1. Exposure Data

1.1. Chemical and physical data

1.1.1. Nomenclature

• Chem. Abst. Serv. Reg. No.: 17117-34-9
• Synonym: 3-Nitro-7H-benzo[d,e]anthracen-7-one

1.1.2. Structural and molecular formulae and relative molecular mass

• C₁₇H₉NO₃
• Relative molecular mass: 275.27 g/mol

1.1.3. Chemical and physical properties of the pure substance

• Description: Yellow powder (Enya et al., 1997)
• Melting-point: 256–257 °C (Enya et al., 1997); 252 °C (Suzuki et al., 1997)
• Boiling-point: 506.2 °C at 760 mm Hg
• Flash-point: 256.6 °C
• Spectrometry data: Infrared and nuclear magnetic resonance data have been reported (Enya et al., 1997; Suzuki et al., 1997).

1.4. Occurrence and exposure

1.4.1. Environmental occurrence

3-Nitrobenzanthrone was first discovered by Suzuki et al. (1997) in organic extracts of both diesel exhaust and airborne particles. It is formed by the combustion of organic material and from reactions of complex PAHs with nitrogen oxides. It has been suggested that 2-nitrobenzanthrone might be formed more specifically by atmospheric processes (Inazu et al., 2008), while 3-nitrobenzanthrone seems to be formed preferentially by combustion processes, such as in a diesel engine (Feilberg et al., 2002; Phousongphouang & Arey, 2003; Tang et al., 2004).

Table 1.1 summarizes the available data on environmental exposure from point sources and on environmental samples of air, soil and rainwater.

Table 1.1

Sources of environmental exposure to 3-nitrobenzanthrone.

(a) Point sources

3-Nitrobenzanthrone has been detected in diesel exhaust particles at concentrations of up to 6.6 µg/g particles (Enya et al., 1997; Murahashi, 2003). Furthermore, it was detected in extracts of particles collected from the chimney of a domestic coal-burning stove at a concentration of 0.23 µg/g particles, suggesting that particles emitted from industrial and domestic coal-burning sources should be considered as possibly minor sources of 3-nitrobenzanthrone in urban air pollution (Taga et al., 2005).

(b) Environmental levels

Airborne concentrations of 3-nitrobenzanthrone in ambient particles have been reported to be in the order of several picgrams per cubic metre (up to > 11.5 pg/m³) in urban and semi-rural areas, and at sites affected by traffic or industrial emissions. Higher levels (up to > 80 pg/m³) have been recorded in ambient air at workplaces exposed to high levels of diesel emissions (Seidel et al., 2002).

More recently, Inazu et al. (2008) measured airborne levels of 3-nitrobenzanthrone in central Tokyo, Japan, in the range of 0.5–3.5 fmol/m³ (0.2–1.0 pg/m³); the highest levels were observed in the winter (average, 0.6 pg/m³) and the lowest levels were found in the spring (average, 0.2 pg/m³).

Murahashi et al. (2003a) detected 3-nitrobenzanthrone in rainwater samples collected at a residential area in Kyoto, Japan, at levels ranging from 0.07 to 2.6 ng/L. It was also detected in surface soil at levels of up to 1200 pg/g soil (Murahashi et al., 2003b; Watanabe et al., 2003).

Substantially higher concentrations of 2-nitrobenzanthrone than of 3-nitrobenzanthrone have been found in ambient air, with a ratio of 37.5–70:1 (Phousongphouang & Arey, 2003; Inazu et al., 2008).

1.4.2. Exposure of the general population

Exposure to 3-nitrobenzanthrone occurs mainly by inhalation and secondarily by oral intake. Inhalation occurs through contamination in air that is formed by combustion. Oral exposure can occur either by mucociliary clearance and subsequent swallowing of the material that has been inhaled or by the consumption of foods that have been affected by dry or wet deposition of airborne 3-nitrobenzanthrone. Overall, inhalation is considered to be the greatest and perhaps most important source of exposure. On the basis of the reported levels of 3-nitrobenzanthrone in diesel exhaust particles and a daily intake by inhalation of 1 µg of particles per cubic metre of air, Arlt (2005) estimated a human lung dose of approximately 90 pg per day, based on a breath intake of about 15 m³ air per day, although the value may be exceeded in highly exposed populations.

