Induction of cytochrome P450 1A in hamster liver and lung by 6-nitrochrysene

MOLECULAR TOXICOLOGY

Ruei-Ming Chen á Ming W. Chou á Tzuu-Huei Ueng

Induction of cytochrome P450 1A in hamster liver and lungby 6-nitrochrysene

Received: 5 January 1998 /Accepted: 25 February 1998

Abstract The present study has determined the e�ect of6-nitrochrysene (6-NC) on hepatic and pulmonary cy-tochrome P450 (P450)-dependent monooxygenasesusing hamsters pretreated with the nitrated polycyclicaromatic hydrocarbon (nitro-PAH) at 5 mg/kg per dayfor 3 days. Pretreatment with 6-NC elevated serumgamma-glutamyltranspeptidase, lactate dehydrogenase,and bilirubin levels. Liver S9 fractions prepared fromcontrols and hamsters pretreated with 6-NC markedlyincreased mutagenicity of the nitro-PAH in Salmonellatyphimurium tester strains TA98, TA100, and TA102.The pretreatment selectively increased 1-nitropyrene re-ductase activities of lung cytosol and liver and lungmicrosomes. Pretreatment with 6-NC resulted in in-creases of microsomal 7-ethoxyresoru®n and methoxy-resoru®n O-dealkylases activities in liver and lungwithout a�ecting the monooxygenase activities in kid-ney. Immunoblot analysis of microsomal proteins usingmouse monoclonal antibody 1±12±3 to rat P450 1A1revealed that 6-NC induced P450 1A-immunorelatedproteins in liver and lung. RNA blot analysis usingmouse P450 1A1 cDNA showed that 6-NC increasedliver and lung P450 1A mRNA. 6-NC had no e�ect onthe kidney P450 protein and mRNA. The present studydemonstrates that the hamster enzymes can support6-NC metabolic activation and the nitro-PAH inducesliver and lung P4501A via a pretranslational mechanism.

Key words 6-Nitrochrysene á Cytochrome P450 áMutagenicity á Nitroreductase

Introduction

The cytochrome P450-dependent monooxygenase systemconsists of multiplicity of P450 hemoproteins, a ¯avo-protein NADPH-cytochrome P450 reductase, and phos-pholipids. The microsomal monooxygenases catalyze theoxidative, reductive, and peroxidative metabolism of avariety of xenobiotics including drugs, carcinogens, andenvironmental pollutants (Estabrook 1996). The P450enzymes are markedly responsive to the stimulatory andinhibitory e�ects of many physiological and environ-mental factors. Such responsiveness plays an importantrole in xenobiotic metabolism and toxicity (Nebert 1994).

The environmental contaminant 6-nitrochrysene(6-NC) demonstrates a remarkable activity as a lung,liver, and colon carcinogen in laboratory animals (Busbyet al. 1985; El-Bayoumy et al. 1989a; Imaida et al. 1992).The tumorigenic activity of the nitrated polycyclic aro-matic hydrocarbon (nitro-PAH) approximates theactivity of certain ultimate carcinogenic metabolites ofPAHs. 6-NC metabolism occurs in bacterial and mam-malian systems (Fu 1990). 6-NC was metabolically acti-vated to mutagens in Salmonella typhimurium TA98 andTA100 tester strains with and without rat liver S9 mix(El-Bayoumy et al. 1989b). Metabolic activation of 6-NCin new born mice involved oxidation to 1,2-dihydrodiol-6-NC, nitroreduction to 1,2-dihydrodiol-6-aminochry-sene, and epoxidation to the proximate metabolite1,2-dihydrodiol-6-aminochrysene-3,4-epoxide (Delcloset al. 1987, 1988; El-Bayoumy et al. 1989b). Human livermicrosomal P450 1A2 and 3A4 participated in oxidationof 6-NC to trans-1,2-dihydro-1,2-dihydroxy-6-NC andreduction of the carcinogen to 6-aminochrysene, res-pectively. Human lung P450 1A1 appeared to play amajor role in the metabolism of 6-NC to trans-1,2-di-hydro-1,2-dihydroxy-6-NC (Chae et al. 1993).

Unlike the rather extensive metabolism studies, re-ports concerning the e�ect of 6-NC on microsomalenzymes are few. Chou et al. (1987) reported that pre-treatment of rats with 6-NC increased benzo(a)pyrene

Arch Toxicol (1998) 72: 395±401 Ó Springer-Verlag 1998

R.-M. Chen á T.-H. Ueng (&)Institute of Toxicology, College of Medicine,National Taiwan University, 1 Jen Ai Road, Section 1,Taipei, Taiwan, ROC

M.W. ChouNational Center for Toxicological Research,Je�erson, Arkansas, USA

hydroxylase, 7-ethoxycoumarin O-deethylase, and 1-ni-tropyrene reductase activities in liver microsomes. Im-aida et al. (1992) showed that pretreatments of rats andmice with 6-NC increased benzo(a)pyrene hydroxylaseactivities in lung, colon, and liver microsomes. Infor-mation regarding the e�ect of 6-NC on P450 beyond thelevel of catalytic activity has not been available.

