Toxic Effects of a Horseradish Extract and Allyl Isothiocyanate in the Urinary Bladder After 13-Week Administration in Drinking Water to F344 Rats

The Journal of Toxicological Sciences (J. Toxicol. Sci.) 763 Vol.36, No.6, 763-774, 2011

Original Article Toxic effects of a horseradish extract and allyl isothiocyanate in the urinary bladder after 13-week administration in drinking water to F344 rats

Mai Hasumura1, Toshio Imai1,2, Young-Man Cho1, Makoto Ueda1, Masao Hirose1,3 Akiyoshi Nishikawa1 and Kumiko Ogawa1

1Division of Pathology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan 2Central Animal Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan 3Present address: Food Safety Commission, Cabinet Ofﬁce, Akasaka Park Building 22nd Floor, 5-2-20 Akasaka, Minato-ku, Tokyo 107-6122, Japan

(Received June 28, 2011; Accepted September 27, 2011)

ABSTRACT — Subchronic toxicity of a horseradish extract (HRE), consisting mainly of a mixture of allyl isothiocyanate (AITC) and other isothiocyanates, was investigated with administration at concentrations of 0, 0.0125, 0.025 and 0.05% of HRE in drinking water for 13 weeks to male and female F344 rats. For comparison, treatment with 0.0425% of AITC was similarly performed. Body weight gain was reduced in the 0.05% HRE and AITC males as compared to the 0% controls, and the cause was considered at least partly related to decreased water consumption due to the acrid smell of the test substance and decreased food consumption. Serum biochemistry demonstrated increased urea nitrogen in 0.025 and 0.05% HRE and AITC males and 0.0125-0.05% HRE and AITC females, along with decreased total cholesterol in 0.0125-0.05% HRE females. On histopathological assessment, papillary/nodular hyperplasia of bladder mucosa was observed in 0.05% HRE and AITC males and females, in addition to simple mucosal hyperplasia found in all treated groups. Based on the above ﬁndings, no-observed-adverse-effect levels (NOAELs) were estimated to be below 0.0125% of HRE for both males and females, corresponding to 9.4 and 8.0 mg/kg body weight/day, respectively, and there appeared to be comparable toxicological properties of HRE to AITC, such as the inductive effect of signiﬁcant proliferative lesions in the urinary bladder.

Key words: Horseradish extract, Bladder toxicity, F344 rats

INTRODUCTION cosinolates, from Brassica vegetables was 75 mg, and it is likely that certain individuals will consume more than Horseradish extract (HRE) is distilled with steam from 300 mg total glucosinolates per day in the UK (Sones et milled horseradish (Armoracia Rusticana) roots, and its al., 1984). However, the daily intake of ITCs from food principal component is allyl isothiocyanate (AITC). HRE additives is uncertain. also includes other isothiocyanates (ITCs), such as phene- ITCs including PEITC and benzyl isothiocyanate thyl isothiocyanate (PEITC), butenyl isothiocyanate and (BITC) have been shown to possess chemopreven- pentenyl isothiocyanate (Tokarska and Karwowska, tive properties against chemically induced carcinogene- 1983). Like AITC and other ITCs, it is used through- sis in animal models (Morse et al., 1991; Nishikawa et out the world as a food additive for its expected antiox- al., 1996; Wattenberg, 1977). These potent effects have idative, antimicrobial and/or ﬂavoring effects. HRE itself been considered partly due to the direct inhibition and/ is also approved as a food additive and widely used for or down-regulation of the cytochrome P-450 enzymes the above-mentioned purposes in Japan (Uematsu et al., involved in carcinogen metabolic activation (Goosen et 2002). ITCs are present in many Brassica vegetables, al., 2001; Nakajima et al., 2001; Nishikawa et al., 1997) and an estimated daily intake of ITC precursors, glu- and the induction of the phase II detoxiﬁcation enzymes Correspondence: Toshio Imai (E-mail: [email protected])

