1 Department of Biochemistry and 2 Department of Organic Chemistry, Faculty of Science, Charles University, Albertov 2030, 128 40 Prague 2, Czech Republic and 3 Division of Molecular Toxicology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
4 To whom correspondence should be addressed Email: stiborov{at}natur.cuni.cz
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Abstract |
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Abbreviations: acetylCoA, acetylcoenzyme A; dAp, deoxyadenosine 3'-monophosphate; CT-DNA, calf thymus DNA; dGp, deoxyguanosine 3'-monophosphate; HPLC, high performance liquid chromatography; NAT, N,O-acetyltransferase; 2-NA, 2-nitroanisole; NQO1, NAD(P)H:quinone oxidoreductase; PEI, polyethylenimine; PAPS, 3'-phosphoadenosyl 5'-phosphosulfate; RAL, relative adduct labeling; r.t., retention time; SULT, sulfotransferase; TLC, thin layer chromatography; XO, xanthine oxidase
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Introduction |
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This is also the case for 2-nitroanisole (2-methoxynitrobenzene, 2-NA) (3). 2-NA is used primarily as a precursor in the synthesis of 2-methoxyaniline (o-anisidine), an intermediate in the manufacture of many azo dyes (10). 2-NA exhibits carcinogenic activity, causing neoplastic transformation in the urinary bladder and, to a lesser extent, in spleen, liver and kidneys in rodents (10,11). 2-NA is also a toxic compound, causing anemia. The anemia is characterized by increased levels of methemoglobin and accelerated destruction of erythrocytes (10). In 1993 an industrial accident in the Hoechst company in Germany caused a large-scale leakage of 2-NA and subsequent local and regional contamination. Various dermatological changes were found among children living in the area of the accident (12). Furthermore, single- and double-strand breaks were induced in DNA of the fire fighters working at the site of the accident (13).
It has not been determined exactly whether 2-NA is a genotoxic or epigenetic carcinogen. In spite of potent rodent carcinogenicity of 2-NA, this chemical is weakly mutagenic in the Ames test with the Salmonella typhimurium TA100 strains (10). This carcinogen also exhibits a low activity in cytogenetic tests. It induces a slight increase in chromosomal aberrations and in sister chromatid exchanges, but only at high concentrations (10).
Most aromatic nitro hydrocarbons require metabolism to reactive species in order to exert their genotoxic activity. The activation of aromatic nitro hydrocarbons to reactive N-hydroxyarylamine intermediates is through nitro reduction, catalyzed primarily by several cytosolic reductases [i.e. xanthine oxidase (XO), NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase) and aldehyde oxidase] and/or microsomal enzymes such as NADPH:cytochrome P450 reductase, whereas cytochrome P450 enzymes are primarily responsible for the oxidative metabolism of these compounds (3,4,7,9,1419). N-hydroxyarylamine intermediates can be further 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-sulfoxyarylamines which undergo heterolysis of the NO or SO bond to produce electrophilic nitrenium ions capable of reacting with DNA to form DNA adducts (2023). The major route of metabolism of 2-NA in vivo is oxidative demethylation to 2-nitrophenol, which appears in urine predominantly as the sulfate conjugate (24). A second pathway involves reduction to 2-methoxyaniline(o-anisidine); at blood concentrations at which the metabolism and elimination of 2-NA are linear, o-anisidine is a minor metabolite formed in liver (24). However, at higher doses the 2-nitrophenyl sulfate pathway may reach saturation, leading to the formation of proportionally more o-anisidine (10). Hence, nitroreduction of 2-NA, which is considered an activation pathway for aromatic nitro compounds, was clearly documented in this in vivo study (24). Recently, we showed that 2-NA was also reductively metabolized in vitro, by buttermilk XO and by hepatic cytosol from rat, rabbit, pig and human. Two major reduction products, N-(2-methoxyphenyl)hydroxylamine and o-anisidine, were generated in these reactions (25,26). XO was found to be the major enzyme responsible for reduction of 2-NA in cytosol (26). In addition, we demonstrated that 2-NA is activated by XO in vitro to species forming DNA adducts detected by 32P-post-labeling (27). Although the structures of the DNA adducts remain to be characterized, we have shown that the nitroreduction pathway is responsible for the formation of these adducts in vitro (25,27). However, in vivo DNA adduct formation by 2-NA has not yet been shown.
