PM2.5 induces cardiac defects via AHR-SIRT1-PGC-1α mediated mitochondrial damage.

Recent evidence indicates that PM2.5 poses a risk for congenital heart diseases, but the mechanisms remain unclear. We hypothesized that AHR activated by PM2.5 might cause mitochondrial damage via PGC-1α dysregulation, leading to heart defects. We initially discovered that the PGC-1α activator ZLN005 counteracted cardiac defects in zebrafish larvae exposed to EOM (extractable organic matter) from PM2.5. Moreover, ZLN005 attenuated EOM-induced PGC-1α downregulation, mitochondrial dysfunction/biogenesis, and apoptosis. EOM exposure not only decreased PGC-1α expression levels, but suppressed its activity via deacetylation, and SIRT1 activity is required during both processes. We then found that SIRT1 expression levels and NAD+/NADH ratio were reduced in an AHR-dependent way. We also demonstrated that AHR directly suppressed the transcription of SIRT1 while promoted the transcription of TiPARP which consumed NAD+. In conclusion, our study suggests that PM2.5 induces mitochondrial damage and heart defects via AHR/SIRT1/PGC-1α signal pathway.

Current Techniques and Models for Assessing the Developmental Toxicity of Environmental Pollutants.

ARTICLES DISCUSSED: Ding, J. et al. Using Alizarin Red staining to detect chemically induced bone loss in zebrafish larvae. Journal of Visualized Experiments. (178), e63251 (2021). Huang, Y. et al. Using immunofluorescence to detect PM2.5-induced DNA damage in zebrafish embryo hearts. Journal of Visualized Experiments. (168), e62021 (2021). Jiang, Q. X., Xu, X. H., DeWitt, J. C., Zheng, Y. X. Using chicken embryo as a powerful tool in assessment of developmental cardiotoxicities. Journal of Visualized Experiments. (169), e62189 (2021). Song, Y., Li, R., Li, L., Ouyang, F., Men, X. Evaluating the effect of pesticides on the larvae of the solitary bees. Journal of Visualized Experiments. (176), e62946 (2021).

AHR-mediated m6A RNA methylation contributes to PM2.5-induced cardiac malformations in zebrafish larvae.

A growing body of evidence indicates that ambient fine particle matter (PM2.5) exposure inhibits heart development, but the underlying mechanisms remain elusive. We hypothesized that m6A RNA methylation plays an important role in the cardiac developmental toxicity of PM2.5. In this study, we demonstrated that extractable organic matter (EOM) from PM2.5 significantly decreased global m6A RNA methylation levels in the heart of zebrafish larvae, which were restored by the methyl donor, betaine. Betaine also attenuated EOM-induced ROS overgeneration, mitochondrial damage, apoptosis and heart defects. Furthermore, we found that the aryl hydrocarbon receptor (AHR), which was activated by EOM, directly repressed the transcription of methyltransferases mettl14 and mettl3. EOM also induced genome-wide m6A RNA methylation changes, which led us to focus more on the aberrant m6A methylation changes that were subsequently alleviated by the AHR inhibitor, CH223191. In addition, we found that the expression levels of traf4a and bbc3, two apoptosis related genes, were upregulated by EOM but restored to control levels by the forced expression of mettl14. Moreover, knockdown of either traf4a or bbc3 attenuated EOM-induced ROS overproduction and apoptosis. In conclusion, our results indicate that PM2.5 induces m6A RNA methylation changes via AHR-mediated mettl14 downregulation, which upregulates traf4a and bbc3, leading to apoptosis and cardiac malformations.

Do shale oil and gas production activities impact ambient air quality? A comprehensive study of 12 years of chemical concentrations and well production data from the Barnett Shale region of Texas.