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(Bogaards et al., 1990; Dingley et al., 2003; Munday and tration in diet or drinking water are possibly preferable Munday, 2004). In vitro molecular studies also have sug- to gavage studies. In the present experiment, subchronic gested chemopreventive effects of ITCs, including AITC toxicity of HRE, consisting mainly of a mixture of AITC (Xu et al., 2006), due to mechanisms other than alteration and other ITCs, was evaluated with 13-week administra- of carcinogen metabolism, e.g., through induction of cel- tion in drinking water to F344 rats. The objective of the lular apoptosis. present study is to estimate no-observed-adverse-effect On the other hand, carcinogenic potential of AITC has levels (NOAELs) of HRE and to compare the toxicologi- also been indicated. Gavage administration in conven- cal properties of HRE to those of AITC, which shows uri- tional 2-year bioassays in rats and mice resulted in urinary bladder toxicities in rats. nary bladder tumor induction limited to the rat (NTP, 1982). PEITC and BITC also showed promoting activi- MATERIALS AND METHODS ty and/or complete carcinogenic potential in the urinary bladder or liver of rats (Hirose et al., 1998; Ogawa et al., 2-Week preliminary study 1998). This carcinogenic and/or tumor promoting activity A total of 35 male F344/DuCrj rats at 6 weeks old of of ITCs in the urinary bladder is considered at least part- age (Charles River Japan; Kanagawa, Japan) were divided ly to be derived from cell proliferation stimuli following into 7 groups, including HRE at concentrations of 0 (con- mucosal damage by free forms of ITCs cleaved from the trol, 0.03% Tween 80), 0.025, 0.05 and 0.1% and AITC conjugated metabolites in urine (Masutomi et al., 2001). at 0.025, 0.05 and 0.1% in drinking water each. Body With regard to genotoxicity, AITC induced DNA-dam- weights and food consumptions were measured week- age/gene mutations in E. coli and Salmonella, and PEITC ly, and water consumption was assessed twice a week. At induced micronucleus, chromosomal aberration or sister the end of the experiment, all animals were euthanized by chromatid exchange in HepG2 and Chinese hamster ova- exsanguination under ether anesthesia, and histopatho- ry cell lines (Kassie and Knasmuller, 2000; Musk et al., logical examination was performed on the liver, kidneys, 1995). These results indicate that carcinogenic effect of stomach and urinary bladder. ITCs in the urinary bladder epithelium may partly reflect their genotoxic potential. Chemicals General toxicological profiles of ITCs except for HRE and AITC were obtained from Yamachu Wasabi AITC have yet to be examined in detail. A pre-chron- (Saitama, Japan). The HRE was distilled with steam from ic toxicity study of AITC was conducted with 4-weeks horseradish roots, and the AITC was synthesized. The gavage administration at doses up to 50 and 400 mg/kg HRE sample was a clear yellow and acrid liquid and dem- body weight/day to mice and rats, respectively, and stom- onstrated contents of AITC, PEITC, butenyl isothiocy- ach mucosa and/or urinary bladder wall thickening were anate and pentenyl isothiocyanate of 82-86, 9, 3 and 1%, detected, but no obvious gross or microscopic chang- respectively (internal data of Yamachu Wasabi). AITC es were found in another 13-week study at doses up to was a clear, colorless and acrid liquid, and the purity was 25 mg/kg body weight/day (NTP, 1982). In a 6-week gav- over 97%. AITC dissolves sparingly in water but is sol- age study of AITC in rats, several toxicological findings, uble in ethanol and miscible with most organic solvents such as decreased body and thymus weights, increased (Tanimura, 1999). For administration in drinking water, liver weight and dilatation of distal tubules in the kidneys, HRE and AITC were individually mixed with Tween 80 were reported at 20 and/or 40 mg/kg body weight/day (Wako Pure Chemical, Osaka, Japan) at an amount so (Lewerenz et al., 1988). A subchronic toxicity study of that the final concentration became 0.03%, and then dilut- PEITC in male rats with 13-weeks dietary administration ed with reverse osmosis (deionized) water. The mixed at concentrations up to 0.1% also revealed degenerative water was prepared just before the treatment twice a week lesions in the liver (Morse et al., 1989). As for pharma- throughout the experiment. The stability of HRE was cokinetics of ITCs, PEITC has been studied extensively evaluated by measurement of AITC concentrations in the to characterize its properties. PEITC has high oral bioa- drinking water and confirmed as being 76, 62 and 40% vailability and shows non linear elimination due at least after still standing for 1, 2 and 4 days, respectively, at partly to saturation of metabolism at doses 0.3-65 mg/kg room temperature. The stability of AITC was also 40-44% body weight in rats (Telang et al., 2009). ITCs are con- after 4 days in the same conditions. No major peaks of the sumed from foodstuff or food additives in man, and the break down products were found on the high-performance absorption is suspected to be slower than that of gavage liquid chromatography charts in the both HRE and AITC cases. Therefore, in vivo toxicity studies with adminis- cases (Fig. 1).

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Experimental animals and housing conditions A total of 50 male and 50 female F344/DuCrj rats at 5 weeks old of age were purchased from Charles River Japan and acclimated for 1 week prior to the com- mencement of the experiment. Rats were allocated with body weight-basis randomization to ﬁ ve groups per sex, each consisting of 10 males and 10 females, and main- tained in a room with a barrier system under the following conditions: temperature of 24 ± 1°C; relative humid- ity 55 ± 5 %; ventilation frequency of 18 times/hr; and a 12-hr light/dark cycle. The animals were housed ﬁ ve rats per plastic cage with sterilized soft wood chips (Sankyo Laboratory Service, Tokyo, Japan) and allowed free access to drinking water containing HRE or AITC and a basal diet CRF-1 (Oriental Yeast, Tokyo, Japan) ad libi- tum. The test substance-supplemented drinking water in clear polycarbonate bottles was replaced twice a week throughout the experiment.

Experimental design The animals of both sexes received HRE at concentrations of 0 (control, 0.03% Tween 80), 0.0125, 0.025 and 0.05% and AITC at 0.0425% in drinking water for 13 weeks, based on a 2-week preliminary study in male rats. In the preliminary study, signifi cantly lowered body weights and water consumption in the 0.1% HRE and AITC groups, obviously reduced water consumption but no signifi cant body weight changes in the 0.025 and 0.05% HRE and AITC groups and simple and papillary/nodular mucosal hyperplasia of the urinary bladder in the 0.025- 0.1% HRE and AITC groups were observed (Table 1). Clinical signs and mortality were checked daily. Body weights and food consumptions were measured weekly, and water consumption was assessed at the time of drinking water replacement. At the end of the experiment, all animals were fasted overnight and euthanized by exsanguination under ether anesthesia after blood sample col- lection from the abdominal aorta for hematology and blood biochemistry. The blood sampling of each group males were performed in rotation at 10:00-14:30 and those of females were at 14:30-16:30 in a day. The experiments were carried out in accordance with the Guide for Animal Experimentation in the National Institute of Health Sciences of Japan. Hematological examinations were performed using an automatic hematology analyzer, M-2000 (Toa Medical Electronics; Hyogo, Japan) and the following parameters Fig. 1. HPLC charts demonstrating a main peak of AITC were determined: red blood cell count (RBC), hemoglob- on day 0 (A), day 1 (B), and a main peak and sev- in concentration (Hb), hematocrit (Ht), mean corpuscular eral small peaks on day 2 (C) after preparation of the 0.01% HRE-mixed water. The small peaks were not volume (MCV), mean corpuscular hemoglobin (MCH), identical with presumed AITC-decomposed materials, mean corpuscular hemoglobin concentration (MCHC), allylcyanid, allyl disulfi de and allyl trisulfi de.

