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Biomedical subjects

Y Funae

Publications and source records attributed to Y Funae.

At least 73 records · Page 4Linked to original sources
PubMed1997

Induction of hepatic heme oxygenase and changes in cytochrome P-450s in response to oxidative stress produced by stilbenes and stilbene oxides in rats.

Both trans- and cis-stilbene oxide (TSO and CSO) markedly induced heme oxygenase-1 (HO-1) at the transcriptional level in rat liver. HO-1 induction by TSO and CSO was preceded by glutathione (GSH) depletion in the liver. Pretreatment of rats with buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis, enhanced GSH depletion evoked by either TSO or CSO and augmented the increase in HO-1 mRNA. In contrast, pretreatment with perfluorodecanoic acid (PFDA), which reduced hepatic GSH S-transferase activity, prevented TSO- and CSO-mediated GSH depletion and abolished HO-1 induction. In addition, TSO and CSO enhanced c-jun but not c-fos mRNA, which is in parallel with the HO-1 mRNA change. These findings indicate that the oxidative stress evoked by GSH depletion after the treatment of rats with stilbene oxides could stimulate both HO-1 and c-jun gene expression. Pretreatment with either BSO or PFDA also affected the induction of CYP2B1/2 mRNA and apoprotein by TSO or CSO, suggesting that not only the change of heme pool size but also some other unknown factor or factors may be involved in the regulation of the CYP2B1/2 and HO-1 gene expression. cis-Stilbene (CS), a parent compound of CSO, also induced HO-1 mRNA, together with hepatic GSH depletion, but trans-stilbene (TS) failed to elevate HO-1 mRNA under the experimental conditions. In addition, CS increased CYP2B1/2 mRNA, whereas TS did not. These results suggest that CS could be rapidly oxidized by cytochrome P-450 (P-450) to CSO, leading to GSH depletion in the liver. Such differences in the hepatic metabolic pathways of CS and TS are attributable to the differential effects on HO and P-450 induction by these compounds. Like other phenobarbital-type P-450 inducers, TSO and CSO also induced CYP2C6 and 3A2 apoproteins in rat liver. Stilbene oxide reduced CYP2E1 mRNA and apoproteins for CYP2E1 and 2C11. All of these findings indicate that stilbene compounds have unique effects on hepatic HO-1 and P-450 regulation in rats.

Animals↗
PubMed1997

Effect of phenobarbital on the pharmacokinetics of lidocaine, monoethylglycinexylidide and 3-hydroxylidocaine in the rat: correlation with P450 isoform levels.

To elucidate the effect of cytochrome P450 levels in hepatic microsomes on the metabolism of lidocaine in vivo, we investigated the metabolism of lidocaine in untreated (UT group) and phenobarbital-treated rats (PB group) in vivo and compared the results with those obtained by immunoblotting of rat hepatic microsomes. There were no differences in pharmacokinetic parameters for lidocaine between the UT and PB groups. The plasma concentrations of the N-deethylated metabolite of lidocaine, monoethyl-glycinexylidide (MEGX), in the PB group were significantly higher than those in the UT group. On the other hand, the plasma concentrations of the aromatic ring hydroxylated metabolite of lidocaine, 3-hydroxylidocaine (3-OH LID), were significantly lower in the PB group than in the UT group. When lidocaine metabolism was studied with hepatic microsomes prepared from rats in the UT and PB groups, the rates of formation of MEGX were higher in the microsomes of the PB group than in those of the UT group. The contents of CYP2B1 and 3A2 in rat hepatic microsomes of the BP group measured by immunoblotting were significantly higher than those of the UT group. Strong correlations were found between the area under the plasma concentration vs. time curve for MEGX and specific contents of CYP2B1 and 3A2. These findings suggest that formation of MEGX in vivo is dependent on the levels of CYP2B1 or 3A2 in rat liver.

Anesthetics, Local↗
PubMed1997

Cytochrome P450 responsible for the stereoselective S-oxidation of flosequinan in hepatic microsomes from rats and humans.

