DNA damage and repair in type 2 diabetes mellitus

DNA damage may be associated with type 2 diabetes mellitus (T2DM) and its complications mainly through oxidative stress. Little is known about DNA repair disturbances potentially contributing to the overall extent of DNA damage in T2DM, which, in turn, may be linked with genomic instability resulting in cancer. To assess whether DNA repair may be perturbed in 2DM we determined: (1) the level of endogenous basal DNA damage, this means damage recognized in the alkaline comet assay (DNA strand breaks and alkali labile sites) as well as endogenous oxidative and alkylative DNA damage (2) the sensitivity to DNA-damaging agents hydrogen peroxide and doxorubicin and the efficacy of removing of DNA damage induced by these agents in peripheral blood lymphocytes of T2DM patients and healthy individuals. The level of DNA damage and the kinetics of DNA repair was evaluated by the alkaline single cell gel electrophoresis (comet assay). Oxidative and alkylative DNA damage were assayed with the use of DNA repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), recognizing oxidized DNA bases and 3-methyladenine-DNA glycosylase II (AlkA) recognizing alkylated bases. The levels of basal endogenous and oxidative DNA damage in diabetes patients were higher than in control subjects. There was no difference between the level of alkylative DNA in the patients and the controls. Diabetes patients displayed higher susceptibility to hydrogen peroxide and doxorubicin and decreased efficacy of repairing DNA damage induced by these agents than healthy controls. Our results suggest that type 2 diabetes mellitus may be associated not only with the elevated level of oxidative DNA damage but also with the increased susceptibility to mutagens and the decreased efficacy of DNA repair. These features may contribute to a link between diabetes and cancer and metrics of DNA damage and repair, measured by the comet assay, may be markers of risk of cancer in diabetes.     

DNA damage and repair in Helicobacter pylori-infected gastric mucosa cells

Helicobacter pylori is a common human pathogen and its infection is believed to contribute to gastric cancer. Impaired DNA repair may fuel up cancer transformation by the accumulation of mutation and increased susceptibility to exogenous carcinogens. To evaluate the role of infection of H. pylori in DNA damage and repair we determined: (1) the level of endogenous basal, oxidative and alkylative DNA damage, and (2) the efficacy of removal of DNA damage induced by hydrogen peroxide and the antibiotic amoxicillin in the H. pylori-infected and non-infected GMCs. DNA damage and the efficacy of DNA repair were evaluated by the alkaline single cell gel electrophoresis (comet assay). Specific damage to the DNA bases were assayed with the DNA repair enzymes formamidopyrimidine-DNA glycosylase (Fpg) recognizing oxidized DNA bases and 3-methyladenine-DNA glycosylase II (AlkA) recognizing alkylated bases. The level of basal and oxidative DNA in the infected GMCs was higher than non-infected cells. H. pylori-infected GMCs displayed enhanced susceptibility to hydrogen peroxide than control cells. There was no difference between the efficacy of DNA repair in the infected and non-infected cells after treatment with hydrogen peroxide and amoxicillin. Our results indicate that H. pylori infection may be correlated with oxidative DNA damage in GMCs. Therefore, these features can be considered as a risk marker for gastric cancer associated with H. pylori infection and the comet assay may be applied to evaluate this marker.     

DNA damage in human colonic mucosa cells induced by bleomycin and the protective action of vitamin E

Using the alkaline comet assay, we showed that bleomycin at 0.1-5 microg/ml induced DNA strand breaks and/or alkali-labile sites, measurable as the comet tail moment, in human colonic mucosa cells. This DNA damage was completely repaired during a 120-minute post-treatment incubation of the cells. Post-treatment of the bleomycin-damaged DNA with 3-methyladenine-DNA glycosylase II (AlkA), an enzyme recognizing alkylated bases, gave rise to a significant increase in the extent of DNA damage, indicating that the drug could induce alkylative bases in DNA. We did not observe any change in the comet tail moment in the presence of catalase. Vitamin E ((+)-alpha -tocopherol) decreased DNA damage induced by bleomycin. The results obtained suggest that hydrogen peroxide might not be involved in the formation of DNA lesions induced by bleomycin in the colonic mucosa cells.     

