Genetically engineered cells stably expressing cytochrome P450 and their application to mutagen assays

Mutation Research 411 Ž1998. 19–43

Genetically engineered cells stably expressing cytochrome P450 and their application to mutagen assays Minoru Sawada a b

a,)

, Tetsuya Kamataki

b

DiÕision of EnÕironmental Hygiene, Hokkaido College of Pharmacy, Katsuraoka-cho 7-1, Otaru, Hokkaido 047-02, Japan DiÕision of Drug Metabolism, Faculty of Pharmaceutical Sciences, Hokkaido UniÕersity, Sapporo, Hokkaido 060, Japan

Abstract Genetically engineered cells transiently and stably expressing cytochrome P450 Ž P450., a key enzyme for biotransformation of a wide variety of compounds, have provided new tools for investigation of P450 functions such as P450-mediated metabolic activation of chemicals. This review will focus on the development of mammalian cell lines stably expressing P450s and application to toxicology testings. Stable expression systems have an advantage over transient ones in that a series of the process from metabolic activation of test compounds to the appearance of toxicological consequences occurs entirely in the same intact cells. Indeed, many cell lines stably expressing a single form of mammalian P450 have been established so far and applied to cytotoxic or genotoxic assays, the endpoints of which contained mutations at hprt and other gene loci, chromosomal aberrations, sister chromatid exchanges, micronuclei, morphological transformation, and 32 P-postlabeling. Analyses of metabolites of toxic substances have also been carried out, using the intact cells or microsomal fractions prepared from the cells. The stable expression systems clearly indicate the form of P450 enzyme capable of activating a certain chemical. More recently, coexpression of P450 together with other components of microsomal electron transfer systems such as NADPH–cytochrome P450 reductase has been successfully performed to increase the metabolic capacity of the heterologously expressed P450. In addition, to reconstruct the entire metabolic activation system for certain heterocyclic amines, cell lines which simultaneously express a form of human P450 and a phase II enzyme, N-acetyltransferase, were established. These cells were highly sensitive to some carcinogenic heterocyclic amines. In genetic toxicology,

Abbreviations: 2-AA, 2-aminoanthracene; 2-AAF, 2-acetylaminofluorene; AFB1 , aflatoxin B1; AFG1 , aflatoxin G1; Bw axP, benzow axpyrene; BPD, benzow axpyrene dihydrodiol; BPDE, benzow axpyrene dihydrodiol epoxide; CP, cyclophosphamide; CYP or P450, cytochrome P450; DMBA, 7,12-dimethylbenzw axanthracene; hprt, hypoxanthine guanine phosphoribosyl transferase; IQ, 2-amino-3methylimidazow4,5-f xquinoline; 3-MC, 3-methylchoranthrene; mEH, microsomal epoxide hydrolase; MeIQ, 2-amino-3,4dimethylimidazow4,5-f xquinoline; MeIQx, 2-amino-3,8-dimethylimidazow4,5-f xquinoxaline; MMC, mitomycin C; NAT, N-acetyltransferase; NDBA, N-nitrosodibutylamine; NDEA, N-nitrosodiethylamine; NDMA, N-nitrosodimethylamine; NNA, 1-Ž N-methyl-N-nitroso.-1-Ž3pyridyl.-4-butanol; NNK, 4-Žmethylnitrosamino.-1-Ž3-pyridyl.-1-butanone; NNN, N-nitrosonornicotine; OAT, O-acetyltransferase; PB, phenobarbital; PCB, polychlorinated biphenyl; PhIP, 2-amino-1-methyl-6-phenylimidazow4,5-b xpyridine; SCE, sister chromatid exchange; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; tk, thymidine kinase; Trp-P-2, 3-amino-1-methyl-5H-pyridow4,3-b xindole; XPA, xeroderma pigmentosum group A ) Corresponding author. 1383-5742r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 4 2 Ž 9 8 . 0 0 0 0 5 - 2

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M. Sawada, T. Kamatakir Mutation Research 411 (1998) 19–43

such a coexpression system for two or more enzymes will provide useful materials which mimic in vivo activation systems. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Genetically engineered cell; Cytochrome P450 ŽCYP.; Stable expression system; Metabolic activation of mutagenrcarcinogen; Cytotoxicity; Mutagenicity; NADPH–cytochrome P450 reductase; Epoxide hydrolase; N-acetyltransferase

1. Introduction Cytochrome P450 Ž P450 or CYP. is a heme-containing enzyme widely distributed from bacteria to mammals, and catalyzes the oxidative and reductive metabolism of a wide variety of compounds including endogenous as well as exogenous compounds. Mammalian P450 present in liver microsomes is characteristic of its nature in metabolizing exogenous compounds including drugs, pesticides, environmental pollutants, mutagens, and carcinogens. In recent studies of P450s, excellent results have been brought about by approaches based on gene technology, including prompt identification of new molecular forms by cDNA cloning, analysis of the gene regulation mechanism leading to ‘enzyme induction’, development of a gene diagnosis of polymorphic P450s, and the generation of animals carrying transgene w1x or lacking a certain P450 gene by gene-knockout technology w2x. Catalytic functions of an individual form of P450 can be analyzed by the method of heterologous expression of its cDNA. In these experiments, the expression vectors which contain the cDNA encoding a specific P450 are introduced to recipient cells Žbacteria, yeast, insect cells, cultured mammalian cells. by an appropriate transfection method. The cDNA expression systems using cultured mammalian cells are divided into transient and stable types. In the transient expression system, although the expression period and lives of the cells are generally short, large amounts of the enzymes can be produced in the transfected cells. Thus, the microsomal fractions from the cells transiently expressing a specific P450 are suitable for investigation of its enzymatic properties such as substrate specificity and kinetic parameters. On the other hand, stable expression systems are, in general, capable of producing lower levels of enzyme proteins, while the gene expression continues stably for longer cell-generations. The stable expression system including mammalian cells has made it possible to evaluate the

relative risk of a chemical in an in vitro toxicology testing. Thus, this review will focus on the usefulness of genetically engineered cells in the in vitro toxicology testings. Mammalian cultured cells have been used as important tools for approaching cellular and molecular mechanisms of chemical toxicity. A variety of systems using immortalized cell lines have been developed in the field of toxicological testings, such as mutagenicity assays. One of the critical points in these assay systems is their capacity to metabolize drugs, since many toxicants, mutagens and carcinogens require metabolic activation to react with intracellular macromolecules and to exert their toxicological consequences. Most of the established cell lines possess actually no or, if any, very low levels of P450 activities. This is an inevitable tendency in cultured cells, even in the cells derived from livers. Thus, to complement the lack of an enzymeŽs. involved in the metabolic activation of chemicals, two metabolic activation systems have been added to the assays. One is an externally added cell-mediated system, in which target cells are co-cultivated with primary-cultured hepatocytes. The other is a method in which so-called S9 mix prepared from liver homogenates is added to the culture medium, expecting the activation of a test compound by enzymes in the S9 mix. Because of feasibility, the latter method has been adopted in the standard assays of mutagens. Although the S9 mix method has been widely used and recognized to be the most common, this method still has some disadvantages to be resolved. First, the S9 mix itself is known to decrease the viability of the cultured cells. Therefore, the periods of exposure and the concentrations of the S9 mix added to the assays are not necessarily suitable to obtain the highest metabolic activation. Second, since the test compounds are metabolized in the culture medium but not inside the cells, it is possible that the shortlived metabolites with high chemical reactivity are expected to bind to the surface of cells, and only a

M. Sawada, T. Kamatakir Mutation Research 411 (1998) 19–43

portion of the metabolites can penetrate through cell membranes to reach and react with target macromolecules to show the toxicities. The S9 used in routine mutagen assays is, in many cases, prepared from the liver of rats pretreated with some P450-inducing agents Že.g., b-naphthoflavone plus phenobarbital, or PCB alone.. Considering species differences in the activities of drug-metabolizing enzymes, the use of human liver S9 is desired to predict human risks of test compounds, but it is virtually impossible to use human livers for such routine research. The population of human enzymes may differ among individual liver specimens by many factors such as induction by drug intake and the genetic polymorphism of certain enzymes. Genetically engineered mammalian cell lines stably expressing P450 enzymes were first established by Doehmer et al. w3x. Since then, many cell lines have been established and the properties reported by many authors. These new cell lines have been evaluated as target cells in mutation assays. The advantages of these cell lines may be as follows: Ž1. The most valuable point is that a series of the process from the metabolic activation of the test compound to the consequences of cytotoxicity andror mutagenicity occur entirely in the cell. There is no assurance that the extracellular activations by the S9 mix always yield information exactly the same as one occurring intracellularly. Some factors, including cell membranes which may prevent the penetration of active metabolites, oxygen tension which may affect active oxygen production as well as the oxidative metabolism of chemicals, and coenzyme concentrations necessary for conjugation reactions, may cause differences from intracellular situations. Ž2. As stated earlier, genetically engineered cells can be expected to show higher sensitivity to short-lived active metabolites, since the metabolites can directly react with target macromolecules in the same cells. Ž3. In studies done to ascertain the roles of human P450s in the activation of mutagens, the cells carrying each human P450 are particularly useful. A panel study using cell lines carrying each human P450 DNA may be conducted to determine a specific formŽs. of P450 involved in the activation. Thus, the aims of this review are to briefly summarize reports on the establishment of the cell lines stably expressing P450 alone or together with a

21

phase II enzyme such as N-acetyltransferase, and to discuss the advantages and disadvantages of their use in cytotoxicity and mutagenicity assays.

