ABSTRACT: Cytochrome P450 (CYP) 1A1 is the most important enzyme activating and detoxifying the human carcinogen benzo[a]pyrene (BaP). In the previous studies, we had shown that not only the canonic NADPH:CYP oxidoreductase (POR) can act as electron donor but also cytochrome b5 and its reductase, NADH:cytochrome b5 reductase. Here, we studied the role of the expression system used on the metabolites generated and the levels of DNA adducts formed by activated BaP. We used an eukaryotic and a prokaryotic cellular system (Supersomes, microsomes isolated from insect cells, and Bactosomes, a membrane fraction of Escherichia coli, each transfected with cDNA of human CYP1A1 and POR). These were reconstituted with cytochrome b5 with and without NADH:cytochrome b5 reductase. We evaluated the effectiveness of each cofactor, NADPH and NADH, to mediate BaP metabolism. We found that both systems differ in catalysing the reactions activating and detoxifying BaP. Two BaP-derived DNA adducts were generated by the CYP1A1-Supersomes, both in the presence of NADPH and NADH, whereas NADPH but not NADH was able to support this reaction in the CYP1A1-Bactosomes. Seven BaP metabolites were found in Supersomes with NADPH or NADH, whereas NADPH but not NADH was able to generate five BaP metabolites in Bactosomes. Our study demonstrates different catalytic efficiencies of CYP1A1 expressed in prokaryotic and eukaryotic cells in BaP bioactivation indicating some limitations in the use of E. coli cells for such studies.

• Klíčová slova
• Coenzymes, DNA, Enzymes, Membranes, Proteins,
• Publikační typ
• časopisecké články MeSH

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 (32)P-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.

• Klíčová slova
• Benzo[a]pyrene, Carcinogen metabolism, Cytochrome P450, DNA adducts, Mouse models, Tumour suppressor p53,
• MeSH
• adukty DNA metabolismus   MeSH
• benzopyren metabolismus   farmakokinetika   MeSH
• cytochrom P-450 CYP1A1 metabolismus   MeSH
• jaterní mikrozomy účinky léků   metabolismus   MeSH
• karcinogeny životního prostředí metabolismus   farmakokinetika   MeSH
• ledviny účinky léků   metabolismus   MeSH
• metabolická aktivace MeSH
• metabolická inaktivace MeSH
• mutantní kmeny myší MeSH
• myši inbrední C57BL MeSH
• NAD(P)H dehydrogenasa (chinon) metabolismus   MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• poškození DNA účinky léků   genetika   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adukty DNA MeSH
• benzo(a)pyrene-DNA adduct MeSH Prohlížeč
• benzopyren MeSH
• cytochrom P-450 CYP1A1 MeSH
• karcinogeny životního prostředí MeSH
• NAD(P)H dehydrogenasa (chinon) MeSH
• nádorový supresorový protein p53 MeSH
• Nqo1 protein, mouse MeSH Prohlížeč

Pulmonary inflammation can contribute to the development of lung cancer in humans. We investigated whether pulmonary inflammation alters the genotoxicity of polycyclic aromatic hydrocarbons (PAHs) in the lungs of mice and what mechanisms are involved. To model nonallergic acute inflammation, mice were exposed intranasally to lipopolysaccharide (LPS; 20 µg/mouse) and then instilled intratracheally with benzo[a]pyrene (BaP; 0.5 mg/mouse). BaP-DNA adduct levels, measured by (32)P-postlabeling analysis, were approximately 3-fold higher in the lungs of LPS/BaP-treated mice than in mice treated with BaP alone. Pulmonary Cyp1a1 enzyme activity was decreased in LPS/BaP-treated mice relative to BaP-treated mice suggesting that pulmonary inflammation impacted on BaP-induced Cyp1a1 activity in the lung. Our results showed that Cyp1a1 appears to be important for BaP detoxification in vivo and that the decrease of pulmonary Cyp1a1 activity in LPS/BaP-treated mice results in a decrease of pulmonary BaP detoxification, thereby enhancing BaP genotoxicity (ie, DNA adduct formation) in the lung. Because less BaP was detoxified by Cyp1a1 in the lungs of LPS/BaP-treated mice, more BaP circulated via the blood to extrapulmonary tissues relative to mice treated with BaP only. Indeed, we observed higher BaP-DNA adduct levels in livers of LPS/BaP-treated mice compared with BaP-treated mice. Our results indicate that pulmonary inflammation could be a critical determinant in the induction of genotoxicity in the lung by PAHs like BaP. Cyp1a1 appears to be involved in both BaP bioactivation and detoxification although the contribution of other enzymes to BaP-DNA adduct formation in lung and liver under inflammatory conditions remains to be explored.

• Klíčová slova
• DNA adducts, benzo[a]pyrene, bronchoalveolar lavage, carcinogen metabolism, cytochrome P450, pulmonary inflammation,
• MeSH
• benzopyren toxicita   MeSH
• cytochrom P-450 CYP1A1 metabolismus   MeSH
• dýchací soustava metabolismus   patologie   MeSH
• karcinogeny toxicita   MeSH
• látky znečišťující vzduch toxicita   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• pneumonie enzymologie   MeSH
• poškození DNA * MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• benzopyren MeSH
• CYP1A1 protein, human MeSH Prohlížeč
• cytochrom P-450 CYP1A1 MeSH
• karcinogeny MeSH
• látky znečišťující vzduch MeSH

UNLABELLED: 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 CYP1A1/2 enzymes was investigated with pure enzymes, enzymes present in subcellular cytosolic and microsomal fractions, selective inhibitors, and animal models (including knock-out and humanized animals). For the theoretical approaches, flexible in silico docking methods as well as ab initio calculations were employed. The results summarized in this review demonstrate that a combination of experimental and theoretical approaches is a useful tool to study the enzyme-mediated reaction mechanisms of 3-NBA and AAI reduction.

• MeSH
• acetyltransferasy metabolismus   MeSH
• adukty DNA chemie   metabolismus   MeSH
• aromatické hydroxylasy metabolismus   MeSH
• benz(a)anthraceny chemie   metabolismus   MeSH
• biokatalýza MeSH
• enzymy metabolismus   MeSH
• kyseliny aristolochové chemie   metabolismus   MeSH
• lidé MeSH
• molekulární modely * MeSH
• NAD(P)H dehydrogenasa (chinon) metabolismus   MeSH
• sulfotransferasy metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• 3-nitrobenzanthrone MeSH Prohlížeč
• acetyltransferasy MeSH
• adukty DNA MeSH
• aristolochic acid I MeSH Prohlížeč
• aromatické hydroxylasy MeSH
• benz(a)anthraceny MeSH
• enzymy MeSH
• kyseliny aristolochové MeSH
• NAD(P)H dehydrogenasa (chinon) MeSH
• sulfotransferasy MeSH