Metabolic activation of the environmental contaminant 3-nitrobenzanthrone by human acetyltransferases and sulfotransferase

Volker M. Arlt1,4, Hansruedi Glatt2, Eva Muckel2, Ulrike Pabel2, Bernd L. Sorg3, Heinz H. Schmeiser3 and David H. Phillips1

1 Institute of Cancer Research, Section of Molecular Carcinogenesis, Cotswold Road, Sutton, Surrey SM2 5NG, UK,
2 German Institute of Human Nutrition, Department of Toxicology, Arthur-Scheunert-Allee 144–166, D-14558 Potsdam-Rehbrücke, Germany and
3 German Cancer Research Center, Division of Molecular Toxicology, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany


    Abstract
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
3-Nitrobenzanthrone (3-NBA) an extremely potent mutagen and suspected human carcinogen identified in diesel exhaust and in airborne particulate matter was shown to form multiple DNA adducts in vitro and in vivo in rats. In order to investigate whether human N,O-acetyltransferases (NATs) and sulfotransferases (SULTs) contribute to the metabolic activation of 3-NBA we used a panel of newly constructed Chinese hamster lung fibroblast V79MZ derived cell lines expressing human NAT1, human NAT2 or human SULT1A1, as well as TA1538-derived Salmonella typhimurium strains expressing human NAT1 (DJ400) or human NAT2 (DJ460) and determined DNA binding and mutagenicity. The formation of 3-NBA-derived DNA adducts was analysed by 32P-postlabelling after exposing V79 cells to 0.01 µM 3-NBA or 0.1 µM N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA), a potential metabolite of 3-NBA. Similarly up to four major and two minor adducts were detectable for both compounds, the major ones being identical to those detected previously in DNA from rats treated with 3-NBA. Comparison of DNA binding between different V79MZ derived cells revealed that human NAT2 and, to a lesser extent, human NAT1 and human SULT1A1, contribute to the genotoxic potential of 3-NBA and N-Ac-N-OH-ABA to form DNA adducts. However, the extent of DNA binding by 3-NBA was higher in almost all V79 cells at a 10-fold lower concentration than by N-Ac-N-OH-ABA, suggesting that N-Ac-N-OH-ABA is not a major intermediate in the formation of 3-NBA-derived adducts. 3-NBA showed a 3.8-fold and 16.8-fold higher mutagenic activity in Salmonella strains expressing human NAT1 and human NAT2, respectively, than in the acetyltransferase-deficient strain, whereas N-Ac-N-OH-ABA was only clearly (but weakly) mutagenic in Salmonella DJ460 expressing human NAT2. This finding suggests that N-Ac-N-OH-ABA is not a major reactive metabolite responsible for the high mutagenic potency of 3-NBA in Salmonella. Collectively our results indicate that O-acetylation and O-sulfonation by human NATs and SULTs may contribute significantly to the high mutagenic and genotoxic potential of 3-NBA. Moreover, the yet-unidentified four major 3-NBA-derived adducts may be DNA adducts without an N-acetyl group.

Abbreviations: 3-ABA, 3-aminobenzanthrone; CYP, cytochrome P450; dA, 2'-deoxyadenosine; dA-N-Ac-ABA, structurally unidentified 2'-deoxyadenosine adduct; dG, 2'-deoxyguanosine; dG-N-Ac-ABA, N-acetyl-3-amino-2-(2'-deoxyguanosin-8-yl)benzanthrone; dGp-N-Ac-ABA, N-acetyl-3-amino-2-(2'-deoxyguanosin-3'-monophosphate-8-yl)benzanthrone; NAT, N,O-acetyltransferase; N-Ac-N-OH-ABA, N-acetyl-N-hydroxy-3-aminobenzanthrone; N-Aco-N-Ac-ABA, N-acetoxy-N-acetyl-3-aminobenzanthrone; 3-NBA, 3-nitrobenzanthrone; N-OH-ABA, N-hydroxy-3-aminobenzanthrone; PAH, polycyclic aromatic hydrocarbon; RAL, relative adduct labelling; SULT, sulfotransferase; TLC, thin-layer chromatography.


    Introduction
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Environmental factors and individual genetic susceptibility play an important role in many human cancers (1). Nitropolycyclic aromatic hydrocarbons (nitro-PAHs) are widely distributed environmental pollutants found in extracts of emissions from diesel and gasoline engines and on the surface of ambient air particulate matter (2). The mutagenic activity of several members of this class in bacterial and mammalian systems as well as the tumorigenic activity in laboratory animals has been clearly documented (35). Their detection in tissues of lung cancer patients has led to considerable interest in assessing their potential cancer risk to humans (6).

