Clastogenicity of chromium contaminated soil samples evaluated by Vicia root-micronucleus assay.

Chromium compounds have been known to be highly toxic in biological systems and to some individuals act as strong allergens. A chromium processing plant in Tianjin city has been abandoned for many years and the chromium residue has been dispersed into the nearby soil. This study was designed to detect the genotoxicity of contaminated soil samples collected at various distances of 100 to 1000 m from the source using the Vicia faba root micronucleus test. Water solutions extracted from the soil samples were used to treat the roots of the Vicia beans. Micronuclei frequencies observed from the root meristems were used to determine the degree of genotoxicity. Micronuclei frequencies of the contaminated soil samples show linear dose responses to chromium contents in the soil, which were inversely proportional to the distance from the source.     

On the mutagenicity of nitroimidazoles

Regarding mutagenicity, metronidazole is one of the best-investigated compounds of the nitroimidazoles. This drug is mutagenic on bacteria, especially if base-pair tester strains are used and bacterial nitroreductases are present. The serum levels attained in man after intake of this drug are sufficient to cause mutations in bacteria. Furthermore, interaction with and binding to DNA occurs under anaerobic conditions and sometimes DNA breaks are observed. However, metronidazole does not show mutagenic activity in mammalian cells in vitro; the micronucleus test is negative and chromosome aberrations are only found under anaerobic conditions. With microbial systems the mutagenicity of 47 nitroimidazoles has been investigated. Only 4 compounds were always negative in the applied test systems. Because with base-pair tester strains mutagenicity was assessed, this class of compounds should be regarded as a base-pair mutagen. In fungi, some compounds (e.g. ZK 26173 and azathioprine) are potent mutagens, whilst with most investigated nitroimidazoles only a weak or no mutagenic activity could be detected. Somewhat similar observations have been made in tests with Drosophila melanogaster, a test for gene mutations in mammalian cells, the micronucleus test, cytogenic tests and the dominant lethal test. The reduction products of metronidazole, misonidazole and 1-methyl-2-nitro-5-vinylimidazole, cause DNA damage if the nitro group is reduced in the presence of DNA. Reduction products are formed by microbes in the gut or by mammalian cells under anaerobic conditions. No teratological effect due to metronidazole or most other nitroimidazoles has been observed. Metronidazole is carcinogenic in mice and rats, and dimetridazole in rats. Up to the present, no carcinogenic effects have been observed in man. Azathioprine is probably carcinogenic for man. It is unlikely that the therapeutic applications of the presently used nitroimidazoles, except for azathioprine, will cause an increase in the tumor incidence in man or will cause other genotoxic effects, although such effects cannot be excluded with certainty.     

In vitro assessment of the toxicity of metal compounds

A review of in vitro mutagenesis assessment of metal compounds in mammalian and nonmammalian test systems has been compiled. Prokaryotic assays are ineffective or inconsistent in their detection of most metals as mutagens, with the notable exception of hexavalent chromium. Mammalian assay systems appear to be similarly inappropriate for the screening of metal compounds based upon the limited number of studies that have employed those compounds having known carcinogenic activity. Although of limited value as screening tests for the detection of potentially carcinogenic metal compounds, the well-characterized in vitro mutagenesis systems may prove to be of significant value as a means to elucidate mechanisms of metal genotoxicity.     

Oral chromium(VI) does not affect the frequency of micronuclei in hematopoietic cells of adult mice and of transplacentally exposed fetuses

