Antipain-mediated suppression of sister chromatid exchanges induced by an inhibitor of poly (ADP-ribose) polymerase

Exposure of mammalian cells to inhibitors of poly(ADP-ribose) polymerase, such as 3-aminobenzamide (3AB) results in the induction of sister chromatid exchanges (SCEs). The mechanism for the induction of SCEs by 3AB is unknown but is thought to be related to the incorporated halogenated pyrimidine used in SCE analysis. In this characteristic, 3AB-mediated SCE induction is similar to the elevated SCE frequency found in Bloom's syndrome (BS) cells. Recently, it has been reported that certain protease inhibitors, such as antipain, will inhibit SCE induction in BS cells. We now report that antipain will also suppress 3AB-induced SCE frequency. As has been reported for BS cells, the effects of antipain on SCE induction are partial, reducing SCE frequency by 0.15 to 0.40 SCE/chromosome (5-25% of the total induced frequency), and 30 microM concentrations of antipain are saturating. Antipain has no effect on baseline SCE frequency. These effects appear to involve free-radical production because dimethylsulfoxide (DMSO), a free-radical scavenger, will mimic the effects of antipain on 3AB-induced SCEs. Both antipain and DMSO will also reduce the elevated SCE frequency found in cells exposed to high (100 microM or more) levels of bromodeoxyuridine (BrdUrd). High exogenous levels of BrdUrd produce some of the same biological effects as 3AB exposure. Thus, a minor fraction of the elevated SCE frequency seen in cells exposed to 3AB or to high levels of BrdUrd appears to be similar to that found in cultured BS cells and is probably due to some free-radical-producing process.     

Induction of sister chromatid exchanges and inhibition of cellular proliferation in vitro. I. caffeine

Cytogenetic assay systems based on the detection of sister chromatid exchanges (SCE) are widely advocated as a sensitive screening method for assessing genotoxic potential. While many agents have been examined for their ability to induce SCE's, complete dose-response information has often been lacking. We have reexamined the ability of one such compound — caffeine — to induce SCEs and also to inhibit cellular proliferation in human peripheral lymphocytes in vitro. An acute exposure to caffeine prior to the DNA synthetic period did not affect either SCE frequency or the rate of cellular proliferation. Chronic exposure to caffeine throughout the culture period lead to both a dose-dependent increase in SCEs (SCE_d or doubling dose = 2.4 mM; SCE₁₀ or the dose capable of inducing 10 SCE = 1.4 mM) and a dose-dependent inhibition of cellular proliferation (IC₅₀ or the 50% inhibition concentration = 2.6 mM). The relative proportion of first generation metaphase cells, an assessment of proliferative inhibition, increased linearly with increasing caffeine concentrations. However, SCE frequency increased nonlinearly over the same range of caffeine concentrations. Examination of the ratio of nonsymmetrical to symmetrical SCEs in third generation metaphase cells indicated that caffeine induced SCEs in equal frequency in each of three successive generations. The dependency of SCE induction and cellular proliferative inhibition on caffeine's presence during the DNA synthetic period suggests that caffeine may act as an antimetabolite in normal human cells. The significance of these results in regard to both caffeine's genotoxic potential and to the reliability of the SCE assay system are discussed.     

Sister chromatid exchange distributions in rabbit lymphocytes treated with streptonigrin

Streptonigrin (NSC-45383), a direct-acting clastogen which induces SCEs in vivo and chromosome aberrations both in vivo and in vitro, was evaluated for SCE induction in both G₀ and stimulated rabbit lymphocytes. Determinations were made for 16 cultures from seven female rabbits. These included controls as well as cells exposed to 90 μg/kg in vivo, cells pulse-treated with 50 ng/ml in vitro, and a culture continuously exposed to 5 ng/ml in vitro. For all cultures the SCE/ cell frequency was determined from 20 complete (44 chromosome) metaphases and, in selected cultures, SCEs on individual chromosomes (880 per culture from 20 cells) were enumerated to determine SCE/chromosome frequency and the chromosomal distribution of SCEs. Analysis of variance and least significant difference tests of the √x transformed SCE/cell data show that cells exposed to Streptonigrin while dividing have significantly higher (P<0.01) frequencies (over double the control 5.3 SCE/cell value) whereas treated G₀ cells were not significantly different from the controls. Dispersion analysis of both SCE/cell and SCE/ chromosome data confirms the adequacy of the Poisson distribution for spontaneous or baseline but not streptonigrin-induced SCEs.     

