125I-induced DNA double strand breaks: use in calibration of the neutral filter elution technique and comparison with X-ray induced breaks

The neutral filter elution assay, for measurement of DNA double strand breakage, has been calibrated using mouse L cells and Chinese hamster V79 cells labelled with [125I]dUrd and then held at liquid nitrogen temperature to accumulate decays. The basis of the calibration is the observation that each 125I decay, occurring in DNA, produces a DNA double strand break. Linear relationships between 125I decays per cell and lethal lesions per cell (minus natural logarithm survival) and the level of elution, were found. Using the calibration data, it was calculated that the yield of DNA double strand breaks after X-irradiation of both cell types was from 6 to 9 X 10(-12) DNA double strand breaks per Gy per dalton of DNA, for doses greater than 6 Gy. Neutral filter elution and survival data for X-irradiated and 125I-labelled cells suggested that the relationships between lethal lesions and DNA double strand breakage were significantly different for both cell types. An attempt was made to study the repair kinetics for 125I-induced DNA double strand breaks, but was frustrated by the rapid DNA degradation which occurs in cells that have been killed by the freezing-thawing process.     

Facilitated detection of chromosome break and repair at low levels of ionizing radiation by addition of wortmannin to G1-type PCC fusion incubation

We have measured rejoining kinetics of chromosome breaks using a modified cell fusion-based premature chromosome condensation (PCC) technique in confluent cultures of normal human fibroblasts irradiated at low doses of X-rays. In order to enhance the sensitivity of the fusion-based PCC assay, we added a DNA double strand break (DSB) repair inhibitor wortmannin during the incubation period for PCC/fusion process resulting in a significantly higher yield of G1-type chromosome breaks. The initial number of chromosome breaks (without repair) gave a linear dose response with about 10 breaks per cell per Gy which is about two times higher than the value with the conventional G1-type PCC method. The chromosome rejoining kinetics at 0.5 and 2.0 Gy X-rays reveal a bi-phasic curve with both a fast and a slow component. The fast component (0-30 min) is nearly identical for both doses, but the slow component for 2 Gy kinetics is much slower than that for 0.5 Gy, indicating that the process occurring during this period may be crucial for the ultimate fate of irradiated cells. The chromosome rejoining kinetics obtained here is similar to that of other methods of detecting DNA DSB repair such as the gammaH2AX assay. Our chromosome repair assay is useful for evaluating the accuracy of other assays measuring DNA DSB repair at doses equal or less than 0.5 Gy of ionizing radiation.     

X-ray induced DNA double strand break production and repair in mammalian cells as measured by neutral filter elution.

This work presents a neutral filter elution method for detecting DNA double strand breaks in mouse L1210 cells after X-ray. The assay will detect the number of double strand breaks induced by as little as 1000 rad of X-ray. The rate of DNA elution through the filters under neutral conditions increases with X-ray dose. Certain conditions for deproteinization, pH, and filter type are shown to increase the assay's sensitivity. Hydrogen peroxide and Bleomycin also induce apparent DNA double strand breaks, although the ratios of double to single strand breaks vary from those produced by X-ray. The introduction of double strand cuts by HpA I restriction endonuclease in DNA lysed on filters results in a rapid rate of elution under neutral conditions, implying that the method can detect double strand breaks if they exist in the DNA. The eluted DNA bands with a double stranded DNA marker in cesium chloride. This evidence suggests that the assay detects DNA double strand breaks. L1210 cells are shown to rejoin most of the DNA double strand breaks induced by 5-10 krad of X-ray with a half-time of about 40 minutes.     

Particularities of blood lymphocyte response to irradiation in vitro in breast cancer patients

DNA breaks and their repair efficiency were analyzed in irradiated in vitro lymphocytes (at doses 1 Gy, gamma-radiation of 60Co, dose rate 1 Gy/min) isolated from peripheral blood of 41 untreated patients with breast cancer and 25 healthy donors using the DNA comet assay under non-denaturing conditions (mainly double-strand DNA breaks (DSB), as well as apoptotic cell death using the DNA halo assay. To estimate the expression of bystander effect, the cells were incubated in a culture medium obtained from lymphocytes irradiated in vitro at doses 1 Gy. The average DSB level in blood lymphocytes of breast cancer patients was shown to be significantly higher (p < 0.05) compared with that in control donors. In general, the following effects were observed in irradiated in vitro lymphocytes of cancer patients: (1) increased sensitivity to y-radiation-induced DNA DSBs compared with lymphocytes from healthy donors, (2) reduced repair efficiency of these damages. Incubation of irradiated blood lymphocytes in a medium from irradiated cells led to an increased relative number of DNA DSBs and an elevated fraction of cells dying through apoptotic pathway both in blood lymphocytes from cancer patients and control donors. However, these non-targeted effects were more expressed for the blood lymphocytes of breast cancer patients.     

