Alcohol - Articles in Press

ER-targeted Bcl-2 and inhibition of ER-associated caspase-12 rescue cultured immortalized cells from ethanol toxicity - Corrected Proof

Andreea G. Balan, Barret J. Myers, Jansi L. Maganti, D. Blaine Moore — 2010-08-23

Abstract: Alcohol abuse, known for promoting apoptosis in the liver and nervous system, is a major public health concern. Despite significant morbidity and mortality resulting from ethanol consumption, the precise cellular mechanism of its toxicity remains unknown. Previous work has shown that wild-type Bcl-2 is protective against ethanol. The present study investigated whether protection from ethanol toxicity involves mitochondrial Bcl-2 or endoplasmic reticulum (ER) Bcl-2, and whether mitochondria-associated or ER-associated caspases are involved in ethanol toxicity. Chinese hamster ovary (CHO695) cells were transiently transfected with cDNA constructs encoding wild-type Bcl-2, mitochondria-targeted Bcl-2, or ER-targeted Bcl-2. MTT assay was used to measure cell viability in response to ethanol. Ethanol treatments of 1 and 2.5M reduced cell viability at 5, 10, and 24h. Wild-type Bcl-2, localized both to mitochondria and ER, provided significant rescue for CHO695 cells treated with 1M ethanol for 24h, but did not rescue toxicity at 2.5M. ER-targeted Bcl-2, however, provided significant and robust rescue following 24h of 1 and 2.5M ethanol. Mitochondria-targeted Bcl-2 offered no protection at any ethanol concentration and generally reduced cell viability. To follow up these experiments, we used a peptide inhibitor approach to investigate which caspases were responsible for ethanol-induced apoptosis. Caspase-9 and caspase-12 are known to be downstream of mitochondria and the ER, respectively. CHO695 cells were treated with a pan-caspase inhibitor, a caspase-9 or caspase-12 inhibitor along with 1.5M ethanol, followed by MTT cell viability assay. Treatment with the pan-caspase inhibitor provided significant rescue from ethanol, whereas inhibition of caspase-9 did not. Inhibition of ER-associated caspase-12, however, conferred significant protection from ethanol toxicity, similar to the pan inhibitor. These findings are consistent with our transfection data and, taken together, suggest a significant role for the ER in ethanol toxicity.

Establishment of steady-state metabolism of ethanol in perfused rat liver: the quantitative analysis using kinetic mechanism-based rate equations of alcohol dehydrogenase - Corrected Proof

Chung-Tay Yao, Ching-Long Lai, Hsiu-Shan Hsieh, Chin-Wen Chi, Shih-Jiun Yin — 2010-08-20

Abstract: Alcohol dehydrogenase (ADH) catalyzes oxidation of ingested ethanol to acetaldehyde, the first step in hepatic metabolism. The purpose of this study was to establish an ex vivo rat liver perfusion system under defined and verified steady states with respect to the metabolites and the metabolic rates, and to quantitatively correlate the observed rates with simulations based on the kinetic mechanism-based rate equations of rat liver ADH. Class I ADH1 was isolated from male Sprague–Dawley rats and characterized by steady-state kinetics in the Krebs–Ringer perfusion buffer with supplements. Nonrecirculating liver perfusion with constant input of ethanol at near physiological hepatic blood flow rate was performed in situ. Ethanol and the related metabolites acetaldehyde, acetate, lactate, and pyruvate in perfusates were determined. Results of the initial velocity, product, and dead-end inhibition studies showed that rat ADH1 conformed to the Theorell–Chance Ordered Bi Bi mechanism. Steady-state metabolism of ethanol in the perfused liver maintained up to 3h as evidenced by the steady-state levels of ethanol and metabolites in the effluent, and the steady-state ethanol disappearance rates and acetate production rates. The changes of the metabolic rates were qualitatively and in general quantitatively correlated to the results from simulations with the kinetic rate equations of ADH1 under a wide range of ethanol, in the presence of competitive inhibitor 4-methylpyrazole and of uncompetitive inhibitor isobutyramide. Preliminary flux control analysis estimated that apparent in the perfused liver may approximate 0.7 at constant infusion with 1–2mM ethanol, suggesting that ADH plays a major but not the exclusive role in governing hepatic ethanol metabolism. The reported steady-state rat liver perfusion system may potentially be applicable to other drug or drug–ethanol interaction studies.

The effects of ethanol administration on brush border membrane glycolipids in rat intestine - Corrected Proof

Ravneet K. Grewal, Akhtar Mahmood — 2010-08-16

Abstract: Ethanol ingestion is well known to induce morphological and biochemical changes in intestine and is responsible for intestinal dysfunctions. Luminal surface of enterocytes is rich in glycolipids, but the effects of ethanol ingestion on membrane glycolipids are not well characterized. In the present study, rats were given 1mL of 30% ethanol daily for 15, 25, 35, and 56 days. Ethanol feeding for 15 days did not affect glycolipid pattern in microvillus membranes, but the levels of cerebrosides (glucosylceramide, lactosylceramide, globotriasyloceramide) were enhanced in rats fed with ethanol for 35 or 56 days compared with controls. In contrast, the content of fucolipids and gangliosides was reduced in rats on ethanol ingestion for 35 or 56 days. The observed changes in membrane glycolipids were substantiated using biotinylated lectins Jacalin (affinity for N-acetylgalactosamine) and Aleuria aurantia (affinity for α-l-fucose). The incorporation of [14C]-mannose and [14C]-glucosamine revealed an increase (P<.01) in glucosamination and reduction (P<.01) in mannosylation of glycolipids from ethanol-fed rats for 45 days compared with controls. These findings were further characterized by autoradiography of the glycolipids separated on thin layer chromatograms. These findings indicate that ethanol ingestion modulates the glycolipids composition of brush borders, resulting in generalized aberration of intestinal glycosylation in chronic alcoholism in rats.

