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Department of Anatomy, University of Occupational and Environmental Health, School of Medicine, Kitakyushu 807-8555, Japan, and * Department of Clinical Pathology, University of Occupational and Environmental Health, School of Health Sciences, Kitakyushu, Japan
1To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • rats • zinc deficiency • glutathione S-transferase • in situ hybridization • olfactory epithelium
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Swenerton and Hurley 1968
) as well as in organogenesis of the nervous system
(Dreosti 1983
). The effect of zinc deficiency on
olfactory system function has been a controversial issue. In humans,
zinc deficiency may contribute to olfactory dysfunction in patients
with eating disorders (Russell et al. 1983
,
Tomita 1990
). Electron microscopic studies have shown
that chronic zinc deficiency in rats leads to marked degeneration of
the olfactory epithelium (Shigihara et al. 1987
). Others
have argued that zinc deficiency does not affect the turnover of cells
in mice olfactory epithelium as examined by
[3H]-thymidine autoradiography
(Mackay-Sim and Dreosti 1989
). However, detailed
analysis of the effect of a zinc-deficient
(ZD)2
diet on olfactory epithelial cells is not available.
Lee and Fong (1986)
were the first to report that
weanling rats fed a ZD diet for 8 wk exhibited reduced total activity
of glutathione S-transferase (GST; EC 2.5.1.18) with
1-chloro-2,4-dinitrobenzene as substrate in the liver, esophagus and
stomach. GST constitutes a gene superfamily of
xenobiotic-metabolizing enzymes that bind various ligands and
catalyze the nucleophilic addition of glutathione to diverse
electrophilic substrates (Jakoby 1978
). On the basis of
its biochemical characteristics, cytosolic GST is usually divided into
the following four classes: alpha, mu, pi and theta (Mannervik and Danielson 1988
, Meyer et al. 1991
). Recent
studies have shown high concentrations of GST in the olfactory
epithelium (Banger et al. 1993
). GST in the olfactory
epithelium may be involved in the termination of odorant signals as
well as protection of the olfactory receptor cells against airborne
toxic compounds and/or residuals of dying cells (Ben-Arie et al. 1993
, Burchell 1991
). Previous
immunohistochemical studies have shown that these GST are localized in
the supporting cells of the olfactory epithelium, and that class mu is
the major GST isoform in the olfactory epithelium of adult rats
(Banger et al. 1994
, Rama-Krishna et al. 1994
). However, few attempts have been made to investigate the
biochemical, molecular biological and immunohistochemical aspects of
the olfactory epithelium in ZD rats. In particular, the effects of ZD
on GST expression in olfactory epithelial cells have not yet been
determined.
In this study, we used immunocytochemistry to determine the distribution of GST mu and its gene in rat olfactory epithelial cells under different dietary zinc conditions. We also used the proliferating cell nuclear antigen (PCNA) and neuron-specific enolase (NSE) for the immunocytochemical identification of mitotic cells and receptor cells, respectively, in olfactory epithelial cells under different dietary zinc conditions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Eight-week-old male Wistar rats weighing 250 ± 20 g were
studied (Seac Yoshitomi, Fukuoka, Japan). The care and use of the rats
strictly followed "The Guiding Principles for the Care and Use of
Animals," as set out by our university in accordance with the
principles of the Declaration of Helsinki. Rats were housed
individually in acid-washed stainless steel cages at 22°C with a
12-h light:dark cycle; they were allowed free access to
double-distilled water (DDW) and fed a semipurified
zinc-adequate diet (58.0 mg zinc/kg) for 1 wk to allow acclimation
to these conditions. The rats were then divided randomly into four
groups. The first group (n = 10) was allowed free
access to a zinc-adequate diet (ad libitum consumption group, AL).
The second group (n = 10) was fed a ZD diet (0.5 mg
zinc/kg). The third group (n = 10) was pair-fed
(PF) a zinc-adequate diet at a level equal to the mean intake of
the ZD group. The above diet regimens were applied for 7 wk. The fourth
group (n = 10) of rats was fed a ZD diet for 7 wk
followed by a zinc-adequate diet for another 5 wk as the
zinc-replacement (ZR) group. These dietary regimens were based on
the Nippon CLEA Mineral Adjective Refined Diet A12551 (Clea Japan,
Osaka, Japan). The composition of each diet is shown in Table 1
.
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Serum zinc analysis.
Blood samples were collected from the left ventricle before perfusion
with physiologic saline solution. Serum samples were prepared by
centrifugation for 10 min at 2000 x g and stored at
-40°C until use. Zinc concentrations in the samples were assayed
after dilution with DDW (1:20) directly in the flame of a Hitachi
Z-8200 Zeeman Atomic Absorption Spectrophotometer (Tokyo, Japan)
according to the modification of Oyama et al. (1994)
.
