Micronuclei test

Teratogenic effects have been noted with 2- and 4-aminophenol in the hamster, but 3-aminophenol was without effect in the hamster and rat (129,130). 4-Aminophenol is known to inhibit DNA synthesis and alter DNA stmcture in human lymphoblasts (131,132) and is mutagenic in mouse micronuclei tests (133). The aminophenols have been shown to be genotoxic, as evidenced by the induction of sister chromatid exchanges (134,135), but they also exert a protective effect against DNA interaction with other noxious chemicals (136). After assessment of available data a recent report stated that the aminophenols were safe as cosmetic ingredients in their present uses and concentrations (137). 

Biscardi, D., Monarca, S., De Fusco, R., Senatore, F., Poli, P., Buschini, A., Rossi, C. and Zani, C. (2003). Evaluation of the migration of mutagens/carcinogens from PET bottles into mineral water by 7>a<7eicawtw/micronuclei test. Comet assay on leukocytes and GC/MS, Sci. Total Environ., 302, 101-108. 

Mouse bone marrow polychromatic-erythrocyte assay (micronucleus test) Micronuclei B Usha Rani et al. 1980... 

Mice exposed to 2,000 ppm of trichloroethylene, 4 hours/day for a 5-day period, had a significant increase in abnormal sperm morphology of 1% 28 days after the exposure (Land et al. 1981). No effect was seen at 200 ppm. A 6% increase in abnormal sperm was observed 4 weeks, but not 4 days or 10 weeks, after mice were exposed to 100 ppm trichloroethylene 7 hours per day for 5 days (Beliles et al. 1980). Based on the time after exposure at which sperm were affected, the study authors indicated that trichloroethylene damages sperm precursor cells but that spermatogonia were either unaffected or were capable of recovery. Reproductive performance was not tested in these studies. Another mouse study tested the effects of a 5-day exposure (6 hours/day) on spermatid micronuclei frequency no effects were observed at exposure levels of up to 500 ppm, the highest concentration tested (Allen et al. 1994). These results were interpreted as evidence that trichloroethylene did not cause meiotic chromosome breakage or loss. No treatment-related reproductive effects were seen in female rats exposed to 1,800 ppm trichloroethylene for 2 weeks (6 hours/day, 7 days/week) before mating (Dorfmueller et al. 1979). 

Monomethylhydrazine-induced mutagenesis was not observed in Ames Salmonella/ microsome with activation (Matheson et al. 1978). In vivo tests in mice (dominant lethal, revertants in host-mediated assay), and dogs (micronuclei) were negative (reviewed in Trochimowicz 1994). However, in vitro chromosomal damage in human and rat tissue has been demonstrated, although in vivo liver DNA damage (as determined by DNA alkaline elution) was equivocal (reviewed in Trochimowicz 1994). 

Damage induced in whole animals can be detected in in vivo chromosome assays in either somatic or germinal cells by examination of metaphases or the formation of micronuclei. The micronucleus test can also detect whole chromosome loss or aneuploidy in the absence of clastogenic activity and is considered comparable in sensitivity to chromosome analysis (Tsuchimoto and Matter, 1979). 

MacGregor, J.T., Wehr, C.M. and Gould, D.H. (1980). Clastogen-induced micronuclei in peripheral blood erythrocytes the basis of an improved micronucleus test. Environ. Mutagen. 2 509-514. 

Disulfoton was negative in a dominant lethal test in male mice treated orally with a single dose ot S mg/kg (Herbold 1980) and in an erythrocyte micronucleus test in mice dosed with 6 or 12 mg/k /day for 2 days (Herbold 1981). Disulfoton also did not induce micronuclei in erythrocytes of mice dosed with 2, 4, or 8 mg/kg disulfoton (EPA 1984a Sandhu et al. ]985), but whether disulfoton was administered by the oral or intraperitoneal route was not clear. Other genotoxicity studies are discussed in Section 2.4. 

The in vivo micronucleus test is used for the detection of damage to chromosomes as well as the mitotic apparatus in bone marrow or peripheral blood cells of rodents. The assay system has been well standardized.14-17 The basic features of the test system are (1) the effect of the test chemical is observed in anucleated polychromatic erythrocytes (PCEs) (2) PCEs have a relatively short lifespan, so that any micronuclei they contain must have been generated as a result of recently induced chromosome damage (3) micronuclei are readily identifiable and their distribution is well defined and (4) the frequency of induced micronuclei in PCEs is dependent on sampling times. 

