Ethanol exposure alters zebrafish development: a novel model of fetal alcohol syndrome

Prenatal exposure to alcohol has been shown to produce the overt physical and behavioral symptoms known as fetal alcohol syndrome (FAS) in humans. Also, it is believed that low concentrations and/or short durations of alcohol exposure can produce more subtle effects. The purpose of this study was to investigate the effects of embryonic ethanol exposure on the zebrafish (Danio rerio) in order to determine whether this species is a viable animal model for studying FAS. Fertilized embryos were reared in varying concentrations of ethanol (1.5% and 2.9%) and exposure times (e.g., 0–8, 6–24, 12–24, and 48–72 h postfertilization; hpf); anatomical measures including eye diameter and heart rate were compared across groups. Results found that at the highest concentration of ethanol (2.9%), there were more abnormal physical distortions and significantly higher mortality rates than any other group. Embryos exposed to ethanol for a shorter duration period (0–8 hpf) at a concentration of 1.5% exhibited more subtle effects such as significantly smaller eye diameter and lower heart rate than controls. These results indicate that embryonic alcohol exposure affects external and internal physical development and that the severity of these effects is a function of both the amount of ethanol and the timing of ethanol exposure. Thus, the zebrafish represents a useful model for examining basic questions about the effects of embryonic exposure to ethanol on development.     

MDMA administration to pregnant Sprague-Dawley rats results in its passage to the fetal compartment

Recent investigations have demonstrated that prenatal 3,4-methylenedeoxymethamphetamine (MDMA; ecstasy) exposure in rats results in significant and persistent changes in the developing brain. However, no published pharmacokinetic studies exist demonstrating that MDMA administered during pregnancy passes to the fetal compartment. This leaves open the question whether MDMA is directly acting on the fetal brain to produce the observed changes in previous studies, or whether such effects are an indirect result of MDMA administration to the pregnant dam. Therefore, pregnant rats were administered a single dose of MDMA (15 mg/kg, subcutaneous) at embryonic day 14 (E14) and the levels of MDMA and its metabolite 3,4-methylenedioxyamphetamine (MDA) were quantified in maternal plasma, amniotic fluid, and fetal brain over 8 h by HPLC. The time course of MDMA and MDA metabolism was reliable and reproducible in all tissues. There was a strong correlation between fetal amniotic fluid and fetal brain suggesting that amniotic fluid could be used to reliably estimate fetal brain levels without directly utilizing fetal brain tissue. These data also provide a framework for subsequent in vitro cell culture studies using biologically relevant MDMA doses.     

Withdrawal symptoms in a long-term model of voluntary alcohol drinking in Wistar rats

Long-term voluntary alcohol drinking with repeated alcohol deprivation episodes has been suggested as animal model for some aspects of alcoholism. Using a radiotelemetric system, the present study investigated the occurrence of withdrawal symptoms in long-term voluntarily alcohol drinking Wistar rats with (repeated alcohol deprivation group) and without (first alcohol deprivation group) prior alcohol deprivation experience. Six days after transmitter implantation, alcohol bottles were removed, and returned 4 days later. Alcohol deprivation induced hyperlocomotion in both groups. In the repeated alcohol deprivation group, hyperlocomotion was increased at the beginning of the alcohol deprivation phase and decreased during the following dark phase, suggesting that removal of the alcohol bottles might have become a conditioned withdrawal stimulus for this group. Both groups showed an enhanced alcohol intake after representation of alcohol bottles compared to preabstinence intakes (alcohol deprivation effect). However, alcohol intake of the repeated alcohol deprivation group was significantly increased compared to the first alcohol deprivation group at the end of the experiment. It is concluded that repeated alcohol deprivation experience might promote the development of alcohol addiction because of its latent stimulating effect on alcohol drinking that can be unveiled by (presumably mildly stressful) experimental situations.     

Assessing the risk of fetal alcohol syndrome: understanding substance use among pregnant women

Fetal alcohol exposure is a common cause of birth defects and developmental disorders. As many as 1 in 100 children in the United States are believed to be affected by fetal alcohol exposure. Characteristics of fetal alcohol syndrome (FAS) include abnormal facial features, growth impairment, problems with learning, memory, attention span, problem solving, speech, and hearing. FAS is 100% preventable. Preliminary data from 1704 pregnant women in Minnesota were assessed: substance use during pregnancy, risk factors related to substance use during pregnancy, and how substance use among pregnant women varies across the state. Of the sample, 19.6% of the women were calculated to have drunk alcohol while pregnant. Nondrinkers were more likely to be married and unemployed than drinkers. The drinkers reported significantly higher levels of depressed mood and greater number of problems with alcohol than their abstaining counterparts. Abstainers reported a greater number of pregnancies and initiated their first prenatal visit earlier than the drinkers. Significant differences in prenatal substance use and related factors also emerged by geographic region in Minnesota. Findings are discussed in relation to prevention and policy efforts.     