Of the compounds found in diesel exhaust and in airborne particulate matter, 3-nitrobenzanthrone (3-NBA, 3-nitro-7H-benz[d,e]anthracene-7-one) (Figure 1Go) is a particularly powerful mutagen (7). It is most likely formed during incomplete combustion or by reaction of the parent aromatic hydrocarbon with nitrogen oxides in the atmosphere (8). 3-NBA was shown to be one of the most potent mutagens in the Ames Salmonella typhimurium assay reported so far, scoring numbers of revertants comparable with 1,8-dinitropyrene in strain TA98 and YG1024 (7). Moreover, preliminary data suggest that 3-NBA is carcinogenic in rats (9). The genotoxicity of this suspected carcinogen was further documented by induction of micronuclei in mouse and human cells as well as by the formation of mutations in human cells (7,10). Recently 3-aminobenzanthrone (3-ABA) was used as diesel-specific urinary biomarker of 3-NBA in mining workers occupationally exposed to diesel exhaust (11).



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Fig. 1. Potential pathways of metabolic activation and DNA adduct formation of 3-NBA. See text for details. Abbreviations are: N-OH-ABA, N-hydroxy-3-aminobenzanthrone; N-Ac-N-OH-ABA, N-acetyl-N-hydroxy-3-aminobenzanthrone; NAT, N,O-acetyltransferase; N,O-AT, N,O-acetyl transfer (also catalysed by NATs); DAase, deacetylase; SULT, sulfotransferase; R = –COCH3 or –SO3H; Me = –CH3.

 
The increased lung cancer risk after exposure to diesel exhaust (12) or to polluted ambient air (13) may be due to the interaction of reactive environmental pollutants with DNA. DNA adducts have been detected in rat lung after exposure to diesel exhaust (14) or extracts of airborne particulate matter (15). The detection of DNA adducts by the ultrasensitive 32P-postlabelling method is widely used in human biomonitoring and several studies have reported higher levels of DNA adducts among subjects heavily exposed to air pollutants and diesel exhaust, such as bus drivers, police officers and residents in heavily polluted areas (1618). This may possibly be predictive of lung cancer risk (19).

3-NBA forms specific DNA adducts after activation by xanthine oxidase and by rat liver S9 in vitro (20), as well as in vivo in Sprague–Dawley rats treated orally with a single dose of 3-NBA (21). Moreover, 3-NBA-derived DNA adducts have been found in rat lung alveolar type II (22) and human hepatoma HepG2 cells (23) treated with 3-NBA. In an attempt to elucidate the structure of adducts formed by 3-NBA, Enya et al. (24) synthesized the N-acetyl-N-hydroxyamino derivative of 3-NBA (N-Ac-N-OH-ABA) (Figure 1Go). After reacting the activated N-acetoxy-N-acetylamino ester (N-Aco-N-Ac-ABA) with deoxyguanosine (dG) and calf thymus DNA, an unusual dG C-8 adduct coupled through a C-C bond to the C-2 of benzanthrone, N-acetyl-3-amino-2-(2'-deoxyguanosin-8-yl)benzanthrone (dG-N-Ac-ABA), was formed and characterized spectroscopically. Therefore, 3-NBA-DNA adducts might be useful as biomarkers to monitor human exposure to diesel exhaust and ambient air particulate matter and thus may help to assess a possible lung cancer risk for humans.

Nitro-PAHs require metabolism to reactive electrophilic species in order to exert their genotoxic activity. The activation of nitroaromatic hydrocarbons is mainly through nitroreduction catalysed primarily by cytosolic reductases, such as xanthine oxidase, DT-diaphorase and aldehyde oxidase, whereas cytochrome P450 (CYP) enzymes are primarily responsible for the oxidative metabolism of these compounds (4). N-Hydroxy arylamine intermediates can further be metabolized by phase II enzymes, such as N,O-acetyltransferases (NATs) or sulfotransferases (SULTs), leading to the formation of reactive esters, e.g. N-acetoxy or N-sulfooxy arylamines which undergo heterolysis of the N-O or S-O bond to produce electrophilic nitrenium ions capable of reacting with DNA to form DNA adducts (25,26). In humans, two isoforms of NAT, designated NAT1 and NAT2, catalyse N- and O-acetylation of various N-hydroxy arylamines (27). A total of 11 different cytosolic SULTs have been detected in man (28). Bioactivation of aromatic hydroxylamines has been primarily observed with human SULT1A1 and SULT1A2 (26,29). Whereas SULT1A1 is highly expressed in numerous tissues including lung (28,30), expression of SULT1A2 appears to be low or absent in the tissues investigated (31).

The objective of this study was to investigate the potential role of human NATs (NAT1, NAT2) and human SULTs (SULT1A1) in the metabolic activation of 3-NBA and N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA), a potential metabolite of 3-NBA. NAT- and SULT-mediated genotoxicity was determined by measuring DNA adduct formation in recombinant V79 Chinese hamster lung fibroblast cells and mutagenicity in genetically modified Salmonella typhimurium strains using the Ames assay.


    Material and methods
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 Material and methods
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Synthesis of 3-NBA and N-Ac-N-OH-ABA
7H-Benz[d,e]anthracen-7-one was purchased from Fluka (Switzerland). 3-NBA was synthesized by a modification of the procedure reported by Enya et al. (24), in which fuming nitric acid was used instead of concentrated nitric acid. The purity and identity of 3-NBA was checked by HPLC and GC-MS. No contamination could be detected with these methods. The results of NMR and mass spectometry confirmed the identity with the data published (7,10). N-Ac-N-OH-ABA was kindly provided by Drs Kawanishi and Enya (24).

