Mutation Research 411Ž. 1998 19±43

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

Minoru Sawada a,), Tetsuya Kamataki b a DiÕision of EnÕironmental Hygiene, Hokkaido College of Pharmacy, Katsuraoka-cho 7-1, Otaru, Hokkaido 047-02, Japan b DiÕision of Drug , Faculty of Pharmaceutical Sciences, Hokkaido UniÕersity, Sapporo, Hokkaido 060, Japan

Abstract

Genetically engineered cells transiently and stably expressing Ž.P450 , a key for biotrans- formation 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 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-acetyltrans- ferase, were established. These cells were highly sensitive to some carcinogenic heterocyclic amines. In genetic toxicology,

wx Abbreviations: 2-AA, 2-aminoanthracene; 2-AAF, 2-acetylaminofluorene; AFB1111 , aflatoxin B ; AFG , aflatoxin G ; B a P, benzowxa pyrene; BPD, benzo wxa pyrene dihydrodiol; BPDE, benzo wxa pyrene dihydrodiol epoxide; CP, cyclophosphamide; CYP or P450, cytochrome P450; DMBA, 7,12-dimethylbenzwxa anthracene; hprt, hypoxanthine guanine phosphoribosyl ; IQ, 2-amino-3- methylimidazowx 4,5-f quinoline; 3-MC, 3-methylchoranthrene; mEH, microsomal epoxide ; MeIQ, 2-amino-3,4- dimethylimidazowx 4,5-f quinoline; MeIQx, 2-amino-3,8-dimethylimidazo wx 4,5-f quinoxaline; MMC, mitomycin C; NAT, N-acetyltransferase; NDBA, N-nitrosodibutylamine; NDEA, N-nitrosodiethylamine; NDMA, N-nitrosodimethylamine; NNA, 1-Ž.ŽN-methyl-N-nitroso -1- 3- pyridyl.Ž.Ž. -4-butanol; NNK, 4- methylnitrosamino -1- 3-pyridyl -1-butanone; NNN, N-nitrosonornicotine; OAT, O-acetyltransferase; PB, phenobarbital; PCB, polychlorinated biphenyl; PhIP, 2-amino-1-methyl-6-phenylimidazowx 4,5-b pyridine; SCE, sister chromatid exchange; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; tk, thymidine kinase; Trp-P-2, 3-amino-1-methyl-5H-pyridowx 4,3-b indole; XPA, xeroderma pigmentosum group A ) Corresponding author.

1383-5742r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S1383-5742Ž. 98 00005-2 20 M. Sawada, T. KamatakirMutation Research 411() 1998 19±43 such a coexpression system for two or more will provide useful materials which mimic in vivo activation systems. q 1998 Elsevier Science B.V. All rights reserved.

Keywords: Genetically engineered cell; Cytochrome P450Ž. CYP ; Stable expression system; Metabolic activation of mutagenrcarcinogen; Cytotoxicity; Mutagenicity; NADPH±cytochrome P450 reductase; Epoxide hydrolase; N-acetyltransferase

1. Introduction relative risk of a chemical in an in vitro toxicology testing. Thus, this review will focus on the useful- Cytochrome P450 Ž.P450 or CYP is a heme-con- ness of genetically engineered cells in the in vitro taining enzyme widely distributed from bacteria to toxicology testings. mammals, and catalyzes the oxidative and reductive Mammalian cultured cells have been used as im- metabolism of a wide variety of compounds includ- portant tools for approaching cellular and molecular ing endogenous as well as exogenous compounds. mechanisms of chemical toxicity. A variety of sys- Mammalian P450 present in liver microsomes is tems using immortalized cell lines have been devel- characteristic of its nature in metabolizing exogenous oped in the field of toxicological testings, such as compounds including drugs, pesticides, environmen- mutagenicity assays. One of the critical points in tal pollutants, mutagens, and carcinogens. these assay systems is their capacity to metabolize In recent studies of P450s, excellent results have drugs, since many toxicants, mutagens and carcino- been brought about by approaches based on gene gens require metabolic activation to react with intra- technology, including prompt identification of new cellular macromolecules and to exert their toxicolog- molecular forms by cDNA cloning, analysis of the ical consequences. Most of the established cell lines gene regulation mechanism leading to `enzyme in- possess actually no or, if any, very low levels of duction', development of a gene diagnosis of poly- P450 activities. This is an inevitable tendency in morphic P450s, and the generation of animals carry- cultured cells, even in the cells derived from livers. ing transgenewx 1 or lacking a certain P450 gene by Thus, to complement the lack of an enzymeŽ. s in- gene-knockout technologywx 2 . Catalytic functions of volved in the metabolic activation of chemicals, two an individual form of P450 can be analyzed by the metabolic activation systems have been added to the method of heterologous expression of its cDNA. In assays. One is an externally added cell-mediated these experiments, the expression vectors which con- system, in which target cells are co-cultivated with tain the cDNA encoding a specific P450 are intro- primary-cultured hepatocytes. The other is a method duced to recipient cellsŽ bacteria, yeast, insect cells, in which so-called S9 mix prepared from liver ho- cultured mammalian cells. by an appropriate trans- mogenates is added to the culture medium, expecting fection method. The cDNA expression systems using the activation of a test compound by enzymes in the cultured mammalian cells are divided into transient S9 mix. Because of feasibility, the latter method has and stable types. In the transient expression system, been adopted in the standard assays of mutagens. although the expression period and lives of the cells Although the S9 mix method has been widely used are generally short, large amounts of the enzymes and recognized to be the most common, this method can be produced in the transfected cells. Thus, the still has some disadvantages to be resolved. First, the microsomal fractions from the cells transiently ex- S9 mix itself is known to decrease the viability of pressing a specific P450 are suitable for investiga- the cultured cells. Therefore, the periods of exposure tion of its enzymatic properties such as substrate and the concentrations of the S9 mix added to the specificity and kinetic parameters. On the other hand, assays are not necessarily suitable to obtain the stable expression systems are, in general, capable of highest metabolic activation. Second, since the test producing lower levels of enzyme , while the compounds are metabolized in the culture medium continues stably for longer cell-gen- but not inside the cells, it is possible that the short- erations. The stable expression system including lived metabolites with high chemical reactivity are mammalian cells has made it possible to evaluate the expected to bind to the surface of cells, and only a M. Sawada, T. KamatakirMutation Research 411() 1998 19±43 21 portion of the metabolites can penetrate through cell phase II enzyme such as N-acetyltransferase, and to membranes to reach and react with target macro- discuss the advantages and disadvantages of their use molecules to show the toxicities. The S9 used in in cytotoxicity and mutagenicity assays. routine mutagen assays is, in many cases, prepared from the liver of rats pretreated with some P450-in- ducing agentsŽ e.g., b-naphthoflavone plus pheno- barbital, or PCB alone. . Considering species differ- 2. Expression systems and cell lines used as ences in the activities of drug-metabolizing enzymes, recipients the use of human liver S9 is desired to predict human risks of test compounds, but it is virtually The stable expressions of P450 in cultured mam- impossible to use human livers for such routine malian cells reported so far are listed in Table 1. To research. The population of human enzymes may establish the cells which stably express P450, avail- differ among individual liver specimens by many able expression vectors containing cDNA or P450 factors such as induction by drug intake and the gene are introduced to recipient cells simultaneously genetic polymorphism of certain enzymes. with vectors containing a drug-resistance gene by an Genetically engineered mammalian cell lines sta- appropriate transfection method. Both the P450 bly expressing P450 enzymes were first established cDNA and the drug-resistance gene may be included by Doehmer et al.wx 3 . Since then, many cell lines in the same vector. The cells transfected with the have been established and the properties reported by vectors are treated with a drug to select cells which many authors. These new cell lines have been evalu- carry the drug-resistance gene, assuming that the ated as target cells in mutation assays. The advan- vector containing the drug-resistance gene is intro- tages of these cell lines may be as follows:Ž. 1 The duced into the cells together with the vector contain- most valuable point is that a series of the process ing the P450 gene. Then, the cell clones resistant to from the metabolic activation of the test compound the drug are isolated and examined for P450 expres- to the consequences of cytotoxicity andror muta- sion. In the system of a human lymphoblastoid cell genicity occur entirely in the cell. There is no assur- line AHH-1Ž. Table 1 , which had been transformed ance that the extracellular activations by the S9 mix with Epstein±Barr virus, the expression vectors always yield information exactly the same as one which contain P450 cDNA and the hygromycin-re- occurring intracellularly. Some factors, including cell sistance gene exist extrachromosomally, and are membranes which may prevent the penetration of replicated because of the presence of a replication active metabolites, oxygen tension which may affect origin of Epstein±Barr virus in the vectorwx 4 . In the active oxygen production as well as the oxidative stable expression systems in Table 1 other than metabolism of chemicals, and coenzyme concentra- AHH-1, the P450 cDNA and drug-resistance gene tions necessary for conjugation reactions, may cause are assumed to be integrated into chromosomal DNA differences from intracellular situations.Ž. 2 As stated of the recipient cells, since the vectors used in those earlier, genetically engineered cells can be expected studies cannot be replicated extrachromosomally. to show higher sensitivity to short-lived active Gene transfer mediated by retroviruses has also metabolites, since the metabolites can directly react been employed to develop a stable expression system with target macromolecules in the same cells.Ž. 3 In wx44 . Briefly, a retroviral vector containing P450 studies done to ascertain the roles of human P450s cDNA and neomycin-resistance gene Žneo r . is trans- in the activation of mutagens, the cells carrying each fected into packaging cells by a calcium phosphate human P450 are particularly useful. A panel study coprecipitation method. A few days after the trans- using cell lines carrying each human P450 DNA fection, the supernatant fraction of the culture may be conducted to determine a specific formŽ. s of medium, which contains the recombinant virus carry- P450 involved in the activation. ing P450 cDNA and neo r, is collected, filtered and Thus, the aims of this review are to briefly sum- used to infect target cells. The infected target cells marize reports on the establishment of the cell lines are selected with G418. The resistant colonies are stably expressing P450 alone or together with a picked up, grown and tested for P450 expression. 22 M. Sawada, T. KamatakirMutation Research 411() 1998 19±43

