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D Sesardic

Publications and source records attributed to D Sesardic.

67 records · Page 4Linked to original sources
PubMed1990

Species variation in the response of the cytochrome P-450-dependent monooxygenase system to inducers and inhibitors.

1. In the safety evaluation of drugs and other chemicals it is important to evaluate their possible inducing and inhibitory effects on the enzymes of drug metabolism. 2. While many similarities exist between species in their response to inducers and inhibitors, there are also important differences. Possible mechanisms of such variation are considered, with particular reference to the cytochrome P-450 system. 3. Differences in inhibition may be due to differences in inhibitory site of the enzyme involved, which is not always the active site of the enzyme, in competing pathways or in the pharmacokinetics of the inhibitor. 4. Differences in induction could be due to differences in the nature of the induction mechanism, in the isoenzyme induced, in tissue- or age-dependent regulation, in competing pathways for the substrate or its products, or in the pharmacokinetics of the inducing agent. 5. Examples of each of these possible differences are considered, often from our own work on the P450 IA subfamily, and results in animals are compared with those in humans, where possible. 6. At present, the differences between species in their response to inducers and inhibitors make extrapolation to humans from the results of animal studies difficult, so that ultimately such effects should be studied in the species of interest, humans.

Allosteric Regulation↗
PubMed1989

Immunohistochemical localization of cytochrome P450b/e in hepatic and extrahepatic tissues of the rat.

The cellular distribution of cytochrome P450b/e has been studied in the liver and a number of extrahepatic tissues in the Wistar rat by immunocytochemistry, using two monoclonal antibodies, 10/1 and 1/4, raised against the major phenobarbitone-inducible form of cytochrome P450. The specificity of these antibodies was verified by a number of techniques. In Western blotting, both antibodies recognised a single band of Mr 52,000 in liver microsomes from rats pre-treated with phenobarbitone, acetone and isosafrole, which co-migrated with a purified preparation of cytochrome P450b. In untreated rats, a weak specific immunostain was visible across the whole of the liver lobule, with stronger staining in a few hepatocytes around the central vein. Immunoreactive cytochrome P450b/e was also found in the Clara cells of the lung and in the enterocytes of the small intestine, with maximal staining at the tips of the villi. No immunoreactive cytochrome P450b/e was detected in kidney, testis or pancreas. Phenobarbitone treatment resulted in a strong, specific immunostain of all hepatic centrilobular cells, antibody titration indicating that induction of cytochrome P450b/e had occurred. Marked induction was also found in the enterocytes of the small intestine, a strong immunostain being apparent in cells along the length of the villus, from crypt to tip. No induction was apparent in lung, kidney, testis or pancreas. Immunoquantification of cytochrome P450b/e, by densitometric scanning of dot blots probed with a monoclonal antibody, 10/1, confirmed these observations. Thus, there are very marked, specific inter- and intra-tissue differences in both the expression and inducibility of cytochrome P450b/e in the rat.

Animals↗
PubMed1989

The specificity of inhibition of debrisoquine 4-hydroxylase activity by quinidine and quinine in the rat is the inverse of that in man.

The kinetics of inhibition of debrisoquine 4-hydroxylase activity by quinidine and quinine in rat and human liver microsomes have been compared. Quinidine is a potent inhibitor of debrisoquine 4-hydroxylase activity of human liver (IC50: 3.6 microM). However, its stereoisomer, quinine, is some 60 times less potent (IC50:223 microM). Both compounds are able to inhibit greater than 95% of 4-hydroxylase activity. In rat liver microsomes quinine is approximately 50 times more potent an inhibitor (IC50:2.4 microM) than quinidine (IC50:137 microM). Again, 4-hydroxylase activity is inhibited by greater than 95%. Inhibition of debrisoquine 4-hydroxylase activity by both quinine and quinidine in human and rat liver is competitive. Values of Ki for quinidine in human and rat were 0.6 microM and 50 microM, whereas with quinidine the Ki values were 13 microM and 1.7 microM, respectively. The data in this paper are consistent with 4-hydroxylation of debrisoquine in both rat and human liver catalysed by a specific form of cytochrome P-450. Although both quinidine and quinine are competitive inhibitors of debrisoquine 4-hydroxylase activity in rat and man, their potency is reversed. This suggests that the nature of the active site of cytochrome P-450dbl differs between the two species, and indicates that data on the specificity of this isoenzyme in the rat should be extrapolated to man with extreme caution.

