Mechanisms of Enzyme-Catalyzed Reduction of Two Carcinogenic Nitro-Aromatics, 3-Nitrobenzanthrone and Aristolochic Acid I: Experimental and Theoretical Approaches

Abstract: This review summarizes the results found in studies investigating the enzymatic activation of two genotoxic nitro-aromatics, an environmental pollutant and carcinogen 3-nitrobenzanthrone (3-NBA) and a natural plant nephrotoxin and carcinogen aristolochic acid I (AAI), to reactive species forming covalent DNA adducts. Experimental and theoretical approaches determined the reasons why human NAD(P)H:quinone oxidoreductase (NQO1) and cytochromes P450 (CYP) 1A1 and 1A2 have the potential to reductively activate both nitro-aromatics. The results also contributed to the elucidation of the molecular mechanisms of these reactions. The contribution of conjugation enzymes such as N,O-acetyltransferases (NATs) and sulfotransferases (SULTs) to the activation of 3-NBA and AAI was also examined. The results indicated differences in the abilities of 3-NBA and AAI metabolites to be further activated by these conjugation enzymes. The formation of DNA adducts generated by both carcinogens during their reductive activation by the NOQ1 and CYP1A1/

Chemical and molecular basis of the carcinogenicity of Aristolochia plants

The herbal drug aristolochic acid (AA), which is derived from the Aristolochia species, has been associated with the development of a novel nephropathy, designated as aristolochic acid nephropathy (AAN), and with human urothelial cancer. The major components of the plant extract AA are nitrophenanthrene carboxylic acids, which, after metabolic activation, are genotoxic mutagens. The major activation pathway of AA involves reduction of the nitro group, primarily catalyzed by NAD(P) H: quinone oxidoreductase (NQO1), to an electrophilic cyclic N-acylnitrenium ion that reacts preferentially with purine bases to form covalent DNA adducts. These specific AA-DNA adducts have been identified and detected in experimental animals exposed to AA or botanical products containing AA, and in urothelial tissues from AAN patients. In rodent tumors induced by AA the predominantly formed DNA adduct 7-(deoxyadenosin-N-6-yl) aristolactam I has been associated with the activation of ras oncogenes through the characteristic transversion mutation AT -> TA. This mutation has been identified in the p53 gene of urothelial tumors of a patient with AAN (induced by use of a herbal product) and in several patients suffering from Balkan endemic nephropathy (BEN) with specific AA-DNA adducts. This is a rare example of a human cancer causally linked to a distinct environmental exposure (ie, use of a herbal product), where the carcinogenic process of initiation is well established, linking formation of carcinogen-specific exposure (specific DNA adduct formation) with the presence of characteristic human tumor mutations

Metabolic activation of carcinogenic aristolochic acid, a risk factor for Balkan endemic nephropathy

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, is associated with tumor development in patients suffering from Chinese herbs nephropathy (now termed aristolochic acid nephropathy, AAN) and may also be a cause for the development of a similar type of nephropathy, the Balkan endemic nephropathy (BEN). Major DNA adducts [7-(deoxyadenosin-N-6-yl)-aristolactam and 7-(deoxyguanosin-N-2-yl)aristolactam] formed from AA after reductive metabolic activation were found in renal tissues of patients with both diseases. Understanding which human enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual's susceptibility to this plant carcinogen. This paper reviews major hepatic and renal enzymes responsible for AA-DNA adduct formation in humans. Phase I biotransformation enzymes play a crucial role in the metabolic activation of AA to species forming DNA adducts, while a role of phase II enzymes in this process is questionable. Most of the activation of AA in human hepatic microsomes is mediated by cytochrome P450 (CYP) 1A2 and, to a lower extent, by CYP1A1; NADPH:CYP reductase plays a minor role. In human renal microsomes NADPH:CYP reductase is more effective in AA activation. Prostaglandin H synthase (cyclooxygenase, COX) is another enzyme activating AA in human renal microsomes. Among the cytosolic reductases, NAD(P)H:quinone oxidoreductase (NQO I) is the most efficient in the activation of AA in human liver and kidney. Studies with purified enzymes confirmed the importance of CYPs, NADPH:CYP reductase, COX and NQO1 in the AA activation. The orientation of AA in the active sites of human CYP1A1, -1A2 and NQO1 was predicted from molecular modeling and explains the strong reductive potential of these enzymes for AA detected experimentally. We hypothesized that inter-individual variations in expressions and activities of enzymes activating AA may be one of the causes responsible for the different susceptibilities to this carcinogen reflected in the development of AA-induced nephropathies and associated urothelial cancer

