Hans-Pietro Eugster

17β-estradiol hydroxylation catalyzed by human cytochrome P450 1A1 : a comparison of the activities induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in MCF-7 cells with those from heterologous expression of the cDNA

Exposure of MCF-7 breast cancer cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes an elevated cytochrome P450 content and a marked increase in the microsomal hydroxylation of 17 beta-estradiol (E2) at the C-2, C-4, C-15 alpha, and C-6 alpha positions. In this study we investigated the involvement of cytochromes P450 of the 1A gene subfamily in this metabolism of E2. Hydroxylation at each of these four positions of E2 was inhibited by P450 1A-subfamily inhibitors, alpha-naphthoflavone, benzo[a]pyrene, and 7-ethoxyresorufin. Northern blots showed that treatment of MCF-7 cells with TCDD resulted in production of the 2.6-kb CYP1A1 mRNA, but not the 3.0-kb CYP1A2 mRNA. Immunoblot analyses with anti-P450 1A antibodies confirmed the production of P450 1A1 protein in TCDD-treated MCF-7 cells. Anti-rat P450 1A IgG inhibited the hydroxylation of E2 at C-2, C-15 alpha, and C-6 alpha, but not hydroxylation at C-4. E2 hydroxylation by human cytochromes P450 1A1 and P450 1A2 was assessed in experiments with microsomes from Saccharomyces cerevisiae after transformation with cDNAs encoding the two cytochromes. The major hydroxylase activities of expressed human P450 1A1 were at the C-2, C-15 alpha, and C-6 alpha positions of E2; expressed human P450 1A2 catalyzed hydroxylation predominately at C-2. While both expressed P450s 1A1 and 1A2 had minor hydroxylase activities at the C-4 position, neither catalyzed a low-Km hydroxylation at C-4 similar to that observed with microsomes from TCDD-treated MCF-7 cells. These results provide strong evidence that P450 1A1 catalyzes the hydroxylations of E2 at the C-2, C-15 alpha, and C-6 alpha in incubations with microsomes from TCDD-treated MCF-7 cells, but suggest TCDD may also induce a cytochrome P450 E2 4-hydroxylase that is distinct from P450 1A1 or P450 1A2.

Constitutive and inducible expression of human cytochrome P450IA1 in yeast Saccharomyces cerevisiae: An alternative enzyme source for in vitro studies

A cDNA of human cytochrome P450IA1 was expressed in yeast Saccharomyces cerevisiae on a multicopy plasmid under the control of the constitutive GAPFL or the inducible PHO5 promoter. Microsomes of transformed yeast contained substantial amounts of the heterologous enzyme as determined by reduced CO-difference spectra (156-68 pmol/mg). Enzyme kinetics with 7-ethoxyresorufin as substrate resulted in a Km value of 92 nM and a Vmax value of 223 pmol/mg/min, which is comparable to data obtained with human liver microsomes. The antimycotic drug ketoconazole (Ki = 22nM) as well as the isozyme specific P450 inhibitor alpha-naphthoflavone (Ki = 1.2 nM) were shown to be strong inhibitors of human P450IA1. Taken together, these data show that heterologous P450 gene expression in yeast is a potent instrument for the study of enzyme specific parameters and might be used to answer further questions with regard to substrate specificity as well as drug interaction in a background with no interfering activities.

Functional co-expression of human oxidoreductase and cytochrome P450 1A1 in Saccharomyces cerevisiae results in increased erod activity

A cDNA coding for human oxidoreductase (NADPH-cytochrome P450 reductase) was expressed in S. cerevisiae on a high copy plasmid under control of a constitutive promoter. Microsomes from a transformed strain lacking endogenous oxidoreductase exhibited cytochrome c reductase activity. An apparent Km of 7.3 microM for the substrate NADPH was determined. Expression of human oxidoreductase complemented a mutation in the yeast oxidoreductase gene CPR1 and fully reversed the ketoconazole sensitive phenotype of the respective strain. The 7-ethoxyresorufin-O-deethylase activity of yeast cells expressing human cytochrome P450 1A1 was increased by more than sixteen-fold upon coexpression of human oxidoreductase. These results strongly suggest that a more efficient coupling between the human enzymes might be responsible for the increase in enzyme activity.

Heterologous expression of human microsomal epoxide hydrolase in saccharomyces cerevisiae: Study of the valpromide-carbamazepine epoxide interaction

A cDNA of human microsomal epoxide hydrolase (hmEH) was constitutively and inducibly expressed in Saccharomyces cerevisiae. The heterologous enzyme was located mainly in the microsomal fraction of yeast cells. Yeast microsomes containing hmEH exerted styrene oxide hydrolase activity (Km = 300 microM; Vmax = 22 nmol/mg min) as well as carbamazepine epoxide hydrolase activity. The hmEH catalysed exclusively the formation of carbamazepine-10,11-transdihydrodiol, since no carbamazepine-10,11-cisdihydrodiol was detected. Inhibition studies using these microsomes revealed unequivocally hmEH as the target for inhibition by the antiepileptic drug valpromide. A Ki value of 27 microM was determined for the inhibitor valpromide with styrene oxide as substrate. For carbamazepine epoxide, a Ki value of 8.6 microM was obtained, which is well in line with data published for hmEH determined with human liver microsomes. Our results demonstrate the potential of heterologous gene expression in S. cerevisiae and its application to the in vitro study of pharmacological and toxicological problems.

Saccharomyces cerevisiae: An alternative source for human microsomal liver enzymes and its use in drug interaction studies

Heterologous expression of human cDNAs in the yeast Saccharomyces cerevisiae represents an attractive alternative source of human enzymes and allows metabolic studies to be performed without the need of human tissue. Here we report on the functional expression of human microsomal epoxide hydrolase (hmEH) and cytochrome P450 1A1 and 1A2 in yeast. Microsomal fractions of corresponding yeast strains exhibited enzyme specific activities which allowed the characterization of the heterologous enzymes. The use of these microsomes enabled us to study drug interactions on the respective enzymes with pharmacologically relevant drugs such as carbamazepine epoxide, valpromide and ketoconazole.