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In vitro carcinogen-protein complex formation Wallick, Carole Ann 1955

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IN VITRO CARCINOGEN - PROTEIN COMPLEX FORMATION  by CAROLE ANN WALLICK  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE  OF  MASTER OF SCIENCE In the Department of CHEMISTRY  We accept t h i s thesis as conforming to the standard required from candidates f o r the degree of MASTER OF SCIENCE  Members of the Department of CHEMISTRY  THE UNIVERSITY OF BRITISH COLUMBIA July, 1955  -liABSTRACT Attempts have been made to form " i n v i t r o " carcinogenprotein and carcinogen-amino acid complexes by various oxidative processes. I r r a d i a t i o n , chemical oxidation, and a combination of the two have proved unsuccessful, A new method, based upon chromatographic separation, has been developed f o r the detection of complex formation. A p p l i c a b i l i t y of the procedure was Investigated using a hydrocarbon3,H-benzpyrene-epidermal  amino acid conjugate and"in vivo" formed protein complex.  Complex formation of 3, *-benzpyrene with several l  purines and nucleic acids was detected i n t h i s way. A series o f hydrocarbons - both carcinogenic and non - carcinogenic - was substituted f o r 3,*t-henzpyrene, hut no c o r r e l a t i o n between carcinogenic a c t i v i t y and complex formation of the hydrocarbons could be found.  In view of the d i f f i c u l t i e s encountered with " i n  v i t r o " complex formation, i t was suggested that further i n v e s t i g ations be made on the " i n vivo" complex.  Chromatographic separat-  ion of the " l n vivo" complex and determination of the fragments a f t e r p a r t i a l hydrolysis looks promising.  ACKNOWLEDGEMENTS The author wishes to express her sincere thanks to Dr. C. Reid f o r h i s assistance and encouragement throughout the course of t h i s research p r o j e c t . Gratitude i s due also to the National Research Council f o r f i n a n c i a l assistance (Bursary, 195 *-19!?5) • l  Table of  Contents  Acknowledgements  i  Abstract  i i 1  Introduction A.  Carcinogens  1  B.  Chemical  1  carcinogens  1.  A c t i v e regions  3  2.  Geometrical considerations  h  3.  Complex f o r m a t i o n  5  C.  The M i l l e r complex  6  D.  Problem t o be c o n s i d e r e d  8 9  E x p e r i m e n t a l Method A.  M i l l e r ' s method f o r p r e p a r i n g " i n v i v o " c a r c i n o g e n - p r o t e i n complex  B.  Methods employed i n the attempted o f " i n v i t r o " hydrocarbon  9  formation 9  complex  1.  P r e p a r a t i o n of,samples  9  2.  I r r a d i a t i o n method  10  3.  I r r a d i a t i o n and oxygen treatment  11  h.  B e n z o y l peroxide treatment  11  5.  Benzoyl peroxide and oxygen treatment  12  6.  Benzoyl peroxide p l u s i r r a d i a t i o n treatment Enzyme method" ......  12 12  Incubation i n a tissue culture whole s k i n samples  13  7. 8.  solution-  9. ~C. D.  Incubation i n a tissue culture solutionamino acids, proteins and purines  M i l l e r ' s method of detecting complex formation  13 1*+  Chromatographic method of detecting complex 15  formation  17  Results and Discussion 1.  Irradiation  18  2.  I r r a d i a t i o n with oxygen treatment  19  3.  Benzoyl peroxide Benzoyl peroxide accompanied by u l t r a v i o l e t irradiation  19 31  5.  Enzyme and incubation treatment  31  6.  Purines and nucleic acids  7.  Chromatographic method f o r detecting complex formation  „  36 ^2  Conclusions and Suggestions f o r Further Work  h7  Appendix  ^9  A.  P u r i f i c a t i o n of solvents  ^9  B.  Preparation of Ringer-Locke Solution  ^9  C.  Synthesis of caproic acid  50  Bibliography  l(l,2-benzanthryl-10-carbamido) 55  INTRODUCTION A.  Carcinogens The i n i t i a l step i n the t r a n s i t i o n from normal to  malignant growth presents an exceedingly complex problem to the investigator.  I n spite of the immense e f f o r t being ex-  pended i n cancer research, the voluminous l i t e r a t u r e shows also that our knowledge of the possible causes of the d i s ease i s i n i t s infancy. As i s to be expected from the comp l e x i t y of the problem, many d i f f e r e n t approaches have been suggested - i n v e s t i g a t i o n of the chemical and biophysical differences between cancerous and normal t i s s u e , research into the chemistry of c e l l metabolism and c e l l reproduction, analysis of the properties and metabolism of chemical c a r c i n ogens, and so f o r t h .  I t i s the l a t t e r approach which has been  adopted i n the research to be described. B.  Chemical  Carcinogens  The discovery of chemical carcinogens l e d to a new and basic method f o r investigating the process r e s u l t i n g i n malignant t i s s u e s .  For i f the investigator can elucidate  the mode of action of the carcinogen, i t s points of attack within the c e l l , or the chemical changes i t brings about, then he w i l l be better equipped to determine a preventive measure and cure. The known carcinogens cover a wide range of compounds (Table M)»  Many attempts have been made to f i n d some property  -2-  Table l a Some carcinogenic compounds compound  3,  structure  benzpyr ene  1,2,7,8-dibenzfluorene  1,2,5,6-dibenzcarbazole  p-aminostilbene  p-dimethylaminoazobenzene CH,  7,9-dimethyl-3,^-benzenacridine  2,5-dimethyl- 3 , ( 1 ' , 2 -naphtho) dibenzthiophene 1  desoxycholic acid  CH)  common to the carcinogens which could be correlated with t h e i r b i o l o g i c a l a c t i v i t y (2, lh  16, 20, 22, 26, 27).  9  However,  although many theories of the mechanism of chemically  induced  carcinoma have been proposed, none are e n t i r e l y s a t i s f a c t o r y . A few of the more important ideas w i l l be 1.  discussed.  Active regions In 19H6,  Pullman (26) presented a c o r r e l -  a t i o n between charge on the IC region (high r e a c t i v i t y of a p a r t i c u l a r carbon-carbon double bond) and carcinogenic a c t i v i t y . The structure of 1,2-benzanthracene i l l u s t r a t e s the region termed the "K region".  K-region  Using wave mechanics to c a l c u l a t e the electron density at each carbon atom and each bond of the molecule, he was  able  to show that f o r those compounds which were b i o l o g i c a l l y a c t i v e , the corresponding ivity.  structures possessed a region of high react-  The r e l a t i v e charge density of the K region f o r a  series of methyl substituted benzanthracenes and benzphenanthrenes proved most i n t e r e s t i n g . Substances with a charge density above a c e r t a i n threshold value were a c t i v e , t h e i r a c t i v i t y increasing as the charge density increased.  Sub-  sequent i n v e s t i g a t i o n of the rate of addition of osmium  -htetroxide (1) to the K region showed good agreement with the Pullman work.  However there were c e r t a i n inconsisten-  c i e s i n the theory.  Some compounds, t h e o r e t i c a l l y i n a c t i v e ,  proved highly a c t i v e .  9-methyl-10-cyano-l,2-benzanthracene,  i n which the cyano group induces a low charge density at the K region, i s as active as the 9,10-dimethyl compound. 2.  Geometrical considerations Geometrical properties of the carcinogens  have been considered as w e l l .  Bergmann (2) suggested that  the disrupting effect of chemical carcinogens might take place through the adsorption of carcinogen by a p a r t i c u l a r c e l l constituent, i n which case surface area and p l a n a r i t y of the foreign molecule might be determining f a c t o r s .  The theory  explained, i n a simple mechanical way, why some molecules were inactivated by the introduction of substitufent groups. Later (19^5) F i e s e r (1?) put f o r t h the idea of selective adsorption by the c e l l walls, which might e a s i l y a f f e c t c e l l permeability.  