Further characterization of loss of heterozygosity enhanced by p53 abrogation in human lymphoblastoid TK6 cells: disappearance of endpoint hotspots

Loss of heterozygosity (LOH) is the predominant mechanism of spontaneous mutagenesis at the heterozygous thymindine kinase locus (tk) in TK6 cells. LOH events detected in spontaneous TK⁻ mutants (110 clones from p53 wild-type cells TK6-20C and 117 clones from p53-abrogated cells TK6-E6) were analyzed using 13 microsatellite markers spanning the whole of chromosome 17. Our analysis indicated an approximately 60-fold higher frequency of terminal deletions in p53-abrogated cells TK6-E6 compared to p53 wild-type cells TK6-20C whereas frequencies of point mutations (non-LOH events), interstitial deletions, and crossing over events were found to increase only less than twofold by such p53 abrogation. We then made use of an additional 17 microsatellite markers which provided an average map-interval of 1.6 Mb to map various LOH endpoints on the 45 Mb portion of chromosome 17q corresponding to the maximum length of LOH tracts (i.e. from the distal marker D17S932 to the terminal end). There appeared to be four prominent peaks (I–IV) in the distribution of LOH endpoints/Mb of Tk6-20C cells that were not evident in p53-abrogated cells TK6-E6, where they appeared to be rather broadly distributed along the 15–20 Mb length (D17S1807 to D17S1607) surrounding two of the peaks that we detected in TK6-20C cells (peaks II and III). We suggest that the chromosomal instability that is so evident in TK6-E6 cells may be due to DNA double-strand break repair occurring through non homologous end-joining rather than allelic recombination.     

Bystander response in human lymphoblastoid TK6 cells

The mechanisms of the medium-mediated bystander response induced by γ-rays in non-irradiated TK6 cells were investigated. Cell cultures were irradiated and the culture medium discarded immediately after irradiation and replaced with a fresh one. In cells incubated with conditioned medium from irradiated cells (CM), a significant decrease in cell viability and cloning efficiency was observed, together with a significant increase in apoptosis, also in directly irradiated cells. To examine whether bystander apoptosis involved the extrinsic pathway, an inhibitor of caspase-8 was added to CM cultures, which significantly decreased apoptosis to control levels. The addition to CM of ROS scavengers, Cu–Zn superoxide dismutase and N-acetylcysteine did not affect the induction of apoptosis. To assess whether CM treatment activates a DNA damage response, also the formation of γ-H2AX foci, as markers of double-strand breaks and their colocalisation with 53-binding protein 1 (53BP1) and the protein mutated in the Nijmegen breakage syndrome 1 (NBS1) was analysed. In cultures treated for 2 h with CM, 9–11% of cells showed γ-H2AX foci, which partially or totally lacked colocalisation with 53BP1 and NBS1 foci. About 85% of irradiated cells were positive for γ-H2AX foci, which colocalised with 53BP1 and NBS1 proteins. At 24 h from irradiation, very few irradiated cells retained foci, fitting DNA repair kinetics. The number of foci-positive bystander cells also decreased to background values 24 h after CM incubation. Our results suggest that irradiated TK6 cells release into the medium some soluble factors, not ROS, which are responsible for the cytotoxic effects induced in bystander cells. In our experimental system, the role of ROS appeared to be of minor importance in inducing cell mortality, but probably critical in activating the DNA damage response in the responsive fraction of bystander cells.     