Chromium(VI) compounds are genotoxic in a variety of cellular systems. Their potential carcinogenicity is affected by toxicokinetic patterns restricting bioavailability to certain targets, and by metabolic pathways affecting interaction of chromate-derived reactive species with DNA. Epidemiological data indicate that chromium(VI) can be carcinogenic to the human respiratory tract following inhalation at doses that are only achieved in certain occupational settings. However, concern has been raised that adverse effects may also result from oral intake. In order to further explore this issue, we performed studies in BDF1 and Swiss mice of both genders and various age. Sodium dichromate dihydrate and potassium dichromate were administered either with the drinking water, up to a concentration of 500 mg chromium(VI)/l for up to 210 consecutive days, or in a single intragastric dose of 17.7 mg/kg body weight. Under these conditions, no increase of the micronucleus frequency was observed in either bone marrow or peripheral blood erythrocytes. Conversely, the same compounds induced a clastogenic damage following intraperitoneal injection, which by-passes detoxification mechanisms. In addition, due to the hypothesis that susceptibility may be increased during the period of embryogenesis, we treated pregnant mice, up to a concentration of 10 mg chromium(VI)/l drinking water. There was no effect on the numbers of fetuses/dam and on body weight of fetuses. Again, no toxic or genotoxic effect was observed either in bone marrow of pregnant mice or in liver and peripheral blood of their fetuses. Thus, even at doses that largely exceed drinking water standards (up to 10,000 times) or by massive intragastric administration, chromium(VI) is not genotoxic to hematopoietic cells of either adult mice or transplacentally exposed fetuses. These conclusions are consistent with the poor toxicity and lack of carcinogenicity of oral chromium(VI), and are mechanistically explained by the high efficiency of chromium(VI) detoxification processes in the gastrointestinal tract.     

Oral chromium(VI) does not affect the frequency of micronuclei in hematopoietic cells of adult mice and of transplacentally exposed fetuses

Chromium(VI) compounds are genotoxic in a variety of cellular systems. Their potential carcinogenicity is affected by toxicokinetic patterns restricting bioavailability to certain targets, and by metabolic pathways affecting interaction of chromate-derived reactive species with DNA. Epidemiological data indicate that chromium(VI) can be carcinogenic to the human respiratory tract following inhalation at doses that are only achieved in certain occupational settings. However, concern has been raised that adverse effects may also result from oral intake. In order to further explore this issue, we performed studies in BDF1 and Swiss mice of both genders and various age. Sodium dichromate dihydrate and potassium dichromate were administered either with the drinking water, up to a concentration of 500 mg chromium(VI)/l for up to 210 consecutive days, or in a single intragastric dose of 17.7 mg/kg body weight. Under these conditions, no increase of the micronucleus frequency was observed in either bone marrow or peripheral blood erythrocytes. Conversely, the same compounds induced a clastogenic damage following intraperitoneal injection, which by-passes detoxification mechanisms. In addition, due to the hypothesis that susceptibility may be increased during the period of embryogenesis, we treated pregnant mice, up to a concentration of 10mg chromium(VI)/l drinking water. There was no effect on the numbers of fetuses/dam and on body weight of fetuses. Again, no toxic or genotoxic effect was observed either in bone marrow of pregnant mice or in liver and peripheral blood of their fetuses. Thus, even at doses that largely exceed drinking water standards (up to 10,000 times) or by massive intragastric administration, chromium(VI) is not genotoxic to hematopoietic cells of either adult mice or transplacentally exposed fetuses. These conclusions are consistent with the poor toxicity and lack of carcinogenicity of oral chromium(VI), and are mechanistically explained by the high efficiency of chromium(VI) detoxification processes in the gastrointestinal tract.     

In vitro genotoxicity data of nanomaterials compared to carcinogenic potency of inorganic substances after inhalational exposure

