Carbamazepine 10,11-epoxidation in human liver microsomes: influence of the CYP3A5*3 polymorphism.

Carbamazepine (CBZ) is a commonly prescribed antiepileptic drug, and is mainly metabolized to 10,11-CBZ epoxide in humans. Its biotransformation is catalyzed by cytochrome P450 (CYP) enzymes, with the predominant isoforms being CYP3A4 and CYP3A5. In the present study, the effects of the CYP3A5*3 (rs776746) polymorphism on CBZ 10,11-epoxidation in human liver microsomes genotyped as CYP3A5*3 were examined using a kinetic analysis. The kinetics for CBZ 10,11-epoxidation fit the Hill model with n of approximately 1.9-2.1 in all liver microsomes of the wild-type (CYP3A5*1/*1) and heterozygous (CYP3A5*1/*3) and homozygous (CYP3A5*3/*3) variants. The S50, Vmax, and CLmax values of wild-type liver microsomes were 263-327 µM, 793-1590 pmol/min/mg protein, and 1.51-2.95 µL/min/mg protein, respectively. The Vmax and CLmax values of liver microsomes of the heterozygous variant were approximately 15-40% those of wild-type liver microsomes. On the other hand, the Vmax and CLmax values of liver microsomes of the homozygous variant were more similar to those of the wild-type than the heterozygous variant. These results suggest that the CYP3A5*3 polymorphism has a negligible effect on CBZ 10,11-epoxidation in an in vitro system using human liver microsomes.

Species and sex differences in propofol glucuronidation in liver microsomes of humans, monkeys, rats and mice.

Propofol (2,6-diisopropylphenol) is a short-acting anesthetic commonly used in clinical practice, and is rapidly metabolized into glucuronide by UDP-glucuronosyltransferase (UGT). In the present study, propofol glucuronidation was examined in the liver microsomes of male and female humans, monkeys, rats, and mice. The kinetics of propofol glucuronidation by liver microsomes fit the substrate inhibition model for humans and mice, the Hill model for monkeys, and the isoenzyme (biphasic) model for rats. The K(m), V(max), and CL(int) values of human liver microsomes were 50 µM, 5.6 nmol/min/mg protein, and 110 µL/min/mg protein, respectively, for males, and 46 µM, 6.0 nmol/min/mg protein, and 130 µL/min/mg protein, respectively, for females. The rank order of the CL(int) or CL(max) (in vitro clearance) values of liver microsomes was mice humans > monkeys > rats (high-affinity phase) rats (low-affinity phase) in both males and females. Although no significant sex differences were observed in the values of kinetic parameters in any animal species, the in vitro clearance values of liver microsomes were males < females in humans, males = females in rats (low-affinity phase), and males > females in monkeys, rats (high-affinity phase), and mice. These results demonstrated that the kinetic profile of propofol glucuronidation by liver microsomes markedly differed among humans, monkeys, rats, and mice, and suggest that species and sex differences exist in the roles of UGT isoform(s), including UGT1A9, involved in its metabolism.

In vitro glucuronidation of propofol in microsomal fractions from human liver, intestine and kidney: tissue distribution and physiological role of UGT1A9.

Propofol (2,6-diisopropylphenol) is intravenously administered for anesthetic induction and maintenance, and is rapidly metabolized into its glucuronide, mainly by UDP-glucuronosyltransferase 1A9 (UGT1A9). In this study, propofol glucuronidation by liver microsomes (HLM), intestinal microsomes (HIM) and kidney microsomes (HKM) of humans were examined. The expression of UGT1A9 protein in HLM, HIM and HKM was analyzed by immunoblotting. The staining band intensities for UGT1A9 of HIM and HKM were 12% and 119% those of HLM, respectively. The kinetics of propofol glucuronidation by HLM and HKM exhibited substrate inhibition, whereas the kinetics by HIM followed the Michaelis-Menten model. The K(m), V(max) and CL(int) values of HLM were 41.8 µM, 5.21 nmol/min/mg protein and 126 µl/min/mg protein, respectively. The K(m) value of HIM was significantly higher (6.7-fold) than that of HLM, and the V(max) and CL(int) values were significantly lower (56% and 8.3%, respectively) than those of HLM. The K(m) value of HKM was comparable to that of HLM, and the V(max) and CL(int) values were significantly higher (2.1- and 3.7-fold, respectively) than those of HLM, respectively. These findings suggest that UGT1A9 expressed in the kidney as well as in the liver plays an important role in propofol glucuronidation. The information gained in this study should contribute to an appropriate use of drugs metabolized by UGT1A9.

