The flame retardant triphenyl phosphate alters the epigenome of embryonic cells in an aquatic in vitro model.

Triphenyl phosphate (TPhP) is an organophosphate flame retardant and plasticizer that is added to a wide variety of consumer and industrial products. It is also a ubiquitous environmental pollutant. Exposure to TPhP has been shown to alter gene expression in metabolic and estrogenic signaling pathways in in vitro and in vivo models of a variety of species, and as such, is considered to be an endocrine disrupting chemical. Exposure to endocrine disrupting chemicals is increasingly being associated with changes to the epigenome, especially during embryonic development. The aim of this study was to evaluate whether TPhP exposure in aquatic ecosystems has the ability to alter the epigenome in two immortal cell lines derived from trout (Oncorhynchus mykiss). This study assessed whether 24 h exposure to TPhP resulted in changes to histone modification and DNA methylation profiles in steelhead trout embryonic cells and rainbow trout gill epithelial cells. Results show that several epigenetic modifications on histone H3 and DNA methylation are altered in the embryonic cells following TPhP exposure, but not in the gill epithelial cells. Specifically, histone H3 acetylation, histone H3 mono-methylation and global DNA methylation were found to be reduced. The alterations of these epigenetic modification profiles in the embryonic cells suggest that exposure to TPhP during fetal development may alter gene expression in the developing embryo, likely in metabolic and estrogenic pathways. The impacts to the epigenome determined in this study may even carry multigenerational detrimental effects on human and ecosystem health, which requires further investigation.

Investigating the effects of valproic acid on placental epigenetic modifications and development in the CD-1 mouse model.

Gestational exposure to the anticonvulsant drug valproic acid (VPA) is associated with congenital malformations and neurodevelopmental disorders through its action as a histone deacetylase inhibitor. VPA can elicit placental toxicity and affect placental growth and development. The objective of this study was to evaluate the impact of maternal exposure to VPA on the mouse placenta following exposure on gestational day (GD) 13 since previous studies have shown that mice exposed at this time during gestation give birth to offspring with an autism spectrum disorder-like phenotype. We exposed CD-1 dams to a teratogenic dose (600 mg/kg) of VPA or saline on GD13 and assessed fetoplacental growth and development on GD18. We evaluated epigenetic modifications, including acetylated histone H4 (H4ac), methylated H3K4 (H3K4me2) using immunohistochemistry, and global DNA methylation in the placenta at 1, 3, and 24 h following maternal exposure on GD13. In utero exposure to VPA on GD13 significantly decreased placental weight and increased fetal resorptions. Moreover, VPA significantly increased the staining intensity of histone H4 acetylation and H3K4 di-methylation across the placenta at 1 and 3 h post maternal dose. Our results also demonstrate that VPA significantly decreased global DNA methylation levels in placental tissue. These results show that gestational exposure to VPA interferes with placental growth and elicits epigenetic modifications, which may play a vital role in VPA-induced developmental toxicity.

Gestational exposure to triphenyl phosphate induces epigenetic modifications in C57Bl/6 fetal liver.

INTRODUCTION: Triphenyl phosphate (TPHP) is a chemical flame retardant and plasticizer which is added to consumer and industrial products. The developmental origins of health and disease hypothesis postulate that in utero exposures can have later-in-life effects on the developing fetus and can alter fetal gene expression. This study aimed to determine whether epigenetic modifications occurred following in utero TPHP exposure in mice and whether these changes were dose and/or sex-dependent. METHODS: Pregnant C57Bl/6 mice were treated with 0, 5, 25, or 50 mg/kg of TPHP on gestational days (GD) 8, 10, 12, and 14 via intraperitoneal injection and fetal livers were collected on GD 19. Changes in the levels of acetylation of H3 and H4, as well as methylation of H3K9 and global DNA methylation were assessed in the fetal livers by western blot. RESULTS: Results showed that there was a significant decrease in fetal DNA methylation following in utero exposure to 50 mg/kg TPHP compared to the control (0 mg/kg) independent of the sex of the fetus. While there were no significant alterations compared to controls in any histone modifications at any dose or sex following in utero TPHP exposure, we did note a decrease (t test, p = .025) in the levels of acetylated H3 in males versus females following a maternal dose of 25 mg/kg. The monomethylated H3K9 levels were also increased in females versus males following exposure to TPHP at 5 mg/kg (p = .018) and 25 mg/kg (p = .027) when analyzed via unpaired t tests, although not significantly different from controls. DISCUSSION: The results suggest that gestational TPHP exposure can induce epigenetic modifications in murine fetal tissue. Specifically, global DNA methylation levels were downregulated in response to TPHP. Additionally, males appear to be more sensitive to TPHP-induced histone modifications than females. These data support the need for further studies investigating the impacts of gestational TPHP exposure on the developing fetus.

