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DNA adduct formation by the environmental contaminant 3-nitrobenzanthrone in V79 cells expressing human cytochrome P450 enzymes
Cancer Letters 200 (2003) 9–18 www.elsevier.com/locate/canlet
DNA adduct formation by the environmental contaminant 3-nitrobenzanthrone in V79 cells expressing human cytochrome P450 enzymes Christian A. Bieler, Volker M. Arlt1, Manfred Wiessler, Heinz H. Schmeiser* Division of Molecular Toxicology, German Cancer Research Center, Im Neuenheimer Feld 280, Heidelberg 69120, Germany Received 1 April 2003; received in revised form 2 June 2003; accepted 4 June 2003
Abstract Diesel exhaust is known to induce tumours in animals. Of the compounds found in diesel exhaust 3-nitrobenzanthrone (3-NBA) is particularly a powerful mutagen. Recently we showed that 3-NBA is genotoxic in vivo in rats by forming specific DNA adducts derived from nitroreduction. In this study a panel of genetically engineered V79 Chinese hamster cell lines expressing various human cytochrome P450 (CYP) enzymes (CYP1A1, CYP3A4) and/or human NADPH:CYP oxidoreductase (CYPOR) was used to identify CYP enzymes involved in the metabolic activation of 3-NBA. We analyzed the formation of specific DNA adducts by 32P-postlabelling after exposing cells to 1 mM 3-NBA. A similar pattern with a total of four distinct 3-NBA– DNA adducts was found in all cells, identical to those detected previously in DNA from rats treated with 3-NBA in vivo. Total adduct levels ranged from 75 to 132 using nuclease P1 and from 103 to 220 adducts per 108 nucleotides, using butanol enrichment. Comparison of DNA binding between different V79MZ derived cells revealed that human CYPOR and CYP3A4 were involved in the metabolic activation of 3-NBA. Furthermore, dose-dependent high adduct levels were detected after exposure to 0.01, 0.1 or 1 mM 3-NBA in the subclone V79NH which exhibits high activities of nitroreductase and N,Oacetyltransferase. Our results suggest that nitroreduction is the major pathway in the human bioactivation of 3-NBA. Moreover, acetylation of the initially formed N-hydroxy arylamine intermediates may contribute to the high genotoxic potential of 3-NBA. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: 3-Nitrobenzanthrone; DNA adducts; 32P-postlabelling; Human metabolism; Nitro-PAH; Diesel exhaust
Abbreviations: 3-NBA, 3-Nitrobenzanthrone; NAT, N,Oacetyltransferase; SULT, Sulfotransferase; CYP, Cytochrome P450; CYPOR, NADPH-dependent cytochrome P450 oxidoreductase; nitro-PAH, Nitro-polycyclic aromatic hydrocarbon; XO, Xanthine oxidase; RAL, Relative adduct labelling; DMSO, Dimethyl sulfoxide. * Corresponding author. Tel.: þ49-6221-4233-48; fax: þ 496221-4233-75. E-mail address: [email protected] (H.H. Schmeiser). 1 Present address: Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Sutton, Surrey SM2 5NG, UK.
1. Introduction Nitro-polycyclic aromatic hydrocarbons (nitroPAH) are widely distributed environmental pollutants found in extracts from diesel and gasoline engines and on the surface of ambient air particulate matter [1]. Mutagenic activities in bacterial and mammalian systems as well as tumorigenic activity in laboratory animals of several members of this class have been clearly documented [2,3]. Although the significance
0304-3835/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0304-3835(03)00418-X
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Fig. 1. Structure of 3-nitrobenzanthrone (3-nitro-7H-benz[d,e ]anthracen-7-one).
of nitro-PAH in the induction of human cancer is presently unknown, their possible link with lung cancer in traffic-congested areas has lead to considerable interest in assessing their potential risk to man [4]. Recently a new member of this group of compounds, 3-nitrobenzanthrone (3-NBA, 3-nitro-7Hbenz[d,e ]anthracen-7-one) (Fig. 1), was discovered in diesel exhaust and bound to the surface of airborne particulate matter [5]. Furthermore, its major metabolite, 3-aminobenzanthrone, was detected in the urine of mining workers occupationally exposed to diesel emissions [6]. 3-NBA was shown to be one of the most potent mutagens in the Ames assay reported so far and to induce micronuclei in mouse and human cells, as well as, mutations in human cells [5,7]. Further evidence for the genotoxic action of this compound was provided by the detection of 3-NBA-derived DNA adducts using the 32 P-postlabelling assay after reductive activation in vitro as well as in vivo in rats treated orally with a single dose of 3-NBA or its metabolites [8 – 10]. Nitro-PAHs require metabolism to reactive electrophilic species in order to exert their genotoxic and carcinogenic activity. The activation of nitroaromatic hydrocarbons is through reduction to N-hydroxy arylamines, catalyzed by both cytosolic and microsomal enzymes such as aldehyde oxidase, xanthine oxidase (XO), DT-diaphorase, cytochrome P450 (CYP) enzymes and nicotinamide adenine dinucleotide phosphate reduced (NADPH) CYP oxidoreductase (CYPOR) [3,11]. Further biotransformation occurs by phase II-enzymes, such as N,O-acetyltransferases (NATs) or sulfotransferases (SULTs), leading to the formation of reactive esters, which undergo
hydrolysis to produce electrophilic nitrenium ions capable of forming DNA adducts [12,13]. Indeed adduct levels induced by 3-NBA or its potential metabolites were significantly increased in V79 cells expressing human phase II enzymes, e.g. NAT2 or SULT1A1, compared to the parental cell line V79MZ lacking such enzymatic activity [14,15]. V79 Chinese hamster lung fibroblasts are widely used in mutagenicity testing but these cells completely lack CYP-dependent enzyme activities [16]. Nevertheless, V79 cells contain detectable amounts of CYPOR, an enzyme known to activate nitroaromatic compounds [17]. With respect to this a panel of V79 cells expressing human CYP enzymes including human CYP1A1 and CYP3A4, required for the biotransformation of many xenobiotics, have been genetically engineered by Doehmer and others [17 – 19]. Previous studies with such recombinant cells have allowed the evaluation of the role of single human isoforms of enzymes in the metabolic activation or detoxication of several carcinogens (e.g. tamoxifen [20] dibenzo[a,l ]pyrene [21 – 24], benzo[a ]pyrene [23,25], 7,12-dimethylbenz[a ]anthracene [26] and benzo[c ]phenanthrene [25] using DNA adduct formation as a suitable biological endpoint. Recently we showed that 3-NBA forms DNA adducts in metabolically competent human lymphoblastoid MCL-5 cells expressing various CYP enzymes [27]. This prompted us to investigate the involvement of human enzymes in the metabolic activation of 3-NBA. For this purpose we have employed V79 cell lines equipped with different enzymes to determine DNA adduct levels by the 32Ppostlabelling method as a means of determining enzyme-mediated genotoxicity. In addition to the V79MZ cell lines that stably express various human CYP enzymes we used V79NH cells, another subclone of the V79 cell line [28]. Like the V79MZ subclone V79NH is devoid of CYP activities but exhibits high nitroreductase and NAT activities, two nitro-PAH activating enzymes. 2. Material and methods 2.1. Chemicals and cell lines 3-NBA was synthesized as described in Ref. [14]. Benzo[a ]pyrene and ellipticine were obtained from
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Sigma Chemical Co. (St Louis, MO). The transfected cells V79MZ –h1A1 [17], V79MZ – h3A4 – CYPhOR [19] were purchased from PharmBioDyn, Freiburg, Germany. Parental V79MZ, V79NH [28] and the transfected cells V79MZ – CYPhOR [19] and V79NH – h1A2 [28] were kindly provided by Prof Doehmer (GenPharmTox BioTech AG, Munich, Germany).
human CYP3A4 V79MZ, V79MZ – CYPhOR and V79MZ –h3A4 –CYPhOR were treated with 1 mM ellipticine. Cells treated with DMSO only were used as a negative control. After an incubation period of 24 h the medium was removed and the cells were harvested and DNA was isolated as described [14]. Cell viability (in % of control) was determined by the trypan blue (Biochrom KG) exclusion assay [14].
2.2. Cell culture
2.4. 32P-Postlabelling of DNA adducts
The parental cells, V79MZ (7-ethoxyresorufin-Odeethylase (EROD) ¼ , 0.2 pmol/min/mg protein; cytochrome c reduction ¼ 8.2 nmol/min/mg protein) [18,19] and the transfected cells, V79MZ – h1A1 expressing human CYP1A1 (EROD ¼ 48.8 pmol/ min/mg protein) [17], V79MZ– CYPhOR expressing human CYPOR (cytochrome c reduction ¼ 272.1 nmol/min/mg protein) [19] and V79MZ – h3A4 – CYPhOR expressing human CYP3A4 in conjunction with CYPOR (CYP3A4 ¼ 6 pmol/mg protein determined by immunochemistry; cytochrome c reduction ¼ 81.0 nmol/min/mg protein) [19], as well as the V79NH (EROD ¼ , 0.2 pmol/min/mg protein) and V79NH – h1A2 expressing human CYP1A2 (EROD ¼ 8.1 pmol/min/mg protein) [18] cells were cultivated in Dulbecco’s modified Eagles’s medium (DMEM, Biochrom KG, Berlin Germany), with high glucose content (4.5 g D -glucose/l), supplemented with 1 mM sodium pyruvate (Biochrom KG), 4 mM L -glutamine (PAA Laboratories, Linz, Austria), 5% fetal calf serum (FCS) (Biochrom KG), 100 U of penicillin/ml and 100 mg streptomycin/ml (Biochrom KG) at 37 8C, 5% CO2 and 95% atmospheric humidity. 2.3. Treatment of V79 cells, cell harvesting and DNA isolation All V79 cells were seeded 24 h prior to treatment at a density of 1 £ 105 cells/ml in 75 cm2 culture flasks in a total volume of 30 ml of medium. 3-NBA dissolved in dimethyl sulfoxide (DMSO) (Merck, Germany) was added to a final concentration of 1 mM to V79MZ cells and to a final concentration of 0.01, 0.1 or 1 mM to V79NH cells. As positive control for oxidative activation by human CYPh1A1 V79MZ and V79MZ– h1A1 were treated with 1 mM benzo[a ] pyrene. As positive control for oxidative activation by
32
P-Postlabelling analyses for 3-NBA-derived adducts were performed using the nuclease P1 enrichment or the butanol extraction method as described previously [8]. 32P-postlabelling analyses for benzo[a ]pyrene- and ellipticine-derived DNA adducts was performed using the nuclease P1 enrichment as described previously [29]. Quantitative analysis was performed using the Canberra Packard Instant Imager system. Relative adduct labelling (RAL) was calculated as cpm adducts per cpm normals. All adduct levels are expressed as mean ^ SD. Comparison was performed by t-test analysis. All P-values are considered significant at the 0.05 level.
