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Jan 30, 2015 - Sophorae radix (SR) is the dried root of Sophorae flavescens Aiton. 55 ...... flavanone derivatives isolated from Sophora flavescens for ...
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Genotoxicity and subchronic toxicity of Sophorae radix in rats: Hepatotoxic and genotoxic potential Jeong-Hwan Che a,b,1, Jun-Won Yun a,1, Yun-Soon Kim a, Seung-Hyun Kim a, Ji-Ran You a, Ja-June Jang c, Hee Chan Kim d, Hyeon Hoe Kim e,⇑, Byeong-Cheol Kang a,b,f,g,⇑ a

Department of Experimental Animal Research, Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea Biomedical Center for Animal Resource and Development, Bio-Max Institute, Seoul National University, Seoul, Republic of Korea Department of Pathology, Seoul National University College of Medicine, Seoul, Republic of Korea d Department of Biomedical Engineering, College of Medicine and Institute of Medical and Biological Engineering, Medical Research Center, Seoul National University, Seoul, Republic of Korea e Department of Urology, Seoul National University College of Medicine, Seoul, Republic of Korea f Graduate School of Translational Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea g Designed Animal and Transplantation Research Institute, Seoul National University, Pyeongchang-gun, Gangwon-do, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 20 November 2014 Available online xxxx Keywords: Sophorae radix Subchronic Genotoxicity Hepatotoxicity Liver Anemia

a b s t r a c t Although Sophorae radix (SR) has been traditionally used as a treatment for various clinical symptoms, a comprehensive investigation of its safety has not yet been carried out. Therefore, we present an evaluation of the toxicity of the SR extract that was performed according to the Organization for Economic Cooperation and Development test guidelines for subchronic toxicity and genotoxicity. In an oral subchronic study for 13 weeks, the repeated treatment of rats with 429 or 1500 mg/kg of the SR extract induced a dose-related change in body weight. In particular, the SR extract was observed to exert a significant increase in liver weight along with an increase in serum alkaline phosphatase and alanine transaminase. A small but statistically significant reduction in red blood cell, hemoglobin, and hematocrit levels in the SR extract-treated rats suggests the possibility that anemia, accompanied by liver injury, was at least partially induced. These findings indicate the no-observed-adverse-effect-level for the SR extract was considered to be 10 mg/kg/d. And, the data obtained from the chromosome aberration assay showed that SR extract might be considered to be a weak clastogen although no significant micronucleus induction was observed in vivo. Despite the benefits that SR extract can exhibit, this study indicates that SR extract may possess hepatotoxic and genotoxic potential. Ó 2015 Published by Elsevier Inc.

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1. Introduction

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Sophorae radix (SR) is the dried root of Sophorae flavescens Aiton (Leguminosae) and is also known as the Chinese drug ‘Kushen’ (Yu et al., 2013; Ou, 1992). For thousands of years, SR has been used in

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East Asian countries – including Korea, China and Japan – as a traditional herbal medicine and as a functional food ingredient. Recently, SR has been observed to have various physiological effects, such as improving mental health (Ko et al., 2007; Hwang et al., 2005), and providing anti-inflammatory (Kim et al., 2002),

Abbreviations: SR, Sophorae radix; OECD, Organization for Economic Co-operation and Development; WBC, white blood cell; RBC, red blood cell; HGB, hemoglobin; HCT, hematocrit; PLT, platelet; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; BUN, blood urea nitrogen; TC, total cholesterol; TP, total protein; TB, total bilirubin; ALP, alkaline phosphatase; AST, aspartate transaminase; ALT, alanine transaminase; TG, triglyceride; H&E, hematoxylin and eosin; CHL, Chinese hamster lung; MNPCEs, micronucleated polychromatic erythrocytes; NCEs, normochromatic erythrocytes; HPLC, high-performance liquid chromatography. ⇑ Corresponding authors at: Department of Urology, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul 110-799, Republic of Korea. Fax: +82 2 742 4665 (H.H. Kim). Graduate School of Translational Medicine, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul 110-744, Republic of Korea. Fax: +82 2 741 7620 (B.-C. Kang). E-mail addresses: [email protected] (H.H. Kim), [email protected] (B.-C. Kang). 1 Contributed equally to this work. http://dx.doi.org/10.1016/j.yrtph.2015.01.012 0273-2300/Ó 2015 Published by Elsevier Inc.

