# Metallothionein as an Anti-Inflammatory Mediator

Authors: Ken-ichiro Inoue; Hirohisa Takano; Akinori Shimada; Masahiko Satoh
Journal: Mediators of Inflammation
 (2009)

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## Abstract

The integration of knowledge concerning the regulation of MT, a highly conserved, low molecular weight, cystein-rich metalloprotein, on its proposed functions is necessary to clarify how MT affects cellular processes. MT expression is induced/enhanced in various tissues by a number of physiological mediators. The cellular accumulation of MT depends on the availability of cellular zinc derived from the diet. MT modulates the binding and exchange/transport of heavy metals such as zinc, cadmium, or copper under physiological conditions and cytoprotection from their toxicities, and the release of gaseous mediators such as hydroxyl radicals or nitric oxide. In addition, MT reportedly affects a number of cellular processes, such as gene expression, apoptosis, proliferation, and differentiation. Given the genetic approach, the apparently healthy status of MT-deficient mice argues against an essential biological role for MT; however, this molecule may be critical in cells/tissues/organs in times of stress, since MT expression is also evoked/enhanced by various stresses. In particular, because metallothionein (MT) is induced by inflammatory stress, its roles in inflammation are implied. Also, MT expression in various organs/tissues can be enhanced by inflammatory stimuli, implicating in inflammatory diseases. In this paper, we review the role of MT of various inflammatory conditions.


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## Body

## 1. Introduction

Metallothioneins (MTs)
discovered as  cadmium-binding protein from horse kidney approximately five
decades ago, and  were later characterized as a low molecular weight protein with
a high cysteine content and a high affinity for divalent important metals, such
as zinc and copper, and unimportant ones, such as cadmium and mercury
(Margoshes) [1].  Because of their high metal content and
unusual bioinorganic structure, they are classified as metalloproteins [2].  MTs are
unusually rich in cysteine residues that coordinate multiple zinc and copper
atoms under physiological conditions.


## 2. Classification

In mice, there are 4 MT genes
that reside in a 50-kb region on chromosome 8 [3].  The mouse MT-I and–II genes are
expressed at all stages of development in many cell types of most organs; they
are coordinately regulated by metals, glucocorticoids, and inflammatory stress [4].  MT-III is
expressed predominantly in not only neurons but also in glia and male reproductive
organs [5–7].  MT-IV is expressed in differentiating
stratified squamous epithelial cells [3].  All four MT genes are expressed in the
maternal deciduum [8].  In humans, whereas, MTs are encoded by a
family of genes consisting of 10 functional MT isoforms, and the encoded
proteins are conventionally subdivided into 4 groups: MT-1, MT-2, MT-3, and
MT-4 proteins [9].  While a single MT-2A gene encodes MT-2
protein, MT-1 protein comprises many subtypes encoded by a set of MT-1 genes
(MT-1A, MT-1B, MT-1E, MT-1F, MT-1G, MT-1H, and MT-1X), accounting for the
microheterogeneity of the MT-1 protein [2].  As shown above, there are multiple MT genes,
expressed in distinct patterns, suggesting that they possess important functions;
however, whether they have redundant or divergent functions under both
physiological and pathological conditions is not fully understood, although the
known functions of MTs include metalloregulatory roles in cell growth,
differentiation, and apoptosis and the enhanced synthesis of MTs in rapidly
proliferating tissues, implying their crucial role in normal and neoplastic
cell growth [10].


## 3. Characteristics

These
intracellular proteins are characterized by their unusually high cysteine
content (30%) and lack of aromatic amino acids.
Because of their thiol rich content, MTs can bind to a number of trace
metals such as cadmium, mercury, platinum, and silver, and protect cells and
tissues against the toxicity of these metals.
Furthermore, MTs are among the most abundant components interacting with
the biologically essential metals zinc and copper.  MT metal-thiolate fractions, being dynamic
and of a high affinity, also facilitate metal exchange in tissues [11].MTs are present in a great
variety of eukaryotes [12], functioning
as antioxidants; they also play a protective role against hydroxyl-free
radicals.  This is relevant in tumors
which are known to be markedly radiosensitive, where radiotherapy is the
treatment of choice [13].


