Induction of cytochrome P450 1B1 in lung, liver and kidney of rats exposed to diesel exhaust

Naoya Hatanaka, Hiroshi Yamazaki, Ryoichi Kizu, Kazuichi Hayakawa, Yasunobu Aoki1, Masashi Iwanari, Miki Nakajima and Tsuyoshi Yokoi,2

Faculty of Pharmaceutical Sciences, Kanazawa University 13-1 Takara-machi, Kanazawa 920-0934 and
1 National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba 305-8506, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have shown previously that diesel exhaust particle (DEP) extracts (DEPE) and 1-nitropyrene were genotoxically activated by human cytochrome P450 1B1 in SOS/umu assay. In this study, the in vivo induction of P450 family 1 enzymes in rats by exposure to diesel exhaust was investigated with regard to mRNA levels, P450 enzyme content, drug oxidation activities in the microsomes and umu gene expression of typical P450 substrates and DEPE itself catalyzed by the microsomes. Male Fischer 344 rats (4 weeks old) were exposed to 0.3 and 3.0 mg/m3 DEP for 12 h per day for 4 weeks; the former dose corresponded to the typical daily airborne particle concentration. The levels of mRNA of rat P450 1B1 and P450 1A1 in the lung and liver were significantly increased 1.1–1.4-fold by exposure to 0.3 mg/m3 DEP. Diesel exhaust particle extracts induced umu gene expression in Salmonella typhimurium TA1535/pSK1002 in the absence of a functional P450 system and were further activated by human recombinant P450 1B1. Using an O-acetyltransferase overexpressing Salmonella strain, genotoxic activation of P450 1B1 marker chemicals (1-nitropyrene, 1-aminopyrene and DEPE) by lung, liver and kidney microsomes was increased 1.7–4.2-, 1.4–1.5- and 1.0–1.3-fold, respectively, by exposure to 0.3 mg/m3 DEP. Activation of 3-amino-1,4-dimethyl-5H-pyrido [4,3-b]indole (Trp-P-1; marker for P450 1A1) by lung microsomes and the P450 1A2 content in liver microsomes were slightly increased by exposure to 3.0 mg/m3 DEP. This is the first report to suggest that typical daily contaminant levels (0.3 mg particle/m3) of diesel exhaust can induce P450 1B1 in rats and that the induced P450 1B1 may catalyze the genotoxic activation of DEP.

Abbreviations: DEP, diesel exhaust particles; DEPE, DEP extracts; MeIQ, 2-amino-3,5-dimethylimidazo[4,5-f]quinoline; NPR, NADPH-cytochrome P450 reductase; P450, cytochrome P450; Trp-P-1, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The risk to human health from air pollutants is an important issue. Many mutagenic and carcinogenic compounds, such as polycyclic aromatic hydrocarbons and nitrated-polycyclic aromatic hydrocarbons, are the main constituents of these pollutants (1). Analyses have indicated that 1-nitropyrene and 1,3-, 1,6- and 1,8-dinitropyrene are major nitroarene components of airborne, surface soil and diesel exhaust particles (DEP) (1–3). Diesel engine emission appears to be a principal source of contaminants and DEP and its crude extracts are often used as model compounds for the investigation of air pollutants. The relationship between chemical carcinogenesis by polycyclic hydrocarbons such as benzo[a]pyrene and drug metabolizing enzymes has been extensively studied (4,5). The induction of drug metabolizing enzymes and the activation of procarcinogens have been also examined (6). Since nitrated-polycyclic aromatic hydrocarbons are widespread environmental contaminants, it can be assumed that humans will ordinarily be exposed to them.

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 (7–9). We have recently reported that DEP extracts (DEPE) and 1-nitropyrene can be effectively activated by human P450 1B1 as well as human P450 1A1 or P450 1A2 in an SOS/umu assay using Salmonella typhimurium TA1535/pSK1002 (10). Rat P450 1B1 has been identified as rat adrenal P450 enzyme active in polycyclic hydrocarbon metabolism (11). Before this identification, Otto et al. (12) demonstrated that a novel adrenocorticotropin-inducible P450 (P450RAP) catalyzes the metabolism of several polyaromatic hydrocarbons. Rat P450 1B1 (P450RAP) has been shown to be distributed in several organs and is induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin and polyaromatic hydrocarbons (12). This P450RAP is probably the same enzyme catalyzing benzo[a]pyrene hydroxylation in rat adrenal microsomes as reported by Guenthner et al. (13). P450 1B1 is expressed in numerous tissues and is inducible by exposure to dioxin and polycyclic aromatic hydrocarbons (14,15). Although topical application or intraperitoneal injection of nitroarenes has been reported to result in a significant induction of aryl hydrocarbon hydroxylase and ethoxyresorufin O-deethylase activities (16,17), there is no information about the P450 forms involved or P450 1B1 induction by exposure to environmental levels of DEP.

