Experimental study of umbilical cord length as a marker of fetal alcohol syndrome.

Umbilical cord length has long been investigated as a potential marker of intrauterine events that may place the neonate at risk for future adverse developmental sequelae. Experimentally, significantly shortened cords have been reported in association with prenatal exposure to common drugs of abuse. This study in rats reports the time course of effects on umbilical cord length of a daily maternal ethanol gavage (3,200 mg/kg) from gestational day 6 through termination of pregnancy at either day 17, 18, 19, or 20. A total of 786 fetuses derived from 60 litters were examined. Control fetuses demonstrated a linear increase in umbilical cord length and body weight gain during late gestation, findings that support previous studies. The body weights of the ethanol-exposed fetuses were reduced significantly on all gestational days examined, indicating intrauterine growth retardation, a characteristic of fetal alcohol syndrome. Similarly, acute fetal akinesia as well as long-term sequelae stemming from impaired neurological development would result from the elevated blood ethanol levels achieved in this study. The umbilical cords of ethanol-exposed fetuses were significantly shorter on gestational days 19 and 20 in comparison to their controls, while cord lengths on days 17 and 18 were not shortened significantly. A stretch hypothesis has been proposed suggesting that the degree of fetal activity is the main determinant of umbilical cord length. In rats, there is a physiologic diminution of the volume of amniotic fluid (oligohydramnios) in late gestation (day 19 to term), which restricts fetal movements but does not appear to alter the linear relationships between gestational age and cord length in controls, thus arguing against the stretch hypothesis. However, cord lengths in the ethanol-exposed fetuses plateaued in late gestation, suggesting possible adherence to a stretch hypothesis. This dichotomy is discussed emphasizing fetal growth and activity as well as intrauterine space.

The incidence of renal anomalies at full term in fetal rats is synergistically increased by estradiol (but not testosterone) supplementation on day 18 of alcoholic gestation.

BACKGROUND/PURPOSE: Fetal alcohol syndrome is characterized by facial dysmorphology, mental and growth retardation, and somatic anomalies including hydronephrosis. The authors sought to determine the influence of exogenous testosterone or estradiol on the incidence of hydronephrosis in a rodent model of fetal alcohol syndrome (FAS). METHODS: Pregnant rats were fed a liquid diet containing 35% ethanol-derived calories from gestation day 6 through 15, with exogenous testosterone or estradiol supplementation on day 18. On day 20, fetal kidneys were examined for evidence of hydronephrosis, and fetal serum estradiol concentrations were determined by radioimmunoassay. RESULTS: Maternal estrogen supplementation resulted in very high fetal serum estradiol levels that were not additionally increased by alcoholism. Despite this fact, the expression of renal malformations was highest in the alcoholic, estradiol-supplemented offspring. Additionally, the rate of renal malformations was significantly higher in the estrogen-supplemented alcoholic group than in the strictly estradiol animals, yet the fetal serum estradiol concentrations did not differ between the two groups. CONCLUSIONS: This suggests that ethanol may act synergistically with estradiol to increase the rate of renal anomalies including hydronephrosis. Such damage may persist via a suppression of normal testosterone-stimulated renal growth and development. FAS includes significant renal anomalies characterized by hydronephrosis in both animal models and affected children. Although the long-term functional sequelae of hydronephrosis and reflux are well known, the progression of renal disease in FAS children remains to be documented.

4-Methylpyrazole blocks acetaminophen hepatotoxicity in the rat.

