Bioactivation of diesel exhaust particle extracts and their major nitrated polycyclic aromatic hydrocarbon components, 1-nitropyrene and dinitropyrenes, by human cytochromes P450 1A1, 1A2, and 1B1

Mutation Research 472 (2000) 129–138

Bioactivation of diesel exhaust particle extracts and their major nitrated polycyclic aromatic hydrocarbon components, 1-nitropyrene and dinitropyrenes, by human cytochromes P450 1A1, 1A2, and 1B1 Hiroshi Yamazaki a , Naoya Hatanaka a , Ryoichi Kizu a , Kazuichi Hayakawa a , Noriaki Shimada b , F. Peter Guengerich c , Miki Nakajima a , Tsuyoshi Yokoi a,∗ a

Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-0934, Japan b Daiichi Pure Chemicals Co., Ibaraki 319-1182, Japan c Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA

Received 31 May 2000; received in revised form 22 August 2000; accepted 2 October 2000

Abstract The genotoxicities of four samples of diesel exhaust particle (DEP) extracts (DEPE) and nine nitroarenes found in DEPE were investigated after activation catalyzed by human cytochrome P450 (P450) family 1 enzymes co-expressed with NADPH-cytochrome P450 reductase (NPR) in Escherichia coli membranes. The DEPE samples induced umu gene expression in Salmonella typhimurium TA1535/pSK1002 without any P450 system and were further activated by human P450 1B1/NPR membranes. Moderate activation of the DEPE sample by P450 1A2/NPR membranes was also observed, but not by either P450 1A1/NPR or NPR membranes. 1-Nitropyrene (1-NP) was strongly activated by human P450 1B1/NPR membranes. 1,8-Dinitropyrene (1,8-DNP) was most highly activated by P450 1A1 and 1B1 systems for the three DNPs tested. In contrast, 1,3-DNP was inactivated by P450 1A1/NPR, 1A2/NPR, and 1B1/NPR systems and slightly activated by NPR membranes. 2-Nitrofluoranthene (2-NF) and 3-nitrofluoranthene (3-NF) showed activities similar to 1-NP after bioactivation by P450 1B1/NPR membranes. However, the genotoxicities of 6-nitrochrysene, 7-nitrobenz[a]anthracene, and 6-nitrobenzo[a]pyrene were all weak in the present assay system. Apparent genotoxic activities of DEPE were very low compared with standard nitroarenes in the presence of P450s, possibly because unknown component(s) of DEPE had inhibitory effects on the bioactivation of 1-NP and 1,8-DNP catalyzed by human P450 1B1. These results suggest that environmental chemicals existing

Abbreviations: 1,3-DNP: 1,3-dinitropyrene; 1,6-DNP: 1,6-dinitropyrene; 1,8-DNP: 1,8-dinitropyrene; 7-NB[a]A: 7-nitrobenz[a]anthracene; 6-NB[a]P: 6-nitrobenzo[a]pyrene; 1-NP: 1-nitropyrene; 2-NF: 2-nitrofluoranthene; 3-NF: 3-nitrofluoranthene; DEP: diesel exhaust particles; DEPE: DEP extracts; NPR: NADPH-cytochrome P450 reductase; P450: cytochrome P450 ∗ Corresponding author. Tel.: +81-76-234-4407; fax: +81-76-234-4407. E-mail address: [email protected] (T. Yokoi). 1383-5718/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 ( 0 0 ) 0 0 1 3 8 - 8

130

H. Yamazaki et al. / Mutation Research 472 (2000) 129–138

in airborne DEP, in addition to 1-NP, 1,6-DNP, 1,8-DNP, 2-NF, and 3-NF, can be activated by human P450 1B1. Biological actions of air pollutants such as nitroarenes to human extrahepatic tissues may be of concern in tissues in which P450 1B1 is expressed. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Diesel exhaust particles extracts; Nitropyrene; Dinitropyrene; Human P450 1B1; umu gene expression; S. typhimurium TA1535/pSK1002

1. Introduction Cytochrome P450 (P450) comprises a superfamily of enzymes that collectively catalyze a great variety of oxidations of endobiotics and xenobiotic chemicals such as drugs, protoxicants, and procarcinogens [1–3]. The relationship between family 1 P450s and chemical carcinogenesis by polycyclic hydrocarbons such as benzo[a]pyrene has been extensively studied [4,5] and the induction of P450s and the activation of procarcinogens have been examined [6]. Human P450s 1A1, 1A2, and 1B1 have overlapping substrate specificities [7,8]. P450s 1A1, 1A2, and 1B1 are regulated in part by the Ah locus; human P450 1A2 is expressed essentially only in the liver, but P450s 1A1 and 1B1 are both expressed in many extrahepatic tissues [7]. P450s 1A1 and 1A2 have been well-characterized in rodents and humans [3]. The other family 1 member, P450 1B1, was discovered more recently. Sutter et al. [9,10] first reported the existence of human P450 1B1 in the early 1990s. The role of human P450 1B1 in breast cancer has been studied following the demonstration of 17␤-estradiol 4-hydroxylation activity [11,12]. The risk to humans from air pollutants and their constituent environmental chemicals is an important issue. 1-Nitropyrene (1-NP) is one of the major components of airborne particles [13]. Analyses have indicated that 1-NP and 1,3-, 1,6-, and 1,8-dinitropyrenes (DNPs) are major nitroarene components of airborne and diesel exhaust particles (DEP) [14–16]. Diesel engine emission appears to be a principal source for contaminants, and crude extracts from DEP can be used as model compounds to investigate air pollutants. 2-Nitrofluoranthene (2-NF), 3-nitrofluoranthene (3-NF), 6-nitrochrysene, 6-nitrobenzo[a]pyrene (6-NB[a]P), and 7-nitrobenz[a]anthracene (7-NB[a]A) have also been detected in airborne particles or diesel exhaust samples [17–20]. Many of these nitroarenes have direct-acting mutagenicity in Salmonella typhimurium tester strains, i.e. the Ames test or the umu

