Hailey Steichen



Project title: Effects of Nanocellulose Exposure on Cytochrome P450 and Glutathione S-Transferase Activity in Wistar Han Rat Livers

Degree: MS | Program: Applied Toxicology (App Tox) | Project type: Thesis/Dissertation
Completed in: 2018 | Faculty advisor: Yue Cui

Abstract:

Activity-based protein profiling (ABPP) is a chemical proteomics technique useful in quantifying the activity of functionally active enzymes. While other proteomics techniques can quantify expression of particular enzymes, the expression of these enzymes is not necessarily indicative of their functional  activity. This study used ABPP to characterize the activity of enzymes we expected to be changed after exposure to nanocellulose (NC). ABPP was used to characterize the activity of glutathione S-transferases (GSTs) and cytochrome P450s (P450s) from microsomal and cytosolic fractions of hepatic lysate from Wistar Han rats exposed to cellulose nanofibrils (CNF) in the presence and absence of a high-fat diet. It is currently unknown how NC is metabolized, but we suspected that the activity of two enzymes involved phase-1 and phase-2 metabolism, P450s and GSTs, could be influenced by exposure to NC. Analysis through SDS-PAGE revealed increased average P450 activity between controls and NC and fat treatments, but significantly decreased average P450 activity with co-exposure to NC + fat. It showed decreased average GST activity in NC-treated rats, increased average GST activity in fat-treated  rats, and increased average GST activity in NC + fat-treated rats. These results suggest the activity of P450s and GSTs could be altered after exposure to NC. Further analysis through mass spectrometry is needed to quantify the activity of these enzymes, and to elucidate which specific isoenzymes  contributed to the observed activity. Precise quantification and isoenzyme detection would reveal the degree to which these detoxification pathways are changed upon NC exposure, and possibly provide information as to how it is metabolized.

Abbreviations: ABPP: activity-based protein profiling; NC: nanocellulose; CYP450/P450: cytochrome P450; GST: glutathione S-transferase; CNF: cellulose nanofibrils; CNC: cellulose nanocrystals; BC: bacterial cellulose; GSH: glutathione; CAR: Constitutive Active/Androstane Receptor; PXR: Pregnane X  Receptor; ABP: activity-based probe; BSH: bile salt hydrolase, LCMS/ MS: liquid chromatography with tandem mass spectrometry; 2EN:  2-ethynylnaphthalene; OC: organochlorine, POP: persistent organic pollutant, BCNU: 1,3-bis-(2-chloroethyl)-1- nitrosourea, NRF2: nuclear factor erythroid 2-related factor 2, KEAP1: Kelch-like-ECHassociated protein 1, JNK: c-Jun NH2-terminal kinase, MAF: small musculoaponeuroticfibrosarcoma virus, AREs: antioxidant response elements, PNNL: Pacific Northwest National Laboratories, BCA: bicinchoninic acid assay; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis, Rho-N3: rhodamine-azide, THPTA: trishydroxypropyltriazolylmethylamine, SDS: sodium dodecyl sulfate, NADPH: Nicotinamide Adenine Dinucleotide Phosphate Hydrogen; qPCR: real-time/quantitative polymerase chain reaction; PAH: polycyclic aromatic hydrocarbon; BSA: bovine serum albumin, DTT: dithiothreitol, DMSO: dimethyl sulfoxide, ROS: reactive oxygen species

Keywords: activity-based protein profiling, nanocellulose, glutathione S-transferase, cytochrome P450, liver, xenobiotic metabolism