Hamster is a widely used model for chemical carci-nogenesis and tumorigenesis studies. The hamster mo-nooxygenase di�ers from the enzymes of other animalsin many aspects. For example, hamster microsomalenzymes are more e�cient than the rat enzymes inmetabolic activation of the chemical carcinogenN-nitrosodimethylamine (Yoo et al. 1987). 3-Methyl-cholanthrene (3-MC) is a potent inducer of P450 1A1 inrat and mouse livers; however, the chemical carcinogenis a poor P450 1A1 inducer in hamster liver (Lai andChiang 1990). Given that nitro-PAH toxicity in ham-sters had not been reported, we have carried out thepresent study to determine 6-NC mutagenicity andhamster enzyme induction property. The present studyhas addressed induction of P450 by 6-NC at the levels ofcatalytic activity, protein, and mRNA.

Materials and methods

Animals and treatments

Male Syrian golden hamsters of 80±100 g body wt. were purchasedfrom the Animal Center of the College of Medicine, NationalTaiwan University, Taipei, Taiwan. Before experiments began, theanimals were allowed a 1-week acclimation period in animalquarters with air conditioning and a 12:12 h light/dark cycle. Theanimals were fed ad libitum a rodent laboratory chow purchasedfrom Purina Mills, St. Louis, Mo., USA. 6-NC was synthesized inthe laboratory of Dr Peter P. Fu, National Center for ToxicologicalResearch, Je�erson, Ark., USA, as described previously (Chou et al.1987). Purity of 6-NC was >99% as determined by high-perfor-mance liquid chromatography (HPLC). 6-NC was dissolved in cornoil and administered intraperitoneally (i.p.) to hamsters at 5 mg/kgper day for 3 days as described previously (Chou et al. 1987).Control hamsters received corn oil only. Animals were killed 24 hafter the last treatment. The blood was collected for clinicalchemistry analysis. Tissues were removed for preparations ofmicrosomes, cytosols, and RNA.

Enzyme assays

Microsomal P450 and cytochrome b5 contents were determined bythe methods of Omura and Sato (1964). NADPH-cytochrome creductase activity was determined following the method of Phillipsand Langdon (1962). Benzo(a)pyrene hydroxylase activity wasdetermined by measuring the formation of the phenolic metaboliteaccording to the ¯uorimetric method of Nebert and Gelboin (1968)with 3-hydroxybenzo(a)pyrene as a standard. Activities of 7-et-hoxyresoru®n and methoxyresoru®n O-dealkylases were deter-mined by measuring the ¯uorescence of the product resoru®n, asdescribed previously (Mayer et al. 1977; Pohl and Fouts 1980).

Microsomal and cytosolic 1-nitropyrene reductase activitieswere determined by measuring the formation of the ¯uorescentproduct 1-aminopyrene following the modi®ed method of Saitoet al. (1984) as described previously (Chou et al. 1987). Preliminarystudies were carried out to determine the linear conditions withrespect to time and enzyme concentration for the hamster 1-ni-

tropyrene reductase assay. Total glutathione content of tissue ho-mogenate was determined with the enzymatic recycling assay basedon glutathione reductase following the method of Tietze (1969).Protein concentration was determined by the method of Lowryet al. (1951) using bovine serum albumin as a standard. Serumclinical chemistry analysis was carried out using a model 7450automatic autoanalyzer system of Hitachi Ltd., Tokyo, Japan.

Gel electrophoresis and immunoblotting

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using the system of Laemmli (1970) asdescribed elsewhere (Ueng et al. 1993). Transfer of microsomalproteins from the slab gel to nitrocellulose membrane was carriedout following the method of Towbin et al. (1979). Immunodetec-tion of P450 1A protein was carried out using a mouse mono-clonal antibody (Mab) 1±12±3 against 3-MC-inducible P450 1A1(Park et al. 1986). The intensities of protein bands in the Westernblots were quantitated with the aid of an IS-1000 Digital ImagingSystem (Alpha Innotech Corporation, San Leandro, Calif., USA)following the instructions in the image analysis software, Alpha-Ease, as provided by the manufacturers.

