Introduction

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1,25(OH)₂D₃, the hormone of the vitamin D₃ endocrine system, is known to mediate its cellular action through an intracellular receptor, the vitamin D receptor, having molecular properties similar to those of the superfamily of steroid receptors. Besides its action on calcium homeostasis, 1,25(OH)₂D₃ can influence the expression of several genes (Silver et al., 1986; Demay et al., 1990; Lemay et al., 1995), as well as the proliferation and differentiation of normal and neoplastic cells in vitro (Bar-Shavit et al., 1983; Provvedini et al., 1983; Mangelsdorf et al., 1984; Manolagas and Deftos, 1984; Abe et al., 1986). Although the liver is the site of the C25-hydroxylation of vitamin D, the liver has been shown to have a very low abundance of vitamin D receptors and, consequently, is not considered a target site of vitamin D action. Recent studies have, however, demonstrated that calcium and/or vitamin D deficiency has significant effects on liver cell physiology, as exemplified by a significant impairment of the normal hepatic compensatory hyperplasia induced by two-thirds partial hepatectomy (Éthier et al., 1990, 1993; Bilodeau et al., 1995) and a lower hepatocyte intracellular calcium response to phenylephrine and epidermal growth factor (Gascon-Barré et al., 1994). In addition, experimental evidence indicate that dietary calcium supplementation can protect against early hepatic changes due to choline deficiency (Ghoshal et al., 1987), whereas both calcium and vitamin D supplementation have been shown to reduce the growth of 7,12-dimethylbenz(a)anthracene-induced mammary tumors (Jacobson et al., 1989; Carroll et al., 1991).

In vivo, most environmentally induced neoplastic transformations are known to occur after low-grade carcinogen exposure over a period of several years, if not decades. Although 1,25(OH)₂D₃ and many of its analogs have been shown to promote the differentiation of cancer cells and cell lines in vitro, the protective effect of normal vitamin D status against an insult known to induce neoplastic growth in vivo has not been investigated. Some epidemiological studies have uncovered possible links between sun exposure and the incidence of colon, breast and prostate cancer in humans (Yang and Newmark, 1987; Gorham et al., 1989; Garland et al., 1990; Bostick et al., 1993; Feldman et al., 1995), but the evidence establishing a cause and effect relationship between the in vivo vitamin D status and neoplastic growth remains tenuous. The purpose of the present study was, therefore, to investigate, in vivo, the influence of the vitamin D status on the response of rat liver to a chemical insult known to induce morphological and functional changes leading to the appearance of a rapidly proliferating, pluripotent, "stem" cell compartment, the oval cells, which are able to differentiate into hepatocytes, ductular intestinal-like or neoplastic cells (Faris et al., 1991; Pack et al., 1993; Nagy et al., 1994; Factor et al., 1994; Golding et al., 1995). In this model, a process of hepatocarcinogenesis develops over a period of several months to 1 year, but preneoplastic foci are known to appear early after the end of the cancer-inducing protocol. We now report that a state of hypocalcemic vitamin D depletion increases the susceptibility of rat liver to the development of preneoplastic foci early after exposure to a potent hepatic carcinogen.

	Materials and Methods

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Animals. All experiments were carried out in male Sprague-Dawley rats (Charles River, St. Constant, Canada) housed in stainless steel wire cages, with a 12-hr light/dark cycle. Normal rats (9-10 weeks of age) were fed a regular rat chow diet containing 2115 IU/kg vitamin D₃ and 0.97% calcium (Purina Rat Chow; Ralston Purina Inc., Mississauga, Canada). They were acclimatized for a period of 5 days before being randomly assigned to the experimental protocol. Vitamin D depletion was performed by feeding a semisynthetic vitamin D-deficient diet containing 0.9% elemental calcium (with normal phosphorus content to avoid the development of rickets) and demineralized water to nursing females; the diet was continued for a period of 6 weeks after weaning, as already described (Haddad et al., 1986; Éthier et al., 1990). Vitamin D depletion was judged by the presence of hypocalcemia, which in these animals was accompanied by low to undetectable serum 25-hydroxyvitamin D and low 1,25(OH)₂D concentrations, as reported previously (Haddad et al., 1986; Éthier et al., 1990, 1993; Beaulieu et al., 1993). All animals (four or five/group) were fed ad libitum throughout the experimental period. All protocols were carried out according to the Standard of Ethics for Animal Experimentation of the Canadian Council on Animal Care and were approved by the local animal ethics committee.