2. Expression systems and cell lines used as recipients The stable expressions of P450 in cultured mammalian cells reported so far are listed in Table 1. To establish the cells which stably express P450, available expression vectors containing cDNA or P450 gene are introduced to recipient cells simultaneously with vectors containing a drug-resistance gene by an appropriate transfection method. Both the P450 cDNA and the drug-resistance gene may be included in the same vector. The cells transfected with the vectors are treated with a drug to select cells which carry the drug-resistance gene, assuming that the vector containing the drug-resistance gene is introduced into the cells together with the vector containing the P450 gene. Then, the cell clones resistant to the drug are isolated and examined for P450 expression. In the system of a human lymphoblastoid cell line AHH-1 ŽTable 1., which had been transformed with Epstein–Barr virus, the expression vectors which contain P450 cDNA and the hygromycin-resistance gene exist extrachromosomally, and are replicated because of the presence of a replication origin of Epstein–Barr virus in the vector w4x. In the stable expression systems in Table 1 other than AHH-1, the P450 cDNA and drug-resistance gene are assumed to be integrated into chromosomal DNA of the recipient cells, since the vectors used in those studies cannot be replicated extrachromosomally. Gene transfer mediated by retroviruses has also been employed to develop a stable expression system w44x. Briefly, a retroviral vector containing P450 cDNA and neomycin-resistance gene Ž neo r . is transfected into packaging cells by a calcium phosphate coprecipitation method. A few days after the transfection, the supernatant fraction of the culture medium, which contains the recombinant virus carrying P450 cDNA and neo r , is collected, filtered and used to infect target cells. The infected target cells are selected with G418. The resistant colonies are picked up, grown and tested for P450 expression.

M. Sawada, T. Kamatakir Mutation Research 411 (1998) 19–43

22

Table 1 Development of cell lines stably expressing cytochromes P450 and their use in toxicological studies P450 Žspecies.

Recipient cell lines

Assays Chemicals assayed

CYP1A1 Žhuman.

Mutation Cytotoxicity Cytotoxicity, mutation

w4x w5x w6x

Metabolism Metabolism Metabolism Cytotoxicity

w7x w8x w9x w10x

Cytotoxicity, mutation Cytotoxicity, mutation, 32 P-postlabeling Cytotoxicity, micronucleus, mutation Hydroxylation Cytotoxicity, mutation Cytotoxicity, mutation Cytotoxicity, chromosomal aberration Cytotoxicity Mutation Micronucleus

w11x w12x w13x w14x w15x w16x w17x w18x w19,20x w21,22x

c-mitosis Chromosomal aberration, SCE

w23x w24x

Hydroxylation Micronucleus SCE Chromosomal aberrations Cytotoxicity Mutation

w14x w25x w26x w27x w28x w29x

Hydroxylation Ames test Cytotoxicity, mutation

w30x w31x w31x

AFB1 NNK AFB1 Acetoaminophen Phenanthrene Bw axP, BPD AFB1 ŽAcetanilide. ŽAlkoxyresorufins. Bw axP Acetaminophen AFB1

Cytotoxicity, mutation, DNA binding Cytotoxicity, mutation Cytotoxicity Metabolism Metabolism Metabolism Metabolism Ž4-Hydroxylation. Ž O-dealkylation. Hydroxylation Cytotoxicity, c-mitosis Cytotoxicity

w32,33x w34x w6,5x w35x w8x w9x w36x w30x w37x w14x w38x w39x

IQ, MeIQx AFB1 , AFG1 Ochratoxin A

Cytotoxicity, mutation Cytotoxicity, DNA-binding Cytotoxicity, mutation

w39x w40x w15x

AFB1 AFB1 AFB1 , Bw axP, NNK, Cyclopentaw c,d xpyrene DMBA Phenanthrene Bw axP, BPD Human skin fibroblasts: XPA, Bw axP, BPD, BPDE DNA-repair normal BPD BPD, BPDE V79 Bw axP, BPD Bw axP NIHr3T3 Ochratoxin A CYP1A1 Žmonkey. CHL AFB1 AFB1 , sterigmatocystin BALB 3T3 A31-1-1 AFB1 , Bw axP CYP1A1 Žrat. V79 Bw axP, BPD Bw axP, sterigmatocystin, tobacco particulate matter, 2-AA, CP Bw axP, BPD AFB1 , Bw axP, CP, DMBA, NDMA Bw axP Hydroquinone, econazole nitrate Hydroxy anthraquinones Hydroxy anthraquinones Adriamycin, MMC Bw axP, BPD, DMBA, chrysene dihydrodiols, 2-AA, 2-AAF, AFB1 , NDBA Cyp1a1 Žmouse. Hepa-1c1c7 c37 Bw axP CHO-derived UV5 BPD, Trp-P-2 Bw axP CYP1A2 Žhuman.

AHH-1 TK q ry

AHH-1 TK q ry

Hepa-1c1c7 c37 V79

CHL-derived CR-68 plus NAT1, NAT2 BEAS-2B NIHr3T3

References Endpoints

M. Sawada, T. Kamatakir Mutation Research 411 (1998) 19–43

23

Table 1 Žcontinued. P450 Žspecies.

Recipient cell lines

CYP1A2 Žrat.

V79

Assays Chemicals assayed

2-AF 2-AA, sterigmatocystin, Bw axP, CP Bw axP, BPD Phenacetin AFB1 , CP, Bw axP, DMBA, NDMA Bw axP Econazole nitrate 1,2-OH anthraquinone, 1,2,4-OH anthraquinone Hydroxy anthraquinones Bw axP, BPD, DMBA, chrysene dihydrodiols, 2-AA, 2-AAF, AFB1 , DMBA 2-AA, 2-AF, 2-AAF,4-AAF, IQ Ad293 Žhuman embryonic kidney. MeIQ, Trp-P-2, PhIP, 2-AA, 2-AF, AFB1 , BPD Cyp1a2 Žmouse. NIHr3T3, Hepa-1, BSC-1, CV-1, HeLa ŽAcetanilide. RLE Žrat liver epithelial. IQ, MeIQx NIHr3T3 IQ, 2-AAF CHO-derived UV5 IQ, PhIP PhIP, N-OH-PhIP

plus NAT2, OAT CYP2A6 Žhuman. AHH-1 TK q ry derived L3 AHH-1 TK q ry

C3Hr10T1r2 CHO-derived AS52 HeLa, NIHr3T3 V79 CYP2B1 Žrat.

V79

References Endpoints Metabolism Micronucleus

w41x w21,22x

w23x c-mitosis w42x Metabolism Chromosomal aberration, SCE w24x Hydroxylation Micronucleus SCE

w14x w25x w26x

Chromosomal aberration Mutation

w27x w29x

SCE Ames test

w29x w43x w44x w45x w46x w47x w48x

PhIP IQ, PhIP

Ž4-Hydroxylation. 32 P-Postlabeling 32 P-Postlabeling Cytotoxicity, mutation Chromosomal aberration, SCE, micronuclesus Mutation spectrum Cytotoxicity, mutation

NDMA, Bw axP AFB1 , Bw axP, NDMA, NDEA AFB1 NNK CP, ifosfamide NNK NDEA, NNK NNK ŽCoumarin. ŽCoumarin.

Cytotoxicity, mutation Mutation Cytotoxicity Cytotoxicity, mutation Cytotoxicity Mutation, cell transformation Cell transformation Mutation, mutation spectrum Ž7-Hydroxylation. Ž7-Hydroxylation.

w51x w52,33x w5x w34x w53x w54x w55x w56x w57x w58x

AFB1 Bw axP, CP, sterigmatocystin, 2-AA CP, ifosfamide, ifosfamide mustard AFB1 , CP, NDMA, Bw axP, DMBA Hydroquinone 1,2-OH anthraquinone, 1,2,4-OH anthraquinone Hydroxy anthraquinones Adriamycin, MMC Bw axP, BPD, DMBA, chrysene dihydrodiols, 2-AA, 2-AAF AFB1 , NDBA

Mutation Micronucleus

w3x w21,22x

Cytotoxicity, mutation

w59,20x

w49x w50x

Chromosomal aberration, SCE w24x Micronucleus SCE

w25x w26x

Chromosomal aberration Cytotoxicity Mutation

w27x w28x w29x

M. Sawada, T. Kamatakir Mutation Research 411 (1998) 19–43

24 Table 1 Žcontinued. P450 Žspecies.

Recipient cell lines

Assays Chemicals assayed

Endpoints

CYP2B1 Žrat.

CYP2B4 Žrabbit. CYP2B5 Žrabbit. CYP2B6 Žhuman.

C3Hr10T1r2 C6 Žrat glioma. 9L Žrat gliosarcoma. MCF-7 HK293 Žhuman kidney. HK293 AHH-1 TK q ry

2-AAF CP, ifosfamide CP, ifosfamide CP, ifosfamide ŽAndrostenedione. ŽAndrostenedione. AFB1 CP, ifosfamide

Cytotoxicity Cytotoxicity Cytotoxicity Cytotoxicity ŽHydroxylation. ŽHydroxylation. Cytotoxicity Cytotoxicity

w61x w62x w63x w64x w65x w65x w5,66x w53x

CYP2C10 Žhuman.

NIHr3T3

Ochratoxin A

Cytotoxicity, mutation

w15x

CYP2D6 Žhuman.

AHH-1 TK q ry

NNK, NNN, NNA NNK Tamoxifen, tamoxifen epoxide, toremifene Ochratoxin A ŽClozapine, fluperlapine. NNK

Cytotoxicity, mutation Cytotoxicity, mutation, metabolism Micronucleus

w34x w67x w68x

Cytotoxicity, mutation ŽMetabolism. Metabolism

w15x w69,70x w71x

Cytotoxicity, mutation Cytotoxicity, mutation Cytotoxicity, mutation Micronucleus

w72x w52x w34x w68x

CYP2E1 Žrabbit.

V79 NIHr3T3 CHL-derived CR-119 CHO-K1

NDMA NDMA, NDEA NNK Tamoxifen, tamoxifen epoxide, toremifen Acetaminophen 1,2-Epoxy-3-butene NDMA Acetaminophen NDMA, p-nitrophenol NDMA Acetoaminophen NDMA, p-nitrophenol Ochratoxin A NDMA ŽChlorzoxazone.

