Comparative studies on the effects of sodium phenobarbital and two other constitutive androstane receptor (CAR) activators on induction of cytochrome P450 enzymes and replicative DNA synthesis in cultured hepatocytes from wild type and CAR knockout rats

Journal Pre-proof Comparative studies on the effects of sodium phenobarbital and two other constitutive androstane receptor (CAR) activators on induction of cytochrome P450 enzymes and replicative DNA synthesis in cultured hepatocytes from wild type and CAR knockout rats Manuela Goettel, Ivana Fegert, Naveed Honarvar, Audrey Vardy, Corinne Haines, Lynsey R. Chatham, Brian G. Lake

PII:

S0300-483X(20)30033-0

DOI:

https://doi.org/10.1016/j.tox.2020.152394

Reference:

TOX 152394

To appear in:

Toxicology

Received Date:

12 November 2019

Revised Date:

31 January 2020

Accepted Date:

1 February 2020

Please cite this article as: Goettel M, Fegert I, Honarvar N, Vardy A, Haines C, Chatham LR, Lake BG, Comparative studies on the effects of sodium phenobarbital and two other constitutive androstane receptor (CAR) activators on induction of cytochrome P450 enzymes and replicative DNA synthesis in cultured hepatocytes from wild type and CAR knockout rats, Toxicology (2020), doi: https://doi.org/10.1016/j.tox.2020.152394

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Comparative studies on the effects of sodium phenobarbital and two other constitutive androstane receptor (CAR) activators on induction of cytochrome P450 enzymes and replicative DNA synthesis in cultured hepatocytes from wild type and CAR knockout rats

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BASF SE, Carl-Bosch-Strasse 38, 67056 Ludwigshafen, Germany

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Manuela Goettela,* [email protected], Ivana Fegerta, Naveed Honarvara, Audrey Vardyb, Corinne Hainesb, Lynsey R. Chathamb, Brian G. Lakeb,c

Concept Life Sciences (formerly CXR Biosciences Ltd.), 2, James Lindsay Place, Dundee Technopole, Dundee DD1 5JJ, United Kingdom c

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Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, GU2 7XH, UK

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Corresponding author at: BASF SE, Carl-Bosch-Strasse 38, 67056 Ludwigshafen, Germany

ABSTRACT

Nongenotoxic chemicals can produce liver tumours in rats and mice by a mitogenic mode of

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action involving activation of the constitutive androstane receptor (CAR). The aim of this study was to evaluate the usefulness of cultured hepatocytes from normal (wild type; WT) and CAR knockout (KO) rats to screen compounds as potential activators of rat CAR and to validate this test system. Cultured hepatocytes from male Sprague-Dawley WT and CAR KO rats were treated with either 100 and 1000 µM sodium phenobarbital (NaPB), 3-100 µM fluquinconazole (FQZ), or 3-300 µM 3-(difluoromethyl)-1-methyl-N-(3´,4´,6-trifluoro[1,1´-biphenyl]-2-yl)-1H1

pyrazole-4-carboxamide (TI1) for 96 h. Induction of cytochrome P450 (CYP) enzymes was monitored by measurement of 7-pentoxyresorufin O-depentylase (PROD), 7-benzyloxyresorufin O-debenzylase (BROD) and 7-benzyloxyquinoline O-debenzylase (BQ) activities. Hepatocytes undergoing replicative DNA synthesis (RDS) were labelled by adding 10 µM 5-bromo-2´deoxyuridine to the culture medium for determination of the hepatocyte labelling index. The treatment of WT, but not of CAR KO, rat hepatocytes with NaPB, FQZ and TI1 increased

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hepatocyte RDS and induced CYP2B-dependent PROD activity. In contrast, all three compounds

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increased CYP2B/3A-dependent BROD and CYP3A-dependent BQ activities in both WT and CAR KO rat hepatocytes. Hepatocyte RDS was increased in both WT and CAR KO rat

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hepatocytes by treatment with 25 ng/ml epidermal growth factor as a positive control. Overall,

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these results demonstrate that the effects of three CAR activators on RDS and CYP2B enzyme induction are abolished in cultured CAR KO rat hepatocytes. As demonstrated by this validation

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study, the CAR KO hepatocyte model is a useful in vitro mechanistic tool for the rapid screening of chemicals as potential activators of rat CAR.

