tBHQ Pretreatment Research Articles

Identification and Validation of Endoplasmic Reticulum Stress-Related Gene in Traumatic Brain Injury.

Endoplasmic reticulum stress (ERS) plays an essential role in the development of traumatic brain injury (TBI). We aimed to identify and validate the potential ERS-related genes of TBI through bioinformatics analysis and in vitro cell experiment. A total of 19 TBI and ERS-related genes were obtained from the GeneCards database and Comparative Toxicogenomics Database (CTD). Enrichment analysis primarily enriched in apoptosis. NFE2L2 was identified as a hub gene based on the protein-protein interactions (PPI) network that combined seven ranked methods included in cytoHubba. To further explore the effect of Nrf2, the protein encoded by NFE2L2, on ERS-induced apoptosis, we conducted cell experiments with tert-butylhydroquinone (tBHQ), the classical inducer of Nrf2. Western blot suggested tBHQ pretreatment could diminish ERS and reduce the protein expressions of apoptosis in the primary cultured neuron injury model. These data may establish some theoretical basis for the treatment of TBI and provide inspiration and innovative ideas for clinicians and pathologists to understand TBI comprehensively.

Activation of NFE2L2 Alleviates Endoplasmic Reticulum Stress-Mediated Pyroptosis in Murine Hippocampus: A Bioinformatics Analysis and Experimental Validation.

Pyroptosis has been implicated in many pathologic processes, including endoplasmic reticulum stress (ERS). However, the underlying mechanisms and molecular targets of ERS affecting pyroptosis still need further exploration. We obtained gene sets associated with ERS and pyroptosis, and the common genes were regarded as crosstalk genes linking ERS and pyroptosis. Protein-protein interaction (PPI) network was constructed, and the hub genes were obtained via Cytoscape. Moreover, to validate the efficacy of the therapeutic target, neurological tests, brain water content measurements, Nissl staining, Western blot, ELISA, TUNEL analyses, and transmission electron microscopy were performed in a mouse model. A total of 13 crosstalk genes were acquired, and enrichment analysis revealed that these genes were mainly enriched in stress-associated cellular processes and pathways, including KEAP1-NFE2L2 pathway. The hub gene, NFE2L2, was identified by Cytoscape, and tert-butylhydroquinone (tBHQ) was screened as candidate drug to activate NFE2L2. Western blot and ELISA results showed that activation of NFE2L2 could attenuate the expression of ERS and pyroptosis-related proteins by promoting nuclear translocation of Nrf2 (encoded by NFE2L2). Pathological evaluation by Nissl staining and TUNEL assay reflected a similar trend. Furthermore, activation of NFE2L2 ameliorated neurological deficits and reduced brain edema. In conclusion, our bioinformatic analysis results established the theoretical foundation of NFE2L2 as a promising therapeutic target. Moreover, in the mouse model, tBHQ pretreatment further confirmed the effectiveness of this target. We hypothesized NFE2L2 may play a key role in the progression of ERS-mediated pyroptosis. These findings may inspire new ideas to treat neurological disorders.

Emodin disrupts the Notch1/Nrf2/GPX4 antioxidant system and promotes renal cell ferroptosis.

Emodin has been demonstrated to possess multiple pharmacological activities. However, emodin has also been reported to induce nephrotoxicity at high doses and with long-term use, and the underlying mechanism has not been fully disclosed. The current study aimed to investigate the roles of oxidative stress and ferroptosis in emodin-induced kidney toxicity. Mice were intraperitoneally treated with emodin, and NRK-52E cells were exposed to emodin in the presence or absence of treatment with Jagged1, SC79, or t-BHQ. Emodin significantly upregulated the levels of blood urea nitrogen, serum creatinine, malondialdehyde, and Fe2+ , reduced the levels of superoxide dismutase and glutathione, and induced pathological changes in the kidneys in vivo. Moreover, the viability of NRK-52E cells treated with emodin was reduced, and emodin induced iron accumulation, excessive reactive oxygen species production, and lipid peroxidation and depolarized the mitochondrial membrane potential (ΔΨm). In addition, emodin treatment downregulated the activity of neurogenic locus notch homolog protein 1 (Notch1), reduced the nuclear translocation of nuclear factor erythroid-2 related factor 2 (Nrf2), and decreased glutathione peroxidase 4 protein levels. However, Notch1 activation by Jagged1 pretreatment, Akt activation by SC79 pretreatment, or Nrf2 activation by t-BHQ pretreatment attenuated the toxic effects of emodin in NRK-52E cells. Taken together, these results revealed that emodin-induced ferroptosis triggered kidney toxicity through inhibition of the Notch1/Nrf2/glutathione peroxidase 4 axis.

