Abstract:Benzo [a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after metabolic activation by cytochrome P450 (CYP) enzymes. Here we investigated the efficiencies of rat hepatic microsomes and rat recombinant CYP1A1 expressed with its reductase, NADPH:CYP oxidoreductase (POR), NADH:cytochrome b 5 reductase, epoxide hydrolase and/or cytochrome b 5 in Supersomes TM to metabolize this carcinogen. We also studied the effectiveness of coenzymes of two of the microsomal reductases, NADPH as a coenzyme of PO… Show more
“…These findings demonstrated that cytochrome b 5 is included in the reduction of CYP3A4 catalyzed both by the NADPH/POR system and a system consisting exclusively of NADH, NADH:cytochrome b 5 reductase and cytochrome b 5 , and thus also without any participation of NADPH and POR. Our data confirmed previous results found for CYP3A4 [ 26 , 28 ] and for CYP1A1 [ 39 , 40 ] that NADH:cytochrome b 5 reductase/cytochrome b 5 /NADH can replace NADPH/POR in the catalytic cycle of the CYP reactions in the monooxygenase system [ 18 , 19 ]. This is an important finding, because substitution of the POR/NADPH system by another reductase system might be clinically relevant for compensation or the lack of defect in POR in patients suffering from diseases caused by mutations of the POR gene.…”
Section: Discussionsupporting
confidence: 92%
“…These results also suggested that NADH can even serve as an exclusive electron donor for CYP in its catalytic cycle independent of NADPH and POR. This suggestion was confirmed by the finding that NADH does not function as a coenzyme of POR when cytochrome c is used as a substrate [ 39 , 40 ].…”
Section: Resultsmentioning
confidence: 86%
“…Recently, we have found that the NADH:cytochrome b 5 reductase/cytochrome b 5 /NADH system can also substitute the POR/NADPH system and act as an electron donor to another CYP enzyme, CYP1A1, for the oxidation of benzo[ a ]pyrene (BaP). This NADH-dependent system can, therefore, function as a sole electron donor for both reduction steps in the reaction cycles of CYP1A1 [ 39 , 40 ] and CYP3A4 [ 26 , 28 ]. Here, we investigated in detail, whether the NADH:cytochrome b 5 reductase/cytochrome b 5 /NADH system can also be the sole electron donor in the CYP3A4-catalyzed oxidation of ellipticine, Another aim of this work was to shed more light on the further functions of cytochrome b 5 in this CYP3A4-mediated reaction.…”
Ellipticine is an anticancer agent that forms covalent DNA adducts after enzymatic activation by cytochrome P450 (CYP) enzymes, mainly by CYP3A4. This process is one of the most important ellipticine DNA-damaging mechanisms for its antitumor action. Here, we investigated the efficiencies of human hepatic microsomes and human recombinant CYP3A4 expressed with its reductase, NADPH:CYP oxidoreductase (POR), NADH:cytochrome b
5 reductase and/or cytochrome b
5 in Supersomes™ to oxidize this drug. We also evaluated the effectiveness of coenzymes of two of the microsomal reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of NADH:cytochrome b
5 reductase, to mediate ellipticine oxidation in these enzyme systems. Using HPLC analysis we detected up to five ellipticine metabolites, which were formed by human hepatic microsomes and human CYP3A4 in the presence of NADPH or NADH. Among ellipticine metabolites, 9-hydroxy-, 12-hydroxy-, and 13-hydroxyellipticine were formed by hepatic microsomes as the major metabolites, while 7-hydroxyellipticine and the ellipticine N
2-oxide were the minor ones. Human CYP3A4 in Supersomes™ generated only three metabolic products, 9-hydroxy-, 12-hydroxy-, and 13-hydroxyellipticine. Using the 32P-postlabeling method two ellipticine-derived DNA adducts were generated by microsomes and the CYP3A4-Supersome system, both in the presence of NADPH and NADH. These adducts were derived from the reaction of 13-hydroxy- and 12-hydroxyellipticine with deoxyguanosine in DNA. In the presence of NADPH or NADH, cytochrome b
