Arch Toxicol (2008) 82:771–777 DOI 10.1007/s00204-008-0293-y

G E N O T O X I C I T Y A N D CA R C I N O G E N I CI T Y

Extremely weak tumor-promoting eVect of troglitazone on splenic hemangiosarcomas in rasH2 mice induced by urethane Meilan Jin · Sayaka Matsumoto · Yasuaki Dewa · Jihei Nishimura · Yukie Saekusa · Keiji Hasumi · Kunitoshi Mitsumori

Received: 22 November 2007 / Accepted: 3 March 2008 / Published online: 9 May 2008 © Springer-Verlag 2008

Abstract To examine the tumor-promoting eVect of troglitazone (TRG), a novel thiazolidinedione insulinsensitizing agent, on splenic hemangiosarcomas in rasH2 mice, histopathological and molecular analyses were performed in the spleen of female rasH2 mice fed a diet containing 6,000 or 0 ppm TRG for 16 weeks after 1,000 or 0 mg/kg urethane (UR) initiation. Histopathologically, splenic hemangiosarcomas were observed in the UR-alone and UR + TRG groups, but there was no signiWcant diVerence in the incidence of splenic hemangiosarcomas between the UR-alone and UR+TRG groups. There were increasing tendencies in the number of positive cells for anti-PCNA antibody and gene expression in the UR + TRG group, but such a change was not statistically signiWcant as compared to that in the UR-alone group. The gene expressions of VEGF, VEGFR1, VEGFC, VEGFR2 and Tie2 related to angiogenesis; c-fos related to MAPK cascade activation; and cyclin D1 related to cell cycle in the URalone and UR + TRG groups were signiWcantly higher than

M. Jin (&) · S. Matsumoto · Y. Dewa · J. Nishimura · Y. Saekusa · K. Mitsumori Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan e-mail: [email protected] M. Jin · K. Hasumi Department of Applied Biological Science, United Graduate School of Agricultural Sciences, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan Y. Dewa · J. Nishimura Pathogenetic Veterinary Science, United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193, Japan

those in the untreated control group. However, only the Tie2 gene in the UR + TRG group was signiWcantly increased as compared to that in the UR-alone group. These results suggest that the vascular tumor-promoting activity of TRG in rasH2 mice is extremely low in the present experimental condition and a part of the gene related to angiogenesis probably contributes to the promotion of splenic hemangiosarcomas in rasH2 mice given TRG. Keywords Troglitazone · PPAR agonist · Urethane · rasH2 mouse

Introduction Thiazolidinediones bind and activate the peroxisome proliferator-activated receptor
(PPAR
), enhance insulin action, and inhibit hepatic gluconeogenesis (Keller et al. 1993; Saltiel et al. 1995; Saltiel et al. 1996; Tontonoz et al. 1994). Troglitazone (TRG), a PPAR
agonist, is a novel thiazolidinedione insulin-sensitizing agent used for the treatment of non-insulin-dependent diabetes mellitus, but it was withdrawn from the market in 2000 because of the occurrence of rare idiosyncratic hepatotoxic eVects with its usage (Ghazzi et al. 1997). In 2-year carcinogenicity studies of TRG in B6C3F1 mice and Wistar rats (Herman et al. 2002), elevated incidences of malignant vascular tumors (hemangiosarcomas) were noted in TRG-treated mice, but not in rats. The rasH2 mice are hemizygous transgenic carrying the human prototype c-Ha-ras gene with its own promoter and enhancer, and they have been widely used in many 6-month short-term carcinogenicity tests. So far, it is well recognized that these mice are not only susceptible to genotoxic carcinogens, but also to some non-genotoxic carcinogens