Table 1 Development of cell lines stably expressing P450 and their use in toxicological studies P450Ž. species Recipient cell lines Assays References Chemicals assayed Endpoints qry wx CYP1A1 Ž.human AHH-1 TK AFB1 Mutation 4 wx AFB1 Cytotoxicity 5 wx wx AFB1 , B a P, NNK, Cytotoxicity, mutation 6 Cyclopentawxc,d pyrene DMBA Metabolismwx 7 Phenanthrene Metabolismwx 8 Bwxa P, BPD Metabolism wx 9 Human skin fibroblasts: XPA, Bwxa P, BPD, BPDE Cytotoxicity w 10 x DNA-repair normal BPD Cytotoxicity, mutationwx 11 BPD, BPDE Cytotoxicity, mutation,32 P-postlabelingwx 12 V79 Bwxa P, BPD Cytotoxicity, micronucleus, mutation w 13 x Bwxa P w 14 x NIHr3T3 Ochratoxin A Cytotoxicity, mutationwx 15 wx CYP1A1 Ž.monkey CHL AFB1 Cytotoxicity, mutation 16 wx AFB1 , sterigmatocystin Cytotoxicity, chromosomal aberration 17 wx w x BALB 3T3 A31-1-1 AFB1 , B a P Cytotoxicity 18 CYP1A1 Ž.rat V79 Bwxa P, BPD Mutation w 19,20 x Bwxa P, sterigmatocystin, tobacco Micronucleus w 21,22 x particulate matter, 2-AA, CP Bwxa P, BPD c-mitosis w 23 x wx w x AFB1 , B a P, CP, DMBA, Chromosomal aberration, SCE 24 NDMA Bwxa P Hydroxylation w 14 x Hydroquinone, econazole nitrate Micronucleuswx 25 Hydroxy anthraquinones SCEwx 26 Hydroxy anthraquinones Chromosomal aberrationswx 27 Adriamycin, MMC Cytotoxicitywx 28 Bwxa P, BPD, DMBA, chrysene Mutation w 29 x dihydrodiols, 2-AA, 2-AAF,

AFB1 , NDBA Cyp1a1 Ž.mouse Hepa-1c1c7 c37 Bwxa P Hydroxylation w 30 x CHO-derived UV5 BPD, Trp-P-2 Ames testwx 31 Bwxa P Cytotoxicity, mutation w 31 x

qry wx CYP1A2 Ž.human AHH-1 TK AFB1 Cytotoxicity, mutation, DNA binding 32,33 NNK Cytotoxicity, mutationwx 34 wx AFB1 Cytotoxicity 6,5 Acetoaminophen Metabolismwx 35 Phenanthrene Metabolismwx 8 Bwxa P, BPD Metabolism wx 9 wx AFB1 Metabolism 36 Hepa-1c1c7 c37Ž. Acetanilide Ž 4-Hydroxylation . wx 30 V79Ž.Ž. Alkoxyresorufins O-dealkylationwx 37 Bwxa P Hydroxylation w 14 x Acetaminophen Cytotoxicity, c-mitosiswx 38 wx CHL-derived CR-68 plus AFB1 Cytotoxicity 39 NAT1, NAT2 IQ, MeIQx Cytotoxicity, mutationwx 39 wx BEAS-2B AFB11 , AFG Cytotoxicity, DNA-binding 40 NIHr3T3 Ochratoxin A Cytotoxicity, mutationwx 15 M. Sawada, T. KamatakirMutation Research 411() 1998 19±43 23

Table 1Ž. continued P450Ž. species Recipient cell lines Assays References Chemicals assayed Endpoints CYP1A2 Ž.rat V79 2-AF Metabolismwx 41 2-AA, sterigmatocystin, Bwxa P, Micronucleus w 21,22 x CP Bwxa P, BPD c-mitosis w 23 x Phenacetin Metabolismwx 42 wx w x AFB1 , CP, B a P, DMBA, Chromosomal aberration, SCE 24 NDMA Bwxa P Hydroxylation w 14 x Econazole nitrate Micronucleuswx 25 1,2-OH anthraquinone, 1,2,4-OH SCEwx 26 anthraquinone Hydroxy anthraquinones Chromosomal aberrationwx 27 Bwxa P, BPD, DMBA, chrysene Mutation w 29 x dihydrodiols, 2-AA, 2-AAF,

AFB1 , DMBA 2-AA, 2-AF, 2-AAF,4-AAF, IQ SCEwx 29 Ad293Ž. human embryonic MeIQ, Trp-P-2, PhIP, 2-AA, Ames testwx 43

2-AF, AFB1 , BPD Cyp1a2 Ž.mouse NIHr3T3, Hepa-1, BSC-1, CV-1, HeLa Ž Acetanilide . Ž 4-Hydroxylation . wx 44 RLEŽ. rat liver epithelial IQ, MeIQx32 P-Postlabelingwx 45 NIHr3T3 IQ, 2-AAF32 P-Postlabelingwx 46 CHO-derived UV5 IQ, PhIP Cytotoxicity, mutationwx 47 PhIP, N-OH-PhIP Chromosomal aberration,wx 48 SCE, micronuclesus PhIP Mutation spectrumwx 49 plus NAT2, OAT IQ, PhIP Cytotoxicity, mutationwx 50

CYP2A6 Ž.human AHH-1 TKqry derived L3 NDMA, Bwxa P Cytotoxicity, mutation w 51 x qry wx w x AHH-1 TK AFB1 , B a P, NDMA, NDEA Mutation 52,33 wx AFB1 Cytotoxicity 5 NNK Cytotoxicity, mutationwx 34 CP, ifosfamide Cytotoxicitywx 53 C3Hr10T1r2 NNK Mutation, cell transformationwx 54 NDEA, NNK Cell transformationwx 55 CHO-derived AS52 NNK Mutation, mutation spectrumwx 56 HeLa, NIHr3T3Ž. Coumarin Ž 7-Hydroxylation . wx 57 V79Ž. Coumarin Ž 7-Hydroxylation . wx 58 wx CYP2B1 Ž.rat V79 AFB1 Mutation 3 Bwxa P, CP, sterigmatocystin, Micronucleus w 21,22 x 2-AA CP, ifosfamide, ifosfamide Cytotoxicity, mutationwx 59,20 mustard wx w x AFB1 , CP, NDMA, B a P, Chromosomal aberration, SCE 24 DMBA Hydroquinone Micronucleuswx 25 1,2-OH anthraquinone, SCEwx 26 1,2,4-OH anthraquinone Hydroxy anthraquinones Chromosomal aberrationwx 27 Adriamycin, MMC Cytotoxicitywx 28 Bwxa P, BPD, DMBA, chrysene Mutation w 29 x dihydrodiols, 2-AA, 2-AAF

AFB1 , NDBA 24 M. Sawada, T. KamatakirMutation Research 411() 1998 19±43

Table 1Ž. continued P450Ž. species Recipient cell lines Assays References Chemicals assayed Endpoints CYP2B1 Ž.rat C3Hr10T1r2 2-AAF Cytotoxicitywx 61 C6Ž. rat glioma CP, ifosfamide Cytotoxicitywx 62 9LŽ. rat gliosarcoma CP, ifosfamide Cytotoxicitywx 63 MCF-7 CP, ifosfamide Cytotoxicitywx 64 CYP2B4 Ž.rabbit HK293 Ž human kidney .Ž . Ž Hydroxylation . wx 65 CYP2B5 Ž.rabbit HK293 Ž Androstenedione . Ž Hydroxylation . wx 65 qry wx CYP2B6 Ž.human AHH-1 TK AFB1 Cytotoxicity 5,66 CP, ifosfamide Cytotoxicitywx 53

CYP2C10 Ž.human NIHr3T3 Ochratoxin A Cytotoxicity, mutationwx 15

CYP2D6 Ž.human AHH-1 TKqry NNK, NNN, NNA Cytotoxicity, mutationwx 34 NNK Cytotoxicity, mutation, metabolismwx 67 Tamoxifen, tamoxifen epoxide, Micronucleuswx 68 toremifene NIH 3T3 Ochratoxin A Cytotoxicity, mutationwx 15 V79, CHOŽ.Ž. Clozapine, fluperlapine Metabolismwx 69,70 CHO NNK Metabolismwx 71

CYP2E1 Ž.human AHH-1 TKqry NDMA Cytotoxicity, mutationwx 72 NDMA, NDEA Cytotoxicity, mutationwx 52 NNK Cytotoxicity, mutationwx 34 Tamoxifen, tamoxifen epoxide, Micronucleuswx 68 toremifen Acetaminophen Metabolismwx 35 1,2-Epoxy-3-butene Metabolismwx 73 NIHr3T3, RLE NDMA DNA-bindingwx 74 PC-12 Acetaminophen Metabolismwx 75 Hep G2 NDMA, p-nitrophenol Oxidationwx 76 NDMA Oxidationwx 77 Acetoaminophen Cytotoxicitywx 78 V79 NDMA, p-nitrophenol Cytotoxicity, mutationwx 116 NIHr3T3 Ochratoxin A Cytotoxicity, mutationwx 15 CHL-derived CR-119 NDMA Cytotoxicitywx 79 CYP2E1 Ž.rabbit CHO-K1 Ž Chlorzoxazone . Ž Hydroxylation . wx 80

qry wx CYP3A4 Ž.human AHH-1 TK AFB1 Cytotoxicity, mutation, 33,5 DNA binding wx AFB1 Metabolism 36 Tamoxifen, tamoxifen epoxide, Micronucleuswx 68 toremifen 1,2-Epoxy-3-butene Metabolismwx 73 r wx NIH 3T3 AFB1 Cytotoxicity, mutation 81,82 Ochratoxin A Cytotoxicity, mutationwx 15 wx CHL-derived CR-119 AFB11 , AFG , sterigmatocystin Cytotoxicity 83 wx V79 AFB1 Cytotoxicity, micronucleus 84,85 V79Ž.Ž. Lisuride, terguride Metabolismwx 86 CHO NNK Metabolismwx 71 wx CYP3A7 Ž.human MCF-7 AFB1 Cytotoxicity 87 wx CHL-derived CR-119 AFB11 , AFG , sterigmatocystin Cytotoxicity 83 wx CHL-derived CR-68 plus AFB1 , IQ, MeIQ, MeIQx Cytotoxicity 88 plus NAT1, NAT2 wx Cyp3a11 Ž.mouse CHL-derived CR-119 AFB1 Cytotoxicity 89 M. Sawada, T. KamatakirMutation Research 411() 1998 19±43 25

