Teratogenesis, Carcinogenesis, and Mutagenesis 16:97-108 (1996)

Teratogenic Effects of Sodium Valproate in Mice and Rats at Midgestation and at Term

Elena Menegola, Maria L. Broccia, Heinz Nau, Mariangela Prati, Rossana Ricolfi, and Erminio Giavini Department of Siologx University of Milan, Milan, Italy (E.M., M. L. B., M.P., R. R., E. G.); Institute of Toxicology and EmbryopharmacologH Free University of Berlin, Berlin, Germany (H.N.)

This experiment was carried out with the aims of comparing the embryotoxic poten- tial of valproic acid (VPA) in rats and mice, better defining the malformation pattern in these species, and comparing the embryotoxic effects detectable in mid-pregnancy to those observed in fetuses at term. Pregnant CD:Crl rats were treated subcutane- ously (s.c.) at 08:OO, 16:00, and 0O:OO on day 9 of gestation with 0, 150, or 300 mg/ kg VPA; pregnant NMRI mice were treated S.C. at 0O:OO on day 7 of gestation, and at 08:OO and 16:OO on day 8 of gestation with 0,75, 150, or 300 mg/kg VPA. Groups of females were killed on day 9 (mice) or day 11 (rats) of pregnancy and their embryos were carefully examined under a dissecting microscope. The remaining females were killed 1 day before parturition and their fetuses were examined for external, visceral, and skeletal malformations. A very high frequency (84%) of malformed embryos was recorded in the group of mice treated with 300 mg/kg, including open brain folds (73%), somite defects (36%), and heart malformations (20%). The rat embryos were less sensitive: only 43% of them were malformed after treatment with 300 mg/ kg, however, the pattern of malformations was quite similar to that observed in mice. The treatment with 150 mg/kg produced about 32% malformed embryos in mice and only 8.5% in rats.

More than 84% of mouse fetuses from mothers treated with the highest dose showed a severe form of exencephaly. The axial skeleton was also severely affected. The postimplantation loss reached 52%. Exencephaly and skeletal malformations were also recorded in mouse fetuses from mothers exposed to 150 mg/kg. The dose of 75 mg/kg was without effects. Exencephaly was not observed in rat fetuses at term. In this species the axial skeleton was the most severely affected region at 300 mg/kg, while the lowest dose produced only sporadic malformations.

These results confirm that the mouse is the more sensitive species for the teratogenic effects of VPA. Furthermore, it has been shown that, in both species, the axial skel- eton is a system which is very sensitive to the teratogenic effects of VPA. The ob- served alterations show a possible link between axial specification and VPA and suggest further studies of embryos exposed to VPA for the expression of genes con- trolling the identity of vertebral segments. @ 1996 Wiley-Liss, Inc.

Address reprint requests to Prof. Erminio Giavini, Department of Biology, University of Milan, Via Celoria 26,I-20133 Milan, Italy.

0 1996 Wiley-Liss, Inc.

98 Menegola et al.

Key words: valproic acid, embryotoxic effects, teratogenicity, midgestation, mice, rats

INTRODUCTION

The antiepileptic drug valproic acid (VPA; sodium valproate, 2-propyl-pentanoic acid) has been implicated as a human teratogen by prospective and retrospective epidemiological studies suggesting that the use of VPA in pregnancy may be associ- ated with an increased incidence of spina bifida along with other malformations [ 1- 41. Neural tube defects (exencephaly) have been observed in vivo after maternal conventional treatments with VPA only in the mouse and hamster [5-81, but an in- creased incidence of spina bifida has been produced in the mouse only after three consecutive administrations of high doses of VPA on day 9 of gestation [ 9 ] . In all tested species (mouse, rat, rabbit, monkey), malformations of the skeletal system are the common effect of prenatal VPA exposure [lo-141.