Evaluation of animal models for intestinal first-pass metabolism of drug candidates to be metabolized by CYP3A enzymes via in vivo and in vitro oxidation of midazolam and triazolam.

1. To search an appropriate evaluation methodology for the intestinal first-pass metabolism of new drug candidates, grapefruit juice (GFJ)- and vehicle (tap water)-pretreated mice or rats were orally administered midazolam (MDZ) or triazolam (TRZ), and blood levels of the parent compounds and their metabolites were measured by liquid chromatography/MS/MS. A significant effect of GFJ to elevate the blood levels was observed only for TRZ in mice. 2. In vitro experiments using mouse, rat and human intestinal and hepatic microsomal fractions demonstrated that GFJ suppressed the intestinal microsomal oxidation of MDZ and especially TRZ. Substrate inhibition by MDZ caused reduction in 1'-hydroxylation but not 4-hydroxylation in both intestinal and hepatic microsomal fractions. The kinetic profiles of MDZ oxidation and the substrate inhibition in mouse intestinal and hepatic microsomal fractions were very similar to those in human microsomes but were different from those in rat microsomes. Furthermore, MDZ caused mechanism-based inactivation of cytochrome P450 3A-dependent TRZ 1'-hydroxylation in mouse, rat and human intestinal microsomes with similar potencies. 3. These results are useful information in the analysis of data obtained in mouse and rat for the evaluation of first-pass effects of drug candidates to be metabolized by CYP3A enzymes.

Determination of polycyclic aromatic hydrocarbons in food samples by automated on-line in-tube solid-phase microextraction coupled with high-performance liquid chromatography-fluorescence detection.

A simple and sensitive automated method, consisting of in-tube solid-phase microextraction (SPME) coupled with high-performance liquid chromatography-fluorescence detection (HPLC-FLD), was developed for the determination of 15 polycyclic aromatic hydrocarbons (PAHs) in food samples. PAHs were separated within 15 min by HPLC using a Zorbax Eclipse PAH column with a water/acetonitrile gradient elution program as the mobile phase. The optimum in-tube SPME conditions were 20 draw/eject cycles of 40 microL of sample using a CP-Sil 19CB capillary column as an extraction device. Low- and high-molecular weight PAHs were extracted effectively onto the capillary coating from 5% and 30% methanol solutions, respectively. The extracted PAHs were readily desorbed from the capillary by passage of the mobile phase, and no carryover was observed. Using the in-tube SPME HPLC-FLD method, good linearity of the calibration curve (r>0.9972) was obtained in the concentration range of 0.05-2.0 ng/mL, and the detection limits (S/N=3) of PAHs were 0.32-4.63 pg/mL. The in-tube SPME method showed 18-47 fold higher sensitivity than the direct injection method. The intra-day and inter-day precision (relative standard deviations) for a 1 ng/mL PAH mixture were below 5.1% and 7.6% (n=5), respectively. This method was applied successfully to the analysis of tea products and dried food samples without interference peaks, and the recoveries of PAHs spiked into the tea samples were >70%. Low-molecular weight PAHs such as naphthalene and pyrene were detected in many foods, and carcinogenic benzo[a]pyrene, at relatively high concentrations, was also detected in some black tea samples. This method was also utilized to assess the release of PAHs from tea leaves into the liquor.

Separate evaluation of intestinal and hepatic metabolism of three benzodiazepines in rats with cannulated portal and jugular veins: comparison with the profile in non-cannulated mice.

Pharmacokinetic analyses of three kinds of benzodiazepines--midazolam (MDZ), triazolam (TRZ) and alprezolam (APZ)--were performed in rats with cannulated portal and jugular veins. Each drug was administered to the double-cannulated rats, and pharmacokinetic data for the parent drugs and their 1'- and 4-hydroxylated metabolites were compared with those obtained in non-cannulated mice. In bioavailability, the drugs ranked APZ >> TRZ = MDZ in rats, and APZ > TRZ >> MDZ in mice, with the values for MDZ remarkably different between rats and mice (19% in rats versus 2.3% in mice). In contrast, hepatic availability (Fh) was similar (APZ > TRZ > MDZ) in both species. Highly significant relationships were found between the ratio of the area under the plasma concentration-time curve (AUC) for the parent drugs in portal blood (AUC(por)) to that in systemic blood (AUC(sys)) and Fh in rats and mice. The double-cannulated rat is useful for estimating the hepatic availability of drug candidates by determining the AUC values for the parent drugs in portal and systemic blood samples.