DNA damage, DNA repair gene expression, and topoisomerase IIα activity in CD-1 mice following in utero benzene exposure.

Benzene is an environmental toxicant and known human carcinogen. Recent epidemiological studies show a relationship between exposure to benzene in pregnant women and increased incidence of childhood leukemias. Studies in murine models demonstrate a relationship between carcinogenicity and in utero benzene exposure which was sex dependent, thus the cellular mechanisms of benzene toxicity by sex require further studies. A hypothesized mechanism of benzene-induced in utero carcinogenicity is through increased DNA damage and reduced fetal DNA repair capacity. This includes the potential inhibition of topoisomerase IIα (topo IIα), in part, to generate double stranded DNA (dsDNA) breaks and induction of error-prone DNA repair. Using a mouse model of transplacental benzene carcinogenicity, gestational day (GD) 14 fetal livers were harvested 2, 6, and 24 h following maternal exposure to 200 mg/kg benzene and used to assess DNA damage, DNA repair gene expression and topo IIα activity. DNA damage, measured by levels of modified histone H2AX (γH2AX), is significantly increased in benzene exposed pups, with sex-dependent significance seen only in female pups. Comet assay results confirmed that benzene exposure in utero induces dsDNA damage in the GD14 fetal liver. Genes involved in DNA repair were assessed, and DNA repair gene expression changes were observed after 24 h in genes related to nucleotide excision repair, homologous recombination, and non-homologous end-joining. There were no significant differences in topo IIα activity in GD14 fetal livers at any timepoint, or between sexes. Overall, this study shows that 200 mg/kg benzene exposure induces dsDNA damage and alters fetal DNA repair gene expression in utero, without perturbing fetal topo IIα in CD-1 mice.

Aberrant inflammation in rat pregnancy leads to cardiometabolic alterations in the offspring and intrauterine growth restriction in the F2 generation.

Children of women with pre-eclampsia have increased risk of cardiovascular (CV) and metabolic disease in adult life. Furthermore, the risk of pregnancy complications is higher in daughters born to women affected by pre-eclampsia than in daughters born after uncomplicated pregnancies. While aberrant inflammation contributes to the pathophysiology of pregnancy complications, including pre-eclampsia, the contribution of maternal inflammation to subsequent risk of CV and metabolic disease as well as pregnancy complications in the offspring remains unclear. Here, we demonstrate that 24-week-old female rats (F1) born to dams (F0) exposed to lipopolysaccharide (LPS) during pregnancy (to induce inflammation) exhibited mild systolic dysfunction, increased cardiac growth-related gene expression, altered glucose tolerance, and coagulopathy; whereas male F1 offspring exhibited altered glucose tolerance and increased visceral fat accumulation compared with F1 sex-matched offspring born to saline-treated dams. Both male and female F1 offspring born to LPS-treated dams had evidence of anemia. Fetuses (F2) from F1 females born to LPS-treated dams were growth restricted, and this reduction in fetal growth was associated with increased CD68 positivity (indicative of macrophage presence) and decreased expression of glucose transporter-1 in their utero-placental units. These results indicate that abnormal maternal inflammation can contribute to increased risk of CV and metabolic disease in the offspring, and that the effects of inflammation may cross generations. Our findings provide evidence in support of early screening for CV and metabolic disease, as well as pregnancy complications in offspring affected by pre-eclampsia or other pregnancy complications associated with aberrant inflammation.