Bacterial mutagenicity assays
The plate incorporation assay without an exogenous metabolizing system for detecting mutagenic activity was performed as described (32). The following strains of Salmonella typhimurium were used: TA1538, which expresses the endogenous S. typhimurium NAT enzyme; the acetyltransferase-deficient strain TA1538/1,8-DNP; and its derivatives DJ400 (TA1538/1,8-DNP pNAT1) and DJ460 (TA1538/1,8-DNP pNAT2), expressing human NAT1 and human NAT2, respectively (33). Bacterial cultures were grown in nutrient broth (with addition of 25 µg/ml ampicillin for DJ400 and DJ460) by a 10 h overnight incubation at 37°C with shaking. Revertant colonies were counted after 2 days (TA1538) or 3 days (TA1538/1,8-DNP, DJ400, DJ460) of incubation at 37°C. Assays were performed in the 0.25–25 ng/plate range. 3-NBA and N-Ac-N-OH-ABA were dissolved in DMSO (100 µl). Control incubations were treated with DMSO only. Mutagenic potencies were obtained from the linear parts of their dose–response curves and expressed as revertants per nanomole (rev/nmol). The bacterial strains TA1538/1,8-DNP, DJ400 and DJ460 were generous gifts from P.D. Josephy (University of Guelph, Ontario, Canada).

Development of V79 cell lines, cell culture of V79 cells, and treatment with 3-NBA and N-Ac-N-OH-ABA
Human NATs (hNAT1, hNAT2) and human SULT1A1 (hSULT1A1) were expressed in the V79 Chinese hamster lung fibroblast subclone V79MZ (34). The names devised for the recombinant V79 cells are composed of the parental cell line (V79MZ) and the short name of the enzyme. Details concerning the construction and characterization of these V79 cell lines will be published elsewhere (E.Muckel, U.Pabel and H.R.Glatt, manuscript in preparation). Briefly, the cDNAs for wild-type forms of human NATs (NAT1*4, NAT2*4) in the plasmid pKK223-3 (35) and that of human SULT1A1*1 in the plasmid pKK233-2 (36) were used for cloning into the eucaryotic expression vectors pSI (Promega, Germany) and pMPSV (37), respectively. The cDNA constructs were introduced into V79MZ using a co-transfection strategy with a vector conferring resistance to puromycin (38). Functional expression of the recombinant enzymes was confirmed by determination of NAT (39) and SULT (40) activities in cytosol preparations using p-aminobenzoic acid, sulfamethazine and p-nitrophenol as substrates of human NAT1, NAT2 and human SULT1A1, respectively.

All V79 cells were cultivated as described recently (41). Briefly, V79 cells were cultivated in 75 cm2 cell culture flasks in a total volume of 25 ml Dulbecco's modified Eagle's medium (DMEM, without pyruvate and L-glutamine), high glucose content (4.5 g D-glucose/l), supplemented with 1 mM sodium pyruvate, 4 mM L-glutamine, 25 mM HEPES, 5% foetal bovine serum (FBS), 100 U of penicillin/ml and 100 µg streptomycin/ml at 37°C, 5% CO2 and 95% atmospheric humidity. For incubations, cells were seeded 24 h prior to treatment at a density of 1 x 105 cells per flask in a total volume of 30 ml of DMEM. 3-NBA and N-Ac-N-OH-ABA dissolved in DMSO (50 µl) were added to a final concentration of 0.01, 0.1 and 1 µM to all V79 cells. In control incubations cells were treated with DMSO only. After an incubation period of 24 h the medium was removed and the cells were washed twice with 5 ml PBS. During this step dead cells were washed away. Cells were harvested by trypsinization with 2 ml of a solution containing 0.025% trypsin and 0.01% EDTA in PBS. Trypsinization was stopped by the addition of DMEM (2 x 4 ml) supplemented with 5% FBS. Cell viability was determined by the trypan blue exclusion assay as described previously (42). Briefly, cell suspensions were gently mixed in an appropriate dilution (1:2–1:10) with trypan blue (0.4% solution in 0.85% saline, Sigma, UK), allowed to stand for 5 min and placed in a haemocytometer. Cells that excluded trypan blue were used as an indicator of cell viability. Viable cells after treatment were expressed as percentage of viable cells without treatment (controls). Subsequently, centrifugation of harvested cells at 2000 rpm for 10 min and one washing step with 10 ml PBS yielded a cell pellet, which was stored at –20°C until DNA isolation.

32P-Postlabelling analysis
DNA from cells was isolated by the phenol extraction method as described recently (43). DNA samples (4 µg) were digested with micrococcal nuclease (120 mU, Sigma, UK) and calf spleen phosphodiesterase (20 mU, Calbiochem, UK) in digestion buffer (16.6 mM sodium succinate, 8.3 mM CaCl2, pH 6.0) overnight at 37°C in a total volume of 4.8 µl.

