Biomedical subjects

K Oguri

Publications and source records attributed to K Oguri.

At least 37 records · Page 2Linked to original sources

PubMed1998

Phenobarbital-mediated induction of the CYP2B subfamily is not antagonized by picrotoxin, a potent antagonist of barbiturates in the central nervous system.

Our previous study indicated that picrotoxin competes with phenobarbital (PB) for the binding to liver microsomes, although the target of binding and the physiological role were not determined (unpublished observation). To seek information on the target site of PB, reflecting the induction of hepatic enzymes, we examined here the effect of picrotoxin on PB-mediated induction of hepatic cytochrome P450 and UDP-glucuronosyltransferase in vivo in rats. The induction of the CYP2B1/2 was estimated by immunoblot analysis and by measuring the activity of testosterone 16beta-hydroxylation. Intraperitoneal injection of picrotoxin alone slightly increased CYP2B1/2 protein. An experiment on co-treatment of picrotoxin and PB showed that picrotoxin enhanced rather than antagonized the inducing effect of PB. The results suggest two possibilities: that 1) picrotoxin increases the CYP2B subfamily by binding to the same site as PB; or 2) the site in microsomes for the competition between PB and picrotoxin does not reflect the induction of P450.

Animals↗

PubMed1998

A new screening method for amphetamine and methamphetamine using dansyl chloride derivatization and cartridge fluorescence.

A new screening method for amphetamines was developed. It consists of derivatization with dansyl chloride, extraction of the derivative using a Sep-Pak C18 or a Bond Elut C18, solid phase extraction columns, and visualization of the fluorescence of the cartridge. A control test using drug-free urine showed no fluorescence. Amphetamine, methamphetamine and the methylenedioxy derivatives exhibited strong fluorescence, while related compounds, such as N-ethylamphetamine and fenetylline, were negative or weakly positive. The disadvantage of the present method is that it is a multi-step procedure and 20-30 min is required for screening. However, since it has a different specificity from the widely used immunochemical technique, it is suggested to be a useful screen for amphetamines.

Amphetamine↗

PubMed1998

Induction of rat hepatic cytochromes P450 by toxic ingredients in plants: lack of correlation between toxicity and inductive activity.

"Animal-Plant Warfare" is one of the hypotheses for the evolution of drug-metabolizing P450s. To address the validity of this hypothesis, we examined the induction of xenobiotic-metabolizing P450s by 12 plant toxins in rats, using hepatic activity for testosterone metabolism as the index. The compounds tested were aconitine, morphine, tubocurarine, physostigmine, pilocarpine, muscarine, cocaine, atropine, amygdalin, digitonin, nicotine and solanine. Drinking water containing a test compound was given to rats for 4 days, and the hepatic activity of testosterone metabolism was determined together with monitoring body weight gain and liver weight as the indices of toxicity. The results showed that while cocaine and nicotine have a minor ability to increase testosterone 16 beta-hydroxylase activity, a marker activity for the CYP2B1 and 2, all other compounds did not have any such effect. No correlation was observed between a change in 16 beta-hydroxylase and toxicity caused by toxins. Therefore, these results did not support the idea that the inducibility of the CYP2B subfamily in animals is acquired through "Animal-Plant Warfare". Several compounds examined here increased or decreased hepatic activities of testosterone 2 alpha-, 6 beta-, 7 alpha- and 16 alpha-hydroxylation and 17-oxidation, indicating a possible effect on the CYP2A, 2C and 3A subfamily. Of these effects, a moderate correlation (r < 0.49) was observed in the changes in the activities of 2 alpha-/16 alpha-hydroxylation and 17-oxidation vs. that in toxicity. It is therefore suggested that inhibition or suppression of the expression of CYP2C11 is one of the mechanisms in the toxicity of plant toxins for rats, although it comes from an examination using limited numbers of compounds.

