Effects of maternal parity and late gestational nutrition on mRNA abundance for growth factors in the liver of postnatal sheep

The liver is a major metabolic and endocrine organ in growing neonates, but the extent to which its hormone receptor (R) sensitivity is potentially determined by maternal parity and the mother's nutritional environment is unknown. This was therefore investigated by sampling livers from postnatal sheep born to nulliparous or multiparous mothers. Offspring were sampled 1 or 30 days after birth from mothers consuming either 100 or 50% [i.e., nutrient-restricted (NR) group] of total metabolizable energy requirements from 110 days gestation to term ( approximately 147 days). Regardless of maternal diet, offspring of nulliparous mothers were lighter at birth and had smaller livers. By 1 mo of age, they exhibited catch-up growth, an adaptation not seen when mothers were NR, but they retained their lighter livers. At both sampling ages, livers from offspring born to nulliparous mothers exhibited increased mRNA abundance for growth hormone (GH) receptor, IGF-IR, plus hepatocyte growth factor (HGF); and at day 1 only IGF-I, but not IGF-IIR mRNA was decreased. In addition, mRNA for IGF-II, the HGFR, c-Met, and Bax were persistently reduced in these offspring. Effects of parity were largely unaffected by maternal nutrient restriction. Maternal parity therefore has a substantial effect on liver size during postnatal development and its receptor population that is not dependent on maternal diet. First-born offspring appear to exhibit a resetting of the endocrine control of hepatic growth within the HGF and GH-IGF axis, which could have later consequences after their growth has caught up.     

Impact of chronic ethanol intake of rat mothers on the seizure susceptibility of their immature male offspring

The aim of present study was to examine the impact of prenatal ethanol exposure on seizure susceptibility of the offspring. Pregnant Wistar rats were compelled to drink either 10% or 20% ethanol solution, as the only drinking fluid since conception up to the weaning of their offspring at the age of 28 days. Pregnant and nursing rats of the control group drank water. Electrophysiological experiments (repeated electrical stimulation and analysis of cortical afterdischarges duration) were than performed on their immature offspring. Rat pups were tested on postnatal day 18, 25, and 35. Shortening of afterdischarges duration was observed in 18-day-old animals (mothers drank 20% ethanol) when compared with age matched controls and failure of post-ictal depression phenomenon was found in 25- and 35-day-old animals. Our findings signalize that ethanol exposure during pregnancy influences seizure susceptibility by acting on excitatory/inhibitory brain systems and this effect is dose- and age-dependent.     

Suckling ability and maternal prolactin levels in hypothyroid rats

Long-Evans rats and their offspring were made hypothyroid by addition of the antithyroid goitrogen 6-N-propylthiouracil (PTU) to the drinking water (0.1%) from the day of parturition. Serum concentrations of prolactin (PRL), thyroid-stimulating hormone (TSH) and thyroxine (T4) were determined by double radioimmunoassay (RIA). From the fifth postnatal day, body weight of PTU-treated pups was significantly lower than that of control rats, and a strikingly elevated serum TSH level and nondetectable amount of T4 were measured both in PTU-exposed mothers and their offspring at Day 10 postpartum. To test the youngs' suckling capability and the amount of maternal milk production, 10- and 15-day-old normal and PTU-treated pups were separated from their mothers for 4 hr in the morning and then reunited and allowed to suckle. Normal pups gained body weight at the end of both the first and second hour postreunion, while PTU pups gained only during the first hour and lost weight in the second hour of testing. When the pups were exchanged between normal and PTU mothers, opposite results were obtained, indicating that the reduced gain in hypothyroid rats was not due to impaired suckling capability, or insufficient sensory stimulation for milk secretion but to a decreased milk production of PTU mothers. In accordance with this, in lactating hypothyroid rats both the basal (presuckling) level and the suckling-induced rise of serum PRL were found significantly depressed.     