For nuclease P1 enrichment digests were incubated with 1.2 µg nuclease P1 (Sigma, UK) in 4.8 ml of buffer containing 125 mM sodium acetate, pH 5.0, 0.4 µM ZnCl2 for 60 min at 37°C. The reaction was terminated by the addition of 1.9 µl Tris base (0.5 M).

For butanol enrichment digests were diluted to 50 µl with water to which was then added 100 µl extraction buffer (15 mM ammonium formate, 1.5 mM tetrabutyl-ammonium chloride, pH 3.5) and the solution was immediately extracted with water-saturated 1-butanol (250 µl). After a second extraction, the organic phases were combined, back-extracted with butanol-saturated water (2 x 400 µl), neutralized with 4 µl 200 mM Tris–HCl (pH 9.5) and evaporated to dryness. The residue was taken up in 11.5 µl water.

The DNA digests or extracted adducts were then 32P-labelled with carrier-free [{gamma}-32P]ATP (50 µCi, ~7000 Ci/mmol, ICN, UK) in a mixture of 4 µl consisting of 80 mM bicine pH 9.0, 40 mM MgCl2, 40 mM DTT, 4 mM spermidine and 6 U T4 polynucleotide kinase (Cambio, Cambridge, UK) for 30 min at 37°C. Resolution of 32P-labelled adducts was carried out by chromatography on polyethyleneimine-cellulose (PEI-cellulose) TLC sheets (10 x 20 cm, Macherey-Nagel, Düren, Germany) using solvents described previously (20): D1, 1.0 M sodium phosphate, pH 6.0 (pH reduced from 6.8); D3, 4 M Li-formate, 7 M urea, pH 3.5; D4, 0.8 M LiCl, 0.5 M Tris, 8.5 M urea, pH 8.0; D5 was omitted.

TLC sheets were scanned using a Packard Instant Imager (Dowers Grove, IL, USA) and DNA adduct levels (RAL, relative adduct labelling) were calculated from the adduct cpm, the specific activity of [{gamma}-32P]ATP and the amount of DNA (pmol of DNA-P) used. Results were expressed as DNA adducts/108 nucleotides.


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Mutagenic activity of 3-NBA and N-Ac-N-OH-ABA in Salmonella expressing recombinant human NAT1 and NAT2
The results of the mutation assays are given in Figure 2Go. 3-NBA was mutagenic in all strains tested (Figure 2AGo). The mutagenic potency was 4.3-fold higher in TA1538 expressing bacterial O-acetyltransferase compared with the acetyltransferase-deficient strain TA1538/1,8-DNP (Table IGo). Likewise, human NAT1 and NAT2, expressed in TA1538/1,8-DNP (DJ400 and DJ460, respectively), enhanced the mutagenicity of 3-NBA 3.8- and 16.8-times, respectively (Table IGo). Under the conditions used, N-Ac-N-OH-ABA was only weakly mutagenic (Figure 2BGo). No mutagenic activity was observed in TA1538/1,8-DNP. In TA1538 and DJ400, N-Ac-N-OH-ABA induced a doubling above the background (spontaneous) mutation frequency at the highest dose used. A dose–response was only observed in DJ460. The ratios of the mutagenic activities of 3-NBA compared with N-Ac-N-OH-ABA were 27.2 in TA1538, 29.2 in DJ400 and 25.8 in DJ460 (Table IGo).



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Fig. 2. Mutagenic activity of 3-NBA (A) or N-Ac-N-OH-ABA (B) in different TA1538-derived Salmonella typhimurium strains. Values are means ± SD of six separate plates determined by two separate incubations (each triplicate plates). For 3-NBA revertants/plate in the presence of DMSO (control) were 28 ± 8, 21 ± 5, 15 ± 5 and 12 ± 2 for TA1538, TA1538/1,8-DNP, DJ400 and DJ460, respectively. For N-Ac-N-OH-ABA revertants/plate in the presence of DMSO were 29 ± 3, 31 ± 13, 19 ± 4 and 17 ± 6 for TA1538, TA1538/1,8-DNP, DJ400 and DJ460, respectively.

 

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Table I. Bacterial mutagenicity of 3-NBA and N-Ac-N-OH-ABA in different TA1538-derived Salmonella typhimurium strains
 
DNA adduct formation of 3-NBA in V79 cells expressing recombinant human NAT1, NAT2 and human SULT1A1
Parental and recombinant V79 cells were treated with 0.01, 0.1 and 1 µM 3-NBA. In the parental cells, V79MZ, viability was only marginally decreased at the highest concentration used (Figure 3AGo). Under the conditions used, cell viability was dramatically decreased in V79 cells expressing human NAT2. In V79 cells expressing human NAT1 and human SULT1A1 viability was strongly decreased at higher concentrations. The high cytotoxicity in these cells compared with the parental cells indicates that human NAT1 and NAT2 as well as human SULT1A1 are strongly involved in the bioactivation of 3-NBA. In order to compare DNA adduct formation between all V79 cells at the highest possible concentration that was not excessively cytotoxic to the most sensitive cell line, DNA binding for 3-NBA was compared at the lowest concentration tested (0.01 µM). For the parental cells, V79MZ, DNA binding was determined at all concentrations.



