Adult honey bee microsomal preparations: how to make them work

Over the last 50 years numerous studies were published by insect toxicologists using native microsomal membrane preparations in order to investigate in vitro the cytochrome P450-driven metabolism of xenobiotics, including insecticides. This protein super family includes many members sitting in microsomal membranes and facilitating oxidative metabolism. Whereas the preparation of active microsomal membranes from many insect species is straightforward, their isolation from Apis mellifera (Hymenoptera: Apidae) worker bees remains difficult if not impossible as the lack of publications using honey bee microsomes obviously suggests. However, investigating the oxidative detoxification of xenobiotics by microsomal membranes offers the advantage of having most P450s present and acting in concert, and thus allowing to develop a better understanding of the metabolic fate of endogenous as well as foreign compounds such as insecticides. 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 workers. We investigated the substrate specificity by incubating honey bee microsomes with 13 different fluorogenic artificial substrates, revealing a strong preference for coumarin over resorufin derivatives. Furthermore, we were able to demonstrate the metabolism of insecticides by honey bee microsomes in comparative studies using an approach coupled to subsequent LC-MS/MS analysis of hydroxylated products.