Neonatal administration of a GABA-T inhibitor alters central GABAA receptor mechanisms and alcohol drinking in adult rats

Long-term effects of chronic treatment with a GABA-T (GABA-transaminase) inhibitor, ethanolamine O-sulphate (EOS) (200 mg/kg/day for the postnatal days 3–21) on the binding parameters of GABA_A receptors, hypothalamic monoamines and subsequent behavior were studied in Wistar rats. At the age of 1 month, EOS-treated rats showed reduced activity in the open-field and, at the age of 4 months, their voluntary alcohol consumption was increased. No changes were seen in Porsolt's swim test or in the plus-maze test. Weight gain was significantly retarded in EOS-treated rats. Maximal stimulation of [³H] flunitrazepam binding by GABA was decreased in the cerebral cortex and the EC₅₀-value for the GABA stimulation increased in the hippocampus in the EOS rats at the age of 4 months. EOS treatment did not alter the cerebellar diazepam sensitive and insensitive binding components of the imidazobenzodiazepine [³H]Ro 15-4513. No changes were observed in the hypothalamic monoamine concentrations. The results are in agreement with the idea that GABA-T inhibitor treatment permanently alters GABA_A mechanisms. Moreover, altering the CNS GABA level during development increases adult alcohol intake in rat.     

Repeated administration of diphenyl diselenide to pregnant rats induces adverse effects on embryonic/fetal development

The present study was carried out to investigate the effects of diphenyl diselenide, (PhSe)(2), on embryo-fetal development. Dams were treated subcutaneously with 1.5, 3.0 and 6.0 mg/kg (PhSe)(2) from days 6 to 15 of pregnancy. After cesarean section at gestation day (GD) 20, external and skeletal abnormalities were evaluated. A decrease in maternal body weight gain was found in (PhSe)(2) groups, indicating maternal toxicity. There was a reduction in the fetal weight and in crown-rump (CR) length of fetuses at three doses tested. The occipito-nasal length decreased in fetuses from dams exposed to 3.0 mg/kg (PhSe)(2). Signs of delayed ossification in the skull, sternebrae and limbs were observed in all (PhSe)(2) groups, revealing a relation between morphological alterations and growth retardation in fetuses, but none of the changes appeared to be dose-dependent. Exposure of dams to (PhSe)(2) resulted in altered placental morphology that may have contributed to adverse reproductive outcomes. We concluded that (PhSe)(2) is toxic to dams and induces developmental delay of the fetal skeleton, but does not cause externally visible malformations in rat fetuses, in this experimental procedure.     

Caffeine disposition after oral administration to pregnant rats

1. Caffeine disposition was studied over 24?h in rats on the 12th day of pregnancy given 80?mg/kg of drug as a single oral dose or in four divided doses every three hours.

2. Peak blood levels of caffeine were reached at three hours after the single dose, and at 10?h (at half the previous value) after the first of the divided doses.

3. At the end of the experiment both caffeine and its dimethylxanthine metabolites were higher in blood, amniotic fluid and fetuses after divided doses than after the single dose. Urinary excretion over 24?h was the same for the two groups.

4. The overall conclusions underline that caffeine per se and not its metabolites are responsible for the teratogenic effects.     

Fluoxetine administration to pregnant rats increases anxiety-related behavior in the offspring

Rationale

Fluoxetine (Prozac?) is the most frequently prescribed drug to battle depression in pregnant women, but its safety in the unborn child has not yet been established. Fluoxetine, a selective serotonin reuptake inhibitor, crosses the placenta, leading to increased extracellular serotonin levels and potentially neurodevelopmental changes in the fetus.

Objectives

The purpose of this study was to elucidate the long-term consequences of prenatal fluoxetine in rats.

Methods

Pregnant rats were injected daily with 12?mg/kg fluoxetine or vehicle from gestational day?11 until birth, and the behavior of the offspring was monitored.

