artigo metformina 3

7/27/2019 Artigo metformina 3

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Mutation Research 611 (2006) 18

Metformin does not prevent DNA damage in lymphocytesdespite its antioxidant properties against cumene

hydroperoxide-induced oxidative stress

Ilhan Onaran a,, Gulgun S. Guven a, Sule Beyhan Ozdas c,Gonul Kanigur a, Suphi Vehid b

a Department of Medical Biology, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkeyb Department of Public Health, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey

c

Department of Chemical and Biological Engineering, Koc University, Istanbul, TurkeyReceived 27 October 2005; received in revised form 29 March 2006; accepted 25 June 2006

Available online 26 September 2006

Abstract

Metformin (1-(diaminomethylidene)-3,3-dimethyl-guanidine), which is the most commonly prescribed oral antihyperglycaemic

drug in the world, was reported to have several antioxidant properties such as the inhibition of advanced glycation end-products. In

addition to its use in the treatment of diabetes, it has been suggested that metformin may be a promisinganti-aging agent. The present

work was aimed at assessing the possible protective effects of metformin against DNA-damage induction by oxidative stress in vitro.

Theeffects of metformin were compared with those ofN-acetylcysteine (NAC). For this purpose, peripheral blood lymphocytes from

aged (n = 10) and young (n = 10) individuals were pre-incubated with various concentrations of metformin (1050M), followed by

incubation with 15M cumene hydroperoxide (CumOOH) for 48 h, under conditions of low oxidant level, which do not induce cell

death. Protection against oxidative DNA damage was evaluated by use of the Comet assay and the cytokinesis-block micronucleus

technique. Changes in the levels of malondialdehyde + 4-hydroxy-alkenals, an index of oxidative stress, were also measured in

lymphocytes. At concentrations ranging from 10 M to 50M, metformin did not protect the lymphocytes from DNA damage,

while 50M NAC possessed an effective protective effect against CumOOH-induced DNA damage. Furthermore, NAC, but not

metformin, inhibited DNA fragmentation induced by CumOOH. In contrast to the lack of protection against oxidative damage

in lymphocyte cultures, metformin significantly protected the cells from lipid peroxidation in both age groups, although not as

effective as NAC in preventing the peroxidative damage at the highest doses. Within the limitations of this study, the results indicate

that pharmacological concentrations of metformin are unable to protect against DNA damage induced by a pro-oxidant stimulus in

cultured human lymphocytes, despite its antioxidant properties.

2006 Elsevier B.V. All rights reserved.

Keywords: Metformin; Oxidative stress; DNA damage; Human lymphocytes; Comet assay; Micronucleus assay

Corresponding author at: Ortaklar Cd. Butan Sk. No: 2, 34394

Mecidiyekoy, Istanbul, Turkey. Fax: +90 2125861548.

E-mail address: [email protected] (I. Onaran).

1. Introduction

Oxidative stress is thought to play a major role in the

etiology of a wide variety of diseases including diabetes

and cancer, as well as in the aging process [1,2]. It

is triggered by exposure to exogenous factors or by

1383-5718/$ see front matter 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.mrgentox.2006.06.036
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2 I. Onaran et al. / Mutation Research 611 (2006) 18

chemicals producing reactive oxygen species (ROS) and

is associated with an overproduction of ROS, as well

as an impairment of antioxidant defense capacity. In

diabetes, oxidative stress seems to arise primarily from

an increase in free radical concentrations in plasma and

a reduction in antioxidant defense [3,4].

ROS are well known to cause DNA damage andinduce cytotoxicity. They induce a variety of lesions

in DNA, including oxidized bases, abasic sites, DNA

strand-breaks and cross-links between DNA and pro-

teins. Growing evidence indicates that oxidative stress

increases DNA damage in diabetics [5,6]. Similarly,

long-lived animals including humans are known to accu-

mulate aberrations in the genome and in cellular compo-

nents during the aging process, which are thought to be

caused by the cumulative effectsof oxidative damage [7].

On the other hand, it has been pointed out that vitamin

antioxidants administered in vitro and in vivo preventDNA damage and increase the DNA-repair capacity of

individuals subject to oxidative stress [8]. Therefore,

agents with antioxidant activity may offer a benefit to

diabetic patients and could be useful in preventing or

delaying the development of diabetic complications.

