Louise M. Winn

Valproic Acid Increases Formation of Reactive Oxygen Species and Induces Apoptosis in Postimplantation Embryos: A Role for Oxidative Stress in Valproic Acid-Induced Neural Tube Defects.

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 oxidative stress. To investigate the effects of VPA on early development, a whole-embryo culture model was used to evaluate the protective effects of antioxidants, measure intracellular reactive oxygen species (ROS) levels, and assess markers of oxidative damage and apoptosis. Furthermore, in vivo teratological evaluations of antioxidant protection were also completed. VPA (0.60 mM in embryo culture, 400 mg/kg in vivo) induced significant decreases in embryonic growth and increases in NTDs. Of the antioxidants tested, catalase provided partial protection against VPA-mediated reductions in morphological and developmental growth parameters in both whole-embryo culture and in vivo systems. VPA exposure resulted in an increase in ROS staining in the head region, as assessed by whole-mount staining with 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate. Markers of embryonic oxidative damage including 8-hydroxyguanosine, 4-hydroxynonenal adducts, and 3-nitrotyrosine were not affected by VPA treatment. Increased ROS levels were correlated with increased staining for apoptotic markers, as assessed by Western blotting and immunohistochemistry. Addition of catalase to the medium attenuated VPA-induced increases in ROS formation and apoptosis. These studies identify regions of the embryo susceptible to ROS and apoptosis induced by VPA, thus establishing a possible molecular pathway by which VPA exerts teratogenicity.

The effect of TiO2 and Ag nanoparticles on reproduction and development of Drosophila melanogaster and CD-1 mice

In the last two decades, nanoparticles (NPs) have found applications in a wide variety of consumer goods. Titanium dioxide (TiO(2)) and silver (Ag) NPs are both found in cosmetics and foods, but their increasing use is of concern due to their ability to be taken up by biological systems. While there are some reports of TiO(2) and Ag NPs affecting complex organisms, their effects on reproduction and development have been largely understudied. Here, the effects of orally administered TiO(2) or Ag NPs on reproduction and development in two different model organisms were investigated. TiO(2) NPs reduced the developmental success of CD-1 mice after a single oral dose of 100 or 1000 mg/kg to dams, resulting in a statistically significant increase in fetal deformities and mortality. Similarly, TiO(2) NP addition to food led to a significant progeny loss in the fruit fly, Drosophila, as shown by a decline in female fecundity. Ag NP administration resulted in an increase in the mortality of fetal mice. Similarly in Drosophila, Ag NP feeding led to a significant decrease in developmental success, but unlike TiO(2) NP treatment, there was no decline in fecundity. The distinct response associated with each type of NP likely reflects differences in NP administration as well as the biology of the particular model. Taken together, however, this study warns that these common NPs could be detrimental to the reproductive and developmental health of both invertebrates and vertebrates.

In Utero Exposure to Benzene Disrupts Fetal Hematopoietic Progenitor Cell Growth via Reactive Oxygen Species

It is hypothesized that the increasing incidence of childhood leukemia may be due to in utero exposure to environmental pollutants, such as benzene, but the mechanisms involved remain unknown. We hypothesize that reactive oxygen species (ROS) contribute to the deregulation of fetal hematopoiesis caused by in utero benzene exposure. To evaluate this hypothesis, pregnant C57Bl/6N mice were exposed to benzene or polyethylene glycol-conjugated catalase (PEG-catalase) (antioxidative enzyme) and benzene. Colony formation assays on fetal liver cells were performed to measure erythroid and myeloid progenitor cell growth potential. The presence of ROS in CD117(+) fetal liver cells was measured by flow cytometric analysis. Oxidative cellular damage was assessed by Western blot analysis of 4-hydroxynonenol (4-HNE) and nitrotyrosine products, as well as reduced to oxidized glutathione ratios. Alterations in the redox-sensitive signaling pathway nuclear factor-kappa-light-chain-enhancer of activated B cells (NF-kappaB) were measured by Western blot analysis of Inhibitor of NF-kB-alpha (IkappaB-alpha) protein levels in fetal liver tissue. In utero exposure to benzene caused a significant increase in ROS production and significantly altered fetal liver erythroid and myeloid colony numbers but did not increase the levels of 4-HNE or nitrotyrosine products or alter reduced to oxidized glutathione ratios. However, in utero exposure to benzene did cause a significant decrease in fetal liver IkappaB-alpha protein levels, suggesting activation of the NF-kappaB pathway. Benzene-induced ROS formation, abnormal colony growth, and decreased IkappaB-alpha levels were all abrogated by pretreatment with PEG-catalase. These results suggest that ROS play a key role in the development of in utero-initiated benzene toxicity potentially through disruption of hematopoietic cell signaling pathways.

