Odorant Metabolism Analysis by an Automated Ex Vivo Headspace Gas-Chromatography Method.

In the olfactory epithelium (OE), odorant metabolizing enzymes have the dual function of volatile component detoxification and active clearance of odorants from the perireceptor environment to respectively maintain the integrity of the tissues and the sensitivity of the detection. Although emphasized by recent studies, this enzymatic mechanism is poorly documented in mammals. Thus, olfactory metabolism has been characterized mainly in vitro and for a limited number of odorants. The automated ex vivo headspace gas-chromatography method that was developed here was validated to account for odorant olfactory metabolism. This method easily permits the measurement of the fate of an odorant in the OE environment, taking into account the odorant gaseous state and the cellular structure of the tissue, under experimental conditions close to physiological conditions and with a high reproducibility. We confirmed here our previous results showing that a high olfactory metabolizing activity of the mammary pheromone may be necessary to maintain a high level of sensitivity toward this molecule, which is critical for newborn rabbit survival. More generally, the method that is presented here may permit the screening of odorants metabolism alone or in mixture or studying the impact of aging, pathology, polymorphism or inhibitors on odorant metabolism.

When the nose must remain responsive: glutathione conjugation of the mammary pheromone in the newborn rabbit.

In insects, xenobiotic-metabolizing enzymes were demonstrated to regulate pheromones inactivation, clearing them from the olfactory periphery and keeping receptors ready for stimulation renewal. Here, we investigate whether similar processes could occur in mammals, focusing on the pheromonal communication between female rabbits and their newborns. Lactating rabbits emit in their milk a volatile aldehyde, 2-methylbut-2-enal, that elicits searching-grasping in neonates; called the mammary pheromone (MP), it is critical for pups which are constrained to find nipples within the 5 min of daily nursing. For newborns, it is thus essential to remain sensitive to this odorant during the whole nursing period to display several actions of sucking. Here, we show that the MP is enzymatically conjugated to glutathione in newborn olfactory epithelium (OE), in accordance with the high mRNA expression of glutathione transferases evidenced by quantitative reverse transcription-PCR. This activity in the nose is higher than in the liver and in OE of newborns compared with weanlings (no more responsive to the pheromone). Therefore, the results pinpoint the existence of a high level of MP-glutathione conjugation activity in the OE of young rabbits, especially in the developmental window where the perceptual sensitivity toward the MP is crucial for survival.

Ciprofibrate regulation of rat hepatic bilirubin glucuronidation and UDP-glucuronosyltransferases expression.

Synthetic fibrates are hypolipidemic drugs known to stimulate hepatic peroxisome proliferation and bilirubin glucuronidation. This study was designed to estimate the effects of ciprofibrate simultaneously on rat hepatic bilirubin glucuronoconjugation and on hepatic expression of UGT1A1, UGT1A2 and UGT1A5, all of which belong to the bilirubin cluster. Hepatic bilirubin glucuronidation activity and UDP-glucuronosyltransferase expression (RT-PCR and Western blotting) were measured after a single-dose ciprofibrate treatment (5 mg/kg by gastric intubation) in 36-h time course experiments. Ciprofibrate regulation of PPARα and UGT1A5 mRNA expression was also investigated in rat hepatocytes. Bilirubin conjugation activity was induced by ciprofibrate, reaching a maximum level (2.4×) 24 h after the treatment. UGT1A1 and UGT1A5 mRNA expression was induced 1.5 times by ciprofibrate, with UGT1A5 reaching the basal level of UGT1A1. Although UGT1A2 mRNA was induced approximately threefold by ciprofibrate, its expression level remained low in comparison with basal or induced levels of UGT1A1 and UGT1A5 mRNA. In the 36-h time course experiment, bilirubin conjugation activity as well as UGT1A5 and PPARα mRNA expression presented a biphasic induction profile. Although a similar level of induction was observed in primary cultured hepatocyte experiments, such biphasic variation was not observed for both UGT1A5 and PPARα, and the induction of UGT1A5 mRNA expression by ciprofibrate required de novo protein synthesis. A single dose of ciprofibrate significantly induces rat liver bilirubin conjugation as well as UGT1A1, UGT1A5 and PPARα expression. The induction mechanism may involve PPARα, at least regarding UGT1A5 regulation.

UDP-glucuronosyltransferases (UGTs) in neuro-olfactory tissues: expression, regulation, and function.

