The genotoxic potential of glutaraldehyde in mammalian cells in vitro in comparison with formaldehyde

Glutaraldehyde (GA) induces DNA-protein crosslinks (DPX), but conflicting results have been reported with regard to other genotoxic and mutagenic effects in mammalian cells in vitro. We, therefore, characterized the genotoxic and mutagenic potential of GA in V79 cells. Using the alkaline comet assay we demonstrated the induction of DPX by GA (reduction of gamma ray-induced DNA migration) at a concentration of 10 microM and above. The standard comet assay did not reveal a significant DNA strand-breaking activity of GA. Cross-linking concentrations of GA were also cytotoxic, i.e. inhibited cell growth of treated V79 cultures. Interestingly, a small but statistically significant increase in sister chromatid exchange (SCE) and micronuclei (MN) was already measured at lower concentrations (2 and 5 microM). FISH analysis revealed that the majority of GA-induced MN was due to chromosome breaks. We also compared the genotoxic activity of GA to that of formaldehyde (FA). Similar to GA, FA-induced DPX, SCE and MN, but distinct differences exist with regard to the sensitivity of the endpoints and the relationship between genotoxicity and cytotoxicity. However, the differences in genotoxicity cannot readily explain the different carcinogenic activities of the two compounds.     

Cytogenetic damage induced in human lymphocytes by sodium bisulfite.

The frequencies of chromosomal aberrations (CA), sister-chromatid exchanges (SCE), and micronuclei (MN) in human blood lymphocytes exposed to sodium bisulfite (sulfur dioxide) at various concentrations ranging from 5 x 10(-5) M to 2 x 10(-3) M in vitro were studied. It was shown that sodium bisulfite (NaHSO3 and Na2SO3, 1:3 M/M) caused an increase in SCE and MN in human blood lymphocytes in a dose-dependent manner, and also induced mitotic delays and decreased mitotic index. For CA, our results indicated that sodium bisulfite induced an increase of chromatid-type aberrations in lymphocytes from three of four donors in a dose-dependent manner. The chemical at low concentrations induced chromatid-type aberrations, but not chromosome-type aberrations; high concentrations induced both chromatid- and chromosome-type aberrations. No cytogenetic damage in human lymphocytes was induced by sodium sulfate. The results have confirmed that sulfur dioxide is a clastogenic and genotoxic agent.     

Low sensitivity of the comet assay to detect acetaldehyde-induced genotoxicity

Acetaldehyde (AA) is known to induce DNA-protein cross-links (DPX) and other genotoxic and mutagenic effects in cultured mammalian cells. Compared to formaldehyde (FA), AA is a very weak inducer of DPX and increased DPX levels are only measured at high, cytotoxic concentrations by different methods. Besides DPX, AA also induces DNA-DNA cross-links. Because the comet assay is increasingly used for the detection of cross-linking agents, we characterized the effects of AA in the comet assay in relation to cytotoxicity and other genetic endpoints such as the induction of sister chromatid exchange (SCE) and micronuclei (MN). The standard alkaline comet assay did not indicate induction of DNA strand-breaks by AA in a range of concentrations from 0.2 to 20 mM. AA at a concentration of 20 mM was clearly cytotoxic and reduced cell growth and population doubling to less than 50% of the control. Using the comet assay modification with proteinase K, slightly enhanced DNA migration was measured in comparison to treatment with AA only. No significant induction of cross-links by AA (measured as reduction of gamma ray-induced DNA migration) was determined by the comet assay. A small and reproducible but statistically not significant effect was measured for the AA concentration 20 mM. A clear and concentration-related increase in the frequency of sister chromatid exchange (SCE) and micronuclei (MN) was already measured at lower concentrations (0.2 and 0.5 mM, respectively). These results suggest that the comet assay has a low sensitivity for the detection of AA-induced DNA lesions leading to the induction of SCE and MN.     