RNA isolation and blotting

Liver, kidney, and lung total RNA were isolated by the acid gu-anidinium thiocyanate-phenol-chloroform method of Chomczynskiand Sacchi (1987). Total RNA preparations were separated onformaldehyde agarose (1.2%) gels and transferred to nylon mem-branes. Transferred membranes were air dried and baked at 80 °Cfor 60 min. The membranes were rinsed in 6 ´ SSC bu�er (0.9 MNaCl, 0.09 M sodium citrate, pH 7.0), preincubated at 42 °C for4 h in a hybridization bu�er containing 50% formamide, 6 ´ SSC,0.5% SDS, 10 mM Na2HPO4, 1 mM ethylenediaminetetraaceticacid (EDTA), 5 ´ Denhardt's solution, and 0.1 mg/ml shearedsalmon sperm DNA. P450 1A1 cDNA probe was prepared from3-MC-inducible mouse P450 1A1 clone in Okayama-Berg vector(Gonzalez et al. 1984). The P450 1A1 cDNA clone was 32P-labeledusing a commercial random primers DNA labeling system (Gibco/BRL, Life Technologies, Gaithersburg, Md., USA). Followingprehybridization, the membranes were reacted with the 32P-labeledP450 1A1 cDNA probe (2 ´ 106 cpm/ml ®nal volume) in hybrid-ization bu�er at 42 °C overnight. The hybridized blots were washedfor 15 min successively in 6 ´ SSC, 2 ´ SSC, and 0.1 ´ SSC con-taining 0.1% SDS at 42 °C, and then subjected to autoradiogra-phy. The blots were deprobed and hybridized to a cDNA probespeci®c for glyceraldehyde 3-phosphate dehydrogenase (GAPDH),as an internal control for the amount of RNA. The intensities ofRNA bands in the Northern blots were quantitated with the aid ofan IS-1000 Digital Imaging System as described previously (Chenand Ueng 1997).

Salmonella mutagenicity test

Mutagenicity of 6-NC was determined by the Ames test employingSalmonella typhimurium TA98, TA100, and TA102 as the testerstrains (Maron and Ames 1983). The metabolic activation systemwas derived from the 9000 g supernatant of liver homogenateprepared from controls and hamsters pretreated with 6-NC. The S9mix, bacteria, and mutagen were preincubated at 37 °C for 30 minprior to plating. The plates were incubated at 37 °C for 2 days andthe number of bacterial revertants was scored as described byMaron and Ames (1983).

Statistical analysis

The statistical signi®cance of the di�erence between control andtreated groups was evaluated by Student's t-test. A P value <0.05was considered as statistically signi®cant.

396

Results

Pretreatment of hamsters with 6-NC had no e�ect onbody, liver, kidney, or lung weights. The results of serumclinical chemistry analysis showed that the pretreatmentcaused fourfold increase of bilirubin and twofold in-creases of levels of the histochemical marker enzymesgamma-glutamyltranspeptidase and lactate dehydroge-nase (Table 1). 6-NC produced 20% increases of alanineand aspartate aminotransferases, 26 to 41% decreases ofurea nitrogen, globulin, and uric acid, and minimale�ect on creatinine and albumin levels. The increases ofthe serum biochemical parameters indicated that 6-NCproduced liver injury and possibly other systemic tox-icity in hamsters. The pretreatment also increased totalglutathione content by 38% in the liver (data notshown).

6-NC showed mutagenicity in S. typhimurium TA98and TA100 tester strains in the absence of hamster liver

S9 (Table 2). These data con®rmed the previous resultsthat 6-NC showed comparable mutagenicity in TA98and TA100 without rat liver S9 (El-Bayoumy et al.1989b). Preincubations of 0.5, 3, and 6 lg 6-NC/platewith control and 6-NC-treated hamster liver S9 causedthree-, six-, and twofold increases of mutagenicity inTA98 as well as ®ve-, two-, and twofold increases inTA100, respectively. Without a bioactivation system,6-NC did not show apparent mutagenicity in TA102, atester strain designed to detect oxidative DNA damage(Levin et al. 1982). In contrast, preincubations of 0.5, 3,and 6 lg 6-NC/plate with control hamster liver S9caused two-, four-, and ®vefold increases of mutageni-city in TA102, respectively. Similarly, preincubations of6-NC with liver S9 from hamsters pretreated with thenitro-PAH produced three- to fourfold increases inmetabolic activation of the mutagen.

Metabolic activation of 6-NC involves reduction ofthe nitro group on the aromatic ring (Fu 1990). The

Table 1 E�ects of 6-nitro-chrysene (6-NC) on serum clin-ical chemistry parameters inhamsters

Assay Control 6-NC

Bilirubin, total (mg/dl) 1.4 � 0.3 5.0 � 0.5*Gamma-glutamyltranspeptidase (U/l) 7 � 1 18 � 1*Lactate dehydrogenase (U/l) 1690 � 222 3928 � 251*Alanine aminotransferase (U/l) 41 � 2 49 � 3*Aspartate aminotransferase (U/l) 139 � 7 169 � 6*Albumin (g/dl) 2.9 � 0.1 3.1 � 0.1*Creatinine (mg/dl) 0.3 � 0.0 0.4 � 0.0Urea nitrogen (mg/dl) 26.2 � 1.4 19.4 � 0.9*Globulin (g/dl) 1.9 � 0.2 1.3 � 0.1*Uric acid (mg/dl) 2.7 � 0.2 1.6 � 0.1*

