Mechanistic investigation of ROS-induced DNA damage by oestrogenic compounds in lymphocytes and sperm using the comet assay.

Past research has demonstrated that oestrogenic compounds produce strand breaks in the DNA of sperm and lymphocytes via reactive oxygen species (ROS). In the current investigation, sperm and lymphocytes were treated in vitro with oestrogenic compounds (diethylstilboestrol, progesterone, 17ß-oestradiol, noradrenaline and triiodotyronine) and several aspects of DNA damage were investigated. Firstly, mediation of DNA damage by lipid peroxidation was investigated in the presence of BHA (a lipid peroxidation blocker). BHA reduced the DNA damage generated by 17ß-oestradiol and diethylstilboestrol in a statistically significant manner. No effects were observed for sperm. Secondly, the presence of oxidized bases employing FPG and EndoIII were detected for lymphocytes and sperm in the negative control and after 24 h recovery in lymphocytes but not immediately after treatment for both cell types. The successful detection of oxidized bases in the negative control (untreated) of sperm provides an opportunity for its application in biomonitoring studies. DNA repair at 24 h after exposure was also studied. A nearly complete recovery to negative control levels was shown in lymphocytes 24 h recovery after oestrogenic exposure and this was statistically significant in all cases. Rapid rejoining of DNA, in a matter of hours, is a characteristic of DNA damaged by ROS.

Incomplete protection of genetic integrity of mature spermatozoa against oxidative stress.

Although DNA damage in human spermatozoa is associated with adverse health effects, its origin is not fully understood. Therefore, we assessed biomarkers in ejaculates that retrospectively reflect processes that occurred in the epididymis or testis. Smoking increased the amount of DNA strand breaks (P<0.01), and enhanced the presence of vitamin C radicals in seminal plasma. In vitro, vitamin C protected mature spermatozoa against DNA damage, but this protection appeared to be insufficient in vivo. CAT and DDIT4 expression in spermatozoa were higher in smokers than in nonsmokers, but were not related to DNA damage. CAT and DDIT4 expression were inversely related with sperm count (P=0.039 and 0.024 resp.), but no effect was observed for SOD2 expression. These data indicate that spermatozoa of smokers encounter higher levels of oxidative stress. Expression of antioxidant enzymes and seminal vitamin C were insufficient to provide full protection of spermatozoa against DNA damage.

Induction of oxidative DNA damage by the marine toxin okadaic acid depends on human cell type.

The marine toxin okadaic acid (OA) is the main representative of diarrhoeic shellfish poisoning (DSP) toxins. Its ingestion induces nausea, vomiting, diarrhoea and abdominal ache. It has also been found to trigger cellular and molecular effects at low concentrations. Its mechanism of action has not been described yet. Results of a previous study showed that OA can induce cytotoxic and genotoxic effects, both directly and indirectly, and modulations in DNA repair processes in three different types of human cells (leukocytes, SHSY5Y neuroblastoma and HepG2 cells). These effects varied depending on the type of cell and the concentration employed (Valdiglesias et al., 2010). On that basis, the ability of OA to induce oxidative DNA damage on the same cell types was investigated in the present study. To this end, the antioxidant enzymes catalase and N-acetylcysteine, and the human DNA- glycosylase hOGG1 were used in combination with the alkaline Comet assay. The cells were treated with a range of OA concentrations (5-1000 nM) in the presence and absence of S9 fraction. The results of this study showed that OA induces oxidative DNA damage directly in leukocytes, directly and indirectly in SHSY5Y cells, while it does not induce oxidative DNA damage in HepG2 cells. Combining the outcomes of both studies, the data showed that OA induces both cytotoxicity and genotoxicity, including DNA strand breaks and oxidative DNA damage, in the cells evaluated. However, the extent of these effects are cell type dependent.

Genotoxicity and cytotoxicity of zinc oxide and titanium dioxide in HEp-2 cells.