Ethanol exposure during the early first trimester equivalent impairs reflexive motor activity and heightens fearfulness in an avian model - Corrected Proof

Susan M. Smith, George R. Flentke, Katherine A. Kragtorp, Laura Tessmer — 2010-08-13

Abstract: Prenatal alcohol exposure is a leading cause of childhood neurodevelopmental disability. The adverse behavioral effects of alcohol exposure during the second and third trimester are well documented; less clear is whether early first trimester-equivalent exposures also alter behavior. We investigated this question using an established chick model of alcohol exposure. In ovo embryos experienced a single, acute ethanol exposure that spanned gastrulation through neuroectoderm induction and early brain patterning (19–22h incubation). At 7 days posthatch, the chicks were evaluated for reflexive motor function (wingflap extension, righting reflex), fearfulness (tonic immobility [TI]), and fear/social reinstatement (open-field behavior). Chicks exposed to a peak ethanol level of 0.23–0.28% were compared against untreated and saline-treated controls. Birds receiving early ethanol exposure had a normal righting reflex and a significantly reduced wingflap extension in response to a sudden descent. The ethanol-treated chicks also displayed heightened fearfulness, reflected in increased frequency of TI, and they required significantly fewer trials for its induction. In an open-field test, ethanol treatment did not affect latency to move, steps taken, vocalizations, defecations, or escape attempts. The current findings demonstrate that early ethanol exposure can increase fearfulness and impair aspects of motor function. Importantly, the observed dysfunctions resulted from an acute ethanol exposure during the period when the major brain components are induced and patterned. The equivalent period in human development is 3–4 weeks postconception. The current findings emphasize that ethanol exposure during the early first trimester equivalent can produce neurodevelopmental disability in the offspring.

Evidence for the role of oxidative stress in the acetylation of histone H3 by ethanol in rat hepatocytes - Corrected Proof

Mahua Choudhury, Pil-Hoon Park, Daniel Jackson, Shivendra D. Shukla — 2010-08-13

Abstract: The relationship between ethanol-induced oxidative stress and acetylation of histone H3 at lysine 9 (H3AcK9) remains unknown and was therefore investigated in primary cultures of rat hepatocytes. Cells were treated with ethanol, and a select group of pharmacological agents and the status of H3AcK9 and reactive oxygen species (ROS) were monitored. Pretreatment of hepatocytes with N-acetyl cystein (ROS reducer), or dietary antioxidants (quercetin, reserveratrol), or NADPH (reduced nicotinamide adenine dinucleotide phosphate) oxidase inhibitor apocynin, significantly reduced ethanol (50 mM, 24 h) induced increases in ROS and H3AcK9. In contrast, l-buthionine sulfoximine (ROS inducer) and inhibitor of mitochondrial complexes I (rotenone) and III (antimycin) increased ethanol-induced H3AcK9 (P<.01). Oxidative stress also affected ethanol-induced alcohol dehydrogenase 1 mRNA expression. These results demonstrate for the first time that oxidative stress is involved in the ethanol-induced histone H3 acetylation in hepatocytes.

Fine mapping and expression of candidate genes within the chromosome 10 QTL region of the high and low alcohol-drinking rats - Corrected Proof

2010-08-13

Abstract: The high and low alcohol-drinking (HAD and LAD) rats were selectively bred for differences in alcohol intake. The HAD/LAD rats originated from the N/Nih heterogeneous stock developed from intercrossing eight inbred rat strains. The HAD×LAD F2 were genotyped, and a powerful analytical approach, using ancestral recombination and F2 recombination, was used to narrow a quantitative trait loci (QTL) for alcohol drinking to a 2-cM region on distal chromosome 10 that was in common in the HAD1/LAD1 and HAD2/LAD2 analyses. Quantitative real-time PCR was used to examine mRNA expression of six candidate genes (Crebbp, Trap1, Gnptg, Clcn7, Fahd1, and Mapk8ip3) located within the narrowed QTL region in the HAD1/LAD1 rats. Expression was examined in five brain regions, including the nucleus accumbens, amygdala, caudate putamen, hippocampus, and prefrontal cortex. All six genes showed differential expression in at least one brain region. Of the genes tested in this study, Crebbp and Mapk8ip3 may be the most promising candidates with regard to alcohol drinking.

Effects of prolonged ethanol vapor exposure on forced swim behavior, and neuropeptide Y and corticotropin-releasing factor levels in rat brains - Corrected Proof

2010-08-13

Abstract: Depressive symptoms in alcohol-dependent individuals are well-recognized and clinically relevant phenomena. The etiology has not been elucidated although it is clear that the depressive symptoms may be alcohol independent or alcohol induced. To contribute to the understanding of the neurobiology of chronic ethanol use, we investigated the effects of chronic intermittent ethanol vapor exposure on behaviors in the forced swim test (FST) and neuropeptide Y (NPY) and corticotropin-releasing factor (CRF) levels in specific brain regions. Adult male Wistar rats were subjected to intermittent ethanol vapor (14h on/10h off) or air exposure for 2 weeks and were then tested at three time points corresponding to acute withdrawal (8–12h into withdrawal) and protracted withdrawal (30 and 60 days of withdrawal) in the FST. The behaviors that were measured in the five-min FST consisted of latency to immobility, swim time, immobility time, and climbing time. The FST results showed that the vapor-exposed animals displayed depressive-like behaviors; for instance, decreased latency to immobility in acute withdrawal and decreased latency to immobility, decreased swim time and increased immobility time in protracted withdrawal, with differences between air- and vapor-exposed animals becoming more pronounced over the 60-day withdrawal period. NPY levels in the frontal cortex of the vapor-exposed animals were decreased compared with the control animals, and CRF levels in the amygdala were correlated with increased immobility time. Thus, extended ethanol vapor exposure produced long-lasting changes in FST behavior and NPY levels in the brain.

