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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 this thesis as conforming to the standard required from candidates for the degree of MASTER OF SCIENCE Members of the Department of CHEMISTRY THE UNIVERSITY OF BRITISH COLUMBIA July, 1955 - l i -ABSTRACT Attempts have been made to form " i n vitro" carcinogen-protein and carcinogen-amino acid complexes by various oxidat-ive processes. Irradiation, chemical oxidation, and a combination of the two have proved unsuccessful, A new method, based upon chromatographic separation, has been developed for the detection of complex formation. Applic-a b i l i t y of the procedure was Investigated using a hydrocarbon-amino acid conjugate and"in vivo" formed 3,H-benzpyrene-epidermal protein complex. Complex formation of 3,l*-benzpyrene with several purines and nucleic acids was detected i n this way. A series of hydrocarbons - both carcinogenic and non - carcinogenic - was substituted for 3,*t-henzpyrene, hut no correlation between car-cinogenic activity 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 vitro" complex formation, i t was suggested that further investig-ations be made on the " i n vivo" complex. Chromatographic separat-ion of the " l n vivo" complex and determination of the fragments after pa r t i a l hydrolysis looks promising. ACKNOWLEDGEMENTS The author wishes to express her sincere thanks to Dr. C. Reid for his assistance and encouragement throughout the course of this research project. Gratitude i s due also to the National Research Council for financial assistance (Bursary, 195l*-19!?5) • Table of Contents Acknowledgements i Abstract i i Introduction 1 A. Carcinogens 1 B. Chemical carcinogens 1 1 . Active regions 3 2 . Geometrical considerations h 3 . Complex formation 5 C. The M i l l e r complex 6 D. Problem to be considered 8 Experimental Method 9 A. M i l l e r ' s method f o r preparing " i n vivo" carcinogen-protein complex 9 B. Methods employed i n the attempted formation of " i n v i t r o " hydrocarbon complex 9 1 . Preparation 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. Benzoyl peroxide treatment 11 5 . Benzoyl peroxide and oxygen treatment 12 6 . Benzoyl peroxide plus i r r a d i a t i o n treatment 12 7 . Enzyme method" ...... 12 8. Incubation i n a tissue culture solution-whole skin samples 13 9 . Incubation in a tissue culture solution-amino acids, proteins and purines 13 ~C. Miller's method of detecting complex formation 1*+ D. Chromatographic method of detecting complex formation 15 Results and Discussion 17 1 . Irradiation 18 2 . Irradiation with oxygen treatment 19 3 . Benzoyl peroxide 19 Benzoyl peroxide accompanied by ultra violet irradiation 31 5 . Enzyme and incubation treatment 31 6 . Purines and nucleic acids „ 36 7 . Chromatographic method for detecting complex formation ^2 Conclusions and Suggestions for Further Work h7 Appendix ^9 A. Purification of solvents ^9 B. Preparation of Ringer-Locke Solution ^9 C. Synthesis of l(l,2-benzanthryl-10-carbamido) caproic acid 50 Bibliography 55 INTRODUCTION A. Carcinogens The i n i t i a l step i n the transition from normal to malignant growth presents an exceedingly complex problem to the investigator. In spite of the immense effort being ex-pended i n cancer research, the voluminous literature shows also that our knowledge of the possible causes of the dis-ease i s i n i t s infancy. As i s to be expected from the com-plexity of the problem, many different approaches have been suggested - investigation of the chemical and biophysical differences between cancerous and normal tissue, research into the chemistry of c e l l metabolism and c e l l reproduction, analysis of the properties and metabolism of chemical carcin-ogens, and so forth. I t i s the latter approach which has been adopted i n the research to be described. B. Chemical Carcinogens The discovery of chemical carcinogens led to a new and basic method for investigating the process resulting i n malignant tissues. 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 find some property - 2 -Table l a Some carcinogenic compounds compound structure 3, benzpyr ene 1,2,7,8-dibenzfluorene 1 ,2 ,5,6-dibenzcarbazole p-aminostilbene p-dimethylaminoazobenzene 7,9-dimethyl-3,^-benzenacridine CH, 2,5-dimethyl- 3 , ( 1 ' , 2 1 -naphtho) dibenzthiophene CH) desoxycholic acid common to the carcinogens which could be correlated with their biological a c t i v i t y (2, lh9 16, 20, 22, 26, 27). However, although many theories of the mechanism of chemically induced carcinoma have been proposed, none are entirely satisfactory. A few of the more important ideas w i l l be discussed. ation between charge on the IC region (high reactivity of a particular carbon-carbon double bond) and carcinogenic a c t i v i t y . The structure of 1,2-benzanthracene illustrates the region termed the "K region". Using wave mechanics to calculate the electron density at each carbon atom and each bond of the molecule, he was able to show that for those compounds which were biologically active, the corresponding structures possessed a region of high react-i v i t y . The relative charge density of the K region for a series of methyl substituted benzanthracenes and benzphen-anthrenes proved most interesting. Substances with a charge density above a certain threshold value were active, their activity increasing as the charge density increased. Sub-sequent investigation of the rate of addition of osmium 1. Active regions In 19H6, Pullman (26) presented a correl-K-region -h-tetroxide (1) to the K region showed good agreement with the Pullman work. However there were certain inconsisten-cies i n the theory. Some compounds, theoretically inactive, proved highly active. 