Metabolism and interaction of bisphenol A in human hepatic cytochrome P450 and steroidogenic CYP17.

The metabolism of bisphenol A (BPA) was determined for 11 forms of human hepatic cytochromes P450 (CYPs) expressed in the yeast Saccharomyces cerevisiae and for human steroidogenic CYP17 expressed in Escherichia coli. Additionally, the effect of BPA on the progesterone 17alpha-chydroxylase activity of CYP17 was investigated. CYP2C18 catalyzed BPA metabolism most efficiently, followed by CYP2C19 and CYP2C9. CYP2C9 and CYP2C18 exhibited the highest affinity (Km=3.9 microM) for BPA metabolism. The Vmax of CYP2C18 (8.10 nmol x min(-1) x nmol CYP(-1)) was 5 times higher than that of CYP2C9. Although the Vmax of CYP2C19 was 1.5 times higher than that of CYP2C18, the affinity of CYP2C19 was 12 times lower than that of CYP2C9 and CYP2C18. Therefore the intrinsic clearance (Vmax/Km) of CYP2C18 was more than 5 times higher than that of CYP2C9 and CYP2C19. On the other hand, BPA exhibited a competitive-type inhibition of the progesterone 17alpha-hydroxylase activity of CYP17 with a Ki value of 71 microM, whereas no metabolism of BPA by CYP17 was detected. These results suggest that BPA is mainly metabolized by the CYP2C subfamily in human liver, and that BPA inhibits human steroidogenic CYP17 activities.

A transgenic mouse expressing human CYP4B1 in the liver.

The human CYP4B1 protein was expressed in the liver of a transgenic mouse line under the control of the promoter of the human apolipoprotein E (apo E) gene. Hepatic microsomes of transgenic mice catalyzed omega-hydroxylation of lauric acid and also activated 2-aminofluorene (2-AF), which is a typical substrate for CYP4B1, to mutagenic compounds detected by an umu gene expression assay. These activities observed in transgenic mouse were efficiently inhibited by CYP4B1 antibody. However, such inhibition was not observed in control mice. This is the first report to indicate catalytic activities of human CYP4B1. For further characterization of human CYP4B1, a fusion protein of CYP4B1 and NADPH-P450 reductase was expressed in yeast cells. It was able to activate 2-AF and was also able to catalyze omega-hydroxylation of lauric acid. This transgenic mouse line and the recombinant fusion protein provide a useful tool to study human CYP4B1 and its relation to chemical toxicity and carcinogenesis.

The amino acid residues affecting the activity and azole susceptibility of rat CYP51 (sterol 14-demethylase P450).

The amino acid residues affecting the function of rat sterol 14-demethylase P450 (CYP51) were examined by means of point mutation. Forty-five mutants with respect to 27 amino acid sites were constructed and expressed in Escherichia coli. Substitution of highly conserved Y131, E369, R372, or R382 decreased the expression of CYP51 protein, indicating some structural importance of these residues. Substitution of H314, T315, or S316 caused considerable effects on the catalytic activity, and T315 was identified as the "conserved threonine" of CYP51. H314 was important for maintenance of the activity of CYP51 and was a characteristic residue of this P450, because the position corresponding to this residue is occupied by an acidic amino acid in most other P450 species. A144 was identified as a residue affecting the interaction of CYP51 with ketoconazole. Substitution of A144 with I, which occupies the corresponding position in fungal CYP51, enhanced the ketoconazole susceptibility of rat CYP51 with little change in the catalytic activity, indicating an important role of this residue in determination of the ketoconazole susceptibility of CYP51. Alteration of the catalytic activity was caused by the substitution at some other sites, whereas substitution of a few highly conserved amino acids caused little alteration of the activity of CYP51.

Coexpression of genetically engineered fused enzyme between yeast NADPH-P450 reductase and human cytochrome P450 3A4 and human cytochrome b5 in yeast.

