[Frontiers in Bioscience E5, 939-946, June 1, 2013]
Mouse models of chemically-induced lung carcinogenesis
Haris G. Vikis1, Amy L. Rymaszewski1, Jay W. Tichelaar1
1Department
of Pharmacology and Toxicology, Medical College of Wisconsin and MCW Cancer Center, Milwaukee, WI, 53202
TABLE OF CONTENTS
1. Abstract
2. Introduction
3. Mouse models of lung cancer
4. A mouse model of inflammation promoted lung carcinogenesis
5. Lung cancer chemoprevention in rodent models of chemical carcinogenesis
6. Conclusions
7. References
1. ABSTRACT
2. INTRODUCTION
Primary pulmonary malignancies remain the
major source of cancer-related deaths in the Western
World. While surgical resection is an efficacious therapy
for those with early stage disease, the majority of patients
present with advanced malignancies and systemic
treatments, such as cytotoxic chemotherapy, have only
limited efficacy in lung cancer.
Furthermore,
chemoprevention for current or former smokers has
demonstrated only limited success using available agents.
The mouse model of primary lung carcinogenesis
represents a very valuable tool for the study of tumor
initiation, promotion, and therapy. Here we discuss several
models of chemically-induced murine lung cancer with a
specific emphasis on translational and clinically-relevant
lines of investigation. We emphasize the pros and cons of
currently available models in order to facilitate further
investigations into the development and treatment of
primary pulmonary malignancies.
Lung cancer is the leading cause of cancer death
in the United States killing an estimated 160,000 people
annually with approximately 200,000 newly diagnosed in
2010 alone (1). The number of deaths caused by lung
cancer exceeds that of colon, breast and prostate cancer
combined. Lung cancer is associated with a dismal 5-year
survival rate of 15% due to the fact that the majority of
patients are diagnosed in the late stages of disease after
metastasis has occurred. Human lung cancer is comprised
of two main histopathologic groups, non-small cell
(NSCLC) and small cell lung cancer (SCLC).
Approximately 80% of lung cancers are NSCLC,
originating from lung epithelial cells. NSCLC is further
subdivided into adeno, squamous, and large cell subtypes.
Adenocarcinomas arise in the periphery and comprise
~40% of all NSCLC. In recent decades the prevalence of
lung adenocarcinoma has been on the rise and is now the
most common cancer type among women and non-
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Mouse models of chemically-induced lung carcinogenesis
smokers. Squamous cell lung cancer (SCC) accounts for
~25% of NSCLC, arises in the central airways and is most
strongly associated with male smokers. The majority of
remaining NSCLCs (~15%) are large cell carcinomas with
a smaller percentages of mixed (e.g. adenosquamous) and
undifferentiated tumors. SCLC is histologically distinct
from NSCLC and originates from the neuroendocrine cells
of the bronchus. SCLC makes up ~15% of lung cancers,
and is associated with extremely poor prognosis because of
high metastatic potential early during tumor progression. In
developed nations smoking rates have dropped
considerably since the 1960s. Nevertheless, cigarette
smoking causes approximately 85% of lung cancer and
there remain millions of individuals who currently smoke
or are former smokers. These individuals have an elevated
risk for developing lung cancer and are targets for
chemoprevention. Additionally, smoking rates in many
developing countries continue to rise and as a result lung
cancer will continue to be a major global health problem
for the foreseeable future.
carcinogens that require metabolic activation to
electrophilic compounds that react with DNA and form
adducts. Subsequent failure of repair or misrepair results in
genetic mutations. Cytochrome P450 enzymes are central
to bioactivation of B(a)P and NNK, and are encoded by a
variety of Cyp genes that exhibit differences in tissue
expression, and chemical specificity (9). P450s are
responsible for activation of B(a)P to a diol-epoxide, that
reacts with deoxyguanine to form a bulky adduct, C8B(a)P-diolepoxide-guanine, which most often results in Gto-T nucleotide transversions. P450s also catalyze αhydroxylation of NNK, which spontaneously decomposes
to aldehydes and diazonium ions, subsequently forming O6methyl-guanine
adducts
and
ultimately
G-to-T
transversions (10). P450 enzymes are expressed most
abundantly in the liver, but are also present in the
peripheral and bronchial epithelia of the lung. Conditional,
lung-specific deletion of NADPH-P450 reductase (the only
mammalian P450 reductase gene and electron donor for
many P450 reactions), demonstrated reduced NNK-induced
tumor load concurrent with lower O6-methylguanine levels
(11). In contrast, the authors demonstrated that conditional
liver-specific deletion of NADPH-P450 reductase increased
lung tumor burden after i.p injection of NNK. This result
suggests that the overall function of liver P450 enzymes in
response to NNK is detoxification and metabolism, while
P450 enzymes in the lung bioactivate the NNK procarcinogen.
