Changes of 8-OH-dG levels in DNA and its base excision repair activity in rat lungs after inhalation exposure to hexavalent chromium

The co-genotoxic effects of cadmium are well recognized and it is assumed that most of these effects are due to the inhibition of DNA repair. We used the comet assay to analyze the effect of low, non-toxic concentrations of CdCl2 on DNA damage and repair-induced in Chinese hamster ovary (CHO) cells by UV-radiation, by methyl methanesulfonate (MMS) and by N-methyl-N-nitrosourea (MNU). The UV-induced DNA lesions revealed by the comet assay are single-strand breaks which are the intermediates formed during nucleotide excision repair (NER). In cells exposed to UV-irradiation alone the formation of DNA strand breaks was rapid, followed by a fast rejoining phase during the first 60 min after irradiation. In UV-irradiated cells pre-exposed to CdCl2, the formation of DNA strand breaks was significantly slower, indicating that cadmium inhibited DNA damage recognition and/or excision. Methyl methanesulfonate and N-methyl-N-nitrosourea directly alkylate nitrogen and oxygen atoms of DNA bases. The lesions revealed by the comet assay are mainly breaks at apurinic/apyrimidinic (AP) sites and breaks formed as intermediates during base excision repair (BER). In MMS treated cells the initial level of DNA strand breaks did not change during the first hour of recovery; thereafter repair was detected. In cells pre-exposed to CdCl2 the MMS-induced DNA strand breaks accumulated during the first 2h of recovery, indicating that AP sites and/or DNA strand breaks were formed but that further steps of BER were blocked. In MNU treated cells the maximal level of DNA strand breaks was detected immediately after the treatment and the breaks were repaired rapidly. In CdCl2 pre-treated cells the formation of MNU-induced DNA single-strand breaks was not affected, while the repair was slower, indicating inhibition of polymerization and/or the ligation step of BER. Cadmium thus affects the repair of UV-, MMS- and MNU-induced DNA damage, providing further evidence, that inhibition of DNA repair is an important mechanism of cadmium induced mutagenicity and carcinogenicity.     

The role of N-nitrosourea-induced changes in the nucleoid structure and activity of repair enzymes in the development of drug resistance in mice with leukemia L1210

Using centrifugation of the nucleoid in a neutral sucrose gradient, the damages in the secondary structure of DNA and the activity of repair enzymes, such as DNA-polymerases alpha and beta and poly(ADP-riboso) polymerase, induced by 1-methyl-nitrosourea (MNU) and 1.3-bis (2-chloroethyl)-1-nitrosourea (BCNU) injected at maximal nonlethal single doses to mice bearing parent leukemia cells (L1210/0) and resistant to MNU and BCNU leukemia L1210 cells (L1210/MNU and L1210/BCNU), were studied. The MNU-induced production of single-strand breaks in L1210/0 and L1210/MNU cells was more conspicuous in newly replicated DNA than in those in preexisting DNA. A more fast repair of the damages in newly replicated DNA was detected in L1210/BCNU and especially in L1210/MNU leukemia cells as compared with L1210/0 cells. The data obtained suggest that there are prone errors in the repair of DNA template, since most of the single-strand breaks were revealed in the newly replicated DNA synthesized on the repaired DNA. The repair of DNA damages in L1210/BCNU and especially in L1210/MNU cells was accompanied by the activation of DNA-polymerases alpha and beta and poly(ADP-riboso)polymerase. Both DNA-polymerases--alpha and beta--were shown to be involved in repair of DNA damages induced by MNU and only DNA-polymerase beta was involved in the repair of damages induced by BCNU.     

