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A chronic toxicity study of diphenylarsinic acid in F344 rats in drinking water for 52 weeks
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Experimental and Toxicologic Pathology xxx (2016) xxx–xxx
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A chronic toxicity study of diphenylarsinic acid in F344 rats in drinking water for 52 weeks Takashi Yamaguchia , Min Gia , Shotarou Yamanoa,b , Masaki Fujiokaa , Kumiko Tatsumia , Satoko Kawachia , Naomi Ishiia , Kenichiro Doia , Anna Kakehashia , Hideki Wanibuchia,* a b
Department of Molecular Pathology, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan Division of Rare Cancer Research National Cancer Center Research Institute, Tsukiji 5-5-1, Chuo-ku Tokyo 104-0045, Japan
A R T I C L E I N F O
Article history: Received 22 July 2016 Received in revised form 13 September 2016 Accepted 6 October 2016 Keywords: Diphenylarsinic acid Chronic toxicity Bile duct toxicity F344 rats
A B S T R A C T
Diphenylarsinic acid (DPAA), a chemical warfare-related neurotoxic organic arsenical, is present in the groundwater and soil in some regions of Japan due to illegal dumping after World War II. The purpose of the present study was to evaluate the potential toxicity of DPAA when administered to rats in their drinking water for 52 weeks. DPAA was administered to groups 1–4 at concentrations of 0, 5, 10, and 20 ppm in their drinking water for 52 weeks. There were no significant differences in the final body weights between the control groups and the treatment groups in male or female rats. In serum biochemistry, in females 20 ppm DPAA significantly increased alkaline phosphatase and g-glitamyl transferase compared to controls, and 10 and 20 ppm DPAA significantly increased total cholesterol compared to controls. Absolute and relative liver weights were significantly increased in females treated with 20 ppm DPAA compared to the control group. Dilation of the common bile duct outside the papilla of Vater and stenosis of the papilla of Vater was observed in all male and female rats administered 20 ppm DPAA. The incidence of intrahepatic bile duct hyperplasia was significantly increased in male and female rats treated with 20 ppm DPAA compared to the control groups. These results suggest that DPAA is toxic to the bile duct epithelium in rats. The no-observed adverse effect levels of DPAA were estimated to be 10 ppm (0.48 mg/kg b.w./day) for males and 5 ppm (0.35 mg/kg b.w./day) for females under the conditions of this study. ã 2016 Elsevier GmbH. All rights reserved.
1. Introduction Diphenylarsine chloride and diphenylarsine cyanide were synthesized as chemical weapons during World War II (Arao et al., 2009; Haas, 1998; Hanaoka et al., 2005). Diphenylarsinic acid (DPAA), an organic arsenical, is an environmental degradation product of these chemical warfare agents. In 2003, inhabitants chronically exposed to DPAA through drinking well water in Kamisu City, Ibaraki, Japan, suffered from neurological symptoms such as vertigo, visual disorder, myoclonus, and tremors (Ishii et al., 2004). Short-term toxicological studies have shown that DPAA induces oxidative and nitrosative stress in Purkinje cells (Kato et al., 2007) and increased exploratory behavior, impaired learning behavior, and decreased cerebellar glutathione concentration in rats
* Corresponding author. E-mail address: [email protected] (H. Wanibuchi).
(Negishi et al., 2013). DPAA also produced behavioral effects in subchronic and chronic toxicity studies in mice (Umezu et al., 2012). In humans, inorganic arsenic is known to cause skin cancer, urinary bladder cancer, and lung cancer and possibly liver and kidney cancers in populations chronically exposed through their drinking water (IARC, 2004). Organic metabolites of inorganic arsenicals in humans, such as dimethylarsenic acid (DMA), monomethylarsonic acid, and trimethylarsine oxide, have been shown to induce urinary bladder cancer and promote liver and kidney carcinogenesis in rats, and enhance skin and lung carcinogenesis in mice (Cohen et al., 2006; Wanibuchi et al., 2004; Wei et al., 2002). We recently demonstrated that DPAA promotes liver carcinogenesis in a medium-term rat liver bioassay (Wei et al., 2013). However, little is known about chronic toxicity of DPAA in rats. Therefore, long-term studies in rats to determine the toxicity of DPAA is required for risk assessment. The purpose of the present study was to evaluate the potential toxicity of DPAA when administered to rats in their drinking water for 52 weeks.
http://dx.doi.org/10.1016/j.etp.2016.10.002 0940-2993/ã 2016 Elsevier GmbH. All rights reserved.
Please cite this article in press as: T. Yamaguchi, et al., A chronic toxicity study of diphenylarsinic acid in F344 rats in drinking water for 52 weeks, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.10.002
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2. Material and methods
2.5. Urinalysis
2.1. Chemicals DPAA was provided by Tri Chemical Laboratories (Yamanashi, Japan). The purity of the DPAA was verified to be more than 99.9%, and its stability in tap water for 28 days at room temperature was validated using an IC (IC7000, Yokogawa Analytic System Inc., Tokyo, Japan)-ICP-MS (HP 4500, DE, USA) system at Osaka City University Graduate School of Medicine.
Urinalysis was conducted for all rats surviving to the end of the study at week 52; the urine of one male rat in the 20 ppm group that died at week 47 was not analyzed. Urine was collected by forced urination at 08:00-10:00 am and measured immediately for pH, protein, glucose, ketone, specific gravity, occult blood, bilirubin, urobilinogen, and nitrite using test paper N-Siemens N-Multistix 1SG-L Urinalysis Test Strips with a Clinitek Status1 Urine Analyzer (Bayer Health Care LLC, NY, USA).
2.2. Animals
2.6. Organ weight
Male and female F344 rats at 5 weeks of age were obtained from Charles River Japan, Inc. (Atsugi, Shiga, Japan). The animals were housed in polycarbonate cages (2 or 3 rats/cage) in experimental animal rooms with a targeted temperature of 24 2 C, relative humidity of 50 10% and a 12-h light/dark cycle. All animals were acclimated for 3 weeks before being used for the experiment. Diet and drinking water were available ad libitum throughout the study. Body weights and food and water consumption were measured weekly until week 13 and every 4 weeks thereafter.
The final body weights and weights of the heart, liver, spleen, kidneys, adrenals, testes, and brain of all rats surviving to the end of the study at week 52 were measured; the body and organs of one male rat in the 20 ppm group that died at week 47 were not measured.
