Pair-fed Control Group Research Articles

Prenatal administration of methadone in the rat: Acoustic startle amplitude and the rest-activity cycle at 30 days of age

Two doses of methadone were administered by osmotic minipump from Day 8 of gestation through parturition, a dosing technique previously shown to produce physical dependence in the dams. A pair-fed control group received sterile water via minipump and was allowed to eat and drink only the amount consumed by the high-dose group on the same gestation days. A nontreated control group was left undisturbed during pregnancy. All treated and control litters were fostered at birth to untreated dams. The effects of methadone on maternal and offspring toxicity replicated our previous findings. At 29–31 days of age each treated and control animal was tested either for changes in acoustic startle amplitude or the rest-activity cycle. Methadone treated offspring were no different from the controls on either measure. These findings support the hypothesis derived from our earlier research that prenatal exposure to methadone produces a prolonged but transitory opioid abstinence. This is evidenced by increased startle amplitude and a disturbed rest-activity cycle that peaks at approximately 3 weeks of age. We demonstrate that these effects are no longer evident at 4 weeks of age. Together, these findings define a state of hyperexcitability in the young rat that resolves by 1 month of age. This transitory state parallels clinical descriptions of human infants undergoing opiate abstinence.

Effects of chronic ethanol consumption on gestation and lactation in rats.

Chronic consumption of ethanol during pregnancy and lactation may lead to abnormalities in the fetus or infant. A group of female Wistar rats was submitted to ethanol treatment over a period of a month. A pair-fed control group received sucrose solution isocaloric to ethanol and the control group received water "ad libitum." Afterward, the females were mated with males over a period of 20 days. At birth, each litter was maximized to eight pups and the remaining ones were decapitated to remove the fetal blood and brains. No significant difference was observed in fetal body and brain weight at birth. During lactation the ethanol and pair-fed groups gained less weight than the control group. After weaning, their weight became similar. Fetal blood glucose levels were decreased in the ethanol-treated group. One hundred percent of the pair-fed and control females delivered live fetuses at term and all survived; only 40% of the females in the ethanol group delivered, and one pup did not survive. Chronic ethanol treatment pointed to a possible reduction in the fertility. It seems likely that the change in body weight of ethanol-fed dams was caused by undernutrition.

Effect of Postnatal Ethanol Exposure on Expression of Differentiation Antigens of Murine Splenic Lymphocytes

Ethanol is a recognized immunosuppressive agent in the chronic alcoholic. However, the effects of ethanol exposure on the developing immune system have not been extensively investigated. This study evaluated the effects of early postnatal ethanol exposure, via breast milk, on splenic lymphocyte differentiation antigen expression in offspring reared by ethanol-fed mice. Maternal mice were fed a liquid diet containing 20% ethanol-derived calories during pregnancy (E-P), pregnancy and lactation (E-PL), or lactation (E-L). Ad libitum-fed (C) and pair-fed (PF) control groups, fed a control liquid diet, were included. Expression of differentiation antigens on splenic lymphocytes from 21-day-old offspring reared by females in 1 of the 3 ethanol exposure conditions was evaluated by flow cytometry. Offspring reared by E-P females had similar numbers of splenic lymphocytes as offspring reared by C and pair-fed during pregnancy (PF-P) females. In contrast, offspring reared by E-PL and E-L females had fewer splenic lymphocytes than both PF-PL and PF-L (respectively), and C offspring. The number of Thy 1.2+, CD4+, CD8+, and IgG+ (B-cell) splenic lymphocytes was reduced in E-PL and E-L offspring compared with PF and C offspring. E-P offspring had fewer CD4+ and IgG+ splenic lymphocytes than C, but not PF-P, offspring. The percentage of Thy 1.2+ splenic lymphocytes was significantly reduced among E-PL and E-L offspring compared with PF-PL and PF-L (respectively), and C offspring.(ABSTRACT TRUNCATED AT 250 WORDS)

