ORIGINAL PAPER

Systemic overexpression of antizyme 1 in mouse reducesornithine decarboxylase activity without major changesin tissue polyamine homeostasis

Marko Pietila • Hiramani Dhungana •

Anne Uimari • Reijo Sironen • Leena Alhonen

Received: 29 July 2013 / Accepted: 21 October 2013 / Published online: 31 October 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Polyamines, spermidine, spermine and

their precursor putrescine, are ubiquitous cell compo-

nents essential for normal cell growth. Increased

polyamine levels and enhanced biosynthesis have been

associated with malignant transformation and tumor

formation, and thus, the polyamines have been con-

sidered to be a meaningful target to cancer therapies.

However, clinical cancer treatment trials using inhib-

itors of polyamine synthesis have been unsuccessful

probably due to compensatory uptake of polyamines

from extracellular sources. The antizyme proteins

regulate both polyamine biosynthesis and transport,

and thus, the antizymes could provide an efficient

approach to control cellular proliferation compared to

the mere inhibition of biosynthesis. To define the role

of antizymes in proliferative processes associated with

the whole animal, we have generated transgenic mice

overexpressing mouse antizyme 1 gene under its own

regulatory sequences. Antizyme 1 protein was abun-

dantly expressed in various organs and the expressed

antizyme protein was functional as ornithine decar-

boxylase activity was significantly reduced in all

tissues analyzed. However, antizyme 1 overexpression

caused only minor changes in tissue polyamine levels

demonstrating the challenges in using the ‘‘antizyme

approach’’ to deplete polyamines in a living animal.

Neither were there any changes in cellular proliferation

in the proliferative tissues of transgenic animals.

Interestingly though, there was occurrence of abnor-

mally high level of apoptosis in the non-proliferating

part of the colon epithelia. Otherwise, the transgenic

founder mice appeared healthy and out of seven

founders six were fertile. However, none of the

founders could transmit the transgene suggesting that

the antizyme 1 overexpression may be deleterious to

transgenic gametes.

Keywords Antizyme � Transgenic � Ornithine

decarboxylase � Polyamine � Proliferation

Introduction

Polyamines spermidine and spermine and their dia-

mine precursor, putrescine, are known to be essential

for cell growth and differentiation. Polyamines are

M. Pietila (&)

Institute of Biomedicine, University of Eastern Finland,

P.O.B 1627, 70211 Kuopio, Finland

e-mail: Marko.Pietila@uef.fi

H. Dhungana � A. Uimari � L. Alhonen

Department of Biotechnology and Molecular Medicine,

A. I. Virtanen Institute for Molecular Sciences, Biocenter

Kuopio, University of Eastern Finland, Kuopio, Finland

R. Sironen

Institute of Clinical Medicine, Pathology and Forensic

Medicine, Cancer Center of Eastern Finland, University of

Eastern Finland, Kuopio, Finland

R. Sironen

Department of Pathology, Imaging Center, Kuopio

University Hospital, Kuopio, Finland

123

Transgenic Res (2014) 23:153–163

DOI 10.1007/s11248-013-9763-y

positively charged at physiologically relevant ionic

and pH conditions and readily bind to negatively

charged macromolecules such as RNA, DNA and

proteins. In cells the polyamines are present in

millimolar concentrations and have been implicated

in many physiological functions including DNA

replication, transcription, translation, posttranslational

protein modification and membrane stability. Some of

these effects are specific for polyamines, while others

are less specific due to the general cationic nature of

these compounds. Irrespective of the specificity of their

effects, polyamines are indispensable cellular compo-

nents because their depletion, either by gene disruption

or inhibitors of their biosynthesis, results in severe

defects in cellular growth (Wallace and Fraser 2004).

The polyamine metabolism has long been an

attractive target for therapy of cancer (Kaiser et al.

2003; Casero et al. 2005) and parasitic diseases (Heby

et al. 2007). Ornithine decarboxylase (ODC) is a key

enzyme in polyamine synthesis and its overexpression

has been associated with cell transformation and

tumor formation. Although ODC is under tight control

in normal cells, its regulation is altered in neoplastic

cells, yielding constitutively high levels of ODC

expression and activity (Shantz and Levin 2007).