Results

Plasma fluoxetine transfer from mother to pup was 83%, and high levels of fluoxetine (13.0???g/g) were detected in the pup brain 5?h after the last injection. Fluoxetine-treated dams gave birth to litters 15% smaller than usual and to pups of reduced weight (until postnatal day?7). Furthermore, prenatal fluoxetine exposure significantly increased anxiety in the novelty-suppressed feeding test, the footshock-induced conditioned place aversion test, and the elevated plus maze test (following footshock pre-exposure) during adulthood, and also significantly decreased components of social play behavior at 4?weeks of age, and a strong tendency for increased self-grooming and making less contact in adults. Behavioral despair, anhedonia, and sexual behavior were not different between treatment groups. Finally, the hypothermic response to the 5-HT_1A agonist flesinoxan was observed at a lower dose in prenatally fluoxetine-exposed rats than in controls.

Conclusions

Prenatal fluoxetine exposure in rats leads to detrimental behavioral outcomes in later life, which may partly be due to altered 5-HT_1A receptor signaling.     

A school-based screening program for fetal alcohol syndrome

Fetal alcohol syndrome (FAS) is a common cause of birth defects and neuropsychiatric impairment. Identification of affected people is crucial for early entry into intervention programs and for the development of prevalence estimates. The objective of this project was to determine if screening for FAS in a community elementary school-based setting was feasible, to estimate prevalence in the screened population, and to determine if a screening program for FAS can be implemented using available personnel from the community.

The FAS Screen was used to screen kindergarten students enrolled in a school system. Students with scores on the FAS Screen above the cutoff for a positive screen (20) were referred to one of several diagnostic clinics for evaluation.

Over a 9-year period, 1384 students were screened and 69 (5%) had a positive screen (20 or above). These 69 children were then seen in a genetics/dysmorphology diagnostic clinic and 7 (10%) were found to have FAS (n=6) or partial FAS (n=1). The prevalence of affected children (FAS and partial FAS) was 1 per 198 students or 4.3 per 1000.

The FAS Screen was completed annually by school staff, teachers, social workers, and psychologists. The test has acceptable epidemiologic performance characteristics in a community setting. The screening takes about 8–10 min. The procedure was well accepted in the community. This screening strategy was inexpensive to implement (less than US$8.00 per student), and can be easily included with the other screens completed at kindergarten entry.     

Ethanol dependence as a determinant of fluid preference

Rats made dependent on ethanol by a schedule-induced polydipsia procedure preferred 5% ethanol to an increasing concentration of dextrose solution to a greater extent than animals on a non-dependent, non-polydipsic procedure which allowed an equivalent opportunity to drink ethanol, confirming a previous study. Two corresponding groups of animals drinking isotonic (0.9%) NaCl rather than 5% ethanol behaved similarly to the latter group, changing to a dextrose preference at a lower dextrose concentration than the ethanol polydipsic group. Therefore, neither the intermittent food regimen (polydipsia-generating procedure) in itself, nor a history of isotonic saline polydipsia biased fluid preference against dextrose solution choices. The enhanced preference for ethanol over dextrose solutions shown by the ethanol polydipsic group can be attributed to physical dependence rather than regiment produced artifacts.     

Reduced alcohol drinking in adult rats exposed to sucrose during adolescence

Intake of sweet-alcoholic drinks during adolescence is believed to favor alcohol abuse and dependence in adulthood. This study examined the influence of early exposure to ethanol with or without sucrose on the consumption of sweet or alcoholic solutions in adulthood. Adolescent rats (from post-natal day 30-46) were given continuous free access to tap water and either 5% sucrose, 5% ethanol or mixed 5% sucrose-5% ethanol. The control group was given access to water only. Upon reaching adulthood (post-natal day 60), rats were tested for saccharin (sweet), quinine (bitter) and ethanol consumption using a two-bottle free-choice paradigm. The results indicated that pre-exposure to ethanol did not alter the intake of sweet or ethanol solutions in adulthood. However, rats exposed to sucrose during adolescence showed a decreased consumption of both sweet and ethanol solutions. Because alcohol has a sweet taste component, an additional group of rats, pre-exposed to either 5% sucrose or water during adolescence, was tested for intravenous ethanol self-administration (preventing oral sensory stimulation) and in a new model of simultaneous access to oral saccharin and intravenous ethanol that results in higher total ethanol intake. Relative to controls, sucrose-exposed rats showed reduced operant self-administration of saccharin, yet no differences were found for intravenous ethanol self-administration. Altogether, these findings indicate that sucrose exposure during adolescence persistently affected the perception of sweet taste reward and thereby alcohol’s acceptance in adulthood.     

