Screening reactive metabolites bioactivated by multiple enzyme pathways using a multiplexed microfluidic system

Area of Science:

• Analytical Chemistry
• Biotechnology
• Toxicology

Background:

• Assessing reactive metabolites is crucial for understanding drug metabolism and predicting genotoxicity.
• Existing methods for detecting reactive metabolites can be time-consuming and complex.
• Mimicking multi-enzyme human drug metabolism pathways is essential for accurate toxicity assessments.

Purpose of the Study:

• To develop and validate a multiplexed, microfluidic platform for detecting reactive metabolites.
• To assess the platform's performance in mimicking human drug metabolism and identifying genotoxic compounds.
• To enable high-throughput screening of enzyme-mediated reactions and DNA damage.

Main Methods:

• A microfluidic device with screen-printed carbon arrays coated with DNA and a ruthenium-polyvinylpyridine (RuPVP) catalyst.
• Integration of multiple enzyme sources: human liver microsomes (HLM), cytochrome P450 (cyt P450) 1B1 supersomes, microsomal epoxide hydrolase (EH), human S9 liver fractions (Hs9), and N-acetyltransferase (NAT).
• Detection of reactive metabolites via DNA adduct formation and measurement using square wave voltammetry (SWV) with RuPVP catalyst.

Main Results:

• The platform successfully detected reactive metabolites from compounds requiring metabolic activation for genotoxicity.
• Metabolism of benzo[a]pyrene (B[a]P) showed enhanced DNA damage when epoxide hydrolase was included with cyt P450s.
• DNA damage rates for arylamines correlated better with rodent genotoxicity metrics when both oxidative and conjugative enzymes were present.

Conclusions:

• The developed microfluidic device is a versatile tool for screening reactive metabolites and assessing potential genotoxicity.
• The platform effectively mimics complex human drug metabolism pathways involving multiple enzymes.
• This technology offers a rapid and efficient method for evaluating chemical safety and predicting drug-induced toxicity.