| ABSTRACT: |
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There is a growing need for short-term assays which can assess the
mechanisms and efficacy of cancer chemopreventive agents. In the present
study we have employed a microsome-mediated test system concomitantly with
DNA adduct detection to assess the efficacy of five chemopreventive
agents, N-acetylcysteine, butylated hydroxytoluene (BHT), curcumin,
oltipraz, and ellagic acid. 32P-Postlabeling analysis of DNA incubated
with benzo[a]pyrene (BP) in the presence of Aroclor 1254-induced
microsomes produced two major adducts: one derived from the interaction of
benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) with deoxyguanosine (dG) and
the other from further activation of 9-OH-BP (309 and 34 adducts/10(7)
nucleotides, respectively). With the exception of N-acetylcysteine, all
test agents significantly altered BP-DNA adduct levels: Intervention with
ellagic acid and oltipraz substantially (64-94%) inhibited both BPDE-dG
and 9-OH-BP adducts, while intervention with curcumin and BHT inhibited
the BPDE-dG adduct (57% and 38%, respectively) and enhanced the 9-OH-BP
adduct (230% and 650%, respectively). Furthermore, ellagic acid was the
only test agent observed to inhibit the anti BPDE-dG adduct in the absence
of microsomal enzymes, which is consistent with the known conjugation of
ellagic acid with BPDE. These results suggest that oltipraz may be acting
as an inhibitor of P4501A1, the isozyme involved in activation of BP to
BPDE, or by conjugation of the electrophilic species by a metabolite of
oltipraz. A plausible mechanism for inhibition of the BPDE-dG adduct and
enhancement of the 9-OH-BP adduct by curcumin and BHT includes inhibition
of epoxide hydrolase. Our results also indicate that N-acetylcysteine does
not act as an electrophilic trapping agent of BP metabolites but may exert
its protective effect in vivo by various other means, including modulation
of detoxification enzymes and altering DNA repair processes. These data
suggest that this cell-free system in conjunction with the sensitive
32P-postlabeling DNA adduct analysis may prove a viable test system for
assessing the mechanisms and efficacy of chemopreventive agents. |