A 13-week subchronic toxicity study of acetaminophen using an obese rat model

Takeshi Toyoda; Young-Man Cho; Jun-ichi Akagi; Yasuko Mizuta; Kohei Matsushita; Akiyoshi Nishikawa; Katsumi Imaida; Kumiko Ogawa

doi:10.2131/jts.43.423

Abstract

Although obesity is increasing worldwide, experimental studies examining the possible association between obesity and susceptibility to chemical toxicity are limited. In the present study, we performed a 13-week toxicity study for acetaminophen (APAP), a well-known drug that exhibits hepatotoxicity as an adverse effect, using an obese rat model to investigate the differences in susceptibility between obese and normal individuals. Male F344 and obese Zucker (lean and fatty) rats were administered 0, 80, 253, 800, 2,530, or 8,000 ppm APAP in the diet for 13 weeks. No significant toxicity related to APAP treatment was observed in terms of clinical signs and hematology in all three strains. Body weight gain in F344 and lean rats was significantly decreased by 8,000 ppm APAP treatment. Significant increases in serum total cholesterol level and relative liver weights were detected in F344 rats in the highest dose group. On histopathological assessment, centrilobular hepatocellular hypertrophy was observed in the 8,000 ppm groups of F344 and lean rats, whereas no histopathological changes were induced by APAP in fatty rats. The no-observed-adverse-effect levels (NOAELs) of APAP were evaluated to be 2,530 ppm in F344 and lean rats (142.1 and 152.8 mg/kg bw/day, respectively) and more than 8,000 ppm in fatty rats (> 539.9 mg/kg bw/day). These results suggested that obese Zucker rats may be less susceptible to APAP-dependent toxicity in the liver than their lean counterparts.

INTRODUCTION

Repeated-dose oral toxicity studies have been performed to determine the no-observed-adverse-effect level (NOAEL) for environmental chemicals including pharmaceuticals and food ingredients, usually using healthy and genetically uniform animals. However, many human adults have underlying diseases, such as obesity, hyperlipidemia, diabetes, and hypertension (Younossi et al., 2016). These diseases can directly or indirectly affect the metabolic and excretory functions of the liver, kidney, and other internal organs (Gray et al., 2014). Indeed, cumulative epidemiological and experimental studies have shown that obesity increases the risk of cancer and cancer-related mortality (Gallagher and LeRoith, 2015). Although obesity is increasing worldwide, experimental studies examining the relationship between obesity and susceptibility to chemical toxicity are limited.

Obese Zucker rats were discovered as a mutant strain in 1961 and have become one of the most widely used animal models of obesity (Zucker and Zucker, 1961; Aleixandre de Artiñano and Miguel Castro, 2009). These animals, called “fatty rats”, are homozygous for the fa allele and harbor a mutation in the leptin receptor, an important factor regulating food intake and energy balance. Thus, fatty rats develop severe obesity associated with hyperphagia after weaning. Although metabolic profiles in the liver of fatty rats have been investigated (Pizarro et al., 2004; Serkova et al., 2006), the results of these studies have sometimes been contradictory, suggesting that the effects of obesity on hepatic xenobiotic metabolism may be substrate-specific (Kim et al., 2004; Chaudhary et al., 1993).

Acetaminophen (APAP, paracetamol) is one of the most commonly used drugs for the treatment of pain and fever in both humans and animals and is mainly metabolized by phase II enzymes into nontoxic glucuronide and sulfate conjugates in the liver (Zhao and Pickering, 2011). However, when APAP is administered at an excessive dose, acute liver failure characterized by hepatocellular necrosis is induced by a highly reactive metabolite produced by CYP2E1 and CYP3A4 (Jaeschke et al., 2012; Michaut et al., 2014). Although several studies have evaluated a single dose of APAP using obese Zucker rats (Blouin et al., 1987; Tuntaterdtum et al., 1993), detailed investigations of the repeated-dose toxicity and dose responses of APAP have not yet been conducted.

We have investigated the toxicity of food additives and contaminants using rodent models (Toyoda et al., 2014a, 2014b; Onami et al., 2014). Recently, we demonstrated that obese Zucker rats are more susceptible to hematopoietic toxicity induced by 3-monochloropropane-1,2-diol, a food contaminant possessing renal carcinogenicity, than their lean counterparts, without any promotion of renal toxicity (Toyoda et al., 2017). These results indicate that obesity can also affect susceptibility to chemical toxicity in organs other than the main target organ. In the present study, we performed a 13-week subchronic toxicity study with dietary administration of APAP using obese Zucker rats in order to evaluate the possible effects of obesity on susceptibility to toxicity.

MATERIALS AND METHODS

Chemicals and experimental animals

APAP (lot no. SLBB2780V, 99.8%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). For administration to rats, APAP was mixed into the basal diet (MF; Oriental Yeast, Tokyo, Japan). A total of 180 5-weeek-old male specific pathogen-free F344 and obese Zucker (lean and fatty) rats were purchased from Charles River Japan (Kanagawa, Japan) and used after 1 week of acclimation. The animals were housed in polycarbonate cages with soft chip bedding (Sankyo Labo Service, Tokyo, Japan) in a room with a barrier system controlled for the light/dark cycle (12 hr), ventilation (air exchange rate 18 times/hr), temperature (23 ± 2°C), and relative humidity (55% ± 5%). The cages and chip bedding were exchanged twice a week. All animals had free access to the diet and water with or without test chemicals.

Study design

At the beginning of the experiment, the animals were randomly allocated to 6 groups of 10 rats each based on their body weights measured just before starting chemical treatment. Animals were administered 0, 80, 253, 800, 2,530, or 8,000 ppm APAP in the diet for 13 weeks. We set the lowest-observed-adverse-effect level (LOAEL) reported in a previous study as the intermediate dose (NTP, 1993), and √10-fold intervals were applied for the descending dose levels to detect 10-fold differences in NOAELs. The diet was changed once per week. General conditions and mortality were checked daily, and body weights were measured once a week during the experimental period. The amounts of supplied and residual diet were weighed weekly in order to calculate the average daily food consumption and chemical intake through the entire treatment period. All rats were fasted overnight at the completion of the treatment, and anesthesia was induced by inhalation of isoflurane. Blood samples were then collected from the abdominal aorta for hematology and serum biochemistry. The experimental design was approved by the Animal Care and Utilization Committee of the National Institute of Health Sciences, Japan, and the animals were cared for in accordance with institutional guidelines as well as the Guidelines for Proper Conduct of Animal Experiments (Science Council of Japan, June 1, 2006).

