Mechanisms Underlying Chronic Binge Alcohol Exposure-Induced Uterine Artery Dysfunction in Pregnant Rat.

BACKGROUND: A cardinal feature of fetal alcohol syndrome is growth restriction. Maternal uterine artery adaptations to pregnancy correlate with birthweight and survival. We hypothesized that gestational binge alcohol exposure impairs maternal uterine vascular function, affecting endothelial nitric oxide (NO)-mediated vasodilation. METHODS: Pregnant rats grouped as pair-fed control or binge alcohol exposed received a once-daily, orogastric gavage of isocaloric maltose-dextrin or alcohol, respectively. On gestational day 20, primary uterine arteries were isolated, cannulated, and connected to a pressure transducer, and functional studies were conducted by dual-chamber arteriography. Uterine arteries maintained at constant intramural pressure (90 mm Hg) were maximally constricted with thromboxane, and a dose-response for acetylcholine (Ach) was recorded. RESULTS: The alcohol group exhibited significantly impaired endothelium-dependent, Ach-induced uterine artery relaxation (↓∼30%). Subsequently, a dose-response was recorded following inhibition of endothelium-derived hyperpolarizing factor (apamin and TRAM-34) and prostacyclin (indomethacin). Ach-induced relaxation in the pair-fed control decreased by ~46%, and interestingly, relaxation in alcohol group further decreased by an additional ~48%, demonstrating that gestational binge alcohol impairs the NO system in the primary uterine artery. An endothelium-independent sodium nitroprusside effect was not observed. Immunoblotting indicated that alcohol decreased the level of endothelial excitatory P-Ser1177 endothelial NO synthase (eNOS) (p < 0.05) and total eNOS expression (p < 0.05) compared to both the normal and pair-fed controls. P-Ser1177 eNOS level was also confirmed by immunofluorescence imaging. CONCLUSIONS: This is the first study to demonstrate maternal binge alcohol consumption during pregnancy disrupts uterine artery vascular function via impairment of the eNOS vasodilatory system.

Chronic binge alcohol exposure during pregnancy impairs rat maternal uterine vascular function.

BACKGROUND: Alcohol exposure during pregnancy results in an array of structural and functional abnormalities called fetal alcohol spectrum disorders (FASD). Alcohol dysregulates the exquisite coordination and regulation of gestational adaptations at the level of the uterine vasculature. We herein hypothesized that chronic binge-like alcohol results in uterine vascular dysfunction and impairs maternal uterine artery reactivity to vasoconstrictors and dilators. METHODS: We utilized a once-daily binge alcohol (4.5 g/kg body weight) exposure paradigm (gestational day 7 to 17) in a pregnant rat model system and investigated primary uterine artery function in response to vasoconstrictors and vasodilators utilizing wire myography. RESULTS: Alcohol (peak blood alcohol concentration, 216 mg/dl) produced uterine vascular dysfunction. Alcohol did not produce altered uterine vascular reactivity to α1 adrenergic agonist phenylephrine or the prostanoid thromboxane. However, alcohol specifically impaired acetylcholine (ACh)-mediated uterine artery vasodilation but exogenous endothelium-independent vasodilators like sodium nitroprusside exhibited no alcohol effect; ACh significantly decreased vessel relaxation (p = 0.003; ↓pD2 [negative log molar ACh concentration producing the half maximum response], -7.004 ± 0.215 vs. -6.310 ± 0.208; EMax [maximal ACh response], 92% vs. 75%). CONCLUSIONS: We conclude that moderate alcohol exposure impairs uterine vascular function in pregnant mothers. Alcohol specifically impairs agonist-induced uterine artery vasodilation. In summary, the maternal uterine compartment may play a significant role in the pathogenesis of FASD. Thus, the mechanistic targets of alcohol at the level of both the mother and the fetus need to be considered in order to develop effective therapeutic treatment strategies for FASD.

Interactive effects of in vitro binge-like alcohol and ATP on umbilical endothelial nitric oxide synthase post-translational modifications and redox modulation.