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 well. Bergmann (2) suggested that the disrupting effect of chemical carcinogens might take place through the adsorption of carcinogen by a particular c e l l con-stituent, i n which case surface area and planarity of the foreign molecule might be determining factors. The theory explained, i n a simple mechanical way, why some molecules were inactivated by the introduction of substitufent groups. Later (19^5) Fieser (1?) put forth the idea of selective adsorption by the c e l l walls, which might easily affect c e l l permeability. Here again, geometrical considerations may be important In determining the strength of the adsorptive forces. 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 vitro" oxidation i s not possible at a l l . -5-3» Complex formation More recent theories involve reaction or complex formation between the carcinogenic agent (or a met-abolite) 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 acid. If 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 sul-phur metabolism (6,10,11). Because of a correlation between carcinogenic act i v i t y and certain substitution reactions, Fieser (l 1*) suggested a reaction of the hydrocarbon with a -S&S- linkage of a peptide giving rise to a hydrocarbon peptide l i n k . Evidence supporting this view has been pro-duced by Crabtree (10,11,12). He found that compounds, having i n common only that they were excreted with sulphur-containing amino acids, inhibit the action of carcinogens. He proposes that "the inhibitors by preferential metabolism and excretion interfere 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 Miller Complex A recent discovery by Elizabeth Miller (25) of the formation of a protein-bound azo dye component i n the l i v e r after oral administration of the carcinogen to rats, has led to renewed interest i n the possible role of complex formation. That the discovered complex was i n some way connected with carcinogenesis, was proposed for the following reasons; 1. Species which are susceptible to l i v e r cancer (e.g. rats) also produce bound azo dye. 2. Species which are not susceptible to l i v e r cancer (e.g. mice) produce no detectible 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 this type of car-cinogen. h. Carcinogenic potency parallels the amount of bound azo dye found. It was conclusively shown that the interaction forces between the carcinogen and the protein were not simple ad-sorption 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 boiling alcohol, ether, benzene, toluene, and after a certain amount of adsorbed carcinogen was removed, no further fluor-escent material could be detected i n the solvent. The complex was further tested by a solution-reprecipitation process. After any adsorbed azo dye had been removed, the l i v e r protein was partially dissolved In a 1:2,5 mixture of ethanol - .IK"., potassium hydroxide and afterwards precipitated with trichloro-acetic acid, Fluor'scence indicating the presence of azo dye or closely related derivatives could s t i l l be detected i n the hydrol-yzate from the reprecipitated protein. Miller extended her work to 3,)*--benzpyrene and epidermal skin cancer. Like the azo dyes, 3,l*-benzpyrene also formed a protein complex. The same method was used by Moodie, Reid and Wallick (2*+) to investigate a series of compounds including potent, weak and non carcinogens. The results substantiated the Miller work. 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 a l l . At present work i s being done on the characterization of the protein involved as well as the determination of the mode of link-age between protein and carcinogen. Using radioactive tracer techniques, Hadler and Heidelberger (17) have been able to show that the hydrocarbon remains bound even after peptic hydrolysis. They are now involved i n characterizing the resulting fractions. Miller, on the other hand, i s striving to identify the azo dye components liberated by hydrolysis of the l i v e r proteins, The detection of small amounts of 3 1 -methyl-U^monomethyl amino azo benzene (5) suggests that the amino nitrogen of the polar dye may be bound to the protein. D. Problem to be Considered One result of the work of Miller (25) and of work i n this laboratory (2U-), Is that the formation of protein carcin-ogen complex was found to take place " i n vivo" only, no complex resulting even when mice were painted immediately after k i l l i n g . The problem of preparing complex from protein and carcinogen " i n vitro" thus arises naturally and i s the subject of this investig-ation. It was hoped that " i n vitro" complex might be prepared i n sufficient quantity for a study of the protein 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 for identifying bound complex led to the de-velopment of chromatographic techniques and to a procedure for separating hydrocarbon-protein complex from either of the uncom-plexed components. EXPERIMENTAL METHOD A, Miller's method for preparing " i n