Effect of oxidative stress on UDP-glucuronosyltransferases in rat astrocytes.

The present work reports data regarding effects of an induced oxidative stress on the mainly expressed isoforms of UDP-glucuronosyltransferases (UGTs) in the brain. UGT1A6 and UGT1A7 expression and enzymatic activities toward the 1-naphthol were analyzed in rat cultured astrocytes following the exposure for 48 h to redox-cycling xenobiotic compounds such as quinones and bipyridinium ions. The expression of NADPH:cytochrome P450 reductase and NAD(P)H:quinone oxidoreductase 1 (NQO1) was also investigated. Oxidative stress induced significant deleterious changes in astrocyte morphology, decreased cell viability and inhibited catalytic function of UGTs as a result of protein oxidation. Alternatively, in the surviving impaired astrocytes, oxidative conditions induced a significant overactivity and overexpression of xenobiotic detoxification enzymes, as adaptive response. These effects were significantly prevented by the presence of melatonin, suggesting its direct antioxidant action on reactive oxygen species, reflected further on the glucuronidation activity and transcriptional regulation of both UGT1A6 and UGT1A7. Results show that both catalytic properties of UGTs and the expression of UGT1A6, UGT1A7, NQO1 and NADPH:cytochrome P450 reductase in rat astrocytes are greatly influenced by the pro-oxidative environment. In conclusion, an experimental increase in oxidative cellular status could have both immediate and long term consequences on detoxification enzymatic system activity and expression.

Expression and differential localization of xenobiotic transporters in the rat olfactory neuro-epithelium.

Transporters, such as multidrug resistance P-glycoproteins (MDR), multidrug resistance-related proteins (MRP) and organic anion transporters (OATs), are involved in xenobiotic metabolism, particularly the cellular uptake or efflux of xenobiotics (and endobiotics) or their metabolites. The olfactory epithelium is exposed to both inhaled xenobiotics and those coming from systemic circulation. This tissue has been described as a pathway for xenobiotics to the brain via olfactory perineural space. Thereby, olfactory transporters and xenobiotic metabolizing enzymes, dedicated to the inactivation and the elimination of xenobiotics, have been involved in the toxicological protection of the brain, the olfactory epithelium itself and the whole body. These proteins could also have a role in the preservation of the olfactory sensitivity by inactivation and clearance of the excess of odorant molecules from the perireceptor space. The goal of the present study was to increase our understanding of the expression and the localization of transporters in this tissue. For most of the studied transporters, we observed an opposite mRNA expression pattern (RT-PCR) in the olfactory epithelium compared to the liver, which is considered to be the main metabolic organ. Olfactory epithelium mainly expressed efflux transporters (MRP, MDR). However, a similar pattern was observed between the olfactory epithelium and the olfactory bulb. We also demonstrate distinct cellular immunolocalization of the transporters in the olfactory epithelium. As previously reported, Mrp1 was mainly found in the supranuclear portions of supporting cells. In addition, Mrp3 and Mrp5 proteins, which were detected for the first time in olfactory epithelium, were localized to the olfactory neuron layer, while Mdr1 was localized to the capillary endothelium of lymphatic vessels in the subepithelial region. The pattern of expression and the distinct localization of the olfactory transporters showed in this work may highlight on their specific function in the whole olfactory epithelium.

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.

Characterization of microsomal cytochrome P450-dependent monooxygenases in the rat olfactory mucosa.

Nasal administration of a drug ensures therapeutic action by rapid systemic absorption and/or the entry of some molecules into the brain through different routes. Many recent studies have pointed out the presence of xenobiotic-metabolizing enzymes in rat olfactory mucosa (OM). Nevertheless, very little is known about the precise identity of isoforms of cytochrome P450 (P450)-dependent monooxygenases (P450) and their metabolic function in this tissue. Therefore, we evaluated mRNA expression of 19 P450 isoforms by semiquantitative reverse transcriptase-polymerase chain reaction and measured their microsomal activity toward six model substrates. For purposes of comparison, studies were conducted on OM and the liver. Specific activities toward phenacetin, chlorzoxazone, and dextromethorphan are higher in OM than in the liver; those toward lauric acid and testosterone are similar in both tissues, and that toward tolbutamide is much lower in OM. There are considerable differences between the two tissues with regard to mRNA expression of P450 isoforms. Some isoforms are expressed in OM but not in the liver (CYP1A1, 2G1, 2B21, and 4B1), whereas mRNA of others (CYP2C6, 2C11, 2D2, 3A1, 3A2, and 4A1) is present only in hepatic tissue. Although expression of CYP1A2, 2A1, 2A3, 2B2, 2D1, 2D4, 2E1, 2J4, and 3A9 is noticed in both tissues, there are a number of quantitative differences. On the whole, our results strongly suggest that CYP1A1, 1A2, 2A3, 2E1, 2G1, and 3A9 are among the main functional isoforms present in OM, at least regarding activities toward the six tested substrates. The implication of olfactory P450-dependent monooxygenases in toxicology, pharmacology, and physiology should be further investigated.

Drug transport into the mammalian brain: the nasal pathway and its specific metabolic barrier.

It is generally accepted that the rate of entry into and distribution of drugs and other xenobiotics within the central nervous system (CNS) depends on the particular anatomy of the brain microvessels forming the blood-brain barrier (BBB), and of the choroid plexus forming the blood-cerebrospinal fluid barrier (CSF), which possess tight junctions preventing the passage of most polar substances. Drug entry to the CNS also depends on the physicochemical properties of the substances, which can be metabolised during this transport to pharmacologically inactive, non-penetrating polar products. Finally, the entry of drugs may be prevented by multiple complex specialized carriers, which are able to catalyse the active transport of numerous drugs and xenobiotics out of the CNS. Nasal delivery is currently considered as an efficient tool for systemic administration of drugs that are poorly absorbed via the oral route, and increasing evidence suggests that numerous drugs and potentially toxic xenobiotics can reach the CNS by this route. This short review summarizes recent knowledge on factors controlling the nasal pathway, focusing on drug metabolising enzymes in olfactory mucosa, olfactory bulb and brain, which should constitute a CNS metabolic barrier.