The intensity of internalization and cytotoxicity of superparamagnetic iron oxide nanoparticles with different surface modifications in human tumor and diploid lung cells.

The human lung adenocarcinoma epithelial (A549) cells and the human embryo lung (HEL 12469) cells were used to investigate the uptake and cytotoxicity of magnetite nanoparticles (MNPs) with different chemically modified surfaces. MNPs uptake was an energy-dependent process substantially affected by the serum concentration in the culture medium. Internalized MNPs localized in vesicle-bound aggregates were observed in the cytoplasm, none in the nucleus or in mitochondria. All MNPs induced a dose- and time-dependent increase in cytotoxicity in both human lung cell lines. The cytotoxicity of MNPs increased proportionally with the particle size. Since the cytotoxicity of MNPs was nearly identical when the doses were equalized based on particle surface area, we suppose that the particle surface area rather than the surface modifications per se underlay the cytotoxicity of MNPs. In general, higher internalized amount of MNPs was found in HEL 12469 cells compared with A549 cells. Accordingly, the viability of the human embryo lung cells was reduced more substantially than that of the adenocarcinoma lung cells. The weak MNPs uptake into A549 cells might be of biomedical relevance in cases where MNPs should be used as nanocarriers for targeted drug delivery in tumor tissue derived from alveolar epithelial cells.

Significance of amino acid substitution variants of DNA repair genes in radiosusceptibility of cervical cancer patients; a pilot study.

The present pilot study was designed to elucidate the functional significance of amino acid substitution variants of DNA repair genes. Using the peripheral blood lymphocytes (PBLs) from healthy donors and cervical cancer patients, the contribution of four non-synonymous single nucleotide polymorphisms (SNPs) in three base excision repair genes (BER), XRCC1 (Arg194Trp and Arg399Gln), hOGG1 (Ser326Cys), and APE1 (Asp148Glu), to the susceptibility to ionizing radiation were evaluated. The level of initial, oxidative and residual DNA damage produced by 2 Gy was measured by the alkaline single cell gel electrophoresis (the comet assay), and the SNPs were determined by PCR-restriction fragment length polymorphism (RFLP) assay. No significant differences in the allele frequencies between cancer patients and controls for any of these four SNPs were detected. Although the initial DNA damage levels were approximately similar, significantly higher level of Fpg-sensitive sites were found in patients compared with controls (p<0.001) irrespective of genotype distribution. A trend towards increased values of EndoIII-sensitive sites was determined in PBLs from cancer patients compared with healthy women, mainly carriers of the XRCC1 and OGG1 variant alleles; however, the mean value of EndoIII-sensitive sites does not reach any significance. A substantial delay in DNA strand-break rejoining was ascertained in patients who carried APE1 Glu variant allele in comparison with healthy donors 15 and 60 minutes after irradiation (p< 0.05 and p< 0.01, respectively). In contrast, slightly higher but statistically significant level of residual DNA damage was estimated in controls (APE1Asp/Asp) compared with patients. An association between single nucleotide polymorphism (SNP) of two DNA repair genes functioning in the same biochemical pathway and susceptibility to radiation was found. In the combined genotype APE1/XRCC1 and APE1/hOGG1, a decreased level of residual DNA damage was detected in carriers of wild type APE1 genotype. In addition, a possible modulating effect of hOGG1 gene on the kinetics of strand-break rejoining was estimated. The lowest residual DNA damage level was determined in subjects with the combined APE1(Asp/Asp)/hOGG1(Ser/Cys+Cys/Cys) genotypes. Based on these preliminary data we suppose that a combination of several amino acid substitution variants of DNA repair genes involved in the same repair pathway rather than one low-penetrance SNP in a single gene may contribute to DNA repair outcomes. Larger study with more subjects is needed to verify these findings.

Radiosensitivity of peripheral blood lymphocytes from healthy donors and cervical cancer patients; the correspondence of in vitro data with the clinical outcome.