Functional characterization of CYP3A4.16: catalytic activities toward midazolam and carbamazepine.

1. To assess the substrate-dependent effects of the low-activity allele of human CYP3A4, CYP3A4*16 (Thr185Ser), a recombinant wild-type (CYP3A4.1) or variant (CYP3A4.16) protein was co-expressed with human NADPH-P450 reductase in Sf21 insect cells using a baculovirus-insect cell system. 2. The holo-CYP3A4 protein level of CYP3A4.16 in insect microsomes was slightly higher than that of CYP3A4.1, while no difference in total (apo- and holo-) CYP3A4 protein levels was observed between them. 3. When midazolam was used as a substrate, K(m) and V(max) for 1'-hydroxylation in CYP3A4.16 were significantly higher and lower, respectively, than those in the wild-type, resulting in a 50% decrease in intrinsic clearance (V(max)/K(m)) of the variant. In contrast, intrinsic clearance for 4-hydroxylation of the variant was decreased by 30% due to a significant increase in K(m) without a difference in V(max). 4. Both the wild-type and variant exhibited sigmoidal kinetic profiles for carbamazepine 10,11-epoxide formation. When the modified two-site equation was applied for the analysis of kinetic parameters, K(m2) and V(max2) of CYP3A4.16 were approximately two times higher and lower than those of the wild-type, resulting in a 74% decrease in intrinsic clearance. 5. These results demonstrated that CYP3A4.16 shows the substrate-dependent altered kinetics compared with CYP3A4.1.

Determination of aflatoxins in food samples by automated on-line in-tube solid-phase microextraction coupled with liquid chromatography-mass spectrometry.

A simple and sensitive automated method for determination of aflatoxins (B1, B2, G1, and G2) in nuts, cereals, dried fruits, and spices was developed consisting of in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-mass spectrometry (LC-MS). Aflatoxins were separated within 8 min by high-performance liquid chromatography using a Zorbax Eclipse XDB-C8 column with methanol/acetonitrile (60/40, v/v): 5mM ammonium formate (45:55) as the mobile phase. Electrospray ionization conditions in the positive ion mode were optimized for MS detection of aflatoxins. The pseudo-molecular ions [M+H](+) were used to detect aflatoxins in selected ion monitoring (SIM) mode. The optimum in-tube SPME conditions were 25draw/eject cycles of 40 microL of sample using a Supel-Q PLOT capillary column as an extraction device. The extracted aflatoxins were readily desorbed from the capillary by passage of the mobile phase, and no carryover was observed. Using the in-tube SPME LC-MS with SIM method, good linearity of the calibration curve (r>0.9994) was obtained in the concentration range of 0.05-2.0 ng/mL using aflatoxin M1 as an internal standard, and the detection limits (S/N=3) of aflatoxins were 2.1-2.8 pg/mL. The in-tube SPME method showed >23-fold higher sensitivity than the direct injection method (10 microL injection volume). The within-day and between-day precision (relative standard deviations) at the concentration of 1 ng/mL aflatoxin mixture were below 3.3% and 7.7% (n=5), respectively. This method was applied successfully to analysis of food samples without interference peaks. The recoveries of aflatoxins spiked into nuts and cereals were >80%, and the relative standard deviations were <11.2%. Aflatoxins were detected at <10 ng/g in several commercial food samples.

Functional characterization of two novel CYP2C19 variants (CYP2C19*18 and CYP2C19*19) found in a Japanese population.

