(CANCER RESEARCH 40, 119-1 26, January 1980)0008-5472/80/0040-0000$02.00

Comparison of the Hydroxylation of Benzo(a)Pyrene with the Metabolismof Vinyl Chloride, N-Nitrosomorpholine, and N-.Nitroso-N'-.Methylpiperazine to Mutagens by Human and Rat LiverMicrosomal Fractions1

Nicole Sabadie,2 Christian Malaveille, Anne-Marie Camus, and Helmut Bartsch3

Unit of Chemical Carcinogenesis, International Agency for Research on Cancer, 69372 Lyon Cedex 2, France

ygenases (e.g. , AHH@),which are responsible for both thedetoxification and metabolic activation of xenobiotics. Sincemany environmental carcinogens are substrates for the cytochrome P450-dependent monooxygenases, these enzymesalso mediate carcinogenesis and toxicity through the formationof electrophilic intermediates via oxidative pathways (31 , 36).Covalent binding of such ultimate carcinogenic metabolites andthe concentrations available for reaction with macromoleculesin the target tissues are determinants for the onset of tumorigenesis. Inherent variations of monooxygenase activity by environmental and genetic factors (12, 49) may thus be associated with the individual differences in susceptibility to cancerseen in both experimental animals and humans. In mice, genetically controlled induction of AHH activity has been shownto influence tumor response to polycyclic aromatic hydrocarbons (31). In humans, a positive association has been suggested between AHH mnducibility in lymphocytes and an increased risk for bronchiocarcinoma in smokers (30), althoughthis has not been confirmed (39). Possible limitations of experimental studies carried out on human lymphocytes have beenpresented (35).

One major difficulty in determining interindividual differencesin rates of metabolism of carcinogens in humans is that suchdata cannot be obtained directly from in vivo studies. Consequently, in vitro and in vivo studies with model (predictor) drugs(themselves nontoxic but metabolized by the same enzymesystems as environmental carcinogens) have been proposedas probes to collect information on the carcinogen-metabolizing ability of individual human subjects. Recent work hasprovided some evidence that such predictor drugs may exist.Kapitulnik et a!. (29) reported significant positive correlationsbetween rates of BP hydroxylation (AHH activity) and in vitrooxidative metabolism of antipyrine, zoxazolamine, and hexobarbital in human livers obtained at autopsy. Kalamegham eta!. (28) and Sotaniemi et a!. (44) found a statistically significantpositive association between antipyrine elimination in vivo andhepatic AHH activity in vitro in 15 human subjects or cytochrome P450 content in human livers.

In our studies with 20 human liver specimens, we extendedthese investigations to include carcinogenic chemicals to whichhumans may be exposed. The animal or human carcinogensN-nitrosomorpholine, N-nitroso-N'-methylpiperazine, VC, andAFB (14, 23—25)(Chart 1) are converted by rodent on humanliver microsomal monooxygenases into electrophiles which can

4 The abbreviations used are: AHH, aryl hydrocarbon [benzo(a)pyrene) hy

droxylase; BP;benzo (a) pyrene; VC, vinyl chloride; AFB. aflatoxin B, ; 5.9, 9000x g liver supernatant fraction; 3-HO-BP. 3-hydroxybenzo(a)pyrene; DM50,dimethyl sulfoxide.

ABSTRACT

Carcinogen metabolism in vitro was studied in 20 surgicalliver specimens from adult male and female human subjects. A60-fold interindividual variation in aryl hydrocarbon [benzo(a)pyrene] hydroxylase activity was seen; the capacity to convert vinyl chloride, N-nitroso-N'-methylpiperazmne, and N-nitrosomorpholine into electrophiles mutagenic to Sa!mone!!a typhimurium varied 9-, 17-, and 35-fold, respectively. The meanenzymic capacity relative to that of liver from untreated ratswas 84% for vinyl chloride, 42% for N-nitrosomorpholine, and380% for N-nitroso-N'-methylpiperazine. When aryl hydrocarbon [benzo(a)pyrene] hydroxyk@seactivity in human liver specimens was plotted against mutagenicity in S. typhimurium mediated by liver microsomes from the same specimens, a positivecorrelation was obtained in the presence of vinyl chloride (r =0.55; p < 0.1), N-nitrosomorpholine (r = 0.86; p < 0.01), orN-nitroso-N'-methylpiperazine (r = 0.94; p < 0.01 ). The resultsare discussed in relation to the possible development of methods for assessing rates of metabolism of environmental carcinogens in human subjects in vivo using nontoxic drugs that aremetabolized by the same enzymes that metabolize carcinogens. Hepatic aryl hydrocarbon [benzo(a)pyrene] hydroxylaseactivity following treatment of rats with phenobarbitone, pregnenolone-16a-carbonitrile, dibenamine, aminoacetonitrile, ordisulfiram, but not after treatment with 3-methylcholanthrene,showed a positive correlation with the mutagenicity of therespective 9000 x g liver supernatant fraction mediated in thepresence of N-nitrosomorpholine, vinyl chloride, or aflatoxinB1. These data lend further support that cytochrome P450-linked monooxygenases in rat liver convert N-nitrosomorpholine, vinyl chloride, and aflatoxin B1 into mutagenic electrophiles.