The reliability of a particular in vitro parameter as potential prognostic biomarker of individual radiosensitivity is still discussed. Therefore, several in vitro radiation-induced cellular endpoints including initial, oxidative and residual DNA damage and the rate of DNA repair were assessed in peripheral blood lymphocytes (PBL) from healthy donors and patients with carcinoma of the cervix using the alkaline single cell gel electrophoresis (the comet assay). PBL from cancer patients were analyzed three times during the course of therapy, prior, in the middle (25-27 Gy) and after the radiotherapy. Interindividual differences in radiation-induced DNA damage and in the kinetics of strand break rejoining were determined within both groups. Significantly higher level of mean background and oxidative DNA damage was estimated in the cancer patient cohort than in the healthy subject group; however similar mean values of the initial DNA damage and the rate of DNA repair kinetics were found in both groups. No adaptive response was determined in PBL from cancer patients due to radiotherapy. The acute radiation toxicity and the clinical outcome were scored according to the criteria as proposed by the National Cancer Institute. A substantial delay in DNA strand break rejoining was determined in cancer patients suffering from adverse side effects (G2+) in comparison to persons with no or very mild radiation toxicity (G0-G1) (p<0.05). The recurrence of disease has been associated with a lower initial DNA damage and slope value of dose-response effect, and increased rate of DNA repair. Results from this pilot study suggest that the residual DNA damage level might be a promising prognostic biomarker of acute radiation morbidity; however, further study is necessary to validate this finding.

Radiosensitivity of cervical cancer cell lines: the impact of polymorphisms in DNA repair genes.

The aim of this study was to evaluate radiosensitivity of cervical cancer cells in vitro and to assess the relationship between genetic polymorphisms in DNA repair genes and the response of cells to ionizing radiation. The alkaline comet assay as a predictive assay of radiosensitivity was used to examine the susceptibility of four human cervical cancer cell lines (CaSki, C-33A, HeLa and SiHa) to radiation damages. The initial DNA damage and the residual DNA damage at 15, 30, 45 and 60 min after irradiation were assessed. Genotypes of DNA repair genes (XRCC1, hOGG1, PARP, XPD, XRCC3 and XRCC4) were analyzed by PCR-RFLP assays. The comet data clearly indicate a variable but dose-dependent increase in the initial DNA damage in all cell lines. The highest slope of dose response curve was observed in C-33A cells and this cell line was assumed to be radiosensitive. All cell lines repaired DNA damage in a similar manner, the level of DNA strand breakage has returned near the background level within 45 min after irradiation. According to the genotype we found that C-33A cells are polymorphic in the majority of analyzed DNA repair genes. This pilot study indicated associations between polymorphisms in DNA repair genes and cell radiosensitivity.

Role of cytoplasmic membrane in the screening of potential tumour promoters.

The functional changes of cytoplasmic membrane of mammalian cells as an alternative approach for evaluating the tumor promoter activity were investigated. The V79 metabolic cooperation assay, as the standard method for detection of tumor promoters in vitro was used. From three known tumor promoters only phenobarbital increased the efflux of [3H]2-deoxy-D-glucose-6-phosphate from the cells in the same concentration range where the inhibition of metabolic cooperation was estimated. Neither TPA, saccharin nor theophylline caused any functional changes of cytoplasmic membrane of V79 cells. On the basis of the results with known tumor promoters we suggest that following the functional changes of cytoplasmic membrane is not a convenient approach for screening potential tumor promoters.

DNA adduct formation in primary mouse embryo cells induced by 7H-dibenzo[c,g]carbazole and its organ-specific carcinogenic derivatives.

The nuclease P1 modification of the 32P-postlabeling technique was used to study the biological activity of 7H-dibenzo[c,g]carbazole (DBC) and some of its derivatives, including N-methyldibenzo[c,g]carbazole (N-MeDBC), 5,9-dimethyldibenzo[c,g]carbazole (5,9-diMeDBC), 5,9,N-trimethyldibenzo[c,g]carbazole (5,9,N-triMeDBC), 6-methoxydibenzo[c,g]carbazole (6-McODBC), N-acetyldibenzo[c,g]carbazole (N-AcDBC), N-hydroxymethyldibenzo[c,g]carbazole (N-HMeDBC) in primary mouse embryo cells. A very good correlation was found between carcinogenic specificity in vivo of these N-heterocyclic aromatic hydrocarbons and their DNA-adduction in vitro. Primary mouse embryo cells were able to metabolize and detect tissue-specific sarcomagens N-MeDBC and 6-MeODBC as well as derivatives with both sarcomagenic and hepatocarcinogenic activity, DBC, N-AcDBC, and N-HMeDBC. The strong specific hepatocarcinogen 5,9-diMeDBC in vivo, did not induce any DNA-adducts in the embryo cells, which suggests that the enzymatic composition of the target tissue probably is the determining factor in the organ specificity of this derivative. 5,9,N-triMeDBC, derivative without any carcinogenic activity in vivo, did not induce any DNA-adducts in primary mouse embryo cells. Pretreatment of cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) apparently stimulated DNA-adduct formation in the cells exposed to DBC, 6-MeODBC, and N-MeDBC. No or a very slight effect of TCDD on DNA-adduct formation was found in cells exposed to N-HMeDBC and N-AcDBC. Preliminary results have shown that TCDD slightly induced cytochrome P4501A1-linked ethoxyresorufin O-deethylase (EROD) activity in primary mouse embryo cells. These data suggest the role of cytochrome P4501A1 in the metabolism of DBC derivatives with sarcomagenic activity.

