Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 133-146).

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

The purpose of the present study was to elucidate if caffeine ingestion reduced perception of effort at submaximal intensities during a maximal exercise test. A secondary purpose of this study was to examine the role of a single nucleotide polymorphism (SNP) at intron 1 of cytochrome P-450 gene in modulating caffeine’s influence on ratings of perceived exertion (RPE) at the same submaximal exercise intensities. Twelve healthy men (age: 24±1 yr., BMI: 23.9±1.2 kg.m2) volunteered to participate in the present study. Subjects consumed 6 mg.kg-1 of USP grade caffeine in 200ml of non-caloric, coloured and flavoured water, or a placebo-matched drink in a single-blind, randomised and crossover style design. Subjects remained seated for 1 hour after consuming the assigned drink, and subsequently completed an incremental maximal exercise test on a bicycle ergometer, which started at 0 Watts for 1 minute and increased by 25 Watts per minute until volitional exhaustion. RPE was reported every third minute during the test. DNA was obtained from whole blood samples and genotypes were determined using previously described methods. Similar to previous studies looking at this SNP, subjects were categorised into groups of AA homozygotes and C allele carriers for statistical analyses between genotypes. Two-way repeated measures ANOVA’s were performed (Treatment × Genotype) for RPE responses at submaximal workloads up to 300 Watts. Significant results were followed up using the bonferroni post-hoc method. There were no significant differences between individuals homozygous for the A variant and C allele carriers for age, height, weight, body mass index (BMI), and VO2max. A significant Time × Treatment interaction was observed (F=5.804, p<0.05) for the rate of increase in RPE between trials. A significant Treatment × Genotype interaction was also found (F=5.714, p<0.05), by which C allele carriers exhibited greater reductions in RPE during the caffeine trial compared to AA homozygotes. The findings of the present study indicate that perception of effort is reduced in individuals who metabolise caffeine at a slower rate (i.e. in C allele carriers). It is postulated that AA homozygotes do not experience reductions in RPE due to a greater cardiovascular workload and enhanced CNS excitability following caffeine ingestion / School of Physical Education, Sport, and Exercise Science

Rheumatoid Arthritis is a chronic inflammatory systemic disease and is one of the autoimmune diseases. In this study, nine candidate genes of the cytochrome P450 system have been analyzed to determine their possible association with the formation of RA. These genes are: CYP1A1, CYP1B1, CYP2B6, CYP2E1, CYP2C9, CYP2D6, CYP2A6, CYP2C19 and CYP3A4.Within these genes, 21 single nucleotide polymorphism, SNPs, in 300 French Caucasian individuals (100 RA trio families) were genotyped using single-base-extensions, SBE, in a mass spectrometric analysis by MALDI-TOF-MS (matrix-assisted Laser Desorption/ Ionization-time-of-flight mass spectometry). The selection of the examined genes was carried out taking into account known associations with RA or other autoimmune disease, as well as known functional variants. Decisive were also the location of the gene and genetic variability. The results of genotyping were used to study polymorphisms on their association with Rheumatoid Arthritis. The statistical analyzes of CYP2C9 rs1799853 (3011) showed, in the family-based single-marker test, a lower transmission (TDT p-value 0.021) for the rare allele (3011-a). The case-control allelic based test shows, there is a protective effect (Odds Ratio 0.58). In the case-control-based genotypic test this issue could be reproduced (p-value 0.046). For the rare allele of the CYP2A6 rs1801272 (3022-a) the family-based single-marker test shows a lower transmittance (TDT p-value 0.037). The case-control allelic based test shows a protective effect of this allele (Odds Ratio 0.32). In the case-control-based genotypic test a statistical trend to protective behavior of this allele occurs. SNPs with predicted functional relevance showed no statistical abnormalities in the studied cohort. In genome-wide studies, the results could not be tracked, at least at the gene level weak associations could be detected. The results of this study should be to replicate in one second independent cohort. Care should be taken specifically to xenobiotic stress, such as job stress, smoking, and medication. In subsequent studies more SNPs of the candidate genes could also genotyped in order to verify the genetic variability with reference to of the haplotypes in more detail. Should the above-mentioned associations be confirmed, functional studies on different gene expression or altered metabolite spectrum are highly interesting.

