N. V. S. Ramakrishna

Synthesis and Structure Determination of the Adducts of Dibenzo[a,l]pyrene Diol Epoxides and Deoxyadenosine or Deoxyguanosine

Abstract (±)−Syn−dibenzo[a,l]pyrene diol epoxide (DB[a,l]PDE) and (±)−anti−DB[a,l]PDE were reacted with deoxyadenosine (dA) or deoxyguanosine (dG) in dimethylformamide at 100 °C for 30 min. The crude products were purified by reverse phase HPLC under gradient and isocratic conditions. The structure of each adduct was assigned by 1D and 2D NMR spectra and by fast atom bombardment mass spectrometry. Five adducts were isolated from the reaction of (±)−syn−DB[a,l]PDE and dA: syn−DB[a,l]PDE−N6dA−1, syn−DB[a,l]PDE−N6dA−2, syn−DB[a,l]PDE−N6dA−3, syn−DB[a,l]PDE−N6dA−4 and syn−DB[a,l]PDE−N7Ade. Four adducts were isolated from the reaction of (±)−anti−DB[a,l]PDE and dA: anti−DB[a,l]PDE−N6dA−1, anti−DB[a,l]PDE−N6dA−2, anti−DB[a,l]PDE−N6dA−3 and anti−DB[a,l]PDE−N6dA−4. Two adducts were isolated from the reaction of (±)−syn−DB[a,l]PDE and dG: (±)−11,12,13−trihydroxy−tetrahydroDB[a,l]P−14−N2dG and (±)−11,12,13−trihydroxy−tetrahydroDB[a,l]P−14−N7Gua. Two adducts were isolated from the reaction of (±)−anti−DB[a,l]PDE and...

Metabolism of 1- and 3-Fluorobenzo[a]pyrene by Cytochrome p450 and Horseradish Peroxidase

Abstract Benzo[a]pyrene (BP) is a good model for elucidating the mechanism of oxygen transfer for substrates that are considered good electron donors. Fluoro substitution of BP represents a suitable probe for studying mechanisms of oxygen transfer in the metabolic formation of BP quinones and BP phenols. By using this strategy with 6-fluoroBP (6-FBP), we have previously demonstrated that the BP quinones are formed metabolically via an initial one-electron oxidation of BP catalyzed by cytochrome P450. Now we have synthesized 1-FBP and 3-FBP with the purpose of elucidating the mechanism of phenol formation. If formation of 3-hydroxyBP (3-OHBP) (major metabolite of BP) and 1-OHBP (minor metabolite of BP) occurs via an initial electron transfer from BP to cytochrome P450, when we use 3-FBP and 1-FBP as substrates, one of the metabolites formed should be BP-3,6-dione and BP-1,6-dione, respectively, with displacement of fluorine. Metabolism of 1-FBP and 3-FBP by rat liver microsomes and by horseradish peroxidas...

Development of Methods for the Production of Monoclonal Antibodies Specific for Depurination Adducts of Benzo[a]pyrene and for Their Use in a Competitive Enzyme-Linked Immunosorbent Assay

Abstract Nearly 80% of benzo[a]pyrene (BP)-DNA adducts are lost by depurination and excreted in the urine. These adducts offer a unique opportunity for biomonitoring BP exposure, although their hydrophobic nature poses challenges for methods development. We have implemented a competitive enzyme-linked immunosorbent assay (ELISA) for detection of 7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua), a major depurination adduct of BP. Linkage of the adduct to keyhole limpet hemocyanin and bovine serum albumin via the heterobifunctional linker succinimidyl-4-(N-maleimido-methyl)-cyclohexane-1-carboxylate produced an effective immunogen and capture complex, respectively. Two cycles of hybridoma production yielded 23 monoclonal antibodies (MAb) specific for the capture complex. One of these MAb was used to optimize the competitive ELISA for BP-6-N7Gua. Conditions of the reaction between specific MAb and the free adduct were extensively modified to produce an assay that detects 50 fmol free adduct/mL reaction mixture.