J.W.I.M. Simons

Linear dose —response relationships after prolonged expression times in V-79 Chinese hamster cells

The expression time for induced mutants resistant to 6-thioguanine, in V-79 Chinese hamster cells, was determined by respreading the cells in the selective medium, at various times after treatment. The length of the expression time for mutants induced by X-rays, ethyl methane sulphonate and ultraviolet irradiation was dose dependent. For the highest dose used this was 7 to 8 days, beyond which there was no further changes in mutant frequency. The dose-response relationship of these agents does not appear to deviate from linearity; this permits the calculation of mutation rate per unit dose. For X-rays this value was 1.35 - 10(-7) per rad per locus, for ethyl methane sulphonate, 2.2 - 10(-2) per mole per locus and for ultraviolet irradiation, 6.3 - 10(-6) per erg per mm2 per locus. The effectiveness of the 3 different mutagens for the induction of mutations was compared by calculating the increase in mutant frequency per unit of decrease in survival (Do). These increments in frequency were: 5.6 - 10(-5) for X-rays, 69.5 - 10(-5) for ethyl methane sulphonate and 16.1 - 10(-5) for ultraviolet irradiation.

DNA strand specificity for UV-induced mutations in mammalian cells

The influence of DNA repair on the molecular nature of mutations induced by UV light (254 nm) was investigated in UV-induced hprt mutants from UV-sensitive Chinese hamster cells (V-H1) and the parental line (V79). The nature of point mutations in hprt exon sequences was determined for 19 hprt mutants of V79 and for 17 hprt mutants of V-H1 cells by sequence analysis of in vitro-amplified hprt cDNA. The mutation spectrum in V79 cells consisted of single- and tandem double-base pair changes, while in V-H1 cells three frameshift mutations were also detected. All base pair changes in V-H1 mutants were due to GC----AT transitions. In contrast, in V79 all possible classes of base pair changes except the GC----CG transversion were present. In this group, 70% of the mutations were transversions. Since all mutations except one did occur at dipyrimidine sites, the assumption was made that they were caused by UV-induced photoproducts at these sites. In V79 cells, 11 out of 17 base pair changes were caused by photoproducts in the nontranscribed strand of the hprt gene. However, in V-H1 cells, which are completely deficient in the removal of pyrimidine dimers from the hprt gene and which show a UV-induced mutation frequency enhanced seven times, 10 out of 11 base pair changes were caused by photoproducts in the transcribed strand of the hprt gene. We hypothesize that this extreme strand specificity in V-H1 cells is due to differences in fidelity of DNA replication of the leading and the lagging strand. Furthermore, we propose that in normal V79 cells two processes determine the strand specificity of UV-induced mutations in the hprt gene, namely preferential repair of the transcribed strand of the hprt gene and a higher fidelity of DNA replication of the nontranscribed strand compared with the transcribed strand.

A mutational assay system for l5178y mouse lymphoma cells, using hypoxanthine-guanine-phosphoribosyl-transferase (hgprt) -deficiency as marker. The occurrence of a long expression time for mutations induced by x-rays and ems.

The development of a system for the detection of somatic cell mutation to hypoxanthine-guanine-phosphoribosyl-transferase (HGPRT) (EC 2.4.2.8) deficiency in L5178Y mouse lymphoma cells is described. The selection of mutant cells was not influenced by the concentration of the selective agent 6-thioguanine (6-TG). In addition, all the mutants selected, spontaneous as well as induced ones, showed a complete loss of HGPRT activity. In reconstruction experiments, in which mutant cells were mixed with wild-type cells, the recovery of mutant cells was only markedly influenced when wild-type cells were seeded in a cell density ten times higher than the one, 5-10(4) cells/ml, used in subsequent induction experiments. X-irradiation and treatment with ethyl methanesulfonate (EMS) increased in the mutation rate above the spontaneous background. A clear-cut dose-dependent mutagenic effect after exposure to X-rays was measured. The rate of induced mutations at the HGPRT locus in lymphoma cells was 1-3-10(-7) per R, as determined after exposures of 200, 300, 400, 500 and 600 R. The time the cells needed to express their mutations was much longer than 48 h. Further study of this phenomenon showed that the optimal expression time for TGr-resistant mutants in L5178Y cells was 6 to 7 days. No indication for a dose-dependent effect on the optimal expression of the mutants was found.