Literature data of epidemiological studies, carcinogenicity studies and in vitro studies on inorganic substances were surveyed with the aim to determine sensitivity and specificity of in vitro tests of nanomaterials. Asbestos, quartz and chromium and cadmium compounds were assigned to classes of highest carcinogenic potency. After 20 years of occupational exposure to long-term average concentrations of 0.5mg/m(3) of these dusts - or to even lower concentrations - an epidemiologically detectable increased lung cancer risk has to be expected. In contrast, diesel engine emissions, some nickel species and "ultrafine" versions (nanomaterials) of titanium dioxide and carbon black were also carcinogenic in inhalation studies, but show varied epidemiological results. The high frequency of lung cancer in the male general population due to cigarette smoking hampers unequivocal detection of occupationally caused lung cancer risks. Based on the experience from the inhalation studies, workers had to be exposed to long-term concentrations of 1mg/m(3) or more to identify epidemiologically a clear cause-and-effect relationship for a specific substance of intermediate potency. Respirable granular biodurable particles without known significant specific toxicity with primary particle sizes of more than 1μm have also shown carcinogenicity in rats. Their potency was even lower; and partially results after instillation rather than inhalation are available. Nearly all types of nanomaterials and control dusts used in the in vitro assays showed genotoxic effects in cell cultures (e.g., CoCr particles, diesel soot, SiO(2) crystalline and amorphous, TiO(2), carbon black), but not consistently in all studies; overall, the proportion of positive results was about 50%. No clear correlation of the probability of a positive in vitro test with particle properties was seen. I recommend trying and calibrating a sensitive in vitro model (e.g., micronucleus assay) against the described rank order of carcinogenic potency by testing a series of inorganic substances.     

Assessment of the utility of the micronucleus test for petroleum-derived materials

The micronucleus test is a commonly used in vivo assay for chromosomal damage and is an integral part of many mutagenicity testing strategies. The present report describes an assessment of the micronucleus test for the detection of mutagenic potential of petroleum-derived materials. To this end, studies were conducted with catalytically cracked clarified oil (CCCO). This material contains high levels of polycyclic aromatic constituents (PAC) and is a very potent inducer of mouse skin tumors. CCCO is also active in the Salmonella assay and other in vitro tests. As CCCO is the most potent of the various petroleum-derived materials in other assays, it was assumed to be the most easily detectable in the micronucleus test. CCCO was tested in standard mouse micronucleus tests utilizing oral and intraperitoneal injection for test material administration. All of these studies were negative, although DMBA, tested at roughly equivalent levels based on potency in the Salmonella assay, produced statistically significant increases in micronucleus frequency. In a second series of studies, aromatic fractions of CCCO were prepared and tested at up to acutely toxic levels. Results of these studies were also negative. Finally, another petroleum-derived material which is carcinogenic and contained PAC was tested in the micronucleus assay. It also produced negative results. Thus, it was concluded that petroleum-derived materials do not produce clastogenic effects in vivo in the mouse micronucleus test, despite the fact that some pure polycyclic aromatic hydrocarbons are quite active in this assay.     

Genotoxic ability of cadmium, chromium and nickel salts studied by kinetochore staining in the cytokinesis-blocked micronucleus assay

The aneugenic and clastogenic ability of cadmium chloride(II), cadmium sulfate(II), nickel chloride(II), nickel sulfate(II), chromium chloride(III) and potassium dichromate(IV) have been evaluated through kinetochore-stained micronucleus test. Traditional genotoxicity assays evaluate DNA damage, gene mutations and chromosome breakage. However, these tests are not adequate to detect aneugenic agents that do not act directly on DNA. Staining kinetochores in the cytokinesis-blocked micronucleus assay is a useful way to discriminate between clastogens and aneuploidogens and may allow a rapid identification of aneuploidy-inducing environmental compounds.Human diploid fibroblasts (MRC-5) were employed. All compounds increased micronuclei frequency in a statistically significant way. However, increases in kinetochore-positive micronuclei frequencies were higher than in kinetochore-negative ones. The present work demonstrates the genotoxic ability of the cadmium and chromium salts studied. Aneugenic as well as clastogenic ability could be observed with this assay. Nickel salts, as it was expected because of their known weak mutagenicity, showed lower genotoxic effects than the other metal salts studied. As the test employed only allows the detection of malsegregation, it is proposed that this mechanism is at least one of those by which the tested metal salts induced aneuploidy. On the other hand, visualization of kinetochores in all experiments suggests that the compounds studied did not act by damaging these structures.     

