Male rat-specific fatty change in liver by DS-1971a: Elevation in phospholipids and adenosine as early responses to the fatty change in male rat-derived primary hepatocytes

Abstract

In a 3-month repeated oral dose toxicity study of DS-1971a, a selective inhibitor of the Nav1.7 voltage-gated sodium channel, fatty change of hepatocytes was observed only in male rats at doses of 100 mg/kg and above. However, this change was not observed in female rats even at the highest dose of 1500 mg/kg. Furthermore, fatty change was not observed in mice and monkeys administered the highest dose of 1000 mg/kg for 6 and 9 months, respectively. To further investigate species differences of this fatty change, lipid accumulation was evaluated by staining with the LipidTOX dye in primary cultured hepatocytes derived from male and female rats, male monkeys, and male and female humans. After exposure to DS-1971a for 72 hr, the staining showed an increase in intensity specifically in male rat-derived hepatocytes in a concentration-dependent manner. Metabolomic analysis using rat-derived primary cultured hepatocytes exposed to DS-1971a for 24 and 72 hr revealed that phospholipids, not neutral lipids like triacylglycerols, and adenosine were elevated in the male-derived hepatocytes. These results suggest that the elevation of phospholipids and adenosine in the hepatocytes may contribute to the specific fatty change observed in male rats.

INTRODUCTION

The extrapolation of toxicity findings obtained in non-clinical toxicity studies using experimental animals to humans is critically important but difficult. It is generally performed by bridging between in vivo and in vitro, and between animals and humans (Mattes, 2020). First, the toxicity is recreated under in vitro conditions. If there is equivalency reflected by the phenotypic or biomarker change between in vivo and in vitro, then the change is confirmed using cultured human cells. Detection of the change in human cells indicates the high probability that there is a risk of the toxicity in humans. However, it is difficult to recreate in vivo toxicity findings under in vitro conditions due to the many limitations of in vitro models, such as a lack of complexity and culture technique.

Hepatic steatosis is characterized by the accumulation of lipids, primarily triacylglycerols, in hepatocytes. Excess and chronic accumulation of lipids in the liver leads to steatohepatitis and consequently hepatic failure. It has been reported that certain drugs, such as valproate, tetracycline, and tamoxifen, induce hepatic steatosis (Schumacher and Guo, 2015). Some in vitro models for detecting the potential risk of hepatic steatosis at early stages of drug development have been proposed (Donato and Gómez-Lechón, 2012). Mechanisms of hepatic steatosis are generally classified into four biological events: increases of fatty acid uptake and de novo lipogenesis, and decreases of fatty acid oxidation and lipid efflux (Gluchowski et al., 2017; Ipsen et al., 2018). Various molecular targets are involved in each of these events. Therefore, phenotypic analysis such as direct colorimetric or fluorescent staining of lipids or hydrophobic vacuoles, not functional analysis, is generally selected to quantitatively evaluate the level of lipid accumulation in cultured cells.

DS-1971a is a selective inhibitor of the Na_v1.7 voltage-gated sodium channel that is under development for the treatment of neuropathic pain (Shinozuka et al., 2020). In a repeated oral dose toxicity study of this compound for 3 months using rats, fatty change of hepatocytes was observed only in male rats at doses of 100 mg/kg and above, despite this change not being observed in female rats even at the highest dose of 1500 mg/kg. This change was also not observed in mice and monkeys administered the highest dose of 1000 mg/kg for 6 and 9 months, respectively. In this study, extrapolation of this male rat-specific fatty change in hepatocytes to humans was confirmed by bridging between in vivo and in vitro, and between rats and humans. Metabolomic analysis was also performed to elucidate the mechanisms behind the fatty change in vitro.

MATERIALS AND METHODS

Compound

DS-1971a (5-chloro-2-fluoro-4-{[(1S,2R)-2-(1-methyl-1H-pyrazol-5-yl)cyclohexyl]oxy}-N-(pyrimidin-4-yl)benzenesulfonamide) was synthesized at Daiichi Sankyo Co., Ltd. (Tokyo, Japan, Fig. 1). For in vivo studies, DS-1971a was suspended with 0.5 w/v% methylcellulose solution. For in vitro studies, DS-1971a was dissolved with dimethyl sulfoxide (DMSO).

Fig. 1

Chemical structure of DS-1971a.