2.3. Experimental design The experimental protocols were approved by the Institutional Animal Care and Use Committee of Osaka City University Graduate School of Medicine. A total of 40 male and 40 female F344 rats were divided into 8 groups of 10 rats of each sex. DPAA was dissolved in the tap water and administered to the rats for 52 weeks at 0 ppm (group 1: control), 5 ppm (group 2), 10 ppm (group 3), or 20 ppm (group 4) in the drinking water. Fresh drinking water containing DPAA was supplied to the animals twice weekly. The highest dose of 20 ppm was determined based on the results of our previous studies in which the 20 ppm dose promoted liver carcinogenesis in a medium-term (8-week) rat liver carcinogenesis assay and the 25 and 50 ppm doses caused more than 20% suppression of body weight gain in a 4-week experiment (Wei et al., 2013). At the end of week 52, rats were fasted overnight, sacrificed by an overdose of sodium pentobarbital i.p. (50 mg/kg SomnopentylTM, Kyoritsu Seiyaku, Japan), and necropsied for macroscopic and histopathological examinations. 2.4. Hematology and serum biochemistry Whole blood samples were collected via the inferior vena cava under deep anesthesia at necropsy. Hematology and serum biochemical parameters were conducted in all rats surviving to the end of the study by LSI Medience Corporation, Tokyo, Japan; the blood and serum of one male rat in the 20 ppm group that died at week 47 was not analyzed. Hematological parameters included erythrocyte count (RBC), white blood cell count (WBC), platelet count (Plt), hemoglobin (Hb), hematocrit (Ht), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), and mean corpuscular concentration (MCHC). Serum biochemical parameters included total protein (TP), albumin/globulin ratio (A/G), albumin (ALB), bilirubin (BIL), triglycerides (TG), total cholesterol (TCHO), blood urea nitrogen (BUN), creatinine (CRE), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), inorganic phosphorus (IP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and g-glitamyl transferase (g-GPT).
2.7. Histopathology The testes from males were fixed in Bouin’s solution. The remaining tissues from all animals were fixed in 10% neutral buffered formalin. Tissues were embedded in paraffin and processed for histopathological examination: The lymph nodes (cervical and mesenteric), intrathoracic aorta, submaxillary, sublingual, thymus, trachea, lung, heart, thyroids, parathyroids tongue, esophagus, forestomach, glandular stomach, duodenum, small intestine (jejunum and ileum), large intestine (cecum, colon and rectum), liver, pancreas, spleen, kidneys, adrenals, urinary bladder, seminal vesicles, prostate, testes, epididymides, ovaries, oviduct, uterus, vagina, brain, pituitary, sciatic nerve, skeletal muscle, spinal cord (cervical and lumbar), eye, Harderian gland, sternum, femur, skull bone (zymbal gland), nasal cavity, and sites of macroscopic abnormality were examined. Intrahepatic bile duct hyperplasia was graded according to the INHAND project as follows: 0 (normal), 1 (minimal hyperplasia), 2 (mild hyperplasia), 3 (moderate hyperplasia), and 4 (severe hyperplasia) (Thoolen et al., 2010). 2.8. Statistical analysis All mean values are reported as mean SD. Statistical analyses were performed using the Statlight program (Yukms Co., Ltd., Tokyo, Japan). The significance of differences between the controls and treated groups in body weight, food consumption, organ weights, hematology, and biochemical parameters was assessed by 2-tailed Dunnett's test when the variance was homogeneous and by 2-tailed Steel's test when variances were heterogeneous: Homogeneity of variance was tested by Bartlett's test. Urinary parameters and histopathological examination results were compared using Fisher’s exact test. P values less than 0.05 were considered significant. 3. Results 3.1. Survival, body weight, food consumption, water and DPAA intake The number of rats surviving to the end of the study, final average body weights, average food consumption, water, and DPAA intake are summarized in Table 1. One rat died in group 4 at week 47. All the remaining animals survived to the end of study in good condition. No clinical signs or symptoms of neurotoxicity were observed in any of the DPAA-treated rats throughout the study. There were no significant differences in final body weights
Please cite this article in press as: T. Yamaguchi, et al., A chronic toxicity study of diphenylarsinic acid in F344 rats in drinking water for 52 weeks, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.10.002
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Table. 1 Survival, food and water consumption, DPAA intake, and final body weights of F344 rats treated with DPAA in their drinking water for 52 weeks. DPAA (ppm)
Initial no. of rats
No. of surviving rats (%)
Final body weight (g)
Average food consumption (g/day/rat)
Average water intake (g/day/rat)
Average DPAA intake (mg/kg b.w./day)
Male 0 5 10 20
10 10 10 10
10 (100) 10 (100) 10 (100) 9 (90)a
422 23 423 13 417 16 418 19
14.3 14.4 13.9 14.1
19.4 18.9 17.3 16.8
0 0.26 0.48 0.95
0 93.73 176.33 344.05
Female 0 5 10 20
10 10 10 10
10 10 10 10
214 14 212 12 219 13 204 7
9.6 9.3 9.6 9.4
14.8 13.4 13.5 12.8
0 0.35 0.70 1.35
0 127.40 256.06 489.72
a
(100) (100) (100) (100)
Total DPAA intake (mg/kg b.w./day)
One rat died at week 47.
3.4. Organ weights
between the DPAA-treated groups and control group; slight body weight suppression was noted in the 20 ppm female group (Fig. 1). The intake of DPAA was approximately proportional to the doses administered in the drinking water, although, average water intake showed a tendency to decrease in the DPAA-treated male and female groups, especially in the 20 ppm male and female groups. There were no apparent differences in food consumption between the DPAA treatment groups and control group.
Organ weights are shown in Table 3. Absolute and relative liver and spleen weights were significantly increased in female rats treated with 20 ppm DPAA compared to the controls. Relative but not absolute heart weight were significantly increased in females treated with 20 ppm DPAA compared to the control group, possibly due to lower body weights in this group.
3.2. Hematology and serum biochemistry
3.5. Histopathology
Data for hematology and serum biochemistry are shown in Table 2. In hematology, 20 ppm DPAA significantly increased Plt in males and decreased Ht in females. In serum biochemistry, ALP and g-GPT activates in the female 20 ppm group, and TCHO in the female 10 and 20 ppm groups were significantly increased compared to the control group. ALT activity was significantly decreased in the 20 ppm female group. In males, AST and g-GPT activates in the 20 ppm group, and ALT activity in the 10 and 20 ppm groups were significantly decreased compared to the controls. There were no significant differences in Na, K, Cl, and Ca between the DPAA-treated groups and the control group (Supplemental Table 1). IP in the female 20 ppm group was significantly increased compared to the control group (Supplemental Table 1).
Histopathology data is shown in Table 4. Dilation of the common bile duct accompanied by stenosis of the papilla of Vater that was caused by simple hyperplasia of bile duct epithelium of the papilla of Vater was observed in all male and female rats administered 20 ppm DPAA (Fig. 2). Dilation of the common bile duct was also observed in one male rat in the 5 ppm DPAA group, but not in the control or 10 ppm DPAA groups. The incidences of dilation of the common bile duct and stenosis of the papilla of Vater were significantly higher in the male and female 20 ppm groups compared with their respective control groups. In the liver, incidences of intrahepatic bile duct hyperplasia of grades 2–4 were significantly increased in male and female rats treated with 20 ppm DPAA compared to the control groups (Fig. 3). One female rat in the 5 ppm group had minimal hyperplasia (grade 1), but no intrahepatic bile duct hyperplasia was observed in the control, 5 ppm, or 10 ppm groups. In the present study, intrahepatic bile duct hyperplasia was characterized by simple hyperplasia, fibrosis, and inflammatory cell infiltrate, but neither atypical nor intrahepatic cholestasis.
