Mutagenesis vol. 19 no. 2 pp. 149-156,
March 2004
© 2004 UK Environmental Mutagen Society/Oxford University Press
Activation of 3-nitrobenzanthrone and its metabolites to DNA-damaging species in human B lymphoblastoid MCL-5 cells
Section of Molecular Carcinogenesis, Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK
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3-Nitrobenzanthrone (3-NBA) is one of the most potent mutagens in the Ames Salmonella typhimurium assay and a suspected human carcinogen recently identified in diesel exhaust and in airborne particulate matter. 3-Aminobenzanthrone (3-ABA), 3-acetylaminobenzanthrone (3-Ac-ABA) and N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA) have been identified as 3-NBA metabolites. In the present study we investigated the genotoxic effects of 3-NBA and its metabolites in the human B lymphoblastoid cell line MCL-5. DNA strand breaks were measured using the Comet assay, chromosomal damage was assessed using the micronucleus assay and DNA adduct formation was determined by 32P-post-labelling analysis. DNA strand-breaking activity was observed with each compound in a concentration-dependent manner (1–50 µM, 2 h incubation time). At 50 µM median comet tail lengths (CTLs) were 25.0 µm for 3-NBA, 48.0 µm for 3-ABA, 54.5 µm for 3-Ac-ABA and 65.0 µm for N-Ac-N-OH-ABA. Median CTLs in control incubations were in the range 7.7–13.1 µm. Moreover, the strand-breaking activity of 3-NBA was more pronounced in the presence of a DNA repair inhibitor, hydroxyurea. Depending on the concentration used (1–20 µM, 24 h incubation time), 3-NBA and its metabolites also showed clastogenic activity in the micronucleus assay. 3-NBA and N-Ac-N-OH-ABA were the most active at low concentrations; at 1 µM the total number of micronuclei per 500 binucleate cells was 4.7 ± 0.6 in both cases, compared with 1.7–3.0 for controls (P < 0.05). Furthermore, multiple DNA adducts were detected with each compound (1 µM, 24 h incubation time), essentially similar to those found recently in vivo in rats treated with 3-NBA or its metabolites. DNA adduct levels ranged from 1.3 to 42.8 and from 2.0 to 39.8 adducts/108 nt using the nuclease P1 and butanol enrichment procedures, respectively. DNA binding was highest for N-Ac-N-OH-ABA, followed by 3-NBA, and much lower for 3-ABA and 3-Ac-ABA. All major 3-NBA-derived DNA adducts produced in MCL-5 cells were found to be formed from reductive metabolites bound to purine bases and lacked an N-acetyl group. These results demonstrate that 3-NBA and its metabolites are effectively activated to DNA-damaging species in human MCL-5 cells, which may reflect the genotoxic potential of 3-NBA in humans. Environmental exposure to 3-NBA may be a health hazard for large sections of the population and the risks associated with such exposure require further assessment.
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Introduction |
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Nitropolycyclic aromatic hydrocarbons (nitro-PAHs) are widely distributed environmental pollutants found in airborne particulate matter, especially that emitted from diesel and gasoline engines (Tokiwa and Ohnishi, 1986
; Yaffe et al., 2001). Many members of this class of compounds are known to be mutagenic in bacterial and mammalian cells and tumorigenic in rodents and some are probably carcinogenic to humans (IARC, 1989; Tokiwa et al., 1993; Purohit and Basu, 2000).
3-Nitrobenzanthrone (3-nitro-7H-benz[de]anthracen-7-one; 3-NBA) (Figure 1) was recently detected in diesel exhaust and in airborne particulate matter and might originate both from incomplete combustion of fossil fuels and from reaction of the parent aromatic hydrocarbon with nitrogen oxides in the atmosphere (Enya et al., 1997; Seidel et al., 2002; Feilberg et al., 2002; Murahashi, 2003). As a likely consequence of atmospheric washout, 3-NBA has also been detected more recently in surface soil (Murahashi et al., 2003; Watanabe et al., 2003). The uptake of 3-NBA in humans has been demonstrated by the detection of 3-aminobenzanthrone (3-ABA) (Figure 1), a major metabolite of 3-NBA, in the urine of salt mining workers occupationally exposed to diesel emissions (Seidel et al., 2002). 3-NBA is an exceptionally potent mutagen in the Ames Salmonella typhimurium assay, with a mutation frequency comparable to that of 1,8-dinitropyrene, a direct acting mutagen which has the highest activity thus far reported in this assay (Enya et al., 1997). Preliminary data suggest that 3-NBA is carcinogenic in rats (Adachi et al., 2000).
