Vanadyl sulfate can differentially damage DNA in human lymphocytes and HeLa cells

Using the comet assay, we showed that vanadyl sulfate induced DNA damage in human normal lymphocytes and in HeLa cells. Vanadyl at 0.5 and 1 mM produced DNA single- and double-strand breaks (SSBs and DSBs) in lymphocytes, whereas in HeLa cells we observed only SSBs. Post-treatment of vanadyl-damaged DNA from lymphocytes with formamidopyrimidine-DNA glycosylase (Fpg), an enzyme recognizing oxidized purines, gave rise to a significant increase in the extent of DNA damage. A similar effect was observed in HeLa cells, but, using endonuclease III, we also detected oxidized pyrimidines in DNA of these cells. There were no differences in the extent of DNA damage in the lymphocytes and HeLa cells in the pH >13 and pH 12.1 conditions of the comet assay, which indicates that strand breaks, and not alkali-labile sites, contributed to the measured DNA damage. Study of DNA repair, determined in the comet assay as an ability of cells to decrease of DNA damage, revealed that HeLa cells retained the ability to repair vanadyl-damaged DNA induced at a ten-fold higher concentration than that in lymphocytes. Incubation of the cells with nitrone spin traps DMPO, POBN and PBN decreased the extent of DNA damage, which might follow from the production of free radicals by vanadyl sulfate. The presence of vitamins A, C or E caused an increase of DNA damage in HeLa cells whereas in lymphocytes such an increase was observed only for vitamin C. Our data indicate that vanadyl sulfate can be genotoxic for normal and cancer cells. It seems to have a higher genotoxic potential for cancer cells than for normal lymphocytes. Vitamins A, C and E can increase this potential.     

Modulation of ethoxyquin genotoxicity by free radical scavengers and DNA damage repair in human lymphocytes

N-tert-butyl-α-phenylnitrone (PBN) and its new derivative N-(Pyridine-4-ylmethylidene)-2-carboxy-tert-butylamine N-oxide (PBNC) were synthesized and used to modulate ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline, EQ) genotoxicity. Ethoxyquin, an antioxidant used mainly as a preservative in animal feeds, was shown to cause DNA breaks in human lymphocytes. The aim of the study was to evaluate the involvement of free radicals in the genotoxicity of EQ and its modulation by cellular repair systems. Human lymphocytes treated with EQ (10–50 μM) and nitrone free radical scavengers (100 μM) were tested with the comet assay. It was shown that both PBN and PBNC reduced the level of EQ-induced DNA damage, but PBN was slightly more effective. The modulation of the level of DNA damage was also observed as a result of DNA repair by cellular repair systems. Moreover, induction of oxidized bases by ethoxyquin was showed; lymphocytes exposed to ethoxyquin and treated with endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (FpG), enzymes recognizing oxidized bases, displayed greater extent of DNA damage than those not treated with the enzymes.     

Suppression of a DNA base excision repair gene, hOGG1, increases bleomycin sensitivity of human lung cancer cell line

Bleomycin (BLM) has been found to induce 8-oxoguanine and DNA strand breaks through producing oxidative free radicals, thereby leading to cell cycle arrest, apoptosis and cell death. Cellular DNA damage repair mechanisms such as single strand DNA break repair/base excision repair (BER) are responsible for removing bleomycin-induced DNA damage, therefore confer chemotherapeutic resistance to bleomycin. In this study, we have investigated if down-regulation of human 8-oxoguanine DNA glycosylase (hOGG1), an important BER enzyme, could alter cellular sensitivity to bleomycin, thereby reducing chemotherapeutic resistance in human tumor cell. A human lung cancer cell line with hOGG1 deficiency (A549-R) was created by ribozyme gene knockdown technique. Bleomycin cellular sensitivity and DNA/chromosomal damages were examined using MTT, colony forming assay, comet assay as well as micronucleus assay. We demonstrated that hOGG1 gene knockdown enhanced bleomycin cytotoxicity and reduced the ability of colony formation of the lung cancer cell lines. We further demonstrated that bleomycin-induced DNA strand breaks resulted in an increase of micronucleus rate. hOGG1 deficiency significantly reduced DNA damage repair capacity of the lung cancer cell lines. Our results indicated that hOGG1 deficiency allowed the accumulation of bleomycin-induced DNA damage and chromosomal breaks by compromising DNA damage repair capacity, thereby increasing cellular sensitivity to bleomycin.     

