DIVERGING OXIDATIVE DAMAGE AND HEAT SHOCK PROTEIN 72 RESPONSES TO ENDURANCE TRAINING AND CHRONIC TESTOSTERONE PROPIONATE TREATMENT IN THREE STRIATED MUSCLE TYPES OF ADOLESCENT MALE RATS

Heat shock proteins (Hsp) play a critical role in maintaining cellular homeostasis. The diverse identified families of Hsp act as molecular chaperones or cochaperones and stress, sensors and bestow direct protection or rescue to the endangered or injured cells (1, 2). Exposure to various stressful conditions, e.g. hypoxia, hyperthermia, or oxidative or mechanical stress typically enhances expression of a variety of Hsp in diverse tissues and organs and may harden them to repeated environmental challenges (3-6). In striated muscles, the most common stressor is physical effort that primarily enhances the expression of the so-called inducible 72 kDa Hsp (Hsp72, also called Hsp70) (7-10). Increased Hsp72 expression may assure complementary protection against exercise-induced increase in oxidative stress (11). Diverse striated muscle types show differing Hsp72 responses to both heat stress and exercise training of various types and intensities (12, 13).

Testosterone and other anabolic-androgenic steroids (AAS) have been used for many years by athletes with the aim to increase muscle volume and strength (14, 15) or just to speed up bodybuilding (16). These effects correlate positively with AAS dosage over a wide range (17), which link promotes AAS abuse at high doses. AAS were also reported to improve submaximal running endurance in male rats when applied concurrently with endurance training (18, 19). Intriguingly, this was despite no difference in maximum oxygen consumption, running economy and red blood cell parameters between the trained rats given AAS and those given the respective vehicle (19). However, no positive effect of AAS on endurance performance was obtained in a human setting (20) and some other rat studies (21).

AAS abuse becomes increasingly popular in adolescents, mainly boys, who often combine it with physical training (15, 22, 23). However, the possible harmful health effects of this combination in the youth, in whom the related oxidative stress and endocrine phenomena may interfere with ongoing somatic growth and the still-developing pattern of sex steroids' milieu (24-26), were given little attention. This was despite both the observation that junior and senior subjects differ in their responses to oxidative stress in at least some types of endurance training (26, 27) and the decreasing age at which teenagers engage in competitive sports. Illicit testosterone supplementation is mostly used in combination with strength training or bodybuilding. Unfortunately, there is no rat model of strength training involving muscle masses comparable (in relative terms) to those engaged in such training in humans. However, endurance exercise is considered an important complement of strength training in some sport disciplines and is also recommended in modern bodybuilding programs (see http://www.bodybuilding.com/fun/wiggy1.htm or http://www.teenbodybuilding.com/locke17.htm). The main goal of this addition is to enhance oxidative capacity and promote body-sculpting by enhancing lipid metabolism. As the data on the effects of combining this exercise type with AAS abuse are scanty, we decided to test this combination in our studies.

We previously modeled (28) the effects of chronic testosterone treatment and endurance training on enzymatic and low-molecular weight nonenzymatic components of anti-oxidative stress myocardial defenses. The present study was aimed at typifying the effects of these factors on yet another line of cellular anti-stress defenses that relies on the expression of Hsp72. Hence, we compared the effects of these treatments on lipid peroxidation, content of the inducible Hsp72 and the expression of androgen receptor (AR) in selected striated muscles of adolescent male rats. While all these muscles are androgen targets well-known for their adaptation to exercise, they greatly differ in metabolic properties. Our choice was based on the fact that the heart left ventricle (LV) is of key importance for proper supply of blood for the working muscles during endurance exercise, and the soleus (Sol) and the extensor digitorum longus (EDL) represent, respectively, skeletal muscles that primarily use oxidative (aerobic) metabolism and those that rely predominantly on glycolytic (anaerobic) metabolism.

