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 TD 50 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.

Figure 1

Reconstructed array data demonstrating ECL from spots of RuPVP/DNA/RLM (labeled RLM) or RuPVP/DNA/h2E1 (labeled h2E1) exposed to 1 mM concentration of denoted substrate using enzymatic NADPH regeneration for denoted time (in s). Data for respective substrates are from same array run. Control is an identical array (RLM; h2E1 was similar (not shown)) not exposed to reaction solutions. Internal controls C1 = 120-s exposure to NADPH solution only; C2 = 120-s exposure to substrate (no NADPH) only.

Figure 2

Percent ECL increase vs reaction time for (a) RLM and (b) h2E1 arrays exposed to NPYR (black circles), NNK (blue squares), NPIP (green diamonds), or styrene (red triangles). Control (open circles, dash) in (a) is the ECL increase on a RLM array not exposed to any xenobiotic solution.

Figure 3

Inhibition by 4-MP. (a) Reconstructed data showing ECL response from RLM spots on same array exposed to 1 mM NPYR without (top) and with (bottom) 50 μM 4-MP as selective cyt P450 2E1 inhibitor for denoted times (s). (b) % ECL change for RLM spots not exposed (black squares) and exposed (blue circles) to 4-MP. (c) Similar reconstructed data as (a) using h2E1 spots. (d) % ECL change for h2E1 spots not exposed (black squares) and exposed (blue circles) to 4-MP.

Figure 4

SRM-MS chromatograms measuring total ion current for predominant guanine adduct (m/z shown in inset) produced via exposure of (a) NPYR, (b) NNK, and (c) NPIP to RLM/DNA films on silica nanospheres followed by DNA hydrolysis. Insets show examples of relevant guanine adducts and fragmentation patterns consistent with each chromatogram. (d) Integrated UV diode array measured peak area increase for NPYR–guanine adducts with reaction time from both RLM and h2E1 films.

Scheme 1. Microsome/DNA Films and Reactions on Nanoreactors (Left) or Array Chip (Right)^a

^a A layer of cationic polymer is initially deposited (blue ribbon in circle, e.g., poly(ethyleneimine (PEI) or [Ru(bpy)₂PVP₁₀]²⁺ (RuPVP), where bpy = bipyridine) followed by layers of negative DNA, polycation, and microsomes (membrane is brown, CPR = red, and cyt P450 = green). NADPH generated by an enzyme reaction in solution reduces CPR, which transfers electrons to cyt P450s. O₂ and Cyt P450s can combine to convert substrate to reactive metabolites that form DNA adducts in the film. ECL is detected with a CCD camera on the array upon application of +1.25 V vs Ag/AgCl and monitors DNA adduct accumulation. Hydrolysis releases labile DNA adducts from the nanoreactors for capLC–MS analysis.