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Nitro-PAHs require metabolism to reactive electrophilic species to exert their genotoxic activity (Purohit and Basu, 2000
). In incubations of rat lung alveolar type II cells with 3-NBA, 3-ABA was identified as the major metabolite, with lesser amounts of 3-acetylaminobenzanthrone (3-Ac-ABA) (Figure 1) also observed (Borlak et al., 2000). Nitroreduction catalysed by cytosolic and microsomal nitroreductases, followed by O-acetylation catalysed by N-acetyltransferases (NATs) and/or O-sulfonation catalysed by sulfotransferases (SULTs), seem to be the major pathways of bioactivation for 3-NBA leading to DNA adduct formation (Bieler et al., 1999, 2003; Arlt et al., 2001a, 2002, 2003a,b,c). Moreover, we found that 3-NBA and its metabolites [3-ABA, 3-Ac-ABA and N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA)] form the same DNA adducts in vivo in rats and in mammalian cells in vitro (Arlt et al., 2003a,b). We demonstrated that all in vivo 3-NBA-derived DNA adducts detected by 32P-post-labelling are formed from reductive metabolites bound to purine bases without carrying an N-acetyl group (Arlt et al., 2001a). However, Kawanishi et al. (2000) reported an unusual acetylated adduct in human hepatoma HepG2 cells resulting from reaction of the activated ester of N-Ac-N-OH-ABA (Figure 1) with DNA (Enya et al., 1998). Collectively, these studies have indicated that 3-NBA can be activated by two pathways leading to DNA adduct-forming species (Figure 1). After nitroreduction to N-OH-ABA the major pathway involves formation of non-acetylated 3-NBA–DNA adducts. The second, but minor, pathway proceeds via the formation of N-Ac-N-OH-ABA, leading to the formation of acetylated 3-NBA–DNA adducts. In order to gain insight into the metabolic pathways by which 3-NBA is activated in human cells, we have treated human B lymphoblastoid MCL-5 cells with 3-NBA, its known metabolites (3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA) and compared their genotoxic potential. DNA damage was measured as DNA strand breaks using the Comet assay, chromosomal damage using the micronucleus assay and DNA adduct formation using 32P-post-labelling.
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Materials and methods |
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Synthesis of 3-NBA, 3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA
3-NBA, 3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA were prepared as described (Enya et al., 1998
; Arlt et al., 2002, 2003b). Their authenticity was confirmed by UV, electrospray mass spectra and high-field proton NMR spectroscopy.
Cell culture of MCL-5 cells and cell viability
Human B lymphoblastoid MCL-5 cells were obtained under license from Gentest Corp. (Woburn, MA). They constitutively express a high level of native CYP1A1 and also four other human CYPs (CYP1A2, CYP2A6, CYP3A4 and CYP2E1) and microsomal epoxide hydrolase that are carried as cDNAs in plasmids (Crespi et al., 1991). The enzyme activities were not estimated in the study. MCL-5 cells were cultivated as described (Martin et al., 1999). Cell viability was determined as recently described (Arlt et al., 2003b).