Animals↗
PubMed1989

Selective destruction of cytochrome P-450d and associated monooxygenase activity by carbon tetrachloride in the rat.

1. The effects of acute treatment of 3-methylcholanthrene (MC)-induced rats with carbon tetrachloride (CCl4) on the content and activity of the polycyclic aromatic hydrocarbon-inducible forms of cytochrome P-450 (P-450c and P-450d) in liver and kidney have been determined. 2. Post-treatment of MC-induced rats with CCl4 in vivo decreased the specific content of total, spectrally determined, P-450 in both hepatic and renal microsomes, by 60% and 40%, respectively. CCl4 treatment destroyed almost all of the hepatic P-450d (specific content after 6 h, less than 2% of control), but had no effect on P-450c, which increased slightly over the 6 h, to 30% above control values. 3. Immunocytochemical measurements demonstrated greater loss of P-450d from the centrilobular and midzonal than from periportal regions of the liver. 4. Hepatic phenacetin O-deethylase, an activity catalysed specifically by P-450d in this tissue, was dramatically decreased following administration of CCl4 to MC-induced rats. Loss of monooxygenase activity was highly correlated with the decrease in P-450d content (r = 0.947, P less 0.001). Aryl hydrocarbon hydroxylase activity of liver, catalysed almost entirely by P-450c, was unchanged and neither activity was affected in kidney. 5. Treatment of MC-induced rats with CCl4 causes a selective loss of hepatic P-450d and associated monooxygenase activities. Phenacetin O-deethylation is catalysed specifically by P-450d in liver, but not in kidney. The mechanism for this destruction of P-450d may be suicide activation of CCl4, but the rate of such activation appears to be much lower than with P-450b. Alternatively, P-450d may be particularly sensitive, and P-450c particularly resistant, to the active metabolite of CCl4 diffusing from a distant site of formation.

Animals↗
PubMed1988

Antibodies to a synthetic peptide that react specifically with a common surface region on two hydrocarbon-inducible isoenzymes of cytochrome P-450 in the rat.

An antibody that reacts with two hydrocarbon-inducible isoenzymes of rat cytochrome P-450 (c and d) in the rat was produced by immunising with a synthetic peptide, Leu-Ile-Ser-Lys-Phe-Gln-Lys-Leu-Met, which has the same primary structure as that of a region of both of these isoenzymes. There was no crossreactivity with hydrocarbon-inducible isoenzymes in liver microsomes from rabbit, mouse or in man. Nor was there any crossreactivity detected with liver microsomes from uninduced rats, or rats induced with phenobarbitone or isonicotinic acid hydrazide. This is consistent with the primary structure of these isoenzymes in the regions aligned with amino acids 174-182 (the immunising peptide) in rat isoenzyme c and demonstrates the ability to produce antibodies of defined specificity against isoenzymes of cytochrome P-450 by using synthetic peptide. As the antibody preparation is able to bind to isoenzymes c and d in their native conformations, either as partially purified enzymes, or in microsomes, it is suggested that this region is present on the surface of these cytochromes P-450.

Animals↗
PubMed1988

A form of cytochrome P450 in man, orthologous to form d in the rat, catalyses the O-deethylation of phenacetin and is inducible by cigarette smoking.