Hematology and serum biochemistry

The following hematological parameters were analyzed using a K-4500 automatic hematology analyzer (Sysmex, Hyogo, Japan): white blood cell count (WBC), red blood cell count (RBC), hemoglobin concentration (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelet count (PLT). Blood smears were processed for May-Giemsa staining and counting of erythroblasts (Ebls) and differential leukocytes using a Microx HEG-50S (Sysmex). Serum biochemical analysis of the following parameters was performed by SRL (Tokyo, Japan): total protein (TP), albumin/globulin ratio (A/G), albumin (Alb), total bilirubin (Bil), glucose, triglyceride (TG), total cholesterol (T-Chol), urea nitrogen (BUN), creatinine (Cre), sodium (Na), chlorine (Cl), potassium (K), calcium (Ca), inorganic phosphorus (IP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and γ-glutamyl transpeptidase (γ-GTP).

Organ weights and histopathological assessment

Complete necropsy was performed for all animals, and the brain, thymus, lungs, heart, spleen, liver, adrenal glands, kidneys, and testes were weighed. These organs and the following tissues were fixed in 10% neutral-buffered formalin, and paraffin-embedded sections were prepared and stained with hematoxylin and eosin for histopathological examination: skin, mammary gland, sternum with marrow, femur with marrow, mandibular and mesenteric lymph nodes, salivary glands, aorta, trachea, tongue, esophagus, stomach, small and large intestines, pancreas, urinary bladder, epididymides, seminal vesicles, prostate gland, bulbourethral glands, pituitary gland, thyroid glands, parathyroid glands, spinal cord with vertebrae, trigeminal nerve, sciatic nerve, Harderian glands, femoral skeletal muscle, and nasal cavity. The testes and eyes were fixed in Bouin’s fixative and Davidson’s solution, respectively. Bony tissues, including the nasal cavity, vertebrae, sternum, and femur, were decalcified with a mixture of 10% formic acid and 10% buffered formalin for up to 2 weeks. Histopathological assessment was first performed on all tissues of the control and highest dose groups and on the liver and kidney of all remaining dose groups. If a chemical treatment-related change appeared at the highest dose, the relevant tissues from the lower dose groups then also underwent examination.

Expression of CYP2E1 was examined in the liver using paraffin-embedded sections with immunohistochemistry. After antigen retrieval by an autoclave in 10 mM citrate buffer (pH 6.0) for 15 min at 121ºC, inhibition of endogenous peroxidase activity by immersion in 3% hydrogen peroxide/methanol solution was performed. Then the sections were incubated with a primary antibody for CYP2E1 (diluted 1:1000; anti-cytochrome P450 2E1 rabbit polyclonal antibody; Abcam, Cambridge, UK) for 12 hr at 4ºC. Visualization of antibody binding was performed using a VectaStain Elite ABC Kit (Vector Laboratories, Burlingame, CA, USA). All sections were counterstained with hematoxylin.

Statistical analysis

Variance in data for body and organ weights and the results of hematology and serum biochemistry were checked using Bartlett’s test for homogeneity. When the data were homogeneous, one-way analysis of variance was conducted. In heterogeneous cases, the Kruskal-Wallis test was applied. When statistically significant differences were indicated, Dunnett’s or nonparametric Dunnett’s multiple tests were employed for comparisons between the control and other groups. For incidences of histopathological findings, Fisher’s exact probability test was applied. Differences with P values of less than 0.05 were considered statistically significant.

RESULTS

In-life parameters

No clinical signs were noted throughout the experimental period, and all animals survived until the scheduled necropsy. Significant reduction of body weight in F344 and lean rats treated with 8,000 ppm APAP was observed at the end of administration (Table 1). In addition, body weight gain in F344 and lean rats was significantly reduced with 8,000 ppm APAP-treatment compared with that of controls from week 1 until the end of the experiment (data not shown). APAP treatment did not change the daily food intake in all 3 strains.

Table 1. Body weight, food intake, and chemical intake data for male F344 and Zucker rats treated with APAP for 13 weeks.

Group	Body weight (g)						Food intake	Chemical intake
Group	Initial			Final^a			(g/rat/day)	(mg/kg bw/day)
F344
0 ppm	113.3	±	5.5	324.1	±	9.6	13.5	0.0
80 ppm	112.8	±	5.5	325.2	±	11.0	13.4	4.4
253 ppm	113.0	±	5.4	316.0	±	11.7	13.3	13.8
800 ppm	113.0	±	5.3	323.9	±	10.9	13.6	44.2
2530 ppm	112.9	±	5.4	324.5	±	15.1	13.8	142.1
8000 ppm	113.1	±	4.7	284.9	±	17.4**	13.0	490.8
Zucker (lean)
0 ppm	138.0	±	7.6	467.9	±	16.9	19.6	0.0
80 ppm	137.5	±	7.7	457.2	±	24.1	18.9	4.7
253 ppm	138.0	±	8.0	465.4	±	16.0	19.2	14.6
800 ppm	138.4	±	6.0	459.3	±	17.1	19.1	46.5
2530 ppm	137.7	±	8.4	454.2	±	23.0	19.6	152.8
8000 ppm	137.8	±	7.1	432.9	±	27.2**	19.5	521.9
Zucker (fatty)
0 ppm	185.8	±	10.5	649.3	±	58.6	31.1	0.0
80 ppm	185.6	±	11.4	670.4	±	49.0	32.6	5.5
253 ppm	185.9	±	9.8	672.2	±	55.4	32.0	17.3
800 ppm	186.6	±	5.6	664.7	±	58.1	31.7	55.4
2530 ppm	185.8	±	8.3	702.6	±	35.1	31.8	169.2
8000 ppm	185.7	±	8.9	673.3	±	53.4	30.9	539.9

Values are means ± SDs. APAP, acetaminophen. **Significantly different from the corresponding control at P < 0.01. ^aAt the end of administration.

Hematology

Data for hematology are summarized in Table 2. Significant increases in MCV and MCH were detected in F344 and lean rats treated with 8,000 ppm APAP (Table 2). Although significant decreases in MCHC and eosinophil counts were observed in APAP-treated fatty rats, the lack of a dose dependency suggested that these changes were not related to test substance exposure.

Table 2. Hematology data for male F344 and Zucker rats treated with APAP for 13 weeks.