MATERIALS AND METHODS

Animals and experimental design

Five-week old male Wistar rats of 100–120 g initial body weight (b.w.) from the stock of the Mossakowski Medical Research Centre in Warsaw were used for the study. The rats were kept on a 12/12-h light/dark cycle (lights on at 7 a.m.) in a temperature (22–24°C) and humidity (45–65%) controlled room, with free access to standard rat chow and purified tap water. Before the start of training, all rats were tested on a motorized rodent treadmill to habituate them to this setting and eliminate rats reluctant to run (28). The preselected rats were randomized into six groups: 1) untrained (sedentary, UTr) controls, 2) untrained rats given 8 mg/kg b.w. of testosterone propionate (TP; Jelfa, Poland) weekly (Utr + TP8), 3) untrained rats given 80 mg/kg b.w. of TP weekly (Utr + TP80), 4) TP-untreated trained rats (Tr), 5) trained rats given 8 mg/kg b.w. of TP weekly (Tr + TP8), and 6) trained rats given 80 mg/kg b.w. of TP weekly (Tr + TP80). The basis for the choice of TP doses was given earlier (28). TP was diluted with sesame oil as needed to ensure equal injection volume for both dosages, and was given intramuscularly once a week, 1 hour after running session; TP-untreated rats received the same volume of the oil by this schedule. The rats scheduled for training were exercised on the treadmill (at 0° inclination) 5 days/week for 6 weeks, starting with a 40 min session at 16 m/min. Exercise duration and intensity were gradually increased over the first 4 weeks and then were kept steady at 60 min daily and 28 m/min. This training scheme has been shown by biochemical, morphological and performance measures to be very effective in terms of endurance improvement (29-31). Core body temperature (Tc) was measured in the trained rats just before and instantly after the last training session, using a flex-tip digital thermometer inserted 1.5–2 cm deep into the rectum; simultaneously, resting Tc was measured in their untrained counterparts. Two days after the session, all rats were anesthetized by intraperitoneal sodium pentobarbital injection (Vetbutal, Polfa, Poland; 50 mg/kg b.w.) and decapitated. Trunk blood was collected and processed for serum that was stored at –80°C for testosterone assay. Chests were opened instantly and the rats were perfused (for 3 min, at 25 ml/min) through the LV with glucose-supplemented (10 mM) O2/CO2 (95%/5%, v/v) mixture-saturated Krebs-Henseleit buffer. Then the LV, the Sol and the EDL from both hind limbs were dissected, snap-frozen in liquid nitrogen and stored at –80°C for Hsp72, AR gene expression and thiobarbituric acid-reactive substances' (TBARS) assays. The study protocol has been accepted by the First Local Ethics Committee for Animal Experimentation, Warsaw, Poland (Certificate of approval No. 251).

Biochemical assays

Tissue samples meant for Hsp72 assay by Western blotting were homogenized with an Ultra-Turrax homogenizer (IKA-Werke, Staufen, Germany) in 3 volumes of an ice-cold 10 mM Tris-HCl buffer (pH 7.5) containing 0.1 mM phenylmethanesulfonyl fluoride and 250 mM sucrose, and then were spun at 100 000 × g for 1 hour. The obtained cytosols were assessed for protein content and next were diluted 1:3 with sample buffer (200 mM Tris-HCl, 10% SDS, 30% glycerol, 0.2% bromophenol blue; pH 6.8) and heated for 4 min in a water bath (90°C). The denatured cytosols (25 µg protein/lane) were mixed with 10–180 kDa Prestained Protein Ladder (Fermentas, Cambridge, UK; 3 µl/lane) and loaded onto 10% acrylamide gel overlaid with 4% stacking gel. Electrophoresis was run at 15 V/cm for 90 min, using a 2 mM Tris, 192 mM glycine, 0.1% SDS buffer pH 8.3. Resolved proteins were transferred onto nitrocellulose membranes with the Mini-Protean 3 Cell/Mini Trans-Blot Module/PowerPac system (BioRad, Hercules, CA), at 4°C. Next, the membranes were blocked with 5% (w/v) solution of nonfat milk powder in Tris-buffered saline (TBS: Tris-HCl 1M, NaCl 2M, pH 7.4), rinsed for 1 min in TBS supplemented with 0.05% (v/v) Tween-20 (TBST), incubated overnight at 4°C with the anti-Hsp72 monoclonal antibody SPA-810 (Stressgen, Ann Arbor, MI) diluted 1:1000 with the milk-supplemented TBS, rinsed again (3 × 10 min) with TBST and incubated for 1 hour with a horseradish peroxidase-conjugated anti-mouse IgG (Vectastain ABC kit, Vector Laboratories Inc., Burlingame, CA) diluted 1:1000 with the milk-supplemented TBS. Then the blots were washed in TBST (3 × 10 min), developed in 3% H2O2/0.2 mM diaminobenzidine solution in 0.5 mM Tris-HCl buffer, pH 7.4, and quantified on a NucleoVision densitometer and the GelExpert 4.0 software (Nucleo-Tech Corp., San Mateo, CA). After the quantitation, the membranes were subjected to a standard stripping process, blocked again with the milk-supplemented TBS, and incubated overnight at 4°C with rabbit polyclonal anti-GAPDH antibody G9545 (Sigma) diluted 1:10,000 with TBST supplemented with 0.1% (w/v) BSA. Next, the membranes were incubated for 1 hour at room temperature with goat anti-rabbit IgG-horseradish peroxidase conjugate (diluted 1:8000 with TBST supplemented with 5% (w/v) nonfat milk powder). GAPDH-containing immune complexes were visualized using an ECL system and quantified with the NucleoVision densitometer and the Gel Expert 4.0 software.

