Shen K. Yang

Inhibitory Monoclonal Antibody to Human Cytochrome P450 2B6

The human cytochrome P450 2B6 metabolizes, among numerous other substrates, diazepam, 7-ethoxycoumarin, testosterone, and phenanthrene. A recombinant baculovirus containing the human 2B6 cDNA was constructed and used to express 2B6 in Sf9 insect cells. The 2B6 was present at 1.8 +/- 0.4% of the total cellular protein and was purified to a specific content of 13.3 nmol/mg protein. Mice were immunized with the purified 2B6, and a total of 811 hybridomas were obtained from the fusion of NS-1 myeloma cells and spleen cells of the immunized mice. Monoclonal antibodies (MAbs) from 24 of the hybrids exhibited immunobinding to 2B6 as determined by ELISA. One of the MAbs, 49-10-20, showed a strong immunoblotting activity and was highly inhibitory to 2B6 enzyme activity. MAb 49-10-20 inhibited cDNA-expressed 2B6-catalyzed metabolism of diazepam, phenanthrene, 7-ethoxycoumarin, and testosterone by 90-91%. MAb 49-10-20 showed extremely high specificity for 2B6 and did not bind to 17 other human and rodent P450s or inhibit the metabolism of phenanthrene catalyzed by human 1A2, 2A6, 2C8, 2C9, 2D6, 2E1, 3A4, and 3A5. MAb 49-10-20 was used to determine the contribution of 2B6 to the metabolism of phenanthrene and diazepam in human liver. In ten liver samples, MAb 49-10-20 inhibited phenanthrene metabolism variably by a wide range of 8-42% and diazepam demethylation by 1-23%. The degree of inhibition by the 2B6 specific MAb 49-10-20 defines the contribution of 2B6 to phenanthrene and diazepam metabolism in each human liver. This technique using inhibitory MAb 49-10-20 determines the contribution of 2B6 to the metabolism of its substrates in a human tissue containing multiple P450s. This study is a prototype for the use of specific and highly inhibitory MAbs to determine individual P450 function.

Role of cDNA-Expressed Human Cytochromes P450 in the Metabolism of Diazepam

The metabolic conversion of diazepam (DZ) to temazepam (TMZ, a C3-hydroxylation product of DZ) and N-desmethyldiazepam (NDZ, an N1-demethylation product of DZ) was studied using cDNA-expressed human cytochrome P450 (CYP) isozymes 1A2, 2B6, 2C8, 2C9, 2C9R144C, 2E1, 3A4, and 3A5 and human liver microsomes from five organ donors. Of the CYPs examined, 3A5, 3A4, and 2B6 exhibited the highest enzymatic activities with turnovers ranging from 7.5 to 12.5 nmol of product formed/min/nmol for the total metabolism of DZ, while 2C8, 2C9, and 2C9R144C showed lesser and moderate activities. 1A2 and 2E1 produced insignificant amounts of metabolites of DZ. The regioselectivity of CYPs was determined, and 2B6 was found to catalyze exclusively and 2C8, 2C9, and 2C9R144C preferentially the N1-demethylation of DZ to form NDZ. 3A4 and 3A5 catalyzed primarily the C3-hydroxylation of DZ, which was more extensive than the N1-demethylation. The ratios of TMZ to NDZ formed in the metabolism of DZ by 3A4 and 3A5 were approximately 4:1. Enzyme kinetic studies indicated that 2B6- and 2C9-catalyzed DZ metabolism followed Michaelis-Menten kinetics, whereas 3A4 and 3A5 displayed atypical and non-linear curves in Lineweaver-Burk plots. Human liver microsomes converted DZ to both TMZ and NDZ at a ratio of 2:1. Our results suggest that hepatic CYP3A, 2C, and 2B6 enzymes have an important role in the metabolism of DZ by human liver.