Genotoxicity of microcystin-LR in human lymphoblastoid TK6 cells

Toxic cyanobacteria (blue-green algae) water blooms have become a serious problem in several industrialized areas of the world. Microcystin-LR (MCLR) is a cyclic heptapeptidic toxin produced by the cyanobacteria. In the present study, we used human lymphoblastoid cell line TK6 to investigate the in vitro genotoxicity of MCLR. In a standard 4h treatment, MCLR did not induce a significant cytotoxic response at <80 microg/ml. In a prolonged 24h treatment, in contrast, it induced cytotoxic as well as mutagenic responses concentration-dependently starting at 20 microg/ml. At the maximum concentration (80 microg/ml), the micronucleus frequency and the mutation frequency at the heterozygous thymidine kinase (TK) locus were approximately five-times the control values. Molecular analysis of the TK mutants revealed that MCLR specifically induced loss of heterozygosity at the TK locus, but not point mutations or other small structural changes. These results indicate that MCLR had a clastogenic effect. We discuss the mechanisms of MCLR genotoxicity and the possibility of its being a hepatocarcinogen.     

Detection of genetic alterations induced by low-dose X rays: analysis of loss of heterozygosity for TK mutation in human lymphoblastoid cells

To elucidate the genetic influence of low-dose ionizing radiation at the chromosome level, we exposed human lymphoblastoid TK6-20C cells to 10 cGy of X rays. The TK mutation frequency was 5.7 +/- 1.3 x 10(-6) at the background level and 6.9 +/- 2.8 x 10(-6) after X irradiation. Although this small increase was not statistically significant (P = 0.40), we applied multilocus analysis using 4 TK locus markers and 12 microsatellite loci spanning chromosome 17 for TK mutants exhibiting loss of heterozygosity (LOH). The analysis demonstrated a clear effect of low-dose ionizing radiation. We observed radiation-specific patterns in the extent of hemizygous LOH in 14 TK mutants among the 92 mutants analyzed. The deleted regions in these patterns were larger than they were in the control mutants, where those restricted to the TK locus. Surprisingly, the radiation-specific LOH patterns were not observed among the 110 nonirradiated TK mutants in this study. They were identified previously in TK6 cells exposed to 2 Gy of X rays. We consider these hemizygous LOH mutants to be a result of end-joining repair of X-ray-induced DNA double-strand breaks.     

Apoptosis induced by MNNG in human TK6 lymphoblastoid cells is p53 and Fas/CD95/Apo-1 related

Agents inducing O(6)-methylguanine (O(6)MeG) in DNA, such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), are not only highly mutagenic and carcinogenic but also cytotoxic because of the induction of apoptosis. In CHO fibroblasts, apoptosis triggered by O(6)MeG requires cell proliferation and MutSalpha-dependent mismatch repair and is related to the induction of DNA double-strand breaks (DSBs). Furthermore, it is mediated by Bcl-2 degradation and does not require p53 for which the cells were mutated [Cancer Res. 60 (2000) 5815]. Here we studied cytotoxicity and apoptosis induced by MNNG in a pair of human lymphoblastoid cells expressing wild-type p53 (TK6) and mutant p53 (WTK1) and show that TK6 cells are more sensitive than WTK1 cells to cell killing (determined by a metabolic assay) and apoptosis. Apoptosis was a late response observed <24h after treatment and was related to accumulation of p53 and upregulation of Fas/CD95/Apo-1 receptor as well as Bax. The data indicate that MNNG induces apoptosis in lymphoblastoid cells by activating the p53-dependent Fas receptor-driven pathway. This is in contrast to CHO fibroblasts in which, in response to O(6)MeG, the mitochondrial damage pathway becomes activated.     

Genotoxicity of acrylamide and glycidamide in human lymphoblastoid TK6 cells

The recent finding that acrylamide (AA), a potent carcinogen, is formed in foods during cooking raises human health concerns. In the present study, we investigated the genotoxicity of AA and its metabolite glycidamide (GA) in human lymphoblastoid TK6 cells examining three endpoints: DNA damage (comet assay), clastogenesis (micronucleus test) and gene mutation (thymidine kinase (TK) assay). In a 4 h treatment without metabolic activation, AA was mildly genotoxic in the micronucleus and TK assays at high concentrations (> 10 mM), whereas GA was significantly and concentration-dependently genotoxic at all endpoints at > or = 0.5 mM. Molecular analysis of the TK mutants revealed that AA predominantly induced loss of heterozygosity (LOH) mutation like spontaneous one while GA-induced primarily point mutations. These results indicate that the genotoxic characteristics of AA and GA were distinctly different: AA was clastogenic and GA was mutagenic. The cytotoxicity and genotoxicity of AA were not enhanced by metabolic activation (rat liver S9), implying that the rat liver S9 did not activate AA. We discuss the in vitro and in vivo genotoxicity of AA and GA.     