Cytochrome P450 2C19 (CYP2C19) plays an important role in the metabolism of a wide range of therapeutic drugs and exhibits genetic polymorphism with interindividual differences in metabolic activity. We have previously described two CYP2C19 allelic variants, namely CYP2C19*18 and CYP2C19*19 with Arg329His/Ile331Val and Ser51Gly/Ile331Val substitutions, respectively. In order to investigate precisely the effect of amino acid substitutions on CYP2C19 function, CYP2C19 proteins of the wild-type (CYP2C19.1B having Ile331Val) and variants (CYP2C19.18 and CYP2C19.19) were heterologously expressed in yeast cells, and their S-mephenytoin 4'-hydroxylation activities were determined. The K(m) value of CYP2C19.19 for S-mephenytoin 4'-hydroxylation was significantly higher (3.0-fold) than that of CYP2C19.1B. Although no significant differences in V(max) values on the basis of microsomal and functional CYP protein levels were observed between CYP2C19.1B and CYP2C19.19, the V(max)/K(m) values of CYP2C19.19 were significantly reduced to 29-47% of CYP2C19.1B. By contrast, the K(m), V(max) or V(max)/K(m) values of CYP2C19.18 were similar to those of CYP2C19.1B. These results suggest that Ser51Gly substitution in CYP2C19.19 decreases the affinity toward S-mephenytoin of CYP2C19 enzyme, and imply that the genetic polymorphism of CYP2C19*19 also causes variations in the clinical response to drugs metabolized by CYP2C19.

Arsenite and arsenate activate extracellular signal-regulated kinases 1/2 by an epidermal growth factor receptor-mediated pathway in normal human keratinocytes.

BACKGROUND: Inorganic arsenic is an environmental contaminant and is associated with the increased risk of human skin cancer. Arsenic has been reported to activate or inhibit a variety of cellular signalling pathways which has effects on cell growth, differentiation and apoptosis. However, the molecular mechanisms of these arsenic-induced biological effects are not completely understood. OBJECTIVES: To understand the molecular basis for the mode of action of arsenicals, we examined the effect of arsenite and arsenate on the activation of mitogen-activated protein kinases (MAPK) and the upstream signalling cascade in normal human epidermal keratinocytes (NHEK). METHODS: NHEK were exposed to arsenite or arsenate. Western blot analysis was performed to determine the activation of extracellular signal-regulated kinases (ERK) 1/2, c-jun N-terminal kinases (JNK), p38, and MAPK or ERK kinases (MEK) 1/2. Epidermal growth factor receptor (EGFR) tyrosine phosphorylation and recruitment of its adaptor proteins, Shc and Grb2, to EGFR were detected by immunoprecipitation and Western blot analysis. RESULTS: Both arsenicals activated ERK1/2, which are most highly activated in response to mitogenic stimulation, in addition to JNK and p38, which show greater activation in response to cellular stresses. The kinetics of ERK1/2 activation differed from those of JNK and p38 activation. Both arsenicals transiently activated ERK1/2 prior to JNK and p38 activation. MEK1/2, upstream kinases of ERK1/2, were also activated by arsenicals with similar time kinetics to that of ERK1/2 activation. To investigate a signalling pathway leading to activation of MEK1/2-ERK1/2, we examined the tyrosine phosphorylation of EGFR and Shc adapter protein. Both arsenicals stimulated tyrosine phosphorylation of EGFR and Shc. After arsenical treatment, Shc immunoprecipitates contained coprecipitated EGFR and Grb2, suggesting that both arsenicals induce the assembly of EGFR-Shc-Grb2 complexes. Both the EGFR inhibitor tyrphostin AG1478 and anti-EGFR blocking antibody markedly attenuated ERK1/2 activation induced by arsenicals, but did not affect JNK and p38 activation. CONCLUSIONS: Our data indicate that both arsenite and arsenate activate the EGFR-Shc-Grb2-MEK1/2-ERK1/2 signalling cascade in NHEK.

Functional characterization of three human cytochrome p450 2E1 variants with amino acid substitutions.

1. Cytochrome p450 (p450) 2E1 is a hepatic enzyme of importance for the metabolism of xenobiotics such as drugs and environmental toxicants. Genetic polymorphisms of CYP2E1 in 5'-flanking and coding regions have been found previously in Caucasian and Chinese populations. 2. In order to investigate the effects of amino acid substitutions on the function of CYP2E1, the enzymes of all known CYP2E1 variants in the coding region (CYP2E1.2, CYP2E1.3 and CYP2E1.4) with Arg76His, Val389Ile and Val179Ile substitutions, respectively, as well as the wild-type CYP2E1 (CYP2E1.1) were expressed in COS-1 cells, and their chlorzoxazone 6-hydroxylation and 4-nitrophenol 2-hydroxylation activities were determined. 3. The protein level of CYP2E1.2 was reduced to 29% compared with that of CYP2E1.1. The profiles of the level of activity relative to CYP2E1.1 for chlorzoxazone 6-hydroxylation (300 microM substrate) and 4-nitrophenol 2-hydroxylation (150 microM substrate) were very similar. 4. Although the K(m) values were not significantly different among wild-type and variant CYP2E1s in any oxidation metabolism, the V(max) and V(max)/K(m) of CYP2E1.2 on the basis of the CYP2E1 protein level were 2.7-3.0-fold higher than those of CYP2E1.1. In contrast, the levels of CYP2E1 protein and catalytic activity of CYP2E1.3 and CYP2E1.4 were not affected by the corresponding amino acid substitutions. 5. The findings suggest that Arg76 is closely associated with the function of CYP2E1, and that the genetic polymorphism of CYP2E1 is one cause of interindividual differences in the toxicity of xenobiotics.