Specificity of arsenite in potentiating cytogenetic damage induced by the DNA crosslinking agent diepoxybutane.

In the present study, the induction of sister chromatid exchanges (SCEs) and chromosomal aberrations were measured in normal human lymphocytes treated with low concentrations of arsenite alone (0.5-2.0 microM) and arsenite in combination with the potent DNA crosslinking agent diepoxybutane (DEB). Experiments were carried out with lymphocytes from blood donors with different sensitivities to SCE induction by DEB. Arsenite, beginning at concentrations as low as 1 microM, increased SCE frequencies; chromosomal aberration frequencies were increased at 2 microM of arsenite. DEB treatments alone increased SCE frequencies and chromosomal aberrations. The yields of chromatid deletions and exchanges in lymphocytes exposed to both arsenite and DEB were markedly increased above the levels expected if the effects of the two agents had been simply additive. The frequencies of chromatid deletions were 4- to 8-fold greater than expected and chromatid exchanges were increased 7- to 40-fold. Chromatid exchanges detected in cells treated with arsenite and DEB were predominately incomplete exchanges. The most dramatic increases in chromatid aberrations were observed in lymphocytes from an individual sensitive to SCE induction by DEB, indicating that individuals may vary in their sensitivity to the co-clastogenic effects of arsenite. At concentrations that dramatically affect aberrations, arsenite had no effect on the induction of SCEs by DEB. These studies suggest a specific interaction of arsenite with the induction or repair of DNA damage produced by DEB that leads to chromosomal aberrations but not to SCEs. Based on the selective chemical reactivity of low concentrations of arsenite with proteins containing vicinal dithiols and the occurrence of these groups within DNA repair proteins, it is proposed that the specific co-clastogenic effects of arsenite may be mediated by its interference with DNA repair activities.     

Low frequency noise and whole-body vibration cause increased levels of sister chromatid exchange in splenocytes of exposed mice

Chronic exposure to low frequency (LF) noise and whole-body vibration (WBV) induces both physiological and psychological alterations in man. Recently, we have shown that long-term occupational exposure to LF noise and WBV produces genotoxic effects in man expressed as an increase in sister chromatid exchange (SCE) levels in lymphocytes. The objectives of the present study were to investigate whether the observed effect could be reproduced in a murine model and, if so, which of the agents, LF noise alone or in combination with WBV, would be instrumental in the SCE induction. SCEs were analyzed in spleen lymphocytes of mice exposed to LF noise alone and in combination with WBV for 300 and 600 hr. An effect at the cell cycle kinetics level was also investigated. The results revealed significant increases in the mean SCE number per cell and in the proportion of cells with high frequency of SCEs (HFCs) in lymphocytes of mice submitted to combined noise and WBV over controls. No significant differences were found between single noise-exposed and control mice. A cell cycle delay was observed exclusively in the noise and WBV exposure groups. In conclusion, we demonstrated that, as in exposed workers, prolonged exposure to the combination of LF noise and WBV determines an increase in SCE level in mice while LF noise alone is not effective in SCE induction.     