INTRODUCTION

Large interindividual differences in the ability of humanbeings to oxidize chemical carcinogens (11) and other foreigncompounds (49) are well documented. The enzymes involvedin most cases are the cytochrome P450-dependent monoox

Received July 19, 1979; accepted September 20, 1979.‘PartiallysupportedbyContractNOI-CP-55630fromtheNationalCancer

Institute, NIH, Bethesda, Md. Someof the data were presented at the InternationalColloquium of CNRS, No. 256, on@ Mécanisrnesd'altórationet de reparation duDNA, relationsavec Ia mutagéneseet Ia cancérogénesechimique'‘, 4—9July,I 976, Menton, France, and at the Seventh International Congress of Pharmacology, 1978, Park,, France (8).

2VisitingscientistfromtheDepartmentofBiochemistry(Director:ProfessorD. Gautheron), University of Lyon, Lyon, France.

3 To whom requests for reprints should be addressed.

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BENZO(a)PYRENE

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prior to the assays. Disulfiram was given@ a dose of 500 mg/kg body weight as a suspension in starct@solution by gavage4 hr before the animals were killed. Dibe4amine was injecteds.c. twice into rats at a dose of 25 mg/kg @8and 24 hr beforethe animals were killed. Rats also receive@an i.p. Injection of3-methylcholanthrene (40 mg/kg) 2 day$ before they werekilled.

Animal and Human Tissue Preparations

5-9 was prepared at 0—4°from the poo'ed livers of 2 to 3rats by centrifugation of a homogenate (3 4@lof 0. 15 M KCI-5mM Sorensen buffer (pH 7.4) per g of wet lfrer). The resultingfractions were kept at 0—4°for less than@ hr and then usedsimultaneously in mutagenicity assays an4l AHH determinations. All procedures were carried out usin@sterile glasswareand solutions. Samples of human liver wi@i no pathologicallesions were obtained from female and mal@adults either fordiagnostic purposes or as surgical specir@iens.The humansubjects listed in Table 1, from whom diagn@sticand surgicalspecimens were taken, were affected by the f4llowing diseases:gastric adenocarcinoma (Subject E); metaskatic colonic adenocarcinoma (Subject F); metastatic pancr4atic adenocarcinoma (Subject G); primary melanoma (Subje$t H); hepatoblastoma (Subject J); pancreatic carcinoma (Sub@ct K); hepatoma(Subject M); colonic adenocarcinoma with \Iiver metastases(Subject N); cholecystitis lithiasis (Subject4 L and Y); andHodgkin's disease (Subjects A, B, C, 0, R, S@T, U, V, and W).Liver samples were kindly provided by: Dr. M4Boiocci, Professor G. Della Porta, and Professor U. Veronesi, @titutoNazionaleper lo Studio e Ia Cura dei Tumori, Milan, Ital$r;Dr. J. Fortner,Memorial Sloan-Kettering Cancer Center, Nevi@York, N. Y.; andDr. P. Berard, HópitalHotel Dieu, Lyon, Frav@ce.Human 5-9was prepared within 3 hr after surgery under @onditionsidentical to those described for rat tissues; 1 to 3 r@ilportions werefrozen in liquid nitrogen and stored at below —@‘0°for less than3 weeks before use. For AHH determinations avid mutagenicityassays, the samples were thawed rapidly in@ water bath at370 and used immediately.

AHH DeterminatIon

In a final volume of 1 ml, 5-9 (if not othe4iise specified,equivalent to 4 to 16 mg wet weight tissue), 5@ @tmolTris-HCIbuffer (pH 7.4), 3 @molMgCI2,35 @smolKCI, 0.4@ @molNADP@,and 3 @smolglucose-6-phosphate were incubat4i at 37°for 10mm (rat liver) or 20 mm (human liver) in the esence of 80

CANCERRESE@RCHVOL. 40

N-NITROSOMORPHOLINE

bind to nucleic acids (5, 7, 15, 16, 32, 45, 47). We thereforeexamined the magnitude of interindividual variations in activityin human liver by making parallel measurements of micnosomemediated mutagenicity and AHH activity. We also examinedthe effect of treating rats with modifiers of the hepatic microsomal monooxygenase system on AHH activity and on 8-9-mediated mutagenicity of N-nitrosomorpholine, VC, and AFB.

The results presented herein on comparative metabolism ofcarcinogens in animal and human tissues may provide a basisfor the extrapolation of carcinogenicity data from animals tohumans, e.g. , for N-nitrosomorpholine (25), to which humansmay be exposed, but for which there are no epidemiologicaldata or case reports of tumor induction.

