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The purpose of this study was to investigate the cytotoxic and genotoxic potential of low intensity laser irradiation (660 nm, 12 mW, 5 kHz) on mammalian cells. Thymidine kinase (TK)-positive and TK-deficient Friend erythroleukaemia (FEL) cells, clone 707 and subclone 707BUF respectively, were used in this investigation. Following irradiation of exponentially growing cells in suspension at doses of 2 and 20 J/cm2 a number of sensitive bioassays were used to facilitate the detection of laser-induced mutations, DNA damage and cell killing. Mutations were assessed by the examination of chromosome spreads, the determination of micronucleus frequency and by the determination of the mutant frequency at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus. DNA damage was quantified using a sensitive ELISA. The cytotoxic effect of laser irradiation was assessed using a cloning assay. The results of this investigation did not show any significant increase in mutation frequency, DNA damage or cell survival in the laser-irradiated cells, compared to sham-irradiated controls. The lack of any demonstrable cytotoxic and genotoxic effects of low intensity laser irradiation on mammalian cells in culture would support it as being a safe modality for clinical use.

In this study the effect of thymidine kinase (TK) deficiency on mutagen sensitivity was examined in the human lymphoblastoid cell line Raji. Wild-type and TK-deficient Raji cells were treated with a range of concentrations of ethyl methanesulphonate (EMS) and a range of doses of ultraviolet (UV) light, then examined for mutagen sensitivity as measured by cell survival and mutation to HGPRT deficiency. Dose-dependent responses were observed and TK-deficient cells exhibited decreased survivals and increased mutant frequencies relative to wild-type cells. TK-deficient Raji cells are also deficient in O6-methylguanine-DNA-methyltransferase. This may partially account for their sensitivity to EMS but does not account for the results obtained with UV. It is therefore likely that an additional factor, such as alterations in supply of deoxyribonucleoside triphosphates, may affect the mutagen sensitivity of Raji cells.

An accumulation of mutations on their own or together with other age-related changes may contribute to aging and the development of age-related pathologies. The aim of this investigation was to assess the extent of DNA mutations as a function of age in humans. The mutant frequency (MF) at the hypoxanthine-guanine phosphoribosyl-transferase (hgprt) locus was assessed in lymphocytes isolated from male volunteers in each of three age groups (35-39, 50-54 and 65-69 years). Results show that the mean MF in the 65-69 years group was approximately twice that in the 35-39 and 50-54 years groups (4.1/10(6) cells, 1.9/10(6) cells and 1.79/10(6) cells, respectively) increasing by about 1.33% per year, after 54 years. In addition, there was an increased frequency of chromosomal aberrations in the 65-69 years group compared to the other two age groups. The results of this investigation show an increase in DNA mutations in cultured human lymphocytes with age. Factors which may influence the extent of DNA damage in human lymphocytes are discussed.

Wild-type Friend mouse erythroleukaemia cells (clone 707) were compared with adenine phosphoribosyltransferase (APRT)-deficient mutant subclones (707DAP8 and 707DAP10) for sensitivity to cell killing and mutagenesis by ethyl methanesulphonate (EMS) and methyl methanesulphonate (MMS). Cells were exposed to 0-300 micrograms/ml EMS and to 0-20 micrograms/ml MMS for a period of 16 h. A slight difference was found between wild-type cells and the two APRT-deficient subclones in terms of sensitivity to cell killing by both mutagens. The APRT-deficient subclones were, however, significantly more sensitive than wild-type cells to mutagenesis to 5-bromo-2-deoxyuridine resistance and 6-thioguanine resistance by EMS and MMS. The APRT-deficient subclones were found to have significantly decreased levels of dATP and dTTP nucleotides and decreased levels of all four ribonucleoside triphosphates (ATP, GTP, CTP and UTP) relative to wild-type cells. Wild-type Friend cells were found to have insignificant levels O6-methylguanine-DNA methyl transferase and it is suggested that the increased mutagen sensitivity of APRT-deficient cells may be due to imbalance of deoxyribonucleoside triphosphate pools during DNA excision-repair processes, or more probably due to deficiency of ATP for ATP-dependent DNA excision-repair enzymes.

Clone 707 of the Friend cell was compared with an APRT-deficient subclone for sensitivity to cell killing and the induction of cytogenetic aberrations by mitomycin C (MMC). Two 16-h doses of MMC were used, 0.1 and 0.15 microgram/ml and cells were scored for aberrations at 16, 33 and 44 h post-treatment. The APRT-deficient subclone showed increased cell killing, a higher frequency of aberrations and a higher frequency of cells with severe cytogenetic damage. It is proposed that APRT may play a role in balancing deoxyribonucleoside triphosphate pools for DNA-repair processes.

Wild-type Friend erythroleukaemia (clone 707) cells and 2 thymidine kinase-deficient subclones, 707BUE and 707BUF, having thymidine kinase activities of 1.4% and 0.7% that of clone 707, were compared for sensitivity to killing and the induction of cytogenetic damage following gamma irradiation. Three doses of gamma irradiation were used (150, 300 and 450 cGy), and cells were harvested for metaphase spreads after 4, 8, 12, 15, 29 and 43 h. G2 delay was evident at 4 h following gamma irradiation in the 3 cell clones examined, and recovery of mitosis was observed to be dose-dependent. G2 delay was found to be most prolonged in subclone 707BUE and most prompt in clone 707. Increased sensitivity to the induction of cytogenetic aberrations at all three doses was apparent in the 2 thymidine kinase-deficient subclones (as compared to wild-type cells) at 15, 29 and 43 h. Th thymidine kinase-deficient subclones also showed increased sensitivity to gamma radiation-induced cell killing. Furthermore, subclone 707BUE consistently exhibited greater resistance to gamma irradiation than did the subclone with lower thymidine kinase activity, 707BUF. The importance of thymidine kinase levels and extended G2 delay for DNA repair processes is discussed.

Spontaneous mutation at the adenine phosphoribosyl transferase (APRT) locus in clone 707 of the Friend cell line was examined. The frequency of cells resistant to 2,6-diaminopurine (DAP) was found to be 2.6 X 10(-5) with a mutation rate of 1.81 X 10(-6) cell-1 generation-1. APRT activities in 9 DAP-resistant clones were found to vary between 0 and 27% the level observed in wild-type cells. It is suggested that clone 707 cells are heterozygous or functionally hemizygous at the APRT locus.

Spontaneous mutation at the thymidine-kinase locus in clone 707 of the Friend cell line has been examined. The rate of mutation in BrdU resistance was found to be 2.6 x 10(-6) cell-1 generation-1. The rate of reversion to HAT resistance was found to vary from 1.1 x 10(-7) to 2.85 x 10(-6) cell-1 generation-1 in 4 BrdU-resistant clones. Of 14 mutant clones assayed for thymidine-kinase activity only one had greater than 13% the activity of wild-type cells. Fluctuation analysis showed that mutations occurred spontaneously and were not induced by the selective agent. The mutation rate at the thymidine-kinase locus is several orders of magnitude greater than those reported for several other cells lines. There does not appear to be a general genetic instability in this cell line as the mutation rate at the HGPRT locus is similar to those found in other established cell lines. It is suggested that the high forward mutation rate at the thymidine-kinase locus may be due to only one functional thymidine-kinase allele being present in cells of this clone.