Analysis of hepatotoxicity in maternal and fetal rats after glucocorticoid administration by lipid histochemistry and thin layer chromatography

Changes in lipid metabolism of fetal and maternal rat livers were investigated on day 20 of pregnancy after administration of either 3 mg/kg or 24 mg/kg triamcinolone-acetonide or 124 mg/kg hydrocortisone in crystalline suspension to the mothers on day 15 of pregnancy. Sudan black B and Nile red as well as the UV-Schiff reaction and thin layer chromatography were used to study qualitatively the response of lipids to these glucocorticoids. Generally, after application of triamcinolone-acetonide fetal livers accumulated more lipids as toxic response to this glucocorticoid than the maternal organ; the degree of lipid accumulation was clearly dose-dependent in the fetuses. After hydrocortisone treatment, lipids in maternal livers were slightly, those in the fetuses were not affected. Histochemistry and thin layer chromatography revealed an accumulation of neutral lipids, especially of triglycerides and fatty acids which both contained increased amounts of ethylene bonds after treatment with triamcinolone-acetonide. The results also show that using combined histochemistry and thin layer chromatography, the analysis of hepatic lipids is a promising tool for the assessment of toxic effects of glucocorticoids on fetal and maternal hepatocytes in rats.     

Efficacy of Ginkgo biloba on Vaginal Estrous and Ovarian Histological Alterations for Evaluating Anti‐Implantation and Abortifacient Potentials in Albino Female Mice

Ginkgo extract, EGb 761 is known as a vasoregulatory variable for the conventional reproduction therapy. EGb 761 was orally administered in 0 (control), 3.7, 7.4, and 14.8 mg/kg bw/day for 28 days (thereafter mated with normal fertile male), from day 1 to day 7 of pregnancy or from the 10th to 18th day of pregnancy, respectively. Vaginal smears were performed daily. On 20th day of pregnancy, the females were killed by cervical dislocation and their kidneys, liver, brain, placenta, spleen and ovaries were removed and weighed. The ovaries were prepared for histological examinations, and then ovarian follicles were counted. Maternal toxicity, estrous cycle, reproductive hormones, ovarian follicle counts, resorption index, implantation index, fetal viability and fetuses, and placenta mean weights were evaluated. There was a dose‐dependent ovarian toxic effect of EGb 761. Ovarian follicle counts, resorption index, implantation index, fetal viability were significantly reduced in 14.8 mg/kg bw/day dose. Treatment with 14.8 mg/kg bw/day EGb 761 induced disruption of estrous cycle and caused maternal toxicity, in addition to fetal toxicity. Therefore, the data obtained indicate that Ginkgo biloba extract at 14.8 mg/kg bw/day dose level exhibit toxic effect on reproductive cyclicity and could have anti‐implantation and abotifacient properties in female mice.     

Effects of the Ay gene on susceptibility to hydrocortisone fetotoxicity and teratogenicity in mice

Effects of the Ay gene, a coat color gene, on susceptibility to hydrocortisone fetotoxicity and teratogenicity were investigated by using the congenic strain of C57BL/6-Ay (Ay/a) which had been maintained by repeated back-crosses of the Ay gene to the C57BL/6 (a/a) background. Matings were conducted as follows (female x male): group I, a/a; group II, a/a x Ay/a; and group III, Ay/a x a/a. Pregnant females were subcutaneously given daily doses of 0, 12.5, 25, or 50 mg/kg of hydrocortisone on days 10-13 of pregnancy. On day 18 of pregnancy, fetuses were sexed, weighed, and examined for external abnormalities. In group I, the mean fetal weight was significantly decreased at a dose of 25 mg/kg or more. The incidences of cleft palate were 3.2 and 22.7% at 25 and 50 mg/kg, respectively. In group II, in which half of the fetuses were expected to carry the Ay gene, the mean fetal weight was decreased significantly at 12.5 mg/kg or more. The incidence of cleft palate in group II at 50 mg/kg was 44.2%, which was significantly higher than that in group I. In group III, in which maternal mice as well as half of their fetuses carried the Ay gene, a decrease in the mean fetal weight was greater than in group II. In addition, the mean percentage of fetal resorptions was significantly increased at 50 mg/kg. The incidence of cleft palate in group III was significantly increased at 25 mg/kg (10.5%) when compared with those in groups I and II. These results indicate that the Ay gene may be associated with susceptibility to hydrocortisone fetotoxicity and teratogenicity in mice.     

