Protective role of metallothionein in acute lung injury
induced by bacterial endotoxin
H Takano, K Inoue, R Yanagisawa, M Sato, A Shimada, T Morita, M Sawada, K Nakamura,
C Sanbongi, T Yoshikawa
See end of article for
Dr H Takano,
Team, National Institute for
16-2 Onogawa, Tsukuba
Received 3 March 2004
Accepted 19 July 2004
Thorax 2004;59:1057–1062. doi: 10.1136/thx.2004.024232
Background: Metallothionein (MT) is a protein that can be induced by inflammatory mediators and
participate in cytoprotection. However, its role in inflammation remains to be established. A study was
undertaken to determine whether intrinsic MT protects against acute inflammatory lung injury induced by
bacterial endotoxin in MT-I/II knock out (2/2) and wild type (WT) mice.
Methods: MT (2/2) and WT mice were given vehicle or lipopolysaccharide (LPS, 125 mg/kg)
intratracheally and the cellular profile of the bronchoalveolar lavage (BAL) fluid, pulmonary oedema, lung
histology, expression of proinflammatory molecules, and nuclear localisation of nuclear factor-kB (NF-kB)
in the lung were evaluated.
Results: MT (2/2) mice were more susceptible than WT mice to lung inflammation, especially to lung
oedema induced by intratracheal challenge with LPS. After LPS challenge, MT deficiency enhanced
vacuolar degeneration of pulmonary endothelial cells and type I alveolar epithelial cells and caused focal
loss of the basement membrane. LPS treatment caused no significant differences in the enhanced
expression of proinflammatory cytokines and chemokines nor in the activation of the NF-kB pathway in the
lung between the two genotypes. Lipid peroxide levels in the lungs were significantly higher in LPS treated
MT (2/2) mice than in LPS treated WT mice.
Conclusions: Endogenous MT protects against acute lung injury related to LPS. The effects are possibly
mediated by the enhancement of pulmonary endothelial and epithelial integrity, not by the inhibition of the
has been proposed that MT may have an important role in
homeostasis and detoxication of heavy metals.1It can react
also with free radicals and electrophils because of its high
sulfhydryl content,2 3and can serve as a sacrificial scavenger
for hydroxyl radicals in vitro4and also protects against free
radical induced DNA damage.5–7In addition, MT is induced
by heavy metals or oxidative stress producing chemicals,8and
exhibits cytoprotection against toxicity of heavy metals or
alkylating anticancer drugs3as well as against oxidative
stress related organ damage.9 10MT-I and MT-II genes are
constitutively expressed in the liver and are highly induced by
Proinflammatory cytokines, including tumour necrosis factor
(TNF)-a, interleukin (IL)-1, IL-6, and interferon-c, induce
hepatic MT gene expression in vivo. However, there are
conflicting reports about the role of MT in inflammatory
processes. In brief, MT plays a pivotal role in mediating the
harmful effects of TNF induced shock11but MT (2/2) mice
have been reported to be more sensitive to lipopolysaccharide
(LPS) induced lethal shock.12
Acute lung injury is characterised by neutrophilic inflam-
mation, increased expression of proinflammatory cytokines,
loss of epithelial and endothelial integrity, and the develop-
ment of interstitial oedema.13–16Intratracheal instillation of
LPS produces a well recognised model of acute lung injury
leading to the activation of alveolar macroghages, tissue infil-
tration of neutrophils, and interstitial oedema.17Although
inhalation of LPS has been reported to induce MT gene and
protein in the lung in vivo,18 19there is no evidence for the
direct contribution of MT in acute lung injury elicited by LPS.
etallothionein (MT) is a highly conserved, low
cysteine residues of MT bind and store metal ions, it
The development of acute lung injury requires several pul-
monary cell populations, transcriptional regulatory factors,
and proinflammatory molecules. Nuclear factor-kB (NF-kB)
activation and the subsequent expression of proinflam-
matory mediators also have an important role.20 21However,
the effects of MT on NF-kB activation is uncertain.22 23
This study was undertaken to determine whether intrinsic
MT has a role in protecting against acute lung injury induced
by LPS using MT-I/II null (MT (2/2)) mice and control wild
type (WT) mice. The mechanisms by which MT protects
against acute lung injury were also studied and its role in the
NF-kB pathway in vivo determined.