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Table 1. Summary of toxicological ﬁndings in rats treated with HRE or AITC for 2 weeks HRE AITC Dose level (%) 0 0.025 0.05 0.1 0.025 0.05 0.1 Average intake (mg/kg body weight/day) 22.8 46.5 69.8 24.2 47.9 83.0 No. of rats 555 5 5 5 5 Final body weight (g) 186.8 ± 6.3 a 181.1 ± 8.6 179.1 ± 6.5 158.4 ± 9.6 ** 180.7 ±9.7 180.1 ± 10.2 157.7 ± 13.4 ** Food consumption (g/rat/day) 13.6 11.2 12.2 10.0 11.4 12.6 11.5 Water consumption (g/rat/day) 19.9 15.3 15.1 10.2 16.3 15.4 12.0 Relative organ weights Liver (g/100 g body weight) 3.9 ± 0.2 a 4.1 ±0.1 4.1 ± 0.1 3.9 ±0.2 4.0 ±0.1 4.1 ± 0.1 3.9 ±0.2 Kidneys (g/100 g body weight) 0.7 ± 0.0 a 0.7 ± 0.0 0.7 ± 0.0 0.9 ± 0.0 ** 0.8 ±0.0 0.8 ± 0.0 1.0 ±0.0 * Histopathology Urinary bladder Simple hyperplasia 05 ** 5 ** 5 ** 5 ** 5 ** 4 * Papillary/nodular hyperplasia 04 *5 ** 5 ** 3 5 ** 4 * Stomach (limiting ridge) Mucosal hyperplasia 0122 0 23 a: Values are means ± S.Ds. *,**: Signiﬁcantly different from the control value at p < 0.05 and p < 0.01, respectively.

platelet count (Plt) and white blood cell count (WBC). of both sexes. In addition, urinary bladder and stomach Differential leukocyte counts and the reticulocyte count specimens of the HRE 0.0125 and 0.025% groups were (Ebl) were obtained with a Microx HEG-120A (Omron also assessed. Tateishi Electronics; Tokyo, Japan). The present study design was basically in accordance Serum biochemistry was conducted for the following with Guidelines for Designation of Food Additives and 18 parameters: total protein (TP), albumin (Alb), albu- for Revision of Standards for Use of Food Additives of min/globulin ratio (A/G), glucose, total bilirubin (T-Bil), Japan (1996). The experiments were carried out in accord- total cholesterol (T-Cho), triglyceride (TG), γ-glutamyl ance with the Guide for Animal Experimentation of the transpeptidase (γ-GTP), aspartate transaminase (AsT), National Institute of Health Sciences of Japan. alanine transaminase (AlT), alkaline phosphatase (ALP), blood urea nitrogen (BUN), creatinine (Cre), calcium Statistics (Ca), inorganic phosphorus (IP), sodium (Na), potassium Variance in data for body weights, hematology, serum (K) and chloride (Cl). biochemistry and organ weights was checked for homoge- All animals were subjected to a complete necrop- neity by Bartlett’s procedure. When the data were homo- sy. Brain, thymus, lungs, heart, spleen, liver, kidneys, geneous, one-way analysis of variance (ANOVA) was adrenals and testes were weighed. In addition to these applied. In the heterogeneous cases, the Kruskal-Wallis organs, nasal cavity, trachea, aorta, pituitary, thyroids, test was used. When statistically significant differences parathyroids, salivary glands, tongue, esophagus, stom- were indicated, Dunnett’s multiple test was employed for ach, duodenum, jejunum, ileum, cecum, colon, rectum, comparison between control and treated groups. For inci- pancreas, urinary bladder, epididymides, prostate, seminal dences of histopathological findings, the Fisher’s exact vesicles, ovaries, uterus, vagina, mammary gland, skin, probability test was applied. Significance was inferred at mesenteric and submandibular lymph nodes, sternum, the 5, 1, and 0.1% levels. femur including bone marrow, sciatic nerve, trigeminal nerve, spinal cord (cervical, thoracic and lumber cords), RESULTS eyes, and thigh muscle were excised. All these organs and tissues were fixed in 10% buffered formalin except for In-life parameters testes fixed in Bouin’s solution. Paraffin-embedded tis- No mortality or obvious clinical signs were found in sue sections of all organs/tissues were routinely prepared any animals throughout the experimental period. No effects and stained with hematoxilin and eosin. Histopatholog- on body weight were observed in females, whereas HRE ical examination was performed on all organs and tis- 0.05% group males from week 1 and AITC group males sues in the control, HRE 0.05% and AITC group animals from week 2 showed lower body weights as compared to

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Hematology/serum biochemistry Although Ebl was increased in AITC group males, no obvious change with a dose-relation was noted in HRP group males (Table 3). In females, Mono was decreased in HRE 0.05% and AITC groups (Table 4). These changes were considered not to be toxicologically signiﬁ cant, since there was no change in other hematological parameters or histopathology of related organs/tissues. Slight increase in MCV found in HRE 0.0125 and 0.05% group females without a dose-relation was not considered test substance related (Table 4). In males, BUN was increased in the HRE 0.025 and 0.05% and AITC groups, whereas Cre was decreased in the AITC group (Table 5). Slight increase in Alb with Fig. 2. Body weight curves of rats treated with HRE or AITC HRE 0.025 and 0.05% and slight increase in A/G for for 13weeks. *,**: Significantly different from the HRE 0.05% was detected, but the toxicological signiﬁ - controls at p < 0.05 and p < 0.01, respectively. cance was not clear, since TP levels were not changed. Cl was slightly increased at HRE 0.05%, but without change in other electrolyte parameters (Table 5). In females, a the controls until the end of the experiment (Fig. 2). decrease of T-Cho and an increase of BUN were observed Food and water consumption was decreased with in HRE-treated groups in a dose-related manner, and a dose-related manner in all HRE treated groups of BUN was also increased in the AITC group (Table 6). both males and females, and also with AITC treatment (Table 2). Average intakes of HRE in the 0.0125, 0.025 Organ weights and 0.05% groups were 12.7, 19.4 and 36.4 mg/kg body In males, increase in relative kidney weight was noted weight/day in males and 10.8, 20.5 and 36.5 mg/kg body in the HRE 0.025 and 0.05% groups. The following alter- weight/day in females, respectively. Average intakes of ations were considered to be related to the body weight AITC in AITC group males and females were 40.0 and changes: decrease in absolute weights of brain, lungs, 37.9 mg/kg body weight/day, respectively. Estimated heart, spleen, liver, kidneys and testes in the HRE 0.05% AITC intakes of HRE 0.05% group were 30.6 and 30.7 group; increase in relative brain weight in the HRE 0.05% mg/kg body weight/day in males and females, respective- and AITC groups; and increase in relative testes weights ly, appearing to be lower than those in AITC group. in the HRE 0.05% group (Table 7). In females, increase in absolute and/or relative kidney weights was observed in