Killing two birds with one stone: Pregnancy is a sensitive window for endocrine effects on both the mother and the fetus.

Pregnancy is a complex process requiring tremendous physiological changes in the mother in order to fulfill the needs of the growing fetus, and to give birth, expel the placenta and nurse the newborn. These physiological modifications are accompanied with psychological changes, as well as with variations in habits and behaviors. As a result, this period of life is considered as a sensitive window as impaired functional and physiological changes in the mother can have short- and long-term impacts on her health. In addition, dysregulation of the placenta and of mechanisms governing placentation have been linked to chronic diseases later-on in life for the fetus, in a concept known as the Developmental Origin of Health and Diseases (DOHaD). This concept stipulates that any change in the environment during the pre-conception and perinatal (in utero life and neonatal) period to puberty, can be "imprinted" in the organism, thereby impacting the health and risk of chronic diseases later in life. Pregnancy is a succession of events that is regulated, in large part, by hormones and growth factors. Therefore, small changes in hormonal balance can have important effects on both the mother and the developing fetus. An increasing number of studies demonstrate that exposure to endocrine disrupting compounds (EDCs) affect both the mother and the fetus giving rise to growing concerns surrounding these exposures. This review will give an overview of changes that happen during pregnancy with respect to the mother, the placenta, and the fetus, and of the current literature regarding the effects of EDCs during this specific sensitive window of exposure.

Characterizing the effects of in utero valproic acid exposure on murine fetoplacental development.

INTRODUCTION: Valproic acid (VPA) is an effective anti-epileptic drug clinically used to treat seizures, bipolar disorders and neuropathic pain in women of reproductive age. Current approval of VPA for psychiatric conditions and migraine has increased the number of VPA exposed pregnancies. VPA crosses the placental barrier and induces birth defects in about 10% of exposed pregnancies. In addition, VPA exposure results in neurodevelopmental disorders in children without any overt birth defects. The current study was designed to investigate the effects of in utero VPA exposure on fetoplacental growth in a mouse model. METHODS: Pregnant CD-1 dams were exposed to a single teratogenic dose of 400 mg/kg VPA or saline via subcutaneous injection on gestational day (GD) 9 and fetuses were harvested on GD 13, 15, 17 and 19, respectively. Resorptions, gross malformations, fetal weight, fetal head weight, fetal crown-rump length, fetal head transverse and anteroposterior diameters, placental weight and placental diameter were noted. RESULTS: VPA exposure led to multiple external deformities including exencephaly, open eye defect, subcutaneous hemorrhage and underdevelopment of tail. All fetoplacental growth parameters fetal weight, fetal head weight, fetal crown-rump length, placental weight and placental diameter were significantly reduced in VPA-exposed fetuses with and without congenital malformations such as exencephaly, compared to control fetuses. DISCUSSION: In conclusion, the effects of in utero VPA exposure on fetal and placental growth persisted throughout pregnancy and our results suggest that the effects of VPA on placental growth may play a role in VPA-induced toxicity.

Gestational valproic acid exposure induces epigenetic modifications in murine decidua.

