Synergistic metabolic toxicity screening using microsome/DNA electrochemiluminescent arrays and nanoreactors.
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.
Krishnan
Sadagopan
S
Department of Chemistry, 55 North Eagleville Road, University of Connecticut, Storrs, Connecticut 06269, USA.
Hvastkovs
Eli G
EG
Bajrami
Besnik
B
Choudhary
Dharamainder
D
Schenkman
John B
JB
Rusling
James F
JF
D000818:Animals; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism; D004247:DNA / Q000032:analysis* / Q000187:drug effects* / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods*; D004357:Drug Synergism; D064420:Drug-Related Side Effects and Adverse Reactions; D004563:Electrochemistry; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D015394:Molecular Structure; D054118:Nanospheres; D051381:Rats; D053719:Tandem Mass Spectrometry; D018675:Toxicity Tests / Q000379:methods*
D004247:DNA; D019392:Cytochrome P-450 CYP2E1