2. Cancer in Humans

No data were available to the Working Group

3. Cancer in Experimental Animals

See Table 3.1

Table 3.1

Studies of the carcinogenicity of 3-nitrobenzanthrone in experimental animals.

4. Mechanistic and Other Relevant Data

4.1. Absorption, distribution, metabolism, and excretion

4.1.1. Humans

Seidel et al. (2002) examined the urinary levels of PAHs and nitrated PAHs (nitroarenes) in the 24-hour urine of 18 (nine smokers and nine non-smokers) underground salt-mine workers occupationally exposed to diesel engine exhaust. 3-Aminobenzanthrone, the major metabolite of 3-nitrobenzanthrone (Borlak et al., 2000; Arlt et al., 2003a; Hansen et al., 2007), and 1-aminopyrene, a reductive metabolite of 1-nitropyrene (El-Bayoumy et al., 1983; Howard et al., 1985; van Bekkum et al., 1998; Chae et al., 1999), were identified in both groups of workers at similar levels of 1–143 ng/24-hour urine and 2–200 ng/24-hour urine, respectively. These results suggest that 3-nitrobenzanthrone and 1-nitropyrene bind to diesel engine exhaust particles and are ingested/inhaled by humans (Seidel et al., 2002), distributed in different organs, metabolized by various enzymes to more polar products (such as 3-aminobenzanthrone and 1-aminopyrene) and excreted in the urine. Thus, 3-aminobenzanthrone and 1-aminopyrene could be used as urinary biomarkers in humans exposed to diesel engine exhaust (Arlt, 2005; Stiborová et al., 2005).

4.1.2. Experimental systems

Several in vivo studies have shown that 3-nitrobenzanthrone is metabolically activated to DNA-binding products in the tissues of animals administered the compound by various routes (Arlt et al., 2001, 2003a, 2004a, 2006a, 2007; Bieler et al., 2005, 2007; Nagy et al., 2005a, 2006, 2007; Schmeiser et al., 2009). These studies have been confirmed by several in-vitro studies which showed that 3-nitrobenzanthrone first requires metabolic activation through nitroreduction by various xenobiotic-metabolizing enzymes, including nicotinamide adenine dinucleotide phosphate (NADPH)-quinone oxidoreductase (NQO1), xanthine/xanthine oxidase, NADPH-P450 oxidoreductase (POR) or cytochrome P450 (CYP) enzymes, leading to the formation of the DNA-binding reactive intermediate, N-hydroxy-3-aminobenzanthrone (Borlak et al., 2000; Arlt et al., 2003a, b, 2004b; Bieler et al., 2003) (Fig. 4.1). N-Hydroxy-3-aminobenzanthrone can be further activated by acetylation and sulfation, which are catalysed by N-acetyltransferases (NATs) and sulfotransferases (SULTs), to form highly reactive N-acetoxy or S-sulfoxy esters, respectively, leading to increased DNA binding (Arlt et al., 2002, 2003c). Although 3-aminobenzanthrone has been found to be the reductive metabolite of 3-nitrobenzanthrone in mammalian cells in culture, including 3-nitrobenzanthrone-treated human lung adenocarcinoma A549 cells (Borlak et al., 2000; Hansen et al., 2007), detailed chemical analyses to identify 3-nitrobenzanthrone and its metabolites in vivo in experimental animals have been scarce.

Fig. 4.1

Metabolic activation of 3-nitrobenzanthrone and DNA-adduct formation

Recently, a mercapturic acid metabolite of 3-aminobenzanthrone, N-acetyl-S-(3-aminobenzanthrone-2-yl)cysteine, as well as 3-aminobenzanthrone and 3-acetylaminobenzanthrone, have been identified in the urine of rats administered 3-nitrobenzanthrone (2 mg/kg bw) by gavage, and it has been suggested that this metabolite could be used as a biomarker in exposed humans (Linhart et al., 2012). The same authors also showed that 2-nitrobenzanthrone, which is reported to be less active in forming DNA adducts (Arlt et al., 2007; Stiborová et al., 2010), does not produce a mercapturic acid metabolite, suggesting that reactive metabolites of 3-, but not those of 2-nitrobenzanthrone, can be trapped by glutathione S-transferase.