Starting around 2008, there was rapid expansion of oil and natural gas (ONG) production into more heavily populated areas within the Dallas-Fort Worth metroplex in the Barnett Shale region of Texas. This colocation raised concerns regarding the effect of ONG activities on chemical levels in the air. In the current study, we examined the potential impacts of ONG activity on the types and concentrations of chemicals in ambient air in the Barnett Shale. Volatile organic compound (VOC) concentrations from 6-12 years (2008-2019) of hourly ambient air monitoring data from 15 monitors (4 monitors had ≥ 10 years of data) were compared to several metrics of ONG activity (number of active wells, natural gas production, condensate production) within a 2-mile radius of each monitor. Monitoring sites were also classified into urban, suburban, and rural areas as a surrogate for nearby vehicular emission sources. Analyses of this huge dataset showed that both peak and mean chemical concentrations of lighter alkane hydrocarbons (e.g., ethane) were most impacted by the number of gas wells. Levels of heavier alkanes (e.g., pentane) were increased by condensate production and at monitors located in areas with greater urbanicity, and therefore higher vehicular emissions. The levels of unsaturated alkynes (e.g., ethylene) were entirely driven by urbanicity and were unaffected by nearby ONG activity. The same pattern was seen with the ratio of iso:n-pentane, which is contrary to the findings of others and suggests an area for future research. Aromatic hydrocarbons were impacted by multiple emissions sources and did not show the same patterns as non-aromatic VOCs. No VOC concentrations were at levels of concern for human health or odor based on comparison to Texas air monitoring comparison values. Overall, ONG activities impact air quality, but this must be evaluated in the context of other emission sources such as automobiles.

Regulation of Cx43 and its role in trichloroethylene-induced cardiac toxicity in H9C2 rat cardiomyocytes.

Trichloroethylene (TCE), a widespread environmental contaminant, has been linked to congenital heart defects. Abnormal regulation of Connexin 43 is closely associated with various cardiac diseases. However, it is yet to be established how Cx43 responds to environmental pollutants. Here, we aim to explore the role of Cx43 in TCE-induced cardiac toxicity using H9C2 cardiomyocytes. EdU incorporation assay and cell cycle analysis revealed that increased number of TCE-treated cells entered into the S stage, indicating that TCE exposure provoked cell proliferation. Additionally, compromised mitochondrial function was observed in TCE-treated cells, and inhibition of mitochondrial permeability transition pore (mPTP) with Cyclosporin A or eliminating mitochondrial ROS by MitoQ alleviated the TCE-induced cardiac toxicity. Importantly, TCE exposure increased the protein expression levels of Cx43 and stimulated the recruitment of Cx43 to the mitochondria. TCE exposure disrupted canonical Wnt signal pathway, resulting in downregulation of antioxidant genes and ß-catenin. The adverse effects of TCE on Wnt signal pathway activation, mitochondrial function and cell proliferation were efficiently counteracted by either Cx43 knockdown or pharmaceutical activator of Wnt signaling, CHIR-99021. Taken together, our results for the first time revealed that dysregulation of Cx43 mediates TCE-induced heart defects via mitochondrial dysfunction and Wnt signaling inhibition, suggesting that Cx43 can be a potential molecular marker or therapeutic target for cardiac diseases caused by environmental pollutants.

PM2.5 induces cardiac malformations via PI3K/akt2/mTORC1 signaling pathway in zebrafish larvae.

Growing evidence indicates that maternal fine particulate matter (PM2.5) exposure is linked with congenital heart diseases in the offspring. To explore the underlying molecular mechanisms, we tested the effects of a number of pharmaceutical inhibitors, and found that suppressing the PI3K/akt signaling pathway had a protective effect against cardiac defects in zebrafish larvae exposed to extractable organic matter (EOM) from PM2.5. Using genetic knockdown and a specific akt2 pharmacological inhibitor, CCT128930, we demonstrated that akt2 activation is essential to EOM-induced heart malformations. Next, we found that the EOM-induced akt2 overactivation enhances intracellular reactive oxygen species (ROS)/mitochondrial ROS production, decreases mitochondrial membrane potential levels, and elicits intrinsic apoptosis in the heart of zebrafish embryos. In addition, EOM-induced akt2 activation decreased active ß-catenin levels and inhibited the expression of Wnt target genes axin2 and nkx2.5. We further demonstrated that mTORC1 phosphorylation mediates the adverse effects of akt2 on intrinsic apoptosis and canonical Wnt signaling in the heart of zebrafish larvae exposed to EOM. Moreover, EOM-induced akt2 activation is mediated via aryl hydrocarbon receptor (AHR)/ROS-induced PTEN inhibition. In conclusion, our results indicate that PM2.5 activates PI3K/akt2/mTORC1 signaling via AHR/ROS-induced PTEN suppression, which leads to mitochondrial-mediated intrinsic apoptosis and Wnt signaling suppression, resulting in cardiac defects in zebrafish larvae.