Mechanisms of radioresistance in terminally differentiated cells of mature retina

Retinopathy of animals is induced by many agents damaging DNA. This fact shows that DNA lesions may initiate retinal degeneration. The aim of our work was to study the effects of gamma and proton irradiation, and methylnitrosourea (MNU) on mice retina. We evaluated morphological changes, DNA damage and repair in retina, and expression of 5 proteins participating in apoptosis: p53, ATM, FasR, PARP and caspase 3 active. Dose of 14 Gy is equitoxic in terms of induction of DNA single strand breaks by both gamma and proton irradiation. But protons were 2 fold more effective than gamma-rays in induction of DNA double strand breaks. All breaks were repaired within < or =10 h. Irradiation resulted in increased expression of p53 and ATM. But no sings of cell death and retinal degeneration were observed during 7 days after irradiation. Proton irradiation in dose of 25 Gy resulted in increasing over time destructive changes localized mainly in photoreceptor layer of retina. These changes were followed by increased expression of proapoptotic proteins. A single systemic administration of MNU (70 mg/kg) increased intracellular levels of p53, PARP, FasR, caspase 3 active, which was followed by destructive changes in retina with sings of apoptosis of photoreceptors. As in the case of irradiation, the 2-fold dose reduction of MNU abrogated cytotoxic effect of MNU on retina. High level of spontaneous DNA damage such as apurine and apyrimidine sites were observed in mouse retina. The results of our study demonstrate the occurrence of genotoxic threshold in the initiation of retinal cell death in vivo. Topoisomerase 2 of retina is suggested to translate primary DNA damage to cytotoxic effect.     

Effect of B polA1- exrA- and recA-gene mutations on the reparation of single-strand DNA breaks induced by N-nitrosomethylurea]

The effect of different doses of N-methyl-N-nitrosourea (MNU) on a viability of bacterial cells with different defects in the systems of repair of UV-damages, and the MNU induction of single-strand DNA breaks (SS) were studied. The kinetics of both processes was investigated. There was a good correlation between the NMU sensitivity of bacterial cells and the number of SS in their DNAs. The most sensitive were the cells defective in DNA polymerase I. The optimal conditions for DNA repair in the strains under investigation were established. 90% of MNU-induced SS are repaired by DNA polymerase I and do not depend on protein synthesis. On the other hand, the exrA and recA dependent ways of SS repair depend on protein synthesis. The existence of an inducible recAexrA-dependent repair system of NMU-induced lesions in bacterial DNA is proposed.     

The formation and repair of single-strand breaks in DNA of cultured mammalian cells treated with UV-light, methylating agents or 4-nitroquinoline-1-oxide.

The technique of sedimentation in alkaline sucrose was used to examine the formation and repair of single-strand (SS) breaks in cultured mammalian cells that were treated with methyl methanesulfonate (MMS), methyl nitrosourea (MNUA), 4-nitroquinoline-1-oxide (4NQO) or UV-light. The SS breaks induced by MMS and 4NQO were largely repaired by HeLa cells during a 5-h post-treatment incubation. The SS breaks induced by MNUA and UV-light were not repaired by HeLa cells. L-cells were not able to repair the SS breaks induced by any of the agents, which correlates with the deficiency of these cells for repair synthesis of DNA. The following conclusions are discussed. MNUA and UV-light produce modifications in DNA which are not repaired but are translated into SS breaks in alkali. MMS produces SS breaks intracellularly but these are not derived from a simple depurination of methylated purines. 4NQO produces a modification in DNA which is translated into an SS break in alkali but which can be removed by an intracellular process.     

Nature of DNA lesions induced in human hepatoma cells, human colonic cells and human embryonic lung fibroblasts by the antiretroviral drug 3'-azido-3'-deoxythymidine

