The mutagenic effect of 1,2-dichloroethane on Salmonella typhimurium. II. Activation by the isolated perfused rat liver.

In this investigation Salmonella typhimurium strain TA 1530 and TA 1535 were combined with isolated perfused rat liver. Samples of perfusate and bile produced were tested for mutagenicity after treatment with 1,2-dichloroethane (DCE), 1,2-dibromoethane (DBE) or 2-chloroethanol. The results are in good agreement with our previous experiments which indicate that both DEC and DBE are activated through conjugation with glutathione (GSH). Most GSH conjugates are normally excreted in bile. Following liver perfusion the bile was highly mutagenic after DCE and DBE treatments, while 2-chloroethanol did not have this effect. The highest mutagenic effect was seen 15--30 min after the addition of DCE or DBE. The production of mutagenic bile also occurred in mice treated in vivo with DCE. One possible metabolic endproduct of a GSH conjugate is the corresponding mercapturic acid. Thus synthetic N-acetyl-S-(2-chloroethyl)-L-cysteine was tested on TA 1535 and found to be as mutagenic as S-(2-chloroethyl)-L-cysteine in the concentration range 0.2--0.6 mumol/plate. Differences and similarities in the metabolism of DCE and vinyl chloride are discussed on the basis of these results.     

Mutagenicity of stereochemical configurations of 1,2-epoxybutene and 1,2:3,4-diepoxybutane in human lymphblastoid cells

The carcinogenicity of 1,3-butadiene (BD) is related to its bioactivation to several DNA-reactive metabolites; accumulating evidence suggests that the stereochemistry of these BD intermediates may play a significant role in the mutagenic and carcinogenic actions of the parent compound. The objective of this study was to evaluate the cytotoxicity and mutagenicity of stereochemical forms of 1,2-epoxybutene (EB) and 1,2:3,4-diepoxybutane (DEB), two genotoxic BD metabolites, in a human lymphoblastoid cell line, TK6. Cytotoxicity was measured by comparing cloning efficiencies in chemical-exposed cells versus those in control cells. The hypoxanthine-guanine phosphoribosyltransferase (HPRT) and thymidine kinase (TK) mutant frequencies (MFs) were measured using a cell cloning assay. HPRT mutants collected from cells exposed to the three forms of DEB were analyzed by PCR to characterize large genetic alterations. All the three stereoisomers of DEB caused increased HPRT and TK MFs compared to the concurrent control samples. There were no significant differences in cytotoxicity or mutagenicity among the three isomers of DEB in TK6 cells. Molecular analysis of HPRT mutants revealed similar distributions of types of mutations among the three isomers of DEB. There were also no statistically significant differences in mutagenic efficiencies between the two isomers of EB in TK6 cells. These results were consistent with the in vivo findings that there was little difference in the mutagenic efficiencies of racemic-DEB versus meso-DEB in rodents. Thus, in terms of mutagenic efficiency, stereochemical configurations of EB and DEB are not likely to play a significant role in the mutagenicity and carcinogenicity of BD.     

In vitro microsome- and cytosol-mediated binding of 1,2-dichloroethane and 1,2-dibromoethane with DNA

Metabolic activation of 1,2-dichloroethane (DCE) and 1,2-dibromoethane (DBE) to forms able to bind covalently with DNA occurs in vitroeither by wat of microsomal or cytosolic pathways. The involvement of these two pathways is variable with respect to species or compound tested. Rat enzymes are generally more efficient than mouse enzymes in bioactivating haloalkanes and DBE is more reactive than DCE. This parallels both the previous report on in vivocomparative interaction and the higher genotoxicity of DBE.Abbreviations DCE dichloroethane - DBE dibromoethane - PB phenobarbitone - ANOVA analysis of variance Supported by grants from Ministero Pubblica Instruzione and from Ministero della Sanità (Contract No. 500.4/RSC/135/L/2353), Rome, Italy.     

