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Fetus-Specific CYP3A7 and Adult-Specific CYP3A4 Expressed in Chinese Hamster CHL Cells Have Similar Capacity to Activate Carcinogenic Mycotoxins1

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Fetus-Specific CYP3A7 and Adult-Specific CYP3A4 Expressed in Chinese Hamster CHL Cells Have Similar Capacity to Activate Carcinogenic Mycotoxins1 [CANCERRESEARCH55,787—791,February15,1995J Fetus-specific CYP3A7 and Adult-specific CYP3A4 Expressed in Chinese Hamster CHL Cells Have Similar Capacity to Activate Carcinogenic Mycotoxins1 HiSaShi Hashimoto, Tetsuya Nakagawa, Tsuyoshi Yokoi, Minoru Sawada,2 Susumu Itoh,3 and Tetsuya Kamataki4 Division of Drug Metabolism, Faculty of PharmaceuticalSciences,Hokkaido University, N12W6, Sapporo,Hokkaido 060, Japan ABSTRACT generally been evaluated by correlating the level of enzyme activities with the contentof specificformsof P450s in multiple samplesor by To assesswhether CYP3A4 and CYP3A7 have a similar capacity to inhibiting theseactivitieswith specificanti-P450 antibodies(13—15). activate carcinogenic rnycotoxins, we established cell lines stably express However, resultswith purified enzymesand antibodies(14, 15) are haghumanCYP3A4and CYP3A7,which are adult- and fetal-specific dependentonthe homogeneityof purified proteinsand the specificity forms of cytochrome P450 In human livers, respectively. Each cDNA was introduced into CR-119 cells which had beenestablishedby introducing of the antibodiesused.Recently, stableexpressionsystemsfor P450 guinea pig NADPH-cytochrome P450 reductase cDNA into Chinese ham by cDNA transfection have been developed by using a variety of sterlung cells. The cell lines (4-line and 7-line) stably expressed the mRNA cultured mammalian cell lines (16_26).6 This approach offers a new andtheproteincorrespondingtoCYP3A4andCYP3A7,respectively.The way to evaluate the catalytic activities of human P450s and is ex concentration-responsefor aflatoxin B, (AFB@)cytotoxicityIn 4-line and pectedto provide valuable information on the intracellularfunctions 7-line, respectively, was compared. 4—10and 7—40cells were approxi of individual P450s.Thus, the cells expressingP450 are applicableas mately 17- and 20 times more sensitive to ATh1 than the parental CR-119 useful tools for assayingof the cytotoxicity of xenobiotics. In previous cells,respectively.Inaddition,thesensitivitiestoAFB@ofboth4-10 and studies, we established cell lines stably expressing monkey CYP1A1 7—40cellswere enhanced approximately seven times by the addition of 10 from CHL fibroblastcellsanddemonstratedthatthe cellswere highly @Ma-naphthoflavone, a known activator of CYP3A enzyme, while the sensitiveto AFB@in both cytotoxicityand mutagenicity(23). Further sensitivities were suppressed approximately four times by the addition of 100 pi@itroleandomycin, which forms a metabolite intermediate complex introduction of guinea pig NADPH-cytochrorne P450 reductase with CYP3A enzyme, Moreover, both cell lines showed approximately 10 cDNA intothecellsexpressingCYP1A1 markedlyenhancedtheP450 and 2 timeshighersensitivitytosterigmatocystinandaflatoxinG1 than activity, indicatingthat simultaneousexpressionof the reductaseand CR-119 cells, respectively. These results indicate that CYP3A4 and P450 cDNAs was a strategyfavorableto enhancingthe activity of the CYP3A7 have essentially similar capacities to activate AFB1, sterigniato P450 system(24). In the presentstudy,by transfectionof CYP3A4 or cystin, and aflatoxin G1 to produce toxic metabolites. CYP3A7 cDNA into CR-119 cells which had been established from CHL cells by introducing the P450 reductase cDNA, we established cell lines carrying human CYP3A4 or CYP3A7 cDNA. Using these INTRODUCTION cells, we investigated the cytotoxicity of several carcinogenic myco Most environmental chemicals are carcinogenic and mutagenic toxins. Furthermore, we compared the capacitiesof CYP3A4 and only after undergoingmetabolic activation by a P450@-linkedmo CYP3A7 to activate each mycotoxin. nooxygenasesystem.P450 is a herne-containingenzymeand corn prises a supergene family containing 12 families in mammals (1). MATERIALS AND METHODS Among the families of P450, CYP3A is one of the major forms of P450in humanlivers. About 25% of the total P450presentin liver Materials. Anti-human P450 HFLa5C4 monoclonal IgG was a kind gift microsomesis composedof the CYP3A subfamily (2). This family from Bio-ScienceLaboratoryin theMochidaPharmaceuticalCompany.Anti consistsof at least four closely related enzymesincluding CYP3A3 rat CYP3A2antibodieswerea generousgiftfrom Dr. Y. Funaeof OsakaCity (3), CYP3A4 (4—6),CYP3A5 (7, 8) and CYP3A7 (9, 10) in human University MedicalSchool(27). ExpressionplasmidpMC1 ricopoly(A) waspurchasedfromStratagene(La livers. We have already found that CYP3A4 and CYP3A7 were Jolla,Ca). Bovinecalf serumwas purchasedfromHyCloneLaboratories expressedspecificallyin adult and fetal humanlivers, respectively,in (Logan, UT). G418 sulfate was purchased from Sigma Chemical Co. (St. spite of their high similarities in a sequencestructure(11); there is Louis, MO). AFB@was obtained from Makor Chemical (Jerusalem, Israel). 