Evaluation of developmental toxicity in rats exposed to the environmental estrogen bisphenol A during pregnancy.

Bisphenol A (BPA) is an essential component of epoxy resins used in the lacquer lining of metal food cans, as a component of polycarbonates, and in dental sealants. The present study was conducted in an attempt to evaluate the adverse effects of the environmental estrogen BPA on initiation and maintenance of pregnancy and embryofetal development after maternal exposure during the entire period of pregnancy in Sprague-Dawley rats. The test chemical was administered by gavage to mated females from days 1 to 20 of gestation (sperm in varginal lavage = day 0) at dose levels of 0, 100, 300, and 1000 mg/kg. All females were subjected to caesarean section on day 21 of gestation and their fetuses were examined for external, visceral and skeletal abnormalities. In the 1000 mg/kg group, significant toxic effects including abnormal clinical signs, decreased maternal body weight and body weight gain, and reduced food consumption were observed in pregnant rats. An increase in pregnancy failure was also found in the successfully mated females. In addition, increased number of embryonal deaths, increased postimplantation loss, reduced litter size and fetal body weight, and decreased number of fetal ossification centers of several skeletal districts were seen. On the contrary, no significant changes induced by BPA were detected in the number of corpora lutea and implantation sites and by fetal morphological examinations. In the 300 mg/kg group, suppressed maternal body weight and body weight gain, decreased food intake and reduced body weight of male fetuses were seen. There were no adverse signs of either maternal toxicity or developmental toxicity in the 100 mg/kg group. It was concluded that BPA administration during the entire period of pregnancy in rats produced pregnancy failure, pre- and postimplantation loss, fetal developmental delay and severe maternal toxicity, but no embryo-fetal dysmorphogenesis at an oral exposure level of 1000 mg/kg.     

Maternal diets enriched in olive oil regulate lipid metabolism and levels of PPARs and their coactivators in the fetal liver in a rat model of gestational diabetes mellitus

In a rat model of gestational diabetes mellitus (GDM) programmed in the offspring of neonatal streptozotocin-induced (nSTZ) diabetic rats, lipids are accumulated in the fetal liver in a sex-dependent way. Here, we evaluated whether maternal diets enriched in olive oil in rats that will develop GDM ameliorate lipid metabolic impairments in the fetal livers. Pregnant offspring of control and nSTZ diabetic rats (F0) were fed a 6% olive oil-supplemented diet throughout the F1 gestation. We evaluated maternal metabolic parameters as well as lipid content, expression of lipid metabolizing enzymes and protein expression of PLIN2, PPARs and PPAR coactivators in the fetal livers. The offspring of nSTZ diabetic rats developed GDM regardless of the maternal treatment. Hypertriglyceridemia in GDM rats was prevented by the olive oil-enriched maternal treatment. In the livers of male fetuses of GDM rats, the maternal olive oil-supplemented diet prevented lipid overaccumulation and prevented the increase in PPARγ and PPARδ levels. In the livers of female fetuses of GDM rats, the maternal olive oil supplementation prevented the increase in PPARδ levels and the reduction in PGC1α levels, but did not prevent the reduced lipid content. Control and GDM rats showed a reduction of lipid metabolic enzymes in the fetal livers, which was associated with reduced levels of the PPAR coactivators PGC-1α and SRC-1 in males and of SRC-1 in females. These results suggest powerful effects of a maternal olive oil-supplemented diet in the fetal liver, possibly providing benefits in the fetuses and offspring from GDM rats.     