Repeated dose toxicity studies in mice, rats, and monkeys treated orally with DS-1971a

To investigate the general toxicity profile of DS-1971a, repeated dose toxicity studies were conducted in mice, rats, and monkeys treated orally with DS-1971a. DS-1971a was orally administered once daily to mice for 6 months at 0, 30, 100, and 1000 mg/kg; to monkeys for 9 months at 0, 10, 100, and 1000 mg/kg; to rats for 14 days at 0, 300, 1000, and 1500 mg/kg; to rats for 3 months at 0, 10, 30, 100, 500, and 1500 mg/kg; and to rats for 6 months at 0, 10, 100, and 1000 mg/kg. Plasma concentrations of DS-1971a were determined in a time course manner. Cmax and AUC_0-24hr were calculated from the values of measured concentrations. Animals were euthanized on the day after the last dose, and livers were collected for histopathological examination.

Cells and serum samples

Primary cultured hepatocytes (PHs) and serum samples of rats and monkeys were isolated from F344/DuCrlCrlj rats and cynomolgus monkeys, purchased from Charles River Laboratories Japan, Inc. (Yokohama, Japan), and Hamri Co., Ltd. (Koga, Japan), or SNBL, Ltd. (Kagoshima, Japan), respectively. These procedures were approved by the Ethics Review Committee for Animal Experimentation of Daiichi Sankyo Co., Ltd., and conducted in compliance with the “Law Concerning the Protection and Control of Animals” (Japan Law No. 105, October 1, 1973, revised on June 22, 2005). Rat PHs were isolated as described previously (Fujimoto et al., 2010). Monkey PHs were isolated from remaining pieces of monkey liver samples used for toxicity studies by a two-step collagenase perfusion procedure. Human cryopreserved hepatocytes and human serum samples were purchased from Thermo Fisher Scientific (Waltham, MA, USA) and Biopredic International (Saint-Grégoire, France), respectively. Cells were cultivated as described previously (Fujimoto et al., 2010) with one modification, namely, a change from FBS to animal species- and sex-matched serum. All in vitro experiments were repeated three times.

In vitro steatosis assay

Hepatocytes were treated with DS-1971a dissolved with modified William’s E medium, the composition of which was described previously (Fujimoto et al., 2020), for 72 hr. Medium including DS-1971a was exchanged every day. Cytotoxicity was assessed using CellTiter-Glo Luminescent Cell Viability Assay (Promega, Tokyo, Japan), in accordance with the manufacturer’s instructions. For in vitro steatosis assay, cells were fixed with 3.3% neutral buffered formalin solution for 30 min at room temperature, and stained with 0.1% HCS LipidTOX Red Neutral Lipid Stain (Thermo Fisher Scientific) and 1.6 μM Hoechst33342 (Thermo Fisher Scientific) in D-PBS for 30 min. Lipid accumulation was measured with Cellomics ToxInsight Reader (Thermo Fisher Scientific) as the total intensity of the staining signal per cell.

Quantitative RT-PCR analysis of Cyp2C11/12 mRNA expression in rat primary hepatocytes

Cells were treated with 0.5% DMSO for 72 hr, after which cDNA was synthesized with FastLane Cell cDNA Kit (Qiagen, Hilden, Germany). Cyp2C11/12 and Actb mRNA expression was measured with TaqMan Fast Advanced MasterMix (Thermo Fisher Scientific) and TaqMan Gene Expression Assay (Thermo Fisher Scientific), having the following assay IDs: Cyp2C11, Rn01502203_m1; Cyp2C12, Rn00755856_m1; and Actb, Rn00667869_m1.

Metabolomic analysis for lipid profiling of rat primary hepatocytes

Metabolomic analyses were performed to clarify the lipid profiles of rat PHs treated with DS-1971a. The cell samples were collected after the exposure of rat PHs to DS-1971a for 24 and 72 hr. The samples were extracted using an automated MicroLab STAR system (Hamilton Company, UT, USA) in methanol, containing the recovery standards. The untargeted metabolic profiling platform employed for this assay was based on a combination of three independent platforms: ultra-high-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) optimized for basic species, UHPLC/MS/MS optimized for acidic species, and gas chromatography/mass spectrometry (GC/MS). The details of this platform, metabolite identification, and data analysis have been described previously (Evans et al., 2009; Ganti et al., 2012; Fujimoto et al., 2020).

Statistical analysis

Welch’s t-tests and two-way ANOVA (treatment and sex) were used to analyze the data. For metabolomic analyses, following normalization to protein, missing values, if any, were imputed with the observed minimum value for that particular compound. The statistical analyses were performed on natural log-transformed data.

