Benzoquinone activates the ERK/MAPK signaling pathway via ROS production in HL-60 cells

Benzene (BZ) is a class I carcinogen and its oxidation to reactive intermediates is a prerequisite of hematoxicity and myelotoxicity. The generated metabolites include hydroquinone, which is further oxidized to the highly reactive 1,4-benzoquinone (BQ) in bone marrow. Therefore, we explored the mechanisms underlying BQ-induced HL-60 cell proliferation by studying the role of BQ-induced reactive oxygen species (ROS) in the activation of the ERK-MAPK signaling pathway. BQ treatment (0.01-30 microM) showed that doses below 10 microM did not significantly reduce viability. ROS production after 3 microM BQ treatment increased threefold; however, catalase addition reduced ROS generation to basal levels. FACS analysis showed that BQ induced a fivefold increase in the proportion of cells in S-phase. We also observed a high proportion of Bromodeoxyuridine (BrdU) stained cells, indicating a higher DNA synthesis rate. BQ also produced rapid and prolonged phosphorylation of ERK1/2 proteins. Simultaneous treatment with catalase or PD98059, a potent MEK protein inhibitor, reduced cell recruitment into the S-phase and also abolished the ERK1/2 protein phosphorylation induced by BQ, suggesting that MEK/ERK is an important pathway involved in BQ-induced ROS mediated proliferation. The prolonged activation of ERK1/2 contributes to explain the increased S-phase cell recruitment and to understand the leukemogenic processes associated with exposure to benzene metabolites. Thus, the possible mechanism by which BQ induce HL-60 cells to enter the cell cycle and proliferate is linked to ROS production and its growth promoting effects by specific activation of regulating genes known to be activated by redox mechanisms.     

Generation of phosphorylated histone H2AX by benzene metabolites

Benzene is a well known environmental carcinogen which causes myeloid leukemia. DNA damage induced by benzene metabolites such as hydroquinone (HQ) and p-benzoquinone (BQ) is one reason for the leukemogenesis. In this study, we showed that treatment with HQ and BQ quickly and clearly generated phosphorylated histone H2AX (gamma-H2AX) which has been recently considered an index of the production of double strand breaks (DSBs). HQ and BQ produced discrete foci of gamma-H2AX within the nucleus of HL-60 cells in a dose-dependent manner. gamma-H2AX appeared after the treatment with HQ and BQ for 2h, and increased time-dependently up to 4-8h. HQ and BQ increased intracellular oxidation, and an antioxidant, N-acetylcysteine, clearly inhibited the phosphorylation, suggesting that reactive oxygen species produced from HQ and BQ contributed to the generation. gamma-H2AX was sensitively detected after treatment with low concentrations of HQ and BQ, compared with the direct detection of DSBs by biased sinusoidal field gel electrophoresis and with the assessment of cytotoxicity based on cell survival. DSBs are the most serious form of DNA damage and are associated with genomic instability leading to myeloid leukemia. gamma-H2AX may be a useful tool for judging the genotoxicity of benzene metabolites sensitively.     

Eriodictyol-7-O-glucoside activates Nrf2 and protects against cerebral ischemic injury

Stroke is a complex disease that may involve oxidative stress-related pathways in its pathogenesis. The nuclear factor erythroid-2-related factor 2/antioxidant response element (Nrf2/ARE) pathway plays an important role in inducing phase II detoxifying enzymes and antioxidant proteins and thus has been considered a potential target for neuroprotection in stroke. The aim of the present study was to determine whether eriodictyol-7-O-glucoside (E7G), a novel Nrf2 activator, can protect against cerebral ischemic injury and to understand the role of the Nrf2/ARE pathway in neuroprotection. In primary cultured astrocytes, E7G increased the nuclear localization of Nrf2 and induced the expression of the Nrf2/ARE-dependent genes. Exposure of astrocytes to E7G provided protection against oxygen and glucose deprivation (OGD)-induced oxidative insult. The protective effect of E7G was abolished by RNA interference-mediated knockdown of Nrf2 expression. In vivo administration of E7G in a rat model of focal cerebral ischemia significantly reduced the amount of brain damage and ameliorated neurological deficits. These data demonstrate that activation of Nrf2/ARE signaling by E7G is directly associated with its neuroprotection against oxidative stress-induced ischemic injury and suggest that targeting the Nrf2/ARE pathway may be a promising approach for therapeutic intervention in stroke.     

Generation of stable ARE- driven reporter system for monitoring oxidative stress

Background

NF-E2-related factor2 (Nrf2)-antioxidant response element (ARE) signaling pathway is the major defensive mechanism against oxidative stress and is up regulated by specific antioxidants and oxidants to comprise the chemoptotective response. Detection of ARE-activating compounds helps to develop new drugs and identify/quantify the tension range of the oxidants.Important reasons promoting this work are high throughput, rapid and inexpensive experiments relative to the in vitro studies for ARE-Nrf2 pathway monitoring of chemicals and environmental samples.

