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Departments of
*
Foods and Nutrition and
Zoology, University of Manitoba, Winnipeg, Manitoba, R3T 2N2 Canada
3To whom correspondence should be addressed. E-mail: ctaylor{at}ms.umanitoba.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • zinc • metallothionein • immunolocalization • small intestine • liver • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, zinc
(Zn)4
can affect many aspects of cellular metabolism, including physiological
processes, such as immune function, antioxidant defense, growth and
development (2
3
4)
. Therefore, an adequate supply of
dietary Zn and the maintenance of Zn homeostasis are crucial for normal
functioning of these systems. Primary mechanisms responsible for Zn
homeostasis involve changes in Zn absorption and excretion in
gastrointestinal tract and hepatic Zn storage and disposal
(5)
. At the cellular level, metallothionein (MT) may be
central to the homeostatic regulation of Zn metabolism
(6
,7)
.
MT is a small (6000- to 7000-Da) metal-binding protein
characterized by high cysteine content and high affinity for heavy
metals (7)
. It has been proposed that MT maintains Zn
homeostasis by controlling cellular Zn uptake, distribution and
excretion and by acting as a short-term storage reservoir for the
metal (6
,8)
. MT is present at basal levels in all major
mammalian organs, and its synthesis can be induced by many factors,
including heavy metals, hormones and chemical and physical stress
(8)
. The regulation of MT gene expression by Zn and other
metals occurs via the metal responsive element on the MT gene promoter
(8)
, and studies indicate that there is an interaction
between dietary Zn and a Zn-sensitive transcription factor that
binds to metal responsive element (9)
. Consistent with
these findings, Zn status has been shown to affect MT concentration and
its mRNA synthesis in various tissues of growing and adult rats and
mice (9
10
11
12
13
14)
. The effect of dietary Zn on tissue MT is
also evident in the maternal-fetal complex (15)
. The
protein is particularly sensitive to dietary Zn in organs of
absorption, storage, secretion and excretion, such as small intestine,
liver, pancreas and kidney, indicating specific roles of MT in Zn
absorption, storage, transport and elimination. In liver, intestine and
kidney, a dose-dependent relationship has been observed between
dietary Zn intake and MT mRNA (9
,12)
. Responses of MT to
changes in dietary Zn supply are very rapid, occurring within the first
few hours or days of dietary treatment (9
10
11)
.
Despite the substantial evidence for a relationship between Zn status and MT tissue concentration, the effects of Zn nutrition on MT tissue distribution have not been studied. Furthermore, knowledge about the effects of short-term Zn repletion on tissue MT concentration and distribution is lacking. Thus, the objective of this study was to investigate the effects of Zn deficiency and 24-h Zn repletion on immunohistochemical localization and concentration of MT in rat small intestine and liver. Immunohistochemistry allows for visualization of MT tissue and intracellular distribution and may provide important information about cell-type-specific responses of MT to dietary Zn. Pair-fed groups were included in the experimental design to separate changes due to Zn from changes due to feed intake during Zn deficiency and repletion.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Weanling 46–56 g male Sprague Dawley rats (Charles River Laboratories,
St. Constant, PQ) were randomly assigned to treatment groups
(n = 8) and housed individually in stainless steel
hanging cages with wire bottoms in a temperature- (21–23°C) and
humidity- controlled (55%) room with lights on from 0700 to 1900 h. After a 3-d acclimation period with nutritionally complete control
diet, rats were fed Zn-deficient (ZD group; < 1 mg Zn/kg) and
control (30 mg Zn/kg) diet, either pair-fed to the Zn-deficient
group (PF group) or consumed food ad libitum (C group) for 16 d.
One-half of ZD and PF rats were repleted with the control diet (ZR
and PFR groups, respectively) for an additional 24 h. Double
deionized water was consumed ad libitum from plastic bottles with
stainless steel sipper tubes. The experimental diets, containing egg
white and additional biotin (2 mg/kg diet) and potassium phosphate (5.4
g/kg diet for the growth formulation), have been previously described
(16)
. Body weight and feed intake data were collected.
Rats were killed by CO2 asphyxiation and trunk blood was
collected after decapitation. Samples of liver and small intestine
(midsection) were excised, washed briefly with phosphate-buffered
saline (pH 7.4) and fixed in 10% phosphate-buffered formalin for
24–48 h. The remaining tissues were frozen in liquid nitrogen and
stored at -80°C. Care and treatment of experimental animals received
approval of the University of Manitoba Protocol and Management
Committee and followed the Canadian Council on Animal Care guidelines
(17)
.
Zn and MT concentrations.
