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Valproic acid increases embryonic p27kip1 and caspase-3 expression: A potential mechanism for valproic acid induced neural tube defects
176
Posters / Reproductive Toxicology 32 (2011) 164–179
liferation related pathways and induced neural related pathways over time. This system appears promising for studying compound effects on neural differentiation in a mechanistic approach. doi:10.1016/j.reprotox.2011.06.107 Valproic acid increases embryonic p27kip1 and caspase-3 expression: A potential mechanism for valproic acid induced neural tube defects Emily W.Y. Tung ∗ , Louise M. Winn Queen’s University, Kingston, Ontario, Canada Introduction: Exposure to the anticonvulsant valproic acid (VPA) during the first trimester of pregnancy is associated with an increased risk of congenital malformations including heart defects, craniofacial abnormalities, skeletal and limb defects, and most frequently, neural tube defects (NTDs). The mechanisms by which VPA induces teratogenic effects are not fully understood, although previous studies support a role for histone deacetylase (HDAC)mediated gene expression changes, that lead to cell cycle arrest and cell death. HDAC1 knockout mice do not live past organogenesis, have reduced proliferation rates, and have increased levels of p21WAF1/CIP1 and p27KIP1 that are attributed to histone hyperacetylation in their promoter regions. The purpose of this study was to measure changes in the expression of proteins involved in cell cycle inhibition and apoptosis to further elucidate the molecular changes that occur following VPA exposure. Methods: CD-1 mice were bred, and females with a vaginal plug were separated and denoted as gestational day (GD) 1. On the morning of neural tube closure (GD 9), pregnant dams were administered a teratogenic dose of VPA (400 mg/kg) and embryos extracted 0.5, 1, 3, 6, and 24 h after injection. Western blotting and immunohistochemistry were performed for p21WAF1/CIP1 , p27KIP1 , and cleaved caspase-3. Results: Expression and localization of p21WAF1/CIP1 were not altered following maternal VPA administration. p27KIP1 and cleaved caspase-3 expression were significantly increased 3 and 6 h following VPA exposure. Immunohistochemistry revealed increased staining for p27KIP1 in the neuroepithelium and mesenchyme, and cleaved caspase-3 was notably increased in the neuroepithelium. Conclusions: Our results support our hypothesis that dysregulation of cell cycle inhibition and increased apoptosis in the head region of the embryo contribute to the mechanism of VPA-induced NTDs. doi:10.1016/j.reprotox.2011.06.108 What if your left leg is longer than your right leg? Fluctuating asymmetry as a sensitive indicator of developmental stress in rabbit fetuses Stefan Van Dongen a , Jessica Bots a , Matteo Breno a , Peter Delille b , Luc De Schaepdrijver b a b
University of Antwerp, Antwerp, Belgium Janssen Research and Development, Beerse, Belgium
Toxicologists are constantly searching for efficient indicators of the possible toxic effects of compounds. Traditionally for reproduction toxicology experiments, model organisms are exposed to different concentrations of a particular molecule under study and visceral and skeletal abnormalities are scored in the offspring. These classic endpoints have shown their merits but have their limitations as well. More specifically, they are scored on a discrete scale and the occurrence of serious yet rare abnormalities may be diffi-
cult to interpret. If for example, one treatment group shows one or two cases of spina bifida, it will be impossible to statistically show a significant increase in the occurrence of this developmental disorder. A potentially useful indicator of stress that has often been applied in ecology and evolutionary biology are subtle increases in asymmetries in bilaterally symmetric traits, so-called fluctuating asymmetry (FA). Some studies have found evidence that FA could act as an early warning system in a variety of applications. In this experiment, we tested for a dose–response association in two traditional embryo–fetal developmental (EFD) toxicity studies conducted in rabbits at Janssen Research and Development. Fetal FA-levels in long bones of the legs (humerus, radius, ulna, femur, tibia and fibula) were recorded to investigate any relationship to the dose to which pregnant female rabbits were exposed. In the first EFD study, there was no increase in FA with exposure, which was not unexpected as the results of the original study only showed adverse changes in maternal condition and no changes on the development of the fetuses. In the second EFD study, there was a significant increase in FA observed in all dose groups including the lowest dose level whereas, based on the traditional