Allicin ameliorates some deoxynivalenol-induced cytotoxic effects in human embryonic kidney (Hek293) cells, but also elicits synergistic and potentiating adverse effects.

Abstract

Introduction: Deoxynivalenol (DON), a type B trichothecene produced by plant pathogenic fungi, especially Fusarium graminearum and F. culmorum, is a highly toxic mycotoxin found throughout South Africa. DON is consumed unintentionally through maize derived products and is rapidly becoming a potential health risk to humans and animals. It is a known immunosuppressant that induces apoptosis and oxidative stress and may cause liver lesions and kidney problems. Recently, dietary therapeutics have demonstrated a role against mycotoxin-induced cytotoxicity. Garlic (Allium sativum) is part of the Alliaceae family. The garlic bulb is used for medicine and as food consumption. The aqueous extract has recently demonstrated the potential to protect against mycotoxin-induced cell death and decrease reactive oxygen species (ROS). Aim: This study investigated the induction of apoptosis and oxidative stress by DON in Hek293 cells, and the ability of allicin to ameliorate these effects. Methods: Hek293 cells were treated with a range of allicin concentrations (0-150mM) over 24hrs. An EC50 of 1.7mM was obtained from the MTT assay and used in all subsequent assays. Hek293 cells were treated with 5μM DON, 1.7mM allicin (A), or a combination (DON+A) for 24hrs; untreated cells served as the control. Lipid peroxidation [malondialdehyde (MDA) and lactate dehydrogenase (LDH) assays] were used to indirectly quantify reactive oxygen species (ROS) and oxidative stress; reactive nitrogen species (RNS) were quantified using the nitrates assay. Apoptotic induction was determined by the detection of phosphatidylserine (annexin V) and DNA fragmentation. Necrotic cells were distinguished by propidium iodide uptake. Luminometric quantification of ATP, reduced glutathione (GSH), and caspase 9, 3/7, were used to verify these events. In addition, antioxidant enzymes protein expression of superoxide dismutase (SOD2), catalase and glutathione peroxidase (GPx1); as well as nuclear factor erythroid 2-related factor 2 (Nrf2) and heat shock protein (Hsp70), and apoptotic markers associated protein expression of p53, Bax, and poly (ADP-ribose) polymerase (PARP) were detected by western blotting. Results: DON-induced ROS production was suggested by the depletion of antioxidants including SOD2 (p < 0.0001), catalase (p < 0.0001) and GSH (p = 0.0886). Decreased lipid peroxidation indicated by the decreased MDA concentration (p < 0.0001) and reduced LDH (p = 0.0342) imply that the Hek293 cells were spared from the membrane-damaging effect of oxidative stress. A reduction in Hsp70 (p = 0.0056) and Nrf2 (p < 0.0001), and upregulation of GPx1 (p = 0.0362) protein expression was noted. In addition, increased nitrate concentration in all treatments compared to the control (p < 0.0001) suggested a shift to RNS production. Notably, allicin maintained Nrf2 protein expression similar to the control. The decrease in MDA concentration (p = 0.0109) by allicin was concurrent with depleted GSH (p = 0.0504)and increased SOD2, catalase and GPx1 (p < 0.0001), and suggests allicin induced an oxidative stress response. Allicin also protected DON-treated cells from oxidative stress by upregulating Hsp70 (p < 0.0001), catalase (p = 0.0006) and GPx1 (p = 0.0018), with concurrent decreased GSH (p = 0.0342) and ATP (p = 0.2028) concentration, which were also decreased by DON. In addition, allicin increased MDA (p < 0.0001) and LDH (p = 0.1267) towards control levels in the combined treatment. Apoptosis was reduced in the DON (p = 0.4631) and DON+A (p < 0.0488) treated cells in comparison to the control, necrosis was not evident in any treatment. The slight induction of p53 (p = 0.0008) and PARP-1 (p = 0.4036) by DON implies an attempt at DNA repair, but the Hek293 cells experienced reduced levels of apoptosis. Indeed, Bax expression was slightly reduced (p = 0.1071), caspases 9 (p = 0.0705) and 3/7 (p = 0.4431) activities were diminished, phosphatidylserine was not externalized, and PARP-1 was not cleaved. A non-fragmented DNA profile in allicin-treated and DON+A-treated Hek293 cells may be explained by increased expression of DNA repair proteins, PARP-1 (p = 0.0048 and p = 0.0004 respectively) and p53 (p < 0.0001). The upregulation of p53 is associated with an increase in Bax expression (p < 0.0001 and p = 0.0026 respectively). However, caspases 9 (p = 0.0596) and 3/7 (p = 0.0311) were not activated and apoptosis did not occur. Conclusion: DON treatment induced oxidative stress but not apoptosis in Hek293 cells at the concentration tested. In addition, its mechanism of toxicity in Hek293 cells appears to be more related to nitrosative stress and induction of DNA damage. Oxidative stress and not apoptosis is the possible mechanism of allicin-induced effects in Hek293 cells. Although allicin ameliorated some of the effects of DON in Hek293 cells, it also elicited synergistically or potentiating adverse effects that require further investigation.