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Comparison of the hepatic and thyroid gland effects of sodium phenobarbital in wild type and constitutive androstane receptor (CAR) knockout rats and pregnenolone-16α-carbonitrile in wild type and pregnane X receptor (PXR) knockout rats
Toxicology 400–401 (2018) 20–27
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Comparison of the hepatic and thyroid gland effects of sodium phenobarbital in wild type and constitutive androstane receptor (CAR) knockout rats and pregnenolone-16α-carbonitrile in wild type and pregnane X receptor (PXR) knockout rats
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Corinne Hainesa, Lynsey R. Chathama, Audrey Vardya, , Clifford R. Elcombea,1, John R. Fosterb, Brian G. Lakea,c a b c
Concept Life Sciences (formerly CXR Biosciences Ltd.), 2, James Lindsay Place, Dundee Technopole, Dundee DD1 5JJ, UK Regulatory Science Associates, Kip Marina, Inverkip, Renfreshire, PA16 0AS, UK Centre for Toxicology, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, GU2 7XH, UK
A R T I C LE I N FO
A B S T R A C T
Keywords: Sodium phenobarbital Pregnenolone-16α-carbonitrile Constitutive androstane receptor Pregnane X receptor Receptor knockout rats Cytochrome P450
A number of chemicals produce liver and thyroid gland tumours in rodents by nongenotoxic modes of action (MOAs). In this study the hepatic and thyroid gland effects of the constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) were examined in male Sprague-Dawley wild type (WT) rats and in CAR knockout (CAR KO) rats and the effects of the pregnane X receptor (PXR) activator pregnenolone-16α-carbonitrile (PCN) were examined in WT and PXR knockout (PXR KO) rats. Rats were either fed diets containing 0 (control) or 500 ppm NaPB or were dosed with 0 (control) or 100 mg/kg/day PCN orally for 7 days. The treatment of WT rats with NaPB and PCN for 7 days resulted in increased relative liver weight, increased hepatocyte replicative DNA synthesis (RDS) and the induction of cytochrome P450 CYP2B and CYP3A subfamily enzyme, mRNA and protein levels. In marked contrast, the treatment of CAR KO rats with NaPB and PXR KO rats with PCN did not result in any increases in liver weight and induction of CYP2B and CYP3A enzymes. The treatment of CAR KO rats with NaPB had no effect on hepatocyte RDS, while PCN produced only a small increase in hepatocyte RDS in PXR KO rats. Treatment with NaPB had no effect on thyroid gland weight in WT and CAR KO rats, whereas treatment with PCN resulted in an increase in relative thyroid gland weight in WT, but not in PXR KO, rats. Thyroid gland follicular cell RDS was increased by the treatment of WT rats with NaPB and PCN, with NaPB also producing a small increase in thyroid gland follicular cell RDS in CAR KO rats. Overall, the present study with CAR KO rats demonstrates that a functional CAR is required for NaPB-mediated increases in liver weight, stimulation of hepatocyte RDS and induction of hepatic CYP enzymes. The studies with PXR KO rats demonstrate that a functional PXR is required for PCN-mediated increases in liver weight and induction of hepatic CYP enzymes; with induction of hepatocyte RDS also being largely mediated through PXR. The hepatic effects of NaPB in CAR KO rats and of PCN in PXR KO rats are in agreement with those observed in other recent literature studies. These results suggest that CAR KO and PXR KO rats are useful experimental models for liver MOA studies with rodent CAR and PXR activators and may also be useful for thyroid gland MOA studies.
1. Introduction A number of nongenotoxic chemicals have been shown to produce liver and thyroid gland tumours in rodents (Gold et al., 2001; Huff et al., 1991). Both rodent liver and thyroid gland tumour formation may be associated with induction of hepatic xenobiotic metabolising enzymes. For example, a number of nongenotoxic chemicals, including
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phenobarbital (PB) and/or its sodium salt (sodium phenobarbital; NaPB), have been shown to produce liver tumours in rats and/or mice (Cohen, 2010; Elcombe et al., 2014; Lake, 2009) by a mode of action (MOA) involving activation of the constitutive androstane receptor (CAR). In an evaluation of the MOA for PB-induced rodent liver tumour formation by Elcombe et al. (2014), the key events were identified as CAR activation, altered gene expression specific to CAR activation,
Corresponding author. E-mail address: [email protected] (A. Vardy). Deceased.
https://doi.org/10.1016/j.tox.2018.03.002 Received 28 November 2017; Received in revised form 5 March 2018; Accepted 6 March 2018 Available online 13 March 2018 0300-483X/ © 2018 Elsevier B.V. All rights reserved.
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under a UK Home Office license and all animal studies were approved by the Ethical Review Committee of the University of Dundee (Dundee, Scotland, UK). Male Sprague-Dawley WT, CAR KO and PXR KO rats (14 weeks old at study start) were implanted subcutaneously with osmotic pumps (Alzet model number 2ML1, Charles River UK Ltd., Margate, Kent, UK) containing 15 mg/ml BrdU in phosphate buffered saline pH 7.4. Rats were then either fed powdered diets containing 0 (control) or 500 ppm (500 mg/kg) NaPB or were dosed with 0 (control) or 100 mg/ kg/day PCN orally for 7 days. For PCN, control animals received corresponding quantities (10 ml/kg body weight) of the corn oil vehicle. Male rather than female rats were used in this study, as male animals are more often used for rodent liver CAR activator MOA studies. At the end of the treatment period rats were killed by exposure to carbon dioxide gas and blood taken for plasma analysis. The liver of each rat was removed, weighed and sampled for morphological and biochemical investigations. The thyroid glands and trachea of each animal were removed intact and fixed in 10% neutral buffered formalin (NBF). After at least 24 h of fixation, the thyroid glands were carefully dissected from the trachea, blotted dry and weighed, prior to returning to the fixation medium. The duodenum of each animal was also excised.
increased cell proliferation, the development of altered hepatic foci and finally liver tumour formation. Associative events for this MOA included liver hypertrophy, induction of cytochrome P450 (CYP) enzymes (particularly CYP2B subfamily enzymes) and inhibition of apoptosis. Some nongenotoxic chemicals, including some CAR activators, have been shown to produce thyroid gland tumours in rodents by a MOA in which circulating thyroid hormone levels are decreased as a result of increased hepatic metabolism and clearance (Capen, 2001; Dellarco et al., 2006; Hill et al., 1998; McClain et al., 1989; Meek et al., 2003). In this MOA, thyroxine (T4) conjugation is enhanced due to the stimulation of hepatic microsomal UDPglucuronosyltransferase (UGT) enzymes towards T4 as substrate resulting in increased biliary excretion of T4, a decrease in serum triiodothyronine (T3) and/or T4 levels and a compensatory increase in serum thyroid stimulating hormone (TSH) levels. In the rodent thyroid gland, the increase in TSH levels results in thyroid follicular cell hypertrophy and hyperplasia, with chronic stimulation subsequently producing thyroid follicular cell tumours (Capen, 2001; Curran and DeGroot, 1991; Hill et al., 1998). In establishing the MOA for rodent liver tumour formation by CAR activators, studies in transgenic mice lacking CAR (i.e. CAR knockout (KO) mice) have been particularly useful. Such studies have demonstrated that PB does not increase liver weight, produce liver hypertrophy, does not induce CYP2B enzymes or replicative DNA synthesis (RDS) and does not promote liver tumours in CAR KO mice (Huang et al., 2005; Scheer et al., 2008; Wei et al., 2000; Yamamoto et al., 2004). Recently, transgenic rat models lacking either the CAR or the pregnane X receptor (PXR), namely CAR KO and PXR KO rats, have been developed and have become commercially available (Forbes et al., 2017). The objective of this study was to evaluate the usefulness of the CAR KO and PXR KO rat models for liver and thyroid gland MOA studies. Male Sprague-Dawley (i.e. wild type; WT) and CAR KO rats were treated with NaPB and male WT and PXR KO rats were treated with pregnenolone-16α-carbonitrile (PCN). NaPB and PCN were selected as known rodent liver CAR and PXR activators, respectively (Elcombe et al., 2014, Lake et al., 1998; Martignoni et al., 2006; Omiecinski et al., 2011).
2.3. Plasma analysis Levels of PB in plasma were determined by protein precipitation followed by quantification by high performance liquid chromatography-mass spectrometry-mass spectrometry (LC–MS/MS) employing a Waters 2795 separation module equipped with a Waters Quattro micro mass spectrometer operated in negative ion mode with electrospray ionisation. Plasma PB levels were determined employing a Kinetex SB column (100 × 4.6 mm, 3 μm), with a mobile phase of 80:20 (v/v) methanol/water at a flow rate of 0.5 ml/min, with detection using a transition of 231.46 → 231.27. Levels of PCN in plasma were determined by protein precipitation followed by quantification by LC–MS/MS employing a Waters 2795 separation module equipped with a Waters Quattro micro mass spectrometer operated in positive ion mode with ACPI ionisation, using a transition of 342.30 → 224.23. Plasma PCN levels were determined employing a Luna C18(2) column (150 × 2 mm, 5 μm) at 35 °C employing mobile phases of methanol and 0.1% (v/v) formic acid in water with gradient elution at a flow rate of 0.4 ml/min.