3.3. Urinalysis There were no differences in urine parameters among the DPAA groups and the control groups (Supplemental Table 2).
Fig. 1. Body weight curves in males (A) and females (B).
Please cite this article in press as: T. Yamaguchi, et al., A chronic toxicity study of diphenylarsinic acid in F344 rats in drinking water for 52 weeks, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.10.002
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Table 2 Hematology and serum biochemistry of F344 rats treated with DPAA in their drinking water for 52 weeks. Male DPAA(ppm) No. of rats Hematology RBC 104/ul Hb g/dl Ht % MCV fl MCH pg Plt 104/ul WBC /ul Biochemistry TP g/dl ALB g/dl A/G AST IU/l ALT IU/l ALP IU/l g-GTP IU/l BUN mg/dl Creatine mg/dl TG mg/dl TCHO mg/dl T-BIL mg/dl a b c
Female
0 10
5 10
10 10
20 9a
0 10
5 10
10 10
20 10
932 25 15.6 0.4 47.6 1.8 51.0 0.9 16.8 0.1 71 4 3620 429
919 22 15.5 0.4 46.8 1.6 50.9 1.0 16.8 0.2 68 6 3480 561
915 35 15.3 0.7 47.2 2.4 51.6 2.0 16.7 0.4 74 13 3830 1293
924 35 15.4 0.5 47.6 1.8 51.6 1.2 16.7 0.2 77 3b 3544 425
870 26 15.7 0.5 49.8 1.6 57.3 1.0 18.1 0.1 58 3 1970 362
874 26 15.7 0.4 50.1 0.9 57.3 1.2 18.0 0.2 60 3 2230 850
869 46 15.8 0.8 50.0 1.8 57.5 1.4 18.1 0.3 61 4 2140 726
784 157 14.1 2.5 45.4 6.7c 59.1 6.9 18.2 1.1 68 23 2650 1362
7.1 0.2 5.0 0.2 2.4 0.1 211 79 123 36 534 68 7.7 2.9 25 6 0.37 0.05 117 30 108 12 0.1 0.0
7.0 0.2 5.0 0.1 2.5 0.1 210 56 115 30 496 61 6.9 3.0 23 3 0.35 0.04 122 28 104 12 0.1 0.0
7.1 0.1 5.2 0.1 2.7 0.1 118 18b 56 5b 495 76 3.4 1.7b 23 4 0.34 0.03 134 107 98 11 0.1 0.0
7.2 0.3 5.5 0.2 3.2 0.2 141 46 60 15 250 37 2.3 0.5 29 7 0.32 0.04 94 32 108 13 0.1 0.0
7.2 0.3 5.5 0.3 3.1 0.3 116 16 49 9 273 50 2.6 1.3 23 3 0.32 0.01 107 49 117 15 0.1 0.0
7.3 0.4 5.5 0.2 3.2 0.3 111 11 47 5 247 59 29 1.5 23 3 0.31 0.03 113 28 112 9c 0.1 0.0
6.8 0.6 5.1 0.5 2.9 0.2 114 16 37 4c 486 117c 5.7 2.0c 25 4 0.35 0.04 85 43 140 27c 0.1 0.0
7.1 0.2 5.0 0.2 2.5 0.3 168 49 88 45b 508 71 8.2 5.3 23 2 0.35 0.03 111 20 108 14 0.1 0.0
One rat died at week 47 Significantly different from 0 ppm male group (control group). Significantly different from 0 ppm female group (control group).
Table 3 Organ weights of F344 rats treated with DPAA in their drinking water for 52 weeks. Male DPAA(ppm) No. of rats
a b
Female
0 10
5 10
10 10
20 9a
0 10
5 10
10 10
20 10
Liver
Absolute (g) Relative (%)
9.60 0.98 2.27 0.13
9.37 0.63 2.21 0.12
9.26 0.68 2.21 0.10
9.93 0.59 2.37 0.08
4.61 0.31 2.13 0.08
4.48 0.38 2.08 0.09
4.78 0.36 2.16 0.08
6.01 0.74b 2.94 0.44b
Kidney
Absolute (g) Relative (%)
2.32 0.16 0.55 0.02
2.36 0.11 0.56 0.02
2.23 0.09 0.53 0.04
2.33 0.11 0.57 0.05
1.49 0.12 0.69 0.03
1.43 0.07 0.67 0.03
1.48 0.15 0.67 0.07
1.38 0.10 0.67 0.05
Spleen
Absolute (g) Relative (%)
0.78 0.08 0.19 0.01
0.79 0.05 0.19 0.01
0.79 0.05 0.19 0.01
0.77 0.03 0.18 0.01
0.46 0.04 0.21 0.01
0.48 0.06 0.22 0.03
0.47 0.04 0.21 0.03
0.61 0.16b 0.30 0.10b
Heart
Absolute (g) Relative (%)
1.18 0.30 0.28 0.07
1.08 0.04 0.26 0.01
1.09 0.05 0.26 0.01
1.11 0.02 0.27 0.02
0.69 0.04 0.32 0.01
0.68 0.05 0.32 0.01
0.69 0.06 0.31 0.03
0.71 0.06 0.34 0.03b
Brain
Absolute (g) Relative (%)
2.12 0.08 0.50 0.03
2.12 0.08 0.50 0.03
2.11 0.06 0.51 0.03
2.07 0.09 0.5 0 0.02
1.89 0.06 0.87 0.05
1.9 0 0.06 0.89 0.06
1.86 0.06 0.84 0.06
1.84 0.05 0.90 0.03
Adrenal
Absolute (g) Relative (%)
0.06 0.04 0.01 0.01
0.05 0.01 0.01 0.00
0.05 0.01 0.01 0.00
0.05 0.00 0.01 0.00
0.05 0.02 0.02 0.01
0.06 0.01 0.03 0.00
0.06 0.01 0.03 0.01
0.05 0.00 0.02 0.00
Testis
Absolute (g) Relative (%)
3.30 0.20 0.78 0.03
3.00 0.70 0.72 0.15
3 .00 0.40 0.73 0.08
3.20 0.50 0.79 0.14
One rat died at week 47. Significantly different from 0 ppm female group (control group).
There were no DPAA treatment-related changes in organs other than the liver (Table 4). In the kidney, chronic nephropathy was observed in two rats in the control group and one rat in the 20 ppm DPAA group. In the pituitary, pseudocysts were observed in three female rats in the control group and in one male and two female rats in the 20 ppm DPAA groups. In the thyroid, follicular cell hyperplasia was observed in one male rat in the 20 ppm DPAA group. C-cell hyperplasia was observed in six male and four female rats in the control groups and in six male and three female rats in the 20 ppm DPAA groups. In the adrenal, pheochromocytoma was observed in one male rat in the control group. In the prostate, inflammatory cell infiltration was observed in one male in the
20 ppm DPAA group. In the testis, interstitial cell tumors were observed in one male rat in the control group and one male rat in the 20 ppm DPAA group, and atrophy was observed in two male rats in the 20 ppm DPAA group. 4. Discussion This is the first study to evaluate the choronic toxicity of DPAA in rats. Our results demonstrate that DPAA is toxic to intrahepatic and extrahepatic bile duct epithelum in male and female rats as evidenced by intrahepatic bile duct hyperplasia and dilation of the common bile duct accompanied by stenosis of the papilla of Vater.