Alcohol dysregulates the regulation of reproductive vascular adaptations. We herein investigated chronic in vitro binge-like alcohol effects on umbilical endothelial nitric oxide synthase (eNOS) multi-site phosphorylation and related redox switches under basal (unstimulated) and stimulated (with ATP) states. Alcohol decreased endothelial excitatory (Pser1177)eNOS (P<0.001), whereas excitatory (Pser635)eNOS exhibited a main effect of alcohol (↓P=0.016) and ATP (↑P<0.001). Alcohol decreased (Pthr495)eNOS (P=0.004) levels, whereas inhibitory (Pser116)eNOS exhibited an alcohol main effect in both basal and stimulated states (↑P=0.005). Total eNOS was reduced by alcohol (P=0.038). In presence of ATP, alcohol inhibited ERK activity (P=0.002), whereas AKT exhibited no alcohol effect. Alcohol main effect on S-nitroso-glutathione reductase (↓P=0.031) and glutathione-S-transferase (↓P=0.027) were noted. Increased protein glutathiolation was noted, whereas no alcohol effect on GSH, GSSG, NOX2 or SOD expression was noted. Thus, alcohol effects on multi-site post-translational modifications and redox switches related to vasodilatory eNOS underscore the necessity for investigating alcohol-induced gestational vascular dysfunction.

Effects of sub-acute and sub-chronic inhalation of 1-bromopropane on neurogenesis in adult rats.

PURPOSE: 1-Bromopropane (1-BP) intoxication is associated with depression and cognitive and memory deficits. The present study tested the hypothesis that 1-BP suppresses neurogenesis in the dentate gyrus, which is involved in higher cerebral function, in adult rats. METHODS: Four groups of 12 male Wistar rats were exposed to 0, 400, 800, 1000 ppm 1-BP, 8 h/day for 7 days. Another four groups of six rats each were exposed to 0, 400, 800 and 1000 ppm 1-BP for 2 weeks followed by 0, 200, 400 and 800 ppm for another 2 weeks, respectively. Another four groups of six rats each were exposed to 0, 200, 400 and 800 ppm 1-BP for 4 weeks. Rats were injected with 5-bromo-2'-deoxy-uridine (BrdU) after 4-week exposure at 1000/800 ppm to examine neurogenesis in the dentate gyrus by immunostaining. We measured factors known to affect neurogenesis, including monoamine levels, and mRNA expression levels of brain-derived neurotrophic factor (BDNF) and glucocorticoid receptor (GR), in different brain regions. RESULTS: BrdU-positive cells were significantly lower in the 800/1000 ppm-4-week group than the control. 1-Week exposure to 1-BP at 800 and 1000 ppm significantly reduced noradrenalin level in the striatum. Four-week exposure at 800 ppm significantly decreased noradrenalin levels in the hippocampus, prefrontal cortex and striatum. 1-BP also reduced hippocampal BDNF and GR mRNA levels. CONCLUSION: Long-term exposure to 1-BP decreased neurogenesis in the dentate gyrus. Downregulation of BDNF and GR mRNA expression and low hippocampal norepinephrine levels might contribute, at least in part, to the reduced neurogenesis.

Effects of exposure to 1-bromopropane on astrocytes and oligodendrocytes in rat brain.

OBJECTIVES: Human cases of 1-bromopropane (1-BP) toxicity showed ataxic gait and cognitive dysfunction, whereas rat studies showed pyknotic shrinkage in cerebellar Purkinje cells and electrophysiological changes in the hippocampus. The present study investigated the effects of 1-BP on astrocytes and oligodendrocytes in the rat cerebellum and hippocampus to find sensitive markers of central nervous system toxicity. METHODS: Forty-eight F344 rats were divided into four equal groups and exposed to 1-BP at 0, 400, 800 and 1,000 ppm for 8 h/day; 7 days/week, for 4 weeks. Nine and three rats per group were used for biochemical and histopathological studies, respectively. RESULTS: Kluver-Barrera staining showed pyknotic shrinkage in the cytoplasm of Purkinje cells and nuclei of granular cells in the cerebellum at 1,000 ppm. Immunohistochemical analysis showed increased length of glial fibrillary acidic protein (GFAP)-positive processes of astrocytes in the cerebellum, hippocampus and dentate gyrus at 800 and 1,000 ppm. The myelin basic protein (MBP) level was lower at 1,000 ppm. The numbers of astrocytes and granular cells per tissue volume increased at 400 ppm or higher. CONCLUSION: The present study showed that elongation of processes of astrocytes accompanies degeneration of granular cells and Purkinje cells in the cerebellum of the rats exposed to 1-BP. The decrease in MBP and number of oligodendrocytes suggest adverse effects on myelination. The increase in astrocyte population per tissue volume in the cerebellum might be a sensitive marker of 1-BP neurotoxicity, but the underlying mechanism for this change remains elusive.

Exposure to 1-bromopropane induces microglial changes and oxidative stress in the rat cerebellum.

