Oscar Hernandez

High mutagenicity and toxicity of a diol epoxide derived from benzo[a]pyrene

Abstract (±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP 7,8-diol-9,10-epoxide) is a suspected metabolite of benzo[a]pyrene that is highly mutagenic and toxic in several strains of Salmonella typhimurium and in cultured Chinese hamster V79 cells. BP 7,8-diol-9,10-epoxide was approximately 5, 10 and 40 times more mutagenic than benzo[a]pyrene 4,5-oxide (BP 4,5-oxide) in strains TA 98 and TA 100 of S. typhimurium and in V79 cells, respectively. Both compounds were equally mutagenic to strain TA 1538 and non-mutagenic to strain TA 1535 of S. typhimurium . The diol epoxide was toxic to the four bacterial strains at 0.5–2.0 nmole/plate, whereas BP 4,5-oxide was nontoxic at these concentrations. In V79 cells, the diol epoxide was about 60-fold more cytotoxic than BP 4,5-oxide.

Synthesis and characterization of 4-dimethylamino-N-triphenylmethylpyridinium chloride, a postulated intermediate in the tritylation of alcohols

Abstract 4-Dimethylamino- N -triphenylmethylpyridinium chloride ( 1 ) reacts with primary but not secondary alcohols to produce trityl ethers in good yield; in addition, amines may be selectively N - tritylated witth 1 in the presence of alcohols.

Regiospecificity and stereospecificity in the enzymatic conjugation of glutathione with (±)-benzo(a)pyrene 4,5-oxide

Abstract 13 C-NMR analysis of the glutathione conjugates formed from (±)-benzo(a)-pyrene 4,5-oxide-4,5- 13 C by a purified glytathione transferase from little skate ( Raja erinacea ) liver demonstrated that equivalent amounts of the positional isomers (4,5-dihydro-4-hydroxy-5-glutathionylbenzo(a)pyrene and 4,5-dihydro-4-glutathionyl-5-hydroxybenzo(a)pyrene) were formed. Separation of these conjugates by HPLC and subsequent 13 C-NME studies showed that only one diastereoisomer of each positional isomer was formed by the skate enzyme, each enantiomer of the arene oxide having produced only one of the two possible positional isomers. The non-enzymic reaction of (±)-benzo(a)pyrene 4,5-oxide with glutathione produced the four possible stereoisomers resulting from trans addition to the epoxide ring. This was also true when rat liver cytosol was used as the source of transferase activity. The data demonstrate that skate liver glutathione transferase 4 has high substrate regiospecificity and stereospecificity for (±)-benzo(a)pyrene 4,5-oxide.

Synthesis and relative stereochemistry of the four mercapturic acids derived from styrene oxide and N-acetylcysteine.

The chemical reaction between (+/-)-styrene oxide and N-acetylcysteine produces both positional isomers (1 and 2) as a mixture of diastereoisomers with a preference for the benzylic thioether isomer 1 (2 : 1). Synthesis of the mercapturic acid conjugates from either (+)- or (-)-styrene oxide produces only two of the four possible stereoisomers. The single diastereoisomers of 1 and 2 were separated by high pressure liquid chromatography (HPLC) and identified by 1H- and 13C-nuclear magnetic resonance (NMR). The relative stereochemistry at the benzylic carbon center of the mercapturic acid conjugates was assigned on the basis of the established chemical correlation between optically pure styrene oxide and its precursor mandelic acid, and considerations on the mechanism of ring opening of epoxides by sulfur nucleophiles. The stereochemical definition of the isomers 3-6 should prove useful in investigations of the biotransformation of the glutathione (GSH) conjugates of styrene oxide.

Synthesis and relative stereochemistry of the benzylic thioether diastereoisomers formed from glutathione and styrene oxide

Abstract The chemical reaction between (±) styrene oxide and glutathione produces both the benzylic and primary thioether positional isomers as a mixture of diastereoisomers ( 2, 5 and 3, 6 ), with a preference for the benzylic thioether isomers (66 : 34). Synthesis of the styrene oxide-glutathione conjugates from either (+)- or (−)- styrene oxide produces both positional isomers as single diastereoisomers. The benzylic thioether isomers ( 2 and 5 ) were prepared from protected 2-bromo-2-phenylethanol ( 8 ) and glutathione and were separated using hplc. The relative stereochemistry of the benzylic thioether isomers was assigned on the basis of the established chemical correlation between the optically pure styrene oxides and their precursors, the mandelic acids, as well as considerations of the mechanism of ring opening of epoxides by sulfur nucleophiles. The availability of the single diastereoisomers of the benzylic thioether isomers and the styrene oxideglutathione conjugates enables investigations concerned with the influence of chirality on the biotransformation and excretion of these conjugates.

