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

Abstract

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 P450 cam , and myoglobin (Mb) were incorporated into thin films with DNA using the electrostatic layer-bylayer (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 DNAenzyme 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.Toxicity is often not identified sufficiently early in developing new pharmaceutical, agricultural, personal care, and dietary products. Roughly 30% of drug development failures result from toxicity issues, driving drug costs up significantly. 1-4 Conventional in vitro biological tests such as Ames, chromosome aberration, mouse lymphoma, and Comet assays provide qualitative answers to toxicity questions based on bulk DNA damage but do not give chemical structure or site-specific DNA damage information. 5,6 Animal testing may not relate to human exposure because of metabolic and physiological differences between humans and animal models. 2,4,7 While all these toxicity tests are valuable, we believe that rapid, high-throughput toxicity screening methods for new chemicals that provide chemical structure information about reactive intermediates at early stages of commercial development are important goals to complement conventional microbiological and animal tests.Bioactivation of xenobiotic molecules by metabolic enzymes often results in reactive metabolites that damage biomolecules, including DNA, in a major toxicity pathway. [8][9][10] An example is the metabolism of lipophilic compounds by liver cytochrome (cyt) P450 (or CYP) enzymes creating reactive electrophilic metabolites. 4,11 These electrophiles attack nucleophilic DNA sites, primarily guanines, potentially resulting in genotoxicity. Depending on the adduct site, additional metabolism, and DNA repair processes, covalent DNA adducts can lead to mutations, teratogenesis, and carcinogenesis. 12-14 Thus, nucleobase adducts are key biomarkers for predicting cancer risk in humans and for exposure to toxic chemicals, and sensitive liquid chromatography-mass spectromet...

Synergistic Metabolic Toxicity Screening Using Microsome/DNA Electrochemiluminescent Arrays and Nanoreactors

“…Similar films formed on 500-nm-diameter silica beads (Polysciences, Warrington, PA) also followed an established protocol. 11 Briefly, 200 μL of silica beads was added to 800 μL of PEI for 20 min. followed by centrifugation and DI water rinsing steps.…”

“…followed by centrifugation and DI water rinsing steps. 11 The beads were then resuspended in 1 mL of DNA solution, allowed to adsorb, and then removed and rinsed. Similar steps were followed for the microsome adsorption.…”

“…6d, e We have also demonstrated LC-MS/MS structural and formation rate analyses, 10 with the most sensitive approach employing DNA/enzyme films on nanoparticles to obtain metabolites and DNA adducts. 11 Common features in these systems are that natural enzyme reactions produce reactive metabolites that are captured by a nucleophile-trapping reaction with DNA. Purified enzymes in these sensors were activated by exploiting the well-known peroxide shunt mechanism by exposure to small quantities of H 2 O 2 .…”

In vitro metabolic study of Rhizoma coptidis extract using liver microsomes immobilized on magnetic nanoparticles

“…The LMMNP bioreactors were prepared by using the layerby-layer self-assembly technique [27,28]. This immobilization approach based on electrostatic adsorption was simple and rapid compared with chemical modification techniques.…”

Sensitively Electrochemical Sensing for Sequence‐Specific Detection of Phosphinothricin Acetyltransferase Gene: Layer‐by‐Layer Films of Poly‐L‐Lysine and Au‐Carbon Nanotube Hybrid

“…The assemblies were characterized using CV. Charge transfer through the multilayer was studied experimentally by using [Fe(CN) 6 ] 3À/4À and [Co(phen) 3 ] 3þ/2þ as the electroactive indicators. In addition, ssDNA probe sequences associated with phosphinothricin acetyltransferase (PAT) gene were immobilized on pLys/Au-CNT multilayers.…”