The forms of cytochrome P450 involved in the stereoselective S-oxidation of flosequinan [(+/-)-7-fluoro-1-methyl-3-methylsulfinyl-4-quinolone] were investigated in vitro using liver microsomes from rats and humans. Rat liver microsomes supplemented with NADPH catalyzed the four different S-oxidations, which were from flosequinan sulfide (FS; 7-fluoro-1-methyl-3-methylthio-4-quinolone) to R(+)- and S(-)-flosequinan (R-FSO and S-FSO, respectively) and from R-FSO and S-FSO to flosequinan sulfone (FSO2; 7-fluoro-1-methyl-3-methylsulfonyl-4-quinolone). The activities of all the S-oxidases in liver microsomes from male rats were higher than those from female rats. The activities of the S-oxidases measured at a high substrate concentration (1 mM) were induced by treatment of rats with phenobarbital and dexamethasone. Treatment of rats with 3-methylcholanthrene also induced the activities, but only at a low substrate concentration (50 microM), except for the S-oxidase catalyzing the reaction from FS to R-FSO. Enzymes induced by clofibrate and ethanol were not involved in the oxidations at a low substrate concentration. The activities of S-oxidases were correlated with the contents of cytochrome P450 (CYP)3A enzymes. Anti-CYP3A2 antisera inhibited the activities of the S-oxidases catalyzing the reactions from FS to R-FSO (40%) and to S-FSO (60%) at the high substrate concentration and inhibited the activities of the S-oxidases, thus catalyzing reactions from R-FSO and S-FSO to FSO2 (70%) at both high and low substrate concentrations. These results suggest that CYP3A is the major enzyme involved in all S-oxidation pathways in flosequinan metabolism in rats. On the other hand, except for the S-oxidation of FS to R-FSO, the rates of the other three S-oxidations by liver microsomes from 30 individual humans correlated highly with each other, suggesting that the same enzyme would be involved in the three S-oxidations. Anti-CYP3A2 antisera inhibited the activities of all the S-oxidases in human liver microsomes ranging from 40 to 80%, suggesting that CYP3A is also involved in all of the S-oxidations of flosequinan in humans.

Animals↗
PubMed1997

Selective deficiency of debrisoquine 4-hydroxylase activity in mouse liver microsomes.

Cytochrome P450 enzymes belonging to the CYP2D subfamily have been shown to be one of determinants of the polymorphic drug oxidations in the human and the rat. Debrisoquine 4-hydroxylation is a typical reaction catalyzed by these enzymes. However, various strains of mice were observed to have much lower debrisoquine 4-hydroxylase activity than Wistar rats, whereas other monooxygenase activities in mice toward bunitrolol, propranolol, imipramine and amitriptyline, which are mediated by the CYP2D enzymes in the rat, were comparable to those of the rats. Immunoblot analysis of mouse liver microsomes with an antibody raised against a rat CYP2D enzyme indicated that the mouse liver contained a P450 enzyme(s) immunochemically related to the rat CYP2D enzyme. The antibody inhibited propranolol ring-hydroxylase and imipramine 2-hydroxylase activities, as well as testosterone 16alpha-hydroxylase activity, a typical reaction of mouse CYP2D9, but not debrisoquine 4-hydroxylase activity in mouse liver microsomes. We partially purified a P450 enzyme (designated P450 ML2d) from livers of male ddY mice by monitoring the cross-reactivity with the antibody. The partially purified enzyme was indicated to belong to the CYP2D subfamily from its N-terminal amino acid sequence, but the homology of the sequence to other CYP2D enzymes of the mouse (CYP2D9-11) was 62%, suggesting that P450 ML2d is a novel P450 enzyme. P450 ML2d had the oxidation activities for the rat CYP2D-substrates, such as propranolol 4-hydroxylation and imipramine 2-hydroxylation, in higher rates than those of the microsomes, but did not exhibit debrisoquine 4-hydroxylase activity. Our result is the first finding that a mouse CYP2D enzyme also metabolizes substrates for the rat CYP2D enzyme, in addition to steroids, but the enzyme had a limited specificity for the substrates of the CYP2D enzymes of the rat and the human.

Amino Acid Sequence↗
PubMed1997

Irreversible inhibition of cytochrome P450 by nitric oxide.

