Objective
Superoxide dismutase, glutathione peroxidase, and catalase prevent cellular damage produced by free radicals. Our objective was to evaluate if prenatal alcohol exposure decreased the expression of antioxidant enzymes in the brain, liver, or placenta of fetal mice.
Study Design
Timed, pregnant C57BL6/J mice were treated on gestational day 8 with intraperitoneal injection of alcohol (0.03 mL/g) or saline (control). Fetuses were harvested on gestational day 18. Fetal brain, liver, and placenta were analyzed for mRNA expression of superoxide dismutase, glutathione peroxidase, and catalase by real-time polymerase chain reaction, with 18S RNA used as reference.
Results
Superoxide dismutase, glutathione peroxidase, and catalase expression was lower in fetal brains exposed to alcohol with no differences detected in the liver or placenta between the 2 groups.
Conclusion
Maternal alcohol consumption causes a decrease in superoxide dismutase, glutathione peroxidase, and catalase expression in the fetal brain. This may explain the long-term neurologic findings in fetal alcohol syndrome.
Alcohol exposure in utero can result in significant fetal effects that are detrimental to offspring throughout their life. In the United States, 0.5 to 3 per 1000 live births each year have fetal alcohol syndrome (FAS). Maternal alcohol consumption is the most commonly identifiable nongenetic cause of mental retardation. Children with FAS have a characteristic set of facial features, microcephaly, growth restriction, and central nervous system impairment. Children with FAS can have mental and learning delay as well as structural brain defects.
The brain oxygen consumption is relatively high, and is estimated to be at about 20% of the total oxygen delivered in humans. In ovine studies of oxygen consumption, fetal cerebral oxygen delivery is increased 70% when compared with adult sheep. Oxygen consumption in the brain results in generation of free radicals, an effect that is magnified with alcohol exposure. Superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) are members of the antioxidant system. They are present in the cerebral cortex, cerebellum, and hypothalamus, and function to prevent the cellular damage produced by free radicals.
SOD is an enzyme that catalyzes the formation of hydrogen peroxide from superoxide radicals. GPx catalyzes a reaction that converts the reduced monomeric glutathione (GSH) to glutathione disulfide (GSSG). CAT functions to catalyze the decomposition of hydrogen peroxide to water and oxygen. In rodent models of alcohol consumption, the enzymatic activity of SOD, GPx, and CAT were all reduced in response to alcohol exposure.
The above studies illustrate that alcohol treatment results in decreased enzymatic activity of SOD, GPx, and CAT. However, it is not known whether this effect is present at the transcriptional level, and whether it is a generalized effect or localized to the brain. Exploring alcohol’s effect on transcription is of importance as a decrease in mRNA transcript may, in part, explain the reduction in enzymatic activity of antioxidant enzymes. Therefore, our aim in this study is to test the hypothesis that alcohol-induced decrease in enzymatic activity is related to reduction in mRNA expression of SOD, GPx, and CAT, and that this effect is specific to the fetal brain.
Materials and Methods
The study protocol and all related procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at The University of Texas Medical Branch, Galveston, TX. The mice were maintained in the animal care facility at The University of Texas Medical Branch. They were housed separately in temperature- and humidity-controlled quarters and kept in a 12-hour light/12-hour dark regimen with food and water available at all times.
Study design
The FAS model by Webster was used. In Webster’s model, pregnant C57BL/6J mice received intraperitoneal (ip) injection of 25% ethyl alcohol on day 7, 8, 9, 10, or 11 of gestation to replicate acute intoxication of alcohol and not chronic consumption. Doses ranged from 0.015 to 0.03 mL/g per body weight, producing maternal blood alcohol levels of 350-800 mg/100 mL. In a dose response fashion, treatment on gestational day 8 with the highest dose of alcohol resulted in the highest rate of fetal anomalies (maxillary hypoplasia, median cleft lip/palate, and mandibular hypoplasia) and an increase in fetal demise. Briefly, pregnant C57BL/6J mice were received from Jackson Laboratory (Bar Harbor, ME) on gestational day 7. On gestational day 8, animals were randomly injected ip with 25% ethyl alcohol in saline (alcohol) solution or vehicle alone (saline solution) at 0.03 mL/gm of body weight. This dose was chosen to represent the severest test of efficacy. Because animals receiving alcohol were incapacitated for 6 hours, water and food were withheld from both groups for 6 hours.
On gestational day 18, pregnant mice were euthanized with carbon dioxide and fetuses were collected. Fetal demise/absorption was recorded. Each litter was weighed as a group to determine the average fetal weight between the groups. Fetal blood was pooled from all pups within each litter from 5 alcohol- and 5 saline-treated mothers that were randomly selected, centrifuged to obtain serum, and stored at −80°C. Fetal brain, liver and placenta were harvested, immediately flash frozen in liquid nitrogen, and stored at −80°C.
Tissue samples from each fetus were analyzed individually. For brain tissue, 4 to 7 brains from 4 alcohol-treated litters and 4 to 6 brains from 5 saline-treated litters were randomly chosen. For liver and placental tissue, 4 individual samples were randomly selected from 3 litters in the alcohol and saline group. Tissues from demised fetuses were excluded.
To measure the degree of systemic oxidative RNA damage, we quantified 8-hydroxyguanosine (8-OHG) levels from pooled fetal serum using OxiSelect RNA damage enzyme linked immunosorbent assay (ELISA) kit (Cell Biolabs, Inc, San Diego, CA) according to the protocol provided by the company.
For RNA extraction, samples were homogenized using Bullet Blender from Next Advance (Averill Park, NY). Reverse transcription into cDNA was performed using High-Capacity cDNA reverse transcription Kit (Applied Biosytems, Foster City, CA) according to manufacturer’s instructions. The cDNA was amplified by real-time PCR with 7500 Fast real-time PCR System (Applied Biosystems) using mouse specific, performed TaqMan probe sets (all purchased from Applied Biosystems) for SOD (catalog no. Mm01344232_g1), GPx (catalog no. Mm00656767_g1), and CAT (catalog no. Mm00437992_m1) according to the manufacturer’s protocol. Target gene expression was expressed in relation to 18S mRNA levels in each specific sample (catalog no. Hs99999901_s1; Applied Biosystems). All PCR reactions for each sample were performed simultaneously on the same plate.
Statistical analysis was performed using Shapiro-Wilk test for normality testing and then Mann-Whitney rank sum test accordingly. The unit of analysis was the litter. When more than 1 pup per litter was evaluated, the average for that litter was used in the analysis. Two-sided P value < .05 was considered significant.
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