Alcohol-induced deterioration in primary antioxidant and glutathione family enzymes reversed by exercise training in the liver of old rats - Corrected Proof

2010-08-13

Abstract: Chronic alcohol consumption causes severe hepatic oxidative damage, particularly to old subjects by decreasing various antioxidant enzymes. In this study, we test the hypothesis that exercise training can protect the aging liver against alcohol-induced oxidative damage. Two different age groups of Wistar albino rats (3 months young, n=24; 18 months old, n=24) were evenly divided into four groups: control (Con), exercise trained (Tr, 23m/min 30min/day, 5 days/week for 2 months), ethanol drinking/treated (Et, 2.0g/kg b.w. orally), and exercise training plus ethanol drinking/treated (Tr+Et). We found significantly (P<.001) lowered hepatic antioxidant enzymes including superoxide dismutase, catalase, selenium (Se)-dependent glutathione peroxidase (Se–GSH-Px), Se–non-dependent glutathione peroxidase (non-Se–GSH-Px), glutathione reductase, and glutathione S-transferase activities in aged rats compared with young. Age-related decrease in antioxidant enzyme status was further exacerbated with ethanol drinking, which indicates liver in aged rats is more susceptible to oxidative damage because of decreased free radical scavenging system in aged/old ethanol-drinking rats. However, the decrease in liver antioxidant enzymes status with ethanol consumption was ameliorated by 2 months exercise training in old and young rats. These results demonstrate that age-associated decrease in hepatic free radical scavenging system exacerbated by ethanol drinking. For the first time, we found that this deterioration was significantly reversed by exercise training in aging liver, thus protects against alcohol-induced oxidative damage.

An improved method for rapidly quantifying fatty acid ethyl esters in meconium suitable for prenatal alcohol screening - Corrected Proof

Janine R. Hutson, Chitra Rao, Netta Fulga, Katarina Aleksa, Gideon Koren — 2010-08-13

Abstract: Fatty acid ethyl esters (FAEEs) are nonoxidative metabolites of ethanol, and elevated levels of FAEE in meconium are a useful biomarker for heavy prenatal alcohol exposure. FAEE in meconium has been recommended as useful and cost-effective for universal screening for prenatal alcohol exposure. To support an efficient universal screening program, an analytical method to detect and quantify FAEE in meconium needs to be accurate, inexpensive, and rapid. The purpose of this study was to develop an analytical method that would satisfy these criteria and to validate this method using established laboratory guidelines. A method was developed and validated to detect and quantify four FAEEs (ethyl palmitate, ethyl linoleate, ethyl oleate, and ethyl stearate) from 0.5g of meconium using d5-ethyl esters as internal standards. The sample undergoes liquid–liquid extraction with heptane:acetone, the heptane layer is isolated and evaporated, and then, the resulting residue undergoes headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry. The detection limits of the four FAEEs ranged from 0.020 to 0.042nmol/g and are 6- to 25-fold lower than the individual FAEE threshold concentrations (0.5nmol/g). This method also has good precision with the coefficient of variation ranging from 2.6 to 19.4% for concentrations of individual FAEE between 0.5 and 2.62nmol/g meconium (n=4). Calculated concentrations of FAEE that underwent extraction from meconium were 100–101% of the expected concentration, demonstrating the accuracy of the method. The peak shape and retention time of each FAEE were unaffected by the presence of the matrix, and there is no carryover at clinically relevant concentrations. This method was also able to produce clean chromatograms from meconium samples that could not be quantified using a previous method because of high chromatographic background. This method provides an optimal approach to detecting and quantifying FAEE in meconium that could be used in a universal screening program for prenatal alcohol exposure.

Subtle decreases in DNA methylation and gene expression at the mouse Igf2 locus following prenatal alcohol exposure: effects of a methyl-supplemented diet - Corrected Proof

2010-08-13

Abstract: C57BL/6J (B6) mice are susceptible to in utero growth retardation and a number of morphological malformations following prenatal alcohol exposure, while DBA/2J (D2) mice are relatively resistant. We have previously shown that genomic imprinting may play a role in differential sensitivity between B6 and D2. The best-characterized mechanism mediating genomic imprinting is differential DNA methylation. In the present study we examined DNA methylation and gene expression, in both embryonic and placental tissue, at the mouse Igf2 locus following in utero ethanol exposure. We also examined the effects of a methyl-supplemented diet on methylation and ethanol teratogenesis. In embryos from susceptible B6 mice, we found small decreases in DNA methylation at four CpG sites in one of the differentially methylated regions of the Igf2 locus; only one of the four sites showed a statistically significant decrease. We observed no significant decreases in methylation in placentae. All Igf2 transcripts showed approximately 1.5-fold decreases following intrauterine alcohol exposure. Placing dams on a methyl-supplemented diet before pregnancy and throughout gestation brought methylation back up to control levels. Methyl supplementation also resulted in lower prenatal mortality, greater prenatal growth, and decreased digit malformations; it dramatically reduced vertebral malformations. Thus, although prenatal alcohol had only small effects on DNA methylation at the Igf2 locus, placing dams on a methyl-supplemented diet partially ameliorated ethanol teratogenesis.

