Detection of Clastogenic and Aneugenic Damage in Newborn Rats Ion Udroiu, 1 Luisa Anna Ieradi, 2 Mauro Cristaldi, 1 and Caterina Tanzarella 3 * 1 Dipartimento di Biologia Animale e dell’Uomo, Universita ` ‘‘La Sapienza,’’ 00161 Rome, Italy 2 Istituto per lo Studio degli Ecosistemi, CNR, 00161 Rome, Italy 3 Dipartimento di Biologia, Universita ` ‘‘Roma Tre,’’ 00146 Rome, Italy The last 25 years have seen an ever-growing use of the erythrocyte micronucleus test for measuring dam- age to mammalian chromosomes in vivo. In addi- tion, staining micronuclei with antikinetochore anti- bodies from CREST serum discriminates aneugenic from clastogenic damage. The use of the micronu- cleus 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 ei- ther X-rays (a clastogen) or vinblastine (an aneugen) and measured micronuclei in erythrocytes from the blood and liver. Each treatment increased the fre- quency of micronuclei in both tissues, with the per- centages of CREST-staining micronuclei reflecting the mechanism of micronucleus induction by the two agents. The results indicate that performing the mi- cronucleus assay in the liver and peripheral blood of 5-day-old rats may be a useful approach for detect- ing the in vivo genotoxicity of chemical and physical agents. Environ. Mol. Mutagen. 47:320–324, 2006. V V C 2006 Wiley-Liss, Inc. Key words: CREST; micronuclei; peripheral blood; liver; newborn rats INTRODUCTION The micronucleus test [Schmid, 1975] is a simple in vivo assay used for detecting cytogenetic damage induced by chemical and physical mutagens in erythrocytes. Mi- cronuclei are formed when an entire chromosome (aneu- genic damage) or a chromosome fragment (clastogenic damage) fails to migrate with one of the two daughter nuclei formed during mitosis. Conventional microscopic analysis, however, cannot determine if micronuclei re- sulted from an aneugenic or clastogenic mechanism. To do this, the presence or absence of centromere proteins can be used to identify micronuclei derived from chromo- some loss or chromosome breakage [Degrassi and Tanzar- ella, 1988; Miller and Adler, 1990]. Many studies have successfully applied CREST-stain- ing of centromeres for analysis of micronuclei from dif- ferent species: humans [Sgura et al., 2001], laboratory mice [Gudi et al., 1990; Cicchetti et al., 1999], hamsters [Gabriele et al., 1995; Sgura et al., 2000], yellow-necked mice, bank voles and Algerian mice [Degrassi et al., 1999; Tanzarella et al., 2001], and sheep [Degen et al., 1997]. There also have been unsuccessful attempts to use the method on rats. In a study of the genotoxicity of tri- chloroethylene, the analysis failed because of high non- specific background staining [Kligerman et al., 1994]. De Stoppelaar et al. [1997] used both serum from patients with CREST syndrome and commercial CREST antibod- ies, but because the latter gave unsatisfactory results, they concluded that this method is not suitable for rat cells. Another problem with measuring micronuclei in rat pe- ripheral blood is that the spleen of adult rats removes micronuclei from circulating erythrocytes [Schlegel and MacGregor, 1983, 1984; Ramirez-Mun ˜oz et al., 1999]. For this reason, the measurement of micronucleus induc- tion in adult rats has been limited to evaluating recent damage using peripheral blood polychromatic erythrocytes [Hayashi et al., 1992; Asanami et al., 1995]. The splenic red pulp (where the removal of micro- nucleated cells takes place) is not yet fully developed in very young rats [Bartoloni Saint Omer, 1964], and micro- nuclei have been observed in the peripheral blood of rats until 30 days after birth [Kojima et al., 1999; Zuniga- Gonzalez et al., 2001; Kasuba et al., 2002]. In addition, the liver is the main hemopoietical organ from the elev- enth day of gestation until one week after birth [Rasso- *Correspondence to: Caterina Tanzarella. E-mail: tanzarel@uniroma3.it Received 7 November 2005; provisionally accepted 2 December 2005; and in final form 3 February 2006 DOI 10.1002/em.20209 Published online 14 March 2006 in Wiley InterScience (www.interscience. wiley.com). V V C 2006 Wiley-Liss, Inc. Environmental and Molecular Mutagenesis 47:320^324 (2006)