Abstract
As part of a comprehensive survey of the impact of the environmental pollutant and hepatocarcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the proteome of hepatic cells, we have performed a high resolution two-dimensional gel electrophoresis study on the rat hepatoma cell line 5L. 78 protein species corresponding to 73 different proteins were identified as up- or down-regulated following exposure of the cells to 1 nm TCDD for 8 h. There was an overlap of only nine proteins with those detected as altered by TCDD in our recent study using the non-gel-based isotope-coded protein label method (Sarioglu, H., Brandner, S., Jacobsen, C., Meindl, T., Schmidt, A., Kellermann, J., Lottspeich, F., and Andrae, U. (2006) Quantitative analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced proteome alterations in 5L rat hepatoma cells using isotope-coded protein labels. Proteomics 6, 2407-2421) indicating a strong complementarity of the two approaches. For the majority of the altered proteins, an effect of TCDD on their abundance or posttranslational modifications had not been known before. Several observations suggest that a sizable fraction of the proteins with altered abundance was induced as an adaptive response to TCDD-induced oxidative stress that was demonstrated using the fluorescent probe dihydrorhodamine 123. A prominent group of these proteins comprised various enzymes for which there is evidence that their expression is regulated via the Keap1/Nrf2/antioxidant response element pathway. Other proteins included several involved in the maintenance of mitochondrial energy production and the regulation of the mitochondrial apoptotic pathway. A particularly intriguing finding was the up-regulation of the mitochondrial outer membrane pore protein, voltage-dependent anion channel-selective protein 2 (VDAC2), which was dependent on the presence of a functional aryl hydrocarbon receptor. The regulatability of VDAC2 protein abundance has not been described previously. In view of the recently discovered central role of VDAC2 as an inhibitor of the activation of the proapoptotic protein BAK and the mitochondrial apoptotic pathway, the present data point to a hitherto unrecognized mechanism by which TCDD may affect cellular homeostasis and survival.
Highlights
As part of a comprehensive survey of the impact of the environmental pollutant and hepatocarcinogen 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) on the proteome of hepatic cells, we have performed a high resolution twodimensional gel electrophoresis study on the rat hepatoma cell line 5L. 78 protein species corresponding to 73 different proteins were identified as up- or down-regulated following exposure of the cells to 1 nM TCDD for 8 h
The molecular mechanisms underlying the tumorigenic activity of the compound are still largely unknown, which strongly hampers the estimation of the risk to humans associated with dioxin exposure and necessitates further studies aimed at the clarification of these mechanisms
Binding of TCDD to the cytosolic receptor results in the translocation of the ligand receptor complex into the cell nucleus and the dimerization with a related protein, the aryl hydrocarbon receptor (AhR) nuclear translocator (ARNT). This heterodimer can act as a ligand-activated transcription factor by binding to a specific DNA sequence known as aryl hydrocarbon response element I (AHRE-I), xenobiotic-responsive element, or dioxin-responsive element, an enhancer located in regulatory regions of aryl hydrocarbon-responsive genes
Summary
TCDD (purity Ͼ99%) in Me2SO was obtained from Okometric (Bayreuth, Germany). All chemicals and solvents used were of the highest purity available. To identify significant differences in spot intensities between the two groups the following rules were established: only spots matched in at least 50% of the gel images in a group were considered, and to get reasonable protein amounts for mass spectrometric identification only spots exceeding an average intensity threshold of 0.2 were taken for further analysis. (iii) The theoretical pI and the molecular mass of the protein suggested by the search result could be correlated with the position of the corresponding spot in the 2-DE gel. If the latter was not the case, posttranslational modifications (PTMs) were taken into consideration. All quantitative data were analyzed by single factor analysis of variance (Microsoft Excel) test for significance (p Ͻ 0.05)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