1. In previous studies (Boobis et al., 1985b) it was shown that a monoclonal antibody (MAb 3/4/2), raised against rat cytochrome P450 form c, reacts with an isoenzyme(s) of cytochrome P450 in human liver. It was predicted that the epitope with which this antibody reacts should be present on both isoenzymes of the P450IA gene sub-family (the orthologues of forms c and d) in man (Edwards et al., 1987). 2. This antibody was used to probe 45 different samples of human liver, by the technique of Western blotting. With one exception, all of the samples contained immunoreactive protein, a single band at Mr 54,000 (orthologous to rat form d), which ranged in content from less than 0.5 to 33.5 pmol mg-1 microsomal protein. The content of the human orthologue of form c was below 0.5 pmol mg-1, the limit of detection of the assay. 3. Thirteen of the samples were from patients of known smoking status. Immunoreactive P450 content was 3.5-fold higher, and phenacetin O-deethylase activity was four-fold higher, in the smokers than in the non-smokers. 4. There was a highly significant correlation between the amount of immunoreactive cytochrome P450 and the high affinity component of phenacetin O-deethylase activity in both smokers and non-smokers. 5. It is concluded that the high affinity component of phenacetin O-deethylase activity in man is catalysed by the orthologue of rat cytochrome P450d, and that this isoenzyme is inducible by cigarette smoking. 6. In a number of previous publications it has been suggested that there is an association between the poor metaboliser (PM) phenotype for debrisoquine and impaired phenacetin O-deethylation. In the present study it was shown that not all subjects PM for debrisoquine are poor metabolisers of phenacetin.

Animals↗
PubMed1986

Immunocytochemical localization of cytochrome P-450 in hepatic and extra-hepatic tissues of the rat with a monoclonal antibody against cytochrome P-450 c.

The cellular distribution of cytochrome P-450 has been studied in the liver and a number of extrahepatic tissues in the rat by immunocytochemistry, using an antibody raised against cytochrome P-450 form c. Immunoreactive cytochrome P-450, most probably form c, was found in the proximal tubules of the kidney, in the Clara cells of the lung, and in the olfactory epithelium and Bowman's glands of the olfactory tissue, in addition to its location in the liver. Immunoreactive cytochrome P-450 was not found in the small intestine, the testes or the adrenal gland, although these organs are known to contain isoenzymes of cytochrome P-450. The use of antibody titration enabled the effects of phenobarbitone, beta-naphthoflavone and clofibrate on the content and distribution of immunoreactive cytochrome P-450 to be studied in both the liver and in the other organs discussed. Phenobarbitone induces epitope-specific cytochrome P-450 in the centrilobular cells of the liver but has no effect in any of the other tissues studied. Clofibrate is without effect on the levels of immunoreactive cytochrome P-450 in any of the tissues studied. In contrast, beta-naphthoflavone induces immunoreactive cytochrome P-450 in the periportal region of the liver, and also in the Clara cells of the lung, in the enterocytes of the small intestine and in the proximal tubules of the kidney. Of all of the tissues studied, in which immunoreactive cytochrome P-450 could be detected, only the olfactory epithelium failed to undergo enzyme induction following treatment with beta-naphthoflavone.

Adrenal Glands↗
PubMed1986

Inter-relatedness of some isoenzymes of cytochrome P-450 from rat, rabbit and human, determined with monoclonal antibodies.

Monoclonal antibodies have been raised to rat liver cytochromes P-450 b and c, and rabbit liver cytochrome P-450 form 4. A total of six antibodies have been studied. Each antibody reacted strongly both with its homologous antigen and with microsomal fractions selectively enriched with that antigen by treatment of animals with inducing compounds. However, several of the antibodies showed cross-reactivity, either within or between species. A combination of enzyme-linked immunosorbent assay, immunoadsorption, Western blotting and competitive radioimmunoassay revealed that each of the antibodies reacted with a different epitope. Proteolytic digestion of antigen followed by Western blotting of the peptide fragments enabled antibodies, otherwise identical in their reactivity, to be distinguished. It is concluded that complex structural relationships exist amongst the different isoenzymes of cytochrome P-450 and that epitope mapping will help in characterizing both animal and human cytochromes P-450.