Relationship between cell killing, chromosomal aberrations, sister-chromatid exchanges and point mutations induced by monofunctional alkylating agents in Chinese hamster cells a correlation with different ethylation products in DNA

Several monofunctional alkylating agents (AA) were compared for their ability to induce chromosomal aberrations, cell killing, sister-chromatid exchanges (SCE) and point mutations in Chinese hamster cells (CHO and V79 cells). The AAs chosen varied in their reaction kinetics as well as their affinity to nucleophilic sites (different s values). AAs with low s values were more mutagenic in comparison to those with high s values, whereas the reverse was true for induction of cytotoxic effects. Neither SCEs nor chromosomal aberrations correlated with the induction of point mutations, indicating that different primary DNA lesions and repair pathways are involved in these biological processes. Molecular dosimetric studies indicate that O6 alkylation of guanine is the most probable cause of lesions in DNA leading to point mutations following treatment with ethyl methanesulphonate and ethyl nitrosourea.

Mutations induced by X-rays at the HPRT locus in cultured Chinese hamster cells are mostly large deletions.

We investigated the molecular basis of 19 X-ray-induced HPRT-deficient mutants of V79 Chinese hamster cells with Southern hybridisation techniques. 12 of those mutants suffer from a big deletion (greater than 10 kb) of HPRT DNA sequences. Cytological studies of chromosome preparations of those 12 deletion mutants showed that in at least 3 of these mutants part of the long arm of the X-chromosome was lost. After correction for spontaneous arising mutations we estimate that at least 70-80% of X-ray-induced mutations are caused by large deletions.

The role of metabolic cooperation in selection of hypoxanthine-guanine-phosphoribosyl-transferase (HG-PRT)-deficient mutants from diploid mammalian cell strains

Abstract A system for the selection of 8-azaguanine-resistant mutants from a diploid human cell strain is described. The selection of mutant cells is largely influenced by a phenomenon, known as metabolic cooperation, which turns mutant cells into phenotypically wild-type cells. As a consequence mutant cells cannot be selected above a certain cell density. Reconstruction experiments in which mutant cells were mixed with wild-type cells and mutant feeder cells showed that a reasonable recovery of mutant cells could only be obtained at a density of about 190 wild-type cells per cm 2 . Under these conditions HG-PRT (hypoxanthine-guanine-phosphoribosyl-transferase) deficient clones were obtained from a diploid human skin fibroblast strain and from a mouse skin fibroblast strain. In order to improve the selection system the mechanism of metabolic cooperation should be understood. Therefore it was investigated whether metabolic cooperation is due to cell-to-cell contact or to factors mediated by the medium. It is shown that when mutant cells and wild-type cells were separated by a fibrin layer, metabolic cooperation did not occur.

Characterization of an X-ray-hypersensitive mutant of V79 Chinese hamster cells.

A V79 Chinese hamster cell line XR-V15B exhibiting hypersensitivity to X-ray has been isolated and characterized. Additionally to increased X-ray-sensitivity (approximately 8-fold, as judged by D10 values), cross-sensitivity to bleomycin (3-fold increase), 4NQO (3-fold), H2O2, EMS, MMS (2-fold) were observed also. No increased sensitivity to UV and MMC was found. Genetic complementation analysis indicates that XR-V15B belongs to the same complementation group as the X-ray-sensitive (xrs) mutants of Chinese hamster ovary (CHO) cells described by Jeggo (1985). Biochemical analysis of XR-V15B confirms this finding: the mutant showed a decreased ability to rejoin double-strand breaks induced by X-ray as measured by neutral elution. After 4 h of repair more than 50% of the double-strand breaks remain in comparison to 3% in V79 cells. No difference was observed between wild-type and XR-V15B cells in the initial number of single-strand breaks induced, in the kinetics of their rejoining and in the final level of unrejoined single-strand breaks. Treatment with 5-azacytidine did not have an effect on the reversion frequency of XR-V15B, contrary to the results obtained with the xrs mutants. XR-V15B has been grown in continuous culture for more than 3 months without evidence of reversion. The mutation induction by X-ray irradiation at the HPRT locus is not significantly increased in the mutant, but at doses giving the same degree of cell killing, XR-V15B cells are hypomutable.

Development of a liquid-holding technique for the study of DNA-repair in human diploid fibroblasts.

Liquid-holding conditions can be obtained for human diploid skin fibroblasts by keeping confluent cultures stationary over periods of 7 days or longer by means of conditioned medium. Under this condition recovery of radiation damage induced by ultraviolet light or X-rays is observed as an increase in cloning efficiency. The amount of recovery when expressed in a dose-modifying-factor appears higher than in bacteria and yeast. The repair-deficient human cell strains XP25Ro and XP7Be (xeroderma pigmentosum from complementation groups A and D respectively) exhibit less but still discernible recovery after UV-irradiation and the same was observed for AT5Bi (ataxia telangiectasia) after X-irradiation. Experiments on mutation induction indicated that the repair which takes place during liquid holding of UV-irradiated XP7Be cells reduces the mutant frequency considerably while after liquid holding of UV-irradiated wild-type cells the same or lower mutant frequencies were found for the lower exposures and the same or higher mutant frequencies for the higher exposures.