endpoints, toxic effects of the compound on the fetus only became apparent in the highest dosage group. This effect was strongest on the bones of the hind-limbs and on the fibula in particular. Although further studies are needed to evaluate the robustness and repeatability of this result across compounds and model-species, this exploratory work show the potential of FA as a measure of developmental problems in toxicological research. doi:10.1016/j.reprotox.2011.06.109 Body weight and auditory startle response in rat offspring permanently changed after reprogramming by diet and early exposure to MeHg Joantine Van Esterik, Celine De Esch, Reinier Kaiser, Henk Hendriks, Roderick Slieker, Ine Waalkens, Andre Wolterbeek, Didima De Groot ∗ TNO, Zeist, The Netherlands People from Dutch Famine studies have been found to be more at risk for development of obesity and related chronic diseases like diabetes, but also neurodegenerative diseases. Following the Developmental Origins of Health and Disease (DOHaD) principle this increased risk is thought to be linked to reprogramming of (neuro)physiological set points caused by both maternal diet deprivation and postnatal diet overload during early development of the nervous system. In the present study it is hypothesized that maternal diet manipulation determines development of body weight (BW) and the vulnerability of the neural system for environmental exposure to the chemical methyl mercury chloride (MeHg) in the offspring. Three groups of female Wistar rats (n = 24) were kept on a semi-synthetic control diet (n = 24), a high-caloric diet (15% butter oil, n = 48) or a caloric restricted diet (75% of control diet, n = 38) during a 6 weeks premating period and during mating (1 week) and gestation (3 weeks). Pups were cross-fostered immediately after birth to obtain 5 different groups, i.e. (gestation/LACTATION): cont/CONT; high/HIGH; def/DEF; def/HIGH and high/DEF. The reprogrammed def/HIGH diet group mimics the situation of the Dutch Famine. A selection of male (n = 100) and female (n = 100) pups was subcutaneously treated with saline (control) or MeHg (3 mg/kg BW) from postnatal day 2 to 21. In time, BW was measured and an auditory startle response (ASR) was performed. The ASR measures anxiety over time; disability to habituate in the ASR test is considered to be indicative of neurodegeneration. BWs of the offspring differed significantly between the diet groups, in particular the def/HIGH group showed ‘catch-up’ growth during
Posters / Reproductive Toxicology 32 (2011) 164–179
liferation related pathways and induced neural related pathways over time. This system appears promising for studying compound effects on neural differentiation in a mechanistic approach. doi:10.1016/j.reprotox.2011.06.107 Valproic acid increases embryonic p27kip1 and caspase-3 expression: A potential mechanism for valproic acid induced neural tube defects Emily W.Y. Tung ∗ , Louise M. Winn Queen’s University, Kingston, Ontario, Canada Introduction: Exposure to the anticonvulsant valproic acid (VPA) during the first trimester of pregnancy is associated with an increased risk of congenital malformations including heart defects, craniofacial abnormalities, skeletal and limb defects, and most frequently, neural tube defects (NTDs). The mechanisms by which VPA induces teratogenic effects are not fully understood, although previous studies support a role for histone deacetylase (HDAC)mediated gene expression changes, that lead to cell cycle arrest and cell death. HDAC1 knockout mice do not live past organogenesis, have reduced proliferation rates, and have increased levels of p21WAF1/CIP1 and p27KIP1 that are attributed to histone hyperacetylation in their promoter regions. The purpose of this study was to measure changes in the expression of proteins involved in cell cycle inhibition and apoptosis to further elucidate the molecular changes that occur following VPA exposure. Methods: CD-1 mice were bred, and females with a vaginal plug were separated and denoted as gestational day (GD) 1. On the morning of neural tube closure (GD 9), pregnant dams were administered a teratogenic dose of VPA (400 mg/kg) and embryos extracted 0.5, 1, 3, 6, and 24 h after injection. Western blotting and immunohistochemistry were performed for p21WAF1/CIP1 , p27KIP1 , and cleaved caspase-3. Results: Expression and localization of p21WAF1/CIP1 were not altered following maternal VPA administration. p27KIP1 and cleaved caspase-3 expression were significantly increased 3 and 6 h following VPA exposure. Immunohistochemistry revealed increased staining for p27KIP1 in the neuroepithelium and mesenchyme, and cleaved caspase-3 was notably increased in the neuroepithelium. Conclusions: Our results support our hypothesis that dysregulation of cell cycle inhibition and increased apoptosis in the head region of the embryo contribute to the mechanism of VPA-induced NTDs. doi:10.1016/j.reprotox.2011.06.108 What if your left leg is longer than your right leg? Fluctuating asymmetry as a sensitive indicator of developmental stress in rabbit fetuses Stefan Van Dongen a , Jessica Bots a , Matteo Breno a , Peter Delille b , Luc De Schaepdrijver b a b