Please cite this article in press as: T. Yamaguchi, et al., A chronic toxicity study of diphenylarsinic acid in F344 rats in drinking water for 52 weeks, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.10.002
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Table 4 Histological findings of F344 rats treated with DPAA in their drinking water for 52 weeks. Organ/Finding
Male
DPAA (ppm) Common bile duct Dilatation, outside of papilla of Vater Stenosis, in papilla of Vater
Female
0 h10i 0 0
5 h10i 1 0
10 h10i 0 0
20 h9i 9a,b 9a,b
0 h10i 0 0
5 h10i 0 0
10 h10i 0 0
20 h10i 10c 10c
h10i 0 7 2 1 0 3
h10i 0 8 2 0 0 2
h10i 0 6 4 0 0 4
h10i 0 0 8 2 0 10b
h10i 10 0 0 0 0 0
h10i 9 1 0 0 0 0
h10i 10 0 0 0 0 0
h10i 0 0 5 4 1 10c
Kidney Chronic nephropathy
h10i 2
–
–
h10i 1
h10i 0
–
–
h10i 0
Pituitary Pseudocyst
h10i 0
h10i 1
h10i 3
Thyroid Follicular cell hyperplasia C-cell hyperplasia
h10i 0 6
– –
– –
h10i 1 6
h10i 0 4
– –
– –
h10i 0 3
Adrenal Pheochromocytoma
h10i 1
–
–
h10i 0
h10i 0
–
–
h10i 0
Prostate Inflammatory cell infiltration
h10i 0
– –
– –
h10i 1
Testis Interstitial cell tumor Atrophy
h10i 1 0
– –
– –
h10i 1 2
Liver Bile duct hyperplasia
Grade 0 1 2 3 4
Total No. of grades of 2–4 align="center"
–
–
–
h10i 2
–
– Not examined. Grade: 0, normal; 1, minimal; 2, mild; 3, moderate; 4, severe. a Number of rats examined. b Significantly different from male 0 ppm group. c Significantly different from female 0 ppm group.
Fig. 2. Stenosis of the papilla of Vater (A) and dilatation of the common bile duct in outside the papilla of Vater (B) was observed in female rats administered 20 ppm DPAA.
There are three known types of bile duct hyperplasia: simple hyperplasia, cystic hyperplasia, and dysplastic hyperplasia (Gary et al., 1990; Greaves, 2012; Hailey et al., 2014). In the present study only the simple type of hyperplasia was observed. It has been reported that intermediates of DPAA generated by SH compounds, such as glutathione (GSH) and dimercaptopropane sulfonate, have enhanced cytotoxic effects (Kinoshita et al., 2006; Ochi et al., 2006, 2004). Generally, GSH conjugated compounds are excreted in the
bile (Curtis, 2013). Therefore, it is probable that DPAA-GSHcomplexes are excreted in the bile and caused the biliary toxicity observed in the present study. However, dysplastic hyperplasia which is a preneoplastic lesion of cholanigiocarcinoma, was not observed (Hailey et al., 2014; Kurashina et al., 1988; Nakanuma, 2012). Our ongoing 2-year carcinogenicity study in rats will clarify the potential carcinogenicity of DPAA.
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Fig. 3. Bile duct hyperplasia in the liver. A: minimal hyperplasia (grade 1) in a female control rat, B: mild hyperplasia (grade 2) in a male rat treated with 20 ppm DPAA, C: moderate hyperplasia (grade 3) in a male rat treated with 20 ppm DPAA, D: severe hyperplasia (grade 4) in a female rat treated with 20 ppm DPAA.
In females, the absolute and relative weights of the liver and bile duct hyperplasia grades 2–4 were significantly increased in female rats treated with 20 ppm DPAA compared to the control group. This suggests that an increase in absolute and relative liver weight in females in the 20 ppm group might be related to the increase in the higher grades of bile duct hyperplasia. In males, however, the increase in the higher grades of bile duct hyperplasia in the 20 ppm group was not accompanied by significant increases in either absolute or relative liver weight. Dilation of the common bile duct outside the papilla of Vater and stenosis of the papilla of Vater were observed in all male and female rats administered 20 ppm DPAA. The alterations in Plt and Ht are considered to lack toxic significance in the present study as the differences were slight and did not appear to be dose-dependent. Decreases in ALT, AST, or g-GPT and the increase in IP activities are also considered to lack toxic significance. Increased ALP and g-GPT activates in the 20 ppm female rats correlated with the toxic changes in the bile duct epithelium. In addition, increased TCHO activity in the 10 and 20 ppm female groups is considered to be treatment-related since it is dose-related. The no-observed adverse effect level for males administered DPAA in their drinking water was 10 ppm and for females it was 5 ppm under the conditions of this study as evidenced by increased TCHO in the 10 ppm female group, increased ALP and g-GPT activities in the 20 ppm female group, and intrahepatic bile duct hyperplasia and dilation of the common bile duct accompanied by stenosis of the papilla of Vater in the male and female 20 ppm groups.
In conclusion, the present study demonstrated that DPAA exhibits biliary toxicity in rats. The no-observed adverse effect levels of DPAA were 10 ppm (0.48 mg/kg b.w./day) for males and 5 ppm (0.35 mg/kg b.w./day) for females under the conditions of this study. Acknowledgments This work was supported by a grant from the Ministry of the Environment of Japan. The authors gratefully acknowledge the technical assistance of Keiko Sakata, Rie Onodera, Yuko Hisabayashi and Yukiko Iura. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.etp.2016.10.002. References Arao T, Maejima Y, Baba K. Uptake of aromatic arsenicals from soil contaminated with diphenylarsinic acid by rice. Environ. Sci. Technol. 2009;43(4):1097–101. Cohen SM, Arnold LL, Eldan M, Lewis AS, Beck BD. Methylated arsenicals: the implications of metabolism and carcinogenicity studies in rodents to human risk assessment. Crit. Rev. Toxicol. 2006;36(2):99–133. Curtis DK. Casarett & Doull’s Toxicology: The Basic Science of Poisons. Eighth ed. Gary AB, Scot LE, Michael RE, Charles AMJ, William FM. Pathology of the Fischer Rat. Greaves P. Histopathology of Preclinical Toxicity Studies. Fourth ed. Haas R. Determination of chemical warfare agents: Gas Chromatographic analysis of chlorovinylarsines (Lewisite) and their metabolites by derivatization with thiols (2nd communication). Environ. Sci. Pollut. Res. Int. 1998;5(1):2–3.