INTRODUCTION: Valproic acid (VPA), a widely prescribed antiepileptic drug and an effective treatment for bipolar disorder and neuropathic pain, results in multiple developmental defects following in utero exposure. Uterine decidua provides nutritional and physical support during implantation and early embryonic development. Perturbations in the molecular mechanisms within decidual tissue during early pregnancy might affect early embryonic growth, result in early pregnancy loss or cause complications in the later gestational stage. VPA is a known histone deacetylase inhibitor and epigenetic changes such as histone hyperacetylation and methylation have been proposed as a mechanism of VPA-induced teratogenesis. METHODS: This study investigated the effects of in utero VPA exposure on histone modifications in murine decidual tissue. Pregnant CD-1 mice were exposed to 400 mg/kg VPA or saline on GD9 via subcutaneous injection. Decidual tissue from each gestational sac was harvested at 1, 3 and 6 h following exposure. Levels of acetylated histones H3, H4 and H3K56, as well as methylated histones H3K9 and H3K27 were acid extracted and assessed by western blotting followed by acid histone extraction. RESULTS: VPA exposure induced a significant increase (p < 0.05) in the levels of acetylated H3 at 1, 3 h; acetylated H4 at 1, 3 and 6 h and trimethylated H3K9 at 6 h. In contrast, no significant perturbations were noted in the levels of monomethylated H3K9, trimethylated H3K27 and acetylated H3K56. DISCUSSION: The results from this study suggest that VPA-induced decidual histone modifications might play an important role as a mechanism of VPA-induced teratogenesis during early embryonic growth.

Characterizing the effects of in utero valproic acid exposure on NF-κB signaling in CD-1 mouse embryos during neural tube closure.

Nuclear factor kappa B (NF-κB) is a heterodimer of protein subunits p65 and p50, that regulates the expression of a large number of genes related to cell growth and proliferation. The p65 subunit is activated after phosphorylation by Pim-1, while the p50 subunit is the cleaved product of its precursor molecule p105. Valproic acid (VPA), an antiepileptic drug, is a known teratogen and its exposure during pregnancy is associated with 1-2% of neural tube defects in the offspring. The current study aimed at investigating the effects of in utero VPA exposure on the key components of the NF-κB signaling pathway including p65, p50, and Pim-1 in CD-1 mouse embryos during the critical period of neural tube closure. Here we report that p65, Pim-1 and p105/p50 mRNA were significantly (p < 0.05) downregulated at 1 and 3 h following in utero exposure to a teratogenic dose (400 mg/kg) of VPA in gestational day (GD)9 exposed embryos. At GD13 heads of control, non-exencephalic and exencephalic embryos were used for analysis and we found significant upregulation of p65 protein expression in non-exencephalic GD13 heads while p50 protein levels were significantly downregulated in both non-exencephalic and exencephalic groups. On the other hand, p65 and p50 protein levels remained unchanged in the nuclear extracts of the VPA-exposed non-exencephalic and exencephalic GD13 embryo heads. The reported results suggest that VPA exposure perturbates p65, p105/p50, Pim-1 transcript and p65/p50 protein levels in mouse embryos.

Role of Cbp, p300 and Akt in valproic acid induced neural tube defects in CD-1 mouse embryos.

Valproic acid (VPA), an antiepileptic and mood-stabilizing drug, is prescribed to women of reproductive age. VPA is associated with a 1-2% increase in neural tube defects in offspring following gestational exposure and results in epigenetic modifications induced by perturbations in transcription cofactors. Cbp and p300, two transcription cofactors, play key roles in embryonic neural development. p300 is a downstream target of Akt, a protein kinase B associated with cell survival and anti-apoptotic mechanisms, as part of the Akt-p300 axis. We examined the effects of in utero VPA exposure on Cbp, p300, and Akt in gestational day (GD)9, GD10 and GD13 CD-1 mouse embryos following a teratogenic maternal dose of 400 mg/kg. Embryos were collected at 0, 1, 3 and 6 h post-dosing on GD9, 24 h post-dosing on GD10 and on GD13. GD10 embryos were grouped according to the status of neural tube closure in control, closed and open groups. GD13 heads were grouped as control, exposed but non-exencephalic and exencephalic. Our data indicate that Cbp, p300 and Akt mRNA levels were downregulated at 1 and 3 h post-exposure in GD9 embryos while Cbp and p300 protein levels remained stable. Akt protein levels were significantly increased 1 h post-exposure. No significant changes were observed in either mRNA or protein expression in embryos with closed or open neural tubes compared to the control group at GD10. Downregulated expression of Cbp, p300, and Akt may play a key role in VPA-induced neural tube defects considering their vitally important role in embryonic development.

