Ether-phosphatidylcholine characterized by consolidated plasma and liver lipidomics is a predictive biomarker for valproic acid-induced hepatic steatosis

Abstract

Valproic acid (VPA) is known to induce hepatic steatosis due to mitochondrial toxicity in rodents and humans. In the present study, we administered VPA to SD rats for 3 or 14 days at 250 and 500 mg/kg and then performed lipidomics analysis to reveal VPA-induced alteration of the hepatic lipid profile and its association with the plasma lipid profile. VPA induced hepatic steatosis at the high dose level without any degenerative changes in the liver on day 4 (after 3 days dosing) and at the low dose level on day 15 (after 14 days dosing). We compared the plasma and hepatic lipid profiles obtained on day 4 between the VPA-treated and control rats using a multivariate analysis to determine differences between the two groups. In total, 36 species of plasma lipids and 24 species of hepatic lipids were identified as altered in the VPA-treated group. Of these lipid species, ether-phosphatidylcholines (ePCs), including PC(16:0e/22:4) and PC(16:0e/22:6), were decreased in both the plasma and liver from the low dose level on day 4, however, neither an increase in hepatic TG level nor histopathological hepatic steatosis was observed at either dose level on day 4. Hepatic mRNA levels of glycerone-phosphate O-acyltransferase (Gnpat), which is a key enzyme for biosynthesis of ePC, was also decreased by treatment with VPA along with the decrease in ePCs. In conclusion, the changes in ePCs, (PC[16:0e/22:4] and PC[16:0e/22:6]), have potential utility as predictive biomarkers for VPA-induced hepatic steatosis.

INTRODUCTION

Valproic acid (VPA) has been widely used as a first-line therapy in the treatment of epilepsy for more than 30 years (Simon and Penry, 1975; Chapman et al., 1982). More recently, some studies have found that VPA is a potential anti-cancer agent, due to its anti-histone deacetylase activity (Chavez-Blanco et al., 2005; Goodyear et al., 2010). However, VPA is known to cause hepatic steatosis frequently and some cases can advance to non-alcoholic steatohepatitis (NASH) or severe hepatitis in humans (Abdel-Dayem et al., 2014; Lewis et al., 1982; Sato et al., 2005). VPA is known to affect the function of mitochondria by various mechanisms including depletion of acetyl coenzyme A (CoA), downregulation of fatty acid beta-oxidation, opening of the mitochondrial permeability transition (MPT) pores and inhibition of carnitine palmitoyl transferase (CPT) 1 (Labbe et al., 2008). In animals, especially in rodents, there are many reports that VPA causes hepatic steatosis, which is considered to be due to the mitochondrial dysfunction induced by VPA and its metabolites (Zhang et al., 2013; Kesterson et al., 1984; Sobaniec-Lotowska, 1997).

Lipids, a class of biological molecules, consist of several categories, which include phosphoglycerolipids, sphingolipids and neutral lipids. These three categories of lipids are components of cellular membranes and blood-circulating lipoproteins. Consequently, their levels in the blood and organs, especially in the liver, are thought to be closely correlated (Kotronen et al., 2010). In addition, these lipids also play a pivotal role in various biological processes including cellular proliferation, apoptosis and inflammation (Mené et al., 1989; Hannun and Linardic, 1993; Blumberg et al., 1995; Pettus et al., 2004). Therefore, blood lipid profiling would be an effective approach to screen and identify biomarkers not only for diseases but also drug toxicity. From these points of view, lipidomics, a mass spectrometry-based approach for the simultaneous assay of lipid molecules in biological samples including blood and organs (Han and Gross, 2003; Houjou et al., 2005) has been also used to identify drug toxicity biomarkers in the blood. For example, lysophosphatidylcholines (LPC), including LPC (20:4), in the serum have been shown to be biomarkers for acetaminophen- and D-galactosamine-induced liver injuries (Cheng et al., 2009; Ma et al., 2014). Furthermore, bis-monophosphatidic acid (BMP) in the serum and glycosylceramide (GCer) and LPC in the plasma were shown to be markers for drug-induced hepatic phospholipidosis (PLD) (Mortuza et al., 2003; Saito et al., 2014). Recently, we reported that hepatic steatosis (fatty change) detected in non-clinical toxicity studies of rodents can be regarded as a critical finding for the estimation of the potential risk to induce drug-induced liver injury (DILI) in humans when the steatosis is induced by mitochondrial dysfunction based on the results of in vitro and in vivo studies including lipidomics analyses (Goda et al., 2017).

