Enhancement of acetaminophen-induced chronic hepatotoxicity in spontaneously diabetic torii (SDT) rats

Abstract

Some patients encounter hepatotoxicity after repeated acetaminophen (APAP) dosing even at therapeutic doses. In the present study, we focused on the diabetic state as one of the suggested risk factors of drug-induced liver injury in humans and investigated the contribution of accelerated gluconeogenesis to the susceptibility to APAP-induced hepatotoxicity using an animal model of type 2 diabetes patients. Sprague-Dawley (SD) rats and spontaneously diabetic torii (SDT) rats were each given APAP at 0 mg/kg, 300 and 500 mg/kg for 35 days by oral gavage. Plasma and urinary glutathione-related metabolites, liver function parameters, and hepatic glutathione levels were compared between the non-APAP-treated SDT and SD rats and between the APAP-treated SDT and SD rats. Hepatic function parameters were not increased at either dose level in the APAP-treated SD rats, but were increased at both dose levels in the APAP-treated SDT rats. Increases in hepatic glutathione levels attributable to the treatment of APAP were noted only in the APAP-treated SD rats. There were differences in the profiles of plasma and urinary glutathione-related metabolites between the non-APAP-treated SD and SDT rats and the plasma/urinary endogenous metabolite profile after treatment with APAP in the SDT rats indicated that hepatic glutathione synthesis was decreased due to accelerated gluconeogenesis. In conclusion, SDT rats were more sensitive to APAP-induced chronic hepatotoxicity than SD rats and the high susceptibility of SDT rats was considered to be attributable to lowered hepatic glutathione levels induced by accelerated gluconeogenesis.

INTRODUCTION

Drug-induced liver injury (DILI) is becoming a significant public health issue because of its potential impact not only on patients but also on the development of new drugs. DILI is the most frequent reason for the withdrawal of a drug from the market and cessation of new drug development in pharmaceutical companies (Lee, 2003; Smith and Schmid, 2006).

There are two types in DILI; one is intrinsic DILI and the other is idiosyncratic DILI. In idiosyncratic DILI, there are responders (patients susceptible to DILI) and non-responders (patients tolerant to DILI) even if drugs are administered under the same regimen and the difference in these two populations is closely related to risk factors which contribute to the susceptibility to DILI. Many risk factors for idiosyncratic DILI have been reported, including nutritional status and concurrent diseases (Chalasani et al., 2014).

Acetaminophen (APAP) has been available on the market for a long time as an analgesic agent and is a relatively safe drug when taken as directed at therapeutic doses even for long-term treatment (Amar and Schiff, 2007). However, it is known that there are responders (susceptibles) and non-responders (tolerators) for APAP-induced chronic hepatotoxicity even though they take APAP at therapeutic doses under a prescribed regimen (Amar and Schiff, 2007; O’Connell and Watkins, 2010; Winnike et al., 2010). Hence, considerable efforts have been made for a long time to identify the risk factors contributing to APAP-induced chronic hepatotoxicity in the clinical field and there are a number of reports (Price and Jollow, 1982; Schiødt et al., 1997). One of the risk factors is the nutritional state of the patients as any undernourishment may lead to low detoxification of the reactive metabolite of APAP.

N-Acetyl-p-benzoquinoneimine (NAPQI), which is produced by cytochrome P450-dependent metabolism, is a causative substance for APAP-induced hepatotoxicity but is quickly detoxified by conjugation with hepatic glutathione under normal conditions (Lee et al., 1996; Vermeulen et al., 1992). Hepatic glutathione is synthesized from several amino acids (Timbrell et al., 1995; Schnackenberg et al., 2009) and undernourishment caused by starvation lowers its stores in the liver through decrease in its synthesis due to a low intake of proteins (Buttar et al., 1976). We previously reported that restrictedly fed rats were more sensitive to APAP-induced chronic hepatotoxicity than ad libitum-fed rats and did not adapt to APAP-induced hepatotoxicity even with the long-term treatment with APAP (Kondo et al., 2012). The higher sensitivity and non-adaptable characteristics of the restrictedly fed rats were considered to be related to their nutritional status; restrictedly fed rats might have too little metabolic capacity to detoxify NAPQI due to lowered hepatic glutathione levels by the preferential use of amino acids for accelerated gluconeogenesis rather than glutathione synthesis. The response of the restrictedly fed rats to APAP treatment was similar to that of patients who are susceptible to APAP-induced chronic hepatotoxicity (Kondo et al., 2012).

Diabetes mellitus is reported as one of the risk factors for idiosyncratic DILI (Chalasani and Björnsson, 2010) and the diabetic patients are susceptible to idiosyncratic DILI caused by methotrexate and antituberculosis drugs (Malatjalian et al., 1996; Chalasani et al., 2014). There is a possibility that diabetic patients are also susceptible to the APAP-induced hepatotoxicity because glutathione levels in individuals with type 2 diabetes are known to be lower than those in healthy subjects (Illing et al., 1951; Lal and Kumar, 1967; Awadallah et al., 1978; Lagman et al., 2015). Many studies have been conducted with diabetes model animals to estimate the risk of DILI in diabetic patients (Wang et al., 2007; Segawa et al., 2018). While some of them were used for APAP-induced hepatotoxicity, the responses of these animal models to APAP were not consistent. Thus, one of the unanswered questions is which animal model would be appropriate to understand the risk of the APAP-induced hepatotoxicity in diabetic patients. The mechanism of development of diabetes mellitus, especially type 2 diabetes mellitus, is very complicated and it is difficult to appropriately mimic human type 2 diabetes mellitus, especially concerning the nutritional status, using animal models.