Investigation of the apoptotic way induced by digallic acid in human lymphoblastoid TK6 cells

ABSTRACT: BACKGROUND: The digallic acid (DGA) purified from Pistacia lentiscus. L fruits was investigated for its antiproliferative and apoptotic activities on human lymphoblastoid TK6 cells. METHODS: We attempt to characterize the apoptotic pathway activated by DGA. Apoptosis was detected by DNA fragmentation, PARP cleavage and by evaluating caspase activities. RESULTS: The inhibition of lymphoblastoid cell proliferation was noted from 8.5 mug/ml of DGA. The induction of apoptosis was confirmed by DNA fragmentation and PARP cleavage. We have demonstrated that DGA induces apoptosis by activating the caspase-8 extrinsic pathway. Caspase-3 was also activated in a dose dependent manner. CONCLUSION: In summary, DGA exhibited an apoptosis inductor effect in TK6 cells revealing thus its potential as a cancer-preventive agent.     

Illegitimate recombination leading to allelic loss and unbalanced translocation in p53-mutated human lymphoblastoid cells.

Allelic loss and translocation are critical mutational events in human tumorigenesis. Allelic loss, which is usually identified as loss of heterozygosity (LOH), is frequently observed at tumor suppressor loci in various kinds of human tumors. It is generally thought to result from deletion or mitotic recombination between homologous chromosomes. In this report, we demonstrate that illegitimate (nonhomologous) recombination strongly contributes to the generation of allelic loss in p53-mutated cells. Spontaneous and X-ray-induced LOH mutations at the heterozygous thymidine kinase (tk) gene, which is located on the long arm of chromosome 17, from normal (TK6) and p53-mutated (WTK-1) human lymphoblastoid cells were cytogenetically analyzed by chromosome 17 painting. We observed unbalanced translocations in 53% of LOH mutants spontaneously arising from WTK-1 cells but none spontaneously arising from TK6 cells. We postulate that illegitimate recombination was occurring between nonhomologous chromosomes after DNA replication, leading to allelic loss and unbalanced translocations in p53-mutated WTK-1 cells. X-ray irradiation, which induces DNA double-strand breaks (DSBs), enhanced the generation of unbalanced translocation more efficiently in WTK-1 than in TK6 cells. This observation implicates the wild-type p53 protein in the regulation of homologous recombination and recombinational DNA repair of DSBs and suggests a possible mechanism by which loss of p53 function may cause genomic instability.     

DNA-damage induction by eight metal compounds in TK6 human lymphoblastoid cells: results obtained with the alkaline Comet assay

Metal compounds are long-lived and can react with different macromolecules, producing a wide range of biological effects, including DNA damage. Since their reactivity is associated with their chemical structure, it is important to obtain information on more than one compound from the same metal. In this study, the DNA-damaging potential of two mercury compounds (mercury chloride and methyl mercury chloride), two nickel compounds (nickel chloride and potassium hexafluoronickelate), two palladium compounds (ammonium tetrachloropalladate and ammonium hexachloropalladate), and two tellurium compounds (sodium tellurite and sodium tellurate) was evaluated in human lymphoblastoid TK6 cells by use of the alkaline version of the Comet assay. As the use of computerized image-analysis systems to collect comet data has increased, the metric used for quantifying DNA damage was the Olive tail moment. Treatments lasted for 3h and the range of concentrations tested was different for each metal compound, depending on its toxicity. Both mercury agents produced DNA damage in TK6 cells, with mercury chloride producing considerably more DNA damage than methyl mercury chloride. Of the two nickel compounds, only nickel chloride (a Ni(II) compound) induced DNA breaks. Similarly, of the two palladium compounds, only the Pd(II) compound (ammonium tetrachloropalladate) was positive in the assay. Sodium tellurite was clearly positive, producing concentration-related increases in DNA damage, while sodium tellurate gave a negative response. In conclusion, the ability of inducing DNA damage by the selected metal compounds in human TK6 cells, when measured with the Comet assay, was dependent on the chemical form and, in general, compounds containing the metal in the lower valence state displayed the greater DNA-damaging ability.     