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Key Words: Sodium phenobarbital; fluquinconazole; fluxapyroxad derivative, constitutive androstane receptor (CAR); CAR knockout rats; cultured hepatocytes 1. Introduction

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A number of nongenotoxic chemicals have been shown to produce liver enlargement, induction of cytochrome P450 (CYP) enzymes and liver tumour formation in the rat and mouse. One such group of nongenotoxic chemicals comprise activators of the constitutive androstane receptor (CAR), which is associated with the induction of CYP2B subfamily enzymes (Omiecinski et al., 2011). Phenobarbital (PB) and its sodium salt (sodium phenobarbital; NaPB) are model CAR activators which produce liver enlargement, stimulation of hepatocyte replicative DNA synthesis 2

(RDS), induction of CYP2B and other CYP subfamily enzymes and liver tumours in rats and mice (Cohen, 2010; Elcombe et al., 2014; IARC, 2001; Lake, 2009, 2018; Whysner et al., 1996). In a recent evaluation of the MOA for PB-induced rodent liver tumour formation, the key events were identified as CAR activation, altered gene expression specific to CAR activation, increased cell proliferation, the development of altered hepatic foci and finally liver tumour formation (Elcombe et al., 2014). Associative events for this MOA included liver hypertrophy, induction of

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CYP2B subfamily enzymes and inhibition of apoptosis.

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In assessing the hepatic effects of rodent liver CAR activators, studies in mice lacking CAR

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(CAR knockout (KO) mice) have been particularly valuable. For example, studies in CAR KO mice have demonstrated that PB does not produce liver hypertrophy, induce Cyp2b enzymes or

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increase RDS (Huang et al., 2005; Wei et al., 2000; Yamamoto et al., 2004). Moreover, PB did not promote liver tumours in CAR knockout mice after initiation with the genotoxic agent

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diethylnitrosamine (DEN), or after treatment with the potent mouse CAR activator 4-bis[2-(3,5dichloropyridyloxy)]benzene (TCPOBOP; Omiecinski et al., 2011), either with or without prior

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DEN administration (Huang et al., 2005; Yamamoto et al., 2004). Rodent CAR activators can produce liver tumours in both the rat and mouse (Elcombe et al., 2014; Lake, 2018). While mice are generally more susceptible to liver tumour formation by CAR

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activators some compounds, including the natural pyrethrins (Osimitz and Lake, 2009) and the synthetic pyrethroids metofluthrin (Yamada et al., 2009) and momfluorothrin (Okuda et al., 2017), have been shown to produce liver tumours only in the rat. Thus, in order to evaluate the hepatic effects of nongenotoxic CAR activators in these two rodent species, both CAR KO rat and mouse models are required.

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Recently, CAR KO rats have become commercially available and have been employed for some in vivo studies on the hepatic effects of NaPB and other CAR activators (Forbes et al., 2017; Haines et al., 2018; Okuda et al., 2017). An investigation by Forbes et al. (2017) demonstrated that TCPOBOP increased hepatic CYP2B mRNA levels in normal (i.e. wild type; WT) rats but not in CAR KO rats; whereas in another study NaPB increased relative liver weight, stimulated RDS and induced CYP2B enzyme activity and mRNA levels in WT but not in

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CAR KO rats (Haines et al., 2018). Similarly NaPB, metofluthrin and momfluorothrin were

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shown to increase relative liver weight, stimulate RDS and to induce CYP2B mRNA levels in

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WT but not in CAR KO rats (Okuda et al., 2017).