This study tried to clarify the question if nuclear genotoxicity played a role in 3'-azido-3'-deoxythymidine (AZT) toxicity. We investigated cytotoxic and DNA-damaging effects of AZT on human hepatoma HepG2 and human colonic CaCo-2 cells as well as on human diploid lung fibroblasts HEL. The amount of induced DNA damage was measured by standard alkaline single cell gel electrophoresis (SCGE). The nature of induced DNA lesions was evaluated (1) by modified SCGE, which includes treatment of lysed cells with DNA repair enzymes Endo III and Fpg and enables to recognize oxidized bases of DNA, and (2) by SCGE processed in parallel at pH 13.0 (standard technique) and pH 12.1, which enables to recognize alkali labile DNA lesions and direct DNA strand breaks. Cytotoxicity of AZT was evaluated by the trypan blue exclusion technique. Our findings showed that 3-h treatment of cells with AZT decreased the viability of all cell lines studied. SCGE performed in the presence of DNA repair enzymes proved that AZT induced oxidative lesions to DNA in all cell types. In hepatoma HepG2 cells and embryonic lung fibroblasts HEL the majority of AZT-induced DNA strand breaks were pH-independent, i.e. they were identified at both pH values (12.1 and 13.0). These DNA lesions represented direct DNA breaks. In colonic Caco-2 cells DNA lesions were converted to DNA strand breaks particularly under strong alkaline conditions (pH>13.0), which is characteristic for alkali-labile sites of DNA. DNA strand break rejoining was investigated by the standard comet assay technique during 48 h of post-AZT-treatment in HepG2 and Caco-2 cells. The kinetics of DNA rejoining, considered an indicator of DNA repair, revealed that AZT-induced DNA breaks were repaired in both cell types slowly, though HepG2 cells seemed to be more repair proficient with respect to AZT-induced DNA lesions.     

DNA damage and repair in embryonic mouse limbs exposed to teratogenic doses of methylnitrosourea

The alkaline elution assay was used to monitor DNA single-strand breaks in embryonic tissue following exposure to the DNA-damaging teratogen N-methyl-N-nitrosourea (MNU, CAS No. 694-93-5). An animal model was developed in which nearly every fetus exposed to the highest dose of MNU had malformations of the hindlimbs while the fetuses exposed to the lowest dose of MNU had none. Hindlimbs pooled within litters were analyzed for DNA single-strand breaks by alkaline elution conducted at rapid (0.35 ml/min) and slow (0.35 ml/min) speeds. Breaks in the DNA of hindlimbs exposed to teratogenic doses of MNU were readily detected by alkaline elution only if slower speeds were used in the assay. Using the more sensitive procedure, DNA breakage was monitored over a 24-h period. DNA breakage peaked in the MNU-exposed hindlimbs in a dose-dependent manner 4 h after injection. While the elution profiles of hindlimbs exposed to the lower doses of MNU returned to control levels 8 h after injection, single-strand breaks persisted in the hindlimbs exposed to the highest dose of MNU for at least 20 h. These latter data suggest that the highly teratogenic dose of MNU induced DNA damage that was more slowly repaired than that produced at lower doses, possibly by saturation of DNA repair systems. Although some necrosis did occur in hindlimbs exposed at teratogenic dose levels, it was not severe and it did not appear to influence the alkaline elution results. These experiments show that alkaline elution is a sensitive assay for the detection of DNA damage in embryonic tissues.     

DNA strand break and rejoining in cultured human fibroblasts exposed to fast neutrons or gamma rays

The production and rejoining of DNA single-strand and double-strand breaks have been monitored in monolayer cultures of proliferating human skin fibroblasts by means of sensitive techniques. Cells were irradiated with low doses of either 60Co gamma-rays or 14.6 MeV neutrons at 0 degrees C (0-5 Gy for measurement of single-strand breaks by alkaline elution and 0-50 Gy for double-strand breaks measured by neutral elution). The yield of single-strand breaks induced by neutrons was 30 per cent of that produced by the same dose of gamma-rays; whilst in the induction of double-strand breaks neutrons were 1.6 times as effective as gamma-rays. Upon post-irradiation incubation of cells at 37 degrees C, neutron-induced single-strand and double-strand breaks were rejoined with a similar time-course to gamma-induced breaks. Rejoining followed biphasic kinetics; of the single-strand breaks, 50 per cent disappeared within 2 min after gamma-rays and 6-10 min after neutrons. Fifty per cent of the double-strand breaks disappeared within 10 min, after gamma-rays and neutrons. Cells derived from patients suffering from ataxia-telangiectasia showed the same capacity for repair of single- and double-strand breaks induced by 14.6 MeV neutrons, as cells established from normal donors. The comparison of neutrons and gamma-rays in the induction of DNA breaks did not explain the elevated r.b.e. on high LET radiation. However, a study of the variation in the spectrum of lesions induced by different radiation sources will probably contribute to the clarification of the relative importance of other radio products.     

