Reaction of a chemotherapeutic agent, 6-mercaptopurine, with a direct-acting, electrophilic carcinogen, benzo[a]pyrene-7,8-diol 9,10-epoxide

Abstract

The chemotherapeutic agent 6-mercaptopurine (6-MP) has been shown to react covalently with the ultimate carcinogenic metabolite of benzo[a]pyrene, 7-r,8-t-dihydroxy-9-t,10-t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), in aqueous solution, forming a single adduct. NMR studies of the HPLC-purified product were consistent with its identification as 10(S)-(6'-mercaptopurinyl)-7,8,9-trihydroxy-7,8,9,10- tetrahydrobenzo[a]pyrene. Reaction kinetics were analyzed by using both HPLC separation of the products formed and a spectrophotometric assay for adduct formation. A simple model in which direct reaction between 6-MP and BPDE takes place without formation of a physical complex was found to adequately predict the dependence of product ratios on 6-MP concentration. Variations in the observed rate constant for this reaction with changes in temperature, pH, and buffer concentration were determined and compared to the effects of these variables on the observed rate constant for BPDE hydrolysis. In each case, the processes were affected quite differently, suggesting that different rate-determining steps are involved. The data suggest that the reaction mechanism involves SN2 attack of the anion of 6-MP, formed by ionization of the sulfhydryl group, on carbon 10 of BPDE, resulting in a trans-9,10 reaction product.

Identification of a Novel, N7-Deoxyguanosine Adduct as the Major DNA Adduct Formed by a Non-Bay-Region Diol Epoxide of Benzo[a]pyrene with Low Mutagenic Potential

“…characteristic of an NH group (Jeffrey et al, 1976b;Roy et al, 1991), while for H8 it is at 5.14 ppm (J = 5.4 Hz), characteristic of an OH group (MacLeod et al, 1991c;MacLeod et al, 1993). Thus, the data indicate attachment of an NH group at C7 and the presence of an OH group at C8 of BPTT.…”

Kinetics of the Reaction of a Potential Chemopreventive Agent, 2,6-Dithiopurine, and Its Major Metabolite, 2,6-Dithiouric Acid, with Multiple Classes of Electrophilic Toxicants

“…Our previous data indicated that purinethiols form adducts with BPDE in vitro , in CHO cell culture, and after topical application to mouse epidermis ( − ). This was correlated with decreases in DNA binding and with decreases in the biological activity of BPDE: cytotoxicity and mutagenicity in CHO cells (), and initiation of tumorigenesis in mouse skin ().…”

“…The adduct concentrations in DTP−melphalan reactions were obtained from the known starting concentrations of melphalan minus the hydrolyzed melphalan concentrations. This allowed us to determine the product ratio, f c , and the binding rate constants were obtained from double reciprocal plots according to eq 3 ( , ).…”

“…Previous studies from this laboratory ( , ) demonstrated that several nucleophilic purine derivatives that contain thiol moieties linked to the 2- and/or 6-carbon positions react facilely with the electrophilic, ultimate carcinogen 7 r ,8 t -dihydroxy-9,10 t -oxy-7,8,9,10-tetrahydrobenzo[ a ]pyrene (BPDE). These reactions were characterized as S N 2 substitutions, with addition of a thiol to the 10-carbon of BPDE.…”

“…One of the most active of these nucleophiles was 2,6-dithiopurine (DTP). Presumably due to this scavenging reaction, several purinethiols, including 6-mercaptopurine and DTP, and the related compound thiopurinol blocked the binding of BPDE to DNA in vitro , in Chinese hamster ovary cells, and in mouse skin ( − ) in a dose-dependent manner. The finding that DNA binding in cells can be virtually abolished by purinethiols and the identification of purinethiol−BPDE adducts in treated cells and tissue ( , ) indicate that purinethiols can be an effective complement to endogenous nucleophiles in reducing DNA damage from electrophilic toxicants.…”