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Experimental Biology and Medicine 231:1379-1397 (2006)
© 2006 Society for Experimental Biology and Medicine

ORIGINAL RESEARCH ARTICLE

Physiologic and Genomic Analyses of Nutrition-Ethanol Interactions During Gestation: Implications for Fetal Ethanol Toxicity

Kartik Shankar*,{ddagger}, Mats Hidestrand*,{ddagger}, Xiaoli Liu*,{ddagger}, Rijin Xiao{dagger},{ddagger}, Charles M. Skinner{ddagger}, Frank A. Simmen{dagger},{ddagger}, Thomas M. Badger*,{dagger},{ddagger} and Martin J. J. Ronis*,{ddagger},1

* Departments of Pharmacology and Toxicology and {dagger} Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205; and {ddagger} Arkansas Children’s Nutrition Center, Little Rock, AR, 72202

1 To whom requests for reprints should be addressed at Arkansas Children’s Nutrition Center, Slot 512–20B, 1120 Marshall Street, Little Rock, AR 72202. E-mail: RonisMartinJ{at}uams.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nutrition-ethanol (EtOH) interactions during gestation remain unclear primarily due to the lack of appropriate rodent models. In the present report we utilize total enteral nutrition (TEN) to specifically understand the roles of nutrition and caloric intake in EtOH-induced fetal toxicity. Time-impregnated rats were intragastrically fed either control or diets containing EtOH (8–14 g/kg/day) at a recommended caloric intake for pregnant rats or rats 30% undernourished, from gestation day (GD) 6–20. Decreased fetal weight and litter size (P < 0.05) and increased full litter resorptions (33% vs. 0%), were observed in undernourished dams compared to adequately fed rats given the same dose of EtOH, while undernutrition alone did not produce any fetal toxicity. Undernutrition led to impairment of EtOH metabolism, increased blood EtOH concentrations (160%), and decreased maternal hepatic ADH1 mRNA, protein, and activity. Microarray analyses of maternal hepatic gene expression on GD15 revealed that 369 genes were altered by EtOH in the presence of undernutrition, as compared to only 37 genes by EtOH per se (±2-fold, P < 0.05). Hierarchical clustering and gene ontology analysis revealed that stress and external stimulus responses, transcriptional regulation, cellular homeostasis, and protein metabolism were affected uniquely in the EtOH-under-nutrition group, but not by EtOH alone. Microarray data were confirmed using real-time RT-PCR. Undernourished EtOH-fed animals had 2-fold lower IGF-1 mRNA and 10-fold lower serum IGF-1 protein levels compared to undernourished controls (P < 0.0005). Examination of maternal GH signaling via STAT5a and -5b revealed significant reduction in both gene and protein expression produced by both EtOH and undernutrition. However, despite significantly elevated fetal BECs, fetal IGF-1 mRNA and protein were not affected by EtOH or EtOH-undernutrition combinations. Our data suggest that undernutrition potentiates the fetal toxicity of EtOH in part by disrupting maternal GH-IGF-1, signaling thereby decreasing maternal uterine capacity and placental growth.

Key Words: ethanol • pregnancy • fetal alcohol syndrome • microarray • undernutrition


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The fetotoxic potential of ethanol (EtOH) has been recognized for nearly three decades, but EtOH continues to be the most common teratogen ingested during pregnancy (1). One of every 29 women who know they are pregnant report EtOH consumption (2). The toxic effects of EtOH are manifested by a constellation of physical, behavioral, and cognitive abnormalities commonly referred to as Fetal Alcohol Spectrum Disorder (FASD). In addition to mental retardation, in utero EtOH exposure results in increased rates of miscarriage, reduced birth weight, growth retardation, and teratogenic effects (3). The incidence of FASD in the general U.S. population ranges from 0.7 to 10 cases per 1000 live births annually (2), which is still a surprisingly small proportion of all children exposed to EtOH during fetal development. The reasons for the low penetrance of FASD and precise mechanisms causing FASD remain elusive. However, it is established that EtOH interferes with nutritional supply to the fetal-placental unit (4). Inadequate maternal diets have been demonstrated to exacerbate the effects of ethanol, and women who consume EtOH during pregnancy are often also malnourished (5). Moreover, chronic EtOH consumption can directly or indirectly compromise nutritional status. Undernutrition resulting in low prepregnancy weight and low gestational weight gain is reported to be a significant independent risk factor for adverse pregnancy outcomes. Hence it is clear from the literature in this area that nutritional aspects of prenatal EtOH exposure are of fundamental importance in determining the toxic effects of EtOH.

Rats administered EtOH via liquid diets (Lieber-Decarli) are often undernourished, consuming as much as 25%–40% less calories than controls fed ad libitum (6, 7). Inadequate nutrition is a particular problem in studies of EtOH consumption during pregnancy due to the increased nutritional requirements imposed by the growing fetal-placental unit. Feeding early formulations of the Lieber-DeCarli diets to pregnant rodents resulted in a 30%–50% reduction in gestational weight gain in pair-fed compared to ad libitum–fed dams (6, 8). While newer formulations of the Lieber-DiCarli diet result in improved body weight gains during gestation, significant deficits persist (8, 9). Lower weight gains during pregnancy are in good part related to decreased dietary intake and independent of EtOH consumption (6). In order to dissect the effects of EtOH and undernutrition in pregnancy on adverse outcomes, we have utilized intragastric infusion models in which isocaloric EtOH-containing liquid diets are administered directly into the stomach via an enteral cannula (10–12).

