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A ninety-day toxicity study of semicarbazide hydrochloride in Wistar Hannover GALAS rats
Food and Chemical Toxicology 47 (2009) 2490–2498
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A ninety-day toxicity study of semicarbazide hydrochloride in Wistar Hannover GALAS rats Miwa Takahashi *, Midori Yoshida, Kaoru Inoue, Tomomi Morikawa, Akiyoshi Nishikawa Division of Pathology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
a r t i c l e
i n f o
Article history: Received 13 March 2009 Accepted 8 July 2009
Keywords: Semicarbazide hydrochloride Osteolathyrism Food contaminant 90-day study GALAS rats
a b s t r a c t A ninety-day toxicity study of semicarbazide hydrochloride (SEM-HCl) was conducted in male and female Wistar Hannover GALAS rats fed diet containing the compound at concentration of 0, 250, 500 and 1000 ppm. Suppression of body weight gain and food consumption was found in both sexes at 1000 ppm throughout the study. Enlargement and deformation of knee joints were obvious at 500 and 1000 ppm from week 3, together with deformation of the thorax and tail. Histopathologically, disarrangement of chondrocytes and fissures in the cartilage matrix were apparent at all doses tested in epiphyseal and articular cartilage. The severity of these lesions increased dose-dependently, accompanied by increased connective tissues and bone deformation at high doses. Additionally, compact bones at 1000 ppm became thin, suggesting loss of bone mass. In the thoracic aorta, the edges of elastic laminae became rough and the interlaminar spaces were altered from a fibrillar to a rod or globular appearance. No abnormalities were detected in any other organs. Taken together, toxicological effects of subchronic exposure to SEM-HCI were mainly observed in bone, cartilage and the aorta, with the no-observedadverse-effect-level estimated from the present histopathological examination of less than 250 ppm in both sexes. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Semicarbazide (SEM) is a metabolite of the banned veterinary antibiotic nitrofurazone which has been used as a marker for abuse of this drug (Effkemann and Feldhusen, 2004; de la Calle and Anklam, 2005). However, it was recently found that SEM can also originate from the thermal breakdown of azodicarbonamide, used as a blowing agent in plastic gaskets (EFSA, 2003; Stadler et al., 2004). Other sources such as from food processing by hypochlorite treatment have further been suggested (Hoenicke et al., 2004; Saari and Peltonen, 2004). In view of possible exposure from various foods, independent of any nitrofurazone use, it has become important to assess the health risk of SEM.
Abbreviations: A/G, albumin: globulin ratio; Alb, albumin; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; Bil, bilirubin; BUN, blood urea nitrogen; BW, body weight; Ca, calcium; Cl, chlorine; CRN, creatinine; c-GTP, gamma glutamyl transpeptidase; Hb, hemoglobin; Ht, hematocrit; IP, inorganic phosphate; K, potassium; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; Na, sodium; NOAEL, no-observed-adverse-effect-level; Plt, platelet; RBC, red blood cell; SEM, semicarbazide; SEM-HCl, semicarbazide hydrochloride; SSAO, semicarbazide-sensitive amine oxidase; TC, total cholesterol; TG, triglyceride; TP, total protein; WBC, white blood cell. * Corresponding author. Tel.: +81 3 3700 9821; fax: +81 3 3700 1425. E-mail address: [email protected] (M. Takahashi). 0278-6915/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2009.07.008
SEM is known to inhibit enzymes, such as lysyl oxidase, semicarbazide-sensitive amine oxidase (SSAO) and glutamic acid decarboxylase (Magyar et al., 2001; Dawson et al., 2002; Macedo et al., 2007). It acts as an osteolathyrogen, and induces osteochondral and vascular lesions in young rats due to impaired cross-linking reactions of collagen and elastin through inhibition of lysyl oxidase or SSAO (Ramamurti and Taylor, 1959; Langford et al., 1999; Dawson et al., 2002; Mercier et al., 2007). In addition, teratogenic effects such as induction of cleft palate and aortic aneurysms have also been reported (Steffek et al., 1972; de la Fuente del Rey, 1986; Gong et al., 2006). Reports have indicated that SEM has weak genotoxicity in vitro but not in vivo (Parodi et al., 1981; Abramsson-Zetterberg and Svensson, 2005; EFSA, 2005; FSC, 2007). SEM hydrochloride (SEM-HCl) at a high dose in the drinking water showed a tendency to increase incidences of lung and blood vessel tumors in mice (Toth et al., 1975). Weisburger et al. reported carcinogenicity study of SEM-HCl using SD rats fed diet containing 500 and 1000 ppm (Weisburger et al., 1981). In this study, the animals in the 500 ppm groups received test diet for 78 weeks, and thereafter received control diet for further 26 weeks. The dosing of 1000 ppm was considered to be over maximal tolerated dose and the treatment was discontinued at week 32 because deaths occurred frequently in males, and they received control diet for the remainder of the study period. Although osteolathyrism was found grossly from week 10 in all treated groups, no carcinogenic effects
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M. Takahashi et al. / Food and Chemical Toxicology 47 (2009) 2490–2498 Table 1 Findings of clinical observation at week 13 in GALAS rats fed diet containing SEM-HCl for 90 days. Sites and findings
Sex
Males
Dose (ppm)
0
250
500
1000
0
250
500
1000
Hindlimb Forelimb Thorax Tail
No. of animals examined Enlargement and deformation of the knee joint Enlargement and deformation of the wrist joint Prominence Stiff flexion
10 0 0 0 0
10 1 0 0 1
10 10 0 3 4
10 10 6 10 9
10 0 0 0 0
10 0 0 0 0
10 10 0 1 0
10 10 8 9 0
were observed in either sex. However, since the treatment was discontinued at week 32 or 78 and no details were provided on the tumor incidences, the carcinogenicity of SEM is still controversial. Accordingly, to examine the subchronic toxicity of SEM prior to conducting the carcinogenicity study, we performed the present 90-day toxicity study of SEM-HCl using rats. 2. Materials and methods 2.1. Animals Male and female Wistar Hannover GALAS rats at 5 weeks of age were obtained from CLEA Japan, Inc. (Tokyo, Japan). Wistar Hannover GALAS rat has been accepted as a suitable strain of toxicology studies (http://www.galas.org/index.html). They received powdered basal diet (CE-2, CLEA Japan, Inc.) and tap water ad libitum, housed 2–4 per plastic cage with sterilized softwood chips as bedding in a barrier-maintained animal room conditioned at 24 ± 1 °C and 55 ± 5% humidity with a 12-h light/dark cycle. After a 1-week acclimatization period, animals showing
500
Females
no abnormalities were used at 6 weeks of age. The animal protocol was reviewed and approved by the Animal Care and Use Committee of the National Institute of Health Sciences, Japan. 2.2. Chemicals Semicarbazide hydrochloride (SEM-HCl, CAS No. 563–41-7) was purchased from Hayashi Pure Chemical Ind., Ltd. (Osaka, Japan), as a white powder with a purity of 99.3%. SEM-HCl was well mixed at concentrations of 0, 250, 500 and 1000 ppm into powdered basal diet (CE-2). Concentrations after storage at room temperature for 4 weeks or at 4 °C for 8 weeks were analyzed at Japan Food Research Laboratories (Osaka, Japan), and more than 89% stability of the test compound was confirmed under both conditions. SEM was not detected in the basal diet (detection limit, 0.01 ppm). Test diets were prepared every 2 weeks, and stored at 4 °C before use. 2.3. Experimental design As a preliminary examination, a 2-week dose finding study was conducted based on a previous report (Weisburger et al., 1981), with 1000 ppm selected as the highest dose for subsequent study. Animals, weighing 166.5 ± 4.5 g for males
Males
45
450
300 250 **
200
**
**
**
**
**
**
**
**
**
**
** 0 ppm
**
150
250 ppm 500 ppm 1000 ppm
100
Food intake (g/animal/day)
Body weight (g)
350
50 0
Males
40
400
35 30 25 20 15
**
10
0 ppm 250 ppm
5
0
1
2
3
4
5
6 7 Weeks
8
9
10 11 12 13
0
1
2
3
4
5
6
7 8 Weeks
9
10
500 ppm 1000 ppm
11
12
13
300
Females
35
200 *
150
**
**
**
**
**
**
**
**
**
0 ppm
100
250 ppm 500 ppm 1000 ppm
50
Food intake (g/animal/day)
Body weight (g)
250
Females
30 25 20 15 10
** 0 ppm 250 ppm
5
0
0
1
2
3
4
5
6 7 Weeks
8
9
10 11 12 13
0
1
2
3
4
5
6
7
8
9
10
500 ppm 1000 ppm
11
12
13
Weeks Fig. 1. Body weight curves for male and female GALAS rats fed diet containing SEMHCl for 90 days. ,: Significantly different from the 0 ppm group at p < 0.05 and p < 0.01, respectively.
Fig. 2. Daily food intake for male and female GALAS rats fed diet containing SEMHCl for 90 days. : Significantly different from the 0 ppm group at p < 0.01.
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and 126.3 ± 4.3 g for females (mean ± SD), were randomly allocated to 4 groups, each consisting of 10 males and 10 females, and given diet containing 0 (control), 250, 500 or 1000 ppm SEM-HCl for 90 days. The test diets were available ad libitum, except for one-night fasting prior to the scheduled sacrifice, and rats had free access to tap water throughout the study. Observations for mortality and clinical signs, including posture and gait abnormalities and deformation of four limbs, thorax and tail, were conducted daily. Body weight and food consumption were recorded every week. At necropsy, all animals were anesthetized with ether, weighed, and blood samples were collected from the abdominal aorta for hematology and serum biochemistry. Relative organ weights were calculated as the values relative to body weights.
2.4. Hematology and serum biochemistry Hematology analysis was performed using an automated hematology analyzer, K-4500 (Sysmex Corp., Hyogo, Japan). Differential leukocyte counts and reticulocyte counts were performed with a MICROX HEG-50S (Sysmex Corp.). Parameters for serum biochemistry shown in Table 4 were analyzed at SRL, Inc. (Tokyo, Japan) using sera frozen after centrifugation of whole blood.
2.5. Histopathological examination After macroscopic examination, the brain, thymus, heart, lungs, liver, spleen, adrenals, kidneys and testes were removed and weighed. In addition, the pituitary, eyes, Harderian glands, salivary glands, tongue, trachea, esophagus, thyroid glands, thoracic aorta, stomach, small intestine (duodenum, jejunum, and ileum), large intestine (cecum, colon, and rectum), pancreas, mesenteric lymph nodes, thigh muscle, sciatic nerve, skin, mammary gland, urinary bladder, epididymides, seminal vesicles, prostate, ovaries, uterus and vagina were similarly resected. All organs were fixed in 10% buffered formalin, except for testes, which were fixed in Bouin’s solution overnight. For examination of osteochondral lesions, the nasal cavity, sternum, right femur, right tibia, left knee joint, right and left ankles, spine (cervical, thoracic, lumbar and caudal vertebrae with corresponding spinal cord) and macroscopic lesions (in wrist joints etc.) were fixed in 10% buffered formalin and then decalcified in EDTA solution at room temperature for a month. Histopathological assessment was performed on the bones, joints and thoracic aorta of all groups. Additionally, all organs of the control and 1000 ppm groups and the heart, lungs, liver, kidneys, thyroid, trachea, testes and prostate of the intermediate dose groups were also examined. The tissues were routinely processed for paraffin embedding, sectioned and stained with hematoxylin and eosin (HE). Victoria blue and HE staining was applied to three transverse sections of the descending thoracic aorta cut at 5 mm intervals to demonstrate elastic fibers.