Nitric oxide (NO) modulates various metabolisms through interaction with thiol proteins and hemoproteins. Although NO interacts reversibly with iron moieties of heme proteins, including cytochrome P450 (P450), dynamic aspects of the formation, catalytic functions and fates of NO-P450 adducts remain to be elucidated. When incubated with NOC7, which spontaneously and stoichiometrically releases NO within 5 min, microsomal P450 rapidly formed nitrosyl-heme adducts as determined by the electron spin resonance method. The signal intensity for the complex increased with time, peaking at 30 min and decreasing to below detectable levels by 60 min of incubation. In contrast, the microsomal levels of low-spin ferric forms of P450 (g = 2.26) rapidly decreased during the initial 30 min but recovered time-dependently thereafter. Analysis by differential spectra (reduced form/CO-reduced form) revealed that on incubation with NOC7, the form of microsomal P450 also changed in a biphasic manner. To elucidate the mechanism for the decrease in the levels of P450, microsomal levels of P450 isozymes (CYPs) were determined by Western blot analysis using specific antibodies against CYP3A2 and CYP2C11, major isoforms found in male rat liver. Kinetic analysis revealed that no appreciable degradation of P450 proteins occurred during the incubation of microsomes with NOC7. The effect of NO on the catalytic activity of the enzymes was determined by using testosterone as substrate because hydroxylation of steroid hormones is one of the major functions of P450. When exposed to NO, the hydroxylation activity in microsomes rapidly decreased during the initial 10 min and then disappeared slowly. These results suggested that NO formed dissociable complexes with P450 isozymes and the catalytic functions of these isozymes were irreversibly inactivated after dissociation from their heme moiety.

Animals↗
PubMed1997

Metabolism of epinastine, a histamine H1 receptor antagonist, in human liver microsomes in comparison with that of terfenadine.

Epinastine is a non-sedative second-generation antiallergic drug, like terfenadine. In the present study, the metabolism of epinastine in human liver microsomes was investigated and compared with that of terfenadine. Terfenadine was extensively metabolized to terfenadine acid with a Km value of 1.78 microM, a Vmax value of 173.8 pmol/min/mg and a metabolic clearance (Vmax/Km) of 103.9. Epinastine, in contrast, was poorly metabolized by microsomes from the same source with a high Km value of 232 microM. Metabolic clearance of epinastine was only 0.832, which was lower by three orders of magnitude than that of terfenadine. Studies with microsomes expressing recombinant cytochrome P450 (CYP) species revealed that the CYP isoforms responsible for epinastine metabolism are CYP3A4, 2D6 and (to a minor extent) 2B6. Epinastine and terfenadine had no effect on CYP1A2 (theophylline 1-demethylation), 2C8/9 (tolbutamide hydroxylation) or 2E1 (chlorzoxazone 6-hydroxylation) activity, but weakly inhibited CYP2D6 (debrisoquine 4-hydroxylation) activity. CYP3A4 (testosterone 6 beta-hydroxylation) activity was strongly inhibited by terfenadine with a Ki value of 25 microM, whereas epinastine had no effect at up to 100 microM. Thus, epinastine is very poorly metabolized compared to terfenadine in human liver microsomes and does not inhibit CYP3A4 activity in vitro, unlike terfenadine.

Aryl Hydrocarbon Hydroxylases↗
PubMed1996

Dose-related increases in quantitative values for altered hepatocytic foci and cytochrome P-450 levels in the livers of rats exposed to phenobarbital in a medium-term bioassay.

The dose-response relationship between liver tumor promoting activity and cytochrome P-450 (CYP) induction by phenobarbital sodium (PB) was investigated using the liver medium-term bioassay system of Ito. Two weeks after a single dose of N-nitrosodiethylamine (DEN) (200 mg/kg body weight, i.p.), rats were given PB at dietary levels of 500, 250, 125, 60, 30, 15 and 8 parts per million (ppm) for 6 weeks. All rats were subjected to partial hepatectomy at week 3, and were killed at week 8. Quantitative values for glutathione S-transferase placental form positive hepatocytic (GST-P+) foci were increased in the high dose groups dose-dependently. In contrast, the values in the low dose groups were rather lower than that of the control. CYP2B1, 2C6 and 3A2 were predominantly immunostainable in hepatocytes around the central vein. While Western blotting revealed CYP2B1 and 2C6 proteins to be increased with strict dose-dependence, CYP3A2 was only elevated at high doses. Thus, a good correlation between increase of GST-P+ foci and CYP3A2 induction was observed, as well as with CYP2B1 and 2C6 in high dose groups.