Previous studies have demonstrated that pregnant rats fed nutritionally adequate diets (220 kcal/day/kg3/4) containing the same dose of EtOH as nonpregnant rats, have significantly greater EtOH metabolism and consequently lower blood ethanol concentrations (BEC) (13). Hence pregnancy tends to provide a degree of protection against EtOH fetal toxicity by enhancing its metabolism and reducing the dose of EtOH reaching target tissues. The present study had three objectives. The first objective was to examine the role of caloric intake on EtOH metabolism in pregnancy. We hypothesized that adequate nutrition is essential for pregnancy-induced enhancement of EtOH metabolism. The second objective was to determine the effect of increasing doses of EtOH on fetal toxicity while maintaining adequate nutrition during pregnancy. Thirdly, these studies aimed to ascertain if undernutrition synergizes with the toxic effects of EtOH. To examine the mechanistic basis of how nutrition and EtOH produce interactive toxicity, we examined the status of ethanol-metabolizing enzymes, alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), and microsomal CYP2E1. Furthermore, microarray-based gene expression analyses of maternal liver were carried out to identify unique changes and novel mechanisms. Modulation of insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 1 and 2 (IGFBP-1 and -2) were studied. Our data strongly suggest that inadequate nutrition significantly increases fetal EtOH toxicity by impairing maternal EtOH metabolism and may also disrupt GH-IGF-1 signaling in the maternal axis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and Experimental Protocol.
Unless otherwise specified, all chemicals were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO). Time impregnated female Sprague-Dawley rats (250–300 g) and weight-matched nonpregnant cohorts were obtained from Charles River Laboratories (Wilmington, MA). Animals were housed in an AAALAC-approved animal facility, and animal maintenance and experimental treatments were conducted in accordance with the ethical guidelines for animal research established and approved by the Institutional Animal Care and Use Committee (IACUC). Rats were surgically cannulated with intragastric cannulae and infused either control or EtOH-containing diets, as described previously (10, 11). Rats were briefly anesthetized with Nembutal (50 mg/kg, ip) and reflexes, breathing, and skin color were monitored. The stomach was exposed and a cannula (0.025 in. i.d. x 0.45 in. o.d.) was inserted and secured with a suture at the interface between the cardiac and pyloric regions. The free end of the cannula was threaded subcutaneously to a headpiece that was anchored on the skull with acrylic dental cement. A swiveled spring acted as a tethering device and was attached to the dental cement. This allowed complete, unrestrained 360° movement for the rats. All animals were infused diets at a constant rate between 1800 and 0800 hrs (during the dark cycle). Animals had access to unlimited water throughout the experiment.

Experimental Treatments: Study 1.
To examine if pregnancy-induced changes in EtOH metabolism are dependent on the level of nutritional intake, nonpregnant and pregnant rats were divided into two groups. Time-impregnated female Sprague-Dawley rats (300 g) were obtained on Day 4 of gestation (GD4) and allowed to acclimatize in our facility for 1 day. On GD5, an intragastric cannula was surgically inserted into pregnant and non-pregnant rats (n = 4–5) and infused with EtOH-containing diets (EtOH, 13 g/kg/day, carbohydrate isocalorically replaced by ethanol) as described previously (10, 11). Two levels of caloric intake were administered to non-pregnant and pregnant rats, 220 kcal/day/kg3/4 (the caloric intake recommended for pregnant rats by the National Research Council, NRC) and 160 kcal/day/kg3/4 (undernourished), keeping minerals and vitamins at NRC-recommended levels while reducing all macronutrient components equally. Since urine ethanol concentrations (UECs) have been observed to be excellent predictors of blood ethanol concentrations in both cycling and pregnant rats (10, 11), 24-hr UECs were measured daily for 15 days using the Analox Instruments GL5 Analyzer fitted with an ampero-metric oxygen electrode sensor (Analox Instruments Ltd., London, UK).

Study 2.
To examine if ethanol-induced fetal toxicity increases with dose, groups of time-impregnated female Sprague-Dawley rats (300 g) were delivered to our animal facility on GD4 and cannulated and fed as described above. The EtOH dose was 0, 8, 9, 10, 10.75, 11.8, 13, or 14 g/kg/ day for groups of animals fed 220 kcal/kg3/4/day, achieved by isocalorically substituting EtOH for carbohydrate calories. Twenty-four hour UECs were measured daily (as described above) from GD6–7 to GD20. At GD20, dams (n = 3–15) were euthanized with 100 mg/kg Nembutol and the fetuses were counted, dissected, and weighed. Uteri were stained with 10% ammonium sulfide to verify embryonic implantation.

Study 3.
To determine if maternal undernutrition potentiates EtOH toxicity, one group of time-impregnated rats was fed 220 kcal/ kg3/4/day and the other group was calorically restricted at 160 kcal/ kg3/4/day (n = 7–8). Both groups received 13 g/kg/day of EtOH, and 24 hour UECs were analyzed. At the end of this experiment on GD20, dams were sacrificed and fetal effects and litter numbers were evaluated as described in Study 2. In addition, activities of the major alcohol metabolizing enzymes, ADH, ALDH, and CYP2E1, were measured in maternal liver.

Study 4.
Finally, to assess the relative contributions of adequate maternal nutrition and in utero ethanol exposure on fetal toxicity parameters and to examine the role of maternal IGF-1 in fetal growth retardation, time-impregnated rats were infused control or EtOH-containing TEN diets at 220 kcal/kg3/4/day or 160 kcal/kg3/4/day from GD6–GD15. The EtOH dose was 12 g/kg/day and was substituted for isocaloric amount of carbohydrate in control diets. UECs were measured daily as described above. On GD15, all dams were sacrificed under anesthesia and serum, livers (maternal), and fetuses were collected. Litter size, weight, and the number of total litter resorptions were recorded. Livers were flash-frozen in liquid nitrogen and stored at –70 °C.

In a separate experiment, dams were fed throughout gestation using the same paradigm as described in Study 4, but continued until GD20. On GD20, all dams were sacrificed under anesthesia and maternal and fetal serum and livers were collected. Serum from all fetuses belonging to one dam were pooled and frozen. Livers were flash-frozen in liquid nitrogen and stored at –70 °C. Amniotic fluid was also pooled from several placental sacs per dam and frozen for EtOH concentration analysis.

Hepatic Alcohol Dehydrogenase and Aldehyde Dehydrogenase Activities.
Liver homogenates from flash frozen livers were prepared in phosphate-sucrose buffer containing 1% Triton X-100 and 1 mM mercaptoethanol and centrifuged at 10,000 g for 30 mins. Supernatants were assayed for ADH and ALDH activities by measuring the formation of NADH at 340 nm (14). ADH (EC 1.1.1.1) activity was assayed at pH 8.5 in pyrophosphate-glycine buffer with 50 mM ethanol and 1 mM 4-methylpyrazole in the reference cuvette. ALDH (EC 1.2.1.3) was measured at pH 8.8 in the presence of 0.5 mM 4-methylpyrazole and 1 mM mercaptoethanol using 0.5 mM acetaldehyde as substrate (14).

Hepatic Microsomal CYP2E1 Activity.
Liver microsomes were prepared from livers of EtOH-fed dams by differential ultracentrifugation. Protein concentration of the microsomes was determined by the Bradford method using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Microsomes were stored at –70° C. Microsomal CYP2E1 activity was assayed by measuring carbon tetrachloride–dependent lipid peroxidation (15).

Quantitation of Hepatic ADH-1, STAT5a, and STAT5b Proteins by Western Blotting.
Hepatic cytosolic fractions were resolved on polyacrylamide gels and transferred to PVDF membranes (Bio-Rad). Immunoblotting was carried out using standard procedures, as previously described (16). Membranes were incubated with primary antibodies for ADH-1, STAT5a, and STAT5b (rabbit anti-rat ADH-1 [16], STAT5a, and STAT5b [1:1000; Upstate Biotechnology, Chicago, IL]) in TBST containing 5% milk powder for 1 hr at room temperature. Following incubation with HRP-conjugated secondary IgG (1:10,000), membranes were washed with TBST and proteins visualized using West Pico ECL chemiluminescence kit (Pierce, Rockford, IL) and detected by autoradiography. Immunoquantitation was performed by densitometric scanning of the resulting autoradiograms using a Bio-Rad GS700 molecular imager.