Repair of bleomycin-induced DNA double-strand breaks in Vicia faba

As detected by neutral DNA elution, bleomycin induced at the concentrations tested (5, 10 and 50 micrograms/ml) DNA double-strand breaks (dsbs) in in vitro cultured embryos of V. faba. Most of these breaks were repaired during a 4-h incubation period after treatment. Dsbs also occurred after treatment with 2.5 and 5 mM of N-methyl-N-nitrosourea (MNU) but in contrast to those induced by bleomycin, these dsbs remained unrepaired during the 4-h incubation period following the treatment.     

The effect of deoxyadenosine plus deoxycoformycin on replicative and repair synthesis of DNA in human lymphoblasts and isolated nuclei

Deoxyadenosine plus deoxycoformycin (dCf) causes increased DNA breaks in lymphoid cells. This study explored the possible inhibition of repair synthesis of DNA by dAdo plus dCf as a cause of DNA breakage. It was shown that DNA breaks accumulated in a human T-lymphoblast cell line, CCRF-CEM, following incubation with dAdo plus dCf and were not fully repaired 20 h after their removal. Analysis of the density distribution of radiolabeled DNA on alkaline CsCl gradient showed that incubation of CCRF-CEM cells with dAdo plus dCf caused inhibition of semiconservative, but not repair synthesis of DNA. Semiconservative synthesis of DNA was also inhibited in CCRF-CEM nuclei isolated from cells pretreated with dAdo and dCf, suggesting damage to DNA replicative machinery. However, no such inhibition was observed in the nuclei of a similarly treated CCRF-CEM mutant that was deficient in adenosine kinase and deoxycytidine kinase. This suggests that dAdo must be phosphorylated in intact cells to exert its effect. Using [3H]dTTP incorporation in isolated CCRF-CEM nuclei to measure DNA synthesis, it was found that a high concentration (greater than 100 microM) of dATP inhibits semiconservative but not repair synthesis of DNA. The present studies thus indicate that accumulation of DNA strand breaks induced by dAdo plus dCf is not the consequence of inhibition of repair DNA synthesis. This implies the mechanism may involve perturbation of DNA ligation or activation of a certain process which causes DNA strand breaks. In addition, dATP may interfere with some steps of semiconservative DNA synthesis, but not the repair synthesis of DNA.     

Mutagenicity and DNA repair of glycidamide-induced adducts in mammalian cells

Glycidamide (GA)-induced mutagenesis in mammalian cells is not very well understood. Here, we investigated mutagenicity and DNA repair of GA-induced adducts utilizing Chinese hamster cell lines deficient in base excision repair (BER), nucleotide excision repair (NER) or homologous recombination (HR) in comparison to parent wild-type cells. We used the DRAG assay in order to map pathways involved in the repair of GA-induced DNA lesions. This assay utilizes the principle that a DNA repair deficient cell line is expected to be affected in growth and/or survival more than a repair proficient cell. A significant induction of mutations by GA was detected in the hprt locus of wild-type cells but not in BER deficient cells. Cells deficient in HR or BER were three or five times, respectively, more sensitive to GA in terms of growth inhibition than were wild-type cells. The results obtained on the rate of incisions in BER and NER suggest that lesions induced by GA are repaired by short patch BER rather than long patch BER or NER. Furthermore, a large proportion of the GA-induced lesions gave rise to strand breaks that are repaired by a mechanism not involving PARP. It is suggested that these strand breaks, which might be the results from alkylation of the backbone phosphate, are misrepaired by HR during replication thereby leading to a clastogenic rather than a mutagenic pathway. The type of lesion responsible for the mutagenic effect of GA cannot be concluded from the results presented in this study.     