Non-synonymous single nucleotide alterations in the microsomal epoxide hydrolase gene and their functional effects.

1. By sequencing genomic DNA from 72 established cell lines derived from Japanese individuals, we detected 25 single nucleotide alterations in the microsomal epoxide hydrolase (EPHX1) gene. Of them, five were exonic alterations resulting in amino acid alterations (77C>G, T26S; 128G>C, R43T; 337T>C, Y113H; 416A>G, H139R; 823A>G, T275A). The T26S, R43T, Y113H and H139R substitutions were found at relatively high frequencies and seemed to be polymorphic, and T26S and T275A were novel. 2. To examine the effects of these amino acid alterations on EPHX1 function, EPHX1 cDNA constructs of wild-type and five variants were transfected into COS-1 cells, and their hydrolytic activities for cis-stilbene oxide were determined in vitro. Although all of the transfectants expressed EPHX1 mRNA and protein at similar levels, the variant H139R protein was expressed at a significantly higher level (128% of the wild-type). K(m) values were not significantly different between the wild-type and variants. 3. Increase (140%) in the enzymatic activity (V(max)) of the variant H139R was accompanied by the increased EPHX1 protein level without any significant change in the intrinsic EPHX1 activity. On the other hand, the variant R43T showed increased values for V(max) and clearance (V(max)/K(m)) (around 130%) both on a microsomal protein basis and on a EPHX1 protein basis. 4. These results suggest that R43T as well as H139R increase epoxide hydrolase activity.

Human liver UDP-glucuronosyltransferase isoforms involved in the glucuronidation of 7-ethyl-10-hydroxycamptothecin.

1. The human liver UDP-glucuronosyltransferase (UGT) isoforms involved in the glucuronidation of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), have been studied using microsomes from human liver and insect cells expressing human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, 2B7, 2B15). 2. The glucuronidation of SN-38 was catalysed by UGT1A1, UGT1A3, UGT1A6 and UGT1A9 as well as by liver microsomes. Among these UGT isoforms, UGT1A1 showed the highest activity of SN-38 glucuronidation at both low (1 microM) and high (200 microM) substrate concentrations. The ranking in order of activity at low and high substrate concentrations was UGT1A1 > UGT1A9 > UGT1A6 > UGT1A3 and UGT1A1 > UGT1A3 > UGT1A6 > or = UGT1A9, respectively. 3. The enzyme kinetics of SN-38 glucuronidation were examined by means of Lineweaver-Burk analysis. The activity of the glucuronidation in liver microsomes exhibits a monophasic kinetic pattern, with an apparent Km and Vmax of 35.9 microM and 134 pmol min(-1) mg(-1) protein, respectively. The UGT isoforms involved in SN-38 glucuronidation could be classified into two types: low-Km types such as UGT1A1 and UGT1A9, and high-Km types such as UGT1A3 and UGT1A6, in terms of affinity toward substrate. UGT1A1 had the highest Vmax followed by UGT1A3. Vmax of UGT1A6 and UGT1A9 were approximately 1/9 to 1/12 of that of UGT1A1. 4. The activity of SN-38 glucuronidation by liver microsomes and UGT1A1 was effectively inhibited by bilirubin. Planar and bulky phenols substantially inhibited the SN-38 glucuronidation activity of liver microsomes and UMT1A9, and/or UGT1A6. Although cholic acid derivatives strongly inhibited the activity of SN-38 glucuronidation by UGT1A3, the inhibition profile did not parallel that in liver microsomes. 5. These results demonstrate that at least four UGT1A isoforms are responsible for SN-38 glucuronidation in human livers, and suggest that the role and contribution of each differ substantially.

High-performance liquid chromatographic assay for glucuronidation activity of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), in human liver microsomes.