AR gene expression was assessed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method. Total RNA was extracted from tissue samples with TRI-reagent (Sigma, St. Louis, MO, USA); contaminating DNA was digested with DNase I (Sigma). Reverse transcription of 2 µg of the DNA-free total RNA was run in a total volume of 20 µl using high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA), in one cycle (25°C for 5 min, 42°C for 45 min, and 85°C for 5 min, with subsequent cooling to 4°C). Multiplex RT-PCR was run in triplicates on a model 7500 real-time PCR system (Applied Biosystems) using rat AR-specific primers/probe set Rn00560747 m1, rat β-actin-specific ACTB endogenous control primers/probe set and TaqMan Universal PCR Master Mix (Applied Biosystems all), and 5 µl of the reverse transcription product, in a total volume of 20 µl. PCR conditions were as follows: 10 min denaturation at 95°C followed by 50 cycles of 95°C for 15 s and 60°C for 1 min. AR gene expression level was calculated using the standard curve method and normalized to actin-β expression.

TBARS were measured by the method of Ohkawa et al. (32), with minor modifications (28). Serum total testosterone (TT) level was assayed with an RIA kit (DSL-4100; Diagnostic System Laboratories, Webster, TX, USA), and tissue protein content was determined by the 2,2'-bicinchoninic acid method using the Sigma protein assay kit BCA1.

Statistical analysis

Data are shown as the mean ± S.D. All data were first tested for homogeneity of variance by the Levene test. TBARS and Hsp72 contents' data were acquired from differing rat numbers for the various muscles studied; hence they were then analyzed by a 3-way ANOVA with TP dose (0, 8, or 80 mg/kg weekly), muscle type (LV, Sol, and EDL) and training status (Tr or UTr) as the main factors, followed by the Tukey test for unequal sample sizes. AR expression data, which were acquired from all the three studied muscle types in each rat, were analyzed by repeated measures 3-way ANOVA with muscle type as the repeated measures factor, followed by the Tukey test for equal sample sizes. Since the 3-way ANOVA of AR data gave a significant muscle type × training × TP dose interaction effect, the significance of between-group differences was tested for each muscle type separately using a 2-way ANOVA followed by the Tukey test. TT levels and exercise-related changes in Tc were analyzed by a 2-way ANOVA with training status and TP dose as the main factors, followed by the Tukey test and the Student-Newman-Keuls test, respectively. Relationships between different variables were tested by Spearman's rank correlation test. In all cases, a p<0.05 was considered significant. All the analyses were performed using the Statistica 7.1 software (StatSoft, Tulsa, OK, USA).

