This study on the toxicity of arsenite compounds includes use of Chinese hamster ovary (CHO) cells: CHO-K1, xrs-5, and xrs-6. The three cell lines used showed various responses to the arsenite compounds and are hypersensitive to X-rays. Xrs-5 is deficient in the activity of glutathione peroxidase and catalase leaving the cells hypersensitive to arsenite. Both enzymes convert hydrogen peroxide (glutathione peroxidase can also convert other peroxides) to less harmful compounds. Xrs-6 cells were much less sensitive to arsenite indicating some use of catalase and glutathione peroxidase.
Previous studies indicate that chronic exposure to arsenic compounds cause among other things, chromosome aberrations, sister-chromatid exchange, and micronuclei formation. Arsenite may react with thiols or generate reactive oxygen species, which as we learned in class is very dangerous to cells due to inhibition of enzymes, cell death, cancer, and lip peroxidation. Previous studies show that arsenite induces lung-specific DNA damage through generation of active oxygen radicals. Superoxide dismutase was found to reduce sister chronmtid exchanges natural crossover events that are enhanced by mutagens. It is the main objective of this study to further prove that arsenite damages DNA through generation of oxygen radicals by studying the effects of catalase and glutathione peroxidase on genotoxicity caused by arsenite.
Cells were grown and cultured after exposure to either X-rays or arsenite. The methods
do not say how long the X-ray exposure was, but the cells were treated with arsenite for four hours. The methods seemed a little vague, since it cross-referenced the methods used in determination of glutathione levels, catalase activity, glutathione S-transferase activity, and
micronuclei assay. Our class discussion of the micronuclei assay clarified that it is a test for a chromosome aberration creating fragments of chromosome found outside of the cell's nucleus.
which is induced by mutagens. Superoxide dismutase activity was measured by the amount of reduction of lucigenin-dependent chemiluminescence.
CHO cells were irradiated with X-rays, and it was observed that CHO-K I cells were not as sensitive as xrs-5 (7.2 fold) and xrs-6 (8.1 fold) cells. Their respective ID50 values are 6.5, 0.9, and 0.8. After 4h arsenite exposure, the ID50 values are as follows: CHO-KI (235), xrs-5 (33), and xrs-6 (108). Again, xrs-5 and xrs-6 cells are more sensitive than CHO-K I cells, but
xrs-5 cells are more sensitive than xrs-6 cells. Micronucleus assay was the endpoint in the study of the genotoxicity of arsenite. The frequency of micronuclei was 10. 8 (xrs-5), 4.5 (xrs-6), and 2.1(CHO-KI). Again, xrs-5 (5-1 fold) and xrs-6 (2.4 fold) cells were more sensitive than CHO-KI cells.
The activity of enzymes involved in the reduction of oxygen radicals were measured, since it was hypothesized that these enzymes were not as active in xrs-5 and xrs-6 cells causing them to be more sensitive to X-rays and arsenite. Only catalase and glutathione peroxidase were found to be lower in these cells than in CHO-K I cells. In XTS-5 cells catalase was 5.8 fold lower and glutathione peroxidase was 5.4 fold lower. In xrs-6 cells, catalase was 3.7 fold lower and glutathione peroxidase was 2.1 fold lower. However, the activities of both enzymes were higher in xrs-6 cells than xrs-5 cells.
The connection between catalase and glutathione peroxidase activity and hypersensitivity of xrs-5 cells to arsenite was determined by the study of arsenite-induced micronuclei. When glutathione peroxidase and catalase were added at the same time as arsenic, micronuclei formation was significantly reduced. Since glutathione peroxidase uses metals as cations, its activity was increased 130% in xrs-5 cells and 1600/o in CHO-K I cells by the addition of sodium selenite. In CHO-K1 cells, micronuclei formation decreased, but not in xrs-5 cells. The paper suggested low glutathione peroxidase activity, even after selenite addition, is one of the reasons that there was no reduction in micronuclei formation. To test this, 3-aminotriazole and mercaptosuccinate inhibited the activities of catalase and glutathione peroxidase. Arsenite-induced micronuclei increased slightly when the two were used independently, but more so when used together.
This study supports the hypothesis that oxygen radicals are involved in the genotoxicity of arsenite. Both xrs-5 and xrs-6 cells are deficient in catalase and glutathione peroxidase and are hypersensitive to arsenite. Since xrs-6 cells had higher activity in those enzymes, it was not as sensitive as xrs-5 cells. The addition of catalase and glutathione peroxidase did reduce the amount of micronuclei formation in xrs-5 cells.
Further experimentation is needed to explore the possibility that glutathione peroxidase is more important than catalase, since xrs-6 cells had a 2.5 fold increase in glutathione peroxidase activity and only 1.6 fold increase in catalase activity. The authors are currently pursuing the role of glutathione peroxidase in preventing micronuclei formation based on the results of selenite addition. Selenite addition improved the activity of glutathione peroxidase in CHO-K1 cells, but not in xrs-5 cells deficient in the enzyme.