DNA adduct formation by the environmental contaminant 3-nitrobenzanthrone after intratracheal instillation in rats
Version of Record online: 26 APR 2005
Copyright © 2005 Wiley-Liss, Inc.
International Journal of Cancer
Volume 116, Issue 6, pages 833–838, 10 October 2005
How to Cite
Bieler, C. A., Cornelius, M. G., Klein, R., Arlt, V. M., Wiessler, M., Phillips, D. H. and Schmeiser, H. H. (2005), DNA adduct formation by the environmental contaminant 3-nitrobenzanthrone after intratracheal instillation in rats. Int. J. Cancer, 116: 833–838. doi: 10.1002/ijc.21095
- Issue online: 28 JUL 2005
- Version of Record online: 26 APR 2005
- Manuscript Accepted: 26 JAN 2005
- Manuscript Received: 28 OCT 2004
- Baden-Württemberg, BWPLUS. Grant Number: BWR 22009
- Cancer Research UK
- DNA adducts;
- diesel exhaust;
- intratracheal instillation
3-Nitrobenzanthrone (3-NBA) is an environmental pollutant and suspected human carcinogen found in emissions from diesel and gasoline engines and on the surface of ambient air particulate matter; human exposure to 3-NBA is likely to occur primarily via the respiratory tract. In our study female Sprague Dawley rats were treated by intratracheal instillation with a single dose of 0.2 or 2 mg/kg body weight of 3-NBA. Using the butanol enrichment version of the 32P-postlabeling method, DNA adduct formation by 3-NBA 48 hr after intratracheal administration in different organs (lung, pancreas, kidney, urinary bladder, heart, small intestine and liver) and in blood was investigated. The same adduct pattern consisting of up to 5 DNA adduct spots was detected by thin layer chromatography in all tissues and blood and at both doses. Highest total adduct levels were found in lung and pancreas (350 ± 139 and 620 ± 370 adducts per 108 nucleotides for the high dose and 39 ± 18 and 55 ± 34 adducts per 108 nucleotides for the low dose, respectively) followed by kidney, urinary bladder, heart, small intestine and liver. Adduct levels were dose-dependent in all organs (approximately 10-fold difference between doses). It was demonstrated by high performance liquid chromatography (HPLC) that all 5 3-NBA-derived DNA adducts formed in rats after intratracheal instillation are identical to those formed by other routes of application and are, as previously shown, formed from reductive metabolites bound to purine bases. Although total adduct levels in the blood were much lower (41 ± 27 and 9.5 ± 1.9 adducts per 108 nucleotides for the high and low dose, respectively) than those found in the lung, they were related to dose and to the levels found in lung. These results show that uptake of 3-NBA by the lung induces high levels of specific DNA adducts in several organs of the rat and an identical adduct pattern in DNA from blood. Therefore, 3-NBA-DNA adducts present in the blood are useful biomarkers for exposure to 3-NBA and may help to assess the effective biological dose in humans exposed to it. © 2005 Wiley-Liss, Inc.
Epidemiological studies have shown that occupational exposure to diesel exhaust is associated with an increased risk of lung cancer1, 2, 3, 4 and environmental exposure to diesel exhaust may also pose a significant cancer risk to the general population.