RESULTS

Effects of testosterone propionate treatment and endurance training on blood serum total testosterone levels and weight of the selected striated muscles

Two-way ANOVA yielded significant effects of TP dose (F2,30=582.7, p<10-3) and training × TP dose interaction (F2,30=5.68, p=0.008), but not of the training (F1,30=2.67, p=0.11), on the TT levels. Blood serum TT levels were significantly and markedly higher in the TP-untreated endurance-trained rats than in their sedentary counterparts, while there was no difference in TT levels between trained and untrained rats given identical TP treatment (Fig. 1). Two-way ANOVA showed no effect of experimental manipulations on LV weight, whereas it yielded significant effect of the training on Sol weight and of the TP treatment on EDL weight. However, these main factor effects did not translate into statistically significant differences between the respective individual study groups (Table 1).

Fig. 1. Blood serum total testosterone level in sedentary (UTr) and endurance-trained (Tr) adolescent male Wistar rats given weekly intramuscular injections of testosterone propionate (TP8 8 mg/kg, TP80 80 mg/kg; 6 doses). The data were taken, under the Creative Commons Attribution limits, from an earlier work of the authors (28). For TP treatment, training and blood collection details see Materials and methods. *** p<0.001 vs. the respective TP-untreated group, # p<0.05 vs. the respective UTr group, ΔΔΔp<0.001 vs. the respective low-dose TP-treated group; the Tukey test. Numbers of rats are shown inside the respective bars.

Table 1. Effects of peri-adolescent testosterone propionate (TP) treatment and endurance training on the weight of selected striated muscles in young male Wistar rats.

Data are means ± S.D. (n); UTr - untrained rats, Tr - endurance-trained rats; TP8 8 mg of TP/kg b.w./week, TP80 80 mg of TP/kg b.w./week. Statistically significant values are shown in bold type.

Effects of testosterone propionate and endurance training on lipid peroxidation

Three-way ANOVA showed significant effects of muscle type, training and TP dose on TBARS content (F2,94=232.6, p<10-3; F1,94=48.0, p<10-3; and F2,94=163.6, p<10-3; respectively), and muscle type × training and muscle type × TP dose interactions (F2,94=3.60, p=0.031, and F4,94=22.6, p<10-3, respectively), but no significant training × TP dose (p=0.55) or training × TP dose × muscle type (p=0.10) interaction. Training alone significantly elevated TBARS only in the Sol, while TP treatment alone (at either dose used) showed much stronger effect, more than doubling TBARS content in the LV and Sol, and almost doubling it in the EDL. Increasing TP dose above 8 mg/kg b.w. did not translate into further significant increases in TBARS level in any muscle studied, either in untrained or in trained rats. There was no difference in TBARS level between the LV and Sol under any experimental condition, whereas the EDL showed significantly lower level of lipid peroxidation than the other studied muscles under all these conditions except in sedentary TP-untreated rats (Fig. 2).

Fig. 2. TBARS content in various muscles of sedentary (UTr) and endurance-trained (Tr) adolescent male Wistar rats given weekly intramuscular injections of testosterone propionate (TP8 8 mg/kg, TP80 80 mg/kg; 6 doses). For TP treatment and training details see Materials and methods. *** p<0.001 vs. the respective TP-untreated group; #p<0.05, ##p<0.01, ●●●p<0.001 vs. the respective Sol value; +++p<0.001 vs. the respective LV value. Numbers of rats are shown inside the respective bars. The LV data were taken, under the Creative Commons Attribution limits, from an earlier work of the authors (28).

Effects of testosterone propionate treatment and endurance training on heat shock protein 72 content

Three-way ANOVA showed significant effects of muscle type, endurance training and TP dose on Hsp72 content (F2,51=242.9, p<10-3; F1,51=3363.6, p<10-3; and F2,51=23.1, p<10-3; respectively), significant muscle type × training and TP dose × training interaction effects (F2,51=177.2, p<10-3, and F2,51=34.4, p<10-3), but not muscle type × TP dose (p=0.48) or muscle type × TP dose × training (p=0.22) interaction effect. Under basal conditions, Hsp72 level was but negligibly higher in the LV and Sol than in the EDL. Endurance training alone elevated Hsp72 relatively more in the LV and Sol than in the EDL (+528, +506 and +302%, respectively). TP treatment dose-dependently up-regulated Hsp72 in all studied muscles in sedentary rats, but not in their endurance-trained counterparts. Contrariwise, the lower TP dose slightly, but significantly, counteracted the effect of the training in the LV and EDL (Fig. 3).

