Proteome Analysis of the Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin on Murine Testicular Leydig and Sertoli Cells.

Abstract

We treated testicular cell lines Leydig TM3 and Sertoli TM4 with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and used two-dimensional electrophoresis to investigate the resulting protein alterations. Cells were cultured in a medium containing 10 -5 to 10 nM TCDD for 4 hr, under which condition viability was not affected. Protein expression was compared semi-quantitatively by silver staining, by autoradiography of [35 S] methionine-labeled proteins, and by anti-phosphotyrosine antibody. In TM3, 34 protein spots were altered by TCDD, 26 of which were increased and 8 of which were decreased; in TM4, the amount of total protein appeared to be reduced and 19 protein spots were altered by TCDD, 12 of which were increased and 7 of which were decreased. Four of these altered proteins were identified by N-terminal protein microsequencing and by a homology search against protein databases. Whereas a pyruvate dehydrogenase E1 beta subunit was decreased by TCDD exposure, ATP synthase beta chain, mitochondrial matrix protein P1 and 78 kDa glucose-regulated protein were relatively increased. The precise role of these proteins in TCDD toxicity remains to be determined, but the observed alterations suggest the proteins to be important in the effects of TCDD on testicular cells.

Effect of a singlein ovo injection of 2,3,7,8-tetrachlorodibenzo-p-dioxin on protein expression in liver and ovary of the one-day-old chick analyzed by fluorescent two-dimensional difference gel electrophoresis and mass spectrometry

“…It thus remains to be investigated if changes in these CYP enzymes are detectable 3 wk postadministration. It is worthwhile to note that in these 2-DE studies on the effects of TCDD in different tissues, no alterations in CYP were mentioned [29,41]. Besides the binding to the AhR, other mechanisms such as oxidative stress and other types of cellular stress mechanisms have been proposed to play an important role in TCDD induced toxicity [42].…”

“…Solvent A: 0.1% FA in HPLCgrade water; solvent B: 0.1% FA in ACN. The column outlet was coupled to the Q-Tof through a stainless steel capillary (Proxeon, Odense, Denmark) at 1.9 kV, with the cone voltage at 30 V. The eluting [M 1 2H] 21 , [M 1 3H] 31 , and [M 1 4H] 41 peptides (in the 400-1200 m/z range) were automatically selected and fragmented using predefined collision energy profiles. For direct infusion MS and MS/MS analysis, the peptide sample was ZipTip-concentrated and loaded into a Au/Pd-coated nanospray needle (Proxeon) and a capillary voltage of 800 V was applied.…”

Liver Proteins that are Sensitive to a Dioxin-Like Toxic Compound, Coplanar Polychlorinated Biphenyl, 3,3',4,4',5-Pentachlorobiphenyl.

“…49) Further, the two-dimensional gel electrophoresis system has been improved and adapted to TOF/MS system for analyzing proteome. 68) Finding of specific AhR ligand(s) 84) and inhibitors 85,86) could also help the interpretation of data. Taken together, it is likely that biochemical approaches which are based on the endpoint of the toxicity caused by dioxins are also important in understanding the pleiotropic effects.…”

Effect of 2,3,7,8-Tetrachlorodibenzo-p-dioxin on Testosterone Production in Isolated Murine Testicular Cells.

“…We previously carried out proteomic 12) and RNA arbitrarily primed polymerase chain reaction analyses 11) of mouse Leydig TM3 cells exposed to TCDD and reported that the synthesis of proteins and expression of mRNAs were markedly altered by TCDD. Taking those findings together with the present results, we believe that TCDD may have an effect on testis function but that it may not affect testosterone production directly.…”

“…Mediation of the toxic effects of TCDD via aryl hydrocarbon receptors (AhR) and AhR nuclear translocators, in particular, have been clarified. 10) To investigate the effects of TCDD on the testis at the mRNA or protein level, experiments using testicular cells derived from the mouse have been carried out, and many interesting genes 11) and proteins 12) have been found. We investigated the direct effects of TCDD on the secretion of testosterone from mouse testicular cells and examined the viability of cells and concentrations of testosterone when primary cultured mouse testicular cells were exposed to TCDD.…”