Genotoxic effects have been reported in animals treated with 3,3 -dichlorobenzidine. A single dose of 3,3 -dichlorobenzidine (1,000 mg/kg) administered to male and pregnant female mice induced micronuclei in polychromatic erythrocytes in the bone marrow of the males and in the liver of the fetuses, but not in bone marrow of the dams (Cihak and Vontorkova 1987). A micronucleus test is performed to detect a chemical s ability to induce chromosomal aberrations. However, the relevance of micronuclei formation to human health is not known. The reason for the lack of effect of 3,3 -dichlorobenzidine on bone marrow micronuclei formation in the mothers is unclear, but it may be related to deficiencies in the metabolic activation of 3,3 -dichlorobenzidine in female mice. The relative importance of pregnancy is unknown since the study did not evaluate nonpregnant females. In another study, an increase in unscheduled deoxyribonucleic acid synthesis (UDS) was observed in cultured liver cells from male mice previously pretreated orally with single doses of 500 mg/kg 3,3 -dichlorobenzidine no response was observed at a dose of 200 mg/kg (Ashby and Mohammed 1988). 

Animals, usually rodents, are exposed to the test substance by an appropriate route, usually by gavage or by intraperitoneal injection. Each treated and control group must include at least five animals per sex. Positive controls should produce micronuclei in vivo at exposure levels expected to give a detectable increase over background. No standard treatment schedule (i.e., 1, 2, or more treatments at 24-h intervals) has been recommended. Three dose levels are generally used these should cover a range from the maximum to little or no toxicity. The erythrocytes are sampled from the bone marrow and/or peripheral blood of the animals. If bone marrow is used, the animals are sacrificed at appropriate times after treatment, the bone marrow extracted, and preparations made and stained. When peripheral blood is used, the blood is collected at appropriate times after treatment and smear preparations are made and stained. Preparations are analyzed for the presence of micronuclei. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage. 

The test is used for the detection of cytogenetic damage to the chromosomes or the mitotic apparatus of erythroblasts by analysis of erythrocytes for formation of micronuclei (small nuclei, separate from and additional to the main nuclei of cells, produced during the telophase of mitosis (meiosis) by lagging chromosome fragments or whole chromosomes). When a bone marrow erythroblast develops into a polychromatic erythrocyte (immature erythrocyte), the main nucleus is extruded any micronucleus that has been formed may remain behind in the otherwise anucleated cytoplasm. 

The test is used to detect DNA repair synthesis and for the detection of micronuclei in the cytoplasm of interphase cells. These micronuclei may originate from acentric fragments (chromosome fragments lacking a centromere) or whole chromosomes that are unable to migrate with the rest of the chromosomes during the... 

Positive results in the mammalian in vivo erythrocyte micronucleus test indicate that a substance produces micronuclei in the immature erythrocytes of the test species, which are the result of chromosomal damage or damage to the mitotic apparatus in the erythroblasts of the test species. 

In genotoxic assays inorganic arsenicals are either inactive or weak mutagens but are able to produce chromosomal effects including aberrations and sister chromatid exchange in most test systems. Studies of exposed human have detected higher incidences of chromosomal aberrations in peripheral lymphocytes and increases in the frequency of micronuclei in the oral mucosa cells, urothelial cells, and peripheral blood lymphocytes. ... 

BA s metabolites are genotoxic in the Ames mutation test and caused unscheduled DNA synthesis in primary rat hepatocytes. In an in vivo mutagenic assay, male CD rats (6/group) were dosed three times with BA over a 24-hour interval by intratracheal instillation. Lung cells were enzymatically separated and used to determine the frequency of DNA adducts, sister chromatid exchanges (SCEs), and micronuclei. BA induced DNA adducts, SCEs, and micronuclei in this rat lung cell system. 

Studies in several test systems have shown DCB to be genotoxic in vitro and in vivo and suggest that this effect most likely mediates the carcinogenicity of the chemical. In vitro, DCB has induced sister chromatid exchanges, unscheduled DNA synthesis, and positive responses in bacterial Salmonella assays in vivo DCB induced micronuclei in polychromatic erythrocytes in male mice and fetuses. ... 

CS has been tested for its ability to cause chromosomal damage in a micronucleus test in mice. Micronucleus tests detect small nuclei that arise from chromosomal fragments or chromosomes that fail to be incorporated into normal daughter nuclei when cells divide. An increased frequency of micronuclei is evidence of chromosomal break-... 

Fortunately, a number of in situ, short-term bioassays to detect genotoxic and related effects have become available. These include a variety of measured endpoints such as aneuploids, chromosal aberrations, DNA damage, dominant lethal mutation, gene mutation, inhibition of intercellular communication, micronuclei, mitotic recombination and gene conversions, and sister chromatid exchange and cell transformation (IARC, 1989). A detailed discussion of these tests is beyond the scope of this book. However, such tests are important from our perspective as atmospheric chemists because, as we shall see, they can be used to detect biologically active compounds in very complex mixtures, and hence serve to focus chemical analysis efforts (IARC, 1989, p. 20). We emphasize in advance the... 

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