Comparison of the effects of arsenic and cadmium on benzo(a)pyrene-induced micronuclei in mouse bone-marrow.

This study was undertaken to investigate the genotoxic interactions between the common environmental pollutants: arsenic (As), cadmium (Cd) and benzo(a)pyrene (BaP), which are known to be human carcinogens. C57BL/6J/Han mice were pre-treated with 100mg cadmium chloride (Cd(2+))/L or 50mg sodium arsenite (As(3+))/L in drinking water for 7 days and then given a single dose of 200mg BaP/kg bw by intra-peritoneal injection. A third group of mice did not receive the pre-treatment and was given BaP alone. Mice were sacrificed before or at 12, 24, 48 or 72h after BaP administration. Chromosome damage in bone-marrow cells was assessed by use of the micronucleus test. The study revealed that BaP induced a statistically significant increase in micronucleus (MN) frequency at 48h after administration. In animals exposed to Cd in drinking water no enhancement of genotoxicity was observed compared with the control group that was given tap water only. In Cd/BaP co-exposed animals, the MN frequency at respective time points did not differ from that for the animals exposed solely to BaP. A statistically higher MN frequency was found in bone marrow of animals exposed to As compared with controls that received tap water (0.92+/-0.29% versus 0.38+/-0.13%, respectively). This effect was even more pronounced after combined exposure to As and BaP. In the co-exposed animals, significantly elevated levels of MN were detected in samples examined at 12, 24 and 48h after BaP administration, compared with animals receiving BaP alone (1.14+/-0.31%, 1.26+/-0.3% and 2.02+/-0.45% versus 0.44+/-0.13%, 0.44+/-0.11% and 1.04+/-0.44%, respectively). These findings imply strong interactions between As and BaP, but not between Cd and BaP, in inducing DNA damage in polychromatic erythrocytes in mouse bone-marrow.

Micronucleus frequency in peripheral blood lymphocytes and buccal mucosa cells of copper smelter workers, with special regard to arsenic exposure.

Occupational exposure in copper smelters may produce various adverse health effects including cancer which, according to available epidemiologic data, is associated mainly with exposure to arsenic. Despite a number of well-documented studies reporting an increased risk of cancer among copper smelters workers, the data on genotoxic effects in this industry are scarce. In view of the above, an assessment of micronuclei (MN) frequency in peripheral blood lymphocytes and buccal epithelial cells from copper smelter workers was undertaken. Additionally, the clastogenic/aneugenic effect in lymphocytes was assessed with the fluorescence in situ hybridization (FISH). The study was conducted in three copper smelters in southwestern Poland. The subjects (n = 72) were enrolled among male workers at departments where As concentration in the air was up to at 80 microg/m(3). Exposure was assessed by measurement of arsenic concentration in urine and toenail samples. The control group (n = 83) was recruited from healthy male individuals living in central Poland who did not report any exposure to known genotoxins. The results of our study showed a significant increase in MN frequency in peripheral blood lymphocytes and in buccal epithelial cells of smelter workers, compared to the controls (7.96 +/- 4.28 vs. 3.47 +/- 1.70 and 0.98 +/- 0.76 vs. 0.50 +/- 0.52, respectively). The FISH technique revealed the presence of clastogenic and aneugenic effects in peripheral blood lymphocytes in both groups. The clastogenic effect was slightly more pronounced in the smelter workers; however, the difference was not statistically significant. The mean arsenic concentrations in urine (total arsenic species) and in toenail samples in the exposed group were 54.04 +/- 42.26 microg/l and 7.63 +/- 7.24 microg/g, respectively, being significantly different from control group 11.01 +/- 10.84 microg/l and 0.51 +/- 0.05 microg/g. No correlation between As content in urine or toenail samples and the genotoxic effect was found under study.

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.

Protective effect of melatonin against in vitro iron ions and 7 mT 50 Hz magnetic field-induced DNA damage in rat lymphocytes.