## 4. Function under Physiological Conditions

The
putative functions of MT include intracellular metal metabolism and/or storage,
metal donation to target apometalloproteins (especially zinc finger proteins
and enzymes), metal detoxification, and protection against oxidants and
electrophils [14].  Evidence for these functions originally came
from traditional animal, cell culture, and in
vitro models.  Furthermore, these
studies have been supported by experiments using murine models with the
targeted deletion or transgenic overexpression of MT genes. MT most
likely functions in the regulation of zinc metabolism [14].  Elevations of dietary zinc induce/enhance
intestinal MT [15], whereas maximal
intestinal Zn accumulation seems to depend on MT synthesis [16].  MT (−/−) mice accumulate less zinc in the
distal gastrointestinal tract when fed a high zinc diet [17].  In most studies, zinc absorption was
inversely related to the intestinal MT content after MT was induced by dietary,
parenteral zinc, or by fasting [14].  Studies using transgenic and knockout mice
have confirmed that MT can alter the processing of zinc taken orally because
the serum zinc concentration was inversely related to the intestinal MT level
in these mice after single oral doses of zinc [17, 18].  In turn, urinary Zn excretion levels measured
during a fast or Zn intake restriction were greater in MT (−/−) mice than in MT
(+/+) mice [19].  As well, the increase in hepatic Zn
concentration after the administration of lipopolysaccharide (LPS) was found
in MT (+/+) but not in MT (−/−) mice [20].  These results suggest that MT has the ability
to retain Zn under physiological and pathological conditions.  On the other hand, tissue Zn concentration
was reduced, and the sensitivity to Zn deficiency during pregnancy was enhanced
in MT (−/−) mice [21], and further, Zn
deficiency caused abnormalities in neonate kidney differentiation in MT (−/−)
mice [22].  Conversely, in MT-transgenic mice, Zn was
accumulated in female organs, and the teratogenicity of Zn deficiency during
pregnancy was significantly ameliorated.
Taken together, MT is likely to have a Zn metabolizing activity in the
individual level [23].Besides,
MT demonstrates strong antioxidant properties.
MT protein levels in rodent liver [24, 25] and mRNA levels in hepatic cell lines [26] are increased following injection with compounds that
result in free radical formation, for example, carbon tetrachloride, menadione, or
paraquat.  An injection of ferric
nitrilotriacetate, which produces reactive oxygen species (ROS), induces
transcriptional level of MT in the liver and kidney [27].  These findings suggest that MT plays a role
in oxidative stress.  Consistent with
this, MT is able to scavenge a wide range of ROS including superoxide, hydrogen
peroxide, hydroxyl radicals, and nitric oxide [19, 28, 29].  In particular, it
has been shown that the ability of MT to capture hydroxyl radicals, which are
primarily responsible for the toxicity of ROS, is three hundred-times greater
than that of glutathione [30], the most
abundant antioxidant in the cytosol [19].  Further, metal-thiolate clusters are
reportedly oxidized in vitro; thus,
they could scavenge deleterious oxygen radicals.  Compelling genetic evidence for this concept
comes from work using yeast.  In brief,
yeast that cannot synthesize copper MTs are more sensitive to oxidative stress
if they also lack superoxide dismutase, suggesting that yeast MT has
antioxidant functions [31].  In addition, the expression of monkey MTs
under the control of the yeast MT promoter also protects against oxidative
stress [31].  Many agents that induce oxidative stress,
such as chloroform, turpentine, diethyl maleate, paraquat, and H2O2,
can also induce MT-I and MT-II in vitro and in vivo [24, 26, 32].  This strongly
suggests that MT is involved in protecting against oxidative damage.  Conversely, mammalian cells that express
excess MTs appear to be resistant to the toxic effects of nitric oxide [33] and many electrophilic antineoplastic agents [34], which are capable of reacting with the cysteines
of MT.  Further, relatively recent
studies have demonstrated that MT is induced by oxidative stress-producing
chemicals [35], and exhibits
cytoprotection against oxidative stress-related organ damage in vivo [36, 37].Despite
the confirmed roles of MT under physical conditions, as mentioned above, a
complete identification of all the functions of this unique protein within an
integrative context has yet to emerge, particularly under pathophysiological
conditions.  Particularly, since
proinflammatory cytokines including interleukin (IL)-1, IL-6, and interferon-γ
also induce hepatic MT gene expression in vivo [38–40], the roles of MT in inflammation have been focused on.  There are conflicting reports about the role
of MT in inflammatory processes.  In
fact, MT (−/−) mice
were resistant to tumor necrosis factor (TNF)-induced lethal shock compared to MT (+/+) mice [38].  MT-I-overexpressing mice are more sensitive
to the lethal effects of TNF than MT (+/+) mice [38].  In contrast, Kimura et al. have
reported that MT (−/−) mice are more susceptible to LPS-induced lethal shock in
D-galactosamine (GalN)-sensitized mice through the reduction of alpha
(1)-acid glycoprotein than MT (+/+) mice [41].  Accordingly, it seems that the roles of MT in
inflammation depend on pathophysiologic conditions (site, route of stimuli, and
type).Whereas, to date, inflammatory diseases such as
systemic inflammatory response syndrome including acute lung injury, allergic
asthma, oxidative lung injury, and acute liver injury are as yet refractory
and/or hindering daily life, possibly due to the incomplete understanding of
molecular targets.  Thus, investigation
for the role of MT in these inflammatory diseases may provide hint for novel
therapeutic options.