Sufficient data are available today on VPA pharmacokinetics to make interspe- cies comparisons for rats, mice, and monkeys. Great differences have been observed in these species in the values of Cmax and area under the curve (AUC) after admin- istration of comparable doses. Cmax is higher in the mouse and AUC in the monkey, while the values measured in the rat are in an intermediate position [12,14-161. In the mouse, steady-state concentrations maintained by drug infusion via implanted osmotic minipumps produced embryolethality and growth retardation, while inter- mittent injections produced a high incidence of neural tube defects [15,17]. This result shows that plasma peak levels are more relevant than AUC in regard to VPA teratogenesis in this species. Embryo concentration values reported in rats and mice indicate a greater accumulation of VPA in embryonic tissues relative to maternal plasma than observed in monkeys [14,18,19]. On the other hand, the higher embryotoxic effects reported in monkeys in comparison to rats for comparable doses cannot be explained only on the basis of kinetics and placental transport. The terato- genic activity of VPA could present aspects of species specificity due to unknown factors, e.g., genetic regulation, because Finnell et al. [20] have shown that suscepti- bility to VPA embryotoxicity in mice is strain dependent.

Neural tube defects together with other abnormalities have been reported in in vitro studies both in rats and in mice showing that the teratogenic activity is due to the drug itself and not to metabolites and that neural tube defects are induced in eariy stages of development in both species [21-241. In order to better understand the process involved in VPA-induced abnormalities, in this paper we compare the teratogenic effects induced by VPA at midgestation and at term in the rat and mouse. To have a comparable schedule of treatment, VPA was administered three times (ev- ery 8 h) starting from about stage 10 as described by Fujinaga et al. [25], corre- sponding to an early phase of gastrulation.

MATERIALS AND METHODS

CD:Crl rats and NMRI mice (Charles River, Calco, Italy) were used for the experiments. After 2 weeks of acclimation the females were caged overnight with males of proven fertility. The morning when the vaginal smears were positive (rats) or vaginal plugs were evident (mice) was considered day 0 of gestation. All animals had free access to tap water and food. They were housed in thermostatically main-

Teratogenic Effects of VPA in Mice and Rats 99

tained rooms (21 k 1°C and 50 k 5% relative humidity), with 12 h/day cycles of light regulated by a time switch.

The female rats were treated subcutaneously (s.c.) with 150 or 300 mg/kg body weight (b.w.) of VPA (Sigma Chemical Co., St. Louis, MO) on day 9 of gestation at 08:00, 16:00, and 0O:OO. The female mice were treated S.C. on day 7 of gestation at OO:OO, and on day 8 at 08:OO and 16:OO with 75, 150, or 300 mg/kg b.w. of VPA dissolved in sterile saline. Controls were treated at the same time with saline. Fifty percent of females were killed on day 11 of gestation (rats) and on day 9.5 of gesta- tion (mice). Their embryos were collected, examined for morphological alterations, and scored according to Brown and Fabro [26]. Head length and crown-rump length were measured and the somite number was recorded. The embryos were fixed in 4% formaldehyde and processed for histological examination. The remaining females were killed on day 21 (rats) or day 18 (mice) of gestation. The number of implanta- tions, resorptions, and live fetuses was recorded. After external examination, some fetuses were fixed in Bouin’s solution and examined for visceral malformations ac- cording to the free sectioning method of Wilson [27], while others were processed for skeletal examination according to the double staining method (alizarin red S, alcian blue) of Kimmel and Trammel [28]. Extra groups of females were treated as described above and sacrificed 30 min after treatment. Blood samples were collected from the vena cava and immediately centrifuged.

The concentrations of VPA and metabolites were determined according to the method of Fisher et al. [29].

Statistical analysis of the results was performed by using analysis of variance (ANOVA), followed by Tukey’s test and chi-square test. The level of significance for all analyses was P < 0.05.

RESULTS Mice at Midgestation

The postimplantation loss was higher than in controls only in the high dose group (Table I). A general dose-related trend of developmental retardation was ob- served in all groups exposed to VPA as shown by reduced crown-rump length, somite number, and total score. The incidence of malformed embryos was higher than in controls only in the groups treated with 150 and 300 mg/kg b.w. (Table I). The most frequent malformations were open brain folds, heart abnormalities, irregular neural suture line (waviness), irregular somite shape, and distribution. Apart from a slight developmental retardation that was not statistically significant, no embryotoxic ef- fects were observed in the group treated with 75 mg/kg b.w.

Mice at Term In the animals killed at term, the resorption rate was increased in all groups

compared to that observed at midgestation, particularly those exposed to VPA (Table 11). Developmental retardation was observed as a reduction of fetal weight in the middle and high dose groups.

