Enhanced NADPH oxidases and reactive oxygen species in the mechanism of methanol-initiated protein oxidation and embryopathies in vivo and in embryo culture.
Methanol (MeOH) teratogenicity in rodents may be mediated in part by reactive oxygen species (ROS), the source of which is unknown. To determine if MeOH enhances embryonic ROS-producing NADPH oxidases (NOXs), p22phox mRNA and protein and oxidatively damaged protein were measured in gestational day 12 MeOH-exposed CD-1 mouse embryos with or without pretreatment with the free radical spin trap phenylbutylnitrone (PBN) or the NOX inhibitor diphenyleneiodonium chloride (DPI). MeOH exposure upregulated p22phox mRNA and protein expression, and enhanced protein oxidation, within 3-6 h. Compared to embryos exposed to MeOH alone, PBN and DPI pretreatment decreased MeOH-enhanced p22phox mRNA expression, DPI but not PBN blocked p22phox protein expression, and both blocked protein oxidation. To assess developmental relevance, mouse embryos were exposed in culture for 24 h to MeOH or vehicle with or without pretreatment with PBN, DPI, or the prostaglandin H synthase (PHS) inhibitor eicosatetraynoic acid (ETYA), and evaluated for abnormalities. ETYA did not prevent MeOH embryopathies, despite blocking phenytoin embryopathies (ROS-initiating positive control), precluding bioactivation of MeOH or its metabolites by PHS. Concentration-dependent MeOH embryopathies were blocked by both DPI and PBN pretreatment, suggesting that enhanced embryonic NOX-catalyzed ROS formation and oxidative stress may contribute to the mechanism of MeOH embryopathies.
Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON, Canada.
Lutfiya
L
Miller-Pinsler
Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada.
Amy
A
Sharma
Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON, Canada. pg.wells@utoronto.ca.|Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada. pg.wells@utoronto.ca.|Division of Biomolecular Sciences, Faculty of Pharmacy, University of Toronto, 144 College St., Toronto, ON, M5S 3M2, Canada. pg.wells@utoronto.ca.
Peter G
PG
Wells
D015117:5,8,11,14-Eicosatetraynoic Acid / Q000494:pharmacology; D000818:Animals; D003573:Cytochrome b Group / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D046149:Embryo Culture Techniques; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D008297:Male; D000432:Methanol / Q000008:administration & dosage / Q000633:toxicity*; D008815:Mice, Inbred Strains; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D009861:Onium Compounds / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects; D011247:Pregnancy; D011506:Proteins / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*
D003573:Cytochrome b Group; D009861:Onium Compounds; D011506:Proteins; D017382:Reactive Oxygen Species; D015117:5,8,11,14-Eicosatetraynoic Acid; C007517:diphenyleneiodonium; D019255:NADPH Oxidases; C515614:Cyba protein, mouse; D000432:Methanol
Embryopathies; Free radical spin trap; Methanol; NADPH oxidases; Oxidative stress; Phenytoin