Animals and study protocol
MT (2/2) mice whose MT-I and MT-II genes had null
mutation and WT mice were provided by Dr Choo (Murdoch
Institute for Research into Birth Defects, Royal Children’s
Hospital, Australia).24They were of a mixed genetic back-
ground of 129 Ola and C57BL/6 strains. F1 hybrid mice were
mated with C57BL/6 mice and their offspring were back-
crossed to C57BL/6 for six generations in the National Insti-
tute for Environmental Studies (NIES; Tsukuba, Japan),
reproducing normally and displaying no overt abnormality in
physical state and behaviour. These mice were routinely bled
in the vivarium of NIES as previously described.10
MT (2/2) and WT mice were treated with vehicle or LPS
(E coli B55:05, Difco Lab, Detroit, MI, USA). For both
Abbreviations: IL, interleukin; KC, keratinocyte chemoattractant; LPS,
lipopolysaccharide; MCP-1, macrophage chemoattractant protein; MIP-
1a, macrophage inflammatory protein-1a; MT, metallothionein; NF-kB,
genotypes the vehicle groups received 100 ml phosphate
buffered saline (PBS) at pH 7.4 (Nissui Pharmaceutical Co,
Tokyo, Japan) by intratracheal inoculation. The LPS groups
received 125 mg/kg LPS dissolved in 100 ml of the same
vehicle. Intratracheal inoculation was conducted as described
Lung water content, bronchoalveolar lavage (BAL),
and measurement of cytokines and lipid peroxides
The lungs were weighed and dried as previously reported.27
The wet lung weight 2 dry lung weight/body weight was
calculated (n=5 in each group). BAL and cell counts were
conducted as previously described (n=5 in each group).26 28
Enzyme linked immunosorbent assays (ELISA) for IL-1b
(Endogen, Cambridge, MA, USA), macrophage inflammatory
protein (MIP)-1a (R&D Systems, Minneapolis, MN, USA),
Systems), and keratinocyte chemoattractant (KC) (R&D
Systems) in the lung tissue supernatants (n=8 in each
group) were carried out as reported elsewhere.26 28Lipid
peroxide in the lung was measured by a Determiner LPO kit
(Kyowa Medics Co Ltd, Tokyo, Japan) (n=8 in each group).
The lungs were fixed and stained with haematoxylin and
eosin as previously described (n=4 in each group).25Lung
tissue was fixed with 2.5% glutaraldehyde and rinsed in
0.1 M phosphate buffer (pH 7.4; n=4 in each group).
Ultrathin sections were stained with uranyl acetate and lead
citrate and examined with a JEM-100CX electron microscope
(JEOL, Tokyo, Japan).
Preparation of nuclear and cytoplasmic protein and
Western blot analysis
Nuclear protein extracts (n=5 in each group) were
examined by Western blot analysis using a rabbit anti-p65
antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA)
and the bands were quantitated as described previously.26
Data were reported as mean (SE) values except for the
cellular profiles of BAL fluid and the lung water content
using Stat View version 4.0 (Abacus Concepts Inc, Berkeley,
CA, USA) as previously described.25Differences in cellular
profiles of BAL fluid, lung water content, and nuclear
localisation of p65 subunit of NF-kB were analysed by a
Kruskal-Wallis test followed by a Mann-Whitney U test using
the SPSS 8.0 statistical package for Windows (Chicago, IL,
USA). Differences in other data were analysed by ANOVA
followed by Fisher’s PLSD test (Stat View version 4.0).
p values of ,0.05 were considered statistically significant.
Role of MT in acute lung injury induced by bacterial
To examine the role of MT in pulmonary oedema related to
bacterial endotoxin we measured the lung water content
24 hours after the intratracheal instillation of vehicle or LPS
(fig 1). LPS increased the lung water content in both
genotypes of mice compared with the vehicle (p,0.01 for
MT (2/2) mice, not significant in WT mice). Following
treatment with LPS, MT (2/2) mice had a significant and
marked increase in the lung water content compared with
WT mice (p,0.05).
To determine the role of MT in neutrophilic lung
inflammation induced by bacterial endotoxin, the cellular
profile of BAL fluid 24 hours after intratracheal instillation
was examined. In both genotypes of mice LPS treatment
induced significant increases in the numbers of total cells
(fig 2A) and neutrophils (fig 2B) compared with vehicle
treatment (p,0.05). LPS treatment caused greater and
significant increases in the number of total cells (fig 2A)
and neutrophils (fig 2B) in the BAL fluid in MT (2/2) mice
than in WT mice (p,0.05).
Effect of MT deficiency on LPS induced
histopathological and ultrastructural changes in the
To determine the differences in the histological changes after
LPS treatment in the presence or absence of MT, lung
???? ??? ?????????? ???????