MATERIALS AND METHODS

Chemicals

VC (purity, 99.9%) was generously provided by RhOne-Poulenc, Lyon, France. Pregnenolone 16a-carbonitrile was a giftfrom Amersham/Searle Inc., Chicago, III. Disulfiram [bis(diethylthiocarbamoyl) disulfide] was provided by the late Dr. F.K. Kruger, German Cancer Research Centre, Heidelberg, Federal Republic of Germany. 3-HO-BP was obtained from theChemical Repository of the National Cancer Institute, Bethesda, Md., through Dr J. N. Keith. The following productswere obtained commercially from the sources indicated: aminoacetonitrile bisulfate (Koch Light Laboratories, Ltd.,Colnbrook, Buckinghamshire, United Kingdom); dibenamine[N-2-chloroethyl)dibenzylammne], 3-methylcholanthrene, andBP (Aldrich Chemical Co., Milwaulkee, Wis.); AFB, N-nitrosomorpholine, and N-nitroso-N'-methylpiperazine (Merck-Schuchardt, Danmstadt,Federal Republic of Germany).

Animals and Pretreatment

Adult male and female BD VI and BD IV rats (110 to 130 gbody weight) were bred in the International Agency for Research on Cancer laboratory and were fed on a Charles RiverCRF diet. Groups of 2 to 3 rats received phenobanbitonesodium (1 mg/mI) in the drinking water for 7 days prior to theexperiment. Other rats were also given either pregnenolone16a-canbonitrile (50 mg/kg body weight) p.o. 5 times at I 2-hrintervals (the last gavage 24 hr before the animals were killed)or s.c. injections of aminoacetonitnile (500 mg/kg body weight)once as a neutral 20% (w/v) solution of the bisulfate 24 hr

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Mutagenicity assays (Procedure B) were carried out with S. typhimurium TA 1530 in the presence of 5or 10 @molof N-nitrosomorphollne or N-nitroso-N'-methylpiperazlne, respectively, and 150 @lof human5-9

per plate. The results are expressed as the number of revertants per I 0 @molN-nitroso compoundperplate.Mutagenicity assays with VC were performed by exposing plates containing S. typhimurium TA1530and

150 ILl human 5-9 to a gaseous mixture of VC In air (Procedure A). Mean values ±SE. forthemutagenicityof N-nitrosomorpholine, N-nitroso-N'-methylplperazmne,and VC in the presence of rat 5-9werecalculated

from 3 to 5 serIes of experiments, each utilizing pooled livers of 2 to 3rats.AHH

activity Mutagenicity(revertants/plate)pmol

3 HO- pmol 3-HO- N-Nitroso- N-NitrosoExperl- Human BP/g liver! BP/mg liver morpho- N'-methylmont subject Sex mm protein/hr line piperazineVC1

U F 30 ND8 35 20302K M 38 30 ND ND203T F 65 75 110 654545 F ND ND 130 55255H M 90 72 ND ND556W M 105 150 180 130507D F ND ND 180 ND708C M ND ND 240 ND809V M 140 150 390 3406010J M 167 147 20 ND4311R M 200 ND 245 ND17512B F ND ND 300 ND9013L M 280 230 390 NDND14A M ND ND 540 ND22015G M 1420 990 ND ND11516F M 1447 919 ND ND11017M F 1500 1072 ND ND15718N F 1500 1006 700 ND9219E M 1595 1120 ND ND7420Y F 1780 1170 ND NDNDMean

690 548 266 12284Rat

BD VI F 1600 ±200 1200 ±150 630 ±70 32 ±6 100 ±14a

ND, not determined.

Carcinogen Activation by Rat and Human Liver Microsoma! Fractions

Table1

AHH activity and S-9-mediated mutagenicity of liver biopsies from adult male and female human subjectsAHH activity was measured following a 20-mm incubation with BP at 37°in the presence of human 5-9

as described in “Materialsand Methods.―Values were calculated from assays performed under conditionsof linearity with respect to tIme and to liver protein concentration. The mean value ±SE. for hepatic AHHactivity in rats was calculated from the results of 3 series of experiments, each utilizing pooled livers of 2rats.

@tmoIBP in 50 @Iacetone, which were added after 1 mm ofpreincubation. The reaction was stopped by the addition of 1ml ice-cold actone; 2 ml of n-hexane were added, and themixture was vortexed for 1 mm and then centrifuged. Theorganic layer was removed, and the phenolic BP metaboliteswere extracted with 2 ml 1 NNaOH. The amounts of fluorescentBP metabolites, predominantly 3-HO-BP and 9-hydroxybenzo(a)pyrene (38) in the alkaline solution, were measured ina Farrand spectrofluorometer at 522 nm with the excitationwavelength set at 396 nm exactly 10 mm after adding theNaOH, using 3-HO-BP as a standard. AHH activity was expressed as nmol or pmol 3-HO-BP formed pen g of wet tissueper mm or per mg protein per hr; the results were calculatedfrom assays performed under conditions of linearity with respect 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 Lowryusing bovine serum albumin (Fraction V; Sigma Chemical Co.,St. Louis, Mo.) as standard.

