Znt. J. Cancer: 20, 607-615(1977)

NEOPLASTIC TRANSFORMATION INDUCED BY FURYLFURAMIDE A N D NITROMETHYLFURAN OF EMBRYONIC HAMSTER CELLS IN TISSUE CULTURE Yoshisuke NISHIl, Masako TAKETOMIand Naomichi INUI Section of Cell Biology and Cytogenetics, Biological Research Center, Japan Tobacco and Salt Public Corporation, Hatano, Kanagawa 257; Department of Experimental Pathology, Cancer Institute, Toshimaku, Tokyo 170, Japan

Secondary cultures of Syrian hamster embryonic fibroblasts were tested, for transformation and neoplastic properties after exposure in vitro to furylfuramide (AF-2) and other nitrofurans. Typical morphological transformation was seen in five of six cultures between 30 and 186 days following treatment with 5-10x106 M AF-2 for 24 h. Transformation was seen in only one of four cultures 145 days after treatment for 6 h with AF-2. Treatment with 5-lox M NMf (5-nitro-2-methylfuran) for 24 h also induced transformation after 50 and 118 days in two cultures. In contrast, untreated cultures, and cultures treated with 5lox M N F T [+(5-nitro-2-furyl)thiazole] for 24 h were not transformed within 200 days. Three of the six lines transformed by AF-2 and both lines transformed by N M F also became tumorigenic 7-24 days after morphologic transformation. The other three transformed lines produced nodules which regressed within a few weeks of transplantation. Untreated and treated non-transformed lines did not produce tumors during an observation period of 6 months. The tumors were classified as fibrosarcomas. The ability to form colonies in soft agar was acquired by only one tumorigenic line

Miyaji (1971) first reported that AF-2 orally given to rats and mice was neither embryotoxic nor tumorigenic, but recently there have been several reports that AF-2 is carcinogenic (Ikeda, personal communication; Nomura, 1975; Kuwabara and Takayama, 1976;Yokoro et al., 1976). Morris et al. (1969) reported the carcinogenic effects of other 5-nitrofuran compounds. As far as we know, there are no studies of in vitro transformation of cells with these compounds, and so this is the first report that furylfuramide induces neoplastic transformation of cells in vitro, when added directly to the culture medium. MATERIAL AND METHODS

Cell culture Near-term embryos of a random-bred Syrian golden hamster were used as the source of embryonic fibroblasts. Whole embryos at days 12-13of gestation were excised and chopped up finely, and the cells Furylfuramide (AF-2), a nitrofuran derivative, was were dispersed by treatment with 0.25 % trypsin widely used in Japan as an antimicrobial food (Difco Lab., Detroit, Mich.) for 30 min at 37" C. additive for almost 10 years until 1974,and various One or two million cells were then inoculated into 5-nitrofuran derivatives are still extensively used as 75 cm2 plastic flasks (Falcon Plastics, Oxnard, clinical and veterinary medicines and as antiseptics Calif.). Primary cells were cultivated in normal medium at 37" C in 5 % C02 in air. Dulbecco's in animal foods. modified Eagle's medium (DMEM; Grand Island Tonomura and Sasaki (1973) found that furylfuramide induced chromosome aberrations in cul- Biological Co., Grand Island, N. Y.)supplemented tured human lymphocytes. Later, the strong muta- with 20% fetal calf serum (Gibco) and 100 ,ug/ml of genicity of AF-2 was demonstrated in bacteria neomycin (Gibco) was used as normal medium. For (Kada, 1973; Kondo and Ichikawa-Ryo, 1973; subcultivation, confluent monolayer cells were Yahagi et al., 1974, 1976), yeasts (Ong and Shahin, detached with 0.25 % trypsin and transferred to 1974; Shahin and von Borstel, 1976), cultured 25 cm2 plastic flasks (Falcon). The medium was mammalian cells (Kuroda, 1974; Wild, 1975; changed twice or three times a week. Umeda et al., 1975), and silk-worm oocytes (Tazima Chemical treatment and Onimaru, 1973, 1974). Furthermore, intraperiThe following nitrofuran compounds were used: toneally or orally administered AF-2 causes chromo2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), 4-(5some abnormalities in rat bone-marrow cells nitro-2-fury1)thiazole (NFT), and 5-nitro-2-methyl(Sugiyama et al., 1975) and is teratogenic in mice (Nomura 1976). We observed that Syrian hamster furan (NMF), kindly provided by Drs. T. Sugimura embryonic cells showed somatic mutations and and T. Kawachi (Biological Division, National chromosome breaks at high frequencies after intraperitoneal application of AFP in the pregnant Received: April 6, 1977 and in revised form June 27, animal (Inui et al., 1976). 1977.

