Different Inhibitory Effects in the Early and Late Phase of Treatment with KAT-681, a Liver-Selective Thyromimetic, on Rat Hepatocarcinogene
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《毒物学科学杂志》
Toxicology Laboratories, Research and Development, Kissei Pharmaceutical Co. Ltd., Hotaka, Minamiazumi-gun, Nagano, Japan
United Graduate School of Veterinary Science, Gifu University, Gifu, Japan
Central Research Laboratories, Research and Development, Kissei Pharmaceutical Co. Ltd., Hotaka, Minamiazumi-gun, Nagano, Japan
Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan
ABSTRACT
We recently reported that short-term treatment with KAT-681 (KAT), a liver-selective thyromimetic, inhibits the development of preneoplastic lesions in rat livers and may be a candidate chemopreventive agent for hepatocarcinogenesis. In this study, time-course observations of hepatocellular proliferative lesions were carried out during short-term and long-term treatment with KAT to investigate its anti-hepatocarcinogenic effects. The hepatocellular proliferative lesions in male F344 rats were induced by the initiation treatment of diethylnitrosamine (DEN), followed by treatment with 2-acetylaminofluorene (2-AAF) and partial hepatectomy (PH). The rats were administered KAT orally at a dose of 0.25 mg/kg/day for 3 weeks (experiment 1) or 0.1 mg/kg/day for 20 weeks (experiment 2). In experiment 1, a serial reduction in the number of altered hepatocellular foci (AHF) with positive expression of glutathione S-transferase placental form (GST-P) was observed until day 14 of the treatment period. The proliferative index (PI) of hepatocytes in the AHF significantly increased in the KAT group throughout the treatment period, with a peak on day 2. KAT treatment showed no obvious effects on GST-P–positive hepatocellular adenomas (HCAs) at any time point. In contrast, long-term KAT treatment in experiment 2 revealed a reduction in the mean size of HCAs in addition to reductions in the number and mean size of AHF. The PIs within the lesions in KAT-treated rats were significantly lower than those in controls. The present study indicates that KAT has different inhibitory effects on hepatocarcinogenesis in the early and late phases of KAT treatment; there is a reduction in AHF with enhanced cell proliferation in the early phase and the inhibition of development of AHF and HCAs with suppression of cell proliferation in the late phase. These results may suggest further potential of KAT as a promising chemopreventive agent for hepatocarcinogenesis.
Key Words: hepatocarcinogenesis; thyromimetic; thyroid hormone; chemoprevention; cell proliferation; rat.
INTRODUCTION
Hepatocellular carcinoma (HCC) ranks fifth in frequency among all human malignant tumors in the world and causes 1 million deaths annually (Yu and Keeffe, 2003). Despite the development of advanced therapies, high recurrence and poor prognosis are frequently observed in HCC patients, and effective chemopreventive agents are needed. Clinical studies of HCC chemoprevention have been performed in Japan with compounds such as interferon (Nishiguchi et al., 1995), sho-saiko-to (Oka et al., 1995), glycyrrhizin (Arase et al., 1997), and acyclic retinoid (Muto et al., 1996). However, all these compounds have been reported to decrease the incidence of lesions, but no consensus on their efficacy has been reached.
Recently, Ledda-Columbano et al. (2000) reported modifying effects of triiodothyronine (T3) on hepatocarcinogenesis induced by carcinogens in rats. They showed that the incidence of hepatocellular carcinomas and metastasis to the lung is reduced in rats fed with a T3-supplemented diet after diethylnitrosamine (DEN) administration followed by 2-acetyl-aminofluorene (2-AAF) treatment and partial hepatectomy (PH). These results are interesting because they suggest a utility for thyromimetics as chemopreventive agents against hepatocarcinogenesis, although the anticarcinogenic mechanisms of T3 remain unclear.
Through their nuclear receptors (TRs), thyroid hormones (THs) influence a variety of physiological functions, including general metabolism, growth, development, and differentiation (Cheng, 1995; Oppenheimer et al., 1994). Although a decrease in plasma cholesterol is one of these effects, thyroid hormone is not used to treat hypercholesterolemia because of its cardiotoxic effects (Toft and Boon, 2000). However, some thyromimetics that decrease plasma cholesterol levels without increasing cardiac activity have been reported to be potent hypocholesterolemic agents (Grover et al., 2003; Taylor et al., 1997; Underwood et al., 1986).
KAT-681 (KAT) is also a novel liver-selective thyromimetic with the hypocholesterolemic property and without the cardiotoxicity. Our previous study showed that KAT treatment for 3 weeks in rats inhibited the development of preneoplastic lesions induced by DEN and 2-AAF treatment along with PH, and it suggested that KAT may be a promising chemopreventive agent for hepatocarcinogenesis (Hayashi et al., 2004). In the present study, to obtain further information regarding the effects of KAT on the preneoplastic lesions and more advanced tumors, we performed time-course observations during short-term and long-term KAT treatments on hepatocellular proliferative lesions in the same rat two-stage hepatocarcinogenesis model.