We have previously shown that simultaneous exposure of rat lymphocytes to iron ions and 50Hz magnetic field (MF) caused an increase in the number of cells with DNA strand breaks. Although the mechanism of MF-induced DNA damage is not known, we suppose that it involves free radicals. In the present study, to confirm our hypothesis, we have examined the effect of melatonin, an established free radicals scavenger, on DNA damage in rat peripheral blood lymphocytes exposed in vitro to iron ions and 50Hz MF. The alkaline comet assay was chosen for the assessment of DNA damage. During pre-incubation, part of the cell samples were supplemented with melatonin (0.5 or 1.0mM). The experiments were performed on the cell samples incubated for 3h in Helmholtz coils at 7mT 50Hz MF. During MF exposure, some samples were treated with ferrous chloride (FeCl2, 10microg/ml), while the rest served as controls. A significant increase in the number of cells with DNA damage was found only after simultaneous exposure of lymphocytes to FeCl2 and 7mT 50Hz MF, compared to the control samples or those incubated with FeCl2 alone. However, when the cells were treated with melatonin and then exposed to iron ions and 50Hz MF, the number of damaged cells was significantly reduced, and the effect depended on the concentration of melatonin. The reduction reached about 50% at 0.5mM and about 100% at 1.0mM. Our results indicate that melatonin provides protection against DNA damage in rat lymphocytes exposed in vitro to iron ions and 50Hz MF (7mT). Therefore, it can be suggested that free radicals may be involved in 50Hz magnetic field and iron ions-induced DNA damage in rat blood lymphocytes. The future experimental studies, in vitro and in vivo, should provide an answer to the question concerning the role of melatonin in the free radical processes in the power frequency magnetic field.

DNA damage in rat lymphocytes treated in vitro with iron cations and exposed to 7 mT magnetic fields (static or 50 Hz).

The present study was undertaken to verify a hypothesis that exposure of the cells to static or 50 Hz magnetic fields (MF) and simultaneous treatment with a known oxidant, ferrous chloride, may affect the oxidative deterioration of DNA molecules. The comet assay was chosen for the assessment of DNA damage. The experiments were performed on isolated rat lymphocytes incubated for 3h in Helmholtz coils at 7 mT static or 50 Hz MF. During MF exposure, part of the cell samples were incubated with 0.01 microM H(2)O(2) and another one with 10 microg/ml FeCl(2,) the rest serving as controls. Lymphocyte exposure to MF at 7 mT did not increase the number of cells with DNA damage in the comet assay. Incubation of lymphocytes with 10 microg/ml FeCl(2) did not produce a detectable damage of DNA either. However, when the FeCl(2)-incubated lymphocytes were simultaneously exposed to 7 mT MF, the number of damaged cells was significantly increased and reached about 20% for static MF and 15% for power frequency MF. In the control samples about 97% of the cells did not have any DNA damage. It is not possible at present to offer a reasonable explanation for the findings of this investigation - the high increase in the number of lymphocytes showing symptoms of DNA damage in the comet assay, following simultaneous exposure to the combination of two non-cytotoxic factors -10 microg/ml FeCl(2) and 7 mT MF. In view of the obtained results we can only hypothesise that under the influence of simultaneous exposure to FeCl(2) and static or 50 Hz MF, the number of reactive oxygen species generated by iron cations may increase substantially. Further studies will be necessary to confirm this hypothesis and define the biological significance of the observed effect.

DNA damage detected by the comet assay in the white blood cells of workers in a wooden furniture plant.

The study was aimed at the assessment of genotoxic effects in workers of a wooden furniture manufacture, based on the level of DNA damage in white blood cells (WBC). The alkaline single cell gel electrophoresis assay (known as the comet assay) in individual cells was adapted for detecting damaged DNA in WBC. The level of DNA damage was determined as the percentage of cells with comets. It was assessed in cells before and after incubation in RPMI 1640 medium and CO(2) at 37 degrees C for 1 h to repair DNA breaks. Thirty-five woodworkers and 41 control subjects were studied. In the woodworkers, significantly more cells with DNA damage (21.5%) were observed than in the control persons (9.7%). A slight but significant decrease in the level of DNA damage was found in the WBC of woodworkers after incubation (17.2%). Significantly higher levels of damaged DNA was observed in woodworkers who either smoked (22.1%) or did not smoke cigarettes (20.8%) than in smokers (13.2%) and non-smokers (7.0%) from the control group. After incubation, a slight decrease in the level of DNA damage was found in both smoking and non-smoking woodworkers compared to the respective subjects in the control group. The increased levels of DNA damage observed in the woodworkers could be associated with the occupational exposure to wood dust in the furniture manufacture.