Aconitine↗

PubMed1997

A highly toxic PCB produces unusual changes in the fatty acid composition of rat liver.

The changes in lipid metabolism produced by a coplanar PCB were studied in rats. Male Wistar rats were given a single intraperitoneal injection of 3,3',4,4',5-pentachlorobiphenyl at a dose of 25 mg/kg. After 5 days of administration, total hepatic lipids were treated with 1 M KOH in methanol at 75 degrees C and the liberated fatty acids were analyzed by HPLC after conversion to fluorescent derivatives. In comparison with free-fed and pair-fed control groups, the proportion of arachidonic acid in the PenCB-treated rats was reduced by about 50%, while oleic and linoleic acids increased significantly. We also examined the individual glycerophospholipids, separated by TLC, to see if they were affected by alteration in the fatty acid composition of the whole liver. In all glycerophospholipids, the proportion of arachidonic acid was reduced significantly to the same degree while linoleic acid increased. Changes in the activity of desaturase isozymes have been postulated to explain this unusual lipid metabolism following administration of a toxic PCB and this may contribute to its toxicity.

Animals↗

PubMed1997

Significant suppression of rat liver aldolase B by a toxic coplanar polychlorinated biphenyl, 3,3',4,4',5-pentachlorobiphenyl.

A toxic coplanar polychlorinated biphenyl, 3,3',4,4',5-pentachlorobiphenyl (PenCB), significantly suppresses the expression of liver aldolase B in rats. Hepatic aldolase activity in PenCB-treated rats was significantly reduced to about 50% of that in free- and pair-fed control groups. The reduced aldolase activity following PenCB-treatment was due to the marked suppression of the expression of aldolase B shown by immunoblot analysis after SDS-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. The suppression of rat liver aldolase B could be a key biochemical lesion caused by PenCB.

Animals↗

PubMed1997

Species-specific alteration of hepatic glucose 6-phosphate dehydrogenase activity with coplanar polychlorinated biphenyl: evidence for an Ah-receptor-linked mechanism.

We examined the in vivo effect of a highly toxic coplanar polychlorinated biphenyl (PCB) on the hepatic activity of glucose 6-phosphate dehydrogenase (G6PDH) in aryl hydrocarbon (Ah)-responsive (C57/BL) and -less-responsive (DBA) strains of mice. The activity in the C57BL strain was moderately increased by 3,3',4,4',5-pentachlorobiphenyl (PCB 126) in a dose dependent manner. However, this was not observed in DBA mice although greater doses were injected. 2,2',5,5'-Tetrachlorobiphenyl (PCB 52) with a non-planar structure did not increase G6PDH activity. The increase in G6PDH activity with PCB 126 was also seen in rats, but not in guinea pigs. The activity in the latter species was decreased rather than increased. These results suggest that the induction of hepatic G6PDH by coplanar PCB is mediated by a mechanism involving the Ah receptor, and the response was highly species-specific.

Animals↗

PubMed1997

Metabolism of 2,4,5,2',4',5'-hexachlorobiphenyl (PCB153) in guinea pig.

1. The in vitro and in vivo metabolism of 2,4,5,2',4',5'-hexachlorobiphenyl (PCB153) in guinea pig has been studied. 2. Seven metabolites were detected in the faeces of PCB153-treated animals and three were identical to those produced by dog liver microsomes. The detection of a metabolite where a chlorine atom was shifted from the 2- to 3-position strongly suggested the involvement of 2,3-arene oxide intermediate, and evidence for the concomitant formation of a 3,4-arene oxide intermediate was provided by identifying other two minor metabolites which were dechlorinated at the 4-position. 3. In vitro studies using liver microsomes from guinea pigs revealed that the 2,3-arene oxide and 3-hydroxylation pathways are the predominant metabolic routes compared with the 3,4-arene oxide pathway. Although the guinea pig is an another species that can metabolize PCB153 mainly to the 2,3-arene oxide intermediate, the rate of formation was only about one-tenth of the dog. 4. These results indicate that the ability to form this unusual 2,3-arene oxide intermediate may not be responsible for high excretion rate of this congener. Our data also suggest that the cytochrome P450-catalysed metabolism of PCB153 in the guinea pig and dog are similar, whereas for post-cytochrome P450 metabolism, the guinea pig resembles the rabbit.