Here again, geometrical considerations may be  important In determining the strength of the adsorptive f o r c e s . One success of such an hypothesis Is the explanation which the hypothesis affords of the metabolic products.  Oxidation  often appeared to take place at points other than active centres; and sometimes i n positions where " i n v i t r o " oxidation i s not possible at a l l .  -53»  Complex formation More recent theories involve reaction or  complex formation between the carcinogenic agent (or a metabolite) and some tissue protein.  Boyland (h) proposes that  i t i s the nucleoproteins that are involved. He has shown how physical, inorganic and organic carcinogens can react with desoxyribonucleic a c i d .  I f metabolism of the carcinogen  takes place during complex formation, i t i s possible that the<-> nucleic acid also i s altered, explaining the chemical mode of chromosome damage. Other workers favor the theory of interference with s u l phur metabolism (6,10,11).  Because of a c o r r e l a t i o n between  carcinogenic a c t i v i t y and c e r t a i n substitution reactions, F i e s e r (l *) suggested a reaction of the hydrocarbon with a 1  -S&S-  linkage  peptide l i n k .  of a peptide giving r i s e to a hydrocarbon Evidence supporting t h i s view has been pro-  duced by Crabtree (10,11,12).  He found that compounds,  having i n common only that they were excreted with sulphurcontaining amino acids, i n h i b i t the action of carcinogens. He proposes that "the i n h i b i t o r s by p r e f e r e n t i a l metabolism and excretion i n t e r f e r e with those c e l l constituents with which the carcinogens must combine i n order to act - those having -SH groups."  -6-  C.  The M i l l e r Complex A recent discovery by E l i z a b e t h M i l l e r (25) of the  formation of a protein-bound azo dye component i n the l i v e r after o r a l administration of the carcinogen to r a t s , has l e d to renewed i n t e r e s t i n the possible r o l e of complex formation. That the discovered complex was carcinogenesis, was 1.  i n some way  proposed f o r the following reasons;  Species which are susceptible to l i v e r  cancer (e.g. rats) also produce bound azo 2.  connected with  dye.  Species which are not susceptible to l i v e r  cancer (e.g. mice) produce no d e t e c t i b l e bound component. 3.  The bound azo dye i s found only i n the  l i v e r which i s the one organ affected by t h i s type of carcinogen. h.  Carcinogenic  potency p a r a l l e l s the amount  of bound azo dye found. I t was  conclusively shown that the i n t e r a c t i o n forces  between the carcinogen and the protein were not simple adsorption forces such as those proposed by Fie.ser and Bergmann, but forces approaching true chemical bonding.  The complex was  subjected to exhaustive extraction with solvents such as b o i l i n g alcohol, ether, benzene, toluene, and a f t e r a c e r t a i n amount of adsorbed carcinogen was  removed, no further f l u o r -  escent material could be detected i n the solvent. was  The complex  further tested by a s o l u t i o n - r e p r e c i p i t a t i o n process.  A f t e r any adsorbed azo dye had been removed, the l i v e r protein was  p a r t i a l l y dissolved  In a 1:2,5  mixture of ethanol - .IK".,  potassium hydroxide and afterwards precipitated with t r i c h l o r o acetic acid,  Fluor'scence i n d i c a t i n g the presence of azo dye  c l o s e l y related derivatives could s t i l l be detected  or  i n the hydrol-  yzate from the reprecipitated protein. M i l l e r extended her work to 3,)*--benzpyrene and epidermal Like the azo dyes, 3, *-benzpyrene also formed a  skin cancer.  l  protein complex. Wallick (2*+)  The same method was used by Moodie, Reid and  to investigate  a series of compounds  potent, weak and non carcinogens. M i l l e r work.  including  The r e s u l t s substantiated  the  The most potent carcinogens, 3,^-henzpyrene and  9,10-dimethyl-l,2-benzanthracene formed appreciable amounts of the protein bound complex; 1,2-benzanthracene formed a small amount of complex; anthracene showed no complex formation at all. At present work i s being done on the characterization of the protein involved as well as the determination of the mode of l i n k age between protein and carcinogen.  Using radioactive tracer  techniques, Hadler and Heidelberger (17) have been able to show that the hydrocarbon remains bound even a f t e r peptic h y d r o l y s i s . They are now  involved i n characterizing the r e s u l t i n g f r a c t i o n s .  M i l l e r , on the other hand, i s s t r i v i n g to i d e n t i f y the azo  dye  components l i b e r a t e d by hydrolysis of the l i v e r proteins,  The  detection of small amounts of 3 -methyl-U^monomethyl amino azo 1  benzene (5)  suggests that the amino nitrogen of the polar dye  may  be bound to the p r o t e i n . D.  Problem to be  Considered  One r e s u l t of the work of M i l l e r (25)  and of work i n  t h i s laboratory (2U-), Is that the formation of protein c a r c i n ogen complex was found to take place " i n vivo" only, no complex resulting even when mice were painted immediately a f t e r k i l l i n g . The problem of preparing complex from protein and carcinogen " i n v i t r o " thus a r i s e s n a t u r a l l y and i s the subject of t h i s i n v e s t i g ation.  I t was hoped that " i n v i t r o " complex might be prepared  i n s u f f i c i e n t quantity f o r a study of  the p r o t e i n linkage i n -  volved, and that a successful method of preparation might e l ucidate the corresponding  " i n vivo" process.  The necessity of  a sensitive method f o r i d e n t i f y i n g bound complex l e d to the development of chromatographic techniques separating hydrocarbon-protein plexed components.  and to a procedure f o r  complex from e i t h e r of the uncom-  EXPERIMENTAL METHOD A,  M i l l e r ' s method f o r preparing " i n vivo" carcinogen-protein complex A/LN mice, 6-10 weeks o l d , sex unsegregated, were treated  with carcinogen.  A portion of the back was shaved with s u r g i c a l  c l i p p e r s and 0.20 ml. of a solution (0.2-0.h% hydrocarbon i n benzene) was applied. The number of applications of carcinogen and the time i n t e r v a l before removal of the epidermis varied with the p a r t i c u l a r experiment.  A f t e r a minimum of twenty-four hours,  the mice were k i l l e d (using ether) and the treated area excised. The skins were immersed i n N/3 ammonia f o r t h i r t y minutes,  after  which the epidermis could e a s i l y be scraped from the dermis. The epidermis was wrapped i n f i l t e r paper and extracted with b o i l i n g ethanol f o r forty-eight hours i n a "multiple" soxhlet extractor.  Controls painted with benzene were run with a l l ex-  periments. (Method f o r detecting the complex w i l l be discussed l a t e r ) • B.  Methods employed i n the attempted formation of " i n v i t r o " hydrocarbon complex (1) Preparation of samples a.  Crude protein A/LN mice, 6-10 weeks o l d , sex unseg-  regated, were used f o r obtaining epidermal protein. the back, approximately h by 2 cm., was  A section of  shaved and the mice  -10k i l l e d with ether.  The shaved regions were excised, immersed i n  N/3 ammonia f o r t h i r t y minutes, and the epidermis scraped from the dermis.  The epidermis was homogenized with 85% ethanol and  10$ t r i c h l o r o a c e t i c acid i n a Waring Blendor.  I t was then dried  and used i n t h i s form. b.  Whole skin samples A/LN mice, 6-10 weeks o l d , sex unseg-  regated, were shaved as described above. using ether and the shaved areas excised.  The mice were k i l l e d The skins were used  i n t h i s form f o r treatment. c.  Intact mice A/LN mice, 6-10 weeks o l d , sex unseg-  regated, were shaved as previously described and k i l l e d using ether.  