Protections Of Metallothionein Against Butenolide-Induced Oxidative Damage

Butenolide (BUT), a water-soluble Fusarium mycotoxin distributing extensively, is a potential heath threat to humans. It has been considered that the contamination of human foodstuffs by Fusarium mycotoxins is correlated with the endemic diseases, Kashin-Beck disease and Keshan disease, and BUT is frequently detected from the cereal grains in endemic areas. Previous studies indicated that BUT, both in vivo and in vitro, could induce hepatic lipid peroxidation, and also induce myocardial oxidative damage manifested by significant lipid peroxidation in myocardial homogenate and cardiomyocytes.Metallothionein (MT) is low-molecular weight protein found in most tissues of a wide variety of vertebrate and invertebrate species, and characterized by their high content of cysteine residues which mainly bind with bivalent metal ions. There are four isoforms of MT in mammalian including MT-I, MT-II, MT-III and MT-IV, of which MT-I/II are the main isoforms. It has been confirmed that MT plays an important role in detoxifying against harmful heavy metals, scavenging oxygen free radicals, anti-oxidative stress, anti-apoptosis and stabilizing biomembranes. Many studies have shown that MT can provide effective protections against oxidative damage induced by free radicals. Therefore, the present study is undertaken to explore whether MT provides protections against BUT-induced hepatic oxidative damage by using two animals, MT-null (MT-/-) mice and the corresponding MT wild type (MT+/+) mice. In addition, the protective effects of MT on the myocardial toxicity and their potential mechanisms were studied using primary cultures of cardiomyocyte of MT+/+ and MT-/- miceThe present study will further reveal the toxic mechanisms of BUT, and provide useful information for prevention and treatment of BUT toxicosis.1. Protections of MT against BUT-Induced Oxidative Damage to Hepatic Tissues of MT+/+ Mice and MT-/- MiceLiver homogenates of MT+/+ mice and MT-/- mice were incubated in vitro with 0-200μg/ml of BUT for 60 min, respectively, and lipid peroxidation was measured using malondialdehyde (MDA) level as a marker. The results showed that BUT induced increases of MDA level in the liver homogenates of both MT+/+ mice and MT-/- mice in a concentration-dependent manner; and the comparisons indicated that the MDA levels in MT-/- mice were significantly higher than those of MT+/+ mice following relatively high concentrations of BUT exposure. For example, incubation of liver homogenates with 200μg/ml of BUT for 60 min, the MDA level in MT-/- mice was about 1.4-fold as that of MT+/+ mice.Liver homogenates of MT+/+ mice and MT-/- mice were incubated with 100μg/ml of BUT for 0.5-4 h, respectively. For 0-2 h of incubation, the MDA levels were increased in an evident time-dependent manner. Following 4 h incubation, the MDA levels in the liver homogenates of MT+/+ mice and MT-/- mice were both reduced. At 0 h, there were no significant difference in MDA levels between MT+/+ mice and MT-/- mice. At 0.5, 1, 2 and 4 h following incubation, the MDA levels in MT-/- mice were about 1.5, 1.42, 1.62 and 1.64-fold as those of MT+/+ mice, respectively.Liver homogenates of MT+/+ mice and MT-/- mice were incubated with a series of concentrations of BUT (0-200μg/ml) for 60 min, the antioxidant capacity was then determined. BUT induced concentration-dependent reductions in the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). By the normalization comparisons, the activities of SOD and GSH-Px in the liver homogenate of MT-/- mice were lower than those of MT+/+ mice. With regard to 200μg/ml of BUT, the activities of GSH-Px in the liver homogenates of MT+/+ mice and MT-/- mice were inhibited by 30% and 70%, respectively, and the GSH-Px activity of MT+/+ mice is2.33 times as that of MT-/- mice; the activities of SOD in MT+/+ mice and MT-/- mice were reduced by 13% and 21%, respectively.BUT induced rapid concentration-dependent depletions of total sulfhydryls (TSH) and non-protein sulfhydryls (NPSH) in the liver homogenates of MT+/+ mice and MT-/- mice. There were significant differences as compared to control values, and the change of NPSH content was particularly obvious. As for 200μg/ml of BUT, the contents of NPSH in the liver homogenates of MT+/+ mice and MT-/- mice were decreased by 95.2% and 97.6%, respectively. By the normalization comparison, there was no significant difference in the contents of TSH between MT+/+ mice and MT-/- mice, whereas the contents of NPSH did. With the concentration of 200μg/ml of BUT, the remaining NPSH in MT+/+ mice is 2.23 times as that of MT-/- mice.BUT at concentrations of 100 and 200μg/ml induced significant increases of nitric oxide (NO) production in the liver homogenates of both MT+/+ mice and MT-/- mice (P<0.01). By the normalization comparison, the content of NO in MT-/- mice was higher than that of MT+/+ mice after exposure to 100 and 200μg/ml of BUT (P<0.05). For the incubation of 200μg/ml of BUT, the contents of NO in the liver homogenates of MT+/+ mice and MT-/- mice were increased by 2.6 and 4.6 times, respectively, and NO content in MT-/- mice is 2.23 times as that of MT+/+ mice.In summary, BUT can induce significant hepatic oxidative damages through breaking the balance of oxidant-antioxidant system, characterized by inhibition of SOD and GSH-Px activities, depletion of TSH and NPSH, increases of MDA and NO contents. MT provides significant antioxidant protections, manifested by enhancement of SOD and GSH-Px activities, decrease the depletion of NPSH and inhibition of lipid peroxidation.2. Protections of MT against BUT-induced Oxidative Damage to Primary Cultures of Cardiomyocytes of MT+/+ mice and MT-/- miceThe primary cultures of cardiomyocytes of MT+/+ mice and MT-/- mice were incubated with 0-64μg/ml of BUT for 12 h. Microscopic observation showed that BUT induced significant morphological changes including cellular swelling, vacuolation, breakage of muscle fibers, nuclear condensation, and coagulative necrosis, all of which are similar to the myocardial damage in Keshan disease patients.BUT induced cell viability decreases in a manner of concentration- and time-dependent. There was significant difference in the viability of cardiomyocytes between MT+/+ mice and MT-/- mice (P<0.01). With cardiomyocytes exposed to 0-32μg/ml of BUT for 12 h, the releases of LDH into the supernatant for MT+/+ mice and MT-/- mice were not different as compared to the controls (0μg/ml), respectively.In order to investigate the oxidative damage of BUT on the cardiomyocytes, as well as the potential protections of MT against BUT-induced cardiotoxicity, intracellular reactive oxygen species (ROS) production and DNA damage were evaluated using DCF-DA fluorescent probe labelling and single cell gel electrophoresis (SCGE), respectively. Results showed that BUT exposure obviously increased ROS production of cardiomyocytes of both MT+/+ and MT-/- mice, which was significantly different from controls, respectively (P<0.05); The normalization comparison indicated that ROS production in MT-/- mice was more than that of MT+/+ mice. SCGE assay revealed that BUT induced DNA damage in the cardiomyocytes of both MT+/+ mice and MT-/- mice in a concentration-dependent manner, and the normalization comparison showed that DNA damage in MT-/- mice was more serious than that of MT+/+ mice. The finding suggested that intracellular antioxidant system could not effectively scavenge ROS, and excessive ROS could cause oxidative damage to DNA of cardiomyocytes. Results also manifested that MT efficiently reduced intracellular ROS and alleviated DNA damage on cardiomyocytes.In conclusion, the present study demonstrated that BUT induced significant oxidative damage, whereas MT could provided potent protections through scavenging ROS overproduction, improving antioxidant capacity and relieving oxidative damage of cellular macromolecules. These findings may conduce to reveal the mechanisms of BUT toxicity and prevent BUT toxicosis.