Matrine-induced nephrotoxicity via GSK-3β/nrf2-mediated mitochondria-dependent apoptosis

IntroductionMatrine (MT), an ingredient extracted from the Chinese herb Sophora flavescens, can result in nephrotoxicity because of long-term exposure. However, the underlying mechanism by which MT leads to kidney injury remains unclear. This study aimed to investigate the roles of oxidative stress and mitochondria in MT-induced kidney toxicity both in vitro and in vivo. MethodsMice were exposed to MT for 20 days, and NRK-52E cells were exposed to MT with or without LiCl (a GSK-3β inhibitor), tert-Butylhydroquinone (t-BHQ, an Nrf2 activator), or small interfering RNA. ResultsThe results showed that MT caused nephrotoxicity accompanied by an increase in reactive oxygen species (ROS) accumulation and mitochondrial dysfunction. Meanwhile, MT significantly upregulated glycogen synthase kinase-3β (GSK-3β) activity, released cytochrome c (Cyt C) and cleaved caspase-3, decreased the activity of nuclear factor-erythroid 2-related Factor 2 (Nrf2), and reduced the expression of heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO-1), which led to the inactivation of antioxidant enzymes and the activation of apoptosis. In addition, GSK-3β inhibition by LiCl or small interfering RNA pretreatment or Nrf2 activation by t-BHQ pretreatment attenuated the toxic effects of MT in NRK-52E cells. ConclusionsTaken together, these results revealed that MT-induced apoptosis triggered kidney toxicity and that GSK-3β or Nrf2 might serve as a promising nephroprotective target for MT-induced kidney injury.

Ethyl carbamate triggers ferroptosis in liver through inhibiting GSH synthesis and suppressing Nrf2 activation

Humans are inevitably exposed to ethyl carbamate (EC) via consumption of fermented food and beverages. EC, known as an environmental toxin, can cause oxidative stress-mediated severe toxicity, but the underlying mechanisms remain unveiled. Ferroptosis is a newly identified ROS-mediated non-apoptotic cell death characterized by iron accumulation and excessive lipid oxidation. In this study, we first found that EC triggered ferroptosis in liver cells by detection of decreased cell viability, GSH, GPX4 and Ferritin levels, as well as increased iron and MDA contents. Ferroptosis inhibitor ferrostatin-1 (Fer-1) pretreatment rescued ferroptotic damage, indicating that ferroptosis was critical for EC-caused cell death. Furthermore, GSH synthesis precursor N-acetylcysteine displayed significant anti-ferroptotic properties and we suggested that GSH depletion might be the main cause of ferroptosis under EC exposure. EC-triggered GSH depletion mainly depended on suppressed GSH synthesis via inhibition of SLC7A11 and GCLC expressions. Notably, EC blocked Nrf2 activation by repression of phosphorylation modification and nuclear translocation, which further resulted in ferroptosis occurrence. We also observed EC-induced liver dysfunction and inflammation, accompanied with oxidative stress, ferroptosis and downregulated Nrf2 signaling in Balb/c mice, which could be effectively reversed by Fer-1 and tBHQ pretreatment. Together, our study indicated that ferroptosis is a new mechanism for EC-caused toxicity, which was attributed to Nrf2 inactivation and GSH depletion.

Tert-butylhydroquinone augments Nrf2-dependent resilience against oxidative stress and improves survival of ventilator-induced lung injury in mice.