## 5. Function of MT under Pathophysiological Conditions in Inflammation

### 5.1. Role of MT in Lung Injury Related to LPS

Previous
as well as our recent studies have shown the expression of MT in the lung [39, 42].
Immunohistopathological examination led to the detection of immunoreactive
MT-I/II proteins in the lungs in
endothelial and alveolar epithelial cells of MT (+/+) mice, whereas they were
not detected in those of MT (−/−) mice.  Furthermore, the expression was confirmed to
be enhanced by oxidative stimuli like LPS and ozone (O3) exposure
(data not shown).The intratracheal
instillation of LPS produces a well-recognized model of acute lung injury,
leading to the activation of alveolar macroghages, tissue infiltration of
neutrophils, and interstitial edema [43].  Although the inhalation of LPS has been
reported to induce MT expression in the lung in vivo [39, 42], there is
no evidence regarding the direct contribution of MT in acute lung injury
related to LPS.MT (−/−) and MT (+/+) mice were administered
vehicle or LPS (125 μg/kg) intratracheally.
Thereafter, the cellular profile of the bronchoalveolar lavage (BAL)
fluid, pulmonary edema, lung histology, expression of proinflammatory
molecules, and nuclear localization of nuclear factor-κ B (NF-κ B) in the lung
were evaluated.  As a result, MT (−/−)
mice were more susceptible than MT (+/+) mice to neutrophilic lung inflammation and
lung edema, which was induced by intratracheal challenge with
LPS.  After LPS challenge, MT deficiency
enhanced the vacuolar degeneration of pulmonary endothelial and type I alveolar
epithelial cells, and caused a focal loss of the basement membrane.  However, unexpectedly, LPS treatment induced
no significant differences neither in the enhanced expression of proinflammatory
cytokines and chemokines, nor in the activation of the NF-κ B pathway in the
lung between the two genotypes.  Lipid
peroxide levels in the lungs were significantly higher in LPS-treated MT (−/−)
than LPS-treated MT (+/+) mice.  These
findings suggest that MT protects against acute lung injury related to
LPS.  The effects are possibly mediated
via the enhancement of pulmonary endothelial and epithelial integrity, not via
inhibition of the NF-κ B pathway [44].Next, MT (−/−) and MT (+/+) mice were administered
vehicle or LPS (30 mg/kg) intraperitoneally.
Thereafter, coagulatory parameters, organ histology (lung, liver, and
kidney), and the local expression of proinflammatory molecules were
evaluated.  As a result,
compared with MT (+/+) mice, MT (−/−)
mice showed a significant prolongation of the prothrombin time (PT)  and activated partial
thromboplastin time (APTT), a significant increase in the levels of fibrinogen and
fibrinogen/fibrin degradation products, and a significant decrease in activated
protein C, after LPS treatment.  LPS
induced inflammatory organ damage in the lung, kidney, and liver in both
genotypes of mice.  The damage including
neutrophil infiltration in the organs was more prominent in MT (−/−) than MT (+/+) mice after LPS treatment.  In both genotypes of mice, LPS enhanced the protein
expression of interleukin (IL)-1β, IL-6,
granulocyte/macrophage-colony-stimulating factor, macrophage inflammatory
protein (MIP)-1α, MIP-2, macrophage chemoattractant
protein-1, and keratinocyte-derived chemoattractant (KC) in the lung, kidney,
and liver and circulatory levels of IL-1β, IL-6, MIP-2,
and KC.  In overall trends, however, the
levels of these proinflammatory proteins were greater in MT (−/−) than in MT (+/+) mice after LPS challenge.  Our results suggest that MT protects against
coagulatory and fibrinolytic disturbance and multiple organ damage including
lung injury induced by LPS, at least partly, via inhibition of the local
expression of proinflammatory proteins in this model (Figure 1) [45].  Nonetheless,
its underlying mechanistic pathways including new ones (e.g., Toll-like
receptors [46], NALP inflammasomes [47], neurotensin [48],
RANK-RANKL [49]) remain to be explored in
future.Figure 1
Hypothesized
mechanisms of cytoprotection of MT in LPS-related inflammation.  Figure reproduced with some modifications
with permission from FASEB journal [45].


