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Oxidative DNA damage is considered to play an important role in pathophysiological processes, ageing and cancer. So far major interest has been on measuring 8-hydroxy-2'-deoxyguanosine (8-OHdG), the preferred methods relying on HPLC or GC-mass spectrometry. The high biological relevance of 8-OHdG is due to its ability to induce G-->T transversions, which are among the most frequent somatic mutations found in human cancers. Effects of workplace exposures on the level of white blood cell 8-OHdG or urinary 8-OHdG have been reported with controversial results. Exposures examined include asbestos, azo-dyes, benzene, fine particulate matter (PM(2.5)), glassworks, polycyclic aromatic hydrocarbons (PAHs), rubber manufacturing, silica, metals, styrene, toluene and xylenes. The available data indicate that there is still a lack of well established dose-response relations between occupational or environmental exposures and the induction of 8-OHdG. Smoking has been most consistently identified as a confounder for 8-OHdG, but various occupational studies did not reveal higher levels of 8-OHdG in smokers. Despite the conflicting results, the reported studies show promise for 8-OHdG as a biomarker of oxidative stress associated with chemical exposure. However, there are critical aspects related to the analytical challenge, artifactual production of 8-OHdG, inter- and intra-individual variation, confounding factors and inter-laboratory differences, implying that further work is needed to reach a consensus on the background level of 8-OHdG.

The issue of adverse health effects of extremely low-frequency electromagnetic fields (ELF-EMFs) is highly controversial. Contradictory results regarding the genotoxic potential of ELF-EMF have been reported in the literature. To test whether this controversy might reflect differences between the cellular targets examined we exposed cultured cells derived from different tissues to an intermittent ELF-EMF (50 Hz sinusoidal, 1 mT) for 1-24h. The alkaline and neutral comet assays were used to assess ELF-EMF-induced DNA strand breaks. We could identify three responder (human fibroblasts, human melanocytes, rat granulosa cells) and three non-responder cell types (human lymphocytes, human monocytes, human skeletal muscle cells), which points to the significance of the cell system used when investigating genotoxic effects of ELF-EMF.

Several studies indicating a decline of DNA repair efficiency with age raise the question, if senescence per se leads to a higher susceptibility to DNA damage upon environmental exposures. Cultured fibroblasts of six healthy donors of different age exposed to intermittent ELF-EMF (50 Hz sinus, 1 mT) for 1-24 h exhibited different basal DNA strand break levels correlating with age. The cells revealed a maximum response at 15-19 h of exposure. This response was clearly more pronounced in cells from older donors, which could point to an age-related decrease of DNA repair efficiency of ELF-EMF induced DNA strand breaks.

Objectives: Epidemiological studies have reported an association between exposure to extremely low frequency electromagnetic fields (ELF-EMFs) and increased risk of cancerous diseases, albeit without dose-effect relationships. The validity of such findings can be corroborated only by demonstration of dose-dependent DNA-damaging effects of ELF-EMFs in cells of human origin in vitro., Methods: Cultured human diploid fibroblasts were exposed to intermittent ELF electromagnetic fields. DNA damage was determined by alkaline and neutral comet assay., Results: ELF-EMF exposure (50 Hz, sinusoidal, 1-24 h, 20-1,000 mu T, 5 min on/10 min off) induced dose-dependent and time-dependent DNA single-strand and double-strand breaks. Effects occurred at a magnetic flux density as low as 35 mu T, being well below proposed International Commission of Non-Ionising Radiation Protection (ICNIRP) guidelines. After termination of exposure the induced comet tail factors returned to normal within 9 h., Conclusion: The induced DNA damage is not based on thermal effects and arouses concern about environmental threshold limit values for ELF exposure.

Results of epidemiological research show low association of electromagnetic field (EMF) with increased risk of cancerous diseases and missing dose-effect relations. An important component in assessing potential cancer risk is knowledge concerning any genotoxic effects of extremely-low-frequency-EMF (ELF-EMF). Human diploid fibroblasts were exposed to continuous or intermittent ELF-EMF (50Hz, sinusoidal, 24h, 1000microT). For evaluation of genotoxic effects in form of DNA single- (SSB) and double-strand breaks (DSB), the alkaline and the neutral comet assay were used. In contrast to continuous ELF-EMF exposure, the application of intermittent fields reproducibly resulted in a significant increase of DNA strand break levels, mainly DSBs, as compared to non-exposed controls. The conditions of intermittence showed an impact on the induction of DNA strand breaks, producing the highest levels at 5min field-on/10min field-off. We also found individual differences in response to ELF-EMF as well as an evident exposure-response relationship between magnetic flux density and DNA migration in the comet assay. Our data strongly indicate a genotoxic potential of intermittent EMF. This points to the need of further studies in vivo and consideration about environmental threshold values for ELF 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.