The genotoxic air pollutant 3-nitrobenzanthrone and its reactive metabolite N-hydroxy-3-aminobenzanthrone lack initiating and complete carcinogenic activity in NMRI mouse skin

3-Nitrobenzanthrone (3-NBA), a genotoxic mutagen found in diesel exhaust and ambient air pollution and its active metabolite N-hydroxy-3-aminobenzanthrone (N-OH-3-ABA) were tested for initiating and complete carcinogenic activity in the NMRI mouse skin carcinogenesis model. Both compounds were found to be inactive as either tumour initiators or complete carcinogens in mouse skin over a dose range of 25-400 nmol. Topical application of 3-NBA and N-OH-3-ABA produced DNA adduct patterns in epidermis, detected by P-32-postlabelling, similar to those found previously in other organs of rats and mice. 24 h after a single treatment of 100 nmol DNA adduct levels produced by 3-NBA (18 +/- 4 adducts/10(8) nucleotides) were 6 times lower than those by 7,12-dimethylbenz[a]anthracene (DMBA; 114 +/- 37 adducts/10(8) nucleotides). In contrast, identical treatment with N-OH-3-ABA resulted in adduct levels in the same range as with DMBA (136 +/- 25 adducts/10(8) nucleotides), indicating that initial DNA adduct levels do not parallel tumour initiating activity. When compounds were tested for tumour initiating activity by a single treatment followed by twice-weekly applications of TPA. DNA adducts formed by DMBA, but not by 3-NBA or N-OH-3-ABA, were still detectable 40 weeks after treatment. When tested for activity as complete carcinogens by twice-weekly topical application, 3-NBA and N-OH-3-ABA produced identical DNA adduct profiles in mouse skin, with adducts still detectable after 40 weeks. Only 3-NBA produced detectable adducts in other organs

The impact of p53 on DNA damage and metabolic activation of the environmental carcinogen benzo[a]pyrene: effects in Trp53(+/+), Trp53(+/–) and Trp53(−/−) mice

The tumour suppressor p53 is one of the most important cancer genes. Previous findings have shown that p53 expression can influence DNA adduct formation of the environmental carcinogen benzo[a]pyrene (BaP) in human cells, indicating a role for p53 in the cytochrome P450 (CYP) 1A1-mediated biotransformation of BaP in vitro. We investigated the potential role of p53 in xenobiotic metabolism in vivo by treating Trp53(+/+), Trp53(+/-) and Trp53(-/-) mice with BaP. BaP-DNA adduct levels, as measured by 32P-postlabelling analysis, were significantly higher in liver and kidney of Trp53(-/-) mice than of Trp53(+/+) mice. Complementarily, significantly higher amounts of BaP metabolites were also formed ex vivo in hepatic microsomes from BaP-pretreated Trp53(-/-) mice. Bypass of the need for metabolic activation by treating mice with BaP-7,8-dihydrodiol-9,10-epoxide resulted in similar adduct levels in liver and kidney in all mouse lines, confirming that the influence of p53 is on the biotransformation of the parent compound. Higher BaP-DNA adduct levels in the livers of Trp53(-/-) mice correlated with higher CYP1A protein levels and increased CYP1A enzyme activity in these animals. Our study demonstrates a role for p53 in the metabolism of BaP in vivo, confirming previous in vitro results on a novel role for p53 in CYP1A1-mediated BaP metabolism. However, our results also suggest that the mechanisms involved in the altered expression and activity of the CYP1A1 enzyme by p53 in vitro and in vivo are different