STUDY OBJECTIVE: To determine whether 4-methylpyrazole inhibits the hepatotoxic effects of acetaminophen in a rat model. DESIGN AND TYPE OF PARTICIPANTS: A nonblinded experiment using male Sprague-Dawley rats. INTERVENTIONS: Animals were divided into four groups. Groups 1 through 3 received 2,000 mg/kg acetaminophen by gavage; group 4 acted as a control. At four or eight hours, group 2 received 400 mg/kg 4-methylpyrazole; group 3 received 50 mg/kg 4-methylpyrazole. Blood samples were taken for measurements of serum AST and ALT levels. Livers were removed for microscopic examination and grading of necrosis. RESULTS: Lower AST and ALT levels were obtained for both the 400-mg/kg (P < .01) and 50-mg/kg (P < .05) doses of 4-methylpyrazole administered four hours after acetaminophen. Although mean AST and ALT levels also were lower when 400 and 50 mg/kg 4-methylpyrazole were administered eight hours after acetaminophen, these results were not statistically significant. Median necrosis scores were 3 for rats receiving acetaminophen alone, 0.5 for those receiving acetaminophen and 400 mg/kg 4-methylpyrazole (P < .05), 1 for those receiving acetaminophen and 50 mg/kg 4-methylpyrazole (P < .05), and 0 for control rats (P < .05). CONCLUSION: When administered four hours after a toxic dose of acetaminophen, 4-methylpyrazole significantly inhibits hepatotoxicity in the rat, as reflected by lower levels of serum transaminases and lesser degrees of hepatic necrosis.

A small dose of morphine leads rats to drink more alcohol and achieve higher blood alcohol concentrations.

Male Sprague-Dawley rats were maintained on a daily regimen of 22 hr of fluid deprivation followed by a 2-hr opportunity to take a sweetened alcoholic beverage and water for over 6 months. During the week before the formal procedures of the experiment described herein, access to the alcoholic beverage was limited to 1.5 hr, but access to water was still for 2 hr. Intakes of ethanol, in terms of g/kg, were tabulated at 30 min for half of the rats and at 90 min for the rest. On the day of formal procedures, half of the rats of the 30- and 90-min measures were given 1 mg/kg of morphine sulfate just before the drinking session, whereas the rest received physiological saline. Morphine increased mean g/kg intakes of ethanol, as compared with controls, at 30 and 90 min. Blood alcohol levels were also increased. These data suggest that the well-documented ability of small doses of morphine to increase rats' intake of ethanol is probably not related to its ability to produce gastrointestinal effects, but rather due to its ability to modulate central motivational mechanisms associated with ingestion.

Prenatal ethanol exposure alters ethanol-induced dopamine release in nucleus accumbens and striatum in male and female rats.

Using in vivo microdialysis, ethanol-induced dopamine release in nucleus accumbens and striatum was examined in adult male and female Long-Evans rats exposed prenatally to ethanol and in controls. Following dialysis, ethanol intake was measured in an operant paradigm. Control rats showed increased dopamine release in nucleus accumbens and striatum in response to 0.5 g/kg ethanol, but not to 1.0 g/kg. Fetal ethanol-exposed rats showed no dopamine response at 0.5 g/kg. At 1.0 g/kg, fetal ethanol-exposed males showed increased dopamine release in both structures. Prenatally exposed females showed no change in accumbens, and decreased release in striatum. Fetal ethanol exposure did not significantly influence ethanol intake. The findings suggest that prenatal ethanol exposure influences subsequent neurochemical responses to ethanol; however, how these neurochemical measures are related to ethanol intake could not be determined in the present study. Data are discussed in terms of sex-specific shifts in the dose-response function for ethanol-induced dopamine release resulting from prenatal ethanol exposure.

Preferential alcoholic embryopathy: effects of liquid diets.

