Effect of pregnane X receptor (PXR) prototype agonists on chemoprotective and drug metabolizing enzymes in mice

European Journal of Pharmacology 660 (2011) 291–297

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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Molecular and Cellular Pharmacology

Effect of pregnane X receptor (PXR) prototype agonists on chemoprotective and drug metabolizing enzymes in mice Wael M. El-Sayed ⁎ King Faisal University, Faculty of Science, Department of Biological Sciences, Al-Hufof 31982, Ahsaa, Saudi Arabia

a r t i c l e

i n f o

Article history: Received 31 October 2010 Received in revised form 4 March 2011 Accepted 22 March 2011 Available online 9 April 2011 Keywords: PXR Dexamethasone Spironolactone Pregnenolone-16alpha-carbonitrile Drug metabolizing enzymes

a b s t r a c t The effects of known PXR inducers; spironolactone [SPL, (i.p.)], pregnenolone-16 alpha-carbonitrile [PCN, (i.g.)] and dexamethasone (i.p.) on hepatic drug metabolizing enzymes in the male CF1 mouse were examined 24 h after 3 daily doses (50, 100, or 200 mg/kg) in corn oil vehicle. All three compounds produced dose-dependent elevations in cytochrome P450 [CYP3A], glutathione S-transferase [GST] and NAD(P)H quinone oxidoreductase [NQO] activities. Only elevations in CYP3A produced after dexamethasone were statistically significant. An elevation in microsomal epoxide hydrolase [mEH, Ephx1] activity was seen after almost all treatments but was erratic with dose. UDP-glucuronosyltransferase and thioredoxin reductase activities were not increased by any agent. Dexamethasone elevated Cyp1a1/2 mRNA at the low dose but reduced the mRNA transcript and activity of the enzyme at the mid and high doses. The mRNA responses of Ephx1 and Nqo1 showed a close parallel to each other with no increases after dexamethasone or SPL treatment, and elevations at the mid dose of PCN. With the exception of dexamethasone at the high dose, elevations in Gst mRNAs were seen with all doses of the three agents. When a large number of hepatic enzymes are examined, the responses to dexamethasone, SPL and PCN are far from identical, and any dose dependency is agent specific. Induction of enzymes seems more complicated to be controlled by one orphan receptor. This study not only filled the void about the murine PXR-induction profile but also will help in the course of drug development research with respect to extrapolation to human risk assessment. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The pregnane X receptor (PXR) binds drugs and xenobiotics affecting the expression of numerous genes involved in the elimination of endobiotics and xenobiotics. Spironolactone (SPL), pregnenolone-16 alpha-carbonitrile (PCN), and dexamethasone are prototypic activators of the PXR in rodents (Johnson and Klaassen, 2002; Buckley and Klaassen, 2009; Martin et al., 2010). Protective enzymes often considered include microsomal epoxide hydrolase (mEH), thioredoxin reductase (TR), and quinone oxidoreductase (NQO), as well as UDPglucuronosyltransferases (UDPGTs), and glutathione transferases (GSTs). Almost all are involved in maintaining cellular components in their appropriate redox status. Cytochrome P450 isoform 3A (CYP3A), UDPGT, and GST are known target genes for PXR agonists in human (Duret et al., 2006). In mice, PCN, dexamethasone and SPL were reported to increase Cyp3a mRNA in liver (Maglich et al., 2002; Xu et al., 2005), while, dexamethasone alone

⁎ Department of Zoology, Faculty of Science, Ain Shams University, Abbassia 11566, Cairo, Egypt. Tel.: + 20 2/2482 1633x253; fax: + 20 2/2684 2123. E-mail address: [email protected]. 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.03.047

increased CYP3A activity (Pirmohamed et al., 1992; Xu et al., 2005). PCN had no effect on the transcript level of Cyp1a1 (Maglich et al., 2002). SPL, dexamethasone, and PCN increased Ugt1a1, Ugt1a6, and Ugt1a9 mRNA levels in mouse liver (Buckley and Klaassen, 2009). In another study, PCN did not affect Ugt1a6 or Ugt2b5 in mice (Maglich et al., 2002; Chen et al., 2003) nor the p-nitrophenol activity (Viollon-Abadie et al., 1999; Chen et al., 2003). Dexamethasone increased Gstm transcripts and protein in murine thymoma cell lines (Briehl et al., 1995), but diminished GST expression and activity in mouse liver (Kim et al., 1998). PCN elevated GST activity and mRNA levels in murine liver (Hammock and Ota, 1983; Gong et al., 2006). Dexamethasone decreased mEH gene expression and activity (Kim et al., 1998), while PCN elevated the enzyme activity (Hammock and Ota, 1983). It is obvious that there is no general consensus or clear understanding of the PXR control of drug metabolizing enzymes in mice, although there is a need for a model better than rats in PXR induction profile, a profile that is very different from a human's (Zhang et al., 1999). In reviewing the literature, it was surprising to discover the paucity of information available on the induction and constitutive expression of murine drug-metabolizing enzymes by PXR agonists. Out of almost a thousand hits on PXR on PubMed.gov, very few were concerned with mice. This lack of information on PXR control of drug metabolizing enzymes especially phase II enzymes may be attributed to the fact that one of the first enzymes linked to this receptor was cytochrome P4503A (CYP3A), a