Diesel exhaust consists of a gaseous phase together with a particulate phase that contains many absorbed chemicals such as nitropolycyclic aromatic hydrocarbons (nitro-PAHs).5 3-NBA (3-nitro-7H-benz[de]anthracen-7-one; Fig. 1), a member of this class of compounds, was identified in organic extracts of both diesel exhaust and ambient air particulate matter.6, 7, 8 The detection of 3-aminobenzanthrone (3-ABA), a major metabolite of 3-NBA, in urine of smoking and nonsmoking salt mining workers occupationally exposed to diesel exhaust,7 demonstrated that exposure to 3-NBA by diesel emissions can be significant and is detectable. 3-NBA has also been detected in surface soil and rain water, suggesting that it is an ubiquitous environmental contaminant.9, 10, 11 This aromatic nitro ketone is one of the most potent mutagens and a suspected human carcinogen.6, 12, 13 Besides its extremely high mutagenicity in Salmonella typhimurium,6, 14 3-NBA induces micronuclei in mouse and human cells,6, 15, 16, 17 as well as mutations in human cells.15
A large body of evidence in experimental systems suggests that DNA adduct formation is a critical event in the initiation stage of carcinogenesis.18 DNA adduct formation in rats treated either orally or intraperitoneally with 3-NBA has been demonstrated using the 32P-postlabeling assay.19, 20 In these studies the same adduct pattern consisting of multiple 3-NBA-specific DNA adducts was detected in various organs of rats. Since inhalation is the major route by which airborne materials gain access to the body, primary exposure of the lungs of rats to 3-NBA would constitute a better model system. However, experimental inhalation exposure systems, with appropriate generation and characterization of exposure atmospheres, are expensive to acquire and maintain.21 Alternatively, direct instillation of a test material into the lungs via the trachea has been employed in many studies as an alternative exposure procedure to inhalation. Instillation has also certain advantages over inhalation, the foremost being that the actual dose delivered to the lungs of each animal can be defined accurately.
Therefore, in the present study we treated rats by intratracheal instillation with 3-NBA. Using the 32P-postlabeling assay we studied the distribution of 3-NBA-DNA adducts in various organs and in blood to identify 3-NBA-specific adduct patterns, in order to provide a basis for the development of sensitive analytical methods to monitor human exposure to environmental sources containing 3-NBA.
Material and methods
Female Sprague-Dawley rats, so-called “ex-breeders” (300–380 g) were treated with 0.2 or 2 mg/kg body weight of 3-NBA by intratracheal instillation under ether anesthesia (3 rats per dose). 3-NBA was dissolved in tricaprylin at a concentration of 0.4 mg/ml for the low dose group, resulting in a volume of approximately 180 μl applied per rat, whereas the high dose group received a similar volume of a homogeneous suspension of 4 mg/ml 3-NBA in tricaprylin. 3-NBA dissolved in tricaprylin was administered to rats at the bronchial bifurcation by injection through a tracheal cannula. A total of 2 control rats were treated with vehicle only (180 μl of tricaprylin). At 48 hr after administration, the animals were sacrificed and blood was collected immediately by puncture of the vena cava inferior and transferred to Paxgene Blood Collection tubes (PreAnalytiX, Qiagen, Hilden, Germany) for subsequent isolation of DNA. A total of 8 organs (lung, kidney, urinary bladder, pancreas, heart, liver and small intestine) were removed, immediately frozen in liquid nitrogen and stored at −80°C until DNA isolation.
Isolation of total DNA from whole blood was performed with the PAXgene Blood DNA Kit (PreAnalytiX, Germany) according to the instructions of the manufacturer. Briefly, 8.5 ml of whole blood collected in PAXgene Blood DNA Tubes was transferred to the processing tube. The solution was mixed to lyze red and white blood cells, centrifuged and the resulting pellet of nuclei and mitochondria was washed and resuspended. After digestion with protease, DNA was precipitated in isopropanol and dissolved in water. Typical yields from 8.5 ml of rat blood were 100–200 μg of DNA.
DNA from organs was isolated by the Qiagen Genomic DNA Purification Procedure. Briefly, 120 mg of tissue was minced and digested by Proteinase K for 3 hr at 50°C. The digest was applied to a Qiagen Tips 100 column and DNA was isolated following the standard column procedure (Blood & Cell Culture DNA Kit; Qiagen, Germany). DNA was precipitated by isopropanol and dissolved in water.
