Abstract
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.
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Abbreviations
- DPI:
-
Diphenyleneiodonium chloride
- ETYA:
-
Eicosatetraynoic acid
- MeOH:
-
Methanol
- NOX:
-
NADPH oxidases
- PBN:
-
Phenylbutylnitrone
- PHS:
-
Prostaglandin H synthase
- ROS:
-
Reactive oxygen species
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This research was supported by Grants from the Methanol Foundation (USA) and the Canadian Institutes of Health Research.
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Miller-Pinsler, L., Sharma, A. & Wells, P.G. Enhanced NADPH oxidases and reactive oxygen species in the mechanism of methanol-initiated protein oxidation and embryopathies in vivo and in embryo culture. Arch Toxicol 90, 717–730 (2016). https://doi.org/10.1007/s00204-015-1482-0
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DOI: https://doi.org/10.1007/s00204-015-1482-0