2.6. Statistical analysis Variance in data for body weights, food consumption, hematology, serum biochemistry and organ weights was checked for homogeneity by Bartlett’s procedure. If the variance was homogeneous, the data were assessed by one-way analysis of variance. If not, the Kruskal–Wallis test was applied. When statistically significant differences were detected, the Dunnett’s multiple test was
Table 2 Food consumption and intake of SEM-HCl in GALAS rats. Sex
Dose (ppm)
No. of animals examined
Food consumption
(g/animal/ day)
(g/kg BW/day)
Intake of SEM-HCl
Daily
Total
(mg/kg BW/day)
(mg/kg BW)
Males
0 250 500 1000
10 10 10 10
23.3 ± 1.2a 24.4 ± 1.7 26.7 ± 2.3** 19.0 ± 1.7**
69.5 ± 16.7 72.6 ± 14.4 81.4 ± 15.0 65.9 ± 13.3
0 18.1 40.7 65.9
0 1633 3662 5929
Females
0 250 500 1000
10 10 10 10
19.2 ± 1.3 17.2 ± 1.0** 17.7 ± 0.9** 12.6 ± 1.1**
96.2 ± 11.2 84.5 ± 11.2* 88.3 ± 13.6 70.5 ± 13.1**
0 21.1 44.2 70.5
0 1901 3975 6344
Abbreviations: SEM-HCl, semicarbazide hydrochloride; BW, body weight. a Mean ± SD. , Significantly different from the 0 ppm group at p < 0.05 and p < 0.01, respectively.
employed for comparison between the control and treatment groups. For histopathological findings, the incidences were compared using the Fisher’s exact probability test.
3. Results 3.1. General condition No deaths occurred throughout the feeding period. The findings of clinical observation at week 13 are summarized in Table 1. Enlargement and deformation of the knee joints were apparent in males and females at 500 and 1000 ppm from week 3, and prominence of the thorax was also found from week 5. In the 1000 ppm group, enlargement and deformation of the wrist joints were observed from week 12 in both sexes, and some showed posture and gait abnormalities. Tails of male rats in the treated groups exhibited stiff flexion from week 4.
Table 3 Hematology data for GALAS rats fed diet containing SEM-HCl for 90 days. SEM-HCl (ppm) 0 No. of animals 10 examined Males 921.1 ± 36.0a RBC (104/ll) Hb (g/dl) 16.5 ± 0.6 Ht (%) 51.9 ± 2.0 MCV (fl) 56.4 ± 1.3 MCH (pg) 18.0 ± 0.4 MCHC (g/dl) 31.9 ± 0.7 95.3 ± 9.2 Plt (104/ll) WBC (102/ll) 45.8 ± 11.9 Differential leukocyte counts Band form neutrophils 0.3 ± 0.4 (%) Segmented 13.8 ± 4.1 neutrophils (%) Eosinophils (%) 0.8 ± 0.5 Basophils (%) 0±0 Lymphocytes (%) 85.0 ± 4.4 Monocytes (%) 0.1 ± 0.2 Reticulocytes 0.1 ± 0.2 (/100 cells) Females 835.7 ± 49.2 RBC (104/ll) Hb (g/dl) 15.5 ± 0.8 Ht (%) 48.6 ± 2.7 MCV (fl) 58.2 ± 1.6 MCH (pg) 18.6 ± 0.5 MCHC (g/dl) 31.9 ± 0.7 93.2 ± 10.3 Plt (104/ll) 24.4 ± 6.2 WBC (102/ll) Differential leukocyte counts Band form neutrophils 0.5 ± 0.6 (%) Segmented 18.3 ± 6.9 neutrophils (%) Eosinophils (%) 1.3 ± 0.9 Basophils (%) 0±0 Lymphocytes (%) 79.4 ± 7.4 Monocytes (%) 0.5 ± 0.3 Reticulocytes 0.2 ± 0.4 (/100 cells)
250
500
1000
10
10
10
929.7 ± 22.2 16.2 ± 0.3 51.8 ± 1.2 55.7 ± 1.1 17.4 ± 0.4* 31.3 ± 0.6 92.2 ± 10.0 43.9 ± 13.7
943.2 ± 40.4 16.4 ± 0.5 52.3 ± 2.4 55.5 ± 1.1 17.4 ± 0.4* 31.3 ± 0.6 93.3 ± 11.6 46.5 ± 15.1
962.6 ± 55.5 17.0 ± 0.7 54.0 ± 2.1 56.1 ± 1.4 17.7 ± 0.6 31.5 ± 0.5 98.6 ± 16.5 35.4 ± 9.8
0.4 ± 0.4
0.6 ± 0.4
0.6 ± 0.5
16.3 ± 4.9
13.4 ± 6.8
7.9 ± 2.2*
1.6 ± 1.0 0±0 81.5 ± 6.1 0.3 ± 0.3 0.1 ± 0.2
1.3 ± 0.9 0±0 84.6 ± 7.3 0.3 ± 0.4 0.1 ± 0.2
0.8 ± 0.8 0±0 90.6 ± 2.4* 0.3 ± 0.4 0.1 ± 0.2
818.9 ± 43.0 15.6 ± 0.8 48.5 ± 2.3 59.2 ± 1.4 19.1 ± 0.7 32.2 ± 0.6 95.2 ± 9.1 22.9 ± 6.2
868.3 ± 64.6 16.2 ± 0.9 50.0 ± 3.1 57.6 ± 1.2 18.7 ± 0.6 32.5 ± 0.6 97.8 ± 11.5 29.8 ± 9.4
845.2 ± 62.3 15.5 ± 0.9 48.6 ± 3.3 57.5 ± 1.2 18.4 ± 0.6 32.0 ± 0.5 104.7 ± 14.7 35.7 ± 15.1
0.5 ± 0.5b
0.6 ± 0.5
0.5 ± 0.5
b
15.8 ± 3.6
18.5 ± 8.8
8.5 ± 3.1**
1.1 ± 0.9b 0 ± 0b 82.2 ± 4.3b 0.4 ± 0.5b 0.4 ± 1.2b
1.5 ± 0.8 0±0 78.9 ± 9.3 0.6 ± 0.5 0.1 ± 0.2
1.0 ± 0.5 0±0 89.8 ± 3.4** 0.2 ± 0.3 0.1 ± 0.2
Abbreviations: SEM-HCl, semicarbazide hydrochloride; RBC, red blood cell; Hb, hemoglobin; Ht, hematocrit; MCV, mean corpuscular volume; MCV, mean corpuscular volume; MCHC, mean corpuscular hemoglobin concentration; Plt, platelet; WBC, white blood cell. a Mean ± SD. b Number of effective animals was reduced to nine due to the failure of blood sampling for differential leukocyte counts. , Significantly different from the 0 ppm group at p < 0.05 and p < 0.01, respectively.
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3.2. Body weight, food consumption and chemical intake
3.3. Hematology and serum biochemistry
Significant suppression of body weight gain was observed at 1000 ppm from week 1 in males and from week 4 in females (Fig. 1). Data for food consumption and SEM-HCl intake are summarized in Fig. 2 and Table 2. In both sexes, food consumption was decreased at 1000 ppm throughout the study, and the mean values for food consumption/animal were significantly lowered compared to the control group. However, there were no intergroup differences in the mean values for food consumption/kg body weight in males, due to suppressed body weight gain. The mean values for food consumption/animal were significantly increased in males at 500 ppm and decreased in females at 250 and 500 ppm. Significant decrease of the mean values for food consumption/kg body weight was found in females of the 250 and 1000 ppm groups.
Hematology data are summarized in Table 3. In both sexes, differential leukocyte counts showed significant decrease and increase in the proportions of segmented neutrophils and lymphocytes, respectively, in the 1000 ppm group. At the same dose, WBC counts were decreased in males and increased in females, although not statistically significant. In males, MCH was significantly decreased at 250 and 500 ppm, but without dose-dependence. In the serum biochemical analysis, significant alterations of CRN, ALT, A/G, total Bil and K were found in males at 500 and/or 1000 ppm (Table 4). In females, significant increases of BUN were observed in all treated groups, and K and IP were statistically increased at 500 and 1000 ppm. Significant alterations of TP, ALT and ALP were also detected at 1000 ppm.
Table 4 Serum biochemical data for GALAS rats fed diet containing SEM-HCl for 90 days.
3.4. Organ weights Final body weights were significantly decreased in both sexes of the 1000 ppm group (Table 5). Absolute weights of the lungs in both sexes and the thymus, heart and liver in males were statistically lowered at 1000 ppm. In males, significant increase in relative
SEM-HCl (ppm)
No. of animals examined Males TP (g/dL) Alb (g/dL) A/G Total Bil (mg/dL) Glucose (mg/dL) TG (mg/dL) TC (mg/dL) BUN(mg/dL) CRN (mg/dL) Na (mEQ/L) Cl (mEQ/L) K (mEQ/L) Ca (mg/dL) IP (mg/dL) AST (IU/L) ALT (IU/L) ALP (IU/L) c-GTP (IU/L) Females TP (g/dL) Alb (g/dL) A/G Total Bil (mg/dL) Glucose (mg/dL) TG (mg/dL) TC (mg/dL) BUN (mg/dL) CRN (mg/dL) Na (mEQ/L) Cl (mEQ/L) K (mEQ/L) Ca (mg/dL) IP (mg/dL) AST (IU/L) ALT (IU/L) ALP (IU/L) c-GTP (IU/L)
0
250
500
1000
10
10
10
10
6.9 ± 0.1a 4.5 ± 0.1 1.9 ± 0.1 0.06 ± 0.02 151.2 ± 18.7 116.9 ± 45.2 72.8 ± 11.7 21.6 ± 1.2 0.33 ± 0.04 147.6 ± 1.3 105.1 ± 1.4 4.3 ± 0.2 10.6 ± 0.3 6.4 ± 0.8 79.9 ± 12.8 37.0 ± 6.0 288.6 ± 48.7 <2
6.7 ± 0.2 4.5 ± 0.1 2.0 ± 0.2 0.06 ± 0.01 136.5 ± 12.2 90.1 ± 43.5 69.7 ± 11.1 21.9 ± 2.8 0.31 ± 0.03 147.5 ± 1.4 105.6 ± 1.2 4.3 ± 0.2 10.3 ± 0.2 6.2 ± 0.6 83.2 ± 11.5 36.7 ± 4.2 329.1 ± 70.7 <2
6.8 ± 0.2 4.5 ± 0.1 1.9 ± 0.2 0.07 ± 0.01 146.7 ± 20.0 104.3 ± 39.4 69.7 ± 12.8 21.5 ± 2.4 0.29 ± 0.03** 146.6 ± 1.3 103.7 ± 1.2 4.7 ± 0.5* 10.5 ± 0.3 7.0 ± 0.8 66.6 ± 10.2 28.7 ± 4.5** 329.2 ± 73.7 <2
6.8 ± 0.2 4.7 ± 0.2 2.2 ± 0.2** 0.09 ± 0.02** 156.2 ± 23.8 74.1 ± 33.2 75.3 ± 11.1 22.1 ± 2.8 0.29 ± 0.03** 147.2 ± 1.1 105.6 ± 2.1 4.4 ± 0.2 10.5 ± 0.4 7.0 ± 0.6 70.9 ± 14.5 27.4 ± 4.4** 318.0 ± 65.7 <2
7.1 ± 0.5 5.1 ± 0.4 2.6 ± 0.3 0.10 ± 0.03 116.1 ± 18.3 41.0 ± 15.5 59.7 ± 11.8 18.3 ± 1.9 0.33 ± 0.04 145.6 ± 1.3 106.4 ± 2.8 3.6 ± 0.2 10.0 ± 0.4 4.5 ± 0.6 74.9 ± 9.5 24.4 ± 4.2 103.6 ± 31.6 <2
7.1 ± 0.5 5.1 ± 0.4 2.6 ± 0.4 0.10 ± 0.02 117.0 ± 29.4 64.8 ± 64.3 66.8 ± 19.6 22.3 ± 3.3* 0.35 ± 0.05 145.4 ± 1.6 105.5 ± 1.5 3.8 ± 0.3 10.1 ± 0.4 4.9 ± 0.8 77.9 ± 16.3 29.0 ± 8.3 115.1 ± 39.1 <2
7.1 ± 0.4 5.0 ± 0.3 2.4 ± 0.3 0.09 ± 0.01 118.9 ± 22.7 38.9 ± 23.2 63.5 ± 12.5 22.3 ± 3.4* 0.35 ± 0.04 145.1 ± 1.5 105.5 ± 0.8 4.0 ± 0.4* 10.2 ± 0.3 5.5 ± 0.9** 70.2 ± 10.1 21.3 ± 3.1 127.1 ± 50.4 <2
6.6 ± 0.3* 4.8 ± 0.2 2.6 ± 0.2 0.10 ± 0.02 111.6 ± 25.5 34.9 ± 13.3 65.6 ± 12.2 23.1 ± 4.2** 0.34 ± 0.04 145.7 ± 1.6 105.9 ± 1.7 3.9 ± 0.3* 10.1 ± 0.3 6.4 ± 0.7** 71.9 ± 11.3 17.9 ± 2.0** 186.7 ± 88.0** <2
Abbreviations: SEM-HCl, semicarbazide hydrochloride; TP, total protein; Alb, albumin; A/G, albumin: globulin ratio; Bil, bilirubin; TG, triglyceride; TC, total cholesterol; BUN, blood urea nitrogen; CRN, creatinine; Na, sodium; Cl, chlorine; K, potassium; Ca, calcium; IP, inorganic phosphate; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase. c-GTP, gamma glutamyl transpeptidase. a Mean ± SD. , Significantly different from the 0 ppm group at p < 0.05 and p < 0.01, respectively.
