Effect of Adrenalectomy on Expression and Induction of UDP-Glucuronosyltransferase 1A6 and 1A7 in Rats

Abstract

Uridine 5′-diphosphate (UDP)-glucuronosyltransferase 1A (UGT1A), which catalyzes major phase II reactions, is regulated by endogenous and exogenous factors via nuclear receptors such as the aryl hydrocarbon receptor (AhR). Glucocorticoid, one of the adrenocortical hormones, regulates AhR and UGT1A expression. We examined the effects of adrenalectomy on the expression and induction of UGT1A via AhR in the rat liver and small intestine. Rats were adrenalectomized bilaterally (ADX) or sham-operated (SHAM) and received intraperitoneal treatment with β-naphthoflavone (BNF) for 4 d. Hepatic UGT1A6 and UGT1A7 mRNA levels were altered by ADX (0.1-fold and 1.6-fold, respectively). BNF treatment increased UGT1A6 and UGT1A7 mRNA expression and the intrinsic clearance of acetaminophen (APAP) glucuronidation, which is primarily catalyzed by UGT1A6 and UGT1A7, in both SHAM and ADX rats. Therefore, ADX rats maintained a functional AhR signaling pathway in the presence of BNF, expressed UGT1A6 and UGT1A7 mRNA, and showed APAP glucuronidation, namely induction by BNF via AhR was not abolished. Our results indicate that adrenal-dependent factors such as glucocorticoids are partially involved in the basal regulation of UGT1A6 and UGT1A7 transcription.

Uridine 5′-diphosphate (UDP)-glucuronosyltransferase (UGT) catalyzes a major phase II reaction involving its conjugation with glucuronic acid. UGT isoforms that are responsible for drug metabolism belong to the UGT1 and UGT2 families in rodents and humans. The UGT1A subfamily is involved in the metabolism of endogenous compounds, including bilirubin,^1–3) serotonin^2,3) and thyroid hormone,³⁾ as well as drugs such as acetaminophen (APAP),²⁾ 7-ethyl-10-hydroxycamptothecin,^2,3) and propofol.^1–3) Human UGT1A is mainly expressed in the liver but has also been detected in the small intestine and kidney.⁴⁾ Similarly, rat UGT1A is expressed in the liver, small intestine, and kidney, with some isoforms expressed at higher levels in the small intestine than in the liver.⁵⁾ Thus, the small intestine is also regarded as an important site of metabolism.

Glucocorticoids, which are pituitary-dependent hormones that are mainly produced in the adrenal cortex, bind to the glucocorticoid receptor (GR). This complex binds to glucocorticoid response elements (GREs) in a target gene to regulate the expression of several genes.⁶⁾ Adrenalectomy, which leads to the depletion of glucocorticoids, reduces the expression of nuclear receptor genes such as the aryl hydrocarbon receptor (AhR).^7,8) AhR modulates the expression and/or induction of drug-metabolizing enzymes.⁹⁾ Gene regulation via AhR involves translocation of AhR from the cytoplasm into the nucleus after activation by a ligand, which is then heterodimerized with AhR nuclear translocator. The heterodimer binds to xenobiotic responsive elements (XREs),^10,11) to enhance the transcription of AhR target genes. The xenobiotic AhR ligands, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3-MC) and β-naphthoflavone (BNF) induce some UGTs and CYPs. A previous study showed that UGT1A6 and UGT1A7 mRNA expression increased after the treatment with TCDD (3.9 µg/kg) or BNF (100 mg/kg/d) for 4 d in rat liver, but not in the duodenum.¹²⁾ However, another report showed that small intestinal UGT1A6 and UGT1A7 mRNA expression also increased after the treatment with BNF (100 mg/kg/d).¹³⁾ CYP1A1 expression was increased by BNF in rat liver and small intestine.¹⁴⁾ These reports indicate that UGT1A6, UGT1A7 and CYP1A1 are target gene of AhR.