Deoxyribonucleoside triphosphate imbalance. 5-Fluorodeoxyuridine-induced DNA double strand breaks in mouse FM3A cells and the mechanism of cell death

The mechanism of cytotoxic action of 5-fluorodeoxyuridine (FdUrd) in mouse FM3A cells was investigated. We observed the FdUrd-induced imbalance of intracellular deoxyribonucleoside triphosphate (dNTP) pools and subsequent double strand breaks in mature DNA, accompanied by cell death. The imbalance of dNTP pools was maximal at 8 h after 1 microM FdUrd treatment; a depletion of dTTP and dGTP pools and an increase in the dATP pool were observed. The addition of FdUrd in culture medium induced strand breaks in DNA, giving rise to a 90 S peak by alkaline sucrose gradient sedimentation. The loss of cell viability and colony-forming ability occurred at about 10 h. DNA double strand breaks as measured by the neutral elution method were also observed in FdUrd-treated cells about 10 h after the addition. These results lead us to propose that DNA double strand breaks play an important role in the mechanism of FdUrd-mediated cell death. A comparison of the ratio of single and double strand breaks induced by FdUrd to that observed following radiation suggested that FdUrd produced double strand breaks exclusively. Cycloheximide inhibited both the production of DNA double strand breaks and the FdUrd-induced cell death. An activity that can induce DNA double strand breaks was detected in the lysate of FdUrd-treated FM3A cells but not in the untreated cells. This suggests that FdUrd induces the cellular DNA double strand breaking activity. The FdUrd-induced DNA strand breaks and cell death appear to occur in the S phase. Our results indicate that imbalance of the dNTP pools is a trigger for double strand DNA break and cell death.     

用脉冲电场凝胶电泳检测电离辐射所致哺乳动物细胞DNA双链断裂

 本文将反向交变电场和六角形电极电场这两种脉冲电场凝胶电泳技术应用于X线照射小鼠乳癌细胞SR-1所致DNA双链断裂的检测,在本实验条件下,用这种电泳都能检测到低至1.5Gy照射所产生的DNA双链断裂,并且用六角形电极电场电泳获得了DNA双链断裂程度与照射剂量之间的良好线性关系,此外,还用此方法观察了不同浓度自由基清除剂DMSO对X线照射SR-1细胞所致DNA双链断裂的保护作用,结果进一步证实本方法的可靠性。     

Measurement of DNA damage induced by irradiation with gamma-rays from a TRIGA Mark II research reactor in human cells using Fast Micromethod.

The Fast Micromethod is a novel quick and convenient microplate assay for determination of DNA single-strand breaks. This method measures the rate of unwinding of cellular DNA upon exposure to alkaline conditions using a fluorescent dye which preferentially binds to double-stranded DNA. Here we applied this method to determine the levels of DNA single-strand breaks in HeLa cells induced by y-irradiation deriving from fission isotopes and activation products at the TRIGA Mark II research reactor in Mainz. An increased strand scission factor (SSF) value, which is indicative for DNA damage, was found at doses of 1 Gy and higher. A similar increase in SSF value, which further increased in a dose-dependent manner, was found in human peripheral blood mononuclear cells after irradiation with 6 MV X-rays from a linear accelerator to give a total exposure of 0.5 to 10 Gy.     

Radiosensitizing effect of gold nanoparticle loaded with small interfering RNA-SP1 on lung cancer: AuNPs-si-SP1 regulates GZMB for radiosensitivity