2. Materials and methods 2.1. Materials
2.4. Hepatic morphological investigations
7-Benzyloxyquinoline and 7-hydroxyquinoline were purchased from Cypex (Dundee, UK). NaPB (purity ≥ 99%), 5-bromo-2′-deoxyuridine (BrdU), and other CYP substrates and metabolites were obtained from Sigma-Aldrich (Poole, Dorset, UK), whereas PCN (purity 99%) was obtained from Enzo Life Sciences Inc. (Farmingdale, NY, USA). For Western immunoblotting, an anti-rat CYP1A antibody was obtained from Corning (Corning B.V., Amsterdam, The Netherlands), whereas anti-rat CYP2B, CYP3A and CYP4A antibodies were obtained from the Biomedical Research Institute (University of Dundee, Scotland, UK). Secondary antibodies were horseradish peroxidase (HRP)-linked antisheep IgG (Abgene, Blenheim Road, Epsom, Surrey, UK) and anti-rabbit IgG (GE Healthcare, Little Chalfont, Buckinghamshire).
Two samples of liver from each rat (one from the left lobe and one from the median lobe) were fixed in NBF, dehydrated, embedded in paraffin wax and approximately 5 μm sections were stained with hematoxylin and eosin. Sections were examined by light microscopy and the degree of liver hypertrophy scored as either minimal (grade 1), slight (grade 2), moderate (grade 3) or marked (grade 4). For each group, the liver hypertrophy severity grades for each animal were summed and a group mean severity grade calculated. 2.5. Replicative DNA synthesis (RDS) Liver, thyroid gland and duodenum (as a methodology positive control) samples were fixed in NBF for approximately 36 h prior to processing into paraffin blocks. BrdU immunocytochemistry was performed as described previously (Ross et al., 2010). Image capture and acquisition were carried out using a Zeiss Imager A1 microscope and Improvision imaging software, Volocity version 4 (Perkin Elmer, Bucks, UK) was used for data analysis. The BrdU labelling index (i.e. percentage of either hepatocyte or thyroid follicular cell nuclei undergoing RDS) for each animal was determined by counting approximately 3000 nuclei in 10 random areas from both the left and median lobes of the liver and at least 3000 nuclei in 20 fields from the thyroid gland.
2.2. Animals and treatment Male Sprague-Dawley WT, CAR KO and PXR KO rats were obtained from Horizon Discovery (Boyertown, PA, USA). Rats were allowed free access to water and powdered or pelleted RM1 laboratory animal diet (Special Diets Services, Witham, Essex, UK) and were housed singly in accommodation with a 12:12 h light:dark cycle. Temperature and relative humidity were maintained between 19 and 23 °C and 40 and 70%, respectively. Rats were allowed to acclimatize to these conditions for at least 5 days before use. All animal procedures were performed 21
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Fig. 1. Effect of treatment of WT and CAR KO rats with 0 (control) and 500 ppm NaPB in the diet and WT and PXR KO rats with 0 (control) and 100 mg/kg/day PCN by gavage for 7 days on body weight (A, B) relative liver weight (C, D) and hepatocyte RDS (E, F). Results are expressed as mean ± SD of 5 animals. Values significantly different from control are: ***p < 0.001.
Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and cDNA was then synthesised using a Quantitect reverse transcription kit (Cat number 205311/205313 Qiagen, Hilden, Germany). Real-time PCR (Life Technologies QuantStudio 6 Flex system) was performed using TaqMan Fast Universal PCR Master Mix (Applied Biosystems, Inchinnan Business Park, Paisley, Scotland, UK) and TaqMan gene expression assays (Applied Biosystems) performed with primer-probe sets for the following genes: CYP1A1 (Rn01418021_g1), CYP1A2 (Rn00561082_m1), CYP2B1 (Rn01457875_m1), CYP2B2 (Rn02786833_m1), CYP3A1 (Rn03062228_m1) and CYP4A1 (Rn00598510_m1). The expression of each gene was normalised against endogenous β-actin (Rn00667869_m1). Data were analysed by generation of the threshold cycle (Ct) and delta Ct values were calculated for all genes. Fold change was analysed by calculating 2−ΔΔCt and results were expressed as fold change relative to control where control values were normalised to 1.00.
2.6. Hepatic biochemical investigations Whole liver homogenates (0.25 g fresh tissue/ml) were prepared in ice-cold 0.25 M sucrose containing 5 mM EDTA and 20 mM Tris-HCl pH 7.4 and liver microsomes prepared by differential centrifugation. Microsomes were resuspended in fresh homogenising medium and aliquots stored at approximately −70 °C. Protein content was determined by the method of Lowry et al. (1951) employing bovine serum albumin as standard. Microsomal total CYP content, 7-ethoxyresorufin O-deethylase (EROD), 7-pentoxyresorufin O-depentylase (PROD), 7-benzyloxyresorufin O-debenzylase (BROD) and 7-benzyloxyquinoline O-debenzylase (BQ) activities were determined as described previously (Choi et al., 2017; Ross et al., 2010).
2.7. Hepatic CYP mRNA levels Liver samples were homogenised using a Precellys 24 lysis and homogenisation control unit. RNA was then extracted using an RNeasy mini kit (Cat number 74104/74106, Qiagen, Hilden, Germany). RNA quantity and quality (260/280 nm ratio) was determined on a 22
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2.8. Western immunoblotting of hepatic CYP enzymes
3.3. Effect on hepatic enzyme activities
For Western immunoblot analysis, microsomal proteins from pooled rat liver microsome samples (n = 5 per group) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, electrophoretically transferred to polyvinylidene difluoride membranes and probed with anti-rat CYP1A, CYP2B, CYP3A and CYP4A antibodies. Protein loadings were 20 μg for CYP1A, 0.5 or 2.5 μg for CYP2B and 40 μg for both CYP3A and CYP4A. Secondary antibodies were HRPlinked anti-sheep IgG (for CYP1A) and anti-rabbit IgG (for CYP2B, CYP3A and CYP4A). Detection of immunoreactive bands was performed by enhanced luminescence detection (GE Healthcare).
The treatment of WT rats with NaPB (Fig. 3A) and PCN (Fig. 3B) for 7 days resulted in statistically significant increases in hepatic microsomal total CYP content. In contrast, the treatment of CAR KO rats with NaPB and PXR KO rats with PCN did not result in any effect on microsomal total CYP content. Hepatic microsomal 7-ethoxyresorufin Odeethylase (EROD), 7-pentoxyresorufin O-depentylase (PROD), 7-benzyloxyresorufin O-debenzylase (BROD) and 7-benzyloxyquinoline Odebenzylase (BQ) activities were determined as markers of induction of CYP1A, CYP2B, CYP2B/3A and CYP3A subfamily enzymes, respectively (Lubet et al., 1990; Renwick et al., 2001). The treatment of WT rats with NaPB (Fig. 3C) and PCN (Fig. 3D) produced some significant increases in microsomal EROD activity. Treatment of WT rats with NaPB produced marked increases in microsomal CYP2B-dependent PROD (Fig. 3E) and CYP2B/3A-dependent BROD (Fig. 4A) activities, with CYP3A-dependent BQ activity also being significantly increased (Fig. 4C). The treatment of WT rats with PCN significantly increased microsomal PROD (Fig. 3F), BROD (Fig. 4B) and BQ (Fig. 4D) activities. In contrast, the treatment of CAR KO rats with NaPB and PXR KO rats with PCN did not result in any increases in the CYP-dependent enzyme activities measured.
2.9. Statistical analysis Results are expressed as mean ± SD for groups of 5 rats. Statistical significance between either control and PB or control and PCN treated groups was determined by two-tailed Student’s t-tests.
3. Results 3.1. Effect on body weight and plasma PB and PCN levels
3.4. Effect on hepatic CYP mRNA levels
Each group of male Sprague-Dawley wild type (WT), CAR KO and PXR KO rats comprised 5 animals. No adverse clinical symptoms were observed and all rats survived to the end of the 7 day treatment period. Compared to WT control rats, the treatment of CAR KO rats for 7 days with 500 ppm NaPB in the diet and PXR KO rats for 7 days with 100 mg/kg/day PCN by gavage had no statistically significant effect on body weight (Fig. 1A and B). From food consumption and body weight data, calculated mean daily NaPB intakes in WT and CAR KO rats given a 500 ppm diet were 31.4 and 30.8 mg/kg/day, respectively. Levels of PB in terminal plasma samples of CAR KO rats given NaPB were significantly higher (p < 0.001) than in WT rats. Analysed PB plasma levels in WT and CAR KO rats were 8.9 ± 4.7 and 43.3 ± 8.7 μg/ml, respectively. While levels of PCN in terminal plasma samples of WT rats given 100 mg/kg/day PCN were below the detection limit of the assay (< 0.1 μg/ml), plasma levels of PCN in PXR KO rats given PCN were determined to be 0.32 ± 0.11 μg/ml.