Oxidative stress caused by mechanical ventilation contributes to the pathophysiology of ventilator-induced lung injury (VILI). A key mechanism maintaining redox balance is the upregulation of nuclear factor-erythroid-2-related factor 2 (Nrf2)-dependent antioxidant gene expression. We tested whether pretreatment with an Nrf2-antioxidant response element (ARE) pathway activator tert-butylhydroquinone (tBHQ) protects against VILI. Male C57BL/6J mice were pretreated with an intraperitoneal injection of tBHQ (n = 10), an equivalent volume of 3% ethanol (EtOH3%, vehicle, n = 13), or phosphate-buffered saline (controls, n = 10) and were then subjected to high tidal volume (HVT) ventilation for a maximum of 4 h. HVT ventilation severely impaired arterial oxygenation ([Formula: see text] = 49 ± 7 mmHg, means ± SD) and respiratory system compliance, resulting in a 100% mortality among controls. Compared with controls, tBHQ improved arterial oxygenation ([Formula: see text] = 90 ± 41 mmHg) and respiratory system compliance after HVT ventilation. In addition, tBHQ attenuated the HVT ventilation-induced development of lung edema and proinflammatory response, evidenced by lower concentrations of protein and proinflammatory cytokines (IL-1β and TNF-α) in the bronchoalveolar lavage fluid, respectively. Moreover, tBHQ enhanced the pulmonary redox capacity, indicated by enhanced Nrf2-depentent gene expression at baseline and by the highest total glutathione concentration after HVT ventilation among all groups. Overall, tBHQ pretreatment resulted in 60% survival (P < 0.001 vs. controls). Interestingly, compared with controls, EtOH3% reduced the proinflammatory response to HVT ventilation in the lung, resulting in 38.5% survival (P = 0.0054 vs. controls). In this murine model of VILI, tBHQ increases the pulmonary redox capacity by activating the Nrf2-ARE pathway and protects against VILI. These findings support the efficacy of pharmacological Nrf2-ARE pathway activation to increase resilience against oxidative stress during injurious mechanical ventilation.

Toxicity of Aqueous L-Selenomethionine and Tert-Butyl Hydroperoxide Exposure to Zebrafish (Danio rerio) Embryos Following Tert-Butyl Hydroquinone Treatment.

Aqueous L-selenomethionine (SeMet) embryo exposures represent a rapid and simplified method for investigating the embryotoxic effects of SeMet. Using zebrafish (Danio rerio) as a model organism, the objective of the present study was to characterize the effects of waterborne exposure to both SeMet and tert-butyl hydroperoxide (tBOOH) to early life stages of zebrafish pre-treated with the antioxidant tert-butyl hydroquinone (tBHQ) in an attempt to investigate the mechanism of Se toxicity as it relates to oxidative stress. During the initial concentration range finding experiment, recently fertilized embryos were exposed for five days to 5, 25, 125, and 625 µg Se/L (as SeMet). These exposures informed the second experiment in which embryos were exposed to two concentrations of SeMet (25 and 125 µg Se/L) and 75 mg/L tBOOH either with (tBOOH-t, 25-t, 125-t) or without (tBOOH, 25, 125) a 4 h 100 µg/L tBHQ pre-treatment. Survival, hatchability, time to hatch, the frequency and severity of deformities (total and type), and changes in the expression of seven antioxidant-associated genes were determined. Exposures to SeMet and tBOOH reduced hatchability, increased time to hatch, decreased survival, increased the incidence and severity of deformities, and increased glutathione-disulfide reductase (gsr) expression in the pre-treated tBOOH treatment group.

The effect of manganese exposure on GnRH secretion via Nrf2/mGluR5/COX-2/PGE2/signaling pathway.

