Development of a rat model of toluene-abuse embryopathy.

A rat model was developed to study toluene-abuse embryopathy, a clinical syndrome which occurs in offspring of women who abuse toluene during pregnancy. On d 6-19 of gestation, eight dams received a daily gavage dose of toluene, 650 mg/kg body weight, diluted in corn oil, whereas eight control dams and eight pair-fed dams received corn oil. The fetuses were delivered on d 19 of gestation. In the toluene-exposed group, the weights of the fetuses were reduced by 21.6% (p < 0.001), and a delay in skeletal ossification was demonstrated. Toluene exposure significantly reduced the weight of the fetal brain by 11.9% (p < 0.001), as well as the weights of the heart, liver, and kidney. Organ weight/body weight ratios did not differ significantly. Morphometric analysis of brain sections demonstrated that toluene exposure resulted in smaller brains together with an increase in the size of the ventricular system and a reduction in the size of the caudate nucleus. Although toluene exposure resulted in a 13.7% reduction in maternal food consumption, the observations made in the pair-fed group did not differ from those made in the control group. These findings suggest that prenatal exposure to toluene results in generalized fetal growth retardation, and that these effects are not due to the reduction in maternal food consumption.

The effects of high-dose toluene on embryonic development in the rat.

Developmental disability, intrauterine growth retardation, renal anomalies, and dysmorphic features have been described in offspring of women who abuse toluene during pregnancy. A Sprague-Dawley rat model was developed to study this clinical syndrome. During d 6-19 of gestation, 11 treated dams received daily gavage doses of toluene, 520 mg/kg body weight, diluted in corn oil, and 11 control dams received corn oil. This dose of toluene simulates the blood toluene levels obtained after an inhalation exposure to 3290 ppm toluene, an inhalation level in the lower end of the range experienced by toluene abusers. Maternal weight gain was 24% less in the toluene-exposed group (p < 0.002); however, there were no maternal deaths. The fetuses were delivered on d 19 of gestation, and 287 fetuses (148 toluene exposed, 139 control) were examined. Toluene treatment did not affect the number of implantations or stillbirths. There were no toluene-induced major congenital malformations or neuropathologic changes noted. In the toluene-treated group, the weights of the fetuses were reduced by 9.4% (p < 0.004) and placental weights were reduced by 10.3% (p < 0.01). Toluene exposure also reduced fetal organ weights as follows: brain 4.6%, heart 5.9%, liver 13.2% (p < 0.02), and kidney 13% (p < 0.05). Organ weight/body weight ratios did not differ significantly, suggesting that prenatal toluene exposure produced a generalized growth retardation.

Nutritional factors and anticonvulsant therapies: effect on growth in children with epilepsy.

Factors influencing growth before puberty were examined in a group of 32 epileptic children in a cross-sectional investigation. Participants in the study were divided into two groups according to what anticonvulsant drug, carbamazepine or valproic acid, they were currently taking. Dietary intakes of energy, iron, and zinc were assessed and hemoglobin and serum zinc concentrations were evaluated. Measurements of height and weight were compared with standard growth charts prepared by the National Center for Health Statistics, Hyattsville, MD. A Student's t test comparison was made along with analyses of covariance and stepwise regression, and no significant differences were found between the two groups of children in terms of height or weight. Linear growth was normal. We found that low dietary zinc intake, 64-87% of the recommended dietary allowance, was not associated with reduced height in children. In addition, neither the number of years taking anticonvulsant medication nor the total amount of drug intake had a significant effect on weight. This was an unanticipated result because valproic acid has been associated with weight gain because of increased appetite.

Effects on the fetal rat eye of maternal benomyl exposure and protein malnutrition.