Human hepatic cytochrome P450 3A4 (CYP3A4) was expressed in yeast Saccharomyces cerevisiae. While the expression level was high as compared with other human hepatic cytochrome P450s, CYP3A4 showed almost no catalytic activity toward testosterone. Coexpression of CYP3A4 with yeast NADPH-P450 reductase did not give a full activity. Low monooxygenase activity of CYP3A4 was attributed to the insufficient reduction of heme iron of CYP3A4 by NADPH-P450 reductase. To enhance the efficiency of electron transfer from NADPH-P450 reductase to CYP3A4, a fused enzyme was constructed between CYP3A4 and yeast NADPH-P450 reductase. The rapid reduction of the heme iron of the fused enzyme by NADPH was observed. The fused enzyme showed a high testosterone 6beta-hydroxylation activity with a sigmoidal velocity saturation curve. However, the coupling efficiency between NADPH utilization and testosterone 6beta-hydroxylation was only 10%. Finally, coexpression of the fused enzyme and human cytochrome b5 was examined. A significant decrease in the Km value and a remarkable increase in the coupling efficiency were observed. Substrate-induced spectra revealed that the dissociation constant of the fused enzyme for testosterone significantly decreased with coexpression of human cytochrome b5. These results strongly suggest that human cytochrome b5 directly interacts with the CYP3A4 domain of the fused enzyme and modifies the tertiary structure of substrate binding pocket, resulting in tight binding of the substrate and high coupling efficiency.

Inhibition of drug-metabolizing enzyme activity in human hepatic cytochrome P450s by bisphenol A.

Effect of bisphenol A on drug-metabolizing enzyme activities by human hepatic cytochrome P450s (CYP) was investigated. We measured aminopyrine N-demethylation by eleven kinds of cDNA-expressed CYPs. CYP2C19 and CYP2B6 catalyzed most efficiently the aminopyrine N-demethylation, followed by CYP2C8 and CYP2D6. Bisphenol A (1 mM) most efficiently inhibited aminopyrine N-demethylation by CYP2C8 and CYP2C19 by 82% and 85%, respectively, whereas inhibition of the activities by CYP 2B6 and 2D6 was less than 40%. Bisphenol A exhibited a noncompetitive-type inhibition of aminopyrine N-demethylase activity by CYP2C8 with Ki value of 97 microM. Additionally, we investigated the inhibitory effect of bisphenol A on CYP2C19-mediated S-mephenytoin 4-hydroxylation. Bisphenol A exhibited a mixed-type inhibition with Ki value of 113 microM. These results suggest that bisphenol A inhibits human hepatic CYP activities, especially CYP2C8 and CYP2C19.

Dynamic mobility of genetically expressed fusion protein between cytochrome P4501A1 and NADPH-cytochrome P450 reductase in yeast microsomes.

A fusion protein of rat liver CYP1A1 with NADPH-cytochrome P450 reductase was expressed genetically in yeast microsomal membranes. This flavo-cytochrome is active in 6-hydroxylation of zoxazolamine. Rotational diffusion of the fusion protein was examined by observing the flash-induced absorption anisotropy r(t) of the P450.CO complex. Theoretical analysis of r(t) was performed based on a "rotation-about-membrane normal" model. The absorption anisotropy decayed within 2 ms to a time-independent value r(3). Forty percent of the fusion protein rotated with a rotational relaxation time phi of 1.35 ms. Treatment with high salt increased the mobile population of the fusion protein to 62% with phi = 0.96 ms. The mobile population of the fusion protein is close to that of CYP1A1 coexpressed with the P450 reductase and greater than that of CYP1A1 alone [Iwase et al. (1991) Biochemistry 30, 8347-8351]. The large mobile population of the fusion protein provides evidence that CYP1A1 is mobilized by forming associations with P450 reductase in microsomal membranes.

Contribution of human hepatic cytochrome P450s and steroidogenic CYP17 to the N-demethylation of aminopyrine.