In this study, the induction of rat P450 family 1 enzymes in vivo, especially P450 1B1, by DEP was determined after exposure to two different doses of DEP in rats. The lower DEP concentration used (0.3 mg/m3 for 4 weeks) was typical of urban contaminant levels. We determined the increased mRNA of P450 1B1 and the role of P450 1B1 in the genotoxic activation of DEPE in lung, liver and kidney prepared from DEP-exposed rats.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
1-Nitropyrene and 1-aminopyrene were purchased from Tokyo Chemical Industry (Tokyo, Japan) and Aldrich (Milwaukee, WI), respectively. 2-Amino-3,5-dimethylimidazo[4,5-f]quinoline (MeIQ) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) were obtained from Wako Pure Chemicals (Osaka, Japan) and 2-aminoanthracene was from Sigma (St Louis, MO). The extraction of DEP prepared at the National Institute for Environmental Studies (Tsukuba, Japan) was carried as previously described (10,18). This crude extract, designated DEPE-5, was dissolved in ethanol to a final concentration of 20 mg/ml. The major ingredients of DEPE-5 were polycyclic hydrocarbons such as benz[a]anthracene, benzo[a]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene and benzo[ghi] perylene (13–102 nmol/mg extract), as determined by the method of Hayakawa et al. (19). The amounts of 1-nitropyrene, 6-nitrochrysene, 7-nitrobenz[a]anthracene, 6-nitrobenzo[a]pyrene and 1,3-, 1,6- and 1,8-dinitropyrene in DEPE-5 were 97, 183, 28, 9, 9, 8 and 7 pmol/mg extract, respectively. Other reagents used in this study were obtained from sources described previously or were of the highest grade commercially available (10).

Exposure of DEP
Four-week-old male Fischer 344 rats (five animals per each group; Nihon Clea, Tokyo, Japan) were exposed to diesel exhaust particles (0, 0.3 or 3 mg/m3) for 4 weeks, 12 h/day at the National Institute for Environmental Studies as previously described (20). Briefly, diesel exhaust was generated by a light duty (2.74 l), four-cylinder, diesel engine (A4JB1-type; Isuzu Automobile, Tokyo, Japan) which was operated using standard diesel fuel. The diesel engine was controlled by a computer to a speed of 1500 r.p.m. under a load of 10 torque (kg/m). There were no apparent toxic effects of the diesel exhaust on the rats under these conditions: the mean body weights (± SD) of 8-week-old rats from the 0, 0.3 and 3 mg/m3 exposed groups were 194 ± 5, 211 ± 10 and 190 ± 7 g, respectively. Livers, kidneys and lungs were obtained from rats and frozen at –80°C until use. Total RNA was isolated from frozen liver, kidney and lung using an ISOGEN RNA extraction reagent (Nippon Gene, Toyama, Japan). Microsomes from liver, kidney and lung were prepared and suspended in 10 mM Tris–HCl buffer (pH 7.4) containing 20% glycerol and 1 mM EDTA (21).

Reverse transcriptase-polymerase chain reaction analysis (RT–PCR)
The measurement of RNA expression levels was carried out using RT–PCR analysis. Total RNA was converted to cDNA by M-MLV reverse transcriptase (Toyobo, Tokyo, Japan). Reverse transcription mixtures were subjected to PCR with 0.5 µM specific primer (22) in a reaction containing 0.25 mM dNTPs, 0.5 U Taq polymerase (Takara, Tokyo, Japan) and 2.5–5.0 mM MgCl2 (2.5 mM for P450 1B1 and ß-actin; 4 mM for P450 1A1; 5 mM for P450 1A2). PCR assays were performed in a Takara Thermal Cycler. Reactions were incubated at 94°C for 3 min and then were amplified using temperature parameters of 94°C for 30 s; 60°C (P450 1A1, P450 1A2 and ß-actin) or 62°C (P450 1B1) for 30 s; 75°C for 30 s. Amplifications were carried out for 25, 25, 35 or 40 cycles for P450 1A2, ß-actin, P450 1A1 and P450 1B1, respectively, followed by a 5 min extension at 75°C. Product formation showed linearity under these conditions. The products were separated by agarose gel electrophoresis and stained with 1 µg/ml ethidium bromide. An image of the stained PCR products was captured as an 8-bit digital TIFF file using a CCD camera and Molecular Analyst software, version 2.1 (Bio-Rad, Hercules, CA). The density of each band was quantified using NIH Image, version 1.59.

