High-Throughput Metabolic Toxicity Screening Using Magnetic Biocolloid Reactors and LC−MS/MS

摘要

An inexpensive, high-throughput genotoxicity screeningnmethod was developed by using magnetic particles coatednwith cytosol/microsome/DNA films as biocolloid reactorsnin a 96-well plate format coupled with liquid chromatography-nmass spectrometry. Incorporation of both microsomalnand cytosolic enzymes in the films provides a broadnspectrum of metabolic enzymes representing a range ofnmetabolic pathways for bioactivation of chemicals. Reactivenmetabolites generated via this process are trappednby covalently binding to DNA in the film. The DNA is thennhydrolyzed and nucleobase adducts are collected usingnfilters in the bottom for the 96-well plate of analysis byncapillary liquid chromatography-tandem mass spectrometryn(LC-MS/MS). The magnetic particles facilitate simplenand rapid sample preparation and workup. Major DNAnadducts from ethylene dibromide, N-acetyl-2-aminofluorenenand styrene were identified in proof-of-conceptnstudies. Relative formation rates of DNA adducts correlatednwell with rodent genotoxicity metric TD50 for thenthree compounds. This method has the potential fornhigh-throughput genotoxicity screening, providingnchemical structure information that is complementarynto toxicity bioassays.