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Fig. 3. Diagram showing cytotoxicity (viable cells as % of control) of 3-NBA (A) and N-Ac-N-OH-ABA (B) in parental and recombinant V79MZ-derived cells. Values represent means ± SD of three separate incubations.

 
32P-postlabelling analysis of DNA isolated from V79MZ cells resulted in the detection of up to eight adducts spots on TLC (Figure 4Go). The pattern was essentially identical in all cell lines (see Figure 6Go), consisting of a cluster of four major adducts (spots 1, 2, 3 and 4) and one minor (spot 5) after enhancement by butanol extraction (Figure 4EGo) and a cluster of three major adducts (spots 1, 2, 3) and one minor (spot 6) after enhancement by nuclease P1 digestion (Figure 4BGo). Adduct spot 3 was always the predominant adduct formed (see Figure 6Go). Moreover, adduct spots 7 and 8 were only detectable at the highest concentration (1 µM) tested after butanol extraction in V79MZ cells. All major adducts were primarily located on a diagonal line on the TLC plates at ~45° to the directions of elution with D3 and D4, typical for DNA adducts derived from extracts of airborne particulate matter and diesel exhaust (44,45). As shown in Figure 4EGo adduct spots 4, 5, 7 and 8 were clearly visible after butanol extraction but not after nuclease P1 digestion. In contrast, adduct spot 6 was only detected after nuclease P1 digestion (Figure 4BGo). 32P-Postlabelling analyses of DNA isolated from cells treated with vehicle (DMSO) only did not show adduct spots (Figure 4A and DGo).



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Fig. 4. Autoradiographic profiles of DNA adducts obtained in parental V79MZ cells treated with 1 µM 3-NBA (B and E), 1 µM N-Ac-N-OH-ABA (C and F) or with vehicle, DMSO, only (A and D), using the nuclease P1 (upper panels) or butanol (lower panels) enrichment version of the 32P-postlabelling assay.

 


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Fig. 6. RAL (relative adduct labelling) of individual DNA adducts in parental and recombinant V79MZ-derived cells after exposure to 0.01 µM 3-NBA (A and C) or 0.1 µM N-Ac-N-OH-ABA (B and D) using the nuclease P1 (upper panels) or butanol (lower panels) enrichment version of the 32P-postlabelling assay. Values represent mean ± SD of three separate incubations each determined by two separate postlabelling analyses. Comparison was performed by t-test analysis: *P < 0.01, #P < 0.05 by comparison with parental V79MZ. n.d. = not detected.

 
DNA adduct formation was concentration-dependent in the parental cell line, V79MZ (Table IIGo), ranging from 0.4 to 47.7 and from 0.5 to 68.4 adducts per 108 nucleotides for total DNA binding after nuclease P1 digestion and butanol extraction, respectively. In the recombinant V79 cells total adduct levels at the lowest concentration (0.01 µM) were in the range from 13.1 to 196.9 and 16.8 to 227.8 adducts per 108 nucleotides after nuclease P1 digestion and butanol extraction, respectively (Figure 5AGo). DNA binding was highest in V79 cells expressing human NAT2. Compared with V79MZ cells total DNA binding by 3-NBA was significantly higher in V79 cells expressing human NAT2 (448–475-fold, P < 0.01), SULT1A1 (62–102-fold, P < 0.01), and NAT1 (30–35-fold, P < 0.01), respectively, demonstrating that human NAT2 strongly contributes to the metabolic activation of 3-NBA to form DNA adducts. Human SULT1A1 and NAT1 also contribute, but to a lesser extent. Levels of individual adduct spots are given in Figure 6A and CGo.


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Table II. Quantitative analysis of DNA binding in V79MZ cells treated with 3-NBA or N-Ac-N-OH-ABA
 


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Fig. 5. RAL (relative adduct labelling) of total DNA adducts in parental and recombinant V79MZ-derived cells after exposure to 0.01 µM 3-NBA (A) or 0.1 µM N-Ac-N-OH-ABA (B) using the nuclease P1 or butanol enrichment version of the 32P-postlabelling assay. Values represent mean ± SD of three separate incubations each determined by two separate postlabelling analyses. Comparison was performed by t-test analysis: *P < 0.01, #P < 0.05 by comparison with parental V79MZ.

 
DNA adduct formation of N-Ac-N-OH-ABA in V79 cells expressing recombinant human NAT1, NAT2 and human SULT1A1
Parental and recombinant V79 cells were treated with 0.01, 0.1 and 1 µM N-Ac-N-OH-ABA. In the parental cells, V79MZ, viability decreased only at the highest concentration used (Figure 3BGo). The same was found for V79 cells expressing human NAT1. In V79 cells expressing human NAT2 and human SULT1A1, cell viability was strongly decreased at higher concentrations. The increased cytotoxicity in these cells compared with the parental cells indicates that human NAT1 and NAT2 as well as human SULT1A1 are involved in the bioactivation of N-Ac-N-OH-ABA. In order to compare DNA adduct formation between all V79 cells at the highest possible concentration that was not excessively cytotoxic to the most sensitive cell line, DNA binding for N-Ac-N-OH-ABA was compared at the medium concentration tested (0.1 µM). For the parental cells, V79MZ, DNA binding was determined at all concentrations.

