GD20 Fetuses Research Articles

Optimal testing time for cerebral heterotopia formation in the rat comparative thyroid assay, a downstream indicator for perinatal thyroid hormone insufficiency.

In a past study, we proposed a modified Comparative Thyroid Assay (CTA) with additional examinations of brain thyroid hormone (TH) concentrations and brain histopathology but with smaller group sizes. The results showed that the modified CTA in Sprague Dawley rats detected 10 ppm 6-propylthiouracil (6-PTU)-induced significant suppressions of serum/brain TH concentrations in offspring. To confirm the reliability of qualitative brain histopathology and identify the optimal testing time for heterotopia (a cluster of ectopic neurons) in the modified CTA, brain histopathology together with serum/brain TH concentrations were assessed in GD20 fetuses and PND2, 4, 21, and 28 pups using a similar study protocol but with a smaller number of animals (N=3-6/group/time). Significant hypothyroidism was observed and brain histopathology revealed cerebral heterotopia formation in PND21 and PND28 pups, with likely precursor findings in PND2 and PND4 pups but not in GD20 fetuses. This study confirmed that the optimal testing time for cerebral heterotopia in rat CTA was PND21 and thereafter. These findings suggest that cerebral heterotopia assessment at appropriate times may be a useful alternative to the original CTA design.

Prenatal test cohort of a modified rat comparative thyroid assay adding brain thyroid hormone measurements and histology but lowering group size appears able to detect disruption by sodium phenobarbital

The Comparative Thyroid Assay (CTA, USEPA) is a screening test for thyroid hormone (TH) disruption in peripheral blood of dams and offspring. Recently, we began investigating feasible improvements to the CTA by adding examination of offspring brain TH concentrations and brain histopathology. In addition, we hypothesize that the number of animals required could be reduced by 50 % while still maintaining sensitivity to characterize treatment related changes in THs. Previously, we showed that the prenatal test cohort of the modified CTA could detect 1000 ppm sodium phenobarbital (NaPB)-induced suppression of brain T3 (by 9 %) and T4 (by 33 %) with no significant changes in serum T3 and T4 (less than 8 %). In the current study we expanded the dose response in a prenatal test cohort. Pregnant SD rats (N = 10/group) were exposed to 0, 1000 or 1500 ppm NaPB in the diet from gestational days (GD) 6 to GD20. Serum THs concentrations in GD20 dams together with serum/brain THs concentrations and brain histopathology in the GD20 fetuses were examined. NaPB dose-dependently suppressed serum T3 (up to −26 %) and T4 (up to −44 %) in dams, with suppression of T3 in serum (up to −26 %) and brain (up to −18 %) and T4 in serum (up to −26 %) and brain (up to −29 %) of fetuses but without clear dose dependency. There were no remarkable findings that deviated significantly from controls in GD20 fetal brain by qualitative histopathology. Overall, the present study suggests that the prenatal test cohort of this modified CTA is able to detect the expected fetal TH disruptions by prenatal exposure to NaPB, while also reducing the number of animals used by 50 %, consistent with the results of our previous study. These findings add to the suggestion that lowering group sizes and adding endpoints may be a useful alternative to the original CTA design.

2,4,6-Tribromophenol Disposition and Kinetics in Pregnant and Nursing Sprague Dawley Rats.

2,4,6-Tribromophenol (TBP, CAS no. 118-79-6) is a brominated chemical used as a precursor, flame retardant, and wood antifungal agent. TBP is detected in environmental matrices and biota, including human breast milk, placenta, and serum. To address reports of TBP accumulation in human placenta and breast milk, studies were conducted to characterize TBP disposition and toxicokinetics in timed-pregnant or nursing Sprague Dawley rats following a single oral dose to the dam. Animals were administered [14C]-TBP (10 μmol/kg, 25 µCi/kg, 4 ml/kg) by gavage on gestation day 12 and 20, or postnatal day 12 and serially euthanized between 15 min and 24 h for collection of blood and tissues from the dam and fetuses/pups. Observed plasma TBP Cmax (3 and 7 nmol/ml) occurred at 15 min in both GD12 and GD20 dams while Cmax (3 nmol/ml) was observed at 30 min for PND12 dams. Concentrations in tissues followed plasma concentrations, with kidneys containing the highest concentrations at 30 min. GD12 litters contained a sustained 0.2%-0.3% of the dose (5-9 nmol/litter) between 15 min and 6 h while GD20 fetuses (2%-3%) and placentas (0.3%-0.5%) had sustained levels between 30 min and 12 h. The stomach contents (approx. 1 nmol-eq/g, 6-12 h), livers (0.04-0.1 nmol-eq/g) and kidneys (0.1-0.2 nmol-eq/g) of PND12 pups increased over time, indicating sustained exposure via milk. Systemic exposure to TBP and its metabolites occurs in both the directly exposed mother and the indirectly exposed offspring and is rapid and persistent after a single dose in pregnant and nursing rats.