3. Results 3.1. DNA adduct formation by 3-NBA in V79MZ cells expressing human CYP enzymes Parental V79MZ, which are devoid of CYP activity, and V79MZ cells expressing human CYP1A1, human CYPOR alone or human CYP3A4 together with human CYPOR were treated with 1 mM 3-NBA to ensure adduct levels easily detectable by the 32P-postlabelling method (RAL range 1 adduct in 106 – 108 nucleotides). Co-expression of human CYP3A4 and CYPOR has been reported to be needed to receive sufficient CYP3A4 activity [19]. Using either enrichment procedure of the 32P-postlabelling assay, nuclease P1 digestion and butanol extraction, DNA adduct analysis revealed a similar pattern of 3NBA-derived adducts in all V79MZ cells as found previously in vivo in different organs of rats treated orally with a single dose of 3-NBA (Fig. 2) [9]. As reported before no adducts were detected in control
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Fig. 2. Autoradiographic profiles of DNA adducts obtained after incubation of V79MZ cells with 1 mM 3-NBA using the nuclease P1 (a –d) or butanol (e– h) enrichment version of the 32P-postlabelling assay. Autoradiograms exemplify digest of V79MZ (a and e), V79MZ-h1A1 (b and f), V79MZ–CYPhOR (c and g) and V79MZ– h3A4–CYPhOR (d and h) cells. Screen enhanced autoradiography was at room temperature for 10–15 h (upper panel) or 4–6 h (lower panel).
cells treated with solvent (DMSO) only [14]. When DNA from 3-NBA-treated cells was analyzed after enrichment by butanol extraction a total of four adducts was detected (Fig. 2(e) –(h)). As shown in the lower panel of Fig. 2 adduct spot 4, derived from deoxyguanosine, was clearly visible after butanol extraction but not after nuclease P1 digestion confirming results reported by us previously [8,9] which is characteristic for N-substituted aryl adducts
bound to the C-8 position of guanine [30]. The spot in the right top corner in Fig. 2(e) –(h) is most likely a false positive signal because it was observed in some analyses only. Quantitative analysis, shown in Fig. 3, revealed adduct levels to be in the range from 75 to 132 adducts per 108 nucleotides for total DNA binding after nuclease P1 digestion with highest total adduct levels obtained in V79 cells expressing human CYPOR
Fig. 3. Bar charts showing RAL (relative adduct labelling; values represent mean ^ SD) of total DNA adducts in V79MZ cells after exposure to 1 mM 3-NBA using the nuclease P1 or butanol enrichment version of the 32P-postlabelling assay. Comparison was performed by t-test analysis: * p , 0:05 by comparison to parental V79MZ; * * p , 0:05 by comparison to V79MZ–CYPhOR.
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alone and lowest in the parental V79MZ cells. Furthermore, compared to V79MZ cells total DNA binding by 3-NBA was significantly higher (1.7-fold, P ¼ 0:024Þ in V79 cells expressing human CYPOR (Fig. 3), indicating that human CYPOR is involved in the bioactivation of 3-NBA. In contrast, no significant change in adduct levels was observed in V79 cells expressing human CYP1A1 or co-expressing both human CYP3A4 and CYPOR (Fig. 3). In V79 cells expressing human CYP1A1 one major adduct was detected after treatment with benzo[a ]pyrene whereas in the parental V79MZ cells no such adduct was found (data not shown). In V79 cells expressing human CYP3A4 in conjunction with human CYPOR high levels of ellipticine-derived DNA adducts were observed after exposure to ellipticine (data not shown). This is in line with reports by Frei et al. [31] where it was shown that ellipticine is mainly activated by human CYP3A4. Using the butanol enrichment version of the assay total DNA binding increased by 1.4- to 2.2-fold in all V79MZ cells in comparison to DNA binding observed after nuclease P1 enrichment (Fig. 3). As expected
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butanol extraction resulted in a 4.6 – 23.2-fold increase of adduct spot 4 (Table 1). Moreover, in V79 cells expressing human CYPOR alone or CYP3A4 in conjunction with CYPOR total DNA binding increased significantly, by 1.4- and 2.1-fold (P ¼ 0.046 and P ¼ 0.001), respectively, in comparison to DNA binding observed in the parental cells V79MZ. No significant change in 3-NBA-adduct levels was found in the V79 cells expressing human CYP1A1 (Fig. 3). Comparison of DNA binding in V79 cells expressing human CYPOR alone versus DNA binding in V79 cells expressing both human CYP3A4 and CYPOR revealed that total DNA binding increased significantly by a factor of 1.5 by co-expression of human CYP3A4 ðP ¼ 0:016Þ (Fig. 3). This implies that human CYP3A4 is also involved in the bioactivation of 3-NBA. In particular adduct levels of spots 2 – 4 increased significantly 1.6-, 1.3- and 2.2-fold, respectively, (Table 1). Cell viability (in % of control) of the V79MZderived cells used for the 32P-postlabelling assay decreased to 45.1% ^ 4.9 in V79MZ, 67.3% ^ 4.9 in V79MZ –h1A1, 62.5% ^ 7.1 in V79MZ –CYPhOR
Table 1 Quantitative analysis of DNA adduct formation in V79MZ cells treated with 1 mM 3-NBA Cell line
Spot no.
RAL mean ^ SD per 108 nucleotidesa Nuclease P1
Butanol extraction
V79MZ (parental)
1 2 3 4
1.1 ^ 0.6 17.8 ^ 11.6 52.4 ^ 18.0 4.0 ^ 3.0
3.0 ^ 0.7 19.7 ^ 1.0 47.4 ^ 5.6 32.8 ^ 4.2
V79MZ–h1A1b
1 2 3 4
1.8 ^ 0.1* 17.8 ^ 0.7 57.1 ^ 5.8 3.8 ^ 0.2
3.6 ^ 0.6 26.5 ^ 5.0* 54.8 ^ 6.4 21.3 ^ 4.6*
V79MZ–CYPhORb
1 2 3 4
2.9 ^ 0.3* 22.3 ^ 6.7 101.0 ^ 19.2* 5.5 ^ 1.9
10.3 ^ 6.1 28.2 ^ 8.4 82.1 ^ 13.7* 25.8 ^ 6.2
V79MZ–h3A4– CYPhORb
1 2 3 4
a
2.0 ^ 0.3* ** 17.7 ^ 4.2 82.7 ^ 15.3* 2.4 ^ 0.8**
7.3 ^ 3.9 46.5 ^ 11.1* ** 108.6 ^ 8.1* ** 55.9 ^ 9.1* **
Mean RAL (relative adduct labelling) ^ SD of three separate incubations analysed once. Comparison was performed by t-test analysis: * p , 0:05 by comparison to parental V79MZ; * * p , 0:05 by comparison to V79MZ– CYPhOR. b
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and 52.0% ^ 2.8 in V79MZ– h3A4 – CYPhOR after 3-NBA-treatment. 3.2. DNA adduct formation by 3-NBA in V79NH cells expressing nitroreductase and NAT In V79NH cells, which are devoid of CYP activity, but exhibit high activities of the two nitro-PAH activating enzymes, nitroreductase and NAT [28] a similar DNA adduct pattern after exposure to 3-NBA was observed to those found in V79MZ cells, irrespective of the enrichment procedure used (Fig. 4). Control incubations with V79NH cells treated with solvent only resulted in autoradiograms essentially free of spots identical with controls from V79MZ cells. Using different concentrations of 3-NBA (0.01, 0.1 and 1 mM) a massive increase in DNA binding as compared to V79MZ cells was found in both V79NH cells yielding remarkably high total adduct levels of up to 1 adduct in 104 nucleotides (Fig. 5). Quantitative analysis revealed that with the two high concentrations of 0.1 and 1 mM 3-NBA no difference in total adduct levels was apparent between
the two V79NH cell lines indicating that human CYP1A2 had no crucial influence on the bioactivation of 3-NBA (Fig. 5) in these cell lines. In contrast, using the low concentration of 0.01 mM 3-NBA expression of human CYP1A2 resulted in a significant decrease by a factor of 12.2 ðP ¼ 0:007Þ and 13.1 ðP ¼ 0:012Þ in adduct levels when analyzed by the nuclease P1 and butanol enrichment procedure, respectively, (Fig. 5). Therefore, these results suggest a detoxifying effect for 3-NBA by human CYP1A2 under these conditions. Viability of the V79NH cells decreased to approximately 10% (in% of control) at the two high concentrations of 3-NBA and was comparable to the cell viability observed in V79MZ cells (about 60 – 70%) at the low concentration of 0.01 mM 3-NBA.