Metabolism Metabolism DNA-binding Metabolism Oxidation Oxidation Cytotoxicity Cytotoxicity, mutation Cytotoxicity, mutation Cytotoxicity ŽHydroxylation.

w35x w73x w74x w75x w76x w77x w78x w116x w15x w79x w80x

CYP3A4 Žhuman.

AHH-1 TK q ry

AFB1

Cytotoxicity, mutation, DNA binding Metabolism Micronucleus

w33,5x

Metabolism Cytotoxicity, mutation Cytotoxicity, mutation Cytotoxicity Cytotoxicity, micronucleus ŽMetabolism. Metabolism Cytotoxicity Cytotoxicity Cytotoxicity

w73x w81,82x w15x w83x w84,85x w86x w71x w87x w83x w88x

Cytotoxicity

w89x

NIH 3T3 V79, CHO CHO CYP2E1 Žhuman.

AHH-1 TK q ry

NIHr3T3, RLE PC-12 Hep G2

CYP3A7 Žhuman.

Cyp3a11 Žmouse.

AFB1 Tamoxifen, tamoxifen epoxide, toremifen 1,2-Epoxy-3-butene NIHr3T3 AFB1 Ochratoxin A CHL-derived CR-119 AFB1 , AFG1 , sterigmatocystin V79 AFB1 ŽLisuride, terguride. V79 CHO NNK MCF-7 AFB1 CHL-derived CR-119 AFB1 , AFG1 , sterigmatocystin CHL-derived CR-68 plus AFB1 , IQ, MeIQ, MeIQx plus NAT1, NAT2 CHL-derived CR-119 AFB1

References

w36x w68x

M. Sawada, T. Kamatakir Mutation Research 411 (1998) 19–43

25

Table 1Žcontinued. P450 Žspecies.

Recipient cell lines

Assays Chemicals assayed

Endpoints

Cyp3a13 Žmouse.

CHL-derived CR-119

AFB1

Cytotoxicity

w90x

CYP4B1 Žrabbit.

C3Hr10T1r2

Ipomeanol, 2-AA,2-AF, 2-AAF, 4-aminobiphenyl, 2-aminonaphthalene

Cytotoxicity

w91x

The choice of recipient cells is one of the important factors determining the usefulness of the cell lines stably expressing P450. Among the cell lines in Table 1, AHH-1 and Chinese hamster cell lines have been widely used as recipient cells. AHH-1 was employed by Crespi et al. to develop a battery of cell lines expressing different forms of human P450 and to apply the cells to hprt mutation assays. Chinese hamster cell lines V79, CHO and CHL were used in several laboratories for the expression of P450 present in the liver of rats, mice, monkeys and humans. In addition to these cell lines, a variety of human cells such as SV40-transformed skin fibroblasts, XPA Žxeroderma pigmentosum group A., HeLa, HepG2, MCF-7 Žmammary tumor., BEAS-2B Žbronchial epithelial cells., and HK293 Žkidney. have also been used. Cell lines isolated from mice, including Hepa1c1c derived from hepatoma, BALB 3T3, NIHr3T3, and C3Hr10T1r2, are also successfully transformed. Only a liver-derived epithelial cell line, RLE, and an adrenal pheochromocytoma cell line, PC-12, have been employed as cell lines from rats. Two points should be considered when a certain cell line is chosen as the recipient. One is whether or not the recipient cells possess other factors necessary for the function of the P450. The activity of the P450 is dependent on components constituting an electron transport system in microsomal membranes, e.g., NADPH–cytochrome P450 reductase, cytochrome b5 and NADPH–cytochrome b5 reductase. It is a matter of course that the level of these factors in the cells modifies the activity of the expressed P450. Doehmer et al. w3x, who used V79 cells, mentioned that the reason for using the V79 cells is that the cells lack P450 activity but, nevertheless, contain NADPH–cytochrome P450 reductase. Although it is assumed that the expression of the P450 reductase is maintained in cultured mammalian cells dissociating with the expression of P450 w92x, the

References

level of the reductase activity varies among cell lines and is generally low as compared to the level in the liver w93x. Accordingly, the cell lines which show high activity of the P450 reductase should be candidates as the recipient cells for P450 cDNA. Mapoles et al. w75x used rat-derived PC-12 cells for the expression of human P450, because the level of the P450 reductase in the cells was of the same order of magnitude as in human liver. To fortify the level of the P450 reductase activity, cDNA coding for the reductase was successfully introduced into Chinese hamster CHL cells. This will be described in detail in a later section. P450 is a heme-containing enzyme. Thus, it may be possible to assume that the formation of the functional holo-P450 enzyme is influenced by the ability of cells to synthesize the heme. Another point to be considered in choosing recipient cells is the purpose of establishing the genetically engineered cells. In general, cytotoxicity tests can be done more sensitively than the analysis of drug metabolite in cell lines established so far, while the feasibility of mutagenicity tests at some endpoints depend on the cell types. In many reported cases, the recipient cell lines have been chosen with the reason that the cell line has been frequently used in mutagen screening assays. This is true for AHH-1 and the Chinese hamster cell lines V79, CHO and CHL. In particular, these Chinese hamster cell lines are the most popular in mutagen screening assays with a variety of endpoints such as hprt mutation, ouabain-resistance mutation, chromosomal aberrations, sister chromatid exchanges, and micronucleus induction. Specific cell lines such as BALB 3T3 and C3Hr10T1r2 have been employed exclusively to detect morphological transformation induced by carcinogens. In some cases, the cell lines showing specific characters have been chosen as the recipients. Thompson et al. w47x used a CHO-derived cell

26

M. Sawada, T. Kamatakir Mutation Research 411 (1998) 19–43

line UV5 which defects the incision step of nucleotide excision repair and is highly sensitive to UV irradiation. The sensitivity to some carcinogens was compared between the excision-repair deficient and proficient cell lines stably transfected with a mouse P450 cDNA w47,48x. A similar study was carried out with human XPA Žrepair deficient. and normal skin fibroblast cells w10x. A human bronchial epithelial cell line BEAS-2B, which had been immortalized by the introduction of the SV-40 T-antigen gene, was chosen because the cells were putative progenitor cells of major types of lung cancer and retained many characteristics useful to study human lung carcinogenesis w40x.

3. Mammalian cells transfected with P450 cDNA and application to toxicological studies The P450 Nomenclature Committee w94,95x has classified P450s present in bioorganisms such as bacteria, fungi, plants, invertebrates, and vertebrates. All P450s in a superfamily are then subdivided into families, subfamilies and individual P450s according to the identity of their amino acid sequences. In the nomenclature rule, a P450 gene is shown by ‘CYP’ Ž‘Cyp’ for mice. denoting cytochrome P450, followed by an Arabic number designating the P450 family, a capital letter Ža small letter for mice. designating the subfamily Žwhen two or more exist., and an Arabic number designating the individual gene. Amino acid sequences of the P450s within the same family are ) 40% identical, although there are several exceptions. Mammalian P450s within the same subfamily have ) 55% identity of amino acid sequences, and their genes appear to be located on the same chromosome to compose a gene cluster. It should be noted that this classification is independent on any catalytic properties including substrate specificity of the P450s, and that the P450s from different species can be included in the same family and subfamily. If two or more individual P450s are included within the same family, then a different Arabic number is assigned to designate an individual P450 in principle. However, in the cases of some P450s such as CYP1A1, CYP1A2 and CYP2E1, the same numbers are assigned to the homologues from different animal species. To date, 14 gene families

comprising 26 subfamilies have been found for the mammalian P450s. The members of P450 which have the capacity to metabolize xenobiotic compounds have been classified mainly into the families from 1 to 4. For this reason, stable expression of P450s have been performed focusing on the members of these four P450 families, especially the families from 1 to 3. 3.1. CYP1A subfamily The CYP1 family contains two forms of CYP1A, 1A1 and 1A2, in all mammalian species so far examined, and a new member, 1B1, recently identified in human, rats and mice w95x. CYP1A1 is probably not expressed constitutively but induced by treatment with some agents in the hepatic and extrahepatic tissues. It is known that TCDD and polycyclic aromatic hydrocarbons ŽPAHs. are potent inducers of CYP1A1 in animals, including rats and mice. The induction of CYP1A1 has been proven to occur by a transcriptional regulation involving the Ah Žaryl hydrocarbon. receptor and xenobiotic responsive elements in the 5X upstream region of the gene. The induction of CYP1A1 by 3-MC and PCB is also observed in the liver of cynomolgus monkeys at the level of mRNA w96x. In humans, CYP1A1 has been detected in the placental tissue of cigarette smokers w97x. On the other hand, CYP1A2 is presumably expressed constitutively at a low level in the liver and can be induced by foreign compounds such as TCDD and 3-MC in experimental animals. Benzow axpyrene is known to be a good substrate for CYP1A1. Studies using the enzyme purified from animals and antibodies to the purified enzyme have proven that CYP1A1 is the major enzyme metabolizing this substrate. This procarcinogen is metabolized to give an ultimate active metabolite, 7,8-dihydrodiol-9,10-epoxide, by sequential metabolic processes catalyzed by CYP1A1 and epoxide hydrolase. CYP1A1 is also involved in the N-hydroxylation of 2-acetylaminofluorene as shown by the experiment using Cos-1 cells transiently expressed human CYP1A1 w98x. CYP1A2 is responsible for the metabolic activation of arylamine carcinogens such as 2-naphthylamine and 4-aminobiphenyl. The primary P450 form that can activate carcinogenic heterocyclic amines isolated from pyrolysates of protein

