Regulation of cytochrome P450 2A5 during hepatic injury
Unlike most cytochrome P450 (CYP) enzymes, murine hepatic CYP2A5 and human CYP2A6 are elevated during hepatitis, fasting, and chemically-induced hepatotoxicity. Since these enzymes metabolize nicotine and activate pro-carcinogens, their upregulation could affect tobacco addiction and carcinogenesis, but the stimulus responsible for their induction during hepatocellular stress remains unclear. This thesis investigated the involvement of pathophysiological changes during liver injury in CYP2A5 overexpression. Using Affymetrix microarrays, we examined the effect of pyrazole on murine hepatic gene expression and identified pathophysiological changes that might upregulate CYP2A5, including altered energy homeostasis, hyperbilirubinemia, endoplasmic reticulum stress, and altered redox status. Based on evidence from the literature, we selected two pathways to investigate further: oxidative stress and altered lipid homeostasis. The involvement of oxidative stress was investigated using an oxidative stress-sensitive glucose-6-phosphate dehydrogenase (G6PD)-deficient mouse model. CYP2A5 overexpression in pyrazole-treated G6PD-deficient mice was greater than in wild-type animals, but DNA, protein, and lipid markers of oxidative stress were unaltered by pyrazole treatment. Furthermore, CYP2A5 expression was not altered in G6PD-deficient mice treated with the pro-oxidant menadione, and the antioxidant polyethylene glycol-conjugated catalase did not prevent the induction of CYP2A5 by pyrazole. These findings did not support a central role for generalized cellular oxidative stress in the regulation of 'Cyp2a5' and suggested that additional factors related to G6PD-deficiency could be involved. Markers of ER stress and several lipid homeostasis genes were differentially expressed in pyrazole-treated G6PD-deficient and wild-type mice, suggesting the involvement of these pathways. The role of altered lipid homeostasis in the regulation of CYP2A5 was also investigated. Pyrazole affected the expression of 109 lipid homeostasis genes and altered the hepatic fatty acid profiles of C57Bl/6 mice, increasing linoleic and linolenic acid levels. While these fatty acids did not upregulate CYP2A5 expression in primary hepatocytes, hepatic lipid extract increased CYP2A5 mRNA levels 2.5-fold, and 250 [mu]M purified arachidonic acid increased CYP2A5 levels 3.5-fold. This indicates that altered lipid homeostasis could play a role in the regulation of 'Cyp2a5' during hepatic injury. This thesis successfully identifies a novel regulator of 'Cyp2a5', and provides a basis for future research into the regulation of 'Cyp2a5 ' by pathophysiological changes in the liver.