AIMS: The rapidly growing industrial and medical use of nanomaterials, especially zinc oxide and titanium dioxide, has led to growing concerns about their toxicity. Accordingly, the intrinsic genotoxic and cytotoxic potential of these nanoparticles have been evaluated. MATERIALS & METHODS: Using a HEp-2 cell line, cytotoxicity was tested along with mitochondrial activity and neutral red uptake assays. The genotoxic potential was determined using the Comet and the cytokinesis-blocked micronucleus assays. In addition, tyrosine phosphorylation events were investigated. RESULTS & CONCLUSION: We found concentration- and time-dependent cytotoxicity and an increase in DNA and cytogenetic damage with increasing nanoparticle concentrations. Mainly for zinc oxide, genotoxicity was clearly associated with an increase in tyrosine phosphorylation. Our results suggest that both types of nanoparticles can be genotoxic over a range of concentrations without being cytotoxic.

Evaluation of the genotoxicity of 10 selected dietary/environmental compounds with the in vitro micronucleus cytokinesis-block assay in an interlaboratory comparison.

Complex exposure to xenobiotics is one of the reasons for the reported increase of respiratory diseases, cancer and immunological disturbances. Among such xenobiotics there are food mutagens whose effects on human health in the low level and/or chronic exposure still remains unknown. In the present manuscript, the compounds ethanol (EtOH), 4-hydroxynonenal (4-HNE), malondialdehyde (MDA), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3',4,4'-tetrachlorobiphenyl (PCB 153), benzo[a]pyrene (BaP), 2-amino-3-methylimidazol[4,5-f]quinoline (IQ), 2-amino-1-methyl-6-phenylimidazol[4,5-b]pyridine (PhIP), N-Nitrosodimethylamine (NDMA) and acrylamide (AA) were evaluated in an interlaboratory comparison in the in vitro cytokinesis-block micronucleus assay (CBMN) with objective of assessing the induction of micronuclei, buds and nucleoplasmic bridges in dose responses. Statistically significant increase in MNBN frequency in binucleated cells was recorded by both laboratories for the compound PhIP (2.5µM). The compounds PCB (250 microM) and AA (500 microM) induced statistically significant increase of MNBN although it was recorded by one of the two laboratories. Induction of buds and nucleoplasmic bridges was only observed for BaP (100 microM) and AA (500 microM) by one of the laboratories. Data generated in this study may assist in the interpretation of the mother/newborn biomonitoring study being carried out within project NewGeneris and will contribute to overall knowledge on the genotoxic potential of dietary/environmental toxicants.

Assessment of okadaic acid effects on cytotoxicity, DNA damage and DNA repair in human cells.

Okadaic acid (OA) is a phycotoxin produced by several types of dinoflagellates causing diarrheic shellfish poisoning (DSP) in humans. Symptoms induced by DSP toxins are mainly gastrointestinal, but the intoxication does not appear to be fatal. Despite this, this toxin presents a potential threat to human health even at concentrations too low to induce acute toxicity, since previous animal studies have shown that OA has very potent tumour promoting activity. However, its concrete action mechanism has not been described yet and the results reported with regard to OA cytotoxicity and genotoxicity are often contradictory. In the present study, the genotoxic and cytotoxic effects of OA on three different types of human cells (peripheral blood leukocytes, HepG2 hepatoma cells, and SHSY5Y neuroblastoma cells) were evaluated. Cells were treated with a range of OA concentrations in the presence and absence of S9 fraction, and MTT test and Comet assay were performed in order to evaluate cytotoxicity and genotoxicity, respectively. The possible effects of OA on DNA repair were also studied by means of the DNA repair competence assay, using bleomycin as DNA damage inductor. Treatment with OA in absence of S9 fraction induced not statistically significant decrease in cell viability and significant increase in DNA damage in all cell types at the highest concentrations investigated. However, only SHSY5Y cells showed OA induced genotoxic and cytotoxic effects in presence of S9 fraction. Furthermore, we found that OA can induce modulations in DNA repair processes when exposure was performed prior to BLM treatment, in co-exposure, or during the subsequent DNA repair process.