Table 2. Water and food consumption in rats treated with HRE or AITC for 13 weeks Dose level No. of rats Water Average intakes Estimated Food (%) consumption of HRE AITC intakes consumption (g/rat/day) (mg/kg body (mg/kg body (g/rat/day) weight/day) weight/day) Male 0 10 26.6 - - 14.1 HRE 0.0125 10 25.0 12.7 10.7 13.7 HRE 0.025 10 18.8 19.4 16.3 13.5 HRE 0.05 10 16.7 36.4 30.6 12.5 AITC 10 22.5 - 40.0 13.2

Female 0 10 19.6 - - 9.3 HRE 0.0125 10 13.1 10.8 9.1 8.9 HRE 0.025 10 12.6 20.5 17.2 8.8 HRE 0.05 10 10.9 36.5 30.7 8.5 AITC 10 13.3 - 37.9 8.6

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Table 3. Hematological changes in male rats treated with HRE or AITC for 13 weeks HRE Dose level (%) 0 (control) AITC 0.0125 0.025 0.05 No. of rats 10 10 10 10 10 RBC 1010/l 937 ± 71 944 ± 162 904 ± 57 960 ± 97 a 905 ± 163 Hb g/dl 16.0 ± 1.3 14.9 ± 2.0 15.5 ± 1.1 16.1 ± 1.8 a 16.1 ± 4.2 Ht % 49.7 ± 3.8 50.1 ± 8.7 48.1 ± 2.9 51.3 ± 5.1 a 48.0 ± 8.7 MCV ﬂ 53.1 ± 0.5 53.1 ± 0.4 53.3 ± 0.4 53.4 ± 0.3 a 53.1 ± 0.3 MCH Pg 17.0 ± 0.3 15.9 ± 1.0 ** 17.1 ± 1.3 16.7 ± 0.4 a 17.8 ± 3.8 MCHC g/dl 32.1 ± 0.4 30.0 ± 1.9 ** 32.2 ± 2.4 31.3 ± 0.8 a 33.5 ± 7.2 Plt 1010/l 70.3 ± 6.5 72.4 ± 6.5 71.1 ± 3.2 70.8 ± 8.4 a 69.8 ± 9.3 WBC 108/l 51.5 ± 6.3 49.8 ± 9.2 43.5 ± 12.2 51.0 ± 11.7 a 46.0 ± 14.8 Ebl count/200 WBC 3.6 ± 2.1 3.4 ± 2.1 7.4 ± 3.6 * 5.6 ± 2.1 9.5 ± 3.7 **

Differential cell count (%) Band 0.0 ± 0.0 0.1 ± 0.2 0.1 ± 0.2 0.0 ± 0.0 0.1 ± 0.2 Seg 11.0 ± 5.3 13.6 ± 6.9 13.2 ± 5.1 15.5 ± 7.4 12.2 ± 6.9 Eosino 1.0 ± 0.3 1.2 ± 0.8 0.9 ± 0.8 0.8 ± 0.5 1.0 ± 0.8 Baso 0.1 ± 0.2 0.0 ± 0.0 0.1 ± 0.2 0.2 ± 0.3 0.3 ± 0.3 Lympho 86.8 ± 5.5 84.3 ± 7.3 84.9 ± 5.6 82.6 ± 7.8 85.4 ± 8.1 Mono 1.2 ± 1.0 0.8 ± 0.9 0.9 ± 0.8 1.0 ± 0.7 1.1 ± 0.9 Values are means ± S.Ds. *,**: Signiﬁcantly different from the controls at p < 0.05 and p < 0.01, respectively. a: The number of samples measured was nine, due to a sampling error.

Table 4. Hematological changes in female rats treated with HRE or AITC for 13 weeks HRE Dose level (%) 0 (control) AITC 0.0125 0.025 0.05 No. of rats 10 10 10 10 10 RBC 1010/l 787 ± 35 820 ± 58 823 ± 83 790 ± 154 806 ± 63 Hb g/dl 13.6 ± 0.7 13.8 ± 1.2 13.6 ± 1.6 13.3 ± 3.2 13.7 ± 1.4 Ht % 43.6 ± 2.0 45.8 ± 3.2 45.8 ± 4.6 44.2 ± 8.5 44.8 ± 3.4 MCV ﬂ 55.4 ± 0.2 55.9 ± 0.2 ** 55.6 ± 0.2 56.0 ± 0.4 ** 55.7 ± 0.3 MCH Pg 17.3 ± 0.3 17.2 ± 0.6 16.6 ± 1.2 16.8 ± 1.0 17.0 ± 0.6 MCHC g/dl 31.3 ± 0.6 30.7 ± 1.4 29.7 ± 2.2 30.0 ± 1.9 30.5 ± 1.1 Plt 1010/l 75.0 ± 5.8 70.4 ± 10.4 74.2 ± 4.8 71.3 ± 7.4 76.3 ± 4.1 WBC 108/l 37.5 ± 4.7 35.6 ± 9.0 39.6 ± 6.2 35.3 ± 12.0 38.5 ± 5.5 Ebl count/200 WBC 3.4 ± 2.1 6.2 ± 4.9 7.7 ± 3.6 8.2 ± 6.7 5.1 ± 3.6