vivo" carcinogen-protein  complex A/LN mice, 6-10 weeks old, sex unsegregated, were treated with carcinogen. A portion of the back was shaved with surgical clippers 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 interval before removal of the epidermis varied with the particular experiment. After 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 for thirty minutes, after which the epidermis could easily be scraped from the dermis. The epidermis was wrapped i n f i l t e r paper and extracted with boiling ethanol for forty-eight hours i n a "multiple" soxhlet extractor. Controls painted with benzene were run with a l l ex-periments. (Method for 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 vitro " hydrocarbon complex (1) Preparation of samples a. Crude protein A/LN mice, 6-10 weeks old, sex unseg-regated, were used for obtaining epidermal protein. A section of the back, approximately h by 2 cm., was shaved and the mice -10-k i l l e d with ether. The shaved regions were excised, immersed i n N/3 ammonia for thirty minutes, and the epidermis scraped from the dermis. The epidermis was homogenized with 85% ethanol and 10$ trichloroacetic acid i n a Waring Blendor. It was then dried and used i n this form. b. Whole skin samples A/LN mice, 6-10 weeks old, sex unseg-regated, were shaved as described above. The mice were k i l l e d using ether and the shaved areas excised. The skins were used in this form for treatment. c. Intact mice A/LN mice, 6-10 weeks old, 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 purification. e. Purines and nucleic acids As i n (d). (2) Irradiation method Solutions were made up of the following for 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, -11-amino acid, or protein preparation plus 1 mg. of 3,l*-benzpyrene. Test tubes containing the solutions were suspended from a dr i l l e d plywood board at positions equidistant from a mercury AH h ultraviolet light source. For most of the experiments solutions were made up i n t r i p l i c a t e for suspension at distances of 3, 6, or 9 cm. from the light source. Varying times of irradiation were used. The shaved area of (c) was painted with 0.2 ml. of »h% 3,V-benzpyrene i n benzene. It was then irradiated with a mercury AH h light source. Samples were irradiated at varying intensities and for varying periods of time. Controls painted with benzene only were subjected to the same treatment. After treatment, the shaved area was excised and the epidermis removed as describ-ed under II A* 3. Irradiation and oxygen treatment (samples (a) T (b) t and (d) ) The irradiation method was carried out while bubbling oxygen through the solutions. *t. Benzoyl peroxide treatment Solutions of the following were prepared for (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. - 1 2 -h 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 or were incubated at 3 0 , HO, and 50° C. for varying periods of time. Sample (c) was painted either with 0 . 2 ml. of a solution containing 3,^-benzpyrene plus benzoyl peroxide or 0 . 2 ml. of a solution containing ,h% benzoyl peroxide alone. Sample (c) was allowed to stand at room temperature for only eight hours or at 0°C. for 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 irradiation treatment Solutions of samples (a), (b), and (d) were prepared as described under benzoyl peroxide treatment. The test tubes were suspended for irradiation as described under irrad-iation method. This treatment was carried out with and without bubbling oxygen through the solutions. Irradiation was carried out for varying periods of time. 7» Enzyme method Duplicate samples of 20 mg. of (a) or 100 mg. - 1 3 -of (b) were made up i n 1% solutions of trypsin, pepsin or pan-creatin. One milligram of S^benzpyrene was added to one of each duplicate sample. Solutions were either incubated at 3 0 , hO or 50°C. for varying periods of time or irradiated as described under irradiation method. 8 . Incubation i n a tissue culture solution (Ringer  Locke solution)(c) A/LN mice, six weeks old, sex unsegregated, were shaved as previously described and k i l l e d with ether. Imm-ediately the back section was excised and floated onto a petri 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 half were used as controls and were painted with 0 . 2 ml. benzene. The treated skins were incubated at 9 8 ° F for 12 , 2*+, 3 6 . and hours. The epidermis was then removed as in II A. 9» Incubation i n a tissue culture solution (Ringer  Locke solution) (d) T (e) Twenty milligrams of samples (d) and (e) were dissolved i n 10 ml. of Ringer-Locke solution. Five milligrams of hydrocarbon was added to the solutions. (Control samples contain-ing no 3,^benzpyrene were run with each experiment.) Air was bubbled through the solutions which were incubated from twelve to eighteen hours. (One experiment was carried out with the addition of 1 ml. of -lH-•*4-N potassium hydroxide and 1 ml. of 95% ethanol to increase the solubility of the protein.) G. Miller'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 entails 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 boiling ethanol i n a "multiple soxhlet ex-tractor" for forty-eight hours. It was then dried and weighed. 