[CANCER RESEARCH 56, 2979-2984, July I, 1996]

Activation of Chemically Diverse Procarcinogens by Human CytochromeP-450 IBI1

Tsutomu Shimada,2 Carrie L. Hayes, Hiroshi Yamazaki, Shantu Amin, Stephen S. Hecht, F. Peter Guengerich,3and Thomas R. Sutler '

Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 IT. S., F. P. G.I; Department ofEnvironmental Health Sciences, The Johns Hopkins School of Hygiene and Public Health, Baltimore, Maryland 21205 [C. L. H., T. R. S.j; Osaka Prefectural Institute of PublicHealth, Nakamichi 1-chome, Higashinari-ku, Osaka 537. Japan ¡H.Y.¡;and American Health Foundation, Valhalla, New York 10595 [S.A., S. S. H.]

ABSTRACT

A human cytochrome P-450 (P450) IBI cDNA was expressed in Sat--

charomyces cerevisiae, and the microsomes containing P450 IBI were usedto examine the selectivity of this enzyme in the activation of a variety ofenvironmental carcinogens and mutagens in Salmonella typhimuriumTA1535/pSK1002 or NM2009 tester strains, using the SOS response as anend point of DNA damage. We also determined and compared theseactivities of P450 IBI with those catalyzed by recombinant human P450s1A1 and 1A2, which were purified from membranes of Escherichia coli.

The carcinogenic chemicals tested included 27 polycyclic aromatic hydrocarbons and their dihydrodiol derivatives, 17 heterocyclic and aryl amines

and aminoazo dyes, three mycotoxins, two nitroaromatic hydrocarbons,iV-nitrosodimethylamine, vinyl carbamate, and acrylonitrile. Among the

three P450 enzymes examined here, P450 IBI was found to have thehighest catalytic activities for the activation of ll,12-dihydroxy-ll,12-dihydrodibenzo[a,/]pyrene, l,2-dihydroxy-l,2-dihydro-5-methylchrysene,(+)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, ll,12-dihydroxy-ll,12-di-hydrobenzo[g]chrysene, 3,4-dihydroxy-3,4-dihydrobenzo[c]phenanthrene,3-amino-l,4-dimcthyl-5/f-pyrido|4,3-A]mdole. 2-aminoanthracene, 3-meth-

oxy-4-aminoazobenzene, and 2-nitropyrene. P450 IBI also catalyzed theactivation of 2-amino-3,5-dirnethylimidazo[4,5-/]quinoline, 2-amino-3,K-di-

mothyliniida/o[4,5-/](|uinoxaline. 2-amino-3-methyUmidazo[4^-/|quinoline,2-aminofluorene, 6-aminochrysene and its 1^-dihydrodiol, (—)-7,8-dihy-

drtixy-7,8-dihydrohenzo|«Jpyreiie, l,2-dihydroxy-l,2-dihydroohrysene, 1,2-diliydroxy-1.2-dihydro-5.6-dimi'thylchrysene. 2,3-dihydroxy-2,3-dihydroflu-

oranthene, 3,4-dihydroxy-3,4-dihydro-7,12-dimethylhenz[a]anthracene, and6-nitrochrysene to appreciable extents. However, P450 IBI did not produce

genotoxic products from benzo[a]pyrene, irans-3,4-dihydroxy-3,4-dihydro-benzo[a]anthracene, <ra/u-8,9-dihydroxy-8,9-dihydrobenzo[a]anthracene,7,12-dimethylbenz[a]anthracene and its cis-5,6-dihydrodiol, 5-methyl-chrysene, ll,12-dihydroxy-ll,12-dihydro-3-methylcholanthrene, 1,2-dihy-droxy-l,2-dihydro-6-niethylchrystne, benzo[c]phenanthrene, 2-amino-6-niethyldipyridi)[l,2-»:3'.2'-</|iinida7ole. 2-acetylaminofluorene, benzidine,

2-naphthylamine, aflatoxin B,. aflatoxin G¡,sterigmatocystìn,.V-nitrosodi-

methylamine, vinyl carbamate, or acrylonitrile in this assay system. P450

IBI is expressed constitutively in extrahepatic organs, including fetal tissuesamples, and is highly inducible in various organs by 2,3,7,8-tetrachlorod-

ibenzo-p-dioxin and related compounds in experimental animal models.

Thus, activation of procarcinogens by P450 IBI may contribute to humantumors of extrahepatic origin.