Glucocorticoids can strongly activate AhR-mediated transcription and subsequent gene expression in rats. Additionally, AhR target genes are synergistically regulated by AhR ligands and glucocorticoids in rats. A GRE-like sequence has been identified in the 5′-flanking region of the rodent AhR gene.¹⁵⁾ Glucocorticoids increased AhR mRNA levels and binding of ligand to AhR, and enhanced expression on CYP1A1 as AhR target gene in H4IIE rat hepatoma cells.^15,16) A GRE is located between base pair −141 and the promoter in the rat UGT1A6 gene.¹⁷⁾ A previous study using rat hepatocytes treated with dexamethasone indicated that glucocorticoids induced the expression of UGT1A6 and p-nitrophenol glucuronidation.¹⁸⁾ Moreover, UGT1A6 protein expression has been shown to increase after treatment with a combination of 3-MC and dexamethasone in rat hepatocytes,^18,19) indicating that glucocorticoids regulate UGT1A6 expression and/or induction.

It is currently unclear whether glucocorticoids affect UGT1A expression and induction in vivo. The purpose of this study was to clarify the effects of adrenalectomy on the basal expression of UGT1A6 and UGT1A7 mRNA and their glucuronidation in the liver and small intestine in rats. Additionally, we examined changes in the induction potency of BNF by adrenalectomy.

METHODS

Materials

APAP and aprotinin were purchased from Nacalai Tesque (Kyoto, Japan). APAP β-D-glucuronide, alamethicin, BNF, 7-ethoxyresorufin, leupeptin, resorufin, trypsin inhibitor and uridine 5′-diphosphoglucuronic acid trisodium salt were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). (p-Amino diphenyl) methanesulfonyl fluoride, β-nicotinamide-adenine dinucleotide phosphate (oxidized form), D-glucose 6-phosphate disodium salt and glucose 6-phosphate dehydrogenase were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other chemicals and solvents were of the highest grade commercially available.

Animal Treatment

The present study was approved by the Institutional Animal Care and Use Committee of Meijo University. Seven-week-old male Sprague-Dawley rats (Japan SLC, Hamamatsu, Japan) were bilaterally adrenalectomized (ADX; n=10) or sham-operated (SHAM; n=10), according to the previous method by Lloyd and Franklin.²⁰⁾ ADX rats were given 0.9% sodium chloride in drinking water for the remainder of the study. Four days after surgery, the rats were treated intraperitoneally once daily for 4 d with BNF at a dose of 40 mg/kg/d (ADX; n=5, SHAM; n=5) or with corn oil as the control (ADX; n=5, SHAM; n=5). One day after the final treatment, the liver and small intestine of the rats were resected.

RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction (PCR)

Total RNA was extracted using TRIzol reagent (Life Technologies, Carlsbad, CA, U.S.A.) and then treated with DNase I (Promega, Madison, WI, U.S.A.). One microgram of total RNA was used for the reverse transcription reactions with ReverTra Ace qPCR RT Kit (TOYOBO, Osaka, Japan). PCR was performed using the Thermal Cycler Dice from TaKaRa Bio (Shiga, Japan) or ABI PRISM 7700 (Applied Biosystems, Foster City, CA, U.S.A.) with SYBR Premix Ex Taq II (TaKaRa Bio) or TaqMan Gene Expression Master Mix (Life Technologies), respectively. The primers used in the present study are described in Table 1 and were commercially synthesized by Greiner Bio-one (Tokyo, Japan), except the primers and a probe for UGT1A6, which were designed and synthesized by Nippon Gene Materials (Toyama, Japan). PCR amplification conditions were as follows: initial 30 s denaturation step at 95°C; 40 cycles of denaturation at 95°C for 5 s; and annealing and extension steps as described in Table 1. The relative expression level of each gene was calculated using the standard curve method. All data were normalized to the expression level of β-actin, which were not different among all samples. Data are shown as the mean values for 5 rats in each group.