However, more lipidomics data are necessary to exclude the possibility that alteration of a biomarker is a consequence of the effects of toxicants/drugs on organs other than the liver. For example, our previous study demonstrated that chronic ocular disease alters several plasma lipid levels (Saito et al., 2016). Especially for drug-induced hepatic steatosis, data correlating serum/plasma-liver lipid levels are limited, although there is an association between the static lipid levels in serum/plasma and in liver (Kotronen et al., 2010).

In the present study, we conducted lipidomics in VPA-treated rats to investigate the VPA-induced alteration of lipids in the liver and its association with mitochondrial toxicity and also alteration of plasma lipids to establish possible predictive biomarkers of the VPA-induced hepatic steatosis.

MATERIALS AND METHODS

Animals

Five-week-old male Crl:CD (SD) rats were purchased from Charles River Japan Inc. (Kanagawa, Japan). The animals were housed individually in wire-mesh cages situated in an air-conditioned room on a 12-hr light-dark cycle (lighting from 7:00 a.m. to 7:00 p.m.) at a temperature of 23 ± 1°C, relative humidity of 55 ± 5%, and a ventilation rate of about 15 times per hour. The rats were quarantined for 1 week and were allowed free access to a commercial pellet diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water ad libitum. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of the Toxicology Research Laboratories, Central Pharmaceutical Research Institute, Japan Tobacco Inc. This study was conducted in accordance with the Japanese Law for the Humane Treatment and Management of Animals (Law No. 105, as revised in 2013, issued in October 1, 1973).

Dosing of VPA

VPA was purchased from Sigma-Aldrich Co. LLC. (St. Louis, MO, USA) and was dissolved in a physiological saline (Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan). VPA solution as well as a control aqueous solution were administrated intraperitoneally once daily for 3 or 14 days with dose volume at 5.0 mL/kg. The dose levels of VPA were set at 250 and 500 mg/kg and 8 animals were assigned to each sampling point at each dose level (sixteen animals per dose level).

Observation of clinical signs and measurements of body weights and food consumption

Animals were observed carefully for any clinical signs twice daily (once before and once immediately after dosing) during the dosing period. Body weights were measured on the day of necropsy after overnight fasting and were shown as the final body weights. Food consumption per animal was calculated between days 2 and 3 or days10 and 14 for 3 or 14 days dosing, respectively.

Sampling of plasma and liver sample

The rats were fasted overnight on the last day of the dosing period (on days 3 and 14), and blood and liver samples were collected the next day (on days 4 and 15). The abdomens of the rats were opened under isoflurane anesthesia and blood samples were collected from the abdominal aorta. The blood samples for the lipidomics were transferred into tubes containing EDTA-2K as an anticoagulant and were centrifuged at 1,750 g for 30 min at room temperature to obtain plasma. The blood samples for the measurements of liver function parameters were transferred into tubes containing heparin as an anticoagulant and were centrifuged at 1,750 g for 30 min at 4°C to obtain plasma.

The livers were removed and weighed, and aliquots of the samples were frozen with liquid nitrogen and stored at –80°C until use. The remaining samples were preserved in neutral buffered formalin for histopathological examination.

Measurements of liver function parameters in plasma

The measurements of the plasma liver function parameters (aspartate aminotransferase (AST) and alanine aminotransferase (ALT)) were conducted by an automated analyzer (TBA-120FR, TOSHIBA Corporation, Tokyo, Japan) using standard reagents for clinical chemistry by the UV kinetic method (Wako Pure Chemicals, Tokyo, Japan).

Measurements of hepatic triglycerides (TG) levels

The liver samples were weighted in 2.0 mL plastic tubes. Methanol was added to the samples at a volume of 1.0 mL per 150 mg wet tissue weight. The mixtures were homogenized with a mixer-mill disruptor (TissueLyser, QIAGEN, Hilden, Germany) at 25 Hz for 2 × 2 min using a zirconia ball (YTZ ball, φ 5 mm, NIKKATO Corporation, Osaka, Japan). After the mixtures were homogenized, 460 μL of the homogenates and 800 μL of chloroform were mixed and stirred sufficiently by a vortex mixer to extract the lipids. The mixtures were centrifuged at 1,000 g for 5 min to obtain the supernatants and a 0.5% sodium chloride aqueous solution, 120 μL, was added to 600 μL of the supernatants and stirred sufficiently by a vortex mixer. The mixtures were centrifuged twice under the same conditions. The lower phase (chloroform phase) was collected and evaporated to dryness with a centrifugal thickener (EZ-2 PLUS, Genevac, Warminster, PA, USA). After evaporation, the residues were dissolved in 200 μL of isopropanol. A 10 μL aliquot of the sample solution was mixed with 90 μL of a 4% bovine serum albumin aqueous solution and stirred sufficiently by a vortex mixer. The measurements of TG, T-CH and PL levels were conducted by an automated analyzer (TBA-120FR, TOSHIBA Corporation) using standard reagents by the glycerol-3-phosphate oxidase-HMMPS, glycerol blanking method (Wako Chemicals).