Protein content and enzyme assays
Escherichia coli membranes expressing recombinant human P450/NPR were prepared as described previously (10,23). The contents of P450 and cytochrome b5 in microsomes from liver and kidney were measured by the method of Omura and Sato (24). The P450 contents in lung were measured by the method of Johannesen and DePierre (25). NADPH-cytochrome P450 reductase (NPR) contents were assumed from the NADPH-dependent cytochrome c reductase activities with a specific activity of 3.0 µmol cytochrome c reduced/min/nmol NPR base on purified human and rabbit NPR preparations. Protein concentrations were measured by the method of Lowry et al. (26). The contents of cytochrome b5 and NPR in liver microsomes were 0.283–0.344 and 0.078–0.096 nmol/mg protein and those in kidney microsomes were 0.022–0.032 and 0.014–0.018 nmol/mg protein, respectively. Recombinant rat P450 1A1 and P450 1A2 in microsomes from insect cells with a baculovirus system co-expressing NPR were obtained from Gentest (Woburn, MA).

Immunoblot analysis was performed according to Laemmli (27) with slight modifications. Microsomal proteins were separated by 7.5% polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Polyclonal goat anti-rat P450 1A1 antisera was obtained from Daiichi Pure Chemicals (Tokyo, Japan). Polyclonal rabbit anti-rat P450 1B1 antisera was obtained from Funakoshi (Tokyo, Japan). The secondary antibodies used were anti-goat IgG for P450 1A enzymes or anti-rabbit IgG for P450 1B1 and detection was carried out with diaminobenzidine staining. The density of stained bands was also quantified using NIH Image.

The activities of benzo[a]pyrene 3-hydroxylation and ethoxyresorufin O-deethylation were determined by the methods of Nebert and Gelboin (28) and Hanioka et al. (29), respectively. P450-dependent activation of procarcinogens and mutagens to reactive metabolites that cause the induction of umu gene expression in Salmonella typhimurium TA1535/pSK1002 and NM2009 were determined (10). TA1535/pSK1002 is a tester strain containing a plasmid with an umuC'–'lacZ fusion gene; NM2009 contains an additional plasmid, pNM12, that overexpresses bacterial O-acetyltransferase and is thus more sensitive to the reactive intermediates of aryl amines (30). Standard incubation mixtures consisted of P450 systems with procarcinogens or DEPE-5 in a final volume of 0.25 ml 200 mM potassium phosphate buffer (pH 7.4) containing an NADPH-generating system and 0.75 ml bacterial suspension. The enzyme sources used were microsomes from rat liver, kidney or lung, insect cell microsomes expressing rat P450/NPR and E.coli membranes expressing human P450/NPR. For enzyme inactivation, heat treatment (100°C, 2 min) of the tubes was conducted before adding the test chemicals and bacteria. Incubations were carried out at 37°C for 2 h and terminated by cooling the mixture on ice. The umu gene expression was monitored by measuring ß-galactosidase activities (30,31). The induction of umu gene expression by the activated carcinogens is presented as umu units of ß-galactosidase activity/min/mg protein. The results presented in this study were the mean of three experiments ± SD.

Statistical analysis
Statistical analysis was performed by the computer program Instat (Graphpad Software, San Diego, CA) designed for Student's t-test after the assumption of equal variance with an F test.