Fig. 3. (a) Hsp72 contents in various muscles of sedentary (UTr) and endurance-trained (Tr) adolescent male Wistar rats given weekly intramuscular testosterone propionate injections (TP8 8 mg/kg, TP80 80 mg/kg; 6 doses). For TP treatment and training details see Materials and methods. * p<0.05, ** p<0.01, *** p<0.001 vs. the respective TP-untreated group; ###p<0.001 vs. the respective UTr group; Δp<0.05 vs. the respective low-dose TP-treated group; ●p<0.05, ●●●p<0.001 vs. the respective Sol value; +++p<0.001 vs. the respective LV value; the Tukey test. (b) Representative Western blots showing Hsp72 and GAPDH expression in the heart left ventricle (LV), in the soleus (Sol) and in the extensor digitorum longus (EDL) of TP-treated and -untreated sedentary and endurance-trained rats. Numbers of rats are shown inside the respective bars.

To verify the possibility that the changes in TBARS and Hsp72 contents may be related to experimentally induced changes in TT or mutually related, correlations between these variables were tested by the Spearman rank correlation test (Table 2).

Table 2. Spearman's rank correlation coefficients (R) between blood serum total testosterone (TT) level and the contents of thiobarbituric acid-reactive substances (TBARS) and Hsp72 in selected muscles of testosterone propionate-treated untrained and endurance-trained young male Wistar rats.

n - number of rats; statistically significant values are shown in bold type.

Effects of testosterone propionate and endurance training on AR mRNA level

Repeated measures 3-way ANOVA yielded significant effects of muscle type and training (F2,36=746.2, p<10-3, and F2,36=283.4, p<10-3; respectively), but not of TP dose (F1,36=2.62, p=0.12), and significant TP dose × training, muscle type × training, muscle type × TP dose and muscle type × TP dose × training interaction effects (F2,36=166.8, p<10-3, F2,36=9.47, p<10-3, F4,36=64.0, p<10-3 and F4,36=34.0, p<10-3, respectively). Due to the triple interaction effect, the data were next analyzed for each muscle separately. Two-way ANOVA gave significant TP dose and TP dose × training interaction effects in the LV (F2,18=5.32, p=0.015, and F2,18=9.12, p=0.002, respectively), Sol (F2,18=122.3, p<10-3, and F2,18=50.3, p<10-3, respectively) and EDL (F2,18=140.6, p<10-3, and F2,18=99.1, p<10-3, respectively), and significant training effect in the EDL only (F1,18=12.4, p=0.0025). AR mRNA level was markedly lower in the LV than in the Sol and EDL. Training alone significantly elevated it in all these muscles (+82%, +54% and +164%, respectively). In sedentary rats, low- and high-dose TP treatment, respectively, significantly increased and reduced AR mRNA in the Sol and EDL (+50% and 22%, and +65% and 53%, respectively), but showed no sizeable effect in the LV (+23% and 2%, respectively, pł0.82). In trained rats, both the low- and high-dose TP treatment significantly reduced AR gene expression in the Sol (26% and 75%, respectively), EDL ( 64% and 89%, respectively) and LV ( 48% and 45%, respectively); (Fig. 4).

Fig. 4. Androgen receptor gene expression in various muscles of sedentary (UTr) and endurance-trained (Tr) young male Wistar rats given weekly intramuscular testosterone propionate injections (TP8 8 mg/kg, TP80 80 mg/kg; 6 doses); N=4 for each group. For TP treatment and training details see Materials and methods. ** p<0.01, *** p<0.001 vs. the respective TP-untreated group; ##p<0.01, ###p<0.001 vs. the respective UTr group, ΔΔΔp<0.001 vs. the respective low-dose TP-treated group; ●●p<0.01, ●●●p<0.001 vs. the respective Sol value; +p<0.05, ++p<0.01, +++p<0.001 vs. the respective LV value; the Tukey test. Please note that the differences between the various muscles were tested for significance by the post-hoc test for 3-way ANOVA, whereas the between-group differences were tested for significance by the post-hoc test for 2-way ANOVA that was run for each muscle separately.