Decemtione (Imidan)-induced single-strand breaks to human DNA, mutations at the hgprt locus of V79 cells, and morphological transformations of embryo cells.

To study the genotoxic activity of Decemtione (Imidan), this substance was subjected to a series of tests. After preliminary cytotoxicity testing, the capacity of Decemtione to damage human DNA was determined by alkaline elution of DNA and DNA unwinding. Both tests gave positive results, suggesting that Decemtione was able to induce single-strand breaks in DNA. This capacity was higher in the absence and lower in the presence of the S9 fraction. The potential mutagenicity of Decemtione was followed on the basis of its ability to induce resistance to 6-thioguanine in V79 hamster cells. Unlike the induction of single-strand breaks, Decemtione showed, in the absence of the metabolic activation system, a very weak mutagenic effect, which was, however, significantly higher in the presence of the S9 fraction. The ability of the substance to transform diploid cells under in vitro conditions was followed on the basis of morphological transformation of Syrian hamster embryo cells. The results showed that Decemtione, like positive carcinogenes, induced a significant elevation in morphologically transformed colonies of embryo cells. The results suggest a carcinogenic potential of this organophosphate insecticide.

Use of repair endonucleases for characterization of DNA damage induced by N-heterocyclic aromatic hydrocarbons.

Several repair endonucleases were used to characterize and quantify various types of DNA damage induced by 7H-dibenzo[c,g]carbazole (DBC) and its methyl derivative, N-methyldibenzo[c,g]carbazole (MeDBC). Differences in the DNA damage profile induced by these two derivatives were found to be related to their chemical structure and dependent on the way of their metabolic activation. Different ways of activation gave rise to different numbers of single strand breaks and DNA modifications or, at least, to different ratios of common modifications. DBC induced the highest level of breaks in human hepatal cell line Hep G2, while MeDBC induced most of the breaks in V79 cell line with stable expression of human cytochrome P4501A1. Our results support the idea of two different pathways of biotransformation of DBC and MeDBC.

The effects of sodium azide on mammalian cells cultivated in vitro.

Sodium azide acted cytostatically to cytotoxically on 2 lines of mammalian cells. After application of the substance in an acid environment the highest cytostatic effect was noted. The results of the DNA-synthesis inhibition test suggest that sodium azide does not damage the DNA of the observed fibroblasts with any of the tested modes of application. In Chinese hamster cells neither 20-h treatment in medium nor 60-min treatment in an acid environment gave rise to significantly increased occurrence of 6-TG-resistant mutations. The results of the DNA-synthesis inhibition test, as well as the mutagenicity testing, do not suggest the possibility that treatment with sodium azide might induce DNA damage in the observed human and Chinese hamster cells. The cytostatic effect of sodium azide on the fibroblasts studied is probably not accompanied by a genotoxic effect.

The influence of multiple mutagenic treatments on the occurrence of 6-thioguanine-resistant mutants in dividing V79 cells.

Repeated treatments of dividing Chinese hamster V79 cells with ultraviolet radiation or a simple monofunctional alkylating agent (methyl methanesulphonate, N-methyl-N'-nitro-N-nitrosoguanidine or N-methyl-N-nitrosourea) were studied with regard to the pattern of cumulative increase of 6-thioguanine-resistant mutants in the surviving population. There were 5-10 divisions of surviving cells between two successive treatments. Repeated treatments with different agents produced different effects. The observed results indicating either a higher or lower than additive effect of single doses may be explained by induced repair processes.

Cytotoxicity and genotoxicity testing of sodium fluoride on Chinese hamster V79 cells and human EUE cells.