Genotoxic ability of cadmium, chromium and nickel salts studied by kinetochore staining in the cytokinesis-blocked micronucleus assay

The aneugenic and clastogenic ability of cadmium chloride(II), cadmium sulfate(II), nickel chloride(II), nickel sulfate(II), chromium chloride(III) and potassium dichromate(IV) have been evaluated through kinetochore-stained micronucleus test. Traditional genotoxicity assays evaluate DNA damage, gene mutations and chromosome breakage. However, these tests are not adequate to detect aneugenic agents that do not act directly on DNA. Staining kinetochores in the cytokinesis-blocked micronucleus assay is a useful way to discriminate between clastogens and aneuploidogens and may allow a rapid identification of aneuploidy-inducing environmental compounds.Human diploid fibroblasts (MRC-5) were employed. All compounds increased micronuclei frequency in a statistically significant way. However, increases in kinetochore-positive micronuclei frequencies were higher than in kinetochore-negative ones. The present work demonstrates the genotoxic ability of the cadmium and chromium salts studied. Aneugenic as well as clastogenic ability could be observed with this assay. Nickel salts, as it was expected because of their known weak mutagenicity, showed lower genotoxic effects than the other metal salts studied. As the test employed only allows the detection of malsegregation, it is proposed that this mechanism is at least one of those by which the tested metal salts induced aneuploidy. On the other hand, visualization of kinetochores in all experiments suggests that the compounds studied did not act by damaging these structures.     

Absence of mutagenic activity of WHR-1142A, lidamidine hydrochloride, in 4 short-term tests for mutagenic/carcinogenic potential

WHR-1142A, lidamidine hydrochloride, an antidiarrhoeal agent, was tested for possible mutagenic/carcinogenic activity in the Ames Salmonella typhimurium/metabolic activation test, the micronucleus test, by analysis of metaphase chromosomes obtained from human lymphocytes grown in culture and in a cell transformation assay. No evidence of mutagenic/carcinogenic activity due to WHR-1142A, lidamidine hydrochloride, was found in any of the 4 tests.     

On the persistence of the radioprotective effect of chlorophyllin (CHLN) in somatic cells of Drosophila

This research was designed to examine the presence of mutagenic/carcinogenic compounds in urban airborne particulates sampled with the inhalable PM-10 high volume sampler in two different streets of Brescia, a heavily industrialized town in northern Italy, using the Tradescantia/micronucleus test and a bacterial mutagenicity test (Kado test, a more sensitive version of the Ames test). In addition, the Tradescantia/micronucleus test was used for in situ monitoring of gaseous pollutants in other urban areas of Brescia and in two car tunnels, one with heavy car traffic in Perugia, a town in central Italy, and one in Brescia with moderate traffic. The Tradescantia-micronucleus test carried out on extracts of airborne particulates gave positive results only for the sample collected in the traffic-congested street where also higher bacterial mutagenicity was found. The in situ monitoring of the urban areas with the Tradescantia/micronucleus test always gave negative results. Monitoring carried out in the two car tunnels showed a significant increase in micronuclei frequency only in flowers exposed in the smaller and more polluted tunnel.     

Characterization of an in vitro micronucleus assay with Syrian hamster embryo fibroblasts

The use of Syrian hamster embryo cells for assessing genotoxicity provides the unique opportunity to determine 5 different end-points (gene mutations, DNA-strand breaks, aneuploidy, DNA repair (unscheduled DNA synthesis, UDS) and neoplastic transformation) in the one cell system. This approach allows direct comparisons of results produced under identical conditions of dose at target, metabolism and bioavailability. We report here on the characterization of an additional end-point in the same cell system: the formation of micronuclei indicating chromosomal changes induced by chemicals. For a preliminary validation of this new test system we have investigated 14 carcinogens and 3 non-carcinogenic structural analogues in order to evaluate the significance of micronucleus induction for carcinogenic properties. All tested carcinogens induced micronuclei in a dose-dependent manner; all non-carcinogens yielded negative results. Correlations between the formation of micronuclei and the Ames test, induction of UDS, cell transformation and the in vivo bone marrow micronucleus test are demonstrated.     