Genetic effects of 2-aminopurine in mammalian cells

The effects of 2-aminopurine (APur) on mutations, sister-chromatid exchanges (SCEs) and proliferation were investigated in V79 cells by means of cytogenetic and flowcytometric experiments. APur did not induce SCEs after a 3-h treatment before the addition of BrdUrd but SCE frequencies were increased after treatment for two cell cycles in the presence of BrdUrd. SCEs were mainly produced during the second cell cycle of the SCE experiment when BrdUrd substituted DNA is replicated. APur also caused a high percentage of polyploid cells. Compared on the basis of DNA content, SCE induction was the same in diploid and tetraploid metaphases. APur-induced SCEs are strongly influenced by nucleosides. The presence of deoxycytidine (dCyd) caused a reduction of AP-induced SCEs to about control level while addition of deoxythymidine (dThd) enhanced SCE induction. Flow cytometric measurements revealed a small increase in S-Phase cells and a strong accumulation in G2/M after APur treatment in the presence of BrdUrd. S-phase delay was strongly enhanced when BrdUrd substituted DNA is replicated. Addition of dCyd removed the APur-induced inhibition of S-phase in both protocols. Using the same treatment protocol, APur also induced mutations at the HPRT locus. In contrast to their effects on SCEs and proliferation neither BrdUrd nor dCyd had an effect on APur-induced mutations, and dThd reduced the mutation frequency. The results demonstrate that APur-induced SCEs and mutations occur independently from each other. APur-induced mutations obviously occur by a mispairing mechanism while SCEs are a consequence of pool imbalances during replication.     

Effect of BrdUrd and low doses of gamma radiation on sister chromatid exchange,chromosome breaks,and mitotic delay in mouse bone marrow cells in vivo

We have measured, in mouse bone marrow cells in vivo, the ability of low doses of gamma radiation to induce sister chromatid exchanges (SCEs) in unifilarly 5-bromodeoxyuridine (BrdUrd)-substituted DNA. Also examined was the effect of BrdUrd incorporation on SCE induction by radiation, the comparative frequency of SCE and chromosome breaks induced by gamma radiation, the ability of ionizing radiation to interfere with normal cellular proliferation, and the relationship between proliferative inhibition and SCE and chromosome break frequency. A direct relationship between the number of SCEs and gamma radiation dose was observed, an effect which was dependent on BrdUrd incorporation. The frequency of SCE was 80 times higher than that of chromosome aberrations in cells with BrdUrd-substituted DNA, and there was no difference between the frequency of SCE in cells with or without chromosome breaks. The mitotic delay increased with the time between irradiation and harvesting. There was no relationship between the extent of mitotic delay and the number of SCEs.     

Contribution of O6-alkylguanine and N-alkylpurines to the formation of sister chromatid exchanges,chromosomal aberrations,and gene mutations: New insights gained from studies of genetically engineered mammalian cell lines

O⁶-methyl- and O⁶-ethylguanine are the major premutagenic and precarcinogenic lesions induced in DNA by monofunctional alkyiating agents, albeit formed in minor amounts. The involvement of these lesions in SCE and aberration formation is less clear. We have analyzed the contribution of O⁶-alkylguanine to SCE and aberration formation, as well as its toxic and point mutation inducing effect in transgenic Chinese hamster ovary (CHO) cell lines that express variable amounts of human O⁶-methylguanine-DNA methyltransferase (MGMT). Cells that overexpress MGMT (or the bacterial Ada protein) gained resistance to the formation of alkylation-induced SCEs and aberrations, as compared to MGMT deficient cells. A correlation was apparent between the level of protection for SCEs and cell killing, indicating that both phenomena are interrelated. The protective effects were dependent on the level of MGMT expression, the agent used for alkylation, and cell cycle progression. Our data suggest that at least 2 kinds of lesions are responsible for SCE and aberration formation, namely, O⁶-alkylguanine and one or various N-alkylation products. The probability that O⁶-methylguanine is converted into cytogenetic effects has been estimated to be about 1:30 for SCEs, and 1:147,000 and 1:22,000 for chromosomal aberrations in the first and second post-treatment mitosis, respectively. The induction of SCEs and likely also of aberrations by O⁶-methylguanine requires two replication cycles and is supposed to involve the formation of secondary DNA lesions. Increased repair of 3-methyladenine and 7-methylguanine in CHO cells that overexpress the N-methylpurine-DNA glycosylase (MPG) after transfection with the human MPG-cDNA did not give rise to protection against methylation-induced SCEs and aberrations, probably because of incomplete excision repair. MPG overexpressing cells reacted even more sensitively to methylating agents, suggesting apurinic sites formed as a result of MPG action to be SCE and aberration-inducing lesions. © 1993 Wiley-Liss, Inc.     

