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Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System
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Screening reactive metabolites bioactivated by multiple enzyme pathways using a multiplexed microfluidic system.

Dhanuka P Wasalathanthri1, Ronaldo C Faria, Spundana Malla

  • 1Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, USA.

The Analyst
|October 26, 2012
PubMed
Summary

A new microfluidic device screens reactive metabolites from drug metabolism, measuring DNA damage from oxidative and bioconjugation enzymes. This platform aids in predicting chemical genotoxicity by mimicking human metabolism.

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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.