4. Discussion Some limited evidence suggests that diesel exhaust and airborne particulates, which contain significant amounts of nitro-PAHs, may be carcinogenic in humans [2,4]. 3-NBA, an aromatic nitroketone, is
Fig. 4. Autoradiographic profiles of DNA adducts obtained after incubation of V79NH cells with 3-NBA using the nuclease P1 (a and b) or butanol (c and d) enrichment version of the 32P-postlabelling assay. Autoradiograms exemplify digest of V79NH (a and c) and V79NH–h1A2 (b and d) cells exposed to 1 mM 3-NBA. Screen enhanced autoradiography was performed for 0.5–1 h.
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Fig. 5. Bar charts showing mean RAL (relative adduct labelling) of total DNA adducts in V79NH cells after exposure to 0.01, 0.1 or 1 mM 3NBA using the nuclease P1 or butanol enrichment version of the 32P-postlabelling assay. The values given are the mean of three separate incubations analysed once within a maximal error range of 40%. Comparison was performed by t-test analysis: * p , 0:05 by comparison to parental V79NH.
a very potent direct acting mutagen recently identified in these environmental sources [5]. Recently, 3-NBA – DNA-adducts were detected in various organs of rats after administration of a single dose of 3-NBA or its metabolites [9,10]. These findings prompted us to determine which human P450 enzymes are the most important in activating 3-NBA to genotoxic products that form DNA adducts. Using the 32P-postlabelling assay a pattern of four 3-NBA-specific adducts was observed in all V79 cells used, similar to that found previously in vivo in rats [9,10]. Moreover, a comparison of chromatographic characteristics among DNA adducts obtained in cell culture with those detected in vivo in rats revealed that these 3-NBA-adducts were chromatographically indistinguishable. Activation of 3-NBA in V79 cells expressing human CYPOR demonstrated that human CYPOR contributes significantly to the bioactivation of 3-NBA. This is in line with our recent report where we showed that 3-NBA is activated by various human hepatic microsomes forming DNA adduct patterns qualitatively similar to those found in the present study [27]. Correlation of CYP- and CYPOR-linked activities with the level of DNA binding indicated that most of the hepatic microsomal activation of 3-NBA was attributed to CYPOR. CYPOR is expressed in human bronchial epithelial cells (BEC) and alveolar
macrophages (AM), a primary defense system against inhaled materials [32]. Therefore metabolic activation of 3-NBA in BEC and AM due to human CYPOR may represent a potential human risk. Furthermore, DNA binding increased significantly in V79 cells expressing human CYP3A4 in conjunction with human CYPOR compared to V79 cells expressing human CYPOR alone. Thus, we conclude that also human CYP3A4 is involved in the bioactivation of 3-NBA. However, DNA binding in V79 cells expressing human CYP3A4 together with CYPOR only increased significantly when analyzed by the butanol extraction version and was associated with the detection of the nuclease P1-sensitive adduct spot 4. CYP3A4 is the most abundant CYP in human liver and small intestine and although CYP3A5 is the predominant CYP3A form in human lung, CYP3A4 is expressed in about 20% of individuals [33,34]. Therefore, metabolic activation of 3-NBA in human lung cells may represent a potential human risk. In contrast, no evidence was seen for the involvement of human CYP1A1 in the biotransformation of 3-NBA in V79MZ cells. Both ring oxidation and nitro reduction are known pathways in the metabolic activation of nitro-PAHs [3,11]. Activation by ring oxidation by CYP enzymes is described for many polycyclic aromatic hydrocarbons
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(PAHs) as well as for nitro-PAHs [3,11,34]. Our results from in vitro incubations of calf thymus DNA with XO and 3-NBA demonstrated that all four 3-NBA-adducts are reaction products derived from nitroreduction [8]. Furthermore, the same 3-NBA-adducts were formed in vitro by rat liver S9 mix, human hepatic microsomes, in vivo in rats, human MCL-5 cells as well as in all V79 cells examined (present study) [8,9,27]. Thus, we assume that all 3-NBA-adducts are formed via nitroreduction and not by ring oxidation. The involvement of human CYP enzymes in the reductive activation of nitro-PAHs was demonstrated [35 – 37] suggesting that human CYP3A4 is involved in the metabolism of 3-NBA via simple nitroreduction. This is also in line with a recent report by Arlt et al. [15] where it was shown that 3-NBA is activated by recombinant human CYP1A2 expressed in V79 cells. Similarly, essentially the same DNA adduct pattern was observed after exposure to 1 mM 3-NBA. Compared with parental V79MZ cells DNA binding by 3-NBA was approximately 2- and 3-fold higher in V79 cells expressing human CYP1A2 cells after nuclease P1 and butanol enrichment, respectively, demonstrating that human CYP1A2 contributes to the metabolic activation of 3-NBA to form DNA adducts derived from nitroreduction [15]. That nitroreduction is the major activation pathway for 3-NBA is further supported by the fact that a 2- to 4-times higher DNA binding by 3-NBA is found in V79NH cells expressing high activities of nitroreductase and NAT after exposure to only 0.01 mM 3-NBA compared to the level of DNA binding observed in V79MZ cells after exposure to a 100-fold higher concentration. Moreover, the massive increased cytotoxicity in V79NH cells supports the view that 3-NBA is strongly activated to toxic products in these cells. Topinka et al. [38] also used V79NH cells to study the genotoxic potential of coke oven emissions extracts in mammalian cells containing a complex mixture of PAHs and nitro-PAHs. In this study no DNA adducts were detected for PAHs tested, including benzo[a ]pyrene, but for nitro-PAHs, including 4-nitropyrene, 6-nitrochrysene and 1,6-dinitropyrene, strong DNA adduct formation was observed. The activation of 3-NBA in V79NH cells most likely occurs by nitroreduction and subsequent acetylation of the resulting N-hydroxy arylamines catalyzed by NAT. However, the NAT genotype in these cells has
not yet been described in the literature. Consistent with this assumption is the finding that 3-NBA had a 30-fold higher mutagenic activity in the Ames S. typhimurium YG1024 strain exhibiting O-acetyltransferase than in TA98 strain [5]. This also matches the findings that human phase II metabolic enzymes significantly increase the level of DNA adducts derived from 3-NBA in V79 cells expressing human NATs and SULTs [14,15]. In V79NH cells expressing human CYP1A2 a decrease in DNA binding was observed at the lowest concentration of 3-NBA used (0.01 mM) suggesting a detoxifying effect by human CYP1A2 in conjunction with NAT in these cells at low concentrations. A similar effect was reported for the inactivation of dinitropyrenes by CYP enzymes and CYPOR in human liver microsomes [36]. It was suggested by the authors that the nitropyrenes are reduced to the corresponding amino compounds, which are rather inactive and not readily oxidized to the corresponding hydroxylamino derivatives. It might also occur that overexpression of human CYP1A2 influences the expression and/or activity of nitroreductases. Indications for this suggestion have been reported by Kappers et al. [39] who showed that in V79NH – h1A2 cells DT-diaphorase enzyme activity was reduced two-fold and that CYPOR activity was six-fold increased, respectively, compared to V79NH cells. The present study demonstrates that human CYPOR and CYP3A4 are able to contribute to the bioactivation of 3-NBA via simple nitroreduction. Moreover, acetylation of the initially formed Nhydroxy arylamine intermediates most likely contributes significantly to the high genotoxic potential of 3-NBA. Catalysis by NAT may therefore be an important step in the bioactivation of 3-NBA in vivo. Acknowledgements Grant sponsor: Baden-Wu¨rttemberg; Grant numbers: BWPLUS, BWB 20003. References [1] H. Tokiwa, Y. Ohnishi, Mutagenicity and carcinogenicity of nitroarenes and their sources in the environment, CRC Crit. Rev. Toxicol. 17 (1986) 23 –60.