The butanol enrichment procedure of the 32P-postlabeling assay was performed as described.19, 22 Briefly, 12.5 μg of DNA were digested using micrococcal endonuclease (750 mU/sample; Sigma, Taufkirchen, Germany) and spleen phosphodiesterase (62.5 mU/sample; Calbiochem, Darmstadt, Germany) for 3 hr at 37°C. A total of 2.5 μg of the digest was removed and diluted for determination of normal nucleotides. Adducts were enriched by butanol extraction and labeled with [γ-32P]ATP (100 μCi/sample; MP Biomedicals, Eschwege, Germany) by incubation with T4-polynucleotide kinase (USB, Freiburg, Germany) for 30 min at room temperature. Efficiency of enrichment and excess of ATP were checked with an aliquot of the radiolabeled sample by 1-dimensional thin layer chromatography (TLC) (PEI-cellulose, 50 mM sodium phosphate, 0.28 M ammonium sulfate, pH 6.5). 32P-labeled adduct nucleoside bisphosphates were separated by chromatography on PEI-cellulose sheets (Macherey & Nagel, Dueren, Germany). The following solvents were used: D1, 1 M sodium phosphate pH 6.5; D3, 3.5 M lithium formate, 8.5 M urea, pH 3.5; D4, 0.8 M lithium chloride, 0.5 M Tris, 8.5 M urea, pH 8.0; D5, 1.7 M sodium phosphate, pH 6.0. Electronic autoradiography was performed using instant imager technology (Instant Imager; Canberra Packard, Dreieich, Germany). DNA adduct levels (relative adduct labeling [RAL]) were calculated as cpm adducts per counts per minute (CPM) normal nucleotides.
HPLC analysis of 32P-labeled 3′, 5′-deoxyribonucleoside bisphosphates
Adduct identification by HPLC analysis was essentially performed as published before.20 Briefly, individual adduct spots or origins after D1 detected by the 32P-postlabeling assay were excised from TLC plates, extracted, dried and redissolved in methanol/phosphate buffer (pH 3.5). Aliquots were run on a phenyl-modified reverse-phase column (Phenomenex, Aschaffenburg, Germany Luna 5 μ phenyl-hexyl) with a linear gradient (from 30–55% in 45 min) of methanol in aqueous sodium phosphate (pH 3.5) at a flow rate of 1 ml/min. Radioactivity eluting from the column was detected by monitoring Cerenkov radiation with a Flow Scintillation Analyzer (Packard, Downers Grove, IL).
Preparation of reference compounds
Deoxyadenosine and deoxyguanosine 3′-monophosphates (4 μmol/ml) (Pharmacia Biotech, Freiburg, Germany) or calf thymus DNA (4 μmol dNps/ml) (Roche, Mannheim, Germany) were incubated with 3-NBA (300 μM) activated by xanthine oxidase (1 U/ml) (Sigma, Deisenhofen, Germany) in the presence of hypoxanthine and 20 μl aliquots of the incubation were directly used for the butanol extraction-mediated 32P-postlabeling method as described.23
DNA adduct analysis in rat tissue
DNA adduct formation in various organs (lung, pancreas, kidney, liver, small intestine, urinary bladder and heart) of female Sprague-Dawley rats treated with a single dose of 3-NBA by intratracheal instillation was analyzed employing the butanol enrichment version of the 32P-postlabeling method. Since oral treatment of female Sprague-Dawley rats with a single dose of 3-NBA resulted in DNA adduct formation in several organs, the same rat strain and the same organs except the stomach were chosen for analysis.19 Likewise, because 3-NBA administered as a single