assay [21–23]. In contrast, some of these nitropolycyclics have been reported to be non-mutagenic in other assay systems [24]. The nitroarenes are also activated by nitroreductase [25] or O-acetyltransferase [26] to N-hydroxylated intermediates in bacterial strains. Much work on the metabolism and mutagenic activation or inactivation of nitropyrenes by human P450s 1A1 and 1A2 was reported in the early 1990s [27–29]. Inactivation of 1,3-, 1,6-, and 1,8-DNPs by human liver microsomal P450s has been reported in the umu assay [29]. A role for human P450 1B1 was not considered in previous studies because the discovery of P450 1B1 was more recent. We have studied genotoxicity by co-incubation of chemicals, human P450 family 1 enzymes, and a bacterial tester strain. The present study was, therefore, undertaken to determine which P450 family 1 enzymes are most important in activating environmental crude samples, as well as nitropyrenes and nitrofluoranthenes, to genotoxic products in S. typhimurium TA1535/pSK1002. Recombinant human P450 1B1 and NPR expressed in Escherichia coli membranes (P450 1B1/NPR membranes) [30] were used as enzyme sources in this study. The inhibitory effects of diesel exhaust particle extracts (DEPE) on P450 1B1-mediated activation of nitropyrenes and 7-ethoxycoumarin O-deethylation are also described.

2. Methods 2.1. Chemicals 1-Nitropyrene (CAS 5522-43-0) (purity 99.9%) was obtained from Tokyo Chemical Industry (Tokyo, Japan) and no dinitropyrenes were detected by HPLC chromatography (<0.01%). Pyrene (129-00-0) (>99%), 1,3-DNP (75321-20-9) (>99%), 1,6-DNP (42397-64-8) (>99%), and 1,8-DNP (42397-65-9) (>99%) were purchased from Aldrich Chemical

H. Yamazaki et al. / Mutation Research 472 (2000) 129–138

Co. (Milwaukee, WI, USA) and 3-NF (892-21-7) (>99%) from Wako Pure Chemicals (Osaka, Japan). 2-NF (13177-29-2) (>99%) and 6-nitrochrysene (7496-02-8) (>99%) were obtained from Chemsyn Science Laboratories (Lenexa, KS, USA). 7-NB[a]A (20268-51-3) (>99%) and 6-NB[a]P (63041-90-7) (>99%) were obtained from the National Cancer Institute (Bethesda, MD, USA). Other chemicals and reagents used in this study were obtained from sources described previously or were of the highest quality commercially available [31,32]. 2.2. Preparation of DEPE Sampling and extraction of DEP were carried out according to the method described previously [16] with minor modifications. Briefly, DEP were collected on glass-fiber filters (55 mm i.d.) at a flow rate of 30 l/min with a low volume air sampler positioned 0.3 m from the end of an exhaust pipe of an idling engine. DEP (10–15 mg from 10 sheets of filters) were extracted with benzene–ethanol (3:1) using ultrasonication. The organic phase was evaporated to dryness and the residue was dissolved in ethanol (20 mg/ml). The crude extracts from DEP samples, designated as DEPE-1, -2, -3, and -4, were prepared from four diesel engine vehicles (1993 model, 2.8 l; 1996 model, 2.5 l; 1990 model, 4.1 l; 1989 model, 7.4 l), respectively. The amounts of 1-NP and 6-nitrochrysene in the DEPE used in this study were determined according to the method of Hayakawa et al. [33] to be 4–63 and 4–160 pmol/mg extract, respectively (Table 1). The amounts of 1,3-, 1,6-, and 1,8-DNP were 0.1–0.4, 0.2–2.4, and 0.5–2.8 pmol/mg extract, respectively. The major components of the DEPE used were polycyclic hydrocarbons such as benz[a]anthracene, benzo[a]pyrene, benzo[ghi]perylene, benzo[b]fluoranthene, and benzo[k]fluoranthene (6–23 nmol/mg extract). 2.3. Enzyme preparations Membranes were prepared from E. coli cells into which P450 1A1, 1A2 or 1B1 cDNAs had been introduced along with NPR as described previously [30,34]. Briefly, single transformed colonies of E. coli strain DH5␣ were used to

131

Table 1 Constituents of DEPEs used in this study Chemicals

DEPE-1 DEPE-2 DEPE-3 DEPE-4

Polycyclic aromatic hydrocarbons (nmol/mg extract) Benz[a]anthracene 12 17 23 Benzo[b]fluoranthene 10 15 11 Benzo[k]fluoranthene 6 9 9 Benzo[a]pyrene 7 9 12 Benzo[ghi]perylene 6 14 14