Benomyl, a benzimidazole fungicide, produced ocular and craniocerebral malformations in fetal rats when administered to the dams by gavage in a dose of 62.4 mg/kg of maternal body weight/day on days 7-21 of gestation. Ocular anomalies included retinal dysplasia, cataracts, microphthalmia, and anophthalmia. These anomalies occurred in 43.3% of fetuses exposed to benomyl and a normal protein diet but increased to 62.5% when benomyl administration was combined with a protein deficient (8% casein) diet. Microscopic examination of the malformed eyes revealed that the most common abnormality, retinal dysplasia, consisted of rosettes of retinal cells and retinal infolding. The majority of rosettes had a single layer and a limiting membrane. Rosettes with two or three layers were also observed, particularly in fetuses exposed both to protein deficiency and benomyl. Although anophthalmia was identified macroscopically in five fetuses, only a single instance of true anophthalmia was found microscopically. These data support the results of previous investigators that benomyl induces ocular malformation and that protein deficiency enhances the teratogenic effects of benomyl. The disorderly proliferation of retinal cells and rosette formation resembled the periventricular cell masses that accumulate in brains exposed to benomyl and certain other teratogenic agents. The anti-tubulin action of benomyl is known to impair microtubule formation and it may produce brain and ocular malformations by disruption of neuronal proliferation and migration.

Relationship of periventricular overgrowth to hydrocephalus in brains of fetal rats exposed to benomyl.

Benomyl, a benzimidazole fungicide, was administered by gavage to pregnant Sprague-Dawley rats in a daily dose of 62.5 mg/kg of maternal body weight beginning at gestational day (GD) 7. Fetuses examined histologically at GD16 or GD20 revealed a high incidence of craniocerebral anomalies--82.6% of those examined at GD16 and 100% of those examined at GD20. Hydrocephalus occurred in 65.2% of fetuses examined at GD16 and in 58.8% at GD20 but was more severe in the GD20 fetuses. A second common anomaly, termed periventricular "overgrowth" (PVO), consisted of subependymal cell masses that in some fetuses obliterated normal subcortical structures. PVO occurred in 34.8% of fetuses examined at GD16 and 76.5% at GD20. The size of the subependymal masses and the regions involved were considerably greater in the GD20 than the GD16 fetuses. Less common anomalies in the GD20 fetuses were periventricular necrosis (41.2%), a single fetus with exencephaly and another with porencephaly. In the majority of malformed fetuses, the severity of hydrocephalus did not parallel the severity of PVO around the lateral and third ventricles. PVO involved tissues surrounding the cerebral aqueduct in 17.4% of GD16 fetuses and 38.2% of GD20 fetuses. This "overgrowth" distorted the cerebral aqueduct in a large number of fetuses with ventriculomegaly, and at GD20 moderate and severe ventriculomegaly was in every instance associated with a narrow or completely occluded cerebral aqueduct. These relationships suggest that PVO in the midbrain may play a role in the production of aqueductal stenosis and hydrocephalus in this experimental model.

Benomyl-induced craniocerebral anomalies in fetuses of adequately nourished and protein-deprived rats.

Benomyl, a benzimidazole fungicide, produced craniocerebral and systemic malformations in fetal rats when administered by gavage in doses of 31.2, 62.5, and 125 mg/kg of maternal body weight on days 7-21 of gestation. Malformations increased in incidence and severity with increasing benomyl dosage and nearly doubled when coupled with a protein-deficient diet. Protein deficiency alone produced only decreased fetal weight. High benomyl doses produced higher percentages of fetal resorptions and late fetal deaths, and these percentages also increased with protein deficiency. A benomyl dose of 62.5 mg/kg in protein-deficiency dams, the optimal combination for a high incidence of anomalies and low fetal wastage, produced hydrocephalus in 69.4% of fetuses, meningocele in 8.2%, encephalocele in 14.3%, exencephaly in 44.9%, anencephaly in 14.3%, corpus callosum agenesis in 26.5%, periventricular necrosis in 26.5%, and periventricular cellular "overgrowth" in 55.1%. The most common combination of anomalies was hydrocephalus, exencephaly, and periventricular "overgrowth." Common systemic malformations included cleft palate, micromelia, hydroureter, and misshapen tails. No fetus was entirely normal at the highest benomyl dose. Benomyl has been shown by others to bind tubulin and inhibit the formation of microtubules that are important in neurulation, mitosis, and cell migration during early brain development. Thus, it is suggested that benomyl, coupled with a protein-deficient diet, offers a teratogenic model with a spectrum of abnormalities similar to hypervitaminosis A but with a higher yield of specific craniocerebral anomalies.