Q1 Please cite this article in press as: Che, J.-H., et al. Genotoxicity and subchronic toxicity of Sophorae radix in rats: Hepatotoxic and genotoxic potential. Regul. Toxicol. Pharmacol. (2015), http://dx.doi.org/10.1016/j.yrtph.2015.01.012

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free radical scavenging (Jung et al., 2005), and antimicrobial activity (Yagi et al., 1989; Kuroyanagi et al., 1999). SR has also been used as a constituent in oriental herb medicine prescriptions for atherosclerosis and arrhythmias (Kwon et al., 2004). It is also known to have anti-allergic activity in pithed rats (Lee et al., 2001) and antioxidant effects in xanthine oxidase/hypoxanthinetreated cardiac endothelial cells (Kwon et al., 2003). Furthermore, SR has the potential for use in therapeutic applications in the clinical treatment of systemic lupus erythematosus progression by correcting the Th1/Th2 balance (Ko et al., 2007). Prior studies of the metabolites from S. flavescens have reported on the isolation and structural determination of the chemical components, including alkaloids, flavonoids, alkylxanthones, quinones, triterpene glycosides, fatty acids, and essential oils (Wang, 1997; State Administration of Traditional Chinese Medicine, 1999). The alkaloids in S. flavescens, including oxymatrine and matrine, have been widely used in China to treat cancer and hepatitis (Yu et al., 2013). S. flavescens is also known to contain numerous flavonoids, such as formononetin, kushenol E, kushenol B, shphoraflavanone G, kushenol L, kushenol M, kuraridin, kurarinone, kushenol N, and kushenol F (Kim et al., 1997; Ryu et al., 1997; Ha et al., 2001). These flavonoids from the root of S. flavescens, including sophoraflavanone G and kurarinone, also present extremely strong tyrosinase inhibitory activity (Ha et al., 2001; Kim et al., 2003; Son et al., 2003). The alkaloids of S. flavescens containing oxymatrine and matrine have been developed in China as anticancer drugs (Sun et al., 2012). Numerous studies have shown that these alkaloids of S. flavescens inhibited the growth of various tumors in vitro and in vivo (Sun et al., 2012; Liu et al., 2014). And, more potent antitumor activities were identified in flavonoids than alkaloids of S. flavescens in vitro and in vivo (Sun et al., 2012). Flavonoids such as (2S)-20 -methoxykurarinone, ()-kurarinone, sophoraflavanone G, and leachianone A exhibited cytotoxic activity against human myeloid leukemia HL-60 (myeloid leukemia) cells (Kang et al., 2000). And, antitumor efficacies were confirmed in several mice models of xenografted tumors (Han et al., 2007; Sun et al., 2012). Paecilomyces tenuipes is widely used as a traditional nutritious medicine for allergic disease, asthma, cancer and tuberculosis in Asian countries (Zhu et al., 1998a,b; Lee et al., 2006), and in a recent study, we found that it has mutagenic potential and can induce kidney abnormalities, including karyomegaly, in the outer medulla of the kidney (Che et al., 2014). An evaluation of the safety of traditional herbs, especially of their subchronic toxicity and genotoxicity, is imperative because these herbs have been continually used for thousands of years not only in medicine, but also as flavors, food colors, tonics, and additives. Although SR has also been commonly used as an herbal medicine and as a functional food ingredient in East Asian countries, the toxicity of the SR extract has not been conclusively established. In this study, we performed measure the genotoxicity and the subchronic oral toxicity in order to provide accurate information on the potential hazards and on the safety concerns associated with the SR extract.

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2. Materials and methods

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2.1. Test substance and animals

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A hot water SR extract was provided by the National Institute of Food and Drug Safety Evaluation (Osong, Korea). In brief, dried S. flavescens roots were collected, homogenized into a powder, and macerated with distilled water at 110 °C. After filtration through filter paper, the suspension was centrifuged at 6000 rpm, and the supernatant from the extract was freeze-dried. For the

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experiments, the SR extract was then resuspended in distilled water (DW). F344 rats (SLC, Hamamatsu, Japan) and ICR mice (Orient bio, Seongnam, Korea) were housed in an environmentally controlled room (22 ± 2 °C, 40–60% humidity, and 12 h light/dark cycle) and were allowed to have access to tap water and a rodent diet (LabDiet 5002 Certified Rodent Diet, Purina, Seoul, Korea) ad libitum. All of the animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of the Biomedical Research Institute at the Seoul National University Hospital, and this study was performed in compliance with the Good Laboratory Practices for toxicity test guidance issued by the Ministry of Food and Drug Safety (MFDS, 2005) and the Organization for Economic Cooperation and Development (OECD) test guidelines.