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subfamily of phase I enzymes that are involved in the metabolism of almost two-thirds of drugs that undergo oxidation (Zhang et al., 1999). Since PXR affects many genes of drug-metabolizing enzymes, extrapolation of results to human in experiments using mice would be inappropriate unless we clearly understand the similarities/differences in responses of these animals to human. To fill the void and to better understand which enzymes are controlled through the PXR in mice, a comprehensive examination was undertaken of the effects of PXR agonists on the mRNAs and activities of a range of drugmetabolizing enzymes in murine liver. 2. Materials and methods 2.1. Chemicals Dexamethasone, spironolactone, pregnenolone-16 alpha-carbonitrile and all other reagents were purchased from Sigma (St. Louis, MO) except where indicated in the specified methods. 2.2. Animal treatment and maintenance Adult male CF-1 albino mice (3 month-old, 25–35 g) were obtained from Charles River Laboratories and were maintained in a humidity (50 ± 5%) and temperature (20–25 °C) controlled environment on a 12-hour light/dark cycle with free access to standard rodent pellet food and tap water. The mice were housed in clear cages, 5–6 animals per cage. The pregnenolone-16 alpha-carbonitrile (PCN) was administered in corn oil by gavage (intragastric, i.g.), while dexamethasone and spironolactone (SPL) were administered in corn oil by intraperitoneal injection (i.p.). The three agents were administered at 50, 100, and 200 mg/kg, daily, for 3 consecutive days. All animal procedures were conducted in concordance with NIH guidelines for the humane care of laboratory animals. 2.3. Biological sample preparation Animals were sacrificed 24 h after the final dose, a blood sample was collected for immediate serum preparation, and the livers were quickly perfused in situ (via the hepatic portal vein) with normal ice cold saline. A 100 mg sample of liver was removed, homogenized in 2 ml of TRIzol solution (Invitrogen; Carlsbad, CA) and frozen at −80 °C for later RNA isolation. The gall bladder was then carefully dissected away, and the remaining liver homogenized in 0.25 M sucrose (20% w/v) and subjected to 3-stage differential centrifugation (9000 ×g for 15 min, 19,000 ×g for 15 min, and 105,000 ×g for 60 min) to prepare the cytosolic and microsomal fractions. Protein content of both fractions was determined by the method of Lowry et al. (1951) using Folin-Ciocalteu's phenol reagent (Sigma; St Louis, MO) and the fractions were stored at −80 °C until assayed for enzyme activity. 2.4. Enzyme activity Serum alanine aminotransferase (sALT) activity was determined according to Wroblewski and LaDue (1956), using a coupled reaction by monitoring the serum dependent change in absorbance of NADH oxidation at 340 nm in the presence of optimized concentrations of Lalanine, α-ketoglutarate and purified lactic dehydrogenase enzyme. Cytosolic quinone oxidoreductase (NQO) activity was determined from the 3,3 -methylene-bis-(4-hydroxycoumarin)-inhibited rate of reduction of 2,6-dichlorophenolindophenol by NADH, monitoring absorbance changes at 600 nm (Benson et al., 1980). Cytosolic glutathione Stransferase (GST) activities were determined from the change in absorbance at 340 nm resulted from conjugation of glutathione with 1chloro-2,4-dinitrobenzene [CDNB] (Habig and Jakoby, 1981). Cytosolic thioredoxin reductase (TR) activity was determined using the aurothioglucose-sensitive rate of reduction of 5,5 dithio-bis-(2-nitroben-

