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Istituto Nazionale della Nutrizione, 00178 Rome, Italy
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENTS
FOOTNOTES
LITERATURE CITED
ABSTRACT 
Structural and functional damage to the intestine and the potential beneficial effects of dexamethasone (Dex) and thyroxine (T4) were examined in zinc-deficient rats. Rats were assigned to zinc deficient (ZD), control (C) or pair-fed (PF ) groups and fed for 40 d a zinc deficient (1 mg/kg) diet (ZD rats) or a similar diet supplemented with 50 mg Zn/kg (C and PF rats). Some rats of the ZD group were treated for the last 10 d with low (250 mg/kg) or high (5 mg/kg) doses of Dex or with T4 (100 mg/kg). Serum corticosterone of T4-treated ZD rats did not differ from untreated ZD rats. Serum T4 of T4-treated ZD rats did not differ from C rats. ZD rats developed ulcerations, inflammation and edema in the small intestine, particularly in the jejunum. PF rats did not show mucosal changes relative to C rats. ZD rats showed significantly lower crypt cell production rate (CCPR) and labeling index (LI) in the three intestinal regions, and lower cell migration rate and higher turnover time in the duodenum relative to C rats. Sucrase and maltase activities of ZD rats were significantly lower than C rats in the three mucosal regions. Treatment with the low dose of Dex resulted in fewer ulcerations compared with ZD rats. In rats administered the high dose of Dex or T4, all morphological alterations disappeared; the CCPR, LI, cell migration rate, cell turnover time and disaccharidase activities did not differ from C rats. In conclusion, Dex and T4 exert beneficial effects on zinc deficiency-induced intestinal alterations in rats.
KEY WORDS: zinc deficiency · dexamethasone · thyroxine · intestine · ratsINTRODUCTION
Deficiency of zinc is frequently observed in human populations. Zinc deficiency can be due to inadequate dietary intake, decreased absorption, increased requirements, decreased utilization, increased loss or genetic disease (Bettger and O'Dell 1993
, Brewer 1995, Sandstead 1995, Vallee and Falchuk 1993). Deficiency of zinc can be observed in many disease states such as alcoholism, liver disease, sickle cell anemia, renal disease and gastrointestinal disorders (Cho 1991, Okada et al. 1995, Vallee and Falchuk 1993).
The intestine is one of the tissues most sensitive to zinc deficiency. Flattening of villi, decreased numbers of crypts, inflammatory cell infiltration of the lamina propria and lesions of intestinal mucosa have been reported in both experimental animal and human zinc deficiency (Elmes and Jones 1979, Southon et al. 1984, Vallee and Falchuk 1993). These morphological alterations are associated with a loss of enterocyte absorptive function. Several studies have shown an impairment of disaccharidase activity after short or long periods of zinc deficiency (Gebhard et al. 1983, Park et al. 1985). However, no effect of zinc deficiency on intestinal architecture and enzyme activity was reported by other authors (Naveh et al. 1990, Zarling et al. 1985).
The biochemical basis of zinc deficiency pathology has not yet been elucidated. Zinc is essential in many biochemical processes and may have a relevant part in the control of both cell proliferation and cell loss (Bettger and O'Dell 1993). Zinc is essential for enzymes involved in DNA synthesis and mitosis (Wu and Wu 1987), is a component of many transcription factors and proteins that control the cell cycle (Endicott et al. 1995, Shields et al. 1996) and can inhibit apoptosis (Lazebnik et al. 1993, Zalewski et al. 1991). Zinc deficiency affects cell cycle progression (Chester 1989, Prasad et al. 1996), although an increase in cell death without alterations in cell cycle has also been reported in embryonal zinc deficiency (Rogers et al. 1995).