Metformin (1-(diaminomethylidene)-3,3-dimethyl-

guanidine) is an anti-hyperglycaemic drug commonly

used for the management of type-2 diabetes. Protec-

tive effects against diabetic complications have been

observed with metformin monotherapy [9]. Previous in

vitroand

in vivostudies have demonstrated that met-formin causes an improvement in antioxidant activities

in various tissues and acts to limit lipid peroxidation

[1013]. Bonnefont-Rousselot et al. [14] also suggested

thatmetformincould directly scavengeROS or indirectly

act by modulating the intracellular production of super-

oxide radicals (O2). Thus, metforminmay help protect

against free radical-induced DNA damage. However, the

ability of metformin to modulate the DNA-damaging

effects of oxidative stress is not known. Therefore, the

aim of the present study is to investigate the in vitro

ability of metformin to protect against oxidative stress-

induced DNA damage in peripheral blood lymphocytesobtained from both elderly and younger subjects, and to

compare it with the activity of N-acetylcysteine (NAC),

a known antioxidant and ROS scavenger. It has been

pointed out that age and diabetes are factors that influ-

ence the generation of DNA damage. However, attempts

to correlate levels of DNA damage with age or dia-

betes have led to contradictory results ranging from no

significant changes [15,16] to either positive [5,6,17]

or negative relationships [18]. In the case of diabetes,

all these reports should be interpreted with caution for

various reasons. Firstly, diabetes is a disease that may

implicate various disturbances with an unknown impact

on DNA. Secondly, glycaemic control seems to play a

role in DNA-damage processing. Another problem fol-

lows from the age onset of this disease. In this study,

the use of cells from diabetics may complicate the eval-

uation of possible protective effects of metformin on

DNA damage induced in vitro. On the other hand, theincidence of type-2 diabetes mellitus increases with age

and leads to significant morbidity and mortality. It has

been suggested that metformin decelerates aging in mice

[19], and that it is the most suitable strategy to prevent

diabetes in the elderly [20]. Hence, studies were under-

taken using lymphocytes in from healthy aged and young

subjects.

2. Materials and methods

2.1. Chemicals

Metformin hydrochloride, N-acetylcysteine, cumene

hydroperoxide, RPMI 1640 growth medium, penicillin,

streptomycin, dimethyl sulfoxide, normal melting point

agarose, low melting point agarose and ethidium bromide

were obtained from Sigma chemicals, Saint Louis, MO,

USA. Giemsa and trypan-blue were obtained from Merck,

Darmstadt, Germany and l-glutamine, fetal calf serum,

phytohaemagglutinin and cytochalasin-B were purchased

from Biological Industries, Israel. Ficoll-Paque was from

Pharmacia, Uppsala, Sweden. All other chemicals and solvents

used were of the highest purity grade available.

2.2. Lymphocyte preparation, cell culture and treatment

Following informed consent, 10 healthy young donors (5

male and 5 female; mean age, 29 5 years; range, 2039) and

10 healthy elderly volunteers (5 male and 5 female; mean age,

79 6 years; range, 7087) were included in the study.

Blood was collected by venipuncture in heparinised tubes,

diluted 1:1 in phosphate-buffered saline (PBS) and separated

by a Ficoll gradient for isolation of lymphocytes. Cell viabil-

ity was measured by use of the trypan-blue exclusion assay.

Approximately 1 106 cells were seeded in RPMI medium

containing l-glutamine (2 mM), fetal calf serum (20%), peni-cillin (100 UI/ml) and streptomycin (100g/ml) and incubated

at 37 C for 72 h. Lymphocyte stimulation was done by addi-

tion of phytohaemagglutinin (1.5%). At 48 h, various concen-

trations of metformin or NAC were added to cultures from

each individual. After pre-incubation with metformin or NAC

for 1 h, oxidative stress was induced by addition of cumene

hydroperoxide (CumOOH) (final concentration 15M) dis-

solved in dimethyl sulfoxide (DMSO). All cell suspensions

not treated with CumOOH were brought to a concentration

of DMSO equivalent to that delivered with CumOOH. Since

pharmacological metformin concentrations are close to 20M

[21], we studied metformin in the concentration range of


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I. Onaran et al. / Mutation Research 611 (2006) 18 3

1050M. NAC (50M), dissolved in PBS and neutralized

with sodium hydroxide, was added to the culture medium 1 h

before CumOOH exposure.

