title: Mitochondrial targeting of bilirubin regulatory enzymes: An adaptive response to oxidative stress.
abstract: The intracellular level of bilirubin (BR), an endogenous antioxidant that is cytotoxic at high concentrations, is tightly controlled within the optimal therapeutic range. We have recently described a concerted intracellular BR regulation by two microsomal enzymes: heme oxygenase 1 (HMOX1), essential for BR production and cytochrome P450 2A5 (CYP2A5), a BR oxidase. Herein, we describe targeting of these enzymes to hepatic mitochondria during oxidative stress. The kinetics of microsomal and mitochondrial BR oxidation were compared. Treatment of DBA/2J mice with 200mgpyrazole/kg/day for 3days increased hepatic intracellular protein carbonyl content and induced nucleo-translocation of Nrf2. HMOX1 and CYP2A5 proteins and activities were elevated in microsomes and mitoplasts but not the UGT1A1, a catalyst of BR glucuronidation. A CYP2A5 antibody inhibited 75% of microsomal BR oxidation. The inhibition was absent in control mitoplasts but elevated to 50% after treatment. An adrenodoxin reductase antibody did not inhibit microsomal BR oxidation but inhibited 50% of mitochondrial BR oxidation. Ascorbic acid inhibited 5% and 22% of the reaction in control and treated microsomes, respectively. In control mitoplasts the inhibition was 100%, which was reduced to 50% after treatment. Bilirubin affinity to mitochondrial and microsomal CYP2A5 enzyme is equally high. Lastly, the treatment neither released cytochrome c into cytoplasm nor dissipated membrane potential, indicating the absence of mitochondrial membrane damage. Collectively, the observations suggest that BR regulatory enzymes are recruited to mitochondria during oxidative stress and BR oxidation by mitochondrial CYP2A5 is supported by mitochondrial mono-oxygenase system. The induced recruitment potentially confers membrane protection.
authors.affiliation: The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland, Australia; Faculty of Pharmacy, University Teknologi Mara, Malaysia. Electronic address: sitinurfadzilah077@ppinang.uitm.edu.my.
authors.forename: Siti Nur Fadzilah
authors.initials: SN
authors.lastname: Muhsain
authors.affiliation: The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland, Australia. Electronic address: m.lang@uq.edu.au.
authors.forename: Matti A
authors.initials: MA
authors.lastname: Lang
authors.affiliation: The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland, Australia. Electronic address: a.abubakar@uq.edu.au.
authors.forename: A'edah
authors.initials: A
authors.lastname: Abu-Bakar
mesh_terms: D021581:Active Transport, Cell Nucleus; D000222:Adaptation, Physiological; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001663:Bilirubin / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000378:metabolism; D051547:Heme Oxygenase-1 / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D008811:Mice, Inbred DBA; D008862:Microsomes, Liver / Q000201:enzymology; D008930:Mitochondria, Liver / Q000187:drug effects / Q000201:enzymology*; D051267:NF-E2-Related Factor 2 / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000378:metabolism; D050050:Protein Carbonylation; D000099148:UGT1A1 Enzyme
chemical_list: D000975:Antioxidants; D001189:Aryl Hydrocarbon Hydroxylases; D001663:Bilirubin; D000072467:Cytochrome P450 Family 2; D004791:Enzyme Inhibitors; D005287:Ferredoxin-NADP Reductase; D014453:Glucuronosyltransferase; D051547:Heme Oxygenase-1; D008565:Membrane Proteins; D051267:NF-E2-Related Factor 2; D016877:Oxidants; D044925:Oxidoreductases Acting on CH-CH Group Donors; D000099148:UGT1A1 Enzyme; C495636:Nfe2l2 protein, mouse; C585520:Cyp2a5 protein, mouse; C496542:Hmox1 protein, mouse; C019789:biliverdin reductase
keywords: Bilirubin; CYP2A5; Endoplasmic reticulum; HMOX1; Mitochondria; Oxidative stress