Genotoxic effects of occupational exposure to lead and cadmium

This study was designed to assess genotoxic damage in somatic cells of workers in a Polish battery plant after high-level occupational exposure to lead (Pb) and cadmium (Cd), by use of the following techniques: the micronucleus (MN) assay, combined with in situ fluorescence hybridization (FISH) with pan-centromeric probes, analysis of sister chromatid exchanges (SCEs), and the comet assay. Blood samples from 44 workers exposed to lead, 22 exposed to cadmium, and 52 unexposed persons were used for SCE and MN analysis with 5'-bromodeoxyuridine (BrdU) or cytokinesis block, respectively. In parallel, the comet assay was performed with blood samples from the same persons for detection of DNA damage, including single-strand breaks (SSB) and alkali-labile sites (ALS). In workers exposed mostly to lead, blood Pb concentrations ranged from 282 to 655 microg/l, while the range in the controls was from 17 to 180 microg/l. Cd concentration in lead-exposed workers fell in the same range as for the controls. In workers exposed mainly to cadmium, blood Cd levels varied from 5.4 to 30.8 microg/l, with respective values for controls within the range of 0.2-5.7 microg/l. Pb concentrations were similar as for the controls. The incidence of MN in peripheral lymphocytes from workers exposed to Pb and Cd was over twice as high as in the controls (P<0.01). Using a combination of conventional scoring of MN and FISH with pan-centromeric probes, we assessed that this increase may have been due to clastogenic as well as aneugenic effects. In Cd- and Pb-exposed workers, the frequency of SCEs as well as the incidence of leukocytes with DNA fragmentation in lymphocytes were slightly, but significantly increased ( P<0.05) as compared with controls. After a 3h incubation of the cells to allow for DNA repair, a clear decrease was found in the level of DNA damage in the controls as well as in the exposed workers. No significant influence of smoking on genotoxic damage could be detected in metal-exposed cohorts. Our findings indicate that lead and cadmium induce clastogenic as well as aneugenic effects in peripheral lymphocytes, indicating a potential health risk for working populations with significant exposures to these heavy metals.     

Quercetin ameliorates gamma radiation-induced DNA damage and biochemical changes in human peripheral blood lymphocytes

We investigated the radioprotective efficacy of quercetin (QN), a naturally occurring flavonoid against gamma radiation-induced damage in human peripheral blood lymphocytes and plasmid DNA. In plasmid study, QN at different concentrations (3, 6, 12, 24 and 48 microM) were pre-incubated with plasmid DNA for 1h followed by exposure of 6 Gy radiation. Among all concentrations of QN used, 24 microM showed optimum radioprotective potential. To establish the most effective protective concentration of QN in lymphocytes, the cells were pre-incubated with 3, 6, 12, 24 and 48 microM of QN for 30 min and then exposed to 4 Gy gamma-radiation. The concentration-dependent effects of QN were evaluated by scoring micronuclei (MN) frequencies. The results showed that QN decreased the MN frequencies dose dependently, but the effect was more pronounced at 24 microM. Thus, 24 microM of QN was selected as the optimum concentration and was further used to evaluate its radioprotective effect in lymphocytes. For that a separate experiment was carried out, in which lymphocytes were incubated with QN (24 microM) for 30 min and exposed to different doses of radiation (1, 2, 3 and 4 Gy). Genetic damage (MN, dicentric aberration and comet attributes) and biochemical changes were measured to evaluate the effect of QN on gamma-radiations (1-4 Gy). Radiation exposed showed significant increases in the genetic damage and thiobarbituric acid reactive substances (TBARS) accompanied by a significant decrease in the antioxidant status. QN pretreatment significantly decreased the genetic damage and TBARS and improved antioxidant status through its antioxidant potential. Altogether, our findings encourage further mechanistic and in vivo studies to investigate radioprotective efficacy of QN.     

