Oxidative stress parameters and anti-apoptotic response to hydroxyl radicals in fish erythrocytes: protective effects of glutamine, alanine, citrulline and proline

Abstract

The present study explored the protective effects of glutamine (Gln), alanine (Ala), citrulline (Cit) and proline (Pro) on hydroxyl radical (·OH)-induced apoptosis in isolated carp erythrocytes. Hydroxyl radicals were generated by ferrous ion (Fe(2+))-mediated decomposition of hydrogen peroxide (H(2)O(2)) (Fenton reaction). In order to select an optimal ·OH concentration to induce apoptosis, cultures were treated with different concentrations of FeSO(4)/H(2)O(2) (0 μM/0 μM-50 μM/25 μM). The results showed that exposure to FeSO(4)/H(2)O(2) (0 μM/0 μM-40 μM/20 μM) increased apoptosis in a dose-dependent manner. Moreover, apoptosis was at its highest level at 40 μM FeSO(4)/20 μM H(2)O(2). We then examined the cytoprotective effects of Gln, Ala, Cit, Pro or the combination of Ala, Cit and Pro under conditions of apoptosis. Carp erythrocytes were treated with the substances listed above in the presence of 40 μM FeSO(4)/20 μM H(2)O(2) for 9 h. The controls were grown in Gln, Ala, Cit, Pro-free culture medium. The results showed that Gln, Ala, Cit, Pro and the combination of Ala, Cit and Pro effectively protected against annexin binding, decrease of forward scatter and DNA fragmentation in carp erythrocytes induced by ·OH. Furthermore, Gln, Ala, Cit, Pro and the combination of Ala, Cit and Pro effectively blocked ·OH-stimulated erythrocyte hemolysis, reduced the increase of superoxide anion and H(2)O(2) concentrations, inhibited the formation of malondialdehyde, protein carbonyls and met-hemoglobin, and prevented the decrease of superoxide dismutase, catalase and glutathione peroxidase activities and glutathione content in carp erythrocytes induced by ·OH. In addition, the results suggest that the combination of Ala, Cit and Pro produces a greater anti-apoptotic and anti-oxidative effect than their individual effects at the same concentrations. Taken together, the results showed that ·OH induces apoptosis and oxidative damage in carp erythrocytes. In addition to inhibiting apoptosis, Gln, Ala, Cit, Pro and the combination of Ala, Cit and Pro protected carp erythrocytes against oxidative damage induced by ·OH, which may be a major factor in the protection of erythrocytes from apoptosis.