Mechanisms involved in mitoquinone-mediated protection of H9C2 cells against anti-cancer drug doxorubicin-induced cardiotoxicity

INTRODUCTION: Due to cardiotoxicity, doxorubicin (DOX), a chemotherapeutic agent, has limited use. Our lab has shown that Mitoquinone (MitoQ) given as a pre-treatment exerted better cardioprotection against DOX-induced cell damage than co-treatment. OBJECTIVE: This study investigated the underlying mechanisms associated with pre-treating cardiomyocytes with MitoQ and why it demonstrated more potent protection against Doxorubicin-induced cardiotoxicity when compared to co-treatment. METHODS: Cultured H9c2 cells were dose-dependently treated with DOX alone, MitoQ alone, MitoQ given concurrently with DOX (i.e., co-treatment), or MitoQ given 24 hours prior to DOX (i.e., pre-treatment). Various factors were explored such as cell viability via CCK assay, DOX accumulation, mitochondrial superoxide anions levels measured by MitoSOX assay, and mitochondrial membrane potential (MMP) as measured by JC-10 assay. Western blotting was used to measure the expression of antioxidant enzymes heme oxygenase 1 and superoxide dismutase 1. RESULTS: DOX (.5-50 μM) dose-dependently increased mitochondrial superoxide levels, and reduced cell viability. DOX also reduced the MMP, but demonstrated a plateau effect (≥ 1μM). Furthermore, when compared to the non-treated control, MitoQ alone (0.005– 5μM) reduced mitochondrial superoxide levels with a maximum reduction of 52% ± 1% at 1 μM. Contrastingly, MitoQ (10 μM) became pro-oxidant. Additionally, higher doses of MitoQ (1-10 μM) depolarized the MMP. Pre-treating with MitoQ (≥ 1μM) showed maximal protection, with a significant reduction of DOX accumulation and superoxide anion levels by 22% ± 8% and 53% ± 7% relative to DOX alone, respectively (both p \u3c 0.05), which was better than that of co- treatment. Western blot also demonstrated that pre-treatment upregulated both antioxidant enzymes. CONCLUSION: The findings presented in this study suggested that MitoQ works as an antioxidant against DOX. Pre-treatment showed significantly better cardioprotection than co- treatment due to its more potent antioxidant effects and improved ability toward reducing intracellular DOX accumulation

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

The effects of ascorbic acid and sodium ascorbate on doxorubicin-induced H9c2 cell damage

INTRODUCTION: Doxorubicin causes high superoxide levels in the mitochondria contributing to cardiotoxicity. Ascorbic acid (AA) and sodium ascorbate (SA) are antioxidants and may mitigate doxorubicin-induced heart cell damage. OBJECTIVE: This study determined the effects of AA and SA on doxorubicin-induced cell damage. METHODS: AA or SA (10-4000 uM) were tested alone or cotreated with doxorubicin (40 uM) on H9c2 cells. Some cells were pretreated with AA and SA for 24 hours before doxorubicin. Cell viability and superoxides were evaluated using a cell counting kit and Mitosox assay, respectively. RESULTS: AA and SA (10-2000 uM) alone increased cell viability by 17-31% (n=3), accompanied by decreased mitochondrial superoxides by 6-29% (n=2) when compared to the non-treated control. However, higher doses of AA and SA (4000 uM, n=2) decreased cell viability and tripled mitochondrial superoxide levels relative to the non-treated control. Furthermore, when compared to doxorubicin-treated cells, cotreatment of SA or AA (100 uM, n=3) showed a maximum increase of cell viability by 21± 6% and 29± 18%, respectively; and pretreatment of SA or AA (100 uM, n=5) showed a maximum increase of cell viability by 46± 2% and 57± 3%, respectively. Pretreatment of AA also reduced mitochondrial superoxide by 47± 4% (n=2) compared to doxorubicin-treated cells. CONCLUSION: Preliminary data suggest that pretreatment of AA and SA exerted better cardiac protection against doxorubicin than cotreatment in H9c2 cells. The effects of pretreatment may be related to the reduction of mitochondrial superoxide. However, high levels of AA and SA showed pro-oxidant properties