Caffeic acid protects human peripheral blood lymphocytes against gamma radiation-induced cellular damage

In the present study, we investigated in vitro radioprotective potential of caffeic acid (CA), a naturally occurring catecholic acid against gamma radiation-induced cellular changes. Different concentrations of CA (5.5, 11, 22, 44, 66, and 88 microM) were incubated with lymphocytes for 30 min prior to gamma-irradiation, and micronuclei (MN) scoring and comet assay were performed to fix the effective concentration of CA against gamma-irradiation. Among all concentrations, 66 microM of CA showed the optimum protection by effectively decreasing the MN frequencies and comet attributes. From the above-mentioned results, 66 microM of CA was selected as the effective concentration and was further used to investigate its radioprotective efficacy. For that purpose, a separate experiment was carried out on the lymphocytes in which lymphocytes were preincubated with CA (66 microM) and were exposed to different doses of radiation (1, 2, 3, and 4 Gy). Genetic damage (MN, dicentric aberration, and comet attributes) and biochemical changes were measured. Gamma-irradiated lymphocytes showed a dose-dependent increase in the genetic damage and thiobarbituric acid reactive substances, accompanied by the significant decrease in the antioxidant status, whereas CA pretreatment positively modulated all the radiation-induced changes through its antioxidant potential. The current study demonstrates that CA is effective in protecting lymphocytes against radiation-induced toxicity and encourages further in vivo study to evaluate radioprotective efficacy of CA.     

Comparative analysis of cytotoxic, genotoxic and antioxidant effects of 2,2,4,7-tetramethyl-1,2,3,4-tetrahydroquinoline and ethoxyquin on human lymphocytes

2,2,4,7-Tetramethyl-1,2,3,4-tetrahydroquinoline (THQ) is a new synthetic compound with potential antioxidant activity. In this study, cytotoxic, genotoxic and antioxidant activities of THQ were studied on human lymphocytes with the use of the trypan blue exclusion assay, the TUNEL method, the comet assay and the micronucleus test. The activities of THQ were compared with those of a structurally similar compound-ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline, EQ), which is used in animal feeds as a preservative. Cytotoxic effects of THQ were observed after 1-h treatment at the concentration of 500 microM and after 24-h treatments at the concentrations of 250-500 microM. Although the micronucleus test did not reveal a genotoxic effect of THQ, in the comet assay the statistically significant increase in DNA damage was observed as compared with the control. On the other hand, the protection of human lymphocytes against DNA damage induced by hydrogen peroxide suggests an antioxidant activity of THQ. The comparative analysis of THQ and EQ activities performed in these studies revealed that THQ was less cytotoxic and less genotoxic than EQ. Slightly lower antioxidant activity of THQ was also shown in the comet assay when it was used at the lower studied doses (1-5 microM), but for the highest one (10 microM) its efficiency was similar to that of EQ. In the micronucleus assay THQ was more effective than EQ in protecting the cultured lymphocytes from clastogenicity of H2O2. We believe that THQ is worthy of further detailed studies on its antioxidant properties to confirm its usefulness as a preservative.     

Effect of epoxide hydrolase and glutathione S-tranferase genotypes on the induction of micronuclei and DNA damage by styrene-7,8-oxide in vitro

Styrene is one of the most important organic chemicals used worldwide. Its main metabolite, styrene-7,8-oxide (SO), is considered responsible for the genotoxic effects associated with exposure to styrene. SO is detoxified by hydrolysis catalyzed by epoxide hydrolase (EH), or, to a minor extent, by conjugation mediated by glutathione S-transferases (GSTs). The purpose of the present study was to investigate whether EH (exons 3 and 4), GSTP1 (exons 5 and 6), GSTM1 and GSTT1 polymorphisms have any influence on the genotoxicity of SO in human leukocytes. Peripheral leukocytes from 30 healthy donors were exposed to SO (50 and 200 micro M) and genotoxicity was evaluated by means of the micronucleus (MN) test and alkaline comet assay, using 1% DMSO as solvent control. When EH genotypes were classified in low, medium, and high with respect to the expected EH activity, an increase in induced comet tail length was observed with decreasing EH activity in SO-exposed cells. An increase was seen in induced MN frequency in EH low-activity donors. These findings are consistent with the detoxifying activity of this enzyme. In addition, increases in MN frequencies for GSTP1 *A/*B and *A/*C genotypes with regard to the wild-type homozygous *A/*A genotype were detected. This may be due to a low detoxifying activity as a consequence of altered SO affinity of the variant protein, but must be confirmed using homozygote variant individuals, not included in this study. No clear results were obtained for GSTM1 or GSTT1 genotypes, even when performing the analysis after grouping individuals with the same expected EH activity, probably due to the minor role that glutathione conjugation plays in styrene metabolism. The present in vitro findings using human leukocytes suggest that polymorphisms in EH, and, to a lesser extent, in GSTP1, may influence induction of cytogenetic and DNA damage by SO.     

