Influence of endogenous female sex-steroids on mutagen metabolism
- Authors: Goold, Richard David
- Date: 1985 , 2013-03-15
- Subjects: Mutagenesis , Drugs -- Metabolism , Steroid hormones -- Receptors , Cytochrome P-450
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3819 , http://hdl.handle.net/10962/d1004919 , Mutagenesis , Drugs -- Metabolism , Steroid hormones -- Receptors , Cytochrome P-450
- Description: Cytochrome P-450, the terminal oxidase of the metabolic mono-oxygenase system, is thought to exist in multiple forms, which have differing substrate specificities, and are variably inducible by different enzyme inducers. Many mutagens, themselves unreactive, require metabolic activation by one or more of these cytochrome P-450-dependent microsomal enzymes for mutagenic activity. Such mutagens may be detected in the Salmonella mutagenicity test only by the incorporation of an hepatic microsomal (59) fraction into the assay (as a first approximation to in vivo metabolism). Induction of the microsomal enzymes by different agents enhances the metabolic activation of mutagens; in fact, many mutagens are only detected when the 59 fraction has been induced by appropriate agents. Inducers of the phenobarbital-type are known to enhance microsomal steroid hydroxylation when administered at supraphysiological levels, inducers of several mono-oxygenase activities. In turn, the steroids, have been reported to be The inductive effects of the female sex-steroids and the combined effects of steroid and phenobarbital (PB) pretreatment on the metabolic activation of four mutagens have been investigated using the Salmonella assay. Female Sprague-Dawley rats were pret reated with 17a-oestradiol (E2) or progesterone (PRG) , at a level of either 1 mg/kg or 20 mg / kg daily for 14 days. A duplicate set of similarly pretreated groups were also induced with PB. Hepatic microsomal fractions were prepared from each group and incubated with each of the te st mutagens in the presence of a tester strain known to detect each particular type of mutagen. Induction of the hepatic metabolizing system by PB increased the activation of the mutagens significantly (as reflected by an increased number of revertant prototrophic S .typhimurium colonies). The administration of PRG also caused significant, and dose-dependent, induction of the activation of af l atoxin B1, benzo(a)pyrene, and dimethylnitrosamine. In general, E2 exhibited no inductive effect, but it did produce an increase in the activation of aflatoxin B1 (a reaction which is known to be catalysed by a mono-oxygenase prefe rentially inducible by PB). When use was made of a microsomal fraction that was prepared from animals which were both steroidpretreated and induced by PB, mutagenic activation was of the same order of magnitude as that observed when induction was brought about by PB alone. The absence of additive effect, taken together with the observations already mentioned, indicate that steroids induce the same cytochrome isozymes that are induced by PB. The implications of sex-hormonal regulation of the metabolic activation of mutagens are briefly discussed. , KMBT_363 , Adobe Acrobat 9.53 Paper Capture Plug-in
- Full Text:
- Date Issued: 1985
- Authors: Goold, Richard David
- Date: 1985 , 2013-03-15
- Subjects: Mutagenesis , Drugs -- Metabolism , Steroid hormones -- Receptors , Cytochrome P-450
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3819 , http://hdl.handle.net/10962/d1004919 , Mutagenesis , Drugs -- Metabolism , Steroid hormones -- Receptors , Cytochrome P-450
- Description: Cytochrome P-450, the terminal oxidase of the metabolic mono-oxygenase system, is thought to exist in multiple forms, which have differing substrate specificities, and are variably inducible by different enzyme inducers. Many mutagens, themselves unreactive, require metabolic activation by one or more of these cytochrome P-450-dependent microsomal enzymes for mutagenic activity. Such mutagens may be detected in the Salmonella mutagenicity test only by the incorporation of an hepatic microsomal (59) fraction into the assay (as a first approximation to in vivo metabolism). Induction of the microsomal enzymes by different agents enhances the metabolic activation of mutagens; in fact, many mutagens are only detected when the 59 fraction has been induced by appropriate agents. Inducers of the phenobarbital-type are known to enhance microsomal steroid hydroxylation when administered at supraphysiological levels, inducers of several mono-oxygenase activities. In turn, the steroids, have been reported to be The inductive effects of the female sex-steroids and the combined effects of steroid and phenobarbital (PB) pretreatment on the metabolic activation of four mutagens have been investigated using the Salmonella assay. Female Sprague-Dawley rats were pret reated with 17a-oestradiol (E2) or progesterone (PRG) , at a level of either 1 mg/kg or 20 mg / kg daily for 14 days. A duplicate set of similarly pretreated groups were also induced with PB. Hepatic microsomal fractions were prepared from each group and incubated with each of the te st mutagens in the presence of a tester strain known to detect each particular type of mutagen. Induction of the hepatic metabolizing system by PB increased the activation of the mutagens significantly (as reflected by an increased number of revertant prototrophic S .typhimurium colonies). The administration of PRG also caused significant, and dose-dependent, induction of the activation of af l atoxin B1, benzo(a)pyrene, and dimethylnitrosamine. In general, E2 exhibited no inductive effect, but it did produce an increase in the activation of aflatoxin B1 (a reaction which is known to be catalysed by a mono-oxygenase prefe rentially inducible by PB). When use was made of a microsomal fraction that was prepared from animals which were both steroidpretreated and induced by PB, mutagenic activation was of the same order of magnitude as that observed when induction was brought about by PB alone. The absence of additive effect, taken together with the observations already mentioned, indicate that steroids induce the same cytochrome isozymes that are induced by PB. The implications of sex-hormonal regulation of the metabolic activation of mutagens are briefly discussed. , KMBT_363 , Adobe Acrobat 9.53 Paper Capture Plug-in
