Benzo[a]pyrene (BaP)
is a human carcinogen that
covalently binds to DNA after activation by cytochrome P450 (P450).
Here, we investigated whether NADH:cytochrome b5 reductase (CBR) in the presence of cytochrome b5 can act as sole electron donor to human P450 1A1 during
BaP oxidation and replace the canonical NADPH:cytochrome P450 reductase
(POR) system. We also studied the efficiencies of the coenzymes of
these reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme
of CBR, to mediate BaP oxidation. Two systems containing human P450
1A1 were utilized: human recombinant P450 1A1 expressed with POR,
CBR, epoxide hydrolase, and cytochrome b5 in Supersomes and human recombinant P450 1A1 reconstituted with
POR and/or with CBR and cytochrome b5 in
liposomes. BaP-9,10-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione,
BaP-3,6-dione, BaP-9-ol, BaP-3-ol, a metabolite of unknown structure,
and two BaP-DNA adducts were generated by the P450 1A1-Supersomes
system, both in the presence of NADPH and in the presence of NADH.
The major BaP-DNA adduct detected by 32P-postlabeling was
characterized as 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP
(assigned adduct 1), while the minor adduct is probably a guanine
adduct derived from 9-hydroxy-BaP-4,5-epoxide (assigned adduct 2).
BaP-3-ol as the major metabolite, BaP-9-ol, BaP-1,6-dione, BaP-3,6-dione,
an unknown metabolite, and adduct 2 were observed in the system using
P450 1A1 reconstituted with POR plus NADPH. When P450 1A1 was reconstituted
with CBR and cytochrome b5 plus NADH,
BaP-3-ol was the predominant metabolite too, and an adduct 2 was also
generated. Our results demonstrate that the NADH/cytochrome b5/CBR system can act as the sole electron donor
both for the first and second reduction of P450 1A1 during the oxidation
of BaP in vitro. They suggest that NADH-dependent
CBR can replace NADPH-dependent POR in the P450 1A1-catalyzed metabolism
of BaP.