Table 1. Primer Sequence and PCR Conditions Used in the Present Study

Gene	Accession No.		Sequence	Annealing temp. (˚C)	Annealing time (s)	Amplicon size (bp)	Reference
UGT1A6	NM_057105	Sense	5′-CTCAGTTCTAGGTGACAA-3′	60	60	157
		Anti-sense	5′-CCTGTAGTATTTGGATTCTC-3′
		Probe	5′-AATTGACTTCTGGCACTAGCACC-3′
UGT1A7	NM_130407	Sense	5′-CAGACCCCGGTGACTATGACA-3′	60	30	73	23)
		Anti-sense	5′-CAACGTGAAGTCTGTGCGTAACA-3′
AhR	U04860	Sense	5′-GCTGTGATGCCAAAGGGCAGCT-3′	60	30	100	8)
		Anti-sense	5′-TGAAGCATGTCAGCGGCGTGGAT-3′
GR	NM_012576	Sense	5′-GTGAAATGGGCAAAGGCGATAC-3′	60	30	97	24)
		Anti-sense	5′-GCAAATGCCATGAGAAACATCC-3′
CYP1A1	NM_12540	Sense	5′-GGGTTCCCAAAGGTCTGAAG-3′	60	30	96	25)
		Anti-sense	5′-GTCAGTGACAGGTGTGGGTTC-3′
ß-Actin	NM_031144	Sense	5′-CTGGCCTCACTGTCCACCTT-3′	60	30	65	23)
		Anti-sense	5′-GGGCCGGACTCATCGTACT-3′

Liver Microsomes

Liver and small intestinal microsomes, which were obtained from the duodenum and jejunum, were prepared as described previously²¹⁾ and stored at −80°C until analysis. Small intestinal microsomes were pooled from 5 rats. Protein concentration was determined using Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA, U.S.A.).

APAP Glucuronidation

APAP glucuronidation was determined using the method described by Kessler et al.²²⁾ with slight modification. The incubation mixture contained 50 mM Tris–HCl buffer (pH 7.4), 10 mM MgCl₂, 25 µg/mL alamethicin, 0.02 mg/mL hepatic or small intestinal microsomes, and 0.05–30 mM APAP. The final concentration of methanol in the reaction mixture was less than 0.25% (v/v). The reaction mixture was incubated at 37°C for 30 min, and the reaction was terminated by adding ice-cold methanol. After the mixture was centrifuged at 14000×g for 10 min, 10 µL of the supernatant obtained was subjected to liquid chromatography-tandem mass spectrometry. Liquid chromatography was performed using a Prominence apparatus (Shimadzu, Kyoto, Japan), which was equipped with an Inertsil ODS-3 column (3.0×150 mm; GL Sciences, Tokyo, Japan). The column temperature was 40°C. The flow rate was 0.2 mL/min. The mobile phase consisted of methanol–0.1% formic acid (40 : 60 [v/v]). The liquid chromatography setup was connected to an API4000 tandem mass spectrometer (Applied Biosystems) operated in the negative electrospray ionization mode. Turbo gas was maintained at 300°C. Mass/charge (m/z) ion transitions were recorded in the multiple reaction-monitoring mode, with m/z 326.0 and 150.0 for APAP β-D-glucuronide. The retention time of APAP β-D-glucuronide was 5.5 min. The limit of detection for APAP β-D-glucuronide was 0.3 pmol. The limit of quantification in the reaction mixture was 30 nM with a CV of less than 10%.

In a preliminary study, we confirmed the linearity of the protein concentrations and incubation times. Kinetic parameters were estimated from the fitted curves using the Michaelis–Menten equation with the Kaleida-Graph computer program (Synergy, Reading, PA, U.S.A.) designed for nonlinear regression analysis. Intrinsic clearance (CL_int) was calculated as V_max/K_m for the Michaelis–Menten kinetics, where K_m is the Michaelis–Menten constant and V_max is the maximum velocity. Data are presented as the mean of measurements from 5 individual liver microsomes and the mean of 3 independent determinations using pooled small intestinal microsomes from 5 rats.

