PROCARCINOGENS BY CYTOCHROMES P 450 1 A 1 AND P 450 1 B 1 ALLELIC VARIANTS AND OTHER HUMAN CYTOCHROMES P 450 IN SALMONELLA TYPHIMURIUM NM 2009

A variety of polycyclic aromatic hydrocarbons and their dihydrodiol derivatives, arylamines, heterocyclic amines, and nitroarenes, were incubated with cDNA-based recombinant (Escherichia coli or Trichoplusia ni ) systems expressing different forms of human cytochrome P450 (P450 or CYP) and NADPH-P450 reductase using Salmonella typhimurium tester strain NM2009, and the resultant DNA damage caused by the reactive metabolites was detected by measuring expression of umu gene in the cells. Recombinant (bacterial) CYP1A1 was slightly more active than any of four CYP1B1 allelic variants, CYP1B1*1, CYP1B1*2, CYP1B1*3, and CYP1B1*6, in catalyzing activation of chrysene-1,2-diol, benz[a]anthracene-trans-1,2-, 3,4-, 5,6-, and 8,9-diol, fluoranthene-2,3-diol, dibenzo[a,l]pyrene, benzo[c]phenanthrene, and dibenz[a,h]anthracene and several arylamines and heterocyclic amines, whereas CYP1A1 and CYP1B1 enzymes had essentially similar catalytic specificities toward other procarcinogens, such as ( )-, ( )-, and ( )-benzo[a]pyrene-7,8-diol, 5-methylchrysene-1,2-diol, 7,12-dimethylbenz[a]anthracene-3,4-diol, dibenzo[a,l]pyrene-11,12-diol, benzo[b]fluoranthene-9,10-diol, benzo[c]chrysene, 5,6-dimethylchrysene-1,2-diol, benzo[c]phenanthrene-3,4-diol, 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene, 5-methylchrysene, and benz[a]anthracene. We also determined activation of these procarcinogens by recombinant (T. ni ) human P450 enzymes in S. typhimurium NM2009. There were good correlations between activities of procarcinogen activation by CYP1A1 preparations expressed in E. coli and T. ni cells, although basal activities with three lots of CYP1B1 in T. ni cells were very high without substrates and NADPH in our assay system. Using 14 forms of human P450s (but not CYP1B1) (in T. ni cells), we found that CYP1A2, 2C9, 3A4, and 2C19 catalyzed activation of several of polycyclic aromatic hydrocarbons at much slower rates than those catalyzed by CYP1A1 and that other enzymes, including CYP2A6, 2B6, 2C8, 2C18, 2D6, 2E1, 3A5, 3A7, and 4A11, were almost inactive in the activation of polycyclic aromatic hydrocarbons examined here. A variety of polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental carcinogens and have been shown to cause cellular transformations only after metabolic activation by so-called drug-metabolizing enzymes, such as cytochrome P450 (P450 or CYP) and epoxide hydrolase, that produce highly reactive carcinogenic electrophiles (Gelboin, 1980; Pelkonen and Nebert, 1982). In most cases, oxidation of PAHs by P450 enzymes is an initial step in the activation process. The resultant epoxide intermediates are usually more reactive than the parent compounds and have been shown to require further metabolism to evoke their critical carcinogenic potentials (Wood et al., 1976; Kapitulnik et al., 1978). These epoxide metabolites have been shown to be readily hydrolyzed to dihydrodiol metabolites (the suffix “dihydrodiol” or “diol” is used in the text to designate the prefix “dihydroxydihydro” for individual polycyclic hydrocarbons) by microsomal epoxide hydrolases and finally oxidized again by P450 enzymes to form highly reactive diol-epoxides (namely, bayand fjord-region epoxides) that interact with DNA to initiate cell transformation (Conney, 1982). In these events, P450 enzymes play key roles, and efforts have been made to identify which P450 species are more active in catalyzing activation of PAHs and PAH dihydrodiols in experimental animals and humans (Pelkonen and Nebert, 1982; Shimada et al., 1989a,b; Gonzalez et al., 1990; Jacob et al., 1996; Shou et al., 1996a,b; Einolf et al., 1997). Among the various forms of P450 determined so far, CYP1A1 and CYP1B1 have been shown to be the most important human P450 enzymes in the metabolic activation of PAHs and PAH dihydrodiols (Hall et al., 1989; Schmalix et al., 1993; Gautier et al., 1996; Shimada et al., 1996, 1999a; Kim et al., 1998; Luch et al., 1998). Both enzymes are expressed predominantly in extrahepatic organs, such as lungs and mammary glands, and it has been suggested that levels of expression and catalytic properties of these P450 enzymes may determine differThis work was supported in part by grants from the Ministry of Education, Science, and Culture of Japan and the Ministry of Health and Welfare of Japan (T.S.); by United States Public Health Service Grants R35 CA44353 and P30 ES00267 (F.P.G.); and by the Kathleen Cuningham Foundation for Breast Cancer Research (E.M.J.G.). 