Differential cell count (%) Band 0.1 ± 0.2 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Seg 19.3 ± 3.6 15.9 ± 3.9 16.2 ± 6.0 17.7 ± 4.7 19.4 ± 4.8 Eosino 1.3 ± 0.8 0.8 ± 0.5 1.1 ± 0.8 0.8 ± 0.5 1.2 ± 0.7 Baso 0.2 ± 0.3 0.1 ± 0.2 0.1 ± 0.2 0.0 ± 0.0 0.1 ± 0.2 Lympho 76.9 ± 4.0 81.1 ± 4.2 80.9 ± 6.3 80.3 ± 4.7 78.2 ± 4.9 Mono 2.3 ± 0.8 2.1 ± 0.7 1.7 ± 1.0 1.2 ± 0.8 * 1.2 ± 0.5 ** Values are means ± S.Ds. *,**: Signiﬁcantly different from the control value at p < 0.05 and p < 0.01, respectively. the HRE 0.025, 0.05% and AITC groups. Slight increase in Histopathology relative spleen weights found with HRE 0.025 and 0.05% HRE- or AITC-related lesions were found in the uri- (Table 8) was considered not to be toxicologically signiﬁ- nary bladder and stomach (Table 9). In the urinary blad- cant, since there were no related-histopathological changes. der, simple mucosal hyperplasia was observed in all HRE

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Table 5. Serum biochemical changes in male rats treated with HRE or AITC for 13 weeks HRE Dose level (%) 0 (control) AITC 0.0125 0.025 0.05 No. of rats 10 10 10 10 10 TP g/dl 6.8 ± 0.1 6.9 ± 0.2 6.9 ± 0.2 6.9 ± 0.2 6.8 ± 0.2 Alb g/dl 4.5 ± 0.1 4.6 ± 0.2 4.7 ± 0.1 * 4.7 ± 0.1 * 4.6 ± 0.1 A/G 2.0 ± 0.1 2.0 ± 0.1 2.1 ± 0.1 2.1 ± 0.1 * 2.1 ± 0.1 Glucose mg/dl 188.7 ± 22.4 177.0 ± 17.8 176.6 ± 27.2 180.8 ± 15.0 172.7 ± 14.8 T-Bil mg/dl 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 T-Cho mg/dl 63.4 ± 3.5 63.6 ± 3.8 66.4 ± 6.7 59.9 ± 4.6 60.4 ± 5.3 TG mg/dl 93.4 ± 31.5 80.5 ± 22.4 77.6 ± 23.3 69.3 ± 17.7 74.1 ± 23.8 γ-GTP IU/l < 2 < 2 < 2 < 2 < 2 AsT IU/l 94.0 ± 11.5 98.0 ± 20.8 105.2 ± 16.7 111.2 ± 32.0 128.3 ± 51.4 AlT IU/l 54.3 ± 5.5 51.1 ± 5.9 56.8 ± 5.7 57.1 ± 11.0 58.1 ± 13.8 ALP IU/l 302.0 ± 23.2 292.9 ± 15.4 293.5 ± 30.9 292.1 ± 25.1 307.0 ± 22.7 BUN mg/dl 18.7 ± 1.6 19.6 ± 1.1 22.0 ± 2.0 ** 23.9 ± 1.8 ** 21.6 ± 1.9 ** Cre mg/dl 0.30 ± 0.03 0.29 ± 0.02 0.28 ± 0.02 0.28 ± 0.01 0.25 ± 0.02 ** Ca mg/dl 10.2 ± 0.5 10.2 ± 0.2 10.3 ± 0.2 10.3 ± 0.2 10.1 ± 0.3 IP mg/dl 6.4 ± 0.7 6.3 ± 0.3 6.7 ± 0.9 6.1 ± 0.4 6.6 ± 0.5 Na mEQ/l 145.7 ± 1.3 146.1 ± 1.3 144.7 ± 1.8 144.5 ± 1.3 144.7 ± 1.3 K mEQ/l 4.3 ± 0.4 4.2 ± 0.3 4.4 ± 0.7 4.2 ± 0.3 4.4 ± 0.3 Cl mEQ/l 103.1 ± 1.0 103.3 ± 0.7 103.1 ± 1.2 104.6 ± 0.8 ** 104.1 ± 1.2 Values are means ± S.Ds. *,**: Signiﬁ cantly different from the control value at p < 0.05 and p < 0.01, respectively.

or AITC treated groups of both sexes, with statistical sig- niﬁ cance noted for HRE 0.025% females and HRE 0.05% and AITC males and females. In addition, papillary/nodular hyperplasia was observed in HRE 0.05% and AITC groups of both sexes (Fig. 3). In the stomach, diffuse mucosal hyperplasia in pylorus was found in both sexes receiving AITC. Other ﬁ ndings in stomach of HRE 0.05% or AITC groups were not considered to be related to the treatment, because similar changes were found in other dose groups including the controls without dose-dependence or they are solitary cases. In other organs, several lesions were found in control, HRE 0.05% or AITC groups, but they are known to occur spontaneously in this strain, and neither increases in their incidences nor specific types of lesions were observed in the HRE 0.05% and AITC groups (Table 10). Therefore, they were not considered test substance related.

DISCUSSION

In the present study of general toxicity of HRE admin- Fig. 3. (A) Normal mucosa of the urinary bladder of a male istered in the drinking water at concentrations of 0, rat treated with 0%, (B) Papillary/nodular mucosal hy- 0.0125, 0.025 and 0.05% to F344 rats for 13 weeks, as perplasia of the urinary bladder of a male rat treated well as 0.0425% of AITC for comparison, test substance– with 0.05% HRE and (C) Papillary/nodular mucosal hyperplasia of the urinary bladder of a male rat treated related changes were observed with regard to body with AITC for 13 weeks, hematoxilin and eosin stain- weights, water and food consumption, serum BUN and ing, original magniﬁ cation is x 180.