Furthermore, a polymorphism in ODC gene associates

with colon adenoma recurrence and the survival of

colon cancer patients (Martinez et al. 2003). Recently,

the inhibition of ODC has been successfully used in

the prevention of basal cell carcinoma of skin (Bailey

et al. 2010) and with the combination of anti-

inflammatory drug sulindac to prevent sporadic colo-

rectal adenomas (Meyskens et al. 2008). In experi-

mental animal studies, inhibition of ODC with its

specific inhibitor a-difluoromethylornithine (DFMO)

has proved to be effective in prevention of prostate

adenocarcinoma. In a study with transgenic adeno-

carcinoma model of mouse prostate (TRAMP),

DFMO treatment decreased malignant prostate growth

and totally prevented the formation of metastases,

which normally arise in these mice upon aging (Gupta

et al. 2000).

Despite these promising results, clinical trials to

treat advanced cancer with inhibitors of polyamine

synthesis have been unsuccessful due to compensatory

uptake of polyamines from extracellular sources and

complex regulation of polyamine metabolism. Even

during drastic changes in the cellular environment,

polyamine levels are maintained within a narrow

optimal range by a complex regulatory circuitry. For

example, the activity of ODC is controlled at the levels

of transcription, translation, and the stability of the

enzyme protein (Perez-Leal and Merali 2012). The

latter is regulated by the antizymes (AZ), and further

by antizyme inhibitor (AZIn) (Kahana et al. 2005).

The expression of AZ is controlled by intracellular

polyamine level whereby increased polyamine level

triggers ?1 ribosomal frameshift on the encoding AZ

mRNA, allowing functional AZ to be expressed

(Matsufuji et al. 1995). From three known mammalian

antizymes, the antizyme 1 (AZ1) is the best known and

most ubiquitously expressed. The AZ1 mRNA con-

tains two potential start codons near the 50 end and

produces a functional protein of either 29 kDa or

24 kDa depending on the start codon used (Mitchell

et al. 1998). AZ2 is also ubiquitously expressed but to

a lesser extent and is less efficient in targeting ODC to

proteosomal degradation. AZ3 is only expressed in

testis. There are also two antizyme inhibitor proteins

(AZIn) known to date, one ubiquitously expressed

(AZIn1) and the other which is restricted to brain and

testis (AZIn2). AZ decreases polyamine levels

through three mechanisms. First, AZ disrupts active

ODC dimers, resulting in decreased polyamine syn-

thesis. Second, AZ targets ODC for degradation by the

26S proteasome in a reaction that does not require

ubiquitination. Third, AZ inhibits polyamine uptake

from the microenvironment through a mechanism that

has yet to be completely characterized (Kahana 2007).

Taking together, the antizymes provide an approach

for cancer therapy which relies on to regulation of both

the synthesis and the uptake of the polyamines.

AZ1 has been shown to have tumor-suppressive

activity in mouse models. Targeted AZ1 overexpres-

sion to the skin using either the bovine keratin 5 or

keratin 6 promoter showed delayed tumor onset and

decreased tumor numbers relative to wild-type controls

after exposure to the chemical carcinogen DMBA

(7,12-dimethylbenz(a)anthracene) and the tumor pro-

moter TPA (12-O-tetradecanoylphorbol-13-acetate)

(Feith et al. 2001). Similarly, the K6/AZ and K5/AZ

mice were also more resistant to forestomach tumors

induced by N-nitrosomethylbenzylamine (NMBA)

(Fong et al. 2003). In the present study, we used mouse

transgenesis to study the physiological consequences

of systemic AZ1 overexpression. A genomic antizyme

gene constructs with a thymidine deletion, correspond-

ing to position 205 of antizyme cDNA was used. This

154 Transgenic Res (2014) 23:153–163

123

converted the inframe UGA stop codon to UGG codon.

As a result, production of antizyme protein was

possible without the need of ribosomal frameshifting

(Matsufuji et al. 1995; Kankare et al. 1997).

Materials and methods

Generation of transgenic mice

The transgenic mice were generated using the standard

pronuclear microinjection technique (Hogan et al.