Hepatic Gene Expression Analysis.
Microarray-Based Analyses.
Hepatic gene expression profiles were assessed using Affymetrix RGU34A GeneChip microarrays (Affymetrix, Santa Clara, CA) containing 8800 genes, with approximately 7000 full-length transcripts and 1800 EST sequences. Livers were excised from dams fed control or EtOH diets (n = 3–4) from GD6 through GD15 containing 220 kcal/kg3/4/day or 160 kcal/kg3/4/day. Total RNA was extracted from livers using TRI reagent and cleaned using RNeasy mini columns (Qiagen, Valencia, CA). RNA quality was ascertained spectrophotometrically (ratio of A260/A280) and also by checking ratio of 18S to 28S ribosomal RNA using the RNA Nano Chip on a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Sample preparation and hybridizations (from individual animals, n = 3–4 per group) were carried out using manufacturer’s instructions. Microarrays were scanned using an Agilent microarray scanner. Raw data intensities acquired from the .cel files using Microarray Suite 5.0 software (Affymetrix) were globally normalized using scaling factor normalization. Genes were filtered based on presence/absence call (either 2 out of 3 or 3 out of 4), and average ± SEM gene expression for each treatment group was computed for present genes. For comparison analysis, genes were filtered based on minimum 2-fold ratio change and P value (<0.05) using Student’s t test. Data analyses were performed using Microsoft Excel and SpotFire DecisionSite for Functional Genomics. Correlation based hierarchical clustering between treatment groups was done using Cluster software (17). A companion software, TreeView, was used for the presentation of data (http://rana.lbl.gov/EisenSoftware.htm). Known biological functions of genes were queried and acquired from Affymetrix online data analysis resource NetAffx (http://www.affymetrix.com/analysis/index.affx) and gene ontology analyses performed using GO Browser (Affymetrix). To analyze and identify relationship of genes and known signaling pathways, commercially available PathwayAssist software (Ariadne Genomics, Rockville, MD) was used. Confirmation of microarray gene expression data was done by real-time RT-PCR.

Real-Time RT-PCR.
Livers were excised from dams (or fetuses) fed control or EtOH diets (n = 4–10) from GD6 through GD15 (or GD20 for fetal livers) containing 220 kcal/kg3/4/day or 160 kcal/kg3/4/day. Total RNA was extracted from livers as previously described. One µg of total RNA was reverse-transcribed using the IScript Reverse Transcription kit (Bio-Rad) according to manufacturer’s instructions. Twenty ng of the reverse transcribed cDNA was utilized for real-time PCR using the 2X SYBR Green master mix and monitored on an ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA). Gene-specific probes were designed using Primer Express Software (Table 1Go; Applied Biosystems). The relative amounts of gene expression were quantitated using a standard curve according to manufacturer’s instructions.


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  		Table 1. Primers sequences for real-time RT-PCR analysesa
 
Free IGF-1 Radioimmunoassay.
Serum from dams (or fetuses) on control or EtOH diets from GD6 through GD15 (or GD20) were analyzed for free IGF-1 using radioimmunoassay (Diagnostic Systems Laboratories, Webster, TX). Prior to immunoradiometric assessment, free IGF-1 was separated from serum samples as described by Lang et al. (18). Briefly, serum samples were diluted 1:5 with Krebs-Ringer bicarbonate buffer (pH 7.4) containing 5% bovine serum albumin. The samples were then centrifuged at 300 g at 37°C using Amicon Centrifree YM-30 tubes (Millipore Corporation, Bedford, MA) for 100 mins. The ultrafiltrate from 40 to 100 mins of centrifugation was used for free IGF-1 analyses. The radioimmunoassay for free IGF-1 was carried out according to manufacturer’s instructions.

Data and Statistical Analysis.
Data are expressed as means ± SEM. Quantitation of Western blot autoradiograms was performed using Quantity One software (Bio-Rad). SigmaStat software package version 3.0 (SPSS Inc., Chicago, IL) was used to perform all statistical tests. The data were tested using Levene’s test for equality of variance. Pearson product moment correlation was performed using SigmaStat software. Comparison between multiple groups was accomplished using either one-way ANOVA followed by Tukey post hoc analysis or two-way ANOVA followed by all-pairwise comparison by Student-Newman-Keuls method to compare differences between EtOH and under-nutrition. P values ≤ 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pregnancy-Induced Changes in EtOH Metabolism.
Ethanol metabolism was indirectly estimated via monitoring 24 hour UECs in nonpregnant and pregnant dams infused EtOH diets (13 g/kg/day) at two levels of caloric intake (220 kcal/day/kg3/4 and 160 kcal/day/kg3/4, hereafter referred to as the 220 group and the 160 group, respectively). Mean UECs were approximately 70% lower in the pregnant dams receiving EtOH in the 220 group compared to the nonpregnant counterparts, consistent with previously reported increased EtOH metabolism and clearance in pregnancy (Table 2Go) (13). However, UECs did not differ statistically between pregnant and non-pregnant rats receiving EtOH in the 160 group, suggesting decreased rate of EtOH metabolism in undernourished pregnant dams. Integrated area under UEC curves (AUC-UEC) were calculated as an estimate of EtOH exposure over the entire experiment. In rats administered ethanol in the 220 group, the AUC-UEC was 75% lower in the pregnant rats compared to nonpregnant dams, consistent with mean UEC estimates (Table 2Go). While undernutrition led to significantly higher AUC-UEC both in cycling and pregnant dams, indicating higher total EtOH burden, the AUC-UEC in pregnant rats in the 160 group was 50% lower than nonpregnant 160 cohorts, indicating a significant pregnancy-induced increase in EtOH clearance even in the face of undernutrition.