Ethoxyresorufin O-Deethylase (EROD) Activity

The incubation mixture contained 100 mM Tris–HCl buffer (pH 7.4), 2.8 mM β-nicotinamide-adenine dinucleotide phosphate (oxidized form), 5 mM D-glucose 6-phosphate disodium salt, 10 mM MgCl₂, 10 U glucose 6-phosphate dehydrogenase, microsomal protein (liver, 0.2 mg/mL; small intestine, 1.0 mg/mL), and 7-ethoxyresorufin (liver, 0.1 µM; small intestine, 1.0 µM). The final concentration of dimethyl sulfoxide in the reaction mixture was less than 0.2% (v/v). The reaction mixture was incubated at 37°C for 10 min, and the reaction was subsequently terminated by the addition of ice-cold methanol. After the mixture was centrifuged at 14000×g for 10 min, 50 µL of the supernatant was subjected to high-performance liquid chromatography with a Cosmosil 5C18-MS-II analytical column (4.6×150 mm; Nacalai Tesque). The column temperature was 35°C and the flow rate was 0.8 mL/min. The mobile phase consisted of methanol–acetonitrile–20 mM phosphate buffer (pH 4.0), 45 : 3 : 52 (v/v). The eluent was monitored fluorometrically (excitation, 574 nm; emission, 596 nm). In a preliminary study, we confirmed the linearity of the protein concentrations, the substrate concentration, and incubation times were confirmed. Data are presented as the mean of data for 5 individual hepatic microsomes and the mean of 3 independent experiments performed using pooled small intestinal microsomes from 5 rats.

Statistical Analysis

Statistical analyses were performed using Student’s paired t-test or Welch t-test with the Kaleida-Graph computer program.

RESULTS

Effect of ADX on Basal Expression of UGT1A6 and UGT1A7 mRNA

The expression of UGT1A6 and UGT1A7 mRNA in the liver and small intestine was compared for SHAM and ADX rats in the control group (Fig. 1). This comparison provided the information regarding the effect of adrenalectomy on basal mRNA expression after 8 d. The basal expression of UGT1A6 and UGT1A7 mRNA in the liver was altered by ADX (0.1-fold and 1.6-fold, respectively). However, the levels in the small intestine were unchanged.

Fig. 1. UGT1A6 (A) and UGT1A7 (B) mRNA Expression in the Liver and Small Intestine from SHAM and ADX Rats

Each column represents the expression level relative to that in the liver in the control SHAM rats, which is shown as the mean±S.D. (n=5). The expression level of mRNA was normalized to that of β-actin. * p<0.05, ** p<0.01, *** p<0.001 compared with the control. ^†† p<0.01 compared with SHAM rats.

Effect of ADX on Induction of UGT1A6 and UGT1A7 mRNA by BNF

Hepatic UGT1A6 mRNA expression was increased by 4-d BNF treatment in both SHAM and ADX rats (9.6-fold and 27.1-fold, respectively). Small intestinal UGT1A6 mRNA expression was also elevated by BNF treatment in SHAM and ADX rats (3.7-fold and 2.4-fold, respectively) (Fig. 1A). Hepatic UGT1A7 mRNA expression was induced by BNF in SHAM and ADX rats (22.6-fold and 16.3-fold, respectively). Small intestinal UGT1A7 mRNA expression was increased by 1.5-fold after treatment with BNF in ADX rats, whereas that in SHAM rats remained unchanged (Fig. 1B).

Effect of ADX on Basal APAP Glucuronidation and Induction of APAP Glucuronidation by BNF

Changes in UGT1A6 and UGT1A7 enzymatic activity following BNF treatment in ADX rats were assessed using APAP as a probe, since APAP glucuronidation is primarily catalyzed by UGT1A6 and UGT1A7²¹⁾ (Fig. 2). In all rats, APAP glucuronidation followed Michaelis–Menten kinetics. As shown in Table 2, kinetic parameters for APAP glucuronidation in the liver and small intestine were not different between ADX and SHAM rats in the control groups. CL_int values were increased by BNF treatment in both SHAM and ADX rats (1.3-fold and 1.5-fold, respectively, in the liver; 1.8-fold and 1.1-fold, respectively, in the small intestine).

Fig. 2. APAP Glucuronidation in the Liver (A) and Small Intestine (B) in SHAM and ADX Rats

The concentration of APAP ranged from 0.05 mM to 30 mM. Data represent the mean values of 3 independent measurements. The Michaelis–Menten equation was fitted to the data for APAP glucuronidation and the kinetic parameters are shown in Table 2.

