Cell proliferation induced by 3,3',5-triiodo-L-thyronine is associated with a reduction in the number of preneoplastic hepatic lesions
G.M. Ledda-Columbano2,
A. Perra,
R. Piga,
M. Pibiri,
R. Loi,
H. Shinozuka1 and
A. Columbano
Dipartimento di Tossicologia, Sezione di Oncologia e Patologia Molecolare, Via Porcell 4, 09124 Cagliari, Italy and
1 Department of Pathology, Pittsburgh, PA, USA
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Abstract
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Previous studies have suggested that liver cell proliferation is fundamental for the growth of carcinogen-initiated cells. To gain further information on the association between cell proliferation and hepatocarcinogenesis, we have examined the effect of the hormone 3,3',5-triiodo-L-thyronine (T3), a strong liver mitogen, on the growth of diethylnitrosamine (DENA)-induced hepatic lesions positive for the placental form of glutathione S-transferase (GSTP). Two weeks after a single initiating dose of DENA (150 mg/kg), cycles of liver cell proliferation were induced in male Fischer rats by feeding a T3-supplemented diet (4 mg/kg) 1 week/month for 7 months. Rats were killed at the end of the seventh cycle or 1 month later. Results indicate that, in spite of an increased labelling index, a 70% reduction in the number/cm2 of GSTP-positive minifoci occurred in T3-treated rats. A decrease in the number of GSTP-positive foci was also observed in T3-treated rats killed 1 month after the last exposure to the hormone (40, versus 67 foci/cm2 in controls), indicating that the reduction was not due to an inhibitory effect on GSTP exerted by the concomitant presence of T3. In a second series of experiments where DENA-treated rats were fed T3 for 1 week and then subjected to the resistant hepatocyte (RH) model, it was found that T3 treatment prior to promotion resulted in a decrease in the number of GSTP-positive foci (16 GSTP+ foci/cm2 in T3-fed animals versus 45 in the control group). The results indicate that cell proliferation associated with T3 treatment: (i) reduces the number of carcinogen-induced GSTP-positive lesions; (ii) does not exert any differential effect on the growth of the remaining foci; (iii) inhibits the capacity of putative DENA-initiated cells to be promoted by the RH model. Data suggest that cell proliferation may not necessarily represent a stimulus for the growth of putative preneoplastic lesions.
Abbreviations: 2-AAF, 2-acetylaminofluorene; BrdU, bromodeoxyuridine; DENA, diethylnitrosamine; GSTP, glutathione S-transferase placental form; LI, labelling index; PH, partial hepatectomy; PP, peroxisome proliferators; PPAR, peroxisome proliferator-activated receptor; RH, resistant hepatocyte; RXR, retinoic acid receptor; T3, 3,3',5-triiodo-L-thyronine; TR, T3 receptor.
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Introduction
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Liver cell proliferation is considered to play an important role in the several steps of the carcinogenic process, initiation, promotion and progression (1). Although the exact mechanism by which cell proliferation plays a role in initiation is not known, its involvement in events such as fixation of a miscoding lesion in the newly made DNA has been entertained (25). In most of the studies supporting a critical role of cell proliferation in the initiation step of chemical hepatocarcinogenesis the proliferative stimulus has been achieved by compensatory regeneration. More recently, however, it was shown that direct hyperplasia induced by primary mitogens fails to support initiation by chemical carcinogens (6,7). Whether the inability is due to the lack of some molecular event required for initiation or whether initiated cells are formed but they are preferentially eliminated by apoptosis during the successive regression of the initial hyperplasia is still unclear. It has been suggested that deletion of a number of carcinogen-altered cells by apoptosis occurs `spontaneously' shortly after initiation (6,812).
A second site at which cell proliferation exerts a critical effect is the promotion of carcinogen-initiated cells. An increased incidence of preneoplastic lesions and tumours has been observed when carcinogen treatment was followed by compensatory regeneration induced by repeated partial hepatectomy (PH) or multiple treatment with necrogenic agents (1315). In contrast, the effect of direct mitogens on the growth of carcinogen-induced preneoplastic hepatocytes is far less clear. The effect of peroxisome proliferators (PPs) is paradigmatic. While a short-term treatment with PPs results in a decrease in the number and size of preneoplastic lesions identified by either adenosine triphosphatase,
-glutamyltranspeptidase or the placental form of glutathione S-transferase (GSTP) staining (1618), longer term treatment produces more and larger tumours (1922). The observation that short-term treatment with PPs results in a significant decrease in enzyme-altered preneoplastic lesions induced by genotoxic compounds (1618) has often been attributed to the inhibitory effect of these agents on the marker enzyme used to identify preneoplastic lesion and not to a real growth inhibitory effect. However, recently Chen et al. have shown that a short-term exposure to the PP ciprofibrate reduced the labelling index of preneoplastic nodules generated by the SoltFarber procedure from 40 to <5% and this effect was associated with a rapid decrease in area and number of nodules (23). This raises the important issue as to whether induction of liver cell proliferation is always a promoting condition for preneoplastic growth or whether the nature of the proliferative stimulus is important in determining the type of response of carcinogen-altered lesions.
