Chromosomal instability in mutagen-sensitive mutants isolated from mouse lymphoma L5178Y cells. I. Five different genes participate in the formation of baseline sister-chromatid exchanges and spontaneous chromosomal aberrations

In a search for cell mutants that show an increase or a decrease in the frequency of baseline sister-chromatid exchanges (SCEs) or spontaneous chromosomal aberrations (CAs), large numbers of mutagen-sensitive clones previously isolated from mouse lymphoma L5178Y cells were analyzed. In addition to two SCE mutants (ES 4 and AC 12) previously reported, three other mutants were identified as an SCE mutant. An ethyl methanesulfonate-sensitive mutant ES 2 and an alkylating agent-sensitive mutant MS 1 exhibited, respectively, 1.4-fold and 1.8-fold higher baseline SCE frequencies than did the parental L5178Y. In contrast, M10, which is sensitive to X-ray and 4-nitroquinoline 1-oxide, showed a reduced frequency of baseline SCEs (0.65-fold). These 5 mutants including ES 4 and AC 12 had 3--9-fold increases in spontaneous CA frequencies. Measurement of baseline SCE formation in inter-mutant hybrids revealed that M10 mutation is dominant, MS 1 and ES 4 mutations are semidominant, and ES 2 and AC 12 mutations are recessive. Because SCE frequencies in hybrids formed between pairs of 4 mutants (ES 2, MS 1, ES 4 and AC 12) were significantly lower than those in the tetraploid mutant cells, these 4 mutants probably belong to different complementation groups. Since M10 behaved dominantly with respect to SCE phenotype, it was not possible to determine by complementation test whether it belongs to a different group from the other mutants. However, the finding that M10 is complemented by other mutants for EMS sensitivity indicates that the M10 mutation is different from the other mutations. From these results, it is concluded that at least 4 different genes participate in the formation of high levels of baseline SCEs. The defects in ES 2, MS 1, ES 4, and AC 12 produce common lesions responsible for the formation of both SCEs and CAs. In contrast, the defect in M10 is associated with a high increase in spontaneous CA frequency, but conversely associated with a decrease in baseline SCE frequency. This suggests that M10 is defective in the process involved in the formation of baseline SCEs.     

Chromosomal instability in mutagen-sensitive mutants isolated from mouse lymphoma L5178Y cells. II. Abnormal induction of sister-chromatid exchanges and chromosomal aberrations by mutagens in an ionizing radiation-sensitive mutant (M10) and an alkylating agent-sensitive mutant (MS1)

To determine the mutual relationships between cell survival and induction of sister-chromatid exchanges (SCEs) as well as chromosomal aberrations (CAs), mutagen-induced SCEs and CAs were analyzed in an ionizing radiation-sensitive mutant (M10) and an alkylating agent-sensitive mutant (MS 1) isolated from mouse lymphoma L5178Y cells. The levels of CA induction in both mutants strictly corresponded to the sensitivity to lethal effects of mutagens, except that caffeine-induced CAs in M10 are considerably lower than those in L5178Y. The results clearly indicate that except for caffeine-induced CAs in M10, mutagen-induced lethal lesions are responsible for CA induction. In contrast, SCE induction in mutants was complicated. In M10, hypersensitive to killing by gamma-rays, methyl methanesulfonate (MMS), and 4-nitroquinoline 1-oxide (4NQO), but not sensitive to UV or caffeine, the frequency of SCEs induced by gamma-rays was barely higher than that in L5178Y, and the frequencies of MMS- and UV-induced SCEs were similar to those in L5178Y, but 4NQO- and caffeine-induced SCEs were markedly lower than those in L5178Y. MS 1, which is hypersensitive to MMS and caffeine, but not sensitive to UV or 4NQO, responded to caffeine with an enhanced frequency of SCEs and had a normal frequency of MMS-induced SCEs, but a reduced frequency of UV- and 4NQO-induced SCEs. Thus, susceptibility to SCE induction by mutagens is not necessarily correlated with sensitivity of mutants to cell killing and/or CA induction by mutagens. Furthermore, the spontaneous levels of SCEs are lower in M10 and higher in MS 1 than that in L5178Y (Tsuji et al., 1987). Based on these results, we speculate that M10 may be partially defective in the processes for the formation of SCEs caused by mutagens. On the other hand, MS 1 may modify SCE formation-related lesions induced by UV and 4NQO to some repair intermediates that do not cause SCE formation. In addition, MMS-induced lethal lesions in MS 1 may not be responsible for SCE induction whereas caffeine-induced lethal lesions are closely correlated with SCE induction. Thus, the lesions or mechanisms involved in SCE production are in part different from those responsible for cell lethality or CA production.     