This thesis describes a study of the substrate selectivity of active site mutants of the monooxygenase cytochrome P450_cam. A range of mutants was constructed which replaced the phenolic side-chain at the Tyr-96 position by various hydrophobic amino acid residues. These 'hydrophobic mutants' were then combined with other mutations around the active site (Val-247, Phe-87, Ile-395 and Phe-193) which altered the space available at different positions in the active site. These mutants were then tested with an in vitro reconstituted P450_cam system with a range of substrates related to diphenylmethane and phenylcylcohexane. All of these large compounds were poor substrates for the wild-type enzyme. It was found that it was necessary to increase both the space available in the active site and the active site hydrophobicity to achieve substrate turnover. The substrates were oxidised preferentially on the aliphatic cyclohexyl ring over the more constrained phenyl ring suggesting that the active site is predisposed to binding the cyclohexyl ring close to the haem. Hydroxylation using the in vitro reconstituted P450_cam system is limited by catalyst lifetime and the need for the expensive cofactor NADH. For P450_cam hydroxylation to become a viable synthetic method it is necessary to find ways to bypass the use of NADH. For this reason various self-sufficient P450_cam system were constructed and expressed in E. coli. The best of these, despite limited protein expression, was found to turnover camphor with the wild-type P450_cam enzyme and other substrates with the Y96A mutant. The in vivo catalytic system was then used to screen many P450_cam mutants for the oxidation of natural products, monoterpenes and sesquiterpenes (e.g. limonene, pinene and valencene). Most of the target substrates are not oxidised by the wild-type enzyme but all are hydroxylated by some if not all of the P450_cam mutants with different degrees of selectivity. Some of the products identified so far are important compounds in the field of flavour and fragrance chemistry (e.g. verbenol and nookatone).

Deficiency of the cytochrome P-450 steroid 21-hydroxylase (21-OHase) which causes Congenital Adrenal Hyperplasia (CAH) is a monogenic autosomal recessive disorder which is linked to HLA. There are two 21-OHase genes in man, A and B, and they are mapped to the HLA class III region ~ 3 kb 3' to the complement genes C4A and C4B, respectively. Two genes encoding 21-OHase were isolated, characterized and sequenced. Both 21-OHase genes are ~ 3.3 kb in length and are split into 10 exons by nine introns. Comparison of the two genes showed that although they are highly conserved, there are three deleterious mutations in the 21-OHase A gene which cause frameshifts and introduce in phase premature termination codons. Thus the 21-OHase A gene is a pseudogene. Comparison of the 21-OHase B gene to the other cytochrome P-450 sequences revealed that although the cysteine-429 was conserved in 21-OHase, there is very little homology with other cytochrome P-450, indicating it belongs to a separate family of genes within the superfamily. Clear evidence of polymorphism in 21-OHase is apparent on comparison with other 21-OHase B sequences. There is a size polymorphism of 494 and 495 amino acids. The differing severities of 21-OHase deficiency in CAH may be due to allelic variants of the 21-OHase B gene, since in most cases the defect is not due to gene deletion (Rumsby et al., 1986). A 21-OHase B gene from a patient with CAH was characterized and sequenced. There were 13 nucleotide alterations in his single 21-OHase B gene, one of which at codon 269 caused a serine to change to a threonine residue. The G → C transversion in the 21-OHase B gene from the patient at codon 269 introduced a new NcoI restriction site into the gene. This restriction fragment length polymorphism (RFLP) was used to study other patients with CAH and normal individuals. The NcoI RFLP was found not to be confined to the 21-OHase B gene but was also present in some 21-OHase A genes. It is likely therefore that the mutation occurred in the pseudogene first and then transferred to some 21-OHase B genes.