Sister chromatid exchange induced by short-lived monoadducts produced by the bifunctional agents mitomycin C and 8-methoxypsoralen

To see if DNA crosslinks are involved in the induction of sister chromatid exchange (SCE), Chinese hamster ovary cells were exposed to two bifunctional alkylating agents, mitomycin C and 8-methoxypsoralen, and their monofunctional derivatives, decarbamoyl mitomycin C and angelicin. The data indicate that monoadducts, rather than crosslinks, are responsible for SCE formation. Furthermore, all agents but angelicin produced short-lived lesions that led to SCEs in the first period of DNA replication after treatment (twin SCEs), but not in the second (single SCEs). In contrast, angelicin, like methyl methanesulfonate and N-acetoxyacetylaminofluorene, produced lesions that lasted more than one cycle, indicating that several different types of DNA lesions are capable of SCE induction.     

SCE induction is uncoupled from mutation induction in mammalian cells following exposure to ethylnitrosourea (ENU)

It has been suggested not only that sister chromatid exchange (SCE) induction might serve as a qualitative indicator of mutagenesis, but also that induced SCE frequencies are linearly related to induced mutation frequencies. The consistency of the relationship between SCEs and mutations was tested in the present work. Confluent Chinese hamster ovary (CHO) cells were exposed to ethylnitrosourea (ENU) and then held at confluency for various times prior to initiation of SCE and mutation assays. Cells held at confluency are typically thought to be arrested in their progression through the cell cycle, so that "S-dependent" processes such as fixation of mutations and formation of SCEs will not occur, while DNA repair processes might continue to operate. If repair processes reduce the number of SCE-inducing lesions during the holding period and, hence, reduce the subsequently determined SCE frequencies, then mutation frequencies should similarly be reduced if SCEs and mutations are related. It was observed, however, that induced SCE frequencies decreased exponentially with holding time, while mutation frequencies remained constant. Qualitatively similar results were obtained in log-phase cells. Cell cycle analysis demonstrates that confluent CHO cells can cycle, and ways are considered in which this might affect SCE and mutation frequencies. It is concluded that the decline in SCE frequency (with time) cannot be attributed solely to the presence of cycling cells in confluent cultures. It appears, therefore, that at least some forms of ENU-induced DNA damage that can lead to SCE were repaired and as such are distinct from those forms that are mutagenic. Thus SCEs are not necessarily related to mutations, because the two events may represent manifestations of different forms of DNA damage. Whether or not this represents a universal phenomenon that would hold true for agents other than ENU remains to be determined.     

Induction of sister chromatid exchanges by coal dust and tobacco snuff extracts in human peripheral lymphocytes

The organic solvent extracts of sub-bituminous coal dust and tobacco snuff, both together and separately, were tested for the induction of sister chromatid exchanges (SCEs) in human peripheral lymphocytes. The results indicate that these extracts induced SCEs, and that when tested together synergistically induced SCEs in two of three donors. Studies with the organic solvent extracts of all five ranks of coal indicate that the extracts of bituminous, lignite, and peat, but not anthracite, induced SCEs. Similar experiments conducted with water extracts show that bituminous, lignite, and peat, but not sub-bituminous extracts, induced SCEs, and that anthracite was equivocal. To determine whether individuals differed in their SCE responses to coal dust extracts, lymphocytes from five donors were tested with organic solvent extracts of bituminous and sub-bituminous coal. An analysis of variance indicates that the SCE response was significantly influenced by the donor (p less than 0.0001) and each of the two coal extracts (p less than 0.0001). From studies of workers occupationally exposed to coal dust, it is known that inhaled coal dust is cleared from the lungs by mucociliary action and introduced into the stomach by swallowing. Coal dust, or coal dust plus snuff, may be responsible for the increased frequency of gastric cancer observed in coal miners. The findings presented here suggest that coal dust, with or without tobacco snuff, may play a role in the elevated incidence of gastric cancer in coal miners. Because water extracts of some ranks of coal induced SCEs, there exists the possibility of adverse environmental effects due to coal leachates.     