Direct resolution of mono- and diol enantiomers of unsubstituted and methyl-substituted benz[a]anthracene and benzo[a]pyrene by high-performance liquid chromatography with a chiral stationary phase

The direct resolution of 86 structurally related monomethyl, mono-ol, and trans- and cis-diol enantiomers of unsubstituted and methyl-substituted benz[a]anthracene and benzo[a]pyrene was investigated by high-performance liquid chromatography with a commercially available column, packed with an (R)-N-(3,5-dinitrobenzoyl)phenylglycine, ionically bonded to gamma-aminopropylsilanized silica. The results indicate that structural factors, such as conformation, presence of a methyl substituent, molecular size and shape, and ring saturation all contribute to chiral interactions between the chiral stationary phase and the solutes. Detailed chiral recognition mechanisms can not yet be established, due to complex structural factors that influence enantiomeric resolutions and the lack of data on the absolute configurations of the resolved enantiomers. Nevertheless, the chromatographic method can be applied to the determination of enantiomeric purity of mono- and diol metabolites of polycyclic aromatic hydrocarbons. The absolute configurations of a limited number of resolved enantiomers have been established.

Evidence for a 2,3-epoxide as an intermediate in the microsomal metabolism of benzo[a]pyrene to 3-hydroxybenzo[a] pyrene

Summary A synthetic benzo[a]pyrene (BP) specifically labeled with deuterium at the 3-position was incubated with rat liver microsomes in the presence of NADPH and oxygen. Mass spectral analysis of the metabolites isolated by high pressure liquid chromatography indicated that the 3-hydroxybenzo[a]pyrene (3-OH-BP) and BP-3,6-quinone retained 29% and 15% of the deuterium label respectively, whereas the BP 4,5-, 7,8-, and 9,10- diols, 9-OH-BP, BP-1,6-quinone, and BP-6,12-quinone retained essentially all the deuterium label. The results indicate that at least 29% of the 3-OH-BP detected as metabolite is formed through a 2,3-epoxide intermediate which rearranges spontaneously via an NIH shift mechanism to 3-OH-BP. At least 15% of the BP-3,6-quinone detected as a metabolite is derived from the enzymatically formed 3-OH-BP.

Stereoselective metabolism of 6-bromobenzo[a]pyrene by rat liver microsomes: Absolute configuration of trans-dihydrodiol metabolites

Abstract Trans -4,5- and trans -7,8-dihydrodiols of 6-bromobenzo[a]pyrene (6-BrBP) were isolated by reversed-phase high performance liquid chromatography (HPLC) as metabolites of 6-BrBP by liver microsomes from 3-methylcholanthrene-treated male Sprague-Dawley rats. 1H NMR spectral analyses indicate that both 6-BrBP dihydrodiols preferentially adopt quasidiaxial conformations which are different from the quasidiequatorial conformations observed with benzo[a]pyrene (BP) trans -4,5- and trans -7,8-dihydrodiols. 6-BrBP trans -4,5-dihydrodiol was converted by catalytic hydrogenolysis to BP trans -4,5-dihydrodiol which had a circular dichroism (CD) spectrum identical to that of BP (−) trans -4R,5R-dihydrodiol. 6-BrBP trans -7,8-dihydrodiol was converted by catalytic hydrogenolysis to BP 7,8,9,10-tetrahydro- trans -7,8-diol which was identical to (+)BP 7,8,9,10-tetrahydro- trans -7R,8R-diol with respect to its CD spectrum and its retention time on a chiral HPLC column. The results indicate that a bromo substituent at the 6-position of BP does not alter the stereoselective preference of rat liver microsomal enzymes toward the formation of 4R,5R- and 7R,8R-dihydrodiol metabolites.

