Decabromodiphenyl ether induces ROS-mediated intestinal toxicity through the Keap1-Nrf2 pathway.

Polybrominated diphenyl ethers (PBDEs) are widely used brominated flame retardants as commercial products. PBDEs have been demonstrated to induce hepatic, reproductive, neural, and thyroid toxicity effects. This study aimed to clarify the potential intestinal toxicity effects of decabrominated diphenyl ether (PBDE-209) in vivo and in vitro. First, we investigated the change of PBDE-209 on oxidative stress in the intestine of mice. Subsequently, the potential toxicity mechanism of PBDE-209 in vitro was investigated. Caco-2 cells were treated with different concentrations of PBDE-209 (1, 5, and 25 µmol/L) for 24 and 48 h. We determined the cell viability, reactive oxygen species (ROS) level, multiple cellular parameters, and relative mRNA expressions. The results showed that PBDE-209 significantly injured the colon of mice, increased the intestinal levels of malondialdehyde (MDA), and changed the antioxidant enzyme activities. PBDE-209 inhibited the proliferation and induced cytotoxicity of Caco-2 cells. The change in ROS production and mitochondrial membrane potential (MMP) revealed that PBDE-209 caused oxidative stress in Caco-2 cells. The real-time PCR assays revealed that PBDE-209 inhibited the mRNA expression level of antioxidative defense factor, nuclear factor erythroid 2-related factor 2 (Nrf2). Furthermore, the FAS and Cytochrome P450 1A1 (CYP1A1) mRNA expression levels were increased in Caco-2 cells. These results suggested that PBDE-209 exerts intestinal toxicity effects in vivo and in vitro and inhibits the antioxidative defense gene expression in Caco-2 cells. This study provides an opportunity to advance the understanding of toxicity by the persistent environmental pollutant PBDE-209 to the intestine.

EGCG drives gut microbial remodeling-induced epithelial GPR43 activation to lessen Th1 polarization in colitis.

Modulation of immune microenvironment is critical for inflammatory bowel disease (IBD) intervention. Epigallocatechin gallate (EGCG), as a natural low toxicity product, has shown promise in treating IBD. However, whether and how EGCG regulates the intestinal microenvironment is not fully understood. Here we report that EGCG lessens colitis by orchestrating Th1 polarization and self-amplification in a novel manner that required multilevel-regulated intestinal microecosystem. Mechanistically, EGCG activates GPR43 on IEC to inhibit Th1 polarization dependently of short chain fatty acid (SCFA)-producing gut microbiota. Inhibition of GPR43 activity weakens the protective effects of EGCG on colitis development. Moreover, we confirm that fecal SCFAs and/or intestinal GPR43 are limited in patients with colitis and are correlated with Th1 cell number. Taken together, our study reveals an intestinal microenvironment-dependent immunoregulatory effects of EGCG in treating IBD and provides insight into mechanisms of EGCG-based novel immunotherapeutic strategies for IBD.

Decabromodiphenyl ether initiates mitochondria-dependent apoptosis by disrupting calcium homeostasis in mice livers.

Decabromodiphenyl ether (BDE-209) tends to accumulate in lipid-rich tissues and targets the liver since its high lipophilicity. This study aimed to investigate the effects of BDE-209 on mouse liver and reveal the underlying toxicological mechanisms. Here we firstly confirmed that treatment of BDE-209 could lead to an imbalance of redox and promote apoptosis with a mitochondria-dependent manner in mice livers. Next, the transmission electron microscope (TEM) image revealed BDE-209 induced changes in mitochondrial morphology and increased endoplasmic reticulum (ER) - mitochondrial contact. ER stress was involved in the apoptosis process, which was displayed by the enhancive ER stress makers . Finally, from the increased abundance of cellular pivotal Ca2+ signals transducer CaM, activating Ca2+ release channel Sig-1R and IP3R1, and the stronger fluorescence density of mitochondria-specifically Ca2+ labeled probe Rhod-2 in vitro, we summarized that there was overloaded mitochondrial Ca2+ in hepatocytes of BDE-209 treated mice. In conclusion, these results partly illustrated evidence to reveal a potential mechanism of BDE-209-induced hepatoxicity, where oxidative stress-induced-ER stress led to the over-release of Ca2+, followed by the overloaded mitochondrial Ca2+, and cell apoptosis initiated. Our findings provided a theoretical basis for further studying.