Investigation on the mechanisms of genotoxicity of butadiene, styrene and their combination in human lymphocytes using the Comet assay.

The toxicity of butadiene and styrene is exerted by their metabolites. Such metabolites have been extensively scrutinized at the in vitro level demonstrating evident genotoxic properties. In monitoring, a diverse range of outcomes has been produced. Additionally, epidemiological studies in rubber workers face difficulties of data interpretation due to the changeability and multiple exposures of the workers as well as to confounding factors inherent to the cohorts. Nevertheless, toxicity has been associated with a significant trend of increasing the risk of leukaemia in employees at the styrene-butadiene rubber industry. Thus, further effort must be made to distinguish the exposures to each chemical over time and to characterize their interrelationships. The present investigation focuses on the effects and mechanisms of damage of the mixture styrene-butadiene by examining its metabolites: styrene oxide (SO), butadiene monoepoxide (BME) and butadiene diepoxide (BDE) respectively. The in vitro Comet assay on frozen lymphocytes has been employed to ascertain the DNA damage patterns for the styrene-butadiene metabolites combined and on their own. Different patterns were observed for the mixture and each of its components. This study has also led to determining the mechanism of damage of the mixture and the compounds. With regard to the presence of reactive oxygen species (ROS), co-treatment with catalase does not modulate the genotoxicity of the mixture but it does modulate its components. The outcomes also indicate that the mixture induces cross-links and this is due to the influence of BDE in the mixture, being more evident as the concentration of BDE increases. An investigation on the sensitivity of lymphocytes from occupationally un/exposed subjects to in vitro exposure of the mixture and its components revealed that occupationally exposed subjects had a substantially higher background of DNA damage and a lower sensitivity to the metabolites of styrene, 1,3-butadiene and its mixture.

Use of spermatozoal mRNA profiles to study gene-environment interactions in human germ cells.

Paternal exposure to genotoxic compounds is thought to contribute to diseases in their offspring. Therefore, it is of importance to develop biomarkers of male germ cell exposure to genotoxins. Unfortunately, the testis cannot be reached for routine biomonitoring, but mRNA-profiles in spermatozoa may reflect the processes that have occurred in the testis after exposures to genotoxins, since spermatozoa are largely transcriptionally inactive. Therefore, mRNA profiles from sperm in ejaculates of cigarette smokers (N=4) were compared with non-smokers (N= 4). Smoking behaviour was verified by assessing cotinine levels in seminal plasma. High expression of the germ cell specific gene protamine 2 (PRM2) was observed in spermatozoal mRNA isolates by Q-PCR, which was absent in reference mRNA isolates obtained from a pool of other organs. Gene-expression analysis was subsequently performed using microarray technology and a total of 781 genes were found to be differentially expressed in spermatozoa of smokers compared to non-smokers (fold change >40%; p < 0.05). To further limit the number of false positive results, genes were additionally selected on basis of the correlation between their expression levels with cotinine concentrations in seminal plasma (r > 0.80 as arbitrary cut-off value, p < 0.05), and a total of 200 transcripts remained, of which the germ cell specific transcription factor SALF was the highest up-regulated gene (5.4-fold) and the zinc finger encoding gene TRIM26 most down regulated (7.4-fold). Although no altered pathways could be identified for the differentially expressed genes, an enrichment was observed for NF-kappaB regulated genes (46% vs. 27%, p = 0.004) playing a central role in stress response. Accordingly, subsequent analysis of transcription factor networks suggests that apoptosis was inhibited in smokers. These data show the feasibility of using gene-expression profiles in mature sperm to elucidate gene-environment interactions in male testis.

