The effect of post-treatment with caffeine on survival and UV-induced mutation frequencies in Chinese hamster and mouse lymphoma cells in vitro

The effect of post-treatment with caffeine on the survival of a number of cell lines after UV-irradiation has been studied. The mouse lymphoma cell lines P388 and L5178YS were sensitized by caffeine but only after UV doses of 50 erg/mm² and above. V79 cells also showed sensitization by caffeine but CHO cells and two cell lines YS and YR derived from Yoshida sarcoma of rats, sensitive and resistant to UV radiation, respectively, showed no effect.P388 and V79 cells were both mutable by UV, and caffeine, when studied at a single expression time (42–48 h) and at a single dose level (0.5 M and 0.75 M, respectively) suppressed the UV-induced mutation frequency in both cell lines. L51788YS cells although sensitized by caffeine showed no increase in frequency of thymidine-resistant (TdR^r) colonies when irradiated with UV.On more detaled examination, caffeine was found to delay the expression of UV-induced mutations inV79 cells, and the delay was dependent on the dose of caffine used. The effect on expression time was less when caffeine was present 0–48 h than when it was present throughout the post-irradiation incubation period. Similar results were obtained in P388 cells.The data are discussed in relation to those of other workers and to the concept that caffeine inhibits an error prone post-replication repair process in mammalian cells     

Mechanism of cytotoxic action of azaguanine and thioguanine in wild-type V79 cell lines and their relative efficiency in selection of structural gene mutants

The cytotoxic effects of azaguanine and thioguanine have been compared in two wild-type V79 cells. To achieve equitoxic effects in both cell lines a 10–20-fold higher concentration of azaguanine than thioguanine was required. Affinity of HGPRT for azaguanine was 10-fold lower than for hypoxanthine in both cell lines and was similar to that for thioguanine in V79S cells. Affinity for thioguanine differed by a factor of 3 in the two cell lines. The rate of cell kill by azaguanine was markedly slower than by thioguanine in both cell lines. Reduction of whole cell uptake of [¹⁴C]hypoxanthine incorporation by unlabelled azaguanine was only demonstrable after prolonged incubation periods as was incorporation of [¹⁴C]azaguanine into acid-insoluble material. Experiments with cell-free extracts indicated that hypoxanthine acts as a non-competitive inhibitor of the enzyme. The slow rate of dissociation of the HGPRT—azaguanine complex is reflected in the slow rate of killing of wild-type cells. Clones resistant to the cytotoxic effects of these analogues have been selected from both cell lines and have been shown to possess HGPRT with altered kinetic properties. Our data suggest that azaguanine and thioguanine may select for mutations at different sites on the HGPRT molecule in V79 cells and provide possible explanations for the differences in effectiveness of these two agents reported in other cell lines.     

DNA excision-repair processes in human cells can eliminate the cytotoxic and mutagenic consequences of ultraviolet irradiation

The ability of DNA excision-repair processes in diploid human fibroblasts to eliminate potentially cytotoxic and mutagenic lesions induced by UV radiation (254 nm) was demonstrated in two ways: (1) Cells with normal rates of excision were compared with cells with an intermediate rate of excision (XP2BE) and cells with an excision rate less than or equal to 1% that of normal (XP12BE) for sensitivity to the killing and mutagenic action of UV radiation. The normal cells proved resistant to doses of UV which reduced the survival of the XP cells to 14% and 0.7%, respectively, and increased the frequency of mutations to 8-azaguanine resistance in the XP cells 5- to 10-fold over background. (2) Cells in confluence were irradiated with cytotoxic and mutagenic doses of UV and allowed to carry out excision repair. After various lengths of time they were replated at lower densities to allow for expression of mutations to 6-thioguanine resistance and/or at cloning densities to assay survival. Normal cells and XP cells with reduced rates of excision repair (from complementation groups C and D) exhibited a gradual increase in survival from an initial level of 15--20% to 100% if held approximately 20 h in confluence. In contrast, XP12BE cells showed no increase from an initial survival of 20% even when held for 7 days. Normal cells irradiated in confluence but prevented from replicating for 7 days exhibited background mutation frequencies, whereas the mutation frequency in XP12BE cells did not change with the time in confluence.     

Relative mutagenicity of antineoplastic drugs and other alkylating agents in V79 chinese hamster cells,independence of cytotoxic and mutagenic responses

The mutagenic and cytotoxic effects of 4 antineoplastic drugs, vinblastine, vincristine, adriamycin and nitrogen mustard and of several monofunctional alkylating agents have been assayed in V79 Chinese hamster cells. Vincristine, vinblastine and nitrogen mustard did not significantly increase the frequency of TG^RHGPRT^? mutants but were all highly cytotoxic. Adriamycin and the monofunctional alkylating agents were all significantly mutagenic even at the lowest doses tested (approx. 70 % survival level). Induced mutant frequency increased linearly with increasing dose whereas dose-response curves for cytotoxicity for these effective mutagens invariably showed a shoulder followed by an exponential decline. At equitoxic doses the relative mutagenic effectiveness was MNU ENU EMS MMS ? DMS. MNU was approx. 20 times more effective than MMS and DMS.Measurement of the total amount of alkylation and the relative amounts of reaction with individual DNA bases at approx. equitoxic doses of MNU and DMS indicated a significantly higher O⁶/N⁷ ratio after MNU (0.15) than after DMS (0.005). However, approx. equal numbers of mutants/10⁵ cells/μM O⁶-Meguanine were induced by these 2 agents. These results support previous conclusions, that mutagenic and cytotoxic responses are independent in V79 cells.     

Cytogenetic and mutagenic effects of DDT and DDE in a Chinese hamster cell line

The mutagenic and cytogenetic effects of the chlorinated hydrocarbon 1,11-trichloro-2,2-bis(p-chlorophenyl)ethane] (DDT), and its metabolite [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene] (DDE) were investigated in vitro using a Chinese hamster cell line. A forward mutation system utilizing the 8-azaguanine sensitive to 8-azaguanine resistant marker was used as the index of mutagenic action. Methyl methanesulfonate (MMS) was used as the positive control. In all experiments, DDE consistently produced a significant increase in the mutation frequency over the control level, while DDT proved inactive.Resultsof the cytogenetic studies indicated that DDE-treated cells had a significant increase in chromosome aberrations over those occuring in the control population; exchange figures and chromatid breaks wre evident. DDT produced no significant increase in chromosome abnormalities. The Chinese hamster cell populations exposed to DDE also manifested an increased number of polyploid cells over the control level.     

Metabolic properties of an azaguanine-resistant variant of Chinese hamster ovary cells (azarts) with normal levels of hypoxanthine-guanine phosphoribosyltransferase activity

Azarts Chinese hamster ovary cells were 20 to 50 times more resistant to 8-azaguanine and 50 to 10 times more resistant to both 6-thioguanine and 6-mercaptopurine than wild-type cells. Resistance correlated with a failure of azarts cells to incorporate 8-azaguanine into the nucleotide pool and into nucleic acids. The uptake of hypoxanthine and guanine, on the other hand, was about the same in both types of cells and the hypoxanthine-guanine phosphoribosyltransferase of the azarts cells as measured in cell lysates was unaltered both in concentration and kinetic properties with hypoxanthine as well as 8-azaguanine as substrate. Plasma membrane permeability to 8-azaguanine and the regulation of intracellular pH were also not altered in azarts cells and there was no significant degradation of 8-azaguanine or azaguanine nucleotides. We conclude therefore that in azarts cells the phosphoribosylation of 8-azaguanine per se is specifically blocked but that this effect is abolished upon cell lysis.