A comparison of the in vitro genotoxicity of anticancer drugs idarubicin and mitoxantrone

Idarubicin is an anthracycline antibiotic used in cancer therapy. Mitoxantrone is an anthracycline analog with presumed better antineoplastic activity and lesser toxicity. Using the alkaline comet assaywe showed that the drugs at 0.01-10 microM induced DNA damage in normal human lymphocytes. The effect induced by idarubicin was more pronounced than by mitoxantrone (P < 0.001). The cells treated with mitoxantrone at 1 microM were able to repair damage to their DNA within a 30-min incubation, whereas the lymphocytes exposed to idarubicin needed 180 min. Since anthracyclines are known to produce free radicals, we checked whether reactive oxygen species might be involved in the observed DNA damage. Catalase, an enzyme inactivating hydrogen peroxide, decreased the extent of DNA damage induced by idarubicin, but did not affect the extent evoked by mitoxantrone. Lymphocytes exposed to the drugs and treated with endonuclease III or formamidopyrimidine-DNA glycosylase (Fpg), enzymes recognizing and nicking oxidized bases, displayed a higher level of DNA damage than the untreated ones. 3-Methyladenine-DNA glycosylase II (AlkA), an enzyme recognizing and nicking mainly methylated bases in DNA, increased the extent of DNA damage caused by idarubicin, but not that induced by mitoxantrone. Our results indicate that the induction of secondary malignancies should be taken into account as side effects of the two drugs. Direct strand breaks, oxidation and methylation of the DNA bases can underlie the DNA-damaging effect of idarubicin, whereas mitoxantrone can induce strand breaks and modification of the bases, including oxidation. The observed in normal lymphocytes much lesser genotoxicity of mitoxantrone compared to idarubicin should be taken into account in planning chemotherapeutic strategies.     

Genotoxicity of acrylamide in human lymphocytes

Acrylamide is used in the industry and can be a by-product in a high-temperature food processing. It is reported to interact with DNA, but the mechanism of this interaction is not fully understood. In the present study, we investigated the DNA-damaging potential of acrylamide (ACM) in normal human lymphocytes using the alkaline-, neutral- and 12.1 versions of the comet assay and pulsed-field gel electrophoresis. We also investigated effect of acrylamide on caspase-3 activity as well as its influence on the repair process of hydrogen peroxide-induced DNA damage. Acrylamide at 0.5-50 microM induced mainly alkali-labile sites. This damage was repaired during a 60-min repair incubation. Post-treatment of the damaged DNA with repair enzymes: thymine glycol DNA N-glycosylase (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), recognizing oxidized DNA bases, as well as 3-methyladenine-DNA glycosylase II (Alk A), recognizing alkylated bases, caused an increase in the extent of DNA damage, indicating the induction of oxidative and alkylative DNA base modifications by acrylamide. Pre-treatment of the lymphocytes with N-tert-butyl-alpha-phenylnitrone (PBN), a spin trap, as well as vitamins C and E decreased the DNA-damaging effect of acrylamide, which suggest that free radicals/reactive oxygen species may be involved in this effect. Acrylamide impaired the repair of DNA damaged by hydrogen peroxide and increased the activity of caspase-3, which may indicate its potential to induce apoptosis. Our results suggest that acrylamide may exert a wide spectrum of diverse effects on DNA of normal cells, including mostly DNA base modifications and apoptosis. Acrylamide may also impair DNA repair. Free radicals may underline these effects and some dietary antioxidants can be considered as protective agents against genotoxic action of acrylamide. As normal lymphocytes contain cyp2e1 and P450, engaged in the bioactivation of ACM to glicidamide it is uncertain whether acrylamide causes all of measured effect per se or this is the result of the action of its metabolites.     

