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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)...

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ABSTRACT:The cytochrome P450 family 1 (CYP1) is considered to be one of the xenobiotic-metabolizing enzyme families and is responsible for oxidative metabolism of polycyclic aromatic hydrocarbons. For example, mouse Cyp1b1 was originally identified as the enzyme responsible for oxidative metabolism of 7,12-dimethylbenz(␣)anthracene (DMBA). A comparison of the kinetics of this metabolism by mouse and human CYP1B1 orthologs revealed the mouse enzyme to have a more favorable metabolism of DMBA, with a catalytic efficiency ratio (CER) of 0.23. However, CYP1 enzymes are also capable of metabolism of endobiotics, and in the present study, the metabolism of retinoids and lipid endobiotics by human CYP1B1 and mouse Cyp1b1 orthologs was compared. Both hemoproteins oxidized retinol to retinal and retinal to retinoate, but did not oxidize retinoate. The CYP1B1 to Cyp1b1 CERs were 13 and 26 for the two steps, respectively; the Cyp1b1 K m(app) values for retinoids were 20-fold higher. Human family 1 cytochromes P450 had unique regional specificities for arachidonate oxidation: the major metabolites of CYP1A1, CYP1A2, and CYP1B1 were 75% terminal hydroxyeicosatetraenoic fatty acids (HETEs), 52% epoxyeicosatrienoic fatty acids (EETs), and 54% midchain HETEs, respectively. CYP1A1 and CYP1B1 K m(app) values for arachidonate were about 30 M, whereas CYP1A2 K m(app) was 95 M. The major metabolites of arachidonic acid by Cyp1b1 were EETs (50%) and midchain HETEs (37%). The mouse ortholog had a CER for metabolite production of 64 due to a K m(app) of 0.5 mM for arachidonate.

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UV/Vis spectroscopy is the major means of identifying intact holocytochrome P450. The carbon monoxide complex of the intact ferrous hemoprotein exhibits a characteristic spectrum between 448 and 452 nm, considerably distinct from the usual Soret absorption peaks of hemoproteins. Methods are described for identification and quantitation of cytochrome P450 (CYP) in membranes, in tissue homogenates, and in purified form, using difference spectroscopy and absolute spectroscopy. CYP are b-type cytochromes, containing protoporphyrin IX as the prosthetic group. Methods are also provided, using alkali and pyridine, for quantitation of the hemoprotein by this prosthetic group. In its oxidized, or ferric state, CYP exists as an equilibrium mixture of high- and low-spin configurations, each with distinctive UV/Vis absorption peaks. Substrate binding causes shifts in the spin equilibrium, and methods are shown for using these shifts for quantitation of substrate binding to CYP.