Stereochemistry and evidence for an arene oxide-NIH shift pathway in the fungal metabolism of naphthalene

The mechanism of naphthalene oxidation by the filamentous fungus, Cunninghamella elegans is described. C. elegans oxidized naphthalene predominately to trans-1,2-dihydroxy-1,2-dihydroxy-1,2-dihydronaphthalene. A trans configuration was assigned for the dihydrodiol by nuclear magnetic resonance (NMR) spectroscopy at 500 MHz which showed a large coupling constant (J1,2) of 11.0 Hz. Comparison of the circular dichroism spectrum of the fungal trans-1,2-dihydroxy-1,2-dihydronaphthalene to that formed by mammalian enzyme systems indicated that the fungal dihydrodiol contained 76% (+)-(1S,2S)-dihydrodiol as the predominant enantiomer. Other naphthalene metabolites formed by C. elegans were identified as 1-naphthol, 2-naphthol and 4-hydroxy-1-tetralone. Incubation of C. elegans with naphthalene and 18O2 indicated that the trans-1,2-dihydroxy-1,2-dihydronaphthalene contained one atom of molecular oxygen which indicated a monooxygenase catalyzed reaction while similar incubations with naphthalene and H182O indicated that the other oxygen atom in trans-1,2-dihydroxy-1,2-dihydronaphthalene was derived from water. Mass spectral analysis of the acid-catalyzed dehydration products of the dihydrodiol indicated that the naphthalene dihydrodiol forms via the addition of water at the C-2 position of naphthalene-1,2-oxide. Fungal metabolism of [1-2H]naphthalene yielded 1-naphthol which retained 78% of the deuterium. NMR analysis of the deuterated 1-naphthol indicated an NIH shift mechanism in which deuterium migrated from the C-1 position to the C-2 position. The above results indicate that naphthalene-1,2-oxide is an intermediate in the fungal metabolism of naphthalene and that the fungal enzymes are highly stereo-selective in the formation of trans-1,2-dihydroxy-1,2-dihydronaphthalene.

Cutaneous metabolism of benzo[a]pyrene: Comparative studies in C57BL/6N and DBA/2N mice and neonatal Sprague-Dawley rats

Abstract The metabolism of the polycyclic aromatic hydrocarbon (PAH) carcinogen benzo[a]pyrene (BaP) was studied using microsomes prepared from the skin of the mouse and rat. Topical application of the polychlorinated biphenyl (PCB) Aroclor 1254 or the PAH 3-methylcholanthrene (3-MC) to the skin of the C57BL/6N and DBA/2N mouse and the Sprague-Dawley rat caused statistically significant enhancement of cutaneous microsomal aryl hydrocarbon hydroxylase (AHH) activity in each animal. PCB was a more potent inducer of the enzyme than was 3-MC. BaP metabolism by skin microsomes from the same animals was assessed using high performance liquid chromatography (HPLC). The skin of untreated animals metabolized BaP into 9,10-, 7,8- and 4,5-dihydrodiols, phenols and quinones. Skin application of PCB caused greater than 16–18-fold enhancement of BaP metabolism in the C57BL/6N mouse and the rat and 2–5-fold enhancement in the DBA/2N mouse. Skin application of 3-MC enhanced BaP metabolism 2–8-fold in the C57BL/6N mouse and 5–10-fold in the rat and had no effect in the DBA/2N mouse. The formation of procarcinogenic metabolite BaP-7, 8-diol was greatly enhanced (4–12-fold) by treatment with the PCB and 3-MC in the tumor susceptible C57BL/6N mouse and in the tumor-resistant neonatal Sprague-Dawley rat. In contrast, the formation of BaP-7,8-diol was either slightly enhanced (2-fold) or unaffected by treatment with the PCB or 3-MC in the tumor-resistant DBA/2N mouse. Our data indicate that neither the patterns of metabolism nor the amount of BaP-7,8-diol formation in the skin are reliable predictors of tumor susceptibility to the PAH in rodent skin.

