Demei Xu

Role of α-lipoic acid in LPS/d-GalN induced fulminant hepatic failure in mice: studies on oxidative stress, inflammation and apoptosis.

This study investigated the protective effect of α-lipoic acid (LA) on lipopolysaccharide (LPS)/d-galactosamine (d-GalN)-induced fulminant hepatic failure in mice. First, we found that LA markedly reduced LPS/d-GalN-induced increases in serum ALT and AST activities, which were supplemented with histopathological examination, suggested that LA has a protective effect on this model of hepatic damage. Livers challenged with LPS/d-GalN exhibited extensive areas of vacuolization with the disappearance of nuclei and the loss of hepatic architecture. On the contrary, these pathological alterations were ameliorated by LA treatment. Next, we found that ROS and TBARS levels were increased in LPS/d-GalN treated liver homogenates, which were attenuated by LA administration. Consistently, decreases in hepatic CAT and GPx activities were observed in LPS/d-GalN group and were significantly restored by LA administration. Moreover, pretreatment with LA markedly reduced LPS/d-GalN-induced iNOS, COX-2, TNF-α, NF-κB, IL-1β and IL-6 expressions. Furthermore, our data showed that TUNEL-positive cells increased in LPS/d-GalN-treated mice liver which was counteracted by LA administration. LPS/d-GalN induced apoptosis of hepatocytes, as estimated by caspase 3, caspase 8 and caspase 9 activations. Also, the increasing of Bax and the decreasing of Bcl-2 expressions also supported LPS/d-GalN induced apoptosis. Interestingly, LA marked relieved these apoptotic features. Taking together, our results indicated that LA plays an important role on LPS/d-GalN-induced fulminant hepatic failure through its antioxidant, anti-inflammatory and anti-apoptotic activities.

Tetrachlorobenzoquinone induces acute liver injury, up-regulates HO-1 and NQO1 expression in mice model: The protective role of chlorogenic acid

Tetrachlorobenzoquinone (TCBQ) is an active metabolite of pentachlorophenol (PCP). Although PCP has been investigated extensively, there are only a few reports describing the toxicity effect of TCBQ, and no report regarding TCBQ-induced liver injury in vivo. In the current study, we aimed to examine the acute hepatic toxicity of TCBQ in the mice model. Chlorogenic acid (CGA) exhibits promising antioxidant activity in the past studies, thus, the second aim of this study was to evaluate the protective effect of CGA on TCBQ-induced liver injury. Our results indicated TCBQ-intoxication caused marked liver cell necrosis and inflammation but not apoptosis, and this damage was alleviated by CGA treatment. Meantime, TCBQ-intoxication enhanced serum ALT, AST activities, TBIL content, hepatic oxidative stress and lipid peroxidation, decreased GSH content and inhibited the activities of antioxidant enzymes. Western blot and immunohistochemical analysis showed that TCBQ marked up-regulated HO-1 and NQO1 expression. On the other hand, pretreatment of CGA reduced TCBQ-induced liver damage remarkably. Taking together, these results revealed that TCBQ has strong hepatic toxic effect, and at least a part of this effect is initiated by free radical and relieved with CGA administration.

Tetrachloro-p-benzoquinone induces hepatic oxidative damage and inflammatory response, but not apoptosis in mouse: The prevention of curcumin

This study investigated the protective effects of curcumin on tetrachloro-p-benzoquinone (TCBQ)-induced hepatotoxicity in mice. TCBQ-treatment causes significant liver injury (the elevation of serum AST and ALT activities, histopathological changes in liver section including centrilobular necrosis and inflammatory cells), oxidative stress (the elevation of TBAR level and the inhibition of SOD and catalase activities) and inflammation (up-regulation of iNOS, COX-2, IL-1β, IL-6, TNF-α and NF-κB). However, these changes were alleviated upon pretreatment with curcumin. Interestingly, TCBQ has no effect on caspase family genes or B-cell lymphoma 2 (Bcl-2)/Bcl-2 associated X (Bax) protein expressions, which implied that TCBQ-induced hepatotoxicity is independent of apoptosis. Moreover, curcumin was shown to induce phase II detoxifying/antioxidant enzymes HO-1 and NQO1 through the activation of nuclear factor erythroid-derived 2-like 2 (Nrf2). In summary, the protective mechanisms of curcumin against TCBQ-induced hepatoxicity may be related to the attenuation of oxidative stress, along with the inhibition of inflammatory response via the activation of Nrf2 signaling.