Effects of tert-butyl acetate on maternal toxicity and embryo-fetal development in Sprague-Dawley rats

This study investigated the potential adverse effects of tert-butyl acetate (TBAc) on maternal toxicity and embryo-fetal development after maternal exposure of pregnant rats from gestational days 6 through 19. TBAc was administered to pregnant rats by gavage at 0, 400, 800, and 1,600 mg/kg/day. All dams were subjected to a Caesarean section on day 20 of gestation, and their fetuses were examined for any morphological abnormalities. At 1,600 mg/kg, maternal toxicity manifested as increases in the incidence of clinical signs and death, lower body weight gain and food intake, increases in the weights of adrenal glands and liver, and a decrease in thymus weight. Developmental toxicity included a decrease in fetal weight, an increase in the incidence of skeletal variation, and a delay in fetal ossification. At 800 mg/kg, only a minimal developmental toxicity, including an increase in the incidence of skeletal variation and a delay in fetal ossification, were observed. In contrast, no adverse maternal or developmental effects were observed at 400 mg/kg. These results show that a 14-day repeated oral dose of TBAc is embryotoxic at a maternally toxic dose (i.e., 1,600 mg/kg/day) and is minimally embryotoxic at a nonmaternally toxic dose (i.e., 800 mg/kg/day) in rats. However, no evidence for the teratogenicity of TBAc was noted in rats. It is concluded that the developmental findings observed in the present study are secondary effects to maternal toxicity. Under these experimental conditions, the no-observed-adverse-effect level of TBAc is considered to be 800 mg/kg/day for dams and 400 mg/kg/day for embryo-fetal development.     

Influence of maternal stress on uranium-induced developmental toxicity in rats

It has been demonstrated that uranium is an embryo/fetal toxicant when given orally or subcutaneously to pregnant mice. On the other hand, maternal stress has been shown to enhance the developmental toxicity of a number of metals. In this study, maternal toxicity and developmental effects of a concurrent exposure to uranyl acetate dihydrate (UAD) and restraint stress were evaluated in rats. Four groups of pregnant animals were given subcutaneous injections of UAD at 0.415 and 0.830 mg/kg/day on Days 6 to 15 of gestation. Animals in two of these groups were also subjected to restraint for 2 hr/day during the same gestational days. Control groups included restrained and unrestrained pregnant rats not exposed to UAD. Cesarean sections were performed on gestation Day 20, and the fetuses were weighed and examined for malformations and variations. Maternal toxicity and embryotoxicity were noted at 0.830 mg/kg/day of UAD, while fetotoxicity was evidenced at 0.415 and 0.830 mg/kg/day of UAD by significant reductions in fetal body weight and increases in the total number of skeletally affected fetuses. No teratogenic effects were noted in any group. Maternal restraint enhanced uranium-induced embryo/fetal toxicity only at 0.830 mg/kg/day, a dose that was also significantly toxic to the dams. As in previous studies with other metals, maternal stress enhances uranium-induced developmental toxicity at uranium doses that are highly toxic to the dams; however, at doses that are less acutely toxic the role of maternal stress would not be significant.     

Evaluation of the developmental toxicity of lenalidomide in rabbits

BACKGROUND: Lenalidomide, a thalidomide analog, is indicated for treatment of patients with deletion-5q myelodysplastic syndromes or multiple myeloma. NZW rabbits were used because of sensitivity to thalidomide's teratogenicity. METHODS: Range-finding and pulse-dosing studies preceded a full developmental toxicity study in New Zealand white (NZW) rabbits (25/group) given lenalidomide (0, 3, 10, or 20 mg/kg/day) or thalidomide (180 mg/kg/day) by stomach tube on gestation days (GD) 7-19. Clinical signs, body weights, and feed consumption were recorded daily from GD 7. On GD 29, standard maternal necropsy, uterine content, and fetal evaluations were carried out. RESULTS: In all studies, thalidomide was selectively toxic to development. In the pulse-dosing study, lenalidomide did not affect development at 100 mg/kg/day. Increases in C(max) and AUC(0-24 hr) values for lenalidomide were slightly less than dose-proportional; lenalidomide occurred in the fetuses. At 10 and 20 mg/kg/day, lenalidomide was maternally toxic (reduced body weight gain and feed consumption; at 20 mg/kg/day, weight loss and one abortion). Developmental toxicity at 10 and 20 mg/kg/day included reduced fetal body weights and increased postimplantation losses and fetal variations (morbidity/purple-discolored skin, undeveloped intermediate lung lobe, irregular nasal-frontal suture, and delayed metacarpal ossification). Thalidomide selectively reduced fetal body weight, increased postimplantation loss and caused characteristic limb and other dysmorphology. CONCLUSIONS: The maternal and developmental NOAELs for lenalidomide are 3 mg/kg/day. Unlike thalidomide, lenalidomide affected embryo-fetal development only at maternally toxic dosages, confirming that structure-activity relationships may not predict maternal or developmental effects. No fetal malformations were attributable to lenalidomide.     