The assessment of DNA damage in lymphocytes of wooden furniture workers.

Single-strand breaks (SSB) and DNA repair were detected in peripheral lymphocytes derived from workers of a furniture factory in a non-polluted region of Poland. The workers were exposed to wood dust (n = 19), or to the dust and varnishes or lacquers together (n = 5). Four groups were studied simultaneously: (a) exposed workers smokers of cigarettes (n = 14), (b) nonexposed smokers--control (n = 14), (c) exposed workers' nonsmokers (n = 14), (d) exposed nonsmokers (n = 10). In exposed workers DNA SSB and DNA repair were statistically significantly increased. DNA SSB was clearly higher in the smoking workers than in the smoking controls. Cigarette smoking itself has produced no evident increase in the frequency of DNA SSB in the control group. Occupational exposure had a significant effect on DNA repair in non stimulated lymphocytes both in smoking and nonsmoking workers.

DNA single-strand breaks and DNA repair in the lymphocytes of wooden furniture workers.

DNA single-strand breaks (DNA SSB) in peripheral lymphocytes of wooden furniture workers and a control group, including smokers and nonsmokers, were detected by the microfiltration method. Our results show that cigarette smoking significantly increases the fragmentation of DNA single strands in the wooden furniture workers (by nearly two times) but not in the control group. Moreover, occupational exposure to wood dust and other wooden plant substances significantly induced DNA SSB only in the lymphocytes of smokers (by about two times). DNA repair in the lymphocytes was investigated as 3H incorporation into DNA. High 3H incorporation in the unstimulated lymphocytes of both smoking and nonsmoking workers, as compared to the references, suggests that besides DNA SSB other DNA damage can be caused by occupational exposure in the wooden plant. Since DNA repair is not always perfect, the possibility is high that the low level of DNA repair in the study group may lead to irreversible DNA damage. We think that the workers exposed to wood dust and the substances emitted by furniture coating materials in the plant may be at higher risk for mutagenesis and/or carcinogenesis than the unexposed population.

Genotoxicity evaluation of trimethylbenzenes.

The three trimethyl isomers of benzene (hemimellitene, 1,2,3-TMB; pseudocumene, 1,2,4-TMB and mesitylene, 1,3,5-TMB) were investigated for different genotoxicity endpoints: in vitro, in the Ames test with Salmonella typhimurium TA97a, TA98, TA100 and TA102 strains in the presence and absence of rat liver S9 metabolic activation; in vivo, in the micronucleus and sister chromatid exchange (SCE) tests with bone marrow cells of Imp:Balb/c mice. Only the isomer of benzene with the methyl-group at position 1, 2, 3 was found to have mutagenic effect on S. typhimurium cells. Increase in bacterial reversions was observed in four conventional strains used in this study, but most clearly in TA97a. The mutagenic responses of 1,2,3-TMB with the SalmonellaL tester strains were observed in the experiments performed in the absence of enzymatic activation. None of the compounds had an influence on the frequency of micronucleated polychromatic erythrocytes in bone marrow cells of mice. However, all the three compounds were observed to have a cytogenetic potential of increasing the SCE level in these cells. Significant responses in SCE induction, compared with the level of those changes in corresponding solvent-administered controls, were obtained at three test doses of 1,2,3-TMB (730, 1470, 2200 mg/kg) and 1,2,4-TMB (900, 1800, 2700 mg/kg) and at two doses of 1,3,5-TMB (1800, 2700 mg/kg). These data provided a limited evidence for the genotoxic activity of 1,2,3-TMB and inadequate evidence for genotoxic activity of 1,2,4-TMB and 1,3,5-TMB.

Genotoxicity of industrial dyes under the inductive effect of ethanol on monooxygenase system in mice.