Isolation of temperature-sensitive mouse FM3A cell mutants exhibiting conditional chromosomal instability

Fourteen mutants exhibiting conditional induction of sister chromatid exchanges (SCEs) and/or chromosomal aberrations (CAs) were selected out of 65 temperature-sensitive (for growth) mutants from mouse FM3A cells treated with N-methyl-N-nitro-N-nitrosoguanidine. The mutants with chromosomal abnormality at the nonpermissive temperature were classified into three groups. The group 1 mutant manifested mainly SCEs at the nonpermissive temperature. The group 2 mutants manifested both SCEs and CAs. The group 3 mutants, including ts-131b, a mutant defective in DNA replication and previously described, manifested only CAs. A substantial number of SCEs accompanied by CAs at the site of SCEs were observed in some of the group 2 mutants at the nonpermissive temperature. The results suggest that there are at least three processes involved in the formation of SCEs and CAs, one is common to both phenomena while the others are independent. Some other mutants belonging to the groups 1 and 2 displayed a high incidence of interchromosomal chromatid exchanges at the nonpermissive temperature, suggesting that the same genetic defects leading to high induction of SCEs correlate with induction of chromosomal rearrangements. From these results, the group 2 mutants were subdivided further into five classes, and the mutants in each class had different cytogenetic properties from one another. This indicates defects in at least several genes that participate in the production of SCEs or CAs. The mutants described should be useful for analyzing the mechanisms of SCE or CA formation, and also the mechanism of chromosomal rearrangements.     

Evidence that SCEs induced by mutagens do not occur at the same locus in successive cell cycles: Lack of cancellation in three-way stained CHO chromosomes

An approach based on the synchronization of CHO cells after a first cell cycle incorporating a relatively low amount of bromodeoxyuridine (BrdUrd) into DNA, followed by mutagenic treatment and subsequent culture for second and third generations of BrdUrd incorporation for the scoring of sister chromatid exchanges (SCEs) per cell cycle in three-way differentially (TWD) stained chromosomes, has been used to investigate the possible concellation of SCEs. Cancellation is expected to occur if two mutagen-induced SCEs occur at exactly the same site in subsequent rounds of replication. Lesions in DNA seem to persist and are able to induce SCE throughout two cell cycles after treatment with the three mutagens tested—mitomycin C (MMC), ethyl methanesulfonate (EMS) and ultraviolet (UV) light—though this latter agent was shown as only moderately persistent. Our results seem to indicate that SCEs induced by these mutagens do not take place at the same locus in successive cell generations, as assessed by a lack of SCE cancellation. © 1994 Wiley-Liss, Inc.     

Trans-stilbene oxide-induced sister chromatid exchange in cultured human lymphocytes: influence of GSTM1 and GSTT1 genotypes

About 50% and 15% of Caucasians lack the glutathione S-transferase M1 (GSTM1) and T1 (GSTT1) genes and the corresponding enzyme activity, respectively. Both of these polymorphisms have been shown to affect the genotoxicity of some epoxides in cultured human lymphocytes. Especially GSTT1 appears to be important in whole-blood cultures, probably because GSTT1 activity is high in erythrocytes. The in vitro genotoxicity of trans-stilbene oxide (TSO), a model substrate for GSTM1, has been shown to depend on individual GSTM1 activity. The potential role of GSTM1 genotype, and the possible interference of GSTT1 genotype, has not previously been examined in this context. We have studied TSO-induced sister chromatid exchanges (SCEs) in 72 h whole-blood lymphocyte cultures from 24 healthy human donors, representing different combinations of GSTM1 and GSTT1 positive and null genotypes. TSO clearly increased SCEs in cultures of all donors. The mean number of SCEs per cell induced by 75 and 150 microM TSO was, respectively, 1.5- and 1.3-times higher in cultures of GSTM1 null than GSTM1 positive donors. In another experiment, GSTM1 null individuals showed, in comparison with GSTM1 positive subjects, a 1.8-fold SCE induction by 50 microM TSO. GSTT1 genotype did not have an unequivocal effect. Our findings suggest that the lack of the GSTM1 gene, resulting in reduced detoxification capacity, increases individual sensitivity to the genotoxic effects of TSO.     