Animals↗
PubMed1985

A monoclonal antibody raised to rat liver cytochrome P-448 (form C) which recognises an epitope common to many other forms of cytochrome P-450.

A murine monoclonal antibody has been raised against a partially purified preparation of hepatic cytochrome P-448 (form c) from beta-naphthoflavone-treated rats. The monoclonal origin of the antibody was established by limiting dilution culture and isoelectricfocusing. The antibody has been designated 3/4/2. It reacts with apparently homogeneous cytochrome P-448 from rat liver in solid phase assay. It also cross reacts with a number of other cytochromes P-450, from rat and rabbit. In addition, a positive reaction was obtained with microsomal fractions from a variety of species, including man. None of the species tested was negative. The antibody does not react appreciably with purified haemoproteins other than cytochromes P-450. Antibody 3/4/2 is not inhibitory, either in reconstituted systems or with intact microsomal fraction. However, evidence was obtained that the antibody does cause some perturbation of the tertiary structure of the apoprotein at or near the haem.

Animals↗
PubMed1985

Mycolipenates and mycolipanolates of trehalose from mycobacterium tuberculosis.

Analysis of the lipids of Mycobacterium tuberculosis, by thin-layer chromatography, revealed the presence of two families of novel glycolipids each having two closely-related members but differing widely in polarity. The least and most polar families of lipids were characterized from M. tuberculosis strains C and H37Rv, respectively; all were based on trehalose, the least polar pair of glycolipids having more long-chain substituents than the more polar pair. The acyl substituents of the least polar of the four glycolipids were mainly straight-chain C16 and C18 acids and 2,4,6-trimethyltetracos-2-enoic (C27-mycolipenic) acid, and the second least polar glycolipid contained major amounts of 3-hydroxy-2,4,6-trimethyltetracosanoic (C27-mycolipanolic) acid in addition to these non-hydroxylated acids. The relatively polar pair of glycolipids were analysed together and released mainly straight-chain C16 and C18 acids, C27-mycolipanolic acid, minor amounts of C25- and C27-mycolipenic acids and major proportions of an acid having the chromatographic properties of 2,4-dimethyldocosanoic acid. The most polar pair of glycolipids co-chromatographed with glycolipid antigens previously detected in Mycobacterium bovis BCG.

Chromatography, Thin Layer↗
PubMed1983

Mycolic acid patterns of four vaccine strains of Mycobacterium bovis BCG.

Thin-layer chromatography of methanolysates of four widely used vaccine strains of Mycobacterium bovis BCG showed that only one organism had the expected pattern of mycolic acid methyl esters characteristic of Mycobacterium bovis and Mycobacterium tuberculosis. The remaining three BCG strains lacked methoxy mycolic acid.

BCG Vaccine↗
PubMed1980

Profile analysis of total mycolic acids from skin corynebacteria and from named Corynebacterium strains by gas-liquid chromatography and gas-liquid chromatography/mass spectrometry.

Gas-liquid chromatography (g.l.c.) was investigated as a potential tool in the classification and identification of Cornyebacterium strains isolated from human skin, on the basis of the g.l.c. profile of the trimethylsilyl derivatives of their mycolic acid methyl esters. The g.l.c. patterns of five skin corynebacteria were compared with those of reference strains Corynebacterium diphtheriae PW8 and Corynebacterium xerosis NCTC 9755 and NCTC 7929. Further compositional information was obtained by gas-liquid chromatography/mass spectrometry (g.l.c./m.s.) of mycolates from C. diphtheriae PW8 and two of the skin isolates. In addition to identifying and examining the individual mycolate species and comparing the differences in mycolate profile between skin corynebacteria and between reference strains, a limited assessment was made of the possibility of distinguishing between organisms at both strain and species levels on the basis of mycolic acid composition as revealed by g.l.c. "fingerprinting".

Chromatography, Gas↗