Spontaneously diabetic torii (SDT) rat is well known as a model animal of type 2 diabetes, which accounts for more than 90% of all the diabetes patients, and shows pre-diabetic states with impaired glucose tolerance at approximately 15 weeks of age followed by type 2 diabetes with hypoinsulinemia associated with decreased insulin secretion and hyperglycemia at around 20 weeks of age. Accelerated gluconeogenesis is also induced in the SDT rats. After onset of hyperglycemia, the atrophic change in the pancreatic islet cells occurs and then the ocular changes, which resemble human diabetic ocular complications (e.g. cataract, proliferative retinopathy and retinal detachment), are also observed (Masuyama et al., 2004). The characteristics of SDT rats are similar to those of type 2 diabetes patients and seem suitable as experimental model animals for human type 2 diabetes.

Since the number of patients with pre-diabetes has been increasing in recent years, the investigation for the DILI risk in this population is very important. In this study, we used SDT rats with pre-diabetic states to investigate the contribution of accelerated gluconeogenesis to the susceptibility to APAP-induced hepatotoxicity, comparing the effects of APAP in intact rats that are more easily adapt to drugs including APAP than humans (O’Brien et al., 2000; Buttar et al., 1976, 1977; Strubelt et al., 1979; Poulsen and Thomsen, 1988; Shayiq et al., 1999).

MATERIALS AND METHODS

Animals

Ten-week-old male Crl:CD (SD) rats and SDT rats were purchased from Charles River Japan (Kanagawa, Japan) and CLEA Japan (Tokyo, Japan), respectively. The animals were housed individually in wire-mesh cages kept in an air-conditioned room with a 12-hr light-dark cycle (lighting from 7:00 a.m. to 7:00 p.m.) at a temperature of 23 ± 1°C, a relative humidity of 55 ± 5% and a ventilation rate of about 15 times per hour. The animals were quarantined for 1 week. Tap water was available for drinking 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 2006, issued in October 1, 1973).

Feeding conditions and measurements of food consumption and body weights

Thirty male SD rats and thirty male SDT rats were used for the study. SD rats were allowed free access to a commercial powder diet (CRF-1, Oriental Yeast, Tokyo, Japan) ad libitum throughout the experimental periods. SDT rats were given CRF-1 at the same amount of the mean food consumption in the SD rats throughout the experimental periods. The animals were acclimated for 8 days after quarantine and divided into 3 groups (n = 10/group) for each strain based on body weights on the day of allocation so that the mean initial body weight of each group was equivalent. Food consumption per day (absolute food consumption) and food consumption relative to body weight (relative food consumption) was calculated on days 3, 10, 17, 24 and 31 during the dosing period for all animals. Body weights were measured on days 1 (just before the initiation of dosing), 10, 17, 24 and 31 in the morning (before dosing APAP).

Dosing of APAP

APAP was purchased from Wako Pure Chemical Industry (Osaka, Japan) and was suspended in 0.5% methylcellulose (MC, Shin-etsu Chemical Co., Ltd., Tokyo, Japan) aqueous solution. The dose levels were set at 300 and 500 mg/kg and the rationale of the dose setting was described previously (Kondo et al., 2012). APAP was given once daily (between 8:00 a.m. and 12:00 a.m.) for 35 days by oral gavage at dosing volume of 5.0 mL/kg body weight.

Blood sampling

Blood samples were collected between 8:30 a.m. and 10:30 a.m. during the pre-dosing period (just before the initiation of dosing, described in the figures as “Pre”) and on days 7, 14, 21, 28 and 35 (before daily APAP dosing). Blood samples were collected from the subclavian vein into sodium heparin-treated syringes. Blood sampling and plasma collection for the measurement of the following parameters were conducted as described previously (Kondo et al., 2012); glucose, insulin, pyruvic acid (PA), lactic acid (LA), creatinine (CRN), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), glutamate dehydrogenase (GLDH) and total bilirubin (T-BIL) levels.

On days 1 and 35 additional blood samples were collected for the measurement of the plasma APAP concentrations at 1 hr after APAP dosing, when the plasma APAP concentrations were considered to reach their maximum level from the results of preliminary studies, and at 24 hr after the dosing. The plasma samples, obtained as described previously (Kondo et al., 2012), were stored at –80°C until use.

Urine sampling

Urine samples were collected once during the pre-dosing period (six days before the initiation of dosing, described in the figures as “Pre”) and on days 6, 13, 20, 27 and 34 of the dosing period as described previously (Kondo et al., 2012). After measurement of the volume, the samples were stored at –80°C until use.

Liver sampling

All the animals were euthanized by exsanguination from the abdominal aorta under ether anesthesia without fasting on the day after the last dosing. The livers were removed and weighed, and aliquots of the samples were stored at –80°C until use. The remaining samples were preserved in neutral buffered formalin (Wako Pure Chemical Industry) for histopathological examination.

Measurements of plasma glucose and insulin levels

Plasma glucose concentrations were measured at 37°C with a TBA-120FR automated analyzer (Toshiba, Tokyo, Japan) using standard reagents by the Hexokinase•G-6-PDH method (Wako Pure Chemical Industry). Plasma insulin concentrations were measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Rat Insulin ELISA Kit, Morinaga Institute of Biological Science, Kanagawa, Japan) according to the manufacturer’s instruction. The optical densities (450 and 630 nm) were measured with a microplate reader (Power Wave X, Bio-Tek Instruments). The data analysis (calculation of the concentrations) was performed using analysis software (KC4, Bio-Tek Instruments).

Measurements of plasma hepatic function parameters

Plasma T-BIL concentrations, and AST, ALT, GLDH and ALP activities were measured at 37°C with a TBA-120FR automated analyzer (Toshiba) using standard reagents by enzyme method for T-BIL (Kanto Chemical, Tokyo, Japan) and UV methods for AST, ALT, ALP (Wako Pure Chemical Industry) and GLDH (RANDOX Laboratories, Antrim, UK).

