This is one of the first studies to demonstrate a possible antioxidant effect of creatine supplementation either in association or not with an RT protocol. It is also one of the few studies to elucidate the antioxidant effect paradigm of creatine in vivo.
In our study, after 8 weeks of RT in squat apparatus adapted for rats, a significant increase in the maximum strength was observed in all groups. However, the strength was higher in the trained group supplemented with creatine. Similar results were observed in other studies that evaluated the gain of maximum strength in humans [26–28]. Although it has not been evaluated in the present study, the muscular content of free creatine and creatine-phosphate storage appear to contribute to an increase in the maximum strength of creatine supplemented individuals submitted to the RT protocol, as demonstrated by Buford and colleagues in 2007 .
In the present work, a lower plasmatic lipoperoxidation, evaluated by MDA, was only observed in those groups which received creatine supplementation. These results are in contrast to those found by Kingsley and colleagues , who sought to evaluate serum lipid peroxidation in humans submitted to two tests until exhaustion using a cycloergometer and did not observe any influence from creatine supplementation on this marker of oxidative stress. In the previous study, the volunteers were physically active and only familiarized to an exhaustive exercise protocol. However, in our work, we submitted the animals to a resistance training program which led to different muscular and metabolic adaptations. Deminice and colleagues  evaluated the acute effect of creatine supplementation for 7 days on plasma oxidative stress in humans submitted to sprint exercise; no antioxidant effect was observed. The divergence from the results presented here might be explained by the different types of exercise, such as the hemodynamic response and the predominant energetic metabolism related to resistance exercise compared to that reported for sprinting or cycling. In another study, rats submitted to 1-h of swimming (load of 4% of body weight) and supplemented with creatine (2% of diet) for a period of 28 days, showed a reduction in plasmatic TBARS immediately after exercise, and 2 h and 6 h after the swimming exercise . It is possible that a longer loading phase of creatine supplementation can increase the antioxidant status, rather than a shorter period of loading. However, when it is associated with a training regimen, higher effects were observed for plasma lipoperoxidation . Interestingly, similar results were observed in the present study. In this way, this antioxidant effect of creatine supplementation associated with RT in plasma oxidative stress corroborated our findings.
Since the SED-Cr group presented a reduction in plasma activity of SOD enzyme and lower lipoperoxidation, it is possible that the creatine may have acted as an ROS scavenger. In the same way, supplemented groups showed no increase in CAT activity; this only occurred in the group submitted to RT. CAT is an enzyme that is highly modulated by physical training, especially by endurance training, where the formation of ROS by the leakage of superoxide radicals in the electron transporter chain is much higher due to the greater utilization of the oxidative pathway [33–35]. Since, in our results, plasmatic CAT activity was higher in the RT group, it is possible that it is necessary to increase this antioxidant enzyme (due to the lack of non-enzymatic antioxidants like creatine) in order to reduce the plasma lipoperoxidation in this group.
Creatine has been considered a cytoplasmic antioxidant of direct action that would mainly promote the scavenging of ROS superoxide radicals . Recently, Lygate and colleagues  sought to assess a possible protective effect of creatine in the ischemia-reperfusion process in mice submitted to acute myocardial infarction. The cardiomyocytes were exposed to an oxidant agent, H2O2, in order to evaluate the antioxidant action in the fluorescent pigment. Creatine treatment was not able to attenuate the damage promoted by H2O2.
However, in our work, a reduction of lipoperoxidation was demonstrated in the heart tissue of creatine supplemented groups, suggesting a possible direct antioxidant effect in the suppression of superoxide radicals in the heart, probably due to its chemical affinity as an antioxidant, being higher with this specific ROS [5, 6].
An interesting finding of our study was that, in the heart, SOD activity was reduced in the sedentary group that was supplemented with creatine, in comparison to both the control group and the RT creatine supplemented group. This was in accordance with Siu and colleagues , where low intensity exercise (walking) for 8 and 20 weeks was not able to increase SOD activity in the heart of rats. Resistance exercise is characterized by a pressure overload in the heart during its execution, causing an increase in cardiac muscle mass . This suggests that, in part, the RT-Cr group increased SOD activity as an adaptive response to a higher formation of anion superoxide in this tissue under physical training conditions, and that the increased production of this ROS occurs through the xanthine oxidase pathway [40, 41]. Creatine supplementation may have exerted a synergistic effect with RT in relation to SOD activity modulation in the heart. In chronic-progressive stress conditions, and in RT, supplementation appears to exert a synergistic effect with regard to adaptation to RT with creatine supplementation, involving the cellular signaling enzymatic adaptation of SOD in cardiac tissue. This mechanism occurs via activation of the NAD(P)H oxidase system that, through vasoactive (angiotensin II) and inflammatory mediators (IL-6, TNF-α), modulates the expression of antioxidant enzymes in a short period [42, 43].
CAT activity in cardiac tissue seems to be modulated by the interaction of creatine supplementation with RT, as observed by McClung and colleagues , who evaluated the effect of the association of creatine with high intensity exercise on cardiac function in rats and found that this interaction was able to up-regulate the cardiac functional capacity. These results indicate a possible direct or indirect enzymatic modulation of creatine in synergism with training.
As creatine is not synthesized exclusively in the kidney and in the pancreas, but at higher proportions in the liver, and is then mainly transported to the skeletal muscle, we investigated the liver with the aim of developing a hypothesis about the redox state of this organ in the presence of supplementation, either associated or not with resistance training. Our results are different to those found by Radak and colleagues , who reported an attenuation of lipoperoxidation levels in the animals submitted to treadmill running training which was adapted for rats. The difference in training protocols, age and animal species may have directly influenced the difference between the results obtained and those of our study.
Studies that have evaluated the effect of creatine supplementation on oxidative stress in different structures are very limited. In our study, we found lower SOD activity in both heart and liver. Botezelli and colleagues  evaluated lipid peroxidation, SOD and CAT activity in the liver following three different training protocols (aerobic, strength and concurrent). However, the training did not have any influence on antioxidant enzymatic activity. Creatine seems to have the same response in different tissues, since the increased production of ROS and RNS at the expense of strength exercise possibly acted upon cellular signaling to increase antioxidant enzymatic defenses .
When we analyzed the lipoperoxidation in skeletal muscle, we observed that only the RT-Cr group showed lower oxidative damage compared to the SED group. Similar results were found by Guimaraes-Ferreira and colleagues , since creatine supplementation associated or not with RT did not change the CAT and SOD activity in skeletal muscle. In this tissue, creatine seems to exert a scavenging antioxidant effect and does not act as an antioxidant enzymatic activity modulator. In a model of spontaneously hypertensive rats submitted to a creatine supplementation protocol, it has been demonstrated that this supplementation does not promote the attenuation of oxidative stress in skeletal muscle .
Lastly, this was one of the first studies to evaluate the effects of isolated creatine supplementation or that associated with RT on oxidative stress. As a limitation of this work, it can be noted that a few antioxidant enzymes (e.g. glutathione peroxidase, glutathione reductase, peroxiredoxin), non-enzymatic antioxidants (e.g. glutathione, GSH/GSSG ratio, total antioxidant capacity), biomarkers of oxidative damage (protein carbonyl, 8-OH-dG) and/or activity of ROS and RNS were not analyzed, but this could clarify certain results obtained in the present study.