Critical evaluation of the human relevance of the mode of action for rodent liver tumor formation by activators of the constitutive androstane receptor (CAR)

Figures & data

Table 1. Examples of compounds where a CAR activation MOA for mouse and/or rat liver tumor formation has been established.

Compound^a	Use	Bioassay		MOA analysis
		Liver tumors	No liver tumors	Causal key event 1: CAR activation^b	Causal key event 2: Hepatocyte proliferation^c	Evidence for CAR-dependency^d	References
Benfluralin	Herbicide	Mouse		Not examined	Not examined	Not examined	Strupp et al. (2020)
		Rat		Increase (CYP2B enzymes)	Increase (BRDU)	CAR KO/PXR KO rat (in vivo study)
Cyproconazole	Fungicide	Mouse	Rat	Increase (Cyp2b enzymes)	Increased (BRDU, MKI67, PCNA)	CAR KO mouse (in vivo study)	Peffer et al. (2007), Tamura et al. (2013), Tamura et al. (2015), and Marx-Stoelting et al. (2020)
Dieldrin	Insecticide	Mouse	Rat, hamster	Increase (Cyp2b enzymes)	Increase (BRDU, [^3H]thymidine)	CAR KO mouse (in vivo study) CAR KO/PXR KO mouse (in vivo study)	Cabral et al. (1979), Kolaja et al. (1996), Stevenson et al. (1999), and Wang et al. (2020)
DDT	Insecticide	Mouse	Hamster	Increase (Cyp2b enzymes)	Not increased	Not examined	Kostka et al. (1996), Kiyosawa et al. (2008), Kazantseva et al. (2013), Harada et al. (2016), and IARC (2018)
		Rat		Increase (CYP2B enzymes)	Increase (PCNA, [^3H]thymidine)	Not examined
Epoxiconazole	Fungicide	Mouse		Increase (Cyp2b enzymes)	Increase (MKI67)	Not examined	Hester et al. (2012) and Marx-Stoelting et al. (2020)
Fluopyram	Fungicide	Rat	Mouse	Increase (CYP2B enzymes)	Increase (MKI67)	Not examined	Tinwell et al. (2014)
Metazachlor	Herbicide	Rat	Mouse	Increase (CYP2B enzymes)	Increase (BRDU)	CAR KO rat (cultured hepatocytes)	Wiemann et al. (2019)
Metofluthrin	Insecticide	Rat	Mouse	Increase (CYP2B enzymes)	Increase (BRDU)	CAR KO rat (in vivo study) CAR RNAi (cultured hepatocytes)	Deguchi et al. (2009), Hirose et al. (2009), Yamada et al. (2009), Yamada et al. (2015), Okuda et al. (2017a), and Yamada (2018)
Methyl isobutyl ketone	Solvent	Mouse	Rat	Increase (Cyp2b enzymes)	Increase (BRDU)	CAR/PXR KO mouse (in vivo study)	Hughes et al. (2016)
Momfluorothrin	Insecticide	Rat	Mouse	Increase (CYP2B enzymes)	Increase (BRDU)	CAR KO rat (in vivo study) CAR RNAi (cultured hepatocytes)	Okuda et al. (2017a, 2017b) and Yamada (2018)
Nitrapyrin	Nitrogen stabilizer	Mouse	Rat	Increase (Cyp2b enzymes)	Increase (BRDU, PCNA)	CAR KO mouse (in vivo study)	Yano et al. (2008) and LaRocca et al. (2017)
Oxazepam^e	Tranquilizer	Mouse	Rat	Increase (Cyp2b enzymes)	Increase (BRDU)	Not examined	Cunningham et al. (1994), Griffin et al. (1995, 1996), and Parkinson et al. (2006)
Piperonyl butoxide	Insecticide synergist	Mouse	Rat	Increase (Cyp2b enzymes)	Increase (BRDU, PCNA)	CAR KO mouse (in vivo study) CAK KO/PXR KO mouse (in vivo study)	Butler et al. (1998), Sakamoto et al. (2013, 2015), and Lake et al. (2020)
Pronamide^e	Herbicide	Mouse	Rat	Increase (Cyp2b enzymes)	Increase (BRDU)	Not examined	LeBaron et al. (2014b)
Propiconazole	Fungicide	Mouse	Rat	Increase (reporter gene assay, Cyp2b enzymes)	Increase (PCNA, BRDU, MKI67)	CAR KO mouse (in vivo study)	Allen et al. (2006), Ward et al. (2006), Currie et al. (2014), Oshida et al. (2015a), and Marx-Stoelting et al. (2020)
Pyrethrins	Insecticide	Rat	Mouse	Increase (CYP2B enzymes)	Increase (BRDU)	Not examined	Price et al. (2007, 2008) and Osimitz and Lake (2009)
Sedaxane	Fungicide	Mouse		Increase (reporter gene assay, Cyp2b enzymes)	Increase (MKI67)	Not examined	Peffer et al. (2018a)
		Rat		Increase (reporter gene assay, CYP2B enzymes)	Increase (BRDU)	Not examined
Sulfoxaflor	Insecticide	Mouse		Increase (Cyp2b enzymes)	Increase (BRDU)	CAR KO/PXR KO mouse (in vivo study)	LeBaron et al. (2013), and LeBaron et al. (2014a)
		Rat		Increase (CYP2B enzymes)	Increase (MKI67)	Not examined
Tetrahydrofuran	Chemical intermediate and solvent	Mouse	Rat	Increase (Cyp2b enzymes)	Increase (BRDU)	CAR KO/PXR KO mouse (in vivo study)	Gamer et al. (2002), Choi et al. (2017), and Dekant (2019)
Toxaphene	Insecticide	Mouse	Rat, hamster	Increase (Cyp2b enzymes)	Increase (BRDU)	CAR KO mouse (in vivo study) CAR KO/PXR KO mouse (in vivo study)	Goodman et al. (2000) and Wang et al. (2015, 2017)
Triadimefon	Fungicide	Mouse	Rat	Increase (Cyp2b enzymes)	Increase (PCNA)	Not examined	Allen et al. (2006) and Ward et al. (2006)