M. Sawada, T. Kamatakir Mutation Research 411 (1998) 19–43

and amino acids is CYP1A2 w99,100x. CYP1A2 also catalyzes the O-deethylation of phenacetin and the N-demethylation of caffeine. As shown in Table 1, many reports on the stable expression of CYP1A have appeared, probably because CYP1A participates in the metabolic activation of well-known mutagens and carcinogens mentioned above. Many cell lines separately expressing the human, rat and mouse CYP1A1 and CYP1A2 and a cell line expressing the cynomolgus monkey CYP1A1 have been successfully established. Human CYP1A1 cDNA was transfected into and expressed in human AHH-1 cells. AFB1-induced mutation at hprt locus was detected in the cells w4,6x. Sister chromatid exchanges ŽSCEs. were also induced by AFB1 in the V79 cells expressing rat CYP1A1 w24x. We introduced monkey CYP1A1 cDNA into CHL cells and established a stably transformed cell line, A-15, which was 25 times more sensitive to the cytotoxicity of AFB1 as compared with parental cells w16x. AFB1-induced gene mutation was seen at the hprt locus in A-15 cells but not in parental CHL cells. Regarding the induction of chromosomal aberrations, the A-15 cells showed a high sensitivity against AFB1 , while in parental CHL cells the aberrations were induced only at very high concentrations of AFB1 w17x. BALB 3T3 cells which had been transfected with the same monkey CYP1A1 cDNA similarly showed a higher sensitivity to AFB1 compared with parental cells w18x. These results coincidentally demonstrate that CYP1A1 is capable of activating AFB1 to a cytotoxic and mutagenic metaboliteŽs.. The cytotoxicity w13x, mutagenicity w19,13x, and micronucleus induction w21x of Bw axP have been sensitively detected in the V79 cells expressing human or rat CYP1A1. Arylhydrocarbon hydroxylase activity is detectable in these transfected cells. BALB 3T3 cells carrying monkey CYP1A1 cDNA were more sensitive to Bw axP than parental cells w18x. Transformation of AHH-1 cells with human CYP1A1 cDNA yielded greater response to Bw axP in the mutation assay w6x. Despite these lines of evidence that CYP1A1 expressed in the transformant cells metabolically activates Bw axP to cause mutations and cytotoxicities, there are some conflicting data compared with the previous results. Monkey CYP1A1 does not seem to activate Bw axP to yield a cytotoxic metaboliteŽs. in CHL cells w16x. States et al. w10x

27

noted that human CYP1A1 expressed in DNA-repair deficient human skin fibroblast XPA and DNA-repair proficient human skin fibroblast did not activate Bw axP to a cytotoxic metaboliteŽs.. It is known that Bw axP is metabolized to Bw axP-7,8-epoxide mainly by CYP1A1 and then converted to Bw axP-7,8-dihydrodiol ŽBPD. by microsomal epoxide hydrolase. Bw axP-7,8-dihydrodiol undergoes further epoxidation by CYP1A1 to form an ultimate carcinogen, Bw axP7,8-dihydrodiol-9,10-epoxide ŽBPDE.. Therefore, it is reasonable to believe that the toxicity of Bw axP occurred depending on the activity of epoxide hydrolase in the recipient cells. Glatt et al. w93x reported that V79 and BALB 3T3 cells possessed the activity of microsomal epoxide hydrolase, although the level was quite low compared with freshly isolated rat hepatocytes. However, it appears that factors other than epoxide hydrolase should also be considered. In fact, AHH-1 cells did not contain any detectable activity of epoxide hydrolase w51x. In addition to DNA damage caused by the reactive intermediate produced by enzymes involving P450, the capacity of cells to repair the DNA damage is also a factor determining the sensitivity of the cells to mutagens such as Bw axP. In this respect, Trinidad et al. w31x showed that Bw axP is highly cytotoxic and mutagenic in repair-deficient CHO cells, but not cytotoxic and less mutagenic in repair-proficient CHO cells, both of which expressed mouse Cyp1a1. Supporting this result, Ž".-Bw axP-trans-7,8-dihydrodiol was more cytotoxic and mutagenic in the repair-deficient XPA cells than in human skin fibroblast cells with normal DNA-repair capacity after both cells were transformed with human CYP1A1 cDNA w10–12x. To account for the complicated data reported so far, many factors, including the expression level of P450, amount of endogenous epoxide hydrolase, DNA-repair capacity of recipient cells, and difference of endpoints, should be considered as factors affecting the sensitivity of the cells to Bw axP. AFB1 is also activated to cytotoxic and mutagenic metabolites by human CYP1A2 expressed in AHH-1 cells w32,33,6x. Gallagher et al. w36x reported that AFB1 was metabolized to AFB1-8,9-epoxide and AFM 1 by microsomes prepared from the AHH-1 cells expressing CYP1A2, and that the formation of these metabolites was strongly inhibited with a selective inhibitor of CYP1A2, furafylline. AFB1 caused

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cytotoxicity and the formation of DNA adducts Žmainly AFB1-N 7 guanine. in human BEAS-2B cells expressing human CYP1A2 w40x. A CHL-derived cell line which expressed simultaneously a guinea-pig NADPH–cytochrome P450 reductase and the human CYP1A2 showed about 300-fold higher sensitivity in the cytotoxicity assay of AFB1 compared with parental cells w39x. Chromosomal aberrations were induced in the cell line at lower concentration of AFB1 ŽSawada et al., unpublished data.. The induction of SCEs was also found in V79 cells transformed with rat CYP1A2 cDNA w24x. A relatively smaller number of papers has appeared on the metabolic activation of cooked foodderived heterocyclic amines by CYP1A2 stably expressed in cultured cells. Battula et al. w45,46x proved by 32 P-postlabeling assays that IQ and MeIQx were activated to form DNA adducts in RLE Žrat liver epithelial. and NIHr3T3 Žmouse embryo fibroblast. cells, which were stably transformed with mouse Cyp1a2 cDNA by the retrovirus-mediated method. According to Thompson et al. w47x, PhIP was highly cytotoxic and mutagenic to CHO-derived repair-deficient cells but less effective to repair-proficient cells, both of which expressed Cyp1a2, lending support to the idea that the repair system as well as the metabolic activation determines the sensitivity of cells to mutagenic chemicals. The experiments performed with IQ showed a tendency similar to PhIP, while the mutation frequency induced by IQ was much lower than that seen with PhIP. The repair-deficient CHO cells expressing Cyp1a2 have been used for the analysis of sequences of PhIP-induced mutations in the adenine phosphoribosyltransferase Ž aprt . gene. It was shown that most of the observed mutations were single-base transversions and that there were three hot-spots w49x. By in vitro studies, it has been clarified that some procarcinogens, including food-derived heterocyclic amines, are activated by certain conjugation enzymes after undergoing oxidation reactions by P450 to yield ultimate reactive forms. In our study, activation systems of IQ and MeIQx to form cytotoxic and mutagenic metabolites were constructed in the CHL-derived cell lines, in which human CYP1A2 and N-acetyltransferase 2 were simultaneously expressed w39x. In the cells expressing CYP1A2 alone, these compounds were not activated to show muta-

genic metabolites. Details of the experiments will be described in a later section. Experiments with a similar idea have been performed by Ellard and colleagues. They introduced rat CYP1A2 cDNA into two V79 cell lines: one was V79-NH possessing endogenous acetyltransferase activity and the other was V79-MZ, which lacked the acetyltransferase. The V79-NH cells expressing CYP1A2 showed a higher level of micronucleus induction by exposure to 2-AA than the V79-MZ cells expressing CYP1A2 w21x. 2-AA also induced gene mutation and SCE in the former cells, while its effect was marginal in the latter cells w29x. Recently, De Groene et al. w15x established NIHr3T3 cell lines expressing human P450s by means of retroviral infection and developed a new mutation assay system. The cells carrying P450 cDNA were transfected with shuttle vectors containing the lacZX gene. After the transfection, the cells were treated with mutagens. The shuttle vectors were then rescued from the cells and again introduced into Escherichia coli DH10B. The mutation frequency was determined by the ratio of white colonies against the total number of colonies. In this mutation assay system, ochratoxin A, which has been reported to induce renal tubular cell tumors in rats, induced mutations at the lacZX gene in the cell lines expressing human CYP1A1 or CYP1A2. V79 cells expressing rat CYP1A1 or CYP1A2 were employed to examine possibility of whether these enzymes are involved in the activation of quinone compounds. Induction of SCE and chromosomal aberrations by seven hydroxy anthraquinones were tested w26,27x. The results did not support the idea that CYP1A was responsible for the activation of the quinone compounds. Similarly, cytotoxicity of adriamycin and mitomycin C was tested with V79 cells expressing CYP1A1 w28x. While adriamycin was equally toxic to the cells expressing CYP1A1 and to parental cells, mitomycin C was less toxic to the former cells. 3.2. CYP2A subfamily Some forms of P450 belonging to this subfamily have been purified andror cloned from the liver of rats, mice, hamsters, rabbits and humans w95x. Among them, two rat enzymes, CYP2A1 and CYP2A2, have