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such as PPAR agonists (Yamamoto et al. 1996; NesWeld et al. 2005; Toyosawa et al. 2001). Furthermore, in our 6-month short-term carcinogenicity test of TRG in rasH2 mice, hemangiomas and hemangiosarcomas were found in six females (30%) in the 6,000 ppm TRG group. However, there was no signiWcant diVerence in the incidence of these vascular tumors between the control and the 6,000 ppm groups because of the spontaneous occurrence of hemangioma in a female of the control group. However, since early short-term carcinogenicity studies of rasH2 mice conducted from 1991 to 1995 reported that the incidences of spontaneous tumors were generally low, the highest incidences being those of splenic hemangiosarcomas in females (7.47%; Mitsumori 2003), we have concluded that vascular tumors in females in the 6,000 ppm group are attributable to the treatment of TRG, while the carcinogenic susceptibility of rasH2 mice to TRG is relatively low (Jin et al. 2007). In addition, it has been reported that single or multiple intraperitoneal injection of urethane (UR), a well-known lung carcinogen, at a dose of 1,000 mg/kg body weight induces lung tumors and splenic hemangiosarcomas in rasH2 mice (Mitsumori et al. 1997, 1998; Mori et al. 2000; Okamura et al. 2006). Accordingly, the vascular tumor-promoting activity of TRG in rasH2 mice can be investigated in a twostage splenic vascular tumorigenesis model of rasH2 mice given UR as an initiator. In the present study, to examine the tumor-promoting eVect of TRG on splenic hemangiosarcomas in rasH2 mice, histopathological and molecular analyses were performed in the spleen of female rasH2 mice fed a diet containing TRG after UR initiation treatment.

Materials and methods

Arch Toxicol (2008) 82:771–777

of the Faculty of Agriculture, Tokyo University of Agriculture and Technology. Experimental design After a 1-week acclimatization period, a total of 30 rasH2 mice were randomly divided into three groups. To initiate spleen vascular tumorigenesis, 25 rasH2 mice were given two intraperitoneal injections of 1,000 mg/kg urethane (Nacalai Tesque Inc., Kyoto, Japan) in physiological saline at 2-day intervals. One week after the urethane injection, mice were fed a diet containing TRG at a concentration of 0 or 6,000 ppm and tap water ad libitum for 16 weeks. Five mice were not subjected to any treatment; these mice served as untreated controls. The dosage in our study was selected based on our previous study (Jin et al. 2007) and the information on 2-year carcinogenicity studies of TRG performed in B6C3F1 mice and Wistar rats (Herman et al. 2002), which described that splenic hemangiosarcomas were induced by the oral administration of 800 mg/kg/day of TRG (equivalent to 6,000 ppm in diet) in both mice sexes. Both body weight and food consumption were measured once a week. The mice were killed under ether anesthesia. Their spleens were removed and weighed. The spleen samples were cut into half and Wxed in 10% neutral buVered formalin. After the Wxation, all the sections were processed routinely, embedded in paraYn and stained with hematoxylin and eosin (HE) for histopathological examinations. We counted the total number of spleen tumors that were found in these specimens. The spleen tumors from the remaining spleen samples were removed and immersed in RNA later (Qiagen, Valencia, CA, USA). The samples were subsequently frozen at ¡80°C until subsequent RNA extraction.

Compounds RNA isolation and real-time RT-PCR analysis Troglitazone was kindly supplied by Daiichi-Sankyo Co., Ltd. (Tokyo, Japan), and stored at 4°C. Animals Female rasH2 mice (aged 6 weeks) were obtained from the Central Institute for Experimental Animals (Kanagawa, Japan), and a maximum of four mice were housed in each polycarbonated cage equipped with absorbent hardwood bedding (White Flakes, Charles River Inc., Tokyo, Japan) in an air-conditioned animal room (12/12 h light/dark cycle at a temperature of 23 § 2°C and a relative humidity of 55 § 5%). They were given a basal diet (CE-2, CLEA Japan Inc., Tokyo, Japan) and tap water ad libitum during the acclimatization period. The experiment was performed in accordance with the guidelines for animal experimentation

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The total RNA sample was isolated from four or Wve animals of each group using TRIzol reagent, according to the manufacturer’s protocol (Invitrogen, USA). Reverse transcription was carried out with 2
g of RNA for cDNA synthesis using a thermoScript RT-PCR system (Invitrogen) according to the manufacturer’s protocol. In the study, quantitative real-time RT-PCR with SYBR Green was performed using an ABI Prism 7000 Sequence Detection System (Applied Biosystems, USA) to validate the microarray (GEArray) results. The PCR primers were designed using Primer Express (Applied Biosystems). Real-time RT-PCR analyses on the angiogenesis, MAPK cascade, and cellcycle-related genes were carried out in the splenic hemangiosarcomas of female rasH2 mice from the two diVerent groups. These genes were reported to be related to the