Automated image analysis of cytokinesis-blocked micronuclei: an adapted protocol and a validated scoring procedure for biomonitoring.

Micronuclei (MN) frequencies in peripheral blood lymphocytes have been used worldwide as a biomarker of chromosomal damage for genotoxicity testing and biomonitoring studies. Automation of MN analysis would provide faster and more reliable results with minimizing subjective MN identification. We developed an automated facility for the scoring of the in vitro MN cytokinesis-block assay for biomonitoring on Giemsa-stained slides, fulfilling the following criteria: applicable to the cytokinesis-block micronucleus methodology, discriminating between mono-, bi- and polynucleated cells, MN scoring according to HUMN scoring criteria, false-negative MN rate <10% and false-positive (FP) MN rate <1%. We first adapted the slide preparation protocol to obtain an optimal cell density and dispersion, which is important for image analysis. We developed specific algorithms starting from the cell as a detection unit. The whole detection and scoring process was separated into two distinct steps: in the first step, the cells and nuclei are detected; then, in the second step, the MN are searched for in the detected cells. Since the rate of FP MN obtained by the automatic analysis was in the range of 0.5-1.5%, an interactive visual validation step was introduced, which is not time consuming and allows quality control. Validation of the automated scoring procedure was undertaken by comparing the results of visual and automated scoring of micronucleated mono- and binucleated cells in human lymphocytes induced by two clastogens (ionizing radiation and methyl methane-sulphonate), two aneugens (nocodazole and carbendazim) and one apoptogen (staurosporine). Although the absolute MN frequencies obtained with automated scoring were lower as compared to those detected by visual scoring, a clear dose response for MNBN frequencies was observed with the automated scoring system, indicating that it is able to produce biologically relevant and reliable results. These observations, together with its ability to detect cells, nuclei and MN in accordance with the HUMN scoring criteria, confirm the usability of the automated MN analysis system for biomonitoring.

Antioxidants and the Comet assay.

It is widely accepted that antioxidants, either endogenous or from the diet, play a key role in preserving health. They are able to quench radical species generated in situations of oxidative stress, either triggered by pathologies or xenobiotics, and they protect the integrity of DNA from genotoxicants. Nevertheless, there are still many compounds with unclear or unidentified prooxidant/antioxidant activities. This is of concern since there is an increase in the number of compounds synthesized or extracted from vegetables to which humans might be exposed. Despite the well-established protective effects of fruit and vegetables, the antioxidant(s) responsible have not all been clearly identified. There might also be alternative mechanisms contributing to the protective effects for which a comprehensive description is lacking. In the last two decades, the Comet assay has been extensively used for the investigation of the effects of antioxidants and many reports can be found in the literature. The Comet assay, a relatively fast, simple, and sensitive technique for the analysis of DNA damage in all cell types, has been applied for the screening of chemicals, biomonitoring and intervention studies. In the present review, several of the most well-known antioxidants are considered. These include: catalase, superoxide dismutase, glutathione peroxidase, selenium, iron chelators, melatonin, melanin, vitamins (A, B, C and E), carotenes, flavonoids, isoflavones, tea polyphenols, wine polyphenols and synthetic antioxidants. Investigations showing beneficial as well as non-beneficial properties of the antioxidants selected, either at the in vitro, ex vivo or in vivo level are discussed.

Quinones are reduced by 6-tetrahydrobiopterin in human keratinocytes, melanocytes, and melanoma cells.