Chromosome aberration and sister chromatid exchange tests in Chinese hamster ovary cells in vitro. V: Results with 46 chemicals

Forty-six coded chemicals were tested for their ability to induce sister chromatid exchanges (SCEs) and chromosomal aberrations (ABs) in cultured Chinese hamster ovary (CHO) cells using a standard protocol with and without exogenous metabolic activation. Sixteen chemicals were negative and 15 were positive in both assays; 15 were positive for SCEs only (one chemical that was positive for SCEs was equivocal for ABs), and no chemicals induced ABs only. The effect of cell harvest time on the ability to detect the induction of ABs was examined for 18 chemicals. Seven chemicals produced a positive response using both standard and extended harvest times, five were positive only using an extended harvest time, and six were negative using both harvest times. The relationship between cell cycle delay and SCE induction was also examined, and the two appear to be unrelated.     

No significant increase in spontaneous and ethyl methane sulfonate-induced sister chromatid exchanges at the Xq27.3 fragile site

The present study was performed to determine if the fra(X)(q27.3) site is inherently a high sister chromatid exchange (SCE) site independent of fragility. Therefore, we studied baseline and ethyl methane sulfonate (EMS)-induced SCEs in peripheral blood lymphocytes from 10 retarded fragile-X male patients and eight retarded nonfragile-X male controls. The distributions of SCE scores per chromosome within each experimental condition showed significant interindividual variability in response to EMS treatment in the fragile-X group. Each fragile-X patient was therefore compared with a matched control. No significant differences were found in the distribution of SCE scores per chromosome. In addition, at the Xq27 site, whatever the degree of expressed fragility, no significant differences between fragile-X and control groups were evident in the spontaneous or EMS-induced SCEs. The fra(X)(q27.3) site therefore is not a hot spot for spontaneous or EMS-induced SCEs. Because fluorodeoxyuridine (FUdR) treatment has been shown to induce SCE at this site, our results indicate that the Xq27.3 site must be structurally different from other nonfragile SCE sites.     

Evaluation of sister chromatid exchange as an indicator of sensitivity to N-ethyl-N-nitrosourea-induced carcinogenesis in rats

Sister chromatid exchange (SCE) frequencies in peripheral lymphocytes are a frequently used endpoint to indicate exposures to genotoxins in groups of humans. The aim of this study was to ascertain, in an experimental design, whether or not SCE rates have any association with the risk of cancer at the individual level in rats exposed to a known carcinogen. Individual SCE rates were determined in three consecutive analyses in cultured blood lymphocytes of 50 adult male Wistar rats. Analyses were done before as well as 24 hr and 7 days after a single intraperitoneal administration of 0, 25, 50, or 75 mg/kg of N-ethyl-N-nitrosourea (ENU). The animals were followed until death; also, the relationship between SCEs and carcinogenic outcome, i.e., the presence or absence of tumors, and their latency period were examined. ENU significantly decreased the life expectancy of the rats. The tumor types most clearly associated with ENU treatment were various gliomas and thyroid-gland and testicular tumors. ENU induced a moderate (maximally 1.6-fold) increase in the mean frequency of SCEs/cell at both sampling times after treatment. The effect was somewhat more pronounced 1 day rather than 1 week after treatment. The mean SCE rates in rats with ENU-specific cancers or in animals with early or multiple tumors did not differ from those in animals that survived no less than 65 weeks or longer without developing tumors. In ENU-treated animals with tumors, no relationship was found between the mean SCE rate and survival time. It is concluded that in outbred Wistar rats the SCE response in cultured lymphocytes does not indicate individual susceptibility to the carcinogenic action of ENU. On a group basis, however, animals with high SCE rates were shown to have increased risk of cancer.     