### 5.2. Role of MT in Allergic Inflammation

Bronchial asthma is a complex syndrome,
characterized by obstruction, hyperresponsiveness, and persistent inflammation
of the airways.  Inflammation in asthma
is characterized by an accumulation of eosinophils, lymphocytes, and
neutrophils in the bronchial wall and lumen [50–52].  The mechanisms via which inflammatory cells
alter airway function in asthmatic conditions include the release of Th2
cytokines (IL-4, IL-5, and IL-13) and chemotactic mediators such as thymus and
activation-regulated chemokine, macrophage-derived chemokine, and eotaxin, and
various proteases as well as the generation of reactive oxygen species.  Thus, next, we determined the role of MT in allergic
airway inflammation induced by ovalbumin (OVA) using MT (−/−) mice.  MT (−/−) and MT (+/+) mice were intratracheally challenged
with OVA (1 μg/body) biweekly 3 times.  Thereafter, the cellular profile of the BAL fluid, lung histology, and
expression of proinflammatory molecules in the lung were evaluated.  After the final OVA challenge, significant
increases were noted in the numbers of total cells, eosinophils, and
neutrophils in BAL fluid in MT (−/−) mice compared to those in MT (+/+) mice.
Histopathologically, in the presence of OVA, the number of inflammatory
cells including eosinophils and neutrophils in the lung was larger in MT (−/−) than
in MT (+/+) mice.  The protein level of IL-1β
was significantly greater in MT (−/−) than in MT (+/+) mice after OVA challenge.  Immunohistochemistry showed that the
formations of 8-hydroxy-2′-deoxyguanosine, a proper marker of oxidative DNA
damage, and nitrotyrosine in the lung were more intense in MT (−/−) than in MT (+/+) mice after OVA challenge.  These results indicate that endogenous MT protects
against allergic airway inflammation induced by OVA, at least partly, via
suppression of the enhanced lung expression of IL-1β and via its
antioxidative potential [53].