zoic acid) by NADPH monitored at 412 nm (modified from the method of Hill et al., 1997). Microsomal UDP-glucuronosyltransferase (UDPGT) activity was determined with 4-nitrophenol as the substrate (Franklin and Finkle, 1986), and microsomal epoxide hydrolase activity (mEH) was determined with cis-stilbene oxide as the substrate (Hammock et al., 1985). 7-methoxyresorufin (Sigma-Aldrich®, St. Louis, MO) Odemethylase activity, determined from the rate of fluorescence increase due to the formation of the resorufin (Ex 544 nm, Em 612 nm) was utilized as a monitor of CYP1A1/2 (Burke et al., 1994). Microsomal CYP3A activity was determined from the 6β-hydroxylation of testosterone (Sigma, St. Louis, MO) where the testosterone metabolite was separated by HPLC and quantified from its absorbance at 236 nm (Guengerich et al., 1986). Any elevation in the activities of the cytosolic or microsomal enzymes in the current study does not distinguish between an activation of existing enzyme and an increase arising from increased amounts of enzyme protein. 2.5. mRNA determination Hepatic mRNA levels were determined by Northern blotting of 20 μg of total RNA isolated by TRIzol extraction. Gel electrophoresis, nucleic acid transfer to membranes and 32P probe labeling, washing stringency, and development conditions were all performed as described previously (El-Sayed et al., 2006a). The sequences and homologies of cDNA probes for the alpha (a), mu (m), and pi (p) glutathione S-transferase (Gst), UDP-glucuronosyltransferase (Ugt-1a1, -1a6, -1a9, -2b5), NAD(P)Hquinone oxidoreductase (Nqo1), thioredoxin reductase (Txnrd1), and microsomal epoxide hydrolase (Ephx1) mRNAs are described in detail elsewhere (El-Sayed et al., 2006a, b). For Cyp1a1/2, the probe spanned the region 1511–2250 of rat CYP1A1 (X00469) which has 92 and 87% homology with mouse Cyp1a1 (BC125444) and Cyp1a2 (BC018298), respectively. The probes used for Cyp1a1 and Cyp1a2 were more than 93% similar and hence reported as Cyp1a1/2. All mRNA bands were normalized to the same-sample cyclophilin mRNA band to control for gel loading and transfer variations. Results are expressed as fold change from the proper corn oil-control animals. 2.6. Statistical analysis of data Results are expressed as the mean± S.E.M. Treated group size was 4– 6 animals. Statistical analyses were performed using ANOVA, followed by Fisher's protected least significant difference multiple range test. The data were compared against those treated with corn oil which in turn were not statistically different from naïve animals. PCN data were compared to those of corn oil administered intragastrically, and the dexamethasone and SPL data were compared to data of corn oil injected intraperitoneally. Differences were considered significant at P values of b0.05. 3. Results None of the treatments caused acute hepatotoxicity, since serum ALT was not elevated by any treatment (Table 1). No significant difference in body weight was recorded (data not shown). During the dosing period, clinical signs of toxicity such as hair loss, bleeding, shortness of breath or reduced food consumption were not reported either. All three treatments caused modest elevations in CYP3A activity but only increases reported after dexamethasone treatment at mid and high doses achieved statistical significance (Table 2). Insignificant reductions were seen in Cyp1a1/2 transcripts and again only dexamethasone treatment caused a significant increase in the enzyme transcript at 50 mg/kg and a significant reduction at the highest dose (Table 2). A reduction was also seen after the mid dose of dexamethasone but it did not achieve statistical significance (P ~ 0.06). These changes in cyp1a1/2 were reflected in enzyme activity, where dexamethasone treatment at both the mid and high doses evoked reductions in CYP1A1/2 (MROD) activities. PCN and SPL at the three

W.M. El-Sayed / European Journal of Pharmacology 660 (2011) 291–297 Table 1 Effect of PXR-agonists on a serum marker of hepatotoxicity. a

Drug/dose (mg/kg)

sALT activity

PCN 50 PCN 100 PCN 200 SPL 50 SPL 100 SPL 200 Dexamethasone 50 Dexamethasone 100 Dexamethasone 200

38.93 ± 5.32 36.32 ± 5.96 38.91 ± 3.84 45.45 ± 5.59 43.89 ± 13.91 49.17 ± 10.28 47.13 ± 3.86 55.98 ± 10.16 73.18 ± 9.98

(mU/ml serum)

No mean sALT value (Mean ± S.E.M., n = 6), was significantly different from the appropriate vehicle control mean value. PCN was compared with corn oil (i.g.), and SPL and dexamethasone were compared with corn oil (i.p.). The mean values for naïve, corn oil (i.g.), and corn oil (i.p.) were 42.23 ± 7.65, 38.73 ± 9.43, and 55.06 ± 8.29 (mU/ml serum), respectively.