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A chronic toxicity study of diphenylarsinic acid in F344 rats in drinking water for 52 weeks Takashi Yamaguchia , Min Gia , Shotarou Yamanoa,b , Masaki Fujiokaa , Kumiko Tatsumia , Satoko Kawachia , Naomi Ishiia , Kenichiro Doia , Anna Kakehashia , Hideki Wanibuchia,* a b
Department of Molecular Pathology, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan Division of Rare Cancer Research National Cancer Center Research Institute, Tsukiji 5-5-1, Chuo-ku Tokyo 104-0045, Japan
A R T I C L E I N F O
Article history: Received 22 July 2016 Received in revised form 13 September 2016 Accepted 6 October 2016 Keywords: Diphenylarsinic acid Chronic toxicity Bile duct toxicity F344 rats
A B S T R A C T
Diphenylarsinic acid (DPAA), a chemical warfare-related neurotoxic organic arsenical, is present in the groundwater and soil in some regions of Japan due to illegal dumping after World War II. The purpose of the present study was to evaluate the potential toxicity of DPAA when administered to rats in their drinking water for 52 weeks. DPAA was administered to groups 1–4 at concentrations of 0, 5, 10, and 20 ppm in their drinking water for 52 weeks. There were no significant differences in the final body weights between the control groups and the treatment groups in male or female rats. In serum biochemistry, in females 20 ppm DPAA significantly increased alkaline phosphatase and g-glitamyl transferase compared to controls, and 10 and 20 ppm DPAA significantly increased total cholesterol compared to controls. Absolute and relative liver weights were significantly increased in females treated with 20 ppm DPAA compared to the control group. Dilation of the common bile duct outside the papilla of Vater and stenosis of the papilla of Vater was observed in all male and female rats administered 20 ppm DPAA. The incidence of intrahepatic bile duct hyperplasia was significantly increased in male and female rats treated with 20 ppm DPAA compared to the control groups. These results suggest that DPAA is toxic to the bile duct epithelium in rats. The no-observed adverse effect levels of DPAA were estimated to be 10 ppm (0.48 mg/kg b.w./day) for males and 5 ppm (0.35 mg/kg b.w./day) for females under the conditions of this study. ã 2016 Elsevier GmbH. All rights reserved.
1. Introduction Diphenylarsine chloride and diphenylarsine cyanide were synthesized as chemical weapons during World War II (Arao et al., 2009; Haas, 1998; Hanaoka et al., 2005). Diphenylarsinic acid (DPAA), an organic arsenical, is an environmental degradation product of these chemical warfare agents. In 2003, inhabitants chronically exposed to DPAA through drinking well water in Kamisu City, Ibaraki, Japan, suffered from neurological symptoms such as vertigo, visual disorder, myoclonus, and tremors (Ishii et al., 2004). Short-term toxicological studies have shown that DPAA induces oxidative and nitrosative stress in Purkinje cells (Kato et al., 2007) and increased exploratory behavior, impaired learning behavior, and decreased cerebellar glutathione concentration in rats
* Corresponding author. E-mail address: [email protected] (H. Wanibuchi).
(Negishi et al., 2013). DPAA also produced behavioral effects in subchronic and chronic toxicity studies in mice (Umezu et al., 2012). In humans, inorganic arsenic is known to cause skin cancer, urinary bladder cancer, and lung cancer and possibly liver and kidney cancers in populations chronically exposed through their drinking water (IARC, 2004). Organic metabolites of inorganic arsenicals in humans, such as dimethylarsenic acid (DMA), monomethylarsonic acid, and trimethylarsine oxide, have been shown to induce urinary bladder cancer and promote liver and kidney carcinogenesis in rats, and enhance skin and lung carcinogenesis in mice (Cohen et al., 2006; Wanibuchi et al., 2004; Wei et al., 2002). We recently demonstrated that DPAA promotes liver carcinogenesis in a medium-term rat liver bioassay (Wei et al., 2013). However, little is known about chronic toxicity of DPAA in rats. Therefore, long-term studies in rats to determine the toxicity of DPAA is required for risk assessment. The purpose of the present study was to evaluate the potential toxicity of DPAA when administered to rats in their drinking water for 52 weeks.
http://dx.doi.org/10.1016/j.etp.2016.10.002 0940-2993/ã 2016 Elsevier GmbH. All rights reserved.
Please cite this article in press as: T. Yamaguchi, et al., A chronic toxicity study of diphenylarsinic acid in F344 rats in drinking water for 52 weeks, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.10.002
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2. Material and methods
2.5. Urinalysis
2.1. Chemicals DPAA was provided by Tri Chemical Laboratories (Yamanashi, Japan). The purity of the DPAA was verified to be more than 99.9%, and its stability in tap water for 28 days at room temperature was validated using an IC (IC7000, Yokogawa Analytic System Inc., Tokyo, Japan)-ICP-MS (HP 4500, DE, USA) system at Osaka City University Graduate School of Medicine.
Urinalysis was conducted for all rats surviving to the end of the study at week 52; the urine of one male rat in the 20 ppm group that died at week 47 was not analyzed. Urine was collected by forced urination at 08:00-10:00 am and measured immediately for pH, protein, glucose, ketone, specific gravity, occult blood, bilirubin, urobilinogen, and nitrite using test paper N-Siemens N-Multistix 1SG-L Urinalysis Test Strips with a Clinitek Status1 Urine Analyzer (Bayer Health Care LLC, NY, USA).
2.2. Animals
2.6. Organ weight
Male and female F344 rats at 5 weeks of age were obtained from Charles River Japan, Inc. (Atsugi, Shiga, Japan). The animals were housed in polycarbonate cages (2 or 3 rats/cage) in experimental animal rooms with a targeted temperature of 24 2 C, relative humidity of 50 10% and a 12-h light/dark cycle. All animals were acclimated for 3 weeks before being used for the experiment. Diet and drinking water were available ad libitum throughout the study. Body weights and food and water consumption were measured weekly until week 13 and every 4 weeks thereafter.
The final body weights and weights of the heart, liver, spleen, kidneys, adrenals, testes, and brain of all rats surviving to the end of the study at week 52 were measured; the body and organs of one male rat in the 20 ppm group that died at week 47 were not measured.