6-Nitrochrysene was administered i.p. to hamsters at 5 mg/kg per day for 3 days. Serum clinicalchemistry parameters were determined as described in the Materials and methods. Each value re-presents mean � SE for 6 animals* Value signi®cantly di�erent from the respective control value, P < 0.05

Table 2 Mutagenicity of 6-NCin Salmonella typhimuriumstrains TA98, TA100, andTA102 with and without ham-ster liver S9

6-NC (lg/plate) 6-NC mutagenicity (histidine revertants/plate)

)S9 + Controlhamster liver S9

+ 6-NC-treatedhamster liver S9

TA980 27 � 2 35 � 1 33 � 30.5 231 � 9 716 � 31 762 � 653.0 354 � 23 2076 � 38 2375 � 2086.0 690 � 50 1511 � 35 1283 � 146

TA1000 112 � 2 121 � 6 125 � 70.5 552 � 7 2595 � 63 2467 � 953.0 1027 � 41 2224 � 68 2206 � 1136.0 825 � 43 1725 � 69 1733 � 87

TA1020 206 � 4 262 � 6 235 � 70.5 205 � 1 335 � 30 673 � 573.0 219 � 3 826 � 38 790 � 486.0 236 � 6 1068 � 45 932 � 32

Metabolic activation of 6-NC to mutagens was determined in the presence and absence of liver S9fractions prepared from controls and hamsters pretreated with 6-NC at 5 mg/kg per day for 3 days.6-NC mutagenicity was determined using TA98, TA100, and TA102 tester strains as described byMaron and Ames (1983). The results are presented as the average numbers of colonies on triplicateplates at each dose of 6-NC

397

following study was carried out to determine the e�ect of6-NC on microsomal and cytosolic nitroreductase ac-tivities using 1-nitropyrene as a test substrate. Underanaerobic conditions, pretreatment with 6-NC caused 62and 31% increases of 1-nitropyrene reductase activitiesin liver and lung microsomes, respectively (Table 3). Thepretreatment had no e�ect on the kidney microsomal1-nitropyrene reductase activity. 6-NC caused a twofoldincrease of 1-nitropyrene reductase activity in lungcytosol without a�ecting the liver or kidney cytosolicenzyme. Under aerobic conditions, 1-nitropyrene re-ductase activity was not detectable in the hamster tissuesmicrosomes, similarly to previous observations with ratliver microsomes (Saito et al. 1984).

The e�ect of 6-NC on microsomal monooxygenasewas determined using benzo(a)pyrene, 7-ethoxyresoru-®n, and methoxyresoru®n as test substrates which are

preferentially metabolized by P450 1A1/2 (Ryan andLevin 1990; Nerurkar et al. 1993). In liver, pretreatmentwith 6-NC caused twofold increases of 7-ethoxyresoru®nO-deethylase and methoxyresoru®n O-demethylase ac-tivities without a�ecting benzo(a)pyrene hydroxylaseactivity (Table 4). In lung, 6-NC resulted in three- and®vefold increases of 7-ethoxyresoru®n and methoxy-resoru®n O-dealkylases activities, respectively. 6-NChad no e�ect on the monooxygenase activities in kidney.Pretreatment with 6-NC was without signi®cant e�ecton P450 and cytochrome b5 contents, or NADPH-cy-tochrome c reductase activity in the hamster tissues(data not shown).

Microsomal proteins from control and 6-NC-treatedhamster tissues were subjected to SDS-PAGE followedby immunoblotting procedures in which a mouse Mab1-12-3 against 3-MC-inducible rat P450 1A1 was used to

Table 3 E�ects of 6-NC on1-nitropyrene reductase inhamster liver, kidney, and lung

1-Nitropyrene reductase (pmol product/min per mg protein)

Anaerobic Aerobic

Microsomes Cytosol Microsomes

LiverControl 123 � 7 114 � 5 n.d.6-N 198 � 15* 115 � 8 n.d.

KidneyControl 19 � 1 9 � 2 n.d.6-NC 20 � 3 13 � 3 n.d.

LungControl 6 � 1 12 � 1 n.d.6-NC 8 � 1* 22 � 1* n.d.

6-Nitrochrysene was administered i.p. to hamsters at 5 mg/kg per day for 3 days. Liver microsomesand cytosols were prepared from individual hamsters. Kidneys or lungs from two hamsters within thesame group were pooled and microsomes and cytosols were prepared. 1-Nitropyrene reductase ac-tivities were determined as described in the Materials and methods. Each value represents mean � SEfor at least 4 determinations* Value signi®cantly di�erent from the respective control value, P < 0.05n.d., Not detectable

Table 4 E�ects of 6-NC onmonooxygenases activities ofhamster liver, kidney,and lung (3OHBP3-Hydroxybenzo(a)pyrene,RF resoru®n)

Benzo(a)pyrenehydroxylase (pmol3OHBP/min per mgprotein)

7-Ethoxyresoru®nO-dealkylase (pmolRF/min per mg protein)

Methoxyresoru®nO-dealkylase (pmolRF/min per mg protein)