Table 2. Kinetic Parameters for APAP Glucuronidation in the Liver and Small Intestine from SHAM and ADX Rats

		Treatment	Km (mM)	Vmax (nmol/min/mg protein)	CLint (µL/min/mg protein)
Liver	SHAM	Control	6.07±1.04	5.53±0.81	0.92±0.07
		BNF	5.25±1.24	6.08±1.59	1.15±0.15*
	ADX	Control	4.07±0.68†	4.41±1.48	0.94±0.27
		BNF	5.25±0.50	7.26±1.03**	1.38±0.13*
Small	SHAM	Control	5.67±0.82	1.41±0.03	0.25±0.04
intestine		BNF	3.95±0.40*	1.75±0.02***	0.45±0.05**
	ADX	Control	6.68±0.84	1.38±0.12	0.21±0.01
		BNF	6.67±0.36	1.63±0.09	0.24±0.01*

* p<0.05, ** p<0.01, *** p<0.001 compared with the control. ^† p<0.05 compared with SHAM.

Effect of ADX on Expression of AhR and GR mRNA Expression

In the control groups, AhR mRNA expression was decreased by 0.5-fold in the small intestine by ADX, and tended to decrease in the liver. Following treatment with BNF, hepatic AhR mRNA expression was increased in SHAM and ADX rats by 2.3-fold and 3.1-fold, respectively. The changes in small intestinal AhR mRNA expression following BNF treatment were smaller than those in the liver.

Hepatic GR mRNA expression was increased by 2.1-fold by ADX in control rats, and tended to increase in the small intestine. Following treatment with BNF, hepatic GR mRNA expression was decreased by 0.5-fold in the liver in ADX rats, but was unchanged in the small intestine in ADX and in SHAM rats (Fig. 3).

Fig. 3. AhR (A) and GR (B) mRNA Expression in the Liver and Small Intestine from SHAM and ADX Rats

Each column represents the expression level relative to that in the liver in the control SHAM rats, which is shown as the mean±S.D. (n=5). The expression level of mRNA was normalized to that of β-actin. * p<0.05, *** p<0.001 compared with the control. ^† p<0.01, ^†† p<0.01 compared with SHAM rats.

Effect of ADX on CYP1A1 mRNA Expression and EROD Activity

As shown in Fig. 4, hepatic CYP1A1 mRNA expression was decreased by 0.7-fold by ADX, which was consistent with the results observed for EROD activity (Fig. 5). However, small intestinal CYP1A1 mRNA expression was unchanged by ADX. Induction of hepatic and small intestinal CYP1A1 mRNA expression by BNF treatment occurred in both SHAM and ADX rats. Moreover, EROD activity increased in all BNF-treated SHAM and ADX rats.

Fig. 4. CYP1A1 mRNA Expression in the Liver and Small Intestine from SHAM and ADX Rats

Each column represents the expression level relative to that in the control SHAM rats, which is shown as the mean±S.D. (n=5). The expression level of mRNA was normalized to that of β-actin. *p<0.05, **p<0.01, ***p<0.001 compared with the control. ^††p<0.01 compared with SHAM rats.

Fig. 5. EROD Activity in the Liver (A) and Small Intestine (B) from SHAM and ADX Rats

Each column represents the mean±S.D. of 3 independent measurements. **p<0.01 compared with control. ^††p<0.01 compared with SHAM rats. N.D., not detected.

DISCUSSION

In the present study, we found that removing the adrenal glands resulted in decreased basal expression of UGT1A6 mRNA (Fig. 1). The degree of this reduction differed among tissues. UGT1A6 is known to be regulated by AhR and GR.^12,17) GR mRNA expression was increased by adrenalectomy (Fig. 3), which is consistent with the results of a previous report demonstrating that decreased glucocorticoid levels can be attributed to autoregulation processes such as transcriptional activation and stabilization of GR mRNA expression.²⁶⁾ Two GRE-like sequences have been reported to be present in the promoter region of rat AhR gene; that one of the glucocorticoids, dexamethasone, induced AhR mRNA expression via GR in rat H4IIE cells.¹⁵⁾ AhR mRNA expression had a tendency to decrease in ADX rats (Fig. 3), indicating that decreased glucocorticoid levels in ADX rats may suppress the signal transduction via GR, which causes the tendency to reduce transcriptional activation for AhR. It has been reported that AhR-null mice show lower basal mRNA expression of Cyp1a2 (target gene of AhR) than wild-type mice.²⁷⁾ The reduction in AhR mRNA expression in ADX rat may be responsible for the decrease UGT1A6 mRNA expression in ADX rats but further studies are needed to clear the contribution of AhR to UGT1A6 expression using adrenalectomized AhR knockout rats.