The aim of this study was to evaluate the use of cultured hepatocytes from normal (WT) and

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CAR KO rats as an in vitro system to screen compounds as potential rat liver CAR activators. The test compounds selected for these studies were the model rodent liver CAR activator NaPB

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(Cohen, 2010; Elcombe et al., 2014; Lake, 2009), the triazole fungicide fluquinconazole (FQZ) and 3-(difluoromethyl)-1-methyl-N-(3´, 4´, 6-trifluoro[1,1´-biphenyl]-2-yl)-1H-pyrazole-4-

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carboxamide (TI1). Previous studies have demonstrated that FQZ is a nongenotoxic agent which produces liver tumours in both rats and mice, with investigative studies demonstrating a CAR activation MOA involving increases in liver weight, centrilobular hypertrophy and induction of CYP2B enzymes (EFSA, 2011). TI1 is structurally related to the nongenotoxic pyrazole

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carboxamide fungicide fluxapyroxad which produces liver tumours in the rat, with investigative studies demonstrating a CAR activation MOA involving increases in liver weight, centrilobular hypertrophy and induction of CYP2B enzymes (EFSA, 2012; EPA, 2012). 2. Materials and methods 2.1. Materials 4

FQZ (molecular weight 376.2; purity 99.1%) and TI1 (molecular weight 381.3; purity >99.5%) were provided by BASF SE (Ludwigshafen am Rhein, Germany). Sodium phenobarbital (NaPB; purity ≥ 99%), 5-bromo-2´-deoxyuridine (BrdU), epidermal growth factor (EGF), CYP substrates and metabolites, Leibowitz L15 culture medium and other tissue culture reagents were purchased from Sigma-Aldrich (Poole, Dorset, UK). Uncoated 6- and 96-well plates and 25 cm2 Falcon flasks were obtained from Greiner Bio-One Ltd. (Stroudwater Business Park, Stonehouse,

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UK).

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2.2. Animals

Male Sprague-Dawley WT and CAR KO (on a Sprague Dawley background) rats (aged 6-8

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weeks) were obtained from Horizon Discovery (PO Box 122, Boyertown, PA, USA). Rats were

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housed in groups on saw-dust in solid-bottom polypropylene cages and were allowed free access to water and powdered RM1 laboratory animal diet (Special Diets Services, Witham, Essex, UK)

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and were housed 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

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allowed to acclimatize to these conditions for at least 5 days before use. All animal procedures were performed 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).

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2.3. Hepatocyte isolation and culture

Hepatocytes from male Sprague- Dawley WT (8-10 weeks old) and CAR KO (10-12 weeks

old) rats were obtained by collagenase perfusion as described previously (Mitchell et al., 1984). Hepatocyte viabilities (determined by trypan blue exclusion) were in excess of 80% and hepatocytes from two independent perfusions were pooled for all studies. Rat hepatocytes were plated in uncoated 96- or 6-well plates (for cytotoxicity and RDS studies, respectively) or in 5

uncoated 25 cm2 Falcon flasks (for CYP enzyme assays) in Leibowitz L15 culture medium with additions as described previously (Plant et al., 1998) and cultured in a humidified incubator at 37°C under air for 4 h. At the end of the attachment period, treatment of hepatocytes was commenced by changing the culture medium to Leibowitz L15 medium containing either 0 (control), 100 or 1000 μM NaPB, 3, 10, 30 or 100 μM FQZ, or 10, 30, 100 and 300 μM TII. All media contained 0.1 % (v/v) dimethyl sulphoxide (DMSO). Subsequently, the medium was

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changed at 24 h intervals to fresh medium containing the above concentrations of either NaPB,

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FQZ or TI1 for a total treatment period of 96 h. To study RDS, 10 µM BrdU was added to the medium for the last 72 h of treatment. Rat hepatocytes were also treated with 25 ng/ml EGF for

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72 h to serve as a positive control for the RDS studies. Six replicate wells at each concentration

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of control, PB, FQZ, TI1 and EGF were examined for cytotoxicity, three replicates at each concentration of control and the test compounds for CYP enzyme activities and five replicates of

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control, each concentration of the test compounds and EGF for RDS studies. At the end of the 96 h treatment period, hepatocyte monolayers were either assayed directly, harvested by scraping

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into SET buffer (0.25 M sucrose containing 5 mM EDTA and 20 mM Tris-HCl buffer, pH 7.4) or were fixed in methanol. 2.4. Cytotoxicity assay