Mutageniclty Assays

Sa!mone!!a typhimurium strains TA 1530 and TA 100 were

provided by Professor B. N. Ames, Berkeley, Calif. The presence of the R-factor in TA 100 was checked by seedingbacteria on ampicillin-containing agar, and the mutability of TA1 530 and TA 1 00 strains was confirmed using N-methyl-N'-

nitroso-N-nitroguanidmne and methyl methanesulfonate, respectively (1).

Procedure A. Mutagenicity assays with VC were carried outin plate incorporation assays adapted to test volatile compounds (6). Plates containing 1 to 2 x 1O@bacteria of TA 1530strain, 150 @tlof nator human S-9, 50 zmolSørensenphosphatebuffer (pH 7.4), 2 @imoINADP@,2.5 j.tmolglucose-6-phosphate,and 4 @tmoIMgCI2 in 2.6 ml of soft agar overlay were exposedto a gaseous mixture of 20% VC in air (v/v) in a desiccator at370 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°,thenumber of revertants was scored (in triplicate). The VC concentration (4 x 1o-@M) in the aqueous phase of the plateswas determined by gas-liquid chromatography (7). In someassays, disulfiram, dissolved in 50 @tIDMSO at a final concentration of 0.1 mM,was added to plates containing all ingredientsdescribed above. Mutagenicity in the presence of rat S-9 andVC was determined from assays in which the increase in thenumber of revertants per plate was proportional to VC concentration and time of exposure. As verified by assays with variousamounts of human 5-9 in the presence of VC (data not in

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N. Sabadie et a!.

cluded), the increase in number of revertant colonies afterexposure to VC was proportional up to 150 @xlof 5-9 per platein most samples. The liver microsome- and NADPH-dependentformation of mutagenic VC metabolites in the TA 1530 strainwas calculated by subtracting the number of revertants onplates in which NADP@and glucose-6-phosphate were omittedafter 4 hr exposure to VC [42 ±2 (S.E.)].

Procedure B. N-Nitrosomorpholineand N-nitroso-N'-methylpiperazine (each added at a concentration of 5 or I 0 @mol/plate) dissolved in 0.1 ml DMSO were added to plates containing 150 @.tIof human or rat S-9 and 1 to 2 X 108 cells of TA1530 strain per plate. AFB was dissolved in 0.1 ml DMSO andwas tested in plates containing 100 @&Iof human or rat 5-9 andI to 2 x 108 cells of TA 100 strain. All other ingredients werethe same as described in Procedure A. Freshly prepared plateswere kept in the dark at room temperature until the soft agarhad hardened; the inverted plates (in triplicate) were incubatedat 37°in the dark, and the number of revertants was scoredafter 48 hr. Control assays, in which the cofactors for microsomal monooxygenases and/or the test compound were omitted, were carried out simultaneously; the number of revertantsper plate (10 to 15 for strain TA 1530; 120 to 150 for strain TA100) have been subtracted from the values reported. In thelinear part of the dose-response curves, none of the testcompounds or their metabolites was grossly toxic to the bactenia,since ‘‘normal'‘background lawns of bacteria were pres

plotted against the respective microsome-rricities in the presence of N-nitrosomorpholmethylpiperazmne,and VC (Chart 2, A andsignificant positive correlation was obtainedBP hydroxylation and mutagenicity in the prsomorpholine (r = 0.86; p < 0.01 ), N-nitrosazine (r = 0.94; p < 0.1), and VC (r = 0.5the microsome-mediated mutagenicity of Vof each of the 12 liver samples (Table 1) wthe respective mutagenicity in the presencipholine (Chart 2C), a positive correlation w@= 0.58; p < 0.05). In 2 human liver specime

L), AHH activity and S-9-mediated mutagenirium TA 100 strain were measured in the pri0.64 nmol AFB per plate. The mutagenic@per nmol per plate) were 400 for liver San@Sample L, corresponding to 5 and 1%, re@mutagenicity observed in the presence of S-@VI rats. In addition, the ratios of AHH activity tmediated mutagenicity in the presence of Ain the 2 samples, the only ones studied.

Carcinogen Metabolism In Liver Fractreated Rodents. We also examined the erats with modifiers of the hepatic microsom

0 100 200 300 400

diated mutagenne, N-nitroso-N'B). A statisticallybetween rates ofsence of N-nitro-N'-methylpiper; p < 0.1). Whenin the presence

5 plotted against

of N-nitrosomoralso obtained Cr

S (Samples Y andity in S. typhimuence of 0.064 tofects (revertantspIe Y and 80 foripectively, of the) from female BDliver microsomeB were identical

ons from Drug@ctof pretreatingmonooxygenase

700ent on each plate.