### 5.3. Role of MT in Oxidative Lung Injury

Ozone (O3) is a highly
toxic principal oxidant found in urban environments throughout the world.  Experimental research has shown that O3 inhalation causes airway inflammation/injury in vivo [54].  Furthermore, O3 is a strong
oxidizing agent that can be rapidly converted into a number of ROS, including
hydrogen peroxide [55, 56].  In fact, O3-induced lung
inflammation/injury comprises oxidative stress-related tissue injury [57–59].
Also, O3 exposure reportedly results in oxidative stress in
the airway, possibly through the disruption of iron homeostasis [59]; iron can increase oxidant generation after O3 interaction with aqueous media and produce hydroxyl radicals [60, 61].  On
the other hand, lung expression of MT is reportedly induced by O3 exposure in vivo [62, 63].  Thus, we next examined
the role of MT in lung inflammation induced by subacute exposure to  O3 using MT (−/−) mice.  After subacute
exposure to O3 (0.3 ppm), the cellular profile of BAL fluid, pulmonary edema, lung histology,
and expression of proinflammatory molecules in the lung were evaluated.  Exposure to O3 induced lung
inflammation and enhanced vascular permeability, which was significantly greater
in MT (−/−) than in MT
(+/+)
mice.  Electron microscopically, O3 exposure induced the vacuolar degeneration of pulmonary endothelial and
epithelial cells, and interstitial edema with focal loss of the basement
membrane, which was more prominent in MT (−/−) than in MT (+/+) mice.
O3-induced lung expression of IL-6 was significantly greater
in MT (−/−) than in MT
(+/+)
mice; however, lung expression of the chemokines such as eotaxin, macrophage
chemoattractant protein-1, and keratinocyte-derived chemoattractant was
comparable between both genotypes of mice in the presence of O3.  Following O3 exposure, the
formation of oxidative stress-related molecules/adducts, such as heme
oxygenase-1, inducible nitric oxide synthase, 8-OHdG, and nitrotyrosine
in the lung was significantly greater in MT (−/−) than in MT (+/+) mice.
Collectively, MT protects against O3-induced lung
inflammation, at least partly, via the regulation of pulmonary endothelial and
epithelial integrity and its antioxidative property (64).


### 5.4. Role of MT in Lethal Liver Injury

Liver has high levels of Zn- and
Cu-bound MT and has a high capacity to regenerate.  MT is reportedly involved in hepatocyte
regeneration after partial hepatectomy [64–67] and chemical injury [68].  Similarly, previous studies have shown that
induction of MT can protect animals from hepatotoxicity of several chemicals,
such as ethanol, carbon tetrachloride, acetaminophen, and cadmium [69].Hepatic dysfunction due to liver disorders
such as viral hepatitis, liver cirrhosis, and hepatocellular carcinoma is
frequently associated with lethal coagulopathy such as DIC.  Kimura et al.  previously reported
that MT is protective against acute liver injury induced by LPS/D- GalN through
the suppression of TNF-α production/release
using MT (−/−) mice [41].  An animal model of acute (lethal) liver injury
using LPS/D-GalN develops severe coagulopathy with histological evidence of DIC
[70] quite similar to that in humans.  Furthermore, most coagulatory factors as well
as MT are produced mainly in the liver, indicating a possible role of MT in the
pathogenesis of hepatic disorder-related coagulopathy.  Besides, our above mentioned study has
implicated MT in pathophysiology of coagulatory disturbance [45].  To expand
the findings by Kimura et al., therefore, we explored the role of MT in
coagulatory disturbance during acute liver injury induced by LPS/D-GalN.  Both MT (−/−) and MT (+/+) mice were injected intraperitoneally with 30 μg/kg of LPS and 800 mg/kg of D-GalN
dissolved in vehicle.  Five hours after
the injection, blood samples were collected and platelet counts and coagulatory
parameters were measured.  LPS/D-GalN
challenge significantly decreased platelet number in both genotypes of mice in
a time-dependent fashion as compared to vehicle challenge.  However, in the presence of LPS/D-GalN, the
decrease was significantly greater in MT (−/−) than in MT (+/+) mice.
LPS/D-GalN challenge caused prolongation of the plasma coagulatory
parameters such as PT and APTT in both genotypes of mice as compared with vehicle challenge.  In the presence of LPS/D-GalN, PT and APTT
were longer in MT (−/−) than in MT (+/+)
mice.  The level of fibrinogen
significantly decreased 5 hours after LPS/D-GalN challenge in both genotypes of
mice as compared to vehicle challenge.
After LPS/D-GalN challenge, the level was significantly lower in MT
(−/−) than in MT (+/+) mice.  As compared to vehicle administration,
LPS/D-GalN administration elicited an increase in the plasma level of von
Willebrand factor in both genotypes of mice.
Further, in the presence of LPS/D-GalN, the level was significantly
greater in MT (−/−) than in MT
(+/+)
mice ([71].