- Full Text:
- Date Issued: 1985
Molecular cloning and expression of equine CYP1A2 in Escherichia coli
- Authors: Mkabayi, Lithalethu
- Date: 2017
- Subjects: Escherichia coli , Escherichia coli infections in animals , Cytochrome P-450 , Cytochromes , Horses -- Effect of drugs on
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/4830 , vital:20734
- Description: Information regarding drug metabolism in veterinary species, especially horses, remains fragmented and incomplete. This information is essential for detection of metabolites of potential performance-enhancing substances in horseracing and for veterinary drug development. Equine liver microsomes have been used to study metabolism of a limited number of drugs, but these provide little information about individual drug metabolizing enzymes. Recombinant CYP enzyme systems are commonly used to determine contribution of individual CYP to metabolism of specific drugs. A limited number of recombinant equine CYPs have been expressed in insect cells and mammalian cell lines. However, there are no reports of recombinant equine CYP1A2 enzyme. In this study, equine CYP1A2 was identified, codon-optimized, cloned and expressed in E. coli BL21 cells. Multiple sequence alignments of equine CYP1A2 revealed an amino acid sequence identity of 83.69% to its human homolog which has previously been expressed in E. coli. The enzyme was expressed using both auto-induction and IPTG induction. Expressed equine CYP1A2 had a size of about 55 kDa, and was insoluble after cell lysis. Sarkosyl- solubilized CYP1A2 was purified using nickel affinity chromatography and gel filtration. For activity reconstitution, yeast NADPH-cytochrome P450 reductase was first expressed in E. coli BL21 cells and exhibited activity of 0.13 U/ml. Activity assay with Glo-P450 CYP1A2 assay kit indicated that CYP1A2 was inactive. Despite numerous attempts to obtain the activity, the CYP1A2 remained inactive. Although expression of equine CYP1A2 in E. coli produced non- catalytically active enzyme, this study could be used as the first step in an effort to fully develop a recombinant equine CYP1A2 system.
- Full Text:
- Date Issued: 2017
- Authors: Mkabayi, Lithalethu
- Date: 2017
- Subjects: Escherichia coli , Escherichia coli infections in animals , Cytochrome P-450 , Cytochromes , Horses -- Effect of drugs on
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/4830 , vital:20734
- Description: Information regarding drug metabolism in veterinary species, especially horses, remains fragmented and incomplete. This information is essential for detection of metabolites of potential performance-enhancing substances in horseracing and for veterinary drug development. Equine liver microsomes have been used to study metabolism of a limited number of drugs, but these provide little information about individual drug metabolizing enzymes. Recombinant CYP enzyme systems are commonly used to determine contribution of individual CYP to metabolism of specific drugs. A limited number of recombinant equine CYPs have been expressed in insect cells and mammalian cell lines. However, there are no reports of recombinant equine CYP1A2 enzyme. In this study, equine CYP1A2 was identified, codon-optimized, cloned and expressed in E. coli BL21 cells. Multiple sequence alignments of equine CYP1A2 revealed an amino acid sequence identity of 83.69% to its human homolog which has previously been expressed in E. coli. The enzyme was expressed using both auto-induction and IPTG induction. Expressed equine CYP1A2 had a size of about 55 kDa, and was insoluble after cell lysis. Sarkosyl- solubilized CYP1A2 was purified using nickel affinity chromatography and gel filtration. For activity reconstitution, yeast NADPH-cytochrome P450 reductase was first expressed in E. coli BL21 cells and exhibited activity of 0.13 U/ml. Activity assay with Glo-P450 CYP1A2 assay kit indicated that CYP1A2 was inactive. Despite numerous attempts to obtain the activity, the CYP1A2 remained inactive. Although expression of equine CYP1A2 in E. coli produced non- catalytically active enzyme, this study could be used as the first step in an effort to fully develop a recombinant equine CYP1A2 system.
- Full Text:
- Date Issued: 2017
Studies on the metabolism of SKF 525 A|
- Authors: Barber, Peter John
- Date: 1978 , 2013-10-14
- Subjects: Drugs -- Metabolism , Cytochrome P-450
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3835 , http://hdl.handle.net/10962/d1007591 , Drugs -- Metabolism , Cytochrome P-450
- Description: Spectrophotometric studies have been carried out to determine the pH dependence of binding of SKF 525 A, Brietal sodium and carbon monoxide to cytochrome P-450. The optimal pH for metabolic conversion of SKF 525 A has been investigated and this agent and its major metabolite, SKF 8742 A, have been metabolised in vitro by swine and rat hepatic microsomes. A suitable gas liquid chromatography assay has been developed and used to analyse metabolic production. The effects of carbon monoxide, dithiothreitol, n-octylamine and of induction of cytochrome P-450 by phenobarbital on metabolism of SKF 525 A and SKF 8742 A have been investigated. Attempts have been made to synthesise SKF 525 AN-oxide. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 1978
- Authors: Barber, Peter John
- Date: 1978 , 2013-10-14
- Subjects: Drugs -- Metabolism , Cytochrome P-450
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3835 , http://hdl.handle.net/10962/d1007591 , Drugs -- Metabolism , Cytochrome P-450
- Description: Spectrophotometric studies have been carried out to determine the pH dependence of binding of SKF 525 A, Brietal sodium and carbon monoxide to cytochrome P-450. The optimal pH for metabolic conversion of SKF 525 A has been investigated and this agent and its major metabolite, SKF 8742 A, have been metabolised in vitro by swine and rat hepatic microsomes. A suitable gas liquid chromatography assay has been developed and used to analyse metabolic production. The effects of carbon monoxide, dithiothreitol, n-octylamine and of induction of cytochrome P-450 by phenobarbital on metabolism of SKF 525 A and SKF 8742 A have been investigated. Attempts have been made to synthesise SKF 525 AN-oxide. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 1978
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