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  		Table 2. Effect of Caloric Intake and Pregnancy on Urine Ethanol Concentrations
 
Dose-Response of EtOH Fetal Toxicity.
To examine the dose response of EtOH-induced fetal toxicity in rats fed NRC-recommended nutrient levels, various fetal parameters affected by EtOH were recorded (Table 3Go). UEC levels positively correlated with EtOH dose (Pearson coefficient r2 = 0.952, P = 0.0009). Fetal toxicity, as indicated by reduced litter weight and pup birth weight, was only evident at EtOH doses exceeding 11.8 g/kg/day where the mean UECs were greater than 169 ± 15 mg/dl. Although whole litter resorptions were observed at >11.8 g/kg ethanol, complete litter resorptions only reached statistical significance at the 14 g/kg/day dose with average UECs > 300 mg/dl (Table 3Go). The importance of nutrition to fetal development was seen when we compared rats fed 220 or 160 kcal/kg/day in a separate experiment (Table 4Go) and found that 63% of dams in the 160 group resorbed their litters at 13 g/kg, whereas none were resorbed in the 220 group. This greater fetal toxicity was associated with a body weight loss (220-EtOH starting body weight 250 ± 5 g and ending body weight 290 ± 9 g vs. 160-EtOH starting body weight 254 ± 4 g and ending body weight 207 ± 7 g, P ≤ 0.05) throughout gestation and higher mean UECs (Table 4Go) and AUC-UEC and compared to dams in the 220 group. UECs correlated positively with BECs at GD15 (r2 = 0.94). Both parameters, UECs and BECs, showed ~30% higher EtOH levels in the undernourished dams compared to optimally fed cohorts (Fig. 1AGo). Consistent with maternal EtOH levels, fetal serum and amniotic fluid EtOH concentrations were also higher (~30%) in EtOH-undernourished dams compared to optimally fed counterparts (Fig. 1AGo). Moreover, undernourished pregnant dams also had greater fetal toxicity as indicated by reduced pup numbers, full litter resorptions, and reduced birth weight (P ≤ 0.05) (Table 4Go).


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  		Table 3. Effect of Increasing Ethanol Dose on Maternal and Fetal Parameters in Rats Fed 220 kcal/kg3/4/daya
 

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  		Table 4. Effect of Undernutrition on Fetal Alcohol Toxicitya
 

Figure 1
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  		Figure 1. (A) Blood, urine and amniotic fluid ethanol concentrations from pregnant dams and fetal blood ethanol concentrations from dams fed diets containing 12 g of EtOH/kg/day throughout gestation (GD5–GD15) with either 220 kcal/kg3/4/day or 160 kcal/kg3/4/day (see Materials and Methods, Study 4, for details). Data represent means ± SEM (n = 7–8). *, P < 0.05 versus 220 EtOH group. (B) Hepatic alcohol dehydrogenase activities from pregnant dams fed diets containing 13 g of EtOH/kg/day throughout gestation (GD5–GD15) with either 220 kcal/kg3/4/day or 160 kcal/kg3/4/day (see Materials and Methods for details). Data represent means ± SEM (n = 7–8). *, P < 0.05 versus 220 EtOH group. (C) Hepatic ADH-1 mRNA. (D) Western blot and densitometric quantitation of ADH-1 protein levels from pregnant dams fed diets containing 12 g of EtOH/ kg/day throughout gestation (GD5–GD15) with either 220 kcal/kg3/4/day or 160 kcal/kg3/4/day (see Materials and Methods, Study 4, for details). Data represent means ± SEM (n = 4–9). *, P < 0.05 versus 220 control group; #, P < 0.05 versus 220 EtOH group.

 
Nutritional Status and Hepatic Alcohol Dehy-drogenase mRNA, Protein, and Activity.
In order to understand the underlying mechanisms of how under-nutrition impairs EtOH metabolism, we examined hepatic EtOH metabolizing enzymes. Activities of hepatic ADH-1 and ALDH were measured in liver homogenates from pregnant dams as described in Study 3 in Materials and Methods. Activity of liver ADH-1 was ~50% lower in 160 group compared to the 220 group (Fig. 1BGo). In concordance with these data, the mean AUC-UEC was greater in the undernourished pregnant dams (P ≤ 0.05), indicating reduced EtOH metabolism. ALDH activities (ALDH nmol/mg/min) did not differ significantly between the 160 and 220 groups (1.3 ± 0.3 in EtOH-220 vs. 0.9 ± 0.3 in EtOH-160). Hepatic microsomal CYP2E1 activity (pmol of TBA formed/mg/min) showed a trend towards an increase in the activity in the undernourished EtOH-fed dams but did not reach statistical significance (86 ± 20 in EtOH-220 vs. 227 ± 88 in EtOH-160).

Furthermore, we examined the decrease in maternal hepatic ADH-1 by measuring gene expression and protein levels. Maternal livers from rats in the 160 or 220 groups receiving control or ethanol diets were used. EtOH-fed 220 dams did not show an induction in ADH-1 compared to controls because the UECs were much below the threshold for ADH-1 induction (>300 mg/dl) (11). Mean mRNA and protein levels of ADH-1 in the 160 group were 50% of those observed in the 220 group (P < 0.05, Fig. 1C and DGo), suggesting that undernutrition may impair EtOH metabolism in pregnancy via decreasing hepatic ADH-1.

EtOH-Fetal Toxicity During Maternal Undernutrition.
Fetal parameters recorded from pregnant dams fed 12 g/kg/day EtOH from Study 4 confirmed the findings from Study 3. Undernourished EtOH-fed rats showed a marked increase in fetal toxicity parameters, including decreased litter size (P ≤ 0.05) and complete litter resorptions (33%), compared to none in the 220 EtOH-fed animals (Table 5Go). Consistent with these data, mean fetal weights at GD20 were significantly lower (1.7 ± 0.04 vs. 1.9 ± 0.04 g, P < 0.05) in the EtOH-fed 160 group compared to the controls in the 160 group. Fetal body weights in both the control (2.07 ± 0.07 g) and the EtOH-fed (1.91 ± 0.04) 220 group were also significantly higher (P < 0.001) than the 160 EtOH group, but not significantly different from each other. Undernutrition alone in the absence of EtOH did not cause fetal toxicity, suggesting a significant EtOH-nutrition interaction.


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  		Table 5. Ethanol/Nutrition Interaction in Fetal Toxicity Parametersa
 
Global Maternal Hepatic Gene Expression and EtOH-Undernutrition Interaction.
Volcano plot of fold change (±2-fold) of gene expression and P value (≤0.05) shows that ~10-fold greater numbers of genes changed in the maternal livers following EtOH treatment in the 160 group compared to the 220 EtOH group (369 genes vs. 37 genes, respectively, Fig. 2AGo). The effects of EtOH alone (220 EtOH vs. 220 control), EtOH-undernutrition (160 EtOH vs. 160 control), and undernutrition (160 control vs. 220 control) are summarized in the Venn diagram (Fig. 2BGo). Undernutrition by itself altered 367 genes (160 control vs. 220 control). Of those genes altered by undernutrition, 164 genes are not affected by EtOH in the undernourished state. On the other hand, 203 genes that are affected by undernutrition alone are also affected by EtOH during undernutrition (Fig. 2BGo). Of the genes altered by the combination of EtOH and undernutrition, almost 44% (166 out of 369) are unique changes only represented in the EtOH-undernutrition interaction (160 EtOH vs. 160 control).