NDa ND ND ND ND

Mononitro compounds (pmol/mg extract) 1-NP 4.3 29 6-Nitrochrysene 4.1 23 7-NB[a]A 0.6 2.4 6-NB[a]P 0.4 5.5

ND ND ND ND

63 160 14 37

Dinitropyrenes 1,3-DNP 1,6-DNP 1,8-DNP

ND ND ND

a

0.16 0.24 0.48

0.12 0.45 0.58

0.37 2.4 2.8

Not determined.

inoculate starter Luria-Bertani media/ampicillin (25 ␮g/ml) cultures. The starter cultures were incubated for 7 or 15 h at 37◦ C with shaking at 170 rpm and then diluted for 1:100 into Terrific Broth/ampicillin (100 ␮g/ml) medium containing additives (0.5 mM ␦-aminolevulinic acid, 1.0 mM isopropyl ␤-d-thiogalactoside, trace salts, and 1.0 mM thiamine) [31]. The expression cultures (100 ml) were grown at 30◦ C with shaking at 120 rpm for 24 h in 500 ml triple-baffled flasks. Membrane fractions were prepared from the bacterial pellets by high speed centrifugation steps [35] and suspended in 10 mM Tris–HCl buffer (pH 7.4) containing 0.10 mM EDTA and 20% (v/v) glycerol. Other recombinant P450 enzymes including P450 1A1, 1A2, and 1B1 in microsomes from B-lymphoblastoid cells or insect cells with baculovirus system co-expressing NPR were obtained from Gentest Co. (Woburn, MA). 2.4. Assay methods The genotoxicities of chemical carcinogens and mutagens were determined by measuring induction of umu gene expression in a Salmonella tester strain as described previously [36,37]. Briefly, overnight cell cultures of S. typhimurium TA1535/pSK1002 were grown in 1% (w/v) bactotryptone, 0.2% (w/v) glucose, 0.5% (w/v) NaCl medium containing 20 ␮g

132

H. Yamazaki et al. / Mutation Research 472 (2000) 129–138

ampicillin/ml until the OD600 of the bacterial culture was ∼0.2. The resulting bacterial suspension was incubated with chemicals dissolved in dimethyl sulfoxide, or an ethanol solution of DEPE at 37◦ C for 2 h, and the expressed ␤-galactosidase activity was determined by the method of Miller [38] and expressed in those units using o-nitrophenyl-␤-d-galactopyranoside as a substrate. Spontaneous background levels of umu gene expression (∼120 ± 20 units/ml) were subtracted when we presented umu gene expression without enzymes activation systems. P450-dependent activation or inactivation of procarcinogens that cause induction of umu gene expression in S. typhimurium TA1535/pSK1002 was carried out with E. coli membranes expressing P450 and NPR (containing 0.01 ␮M P450), as described previously [37]. Incubation mixtures (final volume of 1.0 ml) consisted of E. coli membranes with various concentrations of procarcinogens in 0.25 ml of 200 mM potassium phosphate buffer (pH 7.4) containing an NADPH-generating system and 0.75 ml of bacterial suspension. For inactivation of the enzyme activities, heat treatment (100◦ C, 2 min) of the tubes (before adding test chemicals and the bacteria) was carried out. Incubations were carried out at 37◦ C for 2 h and terminated by cooling the mixtures on ice. The umu gene expression was monitored by measuring ␤-galactosidase activities as described above and expressed as units/min/nmol P450 or NPR. In preliminary experiments, bioactivation of 1-NP (1 ␮M) and 1,8-DNP (0.1 ␮M) was dependent linearly on P450 1B1 concentrations within the range of 0.005–0.015 and 0.002–0.015 ␮M P450 1B1, respectively, using P450 1B1/NPR membranes. When the basic conditions for umu assay with 0.01 nmol P450 and an incubation time of 120 min were used, the calculated metabolic activation (units/min/nmol P450) were similar to the simple subtraction value for the heat blanks unit (from sample units). Results presented in this study were the means of duplicate or triplicate determinations and the S.D. (or ranges). O-deethylation activities of 7-ethoxyresorufin and 7-ethoxycoumarin were determined by the methods described previously [39,40]. These activities of P450 1A1/NPR membranes, P450 1A2/NPR membranes, and 1B1/NPR membranes used in this study were 95 and 32, 6 and 0.5, and 51 and 7.2, respectively.

2.5. Other assays Concentrations of P450 and protein were determined spectrally by the methods of Omura and Sato [41] and Lowry et al. [42], respectively. NADPH-cytochrome c reductase activities were determined as described [43] using 1ε550 =21 mM−1 /cm−1 and an assumed specific activity of 3.0 ␮mol cytochrome c reduced/min/nmol NPR based on purified human and rabbit NPR preparations [34]. Molar ratios for P450 1A1/NPR membranes, P450 1A2/NPR membranes, and 1B1/NPR membranes used in this study were 0.82, 0.28, and 1.03, respectively.

