Bull Vet Inst Pulawy 53, 713-717, 2009 OXIDATIVE STRESS-INDUCED APOPTOSIS IN RAT SKELETAL MUSCLES DURING A SINGLE RUNNING EXERCISE UP TO EXHAUSTION EUGENIA MURAWSKA–CIAŁOWICZ, LIDIA JANUSZEWSKA 1 , MARZENA PODHORSKA–OKOŁÓW 2 , AND PIOTR DZIĘGIEL 2 Physiology and Biochemistry Department, University School of Physical Education, 51-612 Wroclaw, Poland eugenia@awf.wroc.pl 1 Hygiene Department, 2 Histology and Embryology Department, Medical University, 50-368 Wroclaw, Poland Received for publication August 6, 2009 Abstract The aim of this study was to investigate whether the oxidative stress may be the reason for apoptosis in skeletal muscles in rats. Rats were divided into two groups – controls and exposed to physical exercise. Rats were running on the treadmill at the speed of 1km/h until exhaustion. After the exercise, the concentration of lipid peroxidation markers – malonylodialdehyde and 4- hydroxyalkenes (MDA+4-HDA), and the level of reduced glutathione (GSH) was determined in the homogenates of the extensor digitorum longus (EDL) muscle and slow-twitch (ST) fibres in 2, 6, and 96 h of restitution. Aconitase activity as a marker of oxidative protein modification was determined in ST fibres and EDL muscle. Additionally, apoptosis was detected by the TUNEL technique. A significant increase in MDA+4HDA concentrations in comparison to the control group was noticed in both ST fibres and EDL muscle after 6 h. GSH concentration in 2 and 6 h after exercise was significantly decreased in ST fibres and in EDL muscle in all measurements, when compared to the control group. Aconitase activity in ST fibres and EDL muscle was also significantly decreased 2 h after the exercise when compared to the control group, but increased in 6 h of restitution. Apoptotically-changed nuclei were observed only in EDL fibres. On the basis of the results and the suggested mechanism, it can be thought that the oxidative stress triggers apoptosis in ST fibres and in EDL muscle after exercise and it starts in the mitochondria. Key words: rats, exercise, apoptosis, oxidative stress, skeletal muscles. Reactive oxygen species (ROS) are produced only in small amounts in the resting organism. Intense physical exercise increases their production, among other things, due to increased supply of oxygen to working muscles, which in those circumstances can exceed approximately from 100 to 200 fold the resting values (3, 9). There are various sources of ROS in muscles during exertion but most important are increased leakage of electrons in the mitochondrial respiratory chain, where their intensified and accelerated production takes place. The damage of the cell and its death may be the consequence of increased ROS production (9). Until recently, necrosis was thought to be the only way of elimination as far as skeletal muscle fibres were concerned. Currently, it is known that they also can undergo apoptosis, which is programmed cell death. Apoptosis is a process characterised by specific changes at the molecular, biochemical, and morphological level. The process may be activated or inhibited by many factors. ROS are probably one of those factors (15). They break into macromolecule structures, block enzyme activity especially in mitochondria, and disturb energetic equilibrium of the cell, which may turn on the programmed death. Apoptosis in response to a variety of injurious stimuli has been demonstrated in several organs (13, 14, 16) but only few histological investigations confirm the appearance of apoptotically-changed nuclei in fibres of skeletal muscles (16). The mechanisms of apoptosis in skeletal muscles are not known. The knowledge concerning the mechanisms of apoptosis in mononuclear cells does not answer the question, which of those is triggered in polynuclear cells like muscular fibres? It seems quite probable that ROS itself may trigger many processes during intense contraction. Their extremely high production in mitochondria during physical exercise indicates the possibility of their contribution to the induction of the internal pathway of apoptosis, which has its origin in cell mitochondrium (10, 15). The aim of this research was to investigate whether oxidative stress may be the reason for the apoptosis in skeletal muscle fibres of rats exposed to a physical exercise. The other purpose of the study was to investigate whether there are any differences between