Tissues, wet ashed with concentrated nitric acid (18)
, and
serum were analyzed for Zn using a Varian Spectra AA-30
Spectrophotometer (Georgetown, ON). MT concentrations in liver and
small intestine (midsection) were determined by a cadmium saturation
assay based on the method of Eaton and Toal (19)
. Protein
concentration of the homogenized samples was determined using the
bicinchoninic acid protein assay (Pierce, Rockford, IL).
Immunohistochemical localization of MT.
MT localization in liver and small intestine was determined using
standard procedures for indirect immunoperoxidase staining. Briefly,
slides were incubated with monoclonal mouse anti-MT antibody (clone
E9; Dako, Carpinteria, CA; diluted 1:50 in phosphate-buffered
saline) for 1 h at room temperature (small intestine) or overnight
at 4°C (liver), followed by prediluted Dako Envision System goat
anti-mouse/anti-rabbit peroxidase labeled polymer for 30 (small
intestine) or 45 (liver) min. The reaction product was visualized with
3,3'-diaminobenzidine tetrahydrochloride (DAB·4HCl; Polysciences,
Warrington, PA). Tissues were counterstained with Harris hematoxylin
and the small intestine was additionally counterstained with eosin for
easier identification of Paneth cells. The specificity of the reaction
was confirmed with negative and positive control conditions:
substitution of anti-MT antibody with mouse immunoglobulin G (IgG)
49-
(clone DAK-GO1; Dako); omission of anti-MT antibody from the
procedure; and incubation of the anti-MT antibody with small
intestine and liver tissue from a MT null mouse, newborn rat and a
Zn-supplemented rat. Computer images of immunostained sections were
obtained using Northern Eclipse software (Empix Imaging, Toronto, ON).
The intensity of MT staining was estimated using an arbitrary
subjective scale of pluses and minuses according to Jasani and Elmes
(20)
.
Statistical analysis.
One-way analysis of variance (SAS software, Version 6.04; SAS Institute, Cary, NC) followed by Duncan’s multiple range test was used to determine significant differences among treatment groups. Correlation analysis (Zn status vs. MT concentration) was conducted using Pearson’s correlation coefficient. For all analyses, differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Zn-deficient (ZD) and pair-fed (PF) rats had severe growth
retardation as evidenced by significantly lower (43–46%) body weights
(Table 1
). After 24-h repletion with the control diet, the ZD and ZR groups and
PF and PFR groups had similar body weights; however, liver weights of
ZR and PFR groups were greater than the ZD and PF groups, respectively.
During the 24-h repletion, the body weight of the ZR group increased
10% (from 88.5 ± 1.5 to 97.5 ± 1.5 g) and the PRF
group increased 21% (from 98.5 ± 2.7 to 119.1 ± 3.2 g). Similarly, the daily feed intake of the ZR group increased by 137%
(from 5.9 ± 0.4 to 14.0 ± 1.2 g) and the PFR group
increased by 254% (from 5.9 ± 0.4 to 20.9 ± 0.6 g)
during the 24-h repletion phase.
|
Concentrations of Zn and MT in small intestine and liver.
Zn deficiency had a significant effect on MT concentration but not on
Zn concentration in small intestine (Fig. 1A
, and B
) and liver (Fig. 2A
, and D
). However, ZD rats had significantly
less total liver Zn than PF and C rats (19% and 55%, respectively;
Fig. 2B
) and less liver Zn per g body than the other dietary
treatment groups (Fig. 2C
). The intestinal and hepatic MT
concentrations of ZD group were only 43% and 39% of the C group,
respectively. After the 24-h repletion with control diet, the ZR group
had similar MT concentrations as the C group in both organs, but the
hepatic Zn concentration of the ZR group was 16% lower than the C
group. Hepatic Zn content was unaffected in ZR rats, despite an
increase in liver weight during the 24-h Zn repletion (Table 1)
.
However, hepatic Zn per g body was greater than in ZD rats. In small
intestine, neither Zn nor MT concentration was influenced by pair
feeding (Fig. 1)
. In contrast, pair feeding had a significant effect on
Zn and MT concentrations in liver (Fig. 2)
. The hepatic Zn and MT
concentrations of the PF group were significantly higher than other
groups, and 30% and 153% higher, respectively, than the C group.