Assay of hepatic glutathione levels

Hepatic reduced-form (GSH) and oxidized-form (GSSG) glutathione levels were determined by an enzyme recycling method using GSH/GSSG quantification kit (Dojindo Laboratories, Kumamoto, Japan) as described previously (Kondo et al., 2012).

Measurements of plasma and urinary endogenous metabolites

Plasma PA, LA and CRN concentrations were determined as described previously (Kondo et al., 2012) with a TBA-120FR automated analyzer (Toshiba). Urinary creatine (CRE), CRN and taurine (TAU) concentrations were also determined as described previously (Kondo et al., 2012) by liquid chromatography-mass spectrometry (LC/MS): API 4000 QTRAP (AB SCIEX, Tokyo, Japan) equipped with a Prominence series high performance liquid chromatography (HPLC) (Shimadzu, Kyoto, Japan) after deproteination. For the urinary endogenous metabolites, the excretion per day of each metabolite was calculated by multiplying the urine volumes and the concentrations.

Histopathological examination of the liver

The left lobe of the liver was cut into longitudinal sections. The liver slices were embedded in paraffin. Sectioning and hematoxylin-eosin staining was performed according to routine histological procedures (Kobayashi et al., 2009).

Measurements of plasma APAP concentrations

Plasma APAP concentrations at 1 and 24 hr after dosing on days 1 and 35 (C_{1 hr} and C_{24 hr}, respectively) were determined by using a 10A series HPLC (Shimadzu), after deproteination. The area under the concentration-time curve (AUC_{1-24 hr}) values from 1 to 24 hr after APAP dosing were calculated by the trapezoidal method.

Statistical analysis

The mean values and standard deviations in each group were calculated for the food consumption, body weights, plasma glucose and insulin levels, plasma and urinary endogenous metabolite levels and plasma hepatic function parameters for each day of measurement, and liver weights and hepatic GSH and GSSG levels at necropsy and plasma APAP concentrations at 1 and 24 hr after APAP dosing and AUC_{1-24 hr} on day 35. Student’s t test for the comparison of the parameters between untreated SD and SDT rats, and Dunnett’s test was conducted for the comparison of control group and 300 or 500 mg/kg APAP-treated groups each for SD and SDT rats, using a statistical software package, MiTOX version 8 (Mitsui Zosen, Tokyo, Japan). The data were analyzed after logarithmic transformation. The levels of significance were set at 5% and 1% (two-tailed).

RESULTS

Basic characteristics of SD and SDT rats

Food consumption was measured to confirm whether the food consumption in the control SDT rats was comparable to that in the control SD rats. Furthermore, to understand the difference in the metabolism-related characteristics between the control SD and SDT rats, body weights, plasma insulin and glucose levels, liver function parameters, liver weights, hepatic glutathione levels and glutathione-related metabolite levels were measured.

Food consumption and relative food consumption per animal per day in the control SDT rats were comparable to those in the control SD rats throughout the experimental period (Table 1). Body weights were also similar between the control SD and SDT rats throughout the experimental period (Table 1). As expected, the plasma glucose and insulin levels in the control SDT rats were higher than those in the control SD rats throughout the experimental period, except for glucose levels on day 14 (Table 1).

Table 1. Comparison of the basic metabolism-related parameters between SD and SDT rats.