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2.2. Experimental designs for the oral toxicity study

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For the 14-day repeat-dose oral toxicity study, healthy male and female F344 rats were divided into six groups after quarantine and acclimatization. The hot water SR extract was administered orally to rats (5/sex/group) at 125, 250, 500, 1000 and 2000 mg/kg daily for 14 days. The clinical signs and mortality of the rats were observed daily and the food/water consumption and body weights were recorded weekly during the study period. The rats were anesthetized with isoflurane one day after the final gavage, and blood was collected from the anesthetized animals via the posterior vena cava. The 13-week repeat-dose oral toxicity study was performed according to the OECD test guideline 408 (OECD, 1998). The hot water SR extract was orally administered to F344 rats (10/sex/group) at 10, 35, 122, 429 and 1500 mg/kg daily for 13 weeks, and the rats were observed daily for clinical signs and for mortality. The body weights were measured every week during the study period, and at the end of the study, the rats were anesthetized with isoflurane one day after the final gavage, and blood was taken via the posterior vena cava.

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2.3. Urinalysis, hematology and serum biochemistry

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During the last week of treatment, a urine analyzer (Miditron Junior II, Roche, Mannheim, Germany) was used according to the manufacturer’s instructions to perform a urinalysis of 10 rats per group (5 males and 5 females) by using fresh urine to determine the pH, specific gravity, leukocyte, nitrite, protein, ketone body, urobilinogen, bilirubin, protein, glucose, and hemoglobin. Whole blood samples were collected into blood-collecting tubes containing anticoagulant EDTA and were assayed using a MS9-5 Hematology Counter (Melet Schloesing Laboratories, Osny, France) for the following parameters: total white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), platelet (PLT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and differential WBC. For the serum biochemistry analysis, whole blood was centrifuged at 3000 rpm for 15 min, and serum was immediately separated and stored at 80 °C prior to analysis. An automatic chemistry analyzer 7070 (Hitachi, Tokyo, Japan) was used according to the manufacturer’s instructions to evaluate the following serum biochemistry parameters: blood urea nitrogen (BUN), creatinine, creatinine kinase, total cholesterol (TC), total protein (TP), albumin, total bilirubin (TB), alkaline phosphatase (ALP), aspartate transaminase (AST), alanine transaminase (ALT), triglyceride (TG), glucose, K, Cl, and Na.

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2.4. Gross findings, organ weights, and histopathological assessments

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During necropsy, the liver, kidney, adrenal gland, urinary bladder, spleen, pancreas, thymus, thyroid gland, parathyroid gland, trachea, esophagus, lung, heart, salivary gland, lymph node, stomach, duodenum, jejunum, ileum, colon, rectum, preputial gland, clitoral gland, skin, brain, pituitary gland, bone marrow, prostate, seminal vesicle, ovary, uterus, vagina, tongue, larynx, skeletal muscle, and sciatic nerve were removed and fixed in 10% neutral formalin, except for testis and epididymis, which were fixed in Bouin’s solution, and the eyes with the Harderian glands, which were fixed in Davidson solution (30 ml 95% ethyl alcohol + 20 ml formalin + 10 ml glacial acetic acid + 30 ml distilled water). The nasal cavity, spinal cords with bones, sternum, and femora were treated with a decalcification solution for up to 3 weeks. Slices of all tissues were routinely processed for paraffin embedding, sectioning, and hematoxylin and eosin (H&E) staining. After staining, the histological changes were examined via light microscopy.Liver, kidney, adrenal gland, urinary bladder, spleen, pancreas, thymus, thyroid gland, parathyroid gland, trachea, esophagus, lung, heart, salivary gland, lymph node, stomach, duodenum, jejunum, ileum, colon, rectum, preputial gland, clitoral gland, skin, brain, pituitary gland, bone marrow, prostate, seminal vesicle, ovary, uterus, vagina, tongue, larynx, skeletal muscle, sciatic nerve.