Using either enrichment procedure of the 32P-postlabelling assay DNA adduct analysis resulted in the detection of up to nine adducts in the parental V79MZ cells (Figure 4Go). The pattern was essentially similar in all cell lines to those observed after treatment with 3-NBA (see Figure 6Go), consisting of a cluster of four major adducts (spots 1, 2, 3 and 4) and one minor (spot 5) after enhancement by butanol extraction (Figure 4FGo) and a cluster of three major adducts (spots 1, 2, 3) and one minor (spot 6) after enhancement by nuclease P1 digestion (Figure 4CGo). Adduct spot 3 was always the predominant adduct formed (see Figure 6Go). Adduct spots 7 and 8 were only visible at the highest concentration tested in V79MZ cells using butanol extraction. In contrast to the treatment with 3-NBA one additional adduct (spot 9) was found in V79MZ cells treated with N-Ac-N-OH-ABA at the highest concentration (1 µM), both after nuclease P1 digestion and butanol extraction. Adduct spot 9 was chromatographically indistinguishable from N-acetyl-3-amino-2-(2'-deoxyguanosin-3'-monophosphate-8-yl)benzanthrone (dGp-N-Ac-ABA) (Arlt, Schmeiser, Phillips, unpublished data), an identified adduct standard (21,23). However, adduct spot 9 was not observed in recombinant V79 cells at the lower concentration (0.1 µM) analysed (compare Figure 6Go).

A concentration-dependent DNA adduct formation was observed in the parental cell line, V79MZ (Table IIGo), ranging from 0.5 to 24.7 and 0.5 to 87.7 adducts per 108 nucleotides for total DNA binding after nuclease P1 digestion and butanol extraction, respectively. In the recombinant V79 cells total adduct levels at the medium concentration were in the range from 17.0 to 155.8 and 17.3 to 136.2 adducts per 108 nucleotides after nuclease P1 digestion and butanol extraction, respectively (Figure 5BGo). DNA binding was the highest in V79 cells expressing human NAT2. Compared with V79MZ cells total DNA binding by N-Ac-N-OH-ABA was significantly higher in V79 cells expressing human NAT2 (28–30-fold, P < 0.01), SULT1A1 (4–6-fold, P < 0.01), and NAT1 (3–4-fold, P < 0.01), respectively. Thus, human NAT2 strongly contributes to the metabolic activation of N-Ac-N-OH-ABA to form DNA adducts, with lesser involvement of human SULT1A1 and NAT1. Levels of individual adduct spots are shown in Figure 6B and DGo.


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3-NBA is a very potent mutagen and suspected human carcinogen recently identified in diesel exhaust and airborne particulate matter (7,9,11). One major pathway of bioactivation of 3-NBA is simple nitroreduction (11,2022). However, phase II enzymes can also be involved through N- and/or O-acetylation resulting in the formation of highly reactive arylnitrenium ions, which interact with cellular DNA and proteins (7,2224). A previous study showed that 3-NBA had a 30-fold higher mutagenic activity in the Salmonella typhimurium strain YG1024 exhibiting bacterial O-acetyltransferase than in TA98 (7). In the present study the metabolic activation of 3-NBA by human NATs and SULTs was investigated in genetically modified Salmonella strains expressing human NAT1 and NAT2 and recombinant V79 cells expressing human NAT1, NAT2 and human SULT1A1.

We found that the mutagenic potencies of 3-NBA were nearly 4- and 20-fold higher in the Salmonella strains expressing human NAT1 (DJ400) and NAT2 (DJ460), respectively, compared to the Salmonella strain lacking acetyltransferase (TA1538/1,8-DNP) (Table IGo, Figure 2AGo). The very low mutagenic activity in TA1538/1,8-DNP may be due to the reactivity of the unacetylated N-hydroxylamine intermediate, which can, to some extent, form DNA-binding arylamine ions spontaneously. This may be catalysed by cytosolic nitroreductases. Alternatively, Salmonella may contain other, yet unidentified enzymes capable of activating hydroxylamines. Previous work has suggested that the bacterial NAT enzyme may have a greater ability to O-acetylate some N-hydroxylamines than the human enzymes (33). We found that the mutagenic potency in TA1538, expressing the bacterial enzyme, was 4-times higher compared to TA1538/1,8-DNP. This is similar to the mutagenic potency in DJ400 expressing human NAT1, but the mutagenic potency in DJ460 expressing human NAT2 was ~4-fold higher compared with TA1538. This may result from kinetic differences between human and bacterial enzymes. Another possibility is that the expression of the NAT gene varies among these strains. It is noteworthy that O-acetylation of the N-hydroxy arylamine intermediates from 1,8-dinitropyrene, which shows mutagenic activity in the Ames test comparable to that of 3-NBA, was shown to be responsible for its extreme mutagenicity (4). Thus, we conclude that O-acetylation by human NAT2 (and to a lesser extent by human NAT1) strongly contributes to the high mutagenic potential of 3-NBA. However, the relative contributions of N- or O-acetylation to the mutagenicity of 3-NBA by human NATs are still to be determined.