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Table 6. Serum biochemical changes in female rats treated with HRE or AITC for 13 weeks HRE Dose level (%) 0 (control) AITC 0.0125 0.025 0.05 No. of rats 10 10 10 10 10 TP g/dl 6.7 ± 0.2 6.7 ± 0.3 6.6 ± 0.1 6.5 ± 0.2 6.6 ± 0.3 Alb g/dl 4.7 ± 0.1 4.6 ± 0.2 4.6 ± 0.1 4.5 ± 0.1 4.6 ± 0.2 A/G 2.4 ± 0.2 2.3 ± 0.1 2.3 ± 0.1 2.3 ± 0.1 2.3 ± 0.1 Glucose mg/dl 107.3 ± 14.7 127.8 ± 13.8 126.2 ± 30.8 124.5 ± 17.4 132.2 ± 16.4 T-Bil mg/dl 0.1 ± 0.1 0.1 ± 0.1 0.0 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 T-Cho mg/dl 92.6 ± 7.3 84.5 ± 7.4 * 83.2 ± 4.8 * 78.1 ± 8.6 ** 85.8 ± 6.3 TG mg/dl 39.2 ± 19.0 35.1 ± 11.1 38.6 ± 10.8 34.2 ± 22.3 33.0 ± 14.3 γ-GTP IU/l < 2 < 2 < 2 < 2 < 2 AsT IU/l 82.8 ± 5.5 75.0 ± 9.5 72.6 ± 6.6 * 78.5 ± 5.4 75.9 ± 9.8 AlT IU/l 35.0 ± 3.2 35.6 ± 4.0 34.4 ± 4.4 35.5 ± 4.3 35.3 ± 3.5 ALP IU/l 180.5 ± 9.4 191.0 ± 26.6 188.1 ± 25.7 192.3 ± 21.8 187.6 ± 17.8 BUN mg/dl 16.9 ± 1.8 19.6 ± 2.2 * 20.7 ± 1.6 ** 21.6 ± 2.1 ** 21.3 ± 1.9 ** Cre mg/dl 0.32 ± 0.05 0.28 ± 0.03 0.29 ± 0.02 0.31 ± 0.03 0.28 ± 0.01 Ca mg/dl 10.0 ± 0.2 9.9 ± 0.3 9.9 ± 0.2 10.0 ± 0.3 10.1 ± 0.3 IP mg/dl 4.9 ± 0.8 4.9 ± 1.1 5.3 ± 0.8 5.6 ± 1.0 5.5 ± 0.5 Na mEQ/l 144.1 ± 0.7 145.1 ± 1.6 143.8 ± 1.1 144.3 ± 2.4 145.0 ± 1.1 K mEQ/l 3.8 ± 0.3 3.7 ± 0.8 3.8 ± 0.1 4.0 ± 0.5 4.0 ± 0.3 Cl mEQ/l 104.5 ± 1.5 105.4 ± 1.5 104.6 ± 1.4 105.0 ± 1.1 105.0 ± 0.8 Values are means ± S.Ds. *,**: Signiﬁcantly different from the control value at p < 0.05 and p < 0.01, respectively.

Table 7. Final body and organ weights of male rats treated with HRE or AITC for 13 weeks HRE Dose level (%) 0 (control) AITC 0.0125 0.025 0.05 No. of rats 10 10 10 10 10 Body weight (g) 309.4 ± 11.2 297.6 ± 8.4 * 299.4 ± 9.7 271.7 ± 10.7 ** 286.5 ± 6.7 ** Absolute Brain (g) 1.94 ± 0.03 1.94 ± 0.07 1.93 ± 0.04 1.89 ± 0.02 ** 1.92 ± 0.02 Thymus (g) 0.21 ± 0.03 0.21 ± 0.05 0.21 ± 0.04 0.17 ± 0.04 0.19 ± 0.04 Lungs (g) 0.97 ± 0.05 0.95 ± 0.07 0.96 ± 0.05 0.86 ± 0.06 ** 0.93 ± 0.06 Heart (g) 0.90 ± 0.04 0.86 ± 0.04 0.90 ± 0.05 0.82 ± 0.04 ** 0.86 ± 0.06 Spleen (g) 0.61 ± 0.03 0.59 ± 0.03 0.61 ± 0.02 0.54 ± 0.04 ** 0.59 ± 0.03 Liver (g) 7.22 ± 0.34 7.04 ± 0.40 7.11 ± 0.44 6.54 ± 0.42 ** 6.86 ± 0.34 Kidneys (g) 1.83 ± 0.08 1.80 ± 0.07 1.85 ± 0.11 1.72 ± 0.10 * 1.75 ± 0.09 Adrenals (mg) 36.2 ± 4.0 37.6 ± 2.8 39.2 ± 6.4 36.3 ± 5.6 38.0 ± 6.6 Testes (g) 3.16 ± 0.07 2.99 ± 0.26 3.04 ± 0.27 2.95 ± 0.09 ** 3.05 ± 0.12 Relative Brain (g%) 0.63 ± 0.02 0.65 ± 0.03 0.65 ± 0.02 0.70 ± 0.03 ** 0.67 ± 0.02 ** Thymus (g%) 0.07 ± 0.01 0.07 ± 0.01 0.07 ± 0.02 0.06 ± 0.02 0.07 ± 0.02 Lungs (g%) 0.31 ± 0.02 0.32 ± 0.02 0.32 ± 0.02 0.32 ± 0.02 0.33 ± 0.02 Heart (g%) 0.29 ± 0.01 0.29 ± 0.01 0.30 ± 0.02 0.30 ± 0.01 0.30 ± 0.02 Spleen (g%) 0.20 ± 0.01 0.20 ± 0.01 0.20 ± 0.01 0.20 ± 0.01 0.21 ± 0.01 Liver (g%) 2.33 ± 0.08 2.37 ± 0.11 2.37 ± 0.10 2.41 ± 0.10 2.40 ± 0.10 Kidneys (g%) 0.59 ± 0.02 0.60 ± 0.02 0.62 ± 0.03 * 0.63 ± 0.03 ** 0.61 ± 0.02 Adrenals (mg%) 11.8 ± 1.3 12.7 ± 1.1 13.0 ± 2.4 13.6 ± 2.3 13.4 ± 2.5 Testes (g%) 1.02 ± 0.03 1.01 ± 0.09 1.02 ± 0.07 1.09 ± 0.03 ** 1.06 ± 0.04 Values are means ± S.Ds. *,**: Signiﬁcantly different from the control value at p < 0.05 and p < 0.01, respectively.