In order to determine background levels of DNA strand breaks, we examined 80 healthy individuals by comet assay considering age, sex, and smoking as confounding factors. Only age was found to have a significant effect on basal levels. One thousand cells of each donor were graded by eye into 5 categories according to the amount of DNA in the tail: classification group A (no damage) <5%, B (low damage) 5-20%, C (medium damage) 20-40%, D (high damage) 40-95%, and group E (total damage) >95%. The interpretation of the comet assay was modified to achieve a tail factor, which represents the DNA damage of 1000 scored cells as a single number, without the need of an image analysis software package. Hydrogen peroxide and bleomycin used for in vitro exposure of lymphocytes, produced clear dose-related responses in the comet assay. Our data encourage the application of the used classification model for a sensitive and fast quantification of DNA damage. Results in this study are in agreement with most of the earlier investigations.

Unlabelled: Radiation synovectomy is an effective treatment for chronic synovitis refractory to pharmacological treatment in patients with rheumatoid or seronegative arthritis. Concerns persist about possible radiation-induced cytogenetic damage after radiation synovectomy leading to recommendations to use this technique only in the elderly. Micronucleus (MN) frequency in lymphocytes and urinary excretion of 8-hydroxy-2'-deoxyguanosine (8OHdG) as an indicator of cellular oxidative DNA base damage are biomarkers of radiation-induced cytogenetic damage. The course of both biomarkers was studied in patients with different types of chronic synovitis undergoing radiation synovectomy with very short-lived 165Dy-ferric-hydroxide (DFH)., Methods: Radiation synovectomy of the knee was performed in 13 men and 12 women (mean age, 44+/-15 y) using a mean activity of 9.48+/-1.65 GBq 165Dy-DFH in 27 consecutive treatments. MN frequency in lymphocytes and urinary excretion of 8OHdG, measured by high-performance liquid chromatography, were assessed before and 4 (MN only) and 20 h after radiation synovectomy., Results: Urinary excretion of 8OHdG in patients (in micromol/mol creatinine; pretreatment mean, 3.1+/-3.4; median, 2.27) was not significantly different from that in healthy volunteers (mean, 2.0+/-1.2; median, 1.87) and not altered by radiation synovectomy (post-treatment mean, 2.5+/-1.5; median, 2.04, NS). An increase in 8OHdG levels after radiation synovectomy of more than 1 SD was found in only 1 patient, who experienced leakage to the lymph nodes but who already had elevated urinary 8OHdG levels before treatment. The frequency of MN/500 binucleated cells (BNCs) was slightly lower in patients (pretreatment mean, 4.3+/-2.6; median, 4.25) than in healthy volunteers (mean, 5.4+/-2.3; median, 5.3) and did not significantly change after therapy, either (4-h post-treatment mean, 3.9+/-2.1, median, 3.8; 20-h post-treatment mean, 4.1+/-2, median 3.8 MN/500 BNC). In 22 of 27 treatments, no leakage to nontarget organs could be monitored, whereas leakage to the local lymph nodes and the liver was detected after 5 treatments., Conclusion: Radiation synovectomy using 165Dy-DFH causes no significant radiation burden to most patients as indicated by the absence of adverse changes in levels of biomarkers of cytogenetic damage and a low incidence of leakage. These data suggest that the risk of malignancy may not be elevated.

The detection of DNA single-strand breaks (SSB) in human mononucleated white blood cells (MWBC) using a modified version of the nick translation assay is presented. This assay allows rapid and sensitive examination of SSB using only 5 ml heparinized blood for an eightfold determination. The assay was standardized by incubation of MBWC in vitro with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a known genotoxic agent. In vitro incubation of MWBC with MNNG induced a dose-dependent increase in DNA-SSB at doses between 5 and 500 microM MNNG. The detection limit for the assay was 5 microM MNNG. To assess the suitability of this assay to detect SSB in vivo a controlled study was performed in which volunteer smokers (n = 5), nonsmokers (n = 5) exposed to environmental tobacco smoke (ETS), and nonsmokers controls (n = 5) were compared. The study lasted 4 experimental days, 2 control and 2 exposure days. On control days (days 1 and 3) smokers and nonsmokers sat in an unventilated 45 m3 room for 8 h. On the exposure days (days 2 and 4) each of the five smokers smoked 24 cigarettes in 8 h, while the five nonsmokers were exposed to the ETS generated by the smoking volunteers. High exposure to tobacco smoke was confirmed by dosimetry of carboxyhemoglobin (CO-Hb), plasma nicotine and cotinine levels. Blood was drawn before and after each exposure on all 4 experimental days for determination of DNA-SSB in lymphocytes immediately after isolation of blood cells.(ABSTRACT TRUNCATED AT 250 WORDS)