Parameters (unit)	Experiment Day		Strain
			SD				SDT
Food consumption (g/day)	3		21.2	±	1.8		21.7	±	1.6
	10		22.4	±	1.7		21.9	±	1.4
	17		23.1	±	2.0		22.3	±	1.8
	24		22.9	±	2.2		22.3	±	2.4
	31		23.4	±	2.0		22.7	±	2.4
Relative food consumption (g/day/100 g body weight)	3		5.19	±	0.36		5.44	±	0.47
	10		5.25	±	0.28		5.28	±	0.43
	17		5.21	±	0.33		5.12	±	0.37
	24		5.00	±	0.28		4.86	±	0.43
	31		4.97	±	0.23		4.47	±	0.37
Body weights (g)	3		409	±	17		402	±	18
	10		426	±	21		416	±	17
	17		443	±	26		435	±	19
	24		457	±	31		457	±	19
	31		470	±	32		478	±	23
Plasma glucose concentrations (mg/dL)	Pre		135	±	7		153	±	8	##
	7		137	±	7		153	±	8	##
	14		134	±	7		133	±	11
	21		127	±	7		139	±	9	##
	28		121	±	5		145	±	20	##
	35		129	±	8		153	±	13	##
Plasma insulin concentrations (ng/mL)	Pre		2.62	±	0.85		3.44	±	1.25
	7		1.93	±	0.41		2.98	±	0.95	##
	14		1.54	±	0.37		2.84	±	0.68	##
	21		1.76	±	0.51		3.67	±	1.18	##
	28		1.49	±	0.39		3.41	±	1.42	##
	35		1.89	±	0.48		3.21	±	1.56	#
Plasma AST activities (IU/L)	Pre		65.3	±	12.7		71.3	±	6.7
	7		68.3	±	7.4		72.4	±	8.0
	14		65.8	±	11.7		74.5	±	5.4
	21		71.5	±	15.0		72.9	±	4.3
	28		67.7	±	9.0		79.7	±	10.7	#
	35		68.2	±	8.1		78.2	±	6.1	##
Plasma ALT activities (IU/L)	Pre		33.7	±	6.8		53.9	±	6.2	##
	7		39.0	±	3.1		52.4	±	4.4	##
	14		34.3	±	6.9		54.7	±	5.7	##
	21		42.4	±	5.5		53.9	±	5.1	##
	28		44.5	±	4.2		65.0	±	6.5	##
	35		51.4	±	5.5		64.6	±	5.5	##
Plasma GLDH activities (IU/L)	Pre		15.2	±	6.5		19.5	±	6.9
	7		18.8	±	3.7		17.6	±	2.8
	14		15.4	±	4.6		20.4	±	4.5	#
	21		21.0	±	9.6		19.9	±	6.0
	28		18.9	±	6.9		23.0	±	6.5
	35		20.8	±	6.6		26.2	±	6.3
Plasma ALP activities (IU/L)	Pre		1044	±	235		1209	±	143
	7		835	±	192		998	±	210
	14		765	±	168		1042	±	126	##
	21		773	±	184		981	±	90	##
	28		684	±	128		1025	±	125	##
	35		700	±	176		1037	±	116	##
Plasma T-BIL concentrations (mg/dL)	Pre		0.06	±	0.01		0.07	±	0.01	#
	7		0.07	±	0.01		0.07	±	0.01
	14		0.07	±	0.01		0.07	±	0.01
	21		0.07	±	0.02		0.06	±	0.01
	28		0.06	±	0.01		0.07	±	0.02	#
	35		0.06	±	0.01		0.06	±	0.02
Liver weight (g)	36		14.58	±	1.32		15.78	±	1.22	#
Relative liver weight (g/100g body weight)	36		2.95	±	0.22		3.12	±	0.12
Hepatic GSH levels (µmol/g liver)	36		7.78	±	0.92		7.78	±	1.49
Hepatic GSSG levels (µmol/g liver)	36		0.15	±	0.03		0.12	±	0.02	##
Plasma CRN levels (mg/dL)	Pre		0.36	±	0.03		0.36	±	0.02
	7		0.35	±	0.03		0.43	±	0.01	##
	14		0.39	±	0.04		0.42	±	0.02	#
	21		0.41	±	0.05		0.45	±	0.02	#
	28		0.42	±	0.04		0.50	±	0.03	##
	35		0.48	±	0.05		0.49	±	0.05
Urinary CRE excretion (µmol/day)	Pre		1.92	±	0.49		1.97	±	0.49
	6		1.40	±	0.17		3.01	±	2.00	#
	13		1.22	±	0.15		1.62	±	0.35	##
	20		1.24	±	0.19		2.30	±	1.34	##
	27		1.56	±	0.96		1.70	±	0.18
	34		1.47	±	0.63		1.96	±	0.83
Urinary CRN excretion (µmol/day)	Pre		101.6	±	17.9		99.8	±	17.5
	6		113.4	±	8.1		100.7	±	15.6	#
	13		118.2	±	9.6		101.6	±	12.8	##
	20		117.1	±	8.2		119.1	±	7.6
	27		124.2	±	10.3		117.6	±	5.3
	34		132.2	±	17.3		117.5	±	17.9
Plasma PA concentrations (mg/dL)	Pre		0.77	±	0.19		0.89	±	0.18
	7		0.46	±	0.15		0.55	±	0.15
	14		0.36	±	0.13		0.28	±	0.08
	21		0.35	±	0.28		0.31	±	0.10
	28		0.34	±	0.22		0.67	±	0.27	##
	35		0.05	±	0.00		0.71	±	0.16	##
Plasma LA concentrations (mg/dL)	Pre		18.0	±	5.5		19.7	±	4.6
	7		19.0	±	8.4		24.5	±	9.4
	14		17.5	±	6.2		16.6	±	4.8
	21		34.1	±	40.0		20.9	±	7.1
	28		22.6	±	16.0		59.9	±	48.3	#
	35		43.5	±	24.4		43.2	±	18.2
Urinary TAU excretion (µmol/day)	Pre		141.4	±	40.5		73.7	±	26.1	##
	6		111.4	±	23.6		114.9	±	32.6
	13		91.1	±	22.1		107.2	±	24.1
	20		106.1	±	30.3		121.1	±	21.4
	27		100.2	±	30.1		110.5	±	26.9
	34		102.8	±	26.9		123.5	±	41.1

These parameters of each strain (n = 9-10/group/point) were determined as described in the Materials and Methods section. Data are shown as the mean ± S.D. *p < 0.05, **p < 0.01; significantly different from SD rats (Student’s t test).

Plasma AST, ALT and ALP activities of the control SDT rats were higher than those of the control SD rats throughout the experiment period (Table 1), even though there was no statistical significance in AST and ALP levels for some sampling points. Plasma T-BIL and GLDH levels of the control SDT rats were comparable to those of the control SD rats throughout the experiment period (Table 1). Absolute liver weights of the control SDT rats were higher than those of the control SD rats at the end of the experimental period (Table 1), although the change in the relative liver weights was not statistically significant. Hepatic levels of the GSH in the control SDT rats were comparable to those in the control SD rats at the end of the experimental period while hepatic levels of the GSSG in the control SDT rats were lower than those in the control SD rats (Table 1). The plasma CRN levels of the control SDT rats were higher than those of the control SD rats throughout the experiment period except for day 35 (Table 1). The urinary excretion of CRE in the control SDT rats was also higher than that in the control SD rats throughout the experimental period, although the change was not statistically significant for some sampling points (Table 1). In contrast, the urinary excretion of CRN in the control SDT rats was lower than that in the control SD rats throughout the experimental period, although the change was not statistically significant for some sampling points (Table 1). The plasma PA and LA levels of the control SDT rats were higher than those of the control SD rats during the late stage of the experimental period (Table 1). The urinary TAU excretion in the control SDT rats was lower than that in the control SD rats at the pre-experiment period but was comparable to that in the control SD rats during the late phase of the experiment period (Table 1).