Effects of testosterone propionate treatment and endurance training on core body temperature

There was no difference in resting Tc between study groups (one-way ANOVA: F5,42=1.32, p=0.28). The final training session significantly elevated Tc in all endurance-trained rat groups. TP-treated rats showed significantly smaller rises in Tc (ΔTc) than their TP-untreated counterparts, and the rats treated with high TP dose showed larger than those given the lower dose (Table 3). Spearman rank correlation test yielded a moderate negative correlation between serum TT and ΔTc: R= –0.44, p=0.042 (n=22).

Table 3. The effects of 6-week testosterone propionate (TP) treatment and concurrent endurance training on core body temperature (ΔTc) in young male Wistar rats.

Data are means ± S.D.; UTr - untrained rats, Tr - trained rats; TP8 8 mg of TP/kg b.w./week, TP80 80 mg of TP/kg b.w./week; n/a - not applicable. *** p<0.001 vs. the respective resting Tc value, # p<0.05, ##p<0.01, ###p<0.001 vs. the respective TP-untreated group value, †p<0.05, ap=0.052 vs. the (Tr+TP8) group (the Student-Newman-Keuls test).

DISCUSSION

The data on the effects of physical training on antioxidant defenses and oxidative stress are numerous, but inconsistent (33, 34). This is probably due to disparities among various studies in training type and subjects' health, sex, age and hormonal/metabolic status. Notably, adolescent males experience a dramatic testosterone-related expansion of muscle size (35, 36). Our choice of endurance training was due to the fact that it plays a key role in many sports and gets more and more consideration as a health-promoting factor. The main findings of this study are as follows: 1) chronic TP treatment alone enhanced lipid peroxidation markedly more in the LV and Sol than in the EDL; 2) the training alone markedly elevated Hsp72 in all these muscles; 3) the TP treatment alone dose-dependently but slightly upregulated Hsp72 in sedentary, but not in endurance-trained rats; 4) the training alone enhanced AR gene expression in all studied muscles; 5) low-dose TP treatment alone enhanced AR gene expression in the Sol and EDL, whereas high-dose TP treatment alone suppressed it in the EDL, but not in the other muscles; 6) in the trained rats, chronic TP treatment dose-dependently suppressed AR gene expression in all these muscles.

Hsp72 is constitutively expressed at basal levels in all cell types as one of the so-called housekeeping proteins (37). Besides heat stress, the most important factor that enhances its production is oxidative stress (38). In skeletal muscles, Hsp72 upregulation is a typical response to contraction-linked energy and milieu challenges (13). The endurance training-related increases in Hsp72 levels found in the present study are in line with earlier reports (6, 11, 39-42) with some divergence mostly attributable to differences in the type, intensity and duration of the exercise or training.