DNA-damage induction by eight metal compounds in TK6 human lymphoblastoid cells: Results obtained with the alkaline Comet assay

Metal compounds are long-lived and can react with different macromolecules, producing a wide range of biological effects, including DNA damage. Since their reactivity is associated with their chemical structure, it is important to obtain information on more than one compound from the same metal. In this study, the DNA-damaging potential of two mercury compounds (mercury chloride and methyl mercury chloride), two nickel compounds (nickel chloride and potassium hexafluoronickelate), two palladium compounds (ammonium tetrachloropalladate and ammonium hexachloropalladate), and two tellurium compounds (sodium tellurite and sodium tellurate) was evaluated in human lymphoblastoid TK6 cells by use of the alkaline version of the Comet assay. As the use of computerized image-analysis systems to collect comet data has increased, the metric used for quantifying DNA damage was the Olive tail moment. Treatments lasted for 3 h and the range of concentrations tested was different for each metal compound, depending on its toxicity. Both mercury agents produced DNA damage in TK6 cells, with mercury chloride producing considerably more DNA damage than methyl mercury chloride. Of the two nickel compounds, only nickel chloride (a Ni(II) compound) induced DNA breaks. Similarly, of the two palladium compounds, only the Pd(II) compound (ammonium tetrachloropalladate) was positive in the assay. Sodium tellurite was clearly positive, producing concentration-related increases in DNA damage, while sodium tellurate gave a negative response. In conclusion, the ability of inducing DNA damage by the selected metal compounds in human TK6 cells, when measured with the Comet assay, was dependent on the chemical form and, in general, compounds containing the metal in the lower valence state displayed the greater DNA-damaging ability.     

The relative radiosensitivity of TK6 and WI-L2-NS lymphoblastoid cells derived from a common source is primarily determined by their p53 mutational status

The lymphoblastoid cell lines WI-L2-NS and TK6 were derived from a non-clonal pool of cells taken from a human spleen. Despite their common background they exhibit marked differences in radiosensitivities; D₀ values of 93 and 67 cGy have been reported for WI-L2-NS and TK6 cells respectively. We show here that this differential radiosensitivity is due to a decreased ability of the WI-L2-NS cell line to undergo radiation-induced apoptosis. Further, the WI-L2-NS cell line overexpresses the p53 gene product as a result of a mutation in codon 237 of the p53 gene. These data indicate that WI-L2-NS cells through disruption of normal p53 function are unable to engage the radiation-induced apoptosis program and so are relatively radioresistant.     

Combined effects of gamma radiation and arsenite on the proteome of human TK6 lymphoblastoid cells