Animals↗

PubMed1997

Major role of the CYP2C isozymes in deamination of amphetamine and benzphetamine: evidence for the quinidine-specific inhibition of the reactions catalysed by rabbit enzyme.

1. The cytochrome P450 isozymes involved in the deamination of amphetamine (AP) and benzphetamine (BZP) have been studied in liver microsomes from rabbit and rat using isozyme-specific inhibitors. 2. Metabolism of BZP in rat yielding phenylacetone and formaldehyde was moderately inhibited by testosterone and chloramphenicol. N-Debenzylation was thought to be P450-dependent, but all inhibitors except for a non-specific inhibitor, SKF-525A, failed to inhibit this reaction. 3. In rabbit, quinidine and testosterone were potent inhibitors of both BZP deamination and dealkylation. Deamination of AP in rabbit was extensively inhibited only with quinidine. 4. AP deamination with purified rabbit CYP2C3, which was previously identified as the major isozyme responsible for this metabolism, was extensively inhibited with quinidine, previously thought to be a specific inhibitor of CYP2D. 5. These results strongly support the notion that the CYP2C isozymes play a major role in the deamination of both AP and BZP, but not for N-debenzylation of BZP in rat. However, on the basis of different sensitivities toward inhibitors, multiple isozymes seem to be involved in BZP deaminations in both species.

Amphetamine↗

PubMed1997

Deamination of amphetamines by cytochromes P450: studies on substrate specificity and regioselectivity with microsomes and purified CYP2C subfamily isozymes.

Deamination or oxidative cleavage of the carbon-nitrogen bond in various phenylisopropylamines was examined in liver microsomes from rabbits and rats, and in reconstituted systems containing CYP2C subfamily isozymes. Kinetic studies of phenylacetone formation from six amphetamine (AP) derivatives, catalyzed by rabbit liver microsomes, indicated that AP had the highest apparent affinity (lowest K(m)) and increasing the size of the substituent on the nitrogen atom decreased the affinity. The values of maximal velocity increased with increasing size of the substituent. Experiments with purified CYP2C3 from rabbit liver gave similar results: this enzyme showed the highest activity for phenylacetone formation from benzphetamine (BZP) and showed lower activities with compounds having smaller nitrogen substituents. Based on these results, we conclude that among a series of AP derivatives, the parent phenylisopropylamine has the highest affinity for rabbit liver deaminase, where as BZP has the highest turnover. However, the intrinsic clearance (Vmax/K(m)) values for the individual reactions tended to be comparable. The rates of BZP and deprenyl N-demethylation by rat CYP2C11 and 2C13 were far greater than those of the reactions at other N-alpha-positions. This result indicated that rat CYP2C enzymes have a more rigid regioselectivity than rabbit CYP2C3 for the deamination/N-dealkylation of phenylisopropylamines.

Amphetamines↗

PubMed1997

Induction of two UDP-glucuronosyltransferase isoforms sensitive to phenobarbital that are involved in morphine glucuronidation: production of isoform-selective antipeptide antibodies toward UGT1.1r and UGT2B1.