There are limited studies focused on the precise mechanism of gonadotropin-releasing hormone (GnRH) secretion dysfunction after overexposure to manganese (Mn). The objective of the present study was to explore the mechanism of Mn disruption of GnRH synthesis via nuclear factor erythroid-2-related factor-2 (Nrf2)/metabotropic glutamate receptor-5 (mGluR5)/cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) signaling pathway in vitro and in vivo. Primary astrocytes were cultured and treated with different doses of Mn, tert-butylhydroquinonet (tBHQ; Nrf2 agonists), 3-[(2-methyl-4-thaizolyl) ethynyl] pyridine (MTEP; mGluR5 inhibitor), and celecoxib (COX-2 inhibitor) to measure the levels of COX-2, mGluR5, Nrf2, and Nrf2 target genes. Mice were randomly divided into 11 groups, of which included the control group, 12.5-, 25-, and 50-mg/kg MnCl2 group, dimethyl sulfoxide (DMSO) group, tBHQ control group, tBHQ pretreatment group, MTEP control group, MTEP pretreatment group, celecoxib control group, and celecoxib pretreatment group. The injection was administered every day for 2 weeks. Then, levels of GnRH, PGE2, COX-2, mGluR5, Nrf2, Nrf2 target genes, and morphological changes in the hypothalamus of mice were measured. Mn reduced protein levels of Nrf2 and mRNA expression of Nrf2 target genes and increased mGluR5, COX-2, PGE2, and GnRH levels. Meanwhile, injury-related histomorphology changes in the hypothalamus of mice were significantly present. In conclusion, excessive exposure to Mn disrupts GnRH secretion through Nrf2/mGluR5/COX-2/PGE2 signaling pathway.

Activation of the Nrf2-ARE Pathway Ameliorates Hyperglycemia-Mediated Mitochondrial Dysfunction in Podocytes Partly Through Sirt1

Background/Aims: Previously we have shown that activation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-antioxidant response element (ARE) attenuated hyperglycemia-induced damage in podocytes, but the molecular mechanism remains unknown. Methods: Tert-butylhydroquinone (t-BHQ) and small interfering RNAs (siRNAs) were used to regulate Nrf2 expression, while nicotinamide and siRNAs were used to regulate sirtuin 1 (Sirt1) activity and expression, respectively. Mitochondrial superoxide, membrane potential and ATP levels were measured to assess changes in mitochondrial function. Nephrin and synaptopodin expression were measured by western blot analysis. Human podocytes and db/db diabetic mice were used in this study. Results: t-BHQ pretreatment of human podocytes exposed to high glucose (HG) alleviated mitochondrial dysfunction, enhanced the expression of Sirt1, nephrin and synaptopodin and lowered BSA permeability compared with podocytes exposed to HG without t-BHQ pretreatment (p< 0.05). Human podocytes exposed to HG had more severe mitochondrial dysfunction, lower expression of Sirt1, synaptopodin and nephrin and higher BSA permeability than podocytes exposed to HG when Nrf2 expression was downregulated by siRNAs (p< 0.05). The protection provided by activation of the Nrf-ARE pathway in podocytes exposed to HG was partially diminished when Sirt1 expression or activity was decreased by siRNAs or inhibitor compared with podocytes exposed to HG and pretreated with t-BHQ (p< 0.05). When nicotinamide and t-BHQ were both administered to db/db mice, we observed higher levels of urinary albumin/creatinine, lower nephrin and synaptopodin expression, more severe mesangial matrix deposition, collagen deposition on pathological slides and mitochondrial structural damage in podocytes compared to db/db mice treated only with t-BHQ. Conclusions: Our findings suggest that crosstalk between Sirt1 and the Nrf2-ARE anti-oxidative pathway forms a positive feedback loop and that protection provided by t-BHQ activation of the Nrf2-ARE pathway in db/db mice is partly dependent on Sirt1.

Tert-butylhydroquinone protects PC12 cells against ferrous sulfate-induced oxidative and inflammatory injury via the Nrf2/ARE pathway

