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*
Food Science and Human Nutrition Department, Center for Nutritional Sciences, and
Small Animal Clinical Sciences Department, University of Florida, Gainesville, Florida 32611-0370
2To whom correspondence should be addressed at Food Science and Human Nutrition Department, University of Florida, 201 FSHN, P.O. Box 110370, Gainesville, FL 32611-0370. E-mail: rjc{at}gnv.ifas.ufl.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • zinc • metallothionein • oxidative stress • knockout • transgenic • mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In rodents, zinc deficiency is associated with oxidative
stress in some tissues (Bray et al. 1986
, Miceli et al. 1999
, Oteiza et al. 1995
). Also,
lipoproteins from zinc-deficient rats are more sensitive to
oxidation in vitro (DiSilvestro and Blostein-Fujii 1997
). Further, zinc-deficient rodents are more susceptible
to the toxicity of the carbon tetrachloride radical (DiSilvestro and Carlson 1993). This oxidative stress could be due to
depression of a number of zinc-related functions, including
antioxidant functions (Powell 2000
). Potential
antioxidant functions for zinc include: 1) being a
structural factor for the antioxidant enzyme superoxide dismutase,
2) the ability of zinc to protect sulfhydryl groups from
oxidation and 3) the ability of zinc to keep redox active
metals (iron and copper) from binding to and causing oxidative damage
at active sites of zinc metalloenzymes and nonspecific binding sites on
proteins (reviewed in Powell 2000
). Further, zinc
deficiency might influence oxidative stress by impairing signal
transduction pathways, altering gene expression and affecting cell
proliferation, differentiation and apoptosis (reviewed in
Cousins 1996
).
Another result of zinc deficiency is reduced expression of the
zinc-containing protein metallothionein
(MT)3
in some cells and tissues (Davis and Cousins 2000
). MT
is a small cysteine-rich metal-binding protein that can bind up to
seven atoms of zinc per molecule. Expression of MT is induced by zinc
through numerous metal response elements in the MT-1 and MT-2 gene
promoters. MT expression is also induced by oxidants through an
antioxidant response element/electrophile response element and the
metal response elements of MT’s gene promoter (as reviewed in
Andrews 2000
). MT is likely involved in zinc metabolism
and protection against certain heavy metal toxicities (Davis and Cousins 2000
, Klaassen and Liu 1998
). MT also
scavenges free radicals in vitro and is believed to protect cells
against oxidative stress (Sato and Bremner 1993
,
Schroeder and Cousins 1990
). In vitro studies suggest
the hydroxyl radical may have particular reactivity with MT
(Thornalley and Vasak 1985
). The physical interaction of
MT with a number of oxidants also causes release of bound zinc, and as
discussed, the released zinc may also be protective (Berendji et al. 1997
, Fliss and Menard 1992
). As such,
suppression of MT levels during zinc deficiency may predispose tissues
to oxidative damage.
There is evidence that supplemental zinc and overexpression of MT help
protect cells and organisms from a number of stresses. For example, the
administration of pharmacological zinc doses protects rodents from the
toxicity of certain metals and other chemicals, some of which cause
oxidative stress (Blain et al. 1998
, Chvapil et al. 1973
, Dhawan and Goel 1995
). The
mechanism(s) through which supplemental zinc provides protection is
uncertain. Zinc may protect sulfhydryl groups from oxidation or may
limit the redox reactive metal content of tissues (Coppen et al. 1988
, Oteiza et al. 1995
). Many have advanced
the hypothesis that supplemental zinc provides antioxidant protection
through its powerful induction of MT gene expression.
MT induction by a number of metals (including zinc), hormones,
cytokines and other chemicals is associated with protection from the
toxicity of subsequent metal, chemical and other stresses in cell
culture and in vivo (Blain et al. 1998
, Coppen et al. 1988
, Kelley et al. 1988
, Satoh et al. 1988
, Schroeder and Cousins 1990
).