Investigating the effects of functionalized carbon nanotubes on reproduction and development in Drosophila melanogaster and CD-1 mice

Despite numerous applications for functionalized carbon nanotubes (fCNTs) in consumer products, such as electronics, and food packaging, as well as their development as drug delivery vehicles, the consequence of their uptake by living systems has been understudied. In particular, the impact of fCNTs on early development of different species is largely unknown. Here we investigated the effect of ingested hydroxyl-fCNTs on reproduction and development in two model organisms: Drosophila and CD-1 mice. While fCNTs had no measurable impact on Drosophila, a single oral dose of fCNTs (10mg/kg) administered to pregnant CD-1 dams during organogenesis significantly increased the number of resorptions and resulted in fetal morphological and skeletal abnormalities. The observed difference between the responses of these two models likely reflects their physiology and/or differences in administration. This research underscores the need to examine the effects of fCNTs on reproductive health and development before the opportunities for maternal exposure by fCNTs increase further.

Epigenetic modifications in valproic acid-induced teratogenesis.

Exposure to the anticonvulsant drug valproic acid (VPA) in utero is associated with a 1-2% increase in neural tube defects (NTDs), however the molecular mechanisms by which VPA induces teratogenesis are unknown. Previous studies demonstrated that VPA, a direct inhibitor of histone deacetylase, can induce histone hyperacetylation and other epigenetic changes such as histone methylation and DNA demethylation. The objective of this study was to determine if maternal exposure to VPA in mice has the ability to cause these epigenetic alterations in the embryo and thus contribute to its mechanism of teratogenesis. Pregnant CD-1 mice (GD 9.0) were administered a teratogenic dose of VPA (400mg/kg, s.c.) and embryos extracted 1, 3, 6, and 24h after injection. To assess embryonic histone acetylation and histone methylation, Western blotting was performed on whole embryo homogenates, as well as immunohistochemical staining on embryonic sections. To measure DNA methylation changes, the cytosine extension assay was performed. Results demonstrated that a significant increase in histone acetylation that peaked 3h after VPA exposure was accompanied by an increase in histone methylation at histone H3 lysine 4 (H3K4) and a decrease in histone methylation at histone H3 lysine 9 (H3K9). Immunohistochemical staining revealed increased histone acetylation in the neuroepithelium, heart, and somites. A decrease in methylated histone H3K9 staining was observed in the neuroepithelium and somites, METHYLATED histone H3K4 staining was observed in the neuroepithelium. No significant differences in global or CpG island DNA methylation were observed in embryo homogenates. These results support the possibility that epigenetic modifications caused by VPA during early mouse organogenesis results in congenital malformations.

In utero and in vitro effects of benzene and its metabolites on erythroid differentiation and the role of reactive oxygen species

Benzene is a ubiquitous occupational and environmental toxicant. Exposures to benzene both prenatally and during adulthood are associated with the development of disorders such as aplastic anemia and leukemia. Mechanisms of benzene toxicity are unknown; however, generation of reactive oxygen species (ROS) by benzene metabolites may play a role. Little is known regarding the effects of benzene metabolites on erythropoiesis. Therefore, to determine the effects of in utero exposure to benzene on the growth and differentiation of fetal erythroid progenitor cells (CFU-E), pregnant CD-1 mice were exposed to benzene and CFU-E numbers were assessed in fetal liver (hematopoietic) tissue. In addition, to determine the effect of benzene metabolite-induced ROS generation on erythropoiesis, HD3 chicken erythroblast cells were exposed to benzene, phenol, or hydroquinone followed by stimulation of erythrocyte differentiation. Our results show that in utero exposure to benzene caused significant alterations in female offspring CFU-E numbers. In addition, exposure to hydroquinone, but not benzene or phenol, significantly reduced the percentage of differentiated HD3 cells, which was associated with an increase in ROS. Pretreatment of HD3 cells with polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) prevented hydroquinone-induced inhibition of erythropoiesis, supporting the hypothesis that ROS generation is involved in the development of benzene erythrotoxicity. In conclusion, this study provided evidence that ROS generated as a result of benzene metabolism may significantly alter erythroid differentiation, potentially leading to the development of Blood Disorders.

In utero and acute exposure to benzene: investigation of DNA double-strand breaks and DNA recombination in mice.

Benzene, a ubiquitous pollutant, has been identified as a human leukemogen and early exposure to environmental carcinogens such as benzene has been linked to childhood leukemia. It is known that genotoxic agents can increase the frequency of DNA double-strand breaks (DSBs), which can initiate DNA recombinational repair mechanisms. In this study we investigated the induction of micronuclei, the formation of gamma-H2A.X as a marker of DNA DSBs, and the induction of somatic DNA recombination events in hematopoietic tissue from pKZ1 transgenic mice exposed acutely or in utero to benzene. Adult male C57Bl/6N mice were treated with a single i.p. injection of benzene, and timed-pregnant females pKZ1 were treated with daily i.p. injections of 200 mg/kg or 400 mg/kg benzene through gestational days 7-15. Acute exposure to 400 mg/kg benzene resulted in a statistically significant increase in the percentage of micronucleated cells in adult male bone marrow cells and in fetal liver and post-natal day 9 bone marrow cells of mice exposed in utero. Immunoblotting techniques did not detect benzene-induced increases in the formation of gamma-H2A.X in bone marrow cells of adult male mice and in maternal bone marrow, fetal liver, and post-natal bone marrow cells after specific time-point exposures. Finally, no recombination events were detected in adult pKZ1 mouse tissue; however, in post-natal day 9 pups in utero exposure to 400 mg/kg of benzene caused a trend towards increasing recombination frequency although this did not reach statistical significance. These results demonstrate that in utero exposure increases the frequency of micronuclei and DNA recombination events in hematopoietic tissue of fetal and post-natal mice and may be an initiating event in the etiology of childhood leukemias. Further investigations into different types of DNA damage and repair pathways are warranted to fully elucidate the role of genotoxic mechanisms in the etiology of benzene-induced childhood leukemias.