Comet assay in MCL-5 cells
Alkaline cell lysis followed by alkaline gel electrophoresis was employed to detect genotoxin-induced DNA single-strand breaks (Singh et al., 1988; Martin et al., 1999). Aliquots (1 ml) of suspensions of MCL-5 cells (
0.8–1 x 106 cells/ml) were incubated at 37°C for 2 h with 3-NBA, 3-ABA, 3-Ac-ABA or N-Ac-N-OH-ABA (dissolved in 10 µl DMSO) yielding final concentrations of 0.1, 1, 10 and 50 µM. In control incubations cells were treated with DMSO only. In incubations using a DNA repair inhibitor (3-NBA tested only), hydroxyurea (HU) (dissolved in 5 µl water) was added to a final concentration of 5 mM. Benzo[a]pyrene (37 µM) in the presence of HU was used as a positive control. Aliquots of MCL-5 cells (75 µl) were then embedded in agarose (0.85%) on agarose-coated microscope slides (Erie Scientific Co., Portsmouth, UK) on a cold surface. Slides were submerged in cold alkaline lysis solution (2.5 M NaCl, 100 mM EDTA disodium salt, 10 mM Tris, 1% Triton X-100 and 10% DMSO), protected from light and stored at 4°C for 1 h. Under red light, slides were transferred to a horizontal electrophoresis tank, covered in electrophoresis solution (0.3 M NaOH, 1 mM EDTA, assumed to be pH < 13) and stored in a chilled incubator at 10°C for 40 min to allow DNA unwinding, prior to electrophoresis at 0.8 V/cm and 300 mA for 36 min. After electrophoresis, slides were neutralized (0.5 M Tris, pH 7.5), fixed in methanol for 10 min and stained with 25 µl of an aqueous ethidium bromide solution (20 ng/ml). Comets were visualized by epifluorescence using a Leitz Laborlux S microscope. A total of 80 digitized images/data points (Komet 3.1; Kinetic Imaging, Liverpool, UK), 40 from each of two duplicate slides, were measured. All samples were analysed in triplicate and measured blind. Increases in comet tail lengths (CTLs) were assessed for significance using the Mann–Whitney test. Mean median CTL was compared by t-test analysis.
Micronucleus assay in MCL-5 cells
Micronucleus induction in MCL-5 cells, blocked at cytokinesis with cytochalasin B, was used as an indicator of chromosomal damage (Crofton-Sleigh et al., 1993; Fenech, 2000). Aliquots (10 ml) of suspensions of MCL-5 cells (1.5–2.0 x 105 cells/ml) were incubated at 37°C for 24 h with 1, 5, 10 or 20 µM 3-NBA, 3-ABA, 3-Ac-ABA or N-Ac-N-OH-ABA (dissolved in 33.2 µl DMSO). N-Ac-N-OH-ABA was additionally tested at 2.5 µM. Controls were treated with DMSO only. Melphalan (1 µM) was used as a positive control. Cells were then treated with 6 µg/ml cytochalasin B (kept as a 2 mg/ml solution in DMSO; Sigma) for a further 24 h. Aliqouts of MCL-5 cells (100 µl) were centrifuged directly onto slides using a Shandon Cytospin centrifuge (500 r.p.m. for 5 min). Slides were fixed in methanol for 15 min and stained in a solution of acridine orange [40 µg/ml in phosphate-buffered saline (PBS)] (Sigma). The incidence of micronucleated cells was recorded by scoring 500 binucleate cells with intact cytoplasm. The criteria for scoring micronuclei were those listed by Fenech (2000). The percentage of binucleate cells was used as an index of cytotoxicity. All samples were analysed in triplicate and were counted blind. Micronucleated and binucleate cells were compared by t-test analysis.
DNA adduct analysis using the 32P-post-labelling assay
Aliquots (10 ml) of suspensions of MCL-5 cells (0.8–1 x 106 cells/ml) were incubated at 37°C for 24 h with 1 µM 3-NBA, 3-ABA, 3-Ac-ABA or N-Ac-N-OH-ABA (dissolved in 16.6 µl DMSO). Controls were treated with DMSO only. Subsequently, centrifugation of cells at 2000 r.p.m. for 10 min and one washing step with 10 ml PBS yielded a cell pellet, which was stored at –20°C until DNA isolation. DNA from cells was isolated by the phenol extraction method as described previously (Arlt et al., 2001b). 32P-post-labelling analysis using nuclease P1 digestion, butanol extraction and autoradiography were performed as described (Arlt et al., 2002). Chromatographic conditions for thin layer chromatography (TLC) on polyethyleneimine–cellulose (PEI–cellulose) were: D1, 1.0 M sodium phosphate, pH 6.0; D3, 4 M lithium formate, 7 M urea, pH 3.5; D4, 0.8 M LiCl, 0.5 M Tris, 8.5 M urea, pH 8.0. DNA adduct levels (relative adduct labelling, RAL) were calculated from the adduct counts per min, the specific activity of [
-32P]ATP and the amount of DNA (pmol DNA-P) used. Results are expressed as DNA adducts/108 nt.