Oxidation of 3-aminobenzanthrone, a human metabolite of carcinogenic environmental pollutant 3-nitrobenzanthrone, by cytochromes P450-similarity between human and rat enzymes

OBJECTIVES: 3-Aminobenzanthrone (3-ABA) is the main human metabolite of carcinogenic environmental pollutant 3-nitrobenzanthrone (3-NBA). Understanding which cytochrome P450 (CYP) enzymes are involved in metabolism of this toxicant is important in the assessment of individual susceptibility. Characterization of 3-ABA metabolites formed by rat hepatic microsomes containing cytochromes P450 (CYPs) and identification of the major rat and human CYPs participating in this process are aims of this study. METHODS: HPLC with UV detection was employed for the separation and characterization of 3-ABA metabolites. Inducers and inhibitors of CYPs and rat and human recombinant CYPs were used to characterize the enzymes participating in 3-ABA oxidation. RESULTS: Selective CYP inhibitors and hepatic microsomes of rats pre-treated with specific CYP inducers were used to characterize rat liver CYPs metabolizing 3-ABA (measured as consumption of 3-ABA). Kinetics of these reactions catalyzed by rat hepatic microsomes was also evaluated. Based on these studies, we attribute most of 3-ABA metabolism in rat liver to CYP1A and 3A. Among recombinant rat and human CYP enzymes tested in this study, rat CYP3A2 and human CYP3A4/5, followed by CYP I A I of both organisms were the most effective enzymes converting 3-ABA. Rat hepatic CYP enzymes oxidize 3-ABA up to three metabolites. Two of them were identified to be the products formed by oxidation of 3-ABA on its amino group back to the parent compound from which 3-ABA is generated in organisms, 3-NBA. Namely, N-hydroxylation metabolite, N-hydroxy-3-ABA and 3-NBA were identified to be these 3-ABA oxidation products. These metabolites are formed by CYPs of a I A subfamily. Another 3-ABA metabolite, whose structure remains to be characterized, is generated not only by CYP1A but also by other CYP enzymes, predominantly by CYPs of a 3A subfamily. CONCLUSION: The results found in this study, the first report on the metabolism of 3-ABA by human and rat CYPs, clearly demonstrate that CYPs of 3A and 1A subfamilies are the major enzymes metabolizing 3-ABA

Induction of biotransformation enzymes by the carcinogenic air-pollutant 3-nitrobenzanthrone in liver, kidney and lung, after intra-tracheal instillation in rats

3-Nitrobenzanthrone (3-NBA), a carcinogenic air pollutant, was investigated for its ability to induce cytochrome P450 (CYP) 1A1/2 and NAD(P)H:quinone oxidoreductase (NQO1) in liver, kidney and lung of rats treated by intra-tracheal instillation. The organs used were from a previous study performed to determine the persistence of 3-NBA-derived DNA adducts in target and non-target tissues (Bieler et al., Carcinogenesis 28 (2007) 1117-1121, [22]). NQO1 is the enzyme reducing 3-NBA to N-hydroxy-3-aminobenzanthrone (N-OH-3-ABA) and CYP1A enzymes oxidize a human metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA), to yield the same reactive intermediate. 3-NBA and 3-ABA are both activated to species forming DNA adducts by cytosols and/or microsomes isolated from rat lung, the target organ for 3-NBA carcinogenicity, and from liver and kidney. Each compound generated the same five DNA adducts detectable by P-32-postlabelling. When hepatic cytosols from rats treated with 0.2 or 2 mg/kg body weight of 3-NBA were incubated with 3-NBA, DNA adduct formation was 3.2- and 8.6-fold higher, respectively, than in incubations with cytosols from control animals. Likewise, cytosols isolated from lungs and kidneys of rats exposed to 3-NBA more efficiently activated 3-NBA than those of control rats. This increase corresponded to an increase in protein levels and enzymatic activities of NQO1. Incubations of hepatic, pulmonary or renal microsomes of 3-NBA-treated rats with 3-ABA led to an 9.6-fold increase in DNA-adduct formation relative to controls. The highest induction in DNA-adduct levels was found in lung. The stimulation of DNA-adduct formation correlated with expression of CYP1A1/2 induced by the intra-tracheal instillation of 3-NBA. The results demonstrate that 3-NBA induces NQO1 and CYP1A1/2 in livers, lungs and kidneys of rats after intra-tracheal instillation, thereby enhancing its own genotoxic and carcinogenic potential. (c) 2010 Elsevier B.V. All rights reserved