608

NISHI ET AL.

Cancer Center Research Institute, Tokyo). The chemical structures of these nitrofurans are given in Figure 1. Stock solutions (0.1 M) in dimethyl sulfoxide (DMSO) were diluted for use with normal medium containing 20% fetal calf serum.

or X-ray irradiation. Cultivated cells were injected in 0.5 ml of Hanks’ solution into the cheek pouches, and animals were examined for tumors once a week for 6 months. Tumors that developed were examined histologically.

Colony transformation Cytotoxic effects and colony transformation were examined quantitatively. Ten thousand cells from primary cultures in 5 ml medium were incubated in plastic dishes for 24 h. The medium was then removed and the cultures were treated with chemicals for 6 or 24 h, after which the medium was replaced by fresh normal medium and the plates were incubated with one change of medium until colonies became visible; these colonies were fixed in absolute methanol and stained with Giemsa. Cytotoxicity was measured by counting colonies in treated and untreated cultures and expressed as the ratio of the former to the latter. Transformation of colonies was assessed by the criteria of Berwald and Sachs (1 965), Barker and Sanford (1 970), and Umeda and lype (1973), and the transformation rate was expressed as the percentage of transformed colonies among those examined.

Colony fortnation in agar

The methods of Sanders and Burford (1964), MacPherson and Montagnier (1964), and Sat0 et al. (1967) were used. One thousand cells in 3 ml medium, containing 0.33 % Bacto-agar (Difco Lab., Detroit) and 0.1 ”/, Bacto-peptone (Difco Lab.) were seeded onto 3 ml of baselayer (0.5% Bacto-agar) and incubated for several days until colonies formed. Ten plates were used for each experiment.

A

Neoplastic transformation

Chemicals were added to secondary cultures of 4-100 cells/ml prepared 24 h after the first passages. After treatment, the cultures were washed twice with Hanks’ solution and re-fed with fresh medium. Final concentrations of chemicals of both 5 x and 1 x lo-’ M and treatment periods of 6 and 24 h were used. Control cells were treated with DMSO alone at the same concentrations and for the same time as the treated cells. Cultures were subcultivated with split ratios, between 1 :2 and 1 :30 depending on the cell growth, for over 200 days in vitro. The day when transformed cells first appeared was scored as the day of morphological transformation. The tumorigenicity, growth characteristics and chromosomes of the cells were also examined at intervals for 200 days in vitro. Chromosome preparation

B C

C

Cells were arrested in metaphase by treatment with 2-3pg/ml colcemide (Gibco) for 3 h during exponential growth. Chromosomes were then prepared by the procedure of Yoshida e t a / . (1970) with slight modifications. Implantation of cells into host animals

The tumorigenicity of cultures was examined periodically for 200 days after treatment. Cells were inoculated into the cheek pouches of 1-3 young adult male hamsters, weighing 80-130 g, which had received no conditioning such as cortisone treatment

FIGURE 1 Chemical structure of the tested nitrofuran compounds. (A) 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2); (B) 4-(5-nitro-2-furyl)thiazole (NFT); (c)5-nitro2-methylfuran (NMF).

