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
Introduction
Epidemiological studies have shown that occupational exposure to diesel exhaust and ambient air particulate matter is associated with an increased risk of lung cancer [1], [2]. Nitropolycyclic aromatic hydrocarbons (nitro-PAHs) are widely distributed environmental pollutants found in vehicular exhaust from diesel and gasoline engines and on the surface of ambient air particulate matter; their detection in the lungs of nonsmokers with lung cancer has indicated that they may contribute to the aetiology of lung cancer [3], [4], [5]. The aromatic nitroketone 3-nitrobenzanthrone (Fig. 1; 3-nitro-7H-benz[de]anthracen-7-one; 3-NBA) is one of the most potent mutagens and a suspected human carcinogen that is found in diesel exhaust and ambient air pollution [6], [7], [8], [9]. 3-NBA is genotoxic in various short-term bioassays [6], [10], [11], [12] and specific DNA adduct formation was observed in vitro, in cell culture and in vivo in rodents [9], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23]. We found that 3-NBA is activated by cytosolic and microsomal reductases by simple nitroreduction [13], [18], [22], [23]. Moreover, previous work indicated that N-hydroxy-3-aminobenzanthrone (Fig. 1; N-OH-ABA) appears to be the critical intermediate in 3-NBA-derived DNA adduct formation [12], [20], [21], [23], [24] which can be further activated by N,O-acetyltransferases (NATs) and sulfotransferases (SULTs) [19], [21], [23]. The main metabolite of 3-NBA, 3-aminobenzanthrone (Fig. 1; 3-ABA), was recently detected in the urine of smoking and nonsmoking salt mining workers occupationally exposed to diesel emissions [8] demonstrating that exposure to 3-NBA can be significant and is detectable. Moreover, 3-ABA was also the main metabolite of 3-NBA formed in human fetal bronchial cells and rat lung alveolar type II cells [16]. In addition, 3-ABA was evaluated to be suitable for colouration of microporous polyethylene films, which are widely used for practical purposes such as separation of liquid mixtures, in particular, as separation membranes in chemical batteries [25], or an advantageous fluorescent phospholipid membrane label in the form of its N-palmitoyl derivative [26]. This suggests its industrial and/or laboratory utilization. Furthermore, even though the epidemiological study on the toxicity of 3-ABA has not yet been evaluated, formation of DNA adducts by this reductive metabolite of 3-NBA in vitro and in vivo in rodents indicates its potential genotoxicity [12], [20], [21], [24]. Understanding which enzymes are involved in the metabolic activation of 3-ABA is important in the assessment of susceptibility to this 3-NBA metabolite. We recently showed that 3-ABA is activated by cytochrome P450 (CYP) enzymes and human hepatic microsomes forming DNA adduct patterns qualitatively similar to those found in vivo in rodents treated with 3-ABA [20], [24]. The correlation of CYP-linked enzyme activities with the level of DNA binding indicated that most of the hepatic microsomal activation of 3-ABA was attributed to CYP1A1 and -1A2 [24]. Moreover, we found that 3-ABA forms the same DNA adducts as 3-NBA in vitro and in vivo in rodents [12], [20], [22], [24]. While CYP1A-mediated activation of 3-ABA in human liver microsomes [24] and in Chinese hamster lung fibroblast V79 cells expressing human recombinant human CYP1A1 or -1A2 was clearly detected [21], [24], the real participation of these enzymes in the activation process in vivo cannot be evaluated from such experiments. Therefore, we evaluated the contribution of hepatic CYP enzymes to the bioactivation of 3-ABA in vivo in a transgenic mouse model. Furthermore, besides CYP enzymes, 3-ABA might be activated to DNA binding species not only by these enzymes, but in tissues with low expression of CYP enzymes also by others. Peroxidases are known to be involved in the metabolic activation of various procarcinogens including aromatic and heterocyclic amines [27], [28], [29], [30], [31], [32], [33], [34], [35]. Peroxidases can catalyse the formation of reactive intermediates capable of forming DNA adducts. Therefore, we investigated whether different peroxidases (e.g. prostaglandin H synthase and myeloperoxidase) are capable of activating 3-ABA in vitro.
Section snippets
Synthesis of 3-ABA
3-ABA was synthesized as described recently [21] and its authenticity was confirmed by UV, electrospray mass spectra and high field proton NMR spectroscopy.
Enzyme preparations
Plant horseradish peroxidase (HRP; 300 purporogallin units/mg protein, 61 guaiacol units/mg protein), bovine lactoperoxidase (LPO; 117 purporogallin units/mg protein, 13 guaiacol units/mg protein), human myeloperoxidase (MPO; 105 purporogallin units/mg protein, 11 guaiacol units/mg protein), and ovine prostaglandin H synthase 1 (PHS-1; 44371
HRP- and LPO-catalysed 3-ABA activation
3-ABA was activated to reactive DNA-binding intermediates by two model peroxidases plant HRP and bovine LPO peroxidase in the presence of hydrogen peroxide and calf thymus DNA and the 32P-postlabelling assay was used to determine DNA adduct formation (Fig. 2A and B). No 3-ABA-derived adducts were observed when hydrogen peroxide was absent from the incubations or when 3-ABA was incubated with hydrogen peroxide alone (data not shown). The DNA adduct pattern induced by 3-ABA consisted of a cluster
Discussion
3-ABA, the major metabolite of the ubiquitous environmental pollutant 3-NBA, was detected in the urine of smoking and nonsmoking salt mining workers occupationally exposed to diesel emissions at similar concentration (1–143 ng/24 h urine) to 1-aminopyrene (2–200 ng/24 h urine), the corresponding amine of the most abundant nitro-PAH detected in diesel exhaust matter [8]. Recently we found that 3-ABA is activated by human and rat hepatic microsomes generating a DNA adduct pattern identical to that
Acknowledgements
This work was supported by Cancer Research UK and Grant Agency of the Czech Republic (Grant 303/05/2195).
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