The hormone 3,3',5-triiodo-L-thyronine (T3) shares some characteristics with PPs in that: (i) it possesses a nuclear receptor of the same superfamily as the peroxisome proliferator-activated receptor (PPAR) (24); (ii) it is an inducer of peroxisome proliferation (25); (iii) it is a potent hepatomitogen (26,27). However, the effect of T3 on the several steps of the hepatocarcinogenic process is unknown. To gain additional information on the possible association between hepatocyte proliferation induced by primary mitogens and growth of putative carcinogen preneoplastic lesions, we have examined the effect of T3 on growth of GSTP-positive hepatocytes induced by a single diethylnitrosamine (DENA) treatment. We have also examined the effect of T3 on the capacity of GSTP-positive hepatocytes to be promoted when subjected to the resistant hepatocyte (RH) model (28). The results demonstrate that repeated T3 administration leads to a reduction in the number of GSTP-positive lesions with no increase in the size of the remaining ones, in spite of a strong mitogenic effect on the liver. In addition, a 1 week exposure of rats to T3 prior to the RH promotion protocol resulted in a 50% decrease in the number of liver nodules.
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Materials and methods
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Animals
Male Fischer F-344 (100125 g) and Wistar rats (175200 g) purchased from Charles River (Milano, Italy) were maintained on a standard laboratory diet purchased from Ditta (Mucedola, Italy). The animals were given food and water ad libitum with a 12 h light/dark daily cycle and were acclimated for 1 week before the start of the experiment. Guidelines for the Care and Use of Laboratory Animals were followed during the investigation.
Determination of the mitogenic activity of T3
To determine the proliferative effect of T3, male Wistar rats were fed a diet containing 4 mg/kg T3 (Sigma Chemical Co., St Louis, MO) for 1 week. Hepatocyte proliferation was determined immunohistochemically by measuring bromodeoxyuridine (BrdU) incorporation into hepatocyte nuclei. Depending on the experiment, BrdU (20 mg/ml, rate of release 10 µl/h/7 days) was continuously released through osmotic minipumps (Model 2ML1; Alzet Co., Palo Alto, CA) implanted s.c. 7 days before death.
Experimental protocol 1 (Figure 1
)
Thirty Fischer rats were injected i.p. with a single dose of DENA (Sigma Chemical Co.), dissolved in saline, at a dose of 150 mg/kg body wt. Following a 10 week recovery period, rats were exposed to seven weekly cycles of a diet containing 4 mg/kg T3 once every month. During the first and the last T3 cycles, BrdU dissolved in the drinking water (1 mg/ml) was given ad libitum, as previously described (29). Rats were killed at the end of the first and seventh cycles. The remaining animals were switched to the basal diet at the end of the seventh cycle and killed 1 month later.

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Fig. 1. Experimental protocol 1. Ten weeks after administration of a single dose of DENA, rats were subjected to seven cycles of T3-supplemented diet (1 week/month for 7 months). Arrows indicate time of death (end of the first and seventh cycle or 1 month after the last T3 cycle).
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Experimental protocol 2 (Figure 2
)
Fifteen F-344 rats were given a single i.p. dose of DENA (150 mg/kg) and 2 weeks later they were fed a diet containing 4 mg/kg T3 for 1 week. Following an additional 2 week time period on basal diet, rats were subjected to the SoltFarber RH model, consisting of feeding a diet containing 0.02% 2-acetylaminofluorene (2-AAF) for 1 week coupled with a standard two-thirds PH, continuation for an additional week on the 2-AAF containing diet and death.

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Fig. 2. Experimental protocol 2. Male F-344 rats initiated with a single dose of DENA were placed on a T3-supplemented diet for 1 week or maintained on a basal diet prior to exposure to the resistant hepatocyte model. Rats were killed at the end of the promoting procedure.