Ethanol drinking by rhesus monkeys as a function of concentration

Ethanol deliveries maintained fixed-ratio (FR) responding of three rhesus monkeys during daily 3-h sessions. At FR values of 8 or 16, ethanol concentration was varied in the sequence 0 (water), 8, 11.3, 16, 22.6, 32, 8, and 0% (w/v). As the ethanol concentration increased, number of liquid deliveries decreased, although intake of ethanol (g/kg/session) increased somewhat. Blood ethanol levels were usually greater than 200 mg% and occasionally greater than 300 mg%.These data were reported to the Committee on Problems of Drug Dependence (1976) and were part of a doctoral dissertation by J. E. Henningfield     

Body mass index in fetal alcohol syndrome

Introduction: Prenatal alcohol exposure is an important cause of growth impairment. In this study we examined the stability of the growth measurements, including height, weight, and body mass index (BMI) percentiles, from birth to age at the time of diagnosis for subjects who were referred for evaluation to determine if they had fetal alcohol syndrome. Methods: We utilized subjects from the North Dakota Fetal Alcohol Syndrome Registry. Each person referred for assessment was provided a standardized assessment completed by a medical geneticist. We also examined differences in the diagnostic schema from the Institute of Medicine. The population consisted of 315 subjects with paired weight measurements, 234 subjects with paired height measurements, and 322 subjects with current BMI measurements. Results: Increases in weight percentiles and decreases in height percentiles were found. Children with FAS had consistently lower growth measurements. There was significant movement in and out of lower 5th and 10th percentiles by partial and no FAS children from birth to diagnosis. Discussion: The requirement for growth impairment needs to be broad rather than narrow, if most subjects with a diagnosis of FAS and partial FAS/ARND are to be captured.     

Influence of di-(2-ethylhexyl)phthalate on fetal testicular development by oral administration to pregnant rats

Influence of di-(2-ethylhexyl)phthalate (DEHP) on testicular development was studied by oral administration of DEHP at doses of 500 and 1000 mg/kg/day to pregnant rats on gestational days (G) 7 to 18. Ethinyl estradiol (EE) at dose levels of 0.25 and 0.5 mg/kg/day was used as a reference substance. Each 5-6 pregnant rats were sacrificed and their fetuses were examined on G12, 14, 16, 18 and 20. Fetal deaths averaging 20-36% were observed at every examination in the group receiving 1000 mg/kg of DEHP. Increases of fetal deaths over 50% were also observed in the reference group that received 0.5 mg/kg of EE. Microscopic examination of the fetal testis in groups treated with DEHP revealed degeneration of germ cells in G16 fetuses and localized proliferation or hyperplasia of interstitial cells in G18 and 20 fetuses. Germ cells having more than two nuclei were observed in a few cases including the control testes of G14 fetuses. These multinucleated cells were observed frequently in G20 fetuses treated with DEHP. Examination of testes of naturally delivered offspring of dams treated with 1000 mg/kg of DEHP at 7 weeks of age revealed scattered atrophy or dilatation of seminiferous tubules. Another experiment was carried out to confirm the dose of DEHP affecting testicular development and spermatogenesis. DEHP was given to pregnant rats at doses of 125, 250 and 500 mg/kg/day during G7-18. Similar histopathological changes were observed in fetal testes of the group exposed to 500 and 250 mg/kg of DEHP, but not in those exposed to 125 mg/kg. In postnatal examinations, however, no abnormality was found in the testes at 5 and 10 weeks after birth in any of the treated groups. Furthermore, no abnormal findings were observed in the function of sperm, sperm counts and sperm morphology in the offspring of the group treated with DEHP during the fetal period at 10 weeks of age. Thus, 125 mg/kg/day is considered the no-observed-effect-level of DEHP on testicular development of rats by exposure in utero during the period of organogenesis.     