Zinc concentrations were calculated from a standard curve generated
using Zinc Standard Solution (Kanto Chemical, Tokyo, Japan).
Immunohistochemistry.
The olfactory epithelial tissue was fixed with 40 g/L PFA in 0.1 mol/L PB for 72 h at 4°C, rinsed with 0.1 mol/L PB containing 100 g/L sucrose, decalcified with 0.1 mol/L EDTA in 0.1 mol/L PB for 2 wk at 4°C, dehydrated through graded ethanol series and embedded in paraffin (Histosec; Merck, Darmstadt, Germany). Serial 5-µm-thick sections were prepared using a microtome, mounted on glass slides (MAS coated Superfrost; Matsunami, Osaka, Japan) and air-dried at 4°C. After deparaffinization and hydration, sections were digested with 4 g/L pepsin in 0.01 mol/L HCl for 30 min at 37°C and blocked with 3 g/L H2O2 in methanol for 20 min to remove endogenous peroxidase. After being rinsed with PBS, they were incubated with normal goat serum for 15 min followed by incubation in a humid chamber with rabbit anti-GST mu (Novocastra Laboratories, Newcastle upon Tyne, UK) at a dilution of 1:1200 in PBS, or rabbit anti-NSE (ScyTek, Logan, UT) at a dilution of 1:2 in PBS, or mouse anti-PCNA (Dako, Glostrup, Denmark) at a dilution of 1:150 in PBS for 16 h at 4°C. The labeled streptavidin-biotin complex method (DAKO LSAB Kit; Dako, Carpinteria, CA) was used for immunocytochemistry. The peroxidase complex was visualized by treatment with a freshly prepared diaminobenzidine tetrahydrochloride (0.1 g/L) solution with 0.1 g/L H2O2 for 5 min. The specificities of the above immunoreactions were confirmed by replacing each primary antibody with either normal rabbit serum or PBS.
Preparation of cRNA probes.
The template cDNA for polymerase chain reaction (PCR) was synthesized
from poly (A)+ RNA of normal rat olfactory epithelium
according to the procedure described by Kudo et al. (1999)
. Oligonucleotides for rat GST mu and rat ß-actin cDNA
synthesis were as follows: GST upper primer (GST mu 1–24),
5'-ATGCC(CT)ATGA(CT)ACTGGG(ATG)TACTGG-3', GST lower primer (GST mu
600–621), 5'-AGGT(GC)TTG(CT)GAGG(AT)AGCGGCTG-3', actin upper primer
(ß-actin 845–870), 5'-TCATGAAGTGTGACGTTGACATCCGT-3', and actin lower
primer (ß-actin 1104–1129), 5'-CCTAGAAGCATTTGCGGTGCACGATG-3'. These
primers were synthesized at Hokkaido System Science (Sapporo, Japan)
and were designed on the basis of the sequence of the coding region of
rat and human GST mu cDNA (Ding et al. 1985
,
Vorachek et al. 1991
) and rat ß-actin cDNA
(Nudel et al. 1983
). The following conditions were used
for PCR amplification: 35 cycles of incubation, 30 s at 94°C,
30 s at 55°C and 1 min at 72°C, and final extension at 72°C
for 5 min for 3' A overhangs. A 621-bp rat olfactory GST mu cDNA
fragment and a 285-bp rat olfactory ß-actin cDNA fragment were
subcloned separately into a plasmid vector pCRII-TOPO using a TOPO
TA Cloning Kit (Invitrogen, San Diego, CA). The resulting transformed
cells were checked for the sequence of the inserted cDNA using an ABI
PRISM BigDye Terminator Kit and ABI PRISM Model 377 Auto Sequencer
(Applied Biosystems, Foster City, CA). After the plasmids were
linearized, digoxygenin-labeled antisense and sense RNA probes were
prepared with SP6 and T7 RNA polymerase using a DIG RNA labeling kit
(Boehringer Mannheim, Mannheim, Germany) according to the instructions
provided by the manufacturer.
In situ hybridization.