Effects on the fetal rat intestine of maternal malnutrition and exposure to nitrofen (2,4-dichlorophenyl-p-nitrophenyl ether).

The effects of maternal protein-energy malnutrition and exposure to nitrofen on selected aspects of intestinal morphology and function were studied in the fetal rat. Pregnant rats were fed, throughout gestation, diets containing 24% or 6% casein as the sole source of protein. Reduced total food intake produced protein-energy malnutrition (PEM). Each diet group was divided in half and gavaged with either 12.5 mg nitrofen in corn oil/kg/day or corn oil carrier only from days 7 to 21 of gestation. Body weight, intestinal weight, length, and diameter were measured as were villus length (VL), villus width (VW), and number of villi per length of intestine (VMM). Protein (horseradish peroxidase) and lipid absorption were studied histochemically. Lactase and dipeptidase activities were determined in proximal, medial, and distal thirds of the intestine. Results showed that the restricted maternal diet resulted in reduced fetal body weight (BW), intestinal weight (IW) and length (IL), reduced IW/BW and IW/IL ratios, VH, and VMM. The VW was reduced only in the distal third. Protein and lipid absorption were unaffected. Lactase and dipeptidase activities were reduced. Maternal nitrofen exposure resulted in reduced body weight, intestinal size, and lipid absorption, with some evidence of interaction with the diet effects on enzyme activities. It is concluded that effects of maternal malnutrition were extensive, but that nitrofen exposure, at this dosage level, is not likely to contribute to the postnatal fetal mortality rate in either adequately nourished or malnourished rats.

Cell number and size in selected organs of fetuses of rats malnourished and exposed to nitrofen.

The effects of maternal exposure to nitrofen or protein-energy malnutrition on the number and sizes of cells in selected organs of the fetal rat have been studied. Pregnant rats were fed either an adequate (CON) or protein-energy deficient diet (PEM) throughout gestation. Each diet group was divided into two subgroups. One subgroup was gavaged with 25 mg nitrofen/kg body weight on gestational days 7-21 and the other, with corn oil carrier only. Fetal liver, kidneys, intestine, heart, lung, and brain were weighed and assayed for DNA, RNA, and protein. Maternal protein deficiency resulted in a reduction in organ weight and total DNA, RNA, and protein in all six organs. Maternal nitrofen exposure resulted in reduced weight and reduced protein in all organs except the brain. Total DNA and RNA were reduced in intestine, heart, and lung, and total RNA was also reduced in the liver following maternal nitrofen exposure. An interaction between diet and toxin affected lung weight, DNA, RNA, and protein, intestinal total protein, and heart DNA. Protein/DNA ratios were reduced in liver, intestine, and brain in the group fed the inadequate diet and in intestine only following nitrofen exposure. The deficient diet resulted in increased RNA/DNA ratio in the fetal liver and heart and a decreased ratio in the kidney and brain. Nitrofen exposure resulted in a lower RNA/DNA ratio in the liver. The data indicate that maternal protein-energy malnutrition results in smaller organs in the fetuses with fewer cells and containing less protein and RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Kidney morphology and function in the young of rats malnourished and exposed to nitrofen during pregnancy.

