Toxicity Profiling – Acute Liver Failure
Acute liver failure (ALF) can be induced by overdose of drugs such as the paracetamol, adverse reaction to medication (e.g. tetracycline), excessive alcohol intake (severe alcoholic hepatitis) or viral hepatitis (hepatitis A or B - it is extremely uncommon in hepatitis C). ALF is associated with an overwhelming activation of the immune responseand monocytes and macrophages are postulated to play a pivotal role in the initiation, propagation and resolution of ALF. However, due to their plasticity, macrophages are implicated in both tissue destructive and reparatory processes of the inflammatory response. Thus, the presence of effusive macrophage infiltrations seen on biopsy are not good indicators of likely outcome. Better diagnostic and prognostic tools based on molecular protein biomarkers are needed to determine whether liver tissue is likely to become necrotic or whether, with appropriate support, the tissue can be saved.
Regions of necrotic, non-necrotic and healthy liver tissue in patients with ALF and non-ALF controls were prepares by laser capture microdissection. Proteins were extracted, digested with trypsinand labeled with isotopic TMTlight mass tags.A common biological reference was prepared by pooling 10% of each protein extract and labelling this mix with isotopic TMTheavy. Each labelled sample was then mixed with an equal amount of the reference sample and analysed by LC-MS/MS. Biomarkers were identified when the TMTlight signal was 20% greater or lower than the same peptide in the pooled reference.
In total over 3,500 peptides were identified. To date we have identified a panel of 10 promising candidate biomarker proteins from 651 seen in the initial experiments. Candidates map onto different immunological pathways that are consistent with the mechanisms of necrosis in ALF.
Using the isotopic TMT reagents allows us to rapidly develop assays for biomarker validation through TMT-SRM assay formats. This work is currently in progress.
We believe that the markers identified in this ALF study will also be applicable to other forms of drug-induced liver damage. We are looking to establish partnerships with industry consortia interested in evaluating alternate markers of liver toxicity during drug development.