The cytotoxic effects of sodium fluoride (NaF) on hamster V79 cells and human EUE cells were studied by measuring the cloning efficiency and DNA, RNA and protein synthesis in cells cultured in the presence of NaF. Potential mutagenicity of NaF was followed on the basis of induced 6-thioguanine-resistant mutants in treated Chinese hamster V79 cells. The results showed that the addition of 10-150 micrograms of NaF per ml of culture medium induced 10-75% cytotoxic effect on hamster V79 cells but had no toxic effect on human EUE cells. NaF was cytotoxic to human EUE cells at considerably higher concentrations (200-600 micrograms/ml). Growth of both cell types with 100 and 200 micrograms of NaF per ml caused inhibition of 14C-thymidine, 14C-uridine and 14C-L-leucine incorporation. This means that NaF inhibits macromolecular synthesis whereby damaging effects were less drastic in human EUE cells. The results of detailed mutagenicity testing on hamster V79 cells showed that NaF did not show any mutagenic effect after long-term (24-h) incubation of hamster cells in the presence of 10-400 micrograms of NaF per ml of culture medium.

Induction of micronuclei by 7H-dibenzo[c,g]carbazole and its tissue specific derivatives in Chinese hamster V79MZh1A1 cells.

The clastogenicity/aneugenicity of N-heterocyclic polycyclic aromatic pollutant 7H-dibenzo[c,g]carbazole (DBC) and its two synthetic derivatives N-methyl DBC (MeDBC) and 5,9-dimethyl DBC (diMeDBC) was evaluated in the genetically engineered Chinese hamster V79 cell line V79MZh1A1 with stable expression of human cytochrome P4501A1 and in the parental V79MZ cell line without any cytochrome P450 activity. While none of the three carbazoles changed significantly the level of micronuclei in the parental V79MZ cells, a variable, but statistically significant rise of micronucleus frequencies was assessed in V79MZh1A1 cells. DBC induced dose-dependent increase in the number of micronuclei at harvest times of 24 and 48h and MeDBC at sampling time of 48h in V79MZh1A1 cells in comparison to untreated cells, however, no significant time-dependent increase in micronucleus frequencies was found. The use of the antikinetochore immunostaining revealed that DBC and MeDBC induced approximately equal levels of both kinetochore positive (C+) and kinetochore negative (C-) micronuclei. DiMeDBC, a strict hepatocarcinogen, did not manifest any effect on micronucleus induction in V79MZh1A1 cells. These studies suggest that genetically engineered Chinese hamster V79 cell lines expressing individual CYP cDNAs are a useful in vitro model for evaluation the role of particular cytochromes P450 in biotransformation of DBC and its tissue and organ specific derivatives.

Role of cytochrome P4501A1 in biotransformation of a tissue specific sarcomagen N-methyldibenzo[c,g]carbazole.

7H-dibenzo[c,g]carbazole (DBC) is a potent liver and skin carcinogen, while its synthetic methyl derivative N-methyldibenzo[c,g]carbazole (MeDBC) is tissue specific sarcomagen. It is supposed that sarcomagenic activity of DBC depends on biotransformation at ring-carbon atoms, as with PAH, whereas the heterocyclic nitrogen plays an important role in liver carcinogenicity. The objective of this study was to elucidate the role of cytochrome P4501A1 in metabolic activation of sarcomagenic derivatives of DBC and to characterize the DNA damage profiles induced by DBC and MeDBC in relation to the mode of metabolic activation. The genetically engineered V79MZh1A1 cell line with stable expression of cDNA of human cytochrome P4501A1, the parental V79MZ cell line lacking any cytochrome P450 activity and human hepatocarcinoma Hep G2 cells were used as a model cells. Dose-dependent decrease in colony forming ability (CFA) was found in the V79MZh1A1 cell line after treatment of cells with DBC and MeDBC; however, no change in CFA was induced in parental V79MZ cells. These results were in a good correlation with DNA damaging effects of these two derivatives measured by the alkaline DNA unwinding (ADU) and the modified single cell gel electrophoresis (SCGE) techniques. Differences in DNA damage profiles induced by DBC and MeDBC were found in V79MZh1A1 and Hep G2 cells. These differences were probably the result of different reactive metabolite formation depending on chemical structure of the molecule and ways of biotransformation. This study showed that the cytochrome P4501A1 took part in activation of sarcomagenic DBC derivatives. Moreover, V79 cell lines with stable expression of different cytochromes P450 in combination with DNA repair endonucleases should be a useful tool for characterization of the role of individual cytochromes in metabolic activation pathways of DBC and MeDBC.

