Electrochemiluminescent Arrays for Cytochrome P450-Activated Genotoxicity Screening. DNA Damage from Benzo[a]pyrene Metabolites

Abstract

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)...

Enzyme−DNA Biocolloids for DNA Adduct and Reactive Metabolite Detection by Chromatography−Mass Spectrometry

“…[19][20][21] The lower surface coverage on the beads could be a result of the nonfunctionalized nature of the silica versus that of a gold resonator. Also, with regard to cyt P450 cam , the exposed surface area per nanomole is significantly less than when employing gold-coated QCM resonators.…”

“…The diameter increased ∼5 nm after each layer was deposited, which is expected based on layer thicknesses when using high saline polymer solutions and the thickness of Mb layers (∼5 nm) in previous reports. 19,21,46 Myoglobin Activity Assay. The activity of immobilized Mb was initially characterized using the oxidation of classic peroxidase substrate guaiacol activated by hydrogen peroxide to generate the colored dimer product, 3,3′-dimethoxy-4,4′-biphenoquinone (λ max ) 470 nm).…”

“…18 We have developed biosensors 19 (6) Kirkland and arrays based on this principle for early toxicity screening featuring thin films of DNA and cyt P450 enzymes using voltammetric 20 and electrochemiluminescent (ECL) platforms. 21 The enzymes in these films metabolize drug or pollutant substrates to produce possible reactive metabolites that may form DNA adducts. High-throughput electrochemical and ECL arrays can provide simultaneous, rapid assessment of relative DNA damage rates from chemicals and metabolites from a range of enzymes.…”

“…[19][20][21]23 It is well-established that metabolite specificity is a property of the terminal oxidase, cyt P450, and that hydrogen peroxide or organic hydroperoxides activate cyt P450 to yield the same products 32 with possible deviations in product distribution. 11b,33 In cases examined in our laboratory to date, [19][20][21]23,29,34,43 we have not found that Scheme 1. Conceptual Illustration of DNA/ Enzyme Film Formation on Silica Microbeads a a Layer-by-layer electrostatic assembly is used to immobilize positively charged PDDA on the negative microbeads, followed by sequential adsorption of polyanion PSS or DNA and a positively charged enzyme layer (blue circles).…”

Accurate DNA Fragment Sizing by Capillary Electrophoresis with Laser-Induced Fluorescence Array for Detection of Sequence Specificity of DNA Damage

“…The predominant peaks within the range of the size standards are labeled 1-11. Using the correction table, these correspond to DNA fragments that are 29,31,33,40,45,48,50,63,65,69, and 72 bases, respectively. (Figure 2b).…”