Experiments with cultured cells transfected with MT genes (Kaina et al. 1990
, Kelley et al. 1988
, Schwarz et al. 1995
) and cells from MT transgenic and knockout mice or
heterogeneous embryonic cells from these strains (Lazo et al. 1995
, Wang et al. 1999
) produced similar
results. A number of experiments with intact mice of these strains led
to similar conclusions (Kang 1999
, Liu et al. 1998a
, Rofe et al. 1998
). Other reports,
however, contradict the notion that MT is universally protective
(DiSilvestro et al. 1996
, Itoh et al. 1997
, Kaina et al. 1990
, Kelly et al. 1988
, Liu et al. 1999
).
Zinc injections induce MT and protect against various stresses in liver and cultured cells. It is not clear whether supplemental dietary zinc mimics these protective effects and, if so, whether the protection depends on MT production. The experiments reported here were designed to provide a detailed comparison of the relationship of MT genotype to the level of zinc provided in the diet on oxidative stress related to carbon tetrachloride administration. Results confirm the importance of MT expression in protection against oxidative stress but bring into question the impact of supplemental zinc and/or elevated MT expression in such defense.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MT knockout and MT transgenic mice used in these experiments were
derived from founder mice purchased from The Jackson Laboratory (Bar
Harbor, ME). The MT-overexpressing mice (designated TG) were
generated in C57BL/6 mice crossed with SJL mice (Palmiter et al. 1993
), whereas MT knockout mice (KO) were generated in
129/SvCPJ mice (Masters et al. 1994
). C57BL/6 mice
(designated CT) and 129S3/SvImJ mice (designated CK) served as
controls, respectively. All mice used were 8–11 wk of age. Mice were
housed singly in stainless steel hanging cages with a 12-h light/dark
cycle. During experiments, they were given free access to deionized
water and semipurified diet based on the AIN-76A formulation
(American Institute of Nutrition 1977
) with adequate or
supplemental zinc (10 or 500 mg zinc/kg diet, respectively; Research
Diets, New Brunswick, NJ). Care and treatment of the mice received
approval of the University of Florida Institutional Animal Care and Use
Committee.
Experimental design.
Sex-matched male and female TG, KO and control mice were fed a diet containing 10 mg zinc/kg and deionized water for 7 d. For 3 d thereafter, the diet contained 10 mg zinc/kg (Zn10) or 500 mg zinc/kg (Zn500). A sublethal dose of carbon tetrachloride (CCl4) in corn oil (207 µmol/kg body i.p.) or corn oil alone was given between 0800 and 1000 h, and the mice were killed at 0, 12, 24 or 48 h. We used this dose because, in these experiments, it was the lowest dose that consistently produced measurable signs of toxicity (histological and increased serum alanine aminotransferase [ALT] activity) with these genotypes. Food intake was measured for 3 d before the injections. Indices of zinc homeostasis, MT expression and hepatic damage were measured at 0, 12, 24 and 48 h postdosing. Because mice in the 0-h group did not receive injections, data at this time point represent the effects of diet and genotype only.
Analytical methods.
MT concentrations were measured as described previously (Davis et al. 1998
) using the cadmium (109Cd) binding
assay (Eaton and Toal 1982
). Total protein was measured
according to the method of Lowry et al. (1951
). Serum
zinc concentrations were measured by flame atomic absorption
spectrophotometry after dilution with deionized water. Tissue zinc was
measured by atomic absorption spectrophotometry after sections of liver
were digested with HNO3/H2SO4 (3:1)
as described previously (Dunn and Cousins 1989
). Serum
ALT enzyme activity was measured spectrophotometrically (Sigma 505-P;
Sigma Chemical Co., St. Louis, MO). Total thiols were measured
spectrophotometrically after treatment with
5,5'-dithio-bis(2-nitrobenzoic acid) (Jocelyn 1989
),
which generates a yellow chromophore (
max = 412
nm). Nonprotein thiols were measured by the same technique
after first precipitating protein thiols with 5% trichloroacetic acid
(306 mmol/L).
Histology.
Sections of liver were fixed in 10% buffered formalin, embedded in
paraffin and stained with hematoxylin and eosin. These sections were
analyzed visually for necrosis and other signs of hepatotoxicity
(Khoo et al. 1996
). Photomicrographs were obtained with
a Zeiss Axiovert S100 microscope (Carl Zeiss, Thornwood, NY) fitted
with a CCD camera to obtain digital images.
Statistics.