In vitro effect of gliclazide on DNA damage and repair in patients with type 2 diabetes mellitus (T2DM)

Type 2 diabetes mellitus is associated with elevated level of oxidative stress, which is one of the most important factors responsible for the development of chronic complications of this disease. Moreover, it was shown that diabetic patients had increased level of oxidative DNA damage and decreased effectiveness of DNA repair. These changes may be associated with increased risk of cancer in T2DM patients, since DNA damage and DNA repair play a pivotal role in malignant transformation. It was found that gliclazide, an oral hypoglycemic drug with antioxidant properties, diminished DNA damage induced by free radicals. Therefore, the aim of the present study was to evaluate the in vitro impact of gliclazide on: (i) endogenous basal and oxidative DNA damage, (ii) DNA damage induced by hydrogen peroxide and (iii) the efficacy of DNA repair of such damage. DNA damage and DNA repair in peripheral blood lymphocytes of 30 T2DM patients and 30 non-diabetic individuals were evaluated by alkaline single cell electrophoresis (comet) assay. The extent of oxidative DNA damage was assessed by DNA repair enzymes: endonuclease III and formamidopyrimidine-DNA glycosylase. The endogenous basal and oxidative DNA damages were higher in lymphocytes of T2DM patients compared to non-diabetic subjects and gliclazide decreased the level of such damage. The drug significantly decreased the level of DNA damage induced by hydrogen peroxide in both groups. Gliclazide increased the effectiveness of DNA repair in lymphocytes of T2DM patients (93.4% (with gliclazide) vs 79.9% (without gliclazide); P< or =0.001) and non-diabetic subjects (95.1% (with gliclazide) vs 90.5% (without gliclazide); P< or =0.001). These results suggest that gliclazide may protect against the oxidative stress-related chronic diabetes complications, including cancer, by decreasing the level of DNA damage induced by reactive oxygen species.     

Association between DNA damage, DNA repair genes variability and clinical characteristics in breast cancer patients

The cell's susceptibility to DNA damage and its ability to repair this damage are important for cancer induction, promotion and progression. In the present work we determined the level of basal (total endogenous) and endogenous oxidative DNA damage as well as polymorphism of the DNA repair genes: RAD51 (135 G/C), XRCC3 (Thr241Met), OGG1 (Ser326Cys) and XPD (Lys751Gln) in peripheral blood lymphocytes of 41 breast cancer patients and 48 healthy individuals. DNA damage was evaluated by alkaline comet assay with DNA repair enzymes: Endo III and Fpg, preferentially recognizing oxidized DNA bases. The genotypes of the polymorphisms were determined by restriction fragment length polymorphism PCR. We observed a strong association between breast cancer occurrence and the genotypes C/C of the RAD51-135G/C polymorphism, Ser/Ser of the OGG1-Ser326Cys and Lys/Gln of the XPD-Lys751Gln, whereas the genotypes G/C of the RAD51-135G/C and Lys/Lys of the XPD-Lys751Gln exerted a protective effect against breast cancer. We also found that individuals with the G/C genotype of the RAD51-135G/C polymorphism and with the Lys/Lys genotype of the XPD-Lys751Gln polymorphism displayed a lower extent of basal and oxidative DNA damage. A strong association between higher level of oxidative DNA damage and the Lys/Gln genotype of the latter polymorphism was found. We also correlated genotypes with clinical characteristics of breast cancer patients. We observed a strong association between the G/C genotype of the RAD51-135 G/C polymorphism and the expression of the progesterone receptor and between both alleles of the OGG1-Ser326Cys polymorphism and lymph node metastasis. Our results suggest that the polymorphism of the RAD51, OGG1 and XPD genes may be linked with breast cancer by the modulation of the cellular response to oxidative stress and these polymorphisms may be considered as markers in breast cancer along with the genetic or/and environmental indicators of oxidative stress.     