Disparate effects of 5-bromodeoxyuridine on sister-chromatid exchanges and chromosomal aberrations in Bloom syndrome fibroblasts

The existence of a high frequency of spontaneous sister-chromatid exchanges (SCEs) in Bloom syndrome (BS) has thus far been supported by data on a small number of BS cell lines. To examine the cause of baseline SCEs more broadly, the frequencies of SCEs, as well as chromosomal aberrations (CAs) in 4 additional BS fibroblast strains were compared, under different assay and cell culture conditions, with those of normal cells in the range of approximately 0.9-90% 5-bromodeoxyuridine (BrdUrd) substitution into template DNA. SCEs at low levels of BrdUrd substitution were detected by an extremely sensitive immunofluorescent technique. From approximately 0.9% to 4.5% BrdUrd substitution, the SCE frequency in BS cells remained constant, at a level (40/cell) 8 times higher than that of normal cells. As BrdUrd substitution increased further, the SCE frequency in BS cells increased almost linearly, reaching 70-100 per cell at approximately 90% substitution, while the SCE increment in control fibroblasts was less than 5 per cell. Analysis of SCEs in 3 successive replication cycles similarly revealed that the SCE increment in BS cells depended on BrdUrd only at a high BrdUrd substitution level. In contrast to data on SCEs, CA induction by incorporated BrdUrd in BS cells was only slightly higher than that in normal cells. Thus, BS cells are extremely sensitive to BrdUrd for SCE induction, but much less so for CA induction.     

Presence of a Base Line Level of Sister Chromatid Exchanges in Somatic Cells of DROSOPHILA MELANOGASTER IN VIVO and IN VITRO

Sister chromatid exchanges (SCEs) under in vivo and in vitro conditions were examined in ganglion cells of third-instar larvae of Drosophila melanogaster (Oregon-R). In the in vivo experiment, third-instar larvae were fed on synthetic media containing 5-bromo-2'-deoxyuridine (BrdUrd). After two cell cycles, ganglia were dissected and treated with colchicine. In the in vitro experiment, the ganglia were also incubated in media containing BrdUrd for two cell cycles, and treated with colchicine. SCEs were scored in metaphase stained with Hoechst 33258 plus Giemsa. The frequencies of SCEs stayed constant in the range of 25-150 micrograms/ml and 0.25-2.5 micrograms/ml of BrdUrd in vivo and in vitro, respectively. SCEs gradually increased at higher concentrations, strongly suggesting that at least a fraction of the detected SCEs are spontaneous. The constant levels of SCE frequency were estimated, on the average, at 0.103 per cell per two cell cycles for females and 0.101 for males in vivo and at 0.096 for females and 0.091 for males in vitro. No difference was found in the SCE frequency between sexes at any of the BrdUrd concentrations. The analysis for the distribution of SCEs within chromosomes revealed an extraordinarily high proportion of the SCEs at the junctions between euchromatin and heterochromatin; the remaining SCEs were preferentially localized in the euchromatic regions of the chromosomes and in the heterochromatic Y chromosome. These results were largely inconsistent with those of Gatti et al. (1979).     

Analysis of bromodeoxyuridine-induced single and twin sister chromatid exchanges in tetraploid Chinese hamster ovary cells

Culture of cells in high exogenous levels (>10^–4 M) of bromodeoxyuridine (BrdUrd) or thymidine will increase the baseline sister chromatid exchange (SCE) frequency. The effect is thought to be related to the balance of the DNA precursors thymidine and deoxycytidine. Exogenous addition of deoxycytidine will reverse this effect. Single and twin SCEs were analysed in Colcemid-induced tetraploid Chinese hamster ovary cells exposed to different concentrations of BrdUrd to determine at what stage SCEs are induced by high levels of BrdUrd. In cells exposed to low concentrations of BrdUrd (10^–5 M), equal numbers of SCEs were induced in each of the two cell cycles. With increasing concentrations of BrdUrd (10^–4 to 2×10^–4 M), SCE frequency increased in both cell cycles, but far more SCEs were induced in the second cell cycle. Deoxycytidine (2×10^–4 M) reduced the frequency of SCEs primarily by reducing the frequency of SCEs induced in the second cell cycle. Treatment with 3-aminobenzamide (3AB), a potent inhibitor of poly(ADP-ribose) polymerase, produced effects similar to exposure to high levels of BrdUrd including inducing SCEs in the second replication cycle. This suggests a similar mechanism of action. Deoxycytidine had no effect on 3AB-induced SCEs, however, and there was no interaction between 3AB and high exogenous levels of BrdUrd in SCE induction. Thus these two agents probably act through different mechanisms.     

