Peroxiredoxin Was Involved In Metallothionein Protection From Doxorubicin Cardiotoxicity

Doxorubicin (Dox) is an anthracycline antitumor antibiotic and one of the most potent drugs against a wide range of tumors. However, its clinical use was compromised by cardiotoxicity. Several mechanisms are proposed to be involved in doxorubicin-induced heart failure including DNA intercalation, free radical generation, and damage to cell membranes. Among possible mechanisms of doxorubicin-induced cardiotoxicity, oxidative stress has been considered to play an important role. Dox has a high affinity for myocardial cells, and specific accumulation in the heart. In biological systems, doxorubicin is known to produce free radical, resulting in oxidative damage in myocardial cells. There is ample evidence that increased oxidative stress, because of the doxorubicin-induced increase in the production of free radicals and the deficit of antioxidants, plays an important role in the development of cardiomyopathy and congestive heart failure. Therefore, finding an effective method to prevent and treat Dox cardiotoxicity has been a hot research in field of drug toxicology.Metallothionein (MT), a low-molecular weight, cysteine-rich, metal-binding protein. It has been reported that MT is an endogenous protective agent. It is highly inducible in biological systems under such stresses as the presence of heavy metals (Zn), starvation, heat, inflammation, or a diversity of pathological conditions can function as a potent scavenger of reactive oxygen species (ROS). It has a protective effect to resist the damage that caused by exogenous substances. In addition, studies have shown that metallothionein can protect myocardial cells from oxidative damage caused by Dox. Recently, the cardiac-specific MT-overexpressing transgenic (MT-TG) and metallothionein-Ⅰ/Ⅱ(MT-Ⅰ/Ⅱ) null mice provide two useful experimental models for the deep investigation of biological function of MT. Previous studies using a cardiac-specific MT-overexpressing transgenic (MT-TG) mouse model have demonstrated that metallothionein protects from doxorubicin-induced oxidative injury in the heart. Those from our laboratory revealed that MT-Ⅰ/Ⅱ-deficient mice are more sensitive to doxorubicin-induced acute cardiotoxicity. The expression of MT can be highly induced by zinc, by this way zinc protect the heart from the doxorubicin-induced oxidative injury. However, the specific mechanisms need to be further studied.Peroxiredoxins (Prxs) have received considerable attention in recent years as a new and expanding family of thiol-specific antioxidant proteins. It is a ubiquitous family of antioxidant enzymes that help to control intracellular peroxide levels. And the catalytic activity and protein subsequence is different from other antioxidant enzymes. Moreover, Prx-1,-2,-3,-5 and -6 proteins have been detected by immunoblotting in the whole heart of adult rodents. Using a proteomics approach we have observed changes in peroxiredoxin, an important redox regulating molecule, in Wild-type (MT+/+). Although several previous reports confirmed the anti-oxidant effects of peroxiredoxins in the hearts of adult rodents, no study has ever been conducted to specifically examine the molecular mechanisms and the link between metallothionein and peroxiredoxins. Therefore, the aim of this study was to investigate the relationship between metallothionein and peroxiredoxins after doxorubicin induced cardiotoxicity. This find may yield new insights in the treatment of cardiac oxidative stress.First, we assessed a link between metallothionein and peroxiredoxins in vivo. Wild-type (MT+/+) and MT-/- mice were treated intraperitoneally with doxorubicin at a single dose of 15 mg/kg and sacrificed on the 4th day after treatment. MT concentrations in mice heart was determined by the cadmium-hemoglobin affinity assay to confidence the animal model. Doxorubicin induced cardiotoxicity in both MT+/+ and MT-/- mice were manifested with increased serum creatine kinase (CK) and lactate dehydrogenase (LDH) activities, and cardiac morphological changes. These toxic responses were stronger in the hearts of MT-/- mice that were more vulnerable to doxorubicin-induced oxidative injury as exhibited by increased lipid peroxidation and decreased catalase and glutathione peroxidase (GSH-Px) expression. Moreover, in the MT-/- mice, the deficiency of metallothionein inhibited the expression of Cu/Zn Superoxide dismutase (SOD-1) induced by doxorubicin. Doxorubicin significantly increased the mRNA levels and protein expressions of Prx-1,-2,-3,-5, and -6 in the hearts of wild-type