Data were analyzed by ANOVA for a three-way factorial design (2 x 2 x 2) to determine significant main effects and interactions using genotype, dietary zinc and oxidant treatment as independent variables (SAS Institute, Cary, NC). The Tukey-Kramer comparison test was used to determine significant differences between specific groups (P < 0.05). Serum ALT data were log transformed to obtain homogeneous variances. Significance was established at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Food consumption did not differ between adequate zinc
(Zn10) and supplemental zinc
(Zn500) groups and also did not differ among the
genotypes (data not shown). Measures of zinc status were serum zinc
(Figs. 1A
and
2A
), liver zinc (Figs. 1B
and 2B
) and
liver MT (Figs. 1C
and 2C
).
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At 0 h, liver zinc concentration was significantly lower in KO
mice than in control mice fed the Zn500 diet but
not lower than control mice fed the Zn10 diet
(Fig. 1B
). Twelve hours later, CK mice fed the
Zn500 diet and injected with CO
(Zn500 + CO) had 25–45% more liver zinc than
the other CK groups and significantly more (>100%) liver zinc than
all KO groups. This effect may have been due to the stress of the
injection, but all mice received an injection of corn oil vehicle or
CCl4. The CK10 + CO and
CK500 + CCl4 groups had
35–90% more liver zinc than KO groups, but not all differences were
statistically significant (0.03 < P < 0.1). At
24 and 48 h, there was significantly more liver zinc in
CK500 mice than in mice of all other
genotype-diet combinations. The only significant effect of
CCl4 treatment on liver zinc concentrations was
at 24 h, when CCl4-treated mice had lower
liver zinc concentrations than CO-treated mice. In marked contrast,
KO mice did not sequester additional zinc in the liver in response to
the Zn500 diet, CCl4
treatment or the combination.
Liver MT levels depend on genotype and dietary zinc but are also
affected by oxidant treatment and the stress of the injection (Fig. 1C
). These values roughly paralleled hepatic zinc
concentrations. As shown earlier, the level of MT in KO mouse liver
represents assay background and not MT (Davis et al. 1998
). At 0 h, 150% more MT was detected in
CK500 than in CK10 mice. At
12 h after the injections, the CK500 + CO
mice had greater MT concentrations than other mice, and
CK500 mice had greater concentrations than the
CK10 mice. At 48 h, the MT levels in the
CK500 + CCl4 group were
increased, and both CK500 groups had fivefold
greater MT levels than CK10 groups. It appears
that CCl4 toxicity delayed the induction of MT in
these mice. This effect was completely unexpected.
Transgenic overexpressing mice.
Zinc homeostasis was also altered by MT overexpression. Serum zinc was
80–100% greater in CT mice fed the Zn500 diet
than in those fed the Zn10 diet throughout the
experiment (Fig. 2A
). Serum zinc was also greater in TG mice
fed the Zn500 diet, but the difference was less
than that found in CT mice. Serum zinc was still affected by genotype
and diet after injections (TG < CT; Zn10 <
Zn500). Serum zinc was also lower in TG mice than
in CT mice in all genotype-diet and genotype-oxidant
interactions, although not all differences were significant
(TG10 < TG500 +
CT10 < CT500, 0.0001
< P < 0.09; TG + CO and TG +
CCl4 < CT + CCl4 < CT +
CO, 0.0002 < P < 0.08). In contrast, serum zinc
concentrations were significantly greater in CT +
CCl4 mice 12 h after
CCl4 was administered, similar to the serum zinc
response of KO + CCl4 mice. Throughout the time
course of 48 h, MT overexpression in TG mice caused an exaggerated
response of serum zinc to the carbon tetrachloride and corn oil
injections, indicative of the role of MT expression in influencing
serum zinc concentration during stress events.
Liver zinc concentrations were significantly greater at 0 h in the
TG mice fed the Zn500 diet (Fig. 2B
).
From 12 h postinjection, the TG500 mice had
greater concentrations than all other mice, and TG +
CCl4 mice had significantly more zinc compared
with other genotype-oxidant combinations. Of interest is that
CCl4 alone had a minimal effect on liver MT in CT
or TG mice unless combined with the Zn500 diet.