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Comet formation by 3-NBA and its metabolites in MCL-5 cells
When MCL-5 cells were incubated with 0.1, 1, 10 and 50 µM 3-NBA a concentration-dependent increase in median CTLs compared with control values was observed (Figure 2A). In the presence of the DNA repair inhibitor HU, 3-NBA induced greater increases in median CTLs (range 17.8–78.8 µm) than in its absence (range 13.0–33.8 µm) (P < 0.05). Whereas median CTLs for 3-NBA without HU increased only significantly above control values in incubations using 10 and 50 µM 3-NBA, median CTL values were significantly above control values at all 3-NBA concentrations tested (0.1, 1, 10 and 50 µM) in the presence of HU. Moreover, median CTLs in cells treated with 3-NBA in the presence of HU were significantly increased compared with cells treated with 3-NBA alone at all concentrations tested (P < 0.05). Median CTLs in MCL-5 cells treated with solvent (DMSO) only were in the range 7.7–13.1 µm. The median CTL in MCL-5 cells treated with solvent only in the presence of HU was 13.6 µm, indicating that the background level of comet formation was not increased in the presence of the repair inhibitor. Benzo[a]pyrene (37 µM), which was used as a positive control, showed a similar response, with median CTLs ranging from 40 to 60 µm in the presence of HU (data not shown). Representative CTL distributions obtained at 50 µM 3-NBA in the presence of HU or without HU are shown in Figure 2B. Refinements of the Comet assay using repair inhibitors have been found to allow the detection of genotoxic activity that could otherwise have been overlooked (Martin et al., 1999
). However, it was not necessary for 3-NBA and, therefore, the Comet assay for 3-NBA metabolites (3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA) was performed without repair inhibitors. When MCL-5 cells were incubated with 0.1, 1, 10 and 50 µM 3-ABA, 3-Ac-ABA or N-Ac-N-OH-ABA a concentration-dependent increase in median CTLs compared with control values was observed (Figure 3A). Median CTLs ranged from 8.8 to 44.5 µm for 3-ABA, from 8.5 to 48.5 µm for 3-Ac-ABA and from 6.8 to 69.8 µm for N-Ac-N-OH-ABA. Median CTL values increased significantly above control values in all incubations using 10 and 50 µM 3-NBA metabolites (P < 0.05). Representative CTL distributions obtained at 50 µM are shown in Figure 3B. 3-NBA and its metabolites did not induce cytotoxicity in MCL-5 cells used for the Comet assay, as measured by Trypan blue exclusion. Cell viability (as a percentage of the control) was always >80%.
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Micronucleus formation by 3-NBA and its metabolites in MCL-5 cells
Treatment of MCL-5 cells with 3-NBA or its metabolites (3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA) in the range 1–20 µM resulted in cytotoxicity and induction of micronuclei in a concentration-dependent manner (Table I). Total number of micronuclei ranged from 4.7 to 5.3 for 3-NBA, 3.0 to 6.7 for 3-ABA, 2.3 to 22.7 for 3-Ac-ABA and 4.7 to 14.3 for N-Ac-N-OH-ABA. Total number of micronuclei in cells treated with solvent (DMSO) only ranged from 1.7 to 3.0. Cytotoxicity expressed as a reduction in the percentage of binucleate cells was strongest for N-Ac-N-OH-ABA (74.0 to 0%), followed by 3-NBA (72.3 to 46.3%) (Table I). In fact, cytotoxicity for N-Ac-N-OH-ABA was so strong that cells did not survive treatment above 2.5 µM. No strong increase in cytotoxicity was observed for 3-ABA (81.7 to 73.0%) and 3-Ac-ABA (81.7 to 75.0%). Percentage of binucleate cells for MCL-5 cells treated with solvent (DMSO) only ranged from 83.7 to 79.7%. For 3-NBA a significant induction of micronuclei was observed at all concentrations tested (P < 0.05), however, induction of micronuclei stayed constant over the range of concentrations tested (Table I). Micronucleus induction was greatest for N-Ac-N-OH-ABA, in particular at low concentrations (lowest effective concentration 1 µM; P < 0.05); the total number of micronuclei was already 14.3 ± 2.1 at only 2.5 µM. The lowest effective concentration for 3-ABA and 3-Ac-ABA was 5 µM (P < 0.05). Interestingly, in comparison with 3-NBA, micronucleus formation increased
4.5-fold after treatment with 3-Ac-ABA at the highest concentration (20 µM, 5.0 ± 2.6 for 3-NBA versus 22.7 ± 5.7 for 3-Ac-ABA). Melphalan (1 µM), which was used as a positive control (Phousongphouang et al., 2000), also induced a significant number of micronuclei (41.0 ± 10.5; P < 0.05).