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Table 8. Final body and organ weights of female rats treated with HRE or AITC for 13 weeks HRE Dose level (%) 0 (control) AITC 0.0125 0.025 0.05 No. of rats 10 10 10 10 10 Body weight (g) 170.4 ± 6.0 169.2 ± 8.9 168.8 ± 6.8 164.0 ± 3.3 165.0 ± 6.0 Absolute Brain (g) 1.78 ± 0.03 1.71 ± 0.04 * 1.76 ± 0.06 1.77 ± 0.05 1.76 ± 0.05 Thymus (g) 0.16 ± 0.02 0.16 ± 0.03 0.16 ± 0.02 0.14 ± 0.02 0.16 ± 0.03 a Lungs (g) 0.68 ± 0.03 0.66 ± 0.07 0.68 ± 0.04 0.67 ± 0.06 0.66 ± 0.05 Heart (g) 0.55 ± 0.04 0.54 ± 0.04 0.57 ± 0.04 0.54 ± 0.03 0.55 ± 0.03 Spleen (g) 0.37 ± 0.01 0.38 ± 0.02 0.40 ± 0.02 0.39 ± 0.03 0.38 ± 0.02 Liver (g) 3.64 ± 0.15 3.59 ± 0.29 3.64 ± 0.14 3.65 ± 0.22 3.54 ± 0.15 Kidneys (g) 1.04 ± 0.03 1.05 ± 0.07 1.10 ± 0.04 * 1.12 ± 0.05 ** 1.08 ± 0.06 Adrenals (mg) 37.0 ± 2.9 36.8 ± 5.8 41.6 ± 3.4 38.3 ± 4.7 40.5 ± 3.8 Relative Brain (g%) 1.04 ± 0.05 1.02 ± 0.07 1.04 ± 0.05 1.08 ± 0.05 1.07 ± 0.05 Thymus (g%) 0.10 ± 0.01 0.10 ± 0.02 0.09 ± 0.01 0.09 ± 0.01 0.10 ± 0.02 a Lungs (g%) 0.40 ± 0.02 0.39 ± 0.04 0.40 ± 0.02 0.41 ± 0.04 0.40 ± 0.03 Heart (g%) 0.33 ± 0.02 0.32 ± 0.03 0.34 ± 0.02 0.33 ± 0.01 0.33 ± 0.02 Spleen (g%) 0.22 ± 0.01 0.23 ± 0.02 0.23 ± 0.01 * 0.24 ± 0.02 * 0.23 ± 0.01 Liver (g%) 2.14 ± 0.10 2.12 ± 0.14 2.16 ± 0.04 2.23 ± 0.14 2.15 ± 0.08 Kidneys (g%) 0.61 ± 0.02 0.62 ± 0.05 0.65 ± 0.03 * 0.68 ± 0.03 ** 0.66 ± 0.03 * Adrenals (mg%) 21.7 ± 1.9 21.9 ± 4.2 24.4 ± 2.1 23.5 ± 3.1 24.5 ± 3.0 Values are means ± S.Ds. *,**: Signiﬁcantly different from the control value at p < 0.05 and p < 0.01, respectively. a: The number of the samples measured was nine, due to an error in data recording.

Table 9. Histopathological findings in the urinary bladder and stomach of rats treated with HRE or AITC for 13 weeks HRE 0 Dose level (%) AITC (control) 0.0125 0.025 0.05 Sex Organ Findings No. of rats 10 10 10 10 10 Male Urinary bladder Simple hyperplasia +a 0 2 310 ** 10 ** Papillary or nodular hyperplasia + 0 0 0 4 * 4 * Stomach Mononuclear cell infiltration, focal, submucosa, forestomach + 1 0 0 0 0 Basal cell hyperplasia, forestomach + 0 1 0 0 0 Simple hyperplasia, forestomach + 0 0 0 0 1 Mucosal atrophy with thickened foveolar epithelium, focal + 0 0 0 1 0 Erosion, fundus + 0 0 0 2 1 Erosion, pylorus + 0 0 1 1 0 Mucosal hyperplasia, pylorus, diffuse + 0 0 0 0 2 Female Urinary bladder Simple hyperplasia + 0 1 6 ** 10 ** 10 ** Papillary or nodular hyperplasia + 0 0 0 2 4 * Stomach Erosion, forestomach + 1 0 0 0 0 Mononuclear cell infiltration, focal, submucosa, fundus + 0 0 0 1 0 Erosion, pylorus + 1 2 2 1 2 Mucosal hyperplasia, pylorus, diffuse + 0 0 1 0 2 a: Grade of lesions +:minimal. *,**: Significantly different from the control value at p < 0.05 and p < 0.01, respectively (Fisher’s exact test).

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Table 10. Histopathological findings in male and female rats treated with HRE or AITC for 13 weeks Male Female Sex 0 HRE 0 HRE Dose level (%) AITC AITC (control) 0.05 (control) 0.05 Organ Findings No. of rats 10 10 10 10 10 10 Liver Granuloma +a 001 537 Focal necrosis + 0 0 0 0 0 1 Mononuclear cell infiltration, focal + 0 1 0 0 0 0 Kidney Basophilic tubules + 4 2 4 0 0 0 Interstitial mononuclear cell infiltration, focal +001 011 Pancreas Acinar cell atrophy, focal + 1 0 0 0 1 1 Capsular thickening, focal + 0 0 0 1 0 0 Mononuclear cell infiltration, focal + 0 0 0 0 0 1 Hemangioma + 0 1 0 0 0 0 Heart Myocarditis, focal + 7 6 8 2 1 2 ++010 000 Brain Vascular dilatation with thrombosis + 0 0 0 0 1 0 Harderian gland Mononuclear cell infiltration, focal + 0 0 0 1 1 1 ++000 001 Pituitary Cyst, pars intermedia + 0 0 0 0 1 0 Adrenal Accessory adrenocortical tissue + 1 0 0 0 0 0 Duodenum Mucosal hyperplasia, diffuse + 1 0 0 0 0 0 Mononuclear cell infiltration, lamina propria +000 100 Ectopic pancreatic tissue + 0 0 0 1 0 0 Ileum Granuloma, Peyer’s patch + 0 0 0 0 1 0 Cecum Mucosal hyperplasia, diffuse + 1 0 0 0 0 0 Colon Mucosal hyperplasia, diffuse + 0 0 1 0 0 0 Follicular hyperplasia, lymphoid tissue + 0 0 0 0 1 0 Testis Seminiferous tubule atrophy, focal, unilateral +110 - - - Uterus Granuloma, muscular layer + - - - 0 1 0 Endometrial stromal fibrosis + - - - 0 0 1 Vagina Submucosal cell infiltration, focal + - - - 0 0 1 a: Grade of lesions +, minimal; ++, slight