Programming of a developmental imbalance in hypothalamic glutamatergic/GABAergic afferents mediates low basal activity of the hypothalamic-pituitary-adrenal axis induced by prenatal dexamethasone exposure in male offspring rats

This study was intended to demonstrate that prenatal dexamethasone exposure (PDE) can induce low basal activity of the hypothalamic-pituitary-adrenal axis (HPAA) in male offspring rats and explore the underlying mechanism. Pregnant rats were subcutaneously administered 0.2 mg/kg/d dexamethasone from gestational day (GD) 9 to GD20. Male GD20 fetuses and postnatal day 85 adult male offspring rats were sacrificed under anesthesia. Hypothalamic cells were from GD20∼postnatal day (PD) 7 fetal male rats, treated with different concentrations of dexamethasone and the glucocorticoid receptor (GR) antagonist mifepristone for 5 days. The results suggested that dexamethasone enhanced the expression of hypothalamic L-glutamic acid decarboxylase (GAD) 67 by activating GR, further stimulating the conversion of glutamate to gamma-aminobutyric acid (GABA) and inducing an imbalance in glutamatergic/GABAergic afferents in the hypothalamic paraventricular nucleus (PVN). This imbalance change was maintained postnatally, leading to the inhibition of parvocellular neurons, and mediating the low basal activity of the HPAA in PDE offspring rats, which was manifested by decreased levels of blood adrenocorticotropic hormone and corticosterone as well as reduced expression levels of corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) in the hypothalamus. Programming of a developmental imbalance in glutamatergic/GABAergic afferents in the PVN is a potential mechanism responsible for low basal activity of the HPAA in male PDE rats.

Induction of a thoracolumbar supernumerary rib in rat developmental toxicity studies: A short discussion on the critical window.

Thoracolumbar supernumerary ribs (TSRs) are classified as less severe skeletal anomalies in rat developmental toxicity studies, although their incidence is relatively high in rodent studies. To investigate the characteristics of the critical window for chemically-induced TSR, in this study, rats were administered 5-fluorocytocine (5-FC) or sodium salicylate (SAL) at one of three time periods on gestational day (GD) 9, early morning (7:00 am), midday (12:00 pm to 1:00 pm), or late afternoon (4:00 pm or 7:00 pm). The incidence of TSR and other anomalies were assessed in GD20 fetuses. A single treatment with both chemicals on GD9-induced TSR, with the incidence highest when administered at 7:00 Am, decreasing gradually when administered later. This trajectory was clearer in rats treated with 5-FC than with SAL. The critical period of TSR induction is shorter in rats administered 5-FC than SAL. The characteristics of the critical window may cause variability in the incidence of TSR observed in developmental toxicity studies.