20-50 mg. were treated with 2 ml. of ethanol, 5 ml of M-N. pot-assium hydroxide, 5 ml. of toluene and 1.6 g. of activated zinc dust. The mixture was refluxed for three hours. The cooled solution was extracted three times with benzene and the combined extractions set aside for fluorescence determinations. The original solution (water layer) was acidified with dilute hydro-chloric acid and again extracted with benzene. These benzene solutions were also combined. Fluorescence spectra of the "neutral" and "acidic" fractions were obtained using a Hilger E 2 spectrograph combined with a Hilger scanning unit and record-ing system. The light source was an AH 6 high pressure mercury arc with suitable glass and liquid f i l t e r s for isolating the - 1 5 -mercury 3650 , 3100 A lines, D, Chromatographic method of detecting complex formation Because of the limitations and time consumption of the Miller method, another means of detecting complex formation was developed. The new method i s based upon change of solubility properties of protein and hydrocarbon when in a combined state. For this reason, removal of adsorbed hydrocarbon i s not necess-ary. Applicability of the method and detection limits were tested using a hydrocarbon-amino acid conjugate, £ ( 1 , 2 -benzanthryl-10-carbamido)caproic acid. 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. for twelve hours. The solution was then concentrated at 55°C» to 2 ml. The solutions to be investigated were applied to Whatman No. 1 chromatographic paper. In some cases as.-, many as four applications were made. The chromatogram was carefully dried over a hot plate,before eluting, because of the Immiscibility of the solvents. The elutarit used was a water saturated solution of benzene. Because of the almost complete insolubility of protein and protein-hydrocarbon complex i n benzene, both of these components -16-remained at the site of application. The hydrocarbon, on the other hand, being relatively soluble i n benzene, travelled with the solvent front. The solvent moved very rapidly, good separ-ation taking place i n 2-3 hours. Complex formation was detected, after chromatographing the solution, by the characteristic blue-violet fluorescence of the hydrocarbon complex at the site of application. When the complex was to be separated from the pro-tein, the benzene eluted chromatogram was dried and re-eluted with butanol-acetic acid-water (*f:ls!?). In this solvent the R f of the protein (or amino acid) ranged from 0-.6, while the of the complex approached 1. - 1 7 -RESULTS AND DISCUSSION As i s b r i e f l y mentioned i n the introduction, the starting point of this project was the attempted formation of " i n vitro" hydrocarbon-protein complexes. Destructive reduction of the " i n vivo" complex ( 2 5 ) , together with work on hydrocarbon metabol-ism ( 6 , 7 , 8 , 9 , 1 7 , 1 8 , 1 9 , 2 9 , 3 0 , 3 2 , 3 5 ) had given some idea of the possible linkage involved. Wiegert and Mottram ( 3 3 ) were able to separate and tentatively identify four metabolic products of 3,H-benzpyrene found i n various tissues and as excretion prod-ucts after injection of the carcinogenic hydrocarbon. The con-clusions drawn were that the f i r s t pair of metabolites were sub-stituted diols, and that the other pair of metabolites (easily formed from the f i r s t by " i n vitro" treatment with acid) were the f u l l y aromat ized rings produced by the removal of Water or alcohol. That a similar phenolic or hydroxy derivative might be involved in complex formation was suggested by the production of an acidic hydrocarbon fraction after zinc dust reduction of the " i n vivo" hydrocarbon-protein complex, "since the fluorescence spectrum of this derivative i s very similar to that of the parent hydro-carbon, i t was assumed that no radical changes had taken place F, -18-i n the ring skeleton but that substituent groups, possibly hy-droxy 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 vitro" complex formation was attempted through various oxidative processes. 1. Irradiation The f i r s t method selected was that of irradiation of sample with ultra violet l i g h t . Although photo oxidation cannot be envisioned l n the interior 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 biological reactions taking place with the release of energy which might, perhaps, stimulate the same type of transition. Alexander's recent success i n correlating ease of primary photo oxidation and carcinogenic activity suggested that such a method might prove f r u i t f u l i n the problem of "invitro" carcinogen-protein complex formation. The samples to be investigated were suspended i n 3,^benz-pyrene solution and irradiated for different lengths of time at various light intensities. Mouse epidermis and whole skin were chosen as protein samples since they are known to contain the component necessary for " i n vivo" complex formation. However, d i f f i c u l t i e s with irreproducible results led to the investigat-ion of simpler protein and amino acid systems as well. What was lost by specifying the "protein part" of the complex was hoped to be gained by obtaining clear cut, reproducible results. -19-That Irradiation treatment was unsuccessful i s clearly-shown by Table 1. In no instance could complex formation be unequivocally detected. The control samples and 3,Wbenzpyrene treated samples behaved i n identical manner. 