INTRODUCTION

Multiple forms of human P4504 (1) contribute significantly to the

metabolic activation of a number of procarcinogenic chemicals to theirproximate reactive species (2). Among human P450 enzymes examined,P450s 1A1, 1A2, 2E1, and 3A4 are generally recognized to be the majorforms involved in the activation of most of the procarcinogens in humanliver and lung microsomes (3). P450 2E1 activates low molecular weightcancer suspects such as vinyl chloride, benzene, and some nitrosamines(3-5). P450 3A4 is a major P450 enzyme in human liver and some

extrahepatic tissues and has been reported to have important roles in theactivation of a wide range of environmental procarcinogens, includingcarcinogenic mycotoxins, dihydrodiol derivatives of polycyclic aromatichydrocarbons, 6-aminochrysene, and tris(2,3-dibromopropyl)phosphate

(4, 6). P450 1A1 and 1A2 participate principally in the activation ofcarcinogenic polycyclic aromatic hydrocarbons and heterocyclic and arylamines (4,7-9). P450 1Al can be present in human liver but is expressed

predominately in extrahepatic tissues (3, 7). P450 1A2 is expressedprimarily in the liver, and constitutive levels of this enzyme are muchgreater than those of P450 1A1 (7, 10).

Recently, a new P450 family 1 member, P450 IBI, has beenidentified in rodent species (11-14) and in humans (15). P450 IBI

amino acid sequences among human, rat, and mouse are 80% similar;between subfamilies, P450 IBI is equally similar, about 40%, to bothP450 1A1 and 1A2 (14). The mouse and rat P450 IBI enzymes havebeen shown to catalyze the regioselective metabolism of a prototypepolycyclic aromatic hydrocarbon carcinogen, DMBA (16, 17). Furthermore, immunoinhibition studies using microsomes prepared fromhuman cells indicate that such regioselectivity may extend to humanCYP1B1 (18). Because P450 IBI is expressed in numerous tissuesand is inducible by exposure to dioxins and polycyclic aromatichydrocarbons (11, 12, 15), it is of interest to determine the role ofP450 IBI in the oxidation and activation of relevant environmentalcarcinogens.

The present study was designed to examine the role of human P450IB 1 in the activation of a number of procarcinogenic chemicals and tocompare these activities with those of human P450 1A1 and 1A2. Theresults provide evidence that P450 IBI is a very important enzyme inthe activation of a variety of environmental carcinogens and mutagens, including polycyclic aromatic hydrocarbons and aryl amines.

Received 1/30/96; accepted 4/30/96.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported in part by USPHS Grants CA44353, ES00267 (F. P. G.),

ES03819, ES06071 (T. R. S.), and CA44377 (S. S. H.) and by grants from the Ministryof Education, Science, Sports and Culture of Japan (T. S.). C. L. H. is supported byTraining Grant ES07141.

2 Present address: Osaka Prefectural Institute of Public Health, Nakamichi 1-chome,H^gashinari-ku. Osaka 537, Japan.

3 Requests for reprints should be addressed to either of these authors, at Department of

Biochemistry and Center in Molecular Toxicology, Vanderbilt University School ofMedicine, Nashville, TN 37232 (F. P. G.), or Department of Environmental HealthSciences, The Johns Hopkins School of Hygiene and Public Health, Baltimore, MD21205.

MATERIALS AND METHODS

Chemicals. Benz[a]anthracene-l,2-diol,5 benz[a]anthracene-3,4-diol, benz-[a]anthracene-ci'j-5,6-diol, benz[a]anthracene-8,9-diol, 3-methylcholanthrene-

4 The abbreviations used are: P450, cytochrome P-450; IQ, 2-amino-3-methylimi-dazo[4,5-/|quinoline; MelQ, 2-amino-3,5-dimethylimidazo-IQ; MelQx, 2-amino-3,8-di-methylimidazo[4,5--/]quinoxaline; Glu-P-1, 2-amino-6-methyldipyrido[l,2-a:3',2'-i/]imi-

dazole; Trp-P-1, 3-amino-l,4-dimethyl-5//-pyrido[4,3-e]indole; Trp-P-2, 3-amino-l-methyl-5/i-pyrido[4,3-i]indole; PhlP, 2-amino-l-methyl-6-phenylimidazo[4,5-e]pyridine; DMBA.7,12-dimethylbenz[a]anthracene; 3-MeO-AAB, 3-methoxy-4-aminoazobenzene; AFB, afla

toxin B; AFG, aflatoxin G.5 The suffix "dio!" is used in the text to designate the prefix "dihydroxydihydro-" for

individual polycyclic hydrocarbons; all diols are trans unless specifically denoted "ci's."