The effects of heavy metal ions, phthalates and ochratoxin A on oxidation of carcinogenic aristolochic acid I causing Balkan endemic nephropathy

OBJECTIVES: Balkan endemic nephropathy (BEN) is a chronic progressive fibrosis associated with upper urothelial carcinoma (UUC). Aetiology of BEN is still not fully explained. Although carcinogenic aristolochic acid I (AAI) was proven as the major cause of BEN/UUC, this nephropathy is considered to be multifactorial. Hence, we investigated whether other factors considered as potential causes of BEN [a mycotoxin ochratoxin A (OTA), Cd, Pb, Se and As ions and organic compounds (i.e. phthalates) released from lignite deposits in BEN areas] can influence detoxication of AAI, whose concentrations are crucial for BEN development.METHODS: Oxidation of AAI to 8-hydroxyaristolochic acid I (AAIa) in the presence of Cd, Pb, Se, As ions, dibutylphthalate (DBP), butylbenzylphthalate (BBP), bis(2-ethylhexyl)phthalate (DEHP) and OTA by rat liver microsomes was determined by HPLC.RESULTS: Only OTA, cadmium and selenium ions, and BBP inhibited AAI oxidation by rat liver microsomes. These compounds also inhibited activities of CYP1A1 and/or CYP2C6/11 catalysing AAI demethylation in rat livers. Therefore, these CYP inhibitions can be responsible for a decrease in AAIa formation. When the combined effects of these compounds were investigated, the most efficient inhibition was caused by OTA combined with BBP and selenium ions.CONCLUSION: The results show low effects of BBP, cadmium and selenium ions, and/or their combinations on AAI detoxication. No effects were produced by the other metal ions (Pb, As) and phthalates DBP and DEHP. This finding suggests that they do not influence AAI-mediated BEN development. In contrast, OTA might influence this process, by inhibition of AAI detoxication.</p

The effect of aristolochic acid I on expression of NAD(P)H:quinone oxidoreductase in mice and rats:A comparative study

Aristolochic acid is the cause of aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN) and their associated urothelial malignancies. Using Western blotting, we investigated the expression of NAD(P)H:quinone oxidoreductase (NQO1), the most efficient cytosolic enzyme that reductively activates aristolochic acid I (AAI) in mice and rats. In addition, the effect of AAI on the expression of the NQO1 protein and its enzymatic activity in these experimental animal models was examined. We found that NQO1 protein levels in cytosolic fractions isolated from liver, kidney and lung of mice differed from those expressed in these organs of rats. In mice, the highest levels of NQO1 protein and NQO1 activity were found in the kidney, followed by lung and liver. In contrast, the NQO1 protein levels and enzyme activity were lowest in rat-kidney cytosol, whereas the highest amounts of NQO1 protein and activity were found in lung cytosols, followed by those of liver. NQO1 protein and enzyme activity were induced in liver and kidney of AAI-pretreated mice compared with those of untreated mice. NQO1 protein and enzyme activity were also induced in rat kidney by AAI. Furthermore, the increase in hepatic and renal NQO1 enzyme activity was associated with AAI bio-activation and elevated AAI-DNA adduct levels were found in ex vivo incubations of cytosolic fractions with DNA and AAI. In conclusion, our results indicate that AAI can increase its own metabolic activation by inducing NQO1, thereby enhancing its own genotoxic potential