Total RNA was extracted from liver samples from WT, CAR KO and PXR KO rats and CYP1A1, CYP1A2, CYP2B1, CYP2B2, CYP3A1 and CYP4A1 mRNA levels determined. The treatment of WT rats with NaPB for 7 days resulted in marked statistically significant increases in CYP2B1 and CYP2B2 mRNA levels, together with a significant increase in CYP3A1 mRNA levels (Fig. 5A). Treatment of WT rats with PCN for 7 days resulted in statistically significant increases in CYP2B1, CYP2B2 and CYP3A1 mRNA levels (Fig. 5B). The treatment of WT rats with NaPB had no significant effect on CYP1A1, CYP1A2 and CYP4A1 mRNA levels, whereas levels of CYP1A2 and CYP4A1 mRNAs were significantly decreased in WT rats given PCN. The treatment of CAR KO rats with NaPB did not result in any significant effects on any of the CYP mRNA levels examined, whereas the treatment of PXR KO rats with PCN resulted in a significant decrease in CYP2B1 and CYP4A1 mRNA levels. 3.5. Effect on hepatic CYP protein levels
3.2. Effect on relative liver and thyroid gland weights and replicative DNA synthesis (RDS)
Liver microsomes from WT, CAR KO and PXR KO rats were subjected to Western immunoblotting (Fig. 6). The treatment of WT rats with both NaPB and PCN did not result in any induction of CYP1A and CYP4A proteins. In contrast, both NaPB and PCN resulted in a marked induction of CYP2B and CYP3A protein levels in WT rats. The treatment of CAR KO rats with NaPB and PXR KO rats with PCN did not result in any marked effects on hepatic microsomal CYP1A, CYP2B, CYP3A and CYP4A protein levels. Levels of CYP4A protein appeared to be somewhat higher in control CAR KO compared to control WT rats, whereas levels of CYP2B and CYP3A proteins appeared to be somewhat higher in control PXR KO compared to control WT rats.
The treatment of WT rats with both NaPB (Fig. 1C) and PCN (Fig. 1D) for 7 days resulted in statistically significant increases in relative liver weight. Examination of liver sections from NaPB treated WT rats, but not from PCN treated WT rats, revealed a moderate to marked centrilobular hypertrophy (group mean severity grade 3.4). The treatment of CAR KO rats with NaPB and PXR KO rats with PCN had no effect on relative liver weight and did produce any morphological changes. RDS was determined by implanting rats with osmotic pumps to continuously deliver the DNA precursor BrdU over the 7 day treatment period. Treatment with either NaPB (Fig. 1E) or PCN (Fig. 1F) for 7 days resulted in statistically significant increases, in hepatocyte RDS in WT rats. Although treatment with NaPB had no significant effect on hepatocyte RDS in CAR KO rats, a small but statistically significant increase in hepatocyte RDS was observed in PXR KO rats given PCN. While the treatment of WT and CAR KO rats with NaPB rats had no significant effect on relative thyroid gland weight (Fig. 2A), the treatment of WT, but not PXR KO, rats with PCN significantly increased relative thyroid gland weight (Fig. 2B). Treatment with NaPB (Fig. 2C) and PCN (Fig. 2D) significantly increased thyroid gland follicular cell RDS in WT rats, with NaPB also producing a small but statistically significant increase in thyroid gland follicular cell RDS in CAR KO rats.
4. Discussion The objective of this study was to compare the hepatic and thyroid gland effects of NaPB in WT and CAR KO rats and PCN in WT and PXR KO rats. Compared to WT rats, the treatment of CAR KO rats with NaPB and PXR KO rats with PCN for 7 days resulted in increased levels of PB and PCN, respectively, in terminal plasma samples. This effect would appear to be attributable to a reduced capacity of CAR KO and PXR KO rats, compared to WT rats (where CYP enzyme activities are increased), to metabolise these two hepatic CYP enzyme inducers. The hepatic effects of treatment of WT rats with both NaPB and PCN 23
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Fig. 2. Effect of treatment of WT and CAR KO rats with 0 (control) and 500 ppm NaPB in the diet and WT and PXR KO rats with 0 (control) and 100 mg/kg/day PCN by gavage for 7 days on relative thyroid gland weight (A, B) and thyroid gland follicular cell RDS (C, D). Results are expressed as mean ± SD of 5 animals. Values significantly different from control are: *p < 0.05; **p < 0.01; ***p < 0.001. Fig. 3. Effect of treatment of WT and CAR KO rats with 0 (control) and 500 ppm NaPB in the diet and WT and PXR KO rats with 0 (control) and 100 mg/kg/day PCN by gavage for 7 days on hepatic microsomal total CYP content (A, B) and EROD (C, D), and PROD (E,F) enzyme activities. Results are expressed as mean ± SD of 5 animals. Values significantly different from control are: ***p < 0.001.
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Fig. 4. Effect of treatment of WT and CAR KO rats with 0 (control) and 500 ppm NaPB in the diet and WT and PXR KO rats with 0 (control) and 100 mg/kg/ day PCN by gavage for 7 days on hepatic microsomal BROD (A, B) and BQ (C, D) enzyme activities. Results are expressed as mean ± SD of 5 animals. Values significantly different from control are: **p < 0.01; ***p < 0.001.
mRNA levels in WT rats, these effects were abolished in CAR KO rats. Previous studies have demonstrated that there can be considerable crosstalk between hepatic CAR and PXR receptors, with some compounds being activators of both these nuclear receptors (Maglich et al., 2002; Omiecinski et al., 2011; Yoshinari et al., 2008). However, in this study the observation that NaPB did not increase CYP3A enzyme activity, mRNA and protein levels in CAR KO rats suggests that CYP3A induction by NaPB in rat liver is due mainly to activation of CAR and not also to activation of PXR. This finding is in agreement with a previous mouse study where PB was shown to induce CYP2B and CYP3A enzyme activities and protein levels in PXR KO mice, but not in CAR KO mice (Scheer et al., 2008). Compared to WT rats, the treatment of PXR KO rats with PCN did not result in any increase in liver weight, microsomal total CYP content and induction of CYP2B and CYP3A enzymes. These results suggest that functional PXR and not CAR is required for PCN-induced effects on liver weight and induction of CYP2B and CYP3A enzymes in the rat. While PCN produced a marked increase in hepatocyte RDS in WT rats, only a small increase was observed in PXR KO rats. Additional studies would be required to ascertain if this small effect on hepatocyte RDS was due to CAR/PXR receptor crosstalk. The effects of NaPB and PCN on hepatic CYP enzyme activities, mRNA levels and protein levels in CAR KO and PXR KO rats, respectively, are in agreement with the recent study of Forbes et al. (2017), where WT, CAR KO and PXR KO rats were given single oral doses of corn oil (control), 12.5 mg/kg TCPOBOP and 100 mg/kg PCN 16 h prior to necropsy. In the Forbes et al. (2017) study, TCPOBOP increased hepatic CYP2B mRNA levels in WT rats, but not in CAR KO rats, whereas PCN increased hepatic CYP2B and CYP3A mRNA levels in WT rats, but not in PXR KO rats. Moreover, in the present study levels of CYP2B and CYP3A proteins were somewhat higher in liver microsomes from control PXR KO than from control WT rats, which correlates with the study of Forbes et al. (2017) where CYP2B and CYP3A mRNA levels were higher in control PXR KO rats than in control WT rats. In this study, levels of CYP4A proteins appeared to be somewhat higher in liver microsomes from control CAR KO than from control WT rats. Additional studies, including measurement of CYP4A marker enzyme activities, would be required to confirm this finding. Overall, the present study with CAR KO rats demonstrates that a functional CAR is required for NaPB-mediated increases in liver weight, stimulation of hepatocyte RDS and induction of CYP enzymes. In addition, the studies with PXR KO rats demonstrate that a functional PXR is required for PCN-mediated increases in liver weight and induction of