Figure 2
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  		Figure 2. (A) Volcano plot of P value (–log10) versus fold change (log2) of gene expression data from microarray-based analyses in comparisons of the EtOH 220 (blue) and EtOH 160 (red) groups compared to their respective controls. Genes were filtered based on absence/presence criteria, minimum 2-fold change and P ≤ 0.05 in either comparison. Four hundred genes passing these criteria are plotted. Genes to the right (or left) and above the vertical and horizontal green lines, respectively, pass the criteria. (B) Venn diagram representation of overall gene expression changes due to EtOH in adequate nutrition (37 genes), EtOH and undernutrition (369 genes), and undernutrition alone (367 genes). Two hundred-three genes were common between undernutrition alone and EtOH combined with undernutrition, revealing 166 unique genes that were affected by EtOH and undernutrition. Data collected from pregnant dams fed diets containing 12 g of EtOH/kg/day throughout gestation (GD5–GD15) with either 220 kcal/kg3/4/day or 160 kcal/kg3/4/day (see Materials and Methods, Study 4, for details).

 
Known biological functions of genes were queried and acquired from Affymetrix online data analysis resource NetAffx (http://www.affymetrix.com/analysis/index.affx) and gene ontology analyses performed using GO Browser (Affymetrix). Genes belonging to cellular metabolism, response to stress and stimulus, protein, carbohydrate and lipid metabolism, regulators of cell-cycle and DNA transcription, cell communication and development, and morphogenesis were uniquely changed in the 160 EtOH (EtOH and undernutrition) group (Fig. 3A–DGo).


Figure 3
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  		Figure 3. Gene ontology analysis of mRNA expression changes by (A–B) EtOH alone or (C–D) EtOH and undernutrition compared to their respective controls (see Materials and Methods, Study 4, for details). Genes were filtered based on absence/presence criteria, minimum 2-fold change and P ≤ 0.05 in either comparison. Four hundred genes passing these criteria were queried and analyzed using the GO Browser at Affymetrix online data analysis resource NetAffx (http://www.affymetrix.com/analysis/index.affx). Up- and downregulated genes are analyzed separately. Numbers in parentheses denote numbers of genes queried in GO Browser.

 
Correlation-based hierarchical clustering of 194 genes with known biological functions resolved the affected genes into 7 clusters (Fig. 4Go, clusters numbered 1 through 7). Correlation coefficients of changes are given on the left of the dendrogram. Complete gene names can be found in Table 6Go or queried on NetAffx (http://www.affymetrix.com/analysis/index.affx). Genes in clusters 1, 4, and 7 were significantly changed (decreased in 1 and 4 and increased in 7) uniquely in the EtOH 160 group, while remaining unchanged in the EtOH 220 group. On the other hand, all genes in clusters 3 and 5, and some genes in cluster 6, were changed uniquely by EtOH alone and not affected by the EtOH plus under-nutrition. Genes in cluster 2 show a distinct reversal of expression profile in the EtOH 220 and 160 groups.


Figure 4
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  		Figure 4. Cluster analysis of hepatic gene expression profiles in EtOH-fed pregnant dams with either 220 kcal/kg3/4/day or 160 kcal/ kg3/4/day. One hundred and ninety-four genes that showed significant change (± 2-fold, P ≤ 0.05) in either group and had known biological functions (based on annotations from gene ontology biological function, NetAffx, Affymetrix) were selected. Correlation-based hierarchical clustering was performed using Cluster software and data presentation done using Treeview. The gene expression analysis was carried out as described in Materials and Methods. The values represent fold change of each gene transcript in the EtOH-fed group compared to the respective control at the same caloric intake. Gene expression changes are depicted as intensity of color, increase (red), decrease (green), and no change (black). The left columns of the heat map (E + U-nutrn) represent EtOH-induced changes in undernutrition, while the right column (E) represents changes due to EtOH in the adequately fed dams. The dendrogram on the left of the cluster shows relatedness of gene expression change, shorter branches representing closer relationship between gene expression changes. Genes were resolved into seven clusters, with cluster numbers given on the top left side of the cluster diagram based on their gene expression changes. Correlations of their changes are given on the left of the dendrogram.

 

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  		Table 6. Microarray Analyses of EtOH and EtOH-Nutrition Interactionsa
 
A more detailed classification of gene expression changes as based on known biological functions is represented in Table 6Go and discussed below.

Apoptosis and Cell Proliferation.
EtOH combined with undernutrition appeared to shift the status toward a proapoptotic and antiproliferative state. Transforming growth factor-ß inducible early growth response, a primary response for TGF-ß, mimics TGF-ß and regulates differentiation and proliferation and induces apoptosis in many cell types. Proliferative signals such as interleukin-18 (IL-18), IGF-1, growth hormone receptor, and fyn-related kinase were significantly decreased in the 160 EtOH group. Further, met protooncogene (receptor for hepatocyte growth factor), IL-1ß, and annexin-1 were also decreased in the 160 EtOH group. Casein kinase 2, a ubiquitous and highly conserved serine/threonine kinase with important roles in cell growth, proliferation, and suppression of apoptosis, was also decreased in the EtOH 160 group (Table 6Go).

Regulators of DNA Transcription.
Nutrient sensing transcription factors 3-hydroxyanthrinilate 3,4-dioxygenase (involved in tryptophan metabolism), CAAT-enhancer binding protein-ß (C/EBP ß, glucose signaling), and sterol-regulatory element binding protein-1 (SREBP-1, fatty acid biosynthesis) were increased in the 160 EtOH group. The Kruppel-like family member, basic transcription element binding protein (BTEB-1), that regulates expression of pregnancy associated genes was increased in the EtOH 160 group. Nuclear receptor Nr3c1 (glucocorticoid receptor) was decreased specifically in the 160 EtOH group, suggesting changes in glucocorticoid signaling. The stress response transcription factor, early growth response 1, was induced equally in both the 220 and 160 EtOH groups (Table 6Go).

Lipid and Glucose Metabolism.
Undernutrition resulted in a robust increase in gluconeogenic genes. EtOH combined with undernutrition further induced liver serine dehydratase mRNA to 27-fold, compared to the 4-fold induction by EtOH alone. Gene expression of ketohexokinase, slc37a4 (glucose 6-phosphate translocase), and enolase 1 was significantly increased in the EtOH 160 group, indicating increased gluconeogenesis. Genes responsible for biosynthesis of triglycerides (scd1, scd2, FASN) were induced in the EtOH 160 group compared to the 160 controls, suggesting that the acetate generated from the oxidation of EtOH was shunted into fatty acid biosynthesis (Table 6Go).