^a All of these compounds have been shown to have a MOA for rodent liver tumor formation similar to that described for PB (Cohen 2010; Elcombe et al. 2014; Lake 2018; Yamada 2018), with data required to establish a CAR activation MOA for rodent liver tumor formation being obtained as described in the text.

^b CAR activation was determined from reporter gene assay data or in most instances indirectly by measurement of CYP enzymes (mRNA levels and/or enzyme activities).

^c Hepatocyte RDS was determined in most studies by 5-bromo-2′-deoxyuridine (BRDU) labeling and in some studies by either MKI67, proliferating cell nuclear antigen (PCNA), or [³H]thymidine labeling.

^d Evidence for CAR dependency was obtained from in vivo studies with either CAR KO or CAR KO/PXR KO animals and from in vitro studies with hepatocytes from CAR KO rats. In addition, in two studies the RNA interference (siRNA) technique was employed with hepatocytes from wild-type rats to demonstrate the role of CAR in the induction of CYP2B enzymes.

^e MOA considered to involve both CAR and PPARα activation.

Table 2. Some MOAs for hepatocellular carcinogenesis.

I. DNA reactivity

II. Increased cell proliferation

A. Receptor mediated

1. CAR activation

2. PPARα activation

3. Aryl hydrocarbon receptor (AhR) activation

4. Estrogen receptor activation

5. HMG-CoA reductase inhibitors (statins)

6. Porphyrias

B. Non-receptor mediated

1. Cytotoxicity

2. Infection

3. Metal overload (e.g. iron and copper)

4. Increased apoptosis (e.g. fumonisin B1)

C. Inherited disorders leading to cytotoxicity (e.g. porphyrias, alpha-1-antitrypsin abnormality, etc.)

Modified from Cohen (2010) and Cohen and Arnold (2016).

Table 3. Key and associative events for rodent liver tumor formation by PB and other CAR activators.

Key events

Definition: an empirically observable causal precursor step to the adverse outcome that is itself a necessary element of the MOA.