Quinones are potentially dangerous substances generated from quinols via the intermediates semiquinone and hydrogen peroxide. Low semiquinone radical concentrations are acting as radical scavengers while high concentrations produce reactive oxygen species and quinones, leading to oxidative stress, apoptosis, and/or DNA damage. Recently it was recognised that thioredoxin reductase/thioredoxin (TR/T) reduces both p- and o-quinones. In this report we examine additional reduction mechanisms for p- and o-quinones generated from hydroquinone (HQ) and coenzyme Q10 and by 17beta-estradiol by the common cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH(4)). Our results confirmed that TR reduces the p-quinone 1,4 benzoquinone and coenzyme Q10-quinone back to HQ and coenzyme Q10-quinol, respectively, while 6BH(4) has the capacity to reduce coenzyme Q10-quinone and the o-quinone produced from 17beta-estradiol. 6BH(4) is present in the cytosol and in the nucleus of epidermal melanocytes and keratinocytes as well as melanoma cells and colocalises with TR/T. Therefore we conclude that both mechanisms are major players in the prevention of quinone-mediated oxidative stress and DNA damage.

Modulation of the cytotoxicity and genotoxicity of the drinking water disinfection byproduct lodoacetic acid by suppressors of oxidative stress.

Drinking water disinfection byproducts (DBPs) are generated by the chemical disinfection of water and may pose a hazard to the public health. Previously we demonstrated that iodoacetic acid was the most cytotoxic and genotoxic DBP analyzed in a mammalian cell system. Little is known of the mechanisms of its genotoxicity. The involvement of oxidative stress in the toxicity of iodoacetic acid was analyzed with the antioxidants catalase and butylated hydroxyanisole (BHA). lodoacetic acid toxicity was quantitatively measured with and without antioxidants in Salmonella typhimurium strain TA100 and with Chinese hamster ovary (CHO) cells. The endpoints included cytotoxicity in S. typhimurium or in CHO cells, mutagenicity in S. typhimurium, and genotoxicity in CHO cells. Neither catalase nor BHA reduced the level of iodoacetic acid induced cytotoxicity in S. typhimurium. In CHO cells neither antioxidant caused a significant reduction in iodoacetic acid induced cytotoxicity. However, in S. typhimurium, BHA or catalase reduced the mutagenicity of iodoacetic acid by 33.5 and 26.8%, respectively. Likewise, BHA or catalase reduced iodoacetic acid induced genomic DNA damage by 86.5 and 42%, respectively. These results support the hypothesis that oxidative stress is involved in the induction of genotoxicity and mutagenicity by iodoacetic acid.

Estrogens can contribute to hydrogen peroxide generation and quinone-mediated DNA damage in peripheral blood lymphocytes from patients with vitiligo.

To date there is ample in vivo and in vitro evidence for increased epidermal and systemic hydrogen peroxide (H(2)O(2)) levels in vitiligo, which can be reduced with a topical application of a pseudocatalase-K.U. Schallreuter (PC-KUS) leading to the recovery of epidermal catalase levels as well as other enzymes in peripheral blood cells. Recently, the generation of H(2)O(2) by oxidative metabolism of estrogens and other aromatic steroids was documented. Therefore, it was tempting to follow estrogen-generated H(2)O(2) and its possible effect on DNA damage in peripheral blood lymphocytes from patients with vitiligo before and after the reduction of epidermal H(2)O(2) with pseudocatalase PC-KUS compared to controls. For this purpose, 20 Caucasian patients were grouped in treated responders (group A, n=11) and untreated active/acute disease (group B, n=9) and compared to Caucasian healthy controls (group C, n=7). Consequently, epidermal catalase protein expression in full skin biopsies was assessed using immunofluorescence labelling together with determination of basal H(2)O(2) levels in peripheral blood lymphocytes. To test the influence of estrogen on H(2)O(2) generation and DNA damage, freshly prepared peripheral blood lymphocytes from all three groups were used for the alkaline comet assay in the presence and absence of catalase. The results of this study demonstrated that reduction of epidermal H(2)O(2) leads to both increased epidermal catalase protein expression as well as decreased H(2)O(2) concentrations in lymphocytes. Moreover, a direct estrogen-mediated DNA damage was identified in both patient groups, which was absent in healthy controls. This effect was not abolished by catalase pointing to direct quinone-mediated DNA damage by estrogens in peripheral blood lymphocytes in vitiligo.