Electron Transport and Stress Response.
The majority of genes belonging to electron transport were decreased in the EtOH 160 group, while either remaining unchanged or increased in the EtOH 220 group. The rate-limiting step in glutathione biosynthesis, glutamate cysteine ligase, was decreased 3-fold in the 160 EtOH group. Several genes involved in mediating cellular stress response, including hsp70, calpastatin, calnexin, oxidation resistance 1, ubiquitin-conjugating enzyme, and {alpha}2-macroglobin, were decreased in the EtOH 160 group (Table 6Go).

Protein Transcription and Intracellular Transport.
Gene expression of critical kinases such as c-AMP activated protein kinase (AMPK), protein kinase C (PKC), and cAMP regulated nucleotide exchange factor were decreased, accompanied by increases in gene expression of the protein phosphatases Dusp1 and Ptpn21. Significant decreases in mRNAs for at least 8 different factors associated with ribosomal protein synthesis and translation were observed. Genes associated with intracellular transport such as Fabp7, Bet1, Slc25a1 (mitochondrial carrier), Slc16a1 (monocarboxylate transporters), Slc6a1 (GABA transporter), syntaxin-5a, and ralA binding protein were uniquely downregulated in the EtOH 160 group (Table 6Go).

Gene expression of several genes observed in the microarray analyses were confirmed via real-time RT-PCR analyses (Table 7Go). Linear regression analysis of fold changes of 14 genes (Table 7Go, Fig. 6C and DGo) obtained via real-time RT-PCR compared to microarray-based gene expression analyses revealed highly significant positive correlation (P < 0.001, r2 = 0.59, slope = 1.073). Among genes used to confirm microarray results, several genes (rPER2, glucK, PEPCK, PrlR, MT1, rev-erbA-{alpha}, and rev-erbA-ß) altered in the EtOH 160 group, compared to the control 220 group, were mainly due to the undernutrition effect (Table 7Go). In contrast, metallothionein 2 (MT2) mRNA expression was increased (~190%) in the EtOH 160 group compared to the 160 control, revealing a significant EtOH-undernutrition interaction.


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  		Table 7. Real-time RT-PCR Analyses of EtOH and EtOH-Nutrition Interactionsa
 

Figure 6
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  		Figure 6. Hepatic (A) STAT5a and (B) STAT5b protein levels, and (C) STAT5a and (D) STAT5b mRNA levels in pregnant dams fed control diets or diets containing 12 g of EtOH/kg/day throughout gestation (GD5–GD15) with either 220 kcal/kg3/4/day or 160 kcal/kg3/4/day. Data represent means ± SEM (n = 3–8). Top panel shows representative blots. Left and right bottom panels show graphical densitometric quantitation of STAT5a and STAT5b proteins, respectively. *, P < 0.05 versus 220 control group; #, P < 0.05 versus 220 EtOH group.

 
The resulting set of 194 genes from microarray analyses with known biological functions (Table 6Go) was further analyzed using PathwayAssist software (Ariadne Genomics) to identify relationship of genes and known signaling pathways using curated databases. Of the 194 genes queried, 84 genes showed connectivity to other known genes. The top three genes (showing maximum connectivity) were AKT1 (a serine theornine kinase), IL-1ß (cytokine), and IGF-1 (growth factor), all with greater than 2000 interactions with known proteins and signaling molecules. Since IGF-1 has a bona fide role in fetal growth and development and can itself act as a regulator of AKT-1 and IL-1ß, we decided to further examine IGF-1 protein levels and signaling.

IGF-1 Protein and Gene Expression in Undernourished Pregnant Dams Fed EtOH.
To further investigate the mechanisms of intrauterine growth retardation we assessed maternal IGF-1 status, thought to be an important regulator of fetal growth (19–21). Consistent with the fetal growth data, serum IGF-1 concentrations were not significantly reduced by EtOH in the 220 group or by undernutrition alone. However, compromised nutrition in combination with EtOH led to a remarkable decrease (P ≤ 0.005) in free serum IGF-1 concentrations (~90%) compared to control 220 group (Fig. 5AGo).


Figure 5
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  		Figure 5. Maternal (A) serum-free insulin-like growth factor-1 (IGF-1) protein, (B) hepatic IGF-1, (C) IGFBP-1, (D) IGFBP-2, (E) IGFBP-4 mRNA expression normalized to 18S ribosomal RNA gene expression from pregnant dams fed diets containing 12 g of EtOH/kg/day throughout gestation (GD5–GD15) with either 220 kcal/kg3/4/day or 160 kcal/kg3/4/day (see Materials and Methods, Study 4, for details). Data represent means ± SEM (n = 4–9). *, P < 0.05 versus 220 control group; #, P < 0.05 versus 160 control group; @, P < 0.05 versus 220 EtOH group.

 
Changes in hepatic IGF-1 mRNA levels seen in microarrays were confirmed using real-time RT-PCR. Consistent with serum IGF-1 levels, the EtOH-treated 220 group did not have decreased IGF-1 gene expression (Fig. 5BGo). In contrast, undernourished EtOH-fed animals had 40% lower IGF-1 mRNA level compared to the TEN 160 group (P ≤ 0.05). Undernutrition alone did not change IGF-1 mRNA levels (Fig. 5BGo). Expression of 18S ribosomal RNA, assessed as a housekeeping gene, did not change (data not shown).

IGFBP-1, -2 and -4 Gene Expression in Undernourished Pregnant Dams.
Consistent with earlier reports, maternal hepatic mRNA of both IGFBP-1 and -2 were increased ~4-fold due to EtOH in the 220 group (Fig. 5C and DGo). Undernutrition markedly induced IGFBP-1 and -2 gene expression to almost 6- to 9-fold higher than the 220 group (Fig. 5C and DGo). A more modest (~1.8-fold) but significant (P ≤ 0.05) increase was observed in IGFBP-4 mRNA both in the control and EtOH-fed undernourished dams (Fig. 5EGo).

EtOH-Undernutrition and STAT5a and -5b Signaling.
IGF-1 synthesis is known to be controlled in a growth hormone–dependent manner. Recent evidence suggests that both STAT5a and STAT5b are critical for IGF-1 gene expression (22). Since undernourished pregnant dams fed EtOH had lower IGF-1 gene expression, we examined the status of hepatic STAT5a and-5b (Fig. 6A and BGo). Western blot analyses revealed that EtOH-fed undernourished dams had ~75% lower levels (P ≤ 0.05) of STAT5a compared to control 220 group. Undernutrition by itself (without EtOH) also decreased STAT5a levels (P ≤ 0.05). Pregnant dams fed EtOH in the 220 group did not have significant decreases in STAT5a levels. Modulation of STAT5b levels was similar to that of STAT5a. Undernourished EtOH-fed rats produced lower levels of STAT5b compared to the 220 groups, with or without ethanol (P ≤ 0.005). Undernutrition by itself did not significantly change STAT5b levels.

