DNA Damage In Peripheral Blood Lymphocytes Research Articles

Protective role of strawberry tree (Arbutus unedo L.) honey against cyto/genotoxic effects induced by ultraviolet B radiation in vitro

Strawberry tree (Arbutus unedo L.) honey (STH) is widely used as part of traditional medicine in the Mediterranean area, especially in the treatment of wounds, burns and infections. In this study, we investigated the cyto-/genoprotective properties of STH at cell level following short-term exposure to UVB radiation at 2 kJ/m2. Isolated human peripheral blood lymphocytes (PBL) were selected as model cells. Phenolics in STH were determined using an ultra-high-performance liquid chromatograph (UHPLC) coupled to a linear ion trap-Orbitrap hybrid mass spectrometer (LTQ Orbitrap MS), while homogentisic acid (HGA) was quantified using a gas chromatograph-mass spectrometer (GC-MS). Primary DNA damage in PBLs treated with STH before and after irradiation was assessed by alkaline comet assay, while their viability was determined using the apoptosis/necrosis assay. The levels of thiobarbituric acid reactive substances (TBARS) and glutathione (GSH) were determined in plasma samples. STH applied as pre-treatment at a concentration equivalent to its average daily portion efficiently counteracted the cytotoxic and genotoxic effects and diminished UVB-induced oxidative stress. Post-radiation treatment with STH was not as beneficial. Protective effects could be associated to the complex phytochemical profile of STH and the high content of HGA (306.8 mg/kg). It was confirmed that an intake of a complex mixture of bioactive constituents as STH prior to potential cyto/genotoxic insults stimulated a broad-spectrum of protective mechanisms, including intracellular antioxidative defence and DNA repair systems that helped pre-treated cells to counteract harmful exposures more efficiently.

Cisplatin Reduces the Frequencies of Radiotherapy-Induced Micronuclei in Peripheral Blood Lymphocytes of Patients with Gynaecological Cancer: Possible Implications for the Risk of Second Malignant Neoplasms.

Gynaecologic cancers are common among women and treatment includes surgery, radiotherapy or chemotherapy, where the last two methods induce DNA damage in non-targeted cells like peripheral blood lymphocytes (PBL). Damaged normal cells can transform leading to second malignant neoplasms (SMN) but the level of risk and impact of risk modifiers is not well defined. We investigated how radiotherapy alone or in combination with chemotherapy induce DNA damage in PBL of cervix and endometrial cancer patients during therapy. Blood samples were collected from nine endometrial cancer patients (treatment with radiotherapy + chemotherapy—RC) and nine cervical cancer patients (treatment with radiotherapy alone—R) before radiotherapy, 3 weeks after onset of radiotherapy and at the end of radiotherapy. Half of each blood sample was irradiated ex vivo with 2 Gy of gamma radiation in order to check how therapy influenced the sensitivity of PBL to radiation. Analysed endpoints were micronucleus (MN) frequencies, apoptosis frequencies and cell proliferation index. The results were characterised by strong individual variation, especially the MN frequencies and proliferation index. On average, despite higher total dose and larger fields, therapy alone induced the same level of MN in PBL of RC patients as compared to R. This result was accompanied by a higher level of apoptosis and stronger inhibition of cell proliferation in RC patients. The ex vivo dose induced fewer MN, more apoptosis and more strongly inhibited proliferation of PBL of RC as compared to R patients. These results are interpreted as evidence for a sensitizing effect of chemotherapy on radiation cytotoxicity. The possible implications for the risk of second malignant neoplasms are discussed.