DNA damage caused by bisphenol A and estradiol through estrogenic activity

Evidence exists that raises concern about genotoxic effects induced by estrogen: oxidative stress caused by estrogen-derived oxidants, DNA adducts formed by estrogen metabolites and estrogen-induced chromosomal aberration. Estrogen receptors (ER) participate in some of these genotoxic effects by estrogen. In this study, we showed the effects of bisphenol A (BPA), an endocrine-disrupting chemical eliciting weak estrogenic activity, and of 17beta-estradiol (E2), on DNA damage in ER-positive MCF-7 cells by Comet assay. Higher concentrations of BPA, more than 1000 times of E2, were needed to induce the same levels of effects by E2. Immunofluorescence microscopy showed that gammaH2AX, an early marker of DNA breaks, increased after treatment with E2 or BPA in MCF-7 cells. gammaH2AX foci colocalized with Bloom helicase, which is considered to be responsible for the repair of DNA damage after treatment with E2 or BPA. Interestingly, DNA damage was not as severe in ER-negative MDA-MB-231 cells as in MCF-7 cells. The ER antagonist ICI182780 blocked E2 and BPA genotoxic effects on MCF-7 cells. These results together suggest that BPA causes genotoxicity ER dependently in the same way as E2.     

Pro-oxidant Induced DNA Damage in Human Lymphoblastoid Cells: Homeostatic Mechanisms of Genotoxic Tolerance

Oxidative stress contributes to many disease etiologies including ageing, neurodegeneration, and cancer, partly through DNA damage induction (genotoxicity). Understanding the i nteractions of free radicals with DNA is fundamental to discern mutation risks. In genetic toxicology, regulatory authorities consider that most genotoxins exhibit a linear relationship between dose and mutagenic response. Yet, homeostatic mechanisms, including DNA repair, that allow cells to tolerate low levels of genotoxic exposure exist. Acceptance of thresholds for genotoxicity has widespread consequences in terms of understanding cancer risk and regulating human exposure to chemicals/drugs. Three pro-oxidant chemicals, hydrogen peroxide (H(2)O(2)), potassium bromate (KBrO(3)), and menadione, were examined for low dose-response curves in human lymphoblastoid cells. DNA repair and antioxidant capacity were assessed as possible threshold mechanisms. H(2)O(2) and KBrO(3), but not menadione, exhibited thresholded responses, containing a range of nongenotoxic low doses. Levels of the DNA glycosylase 8-oxoguanine glycosylase were unchanged in response to pro- oxidant stress. DNA repair-focused gene expression arrays reported changes in ATM and BRCA1, involved in double-strand break repair, in response to low-dose pro-oxidant exposure; however, these alterations were not substantiated at the protein level. Determination of oxidatively induced DNA damage in H(2)O(2)-treated AHH-1 cells reported accumulation of thymine glycol above the genotoxic threshold. Further, the H(2)O(2) dose-response curve was shifted by modulating the antioxidant glutathione. Hence, observed pro- oxidant thresholds were due to protective capacities of base excision repair enzymes and antioxidants against DNA damage, highlighting the importance of homeostatic mechanisms in "genotoxic tolerance."     

The modulatory effect of melatonin on genotoxicity of irinotecan in healthy human lymphocytes and cancer cells

Drug-target interactions can be modified by adding modulatory agents to increase treatment efficacy and clinical outcome. Combination chemotherapy has become increasingly important because drugs acting synergistically can achieve therapeutic effects at substantially lower doses and with a limited spectrum of side effects. Irinotecan, known as one of the camptothecin analogs, has shown a broad spectrum of antitumor activity against various malignancies. In this study, we evaluated the effect of melatonin on the genotoxic activity of irinotecan in healthy human lymphocytes and a lung cancer cell line (A549) and a colorectal adenocarcinoma cell line (HT29) in vitro. Irinotecan, as a single agent, was shown to induce DNA damage in all types of analyzed cells. The combination of melatonin at concentrations of 50 μM with increasing doses of irinotecan (7.5, 15, 30, and 60 μM) resulted in an increase in the amount of DNA damage in A549 and HT29 cancer cells, but was not effective in inducing DNA damage in healthy human lymphocytes. Analysis of the efficacy of DNA repair, performed after 60 and 120 minutes of postincubation, showed the gradual decrease of DNA percentage in comet tails during repair postincubation in all experimental samples. Our results indicate that melatonin can modulate the genotoxic activity of irinotecan and DNA repair efficacy in human cancer cells in vitro. These findings may be supportive for the optimization of therapeutic efficacy in irinotecan treatment.     