Birds are potentially vulnerable to the toxicity of certain environmental pollutants due to limited detoxification capabilities of their liver microsomal cytochrome P450 (CYP) enzymes. In wild birds, ethoxyresorufin O-deethylation (EROD) activity, a marker of CYP1A activity in mammals and domestic chickens, has been used as a biomarker of exposure to polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs). The aim of the present study was to investigate hepatic CYP activity as an indication of detoxification capacity in New Zealand birds. In addition to the use of conventional in vitro CYP activity assays, the applicability of a noninvasive CYP activity assay was tested using caffeine as the in vivo substrate. The ontogeny of liver microsomal 3-hydroxylation of quinine, a marker of human CYP3A activity, was investigated in Adelie penguins (Pygoscelis adeliae) from Ross Island, Antarctica. The results indicate that chicks (2-4 weeks old) possess a CYP3A-like isoform(s) as active as but not identical to the CYP3A-like isoform(s) in adults. Total CYP content was low at 2 weeks of age and increased rapidly and linearly approaching adult levels by 4 weeks of age implying a rapid development of CYPs other than the CYP3A-like isoform(s). The main study was conducted on adult (and some post-fledging immature) birds of two native species, the herbivorous paradise shelduck (Tadorna variegata) and the omnivorous southern black-backed gull (Larus dominicanus). Birds were shot for liver collection at three sites in the South Island of New Zealand; West Coast, Lake Waipori and Dunedin landfill, in 2001-2002. The results indicate that shelducks posssess multiple CYP isoforms that independently catalyse EROD, p-nitrophenol hydroxylation (p-NP) and erythromycin demethylation (EMD), markers of mammalian CYP1A, CYP2E and CYP3A activity, respectively. In contrast, gulls appear to possess a single isoform catalysing both EROD and p-NP but possess no isoform capable of catalysing EMD. EROD activity was high in shelducks and gulls from the landfill site, although it was not significantly associated with liver concentrations of PCBs (0.079-6.2 and 8.2-310 ng/g in shelducks and gulls, respectively), PCDD/PCDFs, toxic equivalents (TEQs) and dichlorodiphenyldichloroethylene (DDE) (0.85-317 and 44-4800 ng/g in shelducks and gulls, respectively) in either species. In shelduck livers from the landfill site, EROD was positively associated with Pb concentration but negatively associated with Hg concentration. Assessment of PCB congener patterns based on concentration ratios of individual congeners to the reference congener, 2,2�,4,4�,5,5�-hexachlorobiphenyl (IUPAC #153), indicate that the metabolism of 2,4,4�-trichlorobiphenyl (PCB#28) and 2,4,4�,5-tetrachlorobiphenyl (PCB#74) is inducible in shelducks but not in gulls. Hepatic reduced glutathione (GSH) content was higher in gulls than in shelducks suggesting greater resistance to oxidative stress in gulls. The in vivo caffeine metabolism test as a noninvasive method to determine CYP1A activity in shelducks and gulls gave a positive outcome. The test was performed by administration of a single intraperitoneal dose of caffeine (1 mg/kg body weight) followed by blood collection at 2 and 4 h after caffeine administration for determination of the serum concentration ratio of the metabolite, paraxanthine, to caffeine (PX/CA) by HPLC. In both species, the PX/CA ratio was markedly increased by pretreatment with the model CYP1A inducer, β-naphthoflavone (BNF). BNF treatment also increased EROD activity determined after death (80-fold and 20-fold compared to controls in shelducks and gulls, respectively). However, sensitivity of the PX/CA ratio approach was lower in gulls than in shelducks due presumably to the formation of unidentified caffeine metabolites in gulls. Immunoblot analysis failed to reveal increased CYP protein levels caused by BNF treatment in shelducks and gulls due to poor cross-reactivity of avian proteins with polyclonal antibodies raised against mammalian CYPs. EROD activity was also determined in livers of the piscivorous yellow-eyed penguin (Megadyptes antipodes) (1 chick, 3 post-fledging immature, 1 adult) from Otago, South Island of New Zealand, and found to be below the limit of quantitation. The adult liver contained 18.5 ng/g of total PCBs suggesting that EROD in this species is insensitive to induction. Comparison of the PCB congener pattern based on [PCBx]/[PCB#153] between the penguin and its putative source of PCB exposure, New Zealand marine fish, indicates that CYPs in yellow-eyed penguins metabolise 2,2�,5,5�-tetrachlorobiphenyl (PCB#52) and 2,2�,4,5,5�-pentachlorobiphenyl (PCB#101) as in many other avian species. The findings of this study highlight substantial species differences in CYP activity in wild birds. Whether CYP expression in New Zealand birds is genetically distinct from birds in other parts of the world may warrant further investigation.