Group		0 ppm			80 ppm			253 ppm			800 ppm			2530 ppm			8000 ppm
No. of animals		10			10			10			10			10			10
F344
WBC	x 10²/μL	37.5	±	4.6	34.9	±	3.8	40.5	±	5.9	32.5	±	6.0	35.8	±	3.7	37.9	±	6.8
RBC	x 10⁴/μL	947.4	±	48.2	952.1	±	40.1	940.2	±	54.0	917.4	±	98.8	956.5	±	46.5	928.6	±	57.9
HGB	g/dL	16.4	±	0.6	16.5	±	0.5	16.3	±	0.7	15.7	±	1.6	16.5	±	0.7	16.5	±	1.0
HCT	%	51.2	±	2.0	51.5	±	1.8	50.9	±	2.4	49.6	±	5.1	51.5	±	2.2	52.4	±	3.3
MCV	fL	54.1	±	0.9	54.1	±	0.6	54.1	±	0.7	54.1	±	0.5	53.8	±	0.7	56.4	±	0.6**
MCH	pg	17.3	±	0.4	17.3	±	0.4	17.3	±	0.4	17.2	±	0.4	17.2	±	0.4	17.8	±	0.5*
MCHC	g/dL	32.1	±	0.4	32.1	±	0.6	32.1	±	0.6	31.7	±	0.6	32.0	±	0.3	31.5	±	0.6
PLT	x 10⁴/μL	77.3	±	5.8	77.3	±	3.9	76.0	±	5.1	66.9	±	16.7	78.1	±	5.0	69.8	±	11.9
Differential cell count
Band	%	0.1	±	0.2	0.1	±	0.2	0.1	±	0.2	0.1	±	0.2	0.1	±	0.2	0.2	±	0.3
Seg	%	30.3	±	5.7	32.5	±	10.5	34.0	±	4.7	27.5	±	4.7	31.7	±	6.2	24.3	±	2.6
Eosin	%	0.9	±	1.0	1.1	±	1.0	1.3	±	0.9	1.4	±	1.1	1.1	±	0.7	1.0	±	0.9
Baso	%	0.0	±	0.0	0.0	±	0.0	0.0	±	0.0	0.0	±	0.0	0.0	±	0.0	0.0	±	0.0
Lymph	%	68.3	±	6.0	66.0	±	11.0	64.6	±	4.6	70.7	±	4.6	66.9	±	6.6	74.3	±	2.9
Mono	%	0.5	±	0.8	0.3	±	0.3	0.1	±	0.2	0.5	±	0.5	0.3	±	0.5	0.3	±	0.5
Ebl	/200 WBC	0.0	±	0.0	0.0	±	0.0	0.0	±	0.0	0.0	±	0.0	0.0	±	0.0	0.1	±	0.3
Zucker (lean)
WBC	x 10²/μL	49.8	±	13.8	42.9	±	13.7	49.4	±	16.2	38.6	±	6.7	41.6	±	10.6	40.7	±	7.6
RBC	x 10⁴/μL	950.2	±	64.4	985.1	±	64.3	985.2	±	71.5	995.9	±	44.9	998.6	±	73.5	897.6	±	83.8
HGB	g/dL	15.4	±	0.6	15.6	±	1.0	15.5	±	1.0	15.8	±	0.4	15.7	±	1.4	15.3	±	1.3
HCT	%	49.0	±	2.6	50.6	±	3.2	50.2	±	3.2	50.8	±	2.0	50.9	±	3.8	48.4	±	3.9
MCV	fL	51.6	±	1.0	51.4	±	1.2	51.0	±	1.2	51.0	±	1.2	50.9	±	0.5	54.0	±	1.2**
MCH	pg	16.2	±	0.8	15.9	±	0.4	15.8	±	0.5	15.9	±	0.6	15.7	±	0.4	17.0	±	0.7*
MCHC	g/dL	31.4	±	1.1	30.8	±	0.3	30.9	±	0.6	31.1	±	0.8	30.9	±	0.8	31.6	±	1.0
PLT	x 10⁴/μL	121.9	±	18.5	114.5	±	13.2	120.7	±	10.3	115.3	±	19.0	111.1	±	12.3	106.8	±	35.2
Differential cell count
Band	%	0.2	±	0.2	0.4	±	0.5	0.2	±	0.3	0.2	±	0.3	0.4	±	0.4	0.2	±	0.3
Seg	%	23.0	±	6.9	20.9	±	6.0	25.6	±	6.3	22.5	±	6.4	23.3	±	4.2	24.8	±	4.2
Eosin	%	1.6	±	1.4	2.8	±	1.7	1.8	±	2.4	1.6	±	1.1	2.2	±	1.2	1.7	±	0.8
Baso	%	0.0	±	0.0	0.0	±	0.0	0.0	±	0.0	0.0	±	0.0	0.0	±	0.0	0.0	±	0.0
Lymph	%	75.2	±	6.5	75.7	±	6.0	72.2	±	7.5	75.7	±	5.9	73.9	±	5.0	73.1	±	4.0
Mono	%	0.2	±	0.2	0.3	±	0.4	0.3	±	0.3	0.1	±	0.2	0.3	±	0.3	0.2	±	0.3
Ebl	/200 WBC	0.0	±	0.0	0.0	±	0.0	0.0	±	0.0	0.0	±	0.0	0.0	±	0.0	0.1	±	0.3
Zucker (fatty)
WBC	x 10²/μL	72.2	±	17.0	64.0	±	13.1	70.3	±	14.0	67.8	±	24.2	63.2	±	11.5	63.9	±	15.1
RBC	x 10⁴/μL	964.2	±	113.7	1058	±	77.7	1040	±	84.3	1014	±	144.8	1005	±	37.4	988.0	±	38.9
HGB	g/dL	15.4	±	1.5	16.3	±	0.9	16.0	±	1.3	15.6	±	2.0	15.9	±	0.5	16.2	±	0.8
HCT	%	48.1	±	5.0	51.7	±	3.5	51.0	±	4.0	50.3	±	6.6	50.0	±	1.7	50.8	±	2.6
MCV	fL	50.0	±	1.6	48.9	±	0.9	49.1	±	1.2	49.7	±	1.5	49.8	±	1.0	51.5	±	1.7
MCH	pg	16.1	±	0.9	15.4	±	0.6	15.4	±	0.6	15.5	±	0.7	15.8	±	0.6	16.4	±	0.7
MCHC	g/dL	32.1	±	1.2	31.5	±	0.7	31.4	±	0.6	31.1	±	0.9*	31.7	±	0.7	31.9	±	0.7
PLT	x 10⁴/μL	127.9	±	36.4	142.5	±	21.4	136.6	±	15.2	135.9	±	15.7	135.5	±	23.4	140.5	±	20.9
Differential cell count
Band	%	0.6	±	0.8	0.5	±	0.5	1.0	±	0.5	0.4	±	0.4	0.4	±	0.4	0.6	±	0.4
Seg	%	38.9	±	11.1	41.1	±	7.1	36.9	±	15.6	33.9	±	7.2	39.2	±	13.0	37.6	±	8.4
Eosin	%	2.1	±	1.4	1.1	±	0.8	1.0	±	0.9*	0.9	±	1.0*	1.3	±	0.6	1.0	±	0.5*
Baso	%	0.0	±	0.0	0.0	±	0.0	0.0	±	0.0	0.0	±	0.0	0.0	±	0.0	0.0	±	0.0
Lymph	%	58.1	±	10.7	57.2	±	7.0	60.9	±	15.4	64.8	±	7.0	58.9	±	12.6	60.5	±	8.4
Mono	%	0.4	±	0.6	0.1	±	0.2	0.3	±	0.4	0.1	±	0.3	0.3	±	0.5	0.4	±	0.5
Ebl	/200 WBC	0.1	±	0.3	0.1	±	0.3	0.0	±	0.0	0.0	±	0.0	0.0	±	0.0	0.0	±	0.0