Table 1Ž. continued P450Ž. species Recipient cell lines Assays References Chemicals assayed Endpoints wx Cyp3a13 Ž.mouse CHL-derived CR-119 AFB1 Cytotoxicity 90 CYP4B1 Ž.rabbit C3Hr10T1r2 Ipomeanol, 2-AA,2-AF, 2-AAF, Cytotoxicitywx 91 4-aminobiphenyl, 2-aminonaphthalene

The choice of recipient cells is one of the impor- level of the reductase activity varies among cell lines tant factors determining the usefulness of the cell and is generally low as compared to the level in the lines stably expressing P450. Among the cell lines liverwx 93 . Accordingly, the cell lines which show in Table 1, AHH-1 and Chinese hamster cell lines high activity of the P450 reductase should be candi- have been widely used as recipient cells. AHH-1 was dates as the recipient cells for P450 cDNA. Mapoles employed by Crespi et al. to develop a battery of cell et al.wx 75 used rat-derived PC-12 cells for the ex- lines expressing different forms of human P450 and pression of human P450, because the level of the to apply the cells to hprt mutation assays. Chinese P450 reductase in the cells was of the same order of hamster cell lines V79, CHO and CHL were used in magnitude as in human liver. To fortify the level of several laboratories for the expression of P450 pre- the P450 reductase activity, cDNA coding for the sent in the liver of rats, mice, monkeys and humans. reductase was successfully introduced into Chinese In addition to these cell lines, a variety of human hamster CHL cells. This will be described in detail cells such as SV40-transformed skin fibroblasts, XPA in a later section. P450 is a heme-containing en- Ž.xeroderma pigmentosum group A , HeLa, HepG2, zyme. Thus, it may be possible to assume that the MCF-7Ž.Ž mammary tumor , BEAS-2B bronchial ep- formation of the functional holo-P450 enzyme is ithelial cells.Ž. , and HK293 kidney have also been influenced by the ability of cells to synthesize the used. Cell lines isolated from mice, including Hepa- heme. 1c1c derived from hepatoma, BALB 3T3, NIHr3T3, Another point to be considered in choosing recipi- and C3Hr10T1r2, are also successfully trans- ent cells is the purpose of establishing the genetically formed. Only a liver-derived epithelial cell line, engineered cells. In general, cytotoxicity tests can be RLE, and an adrenal pheochromocytoma cell line, done more sensitively than the analysis of drug PC-12, have been employed as cell lines from rats. metabolite in cell lines established so far, while the Two points should be considered when a certain feasibility of mutagenicity tests at some endpoints cell line is chosen as the recipient. One is whether or depend on the cell types. In many reported cases, the not the recipient cells possess other factors necessary recipient cell lines have been chosen with the reason for the function of the P450. The activity of the that the cell line has been frequently used in mutagen P450 is dependent on components constituting an screening assays. This is true for AHH-1 and the electron transport system in microsomal membranes, Chinese hamster cell lines V79, CHO and CHL. In e.g., NADPH±cytochrome P450 reductase, cy- particular, these Chinese hamster cell lines are the tochrome b55and NADPH±cytochrome b reductase. most popular in mutagen screening assays with a It is a matter of course that the level of these factors variety of endpoints such as hprt mutation, in the cells modifies the activity of the expressed ouabain-resistance mutation, chromosomal aberra- P450. Doehmer et al.wx 3 , who used V79 cells, tions, sister chromatid exchanges, and micronucleus mentioned that the reason for using the V79 cells is induction. Specific cell lines such as BALB 3T3 and that the cells lack P450 activity but, nevertheless, C3Hr10T1r2 have been employed exclusively to contain NADPH±cytochrome P450 reductase. Al- detect morphological transformation induced by car- though it is assumed that the expression of the P450 cinogens. In some cases, the cell lines showing reductase is maintained in cultured mammalian cells specific characters have been chosen as the recipi- dissociating with the expression of P450wx 92 , the ents. Thompson et al.wx 47 used a CHO-derived cell 26 M. Sawada, T. KamatakirMutation Research 411() 1998 19±43 line UV5 which defects the incision step of nu- comprising 26 subfamilies have been found for the cleotide excision repair and is highly sensitive to UV mammalian P450s. The members of P450 which irradiation. The sensitivity to some carcinogens was have the capacity to metabolize xenobiotic com- compared between the excision-repair deficient and pounds have been classified mainly into the families proficient cell lines stably transfected with a mouse from 1 to 4. For this reason, stable expression of P450 cDNAwx 47,48 . A similar study was carried out P450s have been performed focusing on the mem- with human XPAŽ. repair deficient and normal skin bers of these four P450 families, especially the fibroblast cellswx 10 . A human bronchial epithelial families from 1 to 3. cell line BEAS-2B, which had been immortalized by the introduction of the SV-40 T-antigen gene, was 3.1. CYP1A subfamily chosen because the cells were putative progenitor cells of major types of lung cancer and retained The CYP1 family contains two forms of CYP1A, many characteristics useful to study human lung 1A1 and 1A2, in all mammalian species so far carcinogenesiswx 40 . examined, and a new member, 1B1, recently identi- fied in human, rats and micewx 95 . CYP1A1 is proba- bly not expressed constitutively but induced by treat- 3. Mammalian cells transfected with P450 cDNA ment with some agents in the hepatic and extrahep- and application to toxicological studies atic tissues. It is known that TCDD and polycyclic aromatic hydrocarbonsŽ. PAHs are potent inducers The P450 Nomenclature Committeewx 94,95 has of CYP1A1 in animals, including rats and mice. The classified P450s present in bioorganisms such as induction of CYP1A1 has been proven to occur by a bacteria, fungi, plants, invertebrates, and vertebrates. transcriptional regulation involving the Ah Žaryl hy- All P450s in a superfamily are then subdivided into drocarbon. receptor and xenobiotic responsive ele- X families, subfamilies and individual P450s accord- ments in the 5 upstream region of the gene. The ing to the identity of their sequences. In induction of CYP1A1 by 3-MC and PCB is also the nomenclature rule, a P450 gene is shown by observed in the liver of cynomolgus monkeys at the `CYP'`Ž.Cyp' for mice denoting cytochrome P450, level of mRNAwx 96 . In humans, CYP1A1 has been followed by an Arabic number designating the P450 detected in the placental tissue of cigarette smokers family, a capital letterŽ. a small letter for mice wx97 . On the other hand, CYP1A2 is presumably designating the subfamilyŽ. when two or more exist , expressed constitutively at a low level in the liver and an Arabic number designating the individual and can be induced by foreign compounds such as gene. Amino acid sequences of the P450s within the TCDD and 3-MC in experimental animals. same family are )40% identical, although there are Benzowxa pyrene is known to be a good substrate several exceptions. Mammalian P450s within the for CYP1A1. Studies using the enzyme purified from same subfamily have )55% identity of amino acid animals and antibodies to the purified enzyme have sequences, and their appear to be located on proven that CYP1A1 is the major enzyme metaboliz- the same to compose a gene cluster. It ing this substrate. This procarcinogen is metabolized should be noted that this classification is independent to give an ultimate active metabolite, 7,8-dihydro- on any catalytic properties including substrate speci- diol-9,10-epoxide, by sequential metabolic processes ficity of the P450s, and that the P450s from differ- catalyzed by CYP1A1 and epoxide hydrolase. ent species can be included in the same family and CYP1A1 is also involved in the N-hydroxylation of subfamily. If two or more individual P450s are 2-acetylaminofluorene as shown by the experiment included within the same family, then a different using Cos-1 cells transiently expressed human Arabic number is assigned to designate an individual CYP1A1wx 98 . CYP1A2 is responsible for the P450 in principle. However, in the cases of some metabolic activation of arylamine carcinogens such P450s such as CYP1A1, CYP1A2 and CYP2E1, the as 2-naphthylamine and 4-aminobiphenyl. The pri- same numbers are assigned to the homologues from mary P450 form that can activate carcinogenic hete- different animal species. To date, 14 gene families rocyclic amines isolated from pyrolysates of M. Sawada, T. KamatakirMutation Research 411() 1998 19±43 27 and amino acids is CYP1A2wx 99,100 . CYP1A2 also noted that human CYP1A1 expressed in DNA-repair catalyzes the O-deethylation of phenacetin and the deficient human skin fibroblast XPA and DNA-re- N-demethylation of caffeine. pair proficient human skin fibroblast did not activate As shown in Table 1, many reports on the stable Bwxa P to a cytotoxic metaboliteŽ. s . It is known that expression of CYP1A have appeared, probably be- Bwxa P is metabolized to B wxa P-7,8-epoxide mainly cause CYP1A participates in the metabolic activation by CYP1A1 and then converted to Bwxa P-7,8-dihy- of well-known mutagens and carcinogens mentioned drodiolŽ. BPD by microsomal epoxide hydrolase. above. Many cell lines separately expressing the Bwxa P-7,8-dihydrodiol undergoes further epoxidation human, rat and mouse CYP1A1 and CYP1A2 and a by CYP1A1 to form an ultimate carcinogen, Bwxa P- cell line expressing the cynomolgus monkey CYP1A1 7,8-dihydrodiol-9,10-epoxideŽ. BPDE . Therefore, it have been successfully established. Human CYP1A1 is reasonable to believe that the toxicity of Bwxa P cDNA was transfected into and expressed in human occurred depending on the activity of epoxide hydro- wx AHH-1 cells. AFB1 -induced mutation at hprt locus lase in the recipient cells. Glatt et al. 93 reported was detected in the cellswx 4,6 . Sister chromatid that V79 and BALB 3T3 cells possessed the activity exchangesŽ. SCEs were also induced by AFB1 in the of microsomal epoxide hydrolase, although the level V79 cells expressing rat CYP1A1wx 24 . We intro- was quite low compared with freshly isolated rat duced monkey CYP1A1 cDNA into CHL cells and hepatocytes. However, it appears that factors other established a stably transformed cell line, A-15, than epoxide hydrolase should also be considered. In which was 25 times more sensitive to the cytotoxic- fact, AHH-1 cells did not contain any detectable wx wx ity of AFB1 as compared with parental cells 16 . activity of epoxide hydrolase 51 . In addition to AFB1 -induced gene mutation was seen at the hprt DNA damage caused by the reactive intermediate locus in A-15 cells but not in parental CHL cells. produced by enzymes involving P450, the capacity Regarding the induction of chromosomal aberrations, of cells to repair the DNA damage is also a factor the A-15 cells showed a high sensitivity against determining the sensitivity of the cells to mutagens wx w x AFB1 , while in parental CHL cells the aberrations such as B a P. In this respect, Trinidad et al. 31 were induced only at very high concentrations of showed that Bwxa P is highly cytotoxic and mutagenic wx AFB1 17 . BALB 3T3 cells which had been trans- in repair-deficient CHO cells, but not cytotoxic and fected with the same monkey CYP1A1 cDNA simi- less mutagenic in repair-proficient CHO cells, both larly showed a higher sensitivity to AFB1 compared of which expressed mouse Cyp1a1. Supporting this with parental cellswx 18 . These results coincidentally result, Ž." -Bwxa P-trans-7,8-dihydrodiol was more demonstrate that CYP1A1 is capable of activating cytotoxic and mutagenic in the repair-deficient XPA