Effects of perinatal exposure to Zamzam water on the teratological studies of the mice offspring

Zamzam water is well documented for plenty of medicinal value for curing illness. In the present study, the effects of perinatal consumption of Zamzam and normal drinking water by the pregnant mice on their offspring’s physical parameters, early sensory motor reflexes, locomotor activities, acetylcholinesterase (AChE) activity in the homogenize brain tissue and blood parameters were compared. To achieve that; Zamzam water was given to female Swiss-Webster strain mice as the only source of drinking fluid and the control animals were administered plain tap water. Treatment started from the first day of pregnancy and continued until the postnatal day fifteen of delivery. All offspring were subjected to various tests. The rate of body weight gain remained relatively unaffected until the second week of weaning period, however; in the last week the offspring exposed to Zamzam water gained significant body weight as compared to their control offspring. Furthermore, the opening of eyes and appearance of body hairs in Zamzam exposed pups remained unaffected as compared to the controls. The sensory motor reflexes in Zamzam exposed pups after birth and during the first two weeks of weaning period were significantly increased. Locomotor Activity Test performed in the male and female offspring after weaning period showed a significant decrease in the male and increase in the female on most of the elements of this test due to Zamzam exposure. AChE activity in the homogenized brain tissue and blood parameters were unaffected as compared to the controls, the present Zamzam effects in the offspring are possibly via in utero action and/or via mother’s milk.     

Effects of uteroplacental insufficiency and reducing litter size on maternal mammary function and postnatal offspring growth

Human intrauterine growth restriction is often associated with uteroplacental insufficiency and a decline in nutrient and oxygen supply to the fetus. This study investigated the effects of uteroplacental insufficiency and intrauterine growth restriction (Restricted) or reducing litter size for normally grown pups (Reduced Litter) on maternal mammary development and function, milk composition, offspring milk intake, and their resultant effects on postnatal growth. Uteroplacental insufficiency was surgically induced by bilateral uterine vessel ligation on day 18 of gestation in the Wistar Kyoto rat. At birth, a group of sham control rats had their litter size reduced to five (Reduced Litter) to match that of the Restricted group. Cohorts of rats were terminally anesthetized on day 20 of gestation or day 6 of lactation, and a third group was studied throughout lactation. Restricted pups had a lower birth weight (by 16%) and litter size (by 36%) compared with controls, as well as reduced mammary parathyroid hormone-related protein content and milk ionic calcium concentrations associated with reduced total pup calcium. Restricted dams with lower circulating progesterone experienced premature lactogenesis, producing less milk per pup with altered composition compared with controls, further slowing growth during lactation. Reducing litter size of pups born of normal birth weight (Reduced Litter) was associated with decreased pup growth, highlighting the importance of appropriate controls. The present study demonstrates that uteroplacental insufficiency impairs mammary function, compromises milk quality and quantity, and reduces calcium transport into milk, further restraining postnatal growth.     

Effect of hypothyroidism induced by propylthiouracil on ovarian function and structure in offspring from treated mothers (Rats)

The aim of the present study was to investigate the effects of hypothyroidism induced during the pre- and postnatal periods of life on ovarian function and structure in offspring (pups) 120 days of age. Three groups were used. In the prenatal group, treatment was given from conception to parturition. In the postnatal group, treatment was given from parturition to 25 days postpartum. Hypothyroidism was induced by administration of 0.1% 6-n-propyl-2-thiouracil (PTU) in the drinking water of mothers. Body weights of the offspring were measured weekly. In each group, ten offspring were sacrificed at 120 days of age. Postnatal PTU treated pups showed delay in eye opening, teething, fur development, and weaning (35-37 days) compared to control animals (28-30 days). Body weight of offspring in the postnatal PTU treatment group was significantly decreased (P < 0.001), while the prenatal PTU treatment group showed a significant increase (P < 0.0001) compared to control animals. There was a significant (P < 0.05) reduction in paired ovarian weight of offspring in the postnatal PTU treatment group compared to control animals. Diameter of the ovaries was not affected by any treatment. Regarding the morphometery, only offspring in the prenatal PTU treatment group showed a significant (P < 0.001) increase in the diameter of graafian follicles. No significant difference was observed in morphometery of the granulosa layer, primary, and developing follicles of control and all treated groups. Number of primary, developing, and graafian follicles of all the treated groups was similar to that of the control group. The corpora lutea of the postnatal PTU treated group contained a population of large numbers of luteal cells compared to the control group. The prenatal PTU treated group did not exhibit a profound effect on ovarian morphology, histology, and morphometery. No difference was found in the serum estradiol concentration of control and PTU treated groups. J. Exp. Zool. 293:407-413, 2002.     