Rat enterocytes are continuously exfoliated from the tips of the villi and are replaced with new cells migrating out of the crypts to reach the villus apex in 2-3 days (Leblond 1981). Reduced crypt cell proliferation may imply a disruption of mucosal structure and function. Only a few studies have investigated whether zinc deficiency could depress the proliferation of mucosal cells. A lower rate of crypt cell division has been shown in jejunum of deficient rats (Southon et al. 1985). However, no direct evidence was reported to indicate whether the reduced mucosal cell proliferation was associated with a decreased cell migration to the villus tip. Moreover, no investigations have been conducted to study whether the three regions of intestine are equally affected by zinc deficiency.
The possibility of repair of damaged tissues in zinc deficiency has been poorly examined. Zinc compounds are used to treat patients with intestinal disorders and a zinc deficient status. However, especially with long term therapy, zinc may inhibit copper absorption and influence the metabolism of iron, calcium, and HDL cholesterol (Brewer 1995, Fox 1989, Hooper et al. 1980.)
Dexamethasone (Dex)4 and thyroxine (T4) are regulators of different intestinal activities, both in normal and pathological conditions. Several studies have shown that glucocorticoids can modulate the proliferation of intestinal epithelial cells. Some authors found a stimulatory effect (Herbst and Koldovsky 1972, Tutton 1973), whereas others reported an inhibitory effect (Wright 1978). Up to now, the role of glucocorticoids in the regulation of epithelial cell proliferation has not been well defined. Thyroid hormones are also regulators of cell proliferation, and their stimulation of epithelial cell production has been demonstrated (Carriere 1966, Tutton 1976).
Both of these hormones can also influence the activity of intestinal brush border enzymes. A role of glucocorticoids and thyroid hormones in the maturation of intestinal enzymes has been reported in several studies (Kedinger et al. 1980, Nanthakumar and Henning 1993, Yeh et al. 1991). Moreover, a synergistic effect of Dex and T4 on enzyme ontogeny has been shown, although no effect was detected when T4 was administered alone (McDonald and Henning 1992).
In this study we investigated whether changes in crypt cell proliferation, cell migration along the villus and turnover time, and enterocyte digestive functions are involved in zinc deficiency-induced damage in the three regions of intestine. Furthermore, we determined whether treatment with Dex or T4 would reverse changes in these parameters.
MATERIALS AND METHODS
Table 1.
Composition of the zinc deficient diet1
80°C for biochemical analysis.
Morphological studies.
The pieces of duodenum, jejunum and ileum were immersed in Bouin's fixative for 12 h, washed in PBS for 24 h, embedded in paraffin at 58°C and sectioned at 7 µm. After Mallory staining, sections were analyzed under a light microscope (Leitz, Germany). Histopathological observations were made on at least four sections of the different regions of the intestine. The variables selected to establish the extent of the most evident tissue modifications were ulcerations, edema, inflammatory cell infiltration (ICI) and dilatation of blood vessels. A score from 0 to 3 was assigned to the histological variables in relation to the severity of disease (0 was normal and 3 was severely damaged). The villus length was measured with a micrometer.
Crypt cell production rate (CCPR) and labeling index (LI).
Rats were injected intraperitoneally with vincristine (1 mg/kg body wt) to arrest cells in metaphase. After 6 h the intestine was removed, and sections of duodenum, jejunum and ileum were prepared as described above. The CCPR was estimated in 20 crypts by the mean number of nuclei arrested in metaphase per crypt column divided by the time of vincristine exposure (cells·crypt
1·h1). Crypt column was defined by the number of epithelial nuclei. The LI, i.e. the proportion of cells entering methaphase, was calculated as (number of metaphase-arrested cells in crypt column·100)/(number of cells in crypt column).
Cell migration rate and turnover time.