Sister chromatid exchange induction and persistence in peripheral blood and spleen lymphocytes of mice treated with ethylnitrosourea

The induction and persistence of sister chromatid exchanges (SCEs) were studied in peripheral blood and spleen lymphocytes of mice given a single i.p. injection of ethylnitrosourea (ENU) of 100, 350, or 600 muMoles ENU/kg. SCE frequencies were measured on days 1, 3, 5, and 7, and at seven additional times up to 172 days post-injection. SCEs were analyzed statistically by comparing the mean frequencies as well as the distribution of SCEs per cell at each time. The latter approach was based on a non-parametric method of identifying high frequency cells (HFCs). The SCE frequencies and proportion of HFCs in each dose and tissue remained elevated for up to 172 days following treatment, although the degree and statistical significance of the increase varied according to the tissue, dose, and statistical test employed. The SCE frequencies were found to oscillate during the first week. Following this, however, the return of the SCE frequencies to control levels was fit to a linear regression model with time as the only independent variable. The persistence of SCE-forming lesions was found to be dose-dependent for the spleen but not for blood. Within each dose the persistence of SCE-forming lesions was significantly greater for the blood relative to the spleen. The results emphasize that tissue, dose, and time since exposure are important factors to consider when quantifying SCEs in vivo; analysis of high frequency cells may be a more sensitive method of detecting exposure than the t-test; and a single determination of SCE frequencies may not be sufficient to quantitatively assess genotoxic damage in the first week following exposure.     

Reduction of sister chromatid exchange frequency with time after mutagen exposure in chinese hamster ovary cells in the presence of 3-aminobenzamide

3-Aminobenzamide (3AB) is a potent inhibitor of poly(ADP-ribose) synthesis and has been reported to inhibit DNA repair. Inhibition of poly(ADP-ribose) synthesis by 3AB results in a potentiation of the cytotoxic effects of alkylating agents and synergistically increases the frequency of sister chromatid exchanges (SCEs) induced in Chinese hamster ovary cells by methyl methanesulfonate (MMS). Experiments were carried out to determine if this synergism was related to an inhibition of the repair or the removal of SCE-forming lesions by 3AB. Cells were treated with MMS or ethyl nitrosourea (ENU) and either held in a nonproliferative state or maintained in exponential growth for up to 48 hr before SCE frequencies were measured. During this time, the SCE frequency decreased greatly. 3AB did not affect this decrease in either cycling or noncycling cells. Furthermore, 3AB appears to exert its effect only in cycling cells when bromodeoxyuridine is present. Therefore, the synergism between 3AB and alkylating agents in the induction of SCEs is unrelated to effects of 3AB on the repair or removal of SCE-forming lesions.     

The in vivo induction of sister chromatid exchange by the demethylating agent 5-aza-2'-deoxycytidine

Previously, we observed that the demethylating agent 5-azacytidine (azaC) induces a constant and limited low frequency of sister chromatid exchange (SCE), seemingly due to a limited number of SCE-prone sites whose expression is related to DNA demethylation. An alternative explanation for the low frequency of SCE induction may be its inefficient incorporation into DNA, as it has a higher incorporation into RNA. The aim of the present work is to determine if the frequency of SCE induction is increased after exposure to 5-aza-2'-deoxycytidine (azadC), a compound with the same mechanism of demethylation as azaC but more efficiently incorporated into DNA. Groups of mice were treated with 2.2, 4.4, 6.6 and 8.8 μmol azadC per kilogram body weight, and the SCE frequency, the mitotic index and the average generation time were determined after two cell division cycles. The dose-response data of SCE induction showed two components: (i) a dose-dependent increase between 0 and 4.4 μmol and (ii) almost a same level of two SCEs per cell at 4.4 and 8.8 μmol. Although azadC is incorporated more efficiently into DNA, as shown by a lower dose required for a maximal effect, the highest frequency of SCE induction is similar to that observed with azaC. These results indicate that the low incorporation of azaC into DNA seems not to be the factor that limits SCE induction, but the limited number of specific SCE-prone sites in demethylated DNA. Perhaps, there are a restricted number of sites prone to homologous recombination due to DNA demethylation.