KE1. CAR activation

KE2. Altered gene expression specific to CAR activation

KE3. Increased hepatocellular proliferation

KE4. Clonal expansion leading to altered hepatic foci

KE5. Hepatocellular adenomas/carcinomas

Associative events^a

Definition: a biological process that is not a causal necessary key event for the MOA, but is a reliable indicator or marker for a key event.

AE1. Hepatic CYP2B induction

AE2. Liver hypertrophy (weight and morphology)

KE: key event; AE: associative event (Andersen et al. 2014).

From Elcombe et al. (2014) and Lake (2018).

^a Other associative events include altered epigenetic changes specific to CAR activation and inhibition of apoptosis, together with inhibition of gap junctional intercellular communication as an associative event or modulating factor. However, data on these endpoints are not required to establish a CAR-dependent MOA for rodent liver tumor formation (Lake 2018; Peffer et al. 2018b).

Table 4. Effect of some CAR activators on RDS in cultured rodent and human hepatocytes.

Hepatocyte RDS^a
Compound	Rodent			Human^b			References
	Animals	Responses to:^c		Number of donors (sex and age (years))	Responses to:^c
		Test compound [concentration tested]	Positive controls^d [concentration tested]		Test compound [concentration tested]	Positive^e/negative controls^f [concentration tested]
Benfluralin	B6C3F1 mouse	[10, 30, 100, and 300 µM] ↑ (10 and 30 µM) ↓ (100 and 300 µM)	PB: [1000 µM] ↑ (NS) EGF: [25 ng/mL] ↑	3 (female 52) 2 (males 51 and 58)	[1, 3, and 10 µM] None	EGF: [25 ng/mL] ↑ PB: [1000 µM] None	Strupp et al. (2020)
	F344 rat	[3, 10, 30, and 100 µM] ↑ (10, 30, and 100 µM)	PB: [1000 µM] ↑ EGF: [25 ng/mL] ↑
	Sprague Dawley rat	[10, 30, 100, and 300 µM)] ↑ (30, 100, and 300 µM)	PB: [1000 µM] ↑ EGF: [25 ng/mL] ↑
	CAR/PXR KO rat	[10, 30, 100, and 300 µM] ↓ (100 and 300 µM)	PB: [1000 µM] ↓ EGF: [25 ng/mL] ↑
Metazachlor	Han Wistar rat	[0.5, 2, 5, 15, and 50 µM] ↑ (0.5, 2, 5, and 15 µM)	PB: [500 µM] ↑ EGF: [25 ng/mL] ↑	2 (males 51 and 58)	[0.5, 1, 3, 10, and 25 µM] None	EGF: [25 ng/mL] ↑ PB: [500 µM] None	Wiemann et al. (2019)
	Sprague Dawley rat	[0.5, 2, 5, 15, and 50 µM] ↑(0.5, 2, 5, and 15 µM), ↓ (50 µM)	PB: [500 µM] ↑ EGF: [25 ng/mL] ↑
	CAR KO rat	[0.5, 2, 5, 15, and 50 µM] ↓ (50 µM)	PB: [500 µM] None EGF: [25 ng/mL] ↑
Metofluthrin	Han Wistar rat	[10, 50, 100, 500, and 1000 µM] ↑ (10, 50, 100, 500, and 1000 µM)	PB: [10, 50, 100, 500, and 1000 µM] ↑ (100 and 500 µM)	2 (females 41 and 63)	[10, 50, 100, 500, and 1000 µM] None	EGF: [1, 2, 5, 10, and 50 ng/mL] ↑ (5, 10, and 50 ng/mL) HGF: [1, 5, 10, 50, and 100 ng/mL] ↑ (5, 10, 50, and 100 ng/mL) PB: [10, 50, 100, 500, and 1000 µM] None	Hirose et al. (2009)
	Han Wistar rat	[7.7, 39, 77, 390, and 770 µM] ↑ (390 and 770 µM) (MKI67 mRNA)	HGF: [100 ng/mL]↑ (MKI67 mRNA)	4 (2 females 38 and 52, 2 males 26 and 66)	[7.7, 39, 77, 390, and 770 µM) ↓ (390 and 770 µM): one experiment with one donor	HGF: [1, 10, and 100 ng/mL] ↑ (10 and 100 ng/mL) PB: [100, 500, 750, and 1000 µM] None	Yamada et al. (2015)
				3 (2 females 31 and 38, male 23)	[3.9, 7.7, 77, 390, and 770 µM)] None (MKI67 mRNA)	HGF: [100 ng/mL] ↑ (MKI67 mRNA) PB: [1000 µM] None (MKI67 mRNA)
Momfluorothrin	Han Wistar rat	[1, 5, 10, 50, 100, 500, and 1000 µM] ↑ (5 and 10 µM) ↓ (100, 500, and 1000 µM)	PB: [500 and 1000 µM] ↑ (500 and 1000 µM) HGF: [10 and 100 ng/mL] ↑ (100 ng/mL)	10 (5 females 0.8, 3, 38, 52 and 80, and 5 males 26, 28, 57, 58 and 66)	[1, 5, 10, 50, 100, and 1000 µM] ↓ (50 µM)	HGF: [10 and 100 ng/mL] ↑ (10 and 100 ng/mL) PB: [500 and 1000 µM] None	Okuda et al. (2017a)
Nitrapyrin	CD-1 mouse	[1, 3, and 10 µM] ↑ (10 µM) (EDU)	EGF: [25 ng/mL]↑ (EDU)	2 (donor information is not reported)	[3, 10, 30, and 100 µM] None (EDU)	EGF: [25 ng/mL] ↑ (EDU)	LaRocca et al. (2017)
Piperonyl butoxide	CD-1 mouse	[5, 10, 20, 50, 200, and 500 µM] ↑ (5, 10, 20, and 50 µM) ↓ (200, and 500 µM)	PB: [10, 100, and 1000 µM] ↑ (10, 100, and 1000 µM) EGF: [25 ng/mL] ↑	2 (female 52 and male 51)	[5, 10, 20, 50, 200, and 500 µM] ↓ (200 and 500 µM)	EGF: [25 ng/mL] ↑ PB: [10, 100, and 1000 µM] None	Lake et al. (2020)
Sedaxane	Han Wistar rat	1, 3 10, 30. 65, and 100 µM ↑ (1, 3 10, and 30 µM) ↓ (65 and 100 µM)	PB: [10, 100, and 1000 µM] ↑ (100 and 1000 µM) EGF: [25 ng/mL)↑	1 (male 51)	[1, 3, 10, 30, 65, and 100 µM] ↓ (30, 65, and 100 µM )	EGF: [25 ng/mL] ↑ PB: [10, 100, and 1000 µM] None	Peffer et al. (2018a)