Nongenotoxic carcinogens: development of detection methods based on mechanisms: a European project

While the accumulation of genetic changes in a somatic cell is considered essential for the genesis of a cancer, it has become clear that not all carcinogens are genotoxic, suggesting that some carcinogens indirectly participate in the generation of genetic changes during carcinogenesis. A European project funded by the European Community was thus conceived to study mechanisms of nongenotoxic aspects of carcinogenesis. Two main strategical approaches were adapted: (i) to study whether and how Syrian hamster embryo (SHE), Syrian hamster dermal (SHD) and BALB/c 3T3 cell transformation systems simulate in vivo carcinogenesis, and to examine whether they can detect nongenotoxic carcinogens; (ii) to study, refine and validate mechanisms-based end-points for detection of nongenotoxic carcinogens. For mechanisms-based research, the proposed end-points included gap junctional intercellular communication (GJIC) inhibition, altered expression of critical genes, immortalization and aberrant cell proliferation. We also selected model compounds commonly usable for various endpoints. Our major results can be summarized as follows: (1) SHE and BALB/c 3T3 transformation systems reflect both genotoxic and nongenotoxic carcinogenic events; they detect not only genotoxic but also many although not all, nongenotoxic carcinogens. This is further supported by the fact that both genotoxic and nongenotoxic carcinogens were able to immortalize SHD cells. (2) Many nongenotoxic carcinogens, although not all, inhibit GJIC in vitro as well as in vivo. Mechanistic studies suggest an important role of blocked GJIC in carcinogenesis and that different mechanisms are involved in inhibition of the communication by different agents used. However, inhibition of GJIC is not a prerequisite for the enhancement (or induction) of transformation of SHE or BALB/c 3T3 cells. (3) Among compounds examined, there was a good correlation between induction of micronuclei and cell transformation in SHE cells while no such correlation was found between the induction of cell transformation and ornithine decarboxylase activity. (4) Two transgenic mouse mutation assays (lacI and lacZ) were established and validated. The genotoxin dimethylnitrosamine was shown to be mutagenic to the liver in both assays. Ortho-anisidine, a bladder-specific carcinogen that was inactive in standard rodent genetic toxicity assays was uniquely mutagenic to the bladder of the transgenic mice. The peroxisome proliferator methyl clofenipate was established as nonmutagenic to the liver of both transgenic mice. That eliminated DNA damage as a cause of the liver tumours produced by this chemical and weakened the idea that induced cell division leads to mutation induction. (5) With an in vitro DNA replication model, it was found that DNA damage induced by genotoxic agents can be responsible for inhibition of DNA replication, while certain nongenotoxic agents such as phorbol esters increase DNA replication. (6) An attempt to use structure-activity relationship for subfamilies of nongenotoxic carcinogens, e.g., receptor-mediated carcinogens, has been initiated with some promising results. Our results support the idea that there are multiple nongenotoxic mechanisms in carcinogenesis, and that working hypothesis-oriented approaches are encouraged rather than simple screening of chemicals in developing test systems for the detection of nongenotoxic carcinogens.

(6-4) Photoproducts and not cyclobutane pyrimidine dimers are the main UV-induced mutagenic lesions in Chinese hamster cells

A partial revertant (RH1-26) of the UV-sensitive Chinese hamster V79 cell mutant V-H1 (complementation group 2) was isolated and characterized. It was used to analyze the mutagenic potency of the 2 major UV-induced lesions, cyclobutane pyrimidine dimers and (6-4) photoproducts. Both V-H1 and RH1-26 did not repair pyrimidine dimers measured in the genome overall as well as in the active hprt gene. Repair of (6-4) photoproducts from the genome overall was slower in V-H1 than in wild-type V79 cells, but was restored to normal in RH1-26. Although V-H1 cells have a 7-fold enhanced mutagenicity, RH1-26 cells, despite the absence of pyrimidine dimer repair, have a slightly lower level of UV-induced mutagenesis than observed in wild-type V79 cells. The molecular nature of hprt mutations and the DNA-strand specificity were similar in V79 and RH1-26 cells but different from that of V-H1 cells. Since in RH1-26 as well as in V79 cells most hprt mutations were induced by lesions in the non-transcribed DNA strand, in contrast to the transcribed DNA strand in V-H1, the observed mutation-strand bias suggests that normally (6-4) photoproducts are preferentially repaired in the transcribed DNA strand. The dramatic influence of the impaired (6-4) photoproduct repair in V-H1 on UV-induced mutability and the molecular nature of hprt mutations indicate that the (6-4) photoproduct is the main UV-induced mutagenic lesion.