for 7 days are in agreement with those observed in previous studies (Elcombe et al., 2014; Lake et al., 1998; Martignoni et al., 2006). Both NaPB and PCN increased relative liver weight, stimulated hepatocyte RDS and induced microsomal total CYP content. Treatment with NaPB produced a marked induction of CYP2B enzyme activities, CYP2B1 and CYP2B2 mRNA levels and CYP2B protein levels. NaPB also produced smaller increases in CYP3A enzyme activity, CYP3A1 mRNA levels and CYP3A protein levels. Treatment with PCN resulted in significant increases in CYP2B and CYP3A enzyme activities, CYP2B1, CYP2B2 and CYP3A1 mRNA levels and also increased CYP2B and CYP3A protein levels. Although treatment with NaPB and to a lesser extent PCN produced small, but statistically significant, increases in hepatic microsomal EROD activity, this was not associated with any increases in CYP1A1 and CYP1A2 mRNA levels and CYP1A protein content. The small increases in hepatic microsomal EROD activity observed in this study are unlikely to represent induction of CYP1A enzymes, but rather that this substrate is also metabolised by other CYP enzymes, including CYP2B and CYP2C enzymes (Burke et al., 1994), which are induced by NaPB and PCN treatment. Neither NaPB nor PCN appeared to increase hepatic microsomal CYP4A protein levels in WT rats, with hepatic CYP4A1 mRNA levels being unaffected by NaPB treatment, but significantly reduced by PCN treatment. Overall, both NaPB and PCN produced the expected marked effects on the hepatic CYP2B and CYP3A parameters measured in WT rats, with only comparatively small effects being observed on the CYP1A and CYP4A markers measured. Compared to effects in WT rats, the treatment of CAR KO rats with NaPB did not result in an increase in relative liver weight, centrilobular hepatocyte hypertrophy and an induction of hepatocyte RDS. In addition, total microsomal CYP content was not increased and (as demonstrated by enzyme activity, mRNA and protein measurements) the induction of CYP2B and CY3A enzymes was completely abolished in CAR KO rats given NaPB. In terms of the key and associative events for the MOA for PB-induced rodent liver tumour formation (Elcombe et al., 2014), the key event of increased hepatocyte RDS and the associative events of liver hypertrophy (both organ weight and morphology) and CYP2B enzyme induction were thus not observed in CAR KO rats treated with NaPB. The lack of effect of NaPB on relative liver weight, hepatocyte RDS and CYP2B enzyme induction in CAR KO rats is in agreement with previous studies conducted in CAR KO mice (Huang et al., 2005; Wei et al., 2000) and in a recent study in CAR KO rats (Okuda et al., 2017). The study of Okuda et al. (2017) also demonstrated that while two other known rat CAR activators, namely metofluthrin and momfluorothrin, induced hepatocyte RDS and CYP2B 25
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Fig. 6. Effect of treatment of WT and CAR KO rats with 0 (control) or 500 ppm NaPB in the diet and WT and PXR KO rats with 0 (control) and 100 mg/kg/day PCN by gavage for 7 days on hepatic microsomal CYP1A, CYP2B, CYP3A and CYP4A proteins. Microsomal fractions (20, 40 and 40 μg protein per lane for CYP1A, CYP3A and CYP4A proteins, respectively) were pooled from groups (n = 5) of WT, CAR KO, and PXR KO control and treated rats. For hepatic microsomal CYP2 B proteins, protein loadings of the pooled microsomal preparations were 0.5 μg protein per lane for WT rats treated with 500 ppm NaPB and 2.5 μg protein per lane for all other groups.
KO rats. Possibly, the small effect of NaPB on thyroid gland follicular cell RDS in CAR KO rats may be attributable to CAR/PXR receptor crosstalk. In order to demonstrate the usefulness of CAR KO and PXR KO rats as suitable animal models for thyroid gland MOA studies, additional studies would be required with known inducers (such as NaPB and PCN) to ascertain effects on serum TSH, T3 and T4 levels, thyroid gland follicular cell hypertrophy and hyperplasia and hepatic microsomal UGT activity towards T4 as substrate. In summary, CAR KO and PXR KO rats appear to be useful animal models for MOA studies on the hepatic effects of rodent liver CAR and PXR activators. While PB/NaPB and other rodent liver CAR activators generally produce liver tumours more readily in the mouse than in the rat (Elcombe et al., 2014), there are exceptions. For example, the synthetic pyrethroids metofluthrin and momflurothrin, the fungicide fluopyram and the natural pyrethrins have been shown to produce liver tumours in the rat, but not in the mouse, by a CAR activation MOA (Deguchi et al., 2009; Okuda et al., 2017; Osimitz and Lake, 2009; Tinwell et al., 2014). Hence it is necessary to have both rat and mouse CAR KO models available for rodent liver MOA studies. Additional studies would be required to ascertain if the rat CAR KO and PXR KO models employed in this study may also be useful for rat thyroid gland MOA studies with CYP2B and CYP3A enzyme inducers.
Fig. 5. Effect of treatment of WT (filled histograms) and CAR KO (hatched histograms) rats with 0 (control) and 500 ppm NaPB in the diet (A) and WT (filled histograms) and PXR KO (hatched histograms) rats with 0 (control) and 100 mg/kg/day PCN by gavage (B) for 7 days on hepatic CYP1A1, CYP1A2, CYP2B1, CYP2B2, CYP3A1 and CYP4A1 mRNA levels. Results for WT, CAR KO and PXR KO rats are expressed as fold change relative to control levels (i.e. animals not treated with either NaPB or PCN) and are presented as mean ± SD of 5 animals. Values significantly different from control are: *p < 0.05; **p < 0.01; ***p < 0.001.
CYP enzymes, with induction of hepatocyte RDS also being largely mediated through PXR. The hepatic effects of NaPB in CAR KO rats and of PCN in PXR KO rats are in agreement with those observed in other recent studies (Forbes et al., 2017; Okuda et al., 2017). This study also investigated the use of CAR KO and PXR KO rat models for MOA studies where treatment with CAR and/or PXR activators leads to thyroid gland tumours in rodents as a consequence of decreased circulating thyroid hormone levels resulting from an increase in hepatic metabolism and clearance of T4 (Capen, 2001; Dellarco et al., 2006; Hill et al., 1998; Meek et al., 2003). Previous studies have demonstrated that the treatment of normal (WT) rats with NaPB/PB and PCN can result in increased thyroid gland weight and induction of thyroid gland follicular cell RDS (Barter and Klaassen, 1992; Finch et al., 2006; Hood et al., 1999a,b; McClain et al., 1989). In the present study, PCN increased relative thyroid gland weight in WT rats and both NaPB and PCN increased thyroid gland follicular cell RDS in WT rats. The treatment of CAR KO rats with NaPB and PXR KO rats with PCN did not result in any effect on relative thyroid gland weight, whereas NaPB produced a small increase in thyroid gland follicular cell RDS in CAR
Acknowledgment This work was funded by Concept Life Sciences (formerly CXR Biosciences Ltd.). References Barter, R.A., Klaassen, C.D., 1992. Rat liver UDP-glucuronosyltransferase activity toward thyroxine: characterization, induction, and form specificity. Toxicol. Appl. Pharmacol. 115, 261–267. 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. Capen, C.C., 2001. Toxic responses of the endocrine system. In: Klaassen, C.D. (Ed.), Casarett and Doull’s Toxicology: the Basic Science of Poisons, 6th edition. McGraw Hill, New York, pp. 711–759. Choi, C.J., Rushton, E.K., Vardy, A., Higgins, L., Augello, A., Parod, R.J., 2017. Mode of action and human relevance of THF-induced mouse liver tumors. Toxicol. Lett. 276, 138–143. Cohen, S.M., 2010. Evaluation of possible carcinogenic risk to humans based on liver tumors in rodent assays: the two-year bioassay is no longer necessary. Toxicol.