UGT1A6 mRNA expression was reduced by adrenalectomy, but UGT1A7 mRNA expression was not (Fig. 1). Although a previous study reported that ligands of AhR, such as TCDD and BNF, regulated both UGT1A6 and UGT1A7 expression,¹²⁾ the expression patterns were changed differently by adrenalectomy between UGT1A6 and UGT1A7 in the present study. Basal AhR and GR mRNA expression was higher in the liver than in the small intestine (Fig. 3), and the expression patterns of AhR and GR were similar to that of UGT1A6. UGT1A6 mRNA was expressed at higher levels in the liver than in the small intestine, but UGT1A7 mRNA expression was higher in the small intestine than in the liver. These results agree with those of previous studies reporting that the expression of UGT1A6 protein is higher in the liver than in the small intestine.²⁸⁾ UGT1A7 has been previously shown to be expressed abundantly in the small intestine, but was either not detected or was present at lower levels in the liver.²⁹⁾ Thus, these findings indicate that UGT1A7 mRNA expression is regulated by another mechanism rather than by glucocorticoids. Hepatocyte nuclear factor 1 (HNF1) has been reported to be involved in UGT1A7 mRNA expression in rats.³⁰⁾ The previous study reported that mutations in the HNF1 gene reduced basal expression of UGT1A7 in primary rat hepatocytes, suggesting that HNF1 is one of the factors regulating UGT1A7 expression.³⁰⁾

Quantification of the UGT1A6 protein is necessary for understanding alterations in UGT1A6 function, but antibodies specific for UGT1A6 are not available. APAP glucuronidation was primarily catalyzed by UGT1A6 and UGT1A7, but is not specific to UGT1A6. Basal APAP glucuronidation in the liver was unchanged by adrenalectomy, although hepatic UGT1A6 was reduced to 10% by adrenalectomy in control rats (Fig. 1). This contradiction may be due to the lack of isoform specificity of APAP glucuronidation. Regarding the involvement of other UGTs, APAP glucuronidation in primary hepatocytes from UGT1A defective Gunn rats was 70% lower than in primary hepatocytes from Sprague-Dawley rats,²²⁾ which suggests that UGT1A6 and UGT1A7 play major roles in APAP glucuronidation. Further studies are required to understand the isoform that catalyzes APAP glucuronidation in rats, and to clarify the changes in UGT1A6 protein or enzyme activity by adrenalectomy.

Following treatment with BNF, UGT1A6 and UGT1A7 mRNA expression was induced both in SHAM and ADX rats (Fig. 1), suggesting that the induction potency of BNF was maintained even when the glucocorticoid was depleted in the body. This may be because AhR is expressed in ADX rats. UGT1A6 and UGT1A7 mRNA expression increased to a greater extent in the liver than in the small intestine. Induction potency may be related to AhR expression level.³¹⁾ AhR mRNA expression was higher in the liver than in the small intestine (Fig. 3), and the induction potencies of BNF for AhR in the liver were larger than those in the small intestine, which is consistent with the induction potency described above. In addition, BNF treatment increased CL_int of APAP glucuronidation equally in both SHAM and ADX rats, indicating that adrenalectomy did not affect the induction potency of BNF on APAP glucuronidation.

For CYP1A1, which is also a target gene of GR,³²⁾ mRNA expression was decreased by 70% after adrenalectomy. EROD activity also decreased, which is consistent with the results of a previous report.⁸⁾ Moreover, ADX rats showed induction of CYP1A1, which agrees with the results of a previous report regarding induction of CYP1A1 mRNA expression and EROD activity in ADX rats.⁸⁾ UGT1A6 mRNA expression showed a pattern similar to that of CYP1A1, but not to that of UGT1A7.

In conclusion, we found that ADX affected the expression of UGT1A6, UGT1A7, CYP1A1 and AhR to varying degrees. ADX rats maintained a functional AhR signaling pathway with normal sensitivity to BNF for UGT1A6 and UGT1A7 mRNAs expression and APAP glucuronidation. These results suggest that the alterations in adrenal-dependent factors such as glucocorticoids are partially involved in the basal regulation of UGT during transcription. Several factors are known to affect the levels of circulating glucocorticoids, developmental stage, stress, and diseases caused by glucocorticoids deficiency and excess. This study contributes to the elucidation of the effect of endogenous factors such as hormones on UGT1A6 and UGT1A7 expression.