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HUMAN P450 IB] ACTIVATION OF CHEMICAL CARCINOGENS

CYP1A2CYP1A1

CYP1B1

ß-ACTIN

- 5.2 kb

- 2.0 kb-1.8kb

Fig. 1. Expression of P450 1 family genes in normal human adult and fetal tissues. Each tissue is identified at the lop. mRNA blots of normal human tissues were hybridizedsuccessively with P450 IBI. P450 ! Al, and ß-aclincDNAs as described in "Materials and Methods." The P450 1A1 cDNA hybridizes to both P450 1A1 (CYP1AI) and to P450 IA2

(CYPIA2) in the liver. The P450 IBI cDNA hybridizes only to P450 IBI (CYP1B1). The specific mRNA and corresponding size in kb are identified at lefi and right, respectively.Two fig of poly(A)* RNA were loaded in each lane except for those of the adrenal, pituitary, uterus, and mammary tissues, which have 0.5 fig poly(A)+ RNAJlane. The exposure

time corresponding to each membrane was 150 h for all P450 1 cDNAs, except P450 l B l kidney 2 and fetal tissue samples, which were exposed for 85 h. The exposure lime for ß-actin

was 2 h.

11,12-diol, DMBA, DMBA-3,4-diol, and DMBA-r/j-5,6-diol were purchased

from the National Cancer Institute Chemical Carcinogen Repository/MidwestResearch Institute (Kansas City, MO). MelQ, MelQx, IQ, PhIP, Trp-P-1, Trp-P-2,4-aminobiphenyl, benzidine, and 2-naphthylamine were provided kindly by Dr. F.F. Kadlubar (National Center for Toxicological Research, Jefferson, AR). Chry-sene-1,2-diol, 5-methylchry sene-1,2-diol, 6-methylchrysene-1,2-diol, 5,6-dimethyl-chrysene-l,2-diol, dibenzo|a,/]pyrene-l 1,12-diol, benzo(g]chrysene-11,12-diol,benzo[c]phenanthrene-3,4-diol, fluoranthene-2,3-diol, benzo[/]fluoranthene-4,5-diol, and benzo[b]fluoranthene-9,10-diol were synthesized as described (19-27).6

Vinyl carbamate was a gift of Prof. J. A. Miller (University of Wisconsin,Madison, WI). Other chemicals used were from the same sources as describedpreviously (4).

Human Tissue Samples. Poly(A)+ mRNA human tissue blots andpoly(A)^ mRNA samples were obtained from Clontech (Palo Alto, CA). All

specimens were from normal Caucasians. Samples represent either a singleindividual or a pool of multiple individuals and were not matched for sex orage. No information was available concerning smoking history or other potential environmental exposures. mRNA (Northern) blot analysis was performed according to the manufacturer's instructions. Blots were hybridized

successively with human cDNA probes for P450 IBI or IAl (15, 28) or forß-actin(Clontech) that were labeled with [a-"P]dCTP to a specific activity of1-2 x IO8 cpm/fig DNA, as described previously (15). Nonspecific hybrid

ization was removed by washing each membrane twice in a solution of 1XSSC buffer (0.15 M sodium chloride and 0.015 M sodium citrate, pH 7.0)containing 0.10% SDS (w/v) at 50°Cfor 60 min. Hybridization signals were

visualized by autoradiography.Enzyme Preparations. Human P450 l B1 cDN A clone was introduced into

Saccharomyces cerevisiae, and microsomes containing P450 l B l protein wereprepared as described (15).7 Recombinant human P450 1A1, 1A2, and 3A4

proteins were purified to homogeneity from membranes of Escherichia coli inwhich modified P4SO genes had been introduced (29-31). Rabbit liverNADPH-P450 reducíase (32) and cytochrome bs (Ref. 33; used in the P450