Values are means ± SDs. APAP, acetaminophen. * and **; significantly different from the corresponding control at P < 0.05 and < 0.01, respectively.

Serum biochemistry

Data for serum biochemistry are summarized in Table 3. In APAP-treated F344 rats, significant increases in T-Chol and K and decreases in Cre, AST, and ALT were observed in the 8,000 ppm group (Table 3). In lean rats, significant increases in A/G and Alb in the 2,530 and 8,000 ppm groups and a significant decrease in Bil in the 8,000 ppm group were noted. There were no significant differences among APAP-treated fatty rats. Although significant fluctuations were also detected in TP, Alb, Na, Cl, and Ca in F344 rats and in T-Chol, Na, and Cl in lean rats, the lack of any dose-dependent changes suggested that there was no association with test substance exposure.

Table 3. Serum biochemistry data for male F344 and Zucker rats treated with APAP for 13 weeks.

Group		0 ppm			80 ppm			253 ppm			800 ppm			2530 ppm			8000 ppm
No. of animals		10			10			10			10			10			10
F344
TP	(g/dL)	6.2	±	0.3	6.2	±	0.3	6.5	±	0.1**	6.3	±	0.2	6.5	±	0.2**	6.4	±	0.2*
A/G		2.0	±	0.1	1.9	±	0.1	1.9	±	0.1	1.9	±	0.1	1.9	±	0.1	1.9	±	0.1
Alb	(g/dL)	4.1	±	0.2	4.1	±	0.2	4.2	±	0.1	4.2	±	0.1	4.2	±	0.2*	4.2	±	0.1
Bil	(mg/dL)	0.04	±	0.01	0.04	±	0.01	0.03	±	0.01	0.04	±	0.01	0.04	±	0.01	0.04	±	0.01
Glucose	(mg/dL)	142.9	±	28.8	143.0	±	19.8	151.4	±	15.9	146.5	±	18.3	149.8	±	26.3	150.1	±	27.0
TG	(mg/dL)	80.1	±	28.6	81.5	±	24.9	80.1	±	28.6	83.7	±	34.6	82.9	±	21.4	67.4	±	24.0
T-Chol	(mg/dL)	50.0	±	3.8	50.2	±	3.8	49.0	±	2.7	51.0	±	4.0	52.1	±	2.3	61.9	±	7.4**
BUN	(mg/dL)	20.6	±	3.0	21.2	±	3.8	22.3	±	3.1	21.0	±	3.2	19.8	±	2.9	17.4	±	2.1
Cre	(mg/dL)	0.29	±	0.03	0.29	±	0.04	0.30	±	0.04	0.28	±	0.03	0.26	±	0.02	0.23	±	0.01**
Na	(mEq/L)	140.0	±	2.7	141.2	±	2.3	144.2	±	0.8**	142.5	±	2.2*	144.3	±	1.1**	143.7	±	0.5**
Cl	(mEq/L)	100.0	±	2.4	100.3	±	2.5	104.1	±	2.0**	102.4	±	2.0	103.7	±	2.3**	103.0	±	1.2*
K	(mEq/L)	4.4	±	0.2	4.2	±	0.2	4.3	±	0.3	4.4	±	0.2	4.4	±	0.2	4.7	±	0.2*
Ca	(mg/dL)	9.7	±	0.4	9.8	±	0.3	10.2	±	0.3*	9.9	±	0.5	10.3	±	0.3**	10.2	±	0.1*
IP	(mg/dL)	5.0	±	0.6	4.8	±	0.8	4.9	±	0.8	5.2	±	0.6	5.5	±	0.7	5.6	±	0.5
AST	(IU/L)	83.3	±	9.3	84.1	±	5.9	79.0	±	4.3	83.4	±	10.4	78.9	±	9.3	66.3	±	5.1**
ALT	(IU/L)	59.8	±	11.0	62.6	±	8.0	58.9	±	4.7	62.5	±	6.7	59.7	±	8.5	36.7	±	4.4**
ALP	(IU/L)	374.9	±	36.9	377.0	±	37.6	389.2	±	28.9	370.3	±	43.5	370.5	±	34.6	345.3	±	55.5
γ-GTP	(IU/L)	< 3			< 3			< 3			< 3			< 3			< 3
Zucker (lean)
TP	(g/dL)	6.2	±	0.2	6.0	±	0.1	6.2	±	0.3	6.1	±	0.2	6.3	±	0.2	6.1	±	0.2
A/G		1.8	±	0.1	1.9	±	0.1	1.8	±	0.2	1.9	±	0.1	2.0	±	0.1*	2.3	±	0.1**
Alb	(g/dL)	4.0	±	0.1	4.0	±	0.1	4.0	±	0.2	4.0	±	0.1	4.1	±	0.2*	4.2	±	0.1**
Bil	(mg/dL)	0.06	±	0.01	0.05	±	0.01	0.05	±	0.02	0.06	±	0.01	0.05	±	0.01	0.05	±	0.01*
Glucose	(mg/dL)	160.2	±	42.2	158.9	±	34.9	149.5	±	30.3	167.3	±	36.7	147.5	±	15.3	154.1	±	24.0
TG	(mg/dL)	95.2	±	25.8	96.6	±	31.6	94.6	±	40.7	97.7	±	21.2	94.8	±	32.0	67.9	±	16.3
T-Chol	(mg/dL)	85.2	±	21.9	71.3	±	6.6	78.3	±	17.1	80.9	±	13.3	73.9	±	5.3	68.0	±	12.3*
BUN	(mg/dL)	14.7	±	2.1	15.2	±	3.3	15.7	±	3.4	14.8	±	3.0	14.1	±	2.0	13.7	±	2.5
Cre	(mg/dL)	0.26	±	0.03	0.26	±	0.03	0.27	±	0.03	0.26	±	0.03	0.25	±	0.04	0.23	±	0.03