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.     

Sudan I induces genotoxic effects and oxidative DNA damage in HepG2 cells

Sudan I, a synthetic lipid soluble azo pigment, is widely used in various industrial fields. However, Sudan I has not been approved at any level of food production, since there are many inconclusive reports relating to its genotoxicity and carcinogenicity in humans. The aim of this study was to assess the genotoxic effects of Sudan I and to identify and clarify the reaction mechanisms by use of human hepatoma HepG2 cells. To study the genotoxic effects of Sudan I, the comet assay and micronucleus test (MNT) were used. In the comet assay and MNT, we found increase of DNA migration and of the micronuclei frequencies at all tested concentrations (25-100 microM) of Sudan I in a dose-dependent manner. The data suggest that Sudan I caused DNA strand breaks and chromosome breaks. To elucidate the underlying mechanism of this difference, we monitored the level of reactive oxygen species (ROS) production with the 2,7-dichlorofluorescein diacetate assay. The level of the oxidative DNA damage and lipid peroxidation was evaluated using immunoperoxidase staining for 8-hydroxydeoxyguanosine (8-OHdG) and by measuring levels of thiobarbituric acid-reactive substances (TBARS). Significantly increased levels of ROS, 8-OHdG and TBARS were observed in HepG2 cells at higher concentrations, the doses being 100, 50-100 and 50-100 microM, respectively. We conclude that Sudan I causes genotoxic effects, probably via ROS-induced oxidative DNA damage at the higher doses.     

Evaluation of the cytogenetic damage induced by the organophosphorous insecticide acephate

The organophosphorous insecticide acephate was tested for its ability to induce in vitro cytogenetic effect in human peripheral lymphocytes by using the chromosomal aberrations (CAs), sister chromatid exchange (SCE) and micronuclei (MN) assay. The level of nuclear DNA damage of acephate was evaluated by using the comet assay. Concentrations of 12.5, 25, 50, 100 and 200 μg mL⁻¹ of acephate were used. All concentrations of acephate induced significant increase in the frequency of CAs and in the formation of MN dose dependently (r = 0.92 at 24 h, r = 0.95 at 48 h for CAs, r = 0.87 for MN). A significant increase was observed in induction of SCE at 50, 100 and 200 μg mL⁻¹ concentrations during 24 h treatment and at all concentrations (except 12.5 μg mL⁻¹) during 48 h treatment period in a dose-dependent manner (r = 0.84 at 24 h, r = 0.88 at 48 h). Acephate did not affect the replicative index and cytokinesis-block proliferation index (CBPI). However, it significantly decreased the mitotic index at all three highest concentrations (50, 100, 200 μg mL⁻¹) for 24 h treatment and at all concentrations (except 12.5 μg mL⁻¹) for 48 h treatment, dose-dependently (r = 0.94 at 24 h, r = 0.92 at 48 h). A significant increase in mean comet tail length was observed at 100 and 200 μg mL⁻¹ concentrations compared with negative control in a concentration-dependent manner (r = 0.94). The mean comet tail intensity was significantly increased at only 200 μg mL⁻¹ concentration. The present results indicate that acephate is a clastogenic, cytotoxic agent and it causes DNA damage at high concentrations in human lymphocytes in culture.     