Resolution of enantiomeric lorazepam and its acyl and O-methyl derivatives and racemization kinetics of lorazepam enantiomers

Abstract Enantiomeric pairs of lorazepam (LZ) and its 3-O-acyl, 1-N-acyl-3-O-acyl, and 3-O-methyl ether derivatives were resolved on high-performance liquid chromatography columns packed with six different chiral stationary phases (CSPs). Resolution was achieved, with several mobile phases of different solvent compositions and with varying chromatographic resolutions, on at least five of the six CSPs tested. Resolved enantiomers of LZ underwent racemization, whereas enantiomers of 3-O-acyl and 3-O-methyl derivatives were stable. Racemization half-lives of LZ enantiomers were determined by monitoring changes in ellipticity as a function of time. Stability of LZ enantiomers vary substantially depending on the solvents used.

Direct separation of non-K-region mono-ol and diol enantiomers of phenanthrene, benz[a]anthracene, and chrysene by high-performance liquid chromatography with chiral stationary phases.

The direct separation of 26 bay region and non-bay region mono-ol and diol enantiomers of phenanthrene, benz[a]anthracene, and chrysene was compared by high-performance liquid chromatography on commercially available columns, packed with gamma-aminopropylsilanized silica to which either (R)-N-(3,5-dinitrobenzoyl)phenylglycine(R-DNBPG) or (S)-N-(3,5-dinitrobenzoyl)leucine(S-DNBL) was either ionically or covalently bonded. In general, enantiomers of bay region mono-ols and diols are more efficiently resolved than those of non-bay region derivatives. Elution orders of enantiomers on either chiral stationary phase are the same, regardless of whether the chiral stationary phase is ionically or covalently bonded. Except for the enantiomers of 4-hydroxy-4-methyl-1,2,3,4-tetrahydrobenz[a]anthracene, 1,2,3,4-tetrahydrobenz[a]anthracene trans-1,2-diol, and benz[a]anthracene trans-1,2-dihydrodiol, elution orders of resolved enantiomers on R-DNBPG are reversed on S-DNBL. The enantiomers are generally more efficiently resolved on R-DNBPG than on S-DNBL. With the exception of the elution order of the enantiomeric 4-hydroxy-1,2,3,4-tetrahydrochrysene, the results of this study are consistent with the chiral recognition mechanisms proposed by Pirkle and co-workers, who developed the chiral stationary phases used in this study.

Stereoselective metabolism of 7-methylbenz[a]anthracene: absolute configuration of five dihydrodiol metabolites and the effect of dihydrodiol conformation on circular dichroism spectra.

7-Methylbenz[a]anthracene (7-MBA) was metabolized stereoselectively by rat liver microsomes to form five optically active dihydrodiols as the predominant metabolites. The dihydrodiols were purified by a combination of reversed-phase and normal-phase high performance liquid chromatography (HPLC). By comparison of their circular dichroism (CD) spectra with the corresponding benz[a]anthracene (BA) dihydrodiols of known absolute stereochemistry, the major dihydrodiol enantiomers of 7-MBA have been determined to have 1R,2R-, 3R,4R- and 10R , 11R - absolute configurations, respectively. Due to their quasi- diaxial conformations, the absolute configuration of trans-5,6- and trans-8,9-dihydrodiols, the two most abundant metabolites of 7-MBA, could not be determined by simple comparisons of their circular dichroism spectra with those of the quasidi -equatorial BA 5R, 6R - and 8R , 9R -dihydrodiols. The major enantiomers of the quasi- diaxial trans-5,6- and trans-8,9-dihydrodiol metabolites of 7-MBA were determined by comparison to the CD spectrum of 7-bromo-BA 5R, 6R -dihydrodiol and by the exciton chirality method to have R,R absolute stereochemistry. This study also revealed that the circular dichroism Cotton effects of an enantiomeric dihydrodiol of polycyclic aromatic hydrocarbons can be drastically altered if the conformation (quasi- diaxial vs. quasi di-equatorial ) of the dihydrodiol is changed.