Identification of Biomarkers for Early Detection of Drug Induced Hepatotoxicity

Abstract

New drug molecules/candidates are subjected to pre-clinical studies in order to predict any potential drug side effects as per the regulatory laws throughout the world. However, in recent decades, the number of new drug approvals has markedly decreased, while the cost of drug development has increased. Main causes for this are, either adverse effects in humans which are not predicted by animal studies leading to the compound’s failure in late phases of the development process (Nefazodone, Pemoline), or intolerable toxic effects in animals during initial drug developmental phases. As far as toxicity is concerns, hepatotoxicity is the most prominent toxicity as after oral intake the compounds are transported to the liver, where they will be metabolised and eliminated. Currently, hepatotoxicity is evaluated in 28-day in vivo repeated-dose toxicity tests by analysis of haematological, histopathological and clinical parameters. However, these parameters are often insensitive and can generate false negative results. Consequently, unexpected hepatotoxicity often appears in the clinical trials or even when the product is already in the market. This emphasises the need for novel screening methods that address toxicological hazards early in the drug developmental process. A better pre- selection of drug candidates could increase the success rate during pre clinical trials since very toxic compounds could be excluded in earlier stages of animal testing. Furthermore, in vitro tests also provide mechanistic information which supports the interpretation of in vivo observations, as well as play a role in human safety assessment, since these tests could be performed in both animal and human cells.In the present study, proteomic analysis was used as a tool to understand the molecular mechanisms underlying Paroxetine-induced liver injury and to define potential bio markers of toxicity. Three groups of male Swiss albino mice were treated with 0.0, 12.33 and 123.33mg/kg of paroxetine for 5, 10 and 15 days (n=5/group). After completion of respective treatments, the animals were euthanised, liver and serum were collected. Serum was used for biochemical parameter investigation Liver tissue was used for histological and proteomic analysis. Primary hepatocyte cells were also treated with the Paroxetine at a dose of 40mM for 24 hours, after completion of cell treatment duration the cells were harvested and protein was isolated for proteomic investigation (SDS-PAGE and 2 D PAGE with LC-MS/MS). Quantitative intensity analysis (Delta 2D Codon) comparing the percentage of alteration of volume of protein spots revealed 118 and 96 protein spots that had increase or decrease in expression in the in vivo and in vitro studies respectively. Briefly, exposure to Paroxetine altered the expression of liver proteins belonging to all the functional categories, especially those related to metabolism and oxidative stress. More pronounced alterations were seen for the high dose group. Proteins that were detected upon exposure to paroxetine or that had their expression induced in the presence of paroxetine are potential biomarkers of toxicity and should be better investigated. Since this is the first study involving proteomic analysis of liver in mice exposed to different doses of paroxetine, these findings indicate important pathways and cellular processes affected by exposure to this element that should be addressed in details in future studies.