In the present study, we examine the in vivo formation of DNA adducts by 2-NA and their tissue distribution in rats treated with this carcinogen.
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Materials and methods |
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Chemicals
Chemicals were obtained from the following sources: 2-NA (>99% based on HPLC) from Fluka Chemical Co. (Switzerland); NADH, NADPH, nuclease P1, deoxyadenosine 3'-monophosphate (dAp), deoxyguanosine 3'-monophosphate (dGp), hypoxanthine, buttermilk XO, acetylcoenzyme A (acetylCoA) and adenosine 3'-phosphate 5'-phosphosulfate (3'-phosphoadenosyl 5'-phosphosulfate, PAPS) from Sigma Chemical Co. (St Louis, MO); bicinchoninic acid from Pierce (Rockford, IL); calf thymus DNA (CT-DNA) from Roche Diagnostics (Mannheim, Germany). All chemicals were of analytical purity or better. N-(2-methoxyphenyl)hydroxylamine was synthesized by a procedure similar to that described earlier (28). Briefly, to a solution of 2 g ammonium chloride and 90 mmol 2-NA in 60% ethanol/water, 180 mmol of zinc powder was added in small portions. After addition of the first portion at room temperature, the reaction starts; this can be monitored by the rising temperature in the flask. The reaction mixture was kept at 1015°C using a cooling bath (ice/sodium chloride mixture) and slowly adding additional doses of zinc powder. After 1 h, excess zinc was removed by filtration and ethanol was removed under reduced pressure. The product was extracted into 100 ml ethyl acetate and crystallized by adding hexane. The yield was 60%.
The 3H-labeled 2-NA (34.5 MBq/mmol) was prepared by the procedure described previously (29) and stored in methanol at 18°C. Radiochemical purity of the compound was >98% [by thin layer chromatography (TLC)]. Enzymes and chemicals for the 32P-post-labeling assay were obtained commercially from sources described previously (30,31).
Animal experiments
Six male Wistar rats (125150 g) were treated once a day for 5 consecutive days with 2-NA dissolved in sunflower oil (0.15 mg/kg body wt i.p. per day). Two control animals received an equal volume of solvent. Rats were placed in cages in temperature and humidity controlled rooms. Standardized diet and water were provided ad libitum. Animals were killed 24 h after the last treatment by cervical dislocation (32). Seven organs (liver, kidney, lung, heart, spleen, brain and urinary bladder) were removed immediately after death, quickly frozen in liquid nitrogen and stored at 80°C until DNA isolation.
Human hepatic cytosolic samples
Ten human hepatic cytosolic fractions were obtained from Gentest Corp. (Woburn, MA) and stored at 80°C. The donors ranged in age from 2 to 71 years and included two men and eight women. Any history of drug and/or alcohol abuse of the samples is described in the Gentest protocols. Each cytosolic preparation was analyzed for reductase (NQO1, XO and aldehyde oxidase) activities (26) and re-analyzed for SULT and NAT activities by assays described in the protocols of the Gentest Corp. The results obtained did not differ practically from those found by Gentest Corp. Two of these cytosolic samples (H806 and H856), exhibiting similar XO activities and high NAT (H806) and SULT (H856) activities, were used to study the activation of 2-NA to metabolites binding to DNA.
Covalent binding to DNA
The deaerated and argon-purged incubation mixtures contained, in a final volume of 0.75 ml: 50 mM sodium phosphate (pH 7.4) containing 0.5 mM [3H]2-NA dissolved in methanol (20 µl/0.75 ml incubation), 1 mM hypoxanthine, 1 mg human hepatic cytosolic protein and 1 mg CT-DNA (4 mM dNp). The reaction was initiated by adding [3H]2-NA. Control incubations were carried out either without cytosol, without DNA, without hypoxanthine or without 2-NA. Incubations with buttermilk XO contained 1 U XO instead of the cytosolic fraction. After incubation (37°C, 60120 min), all reaction mixtures were extracted twice with ethyl acetate (2 x 2 ml). DNA was isolated from the residual water phase by the phenol/chloroform extraction method as described earlier (1519,30,31). The 3H radioactivity of modified DNA was determined by liquid scintillation counting (Packard Tri-Carb 2000 CA). The content of DNA was determined spectrophotometrically (33).