The shaved area was then treated. d.  Amino acids and proteins The best available grades of these  chemicals were used without further p u r i f i c a t i o n . e.  Purines and nucleic acids As i n ( d ) .  (2) I r r a d i a t i o n method Solutions were made up o f the following f o r samples (a), (b), and (d): h ml. of benzene containing 1 mg. of 3,^benzpyrene. h ml. of benzene containing 20 mg. of the protein, or protein preparation. h ml. of benzene containing 20 mg. of the protein,  -11amino acid, or protein preparation plus 1 mg. of 3, *-benzpyrene. l  Test tubes containing the solutions were suspended from a d r i l l e d plywood board at positions equidistant from a mercury AH h u l t r a v i o l e t l i g h t source.  For most of the experiments  solutions were made up i n t r i p l i c a t e f o r suspension at distances of 3, 6, or 9 cm.  from the l i g h t source.  Varying times of  i r r a d i a t i o n were used. The shaved area of (c) was painted with 0.2 ml. of »h% 3,V-benzpyrene i n benzene. AH h l i g h t source.  I t was then i r r a d i a t e d with a mercury  Samples were i r r a d i a t e d at varying i n t e n s i t i e s  and f o r varying periods of time.  Controls painted with benzene  only were subjected to the same treatment.  A f t e r treatment,  the shaved area was excised and the epidermis removed as described  under I I A* 3.  I r r a d i a t i o n and oxygen treatment (samples ( a ) ( b ) T  t  and (d) ) The i r r a d i a t i o n method was carried out while bubbling oxygen through the solutions. *t.  Benzoyl peroxide treatment Solutions of the following were prepared f o r  (a), (b), and (d): h ml. of benzene containing 20 mg. of the protein, amino acid or protein preparation plus 5 mg. of benzoyl peroxide.  -12h ml. of benzene containing 5 mg. of benzoyl peroxide plus 1 mg. of 3,^-benzpyrene. h ml. of benzene containing 20 mg.  of the protein, amino  acid, or protein preparation plus 5 mg. of benzoyl peroxide plus 1 mg. of 3,^benzpyrene. The solutions were allowed to stand at room temperature were incubated at 3 0 , HO,  or  and 50° C. f o r varying periods of time.  Sample (c) was painted either with 0 . 2 ml. of a solution 3,^-benzpyrene plus  containing  benzoyl peroxide or 0 . 2  ml. of a solution containing ,h% benzoyl peroxide alone.  Sample  (c) was allowed to stand at room temperature f o r only eight hours or at 0°C. f o r twenty-four hours. 5.  Benzoyl peroxide and oxygen treatment The benzoyl peroxide method was carried out  simultaneously bubbling oxygen through the solutions. 6.  Benzoyl peroxide plus i r r a d i a t i o n treatment Solutions of samples (a), (b), and (d) were  prepared as described under benzoyl peroxide treatment.  The t e s t  tubes were suspended f o r i r r a d i a t i o n as described under i r r a d i a t i o n method.  This treatment was carried out with and without  bubbling oxygen through the solutions.  I r r a d i a t i o n was  carried  out f o r varying periods of time. 7»  Enzyme method Duplicate samples of 20 mg. of (a) or 100  mg.  -13of (b) were made up i n 1% solutions of t r y p s i n , pepsin or pancreatin.  One milligram of S ^ b e n z p y r e n e was added to one of  each duplicate sample.  Solutions were either incubated at 3 0 , hO  or 50°C. f o r varying periods of time or i r r a d i a t e d as described under i r r a d i a t i o n method. 8.  Incubation i n a tissue culture solution (Ringer Locke solution)(c) A/LN mice, s i x weeks o l d , sex unsegregated,  were shaved as previously described and k i l l e d with ether.  Imm-  ediately the back section was excised and f l o a t e d onto a p e t r i dish containing Ringer Locke solution.  Half the skin samples  were painted with 0 . 2 ml. of a solution of ,h% Sj^benzpyrene i n benzene.  The other h a l f were used as controls and were painted  with 0 . 2 ml. benzene.  The treated skins were incubated at 9 8 ° F  f o r 1 2 , 2*+, 3 6 . and  hours.  The epidermis was then removed as  i n I I A. 9»  Incubation i n a tissue culture solution (Ringer Locke solution) ( d ) (e) T  Twenty milligrams  of samples (d) and (e) were  dissolved i n 10 ml. of Ringer-Locke s o l u t i o n . hydrocarbon was added to the solutions.  F i v e milligrams of  (Control samples contain-  ing no 3,^benzpyrene were run with each experiment.)  A i r was  bubbled through the solutions which were incubated from twelve to eighteen hours. of 1 ml. of  (One experiment was c a r r i e d out with the addition  -lH•*4-N potassium hydroxide and 1 ml. of 95% ethanol to increase the s o l u b i l i t y of the protein.) G.  M i l l e r ' s method of detecting complex formation This method of detection depends upon breakdown of the  complex by complete hydrolysis of the protein with subsequent release of a hydrocarbon fragment.  Presence of complex form-  ation i s detected by fluorescence of the released hydrocarbon derivative.  Success of the scheme e n t a i l s the complete removal  of a l l adsorbed hydrocarbon before hydrolysis. The sample to be investigated was wrapped i n f i l t e r paper and extracted with b o i l i n g ethanol i n a "multiple soxhlet ext r a c t o r " f o r forty-eight hours.  I t was then dried and weighed.  20-50 mg. were treated with 2 ml. of ethanol, 5 ml of M-N. potassium hydroxide, 5 ml. of toluene and 1.6 g. of activated zinc dust.  The mixture was refluxed f o r three hours.  The cooled  solution was extracted three times with benzene and the combined extractions set aside f o r fluorescence determinations.  The  o r i g i n a l solution (water layer) was a c i d i f i e d with d i l u t e hydroc h l o r i c acid and again extracted with benzene. solutions were also combined.  These benzene  Fluorescence spectra of the  "neutral" and " a c i d i c " f r a c t i o n s were obtained using a Hilger E 2 spectrograph combined with a H i l g e r scanning u n i t and recording system.  The l i g h t source was an AH 6 high pressure mercury  arc with suitable glass and l i q u i d f i l t e r s f o r i s o l a t i n g the  -15mercury 3650 , 3100 A l i n e s , D,  Chromatographic method of detecting complex formation Because of the l i m i t a t i o n s and time consumption of the  M i l l e r method, another means of detecting complex formation was developed.  The new method i s based upon change of s o l u b i l i t y  properties of protein and hydrocarbon when i n a combined state. For t h i s reason, removal of adsorbed hydrocarbon i s not necessary.  A p p l i c a b i l i t y of the method and detection l i m i t s were  tested using a hydrocarbon-amino acid conjugate,  £ (1,2-  benzanthryl-10-carbamido)caproic a c i d . I n i t i a l treatment of the sample was necessary i n the case of epidermal protein samples, i n order to dissolve the proteins, which were not soluble i n water or Ringerl-Locke solution. Twenty mg. of epidermal protein was digested i n 10 ml. of l s 2 . 5 ethanol .IN potassium hydroxide at 55°C. f o r twelve hours.  The s o l u t i o n  was then concentrated at 55°C» to 2 ml. The solutions to be investigated were applied to Whatman No. 1 chromatographic paper. applications were made.  In some cases as.-, many as four  The chromatogram was c a r e f u l l y dried  over a hot plate,before e l u t i n g , because of the Immiscibility of the solvents.  The elutarit used was a water saturated  solution  of benzene. Because of the almost complete i n s o l u b i l i t y of protein and protein-hydrocarbon complex i n benzene, both of these components  -16remained at the s i t e of a p p l i c a t i o n .  The hydrocarbon, on the  other hand, being r e l a t i v e l y soluble i n benzene, t r a v e l l e d with the solvent f r o n t .  The solvent moved very r a p i d l y , good separ-  ation taking place i n 2-3 hours.  