Differences between survival, mutagenicity and DNA replication in MMS- and MNU-treated V79 hamster cells.

After treatment with methyl methanesulfonate (MMS) or N-methyl-N-nitrosourea (MNU), the mutagenicity and survival of Chinese hamster V79 cells were investigated, as well as the inhibition of daughter DNA synthesis and, using the DNA unwinding technique and hydroxylapatite chromatography, the character of the newly synthesized DNA was studied. It was found that different cytotoxicity and mutagenicity of MMS and MNU was accompanied by different types of DNA synthesis inhibition. The treatment with the former compound resulted in a longer inhibition of DNA synthesis, while the treatment with the latter showed that as early as 2 h after exposure the percentage of nascent DNA increased. Shortly after the exposure to both alkylating agents, the newly synthesized DNA contained a higher number of gaps than control DNA, in dependence on the concentration used. During culturing after treatment, the character of nascent DNA in MMS-treated cells gradually returned to that of control DNA, while MNU-treated cells, for the whole time of our study, synthesized DNA with a larger number of gaps than control DNA. We suggest that the character of nascent daughter DNA reflects the occurrence of lesions in parental DNA. These are repaired within a shorter time in MMS- than in MNU-treated cells. The long-term persistence of lesions in the DNA of MNU-treated cells might be one of the factors responsible not only for the higher cytotoxic but also for the many times higher mutagenic effect of this alkylating agent.

The genetic effects of the cytostatic drug TS160 on Chinese hamster fibroblasts in vitro.

The cytostatic agent TS160 inhibited DNA synthesis in V79 cells while the course of RNA synthesis and protein synthesis were not changed significantly. DNA inhibition was in direct correlation with the inhibition of growth and reduction of colony-forming ability in the treated cells. The study of the mutagenic consequences of single or repeated TS160 treatments showed that TS160 had a significant mutagenic activity on Chinese hamster V79 cells. In the study of mutagenic effects of TS160, we used the method of repeated treatment of surviving cell fractions with this compound. This method is suitable for those mutagens (e.g. weak mutagens) whose mutagenic activity on mammalian cells in vitro might escape attention after application of a single dose. The possible causes of mutagenic effects of TS160 are discussed.

Results of genotoxicity testing of mazindol (degonan), lithium carbonicum (contemnol) and dropropizine (ditustat) in Chinese hamster V79 and human EUE cells.

We studied inhibition of DNA synthesis, alkaline elution of DNA, cytotoxicity and occurrence of induced 6-thioguanine resistant mutants in mammalian cells, treated with mazindol, lithium carbonicum, and dropropizine, respectively, in the presence and in the absence of microsomal fraction S9. Among the above-mentioned clinically used drugs only dropropizine showed neither mutagenic nor clastogenic effects. Lithium carbonicum manifested a weak and mazindol a medium genotoxic response which was in both cases reduced in the presence of microsomal fraction S9.

Assessment of toxicity, clastogenicity, mutagenicity and transforming activity of pentoxifylline in mammalian cells cultured in vitro.

We tested the possible cytotoxic, clastogenic and genotoxic effects of pentoxifylline on different lines of mammalian cells cultured in vitro. This study was part of the developmental research of agapurin, since pentoxifylline represents an effective compound of this drug. Cells treated for a short time manifested a relatively high resistance to the toxic effects of pentoxifylline. Generally, only cells treated for a long time (18 h) or a short time (2 h) with high concentrations of drug manifested sensitivity to the toxic effects of pentoxifylline. Although the tested drug induced DNA synthesis inhibition in V79 and EUE cells and clastogenic effects in V79 cells, it was not able to induce either 6-TGr mutations in the HGPRT locus of V79 cells or morphological transformation of Syrian hamster embryo cells. Adding of microsomal fraction S9 to the treated cells did not markedly change the effects of pentoxifylline on different studied endpoints. We suggest that pentoxifylline has no genotoxic effects, and that the cytotoxicity and induction of chromosomal aberrations were induced by inhibition of cellular DNA replication.

Detection of MNNG-induced DNA lesions in mammalian cells; validation of comet assay against DNA unwinding technique, alkaline elution of DNA and chromosomal aberrations.