Tissues were fixed with 40 g/L PFA in 0.1 mol/L PB for 72 h at 4°C, decalcified with 0.1 mol/L EDTA in 0.1 mol/L PB for 2 wk at 4°C, dehydrated in graded ethanol series and embedded in paraffin. Serial 5-µm-thick sections were cut, mounted on glass slides and air-dried at 4°C. After deparaffinization and hydration, sections were digested with 20 mg/L proteinase K in 10 mmol/L Tris-HCl, 1 mmol/L EDTA (pH 8.0) for 20 min at 37°C, and treated with 0.2 mol/L HCl for 10 min, followed by 2.5 mL/L acetic anhydrate in 0.1 mol/L triethanolamine-HCl (pH 8.0) for 10 min. Sections were preincubated with a hybridization buffer containing 500 mL/L deionized formamide, 1X Denhardt’s solution (0.2 g/L Ficoll, 0.2 g/L polyvinylpyrrolidone and 0.2 g/L bovine serum albumin), 10 mmol/L Tris-HCl (pH 7.6), 1 mmol/L EDTA, 2.5 g/L SDS, 600 mmol/L NaCl, 200 mg/L yeast transfer RNA and 100 mg/L denatured herring sperm DNA, at room temperature for 1 h in a humidity chamber. The GST mu cRNA probe was diluted in a hybridization buffer containing 5% dextran sulfate and applied to each glass slide with a concentration of 2 mg/L. Sections were incubated for 18 h at 50°C in a humidity chamber. After hybridization, sections were rinsed with 5X standard saline citrate (SSC), washed twice with 500 mL/L formamide/2X SSC for 20 min at 50°C and incubated with RNase A buffer [50 mg/L RNase A (RNaseOUT: Gibco BRL, Rockville, MD), 10 mmol/L Tris-HCl and 500 mmol/L NaCl (pH 7.5)] for 30 min at 37°C. Each section was washed twice in both 2X SSC and 0.2X SSC for 20 min at 50°C. Immunohistochemical detection of hybridized signals was performed using a DIG Nucleic Acid Detection Kit (Boehringer Mannheim). Sections were incubated with alkaline phosphatase–conjugated anti-digoxygenin antibody (1:500) for 2 h at room temperature, followed by color reaction with p-nitro blue tetrazolium chloride and 5-bromo-4-chrolo-3-indolylphosphate p-toluidine salt.
To assess the specificity of the in situ hybridization signals, two negative control procedures were performed. First, RNA was digested in randomly chosen sections by preincubation with the above RNase A buffer. Second, digoxygenin-labeled sense RNA probes were hybridized in parallel to antisense RNA probes in all cases. In addition, the ubiquitously expressed housekeeping gene ß-actin was chosen as a nonrelevant probe.
DNA-fragmentation histochemistry.
We also examined the olfactory epithelium for DNA-fragmentation (i.e., apoptosis of epithelial cells). For this purpose, sections adjacent to those used for the hybridization described above were stained according to the TdT-mediated dUTP-digoxigenin nick end labeling (TUNEL) method (Apop Taq Plus Peroxidase In Situ Apoptosis Detection Kit; Intergen, Purchase, NY) following the manufacturer’s recommendations.
Histological analysis.
Olfactory epithelia from five randomly chosen rats from each group were evaluated histologically. We determined the number of cells that were immunoreactive for NSE, PCNA or GST mu, and positive for GST mu mRNA or TUNEL in 250 µm (for TUNEL, 1 mm) length of four randomly selected areas in each olfactory epithelium using a microscope (BX50, Olympus, Tokyo, Japan) equipped with a Polaroid Digital Camera (Nippon Polaroid, Tokyo, Japan). To quantify the hybridization signal for GST mu mRNA in individual cells, the intensities of the color reaction of at least four cells per each olfactory epithelial section were estimated using the above microscopic compound tools and NIH Image analysis software (version 1.61).
Statistical analysis.
All data were expressed as means ± SEM Differences in serum zinc concentrations, number of cells positive for NSE, PCNA, GST mu and TUNEL, and in the intensity of signals for GST mu mRNA were examined for significance with the use of one-way ANOVA, followed by Bonferroni’s multiple comparison test for post-hoc testing. Statistical analysis was performed using Prism 2.0a (Macintosh version, GraphPad Software, San Diego, CA). A P-value < 0.01 denoted a significant difference.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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); they consumed significantly less food than AL rats and developed
alopecia at the end of ZD treatment.
|
). PCNA-immunoreaction was observed in nuclei of the basal cells in
all experimental groups. The density of immunoreaction appeared to be
similar among AL, PF, ZD and ZR rats (Fig. 2
). PCNA-immunoreactive supporting cells were rarely observed in the
olfactory epithelium, and there were no significant differences in the
number of these cells among AL, PF, ZD and ZR groups. Cells
immunoreactive to GST mu were seen in the supranuclear portion of
supporting and basal cells in all rats. However, the immunoreactivity
of the supranuclear portion of supporting cells was significantly less
in ZD rats (Fig. 3C
) compared with other experimental rats. Table 2
summarizes the
number of NSE, PCNA and GST mu immunoreactive cells in all experimental
groups.