RESULTS

Carcinogen MetabolIsm in Human Liver Specimens. AHHactivity and capacity of liver specimens from different adulthumans to convert N-nitrosomorpholine, N-nitroso-N'-methylpiperazine, and VC into reactive metabolites mutagenic to S.typhimurium TA 1530 in vitro are shown in Table 1. The limitedsize of most samples precluded determination of all biologicalactivities simultaneously. Both activities could always be measured reproducibly in positive control assays, using differentpools of rat liver, which were included in each experiment withhuman tissues (Table 1); therefore, the different figures reported for human liver specimens are unlikely to be attributableto variations in the methodology utilized. Sixtyfold interindividual differences in AHH activity were observed among the 15liver specimens studied; the average AHH activity was 690pmol 3-HO-BP per g of wet weight liver per mm, which isequivalent to about 40% of the hepatic AHH activity observedin untreated female BD VI rats (Table 1). When AHH activity inhuman liver specimens was expressed as pmol 3-HO-BP permg of liver protein per hr, the interindividual variation and theaverage activity relative to that of rat liver remained similar.

Human S-9-mediated mutagenicity in the presence of N-nitrosomorpholine, N-nitroso-N'-methylpiperazine, and VCshowed 35-, 17-, and 9-fold intenindividual variations, respectively, and the average enzymic activity of the human 8-9corresponded to 42% for N-nitrosomorpholine, 380% for N-nitroso-N'-methylpiperazine, and 84% for VC of the activity ofS-9 from untreated female rats, which is given as 100 (Table1).

To investigate whether BP hydroxylation parallels the metabolism of VC and N-nitrosamines to reactive metabolites, theAHH activities of the human liver samples listed in Table 1 were

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Chart 2. RelatIonship between AHH activity and S-@in human liver biopsies in the presence of (A) N-nltrcnitrososo-N'-methylpiperazine (0) or (B) VC. C, plotmutagenicity of VC in the presence of 12 liver sampleswith these samplesof N-nitrosomorpholine. Letters, liversubjects: values for hepatic AHH actMty and mutagenic

CANCERR SEARCHVOL. 40122

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Female BD VI rats were pretreated with the drugs listed, as describedIn.‘Materialsand Methods. “AHH activity was measured following a 10-mm Incu

bation with BP at 37°in the presence of rat 5-9 from untreated andpretreatedrats.The values are taken from assays in which formation of phenolicBPmetabolites

was proportional to 5-9 protein concentration and time ofincubation:thereproducibility of the values for hepatic AHH activity In untreated rats andinratspretreated with phenobarbitone, pregnenolone-16a-carbonltrlle, and 3-meth

ylcholanthrene was ensured in 2 to 3 Independent series ofexperiments.Mutagenicactivity in S. typhimurium TA 1530 in the presence of VCwasdetermined

following exposure of plates to ‘ICin air (Procedure A). Meanvaluesfrom2 to 3 experiments, each using 2 pooled rat livers, are gIven, except inthecaseof 3-methylcholanthrene pretreatment, where only one experimentwascarried

out. Mutagenicity assays (Procedure B) were carried out with eitherS.typhimuriumTA 1530 in the presence of up to 10 @moIN-nitrosomorpholineand1

00 @d5-9 per plate or with S. typhimurium TA 100 In the presence of up to0.08nmolAFB and 100 @I5-9 per plate. Mean values taken from the linear regionofdose-dependent

assays are reported from 2 to 3 series of experiments,eachutilizingpooled livers of 2 to 4 rats.Hepatic5-9-mediated

mutagenicity AHH activityN-Nltro

somorpholine(TA

1530 AFB(TArevert-VC (TA I 00 re- (nmol3-ants/1530 re- vertants/HO-BP/Experl-

@zmol/ vertants/ nmol/ gIiver/mentTreatment plate) plate) plate)mm)1

None 90 100 8,4001.62Phenobarbitone 235 130 13,6007.53Pregnenolone-16a- 265 35 12,10010carbonitnle4

3-Methylcholan- 70 135 6,90032threne5

Dibenamine 70 100 12,05026Aminoacetonitrile 25 50 7,9501.27Disulfiram 20 20 NDa1.28Disulflram (0.1 mM ND 5 ND0.3added

invitro)a

ND, not determined.

Carcinogen Activation by Rat and Human Liver Microsoma! Fractions

system to determine the specificity of the modifiers in alteringmonooxygenase-mediated BP hydroxylase activity and the mutagenicity of N-nitrosomorpholine, VC, and AFB (Table 2).Values for AHH activity in rats given no or different drugtreatments are expressed as nmol 3-HO-BP per g of liver permm and were taken from assays in which formation of phenolicBP metabolites was proportional to incubation time and to 5-9concentration. Mutagenic activities were calculated from linearportions of dose- and time- (in the case of VC) dependentassays.

