Hydrolysis of benzo[a]pyrene diol epoxide and its covalent binding to DNA proceed through similar rate-determining steps.

Abstract

The (6,(9)(10)(11). Reaction at the guanine site is stereoselective both in vivo (6) and in vitro (9, 12) and results from asymmetries in the secondary structure of DNA (12). Minor adducts between racemic anti-BPDE [(+)anti-BPDE] and adenine (9, 10, 13), cytosine (9, 10), and the N-7 position of guanine (14) have been reported.(±)anti-BPDE is unstable in aqueous media and readily undergoes hydrolysis to form isomeric 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrenes (tetrols). Hydrolysis of (+)anti-BPDE and the syn diastereomer (in which the 7-hydroxyl group is cis to the epoxide) occurs by general acid catalysis (15)(16)(17). Furthermore, product analyses and ionic strength effects have led to similar proposals for the mechanism of (±)anti-BPDE hydrolysis (16,17). This mechanism involves a rapid equilibrium between general acid catalyst and (±)anti-BPDE, followed by proton transfer and carbonium ion formation in the rate-determining step (rds).DNA catalyzes hydrolysis of (+)anti-BPDE (18,19), and in a recent study the acid dependence of this reaction was investigated at pH 6.5-7.5 (20). The results indicated that hydrolysis in the presence of DNA was also an acid-catalyzed process (20). A model has been proposed for DNAcatalyzed hydrolysis and covalent binding in which a carbonium ion formed in the rds serves as a common intermediate for both reactions (21). The rate of DNA-catalyzed hydrolysis may be important to the total level of covalent adduct obtained and, thus, to the genesis of tumors induced by chemical carcinogens. We have, therefore, investigated the mechanisms of these reactions in an in vitro model system utilizing calf thymus DNA. Our results suggest that a carbonium ion is formed in the rds for each process, but the activated intermediates for hydrolysis and covalent binding are formed in different domains. Kinetic results support our model that carbonium ion precursors to covalent adducts are derived from physically intercalated hydrocarbons, while tetrols are formed from carbonium ions generated on the outside of the DNA helix.MATERIALS AND METHODS Synthesis. (±)anti-BPDE was synthesized as previously described (10, 12). The preparation of 3H-labeled (±)anti-BPDE has also been reported (10).Chemicals. Calf thymus DNA was obtained from Sigma. All other chemicals were obtained from commercial sources and were reagent quality or higher grade purity.Hydrolysis Kinetics. Hydrolysis of (+)anti-BPDE was followed spectrophotometrically (15-17), both at an absorption band of the hydrocarbon (345.5 nm) and at the long-wavelength red-shifted transition representing the BPDE-DNA intercalation complex (centered at 353 nm) (22, 23). Firstorder plots resulted in straight lines, indicating that hydrolysis was measured under pseudo-first-order conditions. Lines were fitted by least-squares analysis and the resulting slopes were used to determine pseudo-first-order rate constants. Replicate values for rate constants were within 5%.Covalent Binding Assays. Covalent binding of (+)anti-BPDE was meas...

Mechanism of benzo(a)pyrene induction of alpha-human chorionic gonadotropin gene expression in human lung tumor cells.

“…Cycloheximide being an inhibitor of general protein synthesis inhibits the BaP-mediated induction of AHH and thus lowers the effective level of the enzyme in the cell. Flavin mononucleotide inhibits the interaction of the metabolite with nucleic acids (22,23).…”

Non-covalent interactions of the carcinogen (+)-anti-BPDE with exon 1 of the human K-ras proto-oncogene

“…The proposed physical mechanisms leading to eventual major adduct formation have been varied. Some authors 59,60 have favored physical intercalation prior to major adduct formation. However, although physical intercalation is a process clearly established, 59,60 other authors attribute to such mechanism a greater role in (+)-anti-BPDE hydrolysis leading to tetraol formation.…”

“…Some authors 59,60 have favored physical intercalation prior to major adduct formation. However, although physical intercalation is a process clearly established, 59,60 other authors attribute to such mechanism a greater role in (+)-anti-BPDE hydrolysis leading to tetraol formation. In particular, Chen 8,61 has suggested that intercalative covalent adducts originate in intercalative physical binding whereas covalent adducts occurring at external sites in minor grooves derive from external bimolecular associations.…”

Interaction of an ultimate carcinogen, benzo[a]pyrene diol epoxide, with nucleosomal core particles: Apparent lack of protection of DNA by histone proteins

“…The interaction of the ultimate carcinogen benzo[a]pyrene diol epoxide (BPDE-I) with cellular DNA is thought to be important in carcinogenesis [1,2], and consequently the complex interactions of this diol epoxide with purified DNA have been thesubject of numerous in vitro studies [3-131. Reversible physical binding occurs with relatively fast kinetics [9] and has several characteristics associated with intercalation [5,6,13]. On a somewhat slower time scale, the hydrolysis of the epoxide with formation of nonreactive tetrols is catalyzed by DNA [4,5,7-1 I], and the exocyclic amino group of deoxyguanosine has been implicated in this reaction [ I 01.…”