Imatinib (STI571) induces DNA damage in BCR/ABL-expressing leukemic cells but not in normal lymphocytes

Imatinib (STI571) is a 2-phenylaminopyrimidine derivative used mostly in the treatment of chronic myeloid leukaemia. It targets the BCR/ABL oncogenic tyrosine kinase, inhibiting its activity. Using the alkaline comet assay we showed that STI571 at concentrations ranging from 0.2 to 2 microM induced DNA damage in human leukemic K562 and BV173 cells expressing the BCR/ABL oncogene, whereas it had no effect in normal human lymphocytes and leukemic CCRF-CEM cells without the expression of BCR/ABL. Imatinib did not induce DNA strand breaks in the direct interaction with DNA as examined by the circular plasmid relaxation assay. Because the extent of DNA damage observed in the neutral and pH 12.1 versions of the comet assay was much lesser than in the alkaline version, we concluded that the drug induced DNA alkali-labile sites rather than strand breaks. K562 cells were unable to repair H(2)O(2)-induced DNA damage during a 120-min incubation, if they had been preincubated with STI571, whereas normal lymphocytes did so within 60 min. Pre-treatment of K562 cells with Vitamins A, C and E reduced the extent of DNA damage evoked by STI571. Similar results brought experiments with the nitrone spin traps POBN and PBN, suggesting that free radicals may be involved in the formation of DNA lesions induced by STI571 in K562 cells. These cells exposed to imatinib and treated with endonuclease III, formamidopyrimidine-DNA glycosylase and 3-methyladenine-DNA glycosylase II, the enzymes recognizing oxidized and alkylated bases, displayed greater extent of DNA damage than those not treated with these enzymes. Therefore, the mechanism of the anti-leukemic action of STI571 may involve not only the inhibition of BCR/ABL, but also DNA damage in the cells expressing this fusion protein. DNA damage induced by STI571 may follow from oxidative and alkylative base modifications.     

Genotoxicity of streptozotocin in normal and cancer cells and its modulation by free radical scavengers

Streptozotocin (STZ) is an antibiotic which can be used to induce diabetes in experimental animals in order to have an insight into pathogenesis of this disease. To use STZ as a diabetogenic substance, its molecular mode of action should be elucidated. Using the alkaline comet assay, we showed that STZ at concentrations in the range 0.01-100 micromol/L induced DNA damage in normal human lymphocytes and HeLa cancer cells in a dose-dependent manner. Lymphocytes were able to remove damage to their DNA within a 30-min repair incubation, whereas HeLa cells completed the repair in 60 min. Vitamins C and E at 10 and 50 micromol/L diminished the extent of DNA damage induced by 50 micromol/L STZ. Pretreatment of the lymphocytes with the nitrone spin trap, alpha-(4-pyridil-1-oxide)-N-tert-butylnitrone (POBN) or ebselen, which mimics glutathione peroxidase, or pyrrolidine dithiocarbamate (PDTC) reduced the extent of DNA damage evoked by STZ. The cells exposed to STZ and treated with endonuclease III (Endo III), formamidopyrimidine-DNA glycosylase (Fpg) and 3-methyladenine-DNA glycosylase II (AlkA), the enzymes recognizing oxidized and alkylated bases, displayed greater extent of DNA damage than those not treated with these enzymes. These results suggest that free radicals may be involved in the formation of DNA lesions induced by streptozotocin. The drug can also alkylate DNA bases. This broad range of DNA damage induced by STZ indicates that the drug may seriously affect genomic stability in normal and pathological cells.     

DNA damage and repair in children with Down's syndrome

Down's syndrome (DS) is associated with the presence of a third 21 chromosome and is generally considered as a non-cancer-prone genetic disease. However, leukaemias occur more frequently in children with the syndrome than in general population and there is an open question, whether the presence of an additional chromosome may contribute to genomic instability, which, in turn, may play a role in a higher susceptibility to cancer and leukaemias in particular. In order to assess genomic instability associated with the presence of a third 21 chromosome, we determined the level of endogenous DNA damage and susceptibility to a genotoxic stress-inducing factor, hydrogen peroxide and N-methyl-N'-nitro-N-nitrosoguanidyne (MNNG) as well as the ability to remove DNA damage in the peripheral blood lymphocytes of children with DS and healthy kids. The level of DNA damage and the kinetics of DNA repair were evaluated by alkaline comet assay. Oxidative DNA damage was assayed with DNA repair enzymes: endonuclease III-like NTH1 and formamidopyrimidine-DNA glycosylase. The cells taken from children with DS did not display an effective DNA repair after treatment with 10 mM hydrogen peroxide. No difference in the sensitivity to DNA-damaging agents and the efficacy of DNA repair due to age and gender in DS children was observed. These results suggest that children with DS may be characterized by the increased sensitivity to the DNA-damaging agents impaired cellular reaction to DNA damage, which, in turn, may increase the probability of cancers in these children. Therefore, a special care to avoid exposure to potential mutagenic factor my be considered in these children.     