We document here in that two UDP-glucuronosyltransferase (UGT) isoforms sensitive to phenobarbital are involved in morphine glucuronidation in Wistar and Sprague-Dawley rats. The hepatic microsomal morphine UGT activity in untreated Gunn rats was significantly less than that of untreated Wistar rats. Although the morphine UGT activity in the liver of Gunn rats was increased by phenobarbital (PB) treatment, this was significantly less than that in the liver of PB-treated Wistar rats. UGT1.1r is an isoform of morphine UGT in rat, and UGT2B1 is also considered an isoform of morphine UGT, because UGT2B1 (stably expressed in V79 cells) exhibited morphine UGT activity. We prepared specific antipeptide antibodies against UGT1.1r and UGT2B1. Using isoform-specific antipeptide antibodies, both UGT1.1r and UGT2B1 in Wistar and Sprague-Dawley rats were inducible by PB treatment. However, UGT1.1r is not present in the liver from Gunn rats. This study is the first demonstration that protein levels of two morphine UGT isoforms, UGT1.1r and UGT2B1, in the liver of Wistar and Sprague-Dawley rats are inducible by PB treatment.

Animals↗

PubMed1997

[Production of antibody against cytosolic 54 kDa protein in rat liver--evidence of the significant induction by a highly toxic coplanar polychlorinated biphenyl].

We have reported that a 54 kDa protein in rat liver cytosol is highly inducible by treatment with 3,3',4,4',5-pentachlorobiphenyl (PenCB) or 3-methylcholanthrene (MC) using SDS-polyacrylamide gel electrophoresis. Internal amino acid sequences of this protein in the rat liver were highly homologous to those of selenium binding protein (SeBP) or acetaminophen binding protein (APBP) in mouse liver cytosol. In this paper, the purification and characterization of this protein were demonstrated. MC was given at a dose of 20 mg/kg for 3 consecutive days. The liver cytosolic 54 kDa protein was purified twice from the MC-treated male Wistar rats by Rotofore Cell procedure to apparent single on SDS-polyacrylamide gel electrophoresis, and the rabbit antiserum against this protein was obtained. Male Wistar rats were given PenCB in corn oil at a single dose of 25 mg/kg i.p. The liver cytosol was prepared on the 5th day after the treatment and subjected to immunoblot analysis. The 54 kDa protein was markedly induced in the liver cytosol of PenCB-treated rats. Immunoblot analysis after two-dimensional gel electrophoresis suggested that there could be isoforms of 54 kDa protein. The induction of the 54 kDa protein with PenCB was assumed to be mediated through Ah-receptor. The physiological role of the 54 kDa protein was discussed together with SeBP and APBP, the role of which has not yet been elucidated.

Animals↗

PubMed1997

[Effect of co-planar polychlorinated biphenyl on the hepatic glutathione peroxidase redox system in rats and guinea pigs].

The effect of 3,3',4,4',5-pentachlorobiphenyl (PCB 126) on hepatic glutathione peroxidase (GPx) redox system was studied in vivo in rats and guinea pigs. PCB 126 treatment caused significant reduction of Se-dependent and -non-dependent GPx activity in rats. In agreement with this, the content of glutathione (GSH) and the activities of GSH reductase (GR) and gamma-glutamyl transpeptidase (gamma-GTP) were also decreased in this species. On the contrary, guinea pig liver Se-non-dependent GPx activity was significantly enhanced by PCB 126 treatment, while no effect on Se-dependent activity was observed. Neither the content of GSH nor the enzyme activities responsible for GSH supply in guinea pig liver was affected by PCB 126. These result suggested that the damage on GPx redox system is, at least, one of mechanisms by which co-planar PCB induces the toxicity in rats. However, in guinea pigs, this is not the case, and different mechanism from the damage on active oxygen quenching system is likely to be involved.

Animals↗

PubMed1997

Induction of rat hepatic cytochrome P450 2B subfamily by azidophenobarbital, as a possible photoaffinity probe for the putative phenobarbital receptor: comparative study with modified phenobarbitals with different functional groups.