M. Sawada, T. Kamatakir Mutation Research 411 (1998) 19–43

been most extensively studied and characterized. CYP2A1 protein catalyzes the 7a-hydroxylation and the 6 a-hydroxylation of testosterone w101x. CYP2A2 also metabolizes testosterone to form metabolites, mainly at 15 a-hydroxylation w102x. Two enzymes, CYP2A6 and CYP2A7, have been identified to be present so far in human livers. The catalytic activity was examined only for the former one w103x. It was shown by studies using purified enzymes prepared from genetically engineered cells that CYP2A6 catalyzed coumarin 7-hydroxylation and ethoxycoumarin O-deethylation but not testosterone 7a-hydroxylation w103,104x. As shown in Table 1, among the forms of P450 in the CYP2A subfamily, only human CYP2A6 was stably expressed in cultured mammalian cells. Davies et al. w51x and Crespi et al. w52x established a cell line AHH-1 expressing CYP2A6 and observed the increment of sensitivity of the cells to AFB1 , Bw axP, N-nitrosodimethylamine Ž NDMA . , N-nitrosodiethylamine ŽNDEA. w51,52,33x and NNK w34x determined by cytotoxicity andror mutagenicity. The growth rate of the AHH-1 cells expressing CYP2A6 was inhibited by cyclophosphamide and ifosphamide, while the inhibition was much less in the control cells w53x. Both compounds were hydroxylated at 4-position by microsomes prepared from the cells. Tiano et al. w54x established mouse C3Hr10T1r2 cells expressing CYP2A6 by the gene transfer method using retroviruses, and showed that NNK induced ouabain-resistance mutation and morphological transformation in the cells. NDEA also induced morphological transformation in the cells w55x. CHO-derived AS52 cells carrying bacterial gpt gene were stably transfected with CYP2A6 cDNA and the resulting cells showed a high sensitivity in the NNK-induced 6-thioguanine-resistance mutation w56x. PCR amplification of the gpt gene in the mutant cell clones showed that 78.6% of the mutants had putative point mutations and the remaining 21.4% were attributed to deletionsrrearrangements. DNA sequence analysis revealed that 81% of the putative point mutations were G:C to A:T transitions. 3.3. CYP2B subfamily Rat CYP2B1 and CYP2B2 are well known to be induced by phenobarbital ŽPB.. These two enzymes

29

are 97% identical with regard to amino acid sequence deduced from their cDNAs w105x but are distinct from each other in tissue specificity for their constitutive and induced expressions. CYP2B1 is not detectable in the liver of rats without PB-treatment but constitutively expressed in the lung and testis. In the latter tissues, however, CYP2B1 is not inducible by PB. CYP2B2 is constitutively expressed in rat liver, but not in the lung, kidney and testis regardless of PB-treatment w106x. CYP2B3, another member of rat CYP2B, is constitutively expressed but not induced by PB w107x. It is unknown whether human CYP2B6 can be induced by PB or other compounds. Doehmer et al. w3x developed a V79-derived cell line which stably expressed rat CYP2B1, and showed that the cells were sensitive to AFB1 as determined by hprt locus mutation assay. Chromosomal aberrations and SCEs were also induced by AFB1 in the cells w24x. Cyclophosphamide was activated by CYP2B1 and caused cytotoxicity, micronuclei, chromosomal aberrations and SCEs in the cell line w59,20,21,24x. This cell line was used in analysis of CYP2B1-mediated metabolism of androstenedione, testosterone, caffeine and theophylline w108,109x. Cytotoxicity of 2-acetylaminofluorene Ž2-AAF. was seen in mouse C3H10T1r2 cells expressing CYP2B1, developed by Hansen et al. w61x. The C-hydroxylation of 2-AAF was measured using HPLC in the cells expressing CYP2B1. However, the cytotoxicity caused by the metabolic activation of 2-AAF and the metabolic capacity of the cells were seen only in early passage cells. The metabolic capacity was lost in late passages in the absence of G418. The authors noted that one of possible explanations for the loss of enhanced metabolic capacity might be a result of mutations or rearrangements which occurred in prolonged culture. Recently, an application of CYP2B1 expression to cancer chemotherapy has been investigated. Cell lines stably expressing CYP2B1 were established using rat glioma C6 cells w62x, rat gliosarcoma 9L cells w63x, and human breast cancer MCF-7 cells w64x as recipients. The cells expressing CYP2B1 showed a high sensitivity to cyclophosphamide and ifosfamide in vitro. When the tumor cells carrying CYP2B1 were implanted to rats or nude mice, treatment of the animals with cyclophosphamide resulted in efficient inhibition of tumor growth.

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Human CYP2B6 was stably expressed in human AHH-1 cells. The growth inhibition tests showed that the sensitivity of the AHH-1 cells expressing CYP2B6 to AFB1 was much lower than that of the cells expressing human CYP1A2 or CYP3A4 when the relative growth was plotted against AFB1 concentration multiplied by microsomal P450 content w5x. Growth of the cells expressing CYP2B6 was also inhibited by cyclophosphamide and ifosphamide. Both compounds were proven to be 4-hydroxylated by microsomes from the cells w53x. Rabbit CYP2B4 and CYP2B5 were stably expressed in human kidney 293 ŽHK293. cells w65x. Using microsomes prepared from these cells, catalytic activities of CYP2B4 and CYP2B5 toward androstenedione, benzophetamine, 7-ethoxycoumarin and other substrates were assayed. Cytotoxicity and mutagenicity tests using these cell lines have not been reported so far. 3.4. CYP2C subfamily The CYP2C subfamily contains many isoforms and is regarded as a major one existing human and animal livers. Some members of this subfamily are well known to be sex-specific in livers of rats, mice, and Syrian hamsters. Although a large number of P450s belonging to this subfamily have been identified from humans and animals, there are only a few reports on the stable expression of the cDNAs in cultured cells. Human CYP2C8 and CYP2C9 were stably expressed in AHH-1 cells w66x, while data on cytotoxicity or mutagenicity tests using these cell lines have not been reported. De Groene et al. w15x established NIHr3T3 cells expressing human CYP2C10 by means of retroviral infection and applied to the mutation assay, in which a shuttle vector containing the lacZX gene was used as a reporter for mutations. Upon addition of ochratoxin A to the culture medium, the mutation frequency of the cells increased in a dose-dependent manner in the cells expressing CYP2C10. Among forms of P450, CYP2C enzymes are known to be mainly involved in the metabolism of a variety of drugs, and only a few reports have appeared on their roles in the metabolic activation and inactivation of carcinogens. For example, Bw axP 3-hydroxylation was observed with a CYP2C enzyme purified from male rats w110x and with purified human CYP2C8 w111x. Formation of

Bw axP-trans-dihydrodiols from Bw axP was catalyzed by human CYP2C9 which was transiently expressed in Hep G2 cells w9x. 3.5. CYP2D subfamily Forms of P450 in the CYP2D subfamily from rats, mice, cows and humans have been identified. Among the forms, only human CYP2D6 cDNA has been stably expressed in cultured mammalian cells. It is widely known that human CYP2D6 catalyzes the 4-hydroxylation of the antihypertensive drug debrisoquine and N-oxidation of an oxytocic drug sparteine, and that genetic polymorphism in the expression of CYP2D6 is seen. The debrisoquiner sparteine polymorphism has been one of the most extensively studied phenomena in the field of pharmacogenetics. Since a number of drugs are catalyzed by CYP2D6, studies with cells stably expressing CYP2D6 or microsomal fractions prepared from the cells have been concentrated on the metabolism of drugs such as bufuralol w112x, clozapine and fluperlapine w69x, tropisetron and ondansetron w70x; w113x. Only a few papers on cytotoxicity and mutagenicity tests using cells expressing CYP2D have been published so far. A tobacco smoke-derived nitrosamine NNK was cytotoxic and mutagenic in AHH-1 cells expressing CYP2D6 w34,67x. Also, anti-cancer drug tamoxifen increased the frequency of micronuclei in the AHH-1 cells expressing CYP2D6 w68x. Ochratoxin A showed a negative result in the mutation assay using NIHr3T3 cells expressing CYP2D6 w15x. 3.6. CYP2E subfamily CYP2E1 from rats, mice, rabbits, cynomolgus monkeys and humans has been purified andror cloned. Another unique member in this subfamily, CYP2E2, was also found in rabbits. CYP2E1 in rodent livers is not only expressed constitutively but induced by a number of chemicals such as ethanol, acetone, and isoniazid. The induction is not mediated by transcriptional enhancement but caused by the stabilization of CYP2E1 protein after binding with the chemicals w114x. CYP2E1 is able to activate a variety of carcinogens and toxic substances, includ-

M. Sawada, T. Kamatakir Mutation Research 411 (1998) 19–43

ing benzene, carbon tetrachloride, vinyl chloride, trichloroethylene, acrylonitrile and NDMA, with relatively low molecular weights Žreviewed by Koop w115x.. Several lines of cells were stably transformed with human CYP2E1 cDNA and have been used for cytotoxicity and mutagenicity tests. NDMA, NDEA and NNK were shown to be efficiently activated to enhance the cytotoxicity and mutagenicity in the AHH-1 cells expressing human CYP2E1 w72,52,34x. Enhancements of the cytotoxicity and mutagenicity of NDMA were also shown with Chinese hamster V79 cells w116x and CHL cells w79x expressing CYP2E1. Oxidation of NDMA occurred in microsomes from rat PC-12 cells w75x and human HepG2 cells w76x expressing CYP2E1. Formation of DNA adducts with NDMA was confirmed using mouse NIHr3T3 and rat liver epithelial ŽRLE. cells transformed with CYP2E1 cDNA w74x. Tamoxifen increased the frequency of micronuclei in the transformed AHH-1 cells w68x. Cytotoxicity of acetaminophen was seen in the PC12 cells expressing CYP2E1 w75x. The acetaminophen cytotoxicity was increased by addition of buthionine sulfoximine which caused depletion of intracellular glutathione. In agreement with the result, Dai and Cederbaum w78x reported that, when intracellular glutathione was depleted by buthionine sulfoximine, acetaminophen caused severe cytotoxicity in HepG2 cells transformed with the CYP2E1 cDNA. p-Nitrophenol was 10-fold more cytotoxic to the V79 cells expressing CYP2E1 as compared to the parental cells w116x. The CYP2E1-dependent hydroxylation of p-nitrophenol was detectable in the cell homogenates; the activity was enhanced by pretreatment of the cells with ethanol. Relationship between the expression of CYP2E1 and the activation of 1,3-butadien ŽBD. was shown with AHH-1 cells transformed with human CYP2E1 cDNA w73x. BD is known to be carcinogenic in rodents and classified as a probable human carcinogen. BD is metabolized to 1,2-epoxy-3-butene ŽBMO. and then to more mutagenic and carcinogenic metabolite, 1,2:3,4-diepoxybutane ŽBDE.. Studies with microsomes from a series of AHH-1 cell lines expressing human P450 have shown that the oxidation of BMO to BDE is catalyzed by CYP2E1 at a low concentration Ž80 m M. but by