University of Antwerp, Antwerp, Belgium Janssen Research and Development, Beerse, Belgium
Toxicologists are constantly searching for efficient indicators of the possible toxic effects of compounds. Traditionally for reproduction toxicology experiments, model organisms are exposed to different concentrations of a particular molecule under study and visceral and skeletal abnormalities are scored in the offspring. These classic endpoints have shown their merits but have their limitations as well. More specifically, they are scored on a discrete scale and the occurrence of serious yet rare abnormalities may be diffi-
cult to interpret. If for example, one treatment group shows one or two cases of spina bifida, it will be impossible to statistically show a significant increase in the occurrence of this developmental disorder. A potentially useful indicator of stress that has often been applied in ecology and evolutionary biology are subtle increases in asymmetries in bilaterally symmetric traits, so-called fluctuating asymmetry (FA). Some studies have found evidence that FA could act as an early warning system in a variety of applications. In this experiment, we tested for a dose–response association in two traditional embryo–fetal developmental (EFD) toxicity studies conducted in rabbits at Janssen Research and Development. Fetal FA-levels in long bones of the legs (humerus, radius, ulna, femur, tibia and fibula) were recorded to investigate any relationship to the dose to which pregnant female rabbits were exposed. In the first EFD study, there was no increase in FA with exposure, which was not unexpected as the results of the original study only showed adverse changes in maternal condition and no changes on the development of the fetuses. In the second EFD study, there was a significant increase in FA observed in all dose groups including the lowest dose level whereas, based on the traditional endpoints, toxic effects of the compound on the fetus only became apparent in the highest dosage group. This effect was strongest on the bones of the hind-limbs and on the fibula in particular. Although further studies are needed to evaluate the robustness and repeatability of this result across compounds and model-species, this exploratory work show the potential of FA as a measure of developmental problems in toxicological research. doi:10.1016/j.reprotox.2011.06.109 Body weight and auditory startle response in rat offspring permanently changed after reprogramming by diet and early exposure to MeHg Joantine Van Esterik, Celine De Esch, Reinier Kaiser, Henk Hendriks, Roderick Slieker, Ine Waalkens, Andre Wolterbeek, Didima De Groot ∗ TNO, Zeist, The Netherlands People from Dutch Famine studies have been found to be more at risk for development of obesity and related chronic diseases like diabetes, but also neurodegenerative diseases. Following the Developmental Origins of Health and Disease (DOHaD) principle this increased risk is thought to be linked to reprogramming of (neuro)physiological set points caused by both maternal diet deprivation and postnatal diet overload during early development of the nervous system. In the present study it is hypothesized that maternal diet manipulation determines development of body weight (BW) and the vulnerability of the neural system for environmental exposure to the chemical methyl mercury chloride (MeHg) in the offspring. Three groups of female Wistar rats (n = 24) were kept on a semi-synthetic control diet (n = 24), a high-caloric diet (15% butter oil, n = 48) or a caloric restricted diet (75% of control diet, n = 38) during a 6 weeks premating period and during mating (1 week) and gestation (3 weeks). Pups were cross-fostered immediately after birth to obtain 5 different groups, i.e. (gestation/LACTATION): cont/CONT; high/HIGH; def/DEF; def/HIGH and high/DEF. The reprogrammed def/HIGH diet group mimics the situation of the Dutch Famine. A selection of male (n = 100) and female (n = 100) pups was subcutaneously treated with saline (control) or MeHg (3 mg/kg BW) from postnatal day 2 to 21. In time, BW was measured and an auditory startle response (ASR) was performed. The ASR measures anxiety over time; disability to habituate in the ASR test is considered to be indicative of neurodegeneration. BWs of the offspring differed significantly between the diet groups, in particular the def/HIGH group showed ‘catch-up’ growth during