Microscopic examination

The liver was prepared for histopathological examination by embedding in paraffin wax, sectioning and staining with hematoxylin and eosin (HE).

Lipidomics

Plasma lipids were extracted by mixing 20 µL of plasma with 180 µL of methanol:isopropanol (1:1) containing 2 µM phosphatidylcholine (PC[12:0/12:0]; Avanti Polar Lipids, Alabaster, AL, USA) as an internal standard. For liver lipid extraction, a homogenate, 20 mg/mL, was prepared with methanol. Liver lipids were extracted by mixing 100 µL of the liver homogenate with 100 µL of isopropanol containing 2 µM PC(12:0/12:0) as an internal standard. After mixing, the extraction mixture was filtered through a FastRemover for Protein (0.20 µm) 96-well (GL Science, Tokyo, Japan) using Microlab NIMBUS (Hamilton Robotics, Reno, NV, USA) to remove debris. The filtered extracts were collected and stored at a temperature of –80°C until use.

Measurements of the lipid content were performed by LC/MS measurement as previously described (Saito et al., 2017). For lipid ion quantification, the raw full MS data obtained by LC/MS were processed using TraceFinder software 3.3 (Thermo Fisher Scientific, Waltham, MA, USA), which quantifies ion peaks of each biomolecule obtained at a specified m/z with the column retention time (RT). We selected 30,000 (negative ion mode) or 100,000 (positive ion mode) as the quantitative threshold of the ion peak area and the quantified ion peak area less than the threshold was substituted to 30,000 (negative ion mode) or 100,000 (positive ion mode) for statistical analysis. The quantified ion peak area of each biomolecule was normalized to that of the internal standard (PC[12:0/12:0]) and was also adjusted to the median of all samples as 1. The processed lipidomics data are shown in Supplementary Table 1 for plasma and Table 2 for liver. The values of the relative standard deviation of the internal standard (PC[12:0/12:0]) in negative and positive ion modes were 3.42% and 7.35% for the plasma, respectively, and were 4.15% and 6.77% for the liver, respectively, which were monitored for experimental quality control throughout the extraction, measurement, and data processing.

The numbers shown after lipid class represents the total number of carbon atoms and double bonds in the side chain. If more than 2 lipid molecules were identified as isomers (same annotation of class, carbon length, and number of double bond), each lipid molecule is shown with alphabet after class to distinguish each other.

OPLS-DA and lipid identification

The plasma and liver lipidomics data of the control and VPA-treated rats were loaded into SIMCA-P+ 14 (Umetrics, Umea, Sweden), pareto-scaled, and analyzed using OPLS-DA to extract the lipids that contributed to the discrimination between the control and VPA-treated samples. To sort these lipids, |p (corr)| > 0.7 in the loading s-plot of the OPLS-DA score, representing the magnitude of reliability, were selected as cut-off values. Subsequently, the lipids identified as discriminant factors were subjected to lipid molecule identification as previously described (Ishikawa et al., 2014, 2016).

Measurements of hepatic mRNA levels

Tissue sample aliquots were homogenized with a TissueLyser (QIAGEN) and total RNA was extracted using a RNeasy Mini kit (QIAGEN) according to the manufacturer’s instructions. A 2.0 μg aliquot of the isolated total RNA was used to synthesize cDNA with SuperScript VILO Mastermix (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The synthesized cDNA solutions were diluted 5-fold with Tris-EDTA (TE) buffer (pH8.0, NIPPON GENE CO., LTD., Tokyo, Japan). The cDNA solutions were further diluted 10-fold with MILLI-Q water (Millipore Corporation, Darmstadt, Germany) and used for TaqMan probe-based semi quantitative-real time PCR. The mRNA levels were measured in duplicate on a 7300 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) using TaqMan Gene Expression Master Mix (Applied Biosystems) according to the manufacturer’s instructions. The data analysis was performed by a calibration curve method using SDS software (Applied Biosystems) and the results were normalized to actin, beta (Actb) expression.