Increasing evidence had proved the critical role of iron in the pathogenesis of numerous neurodegenerative diseases because of its capacity to promote the formation of reactive oxygen species (ROS). Tert-butylhydroquinone (tBHQ) was a metabolite of butylated hydroxyanisole, a widely used food antioxidant. This study was aimed to investigate the protective effects of tBHQ on a cellular model of neurodegenerative disease, which was established in PC12 cells by exposure to ferrous sulfate (FS), and elucidate the potential protective mechanisms. The results showed that FS exposure increased lactate dehydrogenase (LDH) release and cell apoptosis in PC12 cells, accompanied by significant increases in the bax/bcl-2 ratio, cytochrome c release, and caspase-3 cleavage. It also enhanced the ROS production, malondialdehyde (MDA) content (lipid peroxidation), γ-H2A.X formation (DNA damage), and promoted nuclear factor kappa B (NF-κB) activation and expressions of cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). tBHQ pretreatment alleviated FS-induced LDH release, cell apoptosis, oxidative stress and inflammatory response by promoting Nrf2 nuclear translocation and the protein levels of Nrf2 downstream target genes heme oxygenase-1 (Hmox-1), nicotinamide adenine dinucleotide phosphate (NADPH): quinone oxidoreductase-1 (Nqo1) and glutathione peroxidase-1 (Gpx1). tBHQ alleviated the FS-induced LDH release in control siRNA-treated PC12 cells, but failed to alleviate FS-induced LDH release in Nrf2 siRNA-treated cells. These findings suggested that pretreatment with tBHQ protected PC12 cells from FS-induced oxidative and inflammatory injury via the Nrf2/ARE pathway. tBHQ was promising as a potential therapeutic agent for neurodegenerative diseases induced by iron toxicity and should be encouraged for further research.

Nuclear factor-like factor 2-antioxidant response element signaling activation by tert-butylhydroquinone attenuates acute heat stress in bovine mammary epithelial cells.

Nuclear factor (erythroid-derived 2)-like factor 2 (Nrf2) is a transcription factor that binds to the antioxidant response element (ARE) in the upstream promoter region of many antioxidative genes. The Nrf2-ARE signaling plays a key role in the cellular antioxidant-defense system, but whether Nrf2 activation has protective effects against heat shock (HS) stress in mammary epithelial cells (MEC) remains unclear. The objective of this study was to determine whether tert-butylhydroquinone (tBHQ), a well-known Nrf2 activator, could attenuate heat stress-induced cell damage in MAC-T cells of the bovine MEC line. The MAC-T cells were exposed to HS (42.5°C for 1h) followed by recovery at 37°C to mimic HS. Compared with cells that were consistently cultured at normothermia (37°C), the cell viability levels significantly decreased after HS stress. In parallel, heat stress increased the reactive oxygen species levels and induced cellular apoptosis and endoplasmic reticulum stress. The MAC-T cells that were pretreated with tBHQ (10μM) for 2h followed by HS had a reduction in the loss of cell viability. The tBHQ pretreatment significantly decreased cellular reactive oxygen species levels and stress-related marker gene expression. The tBHQ-treated MAC-T cells showed strong Nrf2-ARE signaling activation and a nuclear accumulation of Nrf2 and upregulated expression of Nrf2-ARE downstream genes. Small interfering RNA silencing of Nrf2 in HS-treated MAC-T cells almost completely abolished the cytoprotective effects by tBHQ. Overall, our results demonstrated that HS could cause cell damage in cultured bovine MEC, and that activation of Nrf2 by tBHQ could attenuate HS-induced cell damage.

Antioxidant tert-butylhydroquinone ameliorates arsenic-induced intracellular damages and apoptosis through induction of Nrf2-dependent antioxidant responses as well as stabilization of anti-apoptotic factor Bcl-2 in human keratinocytes

Human skin is a known target site of inorganic arsenic with effects ranging from hyperkeratosis to dermal malignancies. Tert-butylhydroquinone (tBHQ), approved food-grade phenolic antioxidant, is demonstrated to induce remarkable antioxidant activity in a variety of cells and tissues. The present study aimed at the protective effects of tBHQ on arsenic-induced cytotoxicity and apoptosis in human keratinocytes. Our results demonstrated that tBHQ antagonized arsenic-induced decrease of cell viability, generation of reactive oxygen species (ROS) and lipid peroxidation, as well as reduction of antioxidative enzymes superoxide dismutase (SOD) and catalase (CAT) activities. We also found that tBHQ relieved the G2/M phase arrest by arsenic exposure, which was associated with altering the expression of cell cycle regulators cyclin D1 and CDK4. tBHQ treatment further reduced the numbers of arsenic-induced mitochondrial-mediated apoptotic cells, which occurred concomitantly with the effective recovery of mitochondrial membrane potential (ΔΨm) depolarization, the release of cytochrome c releasing from the mitochondrial as well as the survival signal related factor caspase 3 activation. Our experiments then confirmed that tBHQ activated nuclear factor E2-related factor 2 (NRF2) pathway by increasing NRF2 protein in both nucleus and cytoplasm and upregulating NRF2 downstream targets NAD(P)H: quinine oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1). More interestingly, arsenic-induced decrease of anti-apoptotic factor B-cell lymphoma-2 (Bcl-2) and increase of pro-apoptotic factor Bcl-2-associated X protein (Bax) could all be reversed by tBHQ pretreatment. These results suggested together that tBHQ could ameliorate arsenic-induced cytotoxicity and apoptosis, which might be linked with the induction of Nrf2-dependent antioxidant responses as well as stabilization of anti-apoptotic factor Bcl-2 in human keratinocytes.