In vivo repair during G1 of DNA lesions eliciting sister chromatid exchanges induced by methylnitrosourea or ethylnitrosourea in BrdU substituted or unsubstituted DNA in murine salivary gland cells

The difference in efficiency of methylnitrosourea (MNU) and ethylnitrosourea (ENU) to induce SCE in early or late G1 was determined in synchronized murine salivary gland cells in vivo, as a measure of the capacity of this tissue to repair the lesions involved in SCE formation during G1. The repair during G1 was determined by treating the cells in early or late G1. Treatment was in the first cycle (G1 before incorporation of 5-bromodeoxyuridine (BrdU)) or in G1 of the second cycle (after a single round of BrdU incorporation). It was observed that 50% of the lesions induced by MNU that elicit SCE are repaired during G1. BrdU incorporation into DNA increases the sensitivity of the cell to SCE induction by MNU nearly 40%; however under this circumstance a slightly lower SCE frequency was observed in the cells exposed to MNU at early G1, indicating that during G1 only few lesions are repaired. The ENU-induced DNA-lesions involved in SCE production are nearly 100% persistent along G1; besides, a slight but significantly higher SCE frequency was observed in cells exposed at early G1, suggesting the formation of SCE-inducing lesions during G1. BrdU incorporation to DNA sensitizes the cell to SCE induction by ENU, increasing the SCE frequency to nearly to a 40%, although these additional lesions involved in SCE induction seem to be susceptible to repair during G1.     

Pretreatment with oxygen species increases the resistance of mammalian cells to hydrogen peroxide and gamma-rays

Pretreatment of Chinese hamster ovary (CHO) or H4 (rat hepatoma) cells with low non-toxic doses of H2O2 or xanthine-xanthine oxidase renders the cells more resistant to the toxic effect of H2O2 and gamma-rays. This increased resistance is observed both in exponentially growing and in plateau-phase cells. Cells pretreated with xanthine-xanthine oxidase are less mutated than control cultures when challenged with ionizing radiation. The number of DNA single-strand breaks (measured by nucleoid sedimentation) induced by a high dose of gamma-rays or H2O2 is lower in cells pretreated with xanthine-xanthine oxidase compared to control cultures. However, the pretreatment does not modify the rate of DNA single-strand breaks rejoining in cells challenged with H2O2 or gamma-rays. The catalase activity is not modified in pretreated cells, but the superoxide dismutase activity is increased about 2-fold.     

Zebularine induces replication-dependent double-strand breaks which are preferentially repaired by homologous recombination

Zebularine is a second-generation, highly stable hydrophilic inhibitor of DNA methylation with oral bioavailability that preferentially target cancer cells. It acts primarily as a trap for DNA methyl transferases (DNMTs) protein by forming covalent complexes between DNMT protein and zebularine-substrate DNA. It’s well documented that replication-blocking DNA lesions can cause replication fork collapse and thereby to the formation of DNA double-strand breaks (DSB). DSB are dangerous lesions that can lead to potentially oncogenic genomic rearrangements or cell death. The two major pathways for repair of DSB are non-homologous end joining (NHEJ) and homologous recombination (HR). Recently, multiple functions for the HR machinery have been identified at arrested forks. Here we investigate in more detail the importance of the lesions induced by zebularine in terms of DNA damage and cytotoxicity as well as the role of HR in the repair of these lesions. When we examined the contribution of NHEJ and HR in the repair of DSB induced by zebularine we found that these breaks were preferentially repaired by HR. Also we show that the production of DSB is dependent on active replication. To test this, we determined chromosome damage by zebularine while transiently inhibiting DNA synthesis. Here we report that cells deficient in single-strand break (SSB) repair are hypersensitive to zebularine. We have observed more DSB induced by zebularine in XRCC1 deficient cells, likely to be the result of conversion of SSB into toxic DSB when encountered by a replication fork. Furthermore we demonstrate that HR is required for the repair of these breaks. Overall, our data suggest that zebularine induces replication-dependent DSB which are preferentially repaired by HR.     