Arsenic present in drinking water and mining environments in some areas has been associated with an increased rate of skin and internal cancers. Contrary to the epidemiological evidence in humans, arsenic does not induce cancer in animal models, but is able to enhance the mutagenicity of other agents. In order to achieve a better understanding of the interaction between arsenic and ionising radiation, an investigation was conducted to detect differences at the proteome level of human TK6 lymphoblastoid cells exposed to these agents. Cells were exposed to either a single dose of 1-Gy 137Cs-gamma-rays or to 1 microM arsenite (As(III)) or to both agents in combination. Two-dimensional (2D) electrophoresis and matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) were employed for the screening and identification of proteins, respectively. It proved possible to identify seven proteins with significantly affected abundance, three of which showed increased levels and the remaining four showed decreased levels under at least one of the exposure conditions. Following arsenite treatment or irradiation, a significant increase compared with that of the control was observed for glutathione (GSH) transferase omega 1 and proteasome subunit beta type 4 precursor. The combined exposure did not result in an induction of the enzymes. The expression of electron-transfer flavoprotein subunit alpha was found to be enhanced under all three-exposure conditions. Ubiquinol-cytochrome C reductase complex core protein I, adenine phosphoribosyl transferase and endoplasmic reticulum protein hERp29 showed decreased levels after irradiation or arsenite treatment, but not after the combined exposure. The level of serine/threonine protein phosphatase 1 alpha decreased with all treatments. The main conclusions are that both arsenite and gamma-radiation influence the levels of several proteins involved in major metabolic and regulatory pathways, either directly or by triggering the defence mechanisms of the cell. The combined effect of both exposures on the level of some essential proteins such as glutathione transferase, proteasome or serine/threonine phosphatase may contribute to the co-carcinogenic effect of arsenic.     

Distinct apoptotic phenotypes induced by radiation and ceramide in both p53-wild-type and p53-mutated lymphoblastoid cells

The tumour suppressor gene p53 and the intracellular signalling molecule ceramide have both been shown to play crucial roles in the induction of apoptosis by ionising radiation. In this study we examined whether p53 and ceramide are involved in independent signal pathways, inducing different types of apoptosis. TK6 (p53^wt/wt) and WTK1 (p53^mut/mut) lymphoblastoid cells were treated with ionising radiation or N-acetyl-d-sphingosine (C₂-ceramide). Flow cytometry and fluorescence microscopy studies were performed to characterise the time kinetics and morphological features of induced apoptosis. Ceramide- and radiation-induced apoptotic cells display characteristic differences in morphology and DNA staining and ceramide-induced apoptosis is expressed much faster than radiation-induced apoptosis. Radiation-induced apoptosis is p53-dependent and ceramide-induced apoptosis is p53-independent. The p53 pathway and the ceramide pathway are two independent signal pathways leading to distinct types of apoptosis. Since p53 is very often dysfunctional in tumour cells, modifying the ceramide pathway is a promising strategy to increase tumour sensitivity to radiation and other anticancer agents. Received: 19 April 2001 / Accepted: 15 October 2001     

Both the rate and spectrum of loss of heterozygosity differ between human lymphoblastoid cells derived from various donors

Loss of heterozygosity (LOH) contributes significantly to the inactivation of tumor suppressor genes and may involve a variety of mechanisms. Studying loss of HLA-A2 alleles in human lymphoblastoid cell lines, we previously showed that mitotic recombination and chromosome loss with concomitant duplication of the non-selected chromosome were the most frequent mechanisms of LOH. In the present study we used the HLA system to determine the rate and spectrum of LOH mutations in the EBV transformed lymphoblastoid cell line R83-4915. Spontaneous loss of HLA-A2 in R83-4915 occurred with a rate of 7.9x10-7 which was 5 to 10-times lower compared to the previously observed rate of loss of HLA-A2 in other lymphoblastoid cell lines. Among the HLA-A2 mutants, 27% did not show LOH of additional chromosome 6 markers. Molecular analysis showed that neither large deletion nor gene conversion was the cause for their mutant phenotype. The remaining mutants showed LOH, which was caused by mitotic recombination (40%) and chromosome loss (33%). However, the chromosome loss observed in mutants of R83-4915 was not accompanied by the duplication of the remaining chromosome. Instead 3 out of 5 mutants became polyploid suggesting that different mechanisms exist to compensate for chromosome loss. In conclusion, the rate and types of LOH that can be observed in cell lines obtained from various donors may depend on the genetic make-up or the transformation status of these cells     

Two structurally distinct inhibitors of glycogen synthase kinase 3 induced centromere positive micronuclei in human lymphoblastoid TK6 cells