but not MT-/- mice. Therefore, the present study suggests that metallothionein provides protection against doxorubicin-induced cardiotoxicity, which possibly involves regulation of peroxiredoxins.Secondly, in order to further investigate the possible mechanism of MT against Dox-induced cytotoxicity, the present study was undertaken to establish a primary cardiomyocyte culture system from MT-Ⅰ/Ⅱnull (MT-/-) and corresponding wild type (MT+/+) neonatal mice, and pretreated the cardiomyocyte from wild type (MT+/+) neonatal mice with zinc to establish the cardiomyocyte model with different expression level of MT to investigate the protective effect of MT against Dox-induced cardiac injuries as well as the involved possible mechanisms. MT concentrations in neonatal mice cardiomyocytes was determined by the cadmium-hemoglobin affinity assay to confirm the experimental model. It was shown that zinc administration increased the cardiomyocytes metallothionein concentration (4-fold) in the MT+/+ cardiomyocytes. Cardiomyocyte were treated with Dox at concentrations of 1μM, Dox caused remarkable cytotoxicity in neonatal mice cardiomyocytes, and demonstrated by the increase of LDH leakage and apoptosis. All of these effect were much more servere in MT-/- cardiomyocytes. Zinc pretreatment significantly increased the cardiomyocytes MT levels and inhibited the cardic toxic effects of Dox only in MT+/+ cardiomyocytes, but not in MT-/- cardiomyocytes. Then we observed Dox-induced cardiomyocytes oxidative stress by measuring the generation of intracellular ROS and the activity of antioxidant enzymes. It was observed that Dox induced increase in ROS production, which was exaggerated in MT-/-cardiomyocytes. Furthermore, the activity of antioxidant enzymes in both two type cardiomyocytes, such as superoxide dismutase (SOD) and catalase (CAT), were decreased after treatment with Dox. These effect was also much more servere in MT-/- cardiomyocytes, and in the group of Zn pretreatment cardiomyocytes can resist these changes, indicating that Dox induces more ROS accumulation and oxidative stress in cardiomyocytes when MT is deficient. It has been widely accepted that intracellular ROS accumulation may injury the biological macromolecules, such as lipid and DNA. So we used TBA chromogenic reaction to measure the lipid peroxidation, used the comet assay to detect the DNA damage. It was showned that Dox induced a significant oxidative damage in both MT+/+ and MT-/ cardiomyocytes, including lipid peroxidation and DNA damage. Consistently, the damage to the MT-/- cardiomyocytes were more severe than the damage to the MT+/+ cardiomyocytes, and Zn pretreatment can prevent these damage, which indicated that the protective effect of MT to Dox-induced cardiomyocytes injury may act by alleviating the oxidative damage. Doxorubicin decreased the protein expressions of Prx-1,-2,-3 an d-6 in the cardiomyocytes. And in MT-/- cardiomyocytes were significantly lower than those in MT+/+ cardiomyocytes treated with doxorubicin. Zn induce the expression of Prx-2,-3,-5 and -6, and moreover, it can inhibit the expression of Prxs decrease in the MT+/+ cardiomyocytes, but had no effect in MT-/- cardiomyocytes. From the above result, we speculated that the expression of Prxs was partially affected by the expression level of MT.Finally, we produced oxidative stress by glutathione depletion with BSO, and use Trolox to protect the cardiomyocytes from ROS damage and reduce the oxidative stress. In these two different oxidation states we studied the interaction between MT and Prxs after doxorubicin induced cardiotoxicity. From the results, we can show that Trolox can inhibit the Dox-induced cardiotoxicity, it can remove the ROS, inhibit cardiomyocyte apoptosis and DNA damage. However, BSO trough decreased cellular GSH secondarily reduce GSH-Px activities. Dox-induced cardiotoxicity, as measured by the generation of ROS, cardiomyocyte apoptosis and DNA damage, was dramatically increased in the BSO-treated cardiomyocytes. Trolox abolish the changes of the Prxs expressions induced by Doxorubicin in MT+/+ cardiomyocytes, but had no effect in MT-/- cardiomyocytes. Compare with Dox group, BSO exposure caused a more dramatic reduction of Prx-5 and -6 in MT-/- cardiomyocytes. But In MT+/+ cardiomyocytes, except for Prx-6, the reduction of intracellular glutathione had no significant effect on sensitivity to expression of other Prxs.In summary, the present study clearly demonstrates that metallothionein provides protection against doxorubicin-induced cardiotoxicity, which possibly involves regulation of peroxiredoxins.