## 5.1. Role of MT in Lung Injury Related to LPS

Previous
as well as our recent studies have shown the expression of MT in the lung [39, 42].
Immunohistopathological examination led to the detection of immunoreactive
MT-I/II proteins in the lungs in
endothelial and alveolar epithelial cells of MT (+/+) mice, whereas they were
not detected in those of MT (−/−) mice.  Furthermore, the expression was confirmed to
be enhanced by oxidative stimuli like LPS and ozone (O3) exposure
(data not shown).The intratracheal
instillation of LPS produces a well-recognized model of acute lung injury,
leading to the activation of alveolar macroghages, tissue infiltration of
neutrophils, and interstitial edema [43].  Although the inhalation of LPS has been
reported to induce MT expression in the lung in vivo [39, 42], there is
no evidence regarding the direct contribution of MT in acute lung injury
related to LPS.MT (−/−) and MT (+/+) mice were administered
vehicle or LPS (125 μg/kg) intratracheally.
Thereafter, the cellular profile of the bronchoalveolar lavage (BAL)
fluid, pulmonary edema, lung histology, expression of proinflammatory
molecules, and nuclear localization of nuclear factor-κ B (NF-κ B) in the lung
were evaluated.  As a result, MT (−/−)
mice were more susceptible than MT (+/+) mice to neutrophilic lung inflammation and
lung edema, which was induced by intratracheal challenge with
LPS.  After LPS challenge, MT deficiency
enhanced the vacuolar degeneration of pulmonary endothelial and type I alveolar
epithelial cells, and caused a focal loss of the basement membrane.  However, unexpectedly, LPS treatment induced
no significant differences neither in the enhanced expression of proinflammatory
cytokines and chemokines, nor in the activation of the NF-κ B pathway in the
lung between the two genotypes.  Lipid
peroxide levels in the lungs were significantly higher in LPS-treated MT (−/−)
than LPS-treated MT (+/+) mice.  These
findings suggest that MT protects against acute lung injury related to
LPS.  The effects are possibly mediated
via the enhancement of pulmonary endothelial and epithelial integrity, not via
inhibition of the NF-κ B pathway [44].Next, MT (−/−) and MT (+/+) mice were administered
vehicle or LPS (30 mg/kg) intraperitoneally.
Thereafter, coagulatory parameters, organ histology (lung, liver, and
kidney), and the local expression of proinflammatory molecules were
evaluated.  As a result,
compared with MT (+/+) mice, MT (−/−)
mice showed a significant prolongation of the prothrombin time (PT)  and activated partial
thromboplastin time (APTT), a significant increase in the levels of fibrinogen and
fibrinogen/fibrin degradation products, and a significant decrease in activated
protein C, after LPS treatment.  LPS
induced inflammatory organ damage in the lung, kidney, and liver in both
genotypes of mice.  The damage including
neutrophil infiltration in the organs was more prominent in MT (−/−) than MT (+/+) mice after LPS treatment.  In both genotypes of mice, LPS enhanced the protein
expression of interleukin (IL)-1β, IL-6,
granulocyte/macrophage-colony-stimulating factor, macrophage inflammatory
protein (MIP)-1α, MIP-2, macrophage chemoattractant
protein-1, and keratinocyte-derived chemoattractant (KC) in the lung, kidney,
and liver and circulatory levels of IL-1β, IL-6, MIP-2,
and KC.  In overall trends, however, the
levels of these proinflammatory proteins were greater in MT (−/−) than in MT (+/+) mice after LPS challenge.  Our results suggest that MT protects against
coagulatory and fibrinolytic disturbance and multiple organ damage including
lung injury induced by LPS, at least partly, via inhibition of the local
expression of proinflammatory proteins in this model (Figure 1) [45].  Nonetheless,
its underlying mechanistic pathways including new ones (e.g., Toll-like
receptors [46], NALP inflammasomes [47], neurotensin [48],
RANK-RANKL [49]) remain to be explored in
future.Figure 1
Hypothesized
mechanisms of cytoprotection of MT in LPS-related inflammation.  Figure reproduced with some modifications
with permission from FASEB journal [45].