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2.5. Genotoxicity study

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A bacterial reverse mutation assay (Ames test) was conducted in accordance with the OECD guideline 471 (OECD, 1997a), and the mutagenicity of the SR extract was determined with five Salmonella typhimurium strains – TA98, 100, 102, 1535, and 1537 – provided by the Ministry of Food and Drug Safety (Osong, Korea). The bacterial strains were incubated with SR extract with or without an exogenous metabolic activation (S9 mix) in the dark at 37 °C for 48 h. 2-Nitrofluorene, sodium azide, mitomycin C, 9-aminoacridine, and 2-aminoanthracene (Sigma–Aldrich, St. Louis, MO, USA) were used as positive controls, and the extract was considered to be positive for mutagenicity if there was a twofold increase from negative control or a dose-dependent increase in the number of revertant colonies in one or more strains. The in vitro chromosomal aberration study was conducted according to the OECD guideline 473 (OECD, 1997b). Chinese hamster lung (CHL) fibroblast cells were seeded at a density of 1  105 cells/ml in Minimum Essential medium (GIBCO, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, GIBCO) in a 25 cm2 flask and were incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO2. The cells were exposed to the SR extract for 6 h or for 24 h in the presence or absence of the S9 mix and were washed and then incubated in complete medium for an additional 18 h. For this assay, mitomycin C and cyclophosphoamide (Sigma–Aldrich) were used as positive controls. Next, colcemid (0.2 lg/ml, GIBCO) was added for 2 h, and the cells were then treated with hypotonic solution, fixed in 3:1 methanol/glacial acetic acid, and stained with 4% Giemsa. An in vivo bone marrow micronucleus test was conducted in mice as recommended by the OECD guideline 474 (OECD, 1997c). 8-week-old male ICR mice were orally gavaged daily for 4 d with 0, 500, 1000, and 2000 mg/kg SR extract dissolved in DW. Mitomycin C served as a positive control and was administered as a single intraperitoneal injection at a dose of 2 mg/kg of body weight. The mice were sacrificed 24 h after the last dose of the SR extract, and the femoral bone marrow cells were isolated from each mouse. The cells were centrifuged, smeared onto slides, dried, and fixed in methanol. The fixed slides were stained with 5% Giemsa, and the results were expressed as the number of micronucleated polychromatic erythrocytes (MNPCEs; PCE with one or more micronuclei)

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per 2000 PCEs. Additionally, the PCE/(PCE + NCE) ratio, where the NCEs indicate the normochromatic erythrocytes, was also calculated for detecting the possibility of cytotoxicity (Heddle et al., 1984).

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2.6. Statistical analysis

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All of the values are expressed as mean ± SD, and all of the data were analyzed using SPSS software version 19 (SPSS Inc., Chicago, IL, USA). The statistical analysis was performed using a one-way ANOVA, followed by a multiple comparison procedure with a Tukey/Duncan test. P values of less than 0.05 were considered to be statistically significant.

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3. Results

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3.1. 14-Day repeat-dose oral toxicity study

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High-performance liquid chromatography (HPLC) was performed in order to identify the active components in the SR extract, and the results indicated that 42.7 mg/g of oxymatrine and 18.2 mg/g of matrine were present in the SR extract used in this study. A 14-day repeat-dose oral toxicity study was carried out to select the treatment dose for the SR extract in the 13-week subchronic toxicity study. In this 14-day repeat-dose oral toxicity study, doses of 125, 250, 500, 1000, and 2000 mg/kg of body weight were used for the SR extract, and no mortality or abnormal clinical signs were observed in either vehicle control rats or rats treated with the SR extract. The body weight for the males treated with 2000 mg/kg SR extract were significantly lower than those of the control group starting from Day 11 after the first administration of the SR extract (Fig. 1A). In addition, the body weight gain in males that were administered the SR extract at a dose of 500, 1000 and 2000 mg/kg was significantly lower than that in the control group (Fig. 1B). The body weight gain in females treated with 1000 and 2000 mg/kg SR extract were significantly lower than that of the control group even though there was no statistical significant difference in the absolute body weight among the groups. After the 14-day observation period, the necropsy revealed a significant increase in the absolute and relative liver weights of males treated with 2000 mg/kg of the SR extract and in females treated with 1000 and 2000 mg/kg of the SR extract (Data not shown). In the rats treated with the SR extract, the relative kidney weight significantly increased in males treated with 500, 1000, and 2000 mg/ kg and in females treated with 2000 mg/kg (Data not shown). Based on the results of the 14-day repeat-dose oral toxicity study, the 1500 mg/kg does of the SR extract was selected as the highest dose for the 13-week repeat-dose oral toxicity study.

