(1) Ten monoclonal antibodies have been raised against human hepatic microsomal proteins, seven of which recognised a purified human liver cytochrome P450, P450hA7. (2) Two of the seven anti-P450hA7 monoclonal antibodies, when blotted against control and induced rat liver microsomes, recognised a male-specific, weakly expressed constitutive protein that showed marked induction by pregnenolone-16-carbonitrile (PCN) but not by phenobarbitone (PB). No such P450 has been previously reported. (3) Another of the anti-P450hA7 monoclonal antibodies recognised a protein that was not expressed in control, male or female, rat liver microsomes but was strongly expressed in both PCN- and PB-induced microsomes. This protein may correspond to cytochrome P450PCN1 which exhibits identical induction characteristics. (4) A third rat protein, strongly and constitutively expressed in male liver microsomes, is recognised by other anti-P450hA7 antibodies. This protein may be induced by PCN. (5) The hepatic microsomal level of immunoreactive P450hA7 in fifteen adult individuals showed marked interindividual variation and was approximately ten times higher in an epiletic chronically treated with the drugs PB, phenytoin, carbamazipine and valproate. (6) Foetal human liver microsomes contained a protein that was immunochemically similar, but not identical, to adult P450hA7. The foetal protein exhibited a slightly greater molecular mass than the adult form. The switch from the foetal to the adult form of P450hA7 occurred shortly after birth. (7) P450hA7 was immunochemically detected in HEP G2 human hepatoma cells. The cytochrome was constitutively expressed being present in cells treated with PB, PCN and benzanthracene as well as untreated cells. (8) The anti-P450hA7 antibodies have been put to a variety of applications including the immunohistochemical localisation of cytochrome P450hA7 in human tissue and the screening of human hepatic cDNA libraries in gtll. (9) These monoclonal antibodies constitute a precise and powerful tool for the further characterisation of the human cytochromes P450.

OleTJE (CYP152L1) is a P450 peroxygenase that was first isolated from Jeotgalicoccus sp. 8456 in 2011. OleTJE is primarily a fatty acid decarboxylase, converting mid-chain fatty acids (C10:0 to C22:0) to terminal alkenes, which are industrially useful petrochemicals. Terminal alkenes are hydrophobic with high energy density, and are compatible with existing transportation infrastructure. Thus OleTJE has attracted considerable interest due to potential applications for generating "drop-in" biofuels. As a P450 peroxygenase, OleTJE is able to utilise H2O2 as a sole oxygen and hydrogen donor. This is atypical of P450s, which usually require electron transfer from redox partners to perform substrate oxidation. Other P450 peroxygenases have previously been characterised, including fatty acid hydroxylases P450 Spalpha (CYP152B1) from Sphingomonas paucimobilis and P450 BSβ (CYP152A1) from Bacillus subtilis. In addition to decarboxylation, OleTJE also hydroxylates fatty acids, generating 2-OH and 3-OH fatty acids as minor products. P450 BSβ has also been reported to perform low levels of decarboxylation. However, OleTJE has superior decarboxylase activity, posing questions about the mechanism of OleTJE. This thesis describes initial structural and biochemical characterisation of OleTJE. These data highlighted three amino acid residues thought to be key for effective catalysis: His85, Phe79 and Arg245. We hypothesised that the active site His85 could act as a proton donor to thereactive ferryl-oxo species compound I, allowing homolytic scission of the substrate C-Calpha bond to form the alkene product. Phe79 sandwiches His85 between the heme, and Arg245 co-ordinates the fatty acid carboxylate moiety. I performed mutagenesis studies to probe the roles of these residues, creating H85Q, F79A, F79W, F79Y, R245L and R245E OleTJE mutants, and characterised them by a combination of spectroscopic, analytical and structural methods. I also developed a novel system, where OleTJE was fused to alditol oxidase (AldO) from Streptomyces coelicolor, creating a fusion protein where addition of glycerol drives hydrogen peroxide production and the decarboxylation of fatty acids. Finally, studies showed that OleTJE is capable of performing secondary oxidation of hydroxylated products, which has expanded our knowledge of OleTJE's catalytic repertoire. This thesis also describes the initial characterisation of the OleTJE orthologue P450 KR from Kocuria rhizophila, which is also a terminal alkene-forming fatty acid decarboxylase. The crystal structure of P450 KR revealed an unusual dimeric state, with structural interactions unprecedented for a P450 enzyme. These data thus provide characterisation of two P450 peroxygenases involved in the production of terminal alkenes and which are of great interest as tools for the development of alternative sources of advanced biofuels.