To study the specific target to which phenobarbital (PB) binds, resulting in the induction of cytochrome P450, we prepared two azido-PBs (AZPBs) as photoaffinity ligands. The azido substituent was introduced at the para- or meta-position of the PB aromatic ring. In this study, we estimated the utility of these compounds by examining their inducing activities in vivo in rats. Induction was assessed by immunoblotting with anti-CYP2B1/2 antibody and measuring testosterone-metabolizing activity, using hepatic microsomes. Administration of p-AZPB to rats increased hepatic CYP2B1/2 protein and testosterone 16beta-hydroxylase activity, although the effects were less than those of unmodified PB. m-AZPB showed no effect in the induction of CYP2B1/2. To assess the specificity of the effects of substituents, we compared the inducing activities of p/m-nitro-PBs, p/m-amino-PBs, and p/m-hydroxy-PBs with those of AZPBs. The results showed that p-nitro-PB, m-amino-PB, and p-hydroxy-PB were also potent inducers for CYP2B1/2, with lower activity than that of unmodified PB, whereas the other three isomers had no effect. These results suggest that 1) the absence of any substituents on the aromatic ring of PB is needed for maximal inducing activity and 2) substitution at the meta-position of the PB aromatic ring tends to reduce effectiveness as an inducer more than does substitution at the para-position. Because p-amino-PB and p-acetylamino-PB, the minor and major metabolites of p-AZPB, respectively, were without effect in the induction of CYP2B1/2, the effect of p-AZPB was considered to be due to the unchanged compound itself. The present study demonstrates that, based on the weak but positive ability to induce CYP2B1/2, p-AZPB may be a useful tool for identifying the putative PB receptor.

Animals↗

PubMed1996

Hamster liver cytochrome P450 (CYP2A8) as a 4-hydroxylase for 2,5,2',5'-tetrachlorobiphenyl.

Metabolism of 2,5,2',5'-tetrachlorobiphenyl (TCB) was studied using liver microsomes of hamsters and two hamster P450 isoforms, CYP1A2 and 2A8. CYP2A8 catalyzed selectively 4-hydroxylation of 2,5,2',5-TCB at a rate of 21.7 pmol/min/nmol P450. In contrast, CYP1A2 showed no activity for hydroxylation of 2,5,2',5'-TCB. Immunological study revealed that rabbit antiserum against CYP2A8 almost completely inhibited the microsomal 4-hydroxylation but that against CYP1A2 did not. It was also shown that the induction pattern of CYP2A8 protein by P450 inducer was similar to that of the 4-hydroxylase activity in hamster liver microsomes. These results suggest that CYP2A8 plays a major role in the 4-hydroxylation of 2,5,2',5'-TCB in hamster liver.

Animals↗

PubMed1996

Phosphorylation of a membrane-intercalated proteoglycan, syndecan-2, expressed in a stroma-inducing clone from a mouse Lewis lung carcinoma.

We previously reported that a mouse Lewis lung carcinoma-derived stroma-inducing clone, P29, highly expresses a syndecan-like proteoglycan exhibiting specific binding to fibronectin, a major constituent of the interstitial matrix formed by the induced stromal cells, via its heparan sulphate chains [Itano, Oguri, Nakanishi and Okayama (1993) J. Biochem. (Tokyo) 114, 862-873]. On metabolic labelling of the proteoglycan with [32P]Pi, followed by identification of the radiolabelled material using glycanases, almost all the isotope was found to have been incorporated into a core portion of molecular mass 48 kDa, which was generated by digestion with heparan sulphate lyase I plus chondroitin ABC lyase. Immunoblotting of the core protein with a monoclonal antibody, F58-6G12, demonstrated that the proteoglycan was mouse syndecan-2. CsCl-density-gradient centrifugation after mild treatment of liposome-intercalated 32P-labelled syndecan-2 with trypsin resulted in clear separation of the radioactivity into a bottom fraction containing all the glycosaminoglycans (accounting for 40% of the total radioactivity) and a top fraction containing liposome-associated peptides (60%). The former isotope was shown to be linked covalently to both heparan sulphate and chondroitin sulphate chains, probably at their bridge regions. The latter was mostly attributed to phosphoserine, the one and only phosphorylated amino acid released on acid hydrolysis of this proteoglycan, strongly suggesting that the phosphorylation occurs at a specific serine residue(s) in the cytoplasmic domain of the core protein.