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The cytotoxicity of the test compounds to rat hepatocytes was assessed by ATP depletion. At the end of the 96 h treatment period, rat hepatocyte monolayers were assayed for ATP content using a Promega (Delta House, Southampton Science Park, Southampton, UK) CellTiter-Glo® luminescent cell viability assay kit. Results were expressed as a percentage of the maximum amount of ATP released from control (0.1% (v/v) DMSO only) cultures. 2.5. CYP enzyme assays 6

Hepatocyte monolayers scraped into SET buffer were sonicated and stored at -70ºC until analysis. Protein content was determined by the method of Lowry et al. (1951), employing bovine serum albumin as standard. 7-Pentoxyresorufin O-depentylase (PROD), 7benzyloxyresorufin O-debenzylase (BROD) and 7-benzyloxyquinoline O-debenzylase (BQ) activities were determined as described previously (Choi et al., 2017; Ross et al., 2010).

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2.6. Replicative DNA synthesis (RDS) assay

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Hepatocyte monolayers were fixed in ice-cold methanol for 10 min and then washed with

phosphate buffered saline (PBS) and then again treated with ice cold methanol for 10 min and

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subsequently washed with PBS. Hepatocyte monolayers were stored in PBS at 4°C for 96 h and then processed for BrdU immunocytochemistry as described previously (Ross et al., 2010),

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except that hepatocyte monolayers were not counterstained with hematoxylin. The BrdU labeling

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index (i.e. percentage of hepatocyte nuclei undergoing RDS) was determined by counting approximately 1200-1600 nuclei in 4 random areas from each well.

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2.7. Liver microsomal CYP inhibition assays

Liver microsomes were prepared from male Sprague-Dawley rats treated with either 500 ppm NaPB in the diet for 7 days or with 100 mg/kg/day pregnenolone-16α-carbonitrile (PCN) orally for 7 days (Haines et al., 2018). Microsomes from NaPB-treated rats were assayed for PROD

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activity in the presence of either 0-100 µM FQZ or 0-100 µM TI1 and liver microsomes from PCN-treated rats were assayed for BQ activity in the presence of 0-300 µM FQZ. FQZ and TI1 were added to incubation mixtures in dimethylformamide (maximum concentration 1% (v/v)). IC50 values were calculated using Prism software (GraphPad, San Diego, CA, USA). 2.8. Statistical analysis 7

Results are expressed as mean ± SD. Statistical significance between and control and treated groups was determined by two-tailed Student’s t-tests. 3. Results Male Sprague-Dawley rat WT and CAR KO hepatocytes were cultured in control medium (0.1% (v/v) DMSO only) and in medium containing either 100 or 1000 μM NaPB, 3-100 μM FQZ, or

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10-300 μM TII for 96 h and effects on cytotoxicity, CYP enzyme activities and RDS determined.

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3.1. Effect on hepatocyte cytotoxicity

The cytotoxicity of the test compounds to rat hepatocytes was determined by measurement of

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ATP content. Treatment with 100 and 1000 µM NaPB either had little effect or produced

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statistically significant increases in ATP content of both WT and CAR KO rat hepatocytes (Fig. 1A and 1B). The treatment of WT and CAR KO rat hepatocytes with 3-30 µM FQZ did not

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result in any significant decreases in ATP content, with 100 µM FQZ only producing a small decrease in ATP content in WT and CAR KO rat hepatocytes to 89 and 94% of control,

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respectively (Fig. 1A). While treatment of WT and CAR KO rat hepatocytes with 10-100 µM TI1 did not result in any significant cytotoxicity (reflected by decreases in hepatocyte ATP content), treatment with 300 µM TI1 significantly decreased WT and CAR KO hepatocyte ATP content to 47 and 5% of control, respectively (Fig. 1B). The increases in hepatocyte ATP content

