Thus, almost all experimental protocols were conducted in accordance with ethical rules enforced by French legislation, and were approved by the local Ethical Committee of the University of Burgundy (Comit dEthique de lExprimentation Animale Grand Campus Dijon; C2EA grand campus Dijon N105), and by the French Ministre de lEducation Nationale, de lEnseignement Suprieur et de la Recherche under the no. headspace. These results significantly switch the picture of real-time odorant metabolism and represent a new step forward in the investigation of the function of odorant metabolites in the peripheral olfactory process. Our method allows the systematic identification of odorant metabolites using a validated animal model and permits the screening of olfactory endogenously produced chemosensory molecules. olfactory neurons). The residence time of odorants in the OM environment affects their bioavailability, which is critical Rabbit Polyclonal to Cytochrome P450 39A1 regarding (i) activation the saturation of olfactory receptors, (ii) potential toxicity for the OM and (iii) distribution of odorants to the brain or rest of the body. Odorant bioavailability is usually under the control of perireceptor events, including the action of odorant-metabolizing enzymes (OMEs) involved in odorant biotransformation5. OMEs are xenobiotic-metabolizing enzymes involved in detoxification by the enzymatic deactivation of chemicals and conversion into very easily eliminable hydrophilic metabolites6. Odorants are substrates of these enzymes, which are highly expressed in olfactory TTA-Q6(isomer) tissues (and in comparable concentrations to those in the liver, if measured on a per-cm2 tissue basis)7C10. In addition to some studies conducted with insects11C13, recent studies have exhibited the function of perireceptor OMEs in odorant biotransformation catalysis in vertebrates, as well as olfactory transmission modulation and, consequently, olfactory belief itself14C18. We recently exhibited that odorant-odorant competitive interactions exist at the enzyme level for the odorant 2-methylbut-2-enal (the mammary pheromone) in rabbits. Conceptually, if two odorants compete with the same enzyme in the OM, one odorant is usually metabolized at the expense of the second that accumulates and activates more receptors. Accordingly, in rabbit pups, such metabolic competition with a competitor odorant strikingly enhanced belief of the mammary pheromone14. Enhancement of the transmission consecutively to odorant accumulation was also observed in rats using electrophysiology after exposure to OME chemical inhibitors18. However, the odorant transmission rapidly decreases due to the saturation of the receptors and neuronal adaptation. Nagashima and Touhara (2010) showed that, after exposing mice to odorants, their metabolites were detected in the mucus washed out from the nasal cavity. Moreover, following treatment with the corresponding OME inhibitors, they observed significant changes in both the activated glomerular pattern in the olfactory bulb and olfactory belief in response to odorants. The authors proposed that metabolites, by potentially interacting with receptors, might be involved in the perception initiated by the parent odorant16,17. Additionally, in a single study in humans, the presence of odorant metabolites has been exhibited by an atmospheric pressure chemical ionization (APCI) ion source in exhaled breath after odorant inhalation17. This direct-injection mass spectrometry technique is very suitable for real-time analysis of volatile molecules from biological environments19. Despite these improvements, the significance of OMEs in the process of olfaction remains debatable because few aspects are known about the enzymatic mechanism and its ability to generate odorant metabolites, especially under experimental conditions directly focusing on the tissue involved: the neuroepithelium. We previously set up and validated an automated headspace gas chromatography (GC) method20. Odorants in the gas phase were injected into the headspace of a vial containing a fresh explant of OM, and then the headspace was sampled and injected into the GC for analysis. We measured a decrease in the odorant concentration, which accounts for its metabolism by the tissue explant under near-biological conditions20. Using the same experimental conditions, after a single injection TTA-Q6(isomer) of the odorant in the headspace, we used direct-injection proton transfer reaction-mass spectrometry (PTR-MS) to monitor the metabolism of ethyl acetate and the corresponding ethanol metabolite synthesis in real-time21. However, this device only allowed discontinuous recording that started from 10?seconds and was affected by a slow headspace TTA-Q6(isomer) equilibrium due to the experimental conditions (odorant injection in a 20-mL vial). Here, we developed and validated an innovative technical approach based on continuous direct-injection analysis mass spectrometry using PTR-MS. It was designed to constantly deliver odorants to the OM explants to allow real-time monitoring of the headspace for both odorant TTA-Q6(isomer) uptake and the release.