Table 5 Final body and organ weights of GALAS rats fed diet containing SEM-HCl for 90 days. SEM-HCl (ppm) 0
250
500
1000
No. of animals examined Males Body weight (g) Brain (g) (g%) Thymus (g) (g%) Lungs (g) (g%) Heart (g) (g%) Spleen (g) (g%) Liver (g) (g%) Adrenals (mg) (mg%) Kidneys(g) (g%) Testes (g) (g%)
10
10
10
10
409.3 ± 18.6a 2.06 ± 0.08 0.50 ± 0.02 0.30 ± 0.06 0.07 ± 0.02 1.25 ± 0.13 0.31 ± 0.02 1.05 ± 0.12 0.26 ± 0.02 0.66 ± 0.08 0.16 ± 0.02 10.62 ± 0.99 2.59 ± 0.19 52.40 ± 7.17 12.83 ± 1.92 2.28 ± 0.12 0.56 ± 0.03 3.38 ± 0.34 0.83 ± 0.07
407.8 ± 26.6 2.04 ± 0.08 0.50 ± 0.03 0.32 ± 0.08 0.08 ± 0.02 1.25 ± 0.10 0.31 ± 0.02 1.01 ± 0.10 0.25 ± 0.02 0.66 ± 0.05 0.16 ± 0.01 9.70 ± 0.81 2.38 ± 0.07* 51.30 ± 5.66 12.62 ± 1.45 2.38 ± 0.20 0.58 ± 0.03 3.33 ± 0.27 0.82 ± 0.05
396.9 ± 27.3 2.00 ± 0.08 0.51 ± 0.03 0.31 ± 0.06 0.08 ± 0.01 1.15 ± 0.12 0.29 ± 0.04 1.06 ± 0.06 0.27 ± 0.02 0.62 ± 0.07 0.16 ± 0.02 10.28 ± 1.04 2.58 ± 0.10 55.50 ± 5.40 13.99 ± 1.05 2.35 ± 0.20 0.59 ± 0.04* 3.37 ± 0.30 0.85 ± 0.08
323.8 ± 36.0** 2.00 ± 0.08 0.63 ± 0.06** 0.21 ± 0.04** 0.06 ± 0.01 0.96 ± 0.12** 0.30 ± 0.03 0.89 ± 0.11** 0.27 ± 0.02 0.61 ± 0.10 0.19 ± 0.02** 8.51 ± 1.43** 2.61 ± 0.20 56.10 ± 10.88 17.34 ± 2.78** 2.08 ± 0.28 0.64 ± 0.03** 3.56 ± 0.43 1.10 ± 0.08**
Females Body weight (g) Brain (g) (g%) Thymus (g) (g%) Lungs (g) (g%) Heart (g) (g%) Spleen (g) (g%) Liver (g) (g%) Adrenals (mg) (mg%) Kidneys (g) (g%)
220.9 ± 13.6 1.83 ± 0.09 0.83 ± 0.05 0.26 ± 0.05 0.12 ± 0.03 0.86 ± 0.06 0.39 ± 0.02 0.63 ± 0.06 0.29 ± 0.02 0.47 ± 0.04 0.21 ± 0.02 5.40 ± 0.37 2.45 ± 0.11 69.10 ± 10.34 31.27 ± 4.22 1.42 ± 0.17 0.64 ± 0.04
229.2 ± 22.4 1.82 ± 0.08 0.80 ± 0.09 0.31 ± 0.11 0.13 ± 0.04 0.85 ± 0.07 0.37 ± 0.03 0.67 ± 0.08 0.29 ± 0.03 0.44 ± 0.06 0.19 ± 0.01* 5.84 ± 0.87 2.55 ± 0.29 65.30 ± 12.48 28.52 ± 4.94 1.38 ± 0.16 0.60 ± 0.05
225.2 ± 11.6 1.87 ± 0.09 0.83 ± 0.04 0.25 ± 0.05 0.11 ± 0.02 0.88 ± 0.11 0.39 ± 0.04 0.66 ± 0.06 0.30 ± 0.02 0.45 ± 0.04 0.20 ± 0.02 5.57 ± 0.55 2.47 ± 0.17 68.90 ± 10.21 30.54 ± 3.65 1.47 ± 0.15 0.65 ± 0.04
189.4 ± 7.8** 1.82 ± 0.08 0.97 ± 0.06** 0.20 ± 0.04 0.11 ± 0.02 0.75 ± 0.06* 0.40 ± 0.03 0.61 ± 0.05 0.32 ± 0.03** 0.48 ± 0.05 0.25 ± 0.03 4.77 ± 0.50 2.52 ± 0.23 60.00 ± 7.09 31.73 ± 3.94 1.33 ± 0.11 0.70 ± 0.05*
a
Mean ± SD. Significantly different from the 0 ppm group at p < 0.05 and p < 0.01, respectively. ,
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kidney weights was observed at 500 and 1000 ppm, and relative weights of the brain, spleen, adrenals and testes were also increased at 1000 ppm. In females, relative weights of the brain, heart and kidneys were significantly elevated in the 1000 ppm group. Decreases in relative liver weight in males and relative spleen weight in females were found at 250 ppm, but such changes were not observed in the 500 and 1000 ppm groups. 3.5. Necropsy and histopathology In the macroscopic examination, bowing of the tibia was apparent in males and females at 500 and 1000 ppm. Also, increases in diameter of the femur and tibia were evident due to enlargement of the marrow cavities (Fig. 3A), along with prominence of the sternum (Fig. 3B), thoracic kyphosis and deformation of the wrist joints at 1000 ppm. Histopathological findings for bones and joints are summarized in Table 6. In the femur and tibia, disarrangement of epiphyseal chondrocytes was observed in both sexes at all doses tested. The epiphyseal plate at the proximal end of the tibia was thickened at 500 and 1000 ppm, and degeneration of hypertrophic chondro-
cytes was found in the thickened cartilage plate (Fig. 4A–C). There were fissures in the cartilage matrix, and these were widened and accompanied by increase of connective tissues at 500 and 1000 ppm (Fig. 4D). In the metaphysis, spongy bones were decreased and irregularly-branched. The compact bones in the metaphysis became thin with a rugged surface, and deformed to expand outward, resulting in enlargement of the marrow cavities. Also in the sternum, disarrangement of epiphyseal chondrocytes and fissures in the cartilage matrix were found at all doses tested, and severe fissures with increased connective tissues and bone deformation were more evident at 500 and 1000 ppm in a dosedependent manner. In the animals with deformation of the wrist joint macroscopically, the epiphyseal plates of the radius and ulna exhibited similar histological lesions with the tibia. Deformation and fissures of articular cartilage with disarrangement of chondrocytes were observed in the knee joints and the intervertebral joints (from cervical to caudal) at all doses tested, and synovial inflammation and fibrosis were also found in the 500 and 1000 ppm groups (Fig. 4E and F). In the thoracic vertebrae, fissures occurred in the epiphyseal plate adjacent to the intervertebral disk, and displacement of the vertebra originating in the fissures, as well as
Fig. 3. Macroscopic lesions observed in GALAS rats fed diet containing SEM-HCl for 90 days. (A) Femurs and tibias of male rats at 0 ppm (left), and 1000 ppm (right) showing marked bowing and enlargement of the marrow cavities with treatment. (B) Sternums of male rats given 0 ppm (left), and 1000 ppm (right) with a prominence in the middle of the bone in the latter case.
Table 6 Histopathological findings for the bones and joints of GALAS rats fed diet containing SEM-HCl for 90 days. Organs and findings
Femur
Tibia
Sternum
Knee joint Vertebrae
,
Sex
Males
Dose (ppm)
0
250
500
1000
0
Females 250
500
1000
No. of animals examined Disarrangement of epiphyseal chondrocytes Thickening of epiphyseal plate Fissures of epiphyseal plate Increase of connective tissues Bone deformation Disarrangement of epiphyseal chondrocytes Thickening of epiphyseal plate Fissures of epiphyseal plate Increase of connective tissues Bone deformation Disarrangement of epiphyseal chondrocytes Fissures of epiphyseal plate Increase of connective tissues Bone deformation Deformation and fissures of articular cartilage Synovial inflammation and fibrosis Deformation and fissures of articular cartilage Fissures adjacent intervertebral disk (thoracic vertebra) Displacement of the thoracic vertebra
10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
10 2 0 0 0 0 8** 0 7** 1 0 7** 7** 0 0 6** 2 7** 3 0
10 10** 0 10** 0 0 10** 10** 10** 10** 10** 10** 10** 7** 6** 10** 10** 10** 9** 0
10 10** 2 10** 6** 6** 10** 9** 10** 10** 10** 10** 10** 10** 10** 10** 10** 10** 10** 3
10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
10 1 0 0 0 0 7** 0 0 0 0 4* 2 0 0 1 0 6** 1 0
10 8** 0 0 0 0 10** 4* 10** 9** 4* 10** 9** 3 3 6** 4* 9** 4* 0
10 10** 2 6** 2 0 10** 10** 10** 10** 10** 10** 10** 10** 10** 10** 10** 10** 9** 0
Significantly different from the 0 ppm group at p < 0.05 and p < 0.01, respectively (Fisher’s exact test).
M. Takahashi et al. / Food and Chemical Toxicology 47 (2009) 2490–2498
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Fig. 4. Histopathology of the bones and joints of male GALAS rats fed diet containing SEM-HCl for 90 days. Tibias of the 0 ppm (A) and 1000 ppm groups showing treatmentassociated thickening of the epiphyseal plate and bone deformation (B). At higher power, note disarrangement and degeneration of epiphyseal chondrocytes (C) and increased connective tissue within fissures (D). Deformation and fissures of articular cartilage observed in a knee joint at 250 ppm (E) and in a lumbar intervertebral joint at 1000 ppm (F). Lumbar vertebrae of rats in the 0 ppm (G) and 1000 ppm groups (H). Thinning of bone in the vertebral body is obvious in the 1000 ppm group, suggesting loss of bone mass. HE stain. Bars = 1 mm (A, B, G, H), 100 lm (C, D), 200 lm (E, F).
compression of the spinal cord by displaced vertebra, were seen in males at 1000 ppm. Additionally, thinning of bone in the vertebral body was obvious in the 1000 ppm group, suggesting loss of bone mass (Fig. 4G and H). Overall, the severities of the osteochondral lesions described above were higher in males than females. There were no treatment-related changes in the nasal cavity, ankles and Achilles tendons. Histopathological findings for the thoracic aorta are summarized in Table 7. In both sexes, although the number of elastic laminae was unchanged, their edges became roughened in a
dose-dependent manner (Fig. 5). The interlaminar spaces in the treated groups had a rod or globular appearance, in contrast to the fibrillar appearance in the 0 ppm group. In other organs, the incidences of mineralization in the pulmonary arteries were significantly lowered in males of the 250 and 500 ppm group compared to the controls, but without dose-dependence (Table 8). In males, significant increase of chronic inflammation of the ventral prostate was found at 1000 ppm. The incidence of cysts in the anterior lobe of the pituitary was also significantly elevated in females of the 1000 ppm group.