3. Results 3.1. Genotoxicity of DEPE samples in S. typhimurium TA1535/pSK1002 before and after activation by recombinant human P450 family 1 enzymes Four different DEPE samples, designated DEPE-1– DEPE-4, were used to investigate umu gene expression in S. typhimurium TA1535/pSK1002 induced by crude environmental chemicals. DEPE-1 showed some activities in the absence of activation systems over the range of 40–100 ␮g/ml (Fig. 1A). Two other samples also induced umu gene expression to a small extent, but DEPE-2 did not show genotoxicity under the conditions used. DEPE-1 was activated most effectively by human P450 1B1 co-expressed with human NPR in E. coli membranes (Fig. 1B), followed by P450 1A2/NPR membranes. Genotoxicity of DEPE-1 catalyzed by P450 1A1/NPR or NPR was at the background level of umu gene expression. Recombinant human P450 1B1 activated all the DEPE samples (Fig. 1C). DEPE-2 showed similar activities at high concentrations comparable to those of DEPE-1 at low doses. 3.2. Genotoxicity of 1-NP, DNPs, and NFs in S. typhimurium TA1535/pSK1002 before and after activation by recombinant human P450 family 1 enzymes The above results suggested that these chemical extracts have some direct-acting genotoxicity but were further activated by human P450 1B1. Since nitrated

H. Yamazaki et al. / Mutation Research 472 (2000) 129–138

133

Fig. 1. Genotoxicity of DEPE in S. typhimurium TA1535/pSK1002. (A) umu gene expression of four samples of DEPE in the absence of activation systems; (B) bioactivation of a sample designated DEPE-1 by human P450s 1A1/NPR membranes (䊉), 1A2/NPR membranes (䉱), and 1B1/NPR membranes (䊏) and by human NPR membranes (䊊), and (C) bioactivation of four samples of DEPE by human P450 1B1/NPR membranes. Spontaneous background levels of umu gene expression (∼120 ± 20 units/ml) were subtracted. Results are presented as means of duplicate determinations and S.D. (range).

aromatic hydrocarbons are known to show direct activities and to be metabolically activated, we examined the genotoxicity of nitropyrenes and analyzed umu gene expression of some major nitrated polycyclic hydrocarbons, 1-NP and three DNPs (Fig. 2). 1-NP and 1,3-, 1,6-, and 1,8-DNP showed direct activities in S. typhimurium TA1535/pSK1002; 1,3- and 1,8-DNP induced high activities among those chemicals tested (Fig. 2A and B). P450 1A2/NPR membranes activated 1-NP at low 1-NP concentrations; 1-NP was highly activated by P450 1B1/NPR membranes at high 1-NP concentrations (Fig. 2C). P450 1A1/NPR membranes inactivated 1-NP, and NPR membranes alone weakly activated 1-NP. In contrast, 1,3-DNP was weakly activated by NPR membranes, but P450 1A1/NPR, 1B1/NPR, and 1A2/NPR systems inactivated 1,3-DNP (Fig. 2D). 1,6-DNP was weakly activated by P450 1A1/NPR, 1A2/NPR, and 1B1/NPR membranes (Fig. 2E) and slightly activated by NPR membranes at high concentrations (of 1,6-DNP). The genotoxicity of 1,8-DNP following activation by P450 1A1/NPR and 1B1/NPR oxidation systems was the highest among these individual chemicals (Fig. 2F). P450 1A1/NPR membranes inactivated 1,8-DNP at low concentrations and P450 1A2/NPR membranes also inactivated 1,8-DNP at all concentrations tested. We also analyzed the umu-induced gene expression of 2-NF, 3-NF, 6-nitrochrysene, 7-NB[a]A, and

6-NB[a]P (Fig. 3). 3-NF showed high direct-acting genotoxicity in S. typhimurium TA1535/pSK1002; however, direct activities of 2-NF and 6-nitrochrysene were weak and 7-NB[a]A and 6-NB[a]P did not induce umu gene expression (Fig. 3A). Although P450 1A2/NPR membranes activated 2-NF at a concentration of 0.1 ␮M, 2-NF was highly activated by P450 1B1/NPR membranes at a 2-NF concentration of 1 ␮M (Fig. 3B). P450 1A1/NPR also activated 2-NF. 3-NF was moderately activated by P450 1A2/NPR and 1B1/NPR and slightly activated by P450 1A1/NPR (Fig. 3C). Genotoxic activities of 6-nitrochrysene, 7-NB[a]A, and 6-NB[a]P catalyzed by P450s 1A1/NPR, 1A2/NPR, and 1B1/NPR systems were very low or undetectable in this assay system (Fig. 3D–F). 3.3. Inhibitory effects of DEPE on human P450 1B1-catalyzed activation of 1-NP and 1,8-DNP Since the genotoxic activities of DEPE and standard 1-NP and DNPs were very different, the inhibitory effects of DEPE on P450 1B1-catalyzed activation of 1-NP and 1,8-DNP were investigated. The activities of 1-NP (3 ␮M) and 1,8-DNP (0.3 ␮M) in the presence of heat-inactivated or intact P450 1B1/NPR membranes were plotted against the concentration of DEPE-1 (Fig. 4A). Although DEPE-1 showed weak direct umu gene expression in the presence