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observed at some concentrations of NaPB, FQZ and TI1 are not considered to be associated with cytotoxicity, but could represent increased hepatocyte intermediary metabolism. 3.2. Effect on hepatocyte CYP enzymes Rat hepatocyte PROD, BROD and BQ activities were determined as markers of induction of CYP2B, CYP2B/3A and CYP3A subfamily enzymes, respectively (Lubet et al., 1990; Renwick 8

et al., 2001). The treatment of WT rat hepatocytes with 100 and 1000 µM NaPB significantly increased PROD (to 392 and 557% of control), BROD (to 642 and 1110% of control) and BQ activities (to 161 and 367% of control) (Fig. 2 and 3). In contrast, the treatment of CAR KO rat hepatocytes with NaPB had no statistically significant effect on PROD enzyme activity and produced smaller increases in BROD and BQ activities than those observed in WT rat hepatocytes (Fig. 2 and 3). Treatment of CAR KO rat hepatocytes with 1000 μM NaPB increased

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BROD and BQ enzyme activities to 232 and 246% of control, respectively (Fig. 2) and to 211

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and 253% of control, respectively (Fig. 3).

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The treatment of WT rat hepatocytes with FQZ produced statistically significant increases in PROD (up to 318% of control, Fig. 2A), BROD (up to 539% of control, Fig. 2B) and BQ (up to

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554% of control, Fig. 2C) activities at concentrations of 3 and 10, 3-30 and 3-100 µM, respectively. The induction of CYP enzyme activities was not concentration-dependent with the

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greatest increases in PROD, BROD and BQ activities being observed at FQZ concentrations of 3, 3, and 30 µM, respectively. While treatment with 3-100 µM FQZ had no statistically

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significant effect on PROD activity in CAR KO rat hepatocytes (maximum increase 120 % of control, Fig. 2A), statistically significant increases in BROD (up to 221% of control, Fig. 2B) and BQ (up to 586% of control, Fig. 2C) activities were observed in CAR KO rat

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hepatocytes.

The treatment of WT rat hepatocytes with TI1 produced statistically significant increases in

PROD (up to 529% of control, Fig. 3A), BROD (up to 978% of control, Fig. 3B) and BQ (up to 402% of control, Fig. 3C) activities at concentrations of 10-100, 10-300 and 10-100 µM, respectively. The induction of CYP enzyme activities was not concentration-dependent with the greatest increases in PROD, BROD and BQ activities being observed at TI1 concentrations of 9

10, 10, and 100 µM, respectively. While treatment with 10-300 µM TI1 had no statistically significant effect on PROD activity in CAR KO rat hepatocytes (Fig. 3A) and only produced a small increase in BROD activity to 148% of control at a concentration of 100 μM (Fig. 3B), treatment with 10-300 μM TI1 produced statistically significant increases in BQ activity (up to 316% of control, Fig. 3C).

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3.3. Effect on hepatocyte replicative DNA synthesis (RDS)

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Rat WT and CAR KO hepatocytes were cultured in control medium (0.1% (v/v) DMSO only) and medium containing either NaPB, FQZ or TI1 for 96h, with the DNA precursor BrdU being

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added during the last 72h of treatment. As a positive control for induction of hepatocyte RDS, WT and CAR KO rat hepatocytes were also treated with 25 ng/ml EGF. Treatment with 100 and

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1000 µM NaPB significantly increased RDS in WT rat hepatocytes to 182 and 180% of control,

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respectively, but had no significant effect on RDS in CAR KO rat hepatocytes (Fig. 4A and 4B). The treatment of WT rat hepatocytes with 3 and 10 µM FQZ significantly increased RDS to 160 and 150% of control, respectively, whereas treatment with 30 and 100 µM FQZ significantly

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decreased RDS in CAR KO rat hepatocytes (Fig. 4A). Statistically significant increases in RDS to 190, 174 and 154% of control were observed in WT rat hepatocytes treated with 10, 30 and 100 µM TI1, respectively, whereas treatment with 300 µM TI1 resulted in a significant decrease

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in RDS (Fig. 4B). Treatment with TI1 did not produce any significant increases in RDS in CAR KO rat hepatocytes, with marked cytotoxicity being observed in CAR KO rat hepatocytes treated with 300 µM TI1 (Fig. 4B). The treatment of WT and CAR KO rat hepatocytes with 25 ng/ml EGF significantly increased RDS to 406 and 434 % of control, respectively (Fig. 4A and 4B). 3.4. Effect of FQZ and TI1 on hepatic microsomal PROD and BQ activities