Complex formation was  detected,  a f t e r chromatographing the solution, by the c h a r a c t e r i s t i c bluev i o l e t fluorescence of the hydrocarbon complex at the s i t e of application.  When the complex was to be separated from the pro-  t e i n , the benzene eluted chromatogram was dried and re-eluted with butanol-acetic acid-water  (*f:ls!?).  In t h i s solvent the  of the protein (or amino acid) ranged from 0-.6, the complex approached 1.  while the  R  f  of  -17-  RESULTS AND DISCUSSION As i s b r i e f l y mentioned i n the introduction, the s t a r t i n g point of t h i s project was the attempted formation of " i n v i t r o " hydrocarbon-protein complexes.  Destructive reduction of the " i n  vivo" complex ( 2 5 ) , together with work on hydrocarbon ism ( 6 , 7 , 8 , 9 , 1 7 , 1 8 , 1 9 , 2 9 , 3 0 , 3 2 , 3 5 )  metabol-  had given some idea of the  possible linkage involved. Wiegert and Mottram ( 3 3 ) were able to  separate and t e n t a t i v e l y i d e n t i f y four metabolic products of  3,H-benzpyrene  found i n various tissues and as excretion prod-  ucts a f t e r i n j e c t i o n of the carcinogenic hydrocarbon.  The con-  clusions drawn were that the f i r s t pair of metabolites were substituted diols,  and that the other pair of metabolites ( e a s i l y formed from the f i r s t by " i n v i t r o " treatment with acid) were the f u l l y aromat ized rings produced by the removal of Water or alcohol.  F,  That a similar phenolic or hydroxy d e r i v a t i v e might be involved i n complex formation was suggested by the production of an a c i d i c hydrocarbon f r a c t i o n after zinc dust reduction of the " i n v i v o " hydrocarbon-protein complex, of  "since the fluorescence spectrum  t h i s derivative i s very s i m i l a r to that of the parent hydro-  carbon, i t was assumed that no r a d i c a l changes had taken place  -18i n the ring skeleton but that substituent groups, possibly hydroxy groups, had been introduced.  A linkage between the amino  group of the protein and the hydroxy group of the hydrocarbon was  therefore proposed.  For these reasons " i n v i t r o " complex  formation was attempted through various oxidative processes. 1.  Irradiation The f i r s t method selected was  of sample with u l t r a v i o l e t l i g h t .  that of i r r a d i a t i o n  Although photo oxidation  cannot be envisioned l n the i n t e r i o r organs of an animal where malignant growths are formed, s t i l l , i n any l i v i n g system there are b i o l o g i c a l reactions taking place with the release of energy which might, perhaps, stimulate the same type of t r a n s i t i o n . Alexander's recent success i n c o r r e l a t i n g ease of primary photo oxidation and carcinogenic a c t i v i t y suggested that such a method might prove f r u i t f u l i n the problem of " i n v i t r o "  carcinogen-  protein complex formation. The samples to be investigated were suspended i n 3,^benzpyrene solution and i r r a d i a t e d f o r d i f f e r e n t lengths of time at various l i g h t i n t e n s i t i e s .  Mouse epidermis and whole skin were  chosen as protein samples since they are known to contain the component necessary f o r " i n vivo" complex formation.  However,  d i f f i c u l t i e s with irreproducible r e s u l t s l e d to the i n v e s t i g a t i o n of simpler protein and amino acid systems as w e l l .  What was  l o s t by specifying the "protein part" of the complex was hoped to be gained by obtaining c l e a r cut, reproducible r e s u l t s .  -19That I r r a d i a t i o n treatment was unsuccessful i s clearlyshown by Table 1.  In no instance could complex formation be  unequivocally detected.  The control samples and 3,Wbenzpyrene  treated samples behaved i n i d e n t i c a l manner. 2.  I r r a d i a t i o n with oxygen treatment In view of the key r o l e of oxygen i n many biochemical  processes, the p o s s i b i l i t y that i r r a d i a t i o n need be accompanied by oxygen was next looked i n t o .  Oxygen bleeds were inserted i n  a l l test solutions while i r r a d i a t i o n was carried out.  Table 2  shows that t h i s method also produced no p o s i t i v e r e s u l t s . 3.  Benzoyl peroxide The next step was to t r y a chemical oxidizing  agent.  I f oxidation products are involved, i t i s possible that  photo oxidation does not produce the required products f o r complex formation and that a compound such as benzoyl peroxide might be more successful.  Benzoyl peroxide was chosen c h i e f l y  f o r i t s s o l u b i l i t y properties and i t s known action on 3,*+-benzpyrene.  Conditions such as temperature  and incubation time  were varied and oxidation was repeated i n s e r t i n g oxygen bleeds i n the solutions.  Tables 3 and h  show that under the above  conditions, benzoyl peroxide was unable to stimulate " i n v i t r o " complex formation.  -20-  Table 1 Showing the r e s u l t s of attempted complex formation of 3,U-benzpyrene with various proteins and amino acids. Stimulus - u l t r a violet light. Sample  X  A  t  Results  mouse epidermis  30  7  S  neg.  mouse epidermis  60  7  S  neg.  mouse epidermis  90  7  S  neg.  whole skin  30  7  S  neg.  whole skin  60  7  S  neg.  whole skin  90  7  S  neg.  i n t a c t mouse  30  7  S  neg.  i n t a c t mouse  60  7  S  neg.  i n t a c t mouse  90  7  S  neg.  egg albumin  60  3  s  neg.  egg albumin  60  6  s  neg.  egg albumin  60  9  s  neg.  casein  60  3  s  neg.  casein  60  6  s  neg.  casein  60  9  s  neg.  tryptophane  60  3  c  neg.  tryptophane  60  6  c  neg.  tryptophane  60  9  c  neg.  histidine  60  3  c  neg.  histidine  60  6  c  neg.  histidine  60  9  c  neg.  cystine  60  3  c  neg.  -21-  Sample  A  h  Results  cystine  60  6  C  neg.  cystine  60  9  C  neg.  tyrosine  60  3  C  neg.  tyrosine  60  6  C  neg.  tyrosine  60  9  c  neg.  threonine  60  3  c  neg.  threonine  60  6  c  neg.  threonine  60  9  c  neg.  leucine  60  3  G  neg.  leucine  60  6  C  neg.  leucine  60  9  C  neg.  valine  60  3  e  neg.  valine  60  6  c  neg.  valine  60  9  c  neg.  proline  60  3  c  neg.  proline  60  6  c  neg.  proline  60  9  c  neg.  phenylalanine  60  3  c  neg.  phenylalanine  60  6  c  neg.  phenylalanine  60  9  c  neg.  In which I - irradiation time (min) i j - irradiation distance (cm.) A - analysis by S - spectrograph C - chromatography t  Table 2 Showing the results of attempted complex formation of 3,+-benzpyrene with various proteins and amino acids. Stimulus - u l t r a violet irradiation plus oxygen. l  Sample  V  A  Results  mouse epidermis  30  7  S  neg.  mouse epidermis  60  7  S  neg.  mouse epidermis  90  7  S  neg.  whole skin  30  7  S  neg.  whole skin  60  7  S  neg.  whole skin  90  7  S  neg.  egg albumin  60  3  S  neg.  egg albumin  60  6  S  neg.  egg albumin  60  9  S  neg.  tryptophane  60  3  C  neg.  tryptophane  60  6  neg.  tryptophane  60  9  histidine  60  3  histidine  60  6  histidine  60  9  cystine  60  3  cystine  60  cystine  60  9  phenylalanine  60  3  phenylalanine  60  6  phenylalanine  60  9  tyrosine  60  3  tyrosine  60  6  c c c c c c c c c c c c c  neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg.  -23Sample  A  Results  tyrosine  60  9  C  neg.  proline  60  3  C  neg.  proline  60  6  c  neg.  proline  60  9  c  neg.  casein  60  3  G  neg.  casein  60  6  C  neg.  casein  60  9  C  neg.  In which 1^ - irradiation time (min.) I - irradiation distance (cm.) A - analysis oy S - spectrograph C - chromatography d  -2H-  Table 3 Showing the results of attempted complex formation of 3,H-henzpyrene with various proteins and amino acids. Stimulus - benzoyl peroxide.  