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238 G. KEITH ET AL

in occupational health because it has been reported that they induce hematopoietic dis-orders, and display developmental and reproductive toxicity, especially in małe germ cells, both in human and in laboratory animals (Hardin, 1983; Cheever 1989; Spano, 1991; Smialowicz 1992). Although some EGEs are known to be hematopoietic or tes-ticular toxicants at doses ranging from 100 to 900 mg/kg body weight in rodents, no mutagenic action has been reported so far. The only toxicological data availabie cor-respond to morphological or enzymie changes induced either in whole rodents or in vitro assays (Ghanayem, 1989).

The aim of this work was to study the action of ethylene glycol monobutyl ether (2-butoxyethanol), one of the most widely used compounds of the ethylene glycol ether class (Cicolella, 1992), on the molecular level i.e. on DNA. Indeed, to the best of our knowledge, epigenetic effects of ethylene glycol ethers have never been reported, although this could explain the reproductive disorders or the bonę marrow action of some representatives of this class of Chemicals.

They could act on DNA methylation especially at the m5dCpdG doublets, which are heritable and have various genomie pattems during development and differentiation (Holliday, 1993), and on tumor cells (Gloudemans, 1992; Vachtenheim, 1994). The methylation status of dCpdG is shown in the current work by the use of Hpa II, a methylation sensitive restriction enzyme, and Msp I which digests DNA at the same sequence (CCGG) regardless of whether deoxycytosine is methylated or not.

In addition, the toxicity of 2-butoxyethanol in the target organ (testis, spleen, liver, but also in kidney, bone-marrow and brain) was investigated by studying the overall level of m5dC in rats and mice.

Since 2-butoxyethanol is metabolized into 2-butoxyacetaldehyde, which could react (e.g. other aldehydes: glutaraldehyde and acetaldehyde) on DNA at the molecular level and thus lead to the formation of DNA-adducts which are very often implicated in can-cer pathogenesis (Miller, 1981), we have checked for such DN A-adduct formation upon 2-butoxyethanol treatment in rats and mice by using the ³²P postlabelling method of Randerath (Randerath, 1981; Randerath, 1985; Reddy, 1986).

Transgenic mice should provide valuable bioassays for potential carcinogens test-ing (Adams and Córy, 1991). As these mice can be considered as initiated (namely carrying and expressing an oncogene, a particular animal paradigm has been used, namely v-Ha-ras transgenic mice (Rihn et al., 1995). This transgenic mouse strain carries the viral Harvey ras (v-Ha-ra$) oncogene controlled by the mouse mammary tumor virus (MMTV) promoter (Sinn, 1987). The v-Ha-ras oncogene differs from the rodent c-Ha-ras protooncogene by only two amino acid substitutions leading to its activation.

The p21^ras transducin encoded by the oncogene stimulates continuous celi growth and thus leads to tumor formation. It is therefore a powerful tool for the investigation of predictable carcinogenesis. Both males and females spontaneously develop breast carcinoma, parotid adenocarcinoma as well as Harderian hyperplasia. However the oc-currence of these tumors is significantly inereased following intoxication by initiator or promoter agents. In addition the ras oncogene has been found prevalent in a variety of human carcinomas and is mutated (activated) in 10 to 30% of all human and rodent tumors (Bos, 1989). Our hypothesis was that 2-butoxyethanol could act as a non-geno-

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