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3.2. 13-Week repeat-dose oral toxicity study

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3.2.1. Changes in body weight and daily feed intake During the subchronic trial, 10, 35, 122, 429, and 1500 mg/kg doses of SR extract were orally administered, and no death or abnormal clinical signs were observed during the experimental period. The body weights of males treated with 1500 mg/kg of SR extract were significantly lower than those of the control group during the full period of the experiment (Fig. 2). The body weights of males treated with 429 mg/kg and females treated with 429 and 1500 mg/kg SR extract were also significantly lower than those of the respective control groups, even though these were not always significant at P < 0.05. A significant decrease in the mean daily food intake was often, but not always, observed following treatment with 122, 429, and 1500 mg/kg SR extract in both males and females (Supplementary Table 1). No dose-related differences in

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Fig. 1. Growth curves and body weight gain for male and female F344 rats orally administered with Sophorae radix for 14 days. (A) Growth curve. (B) Body weight gain. Data expressed as means ± SD. ⁄Significantly different from control group (p < 0.05).

Fig. 2. Growth curves for male and female F344 rats orally administered with Sophorae radix for 13 weeks. Data expressed as means ± SD. ⁄Significantly different from control group (p < 0.05).

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water consumption were found among the groups following the subchronic oral exposure to SR extract in either sex for all test groups (Supplementary Table 2). 3.2.2. Urinalysis, hematology, and clinical chemistry During urinalysis, dose-related changes were not detected following the administration of the SR extract in both males and females (Supplementary Table 3). Hematology revealed that the male rats treated with SR extract at a dose of 122 and 1500 mg/kg had a significant decrease in the RBC levels relative to the control group (Table 1). The RBC levels in females treated with 429 and 1500 mg/kg of SR extract were also significantly lower than that in the control group. The HGB levels significantly decreased in males treated with 35, 122, 429 and 1500 mg/kg of SR extract and in females treated with 429 and 1500 mg/kg of SR extract. The HCT levels in females treated with the SR extract with doses of 429 and 1500 mg/kg were significantly lower than those in the control group. The WBC differential showed a significant increase in the percentage of reticulocytes in males treated with the 122 and 429 mg/kg of SR extract

compared to that of the control group (p < 0.05). The females treated with 35, 122, 429 and 1500 mg/kg of SR extract also showed an increasing trend in terms of the percentage of reticulocytes relative to that of the control group. The biochemical analysis of the serum indicated that the ALP levels in both males and females treated with 35, 122, 429, and 1500 mg/kg of SR extract significantly increased relative to those of the respective control groups (Table 2). The repeated oral administration of the SR extract at a dose of 1500 mg/kg induced significant increases in the ALT in both males and females, and the TG levels markedly decreased in males and females treated with 429 and 1500 mg/kg of SR extract. However, the change in the female group did not reach statistical significance. Other serum biochemical parameters showed significant differences among groups, but these were not related to the SR extract treatment since the changes were sporadic.

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3.2.3. Organ weights and histopathological changes Tables 3 and 4 summarize the data from the absolute and relative organ weights. Significant increases in the absolute and

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J.-H. Che et al. / Regulatory Toxicology and Pharmacology xxx (2015) xxx–xxx Table 1 Hematological data for male and female F344 rats orally administered with Sophorae radix for 13 weeks. Dose of Sophorae radix (mg/kg)

*

0

10

35

122

429

1500

Males WBC (103/mm3) RBC (106/mm3) HGB (g/dl) HCT (%) Neutrophils (%) Eosinophils (%) Basophils (%) Lymphocytes (%) Monocytes (%) Reticulocyte (%)

5.4 ± 0.6 7.9 ± 0.3 13.8 ± 0.5 39.1 ± 1.5 16.7 ± 3.0 6.3 ± 2.7 0.5 ± 0.1 70.4 ± 3.2 4.7 ± 0.7 0.4 ± 0.1

5.7 ± 0.7 8.0 ± 0.2 13.9 ± 0.3 40.1 ± 1.6 17.3 ± 2.7 5.7 ± 2.4 0.5 ± 0.1 70.5 ± 4.0 4.4 ± 0.7 0.3 ± 0.0*

5.2 ± 0.7 7.7 ± 0.2 13.4 ± 0.2* 38.8 ± 1.4 13.9 ± 3.7 6.7 ± 2.3 0.5 ± 0.1 73.0 ± 3.1 4.3 ± 0.5 0.5 ± 0.2

5.1 ± 0.5 7.5 ± 0.2* 13.0 ± 0.3* 37.5 ± 1.5 11.2 ± 4.9* 7.4 ± 3.7 0.4 ± 0.1 75.4 ± 2.6* 4.1 ± 0.4* 0.8 ± 0.3*