DNA DAMAGE IN TUMOR CELLS AND PERIPHERAL BLOOD LYMPHOCYTES OF ENDOMETRIAL CANCER PATIENTS ASSESSED BY THE COMET ASSAY

To evaluate the level DNA damage in tumor cells and PBLs of endometrial cancer (EC) patients with regard to clinical and morphological characteristics of the patients. DNA damage was assessed in 106PBLs samples and 42samples of tumor cell suspension from EC patients by comet assay. PBLs from 30healthy women were used as control. The level of DNA damage was expressed as the percentage of DNA in the comet tails (% tail DNA). It was revealed that the amount of DNA damage in PBLs of EC patients was 2.2times higher in comparison with that of healthy donors (8.3 ± 0.7and 3.7 ± 0.4% tail DNA, respectively) (p < 0.05). In this study, no association between the levels of DNA damage in endometrial tumor cells and PBLs was observed (r = 0.11; p > 0.05). The amounts of DNA damage both in tumor cells and PBLs were not related to the degree of tumor differentiation as well as the depth of myometrial invasion, but depended on the body mass index (BMI) of EC patients: high level of lesions was observed in patients with elevated BMI values. Furthermore, the level of DNA damage in tumor cells was associated to familial aggregation of cancer and was significantly higher in endometrial cells from patients with family history of cancer vs that from EC patients with sporadic tumors (32.3 ± 2.9and 22.8 ± 1.8% tail DNA, respectively) (p < 0.05). It was also found that for women who had high level of DNA damage in PBLs, the risk of EC was greater (odds ratio value of 3.5) compared to those with low level of such lesions. Genome instability that appears as an increased level of DNA damage in tumor cells and PBLs of EC patients is associated with BMI and family history of cancer and can reflect a predisposition to cancer.

Unexpected effect of dry olive leaf extract on the level of DNA damage in lymphocytes of lead intoxicated workers, before and after CaNa2EDTA chelation therapy

The CaNa2EDTA chelation therapy is often practiced with antioxidant supplementation. Dry olive leaf extract (DOLE) is natural product with antioxidant and DNA protective properties. The effects of DOLE on the levels of DNA damage were investigated ex vivo in peripheral blood lymphocytes (PBLs) of 19 workers occupationally exposed to lead (Pb), before and after CaNa2EDTA chelation therapy.DOLE demonstrated pronounced radical scavenging activity in concentrations ≥1 mg/mL, and showed no genotoxicity per se, in concentrations 0.125–1 mg/mL. The level of DNA damage in PBLs of workers before chelation therapy was elevated (24.21 ± 14.26) compared to controls (6.0 ± 3.37). The incubation of PBLs before chelation therapy with selected concentration of DOLE lead to a severe increase of DNA damage (64.03 ± 20.96), exhibiting prooxidant rather than antioxidant effect. After the five-day CaNa2EDTA chelation regimen, DNA damage in PBLs of workers decreased (8.26 ± 4.62) significantly compared to baseline. Treatment of PBLs with DOLE after chelation, again produced high level of damage (41.82 ± 23.17) and the acute prooxidant effects of DOLE remained, but, DNA damage was less severe than before chelation. The DOLE exhibits prooxidant effect in presence of Pb in lymphocytes of exposed workers, and its effect is less pronounced following the removal of Pb after standard chelation therapy.

Cytogenetic Damage in the Peripheral Blood Lymphocytes of Thyroid Cancer Patients during Pre and Post 131I Therapy

Aim: 131 I therapy is widely used in patients of differentiated thyroid carcinoma for the ablation of remnant thyroid tissue post thyroidectomy as well as metastasis. These patients are in hypothyroid state with consequent reduction in renal clearance of 131 I thereby increasing the extent of whole body exposure. Present study was carried out to assess the DNA damage due to the presence of thyroid carcinoma as well as posttherapeutic 131 I exposure at 72h by measuring micronuclei (MN) frequency in peripheral blood lymphocytes (PBLs) in patients of differentiated thyroid cancer. Methods: The study group consisted of 25 differentiated thyroid carcinoma patients and 35 healthy donors. The blood samples of the patients were collected pre and post 131 I therapy (1.6-9.3 GBq) and processed for PBL MN frequency by cytokinesis-block MN assay. Results: The mean basal MN frequency was significantly high in thyroid cancer patients as compared to the healthy donors (p< 0.001). 131 I exposed patients demonstrated significant increase in the MN frequency at 72h after therapeutic 131 I exposure compared to their basal MN index ( p<0.001 ). However the percent increase in MN frequency post 131 I therapy did not show any correlation with administered dose in thyroid cancer patients. Conclusion: The study has revealed significant DNA damage in PBLs as indicated by increased PBL MN frequency post 131 I therapy. However marked individual variations were observed in the DNA damage response to 131 I therapy.