MATERIALS AND METHODS
Test substance. KAT, N-(4-{3-[(4-fluorophenyl)hydroxymethyl]-4-hydroxyphenoxy}-3, 5-dimethylphenyl) malonamic acid sodium, was synthesized at Kissei Pharmaceutical Co. Ltd. (Nagano, Japan), as a novel liver-selective thyromimetic.
Animals. Male F344 rats (6 weeks old, 100–125 g body weight) were purchased from Charles River Japan Inc. (Kanagawa, Japan). The animals were housed individually in stainless-steel cages (room temperature 21°–25°C, relative humidity 42–68%, with a 12-h/12-h light/dark daily cycle) and were given access to a basal diet (CE-2; CLEA Japan Inc., Tokyo, Japan) and tap water ad libitum.
Experimental design. According to a well-known protocol for hepatocarcinogenesis (Solt and Farber, 1976), the rats were treated with a single intraperitoneal injection of DEN (Sigma Chemical Co., St. Louis, MO) dissolved in saline, at a dose of 150 mg/kg body weight. After 2 weeks, the rats were given 2-AAF (Sigma) suspended in 0.5% carboxymethylcellulose (CMC) by gavage at a dose of 7.5 mg/kg/day, twice daily for 2 weeks, and were subjected to a standard two-thirds PH on the 8th day after 2-AAF administration. KAT, dissolved in 0.5% CMC, was administered orally starting 5 weeks after the completion of 2-AAF treatment. In experiment 1 (Fig. 1A), 35 animals were allocated randomly to one untreated group and six treated groups, each consisting of five rats. KAT was administered to rats in each treated group at a dose of 0.25 mg/kg/day for 1, 2, 4, 7, 14, or 21 days. In experiment 2 Fig. 1B, 50 animals were allocated randomly to one untreated group and four treated groups, each consisting of ten rats. Two of the four treated groups were administered KAT at a dose of 0.1 mg/kg, 5 days per week, for 10 or 20 weeks, and the two remaining groups acted as controls and
Histopathology and immunohistochemistry. After the rats were sacrificed, the liver obtained from each animal was fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 3-μm thickness, and stained with hematoxylin and eosin (HE). Serial sections of H&E preparations were used for immunohistochemical staining of glutathione S-transferase placental form (GST-P) and proliferating cell nuclear antigen (PCNA).
GST-P staining. Hepatocellular proliferative lesions were detected by immunostaining using an anti-rat GST-P polyclonal antibody (Medical & Biological Laboratories, Aichi, Japan), and a DAKO LSAB Kit (DakoCytomation Inc., Carpinteria, CA). Liver sections were first immersed in 0.3% hydrogen peroxide in methanol for 10 min to block endogenous peroxidase and then incubated at room temperature with primary antibody at a dilution of 1:1000 in 1% bovine serum albumin for 40 min, followed by incubation with biotinylated secondary antibody for 10 min and the streptavidin-biotin peroxidase complex (sABC) for 10 min. Subsequently, 3, 3'-diaminobenzidine (DAB) was applied as a chromogen. The sections were finally counterstained with hematoxylin.
Double staining of GST-P and PCNA. Proliferative activities of hepatocytes within GST-P–positive lesions were detected by immunohistochemical staining with an anti-mouse PCNA monoclonal antibody (DakoCytomation A/S, Glostrup, Denmark). Liver sections were stained immunohistochemically by the procedure described above for GST-P staining, and with anti-PCNA (dilution 1:200) and DAB for color development. After blockage of endogenous biotin with the DAKO biotin-blocking system, the sections were immunostained with an anti–GST-P antibody and a DAKO fuchsin substrate-chromogen kit (DakoCytomation Co. Ltd.) to visualize the binding sites of alkaline phosphatase. The sections were finally counterstained with hematoxylin.
Quantitative analysis of hepatocellular proliferative lesions with GST-P expression. The inhibitory effects of KAT on the proliferative lesions were determined by quantitative analysis of GST-P–positive lesions with a computer-assisted image processor (LUZEX-3; Nikon Corp., Tokyo, Japan). Only lesions larger than 0.05 mm2 were measured. Ratios of the lesion areas to the total observation area were calculated, as were the values for number and mean size.
Determination of proliferative index. To determine whether KAT affects cell proliferation, the number of PCNA-positive cells was measured from the GST-P–positive lesions or from the surrounding GST-P–negative areas. The proliferative index (PI) was expressed as the percentage of positive cells after counting approximately 2000 hepatocytes in each observation area.
Determination of apoptotic index. To determine whether KAT decreases apoptosis, the incidence of apoptotic bodies was measured by microscopic identification from the proliferative lesions or from the surrounding normal areas. The apoptotic index (AI) was calculated as the number of apoptotic bodies per 1000 hepatocytes after counting approximately 2000 hepatocytes in each observation area.
Statistical analysis. All results are presented as mean ± S.E. Data were analyzed by the F-test for homogeneity of variance between the untreated group and each KAT-treated group in experiment 1 and between the control and KAT-treated groups in experiment 2. If the variance was homogeneous, the Student's t-test was applied for comparisons, and if it was heterogeneous, the Aspin-Welch's t-test was used. Differences were considered significant at p < 0.05.
