Document 4Jd8VNNe8dOJOvp8GRDq8819N

R&S 135172 0008-5472/80/0040-0000502.00 Comparison of the Hydroxylation of Benzo(a)Pyrene with the Metabolism 0bf Vinyl Chloride^AZ-Nitrosomorpholinef and A/-Nitroso-A/'- /Methylpiperazinerto Mutagens by Human and Rat Liver Microsomal Fractions1 Nicole Sabadie,*31C* hristian Malaveille,-Anne-Marie Camus; and Helmut Bartsch3 Unit of Chemical Carcinogenesis, International Agency for Research on Cancer. 59372 Lyon Cede* 2. Franca ABSTRACT ygenases (e.g., AHHJ), which are responsible for both the detoxification and metabolic activation of xenobiotics. Since Carcinogen metabolism in vitro was studied in 20 surgical many environmental carcinogens are substrates for the cyto liver specimens from adult male and female human subjects. A chrome P-450-clependent^ monooxygenases,. these enzymes 60-foid' Interindfviduai' variation'\irr^'kfyf!f\ydfocarbon1 [benzo^.'' 'afso mediate carcinogenesis and toxicity through the formatidn' 'CaJpyrene] hydroxylase'activit^ was seen;'the'fcapacity to con- ' of electrophliic intermediates via oxidative pathways (3l36).' sv vert vinyl chloride, N-nitroso-N'-methylpiperazine. and N-nitro- Covalent binding of such ultimate carcinogenic metabolites and somorpholine into electrophiles mutagenic to Salmonella ty- the concentrations available for reaction with macromolecules phimurium varied 9-, 17-, and 35-fold, respectively. The mean in the target tissues are determinants for the onset of tumorienzymic capacity relative to that of liver from untreated rats genesis. Inherent variations of monooxygenase activity by en was 84% for vinyl chloride. 42% for N-nitrosomorpholine, and vironmental and genetic factors (12, 49) may thus be associ 380% for N-nitroso-N'-methylpiperazine. When aryf hydrocar ated with the individual differences in susceptibility to cancer bon [benzo(a)pyrene] hydroxylase activity in human liver spec seen in both experimental animals and humans. In mice, ge imens was plotted against mutagenicity in S. typhimurium me netically controlled induction of AHH activity has been shown diated by liver microsomes from the same specimens, a positive to influence tumor response to polycyclic aromatic hydrocar correlation was obtained in the presence of vinyl chloride (r = bons (31). In humans, a positive association has been sug 0.55; p < 0.1), N-nitrosomorpholine (r = 0.86; p < 0.01), or gested between AHH inducibility in lymphocytes and an in N-nitroso-N'-methylpiperazine (r = 0.94; p < 0.01). The results creased risk for bronchiocarcinoma in smokers (30), although re discussed in relation to the possible development of meth this has not been confirmed (39). Possible limitations of exper ods for assessing rates of metabolism of environmental carcin imental studies carried out on human lymphocytes have been ogens in human subjects in vivo using, nontoxic drugs that are presented (35). metabolized by the same enzymes that metabolize carcino One major difficulty in determining interindividual differences gens. Hepatic aryl hydrocarbon [benzo(a)pyrene] hydroxylase in rates of metabolism of carcinogens in humans is that such activity following treatment of rats with phenobarbitone, preg data cannot be obtained directly from in vivo studies. Conse nenolone-1 6a-carbonitrile, dibenamine, aminoacetonitrile, or quently, in vitro and in vivo studies with model (predictor) drugs disulfiram, but not after treatment with 3-methylcholanthrene. (themselves nontoxic but metabolized by the same enzyme showed a positive correlation with the mutagenicity of the respective 9000 X g liver supernatant fraction mediated in the presence of N-nitrosomorpholine, vinyl chloride, or aflatoxin systems as environmental carcinogens) have been proposed as probes to collect information on the carcinogen-metaboliz ing ability of individual human subjects. Recent work has B,. These data lend further support that cytochrome P-450- provided some evidence that such predictor drugs may exist. linked monooxygenases in rat liver convert N-nitrosomorpho- Kapitulnik ef a/. (29) reported significant positive correlations line, vinyl chloride, and aflatoxin B, into mutagenic electro between rates of BP hydroxylation (AHH activity) and in vitro philes. oxidative metabolism of antipyrine. zoxazolamine, and hexo- barbital in human livers obtained at autopsy. Kalamegham et INTRODUCTION al. (28) and Sotaniemi ef at. (44) found a statistically significant positive association between antipyrine elimination in vivo and Large interindividual differences in the ability of human hepatic AHH activity in vitro in 15 human subjects or cyto beings to oxidize chemical carcinogens (11) and other foreign chrome P-450 content in human livers. compounds (49) are well documented. The enzymes involved In our studies with 20 human liver specimens, we extended in most cases are the cytochrome P-450-dependent monoox these investigations to include carcinogenic chemicals to which humans may be exposed. The animal or human carcinogens Received July 19, 1979; accepted September 20. 