We further investigated whether undernutrition and EtOH affect STAT5a and -5b gene expression. Consistent with protein changes, undernutrition by itself (without EtOH) decreased STAT5a mRNA levels compared to 220 control (P ≤ 0.05, Fig. 6CGo). Undernourished EtOH-fed rats had lower levels of STAT5b mRNA compared to 220 controls (P ≤ 0.05, Fig. 6DGo). These data suggest that undernutrition and EtOH produced STAT5 inhibition, which may at least partly be mediated at the transcriptional or the pretranslational level.

IGF and IGFBP Changes in Fetuses from EtOH-Exposed Dams.
We measured free serum IGF-1 in GD20 fetuses from dams fed EtOH (12 g/kg/day) in the 220 or 160 groups. Neither the serum IGF-1 protein (Fig. 7AGo) nor hepatic mRNA for IGF-1 were significantly different in any of the groups, but there was a tendency for suppression of IGF-1 mRNA (Fig. 7BGo). IGF-2 mRNA was significantly induced by combination of undernutrition and ethanol compared to both the 220 control and ETOH groups (Fig. 7CGo). Serum IGF-2 protein was not measured, since there is no commercially available rat assay. These findings suggest differential regulation of IGF-1 in maternal versus fetal compartments. IGFBP-1 and -2 mRNA expression was also selectively induced (~2-fold) in the livers of undernourished-EtOH fed fetuses (P < 0.05, Fig. 7D and EGo).


Figure 7
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  		Figure 7. (A) Fetal serum-free IGF-1, (B) fetal hepatic IGF-1, (C) IGF-2, (D) IGFBP-1, and (E) IGFBP-2 mRNA expression in fetuses from pregnant dams fed diets containing 12 g of EtOH/kg/day throughout gestation (GD5–GD20) with either 220 kcal/kg3/4/day or 160 kcal/kg3/4/day (see Materials and Methods, Study 4, for details). Blood was pooled from all fetuses per dam. Data represent means ± SEM (n = 4–9). *, P < 0.05 versus 220 control group; #, P < 0.05 versus 160 control group; @, P < 0.05 versus 220 EtOH group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Nutrition plays an important role in assuring a normal pregnancy. Although it has been known for years that there is an important interaction between EtOH and nutrient intake during pregnancy and fetal development, the standard rodent models have not been adequate to study the role of maternal nutrition on the detrimental effects of EtOH on the growing fetus. The TEN model provides a unique opportunity to study the effects of nutrition and EtOH separately and in concert with each other. Using the TEN system, we have demonstrated that ethanol-induced fetal growth retardation is potentiated by undernutrition. The present studies examine the mechanisms involved in the interaction of EtOH and nutrition on in utero growth effects.

The continuous intragastric EtOH infusion model is characterized by well-defined pulses in BECs and UECs that occur with a frequency of 6–7 days (11). EtOH-dependent induction of hepatic ADH-1 underlies the cyclic behavior of blood and urine EtOH concentrations (23). Upon chronic EtOH infusion, hepatic ADH-1 is transcriptionally induced, in part via positive regulation by C/EBP ß (LAP isoform) and EtOH-mediated suppression of SREBP-1 (a negative regulator of ADH-1 transcription) (16, 24). In the present study, pregnant dams receiving EtOH diets at 220 kcal/kg3/4/day did not show an induction in ADH-1 mRNA. The primary reason for the lack of ADH-1 induction may be related to the low UECs (and BECs) attained in the pregnant dams. EtOH-mediated induction of ADH-1 is closely tied to the dose and resultant circulating levels of EtOH. Chronic infusion studies suggest that the threshold for ADH-1 induction is at UECs > 300 mg/dl. Pregnancy enhances whole-body EtOH clearance primarily via increased gastric first-pass metabolism in a manner independent of caloric intake (13). Consistent with earlier reports (11, 13), pregnant dams in the present study also showed increased EtOH clearance and UECs below the threshold for ADH-1 induction.

Several possible mechanisms underlying EtOH-nutrient interactions have been explored in our studies. It is clear that EtOH metabolism itself is decreased in the undernourished state, since undernourished dams had greater mean UECs and BECs compared to pregnant rats fed NRC-recommended calories and infused the same EtOH dose (g/kg/day). In addition, impairment of EtOH metabolism directly translated into greater fetal exposure to EtOH, as evidenced by the higher amniotic fluid and blood EtOH concentrations. Hepatic ADH-1, which was significantly lower in the undernourished pregnant EtOH-fed dams, is responsible for about 75%–90% of in vivo EtOH metabolism (25, 26). The resulting metabolite, acetaldehyde, is rapidly converted to acetate via the ALDH system in the mitochondria. ALDH activities were not affected by undernutrition in pregnancy. Hence, it seems reasonable that the lower rate of EtOH metabolism by ADH-1 in undernutrition significantly increases the potential of fetal toxicity by increasing the EtOH exposure to maternal and fetal tissues. Undernutrition and fasting have previously been shown to decrease ADH-1 activity (27–29). Although the mechanisms of how nutrition modulates ADH-1 levels are not clear, data from the present studies show a decrease of ADH-1 mRNA due to under-nutrition. Several hormones, including growth hormone (GH), thyroid hormone, and androgens that affect ADH mRNA levels (30), might be modulated by nutrition. Several transcription factors, including C/EBP ß, SREBP-1 (16, 26), and STAT5b (31), have been reported to be regulate ADH-1 transcription. C/EBPs and SREBPs are transcription factors that play important roles in energy metabolism and can be modulated by nutritional status (32). Further, data from our microarray analyses (Table 6Go, under Regulators of DNA transcription) showed induction of SREBP-1c, a negative ADH-1 regulator, in undernourished EtOH-fed dams. Also, decreased STAT5b levels (a positive regulator of ADH-1) were observed in the undernourished EtOH-fed dams, consistent with lower ADH-1 levels. Hence the hypothesis that undernutrition impairs EtOH metabolism by decreasing ADH-1 levels via altered C/EBPs, SREBP-1c, or STAT5b signaling is worthy of further investigation.