1. Aminopyrine N-demethylase activity was determined for 11 forms of human hepatic cytochrome P450s (P450s) expressed in yeast Saccharomyces cerevisiae and for human steroidogenic CYP17 expressed in Escherichia coli. 2. Among the hepatic P450s, the N-demethylation of aminopyrine was catalysed most efficiently by CYP2C19, followed by CYP2C8, 2D6, 2C18 and 1A2, whereas the activity with CYP2E1 was negligible. The kinetics of the N-demethylation process by CYP1A2, 2C8, 2C19 and 2D6 were studied by fitting to Michaelis-Menten kinetics by Lineweaver-Burk plots. CYP2C19 exhibited the highest affinity and a high capacity for the aminopyrine N-demethylation. CYP2C8 showed the highest Vmax, followed by CYP2C19, 2D6 and 1A2, whereas the Km for CYP2C8, 2D6 and 1A2 were 10-17 times higher than that for CYP2C19. Accordingly, the Vmax/Km for CYP2C19 was more than nine times higher than that of other P450s. 3. Human steroidogenic CYP17 also catalysed aminopyrine N-demethylation and the activity was comparable with that for CYP3A4 which is a dominant P450 in human liver. The activity was increased 1.5-fold by the addition of cytochrome b5, whereas the activity was not affected by the addition of Mg2+. 4. These results suggest that several human hepatic P450s, especially CYP2C19, and steroidogenic CYP17 exhibit aminopyrine N-demethylase activity.

Enantioselectivity of bunitrolol 4-hydroxylation is reversed by the change of an amino acid residue from valine to methionine at position 374 of cytochrome P450-2D6.

The enantioselectivity of 4-hydroxylation of bunitrolol (BTL), a beta-adrenoceptor blocking drug, was studied in microsomes from human liver, human hepatoma (Hep G2) cells expressing CYP2D6, and lymphoblastoid cells expressing CYP2D6. Kinetics in human liver microsomes showed that the Vmax value for (+)-BTL was 2.1-fold that of (-)-BTL, and that the Km value for (+)-BTL was lower than that for the (-)-antipode, resulting in the intrinsic clearance (Vmax/Km) of (+)-BTL being 2.1-fold over its (-)-antipode. CYP2D6 (CYP2D6-met) expressed in Hep G2 cells had a methionine residue at position 373 of the amino acid sequence and a rat-type N-terminal peptide (MELLNGTGLWSM) instead of the human-type (MGLEALVPLAVIV), and showed enantioselectivity of [(+)-BTL < (-)-BTL] for the rate of BTL 4-hydroxylation. In contrast, enantioselectivity [(+)-BTL > (-)-BTL] for Hep G2-CYP2D6 (CYP2D6-val) with a human-type N-terminal peptide that had a valine residue at 374, which corresponds to the methionine of the CYP2D6-met variant, was the same as that for human liver microsomes. We further confirmed that CYP2D6-met and CYP2D6-val expressed in human lymphoblastoid cells, both of which have methionine and valine, respectively, at position 374 and a human-type N-terminal peptide, exhibited the same enantioselectivities as those obtained from CYP2D6-met and CYP2D6-val expressed in the Hep G2 cell system. These results indicate that the amino acid at 374 of CYP2D6 is one of the key factors influencing the enantioselectivity of BTL 4-hydroxylation.

Contribution of human hepatic cytochrome P450 isoforms to regioselective hydroxylation of steroid hormones.

1. Hydroxylation activities toward steroid hormones were determined for eleven forms of human hepatic cytochrome P450s expressed in yeast Saccharomyces cerevisiae cells. Microsomes were prepared from the yeast cells and assayed for their regioselectivity of hydroxylation toward progesterone, pregnenolone, dehydroepiandrosterone (DHEA) and oestrone. 2. 6 beta-Hydroxylation of progesterone was catalysed most efficiently by CYP3A4, followed by CYP2D6. CYP3A4 showed the highest progesterone 16 alpha-hydroxylation activity, followed by CYP1A1 and CYP2D6. 16 alpha-Hydroxylation of pregnenolone was catalysed efficiently by CYP1A1 and CYP3A4. Only CYP3A4 exhibited 16 alpha-hydroxylase activities toward DHEA and oestrone. 3. Addition of nifedipine, a typical substrate of CYP3A4, inhibited the 6 beta- and 16 alpha-hydroxylation of progesterone by CYP3A4. 4. These results suggest that CYP3A4 and CYP1A1 are responsible for the hydroxylation of these endogenous steroids, as well as xenobiotics, in human liver.