As with CK and KO mice, liver MT concentrations paralleled liver zinc
(Fig. 2C
). TG500 mice had more liver
MT at 0 h than other diet-genotype combinations. When injected
with CCl4 or CO, Zn500
groups showed an increased MT level, which peaked at 24 h and then
returned to near normal at 48 h. MT induction in the
Zn500 group injected with CO was delayed until
24 h. In all cases, the effect was markedly greater in TG mice
than in CT mice. CCl4 alone did not cause marked
MT induction in CT mice.
Hepatotoxicity and oxidative stress
Hepatotoxicity was assessed by measuring serum ALT activity and by histological analysis of liver sections for signs of damage and necrosis. Measurement of liver nonprotein thiols (NPT) (a pool composed mainly of glutathione) and liver total thiols (TT) served as measures of oxidative stress.
KO mice.
All mice had similar and normal serum ALT activity at 0 h
(Fig. 3
). At 12 h after CCl4 was given, all mice had
significantly elevated serum ALT activity, but the levels in KO mice,
compared with actual ALT activity units, were 6–12 times greater than
those in CK mice. At 24 and 48 h after CCl4,
the ALT activities had declined but were still significantly greater in
treated mice. There were no appreciable genotype or diet effects.
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) compared with CK mice (Fig. 4A
). Around
individual central veins, the cytoplasm of the hepatocytes was less
eosinophilic and more amorphous with a general loss of cytologic
structure (Fig. 4B
). The nuclei were pyknotic in many of
these cells. With the presence of localized coagulation necrosis, there
was a collapse of the hepatic sinusoids and disruption of hepatic plate
arrangement. The level of necrosis was not different between CK and KO
mice at 24 and 48 h, however. There were no differences between
dietary groups.
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). Interestingly, NPT levels at 0 h tended
(P = 0.06) to be lower in Zn500
groups than in Zn10 groups. At 12 h
postinjection, there was a general depression of NPT but no differences
among groups. At 24 h, NPT concentrations were significantly lower
in the KO + CCl4 groups, whereas in the KO + CO
groups, they had returned to normal. At 48 h,
CCl4-treated mice had significantly more NPT in
liver than CO-treated mice of all genotypes. Total thiols (TT) are
a measure of both protein thiols and NPT, including the thiols of MT.
There was a trend (P = 0.07) toward greater TT
concentrations in KO mice than CK mice at 0 h (Fig. 5B
). At 12 h posttreatment, however, liver TT
concentrations in KO mice decreased and were significantly lower than
those in CK mice. Thereafter, no differences were detected.
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). Activities rose sharply in all groups treated with
CCl4 by 12 h and remained elevated through
48 h. There were no differences due to genotype. It is also of
interest that activities in these mice did not return to basal levels,
as was observed with the CK and KO strain of mice (Fig. 3)
.
Histological analysis of hematoxylin and eosin–stained sections
revealed necrosis in CCl4-treated mice, but there
were no differences due to genotype (Figs. 7A
nnd B
). Of note is the substantial lymphocyte
infiltration in the area directly surrounding the central vein of mice
of both genotypes fed the higher zinc intake
(Zn500) (Figs. 7C
, D
, E
).
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). At 12 h after treatment, the NPT concentrations were
higher in CCl4-treated mice. Also, NPT
concentrations were lower in TG mice. At 48 h, the CT-CO group
was near the normal level and was significantly above liver NPT levels
of all other genotype-oxidant combinations. TT concentrations did
not differ among groups at 0 h (Fig. 8B
). At 24 h,
CCl4-treated mice had greater TT levels than did
CO-treated mice. Also, TG mice had significantly greater TT
concentrations than CT mice. At 48 h, no significant differences
remained. Because the thiols of MT are included in the TT measurement,
MT induction alone may have accounted for the increased TT in TG mice.
This is supported by the corresponding reduction in NPT (Fig. 8A
).
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Schroeder and Cousins 1990
). Up-regulation of MT expression by cytokines and
dexamethasone is also associated with the same protective effect,
suggesting that this metalloprotein is the species providing
protection. KO and TG mice provide the opportunity to separate the
potential cytoprotective effects of a zinc-dependent mechanism. By
using two zinc intake levels, we were able to generate different
hepatic zinc and MT levels to examine their effects on the toxicity of
a sublethal dose of the hepatotoxin CCl4.