Bisphenol A-glycidyl methacrylate induces a broad spectrum of DNA damage in human lymphocytes

Bisphenol A-glycidyl methacrylate (BisGMA) is monomer of dental filling composites, which can be released from these materials and cause adverse biologic effects in human cells. In the present work, we investigated genotoxic effect of BisGMA on human lymphocytes and human acute lymphoblastic leukemia cell line (CCRF-CEM) cells. Our results indicate that BisGMA is genotoxic for human lymphocytes. The compound induced DNA damage evaluated by the alkaline, neutral, and pH 12.1 version of the comet assay. This damage included oxidative modifications of the DNA bases, as checked by DNA repair enzymes EndoIII and Fpg, alkali-labile sites and DNA double-strand breaks. BisGMA induced DNA-strand breaks in the isolated plasmid. Lymphocytes incubated with BisGMA at 1 mM were able to remove about 50% of DNA damage during 120-min repair incubation. The monomer at 1 mM evoked a delay of the cell cycle in the S phase in CCRF-CEM cells. The experiment with spin trap—DMPO demonstrated that BisGMA induced reactive oxygen species, which were able to damage DNA. BisGMA is able to induce a broad spectrum of DNA damage including severe DNA double-strand breaks, which can be responsible for a delay of the cell cycle in the S phase.     

DNA damage and repair in BCR/ABL-expressing cells after combined action of idarubicin,STI571 and amifostine

STI571 is a specific ABL family tyrosine kinases inhibitor approved for treatment of leukemias. It can differentially modulate the action of other antileukemic drugs. We have recently shown that deregulation of the mechanisms of DNA damage and repair in BCR/ABL-positive cells may be involved in drug resistance of these cells, and thus determine the response of cancer cells to therapy. In the present work we investigated DNA damage and repair induced by idarubicin in the presence of STI571 and amifostine, a normal cell protector, in the BCR/ABL fusion tyrosine kinase-expressing cell line. Amifostine increased the viability of both kinds of cells in the absence of STI571, but had no effect in the presence of the inhibitor. STI571 did not change the response of both BCR/ABL-expressing cells and their control counterparts to idarubicin in terms of DNA damage and repair. However, the presence of amifostine modulated the response of the cells. In the absence of STI571 amifostine decreased the DNA-damaging effect of idarubicin in normal cells and increased it in BCR/ABL-positive cells. STI571 at 2 M abolished the protective effect of amifostine against idarubicin in normal cells and diminished the magnitude of the amifostine-induce increase in cancer cells. These results suggest that amifostine should be applied with special caution in idarubicin-based chemotherapies of BCR/ABL-positive leukemias involving STI571 inhibitor.     

Phthalates inhibit tamoxifen-induced apoptosis in MCF-7 human breast cancer cells

Environmental estrogens represent a class of compounds that can mimic the function or activity of the endogenous estrogen 17 -estradiol (E2). Phthalates including butyl benzyl phthalate (BBP), di(n-butyl) phthalate (DBP), and di(2-ethylhexyl) phthalate (DEHP) are used as plasticizers, and also widely used in food wraps and cosmetic formulations. Phthalates have been shown to mimic estrogen and are capable of binding to the estrogen receptor (ER). It has been demonstrated that estrogen promotes drug resistance to tamoxifen (TAM) in breast cancer. In order to further evaluate the potential role of the phthalates as environmental estrogens, the effect of phthalates was investigated on TAM-induced apoptosis in MCF-7 human breast cancer cells. Our results show that phthalates, BBP (100 M), DBP (10 M), and DEHP (10 M), significantly increased cell proliferation in MCF-7, but not in MDA-MB-231 cells. In addition, BBP, DBP, and DEHP mimicked estrogen in the inhibition of TAM-induced apoptosis in MCF-7 cells. Our data suggest that the inhibitory effect of phthalates on TAM-induced apoptosis involves an increase in intracellular Bcl-2 to Bax ratio. Given that the phthalates are widely used in cosmetics mainly for women, our findings that revealed the promoting effect of BBP, DBP, and DEHP on chemotherapeutic drug resistance to TAM in breast cancer may be of biological relevance.     