Reduction in heavy drinking as a treatment outcome in alcohol dependence

In the field of clinical alcohol disorders treatment in North America, abstinence continues to be largely viewed as the optimal treatment goal; however, there is a growing awareness of limitations when abstinence is considered the only successful outcome. Although this issue has been discussed in research settings, new studies on the public health significance of heavy drinking (defined as five or more standard drinks per drinking day in men, and four or more standard drinks per drinking day in women) in the past 10 years suggest that clinical providers should consider the value of alternative outcomes besides abstinence. A focus on abstinence as the primary outcome fails to capture the impact of treatment on reduction in the pattern and in the frequency of alcohol consumption. In addition, evaluating reduction in drinking as “positive” has value for patients as an indicator of clinical progress. Measurement of continuous variables, such as the quantity and the frequency of alcohol consumption, has provided a clearer understanding of the scope of alcohol-related morbidity and mortality at the societal level, and of the relationship between individual patient characteristics and the naturalistic course of alcohol use, abuse, and dependence. A review of these characteristics suggests that there are clinical benefits associated with reducing heavy drinking in alcohol-dependent patients. Given the significant public health consequences associated with heavy drinking and the benefits associated with its reduction, it is proposed that researchers, public health professionals, and clinicians consider using reduction in heavy drinking as a meaningful clinical indicator of treatment response, and that outcomes be individualized to patients' goals and readiness to change.     

Experimental exposure to alcohol as a model for the evaluation of neurobehavioural tests

The performance characteristics of 4 computerized psychological tests were studied using alcohol as a model compound. Subjects received alcohol (0.5 ml/kg) or placebo in a cross-over design and performance was assessed using the Simple Reaction Time test, the Switching Attention test, the Hand/Eye Coordination test and the Color-Word Vigilance test. Analyses indicated an overall significant effect of alcohol at blood-alcohol levels of 0.03% with response speed on the Color-Word Vigilance test being the most sensitive parameter.     

Efforts to establish an animal model of Fanconi syndrome after ifosfamide administration to rats

About 10% of children develop Fanconi syndrome (FS) a few months after ifosfamide (IFO) treatment. To establish an animal model, IFO was injected as 4 or 5 treatment courses (TCs, once daily for 3 consecutive days), to adult female rats (AF, 8 mg 100 g(-1) body wt, 4 TCs), to young female rats (YF, 8 mg 100 g(-1) body wt, 5 TCs) and to male rats (M, 6 mg 100 g(-1) body wt, 4 TCs). In the adult female rats, polyuria with electrolyte and albumin wasting occurred acutely, 2 days after the first treatment course. After the third treatment course, 30% of the rats died, but survivors showed a reduced excretion of electrolytes and glucose. The body weight increase was significantly diminished in adult female and male rats by about 25% or 70%, respectively. Up to 5 months after 5 TCs in young female rats, 15% of the animals died but the survivors did not show any sign of renal failure. In males, 28% of the rats died and in surviving animals the excretion of electrolytes, proteins and glucose as well as GFR were reduced 7 weeks after the last treatment course. There were no pathomorphological changes in kidney and liver. Determination of renal and hepatic cytochrome P450 activities indicated that results of adult female and male rats could be caused by starving, known as a common side effect of IFO, and not by its nephrotoxicity. Altogether, it was not possible to establish a model of a Fanconi syndrome persisting after cessation of IFO treatment in our rat strain, whereas acute, FS-like IFO effects on the kidney could be shown.     

Effect of short-term administration of lead to pregnant rats.

Lead was administred to adult female rats in drinking water (0;0.1:1 and 10 ppm) for 3 weeks before mating, during pregnancy and during 3 weeks after delivery. On day 21 after delivery the mothers and their newborns were sacrified and various parameters of blood -- lead concentration on (Pb-B), hematocrit (Htc), hemoglobin (Hb), free erythrocyte porphyrins (FEP), delta0aminolevulinate dehydratase (ALAD) -- and tissue -- ALAD, free tissue porphyrins (FTP), lead concentration (Pb-T) -- were determined. In mothers a significant increase in Pb-B and Pb concentration in kidney was found in the 10 ppm group, but this increase in lead concentration was not associated with any statistically significant modification of the biochemical parameters. In newborns, lead concentration in blood and in kidney was also significantly increased in the 10 ppm group and this lead exposure was associated with a decrease of the ALAD activity in blood and an increase of FTP in kidney. On the basis of the biochemical parameters investigated one can therefore conclude that the developing organism is more susceptible to the biological action of lead than the organism of adult animals and that the "no-effect" level of lead administered during pregnancy and in the neonatal period is around 1 ppm.