Benzene and its effects on cell signaling pathways related to hematopoiesis and leukemia.

Benzene is an environmental toxicant found in many consumer products. It is an established human carcinogen and is known to cause acute myeloid leukemia in adults. Epidemiological evidence has since shown that benzene can cross the placenta and affect the fetal liver. Animal studies have shown that in utero exposure to benzene can increase tumor incidence in offspring. Although there have been risk factors established for acute myeloid leukemia, they still do not account for many of the cases. Clearly then, current efforts to elucidate the mechanism by which benzene exerts its carcinogenic properties have been superficial. Owing to the critical role of cell signaling pathways in the development of an organism and its various organ systems, it seems plausible to suspect that these pathways may have a role in leukemogenesis. This review article assesses current evidence of the effects of benzene on critical hematopoietic signaling pathways. Pathways discussed included Hedgehog, Notch/Delta, Wingless/Integrated, nuclear factor-kappaB and others. Following a review of the literature, it seems that current evidence about the effects of benzene on these critical signaling pathways remains limited. Given the important role of these pathways in hematopoiesis, more attention should be given to them.

Gestational exposure to valproic acid upregulates total Stat3 protein expression while downregulating phosphorylated Stat3 in CD-1 mouse embryos with neural tube defects.

Valproic acid (VPA), a widely prescribed antiepileptic drug and an effective treatment for psychiatric disorders, is teratogenic causing neural tube defects (NTDs) and other defects in the exposed embryo. Signal transducer and activator of transcription 3 (Stat3) is a transcription factor that is activated via tyrosine phosphorylation. Stat3, as well as its active form (pYStat3), is expressed during neural tube closure in murine development. This study investigated the effects of in utero VPA exposure on embryonic Stat3 mRNA and protein expression during the critical period of neural tube closure in CD-1 mouse embryos. Following the exposure of CD-1 pregnant mice to the teratogenic dose of 400 mg/kg VPA or saline on gestational day (GD) 9, embryos were harvested at 1, 3, 6, or 24 hr and on GD13. Stat3 mRNA levels remained unchanged at all time points. Total Stat3 protein levels were significantly (p < .05) increased in GD9 embryos at 1 and 6 hr post-exposure and in GD13 exposed nonexencephalic and exencephalic embryo heads. In contrast, phosphorylated Stat3 levels were significantly (p < .05) downregulated in GD9 embryos at the 3 and 6 hr time points with an overall trend of downregulation in the GD10 and GD13 groups. Total and phosphorylated Stat3 protein levels remained unchanged in nuclear extracts of the exposed nonexencephalic and exencephalic GD13 embryo heads. The reported significant downregulation of phosphorylated Stat3 levels suggests its possible role in VPA-induced NTDs in mouse embryos.

Characterizing the effects of in utero exposure to valproic acid on murine fetal heart development.

BACKGROUND: Recently, the use of the antiepileptic drug valproic acid (VPA) for the treatment of psychiatric conditions has been on the rise. However, studies have shown that in utero VPA exposure can affect embryonic development, including being associated with congenital heart defects. One proposed mechanism of VPA-initiated teratogenicity is the inhibition of histone deacetylase, which is involved in the regulation of transcription factors that regulate cardiogenesis. Myocyte enhancing factor 2C (Mef2c), a transcription factor involved in the development of cardiac structure and cardiomyocyte differentiation, has been shown to increase in response to in utero VPA exposure, associating with contractile dysfunction and myocardial disorganization. METHODS: To characterize the effects of VPA on murine heart development, pregnant CD-1 mice were dosed with 400 mg/kg of VPA on gestational day (GD) 9. Using high-resolution ultrasound, we examined the effects of VPA on cardiac contractile function on GD 14-18, with fetal hearts being harvested on GD 19 for histological analysis. Lastly, we conducted quantitative real-time polymerase chain reaction to measure the relative Mef2c gene expression in GD 16 murine hearts. RESULTS: We observed structural anomalies at GD 19 in the hearts of VPA-treated mice. Additionally, our results showed alterations in measures of cardiac contractility, with a decrease or increase in cardiac contractile ability in VPA-treated mice depending on the GD and measurement taken. CONCLUSIONS: These results further characterize the effects of VPA on heart development and suggest that alterations in Mef2c gene expression, at least on GD 16, do not mediate VPA-induced cardiotoxicity in CD-1 mice.

