Effect of fasting/refeeding on the incidence of chemically induced hepatocellular carcinoma in the rat

Cristina Tomasi1, Ezio Laconi1,2,4, Sergio Laconi1, Marianna Greco1, Dittakavi S. R. Sarma3 and Paolo Pani1

1 Istituto di Patologia Sperimentale, Università di Cagliari and
2 Ospedale Oncologico `A. Businco', ASL 8, Cagliari, Italy and
3 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Caloric restriction has been associated with a delay in the development of both spontaneous and induced neoplasia. In contrast, cycles of fasting/refeeding were shown by us and others to enhance the incidence of early lesions during chemical carcinogenesis in rat liver. The present, long-term study was undertaken to establish whether such a diffential effect would also extend to the later phases of cancer development, until the overt appearance of neoplasia. Male Fischer 344 rats were initiated with a single dose of diethylnitrosamine (DENA, 200 mg/kg i.p.) and starting 1 week later they were either exposed to three cycles of fasting (3 days) followed by refeeding (11 days) or were fed continuously. Seven weeks after DENA administration the rats were exposed to the resistant hepatocyte model of the liver tumor promotion protocol. All animals were killed 1 year after initiation. Incidence of hepatocellular carcinoma was 2-fold higher in the fasted/refed group compared with the controls (72 versus 36%). In addition, cancers were also larger and of higher histological grade in the former group, with one animal showing metastases to the lungs, while no metastases developed in control animals. Fasting caused a decrease in total liver DNA (from 25.2 ± 1.1 to 16.5 ± 1.1 mg after 3 days) which was associated with a decrease in hepatocyte labeling index and mitotic activity and high levels of single cell death (apoptosis). In contrast, a sharp increase in hepatocyte proliferation was observed on day 2 of refeeding and this was more pronounced in glutathione S-transferase 7-7 positive foci compared with surrounding liver (10.2 ± 2.3 versus 4.6 ± 0.8%). Such a proliferative wave was associated with a sharp decline in the incidence of cell death. It is concluded that fasting/refeeding performed early after initiation accelerates the development of chemically induced hepatocellular carcinoma in the rat.

Abbreviations: AI, apoptotic index; BrdU, bromodeoxyuridine; DENA, diethylnitrosamine; GST 7-7, glutathione S-transferase 7-7; HCC, hepatocellular carcinoma; H&E, hematoxylin and eosin; LI, labeling index; MI, mitotic index; RH, resistant hepatocyte.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Several lines of evidence, both from clinical as well as experimental studies, indicate that diet plays a major role in neoplasia (17). The quality of food intake appears to be important, although it has been difficult to draw definitive conclusions on the effect of single dietary components on cancer development (8,9). On the other hand, the single most consistent association between diet and cancer is the one that relates a generalized decrease in the incidence of cancer to a decrease in the amount of total caloric intake (1,4,10). The basis for this effect is not clearly understood. Different possibilities have been proposed, including a decrease in growth rate associated with food restriction (4,11), hormonal and/or immunological mechanisms (3,4,12) and an increased incidence of single cell death in precancerous lesions (1315).

Within this context, less information is available on the effect of complete food withdrawal followed by refeeding on various stages of cancer development (1620). Fasting, similar to food restriction, is associated with a suppressive effect on cell growth and cell division and an increase in the rate of cell loss in several organs, including the liver (1315,17). However, these changes are contrasted by the effects of refeeding, which causes a generalized increase in cell proliferation in several organs, including the liver, and a parallel decrease in the rate of cell death (1315,17). Thus, the overall impact of fasting/refeeding on cancer development in a given system will result from the interaction of the above contrasting effects and is likely to be different from that of chronic caloric restriction.