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DNA adduct formation by 3-NBA and its metabolites in MCL-5 cells
When 32P-post-labelling with butanol enrichment was used, the DNA adduct pattern induced in MCL-5 cells by 3-NBA and its metabolites (3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA) on TLC essentially consisted of a cluster of four major adducts (spots 1–4; Figure 4, upper panels). Enrichment by nuclease P1 resulted in a cluster of three major adducts (spots 1–3) and one minor adduct (spot 6; Figure 4, lower panels). No DNA adducts were observed in DNA from MCL-5 cells treated with solvent (DMSO) only (data not shown). The same major DNA adducts (adducts 1–4) have been detected in vivo in rats treated with 3-NBA or its metabolites (Arlt et al., 2003a
). Using the same approach as reported by us previously (Arlt et al., 2001a, 2003a) we found that all of the major 3-NBA-derived DNA adducts detected in human MCL-5 cells are formed by nitroreduction derived from reaction with either adenine (adduct spots 1 and 2) or guanine (adduct spots 3 and 4) in DNA. One additional adduct spot (spot 7) was detected in incubations with 3-ABA (Figure 4B). A similar adduct spot was observed in calf thymus DNA after activation of 3-NBA by reduction with zinc (Bieler et al., 1999) and by human hepatic microsomes (Arlt et al., 2003c), but the low adduct levels prevented HPLC co-chromatographic analysis. DNA adduct levels at 1 µM ranged from 1.3 to 42.2 and from 2.0 to 39.8 adducts/108 nt for total DNA binding after nuclease P1 and butanol enrichment, respectively (Figure 5). DNA binding was highest for N-Ac-N-OH-ABA, followed by 3-NBA, and considerably lower for 3-ABA and 3-Ac-ABA. Comparison of the levels of individual adduct spots are given in Figure 6. In general adduct spot 3 was the predominant adduct found. Cell viability (as a percentage of the control) of MCL-5 cells used for 32P-post-labelling was normally >80%, except that after treatment with N-Ac-N-OH-ABA it decreased to 70%.
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Discussion |
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Some limited evidence suggests that diesel exhaust and airborne particulates, which contain significant amounts of nitro-PAHs, may be carcinogenic to humans (IARC, 1989
; Tokiwa et al., 1993; Farmer, 1997). 3-NBA is a very potent direct acting mutagen in Salmonella typhimurium and a suspected human carcinogen that has been identified recently in these environmental sources (Enya et al., 1997; Adachi et al., 2000; Seidel et al., 2002). In the present study we investigated the genotoxic potential of 3-NBA and its metabolites (3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA) in human lymphoblastoid MCL-5 cells. MCL-5 cells have been used previously in genotoxicity testing using various biological end-points, e.g. mutation frequency, DNA breaks, chromosomal damage and DNA adduct formation, as indicators of DNA damage (Crofton-Sleigh et al., 1993; Pfau et al., 1999; Martin et al., 1999; Phousongphouang et al., 2000; Arlt et al., 2003c). The MCL-5 cell line has proved to be suitable to study the genotoxicity of other nitroaromatics that have also been identified in diesel exhaust and ambient air particulates, including 2-nitropyrene, 2-nitrofluoranthene and 1,6- and 1,8-dinitropyrene (Busby et al., 1994, 1997; Martin et al., 1999).
Previous work by our group has shown that 3-NBA and its metabolites undergo several biotransformations leading to the same specific 3-NBA-derived DNA adducts in vivo in rats (Arlt et al., 2003a). In the present study essentially the same 3-NBA-derived DNA adducts were observed in DNA of human MCL-5 cells treated with 1 µM 3-NBA and its metabolites (Figure 4). Therefore, MCL-5 cells contain enzyme systems capable of catalyzing the activation of 3-NBA and its metabolites leading to DNA adducts. DNA binding was highest for N-Ac-N-OH-ABA, followed by 3-NBA, and much lower for 3-ABA and 3-Ac-ABA (Figure 5). In contrast, DNA binding in vivo in rats was highest for 3-NBA, followed by N-Ac-N-OH-ABA, and much lower for 3-ABA and 3-Ac-ABA (Arlt et al., 2003a).
