «3 #4 15 Dilution < - ) S 9 ( •>S9 <->S9 (+>S9 <-)S9 <+)S9 <->S9 < + >S9 (-)S9 (+)S9 1.0 : 10 2 • ft l t l . O * 1*1.5* l t l . 3 *• *• 211.6* 1*1.0 312.1 110.9 7.5 : 10 3 • ft l t l . O * 111.2* 111.0 *• 111.4 311.9* 141.0 212.0 211.7 5.0 io 3 l t l . 3 * 1*1.0* 211.2* 110.8 211.4* lt0.9 110.9* 141.6 211.9 111.1 2.5 io 3 2*1.S l t l . 4 111.2* 111.0 l l l . S 111.0 312.0 212.1 211.7 211.5 1.0 io 3 l t l . l l t l . l 211.3* 1*1.1 1*1.6 1*1.2 212.4 111.5 212.0 111.6 7.5 io 4 2tl.7 l t l . l 1*1.3 110.9 111.6 111.7 311.9 211.4 2t2.0 1*1.7 S.O : io 4 211.4 lto.e 111.2 110.9 111.2 110.7 111.3 211.6 211.7 211.4 2.5 : io 4 1*1.2 l t l . l l t l . l 1*1.0 l t l . 3 1*1.5 212.3 211.6 2+1.9 111.5 1.0 : io 4 lt0.7 l t l . l 111.5 141.0 211.9 111.6 211.7 211.9 212.0 114.0 S.O : io 5 l t l . 3 l t l . l 110.9 1*1.1 211.5 111.4 212.0 212.4 212.4 211.5 1.0 : 10 5 lt0.7 l±1.2 111.0 141.8 l t l . l 211.3 211.5 2tl.8 211.9 111.0 Control l t l . l 1*1.0 111.4 1*0.7 2*1.8 1*1.S 312.4 312.0 1*1.1 110.9 • S l i g h t l y toxic. **Toxlc. -85-g e n e r a l l y g r e a t e r w i t h e x t r a c t s from sediments c o l l e c t e d from l o c a t i o n s c l o s e r t o the p o i n t o f d i s c h a r g e , but the e f f e c t was l e s s e n e d w i t h the presence o f S9. b) Chromosome a b e r r a t i o n s CHO and Ul-H metaphases were examined f o r chromosome a b e r r a t i o n s f o l l o w i n g exposure t o the sediment e x t r a c t s . For CHO c e l l s (Table 7) c o n t r o l p r e p a r a t i o n s had an a b e r r a t i o n f requency o f 0 t o 2% w h i l e t h e background a b e r r a t i o n frequency from the Spanish Bank e x t r a c t s v a r i e d 0 t o 4%. Treatment w i t h e x t r a c t s from t h e contaminated Sturgeon Bank sediments r e s u l t e d i n a 4 - f o l d i n c r e a s e i n a b e r r a t i o n frequency i n CHO c e l l s . The chromosome a b e r r a t i o n frequency i n Ul-H c o n t r o l p r e p a r a t i o n s was g e n e r a l l y 0 t o 2%. Spanish Bank e x t r a c t t r e a t e d c e l l s e x h i b i t e d chromosome a b e r r a t i o n f r e q u e n c i e s r a n g i n g from 0 t o 10%, w i t h h i g h e r l e v e l s observed from treatment w i t h e x t r a c t #3, t h a t from the c o l l e c t i o n l o c a t i o n c l o s e s t .to Sturgeon Bank. Treatment w i t h Sturgeon Bank sediment e x t r a c t s y i e l d e d a 7 t o 2 0 - f o l d i n c r e a s e i n a b e r r a t i o n frequency over c o n t r o l s , depending on the e x t r a c t and c o n c e n t r a t i o n . O v e r a l l , more fr e q u e n t c y t o t o x i c e f f e c t s were encountered w i t h t h e Ul-H than t h e CHO c e l l s . F o r the S p a n i s h Bank e x t r a c t s some t o x i c e f f e c t s were noted w i t h t h e e x t r a c t c l o s e s t t o Sturgeon Bank. T o x i c e f f e c t s a s s o c i a t e d w i t h the Sturgeon Bank e x t r a c t s g e n e r a l l y i n c r e a s e d toward the sewage tr e a t m e n t p l a n t ' s p o i n t o f d i s c h a r g e but t h e e f f e c t was l e s s e n e d by t h e presence o f S9 i n t h e treatment. Lower Ul-H a b e r r a t i o n f r e q u e n c i e s w i t h e x t r a c t s o f sediments c o l l e c t e d -86-TABLE 7 Frequency o f chromosome a b e r r a t i o n s i n Chinese hamster ovary and Umbra l i m i h e a r t c e l l s t r e a t e d w i t h sediment e x t r a c t s from S p a n i s h Bank and Sturgeon Bank (p e r c e n t metaphase p l a t e s w i t h chromosome a b e r r a t i o n s ) . A c t i v a t i o n p r o v i d e d u s i n g A r o c l o r 1254-injected rainbow t r o u t S9. -87-A. Chromosome aberrations in Chinese hamster ovary cells (assay temperature, 22*C, 3 hr extract treatment; post-exposure period temperature, 37*C) 1. Spanish Bank Spanish Bank 11 Spanish Bank 12 Spanish Bank «3 Dilution <-)S9 (•)S9 <-)S9 (*)S9 (-)S9 (•>S9 1.0 : 102 2 0 3 1 0* 1 7.5 = i o 3 2 1 1 0 3* 1 5.0 : 103 1 1 3 2 1* 2 2.5 : 103 0 0 1 1 1* 0 1.0 : 103 0 1 1 1 2 0 7.5 . i o 4 0 1 2 2 1 0 5.0 : 10< 0 0 1 2 0 0 2.5 : 104 1 0 4 3 2 1 1.0 : 104 2 1 2 0 1 1 S.O : 105 0 4 1 2 2 0 1.0 : 105 2 2 1 2 3 1 Control 2 1 1 1 2 0 •Slightly toxic; decreased • setaphase frequency. -88-T a b l i : 7 ( c o n t ' d ) 2. S t u r t j e o n Hank »1 «2 «3 «4 «5 Dilution (-1S9 (*)S9 (-)S9 (•) S9 (-)S9 C)S9 (-IS9 C1S9 (-)S9 C)S9 1.0 7.5 5.0 2.5 1.0 7.5 5.0 2.5 1.0 7-5 5.0 2.5 1.0 S.0 : 10 : 10 : 10 : 10 : 10 : 10 : 10 : 10 : 10 : 10 5 = 10 5 = io 5 : 10 5 = io 6 Control I 6* • * 2 2 2 1 0 1 6 * • 0 0 0 0 0 1 4* 1* 0* 0* 2 1 0 1 0 2* 1* 5 3 0 1 0 0 0 1* 0* 1 0 1 1 1 0 • S l i g h t l y toxic; decreased metaphase frequency. >*Toxic/mltotic In h i b i t i o n . -89-Table 7 (cont'd) B. Chromosome aberrations in Umbra limi heart cells (assay temperature. 18*C; 3 hr extract treatment; post-exposure period temperature, 18*C) 1. Spanish Bank Spanish Bank t l Spanish Bank «2 Spanish Bank #3 Dilution <-)S9 (•)S9 (-)S9 (•)S9 <-)S9 (•>S9 1.0 = i o 2 6 2 0 4 10* 9 7.5 = io 3 0 4 2 2 6 2 5.0 : 103 4 0 0 6 10 0 2. 5 : 103 4 0 0 2 2 6 1.0 : 103 6 0 4 2 0 2 7.5 : 104 4 4 0 0 0 4 5.0 = i o 4 2 2 2 4 2 6 2.5 : 104 2 0 2 0 4 4 1.0 : : 104 2 4 0 0 0 6 5.0 i : 105 0 2 0 4 0 2 1.0 : ; 105 2 0 0 0 4 2 Control 4 0 2 0 0 -•Slightly toxic; decreased metaphase frequency. -90-Table 7(cont'd) 2. Sturgeon Bank #1 #2 13 »4 «5 Dilution (-)S9 (+)S9 (-)S9 (+)S9 (-)S9 (OS9 (-)S9 (*)S9 (-)S9 (+)S9 1.0 7.5 5.0 2.S 1.0 7.5 5.0 2.5 1.0 7.5 10 10 10 10 10 10 10 10 10 i o 5 5.0 : 10 2.5 : 105 1.0 : 105 5.0 : 106 Control 15 10 5 0 2 0 10 4 4 4 4 4 0 0 0 2 14 15 m> * 8 2 2 4 2 0 0 8 8 4 3 0 2 0 2 2 1 2 0 2 0 2 2 0 2 0 IS* 8* 1 4 2 2 0 0 2 0 40 28 11 6 5 0 23 8 6 2 4 5 3 0 3 20 18 5 0 4 1 2* 7* 10* 1 2 0 2 0 •Slightly toxic; decreased metaphase frequency. ••Toxic/mitotic inhibition. -91-from c l o s e r to the discharge point, i n conjunction with the increase i n t o x i c i t y , tend to indicate a masking of the DNA damaging event by the t o x i c i t y . 2. Micronuclei i n Fish C e l l s I n Vivo C e l l s from the mouth, blood, and l i v e r of s t a r r y flounder (Platichthvs ste l l a t u s ) c o l l e c t e d from Spanish and Sturgeon Banks were examined for the presence of micronuclei. The Sturgeon Bank f i s h were c o l l e c t e d from an area of demonstrated genotoxic a c t i v i t y while the Spanish Bank c o l l e c t i o n area was shown to have no a c t i v i t y (Table 7). Despite t h i s difference i n genotoxic a c t i v i t y the r e s u l t s (Table 8) do not show a marked difference i n micronuclei frequency. -92-TABLE 8 Frequency o f m i c r o n u c l e i i n b u c c a l , l i v e r , and r e d b l o o d c e l l s from s t a r r y f l o u n d e r ( P l a t i c h t h y s s t e l l a t u s ) c o l l e c t e d from S p a n i c h Bank and Sturgeon Bank (mean number o f m i c r o n u c l e i per 1000 c e l l s ± standard d e v i a t i o n ; number o f f i s h examined). - 9 3 -B u c c a l C e l l s B l o o d C e l l s L i v e r C e l l s S p a n i s h Bank : 4.5 ± 2.3; 22 2.9 ± 1.8; 22 5.7 ± 2.3; 17 S t u r g e o n Bank : 5.6 ± 2.2; 25 2.8 ± 1.7; 24 4.6 ± 2.5; 21 -94-DISCUSSION The prime o b j e c t i v e o f the r e s e a r c h r e p o r t e d i n t h i s t h e s i s was t o e v a l u a t e the p o t e n t i a l use o f DNA r e p a i r and breakage assays f o r a s s e s s i n g g e n o t o x i c i t y i n t h e a q u a t i c environment. P r e v i o u s r e s e a r c h i n our Cancer Research L a b o r a t o r y had examined the r e p a i r of DNA and g e n e r a t i o n of chromosome a b e r r a t i o n s i n both HF and CHO c e l l s . The HF c e l l s a r e now p r e f e r r e d f o r DNA r e p a i r assays w h i l e t e s t s f o r chromosome a b e r r a t i o n s are u s u a l l y performed w i t h CHO c e l l s due t o e a s i e r m a n i p u l a t i o n and b e t t e r s e n s i t i v i t y o f these c e l l l i n e s i n the r e s p e c t i v e assays. In attempts t o develop a s s a y s w i t h more r e l e v a n c e t o a q u a t i c organisms and t h e i r environment, f i s h c e l l s were used i n the a s s a y s and the r e s u l t s compared t o these e x i s t i n g model t e s t systems f o r DNA r e p a i r and breakage. 