Benzene-initiated oxidative stress: Effects on embryonic signaling pathways

Approximately 90% of childhood cancers are of unknown etiology; however, it is hypothesized that in utero carcinogen exposure may contribute. Epidemiological studies have correlated parental exposure to benzene with an increased incidence of childhood leukemias. However, mechanisms of benzene-induced carcinogenesis following in utero exposure remain unknown. We hypothesize that in utero exposure to benzene causes alterations in the redox-sensitive signaling pathways involving c-Myb, Pim-1, AKT, ERK-MAPK, p38-MAPK, and NF-kappaB via the production of reactive oxygen species (ROS) as a possible mechanism of in utero-initiated carcinogenesis. Using a CD-1 mouse model we have shown increased oxidative stress in fetal tissue from embryos exposed in utero to benzene by measuring reduced to oxidized glutathione ratios, and increased levels of ROS in male fetuses using flow cytometry and the ROS sensitive fluorescent probe dichlorofluoroscein diacetate (DCFDA). In addition, using Western blotting techniques we observed increased expression of fetal Pim-1, Pim-1 phosphorylation, c-Myb, and phosphorylated p38-MAPK (activated form) and lower protein levels of IkappaBalpha, while phosphorylated ERK-MAPK and AKT protein levels did not change. Interestingly, we found male fetuses more susceptible to benzene-induced oxidative stress, which is in agreement with the literature suggesting that males are more susceptible to benzene toxicity. Further studies evaluating the reason for this gender difference are ongoing.

Transplacental benzene exposure increases tumor incidence in mouse offspring: possible role of fetal benzene metabolism

Childhood cancer is the leading cause of disease-related death in children aged 1-14 years in Canada and the USA and it has been hypothesized that transplacental exposure to environmental carcinogens such as benzene may contribute to the etiology of these cancers. Our objectives were to determine if transplacental benzene exposure increased tumor incidence in mouse offspring and assess fetal benzene metabolism capability. Pregnant CD-1 and C57Bl/6N mice were given intraperitoneal injections of corn oil, 200 mg/kg, or 400 mg/kg benzene on gestational days 8, 10, 12 and 14. A significant increase in tumor incidence was observed in CD-1, but not C57BL/6N, 1-year-old offspring exposed transplacentally to 200 mg/kg benzene. Hepatic and hematopoietic tumors were predominantly observed in male and female CD-1 offspring, respectively. Female CD-1 offspring exposed transplacentally to 200 mg/kg benzene had significantly suppressed bone marrow CD11b(+) cells 1 year after birth, correlating with reduced colony-forming unit granulocyte/macrophage numbers in 2-day-old pups. CD-1 and C57Bl/6N maternal blood benzene levels and fetal liver benzene, t, t-muconic acid, hydroquinone and catechol levels were analyzed by gas chromatography/mass spectrometry. Significant strain-, gender- and dose-related differences were observed. Male CD-1 fetuses had high hydroquinone levels, whereas females had high catechol levels after maternal exposure to 200 mg/kg benzene. This is the first demonstration that transplacental benzene exposure can induce hepatic and hematopoietic tumors in mice, which may be dependent on fetal benzene metabolism capability.

In utero exposure to benzene increases embryonic c-Myb and Pim-1 protein levels in CD-1 mice

Benzene is a known human leukemogen, but its role as an in utero leukemogen remains controversial. Epidemiological studies have correlated parental exposure to benzene with an increased incidence of childhood leukemias. We hypothesize that in utero exposure to benzene may cause leukemogenesis by affecting the embryonic c-Myb/Pim-1 signaling pathway and that this is mediated by oxidative stress. To investigate this hypothesis, pregnant CD-1 mice were treated with either 800 mg/kg of benzene or corn oil (i.p.) on days 10 and 11 of gestation and in some cases pretreated with 25 kU/kg of PEG-catalase. Phosphorylated and total embryonic c-Myb and Pim-1 protein levels were assessed using Western blotting and maternal and embryonic oxidative stress were assessed by measuring reduced to oxidized glutathione ratios. Our results show increased oxidative stress at 4 and 24 h after exposure, increased phosphorylated Pim-1 protein levels 4 h after benzene exposure, and increased Pim-1 levels at 24 and 48 h after benzene exposure. Embryonic c-Myb levels were elevated at 24 h after exposure. PEG-catalase pretreatment prevented benzene-mediated increases in embryonic c-Myb and Pim-1 protein levels, and benzene-induced oxidative stress. These results support a role for ROS in c-Myb and Pim-1 alterations after in utero benzene exposure.