C.A. Bieler et al. / Cancer Letters 200 (2003) 9–18 [2] IARC, Diesel and Gasoline Engine Exhausts and Some Nitroarenes, IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Lyon, No. 46, International Agency for Research on Cancer, Lyon, 1989. [3] V. Purohit, A.K. Basu, Mutagenicity of nitroaromatic compounds, Chem. Res. Toxicol. 13 (2000) 673–692. [4] D.T. Silvermann, L.I. Levin, R.N. Hoover, P. Hartge, Occupational risks of bladder cancer in the United States: I. White men, J. Natl Cancer Inst. 81 (1989) 1472–1480. [5] T. Enya, H. Suzuki, T. Watanabe, T. Hirayama, Y. Hisamatsu, 3-Nitrobenzanthrone, a powerful bacterial mutagen and suspected human carcinogen found in diesel exhausts and airborne particulates, Environ. Sci. Technol. 31 (1997) 2772–2776. [6] A. Seidel, D. Dahmann, H. Krekeler, J. Jacob, Biomonitoring of polycyclic aromatic compounds in the urine of mining workers occupationally exposed to diesel exhaust, Int. J. Hyg. Environ. Health 204 (2002) 333 –338. [7] P.T. Phousongphoung, A.J. Grosovsky, D.A. Eastmond, M. Covarrubias, J. Arey, The genotoxicity of 3-nitrobenzanthrone and the nitropyrene lactones in human lymphoblasts, Mutat. Res. 472 (2000) 93–103. [8] C.A. Bieler, M. Wiessler, L. Erdinger, H. Suzuki, T. Enya, H.H. Schmeiser, DNA adduct formation from the mutagenic air pollutant 3-nitrobenzanthrone, Mutat. Res. 439 (1999) 307–311. [9] V.M. Arlt, C.A. Bieler, W. Mier, M. Wiessler, H.H. Schmeiser, DNA adduct formation by the ubiquitous environmental contaminant 3-nitrobenzanthrone in rats determined by 32 P-postlabeling, Int. J. Cancer 93 (2001) 450 –454. [10] V.M. Arlt, B.L. Sorg, M. Osborne, A. Hewer, A. Seidel, H.H. Schmeiser, D.H. Phillips, DNA adduct formation by the ubiquitous environmental pollutant 3-nitrobenzanthrone and its metabolites in rats, Biochem. Biophys. Res. Commun. 300 (2003) 107–114. [11] P.P. Fu, Metabolism of nitro-polycyclic aromatic hydrocarbons, Drug Metab. Rev. 22 (1990) 209 –268. [12] F.A. Beland, M.M. Marques, DNA Adducts of Nitropolycyclic Aromatic Hydrocarbons, in: DNA Adducts, Identification and Biological Significance, IARC Publications, No. 125, IARC publishers, Lyon, 1994, pp. 229–244. [13] F.F. Kadlubar, G.J. Hammons, The Role of Cytochrome P-450 in the Metabolism of Chemical Carcinogens, in: Mammalian Cytochromes P-450, CRC Press, Boca Raton, 1987, pp. 81 – 130. [14] V.M. Arlt, H. Glatt, E. Muckel, U. Pabel, B.L. Sorg, H.H. Schmeiser, D.H. Phillips, Metabolic activation of the environmental contaminant 3-nitrobenzanthrone by human acetyltransferases and sulfotransferase, Carcinogenesis 23 (2002) 1937–1945. [15] V.M. Arlt, H. Glatt, E. Muckel, U. Pabel, B.L. Sorg, A. Seidel, H. Frank, H.H. Schmeiser, D.H. Phillips, Activation of 3nitrobenzanthrone and its metabolites by human acetyltransferases, sulfotransferases and cytochrome P450 expressed in Chinese hamster V79 cells, Int. J. Cancer 105 (2003) 583–592. [16] J. Doehmer, J.T.M. Buters, A. Luch, V. Soballa, W.M. Baird, H. Morisson, J.J. Stegeman, A.J. Townsend, W.F. Greenlee,
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
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H. Glatt, A. Seidel, J. Jacob, H. Greim, Molecular studies on the toxifying effects by genetically engineered cytochromes P450, Drug Metabol. Rev. 31 (1999) 423–435. W.A. Schmalix, H. Ma¨ser, F. Kiefer, R. Reen, F.J. Wiebel, F. Gonzalez, A. Seidel, H. Glatt, H. Greim, J. Doehmer, Stable expression of human cytochrome P450 1A1 cDNA in V79 Chinese hamster cells and metabolic activation of benzo[a]pyrene, Eur. J. Pharmacol. 248 (1993) 251–261. C. Wo¨lfel, B. Heinrich-Hirsch, T. Schulz-Schalge, A. Seidel, H. Frank, U. Ramp, F. Wa¨chter, F.J. Wiebel, F. Gonzalez, H. Greim, J. Doehmer, Genetically engineered V79 Chinese hamster cells for stable expression of human cytochrome P4501A2, Eur. J. Pharmacol. 228 (1992) 95–102. A. Schneider, W.A. Schmalix, V. Siruguri, E.M. de Groene, G.J. Horbach, B. Kleingeist, D. Lang, R. Bo¨cker, R. Belloc, P. Beaume, H. Greim, J. Doehmer, Stable expression of human cytochrome P450 3A4 in conjunction with human NADPHcytochrome P450 oxidoreductase in V79 Chinese hamster cells, Arch. Biochem. Biophys. 332 (1996) 295–304. H. Glatt, W. Davis, W. Meindl, H. Hermersdo¨rfer, S. Venitt, D.H. Phillips, Rat, but not human, sulfotransferase activates a tamoxifen metabolite to produce DNA adducts and gene mutations in bacteria and mammalian cells in culture, Carcinogenesis 19 (1998) 1709–1713. A. Luch, S.L. Coffing, Y.M. Tang, A. Schneider, V. Soballa, H. Greim, C.R. Jefcoate, A. Seidel, W.F. Greenlee, W.M. Baird, J. Doehemer, Stable Expression of human cytochrome P450 1B1 in V79 Chinese hamster cells and metabolically catalyzed DNA adduct formation of dibenzo[a,l]pyrene, Chem. Res. Toxicol. 11 (1998) 686 –695. A. Luch, S. Kishiyama, A. Seidel, J. Doehemer, H. Greim, W.M. Baird, The K-region trans-8,9.diol does not significantly contribute as an intermediate in the metabolic activation of dibenzo[a,l]pyrene to DNA-binding metabolites by human cytochrome P450 1A1 or 1B1, Cancer Res. 59 (1999) 4603–4609. K. Sundberg, K. Dreij, A. Seidel, B. Jernstro¨m, Glutathione conjugation and DNA adduct formation of dibenzo[a,l]pyrene and benzo[a]pyrene diol epoxides in V79 cells stably expressing different human glutathione transferases, Chem. Res. Toxicol. 15 (2002) 170 –179. B. Mahadevan, W.-M. Dashwood, A. Luch, A. Pecaj, J. Doehmer, A. Seidel, C. Pereira, W.M. Baird, Mutations induced by (-)-anti-11R,12S-dihydrodiol-13S,14R-epoxide of dibenzo[a,l]pyrene in the coding region of the hypoxanthine phosphoribosyltransferase (HPRT) gene in Chinese hamster V79 cells, Environ. Mol. Mutagenesis 41 (2003) 131–139. A. Seidel, T. Friedberg, B. Lo¨llmann, A. Schwierzok, M. Funk, H. Frank, R. Holler, F. oesch, H. Glatt, Detoxication of optically active bay- and fjord-region polycyclic aromatic hydrocarbon dihydrodiol epoxides by human glutathione transferase P1-1 expressed in Chinese hamster V79 cells, Carcinogenesis 19 (1998) 1975–1981. J. Buters, L. Quintanilla-Martinez, W. Schober, V.J. Soballa, J. Hintermair, T. Wolff, F.J. Gonzalez, H. Greim, CYP1B1 determines susceptibility to low doses of 7,12-dimethylbenz[a]anthracene-induced ovarian cancers in mice: correlation of
18
[27]
[28]
[29]
[30]
[31]
[32]
C.A. Bieler et al. / Cancer Letters 200 (2003) 9–18 CYP1B1-mediated DNA adducts with carcinogenicity, Carcinogenesis 24 (2003) 327– 334. V.M. Arlt, M. Stiborova, A. Hewer, H.H. Schmeiser, D.H. Phillips, Human enzymes involved in the metabolic activation of the environmental contaminant 3-nitrobenzanthrone: Evidence for reductive activation by human NADPH:cytochrome P450 reductase, Cancer Res. 63 (2003) 2752– 2761. F. Kiefer, O. Cumpelik, R. Reen, J. Doehmer, F.J. Wiebel, Arylamines suppress their own activation and that of nitroarenes in V79 Chinese hamster cells by competing for acetyltransferases, Environ. Health Perspect. 102 (1994) 95–97. M. Stiborova, C.A. Bieler, M. Wiessler, E. Frei, The anticancer agent ellipticine on activation by cytochrome P450 forms covalent DNA adducts, Biochem. Pharmacol. 62 (2001) 1675–1684. J.E. Gallagher, M.J. Kohan, M.H. George, J. Lewtas, Improvement of the diagnostic potential of 32P-postlabeling analysis demonstrated by the selective formation and comparative analysis of nitrated-PAH-derived adducts arising from diesel particle extracts, Carcinogenesis 12 (1991) 1685– 1691. E. Frei, C.A. Bieler, V.M. Arlt, M. Wiessler, M. Stiborova, Covalent binding of the anticancer drug ellipticine to DNA in V79 cells transfected with human cytochrome P450 enzymes, Biochem. Pharmacol. 64 (2002) 289 –295. J.C. Willey, E. Coy, C. Brolly, M.J. Utell, M.W. Frampton, J. Hammersley, W.G. Thilly, D. Olson, K. Cairns, Xenobiotic metabolism enzyme gene expression in human bronchial epithelial and alveolar macrophage cells, Am. J. Respir. Cell Mol. Biol. 14 (1996) 262–271.
[33] K. Mace, E.D. Bowman, P. Vauttravers, P.G. Shields, C.C. Harris, A.M. Pfeifer, Characterisation of xenobiotic-metabolising enzyme expression in human bronchial mucosa and peripheral lung tissue, Eur. J. Cancer 34 (1998) 914–920. [34] F.P. Guengerich, T. Shimada, Oxidation of toxic and carcinogenic chemicals by human cytochrome P-450 enzymes, Chem. Res. Toxicol. 4 (1991) 391– 407. [35] Y.H. Chae, C.H. Yun, F.P. Guengerich, F.F. Kadlubar, K. El-Bayoumy, Roles of human hepatic and pulmonary cytochrome P450 enzymes in the metabolism of the environmental carcinogen 6-nitrochrysene, Cancer Res. 53 (1993) 2028–2034. [36] 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. [37] Y.H. Chae, B.Y. Ji, J.M. Lin, P.P. Fu, B.P. Cho, K. ElBayoumy, Nitroreduction of 4-nitropyrene is primarily resonsible for DNA adduct formation in the mammary gland of female CD rats, Chem. Res. Toxicol. 12 (1999) 180 –186. [38] J. Topinka, L.R. Schwarz, F. Kiefer, F.J. Wiebel, O. Gajdos, P. Vidova, L. Dobias, M. Fried, R.J. Sram, T. Wolff, DNA adduct formation in mammalian cell culture by polycyclic aromatic hydrocarbons (PAH) and nitro-PAH in coke oven emission extract, Mutat. Res. 419 (1998) 91–105. [39] W.A. Kappers, F.M.M. van Och, E.M. de Groene, G.J. Horbach, Comparison of three different in vitro mutation assays used for the investigation of cytochrome p450mediated mutagenicity of nitro-polycyclic aromatic hydrocarbons, Mutat. Res. 466 (2000) 143– 159.