BRCA1 and BRCA2 protect against oxidative DNA damage converted into double-strand breaks during DNA replication

BRCA1 and BRCA2 mutation carriers are predisposed to develop breast and ovarian cancers, but the reasons for this tissue specificity are unknown. Breast epithelial cells are known to contain elevated levels of oxidative DNA damage, triggered by hormonally driven growth and its effect on cell metabolism. BRCA1- or BRCA2-deficient cells were found to be more sensitive to oxidative stress, modeled by treatment with patho-physiologic concentrations of hydrogen peroxide. Hydrogen peroxide exposure leads to oxidative DNA damage induced DNA double strand breaks (DSB) in BRCA-deficient cells causing them to accumulate in S-phase. In addition, after hydrogen peroxide treatment, BRCA deficient cells showed impaired Rad51 foci which are dependent on an intact BRCA1–BRCA2 pathway. These DSB resulted in an increase in chromatid-type aberrations, which are characteristic for BRCA1 and BRCA2-deficient cells. The most common result of oxidative DNA damage induced processing of S-phase DSB is an interstitial chromatid deletion, but insertions and exchanges were also seen in BRCA deficient cells. Thus, BRCA1 and BRCA2 are essential for the repair of oxidative DNA damage repair intermediates that persist into S-phase and produce DSB. The implication is that oxidative stress plays a role in the etiology of hereditary breast cancer.     

DNA damage and repair in lymphocytes of normal individuals and cancer patients: studies by the comet assay and micronucleus tests

A population study is reported in which the DNA damage induced by g-radiation (2 Gy) and the kinetics of the subsequent repair were estimated by the comet and micronucleus assays in isolated lymphocytes of 82 healthy donors and patients with head and neck cancer before radiotherapy. The parameters of background and radiation-induced DNA damage, rate of repair, and residual non-repaired damage were measured by comet assay, and the repair kinetics for every donor were computer-fitted to an exponential curve. The level of background DNA damage before irradiation measured by comet assay as well as the level of micronuclei were significantly higher in the head and neck cancer patient group than in the healthy donors, while the parameters of repair were widely scattered in both groups. Cancer patient group contained significantly more individuals, whose irradiated lymphocytes showed high DNA damage, low repair rate and high non-repaired DNA damage level. Lymphocytes of donors belonging to this subgroup showed significantly lower inhibition of cell cycle after irradiation.     

In vitro genotoxicity of lead acetate: induction of single and double DNA strand breaks and DNA-protein cross-links

Lead is present in the natural and occupational environment and is reported to interact with DNA, but the mechanism of this interaction is not fully understood. Using the alkaline comet assay we showed that lead acetate at 1-100 microM induced DNA damage in isolated human lymphocytes measured the change in the comet tail length. At 1 and 10 microM we observed an increase in the tail length, whereas at 100 microM a decrease was seen. The former effect could follow from the induction of DNA strand breaks and/or alkali-labile sites (ALS), the latter from the formation of DNA-DNA and/or DNA-protein cross-links. No difference was observed between tail length for the alkaline and pH 12.1 versions of the assay, which indicates that strand breaks and not ALS are responsible for the tail length increase induced by lead. The neutral version of the test revealed that lead acetate induced DNA double-strand breaks at all concentrations tested. The presence of spin traps, 5,5-dimethyl-pyrroline N-oxide (DMPO) and N-tert-butyl-alpha-phenylnitrone (PBN) did not influence the level of DNA damage induced by lead. Post-treatment of the lead-damaged DNA (at 100 microM treatment concentration) by endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), enzymes recognizing oxidized DNA bases, as well as 3-methyladenine-DNA glycosylase II, an enzyme recognizing alkylated bases, gave rise to a significant increase in the extent of DNA damage. Proteinase K caused an increase in comet tail length, suggesting that lead acetate might cross-link DNA with nuclear proteins. Vitamin A, E, C, calcium chloride and zinc chloride acted synergistically on DNA damage evoked by lead. The results obtained suggest that lead acetate may induce single-strand breaks (SSB) and double-strand breaks (DSB) in DNA as well as DNA-protein cross-links. The participation of free radicals in DNA-damaging potential of lead is not important and it concerns other reactive species than could be trapped by DMPO or PBN.     