## 5.2. Role of MT in Allergic Inflammation

Bronchial asthma is a complex syndrome,
characterized by obstruction, hyperresponsiveness, and persistent inflammation
of the airways.  Inflammation in asthma
is characterized by an accumulation of eosinophils, lymphocytes, and
neutrophils in the bronchial wall and lumen [50–52].  The mechanisms via which inflammatory cells
alter airway function in asthmatic conditions include the release of Th2
cytokines (IL-4, IL-5, and IL-13) and chemotactic mediators such as thymus and
activation-regulated chemokine, macrophage-derived chemokine, and eotaxin, and
various proteases as well as the generation of reactive oxygen species.  Thus, next, we determined the role of MT in allergic
airway inflammation induced by ovalbumin (OVA) using MT (−/−) mice.  MT (−/−) and MT (+/+) mice were intratracheally challenged
with OVA (1 μg/body) biweekly 3 times.  Thereafter, the cellular profile of the BAL fluid, lung histology, and
expression of proinflammatory molecules in the lung were evaluated.  After the final OVA challenge, significant
increases were noted in the numbers of total cells, eosinophils, and
neutrophils in BAL fluid in MT (−/−) mice compared to those in MT (+/+) mice.
Histopathologically, in the presence of OVA, the number of inflammatory
cells including eosinophils and neutrophils in the lung was larger in MT (−/−) than
in MT (+/+) mice.  The protein level of IL-1β
was significantly greater in MT (−/−) than in MT (+/+) mice after OVA challenge.  Immunohistochemistry showed that the
formations of 8-hydroxy-2′-deoxyguanosine, a proper marker of oxidative DNA
damage, and nitrotyrosine in the lung were more intense in MT (−/−) than in MT (+/+) mice after OVA challenge.  These results indicate that endogenous MT protects
against allergic airway inflammation induced by OVA, at least partly, via
suppression of the enhanced lung expression of IL-1β and via its
antioxidative potential [53].


## 5.3. Role of MT in Oxidative Lung Injury

Ozone (O3) is a highly
toxic principal oxidant found in urban environments throughout the world.  Experimental research has shown that O3 inhalation causes airway inflammation/injury in vivo [54].  Furthermore, O3 is a strong
oxidizing agent that can be rapidly converted into a number of ROS, including
hydrogen peroxide [55, 56].  In fact, O3-induced lung
inflammation/injury comprises oxidative stress-related tissue injury [57–59].
Also, O3 exposure reportedly results in oxidative stress in
the airway, possibly through the disruption of iron homeostasis [59]; iron can increase oxidant generation after O3 interaction with aqueous media and produce hydroxyl radicals [60, 61].  On
the other hand, lung expression of MT is reportedly induced by O3 exposure in vivo [62, 63].  Thus, we next examined
the role of MT in lung inflammation induced by subacute exposure to  O3 using MT (−/−) mice.  After subacute
exposure to O3 (0.3 ppm), the cellular profile of BAL fluid, pulmonary edema, lung histology,
and expression of proinflammatory molecules in the lung were evaluated.  Exposure to O3 induced lung
inflammation and enhanced vascular permeability, which was significantly greater
in MT (−/−) than in MT
(+/+)
mice.  Electron microscopically, O3 exposure induced the vacuolar degeneration of pulmonary endothelial and
epithelial cells, and interstitial edema with focal loss of the basement
membrane, which was more prominent in MT (−/−) than in MT (+/+) mice.
O3-induced lung expression of IL-6 was significantly greater
in MT (−/−) than in MT
(+/+)
mice; however, lung expression of the chemokines such as eotaxin, macrophage
chemoattractant protein-1, and keratinocyte-derived chemoattractant was
comparable between both genotypes of mice in the presence of O3.  Following O3 exposure, the
formation of oxidative stress-related molecules/adducts, such as heme
oxygenase-1, inducible nitric oxide synthase, 8-OHdG, and nitrotyrosine
in the lung was significantly greater in MT (−/−) than in MT (+/+) mice.
Collectively, MT protects against O3-induced lung
inflammation, at least partly, via the regulation of pulmonary endothelial and
epithelial integrity and its antioxidative property (64).