RESULTS
Experiment 1
Body weight and liver weight. The absolute and relative liver weights gradually decreased in the KAT-treated groups despite the increase in body weights (Table 1). Food consumption was not measured because increased food consumption was confirmed in our previous study, in which rats were treated with KAT for 3 weeks (Hayashi et al., 2004).
Histopathology. The criteria used for histopathological evaluation of the hepatocellular proliferative lesions were the same as those in Guides for Toxicologic Pathology, published by the Society of Toxicologic Pathology (Goodman et al., 1994). In the livers of all rats, most of the proliferative lesions induced by DEN and 2-AAF treatment along with PH were altered hepatic foci (AHF) consisting of eosinophilic hepatocytes. The remainder were hepatocellular adenomas (HCAs), containing large, pale, eosinophilic cells with cellular and nuclear atypia, with compression of the adjacent parenchyma. The abundance of AHF was perceptibly reduced depending on the duration of the KAT treatment period. All of the hepatocytes with normal appearance were small in size because of the complete disappearance of intracytoplasmic glycogen.
Effects of KAT on GST-P–positive lesions. All of the AHF and HCAs observed in histopathological examinations were positive for GST-P–immunostaining, which is known as a stable marker for rat hepatocarcinogenesis (Sato, 1989; Tatematsu et al., 1988). Microscopic observations revealed that GST-P expression progressively decreased in the majority of AHF as a result of KAT treatment. The AHF were finally assimilated into the surrounding hepatocytes because of complete loss of GST-P expression (Fig. 2). Morphometrically, the total area and number, except for the mean size, of the AHF were gradually reduced until day 14 of the treatment period, and their reductions were statistically significant from days 4 or 7 to day 21 in comparison with day 0 (Fig. 3). The values of the GST-P–positive HCAs did not show any significant changes in the KAT-treated groups, except for the reduction of mean size on day 14.
Effects of KAT on PI and AI. The proliferative index was significantly increased in the AHF throughout the KAT treatment period, with a peak on day 2, while there were no changes in the HCAs (Fig. 4). The surrounding GST-P–negative areas also revealed an increase in PI from days 1 to 7. AI showed no remarkable change in all the analyzed areas throughout the observation period (Fig. 4).
Experiment 2
Body weight, food consumption, and liver weight. The expected results of a thyromimetic, increases in body weights and food consumption and decreases in liver weights, were observed on both weeks 10 and 20 (Table 2).
Histopathology. The hepatocellular proliferative lesions observed in experiment 2 were mainly AHF with some HCAs, each consisting of eosinophilic hepatocytes, which were the same as those observed in experiment 1. Hepatocellular carcinomas were observed only in one control animal at week 20. The area of normal-appearing hepatocytes with complete loss of intracytoplasmic glycogen was considerably expanded by KAT treatment after the reduction in the number of AHF.
Effects of KAT on GST-P–positive lesions. Long-term KAT treatment revealed significant decreases in the total areas and the mean sizes of both AHF and HCAs, and in the number of AHF as compared with the controls (Fig. 5). At week 20, although the mean size of the HCAs in the control group was approximately threefold larger than at week 0, the mean size in the KAT-treated group was not changed from week 0.
Effects of KAT on PI and AI. Long-term KAT treatment showed significant decreases in the PI of HCAs and the surrounding areas at week 10 and in AHF, HCAs, and the surrounding areas at week 20 (Fig. 6). With regard to the AI, no noteworthy change was observed in all the analyzed areas throughout the observation period (Fig. 6).
DISCUSSION
Our previous study demonstrated that short-term KAT treatment inhibits the development of preneoplastic lesions but not that of neoplastic lesions in rat liver (Hayashi et al., 2004). In the present study, we examined the anticarcinogenic effects in short-term and long-term KAT treatment of preneoplastic lesions, as well as more advanced tumors in the rat liver, which are induced by DEN and 2-AAF treatment along with PH. In the short-term KAT treatment of 3 weeks duration (experiment 1), the GST-P–positive AHF, which are putative preneoplastic lesions, were continuously reduced in total area and number but not in mean size, from day 1 to day 14 of the treatment period. The time-dependent quantitative reductions in GST-P–positive AHF in the KAT-treated groups were not accompanied by enhanced apoptosis. These results appear to support our previous hypothesis, which was the remodeling of altered hepatocytes through redifferentiation, for explaining mechanisms of the possible anticarcinogenic effects of KAT.