Effects of carcinogenic halogenated aliphatic hydrocarbons on [3H]thymidine incorporation into various organs of the mouse. A comparison between 1,2-dibromoethane and 1,2-dichloroethane

The effects of 1,2-dibromoethane (DBE) and 1,2-dichloroethane (DCE) on the incorporation of [3H]thymidine into DNA were evaluated in various tissues of mice. The compounds were given intraperitoneally 24 h before sacrifice in an equimolar dose (293 mumoles/kg body weight). 2 h before the animals were killed, 0.5 mu Ci [3H]thymidine/g body weight was injected intraperitoneally. Both agents inhibited the [3H]thymidine incorporation in the forestomach, a site for their carcinogenic action. Whereas DBE also suppressed the [3H]thymidine incorporation in the nasal mucosa, the thymus, and the "glandular stomach", DCE was inhibitory only in the kidney. The observed difference in the effect of DBE and DCE on the thymus had its counterpart in a DBE-induced decrease of acid-insoluble radioactivity, demonstrated with whole-body autoradiography. The results indicate that in vivo screening of [3H]thymidine incorporation into various organs of an intact experimental animal is a sensitive technique for comparing cyto- and/or genotoxic effects of chemicals with a similar chemical structure.     

Long-term low-dose mutation studies in human cells: metanil yellow and orange II

We tested the mutagenic effects of two commonly used fold colors, metanil yellow and orange II, in AHH-1 human lymphoblast cells. The cell line, which is competent for oxidative metabolism of various chemicals, was exposed to both compounds in high-dose x short-term (3 day) or high-dose x long-term (10-day) and low-dose x long-term (20-day) treatments. Concentrations of metanil yellow and orange II as low as 22 nM and 12 nM, respectively, were sufficient to induce mutation rates which were equal to twice the spontaneous mutation rate at the HPRT locus in AHH-1 cells.     

Inhalation exposure in Drosophila mutagenesis assays: experiments with aliphatic halogenated hydrocarbons, with emphasis on the genetic activity profile of 1,2-dichloroethane

A series of mutation experiments was carried out with Drosophila melanogaster using inhalation exposure. 1,2-Dichloroethane (DCE) and 1,2-dibromoethane (DBE) were active in the sex-linked recessive lethal assay (SLRLT), whereas dichloromethane, dibromomethane, 1,2-dichloropropane and 1,3-dichloropropane were not. Compared to DBE, DCE is a less potent mutagen in the SLRL system. For both compounds, there is no evidence of a clear-cut dose-rate effect. DCE and dichloromethane were also investigated in the somatic mutation and recombination test (SMART), with results similar to those from the SLRLT. For DCE the genetic activity profile was further analyzed by carrying out a sex-chromosome loss assay and a complementation analysis of a series of induced recessive lethal mutations. A review of the use of inhalation in mutagenicity assays with Drosophila shows that this route of exposure is an effective one. Especially with chronic exposure times, rather low exposure concentrations can be detected. With compounds of intermediate volatility inhalation is not superior to other modes of administration; nor is it likely to be sensitive enough for in situ monitoring.     

Relationship between p53 status and 5-fluorouracil sensitivity in 3 cell lines

Mouse lymphoma L5178Ytk+/- (MOLY) cells and human lymphoblastoid TK6 and WTK-1 cells are widely used to detect mutagens in vitro. MOLY and WTK-1 cells have a p53 mutation, while TK6 cells, which were derived from the same parental line as WTK-1 cells, do not. In this study, we tested the clastogen 5-fluorouracil (5-FU) in the Tk assay and the in vitro micronucleus (MN) assay in MOLY, TK6, and WTK-1 cells to clarify whether differential responses were related to p53 gene status. We also determined the effect of 5-FU on the frequency of apoptotic cells and on cell cycle distribution in each cell line. Furthermore, we measured the activity of the 5-FU metabolizing enzymes (thymidylate synthetase (TS), dihydrouracil dehydrogenase (DPD), orotate phosphoribosyl transferase (OPRT), and thymidine phosphorylase (TP)) in each cell line. We treated MOLY cells with 1.0-8.0 microg/mL 5-FU for 3 h and TK6 and WTK-1 cells with 1.56-25 and 3.13-50 microg/mL, respectively, for 4 h. In MOLY cells, the mutation frequency (MF) and MN frequency increased. In WTK-1 cells, the MN frequency but not the MF increased. In TK6 cells, neither the MF nor the MN frequency increased. Furthermore, the IC50 of 5-FU was lower in MOLY cells than in the human cells. The response to 5-FU treatment differed in other ways as well. At the same level of cytotoxicity, the frequency of apoptotic cell was highest in TK6 cells. The cell cycle was delayed just after treatment in MOLY cells while the delay appeared 24 h later in TK6 and WTK-1 cells. Nothing in our analysis, however, revealed marked differences between the cell lines that could account for the severe cytotoxic and mutagenic responses that 5-FU elicited only in MOLY cells. 5-FU is phosphorylated by OPRT and TP and detoxified by DPD. MOLY cells have higher OPRT activity and markedly lower DPD and TP activity than TK6 and WTK-1 cells. The content of TS, however, the target enzyme of 5-FU, was similar in all cell lines, suggesting that 5-FU was more readily phosphorylated and less readily detoxified in MOLY cells than in TK6 and WTK-1 cells. MOLY cells were more sensitive to 5-FU than WTK-1 cells even though both have a mutated p53 gene, suggesting that the different responses to 5-FU were due to differences in 5-FU metabolism rather than the p53 status.     