2.3. Cytokinesis-block micronucleus assay

The cytokinesis-block micronucleus (MN) assay was car-ried out according to Fenech [22]. Cytochalasin-B (6g/ml)

was added to cultures at 44h to prevent cytokinesis. Cells were

fixed in 3:1 methanol:acetic acid without hypotonic treatment,

and the suspension was dropped onto clean slides and stained

with 5% Giemsa for approximately 10 min. In accordance

with standard criteria [23], 1000 binucleated lymphocytes were

scored for MN identification for each subject. To provide data

regarding proliferation kinetics, the frequencies of mono-, bi-,

tri- and tetra-nucleated cells were determined.

2.4. Comet assay

A slightly modified Comet assay[24], implemented accord-

ing to recent guidelines [25], was used to measure the extent

of DNA damage. After cell culture, the cells were centrifuged

(1000 g, 10 min) and taken up in PBS. The cell suspension

was mixed with low melting-point agarose and placed on a

microscope slide pre-coated with 1% normal melting-point

agarose. The slides with the agarose-embedded cells were then

subjected toa lysis step(1 h incubationat 4 Cin1%N-lauroyl-

sarcosine, 2.5 M NaCl, 100 mM Na2EDTA, 1% Triton X-100,

10% DMSO, pH 10.0). After lysis the slides were washedthree

times for 5 min in endonuclease buffer (40 mM HepesKOH,

0.1 M KCl, 0.5 mM EDTA, 0.2 mg/ml bovine serum albumin,

pH 8.0) and incubated for 30 min with a mixture of repairenzymes (Fpg) at 37 C. Theslides (processed eitherwithout or

with Fpg) were then transferred to an electrophoresis box con-

taining an alkaline solution (300mM NaOH, 1 mM Na2EDTA,

pH 13). They were kept in this solution for a 40 min DNA-

unwinding periodat 4 C.A current of25 V (300mA) was then

applied for 30 min. The slides were removed and neutralized

with TrisHCl (0.4 M, pH 7.5). Cells were stained with 20l

ethidium bromide (5g/ml) and the extent of DNA migration

was evaluated by visual scoring. Slides were scored without

knowledge of the group by only one well-trained scorer. A

Nikon Eclip E600 fluoroscence microscope was used to iden-

tify comets. One thousand cells were graded by eye into fivecategories, according to Anderson et al. [26]. This method

has the advantage of speed; it is calibrated by reference to

computer-image analysis based on fluorometric measurement

of DNA intensities in head and tail. The results were expressed

as comet assay tail factors calculated according to Diem et

al. [27], corresponding to the following amount of DNA frag-

ments in the tail: classification group A < 5%, B < 520%, C

2040%, D 4095% and E > 95%.

Tail factors were then calculated according to the following

formula:

tail factor(%) =AFA + BFB + CFC +DFD + EFE

1000

FA is average of

group A (=2.5)

A is number of cells classified to group A

FB is average of

group B (=12.5)

B is number of cells classified to group B

FC is average of

group C (=30)

Cis number of cells classified to group C

FD is average of

group D (=67.5)

D is number of cells classified to group D

FE is average of group

E (=97.5)

Eis number of cells classified to group E

2.5. Estimation of DNA fragmentation

A cellular DNA fragmentation assay was done by an

ELISA method, using a kit (Roche Molecular Biochemicals,

Mannheim, Germany) according to the recommended proce-

dure. Cells proliferating in the culture were incubated for 2 h at

37 C with the non-radioactive thymidine analog BrdU, which

is incorporated into genomic DNA. After this procedure, cellswere treated with individual agents as described above. After

48 h, cells were lysed and transferred to a microtiter plate

coated with an anti-DNA antibody, and incubated for 75 min

at room temperature. After washing the plate three times, sub-

strate solution was added and the plate was incubated in dark

until color development was sufficient. The reaction was then

stopped by addition 0.56 M H2SO4 and the absorbance of the

samples were measured with a microplate reader at 450nm and

655 nm.