Purification and characterization of recombinant human neutrophil leukotriene B4 omega-hydroxylase (cytochrome P450 4F3).

Recombinant human neutrophil leukotriene B4 (LTB4) omega-hydroxylase (cytochrome P450 4F3) has been purified to a specific content of 14. 8 nmol of P450/mg of protein from yeast cells. The purified enzyme was homogenous as judged from the SDS-PAGE, with an apparent molecular weight of 55 kDa. The enzyme catalyzed the omega-hydroxylation of LTB4 with a Km of 0.64 microM and Vmax of 34 nmol/min/nmol of P450 in the presence of rabbit hepatic NADPH-P450 reductase and cytochrome b5. Furthermore, various eicosanoids such as 20-hydroxy-LTB4, 6-trans-LTB4, lipoxin A4, lipoxin B4, 5-HETE and 12-HETE, and 12-hydroxy-stearate and 12-hydroxy-oleate were efficiently omega-hydroxylated, although their Km values were much higher than that of LTB4. In contrast, no activity was detected toward laurate, palmitate, arachidonate, 15-HETE, prostaglandin A1, and prostaglandin E1, all of which are excellent substrates for the CYP4A fatty acid omega-hydroxylases. This is the first time human neutrophil LTB4 omega-hydroxylase has been isolated in a highly purified state and characterized especially with respect to its substrate specificity.

Rat CYP24 catalyses 23S-hydroxylation of 26,26,26,27,27,27-hexafluorocalcitriol in vitro.

1. Kidney mitochondrial 24-hydroxylase cytochrome P450 (CYP24) catalyses sequential hydroxylation at both C-24 and C-23 positions of calcidiol and calcitriol. Here, we have investigated the in vitro metabolism of a hexafluorinated derivative of calcitriol, 26,26,26,27,27,27-hexafluorocalcitriol (ST-630), in a reconstituted system by using recombinant Escherichia coli membrane fractions containing rat CYP24. 2. When ST-630 was incubated with CYP24 supplemented with bovine adrenodoxin and NADPH-adrenodoxin reductase, a distinct metabolite could be observed. This metabolite was found to be 26,26,26,27,27,27-hexafluoro-23S-hydroxcalcitriol, a biologically active metabolite of ST-630, based on cochromatography on HPLC and mass spectrometric analysis. 3. These results show the direct evidence that CYP24 plays an essential role in the metabolism of ST-630 to yield its 23S-hydroxylated metabolite, as observed in cultured cells and experimental animal studies.

Kinetic analysis of successive reactions catalyzed by bovine cytochrome p450(17alpha,lyase).

Bovine P450(17alpha,lyase) containing an additional four histidine residues at the COOH terminus was expressed in Escherichia coli and purified by one-step column chromatography using Ni-chelate resin. The membrane enzyme was incorporated into liposome membranes having similar lipid composition to that of the endoplasmic reticulum. In the presence of excess substrate, the P450-proteoliposomes metabolize pregnenolone (Delta5-steroid) to 17alpha-hydroxypregnenolone and further to dehydroepiandrosterone. The enzyme catalyzed 17alpha-hydroxylation of progesterone (Delta4-steroid) but did not form androstenedione from progesterone, although the proteoliposomes could catalyze the conversion of 17alpha-hydroxyprogesterone to androstenedione. The kinetic analysis of rapid quenching experiments showed that about 20% of the pregnenolone consumed was converted successively to dehydroepiandrosterone via a fraction of 17alpha-hydroxypregnenolone that did not dissociate from the enzyme. The rapid quenching experiments for progesterone metabolism by the proteoliposomes revealed that the dissociation rate of 17alpha-hydroxyprogesterone was 10 times faster than that of 17alpha-hydroxypregnenolone. The release of the intermediate metabolite of Delta4-steroid is sufficiently faster than the lyase reaction to prevent further reaction by the P450. It is concluded that the dissociation rates of the first hydroxylation metabolites regulate the successive reactions of P450(17alpha,lyase).