Offspring of mothers fed a high fat diet display hepatic cell cycle inhibition and associated changes in gene expression and DNA methylation

The association between an adverse early life environment and increased susceptibility to later-life metabolic disorders such as obesity, type 2 diabetes and cardiovascular disease is described by the developmental origins of health and disease hypothesis. Employing a rat model of maternal high fat (MHF) nutrition, we recently reported that offspring born to MHF mothers are small at birth and develop a postnatal phenotype that closely resembles that of the human metabolic syndrome. Livers of offspring born to MHF mothers also display a fatty phenotype reflecting hepatic steatosis and characteristics of non-alcoholic fatty liver disease. In the present study we hypothesised that a MHF diet leads to altered regulation of liver development in offspring; a derangement that may be detectable during early postnatal life. Livers were collected at postnatal days 2 (P2) and 27 (P27) from male offspring of control and MHF mothers (n = 8 per group). Cell cycle dynamics, measured by flow cytometry, revealed significant G0/G1 arrest in the livers of P2 offspring born to MHF mothers, associated with an increased expression of the hepatic cell cycle inhibitor Cdkn1a. In P2 livers, Cdkn1a was hypomethylated at specific CpG dinucleotides and first exon in offspring of MHF mothers and was shown to correlate with a demonstrable increase in mRNA expression levels. These modifications at P2 preceded observable reductions in liver weight and liver∶brain weight ratio at P27, but there were no persistent changes in cell cycle dynamics or DNA methylation in MHF offspring at this time. Since Cdkn1a up-regulation has been associated with hepatocyte growth in pathologic states, our data may be suggestive of early hepatic dysfunction in neonates born to high fat fed mothers. It is likely that these offspring are predisposed to long-term hepatic dysfunction.     

Developmental toxicity of the HIV-protease inhibitor indinavir in rats

BACKGROUND: Indinavir is an antiviral agent used for the treatment of HIV infection. We studied its developmental toxicity in rats. METHODS: Pregnant animals were treated orally with 500 mg indinavir/kg body weight (bw) from day 6 to 15 of gestation (once daily) or from day 9 to 11 (twice daily). Fetuses were evaluated for external and skeletal anomalies on day 21 of gestation. In addition, 19 rats were treated from day 9 of gestation to day 24 postnatally with 500 mg indinavir/kg bw once daily; a control group of 17 rats was treated with the vehicle accordingly. Developmental landmarks were recorded. Sixteen offspring each were studied on postnatal days 7, 14, 21, and 35 for hepatic enzyme activity. Liver tissue was examined by electron microscopy. RESULTS: Fetal examination on day 21 of pregnancy showed no treatment-related effects on number, weight, and viability of the fetuses; however, an increased incidence was noted in the supernumerary ribs and variations of the vertebral ossification centers in both indinavir-treated groups. Postnatal evaluation showed delayed fur development, eye opening, and descensus testis. The most striking finding was unilateral anophthalmia, observed in 7 pups (3%) from 2 out of 19 litters exposed to indinavir, but not in controls. Only minor changes in hepatic monooxygenase activities occurred in dams. Electron microscopy of liver samples showed hepatocellular inclusions of lipids and myelin figure-like structures in maternal livers and infiltration with granulocytes in offspring livers. CONCLUSIONS: Further studies on reproductive toxicity, including combinations of three or more antiretroviral agents as used therapeutically, are needed to determine the hazards of such a treatment.     

Neonatal behavioral toxicity in rats following prenatal exposure to methanol

Although methanol (MEOH) may assume a significant role as a fuel, which implies wide availability, little is known of its toxicity apart from acute poisoning episodes in human adults. Even less is known about its toxicity in developing organisms. This experiment studied the early behavioral development of rats whose mothers had consumed MEOH during gestation by measuring the responses of suckling (postnatal day 1) and nest-seeking (postnatal day 10). Primigravida Long-Evans rats were divided into three groups (N = 10). Two of the groups consumed drinking solutions of 2% MEOH instead of distilled water either on gestational days 15-17 (MEOH 1) or 17-19 (MEOH 2). No maternal toxicity was apparent as measured by weight gain, gestational duration, and daily fluid intake. Daily MEOH consumption averaged 2.5 gm/kg over the 3-day period in both MEOH groups. Litter size, birth weight, and infant mortality did not differ among the three groups. Postnatal growth and date of eye opening were unaffected. MEOH pups required longer than controls to begin suckling on postnatal day 1. On postnatal day 10, they required more time to locate nesting material from their home cages. These data suggest that prenatal MEOH exposure induces behavioral abnormalities early in life that are unaccompanied by overt toxicity.     

