The effects of high intensity exercise on lymphocyte DNA and antioxidant status in trained athletes.
Govender, Sumentheran Nadarajan.
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Apoptosis (programmed cell death) and exercise immunology have been the focus of research for the past five years. Trained athletes are particularly susceptible to a wide variety of viral and bacterial infections and this has been related to oxidative damage which is a mediator of apoptosis. Apoptosis, a normal physiological mechanism has also been implicated in the pathogenesis of a wide-variety of diseases. To date, the link between apoptosis and exercise has not been shown by established methods or ultrastructurally. The objective of the study was t.o determine the effects of a single bout of high intensity exercise on lymphocyte DNA and antioxidant status in trained athletes. The study was carried out in two phases. In the first phase, 11 trained athletes were subjected to a treadmill run to exhaustion using a ramp protocol to determine their maximum oxygen uptake (V02 max). Fifteen millimetres of blood was collected before exercise, immediately after exercise, 24 hours and 48 hours after exercise. Whole blood (4 ul) was used in the determination of DNA damage in lymphocytes using the single cell gel electrophoresis (SCGE) assay. The remaining blood was centrifuged and used for the following: Vitamin C concentration was determined by the 2,4 dinitrophenylhydrazine method, vitamin E concentration was determined by the High Pressure Liquid Chromatography (HPLC) method and lipid peroxides were determined by the measurement ofhydroperoxides. In the second phase, 3 trained athletes who had participated in phase 1, were subjected to a V02 max. test. Blood samples (10 ml) were collected before and immediately after exercise, 24 hours and 48 hours later. Lymphocytes were isolated using Histopaque 1077. An in situ cell death detection kit, Fluorescein was used for the detection and quantification of apoptosis in lymphocytes at a single cell level, based on labelling of DNA strand breaks. Analysis was carried out using flow cytometry. Lymphocytes were also prepared for Transmission Electron Microscopy (TEM) using conventional techniques. The results showed that immediately after exercise there was a non-significant decrease in vitamin C concentrations (p=o, 16), and a non-significant increase in vitamin E (p=0,82) and lipid peroxide concentrations (p=0,21). There was no significant difference in all 3 levels over the 48 hour period, when compared to the pre-exercise values. The SCGE assay revealed that the immediate post exercise samples showed DNA damage in lymphocytes of all subjects as evidenced by fluorescent strands of DNA outside the cell while DNA damage was observed in only one subsequent sample. In the pre-exercise samples, DNA was visualised as a central core, whereas in all samples taken after exercise, DNA was located at the periphery or confined to one pole of the cell. The pattern of DNA distribution seen in the SCGE assay over the 48 hour period were characteristic features of apoptosis. Flow cytometric analysis showed an increase in apoptosis in lymphocytes immediately after exercise with a further increase after 24 hours. After 48 hours the numbers decreased to control levels. TEM showed that majority of cells were normal before exercise while other lymphocytes were smaller with indented nuclei. Immediately after exercise the lymphocytes displayed features of indented nuclei and microsegregation, cell shrinkage, swelling of the endoplasmic reticulum, mitochondria and Golgi. These changes persisted after 24 hours but were not observed after 48 hours when most of the cells showed normal morphology. The ultrastructural changes observed were also characteristic features of apoptosis. These results suggest that high intensity exercise may cause an increase in apoptosis as evidenced by DNA damage in the SCGE assay and fully supported by the results achieved during flow cytometry and by the ultrastructural changes observed.