3. MOUSE MODELS OF LUNG CANCER
The mouse is the principal animal model for the
study of lung cancer. Widespread adoption of mice as
models for human lung cancer is a consequence of a long
history of use focused around the breadth of genetic
variation, ease of genetic manipulation, and the ability to
induce lung cancer with molecular and histological
similarities to human disease. Mouse lung cancer
models are now frequently used in pre-clinical tests of
therapy and prevention. Engineered models that
replicate specific genetic lesions found in human
tumors, such as expression of activated oncogenes (e.g.
Kras) or inactivated tumor suppressor genes (e.g. p53)
are often used to investigate the genesis of lung cancer.
In addition, chemical carcinogen-induced models
utilizing mouse strains with a predisposition to cancer
have been used to successfully address the genetic
complexity of human lung tumors. Our goal in this
review is to provide a summary of the current state of
these carcinogen-induced models. The reader is referred
to several recent reviews addressing the use of
engineered mouse models for further information on
genetic models of lung cancer (2-4).
Inbred mouse strains, such as A/J and SWR, have
a high incidence of spontaneous lung tumor development.
A/J strain mice have roughly 80-100% incidence of
spontaneous lung tumors after 24 months, and tumors are
often detected within the first 6 months of age (5, 12).
These strains are also very susceptible to carcinogeninduced lung tumors. Other strains such as C57BL6/J,
C3H/J and DBA are very resistant to carcinogen-induced
lung tumors, while strains such as O20 and BALB/c have
intermediate susceptibility. A/J strain mice develop
approximately 25 tumors per lung 14-16 weeks posttreatment with the carcinogen ethyl carbamate (urethane),
while the C57BL/6J strain develops less than 1 tumor per
lung on average (5, 7, 13-15) (Table 1). These straindependent differences have permitted several research
groups to map genetic susceptibility loci that associate with
both carcinogen-induced and spontaneous lung tumor
development (16, 17).
In humans, complex chemical mixtures, in
particular cigarette smoke, are the predominant initiator of
lung cancer. Because cigarette smoking is the primary
cause of human lung cancer, individual cigarette smoke
carcinogens are frequently used to induce lung tumors in
mice. This is commonly performed by intraperitoneal or
dietary administration of carcinogens of the polycyclic
aromatic hydrocarbon (PAH) and nitrosamine class (5-8).
PAHs are largely produced during the combustion of
tobacco, while nitrosamines are already present in
unburned tobacco and are formed as a consequence of the
tobacco curing process. Benzo(a)pyrene (B(a)P), a PAH,
and the nitrosamines, 4- (methylnitrosamino)-1- (3pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine
(NNN), are strong inducers of lung adenomas and
adenocarcinomas in mice. These chemicals are pro-
Murine lung tumors bear similar morphology,
histopathology, and molecular anomalies as those observed
in human tumors. The majority of tumors observed in
murine models are benign pulmonary adenomas that have
clear borders and are comprised of well-differentiated cells.
Although adenomas are rarely observed in humans, likely
because they are asymptomatic and not frequently
diagnosed, murine adenomas do exhibit histological
similarity to non-small cell lung adenocarcinoma derived
from airway type II cells. Murine adenomas are considered
precursors to murine lung adenocarcinomas as they do
progress to malignant adenocarcinomas of various subtypes
(solid, papillary, brochiolo-alveolar) which show signs of
nuclear atypia and invasiveness (18).