5.1 ± 0.7 7.7 ± 0.3 13.3 ± 0.2* 38.5 ± 1.5 16.1 ± 2.3 5.4 ± 1.2 0.5 ± 0.1 72.1 ± 2.5 4.3 ± 0.3 0.6 ± 0.2*

4.4 ± 0.4* 7.5 ± 0.2* 13.1 ± 0.2* 37.6 ± 1.3 19.3 ± 4.7 5.1 ± 1.7 0.5 ± 0.1 68.5 ± 4.2 4.7 ± 0.4 0.4 ± 0.1

Females WBC (103/mm3) RBC (106/mm3) HGB (g/dl) HCT (%) Neutrophils (%) Eosinophils (%) Basophils (%) Lymphocytes (%) Monocytes (%) Reticulocyte (%)

4.2 ± 0.7 7.2 ± 0.2 13.3 ± 0.7 39.0 ± 1.3 10.3 ± 4.6 9.6 ± 3.4 0.5 ± 0.1 73.5 ± 2.6 4.4 ± 0.6 0.7 ± 0.3

4.1 ± 0.6 7.3 ± 0.2 13.7 ± 0.4 39.4 ± 0.9 12.1 ± 2.0 8.0 ± 2.2 0.5 ± 0.2 73.1 ± 3.1 4.6 ± 0.5 0.7 ± 0.2

3.7 ± 0.4 7.1 ± 0.3 13.3 ± 0.4 38.1 ± 1.8 12.6 ± 2.4 7.0 ± 2.6 0.5 ± 0.1 73.9 ± 3.3 4.1 ± 0.4 0.9 ± 0.5

3.6 ± 0.5 7.1 ± 0.3 13.3 ± 0.4 37.6 ± 1.6 11.5 ± 4.0 8.2 ± 1.7 0.6 ± 0.1 73.8 ± 2.9 4.1 ± 0.5 0.9 ± 0.4

4.0 ± 1.0 6.8 ± 0.2* 12.7 ± 0.4* 36.9 ± 1.1* 13.8 ± 2.6 6.9 ± 3.4 0.5 ± 0.1 73.1 ± 3.9 4.0 ± 0.5 1.0 ± 0.4

4.5 ± 0.7 6.8 ± 0.3* 12.5 ± 0.6* 37.0 ± 1.7* 11.4 ± 3.7 7.2 ± 3.7 0.5 ± 0.1 75.2 ± 1.9 4.0 ± 0.3 1.0 ± 0.3

Significantly different from control group (p < 0.05).

Table 2 Serum biochemistry data for male and female F344 rats orally administered with Sophorae radix for 13 weeks. Dose of Sophorae radix (mg/kg)

*

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0

10

35

122

429

1500

Males BUN (mg/dL) TC (mg/dL) TP (g/dL) Albumin (g/dL) ALP (IU/L) AST (IU/L) ALT (IU/L) Creatinine (mg/dL) TG (mg/dL) K (mmol/L) Cl (mmol/L) Na (mmol/L)

20.6 ± 1.9 63.3 ± 8.0 5.9 ± 0.2 2.6 ± 0.1 136.3 ± 18.5 69.8 ± 10.7 47.9 ± 5.5 0.57 ± 0.08 156.9 ± 58.6 4.0 ± 0.2 101.8 ± 2.3 142.5 ± 2.7

19.8 ± 1.7 58.4 ± 6.8 5.9 ± 0.3 2.6 ± 0.1 141.9 ± 18.4 75.0 ± 15.2 45.9 ± 8.0 0.52 ± 0.08 121.1 ± 74.4 3.9 ± 0.2 100.0 ± 2.6 140.2 ± 3.7

19.4 ± 2.9 67.5 ± 4.4 6.2 ± 0.1* 2.6 ± 0.1 226.6 ± 34.8* 72.8 ± 7.9 49.0 ± 4.8 0.51 ± 0.09 173.0 ± 25.8 4.1 ± 0.2 102.5 ± 2.1 144.3 ± 3.7

18.7 ± 2.6 62.8 ± 3.7 6.0 ± 0.2 2.6 ± 0.1 234.3 ± 28.1* 73.6 ± 7.2 50.7 ± 7.6 0.49 ± 0.07 161.7 ± 53.7 4.2 ± 0.1 101.5 ± 2.3 143.1 ± 3.6

19.6 ± 1.4 78.7 ± 10.3* 6.4 ± 0.2* 2.9 ± 0.1* 183.3 ± 27.7* 66.1 ± 8.5 49.3 ± 6.8 0.66 ± 0.07 69.3 ± 17.8* 4.4 ± 0.2 103.0 ± 3.3 151.3 ± 5.8