CCl4 is metabolized to the trichloromethyl
radical by the enzyme cytochrome P4502E1 in the perivenous region of
the liver lobule (McGregor and Lang 1996
). As a result,
this region is most affected by the trichloromethyl radical, which
causes lipid peroxidation and inactivation of enzymes such as the
cytochrome P450 enzymes.
Zinc pretreatment protected against CCl4-induced
hepatotoxicity in some, but not all, in vivo studies (Chvapil et al. 1973
, Dhawan and Goel 1995
, Khoo et al. 1996
, Liu et al. 1998b
). A proposed
mechanism for the protective effect of these pharmacological doses of
zinc is that zinc induces MT and that MT is the real mediator of the
hepatoprotection. Although this might explain some of the
zinc-related protection, large parenteral zinc doses also protect
against CCl4 toxicity in the absence of MT
expression (Itoh et al. 1997
). This might be related to
suppression of CCl4 bioactivation, because
supplementation has been shown to inhibit the activity of some
cytochrome P450 enzymes (Bray et al. 1986
, Coppen et al. 1988
).
Because MT expression has been inversely related to damage after an
oxidative insult, it was expected that the damage in mouse livers in
this experiment would be inversely proportional to MT expression (i.e.,
TG < CT and CK < KO). Enhanced susceptibility to
CCl4 toxicity in KO mice has been reported
recently (Liu et al. 1998b
). We also expected that mice
consuming the Zn500 diet would be protected
compared with those consuming the Zn10 diet,
because zinc supplementation would induce more MT protein and result in
greater cellular zinc accumulation. Finally, because MT expression was
thought to be key to zinc-related cytoprotection, we expected no
protection against hepatotoxicity by zinc supplementation in KO mice.
In support of our hypothesis, we saw greater hepatotoxicity in KO mice (6- to 12-fold higher ALT activities) at the 12- h time point. At later time points, however, toxicity seemed equivalent in these genotypes. This is despite the fact that KO mice had no liver MT and were unable to sequester additional zinc. Also, TG and CT mice did not differ in the level of hepatotoxicity produced despite huge differences in hepatic MT and zinc.
To our knowledge, this is the first report in which the effect of supplemental dietary zinc on CCl4-induced hepatotoxicity in mice was examined. Also, this is the first assessment of the combination of supplemental dietary zinc and toxicity of any kind in TG mice and MT knockout mice. We show for the first time using these models that neither supplemental dietary zinc nor MT overexpression alone protected against CCl4-induced hepatotoxicity in mice. Further, no combination of MT gene expression and either adequate or supplemental dietary zinc provided protection, even though the levels of liver zinc and liver MT varied over a large range among groups.
These results argue against a direct antioxidant role for MT in
CCl4 toxicity, because such protection would
likely be dose dependent (Yao et al. 1994
). Instead, the
data fit better in a plateau model, where MT expression was important
up to a point (equal to or less than the level in CT and CK mice), but
beyond this point further expression is not useful. This is more in
line with a MT-specific function such as regulation of tissue zinc
accumulation and/or intracellular zinc trafficking. Specifically, KO
mice might be less protected against CCl4 than CK
mice because they are unable to regulate zinc homeostasis appropriately
(Davis et al. 1998
, Philcox et al. 1995
).
The most marked differences between genotypes in these experiments were alterations in zinc metabolism. In both the TG and KO experiments, the mice with the lowest MT expression displayed the weakest control over serum zinc levels after CCl4 treatment. In fact, serum zinc rose in KO mice and declined in TG mice. This is coincident with induction of hepatic MT and elevation of hepatic zinc in all except the KO mice. This is also coincident with greater hepatotoxicity in KO mice.
The role of MT in maintaining appropriate hepatic zinc levels might be
especially important under conditions of oxidative stress. The protein
is induced during the acute phase response and during hepatic
regeneration (Arora et al. 1998
, Ohtake 1978
). Also, zinc can be mobilized from MT by oxidants or
shifts in glutathione redox status (Berendji et al. 1997
, Fliss and Menard 1992
). These may be
mechanisms for mobilization of intracellular zinc during oxidative
stress (Maret 1995
). The end result may be enhanced
transfer of zinc from MT to zinc-dependent proteins (Jiang et al. 1998
).