Reduction of DNA damage induced by titanium dioxide nanoparticles through Nrf2 in vitro and in vivo

Titanium dioxide nanoparticles (Nano-TiO2) are widely used to additives in cosmetics, pharmaceutical, paints and foods. Recent studies have demonstrated that Nano-TiO2 induces DNA damage and increased the risk of cancer and the mechanism might relate with oxidative stress. The aim of this study was to evaluate the effects of Nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2), an anti-oxidative mediator, on DNA damage induced by Nano-TiO2. Wildtype, Nrf2 knockout (Nrf2(-/-)) and tert-butylhydroquinone (tBHQ) pre-treated HepG2 cells and mice were treated with Nano-TiO2. And then the oxidative stress and DNA damage were evaluated. Our data showed that DNA damage, reactive oxygen species (ROS) generation and MDA content in Nano-TiO2 exposed cells were significantly increased than those of control in dose dependent manners. Nrf2/ARE droved the downstream genes including NAD(P)H dehydrogenase [quinine] 1(NQO1), heme oxygenase 1 (HO-1) and glutamate-cysteine ligase catalytic subunit (GCLC) expression were significantly higher in wildtype HepG2 cells after Nano-TiO2 treatment. After treatment with Nano-TiO2, the DNA damages were significantly increased in Nrf(-/-) cells and mice whereas significantly decreased in tBHQ pre-treatment cells and mice, compared with the wildtype HepG2 cells and mice, respectively. Our results indicated that the acquired of Nrf2 leads to a decreased susceptibility to DNA damages induction by Nano-TiO2 and decreasing of risk of cancer which would provide a strategy for a more efficacious sensitization of against of Nano-TiO2 toxication.

Protective Role of tert-Butylhydroquinone Against Sodium Fluoride-Induced Oxidative Stress and Apoptosis in PC12 Cells.

The neurotoxicity of fluoride is associated with oxidative stress due to imbalance between production and removal of reactive oxygen species (ROS). In contrast, induction of detoxifying and antioxidant genes through activation of NF-E2-related factor 2 (Nrf2) has been implicated in preventing oxidative stress and apoptosis in neurodegenerative diseases. The present study aimed to investigate the possible neuroprotective role of tert-butylhydroquinone (tBHQ), a general Nrf2 activator, on sodium fluoride (NaF)-induced oxidation damage and apoptosis in neuron-like rat pheochromocytoma (PC12) cells. Pretreatment with tBHQ protected PC12 cells against NaF-induced cytotoxicity as measured by MTT assay and apoptosis detection, simultaneously, inhibited NaF-induced overproduction of intracellular ROS and reduction of total glutathione content. Furthermore, NaF or tBHQ induced the stabilization of Nrf2, and enhanced expression of heme oxygenase-1 (HO-1) and γ-glutamylcysteine synthetase (γ-GCS) as a consequence of Nrf2 inducing. These findings indicated that tBHQ pretreatment conferred protective effect on PC12 cells against NaF-induced apoptotic cell death and oxidation-redox imbalance through stabilization of Nrf2 and elevation of downstream HO-1 and γ-GCS expressions.

