Comparison of DNA‐Reactive Metabolites from Nitrosamine and Styrene Using Voltammetric DNA/Microsomes Sensors

Abstract

Voltammetric sensors made with films of polyions, double-stranded DNA and liver microsomes adsorbed layer-by-layer onto pyrolytic graphite electrodes were evaluated for reactive metabolite screening. This approach features simple, inexpensive screening without enzyme purification for applications in drug or environmental chemical development. Cytochrome P450 enzymes (CYPs) in the liver microsomes were activated by an NADPH regenerating system or by electrolysis to metabolize model carcinogenic compounds nitrosamine and styrene. Reactive metabolites formed in the films were trapped as adducts with nucleobases on DNA. The DNA damage was detected by square-wave voltammetry (SWV) using Ru(bpy)32+ as a DNA-oxidation catalyst. These sensors showed a larger rate of increase in signal vs. reaction time for a highly toxic nitrosamine than for the moderately toxic styrene due to more rapid reactive metabolite-DNA adduct formation. Results were consistent with reported in vivo TD50 data for the formation of liver tumors in rats. Analogous polyion/ liver microsome films prepared on 500 nm silica nanoparticles (nanoreactors) and reacted with nitrosamine or styrene, provided LC-MS or GC analyses of metabolite formation rates that correlated well with sensor response.

Microfluidic Electrochemical Array for Detection of Reactive Metabolites Formed by Cytochrome P450 Enzymes

“…A dense coverage with a distribution of circular well-like structures of variable size was found, with a 2-fold increase in surface roughness over the underlying layer. Microsomes create these well-like vesicular features by partially flattening on surfaces, as concluded from previous electron microscopy and AFM studies. , The horizontal diameter of the circular structures was 141 ± 35 nm, and the vertical height was 12 ± 5 nm. Electron microscopy on RLMs revealed similar circular features with variable diameters of 80–400 nm …”

“…Microsomes create these well-like vesicular features by partially flattening on surfaces, as concluded from previous electron microscopy and AFM studies. 41,42 The horizontal diameter of the circular structures was 141 ( 35 nm, and the vertical height was 12 ( 5 nm. Electron microscopy on RLMs revealed similar circular features with variable diameters of 80À400 nm.…”

Electrochemistry of Nucleic Acids

“…Rusling’s group developed an interesting approach for monitoring reactive metabolites by employing electrodes coated with cytochrome P450/DNA films prepared using a layer-by-layer technique. − In these sensors, enzyme reaction produced metabolites and the resulting DNA damage was detected by either voltammetry using soluble [Ru­(bipy) 3 ] 2+ catalyst (regenerated by A and G residues in damaged DNA), , or with single electrode ECL , and ECL arrays. , The required films could be placed on a single PGE, ,, in a block array format, or on silica nanoparticles for LC-MS analysis . Studies performed on these formats included the metabolism and genotoxicity of styrene, benzo­[a]­pyrene, N -nitrosamines, , and the genotoxicity of arylamines activated by N-acetyltransferase …”

DNA Carbon-Nanodots based Electrochemical Biosensor for Detection of Mutagenic Nitrosamines

“…Amperometric determination of N -nitrosamine at ruthenium-based polymer modified electrodes, enhanced voltammetric stripping analysis of N -nitrosamine at sulfopolyester-modified electrodes, and electrochemical detection of NDPhA using poly­(diallyldimethylammonium chloride) stabilized graphene/platinum nanoparticles have been reported and are sensitive analytical methods for N -nitrosamine detection. Furthermore, DNA adsorbed pyrolytic graphene electrodes were also used to determine N -nitrosamine . All of these have demonstrated that modified electrodes can be used in the development of N -nitrosamine sensors.…”

“…Furthermore, DNA adsorbed pyrolytic graphene electrodes were also used to determine N-nitrosamine. 12 All of these have demonstrated that modified electrodes can be used in the development of N-nitrosamine sensors. Use of DNA in the sensing field has also increased the opportunities leading to various sensing strategies.…”