1979. ' Partially supported by Contract NO l-CP'55630 from the National Cancer institute. NiH. Bethesda. Md, Some of the data were presented at the International Colloquium of CNRS. No. 256. on ''Mecamsmes d'alteration et de reparation du N-nitrosomorpholine. N-mtroso-N'-methylpiperazine, VC, and AFB (14, 23-25) (Chart 1) are converted by rodent or human liver microsomal monooxygenases into electrophiles which can DNA. relations avec la mutagenese et la cancerogenese chimique". 4-9 July. 1976, Menton. France, and at the Seventh International Congress of Pharma cology, 1978, Parr*, France (8). 1 Visiting scientist from the Department pf Biochemistry (Director Professor , Gautheron). University of Lyon. Lyon. France. 3 To whom requests lor reprints should be addressed. * The aDhreviaiione used are; AHH. aryl hydrocartggn (Penzo/a)pyrene| hy droxylase; BP.'benzo (a) pyrene: VC. vinyl chloride; AF8. atlatoxir B,: S-9. 9000 x g liver supernatant fraction; 3-HO-BP, 3-hydroxypenzo(4>pyrene; DMSO. dimethyl sulfoxide. - JANUARY 1980 119 Chart 1. Structures of carcinogens studied in the oresence of hutnan liver samples. BENZOIaiPYRENS OCM, AFLATOXIN B VINYL CHLORIOE -; "V1 h}i.tgKfcr*r+frH>^I R&S 135173 N-NITROSOMORPHOLINE CHj-N N-N . , . ' y_y N-NITROSO -N' METHYLPIPERAZINE bind to nucleic acids (5, 7. 15, 16, 32, 45. 47). We therefore prior to the assays. Disuifiram was given at a dose of 500 mg/ examined the magnitude of interindividual variations in activity. kg body weigh} as a.suspension,in starch-solijtion b/'gavage'^ t , in human liver, by making paralle^measurements di rriicrosorne'- ` ' 4 hr before the animals were killed. Dibenamine was injected'' V mediated: mutagenicity._arid AHH.activity)'We also examined'' ' s.c. twice into rats at a dose of 25 mg/kg 48 and 24 hr before '' 'the effect of treating rats with modifiers of the hepatic micro the animals were killed. Rats also received an i.p, injection of somal monooxygenase system on AHH activity and on S-9- 3-methylcholanthrene (40 mg/kg) 2 days before they were mediated mutagenicity of N-nitrosomorpholine, VC, and AFB. killed. The results presented herein on comparative metabolism of carcinogens in animal and human tissues may provide a basis Animal and Human Tissue Preparations for the extrapolation of carcinogenicity data from animals to humans, e.g.. for N-nitrosomorpholine (25). to which humans may be exposed, but for which there are no epidemiological data or case reports of tumor induction. S-9 was prepared at 0-4 from the pooled livers of 2 to 3 rats by centrifugation of a homogenate (3 ml of 0.15 M KCI-5 mM Sorensen buffer (pH 7.4) per g of wet liver). The resulting fractions were kept at 0-4 for less than 2 hr and then used simultaneously in mutagenicity assays and AHH determina MATERIALS AND METHODS Chemicals tions. All procedures were carried out using sterile glassware and solutions. Samples of human liver with no pathological lesions were obtained from female and male adults either for VC (purity, 99.9%) was generously provided by Rhone-Poulenc, Lyon, France. Pregnenolone 16a-carbonitrile was a gift from Amersham/Searle Inc., Chicago, III. Disuifiram [bis(diethylthiocarbamoyl) disulfide] was provided by the late Dr. F. K. Kruger, German Cancer Research Centre, Heidelberg, Fed eral Republic of Germany. 3-HO-BP was obtained from the Chemical Repository of the National Cancer Institute, Bethesda, Md,. through Dr J. N. Keith. The following products were obtained commercially from the sources indicated: aminoacetonitrile bisulfate (Koch Light Laboratories, Ltd., Colnbrook, Buckinghamshire. United Kingdom): dibenamine [N-2-ch!oroethyl)dibenzylamine], 3-methylcholanthrene, and BP (Aldrich Chemical Co.. Milwaukee, Wis.); AFB, N-nitrosomorpholine, and A/-nitroso-/V'-methylpiperazine (Merck-Schuchardt, Darmstadt, Federal Republic of Germany). diagnostic purposes or as surgical specimens. The human subjects listed in Table 1, from whom diagnostic and surgical specimens were taken, were affected by the following diseases: gastric adenocarcinoma (Subject E); metastatic colonic ade nocarcinoma (Subject F); metastatic pancreatic adenocarci noma (Subject G); primary melanoma (Subject H); hepatoblas toma (Subject J); pancreatic carcinoma (Subject K); hepatoma (Subject M); colonic adenocarcinoma with liver metastases (Subject N); cholecystitis lithiasis (Subjects L and Y): and Hodgkin s disease (Subjects A, B, C, D. R, S, T, U, V, and W), Liver samples were kindly provided by: Dr, M. Boiocci, Profes sor G. Della Porta, and Professor U. Veronesi, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy; Dr. J. Fortner. Memorial Sloan-Kettering Cancer Center, New York, N. Y.; and Dr. P. Berard, Hopital Hotel Dieu, Lyon, France. Human S-9 was prepared within 3 hr after surgery under conditions