Title

Metabolic Toxicity Screening Using Electro-Optical and Bioreactor LC/MS Arrays

Date of Completion

January 2010

Keywords

Health Sciences, Toxicology|Chemistry, Analytical|Chemistry, Biochemistry|Chemistry, Pharmaceutical

Degree

Ph.D.

Abstract

Metabolic toxicity is an important toxicology mechanism causing by foreign chemicals in humans. Metabolic toxicity evaluation represents a major challenge for developing any drugs or chemicals that come in contact with humans. This dissertation focuses on developing new methodologies for screening for reactive metabolite of chemicals using DNA adducts as a detection end point. One theme of this dissertation is to establish a representative electro-optical array platform to provide fast screening of toxic metabolite generated from different metabolic reactions. First, the capability of the established electro-optical array in elucidating species toxicity difference was evaluated using an anti-cancer drug, tamoxifen. A faster bioactivation and a slower detoxication processes were confirmed for rat liver enzymes compared to human enzymes in both ECL array and bioreactor LC-MS experiments. ^ The electro-optical array technique was further advanced by incorporating important metabolic enzymes from cytosolic sources. This project marks the successful fabrication of a standard metabolic toxicity screening platform for reactive metabolite generated from nearly all important metabolic routes. This was demonstrated by using rodent carcinogens, ethylene dibromde and N-acetyl 2-aminotluorene using their known bioactivation pathways involving cytosolic enzymes.^ The other theme of this dissertation is to develop high throughput methodologies for structural elucidation of the chemistry of metabolism and genotoxicity using liquid chromatography and mass spectrometry. Based on previously developed silica particle biocolloid technique, important conjugation enzymes were incorporated into the system. The throughput of this technique was greatly improved by designing the reactions in a 96-well plate format. This was demonstrated by metabolic profiling of several important drugs, such as raloxifene and diclofenac simultaneously.^ A new stage of the bioreactor technology was achieved using magnetic particles to solve some of the key issues associated with silica particle bioreactors, and sample workup and isolation in DNA adduct analysis. For the first time, DNA adduct analysis was conducted in a high throughput manner in a 96-well plate. Major DNA adducts of several known carcinogens were detected using different hydrolysis methods depending on the nature of the DNA adduct. The magnetic handling of the method features the potential for full automation. ^