Richardson ground squirrels (gophers) are pests on the prairies that cause considerable agricultural and ecological damage. Traditional control methods such as the rodenticides strychnine, zinc phosphide, and anticoagulants, have proven ineffective in reducing gopher densities. In additional, current gopher control methods have the significant potential to cause primary and secondary toxicity to non-target animals. Thus, alternative methods for toxicological control of gophers are needed to mitigate these concerns. Present studies focused on the cytochrome P450 (CYP450) enzyme system responsible for xenobiotic detoxification in gophers. In vitro hepatic microsomal systems and HPLC analysis were used to elucidate general metabolic characteristics of major gopher xenobiotic metabolizing pathways. We found that the content and activity of individual components of the CYP450 system including CYP450, cytochrome b5, and NADPH-cytochrome P450 reductase in liver microsomal preparations were higher in gophers exposed to toxins used to control their population than in naïve (unexposed) gophers. When in vitro CYP450 mediated activities for five substrates [coumarin and aniline aromatic hydroxylation, 7-methoxycouamrin O-demethylation, and N-methylaniline, and N,N-dimethylaniline N-demethylation] were measured, naïve gophers were identified to have higher specific activity but similar whole body activity compared to the exposed gophers. Furthermore, there was a clearly identifiable sub-population of poor metabolizers showing considerably lower CYP450 activity within the gopher samples studied. Clotrimazole was found to be a potent inhibitor of several substrates of CYP450 enzyme-mediated reactions, which included aniline aromatic hydroxylation, N-methylaniline and N,N-dimethylaniline N-demethylation, and 7-methoxycoumarin O-demethylation. The cytotoxicity of above compounds was tested using freshly isolated gopher hepatocytes. The results showed that each compound caused considerable cytotoxicity to gopher hepatocytes. Addition of clotrimazole to the freshly isolated hepatocyte suspension increased the cytotoxicity of all tested compounds. <p>In conclusion, gophers may develop resistance to current chemical control methods through the enhancement of CYP450 system content, which can compensate the loss of enzyme activity. Furthermore, clotrimazole is a potent cytochrome P450 inhibitor, which increases the cytotoxicity caused by given compounds in gopher livers. The concept of using CYP450 enzyme inhibitor in combination with another chemical whose elimination depends on CYP450 metabolism to improve current gopher control method has practical importance.