iden Animals and Pretreatment tical to those described (or rat tissues; 1 to 3 ml portions were frozen in liquid nitrogen and stored at below -- 70 for less than Adult male and lemale BD VI and BD IV rats (110 to 130 g 3 weeks before use. For AHH determinations and mutagenicity body weight) were bred in the International Agency for Re assays, the samples were thawed rapidly in a water bath at search on Cancer laboratory and were fed on a Charles River 37 and used immediately. CRF diet. Groups of 2 to 3 rats received phenobarbitone AHH Determination sodium (1 mg/ml) in the drinking water for 7 days prior to the experiment. Other rats were also given either pregnenolone- In a final volume of 1 ml, S-9 (if not otherwise specified, 16a-carbonitrile (50 mg/kg body weight) p.o. 5 times at 12-hr equivalent to 4 to 15 mg wet weight tissue). 50 /imol Tris-HCl intervals (the last gavage 24 hr before the animals were killed) buffer (pH 7.4), 3 /imol MgCI?. 35 pmol KCI, 0.45 /imol NADP*, or s.c. injections of aminoacetonitrile (500 mg/kg body weight) and 3,umol glucose-6-phosphate were incubated at 37 for 10 once as a neutral 20% (w/v) solution of the bisulfate 24 hr min (rat liver) or 20 min (human liver) in the presence of 80 J ! 120 CANCER RESEARCH VOL. 40 AHH activity and S-9-mediated mutagenicity of liver biopsies from adult male and female human subjects AHH activity wasmeasured following a 20-min incubation with BP at 37* in the presence of human S-9 as described In "Materials and Methods/' Values were calculated from assays performed under conditions of linearity with respect to time and to liver protein concentration. The mean value S,E. for hepatic AHH activity in rats was calculated from the results of 3 series of experiments, each utilizing pooled livers of 2 rats. Mutagenicity assays (Procedure B) were carried out with S. typhimurium TA 1530 in the presence of 5 Of 10 j^mol of A/-nitrosomorpholine or A/-nitroso-N'*methylpiperazine, respectively, and 150 pi of human S9 per plate. The results are expressed as the number of revertants per 10 jimol ft~nitroso compound per plate. Mutagenicity assays with VC were performed by exposing plates containing S. typhimurium TA 1530 and 150 jtl human S-9 to a gaseous mixture of VC in air (Procedure A). Mean values S.E. for the mutagenicity of N-nitrosomorpholine. W-nitroso-AT'-methylpiperazine, and VC in the presence of rat S-9 were ^caiculatedjrpfh^to.5 series.of experiments, each utilizing pooled livers of 2 to 3 rats. ........................ .. ' ' AHH activity Mutagenicity (revertants/plate) pmol 3 HO- Experla rtuman'. ' BP/g liver/ ment subject Sex min 1 2 3 . -4 A *. > rYss F- fi .) ' 7, 8 9 10 11 12 13 14 15 16 17 13 19 20 U K T c V J R B L A G F M N E Y F M F v. f M M M M M F M M M M F F M F 30 38 65 ND NO 140 167 200 ND 280 NO 1420 1447 1500 1500 1595 1780 pmol 3-HOSP/mg liver protein/hr no' 30 75 ND ..150 ' 'rlo ' ' ND 150 147 ND ND 230 ND 990 919 1072 1006 1120 1170 N-Nitroso- AANitrosomorpho-" W'-mettlyl- tine piperaxine VC 35 20 ND ND 110 65 130 55 .NO, ./ * 180 ' - 130 '' ''leo- ' JNb`- 240 ND 390 340 20 NO 245 ND 300 NO 390 ND 540 ND NO ND ND ND ND ND 700 ND ND ND ND NO 30 20 45 25 ; >5st\ - . v 7Q 80 60 43 175 90 ND 220 115 110 157 92 74 ND . v.* * Mean 690 54Q 266 122 84 Rat BD VI F * ND, not determined. 1600 200 1 200 150 630 70 32 6 100 14 <U,`> ^ *1'^ * If 'V- R&S 135174 ^mol BP in 50 jtl acetone, which were added after 1 min of preincubation. The reaction was stopped by the addition of 1 ml ice-cold actone; 2 ml of n-hexane were added, and the mixture was vortexed for 1 min and then centrifuged. The organic layer was removed, and the phenolic BP metabolites were extracted with 2 ml 1 n NaOH. The amounts of fluorescent BP metabolites, predominantly 3-HO-BP and 9-hydroxybenzo(a)pyrene (38) in the alkaline solution, were measured in a Farrand spectrofluorometer at 522 nm with the excitation wavelength set at 396 nm exactly 10 min after adding the NaOH, using 3-HO-BP as a standard. AHH activity was ex pressed as nmol or pmol 3-HO-BP formed per g of wet tissue per min or per mg protein per hr; the results were calculated from assays performed under conditions of linearity with re spect to time and liver protein concentration. Tests with rat S9 showed that AHH activity was reduced by less than 10% when the sample was stored at below --70 for 1 month. Protein Determination Protein was determined according to the procedure of Lowry using bovine serum albumin (Fraction V; Sigma Chemical Co., St. Louis. Mo.) as standard. Mutagenicity Assays Salmonella typhimurium strains TA 1530 and TA 100 were provided by Professor B. N. Ames. Berkeley, Calif. The pres ence of the R-factor in TA 100 was checked by seeding bacteria on ampicillin-containing agar, and the mutability of TA 1530 and TA 100 strains was confirmed using /V-methyl-ATnitroso-N-nitroguanidine and methyl methanesulfonate, re spectively (1). Procedure