609

AF-2-INDUCED TRANSFORMATION IN VITRO TABLE I CYTOTOXIC AND TRANSFORMING EFFECTS O F NITROFURANS ON SECONDARY SYRIAN GOLDEN HAMSTER CELLS I N VITRO

Duration of treatment (ht

Chemical (M)

Control AF-2 1 X lo-' 5 x 10-6 1 x 10-5

6 6 6 6 6

NMF 5 ~ 1 0 - ~ 1 x 10-6 NFT 5 ~ 1 0 - ~ I x 10-5 1

~

6 6 24 24 24 24 24 24 24 24

1089 438 568 220 478 405 379 305

6

5 x 10-6 1 x 10-5

~

1098 371 708 46 1 490 528 498 44 1

NMF 5 x 1 x 10-5 NFT 5 ~ 1 0 - ~ 1 x 10-5 Control AF-2 1 X10-6

o

$

~No. ~of dishes ~ ;

c

15

5

14 13 10 10 10 10 15

5 15 15 10 10

10 10

~

~

~

Transformed Transformation colonies ~ ~ ~ frequency i ~ (%) y

1.oo 0.76 0.75 0.58 0.80 0.87 0.82 0.72

0

0.00

0 3

0.00 0.42 0.87

1.oo 0.82 0.69 0.35 0.78 0.67 0.62

0

4

0.61 0.57 0.00 0.00

3 3 0 0

0.50

0 5 3

0.00 0.00 0.88 1.37

4 6

0.84 I .48

0 0

0.00

0.00

Relative number of coloniesldish.

TABLE I1 TRANSFORMING A N D TUMORIGENIC EFFECTS OF NITROFURANS ON HAMSTER EMBRYONIC CELLS

No. of treated cellslml ( x 109

Culture

co1 C03 c02 C04 C05

Treatment COnC. (M)

4 10 4 10 100 ~~

~

t

~

~

~

~ Tumorigenicity ~ ~ $

Duration (h)

6 6 24 24 24

-(49 days) 445) -448) -Continuous 454)

-

_______

AF561 AF563 AF562 AF564 AF565 AF5Ol AF503 AF502 AF504 AF505

4 10 4 10 100 4 10 4 10 100

AF-2 5X10-' 5 x 10-6 5 x 10-6 5 x 10-6 5 x 10-6 J x 10-5 I x 10-5 1 x 10-5 1x 10-5 1 x 10-5

6 6 24 24 24 6 4 24 24 24

-03) -(41) +186 30 42 -(69) +145 42 +140 -Continuous

NM561 NM501 NF561 NF5Ol

100 100 100 100

NMF 5 x 1 x 10-5 NFT 5 ~ 1 0 - ~ 1 x10-5

24 24 24 24

+ 50

+ + +

$118

-Continuous -Continuous

-

-

+-

+ +

-

+ +

-

' Numbers without parentheses indicate when morphological transformation was first detectable. Numbers in parentheses indicate life span in culture; " continuous " means that the cells could grow for more than 200 days after treatment. - For details see Table IV.

~

~

l

610

NlSHl ET AL.

1x

RESULTS

M. N M F caused transformation but NFT

did not.

Cytotoxicity and colony transformation

The cytotoxic effects of AF-2 and nitrofuran derivatives are shown in Table 1. About 40% of the cells were killed by treatment with 1 x M for 6 h and 60% with 1 x M for 24 h. However,

Neoplastic transformarion Control cultures. lnocula of 2.4-10 x lo3 cells, 25 cm2 flask formed complete monolayers after about 10-12 days and surviving cells grew rapidly,

AF 564-8

71

6C04-7

..

n

5-

v

0

,

,

,

,

I

50 Days

I

,

,

,

3

FIGURE 2 Cumulative growth curves of cultured cells treated M AF-2 for 24 h (AF502; continuous line) with 1 x and control cells (C02; broken line). (A) and ( 8 ) represent stages of transformation. (A) ; morphological transformation. (B); neoplastic transformation. NMF and NFT were slightly less toxic. The LD,,, values of these chemicals were approximately 7.3 (AF-2), 14.8 (NMF) and 8.5 (NFT) x M respectively (24 h treatment). Results on colony transformation caused by these chemicals are summarized in Table I. AF-2 caused transformation at higher concentrations, but not at

I

I

50

100

160 (h)

FIGURE3 Growth curves of the cultured cells treated with 5 x 10P M AF-2 for 24 h (AF564; closed circles) and control cells (C04; open circles). These curves were obtained soon after morphological transformation occurred.

forming colonies. After 2-3 weeks, the cells gradually became large flat fibroblasts and they grew more slowly (Figs. 4A and B). Four of five cultures stopped growing after 45-54 days (Table 11) and the cells gradually degenerated (Fig. 2). The fifth culture (C04) continued to grow as a monolayer without losing its doubling potential and was maintained for more than 200 days, in vitro. The cells remained flat and contact-inhibited at confluency. Cultures treated with AF-2 or other nitrofurans. Cytotoxic effects appeared a few days after a 24-h exposure to 5-10 x M AF-2: the cytoplasm