1. Why T e s t f o r A q u a t i c G e n o t o x i c i t y ? I t has been claim e d t h a t 60 t o 90% of a l l c a n c e r s i n man a r e c a u s a l l y r e l a t e d t o chemical c a r c i n o g e n exposure (Boyland, 1969; E p s t e i n , 1974; H e i d e l b e r g e r , 1975). Many o f these c h e m i c a l s , i n c l u d i n g p e s t i c i d e s , h a l o g e n a t e d s o l v e n t s , p o l y c y c l i c aromatic hydrocarbons, and aromatic amines, used by i n d u s t r i a l i z e d s o c i e t i e s become d i s c h a r g e d i n t o and accumulate i n t h e a q u a t i c environment ( K r a y b i l l , 1977; Moore e t a l . , 1980; Nelson e t a l . , 1980; Payne and M a r t i n s , 1980; Van Hoof and Verheyden, 1981; A l i n k , 1982; Sato e t a l . , 1983). G e n o t o x i c a c t i v i t y may a l s o a r i s e from n a t u r a l s o u r c e s such as -95-mycotoxins (Payne and M a r t i n s , 1980), f e c a l m a t e r i a l ( S t i c h e t a l . , 1980), wood (Dunn and Hanham, 1983), and decomposing matter (Payne and M a r t i n s , 1980). A d d i t i o n a l chemical c o m p l e x i t y and g e n o t o x i c i t y r e s u l t s from c h l o r i n a t i o n and o z o n a t i o n o f d r i n k i n g and wastewaters (Payne and M a r t i n s , 1980; A l i n k , 1982; Saxena and Schwartz, 1979; Dolara e t a l . , 1981; Payne and Rahimtula, 1981). I t i s t h e r e f o r e not s u r p r i s i n g t o f i n d e l e v a t e d tumor f r e q u e n c i e s i n f i s h p o p u l a t i o n s i n h a b i t i n g p o l l u t e d environments (Brown e t a l . , 1973; P i e r c e e t a l . , 1978; Smith e t a l . , 1979; Black e t a l . , 1980; Bl a c k e t a l . , 1982; M a l i n s e t a l . , 1984). The h y p o t h e s i s o f a c a u s a l r e l a t i o n s h i p i s f u r t h e r supported by the o b s e r v a t i o n o f tumour f o r m a t i o n i n f i s h f o l l o w i n g i n v i v o exposures t o chemical c a r c i n o g e n s (Meyers and Hendricks, 1982) and by s t u d i e s demonstrating the a b i l i t y o f f i s h t o m e t a b o l i z e c a r c i n o g e n s t o m e t a b o l i t e s which both b i n d t o f i s h DNA and e x h i b i t mutagenic a c t i v i t y ( S t o t t and Sinnhuber, 1978; V a r a n a s i and Gmur, 1980; Ahokas e t a l . , 1979; V a r a n a s i e t a l . , 1981; Balk e t a l . , 1982; S h e l t o n e t a l . , 1983). Of t h e g e n o t o x i c agents used i n my own s t u d i e s here, AFB^, BP, and MNNG have been found t o cause tumors i n f i s h (Meyers and He n d r i c k s , 1982). U l t r a v i o l e t l i g h t has a l s o been found t o cause p a t h o l o g i c a l e f f e c t s on f i s h i n f i s h farms ( B u l l o c k , 1982) and u l t r a v i o l e t l i g h t i r r a d i a t e d f i s h c e l l s i n j e c t e d i n t o t h e Amazon m o l l y ( P o e c i l l a formosa) r e s u l t e d i n a h i g h i n c i d e n c e o f t h y r o i d tumors (Rosen, 1980). The development o f r a p i d , i n e x p e n s i v e a s s a y s f o r g e n o t o x i c a c t i v i t y has p e r m i t t e d the s c r e e n i n g o f many complex -96-mixtures i n a d d i t i o n t o pure chemicals c o n t r i b u t i n g t o the g e n o t o x i c i t y . A t i e r e d t e s t i n g system has evolved whereby chemicals found t o be p o s i t i v e i n simp l e r b a c t e r i a l o r y e a s t assays are reassayed i n more complex but meaningful t e s t s w i t h c u l t u r e d c e l l s (Bridges, 1974; Flamm, 1974; De La I g l e s i a e t a l . , 1980). Although mammalian c e l l s are most commonly used, t h e r e i s i n c r e a s i n g i n t e r e s t i n i n c o r p o r a t i n g f i s h c e l l s and m e t a b o l i z i n g enzymes from f i s h t i s s u e s i n t o these assays. Here the aims are t o d i s c o v e r whether the mammalian assay r e s u l t s are g e n e r a l l y v a l i d , t o determine f i s h c e l l s e n s i t i v i t y t o g e n o t o x i c agents, t o p r o v i d e an assay w i t h i n c r e a s e d r e l e v a n c e t o f i s h and the a q u a t i c ecosystem, and u l t i m a t e l y t o d e v e l o p a t e c h n i q u e t o d i r e c t l y a s s e s s t h e c a r c i n o g e n i c / g e n o t o x i c burden o f a p a r t i c u l a r a q u a t i c environment. S e v e r a l f i s h c e l l l i n e s are a v a i l a b l e from the American Type C u l t u r e C o l l e c t i o n ( R o c k v i l l e , Maryland) o r through the o r i g i n a t o r s o f a p a r t i c u l a r l i n e (Wolf and Mann, 1980). Some f i s h c e l l l i n e s , p a r t i c u l a r l y t h e RTG l i n e , a r e used f o r a s s e s s i n g t o x i c i t y (Marion and Denizeau, 1983; Denizeau and Marion, 1984; B o i s e t a l . , 1985) i n a d d i t i o n t o t h e i r more t r a d i t i o n a l f i s h v i r a l d i s e a s e a p p l i c a t i o n s . Rainbow t r o u t a r e t h e s u b j e c t s o f c o n s i d e r a b l e r e s e a r c h i n a d d i t i o n t o b e i n g a t e s t f i s h f o r a q u a t i c c a r c i n o g e n e s i s (Sinnhuber e t a l . , 1977; Hendricks e t a l . , 1980). T h e r e f o r e because o f t h e i r ready commercial a v a i l a b i l i t y and the a l r e a d y e x i s t i n g data base i t i s advantageous t o i n c o r p o r a t e the RTG c e l l l i n e i n t o g e n o t o x i c i t y assays where p o s s i b l e and p r a c t i c a l . -97-2. The DNA R e p a i r Assay as a T e s t f o r A q u a t i c  G e n o t o x i c i t y a) The DNA r e p a i r response i n mammalian versus f i s h c e l l s In mammalian systems the DNA r e p a i r assay, which measures the e x t e n t o f e x c i s i o n r e p a i r of damaged DNA, has been used i n v i v o and i n v i t r o t o t e s t a d i v e r s e range of p o t e n t i a l g e n o t o x i c agents i n c l u d i n g p e s t i c i d e s , heavy metals, and p o l y c y c l i c a r o m a t i c hydrocarbons ( M i t c h e l l e t a l . , 1983). T h i s assay has both t h e o r e t i c a l and p r a c t i c a l s i g n i f i c a n c e because f a i l u r e t o e f f e c t r e p a i r s t o damaged DNA, as i n the human g e n e t i c d i s o r d e r xeroderma pigmentosum, can l e a d t o an e l e v a t e d tumor frequency ( C l e a v e r and Bootsma, 1975; Kraemer e t a l . , 1984). The comparative approach undertaken here i n d i c a t e d t h a t a t comparable c o n c e n t r a t i o n s of MNNG, 4NQ0, NA2AAF, and AFB 1, f i s h c e l l s e x h i b i t markedly l e s s a u t o r a d i o g r a p h i c a l l y measured DNA r e p a i r than human o r rodent c e l l s ( F i g u r e 2 ) . Woodhead e t a l . (1980) a l s o n o ted low amounts o f i n v i t r o f i s h c e l l e x c i s i o n r e p a i r i n u l t r a v i o l e t - i r r a d i a t e d rainbow t r o u t gonad and Amazon m o l l y c e l l s . Regan e t a l . (1983) compared t h e DNA r e p a i r c a p a c i t y o f primary f i s h c e l l s from Tautocra o n i t l s and T a u t o g o l a b r u s adspersus. w i t h t h a t o f a c u l t u r e d human f i b r o b l a s t c e l l l i n e and found a t l e a s t 1 0 - f o l d l e s s r e p a i r i n t h e f i s h c e l l s f o l l o w i n g treatment w i t h u l t r a v i o l e t l i g h t , 4-n i t r o q u i n o l i n e - l - o x i d e , e t h y l m e t h a n e s u l f o n a t e , m e t h y l methanesulfonate, and N - a c e t o x y - a c e t y l a m i n o f l u o r e n e . However, t h e s e d i f f e r e n c e s a r e m i s l e a d i n g as the comparison made was between primary f i s h c e l l s and an e s t a b l i s h e d human c e l l l i n e . -98-The r e s u l t s here ( F i g u r e s 2, 14, 22) i n d i c a t e t h a t the measured DNA r e p a i r c a p a c i t y i s much lower i n primary than i n c u l t u r e d c e l l s . Furthermore, the magnitude o f the r e p a i r response i n mammalian and f i s h c e l l s v a r i e s w i t h d i f f e r e n t g e n o t o x i c agents ( F i g u r e 2, Table 1), as has a l s o been noted by Regan e t a l . (1983) . However, the r e l a t i v e l y minor d i f f e r e n c e s i n the amount o f r e p a i r measured i n the d i f f e r e n t f i s h c e l l l i n e s ( F i g u r e 3) do not c o n f i r m the r e s u l t s o f Regan e t a l . (1983) who observed a v a r y i n g amount o f r e p a i r between a number o f t e s t chemicals t h a t had been used t o t r e a t primary c e l l s from two f i s h s p e c i e s . But s i n c e Regan e t a l . (1983) r e p o r t r e s u l t s f o r o n l y a s p e c i f i c c o n c e n t r a t i o n , not a dose response, t h i s f i n d i n g may be an a r t e f a c t . b) E x p l a n a t i o n s f o r the low DNA r e p a i r response o f f i s h c e l l s Due t o u n c e r t a i n t y w i t h t h e comparative DNA r e p a i r r e s u l t s , experiments were conducted t o determine i f the low f i s h c e l l r e s p onse r e s u l t e d from d e l a y e d DNA r e p a i r , d i f f e r e n t i a l a b s o r p t i o n o f t e s t chemicals, o r l e s s DNA t a r g e t p e r c e l l . The time course o f r e p a i r ( F i g u r e 5) i n RTG, CHO, and HF c e l l s f o l l o w i n g MNNG or 4NQ0 treatment i s a p p r o x i m a t e l y the same d e s p i t e t h e d i f f e r e n c e i n r e p a i r magnitude. Most o f t h e observed r e p a i r was complete w i t h i n 2Oh and peaked w i t h i n 6h. S i m i l a r l y S t i c h and San (1970) w i t h hamster c e l l s and Warren and S t i c h (1975) w i t h human f i b r o b l a s t s observed t h e r e p a i r r esponse t o peak w i t h i n a few hours f o l l o w i n g c h e m i c a l t r e a t m e n t . The g r a d u a l r e t u r n t o b a s e l i n e r e p a i r l e v e l s s uggests a v a r i a t i o n i n the a c c e s s i b i l i t y o r ease o f r e p a i r o f -99-v a r i o u s DNA l e s i o n s as noted by Kantor and Setlow (1981). The composition of mammalian and f i s h c e l l membranes may d i f f e r , l e a d i n g t o d i f f e r e n t i a l chemical a b s o r p t i o n . T h i s problem was examined by measuring DNA r e p a i r i n RTG c e l l s i r r a d i a t e d w i t h u l t r a v i o l e t l i g h t (Table 1) , which i s not dependent on c e l l u l a r a b s o r p t i o n . R e s u l t s p a r a l l e l i n g those of t h e chemical genotoxin treatments were found, w