Mutagenicity and DNA repair of glycidamide-induced adducts in mammalian cells

Glycidamide (GA)-induced mutagenesis in mammalian cells is not very well understood. Here, we investigated mutagenicity and DNA repair of GA-induced adducts utilizing Chinese hamster cell lines deficient in base excision repair (BER), nucleotide excision repair (NER) or homologous recombination (HR) in comparison to parent wild-type cells. We used the DRAG assay in order to map pathways involved in the repair of GA-induced DNA lesions. This assay utilizes the principle that a DNA repair deficient cell line is expected to be affected in growth and/or survival more than a repair proficient cell.A significant induction of mutations by GA was detected in the hprt locus of wild-type cells but not in BER deficient cells. Cells deficient in HR or BER were three or five times, respectively, more sensitive to GA in terms of growth inhibition than were wild-type cells. The results obtained on the rate of incisions in BER and NER suggest that lesions induced by GA are repaired by short patch BER rather than long patch BER or NER. Furthermore, a large proportion of the GA-induced lesions gave rise to strand breaks that are repaired by a mechanism not involving PARP. It is suggested that these strand breaks, which might be the results from alkylation of the backbone phosphate, are misrepaired by HR during replication thereby leading to a clastogenic rather than a mutagenic pathway. The type of lesion responsible for the mutagenic effect of GA cannot be concluded from the results presented in this study.     

DNA-damaging activity of patulin in Escherichia coli.

At a concentration of 10 micrograms/ml, patulin caused single-strand DNA breaks in living cells of Escherichia coli. At 50 micrograms/ml, double-strand breaks were observed also. Single-strand breaks were repaired in the presence of 10 micrograms of patulin per ml within 90 min when the cells were incubated at 37 degrees C in M9-salts solution without a carbon source. The same concentration also induced temperature-sensitive lambda prophage and a prophage of Bacillus megaterium. When an in vitro system with permeabilized Escherichia coli cells was used, patulin at 10 micrograms/ml induced DNA repair synthesis and inhibited DNA replication. The in vivo occurrence of DNA strand breaks and DNA repair correlated with the in vitro induction of repair synthesis. In vitro the RNA synthesis was less affected, and overall protein synthesis was not inhibited at 10 micrograms/ml. Only at higher concentrations (250 to 500 micrograms/ml) was inhibition of in vitro protein synthesis observed. Thus, patulin must be regarded as a mycotoxin with selective DNA-damaging activity.     

DNA-damaging activity of patulin in Escherichia coli

At a concentration of 10 micrograms/ml, patulin caused single-strand DNA breaks in living cells of Escherichia coli. At 50 micrograms/ml, double-strand breaks were observed also. Single-strand breaks were repaired in the presence of 10 micrograms of patulin per ml within 90 min when the cells were incubated at 37 degrees C in M9-salts solution without a carbon source. The same concentration also induced temperature-sensitive lambda prophage and a prophage of Bacillus megaterium. When an in vitro system with permeabilized Escherichia coli cells was used, patulin at 10 micrograms/ml induced DNA repair synthesis and inhibited DNA replication. The in vivo occurrence of DNA strand breaks and DNA repair correlated with the in vitro induction of repair synthesis. In vitro the RNA synthesis was less affected, and overall protein synthesis was not inhibited at 10 micrograms/ml. Only at higher concentrations (250 to 500 micrograms/ml) was inhibition of in vitro protein synthesis observed. Thus, patulin must be regarded as a mycotoxin with selective DNA-damaging activity.     