2. Irradiation with oxygen treatment In view of the key role of oxygen i n many biochemical processes, the possibility that irradiation need be accompanied by oxygen was next looked into. Oxygen bleeds were inserted i n a l l test solutions while irradiation was carried out. Table 2 shows that this method also produced no positive results. 3 . Benzoyl peroxide The next step was to try a chemical oxidizing agent. If oxidation products are involved, i t i s possible that photo oxidation does not produce the required products for com-plex formation and that a compound such as benzoyl peroxide might be more successful. Benzoyl peroxide was chosen chiefly for i t s solubility properties and i t s known action on 3,*+-benz-pyrene. Conditions such as temperature and incubation time were varied and oxidation was repeated inserting oxygen bleeds in the solutions. Tables 3 and h show that under the above conditions, benzoyl peroxide was unable to stimulate " i n vitro" complex formation. - 2 0 -Table 1 Showing the results of attempted complex formation of 3,U-benz-pyrene with various proteins and amino acids. Stimulus - u l t r a violet l i g h t . Sample X t 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. intact mouse 30 7 S neg. intact mouse 60 7 S neg. intact 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. - 2 1 -Sample h A 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 t - irradiation time (min) i j - irradiation distance (cm.) A - analysis by S - spectrograph C - chromatography Table 2 Showing the results of attempted complex formation of 3,l+-benz-pyrene with various proteins and amino acids. Stimulus - ultra violet irradiation plus oxygen. Sample V A Results mouse epidermis 30 7 S neg. mouse epidermis 60 7 S neg. mouse epidermis 9 0 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 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. cystine 60 c neg. cystine 60 9 c neg. phenylalanine 60 3 c neg. phenylalanine 60 6 c neg. phenylalanine 60 9 c neg. tyrosine 60 3 c neg. tyrosine 60 6 c neg. -23-Sample 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 d - irradiation distance (cm.) A - analysis oy S - spectrograph C - chromatography -2H-Table 3 Showing the results of attempted complex formation of 3,H-henz-pyrene with various proteins and amino acids. Stimulus - benzoyl peroxide. Sample l n c t Inc T A Results mouse epidermis 12 30 S neg. mouse epidermis 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. - 2 5 -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 - 2 6 -Sample l n c 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. - 2 7 -Sample l n c t Inc T A Result phenylalanine 12 30 - 0 neg. 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 t - Incubation time (hrs.) Inc T - Incubation temperature (°C.) A - analysis by S - spectrograph C - chromatography - 2 9 -Table h Showing the results of attempted complex formation of 3,^-benz-pyrene with various proteins and amino acids. Stimulus - benzoyl peroxide plus oxygen. Sample Inc t l n c 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. - 3 0 -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 t - Incubation time (hrs.) Inc T - incubation timperature. (°C.) A - analysis by S - spectrograph G - chromatography - 3 1 -U. Benzoyl peroxide accompanied by ultra violet irradiation A last variation of the oxidative process was made by combining ultra violet irradiation with chemical oxidation. I t i s possible that the " i n vivo" process involves I n i t i a l l y a mol-ecular complex, formed through an excited state of the hydrocarbon, and secondly oxidation of the associated hydrocarbon to form a more firmly bound complex. I f this were the case, irradiation and chemical oxidation might be able to simulate " i n vivo" con-ditions well enough for successful " i n vitro" 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 results i s that i f a simple mechanism of complex formation (such as linkage through an hydroxy group on the hydro-carbon and an amino group of the protein) i s assumed, then failure to produce " i n vitro" complex may be due to other factors. The in a b i l i t y of the hydrocarbon to come i n contact with the necess-ary tissue protein i n a dead system i s a poss i b i l i t y . Experiments were developed i n an attempt to overcome this d i f f i c u l t y . In one case the protein was kept alive by incubation i n a tissue culture solution, while i n the other digestive enzymes were used to render the protein water soluble. Better results were anticipated from the incubation method since hydrocarbon metabolites have reportedly been found in this way. Weigert ( 3 D , using 3,U-benzpyrene and incubation of mouse skin i n Ringer-Locke - 3 2 -Table 5 Showing the results of attempted complex formation of 3,Wbenz-pyrene with various proteins and amino acids. Stimulus - benzoyl peroxide plus ultra violet irradiation. Sample * t X d l n c t A Results mouse epidermis 60 6 12 30 S neg. mouse epidermis 90 6 12 30 s neg. mouse epidermis 90 6 36 30 s neg. mouse epidermis 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. - 3 3 -Sample h I d Inc t Inc T A Results 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^ - irradiation time (min.) I d - irradiation distance (cm.) Inc t - incubation time (hrs.) Incm - incubation temperature (°C.) A - analysis by S - spectrograph G - chromatography Table 6 Showing the results of attempted complex formation of 3,l+-benz-pyrene with various proteins and amino acids. Stimulus - benzoyl peroxide plus ult r a violet irradiation plus