i t h the DNA r e p a i r response v a r y i n g HF > CHO > RTG. Lehninger (1975), i n summarizing DNA q u a n t i t i e s per c e l l i n d i c a t e s 3 - f o l d l e s s DNA i n f i s h than i n mammalian c e l l s , which c o u l d account f o r the low f i s h c e l l DNA r e p a i r response. However, review o f more d e t a i l e d c o m p i l a t i o n s (Sober e t a l . , 1968; Altman and K a t z , 1976) suggests t h i s i s an o v e r s i m p l i f i c a t i o n as some f i s h s p e c i e s , such as t h e rainbow t r o u t , a r e l i s t e d as h aving DNA q u a n t i t i e s per c e l l which approximate t h a t o f mammalian c e l l s . The v a r i a t i o n i n measurements f o r t h e same s p e c i e s suggests t h a t some o f the d i f f e r e n c e s may be due t o the t e c h n i q u e s used. DNA q u a n t i t a t i v e measurements made here showed approximately 10-15% l e s s DNA p e r c e l l f o r the f i s h c e l l s . These measurements were made u s i n g t h e same s t a i n i n g technique throughout and a s s a y i n g a l l c e l l l i n e s s i m u l t a n e o u s l y . Sober e t a l . (1968) i n d i c a t e a l s o t h a t t h e d i s t r i b u t i o n o f p u r i n e s and p y r i m i d i n e s i s remarkably s i m i l a r among most f i s h and mammalian s p e c i e s , so t h a t g r o s s l y , mammalian and f i s h DNA's are q u i t e s i m i l a r . Thus t h e low f i s h c e l l r e p a i r response cannot be a consequence o f a d e l a y i n the r e p a i r o f DNA l e s i o n s , the l a c k o f t e s t c h e m i c a l uptake, o r g r e a t l y reduced DNA t a r g e t . -100-Other f a c t o r s may be important i n i n t e r s p e c i f i c comparisons of the l e v e l s o f DNA r e p a i r . The assay measures e x c i s i o n r e p a i r which i s an e n z y m a t i c a l l y mediated process. The q u a n t i t a t i v e a c t i v i t y o f DNA polymerase, a key enzyme i n the r e p a i r mechanism, has been measured i n mitochondria and the a c t i v i t y was found t o be g r e a t e s t i n human c e l l s , l e s s i n rodent c e l l s , and l e a s t i n f i s h c e l l s ( S c o v a s s i e t a l . , 1979). Damage t o DNA has a l s o been found t o be a nonrandom process, i n d i c a t i n g a s i t e - s p e c i f i c i t y of genotoxic agents (Meyne e t a l . , 1979; O r n s t e i n and Rein, 1979; Werner e t a l . , 1981; P e r i n - R o u s s e l , 1984). P o s s i b l y f i s h DNA simply has fewer s i t e s f o r genotoxin a t t a c k . Regan e t a l . (1983) noted t h a t i n primary f i s h c e l l s o r c u l t u r e d human c e l l s t r e a t e d w i t h methyl m e t h a n e s u l f o n a t e , N - a c e t o x y - a c e t y l a m i n o f l u o r e n e , o r 4-n i t r o q u i n o l i n e - l - o x i d e o r i r r a d i a t e d w i t h u l t r a v i o l e t l i g h t t h a t t h e r e p a i r patches were o f approximately t h e same s i z e but t h e number o f r e p a i r e d s i t e s was u s u a l l y g r e a t e r i n the mammalian c e l l s . P r e f e r e n t i a l b i n d i n g o f g e n o t o x i c agents has been found i n a s s o c i a t i o n w i t h l i n k e r v e r s u s nucleosomal DNA (Jack and Brookes, 1982), s a t e l l i t e DNA v e r s u s t h e main DNA component ( M e l c h i o r and Beland, 1984), and t r a n s c r i p t i o n a l l y a c t i v e v e r s u s i n a c t i v e DNA areas (Yu, 1983). S i t e s e l e c t i v i t y i s a l s o i n f l u e n c e d by pH ( L y l e e t a l . , 1980; Chen, 1984). F u r t h e r study w i l l be r e q u i r e d t o determine the importance o f these f a c t o r s which may v a r y depending on the s p e c i e s o f f i s h , o r c e l l l i n e , examined and the p a r t i c u l a r t e s t c h e m i c a l , c) Enhancing t h e measured f i s h c e l l DNA r e p a i r response When the unexpectedly low f i s h c e l l r e p a i r response f i r s t -101-became apparent i n 1978, c o r r o b o r a t i v e r e s e a r c h (Woodhead e t a l . , 1980; Regan e t a l . , 1983) had not been p u b l i s h e d , so t h a t s e v e r a l assay parameters were i n v e s t i g a t e d t o determine i f the observed r e s u l t s were o n l y a t e c h n i q u e a r t e f a c t . Another g o a l o f t h i s e x e r c i s e was t o determine ways o f a l t e r i n g assay c o n d i t i o n s t o i n c r e a s e the measured f i s h c e l l r e p a i r response, thereby i n c r e a s i n g the assay's u s e f u l n e s s as a t e c h n i q u e t o monitor the a q u a t i c environment f o r g e n o t o x i c i t y . V a r y i n g f o u r assay parameters d i d r e s u l t i n i n c r e a s e s i n t h e measured DNA r e p a i r response. A l o n g e r 3HTdR i n c u b a t i o n time ( F i g u r e 6) a l l o w s more DNA t o be r e p a i r e d , hence more i s o t o p e i s i n c o r p o r a t e d . As the g r e a t e s t amount o f r e p a i r o c c u r s w i t h i n a few hours o f the t e s t c h e m i c a l treatment ( F i g u r e 5 ) , i n c r e m e n t a l g a i n s from i n c r e a s i n g the treatment time d e c r e a s e . I n c r e a s i n g the assay temperature t o 25 C from 18 C ( F i g u r e 8) i n c r e a s e s t h e r e p a i r response, p r o b a b l y through e f f e c t s on the r e p a i r enzymes, r a t h e r than on membrane co m p o s i t i o n changes a f f e c t i n g p e r m e a b i l i t y (Hazel and P r o s s e r , 1974; De Torrengo and Brenner, 1976). 25 C i s t h e upper t o l e r a n c e l i m i t f o r RTG c e l l growth (Plumb and Wolf, 1971). As some k a r y o r r h e x i c n u c l e i were noted i n RTG s t o c k c u l t u r e s m a i n t a i n e d a t 25 C f o r a prolonged p e r i o d o f time perhaps the DNA changes i t s conformation t o a more u n s t a b l e form o r t o one which i s more open t o g e n o t o x i c a t t a c k . Damage by 4NQ0 appears e s s e n t i a l l y t o be immediately upon treatment, w i t h l i t t l e measurable g a i n beyond 30min ( F i g u r e 9) . L a s t l y , a l o n g e r exposure p e r i o d t o the emulsion (Kodak NTB-3) p e r m i t s -102-more o f the i n c o r p o r a t e d HTdR t o degrade (Figure 7) . As 3 HTdR has a h a l f - l i f e o f 12.3 ye a r s (Rogers, 1979) the in c r e m e n t a l response i n c r e a s e s are roughly p r o p o r t i o n a l . Other assay parameters were t e s t e d , none o f which enhanced the RTG r e p a i r response. S i n c e the n u t r i t i o n a l requirements o f RTG c e l l s a re not c l e a r l y d e f i n e d , the e f f e c t s on DNA r e p a i r from h o l d i n g the c e l l s i n a r g i n i n e - d e f i c i e n t c u l t u r e medium, which was a l s o 7.5% lower i n serum, were e v a l u a t e d ( F i g u r e 10). No changes i n the r e p a i r l e v e l were noted i n c e l l s h e l d up t o 7 days i n ADM with 2.5% serum p r i o r t o treatment w i t h MNNG. Warters e t a l . (1985) r e p o r t e d d e c r e a s e d DNA damage r e p a i r i n c u l t u r e d mammary carcinoma c e l l s which were not " f e d " ( i e . c u l t u r e medium was not changed p r i o r t o X - i r r a d i a t i o n ) . The approach taken here ( F i g u r e 11) was t o r a i s e t he n u t r i e n t l e v e l v i a t h e ADM serum c o n c e n t r a t i o n . No e f f e c t on t h e r e p a i r l e v e l was noted from v a r y i n g t he ADM serum c o n c e n t r a t i o n . As e x c i s i o n r e p a i r i n v o l v e s t he i n c o r p o r a t i o n o f n u c l e o t i d e s from c e l l u l a r p o o l s i t i s c r i t i c a l t o t h e assay t o f l o o d t he p o o l s w i t h i s o t o p e . 3HTdR i s not l i m i t i n g i n RTG c e l l DNA r e p a i r , as i n c r e a s e s i n c o n c e n t r a t i o n d i d not i n c r e a s e r e p a i r g r a i n counts ( F i g u r e 12) . I t i s a l s o u n l i k e l y t h a t t he p o o l 3HTdR a v a i l a b l e becomes d i l u t e d as t h e 3HTdR i s added t o excess and Das e t a l . (1983) found t h a t expansion o f Chinese hamster n u c l e o t i d e p o o l s r e q u i r e d 2 t o 4 hours a f t e r MNNG treatment. That t h e r e i s adequate 3HTdR p e n e t r a t i o n i n t o t he RTG c e l l s i s a l s o s u p p o r t e d by the e x i s t e n c e o f a few h e a v i l y - g r a i n e d n u c l e i ( i e . i n S-phase) i n c o n t r o l p r e p a r a t i o n s . S e v e r a l r e s e a r c h e r s -103-have examined v a r i a t i o n s i n DNA r e p a i r c a p a b i l i t y with c e l l a ging. Hart and Setlow (1976) wi t h human c e l l s and Ben-Ishai and P e l e g (1974) w i t h mouse c e l l s have found a decrease i n e x c i s i o n r e p a i r w i t h prolonged time i n c u l t u r e w h i l e Del'Oreo and W h i t t l e (1978) and Hasegawa e t a l . (1984) noted an i n c r e a s e i n human f i b r o b l a s t DNA r e p a i r w i t h p r o g r e s s i v e c e l l l i n e passages. DNA r e p a i r i n RTG c e l l s v a r y i n g from passage 63 t o 96 was examined ( F i g u r e 13) and no d i f f e r e n c e i n the r e p a i r magnitude observed. But the l e v e l s of r e p a i r noted i n c u l t u r e d RTG c e l l s a r e s u b s t a n t i a l l y h i g h e r than found i n the rainbow t r o u t primary