Free radicals-mediated induction of oxidized DNA bases and DNA-protein cross-links by nickel chloride

Using the comet assay, we showed that nickel chloride at 250-1000 microM induced DNA damage in human lymphocytes, measured as the change in comet tail moment, which increased with nickel concentration up to 500 microM and then decreased. Observed increase might follow from the induction of strand breaks or/and alkali-labile sites (ALS) by nickel, whereas decrease from its induction of DNA-DNA and/or DNA-protein cross-links. Proteinase K caused an increase in the tail moment, suggesting that nickel chloride at 1000 microM might cross-link DNA with nuclear proteins. Lymphocytes exposed to NiCl(2) and treated with enzymes recognizing oxidized and alkylated bases: endonuclease III (Endo III), formamidopyrimidine-DNA glycosylase (Fpg) and 3-methyladenine-DNA glycosylase II (AlkA), displayed greater extent of DNA damage than those not treated with these enzymes, indicating the induction of oxidized and alkylated bases by nickel. The incubation of lymphocytes with spin traps, 5,5-dimethyl-pyrroline N-oxide (DMPO) and PBN decreased the extent of DNA damage, which might follow from the production of free radicals by nickel. The pre-treatment with Vitamin C at 10 microM and Vitamin E at 25 microM decreased the tail moment of the cells exposed to NiCl(2) at the concentrations of the metal causing strand breaks or/and ALS. The results obtained suggest that free radicals may be involved in the formation of strand breaks or/and ALS in DNA as well as DNA-protein cross-links induced by NiCl(2). Nickel chloride can also alkylate DNA bases. Our results support thesis on multiple, free radicals-based genotoxicity pathways of nickel.     

Hyperthermia can differentially modulate the repair of doxorubicin-damaged DNA in normal and cancer cells

Hyperthermia can modulate the action of many anticancer drugs, and DNA repair processes are temperature-dependent, but the character of this dependence in cancer and normal cells is largely unknown. This subject seems to be worth studying, because hyperthermia can assist cancer therapy. A 1-h incubation at 37 degrees C of normal human peripheral blood lymphocytes and human myelogenous leukemia cell line K562 with 0.5 microM doxorubicin gave significant level of DNA damage as assessed by the alkaline comet assay. The cells were then incubated in doxorubicin-free repair medium at 37 degrees C or 41 degrees C. The lymphocytes incubated at 37 degrees C needed about 60 min to remove completely the damage to their DNA, whereas at 41 degrees C the time required for complete repair was shortened to 30 min. There was also a difference between the repair kinetics at 37 degrees C and 41 degrees C in cancer cells. Moreover, the kinetics were different in doxorubicin-sensitive and resistant cells. Therefore, hyperthermia may significantly affect the kinetics of DNA repair in drug-treated cells, but the magnitude of the effect may be different in normal and cancer cells. These features may be exploited in cancer chemotherapy to increase the effectiveness of the treatment and reduce unwanted effects of anticancer drugs in normal cells and fight DNA repair-based drug resistance of cancer cells.     