A simple and sensitive assay for glucuronidation activity of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), in human liver microsomes by high-performance liquid chromatography (HPLC) with fluorescence detection is reported. The method was validated for the determination of SN-38 glucuronide (SN-38G) with respect to specificity, linearity, recovery, stability, precision, accuracy, and limits of detection and quantitation. There was no interference from matrix and non-enzymatic reactions. The calibration curve for SN-38G was linear from 5 to 500 nM. Average recoveries ranged from 98 to 100% in spiked human liver microsome samples, and the SN-38G was stable at 4 degrees C for at least 72 h. The newly developed method was found to be more sensitive and selective than previous methods using thin layer chromatography and HPLC. The limit of quantitation for SN-38G was 5 nM (2.5 pmol/assay). The intra- and inter-day precision and accuracy were less than 7 and 4%, respectively. The intra- and inter-day precision of enzyme assay for UDP-glucuronosyltransferase (UGT) activity toward SN-38 in human liver microsomes was less than 4%. With this improved sensitivity, the kinetics of SN-38 glucuronidation in human liver microsomes could be determined more precisely. Therefore, this method is applicable to in vitro study on the side effects and drug interactions of CPT-11 using small amounts of biological sample.

Determination of UDP-glucuronosyltransferase UGT1A6 activity in human and rat liver microsomes by HPLC with UV detection.

A simple and sensitive method for the determination of UDP-glucuronosyltransferase UGT1A6 activity using 4-methylumbelliferone (4-MU) and 4-nitrophenol (4-NP) as substrates in human and rat liver microsomes by high-performance liquid chromatography (HPLC) with uv detection is reported. The method was validated for the determination of 4-methylumbelliferyl beta-D-glucuronide (4-MUG) and 4-nitrophenyl beta-D-glucuronide (4-NPG) with respect to specificity, linearity, detection limit, recovery, stability, precision and accuracy. There was no interference from matrix and non-enzymatic reactions. Calibration curves for 4-MUG and 4-NPG are linear from 0.5 to 500 microM. Average recoveries ranged from 98 to 100% in spiked liver microsomes samples. 4-MUG and 4-NPG were stable at 4 degrees C for at least 72 h in spiked liver microsomes samples. The method was found to be more sensitive than previous methods using a spectrophotometer, a spectrofluorometer and HPLC. The detection limit for 4-MUG and 4-NPG (signal-to-noise ratio of 3) was 14 and 23 nM, respectively. The intra- and inter-day precision (relative S.D. (RSD)) and accuracy (relative mean error (RME)) was <5 and 9%, respectively. The intra- and inter-day reproducibility (RSD) of UGT1A6 enzyme assay in liver microsomes was <6%. With this improved sensitivity, the kinetics of UGT activities toward 4-MU and 4-NP in human and rat liver microsomes could be determined more precisely. In addition, the method could determine the non-inducible, and 3-methylcholanthrene- and phenobarbital-inducible activities of UGT1A6 in rat liver microsomes under the same assay conditions. Therefore, this method is applicable to in vivo and in vitro studies on the interaction of xenobiotic chemicals with UGT1A6 isoform in mammals using small amounts of biological samples.

Non-synonymous single nucleotide alterations found in the CYP2C8 gene result in reduced in vitro paclitaxel metabolism.

By sequencing genomic DNA from 73 established cell lines derived from Japanese individuals, we detected 9 single nucleotide polymorphisms (SNPs) in the CYP2C8 gene. Of them, 3 exonic SNPs resulted in amino acid alterations (g416a, R139K; a1196g, K399R; c1210g, P404A). The first two alterations were detected concurrently in one cell line and thought to be the same as CYP2C8*3. To examine the effects of these amino acid alterations on CYP2C8 function, wild-type and four types of variant CYP2C8 cDNA constructs (R139K, K399R, R139K/K399R and P404A) were transfected into Hep G2 cells and their paclitaxel 6a-hydroxylase activities were determined in vitro. Km values were not significantly different from that of the wild-type in any of the variants studied. The variant R139K/K399R showed reduced values for Vmax and clearance (Vmax/Km) similar to those of its single variant, R139K. The variant P404A also showed a significantly lowered clearance due to reduced level of protein expression. These results suggest that not only the double variant (R139K/K399R, CYP2C8*3) but also our novel variant P404A in the CYP2C8 gene are less efficient in paclitaxel metabolism.

Determination of cytochrome P450 1A activities in mammalian liver microsomes by high-performance liquid chromatography with fluorescence detection.