Presence of abnormally high incidences of sister chromatid exchanges in three successive cell cycles in Bloom's syndrome lymphocytes

The frequencies of sister chromatid exchanges (SCEs) were examined in phytohaemagglutinin-stimulated blood lymphocytes of a normal individual, a Bloom's syndrome heterozygote (bl/+), and two Bloom's syndrome homozygotes (bl/bl). To determine the baseline SCE frequencies, lymphocytes were cultured with various concentrations of 5-bromodeoxyuridine (BrdUrd) for two cell cycles. The incidence of SCEs per two cell cycles inbl/bl lymphocytes levelled off at BrdUrd concentrations below 10 g/ml while that in normal andbl/+ lymphocytes stayed constant below 7.5 g/ml. The baseline SCE frequency in bl/bl cells was ten times higher than that in normal andbl/+ cells. At BrdUrd concentrations above 15 g/ml, SCEs inbl/bl cells were induced more frequently than in normal andbl/+ cells. These results indicate that at low concentrations BrdUrd has a minimal effect on the induction of SCEs in all individuals, while at higher concentrations the BrdUrd incorporated inbl/bl cells has a larger effect than that in normal andbl/+ cells. To elucidate the effect of BrdUrd incorporated into the daughter and parental DNA strands on SCE induction, SCEs occurring during each cell cycle were examined separately in three-way or two-way differentially stained, third-cycle metaphases. The incidence of SCEs detected in each cell cycle at 5 g/ml BrdUrd was constant in all individuals and the rates of SCEs in each cell cycle inbl/bl cells were remarkably higher than those observed in normal andbl/+ cells. These findings strongly indicate that most of the abnormally increased SCEs in thebl/bl cells used in our study occurred independently of any effect of BrdUrd incorporated into both the daughter and parental DNA strands. In addition, an abnormal response ofbl/bl cells to BrdUrd was not found for cell cycle progression or chromosomal aberration induction. Thus, the bl/bl cells did not exhibit an abnormal hypersensitivity to BrdUrd. From these results, it seems quite probable that the abnormally increased SCEs in thebl/bl lymphocytes used here were spontaneous.     

Characterization of the enhancing effect of caffeine on sister-chromatid exchanges induced by ultraviolet radiation in excision-proficient xeroderma pigmentosum lymphoblastoid cells

Cells of some excision-proficient xeroderma pigmentosum (XP) cell lines are highly sensitive to post-UV caffeine treatment in terms of sister-chromatid exchange (SCE) induction as well as cell lethality. In the present study, we conducted a detailed investigation of the enhancing effect of caffeine on SCE frequency induced by UV in excision-proficient XP cells, and obtained the following results. (1) Continuous post-UV treatment with 1 mM caffeine markedly enhances UV-induced SCEs and such enhanced SCEs occur with similar frequency during either the 1st or the 2nd cell cycle in the presence of caffeine and 5-bromodeoxyuridine (BrdUrd). (2) The high sensitivity of the cells to post-UV caffeine treatment persists for at least 2 days after UV when irradiated cells are held in either the proliferating or the nonproliferating state prior to the addition of BrdUrd. (3) Caffeine exerts its effect on cells in S phase. (4) Neither BrdUrd in the medium nor the incorporated 5-bromodeoxyuridine monophosphate (BrdUMP) in DNA plays an appreciable role in the expression of the enhancing effect of caffeine. The most likely explanation for our findings is as follows. In excision-proficient XP cells, the cause of SCE formation such as UV-induced lesions or resulting perturbations of DNA replication persists until the 2nd round or more of post-UV DNA replication. If caffeine is given as post-UV treatment, such abnormalities may be amplified, resulting in a synergistic increase in SCE frequency.     