LiverControl 510 � 46 139 � 4 238 � 176-NC 533 � 23 231 � 28* 481 � 54*

KidneyControl 69 � 2 19 � 1 33 � 46-NC 73 � 6 24 � 2 37 � 4

LungControl n.d.** 6 � 1 7 � 16-NC n.d. 17 � 1* 38 � 6*

6-Nitrochrysene was administered i.p. to hamsters at 5 mg/kg per day for 3 days. Liver microsomeswere prepared from individual hamsters, kidney and lung microsomes were prepared from tissuespooled from two hamsters within the same group. Each value represents mean � SE for 5 and 3determinations in the hepatic and extrahepatic tissues, respectively* Value signi®cantly di�erent from the respective control value, P < 0.05n.d., Not determined

398

analyze for immunorelated proteins (Fig. 1). The inten-sities of the protein bands in the immunoblots werequantitated (Table 5). In liver, the Mab showed crossreactivity with an immunorelated protein with controlhamsters (Fig. 1, lane 1). Pretreatment with 6-NC causeda twofold increase in the intensity of this P450 1A-immunorelated protein (lane 2). In lung, a protein(s) withelectrophoretic mobility slower than that of liver P4501A protein was detected with control hamsters (lane 5).6-NC caused a twofold increase in the intensity of thislung protein (lane 6). 6-NC had no apparent e�ect on theproteins in kidney (lane 4), in contrast to liver and lung.

The immunoblotting data suggested that pretreat-ment with 6-NC induced P450 1A. Previous studies haveshown that pretreatment of hamster with acetone in-creased the level of P450 1A mRNA hybridizable to acDNA probe derived from 3-MC-inducible mouse

Cyp1a1 (Gonzalez et al. 1984; Chen and Ueng 1997). Inthe following study, total RNA preparations from con-trols and 6-NC-treated hamster tissues were subjected toagarose gel electrophoresis and blotting procedures inwhich the mouse P450 1A1 cDNA clone was used toprobe for hybridizable mRNA species (Fig. 2). The in-tensities of the mRNA band in the RNA blots werequantitated (Table 5). In liver, the cDNA probe detecteda P450 1A mRNA band with relative mobility similar to18S RNA in control hamsters (Fig. 2, lane 1). Pre-treatment with 6-NC caused a twofold increase in theintensity of this liver P450 1A mRNA band (lane 2). Inlung, a P450 1A mRNA was detected with controlhamsters, the intensity of this mRNA band was in-creased by twofold in the hamsters pretreated with 6-NC(lanes 5 and 6). Unlike that in liver and lung, 6-NC hadno signi®cant e�ect on the intensity of the mRNA bandin kidney (lanes 3 and 4).

Discussion

The present ®ndings show that 6-NC is mutagenic in allthree S. typhimurium strains tested. The exact cause ofthe oxidative damage to TA102 is not clear. Formationof reactive oxygen species is generally thought to beassociated with reductive metabolism of polycyclic aro-matic hydrocarbon (PAH) quinone derivatives and nitrogroups of nitro-PAHs (Goeptar et al. 1995). Thereforereduction of 6-NC quinone derivative(s) and nitrogroups might have partially caused oxidative damage toDNA in TA102. The present mutagenicity data obtainedwith TA100 have shown that control and 6-NC-treatedhamster liver S9 increased metabolic activation of 6-NC.These data are markedly di�erent from those of El-Bayoumy et al. (1989b), in which Aroclor 1254-treated

Fig. 1 Immunoblots of microsomal P450 1A in tissues fromuntreated and 6-nitrochrysene (6-NC)-treated hamsters. Microsomalproteins were prepared from individual control (C) and 6-NC-treatedhamster tissues and subjected to SDS-PAGE as described in theMaterials and methods. The microsomal proteins were transferred tonitrocellulose membranes and probed with mouse monoclonalantibody Mab 1-12-3 against rat P450 1A1. The microsomal proteinswere loaded at 3 lg (lanes 1 and 2) and 50 lg (lanes 3±6)

Table 5 E�ects of 6-NC on cytochrome P450 1A protein andmRNA in hamster liver, kidney, and lung

Cytochrome P450 (arbitrary unit)

Protein mRNA

LiverControl 323 � 16 699 � 526-NC 674 � 57* 1386 � 112*

KidneyControl 102 � 7 217 � 226-NC 104 � 4 230 � 25

LungControl 203 � 19 217 � 246-NC 494 � 72* 428 � 42*

Microsomal proteins and total RNA were isolated from the tissuesof controls and hamsters pretreated with 6-NC and subjected toprotein and RNA blot analyses, respectively, as described in thelegends to Figs. 1 and 2. Intensity of the protein and mRNA bandsin arbitrary units was obtained from densitometric analyses of theprotein and RNA blots with a digital imaging system. Each valuerepresents the mean � SE for 5 protein and RNA blot analyses