A sensitive method for the determination of cytochrome P450 (P450 or CYP) 1A activities such as ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) in liver microsomes from human, monkey, rat and mouse by high-performance liquid chromatography with fluorescence detection is reported. The newly developed method was found to be more sensitive than previous methods using a spectrofluorimeter and fluorescence plate reader. The detection limit for resorufin (signal-to-noise ratio of 3) was 0.80 pmol/assay. Intra-day and inter-day precisions (expressed as relative standard deviation) were less than 6% for both enzyme activities. With this improved sensitivity, the kinetics of EROD and MROD activities in mammalian liver microsomes could be determined more precisely. EROD activities in human and monkey liver microsomes, and MROD activities in liver microsomes from all animal species exhibited a monophasic kinetic pattern, whereas the pattern of EROD activities in rat and mouse liver microsomes was biphasic. In addition, the method could determine the non-inducible and 3-methylcholanthrene-inducible activities of EROD and MROD in rat and mouse liver microsomes under the same assay conditions. Therefore, this method is applicable to in vivo and in vitro studies on the interaction of xenobiotic chemicals with cytochrome CYP1A isoforms in mammals.

Interaction of bisphenol A with rat hepatic cytochrome P450 enzymes.

The effect of bisphenol A (BPA) on the kinetics of cytochrome P450 (P450)-dependent monooxygenases in rat liver microsomes was studied. Testosterone 16beta-hydroxylase (TS16BH) and testosterone 2alpha-hydroxylase (TS2AH) activities were extensively inhibited by BPA at 100 microM (69% and 74%, respectively). The inhibition type was mixed for both P450-dependent monooxyganases. The Ki of TS16BH and TS2AH from Lineweaver-Burk plots were 25.9 and 24.9 microM, respectively. The activities of acetanilide 4-hydroxylase (AA4H), 7-ethoxycoumarin O-deethylase (ECOD), bufuralol 1'-hydroxylase (BF1'H), chlorzoxazone 6-hydroxylase (CZ6H) and testosterone 6beta-hydroxylase (TS6BH) were also effectively inhibited by BPA at 100 microM (43-52%). The inhibition type of these P450-dependent monooxygenases was mixed or uncompetitive, and the K(i)s (50.5-88.5 microM) were higher than those of TS16BH and TS2AH. By contrast, the values of IC50 and Ki of testosterone 7alpha-hydroxylase (TS7AH) and lauric acid omega-hydroxylase (LAOH) for BPA were >1000 microM. These results suggest that BPA interacts with rat hepatic CYP1A2, CYP2A2, CYP2B2, CYP2C11, CYP2D1, CYP2E1 and CYP3A2 in vitro.

Effect of 4-tert-octylphenol on cytochrome P450 enzymes in rat liver.

The effects were studied of 4-tert-octylphenol (OP) on hepatic cytochrome P450 enzymes in rats. Rats were treated intraperitoneally with OP twice, at doses of 5, 10, and 20 mg/kg. Among the cytochrome P450-dependent monooxygenase activities, testosterone 2alpha-hydroxylase activity, which is associated with CYP2C11, was significantly decreased by OP at all doses. The level relative to control activity was 67-22%. CYP3A2-dependent monooxygenase, testosterone 6beta-hydroxylase activity was also decreased by 51% by OP at 20 mg/kg. Furthermore, immunoblotting showed that OP (10 or 20 mg/kg) significantly decreased CYP2C11/6 and CYP3A2/1 protein levels. However, the reduction ratio of CYP2C11/6 and CYP3A2/1 protein levels by OP treatment was lower than that of testosterone 2alpha-hydroxylase and testosterone 6beta-hydroxylase activities. The Cl(int) (Vmax/Km) value for testosterone 2alpha-hydroxylase was significantly decreased by OP at all doses, whereas the Cl(int) value for testosterone 6beta-hydroxylase was only decreased by OP at 20 mg/kg. In addition, 7-ethoxycoumarin O-deethylase activity was significantly decreased by 32% by the highest dose of OP. By contrast, CYP1A1-, CYP1A2-, CYP2A1-, CYP2B1/2-, CYP2D1-, CYP2E1- and CYP4A1/2/3-dependent monooxygenase activities were not affected by OP at any dose. These results suggest that OP changes the male-specific cytochrome P450 isoforms in rat liver, and that these changes closely relate to the toxicity of OP.