Comparison of geno- and cytotoxicity of methylnitrosourea on MMR-proficient and MMR-deficient human tumor cell lines

Deficient mismatch repair (MMR) is identified as a mutation of one of four major MMR genes and(or) microsatellite instability. These genomic changes are used as markers of MMR status of the heredity nonpolyposis colorectal cancer (HNPCC) spectrum tumors--familial and sporadic tumors of colon and extracolonic cancers fulfilling Amsterdam clinical criteria II. MMR-deficiency results in mutator phenotype and resistance to geno- and cytotoxicity of alkylating agents. The main cytotoxic damage to DNA in response to chemical methylation is O6-methylguanine (O6-mG). The secondary DNA strand breaks, which are formed during the MMR functioning, are proposed to be required for methylation induced cytotoxicity. We have assumed that the secondary double stand breaks (DSB) upon DNA methylation are able to represent functional efficiency of MMR in cells. The purpose of the paper was to test this assumption on human tumor cells differing in MMR-status and pulse-treated with methylnitrosourea (MNU). We used 3 cell lines: HeLa (MMR-competent endometrial tumor cells), HCT116 (MMR-deficient colorectal carcinoma cells), and Colo320 (sigmoid intestine tumor cells with uncharacterized MMR status). DSBs were evaluated with neutral comet assay. Cytotoxicity/viability was evaluated with MTT-asay and apoptotic index (frequency of morphologically determined apoptotic cells). We show that 1) cytotoxic effect of MNU (250 microM) on HeLa cells was exhibited 3 days after pulse-treatment of cells with MNU; 2) DSBs occurred 48 h after the drug treatment but prior to the onset of apoptosis of HeLa cells; 3) MMR-deficient HCT116 cells were resistant to the drug: no decreased viability, DSBs and apoptosis were observed during 3 days after cell treatment. Both cell lines exhibited high sensitivity to etoposide, classical inductor of unrepairable DSBs and p53. Etoposide has been found to induce DSBs in 6-12 h, which was followed by apoptosis (in 24 h). Colo320 cells exhibited intermediate position between HeLa and HCT116 cell lines in regard to sensitivity to MNU according to MTT-assay and the number of secondary DSBs formed in MNU-treated cells. Nevertheless, in contrast to HeLa cells, these breaks did not induce apoptosis in Colo320 cells. Our data confirm the assumption about case/effect relationship between secondary DNA double strand breaks, induced by monofunctional methylating agent MNU, and functioning of MMR in human tumor cells.     

Defective repair of DNA single- and double-strand breaks in the bleomycin- and X-ray-sensitive Chinese hamster ovary cell mutant, BLM-2

Using filter elution techniques, we have measured the level of induced single- and double-strand DNA breaks and the rate of strand break rejoining following exposure of two Chinese hamster ovary (CHO) cell mutants to bleomycin or neocarzinostatin. These mutants, designated BLM-1 and BLM-2, were isolated on the basis of hypersensitivity to bleomycin and are cross-sensitive to a range of other free radical-generating agents, but exhibit enhanced resistance to neocarzinostatin. A 1-h exposure to equimolar doses of bleomycin induces a similar level of DNA strand breaks in parental CHO-K1 and mutant BLM-1 cells, but a consistently higher level is accumulated by BLM-2 cells. The rate of rejoining of bleomycin-induced single- and double-strand DNA breaks is slower in BLM-2 cells than in CHO-K1 cells. BLM-1 cells show normal strand break repair kinetics. The level of single- and double-strand breaks induced by neocarzinostatin is lower in both BLM-1 and BLM-2 cells than in CHO-K1 cells. The rate of repair of neocarzinostatin-induced strand breaks is normal in BLM-1 cells but retarded somewhat in BLM-2 cells. Thus, there is a correlation between the level of drug-induced DNA damage in BLM-2 cells and the bleomycin-sensitive, neocarzinostatin resistant phenotype of this mutant. Strand breaks induced by both of these agents are also repaired with reduced efficiency by BLM-2 cells. The neocarzinostatin resistance of BLM-1 cells appears to be a consequence of a reduced accumulation of DNA damage. However, the bleomycin-sensitive phenotype of BLM-1 cells does not apparently correlate with any alteration in DNA strand break induction or repair, as analysed by filter elution techniques, suggesting an alternative mechanism of cell killing.