1986). Fertilized oocytes were obtained from supe-

rovulated BALB/c 9 DBA/2 (CD2F1) females mated

with CD2F1 males. The microinjected zygotes were

allowed to reach 2-cell stage and were then transferred

into oviducts of pseudopregnant foster females. The

gene construct used for transgenesis was a 5.5-kbp

genomic sequence isolated from 129 SVJ mouse

genomic library (Stratagene, La Jolla, CA) with a

thymidine deletion, corresponding to position 205 of

antizyme cDNA (Kankare et al. 1997) (Fig. 1). As the

transgene construct was of mouse origin, the method

for its detection was based on the integration of

multiple transgene copies in the form of concatamers

(head-to-tail) so that a minimum of two integrated

transgene copies were required for the detection. The

design of the oligonucleotides was based on the head-

to-tail recombination of the transgenes so that the

upper primer recognized the tail region and the lower

one the head region of the AZ1 transgene (Fig. 1). The

following oligonucleotide primers were used in the

PCR: 50-TGCCTATCTTCATGGAGAC-30 (from 30

tail) and 50-ATAGATGGTTGTGAGCCAC-30 (from

50 head). A positive control form the founder animal

was always used when the transgenity of progeny was

tested. The founder animals with no transgenic

offspring were sacrificed at the age of 8–10 months

for the phenotype analysis.

Analytical methods

Western blotting was performed according to a

laboratory manual, Current Protocols in Molecular

Biology (Ausubel et al. 2007). Shortly, 25 lg of

protein was used for SDS-PAGE gel electrophoresis

and transferred onto the PVDF membranes. Mem-

branes were blocked with 2 % ECL advance blocking

reagent (GE Healthcare, Fairfield, CT, USA) in

phosphate-buffered saline containing 0.1 % Tween

and probed with Antizyme 1 [described in Kankare

et al. (1997)], Antizyme inhibitor 1 (Cosmo bio,

Tokyo, Japan), HRP conjugated anti-PCNA (PC-10,

Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit

polyclonal against cleaved Caspase 3 (Cell Signaling

Technology, Danvers, MA) and b-actin (Santa Cruz

Biotechnology) antibodies. DyLight 680 and 800-con-

jugated secondary antibodies (Thermo Fisher Scien-

tific) were used in 1:10,000 dilutions. Antibody-bound

protein was detected by the Odyssey Infrared Imaging

System (Li-Cor Biosciences, Lincoln, NE, USA).

The tissue specimens for histology were fixed in

10 % formalin in phosphate buffer, embedded in

paraffin and cut into 5-lm sections and stained with

hematoxylin/eosin (HE). For immunohistochemistry,

rabbit polyclonal anti-antizyme (1:1,000), HRP con-

jugated anti-PCNA (1:750) and rabbit polyclonal

against cleaved Caspase 3 (1:1,000) were used. PCNA

and cleaved Caspase 3 antigen retrieval was carried

out by boiling samples in 0.01 M citric acid buffer pH

6.0 for 10 min. To visualize the rabbit polyclonal

primary antibodies, we used Power Vision Poly-HRP

Rabbit IgG IHC kit (ImmunoLogic, Duiven, The

Netherlands).

Polyamines were quantified by using high-perfor-

mance liquid chromatography as described earlier

(Hyvonen et al. 1992). The activity of ornithine

decarboxylase was assayed by the method described in

(Janne and Williams-Ashman 1971).

Results

We obtained seven transgenic founder mice out of 57

pups. As the success rate was relatively good, it can be

considered that the transgene, if expressed in the early

embryos of the founders, was compatible with mouse

embryogenesis. All but one of the founders were

fertile, but none of the progeny (185 pups) were

transgenic. The unfertile male founder mouse (406F0)

was first to be sacrificed at the age of 8 months and

analyzed. A macroscopic postmortem examination

and histopathological evaluation of 13 different

tissues was performed but no morphological differ-

ences were detected when compared with age matched

wild type mouse. However, 406F0 overexpressed AZ1

protein in various organs (Fig. 2). Strong overexpres-

sion of full-length AZ1 proteins (24 and 29 kDa) were

Transgenic Res (2014) 23:153–163 155

123

found in testes, epididymes, pancreas and brain. Also

liver, kidneys and salivary glands overexpressed the

transgene. In these organs, however, the majority of

the AZ1 protein was degraded into smaller fragments.

Eventually, the rest of the founder animals were

sacrificed at the age of 10 months and analyzed.