oxygen. Sample h l n c 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 6 0 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 6 0 9 2k 30 c neg. histidine 60 3 2k 30 c neg. histidine 6 0 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. - 3 5 -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 t - irradiation time (mins.) i j - irradiation distance (cms.) Inc t - incubation time (hrs.) Inc„ - incubation temperature (°C.) A - analysis by S - spectrograph G - chromatography -36-solution, was able to isolate one of the metabolites previously discovered after the " i n vivo" injection of 3,U-benzpyrene. The results of these experiments are given i n Tables 7 and 8. In both cases detection of " i n vitro" formed complex could not be made. 6. Purines and nucleic acids The reported increase i n water solubility 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 failure to produce " i n vitro" 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 for which complex formation could be detected. Caffeine, hypoxanthine, and desoxyribonucleic acid showed significant quantities of fluorescent derivative, while complexed u r a c i l and ribose nucleic acid were just detectible. The significance of these complexes i n relation to the can-cer problem was further investigated by substituting a series of carcinogenic and non-carcinogenic hydrocarbons for 3,*+-benzpyrene. Table 10 shows that there does not seem to be any correlation between this type of complex formation of aromatic hydrocarbons -37-Table 7 Showing the results of attempted " i n vitro" complex formation of 3,^-benzpyrene with epidermal protein i n various digestive enzymes. Sample Enzyme Inc t InCrp A Result mouse epidermis T 12 30 C neg. mouse epidermis 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. mouse epidermis 5 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. -38-Sample Enzyme l n c t Inc T I t I d A Results whole skin Pa 2k. 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 - trypsin Pe - pepsin Pa - pancreatin Inc. - incubation time (hrs.) Inc!; - incubation temperature; (°C.) I. i irradiation time (mins.) - irradiation distance (cms.) A a - analysis by S - spectrograph C - chromatography - 3 9 -Table 8 Showing the r e s u l t s of attempted " i n v i t r o " complex formation of 3,^-benzpyTene with epidermal protein i n Ringer-Locke so l u t i o n . Sample l n c t l n c T A Results whole skin 12 98 S neg. whole skin 2k 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. In which Inc. - incubation time (hrs.) Inc« - incubation temperature (°F) A - analysis by S - spectrograph C - chromatography Table 9 Showing the results of attempted complex formation of 3,l«-benz-pyrene with proteins, purines, and amino acids. Sample Inc. t *nc T A Results caffeine 16 37 C pos.*** tryptophane 16 37 C neg. egg albumin 16 37 C neg. ribonucleic acid 16 37 C pos.* desoxyribonucleic acid 16 37 c pos.** cystine 16 37 c neg. adenine 12 39 c neg. thymine 12 39 c neg. hypoxanthine 12 39 c pos.** ur a c i l 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. histidine 18 37 G neg. In which Inc. - incubation time (hrs.) Inc™ - incubation temperature (°C) A - analysis by C - chromatography * * * * * * _ indicates the relative quantity of complex formed. -Hi-Table 10 Showing the results of attempted complex formation of a series of hydrocarbons with proteins and purines in 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.**1 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-benzanthra-cene C pos.* desoxyribonucleic acid 18 37 1,2-benzanthra-cene C neg. hypoxanthine 18 37 1,2-benzanthra-cene C pos.* ribonucleic acid 18 37 1,2-benzanthra-cene 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 methylcholan-threne C neg. desoxyribonucleic acid 17 ho 9,10-dimethyl-1,2-benzanthra-cene C neg. desoxyribonucleic acid 17 ho l , 2 , 5 , 6 Tdibenz-anthracene C neg. 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 car-cinogenic activity of the hydrocarbon. Leiter and Shear's work ( 2 3 ) indicating that various purines and nucleic acids retard tumor formation when injected with 3,U-benzpyrene, correlates well with the results of complex form-ation. 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 result of complex formation. However, from the results 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 biological activity. 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 relatively weakly bound; apparently weaker than the hydro-carbon complexes discussed here. Chromatographic detection or separation would i n these cases be unsuccessful i f the dissoc-iation constants were high enough that elution with a hydrocarbon solvent completely dissociated the complex. The negative results shown i n Tables 9 and 1 0 refer only to "stable" complex formation in which the product does not dissociate during the chromatography i n this manner. 