Genotoxicity of cadmium chloride in human lymphocytes evaluated by the comet assay and cytogenetic tests

Peripheral blood lymphocytes were tested in vitro for genotoxic effects of cadmium chloride. Whole blood samples of four healthy, non-smoking subjects were preincubated with CdCl2 in concentrations of 10(-4), 10(-3), and 5 . 10(-3) mol/L for three hours before the cells were assessed for DNA-damage using the single cell alkaline gel electrophoresis assay (comet assay) or cultivated for chromosomal aberrations (CA), sister chromatid exchanges (SCE), and the micronucleus (MN) test. The comet assay showed notable interindividual differences. The results of the cytogenetic tests showed an increase in the frequency of CA, MN, and SCE with CdCl2 in the treated cultures, yet none was able to show a correlation between concentrations of cadmium chloride and the frequency of damages. The MN slides were stained with Giemsa and with DNA fluorochrome 4', 6'-diamidino-2-phenylindole (DAPI). The frequency of MN in slides stained with DAPI was significantly higher than in those stained with Giemsa, which might be due to an underestimation of small micronuclei in Giemsa-stained slides.     

Genotoxicity studies on the antimicrobial drug sulfamethoxazole in cultured human lymphocytes

The genotoxicity of the antimicrobial drug sulfamethoxazole was evaluated in cultured human peripheral blood lymphocytes. The frequencies of sister-chromatid exchange (SCE) and micronuclei (MN) were scored as genetic endpoints. Both tests cover a wide range of induced genetic damage such as primary DNA damage, clastogenicity and aneugenicity. Cultures were set up with blood samples from two healthy donors and the treatment was done with different sulfamethoxazole concentrations ranging from 10 to 500 microg/ml. From the results obtained it appears that this drug is able to induce weak genotoxic effects, as revealed by the slight increase in the SCE and MN frequencies, at least at one of the two highest concentrations tested. However, the results of the SCE assay should be interpreted with caution because the increase is just significant. In addition, cyotoxic/cytostatic effects of sulfamethoxazole were revealed by a decrease in the proliferative rate index (PRI) and in the cytokinesis block proliferation index (CBPI).     

Chromosomal changes induced in mouse bone marrow cells following six weeks inhalation of cyclohexanol

The effects of low doses of cyclohexanol exposure were studied in mouse bone marrow cells including chromosome aberrations (CA), micronucleus (MN) and sister chromatid exchanges (SCE) as biomarkers. Capillaries with a tested agent that was evaporated continuously were placed in an experimental chamber for six weeks. No clastogenic and/or aneugenic effect of CA and MN induction was observed. A significant elevation of induced damage was achieved in the SCE study (p < 0.001) that has confirmed the early exposure of cyclohexanol to mice.     

Sister-chromatid exchanges in human lymphocytes exposed to 1-p-(3-methyltriazeno)benzoic acid potassium salt

The 1-p-(3-methyltriazeno) benzoic acid potassium salt (MTBA) is a triazeno analogue of dacarbazine, an antineoplastic agent capable of mediating the appearance of new antigenic specificities on cancer cells in mice, a phenomenon described as 'chemical xenogenization' (CX). Recently we reported the clastogenic potential of MTBA on human lymphocytes. Since sister-chromatid exchange (SCE) assay is more sensitive than clastogenic tests, at least at low drug concentrations, we assessed SCE frequencies induced by MTBA on human lymphocytes stimulated by PHA. Drug treatment at 2-500 micrograms/ml was performed in vitro prior to or after PHA addition. SCE values increased significantly in a dose-dependent manner up to 200 micrograms/ml. However, SCE frequencies, as well as chromosome breaks, did not increase dramatically. These data indicate that MTBA concentrations used for CX do not cause severe cytogenetic damage to immune cells at least in vitro.     