REFERENCES

• 1) Tukey RH, Strassburg CP. Human UDP-glucuronosyltransferases: metabolism, expression, and disease. Annu. Rev. Pharmacol. Toxicol., 40, 581–616 (2000).
• 2) de Leon J. Glucuronidation enzymes, genes and psychiatry. Int. J. Neuropsychopharmacol., 6, 57–72 (2003).
• 3) Court MH. Isoform-selective probe substrates for in vitro studies of human UDP-glucuronosyltransferases. Methods Enzymol., 400, 104–116 (2005).
• 4) Harbourt DE, Fallon JK, Ito S, Baba T, Ritter JK, Glish GL, Smith PC. Quantification of human uridine-diphosphate glucuronosyl transferase 1A isoforms in liver, intestine, and kidney using nanobore liquid chromatography-tandem mass spectrometry. Anal. Chem., 84, 98–105 (2012).
• 5) Shelby MK, Cherrington NJ, Vansell NR, Klaassen CD. Tissue mRNA expression of the rat UDP-glucuronosyltransferase gene family. Drug Metab. Dispos., 31, 326–333 (2003).
• 6) Schoneveld OJ, Gaemers IC, Lamers WH. Mechanisms of glucocorticoid signaling. Biochim. Biophys. Acta, 1680, 114–128 (2004).
• 7) Mullen Grey AK, Riddick DS. Glucocorticoid and adrenalectomy effects on the rat aryl hydrocarbon receptor pathway depend on the dosing regimen and post-surgical time. Chem. Biol. Interact., 182, 148–158 (2009).
• 8) Mullen Grey AK, Riddick DS. The aryl hydrocarbon receptor pathway and the response to 3-methylcholanthrene are altered in the liver of adrenalectomized rats. Drug Metab. Dispos., 39, 83–91 (2011).
• 9) Mimura J, Fujii-Kuriyama Y. Functional role of AhR in the expression of toxic effects by TCDD. Biochim. Biophys. Acta, 1619, 263–268 (2003).
• 10) Pollenz RS, Sattler CA, Poland A. The aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator protein show distinct subcellular localizations in Hepa 1c1c7 cells by immunofluorescence microscopy. Mol. Pharmacol., 45, 428–438 (1994).
• 11) Denison MS, Fisher JM, Whitlock JP Jr. The DNA recognition site for the dioxin-Ah receptor complex. Nucleotide sequence and functional analysis. J. Biol. Chem., 263, 17221–17224 (1988).
• 12) Shelby MK, Klaassen CD. Induction of rat UDP-glucuronosyltansferases in liver and duodenum by microsomal enzyme inducers that activate various transcriptional pathways. Drug Metab. Dispos., 34, 1772–1778 (2006).
• 13) Kobayashi T, Yokota H, Ohgiya S, Iwano H, Yuasa A. UDP-glucuronosyltransferase UGT1A7 induced in rat small intestinal mucosa by oral administration of 2-naphthoflavone. Eur. J. Biochem., 258, 948–955 (1998).
• 14) Zhang QY, Wikoff J, Dunbar D, Fasco M, Kaminsky L. Regulation of cytochrome P4501A1 expression in rat small intestine. Drug Metab. Dispos., 25, 21–26 (1997).
• 15) Sonneveld E, Jonas A, Meijer OC, Brouwer A, van der Burg B. Glucocorticoid-enhanced expression of dioxin target genes through regulation of the rat aryl hydrocarbon receptor. Toxicol. Sci., 99, 455–469 (2007).
• 16) Wiebel FJ, Cikryt P. Dexamethasone-mediated potentiation of P450IA1 induction in H4IIEC3/T hepatoma cells is dependent on a time-consuming process and associated with induction of the Ah receptor. Chem. Biol. Interact., 76, 307–320 (1990).
• 17) Falkner KC, Ritter JK, Prough RA. Regulation of the rat UGT1A6 by glucocorticoids involves a cryptic glucocorticoid response element. Drug Metab. Dispos., 36, 409–417 (2008).