7. Chromatographic method for detecting complex formation Several d i f f i c u l t i e s with the spectroscopic method -V3-of detecting complex formation led to the development of a new method. In some cases background fluorescence of uncomplexed pro-tein made interpretation of the spectroscopic traces uncertain. In a l l cases, due to the necessity of removing uncomplexed hydro-carbon, the procedure proved unduly time consuming. For these reasons the possibility of using chromatographic techniques was investigated. The very different solubility properties of poly-peptides and poly cyclic aromatic hydrocarbons suggested that a partition process would be capable of separating both these com-ponents, not only one from the other, but also from a complex of the two. In order to develop this method of separation and to find 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, dilute potassium permanganate and ultra violet irradiation indicated that the conjugate could best be detected by i t s characteristic fluorescence. Dilute potassium permanganate gave positive results, only when the con-jugate 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, ineffective. 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. Ultra violet irradiation was used to locate the conjugate and the free hydrocarbon; while development with nin-hydrin revealed the position of the amino acid. The minimum con-centration of conjugate that could be detected was determined as lying 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 possibility of separation of a protein-hydrocarbon complex from i t s uncomplexed components as well as rapid and clear cut detection. Applicability 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 be applied to chromatographic paper. Here l i e s the d i f f i c u l t y i n work with complex proteins, for the only efficient complex protein solvent ( just recently discovered as such) i s hydrofluoric acid. The work of Miller and Heidelberger with " i n vivo" carcinogen-protein complex has shown that a good proportion of the hydro-carbon remains attached after partial hydrolysis of the protein with dilute sodium hydroxide. This method of "dissolving" the protein was used for the preparation of a solution of " i n vivo" F I G . l . Chromatogram showing the separation of 1,2-ben?,anthracone (1) from caproic acid (2) and conjugate (3)sbenzene elutant. FIG.2. Chromatogram showing the separation of caproic a c i d (2) from 1,2-benzanth-racene (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 to part of the original protein fragment. Separation of the complex from both free hydrocarbon and protein was accomplished by chromatographing the mixture i n two successive solvents. Benzene elutant, which had no effect on the protein or on the complex was used f i r s t to remove any free or adsorbed hydrocarbon. After drying, the chromatogram was re-eluted with butanol-water-acetic acid. The R^ , values for the amino acids and polypeptides present ranged from 0 to 0,6, while that of the complex approached unity. -H7-CONCLUSIONS AND SUGGESTIONS FOR FURTHER WORK The results of this work, while not exactly those I n i t i a l l y hoped for, did produce a clearer picture of the problem and led to a new method for investigation of the " i n vivo" protein-hydro-carbon complex. Failure of oxidative processes to stimulate complex formation i s not without explanation. In systems as complex as biological 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 right enzymes, the physical state of macromolecules i n l i v e or dead c e l l s , and many other questions of biological en-vironment may be involved. As well as this there i s the possible alteration and inactivation of the protein by ultra violet Irrad-iation or chemical oxidants which might prevent complex formation from taking place. Another explanation might be that the original hypothesis - that oxidized derivatives of the hydrocarbons are i n -volved i n complex formation - i s incorrect. I f complex formation takes place before any metabolic process begins, and the hydro-carbon i s oxidized during or after combination with a protein component, then a completely different type of stimulus may be required for " i n vitro" 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 -U8-vitro" complex formation between carcinogenic hydrocarbons and various protein or amino acid components, and because no solution to the problem has been indicated by the experimental results, i t would seem advantageous to carry out further investigations on the " i n vivo" complex. This would of course entail work with much larger numbers of mice i n order to produce the complex i n suffic-ient quantity. Now that a method for the separation of complexed from uncomplexed protein has been devised, analytical work on the protein fragment of the " i n vivo" complex can more easily be accomplished. Column chromatography for isolation of the complex, followed by paper chromatography for identification 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. -U9-APPENDIX A. Purification of solvents 1. Alcohol 100$ ethanol was refluxed for two hours over mag-nesium turnings containing a crystal of iodine. It was then dis-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 re-fluxed for seven hours over fresh 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 dis-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$ CaCl 2 i n d i s t i l l e d water was prepared. Solution B A solution containing 0.^3$ Na2HP0if.12 H20 and 0.0^3$ 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 steril i z e d ; h ml. of solution B was measured into a flask and - 5 0 -s t e r i l i z e d . The two solutions were mixed, the resulting solut-ion having a pH 7 , 5 - 7 .7 . C. Synthesis of £• (l T2-benzanthryl-10-carbamido) caproic acid 1 . 1 0-nitro-l t 2-benzanthracene A solution of l.M- gm. n i t r i c acid (d - 1 . 