Influence of ethanol dose and pigmentation on the incorporation of ethyl glucuronide into rat hair - Corrected Proof

2010-07-05

Abstract: Ethyl glucuronide (EtG) is a minor and specific metabolite of ethanol. It is incorporated into growing hair, allowing a retrospective detection of alcohol consumption. However, the suitability of quantitative EtG measurements in hair to determine the quantity of alcohol consumed has not clearly been demonstrated yet. The purpose of this study was to evaluate the influence of ethanol dose and hair pigmentation on the incorporation of EtG into rat hair. Ethanol and EtG kinetics in blood were investigated after a single administration of ethanol.Eighteen rats were divided into four groups receiving 0 (control group), 1, 2, or 3g ethanol/kg body weight. Ethanol was administered on 4 consecutive days per week for 3 weeks by intragastric route. Twenty-eight days after the initial ethanol administration, newly grown hair was shaved. Pigmented and nonpigmented hair were analyzed separately by gas chromatography coupled to tandem mass spectrometry. Blood samples were collected within 12h after the ethanol administration. EtG and ethanol blood levels were measured by liquid chromatography coupled to tandem mass spectrometry and headspace gas chromatography-flame ionization detector, respectively. No statistically significant difference was observed in EtG concentrations between pigmented and nonpigmented hair (Spearman's rho=0.95). Thus, EtG incorporation into rat hair was not affected by hair pigmentation. Higher doses of ethanol resulted in greater blood ethanol area under the curve of concentration versus time (AUC) and in greater blood EtG AUC. A positive correlation was found between blood ethanol AUC and blood EtG AUC (Spearman's rho=0.84). Increased ethanol administration was associated with an increased EtG concentration in hair. Blood ethanol AUC was correlated with EtG concentration in hair (Pearson's r=0.89). EtG concentration in rat hair appeared to reflect the EtG concentration in blood. Ethanol was metabolized at a median rate of 0.22g/kg/h, and the median elimination half-life of EtG was 1.21h. This study supports that the bloodstream is likely to display a major role in the hair EtG incorporation.

Impact of prenatal alcohol consumption on placenta-associated syndromes - Corrected Proof

2010-07-02

Abstract: The biology of placental and fetal development suggests that alcohol may play a significant role in increasing the risk of feto-infant morbidity and mortality, but study results are inconsistent and the mechanism remains poorly defined. Previous studies have not examined the risk of placenta-associated syndromes (PASs: defined as the occurrence of either placental abruption, placenta previa, preeclampsia, small for gestational age, preterm, or stillbirth) as a unique entity. Therefore, we sought to examine the relationship between prenatal alcohol use and the risk of PAS among singleton births in the Missouri maternally linked data files covering the period 1989–2005. Logistic regression with adjustment for intracluster correlation was used to generate adjusted odds ratios (ORs) and 95% confidence intervals (CIs). Compared with nondrinkers, drinkers were more likely to be smokers, 35 years of age or older, black, and multiparous. Drinkers had an increased risk of PAS (OR=1.26, 95% CI=1.22,1.31) when compared with their nondrinking counterparts. The risk of PAS was progressively amplified with increasing prenatal alcohol consumption (P for trend <.01). Women who reported consuming five or more alcoholic drinks per week had more than twofold increased risk of PASs, whereas women in the lowest drinking category (one to two drinks per week) had only a slight increased risk of PAS (OR=1.09, 95% CI=1.05, 1.14). Enhanced understanding of the mechanism by which prenatal alcohol consumption leads to PAS may aid in the development of more targeted interventions designed to prevent adverse pregnancy outcomes. Screening women for alcohol use may assist providers in protecting developing fetuses from the potential dangers of prenatal alcohol use.

Correlation between drugs of abuse and alcohol by hair analysis: parents at risk for having children with fetal alcohol spectrum disorder - Corrected Proof

Vivian Kulaga, Sarit Shor, Gideon Koren — 2010-06-28

Abstract: The fatty acid ethyl esters (FAEEs) hair test, a biomarker of excessive alcohol exposure, has demonstrated its potential for use in fetal alcohol spectrum disorder (FASD) diagnosis. FASD may be compounded by polydrug exposure. Our objective was to determine the likelihood of positive FAEE test among parents testing positive for other drugs of abuse. Samples submitted for FAEE hair analysis by Children's Aid Societies between October 2005 and May 2007, also concurrently tested for cocaine, cannabinoids, opiates, methamphetamine, amphetamine, benzodiazepines, methadone, and/or oxycodone, were included in our analysis. Subjects consisted of parents suspected of using excessive amounts of alcohol. Parents testing positive for drugs of abuse had a significantly increased risk for testing positive for high FAEE. Mothers testing positive for heavy chronic alcohol use were found to have a threefold increased risk of testing positive for cocaine (odds ratio=3.26, 1.1–9.7). Our results suggest that parents abusing stimulants are at risk of high alcohol exposure, which put their unborn children at risk for FASD.

Rates of fetal alcohol exposure among newborns in a high-risk obstetric unit - Corrected Proof

2010-06-28

Abstract: Meconium fatty acid ethyl esters (FAEEs) are sensitive and specific biomarkers for prenatal alcohol exposure (PAE) in pregnancy. We recently reported a 2.5% rate of FAEE positive meconium in a general population sample of infants born in the region of Grey-Bruce, Ontario. Women in this region with high-risk pregnancies are transferred to a tertiary care facility in London, Ontario. The objective of this study was to determine, in a population-based sample, whether high-risk pregnancies are associated with an increased risk of in utero alcohol exposure. Grey-Bruce residents transferred to the high-risk obstetric unit of St. Joseph's Health Care in London, Ontario were identified and consented to this anonymous prevalence study. Meconium was collected and analyzed for FAEE using gas chromatography with mass spectrometry. The prevalence of FAEE positive meconium was compared with the population-based prevalence in the Grey-Bruce. Fifty meconium specimens were collected from August 1, 2006 to July 31, 2007. Fifteen (30%) specimens tested positive for FAEE. The results indicate that infants born in the high-risk obstetric unit had a 12-fold higher risk of screening positive for second and third trimester alcohol exposure compared with infants born in the general population of Grey-Bruce (relative risk=12.04, 95% confidence interval=6.40–22.65, P<.0001). These results suggest that the high-risk pregnancies should be screened for PAE and followed-up for potential diagnosis of fetal alcohol spectrum disorder.

