Bilirubin metabolism during arsenic toxicity of the liver: involvement of murine cytochrome P450 2a5 and its human orthologue CYP2A6

Dionne Arthur (2010). Bilirubin metabolism during arsenic toxicity of the liver: involvement of murine cytochrome P450 2a5 and its human orthologue CYP2A6 PhD Thesis, School of Medicine, The University of Queensland.

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Author Dionne Arthur
Thesis Title Bilirubin metabolism during arsenic toxicity of the liver: involvement of murine cytochrome P450 2a5 and its human orthologue CYP2A6
School, Centre or Institute School of Medicine
Institution The University of Queensland
Publication date 2010-09
Thesis type PhD Thesis
Total pages 119
Total colour pages 10
Total black and white pages 109
Subjects 11 Medical and Health Sciences
Abstract/Summary Arsenic exposure causes chronic disease and cancer in various organs in humans. The liver is a target organ for arsenic toxicity. The most accepted mechanism of arsenic toxicity involves oxidative stress. The initial response to oxidative stress is defensive, and involves numerous antioxidant defence systems. Two enzyme systems that have been reported to play an important role in cellular defence during metal-induced oxidative stress include haem oxygenase-1 (HO-1) and cytochrome P450 2a5 (Cyp2a5). It was found that both enzymes are involved in bilirubin (BR) homeostasis, where Cyp2a5-dependant BR oxidation is elevated when BR levels increase due to HO-1 induction. The oxidative metabolism of BR is an important pathway of detoxification in addition to glucuronidation however the oxidative products were not identified. In this thesis various bilirubin oxidative metabolites (BOMs) formed as a result of BR oxidation by cytochrome P450 are reported. Bilirubin was incubated with yeast microsomal fractions (wild type, recombinant yeast expressing the murine Cyp2a5 or its human orthologue, CYP2A6). The origin of the products from BR was confirmed by comparing oxidation products of mesobilirubin, an analogue of BR. The products of enzymic incubation were also compared to those of the chemical oxidation of BR. The major products found in both enzymic and chemical oxidation system were identical and were as followed: ion A m/z 301; ion B m/z 333; ion D m/z 315 and ion biliverdin m/z 583. Additionally, in a study using recombinant yeast expressing CYP2A6, BR inhibited coumarin 7-hydroxylation (a CYP2A6 catalysed reaction) in a concentration dependent manner with an IC50 of 4.98 μM BR. The inhibitory effects of BR on the rate of coumarin 7-hydroxylation is that of competitive inhibition as BR increased the Km but not the Vmax for CYP2A6-dependent coumarin 7-hydroxylation. The Ki value of BR was found to be 2.82 μM, which is in the same range as the Km for coumarin 7-hydroxylation (2.44 μM). Furthermore, a monoclonal antibody for Cyp2a5 (which cross-reacts with CYP2A6), caused a dose-dependent inhibition of BR degradation activity by about 70 %. These observations suggest the affinity of BR to CYP2A6 is similar to that of coumarin. In a study using DBA/2J mice, a strain with high Cyp2a5 basal activity, evidence was found that shows the induction of HO-1 and Cyp2a5 is an adaptive response to viii arsenite-mediated oxidative stress to protect the liver against lipid peroxidation. Mice treated with a subacute dose of arsenite showed an induction of HO-1 and Cyp2a5 at both mRNA and protein levels in the liver, which was associated with oxidative stress. This response correlated with a decline in total cytochrome P450; a modest elevation in total bilirubin followed by a decrease in lipid peroxidation; and an increase in microsomal BR degradation rate followed by an increase in urinary elimination of BOMs identified in the enzymic incubations with BR. Furthermore, exposure of human liver cells (HepG2) to arsenic resulted in a time dependant induction of CYP2A6 protein and activity levels. This implies a similar adaptive response towards arsenic may also occur in human liver cells. In sum, these observations suggest that (i) both murine Cyp2a5 and human CYP2A6 enzymes oxidise BR to form BOMs; (ii) concurrent induction of HO-1 and Cyp2a5 during arsenic-mediated oxidative stress may protect the liver against lipid peroxidation; and (iii) production of BOMs may be mediated by Cyp2a5 BR oxidation and subsequently eliminated in the urine.
Keyword arsenic
haem oxygenase-1
cytochrome P450 2a5
cytochrome P450 2A6
bilirubin oxidative metabolites
Additional Notes 6, 14, 39, 40, 42, 44,

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