4.1.3. Role of xenobiotic-metabolizing enzymes in the metabolic activation of 3-nitrobenzanthrone

Several in vitro and in vivo studies have indicated that the metabolic conversion of 3-nitrobenzanthrone is catalysed by xenobiotic-metabolizing enzymes to form DNA-binding metabolites in humans and experimental animals (Fig. 4.1) (reviewed by Arlt, 2005). 3-Nitrobenzanthrone is first bio-activated in humans and experimental animals to N-hydroxy-3-aminobenzanthrone by various nitroreductases, such as NQO1, xanthine/xanthine oxidase and POR (Arlt et al., 2003b, 2004c, 2005; Bieler et al., 2003). Cytosolic NQO1 is the major enzyme involved in the bio-activation of 3-nitrobenzanthrone, based on experiments with an NQO1 inhibitor (dicoumarol) and human recombinant NQO1 (Arlt et al., 2005; Stiborová et al., 2006, 2010). The role of POR in the nitroreduction of 3-nitrobenzanthrone in vivo may be minor, because no difference in the levels of DNA adducts in the liver was observed between 3-nitrobenzanthrone-treated wild-type C57BL/6 and hepatic POR-null mice lacking hepatic CYP enzyme activity (Arlt et al., 2005; Stiborová et al., 2006, 2008). In contrast, the DNA binding induced by 3-aminobenzanthrone in the liver was reduced in POR-null compared with wild-type mice, indicating that the bio-activation of 3-aminobenzanthrone depends on hepatic CYP enzymes (Stiborová et al., 2006).

3-Aminobenzanthrone, the reductive metabolite of 3-nitrobenzanthrone (Borlak et al., 2000; Arlt, 2005; Hansen et al., 2007), can be oxidized by rat CYP enzymes (mainly CYP1A) to N-hydroxy-3-aminobenzanthrone, as detected by HPLC analysis (Mizerovská et al., 2008). Human CYP1A1 and -1A2 (Arlt et al., 2004c, 2006b; Stiborová et al., 2008) and other human CYPs, including CYP2A6, -3A4 and -2B6 (Arlt et al., 2003c; Bieler et al., 2003), have also been shown to be involved in this reaction step. Several peroxidases, such as lactoperoxidase, myeloperoxidase and prostaglandin H synthase, were also able to catalyse the conversion of 3-aminobenzanthrone to N-hydroxy-3-aminobenzanthrone (Stiborová et al., 2005; Arlt et al., 2006b). The reactivity of N-hydroxy-3-aminobenzanthrone in biological systems has been reported in human cell lines, in vivo in rats after intraperitoneal injection (Arlt et al., 2007) and in cultured embryonic fibroblasts from human TP53 knock-in mice (vom Brocke et al., 2009). Arlt et al. (2003b) found that purified POR isolated from rabbit liver microsomes catalysed the activation of 3-nitrobenzanthrone to DNA-binding products in a reconstituted system, indicating that N-hydroxy-3-aminobenzanthrone is the DNA-reactive intermediate.

N-Hydroxy-3-aminobenzanthrone has been shown to be further activated by NATs (NAT1 and NAT2) or SULTs (SULT1A1 and SULT1A2) (Arlt et al., 2002, 2003a, c, 2004b, 2005; Stiborová et al., 2006) (Fig. 4.1). Reactive N-acetoxy or N-sulfoxy esters can bind covalently to DNA (Snyderwine et al., 1988; Arlt, 2005), and evidence has been presented that an aryl nitrenium ion, which is formed through heterolytic cleavage of these ester metabolites, is the ultimate DNA-binding species of 3-nitrobenzanthrone (Arlt, 2005). NATs (i.e. NAT2) may be more important than SULTs in the bio-activation of 3-nitrobenzanthrone (Arlt et al., 2002, 2003c, 2005). By incorporating human SULT1A1, -1A2 and -1A3 into a Salmonella typhimurium strain deficient in O-acetyltransferase activity, Oda et al. (2012) found that the strain that expressed human SULT1A1 was more effective in activating 3-nitrobenzanthrone to genotoxic metabolites than those that harboured SULT1A2 or -1A3, as determined in the umu gene expression assay.