Ethanol-induced downregulation of the angiotensin AT2 receptor in murine fibroblasts is mediated by PARP-1 - Corrected Proof

2010-06-21

Abstract: Molecular mechanisms accompanying ethanol-induced cytotoxicity remain to be defined. The renin–angiotensin system with its respective receptors, the angiotensin AT1 and AT2 receptor (AT1R and AT2R), has been implicated in these processes. The AT2R seems to counteract the pro-inflammatory, pro-hypertrophic, and pro-fibrotic actions of the AT1R and is involved in cellular differentiation and tissue repair. Recently, we identified poly(ADP-ribose) polymerase-1 (PARP-1) as a novel negative transcriptional regulator of the AT2R. However, the complex interactions between ethanol, PARP-1, and the AT2R are largely unknown. In this in vitro study, we aimed to clarify whether acute ethanol treatment modifies AT2R promoter activity or AT2R mRNA and protein levels and whether PARP-1 is involved in ethanol-mediated regulation of the AT2R. Murine fibroblasts of the R3T3 and MEF line (murine embryonic fibroblasts) were exposed to ethanol for 24h. AT2R promoter activity, mRNA and protein levels were analyzed with and without PARP-1 inhibition and in PARP-1 knockout MEF cells. Expression of PARP-1 was analyzed over course of time, and cell viability and DNA fragmentation were measured on single-cell level by flow cytometry. Ethanol exposition induced substantial downregulation of the AT2R on promoter, mRNA and protein levels in a dose-dependent manner. Pharmacological inhibition or ablation of PARP-1 completely abolished this effect. Ethanol treatment did not have any effect on AT1R mRNA and protein levels in MEF cells. Further, acute ethanol treatment promoted DNA fragmentation and caused transcriptional induction of PARP-1. Our findings reveal that PARP-1 is an upstream transcriptional regulator of the AT2 receptor in the context of ethanol exposure and represses the AT2R gene in fibroblasts in vitro. Variations in expression of the potentially tissue-protective AT2R might contribute to ethanol-mediated pathology.

Reduction of ethanol-induced ocular abnormalities in mice through dietary administration of N-acetylcysteine - Corrected Proof

Scott E. Parnell, Kathleen K. Sulik, Deborah B. Dehart, Shao-yu Chen — 2010-06-21

Abstract: N-Acetylcysteine (NAC) is a derivative of the amino acid l-cysteine, which, previously, has been shown to protect against ethanol-induced apoptosis during early development. Ongoing research demonstrates that NAC is also proving clinically beneficial in reducing oxidative stress–mediated lung, liver, and kidney damage, with protection likely resulting from a NAC-mediated increase in glutathione levels. In the present study, the hypothesis that coadministration of NAC and ethanol by means of liquid diet on days 7 and 8 of pregnancy in mice would reduce ethanol's teratogenicity was tested. For this work, adult nonpregnant female mice were acclimated to a liquid diet containing ethanol for 16 days, withdrawn from the ethanol, bred, and then returned to the liquid diet containing 4.8% ethanol and/or either 0.5 or 1-mg NAC/mL diet on their seventh and eighth days of pregnancy. At the concentrations used, the mice received NAC dosages of approximately 300 or 600mg/kg/day and achieved peak blood ethanol concentrations (BEC) that averaged approximately 200mg/dL. There was no difference in BEC between the ethanol-alone and ethanol plus 600mg/kg NAC group. After maternal euthanasia, gestational day (GD) 14 fetuses were removed, fixed, weighed, and examined for the presence and severity of ocular abnormalities, a readily assessed endpoint that results from GD 7 and 8 ethanol exposures. Although the lower dosage of NAC (300mg/kg) resulted in a decrease in the incidence of ocular defects in both the left and right eyes, this reduction was not statistically significant. However, doubling the NAC concentration did yield a significant change; as compared with the group treated with ethanol alone, the incidence of ocular abnormalities was diminished by 22%. These results show the potential of an orally administered compound with proven clinical efficacy to reduce ethanol's teratogenic effects and support the premise that oxidative damage plays an important mechanistic role in fetal alcohol spectrum disorders.

Alcohol-induced facial dysmorphology in C57BL/6 mouse models of fetal alcohol spectrum disorder - Corrected Proof

2010-06-07

Abstract: Alcohol consumption during pregnancy causes fetal alcohol spectrum disorder (FASD), which includes a range of developmental deficits. Fetal alcohol syndrome is the most severe form of FASD and can be diagnosed with pathognomonic facial features such as a smooth philtrum, short palpebral fissure, and thin upper vermilion. However, many children with developmental damage because of prenatal alcohol exposure exhibit none, or only a subset, of the above features, making diagnosis difficult. This study explored novel analyses to quantify the effect of a known dose of alcohol on specific facial measurements in substrains C57BL/B6J (B6J) and C57BL/6NHsd (B6N) mice. Mouse dams were provided alcohol (Alc) consisting of 4.8% (vol/vol) alcohol in a liquid diet for 16 days prepregnancy and chow and water diet during mating, and then the alcohol liquid diet was reinstated on gestational days 7 (E7) to gestational day 17 (E17). Treatment controls included a pair-fed (PF) group given matched volumes of an alcohol-free liquid diet made isocalorically and a group given ad lib access to lab chow and water (Chow). Maternal diet intake (Alc and PF), blood alcohol concentrations (BACs), embryo weights, and 15 morphometric facial measurements for E17 embryos were analyzed. B6N dams drank more alcohol during pregnancy and generated higher BAC than B6J dams. Both the Alc and PF treatments induced significant reductions in embryo weights relative to Chow in both substrains. Alcohol treatments produced significant changes, relative to controls, in 4 of the 15 facial measures for the B6N substrain but only in two measures for the B6J substrain. Discriminant analysis demonstrated successful classification of the alcohol-exposed versus nonalcohol-exposed B6N embryos, with a high sensitivity of 86%, specificity 80%, and overall classification (total correct 83%), whereas B6J mice yielded sensitivity of 80%, specificity 78%, and overall correct classification in 79%. In addition, B6N mice showed significantly more effects of pair feeding on these facial measures than did B6J mice, suggesting that the B6N substrain may be more vulnerable to nutritional stress during pregnancy. Overall, these data indicate that both B6N and B6J mice were vulnerable to alcohol but show differences in the severity and location of alcohol-induced dysmorphic facial features and may parallel findings from human studies comparing different ethnic groups. Furthermore, these findings suggest that discriminant analysis may be useful in predicting alcohol exposure in either mouse substrains.