c e l l s ( F i g u r e 22) . As the RTG c e l l l i n e was e s t a b l i s h e d i n t h e e a r l y 1960's (Wolf and Quimby, 1962) and i s commercially a v a i l a b l e from t h e American Type C u l t u r e C o l l e c t i o n o n l y from passage 60-70, i t was not p o s s i b l e t o i n v e s t i g a t e changes i n DNA r e p a i r c a p a c i t y i n e a r l i e r passages. Ishikawa e t a l . (1978) a u t o r a d i o g r a p h i c a l l y measured i n v i v o DNA r e p a i r i n O r v z i a s l a t i p e s . o f age 4 t o 38 months, th e approximate l i f e span o f the f i s h . T h e i r r e s u l t s note no a g e - a s s o c i a t e d change i n r e p a i r a b i l i t y o f g a n g l i o n c e l l s . I t was d e c i d e d t h a t u n l e s s the r o u t i n e RTG DNA r e p a i r assay procedure c o u l d be a l t e r e d t o y i e l d a response i n excess of f i f t y g r a i n s p e r n u c l e u s , then the m o n i t o r i n g t e c h n i q u e would not be worth p u r s u i n g , due t o i t s low s e n s i t i v i t y i n t h i s c e l l l i n e . I t was found t h a t by conducting t h e assay a t 25 C, i n c u b a t i n g the c e l l s w i t h 3HTdR f o r 6h, and u s i n g a 30 day emulsion exposure p e r i o d , then a 2 t o 3 - f o l d i n c r e a s e i n g r a i n counts c o u l d be a c h i e v e d ( F i g u r e 14), i n c r e a s i n g the -104-assay's s e n s i t i v i t y t o d e s i r e d l e v e l s . d) U s i n g the DNA r e p a i r i n h i b i t i o n assay w i t h f i s h c e l l s A number of chemicals have been found t o i n h i b i t DNA r e p a i r (Balachandran and S r i n i v a s a n , 1982; Charp and Regan, 1985). Routine a p p l i c a t i o n o f the r e p a i r assay has t h e r e f o r e been complemented by i n c o r p o r a t i n g a t e s t f o r r e p a i r i n h i b i t i o n i n t o t he procedure. T h i s t e s t r e q u i r e s an i n i t i a l t reatment o f the c e l l s w i t h a genotoxic agent, t o damage the DNA, p r i o r t o a p p l y i n g a t e s t chemical o r e x t r a c t . The i n i t i a l damaging agent most o f t e n used i s u l t r a v i o l e t l i g h t , not a ch e m i c a l , i n or d e r t o a v o i d i n t e r a c t i v e e f f e c t s o f che m i c a l s w h i l e e n s u r i n g damage t o the DNA. In f i s h c e l l s the r e p a i r i n h i b i t i o n t e s t has l i m i t e d a p p l i c a b i l i t y because, u n l i k e HF c e l l s , thymidine dimers, the primary DNA l e s i o n from u l t r a v i o l e t l i g h t , can be r e p a i r e d by p h o t o r e a c t i v a t i o n (Table 2; Regan and Cook, 1967; Mano e t a l . , 1982; M i t a n i , 1983). e) DMSO and S9 use i n the DNA r e p a i r assay The s p a r i n g l y s o l u b l e n ature o f most o f t h e t e s t c h e m i c a l s i n t i s s u e c u l t u r e medium r e q u i r e d i n i t i a l l y d i s s o l v i n g t he chemical i n d i m e t h y l s u l f o x i d e p r i o r t o d i l u t i o n i n medium. Although N o v i c k i e t a l . (1985) observed no d e c r e a s e i n r a t he p a t o c y t e DNA s y n t h e s i s w i t h DMSO c o n c e n t r a t i o n s o f 0.5% or l e s s , Nestmann e t a l . (1985), r e p o r t e d enhanced m u t a g e n i c i t y o f hexachloroacetone d i s s o l v e d i n DMSO ve r s u s o t h e r s o l v e n t s . The e f f e c t o f DMSO on DNA r e p a i r was t h e r e f o r e examined on u l t r a v i o l e t l i g h t i r r a d i a t e d RTG c e l l s ( F i g u r e 15). DNA r e p a i r i n h i b i t i o n / c y t o t o x i c i t y was noted a t c o n c e n t r a t i o n s i n excess o f 1% DMSO. The f i n a l DMSO -105-c o n c e n t r a t i o n i n the c u l t u r e medium f o r experiments t h e r e f o r e d i d not exceed 1%. C e l l s maintained i n c u l t u r e f o r prolonged p e r i o d s of time l o s e much of t h e i r a b i l i t y t o metabolize t e s t chemicals. As demonstrated i n RTG and FHM c e l l s (Diamond and C l a r k , 1970; Thornton e t a l . , 1982), r e s i d u a l m e t a b o l i c a c t i v i t y may remain. T h i s enzyme a c t i v i t y may be s u f f i c i e n t t o metabolize c h e m i c a l s , such as AFB 1 ( F i g u r e 4) to a c t i v e forms, but a l i v e r microsomal f r a c t i o n or S9 i s r o u t i n e l y added t o chemical d i l u t i o n s t o ensure metabolism of the t e s t chemical o r e x t r a c t . S9 i s u s u a l l y p repared f o l l o w i n g the procedure o f Ames e t a l . (1975) who used A r o c l o r 1254 t o p r e - t r e a t the animals. As the o i l - e x t r a c t o r A r o c l o r 1254 pre-treatment enhances the a c t i v i t y and spectrum of enzymes p r e s e n t and s p e c i e s s p e c i f i c m e t a b o l i c e f f e c t s have been observed, a r e p a i r comparison was made u s i n g rainbow t r o u t and r a t S9s p r e - t r e a t e d w i t h A r o c l o r 1254, and a rainbow t r o u t S9 w i t h o i l - e x t r a c t pre-treatment. The comparison here ( F i g u r e s 18, 19) noted more r e p a i r f o l l o w i n g AFB-^ metabolism w i t h both f i s h S9 p r e p a r a t i o n s than w i t h t h e r a t l i v e r S9. D e s p i t e the d i f f e r e n t pre-treatments, l i t t l e d i f f e r e n c e was noted between the two f i s h S9s. These r e s u l t s may r e f l e c t r a t v e r s u s f i s h S9 d i f f e r e n c e s i n r e a c t i o n k i n e t i c s o r d i f f e r e n c e s i n enzyme a c t i v i t y , as the t r o u t s e n s i t i v i t y t o the c a r c i n o g e n i c e f f e c t s o f AFB^ i s f e l t due t o p a r t i c u l a r m e t a b o l i c pathways (Sinnhuber e t a l . , 1977; W i l l i a m s and Buhler, 1983). f) S c r e e n i n g PAHs f o r g e n o t o x i c i t y i n the DNA r e p a i r assay The DNA r e p a i r assay can be used t o s c r e e n p o s s i b l e -106-a q u a t i c carcinogens f o r gen o t o x i c a c t i v i t y . The p o l y c y c l i c a romatic hydrocarbons were chosen as a r e p r e s e n t a t i v e group of a q u a t i c contaminants some o f which, such as 3,4-benzopyrene, 1,2,5,6-dibenzanthracene, and 1,2-benzanthracene, are known c a r c i n o g e n s ( N a t i o n a l Academy o f S c i e n c e s , 1972). In contaminated a q u a t i c areas PAHs have been i m p l i c a t e d i n the e t i o l o g y o f f i s h tumors (Brown e t a l . , 1973; B l a c k e t a l . , 1980; M a l i n s e t a l . , 1984). F i s h S9 has been shown t o m e t a b o l i z e benzopyrene and dibenzanthracene t o m e t a b o l i t e s c a u s i n g mutagenic a c t i v i t y i n the Ames s a l m o n e l l a t e s t (Ahokas e t a l . , 1977; Stegeman, 1978). Benzo(a)pyrene m e t a b o l i t e s have a l s o been shown t o b i n d t o t h e DNA of salmonid and p l e u r o n e c t i d f i s h (Varanasi e t a l . , 1979; V a r a n a s i e t a l . , 1982), but l i t t l e i s known o f the r e p a i r o f these l e s i o n s . Of t h e f o u r PAHs assayed f o r g e n o t o x i c a c t i v i t y o n l y pyrene i s not c a r c i n o g e n i c , w h i l e 1,2-benzanthracene i s somewhat l e s s c a r c i n o g e n i c than 1,2,5,6-dibenzanthracene and 3,4-benzopyrene ( N a t i o n a l Academy o f S c i e n c e s , 1972). As the HF c e l l s have a g r e a t e r r e p a i r response, and hence are more s e n s i t i v e than RTG c e l l s , t h e assay was f i r s t conducted w i t h b o t h c e l l l i n e s u s i n g the o r i g i n a l assay c o n d i t i o n s and S9 from A r o c l o r 1254 t r e a t e d rainbow t r o u t . DNA r e p a i r was o n l y observed i n HF c e l l s ( F i g u r e 20) t r e a t e d w i t h BP, i n d i c a t i n g t h a t t r o u t S9 can metabolize BP t o forms which damage t r o u t DNA. D e s p i t e the c a r c i n o g e n i c p o t e n t i a l o f DBA, no r e p a i r a c t i v i t y was noted; perhaps because the s o l u b i l i t y o f DBA i n c u l t u r e medium was observed t o be l e s s than BP. P r e v i o u s l y , o f t h e s e f o u r PAHs, onl y BP has been shown t o cause DNA r e p a i r -107-( M i t c h e l l e t a l . , 1983). When the assay was conducted u s i n g the c o n d i t i o n s t h a t enhance the response, r e p a i r was observed i n RTG c e l l s t r e a t e d w i t h BP, but not w i t h the o t h e r PAHs (Figure 21) . Although RTG c e l l s can m e t a b o l i z e BP (Diamond and C l a r k , ' 1970; Thornton e t a l . , 1982) the amount of a p a r t i c u l a r m e t a b o l i t e formed appears i n s u f f i c i e n t t o cause DNA r e p a i r . The amount o f r e p a i r i n BP t r e a t e d c e l l s was g r e a t e r u s i n g S9 from A r o c l o r 1254 t r e a t e d rainbow t r o u t . P o s s i b l y the S9 from the o i l - e x t r a c t t r e a t e d rainbow t r o u t metabolized the BP t o forms o f reduced g e n o t o x i c a c t i v i t y . S h e l t o n e t a l . (1983, 1984) observed a reduced tumor frequency i n rainbow t r o u t g i v e n A r o c l o r 1254 p r i o r t o AFB^ treatment and s p e c u l a t e d t h a t the exposure t o A r o c l o r may have a l t e r e d t he predominance o f m e t a b o l i c pathways l e a d i n g t o d i f f e r e n t m e t a b o l i t e s , g) DNA r e p a i r assays w i t h f i s h primary c e l l s and t i s s u e s l i c e s A g o a l o f t h i s r e s e a r c h was t o develop a tec h n i q u e f o r d i r e c t l y a s s e s s i n g the g e n o t o x i c burden o f an a q u a t i c environment. One means o f acc o m p l i s h i n g t h i s i s t o determine t h e l e v e l o f DNA r e p a i r i n i s o l a t e d primary c e l l s o f f i s h c o l l e c t e d from d i f f e r e n t a r e a s . As a ste p towards t h i s , c e l l s were i s o l a t e d from the stomach, l i v e r , and i n t e s t i n e o f rainbow t r o u t and examined f o r DNA r e p a i r a c t i v i t y f o l l o w i n g treatment w i t h MNNG, 4NQO, and KFB^ ( F i g u r e 22) . D e s p i t e employing t h e assay c o n d i t i o n s t h a t enhance t h e r e p a i r response, o n l y modest l e v e l s o f r e p a i r were observed. S i m i l a r l y K l a u n i g (1984) and Regan e t al.