Modification of hepatic vitamin E stores in vivo. III. Vitamin E depletion by 1,2-dibromoethane may be related to initial conjugation with glutathione

In the companion paper we demonstrated that hepatic vitamin E in rats becomes depleted and extrahepatic pools of vitamin E are altered by treatment with 1,2-dibromoethane (DBE). Vitamin E depletion may be dependent upon initial steps of DBE metabolism that are either oxidative (cytochrome P450 dependent) or conjugative (glutathione transferase dependent). That the liver content of glutathione (GSH) and vitamin E, the plasma concentration of vitamin E, and the serum activities of AST and ALT may be influenced by cytosolic metabolism of DBE was assessed by comparison of findings from rats treated with either 1,2-dichloroethane (DCE) or 1-bromo-2-chloroethane (BCE). The extent of oxidative metabolism was diminished by the use of tetradeutero-DBE (d4-DBE), and the availability of GSH for conjugative metabolism was diminished by pretreatment of rats with L-buthionine-S,R-sulfoximine (BSO) prior to treatment with DBE. Our results indicate that neither DCE nor BCE provokes a liver vitamin E depletion in rats, that d4-DBE treatment hastens but does not enhance the observed hepatic vitamin E depletion by comparison to animals treated with an equimolar dose of DBE, and that BSO pretreatment prevented the hepatic vitamin E depletion observed from animals treated with DBE alone. These results indicate that hepatic vitamin E depletion is the unique sequelae to conjugation of GSH with DBE, and we suggest the reactive episulfonium ion intermediate or a macromolecular adduct of this ion derived from DBE may play a role in liver vitamin E depletion associated with exposure to DBE.     

Genotoxic effects of high-energy iron particles in human lymphoblasts differing in radiation sensitivity.

The effects of (56)Fe particles and (137)Cs gamma radiation were compared in TK6 and WTK1 human lymphoblasts, two related cell lines which differ in TP53 status and in the ability to rejoin DNA double-strand breaks. Both cell lines were more sensitive to the cytotoxic and clastogenic effects of (56)Fe particles than to those of gamma rays. However, the mutagenicity of (56)Fe particles and gamma rays at the TK locus was the same per unit dose and was higher for gamma rays than for (56)Fe particles at isotoxic doses. The respective RBEs for TK6 and WTK1 cells were 1.5 and 1.9 for cytotoxicity and 2.5 and 1.9 for clastogenicity, but only 1 for mutagenicity. The results indicate that complex lesions induced by (56)Fe particles are repaired less efficiently than gamma-ray-induced lesions, leading to fewer colony-forming cells, a slightly higher proportion of aberrant cells at the first division, and a lower frequency of viable mutants at isotoxic doses. WTK1 cells (mutant TP53) were more resistant to the cytotoxic effects of both gamma rays and (56)Fe particles, but showed greater cytogenetic and mutagenic damage than TK6 cells (TP53(+)). A deficiency in the number of damaged TK6 cells (a) reaching the first mitosis after exposure and (b) forming viable mutants can explain these results.     

Human cell mutagenicity of chlorinated and unchlorinated water and the disinfection byproduct 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX).

