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

Abstract

Platforms based on thin enzyme/DNA films were used in two-tier screening of chemicals for reactive metabolites capable of producing toxicity. Microsomes were used for the first time as sources of cytochrome (cyt) P450 enzymes in these devices. Initial rapid screening involved electrochemiluminescent (ECL) arrays featuring spots containing ruthenium poly(vinylpyridine), DNA, and rat liver microsomes or bicistronically expressed human cyt P450 2E1 (h2E1). Cyt P450 enzymes were activated via the NADPH/reductase cycle. When bioactivation of substrates in the films gives reactive metabolites, they are trapped by covalent attachment to DNA bases. The rate of increase in ECL with enzyme reaction time reflects relative DNA damage rates. “Toxic hits” uncovered by the array were studied in structural detail by using enzyme/DNA films on silica nanospheres as “nanoreactors” to provide nucleobase adducts from reactive metabolites. The utility of this synergistic approach was demonstrated by estimating relative DNA damage rates of three mutagenic N-nitroso compounds and styrene. Relative enzyme turnover rates for these compounds using ECL arrays and LC-UV-MS correlated well with TD50 values for liver tumor formation in rats. Combining ECL array and nanoreactor/LC−MS technologies has the potential for rapid, high-throughput, cost-effective screening for reactive metabolites and provides chemical structure information that is complementary to conventional toxicity bioassays.

Evaluating Metabolite-Related DNA Oxidation and Adduct Damage from Aryl Amines Using a Microfluidic ECL Array

“…27 HLM induced faster DNA oxidation and adduction rates than RLM for all the aryl amines, in agreement with previous work using a non-microfluidic manual array. 18 Supersome 1A2 had the largest relative DNA oxidation and adduction rates for 4-ABP, AF, and NA, while for anisidine, the 2E1 isoform caused slightly more bioactivation for DNA oxidation and adduction than 1A2, consistent with other in vitro studies.…”

“…QCM frequency shifts were used to calculate the mass densities and nominal film thicknesses. 14,18,27 Films were nominally 22–48 nm thick depending on layer mass densities (Table S2 and S3). …”

Thin film voltammetry of metabolic enzymes in rat liver microsomes

“…Thicknesses of individual layers were ∼ 3 nm for microsomes and 0.5 nm for PDDA. In experiments to be reported elsewhere, we have shown that these microsome films are catalytically active for substrate oxidation [13]. Fig.…”

Evaluation of chrono-amperometric signal detection for the analysis of genotoxicity by a whole cell biosensor

“…Hvastkovs et al report the use of electrochemiluminescent arrays for genotoxicity testing of metabolites of benzo[a]pyrene that are generated in situ by various immobilized cytochrome (cyt) P450 enzymes [26]. Later on the same group published the use of imbedded microsomes as a source of cyt P450 enzymes [27]. In contrast to the present study the aforementioned electrochemiluminescent arrays detect DNA-damage without any cellular context, i.e.…”