RESULTS

Male rat-specific fatty change of hepatocytes in the repeated dose toxicity studies

The histopathological findings of livers in the repeated dose toxicity studies in mice, rats, and monkeys treated orally with DS-1971a are shown in Table 1. No histopathological findings were observed in the livers of rats upon administration for 14 days. However, midzonal fatty change of hepatocytes, accompanied by increased microgranuloma as a change secondary to the fatty change of hepatocytes, was observed only in male rats upon administration for 3 months and 6 months at 100 mg/kg or more (Fig. 2). In addition, increases in neutral lipids such as triacylglycerols (TAGs), diacylglycerols (DAGs), and cholesterol esters (CEs) were observed in the livers of the male rats (data not shown). This fatty change was not observed in mice, monkeys, and female rats treated with DS-1971a even at the highest doses, 1000 or 1500 mg/kg, for 6 or 9 months. No toxicological changes in blood chemistry were observed in these studies (data not shown). Systemic exposure levels of DS-1971a at lowest observed adverse effect level (LOAEL) for fatty change of hepatocytes on last dosing of the toxicity studies in male rats were comparable or lower than those at no observed adverse effect level (NOAEL) on last dosing of the toxicity studies in mice, monkeys, and female rats (Table 2).

Table 1. Histopathological findings of liver in repeated oral dose toxicity studies using mice, monkeys, and rats.

Animal species	Dosing period	Dose (mg/kg)	Male			Female
			Histopathological finding	NOAEL (mg/kg)		Histopathological finding	NOAEL (mg/kg)
Mouse	6 months	0, 30, 100, 1000	None	1000		None	1000
Monkey	9 months	0, 10, 100, 1000	None	1000		None	1000
Rat	14 days	0, 300, 1000, 1500	None	1500		None	1500
	3 months	0, 10, 30, 100, 500, 1500	Fatty change of hepatocytes	30		None	1500
	6 months	0, 10, 100, 1000	Fatty change of hepatocytes	10		None	1000

NOAEL: no observed adverse effect level

Fig. 2

Fatty change in hepatocytes in male rats dosed with DS-1971a for 3 months. A, Vehicle control; B, 100 mg/kg dosing; C, 1500 mg/kg dosing.

Table 2. Systemic exposure levels of DS-1971a at NOAEL or LOAEL for fatty change of hepatocytes on last dosing of the toxicity studies using mice, monkeys, and rats.

Animal species	Dosing period	Male			Female
		NOAEL/LOAEL (mg/kg)	Cmax (ng/mL) AUC0-24hr (ng*hr/mL)		NOAEL/LOAEL (mg/kg)	Cmax (ng/mL) AUC0-24hr (ng*hr/mL)
Mouse	6 months	NOAEL: 1000	4,400 30,700		NOAEL: 1000	49,100 654,000
Monkey	9 months	NOAEL: 1000	13,100 68,200		NOAEL: 1000	10,500 46,700
Rat	14 days	NOAEL: 1500	14,500 106,000		NOAEL: 1500	13,500 71,300
	3 months	LOAEL: 100	2,890 14,600		NOAEL: 1500	12,900 110,000
	6 months	LOAEL: 100	6,220 29,000		NOAEL: 1000	13,000 109,000

NOAEL: no observed adverse effect level. LOAEL: lowest observed adverse effect level

Male rat-specific accumulation of lipids in primary hepatocytes

Primary hepatocytes from rats, monkeys, and humans were cultivated in medium containing species- and sex-matched serum. To confirm that sex-specific characters of the hepatocytes were maintained under these culture conditions, mRNA expression of male-specific cytochrome P450 Cyp2c11 and female-specific cytochrome P450 Cyp2c12 in the rat PHs was evaluated by TaqMan-qPCR. The results showed that Cyp2c11 and Cyp2c12 mRNAs were expressed predominantly in the PHs from male and female rats, respectively (Table 3). Under these conditions, PHs from rats, monkeys, and humans were exposed to DS-1971a for 72 hr, after which lipid accumulation levels were evaluated with HCS LipidTOX Red Neutral Lipid Stain. The results showed that the lipid accumulation level was increased in the PHs only from male rats, in a concentration-dependent manner (Fig. 3), despite there being no statistically significant difference between the hepatocytes from male and female rats, maybe due to limited sample numbers. No clear cytotoxicity, reflected by a change in cellular ATP content, was observed in the PHs from rats, monkeys, and humans exposed to DS-1971a at up to 1000 μM (Fig. 3).

Table 3. Cyp2c11 and Cyp2c12 mRNA expression in rat primary cultured hepatocytes isolated from male and female rats.

	Male	Female
Cyp2c11	1.15 ± 0.29	N.D.
Cyp2c12	0.11 ± 0.15	0.75 ± 0.80

The expression levels were normalized by the expression level of Actb mRNA.