Animals↗
PubMed1996

Cytochrome P450 enzymes involved in the enhancement of propranolol N-desisopropylation after repeated administration of propranolol in rats.

Repeated oral administration of propranolol (PL, 100 mg/kg daily, for 5, 10 and 15 days) to male Wistar rats increased PL N-desisopropylase and decreased PL 4-,5- and 7-hydroxylase activities in liver microsomes. The increase was highest at the 10 day time point whereas the decrease was relatively constant over the 15 day treatment period. There were no significant changes in the total content of cytochromes P450 (P450) or cytochrome b5 or in NADPH-cytochrome c reductase activity during the PI, treatment. The enhanced N-desisopropylase activities were markedly inhibited by alpha-naphthoflavone (a P450-1A1/2 inhibitor), and moderately by triacetyloleandomycin (a P450-3A1/2 inhibitor) and diethyldithiocarbamate (a P450-2E1 inhibitor). Phenacetin O-deethylase activity, an index of P450-1A2, was significantly increased on day 5, 10 and 15 of the treatment, whereas p-nitrophenol hydroxylase activity was elevated on day 10 only. The PL N-desisopropylation showed a strong and significant correlation with phenacetin O-deethylation, and a weaker but significant correlation with p-nitrophenol hydroxylation. Immunoblot analysis revealed that a protein band corresponding to P450-1A2 was increased by PL pretreatment, and protein band corresponding to P450-3A tended to be increased slightly, but other protein band corresponding to the subfamily of P450-2B, -2C, or -2E was not changed. Pretreatment of rats with P450 inducers (beta-naphthoflavone, phenobarbital, acetone and dexamethasone) increased PL N-dealkylase activity in liver microsomes. Furthermore, antibodies raised against P450-1A and -3A enzymes suppressed PL N-desisopropylation in a concentration-dependent manner, but P450-2E antibody did not. Reconstitution studies showed that P450-1A1, -1A2, -2E1 and -3A2 exhibited catalytic activities for PL N-dealkylation. These results suggest that P450-1A2 is a major PL N-desisopropylase in the PL-treated rats, and P450-3A related enzyme(s) and P450-2E1 as a moderate or minor enzyme are also involved in PL N-dealkylation in native and PL-treated rats.

Administration, Oral↗
PubMed1996

Maintenance and activation of Cyp2e-1 gene expression in mouse hepatocytes in primary culture.

The expression of Cyp2e-1 mRNA and protein was investigated in the C57BL/6NCrj mouse hepatocytes in primary culture, as well as liver and kidney. The mRNA and protein expression in the liver was in the same range in both sexes and was not affected by orchiectomy or ovariectomy. The mRNA expression was enhanced in the kidney of ovariectomized mice, in which the protein contents were not influenced. Orchiectomy decreased the expression of both mRNA and protein. When the hepatocytes were transferred to primary culture, the amounts of the mRNA were not changed within 24 h and about half remained by day 3. However, the expression was low thereafter. The expression of the protein gradually decreased after the start of culture. Dexamethasone showed a potential as an inducer at more than 10(-8) M. Sex hormones increased the expression of this P-450 species a little in culture, but growth hormone did not. These observations indicated that glucocorticoid hormone plays a role in modifying expression of Cyp2e-1 and that the mouse hepatocyte culture is useful for examining its regulation mechanism.

Animals↗
PubMed1996

Multiple forms of human P450 expressed in Saccharomyces cerevisiae. Systematic characterization and comparison with those of the rat.