Prenatal alcohol exposure alters the patterns of facial asymmetry - Corrected Proof

2010-01-11

Abstract: Directional asymmetry, the systematic differences between the left and right body sides, is widespread in human populations. Changes in directional asymmetry are associated with various disorders that affect craniofacial development. Because facial dysmorphology is a key criterion for diagnosing fetal alcohol syndrome (FAS), the question arises whether in utero alcohol exposure alters directional asymmetry in the face. Data on the relative position of 17 morphologic landmarks were obtained from facial scans of children who were classified as either FAS or control. Shape data obtained from the landmarks were analyzed with the methods of geometric morphometrics. Our analyses showed significant directional asymmetry of facial shape, consisting primarily of a shift of midline landmarks to the right and a displacement of the landmarks around the eyes to the left. The asymmetry of FAS and control groups differed significantly and average directional asymmetry was increased in those individuals exposed to alcohol in utero. These results suggest that the developmental consequences of fetal alcohol exposure affect a wide range of craniofacial features in addition to those generally recognized and used for diagnosis of FAS.

Validity of the T-ACE in pregnancy in predicting child outcome and risk drinking - Corrected Proof

2010-01-06

Abstract: Preventing fetal alcohol spectrum disorders (FASDs) requires detection of in-pregnancy maternal risk drinking. The widely used T-ACE screen has been applied in various ways, although the impact of those different uses on effectiveness is uncertain. We examined relations among different T-ACE scoring criteria, maternal drinking, and child outcome. Self-reported across-pregnancy maternal drinking was assessed in 75 African-American women. The different T-ACE criteria used varied the level of drinking that defined tolerance (two or three drinks) and the total T-ACE score cut-points (two or three). Receiver operator curves and regression analysis assessed the significance of relations. Increasing the total T-ACE score cut-point to 3 almost doubled specificity in detecting risk drinking whereas maintaining adequate sensitivity, equivalent to that in the original report, and identified substantially more neurobehavioral deficits in children. Redefining tolerance at three drinks did not improve T-ACE effectiveness in predicting outcomes. This study is among the first to show the ability of an in-pregnancy T-ACE assessment to predict child neurodevelopmental outcome. In addition, increasing the total T-ACE score criterion (from 2 to 3) improved identification of non-drinking mothers and unaffected children with little loss in detection of drinkers and affected children. Efficient in-pregnancy screens for risk drinking afford greater opportunities for intervention that could prevent/limit FASDs.

Diagnosis of fetal alcohol spectrum disorders: a validity study of the fetal alcohol syndrome checklist - Corrected Proof

Larry Burd, Marilyn G. Klug, Qing Li, Jacob Kerbeshian, John T. Martsolf — 2010-01-06

Abstract: Fetal alcohol spectrum disorders (FASD) are a common cause of developmental disability, birth defects, and mortality. The performance characteristics of current diagnostic tools for FASD are not adequately reported. This study examines the performance characteristics of the Fetal Alcohol Syndrome Diagnostic Checklist (FASDC). In a population of 658 subjects from North Dakota, we used the FASDC score to examine the agreement between FASDC score, clinical diagnosis, and the Institute of Medicine criteria for FASD. All subjects were seen for evaluation in the genetic/dysmorphology clinics, which are funded by the state to provide genetic diagnostic services for residents of North Dakota. We compared the clinical diagnosis and the FASDC scores to determine the performance characteristics of the FASDC in the categorical diagnosis of fetal alcohol spectrum (FAS), other-FASD, and a group with No-FASD. Comparisons were made using univariate and logistic models of outcomes using both the presence and the absence of alcohol exposure or FASDC phenotype data. The FASDC performance characteristics for differentiation of the FAS group from non-FASD were excellent (accuracy 99%, sensitivity 99%, and specificity 99%). Logistic models for subjects with scores in the FASD range were differentiated with an accuracy of 82%, sensitivity 85%, and specificity 80% using the data on phenotype and exposure. We were able to delineate subjects with scores in the No-FASD range with an accuracy of 78%, sensitivity 64%, and specificity 81% without including the exposure and phenotype data by use of the other descriptive data (maternal characteristics, birth records, and demographic data) from the FASDC. All diagnostic tools should have performance characteristics assessed and available before adoption for use in clinical settings. The FASDC scores produce diagnostic groupings that approximate expert clinical judgment. The tool may be useful in other clinical settings for the diagnosis of FASD or as an FASD registry or research database.