19.7 ± 3.6 92.0 ± 7.7* 6.0 ± 0.3 2.7 ± 0.1 197.8 ± 56.8* 57.0 ± 6.9* 58.0 ± 8.9* 0.56 ± 0.07 46.9 ± 9.3* 5.3 ± 2.3* 90.4 ± 21.6* 210.6 ± 115.2*

Females BUN (mg/dL) TC (mg/dL) TP (g/dL) Albumin (g/dL) ALP (IU/L) AST (IU/L) ALT (IU/L) Creatinine (mg/dL) TG (mg/dL) K (mmol/L) Cl (mmol/L) Na (mmol/L)

19.6 ± 2.6 96.0 ± 10.1 6.4 ± 0.4 2.9 ± 0.2 135.0 ± 37.8 84.5 ± 16.9 46.6 ± 7.7 0.65 ± 0.08 42.6 ± 18.5 4.6 ± 0.1 107.5 ± 3.6 154.0 ± 9.3

19.9 ± 2.4 97.2 ± 8.4 6.3 ± 0.2 2.9 ± 0.1 120.6 ± 30.2 72.0 ± 3.5* 44.3 ± 6.1 0.65 ± 0.10 42.7 ± 19.6 4.3 ± 0.2* 105.8 ± 0.9 148.5 ± 3.0*

16.4 ± 2.5* 87.1 ± 5.0* 5.8 ± 0.2* 2.7 ± 0.1* 187.7 ± 42.9* 72.8 ± 12.4 47.2 ± 6.6 0.56 ± 0.07 44.0 ± 35.6 3.8 ± 0.1* 100.2 ± 1.6* 137.4 ± 2.8*

15.0 ± 2.0* 80.5 ± 6.7* 5.5 ± 0.3* 2.5 ± 0.1* 207.7 ± 47.8* 66.9 ± 11.5* 45.4 ± 9.1 0.50 ± 0.09* 39.9 ± 13.0 3.7 ± 0.1* 98.2 ± 1.9* 134.4 ± 3.3*

16.6 ± 1.7* 91.9 ± 3.8 5.6 ± 0.2* 2.6 ± 0.1* 210.2 ± 33.1* 69.1 ± 4.5* 50.4 ± 7.2 0.55 ± 0.10* 28.2 ± 12.4 3.8 ± 0.1* 102.3 ± 2.4* 139.3 ± 2.2*

16.6 ± 1.9* 104.0 ± 8.0 5.7 ± 0.2* 2.6 ± 0.1* 213.5 ± 50.6* 70.2 ± 8.5* 67.7 ± 8.7* 0.54 ± 0.05* 22.0 ± 4.1 4.0 ± 0.2* 103.3 ± 1.7* 141.7 ± 2.6*

Significantly different from control group (p < 0.05).

relative liver weights were found in males treated with 35, 122, and 1500 mg/kg and in females treated with 35, 122, 429, and 1500 mg/kg of SR extract. In the male and female rats treated with SR extract at a dose of 1500 mg/kg, the relative kidney weight significantly increased. The organs of the rats were evaluated via gross visual observation and those of the SR extract-treated groups appeared to be similar to those of the control groups. The histopathological findings showed that no dose-related symptoms could be attributed

to the administration of SR extract in the sampled organs because most of the spontaneous changes were sporadic without dosedependent trends (Data not shown).

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3.3. Genotoxicity

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3.3.1. Bacterial reverse mutation assay (Ames test) The results of the Ames test are presented in Table 5. As expected, all of the positive control groups for the respective S.

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Q1 Please cite this article in press as: Che, J.-H., et al. Genotoxicity and subchronic toxicity of Sophorae radix in rats: Hepatotoxic and genotoxic potential. Regul. Toxicol. Pharmacol. (2015), http://dx.doi.org/10.1016/j.yrtph.2015.01.012

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Table 3 Absolute organ weights for male and female F344 rats orally administered with Sophorae radix for 13 weeks. Dose of Sophorae radix (mg/kg)