A. Mutagenicity assays with VC were carried out in plate incorporation assays adapted to test volatile com pounds (6). Plates containing 1 to 2 x 108 bacteria of TA 1530 strain, 150 fil of rat or human S-9, 50 ftmal Sorensen phosphate buffer (pH 7.4), 2 ftmol NADP*, 2.5 fimol glucose-6-phosphate, and 4 jumol MgCI3 in 2.6 ml of soft agar overlay were exposed to a gaseous mixture of 20% VC in air (v/v) in a desiccator at 37 in the dark. After 4 hr of exposure, VC was removed by 2 successive evacuations of the dessicator and refillings with air; after further incubation of the plates for up to 48 hr at 37. the number of revertants was scored (in triplicate). The VC con centration (4 x 1CT3 m) in the aqueous phase of the plates was determined by gas-liquid chromatography (7). In some assays, disulfiram, dissolved in 50 /il DMSO at a final concen tration of 0.1 mw, was added to plates containing all ingredients described above. Mutagenicity in the presence of rat S-9 and VC was determined from assays in which the increase in the number of revertants per plate was proportional to VC concen tration and time of exposure. As verified by assays with various amounts of human S-9 in the presence of VC (data not in- JANUARY 1980 121 eluded), the increase in number of revertant colonies after plotted against the respective microsome-mediated mutagen exposure to VC was proportional up to .150 jrl of S-9 per plate icities in the presence of /V-nitrosomorpholine, W-nitroso-A/'- in most samples. The liver microsome- and NADPH-dependent methylpiperazine, and VC (Chart 2. A and S). A statistically formation of mutagenic VC metabolites in the TA 1530 strain significant.positive correlation was obtained between rates of was calculated by subtracting the number of revertants on BP hydroxylation and mutagenicity in the presence of /V-nitro plates in which NADP* and glucose-6-phosphate were omitted somorpholine (r = 0.86; p < 0.01), /V-nitroso-W'-methylpiper- after 4 hr exposure to VC [42 2 (S.E.)]. azine (r => 0.94; p < 0.1), and VC (r * 0.55; p < 0.1). When Procedure B. /V-Nitrosomorpholine.and W-nitroso-/V'-meth- the microsome-mediated mutagenicity of VC in the presence ylpiperazine (each added at a concentration of 5 or 10 ^mol/ of each of the 12 liver samples (Table 1) was plotted against plate) dissolved in 0.1 mI DMSO were added to plates contain- the respective mutagenicity in `the presenc'e"bf W-hitrosomor-' ing 150 fil of human or rat S-9 and 1 to 2 x 10 cells of TA pholine (Chart 20, a positive correlation was also obtained (r 1530 strain per plate, APB was dissolved in 0.1 ml DMSO and = 0.58; p < 0.05). In 2 human liver specimens (Samples Y and was tested in plates coritainihd 'ioO /tl'ofTiuman or rat S-9 and L), AHH activity and S-9-mediated mutagenicity in S. typhimu- 1 to 2 X 109 cells of TA 100 strain. All other ingredients were rium TA 100 strain were measured in the presence of 0.064 to the same as described in Procedure A. Freshly prepared plates 0.64 nmol AFB per plate. The mutagenic effects (revertants were kept in the dark at room temperature until the soft agar per nmol per plate) were 400 for liver Sample Y and 80 for had hardened; the inverted plates (ir^ triplicate) were incubated Sample L, corresponding to 5. and }%, _resp>ectively,,,qf ,th$, 'at 37. in the- dark;, and the' number'd1*;reverfams' was sd'ored ' ^mutagenicity observed'in the presence of S-9 from female BD/ after 48 hr.' Coritrol assays'/id wriich'the'tofactoYs for micro-1. Vf rats. In' addition, the ratios of AHH activity to liver microsome-' somal monooxygenases and/or the test compound were omit mediated mutagenicity in the presence of AFB were identical ted; were carried out simultaneously; the number of revertants in the 2 samples, the only ones studied. per plate (10 to 15 for strain TA 1530; 120 to 150 for strain TA Carcinogen Metabolism in Liver Fractions from Drug- 100) have been subtracted from the values reported. In the treated Rodents. We also examined the effect of pretreating linear part of the dose-response curves, none of the test rats with modifiers of the hepatic microsomal monooxygenase compounds or their metabolites was grossly toxic to the bac teria, since "normal'' background lawns of bacteria were pres ent on each plate. RESULTS 0 10r 0---------2-0p0.A ?Cr=094 p <01 . ' = 086 0 <001 300 <100 --TTM L 700 N 1500 k 300 Carcinogen Metabolism in Human Liver Specimens. AHH activity and capacity of liver specimens from different adult humans to convert /V-nitrosomorpholine, A/-nitroso-/V'-methylpiperazine, and VC into reactive metabolites mutagenic to S. typhimurium TA 1530 in vitro are shown in Table 1. The limited size of most samples precluded determination of all biological activities simultaneously. Both activities could always be meas ured reproducibly in positive control assays, using different pools of rat liver, which were included in each experiment with human tissues (Table 1); therefore, the different figures re ported for human liver specimens are unlikely to be attributable to variations in the methodology utilized. Sixtyfold interindivid ual differences in AHH activity were observed among the 15 liver specimens studied; the average AHH activity was 690 pmol 3-HO-BP per g of wet weight liver per min, which is equivalent to about 40% of the hepatic AHH activity observed in untreated female BD VI rats (Table 1). When AHH activity in human liver specimens was expressed as pmol 3-HO-BP per mg of liver protein per hr, the interindividual variation and the average activity relative to that of rat liver remained similar. Human S-9-mediated mutagenicity in the presence of Nnitrosomorpholine, W-nitroso-W'-methylpiperazine, and VC showed 35-, 1 7-, and 9-fold interindividual variations, respec tively. and the average enzymic activity of the human S-9 corresponded to 42% for /V-nitrosomorpholine, 380% for Nnitroso-/V'-methylpiperazine, and 84% for VC of the activity of S-9 from untreated female rats, which is given as 100 (Table D. To investigate whether BP hydroxylation parallels the metab olism of VC and /V-nitrosamines to reactive metabolites, the AHH activities of the human liver samples listed in Table 1 were J z R w V d 200 2 r" 100 cLU JJ. 2 |N-NI TflQSAMINESI , .. . 0 u_ REVESTANTS/PlATE *r 0 100 200 1600 B E* o - & G i J> v r eg M a 1500 woo 2p 200 1 iV w* H 7+ e IVmLLHiOiCl 0 100 REVERTA.NTS/P1.ATE 500 c -5 r=05S i,<005 'A =* ^z^_ 'Z > R T 2S2 V 1 ? > > vv 1t 1 RVftTANrS`,PLArg xN-NirR0S0M0*PH0UNe) TOO 100 0 300 200 TOO Chart 2. Relationship between AHH activity and S-9-mediated mutagenicity m human liver biopsies in the presence of (A) W-nitrosomorphoime () or N- nitrososo-N'-methyipioerazme (C) or (S) VC, C. plot of microsome-mediated mutagenicity of VC in the presence of 12 liver samples versus the mutagenicity with these samples ol N-nitrosomorphoime, Letters. liver specimens from different subjects; values for hepatic AHH activity and mutagenicity are taken from Table 1. 1 3 l ; R&S 135175 122 CANCER RESEARCH VOL. 40 R&S 135176 system to oeiermine me specificity of the' mbdifters'in altering mcnooxygenase-mediated BP hydroxylase activity and the mu^Jscenicity of W-nitrosomorpholine; VC,, and AFB (Table 2). ^^k:ues for AHH activity in rats given no or different drug ^^^atments are expressed as nmol 3-HO-BP per g of liver per AHH activity and mutagenicity 0/ N-nitrosomordtiotine, VC. and AFB with fiver from drug-treated and untreated rats Female BO Vl rats were pretreated with.the cirugs listed, as described in "Materials and Methods." AHH activity was measured following a 10-min incu bation with 8P at 37* in the presence of rat S-9 from untreated and pretreated (T-. -i and were taken from assays in which formation of phenolic k r metabolites was proportional to incubation time and to S-9 rats. The values are taken from assays In which formation of phenolic 0P metabolites was proportional to S-9 protein concentration and time of incubation; the reproducibility of the values for hepatic AHH activity in untreated rats and in r ;-icentration. Mutagenic activities were calculated from linear rats pretreated with phenobarbitone. pregnenolone-16-carbonitrile, and 3-mettv tiions of dose- and time- (in the case of VC) dependent _ .ays. yicholanthrene was ensured in 2 to 3 independent series of experiments. Mutagenic activity m S. typhimurium TA 1530 in the presence of VC was determined following exposure of plates to VC in air (Procedure A). Mean values "retreatmem 'oTratS ^ith"3-T^ethyrcholarithren9;'pregneno- -'rfr'TM 2 to 3 experiments, each using 2. pooted rat livers, are given, accept "the,..^. 3-16a-carbonitrile, phenobarbitone, or dibenamine inased the rate of. BP hydrqxy|atjciri,20-J 6-, 5:, and 1.2-fold, ncausae noff 3'tj-nmdefhthuyIlrchhrotllaannfthhfrAenfled MprefMtreaattmmaenfltl, . UwhiherAef-nonnffuy rotnnea ae*xKpnearii^mfflertnntf-was carried out. Mutagenicity assays (Procedure 6) were carried out with either S. typhimurium TA 1530 in the presence of up to 10 pmol N-nitrosomorpholine and * pectively, as compared with assays using liver from un ited animals (Table 2. Experiment 1). Aminoacetonitrile and 100 pi S-9 per plate or with S. typhimurium TA 100 in the presence of up to 0,08nmol AFB and 100 >il &9 per plate. Mean values taken from the finear region of dose-dependent assays are reported from 2 to 3 series of experiments, each ulfiram treatment both lowered AHH activity by 25%. When utilizing pooled livers of 2 to 4 rats. ulfiram at a final concentration of 0.1 him was added to the '-ju . , bbation.-^iiedium,:piontajning.S^9?Jronj untreated,rats, AHH ' ri,.. , ,;ivity,was reduced'by.80%:>with' a,i rtuvi'co'ndentrationj'BP ' "* " '' ` droxylation was inhibited completely. In contrast, hepatic `* ' ` . , '* 1H activity in 3-methylcholanthrene-treated rats was reduced a maximum of only 30 to 40% when.disulfiram was present a concentration of 0.1 to 2 mM (data not included). 