Biochemical, ultrastructural and molecular characterization of the triphenyltin acetate (TPTA)-induced apoptosis in primary cultures of mouse thymocytes.
Triphenyltin acetate (TPTA), a triorganotin compound used in agriculture as a biocide, is immunotoxic in vivo and in vitro. The present study was undertaken to ascertain whether apoptosis might play a role in the TPTA toxicity in vitro. Mouse thymocyte primary cultures were exposed to 0, 4 and 8 micromol/L TPTA; methyl prednisolone (1 micromol/L) was used as a positive control. Cell aliquots were harvested after 0, 1, 2, 4, and 8 h and the presence of early or late apoptotic phenomena was checked by (a) morphological investigations; (b) spectrophotometric quantification of fragmented DNA and agarose gel electrophoresis; (c) cell flow cytofluorometry, using an annexin V-FITC kit; and (d) detection of in situ apoptosis by a colorimetric detection kit (Titer-Tacs). TPTA cytotoxicity was also evaluated using the trypan blue dye exclusion test. Morphological investigation indicated apoptosis and/or necrosis. After 8 h of incubation, cells exposed to 4 micromol/L TPTA showed an increase in DNA fragmentation (on electrophoresis), which was confirmed by spectrophotometry (p < 0.05). Flow cytofluorometry pointed out an early (p < 0.05) increase of annexin V-positive (apoptotic) cells in TPTA-exposed flasks, whereas at least partly contradictory, results were obtained with the Titer-Tacs kit. Overall, these results provide evidence that TPTA, at low concentrations (4 micromol/L) induces early and late apoptotic phenomena, whereas cells exposed to the highest concentrations (8 micromol/L) are likely to undergo necrosis rather than apoptosis.
Bollo
E
E
Dipartimento di Patologia Animale, Sezione di Anatomia Patologica, Università di Torino, Italy.
Guglielmino
R
R
Sant
S
S
Pregel
P
P
Riondato
F
F
Miniscalco
B
B
Cornaglia
E
E
Nebbia
C
C
Dacasto
M
M
D000818:Animals; D017209:Apoptosis; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004587:Electrophoresis, Agar Gel; D005434:Flow Cytometry / Q000379:methods; D005659:Fungicides, Industrial / Q000494:pharmacology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009336:Necrosis; D009947:Organotin Compounds / Q000494:pharmacology*; D013950:Thymus Gland / Q000166:cytology* / Q000187:drug effects*
D005659:Fungicides, Industrial; D009947:Organotin Compounds; C011978:phentin acetate