The activation of nitro-PAHs is mainly through nitroreduction catalysed primarily by cytosolic reductases, such as xanthine oxidase, DT-diaphorase and aldehyde oxidase, whereas CYP enzymes are primarily responsible for the oxidative metabolism of these compounds (Purohit and Basu, 2000). However, the reductive activation of nitroaromatics including 3-NBA by CYP enzymes has also been demonstrated (Chae et al., 1993, 1999; Stiborova et al., 2001; Arlt et al., 2003c). MCL-5 cells express high levels of native CYP1A1 and are transfected with plasmids carrying cDNAs of human CYP1A2, CYP2A6, CYP3A4 and CYP2E1 and the microsomal epoxide hydrolase gene (Crespi et al., 1991). Previous studies demonstrated that nitroreduction catalysed by xanthine oxidase and/or NADPH:CYP reductase is important in the bioactivation of 3-NBA (Figure 1) (Bieler et al., 1999, 2003; Borlak et al., 2000; Arlt et al., 2001a, 2003c). Moreover, we found that 3-NBA can be activated by various human hepatic microsomes forming DNA adduct patterns qualitatively similar to those found in vivo in rats treated with 3-NBA (Arlt et al., 2003c). Correlation of CYP-linked enzyme activities with the level of DNA binding indicated that most of the hepatic microsomal activation of 3-NBA was attributed to NADPH:CYP reductase. Therefore, cytosolic nitroreductases as well as NADPH:CYP reductase may be involved in the metabolic activation of 3-NBA in MCL-5 cells. Using Supersomes and V79 cells expressing recombinant human CYP1A2 we also found that 3-NBA is activated by this CYP isoform due to nitroreduction (Arlt et al., 2003b,c). Human CYP1A2 is expressed in MCL-5 cells and, therefore, might also contribute to the bioactivation of 3-NBA. However, at present we can only speculate on the relative contribution of cytosolic nitroreductases in comparison with microsomal enzymes in the bioactivation of 3-NBA.
Previous data showed that 3-ABA and 3-Ac-ABA can be metabolized to reactive intermediates via N-oxidation by human CYP enzymes (Arlt et al., 2003b) (Figure 1). Using V79 cells expressing recombinant human CYP1A2 we found that expression of this CYP isoform was required for activation of 3-ABA and 3-Ac-ABA in these cells. Preliminary results also suggest that 3-ABA is activated by CYP1A1 in human hepatic microsomes (Arlt, Phillips and Stiborova, unpublished data). Activation of 3-ABA by Supersomes and V79 cells expressing recombinant human CYP1A1 support this finding (Arlt, Phillips and Stiborova, unpublished data). Human CYP1A1 and CYP1A2 are both expressed in MCL-5 cells and, therefore, might be important in the bioactivation of 3-ABA and 3-Ac-ABA.
In MCL-5 cells N-deacetylation seems to be important for activation of 3-Ac-ABA and N-Ac-N-OH-ABA (Figure 1), as observed previously in other mammalian cells as well as in vivo in rats (Arlt et al., 2002, 2003a,b). Alternatively, it was discussed that N,O-acetyltransfer reactions catalysed by NATs may be important for bioactivation of N-Ac-N-OH-ABA. However, NAT activity in MCL-5 cells has not been investigated. Nevertheless, we recently showed that recombinant human NAT1 and NAT2 contribute strongly to the high genotoxicity of 3-NBA and its metabolites (Arlt et al., 2002, 2003b). Collectively, these results indicate that N-OH-ABA (Figure 1) seems to be the critical intermediate for DNA adduct formation by 3-NBA and its metabolites not only in vivo in rats but also in MCL-5 cells. It is of interest to note that 3-NBA induced mutations at the tk and hprt loci in this cell line (Phousongphouang et al., 2000). Therefore, these results also suggest that some or all of the major 3-NBA-derived DNA adducts detected in the present study represent premutagenic lesions involved in the mutagenic process, at least in MCL-5 cells.