Effects of moderate drinking during pregnancy on placental gene expression - Corrected Proof

2010-01-06

Abstract: Many children adversely affected by maternal drinking during pregnancy cannot be identified early in life using current diagnostic criteria for fetal alcohol spectrum disorder (FASD). We conducted a preliminary investigation to determine whether ethanol-induced alterations in placental gene expression may have some utility as a diagnostic indicator of maternal drinking during pregnancy and as a prognostic indicator of risk for adverse neurobehavioral outcomes in affected offspring. Pregnant Long-Evans rats voluntarily consumed either a 0 or 5% ethanol solution 4 h each day throughout gestation. Ethanol consumption produced a mean maternal daily intermittent peak serum ethanol concentration of 84 mg/dL. Placentas were harvested on gestational day 20 for gene expression studies. Microarray analysis of more than 28,000 genes revealed that the expression of 304 known genes was altered twofold or greater in placenta from ethanol-consuming dams compared with controls. About 76% of these genes were repressed in ethanol-exposed placentas. Gene expression changes involved proteins associated with central nervous system development; organ morphogenesis; immunological responses; endocrine function; ion homeostasis; and skeletal, cardiovascular, and cartilage development. To date, quantitative real-time polymerase chain reaction analysis has confirmed significant alterations in gene expression for 22 genes, including genes encoding for three calcium binding proteins, two matrix metalloproteinases, the cannabinoid 1, galanin 2 and toll-like receptor 4, iodothyronine deiodinase 2, 11-β hydroxysteroid dehydrogenase 2, placental growth factor, transforming growth factor alpha, gremlin 1, and epithelial growth factor (EGF)-containing extracellular matrix protein. These results suggest that the expression of a sufficiently large number of placental mRNAs is altered after moderate drinking during pregnancy to warrant more detailed investigation of the placenta as a biomarker system for maternal drinking during pregnancy and as an early indicator of FASD. Furthermore, these results provide new insights into novel mechanisms on how ethanol may directly or indirectly mediate its teratogenic effects through alterations in placental function during pregnancy.

High-throughput caveolar proteomic signature profile for maternal binge alcohol consumption - Corrected Proof

Jayanth Ramadoss, Wu-xiang Liao, Dong-bao Chen, Ronald R. Magness — 2010-01-06

Abstract: Currently, no single marker is sensitive and specific enough to be considered a reliable biomarker for prenatal alcohol exposure. To identify a proteomic signature profile for maternal alcohol consumption, we carried out high-throughput proteomics on maternal endothelial caveolae exposed to moderate binge-like alcohol conditions. In these specialized lipid-ordered microdomains that contain a rich assembly of proteins, we demonstrate that moderate binge-like alcohol resulted in a distinctive maternal caveolar proteomic signature with important proteins being dramatically decreased/knocked out in the alcoholic profile. These proteins span from histones and basic structural proteins like α tubulin to proteins involved in trafficking, deubiquitination, cell signaling, and cell–cell adhesion. The profile also suggests an important role for the mother and the uteroplacental compartment in the pathogenesis of fetal alcohol spectrum disorders (FASD). These data demonstrate that the caveolar proteomic signature created by alcohol shows a promising direction for early detection of FASD.

Collaborative initiative on fetal alcohol spectrum disorders: methodology of clinical projects - Corrected Proof

2009-12-28

Abstract: The Collaborative Initiative on Fetal Alcohol Spectrum Disorders (CIFASD) was created in 2003 to further understanding of fetal alcohol spectrum disorders. Clinical and basic science projects collect data across multiple sites using standardized methodology. This article describes the methodology being used by the clinical projects that pertain to assessment of children and adolescents. Domains being addressed are dysmorphology, neurobehavior, 3-D facial imaging, and brain imaging.

A 14-year retrospective maternal report of alcohol consumption in pregnancy predicts pregnancy and teen outcomes - Corrected Proof

2009-12-28

Abstract: Detecting patterns of maternal drinking that place fetuses at risk for fetal alcohol spectrum disorders (FASDs) is critical to diagnosis, treatment, and prevention but is challenging because information on antenatal drinking collected during pregnancy is often insufficient or lacking. Although retrospective assessments have been considered less favored by many researchers due to presumed poor reliability, this perception may be inaccurate because of reduced maternal denial and/or distortion. The present study hypothesized that fetal alcohol exposure, as assessed retrospectively during child adolescence, would be related significantly to prior measures of maternal drinking and would predict alcohol-related behavioral problems in teens better than antenatal measures of maternal alcohol consumption. Drinking was assessed during pregnancy, and retrospectively about the same pregnancy, at a 14-year follow-up in 288 African-American women using well-validated semistructured interviews. Regression analysis examined the predictive validity of both drinking assessments on pregnancy outcomes and on teacher-reported teen behavior outcomes. Retrospective maternal self-reported drinking assessed 14 years postpartum was significantly higher than antenatal reports of consumption. Retrospective report identified 10.8 times more women as risk drinkers (≥ one drink per day) than the antenatal report. Antenatal and retrospective reports were moderately correlated and both were correlated with the Michigan Alcoholism Screening Test. Self-reported alcohol consumption during pregnancy based on retrospective report identified significantly more teens exposed prenatally to at-risk alcohol levels than antenatal, in-pregnancy reports. Retrospective report predicted more teen behavior problems (e.g., attention problems and externalizing behaviors) than the antenatal report. Antenatal report predicted younger gestational age at birth and retrospective report predicted smaller birth size; neither predicted teen IQ. These results suggest that if only antenatal, in-pregnancy maternal report is used, then a substantial proportion of children exposed prenatally to risk levels of alcohol might be misclassified. The validity of retrospective assessment of prior drinking during pregnancy as a more effective indicator of prenatal exposure was established by predicting more behavioral problems in teens than antenatal report. Retrospective report can provide valid information about drinking during a prior pregnancy and may facilitate diagnosis and subsequent interventions by educators, social service personnel, and health-care providers, thereby reducing the life-long impact of FASDs.

