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Biochemical, ultrastructural and molecular characterization of the triphenyltin acetate (TPTA)-induced apoptosis in primary cultures of mouse thymocytes

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Abstract

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 μmol/L TPTA; methyl prednisolone (1 μmol/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 μmol/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 μmol/L) induces early and late apoptotic phenomena, whereas cells exposed to the highest concentrations (8 μmol/L) are likely to undergo necrosis rather than apoptosis.

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Abbreviations

DMSO:

dimethyl sulfoxide

EC:

European Commission

EDTA:

ethylenediametetraacetic acid

FCS:

fetal calf serum

MPS:

methylprednisolone 21-hemisuccinate, sodium salt

OD:

optical density

OTs:

organotin compounds

PBS:

phosphate buffer solution

PI:

propidium iodide

SD:

standard deviation

TBE:

Tris boric acid and EDTA buffer solution

TBTO:

bis(tri-n-butyltin) oxide

TCDD:

2, 3, 7, 8-tetrachlorodibenzo-p-dioxin

TdT:

deoxynucleotidyltransferase

TOT:

triorganotin

TPTA:

triphenyltin acetate

TUNEL:

deoxynucleotidyltransferase-mediated X-dUTP nick end labeling

UV:

ultraviolet light

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Authors and Affiliations

  1. Dipartimento di Patologia Animale, Sezione di Anatomia Patologica, Università di Torino, Italy

    E. Bollo, P. Pregel & E. Cornaglia

  2. Dipartimento di Patologia Animale, Sezione di Clinica Medica, Università di Torino, Italy

    R. Guglielmino, F. Riondato & B. Miniscalco

  3. Dipartimento di Patologia Animale, Sezione di Farmacologia e Tossicologia, Università di Torino, Italy

    S. Sant & C. Nebbia

  4. Dipartimento di Sanità Pubblica, Patologia Comparata ed Igiene Veterinaria, Area di Farmacologia e Tossicologia, Università di Padova, Italy

    M. Dacasto

  5. Dipartimento di Sanità Pubblica, Patologia Comparata ed Igiene Veterinaria, Area di Farmacologia e Tossicologia, viale dell’Università 16, I-35020, Agripolis Legnaro, PD, Italy

    M. Dacasto

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  1. E. Bollo
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  2. R. Guglielmino
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  3. S. Sant
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  9. M. Dacasto
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Correspondence to M. Dacasto.

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Bollo, E., Guglielmino, R., Sant, S. et al. Biochemical, ultrastructural and molecular characterization of the triphenyltin acetate (TPTA)-induced apoptosis in primary cultures of mouse thymocytes. Cell Biol Toxicol 22, 275–284 (2006). https://doi.org/10.1007/s10565-006-0053-9

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