Stereoselective Synthesis and Reactions of a Diol-Epoxide Derived from Benzo[a]pyrene

In the course of examining the metabolism-induced binding of benzo[a]pyrene (BP) to DNA in the presence of active liver microsomes, Borgen et al. (1) made the intriguing observation that further metabolism of trans-7,8-dihydroxy-7,8-dihydro-BP resulted in much more extensive binding to DNA than that observed with two other metabolic dihydrodiols or with BP itself. Subsequently, Sims et al. (2) suggested that the facile binding of this dihydrodiol was mediated by formation of an oxide at the 9,10-double bond. Synthesis of the suspected binding agent, 7,8-dihydroxy-9,10-epoxy,7,8,9,10-tetrahydro-BP, was claimed to result from the action of m-chloroperoxybenzoic acid on the parent dihydrodiol (2). We have considered the possibility that the suspected high chemical reactivity of this diol-epoxide from BP might well be due to anchimeric assistance (3,4) by the neighboring hydroxyl group at C-7.

The stereoselectivity of four hepatic glutathione S-transferases purifed from a marine elasmobranch (Raja erinacea) with several K-region polycyclic arene oxide substrates

Product analysis by HPLC demonstrated that three hepatic cytosolic glutathione S-transferases of little skate (E-2, E-3 and E-4) were highly stereoselective, if not stereospecific, for thiol reaction at the R-configured oxirane carbons of four K-region arene oxides; pyrene 4,5-oxide, (+/-)-benz[a]anthracene 5,6-oxide, (+/-)-benzo[a]pyrene 4,5-oxide and phenanthrene 9,10-oxide. A fourth transferase, E-5, exhibited no stereopreference with pyrene 4,5-oxide or phenanthrene 9,10-oxide. Immunological and electrophoretic evidence has shown that the three enzymes exhibiting stereospecificity have a common subunit which is absent from enzyme E-5. The high stereoselectivity of the three enzymes E-2, E-3, and E-4 was accompanied by effective catalysis of the reaction of GSH with these K-region epoxides; for example, the calculated turnover number for E-4 with benzo[a]pyrene 4,5-oxide was 550.

Fractionation by High Performance Liquid Chromatography of Microsomal Cytochrome P-450 Induced by Hexachlorobiphenyl Isomers

Abstract High performance liquid chromatography has been employed to fractionate rat liver microsomes under nondenaturing conditions. Selective detection at 405 nm allowed resolution of microsomal heme proteins into three peaks (A, B, and C). Cytochromes in the peaks retain their native property of binding CO after HPLC. Peak-A, first eluting, contains P-450 and is rich in cytochrome P-420. Peak-B is largely hemoglobin and peak-C is a major cytochrome P-450. The ratio of peak-C to A is increased by treatment of rats with phenobarbitone, β-naphthoflavone, 2,3,5,2′,3′,5′-hexachlorobiphenyl and 3,4,5,3′,4′,5′-hexachlorobiphenyl as compared to controls. The highest increment in the ratio is observed on feeding 3,4,5,3′,4′,5′-hexachlorobiphenyl. NADPH cytochrome c reductase elutes earlier than peak-C but cytochrome b5 is not separated from the major cytochrome P-450 peak. The separations obtained are highly reproducible and considerably faster than conventional gel permeation chromatography. The data presented...

High-Performance Liquid Chromatography of Amphibian Peptides. Selectivity Changes Induced by pH

Abstract The effect of pH on the retention behavior under reversed- phase liquid chromatography conditions of a series of peptides was examined. Isocratic conditions were used with either methanol or acetonitrile as organic modifiers. The intrinsic hydrophobicity of the peptides was altered by changes in the pH of the eluent mixture. Increased retention at pH 7 relative to pH 4 was correlated with the presence of a histidine residue in a hydrophobic environment. An experimental parameter, αpH, was defined as the positive quotient of capacity factors at pH 4 and pH 7 for a given eluent. These αpH values are interpreted as reflecting changes in peptide hydrophobicity introduced by variations in solvent and pH. Identical αpH values were obtained for homologous peptides, particularly histidine containing peptides. This approach to selectivity effects yielded diagnostic conditions for the analysis of bombesin, a peptide touted as a potential marker for human small-cell lung carcinoma.

HPLC Separation of Diastereomeric Adducts of Glutatmione with Some K-Region Arene Oxides

Abstract The diastereomeric glutathione (GSH) adducts of the K-region arene oxides phenanthrene 9,10-oxide, pyrene 4,5-oxide, (±)-benz[a]anthracene 5,6-oxide, and (±)-benzo[a]pyrene 4,5-oxide were separated by reversed phase liquid chromatography. Chromatographic conditions involved an organic base, Tris-base or diethylenetriamine (DETA), neutralized to pH 3 with phosphoric acid, and an alcohol, methanol or 1-propanol, as modifier. For (±)-benzo[a]pyrene 4,5-oxide, the use of DETA and 1-propanol provided a complete stereochemical profile of the thioether conjugates derived from this arene oxide. For the GSH adducts of (±)-benz[a]anthracene 5,6-oxide complete separation was achieved under two sets of chroraatographic conditions; methanol and 1-propanol enhanced the selectivity of the system for different sets of diastereomers. For both arene oxides, the GSH adducts with S-configuration eluted earlier than the R-diastereomers.