dose intraperitoneally induced DNA adducts after 24 hr in the pancreas and the heart, both organs were included in this study.20 Enrichment by butanol extraction has been shown to yield more adduct spots and a better recovery of 3-NBA-DNA adducts than using enrichment by nuclease P1 digestion.19 Because highest adduct levels were reached in lung and white blood cells 48 hr after intratracheal exposure of rats with benzo[a]pyrene, the same lag time was used.24 At 48 hr after administration both doses induced the same 3-NBA specific DNA adduct pattern in all organs investigated (Fig. 2). The adduct pattern observed was the same as that found in organs of rats treated with 3-NBA orally or intraperitoneally.19, 20 No adduct spots were detected in DNA isolated from organs of rats treated with the vehicle (tricaprylin) alone. The specific adduct pattern essentially consisted of 5 adduct spots, with spots 1, 3 and 4 being the major adducts. As shown previously, adducts 1 and 2 are derived from deoxyadenosine, whereas spots 3, 4 and 5 are formed from reaction of activated 3-NBA with deoxyguanosine.19, 20
As shown in Table I the highest total DNA binding was found in pancreatic tissue at both doses. In addition, high levels of 3-NBA-derived DNA adducts were also found in the lung, followed by kidney, urinary bladder, heart, small intestine and liver. Due to the bronchial bifurcation the distribution of material administered intratracheally may not be even for both lobes of the lung, which were therefore analyzed individually. Indeed, a 2- to 3-fold difference in adduct levels between lobes was observed. For the low-dose group total adduct levels ranged from 12 ± 1.6 adducts per 108 nucleotides (liver) up to 39 ± 18 adducts per 108 nucleotides in lung or 55 ± 34 per 108 nucleotides in DNA from pancreatic tissue. In all organs examined, spot 3 was the predominant DNA adduct found, accounting for approximately 40% of total DNA binding followed by spots 4 (∼25%), 1 (∼15%), 2 (∼10%) and 5 (∼10%) (Table I). Overall the increase in dose by a factor of 10 resulted in a 5- to 10-fold increase in total adduct levels in the respective organs.
|Dose||Organ||Spot 1||Spot 2||Spot 3||Spot 4||Spot 5||Total RAL|
|0.2 mg/kg body weight||Lung lobe A||6.1 ± 2.0||4.2 ± 4.2||15.6 ± 10.4||9.4 ± 2.6||3.5 ± 1.2||38.7 ± 17.7|
|Lung lobe B||2.9 ± 1.8||1.7 ± 0.9||5.3 ± 2.5||2.6 ± 1.5||1.7 ± 1.5||14.2 ± 7.9|
|Pancreas||11.3 ± 7.9||3.6 ± 1.3||21.0 ± 11.9||14.2 ± 9.7||5.4 ± 4.5||55.4 ± 33.9|
|Kidney||5.9 ± 1.7||3.4 ± 0.8||13.8 ± 0.4||7.5 ± 2.0||2.4 ± 0.3||33.0 ± 2.6|
|Urinary bladder||4.1 ± 1.4||3.7 ± 1.5||12.3 ± 1.6||6.5 ± 3.3||1.3 ± 0.2||27.9 ± 4.9|
|Heart||6.3 ± 2.6||2.4 ± 0.3||10.3 ± 1.3||4.9 ± 2.8||2.0 ± 0.5||26.0 ± 3.4|
|Small intestine||3.3 ± 1.0||2.0 ± 0.2||8.7 ± 0.7||3.8 ± 0.6||1.9 ± 0.6||19.2 ± 1.6|
|Liver||1.7 ± 0.4||2.0 ± 0.5||5.9 ± 0.5||1.8 ± 0.6||0.8 ± 0.3||12.1 ± 1.6|
|Blood||2.1 ± 1.0||2.5 ± 1.2||4.6 ± 2.0||1.8 ± 1.4||ND2||9.5 ± 1.9|
|2 mg/kg body weight||Lung lobe A||38.0 ± 6.2||41.0 ± 26.5||134.9 ± 48.9||118.5 ± 59.7||17.2 ± 2.4||349.6 ± 138.5|
|Lung lobe B||22.2 ± 0.6||26.5 ± 10.8||60.0 ± 18.0||57.9 ± 25.6||8.7 ± 2.7||175.3 ± 49.0|
|Pancreas||82.6 ± 41.7||83.8 ± 48.6||271.6 ± 145.1||278.5 ± 201.2||39.8 ± 31.5||619.9 ± 372.2|