## 5.4. Role of MT in Lethal Liver Injury

Liver has high levels of Zn- and
Cu-bound MT and has a high capacity to regenerate.  MT is reportedly involved in hepatocyte
regeneration after partial hepatectomy [64–67] and chemical injury [68].  Similarly, previous studies have shown that
induction of MT can protect animals from hepatotoxicity of several chemicals,
such as ethanol, carbon tetrachloride, acetaminophen, and cadmium [69].Hepatic dysfunction due to liver disorders
such as viral hepatitis, liver cirrhosis, and hepatocellular carcinoma is
frequently associated with lethal coagulopathy such as DIC.  Kimura et al.  previously reported
that MT is protective against acute liver injury induced by LPS/D- GalN through
the suppression of TNF-α production/release
using MT (−/−) mice [41].  An animal model of acute (lethal) liver injury
using LPS/D-GalN develops severe coagulopathy with histological evidence of DIC
[70] quite similar to that in humans.  Furthermore, most coagulatory factors as well
as MT are produced mainly in the liver, indicating a possible role of MT in the
pathogenesis of hepatic disorder-related coagulopathy.  Besides, our above mentioned study has
implicated MT in pathophysiology of coagulatory disturbance [45].  To expand
the findings by Kimura et al., therefore, we explored the role of MT in
coagulatory disturbance during acute liver injury induced by LPS/D-GalN.  Both MT (−/−) and MT (+/+) mice were injected intraperitoneally with 30 μg/kg of LPS and 800 mg/kg of D-GalN
dissolved in vehicle.  Five hours after
the injection, blood samples were collected and platelet counts and coagulatory
parameters were measured.  LPS/D-GalN
challenge significantly decreased platelet number in both genotypes of mice in
a time-dependent fashion as compared to vehicle challenge.  However, in the presence of LPS/D-GalN, the
decrease was significantly greater in MT (−/−) than in MT (+/+) mice.
LPS/D-GalN challenge caused prolongation of the plasma coagulatory
parameters such as PT and APTT in both genotypes of mice as compared with vehicle challenge.  In the presence of LPS/D-GalN, PT and APTT
were longer in MT (−/−) than in MT (+/+)
mice.  The level of fibrinogen
significantly decreased 5 hours after LPS/D-GalN challenge in both genotypes of
mice as compared to vehicle challenge.
After LPS/D-GalN challenge, the level was significantly lower in MT
(−/−) than in MT (+/+) mice.  As compared to vehicle administration,
LPS/D-GalN administration elicited an increase in the plasma level of von
Willebrand factor in both genotypes of mice.
Further, in the presence of LPS/D-GalN, the level was significantly
greater in MT (−/−) than in MT
(+/+)
mice ([71].


## 6. Conclusion

MTs play important roles in the
physiological condition, such as heavy metal homeostasis and radical
scavenging.  Furthermore, through a
genetic approach, MT has been shown to protect against various types of (including
LPS-related, allergic, and oxidative) inflammatory conditions in mice,
implicating MT-induction/enhancement and/or zinc supplementation to
induce/enhance MT as possible therapeutic options for inflammatory diseases,
although additional research is needed to conclude its clinical
utility.



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*Source: 101659-2009-05-11.xml*