The suitability of rat peripheral blood in suchronic studies for the micronucleus assay

Asanami, Shougo; Shimono, Kazuyuki; Sawamoto, Osamu; Kurisu, Kazunobu; Uejima, Motoo

doi:10.1016/0165-7992(95)90073-x

Cited by 27 publications

(7 citation statements)

References 7 publications

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“…Since there is increased interest in integrating in vivo genotoxicity endpoints, e.g. the erythrocyte micronucleus (MN) test [MacGregor et al, 1990;Asanami et al, 1995;Garriott et al, 1995;Wakata et al, 1998;Hamada et al, 2001] and the liver Comet assay [Rothfuss et al, 2010], into repeat dose studies, we also included the MN and Comet endpoints in our investigation in addition to the Pig-a mutation assessment.…”

Section: Introductionmentioning

confidence: 99%

Assessment of genotoxicity induced by 7,12‐dimethylbenz(a)anthracene or diethylnitrosamine in the Pig‐a, micronucleus and Comet assays integrated into 28‐day repeat dose studies

Shi

Krsmanovic

Bruce

et al. 2011

Environ and Mol Mutagen

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As part of the Stage 3 of the Pig-a international trial, we evaluated 7,12-dimethylbenz(a)anthracene (DMBA) for induction of Pig-a gene mutation using a 28-day repeat dose study design in Sprague-Dawley rats. In the same study, chromosomal damage in peripheral blood and primary DNA damage in liver were also investigated by the micronucleus (MN) assay and the Comet assay, respectively. In agreement with previously published data (Dertinger et al., [2010]: Toxicol Sci 115:401-411), DMBA induced dose-dependent increases of CD59-negative erythrocytes/reticulocytes and micronucleated reticulocytes (MN-RETs). However, there was no significant increase in DNA damage in the liver cells when tested up to 10 mg/kg/day, which appears to be below the maximum tolerated dose. When tested up to 200 mg/kg/day in a follow-up 3 dose study, DMBA was positive in the liver Comet assay. Additionally, we evaluated diethylnitrosamine (DEN), a known mutagen/hepatocarcinogen, for induction of Pig-a mutation, MN and DNA damage in a 28-day study. DEN produced negative results in both the Pig-a mutation assay and the MN assay, but induced dose-dependent increases of DNA damage in the liver and blood Comet assay. In summary, our results demonstrated that the Pig-a mutation assay can be effectively integrated into repeat dose studies and the data are highly reproducible between different laboratories. Also, integration of multiple genotoxicity endpoints into the same study not only provides a comprehensive evaluation of the genotoxic potential of test chemicals, but also reduces the number of animals needed for testing, especially when more than one in vivo genotoxicity tests are required.

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Section: Introductionmentioning

confidence: 99%

Assessment of genotoxicity induced by 7,12‐dimethylbenz(a)anthracene or diethylnitrosamine in the Pig‐a, micronucleus and Comet assays integrated into 28‐day repeat dose studies

Shi

Krsmanovic

Bruce

et al. 2011

Environ and Mol Mutagen

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“…For this reason, the measurement of micronucleus induction in adult rats has been limited to evaluating recent damage using peripheral blood polychromatic erythrocytes [Hayashi et al, 1992;Asanami et al, 1995].…”

Section: Introductionmentioning

confidence: 99%

Detection of clastogenic and aneugenic damage in newborn rats

Udroiu

Ieradi

Cristaldi

et al. 2006

Environ. Mol. Mutagen.

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The last 25 years have seen an ever-growing use of the erythrocyte micronucleus test for measuring damage to mammalian chromosomes in vivo. In addition, staining micronuclei with antikinetochore antibodies from CREST serum discriminates aneugenic from clastogenic damage. The use of the micronucleus test in rats, however, has been problematic because the spleen of adult rats efficiently removes micronucleated erythrocytes from the blood. In the present study, we have treated 5-day-old rats with either X-rays (a clastogen) or vinblastine (an aneugen) and measured micronuclei in erythrocytes from the blood and liver. Each treatment increased the frequency of micronuclei in both tissues, with the percentages of CREST-staining micronuclei reflecting the mechanism of micronucleus induction by the two agents. The results indicate that performing the micronucleus assay in the liver and peripheral blood of 5-day-old rats may be a useful approach for detecting the in vivo genotoxicity of chemical and physical agents.