The proliferative activity was significantly increased in the AHF and the surrounding areas of the KAT-treated group in experiment 1 during the continuous reductions in quantitative values of the AHF. This result is attributed to the thyromimetic action of KAT because T3 is known to be a powerful inducer of hepatocyte proliferation in rats (Francavilla et al., 1994). Ledda-Columbano et al. (2000), have demonstrated anti-hepato carcinogenic effects of a hepatomitogen T3, suggested that cell proliferation per se might play an important role in redifferentiation of preneoplastic cells. Peroxisome proliferators (PPs) and retinoids are also known to be mitogens for hepatocytes (Pibiri et al., 2001; Ohmura et al., 1996) and agents that can reduce carcinogen-induced preneoplastic lesions, identified by GST-P and GGT staining in the rat liver (Bana et al., 1991; Chen et al., 1994; Hosokawa et al., 1989; Moriwaki et al., 1988). In particular, it has been reported that an acyclic retinoid can clinically prevent second primary hepatoma through effects on cellular differentiation (Moriwaki et al., 1997). Considering that all of these receptors, including TRs, belong to the same superfamily of steroid hormone nuclear receptors (Kumar and Thompson, 1999), the proliferative effects per se through these nuclear receptors might cause the reduction of preneoplastic lesions with GST-P expression, probably through the redifferentiation of altered hepatocytes.
In the KAT-treated group of experiment 1, GST-P–positive HCAs were hardly changed in the morphometric analysis and the determination of PI. Considering the relationship between cell proliferation and quantitative reduction of preneoplastic lesions by KAT treatment, it is interesting to note that the proliferative effects, which apparently are among the essential characteristics of KAT, were not present in the HCAs. Although it is unclear why KAT does not provide the proliferative stimuli to hepatocytes progressing from the preneoplastic to the neoplastic phenotype, there appears to be the following possibility: loss or mutation of certain genes related to the signaling pathway of KAT, including TRs themselves. Indeed, high prevalence of mutation of TRs has been found in the tumors of patients with hepatocellular carcinomas (Lin et al., 1999). Further studies are now in progress in our laboratory to clarify the mechanisms responsible for the unresponsiveness of neoplastic hepatocytes to the proliferative stimuli of KAT.
In contrast, experiment 2 showed that long-term KAT treatment for 20 weeks significantly decreased the mean size of HCAs and reduced the number and mean size of AHF. Each PI within these lesions was significantly lower than that of the controls. These results suggest that the antitumorigenic effects of KAT in long-term treatment are caused by suppression of hepatocellular proliferation and are different from the early effects of KAT, evidenced by the relationship between the reduced number of AHF and the enhanced cell proliferation activity observed in the KAT-treated group. In the present study, the hepatocytes with normal appearance were decreased in size because of the loss of intracytoplasmic glycogen as a result of the thyromimetic properties of KAT. These histopathological findings are similar to those of the liver in rats fasted for a long time (Den Otter and De Minjer, 1972; Babcock and Cardell, 1974), while food consumption was increased in the KAT-treated rats. Dietary energy restriction is known to be a potent and reproducible inhibitor of carcinogenesis at a wide variety of sites (Birt et al., 1998). Furthermore, cell replication control as one of the mechanisms of the anticarcinogenic effects has been shown in several tissues, including the liver (Grasl-Kraupp et al., 1994; Lok et al., 1990). Therefore, the late-phase effects of long-term KAT treatment, characterized by the inhibition of cell replication, might be due to long continuance of low energy status in the liver caused by increased glycolysis, which is a thyromimetic property.
In the present study, KAT inhibited the development of hepatocellular proliferative lesions with a histologically eosinophilic phenotype and GST-P expression. Although PPs also have an inhibitory effects on these lesions, as described above, PPs themselves induce GST-P–negative and GGT-negative preneoplastic and neoplastic lesions in rat livers consisting of basophilic cells (Chen et al., 1994; Hosokawa et al., 1989; Rao et al., 1982). Besides, Lee (2000) mentioned that phenobarbital (PB), a representative promoter of hepatocarcinogenesis, has paradoxical effects depending on the histological phenotypes of DEN-induced lesions on mouse hepatocarcinogenesis. Such effects raise serious issues regarding extrapolation of experimental data from laboratory animals to human risk assessment. These results exhibit the necessity for careful consideration in determining the anticarcinogenic effects of KAT, because, in particular, the THs resemble the PPs; they are ligands of a nuclear receptor of the same superfamily of nuclear receptors and require the same dimerization partner that affects gene transcription such as the 9-cis retinoic acid receptor (Corton et al., 2000; Juge-Aubry et al., 1995). In addition, a good correlation exists between the alteration of the TH status and hepatomegaly caused by PPs (Miller et al., 2001; Wang et al., 2004). Nevertheless, in this study, KAT showed no inductions of the proliferative lesions with the basophilic phenotype and without GST-P expression. Additionally, it has been shown that the anticarcinogenic effects of T3 are not exhibited by a PP ciprofibrate (Ledda-Columbano et al., 2003). Thus, the TR-mediated effects appear to be different from those of PPs and PB in this important characteristic.
In conclusion, the present study indicates that a liver-selective thyromimetic KAT induces a reduction in hepatic preneoplastic lesions in rats, probably through redifferentiation of the altered hepatocytes caused by enhanced cell proliferation in the early phase of the KAT treatment and, later, through inhibition of the development of hepatic preneoplastic lesions and hepatocellular adenomas. Such inhibition in the late phase of KAT treatment is probably due to the suppression of cell proliferation. These results suggest that KAT may have potential as a chemopreventive agent for hepatocarcinogenesis.