3A4 system) were purified as described.Assays. P450-dependent activation of procarcinogens to reactive metabo

lites that cause induction of umu gene expression in tester strains Salmonellatyphimurium TAI535/pSK1002 and NM2009 was determined in reconstitutedP450-containing systems as described previously (4, 5, 34). The former is atester strain containing a plasmid with an umuC'lacZ fusion gene; the latter

strain, NM2009, contains an additional plasmid, pNM12, that overexpressesbacterial O-acetyltransferase and thus is more sensitive to the reactive inter

mediates of aryl amines and aminoazo dyes (35). Standard incubation mixturesconsisted of yeast microsomes (5 pmol P450) with 5-10 /UMprocarcinogen in

6 S. Amin, unpublished results.7 C. L. Hayes, D. C. Spink, B. C. Spink, J. C. Cao, N. i. Walker, and T. R. Sutler,

submitted for publication.

a final volume of 1.0 ml of 100 mM potassium phosphate buffer (pH 7.4)containing an NADPH-generating system and 0.75 ml of bacterial suspensionas described previously (5). In the case of yeast microsomes, NADPH-P450reducíase(10 pmol) was added to improve catalytic activities (see "Results").

P450 1A1- and lA2-dependent catalytic activities were determined in reconstituted systems containing P450 (5 pmol) and rabbit NADPH-P450 reducíase(10 pmol) with L-a-dilauroyl-in-glycero-3-phosphocholine as described (5).Incubations were carried out al 37°Cfor 2 h and terminated by cooling the

mixtures on ice. umu gene expression was monitored by measuring ß-galac-

tosidase activities using Ihe method of Miller (36) and expressed in the unilsdescribed. The induclion of umu gene expression by activated carcinogens ispresented as units of ß-galactosidase activity/min/nmol P450 (34, 35). Exper

iments in which results were to be compared directly were done on the sameday with the same enzyme and bacterial preparations, as the absolute values forumu response show some fluctuation due to variables that cannot be strictly

controlled.P450 was estimated spectrally by the method of Omura and Sato (37).

Protein concentrations were estimated by the method of Lowry et al. (38).

RESULTS

RNA Expression of P450 1A1, 1A2, and IBI in Normal HumanTissues. A previous analysis of human P450 IBI RNA expressionrevealed that levels of this 5.2-kb transcript were detectable in mul

tiple adult tissue samples, the kidney sample exhibiting the highestapparent signal relative to the other samples tested ( 15). In this study,we extended these previous results in three ways: (a) analyses of sixadditional adult tissue samples representing five additional organs anda second adult kidney sample; (b) comparative analyses of the relativeexpression of P450 1Al and 1A2 in these same tissue samples; and (c)analyses of tissue samples from five fetal organs.

P450 ¡Al gene expression was detected in 12 of the 21 adult tissueRNA samples but in none of the 5 fetal tissue RNA samples (Fig. 1).The size of the RNA (2.8 kb) was the same as reported initially (39).The most intense hybridization signals occurred in the prostate andmammary tissues. The larger RNA species (3.3 kb) seen in the liveris the result of cross-hybridization of the P450 1A1 cDNA probe with

P450 1A2 RNA (40). The exposure shown here does not distinguishthe two separate bands of 2.8 and 3.3 kilobases, representing P4501A1 and 1A2, but in an autoradiogram from a shorter exposure (datanot shown), two bands were detected at the appropriate sizes. [Theliver P450 1Al mRNA signal (2.8 kb) was about 30% of the intensityof the P450 1A2 mRNA signal (3.3 kb) in this sample. Because a 1A1

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HUMAN P450 IBI ACTIVATION OF CHEMICAL CARCINOGENS

probe was used, the proportion of the 1A2 signal may be underestimated.] P450 IBI RNA was detected in 20 of the 21 adult tissue RNAsamples and in fetal heart, brain, lung, and kidney. The levels of P450IBI RNA in adult kidney of both sexes and fetal kidney, as well as inprostate, uterus, and mammary tissue, are apparently greater than inother tissues (Fig. 1).

The P450 1A1 cDNA hybridized to a larger RNA in the brain; theP450 IBI cDNA hybridized to a larger RNA in adult liver andperipheral blood leukocytes and in fetal heart. Whether these aresample or processing artifacts or actually represent additional P450family 1-related RNAs has not been determined. In the case ofß-actin,this cDNA hybridizes to two /3-actinRNA species in the heartand brain (41). ß-Actinis a positive control for RNA integrity and theblotting procedure. Because levels of this RNA vary between tissues,it is not an accurate control for sample loading (42).

