Influence of fixation and critical point drying in the detection of ultrastructural proliferative changes chemically induced by the diuron in the epithelium of urinary bladder of male Wistar rats

Abstract

Diuron (3-(3,4- Dichloropenyl)- 1,1- dimethylurea) is an urea-derived herbicide widely used in Brazil in soybean crops and sugar cane. It was classified by the USEPA in 2004 as "probably carcinogenic to humans". Scanning Electron Microscopy (SEM) has been used by our group to identify urothelial pro-liferative changes in the male Wistar rats exposed to diuron. Chronic exposure to diuron (> 15 weeks) results in exfoliative necrosis followed by urothelial regenerative hyperplasia; however, there are not conclusive results about the early changes induced by exposure to this herbicide. In a previous study at our laboratory, male Wistar rats exposed to diuron 2500 ppm during 7 days presented urothelial cells with polygonal and flatted aspect considered as normal. However, another study using the same dose and exposure period, but at other laboratory, seemed to induce cellular swelling and appearance of the so-called "swollen cells"meaning early adverse effect of diuron in the urothelium. These differences could be related to different methods of SEM processing which the bladders were submitted. The pur-pose of this study is to determine the influence of Bouin and glutaraldehyde fixatives and the SEM dry-ing procedures (the critical point dry and hexamethyldisilazane) in detecting early chemically induced changes in the urothelium of rats. Eighty four male Wistar rats will be allocated to three groups: control, sodium saccharin 8,3% (as positive control) and diuron 2500 ppm. They will be evaluated at the 7th day and 15th week. The urinary bladders will be submitted to SEM, histological and immuhistochemistry analysis to for identification and classification of citotoxicity and proliferative changes in the urothelium. (AU)