ACKNOWLEDGMENTS
We are grateful to Kaori Kobayashi, Rie Nakano, and Miyuki Uehara for their technical assistance.
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United Graduate School of Veterinary Science, Gifu University, Gifu, Japan
Central Research Laboratories, Research and Development, Kissei Pharmaceutical Co. Ltd., Hotaka, Minamiazumi-gun, Nagano, Japan
Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan
ABSTRACT
We recently reported that short-term treatment with KAT-681 (KAT), a liver-selective thyromimetic, inhibits the development of preneoplastic lesions in rat livers and may be a candidate chemopreventive agent for hepatocarcinogenesis. In this study, time-course observations of hepatocellular proliferative lesions were carried out during short-term and long-term treatment with KAT to investigate its anti-hepatocarcinogenic effects. The hepatocellular proliferative lesions in male F344 rats were induced by the initiation treatment of diethylnitrosamine (DEN), followed by treatment with 2-acetylaminofluorene (2-AAF) and partial hepatectomy (PH). The rats were administered KAT orally at a dose of 0.25 mg/kg/day for 3 weeks (experiment 1) or 0.1 mg/kg/day for 20 weeks (experiment 2). In experiment 1, a serial reduction in the number of altered hepatocellular foci (AHF) with positive expression of glutathione S-transferase placental form (GST-P) was observed until day 14 of the treatment period. The proliferative index (PI) of hepatocytes in the AHF significantly increased in the KAT group throughout the treatment period, with a peak on day 2. KAT treatment showed no obvious effects on GST-P–positive hepatocellular adenomas (HCAs) at any time point. In contrast, long-term KAT treatment in experiment 2 revealed a reduction in the mean size of HCAs in addition to reductions in the number and mean size of AHF. The PIs within the lesions in KAT-treated rats were significantly lower than those in controls. The present study indicates that KAT has different inhibitory effects on hepatocarcinogenesis in the early and late phases of KAT treatment; there is a reduction in AHF with enhanced cell proliferation in the early phase and the inhibition of development of AHF and HCAs with suppression of cell proliferation in the late phase. These results may suggest further potential of KAT as a promising chemopreventive agent for hepatocarcinogenesis.
Key Words: hepatocarcinogenesis; thyromimetic; thyroid hormone; chemoprevention; cell proliferation; rat.
INTRODUCTION
Hepatocellular carcinoma (HCC) ranks fifth in frequency among all human malignant tumors in the world and causes 1 million deaths annually (Yu and Keeffe, 2003). Despite the development of advanced therapies, high recurrence and poor prognosis are frequently observed in HCC patients, and effective chemopreventive agents are needed. Clinical studies of HCC chemoprevention have been performed in Japan with compounds such as interferon (Nishiguchi et al., 1995), sho-saiko-to (Oka et al., 1995), glycyrrhizin (Arase et al., 1997), and acyclic retinoid (Muto et al., 1996). However, all these compounds have been reported to decrease the incidence of lesions, but no consensus on their efficacy has been reached.
Recently, Ledda-Columbano et al. (2000) reported modifying effects of triiodothyronine (T3) on hepatocarcinogenesis induced by carcinogens in rats. They showed that the incidence of hepatocellular carcinomas and metastasis to the lung is reduced in rats fed with a T3-supplemented diet after diethylnitrosamine (DEN) administration followed by 2-acetyl-aminofluorene (2-AAF) treatment and partial hepatectomy (PH). These results are interesting because they suggest a utility for thyromimetics as chemopreventive agents against hepatocarcinogenesis, although the anticarcinogenic mechanisms of T3 remain unclear.
Through their nuclear receptors (TRs), thyroid hormones (THs) influence a variety of physiological functions, including general metabolism, growth, development, and differentiation (Cheng, 1995; Oppenheimer et al., 1994). Although a decrease in plasma cholesterol is one of these effects, thyroid hormone is not used to treat hypercholesterolemia because of its cardiotoxic effects (Toft and Boon, 2000). However, some thyromimetics that decrease plasma cholesterol levels without increasing cardiac activity have been reported to be potent hypocholesterolemic agents (Grover et al., 2003; Taylor et al., 1997; Underwood et al., 1986).
KAT-681 (KAT) is also a novel liver-selective thyromimetic with the hypocholesterolemic property and without the cardiotoxicity. Our previous study showed that KAT treatment for 3 weeks in rats inhibited the development of preneoplastic lesions induced by DEN and 2-AAF treatment along with PH, and it suggested that KAT may be a promising chemopreventive agent for hepatocarcinogenesis (Hayashi et al., 2004). In the present study, to obtain further information regarding the effects of KAT on the preneoplastic lesions and more advanced tumors, we performed time-course observations during short-term and long-term KAT treatments on hepatocellular proliferative lesions in the same rat two-stage hepatocarcinogenesis model.
MATERIALS AND METHODS
Test substance. KAT, N-(4-{3-[(4-fluorophenyl)hydroxymethyl]-4-hydroxyphenoxy}-3, 5-dimethylphenyl) malonamic acid sodium, was synthesized at Kissei Pharmaceutical Co. Ltd. (Nagano, Japan), as a novel liver-selective thyromimetic.