|Kidney||39.2 ± 9.1||38.0 ± 9.2||141.9 ± 29.2||114.8 ± 37.6||15.6 ± 5.5||334.8 ± 50.6|
|Urinary bladder||26.2 ± 8.1||20.3 ± 6.9||84.4 ± 19.4||72.2 ± 19.5||12.2 ± 6.6||215.2 ± 56.4|
|Heart||35.1 ± 16.7||25.6 ± 13.6||89.5 ± 41.3||53.6 ± 26.9||16.2 ± 7.5||220.0 ± 101.1|
|Small intestine||9.2 ± 2.7||12.9 ± 3.5||38.2 ± 2.7||34.1 ± 1.8||3.7 ± 0.6||98.1 ± 5.7|
|Liver||5.0 ± 2.4||6.1 ± 3.8||28.8 ± 15.8||16.8 ± 8.4||2.5 ± 1.7||59.1 ± 31.1|
|Blood||6.3 ± 3.5||10.6 ± 12.0||18.6 ± 11.9||5.2 ± 4.3||ND||41.2 ± 26.9|
HPLC analyses of 3-NBA-DNA adduct spots from rats
To confirm the identity of adducts detected in DNA from organs of rats treated with 3-NBA by intratracheal instillation, HPLC analysis was performed as a second, independent chromatographic system. As illustrated in Figure 3, all 5 DNA adducts derived from 3-NBA were detected under conditions described earlier.20 The retention times (r.t.) of individual adducts were identical to those received using synthetic adduct standards, obtained from incubations of 3-NBA with nucleoside 3′-phosphates under reductive activation by xanthine oxidase.19 Adducts 1 (r.t 40.5 min) and 2 (r.t. 32.5 min) eluted with retention times identical to those derived from incubation of 3-NBA with deoxyadenosine 3′-phosphate. Likewise, spots 3 (r.t. 23.0 min), 4 (r.t. 27.0 min) and spot 5 (r.t. 38.5 min) coeluted with reference compounds prepared from deoxyguanosine 3′-phosphate. When equal amounts of radioactivity of adduct spots found in vivo and the corresponding purine adduct spots obtained in vitro were mixed prior to analysis a single peak was found (data not shown).
DNA adduct analysis of blood from rats treated intratracheally with 3-NBA
Using the butanol enrichment procedure of the 32P-postlabeling method the same 3-NBA-specific adduct pattern as found in tissues was observed in DNA isolated from whole blood of treated rats (Fig. 4). However, adduct spot 5, which has the lowest abundance in other tissues, was not detected. Blood DNA isolated from rats treated with vehicle showed no adduct spots in the region of interest (Fig. 4c). It is clear from Table I and Figure 4 that 48 hr after intratracheal application of 3-NBA total adduct levels in blood were only 10% (for the high dose) and 25% (for the low dose) of those found in the lung but they were clearly detectable and dose-dependent. Overall 3-NBA-DNA adduct levels in blood were related with those found in lung. Therefore blood can be applied as surrogate source of DNA for intratracheal application of 3-NBA.
Although no definite correlation has been established between exposure to nitro-PAHs and the onset of human cancer, some evidence suggests that engine exhausts, which contain significant amounts of these compounds, may be carcinogenic to humans.1, 4 The aromatic nitroketone 3-NBA, an extremely potent direct-acting mutagen and suspected human carcinogen, has been identified as an air pollutant, diesel emissions being the predominant source.6 As a consequence, 3-NBA like other nitro-PAHs is widely distributed in the environment in complex mixtures and human exposure to 3-NBA is thought to occur primarily via the respiratory tract.25, 26 Thus, it is important to study the effect of 3-NBA following administration into the lungs.