The hepatic mRNA levels of three enzymes related to the ether-linkage of lipids, fatty acyl-CoA reductase 1 (Far1), alkyl-DHAP synthase (Agps) and glycerone-phosphate O-acyltransferase (Gnpat) were measured. The following primer and TaqMan probe mixtures were obtained from Applied Biosystems: Agps (Rn00584341_m1), Far1 (Rn01506880_m1) and Gnpat (Rn00584655_m1), Actb (Rn00667869_m1).

Statistical analysis

All numerical data are shown as mean ± or + standard deviation. The statistical differences in the data were determined using one-way analysis of variance (ANOVA), followed by pairwise comparisons (Dunnett’s test). The levels of significance were set at 5% and 1% (two-tailed).

RESULTS

Clinical signs, food consumption, body weights, liver weights, hepatic TG level and histopathology of the liver

A decrease in locomotor activity and ataxic gait were observed immediately after dosing in all the animals at 500 mg/kg in observations of the clinical signs throughout the dosing period. Body weights at necropsy (final body weights) on day 15 (after 14 days dosing) at 500 mg/kg were much lower than those in the control group (p < 0.01) (Table 1). Food consumption was decreased on days 4 and 15 (after 3 and 14 days dosing, respectively) at 500 mg/kg (p < 0.01) (Table 1). VPA treatment at 500 mg/kg for 14 days was considered to be greater than the maximum tolerated dose (MTD) due to deterioration in the animals’ physical conditions. There were no treatment-related changes in body weights or food consumption at 250 mg/kg (Table 1). Relative liver weights did not change at either dose level at either time point. Hepatic TG levels were increased at 500 mg/kg on day 4 (p < 0.01) and at 250 mg/kg on day 15 (p < 0.01) (Table 1). Liver function parameters (AST and ALT) in plasma were not increased at either dose level at either sampling points (Table 1). In the histopathology, fatty change in the hepatocytes in the periportal area of the liver was induced by treatment with VPA at 500 mg/kg on day 4 and at 250 mg/kg on day 15 and this finding correlated with the increase in hepatic TG levels (Fig. 1). This fatty change was considered to be microvesiclular steatosis.

Table 1. Comparison of the parameters in the control and VPA-treated rats.

	Sampling pointa	Control			VPA 250 mg/kg			VPA 500 mg/kg
Final Body Weight	4 days	186.95	±	6.33	186.93	±	11.45	185.70	±	13.22
(g)	15 days	277.08	±	16.91	260.95	±	12.13	235.73	±	12.48**
Food Consumption	2-3 days	24.58	±	1.61	22.89	±	2.34	19.92	±	2.03**
(g/day)	10-14 days	27.05	±	1.64	25.85	±	2.85	22.74	±	1.36**
Relative Liver Weight	4 days	3.216	±	0.110	3.324	±	0.154	3.284	±	0.168
(g/100 g body weight)	15 days	2.984	±	0.164	3.016	±	0.109	2.945	±	0.079
Hepatic TG level	4 days	5.55	±	2.26	6.70	±	2.06	10.85	±	3.45**
(mg/g Liver)	15 days	5.58	±	2.44	10.78	±	4.64**	4.63	±	1.38
AST	4 days	86.8	±	8.2	86.4	±	11.9	74.1	±	4.9*
(IU/dL)	15 days	76.1	±	6.5	73.5	±	5.0	66.5	±	4.0*
ALT	4 days	25.6	±	3.2	22.6	±	3.0	17.4	±	1.8*
(IU/dL)	15 days	24.0	±	2.8	22.0	±	2.8	16.9	±	2.1*

a: Day 4 and 15 mean the day after 3 and 14 days of dosing, respectively.

Eight animals were assigned to each sampling point at each dose level (sixteen animals per dose level).

VPA: valproic acid, TG: triacylglycerol, AST: aspartate aminotransferase, ALT: alanine aminotransferase.

Significantly different from control (Dunnett test): * P < 0.05, ** P < 0.01.

Fig. 1

Fatty change of the hepatocytes in the periportal area induced by treatment with VPA. A: control rats on day 4, B: VPA-treated rats (250 mg/kg) on day 4, C: VPA-treated rats (500 mg/kg) on day 4, D: control rats on day 15, E: VPA-treated rats (250 mg/kg) on day 15. Scale bar = 50 μm, P: portal area. Representative findings for fatty change of hepatocytes are shown.