Although other explanations for the results of this experiment
exist, we can rule out several. First, the results of this experiment
are not likely affected by differences in other antioxidants in these
mice, because the levels of other antioxidant enzymes and molecules are
reported to be similar between genotypes (Iszard et al. 1995
, Lazo et al. 1995
, Rofe et al. 1998
). Also, bioactivation of CCl4 should
not differ between genotypes, because the activity of cytochrome
P4502E1 in TG and KO mouse livers is similar to their respective
controls (Iszard et al. 1995
, Itoh et al. 1997
). Because these results are in opposition to some
experiments conducted in rats, we cannot exclude species difference as
a confounding variable. However, it seems unlikely that a process as
basic as radical scavenging, a simple oxidation-reduction reaction,
would differ between two rodent species. It should be noted that two
different strains of mice were used in these experiments, and
therefore, the results are not likely due to peculiarities of any
individual inbred strain. However, we cannot rule out the possibility
that the KO mice are better protected than we expected due to some
adaptive mechanism or mechanisms.
The lack of protection against oxidative stress by MT overexpression in
this experiment is in line with results from several other experiments
that used models of MT gene overexpression. Early studies using
transfection of the human MT-2a gene into Chinese hamster ovary K1–2
cells and several tumor cell lines found no resistance against free
radical–generating agents (Kaina et al. 1990
,
Kelley et al. 1988
). Further, TG mice were not resistant
to adriamycin cardiotoxicity or 
-irradiation (DiSilvestro et al. 1996
, Liu et al. 1999
). Schwarz et al. (1995
) found that MT overexpression protected NIH-3T3 cells
from nitric oxide–induced cytotoxicity. Viable cell determinations
were not made until 6–7 d after nitric oxide treatment, however, so it
could be argued that the difference in the number of cells at that time
was due to improved cell recovery instead of radical scavenging.
Transfection of SPAEC cells with MT protected against hyperoxia and
tertiary-butyl hydroperoxide, but the determination of viable cell
numbers was not performed until 1–2 d after oxidant exposure was
initiated (Pitt et al. 1997
). Again, it is difficult to
separate the contributions of radical scavenging and cell recovery to
cell survival. Finally, studies in heart-specific TG mice provide
convincing evidence for an antioxidant role for MT (Kang 1999
). When you factor in the astronomical level of MT
expression in these mice (10- to 130-fold of normal), the weaker
antioxidant defenses in mouse heart compared with liver, and the fact
that the murine heart expresses very little MT, it cannot be assumed
that the same results would be seen in the liver.
The lack of protection against acute, sublethal
CCl4 hepatotoxicity by zinc supplementation (500
mg/kg diet) in any of the genotypes used in this experiment strongly
suggests that the required dietary zinc level for mice (10 mg/kg diet)
provides as much protection as is possible by dietary zinc. It also
suggests that the hepatoprotective effects associated with zinc
injections are not readily reproduced with dietary zinc. Combined with
data from zinc deficiency studies, we see a plateau effect of dietary
zinc against oxidative stress in the rodent model, just as we do with
MT. Zinc-deficient diets render rodents more susceptible to
oxidative stress. Zinc-adequate diets alleviate this condition, but
supplemental dietary zinc provides no further protection. These results
do not exclude a role for zinc and/or MT in protection against chronic
CCl4 administration, however, as Zn and MT have
been shown to function in hepatic regeneration (Arora et al. 1998
).
The results of the experiments reported here illustrate the importance of MT expression in protection against oxidative stress but bring into question the impact of supplemental zinc and/or elevated MT expression in defense against oxidative stress. Further, the protection against oxidative stress appears to correlate with changes in zinc metabolism produced by MT expression.
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FOOTNOTES |
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3 Abbreviations used: ALT, alanine aminotransferase; CCl4, carbon tetrachloride; CK, control strain of metallothionein knockout mice; CT, control strain of metallothionein-overexpressing mice; KO, metallothionein knockout mice; MT, metallothionein; NPT, nonprotein thiols; TG, metallothionein overexpressing mice; TT, total thiols; Zn10, AIN-76A diets containing 10 mg Zn/kg; Zn500, AIN-76A diets containing 500 mg Zn/kg.
Manuscript received September 7, 2000. Initial review completed October 6, 2000. Revision accepted November 3, 2000.
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