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Pathol. 38, 487–501. Curran, P.G., DeGroot, L.J., 1991. The effect of hepatic enzyme-inducing drugs on thyroid hormones and the thyroid gland. Endocrin. Rev. 12, 135–150. Deguchi, Y., Yamada, T., Hirose, Y., Nagahori, H., Kushida, M., Sumida, K., Sukata, T., Tomigahara, Y., Nishioka, N., Uwagawa, S., Kawamura, S., Okuno, Y., 2009. Mode of action analysis for the synthetic pyrethroid metofluthrin-induced rat liver tumors: evidence for hepatic CYP2B induction and hepatocyte proliferation. Toxicol. Sci. 108, 69–80. Dellarco, V.L., McGregor, D., Berry Sir, C., Cohen, S.M., Boobis, A.R., 2006. Thiazopyr and thyroid disruption: case study within the context of the 2006 IPCS human relevance framework for analysis of a cancer mode of action. Crit. Rev. Toxicol. 36, 793–801. Elcombe, C.R., Peffer, R.C., Wolf, D.C., Bailey, J., Bars, R., Bell, D., Cattley, R.C., Ferguson, S.S., Geter, D., Goetz, A., Goodman, J.I., Hester, S., Jacobs, A., Omiecinski, C.J., Schoeny, R., Xie, W., Lake, B.G., 2014. Mode of action and human relevance analysis for nuclear receptor-mediated liver toxicity: a case study with phenobarbital as a model constitutive androstane receptor (CAR) activator. Crit. Rev. Toxicol. 44, 64–82. Finch, J.M., Osimitz, T.G., Gabriel, K.L., Martin, T., Henderson, W.J., Capen, C.C., Butler, W.H., Lake, B.G., 2006. A mode of action for induction of thyroid gland tumors by pyrethrins in the rat. Toxicol. Appl. Pharmacol. 214, 253–262. Forbes, K.P., Kouranova, E., Tinker, D., Janowski, K., Cortner, D., McCoy, A., Cui, X., 2017. Creation and preliminary characterization of pregnane X receptor and constitutive androstane receptor knockout rats. Drug Metab. Dispos. 45, 1068–1076. Gold, L.S., Manley, N.B., Slone, T.H., Ward, J.M., 2001. Compendium of chemical carcinogens by target organ: results of chronic bioassays in rats, mice, hamsters, dogs, and monkeys. Toxicol. Pathol. 29, 639–652. Hill, R.N., Crisp, T.M., Hurley, P.M., Rosenthal, S.L., Singh, D.V., 1998. Risk assessment of thyroid follicular cell tumors. Environ. Health Perspect. 106, 447–457. Hood, A., Hashmi, R., Klaassen, C.D., 1999a. Effects of microsomal enzyme inducers on thyroid-follicular cell proliferation, hyperplasia, and hypertrophy. Toxicol. Appl. Pharmacol. 160, 163–170. Hood, A., Liu, J., Klaassen, C.D., 1999b. Effects of phenobarbital, pregnenolone-16αcarbonitrile, and propylthiouracil on thyroid follicular cell proliferation. Toxicol. Sci. 50, 45–53. Huang, W., Zhang, J., Washington, M., Liu, J., Parant, J.M., Lozano, G., Moore, D.D., 2005. Xenobiotic stress induces hepatomegaly and liver tumors via the nuclear receptor constitutive androstane receptor. Mol. Endocrinol. 19, 1646–1653. Huff, J., Cirvello, J., Haseman, J., Bucher, J., 1991. Chemicals associated with site-specific neoplasia in 1394 long-term carcinogenesis experiments in laboratory rodents. Environ. Health Perspect. 93, 247–270. Lake, B.G., Renwick, A.B., Cunninghame, M.E., Price, R.J., Surry, D., Evans, D.C., 1998. Comparison of the effects of some CYP3A and other enzyme inducers on replicative DNA synthesis and cytochrome P450 isoforms in rat liver. Toxicology 131, 9–20. Lake, B.G., 2009. Species differences in the hepatic effects of inducers of CYP2B and CYP4A subfamily forms: relationship to rodent liver tumour formation. Xenobiotica 39, 582–596. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Lubet, R.A., Syi, J.-L., Nelson, J.O., Nims, R.W., 1990. Induction of hepatic cytochrome P450 mediated alkoxyresorufin O-dealkylase activities in different species by
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Comparison of the hepatic and thyroid gland effects of sodium phenobarbital in wild type and constitutive androstane receptor (CAR) knockout rats and pregnenolone-16α-carbonitrile in wild type and pregnane X receptor (PXR) knockout rats
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Corinne Hainesa, Lynsey R. Chathama, Audrey Vardya, , Clifford R. Elcombea,1, John R. Fosterb, Brian G. Lakea,c a b c
Concept Life Sciences (formerly CXR Biosciences Ltd.), 2, James Lindsay Place, Dundee Technopole, Dundee DD1 5JJ, UK Regulatory Science Associates, Kip Marina, Inverkip, Renfreshire, PA16 0AS, UK Centre for Toxicology, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, GU2 7XH, UK
A R T I C LE I N FO
A B S T R A C T
Keywords: Sodium phenobarbital Pregnenolone-16α-carbonitrile Constitutive androstane receptor Pregnane X receptor Receptor knockout rats Cytochrome P450
A number of chemicals produce liver and thyroid gland tumours in rodents by nongenotoxic modes of action (MOAs). In this study the hepatic and thyroid gland effects of the constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) were examined in male Sprague-Dawley wild type (WT) rats and in CAR knockout (CAR KO) rats and the effects of the pregnane X receptor (PXR) activator pregnenolone-16α-carbonitrile (PCN) were examined in WT and PXR knockout (PXR KO) rats. Rats were either fed diets containing 0 (control) or 500 ppm NaPB or were dosed with 0 (control) or 100 mg/kg/day PCN orally for 7 days. The treatment of WT rats with NaPB and PCN for 7 days resulted in increased relative liver weight, increased hepatocyte replicative DNA synthesis (RDS) and the induction of cytochrome P450 CYP2B and CYP3A subfamily enzyme, mRNA and protein levels. In marked contrast, the treatment of CAR KO rats with NaPB and PXR KO rats with PCN did not result in any increases in liver weight and induction of CYP2B and CYP3A enzymes. The treatment of CAR KO rats with NaPB had no effect on hepatocyte RDS, while PCN produced only a small increase in hepatocyte RDS in PXR KO rats. Treatment with NaPB had no effect on thyroid gland weight in WT and CAR KO rats, whereas treatment with PCN resulted in an increase in relative thyroid gland weight in WT, but not in PXR KO, rats. Thyroid gland follicular cell RDS was increased by the treatment of WT rats with NaPB and PCN, with NaPB also producing a small increase in thyroid gland follicular cell RDS in CAR KO rats. Overall, the present study with CAR KO rats demonstrates that a functional CAR is required for NaPB-mediated increases in liver weight, stimulation of hepatocyte RDS and induction of hepatic CYP enzymes. The studies with PXR KO rats demonstrate that a functional PXR is required for PCN-mediated increases in liver weight and induction of hepatic CYP enzymes; with induction of hepatocyte RDS also being largely mediated through PXR. The hepatic effects of NaPB in CAR KO rats and of PCN in PXR KO rats are in agreement with those observed in other recent literature studies. These results suggest that CAR KO and PXR KO rats are useful experimental models for liver MOA studies with rodent CAR and PXR activators and may also be useful for thyroid gland MOA studies.
1. Introduction A number of nongenotoxic chemicals have been shown to produce liver and thyroid gland tumours in rodents (Gold et al., 2001; Huff et al., 1991). Both rodent liver and thyroid gland tumour formation may be associated with induction of hepatic xenobiotic metabolising enzymes. For example, a number of nongenotoxic chemicals, including
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phenobarbital (PB) and/or its sodium salt (sodium phenobarbital; NaPB), have been shown to produce liver tumours in rats and/or mice (Cohen, 2010; Elcombe et al., 2014; Lake, 2009) by a mode of action (MOA) involving activation of the constitutive androstane receptor (CAR). In an evaluation of the MOA for PB-induced rodent liver tumour formation by Elcombe et al. (2014), the key events were identified as CAR activation, altered gene expression specific to CAR activation,
Corresponding author. E-mail address: [email protected] (A. Vardy). Deceased.
https://doi.org/10.1016/j.tox.2018.03.002 Received 28 November 2017; Received in revised form 5 March 2018; Accepted 6 March 2018 Available online 13 March 2018 0300-483X/ © 2018 Elsevier B.V. All rights reserved.
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under a UK Home Office license and all animal studies were approved by the Ethical Review Committee of the University of Dundee (Dundee, Scotland, UK). Male Sprague-Dawley WT, CAR KO and PXR KO rats (14 weeks old at study start) were implanted subcutaneously with osmotic pumps (Alzet model number 2ML1, Charles River UK Ltd., Margate, Kent, UK) containing 15 mg/ml BrdU in phosphate buffered saline pH 7.4. Rats were then either fed powdered diets containing 0 (control) or 500 ppm (500 mg/kg) NaPB or were dosed with 0 (control) or 100 mg/ kg/day PCN orally for 7 days. For PCN, control animals received corresponding quantities (10 ml/kg body weight) of the corn oil vehicle. Male rather than female rats were used in this study, as male animals are more often used for rodent liver CAR activator MOA studies. At the end of the treatment period rats were killed by exposure to carbon dioxide gas and blood taken for plasma analysis. The liver of each rat was removed, weighed and sampled for morphological and biochemical investigations. The thyroid glands and trachea of each animal were removed intact and fixed in 10% neutral buffered formalin (NBF). After at least 24 h of fixation, the thyroid glands were carefully dissected from the trachea, blotted dry and weighed, prior to returning to the fixation medium. The duodenum of each animal was also excised.
