Perspective on prenatal polychlorinated biphenyl exposure and the development of the progeny nervous system (Review).

The developmental origins of health and disease concept illustrates that exposure in early life to various factors may affect the offspring's long­term susceptibility to disease. During development, the nervous system is sensitive and vulnerable to the environmental insults. Polychlorinated biphenyls (PCBs), which are divided into dioxin­like (DL­PCBs) and non­dioxin­like PCBs (NDL­PCBs), are synthetic persistent environmental endocrine­disrupting chemicals. The toxicological mechanisms of DL­PCBs have been associated with the activation of the aryl hydrocarbon receptor and NDL­PCBs have been associated with ryanodine receptor­mediated calcium ion channels, which affect neuronal migration, promote dendritic growth and alter neuronal connectivity. In addition, PCB accumulation in the placenta destroys the fetal placental unit and affects endocrine function, particularly thyroid hormones and the dopaminergic system, leading to neuroendocrine disorders. However, epidemiological investigations have not achieved a consistent result in different study cohorts. The present review summarizes the epidemiological differences and possible mechanisms of the effects of intrauterine PCB exposure on neurological development.

Valproate and folate: Congenital and developmental risks.

Increasing awareness of the congenital and developmental risks associated with the use of sodium valproate (VPA) has led to recent European guidelines designed to avoid the use of this drug in pregnancy if effective alternative treatments are available. In the general population, it is well established that periconceptual folic acid reduces the risk of neural tube defects (NTDs) and possibly other congenital abnormalities. We here review the evidence 1) that VPA interferes with one-carbon metabolism, including the transport of methylfolate into the brain and the placenta by targeting folate receptors; 2) that VPA effects on the folate metabolic system contribute to congenital and developmental problems associated with VPA exposure; and 3) that genetic factors, notably polymorphisms related to one-carbon metabolism, contribute to the vulnerability to these VPA-induced risks. Based on these facts, we propose that the standard periconceptual use of 400 µg of folic acid may not adequately protect against VPA or other antiepileptic drug (AED)-induced congenital or developmental risks. Pending definitive studies to determine appropriate dose, we recommend up to 5 mg of folic acid periconceptually in at-risk women with the caveat that the addition of supplementary vitamin B12 may also be prudent because vitamin B12 deficiency is common in pregnancy in some countries and is an additional risk factor for developmental abnormalities.

Cancer therapy and risk of congenital malformations in children fathered by men treated for testicular germ-cell cancer: A nationwide register study.

BACKGROUND: Because of the potential mutagenic effects of chemo- and radiotherapy, there is concern regarding increased risk of congenital malformations (CMs) among children of fathers with cancer. Previous register studies indicate increased CM risk among children conceived after paternal cancer but lack data on oncological treatment. Increased CM risk was recently reported in children born before paternal cancer. This study aims to investigate whether anti-neoplastic treatment for testicular germ-cell cancer (TGCC) implies additional CM risk. METHODS AND FINDINGS: In this nationwide register study, all singletons born in Sweden 1994-2014 (n = 2,027,997) were included. Paternal TGCC diagnoses (n = 2,380), anti-neoplastic treatment, and offspring CMs were gathered from the Swedish Norwegian Testicular Cancer Group (SWENOTECA) and the Swedish Medical Birth Register. Children were grouped based on +/- paternal TGCC; treatment regimen: surveillance (n = 1,340), chemotherapy (n = 2,533), or radiotherapy (n = 360); and according to time of conception: pre- (n = 2,770) or post-treatment (n = 1,437). Odds ratios (ORs) for CMs were calculated using logistic regression with adjustment for parental ages, maternal body mass index (BMI), and maternal smoking. Children conceived before a specific treatment acted as reference for children conceived after the same treatment. Among children fathered by men with TGCC (n = 4,207), 184 had a CM. The risk of malformations was higher among children of fathers with TGCC compared with children fathered by men without TGCC (OR 1.28, 95% confidence interval [CI] 1.19-1.38, p = 0.001, 4.4% versus 3.5%). However, no additional risk increase was associated with oncological treatment when comparing post-treatment-to pretreatment-conceived children (chemotherapy, OR = 0.82, 95% CI 0.54-1.25, p = 0.37, 4.1% versus 4.6%; radiotherapy, OR = 1.01, 95% CI 0.25-4.12, p = 0.98, 3.2% versus 3.0%). Study limitations include lack of data on use of cryopreserved or donor sperm and on seminoma patients for the period 1995-2000-both tending to decrease the difference between the groups with TGCC and without TGCC. Furthermore, the power of analyses on chemotherapy intensity and radiotherapy was limited. CONCLUSIONS: No additional increased risk of CMs was observed in children of men with TGCC treated with radio- or chemotherapy. However, paternal TGCC per se was associated with modestly increased risk for offspring malformations. Clinically, this information can reassure concerned patients.