Na	(mEq/L)	142.8	±	1.4	143.5	±	2.3	143.7	±	1.4	143.6	±	0.7	144.9	±	0.6**	144.0	±	1.2
Cl	(mEq/L)	102.2	±	1.7	103.5	±	2.4	103.4	±	1.3	103.7	±	1.3	104.7	±	1.3**	103.9	±	1.7
K	(mEq/L)	4.7	±	0.1	4.8	±	0.2	4.7	±	0.2	4.7	±	0.2	4.6	±	0.2	4.7	±	0.1
Ca	(mg/dL)	9.7	±	0.3	9.5	±	0.2	9.7	±	0.4	9.7	±	0.2	10.0	±	0.2	9.9	±	0.2
IP	(mg/dL)	5.7	±	0.5	5.6	±	0.6	5.5	±	0.7	5.7	±	0.5	5.9	±	0.4	5.7	±	0.4
AST	(IU/L)	119.7	±	25.7	104.5	±	12.5	111.7	±	17.9	110.5	±	19.6	113.8	±	17.3	102.6	±	16.5
ALT	(IU/L)	41.5	±	7.2	37.7	±	4.5	35.0	±	5.8	38.9	±	8.1	38.6	±	9.8	36.8	±	7.3
ALP	(IU/L)	199.2	±	30.2	192.0	±	38.7	200.5	±	26.9	205.5	±	25.5	201.7	±	28.9	229.2	±	46.5
γ-GTP	(IU/L)	< 3			< 3			< 3			< 3			< 3			< 3
Zucker (fatty)
TP	(g/dL)	7.0	±	0.5	7.5	±	0.8	7.2	±	0.3	7.1	±	0.7	7.0	±	0.6	7.2	±	0.4
A/G		1.3	±	0.3	1.2	±	0.3	1.3	±	0.3	1.3	±	0.2	1.3	±	0.2	1.4	±	0.2
Alb	(g/dL)	3.9	±	0.5	3.9	±	0.4	4.1	±	0.4	4.0	±	0.6	3.9	±	0.3	4.2	±	0.4
Bil	(mg/dL)	0.06	±	0.04	0.11	±	0.11	0.07	±	0.03	0.07	±	0.03	0.06	±	0.03	0.07	±	0.03
Glucose	(mg/dL)	263.3	±	103.9	319.9	±	88.1	224.0	±	98.7	297.7	±	106.7	293.9	±	120.9	258.4	±	77.2
TG	(mg/dL)	646.4	±	310.2	1102	±	803.2	716.5	±	380.2	749.4	±	296.1	712.1	±	284.5	740.1	±	165.7
T-Chol	(mg/dL)	165.5	±	49.5	204.0	±	71.8	172.9	±	50.7	162.8	±	33.2	176.1	±	38.8	192.2	±	28.5
BUN	(mg/dL)	18.2	±	2.8	19.4	±	5.0	18.7	±	4.3	19.2	±	4.9	17.5	±	2.8	16.1	±	2.5
Cre	(mg/dL)	0.18	±	0.02	0.17	±	0.02	0.17	±	0.03	0.15	±	0.03	0.17	±	0.02	0.15	±	0.02
Na	(mEq/L)	142.1	±	3.1	140.9	±	3.0	143.1	±	2.6	140.3	±	3.2	140.8	±	3.1	141.3	±	2.0
Cl	(mEq/L)	96.2	±	4.6	94.7	±	4.3	96.5	±	4.0	94.9	±	3.3	95.1	±	3.9	96.4	±	2.0
K	(mEq/L)	4.6	±	0.3	4.5	±	0.2	4.6	±	0.2	4.4	±	0.3	4.5	±	0.3	4.7	±	0.3
Ca	(mg/dL)	10.5	±	0.5	10.7	±	0.4	10.8	±	0.5	10.4	±	0.5	10.2	±	0.4	10.5	±	0.5
IP	(mg/dL)	6.4	±	0.6	5.9	±	0.8	6.7	±	0.8	6.2	±	0.6	6.0	±	0.4	6.1	±	0.7
AST	(IU/L)	250.6	±	162.2	259.9	±	199.5	197.1	±	123.7	205.7	±	99.2	261.1	±	310.3	121.5	±	24.4
ALT	(IU/L)	187.8	±	164.8	186.1	±	181.8	124.1	±	99.8	147.0	±	99.9	168.7	±	197.1	65.0	±	20.5
ALP	(IU/L)	444.7	±	178.3	465.0	±	177.0	359.2	±	105.5	484.7	±	162.3	426.5	±	168.3	303.4	±	127.1
γ-GTP	(IU/L)	< 3			< 3			< 3			< 3			< 3			< 3

Values are means ± SDs. APAP, acetaminophen. * and **; significantly different from the corresponding control at P < 0.05 and < 0.01, respectively.

Organ weights

Data for organ weights are summarized in Table 4. In APAP-treated F344 rats, significant decreases in absolute and relative spleen weights and increases in relative weights of the brain, liver, and kidneys were noted in the 8,000 ppm group (Table 4). As significant increase in relative brain weights and a significant decrease in absolute testes weights in lean rats treated with 8,000 ppm APAP were observed. There were no significant differences in organ weights among APAP-treated fatty rats. Although significant changes were observed in the relative weights of the heart and testes in APAP-treated F344 rats and in the absolute brain weights and relative adrenals weights in APAP-treated lean rats, the lack of any dose dependency suggested that these observations were not associated with test substance exposure.

Table 4. Organ weight data for male F344 and Zucker rats treated with APAP for 13 weeks.