Similarly to 406F0, none of the animals showed any

morphological changes in their tissues. The protein

expression in four tissues was analyzed from each

founder mouse (Fig. 3). The testis, small intestine and

colon were chosen for the analysis because we were

interested in the effect of AZ1 on these highly

proliferative tissues. Also kidneys were included

because it is known to have high sex-depended renal

ODC activity in rodents. This sex-dependency was also

seen in our ODC activity measurement where male

mice showed twice as high renal ODC activity than

female mice (Table 1). Similarly to 406F0, the founder

401F0 showed high AZ1 protein expression in the

testis, which was also evident in immunohistochemis-

try as a patchy cytosolic staining in spermatocytes and

intense nuclear staining in the peritubular myoid cells

(Fig. 4). Also the male founders 402F0, 405F0, and

407F0 showed slight AZ1 protein overexpression in

their testis by immunoblotting analysis (Fig. 3). How-

ever, the level of expression was not high enough to

distinguish those animals from the wild type controls

by immunohistochemistry (not shown). Nor did the

ovaries of the two female founders (403F0 and 404F0)

show any AZ1 immunostaining above the wild type

controls (not shown).

Even the relatively weak overexpression of AZ1

protein in colon, kidney and small intestine (Fig. 3)

resulted in significant reduction of ODC activity in

transgenic founder mice. The most dramatic reduction

was seen in the kidneys of both sexes of the transgenic

mice (405F0 being an exception) (Table 1). The

reduction of ODC activity in these organs was not,

however, accompanied with significant depletion of

polyamine pools (Table 2). The only organ showing

reduction of higher polyamines was brain where

spermidine levels in transgenic mice were lower than

in wild type mice. Relevant putrescine concentrations

were only detected in epiditymis where it was also

reduced in transgenic mice. Surprisingly, hepatic

spermine levels were increased in transgenic animals.

A clear compensatory increase in antizyme inhibitor 1

(AZIn1) protein expression (Fig. 3) was wound in

kidney colon and testis. AZ immunohistochemistry

confirmed the results from western analysis and

showed most intense staining in the colon epithelia

of the founder mice 401F0, 402F0 and 403F0 (Fig. 5).

In these animals the staining was evenly spread

through the colonic mucosa whereas in the other

founders and wild type controls the staining was more

restricted to the luminal part of mucosa—as shown

earlier in wild type mice by Gritli-Linde et al. (2001).

Fig. 1 a The transgene was a 5.5-kbp Eco R1- Eco R1 fragment

isolated from 129 SVJ mouse genomic library (Stratagene, La

Jolla, CA) with a thymidine deletion (DT), corresponding to

position 205 of antizyme cDNA. Exon and intron sequences are

specified by boxes and solid lines respectively, and the protein

coding parts in the exons are in black (adopted from Kankare

et al. 1997). b. The PCR detection of transgenic pups based on

integration of multiple transgene copies to the genome in the

form of concatamers. Thus, only pups with two or more

integration were identified as transgenic. The position of PCR

primers are shown as small arrows

Fig. 2 A western blot analysis of a wild type (wt) and a transgenic mouse 406F0. The bands corresponding to the full-length antizyme 1

(29 and 24 kDa) are marked as AZ1. Actin protein was used as a loading control

156 Transgenic Res (2014) 23:153–163

123

The abundant AZ overexpression in the colonic

mucosa of 401F0, 402F0 and 403F0 mice did not

result in changes of morphology or the proliferation

rate of epithelial cells based on HE and PCNA staining

(not shown). However, up to 10–20 % of enterocytes

on the luminal lining of the mucosa were apoptotic

based on Caspase 3 staining and occurrence of

apoptotic bodies. Normally enterocytes are shed of

so rapidly that apoptosis is only seen in detached cells

in bowel lumen (Marshman et al. 2001) (Fig. 5).

Discussion

It was paradoxical that not even the transgenic male

founder mouse 405F0 could give raise to transgenic

progeny although it showed the mildest reduction of

ODC activity (Table 1) and had no visible AZ1

immunostaining in any of its tissues (not shown).

Equally surprising was that both sexes were unable to

transmit the transgene. Thus it is possible that even

moderate AZ1 overexpression is a negative selection

factor for both the transgenic sperm and egg cells.