3-9-mediated mutagenicity of W-nitrosomorpholine in- Experi -eased 2- and 3-fold after pretreatment of rats with pheno- ment Treatment S-9-mediated mutagenicity Hepatic AHH ae- t. V* ' \ ` ' ' * '*) ' * W-Nftro- I ' ' A. ' li . ' \ . * *, somor- pholine (TA 1530 AFB (TA reveri- VC fTA 100 re- ants/ 1530 re- vertantj/ ;imol/ vertants/ nmol/ plate) plate) plate) .' .1 - (nmol 3HO-BP/ g liver/ min) jrbitone and pregnenolone-16a-carbonitrile, while 3-methy(lolanthrene and dibenamine reduced the mutagenic activity y 20%, and aminoacetonitrile and disulfiram reduced the lutagenicity of W-nitrosomorpholine by 70% (Table 2). Micro.ome-dependent conversion of VC into mutagenic metabolites jy rat S-9 was enhanced after treatment with phenobarbitone nd 3-methylcholanthrene; aminoacetonitrile, disulfiram, and regnenolone-16a-carbonitrile decreased the mutagenic ef fects, although the latter compound induced AHH activity. Addition of disulfiram to plates at a final concentration of 0.1 1 None 2 Phenobarbitone 3 Pregnenolone-16o- carbonitnle 4 3-Metbylcholan- threne 5 Dibenamine 6 Aminoacetonitrile 7 Disulfiram 8 Disulfiram (0.1 mM added in vitro) NO, not determined. 90 235 265 70 70 25 20 NO 100 8,400 1.6 130 13.600 7.5 35 12J00 10 135 6,900 32 100 12.050 50 7.950 20 ND'1 5 NO 2 1.2 1.2 0,3 mM lowered liver microsome-mediated mutagenicity of VC by 95%. Changes in the liver microsome-mediated mutagenicity or of other adverse biological effects caused by environmental of AFB after the different drug treatments were qualitatively pollutants. Because data on carcinogen metabolism cannot be similar to those observed in experiments with W-nitrosomor obtained directly from humans, we have used mutagenicity pholine, except that dibenamine increased AFB mutagenicity assays (1) to estimate the concentrations of ultimate reactive by 45% (Table 2). metabolites formed from carcinogens such as W-nitrosomor When the values for hepatic AHH activity in drug-treated rats pholine and W-nitroso-W'-methylpiperazine, VC. and AFB in the were compared with the respective values for liver microsome- presence of a series of human surgical liver specimens. For mediated mutagenicity of W-nitrosomorpholine, VC, and AFB these carcinogens, there is circumstantial evidence that the (Table 2), a positive correlation between the 2 activities was mutagenic metabolites produced in vitro may be the same as noted only when the corresponding values for 3-methylchol those which initiate carcinogenesis by the parent compound in anthrene (an inducer of the cytochrome P^148-dependent experimental animals and probably also in humans. VC is monooxygenase system) were omitted from the calculation of converted by microsomal enzymes into chloroethylene oxide, the correlation coefficients: W-nitrosomorpholine (r =- 0.98; p a highly electrophilic, mutagenic (4. 6). and carcinogenic <0.001); VC (r = 0.55; p <0.1); and AFB (r = 0.72; p <0.D. agent5; AFB undergoes microsomal oxidation to yield aflatoxin These results indicate a proportionality between the amount of B,-2.3-oxide, a compound very probably responsible for the mutagenic metabolites produced and the cytochrome P-450 mutagenic and carcinogenic effect of the parent toxin (1 5, 32, content of the liver microsomal preparations. 47); the heterocyclic W-nitrosamines, W-nitrosomorpholine or W-nitroso-W'-methylpiperazine, are thought to yield alkylating DISCUSSION Most environmental carcinogens are metabolized by micro intermediates following oxidation at the n-carbon atom (14, 33, 37). Large interindividual variations were noted in the capacity of somal cytochrome P-450-dependent monooxygenases (e.g., the human liver specimens to hydroxylate BP (60-fold) and to AHH). Information regarding the inherent variation of this en- 0yme system in different human subjects may eventually facil- ' Zaidela. F., Croisy. A.. Barbin. A.. Mataveiile, C,, Tomalis, L., and Bartsch, te the identification of individuals with a higher risk for cancer H. Submitted (or publication. * JANUARY 1980 123 m' ss 7N-' .Sa*b-1sa1 dmie et agl. m*** activate VC (9-fold), N-nitroso-N'-methylpiperazine (17-fold), and N-nitrosomorpholine (35-fold). A variation of similar mag nitude has previously been reported.iri the oxidative metabolism of BP in human livers (29, 40), and the cytochrome P-450 level could be used for assessing rates of metabolism of other environmental carcinogens, especially those which include th liver as the main target organ, we compared BP hydroxyla activity in human liver samples with their respective ability in livers from 200 human subjects was found to vary 65-fold convert VC. N-nitrosomorpholine. and AFB into mutagenic (44). In our study, the mean AHH activity in 5 females was electrophiles. A statistically significant correlation (p < 0,1) similar to that observed in male human subjects. The average between the rate of BP hydroxylation and liver microsome- BP hydroxylase activity in our 15 specimens was higher than mediated mutagenicity in the presence of N-nitrosomorpholine that reported for 20 liver samples which were processed within or /V-nitroso-/V'-methylpiperazine and VC was obtained (Chart 1 hour of biopsy and the enzymes.pf which.were.assayed.on, , 2, A and B). Similarly, a plot of microsome-mediated mutagen-'^'^ ' "the same day (28)/.These data~suggest that experimental' icity in the presence of VC versus that with N-nitrosomorpholine variations or different lengths of storage do not account for the in 12 human liver specimens (Chart 2C) also revealed a statis . .interindividual differences inactivity (Table 1).