    Results
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 Abstract
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 Materials and methods
 Results
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 References
 
Amounts of P450 family 1 mRNA in liver, kidney and lung from rats exposed to DEP
The relative amounts of P450 family 1 mRNA in liver, kidney and lung were measured by RT–PCR analysis (Table IGo). In rat liver, P450 1B1 mRNA was increased 1.4-fold by exposure to 0.3 mg DEP/m3. P450 1A1 mRNA was slightly induced by 3 mg DEP/m3, but P450 1A2 mRNA was not significantly increased. In rat kidney, the mRNA levels of P450 1A1 and P450 1B1 were not affected by exposure to DEP. In contrast, mRNA levels of P450 1A1 and -1B1 in rat lung were increased 1.2- and 1.1-fold, respectively, by exposure to 0.3 mg DEP/m3. P450 1A1 mRNA was induced 2.3-fold by the high dose of DEP.


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Table I. Relative amounts of mRNA of P450 family 1 enzymes in liver, kidney and lung of DEP-exposed rats
 
P450 contents and drug oxidation activities in liver, kidney and lung microsomes from rats exposed to DEP
The effects of DEP on rat P450 enzymes were investigated with microsomal proteins. The P450 contents and drug oxidation activities in liver, kidney and lung microsomes from rats exposed to DEP are summarized in Table IIGo. Total P450 in rat kidney microsomes were induced 1.5- and 1.4-fold by 0.3 and 3 mg/m3 DEP, respectively. The P450 1A2 levels in liver microsomes from rats exposed to 3 mg/m3 DEP were 1.4-fold higher than those in the control group. Ethoxyresorufin O-deethylase activities in liver microsomes were increased 1.3-fold by 0.3 mg/m3 DEP and those in lung microsomes were induced 2.7-fold by 3 mg/m3 DEP. DEP did not induce benzo[a]pyrene hydroxylation activities in rat liver microsomes. Because of the limited amount of sample, benzo[a] pyrene hydroxylation activities were not determined in kidney or lung microsomes. From these experiments on proteins, the induction of P450 1B1 was not clarified.


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Table II. P450 contents and drug oxidation activities in liver, kidney and lung microsomes from rats exposed to DEP
 
Genotoxicity of DEPE-5 in S.typhimurium TA1535/pSK1002 and NM2009 after activation by recombinant human P450 family 1 enzymes
The genotoxicity of DEPE-5 was investigated in S.typhimurium TA1535/pSK1002 and NM2009 (Figure 1Go). DEPE-5 showed genotoxic activities in the absence of activation systems in the range 0.1–100 µg/ml in TA1535/pSK1002 (Figure 1AGo). DEPE-5 at low concentrations was activated most effectively by human P450 1B1 co-expressed with NPR in E.coli membranes, followed by P450 1A1/NPR membranes (Figure 1BGo). P450 1A2/NPR membranes slightly activated DEPE-5, but NPR membranes did not. In the present study, we used human P450 family 1 enzymes because rat P450 1B1 was not available. When an O-acetyltransferase overexpressing NM2009 was used as the tester strain, DEPE-5 showed high activities in the absence of activation systems within narrow substrate concentration ranges (Figure 1CGo). The metabolic activation of DEPE-5 (5 µg/ml) by P450 1B1/NPR was high in the NM2009 tester strain (309 ± 25 umu units/min/nmol P450 1B1; Figure 1DGo). In addition to DEPE, to select suitable substrates for genotoxic activation assays with rat enzymes, rat and human P450 family 1 enzymes were compared with regard to their abilities to catalyze the activation of five typical procarcinogens to genotoxic metabolites in the O-acetyltransferase-overexpressing S.typhimurium NM2009. Human P450 1B1 was highly active in catalyzing 1 µM 1-aminopyrene (2540 ± 220 umu units/min/nmol P450 1B1) and 0.3 µM 1-nitropyrene (739 ± 35 umu units/min/nmol P450 1B1). Rat and human P450 1A1 activated Trp-P-1 (~1000 umu units/min/nmol P450 1A1). Rat and human P450 1A2 highly activated MeIQ (>6000 umu units/min/nmol P450 1A2). 2-Aminoanthracene (5 µM) was activated by all P450 family 1 enzymes tested in the present study (~1000 umu units/min/nmol P450).



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Fig. 1. Genotoxicity of diesel exhaust particle extract in Salmonella typhimurium TA1535/pSK1002 (A, B) and NM2009 (C, D). (A, C) Direct activity of a sample designated DEPE-5 in the absence of activation systems. Background levels of umu gene expression were subtracted. (B, D) Bioactivation of DEPE-5 by human P450 1A1/NPR membranes (•), P450 1A2/NPR membranes ({blacktriangleup}), P450 1B1/NPR membranes ({blacksquare}) and by human NPR membranes ({circ}). The umu gene expression was expressed as umu units/min/nmol P450 or NPR after subtraction of values with heat-inactivated microsomes. Results are presented as means of triplicate determinations ± SD.