NS: not statistically significant.

^a Hepatocyte RDS was determined by 5-bromo-2′-deoxyuridine (BRDU) labeling unless otherwise indicated, where hepatocyte RDS was determined by either 5-ethynyl-2′-deoxyurideine (EDU) labeling or MKI67mRNA levels.

^b Human hepatocyte donors were all Caucasian subjects except for one donor (Hispanic subject) in the momfluorothrin study.

^c Statistically significant increase in RDS indicated by ↑; whereas ↓ indicates either a statistically significant decrease in RDS or where RDS could not be determined due to cytotoxicity. None represents no statistically significant change in RDS compared to control levels.

^d Positive controls for rodent hepatocytes were sodium phenobarbital (PB), epidermal growth factor (EGF), and/or hepatocyte growth factor (HGF).

^e Positive controls for human hepatocytes were epidermal growth factor (EGF) and/or hepatocyte growth factor (HGF).

^f Negative control for human hepatocytes was sodium phenobarbital (PB).

Figure 1. Effect of CAR activators in different animal models, namely wild-type mice, CAR KO or CAR KO/PXR KO mice, hCAR/hPXR mice and chimeric mice with human hepatocytes. Pathways which do not operate are shown in grey. Thus while Cyp2b enzyme induction and increased cell proliferation are observed in wild-type and hCAR/hPXR mice, these effects are abolished in CAR KO and CAR KO/PXR KO mice due to the absence of the receptor(s). In chimeric mice with human hepatocytes, the human receptors operate through human signaling pathways to produce an induction of CYP2B enzymes but not of cell proliferation.

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