A  Results  30  S  neg.  2h  30  S  neg.  mouse epidermis  36  30  S  neg.  mouse epidermis  12  ho  S  neg.  mouse epidermis  2h  hQ  S  neg.  mouse epidermis  36  ho  S  neg.  mouse epidermis  12  50  S  neg.  mouse epidermis  2h  50  S  neg.  mouse epidermis  36  50  s  neg.  whole skin  12  30  s  neg.  whole skin  2h  30  s  neg.  whole skin  36  30  s  neg.  whole skin  12  ho  s  neg.  whole skin  2h  ho  s  neg.  whole skin  36  ho  s  neg.  whole skin  12  50  s  neg.  whole skin  2*+  50  s  neg.  whole skin  36  50  s  neg.  whole mouse  8  20  s  neg.  whole mouse  2h  0  s  neg.  Sample  lnc  mouse epidermis  12  mouse epidermis  t  Inc  T  -25-  Sample  Inc  t  !nc T  A  Results  egg albumin  12  30  s  neg.  egg albumin  2h  30  s  neg.  egg albumin  36  30  s  neg.  egg albumin  12  HO  s  neg.  egg albumin  2h  h0  s  neg.  egg albumin  36  ho  s  neg.  egg albumin  12  50  s  neg.  egg albumin  2h  50  s  neg.  egg albumin  36  5o  s  neg.  casein  12  30  s  neg.  casein  2h  30  s  neg.  casein  36  30  s  neg.  casein  12  ho  s  neg.  casein  2h  ho  s  neg.  casein  36  ho  s  neg.  casein  12  50  s  neg.  casein  2h  50  s  neg.  casein  36  50  s  neg.  tryptophane  12  30  o  neg.  tryptophane  2h  30  c  neg.  tryptophane  36  30  c  neg.  tryptophane  12  ho  c  neg.  1  -26-  Sample  lnc  t  Ine  T  A  Results  tryptophane  2k-  ko  c  neg.  tryptophane  36  ko  c  neg.  tryptophane  12  50  c  neg.  tryptophane  2k  50  c  neg.  tryptophane  36  50  c  neg.  histidine  12  30  c  neg.  histidine  2H-  30  c  neg.  histidine  36  30  c  neg.  histidine  12  ko  c  neg.  histidine  2k  kO  c  neg.  histidine  36  l+O  c  neg.  histidine  12  50  c  neg.  histidine  2k  50  c  neg.  histidine  36  50  c  neg.  cystine  12  30  c  neg.  cystine  2k-  30  c  neg.  cystine  36  30  c  neg.  cystine  12  ko  c  neg.  cystine  2k  UO  c  neg.  cystine  36  ko  c  neg.  cystine  12  50  c  neg.  cystine  2k  50  c  neg.  cystine  36  50  c  neg.  -27-  Sample  lnc  t  Inc  T  A  Result  0  neg.  phenylalanine  12  30  phenylalanine  2U  30  c  neg.  phenylalanine  36  30  _G  neg.  phenylalanine  12  1+0  c  neg.  phenylalanine  2U  1+0  C  neg.  phenylalanine  36  ko  c  neg.  phenylalanine  12  50  C  neg.  phenylalanine  2U  50  c  neg.  phenylalanine  36  5©  c  neg.  tyrosine  12  30  c  neg.  tyrosine  2k  30  c  neg.  tyrosine  36  30  c  neg.  tyrosine  12  uo  C  neg.  tyrosine  2k  uo  c  neg.  tyrosine  36  uo  c  neg.  tyrosine  12  50  c  neg.  tyrosine  2k  5o  C  neg.  proline  12  30  G  neg.  proline  2k  30  G  neg.  proline  36  30  C  neg.  proline  12  uo  C  neg.  proline  2k  UO  C  neg.  proline  36  UO  C  neg.  -  -28-  Sample  Inc  t  Inc  T  A  Results  proline  12  50  G  neg.  proline  2h  50  C  neg.  proline  36  50  G  neg.  In which Inc Inc A  t T  - Incubation time (hrs.) - Incubation temperature (°C.) - analysis by S - spectrograph C - chromatography  -29Table h Showing the r e s u l t s of attempted complex formation of 3,^-benzpyrene with various proteins and amino acids. Stimulus - benzoyl peroxide plus oxygen.  Sample  Inc  t  lnc  T  A  Result  mouse epidermis  12  30  S  neg.  mouse epidermis  2h  30  s  neg.  mouse epidermis  36  30  s  neg.  whole skin  12  30  s  neg.  whole skin  2h  30  s  neg.  whole skin  36  30  s  neg.  egg albumin  12  50  s  neg.  egg albumin  2h  50  s  neg.  egg albumin  36  50  s  neg.  casein  12  50  s  neg.  casein  2h  50  s  neg.  casein  36  50  s  neg.  tryptophane  12  50  c  neg.  tryptophane  2h  50  c  neg.  tryptophane  36  50  c  neg.  histidine  12  50  c  neg.  histidine  2\  50  c  neg.  histidine  36  50  c  neg.  cystine  12  50  c  neg.  cystine  2h  50  G  neg.  -30-  Sample  Inc  t  Inc  T  A  Results  cystine  36  50  c  neg.  phenylalanine  12  50  c  neg.  phenylalanine  2&  50  c  neg.  phenylalanine  36  50  c  neg.  tyrosine  12  50  c  neg.  tyrosine  2h  50  G  neg.  tyrosine  36  50  C  neg.  proline  12  50  C  neg.  proline  2\  5o  C  neg.  proline  36  50  C  neg.  In which Inc Inc A  t T  - Incubation time (hrs.) - incubation timperature. (°C.) - analysis by S - spectrograph G - chromatography  -31U.  Benzoyl peroxide accompanied by u l t r a v i o l e t i r r a d i a t i o n A l a s t v a r i a t i o n of the oxidative process was made by  combining u l t r a v i o l e t i r r a d i a t i o n with chemical oxidation.  It  i s possible that the " i n vivo" process involves I n i t i a l l y a molecular complex, formed through an excited state of the hydrocarbon, and secondly oxidation of the associated hydrocarbon to form a more f i r m l y bound complex.  I f t h i s were the case, i r r a d i a t i o n  and chemical oxidation might be able to simulate " i n vivo" cond i t i o n s well enough f o r successful " i n v i t r o " complex formation. Tables 5 and 6 reproduce the results that i n no cases were detectible amounts of complex formed. 5.  Enzyme and incubation treatment One possible conclusion to be drawn from the pro-  ceeding negative r e s u l t s i s that i f a simple mechanism of complex formation (such as linkage through an hydroxy group on the hydrocarbon and an amino group of the protein) i s assumed, then f a i l u r e to produce " i n v i t r o " complex may be due to other f a c t o r s .  The  i n a b i l i t y of the hydrocarbon to come i n contact with the necessary tissue protein i n a dead system i s a p o s s i b i l i t y .  Experiments  were developed i n an attempt to overcome t h i s d i f f i c u l t y .  In  one case the protein was kept a l i v e by incubation i n a tissue culture solution, while i n the other digestive enzymes were used to render the protein water soluble.  Better r e s u l t s were  anticipated from the incubation method since hydrocarbon metabolites have reportedly been found i n t h i s way.  Weigert ( 3 D , using  3,U-benzpyrene and incubation of mouse skin i n Ringer-Locke  -32Table 5 Showing the r e s u l t s of attempted complex formation of 3,Wbenzpyrene with various proteins and amino acids. Stimulus - benzoyl peroxide plus u l t r a v i o l e t i r r a d i a t i o n .  A  Results  30  S  neg.  12  30  s  neg.  6  36  30  s  neg.  90  3  36  50  s  neg.  whole skin  60  6  12  30  s  neg.  whole skin  90  6  12  30  s  neg.  whole skin  90  6  36  30  s  neg.  whole skin  90  3  36  50  s  neg.  whole mouse  60  6  8  20  s  neg.  whole mouse  90  6  8  20  s  neg.  whole mouse  90  6 -  2h  0  s  neg.  egg  albumin  60  6  12  30  s  neg.  egg  albumin  90  6  12  30  s  neg.  egg  albumin  90  6  36  30  s  neg.  egg  albumin  90  3  36  50  s  neg.  casein  60  6  12  30  s  neg.  casein  90  6  12  30  s  neg.  casein  90  6  36  30  s  neg.  casein  90  3  36  50  s  neg.  Sample  *t  X  mouse epidermis  60  6  12  mouse epidermis  90  6  mouse epidermis  90  mouse epidermis  d  lnc  t  -33-  Sample  h  I d  Inc t  Inc  A  Results  T  tryptophane  60  6  12  30  C  neg.  tryptophane  90  6  12  30  C  neg.  tryptophane  90  6  36  30  c  neg.  tryptophane  90  3  36  50  c  neg.  histidine  60  6  12  30  c  neg.  histidine  90  6  12  30  c  neg.  histidine  90  6  36  30  c  neg.  histidine  90  3  36  50  c  neg.  cystine  60  6  12  30  c  neg.  cystine  90  6  12  30  c  neg.  cystine  90  6  36  30  c  neg.  cystine  90  3  36  50  c  neg.  phenylalanine  60  6  12  30  c  neg.  phenylalanine  90  6  12  30  c  neg.  phenylalanine  90  6  36  30  c  neg.  phenylalanine  90  3  36  50  c  neg.  In which 1^ I Inc Incm A d  t  i r r a d i a t i o n time (min.) i r r a d i a t i o n distance (cm.) - incubation time (hrs.) - incubation temperature (°C.) - analysis by S - spectrograph G - chromatography  Table 6 Showing the r e s u l t s of attempted complex formation of 3, +-benzpyrene with various proteins and amino acids. Stimulus - benzoyl peroxide plus u l t r a v i o l e t i r r a d i a t i o n plus oxygen. l  Sample  h  lnc  t  Inc  T  A  Results  Mousl epidermis  30  7  2k  30  S  neg.  mouse epidermis  60  7  2U  30  s  neg.  mouse epidermis  90  7  2k  30  s  neg.  whole skin  30  7  2k  30  s  neg.  whole skin  60  7  2k  30  s  neg.  whole skin  90  7  2k  30  s  neg.  egg  albumin  60  3  2k  30  s  neg.  egg  albumin  60  6  2k  30  s  neg.  egg  albumin  60  9  2k  30  s  neg.  casein  60  3  2k  30  s  neg.  casein  60  6  2k  30  s  neg.  casein  60  9  2k  30  s  neg.  tryptophane  60  3  2k  30  c  neg.  tryptophane  60  6  2k  30  c  neg.  tryptophane  60  9  2k  30  c  neg.  histidine  60  3  2k  30  c  neg.  histidine  60  6  2k  30  c  neg.  histidine  60  9  2k  30  c  neg.  cystine  60  3  2k  30  c  neg.  cystine  60  6  2k  30  c  neg.  cystine  60  9  2k  30  c  neg.  -35-  Sample  T  t  X  d  Inc. t  Inc  T  A  Results  phenylalanine  60  3  2h  30  G  neg.  phenylalanine  60  6  2h  30  C  neg.  phenylalanine  60  9  2h  30  C  neg.  tyrosine  60  3  2h  30  C  neg.  tyrosine  60  6  2h  30  C  neg.  tyrosine  60  9  2h  30  C  neg.  proline  60  3  2h  30  C  neg.  proline  60  6  2h  30  C  neg.  proline  60  9  2\  30  C  neg.  In which I ij Inc Inc„ A t  t  irradiation time (mins.) irradiation distance (cms.) - incubation time (hrs.) - incubation temperature (°C.) - analysis by S - spectrograph G - chromatography  -36solution, was able to i s o l a t e one of the metabolites previously discovered a f t e r the " i n vivo" i n j e c t i o n of 3,U-benzpyrene. The r e s u l t s of these experiments are given i n Tables 7 and 8.  In both cases detection of " i n v i t r o " formed complex could  not be made. 6.  Purines and nucleic acids The reported increase i n water s o l u b i l i t y of var-  ious aromatic carcinogens i n purine and nucleic acid solutions by Booth and Boyland (3)  and Weil-Malherbe  (3U)  suggested that  some type of complex formation was taking place.  In view of the  f a i l u r e to produce " i n v i t r o " hydrocarbon-"protein" complex with epidermal protein or with a series of amino acids, focus was directed toward the purines and nucleic acids.  Table 9 shows the  cases f o r which complex formation could be detected. Caffeine, hypoxanthine,  and desoxyribonucleic acid showed s i g n i f i c a n t  quantities of fluorescent derivative, while complexed u r a c i l and ribose nucleic acid were just d e t e c t i b l e . The significance of these complexes i n r e l a t i o n to the cancer problem was further investigated by substituting a series of carcinogenic and non-carcinogenic hydrocarbons f o r 3,*+-benzpyrene. Table 10 shows that there does not seem to be any c o r r e l a t i o n between t h i s type of complex formation of aromatic  hydrocarbons  -37Table 7 Showing the results of attempted " i n v i t r o " complex formation of 3,^-benzpyrene with epidermal protein i n various digestive enzymes. Sample  Enzyme  Inc  mouse epidermis  T  mouse epidermis  InCrp  A  Result  12  30  C  neg.  Pe  12  30  c  neg.  mouse epidermis  Pa  12  30  c  neg.  mouse epidermis  T  2h  1+0  c  neg.  mouse epidermis  Pe  2h  hO  c  neg.  Pa  2h  hO  c  neg.  mouse epidermis  T  36  ho  c  neg.  mouse epidermis  Pe  36  ho  c  neg.  mouse epidermis  Pa  36  ho  c  neg.  mouse epidermis  T  30  7  c  neg.  mouse epidermis  Pe  60  7  c  neg.  mouse epidermis  Pa  90  7  c  neg.  mouse epidermis  T  30  7  c  neg.  mouse epidermis  Pe  60  7  c  neg.  mouse epidermis  Pa  90  7  c  neg.  whole skin  T  36  30  c  neg.  whole skin  Pe  36  30  c  neg.  whole skin  Pa  36  30  c  neg.  whole skin  T  21+  1+0  30  7  c  neg.  whole skin  Pe  21+  1+0  60  7  c  neg.  mouse epidermis  5  t  -38-  Sample  Enzyme  lnc  t  Inc  T  I t  I d  A  Results  whole skin  Pa  2  k.  UO  90  7  G  neg.  whole skin  T  36  30  30  7  C  neg.  whole skin  Pe  36  30  60  7  C  neg.  whole skin  Pa  36  30  90  7  C  neg.  In which T Pe Pa Inc. Inc!; I. i A a  trypsin pepsin pancreatin - incubation time (hrs.) - incubation temperature; (°C.) i r r a d i a t i o n time (mins.) i r r a d i a t i o n distance (cms.) analysis by S - spectrograph C - chromatography  -39-  Table 8 Showing the r e s u l t s o f attempted " i n v i t r o " complex f o r m a t i o n of 3,^-benzpyTene w i t h epidermal p r o t e i n i n Ringer-Locke s o l u t i o n .  A  Results  98  S  neg.  k  98  S  neg.  whole skin  36  98  S  neg.  whole skin  i+8  98  S  neg.  whole skin  12  98  C  neg.  whole skin  2h  98  C  neg.  whole skin  36  98  C  neg.  whole skin  1+8  98  c  neg.  Sample  lnc  whole skin  12  whole skin  2  t  lnc  T  In which Inc. - incubation time (hrs.) Inc« - incubation temperature (°F) A - analysis by S - spectrograph C - chromatography  Table 9 Showing the r e s u l t s of attempted complex formation of 3, «-benzpyrene with proteins, purines, and amino a c i d s . l  Results  Inc. t  *nc  caffeine  16  37  C  pos.***  tryptophane  16  37  C  neg.  egg albumin  16  37  C  neg.  ribonucleic acid  16  37  C  pos.*  16  37  c  pos.**  cystine  16  37  c  neg.  adenine  12  39  c  neg.  thymine  12  39  c  neg.  hypoxanthine  12  39  c  pos.**  uracil  12  39  c  pos.*  thiamine HC1  12  39  c  neg.  riboflavine  12  39  G  neg.  guanine HC1  18  37  C  neg.  tyrosine  18  37  C  neg.  threonine  18  37  C  neg.  cystine  18  37  c  neg.  leucine  18  37  C  neg.  valine  18  37  C  neg.  Sample  desoxyribonucleic  acid  T  A  neg. G 18 histidine 37 In which Inc. - incubation time (hrs.) Inc™ - incubation temperature (°C) A - analysis by C - chromatography * * * * * * _ indicates the r e l a t i v e quantity of complex formed.  -Hi-  Table 10 Showing the results of attempted complex formation of a series of hydrocarbons with proteins and purines i n Ringer-Locke solution. Sample  Inc  t  Inc  T  Hydrocarbon  A  Results  caffeine  18  37  1,2-benzpyrene  C  pos.**  desoxyribonucleic acid  18  37  1,2-benzpyrene  C  tpos.**  egg albumin  18  37  1,2-benzpyrehe  C  neg.  ribonucleic acid  18  37  1«2-benzpyrene  C  pos.*  hypoxanthine  18  37  1,2-benzpyrene  C  pos.*  caffeine  18  37  1,2-benzanthracene C  pos.*  desoxyribonucleic acid  18  37  1,2-benzanthracene C  neg.  hypoxanthine  18  37  1,2-benzanthracene C  pos.*  ribonucleic acid  18  37  1,2-benzanthracene G  neg*  desoxyribonucleic acid  17  ho  naphthacene  C  neg.  desoxyribonucleic acid  17  ho  phenanthrene  C  neg.  desoxyribonucleic acid  17  ho  fluoranthene  C  neg.  desoxyribonucleic acid  17  ho  methylcholanthrene  C  neg.  desoxyribonucleic acid  17  ho  9,10-dimethyl1,2-benzanthracene C  neg.  l,2,5,6 dibenzanthracene C  neg.  desoxyribonucleic acid  17  ho  T  !  1  In which Inc - incubation time (hrs.) Inc>p 6 incubation temperature (°C.) A - analysis by C - chromatography **•,**,* - indicates the relative quantity of complex formed.  -1+2-  with various purines or with desoxyribonucleic acid, and carcinogenic a c t i v i t y of the hydrocarbon. L e i t e r and Shear's work ( 2 3 )  i n d i c a t i n g that various purines  and nucleic acids retard tumor formation when injected with 3,U-benzpyrene, ation.  correlates w e l l with the r e s u l t s of complex form-  The purines - caffeine, hypoxanthine, and desoxyribo-  nucleic acid retard tumor formation and form complex "compounds" with 3,U-benzpyrene.  Retardation of tumor growth i s thus most  probably the r e s u l t of complex formation.  However, from the  r e s u l t s with hydrocarbons other than 3,U-benzpyrene, i t would seem that complex formation i s a function more of the structure of the molecule than of i t s b i o l o g i c a l a c t i v i t y .  Booth and  Boy-  land ( 3 ) have reported molecular complexes between several purines and a series of dibenzacridines and dibenzcarbazoles.  Spectro-  scopic analysis of solutions of the complexes reveals that they are r e l a t i v e l y weakly bound; apparently weaker than the hydrocarbon complexes discussed here.  Chromatographic detection or  separation would i n these cases be unsuccessful i f the dissoci a t i o n constants were high enough that e l u t i o n with a hydrocarbon solvent completely dissociated the complex.  