9i% similarity between5'-flanking sequencesof the CYP3A4and ca-NF and TAO were from Wako Pure Chemical Industries (Tokyo, Japan) and CYP3A7 gene (i2). However, the differences of the function between Sigma, respectively. CYP3A4 and CYP3A7 remainedto be elucidated. Construction of ExpressionPlasmid for CYP3A4 and CYP3A7. Full Purified preparations of P450 have been provided for analyzing lengthhumanCYP3A4andCYP3A7cDNAsclonedin our laboratory(10—12, catalyticpropertiesusingvarioussubstratesbyreconstitutingartificial 28)wereusedtoconstructexpressionplasmids. lipid and NADPH-cytochrome P450 reductase.The roles of the A briefprocedurefortheconstructionofthepSRa3A4(CYP3A4expres individual P450 form for carcinogenmetabolism in the liver have sionplasmid)andthepSRa3A7(CYP3A7expressionplasmid)areshownin Fig. 1. The plasmid,pBCNF1,containinga whole codingregionof CYP3A4 cDNA (NF25) wasconstructedbyligationof thefollowingtwoDNA frag Received10/4/94;accepted12/8/94. ments:(a) a 1.55-kbDdeI-SauIfragmentof NF2S,whosesiteswererendered Thecostsofpublicationofthisarticleweredefrayedinpartby thepaymentofpage charges.Thisarticle mustthereforebeherebymarkedadvertisementinaccordancewith flush by filling in; and (b) the 2.95-kb SmaI-HincII fragment of pBluescript 18 U.S.C.Section1734solely to indicatethis fact. [email protected] 1.57-kbXbaI-XhoIfragmentofpBCNF1wasblunt 1 T'@@work was supported in part by a Grant-in-Aid from the Ministry of Education, endedusingT4 DNA polymeraseandtheninsertedinto Pst I-digestedpUC ScienceandCultureof Japan. SRa plasmid(23,24), whosesiteswererenderedflushby fillingin. 2 Present address: Division of Environmental Hygiene, Hokkaido Institute of PharmaceuticalSciences,7-1Katsuraoka-cho,Otaru,Hokkaido047-02,Japan. The plasmid,pUCHFL1,containingthe whole coding region of CYP3A7 3 Present address: Division of BioMedical and Immunology, Faculty of Pharmaceu cDNA (HFL33) was constructedby ligation of the following two DNA tical Sciences, Osaka University, 1-6 Yasnadaoka, Suita, Osaka 565, Japan. fragments:(a) 1.52-kbSaul fragmentof HFL33, whosesiteswere rendered 4 To whom requests for reprints should be addressed. 5 The abbreviations used are: P450, cytochrome P450; CHL, Chinese hamster lung; AFB1, aflatoxin B,; AFG@,aflatoxin G,; a-NF, a-naphthoflavone (7,8-benzoflavone); 6 T. Yanagimoto, S. Itoh, H. Hashimoto, M. Sawada, and T. Kamataki. Mouse TAO, troleandomycin;kb,kilobase(s);IC@,concentrationofdrugrequiredfor50% cytochrome P450 (Cyp3a-1 1): examination for capacity to activate aflatoxin Bi, inhibition of cell growth. submittedfor publication. 787 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1995 American Association for Cancer Research. @ 1@r@ STABLE EXPRESSION OF CYP3A4 AND CYP3A7 D CYP3A4cDNA(NF25) s CYP3A7cDNA (HFL33) S and its derivative G418 when stably integrated into the genome of the recipient L I, cells. G418-resistantclones were selected in a medium containing G418 sulfate I Blunt-end at a concentration of 500 g.&g/ml.After3 weeks, 0418-resistant colonies were pBluescript SK+ IPstlhnker picked up and used for further investigations. @_—.----@;-;__ pUC18 Preparation of Total RNA and Microsomes. Total RNA was prepared by the acid-guanidinium phenol-chloroform method (30). The preparation of microsomes from transformants was performed by the method of Sawada et a!. (24). Northern Blot Analysis. For Northern blot analysis, total cellular RNA(10 i.@g)waselectrophoresed in a 0.8% agarose gel containing 6% formaldehyde and transferred to a nylon membrane (Nytran; Schleicher & Schuell, Keene, NH). The membranes were prehybridized for more than 12 h in 50 mMsodium phosphate buffer (pH 6.5) containing 50% formamide, 0.5X SSC, and 0.28 mg/ml denatured salmon sperm DNA at 42°Candhybridized overnight in the same buffer with the 32P-labeled hybridization pmbe at 42°C.The membranes Pst I were washed twice at a 30-mm interval with 0.5X SSC containing 0.1% SDS at 50°C. Western Blot Analysis. Western blot analysis was carried out according to the method of Sakuma et al. (31) using polyclonal antibodies to rat CYP3A2 (27) or synthesized peptide to human CYP3A7. Cytotoxicity Assays. Cytotoxicity assays were performed as follows. Cells were seeded at a density of 8 x 104/35-mmtissue culture dish and incubated for 24 h. Then, each mycotoxin dissolved in DMSO (final concentration, 0.5%) SRa promoter SRa promoter was added to the medium, and the cells were incubated for 48 h. The medium was removed, and the cells that were attached to dishes were rinsed with saline, fixed with 3.7% formaldehyde, and stained with 0.1% crystal violet solution for 10 min. The stained cells were washed with water and dried. Crystal violet from the stained cells was eluted with 10% SDS (1 ml).
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