Cytochrome b₅ and epoxide hydrolase contribute to benzo[a]pyrene-DNA adduct formation catalyzed by cytochrome P450 1A1 under low NADPH:P450 oxidoreductase conditions

TY - JOUR

T1 - Cytochrome b5 and epoxide hydrolase contribute to benzo[a]pyrene-DNA adduct formation catalyzed by cytochrome P450 1A1 under low NADPH:P450 oxidoreductase conditions

AU - Stiborová, Marie

AU - Moserová, Michaela

AU - Černá, Vera

AU - Indra, Radek

AU - Dračínský, Martin

AU - Šulc, Miroslav

AU - Henderson, Colin J.

AU - Wolf, C. Roland

AU - Schmeiser, Heinz H.

AU - Phillips, David H.

AU - Frei, Eva

AU - Arlt, Volker M.

PY - 2014/4/6

Y1 - 2014/4/6

N2 - In previous studies we had administered benzo[a]pyrene (BaP) to genetically engineered mice (HRN) which do not express NADPH:cytochrome P450 oxidoreductase (POR) in hepatocytes and observed higher DNA adduct levels in livers of these mice than in wild-type mice. To elucidate the reason for this unexpected finding we have used two different settings for in vitro incubations; hepatic microsomes from control and BaP-pretreated HRN mice and reconstituted systems with cytochrome P450 1A1 (CYP1A1), POR, cytochrome b, and epoxide hydrolase (mEH) in different ratios. In microsomes from BaP-pretreated mice, in which Cyp1a1 was induced, higher levels of BaP metabolites were formed, mainly of BaP-7,8-dihydrodiol. At a low POR:CYP1A1 ratio of 0.05:1 in the reconstituted system, the amounts of BaP diones and BaP-9-ol formed were essentially the same as at an equimolar ratio, but formation of BaP-3-ol was ~1.6-fold higher. Only after addition of mEH were BaP dihydrodiols found. Two BaP-DNA adducts were formed in the presence of mEH, but only one when CYP1A1 and POR were present alone. At a ratio of POR:CYP1A1 of 0.05:1, addition of cytochrome b increased CYP1A1-mediated BaP oxidation to most of its metabolites indicating that cytochrome b participates in the electron transfer from NADPH to CYP1A1 required for enzyme activity of this CYP. BaP-9-ol was formed even by CYP1A1 reconstituted with cytochrome b without POR. Our results suggest that in livers of HRN mice Cyp1a1, cytochrome b and mEH can effectively activate BaP to DNA binding species, even in the presence of very low amounts of POR.

AB - In previous studies we had administered benzo[a]pyrene (BaP) to genetically engineered mice (HRN) which do not express NADPH:cytochrome P450 oxidoreductase (POR) in hepatocytes and observed higher DNA adduct levels in livers of these mice than in wild-type mice. To elucidate the reason for this unexpected finding we have used two different settings for in vitro incubations; hepatic microsomes from control and BaP-pretreated HRN mice and reconstituted systems with cytochrome P450 1A1 (CYP1A1), POR, cytochrome b, and epoxide hydrolase (mEH) in different ratios. In microsomes from BaP-pretreated mice, in which Cyp1a1 was induced, higher levels of BaP metabolites were formed, mainly of BaP-7,8-dihydrodiol. At a low POR:CYP1A1 ratio of 0.05:1 in the reconstituted system, the amounts of BaP diones and BaP-9-ol formed were essentially the same as at an equimolar ratio, but formation of BaP-3-ol was ~1.6-fold higher. Only after addition of mEH were BaP dihydrodiols found. Two BaP-DNA adducts were formed in the presence of mEH, but only one when CYP1A1 and POR were present alone. At a ratio of POR:CYP1A1 of 0.05:1, addition of cytochrome b increased CYP1A1-mediated BaP oxidation to most of its metabolites indicating that cytochrome b participates in the electron transfer from NADPH to CYP1A1 required for enzyme activity of this CYP. BaP-9-ol was formed even by CYP1A1 reconstituted with cytochrome b without POR. Our results suggest that in livers of HRN mice Cyp1a1, cytochrome b and mEH can effectively activate BaP to DNA binding species, even in the presence of very low amounts of POR.

UR - https://www.scopus.com/pages/publications/84896850394

U2 - 10.1016/j.tox.2014.02.002

DO - 10.1016/j.tox.2014.02.002

M3 - Article

AN - SCOPUS:84896850394

SN - 0300-483X

VL - 318

SP - 1

EP - 12

JO - Toxicology

JF - Toxicology

IS - 1

ER -