134

H. Yamazaki et al. / Mutation Research 472 (2000) 129–138

Fig. 2. Genotoxicity of 1-NP and DNPs in S. typhimurium TA1535/pSK1002. (A) umu gene expression of 1-NP without activation systems; (B) umu gene expression of 1,3-DNP (䊊), 1,6-DNP (䉭), and 1,8-DNP (䊐) in the absence of activation systems. (C–F), Bioactivation of 1-NP (C), 1,3-DNP (D), 1,6-DNP (E), and 1,8-DNP (F) by human P450 1A1/NPR membranes (䊉), 1A2/NPR membranes (䉱), and 1B1/NPR membranes (䊏) and by human NPR membranes (䊊). In parts C–F, the umu response was measured in the absence and presence of the enzyme system; negative values indicate inactivation. Results are presented as means of triplicate determinations and S.D.

of heat-inactivated P450 1B1/NPR membranes, umu gene expression caused by 1-NP or 1,8-DNP was not changed by adding DEPE-1. On the other hand, the genotoxic activities of 1-NP and 1,8-DNP following oxidation by P450 1B1/NPR membranes were decreased by adding DEPE-1. In these assay conditions, bacterial growth (monitored at OD600 ) did not decrease in the presence of DEPE-1. These results sug-

gest that chemicals present in DEPE (DEPE-1) might have inhibitory effects on human P450-catalyzed activation of nitropyrenes. Inhibitory effects of DEPE were also observed for 7-ethoxycoumarin O-deethylation activities catalyzed by P450 1B1/NPR membranes (Fig. 4B). Similar inhibitory effects of DEPE-1 were observed for 7-ethoxycoumarin O-deethylation. These results

H. Yamazaki et al. / Mutation Research 472 (2000) 129–138

135

Fig. 3. Genotoxicity of 2-NF, 3-NF, 6-nitrochrysene, 7-NB[a]A, and, 6-NB[a]P in S. typhimurium TA1535/pSK1002. (A) umu gene expression of 2-NF (䊊), 3-NF (䉭), 6-nitrochrysene (䊐), 7-NB[a]A (䊏), and 6-NB[a]P (䉱) in the absence of activation systems. (B–F), Bioactivation of 2-NF (B), 3-NF (C), 6-nitrochrysene (D), 7-NB[a]A (E), and 6-NB[a]P (F) by human P450 1A1/NPR membranes (䊉), 1A2/NPR membranes (䉱), and 1B1/NPR membranes (䊏) and by human NPR membranes (䊊). Background levels of umu gene expression were subtracted. Results are presented as means of triplicate determinations and S.D.

might provide evidence for competition among multiple substrates for effects of DEPE components on P450 1B1 activities.

4. Discussion We demonstrated in this work that crude ethanol– benzene extracts from DEP induce some direct umu gene expression in S. typhimurium TA1535/pSK1002, and further activation was shown with recombinant human P450 1B1. There was some variability of direct-acting activities among four DEPE samples, suggesting that DEP components differ among the engines of the vehicles. In the present studies, genotoxic activities of all DEPE samples (subtracting levels of umu gene expression without activation sys-

tems) were clearly enhanced by recombinant human P450s 1B1 or 1A2 (Fig. 1). We focused on the roles of human P450 1B1 in bioactivation of environmental pollutants from diesel emissions. Human P450 1B1 may have activities with nitroaromatics and account for the activation of diesel exhaust. One of the reasons for the higher activities of the pure nitropyrenes than DEPE may simply be that DEPE has little of the nitropyrenes. However, the other reason for the apparently low activities of DEPE with activation systems (compared with those of standard nitropolycyclics) may be the inhibitory effects of DEPE on catalytic activities of P450 1B1 (Fig. 4). Decreased activities of some DEPE at high substrate concentrations (Fig. 1) might be partly related to this inhibitory effect. The apparent genotoxic activities of DEPE seemed not to be related to the contents of any of the compounds

136

H. Yamazaki et al. / Mutation Research 472 (2000) 129–138

Fig. 4. Inhibitory effects of DEPE-1 on bioactivation of 1-NP and 1,8-DNP (A) and on 7-ethoxycoumarin O-deethylation activities; (B) catalyzed by human P450 1B1/NPR membranes in the presence of S. typhimurium TA1535/pSK1002. (A) 1-NP (3.0 ␮M) or 1,8-DNP (0.3 ␮M) was incubated with DEPE-1 (0–100 ␮g/ml) and heat-inactivated or intact P450 1B1/NPR membranes. (B) Control activity of 7-ethoxycoumarin O-deethylation (at 100 ␮M) without DEPE was 8.2 nmol/min/nmol P450 1B1. Results are presented as means of duplicate determinations and S.D. (range).