Radioresistance is a major challenge that largely limits the efficacy of radiotherapy in lung cancer. Gold nanoparticles (AuNPs) are emerging as novel radiosensitizers for cancer patients. Therefore, this study was designed to explore the radiosensitizing effect and mechanism of AuNPs loaded with small interfering RNA (siRNA)-SP1 (AuNPs-si-SP1) on lung cancer. AuNPs-si-SP1 was prepared by the noncovalent binding between AuNPs and siRNA-SP1. The adsorption capacity of AuNPs to siRNA-SP1 was analyzed by gel electrophoresis. The cell uptake of AuNPs-si-SP1 was observed under a laser confocal microscopy. Silencing efficacy of AuNPs-si-SP1 was validated by RT-qPCR and Western blot analysis. Cell viability was determined by CCK-8 assay, radiosensitization by plate colony formation assay, cell apoptosis and cell cycle by flow cytometry, and DNA double strand breaks by immunofluorescence in the presence or absence of AuNPs-si-SP1 or GZMB. The downstream mechanism of SP1 was predicted by bioinformatics analysis, followed by verification by Western blot analysis. Subcutaneous tumorigenesis in nude mice was established to verify the radiosensitization of AuNPs-si-SP1 and GZMB in vivo. AuNPs-si-SP1 effectively absorbed SP1 siRNA and was highly internalized by A549 cells to reduce SP1 protein expression. AuNPs-si-SP1 or GZMB overexpression promoted cells to G2/M phase, DNA double strand breaks, and enhanced radiosensitivity. SP1 could repress GZMB expression in lung cancer cells. In vivo experiments manifested that AuNPs-si-SP1 could inhibit the growth of solid tumor in nude mice to achieve radiosensitization by inhibiting SP1 to upregulate GZMB. AuNPs-si-SP1 might increase the radiosensitivity of lung cancer by inhibiting SP1 to upregulate GZMB.     

Radiosensitizing effect of gold nanoparticle loaded with small interfering RNA-SP1 on lung cancer: AuNPs-si-SP1 regulates GZMB for radiosensitivity

Radioresistance is a major challenge that largely limits the efficacy of radiotherapy in lung cancer. Gold nanoparticles (AuNPs) are emerging as novel radiosensitizers for cancer patients. Therefore, this study was designed to explore the radiosensitizing effect and mechanism of AuNPs loaded with small interfering RNA (siRNA)-SP1 (AuNPs-si-SP1) on lung cancer. AuNPs-si-SP1 was prepared by the noncovalent binding between AuNPs and siRNA-SP1. The adsorption capacity of AuNPs to siRNA-SP1 was analyzed by gel electrophoresis. The cell uptake of AuNPs-si-SP1 was observed under a laser confocal microscopy. Silencing efficacy of AuNPs-si-SP1 was validated by RT-qPCR and Western blot analysis. Cell viability was determined by CCK-8 assay, radiosensitization by plate colony formation assay, cell apoptosis and cell cycle by flow cytometry, and DNA double strand breaks by immunofluorescence in the presence or absence of AuNPs-si-SP1 or GZMB. The downstream mechanism of SP1 was predicted by bioinformatics analysis, followed by verification by Western blot analysis. Subcutaneous tumorigenesis in nude mice was established to verify the radiosensitization of AuNPs-si-SP1 and GZMB in vivo. AuNPs-si-SP1 effectively absorbed SP1 siRNA and was highly internalized by A549 cells to reduce SP1 protein expression. AuNPs-si-SP1 or GZMB overexpression promoted cells to G2/M phase, DNA double strand breaks, and enhanced radiosensitivity. SP1 could repress GZMB expression in lung cancer cells. In vivo experiments manifested that AuNPs-si-SP1 could inhibit the growth of solid tumor in nude mice to achieve radiosensitization by inhibiting SP1 to upregulate GZMB. AuNPs-si-SP1 might increase the radiosensitivity of lung cancer by inhibiting SP1 to upregulate GZMB.     

Elaboration of cellular DNA breaks by hydroperoxides.

Cellular damage produced by ionizing radiation and peroxides, hydrogen peroxide (HOOH) and the organic peroxides tert-butyl (tBuOOH) or cumene hydroperoxide (CuOOH) were compared. DNA breaks, toxicity, malondialdehyde production, and the rate of peroxide disappearance were measured in a human adenocarcinoma cell line (A549). The alkaline and neutral filter elution assays were used to quantitate the kinetics of single and double strand break formation and repair (SSB and DSB), respectively. Peroxides, at 0.01-1.0 mM, produce multiphasic dose response curves for both toxicity and DNA SSBs. Radiation, 1-6 Gy, produced a shouldered survival curve, and both DNA SSB and DSBs produced in cells x-rayed on ice were nearly linear with dose. The peroxides produced more SSBs than radiation at equitoxic doses. X-ray induced DNA single strand breaks were rejoined rapidly by cells at 37 degrees C with approximately 80% of initial damage repaired in 20 min. Peroxide induced SSBs were maximal after 15 min at 37 degrees C. Rejoining proceeded thereafter, but at a rate less than for x-ray induced strand breaks. Significant DNA DSBs could not be achieved by peroxides even at concentrations 50-fold higher than required to produce SSBs. HOOH treatment of DNA on filters following cell lysis and proteolysis produced SSBs. CuOOH and tBuOOH produced no SSBs in lysed cell DNA. None of the peroxides produced DSBs when incubated with lysed cell DNA. Malondialdehyde was released from cells incubated with organic hydroperoxides, but not HOOH, nor up to 40 Gy of x-rays. HOOH was metabolized three times faster than the organic peroxides. The overall results demonstrate the necessity for a metabolically active cell environment to elaborate maximal DNA strand breaks and cell death at hydroperoxide concentrations of 10(-4) or greater, but prevent strand breaks and stimulate cell growth at 10(-5) M.     

