Effect of prenatal exposure to ethanol on intestinal development of rat fetuses.

BACKGROUND: Chronic alcoholism in pregnant animals and humans lead to general growth impairment in their offspring, which show multiple birth defects and delayed grown (fetal alcohol syndrome). Here we study the maturation of the intestine under the effect of chronic exposure to ethanol in utero together with associated malnutrition. METHODS: Lactase, acid beta-galactosidase, maltase, and alkaline phosphatase activity profiles were monitored in 18-, 19-, 20-, and 21-day-old fetuses from rats kept under three nutritional treatments before and during gestation: alcohol-treated (25% ethanol in drinking water), fiber-treated (50% cellulose-diluted diet) as a control of the malnutrition associated with chronic alcoholism, and control or normal diet. Serum corticosterone determination and lactase immunolocalization were carried out. To detect possible direct effects of ethanol during the period of mucosa development, intestinal explants from 18-, 19-, and 20-day-old control fetuses were cultured either in the basal medium alone or in a medium containing 25 mM ethanol for 72, 48, and 24 h of incubation, respectively. RESULTS: Following chronic ethanol exposure in utero, intestinal weight and brush-border protein content and the specific activities of lactase, acid beta-galactosidase, maltase, and alkaline phosphatase were significantly lower than those of nutritional controls. Organ culture results, under the assay conditions stated, did not show a direct effect of ethanol 25 mM on prenatal mucosal functionality. CONCLUSIONS: All these results suggest that maternal malnutrition is not primarily responsible for the impaired intestinal maturation in rat fetuses from alcohol-treated mothers; indirect effects of ethanol and/or its derivatives throughout embryo-fetal development could be necessary to promote this intestinal delay.

Liver alcohol dehydrogenase activity and ethanol levels during chronic ethanol intake in pregnant rats and their offspring.

The effects of chronic alcohol intake on the ethanol levels in body fluids (blood, amniotic fluid, and fetal intragastric content), hepatic alcohol dehydrogenase (ADH) activity, isoenzyme distribution, and hepatic zinc levels were studied in pregnant rats at term (19 and 21 days), in their offspring at fetal, perinatal, and weaned stages, and in adult virgin rats. Three experimental groups were studied: 1) the alcohol group received ethanol in drinking water (from 10% to 25% over 2 months), 2) the fibre diet group was undernourished on a hypocaloric diet, to assess the effects of malnutrition associated with chronic alcohol intake, and 3) the control group received no alcohol and normal diet. A gradient of increasing ethanol concentrations was found in fetal blood, amniotic fluid, and fetal intragastric contents with respect to maternal blood. A decrease in ADH activity was found in alcohol-consuming pregnant rats compared to controls. This was related neither to liver ADH isoenzyme distribution nor to changes in hepatic zinc levels. Chronic alcohol consumption in pregnant rats produced high ethanol accumulation in fetal fluids and changes in the liver ADH activity depending on the physiological situation (pregnancy, development, virgin state).

Ethanol elimination in alcohol-treated pregnant rats.

The effects of chronic intake of alcohol on ethanol elimination were studied in 20-day pregnant rats, in their foetuses, and in virgin rats. Experimental animals received ethanol in drinking water (from 10% to 25% in 2 months) (alcohol group), whereas controls drank only water. At the end of chronic exposure, alcohol dehydrogenase activity was determined in stomach and liver and cytochrome P-450 was measured in liver. In a complementary assay, the same experimental groups of rats were given an acute dose of ethanol (2 g/kg body wt, 25% w/v) either intragastrically or intraperitoneally, at the end of the chronic exposure, to determine first-pass ethanol metabolism and pharmacokinetic parameters of its elimination. Significant differences were found between alcohol and control groups for liver and stomach alcohol dehydrogenase activity in pregnant rats. Only in virgins did chronic alcohol treatment significantly increase the liver cytochrome P-450 content. In virgin rats, the first-pass metabolism of ethanol was lower in the alcohol group than in control. By contrast, in control rats, the first-pass of ethanol was lower in pregnant, than in virgin, rats. The metabolic rate of ethanol elimination (mg/kg body wt/hr) was clearly enhanced in alcoholic virgin rats, demonstrating that this model of chronic alcoholism induces metabolic tolerance to ethanol. In alcoholic pregnant rats, a surprisingly low theoretical volume of body ethanol distribution (55 ml/100 g body wt vs. 80 ml/100 g body wt in the other groups) masked their metabolic tolerance to ethanol. This preliminary study should be taken into account when evaluating the effects of chronic or/and acute alcohol intake during pregnancy on the circulating ethanol levels in foetuses and on future development of the foetal alcohol syndrome.