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While FQZ and TI1 induced CYP enzyme activities in cultured WT rat hepatocytes, the magnitude of stimulation of PROD and BROD activities was not concentration-dependent, with a reduction in the magnitude of stimulation of BQ activity also being observed at the highest concentrations of FQZ and TI1 examined (Fig. 2 and 3). In order to assess whether these effects were due to enzyme inhibition by the test compounds, the effect of FQZ and TI1 on PROD and BQ activities was determined in liver microsomes prepared from male Sprague-Dawley rats

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treated with either NaPB or PCN to induce CYP2B and CYP3A enzymes, respectively (Elcombe

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et al., 2014; Omiecinski et al., 2011). The addition of either 0.01-100 µM FQZ or 0.01-100 µM TI1 to liver microsomes from NaPB treated rats resulted in a concentration-dependent inhibition

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of hepatic microsomal PROD activity (Fig. 5). Calculated IC50 values for inhibition of

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microsomal PROD activity were 1.4 and 12.9 µM for FQZ and TI1, respectively. The addition of 0.1-300 µM FQZ to liver microsomes from PCN treated rats resulted in a concentration-

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dependent inhibition of hepatic microsomal BQ activity, with a calculated IC50 value of 2.3 µM (Fig. 5). The effect of 0.1-300 µM TI1 on BQ activity in liver microsomes from PCN treated rats

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was not investigated, as in a pilot study enzyme activity was only inhibited by around 50% at a TI1 concentration of 100 µM (data not shown). 4. Discussion

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The treatment of WT male Sprague-Dawley rat hepatocytes with NaPB resulted in the expected effects of induction of CYP2B- and CYP3A-dependent enzyme activities and a stimulation of hepatocyte RDS (Elcombe et al., 2014; Lake, 2009, 2018; Peffer et al., 2018). In contrast, NaPB did not induce CYP2B-dependent PROD activity or stimulate RDS in cultured hepatocytes from CAR KO rats. While treatment with NaPB resulted in some induction of BROD and BQ activities in CAR KO rat hepatocytes, the increase in these enzyme activities are 11

most likely attributable to crosstalk between CAR and the pregnane X receptor (PXR) (Maglich et al., 2002; Moore et al., 2000; Omiecinski et al., 2011; Yoshinari et al., 2008). The treatment of WT rat hepatocytes with FQZ resulted in an induction of PROD activity and a stimulation of RDS, thus confirming that this compound is a CAR activator in rat liver. Treatment with FQZ also induced hepatocyte BROD and BQ activities in WT rat hepatocytes.

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Unlike NaPB, the induction of hepatocyte CYP enzyme activities by FQZ in cultured WT rat hepatocytes was not concentration-dependent. However, at the concentrations examined FQZ

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was not markedly cytotoxic to WT rat hepatocytes. Additional investigations with liver

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microsomes from NaPB- and PCN-treated rats showed that FQZ was an inhibitor of both CYP2B-dependent PROD and CYP3A-dependent BQ enzyme activities. Overall, the lack of

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concentration-dependency in the induction of CYP2B and CYP3A subfamily enzymes by FQZ in cultured WT rat hepatocytes observed in this study appears to be attributable to the inhibition

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of CYP-dependent enzyme activities by the test compound.

The treatment of WT rat hepatocytes with TI1 resulted in an induction of PROD activity and a

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stimulation of RDS, thus confirming that this compound is a CAR activator in rat liver. Treatment with TI1 also induced hepatocyte BROD and BQ activities in WT rat hepatocytes. The induction of hepatocyte CYP enzyme activities by TI1 in cultured WT rat hepatocytes was

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not concentration-dependent, with the highest concentration examined producing some cytotoxicity. Like FQZ, TI1 was also found to be an inhibitor of CTP2B-dependent PROD activity in liver microsomes from NaPB-treated rats. The lack of concentration-dependency of TI1 on CYP enzymes in cultured WT rat hepatocytes appears to be at least partially attributable to enzyme inhibition, with effects on CYP enzymes and hepatocyte RDS at the highest concentration examined being due to cytotoxicity. 12