NATs have also been reported to be involved in the acetylation of N-hydroxy-3-aminobenzanthrone to form N-acetyl-N-hydroxy-3-aminobenzanthrone, and the resultant metabolite is also activated by NATs to form highly reactive N-acetyl-N-acetoxy-3-aminobenzanthrone (Enya et al., 1997; Kawanishi et al., 1998, 2000; Arlt et al., 2002, 2003a). However, this activation step has no relevance to the metabolic activation of 3-nitrobenzanthrone in experimental animals, because all of its major DNA adducts identified in vivo lack an N-acetyl group in the adduct molecules (see Section 4.2.1) (Arlt et al., 2002, 2003a; Kanno et al., 2007; Takamura-Enya et al., 2007); moreover, N-acetyl-N-hydroxy-3-aminobenzanthrone was weakly mutagenic in the Salmonella strain DJ450, which expresses human recombinant NAT2 (Arlt et al., 2002). It has also been reported that N-acetyl-N-hydroxy-3-aminobenzanthrone is readily deacetylated by microsomal enzymes (Arlt et al., 2002, 2003b, c).

4.1.4. Induction of xenobiotic-metabolizing enzymes by 3-nitrobenzanthrone and 3-aminobenzanthrone

3-Nitrobenzanthrone and 3-aminobenzanthrone induce several forms of xenobiotic-metabolizing enzyme in experimental animals in vivo (Stiborová et al., 2006, 2009; Mizerovská et al., 2011). Intraperitoneal administration of 3-nitrobenzanthrone or 3-aminobenzanthrone to rats (at a single dose of 0.4, 4 or 40 mg/kg bw) caused increases in cytosolic menadione reduction and 7-ethoxyresorufin O-deethylation activities with increased levels of DNA adducts in the liver, indicating that these compounds induced NQO1 and CYP1A; the induction of NQO1, and CYP1A1 and -1A2 protein was confirmed by western blot analysis (Stiborová et al., 2006). Intraperitoneal administration of 3-nitrobenzanthrone and 3-aminobenzanthrone to rats also induced CYP1A1 and NQO1 in the lung and kidney (Stiborová et al., 2008, 2009). Because these enzymes play important roles in both the activation and detoxification of PAHs and nitro-PAHs, it is possible that 3-nitrobenzanthrone and 3-aminobenzanthrone not only alter their own toxic responses by inducing xenobiotic-metabolizing enzymes but also modulate the metabolism of related PAHs and nitro-PAHs found in diesel-engine exhaust.

4.2. Genetic and related effects

4.2.1. Humans

No data were available to the Working Group.

4.2.2. Experimental systems

(a) DNA adduct formation

In vivo results in experimental animals show that the levels of 3-nitrobenzanthrone–DNA adducts in different tissues are largely dependent on the route of administration (Arlt et al., 2001, 2003a, 2004a, 2006a, 2007; Bieler et al., 2005, 2007; Nagy et al., 2005a, 2006, 2007; Schmeiser et al., 2009) (Table 4.1). DNA-adduct formation by 3-nitrobenzanthrone has been mainly investigated by ³²P-postlabelling using thin-layer chromatography (TLC) or HPLC. Oral administration of 3-nitrobenzanthrone to rats induced DNA adducts in the small intestine, stomach, liver, kidney and urinary bladder, but relatively fewer in the lung (Arlt et al., 2001; Nagy et al., 2006). A single intraperitoneal dose of 3-nitrobenzanthrone resulted in the distribution of DNA adducts in several organs, such as the pancreas, kidney, liver, lung, urinary bladder, heart and colon (Arlt et al., 2003a, 2005, 2006a, 2007; Bieler et al., 2005, 2007; Nagy et al., 2005a). Intratracheal instillation of 3-nitrobenzanthrone to rats, which induced squamous cell carcinomas in the lung of Fischer 344 rats (Nagy et al., 2005a), resulted in DNA-adduct formation in the lung and other tissues, such as the kidney, pancreas, urinary bladder, heart, small intestine and liver (Bieler et al., 2005, 2007; Nagy et al., 2006). The highest levels of DNA adducts in female Fischer 344 rats that received intratracheal instillations of 3-nitrobenzanthrone (10 mg/kg bw) were observed in the lungs (~250 adducts/10⁸ nucleotides); adduct levels were also high in the kidney (~200 adducts/108 nucleotides), but low in the liver (~30 adducts/10⁸ nucleotides). Although the kidney showed high levels of 3-nitrobenzanthrone–DNA adducts, tumour formation was not reported in this organ (Nagy et al., 2005a). Topical application of 3-nitrobenzanthrone and N-hydroxy-3-nitrobenzanthrone to the skin of NMRI mice was found to produce DNA adducts in the epidermis, but adducts in other organs were only found after treatment with 3-nitrobenzanthrone (Schmeiser et al., 2009). These results suggest that 3-nitrobenzanthrone can largely be metabolized by xenobiotic-metabolizing enzymes near the sites of application to produce DNA adducts, and that 3-nitrobenzanthrone and its metabolites may be distributed via the blood to other organs in which further metabolism may occur (i.e. activation or detoxification).