Zebrafish fetal alcohol syndrome model: effects of ethanol are rescued by retinoic acid supplement - Corrected Proof

2009-12-28

Abstract: This study was designed to develop a zebrafish experimental model to examine defects in retinoic acid (RA) signaling caused by embryonic ethanol exposure. RA deficiency may be a causative factor leading to a spectrum of birth defects classified as fetal alcohol spectrum disorder (FASD). Experimental support for this hypothesis using Xenopus showed that effects of treatment with ethanol could be partially rescued by adding retinoids during ethanol treatment. Previous studies show that treating zebrafish embryos during gastrulation and somitogenesis stages with a pathophysiological concentration of ethanol (100mM) produces effects that are characteristic features of FASD. We found that treating zebrafish embryos with RA at a low concentration (10−9M) and 100mM ethanol during gastrulation and somitogenesis stages significantly rescued a spectrum of defects produced by treating embryos with 100mM ethanol alone. The rescued phenotype that we observed was quantitatively more similar to embryos treated with 10−9M RA alone (RA toxicity) than to untreated or 100mM ethanol-treated embryos. RA rescued defects caused by 100mM ethanol treatment during gastrulation and somitogenesis stages that include early gastrulation cell movements (anterior–posterior axis), craniofacial cartilage formation, and ear development. Morphological evidence also suggests that other characteristic features of FASD (e.g., neural axis patterning) are rescued by RA supplement.

Implementation of a shared data repository and common data dictionary for fetal alcohol spectrum disorders research - Corrected Proof

2009-12-28

Abstract: Many previous attempts by fetal alcohol spectrum disorders researchers to compare data across multiple prospective and retrospective human studies have failed because of both structural differences in the collected data and difficulty in coming to agreement on the precise meaning of the terminology used to describe the collected data. Although some groups of researchers have an established track record of successfully integrating data, attempts to integrate data more broadly among different groups of researchers have generally faltered. Lack of tools to help researchers share and integrate data has also hampered data analysis. This situation has delayed improving diagnosis, intervention, and treatment before and after birth. We worked with various researchers and research programs in the Collaborative Initiative on Fetal Alcohol Spectrum Disorders (CI-FASD) to develop a set of common data dictionaries to describe the data to be collected, including definitions of terms and specification of allowable values. The resulting data dictionaries were the basis for creating a central data repository (CI-FASD Central Repository) and software tools to input and query data. Data entry restrictions ensure that only data that conform to the data dictionaries reach the CI-FASD Central Repository. The result is an effective system for centralized and unified management of the data collected and analyzed by the initiative, including a secure, long-term data repository. CI-FASD researchers are able to integrate and analyze data of different types, using multiple methods, and collected from multiple populations, and data are retained for future reuse in a secure, robust repository.

A neurodevelopmental framework for the development of interventions for children with fetal alcohol spectrum disorders - Corrected Proof

Piyadasa W. Kodituwakku — 2009-12-28

Abstract: Despite considerable data published on cognitive and behavioral disabilities in children with fetal alcohol spectrum disorders (FASD), relatively little information is available on behavioral or pharmacological interventions for alcohol-affected children. The main goals of this article, therefore, are to summarize published intervention studies of FASD and to present a neurodevelopmental framework, based on recent findings from a number of disciplines, for designing new therapies for alcohol-affected children. This framework assumes a neuroconstructionist view, which posits that reciprocal interactions between neural activity and the brain's hardware lead to the progressive formation of intra- and interregional neural connections. In this view, behavioral interventions can be conceptualized as a series of guided experiences that are designed to produce neural activation. Based on evidence from cognitive neuroscience, it is hypothesized that specific interventions targeting executive attention and self-regulation may produce greater generalizable results than those aimed at domain-specific skills in children with FASD. In view of reciprocal interactions between environmental effects and neural structures, the proposed framework suggests that the maximum effects of interventions can eventually be achieved by optimally combining behavioral methods and cognition-enhancing drugs.

Heavy in utero ethanol exposure is associated with the use of other drugs of abuse in a high-risk population - Corrected Proof

Sarit Shor, Irena Nulman, Vivian Kulaga, Gideon Koren — 2009-12-23

Abstract: Many ethanol dependent women also use other drugs of abuse that may affect pregnancy outcome and long-term child neurodevelopment. This study investigated the association between drugs of abuse and concurrent use of ethanol in pregnancy. A study cohort of neonates with FAEE levels above 2nmol per gram meconium, indicative of heavy in utero ethanol exposure, was identified (n=114). Meconium and hair analyses for the presence of other drugs of abuse were obtained for some of these neonates and the rates of drug exposure were compared with the rates in a cohort of neonates who were tested negative (FAEE below 2nmol per gram meconium) for ethanol exposure (n=622). Odds ratios (ORs) for various drugs were calculated with ethanol exposure. A 15.5% positive rate for intrauterine ethanol exposure was detected. A high rate of in utero drug exposure was detected in neonates with and without in utero ethanol exposure, 60.5% versus 62.7% respectively. Neonates with heavy in utero ethanol exposure were almost twice as likely to be exposed to narcotic opiates (OR=1.90; 95% confidence interval [CI]: 1.13–3.20) and 3.3 times as likely to be exposed to amphetamine (OR=3.30; 95% CI 1.06–10.27) when compared to neonates with no ethanol exposure. Exposure to cannabinoids predicted less likely exposure to ethanol (OR=0.61; 95% CI: 0.38–0.98) and no significant difference was noted in the exposure to cocaine (OR=1.24, 95% CI: 0.81–1.91). Neonates suspected of heavy in utero ethanol exposure should be tested for other drugs of abuse and vice versa. Early detection of drug exposures can facilitate early intervention to both the neonate and the mother, thus decreasing the risk of long-term neurodevelopmental outcomes for the child, including secondary disabilities associated with fetal alcohol spectrum disorder.