DNA Damage and Perturbed Topoisomerase IIα as a Target of 1,4-Benzoquinone Toxicity in Murine Fetal Liver Cells.

Benzene is a ubiquitous environmental pollutant. Recent studies have shown a link between the development of childhood leukemias and maternal benzene exposure, suggesting that these leukemias may be initiated in utero. Benzene crosses the placental barrier however the mechanisms behind in utero benzene toxicity have not been well elucidated. This study is the first to show that the benzene metabolite, benzoquinone (BQ), perturbs fetal topoisomerase IIα (Topo IIα), an enzyme essential for DNA repair. Using cultured murine CD-1 fetal liver cells, this study shows that Topo IIα activity decreases following 24 h of exposure to BQ (12.5 and 15.625 µM), with 12.5 µM confirmed to disrupt the c-kit+ Lin- Sca-1- Il7rα- population of cells in culture. Pretreatment with the antioxidant N-acetylcysteine did not prevent the inhibition of Topo IIα by BQ. An increase in Topo IIα-DNA covalent adducts was detected following 24-h exposure to BQ (12.5 and 50 µM). Interestingly, BQ (12.5 µM) exposure did not significantly increase levels of 4-hydroxynonenal (4-HNE), a marker of oxidative stress after 24 h. However, increased levels of the double-stranded DNA break marker γH2AX were detected following 24 h of BQ exposure, confirming that Topo IIα-induced breaks are increased in BQ-treated cells. This study shows that fetal Topo IIα is perturbed by BQ and suggests that this protein is a target of benzene and may be implicated with in utero benzene toxicity.

In Vitro Models in Developmental Toxicology.

Developmental toxicity associated with exposure to exogenous compounds such as drugs and environmental chemicals can be assessed using a variety of different in vitro models, each with their own advantages and disadvantages. These models include cultured cells (Chapters 3 - 6 ), organ and tissue cultures (Chapters 7 and 8 ), and whole embryo cultures (Chapters 13 - 15 ) and typically support the guiding principles of the three Rs: replace, reduce, and refine. These models can be used in early chemical screens and have helped further our understanding into the mechanisms associated with developmental toxicity following exposure to many chemicals.

Mouse Whole Embryo Culture.

The embryotoxicity associated with exposure to exogenous compounds such as drugs and environmental chemicals can be assessed using the mouse whole embryo culture technique. This method has several advantages over traditional in vivo studies including the exclusion of any confounding maternal and placental effects, the selection of embryos that are at similar stages of development, and the control of exposure concentrations of exogenous agents and modifiers of interest. This chapter will detail the steps involved in using this technique to assess embryotoxicity following exposure to a toxicant. Briefly, embryos are explanted from murine dams on gestational day 9.0 (vaginal plug, day 1) and cultured in CO2 saturated male rat serum for up to 24 h at 37 °C in the presence or absence of a specific toxicant. Embryonic morphological and developmental parameters (e.g., anterior neuropore closure) are then evaluated using a dissecting microscope 24 h later. Potential biochemical analyses are also listed and limitations discussed.

Gestational triphenyl phosphate exposure in C57Bl/6 mice perturbs expression of insulin-like growth factor signaling genes in maternal and fetal liver.