MATERNAL ETHANOL CONSUMPTION INDUCES TRANSIENT COMPENSATORY HYPERPLASIA OF DEVELOPING CARDIAC TISSUE IN THE NEONATAL RAT

The effect of continuous exposure to ethanol in utero and postpartum on growth and cell division in developing cardiac tissue was studied in neonatal Fischer rats. Pregnant and lactating females were maintained on three dietary regimens; a control group fed rat chow ad libitum, an experimental group receiving an ethanol-containing (6% by volume) liquid diet, and a pair-fed control group, which received an isocaloric amount of control liquid diet. At days 1, 5, and 10 postpartum, five litters of pups from each control and experimental group were sacrificed and the body weights, heart weights, heart-to-body weight ratios, and mitotic frequency of the ventricular myocardium were measured. When compared to either group of controls, pups continuously exposed to dietary ethanol expressed significantly (P < 0.01) lower body weights. Pups maintained by the pair-fed females had significantly (P < 0.01) lower body weights at days 5 and 10 than pups maintained by the chow-fed females, indicating a pair-fed effect of suboptimal nutrition of the model. As the pups developed, the heart weights of pups maintained by the chow-fed females became progressively greater (P < 0.01) than the heart weights of pups maintained by the pair-fed and ethanol-fed females, which expressed no weight difference. The reduction of heart weight present in the ethanol-fed and pair-fed pups represents a pair-fed effect of suboptimal nutrition and not an obvious effect of exposure to dietary ethanol. The ratio of heart weight to body weight and mitotic frequency were significantly greater (P < 0.01) in 1- to 5-day-old pups exposed to ethanol. Following day 5, these parameters decreased and approached the control values. This indicates that growth of cardiac tissue is not suppressed in the 1- to 5-day-old rat pups exposed continuously to dietary ethanol. These observations further suggest the presence of a mechanism intrinsic to the heart which can provide stage-dependent protection from the adverse effects of ethanol during early development. The decline in heart weight to body weight ratios and mitotic frequency in pups of ethanol-fed females also suggests that ethanol may initiate suppression of the growth of cardiac tissue or may incur stage-dependent injury during the later stages of development. The possible mechanism of this stage-dependent protection during early neonatal development is an increased mitotic activity of the cardiac myocytes.

Impact of chronic alcoholism on the aging rat: changes in nutrition, liver composition, and mortality.

The adverse effects of chronic alcohol consumption (mean 6.68 g/kg/d) were assessed in 150 male Sprague-Dawley rats over their life span (25 months). Evaluations were performed at 2, 3, 8, 13, 19, and 25 months of age for changes in nutrition status, biochemical tests for liver injury, compositional changes in liver, and hepatic regenerative capacity. In spite of nearly identical caloric intake, alcohol treatment was associated with nutritional levels 10-30% lower than controls. Maximal changes were observed at the two extremes of ages (2-3 months and 19-25 months). Hence, a nutritional contribution to other adverse changes could not be excluded. Fatty compositional increases (triglycerides) occurred early (5-fold increases after 1 month of treatment) then declined to levels only slightly above controls. Biochemical tests on sera for liver injury (AST and total bilirubin) were consistently higher with alcohol treatment. Regenerative capacity measured by [3H]thymidine uptake after partial hepatectomy was initially elevated in the alcoholic then rapidly declined beyond 7 months of age. In control animals, an age-related decline was also observed but occurred later beyond 12 months of age. Consistent with these adverse effects, ethanol diet survival was poorer than the pair-fed control groups by 15% (median survival for alcoholics, 17 months vs. 20 months in controls.

Ethanol‐Induced Oxidative Stress and Nutritional Status

The ability of dietary ethanol, administered over a 10-day period, to elevate production rates of reactive oxygen species and to alter glutathione levels has been determined in both liver and cerebellum, a brain region known to be susceptible to ethanol-induced damage. Two groups of ethanol-consuming rats were used. One set of treated animals that received an all-liquid ethanol-containing diet experienced weight gain, and this gain was matched in a pair-fed control group. The other ethanol-treated group that had free access only to solid chow and water containing ethanol lost weight during the exposure period. The corresponding control group that received unlimited water and chow was allowed to gain weight normally. In animals that lost weight as a consequence of ethanol in the drinking water, evidence of oxidative stress was enhanced relative to that in animals receiving ethanol by way of the liquid diet. This latter set gained weight, despite higher blood ethanol levels than the group that lost weight. An excess prooxidant condition prevailed in the liver and cerebellum of the ethanol-dosed malnourished group. In the case of liver, this difference may relate to a greater lability of iron-containing proteins in the rats that experienced weight loss, leading to the appearance of low molecular weight iron in the cytosol.