Ellagic acid prevents gut damage via ameliorating microbe-associated intestinal lymphocyte imbalance.

Inflammatory bowel disease (IBD) pathogenesis involves a sustained microbial-mediated immune response following intestinal stress. Although administration of antibiotics can be an effective therapy, the misuse of antibiotics may risk unknown drug-resistant bacteria. In this study, piglets pretreated with ellagic acid (EA) and Ampicillin (AMP) for 21 days, and were injected intraperitoneally with paraquat (PQ) on 14 and 18 days. We found piglets lost most of their gut microbes in the AMP group, protected from subsequent intestinal damage caused by gut oxidative stress. Hence, we identified some gut microbes that may play a critical role in mediating cellular responses following cytokine stimulation in PQ-induced stress. EA preprocessing exhibited the same performance as AMP. Pretreatment of EA reduced Streptococcus abundance in the gut. Particularly, EA modulated intestinal lymphocyte distribution, reduced the frequency of CD79a+ cells, and alleviated the upward migration of CD3+ cells to the apex of the intestinal villi in the intestinal epithelium. Additionally, the intestinal immune response had been known associated closely with the abundance of Streptococcus in the gut. Thus, we concluded that EA has the potential to replace antibiotics to prevent microbial-mediated immune responses in the gut, and EA can be applied as a supplement candidate to alleviate the development of inflammation caused by intestinal stress.

High-fat diet exacerbated decabromodiphenyl ether-induced hepatocyte apoptosis via intensifying the transfer of Ca2+ from endoplasmic reticulum to mitochondria.

Polybrominated diphenyl ether (PBDE) as the flame retardant is heavily used in daily necessities, causing adverse health effects on humans. This study aimed to evaluate the hepatotoxicity of decabromodiphenyl ether (BDE-209), the most widely used PBDE, in lean and high-fat diet (HFD)-treated obese mice and elucidate the underlying mechanism. Firstly, the increasing levels of TG and proinflammatory factors in the liver and ALT and AST in serum demonstrated the hepatic damage caused by BDE-209 and further exacerbated by HFD. Tunel image revealed that BDE-209 induced more severe hepatocyte apoptosis with the assistant of HFD. Next, the mechanism analysis showed that the pro-apoptotic action of BDE-209 was in an endoplasmic reticulum (ER)/Ca2+ flux/mitochondria-dependent manner, concluded from the impairment of mitochondrial membrane potential, the enhancive protein expression of p-PERK/PERK, p-IRE1/IRE1, ATF6, CHOP, Bax/Bcl-2, cleaved caspase-3/caspase-3, IP3R1 and Sig-1R, and the over-transfer of Ca2+ from ER to mitochondria. Such proposed mechanism was further confirmed by the IP3R1 siRNA transfection cell experiment, where apoptotic rate was reduced in parallel with the reduced mitochondrial Ca2+ level. Finally, the higher expression of PACS-2 protein and the expanded ER contributed to the enriched ER-mitochondria interaction, reflected by the closer distance between ER and mitochondria visually displayed in the TEM image in HFD groups. This change was conducive to the rapid delivery of apoptosis signals via Ca2+, as proven, mechanically explaining the strengthening effect of HFD on BDE-209 hepatotoxicity. These findings detailedly explained the mechanism of BDE-209 hepatotoxicity and clarified the auxiliary effect of HFD, providing a theoretical basis for further studying other analogs.

Hesperidin partly ameliorates the decabromodiphenyl ether-induced reproductive toxicity in pubertal mice.

Many materials use polybrominated diphenyl ethers (PBDEs) as flame retardants. As one of the most common congeners of PBDEs, decabromodiphenyl ether (PBDE-209) is reported to harm reproductive health. However, little is known research on attenuating the reproductive toxicity induced by PBDE-209. The present study aimed to investigate the effects of hesperidin against PBDE-209-induced reproductive toxicity in male mice. Pubertal male C57BL/6 J mice were exposed to PBDE-209 groups (20, 100, 500 mg/kg·bw) and hesperidin groups (100 mg/kg·bw PBDE-209 + 100 mg/kg·bw hesperidin) for 8 weeks. The results showed that PBDE-209 increased the amount of abnormal morphological sperms and decreased the sex hormone levels. PBDE-209 induced the histopathological lesions of seminiferous tubules and blood-testis barrier in mice testis. Expressions of apoptosis-associated proteins and mRNA (Bax, Bcl-2, etc.) were altered by the PBDE-209 treatment. PBDE-209 prominently increased the malondialdehyde (MDA) levels, the biomarker of oxidative stress. Hesperidin treatment partly alleviated PBDE-209-induced histopathological lesions and apoptosis in mice testis. These findings suggested that hesperidin partly protects against PBDE-induced reproductive toxicity in pubertal mice. We conclude that more work needs to be done to explore the appropriate dosage of hesperidin or find other drugs to protect against the reproductive toxicity of PBDEs.