(MT)-I/II knock out (2/2) mice challenged with lipopolysaccharide (LPS,
125 mg/kg). The bilateral lungs were weighed immediately after
exsanguination 24 hours following the intratracheal administration of
LPS or vehicle, dried in an oven at 95˚C for 48 hours, and the wet lung
weight 2 the dry lung weight/body weight was calculated. The results
are shown as mean (SE) values (n=5). *p,0.01 v vehicle treated mice;
#p,0.05 v LPS treated WT mice.
Lung water content in wild type (WT) and metallothionein
????? ????? ?×????? ??? ??????
??????????? ?×????? ??? ??????
(BAL) fluid in WT and MT (2/2) mice challenged with LPS. Twenty four
hours after the intratracheal administration of vehicle or LPS the lungs
were lavaged for the analysis of BAL fluid. The total cell count was
determined on a fresh fluid specimen using a haemocytometer.
Differential cell counts were assessed on cytological preparations stained
with Diff-Quik. The results are shown as mean (SE) values (n=5).
*p,0.05 v vehicle treated mice; #p,0.05 v LPS treated WT mice.
(A) Total cells and (B) neutrophils in bronchoalveolar lavage
1058Takano, Inoue, Yanagisawa, et al
vehicle; MT (2/2) mice: (B, C) LPS, (E) vehicle. The histopathological changes induced by LPS in WT mice showed moderate infiltration of neutrophils
(A; arrows). LPS treatment showed more significant damage in the lungs of MT (2/2) mice which included recruitment of neutrophils (B; arrows) and
alveolar haemorrhage (C). Original magnification 6320 (A, B, D, E), 6250 (C). LPS induced ultrastructural changes included the thickening of the
alveolar basement membrane (arrow) in WT mice (I). LPS treated MT (2/2) mice had large vacuoles in the cytoplasm of endothelial cells and type I
alveolar epithelial cells (F, G). Focal loss of the basement membrane (arrow) was seen in the alveolar walls of LPS challenged MT (2/2) mice (H). RBC,
red blood cell; type I, type I alveolar epithelial cell; En, endothelial cell; Bm, basement membrane. Bar=1 mm.
LPS induced histopathological changes (A–E) and ultrastructural changes (F–I) in the lungs of WT and MT (2/2) mice. WT mice: (A) LPS, (D)
?????α ????????? ????
????β ????????? ????
??????? ??? ??????????
?? ????????? ????
????? ????????? ????
and (D) keratinocyte chemoattractant (KC) in lung tissue supernatants 24 hours after challenge with vehicle or LPS (n=8 in each group) measured by
enzyme linked immunosorbent assays. White bar, WT mice; black bar, MT (2/2) mice. Results are mean (SE) values. *p,0.05 v vehicle treated mice.
Protein levels of (A) interleukin (IL)-1b, (B) macrophage inflammatory protein (MIP)-1a, (C) macrophage chemoattractant protein (MCP)-1,
Protective role of metallothionein in acute lung injury 1059
specimens stained with haematoxylin and eosin were
examined 24 hours after intratracheal instillation. In the
presence of LPS, WT mice showed a moderate infiltration of
neutrophils (fig 3A) while in MT (2/2) mice there was a
marked recruitment of neutrophils and interstitial oedema
with alveolar haemorrhage (fig 3B, C). Vehicle administra-
tion alone caused no histological changes in either genotype
(fig 3D, E).
To elucidate the mechanisms by which MT protects against
acute lung injury, especially against aggravated vascular
permeability related to bacterial endotoxin, the ultrastruc-
tural changes in the lung were examined by electron
microscopy 24 hours after intratracheal instillation. LPS
treated MT (2/2) mice had large vacuoles in the cytoplasm
of the endothelial cells and type I alveolar epithelial cells
(fig 3F, G) accompanied by a focal loss of basement
membrane in the alveolar walls (fig 3H). In LPS treated WT
mice, on the other hand, only thickening of the alveolar
basement membrane occurred (fig 3I). Following vehicle the
alveolar wall was normal in both MT (2/2) and WT mice
(data not shown).
Effect of MT deficiency on lung expression of
proinflammatory cytokines and chemokines related to
To investigate the role of MT in the lung expression of
proinflammatory cytokines and chemokines related to
bacterial endotoxin, we compared the protein levels of IL-
1b, MIP-1a, MCP-1, and KC in lung tissue supernatants from
the four experimental groups 24 hours after intratracheal
instillation. LPS treatment induced significant increases in
these cytokines and chemokines compared with vehicle
treatment in both genotypes (fig 4A–D, p,0.01). In the
presence of LPS treatment, however, the local expression of
these cytokines and chemokines was not significantly
different between the two genotypes (fig 4A–D).