we measured. However, it is necessary to investigate more samples to better establish relationships between genotoxic activities and other chemical ingredients. We did not elucidate the mechanism of inhibition by DEPE and did not directly prove that DEPE components suppress a direct-acting umu response induced by DEPE in this study, although DEPE constituents have been reported to suppress the direct-acting mutagenicity in the Ames test [16]. DEP contains both inhibitors of P450s and promutagens activated by human P450 family 1 enzymes. Cigarette smoke condensate also has many chemical ingredients that both cause DNA damage and inhibit metabolic activation of procarcinogens [44]. 1-NP, 2-NF, and 3-NF showed similar genotoxic activities after bioactivation by human P450 1B1/NPR membranes. 1,6- and 1,8-DNP, which both have two nitro groups in different aromatic rings, showed much higher activities at lower substrate concentrations (catalyzed by P450 1B1/NPR membranes) than those of mononitrated compounds. 1-NP is one of the major genotoxic components of DEPE [15,16]. 1-NP is metabolized essentially via two routes, nitroreduction and P450-mediated C-oxidation [26,27]. Nitroreduction has been demonstrated in bacterial and mammalian cells [24,25]. Human P450-mediated 1-NP activation and C-oxidation have been reported pre-

viously [27,36]. In assays with a human HepG2 cell line, P450-mediated C-oxidative pathways of 1-NP have been shown to be detoxification reactions [24]. Recently, Chae et al. [45] reported the metabolism of mononitropyrenes by human hepatic and pulmonary microsomes. One of their conclusions was that P450 3A4 had a number of activities, including nitroreduction of 4-NP [45]. However, the roles of P450 1B1 involved in activation had not been clarified. In this study, roles of bioactivation of DEPE and standard nitropyrenes by human P450 1B1 were shown using a P450 co-expression system with NPR. Activation of 1-NP was also observed in other P450 1B1 systems including microsomes from insect cells or human B-lymphoblastoid cells expressing P450 1B1 (results not shown). Pyrene did not show direct-acting genotoxicity in S. typhimurium TA1535/pSK1002, and activation by recombinant human P450 family 1 enzymes was lower than for 1-NP (results not shown). These results suggest that human P450 1B1 can metabolize these nitroarenes at the nitro group and produce genotoxic intermediates. However, the possibility of P450 1B1-catalyzed ring hydroxylation and subsequent nitroreduction in the bacterial cells could not be ruled out. Total umu activities should be dependent on metabolism by P450s (and/or NPR) and by bacterial acetyltransferase and/or nitroreductase.

H. Yamazaki et al. / Mutation Research 472 (2000) 129–138

Inactivation of 1,3-, 1,6-, and 1,8-DNPs by human liver microsomal P450s has been reported using a umu assay with the tester strain added after preincubation with microsomal P450s and chemicals [29]. Our present results indicate that environmental chemicals in airborne or diesel exhaust particles, as well as 1-NP, 1,6-DNP, and 1,8-DNP, can be activated by human P450 1B1. Genotoxic potentials of 2-NF and 3-NF were similar to those of 1-NP after activation by human P450 1B1. In conclusion, human P450 1B1 was demonstrated to have activities towards nitroaromatics and this may have relevance to the issue of diesel exhaust. The existence of P450 1B1 in extrahepatic tissues has been shown [7]. Because P450 1B1 is an inducible P450, DEP inhaled into human lungs might cause enzyme induction and DEP ingredients could be activated in target organs. Biological actions of air pollutants such as nitroarenes on human extrahepatic tissues may be of concern in cases where P450 1B1 is expressed constitutively and/or induced by foreign compounds. Further studies of metabolic pathways of 1-NP and related DNPs by human P450 1B1 are in progress. Acknowledgements This work was supported in part by grants from the Ministry of Education, Science, Sports, and Culture of Japan, by a Environmental Health Research Grant from the Ministry of Health and Welfare of Japan, and by United States Public Health Service Grants R35 CA44353 and P30 ES00267. We thank Dr. Yoshimitsu Oda for providing the umu tester strain used in this study. References [1] F.P. Guengerich, Human cytochrome P450 enzymes, in: P.R. Oritiz de Montellano (Ed.), Cytochrome P450, Plenum Press, New York, 1995, pp. 473–535. [2] F.P. Guengerich, T. Shimada, Oxidation of toxic and carcinogenic chemicals by human cytochrome P-450 enzymes, Chem. Res. Toxicol. 4 (1991) 391–407. [3] D.R. Nelson, L. Koymans, T. Kamataki, J.J. Stegeman, R. Feyereisen, D.J. Waxman, M.R. Waterman, O. Gotoh, M.J. Coon, R.W. Estabrook, I.C. Gunsalus, D.W. Nebert, P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature, Pharmacogenetics 6 (1996) 1–42.