N.D.: not determined

Fig. 3

Lipid accumulation and cell viability of the PHs exposed to DS-1971a for 72 hr. A. Lipid accumulation levels were reflected by the fluorescent signal intensity of staining with HCS LipidTOX Red Neutral Lipid Stain, B. Cell viability was reflected by ATP content assessed with CellTiter-Glo Luminescent Cell Viability Assay. Black bar, male rat; white bar, female rat; gray bar, male monkey; striped bar, male human; dotted bar, female human. N = 3 for each group.

Metabolomic analyses for lipid profiling of rat primary hepatocytes

Overall lipid profiles in the rat PHs exposed to DS-1971a for 24 and 72 hr are shown in Fig. 4. Lysophosphatidylcholines (LPCs) and lysophosphatidylethanolamines (LPEs) were more markedly elevated in the male-derived hepatocytes at a lower concentration and an earlier timepoint than in the female-derived hepatocytes. Individual lysophospholipid species were also observed to be increased in the same manner (Fig. 5). Phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), and their individual species were also more markedly elevated in the male-derived hepatocytes than in the female ones (Fig. 6). Meanwhile, phosphatidylinositols (PIs) were observed to be increased in the female-derived hepatocytes. However, no clear change was observed in their individual species (data not shown). Surprisingly, no changes in neutral lipids such as TAGs, DAGs, and CEs were observed in both the male- and female-derived hepatocytes, in contrast to the results of rat repeated dose toxicity studies. Regarding metabolites apart from lipids, adenosine was strongly elevated in the male-derived hepatocytes (Fig. 7).

Fig. 4

Overall lipid profiles in the rat-derived PHs exposed to DS-1971a for 24 and 72 hr. Fold changes relative to the control are expressed by a heat map. Red cells represent increases (fold change of ≥ 2.0) and blue cells represent decreases (fold change of < 0.5).

Fig. 5

Changes in LPC and LPE species in the rat-derived PHs exposed to DS-1971a for 24 and 72 hr. Fold changes relative to the control are expressed by a heat map. Red cells represent increases (fold change of ≥2.0) and blue cells represent decreases (fold change of < 0.5).

Fig. 6

Changes in LPC and LPE species in the rat-derived PHs exposed to DS-1971a for 24 and 72 hr. Fold changes relative to the control are expressed by a heat map. Red cells represent increases (fold change of ≥ 2.0) and blue cells represent decreases (fold change < 0.5).

Fig. 7

Changes in adenosine in the rat-derived PHs exposed to DS-1971a for 24 and 72 hr. Exposure duration, A, 24 hr; B, 72 hr; *, p < 0.05; **, p < 0.01. Black bar, male-derived PHs; white bar, female-derived PHs

DISCUSSION

The extrapolation of toxicity findings obtained in non-clinical toxicity studies using experimental animals to humans is critically important but difficult. In a repeated oral dose toxicity study of DS-1971a for 3 months using rats, the fatty change of hepatocytes was observed only in males at 100 mg/kg or more, despite this change not being observed in females at the highest dose, 1500 mg/kg. This change was also not observed in mice and monkeys treated orally with DS-1971a at the highest dose, 1000 mg/kg, for 6 and 9 months, respectively. The toxicokinetic data suggested that this sex and species difference is not due to differences in the systemic exposure to DS-1971a. Additionally, male rat-specific metabolites were not identified (data not shown), so that the fatty change of hepatocytes was suggested not to be induced by sex and species difference in drug metabolism. To extrapolate this fatty change of hepatocytes observed in the male rats to humans, lipid accumulation was evaluated in rat-, monkey- and human-derived PHs exposed to DS-1971a using HCS LipidTOX Red Neutral Lipid Stain. The results showed an increase in the level of lipid accumulation reflected by the signal intensity of HCS LipidTOX Red Neutral Lipid Stain in the PHs derived only from male rats upon exposure to DS-1971a only for 72 hr, despite the fatty change of hepatocytes not being observed even in male rats treated orally with DS-1971a for 14 days at 1500 mg/kg. This means that this fatty change was expressed earlier under in vitro conditions than under in vivo ones. In addition, no increase in the level of lipid accumulation was observed in the human-derived PHs exposed to DS-1971a for 72 hr. It was thus speculated that the fatty change of hepatocytes might not develop in humans. However, the equivalence of the observed phenotype between male rat livers and male rat-derived PHs is unclear since there is a gap in the onset of this fatty change between them. To clarify this equivalence, metabolomic analysis using rat PHs exposed to DS-1971a for 24 or 72 hr was performed to understand the lipid profile of the cells. Contrary to expectations, the accumulation of phospholipids, but not TAGs, DAGs, and CEs which were increased in the livers of the male rats administered with DS-1971a, was observed in male rat-derived PHs exposed to DS-1971a. Is there an association between the accumulations of phospholipids in male rat-derived PH and neutral lipids in livers of male rats treated with DS-1971a?