Triphenyl phosphate (TPhP) is an organophosphorus flame retardant and plasticizer that has been added to numerous consumer products in recent years. TPhP is not overtly toxic, however recent studies have suggested that it may have metabolic disrupting effects following developmental exposure. The present study aimed to investigate the developmental and potential metabolic effects of TPhP in a murine model. C57Bl/6 dams were exposed on gestational days (GD) 8, 10, 12, and 14 to 0, 5, 25, or 50 mg/kg TPhP via intraperitoneal injection. Dams were euthanized on GD19, maternal organs excised and weighed, fetal measurements taken, and maternal and fetal livers retained for analysis. A significant increase in placenta size of TPhP exposed mice was found. Maternal and fetal liver gene expression of insulin-like growth factor (Igf) 1 and 2, as well as downstream genes involved in Igf signaling were measured. Additionally, Igf1 protein levels were measured in both maternal and fetal liver. A significant decrease in transcript levels of Igf1 and Irs2 was detected in maternal livers, whereas a significant increase in transcript levels of all genes measured was detected in fetal liver. A significant decrease in Igf1 protein levels was detected in maternal liver, however the increase in Igf1 protein levels in fetal livers was not found to be statistically significant. These results support previous findings that TPhP does not cause overt structural developmental toxicity. These data also support the hypothesis that TPhP could disrupt maternal and fetal metabolism, justifying the need for follow-up studies to investigate further.

Valproic acid increases NF-κB transcriptional activation despite decreasing DNA binding ability in P19 cells, which may play a role in VPA-initiated teratogenesis.

The nuclear factor-kappa B (NF-κB) family of transcription factors regulate gene expression in response to diverse stimuli. We previously demonstrated that valproic acid (VPA) exposure in utero decreases total cellular protein expression of the NF-κB subunit p65 in CD-1 mouse embryos with a neural tube defect but not in phenotypically normal littermates. This study evaluated p65 mRNA and protein expression in P19 cells and determined the impact on DNA binding ability and activity. Exposure to 5mM VPA decreased p65 mRNA and total cellular protein expression however, nuclear p65 protein expression was unchanged. VPA reduced NF-κB DNA binding and nuclear protein of the p65 DNA-binding partner, p50. NF-κB transcriptional activity was increased with VPA alone, despite decreased phosphorylation of p65 at Ser276, and when combined with tissue necrosis factor α. These results demonstrate that VPA increases NF-κB transcriptional activity despite decreasing DNA binding, which may play a role in VPA-initiated teratogenesis.

Persistence of risk factors associated with maternal cardiovascular disease following aberrant inflammation in rat pregnancy.

INTRODUCTION: Pre-eclampsia is associated with increased risk of subsequent cardiovascular and metabolic disease in the affected mothers. While aberrant inflammation contributes to the pathophysiology of pre-eclampsia, it is unclear whether maternal inflammation contributes to the increased risk of disease. Here, we determined the effect of aberrant inflammation in pregnancy on cardiovascular and metabolic disease risk factors. METHODS: Wistar rats were administered low doses of lipopolysaccharide (LPS) on gestational days (GD) 13.5-16.5 to induce inflammation. Controls included pregnant rats treated with saline and nonpregnant rats treated with LPS or saline. We previously showed that LPS-treated pregnant rats exhibit key features of pre-eclampsia. Echocardiographic parameters, heart weight, blood pressure, blood lipids, pulse-wave velocity, and glucose tolerance, were assessed at 16 weeks postpartum. Messenger RNA levels of transcription factors associated with cardiac growth were measured in left ventricular tissue; histone modifications and global DNA methylation were determined in hearts and livers at GD 17.5 and at 16 weeks postpartum. RESULTS: Compared with saline-treated pregnant rats and nonpregnant rats treated with LPS or saline, LPS-treated pregnant rats exhibited left ventricular hypertrophy and increased blood cholesterol and low-density lipoprotein levels at 16 weeks postdelivery. LPS-treated rats had increased left ventricular mRNA levels of hypertrophy-associated transcription factors at GD 17.5 and increased levels of modified histones in hearts and livers at GD 17.5 and 16 weeks postpartum. Other parameters remained unchanged. CONCLUSION: Aberrant inflammation during pregnancy results in persistent alterations in maternal physiological parameters and epigenetic modifications that could contribute to the pathophysiology of cardiovascular disease.