Animals. Male F344 rats (6 weeks old, 100–125 g body weight) were purchased from Charles River Japan Inc. (Kanagawa, Japan). The animals were housed individually in stainless-steel cages (room temperature 21°–25°C, relative humidity 42–68%, with a 12-h/12-h light/dark daily cycle) and were given access to a basal diet (CE-2; CLEA Japan Inc., Tokyo, Japan) and tap water ad libitum.
Experimental design. According to a well-known protocol for hepatocarcinogenesis (Solt and Farber, 1976), the rats were treated with a single intraperitoneal injection of DEN (Sigma Chemical Co., St. Louis, MO) dissolved in saline, at a dose of 150 mg/kg body weight. After 2 weeks, the rats were given 2-AAF (Sigma) suspended in 0.5% carboxymethylcellulose (CMC) by gavage at a dose of 7.5 mg/kg/day, twice daily for 2 weeks, and were subjected to a standard two-thirds PH on the 8th day after 2-AAF administration. KAT, dissolved in 0.5% CMC, was administered orally starting 5 weeks after the completion of 2-AAF treatment. In experiment 1 (Fig. 1A), 35 animals were allocated randomly to one untreated group and six treated groups, each consisting of five rats. KAT was administered to rats in each treated group at a dose of 0.25 mg/kg/day for 1, 2, 4, 7, 14, or 21 days. In experiment 2 Fig. 1B, 50 animals were allocated randomly to one untreated group and four treated groups, each consisting of ten rats. Two of the four treated groups were administered KAT at a dose of 0.1 mg/kg, 5 days per week, for 10 or 20 weeks, and the two remaining groups acted as controls and
Histopathology and immunohistochemistry. After the rats were sacrificed, the liver obtained from each animal was fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 3-μm thickness, and stained with hematoxylin and eosin (HE). Serial sections of H&E preparations were used for immunohistochemical staining of glutathione S-transferase placental form (GST-P) and proliferating cell nuclear antigen (PCNA).
GST-P staining. Hepatocellular proliferative lesions were detected by immunostaining using an anti-rat GST-P polyclonal antibody (Medical & Biological Laboratories, Aichi, Japan), and a DAKO LSAB Kit (DakoCytomation Inc., Carpinteria, CA). Liver sections were first immersed in 0.3% hydrogen peroxide in methanol for 10 min to block endogenous peroxidase and then incubated at room temperature with primary antibody at a dilution of 1:1000 in 1% bovine serum albumin for 40 min, followed by incubation with biotinylated secondary antibody for 10 min and the streptavidin-biotin peroxidase complex (sABC) for 10 min. Subsequently, 3, 3'-diaminobenzidine (DAB) was applied as a chromogen. The sections were finally counterstained with hematoxylin.
Double staining of GST-P and PCNA. Proliferative activities of hepatocytes within GST-P–positive lesions were detected by immunohistochemical staining with an anti-mouse PCNA monoclonal antibody (DakoCytomation A/S, Glostrup, Denmark). Liver sections were stained immunohistochemically by the procedure described above for GST-P staining, and with anti-PCNA (dilution 1:200) and DAB for color development. After blockage of endogenous biotin with the DAKO biotin-blocking system, the sections were immunostained with an anti–GST-P antibody and a DAKO fuchsin substrate-chromogen kit (DakoCytomation Co. Ltd.) to visualize the binding sites of alkaline phosphatase. The sections were finally counterstained with hematoxylin.
Quantitative analysis of hepatocellular proliferative lesions with GST-P expression. The inhibitory effects of KAT on the proliferative lesions were determined by quantitative analysis of GST-P–positive lesions with a computer-assisted image processor (LUZEX-3; Nikon Corp., Tokyo, Japan). Only lesions larger than 0.05 mm2 were measured. Ratios of the lesion areas to the total observation area were calculated, as were the values for number and mean size.
Determination of proliferative index. To determine whether KAT affects cell proliferation, the number of PCNA-positive cells was measured from the GST-P–positive lesions or from the surrounding GST-P–negative areas. The proliferative index (PI) was expressed as the percentage of positive cells after counting approximately 2000 hepatocytes in each observation area.
Determination of apoptotic index. To determine whether KAT decreases apoptosis, the incidence of apoptotic bodies was measured by microscopic identification from the proliferative lesions or from the surrounding normal areas. The apoptotic index (AI) was calculated as the number of apoptotic bodies per 1000 hepatocytes after counting approximately 2000 hepatocytes in each observation area.
Statistical analysis. All results are presented as mean ± S.E. Data were analyzed by the F-test for homogeneity of variance between the untreated group and each KAT-treated group in experiment 1 and between the control and KAT-treated groups in experiment 2. If the variance was homogeneous, the Student's t-test was applied for comparisons, and if it was heterogeneous, the Aspin-Welch's t-test was used. Differences were considered significant at p < 0.05.