Effect of MT deficiency on NF-kB activation related to
To determine whether the absence of MT affects the
activation of NF-kB related to bacterial endotoxin in vivo,
we compared nuclear protein levels of the p65 subunit of NF-
kB in the lungs 2 hours following intratracheal instillation.
In both genotypes LPS treatment significantly increased
nuclear localisation of the p65 subunit of NF-kB compared
with vehicle treatment (fig 5, p,0.05). However, there were
no significant differences between the protein levels of MT
(2/2) mice and WT mice after LPS administration (fig 5).
Effects of MT on lipid peroxidation induced by
We also examined the effects of MT on lipid peroxide
formation in the lung. Compared with the vehicle, LPS
increased the lipid peroxide content in both genotypes of
mice (fig 6; p,0.05 for MT (2/2) mice, difference not
significant in WT mice). LPS treatment led to a significantly
greater increase in the lipid peroxide content in MT (2/2)
mice than in WT mice (fig 6; p,0.05).
This study has shown that MT (2/2) mice are more sensitive
to acute lung injury induced by intratracheal administration
of LPS than WT mice, especially to pulmonary oedema.
Electroscopic examination showed that LPS treatment causes
vacuolar degeneration of pulmonary vascular endothelial
cells and type I alveolar epithelial cells, and focal loss of the
basement membrane in MT (2/2) mice but not in WT mice.
The local expression of proinflammatory cytokines and
chemokines and the activation of the NF-kB pathway in
the lung induced by LPS were not significantly different in
the presence or absence of MT. The lipid peroxide content
in the lungs was significantly higher in LPS treated MT
(2/2) mice than in LPS treated WT mice.
MT is a regulator of zinc and copper homeostasis. It has
recently been reported that several inflammatory stimuli
induce hepatic MT. The induction of MT is mediated by
several cytokines such as IL-1, IL-6, TNF-a, and interferon-
c.19 29–33More recently, it has been reported that MT (2/2)
mice are protected from TNF induced lethal shock compared
with WT mice.11In addition, MT-I overexpressing mice are
more sensitive to the lethal effects of TNF than WT mice.11In
contrast, Kimura and colleagues have reported that MT (2/2)
mice are more sensitive to LPS induced lethal shock in
GalN sensitised mice through the reduction of a1-acid
glycoprotein.12The specific role of MT in inflammation
therefore remains controversial.
As for lung inflammation, inhaled LPS induces MT
expression in vivo,18 19which suggests that MT may play a
role in lung inflammation induced by LPS. In particular, LPS
induces MT in epithelial cells in the lung.18We also confirmed
that immunoreactive MT proteins in the lungs are found in
the endothelial cells and alveolar epithelial cells of WT mice
but not in those of MT (2/2) mice (data not shown). In this
study we found that MT (2/2) mice were more susceptible to
acute inflammatory lung injury induced by LPS than WT
mice. In particular, pulmonary oedema induced by LPS was
markedly enhanced by MT deficiency. Based on previous
reports18 19and the results of this study, it is suggested that
(NF-kB) in WT and MT (2/2) mice treated with vehicle or LPS. Nuclear
localisation of the p65 subunit of NF-kB was investigated 2 hours after
intratracheal administration using Western blot analysis. The top panel
shows the actual membrane pictures of p65 and the bottom panel shows
the band density for p65. White bar, WT mice; black bar, MT (2/2)
mice. Each density represents the mean (SE) of at least five animals per
group. *p,0.05 v vehicle treated mice.