AFB1 to a cytotoxic and mutagenic metaboliteŽ. s . cells than in human skin fibroblast cells with normal The cytotoxicitywx 13 , mutagenicity w 19,13 x , and DNA-repair capacity after both cells were trans- micronucleus inductionwx 21 of B wxa P have been formed with human CYP1A1 cDNAwx 10±12 . To sensitively detected in the V79 cells expressing hu- account for the complicated data reported so far, man or rat CYP1A1. Arylhydrocarbon hydroxylase many factors, including the expression level of P450, activity is detectable in these transfected cells. BALB amount of endogenous epoxide hydrolase, DNA-re- 3T3 cells carrying monkey CYP1A1 cDNA were pair capacity of recipient cells, and difference of more sensitive to Bwxa P than parental cells w 18 x . endpoints, should be considered as factors affecting Transformation of AHH-1 cells with human CYP1A1 the sensitivity of the cells to Bwxa P. wx cDNA yielded greater response to B a Pinthe AFB1 is also activated to cytotoxic and mutagenic mutation assaywx 6 . Despite these lines of evidence metabolites by human CYP1A2 expressed in AHH-1 that CYP1A1 expressed in the transformant cells cellswx 32,33,6 . Gallagher et al. wx 36 reported that wx metabolically activates B a P to cause mutations and AFB11 was metabolized to AFB -8,9-epoxide and cytotoxicities, there are some conflicting data com- AFM1 by microsomes prepared from the AHH-1 pared with the previous results. Monkey CYP1A1 cells expressing CYP1A2, and that the formation of does not seem to activate Bwxa P to yield a cytotoxic these metabolites was strongly inhibited with a selec- wx wx metaboliteŽ. s in CHL cells 16 . States et al. 10 tive inhibitor of CYP1A2, furafylline. AFB1 caused 28 M. Sawada, T. KamatakirMutation Research 411() 1998 19±43 cytotoxicity and the formation of DNA adducts genic metabolites. Details of the experiments will be 7 Žmainly AFB1 -N guanine. in human BEAS-2B cells described in a later section. Experiments with a expressing human CYP1A2wx 40 . A CHL-derived similar idea have been performed by Ellard and cell line which expressed simultaneously a guinea-pig colleagues. They introduced rat CYP1A2 cDNA into NADPH±cytochrome P450 reductase and the hu- two V79 cell lines: one was V79-NH possessing man CYP1A2 showed about 300-fold higher sensi- endogenous acetyltransferase activity and the other tivity in the cytotoxicity assay of AFB1 compared was V79-MZ, which lacked the acetyltransferase. with parental cellswx 39 . Chromosomal aberrations The V79-NH cells expressing CYP1A2 showed a were induced in the cell line at lower concentration higher level of micronucleus induction by exposure of AFB1 Ž. Sawada et al., unpublished data . The to 2-AA than the V79-MZ cells expressing CYP1A2 induction of SCEs was also found in V79 cells wx21 . 2-AA also induced gene mutation and SCE in transformed with rat CYP1A2 cDNAwx 24 . the former cells, while its effect was marginal in the A relatively smaller number of papers has ap- latter cellswx 29 . peared on the metabolic activation of cooked food- Recently, De Groene et al.wx 15 established derived heterocyclic amines by CYP1A2 stably ex- NIHr3T3 cell lines expressing human P450s by pressed in cultured cells. Battula et al.wx 45,46 proved means of retroviral infection and developed a new by 32 P-postlabeling assays that IQ and MeIQx were mutation assay system. The cells carrying P450 activated to form DNA adducts in RLEŽ rat liver cDNA were transfected with shuttle vectors contain- X epithelial.Ž and NIHr3T3 mouse embryo fibroblast .ing the lacZ gene. After the transfection, the cells cells, which were stably transformed with mouse were treated with mutagens. The shuttle vectors were Cyp1a2 cDNA by the retrovirus-mediated method. then rescued from the cells and again introduced into According to Thompson et al.wx 47 , PhIP was highly Escherichia coli DH10B. The mutation frequency cytotoxic and mutagenic to CHO-derived repair-defi- was determined by the ratio of white colonies against cient cells but less effective to repair-proficient cells, the total number of colonies. In this mutation assay both of which expressed Cyp1a2, lending support to system, ochratoxin A, which has been reported to the idea that the repair system as well as the metabolic induce renal tubular cell tumors in rats, induced X activation determines the sensitivity of cells to muta- mutations at the lacZ gene in the cell lines express- genic chemicals. The experiments performed with IQ ing human CYP1A1 or CYP1A2. showed a tendency similar to PhIP, while the muta- V79 cells expressing rat CYP1A1 or CYP1A2 tion frequency induced by IQ was much lower than were employed to examine possibility of whether that seen with PhIP. The repair-deficient CHO cells these enzymes are involved in the activation of expressing Cyp1a2 have been used for the analysis quinone compounds. Induction of SCE and chromo- of sequences of PhIP-induced mutations in the ade- somal aberrations by seven hydroxy anthraquinones nine phosphoribosyltransferase Ž.aprt gene. It was were testedwx 26,27 . The results did not support the shown that most of the observed mutations were idea that CYP1A was responsible for the activation single-base transversions and that there were three of the quinone compounds. Similarly, cytotoxicity of hot-spotswx 49 . adriamycin and mitomycin C was tested with V79 By in vitro studies, it has been clarified that some cells expressing CYP1A1wx 28 . While adriamycin procarcinogens, including food-derived heterocyclic was equally toxic to the cells expressing CYP1A1 amines, are activated by certain conjugation enzymes and to parental cells, mitomycin C was less toxic to after undergoing oxidation reactions by P450 to the former cells. yield ultimate reactive forms. In our study, activation systems of IQ and MeIQx to form cytotoxic and 3.2. CYP2A subfamily mutagenic metabolites were constructed in the CHL-derived cell lines, in which human CYP1A2 Some forms of P450 belonging to this subfamily and N-acetyltransferase 2 were simultaneously ex- have been purified andror cloned from the liver of pressedwx 39 . In the cells expressing CYP1A2 alone, rats, mice, hamsters, rabbits and humanswx 95 . Among these compounds were not activated to show muta- them, two rat enzymes, CYP2A1 and CYP2A2, have M. Sawada, T. KamatakirMutation Research 411() 1998 19±43 29 been most extensively studied and characterized. are 97% identical with regard to amino acid se- CYP2A1 protein catalyzes the 7a-hydroxylation and quence deduced from their cDNAswx 105 but are the 6a-hydroxylation of testosteronewx 101 . CYP2A2 distinct from each other in tissue specificity for their also metabolizes to form metabolites, constitutive and induced expressions. CYP2B1 is not mainly at 15a-hydroxylationwx 102 . Two enzymes, detectable in the liver of rats without PB-treatment CYP2A6 and CYP2A7, have been identified to be but constitutively expressed in the lung and testis. In present so far in human livers. The catalytic activity the latter tissues, however, CYP2B1 is not inducible was examined only for the former onewx 103 . It was by PB. CYP2B2 is constitutively expressed in rat shown by studies using purified enzymes prepared liver, but not in the lung, kidney and testis regardless from genetically engineered cells that CYP2A6 cat- of PB-treatmentwx 106 . CYP2B3, another member of alyzed coumarin 7-hydroxylation and ethoxy- rat CYP2B, is constitutively expressed but not in- coumarin O-deethylation but not testosterone 7a-hy- duced by PBwx 107 . It is unknown whether human droxylationwx 103,104 . CYP2B6 can be induced by PB or other compounds. As shown in Table 1, among the forms of P450 Doehmer et al.wx 3 developed a V79-derived cell in the CYP2A subfamily, only human CYP2A6 was line which stably expressed rat CYP2B1, and showed stably expressed in cultured mammalian cells. Davies that the cells were sensitive to AFB1 as determined et al.wx 51 and Crespi et al. wx 52 established a cell line by hprt locus mutation assay. Chromosomal aberra-

AHH-1 expressing CYP2A6 and observed the incre- tions and SCEs were also induced by AFB1 in the wx wx ment of sensitivity of the cells to AFB1 , B a P, cells 24 . Cyclophosphamide was activated by N-nitrosodimethylamineŽ. NDMA , N-nitroso- CYP2B1 and caused cytotoxicity, micronuclei, chro- diethylamineŽ. NDEAwx 51,52,33 and NNK wx 34 de- mosomal aberrations and SCEs in the cell line termined by cytotoxicity andror mutagenicity. The wx59,20,21,24 . This cell line was used in analysis of growth rate of the AHH-1 cells expressing CYP2A6 CYP2B1-mediated metabolism of androstenedione, was inhibited by cyclophosphamide and ifos- testosterone, caffeine and theophyllinewx 108,109 . phamide, while the inhibition was much less in the Cytotoxicity of 2-acetylaminofluoreneŽ. 2-AAF was control cellswx 53 . Both compounds were hydroxyl- seen in mouse C3H10T1r2 cells expressing ated at 4-position by microsomes prepared from the CYP2B1, developed by Hansen et al.wx 61 . The cells. Tiano et al.wx 54 established mouse C-hydroxylation of 2-AAF was measured using C3Hr10T1r2 cells expressing CYP2A6 by the gene HPLC in the cells expressing CYP2B1. However, transfer method using retroviruses, and showed that the cytotoxicity caused by the metabolic activation NNK induced ouabain-resistance mutation and mor- of 2-AAF and the metabolic capacity of the cells phological transformation in the cells. NDEA also were seen only in early passage cells. The metabolic induced morphological transformation in the cells capacity was lost in late passages in the absence of wx55 . CHO-derived AS52 cells carrying bacterial gpt G418. The authors noted that one of possible expla- gene were stably transfected with CYP2A6 cDNA nations for the loss of enhanced metabolic capacity and the resulting cells showed a high sensitivity in might be a result of mutations or rearrangements the NNK-induced 6-thioguanine-resistance mutation which occurred in prolonged culture. Recently, an wx56 . PCR amplification of the gpt gene in the application of CYP2B1 expression to cancer chemo- mutant cell clones showed that 78.6% of the mutants therapy has been investigated. Cell lines stably ex- had putative point mutations and the remaining 21.4% pressing CYP2B1 were established using rat glioma were attributed to deletionsrrearrangements. DNA C6 cellswx 62 , rat gliosarcoma 9L cells wx 63 , and sequence analysis revealed that 81% of the putative human breast cancer MCF-7 cellswx 64 as recipients. point mutations were G:C to A:T transitions. The cells expressing CYP2B1 showed a high sensi- tivity to cyclophosphamide and ifosfamide in vitro. 3.3. CYP2B subfamily When the tumor cells carrying CYP2B1 were im- planted to rats or nude mice, treatment of the animals Rat CYP2B1 and CYP2B2 are well known to be with cyclophosphamide resulted in efficient inhibi- induced by phenobarbitalŽ. PB . These two enzymes tion of tumor growth. 30 M. Sawada, T. KamatakirMutation Research 411() 1998 19±43