Lipidomics for the plasma and liver

To screen for biomarkers for the VPA-induced hepatic steatosis, lipidomics analysis was conducted using the plasma data of rats treated with the vehicle or VPA for 3 days at 500 mg/kg. The lipidomics analysis provided 784 lipids from the plasma. The plasma dataset was loaded into OPLS-DA and the loading s-plot score was calculated to identify the characteristic lipid alterations. The control and VPA-treated rats were clearly discriminated by the OPLS-DA plot of the plasma lipidomics data (Fig. 2A). By screening with |p (corr)| > 0.7 as threshold values in the s-plot, 36 lipids were obtained from the plasma dataset (Table 2). These lipids were mainly ether-phospholipids, including ether-lysophosphatidylcholine (eLPC), ether-phosphatidylcholine (ePC) and ether-phosphatidylethanolamine (ePE), and all of them were decreased by treatment with VPA. To associate alterations in the lipids in plasma with those in the liver, we also performed lipidomics analysis using the data from livers obtained from the same rats as used in the lipidomics analysis for the plasma. The lipidomics analysis provided 748 lipids from the liver. The liver dataset was loaded into OPLS-DA and the loading s-plot score was calculated. The control and VPA-treated rats were clearly discriminated by the OPLS-DA plot of the liver lipidomics data (Fig. 2B). By screening with |p (corr)| > 0.7 as threshold values in the s-plot, 24 lipids were obtained from the liver dataset (Table 3). Seventeen lipids, including cardiolipin (CL), lysophosphatidylcholine, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine and carnitine, were increased by treatment with VPA, while 7 lipids, including phosphatidylcholine (PC), ePC and ePE, were decreased by treatment with VPA.

Fig. 2

OPLS-DA and s-plot of lipidomics data from plasma (A) and liver (B). OPLS-DA score plot (upper) and loading s-plot (lower) using obtained data by LC/MS. Black and White dots in the upper panels represent individual animals of control and VPA-treated rats. White dots in the lower panels represent each lipid. The thresholds are denoted by dotted red lines. Red is for p(corr).

Table 2. Characterized plasma lipids altered by VPA-treatment at 500 mg/kg on day 4 in OPLS-DA.

Class	Name	RT	Response by VPA
eLPC	LPC(16:0e)	6.616	Decreased
eLPC	LPC(18:0e)	8.311	Decreased
eLPC	LPC(18:1e)a	6.864	Decreased
ePC	PC(32:0e)	17.968	Decreased
ePC	PC(36:1e)	19.629	Decreased
ePC	PC(36:2e)a	18.2	Decreased
ePC	PC(36:2e)b	18.507	Decreased
ePC	PC(36:3e)b	16.93	Decreased
ePC	PC(36:3e)c	17.177	Decreased
ePC	PC(38:2e)a	19.906	Decreased
ePC	PC(38:3e)	18.838	Decreased
ePC	PC(38:4e)a	17.518	Decreased
ePC	PC(38:6e)b	15.33	Decreased
ePC	PC(40:4e)b	19.235	Decreased
ePC	PC(40:5e)a	17.618	Decreased
ePC	PC(40:6e)c	17.555	Decreased
ePC	PC(40:7e)a	15.449	Decreased
ePC	PC(42:6e)b	19.246	Decreased
ePE	PE(36:2e)a	18.921	Decreased
ePE	PE(38:2e)a	20.227	Decreased
ePE	PE(38:4e)a	18.004	Decreased
ePE	PE(38:4e)b	18.56	Decreased
ePE	PE(38:5e)a	16.602	Decreased
ePE	PE(40:4e)a	19.566	Decreased
ePE	PE(40:4e)b	19.963	Decreased
ePE	PE(40:5e)b	18.596	Decreased
ePE	PE(40:6e)b	18.006	Decreased
ePE	PE(40:7e)a	15.957	Decreased
ePE	PE(42:4e)a	20.67	Decreased
ePE	PE(42:5e)a	19.972	Decreased
ePE	PE(42:6e)a	19.591	Decreased
PI	PI(32:0)	14.812	Decreased
SM	SM(d36:0)	17.453	Decreased
SM+O	SM(d42:0)+O	20.616	Decreased
SM+O	SM(d42:1)+O	19.348	Decreased
SM+O	SM(d42:2)+O	17.937	Decreased

eLPC; ether-lysophosphatidylcholine, ePC; ether-phosphatidylcholine, ePEe; ether-phosphatidylethanolamine, PI; phosphatidylinositol, SM; sphingomyelin, SM+O; oxidized-sphingomyelin. The numbers shown after lipid class represents the total number of carbon atoms and double bonds in the side chain. If more than 2 lipid molecules were identified as isomers (same annotation of class, carbon length, and number of double bond), each lipid molecule is shown with alphabet after class to distinguish each other. RT: retention time (min).

