In vitro effects of 1-(4-dimethylaminobenzylidene)-2-(2-hydroxybenzylidene) hydrazine and its copper complex on lymphocyte proliferation and function in the presence of free radical generator

Abstract

In this study, the in vitro effects of 1-(4-dimethylaminobenzylidene)-2-(2-hydroxybenzylidene) hydrazone (L₁) and its corresponding copper complex [Cu(L₁)], synthesized in our laboratory, were investigated on the proliferative responses, Th1 (interleukin-2 (IL-2), interferon-γ (INFγ)) and Th2 (IL-4) cytokine secretion, adenosine triphosphate (ATP) levels and intracellular redox status of T lymphocytes submitted to H₂O₂/FeSO₄-mediated oxidative stress. T cells were isolated on histopaque density gradient by differential centrifugation, and were cultured with the mitogen concanavalin A (Con A), free radical generator (H₂O₂/FeSO₄) and with different concentrations of L₁ and [Cu(L₁)] (1 - 100 μM). Proliferation (MTT assay), cytokines (Elisa kits), ATP levels, cytotoxic effect (micronucleus test) and oxidative markers (glutathione, catalase, superoxide dismutase, hydroperoxide and carbonyl protein contents) were investigated after 48-h incubation. Our results showed that H₂O₂/FeSO₄ treatment induced a reduction in T lymphocyte proliferation, cytokine secretion and ATP levels associated to an evident intracellular oxidative stress, inflammatory profile and DNA damage. Addition of L₁ at 100 μM was able to increase cell proliferation, IL-2, IL-4 and INFγ secretion and ATP contents and to reduce hydroperoxide and carbonyl protein contents, catalase activity and micronuclei number in lymphocytes under oxidative stress, with a partial protection. The [Cu(L₁)] exhibited protective effects in T lymphocytes by inhibiting H₂O₂/FeSO₄ - induced cell proliferation suppression, inflammatory status, ATP loss and oxidative stress generation, whatever the concentration used. In conclusion, in the situation of excessive oxidative stress, [Cu(L₁)] treatment improved T lymphocyte proliferation, cytokine production, ATP contents and oxidant/antioxidant status. [Cu(L₁)] could be effective at improving oxidative stress and T cell abnormalities.