Metformin and brown adipose tissue thermogenetic activity in genetically obese Zucker rats

The effect of chronic metformin treatment on brown adipose tissue thermogenetic activity was investigated in young genetically obese Zucker rats. The binding of [ 3H]GDP to brown adipose tissue mitochondria, expression of uncoupling protein mRNA in brown adipose tissue, weight gains and cumulative food intakes were measured in metformin (320 mg/kg orally for 12 days) -treated obese Zucker rats as well as in pair-fed — and in ad libitum — fed control obese rats. The weight gains were identically reduced in the metformin- and pair-fed control group compared to the ad libitum — fed rats. Metformin also significantly reduced cumulative food intake. The binding of [ 3H]GDP to brown adipose tissue mitochondria and the expression of uncoupling protein mRNA in brown adipose tissue were not modified by metformin. It is concluded that the weight gain reducing effect of metformin in obese Zucker rats is mainly due to reduced food intake and does not involve an effect of metformin on brown adipose tissue thermogenetic activity.

Prenatal administration of methadone in the rat increases offspring acoustic startle amplitude at age 3 weeks

Two doses of methadone were administered by osmotic minipump from Day 8 of gestation through parturition, a dosing technique previously shown to produce physical dependence in the dams. A pair-fed control group received saline via minipump and was allowed to eat and drink only the amount consumed by the high dose group on the same gestation days. A nontreated control group was left undisturbed during pregnancy. All treated and control litters were fostered at birth to untreated dams. The effects of methadone on maternal and offspring toxicity replicated our previous findings. At 21–23 days of age, acoustic startle amplitude was measured for each treated and control animal. Because prenatal methadone exposure resulted in reduced body weight at the time of testing, it was necessary to analyze startle amplitude using weight as a covariate. This analysis showed that the methadone treated offspring had a significantly enhanced mean startle amplitude compared with the controls. These findings support the hypothesis derived from our earlier research that prenatal exposure to methadone produces a prolonged state of CNS hyperexcitability similar to clinical descriptions of human infants undergoing opiate abstinence.

Cholesterol Exchange and Lateral Cholesterol Pools in Synaptosomal Membranes of Pair‐Fed Control and Chronic Ethanol‐Treated Mice

Most studies on effects of ethanol on membrane cholesterol have reported on changes in the total or bulk amount of cholesterol. Membrane cholesterol, however, can be described in terms of its kinetics and domains. The kinetics and size of lateral cholesterol exchangeable and nonexchangeable pools were examined in synaptosomes of pair-fed controls and chronic ethanol-treated mice. Effects of sphingomyelin, an exofacial leaflet phospholipid, that has been shown to affect cholesterol pools, were also examined. Radiolabeled small unilamellar vesicles were used to exchange cholesterol with synaptosomes. The total amounts of membrane cholesterol, phospholipid phosphorus, and the ratio of cholesterol to phospholipid did not differ between the pair-fed control and ethanol groups. In control mice, the rate constant (hr-1) and the t1/2 (hr) of cholesterol exchange were 0.065 +/- 0.001 and 10.7 +/- 0.25 (hr), respectively. The rate constant was significantly slower (0.053 +/- 0.001, p < 0.05) and the t1/2 significantly longer (13.33 +/- 0.58, p < 0.05) in synaptosomes of the ethanol group compared with the control group. The size of the exchangeable pool of cholesterol did not differ significantly between the two groups. Sphingomyelinase-induced hydrolysis of sphingomyelin significantly slowed cholesterol exchange with the largest effect in synaptosomes of the control group as compared with the ethanol group (p < 0.05). Hydrolysis of sphingomyelin had significantly (p < 0.05) less of an effect on cholesterol exchange in synaptosomes of the ethanol group. Membrane cholesterol can be described in terms of total content, transbilayer distribution, kinetics, and size of lateral pools.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatic one-carbon metabolism in early folate deficiency in rats