Chemical induction of liver morphological changes. The influence of vitamin D status on liver susceptibility to carcinogenesis was studied after the administration of DEN and 2-AAF according to the Solt-Farber protocol (Solt and Farber, 1976; Evarts et al., 1989). Briefly, a single dose of DEN (Sigma Chemical Co., St. Louis, MO) was administered i.p. (150 mg/kg b.wt. in 0.9% normal saline). All animals were returned to their quarters and left untreated for a period of 2 weeks. 2-AAF (Sigma) was then administered by gavage (1.5 mg/animal, dissolved in carboxymethylcellulose) for a period of 4 days, after which animals were subjected to two-thirds partial hepatectomy (Éthier et al., 1990). 2-AAF administration was continued for the next 4 days. Beginning 24 hr before the end of the 2-AAF regimen, rats were injected daily with 0.5 µCi/g b.wt. [³H]thymidine (specific activity, 73 Ci/mmol; DuPont Chemicals, Boston, MA), for a period of 4 days. Animals were killed under ether anesthesia, after an overnight fast, 1, 2, 3, 4, 5 or 6 weeks after the last dose of 2-AAF. Blood was drawn from the abdominal aorta, and the liver was thoroughly washed with normal saline and processed as described below. Control rats were subjected to a similar protocol but received only normal saline i.p. and carboxymethylcellulose by gavage. They were also subjected to two-thirds partial hepatectomy and [³H]thymidine injection.

Morphological studies. Liver samples were taken from the right and caudate lobes and either immediately frozen in liquid nitrogen for -GT and glucose-6-phosphatase evaluation or fixed in 10% neutral formalin, embedded in paraffin blocks and stained (4-µm sections) with hematoxylin-phloxine-saffron. Each hematoxylin-phloxine-saffron-stained section was evaluated by light microscopy for the detection of preneoplastic foci, nodules and oval cells. Oval cells were determined as cells, in the periportal area of the hepatic acinus, with an oval nucleus and the capacity to incorporate [³H]thymidine in the presence of the cytostatic agent 2-AAF (Alison et al., 1993). Autoradiography was performed as already described (Éthier et al., 1990), on a total of 4000 cells taken at random in each liver section. All evaluations were carried out in a double-blind manner.

Statistical analysis. Data are expressed as means ± S.E.M. Statistically significant differences (P < .05) in the evolution of each parameter over the time period studied were analyzed by the Student t test or a two-way analysis of variance using the Tukey post hoc test (Winer, 1971), as indicated in the figure legends.

	Results

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Serum ionized calcium was found to be significantly influenced by the vitamin D status, with values of 0.78 ± 0.04 and 1.27 ± 0.03 mM in vitamin D-depleted and normal rats, respectively (P < .0001), at the beginning of the experiment. Circulating ionized calcium did not change in either group during the course of the studies, with values of 0.85 ± 0.04 and 1.27 ± 0.01 mM, respectively (P < .0001), at the time of euthanasia.