Previously we and others demonstrated by the 32P-postlabeling technique that 3-NBA forms multiple DNA adducts after metabolic activation in vitro and in cells.14, 17, 19, 23, 27, 28, 29, 30 High levels of DNA adduct levels were detected by the same technique in various organs of rats treated by gavage or intraperitoneal injection with 3-NBA.19, 20 Similarly, 3-NBA-DNA adducts were observed in vivo in mice.13 In all cases the observed adduct pattern was essentially the same, consisting of 5 major spots when analyzed by the butanol enrichment version. These findings prompted us to determine the capability of 3-NBA to form DNA adducts in vivo in rats after intratracheal instillation and whether 3-NBA-specific adduct patterns in blood can serve as biomarkers for exposure to environmental sources containing 3-NBA.
In our study we demonstrated that 3-NBA forms DNA adducts in several organs from rats treated intratracheally with a single dose of 3-NBA. The observed adduct pattern was the same in all organs and qualitatively similar to those obtained from in vitro reactions indicating that no adducts other than those already detected in vitro were detectable in vivo. Due to the intratracheal administration high adduct levels were detected in the lung (Table I), but even higher adduct formation was found in the pancreas, followed by lower levels in kidney, heart, urinary bladder, small intestine and liver, suggesting that 3-NBA or its metabolites are distributed via the blood stream to other organs.
When DNA adduct formation in rats exposed via different modes by a single dose of 2 mg/kg body weight of 3-NBA was compared, the same adduct profile was found in all organs independent of the exposure route. Another study that has shown that adduct profiles are independent of route of administration is that of Godschalk et al.,24 in which adduct formation by benzo[a]pyrene in rats was studied. 3-NBA-DNA adduct levels were substantially higher 48 hr after intratracheal application in lung, kidney, liver and pancreas than levels induced intraperitoneally after 24 hr or orally after 4 hr.19, 20 Even in the small intestine, where 3-NBA-adduct levels were highest after oral treatment, intratracheal administration resulted in comparable adduct levels, indicating that levels depend on the exposure route and that application of 3-NBA via the lungs is more effective in adduct formation than other exposure routes not only in the target organ but in most organs. Thus, exposure to 3-NBA by the respiratory tract may represent the most important route of exposure in lung carcinogenesis.
The much higher 3-NBA-DNA adduct level found in the lung compared to the liver and kidney is consistent with a report by Mitchell31 in which intratracheal instillation of 1-nitropyrene to mice resulted in covalent DNA binding to these 3 organs with the highest level occurring in the lung.
The highest DNA binding for 3-NBA when administered either intratracheally to female Sprague Dawley rats or intraperitoneally to female Wistar rats20 was observed in the pancreas. In this context, it is interesting to note that exposure to nitro-PAHs is thought to be a possible risk factor for pancreatic cancer in humans and that the pancreas exhibits high nitroreductase activity.32
Using two independent chromatographic systems (TLC and HPLC), we showed clearly that adducts 1 and 2 are derived from reaction with deoxyadenosine whereas adducts 3, 4 and 5 are derived from deoxyguanosine (Fig. 3). Although it has been established that these major 3-NBA-derived DNA adducts formed in vivo are products derived from reductive metabolites bound to purine bases without carrying an N-acetyl group,14 the definitive structure of these DNA adducts remains to be characterized. It is interesting to point out that irrespective of the exposure route and the tissue analyzed 3-NBA-DNA adduct profiles in rats were remarkably similar. We therefore assume that these tissues have the metabolic capacity to nitroreduce 3-NBA and, more importantly, that the same reactive species leading to adduct formation are formed.
