Zixuan Liu

Tetrachlorobenzoquinone exhibits neurotoxicity by inducing inflammatory responses through ROS-mediated IKK/IκB/NF-κB signaling

Tetrachlorobenzoquinone (TCBQ) is a joint metabolite of persistent organic pollutants (POPs), hexachlorobenzene (HCB) and pentachlorophenol (PCP). Previous studies have been reported that TCBQ contributes to acute hepatic damage due to its pro-oxidative nature. In the current study, TCBQ showed the highest capacity on the cytotoxicity, ROS formation and inflammatory cytokines release among four compounds, i.e., HCB, PCP, tetrachlorohydroquinone (TCHQ, reduced form of TCBQ) and TCBQ, in PC 12 cells. Further mechanistic study illustrated TCBQ activates nuclear factor-kappa B (NF-κB) signaling. The activation of NF-κB was identified by measuring the protein expressions of inhibitor of nuclear factor kappa-B kinase (IKK) α/β, p-IKKα/β, an inhibitor of NF-κB (IκB) α, p-IκBα, NF-κB (p65) and p-p65. The translocation of NF-κB was assessed by Western blotting of p65 in nuclear/cytosolic fractions, electrophoretic mobility shift assay (EMSA) and luciferase reporter gene assay. In addition, TCBQ significantly induced protein and mRNA expressions of inflammatory cytokines and mediators, such as interleukin-1 beta (IL-1β), IL-6, tumor necrosis factor-alpha (TNF-α), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and the production of nitric oxide (NO) and prostaglandin E2 (PGE2). Pyrrolidine dithiocarbamate (PDTC), a specific NF-κB inhibitor inhibited these effects efficiently, further suggested TCBQ-induced inflammatory responses involve NF-κB signaling. Moreover, antioxidants, i.e., N-acetyl-l-cysteine (NAC), Vitamin E and curcumin, ameliorated TCBQ-induced ROS generation as well as the activation of NF-κB, which implied that ROS serve as the upstream molecule of NF-κB signaling. In summary, TCBQ exhibits a neurotoxic effect by inducing oxidative stress-mediated inflammatory responses via the activation of IKK/IκB/NF-κB pathway in PC12 cells.

The acute exposure of tetrachloro-p-benzoquinone (a.k.a. chloranil) triggers inflammation and neurological dysfunction via Toll-like receptor 4 signaling: The protective role of melatonin preconditioning

This study is aimed to investigate the inflammation and neurological dysfunction induced by tetrachloro-p-benzoquinone (TCBQ) through Toll-like receptor 4 (TLR4) signaling. We also investigated the protective role of melatonin as an antioxidant and anti-inflammatory agent. In vitro model was established by rat pheochromocytoma PC12 cells, meanwhile, TLR4 wild-type (C57BL/6) and knockout mice (C57BL/10ScNJ TLR4-/-) were used as in vivo model. In vitro study showed TCBQ exposure enhanced the expression of TLR4, myeloid differentiation factor 88 (MyD88) at both transcriptional and post-transcriptional levels. By contrast, melatonin decreased TLR4 and MyD88 expressions. Moreover, our result indicated that melatonin disrupted the formation of TLR4/MyD88/MD2/CD14 complex. In addition, melatonin terminated TCBQ-mediated phosphorylation of c-Jun N-terminal kinase (JNK), p38, and extracellular regulated protein kinase (ERK) signaling and hampered its downstream pro-inflammatory cytokine releases. In vivo result also indicated TLR4 deficiency partially protected against TCBQ-induced morphological and neuropathological changes in mice brain, suggested the role of TLR4. In conclusion, melatonin modulates TCBQ-mediated inflammatory genes through TLR4/MyD88-dependent signaling pathway. Our current study, to the best of our knowledge, is the first time show melatonin not only disrupt the binding of TLR4 and MyD88, but also restricted the formation of TLR4/MD2/CD14 complex, suggesting that melatonin supplementary may represent a valuable therapeutic strategy for inflammatory neurological dysfunction.

Tetrachlorobenzoquinone induces Nrf2 activation via rapid Bach1 nuclear export/ubiquitination and JNK-P62 signaling.