Embryogenesis and the later development seem to be

more permissive to the AZ1 overexpression as the

success rate of transgenesis was relatively good and no

pathological changes were detected in any of the

transgenic founder animals. We may speculate, how-

ever, that the expression of microinjected transgene in

a fertilized zygote is different from the transgene

expression in fertilized zygotes derived from trans-

genic sperm cells or oocytes. The mechanism behind

the lack of transgene transmission remains unsolved

Fig. 3 AZ1 and AZIn1 protein expressions in four different

tissues of transgenic founder mice and wild type controls. Full-

length antizyme 1 bands (29 and 24 kDa) are marked as AZ1.

The diffuse bands marked as AZIn1 corresbond to antizyme

inhibitor 1 protein (50 kDa). Actin protein was used as a loading

control. Colon sample from founder mouse 406 was not analysed

Table 1 Tissue ODC activities (pmol/mg tissue wet weight/h) in the AZ1 transgenic founder mice 401–407 compared to wild type

(wt) mice

Colon Kidney Liver Small int. Testis

wt (n$ = 2, n# = 3) 2.88 ± 1.02 36.7 ± 9.78

wt$ 24.1, 30.0

wt# 43.3 ± 4.0#

5.19 ± 3.54 8.44 ± 3.29 3.80 ± 1.43

403$ 0.96 0.14 2.01 1.43

404$ 1.05 1.39 1.91 1.58

401# 0.81 1.96 0.24 0.75 2.15

402# 0.43 1.43 1.63 1.39 2.29

405# 0.48 38.5 1.72 3.44 3.06

406# nd. 1.20 1.40 0.30 1.08

407# 0.72 11.1 1.05 6.55 1.86

Average ± st.dev. 0.74 ± 0.25*** 8.12 ± 14.3** 1.42 ± 0.61* 2.20 ± 2.16** 2.09 ± 0.72*

# The sex-dependency of renal ODC activities in the wild type mice is shown. * P \ 0.05; ** P \ 0.01; *** P \ 0.001 refers to

statistical significances between wild type and transgenic founder mice. Student’s two-tailed t test was used for statistical analyses.

nd. not determined

Transgenic Res (2014) 23:153–163 157

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with the present results and any additional analyses

using gametes of the founder mice are impossible,

leaving the question whether the effect of AZ is

indirect and not polyamine-mediated.

Induction of antizyme proteins has been considered

to be a meaningful approach to deplete cellular

polyamines and thus regulate cellular proliferation.

The present results show, however, that this approach

is problematic in a living animal—as a significant

depletion of higher polyamines could only be seen in

brain tissue even though the ODC activity was

significantly inhibited in all analyzed tissues. Inhibi-

tion of ODC activity should lead to reduced polyamine

biosynthesis which was actually manifested as a

reduction of putrescine in epiditymis. However,

comparative to partial inhibition of ODC, inhibition

Fig. 4 Histological hematoxylin/eosin staining (HE) shows

that there is no overt changes between wild type (Wt) and

transgenic founder mice 401F0 and 406F0 (top row). AZ1

protein was patchily overexpressed in testis of the founder mice

(second row). AZ1 expression did not have any obvious effect

on the number or the distribution of proliferative cells based on

PCNA staining (third row). The non-proliferating peritubular

myoid cells show intense AZ1 staining (arrows). Some sporadic

apoptotic cells could be found both in wild type and transgenic

testis (bottom row). Scale bar 50 lm

Transgenic Res (2014) 23:153–163 159

123

Fig. 5 AZ1 protein was

overexpressed evenly in the

colon epithelia in the

transgenic founders 401F0,

402F0 and 403F0. Some

sporadic apoptotic cells

could be found both in wild

type and transgenic colon

epithelia. However, based

on caspase 3 staining, the

transgenic founders 401F0,

402F0 and 403F0 showed

increased amount of

apoptotic cells on the

epithelial mucosa facing the

lumen of colon

160 Transgenic Res (2014) 23:153–163

123

of polyamine uptake by AZ was probably also only

partial although we have no evidence for it. Therefore,

polyamine uptake from external sources, like from gut

bacteria and diet, is expected to contribute to the

maintenance of polyamine pools in a whole body

setting. The slight but statically significant reduction

of spermidine in brain tissue backs up this assumption

as the transport of polyamines to brain tissue is limited

due to the blood brain barrier (Shin et al. 1985) and

thus the reduction in polyamine biosynthesis is more

readily manifested in brain. The increased hepatic

spermine in transgenic mice may also reflect the

compensatory intake of polyamine from these external

sources. It is also possible that in our animals, during

long-term, ubiquitous and constitutive expression of

the transgene, the tissues have adapted to the meta-

bolic condition and the remaining level of ODC

activity was sufficient to maintain the normal pools of

spermidine and spermine. One explanation for the

unaffected polyamine levels is the compensatory

increase in the AZIn1 protein level in transgenic

founders, which attenuates the effect of AZ1

overexpression.