2.6. Lipid peroxidation assay

Immediately after the incubation with individual agents

of lymphocytes in culture, the cell suspensions in medium

were centrifuged for 10 min at 1000 g. The amounts of

malondialdehyde and 4-hydroxynonenal (MDA + 4-HNE) in

supernatants were determined with CALBIOCHEM Lipid Per-

oxidation Assay kit, exactly as described by the manufacturer.

The level of lipid peroxidation was expressed as the amount (in

106 cells) of MDA + 4-HNE, as major lipid peroxidation end

products. In the assay, we added 2.5 mM (final concentration)

butylated hydroxytoluene to prevent sample auto-oxidation.

2.7. Statistical analysis and data presentation

Values reported are meansS.D. All data were normally

distributed and underwent equal variance testing. The exper-

iments were analyzed with the general linear model of SPSS

11.5 for Windows (SPSS, Chicago, IL). Significance (p < 0.05)

was determined with a one-way ANOVA, Tukeys HSD test.

3. Results

To investigate the protective effects of metformin

against DNA damage in lymphocytes when exposed to

in vitro-induced oxidative stress, we focused on cells


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treated at low oxidant levels that do not decrease cell

viability. In preliminary studies, when lymphocytes were

exposed to differentdoses of CumOOH for 48 h, the dose

response curves showed that a concentration of 15M

CumOOH was the highest at which cell death among

lymphocytes of elderly (n = 4) and younger (n = 5) sub-

jects did not occur. Exposure of cells to this concen-tration revealed insignificant changes in cell-death rate

of 3.4 0.5%. Concentrationresponse determination

for further experiments was not practical and therefore

all samples were studied at a fixed concentration of

CumOOH.

In all subjects, 15M CumOOH induced significant

increasesin MN frequency compared with control values

(p < 0.05). However, a statistically significant difference

was observed in the sensitivity of young and elderly indi-

viduals, as seem by differences in the mean frequency of

MN (p < 0.05) (Table 1). In addition, there was a signifi-cant difference in the MN frequency observed in control

cultures from young and elderly persons. We also deter-

mined the comet assay tail-factor (DNA damage level)

in peripheral lymphocytes. As shown in Table 1, the lev-

els of CumOOH-induced DNA damage in lymphocytes

were again significantly higher in old subjects compared

with younger ones.

In all lymphocyte samples, pre-treatment with met-

formin concentrations ranging from 10M to 50M

did not lead to a significant decrease in CumOOH-

induced micronucleus-forming activity (p

> 0.05). Theresults obtained by the Comet assay on lymphocytes

confirmed the data of the MN test (Table 1). Under the

same experimental conditions, pre-treatment of the cells

with 50M NAC produced a significant reduction in

DNA damage measured by Comet assay and in the fre-

quency of micronuclei, when the agent was present with

CumOOH throughout the incubation period. Table 1

shows the influence of metformin and NAC on MN

frequency and DNA damage induced by CumOOH. In

addition, experiments with Fpg post-treatment further

indicated that there was no reduction in the amount of

oxidative base damage after supplementation with met-

formin (data not shown).

Although under our experimental conditions the invitro treatments of cells did not significantly induce cell

death, the concentration of CumOOH that was applied

may induce apoptosis. To examine this possibility, we

used a cellular DNA fragmentation ELISA assay that

quantifies DNA fragmentation caused by apoptosis. The

basal and CumOOH-induced levels of apoptosis in lym-

phocytes from donors were quantified using this ELISA

procedure, which measures the release of mono- and

oligonucleosomal fragments into the cytoplasm. Treat-

ment of lymphocytes with 15M CumOOH for 48 h

caused a significantincrease in DNA fragmentationcom-pared with that in untreated cells. The DNA fragmenta-

tion in lymphocytes from aging individuals was approx-

imately 1.65 times the amount in untreated cells, similar

to the increase seen with lymphocytes from young

individuals, which was 1.67-fold. As shown in Fig. 1,

treatment with the highest dose of metformin (50 M)

did not significantly affect the levels of DNA fragmen-

tation produced by CumOOH, while NAC treatment at

the same concentration significantly inhibited the DNA

fragmentation.

