Hervé Goudonnet

Glucuronidation of odorant molecules in the rat olfactory system. Activity, expression and age-linked modifications of UDP-glucuronosyltransferase isoforms, UGT1A6 and UGT2A1, and relation to mitral cell activity

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.

Rat olfactory bulb and epithelium UDP-glucuronosyltransferase 2A1 (UGT2A1) expression: in situ mRNA localization and quantitative analysis.

UDP-glucuronosyltransferases (UGTs) form a multigenic family of enzymes involved in the biotransformation and elimination of numerous endo- and xenobiotic compounds. Beside the diverse UGT isoforms present in the liver as well as in other tissues, the UGT2A1 isoform, also called olfactory UGT, was initially thought to be expressed in the nasal epithelium only. In this work, we demonstrate the UGT2A1 mRNA expression in the olfactory bulb, using in situ hybridization and quantitative reverse transcription-polymerase chain reaction (RT-PCR) techniques. Within the epithelium, UGT2A1 mRNA is mainly found in the sustentacular cells and to a lesser extent in Bowmans gland cells. Moreover, in situ hybrization staining reveals UGT2A1 mRNA expression in the olfactory sensory neuron nuclei. Neuronal localization of UGT2A1 mRNA within the olfactory bulb is mainly found in the deeper granular cells. The development of the quantitative multistandard RT-PCR method firstly required characterization of the mouse Ugt2A1 cDNA by rapid amplification of cDNA ends (RACE)-PCR. UGT2A1 mRNA levels appear quantitatively six-fold lower in the olfactory bulb than in the epithelium, in both the rat and mouse. The expression of UGT2A1 in the olfactory bulb, which directly connects the nasal epithelium to the brain, emphasizes the potential role of this enzyme in the protection of the brain against airborne hazardous chemicals.

Opposite regulation of bilirubin and 4-nitrophenol UDP-glucuronosyltransferase mRNA levels by 3,3′,5 triiodo-l-thyronine in rat liver

The effects of 3,3′,5 triiodo‐l‐thyronine (L‐T3) on the constitutive levels of hepatic mRNA encoding two UDP‐glucuronosyltransferase (UGT) isoforms implicated in the glucuronidation of planar phenolic substrates (UGTI∗06) and bilirubin (UGT1∗0) were investigated in rat liver. The amount of UGT mRNA was quantitated by reverse transcription and amplification methods (RT‐PCR). Treatment with L‐T3 significantly increased UGT1∗06 and decreased UGT1∗0 mRNA levels by 41% and 54%, respectively. The opposite situation was observed in thyroidectomised animals. A good relationship observed between UGT activity toward 4‐nitrophenol and bilirubin and mRNA levels emphasizes the key role played by the thyroid hormone L‐T3 on UGT expression.

TRANSCRIPTIONAL REGULATION BY TRIIODOTHYRONINE OF THE UDP-GLUCURONOSYLTRANSFERASE FAMILY 1 GENE COMPLEX IN RAT LIVER : COMPARISON WITH INDUCTION BY 3- METHYLCHOLANTHRENE

This study demonstrates that the expression of the phenol UDP-glucuronosyltransferase 1 gene (UGT1A1) is regulated at the transcriptional level by thyroid hormone in rat liver. Following 3,5,3′-triiodo-l-thyronine (T3) stimulation in vivo, there is a gradual increase in the amount of UGT1A1 mRNA with maximum levels reached 24 h after treatment. In comparison, induction with the specific inducer, 3-methylcholanthrene (3-MC), results in maximal levels of UGT1A1 mRNA after 8 h of treatment. In primary hepatocyte cultures, the stimulatory effect of both T3 and 3-MC is also observed. This induction is suppressed by the RNA synthesis inhibitor actinomycin D, indicating that neither inducer acts at the level of mRNA stabilization. Indeed, nuclear run-on assays show a 3-fold increase in UGT1A1 transcription after T3treatment and a 6-fold increase after 3-MC stimulation. This transcriptional induction by T3 is prevented by cycloheximide in primary hepatocyte cultures, while 3-MC stimulation is only partially affected after prolonged treatment with the protein synthesis inhibitor. Together, these data provide evidence for a transcriptional control of UGT1A1 synthesis and indicate that T3 and 3-MC use different activation mechanisms. Stimulation of the UGT1A1 gene by T3 requiresde novo protein synthesis, while 3-MC-dependent activation is the result of a direct action of the compound, most likely via the aromatic hydrocarbon receptor complex.