DNA adduct formation by the environmental contaminant 3-nitrobenzanthrone in V79 cells expressing human cytochrome P450 enzymes Christian A. Bieler, Volker M. Arlt1, Manfred Wiessler, Heinz H. Schmeiser* Division of Molecular Toxicology, German Cancer Research Center, Im Neuenheimer Feld 280, Heidelberg 69120, Germany Received 1 April 2003; received in revised form 2 June 2003; accepted 4 June 2003
Abstract Diesel exhaust is known to induce tumours in animals. Of the compounds found in diesel exhaust 3-nitrobenzanthrone (3-NBA) is particularly a powerful mutagen. Recently we showed that 3-NBA is genotoxic in vivo in rats by forming specific DNA adducts derived from nitroreduction. In this study a panel of genetically engineered V79 Chinese hamster cell lines expressing various human cytochrome P450 (CYP) enzymes (CYP1A1, CYP3A4) and/or human NADPH:CYP oxidoreductase (CYPOR) was used to identify CYP enzymes involved in the metabolic activation of 3-NBA. We analyzed the formation of specific DNA adducts by 32P-postlabelling after exposing cells to 1 mM 3-NBA. A similar pattern with a total of four distinct 3-NBA– DNA adducts was found in all cells, identical to those detected previously in DNA from rats treated with 3-NBA in vivo. Total adduct levels ranged from 75 to 132 using nuclease P1 and from 103 to 220 adducts per 108 nucleotides, using butanol enrichment. Comparison of DNA binding between different V79MZ derived cells revealed that human CYPOR and CYP3A4 were involved in the metabolic activation of 3-NBA. Furthermore, dose-dependent high adduct levels were detected after exposure to 0.01, 0.1 or 1 mM 3-NBA in the subclone V79NH which exhibits high activities of nitroreductase and N,Oacetyltransferase. Our results suggest that nitroreduction is the major pathway in the human bioactivation of 3-NBA. Moreover, acetylation of the initially formed N-hydroxy arylamine intermediates may contribute to the high genotoxic potential of 3-NBA. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: 3-Nitrobenzanthrone; DNA adducts; 32P-postlabelling; Human metabolism; Nitro-PAH; Diesel exhaust
Abbreviations: 3-NBA, 3-Nitrobenzanthrone; NAT, N,Oacetyltransferase; SULT, Sulfotransferase; CYP, Cytochrome P450; CYPOR, NADPH-dependent cytochrome P450 oxidoreductase; nitro-PAH, Nitro-polycyclic aromatic hydrocarbon; XO, Xanthine oxidase; RAL, Relative adduct labelling; DMSO, Dimethyl sulfoxide. * Corresponding author. Tel.: þ49-6221-4233-48; fax: þ 496221-4233-75. E-mail address: [email protected] (H.H. Schmeiser). 1 Present address: Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Sutton, Surrey SM2 5NG, UK.
1. Introduction Nitro-polycyclic aromatic hydrocarbons (nitroPAH) are widely distributed environmental pollutants found in extracts from diesel and gasoline engines and on the surface of ambient air particulate matter [1]. Mutagenic activities in bacterial and mammalian systems as well as tumorigenic activity in laboratory animals of several members of this class have been clearly documented [2,3]. Although the significance
0304-3835/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0304-3835(03)00418-X
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Fig. 1. Structure of 3-nitrobenzanthrone (3-nitro-7H-benz[d,e ]anthracen-7-one).
of nitro-PAH in the induction of human cancer is presently unknown, their possible link with lung cancer in traffic-congested areas has lead to considerable interest in assessing their potential risk to man [4]. Recently a new member of this group of compounds, 3-nitrobenzanthrone (3-NBA, 3-nitro-7Hbenz[d,e ]anthracen-7-one) (Fig. 1), was discovered in diesel exhaust and bound to the surface of airborne particulate matter [5]. Furthermore, its major metabolite, 3-aminobenzanthrone, was detected in the urine of mining workers occupationally exposed to diesel emissions [6]. 3-NBA was shown to be one of the most potent mutagens in the Ames assay reported so far and to induce micronuclei in mouse and human cells, as well as, mutations in human cells [5,7]. Further evidence for the genotoxic action of this compound was provided by the detection of 3-NBA-derived DNA adducts using the 32 P-postlabelling assay after reductive activation in vitro as well as in vivo in rats treated orally with a single dose of 3-NBA or its metabolites [8 – 10]. Nitro-PAHs require metabolism to reactive electrophilic species in order to exert their genotoxic and carcinogenic activity. The activation of nitroaromatic hydrocarbons is through reduction to N-hydroxy arylamines, catalyzed by both cytosolic and microsomal enzymes such as aldehyde oxidase, xanthine oxidase (XO), DT-diaphorase, cytochrome P450 (CYP) enzymes and nicotinamide adenine dinucleotide phosphate reduced (NADPH) CYP oxidoreductase (CYPOR) [3,11]. Further biotransformation occurs by phase II-enzymes, such as N,O-acetyltransferases (NATs) or sulfotransferases (SULTs), leading to the formation of reactive esters, which undergo
hydrolysis to produce electrophilic nitrenium ions capable of forming DNA adducts [12,13]. Indeed adduct levels induced by 3-NBA or its potential metabolites were significantly increased in V79 cells expressing human phase II enzymes, e.g. NAT2 or SULT1A1, compared to the parental cell line V79MZ lacking such enzymatic activity [14,15]. V79 Chinese hamster lung fibroblasts are widely used in mutagenicity testing but these cells completely lack CYP-dependent enzyme activities [16]. Nevertheless, V79 cells contain detectable amounts of CYPOR, an enzyme known to activate nitroaromatic compounds [17]. With respect to this a panel of V79 cells expressing human CYP enzymes including human CYP1A1 and CYP3A4, required for the biotransformation of many xenobiotics, have been genetically engineered by Doehmer and others [17 – 19]. Previous studies with such recombinant cells have allowed the evaluation of the role of single human isoforms of enzymes in the metabolic activation or detoxication of several carcinogens (e.g. tamoxifen [20] dibenzo[a,l ]pyrene [21 – 24], benzo[a ]pyrene [23,25], 7,12-dimethylbenz[a ]anthracene [26] and benzo[c ]phenanthrene [25] using DNA adduct formation as a suitable biological endpoint. Recently we showed that 3-NBA forms DNA adducts in metabolically competent human lymphoblastoid MCL-5 cells expressing various CYP enzymes [27]. This prompted us to investigate the involvement of human enzymes in the metabolic activation of 3-NBA. For this purpose we have employed V79 cell lines equipped with different enzymes to determine DNA adduct levels by the 32Ppostlabelling method as a means of determining enzyme-mediated genotoxicity. In addition to the V79MZ cell lines that stably express various human CYP enzymes we used V79NH cells, another subclone of the V79 cell line [28]. Like the V79MZ subclone V79NH is devoid of CYP activities but exhibits high nitroreductase and NAT activities, two nitro-PAH activating enzymes. 2. Material and methods 2.1. Chemicals and cell lines 3-NBA was synthesized as described in Ref. [14]. Benzo[a ]pyrene and ellipticine were obtained from
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Sigma Chemical Co. (St Louis, MO). The transfected cells V79MZ –h1A1 [17], V79MZ – h3A4 – CYPhOR [19] were purchased from PharmBioDyn, Freiburg, Germany. Parental V79MZ, V79NH [28] and the transfected cells V79MZ – CYPhOR [19] and V79NH – h1A2 [28] were kindly provided by Prof Doehmer (GenPharmTox BioTech AG, Munich, Germany).
human CYP3A4 V79MZ, V79MZ – CYPhOR and V79MZ –h3A4 –CYPhOR were treated with 1 mM ellipticine. Cells treated with DMSO only were used as a negative control. After an incubation period of 24 h the medium was removed and the cells were harvested and DNA was isolated as described [14]. Cell viability (in % of control) was determined by the trypan blue (Biochrom KG) exclusion assay [14].
2.2. Cell culture
2.4. 32P-Postlabelling of DNA adducts
The parental cells, V79MZ (7-ethoxyresorufin-Odeethylase (EROD) ¼ , 0.2 pmol/min/mg protein; cytochrome c reduction ¼ 8.2 nmol/min/mg protein) [18,19] and the transfected cells, V79MZ – h1A1 expressing human CYP1A1 (EROD ¼ 48.8 pmol/ min/mg protein) [17], V79MZ– CYPhOR expressing human CYPOR (cytochrome c reduction ¼ 272.1 nmol/min/mg protein) [19] and V79MZ – h3A4 – CYPhOR expressing human CYP3A4 in conjunction with CYPOR (CYP3A4 ¼ 6 pmol/mg protein determined by immunochemistry; cytochrome c reduction ¼ 81.0 nmol/min/mg protein) [19], as well as the V79NH (EROD ¼ , 0.2 pmol/min/mg protein) and V79NH – h1A2 expressing human CYP1A2 (EROD ¼ 8.1 pmol/min/mg protein) [18] cells were cultivated in Dulbecco’s modified Eagles’s medium (DMEM, Biochrom KG, Berlin Germany), with high glucose content (4.5 g D -glucose/l), supplemented with 1 mM sodium pyruvate (Biochrom KG), 4 mM L -glutamine (PAA Laboratories, Linz, Austria), 5% fetal calf serum (FCS) (Biochrom KG), 100 U of penicillin/ml and 100 mg streptomycin/ml (Biochrom KG) at 37 8C, 5% CO2 and 95% atmospheric humidity. 2.3. Treatment of V79 cells, cell harvesting and DNA isolation All V79 cells were seeded 24 h prior to treatment at a density of 1 £ 105 cells/ml in 75 cm2 culture flasks in a total volume of 30 ml of medium. 3-NBA dissolved in dimethyl sulfoxide (DMSO) (Merck, Germany) was added to a final concentration of 1 mM to V79MZ cells and to a final concentration of 0.01, 0.1 or 1 mM to V79NH cells. As positive control for oxidative activation by human CYPh1A1 V79MZ and V79MZ– h1A1 were treated with 1 mM benzo[a ] pyrene. As positive control for oxidative activation by
32
P-Postlabelling analyses for 3-NBA-derived adducts were performed using the nuclease P1 enrichment or the butanol extraction method as described previously [8]. 32P-postlabelling analyses for benzo[a ]pyrene- and ellipticine-derived DNA adducts was performed using the nuclease P1 enrichment as described previously [29]. Quantitative analysis was performed using the Canberra Packard Instant Imager system. Relative adduct labelling (RAL) was calculated as cpm adducts per cpm normals. All adduct levels are expressed as mean ^ SD. Comparison was performed by t-test analysis. All P-values are considered significant at the 0.05 level.
