Bioelectrochemical Platform with human liver enzymes for Metabolic Testing of Xenobiotics

Analysis of the metabolic processes of xenobiotics are crucial to understanding their efficacy, toxicity and elimination modes. Traditionally, these studies are conducted using in vivo and in vitro models, such as cell cultures and liver microsomes. However, these techniques have several limitations, including high cost, experimental complexity and the use of living organisms. In this context, electrochemical platforms emerge as innovative and advantageous tools to mimic the metabolic reactions of xenobiotics. In pharmaceutical and toxicological research, electrochemical biosensors play a crucial role in mimicking the metabolic activity of human enzymes, such as cytochrome P450s and flavin-containing monooxygenases (FMOs). The mammalian FMO family consists of a diverse range of microsomal enzymes that contain flavin adenine dinucleotide (FAD), NADPH dependent. Cytochrome P450 monooxygenases (CYPs) are a superfamily of haem-containing proteins that require a NADPH-reductase to conduct their reaction. These enzymes utilize molecular oxygen to oxidize a wide variety of structurally different xenobiotics, including numerous pharmaceutical compounds. Previous study by Sadeghi et al. showed the creation of a tandem bioelectrochemical set up in which the organophosphate pesticide phorate goes through an initial oxidation by hFMO1 (Sulfoxide form) and a subsequent oxidation by CYP3A4 (Sulfone form). Based on this study, in this work a new tandem bioelectrochemical reaction was set up, using hFMO3 and CYP1A2, for the conversion of sulindac, a nonsteroidal, anti inflammatory drug (NSAID) with anticancer activity, in its sulfoxide and sulfone forms. First, transformation, expression and purification of both proteins were performed. Subsequently, both enzyme catalysis was investigated by carrying out chronoamperometry in the presence of the substrate, to identify and measure the quantity of the product generated using HPLC. hFMO3 was initially immobilized on glassy carbon electrodes in the presence of Dimethyldioctadecylammonium bromide (DDAB) for the oxidation of sulindac sulfide into sulindac sulfoxide. Then, CYP1A2 was immobilized with the same procedure to mediate the second oxidation from sulindac sulfoxide into sulindac sulfone. 4 HPLC analysis reveals that, for both reactions, the amount of product generated was very limited and therefore a tandem reaction would be impossible. In conclusion, new in-depth studies will have to be carried out to obtain reaction conditions that will allow a higher yield in the sulindac-sulfide conversion reaction by hFMO3 in such a way as to favor a tandem reaction.