Methods

In this study hepatoma Huh7 reporter cell line was generated which contains a luciferase gene under the control of an ARE. This is the first example of ARE construct containing one copy of extended consensus response element. The cells were treated with hydroquinone (HQ) and p-benzoquinone (BQ) (oxidative stress inducers) and the antioxidant, curcumin.

Results

The luciferase activity was induced in a concentration-dependent manner in a concentration range of 1–2 μM for BQ and HQ. Curcumin was also validated as an ARE inducer in concentration above 10 μM. In addition, this reporter cell line provides a rapid detection as early as 4 h to respond to the ARE inducers.

Conclusion

It is a powerful tool for the sensitive and selective screening of chemicals, drugs and environmental samples for their antioxidant and oxidant activities.     

Acute exposure to 3‐methylcholanthrene induces hepatic oxidative stress via activation of the Nrf2/ARE signaling pathway in mice

Polycyclic aromatic hydrocarbons (PAHs) are the most common contaminants in the environment. The primary focus on the toxicity of PAHs is their ability to activate the aryl hydrocarbon receptor (AhR)‐mediated pathway and lead to carcinogenesis in different organisms. However, the influence of PAHs on the antioxidant system in mammalian systems has received only limited attention. In the present study, we observed that the intraperitoneal injection of 100 mg/kg 3‐methylcholanthrene (3MC) into mice significantly increased reactive oxygen species (ROS) levels and malondialdehyde (MDA) contents and decreased glutathione (GSH) contents and the activity of total antioxidant capacity (T‐AOC), indicating that serious oxidative stress had been induced in the liver of mice. Then, the oxidative stress signal activated the nuclear factor erythroid 2‐related factor 2/antioxidant response element (Nrf2/ARE) pathway by enhancing the mRNA levels of Nrf2, p38, and Erk2. Moreover, the mRNA levels of Nrf2/ARE target genes, including glutathione peroxidase (Gpx), glutathione reductase (GR), glutathione synthetase (GS), NAD(P)H: quinone oxidoreductase 1 (Nqo1), superoxide dismutase 1 (Sod1), and Sod2, increased significantly after treatment with 3MC for 24 hours. The hepatic levels of NQO1 and the activities of GR and GS were also significantly enhanced at 24 hours after 3MC treatment. Because the expression of NQO1 is co‐regulated by Nrf2/ARE and AhR/XRE in mammalian tissues, NQO1 may play an important role in protecting against the oxidative stress induced by 3MC. Taken together, our findings suggested that acute exposure to 3MC altered the cellular redox balance in hepatocytes to trigger Nrf2‐regulated antioxidant responses, which may represent an adaptive cell defense mechanism against the oxidative stress induced by PAHs. © 2013 Wiley Periodicals, Inc. Environ Toxicol 29: 1399–1408, 2014.     

Isorhamnetin protects against oxidative stress by activating Nrf2 and inducing the expression of its target genes

Isorhamentin is a 3′-O-methylated metabolite of quercetin, and has been reported to have anti-inflammatory and anti-proliferative effects. However, the effects of isorhamnetin on Nrf2 activation and on the expressions of its downstream genes in hepatocytes have not been elucidated. Here, we investigated whether isorhamnetin has the ability to activate Nrf2 and induce phase II antioxidant enzyme expression, and to determine the protective role of isorhamnetin on oxidative injury in hepatocytes. In HepG2 cells, isorhamnetin increased the nuclear translocation of Nrf2 in a dose- and time-dependent manner, and consistently, increased antioxidant response element (ARE) reporter gene activity and the protein levels of hemeoxygenase (HO-1) and of glutamate cysteine ligase (GCL), which resulted in intracellular GSH level increases. The specific role of Nrf2 in isorhamnetin-induced Nrf2 target gene expression was verified using an ARE-deletion mutant plasmid and Nrf2-knockout MEF cells. Deletion of the ARE in the promoter region of the sestrin2 gene, which is recently identified as the Nrf2 target gene by us, abolished the ability of isorhamnetin to increase luciferase activity. In addition, Nrf2 deficiency completely blocked the ability of isorhamnetin to induce HO-1 and GCL. Furthermore, isorhamnetin pretreatment blocked t-BHP-induced ROS production and reversed GSH depletion by t-BHP and consequently, due to reduced ROS levels, decreased t-BHP-induced cell death. In addition isorhamnetin increased ERK1/2, PKCδ and AMPK phosphorylation. Finally, we showed that Nrf2 deficiency blocked the ability of isorhamnetin to protect cells from injury induced by t-BHP. Taken together, our results demonstrate that isorhamnetin is efficacious in protecting hepatocytes against oxidative stress by Nrf2 activation and in inducing the expressions of its downstream genes.