Earlier, Sauer et al. (18) reported that fasting was able to increase the growth rate of implanted carcinomas and sarcomas in rats. More recently we investigated the effect of fasting/refeeding on the early stages of experimental liver carcinogenesis in rats (19). One or three cycles of fasting (3 days) followed by refeeding (11 days) were found to increase the size of hepatocyte nodules promoted according to the resistant hepatocyte (RH) model (19). Similar findings have also been described by other investigators (15). However, no studies have been reported so far on the long-term effects of fasting/refeeding on cancer development, until the overt appearance of neoplasia. This information becomes particularly important in the light of the possible reversible nature of early focal lesions in several systems, including rat liver (21). Furthermore, only a small proportion of persistent nodules will eventually progress to overt neoplasia (22) and one could argue that fasting/refeeding may be affecting only the growth of those that do not progress. In order to directly address these issues, the present long-term study was undertaken to investigate the effect of early exposure to cycles of fasting/refeeding on the incidence of chemically induced hepatocellular carcinoma (HCC) in rats. The results indicate that three cycles of fasting/refeeding performed early after initiation result in twice the incidence of HCC at the end of 1 year, compared with control, ad libitum fed animals.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Experiment 1
In the first experiment, 2-month-old male Fischer 344 rats (Charles River Italia, Milan, Italy) were acclimatized for 1 week to a Purina Rodent Chow diet (no. 5001) with food and water available ad libitum and an alternating light/dark cycle of 12 h. Rats were initiated with a single injection of diethylnitrosamine (DENA, 200 mg/kg body wt i.p.; Sigma Chemical Co., St Louis, MO). Starting 1 week later they were divided into two groups. Group 1 was exposed to three cycles of fasting/refeeding, each consisting of 3 days of food withdrawal followed by 11 days of refeeding prior to the next fasting period. Group 2 (control) was fed continuously throughout the experiment. Two weeks after the last fasting cycle both groups were subjected to a modified version of the RH model (23) to selectively stimulate the growth of foci and nodules. Briefly, rats were given three consecutive daily doses of 2-acetylaminofluorene (20 mg/kg body wt intragastrally; Sigma) followed on day 4 by a single administration of CCl4 (0.2 ml/kg body wt intragastrally, mixed in corn oil 1:1 v:v). One rat from each group died between 5 and 7 days after CCl4 dosing. All remaining animals (11 in each group) survived throughout the experiment and were killed at the end of 1 year. Macroscopic examination was performed; samples from liver, lungs, spleen, pancreas, kidneys and other organs that looked abnormal were taken and processed for standard histological (hematoxylin and eosin, H&E) analysis.

Experiment 2
In a second series of studies, liver cell turnover parameters were determined in initiated animals during a complete cycle of 3 days fasting followed by refeeding. Two-month old male Fischer 344 rats were given a single injection of DENA, as above. Starting 1 week later animals were fasted for up to 3 days and then refed ad libitum. Groups of rats were killed at the time of food withdrawal, at 1, 2 and 3 days during fasting and 1, 2 and 3 days after starting refeeding. Each animal received four doses of 5'-bromodeoxyuridine (BrdU; Sigma), 50 mg/kg body wt each, 6 h apart, starting 24 h before killing. Livers were excised and processed for total DNA content, double glutathione S-transferase 7-7 (GST 7-7) (19) and BrdU immunohistochemical staining and standard histological analysis. BrdU staining was performed using a monoclonal antibody (Sigma) followed by biotylinated secondary antibody (Vectastain ABC kit; Vector Laboratories, Burlingame, CA). Labeled nuclei were scored and expressed as percent of total nuclei (labeling index, LI), both in GST 7-7-positive foci and in surrounding non-nodular liver. All foci were considered in each section. Mitotic index (MI) and apoptotic index (AI) were similarly determined on H&E stained sections. Statistical analysis of results was performed with Student's t-test.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Experiment 1
Body weights during the experiment and final liver weights are presented in Table IGo. At 2 months (i.e. 2 weeks after the last cycle of fasting/refeeding) body weights were slightly lower in group 1 compared with ad libitum fed (control) animals, in agreement with our previous studies (19). However, no differences were observed at 6 and 12 months. Food consumption after fasting, measured daily over a period of 1 week, was ~30% higher than the control, ad libitum fed group for the first 5–6 days and returned to baseline levels thereafter (data not shown). Absolute liver weight and percent liver weight/body weight were higher in fasted/refed animals compared with controls (Table IGo). These differences were largely attributable to the presence of larger tumors in the former group. The incidence of hepatocyte nodules and HCC at the end of 1 year is reported in Table IIGo. All rats (11/11) exposed to fasting/refeeding and 9/11 animals in the control group had grossly visible hepatic nodules. However, all animals in both groups had microscopic foci of altered cells in the liver, as determined by histological examination on H&E stained sections. Liver cancer was grossly diagnosed and histologically confirmed as HCC in 8/11 fasted/refed animals (72%), while the incidence was only 36% (4/11) in the group fed throughout. Moreover, cancers were larger (15 ± 3 versus 10 ± 3 mm in maximum diameter) and of higher histological grade (3 poorly differentiated versus 0) in group 1 (Figure 1Go) compared with group 2 (Figure 2Go). One animal in group 1 (fasted/refed) also showed metastasis of HCC to the lungs, based on histological analysis (Figure 3Go).