Permanent abnormal response to a glucose load after prenatal ethanol exposure in rats.

Postnatal development of the glucose and insulin balance in offspring of ethanol-treated and control rats has been studied. Newborn rats were separated from their mothers and placed with normal lactating, nonethanol-treated dams. Prenatal exposure to ethanol led to hypoglycemia on the first day of extrauterine life and a general tendency to hyperinsulinemia during the entire postnatal period studied. The glucose-tolerance test in weaned rats (30 days old) gave a greater and faster increase than controls in levels of both glucose and plasma insulin. At adult age (90 days) the response of blood glucose to an oral glucose load in offspring from ethanol-treated mothers was not different from that in offspring from controls, but the insulin response was higher. This abnormal insulin response, such a long time after the end of ethanol exposure, suggests either a permanent alteration in the pancreatic response, or a peripheral insulin resistance and/or differences in the rate of insulin degradation in these animals.

Free amino acid pools in rat tissues throughout the lactational period.

This is a descriptive work on the changes induced by lactation on the total and essential-free amino acid pools in plasma and several tissues of the rat (liver, kidney, skeletal muscle and skin). Plasma essential-free amino acid levels are increased at the peak of lactation. Significant changes have been also found for free amino acid pools in the liver and the skeletal muscle as well as in the kidney. Conversely, skin levels are very constant throughout lactation.

Effects of chronic ethanol consumption beginning at adolescence: increased numbers of dendritic spines on cortical pyramidal cells in the adulthood.

To increase our understanding of the effects of chronic ethanol consumption beginning at adolescence, 25% ethanol in drinking water (v/v) was administered daily to young rats aged 45-50 days for 5 months. Increased numbers of dendritic spines on the apical dendrite of layer V pyramidal neurons of the somatosensory cortex (U-Mann-Whitney test, P less than 0.01-0.05) were found in almost every 50-micron-long segment over a distance of 500 microns from the cell body in ethanol-treated rats at the age of 195-200 days when compared with age-matched controls. Although the mechanisms leading to this unusual finding are not know, it is suggested that impairment of the naturally occurring elimination of redundant synapses can not be ruled out.

Low intestinal lactase activity in offspring from ethanol-treated mothers.

Some aspects of small intestine maturation have been studied in the newborns from chronic ethanol-treated pregnant rats (25% ethanol in drinking fluid) immediately after birth (before suckling) and after 30 days of life. Litters delivered by mothers fed ad libitum with a standard diet diluted 50% with cellulose were used as a nutritional control. At birth, pups from ethanol-treated mothers showed significant decreases in total intestinal length and thickness, low total lactase activity and low somatostatin intestinal content. The intestinal alterations of these neonatal parameters are not present in newborns from mothers on fiber-diluted diet. From delivery, pups from different experimental groups were nursed by normal lactating dams. At 30 days of age neither of those parameters differed among the groups. We propose that the low levels of total lactase activity in newborns from alcoholic mothers, that are a consequence of a lower intestinal mucosa content, are a direct effect of ethanol in utero on the fetal gastrointestinal system.

High liver lipoprotein lipase activity in hyperlipemic developing rats from undernourished pregnant mothers.