2.3. Experimental design The experimental protocols were approved by the Institutional Animal Care and Use Committee of Osaka City University Graduate School of Medicine. A total of 40 male and 40 female F344 rats were divided into 8 groups of 10 rats of each sex. DPAA was dissolved in the tap water and administered to the rats for 52 weeks at 0 ppm (group 1: control), 5 ppm (group 2), 10 ppm (group 3), or 20 ppm (group 4) in the drinking water. Fresh drinking water containing DPAA was supplied to the animals twice weekly. The highest dose of 20 ppm was determined based on the results of our previous studies in which the 20 ppm dose promoted liver carcinogenesis in a medium-term (8-week) rat liver carcinogenesis assay and the 25 and 50 ppm doses caused more than 20% suppression of body weight gain in a 4-week experiment (Wei et al., 2013). At the end of week 52, rats were fasted overnight, sacrificed by an overdose of sodium pentobarbital i.p. (50 mg/kg SomnopentylTM, Kyoritsu Seiyaku, Japan), and necropsied for macroscopic and histopathological examinations. 2.4. Hematology and serum biochemistry Whole blood samples were collected via the inferior vena cava under deep anesthesia at necropsy. Hematology and serum biochemical parameters were conducted in all rats surviving to the end of the study by LSI Medience Corporation, Tokyo, Japan; the blood and serum of one male rat in the 20 ppm group that died at week 47 was not analyzed. Hematological parameters included erythrocyte count (RBC), white blood cell count (WBC), platelet count (Plt), hemoglobin (Hb), hematocrit (Ht), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), and mean corpuscular concentration (MCHC). Serum biochemical parameters included total protein (TP), albumin/globulin ratio (A/G), albumin (ALB), bilirubin (BIL), triglycerides (TG), total cholesterol (TCHO), blood urea nitrogen (BUN), creatinine (CRE), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), inorganic phosphorus (IP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and g-glitamyl transferase (g-GPT).
2.7. Histopathology The testes from males were fixed in Bouin’s solution. The remaining tissues from all animals were fixed in 10% neutral buffered formalin. Tissues were embedded in paraffin and processed for histopathological examination: The lymph nodes (cervical and mesenteric), intrathoracic aorta, submaxillary, sublingual, thymus, trachea, lung, heart, thyroids, parathyroids tongue, esophagus, forestomach, glandular stomach, duodenum, small intestine (jejunum and ileum), large intestine (cecum, colon and rectum), liver, pancreas, spleen, kidneys, adrenals, urinary bladder, seminal vesicles, prostate, testes, epididymides, ovaries, oviduct, uterus, vagina, brain, pituitary, sciatic nerve, skeletal muscle, spinal cord (cervical and lumbar), eye, Harderian gland, sternum, femur, skull bone (zymbal gland), nasal cavity, and sites of macroscopic abnormality were examined. Intrahepatic bile duct hyperplasia was graded according to the INHAND project as follows: 0 (normal), 1 (minimal hyperplasia), 2 (mild hyperplasia), 3 (moderate hyperplasia), and 4 (severe hyperplasia) (Thoolen et al., 2010). 2.8. Statistical analysis All mean values are reported as mean SD. Statistical analyses were performed using the Statlight program (Yukms Co., Ltd., Tokyo, Japan). The significance of differences between the controls and treated groups in body weight, food consumption, organ weights, hematology, and biochemical parameters was assessed by 2-tailed Dunnett's test when the variance was homogeneous and by 2-tailed Steel's test when variances were heterogeneous: Homogeneity of variance was tested by Bartlett's test. Urinary parameters and histopathological examination results were compared using Fisher’s exact test. P values less than 0.05 were considered significant. 3. Results 3.1. Survival, body weight, food consumption, water and DPAA intake The number of rats surviving to the end of the study, final average body weights, average food consumption, water, and DPAA intake are summarized in Table 1. One rat died in group 4 at week 47. All the remaining animals survived to the end of study in good condition. No clinical signs or symptoms of neurotoxicity were observed in any of the DPAA-treated rats throughout the study. There were no significant differences in final body weights
Please cite this article in press as: T. Yamaguchi, et al., A chronic toxicity study of diphenylarsinic acid in F344 rats in drinking water for 52 weeks, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.10.002
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Table. 1 Survival, food and water consumption, DPAA intake, and final body weights of F344 rats treated with DPAA in their drinking water for 52 weeks. DPAA (ppm)
Initial no. of rats
No. of surviving rats (%)
Final body weight (g)
Average food consumption (g/day/rat)
Average water intake (g/day/rat)
Average DPAA intake (mg/kg b.w./day)
Male 0 5 10 20
10 10 10 10
10 (100) 10 (100) 10 (100) 9 (90)a
422 23 423 13 417 16 418 19
14.3 14.4 13.9 14.1
19.4 18.9 17.3 16.8
0 0.26 0.48 0.95
0 93.73 176.33 344.05
Female 0 5 10 20
10 10 10 10
10 10 10 10
214 14 212 12 219 13 204 7
9.6 9.3 9.6 9.4
14.8 13.4 13.5 12.8
0 0.35 0.70 1.35
0 127.40 256.06 489.72
a
(100) (100) (100) (100)
Total DPAA intake (mg/kg b.w./day)
One rat died at week 47.
3.4. Organ weights
between the DPAA-treated groups and control group; slight body weight suppression was noted in the 20 ppm female group (Fig. 1). The intake of DPAA was approximately proportional to the doses administered in the drinking water, although, average water intake showed a tendency to decrease in the DPAA-treated male and female groups, especially in the 20 ppm male and female groups. There were no apparent differences in food consumption between the DPAA treatment groups and control group.
Organ weights are shown in Table 3. Absolute and relative liver and spleen weights were significantly increased in female rats treated with 20 ppm DPAA compared to the controls. Relative but not absolute heart weight were significantly increased in females treated with 20 ppm DPAA compared to the control group, possibly due to lower body weights in this group.
3.2. Hematology and serum biochemistry
3.5. Histopathology
Data for hematology and serum biochemistry are shown in Table 2. In hematology, 20 ppm DPAA significantly increased Plt in males and decreased Ht in females. In serum biochemistry, ALP and g-GPT activates in the female 20 ppm group, and TCHO in the female 10 and 20 ppm groups were significantly increased compared to the control group. ALT activity was significantly decreased in the 20 ppm female group. In males, AST and g-GPT activates in the 20 ppm group, and ALT activity in the 10 and 20 ppm groups were significantly decreased compared to the controls. There were no significant differences in Na, K, Cl, and Ca between the DPAA-treated groups and the control group (Supplemental Table 1). IP in the female 20 ppm group was significantly increased compared to the control group (Supplemental Table 1).
Histopathology data is shown in Table 4. Dilation of the common bile duct accompanied by stenosis of the papilla of Vater that was caused by simple hyperplasia of bile duct epithelium of the papilla of Vater was observed in all male and female rats administered 20 ppm DPAA (Fig. 2). Dilation of the common bile duct was also observed in one male rat in the 5 ppm DPAA group, but not in the control or 10 ppm DPAA groups. The incidences of dilation of the common bile duct and stenosis of the papilla of Vater were significantly higher in the male and female 20 ppm groups compared with their respective control groups. In the liver, incidences of intrahepatic bile duct hyperplasia of grades 2–4 were significantly increased in male and female rats treated with 20 ppm DPAA compared to the control groups (Fig. 3). One female rat in the 5 ppm group had minimal hyperplasia (grade 1), but no intrahepatic bile duct hyperplasia was observed in the control, 5 ppm, or 10 ppm groups. In the present study, intrahepatic bile duct hyperplasia was characterized by simple hyperplasia, fibrosis, and inflammatory cell infiltrate, but neither atypical nor intrahepatic cholestasis.