31

CYP3A4 and CYP2E1 at a high concentration Ž5 mM. of BMO w73x. Barmada et al. w80x established Chinese hamster CHO cells stably expressing rabbit CYP2E1 by a unique method as follows. CHO-K1 cells were cotransfected with an expression plasmid carrying rabbit CYP2E1 cDNA and a neomycin-resistance gene as well as a plasmid pFR400. The latter plasmid contained mouse dihydrofolate reductase cDNA with a single amino acid substitution which lowers the affinity of the enzyme to bind to methotrexate. The transfected cells were selected with G418 and then further selected with increasing concentrations of methotrexate. Since the cotransfected plasmids could be ligated within the cells and integrated into a chromosome as a unit, the methotrexate selection caused the amplification of the introduced dihydrofolate reductase cDNA accompanied by the CYP2E1 cDNA. In the paper, a high expression of CYP2E1 mRNA could be accomplished in the cotransfected cells which were selected with methotrexate. CYP2E1 expressed in the resulting transformant cells catalyzed the 6-hydroxylation of the muscle relaxant chlorozoxazone at efficient rates. 3.7. CYP3A subfamily CYP3A enzymes are the most abundantly expressed P450s in the liver of humans and experimental animals. This subfamily is comprised of many isoforms w95x; for example, rat CYP3A1, CYP3A2, CYP3A9, CYP3A18 and CYP3A23; mouse Cyp3a11, Cyp3a13 and Cyp3a16; rabbit CYP3A6; dog CYP3A12; human CYP3A4, CYP3A5 and CYP3A7. Most CYP3A enzymes are known to be induced in the liver of experimental animals by chemicals including PB, dexamethazone, and PCN. The expression of individual CYP3A genes, however, appears to be differently regulated. For example, rat liver CYP3A1 is not constitutively expressed but induced by PCN, while CYP3A2 is constitutively expressed in livers of adult male rats but not induced by PCN w117x. Both CYP3A1 and CYP3A2 can be induced by PB. CYP3A4 is the major form among human liver CYP3A enzymes and is able to metabolize a number of steroids and drugs which are structurally varied Žreviewed by Li et al. w60x.. In addition to these

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chemicals, CYP3A4 activates procarcinogens such as AFB1 w118–122x. and Bw axP 7,8-diol w123,121x. Stable expression of CYP3A4 has been successfully performed using several mammalian cell lines ŽTable 1.. The AHH-1 cells expressing CYP3A4 were applied to mutation assays. The result showed that the cells were highly sensitive to AFB1 w33,5x. In the mutation assay using mouse NIHr3T3 cells stably expressing CYP3A4 with a shuttle vector, the mutagenicity of AFB1 and ochratoxin A were shown, supporting again the role of CYP3A4 in the activation of AFB1 w81,15x. CYP3A4 expressed in the cells catalyzed the conversion of AFB1 to give AFB1-8,9epoxide and AFQ1 w36x. The frequency of micronuclei in the AHH-1 cells was increased by exposure to tamoxifen w68x. Metabolic profiles of testosterone and two ergot derivatives were assayed using Chinese hamster V79 cells stably expressing CYP3A4 w86x. Human CYP3A7 cDNA was cloned from fetal cDNA library in our laboratory w124x. Genomic DNA of CYP3A7 was also isolated and the sequence in a possible transcriptional regulatory region was compared with that of CYP3A4 w125,126x. The deduced amino acid sequence of CYP3A7 was 88% identical with that of CYP3A4. Northern blot analysis with specific oligonucleotide probes revealed that CYP3A7 was expressed specifically in fetal livers and that CYP3A4 was expressed in adult livers w127x. Therefore, it seemed of interest to note the physiological and toxicological roles of CYP3A7. The purified protein of CYP3A7 catalyzed the 16 ahydroxylation of dehydroepiandrosterone 3-sulfate w128x and the mutagenic activation of AFB1 w129x. We established a human breast cancer cell line MCF-7 stably transformed with CYP3A7 cDNA and demonstrated that the new cell line was highly sensitive to AFB1 w87x. Furthermore, CYP3A4 and CYP3A7 cDNAs were independently expressed in Chinese hamster CHL-derived cells carrying guineapig cDNA of NADPH–cytochrome P450 reductase w83x. Both of the cells transformed with CYP3A4 and CYP3A7 cDNAs showed higher sensitivity to AFB1 , AFG1 and sterigmatocystin. The activation of AFB1 by CYP3A4 and CYP3A7 in these cell lines was enhanced by a-naphthoflavone, an activator, and inhibited by troleandomycin, a typical inhibitor of CYP3A enzymes. These results show that the CYP3A

enzymes heterologously expressed in cultured cells activated the mycotoxin. Mouse CYP3A, Cyp3a11 and Cyp3a13, were also stably expressed in CHL-derived cells carrying the guinea-pig P450 reductase cDNA w90,89x. Sensitivity to AFB1 and enhancement by a-naphthoflavone were also observed in the new cell lines. 3.8. CYP4B subfamily CYP4B1 cDNAs for humans, rabbits, rats, and mice have been cloned w95x. Northern blot analysis showed that CYP4B1 was expressed in lungs of rabbits, rats, hamsters, guinea pigs w130x and human w131x. CYP4B1 mRNA was also proven to be present in the liver of rabbits and hamsters but not detectable in the liver of guinea pigs and humans. In rat livers, small amounts of mRNA for CYP4B1 were seen; the level was at least 10-fold less than that found in the lung w130x. A naturally occurring pulmonary toxin, 4ipomeanolw1- Ž 3-furyl. -4-hydroxypentanone x, is known as a substrate for rabbit CYP4B1. Conversion of 4-ipomeanol to metaboliteŽs. covalently bound to proteins is catalyzed by rat lung microsomes more effectively than by liver microsomes w132x. 4Ipomeanol was activated by two forms of P450 purified from rabbit lungs, one of which corresponded to CYP4B1 w133x. Czerwinski et al. w134x demonstrated that there was a remarkable difference between rabbit and human CYP4B1 in the capacity to activate 4-ipomeanol. They measured the amounts of metabolites of 4-ipomeanol bound to DNA in HepG2 cells transiently expressing 12 forms of human P450 or rabbit CYP4B1 separately. Rabbit CYP4B1 was the most active enzyme Ž180-fold over the control level. among all P450s tested, while human CYP4B1 had very low activity Ž2-fold over the control level.. Rabbit CYP4B1 was stably expressed in mouse C3Hr10T1r2 cells using retroviral vectors w91x. 4Ipomeanol was highly toxic to the cells expressing CYP4B1. This result coincides with the above-mentioned reports in which 4-ipomeanol is activated by rabbit CYP4B1. Aromatic amine procarcinogens, 2AA, 4-aminobiphenyl, 2-aminonaphthalene, 2-AF and 2-AAF were also examined using the new cell line. It had been previously shown that mutagenic

M. Sawada, T. Kamatakir Mutation Research 411 (1998) 19–43

activations of 2-AA, 2-AF and 2-AAF were possibly associated with rabbit hepatic CYP4B1 w135x. In fact, transiently expressed rabbit CYP4B1 could activate 2-AF w134x. However, only 2-AA exhibited clear cytotoxicity to the C3Hr10T1r2 cells expressing CYP4B1 w91x. As possible reasons for the negative results in the cytotoxicity of 2-AF and 2-AAF, the authors discussed this by pointing out the possibility that the capacities of sulfation and deacetylation needed in the activation of these mutagens might not be sufficient in C3Hr10T1r2 cells and that a metabolite showing cytotoxicity might be different from that showing mutagenicity. Cell lines expressing human CYP4B1 have not yet been reported to the author’s knowledge. 3.9. Other CYPs Cell lines stably expressing P450s belonging to families 11 and 19 were established using cultured mammalian cells. These P450s are physiologically important in that they are needed for the synthesis of the adrenal corticoids or estrogens. Thus, rat CYP11B1 and CYP11B2 cDNAs were stably expressed in rat Leydig tumor cell line MA-10 w136x. Rat CYP11B1 Ž11 b-hydroxylase. and CYP11B2 Žaldosterone synthase. are present in mitochondria in zona fasciculata-reticularis and zona glomerulosa, respectively, of adrenal cortex. The cell line MA-10 was chosen as a recipient, since this cell line produced adrenodoxin and adrenodoxin reductase necessary for the function of mitochondrial P450. The resulting cells expressing CYP11B1 converted deoxycorticosterone ŽDOC. to yield corticosterone, 18OH-DOC and a small amount of 18-OH-corticosterone. On the other hand, the cells expressing CYP11B2 converted DOC to corticosterone, 18OH-corticosterone, aldosterone and a small amount of 18-OH-DOC. CYP19, a single member of family 19, catalyzes the conversion of C 19 androgens to C 18 estrogens and is commonly referred to as aromatase. This enzyme is found in microsomes from the ovary, placenta, testes, adipose tissue, breast tumor, brain and skin. Human placenta CYP19 cDNA was stably transfected into MCF-7 Žhuman breast cancer cells., HBL-100 Žnoncancerous breast cells. and CHO cells w137x. Growth of the MCF-7 cell expressing CYP19

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was stimulated by androstenedione w138x. In order to analyze the key protein sequence to show the enzymatic activity, amino acid substitution was introduced by site-directed mutagenesis. These modified cDNA sequences were stably expressed in CHO cells w139,140x.