Table 4.1

Detection of DNA adducts in tissues of experimental animals treated with 3-nitrobenzanthrone and N-hydroxy-3-aminobenzanthrone by different routes of administration in vivo.

TLC- and HPLC-³²P-postlabelling analyses have been used to characterize 3-nitrobenzanthrone-derived DNA adducts in vitro and in vivo (Kawanishi et al., 1998; Arlt et al., 2001; Bieler et al., 2003; Nagy et al., 2005a; Osborne et al., 2005) (Table 4.1). In TLC, the pattern of DNA adducts induced by 3-nitrobenzanthrone consisted of a characteristic cluster of four major DNA adducts (spots 1, 2, 3 and 4); initial studies showed that all four adducts formed were derived from reductive metabolites bound either to deoxyadenosine (spots 1 and 2) or deoxyguanosine (spots 3 and 4) (Arlt et al., 2001, 2003a, c). Among a series of deoxyguanosine and deoxyadenosine adducts characterized to date, the following have been identified as the major lesions in vivo: 2-(2′-deoxyguanosin-N²-yl)-3-aminobenzanthrone (spot 3), N-(deoxyguanosin-8-yl)-3-aminobenzanthrone (spot 4) and 2-(2’-deoxyadenosin-N⁶-yl)-3-aminobenzanthrone (spot 1) (Nagy et al., 2005a, 2006; Osborne et al., 2005; Arlt et al., 2006a, 2008, 2011; Kanno et al., 2007; Takamura-Enya et al., 2007;) (Fig. 4.1). The formation of 2-(2′-deoxyguanosin-N²-yl)-3-aminobenzanthrone and N-(deoxyguanosin-8-yl)-3-aminobenzanthrone in 3-nitrobenzanthrone-treated rodents has been confirmed by mass spectrometry (Gamboa da Costa et al., 2009). None of the DNA adducts detected in vivo contained an N-acetyl group, suggesting that there is no important role of N-acetyl-N-acetoxy-3-aminobenzanthrone in the reaction with DNA (Arlt, 2005; Kanno et al., 2007). Overall, the pattern of 3-nitrobenzanthrone-derived DNA adducts in experimental animals is the same in vivo and in vitro (Arlt, 2005; Stiborová et al., 2010).

3-Nitrobenzanthrone–DNA adducts have been detected in various organs of rats within 36 weeks after intratracheal instillation of a single dose (0.2 mg/kg bw), including in the target organ, the lung; the most abundant and persistent DNA adduct was 2-(2′-deoxyguanosin-N²-yl)-3-aminobenzanthrone (Bieler et al., 2007). Lukin et al. (2011) showed that this lesion increased the stability of the damaged DNA duplex, which may provide an explanation for its long persistence in vivo.

TP53-dependent adduct formation by 3-nitrobenzanthrone was investigated in two isogenic human colorectal HCT116 cell lines, one that expressed TP53 (p53-wild-type) and another that had this gene knocked out (p53-null) (Hockley et al., 2008; Simoes et al., 2008). The authors showed that the levels of DNA adducts after treatment with benzo[a]pyrene and nitroarene aristolochic acid I was lower in p53-null cells compared with p53-wild-type cells, whereas DNA binding by 3-nitrobenzanthrone was TP53-independent. These results suggest that the cellular TP53 status is linked to the CYP-mediated metabolism of benzo[a]pyrene and nitroarene aristolochic acid I, whereas the nitroreduction of 3-nitrobenzanthrone is not TP53-dependent. The possible lack of protective functions mediated by p53 after the treatment of p53-null cells with 3-nitrobenzanthrone needs to be explored further.