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Mouse models of chemically-induced lung carcinogenesis
Table 1. Selected rodent models of chemically-induced lung carcinogenesis
Model
Mouse
AD/ADC
Strain
Carcinogen
A/J
B (a)P, i.p. 100 mg/kg
Tumor
20 w: 8-10 tumors (AD),
100% incidence (20, 21)
B (a)P, i.g. 100 mg/kg (3X)
20 w: 6-8 tumors (AD),
100% incidence (20, 22, 23)
A/J
A/J
A/J
Swiss albino newborn
A/J
Rat
AD/ADC
Mouse
squamous
NNK, i.p. 100 mg/kg
Urethane, i.p. 1 g/kg
Vinyl carbamate,
i.p. 60 mg/kg
Main-stream cigarette smoke,
120 days
Main- and side-stream cigarette smoke,
5 mos smoke + 4 mos air
52 w: 15 tumors (95% AD, 5% ADC),
70-80% incidence (ADC)
16 w: 20-25 tumors (AD) (21, 24-26)
24w: 25 tumors (AD), 12% incidence (ADC)
52 w: 30% incidence ADC (27, 28)
26-33w: 6-14 tumors (AD), 80% incidence (AD), 5-20% incidence (ADC) (29)
3 tumors (AD) vs 1 spontaneous tumor (AD) (30)
B6C3F1
Mainstream cigarette smoke, lifetime
10X increase in hyperplasia, 4.6X AD and papilloma, 7.3X ADC, 5X metastatic
pulmonary ADC (31)
F344
NNK, s.c. 1.5 mg/kg (3X, 20 w)
98w: 67% incidence (AD), (33% ADC) (32)
F344
Mainstream cigarette smoke, up to 30
months
Incidence increased from 0% in control to 6% (light smoke) to 14% (heavy
smoke) (33)
Swiss – 8 w
NTCU, 3 mol, 2x week (22 w)
24w: 50% hyper/metaplasia, 10% CIS/SCC (34)
i.p. = intraperitoneal, i.g. = intragastric, i.t. = intratracheal, AD = adenoma, ADC = adenocarcinoma
Murine models of lung carcinogenesis exist for
adenocarcinoma and squamous cell subtypes (Table 1),
while currently those for small cell carcinoma rely solely
on genetic ablation of Rb and p53 genes, and large cell
models have yet to be described. The majority of mouse
lung adenoma/adenocarcinoma studies employ single
intraperitoneal injection of the carcinogens B(a)P, NNK,
urethane or vinyl carbamate in a susceptible strain such as
A/J at 5-6 weeks of age. At 20 weeks post injection these
carcinogens cause anywhere from 8-25 tumors of which
almost 100% are histologically adenomas. At 52 weeks
post-initiation, 1-2 adenocarcinomas are typically observed.
frequency to adenocarcinomas, suggesting that the Kras
mutation is an important early event in tumorigenesis (37).
Estimates are that 15-50% of all human lung
adenocarcinomas possess mutations in Kras, most
commonly in codon 12, and less often in codons 13 and 61
(38-40). The tumor suppressor genes Trp53, p16, Rb, Apc,
and p16INK4A (Cdkn2a) are also suppressed and/or
inactivated in both human and mouse tumors, either
through methylation or less frequently by mutation,
suggesting that the molecular mechanisms between species
are relatively well conserved (18, 41).
In mouse, factors influencing Kras mutational
frequency and spectrum include the type of carcinogen, the
type of adduct formed, age of the mouse (fetal or adult),
dose of carcinogen and strain susceptibility. In the A/J
mouse, activating mutations in Kras are observed in 100%
of chemically-induced lung tumors and in greater than 80%
of spontaneous tumors (21, 42). Kras mutations in humans
are typically G-T transversions and correlate with smoking.
Similarly, Kras mutations in mouse lung tumors are G-T
transversions in codon 12 when initiated by the cigarette
smoke carcinogens B(a)P or NNK. Alternatively, urethane
and vinyl carbamate most often cause codon 61 A-T
transversions in A/J mice.