Group		0 ppm			80 ppm			253 ppm			800 ppm			2530 ppm			8000 ppm
No. of animals		10			10			10			10			10			10
F344
Body weight (g)		313.5	±	9.7	314.0	±	9.9	305.3	±	10.7	312.4	±	9.8	312.6	±	14.4	275.0	±	16.4**
Absolute (g)	Brain	1.91	±	0.05	2.03	±	0.27	1.96	±	0.05	1.95	±	0.06	1.92	±	0.06	1.92	±	0.05
	Thymus	0.21	±	0.06	0.18	±	0.02	0.19	±	0.04	0.20	±	0.03	0.20	±	0.03	0.17	±	0.02
	Lung	0.88	±	0.07	0.86	±	0.05	0.84	±	0.10	0.91	±	0.12	0.89	±	0.10	0.79	±	0.09
	Heart	0.86	±	0.05	0.87	±	0.02	0.90	±	0.06	0.89	±	0.07	0.89	±	0.04	0.82	±	0.04
	Spleen	0.61	±	0.04	0.62	±	0.02	0.61	±	0.04	0.62	±	0.02	0.61	±	0.04	0.50	±	0.03**
	Liver	7.03	±	0.40	7.13	±	0.25	7.17	±	0.44	7.09	±	0.28	7.36	±	0.44	7.15	±	0.78
	Adrenals	0.04	±	0.01	0.03	±	0.01	0.04	±	0.01	0.04	±	0.00	0.04	±	0.00	0.03	±	0.00
	Kidneys	1.83	±	0.12	1.86	±	0.10	1.86	±	0.10	1.83	±	0.15	1.84	±	0.11	1.75	±	0.07
	Testes	2.87	±	0.22	2.99	±	0.10	3.01	±	0.15	2.94	±	0.29	3.00	±	0.13	2.77	±	0.09
Relative (%)	Brain	0.61	±	0.02	0.65	±	0.09	0.64	±	0.02	0.63	±	0.03	0.62	±	0.03	0.70	±	0.04**
	Thymus	0.07	±	0.02	0.06	±	0.01	0.06	±	0.01	0.07	±	0.01	0.06	±	0.01	0.06	±	0.00
	Lung	0.28	±	0.02	0.27	±	0.02	0.28	±	0.03	0.29	±	0.04	0.28	±	0.02	0.29	±	0.04
	Heart	0.27	±	0.01	0.28	±	0.01	0.30	±	0.02**	0.29	±	0.02	0.28	±	0.01	0.30	±	0.02**
	Spleen	0.19	±	0.01	0.20	±	0.01	0.20	±	0.01	0.20	±	0.01	0.20	±	0.01	0.18	±	0.01*
	Liver	2.24	±	0.09	2.27	±	0.07	2.35	±	0.13	2.27	±	0.08	2.36	±	0.09	2.60	±	0.17**
	Adrenals	0.01	±	0.00	0.01	±	0.00	0.01	±	0.00	0.01	±	0.00	0.01	±	0.00	0.01	±	0.00
	Kidneys	0.58	±	0.03	0.59	±	0.05	0.61	±	0.04	0.58	±	0.05	0.59	±	0.02	0.64	±	0.04*
	Testes	0.92	±	0.06	0.95	±	0.03	0.99	±	0.03*	0.94	±	0.08	0.96	±	0.04	1.01	±	0.05**
Zucker (lean)
Body weight (g)		450.6	±	15.4	438.2	±	25.3	449.6	±	14.0	443.9	±	15.7	438.8	±	21.6	418.9	±	26.0**
Absolute (g)	Brain	1.96	±	0.09	2.04	±	0.04	2.08	±	0.10**	2.06	±	0.06*	2.03	±	0.07	2.01	±	0.08
	Thymus	0.32	±	0.04	0.29	±	0.06	0.34	±	0.04	0.33	±	0.05	0.29	±	0.05	0.29	±	0.04
	Lung	1.19	±	0.17	1.23	±	0.17	1.18	±	0.20	1.28	±	0.21	1.09	±	0.18	1.21	±	0.25
	Heart	1.16	±	0.10	1.15	±	0.10	1.19	±	0.07	1.18	±	0.06	1.15	±	0.05	1.10	±	0.14
	Spleen	0.59	±	0.11	0.54	±	0.05	0.58	±	0.08	0.55	±	0.05	0.53	±	0.07	0.51	±	0.05
	Liver	12.0	±	0.92	11.8	±	0.67	11.9	±	0.84	11.7	±	0.82	11.7	±	0.92	11.6	±	0.80
	Adrenals	0.04	±	0.00	0.04	±	0.00	0.04	±	0.00	0.04	±	0.01	0.04	±	0.01	0.04	±	0.00
	Kidneys	2.71	±	0.13	2.72	±	0.17	2.73	±	0.27	2.69	±	0.25	2.58	±	0.10	2.66	±	0.19
	Testes	3.12	±	0.12	3.09	±	0.19	3.30	±	0.19	3.32	±	0.24	3.24	±	0.17	2.76	±	0.41**
Relative (%)	Brain	0.44	±	0.02	0.47	±	0.03	0.46	±	0.03	0.46	±	0.01	0.46	±	0.03	0.48	±	0.04**
	Thymus	0.07	±	0.01	0.07	±	0.02	0.08	±	0.01	0.07	±	0.01	0.07	±	0.01	0.07	±	0.01
	Lung	0.27	±	0.04	0.28	±	0.04	0.26	±	0.04	0.29	±	0.05	0.25	±	0.05	0.29	±	0.06
	Heart	0.26	±	0.02	0.26	±	0.02	0.26	±	0.01	0.27	±	0.02	0.26	±	0.02	0.26	±	0.03
	Spleen	0.13	±	0.02	0.12	±	0.01	0.13	±	0.02	0.12	±	0.01	0.12	±	0.02	0.12	±	0.01
	Liver	2.66	±	0.20	2.70	±	0.23	2.64	±	0.19	2.64	±	0.17	2.67	±	0.14	2.78	±	0.13
	Adrenals	0.01	±	0.00	0.01	±	0.00	0.01	±	0.00	0.01	±	0.00	0.01	±	0.00**	0.01	±	0.00
	Kidneys	0.60	±	0.02	0.62	±	0.06	0.61	±	0.06	0.60	±	0.05	0.59	±	0.02	0.64	±	0.05
	Testes	0.69	±	0.04	0.71	±	0.06	0.74	±	0.05	0.75	±	0.05	0.74	±	0.06	0.66	±	0.12
Zucker (fatty)
Body weight (g)		628.3	±	59.6	647.2	±	52.1	652.6	±	59.3	649.4	±	61.5	678.3	±	34.0	657.0	±	57.1
Absolute (g)	Brain	1.84	±	0.06	1.89	±	0.07	1.87	±	0.06	1.87	±	0.06	1.88	±	0.11	1.84	±	0.04
	Thymus	0.26	±	0.08	0.27	±	0.06	0.28	±	0.08	0.27	±	0.07	0.32	±	0.05	0.30	±	0.05
	Lung	1.09	±	0.16	1.07	±	0.15	1.14	±	0.22	1.02	±	0.21	1.20	±	0.14	1.10	±	0.22
	Heart	1.29	±	0.08	1.33	±	0.09	1.30	±	0.05	1.31	±	0.10	1.32	±	0.09	1.31	±	0.06
	Spleen	0.71	±	0.18	0.69	±	0.10	0.68	±	0.06	0.69	±	0.19	0.68	±	0.11	0.63	±	0.06
	Liver	24.2	±	5.74	30.1	±	6.08	25.2	±	5.12	27.0	±	5.75	26.4	±	5.63	24.5	±	2.27
	Adrenals	0.05	±	0.01	0.06	±	0.01	0.05	±	0.01	0.05	±	0.01	0.05	±	0.01	0.05	±	0.01
	Kidneys	3.52	±	0.46	3.74	±	0.46	3.50	±	0.47	3.51	±	0.36	3.67	±	0.33	3.73	±	0.38
	Testes	2.71	±	0.67	3.08	±	0.25	3.14	±	0.35	2.93	±	0.41	2.98	±	0.36	2.49	±	0.46
Relative (%)	Brain	0.29	±	0.02	0.29	±	0.02	0.29	±	0.03	0.29	±	0.02	0.28	±	0.03	0.28	±	0.03
	Thymus	0.04	±	0.01	0.04	±	0.01	0.04	±	0.01	0.04	±	0.01	0.05	±	0.01	0.05	±	0.01
	Lung	0.17	±	0.03	0.17	±	0.03	0.18	±	0.04	0.16	±	0.04	0.18	±	0.03	0.17	±	0.04
	Heart	0.21	±	0.02	0.21	±	0.03	0.20	±	0.02	0.20	±	0.03	0.19	±	0.01	0.20	±	0.02
	Spleen	0.11	±	0.04	0.11	±	0.01	0.11	±	0.02	0.11	±	0.03	0.10	±	0.02	0.10	±	0.02
	Liver	3.88	±	1.01	4.69	±	1.08	3.93	±	1.04	4.15	±	0.72	3.91	±	0.91	3.76	±	0.44
	Adrenals	0.01	±	0.00	0.01	±	0.00	0.01	±	0.00	0.01	±	0.00	0.01	±	0.00	0.01	±	0.00
	Kidneys	0.57	±	0.10	0.58	±	0.10	0.55	±	0.13	0.55	±	0.10	0.54	±	0.06	0.57	±	0.09
	Testes	0.43	±	0.09	0.48	±	0.05	0.48	±	0.06	0.45	±	0.06	0.44	±	0.06	0.39	±	0.09