The negative r e s u l t s  shown i n Tables 9 and 1 0 r e f e r only to "stable" complex formation i n which the product does not dissociate during the chromatography i n t h i s manner. 7.  Chromatographic method f o r detecting complex formation Several d i f f i c u l t i e s with the spectroscopic method  -V3of detecting complex formation l e d to the development of a new method.  In some cases background fluorescence of uncomplexed pro-  t e i n made i n t e r p r e t a t i o n of the spectroscopic traces uncertain. In a l l cases, due to the necessity of removing uncomplexed hydrocarbon, the procedure proved unduly time consuming.  For these  reasons the p o s s i b i l i t y of using chromatographic techniques was investigated.  The very d i f f e r e n t s o l u b i l i t y properties of poly-  peptides and poly c y c l i c aromatic hydrocarbons suggested that a p a r t i t i o n process would be capable of separating both these components, not only one from the other, but also from a complex of the two. In order to develop t h i s method of separation and to f i n d some way of locating the complex once i t had been separated, an amino acid-hydrocarbon conjugate,  £ (l,2-benzanthryl-10-carbam-  ido)caproic acid was synthesized. The synthesis followed was that of Creech et a l (13) where the amino acid was conjugated to the hydrocarbon through the hydrocarbon isocyanate. Tests with amino acid sprays, d i l u t e potassium permanganate and u l t r a v i o l e t i r r a d i a t i o n indicated that the conjugate could best be detected by i t s c h a r a c t e r i s t i c fluorescence.  Dilute  potassium permanganate gave p o s i t i v e r e s u l t s , only when the conjugate was present i n concentrations greater than that required to produce considerable fluorescence.  Amino acid sprays such as  ninhydrin, requiring both a free amino and carboxylic group, were, of course, i n e f f e c t i v e .  Figures 1 and 2 show the chromatograms that were obtained using benzene and butanol-water-acetic  acid elutants upon various  combinations of hydrocarbon, amino acid, and hydrocarbon-amino acid conjugate.  U l t r a v i o l e t i r r a d i a t i o n was used to locate the  conjugate and the f r e e hydrocarbon; while development with n i n hydrin revealed the p o s i t i o n of the amino a c i d .  The minimum con-  centration of conjugate that could be detected was  determined  as l y i n g within the region 2.8 X 10"*^ to .71 X 10"^ gm.1 . -1  Results of the preliminary work with  £  (l,2-benzanthryl-10-  carbamido)caproic acid suggested the p o s s i b i l i t y of  separation  of a protein-hydrocarbon complex from i t s uncomplexed components as well as rapid and c l e a r cut detection.  A p p l i c a b i l i t y of the  method was further tested using " i n vivo" complex i n place of the hydrocarbon-amino acid conjugate.  The one prerequisite of the  method i s that the substances to be separated he soluble i n some solvent i n order that they may paper.  be applied to chromatographic  Here l i e s the d i f f i c u l t y i n work with complex proteins,  f o r the only e f f i c i e n t complex protein solvent ( just recently discovered as such) i s hydrofluoric a c i d . The work of M i l l e r and Heidelberger  with " i n vivo"  carcinogen-  protein complex has shown that a good proportion of the hydrocarbon remains attached a f t e r p a r t i a l hydrolysis of the protein with d i l u t e sodium hydroxide.  This method of " d i s s o l v i n g " the  protein was used f o r the preparation of a solution of " i n vivo"  F I G . l . Chromatogram showing the s e p a r a t i o n o f 1,2-ben?,anthracone (1) from c a p r o i c a c i d (2) and conjugate (3)sbenzene e l u t a n t .  FIG.2. Chromatogram showing t h e s e p a r a t i o n o f c a p r o i c a c i d (2) from 1,2-benzanthracene (1) and conjugate (3)sBu.Ac.H20 elutant.  -U6. complex.  The complex may  have been altered by mild hydrolysis,  but i t s t i l l remains attached fragment.  Separation of the complex from both f r e e hydrocarbon  and protein was two  to part of the o r i g i n a l protein  successive  accomplished solvents.  by chromatographing the mixture i n  Benzene elutant, which had no e f f e c t on  the protein or on the complex was used f i r s t to remove any f r e e or adsorbed hydrocarbon.  After drying, the chromatogram was  eluted with butanol-water-acetic acids and polypeptides  acid.  The R^, values f o r the amino  present ranged from 0 to 0,6,  of the complex approached u n i t y .  re-  while that  -H7CONCLUSIONS AND SUGGESTIONS FOR FURTHER WORK The r e s u l t s of t h i s work, while not exactly those I n i t i a l l y hoped f o r , did produce a clearer picture of the problem and l e d to a new method f o r investigation of the " i n vivo" protein-hydrocarbon complex. F a i l u r e of oxidative processes to stimulate complex formation i s not without explanation.  In systems as complex as b i o l o g i c a l  systems are, i t i s quite possible that a reaction requires the simultaneous fulfilment!-; of a number of conditions.  Questions  such as pH, physical contact between the reacting components, the presence of the r i g h t enzymes, the physical state of macromolecules i n l i v e or dead c e l l s , and many other questions of b i o l o g i c a l environment may be involved. As well as t h i s there i s the possible a l t e r a t i o n and i n a c t i v a t i o n of the protein by u l t r a v i o l e t Irradi a t i o n or chemical oxidants which might prevent complex formation from taking place.  Another explanation might be that the o r i g i n a l  hypothesis - that oxidized derivatives of the hydrocarbons volved i n complex formation - i s i n c o r r e c t .  are i n -  I f complex formation  takes place before any metabolic process begins, and the hydrocarbon i s oxidized during or a f t e r combination with a protein component, then a completely d i f f e r e n t type of stimulus may be required f o r " i n v i t r o " complex formation.  Unfortunately, the  experiments carried out do not indicate which explanation i s correct and the question must remain unsolved unless an oxidizing agent, which brings about complex formation, can be found. Because of the d i f f i c u l t y encountered i n the attempted " i n  -U8v i t r o " complex formation between carcinogenic hydrocarbons and various protein or amino acid components, and because no s o l u t i o n to the problem has been indicated by the experimental r e s u l t s , i t would seem advantageous to carry out further investigations on the " i n vivo" complex.  This would of course e n t a i l work with much  l a r g e r numbers of mice i n order to produce the complex i n s u f f i c ient quantity.  Now that a method f o r the separation of complexed  from uncomplexed protein has been devised, a n a l y t i c a l work on the protein fragment of the " i n vivo" complex can more e a s i l y be accomplished.  Column chromatography f o r i s o l a t i o n of the complex,  followed by paper chromatography f o r i d e n t i f i c a t i o n of the enzyme hydrolyzate would now seem to be the best method of determining the protein fragment of the complex and i t s manner of attachment to the carcinogen.  -U9APPENDIX A. P u r i f i c a t i o n of solvents 1. Alcohol 100$ ethanol was refluxed f o r two hours over magnesium turnings containing a c r y s t a l of iodine.  I t was then d i s -  t i l l e d under a closed system, the f i r s t 200 mis. being discarded. 2.  Benzene and toluene Reagent grade, thiophene free solvents were r e -  fluxed f o r seven hours over f r e s h sodium metal.  They were then  d i s t i l l e d under a closed system, the f i r s t 200 mis. being d i s carded* 3.  