Effects of Rabbit NADPH-P450 Reducíaseon the Activation ofProcarcinogens Catalyzed by P450 IBI in Yeast Microsomes.Yeast microsomes have been shown to contain significant amounts ofNADPH-P450 reducíase,which is active in the oxidation of severalchemicals when reconstituted with mammalian P450 enzymes (43).Activation of 2-aminoanthracene, Trp-P-1, and (—¿�)benzo[a]pyrene-7,8-diol to genotoxic metabolites by P450 IBI in yeast microsomeswas determined in the 5. typhimurium NM2009 strain in the presenceor absence of additional NADPH-P450 reducíase(Table 1). P450 IBI(5 pmol) was found to catalyze the activation of these procarcinogensto reactive metabolites that induced umu gene expression in the testerstrain. Because the addition of rabbit NADPH-P450 reducíase(10pmol) to the yeast microsomes increased carcinogen activation activities by 1.2-1.5-fold in all cases, subsequent experiments with P450lB l in yeast microsomes were conducted in the presence of a 2-foldmolar excess of rabbit NADPH-P450 reducíase.

Comparison of Catalytic Specificities of P450 IBI, 1A1, and1A2 in the Activation of Environmental Carcinogens. Among the27 polycyclic aromatic hydrocarbons and metabolites examined,dibenzo[a, /Jpyrene-11,12-diol, benzo[g]chrysene-11,12-diol, benzo-[c]phenanthrene-3,4-diol, (+)benzo[a]pyrene-7,8-diol, DMBA-3,4-diol, chrysene-1,2-diol, 5-methylchrysene-l,2-diol, and 5,6-dimethyl-chrysene-1,2-diol were most actively catalyzed by P450 IBI when S.typhimurium TA1535/pSK1002 was used as a lesler strain (Table 2).Calalylic aclivilies of P450 IBI and 1A1 were very similar for iheaclivalion of (—)benzo[a]pyrene-7,8-dioland 6-aminochrysene-1,2-diol. Only benzo[e]fluoranlhene-9,10-diol was activaled to a greaterextent by P450 1Al than P450 IBI. P450 l A2 was generally the leasteffective in the activation of the polycyclic aromatic hydrocarbonsexamined.

The three human P450 enzymes were also compared with regard totheir abilities to catalyze the activation of 16 aryl amines and relatedcarcinogenic chemicals to genotoxic metabolites in the S. typhimurium NM2009 tester strain (Table 3). As reported previously (4),P450 1A2 was highly active in catalyzing several aryl amines, par-

Table 1 Effects of rabbit NADPH-P450 reducíaseon the activation of procarcinogens

catalyzed by yeast microsomes containing human P450 IBI

Yeast microsomes conlaining P450 IBI (5 pmol) were incubated with or withoutrabbit NADPH-P-450 reducíase(10 pmol) in the presence of procarcinogen (5 nmol). andumu gene expression in 5. typhimurium NM2009 was determined as described in "Materials and Methods."

Activation of procarcinogen(units/min/nmol P450)

Carcinogen (-)" ( +)2-Aminoanthracene

Trp-P-1( —¿�)Benzo[ci]pyrene-7.8-diol2030

±1001040 ±120720 ±802940

±4601310 ±1901060 ±100

Table 2 Activation of carcinogenic polycyclic aromatic hydrocarbons by human P450IBI expressed in veast and by recombinant P450 IAl and 1A2 in S. typhimurium

TA1535/pSK1002

Procarcinogen aclivalion"

(units/min/nmol P450)

ProcarcinogenBenzo[a]pyrene9-Hydroxybenzo[a]pyreneBenzo[a]pyrene-4.5-diol(

+)Benzo[a]pyrene-7,8-diol(-)Benzo[a]pyrene-7,8-diolDibenzo[a./)pyrene-

11,12-diolBenz[a]anlhracene-1,2-diolBenz[a]anthracene-3.4-diolBenz[a)anthracene-ci'i-5,6-diolBenz[«]anthracene-8,9-diolDMBADMBA-3.4-diolDMBA-ru-5,6-diolBenzo[c]phenanthreneBenzo[c]phenanthrene-3.4-diolFluoranthene-2.3-diolBenzo[/]fluoranthene-4,5-diolBenzo[fc]fluoranthene-9.