Pregnancy in adolescent rats, growth and neurodevelopment in their offspring

Pregnancy, lactation and the relationship between mother and their offspring in adolescence was studied in terms of pups neurosomatic development. The frequency of conception and birth rate were reduced in adolescent dams. Their body weight gain was accelerated during the first two weeks of pregnancy, while a significant delay occurred in the third week. At birth, plasma corticosterone level in the neonates was increased. Adolescent dams ingested less food during the first week of gestation. Following that, till the second week of lactation, the food consumption was equal to that of control group. Although adolescent pups were heavier at birth, the reduction of their number and of their body weight occurred during lactation. Judging by appearance of grasping, righting, placing and of negative geotaxis reflexes, the delay in their neurological maturation was also present. The reason for the growth and neurodevelopmental delay during lactation is probably the result of malnutrition and stress of disturbed mother - infant relationship in adolescent litters, which lasted at least during the two postnatal weeks. It was indicated by the resting plasma corticosterone levels during the first two postnatal weeks. This finding suggests that the pregnancy in adolescent rats induces delay in physical and neurological development, as well as in the increased rate of postnatal mortality of their offspring.     

Drinking desalinated seawater for a long time induces anomalies in the development of new-born albino rats

The present study aimed to elucidate the abnormalities in the development of rat brains, livers, kidney and behaviours after drinking desalinated seawater prenatally. Three types of drinking water were employed as an experimental probe (bottled water, filtered desalinated seawater and tap desalinated seawater) to investigate neurobehavioral and morphological changes in the development of pup rats. Female rats from each group were administered water from their birth until gestation and lactation. The 1st and 2nd generation pups were divided into three groups: Group C, mothers and pups administered with bottled drinking water (the control group); Group F, mothers and pups administered with filtered drinking water; Group T, mothers and pups administered with unfiltered desalinated seawater (tap water). Morphological changes (CNS aberration) and neurobehavioral changes were studied. The aberrations recorded in the tissues (brain, liver, kidney and spinal cord) of rats from groups T and F may be due to oxidative stress in these tissues such as reduced glutathione, lipid peroxidation, peroxidase and super oxide dismutase. In conclusion, drinking desalinated seawater for a long time may cause teratogenic effects in the development of New-born rats.     

Effects of eflornithine hydrochloride (DFMO) on fetal development in rats and rabbits

Eflornithine hydrochloride (DFMO) is a highly selective, enzyme-activated, irreversible inhibitor of the enzyme L-ornithine decarboxylase (ODC). Because of its role in the biosynthetic pathway of polyamines, ODC is essential for the growth and development of newly implanted embryonic tissue. In order to assess its effect on embryonic growth and fetal development, at various stages of gestation, DFMO was administered in the drinking water to pregnant rats and rabbits at several concentrations (from 0.03% to 3.0%) and times (from days 7, 10, or 11 through days 18 or 19). Rats were killed on day 21 and rabbits on day 29 of pregnancy (day 1 = day of insemination), and the implantations and fetuses were examined. At a concentration of 1.0% (approximately 1,270 mg/kg/day) in rats and 3.0% (approximately 915 mg/kg/day) in rabbits, maternal food and water consumption and body weight gain were significantly reduced during the treatment period, and all implantations were aborted or resorbed. At lower doses (approximately 200-600 mg/kg/day) fetuses survived to term, though in reduced numbers, and a marked reduction in average fetal weight was seen. At levels of 60 mg/kg/day or lower, there were no deleterious effects to the dams or their offspring. Few malformations were detected at any dose level by gross teratologic examination; nor were any considered to have been drug induced because of their sporadic incidence. The embryotoxicity and severe growth retardation demonstrated by these studies verify that adequate polyamine levels are essential for normal embryonic and fetal development.     