Differences in the effects of APAP between SD and SDT rats

To investigate the systemic toxicity of APAP, SD and SDT rats were treated with APAP at 300 and 500 mg/kg for 35 days. Absolute and relative food consumption was decreased at 500 mg/kg in both strains on day 3 when compared with the matched control groups, but was comparable to that in the matched control groups on day 10 and thereafter although some statistically higher values were noted in the APAP-treated groups of SD rats. Body weights of the APAP-treated SD rats (at both 300 and 500 mg/kg) were comparable to those in the matched control groups throughout the dosing period. On the other hand, those of the APAP-treated SDT rats at 500 mg/kg but not at 300 mg/kg were lower than those of the matched control groups on day 17 and thereafter (Table 2).

Table 2. Changes in the food consumption and body weights in the APAP-treated SD and SDT rats.

Dosing Day	Strain :	SD													SDT
	Dose (mg/kg) :	0				300				500					0				300				500
Food consumption (g/day)
3		21.2	±	1.8		20.1	±	1.7		16.5	±	3.3	**		21.7	±	1.6		20.7	±	1.7		18.6	±	3.0	**
10		22.4	±	1.7		21.6	±	2.0		21.8	±	2.4			21.9	±	1.4		22.3	±	0.7		21.7	±	1.3
17		23.1	±	2.0		22.6	±	1.4		22.3	±	2.4			22.9	±	2.2		22.9	±	2.3		22.5	±	2.0
24		22.9	±	2.2		23.0	±	1.5		23.8	±	2.5			22.3	±	2.4		22.5	±	3.4		23.1	±	2.0
31		23.4	±	2.0		22.9	±	2.9		25.2	±	2.1			22.7	±	2.4		23.4	±	3.6		22.3	±	3.6
Relative food consumption (g/day/100 g body weight)
3		5.22	±	0.38		4.95	±	0.32		4.06	±	0.73	**		5.44	±	0.47		5.19	±	0.32		4.63	±	0.70	**
10		5.25	±	0.28		5.13	±	0.29		5.31	±	0.34			5.28	±	0.43		5.41	±	0.19		5.38	±	0.21
17		5.21	±	0.33		5.21	±	0.26		5.34	±	0.40			5.12	±	0.37		5.38	±	0.53		5.47	±	0.46
24		5.00	±	0.28		5.16	±	0.35		5.43	±	0.41	*		4.86	±	0.43		5.09	±	0.65		5.43	±	0.40
31		4.97	±	0.23		4.99	±	0.53		5.52	±	0.33	**		4.74	±	0.37		5.08	±	0.72		5.02	±	0.71
Body weights (g)
3		409	±	17		407	±	21		401	±	20			402	±	18		403	±	15		397	±	16
10		426	±	21		420	±	25		410	±	29			416	±	17		412	±	12		404	±	15
17		443	±	26		434	±	27		423	±	27			435	±	19		427	±	14		411	±	14	**
24		457	±	31		447	±	26		438	±	26			457	±	19		442	±	17		426	±	14	**
31		470	±	32		460	±	28		456	±	25			478	±	23		460	±	16		443	±	15	**

Food consumption were measured for each rat (n = 9-10/group/point) on the days indicated. Relative food consumption per day was calculated for each animal on days 1-3, 7-10, 14-17, 21-24 and 28-31 for SD rats and on days 3, 10, 17, 24 and 31 for SDT rats. Data for control groups (0 mg/kg) are from Table 1. Data are shown as the mean ± S.D. *p < 0.05, **p < 0.01; significantly different from the matched control groups (Dunnett’s test).

Next, the effects of APAP on the liver function-related parameters were investigated based on the alteration of liver function parameters. In SD rats, there were no differences in the plasma AST, ALT, GLDH and ALP activities between the APAP-treated and the matched control groups throughout the dosing period (Figs. 1A, 1C, 1E and 1G) and the plasma T-BIL levels were increased slightly on day 35 in the 300 and 500 mg/kg APAP-treated groups (Fig. 1I). In SDT rats, plasma AST activities were increased slightly by 500 mg/kg APAP treatment in the late phase of the dosing period (Fig. 1B). Plasma ALT activities were slightly higher in the 300 and 500 mg/kg APAP-treated groups than those in the matched control groups from day 7 (Fig. 1D). Plasma GLDH activities were much higher in the 300 and 500 mg/kg APAP-treated groups than those in the matched control groups from day 14 (Fig. 1F), although the increase in the 300 mg/kg APAP-treated group was not statistically significant. Plasma ALP activities and T-BIL levels were much higher in the 500 mg/kg APAP-treated group than those in the matched control groups from day 14 (Figs. 1H and 1J). These results indicate that the APAP-induced liver toxicities were more obvious in SDT rats than in SD rats.

Fig. 1

Effects of APAP treatment on the liver function-related parameters in SD and SDT rats. SD rats (A, C, E, G, I) and SDT rats (B, D, F, H, J) were treated with APAP (300 or 500 mg/kg) for 35 days. Blood samples were collected from each rat on the days indicated (n = 8-10/group/time point) under non-fasting conditions in SD and SDT rats. Plasma AST (A, B), ALT (C, D), GLDH (E, F) and ALP (G, H) activities and T-BIL (I, J) levels were measured as described in the Materials and Methods section. “Pre” indicates the pre-dosing period. Data for control groups (0 mg/kg) are from Table 1. Data are shown as the mean. *p < 0.05, **p < 0.01; significantly different from the matched control groups (Dunnett’s test).