Evaluation of N-acetyl-cysteine against tetrachlorobenzoquinone-induced genotoxicity and oxidative stress in HepG2 cells

Tetrachlorobenzoquinone (TCBQ) is an active metabolite of pentachlorophenol (PCP). Although the genotoxic effect of PCP has been comprehensively investigated, there is little known about TCBQs genotoxic effects. In the current study, TCBQ was tested for its genotoxicity using HepG2 cells as experimental model. To select the exposure concentration of interest, cell viability was measured and three concentrations were used for further investigation. In single cell gel electrophoresis (SCGE) assay, concentration-dependent increase in tail length, tail DNA percentage and tail moment were detected following TCBQ exposure. Micronucleus (MN) assay indicated TCBQ gradually increased MN frequency and decreased nuclear division index (NDI). Enzyme-linked immunosorbent assay (ELISA) and western blotting analyses both showed TCBQ caused histone H2AX phosphorylation (γ-H2AX). Furthermore, the elevation of 8-hydroxydeoxyguanosine (8-OHdG) and reactive oxygen species (ROS) level indicated TCBQ-induced genotoxicity is associated with oxidative stress. On the other hand, N-acetyl-cysteine (NAC) administration significantly protected cells from the genotoxic effect of TCBQ. Overall, our data suggested TCBQ exerted genotoxic effect possibly via an oxidative damage mechanism in HepG2 cells and this toxicity is prevented by pretreatment with NAC.

Protective effects of neohesperidin dihydrochalcone against carbon tetrachloride-induced oxidative damage in vivo and in vitro

The purpose of this study was to investigate the possible hepatoprotective effects of neohesperidin dihydrochalcone (NHDC) on carbon tetrachloride (CCl4)-induced acute oxidative injury in vivo and in vitro. In a mouse model, intraperitoneal injection of CCl4 resulted in a significant increase in serum aspartate transaminase (AST) and alanine transaminase (ALT) activities. Histopathological examination revealed severe hepatocyte necrosis and destruction of architecture in liver lesions, and immunohistochemical staining illustrated a remarkable enhancement of COX-2 and iNOS expression. The levels of hepatic antioxidant, such as, catalase (CAT), total superoxide dismutase (T-SOD), glutathione peroxidase (GP-X) and glutathione (GSH) were decreased, compared to the control group. However, pretreatment of NHDC for six consecutive days significantly ameliorated these changes. Moreover, Western blotting assay indicated pretreatment with NHDC also down-regulated CCl4-induced protein expressions of NF-κB, IL-6, caspase 3 and caspase 8. In HepG2 cell model, CCl4-treatment caused significant decrease in cell viability, antioxidant activities and GSH level, increase in intracellular reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) level. Interestingly, pretreatment of NHDC effectively relieved CCl4-induced oxidative damage in a dose-dependent manner. In conclusion, NHDC appeared to possess promising anti-oxidative and anti-inflammatory capacities, it is possible to be used as a hepatoprotective agent.

Polychlorinated biphenyl quinone induces mitochondrial‐mediated and caspase‐dependent apoptosis in HepG2 cells

Polychlorinated biphenyl (PCB) quinones are known to cause toxic effects, but their mechanisms are quite unclear. In this study, we examined whether 2,3,5‐trichloro‐6‐phenyl‐[1,4]benzoquinone, PCB29‐pQ, induces cell death via apoptosis pathway. Our result showed PCB29‐pQ exposure decreased HepG2 cell viability in a time‐dependent manner. Lactate dehydrogenase leakage assay also implied the cytotoxicity of PCB29‐pQ. 4′,6‐Diamidino‐2‐phenylindole dihydrochloride staining and flow cytometry assays both confirmed PCB29‐pQ caused dose‐dependent apoptotic cell death in HepG2 cells. Furthermore, we found that PCB29‐pQ exposure increased cellular reactive oxygen species (ROS) level, decreased mitochondrial membrane potential and induced the translocation of cytochrome c from mitochondria into cytosol in HepG2 cells. Moreover, PCB29‐pQ exposure induced B‐cell lymphoma 2 (Bcl‐2) downregulation and Bcl‐2‐associated X (Bax) upregulation, poly(ADP‐ribose) polymerase cleavage, accompanied with the increased caspase‐3/9 and p53 expressions. Taking together, these results suggested PCB29‐pQ induced HepG2 cells apoptosis through a ROS‐driven, mitochondrial‐mediated and caspase‐dependent pathway.

Tetrachlorobenzoquinone triggers the cleavage of Bid and promotes the cross-talk of extrinsic and intrinsic apoptotic signalings in pheochromocytoma (PC) 12 cells