In this study only NaPB produced concentration-dependent effects on CYP2B enzyme induction, both FQZ and TI1 being shown to produce a concentration-dependent inhibition of hepatic microsomal PROD activity. The effects of FQZ and TI1 are thus, similar to those reported for the CAR activator nitrapyrin (LaRocca et al., 2017) and the combined CAR/peroxisome proliferator-activated receptor alpha (PPARα) activator pronamide (LeBaron et al., 2014), which are both nongenotoxic agents producing liver tumours in the mouse. Inhibition

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of CYP enzyme activity can complicate the interpretation of the effects of a potential CAR

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activator. As an alternative to the measurement of CYP enzyme activities the effects on CYP mRNA levels can be determined in cultured rat hepatocytes, as has been demonstrated for

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several CAR activators (Gährs et al., 2013; Okuda et al., 2017; Price et al., 2008; Soldatow et al.,

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2016). In addition to determining effects on CYP enzyme activities and/or mRNA levels, the RNA interference (siRNA) technique has also been employed in some studies with rat

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hepatocytes to probe the role of CAR in the induction of CYP2B mRNA levels (Deguchi et al., 2009; Gährs et al., 2013; Okuda et al., 2017). Other factors important in interpreting

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concentration-response relationships in cultured hepatocytes include the potential cytotoxicity of the test compound and its solubility in tissue culture medium.

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Unlike effects in WT rat hepatocytes, the treatment of CAR KO rat hepatocytes with both FQZ and TI1 did not result in any induction of CYP2B-dependent PROD activity. While some effects on BROD and BQ activities were observed, these are most likely attributable to effects on PXR rather than on CAR. Treatment with neither FQZ nor TI1 produced any stimulation of RDS in cultured CAR KO rat hepatocytes, with significant decreases in RDS being observed after treatment with 30 and 100 µM FQZ and 300 µM TI1. While the inhibition of RDS in CAR KO 13

rat hepatocytes treated with 300 µM TI1 appears to be attributable to cytotoxicity, the effects of 30 and 100 µM FQZ were not due to cytotoxicity as there was not any marked reduction in hepatocyte ATP levels. The treatment of both WT and CAR KO rat hepatocytes with EGF resulted in significant increases in RDS, thus confirming the potential responsiveness of

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hepatocytes from both WT and CAR KO rats to a known mitogen.

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Overall, this validation study demonstrates that hepatocytes from WT and CAR KO rats can be employed as a rapid in vitro test system to screen compounds for activation of rat hepatic CAR.

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Studies with the model rodent liver CAR activator NaPB and also with two other CAR activators, namely FQZ and TI1, demonstrated that CYP2B enzyme induction and stimulation of

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hepatocyte RDS was only observed in WT and not in CAR KO rat hepatocytes. The stimulation

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of RDS is a pivotal key event in the MOA for liver tumour formation by rodent CAR activators and is the basis for the observed species difference between rodents (rats and mice) and humans in that CAR activators stimulate RDS in rodent hepatocytes, but not in human hepatocytes

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(Cohen, 2010; Elcombe et al., 2014; Lake, 2009, 2018; Peffer et al., 2018). While CAR KO rat hepatocytes are clearly refractory to the mitogenic effects of CAR activators, other mitogenic agents (e.g. PPARα activators) would be expected to stimulate RDS in this model system. The

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responsiveness of CAR KO rat hepatocytes to a mitogenic agent was demonstrated by significant increases in RDS following treatment with EGF. In terms of evaluating the effect of chemicals on RDS in cultured hepatocytes, it is important to utilise concentrations up to those which produce either significant cytotoxicity or an inhibition of RDS in order to confirm whether the test compound can or cannot stimulate RDS. This is particularly important in species differences

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studies where CAR activators, which are mitogenic agents in rodent hepatocytes, have been shown not to stimulate RDS in cultured human hepatocytes (Elcombe et al., 2014; Lake, 2018). The results obtained in this in vitro rat hepatocyte study are in agreement with previous in vivo studies in the rat where NaPB and other CAR activators have been shown not to increase CYP2B enzyme activities and/or mRNA levels and not to stimulate RDS in CAR KO rats (Forbes et al.,

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2017; Haines et al., 2018; Okuda et al., 2017). The WT and CAR KO hepatocyte model offers significant advantages over in vivo studies in terms of a marked reduction in the numbers of

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animals required and the ability to study a wide range of concentrations of the test compounds.