Human CYP2B6 was stably expressed in human Bwxa P-trans-dihydrodiols from B wxa P was catalyzed AHH-1 cells. The growth inhibition tests showed by human CYP2C9 which was transiently expressed that the sensitivity of the AHH-1 cells expressing in Hep G2 cellswx 9 .

CYP2B6 to AFB1 was much lower than that of the cells expressing human CYP1A2 or CYP3A4 when 3.5. CYP2D subfamily the relative growth was plotted against AFB1 con- centration multiplied by microsomal P450 content Forms of P450 in the CYP2D subfamily from wx5 . Growth of the cells expressing CYP2B6 was also rats, mice, cows and humans have been identified. inhibited by cyclophosphamide and ifosphamide. Among the forms, only human CYP2D6 cDNA has Both compounds were proven to be 4-hydroxylated been stably expressed in cultured mammalian cells. by microsomes from the cellswx 53 . Rabbit CYP2B4 It is widely known that human CYP2D6 catalyzes and CYP2B5 were stably expressed in human kidney the 4-hydroxylation of the antihypertensive drug de- 293Ž. HK293 cellswx 65 . Using microsomes prepared brisoquine and N-oxidation of an oxytocic drug from these cells, catalytic activities of CYP2B4 and sparteine, and that genetic polymorphism in the ex- CYP2B5 toward androstenedione, benzophetamine, pression of CYP2D6 is seen. The debrisoquiner 7-ethoxycoumarin and other substrates were assayed. sparteine polymorphism has been one of the most Cytotoxicity and mutagenicity tests using these cell extensively studied phenomena in the field of phar- lines have not been reported so far. macogenetics. Since a number of drugs are catalyzed by CYP2D6, studies with cells stably expressing 3.4. CYP2C subfamily CYP2D6 or microsomal fractions prepared from the cells have been concentrated on the metabolism of The CYP2C subfamily contains many isoforms drugs such as bufuralolwx 112 , clozapine and fluper- and is regarded as a major one existing human and lapinewx 69 , tropisetron and ondansetron wxw 70 ; 113 x . animal livers. Some members of this subfamily are Only a few papers on cytotoxicity and mutagenic- well known to be sex-specific in livers of rats, mice, ity tests using cells expressing CYP2D have been and Syrian hamsters. Although a large number of published so far. A tobacco smoke-derived ni- P450s belonging to this subfamily have been identi- trosamine NNK was cytotoxic and mutagenic in fied from humans and animals, there are only a few AHH-1 cells expressing CYP2D6wx 34,67 . Also, reports on the stable expression of the cDNAs in anti-cancer drug tamoxifen increased the frequency cultured cells. Human CYP2C8 and CYP2C9 were of micronuclei in the AHH-1 cells expressing stably expressed in AHH-1 cellswx 66 , while data on CYP2D6wx 68 . Ochratoxin A showed a negative re- cytotoxicity or mutagenicity tests using these cell sult in the mutation assay using NIHr3T3 cells lines have not been reported. De Groene et al.wx 15 expressing CYP2D6wx 15 . established NIHr3T3 cells expressing human CYP2C10 by means of retroviral infection and ap- plied to the mutation assay, in which a shuttle vector 3.6. CYP2E subfamily X containing the lacZ gene was used as a reporter for mutations. Upon addition of ochratoxin A to the CYP2E1 from rats, mice, rabbits, cynomolgus culture medium, the mutation frequency of the cells monkeys and humans has been purified andror increased in a dose-dependent manner in the cells cloned. Another unique member in this subfamily, expressing CYP2C10. Among forms of P450, CYP2E2, was also found in rabbits. CYP2E1 in CYP2C enzymes are known to be mainly involved in rodent livers is not only expressed constitutively but the metabolism of a variety of drugs, and only a few induced by a number of chemicals such as ethanol, reports have appeared on their roles in the metabolic acetone, and isoniazid. The induction is not mediated activation and inactivation of carcinogens. For exam- by transcriptional enhancement but caused by the ple, Bwxa P 3-hydroxylation was observed with a stabilization of CYP2E1 protein after binding with CYP2C enzyme purified from male ratswx 110 and the chemicalswx 114 . CYP2E1 is able to activate a with purified human CYP2C8wx 111 . Formation of variety of carcinogens and toxic substances, includ- M. Sawada, T. KamatakirMutation Research 411() 1998 19±43 31 ing benzene, carbon tetrachloride, vinyl chloride, CYP3A4 and CYP2E1 at a high concentrationŽ 5 trichloroethylene, acrylonitrile and NDMA, with rel- mM. of BMOwx 73 . atively low molecular weightsŽ reviewed by Koop Barmada et al.wx 80 established Chinese hamster wx115. . CHO cells stably expressing rabbit CYP2E1 by a Several lines of cells were stably transformed unique method as follows. CHO-K1 cells were co- with human CYP2E1 cDNA and have been used for transfected with an expression plasmid carrying rab- cytotoxicity and mutagenicity tests. NDMA, NDEA bit CYP2E1 cDNA and a neomycin-resistance gene and NNK were shown to be efficiently activated to as well as a plasmid pFR400. The latter plasmid enhance the cytotoxicity and mutagenicity in the contained mouse dihydrofolate reductase cDNA with AHH-1 cells expressing human CYP2E1wx 72,52,34 . a single amino acid substitution which lowers the Enhancements of the cytotoxicity and mutagenicity affinity of the enzyme to bind to methotrexate. The of NDMA were also shown with Chinese hamster transfected cells were selected with G418 and then V79 cellswx 116 and CHL cells wx 79 expressing further selected with increasing concentrations of CYP2E1. Oxidation of NDMA occurred in micro- methotrexate. Since the cotransfected plasmids could somes from rat PC-12 cellswx 75 and human HepG2 be ligated within the cells and integrated into a cellswx 76 expressing CYP2E1. Formation of DNA chromosome as a unit, the methotrexate selection adducts with NDMA was confirmed using mouse caused the amplification of the introduced dihydrofo- NIHr3T3 and rat liver epithelialŽ. RLE cells trans- late reductase cDNA accompanied by the CYP2E1 formed with CYP2E1 cDNAwx 74 . Tamoxifen in- cDNA. In the paper, a high expression of CYP2E1 creased the frequency of micronuclei in the trans- mRNA could be accomplished in the cotransfected formed AHH-1 cellswx 68 . Cytotoxicity of ac- cells which were selected with methotrexate. etaminophen was seen in the PC12 cells expressing CYP2E1 expressed in the resulting transformant cells CYP2E1wx 75 . The acetaminophen cytotoxicity was catalyzed the 6-hydroxylation of the muscle relaxant increased by addition of buthionine sulfoximine chlorozoxazone at efficient rates. which caused depletion of intracellular glutathione. In agreement with the result, Dai and Cederbaum 3.7. CYP3A subfamily wx78 reported that, when intracellular glutathione was depleted by buthionine sulfoximine, acetaminophen CYP3A enzymes are the most abundantly ex- caused severe cytotoxicity in HepG2 cells trans- pressed P450s in the liver of humans and experi- formed with the CYP2E1 cDNA. p-Nitrophenol was mental animals. This subfamily is comprised of many 10-fold more cytotoxic to the V79 cells expressing isoformswx 95 ; for example, rat CYP3A1, CYP3A2, CYP2E1 as compared to the parental cellswx 116 . The CYP3A9, CYP3A18 and CYP3A23; mouse CYP2E1-dependent hydroxylation of p-nitrophenol Cyp3a11, Cyp3a13 and Cyp3a16; rabbit CYP3A6; was detectable in the cell homogenates; the activity dog CYP3A12; human CYP3A4, CYP3A5 and was enhanced by pretreatment of the cells with CYP3A7. Most CYP3A enzymes are known to be ethanol. induced in the liver of experimental animals by Relationship between the expression of CYP2E1 chemicals including PB, dexamethazone, and PCN. and the activation of 1,3-butadienŽ. BD was shown The expression of individual CYP3A genes, how- with AHH-1 cells transformed with human CYP2E1 ever, appears to be differently regulated. For exam- cDNAwx 73 . BD is known to be carcinogenic in ple, rat liver CYP3A1 is not constitutively expressed rodents and classified as a probable human carcino- but induced by PCN, while CYP3A2 is constitutively gen. BD is metabolized to 1,2-epoxy-3-butene expressed in livers of adult male rats but not induced Ž.BMO and then to more mutagenic and carcino- by PCNwx 117 . Both CYP3A1 and CYP3A2 can be genic metabolite, 1,2:3,4-diepoxybutaneŽ. BDE . induced by PB. Studies with microsomes from a series of AHH-1 CYP3A4 is the major form among human liver cell lines expressing human P450 have shown that CYP3A enzymes and is able to metabolize a number the oxidation of BMO to BDE is catalyzed by of and drugs which are structurally varied CYP2E1 at a low concentrationŽ. 80 mM but by Žreviewed by Li et al.wx 60. . In addition to these 32 M. Sawada, T. KamatakirMutation Research 411() 1998 19±43 chemicals, CYP3A4 activates procarcinogens such as enzymes heterologously expressed in cultured cells wxwx w x AFB1 118±122. and B a P 7,8-diol 123,121 . Sta- activated the mycotoxin. ble expression of CYP3A4 has been successfully Mouse CYP3A, Cyp3a11 and Cyp3a13, were also performed using several mammalian cell linesŽ Table stably expressed in CHL-derived cells carrying the 1. . The AHH-1 cells expressing CYP3A4 were ap- guinea-pig P450 reductase cDNAwx 90,89 . Sensitiv- plied to mutation assays. The result showed that the ity to AFB1 and enhancement by a-naphthoflavone wx cells were highly sensitive to AFB1 33,5 . In the were also observed in the new cell lines. mutation assay using mouse NIHr3T3 cells stably expressing CYP3A4 with a shuttle vector, the muta- 3.8. CYP4B subfamily genicity of AFB1 and ochratoxin A were shown, supporting again the role of CYP3A4 in the activa- CYP4B1 cDNAs for humans, rabbits, rats, and wx wx tion of AFB1 81,15 . CYP3A4 expressed in the cells mice have been cloned 95 . Northern blot analysis catalyzed the conversion of AFB11 to give AFB -8,9- showed that CYP4B1 was expressed in lungs of wx wx epoxide and AFQ1 36 . The frequency of micronu- rabbits, rats, hamsters, guinea pigs 130 and human clei in the AHH-1 cells was increased by exposure to wx131 . CYP4B1 mRNA was also proven to be present tamoxifenwx 68 . Metabolic profiles of testosterone in the liver of rabbits and hamsters but not detectable and two ergot derivatives were assayed using Chi- in the liver of guinea pigs and humans. In rat livers, nese hamster V79 cells stably expressing CYP3A4 small amounts of mRNA for CYP4B1 were seen; the wx86 . level was at least 10-fold less than that found in the Human CYP3A7 cDNA was cloned from fetal lungwx 130 . cDNA library in our laboratorywx 124 . Genomic DNA A naturally occurring pulmonary toxin, 4- of CYP3A7 was also isolated and the sequence in a ipomeanolw 1-Ž. 3-furyl -4-hydroxypentanonex , is possible transcriptional regulatory region was com- known as a substrate for rabbit CYP4B1. Conversion pared with that of CYP3A4wx 125,126 . The deduced of 4-ipomeanol to metaboliteŽ. s covalently bound to amino acid sequence of CYP3A7 was 88% identical proteins is catalyzed by rat lung microsomes more with that of CYP3A4. Northern blot analysis with effectively than by liver microsomeswx 132 . 4- specific oligonucleotide probes revealed that Ipomeanol was activated by two forms of P450 CYP3A7 was expressed specifically in fetal livers purified from rabbit lungs, one of which corre- and that CYP3A4 was expressed in adult livers sponded to CYP4B1wx 133 . Czerwinski et al. wx 134 wx127 . Therefore, it seemed of interest to note the demonstrated that there was a remarkable difference physiological and toxicological roles of CYP3A7. between rabbit and human CYP4B1 in the capacity The purified protein of CYP3A7 catalyzed the 16a- to activate 4-ipomeanol. They measured the amounts hydroxylation of dehydroepiandrosterone 3-sulfate of metabolites of 4-ipomeanol bound to DNA in wx wx 128 and the mutagenic activation of AFB1 129 . HepG2 cells transiently expressing 12 forms of hu- We established a human breast cancer cell line man P450 or rabbit CYP4B1 separately. Rabbit MCF-7 stably transformed with CYP3A7 cDNA and CYP4B1 was the most active enzymeŽ 180-fold over demonstrated that the new cell line was highly sensi- the control level. among all P450s tested, while wx tive to AFB1 87 . Furthermore, CYP3A4 and human CYP4B1 had very low activityŽ 2-fold over CYP3A7 cDNAs were independently expressed in the control level. . Chinese hamster CHL-derived cells carrying guinea- Rabbit CYP4B1 was stably expressed in mouse pig cDNA of NADPH±cytochrome P450 reductase C3Hr10T1r2 cells using retroviral vectorswx 91 . 4- wx83 . Both of the cells transformed with CYP3A4 and Ipomeanol was highly toxic to the cells expressing