Although there are few studies suggested PCP exposure induced developmental and behavioral disorders, however, the occurrence of neurotoxicity and PCP has not been firmly established. Tetrachlorobenzoquinone (TCBQ) is a reactive metabolite of environmental pollutant pentachlorophenol (PCP). To the best of our knowledge, there has no information regarding to the neurological toxic effect of TCBQ available. Here, we demonstrated that TCBQ induces cytotoxicity in pheochromocytoma PC12 cell line, and the mode-of-action analysis indicated the involvement of apoptotic signalings, such as the activation of caspase family proteins, the increased expressions of Fas and Fas-associated death domain (FADD), the loss of mitochondrial membrane potential (MMP), the release of cytochrome c (Cyt c) and the cleavage of the caspase substrates poly(ADP-ribose) polymerase (PARP). BI-6C9, a specific BH3-interacting domain death agonist (Bid) inhibitor, repressed TCBQ-induced Bid truncation, along with the activation of caspase 3 and the release of Cyt c, suggested the cross-talk of extrinsic and intrinsic apoptotic signalings. Furthermore, the inhibition of caspase 8 impaired TCBQ-induced the activation of caspase 3, as well as the release of Cyt c and the cleavage of Bid, suggesting caspase 8 acting as the upstream molecule of Bid, and TCBQ-induced apoptosis is initiated via caspase 8, leads to the activation of caspase 9/3 through Bid-mediated amplification loop. Finally, the pretreatment of antioxidant NAC ameliorated Fas, FADD and caspase 8/3 expressions, which illustrated that TCBQ-induced apoptotic signaling is ROS dependent. Taken together, these results indicated that the cleavage of Bid may play an important role in TCBQ-induced neurotoxicity which promotes the cross-talk of extrinsic and intrinsic apoptotic signalings in PC12 cells.

Tetrachlorobenzoquinone activates Nrf2 signaling by Keap1 cross-linking and ubiquitin translocation but not Keap1-Cullin3 complex dissociation.

Tetrachlorobenzoquinone (TCBQ), a metabolite of industrial herbicide pentachlorophenol, showed hepatotoxicity and genotoxicity through reactive oxygen species (ROS) mechanism in vivo and in vitro models. Nuclear factor erythroid-derived 2-like 2 (Nrf2) is a cellular sensor of oxidative or electrophilic stress, which controls the expression of detoxifying enzymes and antioxidant proteins. Using the human hepatoma HepG2 cell line as an in vitro model, we demonstrated a significant induction of Nrf2 but not its negative regulator Kelch-like ECH-associated protein 1 (Keap1), following exposure to TCBQ. Also, our results clearly demonstrated the translocation of cytosolic Nrf2 into the nucleus. After translocation, Nrf2 subsequently binds to the antioxidant response element (ARE), up-regulated heme oxygenase-1 (HO-1), and NADH quinone oxidoreductase subunit 1 (NQO1), which may be considered as an antioxidative response to TCBQ-intoxication. The luciferase reporter assay confirmed the formation of the Nrf2-ARE complex. Furthermore, mechanism studies proposed that TCBQ promoted the formation of the Keap1 cross-linking dimer, a ubiquitination switch from Nrf2 to Keap1 but not the dissociation of the Keap1-Cullin3 (Cul3) complex.

Polychlorinated biphenyl quinone induces endoplasmic reticulum stress, unfolded protein response, and calcium release.

Organisms are able to respond to environmental insult to maintain cellular homeostasis, which include the activation of a wide range of cellular adaptive responses with tightly controlled mechanisms. The endoplasmic reticulum (ER) is an organelle responsible for protein folding and calcium storage. ER stress leads to the accumulation of unfolded proteins in the ER lumen. To be against or respond to this effect, cells have a comprehensive signaling system, called unfolded protein response (UPR), to restore homeostasis and normal ER function or activate the cell death program. Therefore, it is critical to understand how environmental insult regulates the ingredients of ER stress and UPR signalings. Previously, we have demonstrated that polychlorinated biphenyl (PCB) quinone caused oxidative stress, cytotoxicity, genotoxicity, and apoptosis in HepG2 cells. Here, we investigated the role of a PCB quinone, PCB29-pQ on ER stress, UPR, and calcium release. PCB29-pQ markedly increased the hallmark genes of ER stress, namely, glucose-regulated protein 78 (GRP78), GRP94, and C/EBP homologous protein (CHOP) on both protein and mRNA levels in HepG2 cells. We also confirmed PCB29-pQ induced ER morphological defects by using transmission electron microscopy. Moreover, PCB29-pQ induced intracellular calcium accumulation and calpain activity, which were significantly inhibited by the pretreatment of BAPTA-AM (Ca(2+) chelator). These results were correlated with the outcome that PCB29-pQ induces ER stress-related apoptosis through caspase family gene 12, while salubrinal and Z-ATAD-FMK (a specific inhibitor of caspase 12) partially ameliorated this effect, respectively. N-Acetyl-l-cysteine (NAC) scavenged ROS formation and consequently alleviated PCB29-pQ-induced expression of ER stress-related genes. In conclusion, our result demonstrated for the first time that PCB quinone leads to ROS-dependent induction of ER stress, and UPR and calcium release in HepG2 cells, and the evaluation of the perturbations of ER stress, UPR, and calcium signaling provide further information on the mechanisms of PCB-induced toxicity.