Developmental toxicity evaluation of Bendectin in CD rats

Bendectin, composed of doxylamine succinate and pyridoxine HCl (1:1), is an antinauseant previously prescribed for nausea and vomiting during pregnancy. The present study examined the maternal and developmental effects of Bendectin (0, 200, 500, or 800 mg/kg/day, po) administered to timed-pregnant CD rats (36-41/group) during organogenesis (gestational days [gd] 6-15). At death (gd 20), all live fetuses were examined for external, visceral, and skeletal abnormalities. At 500 and 800 mg/kg/day, maternal toxicity included reduced food consumption during treatment and for the gestation period, increased water consumption in the posttreatment period, reduced weight gain during treatment, and sedation; water consumption was reduced during treatment and for the gestation period, and maternal mortality (17.1%) was observed only at the high dose. Developmental toxicity included reduced prenatal viability (800 mg/kg/day) and reduced fetal body weight/litter (500 and 800 mg/kg/day). In addition, reduced ossification of metacarpals (800 mg/kg/day), phalanges of the forelimbs (500 and 800 mg/kg/day), and of caudal vertebral centra (all doses) was observed. No increase in percent malformed live fetuses/litter was observed. The proportion of litters with one or more malformed fetuses was higher than vehicle controls only at 800 mg/kg/day, with short 13th rib (to which the test species is predisposed) as the predominant observation. By contrast, a positive control agent (nitrofen, 50 mg/kg/day, po, 14 dams) produced 85% malformed fetuses/litter with the predominant malformation being diaphragmatic hernia. In conclusion, the incidence of litters with one or more malformed fetuses was increased only at a dose of Bendectin which produced maternal mortality (17.1%) and other indices of maternal and developmental toxicity (see Discussion).     

Glucocorticoid teratogenesis in the developing nephron

Although the induction of polycystic kidney disease by neonatal glucocorticoid treatment has been extensively documented, there are no data on induction of polycystic kidney disease with fetal exposure to glucocorticoids. We injected groups of pregnant Swiss Webster albino mice subcutaneously with 250 mg/kg of hydrocortisone acetate on individual days from days 1 to 19 of gestation. A control group received an equal volume of saline. Histologic analysis of 1,522 kidneys from the offspring of these animals revealed no evidence of polycystic kidneys in the control group or in offspring of animals injected before day 11 of pregnancy. A bimodal distribution of cystic kidney disease was noted in the remaining animals, with highest prevalence after injection on day 12 (50.8%) and day 17 (34.3%). We conclude that 250 mg/kg of glucocorticoids may induce polycystic kidney disease in utero, but possibly only during critical periods of metanephric development.     

Embryotoxicity of oral administered chlorothalonil in mice

BACKGROUND: Chlorothalonil (2,4,5,6-tetrachloroisophthalonitril), the nephrotoxic fungicide, was examined for its potential to produce developmental toxicity in mice after oral administration. METHODS: Pregnant ICR (CD-1) mice were given sublethal doses of 0 (corn oil), 100, 400, and 600 mg/kg/day chlorothalonil by gavage on gestation days (GD) 6-15. RESULTS: Maternal effects in 400 and 600 mg/kg/day dose groups included signs of toxicity such as weakness and depression in the maternal activity, and reduction in body weight and weight gain. No maternal toxicity was apparent in the 100 mg/kg/day dose group. Maternal exposure to chlorothalonil during organogenesis significantly affected the number of live fetuses, early resorption, and mean fetal weight in the 400 and 600 mg/kg/day dose groups. No external, visceral, and skeletal abnormalities were observed among any of the treated groups compared to the control. CONCLUSIONS: On the basis of the present results chlorothalonil can produce clinical signs of toxicity and fetotoxicity without teratogenic effects at 400 and 600 mg/kg/day dose groups.     