Impaired DNA Damage Repair Capacity is Associated with an Increased Risk of Esophageal Adenocarcinoma: A Case Control Study

Background: Inherited suboptimal DNA repair capacity in peripheral blood lymphocytes (PBLs) can be unmasked by mutagen challenge and has been associated with susceptibility to cancer. Purpose: To use comet assay to assess the esophageal adenocarcinoma (EAC) risk in relation to mutageninduced DNA damage in PBLs. Materials and methods: In a case-control study, the baseline, benzo[a]pyrene diol epoxide (BPDE)-induced, and γ radiation-induced DNA damage were quantified by the Olive tail moment (TM) in PBLs from 172 Caucasian EAC patients and 154 healthy controls who were frequency matched on age and gender. Logistic regression analysis was used to calculate odds ratios (OR) and 95% confidence intervals (CI) to estimate EAC risk in relation to DNA damage. Results: EAC patients tended to have higher DNA damage than controls, as measured by baseline, net BPDEand net γ radiation-induced TM, but the difference was statistically significant only for net BPDE-induced DNA damage (0.88 ± 0.94 vs. 0.62 ± 0.77, P=0.031). Using the 75th percentile TM in the controls as cutoff point, we found that high levels of net BPDE- and γ radiation-induced DNA damage were associated with significantly increased risks of EAC, with adjusted ORs of 2.15 (95% CI, 1.13-4.10) and 2.27 (95% CI, 1.24–4.16), respectively. EAC risks were further increased among individuals with both net mutagen-induced DNA damages and exposure to gastroesophageal reflux disease or smoking, known risk factors for EAC. Conclusion: Our results suggest impaired repair capacity of mutagen-induced DNA damage in PBLs assessed by comet assay may be a risk factor of EAC.

DNA repair deficiency in peripheral blood lymphocytes of endometrial cancer patients with a family history of cancer.

BackgroundIndividual susceptibility to endogenous and/or exogenous DNA damage depends on DNA repair efficiency and can be evaluated using the comet assay with bleomycin as genotoxic agent. The aim of the study was to evaluate baseline and bleomycin-induced DNA damage and DNA repair capacity in peripheral blood lymphocytes (PBLs) of endometrial cancer (EC) patients considering a family history of cancer.MethodsDNA damage was analyzed in PBLs of 45 EC patients compared to a control group of 10 healthy women, using the comet assay. The level of DNA damage was determined by the% tail DNA.ResultsThe level of baseline DNA damage in PBLs of EC patients was significantly higher (% DNA in tail 9.31 ± 15.32) than in healthy women (% DNA in tail 3.41 ± 4.71) (P <0.01). PBLs of EC patients repaired less bleomycin-induced DNA damage (removed% DNA in tail 63.94 ± 20.92) than PBLs of healthy individuals (removed% DNA in tail 80.24 ± 3.03) (P <0.001). Efficiency of DNA repair in PBLs of EC patients depended on the family history of cancer. The amount of restored damaged DNA was significantly lower (removed% DNA in tail 36.24 ± 14.05%) in EC patients with a family history of cancer compared to patients with sporadic EC (removed% DNA in tail 64.91 ± 19.36%) (P <0.004).ConclusionsLymphocytes of EC patients are characterized by an increased basal level of DNA damage as well as deficiency in DNA repair. DNA repair is less efficient in PBLs of EC patients with a family history of cancer compared to patients with sporadic cancer.

A Decreased Micronucleus Frequency in Human Lymphocytes after Folate and Vitamin B12 Intervention: a Preliminary Study in a Yunnan Population

Micronucleus (MN) is a validated biomarker for DNA damage in peripheral blood lymphocytes (PBL) and is a reflection of the changes of human nutritional status. Folate (FA) and vitamin B(12) are one-carbon metabolism-related micronutrients, which play important roles in maintaining genomic stability. To investigate the correcting effects of FA and B(12) intervention on DNA damage in PBL. One hundred fifty-six volunteers without history of cancer were divided into 5 age groups (20 - 69 y, 47.4 % male) for establishing the baseline of chromosomal damage by means of cytokinesis-block micronucleus assay. Twelve individuals whose MN frequency was higher than the median value in each age group were selected for a four-month FA (200 - 400 μg/day)-B12 (3.125 - 25 μg/day) intervention dosed as to age and MTHFR genotypes. There were significantly positive correlations between age and MN frequency in all groups (p < 0.01). Among all age groups, the baseline MN frequencies were higher in females than that in males. The MN frequencies from 10 volunteers were reduced by 33.5 % after the intervention with the two micronutrients (p < 0.01), and two individuals did not show any changes. Dietary supplement intake of FA and B(12) based on MTHFR genotypes could protect the genome from damage and benefit genome health.