Fig. 2 RNA blots of P450 1A mRNA in tissues from untreated and6-NC-treated hamsters. Total RNA was prepared from the individualcontrol (C) and 6-NC-treated hamster tissues and subjected to agarosegel electrophoresis as described in the Materials and methods. RNAwas electrophoretically transferred to a nylon membrane and probedwith a mouse cDNA clone of 3-methylcholanthrene (3-MC)-inducibleP450 1A1 (upper panel). The membrane was reprobed with a cDNAfor rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as aninternal standard (lower panel). The RNA loading was 20 lg (lanes 1and 2), 30 lg (lanes 3 and 4), and 40 lg (lanes 5 and 6)

399

rat liver S9 failed to increase 6-NC mutagenicity inTA100. These di�erent data suggest, among other pos-sibilities, that the hamster enzymes are more e�cientthan the rat enzymes in metabolic activation of 6-NC.The present mutagenicity data also showed that liver S9from 6-NC-treated hamsters did not increase muta-genicity frequencies compared to the controls. Thusother enzymes, such as P450 3A isoforms, could also beinvolved in metabolic activation of 6-NC in theS. typhimurium tester strains.

To the best of our knowledge, this report is the ®rst toshow that exposure to a nitro-PAH increases the cata-lytic activity, protein, and mRNA levels of P450. Theenzyme induction most likely involves a pretranslationalmechanism, which is initiated by binding of 6-NC to thearylhydrocarbon (Ah) receptor and followed by a seriesof events leading to increases of P450 1A mRNA, pro-tein, and activity (Whitlock et al. 1996). Apparently, thepresence of a nitro-substituent on the PAH-moiety doesnot prevent binding of 6-NC to the receptor. Chou et al.(1987) proposed that a nitro-substituent coplanar to thearomatic ring does not block binding of nitro-PAHs,such as 6-NC and 3-nitrobenzo(a)pyrene to the Ah re-ceptor. However, a nitro group with a perpendicularorientation to the aromatic ring such as that of 6-ni-trobenzo(a)pyrene may interfere with the nitro-PAHand receptor interaction. The regio- and stereo-speci-®cities of the nitro group might be an important factorto consider in assessment of the enzyme inductionproperty and toxicity of a nitro-PAH.

The present data clearly demonstrate that 6-NC in-duces hamster liver and lung P450 1A. Chae et al. (1993)suggested that human liver P450 1A2 and lung 1A1 wereinvolved in 6-NC metabolism. These parallel results in-dicate a possible correlation in 6-NC metabolism andP450 induction. However, it is di�cult to identify thespeci®c forms of P450 1A induced by 6-NC in hamstersbased on the immunoblotting and catalytic activity data.The immunoblotting data revealed that the electropho-retic mobility of 6-NC-inducible liver P450 1A was fasterthan that of lung P450 (Fig. 1). Sagami et al. (1991)reported that the molecular weights of hamster P4501A1 and 1A2 were 59 408 and 58 079, respectively.Therefore it seems reasonable to speculate that 6-NCinduced P450 1A2 in liver and 1A1 in lung. However,such speculation is at variance with the catalytic activitydata: a greater stimulation of P450 1A2-dependent me-thoxyresoru®n O-dealkylase activity was seen in the lungcompared to that of 1A1-dependent 7-ethoxyresoru®nO-deethylase activity (Table 4). Additional studies arerequired to resolve this discrepancy.

Benzo(a)pyrene is a useful substrate for both P4501A1 and 1A2 (Ryan and Levin 1990). In liver, 6-NCinduced 7-ethoxyresoru®n and methoxyresoru®nO-dealkylases activities, whereas benzo(a)pyrenehydroxylase activity remained unchanged. This refrac-tiveness possibly indicates that 6-NC suppressed or hadlittle e�ect on other forms of P450 associated with ben-zo(a)pyrene hydroxylation. Consequently, we did not

observe an increase of total benzo(a)pyrene hydroxylaseactivity in liver microsomes. Yun et al. (1992) reportedthat human liver microsomal P450 3A4, 2C8, and 2C9/10are involved in 3-hydroxylation of benzo(a)pyrene. It willbe of interest to determine the e�ect of 6-NC on theorthologous hamster P450 3A and 2C enzymes. Previousstudies have shown that 3-MC is an e�ective inducer ofP450 2A8 and dependent coumarin 7-hydroxylase ac-tivity in hamster liver (Lai and Chiang 1990; Tohkin et al.1996). In a separate experiment, we found that 6-NCpretreatment had no e�ect on coumarin 7-hydroxylaseactivity in liver microsomes (data not shown). These datado not favor the possibility that pretreatment with 6-NCcaused a marked induction of P450 2A8 in hamster liver.