Phospholipids, including PCs and PEs, are critical components of lipid storage, lipid packages (e.g., in VLDL), and membrane lipids (Ben M’barek et al., 2017). Accordingly, some reports on the association between PCs and hepatic steatosis have been published. PCs can be synthesized via two pathways: a reaction between DAGs and CDP-choline and a conversion from PEs via phosphatidylethanolamine N-methyltransferase (Shields et al., 2001; Fagone and Jackowski, 2013). Conversely, PCs are a quantitatively important source for the synthesis of DAGs (Fagone and Jackowski, 2013). A decrease in PCs, such as by feeding on a choline-deficient diet, reduces the release of lipid droplets from the liver, leading to hepatic steatosis (Yao and Vance, 1988, 1990; Wan et al., 2019). Meanwhile, an increase in PCs also leads to hepatic steatosis by the catabolism of PCs to DAGs (Martínez-Uña et al., 2013). It has also been reported that the PC/PE ratio is important for regulating the release of lipid droplets, and that a decrease in this ratio induces hepatic steatosis (Li et al., 2006; Arendt et al., 2013). In the present study, an elevation in PCs and a decrease in the PC/PE ratio (Fig. 8) were observed in the male rat-derived PHs, despite no increase in DAGs. These changes may be early responses in the male rat-derived PHs, which lead to the hepatic fatty change observed in male rats treated orally with DS-1971a for 3 months. Meanwhile, the increase in lysophospholipids, which is speculated to be due to the increases in PC and PE, may influence the localization of lipid droplets. It has been reported that lysophospholipid-containing lipid droplets with positive curvatures separate from the endoplasmic reticulum and localize in the cytoplasm (Ben M’barek et al., 2017). This localization of lipid droplets may result in lipid accumulation reflected by an elevation in the signal intensity of HCS LipidTOX Red Neutral Lipid Stain, despite no observation of TAG elevation. Considering this reported information, it is speculated that there would be an association between the accumulations of phospholipids in male rat-derived PH and neutral lipids in livers of male rats treated with DS-1971a.

Fig. 8

PC/PE ratios in the rat-derived PHs exposed to DS-1971a for 24 hr. Fold changes relative to the control are expressed by a heat map. Red cells represent increases (fold change of ≥ 2.0) and blue cells represent decreases (fold change of < 0.5).

In addition to the altered lipid profile, remarkable elevation in adenosine was observed only in male rat-derived PHs exposed to DS-1971a. It has been reported that inhibition of the production or signal transduction of adenosine suppresses ethanol-induced hepatic fatty change (Peng et al., 2009), and that a deficiency in adenosine metabolism by disruption of the adenosine kinase (Adk) gene leads to the development of severe hepatic steatosis in neonatal mice (Boison et al., 2002). However, conversely, hepatocyte-specific ADK overexpression was associated with more severe hepatic steatosis in mature mice (Li et al., 2023). Interestingly, LPCs were significantly increased in mice with the hepatocyte-specific overexpression of ADK. These findings suggest that there may be a causal relationship between the elevations in phospholipids and adenosine.

This study demonstrated the phenotypic equivalence of hepatic fatty change between in vivo and in vitro models. However, the lipid profiles exhibited qualitative differences between them. The findings suggest that the elevation of phospholipids and adenosine may represent early responses to the hepatic fatty change, as indicated by the metabolomic analysis. However, it is unclear why phospholipids and adenosine were elevated in male rat-derived PH exposed to DS-1971a and this analysis did not fully cover the underlying reason for the manifestation of hepatic fatty change specifically in male rats. Nonetheless, the observed correlation between these early responses and the hepatic fatty change is supported by male-specific changes observed in both in vivo and in vitro systems, as well as in the literature. A deeper biological understanding of species and sex differences in toxicity changes may contribute to accurate extrapolation of these findings to humans.

ACKNOWLEDGMENTS

The authors would like to express their grateful appreciation to Toshiyuki Watanabe for his encouragement and thoughtful support. We also thank Yoshikazu Nezu, Yoshiko Ohshima, Yukari Shibaya, Mayumi Goto, and Shingo Arakawa for their research support.

Conflict of interest

The authors are employees of Daiichi Sankyo Co., Ltd. All research was funded by Daiichi Sankyo Co., Ltd. with no external funding.

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