3. Results 3.1. DNA adduct formation by 3-NBA in V79MZ cells expressing human CYP enzymes Parental V79MZ, which are devoid of CYP activity, and V79MZ cells expressing human CYP1A1, human CYPOR alone or human CYP3A4 together with human CYPOR were treated with 1 mM 3-NBA to ensure adduct levels easily detectable by the 32P-postlabelling method (RAL range 1 adduct in 106 – 108 nucleotides). Co-expression of human CYP3A4 and CYPOR has been reported to be needed to receive sufficient CYP3A4 activity [19]. Using either enrichment procedure of the 32P-postlabelling assay, nuclease P1 digestion and butanol extraction, DNA adduct analysis revealed a similar pattern of 3NBA-derived adducts in all V79MZ cells as found previously in vivo in different organs of rats treated orally with a single dose of 3-NBA (Fig. 2) [9]. As reported before no adducts were detected in control
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Fig. 2. Autoradiographic profiles of DNA adducts obtained after incubation of V79MZ cells with 1 mM 3-NBA using the nuclease P1 (a –d) or butanol (e– h) enrichment version of the 32P-postlabelling assay. Autoradiograms exemplify digest of V79MZ (a and e), V79MZ-h1A1 (b and f), V79MZ–CYPhOR (c and g) and V79MZ– h3A4–CYPhOR (d and h) cells. Screen enhanced autoradiography was at room temperature for 10–15 h (upper panel) or 4–6 h (lower panel).
cells treated with solvent (DMSO) only [14]. When DNA from 3-NBA-treated cells was analyzed after enrichment by butanol extraction a total of four adducts was detected (Fig. 2(e) –(h)). As shown in the lower panel of Fig. 2 adduct spot 4, derived from deoxyguanosine, was clearly visible after butanol extraction but not after nuclease P1 digestion confirming results reported by us previously [8,9] which is characteristic for N-substituted aryl adducts
bound to the C-8 position of guanine [30]. The spot in the right top corner in Fig. 2(e) –(h) is most likely a false positive signal because it was observed in some analyses only. Quantitative analysis, shown in Fig. 3, revealed adduct levels to be in the range from 75 to 132 adducts per 108 nucleotides for total DNA binding after nuclease P1 digestion with highest total adduct levels obtained in V79 cells expressing human CYPOR
Fig. 3. Bar charts showing RAL (relative adduct labelling; values represent mean ^ SD) of total DNA adducts in V79MZ cells after exposure to 1 mM 3-NBA using the nuclease P1 or butanol enrichment version of the 32P-postlabelling assay. Comparison was performed by t-test analysis: * p , 0:05 by comparison to parental V79MZ; * * p , 0:05 by comparison to V79MZ–CYPhOR.
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alone and lowest in the parental V79MZ cells. Furthermore, compared to V79MZ cells total DNA binding by 3-NBA was significantly higher (1.7-fold, P ¼ 0:024Þ in V79 cells expressing human CYPOR (Fig. 3), indicating that human CYPOR is involved in the bioactivation of 3-NBA. In contrast, no significant change in adduct levels was observed in V79 cells expressing human CYP1A1 or co-expressing both human CYP3A4 and CYPOR (Fig. 3). In V79 cells expressing human CYP1A1 one major adduct was detected after treatment with benzo[a ]pyrene whereas in the parental V79MZ cells no such adduct was found (data not shown). In V79 cells expressing human CYP3A4 in conjunction with human CYPOR high levels of ellipticine-derived DNA adducts were observed after exposure to ellipticine (data not shown). This is in line with reports by Frei et al. [31] where it was shown that ellipticine is mainly activated by human CYP3A4. Using the butanol enrichment version of the assay total DNA binding increased by 1.4- to 2.2-fold in all V79MZ cells in comparison to DNA binding observed after nuclease P1 enrichment (Fig. 3). As expected
13
butanol extraction resulted in a 4.6 – 23.2-fold increase of adduct spot 4 (Table 1). Moreover, in V79 cells expressing human CYPOR alone or CYP3A4 in conjunction with CYPOR total DNA binding increased significantly, by 1.4- and 2.1-fold (P ¼ 0.046 and P ¼ 0.001), respectively, in comparison to DNA binding observed in the parental cells V79MZ. No significant change in 3-NBA-adduct levels was found in the V79 cells expressing human CYP1A1 (Fig. 3). Comparison of DNA binding in V79 cells expressing human CYPOR alone versus DNA binding in V79 cells expressing both human CYP3A4 and CYPOR revealed that total DNA binding increased significantly by a factor of 1.5 by co-expression of human CYP3A4 ðP ¼ 0:016Þ (Fig. 3). This implies that human CYP3A4 is also involved in the bioactivation of 3-NBA. In particular adduct levels of spots 2 – 4 increased significantly 1.6-, 1.3- and 2.2-fold, respectively, (Table 1). Cell viability (in % of control) of the V79MZderived cells used for the 32P-postlabelling assay decreased to 45.1% ^ 4.9 in V79MZ, 67.3% ^ 4.9 in V79MZ –h1A1, 62.5% ^ 7.1 in V79MZ –CYPhOR
Table 1 Quantitative analysis of DNA adduct formation in V79MZ cells treated with 1 mM 3-NBA Cell line
Spot no.
RAL mean ^ SD per 108 nucleotidesa Nuclease P1
Butanol extraction
V79MZ (parental)
1 2 3 4
1.1 ^ 0.6 17.8 ^ 11.6 52.4 ^ 18.0 4.0 ^ 3.0
3.0 ^ 0.7 19.7 ^ 1.0 47.4 ^ 5.6 32.8 ^ 4.2
V79MZ–h1A1b
1 2 3 4
1.8 ^ 0.1* 17.8 ^ 0.7 57.1 ^ 5.8 3.8 ^ 0.2
3.6 ^ 0.6 26.5 ^ 5.0* 54.8 ^ 6.4 21.3 ^ 4.6*
V79MZ–CYPhORb
1 2 3 4
2.9 ^ 0.3* 22.3 ^ 6.7 101.0 ^ 19.2* 5.5 ^ 1.9
10.3 ^ 6.1 28.2 ^ 8.4 82.1 ^ 13.7* 25.8 ^ 6.2
V79MZ–h3A4– CYPhORb
1 2 3 4
a
2.0 ^ 0.3* ** 17.7 ^ 4.2 82.7 ^ 15.3* 2.4 ^ 0.8**
7.3 ^ 3.9 46.5 ^ 11.1* ** 108.6 ^ 8.1* ** 55.9 ^ 9.1* **
Mean RAL (relative adduct labelling) ^ SD of three separate incubations analysed once. Comparison was performed by t-test analysis: * p , 0:05 by comparison to parental V79MZ; * * p , 0:05 by comparison to V79MZ– CYPhOR. b
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and 52.0% ^ 2.8 in V79MZ– h3A4 – CYPhOR after 3-NBA-treatment. 3.2. DNA adduct formation by 3-NBA in V79NH cells expressing nitroreductase and NAT In V79NH cells, which are devoid of CYP activity, but exhibit high activities of the two nitro-PAH activating enzymes, nitroreductase and NAT [28] a similar DNA adduct pattern after exposure to 3-NBA was observed to those found in V79MZ cells, irrespective of the enrichment procedure used (Fig. 4). Control incubations with V79NH cells treated with solvent only resulted in autoradiograms essentially free of spots identical with controls from V79MZ cells. Using different concentrations of 3-NBA (0.01, 0.1 and 1 mM) a massive increase in DNA binding as compared to V79MZ cells was found in both V79NH cells yielding remarkably high total adduct levels of up to 1 adduct in 104 nucleotides (Fig. 5). Quantitative analysis revealed that with the two high concentrations of 0.1 and 1 mM 3-NBA no difference in total adduct levels was apparent between
the two V79NH cell lines indicating that human CYP1A2 had no crucial influence on the bioactivation of 3-NBA (Fig. 5) in these cell lines. In contrast, using the low concentration of 0.01 mM 3-NBA expression of human CYP1A2 resulted in a significant decrease by a factor of 12.2 ðP ¼ 0:007Þ and 13.1 ðP ¼ 0:012Þ in adduct levels when analyzed by the nuclease P1 and butanol enrichment procedure, respectively, (Fig. 5). Therefore, these results suggest a detoxifying effect for 3-NBA by human CYP1A2 under these conditions. Viability of the V79NH cells decreased to approximately 10% (in% of control) at the two high concentrations of 3-NBA and was comparable to the cell viability observed in V79MZ cells (about 60 – 70%) at the low concentration of 0.01 mM 3-NBA.