The genotoxic effects of triarylmethane (Acid Green 16, C.I.44025) and arylmonoazo (Basic Orange 28, developed by Boruta Pigment Plant, Poland, C.I. undisclosed) dyes, were evaluated in Balb/C mice. Animals were fed for 6 days nutritionally adequate Portagen liquid diet (1 kcal/ml) or isocaloric alcoholic diet containing 5% (w/v) ethanol (36% of total calories) in order to induce the cytochrome P-4502E1 monooxygenase. Dye compounds were administered intraperitoneally 30 h before the test at doses: 90 mg/kg of Acid Green 16 and 70 mg/kg of Basic Orange 28. Bone marrow micronucleus test was used for evaluation of genotoxicity of the dyes. Ethanol caused an increase of the level of cytochrome P-450 by 200% and activities of 7-ethoxycoumarin O-deethylase (ECOD) by 650%, 7-ethoxyresorufin O-deethylase (EROD) by 460% and glutathione (GSH)-S-transferase by 60% in the liver. Both dyes exerted genotoxic effect as inferred from a 3-fold increase of frequency of micronucleated polychromatic erythrocytes in bone marrow, and a further increase (2-fold) was caused by ethanol liquid diet combined with Acid Green 16 treatment. Basic Orange 28 genotoxicity remained unaffected by ethanol. It is concluded that: (1) enhancement of genotoxic effect of Acid Green 16 by ethanol is caused by induction of cytochrome P-4502E1 monooxygenases resulting in an increased bioactivation of the dye; (2) lack of enhancement of the genotoxic effect of Basic Orange 28 by ethanol probably results from the dye- and ethanol-mediated stimulation of GSH-S-transferase, bypassing the cytochrome P-4502E1 bioactivation step.

Mutagenic and genotoxic activity detected by the Ames, micronucleus and SCE tests under the influence of samples of dyes manufactured in Poland.

In this study, we evaluated the mutagenic and genotoxic potential of commercial samples of chemicals manufactured by Polish dyestuff industry for their ability to mutate Salmonella typhimurium strains (TA97a, TA98, TA100 and TA102), and to induce formation of micronuclei (MN) and sister chromatid exchanges (SCE) in mice bone marrow cells. This study involved five dyes selected from a list of colorants, considered to be representative of those which were most extensively used in the trade. According to criteria listed by IARC (1984), the results obtained in this work and earlier tests of Basic Blue 26, 44045 and Direct Red 83, 29225 provided no evidence of genetic activity (all 3 tests were negative); for Acid Violet 49, 42640 and Disperse Blue 37, the evidence of genetic activity was inadequate (1 test was positive) and the tests for Acid Black 194 provided limited evidence of genetic activity (2 positive tests).

Cyanuric chloride has no genotoxic and mutagenic properties in bacteria and bone marrow cells.

Three short-term tests were used; Salmonella/microsome assay, in vivo sister chromatid exchange assay (SCE) and micronucleus assay to evaluate mutagenic and genotoxic properties of 2,4,6-trichlorotriazine; cyanuric chloride. Mutagenicity assays were carried out using the standard top agar overplay plate assay described by Maron and Ames (9). Tester strains TA97a, TA98, TA100 and TA102 were used. Compound was dissolved in 0.1 ml of DMSO and doses of 1, 10, 100 and 500 micrograms/plate were tested in the absence and in the presence of the S9-mix. From the results obtained it appeared that incubation of the test substance with the bacteria did not increase the number of His+ revertants with any of the strains of S. typhimurium, either in the absence or in the presence of the S9-mix. At the high dose level used i.e. 100 and 500 micrograms/plate, the test substance appeared to be slightly toxic for strain TA 97a (in the absence of the S9-mix), as was seen from a diminished number of revertant colonies. The SCE test was performed according to the GENE-TOX programme. No significant increase was noted in the incidence of SCE in the groups treated with all tested doses of cyanuric chloride. Thus, in this test cyanuric chloride did not induce chromosomal damage resulting in SCE formation in bone marrow cells of mice. The micronucleus assay in vivo was performed on mice bone marrow cells. The incidence of micronucleated polychromatic erythrocytes after administration of all doses of cyanuric chloride used were not statistically different (p > 0.05) as compared to negative controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Potential carcinogenicity assessment of alpha-aescin and phenbendasol.