The present data indicate that the key factor in Hsp72 upregulation in endurance-trained adolescent rats was the exercise-related increase in Tc. This is because: 1) TP treatment dose-dependently enhanced oxidative stress (as evidenced by increased TBARS content) in most studied muscles, but it counteracted the training-related upregulation of HspSP72 in these rats, as indicated by significant TP dose × training interaction effect (see also Fig. 3); and 2) the effect of TP treatment on Hsp72 contents roughly paralleled its effect on the exercise-related Tc increase (Table 3). Interestingly, some authors (42) reported a sizeable increase in heart Hsp72 level in adolescent male rats only after a relatively long (>12 weeks) period of endurance training, which suggests that the exercise-related increase in Tc may not be a dominant factor in Hsp72 upregulation in this organ. However, TP treatment-related increases in Hsp72 content were also found in all tested muscles of sedentary rats, with no change in resting Tc. This might suggest 'direct' enhancement of Hsp72 expression by testosterone, but this guess lacks a rationale or an experimental support. More likely, this effect was due to the TP treatment-related increase in oxidative stress. No added increase in Hsp72 content by this mechanism was apparent in endurance-trained rats despite significant elevations in TBARS. This could be due to a 'ceiling' effect of the training on Hsp72 content and/or masking of the effect of TP by the much stronger effect of the training. This possibility finds support in the training-related abolition of the correlation between serum TT and tissue Hsp72 levels (Table 2). Still, while the low TP dosage partly counteracted the effect of training on Hsp72 levels, the high-dose TP treatment showed no such action. This suggests a dual effect of TP treatment on Hsp72 expression: 1) an upregulation related to enhanced oxidative stress, and 2) an opposite effect consequent on the TP treatment-related suppression of exercise-related increase in Tc, of which the former apparently played a major role. The fact that the decrease was smaller after the high-dose TP treatment might be the consequence of markedly reduced AR gene expression. This explanation implies also that the effects of TP treatment on lipid peroxidation and Hsp72 expression are mediated by distinct mechanisms, as muscle TBARS contents showed a general tendency for elevation with increased TP dose.

The ΔTc-lowering effect of testosterone was also reported in castrated male Japanese quails (43) and in C57B1/6 mice (44). Altogether, these observations imply that this effect is only at play when Tc is elevated for some reason. Another explanation for the negative impact of high serum TT level on Hsp72 expression could be an AR-mediated suppression of hsp72 gene expression, which effect was found in rat hearts and cultured cardiomyocytes after hyperthermia followed by testosterone treatment (45). Interestingly, the training we used did not affect by itself the exercise-related rise in Tc while it markedly elevated circulating TT. We did not assess the effect of exercise on Tc in untrained rats in this study as this would bias baseline TBARS levels. Yet the exercise-related Tc in the UTr group rats (Table 3) did not significantly differ (P=0.10, two-tailed t-test) from that found in a group of age-matched naive male Wistar rats after a single session of treadmill running at the maximum duration and intensity used in this study (n=10, ΔTc ± S.D: 3.4±0.3°C; unpublished data from our lab).

A common feature of various heat shock factor-activating conditions, which activation is a crucial intermediate step in Hsp72 upregulation, is increased generation of reactive oxygen species (ROS) (46, 47) resulting, inter alia, in enhanced lipid peroxidation and TBARS formation. In this study, the major factor in the damage could be high serum TT level. Significant TP treatment-related elevation of TBARS in the Sol and LV, but not in the EDL, is in an apparent conflict with the known higher capacity of anaerobic muscle fibers for ROS production (48). One reason for the disparity between TP effects on TBARS level in the various muscles might be differences in muscle sensitivity to androgens. There is evidence that endurance training upregulates AR and thus may elevate the sensitivity in rat LV myocytes (49) and in the Sol, but not in the EDL (50). Other data contradict this possibility, see (51), and so do the results of the present study. First, the training alone enhanced AR gene expression in the EDL as well. Second, it was not the AR mRNA-rich EDL, but the AR mRNA-poor LV that showed marked TP treatment-related rises in TBARS. This suggests that AR is not a major determinant of striated muscle reactivity to androgens. Notably, AAS can act via nongenomic pathways that include both AR- and non-AR-mediated mechanisms (52-54). There is also evidence that not all muscle cells within a given striated muscle express AR, see (49) and the respective references therein. Hence, changes in AR expression may not be a reliable predictor of changes in muscle responsiveness to testosterone. There is also evidence that various skeletal muscles differ in the pathways that mediate a given response to androgens (54). However, the differences in TP effect on TBARS level in the various muscles might also be related to higher availability of unsaturated fatty acids that are the major substrate for TBARS formation (55) in aerobic compared with anaerobic muscles (56). Interestingly, as evidenced by TBARs titers, there was no support for a beneficial effect of endurance training on muscular lipid peroxidation in our study, whereas a circumstantial evidence for this effect was found in healthy young adult men (57). This may suggest an elevated risk of exercise-related oxidative damage in adolescence.