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“…Although the integration of the bone marrow micronucleus assay into general toxicology assays in rats has been reported [Henderson et al, 1993;Asanami et al, 1995;Garriott et al, 1995;Asanami and Shimono, 1996], the peripheral blood micronucleus assay is rarely performed in rats because circulating micronucleated erythrocytes are captured and destroyed in the rat spleen [Schlegel et al, 1982[Schlegel et al, , 1986Schlegel and Macgregor, 1984;Hayashi et al, 1992]. Despite this limitation, micronuclei were observed in the peripheral blood of rats by acridine orange supravital staining technique after treatment with mitomycin C (MMC), cyclophosphamide (CP), and benzo[a]pyrene (B[a]P) [Hayashi et al, 1992;Shimada et al, 1992].…”

Section: Introductionmentioning

confidence: 99%

Evaluation of the rodent micronucleus assay by a 28‐day treatment protocol: Summary of the 13th Collaborative Study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Environmental Mutagen Society of Japan (JEMS)–Mammalian Mutagenicity Study Group (MMS)

Hamada¹,

Sutou²,

Morita³

et al. 2001

Environ and Mol Mutagen

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To examine whether micronucleus tests can be incorporated into general toxicology assays, we performed micronucleus tests applying the treatment protocols typically used in such assays. In this 13th Collaborative Study of the CSGMT, both rats and mice were tested, although rats were used in the majority of the studies. Fifteen mutagens were tested in rats, mainly by oral (p.o.) administration. Micronucleus induction was evaluated 2, 3, and 4 days, and 1, 2, 3, and 28 days after the beginning of the treatment in the peripheral blood, and at 28 days in the bone marrow. Of the 15 chemicals that induced micronuclei in rats in short-term assays, two chemicals (1,2-dimethylhydrazine.2HCl and mitomycin C) were negative in all our experiments, possibly because of insufficient dose levels. The remaining 13 were positive within the estimated dose range of a general toxicology assay, suggesting the possibility of integrating the micronucleus assay into general toxicology assays. Three patterns were observed in micronucleus induction during the period of repeated treatment: (1) gradual increases in micronucleus frequency with sequential doses, (2) a peak at 3-5 days followed by gradual decreases in micronucleus frequency with sequential doses, and (3) a rapid increase in micronucleus frequency followed by a plateau. We evaluated factors that might have been involved in those patterns, such as the spleen function, target organ exposure, extramedullary hematopoiesis, hypothermia, and hypoxia. Another factor we considered was dosage. Because the dosages employed in a general toxicity assay are usually lower than those used in short-term micronucleus assays, this discrepancy was considered the greatest potential problem for integrating the micronucleus assay into general toxicology assays. Our results indicate that the integration of the micronucleus assay into a 28-day toxicological assay is feasible. To serve this purpose, blood samples collected 4 days after the beginning of treatment and blood and bone marrow samples collected at autopsy should be examined. Furthermore, although it is recognized that mice may be suitable for performing independent micronucleus assays, we propose that rats can provide biologically important and relevant information regarding potential chemical mutagens that can be evaluated under conditions used in the conduct of general toxicology studies.

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The suitability of rat peripheral blood in suchronic studies for the micronucleus assay

Cited by 27 publications

References 7 publications

Assessment of genotoxicity induced by 7,12‐dimethylbenz(a)anthracene or diethylnitrosamine in the Pig‐a, micronucleus and Comet assays integrated into 28‐day repeat dose studies

Assessment of genotoxicity induced by 7,12‐dimethylbenz(a)anthracene or diethylnitrosamine in the Pig‐a, micronucleus and Comet assays integrated into 28‐day repeat dose studies

Detection of clastogenic and aneugenic damage in newborn rats

Evaluation of the rodent micronucleus assay by a 28‐day treatment protocol: Summary of the 13th Collaborative Study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Environmental Mutagen Society of Japan (JEMS)–Mammalian Mutagenicity Study Group (MMS)

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