Our initial report of a preferential expression of experimental alcoholic embryopathy affecting the male offspring contiguous in utero to male siblings of Long-Evans rats was based on gavage administration of alcohol to pregnant rats without regard to isocaloric, pair-fed exposure paradigms. In this study, pregnant Long-Evans rats were given 35% ethanol-derived calories (EDC) in one of two different liquid diets: 1) a liquid alcohol diet based on Sustacal, a flavored liquid food formulated for human nutritional standards; and 2) a high-protein liquid rodent diet devised by Lieber and DeCarli (L&D). The diets were administered from day 6 to 15 of gestation. Pregnant rats were pair-fed liquid diets containing 0% EDC, but isocalorically balanced to 35% EDC with either sucrose (Sustacal) or maltose-dextrin (L&D). A fifth group of pregnant rats was given access ad libitum to standard certified laboratory rodent diet and served as free-fed controls. On day 20 of gestation, all pregnant rats were euthanized and the products of conception examined by standard teratologic techniques. Pregnant animals fed Sustacal-based diets consistently consumed fewer calories per kilogram body weight per day from day 6 to 15 of gestation (i.e., they were significantly calorie-deprived during pregnancy) as compared with the standard laboratory-diet-fed controls or those consuming L&D diets. Body weights of rats consuming Sustacal diets (both 0 and 35% EDC) were significantly lower throughout gestation when compared with all other groups. Higher (> 150 mg/dl) blood alcohol levels were attained by rats consuming 35% EDC in Sustacal diet as compared with L&D (100 mg/dl) diets.(ABSTRACT TRUNCATED AT 250 WORDS)

Hyperactivity and altered amphetamine sensitivity in premature juvenile rats.

'Premature' rats that were delivered by cesarean section on day 21 of gestation and 'normal' rats that were delivered spontaneously on day 22 of gestation were tested for basal locomotor activity and locomotor stimulation in response to D-amphetamine at 19-21 days of age. Compared to normal rats, premature rats had increased basal levels of locomotor activity and showed enhanced sensitivity to the locomotor stimulant effects of D-amphetamine. Cesarean-delivered premature rats may be a useful animal model for investigating mechanisms of neurobehavioral deficits associated with premature birth in humans.

Alcohol preference and hepatic alcohol dehydrogenase activity in adult Long-Evans rats is affected by intrauterine sibling contiguity.

Alcohol preference and hepatic alcohol dehydrogenase activity in adult rats are known to be sexually dimorphic. Intrauterine sibling contiguity (the intrauterine position of a fetus relative to adjacent siblings of the same or opposite sex) alters selected reproductive, behavioral and enzymatic sexual dimorphisms via intersibling sex hormone transfer. We postulated that sibling contiguity would affect alcohol preference and hepatic alcohol metabolism in adult rats. The results of our study demonstrate that adult mMm male Long-Evans rats (genetic male rat developing in utero between two male siblings) had significantly lower ethanol preference, attained higher blood alcohol levels after standard ethanol "challenge" doses and had significantly lower hepatic alcohol dehydrogenase activity than either male siblings developing in utero between two females (fMf) or genetic females developing between two males or between two females (mFm or fFf). Hepatic cytosolic aldehyde dehydrogenase activity was higher in adult female than male rats regardless of nearest neighbor siblings. It is suggested that the differences in ethanol preference and hepatic alcohol dehydrogenase activity between the adult mMm and fMf male rats is due to differences in prenatal hormonal environment which can modulate sexual dimorphisms in alcohol intake and metabolism in the adult.

Preferential alcoholic embryopathy among contiguous siblings of Long-Evans rats.

Ethanol exposure alters sex steroidogenesis and sexually dimorphic behaviors in rodent offspring. Contiguity to siblings of the same or opposite sex in utero also affects steroidogenesis and sexual dimorphism in rodents. The present study with Long-Evans rats shows that maternal exposure to ethanol during the critical period of rodent organogenesis preferentially affects body weights and increases malformations in offspring dependent on their in utero contiguity to siblings of the opposite sex.

Studies on the embryopathic effects of ethanolamine in Long-Evans rats: preferential embryopathy in pups contiguous with male siblings in utero.