AF-ZINDUCED TRANSFORMATION

IN VITRO

61 1

FIGURE 4 (A) Large fiat fibroblasts in a control culture (C04) after 31 days in vitro. The cells accumulate granules. Phase contrast microscopy; x 100. (B) oriented, monolayer arrangement of cells in a control culture (C02) after 20 days in vitro. A few binucleated cells are seen. Giemsa staining; x 100. (c) Transformed cells in a culture 46 days after M AF-2 for 24 h (AF502). The spindle-shaped cells are randomly arranged, and piled up, treatment with 1 x sometimes with criss-crossing. Giemsa staining; x 100. (D) Transform-ed cells of AF564 (treated with 5 x M AF-2 for 24 h) after 33 days in vitro. Cells are randomly arranged and piled up. They are polygonal, unlike AF502 cells (see Fig. 4c). Phase contrast microscopy; x 100. (E)Typical transformed colony in an AF-2-treated culture. The cells have a swirled arrangement at the periphery, but are densely packed and piled up at the center. Giemsa staining; x 20. (F) Normal colony in an AF-2-treated non-transformed culture. The cells form a monolayer without piling up or crisscrossing. This colony is packed and thinner and more regularly arranged than the transformed colony (see Fig. 4 ~ ) . Giemsa staining; x 20.

612

NlSHl ET AL.

disintegrated or became vacuolated and the cells became detached. Treatment with the same concentration of AF-2 for 6 h, or with NMF or NFT for 24 h, did not induce appreciable toxic effects. Colonies appeared in all treated cultures 10-15 days after treatment. During the next 2-3 weeks, the cell growth became reduced, and morphologically the cells became large flat fibroblasts as in the control cultures. The cells had a well-oriented arrangement and were contact inhibited. Three cultures stopped growing after 41-69 days and the cells degenerated.

shorter doubling time and higher saturation density than control cells (Fig. 3 and Table 111) and their plating efficiency was higher (Table 111). Tuniorigenicity

Untreated and treated cells were inoculated into hamster cheek pouches at various times from 20-225 days after treatment until the cells stopped growing. None of the control cells produced tumors (Table 11) within 6 months during the experiment. However, five of eight transformed cells, AF564, AF503,

TABLE 111 CHARACTERISTICS OF NITROFURAN-INDUCED TRANSFORMED A N D NON-TRANSFORMED CELLS

“Iture

Days after treatment 1

Growth characteristics *

PE (73

DT (h)

Chromosome analysis SD ( x 10’)

Chromosome range

Modal

No. (XJ

Non-transformed cells C04

AF505

41(7) 48(9) 124(20) 163(28) 160(26)

0.6-0.7 0.1-0.2

26.3 86.0

1 .o 0.4

32.0

2.2

14.6

15.3

6.9

38.6-48.9 10.3-17.8

11.2 8.7

12.8 5.3

3 1-73

44

(65.4)

40-62 40-92 65-89

44 44 81

(42.0) (61 .O) (23.1)

43 45 60 44 44 77, 78 50 50 50 50 48 42,44 44

(17.6) (1 5.4) (26.7) (30.3) (23.5) (10.3) (38.5) (23.2) (31.0) (20.0) (25.7) (14.6) (56.4)

Transformed cells AF562 AF564 AF565 AF503 AF502

AF504 NM561

195(16) 222(23) 41 (7) 54(14) I24(29) 159(22) 48(5) 75(10) 970 6) 124(24) 155(33) 175(24) 163(27)

28.7-33.8

15.4

14.9

33.5

12.0

18.5

47.1 42.8

22.6 15.0

10.0 22.0

37-90 37-100 48-66 40-89 36-over 50-over 43-58 42-over 43-93 41 -over 41-over 36-82 30-90

100 100 100

100 100

~

~

Numbers in parentheses are passage numbers. PE, plating efficiency (percentage of colony forming rate); DT, doubling time of exponentially growing cells; SD, saturation density (number of cells per cml). - a More than 50 metaphases of each were examined. Number in parentheses show percentages of cells with the modal number of chromosomes. ~