Our previous studies demonstrated that tetrachlorobenzoquinone (TCBQ), an active metabolite of pentachlorophenol, has effects on the generation of reactive oxygen species (ROS) and oxidative stress in vitro and in vivo. Nuclear factor erythroid-derived 2-like 2 (Nrf2) is a cellular sensor of electrophilic or oxidative stress that regulates the expression of antioxidant enzymes and defensive proteins. We have illustrated that TCBQ activates Nrf2 signaling by promoting the formation of the Kelch-like ECH-associated protein 1 (Keap1) cross-linking dimer and the formation of an ubiquitination switch from Nrf2 to Keap1. The activation of Nrf2 by TCBQ may serve as an adaptive response to a TCBQ-induced oxidative insult. BTB and CNC homolog 1 (Bach1) compete with Nrf2, leading to the negative regulation of the antioxidant response element (ARE). In this report, we propose that TCBQ induces the dynamic inactivation of Bach1. We observed a rapid nuclear efflux of Bach1 and an accumulation of Nrf2 in nuclei upon TCBQ treatment that precedes the binding of Nrf2 with ARE. We found that the nuclear export of Bach1 is dependent on its chromosomal region maintenance 1 (Crm1) interaction and tyrosine phosphorylation. Although TCBQ induces the ubiquitination of Bach1, TCBQ also increases the mRNA and protein levels of Bach1, returning Bach1 to normal levels. Moreover, we found that TCBQ-induced activation of Nrf2 involves c-Jun N-terminal kinase (JNK)-P62 signaling.

Activating Transcription Factor 4 (ATF4)-ATF3-C/EBP Homologous Protein (CHOP) Cascade Shows an Essential Role in the ER Stress-Induced Sensitization of Tetrachlorobenzoquinone-Challenged PC12 Cells to ROS-Mediated Apoptosis via Death Receptor 5 (DR5) Signaling

Tetrachlorobenzoquinone (TCBQ) is a downstream metabolite of pentachlorophenol (PCP). Previously, we demonstrated that TCBQ caused cytotoxicity due to mitochondrial-related apoptosis. Here, we confirmed the upregulation of death receptor 5 (DR5) followed by the construction of the death-inducing signaling complex (DISC). We also detected the activation of the caspase cascade, which was correlated with TCBQ-induced apoptotic cell death in PC12 cells. The upregulation of DR5 included transcriptional activation and de novo protein synthesis in response to TCBQ. We also identified the endoplasmic reticulum (ER) as a new target for the TCBQ challenge in PC12 cells. The protein kinase R-like ER kinase/eukaryotic translation initiation factor 2α (PERK/eIF2α)-mediated activating transcription factor 4 (ATF4)-ATF3-C/EBP homologous protein (CHOP) signaling pathway contributed to the process of TCBQ-induced ER stress. Blocking ATF4, ATF3, or CHOP signaling by gene silencing technology resulted in decreased cell apoptosis after exposure to TCBQ. Finally, NAC ameliorated TCBQ-induced apoptosis and ER stress, which illustrated that TCBQ-induced apoptosis is somehow ROS-dependent. In summary, this study provided important mechanistic insight into how TCBQ utilizes ER stress-related signaling to exhibit pro-apoptotic activity in PC12 cells.

From the Cover: Tetrachlorobenzoquinone Exerts Neurological Proinflammatory Activity by Promoting HMGB1 Release, Which Induces TLR4 Clustering within the Lipid Raft

Tetrachlorobenzoquinone (TCBQ) is a confirmed active metabolite of a well-known environmental pollutant pentachlorophenol (PCP). Unfortunately, there is insufficient knowledge present available on TCBQs toxicity. Our previous studies indicated that TCBQ induces inflammatory response in vivo and in vitro; however, its exact mechanism needs further investigation. Toll-like receptors (TLRs) play a crucial role in conveying of inflammatory signaling, whilst high-mobility group box 1 (HMGB1) functions as a transcription-enhancing nuclear protein that regulates inflammation. Indeed, this study demonstrated that TCBQ induces the secretion/translocation of HMGB1, which in turn activates its receptors, TLR family gene (especially TLR4) and receptor for advanced glycation end-products (RAGE) expressions. Consistently, the binding affinity of HMGB1 with its receptors also increased. In the case of HMGB1 or TLR4 deficiency, there were decreases in TCBQ-induced neuroinflammatory cytokine production and neuropathological changes, eg, neuronal loss, astrocyte and macrophage cells activation. Moreover, we found the mobilization of TLR4 into lipid rafts occurs in response to TCBQ exposure, lipid rafts disruptors weakened this effect, suggested lipid rafts play an essential role for TLR4-mediated signal transduction and target inflammatory cytokines expressions. In summary, our current findings revealed a previously unknown mechanism of TCBQ-induced neurological inflammation related to HMGB1-TLR4 signaling.