dosing regimens used and dexamethasone at the low dose did not cause significant changes in enzyme activity (Table 2). With the exception of dexamethasone at the highest dose, all treatments affected either GST activity or the mRNAs levels, or both. Although PCN at the low dose did not affect GST activity, elevations in Gsta, Gstm, and Gstp transcripts were reported (Table 3 and Fig. 1). A similar scenario was repeated for SPL and dexamethasone at low and mid doses where increases in Gsta, Gstm, or Gstp transcripts were not manifest in the enzyme activity. Dexamethasone at low dose was similar to SPL at the same dose in elevating only Gstp. Both compounds were again similar at the mid dose in increasing both Gsta and Gstm (Table 3 and Fig. 1). The situation was completely different for PCN at mid and high doses and SPL at high dose, where elevations in enzyme activity were accompanied by inductions in mRNA levels; Gsta, Gstm, and Gstp for SPL; Gstm and Gstp for PCN at the mid dose; Gstp only for PCN at the highest dose (Table 3 and Fig. 1). UDPGT (Table 4) and TR (Fig. 2C) activities were not significantly increased by any agent at any dose investigated. Most changes seen for Ugt transcripts were caused by PCN at the mid dose which increased all Ugt1a transcripts (Table 4). PCN and SPL at the high dose elevated only Ugt1a1 mRNA level. Ugt2b5 was only elevated by dexamethasone at the low dose. Dexamethasone caused no elevation in transcripts of any other Ugt1a family at any dose studied (Table 4). At low doses, both PCN and SPL did not result in any significant change in the transcripts of either Ugt2b5 or any member of Ugt1a family, and neither did SPL at the mid dose (Table 4). SPL at the mid dose and dexamethasone at the high dose regimens elevated both mEH and NQO activities without affecting either Ephx1 Table 2 Effect of PXR-agonists on cytochromes P450 (CYP1A1/2) activity and mRNA and CYP3A activity. Drug / dose (mg/kg)

CYP1A1/2 (MROD) activitya Cyp1a1/2b (pmol/mg/min)

CYP3A activitya (pmol/mg/min)

PCN 50 PCN 100 PCN 200 SPL 50 SPL 100 SPL 200 Dexamethasone 50 Dexamethasone 100 Dexamethasone 200

44 ± 9 61 ± 25 36 ± 8 70 ± 43 58 ± 22 30 ± 10 24 ± 10 10 ± 3c 11 ± 5c

1.65 ± 0.10 1.25 ± 0.33 1.72 ± 0.07 1.43 ± 0.24 1.72 ± 0.18 1.87 ± 0.02 1.94 ± 0.04 2.20 ± 0.13c 2.17 ± 0.10c

a

Mean ± S.E.M., n = 6. Fold control (mean ± S.E.M., n = 4) from the proper control value. Indicates a significant difference (P b 0.05) from appropriate control. PCN was compared with corn oil (i.g.), and SPL and dexamethasone were compared with corn oil (i.p.). The mean values of enzyme activities for naïve, corn oil (i.g.), and corn oil (i.p.) were 53 ± 18, 41 ± 8, and 48 ± 11 pmol/mg/min, respectively for CYP1A2, and 1.37 ± 0.45, 1.56 ± 0.32, and 1.38 ± 0.47 pmol/mg/min, respectively for CYP3A. b c

or Nqo1 transcripts (Fig. 2). On the contrary, PCN at the mid dose increased both transcripts without affecting either enzyme activity. PCN at the low dose and SPL at the high dose increased mEH activity (Fig. 2A). SPL at the low dose was the only agent to increase the Txnrd1 transcript level but without affecting the enzyme activity (Fig. 2C and F). 4. Discussion

a

0.56 ± 0.05 0.68 ± 0.15 0.86 ± 0.17 0.91 ± 0.11 0.68 ± 0.13 0.93 ± 0.09 1.46 ± 0.31c 0.54 ± 0.16 0.24 ± 0.06c