Rats were injected intraperitoneally with 100 mg/kg body wt of 5-bromo-2-deoxyuridine (BrdU; Sigma, St. Louis, MO) in saline. After 4 and 24 h, the small intestine was dissected, and pieces of duodenum, jejunum and ileum were embedded in OCT compound (TAAB Laboratories, Berkshire, UK). Tissue sections were prepared in a cryostat (5 µm thick), fixed in 70% ethanol and hydrolyzed in a 95:5 (v/v) solution of formamide:sodium citrate buffer pH 7.4 (0.3 mol/L) for 1 h at 95°C as previously reported (Hamman et al. 1992). BrdU labeling was then detected with an immunohistochemical assay using a BrdU labeling and detection kit (Boehringer Mannheim Biochemica, Milano, Italy). Twenty well-oriented villi per specimen were examined for villus height and distance from the base of the crypts to the leading edge of labeled cells. The migration rate (µm/h) was calculated as (distance at 24 h distance at 4 h)/20, according to Batt and Peters (1976). The cell turnover time (h) was calculated as the ratio of villus height to migration rate (Batt and Peters 1976).
Enzyme and protein assays.
Sucrase (EC 3.2.1.48) and maltase (EC 3.2.1.20) activities were assayed in intestine by the method of Messer and Dahlqvist (1966). Protein concentration was measured by the method of Lowry et al. (1951).
Serum corticosterone and thyroxine analysis.
Corticosterone and thyroxine were assayed in serum with an immunochemical assay using a Corticosterone-DA 125I RIA kit and a Thyroxine CT 125I RIA kit (ICN Biomedicals, Costa Mesa, CA), respectively.
Zinc analysis.
The zinc concentrations of diets and sera were determined by flame atomic absorption spectrophotometry using a SpectrAA-400 model atomic absorption spectrometer (Varian, Sunnyvale, Melbourne, Australia). The diets were first lyophilized and mineralized with nitric acid in the presence of hydrogen peroxide. The accuracy of the method was verified using the appropiate NBS Standard reference (Washington, DC).
Statistical analysis.
Data are the mean ± SEM of at least 6 experiments. The significance of the difference was evaluated by one-way ANOVA with a Fisher's protected least significant difference post hoc test (Winer 1971). Differences with P values < 0.05 were considered significant.
RESULTS
), zinc deficiency had a dramatic effect on body weight; ZD rats weighed 48% less than the C rats (Table 2). Mean body weight of the PF rats was lower than that of C rats, but it was not significantly different from the other groups. Serum Zn concentration of the ZD rats was significantly lower than that of C or PF rats, which did not differ from one another. Treatment of ZD rats with Dex or T4 did not affect body weight or serum zinc concentration. Body weight and serum zinc concentration of hormone-treated C rats did not differ from C rats (not shown).
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Table 2. Body weight and serum zinc concentration of control (C), zinc deficient (ZD), and pair-fed (PF) rats and the effect of dexamethasone (Dex) or thyroxine (T4) treatment on ZD rats1 |
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Table 3. Histologic score of zinc deficient (ZD), pair-fed (PF) and ZD rats treated with dexamethasone (Dex) or thyroxine (T4)1 |
).
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Table 4. Serum corticosterone and thyroxine (T4) concentrations in control (C), zinc deficient (ZD) and T4-treated ZD rats1 |
6. ZD rats were injected with low (250 µg/kg) or high (5 mg/kg) doses of Dex or with T4 (100 µg/kg) for the last 10 d of the experimental period. To measure crypt cell production rate (CCPR) and labeling index (LI), rats were injected with vincristine (1 mg/kg) and killed 6 h later. A: CCPR, expressed as number of cells arrested in metaphase per crypt column divided by the time of vincristine exposure. B : LI representing the proportion of cells entering metaphase. Within each region, significant differences from C rats are represented, a P < 0.01, b P < 0.05.
6. Dex (5 mg/kg) or T4 (100 µg/kg) were administered as daily intraperitoneal injections to ZD rats for the last 10 d of the experimental period. Rats were injected with 5-bromo-2-deoxyuridine (100 mg/kg) and killed 4 or 24 h later. The migration rate (A) was calculated as (distance at 24 h distance at 4 h)/20 h. The cell turnover time (B) is expressed as the ratio of villus height to migration rate. Within each region, significant differences from C rats are represented, a P < 0.01.