Previously it was shown that 3-NBA induced micronuclei in peripheral blood reticulocytes of mice after i.p. treatment with 3-NBA (Enya et al., 1997). In a further study, using the CREST modified micronucleus assay, 3-NBA induced micronuclei in human MCL-5 and h1A1v2 cells (Phousongphouang et al., 2000). In contrast to MCL-5 cells, h1A1v2 cells only have elevated levels of native CYP1A1. However, it is noteworthy that in this study the total percentage of micronuclei was only significantly increased in a dose-dependent manner in h1A1v2 cells. The authors attributed this effect to an apoptotic deficiency in h1A1v2 in comparison with MCL-5 cells, which might be caused by differences in their p53 status and may allow greater survival of damaged h1A1v2 cells (Phousongphouang et al., 2000). In the present study we found that 3-NBA induces micronuclei in MCL-5 cells at all concentrations tested (1–20 µM) (Table I). It seems that in our study MCL-5 cells were more susceptible at low concentrations. However, compared with the previous study, higher concentrations have been used in this study; our highest concentration was 3-fold higher. Micronuclei were also induced by 3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA (Table I). Of all the compounds, N-Ac-N-OH-ABA was again the most active, which correlates with its cytotoxicity and strong DNA adduct formation (Table I and Figure 5).
In the present study we also observed DNA strand-breaking activity by 3-NBA and its metabolites as measured by the Comet assay (Figure 2). The strand-breaking activity of 3-NBA was more pronounced when a DNA repair inhibitor (HU) was included (Figure 3). HU allows the recognition and incision stages of nucleotide excision repair to occur but inhibits subsequent DNA resynthesis, which results in an accumulation of single-strand breaks. Thus, these results also suggest that 3-NBA–DNA adducts are recognized by the nucleotide excision repair system. However, the repair rates and persistence of individual 3-NBA–DNA adducts needs to be elucidated. Chromosomal damage can be induced by both chromosomal loss (aneugenic effect) and chromosomal breakage (clastogenic effect). Since chromosome loss is not detectable in the Comet assay, which detects single- and double-strand breaks as well as alkali-labile sites (Tice et al., 2000), our results suggest that micronucleus formation after treatment with 3-NBA and its metabolites is due to clastogenic effects, although an aneugenic effect is not excluded. This conclusion is in line with earlier data in MCL-5 and h1A1v2 cells, where 3-NBA was classified as a clastogen as well as an aneugen, since 3-NBA induced both chromosomal loss and chromosomal breakage in these cell lines as measured by anti-kinetochore antibody staining (Phousongphouang et al., 2000).
In summary, we found that 3-NBA and its metabolites (3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA) form the same specific DNA adducts in human MCL-5 cells, induce chromosomal damage and exhibit DNA strand-breaking activity. Taken together with previous data in MCL-5 cells, which showed induction of mutations at the tk and hprt loci, this may reflect the genotoxic potential of 3-NBA and its metabolites in humans caused by different cellular interference. Because of the presence of 3-NBA in diesel and ambient air pollution, exposure to 3-NBA may represent a hazard for large sections of the population and the health risks of such exposure to humans need to be further assessed.
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Acknowledgements |
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This work was supported by Cancer Research UK. We are grateful to Dr Albrecht Seidel (Biochemical Institute for Environmental Carcinogens, Prof. Dr Gernot Grimmer-Foundation, Grosshansdorf, Germany) for preparing 3-Ac-ABA. We thank Dr Heinz H.Schmeiser (Division of Molecular Toxicology, German Cancer Research Center, Heidelberg, Germany) for continuous support and many helpful discussions.
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1To whom correspondence should be addressed. Tel: +44 20 8722 4405; Fax: +44 20 8722 4052; Email: volker.arlt{at}icr.ac.uk
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References |
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Arlt,V.M., Bieler,C.A., Mier,W., Wiessler,M. and Schmeiser,H.H. (2001a) DNA adduct formation by the ubiquitous environmental contaminant 3-nitrobenzanthrone in rats determined by 32P-postlabeling. Int. J. Cancer, 93, 450–454.[CrossRef][ISI][Medline]
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Arlt,V.M., Glatt,H.R., Muckel,E., Pabel,U., Sorg,B.L., Schmeiser,H.H. and Phillips,D.H. (2002) Metabolic activation of the environmental contaminant 3-nitrobenzanthrone by human acetyltransferases and sulfotransferase. Carcinogenesis, 23, 1937–1945.
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