Besnik Bajrami

Electrochemical Genotoxicity Screening for Arylamines Bioactivated by N-Acetyltransferase

Genotoxicity screening sensors that measure DNA damage from metabolism of arylamines were developed and evaluated. The sensors feature ultrathin films containing DNA and N-acetyltransferase (NAT) on pyrolytic graphite (PG) electrodes. NAT in the film catalyzed the conversion of the arylamine 2-aminofluorene (2-AF) to 2-acetylaminofluorene (2-AAF) by acetyl coenzyme A (AcCoA) dependent N-acetylation, as verified by liquid chromatography. DNA damage in the films from exposure to reactive 2-AF metabolites was measured subsequent to the enzyme reaction using catalytic voltammetric oxidation with Ru(bpy)32+. Square wave voltammetric (SWV) peaks increased with enzyme reaction time, and relative DNA damage rates at pH 5.8 were measured within 2 min. Control incubations of DNA/NAT films without AcCoA gave no significant sensor response. CapLC-MS/MS analysis of 2-AAF/DNA reaction products was consistent with 2-AF-guanine adducts formed in the films. DNA damage occurred more rapidly under weakly acidic conditions (pH 5.5-5.8) than at neutral pH, suggesting that genotoxicity from arylamine metabolism by NAT could be more significant in slightly acidic environments.

Rapid LC-MS drug metabolite profiling using microsomal enzyme bioreactors in a parallel processing format.

Silica nanoparticle bioreactors featuring thin films of enzymes and polyions were utilized in a novel high-throughput 96-well plate format for drug metabolism profiling. The utility of the approach was illustrated by investigating the metabolism of the drugs diclofenac (DCF), troglitazone (TGZ), and raloxifene, for which we observed known metabolic oxidation and bioconjugation pathways and turnover rates. A broad range of enzymes was included by utilizing human liver (HLM), rat liver (RLM) and bicistronic human-cyt P450 3A4 (bicis.-3A4) microsomes as enzyme sources. This parallel approach significantly shortens sample preparation steps compared to an earlier manual processing with nanoparticle bioreactors, allowing a range of significant enzyme reactions to be processed simultaneously. Enzyme turnover rates using the microsomal bioreactors were 2-3 fold larger compared to using conventional microsomal dispersions, most likely because of better accessibility of the enzymes. Ketoconazole (KET) and quinidine (QIN), substrates specific to cyt P450 3A enzymes, were used to demonstrate applicability to establish potentially toxic drug-drug interactions involving enzyme inhibition and acceleration.

Enzyme-DNA biocolloids for DNA adduct and reactive metabolite detection by chromatography-mass spectrometry.

Silica microbead bioreactors (0.5 microm 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-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.

Microsome biocolloids for rapid drug metabolism and inhibition assessment by LC-MS

Rat liver microsomes attached to nanoparticles were used for LC-MS studies of CYP3A and 2E1 enzymes in metabolism of N-nitroso compounds. Using these biocolloids, turnover rates were measured within 2 min. Inhibitor IC(50) values for ketoconazole (KET) and 4-methylpyrazole (4-MEP) were estimated.

Characterizing Metabolic Inhibition Using Electrochemical Enzyme-DNA Biosensors

Studies of metabolic enzyme inhibition are necessary in drug development and toxicity investigations as potential tools to limit or prevent appearance of deleterious metabolites formed, for example, by cytochrome (cyt) P450 enzymes. In this paper, we evaluate the use of enzyme/DNA toxicity biosensors as tools to investigate enzyme inhibition. We have examined DNA damage due to cyt P450cam metabolism of styrene using DNA/enzyme films on pyrolytic graphite (PG) electrodes monitored via Ru(bpy)(3)(2+)-mediated DNA oxidation. Styrene metabolism initiated by hydrogen peroxide was evaluated with and without the inhibitors, imidazole, imidazole-4-acetic acid, and sulconazole (in micromolar range) to monitor DNA damage inhibition. The initial rates of DNA damage decreased with increased inhibitor concentrations. Linear and nonlinear fits of Michaelis-Menten inhibition models were used to determine apparent inhibition constants (K(I)*) for the inhibitors. Elucidation of the best fitting inhibition model was achieved by comparing correlation coefficients and the sum of the square of the errors (SSE) from each inhibition model. Results confirmed the utility of the enzyme/DNA biosensor for metabolic inhibition studies. A simple competitive inhibition model best approximated the data for imidazole, imidazole-4-acetic acid and sulconazole with K(I)* of 268.2, 142.3, and 204.2 microM, respectively.

Human Cyt P450 Mediated Metabolic Toxicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) Evaluated Using Electrochemiluminescent Arrays

Electrochemiluminescent (ECL) arrays containing polymer ([Ru(bpy)(2)(PVP)(10)](2+), PVP = polyvinylpyridine), DNA, and selected enzymes were employed to elucidate cytochrome (cyt) P450 dependent metabolism of the tobacco specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Bioactivated NNK metabolites formed upon H(2)O(2)-enzymatic activation were captured as DNA adducts and detected simultaneously from 36 spot arrays by capturing and quantifying emitted ECL with an overhead CCD camera. Increased ECL emission was dependent on NNK exposure time. Of the enzymes tested, the activity toward NNK bioactivation was cyt P450 1A2 > 2E1 > 1B1 approximately chloroperoxidase (CPO) > myoglobin (Mb) in accordance with reported in vivo studies. Cyt P450/polyion films were also immobilized on 500 nm diameter silica nanospheres for product analysis by LC-MS. Analysis of the nanosphere film reaction media provided ECL array validation and quantitation of the bioactivated NNK hydrolysis product 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) confirming production of reactive metabolites in the films. Chemical screening in this fashion allows rapid clarification of enzymes responsible for genotoxic activation as well as offering insight into cyt P450-related toxicity and mechanisms.

Rapid LC-MS Drug Metabolite Profiling Using Bioreactor Particles

Enzyme-coated magnetic bioreactor particles enable a fast, convenient approach to metabolic screening. A semi-automated metabolite-profiling technique using these particles in a 96-well plate with liquid chromatography (LC)-mass spectrometry (MS)/MS detection is described. Reactions can be investigated over 1- to 2-min periods, and 96 or more reactions or reaction time points can be processed in parallel. Incorporation of DNA in the particle films facilitates determination of rates of DNA damage and metabolite-DNA adduct structures.