Menadione-induced DNA damage in a human tumor cell line.

The nature and extent of menadione (MD)-induced DNA damage were explored using the human breast cancer cell line MCF-7. Concentration-dependent single-strand (ss) and double-strand (ds) DNA breaks were detected in MD-treated MCF-7 cells using the alkaline- and neutral-elution techniques, respectively. The repair of ss and ds DNA breaks was extensive but not complete after a 6-hr incubation in drug-free medium. Evidence was found for the production of DNA interstrand cross-links in MCF-7 cells treated with the bifunctional alkylating agent, mitomycin C, but not for cells treated with MD. Exposure of MCF-7 cells to etoposide (VP-16), mitoxantrone and camptothecin resulted in the detection of significant amounts of protein-linked DNA breaks, whereas none were found in MD-treated cells. These results support the proposition that MD-induced DNA damage is not likely to be mediated via topoisomerases, nor do significant amounts of protein-linked DNA form in MD-treated cells. Thus, MD serves as a good model for examination of the role of the quinone moiety in DNA damage in relation to redox cycling. Future studies directed at elucidation of the biochemical determinants mediating formation of reactive oxygen species effecting the MD-induced DNA damage are necessary and underway.     

Genotoxicity and cytotoxicity of ZnO and Al2O3 nanoparticles

Abstract

Objectives: Metal oxide nanoparticles (ZnO-NPs and Al₂O₃-NPs) are used in many fields, including consumer products and biomedical applications. As a result, exposure to these NPs is highly frequent, however, no conclusive information on their potential cytotoxicity and genotoxicity mechanisms are available. For this reason, we studied cytotoxic and genotoxic effects of ZnO-NPs and Al₂O₃-NPs on human peripheral blood lymphocytes.

Materials and methods: We obtained our goals by using MTT assay, Annexin V-FITC flow cytometry, and alkaline, neural and pH 12.1 versions of comet assay.

Results: Exposure of lymphocytes to both NPs for 24?h slightly decreased viability of lymphocytes at ≥0.5?mM. For the first time, we revealed using the comet assays that both ZnO-NPs and Al₂O₃-NPs caused a concentration-dependent increase of DNA single-strand breaks, but not alkali-labile sites. Treatment with DNA glycosylases showed that the NPs induced oxidative DNA damage. DNA damage caused by both nanoparticles at 0.05?mM was removed within 120?min, however lymphocytes did not repair DNA damage induced by 0.5?mM NPs. Studied nanoparticles did not induce apoptosis in lymphocytes.

Conclusion: Our results suggest that ZnO-NPs and Al₂O₃-NPs at concentration up to 0.5?mM did not exhibit cytotoxic effect but may exert genotoxic effect on lymphocytes, at least partially by the generation of oxidative DNA damage and strand breaks.     

Amifostine protection against induced DNA damage in γ‐irradiated Escherichia coli cells depend on recN DNA repair gene product activity

Amifostine is the most effective radioprotector known and the only one accepted for clinical use in cancer radiotherapy. In this work, the antigenotoxic effect of amifostine against γ‐rays was studied in Escherichia coli cells deficient in DNA damage repair activities. Assays of irradiated cells treated with amifostine showed that the drug reduced the genotoxicity induced by radiation in E. coli wild‐type genotypes and in uvr, recF, recB, recB‐recC‐recF mutant strains, but not in recN defective cells. Thus, the mechanism of DNA protection by amifostine against γ‐radiation‐induced genotoxicity appears to involve participation of the RecN protein that facilitates repair of DNA double‐strand breaks. The results are discussed in relation to amifostine's chemopreventive potential. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2010.     

Non-homologous end joining pathway is the major route of protection against 4β-hydroxywithanolide E-induced DNA damage in MCF-7 cells

4β-Hydroxywithanolide E is a bioactive withanolide extracted from Physalis peruviana. 4β-Hydroxywithanolide E caused reactive oxygen species production and cell apoptosis in human breast cancer MCF-7 cells. We further found that 4β-hydroxywithanolide E induced DNA damage and regulated the DNA damage signaling in MCF-7 cells. The DNA damage sensors and repair proteins act promptly to remove DNA lesions by 4β-hydroxywithanolide E. The ataxia-telangiectasia mutated protein (ATM)-dependent DNA damage signaling pathway is involved in 4β-hydroxywithanolide E-induced apoptosis of MCF-7 cells. Non-homologous end joining pathway, but not homologous recombination, is the major route of protection of MCF-7 cells against 4β-hydroxywithanolide E-induced DNA damage. 4β-Hydroxywithanolide E had no significant impact on the base excision repair pathway. In this study, we examined the 4β-hydroxywithanolide E-induced DNA damage as a research tool in project investigating the DNA repair signaling in breast cancer cells. We also suggest that 4β-hydroxywithanolide E assert its anti-tumor activity in carcinogenic progression and develop into a dietary chemopreventive agent.     