Previously we demonstrated the formation of specific 3-NBA-adducts in vitro and in vivo in rats (20,21). Using the 32P-postlabelling assay, an essentially similar adduct pattern was observed in all V79 cells treated with 0.01 µM 3-NBA (Figure 6A and CGo), similar to that found in vitro and in rats treated with 3-NBA. Moreover, comparative analyses of the major adducts obtained in cell culture with those detected in vitro and in vivo in rats revealed that these 3-NBA-adducts were chromatographically indistinguishable (data not shown). In an attempt to partially characterize the structure of those DNA adducts generated by 3-NBA we showed that the four major 3-NBA-adducts are products derived from reductive metabolites bound to deoxyadenosine (adduct spots 1 and 2) and deoxyguanosine (adduct spots 3 and 4) (Figure 4B and EGo) (21). Further chemical characterization of these adducts is currently being undertaken. However, besides the known four major 3-NBA-adducts several minor adduct spots (adduct spots 5, 6, 7 and 8) were detected (Figure 4B and EGo), which await further elucidation. Comparison of DNA binding in the parental cells, V79MZ, with DNA binding in the recombinant V79 cells demonstrated that human NAT1, NAT2 as well as human SULT1A1 are strongly involved in the metabolic activation of 3-NBA to form DNA adducts (Figure 5AGo). The involvement of these enzymes was also demonstrated by the increased cytotoxicity in the recombinant V79 cells compared with the parental cells (Figure 3AGo).

As is the case with other nitroPAHs (4,5), metabolic activation of 3-NBA may involve a nitroreduction and/or a ring oxidation pathway followed by esterification. For 3-NBA, we found no indication of ring oxidation in its bioactivation leading to DNA adducts (20,21). Since V79 cells completely lack CYP-dependent enzyme activities (34) our results here suggest that nitroreduction, e.g. catalysed by cytosolic nitroreductases (2022), is the major pathway in the bioactivation of 3-NBA. V79 cells also contain detectable amounts of NADPH:CYP reductase (34), an enzyme known to activate nitroaromatic compounds, explaining the formation of DNA adducts by 3-NBA in the parental cells V79MZ. Moreover, we propose that further O-acetylation and O-sulfonation of the initially formed N-hydroxy-3-aminobenzanthrone (N-OH-ABA) intermediate contributes significantly to the high genotoxic potential of 3-NBA (Figure 1Go). Phase II activation by NATs and SULTs leads to the formation of reactive N-acetoxy or N-sulfooxy esters of aminobenzanthrone which undergo heterolysis of the N-O or S-O bond to produce electrophilic nitrenium ions reacting with DNA to form covalent DNA adducts (Figure 1Go).

Human exposure to 3-NBA is thought to occur primarily via the respiratory tract. NAT1 mRNAs were found in human bronchial mucosa and peripheral lung tissue and NAT2 expression was detected in all peripheral lung tissues, but was not detected in the bronchus (46). Human bronchial epithelial cells and alveolar macrophages belong to the primary defence system against inhaled compounds and SULT1A1 is expressed in these cells (30,47). Therefore, NAT and SULT expression in the human respiratory system could contribute significantly and specifically to the metabolic activation of 3-NBA. Many genes of enzymes metabolizing carcinogens are known to exist in variant forms or show polymorphisms resulting in different activities of the gene products and appear to be important determinants of cancer risk (1). Up to now, 26 NAT1 and 29 NAT2 alleles have been characterized that segregate individuals into slow and rapid acetylator phenotypes (48). Epidemiological studies have suggested that there is a relationship between urinary bladder cancer and slow acetylator NAT2 genotype, and between colorectal cancer and rapid NAT2 acetylator genotype (49). Also for SULT1A1 genetic polymorphisms have been detected in man (28,50,51). Consequently, genetic polymorphisms in the human NAT1, NAT2 as well as in the human SULT1A1 gene could be important determinants of a possible lung cancer risk by 3-NBA. However, the precise kinetics of acetylation and sulfonation of 3-NBA metabolites in human tissue await further investigation.