Hepatic glutathione levels as well as liver weights were measured at the end of the dosing period to investigate the capacity of detoxification for APAP. In both SD and SDT rats, absolute and relative liver weights were increased in the 300 and 500 mg/kg APAP-treated groups (Table 3). In SD rats, hepatic GSH levels were significantly increased in the 500 mg/kg APAP-treated group (Table 3). In contrast, no statistically significant increase in hepatic GSH levels was observed in SDT rats (Table 3). There were no differences in the hepatic GSSG levels between the APAP-treated and their matched control groups both in SD or SDT rats (Table 3).

Table 3. Changes in the liver weights and hepatic glutathione levels after APAP treatment.

Parameters (unit)	Strain	Dose (mg/kg)
		0				300				500
Absolute liver weight (g)	SD	14.6	±	1.30		16.1	±	0.97	*	16.5	±	1.55	**
	SDT	15.8	±	1.22		17.8	±	1.40	**	17.2	±	1.52
Relative liver weight (g/100g body weight)	SD	2.95	±	0.22		3.33	±	0.14	**	3.46	±	0.26	**
	SDT	3.12	±	0.12		3.63	±	0.20	**	3.67	±	0.18	**
Hepatic GSH level (µmol/g liver)	SD	7.78	±	0.92		8.97	±	1.21		9.72	±	0.95	**
	SDT	7.78	±	1.49		8.44	±	0.95		8.91	±	1.13
Hepatic GSSG level (µmol/g liver)	SD	0.15	±	0.03		0.14	±	0.03		0.14	±	0.02
	SDT	0.12	±	0.02		0.12	±	0.03		0.12	±	0.02

SD and SDT rats were treated with APAP (300 or 500 mg/kg) for 35 days, and livers were collected. The samples (n = 9-10/group) were used for the measurement of liver weights and hepatic GSH and GSSG levels. Data for control groups (0 mg/kg) are from Table 1. Data are shown as the mean ± S.D. *p < 0.05, **p < 0.01; significantly different from the matched control groups (Dunnett’s test).

Furthermore, histopathological examination of the liver was conducted to investigate whether histopathological changes were induced after the APAP dosing. In the results, no treatment-related histopathological changes were noted in the liver of any APAP-treated group for either strain (data not shown).

Since there were differences in hepatic glutathione levels after APAP dosing between SD and SDT rats, the plasma and urinary glutathione-related endogenous metabolites were measured to investigate the capacity of glutathione synthesis after treatment with APAP in each strain. The results are shown in Fig. 2.

Fig. 2

Changes in the plasma and urinary glutathione-related metabolites by APAP treatment in SD and SDT rats. SD rats (A, C, E, G, I, K) and SDT rats (B, D, F, H, J, L) were treated with APAP (300 or 500 mg/kg) for 35 days. Blood was collected before dosing from each rat on the days indicated (n = 8-10/group/point). Urines were individually collected for 24 hr from immediately after APAP dosing to before dosing on the next day. Plasma CRN, urinary CRE and CRN concentrations were measured, and urinary CRE and CRN excretions per day were calculated as described in the Materials and Methods section. “Pre” indicates the pre-dosing period. Data for control groups (0 mg/kg) are from Table 1. Data are shown as the mean. *p < 0.05, **p < 0.01; significantly different from the matched control groups (Dunnett’s test).

In SD rats, there were no differences in the plasma CRN levels between the APAP-treated groups and their matched control groups (Fig. 2A). On the other hand, in SDT rats, the plasma CRN levels were lower in the both APAP-treated groups than those in the matched control groups throughout the dosing period (Fig. 2B). The urinary CRE excretion was increased significantly in the APAP-treated groups than with their matched control groups during the dosing period both in SD and SDT rats. In SDT rats, the increase was much more prominent compared to that in SD rats (Figs. 2C and 2D). There were no differences in the urinary CRN excretion between the APAP-treated groups and their matched control groups in either strain (Figs. 2E and 2F).

Plasma PA and LA levels showed no marked changes after APAP treatment in either SD or SDT rats (Figs. 2G, 2H, 2I and 2J). In SD rats, APAP treatment showed no significant influences on the urinary TAU excretion except on day 6, where the levels were decreased at both doses (Fig. 2K). On the other hand, the urinary TAU excretion was significantly decreased or tended to be decreased in the 300 and 500 mg/kg APAP-treated group throughout the dosing period in SDT rats (Fig. 2L).

Next, to investigate the effects of APAP on gluconeogenesis, plasma glucose and insulin levels were measured. However, there were no differences in those levels between the APAP-treated and the matched control groups in either SD or SDT rat (data not shown).

APAP exposure in SD and SDT rats

To investigate the relationship between the systemic exposure to APAP and the effects on the liver, plasma APAP concentrations were measured and the AUC_{1-24 hr} was calculated both in SD and SDT rats on days 1 and 35 (Table 4). Plasma APAP concentrations at 1 and 24 hr after dosing and AUC_{1-24 hr} on days 1 and 35 were higher in the 500 mg/kg groups than in 300 mg/kg groups in both SD and SDT rats, but there were differences between the strains. The plasma APAP concentrations at 1 hr after dosing were comparable between the strains on day 1 while they were lower in SDT rats than in SD rats on day 35 when compared at the same dose levels. The APAP concentrations at 24 hr after dosing on day 1 were also lower in SDT rats than in SD rats with 500 mg/kg APAP although the concentrations were comparable between the strains with 300 mg/kg APAP. APAP was not detected in the plasma of either strain at both doses at 24 hr after dosing on day 35. The AUC_{1-24 hr} values were also lower in SDT rats than in SD rats at 500 mg/kg on both days of measurement. With 300 mg/kg APAP, AUC_{1-24 hr} values on day 35 but not day 1 tended to be lower in SDT rats than in SD rats. These results indicate that SDT rats had relatively lower exposure to APAP than SD rats.