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Table 7 Histopathological findings of the thoracic aorta of GALAS rats fed diet containing SEM-HCl for 90 days. Findings
No. of elastic lamina Edge of elastic fibers Interlaminar space a b
Sex
Males
Dose (ppm)
0
250
500
1000
0
250
500
1000
No. of animals examined
10 9.03 10/0/0b 10/0/0
10 9.20 4/6/0 5/5/0
10 9.03 0/4/6 0/4/6
9a 8.81 0/0/9 0/0/9
9a 8.92 9/0/0 9/0/0
9a 8.59 6/3/0 9/0/0
8a 9.33 2/6/0 6/2/0
10 8.87 0/5/5 0/10/0
Smooth/smooth rough/rough Fibrillar/fibrillar rod/rod
Females
Number of effective animals was reduced due to the failure of sampling. The number of animals showing each finding.
Fig. 5. Transverse sections of the thoracic aorta of male GALAS rats fed diet containing SEM-HCl for 90 days. Aortas of the 0 ppm (A) and 1000 ppm groups showing rough edges of elastic laminae in the latter (B). The interlaminar spaces have a rod to globular appearance, in contrast to the fibrillar appearance in the normal aorta. Victoria blue and HE stain. Bars = 50 lm.
Table 8 Histopathological findings observed in GALAS rats fed diet containing SEM-HCl for 90 days. Organs and findings
Pituitary
Harderian gl. Skeletal muscle Tongue Heart Lung
Thymus Liver
Kidney
Pancreas Thyroid Urinary bladder Testis Prostate Ovary
Sex
Males
Dose (ppm)
0
250
500
1000
0
Females 250
500
1000
No. of animals examined Cyst, anterior lobe Cyst-like structure, anterior lobe Cyst, intermediate lobe Cyst-like structure, intermediate lobe Atrophy Degeneration, focal Erosion Myocardial degeneration/necrosis Ectopic thyroid tissue Mineralization, pulmonary arteries Osseous metaplasia Inflammatory cell infiltration, focal Epithelial cell hyperplasia Microgranuloma Hepatocyte degeneration, focal Brown pigment deposition, periportal Regenerative tubules Mineralization, cortico-medullary junction Renal cyst Mononuclear cell infiltration, perivascular Transitional cell hyperplasia Focal acinar cell atrophy Ultimobranchial cyst Mononuclear cell infiltration, focal Seminiferous tubule atrophy, focal Chronic inflammation, ventral lobe Fat necrosis
10 3 0 0 1 2 0 1 6 0 9 2 0 0 2 1 0 4 0 0 0 0 1 1 0 0 6 –
10 0 0 0 1 – – – 5 0 4* 2 0 – 3 0 0 2 0 0 0 0 – 0 – 1 7 –
10 2 0 0 0 – – – 4 0 4* 2 0 – 3 0 0 1 0 0 1 1 – 0 – 2 7 –
10 2 2 0 1 0 0 0 3 0 5 3 0 1 2 0 0 3 0 0 0 2 2 0 1 0 10* –
10 0 0 0 1 0 0 0 1 0 1 1 0 1 0 0 0 0 9 0 0 0 0 1 1 – – 1
10 1 0 0 0 – – – 0 1 2 1 1 – 0 0 1 1 10 0 0 0 – 0 – – – –
10 0 0 0 1 – – – 0 0 4 0 1 – 2 1 0 0 10 1 0 0 – 0 – – – –
10 4* 0 1 0 0 1 0 1 0 4 1 0 2 2 0 0 1 10 0 0 0 1 3 0 – – 0
–: Not examined. * Significantly different from the 0 ppm group at p < 0.05 (Fisher’s exact test).
4. Discussion In the present study, significant suppression of body weight gain was observed in 1000 ppm group males and females through-
out the experiment. Because continuous decrease of food consumption and deformation of the limbs, thorax and tail were found in both sexes at 1000 ppm from the early part of the study, it was considered that the suppression of body weight gain was
M. Takahashi et al. / Food and Chemical Toxicology 47 (2009) 2490–2498
induced by SEM-HCl treatment. In contrast, body weight increase in the control, 250 and 500 ppm groups were similar. Therefore, the alteration of food consumption found in these groups was regarded as incidental due to variation in measured values. SEM is known to be an inhibitor of the gamma-aminobutyric acid synthesizing enzyme glutamic acid decarboxylase (Macedo et al., 2007). In a recent study to evaluate the effects of SEM administration for 28 days during the juvenile period in rats, the authors performed neurobehavioral tests and reported alterations of motor and exploratory behavior (Maranghi et al., 2009). In the present study, although neurobehavioral tests were not conducted, the posture and gait abnormalities observed in the 1000 ppm group were likely due to deformation of the limbs and osteochondral lesions, because the severities appeared to correlate. In the hematological analysis, differential leukocyte counts showed significant decrease and increase in the proportions of segmented neutrophils and lymphocytes, respectively, in both sexes at 1000 ppm. In addition, WBC counts were decreased in males and increased in females, although not statistically significant. However, neither inflammatory diseases nor histopathological changes in the hematopoietic organs, including the spleen and bone marrow, were found. Therefore, the relation between SEMHCl treatment and the above described changes was not clear. Decreases of CRN and ALT and increases in A/G, total Bil and K were considered to have no toxicological significance, because there were no abnormalities in related parameters and no significant histopathological changes were noted in the liver, kidneys and muscles. In females, decrease of TP at 1000 ppm might be involved in suppression of body weight and decrease in food consumption. Significant increases of BUN in all treated groups might have been caused by low values in the control group, because the values in the treated groups were within the normal range of background data of GALAS rats. Since the increases of IP and ALP, parameters relating to bones, were found only in females despite more severe osteochondral lesions in males, we could not clarify involvement in SEM-HCl treatment-associated pathological lesions. Significant alterations of absolute and relative weights were found in several organs at 1000 ppm, but, these might reflect body weight decrease, since no significant histopathological changes were noted in these organs. SEM-HCl treatment revealed characteristic histopathological anomalies of bone, cartilage and the elastic laminae of arteries, in line with its documented inhibition of cross-linking reactions of collagen and elastin (Dawson et al., 2002) and induction of osteochondral and vascular lesions (Ramamurti and Taylor, 1959; Langford et al., 1999; Mercier et al., 2007). Loss of bone mass and changes in the microarchitecture of the collagen fibers in lathyritic rats were also described in a study of effects of beta-aminopropionitrile, another osteolathyrogen, on the maxillary and mandibular bones (El Rouby et al., 2008). In the aorta, alteration of elastic laminae was found to be induced by SEM due to inhibition of elastin cross-linkage, resulting in increased fragility of arterial walls (Mercier et al., 2007). In our study, a sex difference was obvious in the severity of the osteochondral lesions, with a male predominance. Osteochondral lesions were prominent in the long bones growing rapidly, such as the femur, tibia, radius and ulna. Moreover, the severities of lesions were increased in sites subject to body weight loading, as indicated by more pronounced histopathological changes in the tibia than the femur. These findings suggest reduction in the strength of cartilage due to alteration of matrix properties caused by inhibition of cross-linking reaction of collagen. Differences in body weights of males and females might thus be considered one reason for the more severe lesions in male rats. Except for the bones and joints, the incidences of mineralization in the pulmonary arteries were significantly lowered in males of
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the 250 and 500 ppm group. However, since dose-dependence and significant histopathological changes were lacking in related organs such as the parathyroids and kidneys, no treatment relation could be concluded. Chronic inflammation of the ventral prostate is known to frequently occur spontaneously in male rats, and there were no associated lesions in related organs. Therefore, the increase of chronic inflammation of the ventral prostate found at 1000 ppm can be regarded of low toxicological relevance. In addition, since cysts in the anterior lobe of the pituitary is an incidental finding observed frequently in rats, the difference in the incidence of this lesion noted in females might be of negligible significance. Taken together, toxicological effects of subchronic exposure to SEM-HCI were mainly observed in the bones, cartilages and aorta. From the histopathological examination, since disarrangement, fissures and deformation of the cartilages were found in both sexes from 250 ppm, the no-observed-adverse-effect-levels (NOAELs) estimated from present study were less than 250 ppm in both sexes, equivalent to 18.1 and 21.1 mg/kg/day in males and females. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements This work was supported by Health and Labour Sciences Research Grants (Research on Food Safety) from the Ministry of Health, Labour and Welfare of Japan. We thank Miss Ayako Kaneko for technical assistance in conducting the animal study. References Abramsson-Zetterberg, L., Svensson, K., 2005. Semicarbazide is not genotoxic in the flow cytometry-based micronucleus assay in vivo. Toxicol. Lett. 155, 211–217. Dawson, D.A., Rinaldi, A.C., Pöch, G., 2002. Biochemical and toxicological evaluation of agent-cofactor reactivity as a mechanism of action for osteolathyrism. Toxicology 177, 267–284. de la Calle, M.B., Anklam, E., 2005. Semicarbazide: occurrence in food products and state-of-the-art in analytical methods used for its determination. Anal. Bioanal. Chem. 382, 968–977. de la Fuente del Rey, M., 1986. Teratogenic effect of semicarbazide in Wistar rats. Biol. Neonate 49, 150–157. Effkemann, S., Feldhusen, F., 2004. Triple-quadrupole LC–MS–MS for quantitative determination of nitrofuran metabolites in complex food matrixes. Anal. Bioanal. Chem. 378, 842–844. EFSA, 2003. Statement of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food Updating the Advice Available on Semicarbazide in Packaged Foods (Adopted on 1 October 2003). EFSA, 2005. Opinion of the scientific panel on food additives, flavourings, processing aids and materials in contact with food on a request made from the commission related to semicarbazide in food. EFSA J. 219, 1–36. El Rouby, D.H., Bashir, M.H., Korany, N.S., 2008. Ultrastructural and histomorphometric alterations of rat jaw bones after experimental induction of lathyrism. Arch. Oral Biol. 53, 916–923. FSC, 2007. The 68th Expert Committee of Veterinary Medicines. Available at: (in Japanese). Gong, B., Trent, M.B., Srivastava, D., Boor, P.J., 2006. Chemical-induced, nonlethal, developmental model of dissecting aortic aneurysm. Birth Defects Res. A Clin. Mol. Teratol. 76, 29–38. Hoenicke, K., Gatermann, R., Hartig, L., Mandix, M., Otte, S., 2004. Formation of semicarbazide (SEM) in food by hypochlorite treatment: is SEM a specific marker for nitrofurazone abuse? Food Addit. Contam. 21, 526–537. Langford, S.D., Trent, M.B., Balakumaran, A., Boor, P.J., 1999. Developmental vasculotoxicity associated with inhibition of semicarbazide-sensitive amine oxidase. Toxicol. Appl. Pharmacol. 155, 237–244. Macedo, C.E., Martinez, R.C., Albrechet-Souza, L., Molina, V.A., Brandão, M.L., 2007. 5-HT2- and D1-mechanisms of the basolateral nucleus of the amygdala enhance conditioned fear and impair unconditioned fear. Behav. Brain Res. 177, 100– 108. Magyar, K., Mészáros, Z., Mátyus, P., 2001. Semicarbazide-sensitive amine oxidase. Its physiological significance. Pure Appl. Chem. 73, 1393–1400. Maranghi, F., Tassinari, R., Lagatta, V., Moracci, G., Macrì, C., Eusepi, A., Di Virgilio, A., Scattoni, M.L., Calamandrei, G., 2009. Effects of the food contaminant semicarbazide following oral administration in juvenile Sprague-Dawley rats. Food Chem. Toxicol. 47, 472–479.