Three cultures were maintained for more than 200 days without morphological or neoplastic changes (Table 11). In the remaining eight cultures, transformed cells appeared 30-186 days after treatment (Table 11) being first seen among the large flat fibroblasts. These transformed cells continued to grow indefinitely (Fig. 2). Transformation could easily be detected as changes in cell morphology and growth pattern. Transformed cells were small, compact and fibroblastic with large nuclei, some were bipolar (Fig. 4c) and some were polygonal (Fig. 4u). They grew with a random orientation and piled up. Moreover they had a

AF502, NM561, and NM501, became tumorigenic soon after their morphological transformation (Table 11 and detailed data in Table IV). The animals usually died more than 100 days after inoculation. The tumors were classified as pleomorphic fibrosarcomas. The other transformed cells, AF562, AF565 and AF504, produced soft nodules rather then large tumors, when implanted 204, 130 and 165 days after AF-2 treatment. Cells which were not transformed by treatment, AF561, AF563, AF501, AF505, NF561 and NFSOl, failed to produce either tumors or nodules within 6 months after inoculation when inoculated at various times during culture for 200 days in vitro.

61 3

AF-2-INDUCED TRANSFORMATION IN VITRO

The differences in colony morphology were related to differences in tumorigenicity. Cells showing random orientation with swirling toward the periphery of colonies (Fig. 4 ~ produced ) progressively growing tumors, whereas well-oriented monolayer , not. colonies (Fig. 4 ~ )did Colony formation in agar

AF564 formed colonies in semi-solid agar when the cells had become tumorigenic, but the other lines did not form colonies at any time during the 200-day experiment. Chromosome analysis

The modal number of some transformed cells was aneuploid, varying from near diploid to hypo-

Nitrofuran derivatives were classified by Yahagi et a/. (1974) into four main groups: Group I consisted of 5-thiazole derivatives, Groups I1 and I11 of derivatives with vinyl or acryl residues and azomethin residues, respectively, in position 2 of 5-nitrofurat1, and Group IV of compounds not included in 1-111. In this work the effects of three nitrofuran derivatives, AF-2 (Group 11), NFT (Group I), and N M F (Group VI) were tested (Fig. 1). Of these, AF-2 and N M F were found to be carcinogenic to hamster embryo cells when applied directly to the culture media. NFT induced neither morphological nor neoplastic transformation, which may be due to its thiazole structure, as it previously has been shown that many thiazole derivatives are not carcinogenic (Innes et a/., 1956; Hartwell et a / . , 1951). Alternatively, differences

TABLE IV CAPACITY OF TREATED CELLS TO FORM TUMORS A N D COLONIES IN AGAR Culture

Days after treatment '

AF564

37 ( 5 )

Cell No. inoculated ( x 10')

2.0 0.2 0.02

60 (2) I65 (23) 61 ( 5 ) 74 (10)

1.0 10.0

96 (15) 126 (24) 155 (34)

8.0 10.0 10.0

NM561

65 (10) 142 (27)

NM501

73 (7)

1.0 2.0 2.0

AF503

AF502

No. tumors/ No. inocula

313

212 212

Latency (weeks)

Colonies in agar

0-1 1-2 4-6

t

1-2

-

3-4

-

8.0

4.0 2.0

012 112 212

' Data shown only for tumor-producing cell lines. Similar experiments with the five control cultures and nine experimental cultures were negative (see Table 11). - Numbers in parentheses indicate passage numbers. - a Weeks before progressively growing tumors were produced.