To study the potential relationship between circulating triacylglycerol (TAG) levels and lipoprotein lipase (LPL) activity in the newborn rat liver, pups from undernourished or normal control mothers were nursed by normal dams, and studied at 0, 1, 15 or 30 days of age. Plasma TAG levels and liver TAG concentration increased more in pups from undernourished mothers than they did in controls. At birth, liver LPL activity was similarly high in both groups but, whereas in controls it decreased progressively after birth, in pups from undernourished mothers it remained stable until 15 days of age. Results suggest that the hypertriglyceridemia present in pups from undernourished mothers may be responsible for the sustained high LPL activity in their liver which may enhance the hepatic uptake of circulating TAG.

Morphological recovery of hippocampal pyramidal neurons in the adult rat exposed in utero to ethanol.

Reduced numbers of dendritic spines on the secondary apical dendritic branches and basilar dendrites of CA1 and CA3 pyramidal neurons were observed in ethanol-treated rats during embryonic life aged 15 days when compared with age-matched controls. However, differences were no longer present at the age of 90 days. These results suggest that recovery of some morphological parameters of pyramidal hippocampal neurons may occur in rats exposed in utero to ethanol.

Effects of prenatal ethanol exposure on dendritic spines of layer V pyramidal neurons in the somatosensory cortex of the rat.

The number of dendritic spines on consecutive segments from the cell body along the 400-600 micron proximal region of the apical dendrites of layer V pyramidal neurons, impregnated with the rapid Golgi method, in the somatosensory cortex was counted in ethanol-treated rats during gestation (25% ethanol in drinking water representing 30-35% of the total caloric intake) and in age-matched controls at postnatal ages 4, 15, 30 and 90 days. Although the mean values were lower in ethanol-treated rats than in controls during the first fortnight of postnatal life, significantly lower numbers of spines were observed only in the 15-day-old rat (Student's t-test, P less than 0.01-0.001). Spines with long, thin pedicles were characteristically encountered in ethanol-treated and controls aged 4 days; this sort of spine also predominated in ethanol-treated rats aged 15 days, but not in age-matched controls. The decrease in number and the abnormal morphology of spines was no longer present in ethanol-treated rats aged 30 and 90 days. These data suggest that impaired maturation of dendritic spines on cortical pyramidal cells, followed by recovery of the altered parameters at the end of the first postnatal month, occurs in the offspring of ethanol-treated rats during gestation.

Variations in free amino acids in tissues of rats from birth to puberty.

Variations in concentrations of free amino acids and total protein in tissues of rats have been studied from birth to puberty. In the first 10 days after birth the concentrations of free amino acids in peripheral tissues increased. Only in the liver there was an increase in concentration of essential amino acids. When nutrition changed at weaning, concentration of amino acids decreased. This may be associated with high protein synthesis rates and increased catabolism of amino acids. Even after weaning, when the young rat became nutritionally independent, concentrations of free amino acids in the tissues had not become constant.

Sulphur amino acid levels in some tissues of the rat during pregnancy and lactation.

Variations in free sulphur amino acid content in some rat tissues have been measured during pregnancy and lactation. As a general trend, sulphur amino acids tend to accumulate in tissues in late pregnancy. This accumulation is quantitatively more important in the liver and striated muscle than in the skin and the kidneys. Among sulphur amino acids, taurine shows the most marked changes, with a maximum accumulation in the liver and striated muscle on day 19 of pregnancy. After delivery, the levels of amino acids in the tissue pools show a tendency to drop, especially in liver and striated muscle. As expected, taurine shows the most marked decrease, returning to normal values in muscle but decreasing to depletion in the liver on day 20 after delivery. These results can be interpreted as follows: during pregnancy, increased food intake is sufficient to accommodate the metabolic needs of the mother, but during lactation, the requirements of milk synthesis promote the release of these amino acids from the tissues.

Some aspects of amino acid metabolism in the rat fetus.