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Mercier, N., El Hadri, K., Osborne-Pellegrin, M., Nehme, J., Perret, C., Labat, C., Regnault, V., Lamazière, J.M., Challande, P., Lacolley, P., Fève, B., 2007. Modifications of arterial phenotype in response to amine oxidase inhibition by semicarbazide. Hypertension 50, 234–241. Parodi, S., De Flora, S., Cavanna, M., Pino, A., Robbiano, L., Bennicelli, C., Brambilla, G., 1981. DNA-damaging activity in vivo and bacterial mutagenicity of sixteen hydrazine derivatives as related quantitatively to their carcinogenicity. Cancer Res. 41, 1469–1482. Ramamurti, P., Taylor, H.E., 1959. Skeletal lesions produced by semicarbazide and experimental analysis of the action of lathyrogenic compounds. J. Bone Joint Surg. Brit., 590–599. Saari, L., Peltonen, K., 2004. Novel source of semicarbazide: levels of semicarbazide in cooked crayfish samples determined by LC/MS/MS. Food Addit. Contam. 21, 825–832.
Stadler, R.H., Mottier, P., Guy, P., Gremaud, E., Varga, N., Lalljie, S., Whitaker, R., Kintscher, J., Dudler, V., Read, W.A., Castle, L., 2004. Semicarbazide is a minor thermal decomposition product of azodicarbonamide used in the gaskets of certain food jars. Analyst 129, 276–281. Steffek, A.J., Verrusio, A.C., Watkins, C.A., 1972. Cleft palate in rodents after maternal treatment with various lathyrogenic agents. Teratology 5, 33–38. Toth, B., Shumizu, H., Erickson, J., 1975. Carbamylhydrazine hydrochloride as a lung and blood vessel tumour inducer in Swiss mice. Eur. J. Cancer 11, 17–22. Weisburger, E.K., Ulland, B.M., Nam, J., Gart, J.J., Weisburger, J.H., 1981. Carcinogenicity tests of certain environmental and industrial chemicals. J. Natl. Cancer Inst. 67, 75–88.
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
A ninety-day toxicity study of semicarbazide hydrochloride in Wistar Hannover GALAS rats Miwa Takahashi *, Midori Yoshida, Kaoru Inoue, Tomomi Morikawa, Akiyoshi Nishikawa Division of Pathology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
a r t i c l e
i n f o
Article history: Received 13 March 2009 Accepted 8 July 2009
Keywords: Semicarbazide hydrochloride Osteolathyrism Food contaminant 90-day study GALAS rats
a b s t r a c t A ninety-day toxicity study of semicarbazide hydrochloride (SEM-HCl) was conducted in male and female Wistar Hannover GALAS rats fed diet containing the compound at concentration of 0, 250, 500 and 1000 ppm. Suppression of body weight gain and food consumption was found in both sexes at 1000 ppm throughout the study. Enlargement and deformation of knee joints were obvious at 500 and 1000 ppm from week 3, together with deformation of the thorax and tail. Histopathologically, disarrangement of chondrocytes and fissures in the cartilage matrix were apparent at all doses tested in epiphyseal and articular cartilage. The severity of these lesions increased dose-dependently, accompanied by increased connective tissues and bone deformation at high doses. Additionally, compact bones at 1000 ppm became thin, suggesting loss of bone mass. In the thoracic aorta, the edges of elastic laminae became rough and the interlaminar spaces were altered from a fibrillar to a rod or globular appearance. No abnormalities were detected in any other organs. Taken together, toxicological effects of subchronic exposure to SEM-HCI were mainly observed in bone, cartilage and the aorta, with the no-observedadverse-effect-level estimated from the present histopathological examination of less than 250 ppm in both sexes. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Semicarbazide (SEM) is a metabolite of the banned veterinary antibiotic nitrofurazone which has been used as a marker for abuse of this drug (Effkemann and Feldhusen, 2004; de la Calle and Anklam, 2005). However, it was recently found that SEM can also originate from the thermal breakdown of azodicarbonamide, used as a blowing agent in plastic gaskets (EFSA, 2003; Stadler et al., 2004). Other sources such as from food processing by hypochlorite treatment have further been suggested (Hoenicke et al., 2004; Saari and Peltonen, 2004). In view of possible exposure from various foods, independent of any nitrofurazone use, it has become important to assess the health risk of SEM.
Abbreviations: A/G, albumin: globulin ratio; Alb, albumin; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; Bil, bilirubin; BUN, blood urea nitrogen; BW, body weight; Ca, calcium; Cl, chlorine; CRN, creatinine; c-GTP, gamma glutamyl transpeptidase; Hb, hemoglobin; Ht, hematocrit; IP, inorganic phosphate; K, potassium; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; Na, sodium; NOAEL, no-observed-adverse-effect-level; Plt, platelet; RBC, red blood cell; SEM, semicarbazide; SEM-HCl, semicarbazide hydrochloride; SSAO, semicarbazide-sensitive amine oxidase; TC, total cholesterol; TG, triglyceride; TP, total protein; WBC, white blood cell. * Corresponding author. Tel.: +81 3 3700 9821; fax: +81 3 3700 1425. E-mail address: [email protected] (M. Takahashi). 0278-6915/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2009.07.008
SEM is known to inhibit enzymes, such as lysyl oxidase, semicarbazide-sensitive amine oxidase (SSAO) and glutamic acid decarboxylase (Magyar et al., 2001; Dawson et al., 2002; Macedo et al., 2007). It acts as an osteolathyrogen, and induces osteochondral and vascular lesions in young rats due to impaired cross-linking reactions of collagen and elastin through inhibition of lysyl oxidase or SSAO (Ramamurti and Taylor, 1959; Langford et al., 1999; Dawson et al., 2002; Mercier et al., 2007). In addition, teratogenic effects such as induction of cleft palate and aortic aneurysms have also been reported (Steffek et al., 1972; de la Fuente del Rey, 1986; Gong et al., 2006). Reports have indicated that SEM has weak genotoxicity in vitro but not in vivo (Parodi et al., 1981; Abramsson-Zetterberg and Svensson, 2005; EFSA, 2005; FSC, 2007). SEM hydrochloride (SEM-HCl) at a high dose in the drinking water showed a tendency to increase incidences of lung and blood vessel tumors in mice (Toth et al., 1975). Weisburger et al. reported carcinogenicity study of SEM-HCl using SD rats fed diet containing 500 and 1000 ppm (Weisburger et al., 1981). In this study, the animals in the 500 ppm groups received test diet for 78 weeks, and thereafter received control diet for further 26 weeks. The dosing of 1000 ppm was considered to be over maximal tolerated dose and the treatment was discontinued at week 32 because deaths occurred frequently in males, and they received control diet for the remainder of the study period. Although osteolathyrism was found grossly from week 10 in all treated groups, no carcinogenic effects
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M. Takahashi et al. / Food and Chemical Toxicology 47 (2009) 2490–2498 Table 1 Findings of clinical observation at week 13 in GALAS rats fed diet containing SEM-HCl for 90 days. Sites and findings
Sex
Males
Dose (ppm)
0
250
500
1000
0
250
500
1000
Hindlimb Forelimb Thorax Tail
No. of animals examined Enlargement and deformation of the knee joint Enlargement and deformation of the wrist joint Prominence Stiff flexion
10 0 0 0 0
10 1 0 0 1
10 10 0 3 4
10 10 6 10 9
10 0 0 0 0
10 0 0 0 0
10 10 0 1 0
10 10 8 9 0
were observed in either sex. However, since the treatment was discontinued at week 32 or 78 and no details were provided on the tumor incidences, the carcinogenicity of SEM is still controversial. Accordingly, to examine the subchronic toxicity of SEM prior to conducting the carcinogenicity study, we performed the present 90-day toxicity study of SEM-HCl using rats. 2. Materials and methods 2.1. Animals Male and female Wistar Hannover GALAS rats at 5 weeks of age were obtained from CLEA Japan, Inc. (Tokyo, Japan). Wistar Hannover GALAS rat has been accepted as a suitable strain of toxicology studies (http://www.galas.org/index.html). They received powdered basal diet (CE-2, CLEA Japan, Inc.) and tap water ad libitum, housed 2–4 per plastic cage with sterilized softwood chips as bedding in a barrier-maintained animal room conditioned at 24 ± 1 °C and 55 ± 5% humidity with a 12-h light/dark cycle. After a 1-week acclimatization period, animals showing
500
Females
no abnormalities were used at 6 weeks of age. The animal protocol was reviewed and approved by the Animal Care and Use Committee of the National Institute of Health Sciences, Japan. 2.2. Chemicals Semicarbazide hydrochloride (SEM-HCl, CAS No. 563–41-7) was purchased from Hayashi Pure Chemical Ind., Ltd. (Osaka, Japan), as a white powder with a purity of 99.3%. SEM-HCl was well mixed at concentrations of 0, 250, 500 and 1000 ppm into powdered basal diet (CE-2). Concentrations after storage at room temperature for 4 weeks or at 4 °C for 8 weeks were analyzed at Japan Food Research Laboratories (Osaka, Japan), and more than 89% stability of the test compound was confirmed under both conditions. SEM was not detected in the basal diet (detection limit, 0.01 ppm). Test diets were prepared every 2 weeks, and stored at 4 °C before use. 2.3. Experimental design As a preliminary examination, a 2-week dose finding study was conducted based on a previous report (Weisburger et al., 1981), with 1000 ppm selected as the highest dose for subsequent study. Animals, weighing 166.5 ± 4.5 g for males
Males
45
450
300 250 **
200
**
**
**
**
**
**
**
**
**
**
** 0 ppm
**
150
250 ppm 500 ppm 1000 ppm
100
Food intake (g/animal/day)
Body weight (g)
350
50 0
Males
40
400
35 30 25 20 15
**
10
0 ppm 250 ppm
5
0
1
2
3
4
5
6 7 Weeks
8
9
10 11 12 13
0
1
2
3
4
5
6
7 8 Weeks
9
10
500 ppm 1000 ppm
11
12
13
300
Females
35
200 *
150
**
**
**
**
**
**
**
**
**
0 ppm
100
250 ppm 500 ppm 1000 ppm
50
Food intake (g/animal/day)
Body weight (g)
250
Females
30 25 20 15 10
** 0 ppm 250 ppm
5
0
0
1
2
3
4
5
6 7 Weeks
8
9
10 11 12 13
0
1
2
3
4
5
6
7
8
9
10
500 ppm 1000 ppm
11
12
13
Weeks Fig. 1. Body weight curves for male and female GALAS rats fed diet containing SEMHCl for 90 days. ,: Significantly different from the 0 ppm group at p < 0.05 and p < 0.01, respectively.
Fig. 2. Daily food intake for male and female GALAS rats fed diet containing SEMHCl for 90 days. : Significantly different from the 0 ppm group at p < 0.01.
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and 126.3 ± 4.3 g for females (mean ± SD), were randomly allocated to 4 groups, each consisting of 10 males and 10 females, and given diet containing 0 (control), 250, 500 or 1000 ppm SEM-HCl for 90 days. The test diets were available ad libitum, except for one-night fasting prior to the scheduled sacrifice, and rats had free access to tap water throughout the study. Observations for mortality and clinical signs, including posture and gait abnormalities and deformation of four limbs, thorax and tail, were conducted daily. Body weight and food consumption were recorded every week. At necropsy, all animals were anesthetized with ether, weighed, and blood samples were collected from the abdominal aorta for hematology and serum biochemistry. Relative organ weights were calculated as the values relative to body weights.
2.4. Hematology and serum biochemistry Hematology analysis was performed using an automated hematology analyzer, K-4500 (Sysmex Corp., Hyogo, Japan). Differential leukocyte counts and reticulocyte counts were performed with a MICROX HEG-50S (Sysmex Corp.). Parameters for serum biochemistry shown in Table 4 were analyzed at SRL, Inc. (Tokyo, Japan) using sera frozen after centrifugation of whole blood.
