New approaches to old problems: Removal of phospholipase A2 results in highly active microsomal membranes from the honey bee, Apis mellifera

Abstract

Over the last 50 years numerous studies were published by insect toxicologists using native microsomal membrane preparations in order to investigate in vitro cytochrome P450-(P450) mediated oxidative metabolism of xenobiotics, including insecticides. Whereas the preparation of active microsomal membranes from many pest insect species is straightforward, their isolation from honey bees, Apis mellifera (Hymenoptera: Apidae) remained difficult, if not impossible, due to the presence of a yet unidentified endogenous inhibitory factor released during abdominal gut membrane isolation. Thus hampering in vitro toxicological studies on microsomal oxidative phase 1 metabolism of xenobiotics, including compounds of ecotoxicological concern. The use of microsomal membranes rather than individually expressed P450s offers advantages and allows to develop a better understanding of phase 1 driven metabolic fate of foreign compounds. Here we biochemically investigated the problems associated with the isolation of active honey bee microsomes and developed a method resulting in highly active native microsomal preparations from adult female worker abdomens. This was achieved by removal of the abdominal venom gland sting complex prior to microsomal membrane preparation. Molecular sieve chromatography of the venom sac content leads to the identification of phospholipase A2 as the enzyme responsible for the immediate inhibition of cytochrome P450 activity in microsomal preparations. The substrate specificity of functional honey bee microsomes was investigated with different fluorogenic substrates, and revealed a strong preference for coumarin over resorufin derivatives. Furthermore we were able to demonstrate the metabolism of insecticides by honey bee microsomes using an approach coupled to LC-MS/MS analysis of hydroxylated metabolites. Our work provides access to a new and simple in vitro tool to study honey bee phase 1 metabolism of xenobiotics utilising the entire range of microsomal cytochrome P450s.