Doxorubicin is a chemotherapeutic agent widely used in the treatment of carcinomas, sarcomas, and hematological tumors. However, 40% of treated display liver dysfunction, the main organ responsible for doxorubicin metabolism. The mechanisms involved in doxorubicin-induced hepatotoxicity are still unknown. It is known that doxorubicin accumulates in the liver in a dose-dependent manner, predisposing deleterious effects during doxorubicin regimen. Doxorubicin accumulates in mitochondria due to its high affinity for mitochondrial phospholipids, resulting in bioenergetics dysfunction, accumulation of toxic aldehydes and cell death, which may lead to impaired liver function. Considering the association between mitochondrial dysfunction and doxorubicin-induced hepatotoxicity, pharmacological interventions capable of protecting mitochondrial metabolism are expected to mitigate or prevent the deleterious hepatic effects of doxorubicin. In this context, preliminary results of this project demonstrate that pharmacological activation of the mitochondrial detoxification system via enzyme aldehyde dehydrogenase 2 (ALDH2) is sufficient to remove toxic aldehydes accumulated during mitochondrial dysfunction and, consequently, to protect the liver against toxic effects of doxorubicin. In a proof-of-concept study, we treated ALDH2 deficient transgenic mice with doxorubicin, and we expected greater toxicity. However, we observed these animals are resistant to doxorubicin-induced hepatotoxicity when compared to wild animals exposed to the same conditions. Therefore, we performed an unbiased metabolomics in the liver of ALDH2 deficient mice to identify and better understand compensatory responses capable of neutralizing the degenerative effects of doxorubicin. We found an increased accumulation of fumarate in the liver of ALDH2 deficient transgenic mice when compared with age-matched wild-type animals.Considering that fumarate produced by the Krebs cycle has the potential to directly regulate the functional of proteins by succination (a post-translational modification characterized by the formation of adducts between fumarate and cysteine residues of the target protein), we aim to investigate the role of this fumarate-mediated post-translational modification in cell signal reorganization and its possible contribution to liver protection against doxorubicin. Importantly, there are no reports in the literature on the hepatic cataplerotic action of fumarate. In addition, only two fumarate targets (Keap1-Nrf2 axis and gasdermin D) have been described in others tissues and other conditions, demonstrating the need for exploratory studies capable of identifying new fumarate targets and validating their participation under physiological and pathological conditions.Therefore, this study aims to: 1) Investigate the protective role of the two fumarate targets (Keap1-Nrf2 and gasdermin D) in the liver of ALDH2 deficient transgenic mice; 2) Evaluate the protective effect of fumarate on mitochondrial metabolism and hepatocyte viability in culture against doxorubicin; 3) Test the protective effects of fumarate on hepatic function in wild mice treated with doxorubicin; 4) Identify novel fumarate-mediated succination of proteins in liver samples from animals treated with doxorubicin and/or fumarate(2D-proteomics); and validate these targets in hepatocytes in culture exposed to doxorubicin and/or fumarate. Unraveling the intracellular signals capable of producing an adaptive/protective response against doxorubicin-induced hepatotoxicity is critical for the development of effective and safe therapies for cancer. In addition, fumarate is an FDA approved drug for the treatment of multiple sclerosis; however, its mechanism of action is still poorly understood. Prof. Dr. Daria-Mochly-Rosen (Stanford University), Prof. Dr. Che-Hong Chen (Stanford University) and Prof. Dr. Alicia Kowaltowski (IQ-USP) will collaborate with this project.
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