• 3-aminobenzanthrone, a human metabolite of the carcinogenic environmental pollutant 3-nitrobenzanthrone, induces biotransformation enzymes in rat kidney and lung.

      Stiborova, Marie; Dracinska, Helena; Martinkova, Marketa; Mizerovska, Jana; Hudecek, Jiri; Hodek, Petr; Liberda, Jiri; Frei, Eva; Schmeiser, Heinz H.; Phillips, David H.; et al. (2009-05-31)
      3-aminobenzanthrone (3-ABA) is the metabolite of the carcinogenic air pollutant 3-nitrobenzanthrone (3-NBA). 3-ABA was investigated for its ability to induce cytochrome P450 1A1 (CYP1A1) and NAD(P)H:quinone oxidoreductase (NQO1) in kidney and lung of rats, and for the influence of such induction on DNA adduct formation by 3-ABA and 3-NBA. NQO1 is the enzyme that reduces 3-NBA to N-hydroxy-3-aminobenzanthrone (N-OH-3-ABA) and CYP1A enzymes oxidize 3-ABA to the same intermediate. When activated by cytosolic and and/or microsomal fractions isolated from rat lung, the target organ for 3-NBA carcinogenicity, and kidney, both compounds generated the same DNA-adduct pattern, consisting of five adducts. When pulmonary cytosols isolated from rats that had been treated i.p. with 40 mg/kg bw of 3-ABA were incubated with 3-NBA, DNA adduct formation was up to 1.7-fold higher than in incubations with cytosols from control animals. This increase corresponded to an increase in protein level and enzymatic activity of NQO1. In contrast, no induction of NQO1 expression by 3-ABA treatment was found in the kidney. Incubations of 3-ABA with renal and pulmonary microsomes of 3-ABA-treated rats led to an increase of up to a 4.5-fold in DNA-adduct formation relative to controls. The stimulation of DNA-adduct formation correlated with a higher protein expression and activity of CYP1A1 induced by 3-ABA. These results show that by inducing lung and kidney CYP1A1 and NQO1, 3-ABA increases its own enzymatic activation as well as that of the environmental pollutant, 3-NBA, thereby enhancing the genotoxic and carcinogenic potential of both compounds.
    • The environmental pollutant and carcinogen 3-nitrobenzanthrone induces cytochrome P450 1A1 and NAD(P)H:quinone oxidoreductase in rat lung and kidney, thereby enhancing its own genotoxicity.

      Stiborova, Marie; Dracinska, Helena; Mizerovska, Jana; Frei, Eva; Schmeiser, Heinz H.; Hudecek, Jiri; Hodek, Petr; Phillips, David H.; Arlt, Volker M. (2008-05-02)
      3-Nitrobenzanthrone (3-NBA) is a carcinogen occurring in diesel exhaust and air pollution. Using the (32)P-postlabelling method, we found that 3-NBA and its human metabolite, 3-aminobenzanthrone (3-ABA), are activated to species forming DNA adducts by cytosols and/or microsomes isolated from rat lung, the target organ for 3-NBA carcinogenicity, and kidney. Each compound generated identical five DNA adducts. We have demonstrated the importance of pulmonary and renal NAD(P)H:quinone oxidoreductase (NQO1) to reduce 3-NBA to species that are further activated by N,O-acetyltransferases and sulfotransferases. Cytochrome P450 (CYP) 1A1 is the essential enzyme for oxidative activation of 3-ABA in microsomes of both organs, while cyclooxygenase plays a minor role. 3-NBA was also investigated for its ability to induce NQO1 and CYP1A1 in lungs and kidneys, and for the influence of such induction on DNA adduct formation by 3-NBA and 3-ABA. When cytosols from rats treated i.p. with 40mg/kg bw of 3-NBA were incubated with 3-NBA, DNA adduct formation was up to 2.1-fold higher than in incubations with cytosols from control animals. This increase corresponded to an increase in protein level and enzymatic activity of NQO1. Incubations of 3-ABA with microsomes of 3-NBA-treated rats led to up to a fivefold increase in DNA adduct formation relative to controls. The stimulation of DNA adduct formation correlated with the potential of 3-NBA to induce protein expression and activity of CYP1A1. These results demonstrate that 3-NBA is capable to induce NQO1 and CYP1A1 in lungs and kidney of rats thereby enhancing its own genotoxic and carcinogenic potential.
    • Quantification of 3-nitrobenzanthrone-DNA adducts using online column-switching HPLC-electrospray tandem mass spectrometry.

      Gamboa da Costa, Goncalo; Singh, Rajinder; Arlt, Volker M.; Mirza, Amin; Richards, Meirion; Takamura-Enya, Takeji; Schmeiser, Heinz H.; Farmer, Peter B.; Phillips, David H. (2009-11)
      The aromatic nitroketone 3-nitrobenzanthrone (3-nitro-7H-benz[de]anthracen-7-one; 3-NBA) is an extremely potent mutagen and a suspected human carcinogen detected in the exhaust of diesel engines and in airborne particulate matter. 3-NBA is metabolically activated via reduction of the nitro group to the hydroxylamine (N-OH-3-ABA) to form covalent DNA adducts. Thus far, the detection and quantification of covalent 3-NBA-DNA adducts has relied solely on (32)P-postlabeling methodologies. In order to expand the range of available techniques for the detection and improved quantification of 3-NBA-DNA adducts, we have developed a method based upon online column-switching HPLC coupled to electrospray tandem mass spectrometry, with isotopic dilution of (15)N-labeled internal standards. This methodology was applied to the determination of three 3-NBA-derived adducts: 2-(2'-deoxyguanosin-N(2)-yl)-3-aminobenzanthrone (dG-N(2)-3-ABA), N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG-C8-N-3-ABA) and 2-(2'-deoxyguanosine-8-yl)-3-aminobenzanthrone (dG-C8-C2-3-ABA). Dose-dependent increases were observed for all three adducts when salmon testis DNA was reacted with N-acetoxy-3-aminobenzanthrone (N-AcO-3-ABA). dG-C8-C2-3-ABA was detected at much lower levels (overall 1%) than the other two adducts. DNA samples isolated from tissues of rats treated either intratracheally with 3-NBA or intraperitoneally with N-OH-3-ABA were analyzed by mass spectrometry, and the results compared to those obtained by (32)P-postlabeling. The method required 50 microg of hydrolyzed animal DNA on column and the limit of detection was 2.0 fmol for each adduct. dG-C8-C2-3-ABA was not observed in any of the samples providing confirmation that it is not formed in vivo. Linear regression analysis of the levels of dG-N(2)-3-ABA and dG-C8-N-3-ABA in the rat DNA showed a reasonable correlation between the two methods (R(2) = 0.88 and 0.93, respectively). In summary, the mass spectrometric method is a faster, more automated analytical approach that also provides structural confirmation of the adducts detected by (32)P-postlabeling, and it has sufficient sensitivity and precision to analyze DNA adducts in animals exposed to 3-NBA or its hydroxylamine metabolite.