Glycine N-methyltransferase (GNMT) is inhibited by 5-methyltetrahydrofolate polyglutamate in vitro. It is believed to play a regulatory role in the synthesis de novo of methyl groups. We have used the amino-acid-defined diet of Walzem and Clifford [(1988) J. Nutr. 118, 1089-1096] to determine whether folate deficiency in vivo would affect GNMT activity, as predicted by the studies in vitro. Weanling male rats were fed on the folate-deficient diet or a folate-supplemented diet pair-fed to the deficient group. A third group was fed on the folate-supplemented diet ad libitum. Development of folate deficiency rapidly resulted in decreased levels of S-adenosylmethionine (SAM) and elevation of S-adenosylhomocysteine (SAH). The ratios of SAM to SAH were 1.8, 2.7 and 1.5 in the deficient group for weeks 2, 3 and 4 of the experiment, and the values were 9.7, 7.1 and 8.9 for the pair-fed control group and 10.3, 8.8 and 8.0 for the control group ad libitum fed. The activity of GNMT was significantly higher in the deficient group than in either of the two control groups at each time period. This was not due to increased amounts of GNMT protein, but reflected an increase in specific enzyme activity. Levels of folate in both the cytosol and mitochondria were severely lowered after only 2 weeks on the diet. The distribution of folate coenzymes was also affected by the deficiency, which resulted in a marked increase in the percentage of tetrahydrofolate polyglutamates in both cytosol and mitochondria and a very large decrease in cytosolic 5-methyltetrahydrofolate. The increased GNMT activity is therefore consistent with decreased folate levels and decreased inhibition of enzyme activity.

Erythropoietin metabolism and pharmacokinetics in experimental nephrosis.

We studied erythropoietin (EPO) metabolism, regulation, and pharmacokinetics in rats with nephrotic syndrome. Sprague-Dawley rats were randomized into nephrotic (puromycin-induced) and pair-fed control groups. Animals were studied at baseline and after induction of anemia or exposure to hypobaric conditions (32 cmHg). The nephrotic group showed a reduced hematocrit (P < 0.05), a significant urinary EPO excretion, and an inappropriately low plasma EPO. Induction of anemia and exposure to hypoxia resulted in a less pronounced elevation of plasma EPO in the nephrotic group than in the control group (P < 0.05). The blunted plasma EPO response to hypoxia in nephrotic animals was associated with a marked rise in urinary EPO excretion. Pharmacokinetic studies following intravenous injection of recombinant EPO, 100 U/kg, revealed a shorter plasma half-life (t1/2) (P < 0.05), larger apparent volume of distribution (P < 0.05), and greater clearance (P < 0.02) in the nephrotic group than in the controls. Estimated endogenous EPO production rate in nephrotic rats with severe anemia was significantly lower (P < 0.05) than that of equally anemic controls. Thus puromycin-induced nephrotic syndrome is associated with marked urinary loss of EPO, relatively depressed plasma EPO response to anemia and hypoxia, as well as reduced plasma t1/2, increased volume of distribution, and clearance of exogenous EPO.

Prenatal cocaine exposure in the laboratory mouse: Effects on maternal water consumption and offspring outcome

Pregnant mice were given 50 mg/kg cocaine HCl (1% solution, sc) once daily from gestation days 7 through 18 (sperm positive = day 0; term = day 19). Pair-fed and untreated control groups were also used. The pregnant cocaine-treated females showed normal weight gain and food consumption but had significantly increased water consumption. The cocaine-treated group had a significant increase in embryonic resorptions but no significant effects on stillbirths or postnatal mortality. The offspring of cocaine-treated females had significantly reduced birth weights and postnatal weight gains up to the age of 28 days. There was also a delay in their ear opening but not in other maturational milestones. Increased water consumption following cocaine treatment has been reported by other studies. We speculate that cocaine has a diuretic effect. We discuss the implications of this effect during pregnancy.

Effects of ethanol and carbon tetrachloride upon vitamin A status of rats.

