Gayaram Bhaumik

Enhancement of catalase activity by repetitive low-grade H2O2 exposures protects fibroblasts from subsequent stress-induced apoptosis

Exposure of Chinese hamster V79 fibroblasts to mild and repetitive H2O2 doses in culture for 15 weeks produced no change in lipid peroxidation status, GSH/GSSG ratio and glutathione peroxidase activity of these cells (VST cells). In contrast, in VST cells catalase levels underwent a prominent increase which could be significantly inhibited and brought down to control levels after treatment with the catalase inhibitor 3-aminotriazole (3-AT). When control (VC) cells were exposed to UV radiation (UVC 5 J/m2) or H2O2 (7.5mM, 15 min), intracellular reactive oxygen species (ROS) levels rose prominently with significant activation of caspase-3. Marked nuclear fragmentation and lower cell viability were also noted in these cells. In contrast, VST cells demonstrated a significantly lower ROS level, an absence of nuclear fragmentation and an unchanged caspase-3 activity after exposure to UVC or H2O2. Cell viability was also significantly better preserved in VST cells than VC cells after UV or H2O2 exposures. Following 3-AT treatment of VST cells, UVC radiation or H2O2 brought about significantly higher elevations in intracellular ROS, increases in caspase-3 activity, significantly lowered cell viability and marked nuclear fragmentation, indicating the involvement of high catalase levels in the cytoprotective effects of repetitive stress. Therefore, upregulation of the antioxidant defense after repetitive oxidative stress imparted a superior ability to cope with subsequent acute stress and escape apoptotic death and loss of viability.

Induced resistance in cells exposed to repeated low doses of H2O2 involves enhanced activity of antioxidant enzymes

We have derived cells from the Chinese hamster V79 cell line by conditioning them with repeated low doses of hydrogen peroxide (H2O2). This mimics the physiological condition where cells are repeatedly exposed to low levels of oxidants. In an attempt to characterize such cells, we have exposed both conditioned cells (V79C) and the parental V79 cells (V79P) to different types of cytotoxic agents and compared their sensitivity to cell killing. The V79C cells were found to be stably resistant to killing by agents that produced toxicity through oxidative stress, e.g. H2O2 and cisplatin. It was also found that the lipid peroxidation produced by these agents were considerably lower in the V79C cells. Thus, the difference in sensitivity could be due to lesser extent of damage to these cells. V79C cells had greater antioxidant defense through higher GSH content and greater activity of enzymes such as Cu—Zn superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), which provided protection from damage. Enzyme activities were also assayed at different times after treatment with various cytotoxic agents; there was a relatively large increase in SOD activity which perhaps plays a key role in determining the resistance of the V79C cells to killing.

Supernatant medium from UV-irradiated cells influences the cytotoxicity and mutagenicity of V79 cells

It is known that UV light induces the secretion of some proteins into the extracellular medium. We have carried out experiments to study how the supernatant medium from UV-irradiated cells affects the cytotoxicity and mutagenicity of V79 cells exposed to different damaging agents. So we exposed exponentially growing cells to 20 J/m2 of UV light and then harvested the supernatant medium after 22 h. This supernatant medium was then used to treat a fresh batch of cells for 2 h. After the treatment with this supernatant medium the cells were subsequently exposed to UV light, gamma-rays, hydrogen peroxide or MNNG. We found that exposure to this medium had a protective effect on the survival levels for UV light, gamma-rays and hydrogen peroxide while MNNG-induced killing remained unaffected. With UV light and gamma-rays we found that mutation induction at all doses was increased. Cycloheximide could inhibit this protection and the increase in mutation frequencies was also suppressed. The results indicated a protective role for the UV-induced factor(s). They were probably involved directly or they triggered repair process(es) that were related to oxidative stress.

UV non-mutable mutant from V-79 Chinese hamster cells

SummaryTo get an idea about the response of a living system, exposed to gradually increasing doses of a mutagen for several generations, a population of V-79 Chinese hamster cells was exposed repeatedly to gradually increasing doses of UV radiation. Each dose was followed by a variable period of growth for at least ten generations. After treatment the cells were not mutable by UV radiation, though MNNG was capable of producing mutations with the same efficiency as in the untreated cells. In terms of viability, the treated cells behaved exactly as the untreated ones for both UV and MNNG. The observed behaviour of the treated cells was found to be stable for during the 50 passages studied.

Hyperthermia-induced modulation of killing and mutation by UV and N-methyl-N'-nitro-N-nitrosoguanidine in V79 cells

Hyperthermic exposures of V79 cells did not affect the killing by UV light, whereas it enhanced MNNG-induced killing. Such hyperthermic exposure increased the mutation induction (resistance to 6-thioguanine) by both UV and MNNG. The timing of heat exposure, before or after the treatments, had no effect on the result in cases of cytotoxicity and mutagenesis.