Table 4. Plasma APAP concentrations in SD and SDT rats.

		Strain		Dose (mg/kg)
				300					500
Day 1	C1hr (µg/mL)	SD		81.0	±	14.0			109.7	±	23.5
		SDT		83.4	±	6.3			101.3	±	16.5
	C24hr (µg/mL)	SD		0.7	±	1.0			35.5	±	19.5
		SDT		0.6	±	0.8			10.2	±	4.8	##
	AUC1-24hr (µg•hr/mL)	SD		939	±	170			1670	±	339
		SDT		966	±	75			1283	±	226	##
Day 35	C1hr (µg/mL)	SD		132.8	±	24.9			170.8	±	38.2
		SDT		96.7	±	33.4	#		136.7	±	35.0	#
	C24hr (µg/mL)	SD		0.0	±	0.0			0.0	±	0.0
		SDT		0.0	±	0.0			0.0	±	0.0
	AUC1-24hr (µg•hr/mL)	SD		1527	±	287			1964	±	439
		SDT		1112	±	385			1572	±	403

SD and SDT rats were treated with 300 or 500 mg/kg APAP for 35 days. The plasma was collected at 1 and 24 hr after dosing on days 1 and 35 to determine acetaminophen concentrations, and AUC_{1-24 hr} was calculated as described in the Materials and Methods section. Data are shown as the mean ± S.D. #p < 0.05, ##p < 0.01; significantly different between SD and SDT rats at the same doses (Student’s t test).

DISCUSSION

In this study, we found that SDT rats were much more sensitive to the APAP-induced hepatotoxicity than SD rats. In SD rats, there were no changes in liver function parameters (plasma AST, ALT, GLDH and ALP activities, and T-BIL levels) after APAP treatment at either dose level (Fig. 1), indicating that SD rats are resistant to long-term treatment with APAP at these doses. In SDT rats, on the other hand, plasma ALT and GLDH activities were increased by APAP treatment at both 300 and 500 mg/kg and plasma AST and ALP activities and T-BIL levels were increased by 500 mg/kg APAP, although there were no treatment-related histopathological findings in the liver at the end of the dosing period (Fig. 1). To investigate whether these differences could result from the differences in the systemic exposure to APAP, we determined the plasma concentrations of APAP and AUC_{1-24 hr} in both strains (Table 4). The results showed that SD rats were resistant even at 1964 µg·hr/mL (500 mg/kg APAP) and that the AUC_{1-24 hr} in SDT rats with 300 mg/kg APAP was 1112 µg·hr/mL, which was approximately the half of that in SD rats treated with 500 mg/kg APAP. These results clearly indicate that SDT rats are much more sensitive to the APAP-induced hepatotoxicity than SD rats. We have reported that restrictedly fed rats were more sensitive to APAP-induced hepatotoxicity than ad libitum-fed rats and did not adapt to APAP even during long-term treatment (Kondo et al., 2012). The characteristics in the SDT rats are similar to those of the restrictedly fed rats in terms of response to APAP. There have been many studies reporting the relationships between diabetic states and DILI. For APAP-induced hepatotoxicity, it has been reported that hepatotoxicity is increased in type 1 diabetic rats but is decreased in type 1 or 2 diabetic mice when compared to healthy animals (Price and Jollow, 1982; Sawant et al., 2006; Watkins and Sherman, 1992; Jeffery et al., 1991; Shankar et al., 2003a, 2003b). Taken together, our results indicate that a diabetic state is one of the risk factors for APAP-induced hepatotoxicity.

Plasma glucose and insulin levels were higher in the control SDT rats than in the control SD rats throughout the experiment period, indicating that SDT rats were in a state of abnormal glucose tolerance (i.e. pre-diabetic stage). In such a condition, the liver produces glucose from muscle-derived alanine via the glucose-alanine cycle. The plasma glucose levels in SDT rats were thus considered to be increased mainly by decreased insulin sensitivity of the peripheral tissues and increased hepatic gluconeogenesis using alanine (Garber et al., 1976; Leveille and Chakrabarty, 1968). In fact, it has been also reported that hepatic gluconeogenesis is accelerated in SDT rats (Masuyama et al., 2004). The increased gluconeogenesis might affect the synthesis of glutathione, which is important for APAP detoxification. We have reported that hepatic glutathione levels were lower in restrictedly fed rats than in ad libitum-fed rats (Kondo et al., 2012). Since gluconeogenesis was increased in the restrictedly fed rats, the lowered hepatic glutathione levels might result from a decrease in the supply of amino acids, which were more preferentially used for gluconeogenesis rather than glutathione synthesis (Kondo et al., 2012). In the current study, we determined hepatic glutathione levels in both SDT and SD rats and found that hepatic GSSG levels in the control SDT rats were lower than those in the control SD rats although hepatic GSH levels were comparable between the control SDT and SD rats (Table 1). These results indicate that the pool of glutathione is lower in SDT rats and that the capacity for glutathione synthesis in SDT rats is reduced compared to SD rats, possibly due to accelerated gluconeogenesis.