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Since the cloning of the PXR more than a decade ago (Kliewer et al., 1998), extensive investigations have been performed to discover which key enzymes are controlled by this receptor. PXR is now a target for treatment of many human diseases such as cancer and cholestatic liver diseases (Carnahan and Redinbo, 2005; Raynal et al., 2010). Changes in PXR will affect the metabolism of xenobiotics, endobiotics and drugs by the enzymes controlled through PXR. Thus PXR is a cornerstone in drug development research. Stimulation of phase II drug metabolizing enzymes would be favored because they contribute to the elimination of toxic xenobiotics and improve the antioxidant status of the cell especially GSTs. On the other hand, changes in phase I enzymes especially the CYP3A could lead to drugdrug interactions and/or failure of many drugs such as antiretroviral drugs, cyclosporine A, ethinyl estradiol, digitoxin, lovastatin, tamoxifen, and warfarin (van den Bout-van den Beukel et al., 2006; Ingelman-Sundberg et al., 2007; Andrews et al., 2008; Tirkkonen et al., 2008). Induction of CYPs could also result in the activation of many procarcinogens such as benzo(a)pyrene, dimethylbenz(a)anthracene, NNK and aflatoxin (Kleiner et al., 2004; Peterson et al., 2006). In most of these experiments, rodents especially rats are number one choice but it was shown that rat PXR shares only 79% identity with the human PXR and has a different induction profile (Zhang et al., 1999), and thus extrapolation could not be made. This makes the search for an animal model that has a similar PXR induction profile to that of human an urgent need. In reviewing the literature, few reports were found about the PXR-agonists and PXR-controlled enzymes in mice. This present study seeks to contribute to a better understanding of the murine PXR induction profile. To achieve the goal(s), the effects of three known PXR agonists, spironolactone (SPL), pregnenolone-16 alpha-carbonitrile (PCN), and dexamethasone on the mRNA levels and activities of a range of hepatic drug metabolizing enzymes were examined in CF1 mice. CYP3A expression is known to be controlled through the PXR (Xu et al., 2005). Although all compounds used induced modest dosedependent elevations in CYP3A activity, only elevations seen after dexamethasone treatment both at the mid and high doses were statistically significant. This is in concordance with previous reports by Pirmohamed et al. (1992) and Xu et al. (2005). Dexamethasone at the same doses that elevated CYP3A, caused a 46–76% decrease in the Cyp1a1/2 mRNA levels and about 80% reduction in the enzyme activity. Whether it is due to pharmacological or toxic effect of dexamethasone at these doses, is something that cannot be discerned in the present study. Eken et al. (2006) showed that low and high doses of dexamethasone caused liver damage in rats, but in the present study the 30% increase in sALT caused by dexamethasone at the highest dose was not statistically significant (Table1). The differentiation in control of CYP3A and CYP1A was seen before in rat and human hepatocytes where only CYP3A was elevated and only by dexamethasone (Nishimura et al., 2007; Kishida et al., 2008). This could be attributed to different molecular control of both CYPs. Cross talk between PXR, constitutive androstane receptor, and aryl hydrocarbon receptor is known to regulate many drug metabolizing enzymes such as UDPGT and CYPs (Sugatani et al., 2004; Xu et al., 2005; Tompkins and Wallace, 2007). PXR was suggested to regulate CYP1A1/2 by regulating aryl hydrocarbon receptor (Maglich et al., 2002), and constitutive androstane receptor was reported to regulate CYP3A along with PXR (Xie et al., 2000). Thus, the ability of an agonist

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Table 3 Effect of PXR-agonists on glutathione S-transferase (GST) activity and mRNAs (Gsta, Gstm, and Gstp). Drug / dose (mg/kg)

GST activitya (nmol/mg/min)

Gstab

Gstmb

Gstpb (2.4 kb)

Gstpb (1.2 kb)

PCN 50 PCN 100 PCN 200 SPL 50 SPL 100 SPL 200 Dexamethasone 50 Dexamethasone 100 Dexamethasone 200

4743 ± 457 6147 ± 749c 6329 ± 189c 4966 ± 540 5554 ± 297 6409 ± 681c 4690 ± 118 5199 ± 1073 5226 ± 330

1.95 ± 0.32c 1.01 ± 0.11 0.72 ± 0.10 1.47 ± 0.18 1.75 ± 0.23c 2.05 ± 0.41c 1.21 ± 0.06 2.33 ± 0.39c 0.79 ± 0.10

2.64 ± 0.71c 1.98 ± 0.27c 1.17 ± 0.13 1.58 ± 0.21 2.04 ± 0.21c 1.85 ± 0.23c 1.69 ± 0.10 3.44 ± 0.29c 1.24 ± 0.08

2.77 ± 0.69c 2.52 ± 0.51c 2.23 ± 0.78c 2.53 ± 0.24c 1.77 ± 0.09 2.30 ± 0.55c 1.76 ± 0.12 1.64 ± 0.12 1.04 ± 0.16