Table 3. Characterized liver lipids altered by VPA-treatment at 500 mg/kg on day 4 in OPLS-DA.

Class	Name	RT	Response by VPA
Car	Car(10:0)	3.539	Increased
CL	CL(70:5)	23.082	Increased
CL	CL(70:6)	22.71	Increased
CL	CL(71:5)	23.236	Increased
CL	CL(71:6)	22.9115	Increased
CL	CL(72:5)	23.401	Increased
CL	CL(72:6)	23.0825	Increased
CL	CL(72:7)	22.742	Increased
CL	CL(73:6)	23.265	Increased
LPC	LPC(17:0)	6.7065	Increased
LPC	LPC(19:0)a	8.0895	Increased
PC	PC(34:2)	15.154	Increased
PC	PC(35:2)b	16.2725	Increased
PC	PC(36:6)c	12.3905	Decreased
PC	PC(38:7)c	13.112	Decreased
PC	PC(39:4)a	17.6545	Increased
ePC	PC(36:4e)	16.1	Decreased
ePC	PC(38:4e)a	17.6375	Decreased
ePC	PC(38:6e)b	15.418	Decreased
ePC	PC(40:5e)b	18.268	Decreased
ePE	PE(42:5e)a	20.1	Decreased
PG	PG(36:2)b	16.187	Increased
PI	PI(34:2)b	13.5305	Increased
PS	PS(41:6)a	15.636	Increased

Car; acylcarnitine, CL; cardiolipin, LPC; lysophosphatidylcholine, PC; phosphatidylcholine, ePC; ether-phosphatidylcholine, ePE; ether-phosphatidylethanolamine, PG; phosphatidylglycerol, PI; phosphatidylinositol, PS; phosphatidylserine. The numbers shown after lipid class represents the total number of carbon atoms and double bonds in the side chain. If more than 2 lipid molecules were identified as isomers (same annotation of class, carbon length, and number of double bond), each lipid molecule is shown with alphabet after class to distinguish each other. RT: retention time (min).

Common alterations of characteristic lipids between plasma and liver

After obtaining the characteristic lipids altered in the plasma and liver by treatment with VPA, we investigated common lipids which were altered in both the plasma and liver. Of the obtained lipids, two ePCs (PC[38:4e]a and PC[38:6e]b) and one ePE (PE[42:5e]a) were decreased in both the plasma and liver. By structural analysis, we could characterize PC(38:4e)a as PC(16:0e/22:4) and PC(38:6e)b as PC(16:0e/22:6), while ePE (PE[42:5e]a) could not be characterized. Thus, we chose PC(16:0e/22:4) and PC(16:0e/22:6) and subjected these to further analysis. We evaluated dose- and time-dependency of the changes of PC(16:0e/22:4) and PC(16:0e/22:6) for both the plasma and liver. The plasma and hepatic PC(16:0e/22:4) and PC(16:0e/22:6) levels were decreased dose-dependently from the low dose level of VPA, 250 mg/kg, on day 4 (p < 0.05 or p < 0.01) (Fig. 3). On day 15, the plasma PC(16:0e/22:4) levels also tended to be lower in the VPA-treated groups although this was not statistically significant and the plasma PC(16:0e/22:6) levels were decreased dose-dependently from the low dose level of VPA, 250 mg/kg, as well as on day 4 (p < 0.05 or p < 0.01). The hepatic PC(16:0e/22:4) and PC(16:0e/22:6) levels were decreased at the high dose level of VPA, 500 mg/kg, on both days 4 and 15 (p < 0.01). These levels tended to be lower at 250 mg/kg on both days 4 and 15 although this was not statistically significant.

Fig. 3

Time-dependent changes in level of ePCs in plasma and liver. A: PC(16:0e/22:4) level in plasma, B: PC(16:0e/22:4) level in liver, C: PC(16:0e/22:6) level in plasma, D: PC(16:0e/22:6) level in liver. Day 4 and Day 15 mean the sampling points on days 4 and 15, respectively. Each bar represents mean + S.D. with 8 determinations. Significantly different from control (Dunnett test): * P < 0.05, ** P < 0.01.