Figure 1 illustrates the histological appearance of the liver in both groups of animals after the hepatocarcinogenesis protocol; the complete time course for the morphological data is presented in figure 2. As illustrated, preneoplastic foci (-GT-positive and glucose-6-phosphatase-negative) were already present in both groups of animals 1 week after 2-AAF withdrawal and continued to increase during the subsequent weeks. Livers from vitamin D-depleted rats exhibited a significant increase, over that observed in normal rats, in the number (fig. 2A) of foci at weeks 1 and 5, but the number of foci was significantly lower than in normal rats at week 3 after 2-AAF withdrawal. Focus size (fig. 2B), however, was found to be significantly higher in vitamin D-depleted rat livers at weeks 2, 3, 5 and 6. Finally, due to the increase in focus size, focus area (volume fraction) (fig. 2C) was consistently higher in vitamin D-depleted livers than in normal rat livers. Control rat livers of both groups had normal liver histology, and no foci, nodules or oval cells were detected in either group.

View larger version (K): [in this window] [in a new window]   Fig. 1.   Representative photomicrographs of rat livers treated with DEN and 2-AAF. Animals were killed 3 weeks after the withdrawal of 2-AAF, as indicated in "Materials and Methods." A (normal) and C (hypocalcemic, vitamin D-depleted), histological sections of rat liver specimens stained with hematoxylin-phloxine-saffron (magnification of ×32 for both sections); B (normal) and D (hypocalcemic, vitamin D-depleted), -GT-positive (and glucose-6-phosphatase-negative; data not shown) hepatic foci (magnification of ×20 for both sections).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Morphometric analyses of preneoplastic hepatic focus number (A), size (B) and area (C). Foci were defined as areas presenting -GT-positive and glucose-6-phosphatase-negative staining, as illustrated in figure 1, with n = 3 animals/group. All analyses were done in a double-blind fashion. , normal rats; , vitamin D-depleted rats. Statistically significant differences between group means were analyzed by the Student t test at each time point studied. *P < .05; **P < .01; ***P < .0001.

The oval cell labeling is presented in figure 3. The labeling index was shown to decrease over time in both groups (P < .001), with indices of 7 ± 2.9 and 6 ± 1.8 cells/1000 cells in normal and vitamin D-depleted rat livers, respectively (not significant), 4 weeks after the end of the carcinogenesis induction protocol. However, evaluation of the effect of vitamin D depletion during the period of observation indicated that the labeling index was consistently lower in livers of vitamin D-depleted rats than in those of normal animals (P < .001). Post hoc tests revealed that the significant decrease in oval cell number originated 1 week after 2-AAF withdrawal (P < .001), indicating that the main effect of vitamin D depletion was to impair the creation of oval cells early during the hepatocarcinogenesis protocol.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Labeling of oval cells during induction of hepatic hyperplastic nodules by DEN and 2-AAF. Rats received a single dose of DEN (150 mg/kg i.p.); 2 weeks later, animals were gavaged with 2-AAF (1.5 mg) for 8 days. At the mid-point of 2-AAF administration, animals were subjected to a two-thirds partial hepatectomy. [3H]Thymidine was administered for 4 days beginning 24 hr before the end of 2-AAF administration (n = 4 animals/time point). , normal rats; , vitamin D-depleted rats. Data are presented as means ± S.E.M. Statistically significant differences between group means were evaluated by a two-way analysis of variance. Effect of vitamin D depletion, P < .001; effect of time after 2-AAF withdrawal, P < .001; interaction, P < .04; Tukey post hoc test for effect of vitamin D depletion at 1 week after 2-AAF withdrawal, P < .001; all other time points, P > .05.