32P-post-labeling analysis
The nuclease P1 enrichment version (34) and the 1-butanol extraction-mediated enrichment procedure (35) of the 32P-post-labeling assay were performed as described earlier (36). Labeled DNA digests were separated by two chromatographic methods on polyethylenimine (PEI)cellulose plates. (i) Essentially as described previously (24), except that the D3 solvent was 3.5 M lithium formate, 8.5 M urea (pH 3.5); D4 solvent was 0.8 M lithium chloride, 0.5 M Tris, 8.5 M urea (pH 8.0), followed by a final wash with 1.7 M sodium phosphate (pH 6.0). D2 was omitted (method A). (ii) 32P-labeled adducts were also resolved by the modification described by Reddy et al. (37). This procedure has been shown to be suitable for resolution of benzoquinone-derived adducts (37). The solvents used in this case were: D1, 2.3 M sodium phosphate (pH 5.77); D2 was omitted; D3, 2.7 M lithium formate, 5.1 M urea (pH 3.5); D4, 0.36 M sodium phosphate, 0.23 M TrisHCl, 3.8 M urea (pH 8.0). After D4 development and a brief water wash, the sheets were developed (along D4) in 1.7 M sodium phosphate (pH 6.0) (D5), to the top of the plate, followed by an additional 3040 min development with the TLC tank partially opened, to allow the radioactive impurities to concentrate in a band close to the top edge (method B) (36). Adduct levels were calculated in units of relative adduct labeling (RAL) which is the ratio of c.p.m. of adducted nucleotides to c.p.m. of total nucleotides in the assay.
Preparation of reference compounds and 32P-post-labeling analysis of adducts
Aliquots of 0.3 µmol dAp and 0.3 µmol dGp were incubated with 1 mM 2-NA, 1 U XO and 1 mM hypoxanthine in 50 mM TrisHCl buffer, pH 7.4, at 37°C for 120 min in a total volume of 0.5 ml. Similarly, 0.5 µmol dGp was incubated in 50 mM TrisHCl buffer, pH 7.4, with 20 µmol N-(2-methoxyphenyl)hydroxylamine without further activation at 37°C overnight in a total volume of 0.5 ml. After incubation and extraction by ethyl acetate, 20 µl aliquots were removed and directly used for 32P-post-labeling analysis; the nuclease P1 version of the assay was utilized (34). Control incubations were carried out either without activating system or without deoxynucleotides. Resolution of the adducts on a PEIcellulose TLC plate was carried out by method B.
32P-post-labeling of adducts in DNA from organs of rats treated with 2-NA
Whole rat organs were minced and DNA was isolated by the phenol/chloroform extraction method. 32P-post-labeling analysis was performed as described above. Resolution of DNA adducts on PEIcellulose TLC plate was carried out by method B.
Co-chromatography on PEIcellulose
Adduct spots detected by the 32P-post-labeling assay in incubations with CT-DNA and dGp in vitro and in DNA from treated rats showing similar properties on TLC were excised from the thin layer plates and extracted as described (38). Cut-outs were extracted with two 800 µl portions of 6 M ammonium hydroxide/isopropanol (1:1) for 40 min. The eluent was evaporated in a Speed-Vac centrifuge. For co-chromatographic analyses the extracts were dissolved in water so that equal amounts of radioactivity could be applied for each sample. Development of these adducts was carried out in the D3 and D4 directions using two different solvents systems: (a) D3, 2.7 M lithium formate, 5.1 M urea (pH 3.5) and D4, 0.36 M sodium phosphate, 0.23 M TrisHCl, 3.8 M urea (pH 8.0); (b) D3, 2.7 M lithium formate, 5.1 M urea (pH 3.5) and D4 4 M ammonium hydroxide/isopropanol (1:1).