Polyamine depletion with two different polyamine analogues causes DNA damage in human breast cancer cell lines

It is well known that the positively charged polyamines have a DNA-stabilizing function and that polyamine depletion alters chromatin function. We have previously shown that polyamine depletion causes an S phase prolongation, and others have shown that there is an accumulation of Okazaki-like fragments in polyamine-depleted cells. In the present study, we have used the comet assay to investigate polyamine depletion-induced DNA strand breaks. Three breast cancer cell lines and one normal-like breast cell line were treated with the polyamine analogue N(1),N(11)-diethylnorspermine or with the polyamine biosynthesis inhibitor 4-amidinoindan-1-one 2'-amidinohydrazone (CGP 48664). The comet assay showed that polyamine depletion resulted in DNA strand breaks. We also show that these DNA strand breaks occurred in cells where there was no expression of gamma-H2AX, which is a marker of DNA double-strand breaks. Thus, our conclusion is that polyamine depletion causes DNA single-strand breaks, which may be the cause for the observed delay in S phase progression.     

Quantitative measurement of DNA strand breaks and repair in gamma-irradiated human leukocytes from normal and ataxia telangiectasia donors

Fluorimetric analysis of DNA unwinding, which allows measurement of DNA strand breaks in human leukocytes, has been optimized by reducing the amount of cells required for the test and by modifying the DNA alkali unwinding conditions. This permitted measurement of DNA strand-break induction in cells irradiated with low (0.5-7 Gy) or high doses (5-20 Gy) of gamma rays. Linear dose-response curves were obtained for both dose ranges. Presence of cysteamine during irradiation caused a decrease in the extent of DNA strand breaks. The kinetics of the DNA strand-break rejoining process appeared to be biphasic over the dose range of 2-20 Gy when plotted on a linear vs linear axis (percentage of damage as a function of time). Since the rate of disappearance of damaged DNA was similar for any given dose and for all postirradiation incubation times tested, we have expressed the extent of repair after a given postirradiation incubation as the ratio of the slopes of the regression lines obtained from incubated and nonincubated cells. Leukocytes from 25 healthy donors were analyzed to determine an average value for controls. No difference in the level of DNA strand breaks and the rate of repair of these breaks was observed between leukocytes from three ataxia telangiectasia patients and those from normal donors.     

BRCA1 role in the mitigation of radiotoxicity and chromosomal instability through repair of clustered DNA lesions

Oxidatively-induced clustered DNA lesions are considered the signature of any ionizing radiation like the ones human beings are exposed daily from various environmental sources (medical X-rays, radon, etc.). To evaluate the role of BRCA1 deficiencies in the mitigation of radiation-induced toxicity and chromosomal instability we have used two human breast cancer cell lines, the BRCA1 deficient HCC1937 cells and as a control the BRCA1 wild-type MCF-7 cells. As an additional control for the DNA damage repair measurements, the HCC1937 cells with partially reconstituted BRCA1 expression were used. Since clustered DNA damage is considered the signature of ionizing radiation, we have measured the repair of double strand breaks (DSBs), non-DSB bistranded oxidative clustered DNA lesions (OCDLs) as well as single strand breaks (SSBs) in cells exposed to radiotherapy-relevant γ-ray doses. Parallel measurements were performed in the accumulation of chromatid and isochromatid breaks. For the measurement of OCDL repair, we have used a novel adaptation of the denaturing single cell gel electrophoresis (Comet assay) and pulsed field gel electrophoresis with Escherichia coli repair enzymes as DNA damage probes. Independent monitoring of the γ-H2AX foci was also performed while metaphase chromatid lesions were measured as an indicator of chromosomal instability. HCC1937 cells showed a significant accumulation of all types of DNA damage and chromatid breaks compared to MCF-7 while BRCA1 partial expression contributed significantly in the overall repair of OCDLs. These results further support the biological significance of repair resistant clustered DNA damage leading to chromosomal instability. The current results combined with previous findings on the minimized ability of base clusters to induce cell death (mainly induced by DSBs), enhance the potential association of OCDLs with breast cancer development especially in the case of a BRCA1 deficiency leading to the survival of breast cells carrying a high load of unrepaired DNA damage clusters.     