Arch Toxicol (2008) 82:771–777

potential mechanism of vascular tumors in mice inducted by TRG in the previous studies (Herman et al. 2002; Jin et al. 2007). Histological, histochemical and immunohistochemical evaluations Formalin-Wxed spleen tissues were embedded in paraYn, sectioned and stained with hematoxylin and eosin (H&E) for the histological examinations. The number of hemangiosarcomas in each slide was counted from all rasH2 mice. Additionally, the immunohistochemical staining of proliferating cell nuclear antigen (PCNA) antibody (PC10; DakoCytomation, Japan) was performed by the avidinbiotin-peroxidase complex method. The number of PCNApositive cells per 200–300 cells in each slide was counted from Wve diVerent areas of hemangiosarcomas to obtain the PCNA-positive index (PCNA PI). Statistical analysis Statistical analyses were performed using statistical software (StatLight; Yukms Co., Ltd., Japan), and all results are presented as mean § SD. To identify the diVerences in the frequency of splenic hemangiosarcomas and the gene expression levels of real-time RT-PCR analysis between the tumors of UR-alone and the TRG-treated groups, Student’s and Welch’s t-tests were used after analysis for variances, and the P-values were calculated using MS-Excel.

Results Survival and body weights

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rates of the UR-alone and UR + TRG groups after weeks 9 and 13, respectively, decreased remarkably. The causes of death were mainly lung adenomas and/or splenic hemangiosarcomas. Therefore, we completed the experiment at 16 weeks after initiation of UR to obtain the appropriate number of surviving animals that would be suYcient for histopathological evaluations. Histopathological examinations After 16 weeks, signiWcant increases in the relative spleen weight were observed in the UR-treated groups, as compared to those in the untreated control group. However, there was no signiWcant diVerence in the spleen weight between the UR-alone and UR + TRG groups (Table 1). At necropsy, spleen and lung nodules were observed in all UR-treated mice. Histopathologically, all mice of the URtreated groups demonstrated lung proliferative lesions and splenic hemangiosarcomas (Table 2). Furthermore, the incidence of splenic hemangiosarcomas in each mouse signiWcantly increased in the UR-treated groups as compared to that in the untreated control group, but there was no signiWcant diVerence in the incidence between the UR-alone and UR + TRG groups (Table 2). Evaluation of cell proliferation by immunohistochemistry for PCNA We examined the rate of cell proliferation in the splenic hemangiosarcomas using immunohistochemical staining for PCNA (Fig. 2). The PCNA-positive indices in the UR + TRG group showed an increasing tendency as compared with those in the UR-alone group, but there was no signiWcant diVerence between the UR-alone and UR + TRG groups.

The survival rates of rasH2 mice are shown in Fig. 1. Four mice in the UR-alone group and Wve mice in the UR + TRG group died during the experimental period. The survival Table 1 Body and spleen weights of rasH2 mice in a two-stage spleen vascular tumorigenesis model of troglitazone

Fig. 1 Survival rate of rasH2 mice given TRG after injection of 1,000 mg/kg UR. The initial number of rasH2 mice in untreated, DENalone and DEN + TRG groups were 5, 10 and 15, respectively

No. of animals

Untreated control 5

UR-alone 6

UR + TRG 10

Body weight (g)

25.9 § 2.5

24.8 § 1.6

26.3 § 1.7

Absolute spleen weight (g)

183.2 § 162.1

253.2 § 105.3

203.0 § 49.7

Relative spleen weight (%)

0.4 § 0.1

1.0 § 0.5*

0.8 § 0.2##

Results are mean § SD for indicated number of animals for each group * Aspin-Welch t test signigicantly diVerent from the untreated control value at P < 0.05 ## Student’s test signiWcantly diVerent from the untreated control value at P < 0.01

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Table 2 Incidence (%) of splenic hemangiosarcoma and lung proliferative lesions in a two-stage spleen vascular tumorigenesis model in rasH2 mice given troglitazone No. of animals