THROMBIN RELEASES CALCIUM FROM INTERNAL STORES OF ULTRAVIOLET C-TREATED V79 FIBROBLASTS INDEPENDENT OF PHOSPHATIDYLINOSITOL BISPHOSPHATE HYDROLYSIS : ROLE OF OXIDATIVE STRESS

V79 fibroblasts were treated with ultraviolet (UV) C radiation alone as well as in conjunction with chronic oxidative stress. The effects of these treatments on calcium signaling were observed at 30 min post-irradiation. In the absence of extracellular calcium, thrombin released calcium from internal stores of UVC-irradiated V79 fibroblasts even after exposure to neomycin. In neomycin-treated control and chronic oxidative stress cells, no calcium release by thrombin was observed after chelation of external calcium. Calcium release by thrombin from internal stores of UV-irradiated and neomycin-treated cells was completely abolished by pretreatment with N-acetyl cysteine and dexamethasone. Cellular total soluble thiol content which is a good indicator of cellular reduced glutathione (GSH) level was significantly elevated 30 min after ultraviolet radiation, indicating an adaptive response after oxidative stress. Chronic oxidative stress alone resulted in a much smaller increase in GSH but chronic oxidative stress in conjunction with UVC produced a very prominent elevation in GSH levels. Our data suggest that thrombin can cause calcium release from internal stores of ultraviolet-irradiated fibroblasts which is independent of phosphatidylinositol bisphosphate hydrolysis and is directly related to the level of oxidative stress. Involvement of phopholipase A2 and a role for its products as possible mediators of calcium release from intracellular stores, is strongly indicated.

Influence of hyperthermia on gamma-ray-induced mutation in V79 cells.

Asynchronously growing V79 cells were assayed for mutation induction following exposure to hyperthermia either immediately before or after being irradiated with 60Co gamma rays. Hyperthermia exposures consisted of either 43.5 degrees C for 30 min or 45 degrees C for 10 min. Each of these heat treatments resulted in a survival level of 42%. For all sequences of combined treatment with hyperthermia and radiation, cell killing by gamma rays was enhanced. Mutation induction by gamma rays was enhanced when heat preceded gamma irradiation, but no increase was observed when heat was given after gamma exposures. Treatment at 45 degrees C for 10 min gave a higher yield in mutants at all gamma doses studied compared to treatment at 43.5 degrees C for 30 min. When heat-treated cells were incubated for different periods before being exposed to gamma rays, thermal enhancement of radiation killing was lost after 24 h. In contrast, only 5-6 h incubation was needed for loss of mutation induction enhancement.

Influence of DNA replication on the X-ray sensitivity of bacteria

Abstract It was observed that upon incubation in a medium in which DNA synthesis was allowed but not protein and RNA syntheses (i.e. by incubating the cells in +T−AU medium), UV-irradiated E. coli 15 T-A-U-M-Pr-Tr-Smr (requiring thymine, arginine, uracil, methionine, proline and tryptophan and resistant to streptomycin) became more resistant to X-rays compared to those cells which were irradiated but not incubated. A similar increase in resistance was also observed for UV-irradiated thymine-grown and 5-bromodeoxyuridine (BUdR)-grown cells incubated in +BUdR−AU medium. The results indicated that some of the UV-induced lesions could be repaired on incubation in such restricted media. However, such an increase in X-ray resistance due to the incubation could not be related to the increase in viability; in some cases of incubation, where there was a decrease in viability, an increase in resistance was observed; in other cases, where there was an increase in viability, a decrease in resistance was observed. Incubation of the non-irradiated thymine-grown cells +T−AU medium increased the shoulder of the X-ray survival curve and also decreased the slope, while such cells, on incubation in +BUdR−AU medium, became more sensitive to X-rays than the control non-irradiated and nonincubated cells. For BUdR-grown cells, although there was a marked decrease in X-ray sensitivity due to incubation in +T−AU medium, no significant change in X-ray sensitivity was observed on subsequent incubation in +BUdR−AU medium as compared to the noninncubated cells.

Further studies on tetracycline-induced mutation in V79 Chinese hamster cells

The interaction between ultraviolet light and tetracycline in producing cell killing and mutation has been studied in V79 Chinese hamster cells. It has been established that these agents act independently of each other. Cycloheximide altered the response to tetracycline in the fractionation experiment: when cycloheximide was not present, fractionation of TC treatment resulted in a higher mutation yield but no change in survival level; in the presence of cycloheximide, however, mutation was greatly reduced but survival increased. The results were taken to indicate that for tetracycline action to take place, de novo protein synthesis during tetracycline treatment was necessary. Caffeine had no influence on tetracycline-induced lethality or mutagenicity. This observation was considered to suggest that tetracycline did not affect cellular repair processes.