The separate and combined effects of prenatal protein deficiency (6% casein) and prenatal nitrofen (2,4-dichlorophenyl-p-nitrophenyl ether) exposure (12.5 mg/kg on gestational d 7-21) on renal morphology in the 21-d fetal and postnatal rat were examined. Body weights and kidney weights were reduced in prenatally protein-deprived (PPD) pups at birth and on postnatal day (PND) 10. Numbers of mature glomeruli, creatinine clearance, water diuresis, and response of antidiuretic hormone (ADH), but not the concentrating ability, were lower in the PPD neonates. These changes suggest that prenatal protein deficiency delays renal development and possibly results in a decrease in glomerular clearance and in tubular response to a water load and to antidiuretic hormone. Prenatal nitrofen exposure reduced body weight and kidney size on PND 0 and 10. An increased incidence of hydronephrosis was indicated in the nitrofen-exposed fetus. Prenatal nitrofen exposure depressed the ability to excrete excess water, the response to ADH, and urine-concentrating ability. The functional deficits indicate tubular dysfunction, but little or no effect on glomerular function, as indicated by the absence of an effect on creatinine clearance. Postnatal survival was reduced to 22% by PND in the PPD plus nitrofen pups. Also, prenatal nitrofen exposure increased the susceptibility of the glomeruli in the gestational day (GD) 21 PPD fetus to the adverse effects of prenatal protein deficiency. By PND 10 the toxic effects were of the same order. Renal dysfunction may contribute to the increased mortality in PPD plus nitrofen pups by reducing the ability to respond to stress, but the effects are not sufficiently marked to be considered the primary cause of death.

Effects on the fetus of maternal benomyl exposure in the protein-deprived rat.

The separate and combined effects of protein deprivation and benomyl [(methyl 1-butylcarbomoyl)-2-benzimidazole carbamate] exposure were studied in the pregnant rat fed a diet containing 24% (control) or 8% (deficient) casein throughout gestation. Within each diet group, subgroups were gavaged at 31.2 mg/kg body weight with benomyl or corn-oil carrier only on d 7-16 or 7-21 of gestation. No effects on the skeleton were seen. Benomyl exposure in the last 2 wk in dams fed the 24% casein diet resulted in a high incidence of fetal brain anomalies. This effect did not occur in those with benomyl exposure during the period of organogenesis only and was reduced in groups fed the protein-deficient diet. Exposure to benomyl in the last 2 wk in the protein-deprived rat resulted in a decrease in the weight of the fetal heart in excess of that attributable to diet alone. Lungs were a smaller portion of body weight in fetuses of benomyl-treated dams in both diet groups. The teratogenic effect on the brain in animals exposed to benomyl in wk 2 and 3 of gestation suggests that screening for teratogenic effects during organogenesis only may be insufficient.

Effects on the fetus of maternal nitrofen exposure in the protein-deprived rat.

The separate and combined effects of protein deprivation and nitrofen exposure were studied in the pregnant rat. Animals were fed diets containing 24, 8, 6 or 4% casein throughout gestation. Within each diet group, subgroups were gavage-fed with 12.5 (lower dose) and 25 (higher dose) mg nitrofen/kg body weight or with oil carrier only on days 7-21 of gestation. Dams were weighed and food intake was measured daily. On day 21 of gestation, cesarean-derived pups were examined for congenital anomalies and dissected for determination of organ weights. Skeletons were alizarin-stained and examined for skeletal anomalies and developmental stage. No effects on the skeleton or gross congenital anomalies were seen. Fetal size and weights of liver, kidney, intestine, heart, lung and brain were reduced with decreasing casein content of the diet and as a result of the higher dose of nitrofen. An effect of interaction between diet and nitrofen exposure was shown in the kidney, intestine and lung weights. Specific toxicity affecting organ size was shown to occur in the intestine and lung. An interaction between diet and nitrofen specifically affected kidney and intestine. Brain size tended to be preserved, a possible protective mechanism negated by nitrofen exposure. The data suggest also that kidney, intestine and lung are particularly affected. Effects, however, occur primarily in young of severely malnourished dams receiving a relatively high dose of nitrofen.

Growth and reproduction in the chronically nutrient-deprived rat.