RESULTS
Experiment 1
Body weight and liver weight. The absolute and relative liver weights gradually decreased in the KAT-treated groups despite the increase in body weights (Table 1). Food consumption was not measured because increased food consumption was confirmed in our previous study, in which rats were treated with KAT for 3 weeks (Hayashi et al., 2004).
Histopathology. The criteria used for histopathological evaluation of the hepatocellular proliferative lesions were the same as those in Guides for Toxicologic Pathology, published by the Society of Toxicologic Pathology (Goodman et al., 1994). In the livers of all rats, most of the proliferative lesions induced by DEN and 2-AAF treatment along with PH were altered hepatic foci (AHF) consisting of eosinophilic hepatocytes. The remainder were hepatocellular adenomas (HCAs), containing large, pale, eosinophilic cells with cellular and nuclear atypia, with compression of the adjacent parenchyma. The abundance of AHF was perceptibly reduced depending on the duration of the KAT treatment period. All of the hepatocytes with normal appearance were small in size because of the complete disappearance of intracytoplasmic glycogen.
Effects of KAT on GST-P–positive lesions. All of the AHF and HCAs observed in histopathological examinations were positive for GST-P–immunostaining, which is known as a stable marker for rat hepatocarcinogenesis (Sato, 1989; Tatematsu et al., 1988). Microscopic observations revealed that GST-P expression progressively decreased in the majority of AHF as a result of KAT treatment. The AHF were finally assimilated into the surrounding hepatocytes because of complete loss of GST-P expression (Fig. 2). Morphometrically, the total area and number, except for the mean size, of the AHF were gradually reduced until day 14 of the treatment period, and their reductions were statistically significant from days 4 or 7 to day 21 in comparison with day 0 (Fig. 3). The values of the GST-P–positive HCAs did not show any significant changes in the KAT-treated groups, except for the reduction of mean size on day 14.
Effects of KAT on PI and AI. The proliferative index was significantly increased in the AHF throughout the KAT treatment period, with a peak on day 2, while there were no changes in the HCAs (Fig. 4). The surrounding GST-P–negative areas also revealed an increase in PI from days 1 to 7. AI showed no remarkable change in all the analyzed areas throughout the observation period (Fig. 4).
Experiment 2
Body weight, food consumption, and liver weight. The expected results of a thyromimetic, increases in body weights and food consumption and decreases in liver weights, were observed on both weeks 10 and 20 (Table 2).
Histopathology. The hepatocellular proliferative lesions observed in experiment 2 were mainly AHF with some HCAs, each consisting of eosinophilic hepatocytes, which were the same as those observed in experiment 1. Hepatocellular carcinomas were observed only in one control animal at week 20. The area of normal-appearing hepatocytes with complete loss of intracytoplasmic glycogen was considerably expanded by KAT treatment after the reduction in the number of AHF.
Effects of KAT on GST-P–positive lesions. Long-term KAT treatment revealed significant decreases in the total areas and the mean sizes of both AHF and HCAs, and in the number of AHF as compared with the controls (Fig. 5). At week 20, although the mean size of the HCAs in the control group was approximately threefold larger than at week 0, the mean size in the KAT-treated group was not changed from week 0.
Effects of KAT on PI and AI. Long-term KAT treatment showed significant decreases in the PI of HCAs and the surrounding areas at week 10 and in AHF, HCAs, and the surrounding areas at week 20 (Fig. 6). With regard to the AI, no noteworthy change was observed in all the analyzed areas throughout the observation period (Fig. 6).
DISCUSSION
Our previous study demonstrated that short-term KAT treatment inhibits the development of preneoplastic lesions but not that of neoplastic lesions in rat liver (Hayashi et al., 2004). In the present study, we examined the anticarcinogenic effects in short-term and long-term KAT treatment of preneoplastic lesions, as well as more advanced tumors in the rat liver, which are induced by DEN and 2-AAF treatment along with PH. In the short-term KAT treatment of 3 weeks duration (experiment 1), the GST-P–positive AHF, which are putative preneoplastic lesions, were continuously reduced in total area and number but not in mean size, from day 1 to day 14 of the treatment period. The time-dependent quantitative reductions in GST-P–positive AHF in the KAT-treated groups were not accompanied by enhanced apoptosis. These results appear to support our previous hypothesis, which was the remodeling of altered hepatocytes through redifferentiation, for explaining mechanisms of the possible anticarcinogenic effects of KAT.
The proliferative activity was significantly increased in the AHF and the surrounding areas of the KAT-treated group in experiment 1 during the continuous reductions in quantitative values of the AHF. This result is attributed to the thyromimetic action of KAT because T3 is known to be a powerful inducer of hepatocyte proliferation in rats (Francavilla et al., 1994). Ledda-Columbano et al. (2000), have demonstrated anti-hepato carcinogenic effects of a hepatomitogen T3, suggested that cell proliferation per se might play an important role in redifferentiation of preneoplastic cells. Peroxisome proliferators (PPs) and retinoids are also known to be mitogens for hepatocytes (Pibiri et al., 2001; Ohmura et al., 1996) and agents that can reduce carcinogen-induced preneoplastic lesions, identified by GST-P and GGT staining in the rat liver (Bana et al., 1991; Chen et al., 1994; Hosokawa et al., 1989; Moriwaki et al., 1988). In particular, it has been reported that an acyclic retinoid can clinically prevent second primary hepatoma through effects on cellular differentiation (Moriwaki et al., 1997). Considering that all of these receptors, including TRs, belong to the same superfamily of steroid hormone nuclear receptors (Kumar and Thompson, 1999), the proliferative effects per se through these nuclear receptors might cause the reduction of preneoplastic lesions with GST-P expression, probably through the redifferentiation of altered hepatocytes.