Tert-butylhydroquinone attenuates scrotal heat-induced damage by regulating Nrf2-antioxidant system in the mouse testis

Tert-butylhydroquinone (tBHQ), a widely used nuclear factor erythroid 2-related factor 2 (Nrf2) activator, was always employed to investigate the potential protective role of Nrf2 activation. In this study, to elucidate the effect of tBHQ on scrotal heat-induced damage and Nrf2-antioxidant system in mouse testes, eight-week-old mice were administrated with or without dietary tBHQ (1%w/w) for 1week and afterward subjected to a single scrotal heat treatment (42°C for 25min). Trunk blood and testes were collected 3h or 1, 2, or 7days later. Mice displayed less germ cell loss in testes, higher relative testis weight and lower testosterone concentration on day 2 in tBHQ treatment group. Before heat treatment, there were significant increases in malondialdehyde (MDA) concentration in tBHQ treatment group. After heat treatment, mice in tBHQ treatment group showed lower MDA concentration than those in non-tBHQ treatment group. In addition, both tBHQ pretreatment and scrotal heat treatment induced markedly increased Nrf2 protein expression in cytoplasm and nuclei of interstitial cells, accompanying with elevated mRNA expression of Nrf2 and Nrf2-regulated genes in mice testes. Our data indicated that pretreatment to tBHQ induced a mild oxidative stress, and further enhanced the cellular antioxidative ability to protect testicular cells against scrotal heat-induced damage via a mechanism that might involve the Nrf2-antioxidant system in mice testes.

Tert-butylhydroquinone as a phenolic activator of Nrf2 antagonizes arsenic-induced oxidative cytotoxicity but promotes arsenic methylation and detoxication in human hepatocyte cell line.

Oxidative stress plays crucial roles in exerting a variety of damages upon arsenic exposure. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a master transcriptional regulator protecting cells and tissues from oxidative injuries. The objective of this study was to test whether tert-butylhydroquinone (tBHQ), a well-known synthetic Nrf2 inducer, could protect human hepatocytes against arsenic-induced cytotoxicity and oxidative injuries. Our results showed that 5 and 25μmol/l tBHQ pretreatment suppressed the arsenic-induced hepatocellular cytotoxicity, reactive oxygen species generation, and hepatic lipid peroxidation, while relieved the arsenic-induced disturbances of intracellular glutathione balance. In addition, we also observed that tBHQ treatment promoted the arsenic biomethylation process and upregulated Nrf2-regulated downstream heme oxygenase-1 and NADPH: quinine oxidoreductase 1 mRNA expressions. Collectively, we suspected that Nrf2 signaling pathway may be involved in the protective effects of tBHQ against arsenic invasion in hepatocytes. These data suggest that phenolic Nrf2 inducers, such as tBHQ, represent novel therapeutic or dietary candidates for the population at high risk of arsenic poisoning.

Primary cultured astrocytes from old rats are capable to activate the Nrf2 response against MPP+ toxicity after tBHQ pretreatment

Astrocytes are key players for brain physiology, protecting neurons by releasing antioxidant enzymes; however, they are also susceptible to damage by neurotoxins. Nuclear factor erythroid-derived 2-like 2 (Nrf2) is a central regulator of the antioxidant response, and therefore, pharmacologic inducers are often used to activate this transcription factor to induce cellular protection. To date, it still remains unknown if cells from aged animals are capable of developing this response. Therefore, the purpose of this work was to determine if cortical astrocytes derived from old rats are able to respond to tertbuthyl-hydroquinene (tBHQ) pretreatment and stimulate the Nrf2-antioxidant response pathway to induce an antioxidant strategy against MPP+ toxicity, one of the most used molecules to model Parkinson's disease. Our results show that, although astrocytes from adult and old rats were more susceptible to MPP+ toxicity than astrocytes from newborn rats, when pretreated with tertbuthyl-hydroquinene, they were able to transactivate Nrf2, increasing antioxidant enzymes and developing cellular protection. These results are discussed in terms of the doses used to create protective responses.