The EFSA WEB app based on PROAST: data table generation

Our previous in vivo studies showed that prenatal caffeine exposure (PCE) could restrain the development of chondrogenesis, which may delay fetal articular cartilage development and increase susceptibility to osteoarthritis in adults. So, the goal of the current study is to clarify theincreasing susceptibility to adult osteoarthritis in caffeine-exposed female offspring and its’mechanism. Pregnant rats were treated with 120 mg/kg·d caffeine or equal volumes of saline from gestational day (GD) 9 to 20. knee joints were collected from GD20 female fetuses and 18-week old female offspring which was treated with strenuous running for 6 weeks (55 min/day at 20 m/min) load to induce osteoarthritis. Knee joints from GD20 fetuses and adult offspring were collected for histochemistry and immunohistochemistry. Next, chondrocytes were isolated from 1-day-old newborn rats and in vitro studies were conducted where the cells in primary culture were exposed to 1, 10, and 100 μM caffeine and 250, 500, and 1,250 nM corticosterone. Insulin-like growth factor 1 (IGF-1) signal pathway genes’ expression levels in fetal chondrocytes were studied, and IGF-1 histone acetylation was detected in vitro. Immunohistochemical results showed low expression levels of IGF-1 signaling genes (IGF-1, IRS-1, AKT, and COL2A1) both in fetal and adult cartilage with PCE. For adult offspring, histological results and Mankin score revealed increased cartilage destruction and accelerated osteoarthritis progression in PCE group with strenuous running exercise. Analysis in vitro revealed that caffeine and corticosterone impeded the expression of IGF-1 signaling pathway aggrecan and COL2A1 genes, but only corticosterone decreased H3K9 and H3K27 acetylation in the IGF-1 promoter region. In concluson, PCE low functional programmed cartilage IGF-1 by histone acetylation modification via overexposure to corticosterone and delayed articular cartilage development from fetus to adults. Then, the delayed cartilage development increased susceptibility to osteoarthritis in offsprings.

Prenatal caffeine exprosure increases adult female offspring rat’s susceptibility to osteoarthritis via low-functional programming of cartilage IGF-1 with histone acetylation

Our previous in vivo studies showed that prenatal caffeine exposure (PCE) could restrain the development of chondrogenesis, which may delay fetal articular cartilage development and increase susceptibility to osteoarthritis in adults. So, the goal of the current study is to clarify theincreasing susceptibility to adult osteoarthritis in caffeine-exposed female offspring and its’mechanism. Pregnant rats were treated with 120 mg/kg·d caffeine or equal volumes of saline from gestational day (GD) 9 to 20. knee joints were collected from GD20 female fetuses and 18-week old female offspring which was treated with strenuous running for 6 weeks (55 min/day at 20 m/min) load to induce osteoarthritis. Knee joints from GD20 fetuses and adult offspring were collected for histochemistry and immunohistochemistry. Next, chondrocytes were isolated from 1-day-old newborn rats and in vitro studies were conducted where the cells in primary culture were exposed to 1, 10, and 100 μM caffeine and 250, 500, and 1,250 nM corticosterone. Insulin-like growth factor 1 (IGF-1) signal pathway genes’ expression levels in fetal chondrocytes were studied, and IGF-1 histone acetylation was detected in vitro. Immunohistochemical results showed low expression levels of IGF-1 signaling genes (IGF-1, IRS-1, AKT, and COL2A1) both in fetal and adult cartilage with PCE. For adult offspring, histological results and Mankin score revealed increased cartilage destruction and accelerated osteoarthritis progression in PCE group with strenuous running exercise. Analysis in vitro revealed that caffeine and corticosterone impeded the expression of IGF-1 signaling pathway aggrecan and COL2A1 genes, but only corticosterone decreased H3K9 and H3K27 acetylation in the IGF-1 promoter region. In concluson, PCE low functional programmed cartilage IGF-1 by histone acetylation modification via overexposure to corticosterone and delayed articular cartilage development from fetus to adults. Then, the delayed cartilage development increased susceptibility to osteoarthritis in offsprings.

Reproductive toxicity of linuron following gestational exposure in rats and underlying mechanisms

Linuron is a widely used herbicide in agriculture; its endocrine disruptive toxicity has recently received public attention. This study was designed to examine the developmental toxicity of linuron on the reproductive system of male offspring following maternal exposure. Mother rats received oral gavages of linuron, once daily, at the dose of 0, 50, 100, 150 or 200mg/kg, from gestational day (GD)13 to GD18; gonadal organs from GD20 fetuses were examined. Data indicated that exposed male offspring had a significantly shortened anogenital distance. Pathological examination further revealed a lack of fusion in the urogenital fold in treated fetuses, the damaged seminiferous tubules, and the injured Leydig cell ultrastructure. Analysis of serum testosterone concentrations at postnatal day (PND)2 showed a significant dose-related reduction (about 33.7–58.75%, r=−0.838, p<0.05) as compared to controls. Immunohistochemical results demonstrated a significantly reduced expression of enzymes pertinent to the testosterone production including P450scc, 3β-HSD, and PCNA in Leydig cells (p<0.05). qPCR studies confirmed decreased levels of mRNAs encoding P450scc, 3β-HSD and PCNA (p<0.05). Taken together, these data suggest that maternal exposure to linuron hampers the male gonadal organ development; this appears to be due to linuron’s direct action on the production of testosterone in fetal and postnatal offspring.