5 stimulated the CYP3A4-mediated oxidation of ellipticine, but the stimulation effect differed for individual ellipticine metabolites. This heme protein also stimulated the formation of both ellipticine-DNA adducts. The results demonstrate that cytochrome b
5 plays a dual role in the CYP3A4-catalyzed oxidation of ellipticine: (1) cytochrome b
5 mediates CYP3A4 catalytic activities by donating the first and second electron to this enzyme in its catalytic cycle, indicating that NADH:cytochrome b
5 reductase can substitute NADPH-dependent POR in this enzymatic reaction and (2) cytochrome b
5 can act as an allosteric modifier of the CYP3A4 oxygenase.Graphical abstract
“…These findings demonstrated that cytochrome b 5 is included in the reduction of CYP3A4 catalyzed both by the NADPH/POR system and a system consisting exclusively of NADH, NADH:cytochrome b 5 reductase and cytochrome b 5 , and thus also without any participation of NADPH and POR. Our data confirmed previous results found for CYP3A4 [ 26 , 28 ] and for CYP1A1 [ 39 , 40 ] that NADH:cytochrome b 5 reductase/cytochrome b 5 /NADH can replace NADPH/POR in the catalytic cycle of the CYP reactions in the monooxygenase system [ 18 , 19 ]. This is an important finding, because substitution of the POR/NADPH system by another reductase system might be clinically relevant for compensation or the lack of defect in POR in patients suffering from diseases caused by mutations of the POR gene.…”
Section: Discussionsupporting
confidence: 92%
“…These results also suggested that NADH can even serve as an exclusive electron donor for CYP in its catalytic cycle independent of NADPH and POR. This suggestion was confirmed by the finding that NADH does not function as a coenzyme of POR when cytochrome c is used as a substrate [ 39 , 40 ].…”
Section: Resultsmentioning
confidence: 86%
“…Recently, we have found that the NADH:cytochrome b 5 reductase/cytochrome b 5 /NADH system can also substitute the POR/NADPH system and act as an electron donor to another CYP enzyme, CYP1A1, for the oxidation of benzo[ a ]pyrene (BaP). This NADH-dependent system can, therefore, function as a sole electron donor for both reduction steps in the reaction cycles of CYP1A1 [ 39 , 40 ] and CYP3A4 [ 26 , 28 ]. Here, we investigated in detail, whether the NADH:cytochrome b 5 reductase/cytochrome b 5 /NADH system can also be the sole electron donor in the CYP3A4-catalyzed oxidation of ellipticine, Another aim of this work was to shed more light on the further functions of cytochrome b 5 in this CYP3A4-mediated reaction.…”
Ellipticine is an anticancer agent that forms covalent DNA adducts after enzymatic activation by cytochrome P450 (CYP) enzymes, mainly by CYP3A4. This process is one of the most important ellipticine DNA-damaging mechanisms for its antitumor action. Here, we investigated the efficiencies of human hepatic microsomes and human recombinant CYP3A4 expressed with its reductase, NADPH:CYP oxidoreductase (POR), NADH:cytochrome b
5 reductase and/or cytochrome b
5 in Supersomes™ to oxidize this drug. We also evaluated the effectiveness of coenzymes of two of the microsomal reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of NADH:cytochrome b
5 reductase, to mediate ellipticine oxidation in these enzyme systems. Using HPLC analysis we detected up to five ellipticine metabolites, which were formed by human hepatic microsomes and human CYP3A4 in the presence of NADPH or NADH. Among ellipticine metabolites, 9-hydroxy-, 12-hydroxy-, and 13-hydroxyellipticine were formed by hepatic microsomes as the major metabolites, while 7-hydroxyellipticine and the ellipticine N