Maternal diazepam exposure on brain ornithine decarboxylase and growth of offspring

The prenatal treatment of diazepam on the developmental pattern of brain ornithine decarboxylase and the general growth of offspring were studied. Diazepam (120 mg/kg/day) was administered orally to pregnant Sprague-Dawley rats from day 14 to day 20 of gestation. The activity of brain ornithine decarboxylase and body weight of the offspring were measured from the late fetal stage to the early postnatal stage. It was found that diazepam inhibited both the prenatal and 4-hour postnatal ornithine decarboxylase activities, though the general maturation pattern of the enzyme in the brain was not much altered. It may indicate that diazepam inhibits early brain development. The enzyme activity fell off as it reached maturation. Prenatal treated neonates of 6-hour or older age group had the normal activities of brain ornithine decarboxylase. The general growth of the treated offspring was substantially retarded. Their body weights were very much lower than the control offspring. The results of the present study is an additional evidence that diazepam and other benzodiazepines should be used with great care in pregnant women.     

Growth and liver morphology after long-term ethanol consumption of rats

Ethanol was administered to female and male Wistar rats by mixing it with their drinking water. Ethanol concentrations were gradually increased up to either 8% or 15%. Female rats receiving 8% ethanol in their drinking water consumed 5-13 g, males 4-10 g daily. The ethanol/total food caloric intake percentages were 13 to 20% and 9 to 15% for female and male rats, respectively. There was no difference in body weight and relative liver weight between treated rats and their controls. Female and male rats receiving 15% of ethanol in their drinking water consumed 8-14 g ethanol per kg body weight per day. The percentages of ethanol/total food caloric intake were stabilized at about 25% for both sexes. Growth of the rats differed only slightly from controls; a tendency for a higher increase of body weight of the control rats was found. No difference in relative liver weight between ethanol-treated and control rats was observed. Microscopic examinations revealed that the ethanol treatment resulted in fat accumulation in the liver cells. A proliferation of the Smooth Endoplasmic Reticulum (SER) was more marked in the 15% dosed rats than in the 8% dosed rats and more distinct in female rats than in male rats in both dosage groups.     

Low dose of bisphenol A impairs the reproductive axis of prepuberal male rats

The objective of the present work was to study the effect of a low dose of bisphenol A (BPA), on the reproductive axis of prepuberal male rats exposed to the endocrine disruptor (ED) during gestation and lactation period. Wistar-mated rats were treated with either 0.1 % ethanol or BPA in their drinking water until their offspring were weaned at the age of 21 days. The estimated average dose of exposure to dams was approximately 3 μg/kg/day of BPA. The pups were sacrificed on the 35th day of life. Body weight was measured during the development and at the moment of the sacrifice; testicular and seminal vesicles weight and their respective relative weights were also measured. LH, FSH and testosterone were determined and histological studies of testicular tissue were also performed. Body weight at the moment of the sacrifice was significantly higher in the group exposed to BPA; testicular weight decreased significantly; seminal vesicles weight and relative weights of testes and seminal vesicles were not modified by treatment. LH and FSH serum levels increased significantly after treatment, meanwhile testosterone showed no significant changes. Histological studies showed the lumen of seminal tubes reduced by the presence of immature cells of the spermatic lineage. Our results suggest that pre- and early postnatal exposure to a low dose of BPA disrupts the normal function of the reproductive axis in prepuberal male rats. The effects of the ED may be exerted at different levels of the axis and may be dependent on the dose, manner of administration, and the moment of exposure to the disruptor.     

围产期食物限制对子代成年大鼠海马CA1区突触可塑性的影响

围产期食物限制导致子代大鼠学习和记忆能力等的神经生物学变化,但其机制并不清楚。将成年Wistar雌性大鼠与雄性大鼠同笼,受孕后随机分为对照组 (n=9) 和食物限制组 (n=8) 。对照组母鼠在妊娠期和哺乳期自由进食和饮水,食物限制组母鼠从妊娠的第7天到子代大鼠出生后21天进行食物限制,食物限制量为对照组大鼠的50%。子代雄性大鼠成年后,通过Morris 水迷宫测试空间学习和记忆能力。之后,在海马CA1区在体记录场兴奋性突触后电位 (field excitatory postsynaptic potential,fEPSP),并采用免疫组织化学方法观察海马CA1区神经元型一氧化氮合酶 (nNOS) 阳性细胞密度的变化。结果表明,围产期食物限制降低了子代大鼠出生后第1、7、10、14和21天的体重,并减弱了成年子代大鼠的学习和记忆能力,海马CA1区fEPSP的斜率和nNOS阳性细胞的密度也明显降低。结果提示,围产期食物限制可能通过抑制NO的产生降低了海马突触可塑性,从而影响了子代大鼠的学习和记忆能力。     

