In vivo assessment of epichlorohydrin effects: the chorioallantoic membrane model

BACKGROUND: Previous studies on the effects of the epichlorohydrin (ECH) epoxide demonstrated this compound's toxicity and mutagenicity and suggested a carcinogenic activity also in humans. To gain a better understanding of ECH effects in vivo, the substance was tested on developing tissues utilizing the chick embryo chorioallantoic membrane (CAM) assay. MATERIAL/METHODS: Gelatin sponges adsorbed with ECH were implanted onto nine-day CAMs. After five days the membranes were fixed, cut in serial sections, and stained with toluidine blue. Sections of the ECH-treated CAMs were also submitted to immunocytochemistry for the basal lamina glycoprotein laminin and the gap junction protein connexin 43 (Cx43). Control CAMs were treated with saline solution and submitted to identical procedures. RESULTS: ECH-treated CAMs displayed proliferation of both the epithelial layers and the mesenchyme cells and vessels. The laminin immunolabeling was interrupted beneath the ectoderm thickenings, which penetrated the mesenchyme. The endoderm showed papilloma-like formations and its laminin-positive basal membrane protruded toward the mesenchyme, together with clusters of endodermal cells. The mesenchyme showed increased numbers of cells and microvessels. These reactions were restricted to regions corresponding to the implant. Cx43 expression was strongly decreased in the ECH-treated CAMs compared with the controls, where the connexin punctate pattern regularly decorated the epithelial cell contours. CONCLUSIONS: The study confirms that ECH elicits tissue proliferation at the contact site and corroborates the suggestion of an ECH carcinogenic effect due to hallmarks of tumoral growth, such as angiogenesis, basal membrane alterations, and loss of intercellular communication via gap junctions.