4. Discussion Some limited evidence suggests that diesel exhaust and airborne particulates, which contain significant amounts of nitro-PAHs, may be carcinogenic in humans [2,4]. 3-NBA, an aromatic nitroketone, is
Fig. 4. Autoradiographic profiles of DNA adducts obtained after incubation of V79NH cells with 3-NBA using the nuclease P1 (a and b) or butanol (c and d) enrichment version of the 32P-postlabelling assay. Autoradiograms exemplify digest of V79NH (a and c) and V79NH–h1A2 (b and d) cells exposed to 1 mM 3-NBA. Screen enhanced autoradiography was performed for 0.5–1 h.
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Fig. 5. Bar charts showing mean RAL (relative adduct labelling) of total DNA adducts in V79NH cells after exposure to 0.01, 0.1 or 1 mM 3NBA using the nuclease P1 or butanol enrichment version of the 32P-postlabelling assay. The values given are the mean of three separate incubations analysed once within a maximal error range of 40%. Comparison was performed by t-test analysis: * p , 0:05 by comparison to parental V79NH.
a very potent direct acting mutagen recently identified in these environmental sources [5]. Recently, 3-NBA – DNA-adducts were detected in various organs of rats after administration of a single dose of 3-NBA or its metabolites [9,10]. These findings prompted us to determine which human P450 enzymes are the most important in activating 3-NBA to genotoxic products that form DNA adducts. Using the 32P-postlabelling assay a pattern of four 3-NBA-specific adducts was observed in all V79 cells used, similar to that found previously in vivo in rats [9,10]. Moreover, a comparison of chromatographic characteristics among DNA adducts obtained in cell culture with those detected in vivo in rats revealed that these 3-NBA-adducts were chromatographically indistinguishable. Activation of 3-NBA in V79 cells expressing human CYPOR demonstrated that human CYPOR contributes significantly to the bioactivation of 3-NBA. This is in line with our recent report where we showed that 3-NBA is activated by various human hepatic microsomes forming DNA adduct patterns qualitatively similar to those found in the present study [27]. Correlation of CYP- and CYPOR-linked activities with the level of DNA binding indicated that most of the hepatic microsomal activation of 3-NBA was attributed to CYPOR. CYPOR is expressed in human bronchial epithelial cells (BEC) and alveolar
macrophages (AM), a primary defense system against inhaled materials [32]. Therefore metabolic activation of 3-NBA in BEC and AM due to human CYPOR may represent a potential human risk. Furthermore, DNA binding increased significantly in V79 cells expressing human CYP3A4 in conjunction with human CYPOR compared to V79 cells expressing human CYPOR alone. Thus, we conclude that also human CYP3A4 is involved in the bioactivation of 3-NBA. However, DNA binding in V79 cells expressing human CYP3A4 together with CYPOR only increased significantly when analyzed by the butanol extraction version and was associated with the detection of the nuclease P1-sensitive adduct spot 4. CYP3A4 is the most abundant CYP in human liver and small intestine and although CYP3A5 is the predominant CYP3A form in human lung, CYP3A4 is expressed in about 20% of individuals [33,34]. Therefore, metabolic activation of 3-NBA in human lung cells may represent a potential human risk. In contrast, no evidence was seen for the involvement of human CYP1A1 in the biotransformation of 3-NBA in V79MZ cells. Both ring oxidation and nitro reduction are known pathways in the metabolic activation of nitro-PAHs [3,11]. Activation by ring oxidation by CYP enzymes is described for many polycyclic aromatic hydrocarbons
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(PAHs) as well as for nitro-PAHs [3,11,34]. Our results from in vitro incubations of calf thymus DNA with XO and 3-NBA demonstrated that all four 3-NBA-adducts are reaction products derived from nitroreduction [8]. Furthermore, the same 3-NBA-adducts were formed in vitro by rat liver S9 mix, human hepatic microsomes, in vivo in rats, human MCL-5 cells as well as in all V79 cells examined (present study) [8,9,27]. Thus, we assume that all 3-NBA-adducts are formed via nitroreduction and not by ring oxidation. The involvement of human CYP enzymes in the reductive activation of nitro-PAHs was demonstrated [35 – 37] suggesting that human CYP3A4 is involved in the metabolism of 3-NBA via simple nitroreduction. This is also in line with a recent report by Arlt et al. [15] where it was shown that 3-NBA is activated by recombinant human CYP1A2 expressed in V79 cells. Similarly, essentially the same DNA adduct pattern was observed after exposure to 1 mM 3-NBA. Compared with parental V79MZ cells DNA binding by 3-NBA was approximately 2- and 3-fold higher in V79 cells expressing human CYP1A2 cells after nuclease P1 and butanol enrichment, respectively, demonstrating that human CYP1A2 contributes to the metabolic activation of 3-NBA to form DNA adducts derived from nitroreduction [15]. That nitroreduction is the major activation pathway for 3-NBA is further supported by the fact that a 2- to 4-times higher DNA binding by 3-NBA is found in V79NH cells expressing high activities of nitroreductase and NAT after exposure to only 0.01 mM 3-NBA compared to the level of DNA binding observed in V79MZ cells after exposure to a 100-fold higher concentration. Moreover, the massive increased cytotoxicity in V79NH cells supports the view that 3-NBA is strongly activated to toxic products in these cells. Topinka et al. [38] also used V79NH cells to study the genotoxic potential of coke oven emissions extracts in mammalian cells containing a complex mixture of PAHs and nitro-PAHs. In this study no DNA adducts were detected for PAHs tested, including benzo[a ]pyrene, but for nitro-PAHs, including 4-nitropyrene, 6-nitrochrysene and 1,6-dinitropyrene, strong DNA adduct formation was observed. The activation of 3-NBA in V79NH cells most likely occurs by nitroreduction and subsequent acetylation of the resulting N-hydroxy arylamines catalyzed by NAT. However, the NAT genotype in these cells has
not yet been described in the literature. Consistent with this assumption is the finding that 3-NBA had a 30-fold higher mutagenic activity in the Ames S. typhimurium YG1024 strain exhibiting O-acetyltransferase than in TA98 strain [5]. This also matches the findings that human phase II metabolic enzymes significantly increase the level of DNA adducts derived from 3-NBA in V79 cells expressing human NATs and SULTs [14,15]. In V79NH cells expressing human CYP1A2 a decrease in DNA binding was observed at the lowest concentration of 3-NBA used (0.01 mM) suggesting a detoxifying effect by human CYP1A2 in conjunction with NAT in these cells at low concentrations. A similar effect was reported for the inactivation of dinitropyrenes by CYP enzymes and CYPOR in human liver microsomes [36]. It was suggested by the authors that the nitropyrenes are reduced to the corresponding amino compounds, which are rather inactive and not readily oxidized to the corresponding hydroxylamino derivatives. It might also occur that overexpression of human CYP1A2 influences the expression and/or activity of nitroreductases. Indications for this suggestion have been reported by Kappers et al. [39] who showed that in V79NH – h1A2 cells DT-diaphorase enzyme activity was reduced two-fold and that CYPOR activity was six-fold increased, respectively, compared to V79NH cells. The present study demonstrates that human CYPOR and CYP3A4 are able to contribute to the bioactivation of 3-NBA via simple nitroreduction. Moreover, acetylation of the initially formed Nhydroxy arylamine intermediates most likely contributes significantly to the high genotoxic potential of 3-NBA. Catalysis by NAT may therefore be an important step in the bioactivation of 3-NBA in vivo. Acknowledgements Grant sponsor: Baden-Wu¨rttemberg; Grant numbers: BWPLUS, BWB 20003. References [1] H. Tokiwa, Y. Ohnishi, Mutagenicity and carcinogenicity of nitroarenes and their sources in the environment, CRC Crit. Rev. Toxicol. 17 (1986) 23 –60.