Amino Acid Sequence↗

PubMed1996

Purification of a phenobarbital-inducible UDP-glucuronosyltransferase isoform from dog liver which catalyzes morphine and testosterone glucuronidation.

A morphine UDP-glucuronosyltransferase (UGT) which could belong to the UGT2B subfamily was isolated from liver microsomes of a male beagle dog treated with phenobarbital. Glucuronidation toward morphine in the dog liver microsomes was increased threefold by the treatment. The microsomes were solubilized with Emulgen 911 and applied on a column of hemisuccinate derivative of Sepharose 4B column which has been developed in our laboratory. An isoform of UGT in the eluate was purified further by chromatofocusing and UDP-hexanolamine-affinity chromatography. A purified enzyme, UGTDOG-PB, was homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional electrophoresis and exhibited a subunit molecular weight of 50 kDa. This isoform showed activities toward the 3-hydroxyl group of morphine, 4-hydroxybiphenyl, 4-nitrophenol, 4-methylumbelliferone, and testosterone, but not toward chloramphenicol and the 6-hydroxyl group of morphine. The substrate specificity of UGTDOG-PB is similar to that of stably expressed UGT2B1 which is considered a phenobarbital-inducible morphine UGT in the rat except that UGTDOG-PB is capable of glucuronidating 4-nitrophenol but not chloramphenicol. The NH2-terminus until the 30th residue of UGTDOG-PB is highly homologous to UGT2B subfamily, and the NH2-terminal 15 residues of UGTDOG-PB are completely identical to those of UGT2B1, UGT2B8, and UGT2B15. This is the first report describing the UGT isoform of dog and the purification of morphine UGT which may belong to UGT2B subfamily.

Amino Acid Sequence↗

PubMed1996

A coplanar PCB induces a selenium binding protein as a major cytosolic protein in rat liver.

We obtained evidence that a toxic coplanar polychlorinated biphenyl (PCB) induces a counterpart of murine 56kDa selenium binding protein in rat liver cytosol. A 54kDa protein in the liver cytosol was significantly induced by 3,3',4,4',5-pentachlorobiphenyl and proved to be a major cytosolic protein in the rat liver. The protein exhibited pI of 6.8 on two-dimensional gel electrophoresis. The amino acid sequence of peptide fragments from the protein digested in situ, was highly similar to a 56kDa selenium binding protein and similar to an acetaminophen binding protein in mice.

Amino Acid Sequence↗

PubMed1996

Involvement of cytochrome b5 in the metabolism of tetrachlorobiphenyls catalyzed by CYP2B1 and CYP1A1.

The role of cytochrome b5 in the cytochrome P450 (CYP)-dependent hydroxylation of tetrachlorobiphenyl (TCB) isomers was examined using a reconstituted mixed function oxygenase (MFO) system containing purified CYP2B1 or 1A1, and rat liver microsomes. Hydroxylations of 2,2',5,5'- and 3,3',4,4'-TCBs were catalyzed mainly by CYP2B1 and 1A1, respectively, in the reconstituted MFO system and those of 2,3',4',5- and 2,3',4,4'-TCBs were mediated by both cytochrome P450 systems. The activity toward 2,2',5,5'- and 2,3',4',5-TCB was significantly increased 6.5- and 5.5-fold, respectively, by addition of cytochrome b5 in the reconstituted MFO system containing of CYP2B1. Either hydroxylation activity toward 2,3',4,4'-TCB with the CYP2B1 system was very low or decreased by addition of cytochrome b5. These results suggest that the involvement of cytochrome b5 to the hydroxylation of TCBs is dependent on the TCB congener being metabolized, and the cytochrome P450 isoform involved in its metabolism.

Animals↗