(1983) have noted low -108-l e v e l s of e x c i s i o n r e p a i r i n f i s h primary c e l l s . Such low l e v e l s of r e p a i r i n the rainbow t r o u t primary c e l l s f o l l o w i n g treatment with potent g e n o t o x i c chemicals e f f e c t i v e l y p r e c l u d e d pursuing t h i s l i n e of endeavor, as the amount o f genotoxic a c t i v i t y a n t i c i p a t e d i n f i e l d s i t u a t i o n s would be much lower. A second a p p l i c a t i o n o f the r e p a i r technique t h a t was c o n s i d e r e d was the p o s s i b i l i t y o f d i s t i n g u i s h i n g p h y l o g e n e t i c a l l y d i f f e r e n t c e l l types on t h e b a s i s o f v a r i a t i o n i n DNA r e p a i r c a p a c i t y , assuming the human - rodent - f i s h r e l a t i o n s h i p extended t o o t h e r s p e c i e s . I t has been suggested t h a t the X - c e l l s w i t h i n the f l a t f i s h epidermal p a p i l l o m a s and cod p s e u d o b r a n c h i a l tumors a r e p a r a s i t e s , p o s s i b l y amoebas (Brooks e t a l . , 1969; A l p e r s e t a l . , 1977; Dawe, 1981). Should the f l a t f i s h e p i t h e l i u m s u r r o unding the X - c e l l s e x h i b i t DNA r e p a i r t o the l e v e l o f primary c e l l s and the c e l l s thought t o be p a r a s i t e s have l e s s r e p a i r c a p a c i t y then one might be a b l e t o d i s c r i m i n a t e the d i f f e r e n t c e l l s . However, X - c e l l s and e p i t h e l i a l c e l l s examined i n s e c t i o n s from t i s s u e p i e c e s t r e a t e d w i t h 4NQ0 or MNNG had so few g r a i n s t h a t a dose response was not e v i d e n t . As a l s o observed by Kranz e t a l . (1980), some surrounding e p i t h e l i a l c e l l s a r e h e a v i l y g r a i n e d i n d i c a t i n g both t h a t t h e r e i s c e l l d i v i s i o n i n t h i s area o f the p a p i l l o m a , and t h a t thymidine is. adequately p e n e t r a t i n g the t i s s u e . The X - c e l l s cannot t h e r e f o r e be d i s c r i m i n a t e d from the e p i t h e l i a l c e l l s on the b a s i s o¥ DNA r e p a i r c a p a c i t y . -109-3. Assays f o r DNA Damage as T e s t s f o r A q u a t i c G e n o t o x i c i t y a) Comparison of chromosomal a b e r r a t i o n frequency i n mammalian and f i s h c e l l s The comparatively low amount of DNA r e p a i r i n f i s h c e l l s which have approximately the same c e l l u l a r DNA content as mammalian c e l l s suggested t h a t c o n s i d e r a b l e f i s h DNA damage may not be r e p a i r e d . I f so, then t e s t s f o r d e t e c t i n g damage t o f i s h DNA may be more s e n s i t i v e than r e p a i r assays. I n i t i a l l y a comparative approach was a g a i n undertaken u s i n g the chromosome a b e r r a t i o n t e s t . T h i s t e s t i s r o u t i n e l y performed u s i n g CHO c e l l s because they have r e l a t i v e l y few (2n = 22) l a r g e chromosomes, are s t a b l e i n c u l t u r e , and, as they have a r a p i d r a t e of d i v i s i o n , a l a r g e number of metaphase spreads f o r a n a l y z i n g can be e a s i l y o b tained. Although RTG c e l l s have been used by L a n d o l t e t a l . (1984) and Kocan e t a l . (1985) f o r s t u d y i n g chromosome a b e r r a t i o n s , t h i s c e l l l i n e has a l a r g e and v a r i a b l e number o f chromosomes which c o m p l i c a t e s t h e a n a l y s i s . Gold e t a l . (1979) l i s t chromosome formulae f o r some 306 North American f i s h , o f which o n l y 5 s p e c i e s have fewer than 30 chromosomes. Because c e l l l i n e s had not been e s t a b l i s h e d f o r any o f t h e s e 5 s p e c i e s two l i n e s were i n i t i a t e d from f i n and h e a r t t i s s u e o f Umbra l i m i which has 22 l a r g e chromosomes (Beamish e t a l . , 1971). As found f o r the o t h e r f i s h c e l l l i n e s ( F i g u r e 2) , the Umbra c e l l s a l s o e x h i b i t c o n s i d e r a b l y l e s s DNA r e p a i r ( F i g u r e 23). 3HTdR i n c o r p o r a t i o n was a l s o used w i t h d i v i d i n g c e l l s t o determine p o s t - t r e a t m e n t sampling times f o r chromosome a b e r r a t i o n s -110-(Table 3) . A m i t o t i c d e l a y was noted f o l l o w i n g h a n d l i n g of c e l l s and treatment with 4NQ0. The number of c e l l s found t o be d i v i d i n g was dependent on the c e l l l i n e with comparatively few observed i n the Ul-F and HF p r e p a r a t i o n s . The r e s u l t s showed t h a t Ul-H and CHO c e l l s c o u l d be sampled 12 t o 16h f o l l o w i n g treatment. U l - F c e l l s were excluded from the chromosome a b e r r a t i o n experiments due t o t h e i r low m i t o t i c r a t e . A b e r r a t i o n experiments have p r e v i o u s l y been s u c c e s s f u l l y done with the HF c e l l s u s i n g a post-treatment p e r i o d o f 30h p r i o r t o sampling. L a t e r experience i n d i c a t e d t h a t a h i g h e r r a t e o f c e l l d i v i s i o n was encouraged by a hi g h e r (up t o 20%) serum c o n c e n t r a t i o n i n the c u l t u r e medium and c a r e f u l m a n i p u l a t i o n of the c e l l s i n the procedure. C e l l s growing w e l l i n s t o c k c u l t u r e s a l s o performed b e t t e r i n the experiments. Examination o f chromosome a b e r r a t i o n s i n Ul-H, CHO, and HF c e l l s t r e a t e d w i t h MNNG o r 4NQO i n d i c a t e d a Ul-H dose response a t a lower c o n c e n t r a t i o n than HF o r CHO, w h i l e the g r e a t e s t response o c c u r r e d i n CHO c e l l s ( F i g u r e 24) . C y t o t o x i c i t y i n Ul-H c e l l s prevented a n a l y z i n g f o r a b e r r a t i o n s a t h i g h e r chemical c o n c e n t r a t i o n s . As t h e CHO c e l l s a re the sta n d a r d c e l l l i n e f o r a n a l y z i n g f o r a b e r r a t i o n s and t h e Ul-H c e l l s a l s o performed w e l l f u r t h e r t e s t s compared o n l y these two c e l l l i n e s . b) P r o c e d u r a l d e t a i l s o f the chromosome a b e r r a t i o n t e s t Two a s s a y p r o c e d u r a l d e t a i l s were examined. The a b e r r a t i o n frequency a t p a r t i c u l a r 4NQ0 c o n c e n t r a t i o n s was f a i r l y c o n s i s t e n t a t d i f f e r e n t sampling times (Table 4) . A s l i g h t d e c l i n e i n a b e r r a t i o n frequency was observed i n CHO a t - i l l -the l a t e r sampling times but not RTG, perhaps due t o a h i g h e r l e v e l o f DNA r e p a i r i n CHO c e l l s . As noted with DNA r e p a i r ( F i g u r e 15) , f i n a l c o n c e n t r a t i o n s o f DMSO above 1 t o 2% were found t o be t o x i c t o the f i s h c e l l s (Table 5) . I n t e r e s t i n g l y a s i m i l a r e f f e c t was not e v i d e n t w i t h CHO c e l l s . c) Chromosome a b e r r a t i o n s f o l l o w i n g PAH exposure A demonstration of the p r a c t i c a l use o f t h e a b e r r a t i o n t e s t examined the DNA damaging e f f e c t s o f PAH. In p a r a l l e l w i t h the s i m i l a r DNA r e p a i r experiment ( F i g u r e s 20, 21) , chromosome a b e r r a t i o n s were observed o n l y a f t e r BP treatment ( F i g u r e 27). As p r e v i o u s l y found i n t h e comparative chromosome a b e r r a t i o n s t e s t s the Ul-H dose-response i n d i c a t e s t h e s e c e l l s are more s e n s i t i v e a t lower c o n c e n t r a t i o n s o f the t e s t c h e m i c a l s , but the CHO c e l l s have a h i g h e r a b e r r a t i o n frequency. d) Use o f t h e micronucleus t e s t w i t h f i s h and mammalian c e l l s The observed s e n s i t i v i t y o f t h e f i s h c e l l s t o DNA damaging e v e n t s suggested expanding t h e scope o f t h e a b e r r a t i o n t e s t t o s u r v e y i n g f i s h p o p u l a t i o n s . Both Umbra  l i m i and Umbra pvcrmaea have been t r e a t e d i n v i v o and examined f o r chromosome a b e r r a t i o n s (Mong and B e r r a , 1979; Hooftman and V i n k , 1981). However, v e r y few l o c a l f i s h have a s u i t a b l e k a r y o t y p e and t h e c e n t r a l mudminnow, Umbra l i m i . i s found o n l y i n s o u t h e r n Manitoba and O n t a r i o and t h e n o r t h e a s t e r n U n i t e d S t a t e s ( S c o t t and Crossman, 1973). The m i c r o n u c l e u s