Human cells (VH10 or Hep G2) and hamster cells V79 were exposed to different concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the level of DNA lesions was evaluated by the DNA unwinding technique, alkaline elution of DNA and the comet assay. All three methods were able to detect the effects of MNNG but with a clear difference in sensitivity. At low concentrations of MNNG the most sensitive method appeared to be the comet assay. After the short-term treatment the comet assay was able to detect the lesions induced by MNNG at approx. 0.1 microgram/ml, alkaline elution of DNA at 1 microgram/ml and DNA unwinding at 1-2 micrograms/ml. MNNG treated VH10 cells, human lymphocytes and V79 cells were also tested cytogenetically, confirming that MNNG induced chromosomal aberrations at concentrations > 1 microgram/ml in VH10 cells (short-term treatment): > 0.2 microgram/ml in V79 cells (long-term treatment) and > 8 micrograms/ml in human lymphocytes (long-term treatment). In some experiments we tried to increase the level of MNNG-induced DNA breaks with help of DNA repair inhibitors cytosine arabinoside (Ara C) and hydroxyurea (HU) which were applied either after or during MNNG treatment. Our results showed that the level of MNNG-induced lesions was increased by simultaneous treatment of cells with MNNG and Ara C and HU. 2 x 10(-5) M Ara C and 2 x 10(-3) MHU were as effective as 10-times higher concentrations of inhibitors. Ara C and HU increased the level of MNNG-induced DNA breaks mainly in combination with lower concentrations of MNNG (< 2 micrograms/ml). Rejoining of DNA breaks was observed in human cells VH10 and Hep G2 as well as in Chinese hamster cells V79 damaged by both lower and higher MNNG-concentrations. All methods showed that MNNG-induced DNA breaks had been gradually rejoined.

Preculturing of Chinese hamster V79 cells with sublethal concentration of theophylline sensitizes cells to cytotoxic effects of MNNG.

In our previous work concerning the biologic effects of theophylline, we found that cells incubated during 48 h at low concentrations of theophylline (0.3 mg/ml of medium) manifested short-term deviations in the rate of DNA replication; however, this short-term inhibition of DNA replication did not reduce either the growth rate or the colony-forming ability of cells. In the present study, we concentrated on cytotoxic and DNA-damaging effects of MNNG on V79 cells precultured with sublethal concentration of methylxanthine theophylline. Cytotoxicity was evaluated on the basis of growth rate of treated cells as well as by colony-forming ability (plating efficiency) test and by trypan blue exclusion test. The level of DNA lesions (strand breaks) induced by MNNG was measured by alkaline DNA unwinding and by the comet assay. In an effort to explain higher cytotoxic effects of MNNG on precultured cells, we studied rejoining of damaged parental DNA after 4 h incubation post-MNNG-treatment as well. We found differences as against the controls in theophylline-precultured cells after treatment with the mutagen and carcinogen MNNG. The higher cytotoxic effect of MNNG in precultured cells was accompanied by a higher level of ss breaks of DNA and by more unrepaired lesions which remained after 4 h in parental DNA. Our results demonstrate that theophylline belongs to the group of agents inhibiting repair of potentially lethal DNA lesions.

The nature and origin of DNA single-strand breaks determined with the comet assay.

After treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS) and hydrogen peroxide, the level of alkali-labile sites and single-strand breaks (ssb) in DNA was investigated, using the comet assay. The ability of antioxidant pre-treatment to decrease DNA damage was assessed. Results showed the following. (a) All single-strand (ss) DNA breaks detected immediately after MNNG- and MMS-treatment in hamster V79 cells had the character of alkali-labile sites while true ssb of DNA were represented only as a minor statistically significant (p < 0.01) fraction at the highest MMS concentration. (b) Most ss DNA breaks detected immediately after H2O2-treatment had the character of true breaks in DNA and alkali-labile sites represented only a minor fraction. (c) Pre-treatment of hamster V79 and human CaCo2 cells with vitamin E significantly reduced the number of breaks induced by hydrogen peroxide, but has no effect on the level of breaks induced by MNNG or MMS. We suggest that MNNG and MMS do not induce significant oxidative damage of DNA. Most of breaks induced by hydrogen peroxide have the nature of oxidative lesions of DNA. (d) In contrast to the effect of vitamin E, stobadine (STB) decreased not only the breaks induced by hydrogen peroxide but also those induced by MNNG and MMS. The reduced level of DNA damage in STB pre-treated samples could be due to inactivation of these alkylating agents by STB.