10-diol3-Methylcholanthrene-l1,12-diolDibenz[(j,/i]anlhracene-5,6-diolChrysene-

1.2-diol5-Methylchrysene5-Methylchrysene-

1,2-diol6-Methylchrysene-1,2-diol5.6-

Dimethylchrysene-1.2-diolBenzo[g]chrysene-11.12-diol6-

Aminochrysene- 1.2-diolP450

IBI<10<1087

±341260±180940±1302560±5042±12<1035

±15<10<10870

±280<10<10126

±4055±3<10140

±30<10<IO250

±60<102000

±150<10220

±25575±63600±140P450

IAI130

±20<10ND*630

±771100±120<10<10<10<10<10<10<10<10<10<10<10<10500

±42NDND<10<10260

±20<10<10<10690

±40P450

1A239

±29<IOND210

±22310±41<10<10<10<10<10<10<10<10<10<10<10<10180

±31NDND<10<10110±

10<10<10<10880

±110" P450 IBI (5 pmol) in yeast microsomes fortified with rabbit NADPH-P450 reducíase

(10 pmol) was incubated with 5 nmol of each procarcinogen in 5. typhimurium TA1535/pSK1002. and the induction of umu gene expression was determined by the methoddescribed in "Materials and Methods." Reconstitution of enzymes involved in activation

of procarcinogens was carried out as described. Results are presented as means ofduplicate or triplicate determinations ±SD.

ND. assay not done.

Table 3 Activation of carcinogenic aryl amines by human P450 IBI expressed in yeastand by recombinant P450s IA¡and 1A2 in S. typhimurium NM2009

Procarcinogen activation"

(units/min/nmol P450)

ProcarcinogenMelQMelQxIQTrp-P-1Trp-P-2Glu-P-1PhIP2-Aminoanthracene2-Aminofluorene2-Acetylaminofluorene4-Aminobiphenyl3-MeO-AAB«-Aminoazotoluene6-

AminochryseneBenzidine2-NaphthylamineP450

IBI460

±30230±30380±201250±130140

±64<1049

±93000±3001020±190<IO93

±241300±6075±111600±210<10<IOP450

1A1660

±40510±50300

±401250±10052

±25180±30180

±20430±50232±40180

±43180±211140±

120188±191930±110<10<10P450

1A23140

±3001700±1202500±220370±20160±302600±210122±101700±1001580±100820±11091±32440±60150±20760±30<10<10

' —¿�,no reducíaseadded; +, reducíaseadded.

" P450 IBI (5 pmol) in yeast microsomes fortified with rabbit NADPH-P450 reducíase

(10 pmol) was incubaled with 5 nmol of each procarcinogen in S. typhimurium NM2009.and the induction of umu gene expression was determined as described in "Materials andMethods." Reconstilulion of enzymalic aclivation of procarcinogens was carried out as

described. Results are presenied as means of duplicale or triplicate determinations ±SD.

ticularly MelQ, MelQx, IQ, Glu-P-1, and 2-acetylaminofluorene.Interestingly, P450 l Bl was the most active of the three enzymes incatalyzing the activation of 2-aminoanthracene and 3-MeO-AAB.P450 IBI had catalytic activity similar to P450 1A2 in the activationof 2-aminofluorene. The activities of P450 IBI toward Trp-P-1 and6-aminochrysene were very similar to those of P450 1A1. The catalytic specificities of P450 IBI were very similar toward several arylamines, including MelQ, IQ, and 6-aminochrysene-1,2-diol. P450

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Table 4 Aclivalinn of other procarcinogens b\ human P450 IB! expressed in yeastand by recombinant P450 IAl and 1A2 in S. typhimurium TA1535/pSK1002

Procarcinogen activation0

(units/min/nmol P450)

Procarcinogen2-Nitropyrene6-NitrochryseneAFB,AFG,Sterigmatocystin/V-Nitrosodimethy

lamineVinylcarbamateAcrylonitrileP450

IBI P4501A1610

±40 110±12110±18<10<10

45 ±31<1011±10<1072 ±19<10

ND*<10

ND<10NDP450

1A2<10<10150

±1924±11160

±33NDNDND

" P450 IBI (5 pmol) in yeast microsomes. fortified with rabbit NADPH-P450 reduc

íase(10 pmol). was incubated with 5 nmol of each procarcinogen in 5. typhimuriumTA1535/pSK1002, and the induction of umu gene expression was determined as describedin "Materials and Methods." Reconstitution of enzymatic activation of procarcinogens

was carried out as described. Results are presented as means of duplicate or triplicatedeterminations ±SD.

h ND, not done.