Copies of author's previously published articles inserted. / Bibliography: leaves 103-119. / viii, 199, [87] leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Aims to understand at the transcriptional level how 1,25-dihydroxyvitamin D3 up-regulates the mitochondrial cytochrome P450 enzyme. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1998?

The first step in the synthesis of steroid hormones occurs in the mitochondria where cholesterol is converted to pregnenolone by cytochrome P450scc (CYP11A1). Cholesterol is insoluble in water and is supplied to the CYP11A1 directly from the inner mitochondrial membrane to which the enzyme is bound. The aim of this study was to characterise the interaction of bovine CYP11A1 with the phospholipid membrane. The effect of osmotic stress provided by glycerol on the spin-state, activity and degree of hydration of CYP11A1 was also investigated. Multiple sequence alignment of mitochondrial P450s revealed that there are 46 absolutely conserved residues, with the highest conservation in the heme-binding region at the C-terminal. The greatest variablility between subfamilies is in the regions believed to be involved in substrate binding (SRSs), as defined for the CYP2B family. The secondary structure prediction for CYP11A1 suggests that there is strong similarity in secondary structure to P450s of known structure. A model structure of CYP11A1 was built from primary sequence alignment to template P450 structures using the SwissModel automated server. From the model and other bioinformatic analyses, the distal face of the P450 which includes the A’ helix, F-G loop and beta sheet 1 regions, were predicted to interact with the membrane. Tryptic digests of CYP11A1 were performed with the aim of identifying membrane bound peptides that may be protected from protease activity. HPLC tryptic maps were similar in profile between soluble and vesicle-bound P450 which suggests that there is not a large region of CYP11A1 protected from protease digestion when the enzyme is attached to a membrane. Mass spectrometric analysis of peptides resulting from tryptic digestion revealed a number of peptides in the soluble digest that were not present in the digest of vesicle-bound P450. These peptides were located at the N-terminal and the J to J’ helix and interestingly, there was an absence of C-terminal peptides for both digests. This C-terminal peptide could be detected in digests of vesicle-bound P450 but not in digests of soluble P450 by tricine SDS polyacrylamide gel electrophoresis, Western transfer and N-terminal sequence analysis. Based upon the bioinfomatic and tryptic digestion data, a set of N- and C-terminal deletion mutants of CYP11A1 were expressed in E. coli and fractionated based on their association with the soluble or membrane fraction of the cells. The N-terminal deletion of the A’ helix resulted in an increase in the proportion of CYP11A1 in the soluble fraction while the C-terminal deletion did not alter membrane localisation. There are eight tryptophan residues in mature CYP11A1. The accessibility of these tryptophans to a water-soluble fluorescence quencher was determined for soluble and vesicle-bound enzyme. When CYP11A1 was associated with the vesicle membrane an average of 2 tryptophan residues were protected from quenching compared to soluble CYP11A1. This suggests that these tryptophan residues become buried within the membrane following association of CYP11A1 with the vesicles and are no longer accessible to quencher. The only free cysteine (C265S) of bovine CYP11A1 was removed by site directed mutagenesis and new cysteine residues introduced at selected sites based upon earlier results and the modelled CYP11A1 structure. The cysteine mutants were expressed, purified and labelled with the environmentally sensitive fluorescent probe, N-(7-nitrobenz-2-oxal-3-diazol-4-yl)ethylenediamine (NBD). There was an increase in the hydrophobicity of the NBD environment following the association of CYP11A1 with vesicles for the labeled mutants V212C and L219C. This indicates that these residues which are in the F-G loop, become localized to a more hydrophobic environment following membrane binding. Labeled cysteine residues introduced into the A’, B’ and G helices and β4-2 did not show major changes in hydrophobicity following membrane integration of CYP11A1. Osmotic stress of CYP11A1 induced by glycerol resulted in a low-spin spectral response and inhibition of activity. The change to low spin correlated with the dissociation of five or six water molecules from CYP11A1 and the inhibition of activity with cholesterol as substrate correlated with the dissociation of two molecules of water. In conclusion, this study shows that CYP11A1 is held to the membrane, at least in part, by the F-G loop region, and that the removal of water from the active site of CYP11A1 by osmotic stress causes a low spin spectral response and inhibition of activity.

Thesis (Ph. D.)--University of Nevada, Reno, 2007. / "August 2007." Includes bibliographical references. Online version available on the World Wide Web.