Antioxidant activity of diphenyl diselenide prevents the genotoxicity of several mutagens in Chinese hamster V79 cells

Diphenyl diselenide (DPDS) is an electrophilic reagent used in the synthesis of a variety of pharmacologically active organic selenium compounds. Studies have shown its antioxidant, hepatoprotective, neuroprotective, anti-inflammatory, and antinociceptive effects. We recently showed the antioxidant effect of DPDS in V79 cells, and established the beneficial and toxic doses of this compound in this cell line. Here, we report the antigenotoxic and antimutagenic properties of DPDS, investigated by using a permanent lung fibroblast cell line derived from Chinese hamsters. We determined the cytotoxicity by clonal survival assay, and evaluated DNA damage in response to several mutagens by comet assay and micronucleus test in binucleated cells. In the clonal survival assay, at concentrations ranging from 1.62 to 12.5microM, DPDS was not cytotoxic, while at concentrations up to 25microM, it significantly decreased survival. The treatment with this organoselenium compound at non-cytotoxic dose range increased cell survival after challenge with hydrogen peroxide, methyl-methanesulphonate, and UVC radiation, but did not protect against 8-methoxypsoralen plus UVA-induced cytotoxicity. In addition, the treatment prevented induced DNA damage, as verified in the comet assay. The mutagenic effect of these genotoxins, as measured by the micronucleus test, similarly attenuated or prevented cytotoxicity and DNA damage. Treatment with DPDS also decreased lipid peroxidation levels after exposure to hydrogen peroxide MMS, and UVC radiation, and increased glutathione peroxidase activity in the extracts. Our results clearly demonstrate that DPDS at low concentrations presents antimutagenic properties, which are most probably due to its antioxidant properties.     

Genotoxic effect and nitrative DNA damage in HepG2 cells exposed to aristolochic acid

Aristolochic acid (AA), extensively used as a traditional herbal medicine, was withdrawn from the market in the last century because it was found to be a potent carcinogen in humans and animals. The aim of this study was to evaluate the genotoxic effect of AA and obtain further insight into whether the nitrative DNA damage can be induced by reactive nitrogen species (RNS), including nitric oxide (NO) and its derivative peroxynitrite (ONOO(-)) using human hepatoma HepG2 cells. To identify the genotoxic effect, the comet assay and micronucleus test (MNT) were performed. In the comet assay, 25-200microM of AA caused a significant increase of DNA migration in a dose-dependent manner. A significant increase of the frequency of micronuclei was found in the range between 12.5 and 50microM in the MNT. The results showed that AA caused DNA and chromosome damages. To elucidate the nitrative DNA damage mechanism, the level of nitrite and 8-hydroxydeoxyguanosine (8-OHdG), which can be generated by ONOO(-), were monitored with the 2,3-diaminonaphthalene (DAN) assay and immunoperoxidase staining, respectively. The results showed that AA causes a significant increase in the levels of NO and formation of 8-OHdG at concentrations >/=50microM. This observation supports the assumption that AA could exert genotoxicity probably via NO and its derivatives at higher concentrations in HepG2 cells.     

Induction of micronuclei and sister chromatid exchange in bone-marrow cells and abnormalities in sperm of Algerian mice (Mus spretus) exposed to cadmium, lead and zinc

As a consequence of human activities, large amounts of cadmium, lead and zinc are released in the environment, often simultaneously. The aim of this study was to investigate under experimental conditions the DNA damage induced in Algerian mice (Mus spretus) exposed to cadmium (Cd), lead (Pb) and zinc (Zn) separately, or in selected combinations. Three cytogenetic end points were considered: the frequencies of micronucleated cells (MN) and sister chromatid exchange (SCE) in the bone marrow and the frequency of sperm abnormalities. Mice were treated by intraperitoneal (i.p.) injections with 5 or 10 doses of aqueous solutions of cadmium acetate, lead acetate and zinc acetate in concentrations corresponding to 1/10 of the LD50, respectively, 21.5, 0.46 and 1.5 mg/kg bw. The control groups were injected in the same way with distilled water.With only one exception (Cd + Zn group treated with 5 doses), the results show a significant increase of MN in all groups for both treatments (5 and 10 doses). Similarly, the results concerning the SCE revealed a statistically significant increase in all treated animals, with the exception of the Zn group treated with 5 doses. The number of sperm abnormalities was significantly higher in animals treated with 5 doses, except in the group Pb + Zn. In animals treated with 10 doses the number of sperm abnormalities was always statistically higher compared with controls.This study indicates that cadmium, lead and zinc can induce MN, SCEs and sperm abnormalities in Algerian mice and that the clastogenic potential is dependent on the time of exposure and the interaction between the three elements, confirming the environmental damage that may result from the simultaneous action of several metals. Most relevant is the toxic potential for Zn, related with the dose, which may compromise its protective effect against other metal contaminations, such as cadmium.     