Nuclear localisation of the p65 subunit of nuclear factor-kB
determined by Determiner LPO kit. Values are mean (SE) of six animals
per group. White bar, WT mice; black bar, MT (2/2) mice. *p,0.05 v
Formation of lipid peroxides in lung tissue supernatants
1060Takano, Inoue, Yanagisawa, et al
MT may have protective properties against acute inflamma-
tory lung injury induced by bacterial endotoxin, predomi-
nantly by protecting the integrity of the pulmonary vascular
Despite the sensitivity of the vascular endothelium to both
heavy metal toxicity and oxidative stress,34 35little is known
about the role of MT in vascular endothelial/epithelial
integrity. In human lungs immunoreactive MT is detected
in pleural endothelial cells and basal cells from the bronchial
epithelium.36On the other hand, cultured sheep pulmonary
artery endothelial cells which overexpress MT are resistant to
pro-oxidant stimuli in a metal independent fashion.37It has
also been postulated that MT protects endothelial cells
against oxidative stress with or without subsequent increased
expression of cytokines, thrombin, and endothelin-1 in
human venous endothelial cells.38
In the present study, electron histological examination of
the lungs showed that LPS caused more pronounced damage
of the vascular endothelial cells, endothelial basement
membrane, and alveolar epithelial cells in MT (2/2) mice
than in WT mice. It is likely that the protective role of MT in
this model is mediated through the maintenance of
endothelial and epithelial integrity. Since enhanced expres-
sions of cyclo-oxygenase (COX)-239and Rho kinase40have
been reported to play a role in endothelial retraction and
permeability, we examined the local expression of these
proteins by Western blotting (data not shown). In both
genotypes LPS treatment increased COX-2 and Rho kinase
proteins in the lung compared with vehicle treatment.
However, there were no significant differences in the levels
of these two proteins between MT (2/2) and WT mice after
LPS administration (data not shown). Further studies are
needed to elucidate the molecular mechanism by which MT
protects the vascular integrity.
Activation of transcription factors such as NF-kB and the
subsequent production of proinflammatory mediators play a
critical role in the development of acute lung injury. NF-kB
activation in the lung after intratracheal instillation of LPS is
correlated with cytokine mRNA expression and neutrophilic
alveolitis, supporting the idea that NF-kB activation is a
pivotal event in the generation of neutrophilic lung inflam-
mation.41IL-1b, MIP-1a, MCP-1 and IL-8 have also been
shown to participate in the pathogenesis of acute lung
injury.42–45Intriguingly, MT is observed in the nucleus and/or
the cytoplasm of cells,46suggesting that it can interact with
nuclear transcription factors. In fact, TNF induced NF-kB
activation in WT cells has been reported to be lower than that
in MT (2/2) cells.23Transfection of the MT-I gene to MT
(2/2) cells reduces NF-kB activation by suppressing the
degradation of I-kB, which suggests that MT functions as a
negative regulator of NF-kB activation. In contrast, Abdel-
Mageed and Agrawal22have shown that MT potentiates the
activation of NF-kB, so the apparent correlation between MT
and NF-kB has not been established. In the present study MT
deficiency did not alter the nuclear localisation of NF-kB.
Although the levels of NF-kB induced proinflammatory
molecules were increased in the lungs after LPS challenge,
the differences between MT (2/2) and WT mice were not
statistically significant. Our results indicate that the protec-
tive effect of MT on acute lung injury related to bacterial
endotoxin may not be mediated via NF-kB dependent
pathways. Further studies are needed to elucidate the role
of MT in NF-kB mediated gene expression in other
We have previously confirmed the expression of pro-
inflammatory cytokines and chemokines including IL-1b,
MIP-1a, MCP-1, and KC in the lung 24 hours after the
intratracheal administration of LPS,26 28and the localisa-
tion of NF-kB 2 hours after LPS administration.26Close
correlations were found between NF-kB activation, the
enhanced local expression of proinflammatory mediators,
and the magnitude of acute lung injury.26We therefore
examined the expression of proinflammatory mediators
24 hours after intratracheal LPS challenge and the activation
of NF-kB 2 hours after LPS challenge in the present study. In
addition, the overall trends for the expression of these
proinflammatory proteins in the lung at other time points
(2 and 6 hours after challenge) were similar to those at
24 hours after the intratracheal challenge in the present
study (data not shown).
Oxidative stress is thought to play a role in the pathogen-
esis of acute lung injury.47It has been reported that endotoxin
treatment results in reduced glutathione levels in the
lungs.48 49Augmentation of the pulmonary antioxidant status
can ameliorate LPS induced lung injury.50MT can also
assume the function of superoxide dismutase in yeast51and
protect against lipid peroxidation in erythrocyte ghosts
produced by xanthine oxidase derived superoxide anion
and hydrogen peroxide.52One possibility is that the increased
lung inflammation and pulmonary oedema in MT (2/2)
mice result from the loss of antioxidative effects caused by
MT deficiency. We therefore looked at the contribution of
oxidative stress to the aggravation of LPS induced injury in
the absence of MT. The immunoreactivity of 8-OhdG,
pentosidine, N-(carboxymethyl) lysine (CML), and nitro-
tyrosine in the lungs of the four experimental groups
were compared by immunohistochemistry (data not shown).
8-OhdG is a proper marker of oxidative DNA damage.