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Histology and immunohistochemistry
Immediately after death, liver sections were fixed in 10% formalin, embedded in paraffin and routinely stained with haematoxylin and eosin. Other sections were employed for immunohistochemical detection of BrdU and GSTP.
BrdU staining
Mouse monoclonal anti-BrdU antibody was obtained from Becton Dickinson (code 347580; Becton Dickinson, San Jose, CA) and the peroxidase method was used to stain BrdU-positive hepatocytes. Peroxidase-conjugated goat anti-mouse immunoglobulin was obtained from Dako (EnVisionTM Peroxidase Mouse, code K4001; Dako Corp., Carpinteria, CA). Four micron thick sections were deparaffinized, treated with 2 N HCl for 20 min, then with 0.1% trypsin type II (crude from porcine pancreas, code SI T8128; Sigma, Milano, Italy) for 20 min and treated sequentially with normal goat serum 1:10 (Dako, code X0907), mouse anti-BrdU 1:200 for 90 min and Dako EnVisionTM Peroxidase Mouse ready-to-use. The sites of peroxidase binding were detected with 3,3'-diaminobenzidine.
GSTP staining
The location of GSTP in the liver was determined immunohistochemically by the phosphatase method using an anti-rat GSTP polyclonal antibody (MBL, Nagoya, Japan). Alkaline phosphatase-conjugated goat anti-rabbit/mouse immunoglobulin was obtained from Dako (Dako EnVisionTM alkaline phosphatase rabbit/mouse; Dako Corp.). Briefly, tissue sections were deparaffinized, exposed to 0.3% hydrogen peroxide in ethanol for 10 min to block endogenous peroxidase and incubated with normal goat serum for 20 min at room temperature. The sections were then incubated overnight with a rabbit anti-GSTP polyclonal antibody (1:1000), followed by Dako EnVisionTM alkaline phosphatase rabbit/mouse ready-to-use. The binding sites of phosphatase were determined using BCIP/NBT substrate (Dako Corp., code K598). Sections were then counterstained with methyl green.
Measurement of BrdU labelling index and determination of DNA content
At least 5000 hepatocyte nuclei per rat were scored. Labelling index (LI) was expressed as the number of positive hepatocyte nuclei/100 nuclei. Total hepatic DNA content was quantitatively assayed by Burton's diphenylamine method (30).
Measurement of GSTP-positive foci
GSTP-positive foci were measured with a computer-assisted image processor, programmed for the three dimensional calculation of Campbell et al. (31). Only foci >76 µm in diameter were measured.
Statistical analysis
Comparisons between treated and control groups were performed by Student's t-test.
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Results
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Mitogenic effect of a T3-supplemented diet on normal rat liver
In our previous studies aimed to determine a dose of T3 in the diet that could induce hepatocyte DNA synthesis in male Wistar rats, it was found that feeding rats a T3-supplemented diet (4 mg/kg diet) for 1 week induced a several-fold increase in labelled thymidine incorporation into liver DNA (data not shown). Therefore, the latter concentration of T3 was selected for all future experiments. Further studies aimed to determine the exact extent of hepatocyte proliferation showed that this dosage of T3 induced a hepatocyte LI of ~29%, versus 3% in control liver (Table I
). The increased LI was associated with an increase in the number of mitotic figures, especially between 2 and 4 days, and, most importantly, by an increase in liver DNA concentration and total hepatic DNA content (Table I
). Interestingly, T3 treatment, unlike other hepatomitogens previously tested (lead nitrate, PPs, cyproterone acetate, phenobarbital and TCPOBOP), was not associated with cellular hypertrophy. In contrast, microscopic analysis of liver from T3-treated rats did reveal the presence of hepatocytes with an increased nuclear/cytoplasmic ratio and an overall decrease in cell size. No evidence of liver cell damage could be observed during 7 days of T3 feeding. However, treatment with T3 did cause a loss in body weight, due to the catabolic effects of the hormone, which accounted for ~1015% when compared with controls.
Repeated mitogenic cycles with T3 lead to a reduction in the number of GSTP-positive hepatocytes
Treatment with 150 mg/kg DENA is known to produce single or doublets of GSTP-positive hepatocytes (putative initiated cells) that are considered to be precursors of preneoplastic and neoplastic lesions (9). Accordingly, 9 months after DENA administration several small GSTP-positive foci, most likely deriving from single GSTP-positive hepatocytes, were observed (~55 foci/cm2) (Table II
). Cyclic exposure of rats to a T3-supplemented diet resulted in a severe reduction in number (~14 foci/cm2) and size of GSTP-positive foci. Consequently, a 6-fold reduction in the percentage of liver section occupied by GSTP-positive hepatocytes was observed in T3-fed animals (0.23%, versus 1.35% in control liver; Table II
).