* a

0

10

35

122

429

1500

Males Liver Kidney Testis Thymus Heart Lung

9.36 ± 0.56a 1.01 ± 0.06 1.46 ± 0.05 0.18 ± 0.02 0.83 ± 0.26 1.08 ± 0.11

9.03 ± 0.85 0.97 ± 0.07 1.42 ± 0.08 0.19 ± 0.01 0.90 ± 0.03 1.06 ± 0.10

11.20 ± 1.17* 1.01 ± 0.07 1.45 ± 0.03 0.22 ± 0.02 0.89 ± 0.04 1.10 ± 0.11

11.49 ± 0.83* 1.00 ± 0.07 1.41 ± 0.07 0.21 ± 0.02 0.88 ± 0.04 1.11 ± 0.07

9.77 ± 0.70 0.99 ± 0.07 1.41 ± 0.06 0.19 ± 0.02 0.87 ± 0.04 1.05 ± 0.05

11.28 ± 0.67* 1.05 ± 0.06 1.43 ± 0.07 0.16 ± 0.01 0.83 ± 0.04 1.05 ± 0.12

Females Liver Kidney Thymus Heart Lung

4.80 ± 0.30 0.60 ± 0.03 0.18 ± 0.03 0.62 ± 0.04 0.81 ± 0.05

4.94 ± 0.53 0.63 ± 0.08 0.19 ± 0.03 0.61 ± 0.03 0.84 ± 0.10

5.65 ± 0.56* 0.61 ± 0.03 0.19 ± 0.02 0.59 ± 0.03 0.81 ± 0.06

5.80 ± 0.45* 0.63 ± 0.04 0.18 ± 0.02 0.59 ± 0.04 0.82 ± 0.06

5.65 ± 0.51* 0.63 ± 0.05 0.17 ± 0.03 0.60 ± 0.04 0.84 ± 0.08

6.87 ± 0.69* 0.74 ± 0.14* 0.17 ± 0.04 0.62 ± 0.05 0.81 ± 0.13

Significantly different from control group (p < 0.05). Unit, g.

Table 4 Relative organ weights for male and female F344 rats orally administered with Sophorae radix for 13 weeks. Dose of Sophorae radix (mg/kg)

* a

357 358 359 360 361 362 363

364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380

0

10

35

122

429

1500

Males Liver Kidney Testis Thymus Heart Lung

2.95 ± 0.11a 0.32 ± 0.02 0.46 ± 0.02 0.06 ± 0.01 0.26 ± 0.08 0.34 ± 0.03

2.85 ± 0.19 0.31 ± 0.01 0.45 ± 0.02 0.06 ± 0.00 0.29 ± 0.01 0.34 ± 0.02

3.36 ± 0.18* 0.31 ± 0.01 0.44 ± 0.03 0.07 ± 0.00 0.27 ± 0.01 0.33 ± 0.02

3.54 ± 0.11* 0.31 ± 0.01 0.44 ± 0.02 0.06 ± 0.00 0.27 ± 0.01 0.34 ± 0.02

3.29 ± 0.14* 0.33 ± 0.02 0.48 ± 0.02 0.06 ± 0.01 0.29 ± 0.01 0.35 ± 0.02

4.25 ± 0.19* 0.40 ± 0.03* 0.54 ± 0.02* 0.06 ± 0.00 0.31 ± 0.02* 0.40 ± 0.05*

Females Liver Kidney Thymus Heart Lung

2.57 ± 0.11 0.32 ± 0.01 0.10 ± 0.01 0.33 ± 0.02 0.43 ± 0.03

2.68 ± 0.19 0.34 ± 0.03 0.10 ± 0.02 0.33 ± 0.02 0.46 ± 0.03

2.96 ± 0.20* 0.32 ± 0.01 0.10 ± 0.01 0.31 ± 0.02* 0.42 ± 0.03

3.05 ± 0.14* 0.33 ± 0.01 0.09 ± 0.01 0.31 ± 0.01 0.43 ± 0.03

3.15 ± 0.20* 0.35 ± 0.02 0.10 ± 0.02 0.33 ± 0.02 0.47 ± 0.04

3.75 ± 0.23* 0.40 ± 0.07* 0.09 ± 0.02 0.34 ± 0.01 0.44 ± 0.07

Significantly different from control group (p < 0.05). Unit, percentage of body weight.

typhimurium strains in the presence or absence of the S9 mix showed a twofold or greater increase above the negative control in the number of His+ mutants, indicating that the test conditions and the metabolic activation system were adequate. In contrast, no significant increase in the number of revertant colonies could be observed in the SR extract-treated groups in the presence or absence of the S9 mix in the Ames test.

3.3.2. In vitro chromosomal aberration assay An in vitro chromosome aberration assay was performed in order to evaluate the chromosomal aberrations, including breaks, fragments, exchanges, and other multiple damage. A cytotoxicity experiment was first conducted at concentrations 625–5000 lg/ ml for 24 h exposure. Low levels (