2.5. Histopathological examination After macroscopic examination, the brain, thymus, heart, lungs, liver, spleen, adrenals, kidneys and testes were removed and weighed. In addition, the pituitary, eyes, Harderian glands, salivary glands, tongue, trachea, esophagus, thyroid glands, thoracic aorta, stomach, small intestine (duodenum, jejunum, and ileum), large intestine (cecum, colon, and rectum), pancreas, mesenteric lymph nodes, thigh muscle, sciatic nerve, skin, mammary gland, urinary bladder, epididymides, seminal vesicles, prostate, ovaries, uterus and vagina were similarly resected. All organs were fixed in 10% buffered formalin, except for testes, which were fixed in Bouin’s solution overnight. For examination of osteochondral lesions, the nasal cavity, sternum, right femur, right tibia, left knee joint, right and left ankles, spine (cervical, thoracic, lumbar and caudal vertebrae with corresponding spinal cord) and macroscopic lesions (in wrist joints etc.) were fixed in 10% buffered formalin and then decalcified in EDTA solution at room temperature for a month. Histopathological assessment was performed on the bones, joints and thoracic aorta of all groups. Additionally, all organs of the control and 1000 ppm groups and the heart, lungs, liver, kidneys, thyroid, trachea, testes and prostate of the intermediate dose groups were also examined. The tissues were routinely processed for paraffin embedding, sectioned and stained with hematoxylin and eosin (HE). Victoria blue and HE staining was applied to three transverse sections of the descending thoracic aorta cut at 5 mm intervals to demonstrate elastic fibers.
2.6. Statistical analysis Variance in data for body weights, food consumption, hematology, serum biochemistry and organ weights was checked for homogeneity by Bartlett’s procedure. If the variance was homogeneous, the data were assessed by one-way analysis of variance. If not, the Kruskal–Wallis test was applied. When statistically significant differences were detected, the Dunnett’s multiple test was
Table 2 Food consumption and intake of SEM-HCl in GALAS rats. Sex
Dose (ppm)
No. of animals examined
Food consumption
(g/animal/ day)
(g/kg BW/day)
Intake of SEM-HCl
Daily
Total
(mg/kg BW/day)
(mg/kg BW)
Males
0 250 500 1000
10 10 10 10
23.3 ± 1.2a 24.4 ± 1.7 26.7 ± 2.3** 19.0 ± 1.7**
69.5 ± 16.7 72.6 ± 14.4 81.4 ± 15.0 65.9 ± 13.3
0 18.1 40.7 65.9
0 1633 3662 5929
Females
0 250 500 1000
10 10 10 10
19.2 ± 1.3 17.2 ± 1.0** 17.7 ± 0.9** 12.6 ± 1.1**
96.2 ± 11.2 84.5 ± 11.2* 88.3 ± 13.6 70.5 ± 13.1**
0 21.1 44.2 70.5
0 1901 3975 6344
Abbreviations: SEM-HCl, semicarbazide hydrochloride; BW, body weight. a Mean ± SD. , Significantly different from the 0 ppm group at p < 0.05 and p < 0.01, respectively.
employed for comparison between the control and treatment groups. For histopathological findings, the incidences were compared using the Fisher’s exact probability test.
3. Results 3.1. General condition No deaths occurred throughout the feeding period. The findings of clinical observation at week 13 are summarized in Table 1. Enlargement and deformation of the knee joints were apparent in males and females at 500 and 1000 ppm from week 3, and prominence of the thorax was also found from week 5. In the 1000 ppm group, enlargement and deformation of the wrist joints were observed from week 12 in both sexes, and some showed posture and gait abnormalities. Tails of male rats in the treated groups exhibited stiff flexion from week 4.
Table 3 Hematology data for GALAS rats fed diet containing SEM-HCl for 90 days. SEM-HCl (ppm) 0 No. of animals 10 examined Males 921.1 ± 36.0a RBC (104/ll) Hb (g/dl) 16.5 ± 0.6 Ht (%) 51.9 ± 2.0 MCV (fl) 56.4 ± 1.3 MCH (pg) 18.0 ± 0.4 MCHC (g/dl) 31.9 ± 0.7 95.3 ± 9.2 Plt (104/ll) WBC (102/ll) 45.8 ± 11.9 Differential leukocyte counts Band form neutrophils 0.3 ± 0.4 (%) Segmented 13.8 ± 4.1 neutrophils (%) Eosinophils (%) 0.8 ± 0.5 Basophils (%) 0±0 Lymphocytes (%) 85.0 ± 4.4 Monocytes (%) 0.1 ± 0.2 Reticulocytes 0.1 ± 0.2 (/100 cells) Females 835.7 ± 49.2 RBC (104/ll) Hb (g/dl) 15.5 ± 0.8 Ht (%) 48.6 ± 2.7 MCV (fl) 58.2 ± 1.6 MCH (pg) 18.6 ± 0.5 MCHC (g/dl) 31.9 ± 0.7 93.2 ± 10.3 Plt (104/ll) 24.4 ± 6.2 WBC (102/ll) Differential leukocyte counts Band form neutrophils 0.5 ± 0.6 (%) Segmented 18.3 ± 6.9 neutrophils (%) Eosinophils (%) 1.3 ± 0.9 Basophils (%) 0±0 Lymphocytes (%) 79.4 ± 7.4 Monocytes (%) 0.5 ± 0.3 Reticulocytes 0.2 ± 0.4 (/100 cells)
250
500
1000
10
10
10
929.7 ± 22.2 16.2 ± 0.3 51.8 ± 1.2 55.7 ± 1.1 17.4 ± 0.4* 31.3 ± 0.6 92.2 ± 10.0 43.9 ± 13.7
943.2 ± 40.4 16.4 ± 0.5 52.3 ± 2.4 55.5 ± 1.1 17.4 ± 0.4* 31.3 ± 0.6 93.3 ± 11.6 46.5 ± 15.1
962.6 ± 55.5 17.0 ± 0.7 54.0 ± 2.1 56.1 ± 1.4 17.7 ± 0.6 31.5 ± 0.5 98.6 ± 16.5 35.4 ± 9.8
0.4 ± 0.4
0.6 ± 0.4
0.6 ± 0.5
16.3 ± 4.9
13.4 ± 6.8
7.9 ± 2.2*
1.6 ± 1.0 0±0 81.5 ± 6.1 0.3 ± 0.3 0.1 ± 0.2
1.3 ± 0.9 0±0 84.6 ± 7.3 0.3 ± 0.4 0.1 ± 0.2
0.8 ± 0.8 0±0 90.6 ± 2.4* 0.3 ± 0.4 0.1 ± 0.2
818.9 ± 43.0 15.6 ± 0.8 48.5 ± 2.3 59.2 ± 1.4 19.1 ± 0.7 32.2 ± 0.6 95.2 ± 9.1 22.9 ± 6.2
868.3 ± 64.6 16.2 ± 0.9 50.0 ± 3.1 57.6 ± 1.2 18.7 ± 0.6 32.5 ± 0.6 97.8 ± 11.5 29.8 ± 9.4
845.2 ± 62.3 15.5 ± 0.9 48.6 ± 3.3 57.5 ± 1.2 18.4 ± 0.6 32.0 ± 0.5 104.7 ± 14.7 35.7 ± 15.1
0.5 ± 0.5b
0.6 ± 0.5
0.5 ± 0.5
b
15.8 ± 3.6
18.5 ± 8.8
8.5 ± 3.1**
1.1 ± 0.9b 0 ± 0b 82.2 ± 4.3b 0.4 ± 0.5b 0.4 ± 1.2b
1.5 ± 0.8 0±0 78.9 ± 9.3 0.6 ± 0.5 0.1 ± 0.2
1.0 ± 0.5 0±0 89.8 ± 3.4** 0.2 ± 0.3 0.1 ± 0.2
Abbreviations: SEM-HCl, semicarbazide hydrochloride; RBC, red blood cell; Hb, hemoglobin; Ht, hematocrit; MCV, mean corpuscular volume; MCV, mean corpuscular volume; MCHC, mean corpuscular hemoglobin concentration; Plt, platelet; WBC, white blood cell. a Mean ± SD. b Number of effective animals was reduced to nine due to the failure of blood sampling for differential leukocyte counts. , Significantly different from the 0 ppm group at p < 0.05 and p < 0.01, respectively.
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3.2. Body weight, food consumption and chemical intake
3.3. Hematology and serum biochemistry
Significant suppression of body weight gain was observed at 1000 ppm from week 1 in males and from week 4 in females (Fig. 1). Data for food consumption and SEM-HCl intake are summarized in Fig. 2 and Table 2. In both sexes, food consumption was decreased at 1000 ppm throughout the study, and the mean values for food consumption/animal were significantly lowered compared to the control group. However, there were no intergroup differences in the mean values for food consumption/kg body weight in males, due to suppressed body weight gain. The mean values for food consumption/animal were significantly increased in males at 500 ppm and decreased in females at 250 and 500 ppm. Significant decrease of the mean values for food consumption/kg body weight was found in females of the 250 and 1000 ppm groups.
Hematology data are summarized in Table 3. In both sexes, differential leukocyte counts showed significant decrease and increase in the proportions of segmented neutrophils and lymphocytes, respectively, in the 1000 ppm group. At the same dose, WBC counts were decreased in males and increased in females, although not statistically significant. In males, MCH was significantly decreased at 250 and 500 ppm, but without dose-dependence. In the serum biochemical analysis, significant alterations of CRN, ALT, A/G, total Bil and K were found in males at 500 and/or 1000 ppm (Table 4). In females, significant increases of BUN were observed in all treated groups, and K and IP were statistically increased at 500 and 1000 ppm. Significant alterations of TP, ALT and ALP were also detected at 1000 ppm.
Table 4 Serum biochemical data for GALAS rats fed diet containing SEM-HCl for 90 days.