4. Coexpression of P450 and other enzymes To develop cell lines possessing a high capacity to activate procarcinogens, several factors besides P450 enzyme must be considered. These factors can be divided broadly into two groups. One group contains components of the electron transfer system in microsomal membranes, i.e., NADPH–cytochrome P450 reductase, NADH–cytochrome b5 reductase and cytochrome b5 . Another group contains certain drug-metabolizing enzymes such as epoxide hydrolase and conjugation enzymes, since these enzymes are also required in the sequential processes of metabolic activation to form ultimate carcinogens which are capable of binding to DNA. Amounts of these enzymes are not always sufficient in cultured mammalian cells. Therefore, it is expected that coexpression of cDNAs for P450 and other enzymes provides a high ability to activate procarcinogens. 4.1. NADPH – cytochrome P450 reductase, NADH–cytochrome b5 reductase and cytochrome b5 NADPH–cytochrome P450 reductase mediates an electron transfer from NADPH to P450. Thus, this enzyme is essential for the catalytic function of P450. Activity of the reductase is detectable even in long-term cultured cells regardless of their origins, but the level is generally low as compared with liver tissues w92,93x. We used a Chinese hamster cell line CHL as a recipient for the transfection of P450 cDNA. The CHL cells have been widely used in mutagen screening assays w141,142x, but have essentially no activity of P450. To establish CHL cells expressing the reductase together with P450, the cells were first transfected with an expression vector carrying CYP1A1 cDNA of the cynomolgus monkey. The resulting cell line Ždesignated A-15. showed a higher Ž25-fold. sensitivity in the cytotoxicity assay with AFB1 as compared with parental CHL cells

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M. Sawada, T. Kamatakir Mutation Research 411 (1998) 19–43

w16x. As a second step, A-15 cells were further transfected with cDNA encoding guinea-pig NADPH–cytochrome P450 reductase. Three cell lines Ždesignated AR-10, -13, -18. showing high expression of mRNA for the P450 reductase were cloned w17x. Specific activities of the microsomal P450 reductase determined by using cytochrome c as an electron acceptor were 110, 180, and 75 nmol miny1 mgy1 protein for AR-10, -13, and -18 cells, respectively, while the value was 16 nmol miny1 mgy1 protein for the parental A-15 cells. The introduction of the reductase cDNA resulted in enhanced sensitivity to AFB1. Thus, IC 50 values obtained from three AR- cell lines were about 10-times lower than those from A-15 cells ŽFig. 1.. The cell line CR-68, which was established by introducing the same expression vector carrying the P450 reductase cDNA directly into the original CHL cells, showed enhanced activity of the P450 reductase Ž129 nmol miny1 mgy1 .. As expected, the CR-68 cells did not show enhancement of sensitivity to AFB1 , indicating that the P450 reductase alone did not activate the mycotoxin. These results again demonstrate that the coexpression of cDNAs for P450 and the P450 reductase is necessary for P450 to show its full activity in cultured mammalian cells. The cell lines CR-68 or CR-119 stably expressing the guinea-pig P450 reductase have been used as a recipient of other P450s in further studies: human CYP1A2 w39x, CYP2E1 w79x, CYP3A4 and CYP3A7 w83x, and

mouse Cyp3a11 and Cyp3a13 w89,90x. Recently, Schnieder et al. w85x also established V79 cells stably coexpressing human CYP3A4 and P450 reductase cDNAs, and showed that the cells were much more sensitive to AFB1 than the cells expressing CYP3A4 alone in cytotoxicity and micronucleus formation assays. Although the exact mechanism is not known, cytochrome b5 potentiates the reactions mediated by microsomal P450. The enhancement of drug oxidation by cytochrome b5 has been accounted for by the potentiation of the flow of the second electron from NADH to P450 via NADH–cytochrome b5 reductase and cytochrome b5 or from NADPH to P450 via NADPH–cytochrome P450 reductase and cytochrome b5 . The effect of cytochrome b5 appears to vary with P450 forms and substrates w143x. A reconstitution study showed that nifedipine oxidation by the purified human CYP3A enzyme was inhibited by anti-cytochrome b5 antibody and enhanced by purified human liver cytochrome b5 w144x. The effects of cytochrome b5 on the activity of CYP3A7 in cultured cells were studied in our laboratory. We introduced cDNAs coding for human cytochrome b5 and NADH–cytochrome b5 reductase into the CR-119 cells which were expressing CYP3A7 ŽHashimoto et al., unpublished results.. As compared to the parental cells, the coexpression of the cDNAs brought a 5-fold increase in NADH-dependent ferricyanide reductase activity and a 2-fold increase in sensitivity to AFB1 , suggesting that the electron transfer mediated by the b5 reductase and cytochrome b5 was constructed in the cells. 4.2. Microsomal epoxide hydrolase (mEH)

Fig. 1. Sensitivity to AFB1 of CHL-derived cell lines expressing monkey CYP1A1 andror guinea-pig NADPH–cytochrome P450 reductase Žsee text.. Cells Ž8=10 4 . were seeded in a 60-mm dish and, on the next day, treated with AFB1 . After a 48-h exposure, the relative survival rate was measured by staining the cells on the dishes with crystal violet. `, CHL; v, A-15; B, AR-10; I, AR-13; ', AR-18; ^, CR-68 cells.

Epoxide hydrolases, which catalyze the conversion of epoxides to glycols, play an important role in the activation and detoxification of xenobiotic compounds. The role of mEH in the activation of polycyclic aromatic hydrocarbon ŽPAH. procarcinogens such as Bw axP has been particularly studied. Bw axP and some other PAHs are primarily activated by P450-dependent epoxidation at bay regions, and then the epoxide is hydrated by mEH. Glatt et al. w93x measured the mEH activity in many cell lines, using w 3 Hx-Bw axP-4,5-oxide as a substrate, and showed that the mEH activity varied among cell lines; variation

M. Sawada, T. Kamatakir Mutation Research 411 (1998) 19–43

of the activity was greater than that of NADPH–cytochrome P450 reductase and glutathione-S-transferase. A cDNA-mediated stable expression of human mEH in combination with P450 was accomplished by Davies et al. w51x. An expression vector containing human CYP2A6 and mEH cDNAs was transfected into cells of an AHH-1 subline L3, in which CYP1A1 activity had been enhanced by mutation and selection. The resulting cells, designated MCL-1, were shown to possess mEH activity, which was not detectable in the parental cells. The MCL-1 cells showed increased sensitivity to Bw axP in the mutation assay. The analysis of the metabolite revealed that the overall metabolism of Bw axP was elevated by the expression of the P450 and mEH cDNAs in the MCL-1 cells and that the major metabolites were dihydrodiols in the MCL-1 cells but phenols in the parental AHH-1 cells. Bw axP metabolism was also analyzed using microsomes from the AHH-1-derived cells which were stably transformed with human CYP1A1 and CYP1A2 cDNAs with and without mEH cDNA w9x. CYP1A1 produced phenols Ž3-, 7-, 9-., quinones Ž1,6-, 3,6-, 6,12-. and a minor amount of trans-dihydrodiols Ž4,5-, 7,8-, 9,10-.. Microsomes from cells coexpressing CYP1A1 and mEH produced lesser amounts of 7- and 9-phenols and a remarkably increased amounts of 7,8- and 9,10-diols. Formation of the Bw axP metabolites was enhanced by introduction of CYP1A2 cDNA, and no substantial effects of simultaneous introduction of mEH cDNA were observed. Human mEH cDNA was also introduced into AHH-1 cells together with cDNAs of multiple human P450s w145x. Properties of the resulting cell line will be mentioned in a later section. 4.3. N-acetyltransferase Enzymes catalyzing the conjugation reactions, such as acetylation, sulfation, glucuronidation and glutathione conjugation are also playing key roles in the detoxification of reactive intermediates produced by the metabolic activation of chemicals. However, it is now known that such enzymes are also involved in the activation of aromatic amine procarcinogens. Heterocyclic amines derived from cooked foods, for example, are assumed to be N-hydroxylated mainly

35

by CYP1A2. The resulting N-hydroxylated metabolites are further converted to yield O-acetyl or Osulfonyl esters which readily react with protein and DNA Õia the formation of arylnitrenium ions w146,147x. The O-acetylation is mediated by acetyl CoA-dependent N-acetyltransferase ŽNAT.. To date two molecular forms of the enzyme, NAT1 and NAT2, have been found in the human liver. NAT1 and NAT2 share 80% homology in cDNA-deduced amino acid sequences, while differences in substrate specificity between these two NATs have been noted. To clarify the roles of CYP1A2 and NAT in the activation of heterocyclic amines within cells, we developed cell lines which stably expressed human CYP1A2 and NAT alone or in combinations, using the above-mentioned CR-68 cells as the recipient w39x. The expression of the NATs was monitored using p-aminobenzoic acid Žfor NAT1. and sulfamethazine Žfor NAT2. as typical substrates. No endogenous activities for either substrate were detected in the parental cells. Newly developed cell lines were as follows: A2R-5 ŽCYP1A2 into CR-68 cells., ANM-13 ŽNAT1 into A2R-5 cells., ANP-25 ŽNAT2 into A2R-5 cells., CNM-4 ŽNAT1 into CR-68 cells., and CNP-40 ŽNAT2 into CR-68 cells.. Using these new cell lines, cytotoxicity and mutagenicity of IQ and MeIQx were examined w39x. In the ANP-25 cells expressing CYP1A2 and NAT2 in addition to the P450 reductase, these heterocyclic amines showed remarkably higher cytotoxicity and mutagenicity ŽFig. 2.. In the ANM-13 cells expressing CYP1A2 and NAT1 together with the P450 reductase, on the contrary, these compounds showed only a low level of cytotoxicity and actually no mutagenicity. A2R-5 cells which expressed CYP1A2 and the P450 reductase did not activate the heterocyclic amines to induce cytotoxicity and mutagenicity. From these results, it was confirmed that human CYP1A2 in combination with NAT2 could efficiently activate IQ and MeIQx by sequential reactions in the cells, as proposed by in vitro studies. In similar experiments, we found that 2-AA was also activated in only ANP-25 cells to produce cytotoxic and a mutagenic metaboliteŽs. Žunpublished data.. Thompson et al. w50x reported results of experiments in which human NAT2 and Salmonella Oacetyltransferase ŽOAT. were separately expressed in the UV5P3 cells which had been previously devel-