High performance liquid chromatography (HPLC) analysis of 32P-labeled 3',5'-deoxyribonucleoside bisphosphate adducts
HPLC analysis was performed essentially as described previously (39,40). Individual spots detected by the 32P-post-labeling assay were excised from thin layer plates and extracted (38,41). The dried extracts were redissolved in 100 µl of methanol/phosphate buffer (pH 3.5) 1:1 (v/v). Aliquots (50 µl) were analysed on a phenyl-modified reversed phase column (250 x 4.6 mm, 5 µm Zorbax Phenyl; Säulentechnik Dr Knauer, Berlin, Germany) with a linear gradient of methanol (from 40 to 80% in 45 min) in aqueous 0.5 M sodium phosphate and 0.5 M phosphoric acid (pH 3.5) at a flow rate of 0.9 ml/min. Radioactivity eluting from the column was measured by monitoring Cerenkov radiation with a Berthold LB 506 C-1 flow-through radioactivity monitor (500 µl cell, dwell time 6 s).
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Results |
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Two different enhancement versions of the 32P-post-labeling assay (the nuclease P1 version and enrichment of DNA adducts by 1-butanol extraction) were initially used to analyze 2-NADNA adducts. In addition, two variant multidirectional chromatographic systems were utilized to separate 32P-labeled adducts by TLC: (i) method A is essentially the chromatographic system originally described by Randerath et al. (42) and resolves bulky adducts; (ii) method B is a modification developed by Reddy et al. (37) for smaller more polar adducts, for example those containing only one benzene ring (37,43). This method should be suitable to detect adducts in DNA formed directly by reactive species of 2-NA consisting of one benzene ring [i.e. nitrenium or carbenium ions formed from one of the metabolites of 2-NA, N-(2-methoxyphenyl)hydroxylamine]. We could show that 2-NA-derived DNA adducts detectable by 32P-post-labeling are formed after activation of 2-NA with human hepatic cytosols. The nuclease P1 version of the assay allowed the detection of 2-NA-derived DNA adducts (Figure 1A), while no adducts were visible after 1-butanol extraction. One major and one minor adduct spots were detected when TLC plates were developed using method B (spots 1 and 2 in Figure 1A). Although the exact nature of these adducts has not yet been elucidated, the chromatographic properties of the adducts on TLC indicate that 2-NA metabolites containing only one benzene ring are covalently linked to DNA (37,43,44), because no adducts were detectable using method A, which resolves more hydrophobic bulky DNA adducts (36,42), or by using the butanol extraction procedure, which also extracts predominantly hydrophobic bulky adducts (35). Control incubations performed without hypoxanthine or without cytosolic preparation were free of adduct spots (not shown). The quantitative analyses of adducts revealed that the amount of adducts detectable by 32P-post-labeling was one order of magnitude lower than that detected by methods utilizing [3H]2-NA (Tables I and II).
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During the enzymatic reduction of 2-NA with human hepatic cytosol or XO an N-(2-methoxyphenyl)hydroxylamine metabolite is formed (26). Such a reactive compound is assumed to undergo decomposition to produce the nitrenium ion and might, therefore, be the proximate 2-NA species responsible for modification of DNA. To confirm this hypothesis, N-(2-methoxyphenyl)hydroxylamine was synthesized and reacted with dGp. As shown in Figure 1D, 32P-post-labeling analysis resulted in detection of two dGp adduct spots formed in this reaction, migrating similarly to those generated in DNA or dGp modified by 2-NA activated with XO (Figure 1C and D). To determine whether the adducts are identical to the adducts formed between dGp and N-(2-methoxyphenyl)hydroxylamine (dGp standard), these were analyzed by co-chromatography on PEIcellulose plates. The adduct spots 1 and 2 obtained from DNA modified by 2-NA activated with XO (Figure 1B) and those obtained from the dGp standard (Figure 1D) were excised, extracted from the thin layer plates and analyzed by co-chromatography on PEIcellulose TLC plates in directions D3 and D4 using two different solvent systems. These experiments showed that the 32P-labeled 2-NA adducts were stable under the alkaline extraction conditions used and that both major adducts in DNA formed by 2-NA were indistinguishable from those of the dGp standard [dGp reacted with N-(2-methoxyphenyl)hydroxylamine] (data not shown).