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Table I. Body weights during the experiment and final liver weight following three cycles of fasting/refeeding
 

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Table II. The effect of fasting/refeeding on the incidence of HCC in rats initiated with DENA
 


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Fig. 1. Poorly differentiated HCC developing in a rat from the group exposed to three cycles of fasting/refeeding. No defined tissue pattern is present; cells are extremely pleomorphic; numerous irregular mitoses and extensive cell death (apoptosis) are present. H&E, 100x.

 


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Fig. 2. Well-differentiated HCC with trabecular pattern developing in a rat from the control group, regularly fed throughgout the experiment. Cell size and shape is relatively regular; mitoses were rare, while apoptotic figures were common. H&E, 100x.

 


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Fig. 3. Lung metastasis of HCC in a rat from the group exposed to three cycles of fasting/refeeding. Numerous other metastatic nodules were present in this animal. H&E, 100x.

 
Experiment 2
In this second series of experiments the effect of a single cycle of fasting/refeeding on liver cell turnover was studied in initiated rats. Changes in body weight, liver weight and liver DNA content during a cycle of fasting/refeeding are reported in Table IIIGo. As expected, all parameters decreased during the fasting period and increased upon refeeding (14,15). However, total liver DNA was still significantly lower than control levels after 3 days of ad libitum refeeding. BrdU LI (Figure 4Go) decreased during the second and third day of fasting in both surrounding liver and in GST 7-7-positive foci. Conversely, a sharp increase in LI was seen on day 2 post-refeeding and this was more pronounced in GST 7-7-positive foci compared with surrounding parenchyma. These data are in agreement with those reported in other studies (14,15,24). Basal rates of mitosis (Table IVGo) were high compared with values observed in normal resting liver, possibly due to a residual compensatory response to the necrogenic dose of DENA, which was given 1 week before killing. MI decreased during fasting and increased sharply on day 2 after refeeding (Table IVGo). An opposite trend was observed in AI, which remained high throughout the fasting period and declined to a minimum level on day 2 post-refeeding (Table IVGo).


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Table III. Body weight, liver weight and liver DNA content in rats exposed to fasting/refeeding following administration of DENA
 


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Fig. 4. Rats were given DENA (200 mg/kg body wt) and 1 week later they were exposed to 3 days of fasting followed by refeeding. Animals were killed as indicated in the figure. Each animal received four injections of BrdU, (50 mg/kg each, 6 h apart), starting 24 h before killing. Labeling index was determined in both GST 7-7-positive foci and surrounding liver. Details are reported in Materials and methods. Data are means ± SE of three or four animals.