increased cell proliferation, the development of altered hepatic foci and finally liver tumour formation. Associative events for this MOA included liver hypertrophy, induction of cytochrome P450 (CYP) enzymes (particularly CYP2B subfamily enzymes) and inhibition of apoptosis. Some nongenotoxic chemicals, including some CAR activators, have been shown to produce thyroid gland tumours in rodents by a MOA in which circulating thyroid hormone levels are decreased as a result of increased hepatic metabolism and clearance (Capen, 2001; Dellarco et al., 2006; Hill et al., 1998; McClain et al., 1989; Meek et al., 2003). In this MOA, thyroxine (T4) conjugation is enhanced due to the stimulation of hepatic microsomal UDPglucuronosyltransferase (UGT) enzymes towards T4 as substrate resulting in increased biliary excretion of T4, a decrease in serum triiodothyronine (T3) and/or T4 levels and a compensatory increase in serum thyroid stimulating hormone (TSH) levels. In the rodent thyroid gland, the increase in TSH levels results in thyroid follicular cell hypertrophy and hyperplasia, with chronic stimulation subsequently producing thyroid follicular cell tumours (Capen, 2001; Curran and DeGroot, 1991; Hill et al., 1998). In establishing the MOA for rodent liver tumour formation by CAR activators, studies in transgenic mice lacking CAR (i.e. CAR knockout (KO) mice) have been particularly useful. Such studies have demonstrated that PB does not increase liver weight, produce liver hypertrophy, does not induce CYP2B enzymes or replicative DNA synthesis (RDS) and does not promote liver tumours in CAR KO mice (Huang et al., 2005; Scheer et al., 2008; Wei et al., 2000; Yamamoto et al., 2004). Recently, transgenic rat models lacking either the CAR or the pregnane X receptor (PXR), namely CAR KO and PXR KO rats, have been developed and have become commercially available (Forbes et al., 2017). The objective of this study was to evaluate the usefulness of the CAR KO and PXR KO rat models for liver and thyroid gland MOA studies. Male Sprague-Dawley (i.e. wild type; WT) and CAR KO rats were treated with NaPB and male WT and PXR KO rats were treated with pregnenolone-16α-carbonitrile (PCN). NaPB and PCN were selected as known rodent liver CAR and PXR activators, respectively (Elcombe et al., 2014, Lake et al., 1998; Martignoni et al., 2006; Omiecinski et al., 2011).
2.3. Plasma analysis Levels of PB in plasma were determined by protein precipitation followed by quantification by high performance liquid chromatography-mass spectrometry-mass spectrometry (LC–MS/MS) employing a Waters 2795 separation module equipped with a Waters Quattro micro mass spectrometer operated in negative ion mode with electrospray ionisation. Plasma PB levels were determined employing a Kinetex SB column (100 × 4.6 mm, 3 μm), with a mobile phase of 80:20 (v/v) methanol/water at a flow rate of 0.5 ml/min, with detection using a transition of 231.46 → 231.27. Levels of PCN in plasma were determined by protein precipitation followed by quantification by LC–MS/MS employing a Waters 2795 separation module equipped with a Waters Quattro micro mass spectrometer operated in positive ion mode with ACPI ionisation, using a transition of 342.30 → 224.23. Plasma PCN levels were determined employing a Luna C18(2) column (150 × 2 mm, 5 μm) at 35 °C employing mobile phases of methanol and 0.1% (v/v) formic acid in water with gradient elution at a flow rate of 0.4 ml/min.
2. Materials and methods 2.1. Materials
2.4. Hepatic morphological investigations
7-Benzyloxyquinoline and 7-hydroxyquinoline were purchased from Cypex (Dundee, UK). NaPB (purity ≥ 99%), 5-bromo-2′-deoxyuridine (BrdU), and other CYP substrates and metabolites were obtained from Sigma-Aldrich (Poole, Dorset, UK), whereas PCN (purity 99%) was obtained from Enzo Life Sciences Inc. (Farmingdale, NY, USA). For Western immunoblotting, an anti-rat CYP1A antibody was obtained from Corning (Corning B.V., Amsterdam, The Netherlands), whereas anti-rat CYP2B, CYP3A and CYP4A antibodies were obtained from the Biomedical Research Institute (University of Dundee, Scotland, UK). Secondary antibodies were horseradish peroxidase (HRP)-linked antisheep IgG (Abgene, Blenheim Road, Epsom, Surrey, UK) and anti-rabbit IgG (GE Healthcare, Little Chalfont, Buckinghamshire).
Two samples of liver from each rat (one from the left lobe and one from the median lobe) were fixed in NBF, dehydrated, embedded in paraffin wax and approximately 5 μm sections were stained with hematoxylin and eosin. Sections were examined by light microscopy and the degree of liver hypertrophy scored as either minimal (grade 1), slight (grade 2), moderate (grade 3) or marked (grade 4). For each group, the liver hypertrophy severity grades for each animal were summed and a group mean severity grade calculated. 2.5. Replicative DNA synthesis (RDS) Liver, thyroid gland and duodenum (as a methodology positive control) samples were fixed in NBF for approximately 36 h prior to processing into paraffin blocks. BrdU immunocytochemistry was performed as described previously (Ross et al., 2010). Image capture and acquisition were carried out using a Zeiss Imager A1 microscope and Improvision imaging software, Volocity version 4 (Perkin Elmer, Bucks, UK) was used for data analysis. The BrdU labelling index (i.e. percentage of either hepatocyte or thyroid follicular cell nuclei undergoing RDS) for each animal was determined by counting approximately 3000 nuclei in 10 random areas from both the left and median lobes of the liver and at least 3000 nuclei in 20 fields from the thyroid gland.
2.2. Animals and treatment Male Sprague-Dawley WT, CAR KO and PXR KO rats were obtained from Horizon Discovery (Boyertown, PA, USA). Rats were allowed free access to water and powdered or pelleted RM1 laboratory animal diet (Special Diets Services, Witham, Essex, UK) and were housed singly in accommodation with a 12:12 h light:dark cycle. Temperature and relative humidity were maintained between 19 and 23 °C and 40 and 70%, respectively. Rats were allowed to acclimatize to these conditions for at least 5 days before use. All animal procedures were performed 21
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Fig. 1. Effect of treatment of WT and CAR KO rats with 0 (control) and 500 ppm NaPB in the diet and WT and PXR KO rats with 0 (control) and 100 mg/kg/day PCN by gavage for 7 days on body weight (A, B) relative liver weight (C, D) and hepatocyte RDS (E, F). Results are expressed as mean ± SD of 5 animals. Values significantly different from control are: ***p < 0.001.
Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and cDNA was then synthesised using a Quantitect reverse transcription kit (Cat number 205311/205313 Qiagen, Hilden, Germany). Real-time PCR (Life Technologies QuantStudio 6 Flex system) was performed using TaqMan Fast Universal PCR Master Mix (Applied Biosystems, Inchinnan Business Park, Paisley, Scotland, UK) and TaqMan gene expression assays (Applied Biosystems) performed with primer-probe sets for the following genes: CYP1A1 (Rn01418021_g1), CYP1A2 (Rn00561082_m1), CYP2B1 (Rn01457875_m1), CYP2B2 (Rn02786833_m1), CYP3A1 (Rn03062228_m1) and CYP4A1 (Rn00598510_m1). The expression of each gene was normalised against endogenous β-actin (Rn00667869_m1). Data were analysed by generation of the threshold cycle (Ct) and delta Ct values were calculated for all genes. Fold change was analysed by calculating 2−ΔΔCt and results were expressed as fold change relative to control where control values were normalised to 1.00.
2.6. Hepatic biochemical investigations Whole liver homogenates (0.25 g fresh tissue/ml) were prepared in ice-cold 0.25 M sucrose containing 5 mM EDTA and 20 mM Tris-HCl pH 7.4 and liver microsomes prepared by differential centrifugation. Microsomes were resuspended in fresh homogenising medium and aliquots stored at approximately −70 °C. Protein content was determined by the method of Lowry et al. (1951) employing bovine serum albumin as standard. Microsomal total CYP content, 7-ethoxyresorufin O-deethylase (EROD), 7-pentoxyresorufin O-depentylase (PROD), 7-benzyloxyresorufin O-debenzylase (BROD) and 7-benzyloxyquinoline O-debenzylase (BQ) activities were determined as described previously (Choi et al., 2017; Ross et al., 2010).
2.7. Hepatic CYP mRNA levels Liver samples were homogenised using a Precellys 24 lysis and homogenisation control unit. RNA was then extracted using an RNeasy mini kit (Cat number 74104/74106, Qiagen, Hilden, Germany). RNA quantity and quality (260/280 nm ratio) was determined on a 22
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2.8. Western immunoblotting of hepatic CYP enzymes
3.3. Effect on hepatic enzyme activities
For Western immunoblot analysis, microsomal proteins from pooled rat liver microsome samples (n = 5 per group) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, electrophoretically transferred to polyvinylidene difluoride membranes and probed with anti-rat CYP1A, CYP2B, CYP3A and CYP4A antibodies. Protein loadings were 20 μg for CYP1A, 0.5 or 2.5 μg for CYP2B and 40 μg for both CYP3A and CYP4A. Secondary antibodies were HRPlinked anti-sheep IgG (for CYP1A) and anti-rabbit IgG (for CYP2B, CYP3A and CYP4A). Detection of immunoreactive bands was performed by enhanced luminescence detection (GE Healthcare).