Role of thyroid state on induction by ciprofibrate of laurate hydroxylase and peroxisomal enzymes in rat liver microsomes

The effects of hypothyroidism and hyperthyroidism upon liver microsomal omega-laurate hydroxylase activity (cytochrome P450 IV A1-dependent), peroxisome proliferation marker enzyme activities and acyl CoA oxidase (AOX) expression induced by ciprofibrate (2 mg/kg/day during 8 days) were studied in the male Wistar rat so as to clarify firstly the possible involvement of thyroid hormones in the modification of peroxisomal ciprofibrate-induced enzyme activities in relation to hepatic microsomal cytochrome P450 IV A1 induction, and secondly the possible direct effect of thyroid hormones on the gene expression of specific peroxisomal enzymes. No significant change was found in the ciprofibrate-induced omega-laurate hydroxylase activity in hypothyroid rats or in rats that had received a large dose of triiodothyronine (LT3), suggesting that the thyroid hormone does not interfere with the peroxisome proliferation process through such an indirect mechanism. The induction by ciprofibrate [2-(4-(2-2dichlorocyclopropyl)phenoxyl-2methyl-propion ic acid)] of mitochondrial alpha-glycerolphosphate dehydrogenase and microsomal bilirubin UDPGT was decreased about 3-fold and 1.5-fold, respectively, while the induction of peroxisomal AOX, carnitine acetyl transferase and enoyl CoA hydratase enzyme activities was decreased by 36%, 34% and 22% in thyroidectomized animals, as compared to euthyroid animals. However, no significant changes in the quantity of peroxisomal proteins and in the AOX mRNA level were noted. The administration of large doses of LT3 to normal rats decreased the peroxisomal ciprofibrate AOX enzyme induction with a marked concomitant decrease in the AOX mRNA level. This suggests that high doses of LT3 enhance the turnover of some specific mRNAs or down regulate the peroxisome proliferator receptor. Our results also do not exclude inhibition of catabolic activity towards AOX which depends on thyroid hormone.

Liver microsome phospholipids and cytochrome P.450 conceptration in phenobarbital treated rats fed on different diets

Liver microsomal concentration of cytochrome P.450 is increased in animals which are fed diets rich in polyunsaturated fatty acids. On the other hand, the effects of phenobarbital are more important when the dietary fat is more unsaturated. The unsaturation index in liver microsomal phosphatidylcholines depends on the unsaturation of the dietary fats. The treatment with phenobarbital constantly results in a decrease of the unsaturation index of fatty acids both in lecithins and cephalins. The importance of the liver microsomal cytochrome P.450 increase and the importance of the unsaturation index decrease in liver microsomal lecithins, both promoted by phenobarbital, are in good agreement.

Effect of thyroid status and cold stress on tyrosine hydroxylase activity in adrenal gland and brown adipose tissue

Abstract A tyrosine hydroxylase activity (THa) of 1.4 nanomoles DOPA formed per hour per mg of protein has been found in brown adipose tissue homogenate; a value of 30.3 nanomoles was found in the corresponding adrenal homogenate. The THa of brown adipose tissue of normal rat increases markedly upon intermittent exposure to cold temperature and is greatly increased in the thyroidectomized rat. In adrenal gland there is, upon intermittent cold stress, an increase in the THa of normal and of thyroidectomized rats, but no increase in the THa of animal receiving triiodothyronine.

9-cis-Retinoic Acid Regulation of Four UGT Isoforms in Hepatocytes from Rats with Various Thyroid States

AbstractPurpose. 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 · 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, β-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 · 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.

Vitamin A modulates the effects of thyroid hormone on UDP-glucuronosyltransferase expression and activity in rat liver.

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.

Isopropyldiiodothyronine and α-methylthyroxine: Comparison of their in vitro and in vivo effects with those of thyroid hormones

Abstract The in vivo and d in vitro effects of the two thyroid hormone analogs 3,5-diiodo-3′-isopropyl- L -thyronine (IPT 2 ) and of α-methyl- DL -thyroxine (MT 4 ) have been compared to those of the thyroid hormones 3,5,3′-triiodo- L -thyronine (T 3 ) and thyroxine (T 4 ). Against purified glutamate, isocitrate and alcohol dehydrogenases IPT 2 and MT 4 exert inhibitory effects which are quite similar to those exerted by T 3 and T 4 . The effects of the analogs on isolated mitochondria follow closely those of the hormones i.e. they uncouple phosphorylations and inhibit electron transfer along the respiratory chain. When thyroidectomized animals are treated with analogs (5 nmoles/100 g/day for 15 days) their effects on two accepted parameters of thyromimetic activity, metabolic rate (BMR) and α-glycerophosphate (GPD) induction, are quite different. After IPT 2 -treatment, the rise in MR and GPD indicates a hyperthyroid state, whereas MT 4 -treatment results in very incomplete compensation of th hypothyroid state. However, treatment of the thyroidectomized animal with IPT 2 or MT 4 has the same effect on hepatic mitochondrial glutamate dehydrogenase as treatment with T 4 . A relation between thyroid state and adrenal tyrosine hydroxylase (TH) activity has been established; TH activity which decreases in the thyroidectomized animal, is brought back to normal by administration of T 4 and above normal by that of IPT 2 .