Radiation-induced genomic instability in Caenorhabditis elegans

Currently, the cosmetics industry relies on the results of in vitro genotoxicity tests to assess the safety of chemicals. Although the cytokinesis-block micronucleus (CBMN) test for the detection of cells that have divided once is routinely used and currently accepted by regulatory agencies, it has some limitations. Reconstituted human epidermis (RHE) is widely used in safety assessments because its physiological properties resemble those of the skin, and because it allows testing of substances such as hydrophobic compounds. Thus, the micronucleus test is being adapted for application in RHE-reconstructed tissues. Here we investigated whether two different reconstructed epidermis models (EPI/001 from Straticell, and RHE/S/17 from Skinethic) are suitable for application of the micronucleus test. We found that acetone does not modify micronucleus frequency, cell viability, and model structure, compared with non-treated RHE. Treatment of the EPI/001 model with mitomycin C and vinblastine resulted in a dose-dependent increase of micronucleus frequency as well as a decrease of tissue viability and of binucleated cell rate, while no changes of the epidermal structure were observed. The number of binucleated cells obtained with the RHE/S/17 model was too small to permit micronucleus testing. These results indicate that the proliferative rate of the tissue used is a critical parameter in performing the micronucleus test on a 3D model.     

铬渣堆场渗滤液对蚕豆根尖细胞微核的影响

利用蚕豆根尖微核技术监测化工铬渣堆场渗出液的遗传毒性,结果表明,化工铬渣渗出液含有较高浓度的Cr6+,能明显诱导蚕豆根尖细胞微核的形成,具有较强的遗传毒性。当铬渣渗出液中Cr6+浓度为186.6460 mg/L时,微核率达到最大。铬渣渗出液所诱导的微核率与阴性对照组相比有极显著差异。     

An evaluation of the E. coli K-12 uvrB/recA DNA repair host-mediated assay. II. In vivo results for 36 compounds tested in the mouse.

The aim of this study was to further evaluate the E. coli K-12 DNA repair host-mediated assay, as a short-term in vivo genotoxicity test, to be used as a complement to the micronucleus test in the routine testing of chemicals and drugs. The assay involves the administration of the test substance to mice by the route of choice, followed by the intravenous administration of a mixture of DNA repair deficient and proficient derivatives of E. coli K-12. After an incubation period the relative survival of the two strains was determined in blood, liver, lungs, kidneys and testes of the host. A significant preferential reduction of the DNA repair deficient strain in any organ indicates that the test substance possesses genotoxic properties. A total of 36 substances, 26 carcinogens, 4 weak or non-carcinogens and 6 unclassified substances, were tested in this assay. Positive results were obtained for 23 compounds. Of the carcinogens 18 were positive and of the non-carcinogens 3 were negative. The overall concordance between the assay and carcinogenicity was 72%. In general, alkylating agents and direct-acting nitroso compounds showed genotoxic activity in all organs tested, while the other substances were positive in a limited number of organs. With oral administration, which was the most commonly used administration route in the study, the organ showing a positive response most often was the blood. The results from the present study were compared with results from the micronucleus test, which were available for 26 of the substances. Results were in agreement for 15 of the substances, while 8 substances were positive in the present assay and negative in the micronucleus test: 4-aminobiphenyl, 2-anisidine, epichlorohydrin, formaldehyde, 1- and 2-naphthylamine, 2-nitrophenylenediamine and 4-nitroquinoline-N-oxide. The substances negative in the E. coli DNA repair host-mediated assay, but positive in the micronucleus test were: benzene, catechol and cyclophosphamide. It is concluded from this evaluation that the E. coli K-12 DNA repair host-mediated assay detects a number of carcinogens that are negative in the micronucleus test, while detecting most of the compounds that are positive in the latter. The advantages of this test are that differential DNA repair measures a broad spectrum of genetic damage, an in vitro/in vivo comparison is possible with the same test organisms, results can be obtained from various organs and the test is rapid.(ABSTRACT TRUNCATED AT 400 WORDS)     