After 24-h repletion with the control diet, the PFR group had liver MT
concentrations not different from the C group. Total hepatic Zn content
was unaffected but the hepatic Zn concentration of the PFR group was
19–38% lower than the PF and C groups. A significant positive
correlation was found between tissue Zn and MT concentrations in liver
(r = 0.60, P = 0.0001) but not in small
intestine. When MT was expressed as µg/g tissue (data not shown),
µg/liver (Fig. 2E
) or µg/g body (data not shown),
results were similar to MT expressed as µg/mg protein (Figs. 1B
and 2D
, respectively).
|
|
Results of the immunoperoxidase staining for rat small intestine and
liver are depicted in Figures 3
and
4
, respectively. The staining was specific for MT because the
negative control procedures gave no staining (Figs. 3H
and 4H
) and there was strong positive staining in tissues from
Zn-supplemented rat (Figs. 3G
and 4G
) and
newborn rat (data not shown).
|
|
. In the ZD group, the staining was absent or
weak and limited to very few cells (Fig. 3A
). Similarly,
cytoplasm and nuclei of surface columnar epithelial cells, with the
exception of goblet cells, stained with varying intensity in all groups
but the ZD group. The strongest and most consistent staining of
epithelial cells was present in PF (Fig. 3C
) and C rats
(Fig. 3E
). In ZR (Fig. 3B
) and PFR rats (Fig. 3D
), there was some individual variability with staining
ranging from very weak to strong. Staining of epithelial cells was
predominantly localized in proliferative regions of villi. No MT
staining was detected in lamina propria, submucosa, muscularis and
vasculature of small intestine in any of the treatment groups. Figure 3F
is a higher magnification of the PF intestinal section
and depicts strong MT staining in Paneth and epithelial cells and
the lack of MT staining in goblet cells.
In liver, strong and moderate MT staining was demonstrated in
hepatocytes of PF (Fig. 4C
) and PFR (Fig. 4D
)
rats, respectively. MT staining was weak in C group (Fig. 4E
). In ZD (Fig. 4A
) and ZR (Fig. 4B
)
groups, MT staining was absent, or weak to moderate staining appeared
in only a few cells. In the PF group (Fig. 4C
), and to a
lesser extend in the PFR group (Fig. 4D
), MT staining was
concentrated predominantly around central veins. In contrast, the MT
staining was scattered throughout the liver of C group (Fig. 4E
). In C, PF and PFR groups, the staining was localized in
cytoplasm and nuclei of hepatocytes. Figure 4F
demonstrates
strong MT staining around central vein in the liver of PF group under
high magnification. The differences among treatment groups using an
arbitrary scale for the intensity of MT staining are summarized in
Table 2
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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22
23)
, injury (24)
or tumor-related
changes in tissue MT localization (25
,26)
or have
investigated MT distribution in tissues from cadmium-treated rats
(27)
. This study is the first to report differential MT
localization in rat tissues in response to dietary deficiency and
repletion of Zn and energy.
The absence of MT staining in surface epithelial cells observed in
response to dietary Zn deficiency (Fig. 3A
) and the presence
of strong MT staining after Zn repletion (Fig. 3B
) supports
the role of MT in Zn storage and toxin or heavy metal protection in the
gut (8)
. The pattern of MT distribution in epithelial
cells, where it was predominantly localized in the proliferative
regions of villi as opposed to the tip of villi, does not favor the
idea of MT function in Zn absorption but may imply a role for MT and Zn
in cell proliferation and mucosal turnover. The lack of MT staining in
Paneth cells in ZD rats (Fig. 3A
) and the appearance of
strong MT staining in ZR rats (Fig. 3B
) further supports the
role of MT in the heavy metal protection and also suggests the
involvement of Zn and MT in gut immunity. Selective localization of MT
in Paneth and surface epithelial cells similar to that observed in our
study in all groups but the ZD group has been reported in the small
intestine of cadmium-treated (27)
and developing rats
(22)
. It is believed that Paneth cells play an important
role in the elimination and storage of heavy metals and the control of
intestinal pathogens (28)
. Furthermore, patients with
Acrodermatitis enteropathica, a genetic disorder of Zn
absorption, show structural abnormalities in Paneth cells that
disappear after Zn supplementation (29)
.
In liver, the weak MT staining in hepatocytes of the control rats that
consumed food ad libitum (Fig. 4E
) indicates a low basal
level of MT in adult liver and is consistent with other
immunohistochemical studies (21
,22)
. The absence of MT
staining in the liver of ZD rats (Fig. 4A
) is likely due to
reduced MT synthesis in Zn deficiency and is in agreement with the
study by Sato et al. (11)
that did not detect MT in the
liver of Zn-deficient rats using radioimmunoassay. Strong MT
staining in the liver of PF rats that diminished after repletion with
the control diet (Fig. 4C
, and D
) may be
explained by food restriction. It is known that physical stress,
including starvation and energy restriction, results in the induction
of hepatic MT synthesis, which is believed to be mediated via a stress
hormone response (8
,11
,30)
.