Cadmium chloride-induced DNA and lysosomal damage in a hepatoma cell line.

Cadmium is a toxic metal and no uniform mechanism of toxicity has so far been proposed. The aim of this study was to investigate the biochemical effects of cadmium chloride in a rat hepatoma cell line (HTC cells) and the cellular events mediating DNA damage. HTC cells were exposed to various concentrations of cadmium chloride for 5 and 8 h and lysosomal damage was assessed with the neutral red assay (NR) and fluorescence microscopy. Mitochondrial integrity was assessed from ATP levels and DNA damage determined with the single cell gel electrophoresis/comet assay. The formation of reactive oxygen species (ROS) was also determined under the same experimental conditions with the dichlorofluorescein assay. Cytotoxicity was assessed with the LDH leakage assay and the levels of glutathione were measured and correlated with the other effects. The results indicate that lysosomal damage occurs at a lower concentration of cadmium chloride (20 microM) than DNA damage (500 microM) in HTC cells. The latter effect was accompanied by an increase of reactive oxygen species without any significant LDH leakage whereas lysosomal damage was significant as determined by the neutral red assay and confirmed with fluorescence microscopy. The effect of CdCl2 on mitochondria and glutathione levels were observed at concentrations or incubation times higher than the ones required to induce lysosomal damage. The data suggest that DNA damage may be due to the formation of reactive oxygen species. It is possible that cadmium induced lysosomal damage is an earlier event than DNA damage and can mediate other cellular events that lead to cell death.

Modulation by flavonoids of DNA damage induced by estrogen-like compounds.

Reactive oxygen species (ROS) are produced by a wide variety of exogenous chemicals and metabolic processes and cause a broad spectrum of damage to biological systems. As a consequence, ROS react with DNA, among many other biological targets, disrupting its structure and functionality. Estrogen-like compounds mediate DNA damage by ROS generation, implying that their effects can be modulated by antioxidants such as catalase, superoxide dismutase, and vitamin C. We examined DNA damage in human lymphocytes and sperm after treatment with four estrogen-like compounds (beta-estradiol, diethylstilbestrol, daidzein, and genistein) and its modulation by flavonoids (quercetin and kaempferol) using the Comet assay. The results indicated that quercetin and kaempferol reduced the DNA damage produced in sperm and lymphocytes by the four estrogenic compounds. The flavonoids also reduced the DNA damage induced by hydrogen peroxide, which was used as a positive control. Our results demonstrate that the antioxidant properties of flavonoids can protect the integrity of human sperm and lymphocyte DNA from ROS induced by estrogenic compounds.

Genotoxicity studies on DNA-interactive telomerase inhibitors with application as anti-cancer agents.

Telomerase-targeted strategies have aroused recent interest in anti-cancer chemotherapy, because DNA-binding drugs can interact with high-order tetraplex rather than double-stranded (duplex) DNA targets in tumour cells. However, the protracted cell-drug exposure times necessary for clinical application require that telomerase inhibitory efficacy must be accompanied by both low inherent cytotoxicity and the absence of mutagenicity/genotoxicity. For the first time, the genotoxicity of a number of structurally diverse DNA-interactive telomerase inhibitors is examined in the Ames test using six Salmonella typhimurium bacterial strains (TA1535, TA1537, TA1538, TA98, TA100, and TA102). DNA damage induced by each agent was also assessed using the Comet assay with human lymphocytes. The two assay procedures revealed markedly different genotoxicity profiles that are likely to reflect differences in metabolism and/or DNA repair between bacterial and mammalian cells. The mutational spectrum for a biologically active fluorenone derivative, shown to be mutagenic in the TA100 strain, was characterised using a novel and rapid assay method based upon PCR amplification of a fragment of the hisG46 allele, followed by RFLP analysis. Preliminary analysis indicates that the majority (84%) of mutations induced by this compound are C --> A transversions at position 2 of the missense proline codon of the hisG46 allele. However, despite its genotoxic bacterial profile, this fluorenone agent gave a negative response in the Comet assay, and demonstrates how unwanted systemic effects (e.g., cytotoxicity and genotoxicity) can be prevented or ameliorated through suitable molecular fine-tuning of a candidate drug in targeted human tumour cells.