Testosterone can shift prooxidant/antioxidant balance toward the prooxidant state and increase lipid peroxidation in various AR-positive cells, including myocytes (58-60), but this effect may not be AR-mediated (61). It seems also doubtful to result from testosterone aromatization and the consequent stimulation of ROS formation, as it can be induced with the non-aromatizable androgen 5a-dihydrotestosterone (62) and the TP treatment caused no major elevation of circulating estradiol (28). On the other hand, the synthetic nonaromatizable AAS stanozolol that compared to testosterone shows low affinity for AR (63) and very low androgenic/anabolic ratio (15) was found to cause no increase in Sol and EDL TBARS contents when given at a dose of 2 mg/kg b.w., 5 days/week, for 8 weeks, to adolescent male rats (21). Whereas chronic TP treatment elevated TBARS considerably more in the LV and Sol than in the EDL, there were only minor differences in relative magnitudes of this effect for the two TP doses used. This may be due to the fact that the blood TT levels attained with either TP dose exceeded by an order of magnitude the rat AR dissociation constant (64), suggesting near-saturation of the receptor. More importantly, the effects of TP treatment on AR gene expression showed different dose-effect relationship in sedentary compared to endurance-trained rats, whereas there was no such difference in the respective changes in TBARS contents. This suggests that the effect of TP treatment on lipid peroxidation could be mediated by nongenomic pathways.

Our finding of positive association between tissue TBARS and serum TT levels confirms pro-oxidant action of elevated testosterone. AR is present in both cardiomyocytes (65) and in slow-twitch (aerobic) and fast-twitch (anaerobic) muscle fibers in rodents (54). Testosterone stimulates hormone-sensitive lipase and enhances lipolysis in rat cardiomyocytes (66) and likely also in the Sol, but less so in the EDL (67). The possible rise in fatty acids availability for ATP synthesis suggests increased oxygen use and enhanced ROS generation and oxidative stress. It is thus reasonable to assume that the latter effect would be more evident in aerobic (e.g. Sol) than in anaerobic (e.g. EDL) muscles (68).

The endurance training-related increases in muscle TBARS contents were relatively low in this study compared with studies on exercise-related oxidative damage in skeletal muscles of young adult rats (27, 69). This disparity may be due, at least partly, to differences in exercise/training protocols. In rats treated with the lower TP dose (8 mg/kgBW), the effect of endurance training might have been further obscured by the apparently near-maximal lipid peroxidation damage for a given muscle type. On the other hand, the significant TBARS-elevating effect of the increase in TP dosage, which was apparent in the LV and Sol, could be related to the aforementioned effect of testosterone on lipolysis and the resulting increased supply of substrate for lipid peroxidation. This possibility is supported by little impact of endurance training on the correlation between TBARS and serum TT in the LV and Sol that remained close to that found in sedentary rats (Table 2). The latter was not true for the EDL that has relatively small supply of unsaturated fatty acids (56), and thus endurance training alone might be sufficient for maximal enhancement of ROS formation and TBARS production in this muscle.

The question arises whether and which of the effects observed in our study could be related to the influence of endurance training on maturational development of the constituent fiber types in the various studied muscles, as such influence was observed in adolescent male mice (70) and the muscles considered in our study differed considerably in fiber type composition. Answering this question warrants an additional investigation.

In summary, the results of this study indicate that chronic treatment with suprapharmacological TP doses may exert, due to upregulation of Hsp72 expression, a protective effect on muscle cell proteins in sedentary, but not in endurance-trained adolescent male rats. The up-regulation of Hsp72 in sedentary male adolescent rats subject to high-dose TP treatment is stimulated by androgen-induced oxidative stress, but it seems unlikely that this effect results from AR-mediated direct genomic action of testosterone. Exercise-related heat stress, and not oxidative stress, is the main factor responsible for up-regulation of Hsp72 expression in the myocardium and skeletal muscles of endurance-trained and TP-treated adolescent male rats.

Acknowledgments: This study was supported by the grant #6P05A-086-21 from the State Committee for Scientific Research (Warsaw, Poland) and by statutory funds from the Jerzy Kukuczka Academy of Physical Education, Katowice, Poland.

Conflict of interests: None declared.

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