IBI did not catalyze the activation of Glu-P-1, 2-acetylaminoflu-orene, benzidine, or 2-naphthylamine.

Several other environmental carcinogens were also tested (Table 4);P450 IBI did not activate AFB,, AFG,, Sterigmatocystin, /V-ni-trosodimethylamine, vinyl carbamate, or acrylonitrile. 2-Nitropyreneand 6-nitrochrysene activations were catalyzed selectively by P450

IBI. Of the three enzymes examined, P450 1A2 was the most activein the activation of AFB,, AFG,, and Sterigmatocystin.

Activation of 5-methylchrysene-l,2-diol by P450 IBI, IVI, 1A2,

and 3A4. The above results suggested that P450 IBI has very highcatalytic activity for the activation of 5-methylchrysene-l,2-diol. Con

centration dependence studies were conducted with yeast microsomalP450 IBI and recombinant P450s 1A1, 1A2, and 3A4 (Fig. 2). Ofthese P450 enzymes, P450 IBI was the most effective in the activation of 5-methylchrysene-l,2-diol in the range of substrate concentra

tions examined. P450 3A4 was the least active among these four P450enzymes.

DISCUSSION

P450s 1A1 and 1A2 are well-characterized enzymes that are known

to metabolize many commonly occurring carcinogens (3, 29, 44).Because the predicted human P450 IBI protein is similar to both ofthese enzymes (15), it was of interest to compare the expression andactivity of the P450 family 1 members.

The human tissue RNA blot (Fig. 1) demonstrates the organ-

specific distribution of this P450 gene family. Although P450 1A2 isdetected only in the liver, P450 1A1 and IBI are expressed widelyand found in many of the same tissues. Although qualitative, therelative expression of P450 IBI appears greatest in adult kidney,prostate, uterus, and mammary tissue. Furthermore, recent studies ina variety of human cells of epithelial origin indicate that, like P4501A1, levels of P450 IBI are highly inducible in response to Ahreceptor agonists, including polycyclic aromatic hydrocarbons, diox-

ins and related compounds, and certain dietary indole carbinols. Inaddition, P450 IBI appears to be the predominant family l P450expressed in fetal tissues (Fig. 1). Current knowledge of fetal P450content and activities is much less than that of adult tissues. However,several groups have demonstrated the presence of aryl hydrocarbonhydroxylation activity in fetal tissues, including the kidney (45), andthis activity has been distinguished from P450 1A1 (46-48), indicat

ing that additional studies of fetal expression of P450 IBI are warranted.

Using expressed P450 1A1, 1A2, and IBI proteins and the S.typhimurium umu gene expression assay, we determined the relative

capacity of each family 1 P450 to activate 51 procarcinogenic chemicals. A caveat is that this comparison can only be considered semi-

quantitative because of the differences in the expression systems, i.e.,yeast microsomes for P450 IBI versus bacterial expression and reconstitution for P450s 1A1 and 1A2. Among the three human P450 enzymes examined, P450 IBI had the highest catalytic activities towardseveral polycyclic aromatic hydrocarbons, including dibenzo[o,/]pyrene-11,12-diol, benzo[g]chrysene-11,12-diol, benzo[c]phenanthrene-3,4-diol, (±)benzo[a]pyrene-7,8-diol, DMBA-3,4-diol, chrysene-1,2-diol, 5-methylchrysene-1,2-diol, and 5,6-dimethylchrysene-l,2-diol, the ni-tropolycyclic hydrocarbon 2-nitropyrene, and several aryl amines,including 2-aminoanthracene, 3-MeO-AAB, 6-aminochrysene, and theheterocyclic amine Trp-P-1. P450 IBI was also found to catalyze the

activation of other potentially important carcinogens at a considerablerate, including (-)benzo[a]pyrene-7,8-diol, 2-aminofluorene, MelQ, IQ,

6-aminochrysene-1,2-diol, and 6-nitrochrysene. In contrast, P450 IBIdid not catalyze activation of Glu-P-1, 2-acetylaminofluorene, benzidine,2-naphthylamine, AFB,, AFG,, Sterigmatocystin, N-nitrosodimethyl-

amine, vinyl carbamate, or acrylonitrile.We chose to do these initial screening studies in the absence of

other enzymes involved in carcinogen metabolism to simplify thesystem. The 5. typhimurium strains have significant levels of (bacterial) W-acetyltransferase. Sulfotransferases and epoxide hydrolase