The most frequent external malformation in live fetuses was exencephaly (Table 111), in some cases associated to ablepharia, facial cleft, and protrusion of the tongue. Visceral examination revealed a few cases of cardiovascular and urogenital malfor- mations. A great number of alterations were observed at the level of the axial skel-

100 Menegola et al.

TABLE I. Reproductive and Litter Parameters at Midgestation in Mice Treated With Sodium Valproate

Control 75 mgkg 150 mgkg 300 mg/kg

Mated females 9 12 11 12 Pregnant females 8 10 10 8 Live embryos (mean f SD) 12.50f2.51 13.3f4.19 12.40f4.50 10.37f3.89 Postimplantation loss (%) 3.9 6.0 5.3 1 9.4A,b.L Crown-rump length (mm) 3.1 1 f 0.39 2.84 f 0.30 2.82 f 0.35 2.67 f 0.20

Head length (mm) (mean f SD) 1.57 f 0.30 1.44 f 0.15 1.38 f 0.18 1.13 f 0.12" Somite number (mean f SD) 23.81 f 3.30 23.02 f 2.20 22.11 f 2.93 21.50 f 2.05 Total score (mean f SD) 38.19 f 2.79 37.97 f 2.82 37.05 * 3.60 29.53 f 3.25".b Malformed embryos (8) 9.68 (6/62) 6.14 (7/114) 32.26 (40/124)" 84.34 (70/83)".b

(mean f SD)

Cardiac defects - 0.88 0.81 20.48

Irregular neural suture line 1.61 - 1.61 10.84 Irregular somite shape 1.61 - 13.71 36.14

Reduced branchial arches 1.61 - 3.23 8.43

Open brain folds 6.45 2.63 16.12 73.49

Microcephal y - 2.63 8.06 1.20

"Significantly different from control group. hSignificantly different from 75 mgkg group. 'Significantly different from 1 SO mgkg group.

eton: extra vertebrae (high dose) and fused vertebrae (middle dose) in the cervical region (Fig. la), fused thoracic vertebrae and ribs, asymmetric sterum, and absence of the first lumbar vertebra (Fig. lb). A high percentage of fetuses had an extra tho- racic vertebra with corresponding ribs inserted in the sternum (Fig. lc). As a conse- quence, these fetuses had eight pairs of ribs attached to the stemum with an extra stemebra (Fig. Id). We interpreted this malformation as a duplication of one thoracic metamere. The extra lumbar ribs that were present at high frequency in this strain of mice (more than 60%) were significantly reduced in the fetuses of the high dose group.

Rats at Midgestation

The treatment with 300 mg/kg b.w. VPA resulted in a significant increase of malformed embryos (Table IV). More than 20% had open brain folds, irregular

TABLE 11. Reproductive and Litter Parameters Near Term in Mice Treated with Sodium Valproate

Control 75 m g k g 150 mgkg 300 mg/kg

Mated females 14 16 18 17 Pregnant females 11 13 9 13 Totally bed litters 0 0 0 4 Implantation (mean f SD) 1.5.27 f 1.74 15.08 f 2.22 12.64 f 4.10 11.15 f 5.53 Live fetuses (mean f SD) 14.27 k 2.41 12.77 f 2.77 10.18 f 4.58 5.31 f 5.36",'.' Postimplantation loss (%) 6.5 15.3 19.4" 52.4a,b.' Fetal weight (8) (mean f SD) 1.26 f 0.09 1.28 f 0.08 1.17 f 0.19 1.05 f O.lVb Placental weight (g) (mean * SD) 0.088 f 0.013 0.090 f 0.016 0.094 f 0.031 0.091 f 0.011

"Significantly different from control group. bSignificantly different from 75 mgkg group. 'Significantly different from 150 mg/kg group.

