Effects of hydroxytyrosol dose on the redox status of exercised rats: the role of hydroxytyrosol in exercise performance

We aimed to describe the effects of a low and a high HT dose on the physical capacity and redox state of both sedentary and exercised rats. Our results suggest that 20 mg/kg/d of HT intake over 10 weeks may hinder training-induced physical capacity enhancement, whereas a dosage of 300 mg/kg/d HT does not. This effect may be related to changes in the systemic redox environment, as the 300 mg/kg/d dosage increases plasma HPx levels. All the supplemented groups (i.e., exercised and sedentary) showed similar weight gain, suggesting no evidence for potential toxicity for the doses used [28]. However, the lower levels of HGB together with the higher HPx levels reported in the EXE300 group should be further studied as they may reflect a harmful effect of the higher HT dose when combined with exercise.

The antioxidant capacity of the endurance exercise is well recognized. This systemic effect is achieved because within contracting muscles, there are multiple sites of ROS production, including the mitochondrial respiratory chain [34] and the plasma membrane [35]. These ROS act as signaling molecules that activate molecular pathways, which chronically lead to improvements in the function and content of endogenous antioxidants [15, 36]. It should be highlighted that the exercise protocol applied induces a strong antioxidant effect within skeletal muscles, which is reflected in blood plasma as a decrease in HPx concentration [29]. In this scenario, the loss of performance reported in the EXE20 group may be a consequence of the antioxidant effect of this dose. However, we have not found statistical evidence supporting that the 20 mg/kg/d dosage of HT has antioxidant effects. This occurred even though this dose was previously described as an antioxidant in rodents [21]. A plausible explanation is that the antioxidant effect induced by endurance exercise may mask any additional antioxidant effects induced by the low HT dose. Moreover, Feng et al. [20] showed that 25 mg/kg/d HT blunts the autophagic response of exercised rats. Importantly, the autophagic response to exercise is known to be ROS-dependent [37, 38]. Taken together, our results and those previously published suggest that dosages close to 20 mg/kg/d may hinder some exercise adaptations, probably due to their antioxidant effect.

An important finding of the present study is that 300 mg/kg/d of HT decreased plasma HPx concentrations in sedentary rats while it increased plasma HPx concentrations in exercised rats. It is unclear why this redox change toward a pro-oxidant effect occurs. However, a similar effect has been described for other polyphenols in vitro. As a by-product of their antioxidant activity, polyphenols can be oxidized [22, 24], and if the glutathione concentration is not high enough, the potential oxidative reactions may be focused on protein thiols [23]. Therefore, during the antioxidant activity of polyphenols such as HT, several metabolites are produced, and some of them may turn to pro-oxidant agents. This effect could be more easily manifested in the exercised rats consuming a high HT dose because exercise adaptations result are preceded by transient increases in oxidative stress. Indeed, it has been recently reported that subjects who show a higher oxidative response to a given training have a greater adaptation than those who show a lower level of oxidative stress [19]. Under this scenario, the polyphenol paradox may have increased the oxidative bursts for each training session and may explain the higher performance exhibited by the EXE300 animals.

It should be highlighted that we have not found evidence for mitochondrial redox alterations within exercised animals. Several potential explanations for the differences found between the plasma and mitochondrial compartments, especially for the EXE300 group, are suggested. First, mitochondria can alter their inner membrane structure in response to exercise in order to prevent their oxidative damage. Recent findings from our group showed that physical exercise stimulates the assembly of mitochondrial complexes in Supercomplexes (SCs), and prevents excessive ROS production [29]. Exercise can increase mitochondria resilience to excessive oxidative stress. In addition, previous studies on contracting muscles have reported that the main source of ROS production in response to contractile activity is the enzyme NADPH-oxidase, wich is mainly located in the sarcoplasmic reticulum [35]. Finally, data on other polyphenols suggest that plasma proteins and lipids can be rapidly oxidized in response to high polyphenol concentrations [39, 40]. Altogether, these data suggest that exercise training may adapt mitochondria to be protected from excessive oxidative stress. However, the high polyphenol dose in conjunction with the ROS produced by the extra mitochondrial enzymes may result in increases in circulating lipid peroxide.

Table 1 shows that sedentary animals consuming HT may undergo hematological dysfunction. However, Wistar rats are known to show high variability in response along hematological parameters. Previous studies on sedentary rats subjected to an intake of 25 mg/kg/d of the polyphenol quercetin showed lower hematological values than exercised rats, wich is consistent with the data of the present study. Indeed, HCT values below 35% and HGB values below 11 g/dL have been reported [41]. These results are consistent with the concept that polyphenols may interfere with circulating proteins [39, 40]. However, it seems that exercise can prevent such an effect but only when a low dose of HT is administered. In fact, we found a lower HGB concentration in the EXE300 group compared to the EXE20 group. Therefore, even though the EXE300 rats may have maintained their physical capacity, if HPx and HGB are considered in conjunction, these data may reflect the beginning of a harmful process. The optimal HT dose and the supplementation period required to maximize benefits (i.e., endurance performance) without inducing harmful effects must be elucidated in future studies. Furthermore, the high variability found in HGB and HCT data reported here and previously in response to polyphenol intake [41] suggests that Wistar rats may not respond homogenously to HT intake.