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.

Genotoxicity evaluation of tetramethylbenzenes.

The three tetramethyl isomers of benzene (prenitene, 1,2,3,4-; izodurene, 1,2,3,5-; and durene, 1,2,4,5-tetramethylben- zene) were studied using in vitro mutagenicity and in vivo genotoxicity tests. Potency of mutate induction by these solvents was evaluated in Salmonella typhimurium cells with, and without S9-mix made from Aroclor 1254-induced rat liver S9. The potency of induction of micronuclei (MN) and sister chromatid exchanges (SCEs) by solvents was evaluated in bone marrow of mice. Izodurene displayed mutagenic potency in strains TA97a, TA98 and TA100 only in the absence of the S9-mix. In MN tests, all three tetramethylbenzenes demonstrated no clastogenic activity on the bone marrow cells. All the tested solvents were active as genotoxic compounds in the SCE tests, demonstrating a dose-response relationships.

Genotoxicity of Farbasol and its component: 4-ethyltoluene.

A combination of assays for gene mutations in Salmonella typhimurium TA97a, TA98, TA100 and TA102 strains with and without rat liver activation, and for micronucleus and sister chromatid exchange (SCE) in bone marrow cells of Imp:Balb/c mice was used to provide data on the mutagenic and genotoxic properties of the mixture of aromatic solvents, known under the trade name of Farbasol. In addition, 4-ethyltoluene (the main ethylmethylbenzenic component of Farbasol) was also tested for muta- and genotoxicity. The results revealed that neither Farbasol nor 4-ethyltoluene induced an increased reverse mutation in bacterial cells or the formation of micronucleated polychromatic erythrocytes in bone marrow. However, those compounds were found to be active as sister chromatid exchange (SCE) agents.

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).

Cytochrome P-450 mediated genotoxicity of triarylmethane dye in mice fed ethanol liquid diet.

Genotoxic effect of synthetic triarylmethane dye (Acid Green 16) was evaluated in Balb C mice fed nutritionally adequate liquid diet (1 kcal/ml) or isocaloric alcoholic diet containing 5% (w/v) ethanol (36% of total calories) for 6 days. Dye compound was given intraperitoneally at dose 150 mg/kg body wt. 30 h before test. The micronucleus test was used for evaluation of genotoxicity of the dye. The level of cytochrome P-450 and the activity of 7-ethoxycoumarin O-deethylase (ECOD) and 7-ethoxyresorufin O-deethylase (EROD) in microsomes were determined to assess the metabolic efficiency of the microsomal system. Acid Green 16 dye provoked an increased frequency of micronucleated polychromatic erythrocytes in bone marrow and ethanol enhanced this genotoxic effect through induction of cytochrome P-4502E1 and stimulation of activities of microsomal monooxygenases (ECOD and EROD), presumably catalyziung bioactivation of the dye.

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)

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.

Experimental carcinogenicity and mutagenicity of non-asbestos natural fibres--preliminary report.

The aim of this investigation was to quantify the carcinogenic and mutagenic activity of antigorite occurring in the form of admixtures in different mineral raw materials (serpentinite, magnesite, dolomite and nickel ore). The carcinogenicity of dusts was evaluated after intraperitoneal injections of 5 mg (mice) or 20 mg (rats) of dust suspended in saline. A pathomorphological examination was performed in all the dead animals. For two raw materials--serpentinite and nickel ore--their mutagenic potency was investigated (SCE test was used in this study). Results obtained in the experiments on animals (rats and mice) showed that the biological aggressiveness of the mineral raw materials tested was associated with the content of antigorite fibres. Particularly the frequency of mesothelioma (5-85%) was related to the number of antigorite fibres longer than 5 microns. Both of the investigated raw materials (serpentinite and nickel ore) were mutagenic in the SCE test.

Increased urinary excretion of the oxidative DNA adduct, 8-hydroxydeoxyguanosine, as a possible early indicator of occupational cancer hazards in the asbestos, rubber, and azo-dye industries.

Oxidative damage to DNA has been suggested to contribute to a number of diseases including cancer. In order to study the relationship between oxidative damage to DNA and occupational exposures, urinary excretion of the oxidative DNA adduct, 8-hydroxydeoxyguanosine (8-OHdG), was determined in asbestos workers, rubber workers, azo-dye workers and controls. Levels of 8-OHdG in urinary samples were quantified by automated coupled-column high-performance liquid chromatography with electrochemical detection (HPLC-EC). The registered 8-OHdG levels were 1.40 +/- 0.56 mumol/mol creatinine in asbestos workers, 1.48 +/- 0.57 in rubber workers, 1.92 +/- 0.85 in azo-dye workers and 1.07 +/- 0.41 in controls (means +/- SD). Thus, 8-OHdG levels appeared to be significantly higher (p < 0.05) in each of the exposed groups than in the control group. Regression analysis revealed no important association between 8-OHdG excretion, age and smoking. These findings suggest that occupational exposures may contribute to an increased oxidative damage to human DNA and point to the possible use of urinary 8-OHdG assays in biomonitoring of biological effects of chemicals in selected industrial workplaces.

[Identification of potential carcinogenic dyes and intermediates on the basis of their genotoxicity]. / Identyfikacja potencjalnie rakotwórczych barwników i pólproduktów na podstawie oceny ich genotoksycznosci.