Effect of exogenous thymidine on sister-chromatid exchange frequency in Chinese hamster ovary cells with bromodeoxyuridine- and chlorodeoxyuridine-substituted chromosomes

There are conflicting reports on the effect of exogenous thymidine (dThd) on the frequency of sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. Thymidine has been reported either to increase or to have no effect on SCE frequency under similar experimental conditions. To resolve this controversy, we have carried out a series of experiments to examine the effect of dThd on CHO cells cultured with 5-bromodeoxyuridine (BrdUrd). In addition, we have examined the effect of dThd on CHO cells cultured with 5-chlorodeoxyuridine (CldUrd), a much more potent inducer of SCEs than BrdUrd. The addition of 100 microM dThd to the culture medium caused a consistent decrease in the yield of SCEs in cells grown in BrdUrd for two cell cycles. The decrease was even greater when cells were grown in dThd and CldUrd. Analysis of twin and single SCEs indicated that dThd must be present during the first cell cycle to reduce the frequency of SCEs. Because excess dThd is thought to have an effect when DNA replicates on a template substituted with a halogenated nucleoside, dThd at concentrations from 100 microM to 9 mM was added to cultures for the second cell cycle after a first cell cycle in BrdUrd. In this experiment, the presence of dThd increased SCE frequency in a dose-dependent manner. The results suggest that if dThd competes with halogenated nucleosides and thus decreases their incorporation into DNA, SCEs are suppressed in the subsequent cell cycle, whereas if excess dThd creates a dNTP pool imbalance, SCEs can be increased.     

Cytogenetic damage in workers exposed to ethylene oxide

Sister-chromatid exchanges (SECs) and chromosomal aberrations (CAs) were detected in the peripheral lymphocytes of 41 sanitary workers exposed to ethylene oxide (EO) in the sterilizing units of 8 hospitals in the Venice Region. The first group (19 workers) was exposed to 10.7 +/- 4.9 ppm EO, expressed as the time-weighted average concentration for an 8-h working day (TWA/8 h conc.), and the second group (22 workers) to 0.35 +/- 0.12 ppm. Each exposed worker was paired with a control of similar age and smoking habits. A highly significant (P less than 0.001) increase in the mean frequency of SCEs was found in the higher exposure group, 14 (74%) exposed subjects having significantly increased levels of SCEs compared to their matched controls. In the lower exposure group, the increase in mean frequency of SCEs was lower, though still significant (P less than 0.05): 7 (33%) exposed subjects had higher and 1 (5%) had a lower SCE level than the matched controls. From the first group, 10 subjects, 7 of whom had increased SCE levels, were reanalysed 12-18 months after their exposure had been lowered or interrupted: in only 2 of them the SCE level was significantly decreased. A statistically significant correlation between SCE frequency and level of EO exposure (TWA/8 h conc.), as well as a multiple correlation between SCE level and EO exposure, smoking and age were found. However, no interaction could be detected between EO exposure and smoking in the induction of SCEs. In controls, SCE frequency was correlated with smoking and age. In the higher exposure group, the number of both chromatid- and chromosome-type aberrations, independent of gaps, was significantly increased, whereas in the lower exposure group only the frequency of chromosome-type aberrations, excluding gaps, was statistically higher than in controls. The level of CAs remained to a great extent unchanged in the 10 subjects re-examined at a later stage after lowering or halting exposure. Taking the group as a whole, the frequency of cells with total CAs was found to be weakly (P = 0.05) correlated with EO exposure, and was not correlated with smoking, age or SCE frequency.     

Both cross-links and monoadducts induced in DNA by psoralens can lead to sister chromatid exchange formation

The relative importance of DNA-DNA cross-links and bulky monoadducts in sister chromatid exchange (SCE) formation was investigated in three human fibroblast cell lines with different repair capabilities. These cell lines included normal cells, which can repair both classes of lesions; xeroderma pigmentosum (XP) cells, which cannot repair either psoralen-induced cross-links or monoadducts; and an XP revertant that repairs only cross-links and not monoadducts. SCEs were induced by two psoralen derivatives, 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and 5-methylisopsoralen (5-MIP). After activation with long-wave ultraviolet light, HMT produces cross-links and monoadducts in DNA, whereas 5-MIP produces only monoadducts. In normal human cells both psoralens induced SCEs, but if cells were allowed to repair for 18 h before bromodeoxyuridine (BrdUrd) was added for SCE analysis, the SCE frequency was significantly reduced. XP cells showed an SCE frequency that remained high regardless of whether SCEs were analyzed immediately after psoralen exposure or 18 h later. In the XP revertant that repairs only cross-links, both psoralens induced a high yield of SCEs when BrdUrd was added immediately after psoralen treatment. When XP revertant cells were allowed 18 h to repair before addition of BrdUrd, the SCEs induced by HMT were greatly reduced, whereas those induced by 5-MIP were only slightly reduced. These observations indicate that both cross-links and monoadducts are lesions in DNA that can lead to SCE formation.     