Extracts of three water samples--humic acid-enriched water-both peatland water and drinking water, both with and without chlorination were tested for mutagenicity at the tk locus in MCL-5 cells, a line of human B-lymphoblastoid cells that express cytochrome P450 enzymes and microsomal epoxide hydrolase. Our results show that chlorination caused a 5.5-fold increase (P<0.0001) in the mutagenicity of the humic acid-enriched water. The unchlorinated peatland water was mutagenic at the two highest doses (240 and 480 microg equivalent total organic carbon (TOC)/ml), possibly due to polycyclic aromatic hydrocarbons (PAH) that were measured in the peat. In contrast, the chlorinated peatland water was non-mutagenic at low doses, while at the highest dose (240 microg equivalent TOC/ml) the sample was so toxic that an insufficient number of cells survived treatment to allow plating. The chlorinated and unchlorinated drinking water were both non-mutagenic. 3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a potent bacterial mutagen and chlorine-disinfection byproduct, was also tested in MCL-5 cells as well as in two other human B-lymphoblastoid cell-lines, AHH-1 TK+/- and h1A1v2 cells, which differ from each other and from MCL-5 cells in the amounts of cytochrome P450 enzymes they can express. MX was mutagenic to all three cell-lines, but there was no apparent correlation between cytochrome P450 enzyme expression and the mutagenicity of MX. Overall, our results show that samples of chlorinated humic acid-enriched water and MX, a model chlorine-disinfection byproduct, are moderately mutagenic to human cells.     

Mutagenic adaptive response to high-LET radiation in human lymphoblastoid cells exposed to X-rays

The ability of cells to adapt low-dose or low-dose rate radiation is well known. High-LET radiation has unique characteristics, and the data concerning low doses effects and high-LET radiation remain fragmented. In this study, we assessed in vitro the ability of low doses of X-rays to induce an adaptive response (AR) to a subsequent challenging dose of heavy-ion radiation. Lymphoblastoid cells (TK6, AHH-1, NH32) were exposed to priming 0.02-0.1Gy X-rays, followed 6h later by challenging 1Gy heavy-ion radiation (carbon-ion: 20 and 40keV/μm, neon-ion: 150keV/μm). Pre-exposure of p53-competent cells resulted in decreased mutation frequencies at hypoxanthine-guanine phosphoribosyl transferase locus and different H2AX phosphorylation kinetics, as compared to cells exposed to challenging radiation alone. This phenomenon did not seem to be linked with cell cycle effects or radiation-induced apoptosis. Taken together, our results suggested the existence of an AR to mutagenic effects of heavy-ion radiation in lymphoblastoid cells and the involvement of double-strand break repair mechanisms.     

Are endogenous clustered DNA damages induced in human cells?

Although clustered DNA damages are induced in cells by ionizing radiation and can be induced artifactually during DNA isolation, it was not known if they are formed in unirradiated cells by normal oxidative metabolism. Using high-sensitivity methods of quantitative gel electrophoresis, electronic imaging, and number average length analysis, we found that two radiosensitive human cell lines (TK6 and WI-L2-NS) accumulated Fpg-oxidized purine clusters and Nth-oxidized pyrimidine clusters but not Nfo-abasic clusters. However, four repair-proficient human lines (MOLT 4, HL-60, WTK1, and 28SC) did not contain significant levels (<5/Gbp) of any cluster type. Cluster levels were independent of p53 status. Measurement of glycosylase levels in 28SC, TK6, and WI-L2-NS cells suggested that depressed hOGG1 and hNth activities in TK6 and WI-L2-NS could be related to oxybase cluster accumulation. Thus, individuals with DNA repair enzyme deficiencies could accumulate potentially cytotoxic and mutagenic clustered DNA damages. The absence of Nfo-detected endogenous clusters in any cells examined suggests that abasic clusters could be a signature of cellular ionizing radiation exposure.     

Mutations and chromosomal aberrations in hMTH1-transfected and non-transfected TK6 cells after exposure to low dose rates of gamma radiation

The aim of the present study was to analyse the dose rate effect of gamma radiation at the level of mutations, chromosomal aberrations, and cell growth in TK6 cells with normal as well as reduced levels of hMTH1 protein. TK6 cells were exposed to gamma radiation at dose rates ranging from 1.4 to 30.0 mGy/h (chronic exposure) as well as 24 Gy/h (acute exposure). Cell growth, frequency of thymidine kinase mutants, and of chromosomal aberrations in painted chromosomes 2, 8, and 14 were analysed. A decline in cell growth and an increase in unstable-type chromosomal aberrations with increasing dose rate were observed in both cell lines. A dose rate effect was not seen on mutations or stable-type chromosomal aberrations in any of the two cell lines. Reduction in the hMTH1 protein does not influence the sensitivity of TK6 cells to gamma radiation. This result fits well with data of others generated with the same cell line.     