The embryopathic effects of high doses of ethanolamine were evaluated in pregnant Long-Evans rats during the "critical period" or organogenesis. Ethanolamine was given by gavage at levels of 0, 500, 300, or 50 mg/kg/day (24%, 14.4%, or 2.4% of the LD50 value). Ethanolamine caused dose-dependent increases in intrauterine deaths, malformations, and intrauterine growth retardation. Embryolethality caused by 500 mg/kg of ethanolamine was not random: male pups contiguous to two male siblings (designated mMm) were almost quantitatively replaced by resorptions that were contiguous to two live male pups (designated mRm) (mMm pups constituted 6.7% of control implants and decreased to only 0.9% of group II implants while mRm resorptions increased from 0.3% in controls to 5.6% in group II dams). Intrauterine growth retardation and increases in gross structural anomalies (considered indicative of depressed fetal growth) more severely affected male than female offspring at all dose levels. Pups of either sex who were contiguous to male siblings were more adversely affected than those offspring contiguous to one or more female siblings. As ethanolamine was given prior to the period of greatest fetal growth and fetal sex steroidogenesis, it is suggested that intrauterine levels of female sex steroids (estradiol) enhance fetal repair of cellular damage while testosterone inhibits fetal repair or exacerbates previous embryonic damage by some unknown mechanism. Such interaction furthers the concept that intrauterine position affects the endpoints of developmental toxicity, as expressed at parturition.

Effects of ethanol on reproduction and arterial hypertension in spontaneously hypertensive and normotensive rats: a preliminary communication.

Ethanol consumption and spontaneous (essential) hypertension are important fetal and maternal risk factors. Alone, they contribute to embryopathy (fetal alcohol syndrome) or maternal organ pathology and fetal loss in hypertensive pregnancies. Combined, the effects of ethanol consumption on the progress of a hypertensive pregnancy have not been adequately investigated. In the present study, groups of O-A strain genetic hypertensive (SHR: groups 1 and 2) and Wistar-Kyoto normotensive (WKY: groups 3 and 4) pregnant rats were given 20 ml/kg of distilled water by gavage to serve as controls [groups 1 (SHR) and 3 (WKY)] or 3.2 g/kg of ethanol [groups 2 (SHR) and 4 (WKY)] from days 6 to 15 of gestation. During acclimation, hypertension developed in SHR rats (WKY pressures were 105 to 114 mm Hg; SHR pressures were 137 to 148 mm Hg). From day 6 to 15 of gestation, ethanol-consuming rats (groups 2 and 4) had higher arterial pressures than controls (groups 1 and 3). Pregnant SHR rats given ethanol did not experience a prebirthing hypotension. On gestation day 20, most offspring (84%, group 2; 86%, group 4) of alcoholic dams were dead or malformed. Intrauterine growth retardation occurred in group 4. Hydrocephalus, microphthalmia, and mild hydronephrosis and hydroureter were common in live offspring of group 2 dams. Hydronephrosis and hydroureter were increased in group 4 pups. Variant cranial ossification was noted in group 2 and 4 pups. These preliminary data suggest an altered hypertensive response during pregnancy in alcohol-consuming rats and confirm the embryopathic effects of relatively high levels of ethanol consumed during the critical period of organogenesis in two additional strains of rats.

Effects of various exposure levels of 2-phenylethanol on fetal development and survival in Long-Evans rats.

Phenylethanol was given at different levels (432, 43, or 4.3 mg/kg) by gavage to pregnant Long-Evans rats during the "critical period" of organogenesis. Examination of offspring revealed adverse reproductive and teratogenic effects in a dose-related manner. Intrauterine growth retardation occurred at levels of 432 and 4.3 mg/kg. Embryolethality was 18% at 43 mg/kg and 10% at 4.3 mg/kg. Malformations occurred in the following sequence: 100% at 432 mg/kg; 93% at 43 mg/kg, and 50% at 4.3 mg/kg. Noteworthy dose-related teratogenic effects of phenylethanol in offspring manifested themselves in increased incidences of malformed eyes, neural-tube defects, hydronephrosis, and limb defects.