To further understand the molecular mechanisms of EtOH-undernutrition interactive toxicity, we utilized micro-arrays to generate mechanistic leads. Although expression of a large number of genes was altered, a closer examination of the data revealed that IGF-1 was decreased by EtOH during undernutrition and several genes across different functional families known to be regulated by IGF-1 were also altered, making it a candidate for further investigation. For example, casein kinase activity, which is directly increased with IGF-1, was decreased in correlation with IGF-1 (33). Expression of other IGF-1 regulated genes such as AMPK, MAPKK2, PKC-ß, and several members of the ribosomal proteins and translation elongational factors involved in protein synthesis were also decreased. Furthermore, gene expression of the growth hormone receptor and IGFBP-3 was significantly altered so as to decrease IGF-1 signaling.

Several growth factors, including IGF-1, are important in placental and fetal development (19–21). Exposure to EtOH decreases circulating IGF-1 levels in both animal models and humans (34, 35). EtOH also inhibits the autophosphorylation of the IGF-1 receptor (36). In addition, EtOH feeding via oral liquid diets to pregnant dams (which also decrease food intake) has been shown to result in ~50% reduction in serum IGF-1 levels (37). These data are consistent with our findings that show ~50% decrease in hepatic IGF-1 mRNA and freely dissociable IGF-1 protein levels in undernourished EtOH-fed dams. The liver is the major source (75%–80%) of IGF-1 in the general circulation (38, 39). It is likely, therefore, that a 50% decrease in hepatic IGF-1 mRNA in the undernourished EtOH dams is responsible for the reduced serum IGF-1 levels. However, it is not clear what degree of IGF-1 decrease in the circulation of the dam is required for fetal growth retardation.

IGF binding proteins (IGFBPs) control the minute-to-minute bioavailability and tissue-targeting of IGF-1 (40). Circulating IGF-1 is mainly found complexed to either IGFBP-3 or IGFBP-1 (41). Consistent with our data, previous studies have shown induction of IGFBP-1 by EtOH (18). This increase in IGFBP-1 may be mediated by tumor necrosis factor-{alpha} (41). It is interesting to note that IGFBP-1 and -2 levels were induced following reduced caloric intake. IGF-1 gene expression is controlled by growth hormone (GH) levels (18). Chronic EtOH-infusion decreases GH levels (10) and suppresses STAT5b expression in male rats (43). Since the present studies demonstrated decreased IGF-1 gene expression, we investigated GH signaling and found that the combination of EtOH and undernutrition decreased STAT5b protein and mRNA in pregnant rats. Since Woelfle et al. (22) recently implied that IGF-1 gene expression is acutely controlled by GH through STAT5b, our data suggest that reduced STAT5b may mediate the decrease in IGF-1 gene expression. Srivastava et al. (44), using rats overexpressing bovine GH, reported that EtOH-induced suppression of IGF-1 gene expression was dependent on post-GH-receptor signaling mechanisms. Our data are consistent with these findings and suggest that the post-GH-receptor events, such as decreased STAT5b activation or transcription, may be involved. To our knowledge, this is the first report to demonstrate interactive effects of EtOH and nutrition on STAT5 mRNA levels. However, the mechanisms behind EtOH-induced decreased STAT5b mRNA levels in undernourished EtOH dams require further study.

The present data address the question of whether EtOH and undernutrition affect fetal circulating (serum) IGF-1 levels in relation to fetal growth. Elegant studies by Yakar et al. (39) suggest that even in the complete absence of maternal hepatic IGF-1, other sources of IGF-1, such as the adipose tissue, kidney, and uterus, can sustain normal fetal growth. The evidence on modulation of fetal IGF-1 levels by EtOH is split, as Singh et al. (45) found modest (17%) decrease in serum IGF-1 in fetuses of EtOH-fed dams and Mauceri et al. (46) found no change in circulating fetal IGF-1 levels. Our data are consistent with the findings of Mauceri et al. (46). However, in agreement with the modulation of IGFBPs in the maternal liver, fetal livers also showed an increase in IGFBP-1 and -2. These data raise the possibility that increased IGFBP-1 levels in the dam and the fetus may cause growth retardation in an IGF-1-independent fashion. This possibility has been studied in transgenic-mice overexpressing hIGFBP-1, specifically in the maternal decidua (47).

IGF-1 and -2 and IGFBPs have important roles in feto-placental development and growth (48, 49). Chronic EtOH exposure has significant adverse effects on placental morphology and function. Abnormal trophoblast invasion in decidua, nuclear disorder in trophoblastic giant cells, derangement in the labyrinth, changes in blood sinuses, and extracellular matrix deposition have been recently described (50). Further, EtOH inhibits IGF-1-stimulated amino acid uptake in human placental trophoblasts (51). Since fetal growth retardation in the EtOH-undernutrition combination occurs in the absence of changes in fetal IGF-1, this is consistent with the hypothesis that EtOH-induced fetal growth retardation occurs primarily as a result of impaired placental development and transport of nutrients resulting primarily from disruption of maternal GH-IGF axis.

In addition, other mechanisms involving induced MT-2 expression in the 160 EtOH group, as observed by real-time RT-PCR, may alter maternal-fetal zinc homeostasis and lead to fetal growth retardation. Metallothionein overexpression protects against EtOH-induced liver damage, oxidative stress, and apoptosis (52, 53). Zinc treatment in MT-knockout mice also attenuates EtOH-induced liver necrosis via its antioxidant properties (54, 55). Further, in a murine model of intraperitoneal EtOH challenge on GD8, fetal zinc transfer was impaired in WT but not in MT-knockout mice, linking MT-induction to impairment of maternal-fetal zinc transfer (56). Using the same model, supplementation of zinc ameliorated both overt EtOH-induced fetal tertogenicity and spatial memory impairments in the offspring (57, 58). It is likely that the marked induction of MT genes in 160 EtOH group (over that of the undernutrition effect itself) has important mechanistic implications that remain to be examined.

In conclusion, we report marked enhancement of EtOH metabolism in pregnancy, an effect abolished by under-nutrition due to suppression of maternal ADH-1. Consistent with higher UECs, fetuses from undernourished EtOH-fed dams suffered higher toxicity. Hepatic IGF-1 mRNA and serum IGF-1 levels were downregulated in undernourished EtOH-fed dams accompanied with increased IGFBP-1 and -2 gene expression and decreased STAT5b protein and gene expression. Undernutrition appears to be a significant risk factor in EtOH-associated fetal growth retardation, which may be in part due to the altered maternal hepatic gene expression profile. The data suggest that optimal nutritional management during pregnancy may be an effective way to reduce the penetrance of fetal alcohol toxicity in high-risk individuals.


   Acknowledgments
 
We thank Brandi Yarberry, Janelle C. Johnson, Ying Chen, Matt Ferguson, Tammy Dallari, Pamela Treadaway, and Michele Perry for their technical assistance.


   Footnotes
 
These studies were supported in part by R01AA012819 to M.J.J.R.

Received for publication January 9, 2006. Accepted for publication March 24, 2006.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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