3.4. Organ weights Final body weights were significantly decreased in both sexes of the 1000 ppm group (Table 5). Absolute weights of the lungs in both sexes and the thymus, heart and liver in males were statistically lowered at 1000 ppm. In males, significant increase in relative
SEM-HCl (ppm)
No. of animals examined Males TP (g/dL) Alb (g/dL) A/G Total Bil (mg/dL) Glucose (mg/dL) TG (mg/dL) TC (mg/dL) BUN(mg/dL) CRN (mg/dL) Na (mEQ/L) Cl (mEQ/L) K (mEQ/L) Ca (mg/dL) IP (mg/dL) AST (IU/L) ALT (IU/L) ALP (IU/L) c-GTP (IU/L) Females TP (g/dL) Alb (g/dL) A/G Total Bil (mg/dL) Glucose (mg/dL) TG (mg/dL) TC (mg/dL) BUN (mg/dL) CRN (mg/dL) Na (mEQ/L) Cl (mEQ/L) K (mEQ/L) Ca (mg/dL) IP (mg/dL) AST (IU/L) ALT (IU/L) ALP (IU/L) c-GTP (IU/L)
0
250
500
1000
10
10
10
10
6.9 ± 0.1a 4.5 ± 0.1 1.9 ± 0.1 0.06 ± 0.02 151.2 ± 18.7 116.9 ± 45.2 72.8 ± 11.7 21.6 ± 1.2 0.33 ± 0.04 147.6 ± 1.3 105.1 ± 1.4 4.3 ± 0.2 10.6 ± 0.3 6.4 ± 0.8 79.9 ± 12.8 37.0 ± 6.0 288.6 ± 48.7 <2
6.7 ± 0.2 4.5 ± 0.1 2.0 ± 0.2 0.06 ± 0.01 136.5 ± 12.2 90.1 ± 43.5 69.7 ± 11.1 21.9 ± 2.8 0.31 ± 0.03 147.5 ± 1.4 105.6 ± 1.2 4.3 ± 0.2 10.3 ± 0.2 6.2 ± 0.6 83.2 ± 11.5 36.7 ± 4.2 329.1 ± 70.7 <2
6.8 ± 0.2 4.5 ± 0.1 1.9 ± 0.2 0.07 ± 0.01 146.7 ± 20.0 104.3 ± 39.4 69.7 ± 12.8 21.5 ± 2.4 0.29 ± 0.03** 146.6 ± 1.3 103.7 ± 1.2 4.7 ± 0.5* 10.5 ± 0.3 7.0 ± 0.8 66.6 ± 10.2 28.7 ± 4.5** 329.2 ± 73.7 <2
6.8 ± 0.2 4.7 ± 0.2 2.2 ± 0.2** 0.09 ± 0.02** 156.2 ± 23.8 74.1 ± 33.2 75.3 ± 11.1 22.1 ± 2.8 0.29 ± 0.03** 147.2 ± 1.1 105.6 ± 2.1 4.4 ± 0.2 10.5 ± 0.4 7.0 ± 0.6 70.9 ± 14.5 27.4 ± 4.4** 318.0 ± 65.7 <2
7.1 ± 0.5 5.1 ± 0.4 2.6 ± 0.3 0.10 ± 0.03 116.1 ± 18.3 41.0 ± 15.5 59.7 ± 11.8 18.3 ± 1.9 0.33 ± 0.04 145.6 ± 1.3 106.4 ± 2.8 3.6 ± 0.2 10.0 ± 0.4 4.5 ± 0.6 74.9 ± 9.5 24.4 ± 4.2 103.6 ± 31.6 <2
7.1 ± 0.5 5.1 ± 0.4 2.6 ± 0.4 0.10 ± 0.02 117.0 ± 29.4 64.8 ± 64.3 66.8 ± 19.6 22.3 ± 3.3* 0.35 ± 0.05 145.4 ± 1.6 105.5 ± 1.5 3.8 ± 0.3 10.1 ± 0.4 4.9 ± 0.8 77.9 ± 16.3 29.0 ± 8.3 115.1 ± 39.1 <2
7.1 ± 0.4 5.0 ± 0.3 2.4 ± 0.3 0.09 ± 0.01 118.9 ± 22.7 38.9 ± 23.2 63.5 ± 12.5 22.3 ± 3.4* 0.35 ± 0.04 145.1 ± 1.5 105.5 ± 0.8 4.0 ± 0.4* 10.2 ± 0.3 5.5 ± 0.9** 70.2 ± 10.1 21.3 ± 3.1 127.1 ± 50.4 <2
6.6 ± 0.3* 4.8 ± 0.2 2.6 ± 0.2 0.10 ± 0.02 111.6 ± 25.5 34.9 ± 13.3 65.6 ± 12.2 23.1 ± 4.2** 0.34 ± 0.04 145.7 ± 1.6 105.9 ± 1.7 3.9 ± 0.3* 10.1 ± 0.3 6.4 ± 0.7** 71.9 ± 11.3 17.9 ± 2.0** 186.7 ± 88.0** <2
Abbreviations: SEM-HCl, semicarbazide hydrochloride; TP, total protein; Alb, albumin; A/G, albumin: globulin ratio; Bil, bilirubin; TG, triglyceride; TC, total cholesterol; BUN, blood urea nitrogen; CRN, creatinine; Na, sodium; Cl, chlorine; K, potassium; Ca, calcium; IP, inorganic phosphate; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase. c-GTP, gamma glutamyl transpeptidase. a Mean ± SD. , Significantly different from the 0 ppm group at p < 0.05 and p < 0.01, respectively.
Table 5 Final body and organ weights of GALAS rats fed diet containing SEM-HCl for 90 days. SEM-HCl (ppm) 0
250
500
1000
No. of animals examined Males Body weight (g) Brain (g) (g%) Thymus (g) (g%) Lungs (g) (g%) Heart (g) (g%) Spleen (g) (g%) Liver (g) (g%) Adrenals (mg) (mg%) Kidneys(g) (g%) Testes (g) (g%)
10
10
10
10
409.3 ± 18.6a 2.06 ± 0.08 0.50 ± 0.02 0.30 ± 0.06 0.07 ± 0.02 1.25 ± 0.13 0.31 ± 0.02 1.05 ± 0.12 0.26 ± 0.02 0.66 ± 0.08 0.16 ± 0.02 10.62 ± 0.99 2.59 ± 0.19 52.40 ± 7.17 12.83 ± 1.92 2.28 ± 0.12 0.56 ± 0.03 3.38 ± 0.34 0.83 ± 0.07
407.8 ± 26.6 2.04 ± 0.08 0.50 ± 0.03 0.32 ± 0.08 0.08 ± 0.02 1.25 ± 0.10 0.31 ± 0.02 1.01 ± 0.10 0.25 ± 0.02 0.66 ± 0.05 0.16 ± 0.01 9.70 ± 0.81 2.38 ± 0.07* 51.30 ± 5.66 12.62 ± 1.45 2.38 ± 0.20 0.58 ± 0.03 3.33 ± 0.27 0.82 ± 0.05
396.9 ± 27.3 2.00 ± 0.08 0.51 ± 0.03 0.31 ± 0.06 0.08 ± 0.01 1.15 ± 0.12 0.29 ± 0.04 1.06 ± 0.06 0.27 ± 0.02 0.62 ± 0.07 0.16 ± 0.02 10.28 ± 1.04 2.58 ± 0.10 55.50 ± 5.40 13.99 ± 1.05 2.35 ± 0.20 0.59 ± 0.04* 3.37 ± 0.30 0.85 ± 0.08
323.8 ± 36.0** 2.00 ± 0.08 0.63 ± 0.06** 0.21 ± 0.04** 0.06 ± 0.01 0.96 ± 0.12** 0.30 ± 0.03 0.89 ± 0.11** 0.27 ± 0.02 0.61 ± 0.10 0.19 ± 0.02** 8.51 ± 1.43** 2.61 ± 0.20 56.10 ± 10.88 17.34 ± 2.78** 2.08 ± 0.28 0.64 ± 0.03** 3.56 ± 0.43 1.10 ± 0.08**
Females Body weight (g) Brain (g) (g%) Thymus (g) (g%) Lungs (g) (g%) Heart (g) (g%) Spleen (g) (g%) Liver (g) (g%) Adrenals (mg) (mg%) Kidneys (g) (g%)
220.9 ± 13.6 1.83 ± 0.09 0.83 ± 0.05 0.26 ± 0.05 0.12 ± 0.03 0.86 ± 0.06 0.39 ± 0.02 0.63 ± 0.06 0.29 ± 0.02 0.47 ± 0.04 0.21 ± 0.02 5.40 ± 0.37 2.45 ± 0.11 69.10 ± 10.34 31.27 ± 4.22 1.42 ± 0.17 0.64 ± 0.04
229.2 ± 22.4 1.82 ± 0.08 0.80 ± 0.09 0.31 ± 0.11 0.13 ± 0.04 0.85 ± 0.07 0.37 ± 0.03 0.67 ± 0.08 0.29 ± 0.03 0.44 ± 0.06 0.19 ± 0.01* 5.84 ± 0.87 2.55 ± 0.29 65.30 ± 12.48 28.52 ± 4.94 1.38 ± 0.16 0.60 ± 0.05
225.2 ± 11.6 1.87 ± 0.09 0.83 ± 0.04 0.25 ± 0.05 0.11 ± 0.02 0.88 ± 0.11 0.39 ± 0.04 0.66 ± 0.06 0.30 ± 0.02 0.45 ± 0.04 0.20 ± 0.02 5.57 ± 0.55 2.47 ± 0.17 68.90 ± 10.21 30.54 ± 3.65 1.47 ± 0.15 0.65 ± 0.04
189.4 ± 7.8** 1.82 ± 0.08 0.97 ± 0.06** 0.20 ± 0.04 0.11 ± 0.02 0.75 ± 0.06* 0.40 ± 0.03 0.61 ± 0.05 0.32 ± 0.03** 0.48 ± 0.05 0.25 ± 0.03 4.77 ± 0.50 2.52 ± 0.23 60.00 ± 7.09 31.73 ± 3.94 1.33 ± 0.11 0.70 ± 0.05*
a
Mean ± SD. Significantly different from the 0 ppm group at p < 0.05 and p < 0.01, respectively. ,
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kidney weights was observed at 500 and 1000 ppm, and relative weights of the brain, spleen, adrenals and testes were also increased at 1000 ppm. In females, relative weights of the brain, heart and kidneys were significantly elevated in the 1000 ppm group. Decreases in relative liver weight in males and relative spleen weight in females were found at 250 ppm, but such changes were not observed in the 500 and 1000 ppm groups. 3.5. Necropsy and histopathology In the macroscopic examination, bowing of the tibia was apparent in males and females at 500 and 1000 ppm. Also, increases in diameter of the femur and tibia were evident due to enlargement of the marrow cavities (Fig. 3A), along with prominence of the sternum (Fig. 3B), thoracic kyphosis and deformation of the wrist joints at 1000 ppm. Histopathological findings for bones and joints are summarized in Table 6. In the femur and tibia, disarrangement of epiphyseal chondrocytes was observed in both sexes at all doses tested. The epiphyseal plate at the proximal end of the tibia was thickened at 500 and 1000 ppm, and degeneration of hypertrophic chondro-
cytes was found in the thickened cartilage plate (Fig. 4A–C). There were fissures in the cartilage matrix, and these were widened and accompanied by increase of connective tissues at 500 and 1000 ppm (Fig. 4D). In the metaphysis, spongy bones were decreased and irregularly-branched. The compact bones in the metaphysis became thin with a rugged surface, and deformed to expand outward, resulting in enlargement of the marrow cavities. Also in the sternum, disarrangement of epiphyseal chondrocytes and fissures in the cartilage matrix were found at all doses tested, and severe fissures with increased connective tissues and bone deformation were more evident at 500 and 1000 ppm in a dosedependent manner. In the animals with deformation of the wrist joint macroscopically, the epiphyseal plates of the radius and ulna exhibited similar histological lesions with the tibia. Deformation and fissures of articular cartilage with disarrangement of chondrocytes were observed in the knee joints and the intervertebral joints (from cervical to caudal) at all doses tested, and synovial inflammation and fibrosis were also found in the 500 and 1000 ppm groups (Fig. 4E and F). In the thoracic vertebrae, fissures occurred in the epiphyseal plate adjacent to the intervertebral disk, and displacement of the vertebra originating in the fissures, as well as
Fig. 3. Macroscopic lesions observed in GALAS rats fed diet containing SEM-HCl for 90 days. (A) Femurs and tibias of male rats at 0 ppm (left), and 1000 ppm (right) showing marked bowing and enlargement of the marrow cavities with treatment. (B) Sternums of male rats given 0 ppm (left), and 1000 ppm (right) with a prominence in the middle of the bone in the latter case.
Table 6 Histopathological findings for the bones and joints of GALAS rats fed diet containing SEM-HCl for 90 days. Organs and findings
Femur
Tibia
Sternum
Knee joint Vertebrae
,
Sex
Males
Dose (ppm)
0
250
500
1000
0
Females 250
500
1000
No. of animals examined Disarrangement of epiphyseal chondrocytes Thickening of epiphyseal plate Fissures of epiphyseal plate Increase of connective tissues Bone deformation Disarrangement of epiphyseal chondrocytes Thickening of epiphyseal plate Fissures of epiphyseal plate Increase of connective tissues Bone deformation Disarrangement of epiphyseal chondrocytes Fissures of epiphyseal plate Increase of connective tissues Bone deformation Deformation and fissures of articular cartilage Synovial inflammation and fibrosis Deformation and fissures of articular cartilage Fissures adjacent intervertebral disk (thoracic vertebra) Displacement of the thoracic vertebra
10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
10 2 0 0 0 0 8** 0 7** 1 0 7** 7** 0 0 6** 2 7** 3 0
10 10** 0 10** 0 0 10** 10** 10** 10** 10** 10** 10** 7** 6** 10** 10** 10** 9** 0
10 10** 2 10** 6** 6** 10** 9** 10** 10** 10** 10** 10** 10** 10** 10** 10** 10** 10** 3
10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
10 1 0 0 0 0 7** 0 0 0 0 4* 2 0 0 1 0 6** 1 0
10 8** 0 0 0 0 10** 4* 10** 9** 4* 10** 9** 3 3 6** 4* 9** 4* 0
10 10** 2 6** 2 0 10** 10** 10** 10** 10** 10** 10** 10** 10** 10** 10** 10** 9** 0
Significantly different from the 0 ppm group at p < 0.05 and p < 0.01, respectively (Fisher’s exact test).