We chose to use a mouse-derived transgene con-

struct with its own regulatory sequences, as this

approach can give valuable information about the

tissue dependency of AZ1 action. For example, it is

remarkable how strongly AZ1 expression was directed

to the testes of the transgenic mice. It has been shown

earlier that polyamine synthesis follows a fine tuned

temporal and spatial pattern during spermatogenesis

where ODC transcripts are especially abundant in late

pachytene of the first meiotic division and in the early-

stage round spermatids (Kaipia et al. 1990). The

polyamine biosynthesis needs to be strictly regulated

as disturbance of this regulation e.g. by ODC overex-

pression leads to reduction of meiotic cells and to

disruption of spermatogenesis (Halmekyto et al.

1991). Remarkably, a testis specific antizyme (AZ3)

has specially evolved to shut down the polyamine

synthesis at the time of late meiosis and spermiogen-

esis. It has also been shown that there is a transition of

AZ expression from AZ1 in spermatogonias and

spermatocytes to AZ3 in haploid spermatids (Tosaka

et al. 2000; Ivanov et al. 2000). Our results support

these findings as the AZ1 overexpression was mainly

directed to the basal compartment of semiferous

tubules (Fig. 4). However, a mysterious patchy

expression pattern was found where some of the

tubular cross sections were devoid of staining and

some had patches of more and less intense staining.

The latter can be partly explained by the syncytium

where developing sperm cells, originated from single

spermatogonia, are attached by cytoplasmic bridges

forming clusters of cells shearing their cytoplasmic

proteins. It is also possible, but highly unlikely, that

each founder animal was a gonadal mosaic due to late

transgene integration occurring after the one cell stage

of the embryo, which could easily lead to uneven

distribution of the transgene. However, in other

organs, such as in intestine, kidney and liver, the

overexpressed transgene resulted to even staining

without any visible pattern of somatic mosaicism.

We and others have shown that it is possible to

hinder (Murakami et al. 1994) or halt (Pietila et al.

2012) the cellular proliferation by overexpressing AZ1

in vitro. Moreover, AZ1 overexpression inhibits

malignant growth in chemical skin carcinoma model

(Feith et al. 2001). There is also evidence that AZ1 can

induce apoptosis through mitochondrial membrane

depolarization in haematopoietic cells (Liu et al.

2006). Based on this background, we were interested

to see whether AZ overexpression could have an

impact on highly proliferative intestinal epithelia

where apoptosis has also an important role in the

mucosal regeneration. Based on histological exami-

nation, no obvious changes were seen in proliferative

base of intestinal epithelia. However, increased apop-

tosis was evident on the luminal lining of the mucosa

of transgenic animals. This can be regarded as

abnormal, as in normal undamaged epithelia, apopto-

sis is triggered by the detachment of epithelial cells

from the underlying basement membrane and is rarely

seen in cells still attached to the epithelia (Marshman

et al. 2001). Further research is needed to conclude

whether this observation is due premature apoptosis or

defect in detachment of cells, as the polyamines have

been linked both to regulation of apoptosis of intes-

tinal epithelia by HuR dependent p53 mRNA stabil-

ization (Wang et al. 2007) and to integrity of epithelia

by c-Myc regulated E-catherin expression (Liu et al.

2009).

Acknowledgments We thank Prof. Olli Janne for providing

the mutated mouse AZ1 gene construct and the rabbit polyclonal

anti-AZ1 antibody. We also thank Ms. Marita Heikkinen, Sisko

Juutinen, Arja Korhonen, Anne Karppinen and Tuula Reponen

for their skillful technical assistance. This work was financially

supported by the Academy of Finland.

Transgenic Res (2014) 23:153–163 161

123

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