Enzymes known to catalyze nitro-reduction of nitro-PAHs include microsomal P450 and NADPH-cyto-chrome P450 reductase and cytosolic quinone reductase,aldehyde oxidase, and xanthine oxidase (Saito et al.1984; Goeptar et al. 1995). Pretreatment of rats with1-nitropyrene caused a twofold increase of 1-nitropyrenereductase activity in liver cytosol (Djuric et al. 1988).The e�ect of nitro-PAH on nitroreductase activity hadnot been reported with an extrahepatic tissue. Thepresent ®nding showed that pretreatment with 6-NCincreased cytosolic 1-nitropyrene reductase activity inhamster lung, but not liver (Table 3). Possible explana-tions for this tissue variation include too low a concen-tration of the nitoreductase inducer or a tissue-speci®ctranscription factor required for induction is absent inthe liver. In any event, the simultaneous induction ofmicrosomal P450 1A and cytosolic nitroreductase ac-tivity in the lung may be an important determinant insusceptibility of the pulmonary tissue to 6-NC-inducedtumorigenicity. In conclusion, the present study dem-onstrates that 6-NC can induce hamster P450 1A cata-lytic activity, protein, and mRNA and increase1-nitropyrene reductase activity. The hamster metabolicenzymes are e�cient in activation of 6-NC to mutagensin S. typhimurium TA98, TA100, and TA102.

Acknowledgements The authors thank Dr Peter P. Fu for 6-NC,Dr Frank J. Gonzalez for the P450 1A1 cDNA clone, and Dr SangS. Park for the monoclonal antibody to P450 1A1. This work wassupported by grants NSC85-2331-B002-056 and NSC86-2314-B002-305 from the National Science Council, ROC.

References

Busby WF Jr, Garner RC, Chow FL, Martin CN, Stevens EK,Newberne PM, Wogan GN (1985) 6-Nitrochrysene is a potenttumorigen in newborn mice. Carcinogenesis 6: 801±803

Chae Y-H, Yun C-H, Guengerich FP, Kadlubar FH, El-BayoumyK (1993) Roles of human hepatic and pulmonary cytochromeP450 enzymes in the metabolism of the environmental carcin-ogen 6-nitrochrysene. Cancer Res 53: 2028±2034

Chen R-M, Ueng T-H (1997) Induction of cytochrome P450 1A,2B and 2E in hamster tissues by acetone. Arch Toxicol 71: 489±495

Chomczynski P, Sacchi N (1987) Single-step method of RNA iso-lation by acid guanidinium thiocyanate-phenol-chloroform ex-traction. Anal Biochem 162: 156±159

400

Chou MW, Wang B, Von Tungeln LS, Beland FA, Fu PP (1987)Induction of rat hepatic cytochromes P-450 by environmentalnitropolycyclic aromatic hydrocarbons. Biochem Pharmacol36: 2449±2454

Delclos KB, Walker RP, Dooley KL, Fu PP, Kadlubar FF (1987)Carcinogen-DNA adduct in the lungs and livers of preweanlingCD-1 male mice following administration of [3H]-6-nitrochry-sene, [3H]-6-aminochrysene, and [3H]-1,6-dinitropyrene. CancerRes 47: 6272±6277

Delclos KB, El-Bayoumy K, Hecht SS, Walker RP, Kadlubar FF(1988) Metabolism of the carcinogen [3H]6-nitrochrysene in thepreweanling mouse: identi®cation 6-aminochrysene-1,2- dihy-drodiol as the probable proximate carcinogenic metabolite.Carcinogenesis 9: 1875±1884

Djuric Z, Fifer EK, Yamazoe Y, Beland FA (1988) DNAbinding by 1-nitropyrene and 1,6-dinitropyrene in vitro andin vivo: e�ects of nitroreductase induction. Carcinogenesis 9:357±364

El-Bayoumy K, Shiue G-H, Hecht SS (1989a) Comparative tu-morigenicity of 6-nitrochrysene and its metabolites in newbornmice. Carcinogenesis 10: 369±372

El-Bayoumy K, Delclos KB, He¯ich RH, Walker R, Shiue G-H,Hecht SS (1989b) Mutagenicity, metabolism and DNA adductformation of 6-nitrochrysene in Salmonella typhimurium. Mu-tagenesis 4: 235±240

Estabrook RW (1996) The remarkable P450s: a historical overviewof these versatile hemeprotein catalysts. FASEB J 10: 202±204

Fu PP (1990) Metabolism of nitro-polycyclic aromatic hydrocar-bons. Drug Metab Rev 22: 209±268

Goeptar AR, Scheerens H, Vermeulen PE (1995) Oxygen andxenobiotic reductase activities of cytochrome P450. Crit RevToxicol 25: 25±65

Gonzalez FJ, Mackenzie PI, Kimura S, Nebert DW (1984) Isola-tion and characterization of full-length mouse cDNA and ge-nomic clones of 3-methylcholanthrene-inducible cytochromeP1- 450 and P3±450. Gene 29: 281±292