The effects of ethanol and carbon tetrachloride (CCl4) upon tissue vitamin A, liver lipids, liver cytochrome P450 and hepatic morphology were investigated. It was anticipated that CCl4 treatment would have more severe effects upon vitamin A status because CCl4 provides greater hepatic injury than does ethanol. After a 2-week standardization feeding period, young male rats were divided into four groups. For 5 weeks one group of rats (n = 17) received ethanol in liquid diets (30% of calories) while another (n = 8) was exposed to CCl4 inhalation twice a week along with phenobarbital in the diet. All groups received the National Regulatory Commission recommended level for vitamin A. Comparison of ethanol and its pair-fed control group (n = 17) revealed: decreased hepatic vitamin A, no change in serum vitamin A, increased percentage of liver lipid, and cytochrome P450 with moderate fat accumulation in hepatocytes. Comparison of the CCl4-phenobarbital group with pair-fed controls (n = 8) showed: increased serum vitamin A, decreased hepatic vitamin A, increased cytochrome P450, marked hepatic fat accumulation, hepatic cell necrosis, and early cirrhosis. Thus, CCl4 (with phenobarbitol), which is a more potent hepatotoxin as evidenced by a more elevated cytochrome P450 and distorted liver morphology, not only reduced liver vitamin A, but also increased serum vitamin A. The extent of substrate and/or organ specificity remains to be elucidated.

Effects of β-carotene and retinol repletion on antibody production in vitamin A-deficient rats immunized with pneumococcal polysaccharide

Experiments were conducted in male Lewis rats to compare the efficacy with which β-carotene (βC) and retinol restore antibody production in the previously vitamin A-depleted rat immunized with pneumococcal polysaccharide (SSS-III). Weanling rats were fed a semipurified vitamin A-free diet for three weeks and were then repleted with either retinol or βC provided in various doses and by different routes. Pairfed control rats were fed the same diet with adequate vitamin A. When 45–46 days old, rats were immunized with SSS-III and antibody production was measured 5 d later. In rats beginning to manifest vitamin A deficiency, the splenic antibody response was reduced significantly in comparison to that of the pairfed control group as well as to groups repleted with retinyl palmitate or with βC [1500 retinol equivalents (R.E.) each]. When βC was provided in an aqueous dispersion and delivered either orally or intravenously, the splenic antibody response of both βC-repleted groups were similar to each other and both were significantly greater than the response of the vitamin A-deficient group. The antibody response of rats repleted intravenously with βC or retinol, provided in nutritionally equivalent amounts (1000 R.E.), was also similar in magnitude. These observations indicate that βC functions approximately equally well as retinol in enabling the previously vitamin A-deficient rat to respond to pneumococcal polysaccharide; however, βC did not show any superior effect to retinol in restoring this antibody response in the previously vitamin A-depleted rat.

Methyl Group Metabolism in the Pancreas of Folate-Deficient Rats

Several studies have suggested that the metabolism of one-carbon compounds may have a special role in the function of the exocrine pancreas. An amino acid-defined diet was used to produce folate deficiency in a group of male rats. These rats were compared with a group of rats pair-fed the same diet supplemented with adequate folate and with a third group fed the folate-supplemented diet with ad libitum access. Pancreatic folate concentrations were already severely depleted after 4 wk of feeding the deficient diet (0.95 +/- 0.10, 5.81 +/- 0.29 and 4.58 +/- 0.30 nmol/g for the deficient, pair-fed control and ad libitum-fed control groups, respectively). The level of folate present in the pancreas of nondeficient animals was second only to that reported for liver. Urinary amylase excretion by animals in the deficient group was higher than that by the other groups (245.5 +/- 21.9, compared with 181.9 +/- 14.5 and 195.3 +/- 10.9 units/mg creatinine for the deficient, pair-fed control and ad libitum-fed control groups, respectively) after 4 wk. The ratio of S-adenosylmethionine to S-adenosylhomocysteine was 18.6 +/- 1.6 and 14.5 +/- 1.0 after 4 wk for the ad libitum-fed control and pair-fed control groups, respectively, but was significantly lower at 6.3 +/- 1.1 for the deficient group. These results indicate a profound effect of folate deficiency upon methyl group metabolism of the pancreas and suggest that this may result in decreased pancreatic function.

Vitamin A deficiency and colonic electrogenic absorption and secretion in the rat.