Oxidatively modified proteins and DNA in digestive gland cells of the fresh-water mussel Unio tumidus in the presence of tannic acid and its derivatives

The oxidative effect of tannic acid and its two derivatives (ellagic and gallic acid), naturally occurring plant polyphenols, has been studied on digestive gland cells of the fresh-water mussel Unio tumidus. A spectrophotometric method was used to determine the protein thiol groups after incubation of the cells with the polyphenols at concentrations of 1, 15 and 60 microM. The results showed that the oxidative modification of proteins increased in a concentration-dependent manner but no changes were observed at the concentration of 1 microM. The comet assay (single-cell gel electrophoresis assay) with the formamido-pyrimidine glycosylase (FPG) protein was used to assess oxidative DNA base damage. The cells were treated with polyphenols at the concentrations of 30 and 60 microM and post-incubated with FPG. FPG strongly enhanced DNA damage induced by the polyphenols, indicating that N-7 guanine oxidation is responsible for the observed effect. Using the comet assay in combination with proteinase K we were able to demonstrate the presence of DNA-protein cross-links as the probable cause of the decrease in DNA migration. After treatment of the cells with tannic acid and its metabolites at concentrations of 120, 180 and 240 microM, they were post-incubated with proteinase K. After this treatment an increased DNA migration was observed, indicating the presence of DNA-protein cross-links. We have also used a fluorescence method with Hoechst 33258/propidium iodide DNA-binding dyes to study the extent of DNA fragmentation after exposure of the cells to polyphenols at concentrations of 1, 5 and 60 microM. The results demonstrate that the polyphenols can induce apoptosis and necrosis at higher concentrations (5 and 60 microM). All experimental data suggest that tannic, ellagic and gallic acids at concentrations above 1 microM are able to interact with proteins and DNA, which leads to their degradation or changes in their function.     

Study of the biological effects and DNA damage exerted by a new dipalladium-Hmtpo complex on human cancer cells

The new dipalladium complex [Pd(2)(mu-mtpo-N(3),N(4))(2)(phen)(2)](NO(3))(2) (where phen=1,10-phenantroline; Hmtpo=5,7-dihydro-7-oxo-5-methyl[1,2,4]triazolopyrimidine), (Pd(2)-Hmtpo, or complex I), interacts effectively with DNA plasmid (pBS), as studied by circular dichroism spectroscopy (CD), causing large helix distortions, altering the direction of the main DNA helix axis and producing unwinding of the DNA double helix. DNA damage induced by complex I was highly significant at 2.81 microM (ovarian carcinoma TG cell line), as assessed by comet assay, a dose at which all treated nuclei showed more than 30% DNA migration to the comet tail. DNA damage effect is a consequence of genotoxicity and not a false positive response caused by cytotoxicity. In vitro cytotoxic assay on the two human tumor cell lines TG and BT-20 (breast carcinoma), shows that doses of 0.47, 1.41 and 2.81 microM produce significant antiproliferative effects after 4 days of treatment compared with control. Complex I was highly cytotoxic at 2.81 microM causing an inhibition of viable cells of 65.5%. Cisplatin (cis-DDP) exhibits lower cytotoxic activity in TG cells than dipalladium complex (a cisplatin dose of 6.67 microM inhibits 30.3%) and does not cause migration of DNA to comet tail.