T-Cho levels, kidney weights and histopathology of the food consumption might relate the decreased water con- urinary bladder. sumption, but the possibility remains that HRE and AITC Food and water consumption were decreased in a directly affect the function of digestive organs or physio- dose-related manner in all HRE treated groups of both logical metabolism resulting in decreased food appetite. sexes and also in AITC treated males and females. The Decreased body weight gain observed in HRE 0.05% and decreased water consumption was considered to be due to AITC group males was more than 10% relative to the con- the acrid smell of HRE and AITC. The cause of decreased trol, and it was considered at least partly to be related to

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Bladder toxicity of horseradish extract the decreased food and water consumption. From anoth- ing AITC could be strongly related to the urinary bladder er viewpoint, the decreased body weight gain was limit- toxicity in rats. ed to the HRE 0.05% and AITC group males despite the In a NTP report, AITC administration by 4-weeks gav- marked decrease in water consumption with a dose-rela- age at doses up to 50 and 400 mg/kg body weight/day to tion in all HRE treated groups of both sexes, partly dem- mice and rats, respectively, was documented to induce onstrating a physiological adaptation in HRE 0.025 and mucosal thickening of gastric and/or urinary bladder 0.0125% groups. The decreased water consumption in the (NTP, 1982). Carcinogenic potential of AITC was previ- AITC group was comparable to those in the HRE 0.025 ously examined by gavage administration in a conven- and 0.0125% group males and females, respectively, sug- tional 2-year bioassay in rats and mice, and urinary blad- gesting the repellent effect of HRE might be stronger than der papillomas were induced in male rats at 12 and 25 mg/ that of AITC. kg body weight/day (NTP, 1982). Because HRE adminis- In serum biochemistry, BUN was increased in males tration caused urothelial proliferative lesions, i.e. simple of HRE 0.025 and 0.05% groups and females of 0.0125- mucosal hyperplasia and papillary/nodular hyperplasia 0.05% groups and AITC group of both sexes. In addition, in the present study at comparable doses to the previous absolute and/or relative kidney weights were increased AITC cases, HRE might also be expected to possess car- in HRE 0.025 and 0.05% groups of both sexes and AITC cinogenic effects in the rat urinary bladder. As compared group females. There is a possibility that dehydration sec- to the results of our 2-week preliminary study in male rats ondary to the obviously decreased water consumption administered 0.025-0.1% HRE and AITC each (Table 1), caused the BUN elevation. However, no significant chang- however, the incidence of papillary/nodular hyperplasia es were found in other parameters indicating dehydration, was obviously lower in the 0.05% HRE and AITC group such as increased RBC, Ht and TP levels. These results males of the present 13-week study (p < 0.05). Akagi et indicate that HRE and AITC might possess renal toxico- al. (2003) earlier demonstrated that dietary administration logical potentials in rats or physiological adaptation to the of other ITCs, such as PEITC and BITC, induced marked decreased water consumption might partly be related to inflammatory changes along with mucosal damages in the organ weight and BUN changes, but no microscopic the urinary bladder of rats, with peaks apparent on days changes were found in the kidneys of any of the HRE- or 2 or 3, and these changes were considered to be directly AITC-treated animals. On the other hand, AITC was pre- correlated with subsequent tumor promotion/formation. viously reported to induce renal microscopic changes such However, Sugiura et al. (2003) reported simple and pap- as dilatation of distal tubules and increased desquamation illary/nodular hyperplasia induced by PEITC in the uri- in rats given a dose of 20 mg/kg body weight/day by gav- nary bladder of rats to be reversible. Both in the present age for 6 weeks (Lewerenz et al., 1988). T-Cho was also 2-week preliminary and 13-week studies, no obvious found to be decreased with a dose-dependence in females inflammatory changes/mucosal damage were observed, given HRE, whereas among ITCs, BITC was reported to and it was not clear whether such toxicological changes increase serum cholesterol levels (Lewerenz et al., 1992). in early stages related induction of proliferative lesions Although the composition of the test substance and the pro- in the urinary bladder of rats treated with HRE/AITC. cedure for administration were different, the cause of these Therefore, further studies are needed to clarify wheth- discrepancies may be due to some other factors. er HRE/AITC induces mucosal inflammatory damages in Regarding urinary bladder toxicity, simple mucos- the shorter term and HRE actually shows tumor promo- al hyperplasia was microscopically observed in all HRE tional and/or carcinogenic effects in the rat urinary blad- or AITC treated groups of both sexes, together with der in the longer term. papillary/nodular hyperplasia in the HRE 0.05% and In conclusions, NOAELs were estimated to be below AITC groups. The incidence and degree of the prolifer- 0.0125% of HRE for both males and females, correspond- ative lesions observed in AITC groups were comparable ing to 12.7 and 10.8 mg/kg body weight/day, respectively, to those in HRE 0.05% group of both sexes, and there and HRE appeared to possess comparable toxicological appeared no more significant urinary bladder toxicologi- properties to AITC, such as the inductive effect of signif- cal properties with HRE than with AITC. Urine is report- icant proliferative lesions in the urinary bladder. Consid- ed to be the major route of excretion of AITC (Bollard et ering the stability data of the HRE-mixed drinking water, al., 1997), and free forms of ITCs cleaved from the conju- the NOAELs were revised to 9.4 and 8.0 mg/kg body gated metabolites in urine were shown to induce mucos- weight/day for males and females, respectively. al damage in urinary bladder of rats (Masutomi et al., 2001). Therefore, the distribution profile of ITCs includ-

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