DNA adduct and tumor formations in rats after intratracheal administration of the urban air pollutant 3-nitrobenzanthrone
Eszter Nagy,
Magnus Zeisig,
Ken Kawamura 1,
Yoshiharu Hisamatsu 2,
Akiko Sugeta 3,
Shuichi Adachi 3 and
Lennart Möller *
Department for Biosciences at Novum, Karolinska Institutet, SE-141 57 Huddinge, Stockholm, Sweden, 1 Department of Pathology, Kagawa Nutrition University, Sakado, Saitama 350-0288, Japan, 2 Department of Community Environmental Science, National Institute of Public Health, Shirokanedai, Tokyo 108-8638, Japan and 3 Department of Public Health, Sagami Women's University, Sagamihara, Kanagawa 228-8533, Japan
* To whom correspondence should be addressed. Tel: +46 8 608 9233; Fax: +46 8 774 6833; Email: lennart.moller{at}biosci.ki.se
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Abstract
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3-Nitrobenzanthrone (3-NBA) has been isolated from diesel exhaust and airborne particles and identified as a potent direct-acting mutagen in vitro and genotoxic agent in vivo. In order to evaluate the in vivo toxicity and carcinogenicity of 3-NBA in a situation corresponding to inhalation, a combined short-term and lifetime study with intratracheal (i.t.) instillation in female F344 rats was performed. DNA adduct formation, as a marker for the primary effect and analyzed by 32P-HPLC after single instillation, showed a few major DNA adducts and a rapid increase with a peak after 2 days, followed by a decline. No DNA adducts above the background level were observed after 16 days. The highest DNA adduct formation was observed in lung [
250 DNA adducts/108 normal nucleotides (NN)] closely followed by kidney (
200 DNA adducts/108 NN), whereas liver contained only 12% (
30 DNA adducts/108 NN) of the levels of DNA adducts found in lung. In the tumor study, squamous cell carcinomas were found after 79 months in the high-dose group (total dose of 2.5 mg 3-NBA) and after 1012 months in the low-dose group (total dose of 1.5 mg 3-NBA). The fraction of squamous cell carcinoma out of the total amount of tumors observed at the end of experiment at 18 months, corresponded to 3/16 and 11/16 in the low- and high-dose group, respectively. A single case of adenocarcinoma was also observed in each group. In the control group, no tumors were observed during the entire study of 18 months. In addition, a few cases of squamous metaplasia were also observed in the lung in both dose groups but not in the controls. In conclusion, 3-NBA forms DNA adducts in the lung immediately after i.t. administration but almost all DNA adducts were eliminated after 16 days. Tumor formation in two dose groups was observed in a dose-dependent manner with squamous cell carcinomas as the predominant tumor type at high exposure.
Abbreviations: 3-ABA, 3-aminobenzanthrone; AUC, area under curve; B[a]f, benzo[a]pyrene; dG-C8-C2-ABA, 8-(3-amino-7H-benz[de]anthracen-7-one-2-yl)-2'-deoxyguanosine; dG-C8-N-ABA, 8-(3-amino-7H-benz[de]anthracen-7-one-N-yl)-2'-deoxyguanosine; dG-N2-C2-ABA, N2-(3-amino-7H-benz[de]anthracen-7-one-2-yl)-2'-deoxyguanosie; i.p., intraperitoneal; i.t., intratracheal; NATs, N,O-acetyltransferases; 3-NBA, 3-nitrobenzanthrone; NuP1 Nuclease P1; SULTs, sulfotransferases; TBA, tetrabutyl ammonium chloride; XMEs, xenobiotic metabolizing enzymes
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Introduction
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3-Nitrobenzanthrone (3-NBA) has been isolated from diesel exhaust and airborne particles and characterized both as a potent direct-acting mutagen in bacterial mutagenicity tests and as an inducer of micronuclei in mouse peripheral reticulocytes (11
). Both in vitro and in vivo studies suggest that 3-NBA reacts with DNA through reduction of the nitro-group as a first step, then further metabolized by involvement of both phase I and phase II enzymes, which gives rise to a variety of 3-NBA derived DNA adducts (Figure 1) (27). When female SpragueDawley rats were orally administered 3-NBA, high levels of 3-NBA-specific DNA adducts were detectable in all gastrointestinal tissues analyzed (4). The results from this and other studies, suggest that 3-NBA is genotoxic to mammals and that the main targets of reactive intermediates are the purine bases (
70% dG and
30% dA) (4,8). Repeated intraperitoneal (i.p.) treatment of transgenic mice (Muta Mouse) with 3-NBA has been shown to result in a variety of mutations, suggesting that the observed G:C
T:A transversions are most probably caused by misreplication of adducted guanine residues through incorporation of adenine opposite the adduct, referred to as the A-rule (8). In addition to the expression of different xenobiotic metabolizing enzymes (XMEs), polymorphism of these genes has also been focused on, since polymorphism may affect protein expression, stability and catalytic activity of XMEs (9). This assumption may be true for 3-NBA as well, since a variety of XMEs that are known to take part in the activation of 3-NBA and its metabolites are also polymorphic (5,7,10,11). However, the effects of polymorphism on 3-NBA activation have to be more closely investigated.

