Sukhendu B. Bhattacharjee

Induction of repair functions by hydrogen peroxide in Chinese hamster cells.

Hydrogen peroxide has been found to kill Chinese hamster V79 cells as an exponential function of dose. When a small dose (0.9 microgram/ml for 1 h) was used as a pretreatment, before exposure to higher concentrations of the same agent, the cells became more resistant to killing than those which were not so pretreated. The presence of cycloheximide or benzamide, during this pretreatment, inhibited this observed increase in resistance. This pretreatment also resulted in decreased killing efficiency by MNNG and gamma-rays, but had no effect upon UV-light-induced killing. The results suggest that proteins (repair enzymes?) are synthesized after treatment with the small dose of hydrogen peroxide, and that these induced proteins enhance the cellular repair functions for agents causing DNA breaks.

Tetracycline-induced mutation in cultured Chinese hamster cells.

After tetracycline hydrochloride (TC) treatment for 48 h, we obtained 8-azaguanine-resistant mutants in a population of cultured Chinese hamster cells. Optimal expression of mutation was reached after about 5 days, and mutation frequency remained constant thereafter. There was a gradual increase in the yield of mutants with increasing doses of TC, and measurable yield was observed at a dose as low as 10 microgram of TC per ml. When the cells, before or after the TC treatment, were grown in presence of L-ascorbic acid (10 or 20 microgram/ml) for 48 h, there was significant decrease in the frequency of mutation induced by TC. If L-ascorbic acid were present at the same concentration during the treatment with TC, no significant influence on the induction of mutants by TC was detected.

Response of MTX-resistant V79 cells to some DNA-damaging agents

Three methotrexate (MTX)-resistant clones selected at 200 nM MTX have been isolated from UV-irradiated V79 cells and were marked M1, M2 and M3. Two further clones were also isolated by gradually exposing M1 to higher concentrations of MTX and marked M4 and M5, isolated at 1200 nM and 2400 nM MTX respectively. The frequency of the induction of MTX-resistant clones by UV light was found to increase significantly on treatment of the irradiated cells with either 3-aminobenzamide or caffeine. The resistant clones had the same sensitivity as the parental cells to UV light, but were resistant to gamma-rays, MNNG and H2O2. In MTX-resistant cells low-dose H2O2-induced repair function was absent. UV-induced mutation at the HGPRT locus was the same in MTX-resistant as sensitive cells, whereas for MNNG-induced mutation it was higher in resistant cells. The rate of SCE induction by UV light was higher in MTX-resistant cells compared to the parental ones, but was not significantly different for induction by MNNG and H2O2.

Characterisation of methotrexate-resistant clones

Three methotrexate (MTX)-resistant clones M1, M2 and M3 have been isolated from Chinese hamster V79 cells and characterised for aneuploidy, chromosomal aberrations, sister-chromatid exchange (SCE) mutation and transfection. Amplification of the dihydrofolate reductase (DHFR) gene in these clones has been established from (a) direct measurement of DHFR activity, (b) existence of double minute chromosomes and (c) homogeneously staining region (HSR) in chromosome number 2 by G-banding technique. Clone M1 on further exposure to gradually increasing concentrations of MTX gave rise to two more clones M4 and M5, resistant to 1200 nM and 2400 nM MTX, respectively. The levels of folate reductase activity in clones M4 and M5 were 21.90 units per 10(7) cells and 33.30 units per 10(7) cells, whereas the value was 13.90 units per 10(7) cells in clone M1 and 1.1 units per 10(7) cells in normal V79 cells. Increased chromosomal aberrations were observed in each of these clones in comparison to those in normal V79 cells. There was a correlation between the increase in DHFR activity and the increase in the level of background SCE as well as the increase in aneuploidy. However, background mutation frequency at the HGPRT locus remained unaffected though transfection frequency decreased.

Influence of caffeine on X-ray-induced killing and mutation in V79 cells.

Effects produced by caffeine on X-irradiated Chinese hamster V79 cells depended on the growth conditions of the cells. For exponentially growing cells, nontoxic concentrations of caffeine decreased the shoulder width from the survival curve, but the slope remained unchanged. The yield of mutants under the same conditions also remained unaffected. In case of density-inhibited cells, delaying trypsinization for 24 h after X irradiation increased the survival and decreased the yield of mutants. The presence of caffeine during this incubation period inhibited such recovery and significantly increased the yield of X-ray-induced mutants.

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.

Response of V79 cells to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment: inhibition of poly(ADP-ribose) and topoisomerase activity

MNNG-induced killing of V79 cells has been found to be enhanced on inhibition of topoisomerase II activity by nalidixic acid and poly(ADP-ribose) polymerase synthesis by benzamide. Using these 2 inhibitors in conjunction after MNNG treatment, some overlap in the functions of these 2 enzymes was observed. Nalidixic acid and benzamide were found to suppress the yields of mutations and SCEs induced by MNNG. Benzamide was more effective in suppressing the mutation yield whereas nalidixic acid was more effective in suppressing SCEs. A model based on the relative requirement of topoisomerase and poly(ADP-ribose) for the repair of different types of damage has been proposed to explain the results.

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.

Photoreactivation of thymine-starved Escherichia coli Bs-1.

Abstract—Thymine starvation prior to 254 nm ultraviolet light (UV) exposures has been found to decrease the level of maximum photoreactivation in Escherichia coliBs‐1. The dark equilibrium level of photoreactivating enzyme‐substrate complexes was determined from the levels of photoreactivation obtained with exposures to single flashes of high‐intensity light. The kinetics indicate that photoreactivating enzyme concentration does not decrease as a result of thymine starvation. The UV sensitivities of normal and thymine‐starved cells are found to be the same. Photoreactivation by sequential flashes shows a lesser number of total photorepairable lesions in starved cells. It is concluded that thymine starvation renders a portion of the dimers inaccessible to the photoreactivating enzyme, thus lowering the level of maximum photoreactivation.

Photodynamic inactivation of Chinese hamster cells.

Abstract— Visible light exposures have been shown to kill acriflavine bound Chinese hamster cells. Such killing was enhanced when (a) dye was present in the medium during irradiation and (b) the pH of the medium was 8.5, instead of the normal 7.5 during the exposure. The induced killing could be suppressed by the presence of sodium azide during exposure. The results were taken to indicate that both DNA and non‐DNA sites were involved in the cellular inactivation by visible light and that singlet oxygen was involved in the process.