Previous work in our laboratory indicated that N-hydroxy-3-aminobenzanthrone is the critical intermediate in 3-NBA-derived DNA adduct formation in vivo and in vitro.13, 17, 19, 20, 33, 34 In mammalian cells, both cytosolic and microsomal subfractions contain enzymes that catalyze the reduction of nitroaromatic compounds.20, 28, 29, 35, 36 In rat and human hepatic microsomes we have already identified NADPH:cytochrome P450 reductase as the enzyme activating 3-NBA, generating 3-NBA-DNA adduct profiles identical to those found in liver tissue of 3-NBA-treated rodents.13, 19, 20, 33 We also found that 3-NBA is activated by NAD(P)H:quinone oxidoreductase (NQO1) in rat and human hepatic cytosols.37 Using genetically engineered V79 cells expressing human N,O-acetyltransferase (NAT) NAT1, NAT2, sulfotransferase (SULT) SULT1A1 or SULT1A2, we showed previously that these enzymes strongly contribute to the metabolic activation of 3-NBA.14, 30 NADPH:cytochrome P450 reductase, NQO1, NATs and SULTs are expressed in the respiratory tract,38, 39 suggesting that all these enzymes may contribute to the metabolic activation of 3-NBA. More importantly, the results of the present study support the conclusion that some or all of the detected 3-NBA-DNA adducts represent premutagenic lesions involved in mutagenesis and/or carcinogenesis. Indeed, preliminary data suggest that 3-NBA is carcinogenic in F344 rats after intratracheal administration of 3-NBA.12 In preliminary findings on moribund rats, the authors describe squamous metaplasia from trachea to bronchial epithelium. In lung parenchyma, advanced squamous metaplasia from bronchiole to the alveolar region was observed. Moreover, recent data indicate that G:C to T:A transversion mutations induced by 3-NBA in the transgenic MutaMouse assay after intraperitoneal treatment with 3-NBA are caused by misreplication of adducted guanine residues through incorporation of adenine opposite the adduct (“A”-rule).13
In general, DNA adducts are useful biomarkers for detecting exposures to genotoxic compounds, such as those in diesel emissions.40, 41 Several human biomonitoring studies using the detection of DNA adducts in peripheral blood cells have reported higher levels of bulky DNA adducts detected by 32P-postlabeling among subjects heavily exposed to diesel exhaust.42, 43 This may be predictive of cancer risk. After intratracheal treatment of rats with 3-NBA an overall relationship was observed between DNA adducts in blood and the lung. However, adduct levels in blood were significantly lower as compared with the lung tissue, which suggests that at low 3-NBA exposures, adducts in blood might be below the detection limit of the 32P-postlabeling assay, while adducts may still be detectable in lung.
In summary, a single intratracheal administration of 3-NBA to rats resulted in the formation of a specific adduct pattern in each of the seven organs under investigation and in blood. These TLC 32P-fingerprints consisted of multiple adducts chromatographically identical to those obtained in vitro via nitroreduction clearly indicating the importance of the nitroreduction pathway in the bioactivation of 3-NBA. Our study can form the basis for development of methods to monitor human exposure. To better understand the potential role of 3-NBA-DNA adducts in induction of mutations and cancer, our results will require confirmation in animal studies monitoring the dose-dependent formation and persistence of 3-NBA-DNA adducts in susceptible target tissues and in blood after intratracheal exposure.
- 1IARC. Monographs on the evaluation of carcinogenic risks to humans. Diesel and gasoline engine exhausts and some nitroarenes. vol. 46. Lyon: IARC, 1989.
- 4U.S. Environmental Protection Agency. Health assessment document for diesel engine exhaust, EPA/600/8-90/057F, 2002.
- 12Carcinogenicity of 3-nitrobenzanthrone, a potent mutagen in diesel exhaust-preliminary results in F344 rats after intratracheal administration. In: HeinrichU, MohrU, eds. Relationships between respiratory disease and exposure to air pollution. Washington, DC: ILSI Press, 2000; 315–9., , , , .
- 343-Aminobenzanthrone, a human metabolite of the environmental pollutant 3-nitrobenzanthrone, forms DNA adducts after metabolic activation by human and rat liver microsomes: evidence for activation by cytochrome P450 1A1 and P450 1A2. Chem Res Toxicol 2004; 17: 1092–1101., , , , , .
- 37The environmental pollutant and potent mutagen 3-nitrobenzanthrone forms DNA adducts after reduction by NAD(P)H:quinone oxidoreductase and conjugation by acetyltransferases and sulfotransferases in human hepatic cytosols. Cancer Res 2005; 65: 2644–52., , , , , , , , , , .