Radiation-induced DNA base damage detected in individual aerobic and hypoxic cells with endonuclease III and formamidopyrimidine-glycosylase.

X-ray-induced DNA base damage can be detected using endonuclease III and formamidopyrimidine-glycosylase, which create DNA strand breaks at enzyme-sensitive sites. Strand breaks can then be measured with excellent sensitivity using the alkaline comet assay, a single-cell gel electrophoresis method that detects DNA damage in individual cells. In using this approach to measure the oxygen enhancement ratio (OER) for radiation-induced base damage, we observed that the number of enzyme-sensitive sites increased with dose up to 4 Gy in air and 12 Gy in hypoxic WIL2NS cells. After rejoining of radiation-induced strand breaks, base damage was detected more easily after higher doses. The number of radiation-induced enzyme-sensitive sites was similar under both air and nitrogen. Base damage produced by hydrogen peroxide and 4-nitroquinoline-N-oxide (4NQO) was also measured. Results with hydrogen peroxide (20 min at 4 degrees C) were similar to those observed for X rays, indicating that enzyme-sensitive sites could be detected most efficiently when few direct strand breaks were present. Removing DNA-associated proteins before irradiation did not affect the ability to detect base damage. Base damage produced by 4NQO (30 min at 37 degrees C) was readily apparent after treatment with low concentrations of the drug when few 4NQO-induced strand breaks were present, but the detection sensitivity decreased rapidly as direct strand breaks increased after treatment with higher concentrations. We conclude that: (1) the OER for base damage is approximately 1.0, and (2) the presence of direct DNA strand breaks (>2000-4000 per cell) prevents accurate detection of base damage measured as enzyme-sensitive sites with the alkaline comet method.     

miR‐200c enhances radiosensitivity of human breast cancer cells

Due to the intrinsic resistance of many tumors to radiotherapy, current methods to improve the survival of cancer patients largely depend on increasing tumor radiosensitivity. It is well‐known that miR‐200c inhibits epithelial–mesenchymal transition (EMT), and enhances cancer cell chemosensitivity. We sought to clarify the effects of miR‐200c on the radiosensitization of human breast cancer cells. In this study, we found that low levels of miR‐200c expression correlated with radiotolerance in breast cancer cells. miR‐200c overexpression could increase radiosensitivity in breast cancer cells by inhibiting cell proliferation, and by increasing apoptosis and DNA double‐strand breaks. Additionally, we found that miR‐200c directly targeted TANK‐binding kinase 1 (TBK1). However, overexpression of TBK1 partially rescued miR‐200c mediated apoptosis induced by ionizing radiation. In summary, miR‐200c can be a potential target for enhancing the effect of radiation treatment on breast cancer cells. J. Cell. Biochem. 114: 606–615, 2013. © 2012 Wiley Periodicals, Inc.     

DNA repair patch-mediated double strand DNA break formation in human cells

To investigate the mechanism of double strand DNA break formation in mammalian cells, an in vitro assay was established using closed circular DNA containing two uracils on opposite DNA strands (18 and 30 base pairs apart) and extracts prepared from human cells. In this assay, formation of double strand breaks was detected by the conversion of circular DNA to linear DNA. Approximately 4-fold more double strand DNA breaks were produced by extracts from cells deficient in DNA ligase I (46BR) relative to those produced by extracts from control cells (MRC5, derived from a clinically normal individual). In parallel with the amount of double strand DNA breaks, extracts from 46BR cells produced longer repair patches (up to 24 bases in length) than those from MRC5 cells (typically <5 bases long). When purified DNA ligase I was added to 46BR extracts to complement the DNA ligase deficiency, only a negligible difference was found between the amount of doublestrand DNA breaks or the repair patch size generated in the assay relative to MRC5 extracts. Together, our data demonstrate that double strand DNA breaks are produced through formation of DNA repair patches. We refer to this process of double strand break formation as the "DNA repair patch-mediated pathway."     

