Frances J. Zeman

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.

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.

Early Postnatal Development of the Intestine in Progeny of Protein-Deprived Rats

Development of the small intestine was studied in newborn, and 4-, 8-, and 12-day-old young of rats fed diets, during gestation, containing 24 or 4% casein as the source of protein. Newborn prenatally protein-deprived rats had shorter, narrower intestines, reduced numbers of villi per unit length, shorter villi, and reduced numbers of absorptive cells and crypt cells. These differences had disappeared by the age of 12 days in adequately fed pups that had survived to that age.

Protein, dipeptide, and amino acid absorption in the young of protein-deprived rats.

Summary: Intestines of newborn and postnatal young of female rats fed diets containing 4% or 24% casein during pregnancy were infused in vivo with 50 mM 14C-labeled 1-aminocyclopentane-1-carboxylic acid (ACC), α-aminoisobutyric acid (AIB), glycyl-1-L-leucine (GL), or 8% 3H-histidine-labeled casein in 5% glucose and physiologic saline. The amounts of each material removed from the lumen or absorbed and the amount retained by the intestinal tissue were expressed as a total amount and per g body weight and per enterocyte.Casein absorption was significantly reduced per absorptive cell in prenatally protein-deprived (PPD) young at birth, but there were no differences at 8 days of age. AIB absorption and retention were decreased in PPD newborn young compared to controls whether expressed as a total, or on the basis of body weight or per enterocyte. At 12 days, total retention and absorption were reduced in PPD young, but all other differences no longer existed. ACC absorption was reduced in PPD young, compared to controls, regardless of the means of expression. Retention of ACC per enterocyte was increased at birth compared to controls. These differences had disappeared by 8 days. GL retention was increased in PPD young compared to controls at birth, but not at 8 days.The data on casein absorption indicate that newborn PPD young are handicapped at some point in the process of digestion and absorption of protein. At birth, amino acid absorption is also affected. Active transport of amino acids may be affected in some way, such as by an effect on carrier protein, by a deficit of available energy for active transport. The high retention of dipeptide per enterocyte may be the consequence of a decreased ability of the enterocytes of PPD young to hydrolyze the dipeptide or may be the result of increased incorporation of these materials into mucosal protein. There is apparently a marked difference between the effects of intestinal absorption of prenatal protein deprivation and those of postnatal malnutrition. It is suggested that the competence of the enterocytes are reduced in newborn PPD young whereas animals whose prenatal nutrition was normal would begin life with a larger complement of fully differentiated absorptive cells and might therefore be capable of greater adaptation to the nutritional deficit.Speculation: The data presented demonstrate that maternal protein deficiency results in decreased absorption of protein and its digestion products in the intestine of newborn rat pups. This may contribute to an explanation of the postnatal growth retardation, high mortality rate, and reduced immunocompetence in these offspring. Further investigation is needed on the specific steps in the protein digestion and absorption process which are affected and on the procedures which might be used to compensate for the depressed absorption.

The Relationship Between Alkaline Phosphatase and Pyrophosphatase Activity in Quail Bone

Summary The pyro- and alkaline phosphatase activities of quail bone appear to be catalyzed by a single enzyme. The activities could not be separated after ion-exchange chromatography or gel filtration. The pH optima for pyro- and alkaline phosphatase were 8.0 and 10.7, respectively. Alkaline phosphatase activity was stimulated by magnesium. Optimal stimulation of pyrophosphatase activity occurred at a pyrophosphate to magnesium ratio of 2.5. Both enzymatic activities were not markedly affected by high concentrations of fluoride.

Thyroid, pituitary, and hypothalamic hormone levels in neonatal rat pups following maternal protein deficiency.

Summary Thyroidal, pituitary, and hypothalamic hormones in the plasma and tissues of newborn young of rats fed diets containing 24 or 4% casein as the sole source of protein were quantitated by radioimmunoassay. Circulating levels of total plasma triiodothyronine (T3) were significantly reduced and the free fraction of T3 was lower in young of protein-deprived rats than in control pups. Total plasma T4, free T4, plasma TSH, pituitary TSH per milligram of pituitary, the metabolic clearance rate of exogenous TSH per 100 g of body weight, and the TRH content of the hypothalamus were not significantly affected by the maternal diet.

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.

Lipid absorption in newborn rat intestine.

Abstract There appear to be three distinct regions of cellular maturation within the newborn intestinal epithelium. As crypt cells move onto the lower villus surface, they become transition cells which in turn differentiate into mature villus-absorptive cells. In order to study lipid absorption in cells of each region, proximal segments of intestines of both suckled and corn oil-infused newborn rats were prepared by routine light and electron microscopic methods. Crypt cells were capable of division and, perhaps, of some absorption, whereas villus cells were capable only of absorption. Transition cells were capable of both division and lipid absorption.

Metabolic effects of a “carbohydrate-free” diet in the pregnant rat

Abstrac The effects on selected metabolic indicators of carbohydrate deprivation in pregnant rats was studied. Pregnant rats were fed a diet containing 13% casein, either 69.5% (CON) or 0% (CD) carbohydrate, and oleic acid to make the diets isocaloric. Diets were fed beginning on Day 0 of pregnancy. A restricted intake group (RI) was also included to indicate the effect of reduced food intake. On days 8, 10, and 12 of pregnancy, animals were killed and liver, uterus and blood were harvested. Glycogen and glucose-6-phosphatase in endometrial tissue, glycogen in liver, ketone bodies in whole blood, and glucose, total lipid, cholesterol, urea nitrogen and alanine in plasma were determined. In CD dams, fetal resorption reached 75% by day 12 of gestation but only 35% in RI dams. Carbohydrate deprivation and restricted intake resulted in depleted liver glycogen. CD dams were hypoglycemic and hyperlipemic. Plasma alanine did not differ among the 3 groups and plasma urea was elevated in the CD group only on day 12. CD dams were markedly hyperketonemic. Neither uterine glycogen nor uterine glucose-6-phosphatase activity differed between the 3 groups, suggesting that uterine glycogen utilization was not the limiting factor in embryonic survival. Hypoglycemia, hyperketonemia or both may be related to the reproductive failure in CD rats.