Detecting DNA damage of human lymphocytes exposed to 1,2-DCE with γH2AX identified antibody using flow cytometer assay

To study DNA damage of human peripheral blood lymphocytes exposed to 1,2-dichloroethane (1,2-DCE) with flow cytometry (FCM) assay. The lymphocytes were obtained from 21 workers who are occupationally exposed to 1,2-DCE (exposed group) and 27 workers who were not exposed to 1,2-DCE in the same factory (inner control) and 28 island residents who had never been occupationally exposed to adverse factors (external control). FCM assay was adopted to detect DNA damage of the lymphocytes of each group. Lymphocytes of the health people were incubated with 1,2-DCE at different doses, and FCM assay was used to detect DNA damage. DNA damage rate (%) of the exposed group of exposed workers (4.05% ± 2.55%) was significantly higher than the inner control group of workers (1.97% ± 1.40%) and external control groups of island residents (0.23% ± 0.13%), and the DNA damage of inner control was higher than the external control, all the differences were statistically significant (P < 0.01 or P < 0.05). The geometric mean fluorescence intensity of the workers in the exposed group (3.33 ± 3.01) was significantly higher than the (2.07 ± 0.58) only (P < 0.05). There was no significant difference in the DNA damage rate as well as the geometric mean fluorescence intensity among the exposed group of workers with different years of working period (P > 0.05). In vitro, the fluorescence intensity at the dose of 20, 30 µmol/L for 0.5 h exposure showed statistical significance compared with the negative control group (P < 0.01). The DNA damage rate at the dose of 20, 30 µmol/L for 1.0 h exposure was statistically significant compared with the negative control group (P < 0.05, P < 0.01); The fluorescence intensity at the dose of 10, 20, 30 µmol/L for 1.0 h exposure was statistically significant compared with the negative control group (P < 0.05, P < 0.01). 1,2-DEC can cause DNA damage. And γH2AX FCM assay can be a sensitive, objective and effective method of detecting DNA damage of peripheral blood lymphocytes.

Prevention of DNA damage by polyphenol‐rich food

Epidemiological and experimental data suggest that an increased intake of plant food rich in polyphenols can reduce the risk of cancers. DNA damage is an important step in the initiation of carcinogenesis. Here we report data on the effect of polyphenol rich food such as apples and apple juice on DNA damage in colon epithelial cells from animal studies and in peripheral blood lymphocytes (PBL) from human intervention studies.1,2‐Dimethylhydrazine‐induced DNA strand breaks in colon epithelial cells in rats were significantly inhibited by consumption of cloudy and clear apple juice. In a human intervention study, consumption of 1 kg apples caused no changes in endogenous DNA strand breaks, in Fpg‐sensitive sites (oxidative purine damage) or in the capacity to protect DNA against damage induced by hydrogen peroxide in PBL. But, a statistically significant decrease in the levels of endonuclease III sensitive sites (oxidative pyrimidine damage) and an increase in capacity to protect DNA against strand breaks induced by iron chloride were identified. In contrast to the effects of apples, a human intervention with 2, 5 or 8 servings of fruit and vegetables/day had no effect on DNA damage in PBL. Thus, we have identified apples as another fruit to add to the already known types of fruit (kiwi) and vegetables (tomato) with antigenotoxic properties and partly characterized their antigenotoxic mechanism in humans.Supported by the Deutsche Forschungsgemeinschaft and the Federal Ministry of Research and Education, Germany

High protein-high red meat versus high carbohydrate weight loss diets do not differ in effect on genome stability and cell death in lymphocytes of overweight men

The importance of diet in DNA damage prevention is well established; however, the comparison of weight loss diets with different micronutrient and macronutrient profiles on genome stability in peripheral blood lymphocytes (PBLs) has not been studied. This study tested the hypothesis that genome stability in PBLs of overweight men who consume a high protein-high red meat (HP) weight loss diet is different from that of overweight men who consume a high carbohydrate (HC) weight loss diet. Thirty-three male subjects were randomly assigned to an HP or HC isocaloric energy-restricted dietary intervention for 12 weeks intensive weight loss and weight maintenance up to 52 weeks. Blood samples were collected at 0, 12 and 52 weeks. DNA damage in PBLs was assessed using the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay. Average weight loss after 12 weeks was 9.3 +/- 0.7 kg for both diets, with no further change at 52 weeks. Two-way analysis of variance showed no time or diet effect on micronucleus frequency (chromosome loss/breaks). There was a significant trend with time (P = 0.03) but not diet, for reduction of nuclear buds (gene amplification). There was a positive trend with time for increased nucleoplasmic bridges (chromosome rearrangement) (P = 0.051). Necrosis and apoptosis both significantly decreased with time (P = 0.037 and P = 0.007, respectively) with no diet effect. There was no significant effect of time or diet for nuclear division index, a biomarker of immune response. The results suggest that the effect of the HP weight loss diet on DNA damage measured using the CBMN-Cyt assay in PBLs was not different from that observed for the HC weight loss diet.