5 ) , diluted with 30 ml. of glacial acetic acid was added to a sus-pension of H.6 gm. of 1,2-benzanthracene i n 30 ml. of glacial acetic acid. After ten minutes stir r i n g , almost a l l the benz-anthracene dissolved. The f i l t e r e d solution, on refrigeration, deposited fine yellow needles. The crude yield was H.55 gm. ( 8 5 $ ) . Purification from aqueous pyridine yielded 10-nitro-1,2-benzanthracene, m.p. 16H.5 - 165°C. 2 . 10-amino-lT 2-benzanthracene A suspension of H.5 gm. of 10-nitro-l,2-benz-anthracene i n 700 ml. of hot glacial acetic acid was treated with 55 gm. of stannous chloride i n 70 ml. of concentrated hydro-chloric acid. The mixture was then refluxed for twenty minutes while the suspension turned to a yellow solution. The addition of 70 ml. of concentrated hydrochloric acid caused the formation of a voluminous yellow precipitate. The flask was cooled to H0°C. and water added to complete the precipitation. The mixture was f i l t e r e d and the solid washed with dilute hydrochloric and acetic acids. It was then washed with alcohol, ether, and then dried. After drying, the solid was stirred at room temperature with - 5 1 -280 mi. of normal ammonium hydroxide for two hours. One and one half l i t r e s of benzene were added and the solid rapidly went into solution i n the benzene layer. The benzene layer was separated, washed, and dried. The solution was concentrated, l i g r o i n added, and upon cooling crystals of amine were deposited. The crude yield was 3*7 gm. (90$)• Recrystallization from benzene-ligroin yielded 10-amino-l,2-benzanthracene, m.p, 175 .5 - 1 7 6 ° C, 3 . 1 f2-benzanthracene-lO-isocyanate A solution of 3-65 gm. of 10-amino-l,2-ben»-anthracene i n 1 1 . of warm benzene was treated with phosgene. Twenty five grams of phosgene were f i r s t introduced into an acetone-dry ice cooled test tube, enabling controlled addition of phosgene to the amine solution. The phosgene was added slowly to the warm benzene solution producing an immediate gelatinous precipitate. Upon refluxing for ten minutes, the precipitate disappeared leaving a slightly brown solution. Approximately 1 1 , of solvent was rempved by d i s t i l l a t i o n at atmospheric pressure, Ligroin was added, and upon cooling shiny yellow needles of isocyanate appeared, Recrystallization from benzene-ligroin yielded l,2-benzanthracene-10-isocyanate, m.p, Ihh - l H H . 5 0 0* The crude yield was 3 . 5 gm. (85/0* H . £(lT2-benzanthryl-10-carbamido) canroic acid A solution of 0 . 5 gm of l , 2-benzanthryl - 1 0 -isocyanate i n 200 ml. ofl dioxane was added dropwise to a stirred solution containing the sodium salt from 1.17 gm. of t amino caproic acid i n 200 ml, of 1:1 dioxane-water. The solution was stirred for twenty minutes, acidified and diluted with water to about 1 1. • Refrigeration and f i l t r a t i o n yielded 0.53 gm. of fawn crystals. As purification of such a conjugate i s a long and involved process, and was not necessary i n this case, the solid was simply washed with benzene to remove any contaminating benzathracene-isocyanate v and with water to remove any adhering caproic acid. The resulting substance fluoresced under u l t r a violet light (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 similarity of properties of the above product and the similarly synthesized t (9,10-dimethyl-l,2-benz-anthryl-3-carbamido) caproic acid, the product was assumed to be a hydrocarbon-amino acid conjugate, most probably d(l,2-benz-anthryl-10-carbamido)caproic acid. ^m5m Insulated chromatographic t a n k and stand u s e d f o r p a p e r c h r o m a t o g r a p h y . F I G « 6 # Typical chrorantograr-i (chron*» a t o g r a m o f t h e h y * r o l y s a t © f r o m popsln-dlgested nouso epidermis.) - 5 4 -H i l g e r spectrograph, scanning u n i t and reoowar* PIG.**. Typical spectrographs trace (trace of fluorescence of the hydrocarbon r e -leased by the reduction of *ln vivo* 3 j'-wbenzpyrene-protoin complex.* -55-BIBLIOGRAPHY 1 . Badger, G. M., J . Chem. S o c , H56. 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., 12 :77 . 1952. 5. Brown, R. R., Miller, J. A. and Miller, 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 :55H. 19 5H. 8 . Calcutt, G. and Payne, S., B r i t . J . Cancer, 8 : 5 6 1 . 195H. 9 . Calcutt, G. and Payne, S., B r i t . J . Cancer, 8 :710. 195H. 1 0 . Crabtree, H. G., Cancer Res., 5:3^6. 19^5. 11 . 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 . 19^8. 1 3 . Creech, H., J . Am. Chem. Soc, 73:319. 1951. lH. Fieser, L., "Production of Cancer by Polynuclear Hydrocarbons", University of Pennsylvania, Philadelphia. 19^-1. 15. Fieser, 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 . 1953. 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., 11:511. 1951 2 0 . Hewett, C. L., J. Chem. S o c , 2 9 3 . 19^0. - 5 6 -2 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 . Leiter, J . and Shear, M., J. Natl. Cancer Inst., 3:^55. 19^2. 2*f. Moodie, M., Reid, C. and Wallick, C , Cancer Res., lH : 3 6 7 . 195H. 2 5 . Miller, E.C., Cancer Res., 12:5^7• 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. Soc , 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 , Cancer Res., 6 : 9 7 . 1 0 9 . 19^6. 3H. Weil-Malherbe, H., Biochem. J., HO:351. 19H6a. 3 5 . Wolf, G. and Heidelberger, C , Cancer Res., 1 1 :290. . 1951. 

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