- Since no data tically significant positive correlation (r => 0.58; p < 0.05). The on drug intake or smoking habits of the subjects were available, lack of a perfect relationship suggests that human livers from R&S 135177 the reasons for the differences in the rate of oxidative metab different individuals have different relative proportions of var olism noted in our human samples cannot be established. The ious forms of cytochrome P-450; multiple P-450 cytochromes average capacities of all liver samples from male and female are known to occur in rats, mice, and rabbits (19, 21 ( 42). A ; human subjects ta.actLvate'A/-nitrosomorpholrne.and:.VC into. proportionality has'previously been' reported* between/cyjto- ..t ' mutagens 'were^42 and B4%,. respectively,.- of, that of liver of v chrome -P-450 conteht'and the ability "of human river micro- untreated female BD VI rats. In contrast, the mean activity of somes to activate N-nitrosodimethylamine to a bacterial muta the human livers in converting N-nitroso-N'-methylpiperazine gen (13). into mutagens was 4 times (in some individuals up to 10 times) Evidence that N-nitr.osomorpholine, VC. and AFB are also higher than that in control rat liver. In 2 human liver samples, activated metabolically by cytochrome P-450-dependent mi mutagenicity in the presence of AFB was <5% of that observed crosomal monooxygenases in rat liver (6, 17, 26, 45, 47) is for rat liver; similar mean values for liver-mediated mutagenicity provided by our studies in which rats were treated with inducers of AFB (on the order of <10% that of rat liver) have been or inhibitors of the hepatic monooxygenase system (Table 2); reported elsewhere (20, 48). These observed species differ changes in AHH activity and microsome-mediated mutagenicity ences may indicate that the monooxygenases in rat liver which in vitro were recorded. A comparison of hepatic AHH activities oxidize AFB into aflatoxin Bt-2,3-oxide may be different from in the livers of rats treated with different drugs versus the those in human liver. The differential stimulation of microsomal respective values for microsome-mediated mutagenicity in the enzymes of rat and of human origin by 7,8-benzoflavone to presence of either N-nitrosomorpholine, VC, or AFB revealed activate AFB into mutagens in vitro supports this assumption positive correlations (which were significant in the case of N- (10). nitrosomorpholine) only when the values for 3-methylcholan- Although great caution must be exercised when extrapolat threne, an inducer of the cytochrome-P-448-dependent mono- ing from in vitro data to the mechanisms in the intact organism, oxygenase system, were omitted from the calculations. These the validity of in vitro mutagenicity data for predicting species results suggest that there is a relationship between the amount susceptibility to certain hepatocarcinogens is supported by the of mutagenic metabolites produced from these 3 carcinogens observation that the efficiency with which livers of different and the activity of cytochrome P-450-dependent monooxygen animal species activate aflatoxins corresponds to their suscep ases in the rat liver preparations. tibility to the hepatocarcinogenic effect of these mycotoxins The effect of treatment of rats with phenobarbitone, preg (20). Similarly, although our data revealed some species and nenolone-1 6a-carbonitrile, 3-methylcholanthrene, and diben- interindividual variations, the average capacity of all human amine on liver microsome-dependent mutagenicity in the pres liver specimens to convert N-nitrosomorpholine or VC into ence of N-nitrosomorpholine. VC. and AFB was substrate spe reactive intermediates was close to that of rat liver (Table 1). cific (Table 2). Administration of 3-methylcholanthrene and Such data would indicate that humans are probably not resist phenobarbitone enhanced hepatic AHH activity severalfold, ant to the adverse biological effects caused by these carcino while aminoacetonitrile and disulfiram led to a reduction. The gens. an assumption which is supported by the finding that VC latter 2 drugs and diethyldithiocarbamate. a cleavage product induces angiosarcomas in human liver (25). readily formed