DNA adduct formation by 2-NA in rats detected by 32P-post-labeling
In order to resolve whether 2-NA induces the formation of DNA adducts in vivo, samples of DNA isolated from several organs of Wistar rats treated with a total dose of 0.75 mg/kg body wt 2-NA were analyzed by the 32P-post-labeling assay. The nuclease P1 version of the 32P-post-labeling assay was used to detect and quantify DNA adducts formed in vivo. 2-NA-specific DNA adduct patterns, similar to those found in vitro, were detected in the target organ for the 2-NA carcinogenic effect, the urinary bladder (Figure 1E), and also in liver, kidney and spleen. In contrast, no such DNA adducts were detected in lung, heart and brain. Likewise, DNA samples of control (solvent-treated) rats were free of these adduct spots even after prolonged exposure of the autoradiograms (not shown). Besides these adducts, two additional minor adducts, which were located close to the bottom of the plate, were detected in DNA of most rat organs. These radioactive spots are background spots that were also observed in the DNA samples of lung, heart and brain, in which no 2-NA-derived DNA adducts (spots 1 and 2) were detected, and in DNA of organs from control rats (not shown).
DNA adducts were quantified to compare adduct formation in DNA of individual organs. The levels of adducts were determined by measurement of the adduct count rates and expressed as RAL (Table III). Quantitative analyses revealed the highest level of 2-NA-derived DNA adducts in urinary bladder, followed by liver, kidney and spleen (Table III). Total adduct levels were in the range 0.23.4 adducts/107 nt.
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As a second, independent chromatographic procedure to confirm the identities of the adduct spots we employed reversed phase HPLC analysis (Figure 2). Individual spots were isolated from the PEI plates and subjected to HPLC on a phenyl-modified column eluted with a gradient of methanol in phosphate buffer of high molarity and low pH. These results confirmed the co-chromatography on PEIcellulose TLC plates. As shown in Figure 2, the major adduct in DNA exposed to 2-NA activated with XO in vitro (spot 1 in Figure 1B) eluted with a retention time (r.t.) of 4.05 min (Fig. 2C), corresponding to the r.t. of 4.15 min (Figure 2A) of adduct spot 1 found in the DNA of the urinary bladder of rats treated with 2-NA (Figure 1E). When equal amounts of radioactivity of adduct spots 1 from Figure 1B and Figure 1E were co-injected, a single peak was found (Figure 2B). Spot 2 in DNA isolated from urinary bladder (Figure 1E) produced one peak of radioactivity (r.t. 5.20 min) (Figure 2D), but its HPLC co-chromatography with adduct spot 2 generated by 2-NA activated by XO in vitro was precluded by the low levels of these adducts in DNA.
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Discussion |
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We also investigated the capacity of 2-NA to form DNA adducts in vivo, by the 32P-post-labeling assay. Two DNA adducts generated by activated 2-NA in vitro exhibited the same chromatographic properties as adducts formed by 2-NA in DNA of rats treated i.p. with 2-NA (0.15 mg/kg body wt daily for 5 days). These results demonstrate a genotoxic mechanism of 2-NA carcinogenicity. The dose used was three orders of magnitude lower than that causing malignant neoplasms of the urinary bladder, spleen and liver and transitional cell neoplasms of the kidney in rodents. In those studies, the animals received diets containing 6000 p.p.m. 2-NA (average daily consumption 300 mg/kg body wt) for 6 months (10,11). The 2-NA-derived DNA adducts were identified as deoxyguanosine adducts derived from a reductive metabolite of 2-NA, N-(2-methoxyphenyl)hydroxylamine. The structure of these adducts needs further characterization. The highest level of DNA adducts was found in the target organ of 2-NA carcinogenicity, the urinary bladder, however, DNA adduct formation was also observed in liver, kidney and spleen, but at one order of magnitude lower levels than in the urinary bladder. This is highly consistent with the carcinogenic activity of 2-NA; it causes neoplastic transformation in urinary bladder and, to a lesser extent, in spleen, liver and kidney (10).