Effect of in utero exposure to endocrine disruptors on fetal steroidogenesis governed by the pituitary-gonad axis: a study in rats using different ways of administration.

The effects of endocrine disruptors on testicular steroidogenesis in fetal rats were investigated in a study involving in utero exposure. In the major part of this study, pregnant rats at gestational day (GD)15 were given a single oral administration of the test substance, and then the expression of the following mRNAs in GD20 fetuses was determined: testicular steroidogenic acute-regulatory protein (StAR), a cholesterol transporter mediating the rate-limiting step of steroidogenesis, a ß-subunit of pituitary luteinizing hormone (LH), and a regulator of gonadal steroidogenesis. Among the substances tested, only di(2-ethylhexyl)phthalate (DEHP) reduced the expression of fetal testicular StAR. The others listed below exhibited little effect on fetal StAR: 2,2',4,4'-tetrabromodiphenylether, tributyltin chloride, atrazine, permethrin, cadmium chloride (Cd), lead acetate (Pb) and methylmercury (CH3HgOH). None of them, including DEHP, lacked the ability to reduce the expression of pituitary LHß mRNA. The present study also examined the potential of metals as modifiers of fetal steroidogenesis by giving them to pregnant dams in drinking water during GD1 and GD20. Under these conditions, Cd and Pb at a low concentration (0.01 ppm) significantly attenuated the fetal testicular expression of StAR mRNA without a concomitant reduction in LHß. No such effect was detected with CH3HgOH even at 1 ppm. These results suggest that: 1) DEHP, Cd and Pb attenuate the fetal production of sex steroids by directly acting on the testis, and 2) chronic treatment during the entire gestational period is more useful than a single administration for determining the hazardous effect of a suspected endocrine disruptor on fetal steroidogenesis.

Effects of Oral Exposure to Arsenobetaine During Pregnancy and Lactation in Sprague-Dawley Rats

Arsenobetaine (ASB) is the major form of arsenic (As) in seafood sources such as molluscs and fish. Limited data demonstrated that ASB toxicity in mammals is minimal; however, data on possible reproductive effects are lacking. This study investigated the tissue distribution and developmental effects of ASB during pregnancy, early postnatal life, and development to adulthood. Pregnant rats were randomly assigned to 3 cohorts and gavaged daily from gestational day 8 (GD8) with ASB in deionized water at 0, 0.1, 1, or 10 mg/kg body weight (bw)/d. Cohort 1 dams were sacrificed on GD20 (n = 6 per dose group), cohort 2 dams and pups were sacrificed on postnatal day 13 (PND13; n = 4 dams per dose group), and cohort 3 pups (n = 2 dams per dose group) were sacrificed on PND90. Residue analysis detected significant levels of ASB in livers of cohort 1 dams and lower levels in cohort 1 GD20 fetuses, as well as in cohort 2 male and female offspring, indicating placental transfer from the maternal circulation in utero. Trace amounts of ASB in dams’ milk were found only in the 10-mg/kg bw/d dose cohort 2 (PND13), demonstrating that lactational transfer was limited. ASB levels in liver varied during pregnancy, lactation, and postweaning, with levels falling rapidly as these physiological states progress. Although transfer of ASB through the placenta to the fetuses and to a limited extent through milk was confirmed, ASB exposure during pregnancy and lactation appeared to produce no teratogenic or deleterious effects on reproductive development.

