The role of NOX enzymes in ethanol-induced oxidative stress and apoptosis in mouse embryos

Abstract

Reactive oxygen species (ROS) play an important role in ethanol-induced apoptosis and teratogenesis. However, the major sources of ROS in ethanol-exposed embryos have remained undefined. This study was conducted to determine the role of NADPH oxidase (NOX) in ethanol-induced oxidative stress and apoptosis in mouse embryos. Analyses of mRNA expression indicated that ethanol treatment resulted in a significant increase in mRNA expression of NOX catalytic subunit Duox-1 in gestational day 9 (GD 9:0) mouse embryos. Ethanol exposure also resulted in significant increases in mRNA expression of NOX regulatory subunits, p22phox, p67phox, NOXA1 and NOXO1. In addition, a significant increase in NOX enzyme activity was found in the ethanol-exposed embryos as compared to controls. Co-treatment with the NOX inhibitor, diphenyleneiodonium (DPI), significantly prevented ethanol-induced increases in NOX enzyme activity, ROS generation and oxidative DNA damage in ethanol-exposed embryos. DPI treatment also resulted in a reduction in caspase-3 activation, decreased caspase-3 activity and diminished prevalence of apoptosis in ethanol-exposed embryos. These results support the hypothesis that NOX is a critical source of ROS in ethanol-exposed embryos and that it plays an important role in ethanol-induced oxidative stress and pathogenesis.

Figure 1

Ethanol exposure results in a significant increase in mRNA expression of NOX catalytic subunit Duox1 in mouse embryos. mRNA expression of NOX catalytic subunits was determined by RT-PCR in GD 9 mouse embryos treated with Ringer's solution (Control) or 2.9 g/kg ethanol (EtOH). All data are expressed as fold change over control and represent the mean ± SEM of three separate experiments. * p < 0.05 vs. control.

Figure 2

Ethanol exposure significantly increases mRNA expression of NOX regulatory subunits in mouse embryos. mRNA expression of NOX regulatory subunits was determined by RT-PCR in GD 9 mouse embryos treated with Ringer's solution (Control) or 2.9 g/kg ethanol (EtOH). All data are expressed as fold change over control and represent the mean ± SEM of three separate experiments. * p < 0.05 vs. control.

Figure 3

Co-treatment with DPI significantly prevents ethanol-induced increases in NOX activity in mouse embryos. NOX activity was measured in GD 9 mouse embryos 6 hours after exposure to Ringer's solution (Control), 2.9 g/kg ethanol (EtOH), treatment with 4 mg/kg DPI (DPI) or co-treatment with both ethanol and DPI (EtOH+DPI). Data are expressed as relative light units (RLU) and represent the mean ± SEM of three separate experiments.

Figure 4

DPI significantly prevents ethanol-induced increase in ROS generation in mouse embryos exposed to ethanol in vivo. ROS generation was measured in GD 9 mouse embryos 6 hours after exposure to Ringer's solution (Control), 2.9 g/kg ethanol (EtOH), treatment with 4 mg/kg DPI alone (DPI) or treatment with both ethanol and DPI (EtOH+DPI). Data are expressed as relative fluorescence intensity units (FIU) and represent the mean ± SEM of three separate experiments. * p < 0.05 vs. control. ♣ p < 0.05 vs. EtOH.

Figure 5

Co-treatment with DPI significantly decreases oxidative DNA damage in mouse embryos exposed to ethanol in vivo. Level of 8-OHdG, a sensitive index of oxidative DNA damage, was determined in GD 9 mouse embryos 6 hours after exposure to Ringer's solution (Control), 2.9 g/kg ethanol (EtOH), treatment with 4 mg/kg DPI alone (DPI) or treatment with both ethanol and DPI (EtOH+DPI). Data are expressed as the mean ± SEM of three separate experiments. * p < 0.05 vs. control. ♣ p < 0.05 vs. EtOH.

Figure 6

Co-treatment with DPI decreases the activation and activity of caspase-3 in mouse embryos exposed to ethanol in vivo. Western blot analyses and fluorimetric assay were performed to examine the activation (A) and activity (B) of caspase-3, respectively, in GD 9 mouse embryos 12 hours after exposure to Ringer's solution or ethanol. Embryo lysates were prepared from embryos treated with Ringer's solution (Control), 2.9 g/kg ethanol (EtOH), treated with 4 mg/kg DPI alone (DPI) or treated with both ethanol and DPI (EtOH+DPI). Data are expressed as fold change over control and represent the mean ± SEM of three separate experiments. * p < 0.05 vs. control. ♣ p < 0.05 vs. EtOH.

Figure 7

DPI treatment reduces ethanol-induced apoptosis. Nile blue sulfate vital staining of comparably staged mouse embryos treated with Ringer's solution (Control) (a), DPI (b), 2.9 g/kg ethanol (c), or treated with both ethanol and DPI (d) illustrates excessive stain uptake in mouse embryos exposed to ethanol alone (arrows are directed toward sites of specific uptake in the brain and facial region of GD 9 embryos). Co-treatment with DPI results in diminished staining in ethanol-exposed mouse embryos. Shown are representatives of 10-15 embryos from several different litters of each group.