Strategy for Hepatotoxicity Prediction Induced by Drug Reactive Metabolites Using Human Liver Microsome and Online 2D-Nano-LC-MS Analysis

Abstract

Hepatotoxicity is a leading cause of drug withdrawal from the market; thus, the assessment of potential drug induced liver injury (DILI) in preclinical trials is necessary. More and more research has shown that the covalent modification of drug reactive metabolites (RMs) for cellular proteins is a possible reason for DILI. Unfortunately, so far no appropriate method can be employed to evaluate this kind of DILI due to the low abundance of RM-protein adducts in complex biological samples. In this study, we proposed a mechanism-based strategy to solve this problem using human liver microsomes (HLMs) and online 2D nano-LC-MS analysis. First, RM modification patterns and potential modified AA residues are determined using HLM and model amino acids (AAs) by UHPLC-Q-TOF-MS. Then, a new online 2D-nano-LC-Q-TOF-MS method is established and applied to separate the digested modified microsomal peptides from high abundance peptides followed by identification of RM-modified proteins using Mascot, in which RM modification patterns on specific AA residues are added. Finally, the functions and relationship with hepatotoxicity of the RM-modified proteins are investigated using ingenuity pathway analysis (IPA) to predict the possible DILI. Using this strategy, 21 proteins were found to be modified by RMs of toosendanin, a hepatotoxic drug with complex structure, and some of them have been reported to be associated with hepatotoxicity. This strategy emphasizes the identification of drug RM-modified proteins in complex biological samples, and no pretreatment is required for the drugs. Consequently, it may serve as a valuable method to predict potential DILI, especially for complex compounds.

A cell lines derived microfluidic liver model for investigation of hepatotoxicity induced by drug-drug interaction

“…As the APAP concentration increased, the cell mortality rate went up gradually. In addition, after exposure to the same concentration (5,15, and 25 mM) of APAP, the mortality rate of HepG2 cells in the chip model (39%, 54%, and 87%) was higher than that in the 96-well plate HepG2 model (18%, 27%, and 42%), presumably because of the higher drug metabolism ability, more consistent oxygen content of the media, cross talk between multiple cell types, the impact from shear stress, and the rapid removal of cellular by-products in the chip. The release of biochemical markers (LDH, AKP, AST, and ALT) in the "artificial blood" also showed the same trend as mortality rate (Fig.…”

“…In vitro models, such as a recombinant enzyme, 3,4 liver microsome, 5 and primary hepatocytes (HCs), 6 have a major role in evaluating the hepatotoxicity of the drug-drug combination. Specifically, primary hepatocytes are widely used at the preclinical stage because of their considerable metabolic ability.…”

IKKβ mediates homeostatic function in inflammation via competitively phosphorylating AMPK and IκBα

“…Finally, the gel piece was incubated with 10 ng/μL trypsin in 40 mmol/L ammonium bicarbonate with 10% acetonitrile (ACN) for 30 min on ice and replenished with 40 mmol/L ammonium bicarbonate with 10% ACN at 37 °C overnight. For immunoprecipitated proteins, the process of protein digestion was the same with the description in the previous study 18 . Briefly, the protein contents were firstly quantitated using the detergent compatible (DC) Protein Assay Reagent (Bio-Rad).…”

“…The methionine oxidation (M), phospho (C), phospho (D), phospho (H), phospho (R), phospho (ST), and phospho (Y) were selected as variable modifications, while cysteine carbamidomethyl (C) was selected as fixed modification. The parameters selected for database searches were as previously described 18 .…”

Nanomaterials‐Based Surface Protein Imprinted Polymers: Synthesis and Medical Applications

“…[ 147,148 ] By combining with the chromatrography separation, MS has gradually become the core technology for sample separation, structure identification, and quantitative analysis. [ 149–151 ] However, MS is poorly tolerant of matrix interferences in the test sample and needs to enrich the target protein due to their low content in samples. [ 152 ] Therefore, before the sample enters the MS system, an additional sample preparation step is usually needed for the enrichment of the target protein or peptide and effectively removing impurities in the sample.…”