1.77 ± 0.12c 1.90 ± 0.37c 1.63 ± 0.35c 1.93 ± 0.24c 1.17 ± 0.25 1.61 ± 0.39 2.33 ± 0.25c 1.62 ± 0.03 0.82 ± 0.15

a

Mean ± S.E.M., n = 6. Fold control (mean ± S.E.M., n = 4) from the proper control value. Indicates a significant difference (P b 0.05) from appropriate control. PCN was compared with corn oil (i.g.), and SPL and dexamethasone were compared with corn oil (i.p.). The mean values of enzyme activities for naïve, corn oil (i.g.), and corn oil (i.p.) were 5019 ± 958, 4413 ± 467, and 4201 ± 186 nmol/mg/min, respectively. b c

to induce an enzyme expression/activity depends on its capability of acting on more than a single orphan receptor. Interestingly, dexamethasone at the low dose caused about 50% increase in Cyp1a1/2 mRNA level without affecting the enzyme activity. Despite the fact that PCN and SPL are also known PXR activators, both failed to significantly affect either the activity or mRNA level of CYP1A1/2 and CYP3A at any dose examined (Table 2). It seems that the response of CYPs is very much dependent on the agonist and the dose. As for the phase II-drug metabolizing enzymes, GSTs were the most responsive enzymes and PCN was the most effective treatment. With the exception of the treatment at the low dose, PCN at all doses evoked elevations in both GST gene expression and enzyme activity (Table 3 and Fig. 1). Similarly, SPL elevated the mRNA levels and enzyme activity but only at the highest dose. Dexamethasone and SPL at low and mid doses caused very similar patterns of changes, both elevated Gstp at the low dose and elevated Gsta and Gstm at the mid dose without affecting the enzyme activity (Table 3). The unique effectiveness of PCN in inducing GST activity and mRNA levels was previously reported in mice (Hammock and Ota, 1983; Maglich et al., 2002; Gong et al., 2006; Knight et al., 2008). GST is known to be controlled by an antioxidant response element (ARE) and nuclear related factor 2 (Nrf2) which is known to regulate alpha and mu GST subunits (Hayes et al., 2000), but PXR is now known to play a major role in the control of some GST isozymes through possible interaction with ARE (Falkner et al., 2001; Knight et al., 2008). For UDPGT, PCN was again the most effective agent in inducing elevations in mRNA levels. PCN at the mid dose caused ~(3–4)-fold elevations in all Ugt1a family members examined; Ugt -1a1, -1a6, and -1a9. PCN and SPL at the highest doses only caused significant elevations in Ugt1a1 and in both situations without manifest in enzyme activity (Table 4). Indeed,

no agent at any dose significantly elevated the p-nitrophenol (PNP) activity. Dexamethasone was the only agent to increase mRNA level of Ugt2b5 but was devoid of any significant effect on any Ugt1a family members at any dose (Table 4). UDPGT has been the most studied enzyme in mice and there is a general agreement that PCN is an inducer of Ugt mRNAs through PXR (Maglich et al., 2002; Chen et al., 2003; Buckley and Klaassen, 2009) without affecting the PNP activity (Hazelton and Klaassen, 1988; Viollon-Abadie et al., 1999; Chen et al., 2003). To a much lesser extent, dexamethasone and SPL have been studied and reported to elevate Ugt1a family transcripts in mice (Buckley and Klaassen, 2009). In humans, UDPGT genes are suggested to be regulated by constitutive androstane receptor, aryl hydrocarbon receptor and PXR (Mackenzie et al., 2003; Sugatani et al., 2005), but the differential expression of UDPGT isoforms between subjects may be attributed to individual variation in PXR expression (GardnerStephen et al., 2004). Dexamethasone was reported to elevate UGT1A1 through PXR in human (Sugatani et al., 2005), while in the present study, dexamethasone at all doses had no effect on any genes of the family Ugt1a investigated. Similarly, Usui et al. (2006) reported that PCN was also without any effect on UGT1A1, while in the present study, PCN elevated Ugt1a1. Usui et al. (2006) found that dexamethasone had no effect on UGT1A1 expression, which is similar to the present findings. Overexpression of PXR in hPXR transfected HepG2 cells resulted in increase in the mRNA of UGT1A6, GST alpha and mu subunits in comparison to HepG2 cells which are devoid of PXR (Naspinski et al., 2008). In the current study, PCN elevated Ugt1a6 mRNA and PCN, SPL, and dexamethasone elevated the mRNA of GST alpha and mu subunits. These similarities suggest that some GST and UDPGT isoforms are regulated by PXR and the inducers used in the present study could be PXR agonists. The mechanism of induction

Fig. 1. Effect of PXR agonists on mRNA transcript levels of glutathione S-transferase (Gsta, Gstm, and Gstp (2.4 kb and 1.2 kb)). All mRNA bands were normalized to the same-sample cyclophilin (CP) mRNA band. Pregnenolone-16alpha-carbonitril (PCN) was compared to corn oil (i.g.), and spironolactone (SPL) and dexamethasone were compared to corn oil (i.p.). Lane 1: naïve. Lane 2: corn oil control (i.g.). Lane 3: corn oil control (i.p.). Lanes 4, 5, and 6: PCN at 50, 100, and 200 mg, respectively. Lanes 7, 8, and 9: SPL at 50, 100, and 200 mg, respectively. Lanes 10, 11, 12: Dexamethasone at 50, 100, and 200 mg, respectively. ⁎Bands were cut and rearranged to keep the same order for clarity.