Butanol Reagent grade butanol was refluxed f o r two hours  over magnesium turnings and then d i s t i l l e d under a closed system. The f i r s t 200 mis. were discarded. B. Preparation of Ringer-Locke solution Solution A A solution of 8.00$ NaCl, 0.U2 $ KC1 and 0.20$ C a C l  2  i n d i s t i l l e d water was prepared. Solution B A solution containing 0.^3$ Na HP0i .12 H 0 and 0.0^3$ 2  f  2  NaHgPO^.UH^ i n d i s t i l l e d water was also prepared. 8 ml. of solution A was added to 88 ml. of d i s t i l l e d water and s t e r i l i z e d ; h ml. of solution B was measured into a f l a s k and  -50sterilized.  The  two  i o n having a pH 7 , 5  C.  - 7.7  .  £• (l 2-benzanthryl-10-carbamido) caproic  Synthesis of 1.  solutions were mixed, the r e s u l t i n g solut-  1 0 - n i t r o - l 2-benzanthracene t  A solution of l.M- gm.  pension of H.6 acetic a c i d .  gm.  added to a sus-  of 1,2-benzanthracene i n 30 ml. of g l a c i a l  A f t e r ten minutes s t i r r i n g , almost a l l the benz-  anthracene dissolved.  The f i l t e r e d solution, on r e f r i g e r a t i o n ,  deposited f i n e yellow needles.  The crude y i e l d was  H.55  1,2-benzanthracene, m.p. 2.  16H.5  - 165°C.  10-amino-l 2-benzanthracene T  A suspension of H.5  gm.  of 10-nitro-l,2-benz-  anthracene i n 700 ml. of hot g l a c i a l acetic acid was  treated  of stannous chloride i n 70 ml. of concentrated hydro-  chloric acid.  The mixture was  then refluxed f o r twenty minutes  while the suspension turned to a yellow solution. of 70 ml. of concentrated hydrochloric of a voluminous yellow p r e c i p i t a t e .  The  The f l a s k was  cooled to H0°C.  The mixture  f i l t e r e d and the s o l i d washed with d i l u t e hydrochloric I t was  addition  acid caused the formation  and water added to complete the p r e c i p i t a t i o n .  acids.  gm.  10-nitro-  P u r i f i c a t i o n from aqueous pyridine yielded  with 55 gm.  1.5),  n i t r i c acid (d -  d i l u t e d with 30 ml. of g l a c i a l acetic acid was  (85$).  acid  T  and  was acetic  then washed with alcohol, ether, and then d r i e d .  A f t e r drying, the s o l i d was  s t i r r e d at room temperature with  -51280 mi. of normal ammonium hydroxide f o r two hours.  One and one  h a l f l i t r e s of benzene were added and the s o l i d r a p i d l y went into solution i n the benzene l a y e r . washed, and d r i e d .  The benzene layer was separated,  The solution was concentrated, l i g r o i n added,  and upon cooling c r y s t a l s of amine were deposited. The crude y i e l d was 3*7 gm. (90$)•  R e c r y s t a l l i z a t i o n from benzene-ligroin  yielded 10-amino-l,2-benzanthracene, m.p, 1 7 5 . 5 - 1 7 6 ° C, 3.  1 2-benzanthracene-lO-isocyanate f  A solution of 3 - 6 5 gm. of 10-amino-l,2-ben»anthracene i n 1 1 . of warm benzene was treated with phosgene. Twenty f i v e grams of phosgene were f i r s t introduced into an acetone-dry i c e cooled t e s t tube, enabling controlled of phosgene to the amine solution.  addition  The phosgene was added slowly  to the warm benzene solution producing an immediate gelatinous precipitate.  Upon refluxing f o r ten minutes, the p r e c i p i t a t e  disappeared leaving a s l i g h t l y brown solution.  Approximately  11,  of solvent was rempved by d i s t i l l a t i o n at atmospheric pressure, L i g r o i n was added, and upon cooling isocyanate appeared,  shiny yellow needles of  R e c r y s t a l l i z a t i o n from benzene-ligroin  yielded l,2-benzanthracene-10-isocyanate, m.p, Ihh - l H H . 5  0  0*  The crude y i e l d was 3 . 5 gm. ( 8 5 / 0 * H.  £(l 2-benzanthryl-10-carbamido) canroic acid T  A solution of 0 . 5 gm of l , 2 - b e n z a n t h r y l - 1 0 isocyanate i n 200 ml. ofl dioxane was added dropwise to a s t i r r e d solution containing the sodium s a l t from 1.17 gm. of t amino  caproic acid i n 200 ml, of 1:1 dioxane-water.  The solution was  s t i r r e d f o r twenty minutes, a c i d i f i e d and d i l u t e d with water to about 1 1. • Refrigeration and f i l t r a t i o n yielded 0.53 gm. of fawn c r y s t a l s .  As p u r i f i c a t i o n of such a conjugate i s a long and  involved process, and was not necessary i n t h i s case, the s o l i d was simply washed with benzene to remove any contaminating benzathracene-isocyanate v and with water to remove any adhering caproic a c i d .  The resulting substance fluoresced under u l t r a  v i o l e t l i g h t (blue fluorescence), was insoluble i n benzene, and soluble i n water pH 8.  No further characterization was made.  However, because of the known reaction between isocyanates and amines and because of the s i m i l a r i t y of properties of the above product and the s i m i l a r l y synthesized t (9,10-dimethyl-l,2-benzanthryl-3-carbamido) caproic a c i d , the product was assumed to be a hydrocarbon-amino acid conjugate, most probably d(l,2-benzanthryl-10-carbamido)caproic a c i d .  ^m5m  Insulated chromatographic t a n k a n d  stand u s e d f o r p a p e r  chromatography.  FIG«6  #  T y p i c a l chrorantograr-i (chron*»  atogram o f t h e hy*rolysat©  from  popsln-dlgested nouso epidermis.)  -54-  and  H i l g e r spectrograph, scanning u n i t reoowar*  PIG.**. T y p i c a l s p e c t r o g r a p h s t r a c e ( t r a c e of f l u o r e s c e n c e o f the hydrocarbon r e leased by the reduction of * l n v i v o * 3 j'-wbenzpyrene-protoin complex.*  -55BIBLIOGRAPHY 1 . Badger, G. M., J . Chem. S o c , H 5 6 . I 9 H 9 . 2 . Bergmann, F., Cancer Res., 2:660.  19H-2.  3 . Booth, J . and Boyland, E., Biochim. et Biophys. Acta, 1 2 : 7 5 . 1953. H. Boyland, E., Cancer Res., 1 2 : 7 7 .  1952.  5. Brown, R. R., M i l l e r , J . A. and M i l l e r , E. C , Proc. Am. Assoc. Cancer Res., 1 : 5 .  1953.  6 . Calcutt, G., B r i t . J . Cancer, 3 : 3 0 6 .  19^-9.  7 . Calcutt, G. and Payne, S.,  B r i t . J . Cancer, 8 : 5 5 H . 19 5H.  8 . Calcutt, G. and Payne, S.,  B r i t . J . Cancer, 8 : 5 6 1 . 1 9 5 H .  9 . Calcutt, G. and Payne, S.,  B r i t . J . Cancer, 8 : 7 1 0 . 195H.  1 0 . Crabtree, H. G., Cancer Res., 5:3^6.  19^5.  1 1 . Crabtree, H. G., Cancer Res., 6 : 5 5 3 .  19^6.  1 2 . Crabtree, H. G., B r i t . J . Cancer, 2 : 2 8 1 . 1 3 . Creech, H., J . Am. Chem. S o c , 7 3 : 3 1 9 .  19^8. 1951.  lH. F i e s e r , L., "Production of Cancer by Polynuclear Hydrocarbons", University of Pennsylvania, Philadelphia.  19^-1.  15. F i e s e r , L., Report of the A.A.A.S. Conference on Cancer, Washington, 19hh,  Page 1 0 8 .  1 6 . Haddow, A., Endeavour, 2 : 2 7 . 19^3. 1 7 . Hadler, H. I.,and Heidelberger, C , Proc. Am. Assoc. Cancer Res., 1 : 2 2 . 1 9 5 3 . 1 8 . Heidelberger, C. and Weist, W. S., Cancer Res., 1 0 : 2 2 3 .  1950.  1 9 . Heidelberger, C. and Weist, W. S., Cancer Res., 1 1 : 5 1 1 .  1951  2 0 . Hewett, C. L., J . Chem. S o c , 2 9 3 . 19^0.  -562 1 . Iverson, S., B r i t . J . Cancer, 2 : 3 0 1 .  19^8.  2 2 . Lacassagne, A. et a l . , B r i t . J . Exp. Path., 2 6 : 5 .  19^5.  2 3 . L e i t e r , J . and Shear, M., J . N a t l . Cancer Inst., 3 : ^ 5 5 . 2*f. Moodie, M., Reid, C. and Wallick, C , 2 5 . M i l l e r , E.C., Cancer Res., 12:5^7•  19^2.  Cancer Res., l H : 3 6 7 . 195H. 1952.  26. Pullmann, A. and Pullmann, B., La Revue Scientifque, 3:1^-5. 19H6. 2 7 . Pullmann, A., Ann. Chim., 2 : 5 .  19*+7.  2 8 . Smith, W. M., Pratt, E. F. and Creech, H. J . , J . Am. Chem. S o c , 73:319.  1951.  2 9 . Weigert, F., Cancer Res., 6 : 1 0 9 .  19H6.  3 0 . Weigert, F., Cancer Res., 8 : 1 6 9 .  19^6.  3 1 . Weigert, F., Calcutt, G. and Powell, A. K., Nature, 158:^17. 19H6. 3 2 . Weigert, F., Calcutt, G. and Powell, A. K., B r i t . J . Cancer, 1:H05.  19H-7.  3 3 . Weigert, F. and Mottram, J . C , 3H. Weil-Malherbe, H.,  Cancer Res., 6 : 9 7 . 1 0 9 .  Biochem. J . , HO:351.  3 5 . Wolf, G. and Heidelberger, C ,  19^6.  19H6a.  Cancer Res., 1 1 : 2 9 0 . . 1951.  

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