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Fig. 1. The hypothesized metabolic pathway of 3-NBA through nitro-reduction as partly suggested by Arlt et al. (8). Through the reaction with DNA, primarily dG, three non-acetylated DNA adducts are thought to form. The first, (Adduct 1) through a C8/C2 coupling between the purine and adducted moiety, the second, (Adduct 2) through a C8/N coupling and the third possible adduct would form through the attachment of the adducted moiety to the exocyclic amino group on the purine, N2/C2 coupling.
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Since 3-NBA is mainly present in diesel exhaust, the aim of this study was to investigate the in vivo genotoxicity and carcinogenicity of 3-NBA corresponding to inhalation. Inhalation experiments are the best way to extrapolate the effects of inhaled gas, mist, dust or chemicals, but require not only considerable amounts of material, but also a safe and well-controlled system for exposure and maintenance of animals. Therefore, intratracheal (i.t.) administration was used as a model for inhalation and as a way to observe the effect of deposited materials from trachea to alveoli.
Furthermore, the main DNA adducts found in vivo were characterized by using synthesized 3'-monophosphate standards, 8-(3-amino-7H-benz[de]anthracen-7-one-2-yl)-2'-deoxyguanosine (dG-C8-C2-ABA), 8-(3-amino-7H-benz[de]anthracen-7-one-N-yl)-2'-deoxyguanosine (dG-C8-N-ABA) and N2-(3-amino-7H-benz[de]anthracen-7-one-2-yl)-2'-deoxyguanosine (dG-N2-C2-ABA) by cochromatography, using a 32P-HPLC plateau method, to increase separation.
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Material and methods
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Reagents and chemicals
Reagents and enzymes used were purchased from the following sources: RNAse A and Spleen Phosphodiestrase (SPD; Boehringer Mannheim GmbG, Mannheim, Germany), RNAse T1 (from Aspergillus oryzae) and Micrococcal Nuclease (MN; Sigma Chemical, Cleveland, OH, USA), adenosine 5'-[
-32P] triphosphate ([32P]ATP, 32P-activity of 3000 Ci/mmol; Amersham International, Little Chalfont, UK), Dithiothreitol (DTT; Merck, Darmstadt, Germany) and Polynucleotide Kinase (PNK; USB, Cleveland, OH, USA). All chemicals were of analytical grade.
3-NBA was synthesized and kindly provided by Prof. Hitomi Suzuki (Department of Chemistry, Kyoto University, Kyoto, Japan) and Dr Takeji Takamura Enya (Cancer Prevention Division, National Cancer Center, Tsukiji, Tokyo, Japan).
Warning: The substances 3-NBA, phenol and [32P]ATP that are used in the experimental procedures are extremely hazardous and proper precautions and guidelines should be followed when handling and discarding the chemicals.
Short-term study for DNA adduct analysis
Six-week-old female F344 rats (
100 g), purchased from Nihon SLC (Shizuoka, Japan), were i.t. administered with 1 mg [10 mg/kg body weight (body wt)] of 3-NBA dissolved in 0.1 ml propylene glycol:saline (1:9). The animals were killed at 6 h, 1, 2, 3, 5, 10, 16 days and 1, 2, 3 months after administration (n = 3 for each time point, except 2 months where n = 2). Control animals (n = 5) received a single i.t. administration of 0.1 ml vehicle. Lung, liver and kidney were removed and the tissues were immediately frozen at 80°C until analysis. The animal experiment was performed at the Animal Experiment Center at Saitama Medical University (Saitama, Japan) and the tissues were air transported on dry ice to Karolinska Institutet, Stockholm, Sweden, for DNA adduct analysis. Ethical permission, No. 000135, was provided by the Animal Research Committee of Saitama Medical University in Japan.
DNA adduct analysis
The frozen tissues were DNA extracted by a standard phenolchloroform methodology as previously described (12). For DNA adduct analysis, 10 µg aliquots of DNA were dissolved in Millipore water and digested by 4 µl micrococcal nuclease (0.2 U/µl) and SPD (1 mU/µl) with a total incubation time of 4 h at 37°C.
The hydrolyzed DNA and nucleotide samples were adduct-enriched by butanol extraction. Owing to suspected nuclease P1 (NuP1) sensitive dG-C8-bound DNA adducts, butanol enrichment was chosen before NuP1 treatment. Since characterization was performed on the DNA adducts, comparison between the butanol and NuP1 enrichment was not considered necessary. The phase transfer agent 10 mM tetrabutyl ammonium chloride (TBA), together with 100 mM ammonium formate buffer (pH 3.5), facilitated the transfer of lipophilic adducts to the butanol phase, whereas unmodified hydrophilic nucleotides remained in the water phase. The organic phase was then evaporated to dryness.
