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Trichloroethylene (TCE), a widely used industrial solvent, is a common environmental contaminant. We previously reported that TCE-induced changes in DNA methylation and miRNA expression contributed to the development of a liver tumor in mice. In this study, we investigated the role of long intergenic noncoding RNA (LincRNA), another type of epigenetic modification, in TCE hepatocarcinogenesis. Male B6C3F1 mice were gavaged with TCE at dose levels of 0, 100, 500, and 1000 mg/kg b.w. for 5 days. The expression changes of LincRNAs in liver samples from control and TCE-exposed mice were screened by microarray. When compared to the control group, 21 and 29 LincRNAs were upregulated and downregulated, respectively, in the liver of mice exposed to TCE at 1000 mg/kg b.w. In addition, TCE treatment increased the expression levels of LincRNA-GM8704 but decreased the expression levels of LiverLincs_chr17_4383_2 in a dose-dependent manner. We further found that the mRNAs that are highly correlated with the expression of LiverLincs_chr17_4383_2 are involved in a number of cancer-related signaling pathways including PPARs, cell cycle, and ErbB and p53 signaling pathways. Among the expression-correlated mRNAs, Cdkn1a was found to be a downstream target gene of LiverLincs_chr17_4383_2. To follow up on that, we also found that miR-182-5p might mediate the association between downregulation of LiverLincs_chr17_4383_2 and upregulation of Cdkn1a, leading to increased cell proliferation in TCE exposed liver cells. In conclusion, TCE induced extensive LincRNA expression changes in mouse liver, and the downregulation of LiverLincs_chr17_4383_2 might contribute to TCE hepatocarcinogenesis by interacting with miR-182-5p and Cdkn1a.

Benzo[a]pyrene (BaP), a prototypical polycyclic aromatic hydrocarbon, is widely present in the environment. BaP-induced heart defects have been frequently reported, but the underlying molecular mechanisms remain elusive. Here, we found that BaP increased heart malformations in zebrafish embryos in a concentration-dependent manner, which were attenuated by supplementation with either CH223191 (CH), an aryl hydrocarbon receptor (AHR) inhibitor, or N-acetyl-l-cysteine (NAC), a reactive oxygen species (ROS) scavenger. While CH and NAC both inhibited BaP-induced ROS generation, NAC had no effect on BaP-induced AHR activation. We further demonstrated that BaP increased mitochondrial ROS, decreased mitochondrial membrane potential, and caused endogenous apoptosis, with all these effects being counteracted by supplementation with either CH or NAC. Resveratrol (RSV), a natural AHR antagonist and ROS scavenger, also counteracted the heart malformations caused by BaP. Further experiments showed that RSV attenuated BaP-induced oxidative stress, mitochondrial damage and apoptosis, but had no significant effect on AHR activation. In conclusion, our findings show that BaP induces oxidative stress via AHR activation, which causes mitochondria-mediated intrinsic apoptosis, resulting in heart malformations in zebrafish embryos, and that RSV had a protective effect against BaP-induced heart defects mainly by inhibiting oxidative stress rather than through antagonism of AHR activity.

Ambient fine particulate matter (PM2.5) has been found to be associated with congenital heart defects, but the molecular mechanisms remain to be elucidated. Our previous study revealed that extractable organic matter (EOM) from PM2.5 exerted cardiac developmental toxicity in zebrafish embryos. The aim of the current study is to explore the effects of EOM on cardiac differentiation of P19 mouse embryonic carcinoma stem cells. We found that EOM at 10 μg/ml (a non-cytotoxic dose level) significantly reduced the proportion of cardiac muscle troponin (cTnT) positive cells and the percentage of spontaneously beating embryoid bodies, indicating a severe inhibition of cardiac differentiation. Immunofluorescence and qPCR data demonstrated that EOM increased the expression levels of the aryl hydrocarbon receptor (AhR) and its target gene Cyp1A1 and diminished the expression level of β-catenin. Furthermore, EOM treatment significantly upregulated cell proliferation rate and elevated the percentage of γH2A.X positive cells without affecting apoptosis. It is worth noting that the EOM-induced changes in gene expression, cellular proliferation and DNA double strain breaks were attenuated by the AhR antagonist CH223191. In conclusion, our data indicate that AhR mediates the inhibitory effects of EOM (from PM2.5) on the cardiac differentiation of P19 cells.