137

[4] A. Dipple, C.J. Michejda, E.K. Weisburger, Metabolism of chemical carcinogens, Pharmacol. Ther. 27 (1985) 265–296. [5] C. Heidelberger, Chemical carcinogenesis, Annu. Rev. Biochem. 44 (1975) 79–121. [6] A.H. Conney, Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbons: G.H.A. Clowes memorial lecture, Cancer Res. 42 (1982) 4875–4917. [7] T. Shimada, C.L. Hayes, H. Yamazaki, S. Amin, S.S. Hecht, F.P. Guengerich, T.R. Sutter, Activation of chemically diverse procarcinogens by human cytochrome P450 1B1, Cancer Res. 56 (1996) 2979–2984. [8] T. Shimada, E.M.J. Gillam, T.R. Sutter, P.T. Strickland, F.P. Guengerich, H. Yamazaki, Oxidation of xenobiotics by recombinant human cytochrome P450 1B1, Drug Metab. Dispos. 25 (1997) 617–622. [9] T.R. Sutter, K. Guzman, K.M. Dold, W.F. Greenlee, Targets for dioxin: genes for plasminogen activator inhibitor-2 and interleukin-1␤, Science 254 (1991) 415–418. [10] T.R. Sutter, Y.M. Tang, C.L. Hayes, P. Wo Y-Y, W. Jabs, X. Li, H. Yin, C.W. Cody, W.F. Greenlee, Complete cDNA sequence of a human dioxin-inducible mRNA identifies a new gene subfamily of cytochrome P450 that maps to chromosome 2, J. Biol. Chem. 269 (1994) 13092–13099. [11] C.L. Hayes, D.C. Spink, B.C. Spink, J.Q. Cao, N.J. Walker, T.R. Sutter, 17␤-Estradiol hydroxylation catalyzed by human cytochrome P450 1B1, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 9776–9781. [12] D.C. Spink, C.L. Hayes, N.R. Young, M. Christou, T.R. Sutter, C.R. Jefcoate, J.F. Gierthy, The effects of 2,3,7,8tetrachlorodibenzo-p-dioxin on estrogen metabolism in MCF-7 breast cancer cells: evidence for induction of a novel 17␤-estradiol 4-hydroxylase, J. Steroid Biochem. Mol. Biol. 51 (1994) 251–258. [13] P.T. Scheepers, M.H. Martens, D.D. Velders, P. Fijneman, M. van Kerkhoven, J. Noordhoek, R.P. Bos, 1-Nitropyrene as a marker for the mutagenicity of diesel exhaust-derived particulate matter in workplace atmospheres, Environ. Mol. Mutagen. 25 (1995) 134–147. [14] K. Hayakawa, M. Butoh, Y. Hirabayashi, M. Miyazaki, Detemination of 1,3-, 1,6-, 1,8-dinitropyrene and 1-nitropyrene in vehicle exhaust particulates, Jpn. J. Toxicol. Environ. Health 40 (1994) 20–25. [15] K. Hayakawa, Y. Kawaguchi, T. Murahashi, M. Miyazaki, Distributions of nitropyrenes and mutagenicity in airborne particulates collected with an Anderson sampler, Mutat. Res. 348 (1995) 57–61. [16] K. Hayakawa, A. Nakamura, N. Terai, R. Kizu, K. Ando, Nitroarene concentrations and direct-acting mutagenicity of diesel exhaust particulates fractioned by silica-gel column chromatography, Chem. Pharm. Bull. 45 (1997) 1820–1822. [17] R. Nakagawa, S. Kitamori, K. Horikawa, K. Nakashima, H. Tokiwa, Identification of dinitropyrenes in diesel-exhaust particles. Their probable presence as the major mutagens, Mutat. Res. 124 (1983) 201–211. [18] T. Murahashi, K. Hayakawa, A sensitive method for the determination of 6-nitrochrysene, 2-nitrofluoranthene and 1-,

138

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

H. Yamazaki et al. / Mutation Research 472 (2000) 129–138 2-, and 4-nitropyrenes in airborne particles using highperfomance liquid chromatography with chemiluminescence detection, Anal. Chim. Acta 343 (1997) 251–257. J.N. Pitts Jr., D.M. Lokensgard, W. Harger, T.S. Fisher, V. Mejia, J.J. Schuler, G.M. Scorziell, Y.A. Katzenstein, Mutagens in diesel exhaust particulate. Identification and direct activities of 6-nitrobenzo[a]pyrene, 9-nitroanthracene, 1-nitropyrene and 5h-phenanthro[4,5-bcd]pyran-5-one, Mutat. Res. 103 (1982) 241–249. M.W. Chou, B.X. Wang, L.S. Von Tungeln, F.A. Beland, P.P. Fu, Induction of rat hepatic cytochromes P-450 by environmental nitropolycyclic aromatic hydrocarbons, Biochem. Pharmacol. 36 (1987) 2449–2454. P. Einistö, M. Watanabe, M.T. Nohmi, M. Ishidate Jr., Mutagenicity of 30 chemicals in Salmonella typhimurium strains possessing different nitroreductase or O-acetyltransferase activities, Mutat. Res. 259 (1991) 95–102. S. Nakamura, Y. Oda, T. Shimada, I. Oki, K. Sugimoto, SOS-inducing activity of chemical carcinogens and mutagens in Salmonella typhimurium TA1535/pSK1002: examination with 151 chemicals, Mutat. Res. 192 (1987) 239–246. V. Mersch-Sundermann, S. Mochayedi, S. Kevekordes, S. Kern, F. Wintermann, The genotoxicity of unsubstituted and nitrated polycyclic aromatic hydrocarbons, Anticancer Res. 13 (1993) 2037–2044. K.J. Silvers, L.H. Couch, E.A. Rorke, P.C. Howard, Role of nitroreductases but not cytochromes P450 in the metabolic activation of 1-nitropyrene in the HepG2 human hepatoblastoma cell line, Biochem. Pharmacol. 54 (1997) 927–936. Y. Oda, T. Shimada, M. Watanabe, M. Ishidate Jr., T. Nohmi, A sensitive umu test system for the detection of mutagenic nitroarenes in S. typhimurium NM1011 having a high nitroreductase activity, Mutat. Res. 272 (1992) 91–99. Y. Oda, H. Yamazaki, M. Watanabe, T. Nohmi, T. Shimada, Highly sensitive umu test system for the detection of mutagenic nitroarenes in Salmonella typhimurium NM3009 having high O-acetyltransferase activities, Environ. Mol. Mutagen. 21 (1993) 357–364. P.C. Howard, T. Aoyama, S.L. Bauer, H.V. Gelboin, F.J. Gonzalez, The metabolism of 1-nitropyrene by human cytochromes P450, Carcinogenesis 11 (1990) 1539–1542. K.J. Silvers, T. Chazinski, M.E. McManus, S.L. Bauer, F.J. Gonzalez, H.V. Gelboin, P. Maurel, P.C. Howard, Cytochrome P-450 3A4 (nifedipine oxidase) is responsible for the C-oxidative metabolism of 1-nitropyrene in human liver microsomal samples, Cancer Res. 52 (1992) 6237–6243. T. Shimada, F.P. Guengerich, Inactivation of 1,3-, 1,6-, and 1,8-dinitropyrene by cytochrome P-450 enzymes in human and rat liver microsomes, Cancer Res. 50 (1990) 2036–2043. T. Shimada, R.M. Wunsch, I.H. Hanna, T.R. Sutter, F.P. Guengerich, E.M.J. Gillam, Recombinant human cytochrome P450 1B1 expression in Escherichia coli, Arch. Biochem. Biophys. 357 (1998) 111–120. H. Yamazaki, M. Nakajima, M. Nakamura, S. Asahi, N. Shimada, E.M.J. Gillam, F.P. Guengerich, T. Shimada, T. Yokoi, Enhancement of cytochrome P450 3A4 catalytic activities by cytochrome b5 in bacterial membranes, Drug Metab. Dispos. 27 (1999) 999–1004.