(presumably not present in the yeast microsomes) might facilitate theformation of some reactive products of the polycyclic hydrocarbonsand aryl amines [although several of the polycyclic hydrocarbon diolepoxides have not been found to be good substrates for epoxidehydrolases (49, 50)]. The presence of epoxide hydrolase could complicate the results, by detoxication of genotoxic products. Ultimately,studies with several "Phase II" enzymes will be required with P450

IBI and the lead compounds identified in this study.It is interesting to note that P450 IBI was able to catalyze the

activation of both polycyclic aromatic hydrocarbons and aryl amines.It has been a general view that carcinogenic polycyclic aromatichydrocarbons are activated mainly by P450 1A1 and aryl aminesmainly by P450 1A2 in experimental animal models and humans (2).Why P450 IBI activates such a wide range of carcinogenic chemicalsis not known, and additional work will be necessary to elucidate thecatalytic mechanisms of P450 IBI at the molecular level. We shouldemphasize that the studies reported here deal only with the humanP450s 1A1, 1A2, and IBI. At this time, we cannot make exactpredictions of the relative contributions of the orthologues of theseenzymes in experimental animals, although some studies have shownhigh activity of rodent P450 l B l enzymes toward certain polycyclichydrocarbons (18).

P450 IBI exhibited remarkable activity for the metabolic activationof dihydrodiols, in numerous cases being the only enzyme of the three

O 2 4 6 8 105-Methylchrysene-1,2-diol (uM)

Fig. 2. Dose-response curves for the activation of 5-methylchrysene-1,2-diol byrecombinant P450 IBI (•).P450 1A1 (O), P450 1A2 (A), and P450 3A4 (A). Experimental details are described in "Materials and Methods."

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to activate the test compound (Table 1). This was especially notablefor the dihydrodiols in which the epoxide ring would be formed in asterically hindered bay region or fjord region. Examples include5-methylchrysene-1,2-diol, 5,6-dimethylchrysene-1,2-diol, dibenzo-[o,/]pyrene-l 1,12-diol, benzo[c]phenanthrene-3,4-diol, and benzo-[g]chrysene-l 1,12-diol. The resulting diol epoxides are potent carcinogens in several tumor models, including mouse lung and ratmammary gland (22, 51-55).

5-Methylchrysene, one of the components of tobacco smoke condensate, is a strong carcinogen in experimental animals, whereas6-methylchrysene is a weak carcinogen (49, 56-59). Although thethree human P450 enzymes examined (in the absence of epoxidehydrolase) would have catalyzed activation of 5-methylchrysene todihydrodiols too slowly to utilize assays directly with this parentcompound, P450 IBI showed the highest activation of the majorproximate carcinogen, 5-methylchrysene-1,2-diol, but not of 6-methylchrysene- 1,2-diol. These results are of interest, because 5-methylchrysene is a strong pulmonary carcinogen in mice, and recent resultssuggest that P450 IBI is expressed in various organs including lung(Fig. 1; Refs. 14, 15, 17, and 60).

The activation of the dihydrodiols examined in this study is ofparticular interest with respect to the high expression of P450 IBIin human mammary tissue. In several cases—fluoranthene-2,3-diol, benzo[c]phenanthrene-3,4-diol, dibenzo[o,/]pyrene-l 1,12-diol, and benzo[g]chrysene-l 1,12-diol—theresulting diol epoxideshave been shown to be potent mammary carcinogens in the rat (52,53, 61). Human exposure to some of the parent compounds, particularly fluoranthene and benzo[c]phenanthrene, is extensive, andthe stable dihydrodiol metabolites could be formed in the liver andtransported to the mammary tissue, where oxidation to the diolepoxides could occur. In fact, the formation of fluoranthene-2,3-diol has already been demonstrated in human liver (62). Alternatively, P450 IBI may activate such procarcinogens in situ. Forsuch compounds, including certain heterocyclic and aryl amines(Table 2), P450 IBI represents an extrahepatic route of metabolicactivation. Such local activation lessens the need to invoke complex pharmacokinetic schemes for the redistribution of more polarand/or conjugated metabolites, subsequent to metabolic activationin the liver, and may provide important insights into organ-specificcancers.

In conclusion, the present results demonstrate for the first time thatP450 IBI is an important enzyme involved in the activation of anumber of environmental carcinogens and should be considered regarding mechanisms of development of human cancers.

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