The treatment of WT rats with NaPB (Fig. 3A) and PCN (Fig. 3B) for 7 days resulted in statistically significant increases in hepatic microsomal total CYP content. In contrast, the treatment of CAR KO rats with NaPB and PXR KO rats with PCN did not result in any effect on microsomal total CYP content. Hepatic microsomal 7-ethoxyresorufin Odeethylase (EROD), 7-pentoxyresorufin O-depentylase (PROD), 7-benzyloxyresorufin O-debenzylase (BROD) and 7-benzyloxyquinoline Odebenzylase (BQ) activities were determined as markers of induction of CYP1A, CYP2B, CYP2B/3A and CYP3A subfamily enzymes, respectively (Lubet et al., 1990; Renwick et al., 2001). The treatment of WT rats with NaPB (Fig. 3C) and PCN (Fig. 3D) produced some significant increases in microsomal EROD activity. Treatment of WT rats with NaPB produced marked increases in microsomal CYP2B-dependent PROD (Fig. 3E) and CYP2B/3A-dependent BROD (Fig. 4A) activities, with CYP3A-dependent BQ activity also being significantly increased (Fig. 4C). The treatment of WT rats with PCN significantly increased microsomal PROD (Fig. 3F), BROD (Fig. 4B) and BQ (Fig. 4D) activities. In contrast, the treatment of CAR KO rats with NaPB and PXR KO rats with PCN did not result in any increases in the CYP-dependent enzyme activities measured.
2.9. Statistical analysis Results are expressed as mean ± SD for groups of 5 rats. Statistical significance between either control and PB or control and PCN treated groups was determined by two-tailed Student’s t-tests.
3. Results 3.1. Effect on body weight and plasma PB and PCN levels
3.4. Effect on hepatic CYP mRNA levels
Each group of male Sprague-Dawley wild type (WT), CAR KO and PXR KO rats comprised 5 animals. No adverse clinical symptoms were observed and all rats survived to the end of the 7 day treatment period. Compared to WT control rats, the treatment of CAR KO rats for 7 days with 500 ppm NaPB in the diet and PXR KO rats for 7 days with 100 mg/kg/day PCN by gavage had no statistically significant effect on body weight (Fig. 1A and B). From food consumption and body weight data, calculated mean daily NaPB intakes in WT and CAR KO rats given a 500 ppm diet were 31.4 and 30.8 mg/kg/day, respectively. Levels of PB in terminal plasma samples of CAR KO rats given NaPB were significantly higher (p < 0.001) than in WT rats. Analysed PB plasma levels in WT and CAR KO rats were 8.9 ± 4.7 and 43.3 ± 8.7 μg/ml, respectively. While levels of PCN in terminal plasma samples of WT rats given 100 mg/kg/day PCN were below the detection limit of the assay (< 0.1 μg/ml), plasma levels of PCN in PXR KO rats given PCN were determined to be 0.32 ± 0.11 μg/ml.
Total RNA was extracted from liver samples from WT, CAR KO and PXR KO rats and CYP1A1, CYP1A2, CYP2B1, CYP2B2, CYP3A1 and CYP4A1 mRNA levels determined. The treatment of WT rats with NaPB for 7 days resulted in marked statistically significant increases in CYP2B1 and CYP2B2 mRNA levels, together with a significant increase in CYP3A1 mRNA levels (Fig. 5A). Treatment of WT rats with PCN for 7 days resulted in statistically significant increases in CYP2B1, CYP2B2 and CYP3A1 mRNA levels (Fig. 5B). The treatment of WT rats with NaPB had no significant effect on CYP1A1, CYP1A2 and CYP4A1 mRNA levels, whereas levels of CYP1A2 and CYP4A1 mRNAs were significantly decreased in WT rats given PCN. The treatment of CAR KO rats with NaPB did not result in any significant effects on any of the CYP mRNA levels examined, whereas the treatment of PXR KO rats with PCN resulted in a significant decrease in CYP2B1 and CYP4A1 mRNA levels. 3.5. Effect on hepatic CYP protein levels
3.2. Effect on relative liver and thyroid gland weights and replicative DNA synthesis (RDS)
Liver microsomes from WT, CAR KO and PXR KO rats were subjected to Western immunoblotting (Fig. 6). The treatment of WT rats with both NaPB and PCN did not result in any induction of CYP1A and CYP4A proteins. In contrast, both NaPB and PCN resulted in a marked induction of CYP2B and CYP3A protein levels in WT rats. The treatment of CAR KO rats with NaPB and PXR KO rats with PCN did not result in any marked effects on hepatic microsomal CYP1A, CYP2B, CYP3A and CYP4A protein levels. Levels of CYP4A protein appeared to be somewhat higher in control CAR KO compared to control WT rats, whereas levels of CYP2B and CYP3A proteins appeared to be somewhat higher in control PXR KO compared to control WT rats.
The treatment of WT rats with both NaPB (Fig. 1C) and PCN (Fig. 1D) for 7 days resulted in statistically significant increases in relative liver weight. Examination of liver sections from NaPB treated WT rats, but not from PCN treated WT rats, revealed a moderate to marked centrilobular hypertrophy (group mean severity grade 3.4). The treatment of CAR KO rats with NaPB and PXR KO rats with PCN had no effect on relative liver weight and did produce any morphological changes. RDS was determined by implanting rats with osmotic pumps to continuously deliver the DNA precursor BrdU over the 7 day treatment period. Treatment with either NaPB (Fig. 1E) or PCN (Fig. 1F) for 7 days resulted in statistically significant increases, in hepatocyte RDS in WT rats. Although treatment with NaPB had no significant effect on hepatocyte RDS in CAR KO rats, a small but statistically significant increase in hepatocyte RDS was observed in PXR KO rats given PCN. While the treatment of WT and CAR KO rats with NaPB rats had no significant effect on relative thyroid gland weight (Fig. 2A), the treatment of WT, but not PXR KO, rats with PCN significantly increased relative thyroid gland weight (Fig. 2B). Treatment with NaPB (Fig. 2C) and PCN (Fig. 2D) significantly increased thyroid gland follicular cell RDS in WT rats, with NaPB also producing a small but statistically significant increase in thyroid gland follicular cell RDS in CAR KO rats.
4. Discussion The objective of this study was to compare the hepatic and thyroid gland effects of NaPB in WT and CAR KO rats and PCN in WT and PXR KO rats. Compared to WT rats, the treatment of CAR KO rats with NaPB and PXR KO rats with PCN for 7 days resulted in increased levels of PB and PCN, respectively, in terminal plasma samples. This effect would appear to be attributable to a reduced capacity of CAR KO and PXR KO rats, compared to WT rats (where CYP enzyme activities are increased), to metabolise these two hepatic CYP enzyme inducers. The hepatic effects of treatment of WT rats with both NaPB and PCN 23
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Fig. 2. Effect of treatment of WT and CAR KO rats with 0 (control) and 500 ppm NaPB in the diet and WT and PXR KO rats with 0 (control) and 100 mg/kg/day PCN by gavage for 7 days on relative thyroid gland weight (A, B) and thyroid gland follicular cell RDS (C, D). Results are expressed as mean ± SD of 5 animals. Values significantly different from control are: *p < 0.05; **p < 0.01; ***p < 0.001. Fig. 3. Effect of treatment of WT and CAR KO rats with 0 (control) and 500 ppm NaPB in the diet and WT and PXR KO rats with 0 (control) and 100 mg/kg/day PCN by gavage for 7 days on hepatic microsomal total CYP content (A, B) and EROD (C, D), and PROD (E,F) enzyme activities. Results are expressed as mean ± SD of 5 animals. Values significantly different from control are: ***p < 0.001.