The effects of vitamin A deficiency on electrogenic transport in the colon were examined in rats made vitamin A deficient at weaning by feeding a vitamin A deficient diet for 40 days. A pair fed control group was given the same diet but supplemented with soluble vitamin A in their drinking water. The basal and stimulated electrogenic secretory and absorptive functions of the muscle stripped proximal, mid, and distal colon were examined in vitro using the short circuit current (Isc) as the index of net ion transport. A significant increase in the basal and secretory Isc (mainly Cl-ions) induced by the cholinergic agonist bethanechol was observed in the mid-colon of the vitamin A deficient rats. In the distal colon, however, vitamin A deficiency caused a significant reduction in both the basal and secretory Isc response to bethanechol compared with the vitamin A supplemented pair fed control. Secretory Isc induced by dibutyryl cyclic adenosine monophosphate was not significantly altered by vitamin A deficiency. The condition abolished the response of the distal colon to luminal amiloride (0.1 mmol/l). Thyroid hormone induced reduction in the distal colonic response to aldosterone is implicated in this lack of response. This is the first experimental linkage between vitamin A action, the thyroid hormone and aldosterone on colonic function. The colonic changes induced by vitamin A deficiency, namely hypersecretion and a reduced electrogenic distal absorptive function, together with the previously described small intestine hypersecretion may be the underlying basis for the diarrhoea observed in human and animal vitamin A deficiency.

BENZODIAZEPINE METABOLISM IN ETHANOL-TREATED MALE RATS: USE OF PAIR-FED AND AGE-MATCHED CONTROLS

The effects of chronic moderate (15%) ethanol consumption and ageing on rat hepatic cytochrome P450 monooxygenase activities were examined using diazepam, nordazepam, d-benzphetamine, erythromycin, ethylmorphine and nitrosodimethylamine (NDMA) as substrates. In addition, the effects of moderate ethanol alone on the oxidation of metoprolol, morphine and temazepam were examined. Cytochrome P450 specific content increased significantly only in the 6-week ethanol-treated rats, and no changes in percentage liver to body weights were apparent in any of the ethanol-treated animals compared with pair-fed controls. Only cytochrome P450IIIA enzyme activities displayed age-related decreases, these being identified in the pair-fed animals. C3-hydroxylation of diazepam and nordazepam (36% of controls) and N-demethylation of erythromycin and ethylmorphine (58% and 64% of controls) were decreased in 6-week ethanol-treated animals, these effects being less pronounced in the 12, 24 and 48-week ethanol-treated groups. The decrease seen for diazepam and d-benzphetamine N-demethylation caused by ethanol consumption was approximately 80% of control groups for the duration of the treatment. NDMA and morphine N-demethylations were increased to 120% of control activities and metoprolol alpha-hydroxylase was increased to 140% of control activities at 6 weeks, whilst metoprolol O-demethylase activity remained unaltered. NDMA N-demethylase activity showed a two-fold induction at 24 and 48 weeks of ethanol treatment, compared with corresponding pair-fed control groups. These results support previous findings from this laboratory showing that the same or similar P450IIIA family isozymes are involved in the C3-hydroxylation of diazepam and nordazepam.(ABSTRACT TRUNCATED AT 250 WORDS)

Prenatal administration of methadone using the osmotic minipump: Effects on maternal and offspring toxicity, growth, and behavior in the rat

Two doses of methadone were administered by osmotic minipump from day 8 of gestation through parturition. A pair-fed control group received saline via minipump and was allowed to eat and drink only the amount consumed by the high dose group on the same gestation days. A nontreated control group was left undisturbed during pregnancy. All treated and control litters were fostered at birth to untreated dams. Naloxone challenge of the dams after parturition showed that drug treatment produced physical dependence. Methadone treatment reduced maternal weight gain but had no effect on either the frequency of resorptions or birthweight. Both doses of methadone increased perinatal mortality but only the high dose produced a decrement in postnatal growth. To examine the effects of methadone on the rest-activity cycle of the offspring, groups of three littermates from each of the treated and control groups were tested for an 8 h observation period on electronic activity monitors at 22 days of age. No behavioral effects were observed for either control group or the low dose methadone group. The high dose methadone offspring, however, spent less time resting, showed disrupted rhythmicity, and poor state regulation. These findings are discussed in relation to earlier studies using once per day methadone administration as well as clinical descriptions of infants undergoing opiate abstinence.