 

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Table IV. MI and AI in rats exposed to fasting/refeeding following administration of DENA
 
The effect of fasting/refeeding on the size of GST 7-7-positive areas was directly assessed by counting the number of cells in individual foci at various time points. As reported in Figure 5Go, incidence of foci of larger size (4–6 cells/cross section and higher) decreased during fasting by >60% compared with basal levels, while it gradually increased during 3 days of refeeding.



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Fig. 5. Experimental details are reported in the legend to Figure 4Go and in Materials and methods.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study for the first time demonstrates the effect of fasting/refeeding on cancer development in a well-characterized experimental setting. Sauer et al. reported that the growth rate of transplanted rat carcinomas and sarcomas was enhanced by fasting (18). More recently, we (19) and others (15) have observed that cycles of fasting/refeeding caused an increased incidence of early focal lesions arising during carcinogenesis in rat liver. Our present findings clearly indicate that the effect of an early exposure to fasting/refeeding is able to influence the entire carcinogenic sequence, until the appearance of invasive cancer, which is greatly enhanced at the end of 1 year.

The mechanism(s) underlying this effect is unclear at the present time. Analysis of cell turnover parameters in rat liver during a full cycle of fasting/refeeding given after the administration of DENA indicated that fasting caused a decrease in the proliferative rate and an increase in single cell death in both GST 7-7-positive foci and in surrounding liver. The number of large foci/section was greatly reduced following complete food withdrawal, implying that significant cell loss was also present in focal lesions. These findings are in agreement with those reported by Grasl-Kraupp et al. (14) and by Hikita et al. (15,24).

It is important to note that, despite the decrease in the size of foci induced by fasting, the overall impact of fasting/refeeding is stimulatory, rather than inhibitory, for the growth of focal lesions (19), leading to an increased incidence of cancer, as shown in the present study. This is possibly explained, at least in part, by the alterations in cell turnover observed during refeeding, which caused a wave of hepatocyte proliferation peaking at day 2, the latter being more sustained in GST 7-7-positive foci. In fact, previous reports in the literature have described a differential proliferative response to refeeding in cancer cells compared with normal tissues in the host (16,17). Furthermore, hepatocyte foci developing early in carcinogenesis were found to have a lower threshold (25) and to be more responsive (26,27) to growth stimuli compared with surrounding parenchyma. Thus, the enhancing effect of fasting/refeeding on cancer development in this system could be linked, at least in part, to this phenotypic property of early focal lesions (19).

Irrespective of the mechanism(s) involved, it is noteworthy that a few cycles of complete food withdrawal followed by refeeding have the ability to exert such a profound effect on the long-term process of cancer development. In fact, our previous observations indicated that even a single cycle of fasting/refeeding significantly increased the growth of hepatocyte nodules (19). Thus, in addition to the quantity and the quality of dietary intake, the pattern of food consumption can also affect the rate of progression during cancer development. While environmental factors, including diet, are known to play a major role in the etiology and pathogenesis of neoplasia, the magnitude of the effect observed in this study is certainly worth considering. Such findings reinforce the concept that cancer development is a process extremely susceptible to modulation by exogenous factors, among which nutrition appears to be of major impact. As an important corollary, these considerations support the view that identification of such risk factors, which are largely avoidable, represents a realistic and rational means for the design of effective strategies for the prevention of cancer in humans.


    Acknowledgments
 
We thank Giovanna Milia and Marinella Boi for their expert technical support and Bruna Melis, Tiziana Puxeddu and Alessandro Medas for their excellent secretarial assistance. This work was supported in part by funds from Progetto Finalizzato CNR-ACRO, Italy (P.P.) and by US PHS grant CA37077 from NCI, USA (D.S.R.S.)


    Notes
 
4 To whom correspondence should be addressed at: Istituto di Patologia Sperimentale, Università di Cagliari, Via Porcell 4, 09125 Cagliary, ItalyEmail: elaconi{at}unica.it Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 1, 1999; revised May 27, 1999; accepted June 17, 1999.