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Table II. Effect of repeated T3 treatments on number and area of GSTP-positive foci in rat liver initiated with DENAa
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The inhibitory effect of T3 on the number and size of GSTP-positive foci occurred in spite of its mitogenic effect. Indeed, immunohistochemical determination of BrdU-positive hepatocytes revealed that although the proliferative response of the liver to T3 declined with the number of treatments, hormone administration was able to stimulate a significant mitotic activity of hepatocytes even after the seventh cycle (LI of 8.1%, versus 3.0% in control liver) (Figure 3
).

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Fig. 3. Labelling index of rat hepatocytes following T3. DENA-initiated rats were exposed to either one or seven cycles of a T3-supplemented diet (4 mg/kg diet). All rats were given BrdU (1 mg/ml in drinking water) for 7 days. At least 5000 hepatocyte nuclei/liver were scored. Labelling index was expressed as number of BrdU-positive hepatocyte nuclei/100 nuclei. Results are expressed as means ± SE of 68 animals/group.
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The possibility that the reduction in the number of GSTP-positive hepatocytes could be due to a loss of GSTP positivity caused by a possible inhibitory effect on GSTP gene expression by T3, as previously suggested for PPs (18), was considered. To examine this possibility, two extra groups of animals initiated with DENA and fed T3 or basal diet were killed 1 month after the last T3 cycle. The results (Table III
) showed that a 40% reduction in the number of GSTP-positive foci (40 foci/cm2 in T3-treated rats versus 67 in control rats) was still present 1 month after T3 removal, demonstrating that loss of GSTP-positive foci was not the consequence of any phenotypic change due to the presence of T3.
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Table III. Number and area of GSTP-positive foci in DENA-initiated rat liver one month after the last exposure to the T3-supplemented dieta
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A single exposure to T3 inhibits the capability of GSTP-positive hepatocytes to be promoted by the RH model
From the previous studies it is evident that exposure of rats to repeated T3 cycles leads to a reduction in GSTP-positive foci. To determine whether multiple T3 treatments are necessary to exert such an inhibitory effect or whether a single exposure would lead to similar results, we designed an experimental protocol where following a 1 week exposure to T3 diet, DENA-initiated rats were subjected to the RH model of liver tumor promotion (Figure 2
). The results (Table IV
) indicate that 7 days of T3 diet are sufficient to inhibit the promoting ability of the RH model. Indeed, while ~45 foci/cm2 were observed in DENA-initiated animals, the number of foci was severely reduced (16 GSTP-positive foci/cm2) in rats exposed to T3 prior to promotion with the RH model. Consistent with the immunohistochemical analysis, a reduction in foci number was also observed by examination of sections stained with haematoxylin and eosin. The decrease in the number of foci was not accompanied by an increase in the size of the remaining foci nor by significant differences in the size distribution of foci betweeen the two groups (Table IV
and Figure 4
).
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Discussion
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The role of enhanced cell proliferation on the genesis of cancer is somewhat controversial. While there is strong evidence suggesting that cell proliferation is essential for development of many types of cancer, certain experimental and clinical observations cast some doubt about the validity of their generalization (3235). It is evident from the results of the present experiment that hepatocyte proliferation induced by T3 did not stimulate the growth of carcinogen-initiated cells and it significantly reduced the number of putative preneoplastic lesions. These effects were similar to those reported previously by other investigators using PPs as inducers of hepatocyte proliferation in carcinogen-treated animals (1618,23), thus it is reasonable to suggest that enhanced liver cell proliferation is not always a promoting condition of liver carcinogenesis and that the nature of the proliferative stimulus is the determining factor in modulating carcinogenesis.