Imaida K, Uneyama C, Ogasawara H, Hayashi S, Fukuhara K,Miyata N, Takahashi M (1992) Induction of colon adenocar-cinomas in CD rats and lung adenomas in ICR mice by6-nitrochrysene: comparison of carcinogenicity and arylhydrocarbon hydroxylase induction in the target organs of eachspecies. Cancer Res 52: 1542±1545

Laemmli UK (1970) Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature 227: 680±685

Lai TS, Chiang JYL (1990) Cloning and characterization of twomajor 3-methylcholanthrene inducible hamster liver cyto-chrome P450s. Arch Biochem Biophys 283: 429±439

Levin DE, Hollstein M, Christman MF, Schwiers EA, Ames BN(1982) A new Salmonella tester strain (TA102) with A:T basepairs at the site of mutation detects oxidative mutagens. ProcNatl Acad Sci USA 79: 7445±7449

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Proteindetermination with Folin phenol reagent. J Biol Chem 193:265±275

Maron DM, Ames BN (1983) Revised methods for the Salmonellamutagenicity test. Mutat Res 113: 173±215

Mayer RT, Jermyn JW, Burke MD, Prough RA (1977) Methoxy-resoru®n as a substrate for the ¯uorometric assay of insectmicrosomal O-dealkylase. Pest Biochem Physiol 7: 349±354

Nebert DW (1994) Drug-metabolizing enzymes in ligand-modu-lated transcription. Biochem Pharmacol 47: 25±37

Nebert DW, Gelboin HV (1968) Substrate-inducible microsomalaryl hydroxylase in mammalian cell culture. J Biol Chem 243:6242±6249

Nerurkar PV, Park SS, Thomas PE, Nims RW, Lubet RA (1993)Methoxyresoru®n and benzyloxyresoru®n: substrates preferen-tially metabolized by cytochromes P4501A2 and 2B, respec-tively, in the rat and mouse. Biochem Pharmacol 46: 933±943

Omura T, Sato R (1964) The carbon monoxide-binding pigment ofliver microsomes. I. Evidence for its hemoprotein nature. J BiolChem 239: 2370±2378

Park SS, Miller H, Klotz AV, Kloppers-Sams PJ, Stegeman JJ,Gelboin HV (1986) Monoclonal antibodies to liver microsomalcytochrome P450-E of the marine ®sh Stenotomus chrysops(scup): cross reactivity with 3-methylcholanthrene induced ratcytochrome P-450. Arch Biochem Biophys 249: 339±350

Phillips AH, Langdon RG (1962) Hepatic triphosphopyridine nu-cleotide-cytochrome c reductase: isolation, characterization,and kinetic studies. J Biol Chem 237: 2652±2660

Pohl RJ, Fouts JR (1980) A rapid method for assaying the me-tabolism of 7-ethoxyresoru®n by microsomal subcellular frac-tions. Anal Biochem 107: 150±155

Ryan DE, Levin W (1990) Puri®cation and characterization ofhepatic microsomal cytochrome P-450. Pharmacol Ther 45:153±239

Sagami I, Ohmachi T, Fujii H, Kikuchi H, Watanabe M (1991)Hamster cytochrome P-450-IA1 and P-450-IA2 in lung andliver: cDNA cloning and sequence analysis. J Biochem 110:641±647

Saito K, Kamataki T, Kato R (1984) Participation of cytochromeP450 in the reductive metabolism of 1-nitropyrene by rat livermicrosomes. Cancer Res 44: 3169±3173

Tietze F (1969) Enzymic method for qualitative determination ofnanogram amounts of total and oxidized glutathione. AnalBiochem 27: 502±522

Tohkin M, Kurose K, Fukuhara M (1996) Okadaic acid potenti-ates 3-methylcholanthrene-induced CYP2A8 gene expression inprimary cultures of Syrian golden hamster hepatocytes: possibleinvolvement of activator protein-1. Mol Pharmacol 50: 556±564

Towbin H, Staehlin T, Gordon J (1979) Electrophoretic transfer ofproteins from polyacrylamide gels to nitrocellulose sheets:procedure and some applications. Proc Natl Acad Sci USA 76:4350±4354

Ueng TH, Ueng YF, Chen TL, Park SS, Iwasaki M, GuengerichFP (1993) Induction of cytochrome P450-dependent monoox-ygenases in hamster tissues by fasting. Toxicol Appl Pharmacol119: 66±73

Whitlock JP Jr, Okino ST, Dong L, Ko HP, Clarke-Katzenberg R,Ma Q, Li H (1996) Induction of cytochrome P450 1A1: a modelfor analyzing mammalian gene transcription. FASEB J 10: 809±818

Yoo J-S H, Ning SM, Patten CJ, Yang CS (1987) Metabolism andactivation of N-nitrosodimethylamine by hamster and rat mic-rosomes: comparative study with weanling and adult animals.Cancer Res 47: 992±998

Yun C-H, Shimada T, Guengerich FP (1992) Roles of human livercytochrome P4502C and 3A enzymes in the 3-hydroxylation ofbenzo(a)pyrene. Cancer Res 52: 1868±1874

401