Tert-Butylhydroquinone pretreatment protects kidney from ischemia-reperfusion injury

It is generally accepted that an excessive production of reactive oxygen species plays an important role in acute renal failure secondary to ischemia and reperfusion. tert-Butylhydroquinone (tBHQ) is a well-known antioxidant. In this study, we evaluated whether tBHQ pretreatment prevented renal damage induced by ischemia and reperfusion (I/R). Four groups of rats were studied: (a) control-sham (CT), (b) tBHQ-sham (tBHQ), (c) I/R and (d) tBHQ + I/R. Intraperitoneal (i.p.) injections of tBHQ (50 mg/kg) were given to the tBHQ and tBHQ + I/R groups and 3% ethanol/isotonic saline solution to the CT and I/R groups. Animals were killed 24 hours after I/R. tBHQ attenuated I/R-induced renal dysfunction, structural damage, oxidative/nitrosative stress, glutathione depletion and the decrease in several antioxidant enzymes. The renoprotective effect of tBHQ on I/R injury was associated with the attenuation in oxidative/nitrosative stress and the preservation of antioxidant enzymes.

Protective effect of tert-butylhydroquinone on PC12 cells from neurotoxicity induced by manganese in vitro

To investigate the protective effect of the tert-butylhydroquinone (tBHQ) on PC12 cells from neurotoxicity induced by manganese. Cytotoxicity of PC12 cells was measured by MTT assay, following the PC12 cells treatment with different concentrations of MnCl₂ (300, 600, 900 μmol/L) for 24, 48 or 72 h. PC12 cells were pretreated with 40 μmol/L tBHQ for 12 h, followed by the treatment of 600 micromol/L or 300 μmol/L MnCl₂ for 72 h. Cytotoxicity of PC12 cells was measured by MTT assay, and cell apoptosis was examined by the method of Annexin V-FITC/PI in flow cytometry (FCM). The proliferation of PC12 cells treated with 300, 600, 900 μmol/L MnCl2 was suppressed in the dose dependent pattern (P < 0.01). Proliferation of PC12 cells treated with 600 μmol/L MnCl₂ was suppressed to 40% of that in control group (P < 0.01), but the proliferation rate of PC12 cell pretreated with 40 μmol/L tBHQ was 180% of that in control group (P < 0.01). Apoptotic rate of PC12 cells treated with 300 micromol/L MnCl₂ was higher than the vehicle control group (P < 0.01). Apoptotic rate of 40 μmol/L tBHQ pretreatment followed by 300 μmol/L MnCl₂ treatment was lower than that of MnCl2 treatment group (P < 0.01). The inhibition rate of apoptosis was 61%. Manganese may suppress PC12 cells proliferation and induce apoptosis. tBHQ can reduce PC12 cells proliferation suppressed by manganese and attenuate the apoptosis induced by manganese.

Microarray Analysis Reveals an Antioxidant Responsive Element-driven Gene Set Involved in Conferring Protection from an Oxidative Stress-induced Apoptosis in IMR-32 Cells

The present study was designed to investigate how tert-butylhydroquinone (tBHQ) prevents hydrogen peroxide-induced apoptosis in IMR-32 cells. tBHQ pretreatment (10 microm) attenuated hydrogen peroxide-induced cell death and reduced the number of TUNEL (terminal deoxynucleotidyltransferase-mediated, dUTP-incorporated nick end labeling)-positive cells. We hypothesize that tBHQ-mediated activation of the antioxidant responsive element is critical for generating this protective response. Addition of LY294002, a selective inhibitor of phosphatidylinositol 3-kinase (PI3K), 30 min prior to tBHQ treatment completely reversed the protective effect of tBHQ. Oligonucleotide microarrays were used to analyze the gene expression profile associated with tBHQ treatment in the absence and presence of LY294002. Ranking analysis using Affymetrix's difference call indicated that the expression of 137 genes changed with tBHQ treatment. Further analysis using the coefficient of variation for -fold change or average difference change reduced the list to 63 increased and 0 decreased genes. Reverse transcription-PCR for selected genes also confirmed the gene expression pattern. Many of these genes function to combat oxidative stress and increase the detoxification potential of the cells. Inhibition of PI3K significantly blocked the enhanced expression of 49 of the 63 genes induced by tBHQ. These data are the first to show a set of programmed cell life genes involved in conferring protection from an oxidative stress-induced apoptosis.