Analysis of trifluorothymidine-resistant (TFTr) mutants of L5178Y/TK+/- mouse lymphoma cells

Three classes of TFTr variants of L5178Y/TK+/- -3.7.2C mouse lymphoma cells can be identified--large colony (lambda), small colony (sigma), and tiny colony (tau). The sigma and lambda mutants are detectable in the routine mutagenesis assay using soft agar cloning. The tau mutants are extremely slow growing and are quantitated only in suspension cloning in microwells. Variants of all three classes have been analyzed in the process of evaluating the usefulness of the thymidine kinase locus in L5178Y/TK+/- mouse lymphoma cells for detecting induced mutational damage. 150 of 152 variants from mutagen treated cultures and 163 of 168 spontaneous mutants were TFTr when rechallenged approximately 1 week after isolation (3 weeks after induction). All of the 41 mutants assayed for enzyme activity were TK-deficient. The sigma and tau phenotypes were found to correlate with slow cellular growth rates (doubling time greater than 12 h), rather than from effects of the TFT selection or mutagen toxicity. Cytogenetic analysis of sigma mutants approximately 3 weeks after induction shows an association between the sigma phenotype and readily observable (at the 230-300 band level) chromosomal abnormalities (primarily translocations involving that chromosome 11 carrying the functional TK gene) in 30 of 51 induced mutants studied. Using an early clonal analysis of mutants (approximately 2 weeks after induction) 28 of 30 sigma mutants showed chromosome 11 rearrangements. All lambda mutants studied (17 of 17 evaluated 3 weeks after induction and 8 of 8 evaluated 2 weeks after induction) showed normal karyotypes (at the 230-300 band resolution level), including the chromosome 11s. These observations support the hypothesis that sigma (and likely tau) mutants represent chromosomal mutations and lambda mutants represent less extensive mutations affecting the TK locus. The inclusion of sigma mutants in the total induced mutant frequency, as well as distinguishing them as a separate subpopulation of TK-deficient mutants, is, therefore, essential in obtaining maximum utility of the information provided by the L5178Y/TK+/- mouse lymphoma assay.     

Induction of sister-chromatid exchanges and chromosomal aberrations in hematopoietic tissue of a marine fish following in vivo exposure to genotoxic carcinogens

The development of procedures to assess genetic damage in fish exposed in situ to point sources of aquatic pollution can be expected to contribute to the evaluation of the role of genotoxic contaminants in epizootic neoplasia in fish populations. To this end methods have been developed for assessing the in vivo induction of chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) in tissues of a marine teleost, the oyster toadfish, which may be applicable to other species. An alternative to the solid tissue and squash techniques for metaphase preparation permits the resolution of more than 100 SCEs/metaphase in toadfish kidney cells, which have moderately large chromosomes (0.122 pg DNA/chromosome). The bleeding of toadfish which have been injected with 5-bromodeoxyuridine (BrdUrd) and the subsequent use of hematopoietic tissue (kidney) for cytogenetic analysis was shown to increase the metaphase yield and provide a more predictable production of second-division metaphases required for SCE analysis. With these methods linear dose-dependent increases in chromatid-type exchange CAs and SCEs were obtained with i.p. exposure to ethyl methanesulfonate (EMS) and cyclophosphamide (CP). The doses required to double the observed control SCE frequencies (least effective doses) were 170 mg/kg for EMS and 7.4 mg/kg for CP. which are comparable to those reported for rodent bone marrow assays. A BrdUrd-sensitive site for chromatid breakage was observed on a pair of apparently homologous acrocentric chromosomes for the toadfish.     