Richardson ground squirrels (gophers) are pests on the prairies that cause considerable agricultural and ecological damage. Traditional control methods such as the rodenticides strychnine, zinc phosphide, and anticoagulants, have proven ineffective in reducing gopher densities. In additional, current gopher control methods have the significant potential to cause primary and secondary toxicity to non-target animals. Thus, alternative methods for toxicological control of gophers are needed to mitigate these concerns. Present studies focused on the cytochrome P450 (CYP450) enzyme system responsible for xenobiotic detoxification in gophers. In vitro hepatic microsomal systems and HPLC analysis were used to elucidate general metabolic characteristics of major gopher xenobiotic metabolizing pathways. We found that the content and activity of individual components of the CYP450 system including CYP450, cytochrome b5, and NADPH-cytochrome P450 reductase in liver microsomal preparations were higher in gophers exposed to toxins used to control their population than in naïve (unexposed) gophers. When in vitro CYP450 mediated activities for five substrates [coumarin and aniline aromatic hydroxylation, 7-methoxycouamrin O-demethylation, and N-methylaniline, and N,N-dimethylaniline N-demethylation] were measured, naïve gophers were identified to have higher specific activity but similar whole body activity compared to the exposed gophers. Furthermore, there was a clearly identifiable sub-population of poor metabolizers showing considerably lower CYP450 activity within the gopher samples studied. Clotrimazole was found to be a potent inhibitor of several substrates of CYP450 enzyme-mediated reactions, which included aniline aromatic hydroxylation, N-methylaniline and N,N-dimethylaniline N-demethylation, and 7-methoxycoumarin O-demethylation. The cytotoxicity of above compounds was tested using freshly isolated gopher hepatocytes. The results showed that each compound caused considerable cytotoxicity to gopher hepatocytes. Addition of clotrimazole to the freshly isolated hepatocyte suspension increased the cytotoxicity of all tested compounds. <p>In conclusion, gophers may develop resistance to current chemical control methods through the enhancement of CYP450 system content, which can compensate the loss of enzyme activity. Furthermore, clotrimazole is a potent cytochrome P450 inhibitor, which increases the cytotoxicity caused by given compounds in gopher livers. The concept of using CYP450 enzyme inhibitor in combination with another chemical whose elimination depends on CYP450 metabolism to improve current gopher control method has practical importance.

No description available.

Although dialkylnitrosamines are environmentally significant carcinogens, the use of short-term bioassays to assess the mutagenic potential of these compounds remains problematic. The Ames test, a mutagenicity assay based on the reversion of Salmonella typhimurium histidine auxotrophs, is the most widely used bioassay in genetic toxicology, but the traditional Ames tester strains are largely insensitive to dialkylnitrosamine mutagenicity. I have constructed several mutagenicity tester strains that co-express combinations of full-length human cytochrome P450 2E1, rat cytochrome P450 reductase, and human cytochrome b5 in S. typhimurium lacking ogt and ada methyltransferases (YG7104ER, ogt-; and YG7108ER, ogt-, ada-). These new strains are susceptible to dialkylnitrosamine mutagenicity in the absence of an exogenous metabolic activating system (S9 fraction). Mutagenicity is dependent upon the coexpression of P450 2E1 with P450 reductase and is similar or greater than that obtained with the parental strains in the presence of S9 fraction from ethanol-induced rat liver. Coexpressing human cytochrome b5 with cytochrome P450 2E1 and cytochrome P450 reductase potentiates the mutagenicity observed with dialkylnitrosamines. These strains were sensitive to nitrosamines with varying alkyl side chains, including dimethylnitrosamine, diethylnitrosamine, dipropylnitrosamine, and dibutylnitrosamine. Mutagenicity decreased with alkyl chain length, consistent with the stringency of the ada-encoded enzyme for methyl and ethyl DNA adducts. These new strains may prove useful in the evaluation of nitrosamine contamination of food and environmental samples, and may serve as useful tools in investigating the molecular properties of proteins in the cytochrome P450 monooxygenase system.

The cytochromes P450 (P450s) are important enzymes involved in metabolic pathways, which use exogenous and endogenous substances as their substrate for various enzymatic reactions. These enzymes can also use precarcinogens as their substrate and activate them into carcinogens, which leads to a cancer development. If the P450s are induced, the cancer risk increases. Some chemopreventive compounds may induce the P450s and thus be harmful to the human body. Therefore it is necessary to pay enough attention to a study of the mechanism of action of P450s and the influence of the chemopreventive compounds on the activity of cytochromes P450. mRNA expression of most of the P450s isoforms is detected in a number of healthy (nontransformed) tissues, viz. liver, brain, heart, colon, kidney or placenta. Nevertheless there are a few P450s isoforms which mRNAs are expressed at relatively low levels in the nontransformed tissues, whereas the expression in the transformed tissues is significantly higher. One of these P450s is CYP2W1, which can be used as a prognostic marker for colorectal cancer - therefore it is useful to be able to detect a presence of this enzyme in various tissues. A detection of P450s can be accomplished by using a method Western blot. In this method, the immunodetection is achieved by using...

