title: Use of a microsome-mediated test system to assess efficacy and mechanisms of cancer chemopreventive agents.
abstract: 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.
authors.affiliation: Graduate Center for Toxicology and Preventive Medicine, University of Kentucky Medical Center, Lexington, KY 40536-0305, USA.
authors.forename: W A
authors.initials: WA
authors.lastname: Smith
authors.forename: R C
authors.initials: RC
authors.lastname: Gupta
mesh_terms: D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000378:metabolism / Q000633:toxicity; D000818:Animals; D016588:Anticarcinogenic Agents / Q000494:pharmacology*; D001564:Benzo(a)pyrene / Q000378:metabolism* / Q000633:toxicity; D002273:Carcinogens / Q000378:metabolism / Q000633:toxicity; D002417:Cattle; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D018736:DNA Adducts / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D008297:Male; D008861:Microsomes / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D016588:Anticarcinogenic Agents; D002273:Carcinogens; D018736:DNA Adducts; C043931:benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-DNA; C056612:benzo(a)pyrene-DNA adduct; D001564:Benzo(a)pyrene; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D004247:DNA