The combined impact of decabromodiphenyl ether and high fat exposure on non-alcoholic fatty liver disease in vivo and in vitro.

Non-alcoholic fatty liver disease (NAFLD) is considered a public health concern. Decabromodiphenyl ether (BDE-209) and high fat (HF) exposure cause liver injury, yet the combined impact on NAFLD development remains unclear. HepG2 cells were incubated with BDE-209 or/and HF reagent (Csodium oleate/Csodium palmitate = 2/1) for establishing the in vitro model, while C57BL/6 mice fed BDE-209 or/and HF diet (HFD) was the in vivo model. Oil Red O staining and the determination of triglyceride, malondialdehyde, and reactive oxygen species (ROS) contents proved the elevated lipid accumulation and oxidative stress by the mixture of BDE-209 and HF in HepG2 cells, consistent in C57BL/6 mice. Importantly, the action analysis showed the synergistic effect between BDE-209 and HF, suggesting that the population preferring the HFD is more susceptible to BDE-209 to aggravate the progression of NAFLD. Further, the increased protein expression of sterol regulatory element-binding protein 1, fatty acid synthase, and stearoyl-CoA desaturase 1 was considered to be responsible for hepatic steatosis. The impairment of antioxidant system was reflected by the lower hepatic superoxide dismutase and glutathione transferase activities and reduced glutathione level, explaining the detected excessive ROS production. Besides, using high content analysis, the decline of mitochondrial mass and membrane potential, which was closed to the NAFLD pathogenesis, was also demonstrated in HepG2 cells.

Reparative Effects of Ethanol-Induced Intestinal Barrier Injury by Flavonoid Luteolin via MAPK/NF-κB/MLCK and Nrf2 Signaling Pathways.

Luteolin, a dietary flavonoid, has gained increasing interest as an intestinal protectant. This study aimed to evaluate the reparative effect of luteolin against ethanol-induced intestinal barrier damage in a Caco-2 cell monolayer model and the potential mechanisms. Luteolin attenuated ethanol-induced intestinal barrier injury, by increasing transepithelial monolayer resistance (TEER, 27.75 ± 14.75% of the ethanol group, p < 0.01), reducing Lucifer yellow flux (13.21 ± 1.23% of ethanol group, p < 0.01), and upregulating the expression of tight junction (TJ) proteins zonulin occludin-1 (ZO-1), occludin, and claudin-1 (37.963 ± 8.62%, 17.69 ± 7.35%, and 29.40 ± 8.08% of the ethanol group, respectively, p < 0.01). Further mechanistic studies showed that luteolin suppressed myosin light chain 2 (MLC) phosphorylation, myosin light chain kinase (MLCK) activation, nuclear factor kappa-B (NF-κB) nuclear translocation, and mitogen-activated-protein-kinase (MAPK) phosphorylation. Moreover, luteolin also acted as antioxidants indirectly by upregulating antioxidant-responsive-element (ARE) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) nuclear translocation to relieve ethanol-induced oxidative damage and TJ dysfunction. The results of the study indicate that luteolin may play an effective role in relieving intestinal barrier damage, and this effect is at least partially due to its indirect antioxidant capacity.

Assessing the combinatorial cytotoxicity of the exogenous contamination with BDE-209, bisphenol A, and acrylamide via high-content analysis.