CYP3A7 cDNAs showed higher sensitivity to AFB1 , CYP4B1. This result coincides with the above-men- AFG11 and sterigmatocystin. The activation of AFB tioned reports in which 4-ipomeanol is activated by by CYP3A4 and CYP3A7 in these cell lines was rabbit CYP4B1. Aromatic amine procarcinogens, 2- enhanced by a-naphthoflavone, an activator, and AA, 4-aminobiphenyl, 2-aminonaphthalene, 2-AF inhibited by troleandomycin, a typical inhibitor of and 2-AAF were also examined using the new cell CYP3A enzymes. These results show that the CYP3A line. It had been previously shown that mutagenic M. Sawada, T. KamatakirMutation Research 411() 1998 19±43 33 activations of 2-AA, 2-AF and 2-AAF were possibly was stimulated by androstenedionewx 138 . In order to associated with rabbit hepatic CYP4B1wx 135 . In fact, analyze the key protein sequence to show the enzy- transiently expressed rabbit CYP4B1 could activate matic activity, amino acid substitution was intro- 2-AFwx 134 . However, only 2-AA exhibited clear duced by site-directed mutagenesis. These modified cytotoxicity to the C3Hr10T1r2 cells expressing cDNA sequences were stably expressed in CHO CYP4B1wx 91 . As possible reasons for the negative cellswx 139,140 . 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 4. Coexpression of P450 and other enzymes needed in the activation of these mutagens might not be sufficient in C3Hr10T1r2 cells and that a To develop cell lines possessing a high capacity metabolite showing cytotoxicity might be different to activate procarcinogens, several factors besides from that showing mutagenicity. Cell lines express- P450 enzyme must be considered. These factors can ing human CYP4B1 have not yet been reported to be divided broadly into two groups. One group the author's knowledge. contains components of the electron transfer system in microsomal membranes, i.e., NADPH±cyto-

3.9. Other CYPs chrome P450 reductase, NADH±cytochrome b5 re- ductase and cytochrome b5. Another group contains Cell lines stably expressing P450s belonging to certain drug-metabolizing enzymes such as epoxide families 11 and 19 were established using cultured hydrolase and conjugation enzymes, since these en- mammalian cells. These P450s are physiologically zymes are also required in the sequential processes important in that they are needed for the synthesis of of metabolic activation to form ultimate carcinogens the adrenal corticoids or . Thus, rat which are capable of binding to DNA. Amounts of CYP11B1 and CYP11B2 cDNAs were stably ex- these enzymes are not always sufficient in cultured pressed in rat Leydig tumor cell line MA-10wx 136 . mammalian cells. Therefore, it is expected that coex- Rat CYP11B1Ž.Ž 11b-hydroxylase and CYP11B2 al- pression of cDNAs for P450 and other enzymes dosterone synthase. are present in mitochondria in provides a high ability to activate procarcinogens. zona fasciculata-reticularis and , respectively, of . The cell line MA-10 4.1. NADPH±cytochrome P450 reductase, was chosen as a recipient, since this cell line pro- NADH±cytochrome b55 reductase and cytochrome b duced adrenodoxin and adrenodoxin reductase neces- sary for the function of mitochondrial P450. The NADPH±cytochrome P450 reductase mediates an resulting cells expressing CYP11B1 converted de- electron transfer from NADPH to P450. Thus, this oxycorticosteroneŽ. DOC to yield , 18- enzyme is essential for the catalytic function of OH-DOC and a small amount of 18-OH-cortico- P450. Activity of the reductase is detectable even in sterone. On the other hand, the cells expressing long-term cultured cells regardless of their origins, CYP11B2 converted DOC to corticosterone, 18- but the level is generally low as compared with liver OH-corticosterone, and a small amount tissueswx 92,93 . We used a Chinese hamster cell line of 18-OH-DOC. CHL as a recipient for the transfection of P450 CYP19, a single member of family 19, catalyzes cDNA. The CHL cells have been widely used in wx the conversion of C19 to C 18 estrogens mutagen screening assays 141,142 , but have essen- and is commonly referred to as . This tially no activity of P450. To establish CHL cells enzyme is found in microsomes from the ovary, expressing the reductase together with P450, the placenta, testes, adipose tissue, breast tumor, brain cells were first transfected with an expression vector and skin. Human placenta CYP19 cDNA was stably carrying CYP1A1 cDNA of the cynomolgus mon- transfected into MCF-7Ž. human breast cancer cells , key. The resulting cell lineŽ. designated A-15 showed HBL-100Ž. noncancerous breast cells and CHO cells a higherŽ. 25-fold sensitivity in the cytotoxicity as- wx 137 . Growth of the MCF-7 cell expressing CYP19 say with AFB1 as compared with parental CHL cells 34 M. Sawada, T. KamatakirMutation Research 411() 1998 19±43 wx16 . As a second step, A-15 cells were further mouse Cyp3a11 and Cyp3a13wx 89,90 . Recently, transfected with cDNA encoding guinea-pig Schnieder et al.wx 85 also established V79 cells stably NADPH±cytochrome P450 reductase. Three cell coexpressing human CYP3A4 and P450 reductase linesŽ. designated AR-10, -13, -18 showing high cDNAs, and showed that the cells were much more expression of mRNA for the P450 reductase were sensitive to AFB1 than the cells expressing CYP3A4 clonedwx 17 . Specific activities of the microsomal alone in cytotoxicity and micronucleus formation P450 reductase determined by using cytochrome c assays. as an electron acceptor were 110, 180, and 75 nmol Although the exact mechanism is not known, y1 y1 min mg protein for AR-10, -13, and -18 cells, cytochrome b5 potentiates the reactions mediated by respectively, while the value was 16 nmol miny1 microsomal P450. The enhancement of drug oxida- y1 mg protein for the parental A-15 cells. The intro- tion by cytochrome b5 has been accounted for by the duction of the reductase cDNA resulted in enhanced potentiation of the flow of the second electron from sensitivity to AFB150 . Thus, IC values obtained from NADH to P450 via NADH±cytochrome b5 reduc- three AR- cell lines were about 10-times lower than tase and cytochrome b5 or from NADPH to P450 those from A-15 cellsŽ. Fig. 1 . The cell line CR-68, via NADPH±cytochrome P450 reductase and cy- which was established by introducing the same ex- tochrome b55. The effect of cytochrome b appears to pression vector carrying the P450 reductase cDNA vary with P450 forms and substrateswx 143 . A recon- directly into the original CHL cells, showed en- stitution study showed that nifedipine oxidation by hanced activity of the P450 reductaseŽ 129 nmol the purified human CYP3A enzyme was inhibited by y1 y1 min mg. . As expected, the CR-68 cells did not anti-cytochrome b5 antibody and enhanced by puri- wx show enhancement of sensitivity to AFB1 , indicating fied human liver cytochrome b5 144 . The effects of that the P450 reductase alone did not activate the cytochrome b5 on the activity of CYP3A7 in cul- mycotoxin. These results again demonstrate that the tured cells were studied in our laboratory. We intro- coexpression of cDNAs for P450 and the P450 duced cDNAs coding for human cytochrome b5 and reductase is necessary for P450 to show its full NADH±cytochrome b5 reductase into the CR-119 activity in cultured mammalian cells. The cell lines cells which were expressing CYP3A7Ž Hashimoto et CR-68 or CR-119 stably expressing the guinea-pig al., unpublished results. . As compared to the parental P450 reductase have been used as a recipient of cells, the coexpression of the cDNAs brought a other P450s in further studies: human CYP1A2wx 39 , 5-fold increase in NADH-dependent ferricyanide re- CYP2E1wx 79 , CYP3A4 and CYP3A7 wx 83 , and ductase activity and a 2-fold increase in sensitivity to

AFB1 , suggesting that the electron transfer mediated by the b55 reductase and cytochrome b was con- structed in the cells.

