Overexpression of glutamate-cysteine ligase protects human COV434 granulosa tumour cells against oxidative and gamma-radiation-induced cell death.

Ionizing radiation is toxic to ovarian follicles and can cause infertility. Generation of reactive oxygen species (ROS) has been implicated in the toxicity of ionizing radiation in several cell types. We have shown that depletion of the antioxidant glutathione (GSH) sensitizes follicles and granulosa cells to toxicant-induced apoptosis and that supplementation of GSH is protective. The rate-limiting reaction in GSH biosynthesis is catalysed by glutamate-cysteine ligase (GCL), which consists of a catalytic subunit (GCLC) and a regulatory subunit (GCLM). We hypothesized that overexpression of Gclc or Gclm to increase GSH synthesis would protect granulosa cells against oxidant- and radiation-induced cell death. The COV434 line of human granulosa tumour cells was stably transfected with vectors designed for the constitutive expression of Gclc, Gclm, both Gclc and Gclm or empty vector. GCL protein and enzymatic activity and total GSH levels were significantly increased in the GCL subunit-transfected cells. GCL-transfected cells were resistant to cell killing by treatment with hydrogen peroxide compared to control cells. Cell viability declined less in all the GCL subunit-transfected cell lines 1-8 h after 0.5 mM hydrogen peroxide treatment than in control cells. We next examined the effects of GCL overexpression on responses to ionizing radiation. ROS were measured using a redox-sensitive fluorogenic dye in cells irradiated with 0, 1 or 5 Gy of gamma-rays. There was a dose-dependent increase in ROS within 30 min in all cell lines, an effect that was significantly attenuated in Gcl-transfected cells. Apoptosis, assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling and activated caspase-3 immunoblotting, was significantly decreased in irradiated Gclc-transfected cells compared to irradiated control cells. Suppression of GSH synthesis in Gclc-transfected cells reversed resistance to radiation. These findings show that overexpression of GCL in granulosa cells can augment GSH synthesis and ameliorate various sequelae associated with exposure to oxidative stress and irradiation

Overexpression of glutamate–cysteine ligase protects human COV434 granulosa tumour cells against oxidative and γ-radiation-induced cell death

Ionizing radiation is toxic to ovarian follicles and can cause infertility. Generation of reactive oxygen species (ROS) has been implicated in the toxicity of ionizing radiation in several cell types. We have shown that depletion of the antioxidant glutathione (GSH) sensitizes follicles and granulosa cells to toxicant-induced apoptosis and that supplementation of GSH is protective. The rate-limiting reaction in GSH biosynthesis is catalysed by glutamate–cysteine ligase (GCL), which consists of a catalytic subunit (GCLC) and a regulatory subunit (GCLM). We hypothesized that overexpression of Gclc or Gclm to increase GSH synthesis would protect granulosa cells against oxidant- and radiation-induced cell death. The COV434 line of human granulosa tumour cells was stably transfected with vectors designed for the constitutive expression of Gclc, Gclm, both Gclc and Gclm or empty vector. GCL protein and enzymatic activity and total GSH levels were significantly increased in the GCL subunit-transfected cells. GCL-transfected cells were resistant to cell killing by treatment with hydrogen peroxide compared to control cells. Cell viability declined less in all the GCL subunit-transfected cell lines 1–8 h after 0.5 mM hydrogen peroxide treatment than in control cells. We next examined the effects of GCL overexpression on responses to ionizing radiation. ROS were measured using a redox-sensitive fluorogenic dye in cells irradiated with 0, 1 or 5 Gy of γ-rays. There was a dose-dependent increase in ROS within 30 min in all cell lines, an effect that was significantly attenuated in Gcl-transfected cells. Apoptosis, assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling and activated caspase-3 immunoblotting, was significantly decreased in irradiated Gclc-transfected cells compared to irradiated control cells. Suppression of GSH synthesis in Gclc-transfected cells reversed resistance to radiation. These findings show that overexpression of GCL in granulosa cells can augment GSH synthesis and ameliorate various sequelae associated with exposure to oxidative stress and irradiation

Dichlorvos exposure results in large scale disruption of energy metabolism in the liver of the zebrafish, Danio rerio

BACKGROUND: Exposure to dichlorvos (DDVP), an organophosphorus pesticide, is known to result in neurotoxicity as well as other metabolic perturbations. However, the molecular causes of DDVP toxicity are poorly understood, especially in cells other than neurons and muscle cells. To obtain a better understanding of the process of non-neuronal DDVP toxicity, we exposed zebrafish to different concentrations of DDVP, and investigated the resulting changes in liver histology and gene transcription. RESULTS: Functional enrichment analysis of genes affected by DDVP exposure identified a number of processes involved in energy utilization and stress response in the liver. The abundance of transcripts for proteins involved in glucose metabolism was profoundly affected, suggesting that carbon flux might be diverted toward the pentose phosphate pathway to compensate for an elevated demand for energy and reducing equivalents for detoxification. Strikingly, many transcripts for molecules involved in β-oxidation and fatty acid synthesis were down-regulated. We found increases in message levels for molecules involved in reactive oxygen species responses as well as ubiquitination, proteasomal degradation, and autophagy. To ensure that the effects of DDVP on energy metabolism were not simply a consequence of poor feeding because of neuromuscular impairment, we fasted fish for 29 or 50 h and analyzed liver gene expression in them. The patterns of gene expression for energy metabolism in fasted and DDVP-exposed fish were markedly different. CONCLUSION: We observed coordinated changes in the expression of a large number of genes involved in energy metabolism and responses to oxidative stress. These results argue that an appreciable part of the effect of DDVP is on energy metabolism and is regulated at the message level. Although we observed some evidence of neuromuscular impairment in exposed fish that may have resulted in reduced feeding, the alterations in gene expression in exposed fish cannot readily be explained by nutrient deprivation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-1941-2) contains supplementary material, which is available to authorized users