• 18) Jemnitz K, Veres Z, Vereczkey L. Coordinate regulation of UDP-glucuronosyltransferase UGT1A6 induction by 3-methylcholanthrene and multidrug resistance protein MRP2 expression by dexamethasone in rat primary rat hepatocytes. Biochem. Pharmacol., 63, 2137–2144 (2002).
• 19) Jemnitz K, Veres Z, Monostory K, Vereczkey L. Glucuronidation of thyroxine in primary monolayer cultures of rat hepatocytes: in vitro induction of UDP-glucuronosyltransferases by methylcholanthrene, clofibrate, and dexamethasone alone and in combination. Drug Metab. Dispos., 28, 34–37 (2000).
• 20) Lloyd SA, Franklin MR. Modulation of carbon tetrachloride hepatotoxicity and xenobiotic-metabolizing enzymes by corticosterone pretreatment, adrenalectomy and sham surgery. Toxicol. Lett., 55, 65–75 (1991).
• 21) Shiratani H, Katoh M, Nakajima M, Yokoi T. Species differences in UDP-glucuronosyltransferase activities in mice and rats. Drug Metab. Dispos., 36, 1745–1752 (2008).
• 22) Kessler FK, Kessler MR, Auyeung DJ, Ritter JK. Glucuronidation of acetaminophen catalyzed by multiple rat phenol UDP-glucuronosyltransferases. Drug Metab. Dispos., 30, 324–330 (2002).
• 23) Tian H, Ou J, Strom SC, Venkataramanan R. Activity and expression of various isoforms of uridine diphosphate glucuronosyltransferase are differentially regulated during hepatic regeneration in rats. Pharm. Res., 22, 2007–2015 (2005).
• 24) Lukaszewski MA, Mayeur S, Fajardy I, Delehaye F, Dutriez-Casteloot I, Montel V, Dickes-Coopman A, Laborie C, Lesage J, Vieau D, Breton C. Maternal prenatal undernutrition programs adipose tissue gene expression in adult male rat offspring under high-fat diet. Am. J. Physiol. Endocrinol. Metab., 301, E548–E559 (2011).
• 25) Kransler KM, McGarrigle BP, Swartz DD, Olson JR. Lung development in the Holtzman rat is adversely affected by gestational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Sci., 107, 498–511 (2009).
• 26) Kalinyak JE, Dorin RI, Hoffman AR, Perlman AJ. Tissue-specific regulation of glucocorticoid receptor mRNA by dexamethasone. J. Biol. Chem., 262, 10441–10444 (1987).
• 27) Aleksunes LM, Klaassen CD. Coordinated regulation of hepatic phase I and phase II drug metabolizing genes and transporters using AhR-, CAR-, PXR-, PPARα-, and Nrf2-null mice. Drug Metab. Dispos., 40, 1366–1379 (2012).
• 28) Miles KK, Kessler FK, Smith PC, Ritter JK. Characterization of rat intestinal microsomal UDP-glucuronosyltransferase activity toward mycophenolic acid. Drug Metab. Dispos., 34, 1632–1639 (2006).
• 29) Webb LJ, Miles KK, Auyeung DJ, Kessler FK, Ritter JK. Analysis of substrate specificities and tissue expression of rat UDP-glucuronosyltransferases UGT1A7 and UGT1A8. Drug Metab. Dispos., 33, 77–82 (2005).
• 30) Metz RP, Auyeung DJ, Kessler FK, Ritter JK. Involvement of hepatocyte nuclear factor 1 in the regulation of the UDP-glucuronosyltransferase 1A7 (UGT1A7) gene in rat hepatocytes. Mol. Pharmacol., 58, 319–327 (2000).
• 31) Carver LA, Hogenesch JB, Bradfield CA. Tissue specific expression of the rat Ah-receptor and ARNT mRNAs. Nucleic Acids Res., 22, 3038–3044 (1994).
• 32) Monostory K, Kohalmy K, Prough RA, Kóbori L, Vereczkey L. The effect of synthetic glucocorticoid, dexamethasone on CYP1A1 inducibility in adult rat and human hepatocytes. FEBS Lett., 579, 229–235 (2005).