Interaction of amoxicillin with DNA in human lymphocytes and H. pylori-infected and non-infected gastric mucosa cells

Amoxicillin is a penicillin derivative belonging to a group of beta-lactam antibiotics used in Helicobacter pylori eradication. Clinical application of amoxicillin is underlined by its antibacterial activity, but little is known about its interaction with DNA of human cells. Using the alkaline comet assay we investigated the genotoxicity of amoxicillin in human peripheral blood lymphocytes as well as in H. pylori-infected and non-infected human gastric mucosa cells. To assess the role of reactive oxygen species in the genotoxicity of amoxicillin we employed a set of antioxidant and free radical scavengers, including Vitamins C and E, melatonin and the nitrone spin trap N-tert-butyl-alpha-phenyl-nitrone (PBN). Amoxicillin-induced DNA damage was completely repaired after 60 min. The vitamins, melatonin and the spin trap decreased the extent of the damage. The cells exposed to amoxicillin and treated with endonuclease III and 3-methyladenine-DNA glycosylase II, the enzymes recognizing oxidized bases displayed greater extent of DNA damage than those not treated with these enzymes. H. pylori non-infected gastric mucosa cells exposed to hydrogen peroxide repaired their DNA in a 60 min incubation, but the infected cells were not able to do so. The action of DNA repair enzymes, the vitamins, melatonin and PBN indicated that amoxicillin-induced oxidative DNA damage. The drug did not induce DNA strand breaks in isolated pUC19 plasmid DNA. Our results suggest that amoxicillin can induce DNA damage in human lymphocytes and gastric mucosa cells and this effect may follow from the production of reactive oxygen species. Cellular activation of the drug is needed to induce DNA damage. Free radical scavengers and antioxidants may be used to assist H. pylori eradication with amoxicillin to protect DNA of the host cells. Our results suggest also that H. pylori infection may alter gastric mucosa cells response to DNA-damaging agents and in this way contribute to initiation/promotion of cancer transformation of these cells induced by external or internal carcinogens.     

DNA damage in peripheral blood lymphocytes in patients during combined chemotherapy for breast cancer

Combined chemotherapy is used for the treatment of a number of malignancies such as breast cancer. The target of these antineoplastic agents is nuclear DNA, although it is not restricted to malignant cells. The aim of the present study was to assess DNA damage in peripheral blood lymphocytes (PBLs) of breast cancer patients subjected to combined adjuvant chemotherapy (5-fluorouracil, epirubicin and cyclophosphamide, FEC), using a modified comet assay to detect DNA single-strand breaks (SSB) and double-strand breaks (DSB).

Forty-one female patients with advanced breast cancer before and after chemotherapy and 60 healthy females participated in the study. Alkaline and neutral comet assays were performed in PBLs according to a standard protocol, and DNA tail moment was measured by a computer-based image analysis system.

Breast cancer patients before treatment had higher increased background levels of SSB and DSB as compared to healthy women. During treatment, a significant increase in DNA damage was observed after the 2nd cycle, which persisted until the end of treatment. Eighty days after the end of treatment the percentage of PBLs with SSB and DSB remained elevated, but the magnitude of DNA damage (tail moment) returned to baseline levels. There was no correlation between PBL DNA damage and response to chemotherapy.

DNA-SSB and DSB in PBLs are present in cancer patients before treatment and increase significantly after combined chemotherapy. No correlation with response to adjuvant chemotherapy was found. Biomonitoring DNA damage in PBLs of cancer patients could help prevent secondary effects and the potential risks of developing secondary cancers.     

Detection of alkylation damage in human lymphocyte DNA with the comet assay

The enzyme 3-methyladenine DNA glycosylase II (AlkA) is a bacterial repair enzyme that acts preferentially at 3-methyladenine residues in DNA, releasing the damaged base. The resulting baseless sugars are alkali-labile, and under the conditions of the alkaline comet assay (single cell gel electrophoresis) they appear as DNA strand breaks. AlkA is no t lesion-specific, but has a low activity even w ith undamagedbases. We have tested the enzyme at different concentrations to find conditions that maximise detection of alkylated bases with minimal attack on normal, undamaged DNA. AlkA detects damage in the DNA of cells treated with low concentrations of methyl methanesulphonate. We also find low background levels of alkylated bases in normal human lymphocytes.