Specific binding of 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl propyl) piperazine (GBR-12935), an inhibitor of the dopamine transporter, to human CYP2D6.

The binding of [3H]1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl propyl) piperazine (GBR-12935), an antagonist of the dopamine transporter, to human P450s expressed in yeast cells was investigated. Among the ten forms of human P450 tested (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2D6, 2E1, and 3A4), [3H]GBR-12935 bound most strongly to CYP2D6. The calculated Kd of [3H]GBR-12935 binding to CYP2D6 was 42.2 nM, indicating that GBR-12935 has a high affinity for CYP2D6. The characteristics of [3H]GBR-12935 binding to CYP2D6 were investigated by competitive studies using several chemicals. The binding of [3H]GBR-12935 to CYP2D6 was not changed by dopamine, suggesting that these binding sites are not dopamine-sensitive binding sites. The binding of [3H]GBR-12935 to CYP2D6 was decreased partially by substrates or inhibitors of CYP2D isoforms (quinine, quinidine, propranolol, bufuralol, imipramine, and desipramine). By means of binding studies using several forms of expressed human P450, we demonstrated that the CYP2D isoform is one GBR-12935 binding site that is insensitive to dopamine.

Acetaldehyde as well as ethanol is metabolized by human CYP2E1.

Acetaldehyde was oxidized by rat and human hepatic microsomes in the presence of NADPH. We designated this NADPH-dependent oxidation system MAOS (microsomal acetaldehyde-oxidizing system), to distinguish it from the NAD-dependent acetaldehyde oxidation system of acetaldehyde dehydrogenase in mitochondria and cytosol. This activity was increased 2.3-fold by giving rats ethanol. Judging from the Vmax/Km values, the metabolic capacity of rat hepatic microsomes for MAOS activity was increased 24-fold by ethanol. The acetaldehyde oxidation activity of eight forms of purified rat cytochrome P450 was investigated in a reconstituted system. CYP2E1 had the highest level, followed by CYP1A2 and 4A2. Immunoinhibition studies showed that an anti-CYP2E1 antibody inhibited 90% of the MAOS activity in rats given ethanol. NADPH-dependent acetate formation was 12% or 33.6% of the NAD-dependent acetate formation in liver homogenates of control rats and those treated with ethanol, respectively. We investigated human MAOS activity further. Among the 10 forms of human cytochrome P450 expressed in yeast, CYP2E1 had especially high acetaldehyde oxidation activity. The correlation of MAOS activity with the levels of immunoreactive CYP2E1 in individual human microsomes was highly significant (r2 = 0.88, P < .01). These results indicate that hepatic CYP2E1 mainly contributes to MAOS in rats and humans, the pathway of which may play an alternative role against acetaldehyde in the liver after alcohol consumption together with acetaldehyde dehydrogenase in the metabolism of acetaldehyde.

A comparison of hepatic cytochrome P450 protein expression between infancy and postinfancy.

We immunochemically measured the contents of 9 different cytochrome P450 (CYP) isoenzymes expressed in the liver and compared them between two groups: one group of 6 infant and 4 perinatal patients and one group of 10 patients after infancy (over 1 year old). CYP protein expressed in human liver can be divided into three groups on the basis of expression pattern: (a) CYP2A6, 2C9, 2D6, 2E1, and 3A were present in all samples and no difference was observed between the two groups; (b) CYP1A2, 2B6, and 2C8 were expressed more after infancy than during infancy; and (c) CYP3A7, which has been considered a major CYP enzyme in fetal liver microsomes, was expressed in all infants as well as the four perinatal patients, whereas it was detected in only 2 patients after infancy. These results implied that CYP2A6, 2C9, 2D6, 2E1, and 3A are already expressed during perinatal and infant period, while CYP1A2, 2B6, and 2C8 are expressed highly in subjects over 1 year old, and CYP3A7 disappeared after infancy.

Expression of four rat CYP2D isoforms in Saccharomyces cerevisiae and their catalytic specificity.