Interactions of caffeine and restraint stress during pregnancy in mice

The maternal and developmental toxicity of combined exposure to restraint stress and caffeine was assessed in mice. On gestational Days 0-18, three groups of plug-positive females (n = 13-15) were given by gavage caffeine at 30, 60, and 120 mg/kg/day. Three additional groups received the same caffeine doses and were restrained for 2 hr/day. Control groups included restrained and unrestrained plug-positive mice not exposed to caffeine. All animals in the group concurrently exposed to 120 mg/kg/day of caffeine and restraint died during the experimental period. In the remaining groups, cesarean sections were performed on Day 18 of gestation, and the fetuses were weighed and examined for external, internal, and skeletal malformations and variations. Although maternal and embryo/fetal toxicity were observed at all caffeine doses, the adverse maternal and developmental effects were significantly enhanced in the groups concurrently exposed to caffeine and restraint. It was especially remarkable at 60 and 120 mg/kg/day. The results of this study suggest that maternal and developmental toxic effects might occur if high amounts of caffeine were consumed by women under a notable stress during pregnancy.     

Chronic alterations in ovine maternal corticosteroid levels influence uterine blood flow and placental and fetal growth

Previous work from this laboratory demonstrated that the elevation of maternal plasma corticosteroid concentrations during pregnancy is important for the support of fetal development. Reducing ovine maternal plasma cortisol concentrations to nonpregnant levels stimulates homeostatic responses that defend fetal blood volume. The present study was designed to test the hypothesis that chronic decreases or increases in maternal plasma cortisol concentration alter uterine and placental blood flow and morphology. Three groups of pregnant ewes and their fetuses were chronically catheterized and studied: ewes infused with cortisol (1 mg.kg(-1).day(-1); high cortisol), ewes adrenalectomized and underreplaced with cortisol (0.5 mg.kg(-1).day(-1); low cortisol), and control ewes. The normal increment in uterine blood flow between 120 and 130 days was eliminated in the low-cortisol ewes; conversely, uterine blood flow was increased in the high-cortisol group compared with the control group. Fetal arterial blood pressure was increased in the high-cortisol group compared with controls, but there was no increase in fetal arterial pressure from 120 to 130 days of gestation in the low-cortisol group. The fetuses of both low-cortisol and high-cortisol groups had altered placental morphology, with increased proportions of type B placentomes, and overall reduced fetal placental blood flow. The rate of fetal somatic growth was impaired in both low-cortisol and high-cortisol groups compared with the fetuses in the intact group. The results of this study demonstrate that maternal plasma cortisol during pregnancy is an important contributor to the maternal environment supporting optimal conditions for fetal homeostasis and somatic growth.     

Endocrine Regulation of Fetal Adipose Tissue Metabolism in the Pig: Role of Hydrocortisone

Glucocorticoids have been shown to be essential for the excessive fat deposition and development of obesity in several animal models. This study was performed to characterize the role of glucocorticoids in the developmental regulation of adipose tissue metabolism. On day 70 of gestation, pig fetuses were hypophysectomized by micro-cauterization. Hypophysectomized fetuses were implanted subcutaneously with hydrocortisone pellets or received no hormone replacement. Fetuses were removed by laparotomy on day 90 of gestation. Additional fetuses were hypophysectomized on day 70, implanted with hydrocortisone pellets on day 90 and removed on day 105 of gestation. Several intact fetuses were also implanted subcutaneously with hydrocortisone pellets during this later gestational period. Serum cortisol concentrations were reduced in hypophysectomized pigs at both fetal ages and were restored to intact levels by hydrocortisone treatment. Hydrocortisone supplementation enhanced lipolytic response to isoproterenol in intact fetuses but failed to restore lipolytic response to isoproterenol in hypophysectomized animals at either fetal age. Hydrocortisone induced a slight increase in lipogenesis in hypophysectomized fetuses when administered from 70 to 90 days of gestation and a more dramatic increase when administered from days 90 to 105 of gestation. However, hydrocortisone had no effect on basal or insulin stimulated lipogenesis in intact fetuses when administered from days 90 to 105 of gestation. These results indicate that hydrocortisone may have a primary influence on adipose tissue metabolism during late fetal development only in the absence of inhibition from counterregulatory hormones of pituitary origin.     