MRI-guided intracerebral convection-enhanced injection of gliotoxins to induce focal demyelination in swine.

Demyelinating disorders such as multiple sclerosis (MS) or transverse myelitis are devastating neurological conditions with no effective cure. Prevention of myelin loss or restoration of myelin are key for successful therapy. To investigate the disease and develop cures animal models with good clinical relevance are essential. The goal of the current study was to establish a model of focal demyelination in the brain of domestic pig using MRI-guided gliotoxin delivery. The rationale for developing a new myelin disease model in the domestic pig was based on the fact that the brain in pigs is anatomically and histologically much more similar to that of humans compared to the rodent brain. For MRI-assisted gliotoxin injection, eight 30 kg pigs were subjected to treatment with lysolecithin (20, 30 mg/ml); or with ethidium bromide (0.0125, 0.05, 0.2 mg/ml). Animals were placed in an MRI scanner for intraparenchymal targeting of gliotoxin into the corona radiata (250 µl over 1h), with real-time monitoring of toxin distribution on T1 scans and monitoring of lesion evolution over seven days using both T1 and T2 scans. After the last MRI, animals were transcardially perfused and brains were processed for histological and immunofluorescent analysis. Gadolinium-enhanced T1 MRI during injection demonstrated biodistribution of the contrast (as a surrogate marker for toxin distribution) and its diffusion through the brain parenchyma. Lesion induction was confirmed on T2-weighted MRI and histopathology, thus enabling the establishment of optimal doses of gliotoxins. To conclude, MRI-guided focal demyelination in swine is accurate and provides real-time confirmation of gliotoxin, thus facilitating placement of focal lesions with high precision. This new model of focal demyelination can be used for further investigation and development of novel therapeutic approaches.

Brain microglia activation induced by intracranial administration of oligonucleotides and its pharmacological modulation.

Oligonucleotide overloading results in type I interferonopathies such as the Aicardi-Goutiéres Syndrome, a progressive encephalopathy determined by an immune response against endogenous DNA/RNA molecules. No therapy targeting pathogenic mechanisms is available for affected patients. Accordingly, we set up an in vitro/in vivo experimental model aimed at reproducing the pathogenic mechanisms of type I interferonopathies, in order to develop an effective pharmacological modulation and toxicological alterations caused by intracranial delivery of encapsulated CpG. The in vitro model used Aicardi-Goutiéres Syndrome immortalized lymphocytes activated by interferon I and co-cultured with human astrocytes; lymphocyte neurotoxicity was attenuated by the calcineurin-inhibitor Tacrolimus and by the anti-interferon monoclonal antibody Sifalimumab. The in vivo model was set up in mice by subcutaneous injection of encapsulated CpG oligonucleotides; the immune-stimulating activity was demonstrated by cytometric analysis in the spleen. To mime pathogenesis of type I interferonopathies in the central nervous system, CpG oligonucleotides were administered intracranially in mice. In the brain, CpG overload induced a rapid activation of macrophage-like microglial cells and focal accumulation mononuclear cells. The subcutaneous administration of Tacrolimus and, more potently, Sifalimumab attenuated CpG-induced brain alterations. These findings shed light on molecular mechanisms triggered by oligonucleotides to induce brain damage. Monoclonal antibodies inhibiting interferon seem a promising therapeutic strategy to protect brain in type I interferonopathies.

Repeated prenatal exposure to valproic acid results in cerebellar hypoplasia and ataxia.