M. Takahashi et al. / Food and Chemical Toxicology 47 (2009) 2490–2498
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Fig. 4. Histopathology of the bones and joints of male GALAS rats fed diet containing SEM-HCl for 90 days. Tibias of the 0 ppm (A) and 1000 ppm groups showing treatmentassociated thickening of the epiphyseal plate and bone deformation (B). At higher power, note disarrangement and degeneration of epiphyseal chondrocytes (C) and increased connective tissue within fissures (D). Deformation and fissures of articular cartilage observed in a knee joint at 250 ppm (E) and in a lumbar intervertebral joint at 1000 ppm (F). Lumbar vertebrae of rats in the 0 ppm (G) and 1000 ppm groups (H). Thinning of bone in the vertebral body is obvious in the 1000 ppm group, suggesting loss of bone mass. HE stain. Bars = 1 mm (A, B, G, H), 100 lm (C, D), 200 lm (E, F).
compression of the spinal cord by displaced vertebra, were seen in males at 1000 ppm. Additionally, thinning of bone in the vertebral body was obvious in the 1000 ppm group, suggesting loss of bone mass (Fig. 4G and H). Overall, the severities of the osteochondral lesions described above were higher in males than females. There were no treatment-related changes in the nasal cavity, ankles and Achilles tendons. Histopathological findings for the thoracic aorta are summarized in Table 7. In both sexes, although the number of elastic laminae was unchanged, their edges became roughened in a
dose-dependent manner (Fig. 5). The interlaminar spaces in the treated groups had a rod or globular appearance, in contrast to the fibrillar appearance in the 0 ppm group. In other organs, the incidences of mineralization in the pulmonary arteries were significantly lowered in males of the 250 and 500 ppm group compared to the controls, but without dose-dependence (Table 8). In males, significant increase of chronic inflammation of the ventral prostate was found at 1000 ppm. The incidence of cysts in the anterior lobe of the pituitary was also significantly elevated in females of the 1000 ppm group.
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Table 7 Histopathological findings of the thoracic aorta of GALAS rats fed diet containing SEM-HCl for 90 days. Findings
No. of elastic lamina Edge of elastic fibers Interlaminar space a b
Sex
Males
Dose (ppm)
0
250
500
1000
0
250
500
1000
No. of animals examined
10 9.03 10/0/0b 10/0/0
10 9.20 4/6/0 5/5/0
10 9.03 0/4/6 0/4/6
9a 8.81 0/0/9 0/0/9
9a 8.92 9/0/0 9/0/0
9a 8.59 6/3/0 9/0/0
8a 9.33 2/6/0 6/2/0
10 8.87 0/5/5 0/10/0
Smooth/smooth rough/rough Fibrillar/fibrillar rod/rod
Females
Number of effective animals was reduced due to the failure of sampling. The number of animals showing each finding.
Fig. 5. Transverse sections of the thoracic aorta of male GALAS rats fed diet containing SEM-HCl for 90 days. Aortas of the 0 ppm (A) and 1000 ppm groups showing rough edges of elastic laminae in the latter (B). The interlaminar spaces have a rod to globular appearance, in contrast to the fibrillar appearance in the normal aorta. Victoria blue and HE stain. Bars = 50 lm.
Table 8 Histopathological findings observed in GALAS rats fed diet containing SEM-HCl for 90 days. Organs and findings
Pituitary
Harderian gl. Skeletal muscle Tongue Heart Lung
Thymus Liver
Kidney
Pancreas Thyroid Urinary bladder Testis Prostate Ovary
Sex
Males
Dose (ppm)
0
250
500
1000
0
Females 250
500
1000
No. of animals examined Cyst, anterior lobe Cyst-like structure, anterior lobe Cyst, intermediate lobe Cyst-like structure, intermediate lobe Atrophy Degeneration, focal Erosion Myocardial degeneration/necrosis Ectopic thyroid tissue Mineralization, pulmonary arteries Osseous metaplasia Inflammatory cell infiltration, focal Epithelial cell hyperplasia Microgranuloma Hepatocyte degeneration, focal Brown pigment deposition, periportal Regenerative tubules Mineralization, cortico-medullary junction Renal cyst Mononuclear cell infiltration, perivascular Transitional cell hyperplasia Focal acinar cell atrophy Ultimobranchial cyst Mononuclear cell infiltration, focal Seminiferous tubule atrophy, focal Chronic inflammation, ventral lobe Fat necrosis
10 3 0 0 1 2 0 1 6 0 9 2 0 0 2 1 0 4 0 0 0 0 1 1 0 0 6 –
10 0 0 0 1 – – – 5 0 4* 2 0 – 3 0 0 2 0 0 0 0 – 0 – 1 7 –
10 2 0 0 0 – – – 4 0 4* 2 0 – 3 0 0 1 0 0 1 1 – 0 – 2 7 –
10 2 2 0 1 0 0 0 3 0 5 3 0 1 2 0 0 3 0 0 0 2 2 0 1 0 10* –
10 0 0 0 1 0 0 0 1 0 1 1 0 1 0 0 0 0 9 0 0 0 0 1 1 – – 1
10 1 0 0 0 – – – 0 1 2 1 1 – 0 0 1 1 10 0 0 0 – 0 – – – –
10 0 0 0 1 – – – 0 0 4 0 1 – 2 1 0 0 10 1 0 0 – 0 – – – –
10 4* 0 1 0 0 1 0 1 0 4 1 0 2 2 0 0 1 10 0 0 0 1 3 0 – – 0
–: Not examined. * Significantly different from the 0 ppm group at p < 0.05 (Fisher’s exact test).
4. Discussion In the present study, significant suppression of body weight gain was observed in 1000 ppm group males and females through-
out the experiment. Because continuous decrease of food consumption and deformation of the limbs, thorax and tail were found in both sexes at 1000 ppm from the early part of the study, it was considered that the suppression of body weight gain was
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induced by SEM-HCl treatment. In contrast, body weight increase in the control, 250 and 500 ppm groups were similar. Therefore, the alteration of food consumption found in these groups was regarded as incidental due to variation in measured values. SEM is known to be an inhibitor of the gamma-aminobutyric acid synthesizing enzyme glutamic acid decarboxylase (Macedo et al., 2007). In a recent study to evaluate the effects of SEM administration for 28 days during the juvenile period in rats, the authors performed neurobehavioral tests and reported alterations of motor and exploratory behavior (Maranghi et al., 2009). In the present study, although neurobehavioral tests were not conducted, the posture and gait abnormalities observed in the 1000 ppm group were likely due to deformation of the limbs and osteochondral lesions, because the severities appeared to correlate. In the hematological analysis, differential leukocyte counts showed significant decrease and increase in the proportions of segmented neutrophils and lymphocytes, respectively, in both sexes at 1000 ppm. In addition, WBC counts were decreased in males and increased in females, although not statistically significant. However, neither inflammatory diseases nor histopathological changes in the hematopoietic organs, including the spleen and bone marrow, were found. Therefore, the relation between SEMHCl treatment and the above described changes was not clear. Decreases of CRN and ALT and increases in A/G, total Bil and K were considered to have no toxicological significance, because there were no abnormalities in related parameters and no significant histopathological changes were noted in the liver, kidneys and muscles. In females, decrease of TP at 1000 ppm might be involved in suppression of body weight and decrease in food consumption. Significant increases of BUN in all treated groups might have been caused by low values in the control group, because the values in the treated groups were within the normal range of background data of GALAS rats. Since the increases of IP and ALP, parameters relating to bones, were found only in females despite more severe osteochondral lesions in males, we could not clarify involvement in SEM-HCl treatment-associated pathological lesions. Significant alterations of absolute and relative weights were found in several organs at 1000 ppm, but, these might reflect body weight decrease, since no significant histopathological changes were noted in these organs. SEM-HCl treatment revealed characteristic histopathological anomalies of bone, cartilage and the elastic laminae of arteries, in line with its documented inhibition of cross-linking reactions of collagen and elastin (Dawson et al., 2002) and induction of osteochondral and vascular lesions (Ramamurti and Taylor, 1959; Langford et al., 1999; Mercier et al., 2007). Loss of bone mass and changes in the microarchitecture of the collagen fibers in lathyritic rats were also described in a study of effects of beta-aminopropionitrile, another osteolathyrogen, on the maxillary and mandibular bones (El Rouby et al., 2008). In the aorta, alteration of elastic laminae was found to be induced by SEM due to inhibition of elastin cross-linkage, resulting in increased fragility of arterial walls (Mercier et al., 2007). In our study, a sex difference was obvious in the severity of the osteochondral lesions, with a male predominance. Osteochondral lesions were prominent in the long bones growing rapidly, such as the femur, tibia, radius and ulna. Moreover, the severities of lesions were increased in sites subject to body weight loading, as indicated by more pronounced histopathological changes in the tibia than the femur. These findings suggest reduction in the strength of cartilage due to alteration of matrix properties caused by inhibition of cross-linking reaction of collagen. Differences in body weights of males and females might thus be considered one reason for the more severe lesions in male rats. Except for the bones and joints, the incidences of mineralization in the pulmonary arteries were significantly lowered in males of
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the 250 and 500 ppm group. However, since dose-dependence and significant histopathological changes were lacking in related organs such as the parathyroids and kidneys, no treatment relation could be concluded. Chronic inflammation of the ventral prostate is known to frequently occur spontaneously in male rats, and there were no associated lesions in related organs. Therefore, the increase of chronic inflammation of the ventral prostate found at 1000 ppm can be regarded of low toxicological relevance. In addition, since cysts in the anterior lobe of the pituitary is an incidental finding observed frequently in rats, the difference in the incidence of this lesion noted in females might be of negligible significance. Taken together, toxicological effects of subchronic exposure to SEM-HCI were mainly observed in the bones, cartilages and aorta. From the histopathological examination, since disarrangement, fissures and deformation of the cartilages were found in both sexes from 250 ppm, the no-observed-adverse-effect-levels (NOAELs) estimated from present study were less than 250 ppm in both sexes, equivalent to 18.1 and 21.1 mg/kg/day in males and females. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements This work was supported by Health and Labour Sciences Research Grants (Research on Food Safety) from the Ministry of Health, Labour and Welfare of Japan. We thank Miss Ayako Kaneko for technical assistance in conducting the animal study. References Abramsson-Zetterberg, L., Svensson, K., 2005. Semicarbazide is not genotoxic in the flow cytometry-based micronucleus assay in vivo. Toxicol. Lett. 155, 211–217. Dawson, D.A., Rinaldi, A.C., Pöch, G., 2002. Biochemical and toxicological evaluation of agent-cofactor reactivity as a mechanism of action for osteolathyrism. Toxicology 177, 267–284. de la Calle, M.B., Anklam, E., 2005. Semicarbazide: occurrence in food products and state-of-the-art in analytical methods used for its determination. Anal. Bioanal. Chem. 382, 968–977. de la Fuente del Rey, M., 1986. Teratogenic effect of semicarbazide in Wistar rats. Biol. Neonate 49, 150–157. Effkemann, S., Feldhusen, F., 2004. Triple-quadrupole LC–MS–MS for quantitative determination of nitrofuran metabolites in complex food matrixes. Anal. Bioanal. Chem. 378, 842–844. EFSA, 2003. Statement of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food Updating the Advice Available on Semicarbazide in Packaged Foods (Adopted on 1 October 2003). EFSA, 2005. Opinion of the scientific panel on food additives, flavourings, processing aids and materials in contact with food on a request made from the commission related to semicarbazide in food. EFSA J. 219, 1–36. El Rouby, D.H., Bashir, M.H., Korany, N.S., 2008. Ultrastructural and histomorphometric alterations of rat jaw bones after experimental induction of lathyrism. Arch. Oral Biol. 53, 916–923. FSC, 2007. The 68th Expert Committee of Veterinary Medicines. Available at:
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