Low iron diet and parenteral cadmium exposure in pregnant rats: the effects on trace elements and fetal viability

The effects of latent iron deficiency combined with parenteral subchronic or acute cadmium exposure during pregnancy on maternal and fetal tissue distribution of cadmium, iron and zinc, and on fetal viability were evaluated. Timed-pregnant Sprague-Dawley rats were fed on semisynthetic test diets with either high iron (240 mg kg) or low iron (10 mg kg), and concomitantly exposed to 0, 3 or 5 mg cadmium (as anhydrous CdCl₂) per kilogram body weight. Animals were exposed to cadmium from gestation day 1 through 19 by subcutaneously implanted mini pumps (Subchronic exposure) or on gestation day 15 by a single subcutaneous injection (Acute exposure). All rats were killed on gestation day 19. Blood samples, selected organs and fetuses were removed and prepared for element analyses by atomic absorption spectrometry. Low iron diet caused decreases in maternal body weight, maternal and fetal liver weights, placental weights and tissue iron concentrations. By cadmium exposure, both subchronic and acute, tissue cadmium concentrations were increased and the increase was dose-related, maternal liver and kidney zinc concentrations were increased, and fetal zinc concentration was decreased. Cadmium concentration in maternal liver was additionally increased by low iron diet. Acute cadmium exposure caused lower maternal body and organ weights, high fetal mortality, and decreased fetal weights of survivors. In conclusion, parenteral cadmium exposure during pregnancy causes perturbations in essential elements in maternal and fetal compartments. Acute cadmium exposure in the last trimester of gestation poses a risk for fetal viability especially when combined with low iron in maternal diet.     

Amelioration of some metabolic effects produced by hyperthyroidism in late pregnant rats and their fetuses. Effects on lipids and proteins.

We have studied the effect of 40-45 days administration of 1 mg/kg thyroxine on protein and lipid metabolism in liver, heart, lungs, kidneys and adrenal glands of virgin and 21-day pregnant rats and their fetuses and placentae. The chronic administration of thyroid hormone produced significant increases in serum T3 and T4 in both groups as well as in organ weights and protein concentrations in virgin rats, but much smaller modifications in pregnant ones. Hyperthyroidism decreased the weight of fetal livers and increased that of placentae; protein content was increased in all fetal organs. Hyperthyroidism induced increases in phospholipid concentrations in all the organs and in total lipids only in liver and heart of adult rats, which were not counteracted by pregnancy. Pregnant rats had increases in total lipids in liver and kidneys and in adrenal phospholipids. In hyperthyroid fetuses there was an increase in hepatic total lipids and no changes in phospholipids. Hepatic lipogenesis (measured by in vivo incorporation of 3H2O into lipids) was increased by hyperthyroidism in virgin and pregnant rats, but the increase was significantly smaller in the pregnant hyperthyroid rats compared with the virgin ones. Fetal lipogenesis in liver and lung was not changed. In addition, an increase was observed in lipogenic enzyme (fatty acid synthetase and glucose-6-phosphate dehydrogenase) activities in hyperthyroid virgin rats which was prevented by pregnancy. In fetuses only pulmonary glucose-6-phosphate dehydrogenase was increased when expressed in terms of tissue weight. Our results indicate that the metabolic effect of hyperthyroidism is attenuated in pregnant rats and their fetuses, when compared with adult virgin rats, in most of the parameters studied.(ABSTRACT TRUNCATED AT 250 WORDS)