[32] Y. Oda, H. Yamazaki, T. Shimada, Role of human N-acetyltransferases, NAT1 or NAT2, in genotoxicity of nitroarenes and aromatic amines in Salmonella typhimurium NM6001 and NM6002, Carcinogenesis 20 (1999) 1079–1083. [33] K. Hayakawa, T. Murahashi, M. Butoh, M. Miyazaki, Determination of 1,3-, 1,6-, and 1,8-dinitropyrenes and 1-nitropyrene in urban air high-performance liquid chromatography using chemiluminescence detection, Environ. Sci. Technol. 29 (1995) 928–932. [34] A. Parikh, E.M.J. Gillam, F.P. Guengerich, Drug metabolism by Escherichia coli expressing human cytochromes P450, Nature Biotechnol. 15 (1997) 784–788. [35] F.P. Guengerich, M.V. Martin, Z. Guo, Y.-J. Chun, Purification of functional recombinant P450s from bacteria, Methods Enzymol. 272 (1996) 35–44. [36] T. Shimada, M. Iwasaki, M.V. Martin, F.P. Guengerich, Human liver microsomal cytochrome P-450 enzymes involved in the bioactivation of procarcinogens detected by umu gene response in Salmonella typhimurium TA1535/pSK1002, Cancer Res. 49 (1989) 3218–3228. [37] T. Shimada, Y. Oda, H. Yamazaki, M. Mimura, F.P. Guengerich, SOS function tests for studies of chemical carcinogenesis using Salmonella typhimurium TA1535/ pSK1002, NM2009, and NM3009, in: K.W. Adolph (Ed.), Methods in Molecular Genetics, Gene and Chromosome Analysis Part C, Vol. 5, Academic Press, San Diego, New York, 1994, pp. 342–355. [38] J.H. Miller, in: Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1972, pp. 352–355. [39] T. Shimada, H. Yamazaki, M. Foroozesh, N.E. Hopkins, W.L. Alworth, F.P. Guengerich, Selectivity of polycyclic inhibitors for human cytochrome P450s 1A1, 1A2, and 1B1, Chem. Res. Toxicol. 11 (1998) 1048–1056. [40] H. Yamazaki, M. Tanaka, T. Shimada, Highly sensitive HPLC assay for coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation by human liver cytochrome P450 enzymes, J. Chromatogr. B. Biomed. Appl. 721 (1999) 13–19. [41] T. Omura, R. Sato, The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature, J. Biol. Chem. 239 (1964) 2370–2378. [42] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275. [43] C.H. Williams Jr., H. Kamin, Microsomal triphosphopyridine nucleotide-cytochrome c reductase of liver, J. Biol. Chem. 237 (1962) 587–595. [44] T. Shimada, F.P. Guengerich, Activation of amino-␣carboline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, and a copper phthalocyanine cellulose extract of cigarette smoke condensate by cytochrome P-450 enzymes in rat and human liver microsomes, Cancer Res. 51 (1991) 5284–5291. [45] Y.H. Chae, T. Thomas, F.P. Guengerich, P.P. Fu, K. El-Bayoumy, Comparative metabolism of 1-, 2-, and 4-nitropyrene by human hepatic and pulmonary microsomes, Cancer Res. 59 (1999) 1473–1480.