from disulfiram in the presence of rat liver Model drugs which are nontoxic but are metabolized by the cytosol (46), impair monooxygenase activity of microsomes in same enzyme system as carcinogens have been proposed as vitro (18, 22, 53); the marked inhibition of AHH activity could probes to gather information on the carcinogen-handling ca be explained by such a mechanism. Interestingly, the inhibitory pacity in human subjects. Kapitulnik et al, (29) found positive effect of disulfiram was greater with liver microsomes from correlations between the rates of hydroxylation of BP and those control rats than with liver microsomes from 3-methycholan- of antipyrine, hexobarbital, and zoxazolamine in human au threne-treated rats. In rats treated with 3-methylcholanthrene topsy livers. These observations were expanded by Kala- (an inducer of the microsomal cytochrome P-448-linked mon megham er al. (28) and Sotaniemi et al. (44) who reported a ooxygenases). hepatic AHH activity was inhibited by only 30% positive association between antipyrine metabolism in vivo and after in vitro addition of 0,1 to 2 mM disulfiram (data not hepatic AHH activity or cytochrome P-450 content determined included), suggesting that disulfiram (or its cleavage product) in vitro. The observed correlation, although statistically signifi may interact preferentially with the cytochrome P-450 in liver cant, was not sufficiently strong to have clinically significant from control rats. predictive value. To further explore whether BP hydroxylation Administration of inducers and inhibitors of hepatic micro- 124 CANCER RESEARCH VOL. 40 pendent mutagenicity of many carcinogens, and these treatnts also modify carcinogenicity in vivo, possibly by changing 67: 596-603, 1975, 5. Bartsch, H,, Camus, A., and MalaveiUe. C. Comparative mutagenicity of /V* nitrosamines in a semi-solid and in a liquid incubation system in the presence | balance between activation and detoxification reactions of rat or human tissue fractions. Mutat. Res.. 37: 149-162. 1976. ). Comparison of the drug-induced alterations in vitro and 6. Bartsch. H., Malaveille. C., Barbin, A., and Planche, G. Mutagenic and alkylating metabolites of halo-ethylenes. chlorobutadienes and dichlorobu- in avo showed a good, but not perfect, correlation; although tones produced by rodent or human liver tissues; evidence for oxirane phcnobarbitone treatment increased AFB mutagenesis, it is formation by P-450-llnked microsomal monooxygenase. Arch. Toxicol., 4f. Known to be inhibitory to AFB carcinogenicity in the rat liver 249-277. 1979. 7. Bartsch, H.. Malaveille, C., and Montesano. R. Human, rat and mouse liver- On the other hand, the inhibitory effect of disulfiram on mediated mutagenicity of vinyl chloride in 5. typhimunum strains. Int. J, A;-:i activity and on liver microsome-mediated mutagenicity in f ' Cancer. 75:429-437. 1975. ' ' * - th" presence of AAnitrosomorpholine was matched by-an inhib 8. Bartsch, H., Sabadie. N.. Malaveille, C.. Camus. A-M,, and Brun, G. Tissue specificity fn metabolic actfvatten; frrrYirCohen (ed.), Advances in pharma-- it in vivo action of disulfiram on the induction of forestomach coiogy and therapeutics. Vol.'9, Toxicology Adv`. Pharmacol. Chemother./ : ors by BP in mice (9. 50) and of /V-nitrosodialkylamine ction of liver tumors in rats' {43)'.* The Hepafbtoxicity of VC 9:93-102, 1978. 9. Borchert, P,, and Wattenberg, L W. Inhibition of macromolecular binding of benzo(a}pyrene and'inhibition of neoplasia by disulfiram' in the mouse its has been shown, to be increased by administration of nobarbitone and to be decreased by pregnenolone-16a;onitrile (27, 41). These changes are in accord with our forestomach, J. Natl. Cancer Inst-. 57: 173-179, 1976. 10. Boening, M. K.. Fortner, J. G.. Kappas, A., and Conney, A. H. 7,8-Benzo- flavone stimulates the metabolic activation of aflatoxin B to mutagens by human liver. Biochem. Blophys. Res. Commun,, 82: 348-355, 1978. agenicity data,(Table 2)..ln;c.onclusio^( our studies on the 11. Conney, A. H.. and Levin, W. Carcinogen metabolism in experimental animals :la`tive':mefaboiism bf.BP'r^C^Are^an^cycItcf'A^riitrbsa-,# ^ * ^ ' and man*Jn: R..Montesano ant^L- Jomati>(eds.), Chemical Carcinogenesis^ Essays, IARC Scientific:Publicatidn No. fO.'pp. 3-^24Lyoh,*France: Inter-- 'as revealed'lafge fnterihdivid'Qat''differehces''irr1he 'activity ** ^x-nationaPAgency for Research on Cancer. 1974^/ licrosomal cytochrome P-450-dependent monooxygenases uman liver specimens. The relevance of these observations 12. Conney, A. H., Pantuck, E. J.. Hsiao. K-C., Kuntzman. R.. Afvares, A. P., and Kappas. A. Regulation of drug metabolism in man by environmental chemicals and diet. Fed. Proc., 36; 1647-1652, 1977. nhanced by the consistency with which interindividual var 13. Czygan. P., Greim, H.. Garro, A, J.. Hutterer, F., Rudick, J.. Schaffner. 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