Hepatic mRNA levels related to ether-linkage of fatty acids

Hepatic Far1 mRNA levels were slightly decreased by treatment with VPA at 500 mg/kg only on day 15 (Fig. 4). Hepatic Agps mRNA levels did not change at either dose level of VPA at either sampling point. Hepatic Gnpat mRNA levels were decreased dose-dependently from the low dose level of VPA, 250 mg/kg, on day 4, while increased at 500 mg/kg on day 15.

Fig. 4

Hepatic relative mRNA levels of Far1, Agps and Gnpat. Far1: fatty acyl-CoA reductase 1, Agps: alkyl-DHAP synthase, Gnpat: glycerone-phosphate O-acyltransferase, Actb: Actin, beta. Day 4 and Day 15 mean the sampling points on days 4 and 15, respectively. Each bar represents mean + S.D. with 5 determinations. Significantly different from control (Dunnett test): * P < 0.05, ** P < 0.01.

DISCUSSION

In the development of drugs, we sometimes encounter fatty change in the hepatocytes, which is not accompanied by any degenerative changes in the liver, in non-clinical toxicity studies, especially in rodents. There are many drugs that cause hepatic fatty change (steatosis) in rodents and some of them have the potential to induce DILI in humans through mitochondrial toxicity (McCarthy et al., 2004; Gudbrandsen et al., 2006; Ulrich et al., 2001; Silva et al., 2008; Le Dinh et al., 1988; Luiken et al., 2009; Choi et al., 2015; Sahini et al., 2014; Goda et al., 2016; Sahi et al., 2010). This is reasonable because mitochondria take part in the metabolism of lipids, including the oxidation of fatty acids, and fatty acids or other endogenous metabolites related to lipid metabolism will accumulate in the hepatocytes when the oxidation of fatty acids is inhibited by compounds as a consequence of their mitochondrial toxicity. Therefore, investigation of the mechanism of steatosis, especially in terms of mitochondrial toxicity, is necessary to estimate the potential of drug candidates to induce DILI in humans when steatosis, even without any degenerative changes in the liver, is observed in the non-clinical toxicity studies in rodents. The biomarkers for hepatic steatosis in rodents are also considered to be useful for the estimation of the risk of DILI in humans.

VPA has been widely used as a first-line therapy in the treatment of epilepsy for more than 30 years. However, VPA is known to cause hepatic steatosis frequently and some cases can advance into NASH or severe hepatitis in humans (Abdel-Dayem et al., 2014; Lewis et al., 1982; Sato et al., 2005). In the animals, especially in rodents, there are many reports that VPA causes hepatic steatosis possibly following the mitochondrial dysfunction induced by VPA and its metabolites (Zhang et al., 2013; Kesterson et al., 1984; Sobaniec-Lotowska, 1997). Therefore, in the present study, we selected VPA as a representative compound that causes hepatic steatosis in rodents and also has the potential to cause DILI in humans.

In the present study, VPA was dosed to rats at the dose levels of 250 and 500 mg/kg for 3 or 14 days and lipidomics analyses for the plasma and liver were applied to investigate biomarkers for the VPA-induced hepatic steatosis. VPA treatment at 500 mg/kg for 14 days was considered to be greater than the MTD due to deterioration in the animals’ physical conditions. Fatty change of the liver assessed by histopathological examination was noted in rats treated with VPA at 500 mg/kg for 3 days and at 250 mg/kg for 14 days. Hepatic TG levels were correlated to be increased in rats treated with VPA at the same dose level and with the same dosing period as for the hepatic fatty change. These changes in the liver were not accompanied by any degenerative changes in the liver or elevation of the plasma liver function parameters. From the results of lipidomics on the liver, cardiolipin, which is a component of the mitochondrial membrane, was increased even on day 4 at 250 mg/kg, at which neither an increase in hepatic TG level nor histopathological hepatic steatosis was observed, indicating that VPA primarily affected the mitochondria to induce hepatic steatosis. The results obtained in the present study are consistent with those previously reported in VPA-treated rodents and indicate steatosis (hepatic fatty change) probably due to a decrease in beta-oxidation of fatty acids as a consequence of mitochondrial toxicity (Zhang et al., 2013; Abdel-Dayem et al., 2014; Lewis et al., 1982; Kesterson et al., 1984; Sobaniec-Lotowska, 1997).