PM2.5 induces mitochondrial dysfunction via AHR-mediated cyp1a1 overexpression during zebrafish heart development.

Accumulating evidence suggests an association between maternal PM2.5 exposure and congenital heart diseases, but the underlying mechanisms remain unclear. We previously reported that PM2.5 induces cardiac malformations in zebrafish embryos via the aryl hydrocarbon receptor (AHR) pathway, which mediates the generation of reactive oxygen species (ROS). Since mitochondria are not only the main source of ROS but also sensitive to oxidative damage, we hypothesize that mitochondria may play an important role in the cardiac developmental toxicity of PM2.5. In this study, we demonstrated that extractable organic matter (EOM) from PM2.5 caused mitochondrial dysfunction in the heart of zebrafish embryos, including increased mitochondrial ROS (mtROS) levels, mitochondrial permeability transition pore (mPTP) opening, mitochondrial membrane potential (MMP) collapse, reduced mitochondrial ATP levels, and decreased expression levels of the mRNAs encoding mitochondrial proteins, which were attenuated by either pharmacological or genetic inhibition of AHR. We further demonstrated that improving mitochondrial function by inhibiting mPTP opening with Cyclosporin A suppressed the EOM-induced intracellular ROS and mtROS generation, MMP collapse, intrinsic apoptosis, and heart defects. Moreover, the EOM-induced mPTP opening was counteracted by inhibiting mtROS with mitoquinone mesylate (MitoQ). Supplementation with MitoQ also attenuated the EOM-induced mitochondrial dysfunction, apoptosis and heart defects. Additionally, knockdown of cyp1a1 but not cyp1b1 attenuated the EOM-induced mtROS generation and heart defects. Taken together, this study indicates that PM2.5 triggers mtROS generation via AHR-mediated cyp1a1 overexpression, which then causes mPTP opening and mitochondrial dysfunction, leading to apoptosis and heart defects.

Synergistic protective effects of folic acid and resveratrol against fine particulate matter-induced heart malformations in zebrafish embryos.

Ambient fine particulate matter (PM2.5) is a major environmental health problem worldwide, and recent studies indicate that maternal PM2.5 exposure is closely associated with congenital heart diseases (CHDs) in offspring. We previously found that supplementation with folic acid (FA) or Resveratrol (RSV) could protect against heart defects in zebrafish embryos exposed to extractable organic matter (EOM) from PM2.5 by targeting aryl hydrocarbon receptor (AHR) signaling and reactive oxygen species (ROS) production respectively. Thus, we hypothesized that FA combined with RSV may have a synergistic protective effect against PM2.5-induced heart defects. To test our hypothesis, we treated zebrafish embryos with EOM in the presence or absence of FA, RSV or a combination of both. We found that RSV and FA showed a clear synergistic protection against EOM-induced heart defects in zebrafish embryos. Further studies showed that FA and RSV suppressed EOM-induced AHR activity and ROS generation respectively. Although only RSV inhibited EOM-induced apoptosis, FA enhanced the inhibitory effect of RSV. Moreover, vitamin C (VC), a typical antioxidant, also exhibits a synergistic inhibitory effect with FA on EOM-induced apoptosis and heart defects. In conclusion, supplementation with FA and RSV have a synergistic protective effect against PM2.5-induced heart defects in zebrafish embryos by targeting AHR activity and ROS production respectively. Our results indicate that, in the presence of antioxidants, FA even at a low concentration level could protect against the high risk of CHDs caused by air pollution.

Paternal acrylamide exposure induces transgenerational effects on sperm parameters and learning capability in mice.

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.

Cx43 overexpression is involved in the hyper-proliferation effect of trichloroethylene on human embryonic stem cells.

