Co-localized expression of FasL, Fas, Caspase-3 and apoptotic DNA fragmentation in mouse testis after oral exposure to di(2-ethylhexyl)phthalate.

Expression of apoptosis-related proteins FasL, Fas and Caspase-3, as well as DNA fragmentation were examined in mouse testis 12 h after exposure to 4-0.004 mg/g di(2-ethylhexyl)phthalate (DEHP). Immunocytochemical examination of the highest dose (4 mg/g DEHP) mouse revealed a distribution of FasL in Sertoli cell and Fas in nearby spermatocyte, and Fas and Caspase-3 in the same spermatocyte. Fas-positive spermatocytes had a DNA-fragmented nucleus detectable by terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling (TUNEL) method. After exposure to 4, 0.4, 0.04 or 0.004 mg/g DEHP, the maximum number of nuclei with fragmented DNA per 0.5 microm testis section was 22, 7, 5 and 3, respectively. In unexposed control the maximum number was 3. To further estimate total amount of the fragmented DNA in testis of the exposed mouse, the extracted DNA fragments were analyzed by agarose gel electrophoresis. The amount of fragments in the first three steps of the DNA ladder was estimated by a photo-densitometry. In the highest dose mouse (4 mg/g DEHP), the fragmented DNA was 2.2 times as much in the control. In lower dose mouse (0.4, 0.04 or 0.004 mg/g DEHP), it was 1.1 times as much in the control. Taken together, these observations suggest that a single oral exposure to DEHP as low as 0.04 mg/g might be effective to testicular DNA fragmentation and apoptosis.