Xenobiotic metabolism and mutation in a human lymphoblastoid cell line

Aryl hydrocarbon hydroxylase-1 (AHH-1) cells are a human lymphoblastoid cell line competent in some aspects of xenobiotic metabolism. This cell line contains stable mixed function oxidase activity which is inducible by polycyclic aromatic hydrocarbons (PAHs) but not by phenobarbital or Arochlor 1254. Two substrates for the cellular mixed function oxidase activity, benzo[a]pyrene (B[a]P) and 7-ethoxyresorufin, have been examined. The basal and induced activities have different kinetic parameters toward these two substrates. In contrast, basal and induced activities had similar sensitivities to two cytochrome P-450 suicide substrates. B[a]P metabolism and mutagenicity were studied in this cell line. AHH-1 cells were found to produce predominantly B[a]P phenols and quinones. The major phenol metabolite cochromatographed with authentic 9-hydroxy B[a]P. AHH-1 cells were capable of forming glucuronic acid conjugates of B[a]P phenols; the major product after hydrolysis cochromatographed with 3-hydroxy B[a]P standard. AHH-1 cells did not contain detectable epoxide hydrolase activity using B[a]P-4,5-oxide as substrate. This observation is consistent with the absence of trans-dihydrodiol B[a]P metabolites in the metabolic profile. B[a]P-induced mutagenicity at the hypoxanthine guanine phosphoribosyl transferase (hgprt) locus in AHH-1 cells was found to be linearly related to phenol production during treatment and inhibited by alpha-naphthoflavone (ANF).     

Evaluation of the genotoxicity of the phytoestrogen, coumestrol, in AHH-1 TK(+/-) human lymphoblastoid cells

Coumestrol, a phytoestrogen found in high levels in alfalfa and red clover, is of concern since endocrine disorders have been observed in farm animals exposed to high levels of phytoestrogens. Previous studies found that coumestrol was an effective inducer of DNA strand breaks, micronuclei, and mutations in the Hypoxanthine phosphoribosyl transferase (HPRT) gene of Chinese hamster ovary cells. In the experiments presented here, we extended the previous studies to examine the effect of coumestrol exposure on AHH-1 TK(+/-) human lymphoblastoid cells. Micronuclei were induced with the highest frequency occurring at day 2 after exposure. Flow cytometric analysis of annexin V-FITC-7-aminoactinomycin D stained cells indicated that the primary pathway of cell death was by apoptosis. Mutations were induced in the Thymidine Kinase (TK) gene and were due primarily to the induction of clones with the slow-growth phenotype. Subsequent molecular analysis revealed the loss of exon 4 in the coumestrol-induced clones, indicative of loss-of heterozygosity and consistent with a proposed inhibition of topoisomerase-II activity as a mechanism of action for coumestrol. Taken together, these results suggest that coumestrol exhibits both mutagenic and clastogenic properties in cultured human lymphoblastoid cells.     

The role of human O(6)-alkylguanine-DNA alkyltransferase in promoting 1,2-dibromoethane-induced genotoxicity in Escherichia coli

The expression of the DNA repair protein human O(6)-alkylguanine-DNA alkyltransferase (AGT) in Escherichia coli strains GWR109 or TRG8 that lack endogenous AGT greatly increased the toxicity and mutagenicity of 1,2-dibromoethane (DBE). Pretreatment of strain TRG8 expressing human AGT, which is permeable to exogenous drugs, with the AGT inhibitor O(6)-benzylguanine (BG) abolished the lethal and mutagenic effects of DBE, indicating that an active AGT is required for promoting DBE genotoxicity. This was confirmed by the observation that E. coli expressing either the C145A AGT mutant, which is inactive due to loss of the alkyl acceptor site, or mutants Y114E and R128A, which are inactive due to alteration of the DNA binding domain, did not enhance the action of DBE. However, the AGT mutant protein P138M/V139L/P140K, which is active in repairing methylated DNA but is totally resistant to inactivation by BG due to alterations in the active site pocket, was unable to enhance the genotoxicity of DBE. Similarly, other mutants, G156P, Y158H and K165R that are strongly resistant to BG, were much less effective than wild type AGT in mediating the genotoxicity of DBE. Mutant P140A, which is moderately resistant to BG, did increase mutations in response to DBE but was less active than wild type. These results suggest that human AGT is able to interact with a DNA lesion produced by DBE but, instead of repairing it, converts it to a more genotoxic adduct. This interaction is prevented by mutations that modify the active site of AGT to exclude BG.     