Advanced glycation end products such as pentosidine and
CML are reported to be the final products of oxidative
stress.53–55In addition, reactive nitrogen species have a
number of inflammatory actions and their products such as
nitrotyrosine are accurate biomarkers of oxidation of amino
acids.56In the presence of LPS challenge, however, the
expression of these molecules did not differ between the two
genotypes. Lipid peroxidation is another biomarker for
oxidative stress. We have previously reported that lung injury
caused by LPS is concomitant with the enhancement of lipid
peroxidation in the lung.57We therefore studied the effects of
MT on lipid peroxide formation in the lung and found that
the lipid peroxide content was higher in the lungs of LPS
treated mice than in those of vehicle treated mice, both in the
presence and absence of MT. In particular, LPS treatment
resulted in significantly higher levels of lipid peroxides in the
lungs of MT (2/2) mice than in those of WT mice. It is
suggested that the enhancement of lipid peroxidation is
involved, at least partly, in the aggravation of acute lung
injury induced by LPS in MT (2/2) mice. Lipid peroxidation
might also be related to the ultrastructural changes in the
endothelial and epithelial cells seen in LPS treated MT (2/2)
In conclusion, this study has shown that endogenous MT
protects against acute lung injury induced by bacterial
endotoxin possibly via a protective effect on the vascular
integrity. The effect does not appear to be mediated through
inhibition of the NF-kB pathway and subsequent pro-
inflammatory cytokine expression. Augmentation of MT
may provide alternative therapeutic strategies for acute lung
injury, especially when treatments for inhibition of the NF-
kB pathway are ineffective.
H Takano, K Inoue, R Yanagisawa, National Institute for Environmental
Studies, Tsukuba, Japan
H Takano, T Yoshikawa, Department of Medicine, Kyoto Prefectural
University of Medicine, Kyoto, Japan
M Sato, Department of Hygienics, Gifu Pharmaceutical University, Gifu,
Protective role of metallothionein in acute lung injury1061
A Shimada, T Morita, M Sawada, K Nakamura, Department of
Veterinary Pathology, Faculty of Agriculture, Tottori University, Tottori,
C Sanbongi, Meiji Seika Kaisha Co, Saitama, Japan
1 Klaassen CD, Liu J. Metallothionein transgenic and knock-out mouse models in
the study of cadmium toxicity. J Toxicol Sci 1998;23:97–102.
2 Klaassen CD, Cagen SZ. Metallothionein as a trap for reactive organic
intermediates. Adv Exp Med Biol 1981;136:633–46.
3 Lazo JS, Pitt BR. Metallothioneins and cell death by anticancer drugs. Annu
Rev Pharmacol Toxicol 1995;35:635–53.
4 Thornalley PJ, Vasak M. Possible role for metallothionein in protection against
radiation-induced oxidative stress. Kinetics and mechanism of its reaction with
superoxide and hydroxyl radicals. Biochim Biophys Acta 1985;827:36–44.
5 Abel J, de Ruiter N. Inhibition of hydroxyl-radical generated DNA
degradation by metallothionein. Toxicol Lett 1989;47:191–6.
6 Chubatsu LS, Meneghini R. Metallothionein protects DNA from oxidative
damage. Biochem J 1993;291:193–8.
7 Schwarz MA, Lazo JS, Yalowich JC, et al. Metallothionein protects against the
cytotoxic and DNA-damaging effects of nitric oxide. Proc Natl Acad Sci USA
8 Bauman JW, Liu J, Liu YP, et al. Increase in metallothionein produced by
chemicals that induce oxidative stress. Toxicol Appl Pharmacol
9 Sato M, Bremner I. Oxygen free radicals and metallothionein. Free Radic Biol
10 Takano H, Satoh M, Shimada A, et al. Cytoprotection by metallothionein
against gastroduodenal mucosal injury caused by ethanol in mice. Lab Invest
11 Waelput W, Broekaert D, Vandekerckhove J, et al. A mediator role for
metallothionein in tumor necrosis factor-induced lethal shock. J Exp Med
12 Kimura T, Itoh N, Takehara M, et al. Sensitivity of metallothionein-null mice to
LPS/D-galactosamine-induced lethality. Biochem Biophys Res Commun
13 Ware LB, Matthay MA. The acute respiratory distress syndrome. N Engl J Med
14 Chollet-Martin S, Montravers P, Gibert C, et al. Subpopulation of
hyperresponsive polymorphonuclear neutrophils in patients with adult
respiratory distress syndrome. Role of cytokine production. Am Rev Respir Dis
15 Goodman RB, Strieter RM, Martin DP, et al. Inflammatory cytokines in patients
with persistence of the acute respiratory distress syndrome. Am J Respir Crit
Care Med 1996;154:602–11.