The butanol extracted samples were redissolved in water. A mixture of 0.25 µl 400 mM PNK buffer, 0.5 µl T4-PNK enzyme (0.5 U/µg DNA) and 1.8 µl (0.06 pmol/µg DNA) [32P]ATP was added and the samples were incubated 30 min at 37°C. After incubation the samples were stored at 20°C until analysis.
DNA adduct analysis was performed on a 32P-HPLC system, which consisted of a Waters 600 E pump (Waters, Milford, MA, USA), a Hichrom, RP 5-C18, K-100 guard column (Hichrom Reading, UK), two serial reverse-phase DeltaPak C18 150 mm x 3.9 mm i.d., 5 µm 100 A main columns (Waters, Milford, MA, USA), a Packard 500 TR flow scintillation detector (Packard Instrument Meriden, CT, USA). Labeled samples were diluted with
150 µl of Milli-Q water immediately before injection into the 32P-HPLC system. A linear gradient of 040% of 87.5% acetonitrile and 12.5% water, during 070 min in 2 M ammonium formate (pH 4.5) was used eluting at a rate of 0.5 ml/min. Most of the polar compounds in the sample were separated by the guard column and removed by a switch valve, which was opened for 1 min after injection. The limit of detection for the 32P-post-labeling method ranges between 0.01 and 1 fmol, depending on the amount of DNA used, which is sufficient for this type of analysis (13).
Characterization of DNA adducts in vivo
The standards, dG-C8-C2-ABA, Std 1, dG-C8-N-ABA, Std 2 and dG-N2-C2-ABA, Std 3 (Figure 4), were kindly donated by Dr Takeji Takamura Enya, Cancer Prevention Division, National Cancer Center Research Institute, Tsukiji, Tokyo, Japan. Using a plateau method, the standards were analyzed as single substances, mixed, and both with and without tissue samples (liver, lung and kidney).
The plateau method for the analysis of standards with selected tissues with a flow rate of 0.5 ml/min was used as follows: 019% of 87.5% acetonitrile: water, during 033 min in 2 M ammonium formate and 0.4 M formic acid (pH 4.5). A plateau followed holding this mixture for 15 min. Then the amount of 87.5% acetonitrile:water was increased to 40% during 4890 min.
Most of the polar compounds in the samples were separated by the guard column and removed by a switch valve, which was opened for 1 min after injection. The method is described in detail elsewhere (14).
Long-term in vivo study for carcinogenicity assay
Four-week-old F344 rats (female n = 79) were purchased from Nihon SLC (Shizuoka, Japan) and were divided into three groups after a 1 week adaptation period in the laboratory. 100 µl of 3-NBA suspension (5 mg/ml) in 10% propylene glycolsaline was administered (i.t.) to the rats with 0.5 ml of air under ether anesthesia, once a week. The average weight of the rats during the administration period was 99 and 112 g for the low- and high-dose groups, respectively. The total dose of the low dose group was 1.5 mg (15 mg/kg body wt, n = 21) and for the high dose group it was 2.5 mg (22 mg/kg body wt, n = 33). Control rats (n = 25) were administered vehicle alone (100 µl) similar to the highest dose group.
Moribund rats were killed by the exanguination of postcaval vein after i.p. injection of sodium pentobarbital. Organs and tissues were fixed in 10% neutral formalin. Deceased rats were autopsied and the tissues were treated as above. The respiratory area was divided into trachea/bronchus and lung and the lesions examined were classified as papilloma, squamous metaplasia, squamous cell carcinoma and adenocarcinoma. Formalin-fixed organs were prepared for histopathological examinations using established methods.
Statistical analysis
DNA adduct levels were calculated as the average from all animals in a dose or control group killed at a certain time point with single analysis of each tissue from each animal. Statistical analysis of the DNA adduct formation was performed by comparing the area under curve (AUC) for the different tissues (liver, lung and kidney) over a time range of 6 h to 16 days. To evaluate statistical significance an unpaired, two-tailed Student's t-test with unequal variances was performed.
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Results
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DNA adducts
Tissues obtained after 16 days were excluded from the statistical analysis since no DNA adducts above the control values were seen (data not shown). In the tissues, liver, lung and kidney, that were analyzed for DNA adducts, four major DNA adducts were detected (Figure 2A). The shaded peaks in Figure 2A represent the dominating DNA adducts from 3-NBA exposure. The major peak, consisting of two DNA adducts aligning very closely, was varying between 50 and 90% of the total DNA adduct levels in all three tissues (data not shown). There were other minor DNA adducts present, which were similar to the normal background level of DNA adducts and occurred in untreated animals as well (Figure 2B), and therefore not considered 3-NBA-related.

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Fig. 2. Representative 32P-HPLC chromatograms of DNA adduct formation in vivo in lung, kidney and liver of female Fischer 344 rats instilled (i.t.) with 1 mg (10 mg/kg body wt) of 3-NBA (A) and vehicle alone (B). The shaded peaks represent the most dominant DNA adducts in the different tissues. The major peaks consist of two fairly unresolved DNA adducts.
