Mechanisms Of Antioxygen Protective Protein Metallothionein Protect Against DOX-induced Cadiocytotoxicity

Doxorubicin (DOX) is a highly effective anticancer drug that is frequently employed totreat hematological and solid tumors including leukemia, breast cancer, and soft tissuesarcomas. However, the clinical use of DOX is limited by its concurrent dose-dependentcardiotoxicity. It has been reported that the mechanism of Dox-induced cardiotoxicity ismainly attributed to the formation of reactive oxygen species (ROS) and promotion ofmyocardial oxidative stress. Metallothionein (MT) is a ubiquitous, low-molecular-weight,and thiol-rich protein which is highly inducible in response to oxidative stress. MT canfunction as antioxidant on scavenging free radicals though the precise biological function ofMT still remains indistinct and controversial. We previously found metallothionein-I/II(MT-I/II) null mice are more vulnerable to Dox cardiomyopathy, but it is unknown whetherdepletion of MT would sensitize cardiomyocytes to DOX toxicity in vitro. To this end and toexamine directly the effects of MT deficiency on Dox-induced toxicity in cardiomyocytes,the present study was undertaken to establish a primary cardiomyocyte culture system fromMT-I/II null (MT-/-) and corresponding wild type (MT+/+) neonatal mice, and to investigatethe possible mechanism of MT against Dox-induced cytotoxicity focusing on oxidativedamage.At first, MT concentrations in neonatal mice cardiomyocytes was determined by thecadmium-hemoglobin affinity assay to confirm the experimental model. It was shown thatMT concentrations in the MT-/- cardiomyocytes were about 2.5-fold lower than those inMT+/+ cardioymyocytes. Cardiomyocytes from MT-I/II null (MT-/-) and corresponding wildtype (MT+/+) neonatal mice were treated with DOX at concentrations of 0, 0.1, 1, 10μM,respectively, and then cardiomyocytes morphological alterations, LDH leakage, cell viability, beating frequency and apoptosis were observed. The results showed DOXconcentration-dependently induced cytotoxicity in neonatal mice cardiomyocytes, increasedLDH leakage and cell apotosis, decreased cell viability and beating frequency, all of theseeffect were much more servere in MT-/- cardiomyocytes.Then we observed DOX-induced cardiomyocytes oxidative stress by measuring thegeneration of intracellular ROS and the activity of antioxidant enzymes. It was observedthat DOX induced a concentration- and time-dependent increase in ROS production, whichwas exaggerated in MT-/- cardiomyocytes. Furthermore, the activity of antioxidant enzymesin 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, indicating that DOX induces more ROS accumulation and oxidative stressin cardiomyocytes when MT is deficient.It has been widely accepted that intracellular ROS accumulation may injury thebiological macromolecules, such as lipid, DNA and protein, which eventually leads to cellnecrosis as well as apoptosis. So we used TBA chromogenic reaction to measure the lipidperoxidation, used the comet assay and immunoperoxidase staining for 8-OHdG to detectthe DNA damage, and used the DNPH colorimetric test to determine the proteincarbonylation. It was showned that DOX induced a significant oxidative damage in bothMT+/+ and MT-/- cardiomyocytes, including lipid peroxidation, DNA damage and proteincarbonylation. Consistently, the damage to the MT-/- cardiomyocytes were more severe thanthe damage to the MT+/+ cardiomyocytes, which indicated that the protective effect of MT toDOX-induced cardiomyocytes injury may act by alleviating the oxidative damage.Furthermore, additional antioxidant N-acetylcysteine (NAC) and glutathione (GSH)significantly rescued MT+/+ but not MT-/-cardiomyocytes from DOX-induced cell death,ROS generation, and oxidative stress. These findings suggest that basal MT provideprotection against Dox-induced toxicity in cardiomyocytes, particularly highlight theimportant role of MT as a cellular antioxidant on scavenging ROS.It is now well recognized that mitochondrial dysfunction plays a crucial role in thepathogenesis of multiple cardiac diseases. So we used relevant fluorescent probe, such asMitoSox Red and Rhodamine123, to detect the ROS (O₂ -) generation and transmembranepotential in mitochondrion. The photofluorograms were then analyzed and showed as meanfluorescence intensity. The results showed DOX significantly increased mitochondrialproduction of O₂ -, while decreased the transmembrane potential of mitochondrion in bothMT+/+ and MT-/-cardiomyocytes, the latter were much more sensitive to these effect. We alsofound NAC and GSH could completely inhibit DOX-induced mitochondrial injury in MT+/+but not MT-/-cardiomyocytes, which indicated that the protective effect of MT to DOX-induced cardiomyocytes injury may act by alleviating the mitochondrial oxidativedamage.In summary, the present study clearly demonstrates that Dox-induced oxidativedamage are enhanced in the primary cultures of cardiomyocytes derived from neonatalMT-I/II null mice, and MT as a cellular antioxidant protected against these effect byscavenging intracellular and mitochondrial ROS.KeywordKeywords: Metallothionein, doxorubicin, MT-I/II null mice, cardiomyocytes, oxidativedamageSupported by grant of National Natural Science Foundation of China (30873130).