Effects of prenatal X-irradiation on the 14th-18th days of gestation on postnatal growth and development in the rat

Thirty-nine pregnant adult Wistar strain rats were randomly assigned to one of three exposure groups: 0, 0.75, or 1.50 Gy X-radiation total exposure. Animals were exposed from the 14th to the 18th days of gestation at 0, 0.15, or 0.30 Gy per day. At term, 15 rats were killed and morphologic analyses were completed. Twenty-four rats were allowed to deliver their offspring. On the first day of postnatal life, litters were reduced to a maximum of eight pups per litter, with equal numbers of male and female offspring wherever possible. A total of 187 pups were observed for the age of acquisition of five reflexes (air righting, surface righting, visual placing, negative geotaxis, auditory startle) and the appearance of four physiologic markers (pinna detachment, eye opening, vaginal opening, testes descent). There was significant dose-related weight reduction in term fetuses and offspring throughout the 86-day postnatal period. Postnatal growth rate (g gained/day) was unaffected. Adult offspring brain and gonadal weight and organ weight:body weight ratios were reduced. Using the PAC50 methodology, dose-related alterations occurred in the acquisition of several reflexes. All physiologic markers exhibited a dose-related delay in appearance. These results indicate that fractionated exposure to X-radiation during the fetal period in the rat results in dose-dependent alterations in postnatal growth and physiologic development. These studies are important for our understanding of the long-range effects of prenatal exposure to ionizing radiation late in gestation.     

Probable gamma-aminobutyric acid involvement in bisphenol A effect at the hypothalamic level in adult male rats

The aim of the present study was to investigate the effects of bisphenol A (BPA) on the neuroendocrine mechanism of control of the reproductive axis in adult male rats exposed to it during pre- and early postnatal periods. Wistar mated rats were treated with either 0.1% ethanol or BPA in their drinking water until their offspring were weaned at the age of 21 days. The estimated average dose of exposure to dams was approximately 2.5 mg/kg body weight per day of BPA. After 21 days, the pups were separated from the mother and sacrificed on 70 day of life. Gn-RH and gamma-aminobutyric acid (GABA) release from hypothalamic fragments was measured. LH, FSH, and testosterone concentrations were determined, and histological and morphometrical studies of testis were performed. Gn-RH release decreased significantly, while GABA serum levels were markedly increased by treatment. LH serum levels showed no changes, and FSH and testosterone levels decreased significantly. Histological studies showed abnormalities in the tubular organization of the germinal epithelium. The cytoarchitecture of germinal cells was apparently normal, and a reduction of the nuclear area of Leydig cells but not their number was observed. Taken all together, these results provide evidence of the effect caused by BPA on the adult male reproductive axis when exposed during pre- and postnatal period. Moreover, our findings suggest a probable GABA involvement in its effect at the hypothalamic level.     

Behavioral and reproductive effects of chronic developmental exposure to brominated vegetable oil in rats

Adult Sprague-Dawley rats were fed diets containing 0, 0.25, 0.5, 1.0, or 2.0% of the food additive brominated vegetable (soybean) oil (BVO) for 2 weeks prior to mating. After conception, the diets were continued throughout gestation and lactation for the females. The same diets were also provided to the dams' offspring throughout their development (up to 90-120 days of age). BVO at 2.0% of the diet completely blocked reproduction. BVO at 1.0% of the diet severely impaired conception, reduced maternal body weight, and produced slightly reduced litter sizes but no evidence of malformations. At this dose postnatal mortality was high, and survivors showed impaired growth and severe behavioral impairments on a battery of standardized tests of functional development. After weaning, adequate data could not be obtained because of the high mortality rate in this group. BVO at 0.5% of the diet produced less reproductive interference and much less offspring mortality or impairment of growth, but produced behavioral impairments almost as severe as seen in the BVO 1.0% group. In addition, this group exhibited severely reduced postweaning activity, delayed vaginal patency development, and reduced day-90 weight. BVO at 0.25% of the diet produced reproductive deficits similar to the BVO 0.5% group, but less severe effects on growth and behavioral development. This group showed no significant increase in offspring mortality. The data demonstrate clear evidence of dose-related physical and behavioral developmental toxicity.