Most lung carcinogens are ‘complete’ and thus
both initiate tumorigenesis and promote tumor progression
through dysregulated proliferation of Clara cells and Type
II pneumocytes. Soon after initiation, hyperplastic foci in
the bronchioles and alveoli are observed. Which of these
foci progress to adenomas, and which of these
spontaneously regress is currently unknown. However,
progression of adenoma to invasive adenocarcinoma may
be infrequent (< 10%, 1 year post carcinogen) and
metastasis to other organs is extremely rare in carcinogenbased models (19). Thus, the majority of tumors remain as
benign adenomas even 1 year post administration of
carcinogen. Immunohistochemical staining of tumors is
typically positive for surfactant protein C (SPC, an
immunohistochemical marker for type II cells), but not
secretoglobin 1a1 (also known as Clara cell secretory
protein (CCSP) or CC10, a marker for Clara cells),
suggesting
that
most
mouse
lung
adenoma/adenocarcinomas originate from Type II cells, or
potentially through loss of expression of CC10 in a Clara
cell transdifferentiation process.
Approximately 60 known carcinogens are present
in both mainstream and sidestream cigarette smoke, in
addition to several co-carcinogens and tumor promoting
compounds. Initial attempts using cigarette smoke to
produce pulmonary malignancies with high incidence in
mice indicated cigarette smoke was weakly tumorigenic in
mouse models (43). Subsequently, a protocol was
developed by which tumors could be induced in A/J strain
mice through a 5 month exposure of a combined
mainstream and sidestream smoke followed by a 4 month
exposure to normal air (30). This exposure to air was
essential to the development of tumors. Multiple groups
have used this protocol (generally in A/J and SWR strains)
and have observed an increase in tumor multiplicity from
Activating mutations in Kras are a prominent
early events observed in both human and mouse lung
tumors induced by carcinogen (35, 36). The human
precursor lesion to adenocarcinoma, atypical adenomatous
hyperplasia (AAH), possesses Kras mutations at similar
941
Mouse models of chemically-induced lung carcinogenesis
approximately 1 to 3 tumors (adenomas) per lung. The
effect on malignant lesions (adenocarcinomas) was
inconclusive. Tests on nine different inbred strains
demonstrated that strain sensitivity to smoke-induced lung
tumors was similar to purified carcinogen-induced tumors
(44). An alternative assay has since been developed using
mainstream cigarette smoke for 920 days (6 h/day, 5
days/week) and demonstrated more robust enhancement of
lung tumor incidence and multiplicity (31). This was
observed through measurement of a variety of lesions
(hyperplastic, benign, and malignant) in a mouse strain
(B6C3HF1) that normally has a very low basal level of
pulmonary neoplasia. Incidence of lung adenoma (28% vs.
7%), adenocarcinoma (20% vs. 3%) and distant metastases
(1.5% vs. 0.3%) was enhanced by smoke versus normal air.
Exposure of newborn Swiss albino mice to mainstream
cigarette smoke for 120 days induced lung tumor
multiplicities of 6 and 14 tumors in males and females,
respectively, when mice reach 180 to 230 days of age,
demonstrating enhanced susceptibility in early life (29).
administration of non-carcinogenic lung inflammatory
agents, such as the chemical butylated hydroxytoluene
(BHT) (50-52). BHT undergoes metabolism by lung
specific P450s to a very reactive BHT-quinone methide,
which subsequently forms adducts with cellular proteins
and creates an environment of chronic tissue damage and
compensatory epithelial cell proliferation. This includes
type I cell necrosis followed by type II cell hyperplasia and
differentiation to replace lost type I cells (53-55). Repeated
delivery of BHT causes massive inflammatory cell
infiltration in the alveolar spaces of inflammation
susceptible BALB/cByJ (BALB) strain mice. Importantly,
if BHT is administered weekly for 6 weeks after an initial
single dose of MCA, the result is a 10-fold enhancement in
observed lung tumors (54, 55). It is important to note that
there is strong genetic control of these inflammatory and
tumor responses, as different inbred mouse strains differ in
the degree of inflammation and degree of tumor promotion
caused by BHT. C57BL/6J (B6) strain mice exhibit low
levels of BHT-induced inflammation and are also resistant
to tumor promotion by BHT, while strains such as A/J and
BALB are considered susceptible. BHT elicits similar
injury as other lung irritants, such as ozone, crystalline
silica, hyperoxia, and vanadium pentoxide (50). Several
genetic susceptibility mapping studies have identified
genetic loci for susceptibility to inflammation that are
common to different lung inflammatory agents, suggesting
they operate by similar mechanisms. Interestingly, many of
these loci overlap known lung cancer susceptibility loci,
suggesting common mechanisms of action between lung
injury/inflammation and carcinogenesis (56).