Effects of CCNU therapy on human chromosomes

Peripheral blood lymphocytes of 9 patients under CCNU therapy were examined for frequency of sister-chromatid exchanges (SCEs) and chromosomal aberrations (CAs). 7 out of 9 patients were treated with only CCNU, whereas the remaining 2 were treated with other chemotherapeutic agents in combination with CCNU. Compared to normal individuals, a significantly increased frequency of SCE was observed in the patients before starting anticancer therapy (P less than 0.001). Increased incidences of structural changes in chromosomes were observed in cells from all the treated patients. The most frequent aberrations were of chromatid type. After administration of a single dose of CCNU, an increase in SCE frequencies was observed which remained elevated even after 6 weeks. It was concluded that increases in SCEs and CAs in lymphocytes were caused by CCNU treatment. Further studies are needed to elucidate whether any CAs observed in the present study could participate in the induction of second neoplasm.     

The relationship between sister-chromatid exchange and perturbations in DNA replication in mutant EM9 and normal CHO cells

The majority of the high (12-fold elevated) baseline sister-chromatid exchanges (SCEs) that occur in the CHO mutant line EM9 appear to be a consequence of incorporated BrdUrd, and they arise during replication of DNA containing BrdUrd in a template strand. In normal CHO cells the alkaline elution patterns of DNA newly replicated on a BrdUrd-containing template are significantly altered compared with those seen during the replication on an unsubstituted template. The nascent DNA synthesized on such an altered template is delayed in reaching mature size, possibly because replication forks are temporarily blocked at sites occurring randomly along the template. Transient blockage of replication forks may be a prerequisite for SCE. The delay in replication on BrdUrd-substituted templates was greater in EM9 cells than in parental AA8 cells and was also greater in AA8 cells treated with benzamide, an inhibitor of poly(ADPR) polymerase, than in untreated AA8 cells. Under these conditions, treatment with benzamide also produced a 7-fold increase in SCEs in AA8. An EM9-derived revertant line that has a low baseline SCE frequency showed less delay in replication on BrdUrd-substituted templates than did EM9. However, under conditions where the template strand contained CldUrd, which was shown to produce 4-fold more SCEs than BrdUrd in AA8 cells, the replication delay in AA8 was not any greater in the CldUrd-substituted cells. Thus, other factors besides the delay appear to be involved in the production of SCEs by the template lesions resulting from incorporation of the halogen-substituted pyrimidine molecules.     

Sister-chromatid exchanges induced by ultraviolet light in Bloom's syndrome fibroblasts

The relationships between the cytotoxic effect of ultraviolet light and the UV-induced sister-chromatid exchanges (SCEs) were compared among fibroblast cell strains from two unrelated Bloom's syndrome (BS) patients, one xeroderma pigmentosum (XP) patient belonging to complementation group A and two unrelated normal controls. The "net" induced SCEs as a function of UV fluence, obtained by subtracting spontaneous SCEs from observed SCEs, were much higher in both BS cells and XP group A cells than in normal cells. The relative efficiency of induced SCE, defined as the "net" induced SCEs as a function of surviving fraction after UV irradiation, was higher in BS cells than in normal and XP cells, and there was essentially no difference between XP and normal cells. These results imply that in addition to the extremely high frequency of spontaneous SCEs, the increased efficiency in UV induction of SCEs may reflect the intrinsic defect(s) in BS cells.     

Cytological characterization of Chinese hamster ovary X-ray-sensitive mutant cells, xrs 5 and xrs 6. II. Induction of sister-chromatid exchanges and chromosomal aberrations by X-rays and UV-irradiation and their modulation by inhibitors of poly(ADP-ribose) synthetase and alpha-polymerase