Food is often exposed to multiple types of contaminants, and the coexistence of contaminants may have antagonistic, additive or synergistic effects. This study investigated the combinatorial toxicity of the three most widespread exogenous contaminants, decabrominated diphenyl ether (BDE-209), bisphenol A (BPA), and acrylamide (ACR) to HepG2 cells. A mathematical model (Chou-Talalay) and high-content analysis (HCA) were used to probe the nature of the contaminants' interactions and their cytotoxicity mechanisms, respectively. The results highlighted that for the individual pollutants, the cytotoxicity order was BDE-209> BPA > ACR, and varying combinations of contaminants exhibited additive/synergistic effects. In general, combining multiple contaminants significantly increased intracellular reactive oxygen species (ROS), Ca2+ flux, DNA damage and Caspase-3, and decreased mitochondrial membrane potential (MMP) and nucleus roundness, indicating that the additive or synergistic mechanism of the combined contaminations was disturbance to multiple organelles. This study emphasizes the complexity of human exposure to food contaminants and provides a scientific basis for formulating strict regulatory standards.

Hesperidin prevents the combined toxicity of decabromodiphenyl ether and sodium nitrite in vitro.

Decabromodiphenyl ether (BDE-209) and Sodium nitrite (SN) coexist in the processing meat and fish foods, but there is no research considering them together. The present study aimed to investigate the binary mixture's toxicity of BDE-209 and SN and explore the protective effect of hesperidin (Hsp) on the combined toxicity. Results showed that compared with the impact of BDE-209 or SN alone, the binary mixture had a synergistic toxic effect on impairing the viability of HepG2 cells, accompanied by oxidative stress, Ca2+ accumulation, mitochondrial dysfunction. The increase of γ-H2AX fluorescent foci and micronuclei number also indicated its genotoxicity. Pretreatment of Hsp could significantly alleviate the above damage caused by the binary combination. These findings revealed the toxicological interaction of BDE-209 and SN and highlighted that food containing abundant natural flavonoids, as hesperidin, could reduce this toxicological risk.

AhR-mediated CYP1A1 and ROS overexpression are involved in hepatotoxicity of decabromodiphenyl ether (BDE-209).

Polybrominated diphenyl ethers (PBDEs) are persistent organic pollutants. They are constantly detected in terrestrial, ocean, and atmospheric systems, and it is of particular concern that these fat-soluble xenobiotics may have a negative impact on human health. This study aimed to evaluate the toxic effect and underlying mechanism of decabromodiphenyl ether (BDE-209) on human liver in a HepG2 cell model. The results showed that BDE-209 significantly induced HepG2 cells apoptosis, increased intracellular reactive oxygen species (ROS), disturbed [Ca 2+] homeostasis and mitochondrial membrane potential (MMP), and caused nuclear shrinkage and DNA double-strand breaks. BDE-209 also significantly decreased the activities of antioxidant parameters, superoxide dismutase (SOD), total antioxygenic capacity (T-AOC), glutathione (GSH), and total glutathione (T-GSH). The up-regulation of the Aryl hydrocarbon receptor (AhR)/cytochrome P4501A1 (CYP1A1) signaling pathway indicates that after long-term and high-dose exposure, BDE-209 may be a liver carcinogen. Interestingly, HepG2 cells attempt to metabolize BDE-209 through the Nrf2-mediated antioxidant pathway. These findings help elucidate the mechanisms of BDE-209-induced hepatotoxicity in humans.

Regulatory effects of flavonoids luteolin on BDE-209-induced intestinal epithelial barrier damage in Caco-2 cell monolayer model.

Polybrominated diphenyl ethers (PBDEs) are persistent organic pollutants (POPs). They are constantly detected in foods. PBDEs can disrupt the intestinal flora, but enterotoxicity is unknown. Luteolin, one kind of flavonoid, has drawn increasing interest as an agent that strengthens the intestinal barrier. This study aimed to evaluate the mitigating effect of luteolin on damage to the intestinal barrier induced by decabromodiphenyl ether (BDE-209) in a Caco-2 cell monolayer model. Results showed that luteolin mitigated BDE-209-induced damage to intestinal epithelial barrier by reducing the levels of reactive oxygen species (ROS), increasing the activity of superoxide dismutase (SOD) and glutathione (GSH), suppressed the secretion of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß), and increased the expression of tight junction (TJ) proteins (ZO-1, occludin, and claudin-1). Furthermore, the protective effects were related to the inhibition of extracellular regulated protein kinases (ERK) and nuclear factor kappa-B (NF-κB)/myosin light chain kinase (MLCK) signaling pathways, and the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways. This study is the first to provide strong evidence that BDE-209 can damage the intestinal barrier, and we here investigated the important protective effect of luteolin, which may lay the foundation for the development of luteolin as a dietary supplement to strengthen the intestinal barrier.