The effects on growth and reproduction of chronic, moderate multiple deficiencies of protein, riboflavin, pyridoxine and vitamin A were studied in the rat. The effects of combined deficits on feed intake and feed efficiency and on the young were then determined. All deficits, singly or in combination, interfered with the increase in feed efficiency in pregnancy seen in the control rats. Rats fed diets deficient in both protein and vitamin A did not survive to breeding weight. Protein deficiency resulted in reduced number of live young which were of reduced weight. When deficits of riboflavin or pyridoxine or both were also imposed, even fewer live young were produced, but there was no further effect on weights at birth. Reduction in feed intake in pair-fed animals was not totally responsible for these effects. Production of live young was not as depressed by combined vitamin deficiencies when dietary protein was adequate. The most severe effects of any of the deficiencies appeared to occur early in the reproductive process, possibly involving ovulation, embryo survival, implantation and the very early post-implantation period during the development of vital organs. It is suggested that the nutrient deficit interferes with the action of progesterone or other hormones important to the physiological adaptations to pregnancy.

In vitro amino acid transport by renal tubules of prenatally protein-deprived fetal rats.

Amino acid transport in the renal tubules of 21-day fetal offspring of protein-deprived (4% casein diet) and adequately fed (24% casein diet) rats was studied by measuring the in vitro accumulation of glycine and the amino acid analog, alpha-aminoisobutyric acid (AIB). The prenatally protein-deprived (PPD) fetuses were smaller than the controls with a mean weight of 3.8 g compared to 5.5 g, and their kidneys were 0.4% of body weight, compared to 0.5% of body weight in the control group. Assessment of the renal proximal tubule transport mechanisms indicated that they were capable of transporting glycine and AIB at day 21 of gestation. There were no differences between the two groups in total water content or extracellular fluid content of the renal cortical tissue slices. The samples from PPD fetuses were able to accumulate concentrations of amino acids equal to or greater than those of control fetuses. The accumulation of AIB per gram of tissue was almost twice as great in the PPD tissue slices as in the control slices, whereas the accumulation of glycine per gram of tissue was equal in the two groups. Comparison of the fluid content and extracellular space of the PPD and control tissues suggests that this difference in uptake was not due to changes in the fluid distribution. It is proposed that the increase in AIB absorption by PPD renal tissue is related to retardation in maturation of the tissue. Transport of glycine, which matures later in development, may be immature in both groups. The greater amino acid uptake may conserve amino acids and thus have survival value in PPD young.

Maternal and embryonic response to a "carbohydrate-free" diet fed to rats.

The effects of a carbohydrate-free, fatty acid (CF) diet on pregnant rats and their progeny were examined. A pregnant pair-fed group was included to compensate for the 40% reduction in food intake of the CF dams. Control and CF dams were killed on days 6, 8, 10, 12, or 14 of gestation. Pair-fed dams were killed on day 14. Maternal CF rats were hypoglycemic and hyperketonemic as compared to pair-fed or control dams. Both CF and pair-fed dams had significantly reduced liver glycogen. Diet had no effect on the number of implantation sites. Conceptuses were classified histologically as normal, retarded, malformed, degenerating or resorbed. Numbers of normal CF embryos were significantly reduced from day 6 and, by day 12, all CF embryos had been resorbed. Control and pair-fed dams showed 5.2% and 43.8% resorptions, respectively, on day 14. These data suggest that approximately half of the embryonic loss can be attributed to the reduction in food intake whereas the remaining embryos succumbed to embryolethal conditions more directly related to the metabolic consequences of carbohydrate deprivation.

The effect of prenatal protein-calorie malnutrition on kidney development in the rat.