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Table 4 Effect of PXR-agonists on UDP-glucuronosyltransferase (UDPGT) activity and mRNAs (Ugt-1a1, -1a6, -1a9, and -2b5). Drug / dose (mg/kg)

UDPGT activitya (nmol/mg/min)

Ugt1a1b

Ugt1a6b

Ugt1a9b

Ugt2b5b

PCN 50 PCN 100 PCN 200 SPL 50 SPL 100 SPL 200 Dexamethasone 50 Dexamethasone 100 Dexamethasone 200

5.35 ± 0.43 4.19 ± 0.05 4.70 ± 1.00 6.04 ± 0.90 6.23 ± 1.22 5.14 ± 0.37 4.78 ± 1.74 4.38 ± 0.74 5.23 ± 0.88

1.03 ± 0.36 3.00 ± 1.17c 1.93 ± 0.26c 1.70 ± 0.13 1.16 ± 0.24 2.04 ± 0.48c 1.15 ± 0.21 1.22 ± 0.29 0.77 ± 0.22

1.20 ± 0.44 3.42 ± 1.08c 1.89 ± 0.27 1.49 ± 0.21 1.87 ± 0.45 1.73 ± 0.27 1.53 ± 0.34 1.29 ± 0.29 0.84 ± 0.26

1.40 ± 0.64 3.73 ± 1.42c 1.44 ± 0.32 1.93 ± 0.13 1.84 ± 0.21 0.32 ± 0.09 0.72 ± 0.25 1.11 ± 0.35 0.65 ± 0.10

0.95 ± 0.20 1.45 ± 0.23 1.65 ± 0.31 1.19 ± 0.33 1.11 ± 0.24 1.08 ± 0.06 2.06 ± 0.57c 0.99 ± 0.18 0.69 ± 0.15

a

Mean ± S.E.M., n = 6. Fold control (mean ± S.E.M., n = 4) from the proper control value. Indicates a significant difference (P b 0.05) from appropriate control. PCN was compared with corn oil (i.g.), and SPL and dexamethasone were compared with corn oil (i.p.). The mean values of enzyme activities for naïve, corn oil (i.g.), and corn oil (i.p.) were 4.39 ± 0.69, 5.23 ± 1.92, and 5.01 ± 0.36 nmol/mg/min, respectively. b c

may include both transcriptional and post-transcriptional control and the lack of concordance in the changes in mRNA levels and enzyme activity suggests a post-translational regulation as well. For the mechanistic regulation of UDPGT genes, there is no general agreement and the regulation process seems to be very complex and due to

interactions between many nuclear receptors and transcription factors to be determined making the comparison not only to mice but also to any experimental animal model not possible. Sporadic changes were seen in activity and mRNA levels for mEH, NQO, and TR. For mEH, only the mid dose of PCN induced mRNA but

Fig. 2. A: Effect of PXR agonists on microsomal epoxide hydrolase (mEH) activity. ⁎Indicates a significant difference (P b 0.05) from appropriate control. PCN was compared with corn oil (i.g.), and SPL and dexamethasone were compared with corn oil (i.p.). Data are expressed as Mean ± S.E.M., n = 6. Fig. 2B: Effect of PXR agonists on quinone oxidoreductase (NQO) activity. ⁎ indicates a significant difference (P b 0.05) from appropriate control. PCN was compared with corn oil (i.g.), and SPL and dexamethasone were compared with corn oil (i.p.). Data are expressed as Mean ± S.E.M., n = 6. Fig. 2C: Effect of PXR agonists on thioredoxin reductase (TR) activity. PCN was compared with corn oil (i.g.), and SPL and dexamethasone were compared with corn oil (i.p.). Data are expressed as Mean ± S.E.M., n = 6. Fig. 2D: Effect of PXR agonists on microsomal epoxide hydrolase (Ephx1) mRNA. Data are expressed as fold control (Mean ± S.E.M., n = 4) from the proper control value. PCN was compared with corn oil (i.g.), and SPL and dexamethasone were compared with corn oil (i.p.). ⁎Indicates a significant difference (P b 0.05) from appropriate control. Fig. 2E: Effect of PXR agonists on quinone oxidoreductase (Nqo1) mRNA. Data are expressed as fold control (Mean ± S.E.M., n = 4) from the proper control value. PCN was compared with corn oil (i.g.), and SPL and dexamethasone were compared with corn oil (i.p.). ⁎Indicates a significant difference (P b 0.05) from appropriate control. Fig. 2F: Effect of PXR agonists on thioredoxin reductase (Txnrd1) mRNA. Data are expressed as fold control (Mean ± S.E.M., n = 4) from the proper control value. PCN was compared with corn oil (i.g.), and SPL and dexamethasone were compared with corn oil (i.p.). ⁎Indicates a significant difference (P b 0.05) from appropriate control.

