Gender specific differences in the liver proteome of rats exposed to short term and low-concentration hexabromocyclododecane (HBCD)

Gender specific impact of HBCD on rat liver proteome, determined by 2D-DIGE.


Specifications
Value of the data  Identification of liver proteins from male rats altered due to HBCD exposure  Identification of liver proteins from male rats changed in hypothyroid status  Comparison of male and female rats exposed in a similar exposure experiment, but showing different response in the liver proteome  Identified liver proteins form the basis for a more detailed understanding of involved mechanisms for the investigated compound, of differences in gender susceptibility and of gender dimorphic liver protein composition.

Data
Rat liver protein lysates show a complex spot pattern in high-resolution two-dimensional electrophoresis (about 3000 spots per gel). Patterns of 24 gels from different exposures of eu-and hypothyroid male rats were evaluated quantitatively. The same number of gels from an identical experiment with female rats from a previously described study [1,3] was re-evaluated together with the newly obtained males' data, and additional MS identifications of regulated spots in females vs. males were performed. Data from different animals groups in both genders were compared, taking different aspects into account (HBCD exposure, thyroid status, gender). Statistically significant fold-changes of at least 30 % between groups (P<0.05 within group) were considered to be relevant.
Suppl. Figure 1 presents the master gel of the combined gel set, and all spots with significant abundance changes in any of the performed comparisons are labelled. Overall, single proteins in 496 spots, selected by ANOVA evaluation, were successfully identified by MS analyses. Their spot numbers together with relevant peptide identifications are listed in Suppl. Table 1. Abundance changes of the various animal groups are compiled in Suppl. Table 2. Hierarchical clustering gave a clear grouping of regulated spots according to gender, and in females also according to thyroid state (Suppl. Figure 2). This is in line with PCA results (score plot of experimental groups) reported in [2]. The loading plot of this PCA is shown in more detail in Figure 1, with additional identification of single proteins in the border area or close to the main cluster.

Animals, treatment and experimental protocol
The animal experiment was detailed in [1,2] and approved under number 2006-051 by the Animal Welfare Committee of Wageningen University. In brief, male Wistar WU (HsdCpbWU) rats with normal or reduced thyroid function (hypothyroid) were orally exposed to 0, 3 or 30 mg/kg bw / d HBCD, respectively, for 7 consecutive days. Four liver samples per group were analyzed by proteomic methods.
The experimental setup was identical to the one used in [1], but applying it on male rats.

Proteomic Analysis
Rat liver lysates were subjected to two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) as previously described, without modifications, to make it compatible to the previously performed study in females [1][2][3]. This comprised a classical 2D-DIGE experiment, by separating CyDye-labeled proteins from liver lysates in a non-linear 3-10 pH-range and subsequently in large-format SDS-PAGE gels (260 x 200 x 1 mm). Gel images (acquired on a Typhoon 9400) were evaluated with the DeCyder 7.0 software package (both GE Healthcare, Diegem, Belgium), including all gels of the previous study on females in matching and statistics [1,3]. Univariate and multivariate analyses were applied to highlight differentially regulated spots (fold change at least 1.3) with a P-value in the respective univariate ANOVA or two way ANOVA <0.05.
Spots differentially regulated in any of the group comparisons (including samples/gels from males and females) were automatically picked, trypsin digested and proteins identified by MALDI-TOF/TOF 5800 (Sciex) as previously described [1][2][3]. This included also spots from female rat livers previously not analyzed, as only now they were found regulated in the comparison with male rats' data. Protein identification was based on searches of mass fingerprints (PMF) and MS/MS spectra against the SwissProt database with "Rattus norvegicus" as taxonomy, using the ProteinPilot software (Sciex, Nieuwerkerk aan den Ijssel, The Netherlands) and the searching algorithm MASCOT (Matrix Science, www.matrixscience.com, London, UK). For each spot one protein mass fingerprint and up to 8 MS/MS spectra were generated. Parameters for the search were set as in the previous study [1,3]: up to two missed cleavages allowed, 100 ppm tolerance in PMF, 0.75 Da mass tolerance for precursor ion mass, carbamidomethyl cysteine as fixed modification, oxidation of methionine and oxidation of tryptophan (single oxidation, double oxidation and kynurenin) as variable modifications. Identifications were considered to be significant when the combined MOWSE score had P<0.05.
For statistics the Extended Data Analysis (EDA) module as well as univariate analysis (ANOVA and t-test) and multivariate analysis (two way ANOVA), all part of the Decyder 7.0 software package, were applied.