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Fig. 4. Effect of treatment of WT and CAR KO rats with 0 (control) and 500 ppm NaPB in the diet and WT and PXR KO rats with 0 (control) and 100 mg/kg/ day PCN by gavage for 7 days on hepatic microsomal BROD (A, B) and BQ (C, D) enzyme activities. Results are expressed as mean ± SD of 5 animals. Values significantly different from control are: **p < 0.01; ***p < 0.001.
mRNA levels in WT rats, these effects were abolished in CAR KO rats. Previous studies have demonstrated that there can be considerable crosstalk between hepatic CAR and PXR receptors, with some compounds being activators of both these nuclear receptors (Maglich et al., 2002; Omiecinski et al., 2011; Yoshinari et al., 2008). However, in this study the observation that NaPB did not increase CYP3A enzyme activity, mRNA and protein levels in CAR KO rats suggests that CYP3A induction by NaPB in rat liver is due mainly to activation of CAR and not also to activation of PXR. This finding is in agreement with a previous mouse study where PB was shown to induce CYP2B and CYP3A enzyme activities and protein levels in PXR KO mice, but not in CAR KO mice (Scheer et al., 2008). Compared to WT rats, the treatment of PXR KO rats with PCN did not result in any increase in liver weight, microsomal total CYP content and induction of CYP2B and CYP3A enzymes. These results suggest that functional PXR and not CAR is required for PCN-induced effects on liver weight and induction of CYP2B and CYP3A enzymes in the rat. While PCN produced a marked increase in hepatocyte RDS in WT rats, only a small increase was observed in PXR KO rats. Additional studies would be required to ascertain if this small effect on hepatocyte RDS was due to CAR/PXR receptor crosstalk. The effects of NaPB and PCN on hepatic CYP enzyme activities, mRNA levels and protein levels in CAR KO and PXR KO rats, respectively, are in agreement with the recent study of Forbes et al. (2017), where WT, CAR KO and PXR KO rats were given single oral doses of corn oil (control), 12.5 mg/kg TCPOBOP and 100 mg/kg PCN 16 h prior to necropsy. In the Forbes et al. (2017) study, TCPOBOP increased hepatic CYP2B mRNA levels in WT rats, but not in CAR KO rats, whereas PCN increased hepatic CYP2B and CYP3A mRNA levels in WT rats, but not in PXR KO rats. Moreover, in the present study levels of CYP2B and CYP3A proteins were somewhat higher in liver microsomes from control PXR KO than from control WT rats, which correlates with the study of Forbes et al. (2017) where CYP2B and CYP3A mRNA levels were higher in control PXR KO rats than in control WT rats. In this study, levels of CYP4A proteins appeared to be somewhat higher in liver microsomes from control CAR KO than from control WT rats. Additional studies, including measurement of CYP4A marker enzyme activities, would be required to confirm this finding. Overall, the present study with CAR KO rats demonstrates that a functional CAR is required for NaPB-mediated increases in liver weight, stimulation of hepatocyte RDS and induction of CYP enzymes. In addition, the studies with PXR KO rats demonstrate that a functional PXR is required for PCN-mediated increases in liver weight and induction of
for 7 days are in agreement with those observed in previous studies (Elcombe et al., 2014; Lake et al., 1998; Martignoni et al., 2006). Both NaPB and PCN increased relative liver weight, stimulated hepatocyte RDS and induced microsomal total CYP content. Treatment with NaPB produced a marked induction of CYP2B enzyme activities, CYP2B1 and CYP2B2 mRNA levels and CYP2B protein levels. NaPB also produced smaller increases in CYP3A enzyme activity, CYP3A1 mRNA levels and CYP3A protein levels. Treatment with PCN resulted in significant increases in CYP2B and CYP3A enzyme activities, CYP2B1, CYP2B2 and CYP3A1 mRNA levels and also increased CYP2B and CYP3A protein levels. Although treatment with NaPB and to a lesser extent PCN produced small, but statistically significant, increases in hepatic microsomal EROD activity, this was not associated with any increases in CYP1A1 and CYP1A2 mRNA levels and CYP1A protein content. The small increases in hepatic microsomal EROD activity observed in this study are unlikely to represent induction of CYP1A enzymes, but rather that this substrate is also metabolised by other CYP enzymes, including CYP2B and CYP2C enzymes (Burke et al., 1994), which are induced by NaPB and PCN treatment. Neither NaPB nor PCN appeared to increase hepatic microsomal CYP4A protein levels in WT rats, with hepatic CYP4A1 mRNA levels being unaffected by NaPB treatment, but significantly reduced by PCN treatment. Overall, both NaPB and PCN produced the expected marked effects on the hepatic CYP2B and CYP3A parameters measured in WT rats, with only comparatively small effects being observed on the CYP1A and CYP4A markers measured. Compared to effects in WT rats, the treatment of CAR KO rats with NaPB did not result in an increase in relative liver weight, centrilobular hepatocyte hypertrophy and an induction of hepatocyte RDS. In addition, total microsomal CYP content was not increased and (as demonstrated by enzyme activity, mRNA and protein measurements) the induction of CYP2B and CY3A enzymes was completely abolished in CAR KO rats given NaPB. In terms of the key and associative events for the MOA for PB-induced rodent liver tumour formation (Elcombe et al., 2014), the key event of increased hepatocyte RDS and the associative events of liver hypertrophy (both organ weight and morphology) and CYP2B enzyme induction were thus not observed in CAR KO rats treated with NaPB. The lack of effect of NaPB on relative liver weight, hepatocyte RDS and CYP2B enzyme induction in CAR KO rats is in agreement with previous studies conducted in CAR KO mice (Huang et al., 2005; Wei et al., 2000) and in a recent study in CAR KO rats (Okuda et al., 2017). The study of Okuda et al. (2017) also demonstrated that while two other known rat CAR activators, namely metofluthrin and momfluorothrin, induced hepatocyte RDS and CYP2B 25
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Fig. 6. Effect of treatment of WT and CAR KO rats with 0 (control) or 500 ppm NaPB in the diet and WT and PXR KO rats with 0 (control) and 100 mg/kg/day PCN by gavage for 7 days on hepatic microsomal CYP1A, CYP2B, CYP3A and CYP4A proteins. Microsomal fractions (20, 40 and 40 μg protein per lane for CYP1A, CYP3A and CYP4A proteins, respectively) were pooled from groups (n = 5) of WT, CAR KO, and PXR KO control and treated rats. For hepatic microsomal CYP2 B proteins, protein loadings of the pooled microsomal preparations were 0.5 μg protein per lane for WT rats treated with 500 ppm NaPB and 2.5 μg protein per lane for all other groups.
KO rats. Possibly, the small effect of NaPB on thyroid gland follicular cell RDS in CAR KO rats may be attributable to CAR/PXR receptor crosstalk. In order to demonstrate the usefulness of CAR KO and PXR KO rats as suitable animal models for thyroid gland MOA studies, additional studies would be required with known inducers (such as NaPB and PCN) to ascertain effects on serum TSH, T3 and T4 levels, thyroid gland follicular cell hypertrophy and hyperplasia and hepatic microsomal UGT activity towards T4 as substrate. In summary, CAR KO and PXR KO rats appear to be useful animal models for MOA studies on the hepatic effects of rodent liver CAR and PXR activators. While PB/NaPB and other rodent liver CAR activators generally produce liver tumours more readily in the mouse than in the rat (Elcombe et al., 2014), there are exceptions. For example, the synthetic pyrethroids metofluthrin and momflurothrin, the fungicide fluopyram and the natural pyrethrins have been shown to produce liver tumours in the rat, but not in the mouse, by a CAR activation MOA (Deguchi et al., 2009; Okuda et al., 2017; Osimitz and Lake, 2009; Tinwell et al., 2014). Hence it is necessary to have both rat and mouse CAR KO models available for rodent liver MOA studies. Additional studies would be required to ascertain if the rat CAR KO and PXR KO models employed in this study may also be useful for rat thyroid gland MOA studies with CYP2B and CYP3A enzyme inducers.
Fig. 5. Effect of treatment of WT (filled histograms) and CAR KO (hatched histograms) rats with 0 (control) and 500 ppm NaPB in the diet (A) and WT (filled histograms) and PXR KO (hatched histograms) rats with 0 (control) and 100 mg/kg/day PCN by gavage (B) for 7 days on hepatic CYP1A1, CYP1A2, CYP2B1, CYP2B2, CYP3A1 and CYP4A1 mRNA levels. Results for WT, CAR KO and PXR KO rats are expressed as fold change relative to control levels (i.e. animals not treated with either NaPB or PCN) and are presented as mean ± SD of 5 animals. Values significantly different from control are: *p < 0.05; **p < 0.01; ***p < 0.001.
CYP enzymes, with induction of hepatocyte RDS also being largely mediated through PXR. The hepatic effects of NaPB in CAR KO rats and of PCN in PXR KO rats are in agreement with those observed in other recent studies (Forbes et al., 2017; Okuda et al., 2017). This study also investigated the use of CAR KO and PXR KO rat models for MOA studies where treatment with CAR and/or PXR activators leads to thyroid gland tumours in rodents as a consequence of decreased circulating thyroid hormone levels resulting from an increase in hepatic metabolism and clearance of T4 (Capen, 2001; Dellarco et al., 2006; Hill et al., 1998; Meek et al., 2003). Previous studies have demonstrated that the treatment of normal (WT) rats with NaPB/PB and PCN can result in increased thyroid gland weight and induction of thyroid gland follicular cell RDS (Barter and Klaassen, 1992; Finch et al., 2006; Hood et al., 1999a,b; McClain et al., 1989). In the present study, PCN increased relative thyroid gland weight in WT rats and both NaPB and PCN increased thyroid gland follicular cell RDS in WT rats. The treatment of CAR KO rats with NaPB and PXR KO rats with PCN did not result in any effect on relative thyroid gland weight, whereas NaPB produced a small increase in thyroid gland follicular cell RDS in CAR
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