16 Suter PM, Suter S, Girardin E, et al. High bronchoalveolar levels of tumor
necrosis factor and its inhibitors, interleukin-1, interferon, and elastase, in
patients with adult respiratory distress syndrome after trauma, shock, or
sepsis. Am Rev Respir Dis 1992;145:1016–22.
17 Brigham KL, Meyrick B. Endotoxin and lung injury. Am Rev Respir Dis
18 Hur T, Squibb K, Cosma G, et al. Induction of metallothionein and heme
oxygenase in rats after inhalation of endotoxin. J Toxicol Environ Health A
19 De SK, McMaster MT, Andrews GK. Endotoxin induction of murine
metallothionein gene expression. J Biol Chem 1990;265:15267–74.
20 Xing Z, Jordana M, Kirpalani H, et al. Cytokine expression by neutrophils and
macrophages in vivo: endotoxin induces tumor necrosis factor-alpha,
macrophage inflammatory protein-2, interleukin-1 beta, and interleukin-6 but
not RANTES or transforming growth factor-beta 1 mRNA expression in acute
lung inflammation. Am J Respir Cell Mol Biol 1994;10:148–53.
21 Carter AB, Monick MM, Hunninghake GW. Lipopolysaccharide-induced NF-
kappaB activation and cytokine release in human alveolar macrophages is
PKC-independent and TK- and PC-PLC-dependent. Am J Respir Cell Mol Biol
22 Abdel-Mageed AB, Agrawal KC. Activation of nuclear factor kappaB:
potential role in metallothionein-mediated mitogenic response. Cancer Res
23 Sakurai A, Hara S, Okano N, et al. Regulatory role of metallothionein in NF-
kappaB activation. FEBS Lett 1999;455:55–8.
24 Michalska AE, Choo KH. Targeting and germ-line transmission of a null
mutation at the metallothionein I and II loci in mouse. Proc Natl Acad Sci USA
25 Takano H, Yoshikawa T, Ichinose T, et al. Diesel exhaust particles enhance
antigen-induced airway inflammation and local cytokine expression in mice.
Am J Respir Crit Care Med 1997;156:36–42.
26 Takano H, Yanagisawa R, Ichinose T, et al. Diesel exhaust particles enhance
lung injury related to bacterial endotoxin through expression of
proinflammatory cytokines, chemokines, and intercellular adhesion molecule-
1. Am J Respir Crit Care Med 2002;165:1329–35.
27 Ichinose T, Furuyama A, Sagai M. Biological effects of diesel exhaust particles
(DEP). II. Acute toxicity of DEP introduced into lung by intratracheal instillation.
28 Yanagisawa R, Takano H, Inoue K, et al. Enhancement of acute lung injury
related to bacterial endotoxin by components of diesel exhaust particles.
29 DiSilvestro RA, Cousins RJ. Glucocorticoid independent mediation of
interleukin-1 induced changes in serum zinc and liver metallothionein levels.
Life Sci 1984;35:2113–8.
30 Cousins RJ, Leinart AS. Tissue-specific regulation of zinc metabolism and
metallothionein genes by interleukin 1. FASEB J 1988;2:2884–90.
31 Schroeder JJ, Cousins RJ. Interleukin 6 regulates metallothionein gene
expression and zinc metabolism in hepatocyte monolayer cultures. Proc Natl
Acad Sci USA 1990;87:3137–41.
32 Sato M, Sasaki M, Hojo H. Differential induction of metallothionein synthesis
by interleukin-6 and tumor necrosis factor-alpha in rat tissues.
Int J Immunopharmacol 1994;16:187–95.
33 Friedman RL, Stark GR. alpha-Interferon-induced transcription of HLA and
metallothionein genes containing homologous upstream sequences. Nature
34 Crapo JD. Morphologic changes in pulmonary oxygen toxicity. Annu Rev
35 Nelson JM, Duane PG, Rice KL, et al. Cadmium ion-induced alterations of
phospholipid metabolism in endothelial cells. Am J Respir Cell Mol Biol
36 Courtade M, Carrera G, Paternain JL, et al. Metallothionein expression in
human lung and its varying levels after lung transplantation. Toulouse Lung
Transplantation Group. Chest 1998;113:371–8.
37 Pitt BR, Schwarz M, Woo ES, et al. Overexpression of metallothionein
decreases sensitivity of pulmonary endothelial cells to oxidant injury.