Autism spectrum disorder (ASD) is a developmental brain disorder characterized by restricted and repetitive patterns of behavior, social and communication defects, and is commonly associated with difficulties with motor coordination. The etiology of ASD, while mostly idiopathic, has been linked to hereditary factors and teratogens, such as valproic acid (VPA). VPA is used clinically to treat epilepsy, mood disorders, and in the prevention of migraines. The use of VPA during pregnancy significantly increases the risk of ASD in the offspring. Neuropathological studies show decreased cerebellar function in patients with ASD, resulting in gait, balance and coordination impairments. Herein, we have exposed pregnant rats to a repeated oral dose of VPA on embryonic days 10 and 12 and performed a detailed investigation of the structure and function of the cerebellar vermis. We found that throughout all ten lobules of the cerebellar vermis, Purkinje cells were significantly smaller and expression of the calcium binding protein calbindin (CB) was significantly reduced. We also found that dendritic arbors of Purkinje cells were shorter and less complex. Additionally, animals exposed to a repeated dose of VPA performed significantly worse in a number of motor tasks, including beam walking and the rotarod. These results suggest that repeated embryonic exposure to VPA induces significant cerebellar dysfunction and is an effective animal model to study the cerebellar alterations in ASD.

Effects of lacosamide "a novel antiepileptic drug" in the early stages of chicken embryo development.

INTRODUCTION: Antiepileptic drugs (AEDs) are teratogens and confer a risk of congenital malformation. The estimated prevalence of major congenital malformations such as cardiac defects, facial clefts, hypospadias, and neural tube defects in epileptic women is 4-10 %, which represents a two- to fourfold increase in pregnant women compared to the general population. However, there are no clear data for newer drugs. Lacosamide (LCM), a novel AED, is the first of the third-generation AEDs to be approved as adjunctive therapy for the treatment of partial-onset seizures. There are no data on the pharmacokinetics of LCM during pregnancy, and only some published data have reported on its effects during pregnancy. METHODS: In this study, three different doses of LCM (0.12, 0.5, and 1.60 mg in 0.18 mL) were applied under the embryonic disks of specific pathogen-free Leghorn chicken embryos after a 30-h incubation. Incubation was continued for 80 h, at which time all embryos were evaluated macroscopically and microscopically. RESULTS: There was growth retardation in all of the LCM-treated groups. Major malformations increased in a dose-dependent manner and were mostly observed in the supratherapeutic group. CONCLUSION: Based on our data, LCM may cause growth retardation or major congenital malformations. Nevertheless, more extensive investigations of its reliability are needed.

Smaller Cerebellar Growth and Poorer Neurodevelopmental Outcomes in Very Preterm Infants Exposed to Neonatal Morphine.

OBJECTIVE: To examine the relationship between morphine exposure and growth of the cerebellum and cerebrum in very preterm neonates from early in life to term-equivalent age, as well as to examine morphine exposure and brain volumes in relation to neurodevelopmental outcomes at 18 months corrected age (CA). STUDY DESIGN: A prospective cohort of 136 very preterm neonates (24-32 weeks gestational age) was serially scanned with magnetic resonance imaging near birth and at term-equivalent age for volumetric measurements of the cerebellum and cerebrum. Motor outcomes were assessed with the Peabody Developmental Motor Scales, Second Edition and cognitive outcomes with the Bayley Scales of Infant and Toddler Development, Third Edition at 18 months CA. Generalized least squares models and linear regression models were used to assess relationships between morphine exposure, brain volumes, and neurodevelopmental outcomes. RESULTS: A 10-fold increase in morphine exposure was associated with a 5.5% decrease in cerebellar volume, after adjustment for multiple clinical confounders and total brain volume (P = .04). When infants exposed to glucocorticoids were excluded, the association of morphine was more pronounced, with an 8.1% decrease in cerebellar volume. Morphine exposure was not associated with cerebral volume (P = .30). Greater morphine exposure also predicted poorer motor (P < .001) and cognitive outcomes (P = .006) at 18 months CA, an association mediated, in part, by slower brain growth. CONCLUSIONS: Morphine exposure in very preterm neonates is independently associated with impaired cerebellar growth in the neonatal period and poorer neurodevelopmental outcomes in early childhood. Alternatives to better manage pain in preterm neonates that optimize brain development and functional outcomes are urgently needed.

Teratogenic effect of Carbamazepine use during pregnancy in the mice.

