Mitochondrial Critically Involved In MT-mediated Protection Against Dox-induced Cardiotoxicity

Doxorubicin (DOX) is an anthracycline antibiotic, it is one of the most effective andimportant anticancer agents widely used in the treatment of a variety of human cancers includingacute leukaemia and malignant lymphoma as well as a number of solid tumor. However,cardiotoxicity greatly compromises the clinical application of DOX. In the past years, severalmechanisms such as free radical hypothesis, calcium overload hypothesis, anthracyclinemetabolite hypothesis, histamine hypothesis have been proposed to account for DOX-inducedcardiotoxicity. Among these, the free radical hypothesis is the most popular one that has beenthoroughly investigated. DOX produces free radicals during its intracellular metabolismincluding hydroxide radical, superoxide anion and hydrogen peroxide. superoxide has beenreported to be the major oxidant that mediates DOX toxicity. These highly toxic free radicalsreact with various intracellular components such as lipids, protein and DNA, resulting inoxidative damages which appear to play the major role in the development of cardiomyopathy.Generally, the most important protective mechanism for heart is the antioxidant defense system.However, oxidative injury occurs when excessive free radicals were generated that exceeded theprotective capability of antioxidant defense system. Glutathione (GSH) is one of the maincomponents of cardiac antioxidant defense system and plays important role in protection theheart against oxidative attacks.Metallothionein (MT) is low-molecular weight protein found in most tissues of a widevariety of vertebrate and invertebrate species, Recent studies have indicated that MT activelyparticipate in an array of protective stress responses and could function as a potent free radicalscavenger. Many MT inducers have been found protect the heart from DOX-inducedcardiotoxicity. However, the mechanisms still remain obscure. Transgenic technology providesuseful mouse model for deep investigation of pharmacology and toxicology. Therefore, thisstudy was undertaken to investigate the effects and potential mechanisms of MT onDOX-induced cardiotoxicity using MT wild type mice (MT+/+) and MT-null (MT-/-) mice. 1. Role of mitochondria in MT-mediated protection against Dox-induced cardiotoxicityMitochondrial are believed to be the primary target and early indicator forDOX-induced cardiotoxicity. We hypothesized that MT plays a protective role fromDOX-induced mitochondrial injury. The present study was undertaken to investigate the effectsand potential mechanism of MT on DOX-induced cardiotoxicity and determine the distinct roleof mitochondria in that protective action. using wild-type (MT+/+) and MT-null (MT-/-) micewere randomly divided into four groups:5times group(NS+DOX),10times group (NS+DOX),5control group (NS+NS),10control groups(NS+NS). received0,2.0mg/kg Dox by intraperitonealinjections. Dox was administered twice a week for5wk for a total of10treatments. A2-wk"washout" period was allowed after the last treatment, Mice were then killed and cardiacspecimens were analyzed.morphological changes as examined by light microscopy and electronmicroscopy, myocardial lipid peroxidation. After5times chronic administration, myocardial celldamage observed only in DOX group MT-/-mice through pathological observation,there wasno obvious damage of DOX group MT+/+mice and the control group; Electron microscopyshowed the DOX group MT+/+and MT-/-mice are mitochondrial damage in MT-/-mousecardiac mitochondrial damage greater than MT+/+mice; of DOX group than the control group,mitochondrial MDA content increase in, GSHpx decreased activity, MT-/-mice than in the MT+/+mice changes more obvious pathological observation DOX group MT-/-mice myocardialcell morphology damage is more serious;10times after administration of DOX group MT+/+mice myocardialcell injury phenomenon also appeared; Compared to mice administered5times,the10times group observed more pronounced mitochondrial damage by electron microscope,mitochondrial MDA content and GSHpx activity decreased more.2. Protection Effects of Metallothionein and Dox-induced mitochondrial injurycharacteristicsMT+/+mice and MT-/-mice were randomly divided into two groups, namelythe control group and treatment group. Treatment group animals by a single intraperitonealinjection of DOX (15mg/kg), the control group was given the same dose of NS.4days afteradministration animals were killed and get the hearts. Identified Dox-induced cardiotoxicity byTTC staining; isolated mitochondria by differential centrifugation, mitochondrial lipid oxidation MDA and protein carbonyl product content to observe the myocardial tissue lipid oxidation andprotein oxidative damage; determination of the antioxidant glutathione (GSH)content andantioxidant activity of glutathione peroxidase (GSHpx); analysis of myocardial mitochondrialanti-oxidant defense enzyme manganese superoxide dismutase MnSOD and apoptosis-relatedsignaling factor of Caspase-3protein expression by Western blot.The results showed that thetreatment of acute administration of DOX group of mitochondrial oxidative damage, MDA andprotein carbonyl product content increase in the myocardial mitochondria; mitochondrialantioxidant GSH content decreased, the GSHpx activity; MnSOD expression was decreased andthe apoptotic factors caspase-3expression increased;...