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The total DNA adduct level (sum of DNA adducts between 50 and 70 min) in the three different tissues showed that there was a rapid formation of DNA lesions (Figure 3). In the lung, which was the site of administration, DNA adducts were present at high levels already 6 h after exposure. The kidney showed a similar rapid response. No statistically significant differences were found between the lung and the kidney. Both lung and kidney were significantly higher (P < 0.05) in DNA adduct formation when compared with liver. All the tissues reached a maximum of DNA adduct formation around 2 days after a single administration (Figure 3).

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Fig. 3. Comparison of DNA adduct formation in the lung, kidney and liver of female Fischer 344 rats over time after a single instillation (i.t.) of 1 mg (10 mg/kg body wt) of 3-NBA. Each point is the mean value ± SD of three animals. There was a statistically significant difference between the liver versus lung and kidney (P < 0.05 based on AUC with all values included). The amounts depicted at time 0 are the controls with a mean value ± SD of five animals.
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The sum of DNA adducts in the control tissues was on average 12 ± 6, 8 ± 3 and 11 ± 5 per 108 NN for the liver, lung and kidney, respectively. Significantly higher levels of DNA adducts (P < 0.05) of the treated livers were observed only on days 1 and 2 when compared with the control livers (data not shown).
Characterization of DNA adducts in vivo
The synthsesized standards used for characterization of DNA adducts in vivo are shown in Figure 4 with their corresponding 32P-HPLC chromatograms. The peak corresponding to dG-C8-C2-ABA had a retention time of 55 min and did not align with any of the in vivo adducts found in any of the samples. The standards dG-C8-N-ABA at
65 and dG-N2-C2-ABA at 66 min, respectively, did align with the largest peaks found in the in vivo samples (Figure 5). With the plateau method there were >10 different DNA adduct peaks detected that were most probably formed from 3-NBA in vivo (data not shown). The DNA adducts varied in intensities but were outside the retention time window of the standard characterization.

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Fig. 4. Structure of the three synthesized 3-NBA derived DNA adduct standards and their respective 32P-HPLC chromatograms analyzed by the plateau method. Adduct 1 (dG-C8-C2-ABA) has a retention time of 55 min, whereas adducts 2 (dG-C8-N-ABA) and 3 (dG-N2-C2-ABA) have a retention time of 65 and 66 min, respectively.
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Fig. 5. Representative 32P-HPLC chromatograms of the synthesized 3-NBA derived DNA adduct standards and the in vivo DNA adducts from lung, kidney and liver analyzed by the plateau method. Characterization has shown that the synthesized DNA adducts labeled 2 and 3 align well with the peaks seen in vivo, whereas DNA adduct 1 is not found in the in vivo samples.
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Carcinogenicity
Owing to the increased number of animals killed in the high-dose group, it was decided to terminate the tumor study at 18 months. The pathological findings observed during 18 months after the first administration (i.t.) are presented in Table I. None of the animals in the control group developed any kind of tracheal or lung lesion of pretumor or tumor character.
In the lower dose group (1.5 mg), the first cases of tissue damage, in the form of squamous metaplasia, were observed in the trachea as well as lung of three moribund rats, autopsied within 9 months after administration. Within 12 months, besides the occurrence of metaplasia in the trachea and lung of autopsied rats, squamous cell carcinoma and adenocarcinoma were also observed. After 18 months, the remaining rats were killed and two additional cases of squamous cell carcinoma were found.
In the highest dose group (2.5 mg) three moribund rats were autopsied within 9 months after administration of 3-NBA and two cases of squamous metaplasia as well as three cases of squamous cell carcinoma were observed. Later, additional cases of squamous cell carcinoma and adenocarcinoma were found 12 months after administration. At the end of experiment (18 months) the remaining 19 animals were killed and an additional six cases of squamous cell carcinoma were found. So the high-dose group developed lung lesions at an earlier stage when compared with the low-dose group (Figure 6).

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Fig. 6. Formation of squamous cell carcinoma in the lung of female Fischer 344 rats as a function of time and total dose of 1.5 and 2.5 mg of 3-NBA. The diagram depicts the accumulative formation of squamous cell carcinoma in a dose-dependant manner with no lesions present in the controls throughout the entire study period of 18 months.
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Additional lesions observed were severe alveolitis with hemorrhage, and exudation of alveolar fluids was found in some of the deceased rats. No other changes were seen in organs and tissues, except for the respiratory system. In the trachea and bronchus, the loss of ciliated cells and metaplasic changes were observed.
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Discussion
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DNA adducts
In a previous study with rats orally administered 3-NBA, the highest levels of DNA adducts were observed in the GI-tract (4). In this study, the highest levels of 3-NBA derived DNA adducts were found in the lung followed by the kidney and a minor amount in the liver. High levels in the lung could be expected owing to the route of administration. High levels in the kidney, rather than in liver, suggests that excretion could mainly occur through the kidneys and only to a limited extent via the liver/bile. In addition, the concentration of metabolites in the kidneys before excretion, and further metabolism on site, could also partly add to the high levels of DNA adducts seen in the organ.
