1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 DOI: 10.1002/elan.201600713 Human Cytochrome P450 (CYP1A2)-dsDNA Interaction in situ Evaluation Using a dsDNA-electrochemical Biosensor Ilanna Campelo Lopes [a] and Ana Maria Oliveira-Brett* [a] Abstract: Human cytochrome CYP1A2 is one of the major hepatic cytochrome P450s involved in many drugs metabolism, and chemical carcinogens activation. The CYP1A2-dsDNA interaction in situ evaluation using a DNA-electrochemical biosensor and differential pulse voltammetry was investigated. A dsDNA-electrochemical biosensor showed that CYP1A2 interacted with dsDNA causing conformational changes in the double helix chain and DNA oxidative damage. A preferential interaction between the dsDNA guanosine residues and CYP1A2 was found, as free guanine and 8-oxoguanine, a DNA oxidative damage biomarker, oxidation peaks were de- tected. This was confirmed using guanine and adenine homopolynucleotides-electrochemical biosensors. The CYP1A2-dsDNA interaction and dsDNA conformation changes was also confirmed by UV-Vis spectrophotom- etry. Keywords: CYP1A2 · Cytochrome P450 oxidation · CYP1A2-dsDNA interaction · DNA-electrochemical biosensor · DNA oxidative damage · glassy carbon electrode 1 Introduction Cytochromes P450 are ubiquitously distributed enzymes that were discovered about 50 years ago, possess high complexity and display a large field of activity. They are hemoproteins encoded by a superfamily of genes convert- ing a broad variety of substrates and catalysing many important chemical reactions. They are involved in the biotransformation of drugs, the bioconversion of xeno- biotics, the metabolism of chemical carcinogens, the biosynthesis of physiologically important compounds, such as steroids, fatty acids, eicosanoids, fat-soluble vitamins, bile acids, the conversion of alkanes, terpenes, and aromatic compounds, as well as in the degradation of herbicides and insecticides, and a great variety of catalysed reactions, like carbon hydroxylation, heteroatom oxygenation, dealkylation, epoxidation, aromatic hydrox- ylation, reduction, and dehalogenation [1–5]. The P450 classical reaction is the introduction of an oxygen atom (derived from molecular oxygen) to facili- tate hydroxylation at an inactivated carbon centre on a molecule. To achieve this reaction, oxygen is bound to the ferrous heme at the centre of a P450 bound to a b-type heme. The present P450 activity model involves further reductive activation and protonation of the oxyferrous species, ultimately leading to the formation of a highly reactive ferryl-oxo intermediate and the production of a water molecule. The ferryl-oxo intermediate attacks a substrate molecule bound proximal to the heme iron to perform the oxygenation and to restore the resting state of the P450 [6–8]. Since many of the individual P450 catalyse multiple reactions, the usual method of naming enzymes is inadequate for this group of proteins, and a systematic nomenclature has been derived based on structural homology [9, 10]. P450 genes, for example CYP1A2, are identified by the abbreviation CYP followed by a number denoting the family (proteins with more than 40 % sequence identity), a letter designating a subfamily (more than 55% identity) and a number representing the individual gene within the subfamily, and 267 families with more than 5000 genes were notified [11, 12]. The crystal structure of human microsomal P450 CYP1A2, with twelve a-helices, four b-sheets and heme prostetic group, in a complex with alpha-naphthoflavone is in Scheme 1, [13, 14]. CYP1A2, a member of the P450 superfamily of proteins, is one of the major inducible P450 proteins in the human liver (~ 13–15 %), where it has a basal expression level [15]. CYP1A2 is involved not only in the metabolism of caffeine [16] and therapeutic drugs, such as acetaminophen, theophylline, and propranolol [17–18], but also in the activation of food contaminating carcino- gens, like aflatoxin B1 [19, 20] and heterocyclic amines [21–23]. Furthermore, CYP1A2 is considered to participate in the metabolism of endogenous steroids [24, 25] and is inducible by a wide variety of compounds present in the environment, including constituents of cooked meat, cruciferous vegetables and cigarette smoke [26], catalyses carcinogens, to highly reactive intermediates that cova- [a] I. C. Lopes, A. M. Oliveira-Brett Chemistry Department, Faculty of Sciences and Technology, University of Coimbra, 3004-535 Coimbra, Portugal E-mail: brett@ci.uc.pt Full Paper www.electroanalysis.wiley-vch.de # 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2017, 29, 1674 – 1682 1674