Glycogen synthase kinase 3 (GSK3) is an attractive novel pharmacological target. Inhibition of GSK3 is recently regarded as one of the viable approaches to therapy for Alzheimer's disease, cancer, diabetes mellitus, osteoporosis, and bipolar mood disorder. Here, we have investigated the aneugenic potential of two potent and highly specific inhibitors of GSK3 by using an in vitro micronucleus test with human lymphoblastoid TK6 cells. One inhibitor was a newly synthesized maleimide derivative and the other was a previously known aminopyrimidine derivative. Both compounds elicited statistically significant and concentration-dependent increases in micronucleated cells. One hundred micronuclei (MN) of each were analyzed using centromeric DNA staining with fluorescence in situ hybridization. Both the two structurally distinct compounds induced centromere-positive micronuclei (CMN). Calculated from the frequency of MN cells and the percentage of CMN, CMN cell incidence after treatment with the maleimide compound at 1.2muM, 2.4muM, and 4.8muM was 11.6, 27.7, and 56.3 per 1000 cells, respectively; the negative control was 4.5. CMN cell incidence after the treatment with the aminopyrimidine compound at 1.8muM, 3.6muM, and 5.4muM was 6.7, 9.8 and 17.2 per 1000 cells, respectively. Both compounds exhibited concentration-dependent increase in the number of mitotic cells. The frequency of CMN cells correlated well with mitotic cell incidence after treatment with either compound. Furthermore, both inhibitors induced abnormal mitotic cells with asymmetric mitotic spindles and lagging anaphase chromosomes. These results lend further support to the hypothesis that the inhibition of GSK3 activity affects microtubule function and exhibits an aneugenic mode of action.     

Involvement of TP53 in apoptosis induced in human lymphoblastoid cells by fast neutrons

We investigated the involvement of TP53 in apoptosis induced by fast neutrons in cells of three human B-lymphoblast cell lines derived from the same donor and differing in TP53 status: TK6 (wild-type TP53), WTK1 (mutant TP53) and NH32 (knockout TP53). Cells were exposed to X rays or to fast neutrons at doses ranging from 0.5 to 8 Gy. Apoptosis was determined by measurements of the sub-G0 /G1-phase DNA content and by the externalization of phosphatidylserine. Fast neutrons induced extensive apoptosis in TK6 cells, as shown by the formation of hypodiploid particles, the externalization of phosphatidylserine, and the activation of caspases. In contrast, cell death was triggered at a significantly lower rate in cells lacking functional TP53. However, TP53-independent cell death also expressed the morphological and biochemical hallmarks of apoptosis. Proliferation tests and clonogenic assays showed that fast neutrons can nevertheless kill WTK1 and NH32 cells efficiently. The absence of functional TP53 only delays radiation-induced cell death, which is also mediated by caspases. These results indicate that fast-neutron irradiation activates two pathways to apoptosis and that the greater relative biological effectiveness of fast neutrons reflects mainly an increase in clonogenic cell death.     

Validation of a genotoxicity test based on p53R2 gene expression in human lymphoblastoid cells

Genotoxicity assessment is important for predicting the carcinogenicity of chemical substances. p53R2 is a p53-regulated gene that is induced by various genotoxic stresses. We previously developed a p53R2-dependent luciferase reporter gene assay in the MCF-7 human breast adenocarcinoma cell line, and demonstrated its ability to detect genotoxic agents. In this paper, we investigate the applicability of the p53R2-based genotoxicity test in the human lymphoblastoid cell line TK6. TK6 cells that express wild-type p53 have been widely used for genetic toxicology studies. To evaluate the performance of the test system in TK6 cells, we referred to 61 of the chemicals on the list of 20 genotoxic and 42 non-genotoxic chemicals recommended for the evaluation of modified or new mammalian cell genotoxicity tests by the European Centre for the Validation of Alternative Methods. The overall accordance, sensitivity, and specificity of our results with the ECVAM list were 90% (55/61), 85% (17/20), and 93% (38/41), respectively. These results indicate that the p53R2-based genotoxicity test can detect various types of genotoxic chemicals without compromising its specificity. This test will be a valuable tool for rapid screen for identifying chemicals that may be genotoxic to humans.     

