Drug-Induced Liver Injury (DILI) is the most common event causing during the drug withdrawals. But the causes of the hepatic failure provoked by the chemical compounds could be several. Due to this reason, a multiparametric assay is needed to predict toxicities that could be hidden in a simple one parameter assay. We analyze the following parameters in the hepatotoxicity Assay:
- Oxidative Stress
- Mitochondrial damage
- Genotoxicity (DNA damage)
- Cell Viability
In this assay we predict the hepatotoxicity of the compounds measuring the most important parameters affected by the drugs: Oxidative stress, ATP production, mitochondrial damage and genotoxicity. Human or animal primary hepatocytes are treated with the drugs at several concentrations and the results are compared with the positive controls of hepatotoxicants for each parameter. We measure the mitochondrial damage with the CM-H2DCFDA probe. This probe measure mitochondrial membrane potential and the results are correlated with the mitochondrial integrity. The oxidative stress is measured quantifying the reactive oxygen strains using the TMRM. Meanwhile, we use an antibody against phosphorilated H2AX protein to measure DNA damage. The H2AX is a member of the histone H2A family which phosphorilate when the double strand of DNA is broken. The presence of phosphorilated H2AX is correlated with DNA damage & subsequently with genotoxic agents presence.
Cell line used: Human & Animal Hepatocytes
Readout: Oxidative Stress, DNA Damage, Caspase Activation, Cell Viability
Readout: ATP Production, LDH Release, Caspase Activation, Cell Viability