Tetrachlorobenzoquinone Stimulates NLRP3 Inflammasome-Mediated Post-Translational Activation and Secretion of IL-1β in the HUVEC Endothelial Cell Line

Our previous studies suggested that tetrachlorobenzoquinone (TCBQ) elicits pro-inflammatory activities; however, the mechanism of its toxicity toward vascular endothelial cell has not been characterized. Although TCBQ has been shown to stimulate interleukin-1 beta (IL-1β) expression, it is unknown whether TCBQ regulates post-translational IL-1β activation. Using human umbilical vein endothelial cells, we discovered that TCBQ not only promotes the expression of NOD-like receptor family, pyrin domain-containing protein 3 (NLRP3) components [composed of NLRP3, adaptor molecule apoptosis-associated speck like protein containing a caspase activation and recruitment domain (ASC), and pro-caspase 1] but also participates in priming the NLRP3 inflammasome. Activation of the NLRP3 inflammasome results in the maturation and release of IL-1β. Further experiments showed that K(+) efflux, reactive oxygen species (ROS) production, and mitochondrial DNA damage may be involved in NLRP3 inflammasome activation mediated by TCBQ. Moreover, TCBQ downregulates the ubiquitination of NLRP3, further facilitating the activation of the NLRP3 inflammasome. These results suggest that the NLRP3/IL-1β signaling pathway plays an important role in TCBQ-induced endothelial cell pro-inflammatory responses, which may point to potential therapeutic approaches against TCBQ-mediated toxicity.

Tetrachlorobenzoquinone-induced Nrf2 Confers Neuron-Like PC12 Cells Resistance to Endoplasmic Reticulum Stress via Regulating Glutathione Synthesis and Protein Thiol Homeostasis

Our previous studies demonstrated that tetrachlorobenzoquinone (TCBQ) is toxic to neuron-like cells, which is related to endoplasmic reticulum (ER) stress-induced apoptosis. However, it remains unclear whether TCBQ causes the opening of cellular defense responses. Here we found that activation of nuclear factor erythroid-derived 2-like 2 (Nrf2) triggered an adaptive response against the neurotoxicity induced by TCBQ through the upregulation of intracellular glutathione (GSH) levels in rat pheochromocytoma PC12 cells. TCBQ upregulated the levels of GSH mainly by the following two ways: (i) Nrf2 activation induced the expression of cystine/glutamate antiporter solute carrier family 7 member 11 (SLC7A11, also called xCT); (ii) Nrf2 activation resulted in increased the expression of glutamylcysteine ligase. GSH is involved in cell antioxidant ability and protein thiol homeostasis, especially in the ER. Therefore, GSH has the ability to inhibit ER stress and promote cell survival. Our data showed that decreasing GSH levels exacerbated TCBQ-induced depletion of protein-SH, particularly in the ER. Conversely, increasing GSH levels attenuated TCBQ-induced protein damage, degree of ER stress, and cell death. These findings demonstrated that GSH-inhibited cells were vulnerable to TCBQ-induced ER stress and apoptosis. Overall, our results analyzed the relationships between Nrf2 and ER stress in response to TCBQ and showed that activation of Nrf2-GSH played a protective role against TCBQ-induced ER stress-associated neurotoxicity via regulating GSH synthesis and protein thiol homeostasis.

The electrophilic character of quinones is essential for the suppression of Bach1

The activation of nuclear factor erythroid 2-related factor 2 (Nrf2) is the most important cellular defense mechanisms against oxidative attack. BTB and CNC homology-1 (Bach1), like Kelch-like ECH-associated protein 1 (Keap1), is one of a negative regulator of Nrf2 that control antioxidant response elements (ARE)-dependent gene expressions. In the current study, we found that quinones show greater capacity than hydroquinones in nuclear Bach1 export, as well as ubiquitin-dependent Bach1 degradation in our experimental time frame. Consistently, quinones are easier than hydroquinones in Nrf2 activation and ARE-driven antioxidant protein expressions. Considering the redox cycling potential of quinone-hydroquinone couple, we investigated the effect of transit metal oxidation on the regulation of Nrf2 activity. As shown, Fe3+ enhanced hydroquinone-induced Nrf2 activation and ARE-driven gene expressions, suggesting quinones rather than hydroquinone activate Nrf2 through Bach1 arylation. Taking together, our investigation illustrated that the electrophilic character of quinones ensure their conjugation with Bach1, which is important for the downregulation of Bach1 and the upregulation of Nrf2 signaling.