Enzyme−DNA Biocolloids for DNA Adduct and Reactive Metabolite Detection by Chromatography−Mass Spectrometry

• Published in: Analytical chemistry (Washington) Vol. 80; no. 4; pp. 922 - 932
• Main Authors: Bajrami, Besnik, Hvastkovs, Eli G, Jensen, Gary C, Schenkman, John B, Rusling, James F
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 15.02.2008
• Subjects: Analytical chemistry; Camphor 5-Monooxygenase - metabolism; Catalysis; Chemistry; Chromatographic methods and physical methods associated with chromatography; Chromatography, Liquid - methods; Cytochrome P-450 CYP2E1 - metabolism; Deoxyribonucleic acid; DNA; DNA - chemistry; DNA Adducts - analysis; Enzymes; Epoxy Compounds - chemistry; Exact sciences and technology; Gas chromatographic methods; Humans; Liver; Liver - enzymology; Mass spectrometry; Mass Spectrometry - methods; Measurement; Medical screening; Myoglobin - metabolism; Other chromatographic methods; Spectrometric and optical methods; Static Electricity; Time Factors; Human; Enzyme; Metabolite; Liquid chromatography; Silica; Thin film; Hydrolysis; Gas chromatography; Improvement; Bioreactor; DNA; Mass spectrometry MS/MS; Styrene; Oxidation; Surface area; Mass spectrometry; Adduct
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac702025f

Silica microbead bioreactors (0.5 μm diameter) coated with DNA and enzymes were fabricated to measure reactive metabolite and DNA−adduct formation rates relevant to genotoxicity screening. Cytochrome (cyt) P450 2E1, cyt P450cam, and myoglobin (Mb) were incorporated into thin films with DNA using the electrostatic layer-by-layer (LbL) method. The utility of these biocolloids was demonstrated by oxidation of guaiacol, styrene, and (4-methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK). Enzyme turnover rates for formation of reactive metabolites were monitored using gas chromatography/mass spectrometry (GC/MS) and liquid chromatography−mass spectrometry (LC−MS). Capillary LC−MS/MS was employed to determine DNA nucleobase adducts after catalyzing the reactive metabolite formation with DNA−enzyme biocolloids and then using neutral thermal hydrolysis on the biocolloids. Dramatic improvements in surface area to volume ratio over similar films on macroscopic surfaces opens new avenues for genotoxicity screening and enabled the first use of pure cyt P450 enzymes in enzyme−DNA films to produce DNA adducts. The method makes possible identification and formation rate measurements of major and minor DNA adducts as well as the metabolites themselves in <5 min of reaction time using relevant human liver enzymes.