This work aims to review uridine diphosphate (UDP)-glucuronosyltransferase (UGT) expression and activities along different neuronal structures involved in the common physiological process of olfaction: olfactory epithelium, olfactory bulb, and olfactory cortex. For the first time, using high-throughput in situ hybridization data generated by the Allen Brain Atlas (ABA), we present quantitative analysis of spatial distribution of UGT genes in the mouse brain. The olfactory area is a central nervous system site with the highest expression of UGTs, including UGT isoforms not previously identified in the brain. Since there is evidence of the transfer of xenobiotics to the brain through the nasal pathway, circumventing the blood-brain barrier, olfactory UGTs doubtlessly share the common function of detoxification, but they are also involved in the metabolism and turnover of exogenous or endogenous compounds critical for physiological olfactory processing in these tissues. The function of olfactory UGTs will be discussed with a special focus on their participation in the perireceptor events involved in the modulation of olfactory perception.

Effects of typical inducers on olfactory xenobiotic-metabolizing enzyme, transporter, and transcription factor expression in rats.

Several xenobiotic-metabolizing enzymes (XMEs) have been identified in the olfactory mucosa (OM) of mammals. However, the molecular mechanisms underlying the regulation of these enzymes have been little explored. In particular, information on the expression of the transcriptional factors in this tissue is quite limited. The aim of the present study was to examine the impact of five typical inducers, Aroclor 1254, 3-methylcholanthrene, dexamethasone, phenobarbital, and ethoxyquin, on the activities and mRNA expression of several XMEs in the OM and in the liver of rats. We also evaluated the effects of these treatments on the mRNA expression of transcription factors and transporters. On the whole, the intensities of the effects were lower in the OM than in the liver. Dexamethasone was found to be the most efficient treatment in the OM. Dexamethasone induced the transcription of several olfactory phase I, II, and III genes [such as cytochromes P450 2A3 and 3A9, UDP-glucuronosyltransferase (UGT) 2A1, and multidrug resistance-related protein type 1] and increased UGT activities. We observed that dexamethasone up-regulated sulfotransferase 1C1 expression in the OM but down-regulated it in the liver. Aroclor and ethoxyquin induced the gene expression of CYP1A and quinone reductase, respectively, in the OM. The transcription factors aryl hydrocarbon receptor, nuclear factor E2-related factor 2 (Nrf2), peroxisome proliferator-activated receptor α, pregnane X receptor, and glucocorticoid receptor were detected in the OM, but no constitutive androstane receptor expression was observed. Dexamethasone and Aroclor enhanced olfactory Nrf2 expression. These results demonstrate that olfactory XME can be modulated by chemicals and that the mechanisms involved in the regulation of these enzymes are tissue-specific.

Modulation of Sex Pheromone Discrimination by A UDP-Glycosyltransferase in Drosophila melanogaster.

The detection and processing of chemical stimuli involve coordinated neuronal networks that process sensory information. This allows animals, such as the model species Drosophila melanogaster, to detect food sources and to choose a potential mate. In peripheral olfactory tissues, several classes of proteins are acting to modulate the detection of chemosensory signals. This includes odorant-binding proteins together with odorant-degrading enzymes (ODEs). These enzymes, which primarily act to eliminate toxic compounds from the whole organism also modulate chemodetection. ODEs are thought to neutralize the stimulus molecule concurrently to its detection, avoiding receptor saturation thus allowing chemosensory neurons to respond to the next stimulus. Here, we show that one UDP-glycosyltransferase (UGT36E1) expressed in D. melanogaster antennal olfactory sensory neurons (OSNs) is involved in sex pheromone discrimination. UGT36E1 overexpression caused by an insertion mutation affected male behavioral ability to discriminate sex pheromones while it increased OSN electrophysiological activity to male pheromones. Reciprocally, the decreased expression of UGT36E1, controlled by an RNAi transgene, improved male ability to discriminate sex pheromones whereas it decreased electrophysiological activity in the relevant OSNs. When we combined the two genotypes (mutation and RNAi), we restored wild-type-like levels both for the behavioral discrimination and UGT36E1 expression. Taken together, our results strongly suggest that this UGT plays a pivotal role in Drosophila pheromonal detection.

The Radioprotective Effect of Procaine and Procaine-Derived Product Gerovital H3 in Lymphocytes from Young and Aged Individuals.