1. In spite of an eventual catabolic phase during the last third of pregnancy, nitrogen retention seems to increase in pregnant rats. Furthermore, the high uterine blood flow and the high placental transfer of amino acids maintains an adequate nutrient supply to the fetuses. 2. The terminal rat fetus has a high circulating plasma amino acid level, as well as an increased free amino acid tissue pool when compared to its mother's. 3. In the rat fetus the development of enzymatic capabilities shows a sudden emergence (also denomined clustering) in late fetal life. In a general trend, the activities of enzymes related with amino acid metabolism are not well developed during rat fetal life. 4. The rate of amino nitrogen excretion in rat fetus is low, mainly due to the low development of urea cycle enzyme activities. 5. The rates of protein synthesis in many tissues are high in the rat fetus and they show a progressive decrease until delivery. On the other hand, the rates of protein breakdown are also higher during fetal life than in the adult.

The appearance of 2,3-butanediol in the chronic ethanol treated pregnant rat.

The chronic administration of ethanol to pregnant rats at term results in the appearance of high concentrations of glycols such as 2,3-butanediol and 1,2-propanediol in their blood and urine. The results support the idea that 2,3-butanediol formation could be a mechanism for ethanol detoxication. The clinical implications of the findings are referred to.

Free amino acid pools in some tissues of the pregnant rat.

Plasma amino acid pools show important variations throughout the gestational period in the rat, with decreased values at mid-pregnancy and recovered levels before parturition. This decrease in amino acid levels at mid-pregnancy is mainly due to changes in the gluconeogenic amino acid group. During pregnancy, whole tissue amino acid pools increase in the liver but no changes appear in other studied tissues. However, individual gluconeogenic amino acid concentrations change significantly in skeletal muscle, skin and liver through the gestational period. Pregnancy may be understood as a challenge to the mother's metabolism but the pattern of tissue amino acid changes is not similar to that found in some stressant situations and it should be the net consequence of maternal adaptations to the increased metabolic needs.

Effects of prenatal ethanol exposure on physical growth, sensory reflex maturation and brain development in the rat.

In the offspring of ethanol-treated rats during gestation (25% ethanol in drinking water) decreased litter size, increased postnatal mortality rate, reduced body weight and body size, delayed ear opening, eyelid opening and teeth eruption, retarded air righting reflex acquisition, impaired brain growth, reduced cortical thickness and delayed maturation of layer Vth's pyramidal neurons: reduced basilar dendritic arborization and decreased number of spines in the apical dendrite, were observed when compared with age-matched controls fed with a standard diet. Minimal effects were found in the offspring of fibre-treated rats during gestation (standard diet mixed with cellulose) in which the body weight was similar to that of controls, although both the calorific intake from food and the mother's weight gain during pregnancy were similar to those of the ethanol-treated group. All these abnormal parameters became normal at the end of the first month of postnatal life, indicating recovery of these developmental defects produced by prenatal ethanol consumption.

Tissue amino acid pool changes during the perinatal period of the rat.

Total free amino acid content in foetal liver, kidney, skin and striated muscle increases sharply during pregnancy. After delivery, there is no significant change in tissue total amino acid pools. The essential free amino acid pool in striated muscle decreases after delivery. This decrease suggests a relationship with the increased protein content in striated muscle.

Blood amino acid compartmentalization during pregnancy and lactation in the rat.

The blood amino acid compartmentalization during pregnancy and lactation of the rat have been studied. Cellular amino acid levels are similar to those of the plasmatic fraction. Important generalized decreases are detected on days 12 and 21 of the pregnancy; Glu + Gln, Ala and essential and semiessential amino acids being the most affected. During lactation, after a transient phase on day 1 after delivery, there is a generalized increase in blood levels, especially in the plasma fraction, that affects the whole essential values. The different patterns shown by amino acids during pregnancy and lactation confirm that the measurement of plasmatic levels underestimates the actual capacity of whole blood to transport amino acids.