Values are means ± SDs. APAP, acetaminophen. * and **; significantly different from the corresponding control at P < 0.05 and 0.01, respectively.

Histopathological findings

Histopathological findings are summarized in Table 5. Focal glomerulosclerosis and chronic nephropathy characterized by tubular basophilia, inflammatory cell infiltration, and hyaline cast, which are known to occur spontaneously in obese Zucker rats (Coimbra et al., 2000), were noted in the kidneys of fatty rats (Table 5). Moderate to severe vacuolar degeneration of hepatocytes was diffusely observed in the livers of fatty rats. There were no modifying effects of APAP on these spontaneous lesions. Centrilobular hypertrophy of hepatocytes was observed in a single case in both F344 and lean rats treated with 8,000 ppm APAP. In immunohistochemical analysis, CYP2E1 expression in hepatocytes was increased in 8,000 ppm APAP-treated F344 rats compared to the controls (Fig. 1). In both the control and APAP-treated groups, the expression levels of CYP2E1 in lean and fatty rats were lower than that in F344 rats. Although several other lesions were sporadically detected in APAP-treated rats, no significant treatment-dependent alterations in their incidences were observed.

Table 5. Histopathological findings in male F344 and Zucker rats treated with APAP for 13 weeks.

Organs and findings	Group (ppm)	0	80	253	800	2530	8000
Organs and findings	No. of animals	10	10	10	10	10	10
F344
Liver	Microgranuloma	4	2	3	2	3	3
	Hepatocellular hypertrophy, centrilobular	0	0	0	0	0	1
Kidney	Tubular basophilia	0	1	2	0	0	1
	Infiltrate, inflammatory cell, interstitium	1	2	1	1	0	2
	Hyaline cast	0	0	0	1	0	0
	Mineralization	1	0	1	0	0	2
Heart	Mononuclear cell infiltration, focal	5	-	-	-	-	3
Pancreas	Mononuclear cell infiltration, focal	1	-	-	-	-	1
Tongue	Inflammation, focal, mild	0	-	-	-	-	1
Testis	Degeneration/atrophy, tubular, unilateral	1	-	-	-	-	0
	Degeneration/atrophy, tubular, bilateral	1	-	-	-	-	0
Epididymis	Cell debris, luminal	2	-	-	-	-	1
Prostate	Infiltrate, inflammatory cell	3	-	-	-	-	5
	Atrophy	0	-	-	-	-	2
Zucker (lean)
Liver	Microgranuloma	7	5	5	6	3	5
	Hepatocellular hypertrophy, centrilobular	0	0	0	0	0	1
Kidney	Tubular basophilia	1	1	1	1	0	0
	Infiltrate, inflammatory cell, interstitium	1	2	1	1	4	0
	Hyaline cast	1	1	2	3	1	0
	Mineralization	0	1	0	0	0	0
	Dilation of pelvis	1	0	2	0	1	0
	Cyst	0	0	1	0	0	1
	Papillary necrosis	0	1	0	0	0	0
Heart	Mononuclear cell infiltration, focal	1	-	-	-	-	0
Lung	Osseous metaplasia	1	-	-	-	-	1
Pancreas	Mononuclear cell infiltration, focal	0	-	-	-	-	1
Testis	Degeneration/atrophy, tubular, unilateral	2	-	-	-	-	1
	Dilation, tubular, unilateral	1	-	-	-	-	0
Epididymis	Cell debris, luminal	2	-	-	-	-	1
	Atrophy, ductal	1	-	-	-	-	1
	Sperm granuloma	2	-	-	-	-	0
Prostate	Infiltrate, inflammatory cell	7	-	-	-	-	9
Harderian gland	Mononuclear cell infiltration, focal	0	-	-	-	-	4
Zucker (fatty)
Liver	Microgranuloma	1	0	3	0	0	1
	Vacuolar degeneration, moderate	4	4	6	3	6	5
	Vacuolar degeneration, severe	5	6	4	7	4	3
	Necrosis, focal	0	1	0	0	0	0
Kidney	Glomerulosclerosis, focal	7	6	5	5	7	6
	Tubular basophilia	7	9	7	7	8	8
	Infiltrate, inflammatory cell, interstitium	2	2	2	1	3	1
	Hyaline cast	9	10	8	8	9	10
	Mineralization	8	4	5	4	6	3
	Dilation of pelvis	0	0	1	0	2	0
	Pyelonephritis	0	0	1	0	0	0
	Papillary necrosis	0	0	1	0	0	1
Heart	Mononuclear cell infiltration, focal	1	-	-	-	-	0
Lung	Osseous metaplasia	1	-	-	-	-	0
Pancreas	Mononuclear cell infiltration, focal	1	-	-	-	-	2
Testis	Degeneration/atrophy, tubular, unilateral	2	-	-	-	-	1
	Degeneration/atrophy, tubular, bilateral	2	-	-	-	-	4
	Dilation, tubular, unilateral	1	-	-	-	-	0
Epididymis	Cell debris, luminal	3	-	-	-	-	4
	Atrophy, ductal	4	-	-	-	-	3
	Sperm granuloma	1	-	-	-	-	1
Prostate	Infiltrate, inflammatory cell	4	-	-	-	-	9
	Atrophy	1	-	-	-	-	0