Genotoxicity of tryptophol in a battery of short-term assays on human white blood cells in vitro

The genotoxic effects of tryptophol (indole-3-ethanol), an aromatic alcohol and known secondary metabolite of the opportunistic yeast Candida albicans and other Candida spp., were studied using a battery of short-term assays on human white blood cells in vitro. The concentration range of tryptophol tested was 0.25 mM to 2.00 mM. Lymphocyte viability and induction of apoptosis/necrosis were studied by simultaneous use of a fluorescent assay with ethidium bromide and acridine orange. Levels of primary DNA damage and dynamics of DNA repair were evaluated using the alkaline comet assay while the levels and nature of residual DNA damage were assessed by the analysis of structural chromosome aberrations, the sister chromatid exchange test and the cytokinesis-block micronucleus assay. The results obtained suggest cytotoxic, cytostatic and genotoxic effects of the tryptophol treatment in vitro that were mainly dose-dependent. The type and the extent of DNA lesions detected in tryptophol-treated samples indicate the possibility that observed damage is mediated by highly reactive aldehyde metabolite and/or free radicals produced by treatment. The results show that mortality of lymphocytes in tryptophol-treated samples was primarily caused by apoptosis. The generation of additional DNA strand breaks and cytogenetic consequences (chromosome aberrations, sister chromatid exchanges and micronuclei), as observed in this study, sustain the possibility that tryptophol toxicity is mediated by the formation of DNA cross-links and aldehyde-protein adducts. In conclusion, this preliminary study elucidates only a part of tryptophol toxicity to human cells. Because current evidence is not sufficient to obtain information relevant for human risk assessment, further in vitro and in vivo studies are essential in order to clarify remaining issues, especially to elucidate the exact mechanisms and nature of the damage produced following treatment as well to estimate possible interindividual variability in genotoxic responses to the chemical.     

Oxoguanine glycosylase 1 (OGG1) protects cells from DNA double-strand break damage following methylmercury (MeHg) exposure

Methylmercury (MeHg) is a potent neurotoxin, teratogen, and probable carcinogen, but the underlying mechanisms of its actions remain unclear. Although MeHg causes several types of DNA damage, the toxicological consequences of this macromolecular damage are unknown. MeHg enhances oxidative stress, which can cause various oxidative DNA lesions that are primarily repaired by oxoguanine glycosylase 1 (OGG1). Herein, we compared the response of wild-type and OGG1 null (Ogg1(-/-)) murine embryonic fibroblasts to environmentally relevant, low micromolar concentrations of MeHg by measuring clonogenic efficiency, cell cycle arrest, DNA double-strand breaks (DSBs), and activation of the DNA damage response pathway.Ogg1(-/-) cells exhibited greater sensitivity to MeHg than wild-type controls, as measured by the clonogenic assay, and showed a greater propensity for MeHg-initiated apoptosis. Both wild-type and Ogg1(-/-) cells underwent cell cycle arrest when exposed to micromolar concentrations of MeHg; however, the extent of DSBs was exacerbated in Ogg1(-/-) cells compared with that in wild-type controls. Pretreatment with the antioxidative enzyme catalase reduced levels of DSBs in both wild-type and Ogg1(-/-) cells but failed to block MeHg-initiated apoptosis at micromolar concentrations. Our findings implicate reactive oxygen species mediated DNA damage in the mechanism of MeHg toxicity; and demonstrate for the first time that impaired DNA repair capacity enhances cellular sensitivity to MeHg. Accordingly, the genotoxic properties of MeHg may contribute to its neurotoxic and teratogenic effects, and an individual's response to oxidative stress and DNA damage may constitute an important determinant of risk.     