Ambient fine particulate matter (PM2.5) exposure can lead to cardiac developmental toxicity but the underlying molecular mechanisms are still unclear. 8-hydroxy-2'deoxygenase (8-OHdG) is a marker of oxidative DNA damage and γH2AX is a sensitive marker for DNA double strand breaks. In this study, we aimed to detect PM2.5-induced 8-OHdG and γH2AX changes in the heart of zebrafish embryos using an immunofluorescence assay. Zebrafish embryos were treated with extractable organic matters (EOM) from PM2.5 at 5 μg/mL in the presence or absence of antioxidant N-acetyl-L-cysteine (NAC, 0.25 μM) at 2 h post fertilization (hpf). DMSO was used as a vehicle control. At 72 hpf, hearts were dissected from embryos using a syringe needle and fixed and permeabilized. After being blocked, samples were probed with primary antibodies against 8-OHdG and γH2AX. Samples were then washed and incubated with secondary antibodies. The resulting images were observed under fluorescence microscopy and quantified using ImageJ. The results show that EOM from PM2.5 significantly enhanced 8-OHdG and γH2AX signals in the heart of zebrafish embryos. However, NAC, acting as a reactive oxygen species (ROS) scavenger, partially counteracted the EOM-induced DNA damage. Here, we present an immunofluorescence protocol for investigating the role of DNA damage in PM2.5-induced heart defects that can be applied to the detection of environmental chemical-induced protein expression changes in the hearts of zebrafish embryos.

Ambient fine particulate matter (PM2.5) exposure can lead to cardiac developmental toxicity but the underlying molecular mechanisms are still unclear. 8-hydroxy-2'deoxygenase (8-OHdG) is a marker of oxidative DNA damage and γH2AX is a sensitive marker for DNA double strand breaks. In this study, we aimed to detect PM2.5-induced 8-OHdG and γH2AX changes in the heart of zebrafish embryos using an immunofluorescence assay. Zebrafish embryos were treated with extractable organic matters (EOM) from PM2.5 at 5 μg/mL in the presence or absence of antioxidant N-acetyl-L-cysteine (NAC, 0.25 μM) at 2 h post fertilization (hpf). DMSO was used as a vehicle control. At 72 hpf, hearts were dissected from embryos using a syringe needle and fixed and permeabilized. After being blocked, samples were probed with primary antibodies against 8-OHdG and γH2AX. Samples were then washed and incubated with secondary antibodies. The resulting images were observed under fluorescence microscopy and quantified using ImageJ. The results show that EOM from PM2.5 significantly enhanced 8-OHdG and γH2AX signals in the heart of zebrafish embryos. However, NAC, acting as a reactive oxygen species (ROS) scavenger, partially counteracted the EOM-induced DNA damage. Here, we present an immunofluorescence protocol for investigating the role of DNA damage in PM2.5-induced heart defects that can be applied to the detection of environmental chemical-induced protein expression changes in the hearts of zebrafish embryos.

Trichloroethylene (TCE), a prevalent environmental contaminant, has been shown to induce cardiac malformations. Resveratrol (RSV) is a natural polyphenolic compound exhibiting protective effects on heart development. To investigate if RSV could protect against TCE-induced heart defects, we exposed zebrafish embryos to TCE (10 ppb) in the presence or absence of RSV (1 μg/mL). Our results showed that RSV significantly attenuated TCE-induced heart defects in zebrafish embryos. The TCE-induced ROS (reactive oxygen species) generation, 8-OHdG (8-hydroxy-2`-deoxyguanosine) formation and cell proliferation were significantly counteracted by RSV. Moreover, RSV attenuated the TCE-induced changes in mRNA expression or activity of genes involved in AHR and Nrf2 signal pathways. We further showed that RSV might inhibit TCE-enhanced cell proliferation by rescuing the downregulation of the p53/p21 axis. In conclusion, our data demonstrates that RSV protects against the cardiac developmental toxicity of TCE by inhibiting AHR activity, oxidative stress and cell proliferation.

Acrylamide (AA) has been shown to have neurological and reproductive toxicities, but little is known about transgenerational effects of AA. In this study, male C57BL/6 mice were exposed to AA (0.01, 1, 10 μg/mL) and its metabolite glycidamide (GA, 10 μg/mL) in drinking water, which were then mated with unexposed female mice to produce F1 and F2 generations. We found that both AA and GA at high concentrations decreased sperm motility in F0 mice and increased sperm malformation rates in mice from all the three generations. In addition, AA and GA increased sperm reactive oxygen species as well as decreased serum testosterone levels, and increased the escape latency time in exposed mice and their offspring. We further found that AA-induced mRNA expression changes in the hippocampus of F0 mice persist to the F2 generation. In the sperm of F0 mice, AA induced significant DNA methylation changes in genes involved in neural and reproduction; the mRNA expression levels of Dnmt3b, a DNA methyltransferase, were dramatically decreased in the testes of F0 and F1 mice. In conclusion, our study indicates that paternal AA exposure leads to DNA methylation-mediated transgenerational adverse effects on sperm parameters and leaning capability in mice.

Resveratrol (RSV), a natural polyphenolic compound commonly found in food, has antioxidant and aryl hydrocarbon receptor (AHR) antagonist effects. We have recently demonstrated that AHR mediated reactive oxygen species (ROS) generation contributes to the cardiac developmental toxicity of ambient fine particle matter (PM2.5). Thus, we hypothesized that RSV protects against the cardiac developmental toxicity of PM2.5 by inhibiting ROS generation and AHR activity. To test this concept, we exposed zebrafish embryos to extractable organic matter (EOM) from PM2.5 in the presence or absence of RSV. We found that RSV significantly counteracted EOM-induced cardiac malformations in zebrafish embryos. The EOM-induced ROS production, DNA damage and apoptosis in the heart of zebrafish embryos were also counteracted by RSV supplementation. Furthermore, RSV attenuated EOM-induced changes in the expression of genes involved in cardiac development (nkx2.5, sox9b, axin2), oxidative stress (nrf2a, nrf2b, gstp1, gstp2, sod1, sod2, cat) and apoptosis (p53, bax). However, RSV did not suppress EOM-induced AHR activity. In conclusion, our data indicates that RSV protects against the PM2.5-induced heart malformations by inhibiting oxidative stress rather than through AHR antagonism.

