Benzoquinone toxicity is not prevented by sulforaphane in CD-1 mouse fetal liver cells.

Benzene is an environmental pollutant known to cause leukemia in adults, and may be associated with childhood leukemia. While the mechanisms of benzene-mediated carcinogenicity have not been fully elucidated, increased reactive oxygen species (ROS) and DNA damage are implicated. Sulforaphane (SFN) induces nuclear factor erythroid 2-related factor 2 (Nrf2), which contributes to SFN-mediated protection against carcinogenesis. We exposed cultured CD-1 mouse fetal liver cells to the benzene metabolite, benzoquinone, to determine its potential to cause DNA damage and alter DNA repair. Cells were also exposed to SFN to determine potential protective effects. Initially, cells were exposed to benzoquinone to confirm increased ROS and SFN to confirm Nrf2 induction. Subsequently, cells were treated with benzoquinone (with or without SFN) and levels of ROS, 8-hydroxy-2-deoxyguanosine (8-OHdG; marker of oxidative DNA damage), gamma histone 2A variant X (γH2AX; marker of DNA double-stranded breaks; DSBs) and transcript levels of genes involved in DNA repair were measured. Benzoquinone exposure led to a significant increase in ROS, which was not prevented by pretreatment with SFN or the antioxidative enzyme, catalase. DNA damage was increased after benzoquinone exposure, which was not prevented by SFN. Benzoquinone exposure significantly decreased the transcript levels of the critical base excision repair gene, 8-oxoguanine glycosylase (Ogg1), which was not prevented by SFN. The findings of this study demonstrate for the first time that DNA damage and altered DNA repair are a consequence of benzoquinone exposure in CD-1 mouse fetal liver cells and that SFN conferred little protection in this model. Copyright © 2015 John Wiley & Sons, Ltd.

Investigating the effects of in utero benzene exposure on epigenetic modifications in maternal and fetal CD-1 mice.

Exposure to the ubiquitous environmental pollutant benzene is positively correlated with leukemia in adults and may be associated with childhood leukemia following in utero exposure. While numerous studies implicate oxidative stress and DNA damage as playing a role in benzene-mediated carcinogenicity, emerging evidence suggests that alterations in epigenetic regulations may be involved. The present study aimed to determine whether DNA methylation and/or various histone modifications were altered following in utero benzene exposure in CD-1 mice. Global DNA methylation and promoter-specific methylation of the tumor suppressor gene, p15, were assessed. Additionally, levels of acetylated histones H3, H4, and H3K56, as well as methylated histones H3K9 and H3K27 were assessed by Western blotting. A significant decrease in global DNA methylation of maternal bone marrow was observed following benzene exposure; however no effect on global DNA methylation was detected in fetal livers. Additionally, no effect of benzene exposure was observed on p15 promoter methylation or any measured histone modifications in both maternal bone marrow and fetal livers. These results suggest that the methodology used in the present study did not reveal alterations in DNA methylation and histone modifications following in utero exposure to benzene; however further experimentation investigating these modifications at the whole genome/epigenome level, as well as at later stages of benzene-induced carcinogenesis, are warranted.

Benzo[a]pyrene increases DNA double strand break repair in vitro and in vivo: a possible mechanism for benzo[a]pyrene-induced toxicity.

Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon and carcinogen that is released into the environment through natural and anthropogenic sources. BaP toxicity is dependent on its metabolism by cytochrome P450s to the reactive metabolite benzo[a]pyrene diol epoxide (BPDE), which is strongly associated with increased mutation frequency. BaP can also be metabolized to benzo[a]pyrene quinones that can undergo redox cycling and induce oxidative stress. The purpose of this study was to examine if BaP exposure induces DNA double strand breaks (DSBs) and subsequently activate DNA DSB repair pathways in the CHO 3-6 cell line and pKZ1 mouse model. In vitro assessment of homologous recombination (HR) showed significantly increased HR frequency following exposure to 10µM of BaP. In vivo evaluations of BaP-induced DNA DSB repair demonstrated positive staining for intrachromosomal recombination events, which are associated with non-homologous end joining (NHEJ), in the lung and thymus of exposed animals that were statistically significant in the thymus when quantified by Western blotting. Gene expression analyses from mouse tissues showed significantly decreased expression of ATM and Xrcc6 in BaP-treated liver and lung. In addition, BaP exposure significantly reduced the expression of Xrcc5, p53, and DNA-PKcs in lung. Taken together, our results demonstrate that BaP increases DNA DSB repair in vitro and in vivo, and induces expression changes in DNA repair pathway genes. As repair of DNA DSBs is not error-free, aberrant DNA repair may be contributing to the mechanism of BaP-induced 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.

The effect of TiO(2) 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.

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.

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.

Sub-chronic sulforaphane exposure in CD-1 pregnant mice enhances maternal NADPH quinone oxidoreductase 1 (NQO1) activity and mRNA expression of NQO1, glutathione S-transferase, and glutamate-cysteine ligase: potential implications for fetal protection against toxicant exposure.

The study objective was to determine if maternal administration of sulforaphane (SFN) induced Nrf2-controlled genes. In acute studies, when non-pregnant and pregnant mice were orally exposed to SFN (50 or 100 mg/kg) on gestational day (GD) 14 and euthanized after 2, 6 or 24h, results demonstrated increased GSTM1, NQO1, HO-1, and Gclc mRNA transcript levels in adult liver, but no change in NQO1 activity. In sub-chronic studies, when non-pregnant and pregnant mice were orally exposed to SFN (65 mg/kg) daily for 30 days and euthanized on GD14, results demonstrated a 2- to 3-fold increase in GSTM1, Gclc and NQO1 transcript levels, and a 2-fold increase in NQO1 activity in adult livers. No effects of maternal treatment on fetal liver gene transcript levels or enzyme activity were observed. Demonstration that SFN induces maternal gene expression and activity supports further investigation of SFN as a preventative agent against transplacental toxicity.

DNA double-strand breaks and DNA recombination in benzene metabolite-induced genotoxicity.

In utero exposure to environmental carcinogens, including the ubiquitous pollutant benzene, may cause DNA damage in the fetus, leading to an increased risk for the development of childhood cancer. Benzene metabolite-induced DNA double-strand breaks (DSBs) may undergo erroneous repair, leading to chromosomal aberrations including chromosomal inversions and translocations. In this study, fetal murine hematopoietic cells from pZK1 transgenic mice were exposed to p-benzoquinone (BQ), a toxic metabolite of benzene, and assessed for DNA recombination, DNA damage including DNA DSBs as measured by γ-H2A.X foci and oxidative DNA damage, and reactive oxygen species (ROS) production. The pZK1 transgenic mouse model contains a DNA construct allowing for the detection of intrachromosomal recombination events. Using this model, a significant increase in recombination was observed following exposure to BQ (25 and 50µM) at various time points. Additionally, increased γ-H2A.X foci were observed following exposure to 25µM BQ for 30 min, 45 min, and 1 h, whereas this exposure did not significantly increase oxidative DNA damage. Pretreatment with 400 U/ml polyethylene glycol-conjugated-catalase attenuated increases in DNA recombination as compared with treatment with BQ alone. An increase in ROS production (30 min and 1 h), as measured by dichlorodihydrofluorescein diacetate fluorescence, was also observed following exposure to 25µM BQ. These studies indicate that BQ is able to induce DNA damage and recombination in fetal liver cells and that ROS may be important in the mechanism of toxicity.

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.