Trichloroethylene (TCE) is a major environmental contaminant. Maternal exposure of TCE is linked to developmental defects, but the mechanisms remain to be elucidated. Along with a strategy of 3Rs principle, human embryonic stem cells (hESCs) are regarded as most promising in vitro models for developmental toxicity studies. TCE interfered with hESCs differentiation, but no report was available for TCE effects on hESCs proliferation. Here, we aimed to explore the toxic effects and mechanisms of TCE on hESCs proliferation. Treatment with TCE, did not affect the pluripotency genes expression. However, TCE enhanced hESCs proliferation, manifested by increased cell number, PCNA expression and EdU incorporation. Moreover, TCE exposure upregulated the protein expression levels of Cx43 and cyclin-dependent kinases. Knockdown of Cx43 attenuated the TCE-induced cell hyper-proliferation and CDK2 upregulation. Furthermore, TCE increased Akt phosphorylation, and the inhibition of Akt blocked the TCE-induced Cx43 overexpression and cell proliferation. In conclusion, TCE exposure resulted in upregulation of Cx43 via Akt phosphorylation, consequently stimulated CDK2 expression, contributing to hyper-proliferation in hESCs. Our study brings to light that TCE stimulated the proliferation of hESCs via Cx43, providing a new research avenue for the causes of TCE-induced developmental toxicity.

AHR/ROS-mediated mitochondria apoptosis contributes to benzo[a]pyrene-induced heart defects and the protective effects of resveratrol.

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.

Using Immunofluorescence to Detect PM2.5-induced DNA Damage in Zebrafish Embryo Hearts.

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.

Protective effects of resveratrol against the cardiac developmental toxicity of trichloroethylene in 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.

Resveratrol protects against PM2.5-induced heart defects in zebrafish embryos as an antioxidant rather than as an AHR antagonist.

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.

Effects of Trichloroethylene on the Expression of Long Intergenic Noncoding RNAs in B6C3F1 Mouse Liver.

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.

AHR-mediated ROS production contributes to the cardiac developmental toxicity of PM2.5 in zebrafish embryos.

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.

AHR-mediated oxidative stress contributes to the cardiac developmental toxicity of trichloroethylene in zebrafish embryos.

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.

PM2.5-induced extensive DNA methylation changes in the heart of zebrafish embryos and the protective effect of folic acid.

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.

Aryl hydrocarbon receptor mediates the cardiac developmental toxicity of EOM from PM2.5 in P19 embryonic carcinoma cells.

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 ß-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.

Changes in gene expression and assessment of DNA methylation in primary human hepatocytes and HepG2 cells exposed to the environmental contaminants-Hexabromocyclododecane and 17-beta oestradiol.

We evaluated the effects of two putative non-genotoxic hepatic carcinogens, hexabromocyclododecane (HBCD) and 17-beta oestradiol (E(2)) on global and CpG promoter DNA methylation in both primary human hepatocytes and hepatocellular carcinoma (HepG2) cells. The mRNA gene expression levels of genes involved particularly in cell cycle were also evaluated and potential correlation with DNA methylation status examined. HBCD at 0.03 and 0.3 ng/mL did not produce statistically significant differences in global genomic methylation. However, E(2) (0.1 ng/mL) significantly lowered global DNA methylation levels in HepG2 cells by approximately 65% (P<0.01). In primary hepatocytes, the promoter regions of N-cym and ERalpha were methylated in both control and treated groups, signifying lack of promoter demethylation by both HBCD and E(2). Furthermore, CpG promoter methylation of RB1 was observed in HepG2 cells but this was unaffected by treatments. The remaining genes (p16, C-myc, H-ras, THRalpha, histone H3, TBK1 and TNFRalpha) were unmethylated in their CpG promoter regions in both test systems. Quantitative RT-PCR showed that HBCD at 0.03 ng/mL up-regulated the expression of N-cym whereas E(2) up-regulated the expression of ERalpha and THRalpha genes in primary hepatocytes. In HepG2 cells, the mRNA gene expression levels of p16, RB1 and N-cym were significantly down regulated by HBCD (0.03 ng/mL) and E(2) (0.1 ng/mL) while HBCD at 0.3 ng/mL, significantly down regulated the expression levels of N-cym, ERalpha and ERbeta genes. Thus, while both HBCD and E(2) may alter the expression of certain genes involved in proliferation, the mechanisms appear unrelated to DNA methylation.