Spleen

Lung

Hemangiosarcoma incidence (%)

Incidence (%) Hyperplasia

Untreated control

5

0

0

Adenoma 0

Carcinoma 0

UR-alone

6

100**

66.7

100**

0

UR + TRG

10

100***

70*

100***

10

*, **, *** SigniWcantly diVerent from the untreated control value at P < 0.05, 0.001, 0.0001 (Fisher’s exact test)

Fig. 2 EVects of TRG on the formation of hemangiosarcomas and cell proliferation in the splenic hemangiosarcomas of rasH2 mice given TRG for 16 weeks after UR initiation. a Splenic hemangiosarcoma in a rasH2 mouse of the UR-alone group. b Splenic hemangiosarcoma in a rasH2 mouse of the UR + TRG group with H&E. c, d PCNA immunostaining of splenic hemangiosarcomas. The number of PCNA-positive cells (arrows) increased in a hemangiosarcoma of the UR + TRG group (d), as compared to that of the UR-alone group (c). e Quantitative analysis of the number of PCNA-positive cells in hemangiosarcomas; values are expressed as mean § SD in 5 rasH2 mice

Gene expression analysis by real-time RT-PCR The results of real-time RT-PCR are shown in Fig. 3. The expressions of the following genes were signiWcantly up-regulated in the UR-alone and UR + TRG groups as compared to those in the untreated control group: vascular endothelial growth factor (VEGF); vascular endothelial

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growth factor receptor 1 (VEGFR1); vascular endothelial growth factor C (VEGF-C); angiopoietin 2 (Angpt2); protein tyrosine kinase Tie 2 (Tie2) related to blood vessel formation; Ha-ras and c-fos related to MAPK cascade activation; and cyclin D1 and transforming growth factor (TGF-) related to cell cycle or cell proliferation. However, only the Tie2 gene in the UR + TRG group

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Fig. 3 The expression level, which was obtained from real-time RTPCR data of genes selected as candidates involved in the enhanced carcinogenesis in rasH2 mice. The gene expression level was normalized by -actin. The data are given as mean § SD. *, **, ***: signiWcantly

diVerent from the normal spleen tissue at P < 0.05, P < 0.01 or P < 0.001, respectively, by the Student’s t-test. #, ##, ###: signiWcantly diVerent from the normal spleen tissue at P < 0.05, P < 0.01 or P < 0.001, respectively, by the Welch’s t-test

increased signiWcantly as compared to that in the UR-alone group.

margins of safety to support continued clinical development with some members of this class of compounds (Elangbam et al. 2002). Based on this information, the new guidelines provided by the FDA for compounds in this class indicate that the carcinogenic potential of these PPAR agonists cannot be evaluated in TG or KO mice, and clinical studies longer than 6 months in duration cannot be initiated until 2-year rodent carcinogenicity studies are completed and submitted for agency review (Jeri El-Hage 2004). On the other hand, it is unknown whether rasH2 mice were susceptible to such PPAR
or /
agonists, although it has been demonstrated that they are susceptible to PPAR agonists. This means that in the absence of a database on rasH2 mice, the FDA recognizes the insusceptibility of these TG and KO mice to PPAR agonists. The carcinogenesis of TRG has already been evaluated in a 2-year carcinogenicity study of TRG by Herman et al. (2002). According to that study, the incidences of hemangiosarcomas and hepatocellular carcinomas increased in both sexes given 800 mg/kg and females given 400 mg/kg, respectively. Furthermore, the incidence of vascular tumors was observed to increase in females of the 6,000 ppm group in our previous 6-month carcinogenicity study of rasH2 mice, although the carcinogenic potential of TRG was

Discussion According to the new guidelines of the International Conference on Harmonization of Technical Requirements of Pharmaceuticals for Human Use (ICH), the carcinogenicity of drugs can be evaluated based on the data of 6-month carcinogenicity studies using transgenic (TG) or knockout (KO) mice in addition to using data of a 2-year conventional carcinogenicity study of one rodent species (D’Arcy and Harron 1998). Thus far, many 6-month carcinogenicity studies using p53 KO mice have been conducted for newly developed drugs in accordance with FDA recommendations. The FDA conWrmed that p53 KO mice are not susceptible to non-genotoxic carcinogens, but rather to genotoxic carcinogens, despite the fact that positive results were obtained in 2-year carcinogenicity studies that were conducted on these non-genotoxic carcinogens in rodents (Storer et al. 2001). Accumulated rodent data reviewed by the FDA for a number of PPAR
or /
agonists showed carcinogenicity Wndings that did not demonstrate adequate