APAP, acetaminophen; -, not evaluated.

Fig. 1

Immunohistochemistry for CYP2E1 in the livers of male F344 and obese Zucker (fatty) rats. Bars = 100 μm. (A) An untreated control F344 rat. (B) A F344 rat treated with 8,000 ppm acetaminophen (APAP). Note increased expression of CYP2E1 in hepatocytes. (C) An untreated fatty rat. (D) A fatty rat treated with 8,000 ppm APAP. There was no increase in CYP2E1 expression.

DISCUSSION

The present 13-week subchronic toxicity study demonstrated that dietary administration of 8,000 ppm APAP caused decreased body weight gain and centrilobular hepatocellular hypertrophy in F344 and lean rats. In addition, increases in serum T-Chol levels and relative liver weights in the 8,000 ppm group of F344 rats were observed as adverse effects. Increases in MCV and MCH in F344 and lean rats and an increase in K in F344 rats were considered to have no toxicological significance because there were no abnormalities in related parameters and significant histopathological changes in related organs. Among the changes observed in serum biochemistry in F344 and lean rats, decreases in Cre, AST, ALT, and Bil and increases in A/G and Alb were the opposite of toxic signs. Although significant decreases in absolute and relative spleen weights were noted in F344 rats treated with 8,000 ppm APAP, there were no treatment-related histopathological findings in the spleen. Increases or decreases in organ weights of the brain, kidneys, and testes in F344 and lean rats were thought to be associated with the reduced body weight gain.

Although fatty rats showed higher APAP intake and higher serum TG, T-Chol, AST, ALT, and ALP levels than lean and F344 rats, no significant changes in body weight gain and APAP-induced hepatotoxicity were observed. From our analysis, the NOAELs for APAP were 2,530 ppm for F344 and lean rats (142.1 and 152.8 mg/kg bw/day, respectively) and more than 8,000 ppm for fatty rats (> 539.9 mg/kg bw/day).

Several studies using rodent models of obesity, including animals fed a high-fat diet, have reported that obesity-related nonalcoholic fatty liver disease is associated with a higher risk of APAP-induced liver injury (Michaut et al., 2014). In the present study, although centrilobular hepatocellular hypertrophy was observed in both F344 and lean rats treated with the highest dose of APAP, no APAP-related hepatotoxicity was detected in fatty rats despite the existence of diffuse vacuolar degeneration of hepatocytes. Previous studies using obese Zucker rats also showed that the degree of liver injury induced by a single dose of APAP was lower in fatty rats than in lean rats (Blouin et al., 1987; Tuntaterdtum et al., 1993). In addition, Ito et al. demonstrated that a high-fat and high-carbohydrate diet attenuated APAP-induced acute hepatotoxicity in male C57BL/6 mice through the inhibition of CYP2E1 expression (Ito et al., 2006). Unlike in cases of other genetic obesity models, such as KK-A^y and db/db mice (Aubert et al., 2012), hepatic CYP2E1 activity has also been shown to be downregulated in obese Zucker rats compared with that in lean rats (Carmiel-Haggai et al., 2003; Irizar et al., 1995). In the present study, the expression level of CYP2E1 in fatty rats was lower than that in F344 rats, and this may explain the lower hepatotoxicity of APAP-treated fatty rats at least in part. Although there were no obvious differences in CYP2E1 expression between lean and fatty rats, it may be due to the relatively long administration period.

In the present study, the dose of 800 ppm was set as the intermediate dose since it was reported to be the LOAEL with a significant increase in relative liver weights in a previous 13-week toxicity study using F344 rats (NTP, 1993). This previous study has reported that liver lesions, including hepatocellular hypertrophy and chronic-active inflammation, were found with a high frequency in rats treated with 12,500 ppm APAP, whereas there were no hepatic lesions in the lower dose (6,200, 3,200, 1,600, and 800 ppm) groups (“sharp dose response”). Therefore, increased liver weights in the lower dose groups of the previous study may be an adaptive change. In the present study, because a single case of hepatocellular hypertrophy was observed in the 8,000 ppm group, the dose response of liver lesions was considered similar to that of the previous report.

In the risk assessment of chemicals, the concept of uncertainty factor (UF) has been used to convert the NOAEL into the tolerable or acceptable daily intake (TDI/ADI), a level of exposure “without appreciable health risk” (Burin and Saunders, 1999). The 100-fold UF represents the product of 2 separate 10-fold factors that allow for interspecies differences and human variability (Renwick and Lazarus, 1998). Intraspecies variations include age, genetic polymorphism, lifestyle, diseases, and various other factors, and studies have investigated whether the current 10-fold UF for intraspecies differences can account for human variability in these factors (Dorne, 2010). Although additional data using various types of chemicals are needed, the present study demonstrated that the NOAEL of obese rats was not less than one tenth of that of healthy counterparts and thus supported the validity of current UFs for intraspecies variation.

In conclusion, our results suggested that obese Zucker rats may be less susceptible to APAP-induced toxicity than their lean counterparts. However, obesity-related changes in susceptibility to toxicity can be specific to certain types of chemicals. Other underlying diseases, including diabetes and hypertension, could be investigated for further evaluation of the effects of basic diseases on susceptibility to toxicity.

ACKNOWLEDGMENTS

The authors thank Ayako Saikawa and Yoshimi Komatsu for expert technical assistance in processing histological materials. This work was supported by a grant from the Food Safety Commission of Japan (grant no. 1204).

Conflict of interest

The authors declare that there is no conflict of interest.

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