In a controlled study, ten male volunteers (five smokers and five nonsmokers) were subjected to different smoking conditions and compared to five nonsmokers, not exposed to cigarette smoke. During the 4 days of the study, nonsmoking periods were strictly controlled. On the first day the ten subjects were sham exposed. On the second day the five smokers smoked 24 cigarettes in 8 h, while the five nonsmokers were exposed to the environmental tobacco smoke. After another day of sham exposure the smoke exposure was repeated under the same conditions. Blood was drawn before and after exposure and DNA single-strand breaks (SSBs) were analyzed in lymphocytes immediately (1 h) after isolation of cells and after 4 h incubation at 37 degrees C, using a modified assay based on the nick translation reaction. Base levels of unscheduled DNA synthesis (UDS) and UDS levels were determined after 1 h incubation with methyl methanesulfonate. Duplicate analysis using the same method was performed in a second laboratory after transportation of blood samples at 0 degree C on a train from Munich to Hamburg. Tobacco smoke exposure of the subjects increased COHb and plasma cotinine levels. SSBs could be detected in all probands with some interindividual day-to-day and morning-to-evening variations. In four of five active smokers, SSB increases were found after smoking. In nonsmokers exposed to tobacco smoke no exposure-related variation in SSB levels could be detected.(ABSTRACT TRUNCATED AT 250 WORDS)

A modified assay for the detection of DNA single-strand breaks (SSBs) in human mononucleated white blood cells (MWBCs) based on the nick translation (NT) reaction was developed and combined with the test for unscheduled DNA synthesis (UDS). Both assays were performed on disposable 96-well filtration plates and therefore allowed rapid and sensitive examination of SSBs and UDS. Only 5-8 ml of heparinized blood is required for an eightfold determination in both assays. The uptake of radioactive nucleotide precursors was demonstrated to depend linearly upon the NT reaction time and in both assay systems on the number of investigated cells. The best results and the lowest signal to noise ratio were obtained when the NT assay was performed at 25 degrees C for 20 min. The test was standardized for 150,000 MWBCs/well and a polymerase I concentration of 20 U/ml. The same number of cells were used to measure UDS during a 4-h incubation at 37 degrees C. We observed a dose-dependent increase in SSBs after in vitro incubation with N-methyl-N-nitrosoguanidine (MNNG), with a detection limit of 50 microM when MNNG was present for 1 h and of 5 microM after 20-h incubation period. UDS in MWBCs was increased after treatment for 1 h with MNNG (200 microM) only if poly(ADP)ribose synthesis was inhibited by 3-aminobenzamide. UDS was induced by 320 microM methyl methanesulfonate, but SSBs could only be detected after inhibition of UDS by 100 microM hydroxyurea.(ABSTRACT TRUNCATED AT 250 WORDS)

It was the aim of this study to compare the formation of DNA adducts in different human white blood cells. Lymphocytes and monocytes were isolated from peripheral blood by density centrifugation and by a monocyte-specific antibody linked to magnetic beads. DNA adducts were determined by 32P-postlabelling. After in vitro incubation with benzo[a]-pyrene (B[a]P) we found DNA adducts in monocytes, but not in unstimulated blood lymphocytes. Only after growth stimulation by phytohaemagglutinin did lymphocytes show an adduct pattern similar to monocytes. In contrast, B[a]P-7,8-dihydro-9,10-diolepoxide, an activated intermediate of B[a]P metabolism leads to similar DNA adducts in both cell types. When lymphocytes were incubated in the presence of B[a]P together with monocytes, then after subsequent separation lymphocytes exhibited most of the adducts found in monocytes, though to a lower extent. Our results suggest that unstimulated blood lymphocytes--which are unable to activate metabolically promutagens like B[a]P--receive the genotoxic material to form DNA adducts from other blood cells or from cells in the vessel lining. We conclude that the amount and the pattern of DNA adducts formed in whole white blood cells may be influenced considerably by a variable proportion of monocytes.

The clinical radiosensitizer bromodeoxyuridine (BrdU) was shown to enhance oxygen free radical-mediated growth inhibition. Cells from Bloom's syndrome, a rare autosomal recessive disorder characterized by pre- and post-natal growth deficits, telangiectatic erythema, recurrent respiratory infections and a high incidence of cancer, exhibit in culture a hypersensitivity to BrdU. We analysed disturbed cell kinetics of Bloom's syndrome fibroblasts and permanent B-cell lines with a novel cell kinetic method: BrdU/Hoechst flow cytometry. Fibroblasts show a pattern similar to that of normal cells exposed to a breakdown product of lipid peroxides, whereas B-cells exhibit the cell kinetic disturbance provoked by elevated oxygen concentrations in normal cells. In both cell types the cell kinetic pattern was dependent upon the BrdU concentration in the culture medium. These data suggest an elevated endogenous generation of oxygen free radicals in Bloom's syndrome cells, which may relate to the elevated incidence of malignancies in these patients.