In an attempt to elucidate the structure of adducts formed by 3-NBA, Enya et al. (24) identified an unusual dG C-8 adduct, N-acetyl-3-amino-2-(2'-deoxyguanosin-8-yl)benzanthrone (dG-N-Ac-ABA). This adduct is formed after reaction of the N-acetoxy-N-acetylamino derivative of 3-NBA (N-Aco-N-Ac-ABA) with DNA (Figure 1Go). Recently this adduct was identified in human hepatoma HepG2 cells treated with 3-NBA by 32P-postlabelling as a minor adduct (23). Moreover, Enya et al. (24) reported the detection of a deoxyadenosine (dA) adduct after reacting N-Aco-N-Ac-ABA with dA and calf thymus DNA, which was assigned as dA-N-Ac-ABA. This adduct was also identified in human hepatoma HepG2 cells treated with 3-NBA by 32P-postlabelling as a minor adduct (23), but has not been structurally characterized. In order to examine whether human NAT1 and NAT2 contribute to the mutagenic activity and DNA adduct formation of N-Aco-N-Ac-ABA we incubated the Salmonella strains and V79 cells with the immediate metabolic precursor of N-Aco-N-Ac-ABA, N-Ac-N-OH-ABA (Figure 1Go).

N-Ac-N-OH-ABA was only clearly mutagenic in the Salmonella strain DJ460 expressing human NAT2 (Figure 2BGo), but 3-NBA was ~30-fold more mutagenic in this strain (Table IGo). Therefore, these results indicate that N-Ac-N-OH-ABA is not a major reactive metabolite responsible for the high mutagenic potency of 3-NBA in Salmonella. In V79 cells DNA binding by N-Ac-N-OH-ABA increased significantly in V79 cells expressing human NAT1, NAT2 and human SULT1A1 (Figure 5BGo). Thus, we conclude that human NAT1, NAT2 and human SULT1A1 are involved in the metabolic activation of N-Ac-N-OH-ABA leading to DNA adducts. That human NAT1, NAT2 and human SULT1A1 contribute to the bioactivation of N-Ac-N-OH-ABA was also demonstrated by the increased cytotoxicity in the recombinant V79 cells compared with the parental cells (Figure 3BGo). A similar adduct pattern was observed in all V79 cells treated with 0.1 µM N-Ac-N-OH-ABA (Figure 6B and DGo). A comparison of chromatographic characteristics among DNA adducts obtained with N-Ac-N-OH-ABA with those detected in V79 cells treated with 3-NBA revealed that the major N-Ac-N-OH-ABA-derived adducts were indistinguishable from the major 3-NBA-derived adducts (Figure 4Go). Since previous in vitro data demonstrated that at least all four major 3-NBA-adducts are products derived from nitroreduction bound to purine bases without an N-acetyl group (20,21) we assume that all four major N-Ac-N-OH-ABA-derived adducts obtained in this study are also bound to the corresponding purine base without carrying an N-acetyl group. Moreover, since DNA binding by 3-NBA was higher in almost all V79 cells at a 10-fold lower concentration than by N-Ac-N-OH-ABA we conclude that N-Ac-N-OH-ABA does not seem to be a major intermediate in the formation of 3-NBA-derived adducts.

The data presented here suggest that N-Ac-N-OH-ABA is readily deacetylated to N-OH-ABA which is further esterified to the corresponding N-acetoxy- or N-sulfooxyester, which can react directly with DNA (Figure 1Go). An essential role of microsomal deacetylase in adduct formation by N-hydroxy-2-acetylaminofluorene has been demonstrated (52). It is also known that human NATs catalyse, to limited extent, N,O-acetyltransfer reactions (25). It may be possible that N-Ac-N-OH-ABA is converted to the corresponding N-acetoxyester by N,O-acetyltransfer, which again can react directly with DNA (Figure 1Go). As described above the formation of both these reactive esters (N-acetoxy- or N-sulfooxyester) after initial nitroreduction of 3-NBA are presumably mainly responsible for DNA adduct formation by 3-NBA. This explains why treatment with 3-NBA and N-Ac-N-OH-ABA lead to the same DNA adducts. On the other hand N-Ac-N-OH-ABA may be rapidly detoxified in V79 cells by pathways such as glutathione conjugation (34), which could explain the lower DNA binding by N-Ac-N-OH-ABA compared to 3-NBA.

In contrast to incubations with 3-NBA, one additional adduct spot (spot 9) was observed in the parental cells, V79MZ, after treatment with 1 µM N-Ac-N-OH-ABA (Figure 4Go). Adduct spot 9 was chromatographically indistinguishable from dG-N-Ac-ABA (21,23), which was previously identified also in human hepatoma HepG2 cells treated with 3-NBA as a minor adduct (23). Despite the fact that dG-N-Ac-ABA was not detected in vitro in V79 cells, rat lung alveolar type II cells (22) and in vivo in rats (21) after treatment with 3-NBA it cannot be excluded that N-Ac-N-OH-ABA does contribute to DNA adduct formation by 3-NBA. However, the available data (present paper, ref. 23) indicate that the structurally yet-unidentified major 3-NBA-adducts are deoxyguanosine and deoxyadenosine adducts formed with aminobenzanthrone that lack the N-acetyl group.


    Notes
 
4 To whom correspondence should be addressed Email: v.arlt{at}icr.ac.uk Back


    Acknowledgments
 
This work was supported financially by Cancer Research UK, the European Union (QLK1-1999-01197) and Baden-Württemberg (BWPLUS, BWB 20003).


    References
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 

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Received May 23, 2002; revised August 22, 2002; accepted August 22, 2002.