Can exercise training counteract doxorubicin-induced oxidative damage of testis proteome?

The use of the chemotherapeutic drug doxorubicin (DOX) is limited by its toxicity in several organs such as testes. So, we analyzed the effect of endurance treadmill exercise training (EX) performed before sub-chronic DOX treatment on sperm count and motility, testes markers of oxidative damage and apoptosis. Tissue profiling of proteins more susceptible to oxidation was made to identify the molecular pathways regulated by oxidative modifications, as nitration and carbonylation. Twenty-four adult male rats were divided into four groups (n=6/group): sedentary saline (SED+SAL), sedentary sub-chronically injected with DOX (2mg-kg-1 per week, during 7 weeks; SED+DOX), 12 weeks trained saline (EX+SAL) and trained treated with DOX (EX+DOX). DOX treatment started 5 weeks after the beginning of the exercise program. Testes caspase-3, -8 and -9, as well as aconitase activities, the content of malondialdehyde (MDA), sulfhydryl groups (-SH), carbonyl and nitrotyrosine derivatives were determined. Modified proteins were identified by 2D-Western blot followed by MALDI-TOF/TOF mass spectrometry, and bioinformatic analysis was performed to assess the biological processes regulated by these chemical modifications. The decreased sperm motility induced by DOX was not modified by exercise. Significant increases in MDA content in SED+DOX and in caspase-3 and -9 activities in EX+DOX were found. Despite no significant differences in the levels of carbonylated and nitrated proteins, exercise modulated testis proteome susceptibility to oxidation in DOX-treated group, with less modified proteins identified. Zinc finger Ran-binding domain-containing protein 2 (ZRAB2) and AN1-type zinc finger protein 3 (ZFAN3) were among the proteins found oxidativelly modified. Although no marked alterations in testes oxidative damage were noticed, proteomic analysis of oxidativelly modified proteins highlighted the protective role of exercise against oxidative damage of some proteins involved in metabolism and stress response against DOX.

Physical exercise prior and during treatment reduces sub-chronic doxorubicin-induced mitochondrial toxicity and oxidative stress.

Doxorubicin (DOX) is an anti-cancer agent whose clinical usage results in a cumulative and dose-dependent cardiotoxicity. We have previously shown that exercise performed prior to DOX treatment reduces the resulting cardiac(mito) toxicity. We sought to determine the effects on cardiac mitochondrial toxicity of two distinct chronic exercise models (endurance treadmill training-TM and voluntary free-wheel activity-FW) when used prior and during DOX treatment. Male-young Sprague-Dawley rats were divided into six groups (n=6 per group): SAL+SED (saline sedentary), SAL+TM (12-weeks TM), SAL+FW (12-weeks FW), DOX+SED (7-weeks of chronic DOX treatment 2mg/kg per week), DOX+TM and DOX+FW. DOX administration started 5weeks after the beginning of the exercise protocol. Heart mitochondrial ultrastructural alterations, mitochondrial function (oxygen consumption and membrane potential), semi-quantification of oxidative phosphorylation (OXPHOS) proteins and their in-gel activity, as well as proteins involved in mitochondrial oxidative stress (SIRT3, p66shc and UCP2), biogenesis (PGC1α and TFAM), acetylation and markers for oxidative damage (carbonyl groups, MDA,SH, aconitase, Mn-SOD activity) were evaluated. DOX treatment resulted in ultrastructural and functional alterations and decreased OXPHOS. Moreover, DOX decreased complex I activity and content, mitochondrial biogenesis (TFAM), increased acetylation and oxidative stress. TM and FW prevented DOX-induced alteration in OXPHOS, the increase in oxidative stress, the decrease in complex V activity and in complex I activity and content. DOX-induced decreases in TFAM and SIRT3 content were prevented by TM only. Both chronic models of physical exercise performed before and during the course of sub-chronic DOX treatment translated into an improved mitochondrial bioenergetic fitness, which may result in part from the prevention of mitochondrial oxidative stress and damage.

Mitochondria proteome profiling: a comparative analysis between gel- and gel-free approaches.

Mitochondrial proteomics emerged aiming to disclose the dynamics of mitochondria under various pathophysiological conditions. In the present study we investigated the relative merits of gel-based (2DE and SDS-LC) and gel-free (2D-LC) protein separation approaches and protein identification algorithms (Mascot and Paragon) in the proteome profiling of mitochondria isolated from cultured fibroblasts, a sample traditionally used for diagnosis purposes. Combining data retrieved from 2DE, 2D-LC and SDS-LC and search methods, a total of 696 non-redundant proteins were identified. An overlap of only 19% between the proteins identified by the three different methods was observed when Mascot and Paragon were used. Regarding protein ID, a consistency in the number of identified proteins per sample was noticed for 2DE approach. Independent of the methodological approach chosen, it was noticed that the predominance in mitochondria of hydrophilic proteins with 20-50 kDa and pI 5-6 and 8-9; however, 2D-LC and SDS-LC allowed the enrichment of proteins with a mass below 30 kDa and of basic proteins with pI values above 8. In conclusion, data from the present study highlight the power of integrating different separation technologies and protein identification algorithms.

iTRAQ-based quantitative proteomic analysis of submandibular glands from rats with STZ-induced hyperglycemia.

The impairment of salivary glands activity is often connected to the complaints of dry-mouth and subsequent degradation of the periodontium of diabetic patients. In this context, submandibular glands (SMGs) play a central role in saliva production and so the understanding of the molecular pathways affected is of paramount importance. Using a streptozotocin-induced hyperglycemia rat model and two different time points (2 and 4 months), we applied mass spectrometry-based proteomic techniques, validated with standard western blot analysis, to identify and quantify the effect of chronic hyperglycemia on the proteome of SMGs. We observed significant variations of proteins such as kallikreins, protein S100A6 or annexins. After 2 months of hyperglycemia, we observed an early phase response characterized by a significant increase of protein S100A6, linked to the inflammatory response, together with the impairment of metabolic and energy production processes. On the other hand, vesicular transport appeared to be favoured in such conditions. Interestingly, in a long-term response to hyperglycemia after 4 months of exposure, we observed a general attenuation of the variations. In conclusion, we present data that support the existence of an adaptation of the gland to long-term stress.