The major DNA adduct formation pathway of 3-NBA is through the reduction of the nitro function. It has been suggested that there would occur a complete reduction of 3-NBA to its metabolite 3-aminobenzanthrone (3-ABA), which was identified as the major metabolite in different tissue derived cell lines (15). However, the lack of acetylated 3-NBA-derived adducts found in hamster V79 cells pointed to a partially reduced intermediate of 3-NBA, N-OH-3-ABA, as a probable reactive metabolite that can be further metabolized to form DNA adducts (Figure 1) (3). This intermediate is most probably formed in all the organs analyzed, since the pattern of DNA adducts are very similar in the tissues with high levels of DNA adducts, dominating
58 min retention time of the chromatograms (Figure 2A). The major peaks consist of two non-separated DNA adducts that can be distinguished with a plateau method of 32P-HPLC analysis, but not completely resolved, and have been characterized as dG-C8-N-ABA and dG-N2-C2-ABA (Figure 5). We have also confirmed a previous finding stating that the dG-C8-C2-ABA adduct, which was hypothetically proposed as a major DNA adduct, was not present in the in vivo samples in our analysis (2,4).
The distribution of DNA adducts in the different tissues analyzed in this study suggests that reactive metabolites can be formed in the lung. Studies have shown that NADPH:Cytochrome P450 reductase, which is present in lung, can reduce 3-NBA to a reactive intermediate (11). However, a number of other reductive enzymes have been found in different in vitro and in vivo systems (1618). Following this, different oxidative processes can occur. The activation of both 3-NBA and 3-ABA has shown to be greatly mediated by CYP1A1 and CYP1A2 enzymes in different cell lines and in hamster lung (3,5,17). Similar observations have been made when human lung cells were treated with different kinds of nitro-PAHs (19). However, the induction of CYP1A2 was not detected in the study, most probably because in humans this enzyme is said to be liver-specific (20) and the expression of CYP1A2 is exceptionally low, if not absent, in tissue-derived cell lines (21). CYP1A2 was not detected in kidney cells either. The liver cells, however, displayed an elevated expression of all three CYPs tested (CYP1A1/1A2 and 1B1) (19).
Further metabolism of 3-NBA metabolites is most probably through phase II biotransformation involving GSTs, N,O-acetyltransferases (NATs) and sulfotransferases (SULTs) (3,10,22). It is known that phase II enzymes can activate chemical carcinogens just as well as phase I enzymes (23). Hence, since human exposure to 3-NBA is suspected to occur primarily via the respiratory tract, polymorphism of genes involved in xenobiotic metabolism may contribute significantly and specifically to the activation of 3-NBA and its metabolites. In addition, studies have also shown that there is a gender factor as well as hormonal-related factors that influence metabolism of carcinogens in rodents, which are also of importance and need to be taken into account (24,25).
It is evident that there is a fast reduction of DNA adducts, most probably because of DNA repair, occurring after a single administration and that the 3-NBA derived DNA adducts are not persistent (Figure 3). Maximum levels are reached merely after 23 days after which a rapid decline is seen. However, it is difficult to draw any conclusions on how the DNA adduct levels would develop in case of repeated administration, similar to real life situations where individuals are exposed to air pollutants on a daily basis.
Carcinogenicity
Acute or subacute changes in the respiratory tract are not common features in experimental studies on lung carcinogens. Mustard gas and some nitrosamines, which are known as very potent lung carcinogens, have been shown to induce squamous metaplasia and hyperplasia in lung tissues including trachea and bronchus, at early stages of exposure (26,27). In this study, metaplasic and hyperplasic changes developed in rat lungs within a few weeks after the administration of 3-NBA. A dose-dependent development of squamous cell carcinoma could be observed (Figure 6). Tumors first appeared in the respiratory tract after 79 months in the highest dose group and after 1012 months in the low-dose group (Table I). No tumors were observed in the control animals. The rapid development of these precancerous lesions by 3-NBA suggests a high carcinogenic potential of 3-NBA.