 
Activation of chemicals by liver, kidney and lung microsomes from rats exposed to DEP
Liver, kidney and lung microsomes from rats exposed to DEP were compared with regard to their abilities to catalyze the activation of five procarcinogens and DEPE-5 to genotoxic metabolites in the S.typhimurium NM2009. Those of umu gene expression were shown as umu units/min/mg protein in Table IIIGo. In the 0.3 mg/m3 DEP exposed group, genotoxic activation of 1-aminopyrene and 1-nitropyrene by rat liver microsomes was increased 1.4–1.5-fold. Genotoxic activation of DEPE-5 by rat liver microsomes was also increased 1.5-fold in the 0.3 mg/m3 DEP exposed groups. The activation of Trp-P-1 and MeIQ in liver microsomes was not affected by exposure to 0.3 mg/m3 DEP; however, activation of 2-aminoanthracene was increased 1.3-fold. In the 3 mg/m3 DEP exposed group, the activities in rat liver microsomes were similar to those in the 0.3 mg/m3 DEP exposed group.


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Table III. Genotoxic activation of chemicals by liver, kidney, and lung microsomes from rats exposed to DEP
 
The genotoxic activation of 1-aminopyrene and DEPE-5 by kidney microsomes increased 1.1- and 1.3-fold, respectively, in the 0.3 mg/m3 DEP exposed group (Table IIIGo). The activation of 2-aminoanthracene was also increased 1.1-fold. In the 3 mg/m3 DEP exposed group, the activation of 1-aminopyrene, 1-nitropyrene and DEPE-5 was increased 1.1–1.5-fold. The activation of 2-aminoanthracene was also increased 1.3-fold. The genotoxic activation of Trp-P-1 and MeIQ by rat kidney microsomes was not increased by exposure to DEP.

The genotoxic activation of 1-aminopyrene, 1-nitropyrene and DEPE-5 in pooled lung microsomes increased 1.7–4.2-fold in the 0.3 mg/m3 DEP exposed groups (Table IIIGo). Although the activation of MeIQ was not affected, the activities of Trp-P-1 were increased by DEP. Activation of 2-aminoanthracene was also increased 1.7-fold. In the 3 mg/m3 DEP exposed group, Trp-P-1 activation was highly induced.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The risk to human health associated with exposure to diesel exhaust has generally been considered a particle problem and the role of chemicals bound to the particles has been underestimated (32,33). Previously, biochemical and cytological changes in lung tissue by inhaled diesel exhaust have been investigated (20,34,35). However, limited information regarding the effects of in vivo exposure to diesel exhaust on rat liver and lung microsomal activities is available (36,37). Topical application of nitroarenes found in DEP including 1-nitropyrene (10 mg/kg) to newborn rats has been shown to induce P450-dependent benzo[a]pyrene 3-hydroxylation and ethoxyresorufin O-deethylase activities in both skin and liver microsomes (16). However, there is no information on the P450 forms involved in this induction by nitroarenes.

In the present study, we demonstrated the induction of P450 family 1 in male Fischer rats exposed to diesel exhaust (12 h/day, 4 weeks), the DEP contents of which were maintained at an environmental level (0.3 mg/m3) and a level 10-fold higher. The analyses conducted in this study were quantification of mRNA, immunochemical determinations of P450 enzymes, and those of drug oxidation activities and genotoxic activation of typical P450 substrates including DEPE itself by the microsomes. This is the first report, to our knowledge, of the induction of DEP-activating enzymes by diesel exhaust (apparent autoinduction) using an umu assay in the experimental design. The mRNA levels of P450 1B1 and P450 1A1 in lung and/or liver were increased by exposure to diesel exhaust at environmental levels (0.3 mg/m3) (Table IGo). Total P450 content in rat kidney microsomes and P450 1A2 determined immunochemically was induced in liver microsomes (Table IIGo). 7-Ethoxyresorufin O-deethylase activities in lung and liver microsomes were also increased. These results would indicate weak induction of the P450 family 1 enzymes including P450 1B1 by DEP in rats.