|
|
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). The hybridized signal from the supranuclear portion of supporting
cells was significantly less in ZD rats (Fig. 4D
), although
that in ZR rats was not different from AL and PF rats (Fig. 4E
). In the RNase pretreated control sections, signals were
abolished in all cases (not shown). Hybridization using labeled sense
control probe in adjacent sections of Figure 4C
showed no detectable signals (Fig. 4A
).
|
). Furthermore, the intensity of GST mu mRNA signal per supporting cell
in ZD rats was significantly weaker than that in other rats (Fig. 5B
). On the other hand, we found no significant differences
in the numbers of supporting cells in the olfactory epithelia among AL,
PF, ZD and ZR rats in sections stained with hematoxylin and eosin (not
shown).
|
). Hybridization using a labeled sense control probe in adjacent
sections of Figure 6C
showed no detectable signals
(Fig. 6A
).
|
). There were no significant differences in numbers of those cells among
AL, PF, ZD and ZR rats (Table 2)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Pellier and Astic (1994)
demonstrated a rapid
disappearance of NSE immunoreactivity after degeneration of receptor
cells. In this study, there was no marked reduction in NSE
immunoreactivity in receptor cells of ZD rats compared with other
groups, indicating that zinc deficiency does not lead to degeneration
of olfactory receptor cells. PCNA is an intranuclear polypeptide
synthesized in the late G1 and S phase
(Hall et al. 1990
, Mathews et al. 1984
).
Immunohistochemical analysis in this study showed localization of
PCNA-immunoreactivity in the basal cells of the olfactory
epithelium of ZD rats as well as in other groups. These result are
similar to those reported by Mackay-Sim and Dreosti (1989)
who demonstrated similar uptakes of
[3H]-thymidine in basal cells of ZD and PF
mice. Together, these results suggest that zinc deficiency in this
study did not affect receptor cell turnover. Moreover, our analysis of
the extent of apoptosis with the use of the TUNEL method showed that
the number of apoptotic cells in the olfactory epithelia of ZD rats was
not different from that in other control rats, indicating that zinc
deficiency does not induce apoptosis of olfactory epithelial cells.
GST in supporting cells showed distinct differences; both
immunoreactivity for GST protein and strong hybridization signals for
GST mRNA were observed in AL, PF and ZR rats but were rare in ZD rats.
These cells in ZD rats showed hybridization signals for ß-actin mRNA,
but GST immunoreactivity and its hybridization signals were hardly
detectable. These findings suggest that zinc deficiency directly and/or
indirectly reduced expression of GST in the supporting cells of the
olfactory epithelium. At the same time, PCNA-immunoreactivity in
supporting cells was similar in AL, PF, ZD and ZR rats, and
DNA-fragmentation was not detected in supporting cells in any
group. Although intestinal villi of ZD rats contain a considerable
number of apoptotic-positive cells (Cui et al. 1999
), zinc deficiency in our rats did not affect the turnover
and apoptosis of supporting cells in the olfactory epithelium. This
discrepancy may be due to the long life span (Farbman et al. 1988
, Weiler and Farbman 1998
) and low
rate of apoptosis (Deckner et al. 1997
) of the
supporting cells in normal adult rats.
Immunoreactivity for GST has already been examined in olfactory
supporting cells in adult rats (Banger et al. 1994
) as
well as perinatal rats (Rama-Krishna et al. 1994
). These
studies suggested that GST may be involved in xenobiotic metabolism of
various substances such as odorants and cellular debris of dying cells
in olfactory epithelium. Several groups have indicated that xenobiotic
enzymes terminate the odorant response with a simple mechanism that
involves the removal of the above substances by bioactivation and
accelerated excretion of the modified compounds (Ben-Arie et al. 1993
, Burchell 1991
, Hatt 1996
,
Lazard et al. 1991
). Thus, the reduced expression of GST
in the supporting cells might induce abnormal xenobiotic metabolism in
our ZD rats. However, the exact mechanism that down-regulates the
GST synthesis rate is unknown at present. Further molecular biological
analyses of the regulatory elements of GST genes and their
transcription factors should be performed using our model of ZD rats.
Recent studies have reported that zinc transporters and divalent cation
transporter-1 regulate zinc absorption and homeostasis in various
organs of mammals (for review see McMahon and Cousins 1998
). Because the involvement of xenobiotic metabolism of
these transporter families has not yet been identified in the olfactory
epithelium, further studies are required to elucidate their function in
olfactory supporting cells in rats fed a zinc-deficient diet.
In conclusion, we have demonstrated that zinc deficiency in rats is associated with a marked reduction in the expression of GST mu in the supporting cells of the rat olfactory epithelium.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received February 16, 1999. Initial review completed March 24, 1999. Revision accepted September 27, 1999.
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