	Discussion

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In the present studies, the well characterized "resistant" hepatocyte model developed by Solt and co-workers (Solt and Farber, 1976; Solt et al., 1977) was used to evaluate the influence of vitamin D status on the early response to carcinogenesis. In this model, exposure to the genotoxic carcinogen DEN, followed by partial hepatectomy during treatment with the cytostatic agent 2-AAF, inhibits the replication of normal hepatocytes (resistant hepatocytes) and promotes the expansion of a novel, pluripotent, stem cell compartment (oval cells) originating in the portal triad. In the early stages of hepatocarcinogenesis, oval cells rapidly proliferate in the periportal area; they finally invade other areas of the liver acinus (Nagy et al., 1994). These cells show some common features with ductular cells in electron microscopy (Grisham, 1962; Evarts et al., 1987), but functionally they also express several phenotypic markers of normal hepatocytes, such as albumin, glucose-6-phosphatase and -fetoprotein (Evarts et al., 1989; Hsia et al., 1992; Alpini et al., 1992). The ultimate fate of the oval cell population is, however, still a subject of debate. Gerlyng et al. (1994) have proposed no precursor-product relationship between oval cells and hepatocytes, and the suggestion has been made that they play a role in the genesis of hepatocellular carcinoma (Faris et al., 1991). Others, however, have presented strong evidence showing that oval cells are precursors of both normal mature hepatocytes (Evarts et al., 1989; Lemire et al., 1991) and ductular cells (Golding et al., 1995) and that the early foci and nodules observed in this model are derived from resistant hepatocytes, rather than from the pluripotent oval cell population (Anilkumar et al., 1995).

Our data revealed the presence of a significant number of oval cells after 2-AAF withdrawal in both groups of animals. They also showed, however, that the formation of oval cells was significantly decreased by vitamin D depletion. This observation is significant, particularly because accumulating evidence supports the notion that oval cells belong to the compartment responsible for the normal repopulation of the liver parenchyma during carcinogen insult (Nagy et al., 1994; Factor et al., 1994; Frenkel et al., 1996), whereas neoplastic nodules are now thought to originate from resistant hepatocytes (Anilkumar et al., 1995). If this is the case, then the decrease in oval cell number associated with vitamin D depletion would put these animals in a position of increased vulnerability when confronted with situations requiring normal compensatory growth, such as those observed after loss of liver mass and toxic aggression leading to necrosis or neoplastic transformation. In fact, vitamin D depletion has already been shown to decrease the normal regeneration process after partial hepatectomy (Éthier et al., 1990) and the density of basophilic hepatocytes (a marker of regeneration) after bromobenzene intoxication (Haddad et al., 1987). Our observation suggests that oval cells may also be influenced by the in vivo vitamin D status.

The early morphological changes observed during this study also indicate that vitamin D depletion seems to promote the development of early putative preneoplastic foci. Indeed, although foci numbers were found to be quite similar in the two groups of animals, the size and volume fraction of the foci were significantly larger in livers of vitamin D-depleted rats than in those of normal rats. Collectively, these data suggest that vitamin D depletion in association with hypocalcemia leads to increased susceptibility to chemicals known to induce hepatocarcinogenesis with a reduction in oval cell number, possibly further inhibiting the normal repopulation of the liver parenchyma. Although the mechanism involved in the protective effect of normal vitamin D status on the early manifestation of tumor growth is not presently known, others have suggested that vitamin D and calcium may increase intracellular calcium bioavailability and reduce lipid peroxidation (Ghoshal et al., 1987). Such a mechanism could also be present in the model system, because a similar dietary regimen of vitamin D depletion accompanied by hypocalcemia has already been shown to reduce basal as well as stimulated intracellular calcium levels in hepatocytes (Gascon-Barré et al., 1994; Bilodeau et al., 1995). However, vitamin D alone [through the action of its active metabolite 1,25(OH)₂D₃] may also be the protective agent against the observed putative preneoplastic foci, because several nonhypercalcemiant analogs of 1,25(OH)₂D₃ are known to be potent antitumor molecules (Kawa et al., 1996). Further studies will need to be conducted to investigate the longer-term protective effects of vitamin D on the genesis of hepatic tumors, as well as its influence on oval cells, because they are a common feature of several experimental models of hepatocarcinogenesis (Pack et al., 1993; Factor et al., 1994) and have also been observed in human hepatitis B virus-associated hepatocellular carcinoma (Hsia et al., 1992).