1 Abbreviations used are: PAH, polycyclic aromatic hydrocarbon; P450, general term for cytochrome P450; CYP, individual forms of P450; B[a]P, benzo[a]pyrene; 7,12-DMBA, 7,12-dimethylbenz[a]anthracene; DB[a,l]P, dibenzo[a,l]pyrene; MeIQ, 2-amino-3,5-dimethylimidazo[4,5-f]quinoline. Address correspondence to: Dr. Tsutomu Shimada, Osaka Prefectural Institute of Public Health, 3-69 Nakamichi 1-chome, Higashinari-ku, Osaka 537-0025, Japan. E-mail: shimada@iph.pref.osaka.jp 0090-9556/01/2909-1176–1182$3.00 DRUG METABOLISM AND DISPOSITION Vol. 29, No. 9  2017 dm d.aspurnals.org D ow nladed from ent susceptibilities of individuals toward lung and breast cancers caused by these procarcinogens (Huang et al., 1996; Shimada et al., 1996; Shimada, 2000). Induction of enzymes by environmental chemicals and polymorphisms in P450 genes have been shown to be major factors causing differences in expression and altered catalytic activities in P450 enzymes, and the latter has been shown to be more important (Coughlin and Piper, 1999; Guengerich, 2000; Shimada, 2000). In fact, numerous epidemiological studies have suggested that genetic polymorphisms in CYP1A1 and CYP1B1 genes may produce differences in lung and breast cancer susceptibilities in humans (Kawajiri et al., 1990; Hayashi et al., 1991; Kawajiri and FujiiKuriyama, 1991; Nebert et al., 1999; Watanabe et al., 2000). Catalytic specificities of mutated enzymes have been determined in many laboratories using cDNA-based recombinant P450 systems to examine whether genetic polymorphisms cause alterations in protein catalytic activities toward environmental PAH procarcinogens (Guengerich et al., 1996; Doehmer et al., 1999; Shimada et al., 1999c, 2000). For example, replacement of isoleucine by valine at residue 462 in CYP1A1 produced a slight increase in the activity of B[a]P oxidation (Hayashi et al., 1991; Zhang et al., 1996). We have recently compared activities of activation of 12 PAHs, five heterocyclic amines, 2-aminofluorene, and 3-methoxy-4-aminoazobenzene by CYP1B1*1 and CYP1B1*3 in a tester strain Salmonella typhimurium NM2009 and found that the Leu432Val substitution causes small changes in the catalytic properties (Shimada et al., 1999c). However, it remains unclear whether other types of CYP1B1 genetic polymorphisms change catalytic specificities toward a variety of environmental procarcinogens. In this study, we further compared activities of metabolic activation of a number of PAHs and PAH dihydrodiols and other procarcinogens by recombinant (Escherichia coli and Trichoplusia ni cells) human P450 enzymes using a genotoxicity assay based on S. typhimurium NM2009 tester strain (Shimada et al., 1994a). The aims of the current study were 1) to determine differences in catalytic activities of CYP1A1 and CYP1B1 toward activation of seven PAHs, 21 PAH dihydrodiols, and 9-hydroxy-B[a]P to reactive metabolites in this assay system; 2) to compare activities of procarcinogen activation in CYP1B1*1 (ArgAlaLeuAsn), CYP1B1*2 (GlySerLeuAsn), CYP1B1*3 (ArgAlaValAsn), and CYP1B1*6 (GlySerValAsn); 3) to examine differences in catalytic activities of human P450 enzymes using recombinant systems in E. coli and T. ni cells; and 4) to determine activities of other recombinant (T. ni) human P450s, including CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, 3A7, and 4A11 with PAHs and PAH dihydrodiols.  B[a]P and benz[a]anthracene were purchased from Sigma (St. Louis, MO) and Aldrich (Milwaukee, WI) respectively. 7,12-DMBA, DMBA-trans-3,4-diol, DMBA-cis-5,6-diol, chrysene-1,2-diol, 5-methylchrysene-1,2-diol, 5,6-dimethylchrysene-1,2-diol, dibenzo[a,l]pyrene-11,12diol, benzo[g]chrysene-11,12-diol, and benzo[c]phenanthrene-3,4-diol were kindly donated by Dr. Stephen Hecht (University of Minnesota, Minneapolis, MN). Other chemicals and reagents used in this study were obtained from sources described previously or were of the highest qualities commercially available (Shimada et al., 1994b, 1999a,c).  CYP1A1, CYP1A2, and four allelic variants of human CYP1B1, namely, CYP1B1*1 (ArgAlaLeuAsn), CYP1B1*2 (GlySerLeuAsn), CYP1B1*3 (ArgAlaValAsn), and CYP1B1*6 (GlySerValAsn) were expressed together with human NADPH-P450 reductase in E. coli, and the enzymes thus produced in membranes of the bacteria were used as described previously (Shimada et al., 2000, 2001). Yields of P450, as determined by the original spectral method (Omura and Sato, 1964), ranged between 40 and 250 nmol/l of medium, respectively, and all of the CYP1B1 preparations showed wavelength maxima at 446 nm for the reduced CO complex. NADPH-P450 reductase expression in these bacterial membranes ranged from 25 to 75 nmol/l of culture, and the P450/reductase ratio was determined to be 2 to 5. Recombinant CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, 3A7, and 4A11 expressed in microsomes of T. ni cells infected with a baculovirus containing human P450 and NADPH-P450 redu