It has been shown that 1,6-dinitropyrene, that produces a dose-dependent induction of lung tumors after i.t. administration, also causes a dose-dependent formation of DNA adducts and induction of lymphocyte mutations, but that the doseresponse curves for DNA binding and mutations are different (28,29). A high incidence of lung carcinoma in Syrian golden hamsters after repeated i.t. administrations of 1,6-dinitropyrene (0.5 mg/week, during 26 weeks) has been reported (30). In other studies, the carcinogenicity of nitropyrenes was determined after a single injection to the thoracic cavity (3133). The reason for the high incidences of tumors at the injection site was owing to the relatively high concentration of the test compound. It is not possible to directly compare the carcinogenicity of 3-NBA and 1,6-dinitropyrene by the same protocol, because of the high acute toxicity of 3-NBA. In a pilot study, repeated administrations of the suspended solution of 3-NBA induced severe acute inflammation in the lung parenchyma. These lesions were so fatal that the experimental protocol had to be changed to lower doses than the protocol for 1,6-dinitropyrene (data not shown). We suggest, based on the early development of precancerous lesions in the respiratory tract of rats administered 3-NBA, that the carcinogenicity of 3-NBA is similar to, or stronger than, that of 1,6-dinitropyrene, and that 3-NBA has a high potency to induce the first critical steps of chemically induced carcinogenesis. Presently, it is difficult to estimate the level of exposure to human populations since there is a limited number of data on distribution and concentration in the urban environment. The inhalation of 3-NBA has been estimated to
90 pg/day (34). Human exposure to elevated levels of 3-NBA, owing to occupancy, has also been documented (35). Generally, 3-NBA has been found in ambient air in concentrations of pg/m3 (comparable with dinitropyrenes), whereas benzo[a]pyrene (B[a]P) is present in ng/m3 in urban areas. However, when the genotoxicity of 3-NBA was compared with that of benzo[a]pyrene (B[a]P), a 10002000-fold higher potency was observed for this nitro-PAH compound (34). Therefore if 3-NBA is present even in modest levels in ambient air, it is necessary to assess the carcinogenicity of 3-NBA for the evaluation of risk to humans by exposure of particulate matter in urban air.
Carcinogenicity versus DNA adducts
Considering the rather similar DNA adduct levels observed in lung and kidney, it could be seen as unexpected that carcinogenicity was observed only in the lung. However, because of the other responses found in the respiratory tract, a local promoting effect owing to the high concentration of 3-NBA during instillation could be suspected, similar to what have been seen after injection of nitropyrenes (32,33).
The correlation of the initially high DNA adduct levels and the high carcinogenicity effect seen in this study is also consistent with a previous study on the nitro-PAH 2-nitrofluorene in rats (36). It has been shown that 3-NBA induces mainly base substitutions but also transversions and transitions in Muta Mouse after i.p. treatment (8), and the 3-NBA derivative N-acetoxy-N-acetyl-3-aminobenzanthrone induces base substitutions, transversion and transitions in a shuttle vector system based on human cells (37). These base substitutions are similar to aminofluorene, acetylaminofluorene, nitro- and nitroso-PAHs (38,39), suggesting a similar metabolism and DNA adduct formation of 3-NBA.
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Conclusions
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The present results suggest that the 3-NBA-specific DNA adducts could be used as biomarkers of exposure to 3-NBA and possibly diesel emissions. Therefore, profiles of DNA adduct formation after repeated and chronic exposure to 3-NBA should be investigated in future studies. Based on data discussed in this paper it is reasonable to assume that 3-NBA is a potent carcinogen to mammals.
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Acknowledgments
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We wish to express our gratitude to Mary-Ann Zetterqvist for her skilful technical assistance. We would also like to acknowledge Dr Takeji Takamura Enya, Cancer Prevention Division, National Cancer Center Research Institute, Japan for the provision of the DNA adduct standards. The research group is a member of the European ECNIS (Environmental Cancer risk, Nutrition and Individual Susceptibility) project. This study was partly supported by a grant for Global Environment Protection from Ministry of the Environment, Japan.
Conflict of Interest Statement: None declared.
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References
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- Enya,T., Suzuki,H., Watanabe,T., Hirayama,T. and Hisamatsu,Y. (1997) 3-Nitrobenzanthrone, a powerful bacterial mutagen and suspected human carcinogen found in diesel exhaust and airborne particulates. Environ. Sci. Technol., 30, 27722776.[CrossRef]
- Enya,T., Kawanishi,M., Suzuki,H., Matsui,S. and Hisamatsu,Y. (1998) An unusual DNA adduct derived from the powerfully mutagenic environmental contaminant 3-nitrobenzanthrone. Chem. Res. Toxicol, 11, 14601467.[CrossRef][ISI][Medline]