Levodopa therapy reduces DNA damage in peripheral blood cells of patients with Parkinson’s disease

Oxidative stress seems to play a major role in the pathogenesis of neurodegeneration. In Parkinson's disease (PD) patients, the dopaminergic neurons are subjected to oxidative stress resulting from reduced levels of antioxidant defenses such as glutathione and high amount of intracellular iron. Levodopa (LD) is widely used for the symptomatic treatment of PD, but its role in oxidative damage control is still unclear. The aim of this study was to analyze the presence of DNA damage in peripheral blood lymphocytes (PBL) of PD patients, during a washout and a controlled LD dosage and to evaluate the oxidative damage fluctuation after LD intake. The standard and the Fpg-modified version of Comet assay were applied in analyzing DNA damage in PBL from blood samples of nine PD patients and nine matched controls. Due to the limited number of patients we cannot reach definite conclusions even if our data confirm the accumulation of DNA lesions in PD patients; these lesions decrease after LD intake.

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.

Noninvasive Prediction of Prostatic DNA Damage by Oxidative Stress Challenge of Peripheral Blood Lymphocytes

To move closer to the goal of individualized risk prediction for prostate cancer, we used an in vivo canine model to evaluate whether the susceptibility of peripheral blood lymphocytes (PBLs) to oxidative stress-induced DNA damage could identify those individuals with the highest prostatic DNA damage. This hypothesis was tested in a population of 69 elderly male beagle dogs after they had completed a 7-month randomized feeding trial to achieve the broad range of dietary selenium status observed in U.S. men. The alkaline Comet assay was used to directly compare the extent of DNA damage in PBLs with prostatic DNA damage in each dog. Using stepwise logistic regression, the sensitivity of PBLs to oxidative stress challenge with hydrogen peroxide (H(2)O(2)) predicted dogs in the highest tertile of prostatic DNA damage. Dogs with PBLs highly sensitive to H(2)O(2) were 7.6 times [95% confidence interval (95% CI), 1.5-38.3] more likely to have high prostatic DNA damage than those in the H(2)O(2)-resistant group. This risk stratification was observed in multivariate analysis that considered other factors that might influence DNA damage, such as age, toenail selenium concentration, and serum testosterone concentration. Our data show that the sensitivity of PBLs to oxidative stress challenge, but not endogenous DNA damage in PBLs, provides a noninvasive surrogate marker for prostatic DNA damage. These findings lend support to the concept that oxidative stress contributes to genotoxic damage, and that oxidative stress challenge may stratify men for prostate cancer risk.

DNA damage in peripheral lymphocytes of untreated breast cancer patients

Using the alkaline comet assay, DNA single-strand breaks (ssb) have been described in peripheral blood lymphocytes (PBL) from breast cancer patients without treatment, but there is no information concerning the occurrence of double-strand breaks (dsb) in the same patients. The purpose of this study was to investigate whether PBL of untreated sporadic breast cancer patients harbor an elevated number of both DNA strand breaks. Forty breast cancer patients without family history of cancer in clinical stage III devoid of treatment and 60 age-matched healthy subjects without history of breast cancer were included. PBL from freshly drawn blood were processed following two different methods: the alkaline and the neutral comet assay. Percentage and tail moment were assessed under fluorescence microscopy and a computer-based image analysis system. In controls, ssb were found in 18 +/- 4.67% of PBL, and dsb at 7.99 +/- 3.67% of PBLs. Cancer patients had higher values of both ssb 24.08 +/- 4.96 (p <0.05) and dsb 13.11 +/- 3.2% (p <0.01). Tail moment for ssb was 6.23 +/- 1.44 and dsb, 2.31 +/- 1.09 for controls. For breast cancer patients, tail moment for ssb was 11.73 +/- 2.40 and for dsb, 6.33 +/- 1.75 (p <0.01). On plotting individual measurements of comet percentage against tail moment for alkaline and neutral comet assays, clear separation of control group from cancer patients can be seen in both assays. Demonstration of two types of DNA damage in PBL of breast cancer patients devoid of treatment or without exposure to environmental genotoxic agents provides a better picture of the degree of DNA damage present in somatic cells of these patients.