Bleomycin genotoxicity and amifostine (WR-2721) cell protection in normal leukocytes vs. K562 tumoral cells

Recent advances in chemotherapy have focused on the benefit of high dose regimens, increasing the dose intensity of conventional chemotherapy. However, unacceptable cytotoxicity and genotoxicity on normal cells often impairs the proper management of patients. Phosphoaminothiol WR-1065, the active metabolite of amifostine, appears to protect normal cells and tissues against cytotoxic exposure to radiation or chemotherapeutic agents. Nevertheless, there is disagreement in findings on amifostine protection against bleomycin-induced severe side effects which have suggested that amifostine effectiveness against bleomycin-induced genotoxicity in normal leukocytes and tumour line cells K562 be studied. DNA damage was detected by single cell gel electrophoresis (or Comet) assay, a technique able to detect DNA strand breaks, alkali-labile sites and incomplete excision repair events in individual cells and which appears to be an ideal tool for assessing variability in response of different cell types in vitro. WR-2721 appears to selectively protect healthy leukocytes but not K562 tumoral cells. On the other hand, data on the inter- and intra-individual sensitivity to bleomycin and amifostine suggest that individual metabolic/genetic differences and other factors relating to lifestyle may be responsible for response variability. Application of the Comet assay in appropriate clinical settings to test the sensitivity of patients when undergoing chemotherapy appears possible.     

γH2AX:DNA双链断裂的标志

γH2AX是目前国内外研究细胞DNA损伤应激反应的热点之一。细胞在电离辐射或其他因素作用下直接诱导或是通过复制压力诱导形成的双链断裂(DSBs) ,以及细胞自身调控的程序性DSBs和逆转录病毒转染细胞过程中产生的DSBs均可诱导H2AX的磷酸化(γH2AX)和簇集。本文对H2AX及其组蛋白家族,以及γH2AX与DSBs之间的关系及可能作为一个探测DNA损伤的新分子探针来利用作一简要综述。     

The secretome of non-tumorigenic mammary cells MCF-10A elicits DNA damage in MCF-7 and MDA-MB-231 breast cancer cells

Radiotherapy is used in breast cancer to destroy tumor cells lingering after surgery. It is accepted that lethal effects of ionizing radiation occur as a result of damage to DNA in irradiated (IR) cells. However, response mechanisms may promote cell survival with efficient DNA repair or genomic alterations. Chromosomal aberrations are frequent in surviving cells and may enhance chromosomal instability (CIN) which is associated with increased risk of recurrence and metastasis. Intercellular communication can affect the response in IR cells and cause damage in non-irradiated (N-IR) cells. We evaluated the effect of the secretome of non-tumorigenic mammary cells (MCF-10A) on proliferation and DNA damage in breast cancer cells (MCF-7 and MDA-MB-231). Results showed that conditioned media from IR and N-IR MCF-10A cells produced cycles of DNA double-strand breaks in N-IR and IR tumor cells leaving them with residual damage. CIN markers (micronuclei, nucleoplasmic bridges, nuclear buds) were also increased in IR and N-IR tumor cells, being the effect of conditioned media from IR MCF-10A greater in many cases. The inhibition of phosphorylation/activation of Src kinase in cancer cells hindered CIN markers' increment. Besides, clonogenic survival of tumor cells was differentially modulated by conditioned media from MCF-10A: decreased in MCF-7 and enhanced in MDA-MB-231 cells. These results signal the relevance of tumor-host interaction in tumor behavior and the response to radiotherapy.     

Amifostine (WR2721) Confers DNA Protection to In Vivo Cisplatin-Treated Murine Peripheral Blood Leukocytes

Amifostine [S-2-3-aminopropil amino ethyl phosphorotioic acid], a modulator agent for antineoplastic drugs involved in free radicals generation has given controversial results in cisplatin treated leukocytes in vitro. We have evaluated the amifostine protection over leukocytes in vivo, using comet assay. Groups of five OF1 male mice were given one of three doses of amifostine (56, 105 and 200 mg/Kg) after a cisplatin single injection (10 mg/Kg). Serum malonyldialdehide levels, catalase and superoxide dismutase activity were also evaluated. Amifostine showed significant DNA protection (p< 0.01) at the two lower doses evaluated. Malonyldildehide decreased in all amifostine treatments with respect to cisplatin while antioxidant enzyme activities remained unchanged. However, DNA migration increased with the highest amifostine dose; in fact highest dose of amifostine did no protect damage caused by cisplatin this result have implications on amifostine treatment schedules in clinical practice.