Evidence is accumulating to indicate that signal pathways leading to hepatocyte proliferation induced by direct mitogens such as PPs and T3 are distinctly different from those elicited by compensatory regeneration seen after PH and necrogenic chemicals (36). Compensatory regeneration, which exerts a strong promoting stimulus on liver carcinogenesis, is associated with induction of a number of early growth response genes as well as induction of certain growth factors (hepatocyte growth factor, trasforming growth factor-
) and cytokines such as TNF-
and IL-6 (3739). These growth factors/cytokines may play a role in stimulating growth of preneoplastic lesions. In contrast, liver cell proliferation induced by primary mitogens is not associated with induction of these immediate early genes, growth factors and cytokines (29,40,41). Although precise mechanisms of induction of hepatocyte proliferation by primary mitogens are not well defined, some of the mitogens identified so far share the common property of being ligands for nuclear receptors of the superfamily of steroid receptors [PPAR, T3 receptor (TR), retinoic acid receptor and retinoid X receptor (RXR)]. Activated nuclear receptors of this group dimerize with the RXR and act as transcription factors for induction of specific genes (24). The recent finding that the presence of PPAR is essential for induction of hepatocyte proliferation by PPs (42) supports the notion that nuclear receptors are important mediators of cell growth induced by primary mitogens. One plausible explanation for the failure of T3-induced hepatocyte proliferation to stimulate growth of preneoplastic lesions is quantitative and/or qualitative changes in nuclear receptors in carcinogen-initiated hepatocytes. It has been reported that carcinogen-induced liver nodules are less responsive for the induction of peroxisomal enzymes and cell proliferation than the surrounding non-nodular liver in response to PPs (18,23), suggesting diminished PPAR expression in carcinogen-induced nodules. Although, at present, no information is available as to the changes in levels of TRs in carcinogen-initiated liver cells, it is possible that reduction and/or loss of T3 receptors may be responsible for the inability of T3 to stimulate the growth of preneoplastic lesions, in spite of its mitogenic effect on normal hepatocytes. From the present study, it is also evident that a large fraction of GSTP-positive lesions is lost following exposure to T3. The possibility that the partial loss of GSTP-positive foci could be the consequence of inhibition of GSTP expression by T3, similar to that seen with PPs (18,22), rather than a true reduction in number, was considered. However, although this possibility cannot be excluded, it appears to be unlikely in view of the following: (i) in experimental protocol 2, where exposure to the promoting procedure allowed rapid expansion of the foci and therefore their easy detection, the presence of a reduced number of foci was confirmed by haematoxylin and eosin analysis; (ii) in spite of the fact that full reversion of inhibition of GSTP expression in preneoplastic lesions has been shown to occur within a month (43), the number of GSTP-positive foci was reduced, compared with controls, even 1 month after release from T3 diet (Table III
); (iii) short-term T3 feeding does not inhibit GSTP transcription or GSTP activity induced in rat liver by lead nitrate (submitted for publication); (iv) preliminary results indicate that repeated treatment with T3 reduce the incidence of hepatocellular carcinoma (data not shown).
This finding suggests that among the pleiotropic effects of T3, some could be potentially relevant for apoptosis and/or differentiation; as to the former, it is well known that in the course of chemical carcinogenesis, preneoplastic lesions undergo extensive cell turnover (4447), implying that they contain a significant proportion of cells whose survival is compromised. Under such circumstances, additional stimulation with T3 could further upset the finely tuned balance between cell proliferation and cell death. Although no evidence of apoptosis was observed in normal hepatocytes following T3 treatment, specific deletion of carcinogen-altered hepatocytes induced by T3 could have occurred. That T3 might exert a differential effect between normal hepatocytes and carcinogen-altered cells is supported by preliminary findings from our laboratory showing that T3 is apoptogenic for Reuber hepatoma cell line FaO, but not for primary cultures of hepatocytes (data not shown). The occurrence of massive apoptosis after treatment with another ligand of nuclear receptors, the PP clofibrate, has also been reported in HepG2 cells (48), suggesting that ligand-activated nuclear receptors may mediate opposite processes (i.e. proliferation and death). Finally, in consideration of the active remodelling process occurring in the several steps of hepatocarcinogenesis (49), the possibility that the loss of GSTP-positive lesions could be due to induction of a differentiative process of carcinogen-altered hepatocytes by T3 cannot be disregarded. Studies addressed to determining the precise mechanisms responsible for the inhibitory effect of T3 on the growth of early preneoplastic lesions and whether this hormone could exert a similar effect also at later stages of the carcinogenic process are currently in progress.
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Acknowledgments
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This study was supported by grants from the Associazione Italiana Ricerca sul Cancro (AIRC) and Ministero Università e Ricerca Scientifica (MURST 40% and 60%), Italy.
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Notes
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2 To whom correspondence should be addressed Email: gmledda{at}vaxca1.unica.it 
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Received April 19, 1999;
revised August 6, 1999;
accepted August 16, 1999.