Technical aspects of automatic micronucleus analysis in rodent bone marrow assays

The mouse bone marrow micronucleus assay is anin vivo test commonly used in the pharmaceutical industry to evaluate the genotoxic potential of new compounds. The test detects agent-induced chromosomal damage or damage of the mitotic spindle apparatus. In this paper the state-of-the-art in automated rodent micronucleus evaluation using computerized image analyis in combination with high-quality slides obtained by the cellulose column fractionation technique is reviewed. The latter allows the effective removal of nucleated cells from rodent bone marrow. It has been found that automatic micronucleus scoring with the Leitz MIAC image analyzer is substantially faster than labor-intensive manual analysis. Automatic scoring can be performed overnight for up to 16 slides. We have been successfully using automatic micronucleus analysis for the testing of new pharmaceutical drugs for more than 3 years.Abbreviations MNE NCE containing micronuclei - MPE PCE containing micronuclei - NCE normochromatic erythrocyte - PCE polychromatic erythrocyte deceased on 25 May 1994     

In vivo and in vitro cytogenetic effects of the anti-tumor agent amsacrina (AMSA)

The genotoxic effect of AMSA, an anti-tumor agent, was evaluated using the micronucleus and anaphase-telophase tests. The doses assayed by the in vivo micronucleus test were 1.5, 3 and 6 mg/kg: they are within the range of those used in clinical trials. A significant increase of micronucleated cells (P less than 0.01) was observed in the three assayed doses, with a linear dose response (r 0.98). In the in vitro test, 3 drug concentrations, i.e. 10, 1 and 0.1 microgram/ml, were analyzed with the 2 higher doses. AMSA showed a marked inhibition of cellular replication, but with 0.1 microgram/ml it was possible to determine an increase (P less than 0.01) in aberrations in anaphase-telophase cells. Both studies clearly demonstrate the clastogenic effect of the drug, which should be taken into account when considering its carcinogenic risk.     

Mutagenicity of sulphonylureas

The 11 derivatives of β-cytotropic sulphonylureas commonly used in the treatment of diabetes mellitus were tested in vivo in the highly sensitive sister-chromatid exchange test. Chlorpropamide and tolbutamide gave a positive reaction with a clear dose—response in Chinese hamsters and mice. The two compounds gave a mutagenic response neither in the Salmonella/mammalian-microsome mutagenicity test (with and without microsomal activation) nor in the chromosomal aberration test. In the micronucleus test, chlorpropamide was positive in 3 strains of mouse, tolbutamide in one strain. In Chinese hamsters and in rats the micronucleus test was negative with both compounds.     

Induction by mycotoxins of somatic mosaicism in Drosophila and DNA repair in mammalian liver cell cultures

The genotoxic activity of four mycotoxins has been studied. High level of somatic mutagenesis in imaginal discs of Drosophila melanogaster larvae and DNA repair synthesis in human embryo and adult rat liver cell cultures were inducible only by highly carcinogenic aflatoxin B1. Patulin, a weak direct-action carcinogenic substance, slightly elevated the mutagenesis in somatic cells of Drosophila but did not induce DNA repair synthesis in liver cell cultures. Citrinin that did not exhibit any carcinogenic properties when used alone and stachybotrotoxin with non-reported carcinogenic activity appeared inactive in the test-systems applied. The possibilities of rapid recognition of carcinogenic mycotoxins by detecting their genotoxic properties are discussed.