Arrays suitable for genotoxicity screening are reported that generate metabolites from cytochrome P450 enzymes (CYPs) in thin-film spots. Array spots containing DNA, various human cyt P450s, and electrochemiluminescence (ECL) generating metallopolymer [Ru(bpy) 2 PVP 10 ] 2+ were exposed to H 2 O 2 to activate the enzymes. ECL from all spots was visualized simultaneously using a CCD camera. Using benzo [a]pyrene as a test substrate, enzyme activity for producing DNA damage in the arrays was found in the order CYP1B1 > CYP1A2 > CYP1A1 > CYP2E1 > myoglobin, the same as the order of their metabolic activity. Thus, these arrays estimate the relative propensity of different enzymes to produce genotoxic metabolites. This is the first demonstration of ECL arrays for highthroughput in vitro genotoxicity screening.Bioactivation of xenobiotic molecules by cytochrome P450 (cyt P450, or CYP) enzymes in the human liver is a major source of genotoxicity. Metabolites formed in this way can cause damage to genetic material. 1,2 Since levels of cyt P450 enzyme expression vary dramatically in different individuals, subpopulations may be subject to varying degrees of chemical or drug toxicity. 3 Thus, knowledge of which isoforms of cyt P450 produce toxic metabolites is critical in the development of new drugs, agricultural chemicals, and other substances that impact the public.Genotoxic metabolites and their nucleobase adducts can be detected by separation methods such as LC-MS. 4,5 These methods are very sensitive and provide specific and detailed molecular information, but may be limited for screening by throughput, analysis time, and cost. On the other hand, alternative, relatively rapid array technologies have been very successful in genomics and proteomics and are in principle capable of many thousands of measurements on a single chip. 6We recently demonstrated rapid, inexpensive, voltammetric genotoxicity screening sensors assembled from films of DNA and cyt P450s in single-electrode 7 and eight-electrode formats. 8 In a two-step process, test molecules are first bioactivated by the enzymes. Then, possible adducts with DNA nucleobases are detected by voltammetry using ruthenium tris(2,2′-bipyridyl)Ru II (Ru(bpy) 3 2+ ) to catalytically oxidize the guanine bases 9 in DNA. Nucleobase adducts formed as a consequence of the enzyme reaction in the enzyme/DNA films are not detected directly, but increases in voltammetric peaks result because the adducts cause DNA * To whom correspondence should be addressed. E-mail: james.rusling@uconn.edu. † University of Connecticut. ‡ University of Connecticut Health Center. [Ru(bpy) 2 (PVP) 10 ] 2+ contains six N-bonds to Ru, and is thought to produce ECL upon reaction with guanines in DNA according to the following pathway: NIH Public AccessECL generated from adsorbed polymer films is more efficient and intense compared to ECL produced by solution species. 15 The Ru II center is oxidized by the electrode (eq 1) and subsequently oxidizes a guanine in DNA to form a guanine radical (eq 2)...
Neutral hydrolysis and LC-MS/MS analysis of 6-nm-thick DNA-polyion films used in voltammetric genotoxicity screening sensors showed that concentrations of N7-guanine DNA adducts with methyl methanesulfonate and styrene oxide increased with incubation time with the same trends as found for sensor response. Results show that the genotoxicity sensors can be used to estimate relative DNA damage rates for chemical toxicity screening. Neutral thermal hydrolysis provided a relatively clean sample matrix allowing quantitative estimates of nucleobase adducts after several minutes of incubation with damage agents. In addition, an approximate standardization procedure for neutral thermal hydrolysis was developed and validated that avoids need for a pure standard and should be useful in cases where nucleobase adduct standards are unavailable or where their identities are unknown.
Detection of DNA adducts can serve as a basis for genotoxicty screening of new chemicals and drugs. We report here a simple, sensitive procedure for this purpose using films containing DNA and a biocatalyst to mimic the metabolic action of human liver cytochrome P450s. DNA adducts formed from an in-situ-generated toxic metabolite (styrene oxide) were detected at subpicomole levels after neutral thermal hydrolysis of the DNA films and analysis with capillary liquid chromatography with on-line column preconcentration and MS/MS detection. An on-line column switching system allowed for increased sample loading volume and analyte preconcentration. This approach provides an estimate of the relative rate of DNA damage.
The detection and identification of DNA adducts is important for predicting human cancer risk posed by chemicals and for uncovering potential genotoxicity of new drug and agricultural chemical candidates. For compounds that react with DNA to form N7-guanine and/or N3-adenine adducts, neutral thermal hydrolysis provides a simple procedure for sample preparation. The N7-guanine and N3-adenine adducts are selectively ejected from the DNA chain, resulting in a clean sample matrix enriched in nucleobase adducts. Coupling neutral thermal hydrolysis with liquid chromatography-mass spectrometry (LC-MS) provides sensitive methods to detect and quantitate DNA adducts, and structural information is provided by MS. Combining these technologies with capillary liquid chromatography sample preconcentration systems can provide exquisitely sensitive detection. In this review, we first summarize the chemistry of nucleobase adduct formation, briefly summarize modern methods to detect DNA adducts, and then describe neutral thermal hydrolysis coupled to LC-MS/MS and some of its applications to DNA damage studies. Finally, we review recent applications of neutral thermal hydrolysis and LC-MS to toxicity screening of chemicals.
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