Prenatal ethanol exposure during the last third of gestation in rat reduces hippocampal NMDA agonist binding site density in 45-day-old offspring

The effect of ethanol exposure during different periods of prenatal or postnatal development on hippocampal N-methyl-D-aspartate (NMDA) receptor binding was studied in rat. Fetal rat pups were exposed to ethanol for different periods of time during gestation via maternal consumption of a 3.35% ethanol liquid diet. In a separate experiment, neonatal pups were fed 2.51 g ethanol/kg body weight/day from Postnatal Day (PD) 4 to PD 10 via intragastric feeding tube. These two ethanol administration paradigms produced average peak maternal and pup blood ethanol concentrations of 39 mg/dl and 57 mg/dl, respectively. At 45 days of age, offspring from each treatment group were sacrificed for measurements of hippocampal NMDA-sensitive [ 3H]-glutamate binding site density using in vitro radiohistochemical techniques. As observed previously, prenatal ethanol exposure throughout gestation resulted in NMDA-sensitive [ 3H]-glutamate binding site reductions in the apical dendritic field regions of dentate gyrus, hippocampal CA 1 and subiculum of dorsal hippocampal formation compared to the ad lib or pair-fed control groups. NMDA-sensitive [ 3H]-glutamate binding was not different than control in rats exposed to ethanol during the first half of gestation only. Prenatal ethanol exposure during the last half or the last third of gestation resulted in NMDA-sensitive [ 3H]-glutamate binding site reductions comparable to the binding site reductions observed in rats exposed to ethanol throughout gestation. Hippocampal NMDA-sensitive [ 3H]-glutamate binding site density in postnatal ethanol-exposed rats was not different than the suckling or gastrostomy control groups. These results suggest that the critical period of vulnerability to ethanol-induced reductions in hippocampal NMDA receptor binding occurs during the last third of gestation in rat, which, in terms of human hippocampal development, is a period of time roughly equivalent to the fifth and six months of human gestation.

The role of metallothionein induction and altered zinc status in maternally mediated developmental toxicity: Comparison of the effects of urethane and styrene in rats

We hypothesize that maternal metallothionein (MT) induction by toxic dosages of chemicals may contribute to or cause developmental toxicity by a chain of events leading to a transient but developmentally adverse decrease in Zn availability to the embryo. This hypothesis was tested by evaluating hepatic MT induction, maternal and embryonic Zn status, and developmental toxicity after exposure to urethane, a developmental toxicant, or styrene, which is not a developmental toxicant. Pregnant Sprague-Dawley rats were given 0 or 1 g/kg urethane ip, or 0 or 300 mg/kg styrene in corn oil po, on Gestation Day 11 (sperm positive = Gestation Day 0). These were maternally toxic dosages. As both treatments decreased food consumption, separate pair-fed control groups were also evaluated for effects on MT and Zn status and development. In addition, Gestation Day 11 rat embryos were exposed to urethane in vitro in order to determine whether urethane has the potential to be directly embryotoxic. Urethane treatment induced hepatic MT 14-fold over control; styrene treatment induced MT 2.5-fold. The MT induction by styrene could be attributed to decreased food intake, as a similar level of induction was observed in a pair-fed untreated control group. However, the level of MT induction by urethane was much greater than that produced by decreased food intake alone. Hepatic Zn concentration, particularly in the cytosol, was increased in the presence of increased hepatic MT concentration. Plasma Zn concentration was significantly decreased (approximately 30%) by urethane treatment, but not by styrene or food restriction (pair-feeding). Distribution of 65Zn to the liver of urethane-treated dams was significantly greater (by 30%), while distribution to embryonic tissues was significantly lower (by at least 50%) than in pair-fed or ad lib.-fed controls. Styrene treatment had no effect on 65Zn distribution. Urethane was developmentally toxic, causing an 18% decrease in fetal weight and a significant delay in skeletal ossification, but was not toxic to rat embryos in vitro. Styrene was not developmentally toxic. The changes observed after urethane treatment, namely substantial hepatic MT induction and altered maternal and embryonic Zn status, along with the lack of direct embryotoxicity of urethane in vitro, support the hypothesis that these maternal effects contribute to developmental toxicity. The lack of similar changes in styrene-intoxicated dams provides one explanation for its low developmental toxicity at maternally toxic dosages.