Am J Physiol 1997;273:L856–65.
38 Apostolova MD, Chen S, Chakrabarti S, et al. High-glucose-induced
metallothionein expression in endothelial cells: an endothelin-mediated
mechanism. Am J Physiol Cell Physiol 2001;281:C899–907.
39 Falk S, Goggel R, Heydasch U, et al. Quinolines attenuate PAF-induced
pulmonary pressor responses and edema formation. Am J Respir Crit Care
40 Essler M, Hermann K, Amano M, et al. Pasteurella multocida toxin increases
endothelial permeability via Rho kinase and myosin light chain phosphatase.
J Immunol 1998;161:5640–6.
41 Blackwell TS, Blackwell TR, Holden EP, et al. In vivo antioxidant treatment
suppresses nuclear factor-kappa B activation and neutrophilic lung
inflammation. J Immunol 1996;157:1630–7.
42 Shanley TP, Schmal H, Friedl HP, et al. Role of macrophage inflammatory
protein-1 alpha (MIP-1 alpha) in acute lung injury in rats. J Immunol
43 Standiford TJ, Kunkel SL, Lukacs NW, et al. Macrophage inflammatory
protein-1 alpha mediates lung leukocyte recruitment, lung capillary leak, and
early mortality in murine endotoxemia. J Immunol 1995;155:1515–24.
44 Yokoi K, Mukaida N, Harada A, et al. Prevention of endotoxemia-induced
acute respiratory distress syndrome-like lung injury in rabbits by a monoclonal
antibody to IL-8. Lab Invest 1997;76:375–84.
45 Jones ML, Mulligan MS, Flory CM, Warren JS, et al. Potential role of
monocyte chemoattractant protein 1/JE in monocyte/macrophage-dependent
IgA immune complex alveolitis in the rat. J Immunol 1992;149:2147–54.
46 Woo ES, Kondo Y, Watkins SC, et al. Nucleophilic distribution of
metallothionein in human tumor cells. Exp Cell Res 1996;224:365–71.
47 Gonzalez PK, Zhuang J, Doctrow SR, et al. Role of oxidant stress in the adult
respiratory distress syndrome: evaluation of a novel antioxidant strategy in a
porcine model of endotoxin-induced acute lung injury. Shock 1996;6:S23–6.
48 Suntres ZE, Shek PN. Treatment of LPS-induced tissue injury: role of liposomal
antioxidants. Shock 1996;6:S57–64.
49 Liaudet L, Murthy KG, Mabley JG, et al. Comparison of inflammation, organ
damage, and oxidant stress induced by Salmonella enterica serovar
Muenchen flagellin and serovar Enteritidis lipopolysaccharide. Infect Immun
50 Suntres ZE, Shek PN. Prophylaxis against lipopolysaccharide-induced acute
lung injury by alpha-tocopherol liposomes. Crit Care Med 1998;26:723–9.
51 Tamai KT, Gralla EB, Ellerby LM, et al. Yeast and mammalian metallothioneins
functionally substitute for yeast copper-zinc superoxide dismutase. Proc Natl
Acad Sci USA 1993;90:8013–7.
52 Thomas JP, Bachowski GJ, Girotti AW. Inhibition of cell membrane lipid
peroxidation by cadmium- and zinc-metallothioneins. Biochim Biophys Acta
53 Miyata T, Taneda S, Kawai R, et al. Identification of pentosidine as a native
structure for advanced glycation end products in beta-2-microglobulin-
containing amyloid fibrils in patients with dialysis-related amyloidosis. Proc
Natl Acad Sci USA 1996;93:2353–8.
54 Dunn JA, Patrick JS, Thorpe SR, et al. Oxidation of glycated proteins: age-
dependent accumulation of N epsilon-(carboxymethyl)lysine in lens proteins.
55 Fu MX, Requena JR, Jenkins AJ, et al. The advanced glycation end product,
Nepsilon-(carboxymethyl) lysine, is a product of both lipid peroxidation and
glycoxidation reactions. J Biol Chem 1996;271:9982–6.
56 Stadtman ER, Oliver CN. Metal-catalyzed oxidation of proteins. Physiological
consequences. J Biol Chem 1991;266:2005–8.
57 Yoshikawa T, Takano H, Takahashi S, et al. Changes in tissue antioxidant
enzyme activities and lipid peroxides in endotoxin-induced multiple organ
failure. Circ Shock 1994;42:53–8.
1062Takano, Inoue, Yanagisawa, et al