Dosing of VPA at 500 mg/kg for 14 days was considered not to be appropriate for lipidomics analyses. Lipidomics data obtained from the plasma and liver on day 4 (after 3 days dosing) were analyzed at 500 mg/kg, accordingly, with multivariate statistics with an OPLS-DA model because the hepatic TG levels were clearly increased and the hepatic steatosis was obvious on day 4. From the OPLS-DA and its s-plot analysis, 36 plasma and 24 liver lipid species were characterized as the major factors discriminating between the control and VPA-treated rats. Among these lipid species, two ePCs (PC[16:0e/22:4] and PC[16:0e/22:6]) were identified as the common discriminating factors in both the plasma and liver. These ePCs in plasma were decreased even on day 4 at 250 mg/kg, where neither an increase in hepatic TG level nor histopathological hepatic steatosis was observed. Therefore, these ePCs could be predictive biomarkers for development of hepatic steatosis in VPA-treated rats in terms of dose- and time-sequence of a series of findings observed after treatment with VPA. This is the first report which indicates that ePCs are predictive biomarkers for the VPA-induced hepatic steatosis.

From the results of the measurement of hepatic mRNA levels of enzymes related to ether-linkage of lipids, down-regulation of hepatic Gnpat was detected in the VPA-treated rats. Gnpat is an enzyme localized in peroxisomes and takes part in the first step of synthesis of ether lipids, indicating that this enzyme plays a crucial role in the synthesis of ether lipids. Therefore, the decrease in ePCs was considered to be related to the down-regulation of Gnpat in the peroxisomes.

Mitochondria and peroxisomes have a number of common essential biochemical pathways including beta-oxidation of fatty acids (Mohanty and McBride, 2013). Recently, it has been reported that peroxisomes are generated from the mitochondrial membrane in mammalian cells and that pre-peroxisome fuses with the endoplasmic reticulum vesicles to become mature peroxisomes (Sugiura et al., 2017). Mitochondrial toxicity caused by treatment with VPA is known to be related to inhibition of beta-oxidation caused by the metabolites of VPA. CoA esters of VPA, the metabolites of VPA, inhibit beta-oxidation of fatty acids in mitochondria (Aires et al., 2010). VPA is metabolized to 2-propylpent-4-enoic acid (4-en-VPA) and further to 2-propyl-penta-2,4-dienoic acid (2,4-dien-VPA) (Vamecq et al., 1993). The 4-en-VPA induces microvesiclular steatosis which is accompanied by ultrastructural changes characterized by myeloid bodies, lipid vacuoles and mitochondrial abnormalities in rats treated with this metabolite (Kesterson et al., 1984). The fatty change observed in the present study was also microvesiclular steatosis. The inhibition of beta-oxidation of fatty acids induced by the metabolites of VPA is considered to lead to acceleration of glycolysis. In the present study, down-regulation of Gnpat was observed in the liver and the substrate of Gnpat is dihydroxyacetone phosphate (DHAP). DHAP and glyceraldehyde 3-phosphate (G3P) are synthesized from fructose-1,6-bisphosphate (FBP) which is an intermediate in the glycolysis pathway. The synthesis of DHAP from FBP is considered to be decreased when glycolysis is enhanced because G3P is preferentially synthesized from FBP. The decrease in synthesis of DHAP is considered to lead to down-regulation of Gnpat. From these, the decreases in ePCs and ePEs, both of which are the products of Gnpat, are considered to reflect the enhancement of glycolysis as the consequence of mitochondrial dysfunction (inhibition of beta-oxidation) caused by the metabolite of VPA. This idea can be extended that decreases in ePCs and ePEs could be a common phenomenon in the condition of mitochondrial dysfunction.

In conclusion, the changes in ePCs, (PC[16:0e/22:4] and PC[16:0e/22:6]), were considered to be related to the VPA-induced mitochondrial dysfunction and ePCs have potential utility as predictive biomarkers for VPA-induced hepatic steatosis.

ACKNOWLEDGMENTS

We would like to thank Ms. Katsuko Toyoshima, Ms. Yuriko Sato, and Ms. Mai Kojima (National Institute of Health Sciences; NIHS) for experimental/analytical assistance of lipidomics studies; Dr. Taku Masuyama, Mr. Naohito Yamada, Mr. Yusuke Kemmochi and Mr. Katsunori Ryoke (Japan Tobacco Inc.) for experimental/analytical assistance of animal studies.

Conflict of interest

The authors declare that there is no conflict of interest.

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