Master of Philosophy / Clozapine is a drug of choice for treatment of refractory schizophrenia, which is primarily metabolized by Cytochrome P450 1A2 (CYP1A2). Norclozapine is its main metabolite. There are reports of wide ranging gastrointestinal side effects associated with clozapine therapy, that result in concomitant administration of proton pump inhibitors to treat acid-related disorders. Omeprazole is an established CYP1A2 inducer, while an in vitro study has shown that rabeprazole is much less potent in this regard. There is no available information about the impact of rabeprazole on CYP1A2 activity in patients. Firstly, this information is essential when prescriptions are changed from omeprazole to rabeprazole to reduce medication costs. Therefore, the aim of this study was to compare the effects of rabeprazole and omeprazole on CYP1A2-mediated clearance (CL/F) of clozapine. Secondly, the effective dosage of clozapine varies widely among patients, making it necessary to individualize drug therapy with clozapine. The reason for dosage variation could be due to the influence of patient-related variables on clozapine plasma concentrations. Therefore, another aim of this study was to investigate the relationship between patient variables, such as age, gender, cigarette smoke, weight and body mass index and clozapine clearance (CL/F). A cross-over study design was used for this study. Twenty patients from Macquarie hospital who were receiving clozapine and rabeprazole (with no other interacting medications) were recruited in this study. Blood samples were taken at 30 min, 1 hr, 2 hr and 12 hr after a dose of clozapine. Rabeprazole was then replaced with omeprazole. After at least 1 month blood samples were again collected at the above corresponding intervals after clozapine. The plasma concentrations of clozapine and norclozapine were determined by high performance liquid chromatography. Abbottbase Pharmacokinetic Systems Software, which utilizes Bayesian forecasting, was used to estimate pharmacokinetic parameters of clozapine. The ratio of plasma norclozapine/clozapine concentrations at trough level was used to reflect CYP1A2 activity. No difference was observed in clozapine clearance (CL/F) and CYP1A2 activity during concurrent therapy with either rabeprazole or omeprazole. According to some studies CYP1A2 induction by omeprazole is dose dependent. Furthermore, since rabeprazole is a weak CYP1A2 inducer in vitro, we conclude that omeprazole and rabeprazole may not induce CYP1A2 activity when used at conventional therapeutic dosage (<40 mg/day). Hence, replacement of omeprazole with rabeprazole at conventional therapeutic dosages (20 or 40 mg daily) offers no advantages in the management of patients with schizophrenia on clozapine and no dose adjustment is required. Consistent with previous studies, clozapine concentrations were found to be significantly lower in cigarette smokers due to CYP1A2 induction. No relationship was found between age, gender, or weight and clozapine clearance (CL/F). However, body mass index showed a significant negative correlation with clozapine clearance (CL/F). Since weight gain and lipid accumulation are common side effects of clozapine they may be associated with a reduction of CYP1A2 activity and clozapine clearance (CL/F). Moreover, high lipoprotein levels may decrease the unbound fraction of clozapine and decrease the availability of clozapine for oxidation by cytochrome P450 enzymes. Therefore, it is concluded that omeprazole and rabeprazole may not induce CYP1A2 activity when used at conventional therapeutic dosage (<40mg/day). Hence, replacement of omeprazole with rabeprazole does not require the dose of clozapine to be adjusted. Moreover, the negative correlation between clozapine clearance (CL/F) and BMI is informative. Further studies are now required to clarify the relationship between BMI, lipoprotein levels and clozapine clearance in patients with schizophrenia.