Oxygen-induced DNA damage in freshly isolated brain cells compared with cultured astrocytes in the Comet assay.

Brain cells are continuously exposed to reactive oxygen species generated by oxidative metabolism, and in certain pathological conditions defence mechanisms against oxygen radicals may be weakened and/or overwhelmed. DNA is a potential target for oxidative damage, and genomic damage can contribute to neuropathogenesis. It is important, therefore, to identify tools for the quantitative analysis of DNA damage in models of neurological disorders. The aim of this study was to compare the susceptibility of DNA to oxidative stress in cells freshly dissociated from the mouse brain, to that in cultured brain cells. Both primary cultures and a continuous cell line of astrocytes were considered. All cells were treated by xanthine/xanthine oxidase, a superoxide generator or hydrogen peroxide, applied alone or in the presence of the oxygen radical scavengers, superoxide dismutase, catalase, or ascorbic acid. DNA damage, quantified with the Comet assay, was consistent in all the different cell preparations exposed to oxidative stress, and was attenuated in similar ways by superoxide dismutase and catalase, scavengers of superoxide anion and hydrogen peroxide, respectively. The results with ascorbic acid were more variable, presumably because this compound may switch from anti- to pro-oxidant status depending on its concentration and other experimental conditions. Overall, similar responses were found in freshly dissociated and cultured brain cells. These results suggest that the Comet assay can be directly applied to cells freshly dissociated from the brain of rodents, including models of neurological disorders, such as stroke models and animals with targeted mutations that mimic human diseases.

Antigenotoxic properties of selenium compounds on potassium dichromate and hydrogen peroxide.

Selenium is an environmental metal that occurs ubiquitously and is produced throughout the world for various industrial activities. Selenium has been reported to have anticarcinogenic and preventive effects in clinical and epidemiological studies. Selenium supplements can inhibit chemically-induced tumours. From the viewpoint of genotoxicity, selenium has not been adequately studied and an IARC review concluded that there were not sufficient data to consider it a carcinogen for man. In contrast, hexavalent chromium is classified as a known respiratory carcinogen producing DNA damage through free oxygen radicals. In the present study, a collaborative study has been carried out to evaluate the genotoxicity of selenium compounds and their possible interactions with potassium dichromate and hydrogen peroxide. Thus, in laboratory 1 (U.K.), the genotoxic effects of three selenium compounds were examined. Sodium selenate, sodium selenite, and selenous acid were investigated in the Ames test using strain TA102 and in the Comet assay using human lymphocytes, and also investigated for their interaction with potassium dichromate. In the Ames test, it was shown that potassium dichromate produced a highly mutagenic response, whilst the three selenium compounds did not. In combination, sodium selenate reduced the genotoxicity of potassium dichromate, but sodium selenite and selenous acid had no effect. In the Comet assay, potassium dichromate induced DNA damage, but so did the selenium compounds. In combination with potassium dichromate, however, only sodium selenate reduced its effect, whereas sodium selenite and selenous acid exacerbated DNA damage. In laboratory 2 (Spain), in the TK6 lymphoblastoid cell line, the Comet assay showed that sodium selenite was non-genotoxic, while potassium dichromate and hydrogen peroxide induced DNA damage. It was also shown that sodium selenite did not decrease the genotoxicity of potassium dichromate or hydrogen peroxide when administered as a pre-treatment or at the same time, or when potassium dichromate and sodium selenite treatments were for different time periods. Thus, only sodium selenate has shown antigenotoxic properties against potassium dichromate in the Ames test and in human lymphocytes in the Comet assay.