Pretreatment of rats with 3-methylcholanthrene, pregnenolone-i 6a-canbonitrile, phenobarbitone, or dibenamine increased the rate of BP hydroxylation 20-, 6-, 5-, and 1.2-fold,respectively, as compared with assays using liver from untreated animals (Table 2, Experiment 1). Aminoacetonitrile anddisulfiram treatment both lowered AHH activity by 25%. Whendisulfiram at a final concentration of 0. 1 m@iwas added to theincubation medium containing 5-9 from untreated rats, AHHactivity was reduced by 80%; with a I mM concentration, BPhydroxylation was inhibited completely. In contrast, hepaticAHH activity in 3-methylcholanthrene-treated rats was reducedby a maximum of only 30 to 40% when disulfiram was presentat a concentration of 0. 1 to 2 m@(data not included).

S-9-mediated mutagenicity of N-nitnosomorpholine increased 2- and 3-fold after pretreatment of rats with phenobanbitone and pnegnenolone-i 6a-carbonitrile, while 3-methylcholanthrene and dibenamine reduced the mutagenic activityby 20%, and aminoacetonitrile and disulfiram reduced themutagenicity of N-nitrosomorpholine by 70% (Table 2). Microsome-dependent conversion of VC into mutagenic metabolitesby rat 5-9 was enhanced after treatment with phenobanbitoneand 3-methylcholanthrene; aminoacetonitrile, disulfiram, andpregnenolone-i 6a-carbonitrile decreased the mutagenic effects, although the latter compound induced AHH activity.Addition of disulfiram to plates at a final concentration of 0.1mM lowered liver microsome-mediated mutagenicity of VC by

95%. Changes in the liver microsome-mediated mutagenicityof AFB after the different drug treatments were qualitativelysimilar to those observed in experiments with N-nitrosomorpholine, except that dibenamine increased AFB mutagenicityby 45% (Table 2).

When the values for hepatic AHH activity in drug-treated ratswere compared with the respective values for liver microsomemediated mutagenicity of N-nitnosomorpholine, VC, and AFB(Table 2), a positive correlation between the 2 activities wasnoted only when the corresponding values for 3-methylcholanthrene (an inducer of the cytochrome P448-dependentmonooxygenase system) were omitted from the calculation ofthe correlation coefficients: N-nitrosomorpholine (r = 0.98; p<0.001); VC (r = 0.55; p <0.1); and AFB (r = 0.72; p <0.1).These results indicate a proportionality between the amount ofmutagenic metabolites produced and the cytochrome P450content of the liver microsomal preparations.

DISCUSSION

Most environmental carcinogens are metabolized by microsomal cytochrome P-450-dependent monooxygenases (e.g.,AHH). Information regarding the inherent variation of this enzyme system in different human subjects may eventually facilitate the identification of individuals with a higher risk for cancer

Table2

AHH activity and 5-9-mediated mutagenicity of N-nitrosomorpholine, VC, andAFB with liver from drug-treated and untreated rats

or of other adverse biological effects caused by environmentalpollutants. Because data on carcinogen metabolism cannot beobtained directly from humans, we have used mutagenicityassays (1) to estimate the concentrations of ultimate reactivemetabolites formed from carcinogens such as N-nitrosomorpholine and N-nitroso-N'-methylpiperazine, VC, and AFB in thepresence of a series of human surgical liver specimens. Forthese carcinogens, there is circumstantial evidence that themutagenic metabolites produced in vitro may be the same asthose which initiate cancinogenesis by the parent compound inexperimental animals and probably also in humans. VC isconverted by microsomal enzymes into chloroethylene oxide,a highly electrophilic, mutagenic (4, 6), and carcinogenicagent5; AFB undergoes microsomal oxidation to yield aflatoxinB1-2,3-oxide, a compound very probably responsible for themutagenic and carcinogenic effect of the parent toxin (15, 32,47); the heterocyclic N-nitrosamines, N-nitrosomorpholine orN-nitroso-N'-methylpiperazine, are thought to yield alkylatingintermediates following oxidation at the a-carbon atom (14, 33,37).

