Influence of glutathione levels and heat-shock on the steady-state levels of oxidative DNA base modifications in mammalian cells

The effects of thiols, ascorbic acid and thermal stress on the basal (stead y-state) levels of oxidative DNA base modifications were studied. In variou s types of untreated cultured mammalian cells, the levels of total glutathi one were found to be inversely correlated with the levels of DNA base modif ications sensitive to the repair endonuclease Fpg protein, which include 8- hydroxyguanine (8-oxoG), A depletion of glutathione by treatment with buthi onine sulphoximine increased the steady-state level in AS52 Chinese hamster cells by similar to 50%. However, additional thiols in the culture medium did not reduce the level of Fpg-sensitive base modifications: 0-10 mM N-ace tylcysteine had no effect, whereas cysteine ethylester even increased the o xidative DNA damage at concentrations >0.1 mM. Similarly, ascorbic acid (0- 20 mM) failed to reduce the steady-state levels. When AS52 cells were grown at elevated temperature (41 degrees C), the steady-state level of the oxid ative DNA modifications increased by 40%, in spite of a concomitant 1.6-fol d increase of the cellular level of total glutathione, Depletion of glutath ione at 41 degrees C nearly doubled the already elevated level of oxidative damage. A constitutive expression of the heat-shock protein Hsp27 in L929 mouse fibrosarcoma cells at 37 degrees C increased the glutathione level by 60%, but had little effect on the level of oxidative DNA damage.