t e s t which a l s o examines DNA damage, but i s not - dependent on low numbers o f chromosomes was t h e r e f o r e e x p l o r e d as an a l t e r n a t i v e t o t h e chromosome a b e r r a t i o n t e s t . In metaphase -112-p l a t e s damaged DNA may be observed as fragmented chromosomes, but i f the c e l l s d i v i d e , these fragments aggregate t o form so-c a l l e d m i c r o n u c l e i which can be enumerated t o produce dose responses. J n v i t r o comparisons i n d i c a t e d dose responses i n HF, CHO, Ul-H, and U l - F c e l l s f o l l o w i n g treatment w i t h both 4NQ0 and MNNG ( F i g u r e s 25, 26) . As the g e n e r a t i o n o f m i c r o n u c l e i i s dependent on c e l l d i v i s i o n the h i g h e r frequency noted i n CHO and Ul-H i s l i k e l y , i n p a r t , r e l a t e d t o the f a s t e r d i v i s i o n o f c e l l s i n these l i n e s . M i c r o n u c l e i have been observed i n p e r i p h e r a l b l o o d e r y t h r o c y t e s o f t h e e a s t e r n mudminnow Umbra pygmaea f o l l o w i n g i n v i v o treatment w i t h e t h y l methanesulphonate (Hooftman and de Raat, 1982). In a f i e l d survey of i n s h o r e and o f f s h o r e p o p u l a t i o n s o f windowpane f l o u n d e r , w i n t e r f l o u n d e r , and A t l a n t i c mackerel along the U n i t e d S t a t e s 1 e a s t e r n seaboard, Longwell e t a l . (1983) noted a h i g h e r i n c i d e n c e o f m i c r o n u c l e i i n t h e i n s h o r e f i s h . However, the m i c r o n u c l e i f r e q u e n c i e s r e p o r t e d i n these papers are q u i t e low. F u r t h e r c o m p l i c a t i o n s may a l s o a r i s e from m i s - i d e n t i f i c a t i o n o f m i c r o n u c l e i , e s p e c i a l l y i n r e d b l o o d c e l l s . C y t o p l a s m i c i n c l u s i o n s d i a g n o s t i c o f v i r a l e r y t h r o c y t i c n e c r o s i s ( S m a i l , 1982) resemble and may be mistaken f o r m i c r o n u c l e i . M i c r o n u c l e u s -l i k e c h a r a c t e r s may a l s o r e s u l t from e r y t h r o c y t e f r a g m e n t a t i o n and a m i t o s i s which has been observed i n A t l a n t i c salmon (Benfey and S u t t e r l i n , 1984) and p l a i c e ( E l l i s , 1984). A n a l y z i n g f o r m i c r o n u c l e i i n c e l l s from t i s s u e s such as l i v e r o r stomach r a t h e r than b l o o d may t h e r e f o r e be p r e f e r a b l e . -113-4. F i e l d Testing the Genotoxicity Assays a) Overview Gi l b e r t s o n (1984) expressed the opinion that f i s h populations f i r s t be surveyed for pathological anomalies p r i o r to developing further laboratory t o x i c o l o g i c a l techniques for exploring a problem. S i m i l a r l y the work here follows e a r l i e r studies i n our laboratory which linked a high incidence of epidermal papillomas i n f l a t f i s h with environmentally degraded areas (Stich et a l . , 1977a,b). More recent consideration of these tumors has suggested they are parasite i n f e c t i o n s (Alpers et a l . , 1977; Dawe, 1981) and a 1984 survey of tumorous f i s h from the coastal waters of Hokkaido, Japan f a i l e d to f i n d a d i r e c t association between municipal and i n d u s t r i a l discharges with f l a t f i s h having the epidermal papillomas (Katsura et a l . , 1984). However, experiments t r e a t i n g rainbow trout eggs with BP l a t e r resulted i n the formation of two integumental nodules i n alevins, one of which resembled an a n g i o e p i t h e l i a l nodule, a precursor to the f l a t f i s h epidermal papillomas (Hose et a l . , 1984). Elevated 3,4-benzopyrene concentrations were also found i n sediments from Sturgeon Bank, near Vancouver (Dunn and St i c h , 1976), where S t i c h and co-workers found the high incidence of f l a t f i s h tumors. The Sturgeon Bank area i s contaminated with e f f l u e n t from the Iona Island sewage treatment plant which serves Vancouver. The plant discharges approximately 1,178,000 m3/d of primary treated sewage which i s chlorinated during the summer months. A wide range of chemicals has been i d e n t i f i e d i n the Sturgeon Bank sediments and bio t a (Bawden et -114-a l . , 1973; Koch e t a l . , 1977) and a seasonal v a r i a t i o n may be expected due to c h l o r i n a t i o n e f f e c t s . Elsewhere a v a r i e t y of c a r c i n o g e n s such as n i t r o s a m i n e s and p o l y c y c l i c aromatic hydrocarbons have been i s o l a t e d from sewage and sewage sludge (Grzybowski e t a l . , 1983; Brewer e t a l . , 1980; Babish e t a l . , 1983; Hopke e t a l . , 1984), g e n o t o x i c a c t i v i t y has been found i n e x t r a c t s o f f e c a l m a t e r i a l ( S t i c h e t a l . , 1980), and g e n o t o x i c a c t i v i t y was observed i n e x t r a c t s o f sediments c o l l e c t e d near sewage o u t f a l l s (Moore e t a l . , 1980; Van Hoof, 1981; Sato e t a l . , 1983). Tumorous growths i n salamanders have p r e v i o u s l y been a s s o c i a t e d w i t h t r e a t e d sewage e f f l u e n t (Rose, 1976; Rose and Harshbarger, 1977). More r e c e n t l y Murchelano and Wolke (1985) found an e l e v a t e d i n c i d e n c e o f l i v e r tumors i n f i s h caught a t a raw sewage o u t f a l l and G r i z z l e e t a l . (1984) found a h i g h p r e v a l e n c e of o r a l p a p i l l o m a s i n f i s h l i v i n g i n c h l o r i n a t e d e f f l u e n t from a sewage treatment f a c i l i t y . b) In v i t r o g e n o t o x i c i t y t e s t i n g o f the contaminated area The g e n o t o x i c i t y t e s t s used here w i t h f i s h and mammalian c e l l s were f i e l d t e s t e d w i t h sediment samples and f i s h c o l l e c t i o n s from the Sturgeon Bank - Iona I s l a n d sewage treatment p l a n t o u t f a l l a r e a. A nearby Spanish Bank area i n E n g l i s h Bay was s e l e c t e d as a c o n t r o l s i t e . V i r t u a l l y no DNA r e p a i r a c t i v i t y was noted i n e i t h e r HF o r RTG c e l l s t r e a t e d w i t h e x t r a c t s o f Sturgeon o r Spanish Bank sediment e x t r a c t s ( T a b l e 6) . Some c y t o t o x i c i t y was observed i n RTG c e l l s t r e a t e d w i t h Sturgeon Bank sediment e x t r a c t s but t h i s t o x i c i t y was d i m i n i s h e d i n the presence o f S9. The f a i l u r e t o d e t e c t -115-any DNA r e p a i r a c t i v i t y may be due to components of the extract which i n h i b i t DNA repair, genotoxic agents not being i n high enough concentrations i n the extracts, or a lack of s e n s i t i v i t y of the assay. In the chromosome aberration t e s t some DNA damage was caused i n CHO c e l l s by treatment with extracts from sediment samples c o l l e c t e d near the o u t f a l l (Table 7). A more pronounced e f f e c t was noted with Ul-H c e l l s where peak aberration frequencies of 10 to 23% were noted from treatment with extracts from the o u t f a l l area. S l i g h t c y t o t o x i c i t y and DNA damaging a c t i v i t y were noted i n both c e l l l i n e s following treatment with the Spanish Bank #3 sediment extract. This may be due to the observed c i r c u l a t i o n pattern of the sewage e f f l u e n t toward English Bay, or contaminant deposition from i n d u s t r i a l discharges into the North Arm of the Fraser River in f l u e n c i n g t h i s control s i t e (Greater Vancouver Sewerage and Drainage D i s t r i c t , 1983). Generally more c y t o t o x i c i t y was noted with extracts from sediments c o l l e c t e d c l o s e r to the o u t f a l l but the t o x i c e f f e c t i s reduced i n the presence of S9. Because PAHs, such as BP, require a c t i v a t i o n to DNA damaging forms, and S9 generally reduced the damaging a c t i v i t y , i t i s implied that an array of chemicals are present i n the extracts, some of which do not require metabolic a c t i v a t i o n , c) JJQ vi v o genotoxicity assessment of the contaminated area Despite the difference i n DNA damaging a c t i v i t y of the Sturgeon Bank versus - Spanish Bank sediments, no increase i n micronuclei frequency was found i n buccal, blood, or l i v e r c e l l s from s t a r r y flounder (Table 8). Possibly the Sturgeon -116-Bank f i s h were c o l l e c t e d too far out on the Bank. However, DNA damaging a c t i v i t y was detected at the c o l l e c t i o n s i t e and the muddy nature of the sediments precluded beach seine c o l l e c t i o n s closer to the point of discharge. The f i s h may be t r a n s i e n t , and hence receive only small, perhaps inconsequential exposures to the contaminated sediments. But the contents of the Sturgeon Bank beach seines did show a larger food resource, such as shrimp, and starry flounder are not known for being migratory (Hart, 1973). The s t a r r y flounder micronuclei frequencies are also low i n comparison to those found i n eastern mudminnows t r e a t e d with methanesulphonate (Hooftman and de Raat, 1983). I t may be that the e x t r a c t i o n of chemicals from the sediments concentrates the a c t i v i t y to a detectable l e v e l which, though not detected i n vivo, i s s t i l l damaging the s t a r r y flounder DNA but to a much smaller degree. Possibly starry flounder are not a s e n s i t i v e i n d i c a t o r species. However, beach seines d i d not c o l l e c t substantial numbers of other f i s h species and examination of the few specimens c o l l e c t e d d i d not indicate a d i f f e r e n c e i n micronuclei frequency. Future research may expand the ap p l i c a t i o n s of these assays, examine a broader range of t e s t chemicals, and other f i e l d s i t u a t i o n s . -117-SUMMARY T h i s study's primary o b j e c t i v e was t o i n c o r p o r a t e f i s h c e l l s and m e t a b o l i z i n g enzymes i n t o g e n o t o x i c i t y assays commonly used w i t h mammalian c e l l s i n order t o : a) p r o v i d e assays w i t h i n c r e a s e d r e l e v a n c e t o f i s h and t h e a q u a t i c ecosystem b) v a l i d a t e mammalian assay r e s u l t s c) determine f i s h c e l l s e n s i t i v i t y t o e n v i r o n m e n t a l l y important g e n o t o x i c agents such as PAH d) develop techniques t o d i r e c t l y assess the c a r c i n o g e n i c / g e n o t o x i c burden o f contaminated environments, such as Sturgeon Bank, near Vancouver. 