In addition, earlier (26) and the present studies document the role of specific human cytosolic enzymes in activation pathways of 2-NA. XO seems to be the principal enzyme responsible for the reductive activation of 2-NA. The role of XO in 2-NA activation was supported by strong correlation coefficients between the levels of XO activities in human hepatic cytosolic samples and the levels of o-anisidine formed by the same samples (26). The reduction of 2-NA by isolated XO mediated the formation of the same two 2-NA metabolites as human hepatic cytosol (26). Using this enzyme, 2-NA was activated to form DNA adducts chromatographically indistinguishable from those generated after its activation with human hepatic cytosol and with those formed in vivo in rats.
In mammals, the liver and intestine have the highest XO activities of all tissues. XO activity, however, can differ substantially between mammalian species, with human organs showing relatively low XO (47,48). The high XO activity of human liver and intestine is largely due to their XO-rich parenchyme cells. The data on lung and kidney tissues are inconclusive as some samples appeared to have some XO activity while other samples tested had none (48,49). However, several recent immunohistochemical studies have shown the presence of XO in most human tissues and provided answers to some of the discrepancies between the studies. These studies identified microvascular endothelial cells from several human tissues as being rich in XO activity (4852). These XO-rich but relatively small subpopulations of cells could account for the extremely low tissue activity found when relatively large pieces of tissue are homogenized for enzymatic activity or blotting (48). Because the XO enzyme is present in blood vessels in many human tissues, 2-NA transported in blood could be effectively reduced to its reactive metabolite N-(2-methoxyphenyl)hydroxylamine, which generates DNA adducts. Higher amounts of the DNA adducts are, however, formed if high levels of the phase II enzymes SULTs are present in the human hepatic cytosol. O-sulfonation of the hydroxylamine results in the formation of a highly reactive electrophilic nitrenium ion after hydrolysis of the sulfate conjugate. In contrast, NAT had no effect on DNA adduct levels.
These results are consistent with the finding that the highest level of 2-NADNA adducts were found in the urinary bladder of rats treated with this carcinogen. N-sulfonyloxyesters of many N-hydroxyarylamine intermediates formed from aromatic nitro hydrocarbons or arylamines in the liver decompose under acidic conditions in the urinary bladder to electrophilic nitrenium ions capable of reacting with DNA to form DNA adducts in this target organ (53).
Human exposure to 2-NA is thought to occur primarily via the respiratory tract. Human bronchial epithelial cells and alveolar macrophages belong to the primary defense system against inhaled compounds and SULT1A1 and SULT1A2 are expressed in these cells (54,55). Therefore, SULT expression in the human respiratory system could contribute significantly to the metabolic activation of 2-NA. Furthermore, the participation of SULTs in conjugation of 2-NA metabolites leading to higher levels of 2-NADNA adducts in the urinary bladder might be important not only for rats (the present paper), but also for humans. However, the precise kinetics of sulfonation of 2-NA metabolites in human tissue await further investigation.
One of the most important results found in our present study is that reductive activation of 2-NA by the human enzymatic system is analogous to that observed in rats. Human hepatic cytosol activated 2-NA to form DNA adducts chromatographically indistinguishable from adducts formed in rats in vivo. Taken together, these results suggest that studies in rats can predict human susceptibility to 2-NA. While 2-NA is carcinogenic for rats (10), its carcinogenicity for humans has not yet been proven. Our results, showing for the first time analogy between the 2-NA activation to species forming DNA adducts by human enzymes and in rats in vivo, strongly suggest a carcinogenic potential of this rodent carcinogen for humans. An increased cancer risk should be taken into account mainly for individuals working in the chemical industry and exposed to 2-NA during production of this chemical in the manufacture of azo dyes.
Finally, our study can form the basis for structural characterization of the 2-NA adducts found in vivo and subsequently for the development of methods to monitor human exposure. To better understand the potential role of 2-NA-derived DNA adducts in the induction of cancer, our results require confirmation by larger animal studies, monitoring the dose-dependent formation and persistence of these DNA adducts in susceptible target tissues after inhalation and oral exposure.
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Acknowledgments |
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