Ionizing radiation induces genomic instability in living organisms, and several studies reported an ageing-dependent radiosensitivity. Chemical compounds, such as scavengers, radioprotectors, and modifiers, contribute to reducing the radiation-associated toxicity. These compounds are often antioxidants, and therefore, in order to be effective, they must be present before or during exposure to radiation. However, not all antioxidants provide radioprotection. In this study, we investigated the effects of procaine and of a procaine-based product Gerovital H3 (GH3) on the formation of endogenous and X-ray-induced DNA strand breaks in peripheral blood mononuclear cells (PBMCs) isolated from young and elderly individuals. Interestingly, GH3 showed the strongest radioprotective effects in PBMCs from young subjects, while procaine reduced the endogenous amount of DNA strand breaks more pronounced in aged individuals. Both procaine and GH3 inhibited lipid peroxidation, but procaine was more effective in inhibiting mitochondria free radicals' generation, while GH3 showed a higher antioxidant action on macrophage-induced low-density lipoprotein oxidation. Our findings provide new insights into the mechanisms underlying the distinct effects of procaine and GH3 on DNA damage.

Drug metabolizing enzyme expression in rat choroid plexus: effects of in vivo xenobiotics treatment.

The presence of drug metabolizing enzymes in extrahepatic tissues such as the choroid plexus (CP) suggests that the CP, like the blood-brain barrier, affords a metabolic protection to the brain against xenobiotics. The CP, which is the principal site of formation of the cerebrospinal fluid (CSF), controls the exchange of many endogenous compounds and exogenous molecules between brain tissue and CSF. We present the changes in mRNA expression and enzymatic activities of UDP-glucuronosyltransferase, UGT1A6 isoform and NADPH-cytochrome P450 reductase, after in vitro treatment with xenobiotic molecules known to act in the liver as inducers or inhibitors of these drug metabolizing enzymes. Five study groups of male Sprague-Dawley rats were treated separately with 3-methylcholantrene (3-MC), phenobarbital (PB), dexamethasone (DEX), cyclosporine (CsA) or paraquat (PQ). Choroidal 1-naphthol glucuronidation activities were significantly induced by 3-MC and PQ administration (354 +/- 85 and 257 +/- 49 vs. 115 +/- 24 nmol/h per mg protein, in control group), whereas the other molecules were without effect. Accordingly, UGT1A6 mRNA expression, measured by RT-PCR, was 2.3-fold higher after 3-MC treatment and 2.1-fold higher after PQ administration. By contrast, reductase activities and mRNA expression remained unchanged in the isolated choroids plexus in these experimental conditions. We present for the first time evidences that the choroids plexus express transcripts for both UGT1A6 and NADPH-cytochrome P450 reductase, and their mRNA expression can be differently regulated by exogenous factors. These results emphasize that xenobiotics could modulate the biotransformation of exogenous and/or endogenous compounds in the choroids plexus, and underline the role of UGTs in the maintenance of brain homeostasis.

Nasal mucus glutathione transferase activity and impact on olfactory perception and neonatal behavior.

In olfaction, to preserve the sensitivity of the response, the bioavailability of odor molecules is under the control of odorant-metabolizing enzymes (OMEs) expressed in the olfactory neuroepithelium. Although this enzymatic regulation has been shown to be involved in olfactory receptor activation and perceptual responses, it remains widely underestimated in vertebrates. In particular, the possible activity of OMEs in the nasal mucus, i.e. the aqueous layer that lined the nasal epithelium and forms the interface for airborne odorants to reach the olfactory sensory neurons, is poorly known. Here, we used the well-described model of the mammary pheromone (MP) and behavioral response in rabbit neonates to challenge the function of nasal mucus metabolism in an unprecedented way. First, we showed, in the olfactory epithelium, a rapid glutathione transferase activity toward the MP by ex vivo real-time mass spectrometry (PTR-MS) which supported an activity in the closest vicinity of both the odorants and olfactory receptors. Indeed and second, both the presence and activity of glutathione transferases were evidenced in the nasal mucus of neonates using proteomic and HPLC analysis respectively. Finally, we strikingly demonstrated that the deregulation of the MP metabolism by in vivo mucus washing modulates the newborn rabbit behavioral responsiveness to the MP. This is a step forward in the demonstration of the critical function of OMEs especially in the mucus, which is at the nasal front line of interaction with odorants and potentially subjected to physiopathological changes.

Publisher Correction: Nasal mucus glutathione transferase activity and impact on olfactory perception and neonatal behavior.

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