Squamous cell carcinoma is the second most
common lung neoplasm, however, reproducible mouse
models of this disease were lacking until the last 10 years.
Early models using intratracheal inhalation of B(a)P and 3methylcholanthrene (3-MCA) were technically difficult and
not easily replicated (45-47). It was not until experiments
involving topical application of nitrosoalkylureas to induce
skin cancer in female Swiss mice revealed that such
compounds could induce a broad spectrum of cancers that a
reproducible model of squamous cell lung cancer was
developed (48). Since then a protocol has been established
using twice weekly application of N-nitroso-trischloroethylurea (NTCU) on a shaved dorsal patch of skin
in female NIH Swiss mice for the duration of the
experiment, typically 22-24 weeks. NTCU causes
bronchiolar basal cell hyperplasia, and continues through
squamous metaplasia, dysplasia, carcinoma in situ (CIS)
and invasive squamous cell carcinoma, in a sequence of
events comparable to the human condition. In contrast to
mouse lung adenomas, SCC tumors are not nodular and
appear as more opaque patches with indistinct boundaries.
At 24 weeks after the initial NTCU dose there is a 50%
incidence of hyperplasia/metaplasia and 10% incidence of
CIS/SCC. Eight months of treatment results in an 80%
incidence of squamous cell lung carcinoma in these mice
(34). Further refinement of the timing and dose of NTCU
administration in this model may be necessary to generate
the full spectrum of pre-malignant and malignant lesions
without overt toxicity (49).
Recent studies have demonstrated that neutrophils
play a seminal pro-tumorigenic role in mediating tumor
promotion by BHT (57). When compared to tumor
promotion in control IgG-treated BALB mice, antibodymediated depletion of neutrophils in BALB/cByJ mice,
reduced tumor multiplicity by 71%. BHT induces both
neutrophil numbers and levels of the neutrophil chemokine
KC in the airways of susceptible BALB/cByJ mice.
Furthermore, data from this study suggests that KC
expression by lung tissue-resident CD11c+ cells may play
an important role in susceptibility to pulmonary
carcinogenesis by maintaining high levels of neutrophil
trafficking into the lung. This is consistent with other
studies showing similar kinetics of KC and neutrophil
levels, caused by V2O5, another tumor promoter of MCA
tumorigenesis (58). The requirement for neutrophils in this
model is consistent with enhanced neutrophil and KC levels
observed in bronchoalveolar carcinoma patients with poor
outcome (59). These data indicate that neutrophils and their
effector functions are potential targets for prevention and
therapy.
4. A MOUSE MODEL OF INFLAMMATION
PROMOTED LUNG CARCINOGENESIS
Chemical carcinogenesis models in mouse have
been central to elucidating the stages of tumor evolution,
namely initiation, promotion, and progression. One
commonly used model in the mouse lung, involves
initiation of tumorigenesis with the PAH, 3-MCA. 3-MCA
initiates tumorigenesis through mutational activation of the
proto-oncogene K-Ras.
Subsequent promotion and
progression of tumorigenesis can be accelerated by chronic
5. LUNG CANCER CHEMOPREVENTION IN
RODENT
MODELS
OF
CHEMICAL
CARCINOGENESIS
Chemically-induced lung tumors have been
widely used to identify drugs and botanically-derived
agents that may be effective for chemoprevention.
Chemoprevention can be defined as the use of chemo- or
942
Mouse models of chemically-induced lung carcinogenesis
dietary agents to prevent tumor formation or progression.
As mentioned above, chemically-induced lung tumors in
the mouse share many characteristics with human lung
cancer, both genetic and histological. These properties also
make them a suitable model to use for chemoprevention.
Administration of a test compound can begin anywhere
from pre-initiation to late in the tumorigenesis process .