Assay for gene mutation in a human lymphoblast line, AHH-1, competent for xenobiotic metabolism

A novel quantitative gene-locus mutation assay has been developed using a line of human lymphoblast cells, designated AHH-1, competent in oxidative xenobiotic metabolism. AHH-1 cells are sensitive to the mutagenic action of both chemically reactive mutagens and mutagens which require oxidative metabolism to exert their mutagenicity. These cells are readily mutated by direct exposure to ethyl methanesulfonate, ICR-191, 2-acetoaminofluorene, aflatoxin B1, benzo[a]pyrene (BP), cyclopenta[c, d]pyrene, dimethylnitrosamine, lasiocarpine, and 1-methylphenanthrene.     

Toxicity of 1,2-dibromoethane in isolated hepatocytes: Role of lipid peroxidation

Treatment of isolated hepatocytes with 1,2-dibromoethane (DBE) caused a concentration dependent depletion of cellular glutathione (GSH) content and a parallel increase in the covalent binding of reactive intermediates to cell proteins, as a consequence of the haloalkane activation. The reduction of the hepatocyte GSH content, induced by DBE, stimulated the onset of lipid peroxidation, as measured by malondialdehyde (MDA) accumulation. N-Acetylcysteine (1 mM) was found to partially prevent GSH loss and to inhibit MDA formation, whereas equal concentrations of cysteine and methionine were ineffective on these respects. The stimulation of the peroxidative reactions appeared to be also associated with an increase in the leakage of lactate dehydrogenase (LDH) from the cells, indicative of a severe hepatocyte injury. Antioxidants such as -tocopherol, N,N′-phenyl-phenylenediamine (DPPD) and promethazine, as well as N-acetylcysteine reduced MDA formation to various extents and also protect against LDH release, yet without interfering with the covalent binding of DBE reactive intermediates to hepatocyte proteins. These results suggest the involvement of lipid peroxidation, consequent to GSH depletion, in the pathogenesis of liver cell necrosis due to DBE.     

Characteristics of somatic cell hybrids (mouse x Chinese hamster) with a different ratio of chromosome sets from the parent species. II. Thymidine kinase activity]

The activity of thymidine kinase (TK) was studied in series of somatic cell hybrids between the mouse cell line 3T3-4E (TK-) and Chinese hamster cells M-15-1 (HGPRT-). Four groups of hybrid lines with different ratio of parental chromosome sets have been investigated: 1) three lines containing one hamster and one mouse chromosome set (1 hs+1 ms); 2) one line with 2 hs+1 ms; 3) one line containing 3 hs+1 ms and 4) one line containing 1 hs+2 ms. Mixtures of extracts from the parental cells were shown to possess the expected TK activity. The calculation of the activity per cell revealed that the 1 hs+1 ms and 2 hs+1 ms hybrid lines possessed about 50% of the initial hamster cell TK activity. The decreased TK activity in these hybrids might be due either to a loss of hamster chromosomes or to some inhibitory effect of mouse genome in cells with the studied ratio of parental sets. The enzyme activity in the 3 hs+1 ms hybrid was as expected, about three times greater than that of hamster cells.     

The TP53 dependence of radiation-induced chromosome instability in human lymphoblastoid cells

The dose and TP53 dependence for the induction of chromosome instability were examined in cells of three human lymphoblastoid cell lines derived from WIL2 cells: TK6, a TP53-normal cell line, NH32, a TP53-knockout created from TK6, and WTK1, a WIL2-derived cell line that spontaneously developed a TP53 mutation. Cells of each cell line were exposed to (137)Cs gamma rays, and then surviving clones were isolated and expanded in culture for approximately 35 generations before the frequency and characteristics of the instability were analyzed. The presence of dicentric chromosomes, formed by end-to-end fusions, served as a marker of chromosomal instability. Unexposed TK6 cells had low levels of chromosomal instability (0.002 +/- 0.001 dicentrics/cell). Exposure of TK6 cells to doses as low as 5 cGy gamma rays increased chromosome instability levels nearly 10-fold to 0.019 +/- 0.008 dicentrics/cell. There was no further increase in instability levels beyond 5 cGy. In contrast to TK6 cells, unexposed cultures of WTK1 and NH32 cells had much higher levels of chromosome instability of 0.034 +/- 0.007 and 0.041 +/- 0.009, respectively, but showed little if any effect of radiation on levels of chromosome instability. The results suggest that radiation exposure alters the normal TP53-dependent cell cycle checkpoint controls that recognize alterations in telomere structure and activate apoptosis.