4.2. Microsomal epoxide hydrolase() mEH

Epoxide , which catalyze the conver- sion of epoxides to glycols, play an important role in the activation and detoxification of xenobiotic com- pounds. The role of mEH in the activation of poly- cyclic aromatic hydrocarbonŽ. PAH procarcinogens such as Bwxa P has been particularly studied. B wxa P

Fig. 1. Sensitivity to AFB1 of CHL-derived cell lines expressing and some other PAHs are primarily activated by monkey CYP1A1 andror guinea-pig NADPH±cytochrome P450 P450-dependent epoxidation at bay regions, and then 4 reductaseŽ.Ž see text . Cells 8=10. were seeded in a 60-mm dish the epoxide is hydrated by mEH. Glatt et al.wx 93 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 measured the mEH activity in many cell lines, using w 3 xwx dishes with crystal violet. `, CHL; v, A-15; B, AR-10; I, H-Ba P-4,5-oxide as a substrate, and showed that AR-13; ', AR-18; ^, CR-68 cells. the mEH activity varied among cell lines; variation M. Sawada, T. KamatakirMutation Research 411() 1998 19±43 35 of the activity was greater than that of NADPH±cy- by CYP1A2. The resulting N-hydroxylated metabo- tochrome P450 reductase and glutathione-S-trans- lites are further converted to yield O-acetyl or O- ferase. sulfonyl esters which readily react with protein and A cDNA-mediated stable expression of human DNA Õia the formation of arylnitrenium ions mEH in combination with P450 was accomplished wx146,147 . The O-acetylation is mediated by acetyl by Davies et al.wx 51 . An expression vector contain- CoA-dependent N-acetyltransferaseŽ. NAT . To date ing human CYP2A6 and mEH cDNAs was trans- two molecular forms of the enzyme, NAT1 and fected into cells of an AHH-1 subline L3, in which NAT2, have been found in the human liver. NAT1 CYP1A1 activity had been enhanced by mutation and NAT2 share 80% in cDNA-deduced and selection. The resulting cells, designated MCL-1, amino acid sequences, while differences in substrate were shown to possess mEH activity, which was not specificity between these two NATs have been noted. detectable in the parental cells. The MCL-1 cells To clarify the roles of CYP1A2 and NAT in the showed increased sensitivity to Bwxa P in the muta- activation of heterocyclic amines within cells, we tion assay. The analysis of the metabolite revealed developed cell lines which stably expressed human that the overall metabolism of Bwxa P was elevated by CYP1A2 and NAT alone or in combinations, using the expression of the P450 and mEH cDNAs in the the above-mentioned CR-68 cells as the recipient MCL-1 cells and that the major metabolites were wx39 . The expression of the NATs was monitored dihydrodiols in the MCL-1 cells but phenols in the using p-aminobenzoic acidŽ. for NAT1 and sul- parental AHH-1 cells. famethazineŽ. for NAT2 as typical substrates. No Bwxa P metabolism was also analyzed using micro- endogenous activities for either substrate were de- somes from the AHH-1-derived cells which were tected in the parental cells. Newly developed cell stably transformed with human CYP1A1 and lines were as follows: A2R-5Ž CYP1A2 into CR-68 CYP1A2 cDNAs with and without mEH cDNAwx 9 . cells.Ž , ANM-13 NAT1 into A2R-5 cells . , ANP-25 CYP1A1 produced phenolsŽ. 3-, 7-, 9- , quinones Ž.ŽNAT2 into A2R-5 cells , CNM-4 NAT1 into CR-68 Ž.1,6-, 3,6-, 6,12- and a minor amount of trans-dihy- cells.Ž , and CNP-40 NAT2 into CR-68 cells . . Using drodiolsŽ. 4,5-, 7,8-, 9,10- . Microsomes from cells these new cell lines, cytotoxicity and mutagenicity of coexpressing CYP1A1 and mEH produced lesser IQ and MeIQx were examinedwx 39 . In the ANP-25 amounts of 7- and 9-phenols and a remarkably in- cells expressing CYP1A2 and NAT2 in addition to creased amounts of 7,8- and 9,10-diols. Formation of the P450 reductase, these heterocyclic amines the Bwxa P metabolites was enhanced by introduction showed remarkably higher cytotoxicity and muta- of CYP1A2 cDNA, and no substantial effects of genicityŽ. Fig. 2 . In the ANM-13 cells expressing simultaneous introduction of mEH cDNA were ob- CYP1A2 and NAT1 together with the P450 reduc- served. Human mEH cDNA was also introduced into tase, on the contrary, these compounds showed only AHH-1 cells together with cDNAs of multiple hu- a low level of cytotoxicity and actually no muta- man P450swx 145 . Properties of the resulting cell line genicity. A2R-5 cells which expressed CYP1A2 and will be mentioned in a later section. the P450 reductase did not activate the heterocyclic amines to induce cytotoxicity and mutagenicity. From 4.3. N-acetyltransferase these results, it was confirmed that human CYP1A2 in combination with NAT2 could efficiently activate Enzymes catalyzing the conjugation reactions, IQ and MeIQx by sequential reactions in the cells, as such as acetylation, sulfation, glucuronidation and proposed by in vitro studies. In similar experiments, glutathione conjugation are also playing key roles in we found that 2-AA was also activated in only the detoxification of reactive intermediates produced ANP-25 cells to produce cytotoxic and a mutagenic by the metabolic activation of chemicals. However, metaboliteŽ.Ž s unpublished data . . it is now known that such enzymes are also involved Thompson et al.wx 50 reported results of experi- in the activation of aromatic amine procarcinogens. ments in which human NAT2 and Salmonella O- Heterocyclic amines derived from cooked foods, for acetyltransferaseŽ. OAT were separately expressed in example, are assumed to be N-hydroxylated mainly the UV5P3 cells which had been previously devel- 36 M. Sawada, T. KamatakirMutation Research 411() 1998 19±43