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relatively weak in rasH2 mice (Jin et al. 2007). In the present study, the incidence of splenic hemangiosarcomas signiWcantly increased in the UR-treated groups, but there was no signiWcant diVerence in their incidence between the URalone and UR + TRG groups. In addition, the gene expressions of VEGF, VEGFR1, VEGFC, VEGFR2 and Tie2 related to angiogenesis; c-fos related to MAPK cascade activation; and cyclin D1 related to cell cycle in the URtreated groups were signiWcantly higher than those in the untreated control group. However, only the Tie2 gene in the UR + TRG group signiWcantly increased as compared to that in the UR-alone group. Angiopoietins (Angpt), which were recently described as growth factors for the vascular endothelium, bind to Tie receptors (Tie1 or Tie2) and play a role in the pathobiology of angiosarcomas (Brown et al. 2000). In addition, a previous study provided evidence that VEGF regulates angiopoetin-Tie2 signaling by inducing proteolytic cleavage and Tie2 shedding via a novel PI3k/ akt-dependent pathway and that it may inhibit vascular stabilization to promote angiogenesis and vascular remodeling (Findley et al. 2007). This up-regulation of Tie2 in the UR + TRG group suggests that the induction of splenic hemangiosarcomas in rasH2 mice induced by UR is slightly enhanced by the administration of TRG, although the vascular tumor-promoting eVect of TRG is extremely low. In gene expression analysis by real-time RT-PCR, the gene expressions of VEGF, VEGFR1, VEGF-C and VEGFR2 related to blood vessel formation, increased signiWcantly in the microarray analysis of a 6-month carcinogenicity study using rasH2 mice. However, the expressions did not show any signiWcant increase in the UR + TRG group as compared to those in the UR-alone group. Herman et al. (2002) reported that one potential mechanism of vascular tumor induction is based on the constitutive synthesis of angiogenic growth factors, such as the basic Wbroblast growth factor (bFGF) and VEGF in adipocytes. Furthermore, these angiogenic factors, in turn, could exert trophic eVects on endothelial cells, leading to angiogenesis, endothelial cell proliferation, increased mutational frequency and eventually the neoplastic transformation of endothelial cells. These could result in clonal expansion and tumor formation. Accordingly, the Wndings of the literature described above may suggest that the increased expressions of these angiogenic growth factors are probably responsible for the development of splenic hemangiosarcomas in mice given TRG. However, since such genes relating to angiogenic growth factors in the UR + TRG group were not signiWcantly higher than those in the UR-alone group, the reason why vascular tumorpromoting eVect is extremely low in the present study should be clariWed. In the present study, multiple lung adenomas were observed in all rasH2 mice of UR-treated groups, and some

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of the mice died by the progression of these lung tumors. It has been reported that splenic hemangiosarcomas are also frequently induced by UR and its metabolite vinyl carbamate (Mitsumori et al. 1997; Ozaki et al. 2005). Furthermore, Okamura et al. (2006) reported that splenic hemangiosarcomas were observed in 80% of the mice that were reared without any treatment for 10 weeks after an i.p. injection of UR. These results suggest that the initiation activity of UR, inducing lung and splenic tumors, is too strong to examine the vascular tumor-promoting eVects of TRG. Therefore, further studies by changing the initiator are necessary to clarify the vascular tumor-promoting mechanism of TG in rasH2 mice. In conclusion, the results of the present study suggest that the vascular tumor-promoting eVect of TRG in rasH2 mice is extremely low in the present experimental condition. Although this Wnding indicates that TG and KO mice are not susceptible to PPAR
agonists, as pointed out by the FDA, further studies are necessary to increase the number of validation studies on rasH2 mice using other PPAR agonists. Acknowledgments This work was supported in part by grants-in-aid from the Ministry of Health, Labor and Welfare and the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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