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all PXR agonists at some dose were able to significantly increase the enzyme activity; PCN at low dose, SPL at both mid and high doses, and dexamethasone at high dose (Fig. 2). In a study by Hammock and Ota (1983), PCN was also able to elevate mEH activity. A lack of concordance between the gene expression and activity was reported in human hepatocytes (Hassett et al., 1998). The scenario was repeated for NQO, where again PCN at the mid dose elevated the Nqo1 transcript level without affecting the activity, while, SPL at the mid dose and dexamethasone at the high dose elevated the activity but caused no increase in gene expression (Fig. 2). In fact, most treatments caused statistically insignificant reductions in Nqo1 mRNA levels and the highest reduction of ~58% was caused by dexamethasone at the mid dose. Dexamethasone was reported to decrease the NQO activity and gene expression in rats through the glucocorticoid receptor not through PXR (Schuetz and Guzelian, 1984; Pinaire et al., 2004). Similar to UDPGT activity, TR activity was not affected by any treatment at any dose and SPL at the low dose was the only treatment that evoked an increase in the Txnrd1 mRNA level. 5. Conclusions To summarize, not all changes in mRNA levels were accompanied by similar changes in enzyme activity, suggesting various molecular regulatory mechanisms. Among the three PXR agonists, dexamethasone was the most efficacious inducer of CYP3A. PCN was the most effective inducer of almost all enzymes suggesting that it might act through many orphan receptors in mice. From this 3-compound, 3-dose study, no prototypical murine PXR agonist response in drug metabolizing enzymes could be discerned. It seems that every individual agonist has its own distinct induction profile and the response of these enzymes to PXR agonists was very much dependent on the individual agonist and sometimes on the dose. It is possible that some responses may be related to other pharmacological activities outside their ability to act as PXR agonists, or may be due to their potential capability of affecting orphan receptors other than PXR, an overlapping property that is known for many prototypical inducers and for many enzymes. PXR induction profile in mice in the current study has shown some similarities to that of human such as the complex interaction in the regulation of UDPGT. Dexamethasone was devoid of effect on Ugt1a family of genes and in both human and mice, PCN had similar effects elevating Gsta and Gstm subunits and Ugt1a6. In addition, dexamethasone elevated CYP3A in both human and mice. mEH responses in humans as well as in mice were modest and a lack of concordance between protein and mRNA was common. The results also suggest that PCN could act as PXR ligand in both mice and humans in a very much similar way. With the paucity of information in the literature about the response of the hepatic drug metabolizing enzymes to PXR agonists in mice, more studies are needed. References Andrews, E., Damle, B.D., Fang, A., Foster, G., Crownover, P., LaBadie, R., Glue, P., 2008. Pharmacokinetics and tolerability of voriconazole and a combination oral contraceptive co-administered in healthy female subjects. Br. J. Clin. Pharmacol. 65, 531–539. Benson, A.M., Hunkeler, M.J., Talalay, P., 1980. Increase of NAD(P)H:quinone reductase by dietary antioxidants: possible role in protection against carcinogenesis and toxicity. Proc. Natl. Acad. Sci. U. S. A. 77, 5216–5220. Briehl, M.M., Cotgreave, I.A., Powis, G., 1995. Downregulation of the antioxidant defence during glucocorticoid-mediated apoptosis. Cell Death Differ. 2, 41–46. Buckley, D.B., Klaassen, C.D., 2009. Induction of mouse UDP-glucuronosyltransferase mRNA expression in liver and intestine by activators of aryl-hydrocarbon receptor, constitutive androstane receptor, pregnane X receptor, peroxisome proliferatoractivated receptor alpha, and nuclear factor erythroid 2-related factor 2. Drug Metab. Dispos. 37, 847–856. Burke, M.D., Thompson, S., Weaver, R.J., Wolf, C.R., Mayer, R.T., 1994. Cytochrome P450 specificities of alkoxyresorufin O-dealkylation in human and rat liver. Biochem. Pharmacol. 48, 923–936. Carnahan, V.E., Redinbo, M.R., 2005. Structure and function of the human nuclear xenobiotic receptor PXR. Curr. Drug Metab. 6, 357–367.

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