Recent studies have shown an association between maternal exposure to ambient fine particle matter (PM2.5) and congenital heart defects in the offspring, but the underlying molecular mechanisms are yet to be elucidated. Previously, we demonstrated that extractable organic matter (EOM) from PM2.5 induced heart defects in zebrafish embryos by activating the aromatic hydrocarbon receptor (AHR). Hence, we hypothesized that AHR mediates excessive reactive oxygen species (ROS) production, leading to the cardiac developmental toxicity of PM2.5. To test our hypothesis, we examined AHR activity and ROS levels in the heart of zebrafish embryos under a fluorescence microscope. mRNA expression levels were then quantified using qPCR whereas DNA damage and apoptosis were detected by immunofluorescence. Our results showed that the AHR inhibitor, CH223191 (CH) as well as the ROS scavenger, N-Acetyl-L-cysteine (NAC), significantly mitigated the PM2.5-induced cardiac malformations in zebrafish embryos. Furthermore, both CH and NAC diminished the EOM-elevated ROS generation, DNA damage and apoptosis in the test system. Incidentally, both CH and NAC attenuated the EOM-induced changes in the mRNA expression of genes involved in cardiac development (nkx2.5, sox9b), oxidative stress (nrf2a, nrf2b, gstp1, gstp2, sod2, ho1, cat) and apoptosis (p53, bax). We further confirmed that AHR activity is a necessary condition for EOM-induced ROS generation, DNA damage and apoptosis, through AHR knockdown. However, the ROS scavenger NAC did not counteract the EOM-induced AHR activity. In conclusion, our findings suggest that AHR mediates EOM-induced oxidative stress, resulting in DNA damage and apoptosis, thereby contributing to the cardiac developmental toxicity of PM2.5.

Trichloroethylene (TCE), a widely used chlorinated solvent, is a common environmental pollutant. Current evidence shows that TCE could induce heart defects during embryonic development, but the underlining mechanism(s) remain unclear. Since activation of the aryl hydrocarbon receptor (AHR) could induce oxidative stress, we hypothesized that AHR-mediated oxidative stress may play a role in the cardiac developmental toxicity of TCE. In this study, we found that the reactive oxygen species (ROS) scavenger, N-Acetyl-L-cysteine (NAC), and AHR inhibitors, CH223191 (CH) and StemRegenin 1, significantly counteracted the TCE-induced heart malformations in zebrafish embryos. Moreover, both CH and NAC suppressed TCE-induced ROS and 8-OHdG (8-hydroxy-2' -deoxyguanosine). TCE did not affect ahr2 and cyp1a expression, but increased cyp1b1 expression, which was restored by CH supplementation. CH also attenuated the TCE-induced mRNA expression changes of Nrf2 signalling genes (nrf2b, gstp2, sod2, ho1, nqo1) and cardiac differentiation genes (gata4, hand2, c-fos, sox9b). In addition, the TCE enhanced SOD activity was attenuated by CH. Morpholino knockdown confirmed that AHR mediated the TCE-induced ROS and 8-OHdG generation in the heart of zebrafish embryos. In conclusion, our results suggest that AHR mediates TCE-induced oxidative stress, leading to DNA damage and heart malformations in zebrafish embryos.

Ambient fine particulate matter (PM

We previously found that folic acid (FA) attenuated cardiac defects in zebrafish embryos exposed to extractable organic matter (EOM) from PM2.5, but the underlining mechanisms remain to be elucidated. Since DNA methylation is crucial to cardiac development, we hypothesized that EOM-induced aberrant DNA methylation changes could be diminished by FA supplementation. In this study, zebrafish embryos were exposed to EOM in the absence or presence of FA. Genomic-wide DNA methylation analysis identified both DNA hypo- and hyper-methylation changes in CCGG sites in zebrafish embryos exposed to EOM, which were attenuated by FA supplementation. We identified a total of 316 genes with extensive DNA methylation changes in EOM samples but little or no DNA methylation changes in EOM plus FA samples. The genes were involved in critical cellular processes and signaling pathways important for embryo development. In addition, the EOM-decreased SAM/SAH ratio was counteracted by FA supplementation. Furthermore, FA attenuated the EOM-induced changes in the expression of genes involved in the regulation of DNA methylation and in folate biosynthesis. In conclusion, our data suggest that FA supplementation protected zebrafish embryos from the cardiac developmental toxicity of PM2.5 by alleviating EOM-induced DNA methylation changes.