Because the stages of lung tumor progression are well
characterized in chemically induced mouse lung tumors,
the efficacy of chemo- or dietary agents can be determined
at each step in the tumorigenic process, an important
consideration for translating findings into humans..
chemoprevention agent must effectively block or slow the
progression of pre-cancerous lesions to cancerous ones. In
many early studies, the administration of a
chemopreventive agent began prior to initiation with the
carcinogen. This raises the possibility that the effect of the
agent is on expression of metabolic enzymes, agent uptake
or agent excretion rather then effects on tumor progression
per se. Endpoints such as incidence, multiplicity and tumor
size are frequently measured. In addition, the pathology and
histopathology of lesions is frequently investigated. The
detection and localization of cells expressing a particular
protein in lung tumors can provide clues as to molecular
mechanisms of the agent and has the potential for
identifying biomarkers.
Because of its susceptible nature, the most
common pre-clinical chemoprevention model for the
lung is the A/J strain mouse. The cigarette smoke
carcinogens B(a)P and NNK have been the most widely
used and are typically administered by i.p. injection,
although other carcinogens such as urethane and vinyl
carbamate and other routes of administration such as
oral gavage have been used. The types of
chemoprevention schedules used can be divided into
three general categories: 1) complete, where the agent is
administered
beginning
before
carcinogen
administration and continues throughout the experiment;
2) initiation, where the agent is administered from just
before carcinogen administration and is terminated
within a few weeks of initiation, and 3) progression,
where
treatment
is
begun
after
carcinogen
administration and continued until experiment
termination. Obviously, there are many variations of
experimental design that will depend on the goals of the
investigators.
Several
notable
limitations
for
testing
chemoprevention agents in mouse models exist. As
mentioned above, beginning treatment prior to initiation of
carcinogenesis has the potential to cause changes in tumor
formation that are based on alterations in carcinogen
metabolism. Notable differences in key metabolic enzymes
such as cytochrome p450s exist between mice and humans
and could ultimately affect the disposition of the
carcinogen. This is another reason to avoid starting
chemoprevention prior to initiation of carcinogenesis. The
metabolic differences between rodents and humans can of
course also have effects on the metabolism and ultimate
excretion of preventive agents. The route of administration
of a chemoprevention agent can also have a major effect on
the bioavailability of that agent. Ideally, pharmacokinetic
/phamacodynamic studies should be performed in
conjunction with initial characterization of a chemoprentive
agent.
To date, over 200 pre-clinical studies of lung
cancer chemoprevention have been published and it is
beyond the scope of this brief review to begin to discuss
them all. An ideal chemoprevention agent will combine
efficacy with a very favorable safety profile. Because
chemoprevention agents would be administered to patients
essentially free of overt disease, the presence of even
relatively mild effects could limit the usefulness of a
compound due to low levels of patient compliance as well
as concerns over patient welfare. This is a major reason
why many chemoprevention studies have focused on using
botanically derived agents, sometimes referred to as
neutraceuticals. These include both complex mixtures
isolated from a given plant type (e.g. tea polyphenol
fractions or freeze dried berries) and purified compounds
that are thought to be the main active ingredients present in
these preparations. The compound (-)-epigallocatechin
gallate (EGCG) is generally considered to be the most
potent polyphenolic compound in green tea extracts.
However, chemoprevention with purified EGCG has not
yielded as large of an effect as the complex mixture (60).
The use of complex mixtures may provide higher degrees
of efficacy or alternatively improve stability and
bioavailability.
6. CONCLUSIONS
Mice have been, and will continue to be, a valuable
resource in the modeling of human lung neoplasms.
Critiques of the models discussed is based mainly on the
predominant use of large doses of single carcinogens that
far exceed the human situation in which the carcinogen
dose is accumulated over time, and via a mixture of
carcinogens through cigarette smoke.
Particularly
promising in addressing this issue is the development of
cigarette smoke-induced models that has hastened over the
last decade.
Nevertheless, mouse models of lung
carcinogenesis as a whole have been seminal to the
discovery of etiological factors of disease, and are now
frequently used to assess efficacy of various treatments and
preventive agents that may benefit the human condition.
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Key Words: Carcinogen,
Chemoprevention, Review
Adenoma,
A/J
strain,
Send correspondence to: Haris G. Vikis, Department of
Pharmacology and Toxicology, Medical College of
Wisconsin and MCW Cancer Center, Milwaukee, WI,
53202, Tel: 414-955-7588, Fax: 414-955-6059 E-mail:
Haris Vikis, hvikis@mcw.edu
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