Quantification of the activity of detoxifying enzymes in terrestrial invertebrates: Optimization, evaluation and use of in vitro and ex vivo methods

Abstract

1. The negative effects of pesticides on beneficial terrestrial invertebrates have received much attention, and in this context quantification of the activity of metabolic enzymes provides an important tool. Unfortunately, current methodologies are limited in their use.2. In this study, the grain beetle, Tenebrio molitor was used to illustrate why in vitro methods are ineffective to quantify metabolic enzymes in some insects. It was further used to optimize an ex vivo assay that can replace in vitro methods when these are ineffective. The ex vivo method can quantify metabolic enzymatic activities of cytochrome P450 mixed function oxidases (CYPs), general esterases (EST) and glutathione S-transferases (GST).3. In detail we show, that T. molitor produces inhibitory and/or degrading agents, which are released during tissue homogenization. As these agents inhibit the in vitro CYP activity of rat liver microsomes we suggest, that they are also responsible for the lack of in vitro CYP activity of T. molitor. We tested the effectiveness of broad-spectrum protease inhibitors as protective agents and found, that they were only effective in concentrations that also interfered directly with the activity of CYP enzymes. As an alternative to in vitro measurements, we optimized an ex vivo method for quantification of CYP, EST and GST on individual gut systems of T. molitor. Our ex vivo method advances previous ex vivo methods, by allowing quantification of three major enzyme families, all in a single individual, and by exploiting real-time kinetic measurements of the enzymatically produced product.4. Subsequently, our method was successfully tested for its use on three different life stages: larvae, pupae, and adult insects from T. molitor and Apis mellifera. The method shows promise for expansion to other species and potentially other enzyme families and/or substrates. K E Y W O R D SApis mellifera, detoxification, enzymatic activity, ex vivo, in vitro, Tenebrio molitor, terrestrial insect | 727Methods in Ecology and Evoluঞon PEDERSEN Et al.