Acute embryopathic effects of ethanol in the Long-Evans rat.

Thirty-two pregnant Long-Evans rats were divided into 10 groups of 3 or 4 pregnant rats, and each rat was given a single dose of 4 ml ethanol/kg (20 ml/kg of a 20% solution) between d 6 and 15 of gestation. An 11th group of 50 pregnant rats received distilled water and served as controls. Offspring body weights were decreased in groups of rats given ethanol as compared to controls (3.0-3.6 g, versus 3.9 g for controls). Total litter weight was decreased in dams given ethanol on d 6. Skeletal variants were seen in 13-78% of the offspring given ethanol, compared to 0.6% of the controls. Variations may be considered as additional signs of embryotoxicity. Malformations such as hydronephrosis, pelvic kidney, microcephalus, cranioschisis, and microphthalmia occurred in 72-100% of the ethanol treated offspring, as compared to 12% of controls. Hydronephrosis was most frequent on d 9 or 14, pelvic kidney on d 8 and 11, and microphthalmia from d 10-12. Cranioschisis was maximal on d 7, 11, and 15, and microcephalic offspring were most frequently born to dams given ethanol on d 7 or 14. Skeletal defects were usually single entities, while soft-tissue anomalies occurred in a consistent pattern. These results suggest that ethanol is a stage-specific teratogen in the rat at comparable exposure levels attained by many humans.

Paternal effects of ethanol in the long-evans rat.

Ten male Long-Evans rats were given 20% v/v ethanol in the drinking water for 60 consecutive days. Ten other males were given distilled water and served as controls. Each male was then allowed to mate with three virgin female Long-Evans rats, once per week for three consecutive weeks. The males were necropsied after the third mating, the females were killed on d 20 of gestation, and the offspring were examined for parameters of fetal growth, skeletal ossification, and soft-tissue anomalies. Ethanol caused testicular weight reductions and gross testicular atrophy in 1 of 10 males. Five matings of alcoholic male rats proved infertile. Total embryonic deaths (resorptions and preimplantation loss) were increased by ethanol, while implantations and litter size were significantly decreased. Fetuses fathered by alcoholic male rats were malformed: 55% had soft-tissue anomalies (microcephalus, microphthalmia, cranial fissure, and hydronephrosis). Litter weight and average pups weights were also reduced by paternal ethanol consumption. No recovery in reproductive function was evident over the 21-d post-ethanol mating period.

Renal effects of indoprofen in rhesus monkeys.

The non-steroidal anti-inflammatory analgesic alpha-[4-(1-oxo-2-iso-indolinyl)-phenyl]propionic acid (indoprofen) was given p.o. in gelatin capsules to six groups of six Rhesus monkeys (three males and three females/group) for 15 months. Group I (five males and five females) served as control and was given empty gelatin capsules. The following daily doses were used: Group 2--20 mg/kg b.i.d.; Group 3--40 mg/kg q.d.; Group 4--30 mg/kg b.i.d.; Group 5--60 mg/kg q.d.; Group 6--40 mg/kg b.i.d. and Group 7--80 mg/kg q.d. Except for some elevated blood urea nitrogen levels after 12 months in the animals of Group 7, no changes in clinical function test values, absolute and relative kidney weights or renal gross abnormalities were found. The renal papillae and pelves were normal and not affected in all monkeys. The microscopic examination revealed a focal chronic interestitial nephritis in some of the monkeys. In the survivors the incidence of this disease increased from one out of ten control animals to three out of five monkeys in Group 7 (80 mg/kg/d). However, this quantal, statistically significant difference became biologically irrelevant and no longer statistically significant when these microscopic changes were quantified. The most severe case was seen in the control animal and the least pronounced in monkeys having received 80 mg/kg/d. When these cases of interstitial nephritis were further evaluated using a multifactorial point sampling procedure, no significant quantitative changes in renal tissue components, indicative of analgesic-induced nephropathy, were detectable.