We cloned four cDNAs belonging to the CYP2D subfamily to express these enzymes in yeast cells and to compare their catalytic activities simultaneously. Three are believed to be alleles of CYP2D1, 2D2, and 2D3, respectively, based on high nucleotide sequence similarity, while CYP2D4 had both sequences of CYP2D4 and CYP2D18. Expression plasmids carrying CYP2D cDNAs were transformed into Saccharomyces cerevisiae. Typical P450 CO-difference spectra with absorbance maximum at 448 nm were recorded with microsomal preparations from the yeast cells expressing the four CYP2D forms. A catalytic study of these CYP2D forms was done with debrisoquine, bufuralol, and lidocaine. CYP2D2 had the highest debrisoquine 4-hydroxylation (2.2 nmol/min/nmol P450) activity, similar to that (2.2 nmol/min/nmol) of human CYP2D6 expressed in yeast cells. CYP2D3 had high lidocaine N-deethylation (43 nmol/min/nmol P450) activity, and both CYP2D3 and 2D2 exhibited high lidocaine 3-hydroxylation (2.4 and 1.6 nmol/min/nmol P450, respectively) activity. Bufuralol 1'-hydroxylation catalytic capabilities were comparable among the four isoforms. The activity of CYP2D1 was relatively low toward the three substrates (debrisoquine, 0.091; bufuralol, 1.5; lidocaine 3-hydroxylation, 0.019; lidocaine N-deethylation, 2.8 nmol/min/nmol P450). These findings indicate that debrisoquine, a typical substrate for CYP2D forms, was mainly metabolized by CYP2D2 but not CYP2D1 in rat liver and that the CYP2D forms have different substrate specificity.

Molecular engineering study on electron transfer from NADPH-P450 reductase to rat mitochondrial P450c27 in yeast microsomes.

We have reported the localization on yeast microsomes for a modified P450c27 (mic-P450c27) that contains the microsomal targeting signal of bovine P450c17 in front of the mature form of rat mitochondrial P450c27 (Sakaki, T., Akiyoshi-Shibata, M., Yabusaki, Y., and Ohkawa, H. (1992) J. Biol. Chem. 267, 16497-16502). In this study, we found that mic-P450c27 could be reduced by NADPH in the yeast microsomes without supplement of its physiological redox partners, adrenodoxin and NADPH-adrenodoxin reductase. In order to elucidate the direct electron transfer from NADPH-P450 reductase to mic-P450c27, we carried out simultaneous expression of mic-P450c27 and yeast P450 reductase. The reduction rate of mic-P450c27 was increased by overproduction of yeast P450 reductase, roughly in proportion to the reductase content in the microsomes. In addition, we constructed a fused enzyme between mic-P450c27 and yeast P450 reductase. The reduction rate of heme iron in the fused enzyme was too rapid to be measured. These recombinant yeast microsomes showed a notable 27-hydroxylation activity toward 5beta-cholestane-3alpha,7alpha, 12alpha-triol in the absence of adrenodoxin and adrenodoxin reductase. Finally, we purified mic-P450c27 from the recombinant yeast microsomes and reconstituted the hydroxylation system in liposomal membranes using the purified mic-P450c27 and yeast NADPH-P450 reductase. Mic-P450c27 was reduced by NADPH and showed its monooxygenase activity on the reconstituted system. Therefore, yeast NADPH-P450 reductase alone was found to transfer two electrons from NADPH to mic-P450c27. These results clearly show that mic-P450c27 not only localizes on the microsomes but also functions as a microsomal cytochrome P450 that accepts electrons from NADPH-P450 reductase.

Expression of 135-kDa insecticidal protein gene from Bacillus thuringiensis in the yeast Saccharomyces cerevisiae.

Bacillus thuringiensis subsp. aizawai produces 130-kDa and 135-kDa (CryIA(a)) insecticidal proteins. When Saccharomyces cerevisiae was transformed by the vector carrying a cryIA(a) gene, the gene expression could not be observed. When the 5'-upstream region from the initiation codon was removed using a synthetic oligonucleotide, the CryIA(a) protein was successfully synthesized in yeast. The yeast extract containing CryIA(a) protein had insecticidal activity against Plutella xylostella larvae.