ABSTRACT
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.
Subject(s)
Aryl Hydrocarbon Hydroxylases/metabolism , Olfactory Mucosa/metabolism , Animals , Aryl Hydrocarbon Hydroxylases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Kinetics , Male , Microsomes/enzymology , Microsomes/metabolism , Microsomes, Liver/enzymology , Microsomes, Liver/metabolism , Olfactory Mucosa/enzymology , RNA, Messenger/metabolism , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Substrate SpecificityABSTRACT
PURPOSE: To investigate the influence of thyroid hormone status on the regulation of UGTs expression by 9-cis-retinoic acid in cultured rat primary hepatocytes. METHODS: Hepatocytes from rats with various thyroid states were isolated and treated with 9-cis retinoic acid (1 x 10(-6) M). mRNA was amplified by reverse transcription and polymerase chain reaction (RT-PCR) and quantified by UV light densitometry. Variations in the expression levels of four different UGT isoforms (UGT1A1, 1A2, 1A5, and 1A6) that are involved in the glucuronication of bilirubin and phenols were determined by comparison with those of an internal standard, beta-actin, which is known to be insensitive to nutritional and hormonal conditions. RESULTS: Primary hepatocyte cultures from rats with various thyroid states present similar metabolite characteristics to those from hypo- or hyperthyroid animals. The treatment of hepatocytes from hypothyroid rats with 9-cis-retinoic acid (1 x 10(-6) M) did not significantly modify bilirubin and phenol-UGT isoform expression. In contrast, in hepatocytes from normal and specially hyperthyroid rats treated with 9-cis-retinoic acid, UGT mRNA levels were modified. This suggests that the effect of retinoic acid on UGT mRNA expression requires the presence of thyroid hormone. This was confirmed by the treatment of cultured hepatocytes from hypothyroid rats with both retinoic acid and L-T3. CONCLUSIONS: This study demonstrates that in cultured hepatocytes, the thyroid status can differentially modulate the expression of four UGT isoforms, and the regulation of their expression can be affected by 9-cis-retinoic acid.
Subject(s)
Glucuronosyltransferase/biosynthesis , Hepatocytes/drug effects , Thyroid Gland/physiology , Thyroid Hormones/physiology , Tretinoin/pharmacology , Alitretinoin , Animals , Cells, Cultured , Gene Expression , Glucuronosyltransferase/genetics , Hepatocytes/enzymology , Isoenzymes/biosynthesis , Isoenzymes/genetics , Male , RNA, Messenger/biosynthesis , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Thyroidectomy , Time Factors , Triiodothyronine/pharmacologyABSTRACT
The aim of the present study was to examine the glucuronidation of a series of odorant molecules by homogenates prepared either with rat olfactory mucosa, olfactory bulb or brain. Most of the odorant molecules tested were efficiently conjugated by olfactory mucosa, whereas olfactory bulb and brain homogenates displayed lower activities and glucuronidated only a few molecules. Important age-related changes in glucuronidation efficiency were observed in olfactory mucosa and bulb. Therefore, we studied changes in expression of two UDP-glucuronosyltransferase isoforms, UGT1A6 and UGT2A1, in 1-day, 1- and 2-week-, 3-, 12- and 24-month-old rats. UGT1A6 was expressed at the same transcriptional level in the olfactory mucosa, bulb and brain, throughout the life period studied. UGT2A1 mRNA was expressed in both olfactory mucosa and olfactory bulb, in accordance with previous results [Mol. Brain Res. 90 (2001) 83], but UGT2A1 transcriptional level was 400-4000 times higher than that of UGT1A6. Moreover, age-dependent variations in UGT2A1 mRNA expression were observed. As it has been suggested that drug metabolizing enzymes could participate in olfactory function, mitral cell electrical activity was recorded during exposure to different odorant molecules in young, adult and old animals. Age-related changes in the amplitude of response after stimulation with several odorant molecules were observed, and the highest responses were obtained with molecules that were not efficiently glucuronidated by olfactory mucosa. In conclusion, the present work presents new evidence of the involvement of UGT activity in some steps of the olfactory process.
Subject(s)
Glucuronosyltransferase/metabolism , Monosaccharide Transport Proteins , Neurons/metabolism , Olfactory Pathways/enzymology , Receptors, Odorant/metabolism , Smell/physiology , Telencephalon/enzymology , Uridine Diphosphate Glucuronic Acid/metabolism , Action Potentials/drug effects , Action Potentials/physiology , Aging/metabolism , Animals , Animals, Newborn , Cerebral Cortex/drug effects , Cerebral Cortex/enzymology , Gene Expression Regulation, Enzymologic/physiology , Glucuronosyltransferase/drug effects , Male , Neurons/drug effects , Odorants , Olfactory Bulb/drug effects , Olfactory Bulb/enzymology , Olfactory Pathways/drug effects , Olfactory Receptor Neurons/drug effects , Olfactory Receptor Neurons/enzymology , Protein Isoforms/drug effects , Protein Isoforms/metabolism , Rats , Rats, Wistar , Receptors, Odorant/drug effects , Smell/drug effects , Telencephalon/drug effectsABSTRACT
We studied the influence of thyroid hormones and vitamin A status on the regulation of UDP-glucuronosyltransferase (UGT) expression and the glucuronidation of thyroid hormones by UGTs. For this, we used an original model of rats fed with different vitamin A diets and implanted subcutaneously by osmotic minipumps delivering vehicle or thyroid hormones, which permitted the control of plasma thyroid hormone concentrations. The activity and expression of family 1 UGTs are correlated and were significantly modified by both thyroid status and amounts of retinol in the diet. Dietary vitamin A did not perturbe the UGT1A expression in thyroidectomized animals. Thyroid hormones and dietary vitamin A did not affect the activity and expression of family 2 UGTs. We conclude that thyroid hormones and vitamin A are co-regulator of the UGT1 family expression, without affecting the UGT2 family; by modifying activity and expression of the bilirubin UOT isoform, a member of UGT1 family, thyroid hormone reduced the glucuronidation of T4 and rT3.