Using the umu gene expression test system, clear information was obtained with regard to P450 family 1 enzyme induction in rats in vivo after DEP exposure. DEPE-5 at a low concentration was further activated to the highest extent by human P450 1B1/NPR membranes when O-acetyltransferase-overexpressing NM2009 was used as a tester strain (Figure 1Go). We previously demonstrated that a DEPE (designated DEPE-1) prepared from an automobile exhaust induced the highest umu gene expression after metabolism by human P450 1B1, followed by P450 1A2 (10) using TA1535/pSK1002. This finding is consistent with the present results showing the highest catalytic activity by P450 1B1. Although recombinant rat P450 1B1 was not available, the counterparts of P450 1A1 and P450 1A2 were activated with typical procarcinogens to a similar extent. Although it is well-known that there are species differences among rat and human P450 enzymes with regard to substrate specificities (8), these differences have been thought to be relatively small in P450 family 1 enzymes, especially P450 1B1 (38).

The roles of rat and human P450 1A1, P450 1A2 or P450 1B1 in the genotoxic activation of typical procarcinogens have been reported using recombinant P450 enzymes or liver and lung microsomes (14,33,39). Genotoxic activation of 1-nitropyrene, 1-aminopyrene and DEPE-5 (marker for P450 1B1) by liver, kidney and lung microsomes was significantly induced by exposure to 0.3 mg/m3 DEP, which corresponds to ordinary environmental levels (40). Genotoxic activation of Trp-P-1 (marker for P450 1A1) by pooled lung microsomes and that of MeIQ (marker for P450 1A2) by liver microsomes were increased by exposure to DEP (Table IIIGo). These results suggest that diesel exhaust can strongly induce P450 1B1 and moderately induce P450 1A1 and P450 1A2 in rats and that the induced P450 enzymes, especially P450 1B1, may catalyze the genotoxic activation of components of DEP. This is the first report, to our knowledge, to show that diesel exhaust induces P450 1B1 in rats in vivo.

The genotoxic activation of 1-nitropyrene by lung microsomes was higher in the 0.3 mg/m3 DEP group than in the 3 mg/m3 DEP group (Table IIIGo). Other marker activities were generally higher in the 3 mg/m3 DEP group than in the 0.3 mg/m3 DEP group. In this condition, P450 1A1 was also induced judging from the Trp-P-1 activation. 1-Nitropyrene was activated by human P450 1B1 and appeared to be inactivated by human P450 1A1 (41). We have recently demonstrated that 1-nitropyrene can be genotoxically activated mainly by human P450 1B1 by way of nitroreduction/O-acetylation (41). In addition, the apparent inactivation of 1-nitropyrene was observed after metabolism by P450 1A1/NPR which had high catalytic activities of 1-nitropyrene 8-hydroxylation (41). Taking these results into consideration, induced P450 1A1 in rat lung microsomes might inactivate the direct-acting genotoxicity of 1-nitropyrene, the activities of which may have been decreased in the 3 mg/m3 DEP group.

P450 1B1 is expressed in numerous tissues and is inducible (14). P450 1B1 is an important enzyme involved in the activation of a number of environmental carcinogens and should be considered regarding the mechanisms by which the development of human cancer occurs (18). In carcinogenic experiments with animals, nitroarenes found in diesel exhaust generally induce tumors in topical areas (41). In the present study, the inducibility of P450 1B1 was demonstrated by exposure to DEP in lung, which is the first organ exposed to diesel exhaust, and in livers and kidneys. In conclusion, the present results suggest that diesel exhaust can induce P450 1B1 in rats and that the induced P450 1B1 may catalyze the genotoxic activation of DEP components. This is the first report to show that a daily contaminant level of diesel exhaust particles for 4 weeks can induce P450 1B1 in rats in vivo.


    Notes
 
2 Email: tyokoi{at}kenroku.kanazawa-u.ac.jp Back


    Acknowledgments
 
This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan and by an Environmental Health Research Grant from the Ministry of Health and Welfare of Japan. We thank Drs F.Peter Guengerich and Yoshimitsu Oda for providing bicistronic plasmids of human P450/NPR and umu tester strains, respectively. We also thank Kei Takemoto and Tomo Kobayashi for their technical assistance and Mr Brent Bell for critical reading of the manuscript.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 24, 2001; revised September 6, 2001; accepted September 7, 2001.