Abstract
One characteristic feature of acute 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity is dramatic interspecies and interstrain variability in sensitivity. This complicates dioxin risk assessment for humans. However, this variability also provides a means of characterizing mechanisms of dioxin toxicity. Long-Evans (Turku/AB) rats are orders of magnitude more susceptible to TCDD lethality than Han/Wistar (Kuopio) rats, and this difference constitutes a very useful model for identifying mechanisms of dioxin toxicity. We adopted a proteomic approach to identify the differential effects of TCDD exposure on liver protein expression in Han/Wistar rats as compared with Long-Evans rats. This allows determination of which, if any, protein markers are indicative of differences in dioxin susceptibility and/or responsible for conferring resistance. Differential protein expression in total liver protein was assessed using two-dimensional gel electrophoresis, computerized gel image analysis, in-gel digestion, and mass spectrometry. We observed significant changes in the abundance of several proteins, which fall into three general classes: (i) TCDD-independent and exclusively strain-specific (e.g. isoforms of the protein-disulfide isomerase A3, regucalcin, and agmatine ureohydrolase); (ii) strain-independent and only dependent on TCDD exposure (e.g. aldehyde dehydrogenase 3A1 and rat selenium-binding protein 2); (iii) dependent on both TCDD exposure and strain (e.g. oxidative stress-related proteins, apoptosis-inducing factor, and MAWD-binding protein). By integrating transcriptomic (microarray) data and genomic data (computational search of regulatory elements), we found that protein expression levels were mainly controlled at the level of transcription. These results reveal, for the first time, a subset of hepatic proteins that are differentially regulated in response to TCDD in a strain-specific manner. Some of these differential responses may play a role in establishing the major differences in TCDD response between these two strains of rats. As such, our work is expected to lead to new insights into the mechanism of TCDD toxicity and resistance.
Highlights
One characteristic feature of acute 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity is dramatic interspecies and interstrain variability in sensitivity
Proteome scale approach to investigate the extent to which TCDD exposure alters liver protein expression in H/W versus L-E rats to determine which, if any, protein markers are indicative of differences in dioxin susceptibility and are candidates for conferring resistance or sensitivity to TCDD
Data were preprocessed with the GCRMA algorithm [26] as implemented in the affy package [27] for R
Summary
2,3,7,8-TCDD was purchased from the UFA-Oil Institute (Ufa, Russia) and was found to be over 99% pure by gas chromatographymass spectrometry It was dissolved in diethyl ether: adjusted volumes of the solution were mixed with corn oil after which the ether was allowed to evaporate. For each rat liver sample, 150 g of total protein were diluted to a final volume of 250 l in the rehydration solution (5 M urea, 2 M thiourea, 2% CHAPS, 2% Zwittergent, 100 mM DeStreak, and 0.5% IPG buffer pH 3–10 linear (Amersham Biosciences) and applied on immobilized pH 3–10 linear gradient strips (IPG strips; Amersham Biosciences). After the first dimension was run, proteins were reduced by incubating individual strips for 15 min in a solution containing 50 mM Tris-Cl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 60 mM DTT (Amersham Biosciences).
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