- Arlt,V.M., Glatt,H., Muckel,E., Pabel,U., Sorg,B.L., Seidel,A., Frank,H., Schmeiser,H.H. and Phillips,D.H. (2003) Activation of 3-nitrobenzanthrone and its metabolites by human acetyltransferases, sulfotransferases and cytochrome P450 expressed in Chinese hamster V79 cells. Int. J. Cancer, 105, 583592.[CrossRef][ISI][Medline]
- Arlt,V.M., Bieler,C.A., Mier,W., Wiessler,M. and Schmeiser,H.H. (2001) DNA adduct formation by the ubiquitous environmental contaminant 3-nitrobenzanthrone in rats determined by (32)P-postlabeling. Int. J. Cancer, 93, 450454.[CrossRef][ISI][Medline]
- Arlt,V.M., Hewer,A., Sorg,B.L., Schmeiser,H.H., Phillips,D.H. and Stiborova,M. (2004) 3-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., 17, 10921101.[CrossRef][ISI][Medline]
- Bieler,C.A., Wiessler,M., Erdinger,L., Suzuki,H., Enya,T. and Schmeiser,H.H. (1999) DNA adduct formation from the mutagenic air pollutant 3-nitrobenzanthrone. Mutat. Res., 439, 307311.[ISI][Medline]
- Bieler,C.A., Arlt,V.M., Wiessler,M. and Schmeiser,H.H. (2003) DNA adduct formation by the environmental contaminant 3-nitrobenzanthrone in V79 cells expressing human cytochrome P450 enzymes. Cancer Lett., 200, 918.[CrossRef][ISI][Medline]
- Arlt,V.M., Zhan,L., Schmeiser,H.H., Honma,M., Hayashi,M., Phillips,D.H. and Suzuki,T. (2004) DNA adducts and mutagenic specificity of the ubiquitous environmental pollutant 3-nitrobenzanthrone in Muta Mouse. Environ. Mol. Mutagen, 43, 186195.[CrossRef][ISI][Medline]
- Turesky,R.J. (2004) The role of genetic polymorphisms in metabolism of carcinogenic heterocyclic aromatic amines. Curr. Drug Metab., 5, 169180.[CrossRef][ISI][Medline]
- Arlt,V.M., Glatt,H., 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, 19371945.[Abstract/Free Full Text]
- Arlt,V.M., Stiborova,M., Hewer,A., Schmeiser,H.H. and Phillips,D.H. (2003) Human enzymes involved in the metabolic activation of the environmental contaminant 3-nitrobenzanthrone: evidence for reductive activation by human NADPH:Cytochrome P450 reductase. Cancer Res., 63, 27522761.[Abstract/Free Full Text]
- Carlberg,C.E., Möller,L., Paakki,P., Kantola,M., Stockmann,H., Purkunen,R., Wagner,P., Lauper,U., Kaha,M., Elovaara,E., Kirkinen,P. and Pasanen,M. (2000) DNA adducts in human placenta as biomarkers for environmental pollution, analysed by the 32P-HPLC method. Biomarkers, 5, 182191.[CrossRef][ISI]
- Garner,R.C. (1998) The role of DNA adducts in chemical carcinogenesis. Mutat. Res., 402, 6775.[ISI][Medline]
- Zeisig,M. and Moller,L. (1995) 32P-HPLC suitable for characterization of DNA adducts formed in vitro by polycyclic aromatic hydrocarbons and derivatives. Carcinogenesis, 16, 19.[Abstract]
- Borlak,J., Hansen,T., Yuan,Z., Sikka,H.C., Kumar,S., Schmidbauer,S., Frank,H., Jakob,J. and Seidel,A. (2000) Metabolism and DNA-binding of 3-nitrobenzanthrone in primary rat alveolar type II cells, in human fetal bronchial, rat epithelial and mesenchymal cell lines. Polycyclic Aromat. Compds, 21, 7386.
- Arlt,V.M., Stiborova,M., Henderson,C.J., Osborne,M.R., Bieler,C.A., Frei,E., Martinek,V., Sopko,B., Wolf,C.R., Schmeiser,H.H. and Phillips,D.H. (2005) 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., 65, 26442652.[Abstract/Free Full Text]
- Arlt,V.M., Henderson,C.J., Wolf,C.R., Schmeiser,H.H., Phillips,D.H. and Stiborova,M. (2005) Bioactivation of 3-aminobenzanthrone, a human metabolite of the environmental pollutant 3-nitrobenzanthrone: evidence for DNA adduct formation mediated by cytochrome P450 enzymes and peroxidases. Cancer Lett., 7, (Epub ahead of print).
- 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, 26, (Epub ahead of print).
- Iwanari,M., Nakajima,M., Kizu,R., Hayakawa,K. and Yokoi,T. (2002) Induction of CYP1A1, CYP1A2, and CYP1B1 mRNAs by nitropolycyclic aromatic hydrocarbons in various human tissue-derived cells: chemical-, cytochrome P450 isoform-, and cell-specific differences. Arch. Toxicol., 76, 287298.[CrossRef][ISI][Medline]
- Shimada,T., Hayes,C.L., Yamazaki,H., Amin,S., Hecht,S.S., Guengerich,F.P. and Sutter,T.R. (1996) Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1. Cancer Res., 56, 29792984.[Abstract]
- Li,W., Harper,P.A., Tang,B.K. and Okey,A.B. (1998) Regulation of cytochrome P450 enzymes by aryl hydrocarbon receptor in human cells: CYP1A2 expression in the LS180 colon carcinoma cell line after treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin or 3-methylcholanthrene. Biochem. Pharmacol., 56, 599612.[CrossRef][ISI][Medline]
- Arlt,V.M., Sorg,B.L., Osborne,M., Hewer,A., Seidel,A., Schmeiser,H.H. and Phillips,D.H. (2003) DNA adduct formation by the ubiquitous environmental pollutant 3-nitrobenzanthrone and its metabolites in rats. Biochem. Biophys. Res. Commun., 300, 107114.[CrossRef][ISI][Medline]
- Guengerich,F.P. (2000) Metabolism of chemical carcinogens. Carcinogenesis, 21, 345351.[Abstract/Free Full Text]
- Spivack,S.D., Hurteau,G.J., Fasco,M.J. and Kaminsky,L.S. (2003) Phase I and II carcinogen metabolism gene expression in human lung tissue and tumors. Clin. Cancer Res., 9, 60026011.[Abstract/Free Full Text]
- Hein,D.W. (2002) Molecular genetics and function of NAT1 and NAT2: role in aromatic amine metabolism and carcinogenesis. Mutat. Res., 506507, 6577.