41 dyes, pigments and surface-active compounds were tested for mutagenic and genotoxic properties. It was found that 43% of the studied dyes of Polish make which belonged to the azo, triarylmethane and anthraquinone compounds were mutagenic or genotoxic. All the studied pigments and surface active compounds did not reveal potential carcinogenic activity except for NNO Dyspergator whose possible genotoxic activity needs confirmation in further study.

[Methods for the evaluation of the mutagenic and genotoxic properties of chemical compounds. III. The sister chromatid exchange test in vivo (an abbreviated method)]. / Metodyki stosowane do oceny wlasciwosci mutagennych i genotoksycznych zwiazków chemicznych. Czesc III. Test wymian chromatyd siostrzanych (SCE) in vivo.

The principle and experimental procedure of the in vivo sister chromatid exchange (SCE) test applied in the screening of chemical compounds for their mutagenic properties are presented. The objective of this assay is to evaluate the ability of a test chemical to induce SCE in bone marrow cells of laboratory rodents. Animals are exposed to test substances by appropriate means and are sacrificed at sequential intervals. Chromosome preparations from bone marrow cells of the exposed animals are stained and examined for the presence sister chromatid exchange. The criterion of mutagenic effect of the test chemical is the dose-related increase in SCE frequency and/or statistically significant and reproducible positive response (beyond the level of the control group) for at least one dose of the test chemical.

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.

[Evaluation of the mutagenic properties of mitomycin C by the sister chromatid exchange test in human blood lymphocytes in vitro]. / Ocena wlasci mutagennych mitomycyny C testem wymian chromatyd siostrzanych (SCE) w limfocytach krwi ludzkiej in vitro.

By means of the test of sister chromatid exchanges the SCE rate induced by 6 mitomycin C (MMC) doses was evaluated in lymphocytes of human blood collected from two donors and cultured in vitro for 72 hours in the presence of BrdU. A clear curvilinear dose-response relationship was found. The rate of changes of 4.1 cell in control cultures, after the 0.1 micrograms/ml dose, came to the mean value of 68.0/cell. The findings were consistent with those of other authors. In addition, the consistence of the results obtained for the two donors and minimum, variability between the cultures for the same dose indicate the method is reproducible and may be used to test chemical compounds of unknown mutagenicity.

Dose-response relationships for chromosome aberrations in peripheral blood lymphocytes after whole- and partial-body irradiations. I. Effects immediately after irradiation.

Dose-response relationships were established for yield of dicentrics and for a fraction of damaged metaphases in lymphocytes after gamma-irradiation of rabbits' whole blood in vitro. These relationships were based on the scoring of cells only in their first post-stimulation division and they served as a reference system for comparison with results of 60Co gamma-irradiation in vivo, either of the whole or of predetermined parts of an animal's body. There was a statistically acceptable agreement between dose-response data established for dicentric yield after whole-body irradiation in vivo and the reference dose-response curve derived from exposure of rabbit's blood in vitro. For partial-body (1/2) irradiations there was a satisfactory agreement between the dose-response curves in vitro for dicentric yield and fraction of metaphases damaged on the one hand and the response in vivo when the latter was related to mean doses to circulating blood. However, there was a drastic disagreement with the dose responses in vitro when measured cytogenetic quantities were plotted versus mean doses to body mass. When the latter were substituted for by comparable doses to circulating blood the in vivo-in vitro agreement was acceptable after irradiation of 'cerebral' 1/2 mass and very much improved for the 'caudal' 1/2 exposures. Therefore, it is concluded that, after partial-body irradiations, the dicentric yield or fraction of damaged metaphases in lymphocytes sampled immediately after exposure reflect mean dose to circulating blood irrespective of whether or not this dose coincides with mean dose to body mass.

Dose-response relationships for chromosome aberrations in peripheral blood lymphocytes after whole- and partial-body irradiations. II. Decline of aberration-carrying cells in blood with time post-exposure.

Dicentric yield and fractionation of damaged metaphases and lymphocyte count were followed for an interval of up to 3 months in rabbits after partial- and whole-body irradiations. Some partial-body exposures led to less-pronounced decline of the lymphocyte count in peripheral blood than others; in the former case the reduction of dicentric yield in peripheral blood lymphocytes over the first 24 h post exposure was significantly faster than in the latter. The decline was also faster than after uniform whole-body irradiation where it was virtually absent. However, the results suggest that, after whole-body exposures, the decline of the fraction of damaged metaphases with post-exposure time was much slower than that of the dicentric yield.

The yield of radiation-induced chromosomal aberrations in lymphocytes as related to the time of arrival at first post-stimulation mitosis.

Blood from 3 donors of each species, man, rabbit and pig, were irradiated with a dose of 2.5 Gy 60Co gamma-rays. Micro-cultures of lymphocytes, established in presence of BrdUrd, were harvested at 6 different times after stimulation by PHA. The preparations containing metaphase figures were stained according to Perry and Wolff to permit differentiation of the cells in first and later mitoses. In all individuals and species studied there was a highly significant negative correlation between dicentric yield and time from stimulation to harvest. The decline of the yield with time of harvest varied in 3 species between 1.0 and 3.6% per hour. Implications for biological dosimetry are discussed.