(b) Mutagenicity of 3-nitrobenzanthrone and its metabolites

(i) In-vivo studies

Arlt et al. (2004a) reported that intraperitoneal administration of 3-nitrobenzanthrone to the MutaMouse increased the mutant frequency in the cII gene in the colon, liver and urinary bladder, but not in the lung, kidney, spleen or testis. In addition, the percentage of G:C to T:A transversions in the liver was found to be higher in 3-nitrobenzanthrone-treated mice than in untreated mice (49% versus 6%). The authors also showed preferential DNA binding at deoxyguanosine (70–80%), which correlated with the preferential occurrence of G:C to T:A transversions; 2-(2′-deoxyguanosin-N²-yl)-3-aminobenzanthrone and N-(deoxyguanosin-8-yl)-3-aminobenzanthrone appeared to be the pre-mutagenic lesions. LacZ mutant frequencies were also investigated in the MutaMouse strain treated with 3-nitrobenzanthrone and 3-aminobenzanthrone (0, 2 and 5 mg/kg bw per day for 28 days) (Arlt et al., 2008). Dose-related increases in mutant frequency were seen in the liver and bone marrow, but not in the lung; the mutagenic activity of 3-aminobenzanthrone was approximately twofold lower than that of 3-nitrobenzanthrone. Chen et al. (2008) showed that 3-nitrobenzanthrone induced lacZ mutations in the bone marrow and liver, but not in the lung or intestinal epithelium, in the MutaMouse after single and repeated oral administration. High 3-nitrobenzanthrone nitroreductase activity was found in all tissues examined, whereas no NAT activity was observed in bone marrow.

(ii) In-vitro studies

3-Nitrobenzanthrone was highly mutagenic in S. typhimurium strain TA98, only in the absence of metabolic activation. It was less mutagenic in S. typhimurium TA100 than in TA98, suggesting that it causes frameshift-type mutations in the bacteria. In strain TA98, the mutagenic potency of 3-nitrosobenzanthrone was comparable with that of 1,8-dinitropyrene, and both compounds were found to be highly mutagenic in S. typhimurium YG1024, an O-acetyltransferase-overexpressing strain (Enya et al., 1997; Watanabe et al., 2005). It should be noted that 3-nitrosobenzanthrone induced 6.3 million revertants/nmol in strain YG1024, and is thus one of the most potent bacterial mutagens known to date. Takamura-Enya et al. (2006) compared the mutagenic activities of several derivatives of mono-, di- and trinitrobenzanthrone in S. typhimurium TA98, and found that 3-nitrobenzanthrone was more mutagenic than 1-, 2-, 9- and 11-nitrobenzanthrone, 1,9-, 3,9- and 3,11-dinitrobenzanthrone, and 3,9,11-trinitrobenzanthrone. In comparison, 3-nitrobenzanthrone showed high mutagenic potency, similar to that of 1,3-, 1,6- and 1,8-dinitropyrene, and 3,7- and 3,9-dinitrofluoranthene, in S. typhimurium TA98 in the absence of metabolic activation. In general, these nitroarenes have been less active in inducing reverse mutations in S. typhimurium TA100. It should be mentioned that the nitrenium ion of 3-nitrobenzanthrone is more stable than comparable ions derived from other nitrated benzanthrone derivatives, which could provide a possible explanation for its potent mutagenic and DNA adduct-forming activities (Arlt et al., 2007, 2011; Reynisson et al., 2008).

Watanabe et al. (2005) found that 3-aminobenzanthrone and 3-acetyl-3-aminobenzanthrone, two metabolites of 3-nitrobenzanthrone, were activated by a rat-liver metabolic activation system to reactive metabolites that caused the induction of reverse mutations in S. typhimurium TA98, TA100, YG1024 and YG1029; the highest activity was seen in the YG1024 strain, which overexpresses O-acetyltransferase. They also showed that 3-nitrobenzanthrone itself was deactivated by the metabolic activation system in this assay, suggesting that enzymes present in this fraction may detoxify 3-nitrobenzanthrone or its metabolites.

Oda et al. (2007) reported that a very low concentration (~2 nM) of 3-nitrobenzanthrone induced umu gene expression in S. typhimurium NM2009 and NM3009, which express O-acetyltransferase and exhibit both nitroreductase and O-acetyltransferase activities, respectively. They also showed that 3-nitrobenzanthrone was cytotoxic in these tester strains, suggesting the formation of active metabolites. More recently, Oda et al. (2012) reported that 3-nitrobenzanthrone induced umu gene expression more actively in S. typhimurium NM7001, which expresses SULT1A1, than in strains NM7002 and 7003, which express human SULT1A2 and 1A3, respectively.