Large interindividual variations were noted in the capacity ofthe human liver specimens to hydroxylate BP (60-fold) and to

5 Zajdela. F., Crolsy, A.. Barbin, A., Malaveille, C., Tomatis, L., and Bartsch,

H. Submitted for publication.

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activate VC (9-fold), N-nitroso-N'-methylpiperazine (17-fold),and N-nitrosomorpholine (35-fold). A variation of similar magnitude has previously been reported in the oxidative metabolismof BP in human livers (29, 40), and the cytochrome P450 levelin livers from 200 human subjects was found to vary 65-fold(44). In our study, the mean AHH activity in 5 females wassimilar to that observed in male human subjects. The averageBP hydroxylase activity in our 15 specimens was higher thanthat reported for 20 liver samples which were processed within1 hour of biopsy and the enzymes of which were assayed onthe same day (28). These data suggest that experimentalvariations or different lengths of storage do not account for theintenindividual differences in activity (Table 1). Since no dataon drug intake or smoking habits of the subjects were available,the reasons for the differences in the rate of oxidative metabolism noted in our human samples cannot be established. Theaverage capacities of all liver samples from male and femalehuman subjects to activate N-nitrosomorpholine and VC intomutagens were 42 and 84%, respectively, of that of liver ofuntreated female BD VI rats. In contrast, the mean activity ofthe human livers in converting N-nitroso-N'-methylpiperazineinto mutagens was 4 times (in some individuals up to 10 times)higher than that in control rat liver. In 2 human liver samples,mutagenicity in the presence of AFB was @5%of that observedfor rat liver; similar mean values for liver-mediated mutagenicityof AFB (on the order of <1 0% that of rat liver) have beenreported elsewhere (20, 48). These observed species differences may indicate that the monooxygenases in rat liver whichoxidize AFB into aflatoxin B1-2,3-oxide may be different fromthose in human liver. The differential stimulation of microsomalenzymes of rat and of human origin by 7,8-benzoflavone toactivate AFB into mutagens in vitro supports this assumption(10).

Although great caution must be exercised when extrapolating from in vitro data to the mechanisms in the intact organism,the validity of in vitro mutagenicity data for predicting speciessusceptibility to certain hepatocarcinogens is supported by theobservation that the efficiency with which livers of differentanimal species activate aflatoxins corresponds to their susceptibility to the hepatocarcinogenic effect of these mycotoxins(20). Similarly, although our data revealed some species andinterindividual variations, the average capacity of all humanliven specimens to convert N-nitrosomorpholine or VC intoreactive intermediates was close to that of rat liver (Table 1).Such data would indicate that humans are probably not resistant to the adverse biological effects caused by these carcinogens, an assumption which is supported by the finding that VCinduces angiosarcomas in human liver (25).

Model drugs which are nontoxic but are metabolized by thesame enzyme system as carcinogens have been proposed asprobes to gather information on the carcinogen-handling Capacity in human subjects. Kapitulnik et a!. (29) found positivecorrelations between the rates of hydroxylation of BP and thoseof antipynine, hexobarbital, and zoxazolamine in human autopsy livers. These observations were expanded by Kalamegham et a!. (28) and Sotaniemi et a!. (44) who reported apositive association between antipyrine metabolism in vivo andhepatic AHH activity or cytochrome P450 content determinedin vitro. The observed correlation, although statistically significant, was not sufficiently strong to have clinically significantpredictive value. To further explore whether BP hydroxylation

could be used for assessing rates of@ metabolism of otherenvironmental carcinogens, especially tt$se which include theliver as the main target organ, we com@aredBP hydroxylaseactivity in human liver samples with their respective ability toconvert VC, N-nitrosomorpholine, and AFB into mutagenicelectrophiles. A statistically significant 4orrelation (p < 0.1)between the rate of BP hydroxylation and liver microsomemediated mutagenicity in the presence o N-nitrosomorpholineor N-nitroso-N'-methylpiperazine and V( was obtained (Chart2, A and B). Similarly, a plot of microsom h-mediatedmutagenicity in the presence of VC versus that witi N-nitrosomorpholinein 12 human liver specimens (Chart 2C)@ Iso revealed a statistically significant positive correlation (r = 0.58; p < 0.05). Thelack of a perfect relationship suggests ti it human livers fromdifferent individuals have different relati%@ proportions of various forms of cytochrome P450; multiph P450 cytochromesare known to occur in rats, mice, and ra bits (19, 21 , 42). Aproportionality has previously been rep4rted between cytochrome P450 content and the ability ot human liver microsomes to activate N-nitrosodimethylaminL@to a bacterial mutagen (13).

Evidence that N-nitrosomorpholine, Vi , and AFB are alsoactivated metabolically by cytochrome I 450-dependent microsomal monooxygenases in rat liver (i , 17, 26, 45, 47) isprovided by our studies in which rats were reated with inducersor inhibitors of the hepatic monooxygena e system (Table 2);changes in AHH activity and microsome-m diated mutagenicityin vitro were recorded. A comparison of h patic AHH activitiesin the livers of rats treated with differe t drugs versus therespective values for microsome-mediate mutagenicity in thepresence of either N-nitrosomorpholine,@ C, or AFB revealedpositive correlations (which were signific nt in the case of N-

or 3-methylcholan@-dependentmono

calculations. Theseetween the amountese 3 carcinogens

@ ident monooxygenases in the rat liver preparations.