The specific distribution of MT around the central veins in the livers
of PF and PFR groups (Figs. 4C
, and D
,
respectively) may be unique to energy restriction because it has not
been observed in other immunohistochemical studies
(21
22
23
,27)
. The localization of MT around central veins
may be related to MT function in hepatic Zn storage or may reflect
functional aspects of the zonation of hepatic lobules. Hepatocytes in
the area surrounding central veins are specialized in drug and lipid
metabolism and glycolysis (31)
. Altered energy metabolism
and obesity have been reported in MT-1 and -2 null mice
(32)
. The MT knockout mice are also more sensitive to drug
and cadmium hepatotoxicity than wild-type controls
(33
34
35)
. Therefore, the localization of MT around the
central veins may be linked to detoxification processes or energy
metabolism. Furthermore, hepatic cells proximal to central veins have
lower levels of nutrients and oxygen and, thus, are more sensitive to
injury (31)
. Accordingly, centrilobular necrosis is
observed in the conditions involving oxidative stress or hypoxia
(31)
. MT knockout mice exhibit more extensive
acetaminophen-induced hepatic damage and more intense staining for
lipid peroxidation adducts around central veins than do normal
controls, and it has been concluded that MT may offer protective
mechanism at times of increased oxidative stress (33)
.
Similarly, because food restriction results in increased oxidative
processes, strong MT staining detected around central veins in the PF
rats may indicate a protective role for MT during stress imposed by
energy restriction.
Tissue MT concentrations determined by the
109Cd-binding assay reflected the
immunohistochemical findings in C, PF and PFR groups but not in ZD and
ZR groups (Figs. 1B
, 2D—F
, 3
and 4
). Although MT staining in small intestine and liver of ZD rats
was not detected by immunohistochemistry, reduced MT concentrations
were quantified by the Cd-binding assay. Similarly, after Zn
repletion, hepatic MT staining was still not detectable, although MT
concentrations were 25% higher in ZR rats. These discrepancies may be
due to an overestimation of MT concentration by the
109Cd-binding assay (36)
.
Although Zn deficiency resulted in the absence of MT staining (Figs. 3A
and 4A
) and reduction of MT concentration in
small intestine and liver (Figs. 1B
and 2C
), it
did not alter intestinal or hepatic Zn concentrations (Figs. 1A
and 2A
) despite impaired growth, reduced serum
and femur Zn concentrations (Table 1)
and reduced hepatic Zn content
(Fig. 2B
, and C
). It has been shown that that
65Zn tracer binds predominantly to intestinal MT
when rats are fed supplemental Zn levels, but the majority of
65Zn binds to high-molecular-weight proteins when
a Zn-deficient diet is fed (10)
. Similarly, it has
been reported that Zn accumulated by liver is associated with MT only
in response to very high dietary Zn levels (>500 mg/kg)
(37)
. This may explain the reduction in hepatic MT
concentration without changes in Zn concentration in Zn-deficient
rats observed in this and other studies (11
,13)
and the
absence of immunologically detectable MT in the liver of the ZR group
after 24-h repletion with 30 mg Zn/kg diet. Furthermore, the present
study suggests that reduced hepatic MT concentration and content in Zn
deficiency may be related to a lower liver Zn content. Our findings
together with the previous studies indicate that at low dietary Zn
levels, Zn concentration is not a determinant of MT concentration in
rat intestine and liver, but the study suggests that MT plays a role in
transient Zn storage when excessive amounts of Zn are present. These
findings also imply the possible existence of other proteins involved
in the absorption and/or transport of Zn in small intestine.
In summary, MT immunolocalization and concentration in small intestine and liver were responsive to changes in Zn status, supporting a role for MT in the regulation of Zn homeostasis. The differential localization of MT in intestinal Paneth cells and in surface epithelial cells of the proliferative region of the villi after dietary Zn manipulation implies a function of MT and Zn in gut immunity and intestinal mucosal turnover. The localization of hepatic MT around central veins in pair-fed rats may be unique to energy restriction and may be associated with oxidative processes and/or Zn transport. However, these findings in the young Zn-deficient rat model may not be applicable to the adult Zn-deficient rat model. Future nutrition studies of MT localization in other tissues and under various physiological and pathological conditions will increase our understanding of relationships between dietary intake and nutrient function at a tissue and cellular level.
|
ACKNOWLEDGMENTS |
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|
FOOTNOTES |
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2 Supported by Natural Sciences and Engineering Research Council (NSERC) Operating Grant PIN 186434 (to C.G.T.) and NSERC Postgraduate Scholarship (to E.I.S.).
4 Abbreviations used: C, control group; IgG, immunoglobulin G; MT, metallothionein; PF, group pair-fed to zinc-deficient group; PFR, energy-repleted group (pair-fed group repleted with control diet ad libitum); ZD, zinc-deficient group; Zn, zinc; ZR, zinc-repleted group (zinc-deficient group repleted with control diet ad libitum).
Manuscript received February 14, 2001. Initial review completed March 14, 2001. Revision accepted May 8, 2001.
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