3-Nitrobenzanthrone induced base-substitution mutations in mammalian cell systems (Phousongphouang et al., 2000; Arlt et al., 2008; Nishida et al., 2008). The induction of base-substitution mutations in mammalian cells compared with frameshift mutations in bacteria is probably due to differences in the target genes or in the mechanisms of mutagenesis between bacterial and mammalian systems. In human B-lymphoblastoid cell lines, 3-nitrobenzanthrone induced mutations at the thymidine kinase^+/– and hypoxanthine phosphoribosyltransferase loci (Phousongphouang et al., 2000). 3-Nitro- and 3-aminobenzanthrone produced a dose-dependent increase in mutant frequency in lung epithelial FE1 cells derived from the MutaMouse (in the presence and absence of metabolic activation), which correlated with an increase in DNA-adduct formation (Arlt et al., 2008).

Nishida et al. (2008) determined the mutagenic specificity of N-acetoxy-3-aminobenzanthrone, probably the most active metabolite of 3-nitrobenzanthrone, in the supF system using human fibroblast cell lines. They found that N-acetoxy-3-aminobenzanthrone bound to guanine rather than adenine and preferentially induced G:C to T:A transversions; deoxyadenosine adducts may be repaired more efficiently by nucleotide excision repair than deoxyguanosine adducts.

3-Nitrobenzanthrone also induced G:C to T:A transversions in the TP53 gene in immortalized human TP53 knock-in murine embryonic fibroblasts (vom Brocke et al., 2009; Kucab et al., 2012), suggesting that G:C to T:A transversions in TP53 could be used as a signature mutation for exposure to 3-nitrobenzanthrone in human lung tumours, when exposure to this carcinogen has been documented (Kucab et al., 2010).

(c) Other genetic effects of 3-nitrobenzanthrone in vivo and in vitro

3-Nitrobenzanthrone induced micronuclei in mouse peripheral blood reticulocytes after intraperitoneal injection (25 or 50 mg/kg bw) into male ICR mice (Enya et al., 1997). Similar results have been reported by Arlt et al. (2004a). Intraperitoneal administration of 3-nitrobenzanthrone (25 mg/kg bw once a week for 4 weeks) increased the frequency of micronuclei in the peripheral reticulocytes of the male MutaMouse.

In vitro induction of micronucleus formation and DNA damage following treatment with 3-nitrobenzanthrone has also been reported in human hepatoma HepG2 cells (Lamy et al., 2004), human B-lymphoblastoid MCL-5 cells (Arlt et al., 2004b) and human A549 lung cells (Nagy et al., 2005b). Both 3-nitrobenzanthrone and 3-aminobenzanthrone increased the tail moment in the alkaline comet assay in human lung epithelial A549 cells and in the liver, kidney, spleen, lung and bone marrow of male ICR mice (Watanabe et al., 2005; Hansen et al., 2007).

N-Hydroxy-3-aminobenzanthrone induced oxidative DNA damage through the formation of 8-hydroxydeoxyguanosine in the presence of divalent copper and nicotinamide adenine dinucleotide in vitro (Murata et al. 2006; Hansen et al., 2007). Using ³²P-labelled DNA fragments from human TP53, Murata et al. (2006) showed that the DNA damage caused by N-hydroxy-3-aminobenzanthrone could be inhibited by catalase and bathocuproline, suggesting the involvement of hydrogen peroxide and monovalent copper in the induction of DNA damage; DNA damage occurred at the cytosine and guanine residues of an ACG sequence complementary to codon 273, which is a well known hotspot for TP53 mutations (Murata et al., 2006). Shimohara et al. (2008) reported that 3-nitrobenzanthrone caused double-strand DNA breaks by analysing the phosphorylation of histone H2AX in human Hela cells.

3-Nitrobenzanthrone induced apoptosis, DNA-adduct formation and DNA damage in human bronchial epithelial BEAS-2B cells (Ovrevik et al., 2010; Oya et al., 2011). In mouse hepatoma Hepa1c1c7 cells, Landvik et al. (2010) reported that 3-nitrobenzanthrone caused cell death with increasing levels of DNA adducts, single-strand DNA breaks and oxidative DNA damage (measured in the comet assay).

5. Summary of Data Reported

6. Evaluation

References

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