We systematically characterized the levels and substrate specificity of P450s from humans and rats to extrapolate drug metabolism data from experimental animals to humans. Human P450s (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2D6, 2E1, and 3A4) were expressed in Saccharomyces cerevisiae and purified. Rat P450s were purified from hepatic microsomes of rats. We investigated the catalytic activities of purified P450s in a reconstituted system. Human CYP2B6 and rat CYP2B1 had high lidocaine N-deethylation activity. Human and rat CYP2D forms had high debrisoquine 4-hydroxylation activity. Human CYP3A4 and rat CYP3A2 had high testosterone 2 beta- and 6 beta-hydroxylation activities in a modified reconstituted system with a lipid mixture. The hydroxylation site of testosterone by CYP2B6 (16 alpha- and 16 beta-positions) agreed with that by rat CYP2B1. Human CYP2E1 had the highest lauric acid (omega-1)-hydroxylation activity and also had catalytic properties similar to those of rat CYP2E1. Human CYP2A and 2C forms had catalytic properties in testosterone metabolism different from those of rats. Antibodies raised against purified P450s were used to measure the levels of hepatic P450s. The level of CYP3A4 was the highest in human hepatic microsomes, comprising 30-40% of the total P450. CYP2C9 comprised 10-20% of the total. The levels of CYP1A2, 2A6, 2C8, 2D6, and 2E1 were moderate (5-15% of total P450). CYP2B6 content was very low. The information of this study is useful for drug metabolism and toxicological studies.

Animals↗
PubMed1996

Atypical adenomatous hyperplasia and adenocarcinoma of the human lung: their heterology in form and analogy in immunohistochemical characteristics.

BACKGROUND: In a previous study, morphometry and multivariate cluster analysis was performed on 97 lesions. These consisted of atypical adenomatous hyperplasia (AAH), considered to be an important lesion corresponding to a step of carcinogenesis for adenocarcinoma of the human lung, Clara cell type, and type 2 pneumocyte type adenocarcinomas. Although AAH and the two types of adenocarcinoma were re-classified into three clusters and AAH was defined in clear morphologic terms, the biologic nature of AAH has yet to be clarified. In the present study, immunohistochemical analysis was performed to gain a deeper understanding of the relationship between AAH and the two types of adenocarcinoma, and to compare the results with those obtained by morphometry. METHODS: The 97 lesions analyzed by morphometry were submitted to immunohistochemical analyses using antibodies against surfactant apoprotein A, urine protein 1, carcinoembryonic antigen and cytochrome P-450s (1A1-2, 2B1-2, 2E1). Also examined, as controls, were 17 lesions with adenomatous hyperplasia (AH), a non-neoplastic reactive change of bronchiolo-alveolar cells, 30 areas of normal Clara cells, and 36 areas of normal type 2 pneumocytes. The immunoreactivity was graded by introducing a semi-quantitative scoring system. RESULTS: Immunohistochemically, AAHs behaved quite similarly to the lesions classified as Clara cell type or type 2 pneumocyte type adenocarcinomas. For any of the antibodies employed, no significant difference in immunoreactivity was demonstrated among these lesions. CONCLUSIONS: The results suggest, in accordance with our previous morphometry, that AAH is a lesion closely related with Clara cell type and type 2 pneumocyte type adenocarcinomas, probably as their common precursor. However, the two types of adenocarcinomas, despite their characteristic morphologic features, are indistinguishable using the biological indicators applied in this study.

Adenocarcinoma↗
PubMed1996

Sterol 14-demethylase P450 (P45014DM*) is one of the most ancient and conserved P450 species.

To determine the orthology of sterol 14-demethylase (P45014DM), the only known P450 enzyme distributed widely in eukaryotes with a conserved metabolic role, the full-length amino acid sequences of rat and human P45014DMs were determined from the cloned cDNA sequences, and compared with those of the corresponding fungal proteins (CYP51). The amino acid identity value between given pairs of P45014DMs ranged from 93% (human/rat) to 39% (human or rat/Saccharomyces cerevisiae). All the P45014DMs formed a single cluster in a phylogenetic tree constructed from representative P450 protein sequences currently available. The nearest neighbors to the P45014DM cluster in the phylogenetic tree were CYP7 (cholesterol 7 alpha-hydroxylase) and CYP8 (prostacyclin synthase), and the divergence point of fungal and mammalian P45014DMs was clearly more recent than that of P45014DM and CYP7/CYP8. These lines of evidence show that fungal and mammalian P45014DMs are really orthologous. This is the first example of orthologous P450s occurring in distinct kingdoms. P45014DM may be an ancient P450 which arose before the divergence of major eukaryotic branches and has been conserved throughout evolution. The amino acid identity value (93%) between human and rat P45014DMs was comparable to those observed for some housekeeping enzymes. In addition, a processed pseudogene of P45014DM was found in a rat genomic DNA library, suggesting the expression of P45014DM in germ line cells. These facts suggest that P45014DM may be a housekeeping enzyme essential for the viability of mammals.