determining the form of P450 catalyzing the activa- tion of a particular chemical to a cytotoxic or muta- genic metaboliteŽ. s . However, to specify the form Ž. s 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 alterna- tive strategy for mutagen screening is to use a cell line which coexpresses multiple P450 enzymes si- multaneously. Crespi et al.wx 145 introduced two Fig. 2. Mutagenic activation of IQŽ. A and MeIQx Ž. B in the cell lines expressing human CYP1A2 and NAT. The cell line A2R-5 distinct expression vectors which contained human Ž.` was established by transfecting CYP1A2 cDNA into CR-68 cDNAs encoding CYP1A2, CYP2A6, CYP2E1, cells. The cell lines ANM-13 Ž.B and ANP-25 Ž.' were estab- CYP3A4 and mEH into AHH-1-derived cell line L3 lished from A2R-5 cells by the transfection of NAT1 or NAT2 possessing a higher endogenous CYP1A1 activity. In cDNA, respectively. Top, relative survival rates after a 24-h the resulting MCL-5 cells, enzyme activities of the treatment with the test compounds determined by the colony formation assay. Bottom, mutant frequencies expressed as the introduced P450s and mEH were detected. Muta- number of 6-thioguanine-resistant cells per 106 clonable cells. genicity tests at the hprt and tk loci showed that the MCL-5 cells were much more sensitive to Bwxa P, oped by introducing mouse Cyp1a2 cDNA into the 3-MC, NDMA, NDEA, AFB1 and 2-AAF than the CHO-derived repair-deficient cell line UV5wx 47 . The AHH-1 cells which showed only low level of en- new cell lines, 5P3NAT2Ž. with Cyp1a2 and NAT2 dogenous CYP1A1 activitywx 145 . Regarding mi- and 5P3YGŽ. with Cyp1a2 and Salmonella OAT , cronucleus formation, the MCL-5 cells responded to showed extremely high sensitivity to IQ. There was a number of promutagens, including AFB1 , sterigma- no marked differences in the sensitivity between tocystin, Bwxa P, dibenz wa,h xanthracene, 3-MC, both new cell lines. cyclophosphamide, NDMA, NDEA, MeIQx, benzi- We established cell lines coexpressing human fe- dine, 2-AF, 2-AAF, benzene, tamoxifen, and tus-specific CYP3A7 and NAT, and examined the omeprazolewx 150 . These results indicate that the capacity of the cells for sensitivity to heterocyclic MCL-5 cells simultaneously expressing multiple amineswx 88 . The sensitivities to IQ, MeIQ, and forms of P450 are useful in screening assays for a MeIQx were enhancedŽ 4-, 30-, and 14-fold, respec- wide range of mutagens. MCL-5 cells are the sole tively, as compared with parental CR-68 cells. by the example expressing multiple forms of P450 to our introduction of CYP3A7 and NAT2Ž. but not NAT1 knowledge. cDNAs into the cells. These results are noteworthy When genetically engineered cells are used for from the point of view of developmental toxicology, genotoxicity assays, one should keep in mind a since the capacity to N-acetylate p-aminobenzoic chromosomal instability due to integrated DNA. El- acid and procainamideŽ. a substrate for NAT2 is at a lard et al.wx 151 observed in micronucleus assay that considerable level in human fetal liverswx 148,149 . a spontaneous frequency of micronuclei in V79-de- rived SD1 cells expressing rat CYP2B1 was higher than that in parental V79 cells, and that a marker 5. Future perspectives on the use of genetically chromosome with elongated p-arms was found in engineered cells SD1 cells but not in V79 cells. Fluorescence in situ hybridization revealed that this marker chromosome The genetically engineered cells expressing a spe- was associated with DNA amplification at the inte- cific form of P450 will become a powerful tool in gration site of the transfected CYP2B1-expression M. Sawada, T. KamatakirMutation Research 411() 1998 19±43 37 vector. There is a possibility that the integration of wx2 T. Pineau, P. Fernandez-Salguero, S.S.T. Lee, T. McPhail, externally added DNA may stimulate chromosomal J.M. Ward, F.J. Gonzalez, Neonatal lethality associated instability in the absence andror presence of geno- with respiratory distress in mice lacking cytochrome P4501A2, Proc. Natl. Acad. Sci. U.S.A. 92Ž. 1995 5134± toxic materials. In such a situation, specific P450 5138. inhibitors might be useful tools to confirm that the wx3 J. Doehmer, S. Dogra, T. Friedberg, S. Monier, M. Adesnik, effects of test compounds observed in genotoxic H. Glatt, F. Oesch, Stable expression of rat cytochrome assays are actually mediated by increase of catalytic P-450IIB1 cDNA in Chinese hamster cellsŽ. V79 and activity in the cells. Known in vitro specific in- metabolic activation of aflatoxin B1 , Proc. Natl. Acad. Sci. U.S.A. 85Ž. 1988 5769±5773. hibitors of P450 have been shown to inhibit the wx4 C.L. Crespi, R. Langenbach, K. Rudo, Y.-T. Chen, R.L. P450 activity in genetically engineered cells. a- Davies, Transfection of a human cytochrome P-450 gene into the human lymphoblastoid cell line, AHH-1, and use of Naphthoflavone inhibited the cytotoxicity of AFB1 in the cells expressing monkey CYP1A1wx 16 and the recombinant cell line in gene mutation assays, Carcino- Ž. human CYP1A2wx 39 . Fluvoxamine, sulfaphenazole, genesis 10 1989 295±301. wx5 C.L. Crespi, B.W. Penman, F.J. Gonzalez, H.V. Gelboin, quinidine, and 3-amino-1,2,4-triazole inhibited the M. Galvin, R. Langenbach, Genetic toxicology using human function of CYP1A2wx 152 , CYP2C10 wx 15 , CYP2D6 cell lines expressing human P-450, Biochem. Soc. Trans. wx67 , and CYP2E1 wx 79 , respectively. Ketoconazole 21Ž. 1993 1023±1028. wx wx81,15 and tiamulin wx 82 were shown to be effective 6 B.W. Penman, L. Chen, H.V. Gelboin, F.J. Gonzalez, C.L. against CYP3A4. Crespi, Development of a human lymphoblastoid cell line constitutively expressing human CYP1A1 cDNA: substrate In future studies, stable expression systems for specificity with model substrates and promutagens, Carcino- P450 enzymes are expected to be used more com- genesis 15Ž. 1994 1931±1937. prehensively and more deeply to predict human wx7 M. Christou, U.È Savas, D.C. Spink, J.F. Gierthy, C.R. genotoxicity of chemicals. For example, the systems Jefcoate, Co-expression of human CYP1A1 and a human will be valuable for a detailed comparison among a analog of cytochrome P450-EF in response to 2,3,7,8-tetra- chloro-dibenzo-p-dioxin in the human mammary carci- human P450 molecule and its animal counterparts. noma-derived MCF-7 cells, Carcinogenesis 15Ž. 1994 725± Although the species-difference in the metabolic ac- 732. tivation of promutagensrprocarcinogens still re- wx8 M. Shou, K.R. Korzekwa, K.W. Krausz, C.L. Crespi, F.J. mains to be studied, only a few reports in which a Gonzalez, H.V. Gelboin, Regio- and stereo-selective comparison was carried out using the stable expres- metabolism of phenanthrene by twelve cDNA-expressed human, rodent, and rabbit cytochromes P-450, Cancer Lett. sion system have been published. The stable expres- 83Ž. 1994 305±313. sion systems will also be applied to the comparative wx9 M. Shou, K.R. Korzekwa, C.L. Crespi, F.J. Gonzalez, H.V. study for human polymorphic P450 forms. This may Gelboin, The role of 12 cDNA-expressed human, rodent, and rabbit cytochromes P450 in the metabolism of provide valuable information concerning relation- wx wx ships between the genetic polymorphisms of P450 benzo a pyrene and benzo a pyrene trans-7,8-dihydrodiol, Mol. Carcinogen 10Ž. 1994 159±168. and cancer risks. In addition, coexpression systems wx10 J.C. States, T. Quan, R.N. Hines, R.F. Novak, M. Runge- for P450s and other enzymes which are possibly Morris, Expression of human cytochrome P450 1A1 in involved in the activation or inactivation of toxic DNA repair deficient and proficient human fibroblasts sta- substances are also expected to be developed for the bly transformed with an inducible expression vector, Car- Ž. estimation of the toxicological significance of the cinogenesis 14 1993 1643±1649. wx11 T. Quan, J.J. Reiners Jr., A.O. Bell, N. Hong, J.C. States, enzymes. These trials will provide valuable new Cytotoxicity and genotoxicity of Ž." -benzowxa pyrene-trans- information to predict the human toxicity of chemi- 7,8-dihydrodiol in CYP1A1-expressing human fibroblasts cals. quantitatively correlate with CYP1A1 expression level, Car- cinogenesis 15Ž. 1994 1827±1832. wx12 T. Quan, J.J. Reiners Jr., S.J. Culp, P. Richter, J.C. States, References Differential mutagenicity and cytotoxicity of Ž." - benzowxa pyrene-trans-7,8-dihydrodiol and Ž." -anti- wx1 Y. Li, T. Yokoi, R. Kitamura, M. Sasaki, M. Gunji, M. benzowxa pyrene-trans-7,8-dihydrodiol-9,10-epoxide in ge- Katsuki, T. Kamataki, Establishment of transgenic mice netically engineered human fibroblasts, Mol. Carcinogen 12 carrying human fetus-specific CYP3A7, Arch. Biochem. Ž.1995 91±102. Biophys. 329Ž. 1996 235±240. wx13 W.A. Schmalix, H. Maser,È F. Kiefer, R. Reen, F.J. Wiebel, 38 M. Sawada, T. KamatakirMutation Research 411() 1998 19±43

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2B1 cDNAs in micronucleus assays, Mutagenesis 6Ž. 1991 of aflatoxin B1 and comparison to CYP1A2 and CYP2A3, 461±470. Carcinogenesis 12Ž. 1991 355±359. wx22 S. Ellard, J.M. Parry, A comparative study of the use of wx34 C.L. Crespi, B.W. Penman, H.V. Gelboin, F.J. Gonzalez, A primary Chinese hamster liver cultures and genetically engi- tobacco smoke-derived nitrosamine, 4-Ž. methylnitrosamino - neered immortal V79 Chinese hamster cell lines expressing 1-Ž. 3-pyridyl -1-butanone, is activated by multiple human rat liver CYP1A1, 1A2 and 2B1 cDNAs in micronucleus cytochrome P450s including the polymorphic human cy- assays, Toxicology 82Ž. 1993 131±149. tochrome P4502D6, Carcinogenesis 12Ž. 1991 1197±1201. wx23 K.G. Jensen, A. Onfelt,È H.E. Poulsen, J. Doehmer, S. Loft, wx35 J.E. Snawder, A.L. Roe, R.W. Benson, D.A. Casciano, Effects of benzowxa pyrene and Ž." -trans-7,8-dihydroxy- D.W. Roberts, Cytochrome P450-dependent metabolism of 7,8-dihydrobenzowxa pyrene on mitosis in Chinese hamster acetaminophen in four human transgenic lymphoblastoid V79 cells with stable expression of rat cytochrome P4501A1 cell lines, Pharmacogenetics 4Ž. 1994 43±46. or 1A2, Carcinogenesis 14Ž. 1993 2115±2118. wx36 E.P. Gallagher, L.C. Wienkers, P.L. Stapleton, K.L. Kunze, wx24 U. Kulka, J. Doehmer, H.-R. Glatt, M. Bauchinger, Cytoge- D.L. Eaton, Role of human microsomal and human comple- netic effects of promutagens in genetically engineered V79 mentary DNA-expressed cytochromes P4501A2 and

Chinese hamster cells expressing cytochrome P450, Eur. J. P4503A4 in the bioactivation of aflatoxin B1 , Cancer Res. Pharmacol. 228Ž. 1993 299±304. 54Ž. 1994 101±108. wx25 S. Ellard, E.M. Parry, Induction of micronuclei in V79 wx37 C. Wolfel,È B. Heinrich-Hirsch, T. Schulz-Schalge, A. Sei- M. Sawada, T. KamatakirMutation Research 411() 1998 19±43 39

del, H. Frank, U. Ramp, F. Wachter,È F.J. Wiebel, F. bxpyridineŽ. PhIP in Chinese hamster ovary cells expressing Gonzalez, H. Greim, J. Doehmer, Genetically engineered murine cytochrome P450IA2, Mutagenesis 6Ž. 1991 253± V79 Chinese hamster cells for stable expression of human 259. cytochrome P450IA2, Eur. J. Pharmacol. 228Ž. 1992 95± wx49 R.W. Wu, E.M. Wu, L.H. Thompson, J.S. Felton, Identifi- 102. cation of aprt gene mutations induced in repair-deficient wx38 K.G. Jensen, H.E. Polsen, J. Doehmer, S. Loft, Paraceta- and P450-expressing CHO cells by the food-related muta- mol-induced spindle disturbances in V79 cells with and genrcarcinogen, PhIP, Carcinogenesis 16Ž. 1995 1207± without expression of human CYP1A2, Pharmacol. Toxicol. 1213. 78Ž. 1996 224±228. wx50 L.H. Thompson, R.W. Wu, J.S. Felton, Genetically modi- wx39 Y. Yanagawa, M. Sawada, T. Deguchi, F.J. Gonzalez, T. fied Chinese hamster ovaryŽ. 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