Mutagenicity of tamoxifen DNA adducts in human endometrial cells and in silico prediction of p53 mutation hotspots

Tamoxifen elevates the risk of endometrial tumours in women and alpha-(N(2)-deoxyguanosinyl)-tamoxifen adducts are reportedly present in endometrial tissue of patients undergoing therapy. Given the widespread use of tamoxifen there is considerable interest in elucidating the mechanisms underlying treatment-associated cancer. Using a combined experimental and multivariate statistical approach we have examined the mutagenicity and potential consequences of adduct formation by reactive intermediates in target uterine cells. pSP189 plasmid containing the supF gene was incubated with alpha-acetoxytamoxifen or 4-hydroxytamoxifen quinone methide (4-OHtamQM) to generate dG-N(2)-tamoxifen and dG-N(2)-4-hydroxytamoxifen, respectively. Plasmids were replicated in Ishikawa cells then screened in Escherichia coli. Treatment with both alpha-acetoxytamoxifen and 4-OHtamQM caused a dose-related increase in adduct levels, resulting in a damage-dependent increase in mutation frequency for alpha-acetoxytamoxifen; 4-OHtamQM had no apparent effect. Only alpha-acetoxytamoxifen generated statistically different supF mutation spectra relative to the spontaneous pattern, with most mutations being GC-->TA transversions. Application of the LwPy53 algorithm to the alpha-acetoxytamoxifen spectrum predicted strong GC-->TA hotspots at codons 244 and 273. These signature alterations do not correlate with current reports of the mutations observed in endometrial carcinomas from treated women, suggesting that dG-N(2)-tam adduct formation in the p53 gene is not a prerequisite for endometrial cancer initiation in women.     

Study of oxidative DNA damage in TK6 human lymphoblastoid cells by use of the in vitro micronucleus test: Determination of No-Observed-Effect Levels

The existence of thresholds for indirect DNA-damaging agents has been widely accepted in the last few years. In contrast, DNA-reactive agents have been assumed to have a non-threshold mode of action, as they directly induce DNA lesions that have the potential to be converted into mutations. However, this does not take into account protective factors acting to reduce or repair genotoxic damage. Among the compounds acting through possible threshold-mechanisms, some of them induce DNA damage by oxidative stress. In this context, the aim of our study was to investigate the dose–response relationship of well-known DNA-oxidizing agents acting through different mechanisms of oxidative stress, viz. potassium bromate, bleomycin and hydrogen peroxide (by the action of glucose oxidase) by assessing the induction of chromosomal damage using the in vitro micronucleus test (MNT) on the human lymphoblastoid cell line TK6. In order to provide a first characterization of their genotoxic mechanism, two treatment schedules were applied. Cells received both short-term treatment followed by a recovery time (1 + 23 h, 2 + 22 h, 3 + 21 h or 6 + 18 h) and long-term treatment (24 h continually). Our results show interesting non-linear dose–effect relationships starting with a range of non-mutagenic very low doses allowing the determination of a No-Observed-Effect Level (NOEL) and going step-wise up to higher doses. After a short exposure, three different plateaus were observed suggesting complex activations and interactions of different cellular mechanisms whose nature and efficiency were dose-dependent. In contrast, after a long treatment, the dose–response curves were different depending on the test compound investigated. Therefore, the in vitro MNT seems to be an appropriate predictive test to establish the NOEL(s) of DNA-oxidizing agents. In order to confirm and to determine the origin of the different cellular step-wise responses observed, additional mechanistic studies would be required, especially by means of other genotoxicity endpoints and gene-expression profiling.