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M. Sawada, T. Kamatakir Mutation Research 411 (1998) 19–43

Fig. 2. Mutagenic activation of IQ ŽA. and MeIQx ŽB. in the cell lines expressing human CYP1A2 and NAT. The cell line A2R-5 Ž`. was established by transfecting CYP1A2 cDNA into CR-68 cells. The cell lines ANM-13 ŽB. and ANP-25 Ž'. were established from A2R-5 cells by the transfection of NAT1 or NAT2 cDNA, respectively. Top, relative survival rates after a 24-h treatment with the test compounds determined by the colony formation assay. Bottom, mutant frequencies expressed as the number of 6-thioguanine-resistant cells per 10 6 clonable cells.

oped by introducing mouse Cyp1a2 cDNA into the CHO-derived repair-deficient cell line UV5 w47x. The new cell lines, 5P3NAT2 Žwith Cyp1a2 and NAT2. and 5P3YG Žwith Cyp1a2 and Salmonella OAT., showed extremely high sensitivity to IQ. There was no marked differences in the sensitivity between both new cell lines. We established cell lines coexpressing human fetus-specific CYP3A7 and NAT, and examined the capacity of the cells for sensitivity to heterocyclic amines w88x. The sensitivities to IQ, MeIQ, and MeIQx were enhanced Ž4-, 30-, and 14-fold, respectively, as compared with parental CR-68 cells. by the introduction of CYP3A7 and NAT2 Žbut not NAT1. cDNAs into the cells. These results are noteworthy from the point of view of developmental toxicology, since the capacity to N-acetylate p-aminobenzoic acid and procainamide Ža substrate for NAT2. is at a considerable level in human fetal livers w148,149x.

5. Future perspectives on the use of genetically engineered cells The genetically engineered cells expressing a specific form of P450 will become a powerful tool in

determining the form of P450 catalyzing the activation of a particular chemical to a cytotoxic or mutagenic metaboliteŽs.. However, to specify the formŽs. of P450 involved in the activation of a certain chemical, a set of cell lines individually expressing the different P450 enzyme is needed. As shown in Table 1, several sets derived from AHH-1, V79 and NIHr3T3 cells are usable for this purpose. While analysis for the above-mentioned purpose is possible at present, the method using many cell lines is laborious and costly as a screening assay. An alternative strategy for mutagen screening is to use a cell line which coexpresses multiple P450 enzymes simultaneously. Crespi et al. w145x introduced two distinct expression vectors which contained human cDNAs encoding CYP1A2, CYP2A6, CYP2E1, CYP3A4 and mEH into AHH-1-derived cell line L3 possessing a higher endogenous CYP1A1 activity. In the resulting MCL-5 cells, enzyme activities of the introduced P450s and mEH were detected. Mutagenicity tests at the hprt and tk loci showed that the MCL-5 cells were much more sensitive to Bw axP, 3-MC, NDMA, NDEA, AFB1 and 2-AAF than the AHH-1 cells which showed only low level of endogenous CYP1A1 activity w145x. Regarding micronucleus formation, the MCL-5 cells responded to a number of promutagens, including AFB1 , sterigmatocystin, Bw a xP, dibenzw a, h xanthracene, 3-MC, cyclophosphamide, NDMA, NDEA, MeIQx, benzidine, 2-AF, 2-AAF, benzene, tamoxifen, and omeprazole w150x. These results indicate that the MCL-5 cells simultaneously expressing multiple forms of P450 are useful in screening assays for a wide range of mutagens. MCL-5 cells are the sole example expressing multiple forms of P450 to our knowledge. When genetically engineered cells are used for genotoxicity assays, one should keep in mind a chromosomal instability due to integrated DNA. Ellard et al. w151x observed in micronucleus assay that a spontaneous frequency of micronuclei in V79-derived SD1 cells expressing rat CYP2B1 was higher than that in parental V79 cells, and that a marker chromosome with elongated p-arms was found in SD1 cells but not in V79 cells. Fluorescence in situ hybridization revealed that this marker chromosome was associated with DNA amplification at the integration site of the transfected CYP2B1-expression

M. Sawada, T. Kamatakir Mutation Research 411 (1998) 19–43

vector. There is a possibility that the integration of externally added DNA may stimulate chromosomal instability in the absence andror presence of genotoxic materials. In such a situation, specific P450 inhibitors might be useful tools to confirm that the effects of test compounds observed in genotoxic assays are actually mediated by increase of catalytic activity in the cells. Known in vitro specific inhibitors of P450 have been shown to inhibit the P450 activity in genetically engineered cells. aNaphthoflavone inhibited the cytotoxicity of AFB1 in the cells expressing monkey CYP1A1 w16x and human CYP1A2 w39x. Fluvoxamine, sulfaphenazole, quinidine, and 3-amino-1,2,4-triazole inhibited the function of CYP1A2 w152x, CYP2C10 w15x, CYP2D6 w67x, and CYP2E1 w79x, respectively. Ketoconazole w81,15x and tiamulin w82x were shown to be effective against CYP3A4. In future studies, stable expression systems for P450 enzymes are expected to be used more comprehensively and more deeply to predict human genotoxicity of chemicals. For example, the systems will be valuable for a detailed comparison among a human P450 molecule and its animal counterparts. Although the species-difference in the metabolic activation of promutagensrprocarcinogens still remains to be studied, only a few reports in which a comparison was carried out using the stable expression system have been published. The stable expression systems will also be applied to the comparative study for human polymorphic P450 forms. This may provide valuable information concerning relationships between the genetic polymorphisms of P450 and cancer risks. In addition, coexpression systems for P450s and other enzymes which are possibly involved in the activation or inactivation of toxic substances are also expected to be developed for the estimation of the toxicological significance of the enzymes. These trials will provide valuable new information to predict the human toxicity of chemicals. References w1x Y. Li, T. Yokoi, R. Kitamura, M. Sasaki, M. Gunji, M. Katsuki, T. Kamataki, Establishment of transgenic mice carrying human fetus-specific CYP3A7, Arch. Biochem. Biophys. 329 Ž1996. 235–240.

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w2x T. Pineau, P. Fernandez-Salguero, S.S.T. Lee, T. McPhail, J.M. Ward, F.J. Gonzalez, Neonatal lethality associated with respiratory distress in mice lacking cytochrome P4501A2, Proc. Natl. Acad. Sci. U.S.A. 92 Ž1995. 5134– 5138. w3x J. Doehmer, S. Dogra, T. Friedberg, S. Monier, M. Adesnik, H. Glatt, F. Oesch, Stable expression of rat cytochrome P-450IIB1 cDNA in Chinese hamster cells ŽV79. and metabolic activation of aflatoxin B1 , Proc. Natl. Acad. Sci. U.S.A. 85 Ž1988. 5769–5773. w4x C.L. Crespi, R. Langenbach, K. Rudo, Y.-T. Chen, R.L. Davies, Transfection of a human cytochrome P-450 gene into the human lymphoblastoid cell line, AHH-1, and use of the recombinant cell line in gene mutation assays, Carcinogenesis 10 Ž1989. 295–301. w5x C.L. Crespi, B.W. Penman, F.J. Gonzalez, H.V. Gelboin, M. Galvin, R. Langenbach, Genetic toxicology using human cell lines expressing human P-450, Biochem. Soc. Trans. 21 Ž1993. 1023–1028. w6x B.W. Penman, L. Chen, H.V. Gelboin, F.J. Gonzalez, C.L. Crespi, Development of a human lymphoblastoid cell line constitutively expressing human CYP1A1 cDNA: substrate specificity with model substrates and promutagens, Carcinogenesis 15 Ž1994. 1931–1937. ¨ Savas, D.C. Spink, J.F. Gierthy, C.R. w7x M. Christou, U. Jefcoate, Co-expression of human CYP1A1 and a human analog of cytochrome P450-EF in response to 2,3,7,8-tetrachloro-dibenzo-p-dioxin in the human mammary carcinoma-derived MCF-7 cells, Carcinogenesis 15 Ž1994. 725– 732. w8x M. Shou, K.R. Korzekwa, K.W. Krausz, C.L. Crespi, F.J. Gonzalez, H.V. Gelboin, Regio- and stereo-selective metabolism of phenanthrene by twelve cDNA-expressed human, rodent, and rabbit cytochromes P-450, Cancer Lett. 83 Ž1994. 305–313. w9x M. Shou, K.R. Korzekwa, C.L. Crespi, F.J. Gonzalez, H.V. Gelboin, The role of 12 cDNA-expressed human, rodent, and rabbit cytochromes P450 in the metabolism of benzow axpyrene and benzow axpyrene trans-7,8-dihydrodiol, Mol. Carcinogen 10 Ž1994. 159–168. w10x J.C. States, T. Quan, R.N. Hines, R.F. Novak, M. RungeMorris, Expression of human cytochrome P450 1A1 in DNA repair deficient and proficient human fibroblasts stably transformed with an inducible expression vector, Carcinogenesis 14 Ž1993. 1643–1649. w11x T. Quan, J.J. Reiners Jr., A.O. Bell, N. Hong, J.C. States, Cytotoxicity and genotoxicity of Ž".-benzow axpyrene-trans7,8-dihydrodiol in CYP1A1-expressing human fibroblasts quantitatively correlate with CYP1A1 expression level, Carcinogenesis 15 Ž1994. 1827–1832. w12x T. Quan, J.J. Reiners Jr., S.J. Culp, P. Richter, J.C. States, Differential mutagenicity and cytotoxicity of Ž" .benzo w a xpyrene-trans-7,8-dihydrodiol and Ž" .-antibenzow axpyrene-trans-7,8-dihydrodiol-9,10-epoxide in genetically engineered human fibroblasts, Mol. Carcinogen 12 Ž1995. 91–102. w13x W.A. Schmalix, H. Maser, F. Kiefer, R. Reen, F.J. Wiebel, ¨

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