Carbamazepine use is the first choice of antiepileptic drugs among epileptic pregnant females. There are many inconclusive studies regard the safety of carbamazepine use during pregnancy. This study aims to investigate the morphological and histopathological teratogenic effects of carbamazepine use during pregnancy. The healthy pregnant females mice divided into equal five groups (each n=20). The first (control) group received distilled water/day. Second, third, fourth and fifth group received 8.75, 22.75, 52.5, 65 mg of carbamazepine/day respectively. Carbamazepine and water were given by gastric gavage throughout gestational period. Fetuses were delivered on the 18th day of gestation by hysterectomy. Fetal measurements and appearance were assessed with investigation the histopathological changes of brain and spinal cord. There was a significant decrease of weight, different organs weight, length, upper and lower limb length of mice in the first day of delivery in fifth group. There was a significant increase of weight, different organs weight, length, upper and lower limb length in the third group. Many congenital anomalies such as spina bifida, meromelia, microphalmia, oligodactyly, anencephaly, neurodegeneration of brain and spinal cord were noticedin fifth group. Teratogenic effect of carbamazepine represented as growth retardation and neurodevelopmental toxicity depending on its overdose degree.

Comment: valproate dose effects differ across congenital malformations.

Fetal valproate exposure has been associated with the highest risk of congenital malformations among antiepileptic drugs.(1) Valproate's effect is dose-dependent(1) and has been associated with multiple specific malformations.(2,3) Vadja et al.(4) examined data from the Australian Pregnancy Registry (1999-2012 data), which included 1,705 pregnancies with 436 valproate exposures.(4) They found that the use and dosages of valproate have fallen over the last 5 years. The rates of spina bifida and hypospadius in those exposed dropped with reducing dosages of valproate, but the rates of other malformations did not. Mean dosages for malformations were higher for spina bifida (2,000 mg/d) and hypospadius (2,417 mg/d) than all other malformations (1,083 mg/d).

Exposure to the JNK inhibitor SP600125 (anthrapyrazolone) during early zebrafish development results in morphological defects.

SP600125 (anthrapyrazolone) is a synthetic polyaromatic chemical that inhibits c-Jun N-terminal kinase (JNK) signaling by interfering with phosphorylation of c-Jun. To determine the pharmacological impact of SP600125 on zebrafish development, we incubated embryos in various concentrations of SP600125 from 18 h postfertilization (hpf) to 48 hpf. Embryos treated with 1.25 µm appeared with occasional pericardium edema. Treatment with 12.5 µm resulted in complete mortality by 120 hpf, preventing an assessment of physiological defects. Embryos treated with 5 µm exhibited slowed overall growth, a delay in hatching and numerous morphological defects such as pericardium edema, yolk sac edema, swim bladder deflation, bent vertebrae and eye and jaw malformations. Whole-mount immunohistochemical studies using an anti-acetylated ß-tubulin antibody confirmed developmental defects in the nervous system. Within the retina, fish treated with 1.25 µm showed a mild reduction of immunoreactivity. Immunoreactivity in the retina was further reduced in fish treated with 5 µm of SP600125. In these fish, eyes and olfactory organs were half the size compared with other groups. Multiple lenses were observed in 67% of these fish. A second experiment with a shorter exposure period of SP600125 (6 h) presented significantly fewer morphological defects. The treatment led to a delay in hatching, and increased incidences of swim bladder deflation and pericardium edema with increasing concentrations. In summary, SP600125 caused developmental abnormalities during zebrafish organogenesis starting at 1.25 µm and the defects were exacerbated with increasing concentrations. Our study suggests that SP600125 at 1.25 µm and beyond has devastating consequences for zebrafish development.

Non-occupational exposure to paint fumes during pregnancy and risk of congenital anomalies: a cohort study.

BACKGROUND: Occupational exposure to organic solvents during the 1st trimester of pregnancy has been associated with congenital anomalies. Organic solvents are also used in the home environments in paint products, but no study has investigated the effect of such exposure in a general population. METHODS: We studied associations between residential exposure to paint fumes during the 1st trimester of pregnancy and predefined subgroups of congenital anomalies, using data from the Danish National Birth Cohort (DNBC). During 2001 and 2003, a total of 20,103 pregnant women, enrolled in the DNBC, were interviewed in the 30th week of gestation about the use of paint in their residence during pregnancy. By the end of first trimester, information about smoking habits, alcohol consumption and occupation were collected. Information on congenital anomalies was obtained from national registers. Associations were examined by estimating odds ratios (OR) using logistic regression. RESULTS: In total 1404 women (7%) had been exposed to paint fumes during the 1st trimester of pregnancy and 1086 children were diagnosed with congenital anomalies; 73 children with congenital anomalies had been exposed to paint fumes in utero. Exposure to paint fumes seemed positively associated with congenital anomalies of the nervous system (OR 2.19, 95% confidence interval (CI) 0.76 to 6.32), ear, face and neck (OR 2.15, 95% CI 0.84 to 5.55) and the renal system (OR 2.16, 95% CI 1.02 to 4.58) after adjustment for maternal age, smoking, alcohol consumption and occupational solvent exposure. Congenital anomalies in the remaining subgroups were not associated with the exposure. CONCLUSIONS: Our results suggest that in the general population, exposure to paint fumes during the 1st trimester of pregnancy may increase the risk of some types of congenital anomalies, but the findings need to be confirmed.