2-oxide were the minor ones. Human CYP3A4 in Supersomes™ generated only three metabolic products, 9-hydroxy-, 12-hydroxy-, and 13-hydroxyellipticine. Using the 32P-postlabeling method two ellipticine-derived DNA adducts were generated by microsomes and the CYP3A4-Supersome system, both in the presence of NADPH and NADH. These adducts were derived from the reaction of 13-hydroxy- and 12-hydroxyellipticine with deoxyguanosine in DNA. In the presence of NADPH or NADH, cytochrome b
5 stimulated the CYP3A4-mediated oxidation of ellipticine, but the stimulation effect differed for individual ellipticine metabolites. This heme protein also stimulated the formation of both ellipticine-DNA adducts. The results demonstrate that cytochrome b
5 plays a dual role in the CYP3A4-catalyzed oxidation of ellipticine: (1) cytochrome b
5 mediates CYP3A4 catalytic activities by donating the first and second electron to this enzyme in its catalytic cycle, indicating that NADH:cytochrome b
5 reductase can substitute NADPH-dependent POR in this enzymatic reaction and (2) cytochrome b
5 can act as an allosteric modifier of the CYP3A4 oxygenase.Graphical abstract
“…The 10-(deoxyguanosin- N 2 -yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP (dG- N 2 -BPDE) adduct (Fig. 1 ) is the major product of the reaction of BPDE with DNA in vitro and in vivo [ 4 , 5 , 7 – 11 ]. Fig.…”
Section: Introductionmentioning
confidence: 99%
“…One of these metabolites, 9-hydroxy-BaP (BaP-9-ol), is formed predominantly by CYP1A1 and is a precursor of 9-hydroxy-BaP-4,5-epoxide, which can also form an adduct with deoxyguanosine in DNA (Fig. 1 ) [ 4 , 5 , 10 , 11 , 17 – 19 ]. Many other CYP enzymes, CYP1B1, 2B6, 2C8, 2C9, 2C19, and 3A4, have all been reported to oxidise BaP, but their efficiencies are about one order of magnitude lower than CYP1A1 [ 3 , 9 , 14 , 20 ].…”
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 b
5 and its reductase, NADH:cytochrome b
5 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 b
5 with and without NADH:cytochrome b
5 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.Graphical abstract
Benzo[a]pyrene (BaP) is an environmental pollutant that, based on evidence largely from in vitro studies, exerts its genotoxic effects after metabolic activation by cytochrome P450s. In the present study, Hepatic Reductase Null (HRN) and Hepatic Cytochrome b5/P450 Reductase Null (HBRN) mice have been used to study the role of P450s in the metabolic activation of BaP in vivo. In HRN mice, cytochrome P450 oxidoreductase (POR), the electron donor to P450, is deleted specifically in hepatocytes. In HBRN mice the microsomal haemoprotein cytochrome b5, which can also act as an electron donor from cytochrome b5 reductase to P450s, is also deleted in the liver. Wild-type (WT), HRN and HBRN mice were treated by i.p. injection with 125 mg/kg body weight BaP for 24 h. Hepatic microsomal fractions were isolated from BaP-treated and untreated mice. In vitro incubations carried out with BaP-pretreated microsomal fractions, BaP and DNA resulted in significantly higher BaP–DNA adduct formation with WT microsomal fractions compared to those from HRN or HBRN mice. Adduct formation (i.e. 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP [dG-N2-BPDE]) correlated with observed CYP1A activity and metabolite formation (i.e. BaP-7,8-dihydrodiol) when NADPH or NADH was used as enzymatic cofactors. BaP–DNA adduct levels (i.e. dG-N2-BPDE) in vivo were significantly higher (~ sevenfold) in liver of HRN mice than WT mice while no significant difference in adduct formation was observed in liver between HBRN and WT mice. Our results demonstrate that POR and cytochrome b5 both modulate P450-mediated activation of BaP in vitro. However, hepatic P450 enzymes in vivo appear to be more important for BaP detoxification than its activation.Electronic supplementary materialThe online version of this article (10.1007/s00204-018-2162-7) contains supplementary material, which is available to authorized users.
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