In the KAT-treated group of experiment 1, GST-P–positive HCAs were hardly changed in the morphometric analysis and the determination of PI. Considering the relationship between cell proliferation and quantitative reduction of preneoplastic lesions by KAT treatment, it is interesting to note that the proliferative effects, which apparently are among the essential characteristics of KAT, were not present in the HCAs. Although it is unclear why KAT does not provide the proliferative stimuli to hepatocytes progressing from the preneoplastic to the neoplastic phenotype, there appears to be the following possibility: loss or mutation of certain genes related to the signaling pathway of KAT, including TRs themselves. Indeed, high prevalence of mutation of TRs has been found in the tumors of patients with hepatocellular carcinomas (Lin et al., 1999). Further studies are now in progress in our laboratory to clarify the mechanisms responsible for the unresponsiveness of neoplastic hepatocytes to the proliferative stimuli of KAT.
In contrast, experiment 2 showed that long-term KAT treatment for 20 weeks significantly decreased the mean size of HCAs and reduced the number and mean size of AHF. Each PI within these lesions was significantly lower than that of the controls. These results suggest that the antitumorigenic effects of KAT in long-term treatment are caused by suppression of hepatocellular proliferation and are different from the early effects of KAT, evidenced by the relationship between the reduced number of AHF and the enhanced cell proliferation activity observed in the KAT-treated group. In the present study, the hepatocytes with normal appearance were decreased in size because of the loss of intracytoplasmic glycogen as a result of the thyromimetic properties of KAT. These histopathological findings are similar to those of the liver in rats fasted for a long time (Den Otter and De Minjer, 1972; Babcock and Cardell, 1974), while food consumption was increased in the KAT-treated rats. Dietary energy restriction is known to be a potent and reproducible inhibitor of carcinogenesis at a wide variety of sites (Birt et al., 1998). Furthermore, cell replication control as one of the mechanisms of the anticarcinogenic effects has been shown in several tissues, including the liver (Grasl-Kraupp et al., 1994; Lok et al., 1990). Therefore, the late-phase effects of long-term KAT treatment, characterized by the inhibition of cell replication, might be due to long continuance of low energy status in the liver caused by increased glycolysis, which is a thyromimetic property.
In the present study, KAT inhibited the development of hepatocellular proliferative lesions with a histologically eosinophilic phenotype and GST-P expression. Although PPs also have an inhibitory effects on these lesions, as described above, PPs themselves induce GST-P–negative and GGT-negative preneoplastic and neoplastic lesions in rat livers consisting of basophilic cells (Chen et al., 1994; Hosokawa et al., 1989; Rao et al., 1982). Besides, Lee (2000) mentioned that phenobarbital (PB), a representative promoter of hepatocarcinogenesis, has paradoxical effects depending on the histological phenotypes of DEN-induced lesions on mouse hepatocarcinogenesis. Such effects raise serious issues regarding extrapolation of experimental data from laboratory animals to human risk assessment. These results exhibit the necessity for careful consideration in determining the anticarcinogenic effects of KAT, because, in particular, the THs resemble the PPs; they are ligands of a nuclear receptor of the same superfamily of nuclear receptors and require the same dimerization partner that affects gene transcription such as the 9-cis retinoic acid receptor (Corton et al., 2000; Juge-Aubry et al., 1995). In addition, a good correlation exists between the alteration of the TH status and hepatomegaly caused by PPs (Miller et al., 2001; Wang et al., 2004). Nevertheless, in this study, KAT showed no inductions of the proliferative lesions with the basophilic phenotype and without GST-P expression. Additionally, it has been shown that the anticarcinogenic effects of T3 are not exhibited by a PP ciprofibrate (Ledda-Columbano et al., 2003). Thus, the TR-mediated effects appear to be different from those of PPs and PB in this important characteristic.
In conclusion, the present study indicates that a liver-selective thyromimetic KAT induces a reduction in hepatic preneoplastic lesions in rats, probably through redifferentiation of the altered hepatocytes caused by enhanced cell proliferation in the early phase of the KAT treatment and, later, through inhibition of the development of hepatic preneoplastic lesions and hepatocellular adenomas. Such inhibition in the late phase of KAT treatment is probably due to the suppression of cell proliferation. These results suggest that KAT may have potential as a chemopreventive agent for hepatocarcinogenesis.
ACKNOWLEDGMENTS
We are grateful to Kaori Kobayashi, Rie Nakano, and Miyuki Uehara for their technical assistance.
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