Cytochrome P450 3A4 (CYP3A4) is a key enzyme responsible for the metabolism of drugs and endogenous compounds in human liver and intestine. CYP3A4 gene expression is mainly regulated by Pregnane X receptor (PXR) which is a ligand-dependent nuclear receptor. It is a long-standing observation that inflammatory responses and infections decrease drug metabolism capacity in human and experimental animals. In this study, I reported that NF-κB activation by LPS and TNF-α plays a pivotal role in the suppression of CYP3A4 through interactions of NF-κB with PXR/RXR complex. Inhibition of NF-κB by NF-κB specific suppressor SRIκBα reversed the suppressive effects of LPS and TNF-α. Furthermore, I showed that NF-κB p65 disrupted the association of PXR/RXRα complex with DNA sequences as determined by EMSA and chromatin immunoprecipitation assays. NF-κB p65 directly interacted with DNA binding domain of RXRα and DNA binding domain, hinge domain and ligand-binding domain of PXR and may prevent its binding to the consensus DNA sequences, thus inhibiting the transactivation by PXR/RXRα complex. This mechanism of suppression by NF-κB activation may be extended to other nuclear receptor-regulated systems where RXRα is a dimerization partner. Many genes regulated by PXR and AhR are important for phase I, II and III drug metabolism. In this study I reported a crosstalk between PXR and AhR pathways. AhR physically and functionally interacted with PXR and enhanced the PXR transcriptional activity, and the interaction repressed the AhR transcriptional activity. AhR also physically interacted with RXRα. The synergistic induction of Gsta1 in the liver of mice by PCN and TCDD might assume a different mechanism. The results suggested the metabolism kinetics of mixture drugs was different from and more complicated than that of single compound. Using a HepG2 cell-based PXR-driven CYP3A4-Luciferase assay, I reported that E/F domain of PXR was responsible for ligand-dependant activation. A/B domain was necessary for co-activating the ligand-dependent activation and D domain was suppressive. High doses of Valerian Root extraction were PXR-dependent CYP3A4 inducers. Green tea polyphenols, aflatoxin B1, CuSO4 and MnCl2 enhanced the PXR transcription activity activated by rifampicin. The results suggested PXR-mediated drug metabolism kinetics altered on xenobiotic exposure.

Cytochrome P450 3A4 (CYP3A4) is a key enzyme responsible for the metabolism of drugs and endogenous compounds in human liver and intestine. CYP3A4 gene expression is mainly regulated by Pregnane X receptor (PXR) which is a ligand-dependent nuclear receptor. It is a long-standing observation that inflammatory responses and infections decrease drug metabolism capacity in human and experimental animals. In this study, I reported that NF-κB activation by LPS and TNF-α plays a pivotal role in the suppression of CYP3A4 through interactions of NF-κB with PXR/RXR complex. Inhibition of NF-κB by NF-κB specific suppressor SRIκBα reversed the suppressive effects of LPS and TNF-α. Furthermore, I showed that NF-κB p65 disrupted the association of PXR/RXRα complex with DNA sequences as determined by EMSA and chromatin immunoprecipitation assays. NF-κB p65 directly interacted with DNA binding domain of RXRα and DNA binding domain, hinge domain and ligand-binding domain of PXR and may prevent its binding to the consensus DNA sequences, thus inhibiting the transactivation by PXR/RXRα complex. This mechanism of suppression by NF-κB activation may be extended to other nuclear receptor-regulated systems where RXRα is a dimerization partner. Many genes regulated by PXR and AhR are important for phase I, II and III drug metabolism. In this study I reported a crosstalk between PXR and AhR pathways. AhR physically and functionally interacted with PXR and enhanced the PXR transcriptional activity, and the interaction repressed the AhR transcriptional activity. AhR also physically interacted with RXRα. The synergistic induction of Gsta1 in the liver of mice by PCN and TCDD might assume a different mechanism. The results suggested the metabolism kinetics of mixture drugs was different from and more complicated than that of single compound. Using a HepG2 cell-based PXR-driven CYP3A4-Luciferase assay, I reported that E/F domain of PXR was responsible for ligand-dependant activation. A/B domain was necessary for co-activating the ligand-dependent activation and D domain was suppressive. High doses of Valerian Root extraction were PXR-dependent CYP3A4 inducers. Green tea polyphenols, aflatoxin B1, CuSO4 and MnCl2 enhanced the PXR transcription activity activated by rifampicin. The results suggested PXR-mediated drug metabolism kinetics altered on xenobiotic exposure.