Nonlinear imaging study of extracellular matrix in chemical‐induced, developmental dissecting aortic aneurysm: Evidence for defective collagen type III

Using a recent model of dissecting aortic aneurysm (DAA) caused by in utero exposure to semicarbazide, we examined the elastin and collagen using standard methods and two nonlinear imaging techniques, multiphoton fluorescence (MPF) and second harmonic generation (SHG) microscopy. Sprague-Dawley rat dams were given semicarbazide (6.13 mg/kg/day i.p.) from gestational days 14 to 20 (GD14-20). Fetuses were harvested on GD20 and pups on postnatal day 1 (PND1), PND7, and PND28; matched controls were from dams treated with saline. Aortic immunohistopathology and collagen/elastin signal intensity via MPF and SHG microscopy at an excitation wavelength of 800 nm were studied. Massive DAA of the aortic arch occurred in nearly 100% of pups at birth (i.e., no GD20 fetuses showed lesions). MPF and SHG demonstrated that collagen was significantly degraded at GD20 and in newborns, but normalized by PND28. GD20 fetuses and newborn pups showed a decreased content of medial and adventitial collagen type III in pooled aortas by Western blot and immunohistochemistry. In 7- and 28-day-old pups resolution of DAA blood in vascular media and a recovery of stainable collagen type III was found. Elastin in healed DAA (PND28 pups) was focally disorganized. MPF and SHG microscopy provide sensitive and high-resolution information on aortic elastin and collagen. In this model of DAA, collagen displays aberrant imaging quality likely linked to a marked decrease in collagen type III in the developing extracellular matrix. Birth Defects Research (Part A) 2008.

Sex differences and androgen-dependent regulation of aromatase (CYP19) mRNA expression in the developing and adult rat brain

Sex differences, androgen dependence and asymmetries of aromatase activity have been reported during ontogeny of the rat. It remains to be elucidated, however, whether the changes in aromatase activity are reflected by similar changes in specific mRNA levels. In addition, very little is known regarding mechanism(s) underlying such differential regulation of aromatase expression. To address these questions, we have employed the in situ hybridization (ISH) technique to examine specific mRNA levels in the brain of both male and female rats at selected stages of development. In prenatal stages of development, at gestational day (GD) 18 and 20, aromatase mRNA was detected in several hypothalamic and limbic brain regions. Semiquantitative analysis of aromatase mRNA did not reveal statistically significant sex differences in any of these regions (except in one experiment at GD20, when a sex difference was found in the medial preoptic nucleus). In contrast, clear sex differences were determined at postnatal day (PN) 2; male animals contained significantly more aromatase mRNA in the bed nucleus of the stria terminalis (BST) and thesexually dimorphic nucleus of the preoptic area (SDN) compared to female rats. Four days later in development, at PN6, sex differences of aromatase mRNA signals were observed in the BST, but were no longer detectable in the SDN. At PN15 and in adult animals, no sex differences could be determined. The effect of flutamide treatment (50 mg/kg/day) was investigated in GD20 fetuses as well as in adult rats. No statistically significant changes in aromatase mRNA expression were found in either case. In summary, our results suggest that differential regulation of aromatase mRNA expression during the critical period of sexual differentiation might, in part, account for the establishment of some of the many sexually dimorphic parameters of the rat brain. The role of androgens in the regulation of the sex-specific and developmental expression of aromatase mRNA in the rat brain remains to be clarified.

In utero ethanol exposure retards growth and alters morphology of cortical cultures: GM1 reverses effects.

Ethanol, a developmental neurotoxin, alters plasma membranes' physicochemical properties affecting embryogenesis, cell migration, differentiation, and synaptogenesis. In a previous study using a model for fetal alcohol effects, GM1 ganglioside treatment was shown to reduce ethanol-induced accumulation of endogenous GM1 and fatty acid ethyl esters in rat fetuses. The present study was initiated to define further the in utero effects of ethanol and the capacity of GM1 treatment to ameliorate such effects. Wistar dams were exposed to ethanol (intragastrically) on gestation day (GD) 7 and GD8 and GD13 and GD14. GM1 ganglioside (10 mg/kg, im) was given 24 hr before ethanol administration. Cortical cultures were derived from GD15 and GD20 fetuses. GM1, which is highly localized on the cellular plasma membrane outer surface of CNS cells, was used as a marker molecule to assess cell integrity. Cholera toxin/antitoxin/fluorescence immunohistochemistry was used to localize GM1. Results indicate that the brief in utero exposure to ethanol affected cell growth and morphology. A marked retardation of cell development and arborization was observed as early as 24 hr after plating. Ethanol-exposed cells evidenced considerably altered GM1 localization. Such alterations likely reflect losses of membrane integrity. These in utero ethanol-induced pathologies are remarkably diminished in cultures derived from ethanol-exposed fetuses of dams treated with GM1.

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.