Reduction in DNA damage in brain and peripheral blood lymphocytes of elderly dogs after treatment with dehydroepiandrosterone (DHEA)

Steady state levels of DNA damage are substantial in vertebrate animals as a consequence of exposure to endogenous and environmental mutagens. DNA damage may contribute to organismal senescence and an increased risk for specific age-related diseases. In this study, we determined if treatment with the neuroactive adrenal steroid, dehydroepiandrosterone (DHEA), which exhibits antioxidant and anticarcinogenic properties in rodents, would reduce DNA damage in the brain and peripheral blood lymphocytes (PBLs) of elderly dogs. Elderly male dogs, physiologically equivalent to 59–69-year-old men, were randomly assigned to receive no treatment ( n=9 dogs) or DHEA at 100 mg/kg PO daily ( n=8 dogs). Extent of DNA damage in brain cells and PBLs was measured using alkaline comet assay. The effect of DHEA treatment on the susceptibility of PBLs to H 2O 2-induced DNA damage was also measured. We found that elderly male dogs receiving daily DHEA treatment for 7 months had significantly less DNA damage detectable in their brain compared to age-matched control dogs. After 7 months treatment, DHEA-treated dogs also had a significant reduction in DNA damage in PBLs compared to pre-treatment levels. We also found that PBLs of dogs treated with DHEA were more resistant to H 2O 2-induced DNA damage than PBLs of untreated dogs. Our results did not show that basal DNA damage in PBLs was strongly correlated with DNA damage within the brain. The results of this study suggest that DHEA supplementation can significantly reduce steady state levels of DNA damage in the mammalian brain. Further evaluation of DHEA as a neuroactive agent and its effects on DNA damage and gene expression in other tissues and species is warranted.

Ifosfamide metabolism and DNA damage in tumour and peripheral blood lymphocytes of breast cancer patients.

This study was designed to determine individual variation in the metabolism of ifosfamide (IF) and any influence this may have on the degree of DNA damage produced in both peripheral blood lymphocytes (PBL) and in tumour tissue. The pharmacokinetics and metabolism of IF and also of doxorubicin (DOX) were determined in patients receiving IF/DOX neoadjuvant chemotherapy for the treatment of advanced breast cancer. The DNA-damaging effects of this regimen were measured using the comet assay in PBL and in breast tumour tissue obtained by fine needle aspirate. Parallel in vitro studies were carried out in order to establish if DNA damage caused by IF metabolites or DOX was predictive of cytotoxicity in breast cancer cell lines. The median AUC, half-life and clearance of IF were found to be 291 microM.min, 5.2 h and 66 ml/min per m2, respectively. A high degree of interpatient variability (up to sevenfold) was observed in the metabolism of both IF and DOX and also in their metabolites. Treatment-related changes in the amount of DNA damage were observed in both PBL and tumour cells. That in PBL peaked 48 h after the end of IF infusion (median 17% damaged cells at 48 h compared to 4% damaged before treatment). DNA damage in tumour cells was not elevated above low pretreatment values (median 1.5% damaged cells) until 3 weeks after IF and DOX treatment (median 30% damaged cells), by which time damage in PBL showed almost complete resolution to basal levels. The DNA damage in PBL determined 24 h after the start of chemotherapy was found to be related to the AUC of 4-hydroxyifosfamide (4OHI; P = 0.05). The amount of damage in either tissue did not significantly correlate with clinical response or toxicity, but lower amounts of damage were observed in the tumour cells 3 weeks after treatment in those patients that subsequently relapsed, compared to those that remained disease free. DNA damage (more than 20% damaged cells) was observed after exposure to active IF metabolites at concentrations equal to or greater than the IC50 in MCF-7 and MDA-MB231 cell lines. At concentrations of 4OHI similar to those determined in vivo, an equivalent level of DNA damage was observed in PBL and in cell lines and was associated with significant growth inhibition. DNA damage induced by DOX was not predictive of cytotoxicity. Systemic DNA damage appeared to be related to levels of the active metabolite, consistent with the results of in vitro investigations of DNA damage. Further studies are warranted to substantiate this observation and to explore the relationship between metabolism, DNA damage and antitumour activity.