1. DNA R e p a i r Assay a) F i s h and mammalian c e l l e x c i s i o n r e p a i r was measured i n t h e DNA r e p a i r assay f o l l o w i n g treatment w i t h f o u r g e n o t o x i c c h e m i c a l s . The amount o f measured r e p a i r v a r i e d i n t h e o r d e r o f human c e l l s > rodent c e l l s > s e v e r a l f i s h c e l l l i n e s . R e l a t i v e l y l i t t l e v a r i a t i o n i n the r e p a i r response was noted among t h e f i s h c e l l l i n e s . b) S e l e c t i n g t h e RTG c e l l l i n e as b e i n g r e p r e s e n t a t i v e o f f i s h c e l l s , i t was determined t h a t t h e i r r e p a i r response was no t due t o d e l a y e d r e p a i r o f damage, l a c k o f t e s t c h e m i c a l uptake, o r s m a l l e r q u a n t i t y p e r c e l l o f DNA t a r g e t . c) I t was found t h a t adequate RTG DNA r e p a i r s e n s i t i v i t y c o u l d be a t t a i n e d by r a i s i n g t h e assay temperature, i n c r e a s i n g -118-the HTdR i n c u b a t i o n time, and l e n g t h e n i n g the HTdR de g r a d a t i o n p e r i o d i n the a u t o r a d i o g r a p h i c workup. L i t t l e o r no e f f e c t on RTG DNA r e p a i r was noted from v a r y i n g the 3HTdR c o n c e n t r a t i o n , d u r a t i o n i n ADM, ADM serum c o n c e n t r a t i o n , and c e l l l i n e passage. d) Treatment o f HF and RTG c e l l s with a s e r i e s o f PAH i n d i c a t e d t h a t o n l y BP c o u l d be a c t i v a t e d by f i s h S9 t o r e s u l t i n DNA r e p a i r i n HF c e l l s and such r e p a i r c o u l d o n l y be d e t e c t e d i n RTG c e l l s when u s i n g the assay c o n d i t i o n s which enhance the f i s h c e l l r e p a i r response. e) In a p r o g r e s s i o n towards i n v i v o measurements, DNA r e p a i r was measured i n rainbow t r o u t primary c e l l s and s t a r r y f l o u n d e r t i s s u e s l i c e s but the measured r e p a i r was even l e s s t h an t h e s m a l l amounts observed i n f i s h c e l l s i n v i t r o . F u r t h e r r e p a i r assays were abandoned i n f a v o u r o f t h e h y p o t h e s i s t h a t f i s h c e l l DNA damage may not be r e p a i r e d , hence t e s t s examining DNA damage may be c o n s i d e r a b l y more s e n s i t i v e . 2. T e s t s f o r DNA Damage a) As t h e RTG c e l l l i n e i s not i d e a l l y s u i t e d f o r chromosome work, two c e l l l i n e s from the c e n t r a l mudminnow, Umbra l i m i . were e s t a b l i s h e d . b) The DNA r e p a i r response o f U l - F and Ul-H l i n e s i s comparable t o t h a t observed i n the o t h e r f i s h c e l l l i n e s . c) Ul-H c e l l s were more s e n s i t i v e than CHO o r HF c e l l s i n chromosome a b e r r a t i o n t e s t s but t h e g r e a t e s t a b e r r a t i o n frequency was observed i n CHO c e l l s . - 1 1 9 -d) Of the PAH s e r i e s examined on l y BP metabolized with rainbow t r o u t S9 r e s u l t e d i n chromosome a b e r r a t i o n s i n Ul-H and CHO c e l l s . e) A n a l y z i n g f o r DNA damage was extended from examining f o r chromosome a b e r r a t i o n s , t o m i c r o n u c l e i as t h i s l a t t e r t e s t has wider i n v i v o a p p l i c a b i l i t y . 3. F i e l d T e s t i n g the Developed G e n o t o x i c i t y T e s t s a) P r i o r r e s e a r c h had shown an e l e v a t e d frequency of epidermal papillomas i n f l a t f i s h c o l l e c t e d from Sturgeon Bank, an a r e a contaminated with a v a r i e t y o f chemicals, i n c l u d i n g PAHs, from a sewage treatment p l a n t (Dunn and S t i c h , 1976; S t i c h e t a l . , 1977a,b). b) Sediments and f l a t f i s h were c o l l e c t e d from Sturgeon Bank and Spanish Banks, the l a t t e r o f which was s e l e c t e d as a c o n t r o l s i t e . c) No DNA r e p a i r was observed i n HF o r RTG c e l l s t r e a t e d w i t h sediment e x t r a c t s but some RTG c e l l c y t o t o x i c i t y was noted a t h i g h c o n c e n t r a t i o n s o f the Sturgeon Bank sediment e x t r a c t s . d) Chromosome a b e r r a t i o n s and c y t o t o x i c i t y were found i n CHO and Ul-H c e l l s t r e a t e d w i t h Sturgeon Bank sediment e x t r a c t s . 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DNA damage r e p a i r i n quiescent murine mammary carcinoma c e l l s i n culture. Biochim. et Biophys. Acta. 824: 357-364. Werner, D., D. Hadjioloo, and H. Hershey. 1981. Non-random d i s t r i b u t i o n of N-methyl-N-nitrosourea s e n s i t i v e s i t e s i n a eukaryotic genome. Chem.-Biol. Inter. 37: 279-287. Williams, D. and D. Buhler. 1983. P u r i f i e d form of cytochrome P-450 from rainbow trout with high a c t i v i t y toward conversion of a f l a t o x i n B± to a f l a t o x i n B-L-2,3-epoxide. Cane. Res. 43: 4752-4756. Wolf, K. 1979. Cold-blooded vertebrate c e l l and t i s s u e cul t u r e . In Jakoby, W. and I. Pastan (Eds.) Methods i n Enzymology: * Volume LVIII C e l l Culture. Academic Press, New York, 1979, pp.466-477. Wolf, K. and J . Mann. 1980. Poikilotherm vertebrate c e l l l i n e s and viruses: a current l i s t i n g f o r f i s h e s . In V i t r o 16: 168-179. Wolf, K. and M. Quimby. 1962. Established eurythermic l i n e of f i s h c e l l s i n v i t r o . Science 135: 1065-1066. -131-Wolf, K. and M. Quimby. 1976. Primary monolayer culture of f i s h c e l l s i n i t i a t e d from minced tissues. TCA Manual 4: 445-448. Woodhead, A., R. Setlow, and E. G r i s t . 1980. DNA repair and longevity i n three species of cold-blooded vertebrates. Exp. Geront. 15: 301-304. Yu, F. 1983. Preferential binding of a f l a t o x i n B 1 to the t r a n s c r i p t i o n a l l y active regions of rat l i v e r nucleolar chromatin i n vivo and i n v i t r o . Carcino. 4: 889-893. -132-APPENDIX I Publications from the Thesis Material a) Accepted 1. Walton, D. , A. Acton, and H. Stich. 1983. DNA repair synthesis i n cultured mammalian and f i s h c e l l s following exposure to chemical mutagens. Mut. Res. 124: 153-161. 2. Walton, D. , A. Acton, and H. Stich. 1984. DNA repair synthesis following exposure to chemical mutagens i n primary l i v e r , stomach, and i n t e s t i n a l c e l l s i s o l a t e d from rainbow trout. Cane. Res. 44: 1120-1121. 3. Walton, D., A. Acton, and H. S t i c h . 1984. Comparison of DNA-repair synthesis, chromosome aberrations and induction of micronuclei i n cultured human f i b r o b l a s t , Chinese hamster ovary and c e n t r a l mudminnow (Umbra limi) c e l l s exposed to chemical mutagens. Mut. Res. 129: 1129-136. 4. Walton, D., A. Acton, and H. S t i c h . 1985. Increased response of the rainbow trout gonad c e l l unscheduled DNA repa i r assay. B u l l . Environ. Contam. Toxicol. 34: 340-348. 5. Walton, D. In press. In v i t r o f i s h c e l l DNA breakage and repa i r assays for detecting mutagenic a c t i v i t y . Aquatic T o x i c i t y Conference Proceedings. Can. Tech. Rep. Fish. Aquat. S c i . b) Submitted 1. Walton, D., A. Acton, and H. S t i c h . DNA re p a i r synthesis i n cultured f i s h and human c e l l s exposed to f i s h S9-activated aromatic hydrocarbons. 