MDA + 4-HNE levels were used to measure theoxidative damage in lymphocytes treated with indi-

vidual agents. Despite the differences in inhibition

profiles of the MDA + 4-HNE formation in lymphocytes

from aged and young subjects, the increased lipid

peroxidation induced by CumOOH was attenuated

by co-incubation with metformin or NAC. Treat-

ment of the cells with the highest dose of metformin

Table 1

Effect of metformin on CumOOH-induced DNA damage estimated by MN and Comet assay in peripheral lymphocytes from young and elderly

individuals

Treatment MN/1000 binucleated lymphocytes Comet assay tail factor (%)

Aging group (n = 1 0) Young group (n = 10) Aging group (n = 1 0) Young group (n =10)

Basal 14.80 4.98a 8.80 2.39 6.53 1.88a 3.19 0.33

0M Metformin+ CumOOH 25.20 8.59a,b 16.80 5.59b 11.99 3.21a,b 7.32 1.90b




50M l-NAC + CumOOH 17.34 5.44a 9.71 2.02 6.01 1.31a 3.70 0.67

Cells were treated with metformin (1050M) or NAC (50M) for 1 h before addition of 15M CumOOH and were incubated in culture medium

for 48 h.a Significant difference (p < 0.05) with regard to young group.b

Significant difference (p < 0.05) with regard to the basal values.


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Fig. 1. Effect of metformin and NAC on CumOOH-induced cellular DNA fragmentation in lymphocytes from elderly ( n = 10) and young (n =10)

individuals as assessed by ELISA. The following conditions were tested: Basal; 15M CumOOH; 50M Metformin + 15M CumOOH; 50M

N-acetylcysteine + 15M CumOOH. The results are the mean S.D. *Significant difference (p < 0.05) with regard to the cells incubated with

CumOOH alone.

(50M) significantly decreased CumOOH-induced

lipid peroxidation compared with CumOOH alone

(Table 2). At this dose of metformin, the inhibition

percentages of CumOOH-induced lipid peroxidation in

lymphocytes from aged and young subjects were

61%and 56%, respectively. Under the same experimental

conditions, the protective effect of metformin against

lipid peroxidation in lymphocytes was smaller than that

in lymphocytes treated with NAC (Table 2).

Table 2

Effect of metformin on lipid peroxidation (expressed as amount

of MDA+4-HNE in M/106 cells) in 15M CumOOH-induced

oxidative stress in peripheral lymphocytes from elderly and young

individuals

In vitro treatment M MDA+ 4-HNE/106 cells

Aging group

(n =10)

Young group

(n =10)

Basal 0.969 0.162 0.851 0.088

0M Metformin + CumOOH 1.989 0.270a 1.768 0.224a

50M Metformin + CumOOH 1.363 0.208a,b 1.255 0.178a,b

50M NAC + CumOOH 1.182 0.182b 1.095 0.178b

MDA, malondialdehyde, lipid peroxidation product; 4-HNE, 4-

hydroxynonenal, lipid peroxidation product.a Significant difference (p < 0.05) with regard to the basal values.b Significant difference (p < 0.05) with regard to the cells incubated

with CumOOH.

4. Discussion

To test whether metformin could inhibit DNA damage

and apoptosis induced by oxidative stress in lympho-

cytes as target cells we used cumene hydroperoxide, aknown genotoxic agent. CumOOH, which is a strong

non-polar oxidizing agent used in industry, is easily

taken up by cells and is not metabolized by catalase [28].

Oxidative stress generated by CumOOH caused certain

aspects of aging as well as induction of DNA damage

[29]. In the present study, in vitro anti-genotoxic effects

of metformin were compared with those of NAC, com-

monly used by the pharmaceutical industry. Numerous

studies have demonstrated that NAC is able to inhibit

chemically induced oxidative stress and DNA damage

[30,31].