Potential carcinogenic activity of alpha-aescin and phenbendasole made by "Polfa" (Poland) as well as phenbendasole produced by "Hoechst" (Germany) was studied using Salmonella/microsome test, DNA repair test and micronucleus assay. None of tested preparations were mutagenic or genotoxic what suggest that none of them possess potential carcinogenic activity. Besides, it was established that alpha-aescin exhibits strong and phenbendasol weak acute systemic toxicity for mice. Alpha-aescin and phenbendasole produced in Poland have been found to be toxic for bone marrow cells of mice but only when administered at a high dose of 80% LD50.

Genotoxicity assessment of Rokanol B2 and Rokamid R1.

Two surface-active compounds, Rokanol B2 and Rokamid R1, were examined with three types of screening tests: 1. standard Ames test in vitro using S. typhimurium TA97a, TA98, TA100 and TA102 strains; 2. micronucleus test in vivo; 3. sister chromatid exchange (SCE) on bone marrow cells of Balb C mice. Negative results of the testing, in terms of the effects of activity of both the technical preparations on S. typhimurium cells and bone marrow of Balb C mice, were found for both tested chemicals.

Genotoxicity assessment of sulfosuccinate IO-5, Rokamid MRZ 17, Rokacet RZG7P2 and Roksol TL-7 using bacteria and bone marrow rodent cells.

Three short-term tests were used for evaluation of the genotoxic activity of four surface active agents. These were: Ames, Salmonella reversion assay using 4 tester strains (TA97a, TA98, TA100 and TA102), the micronucleus test int the bone marrow of Balb C mice and in vivo sister chromatid exchange (SCE) analysis in SFIS or Balb C mice. The frequency of micronucleated PCEs did not exceed the control values in mice of both sexes after intraperitoneal administration of all four compounds. Three preparations--Sulfosuccinate IO-5, Rokamid MRZ 17 and Rokacet RZG7P2 produced a negative response in Salmonella strains gene mutation assay and SCE induction test in mouse bone marrow cells. The Roksol TL-7 induced frameshift and base-pair substitution mutations in Salmonella tester strains both with and without S9 (prepared from the liver of rats which had been pretreated with Aroclor 1254). Evidently positive results (more than a twofold increase in the number of revertants per plate) were observed in tester strain S. typhimurium TA97a (with and without S9 metabolic activation) and S. typhimurium TA100 (with S9 metabolic activation) at a dose of 0.2 microliter Roksol TL-7 per plate. Roksol TL-7 caused slight increase in the SCE level in mouse bone marrow cells. A significant increase in SCE frequency was observed at doses of 50, 75 and 100 mg/kg.

Genotoxic effects of dioxolane and trioxane in mice evaluated by the micronucleus test.

The possible genotoxic effects of dioxolane and trioxane were studied using the micronucleus test. Mice were given dioxolane and trioxane i.p. in two doses at 24-h intervals. Dioxolane at doses from 1500 mg/kg to 6000 mg/kg caused a significant increase of micronucleated polychromatic erythrocytes in bone marrow of mice as compared to the negative control value. Positive results in the micronucleus test provide evidence of genotoxic activity of dioxolane. I.p. administration of trioxane produced no chromosomal damage resulting in erythrocyte micronucleus formation, even at highly toxic doses.

Genotoxic effects of ethyl acrylate and methyl acrylate in the mouse evaluated by the micronucleus test.

The micronucleus test was used to study the mutagenic effects of ethyl acrylate and methyl acrylate. Investigated compounds were administered to Balb C male mice by i.p. injection in two doses, separated by a period of 24 h. Ethyl acrylate at doses of 225-1800 mg/kg, and methyl acrylate at doses of 37.5-300 mg/kg significantly induce chromosome damage resulting in micronuclei formation in bone marrow polychromatic erythrocytes. Both investigated compounds significantly decrease the ratio of polychromatic to normochromatic erythrocytes. Positive results provide evidence of clastogenic activity of ethyl acrylate and methyl acrylate.