3.3. Urinalysis There were no differences in urine parameters among the DPAA groups and the control groups (Supplemental Table 2).
Fig. 1. Body weight curves in males (A) and females (B).
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Table 2 Hematology and serum biochemistry of F344 rats treated with DPAA in their drinking water for 52 weeks. Male DPAA(ppm) No. of rats Hematology RBC 104/ul Hb g/dl Ht % MCV fl MCH pg Plt 104/ul WBC /ul Biochemistry TP g/dl ALB g/dl A/G AST IU/l ALT IU/l ALP IU/l g-GTP IU/l BUN mg/dl Creatine mg/dl TG mg/dl TCHO mg/dl T-BIL mg/dl a b c
Female
0 10
5 10
10 10
20 9a
0 10
5 10
10 10
20 10
932 25 15.6 0.4 47.6 1.8 51.0 0.9 16.8 0.1 71 4 3620 429
919 22 15.5 0.4 46.8 1.6 50.9 1.0 16.8 0.2 68 6 3480 561
915 35 15.3 0.7 47.2 2.4 51.6 2.0 16.7 0.4 74 13 3830 1293
924 35 15.4 0.5 47.6 1.8 51.6 1.2 16.7 0.2 77 3b 3544 425
870 26 15.7 0.5 49.8 1.6 57.3 1.0 18.1 0.1 58 3 1970 362
874 26 15.7 0.4 50.1 0.9 57.3 1.2 18.0 0.2 60 3 2230 850
869 46 15.8 0.8 50.0 1.8 57.5 1.4 18.1 0.3 61 4 2140 726
784 157 14.1 2.5 45.4 6.7c 59.1 6.9 18.2 1.1 68 23 2650 1362
7.1 0.2 5.0 0.2 2.4 0.1 211 79 123 36 534 68 7.7 2.9 25 6 0.37 0.05 117 30 108 12 0.1 0.0
7.0 0.2 5.0 0.1 2.5 0.1 210 56 115 30 496 61 6.9 3.0 23 3 0.35 0.04 122 28 104 12 0.1 0.0
7.1 0.1 5.2 0.1 2.7 0.1 118 18b 56 5b 495 76 3.4 1.7b 23 4 0.34 0.03 134 107 98 11 0.1 0.0
7.2 0.3 5.5 0.2 3.2 0.2 141 46 60 15 250 37 2.3 0.5 29 7 0.32 0.04 94 32 108 13 0.1 0.0
7.2 0.3 5.5 0.3 3.1 0.3 116 16 49 9 273 50 2.6 1.3 23 3 0.32 0.01 107 49 117 15 0.1 0.0
7.3 0.4 5.5 0.2 3.2 0.3 111 11 47 5 247 59 29 1.5 23 3 0.31 0.03 113 28 112 9c 0.1 0.0
6.8 0.6 5.1 0.5 2.9 0.2 114 16 37 4c 486 117c 5.7 2.0c 25 4 0.35 0.04 85 43 140 27c 0.1 0.0
7.1 0.2 5.0 0.2 2.5 0.3 168 49 88 45b 508 71 8.2 5.3 23 2 0.35 0.03 111 20 108 14 0.1 0.0
One rat died at week 47 Significantly different from 0 ppm male group (control group). Significantly different from 0 ppm female group (control group).
Table 3 Organ weights of F344 rats treated with DPAA in their drinking water for 52 weeks. Male DPAA(ppm) No. of rats
a b
Female
0 10
5 10
10 10
20 9a
0 10
5 10
10 10
20 10
Liver
Absolute (g) Relative (%)
9.60 0.98 2.27 0.13
9.37 0.63 2.21 0.12
9.26 0.68 2.21 0.10
9.93 0.59 2.37 0.08
4.61 0.31 2.13 0.08
4.48 0.38 2.08 0.09
4.78 0.36 2.16 0.08
6.01 0.74b 2.94 0.44b
Kidney
Absolute (g) Relative (%)
2.32 0.16 0.55 0.02
2.36 0.11 0.56 0.02
2.23 0.09 0.53 0.04
2.33 0.11 0.57 0.05
1.49 0.12 0.69 0.03
1.43 0.07 0.67 0.03
1.48 0.15 0.67 0.07
1.38 0.10 0.67 0.05
Spleen
Absolute (g) Relative (%)
0.78 0.08 0.19 0.01
0.79 0.05 0.19 0.01
0.79 0.05 0.19 0.01
0.77 0.03 0.18 0.01
0.46 0.04 0.21 0.01
0.48 0.06 0.22 0.03
0.47 0.04 0.21 0.03
0.61 0.16b 0.30 0.10b
Heart
Absolute (g) Relative (%)
1.18 0.30 0.28 0.07
1.08 0.04 0.26 0.01
1.09 0.05 0.26 0.01
1.11 0.02 0.27 0.02
0.69 0.04 0.32 0.01
0.68 0.05 0.32 0.01
0.69 0.06 0.31 0.03
0.71 0.06 0.34 0.03b
Brain
Absolute (g) Relative (%)
2.12 0.08 0.50 0.03
2.12 0.08 0.50 0.03
2.11 0.06 0.51 0.03
2.07 0.09 0.5 0 0.02
1.89 0.06 0.87 0.05
1.9 0 0.06 0.89 0.06
1.86 0.06 0.84 0.06
1.84 0.05 0.90 0.03
Adrenal
Absolute (g) Relative (%)
0.06 0.04 0.01 0.01
0.05 0.01 0.01 0.00
0.05 0.01 0.01 0.00
0.05 0.00 0.01 0.00
0.05 0.02 0.02 0.01
0.06 0.01 0.03 0.00
0.06 0.01 0.03 0.01
0.05 0.00 0.02 0.00
Testis
Absolute (g) Relative (%)
3.30 0.20 0.78 0.03
3.00 0.70 0.72 0.15
3 .00 0.40 0.73 0.08
3.20 0.50 0.79 0.14
One rat died at week 47. Significantly different from 0 ppm female group (control group).
There were no DPAA treatment-related changes in organs other than the liver (Table 4). In the kidney, chronic nephropathy was observed in two rats in the control group and one rat in the 20 ppm DPAA group. In the pituitary, pseudocysts were observed in three female rats in the control group and in one male and two female rats in the 20 ppm DPAA groups. In the thyroid, follicular cell hyperplasia was observed in one male rat in the 20 ppm DPAA group. C-cell hyperplasia was observed in six male and four female rats in the control groups and in six male and three female rats in the 20 ppm DPAA groups. In the adrenal, pheochromocytoma was observed in one male rat in the control group. In the prostate, inflammatory cell infiltration was observed in one male in the
20 ppm DPAA group. In the testis, interstitial cell tumors were observed in one male rat in the control group and one male rat in the 20 ppm DPAA group, and atrophy was observed in two male rats in the 20 ppm DPAA group. 4. Discussion This is the first study to evaluate the choronic toxicity of DPAA in rats. Our results demonstrate that DPAA is toxic to intrahepatic and extrahepatic bile duct epithelum in male and female rats as evidenced by intrahepatic bile duct hyperplasia and dilation of the common bile duct accompanied by stenosis of the papilla of Vater.