The discussion that gluconeogenesis is accelerated in SDT rats is supported by the profiles of glutathione-related metabolites as well as plasma glucose, insulin, AST, ALT and GLDH levels. Plasma PA and LA levels were higher in the control SDT rats than in the control SD rats (Table 1). PA is produced from muscle-derived alanine in the liver and is necessary to produce glucose under fasting conditions (Fig. 3). LA is produced from PA by anaerobic metabolism (Fig. 3). Therefore, plasma PA and LA levels are increased when gluconeogenesis is accelerated. Additionally, urinary levels of TAU, a by-product of cysteine (Fig. 3), were lower in the control SDT rats than in the control SD rats (Table 1). We have reported that low urinary TAU levels reflect the reduced productivity of hepatic glutathione in restrictedly fed rats (Kondo et al., 2012). Therefore, the lower TAU levels in SDT rats indicate the decreased capability for glutathione production in the liver. Moreover, plasma AST, ALT and GLDH activities were higher in the control SDT rats than in the control SD rats without APAP treatment (Table 1). These higher values of the hepatic enzymes noted in the control SDT rats were not accompanied by increases in plasma T-BIL levels or any histopathological changes in the liver, indicating that the increases were not related to hepatotoxicity. AST, ALT and GLDH are involved in the alanine-glucose cycle and the activities of these enzymes are increased in the liver or intestine when gluconeogenesis and protein catabolism are accelerated (Kobayashi et al., 2009, 2011; Hagopian et al., 2003). Based on the profiles of those parameters, we assume that in the liver of SDT rats gluconeogenesis is accelerated and glutathione synthesis is reduced, probably due to the preferential use of amino acids for gluconeogenesis rather than glutathione synthesis.

Fig. 3

The metabolisms of APAP and glutathione-related metabolites.

In addition to the basal differences in the capability for glutathione synthesis between SDT and SD rats, the changes in the endogenous metabolites after APAP treatment also suggest that SDT rats scarcely have the metabolic capacity to detoxify formed NAPQI. In SD rats, plasma levels of PA and LA, by-products of glutathione synthesis (Fig. 3), and hepatic GSH levels were increased after treatment with APAP at 500 mg/kg. This phenomenon is considered to be due to the adaptation to NAPQI-induced oxidative stress by upregulation of glutathione synthesis in the liver (O’Brien et al., 2000). These results indicate that hepatic glutathione synthesis was enhanced for NAPQI detoxification in SD rats. On the other hand, in SDT rats, APAP treatment tended to decrease plasma PA and LA levels in the late stage of the dosing period (Fig. 2). Additionally, plasma CRN levels and urinary TAU levels were decreased after repeated dosing with APAP at both dose levels in SDT rats. These endogenous metabolite profiles in the APAP-treated SDT rats indicate that this strain does not have the metabolic capacity enough to detoxify NAPQI due to lowered hepatic glutathione synthesis.

In this study, no histopathological changes were observed in the liver of SDT rats after APAP treatment. This is probably because that the magnitude of increase in gluconeogenesis and decrease in GSH production was small in the animals at the age of 16 weeks old (pre-diabetes state). After onset of diabetes, the magnitude of these changes might be more prominent and APAP treatment might induce histopathological changes in the liver.

The endogenous metabolites whose basal levels were different between SD and SDT rats could become predisposition biomarkers for the APAP-induced hepatotoxicity. Among the metabolites investigated in this study, we suggest the urinary TAU level as a possible candidate. The urinary TAU levels were lower in SDT rats than in SD rats before treatment with APAP. This is consistent with the results of a study reported by Clayton et al. (2006). They demonstrated that the pre-dose levels of urinary TAU were lower in rats that were sensitive to APAP-induced acute hepatotoxicity than in rats that were less sensitive to the toxicity. We have previously reported that the pre-dose urinary TAU levels can be a predisposition biomarker for the APAP-induced chronic hepatotoxicity based on a study to compare sensitivity to the APAP-induced chronic hepatotoxicity between ad libitum-fed rats and restrictedly fed rats (Kondo et al., 2012). Moreover, taurine could become the disposition biomarker for hepatotoxicity other than APAP (Clayton et al., 2004). From these observations, the urinary TAU levels could become a predisposition biomarker to predict susceptibility to DILI including the APAP-induced chronic hepatotoxicity not only in healthy rats but also in diabetic rats. In a future study, it is necessary to investigate the changes of plasma TAU levels using animals in which the pathological condition of diabetes progressed to establish this as a predisposition biomarker.

APAP undergoes detoxifying metabolism by sulfotransferases and glucuronosyltransferases at normal doses but overdosing results in its metabolic activation to NAPQI by cytochrome P450s. At day 35, the plasma APAP concentrations were higher in SD rats than in SDT rats. These results suggest changes in the metabolism of APAP in SDT rats, although we have not determined them. In fact, there are several reports that the expression of cytochrome P450s and phase II enzymes changes during the onset diabetes in model animals (Yoshinari et al., 2006; Sindhu et al., 2006; Oh et al., 2012; Zhou et al., 2016). The differences in the activity of these drug-metabolizing enzymes between SD and SDT rats need to be investigated in a future study to fully understand the causes of the increased susceptibility of SDT rats to APAP.

In conclusion, our present results suggest that the accelerated gluconeogenesis contributes to the susceptibility to APAP-induced chronic hepatotoxicity in SDT rats, which are not capable of adapting to APAP-induced hepatotoxicity even with the long-term treatment with APAP, compared to SD rats, because SDT rats do not have metabolic capacity sufficient to detoxify the APAP-derived reactive metabolite, NAPQI, due to lower capacity for synthesizing glutathione in the liver by the preferential use of amino acids for accelerated gluconeogenesis. These results thus support the possibility that type 2 diabetic patients are susceptible to DILI. The SDT rat might be a useful model to estimate the contribution of the diabetic state, as a risk factor for DILI, to the hepatotoxicity induced by substances producing electrophilic reactive metabolites that are detoxified by glutathione conjugation.

ACKNOWLEDGMENTS

The authors would like to thank the invaluable contributions of the staff at the Toxicology Research Laboratories, Central Pharmaceutical Research Institute, JAPAN TOBACCO INC to technical supports and suggestions.

Conflict of interest

The authors declare that there is no conflict of interest.

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