Our studies show that prenatal protein deprivation has a long-term (if not permanent) effect on kidney morphology in the young and effects on renal function that persist at least to the end of weaning. Since animals are still alive and growing, we might ask whether this has any real physiological significance. At this point, it is important to mention that 50% of the young born of protein-deprived dams in these studies did not survive more than 3 d. Those animals that were the subject of the study of postnatal kidneys were presumably less affected. We do not know the extent to which compromised renal function contributed to neonatal death in the others. These results can be extrapolated to other species only with great caution, since the rat kidney is relatively immature at birth. It is probable that increased postnatal feeding has a more beneficial effect on these rapidly developing tissues than it would have in species in which the kidneys are more mature at birth. Yet, kidney function did not reach the control level in spite of improved postnatal nutrition. Compromised renal function in the more mature kidneys of other species may be less amenable to postnatal recovery and--at the very least--leave the kidneys less able to respond to postnatal stress factors.

Hematologic and trace element alterations following chronic maternal ingestion of propylthiourea.

Rats with diets containing 0.2% propylthiouracil (PTU) throughout gestation had progeny with persistent cyanosis and high neonatal mortality. Histological and histochemical studies failed to reveal lung abnormalities in these pups. Studies of the blood of PTU-fed dams demonstrated that hemoglobin, packed cell volume, and numbers of erythrocytes were significantly reduced. In their 21-day fetal young, erythrocytopenia was accompanied by an elevated mean corpuscular volume and a reduced mean corpuscular hemoglobin concentration. Imprints of marrow from dams and of liver and spleens of the young showed normoblastic erythropoiesis. A granulocytic leucocytosis was present in the blood of the PTU-fed dams, whereas their progeny had a granulocytopenic leucopenia. Tissue concentrations of copper, zinc, manganese, magnesium and iron were determined. The most striking changes observed were the significant elevations of copper in the dams' brain, liver and kidneys. No changes in the concentration of any of the trace minerals were found in the livers of the pups. Food restriction to the dam failed to significantly alter maternal or fetal hematologic or trace element concentrations as compared with controls. It is evident that PTU, when fed to pregnant rats, has demonstrable effects on erythropoiesis, granulocytopoiesis, and maternal trace element distribution. It is not presently known whether these phenomena are interrelated.

Effect of maternal protein-calorie malnutrition on fetal rat cerebellar neurogenesis.

Pregnant Sprague-Dawley rats were fed, ad libitum, diets containing either 24% (control) or 4% casein (deprived) from conception through gestation. On the 13th, 14th, 15th, 16th, 17th or the 18th day of gestation the dams of both dietary groups were injected with 2.5 mCi [3H]thymidine per gram of body weight. Pups born to these dams were reared by stock diet-fed foster mothers to insure normal postnatal nutrition and were killed on postnatal day 28. Retention of isotope in the nuclei of four types of macroneurons, as visualized in autoradiographs prepared from cerebellar tissues, was used to determine the time of final DNA synthesis in these cells. In the brains of animals malnourished in utero, the time of administration of the label which resulted in maximal retention and peak cerebellar neurogenesis had occurred as early as the 13th day of gestation.

Production and metabolism of thyroid hormones in protein-deficient and food-restricted pregnant rats.

Thyroxine (T4) and triiodothyronine (T3) concentrations in the plasma and their metabolic clearance and production rates were studied in pregnant protein-deficient and food-restricted rats on the 20th day of gestation. Total T3 levels in the plasma were significantly reduced in malnourished dams, with those of food-restricted dams being consistently lower than those of protein-deficient dams. There were no changes in plasma total T4 concentrations as a result of dietary treatment. Unbound T4 and T3 in the plasma were significantly reduced in both protein-deficient and food-restricted dams. Maternal protein deficiency significantly lowered fetal T4, whereras food restriction caused an elevation. Metabolic clearance rates of T3 and T4 and production rate of T4 were unaffected by diet, while the production rate of T3 was significantly increased in protein deficient dams. Thyroxine to triiodothyronine ratios in malnourished dams were elevated suggesting depressed peripheral conversion of T4 to T3. Alternative factors which may lead to these effects are discussed.