The cell killing and induction of sister-chromatid exchanges (SCEs) by X-rays and short-wave ultraviolet (UV) irradiation in combination with inhibitors of DNA repair, 3-aminobenzamide (3AB), cytosine arabinoside (ara-C) or aphidicolin (APC) were studied in wild-type CHO-K1 and two X-ray-sensitive mutants, xrs 5 and xrs 6 cells. The spontaneous frequency of SCEs was similar in the mutants and the wild-type CHO-K1 cells (8.4-10.3 SCEs/cell). Though X-rays are known to be poor inducers of SCEs, a dose-dependent increase in the frequency of SCEs in xrs 6 cells (doubling at 150 rad) was found in comparison to a small increase in xrs 5 and no increase in wild-type CHO-K1 cells. 3AB, an inhibitor of poly(ADP-ribose) synthetase increased the spontaneous frequency of SCEs in all the cell types. 3AB did not potentiate the X-ray-induced frequency of SCEs in any of the cell lines. Ara-C, an inhibitor of DNA polymerase alpha, increased the frequency of SCEs in all the cell lines. In combined treatment with X-rays, ara-C had no synergistic effect in xrs 5 and xrs 6 cells, but the frequency of SCEs increased in X-irradiated wild-type CHO-K1 cells post-treated with ara-C. For the induced frequency of SCEs, CHO-K1 cells treated with X-rays plus ara-C behaved like xrs 6 cells treated with X-rays alone, suggesting a possible defect in DNA base damage repair in xrs 6 cells, in addition to the known defective repair of DNA double-strand breaks (DSBs). Survival experiments revealed higher sensitivity of xrs 5 and xrs 6 mutant cells to the cell killing effect of X-rays in S-phase when compared to wild-type CHO-K1 cells. The mutants responded with lesser sensitivity to cell killing effect of ara-C and APC than CHO-K1 cells, the relative sensitivity to ara-C or APC being CHO-K1 greater than xrs 5 greater than xrs 6 cells. When X-irradiation was coupled with ara-C, the results obtained for survival were similar to those of the SCE test, i.e., unlike wild-type CHO-K1, no synergistic effect was observed in xrs 5 or xrs 6 cells. After UV-irradiation, the frequency of SCEs increased similarly in wild-type CHO-K1 and xrs 6 cells, but xrs 5 cells responded with lower frequency of SCEs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biomarkers of DNA damage in marine mammals.

Certain environmental contaminants found in marine mammals have been shown to cause DNA damage and cancer. The micronuclei (MN), sister chromatid exchange (SCE) and/or chromosome aberration (CA) assays were used to assess baseline (spontaneous) levels of DNA damage in blood lymphocytes of individuals of the relatively healthy and lightly contaminated Arctic beluga whale (Delphinapterus leucas), Sarasota Bay, FL, bottlenose dolphin (Tursiops truncatus) and Northwestern Atlantic grey (Halichoerus grypus) and harp (Phoca groenlandicus) seal populations. MN cell (MNC) frequencies ranged between 2 and 14/1000 binucleated (BN) cells and were statistically similar between species. In bottlenose dolphins, MNC frequency was correlated with age and was significantly higher in females than in males. No intraspecific variation in MNC frequency was found in beluga whales. Intraspecific variation was not tested in seals due to the small sample size. Frequencies of SCEs and total CAs, excluding gaps, ranged, respectively, between 1 and 15 SCE(s)/per cell and 4-6 CAs/100 cells in beluga whales. SCE and CA frequencies did not vary with age or sex in beluga whales. The MN, SCE and CA assays were found to be practical tools for the detection of DNA damage in marine mammals and could be used in the future to compare DNA damage between relatively lightly and highly contaminated populations.     

Incorporated bromodeoxyuridine enhances the sister-chromatid exchange and chromosomal aberration frequencies in an EMS-sensitive Chinese hamster cell line

The mutant Chinese hamster cell line, EM9, is characterized by a high baseline sister-chromatid exchange (SCE) frequency, increased sensitivity to cell killing, and a defect in DNA strand-break repair. The molecular basis for this pleotrophic phenotype is not known. We examined, at the chromosomal level, the increased sensitivity of this mutant to incorporated BrdUrd. By varying the amount of BrdUrd in template DNA and measuring the frequency of SCEs and chromosomal aberrations, we demonstrated the enhanced sensitivity of EM9 to BrdUrd present in the template strand of DNA. Our results show that a 6-fold increase in SCEs occurs due to DNA replication over a BrdUrd-substituted template relative to a dThd-substituted template. With regard to aberration production in EM9, there is a significant enhancement of aberrations and a specific bias toward damage for the chromatid with Brdurd in the template strand. While these cells share some phenotypic properties with cells from patients with Bloom's syndrome, the genotypic similarities have not yet been established.     

Influence of incorporated 5-bromodeoxyuridine on the frequencies of spontaneous and induced sister-chromatid exchanges, detected by immunological methods

We have utilized monoclonal antibody against BrdUrd to detect sister-chromatid exchanges in CHO cells. This technique allows detection of SCEs at very low levels of BrdUrd incorporation. At incorporation level of 0.5%, a frequency of about 2 SCEs/cell/cycle was found. In a UV-sensitive mutant (43-3B) which has an increased spontaneous frequency of SCEs, it is found that this increase is due to incorporated BrdUrd. In MMS- and MMC-treated cells, an influence of BrdUrd on the frequencies of induced SCEs was found only when high concentrations of mutagens were employed.