The effect of vitamin C or vitamin E supplementation on basal and H2O2-induced DNA damage in human lymphocytes.

There is a wealth of epidemiological information on antioxidants and their possible prevention of disease progression but very little of the research on antioxidants has involved intervention studies. In this study, the potential protective effect of vitamin C or E supplementation in vivo against endogenous and H2O2-induced DNA damage levels in lymphocytes was assessed. The supplementation involved fourteen healthy male and female non-smokers mean age 25-53 (SD 1.82) years, who were asked to supplement an otherwise unchanged diet with 1000 mg vitamin C daily for 42 d or 800 mg vitamin E daily for 42 d. DNA damage in H2O2-treated peripheral blood lymphocytes (PBL) and untreated PBL before and after supplementation, and during a 6-week washout period was assessed using an ELISA. At each sampling time-point, the red cell concentrate activities of superoxide dismutase, catalase and glutathione peroxidase were also determined. Supplementation with vitamin C or vitamin E decreased significantly H2O2-induced DNA damage in PBL, but had no effect on endogenous levels of DNA damage. The activities of the antioxidant enzymes superoxide dismutase and glutathione peroxidase were suppressed during the supplementation period. These supplementation regimens may be used to limit the possible adverse effects of reactive oxygen species (including those produced during the course of an immune response) on lymphocytes in vivo, and so help to maintain their functional capacity.

Clinical evaluation of chelation therapy: Is there any wheat amidst the chaff?

The CaNa2EDTA chelation therapy is often practiced with antioxidant supplementation. Dry olive leaf extract (DOLE) is natural product with antioxidant and DNA protective properties. The effects of DOLE on the levels of DNA damage were investigated ex vivo in peripheral blood lymphocytes (PBLs) of 19 workers occupationally exposed to lead (Pb), before and after CaNa2EDTA chelation therapy.DOLE demonstrated pronounced radical scavenging activity in concentrations ≥1 mg/mL, and showed no genotoxicity per se, in concentrations 0.125–1 mg/mL. The level of DNA damage in PBLs of workers before chelation therapy was elevated (24.21 ± 14.26) compared to controls (6.0 ± 3.37). The incubation of PBLs before chelation therapy with selected concentration of DOLE lead to a severe increase of DNA damage (64.03 ± 20.96), exhibiting prooxidant rather than antioxidant effect. After the five-day CaNa2EDTA chelation regimen, DNA damage in PBLs of workers decreased (8.26 ± 4.62) significantly compared to baseline. Treatment of PBLs with DOLE after chelation, again produced high level of damage (41.82 ± 23.17) and the acute prooxidant effects of DOLE remained, but, DNA damage was less severe than before chelation. The DOLE exhibits prooxidant effect in presence of Pb in lymphocytes of exposed workers, and its effect is less pronounced following the removal of Pb after standard chelation therapy.

ROLE OF INTRACELLULAR DEOXYNUCLEOSIDE TRIPHOSPHATES IN LEVELS IN DNA DAMAGE IN HUMAN PERIPHERAL BLOOD LYMPHOCYTES: 37

The effect of deoxynucleosides on single strand chromosomal breaks in peripheral blood lymphocytes (PBL) were measured both by the sucrose gradient nucleoid sedimentation method and by alkaline unwinding followed by fluorimetric measurement of etidium bromide binding to double stranded DNA. Extracellular deoxyadenosine (0.3-10 μM) in the presence of deoxycoformycin (1 μM) cause extensive DNA damage in PBL in a concentration dependent manner. The appearance of DNA damage following deoxyadenosine addition to PBL correlated with the accumulation of intracellular dATP levels. Extracellular deoxycytidine (50 μM) inhibited the accumulation of intracellular dATP from deoxyadenosine and in parallel prevented the DNA damage caused by deoxyadenosine in PBL. On the other hand, addition of extracellular deoxyguanosine (50 μM) or thymidine (50 μM) also completely prevented the DNA damage by deoxyadenosine but without interferring with dATP accumulation. Measurements of intracellular deoxynucleoside triphosphates levels in PBL show extremely low levels of dGTP, dCTP and TTP and higher levels of dATP. It is possible that the marked increase of intracellular dATP levels combined with the extremelylow levels of the other deoxynucleoside triphosphates interfer with DNA repair process in resting PBL resulting in the appearance of single stranded breaks. Thymocytes, which contain higher levels of deoxynucleoside triphosphates, did not show DNA damage in the presence of deoxyadenosine.