2. Walton, D. The use of f i s h c e l l s i n short-term mutagenicity t e s t s . -133-APPENDIX II L i s t of Abbreviations ADM AFB, BA BP CH CHO DBA DMSO FHM HF HTdR MEM MNNG NA2AAF 4NQO PAH PY RTG RTO S9 Ul-F Ul-H arginine-deficient medium aflat o x i n B, 1,2-benzantnracene 3,4-benzopyrene chum salmon heart c e l l l i n e Chinese hamster ovary c e l l l i n e 1,2,5,6-dibenzanthracene dimethylsulfoxide fathead minnow c e l l l i n e human f i b r o b l a s t c e l l l i n e [methyl- 3H]-thymidine Eagle's minimal e s s e n t i a l medium N-methyl-N•-nitro-N-nitrosoguanidine N-acetoxy-2-acetylaminoflourene 4-nitroquinoline 1-oxide p o l y c y c l i c aromatic hydrocarbon pyrene rainbow trout gonad c e l l l i n e rainbow trout ovary c e l l l i n e l i v e r microsomal f r a c t i o n , 9000g supernatant Umbra l i m i f i n c e l l l i n e Umbra l i m i heart c e l l l i n e -134-APPENDIX III Theoretical Aspects and S t a t i s t i c a l Analysis of the  DNA Repair and Breakage Assays 1. DNA Repair Assay The DNA rep a i r assay measures the amount of nucleotide excision repair by the incorporation of 3HTdR i n damaged DNA. In the autoradiographic workup beta-particles emitted from 3HTdR react with the nuclear track emulsion coating on the s l i d e s . The s i l v e r grains formed over the nu c l e i (Figure 28) are enumerated and the data graphed to produce dose response curves. In the grain counting process the background grain count, determined from an area equivalent i n s i z e and adjacent to each nucleus, was subtracted to give a net grain count which was plotted. The factors noted produce a 2 to 3 - f o l d increase of the nuclear grain counts (Results, section A-l-c) also increased negative control (no chemical) and background grain counts. These increases i n negative c o n t r o l counts were f a r overshadowed by the increases i n experimental counts (eg. 1 — > 3 vs. 20 —> 60) . Background grain counts were also found to increase but the increase was minimized by thorough r i n s i n g of 3HTdR from the coverslips and using a low c e l l density on the cov e r s l i p s . However, occasionally, despite these e f f o r t s , background grain counts i n some experiments, f o r unknown reasons, were s u f f i c i e n t l y high that enumeration was aborted. - 1 3 5 -FIGURE 28 Assay preparations showing (A) DNA rep a i r i n rainbow trout gonad c e l l s (arrows in d i c a t e n u c l e i ) , (B) chromosome aberrations i n an Umbra l i m i heart c e l l (arrow indicates chromatid breads) , and (C) a micronucleus i n an Umbra l i m i heart c e l l (arrow indicates a micronucleus). -136--137-I n i t i a l experiments scored 60 nuclei while l a t e r experiments scored 30, as l i t t l e improvement i n the data was found with the additional counts. Only nuclei of the same s t a i n i n g i n t e n s i t y and s i z e were scored f o r a p a r t i c u l a r s l i d e . Comparison of po s i t i v e (4NQ0-exposed) controls from i n v i t r o sediment extract exposure experiments (Results, section C-l-a) indicated a 20-25% v a r i a t i o n i n counts between experiments with HF or RTG c e l l s . Negative control grain counts (no chemical) are noted i n the Figures. T y p i c a l l y these net gra i n counts were les s than 3 with a standard deviation of 1 to 2. H i s t o r i c a l l y discrimination of data s i g n i f i c a n c e has r e l i e d on observing a dose response. More rec e n t l y s t a t i s t i c a l t e s t s such as the t - t e s t have been used (Klaunig, 1984) . For the DNA r e p a i r r e s u l t s presented here, data s i g n i f i c a n c e was viewed i n three ways. F i r s t l y , the gross appearance of the dose response was appraised to see i f a response was obviously present. Secondly, counts of 5-10 grains per nucleus were viewed as representing only nominal amounts of r e p a i r and l i t t l e credance was placed on data s i g n i f i c a n c e . Grain counts i n excess of 10 grains per nucleus were deemed s i g n i f i c a n t . Thirdly, the number of data points i n excess of the 5-10 l e v e l were considered. I f only 1 or 2 data points were above t h i s l e v e l again l i t t l e credance was placed on data s i g n i f i c a n c e . S t a t i s t i c a l s i g n i f i c a n c e associated with the 5-10 and -138-FIGURE 29 Plots of the data from Figure 22 B showing v a r i a t i o n on the mean as represented with (A) standard deviations, (B) 99% confidence i n t e r v a l s , and (C) standard errors. The stars indicate s t a t i s t i c a l s i g n i f i c a n c e as calculated using a two-t a i l e d t - t e s t P 0.10;^- p < 0.001). - 1 3 9 -greater than 10 l e v e l s of grain counts were considered (Figure 29) using a two-tailed t - t e s t . 0.01 and < 0.001 lev e l s of s i g n i f i c a n c e were associated with grain counts i n the 5-10 range while the counts i n excess of 10 grains per nucleus had less than the 0.001 l e v e l of s i g n i f i c a n c e . Graphical representation of v a r i a t i o n on the mean was provided using standard deviations although confidence i n t e r v a l s and standard errors were also considered (Figure 29) . As the standard errors proved to be so small that they were d i f f i c u l t to plo t and standard deviations were a c a l c u l a t o r function, hence e a s i l y and immediately available, the l a t t e r was chosen. 2. Chromosome Aberration Test This t e s t detects unrepaired or misrepaired damage to the DNA. At approximately 20-24 h post-exposure to a t e s t chemical the mitotic-spindle poison, c o l c h i c i n e , was added to the culture medium for a 4 h period. C e l l d i v i s i o n cannot therefore proceed and i s arrested i n metaphase. The resultant spreads of chromosomes were examined for damage which, i n CHO and Ul-H c e l l s , was noted to most often be chromatid breaks and exchanges (Figure 28). Although not s t a t i s t i c a l l y proven, the frequency of exchanges i n Ul-H c e l l s was observed to be considerably lower than i n CHO c e l l s . H i s t o r i c a l l y data s i g n i f i c a n c e has been assessed by observing a dose-response. V a r i a t i o n between experiments occurs and, from p o s i t i v e (4NQ0-exposed) controls i n the chromosome aberration experiments with the sediment extracts -141-(Results, section C-l-b), was found to be 10-15% with CHO c e l l s and 20-25% with the Ul-H c e l l s . For the research reported here negative control (no chemical) l e v e l s of aberrations were t y p i c a l l y less than 3%. As with the DNA repa i r data, significance of the chromosome aberration data was f i r s t assessed by discriminating a dose-response. Nominal amounts of breakage were ascribed to l e v e l s of 5-10% breakage while t r u e l y s i g n i f i c a n t amounts of damage were at t r i b u t e d to incidences i n excess of 10%. Added credance i n data s i g n i f i c a n c e was f e l t by having more than 1 or 2 data points i n excess of the 5-10% l e v e l of breakage. 3. Micronucleus Test The micronucleus t e s t i s a gross measure of DNA breakage hence i s somewhat of a proxy f o r chromosome aberrations. In comparison to the aberration t e s t the micronucleus assay detects DNA strand breakage but not exchanges. Micronuclei are therefore pieces of broken DNA material which do not migrate to the poles i n mitosis, rather they remain as small b a l l s of nuclear material i n the daughter c e l l s (Figure 28) . In the experiments, micronuclei were distinguished by s i z e , shape, st a i n i n g r e l a t i v e to the nucleus, and by focusing up and down through the c e l l . As demonstrated (Results, section B-l-a) the i n v i t r o micronucleus dose-response was influenced by m i t o t i c delay following chemical exposure, the rate of c e l l d i v i s i o n , and the time u n t i l sampling a f t e r exposure. The s i g n i f i c a n c e of the i n v i t r o data can only be judged by observation of - 1 4 2 -a dose-response and discrimination of the incidence of micronuclei at a p a r t i c u l a r chemical concentration r e l a t i v e to the negative control (no chemical). For these experiments (Figures 25, 26) a strong dose-response with a 5-10% incidence of micronuclei was deemed s i g n i f i c a n t . When evaluating the i n vivo data (Table 8) the t - t e s t can be applied. - 1 4 3 -Publications from the Thesis Materia 1 Accepted 1. Walton, D. , A. Acton, and H. S t i c h . 1983. DNA r e p a i r s y n t h e s i s i n c u l t u r e d mammalian and f i s h c e l l s f o l l o w i n g exposure t o c h e m i c a l mutagens. Mut. Res. 124: 153-161. 2. Walton, D. , A. Acton, and H. S t i c h . 1984. DNA r e p a i r s y n t h e s i s f o l l o w i n g exposure t o c h e m i c a l mutagens i n primary l i v e r , stomach, and i n t e s t i n a l c e l l s i s o l a t e d from rainbow t r o u t . Cane. Res. 44: 1120-1121. 3. Walton, D., A. Acton, and H. S t i c h . 1984. Comparison o f DNA-repair s y n t h e s i s , chromosome a b e r r a t i o n s and i n d u c t i o n o f m i c r o n u c l e i i n c u l t u r e d human f i b r o b l a s t , Chinese hamster ovary and c e n t r a l mudminnow (Umbra l i m i ) c e l l s exposed t o che m i c a l mutagens. Mut. Res. 129: 1129-136. 4. Walton, D. , A. Acton, and H. S t i c h . 1985. I n c r e a s e d response of the rainbow t r o u t gonad c e l l u n scheduled DNA r e p a i r assay. B u l l . E n v i r o n . Contam. T o x i c o l . 34: 340-348. 5. Walton, D. In p r e s s . In v i t r o f i s h c e l l DNA breakage and r e p a i r assays f o r d e t e c t i n g mutagenic a c t i v i t y . A q u a t i c T o x i c i t y Conference Proceedings.. Can. Tech. Rep. F i s h . Aquat. S c i . Submitted 1. Walton, D., A. Acton, and H. S t i c h . DNA r e p a i r s y n t h e s i s i n c u l t u r e d f i s h and human c e l l s exposed t o f i s h S 9 - a c t i v a t e d aromatic h y d r o c a r b o n s . 2. Walton, D. The use o f f i s h c e l l s i n s h o r t - t e r m m u t a g e n i c i t y t e s t s . 

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