Please cite this article in press as: T. Yamaguchi, et al., A chronic toxicity study of diphenylarsinic acid in F344 rats in drinking water for 52 weeks, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.10.002
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Table 4 Histological findings of F344 rats treated with DPAA in their drinking water for 52 weeks. Organ/Finding
Male
DPAA (ppm) Common bile duct Dilatation, outside of papilla of Vater Stenosis, in papilla of Vater
Female
0 h10i 0 0
5 h10i 1 0
10 h10i 0 0
20 h9i 9a,b 9a,b
0 h10i 0 0
5 h10i 0 0
10 h10i 0 0
20 h10i 10c 10c
h10i 0 7 2 1 0 3
h10i 0 8 2 0 0 2
h10i 0 6 4 0 0 4
h10i 0 0 8 2 0 10b
h10i 10 0 0 0 0 0
h10i 9 1 0 0 0 0
h10i 10 0 0 0 0 0
h10i 0 0 5 4 1 10c
Kidney Chronic nephropathy
h10i 2
–
–
h10i 1
h10i 0
–
–
h10i 0
Pituitary Pseudocyst
h10i 0
h10i 1
h10i 3
Thyroid Follicular cell hyperplasia C-cell hyperplasia
h10i 0 6
– –
– –
h10i 1 6
h10i 0 4
– –
– –
h10i 0 3
Adrenal Pheochromocytoma
h10i 1
–
–
h10i 0
h10i 0
–
–
h10i 0
Prostate Inflammatory cell infiltration
h10i 0
– –
– –
h10i 1
Testis Interstitial cell tumor Atrophy
h10i 1 0
– –
– –
h10i 1 2
Liver Bile duct hyperplasia
Grade 0 1 2 3 4
Total No. of grades of 2–4 align="center"
–
–
–
h10i 2
–
– Not examined. Grade: 0, normal; 1, minimal; 2, mild; 3, moderate; 4, severe. a Number of rats examined. b Significantly different from male 0 ppm group. c Significantly different from female 0 ppm group.
Fig. 2. Stenosis of the papilla of Vater (A) and dilatation of the common bile duct in outside the papilla of Vater (B) was observed in female rats administered 20 ppm DPAA.
There are three known types of bile duct hyperplasia: simple hyperplasia, cystic hyperplasia, and dysplastic hyperplasia (Gary et al., 1990; Greaves, 2012; Hailey et al., 2014). In the present study only the simple type of hyperplasia was observed. It has been reported that intermediates of DPAA generated by SH compounds, such as glutathione (GSH) and dimercaptopropane sulfonate, have enhanced cytotoxic effects (Kinoshita et al., 2006; Ochi et al., 2006, 2004). Generally, GSH conjugated compounds are excreted in the
bile (Curtis, 2013). Therefore, it is probable that DPAA-GSHcomplexes are excreted in the bile and caused the biliary toxicity observed in the present study. However, dysplastic hyperplasia which is a preneoplastic lesion of cholanigiocarcinoma, was not observed (Hailey et al., 2014; Kurashina et al., 1988; Nakanuma, 2012). Our ongoing 2-year carcinogenicity study in rats will clarify the potential carcinogenicity of DPAA.
Please cite this article in press as: T. Yamaguchi, et al., A chronic toxicity study of diphenylarsinic acid in F344 rats in drinking water for 52 weeks, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.10.002
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Fig. 3. Bile duct hyperplasia in the liver. A: minimal hyperplasia (grade 1) in a female control rat, B: mild hyperplasia (grade 2) in a male rat treated with 20 ppm DPAA, C: moderate hyperplasia (grade 3) in a male rat treated with 20 ppm DPAA, D: severe hyperplasia (grade 4) in a female rat treated with 20 ppm DPAA.
In females, the absolute and relative weights of the liver and bile duct hyperplasia grades 2–4 were significantly increased in female rats treated with 20 ppm DPAA compared to the control group. This suggests that an increase in absolute and relative liver weight in females in the 20 ppm group might be related to the increase in the higher grades of bile duct hyperplasia. In males, however, the increase in the higher grades of bile duct hyperplasia in the 20 ppm group was not accompanied by significant increases in either absolute or relative liver weight. Dilation of the common bile duct outside the papilla of Vater and stenosis of the papilla of Vater were observed in all male and female rats administered 20 ppm DPAA. The alterations in Plt and Ht are considered to lack toxic significance in the present study as the differences were slight and did not appear to be dose-dependent. Decreases in ALT, AST, or g-GPT and the increase in IP activities are also considered to lack toxic significance. Increased ALP and g-GPT activates in the 20 ppm female rats correlated with the toxic changes in the bile duct epithelium. In addition, increased TCHO activity in the 10 and 20 ppm female groups is considered to be treatment-related since it is dose-related. The no-observed adverse effect level for males administered DPAA in their drinking water was 10 ppm and for females it was 5 ppm under the conditions of this study as evidenced by increased TCHO in the 10 ppm female group, increased ALP and g-GPT activities in the 20 ppm female group, and intrahepatic bile duct hyperplasia and dilation of the common bile duct accompanied by stenosis of the papilla of Vater in the male and female 20 ppm groups.
In conclusion, the present study demonstrated that DPAA exhibits biliary toxicity in rats. The no-observed adverse effect levels of DPAA were 10 ppm (0.48 mg/kg b.w./day) for males and 5 ppm (0.35 mg/kg b.w./day) for females under the conditions of this study. Acknowledgments This work was supported by a grant from the Ministry of the Environment of Japan. The authors gratefully acknowledge the technical assistance of Keiko Sakata, Rie Onodera, Yuko Hisabayashi and Yukiko Iura. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.etp.2016.10.002. References Arao T, Maejima Y, Baba K. Uptake of aromatic arsenicals from soil contaminated with diphenylarsinic acid by rice. Environ. Sci. Technol. 2009;43(4):1097–101. Cohen SM, Arnold LL, Eldan M, Lewis AS, Beck BD. Methylated arsenicals: the implications of metabolism and carcinogenicity studies in rodents to human risk assessment. Crit. Rev. Toxicol. 2006;36(2):99–133. Curtis DK. Casarett & Doull’s Toxicology: The Basic Science of Poisons. Eighth ed. Gary AB, Scot LE, Michael RE, Charles AMJ, William FM. Pathology of the Fischer Rat. Greaves P. Histopathology of Preclinical Toxicity Studies. Fourth ed. Haas R. Determination of chemical warfare agents: Gas Chromatographic analysis of chlorovinylarsines (Lewisite) and their metabolites by derivatization with thiols (2nd communication). Environ. Sci. Pollut. Res. Int. 1998;5(1):2–3.
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Please cite this article in press as: T. Yamaguchi, et al., A chronic toxicity study of diphenylarsinic acid in F344 rats in drinking water for 52 weeks, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.10.002