Individual cytogenetic and molecular-biological characteristics of lymphocytes in blood of pilots and cosmonauts

The purpose of this paper was the investigation of the pilots and of cosmonauts individual sensitivity to the fly conditions, to the additional irradiation (in the dose of 1 Gy), the adaptive response manifestation (in the doses 0.05 and 0.5 Gy). The DNA comet assay (the double strand DNA breaks was determined) and the method of unstable chromosome aberrations in metaphase was used. The human blood lymphocytes was the object of investigation. The significant individual differences were discovered in pilots and in cosmonauts in the initial DNA damage; in the sensitivity to the additional irradiation. The frequency of the adaptive response induction was decreased in the pilots in the comparison with the control group. The adaptive response was registered in cosmonauts (3 men). It is supposed that DNA damage, chromosome aberrations, sensitivity to the additional irradiation, the adaptive response manifestations can be used as biological markers of individual risk disease.     

CHK2在肿瘤中作用的研究进展

细胞周期检测点激酶2(cell cycle checkpoint kinase 2,CHK2)是抑癌基因CHK2编码的丝氨酸/苏氨酸激酶,广泛存在于哺乳动物中。它可在DNA双链断裂后参与DNA修复应答,并作为重要的信号转导蛋白,使细胞周期进程发生阻滞,从而促进DNA修复或诱导细胞凋亡。目前在肺癌、乳腺癌、结直肠癌等多种恶性肿瘤均发现CHK2的缺失,研究者发现CHK2是增强DNA损伤治疗在癌症中治疗效果的良好靶标。CHK2基因作为肿瘤治疗的重要研究靶点,为肿瘤的治疗研究提供了新的方向。本文就CHK2目前在肿瘤中的研究进展做简要综述。     

Radiosensitization of Chinese hamster V79 cells by bromodeoxyuridine substitution of thymidine: enhancement of radiation-induced toxicity and DNA strand break production by monofilar and bifilar substitution

Bromodeoxyuridine (BrdU) competes with thymidine (TdR) for incorporation into DNA of exponentially growing V79-171 cells. Such cells show an enhancement of the radiation response as determined by clonogenic survival and DNA damage measured by filter elution techniques after doses up to 15 Gy. The degree of radiosensitization for both survival and rates of alkaline and neutral elution are dependent on percentage BrdU substitution and independent of whether BrdU is in one strand only (monofilar) or both strands (bifilar) of the DNA duplex: e.g., for 16% BrdU substitution distributed either monofilarly or partially bifilarly, there is an enhancement factor for Do of 1.55. At this percentage substitution, the enhancement factor for the rate of alkaline elution is 1.75 and that for the rate of neutral elution is 1.54. The greater the percentage BrdU substitution, the larger was the enhancement ratio for survival and radiation-induced strand breaks in both monofilarly and bifilarly substituted cells. The increase in cell radiosensitivity caused by BrdU substitution shows a better correlation with the increase in radiation-induced double-strand breaks than with the increase in radiation-induced single-strand breaks.     

Vanadate induces DNA strand breaks in cultured human fibroblasts at doses relevant to occupational exposure

To study possible genotoxic effects of occupational exposure to vanadium pentoxide, we determined DNA strand breaks (with alkaline comet assay), 8-hydroxy-2'deoxyguanosine (8-OHdG) and the frequency of sister chromatid exchange (SCE) in whole blood leukocytes or lymphocytes of 49 male workers employed in a vanadium factory in comparison to 12 non-exposed controls. In addition, vanadate has been tested in vitro to induce DNA strand breaks in whole blood cells, isolated lymphocytes and cultured human fibroblasts of healthy donors at concentrations comparable to the observed levels of vanadium in vivo. To investigate the impact of vanadate on the repair of damaged DNA, co-exposure to UV or bleomycin was used in fibroblasts, and DNA migration in the alkaline and neutral comet assay was determined. Although, exposed workers showed a significant vanadium uptake (serum: median 5.38microg/l, range 2.18-46.35microg/l) no increase in cytogenetic effects or oxidative DNA damage in leukocytes could be demonstrated. This was consistent with the observation that in vitro exposure of whole blood leukocytes and lymphocytes to vanadate caused no significant changes in DNA strand breaks below concentrations of 1microM (50microg/l). In contrast, vanadate clearly induced DNA fragmentation in cultured fibroblasts at relevant concentrations. Combined exposure of fibroblasts to vanadate/UV or vanadate/bleomycin resulted in non-repairable DNA double strand breaks (DSBs) as seen in the neutral comet assay. We conclude that exposure of human fibroblasts to vanadate effectively causes DNA strand breaks, and co-exposure of cells to other genotoxic agents may result in persistent DNA damage.