tetraploid (Table III), but some cells were diploid between these nitrofurans might affect the activities of (n = 44). It is interesting that the mainly diploid reducing or oxidating enzymes. NM561 cells (56.4 %) produced tumors after 163 Spontaneous neoplastic transformation occurs in days in vitro. Moreover, r~on-transformedcells, with , mouse and rat cells, but very rarely in hamster cells or without treatment, tended to have diploid or (Sanford and Hoemann, 1967; Yaniv and Gottliebtetraploid modal numbers. It is especially noteworthy Stematsky, 1970). In this work, during the 2W-day that 163 days after DMSO treatment, 61 % of the experiment no control cells became transformed metaPhases from co4 were diploid, whereas Over which excludes the uossibilitv that transformation 95 % of the cells showed a normal karYotYPe at the occurred spontaneously in treated cultures. beginning of the experiment. There are several views concerning the time-lag between morphological transformation and tumorDISCUSSION igenicity (Huberman et al., 1968; DiPaolo et al., This work seems to be the first test of the carcino1969; Inui et al., 1972; Borek and Hall, 1974; genicity of nitrofurans in vitro, and shows that AF-2 Tsuda et al., 1976). DiPaolo et a/. (1969) reported causes neoplastic transformation in Syrian hamster that fibrosarcomas were produced from early transembryonic cells. formed colonies. Huberman et a/. (1968) found that

614

NISHI ET AL.

transformation occurred in hamster embryonic cells 5 months after treatment with dimethylnitrosoamine and not until 3 months later did the cells become neoplastic. Borek and Hall (1974) reported that morphological transformation was induced in hamster embryonic cells by X-ray irradiation and the cells were not neoplastic until after more than 30 passages. Under our conditions, tumorigenicity was detected 7-24 days after morphological transformation. Previous results on the carcinogenicity of AF-2 have been inconsistent and confusing: Miyaji (1971) did not detect tumorigenicity in mice and rats. However, Ikeda (1974; personal communication) found tumors in the forestomach of ddy/SLC mice fed a diet containing AF-2 for more than 18 months. Nomura (1975) reported that AF-2 induced lung tumors in ICR/CL mouse fetuses when injected subcutaneously into pregnant mothers, although at a lower frequency. Yokoro et al. (1976) and Kuwabara

and Takayama (1976) found that AF-2 was carcinogenic when given orally to mice and rats. These varying findings on the carcinogenicity of AF-2 reflect the difficulty in finding suitable in vivo systems for testing the carcinogenicity of such chemicals. We concluded on the basis of the present work that even weak carcinogens, such as nitrofurans, can be detected more easily and rapidly by using in vitvo systems than by tests in vivo. ACKNOWLEDGEMENTS

This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture and a Grant from the Ministry of Health and Welfare. We wish to thank Drs. T. Sugimura and T. Kawachi for kindly supplying nitrofuran compounds, and for helpful suggestions.

TRANSFORMATION NEOPLASIQUE, I N D U l TE PAR LE FURYLFURAMIDE ET LE NITROMETHYLFURANE, DES CELLULES EMBRYONNAIRES D E HAMSTER EN CULTURE D E TISSU Des cultures secondaires de fibroblastes embryonnaires d e hamster syrien o n t et6 testees d u point d e vue de leurs proprietbs nkoplasiques et de leur transformation apres exposition in vitro au furylfuramide (AF-2) et d’autres nitrofuranes. O n a observe une transformation morphologique typique dans cinq cultures sur six 30 a 186 jours apres un traitement d e 24 h a I’AF-2 (5-10 x lo6 M). Une telle transformation ne s’est produite que dans une culture sur quatre I45 jours aprks un traitement de 6 h i I’AF-2. Un traitement de 24 h au N M F (5-nitro-2-mtthylfurane), une concentration d e 5 - l o x l o n M, a kgalement induit une transformation dans deux cultures aprbs 50 et 11 8 jours. Par contre, les cultures non traitees, et les cultures traittes a une concentration de 5-10 x lo-’ M, n’etaient pas transformkes au bout de pendant 24 h au N F T [4-(5-nitro-2-furyl)thiazole] 200 jours. Trois des six lignees transformees par I’AF-2 et les deux lignkes transformees par le N M F sont aussi devenues tumorigbnes 7 A 24 jours aprks la transformation morphologique. Les trois autres ligntes transformees ont produits des nodules qui ont rkgressk en I’espace de quelques semaines apres la transplantation. Les lignees non transformees, t r ai t k s ou non, n’ont pas produit de tumeurs au cours d’une periode d’observation de six mois. Les tumeurs ont ete classtes dans la cattgorie des fibrosarcomes. Une seule lignee tumorigbne a acquis la capacite de former des colonies dans la gelose molle.

REFERENCES

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AF-2-INDUCED TRANSFORMATION IN VlTRO

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