Enzyme activities. The sucrase and maltase activities in all three regions of intestine were significantly lower in the ZD than in the C rats (Fig. 4). The most dramatically affected was the jejunal sucrase activity. Treatment of the ZD rats with the high dose of Dex or with T4 reversed the negative effect of zinc deficiency on these activities, resulting in activities that did not differ from the C rats in the three intestinal regions.
6. Dex (5 mg/kg) or T4 (100 µg/kg) were injected to ZD rats for the last 10 d of the experimental period. Within each region, significant differences from C rats are represented, aP < 0.05, bP < 0.01.
DISCUSSION
, Lionetti et al. 1993). Moreover, there are no data on the use of a physiological dose of T4 to protect intestine from damage. To our knowledge, this is the first time that Dex and T4 have been shown to induce a complete recovery of intestinal damage caused by zinc deficiency.
). Therefore, the results obtained in this study in ZD rats administered T4 could be ascribed to an increase in circulating corticosterone. However, only a small and insignificant increase was detected in serum corticosterone of the T4-treated ZD rats compared with the ZD rats, indicating that the beneficial effects exerted by T4 are likely not mediated by corticosterone. The lack of an increase in circulating corticosterone after T4 administration can be explained by the inability of the ZD rats to increase the production of corticosteroid-binding globulin or by a negative feedback regulation due to the high serum corticosterone concentration of the ZD rats.
). In this study we have found that the CCPR and LI were markedly lower in zinc deficient rats. However, the extent of difference between ZD and C rats was similar in the three tracts of intestine or, in the case of LI, less evident in the jejunum. In addition, both the migration rate and the cell turnover time were significantly affected by zinc deficiency only in the duodenum. Since the epithelium of the jejunum is the most damaged by zinc deficiency, our results suggest that the changes in cell kinetics contribute to villus ulcerations but are not the only cause.
, Tutton 1973) and that adrenalectomy reduced the epithelial cell migration rate of fetal sheep and adult rats (Trahair et al. 1987, Tutton 1973). Moreover, studies conducted both in vivo and in vitro showed a stimulation of rat intestinal cell proliferation by T4 (Carriere 1966, Tutton 1976), although it was not clear whether this effect was mediated by an increase in corticosterone level (D'Agostino and Henning 1982). However, it has also been reported that glucocorticoids inhibit mitotic activity of crypt cells of adult rat jejunum (Wright et al. 1978). This discrepancy could be explained by the different dose and length of Dex treatment or, more likely, by the different physiological conditions of the animals.
). It is possible that Dex and T4 can also act by blocking one particular event of the cellular sequela thus preventing cellular injury. Support of this hypothesis is given by the disappearence not only of the ulcerations but also of all the morphological alterations induced by zinc deficiency after Dex or T4 treatment.
). On the contrary, Naveh el al. (1990) reported that feeding rats a zinc deficient diet did not result in a reduced disaccharidase activity in the jejunum. However, in their study the duration of zinc deficiency was shorter than in our study and the intestinal morphology was not analyzed to indicate whether intestinal alterations were present.
). More recent studies have shown a synergistic effect of T4 and Dex on enzyme ontogeny in rat small intestine (McDonald and Henning 1992, Yeh et al. 1991) and a partial stimulation by Dex alone (McDonald and Henning 1992). On the other hand, it has been shown that glucocorticoids have no effect on intestinal enzymes such as sucrase, maltase, lactase and acid 
-galactosidase of adult rats (Henning and Leeper 1982, Nanthakumar and Henning 1993). The present results show that in particular conditions such as zinc deficiency, the disaccharidase activities of adult rats can respond to Dex, indicating that the lack of glucocorticoid responsiveness occurs in normal control conditions but not necessarily in nonphysiological or pathological conditions.
). In fact, the promoter region of human and mouse sucrase-isomaltase genes do not contain the glucocorticoid response elements.
ACKNOWLEDGMENTS
FOOTNOTES
Manuscript received 22 January 1997. Initial reviews completed 20 February 1997. Revision accepted 13 May 1997.
LITERATURE CITED
-producing cells in the intestinal mucosa of children with inflammatory bowel disease.
Gastroenterology
1994;
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