The mammalian cytokinesis-block micronucleusassay and the alkaline Comet assay (or single-cell

gel electrophoresis assay) were used to investigate the

modifying potential of metformin on CumOOH-induced

DNA damage. The Comet assay is a simple, sensitive

and reliable method for detecting DNA single- and

double-strand breaks and alkali-labile sites at the single-

cell level. However, the Comet assay is not a direct

indicator of the amount of DNA adducts formed. On the

other hand, MN is a well-known cytogenetic technique

to quantify DNA damage induced by chemical com-

pounds and complex mixtures [23]. A good correlation


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between micronucleus formation used as a biomarker of

DNA damage and oxidative stress in cells was obtained

in several experiments [32,33]. It is documented that one

of the effects of oxidative stress-induced cytotoxicity

in cells is DNA fragmentation caused by apoptosis

[3436]. Therefore, this parameter was also determined

to see if metformin affects this event associated withDNA damage.

From results reported in Tables 1 and 2 and Fig. 1

it is clear that exposure of peripheral lymphocytes in

culture to 15M CumOOH leads to substantial DNA

damage and an increase in lipid peroxidation, although

there were significant differences between elderly and

younger groups of donors. Using MN and Comet assay

techniques, evaluation of DNA damage of 48 h cultures

of cells treated with CumOOH and three different con-

centrations of metformin(1050M)failedtorevealany

significant differences by comparison with CumOOHalone, in either group. However, the highest dose of

metformin showed a partial inhibition of lipid peroxi-

dation as manifested by the decreased concentrations of

MDA + 4-HNE. Higher concentrations of metformin in

preliminary experiments not only did not protect against

DNA damage, but even increased the CumOOH-induced

DNA damage (data not shown). In contrast, treatment

with 50M NAC resulted in a marked reduction of

DNA damage and in a more effective protection against

lipid peroxidation. In the present study, DNA fragmen-

tation showed a similar trend. No significant differencein DNA fragmentation in lymphocytes from either aged

and young individuals was observed between 50M

metformin-treated and untreated cells under oxidative

stress. This indicates that metformin did not protect

the lymphocytes from apoptotic cell death. The present

study confirms previous findings that NAC provides

protection against diverse oxidative insults [3739],

which correlates with a reduction in chemical-induced

apoptosis. Therefore, the results from all three widely

used assays indicate that we were unable to find dose-

dependent effects of metformin against oxidative DNA

damage in cultured lymphocytes of young and elderlyindividuals. However, we should be aware of the lim-

itations of this in vitro study, which does not analyze:

(a) the protective effects of metformin on DNA damage

induced by other free radical-producing agents such as

H2O2, which has a different action mechanism, and (b)

short-term protective effects of metformin against free

radical-induced DNA damage. Also, this study was con-

ducted on peripheral blood cells, which may not fully

represent changes that occur in all tissues. Furthermore,

our experimental conditions may not necessarily reflect

the in vivo situation.

At present, thebasisforthe lack of effect of metformin

on oxidatively-induced DNA damage remains unknown,

although the compound has partial protective effect on

lipid peroxidation. Lack of protective effect of met-

formin may be simply attributed to the fact that it has a

partial inhibitory effect on oxidative stress. Studies about

the action of metformin on free radicals have shown thatit hasa directscavenging effect on the hydroxyl free radi-

cals (OH), but no direct scavenging effect on O2 free

radicals, which have an indirect role in DNA damage

[14,40]. Therefore, it may not have a sufficiently protec-

tive effect against damage in an environment in which

we know that CumOOH has the potential to produce free

radicals suchasOHandO2 [41]. SinceNACisawell-

known O2 scavenger [42], under our experimental

conditions it may appear as a protective anti-genotoxic

factor. Another possibility is that metformin can modu-

late the anti-oxidant systems through an increased activ-ity of certain genes involved in the stress response.

However, under our experimental conditions it may not

modulate DNA repair against CumOOH-induced DNA

damage. This possibility could have been checked by

alterations in the protocol, as noted by Glei et al. [43].

On the other hand, it has been reported that the concen-

tration of metformin recovered in the cytoplasm after an

incubation period of 60 min with Xenopus oocytes was

only


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artigo metformina 3

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