The effect of treatment of rats with ph@nobarbitone, pregnenolone-i 6a-carbonitrile, 3-methyIcholar@threne,and dibenamine on liver microsome-dependent muta@enicityin the presence of N-nitrosomorpholine, VC, and AF@was substrate specific (Table 2). Administration of 3-meth@rlcholanthreneandphenobarbitone enhanced hepatic AHH @ctivityseveralfold,while aminoacetonitrile and disulfiram led to a reduction. Thelatter 2 drugs and diethyldithiocarbamate,@ cleavage productreadily formed from disulfiram in the pr@sence of rat livercytosol (46), impair monooxygenase activity of microsomes invitro (18, 22, 53); the marked inhibition ofi AHH activity couldbe explained by such a mechanism. Interestingly, the inhibitoryeffect of disulfiram was greater with liver microsomes fromcontrol rats than with liver microsomes from 3-methycholanthrene-treated rats. In rats treated with 3-methylcholanthrene(an inducer of the microsomal cytochrome P448-linked monooxygenases), hepatic AHH activity was ml ibited by only 30%after in vitro addition of 0.1 to 2 m@,id sulfiram (data notincluded), suggesting that disulfiram (or itt cleavage product)may interact preferentially with the cytoch me P450 in liverfrom control rats.

Administration of inducers and inhibitor

nitrosomorpholine) only when the valuesthrene, an inducer of the cytochrome-P4oxygenase system, were omitted from theresults suggest that there is a relationshipof mutagenic metabolites produced fromand the activity of cytochrome P450-depe

of hepatic micro

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Carcinogen Activation by Rat and Human Liver Microsomal Fractions

somal monooxygenases to animals alters the microsome-dependent mutagenicity of many carcinogens, and these treatments also modify carcinogenicity in vivo, possibly by changingthe balance between activation and detoxification reactions(51). Comparison of the drug-induced alterations in vitro andin vivo showed a good, but not perfect, correlation; althoughphenobarbitone treatment increased AFB mutagenesis, it isknown to be inhibitory to AFB carcinogenicity in the rat liver(34). On the other hand, the inhibitory effect of disulfiram onAHH activity and on liver microsome-mediated mutagenicity inthe presence of N-nitrosomorpholine was matched by an inhibitory in vivo action of disulfiram on the induction of forestomachtumors by BP in mice (9, 50) and of N-nitrosodialkylamineinduction of liver tumors in rats (43). The hepatotoxicity of VCin rats has been shown to be increased by administration ofphenobarbitone and to be decreased by pregnenolone-i 6a-canbonitrile (27, 41). These changes are in accord with ourmutagenicity data (Table 2). In conclusion, our studies on theoxidative metabolism of BP, VC, AFB, and cyclic N-nitrosamines revealed large intenindividual differences in the activityof microsomal cytochrome P450-dependent monooxygenasesin human liver specimens. The relevance of these observationsis enhanced by the consistency with which intenindividual variations of drug- and carcinogen-metabolizing capacity are observed, irrespective of whether the data are obtained in humansin vivo (11, 49) or by in vitro studies using human tissuefractions (13, 40), cells (2, 30, 52), or organ cultures (3).Statistically significant positive correlations have been foundbetween the rates of hydnoxylation of BP and those of antipyrme in human livers (28, 29), on the other hand, and the capacityto convert structurally diverse carcinogens, such as VC, N-nitrosamines, and AFB (Chart 1) into reactive metabolites, onthe other. Although statistically significant correlations wereobserved in several studies for the metabolism of substratepairs, using multiple liver samples (29) or in in vivo studies (28,44), the degree of correlation is not good enough for use in theclinical assessment of carcinogen-metabolizing capacity. Furthen research is needed to find nontoxic drugs that may beused for assessing the rate of metabolism of environmentalcarcinogens in vivo. Such an approach might eventually facilitate the identification of individuals or population groups athigh risk, as well as indicate preventive measures.

ACKNOWLEDGMENTS

The authors are grateful to G. Brun for skilled technical assistance, E. Wardfor editorial help, L. Kitchen for secretarial assistance, and Drs. R. Montesano.L. Tomatis, and H. Yamasaki for the critical reading of the manuscript. Theauthors wish to thank in particular Dr. A. H. Conney, Department of Biochemistryand Drug Metabolism, Hoffman-La Roche Inc., Nutley, N. J. for valuable suggestions and for collaboration during different phases of the work presented.

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1980;40:119-126. Cancer Res   Nicole Sabadie, Christian Malaveille, Anne-Marie Camus, et al.   Liver Microsomal Fractions

-Methylpiperazine to Mutagens by Human and Rat′N-Nitroso-N-Nitrosomorpholine, and NMetabolism of Vinyl Chloride,

)Pyrene with theaComparison of the Hydroxylation of Benzo(

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