Protective effect of oxidative stress in HaCaT keratinocytes expressing E7 oncogene.

In a previous study, we established a stable cell line which constitutively expresses E7 in HaCaT human keratinocyte cell line and identified various relevant factors including oxygen modulators affected by the E7 oncogene. E7-expressing HaCaT cells (HaCaT/E7) appeared to be more resistant to H2O2-induced cell death. Here, we demonstrate how E7 oncogene would modulate oxidative stress-induced cell death. In addition, we verified the increased expression of catalase in the HaCaT/E7 by Western blot analysis. The results suggest that the E7 oncogene would induce higher resistance to ROS-induced cell injury in the E7-infected cells via the upregulation of catalase. To investigate these paradoxical effects of high concentrations of H2O2 (500 microM-1 mM), we examined their effects on receptor mediated apoptosis, cell death via the mitochondrial pathway and modulation of apoptosis related factors. Our results revealed that HaCaT keratinocytes infected with HPV 16 E7 oncogene modulated expressions of catalase, Bcl-xL, IL-18, Fas, Bad, and cytochrome c as well as NF-kappaB, resulting in the resistance to oxidative stress-induced cell death.

Ascorbate attenuates trimethyltin-induced oxidative burden and neuronal degeneration in the rat hippocampus by maintaining glutathione homeostasis.

The specific role of endogenous glutathione in response to neuronal degeneration induced by trimethyltin (TMT) in the hippocampus was examined in rats. A single injection of TMT (8 mg/kg, i.p.) produced a rapid increase in the formation of hydroxyl radical and in the levels of malondialdehyde (MDA) and protein carbonyl. TMT-induced seizure activity significantly increased after this initial oxidative stress, and remained elevated for up to 2 weeks post-TMT. Although a significant loss of hippocampal Cornus Ammonis CA1, CA3 and CA4 neurons was observed at 3 weeks post-TMT, the elevation in the level of hydroxyl radicals, MDA, and protein carbonyl had returned to near-control levels at that time. In contrast, the ratio of reduced to oxidized glutathione remained significantly decreased at 3 weeks post-TMT, and the glutathione-like immunoreactivity of the pyramidal neurons was decreased. However glutathione-positive glia-like cells proliferated mainly in the CA1, CA3, and CA4 sectors and were intensely immunoreactive. Double labeling demonstrated the co-localization of glutathione-immunoreactive glia-like cells and reactive astrocytes, as indicated by immunostaining for glial fibrillary acidic protein. This suggests that astroglial cells were mobilized to synthesize glutathione in response to the TMT insult. The TMT-induced changes in glutathione-like immunoreactivity appear to be concurrent with changes in the expression levels of glutathione peroxidase and glutathione reductase. Ascorbate treatment significantly attenuated TMT-induced seizures, as well as the initial oxidative stress, impaired glutathione homeostasis, and neuronal degeneration in a dose-dependent manner. These results suggest that ascorbate is an effective neuroprotectant against TMT. The initial oxidative burden induced by TMT may be a causal factor in the generation of seizures, prolonged disturbance of endogenous glutathione homeostasis, and consequent neuronal degeneration.

Cloning and sequencing of a 2,5-dichlorohydroquinone reductive dehalogenase gene whose product is involved in degradation of gamma-hexachlorocyclohexane by Sphingomonas paucimobilis.

Sphingomonas (formerly Pseudomonas) paucimobilis UT26 utilizes gamma-hexachlorocyclohexane (gamma-HCH), a halogenated organic insecticide, as a sole carbon and energy source. In a previous study, we showed that gamma-HCH is degraded to 2,5-dichlorohydroquinone (2,5-DCHQ) (Y. Nagata, R. Ohtomo, K. Miyauchi, M. Fukuda, K. Yano, and M. Takagi, J. Bacteriol. 176:3117-3125, 1994). In the present study, we cloned and characterized a gene, designated linD, directly involved in the degradation of 2,5-DCHQ. The linD gene encodes a peptide of 343 amino acids and has a low level of similarity to proteins which belong to the glutathione S-transferase family. When LinD was overproduced in Escherichia coli, a 40-kDa protein was found after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Northern blot analysis revealed that expression of the linD gene was induced by 2,5-DCHQ in S. paucimobilis UT26. Thin-layer chromatography and gas chromatography-mass spectrometry analyses with the LinD-overexpressing E. coli cells revealed that LinD converts 2,5-DCHQ rapidly to chlorohydroquinone (CHQ) and also converts CHQ slowly to hydroquinone. LinD activity in crude cell extracts was increased 3.7-fold by the addition of glutathione. All three of the Tn5-induced mutants of UT26, which lack 2,5-DCHQ dehalogenase activity, had rearrangements or a deletion in the linD region. These results indicate that LinD is a glutathione-dependent reductive dehalogenase involved in the degradation of gamma-HCH by S. paucimobilis UT26.