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In conclusion, the CAR KO hepatocyte model is a useful in vitro mechanistic tool for the rapid

Disclosure statement

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screening of chemicals as potential activators of rat CAR.

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M. Goettel, I. Fegert, and N. Honarvar are all employed by BASF, which manufactures FQZ and the fluxapyroxad derivative TI1. All other authors have been involved in studies performed for

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BASF. The authors alone are responsible for the content and writing of this article.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Acknowledgment

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This work was sponsored by BASF (Ludwigshafen am Rhein, Germany).

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Legends to Figures

Fig. 1. Effect of NaPB, FQZ and TI1 on rat hepatocyte cytotoxicity employing the ATP assay.

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Male Sprague-Dawley WT and CAR KO rat hepatocytes were treated with control medium (0.1% (v/v) DMSO only), medium containing 100 or 1000 µM NaPB and either 3-100 µM FQZ

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(A) or 10-300 µM TI1 (B) for 96 h. Results are expressed as mean ± SD of 6 wells per treatment.

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Values significantly different from control (0.1% (v/v) DMSO only) are: **p<0.01; ***p<0.001.

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Fig. 2. Effect of NaPB and FQZ on rat hepatocyte PROD (A), BROD (B) and BQ (C) enzyme activities. Male Sprague-Dawley WT and CAR KO rat hepatocytes were treated with control medium (0.1% (v/v) DMSO only) and medium containing either 100 or 1000 µM NaPB or 3-100 µM FQZ for 96 h. Results are expressed as mean ± SD of 3 wells per treatment. Values

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significantly different from control (0.1% (v/v) DMSO only) are: *p<0.05; **p<0.01;

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***p<0.001.

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Fig. 3. Effect of NaPB and TI1 on rat hepatocyte PROD (A), BROD (B) and BQ (C) enzyme activities. Male Sprague-Dawley WT and CAR KO rat hepatocytes were treated with control medium (0.1% (v/v) DMSO only) and medium containing either 100 or 1000 µM NaPB or 10300 µM TI1 for 96 h. Results are expressed as mean ± SD of 3 wells per treatment. Values significantly different from control (0.1% (v/v) DMSO only) are: *p<0.05; **p<0.01;

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***p<0.001.

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Fig. 4. Effect of NaPB, FQZ and TI1 on rat hepatocyte RDS. Male Sprague-Dawley WT and CAR KO rat hepatocytes were treated with control medium (0.1% (v/v) DMSO only), medium containing 100 or 1000 µM NaPB, medium containing 25 ng/ml EGF and medium containing either 3-100 µM FQZ (A) or 10-300 µM TI1 (B) for 96 h. Results are expressed as mean ± SD of 5 wells per treatment. Due to marked cytotoxicity (#), no results were obtained for CAR KO

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hepatocytes treated with 300 µM TI1. Values significantly different from control (0.1% (v/v)

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DMSO only) are: *p<0.05; **p<0.01; ***p<0.001.

Fig. 5. Inhibition of PROD activity by FQZ and TI1 and BQ activity by FQZ in rat liver microsomes. The effect of 0.01-100 µM FQZ and 0.01-100 µM TI1 on PROD activity was determined in NaPB-induced rat liver microsomes and the effect of 0.1-300 µM FQZ on BQ

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activity was determined in PCN-induced rat liver microsomes. Results are presented as mean values of 2-3 control (no test compound) and 1 or more replicates at each concentration of either FQZ or TI1, with IC50 values shown in parentheses. Control rates of PROD activity in NaPBtreated liver microsomes and BQ activity in PCN-treated liver microsomes were 47 pmol/min/mg protein and 8.1 nmol/min/mg protein, respectively.

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