Amino Acid Sequence↗
PubMed1996

Metabolism of acetaldehyde to acetate by rat hepatic P-450s: presence of different metabolic pathway from acetaldehyde dehydrogenase system.

NADPH-dependent activity of acetaldehyde oxidation was investigated in microsomes by assaying [14C]acetic acid produced from [14C]acetaldehyde with ion-exchange column. Rat hepatic microsomes exhibited acetaldehyde oxidation activity in the presence of NADPH. This activity was induced 2-fold by the treatment of rats with ethanol. We designated this NADPH-dependent oxidation system as microsomal acetaldehyde-oxidizing system (MAOS), to distinguish from the NAD-dependent acetaldehyde oxidation system by acetaldehyde in mitochondria and cytsol. We further investigated essential enzymes contributing to MAOS activity. Acetaldehyde oxidation activity was investigated in eight forms of purified P-450 in a reconstituted system. Cytochrome P-450 (CYP) 2E1 had the highest oxidation activity and CYP1A2 and CYP4A2 had the next highest activity. Other forms had low activity. To assess the contribution of these forms to MAOS activity, immunoblot was done. CYP2E1 was induced 2-fold by ethanol treatment, but CYP1A2 and CYP4A2 were not reflecting the MAOS activity increased by ethanol treatment. These results suggest that CYP2E1 is the essential enzyme in the MAOS of rats.

Acetaldehyde↗
PubMed1996

Differential effects of adrenocorticotropin in vivo on cytochromes P4502D16 and P450c17 in the guinea pig adrenal cortex.

Studies were performed to compare the effects of ACTH treatment in vivo on cytochromes P4502D16 and P450c17 in the guinea pig adrenal cortex. In untreated animals, CYP2D16 protein and messenger RNA (mRNA) expression as well as xenobiotic-metabolizing activities (bufuralol 1'-hydroxylase, benzphetamine N-demethylase, and benzo(a)pyrene hydroxylase) were far greater in the inner (zona reticularis) than the outer (zona fasciculata plus zona glomerulosa) zones of the cortex. ACTH treatment for 3 or 7 days significantly decreased the rates of xenobiotic metabolism in both the inner and outer adrenal zones. Western and Northern blot analyses revealed that adrenal CYP2D16 protein and mRNA concentrations were significantly decreased by ACTH. In contrast to its inhibitory effects on CYP2D16, ACTH treatment increased steroid 17 alpha-hydroxylase activity in the adrenal inner zone, but did not affect outer zone activity. Microsomal CYP17 protein concentrations were not affected by ACTH despite increases in CYP17 mRNA levels in both zones. The results indicate that ACTH causes down-regulation of adrenal CYP2D16, probably at the transcriptional level. Thus, modulation of CYP2D16 by ACTH is opposite that for the steroidogenic P450 isozymes, suggesting unique regulatory mechanisms. In addition, the data suggest that posttranscriptional mechanisms contribute to ACTH regulation of 17 alpha-hydroxylase activity in the guinea pig adrenal cortex.

Adrenal Cortex↗
PubMed1996

[Autoradiographic studies of oxalate distribution in rat kidney].