C.A. Bieler et al. / Cancer Letters 200 (2003) 9–18 [2] IARC, Diesel and Gasoline Engine Exhausts and Some Nitroarenes, IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Lyon, No. 46, International Agency for Research on Cancer, Lyon, 1989. [3] V. Purohit, A.K. Basu, Mutagenicity of nitroaromatic compounds, Chem. Res. Toxicol. 13 (2000) 673–692. [4] D.T. Silvermann, L.I. Levin, R.N. Hoover, P. Hartge, Occupational risks of bladder cancer in the United States: I. White men, J. Natl Cancer Inst. 81 (1989) 1472–1480. [5] T. Enya, H. Suzuki, T. Watanabe, T. Hirayama, Y. Hisamatsu, 3-Nitrobenzanthrone, a powerful bacterial mutagen and suspected human carcinogen found in diesel exhausts and airborne particulates, Environ. Sci. Technol. 31 (1997) 2772–2776. [6] A. Seidel, D. Dahmann, H. Krekeler, J. Jacob, Biomonitoring of polycyclic aromatic compounds in the urine of mining workers occupationally exposed to diesel exhaust, Int. J. Hyg. Environ. Health 204 (2002) 333 –338. [7] P.T. Phousongphoung, A.J. Grosovsky, D.A. Eastmond, M. Covarrubias, J. Arey, The genotoxicity of 3-nitrobenzanthrone and the nitropyrene lactones in human lymphoblasts, Mutat. Res. 472 (2000) 93–103. [8] C.A. Bieler, M. Wiessler, L. Erdinger, H. Suzuki, T. Enya, H.H. Schmeiser, DNA adduct formation from the mutagenic air pollutant 3-nitrobenzanthrone, Mutat. Res. 439 (1999) 307–311. [9] V.M. Arlt, C.A. Bieler, W. Mier, M. Wiessler, H.H. Schmeiser, DNA adduct formation by the ubiquitous environmental contaminant 3-nitrobenzanthrone in rats determined by 32 P-postlabeling, Int. J. Cancer 93 (2001) 450 –454. [10] V.M. Arlt, B.L. Sorg, M. Osborne, A. Hewer, A. Seidel, H.H. Schmeiser, D.H. Phillips, DNA adduct formation by the ubiquitous environmental pollutant 3-nitrobenzanthrone and its metabolites in rats, Biochem. Biophys. Res. Commun. 300 (2003) 107–114. [11] P.P. Fu, Metabolism of nitro-polycyclic aromatic hydrocarbons, Drug Metab. Rev. 22 (1990) 209 –268. [12] F.A. Beland, M.M. Marques, DNA Adducts of Nitropolycyclic Aromatic Hydrocarbons, in: DNA Adducts, Identification and Biological Significance, IARC Publications, No. 125, IARC publishers, Lyon, 1994, pp. 229–244. [13] F.F. Kadlubar, G.J. Hammons, The Role of Cytochrome P-450 in the Metabolism of Chemical Carcinogens, in: Mammalian Cytochromes P-450, CRC Press, Boca Raton, 1987, pp. 81 – 130. [14] V.M. Arlt, H. Glatt, E. Muckel, U. Pabel, B.L. Sorg, H.H. Schmeiser, D.H. Phillips, Metabolic activation of the environmental contaminant 3-nitrobenzanthrone by human acetyltransferases and sulfotransferase, Carcinogenesis 23 (2002) 1937–1945. [15] V.M. Arlt, H. Glatt, E. Muckel, U. Pabel, B.L. Sorg, A. Seidel, H. Frank, H.H. Schmeiser, D.H. Phillips, Activation of 3nitrobenzanthrone and its metabolites by human acetyltransferases, sulfotransferases and cytochrome P450 expressed in Chinese hamster V79 cells, Int. J. Cancer 105 (2003) 583–592. [16] J. Doehmer, J.T.M. Buters, A. Luch, V. Soballa, W.M. Baird, H. Morisson, J.J. Stegeman, A.J. Townsend, W.F. Greenlee,
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
17
H. Glatt, A. Seidel, J. Jacob, H. Greim, Molecular studies on the toxifying effects by genetically engineered cytochromes P450, Drug Metabol. Rev. 31 (1999) 423–435. W.A. Schmalix, H. Ma¨ser, F. Kiefer, R. Reen, F.J. Wiebel, F. Gonzalez, A. Seidel, H. Glatt, H. Greim, J. Doehmer, Stable expression of human cytochrome P450 1A1 cDNA in V79 Chinese hamster cells and metabolic activation of benzo[a]pyrene, Eur. J. Pharmacol. 248 (1993) 251–261. C. Wo¨lfel, B. Heinrich-Hirsch, T. Schulz-Schalge, A. Seidel, H. Frank, U. Ramp, F. Wa¨chter, F.J. Wiebel, F. Gonzalez, H. Greim, J. Doehmer, Genetically engineered V79 Chinese hamster cells for stable expression of human cytochrome P4501A2, Eur. J. Pharmacol. 228 (1992) 95–102. A. Schneider, W.A. Schmalix, V. Siruguri, E.M. de Groene, G.J. Horbach, B. Kleingeist, D. Lang, R. Bo¨cker, R. Belloc, P. Beaume, H. Greim, J. Doehmer, Stable expression of human cytochrome P450 3A4 in conjunction with human NADPHcytochrome P450 oxidoreductase in V79 Chinese hamster cells, Arch. Biochem. Biophys. 332 (1996) 295–304. H. Glatt, W. Davis, W. Meindl, H. Hermersdo¨rfer, S. Venitt, D.H. Phillips, Rat, but not human, sulfotransferase activates a tamoxifen metabolite to produce DNA adducts and gene mutations in bacteria and mammalian cells in culture, Carcinogenesis 19 (1998) 1709–1713. A. Luch, S.L. Coffing, Y.M. Tang, A. Schneider, V. Soballa, H. Greim, C.R. Jefcoate, A. Seidel, W.F. Greenlee, W.M. Baird, J. Doehemer, Stable Expression of human cytochrome P450 1B1 in V79 Chinese hamster cells and metabolically catalyzed DNA adduct formation of dibenzo[a,l]pyrene, Chem. Res. Toxicol. 11 (1998) 686 –695. A. Luch, S. Kishiyama, A. Seidel, J. Doehemer, H. Greim, W.M. Baird, The K-region trans-8,9.diol does not significantly contribute as an intermediate in the metabolic activation of dibenzo[a,l]pyrene to DNA-binding metabolites by human cytochrome P450 1A1 or 1B1, Cancer Res. 59 (1999) 4603–4609. K. Sundberg, K. Dreij, A. Seidel, B. Jernstro¨m, Glutathione conjugation and DNA adduct formation of dibenzo[a,l]pyrene and benzo[a]pyrene diol epoxides in V79 cells stably expressing different human glutathione transferases, Chem. Res. Toxicol. 15 (2002) 170 –179. B. Mahadevan, W.-M. Dashwood, A. Luch, A. Pecaj, J. Doehmer, A. Seidel, C. Pereira, W.M. Baird, Mutations induced by (-)-anti-11R,12S-dihydrodiol-13S,14R-epoxide of dibenzo[a,l]pyrene in the coding region of the hypoxanthine phosphoribosyltransferase (HPRT) gene in Chinese hamster V79 cells, Environ. Mol. Mutagenesis 41 (2003) 131–139. A. Seidel, T. Friedberg, B. Lo¨llmann, A. Schwierzok, M. Funk, H. Frank, R. Holler, F. oesch, H. Glatt, Detoxication of optically active bay- and fjord-region polycyclic aromatic hydrocarbon dihydrodiol epoxides by human glutathione transferase P1-1 expressed in Chinese hamster V79 cells, Carcinogenesis 19 (1998) 1975–1981. J. Buters, L. Quintanilla-Martinez, W. Schober, V.J. Soballa, J. Hintermair, T. Wolff, F.J. Gonzalez, H. Greim, CYP1B1 determines susceptibility to low doses of 7,12-dimethylbenz[a]anthracene-induced ovarian cancers in mice: correlation of
18
[27]
[28]
[29]
[30]
[31]
[32]
C.A. Bieler et al. / Cancer Letters 200 (2003) 9–18 CYP1B1-mediated DNA adducts with carcinogenicity, Carcinogenesis 24 (2003) 327– 334. V.M. Arlt, M. Stiborova, A. Hewer, H.H. Schmeiser, D.H. Phillips, Human enzymes involved in the metabolic activation of the environmental contaminant 3-nitrobenzanthrone: Evidence for reductive activation by human NADPH:cytochrome P450 reductase, Cancer Res. 63 (2003) 2752– 2761. F. Kiefer, O. Cumpelik, R. Reen, J. Doehmer, F.J. Wiebel, Arylamines suppress their own activation and that of nitroarenes in V79 Chinese hamster cells by competing for acetyltransferases, Environ. Health Perspect. 102 (1994) 95–97. M. Stiborova, C.A. Bieler, M. Wiessler, E. Frei, The anticancer agent ellipticine on activation by cytochrome P450 forms covalent DNA adducts, Biochem. Pharmacol. 62 (2001) 1675–1684. J.E. Gallagher, M.J. Kohan, M.H. George, J. Lewtas, Improvement of the diagnostic potential of 32P-postlabeling analysis demonstrated by the selective formation and comparative analysis of nitrated-PAH-derived adducts arising from diesel particle extracts, Carcinogenesis 12 (1991) 1685– 1691. E. Frei, C.A. Bieler, V.M. Arlt, M. Wiessler, M. Stiborova, Covalent binding of the anticancer drug ellipticine to DNA in V79 cells transfected with human cytochrome P450 enzymes, Biochem. Pharmacol. 64 (2002) 289 –295. J.C. Willey, E. Coy, C. Brolly, M.J. Utell, M.W. Frampton, J. Hammersley, W.G. Thilly, D. Olson, K. Cairns, Xenobiotic metabolism enzyme gene expression in human bronchial epithelial and alveolar macrophage cells, Am. J. Respir. Cell Mol. Biol. 14 (1996) 262–271.
[33] K. Mace, E.D. Bowman, P. Vauttravers, P.G. Shields, C.C. Harris, A.M. Pfeifer, Characterisation of xenobiotic-metabolising enzyme expression in human bronchial mucosa and peripheral lung tissue, Eur. J. Cancer 34 (1998) 914–920. [34] F.P. Guengerich, T. Shimada, Oxidation of toxic and carcinogenic chemicals by human cytochrome P-450 enzymes, Chem. Res. Toxicol. 4 (1991) 391– 407. [35] Y.H. Chae, C.H. Yun, F.P. Guengerich, F.F. Kadlubar, K. El-Bayoumy, Roles of human hepatic and pulmonary cytochrome P450 enzymes in the metabolism of the environmental carcinogen 6-nitrochrysene, Cancer Res. 53 (1993) 2028–2034. [36] 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. [37] Y.H. Chae, B.Y. Ji, J.M. Lin, P.P. Fu, B.P. Cho, K. ElBayoumy, Nitroreduction of 4-nitropyrene is primarily resonsible for DNA adduct formation in the mammary gland of female CD rats, Chem. Res. Toxicol. 12 (1999) 180 –186. [38] J. Topinka, L.R. Schwarz, F. Kiefer, F.J. Wiebel, O. Gajdos, P. Vidova, L. Dobias, M. Fried, R.J. Sram, T. Wolff, DNA adduct formation in mammalian cell culture by polycyclic aromatic hydrocarbons (PAH) and nitro-PAH in coke oven emission extract, Mutat. Res. 419 (1998) 91–105. [39] W.A. Kappers, F.M.M. van Och, E.M. de Groene, G.J. Horbach, Comparison of three different in vitro mutation assays used for the investigation of cytochrome p450mediated mutagenicity of nitro-polycyclic aromatic hydrocarbons, Mutat. Res. 466 (2000) 143– 159.