A prospective cohort study of the prevalence of growth, facial, and central nervous system abnormalities in children with heavy prenatal alcohol exposure.

BACKGROUND: Most children who are exposed to large quantities of alcohol in utero do not develop fetal alcohol syndrome (FAS). Population-based prospective data on the risk of developing components of fetal alcohol spectrum disorders (FASD), however, are limited. METHODS: This was a prospective cohort study of 9,628 women screened during their first prenatal appointment in Chile, which identified 101 who consumed at least 4 drinks/d (exposed) matched with 101 women with no reported alcohol consumption during pregnancy (unexposed). Detailed alcohol consumption data were collected during the pregnancy. Children were evaluated up to 8.5 years of age by clinicians masked to exposure status. RESULTS: One or more functional central nervous system abnormalities were present in 44.0% (22/50) of the exposed children compared to 13.6% (6/44) of the unexposed (p = 0.002). Growth restriction was present in 27.2% (25/92) of the exposed and 12.5% (12/96) of the unexposed (p = 0.02). Abnormal facial features were present in 17.3% (14/81) of the exposed children compared to 1.1% (1/89) of the unexposed children (p = 0.0002) by direct examination. Of the 59 exposed children with data available to detect at least 1 abnormality, 12 (20.3%) had no abnormalities. Binge drinking from conception to recognition of pregnancy (OR = 1.48 per day, 95% CI: 1.15 to 1.91, p = 0.002) and after recognition of pregnancy (OR= 1.41 per day, 95% CI: 1.01 to 1.95, p = 0.04) and total number of drinks consumed per week from conception to recognition of pregnancy (OR = 1.02 per drink, 95% CI: 1.01 to 1.04, p = 0.0009) were significantly associated with abnormal child outcome. CONCLUSIONS: After exposure to heavy alcohol consumption during pregnancy, 80% of children had 1 or more abnormalities associated with alcohol exposure. Patterns of alcohol use that posed the greatest risk of adverse outcomes were binge drinking and high total weekly intake. Functional neurologic impairment occurred most frequently and may be the only sign to alert physicians to prenatal alcohol exposure.

Brain anomalies in children exposed prenatally to a common organophosphate pesticide.

Prenatal exposure to chlorpyrifos (CPF), an organophosphate insecticide, is associated with neurobehavioral deficits in humans and animal models. We investigated associations between CPF exposure and brain morphology using magnetic resonance imaging in 40 children, 5.9-11.2 y, selected from a nonclinical, representative community-based cohort. Twenty high-exposure children (upper tertile of CPF concentrations in umbilical cord blood) were compared with 20 low-exposure children on cortical surface features; all participants had minimal prenatal exposure to environmental tobacco smoke and polycyclic aromatic hydrocarbons. High CPF exposure was associated with enlargement of superior temporal, posterior middle temporal, and inferior postcentral gyri bilaterally, and enlarged superior frontal gyrus, gyrus rectus, cuneus, and precuneus along the mesial wall of the right hemisphere. Group differences were derived from exposure effects on underlying white matter. A significant exposure × IQ interaction was derived from CPF disruption of normal IQ associations with surface measures in low-exposure children. In preliminary analyses, high-exposure children did not show expected sex differences in the right inferior parietal lobule and superior marginal gyrus, and displayed reversal of sex differences in the right mesial superior frontal gyrus, consistent with disruption by CPF of normal behavioral sexual dimorphisms reported in animal models. High-exposure children also showed frontal and parietal cortical thinning, and an inverse dose-response relationship between CPF and cortical thickness. This study reports significant associations of prenatal exposure to a widely used environmental neurotoxicant, at standard use levels, with structural changes in the developing human brain.

Perinatal dexamethasone-induced alterations in apoptosis within the hippocampus and paraventricular nucleus of the hypothalamus are influenced by age and sex.