- IARC (1975) Some aziridines, N-, S-, O-mustards and selenium. International Agency for Research on Cancer, vol. 9, pp. 181192.
- Taylor,H.W. and Lijinsky,W. (1985) Squamous-cell carcinoma of the lung. NHMI-induced squamous-cell carcinoma in the lungs of rats. Am. J. Pathol., 119, 168170.[ISI][Medline]
- Beland,F.A., Fullerton,N.F., Smith,B.A. and Heflich,R.H. (1994) Formation of DNA adducts and induction of mutations in rats treated with tumorigenic doses of 1,6-dinitropyrene. Environ. Health Perspect., 102 (Suppl 6), 185189.[ISI][Medline]
- Smith,B.A., Fullerton,N.F., Heflich,R.H. and Beland,F.A. (1995) DNA adduct formation and T-lymphocyte mutation induction in F344 rats implanted with tumorigenic doses of 1,6-dinitropyrene. Cancer Res., 55, 23162324.[Abstract]
- Takayama,S., Ishikawa,T., Nakajima,H. and Sato,S. (1985) Lung carcinoma induction in Syrian golden hamsters by intratracheal instillation of 1,6-dinitropyrene. Jpn. J. Cancer Res., 76, 457461.[ISI][Medline]
- Horikawa,K., Sera,N., Otofuji,T., Murakami,K., Tokiwa,H., Iwagawa,M., Izumi,K. and Otsuka,H. (1991) Pulmonary carcinogenicity of 3,9- and 3,7-dinitrofluoranthene, 3-nitrofluoranthene and benzo[a]pyrene in F344 rats. Carcinogenesis, 12, 10031007.[Abstract]
- Iwagawa,M., Maeda,T., Izumi,K., Otsuka,H., Nishifuji,K., Ohnishi,Y. and Aoki,S. (1989) Comparative doseresponse study on the pulmonary carcinogenicity of 1,6-dinitropyrene and benzo[a]pyrene in F344 rats. Carcinogenesis, 10, 12851290.[Abstract]
- Maeda,T., Izumi,K., Otsuka,H., Manabe,Y., Kinouchi,T. and Ohnishi,Y. (1986) Induction of squamous cell carcinoma in the rat lung by 1,6-dinitropyrene. J. Natl Cancer Inst., 76, 693701.[ISI][Medline]
- Lamy,E., Kassie,F., Gminski,R., Schmeiser,H.H. and Mersch-Sundermann,V. (2004) 3-Nitrobenzanthrone (3-NBA) induced micronucleus formation and DNA damage in human hepatoma (HepG2) cells. Toxicol. Lett., 146, 103109.[CrossRef][ISI][Medline]
- Seidel,A., Dahmann,D., Krekeler,H. and Jacob,J. (2002) Biomonitoring of polycyclic aromatic compounds in the urine of mining workers occupationally exposed to diesel exhaust. Int. J. Hyg. Environ. Health, 204, 333338.[ISI][Medline]
- Cui,X.S., Torndal,U.B., Eriksson,L.C. and Moller,L. (1995) Early formation of DNA adducts compared with tumor formation in a long-term tumor study in rats after administration of 2-nitrofluorene. Carcinogenesis, 16, 21352141.[Abstract]
- Kawanishi,M., Enya,T., Suzuki,H., Takebe,H., Matsui,S. and Yagi,T. (1998) Mutagenic specificity of a derivative of 3-nitrobenzanthrone in the supF shuttle vector plasmids. Chem. Res. Toxicol., 11, 14681473.[CrossRef][ISI][Medline]
- Shelton,M.L. and DeMarini,D.M. (1995) Mutagenicity and mutation spectra of 2-acetylaminofluorene at frameshift and base-substitution alleles in four DNA repair backgrounds of Salmonella. Mutat. Res., 327, 7586.[CrossRef][ISI][Medline]
- Shibutani,S. and Grollman,A.P. (1997) Molecular mechanisms of mutagenesis by aromatic amines and amides. Mutat. Res., 376, 7178.[ISI][Medline]
Received March 21, 2005;
revised May 20, 2005;
accepted May 22, 2005.