1-Bromopropane (1-BP), an alternative to ozone-depleting solvents, is reported to exhibit neurotoxicity and reproductive toxicity in animals and humans. However, the underlying mechanism of the toxicity remains elusive. This study was designed to identify the microglial changes and oxidative stress in the central nervous system (CNS) after 1-BP exposure. Four groups of Wistar-ST rats (n=12 each) were exposed to 0, 400, 800 and 1000ppm of 1-BP, 8h/day for 28 consecutive days. The cerebellum was dissected out in 9 rats of each group and subjected to biochemical analysis, while the brains of the remaining 3 rats were examined immunohistochemically. Exposure to 1-BP increased the levels of oxidative stress markers [thiobarbituric acid reactive substances (TBARS), protein carbonyl and reactive oxygen species (ROS)] in a dose-dependent manner. Likewise, there was also 1-BP dose-dependent increase in nitric oxide (NO) and dose-dependent decrease in protein concentrations in the cerebellum. Immunohistochemical studies showed 1-BP-induced increase in cd11b/c-positive microglia area in the white matter of the cerebellar hemispheres. The results showed that exposure to 1-BP induced morphological change in the microglia and oxidative stress, suggesting that these effects are part of the underlying neurotoxic mechanism of 1-BP in the CNS.

Proteomic identification of carbonylated proteins in F344 rat hippocampus after 1-bromopropane exposure.

1-Bromopropane (1-BP) is neurotoxic in both experimental animals and humans. Previous proteomic analysis of rat hippocampus implicated alteration of protein expression in oxidative stress, suggesting that oxidative stress plays a role in 1-BP-induced neurotoxicity. To understand this role at the protein level, we exposed male F344 rats to 1-BP at 0, 400, or 1000 ppm for 8h/day for 1 week or 4 weeks by inhalation and quantitated changes in hippocampal protein carbonyl using a protein carbonyl assay, two-dimensional gel electrophoresis (2-DE), immunoblotting, and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-TOF/MS). Hippocampal reactive oxygen species and protein carbonyl were significantly increased, demonstrating 1-BP-associated induction of oxidative stress and protein damage. MALDI-TOF-TOF/MS identified 10 individual proteins with increased carbonyl modification (p < 0.05; fold-change ≥ 1.5). The identified proteins were involved in diverse biological processes including glycolysis, ATP production, tyrosine catabolism, GTP binding, guanine degradation, and neuronal metabolism of dopamine. Hippocampal triosephosphate isomerase (TPI) activity was significantly reduced and negatively correlated with TPI carbonylation (p < 0.001; r = 0.83). Advanced glycation end-product (AGE) levels were significantly elevated both in the hippocampus and plasma, and hippocampal AGEs correlated negatively with TPI activity (p < 0.001; r = 0.71). In conclusion, 1-BP-induced neurotoxicity in the rat hippocampus seems to involve oxidative damage of cellular proteins, decreased TPI activity, and elevated AGEs.

Proteomic analysis of hippocampal proteins of F344 rats exposed to 1-bromopropane.

1-Bromopropane (1-BP) is a compound used as an alternative to ozone-depleting solvents and is neurotoxic both in experimental animals and human. However, the molecular mechanisms of the neurotoxic effects of 1-BP are not well known. To identify the molecular mechanisms of 1-BP-induced neurotoxicity, we analyzed quantitatively changes in protein expression in the hippocampus of rats exposed to 1-BP. Male F344 rats were exposed to 1-BP at 0, 400, or 1000 ppm for 8h/day for 1 or 4 weeks by inhalation. Two-dimensional difference in gel electrophoresis (2D-DIGE) combined with matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) were conducted to detect and identify protein modification. Changes in selected proteins were further confirmed by western blot. 2D-DIGE identified 26 proteins with consistently altered model (increase or decrease after both 1- and 4-week 1-BP exposures) and significant changes in their levels (p<0.05; fold change ≥ ± 1.2) at least at one exposure level or more compared with the corresponding controls. Of these proteins, 19 were identified by MALDI-TOF-TOF/MS. Linear regression analysis of 1-BP exposure level identified 8 differentially expressed proteins altered in a dose-dependent manner both in 1- and 4-week exposure experiments. The identified proteins could be categorized into diverse functional classes such as nucleocytoplasmic transport, immunity and defense, energy metabolism, ubiquitination-proteasome pathway, neurotransmitter and purine metabolism. Overall, the results suggest that 1-BP-induced hippocampal damage involves oxidative stress, loss of ATP production, neurotransmitter dysfunction and inhibition of ubiquitination-proteasome system.