Exposure to high levels of glucocorticoids (GCs) during development leads to long-term changes in hypothalamic-pituitary-adrenal (HPA) axis regulation, although little is known about the neural mechanisms that underlie these alterations. In this study, we investigated the effects of late gestational (days 18-22) or postnatal (days 4-6) administration of the GC receptor agonist dexamethasone (DEX) on an apoptosis marker in two brain regions critical to HPA axis regulation, the hippocampus and the hypothalamic paraventricular nucleus (PVN). One day after the final DEX injection, male and female rats were sacrificed, and brains were processed for immunohistochemical detection of cleaved caspase-3, an apoptotic cell death indicator. DEX increased cleaved caspase-3 immunoreactivity in the CA1 hippocampal region of both sexes following prenatal but not postnatal treatment. Prenatal DEX also increased caspase-3 immunoreactivity in the CA3 region, an elevation that tended to be greater in females. In contrast, postnatal DEX resulted in a much smaller, albeit significant, induction in CA3 caspase-3 compared with prenatal treatment. Quantitative real-time PCR analysis revealed that prenatal but not postnatal DEX-induced hippocampal cleaved caspase-3 correlated with elevated mRNA of the proapoptotic gene Bad. Few caspase-3-ir cells were identified within the PVN regardless of treatment age, although postnatal but not prenatal DEX increased this number. However, the region immediately surrounding the PVN (peri-PVN) showed significant increases in caspase-3-ir cells following pre- and postnatal DEX. Together these findings indicate that developmental GC exposure increases apoptosis in HPAaxis-associated brain regions in an age- and sex-dependent manner.

Neuropathology of the area postrema in sudden intrauterine and infant death syndromes related to tobacco smoke exposure.

The area postrema is a densely vascularized small protuberance at the inferoposterior limit of the fourth ventricle, outside of the blood-brain barrier. This structure, besides to induce emetic reflex in the presence of noxious chemical stimulation, has a multifunctional integrative capacity to send major and minor efferents to a variety of brain centers particularly involved in autonomic control of the cardiovascular and respiratory activities. In this study we aimed to focus on the area postrema, which is so far little studied in humans, in a large sample of subjects aged from 25 gestational weeks to 10 postnatal months, who died of unknown (sudden unexplained perinatal and infant deaths) and known causes (controls). Besides we investigated a possible link between alterations of this structure, sudden unexplained fetal and infant deaths and maternal smoking. By the application of morphological and immunohistochemical methods, we observed a significantly high incidence of alterations of the area postrema in fetal and infant victims of sudden death as compared with age-matched controls. These pathological findings, including hypoplasia, lack of vascularization, cystic formations and reactive gliosis, were related to maternal smoking. We hypothesize that components from maternal cigarette smoke, particularly in pregnancy, could affect neurons of the area postrema connected with specific nervous centers involved in the control of vital functions. In conclusion, we suggest that the area postrema should be in depth examined particularly in victims of sudden fetal or infant death with smoker mothers.

Studying significance of apoptosis in mediating tolbutamide-induced teratogenesis in vitro.

The incidence of type 2 diabetes mellitus (non-insulin-dependent diabetes mellitus) is growing worldwide and poses a serious public health problem in a current paradigm of changing life style and food habits. Tolbutamide (sulfonylurea) is among the commonly used anti-diabetic drugs worldwide for treating type 2 diabetes and is known to cause congenital malformations in animals. In this study, the effect of tolbutamide on major organogenesis period and the possible involvement of apoptosis in mediating congenital malformations have been carried out. In the present study design, post-implantation rat embryos of day 11 were cultured for 24 h with various concentrations of tolbutamide, i.e., 10, 100, and 1000 µg/mL cultures, respectively. The growth and developmental of each embryo was evaluated and compared with control ones for the presence of any malformations. The tolbutamide decreased all growth and developmental parameters in a concentration-dependent manner, when compared with control. However, exposure to tolbutamide at 10 µg/mL culture did not show any significant effect on embryonic growth and development in vitro. In parallel to this, flow cytometric analysis (cell cycle and annexin V binding) and DNA fragmentation assay were carried out followed by quantitation by 3'-OH labeling of cultured rat embryos to examine the role of apoptosis in bringing about tolbutamide-induced teratogenesis. All results were found to be dose dependent and an increase in apoptosis in embryonic tissues may be related to the increased risk of congenital malformations. The outcome of the research suggested that apoptosis might be involved in mediating teratogenesis of tolbutamide in vitro. Further research is warranted to fully understand this mechanism.