BACKGROUND: We performed macro and micro-autoradiographic studies using 14C-oxalate in normal and hyperoxaluric rats in order to elucidate intrarenal distribution of oxalate and to determine where calcium oxalate crystal can be adhere in the kidney. METHODS: Macro and micro-autoradiographic studies of the kidney in normal rats were carried out at intervals of 15, 30 and 90 min after intravenous administration of 14C-oxalate (37 MBq/kg BW). Hyperoxaluria was induced by vitamin B6 deficient diet. The procedure of macro and micro autoradiography is the same as that in normal rat. RESULTS: In normal rats, macroautoradiogram showed that the radioactivity of 14C-oxalate exists in the whole kidney, and microautoradiogram identified this radioactivity mainly in the extraluminal space of renal tubules at 15 min. Macro and micro-autoradiograms taken 90 min after the injection showed practically no radioactivity of 14C-oxalate in the cortex or the medulla, but it was located in the extraluminal space of the papilla. In hyperoxaluric rats, macroautoradiogram showed some spotty accumulations of 14C-oxalate in the inner medulla and papilla. Microautoradiogram revealed that these accumulations are mainly seen in the extraluminal space. CONCLUSION: These results indicate that in normal rats the injected 14C-oxalate remains in the renal papilla, especially in the extraluminal space, when nearly all oxalate was excreted by urine, and in hyperoxaluric rats 14C-oxalate exists there as calcium oxalate crystal or microlith.

Animals↗
PubMed1996

Microsomal ethanol oxidizing system activity by human hepatic cytochrome P450s.

To assess the contribution of cytochrome P450 (P450) to the microsomal ethanol oxidation system (MEOS) in humans, we examined ethanol oxidization activity in human hepatic microsomes and multiple forms of human hepatic P450s expressed in B-lymphoblastoid cells. Acetaldehyde produced by the MEOS was converted into a fluorescent derivate with cyclohexane-1,3-dione and analyzed by high-performance liquid chromatography using a fluorescence detector. The ethanol oxidation activity of seven forms of human P450s was investigated. P450s 2E1 and 1A2 formed acetaldehyde at high rates. In immunoinhibition studies, anti-P450 2E1 antibody inhibited the ethanol oxidation activity of human hepatic microsomes by 54%, and anti-P450 1A2 antibody inhibited ethanol oxidation activity by 21%. 7,8-Benzoflavone, an inhibitor of P450 1A forms, also inhibited this activity by 38%. The correlation of ethanol oxidation activity with the levels of immunoreactive P450 2E1 in individual human microsomes was highly significant (r = 0.91, P < .001), and the correlation of ethanol oxidation activity with the levels of P450 1A2 was also highly significant (r = 0.87, P < .001). The Km values of P450s 2E1 and 1A2 for ethanol oxidation were 16.5 and 23.6 mM, respectively. These results indicated that P450 2E1 was a major contributor to the MEOS in humans; however, P450 1A2 was considered to play an important role in the MEOS.

Adolescent↗
PubMed1996

Characterization of CYP2A6 involved in 3'-hydroxylation of cotinine in human liver microsomes.

Nicotine is primarily metabolized to cotinine, and cotinine is further metabolized to trans-3'-hydroxycotinine in human liver, which is a major metabolite of nicotine in humans. We studied the formation of trans-3'-hydroxycotinine from cotinine in human liver microsomes. trans-3'-Hydroxycotinine formation demonstrated single enzyme Michaelis-Menten kinetics (Km, 234.5 +/- 26.8 MicroM; Vmax, 37.2 +/- 2.4 pmol/min/mg protein). Significant correlation (r = .967, P < .001) between cotinine 3'-hydroxylase activities at low (50 microM) and high (1 microM) cotinine concentrations in 20 human liver microsomes suggested the contribution of a single enzyme to cotinine 3'-hydroxylation. The cotinine 3'-hydroxylase activity correlated significantly with immunoreactive cytochrome P450 (CYP)2A6 contents (r = .756, P < .01) and coumarin 7-hydroxylase activity (r = .887, P < .001). The cotinine 3'-hydroxylase activity was inhibited by coumarin, alpha-naphthoflavone, chlorzoxazone and anti-rat CYP2A1 antibodies. Microsomes of B-lymphoblastoid cells expressing human CYP2A6 exhibited cotinine 3'-hydroxylase activity. The Km value of the expressed CYP2A6 (264.7 microM) was almost identical to that of human liver microsomes. In conclusion, cotinine 3'-hydroxylation appears to be catalyzed solely by CYP2A6 in humans. Cotinine is a candidate for a new substrate for CYP2A6 in humans.

Aryl Hydrocarbon Hydroxylases↗