TABL

E 11

1. M

alfo

rmat

ions

in F

etus

es F

rom

Mic

e T

reat

ed W

ith S

odiu

m V

alpr

oate

Con

trol

75 m

gkg

150

mgk

g 30

0 m

gkg

Exte

rnal

exa

min

atio

n

Abl

epha

ria

Exen

ceph

alia

Fa

cial

cle

fting

H

indl

imb

mal

rota

tion

Mic

rogn

athi

a O

mph

aloc

ele

Pala

tosc

hisi

s To

ngue

pro

trusi

on

Vis

cera

l exa

min

atio

n M

alfo

rmed

fet

uses

(%

)

Mal

form

ed f

etus

es (

%)

Car

diov

ascu

lar

mal

form

atio

n U

roge

nita

l m

alfo

rmat

ion

Skel

etal

exa

min

atio

n

Cer

vica

l rib

Ex

tra c

ervi

cal

verte

bra

Fuse

d ce

rvic

al v

erte

brae

La

ck 7

th c

ervi

cal v

erte

bra

Age

nesi

s 13

th ri

b A

gene

sis

11 th

rib

and

Asy

mm

etric

ste

rnum

Fu

sed

ribs

Fuse

d th

orac

ic v

erte

brae

D

uplic

atio

n of

1 th

orac

ic

Lack

1st

lum

bar

verte

bra

Mal

form

ed f

etus

es (

%)

agen

esis

em

iver

tebr

a

verte

bra

+ rib

+ st

erne

bra

1.2

(2/1

66)

0.6 -

-

-

-

-

0.6 -

1.19

(1/8

4)

1.19

65.8

5 (5

4/82

) 17

.07

-

-

-

-

-

-

12.1

9 -

-

4.88

2.44

15.7

8 (1

5/95

)".b

14

.73a

.b

14.7

3".b

6.

3 1 a

,b

-

-

-

2.04

5.

26

4.08

(2/

49)

2.04

4.

08

84.7

8 (3

9/46

)".b

15.2

2

15.2

2a.b

2.

17

4.35

4.

35

17.3

9 10

.87

8.69

19

.56

8.69

-

88.4

0 (6

1/69

)"3b.

' 73

.9 1

84

.05a

.b.c

1 8

.73a

,b,"

1.44

1.

44

1.44

16.6

6 (6

/36)

"3b*

' 2.

78

1 3

96.9

6 (3

2/33

)a.b

.' 12

.12

24.2

Pb.

'

-

9.0V

b

30.3

a,b,

'

18.1

Vb

54.5

Pb.C

5 7. 5

7a,b

,"

36.3

6a'.b

." Lu

mba

r rib

s 66

.67

57.3

2 58

.69

15. 1

5a.b

.c

"Sig

nific

antly

diff

eren

t fr

om c

ontro

l gro

up.

bSig

nific

antly

diff

eren

t fro

m 7

5 m

gkg

grou

p.

"Sig

nific

antly

diff

eren

t fro

m 1

50 m

g/kg

gro

up.

102 Menegola et al.

Fig. 1 . a: Cervical region of a mouse fetus at term: the atlas is fused with basioccipital (open arrow) and the sixth and seventh vertebrae are fused together (solid arrow) (150 mgkg VPA) b: Lumbar region of a mouse fetus at term showing only five lumbar vertebrae (I-V) (300 mgkg VPA). c: Lateral view of the thorax of a mouse fetus at term. It is possible to see the duplication of one thoracic metamere with consequent fusion of ribs (arrow) (300 mgkg VPA). d: Stemum of a mouse fetus at term: eight ribs are connected to the sternum and induce an extra stemebra (I-VIII, 300 mgkg VPA; control sternum on the left, I-VII).

neural suture lines, and reduced branchial bars. Also a reduction of the telen- cephalic vesicles was observed at high frequency. Other typical alterations ob- served in these embryos were irregular distribution and/or shape of somites and cardiac abnormalities. The low dose produced only a few malformed embryos:

Teratogenic Effects of VPA in Mice and Rats 103

TABLE IV. Reproductive and Litter Parameters at Midgestation in Rats Treated with Sodium Valproate

Control

Mated Females 10 Pregnant females Live embryos (mean f SD) Postimplantation loss (%) Crown-rump length

(mm) (mean f SD) Head length (mm) (mean f SD) Somite number (mean f SD) Total score (mean f SD) Malformed embryos (%)

Cardiac defects Open brain folds Irregular neural suture line Irregular somite shape Microcephaly Reduced branchial arches

9 14.11 f 4.08

7.3 3.55 f 0.16

1.73 f 0.12 24.09 f 0.37 40.10 f 0.99 - (0/127)

150 mgkg 300 mgkg

10 10 10 7

15.20f 1.23 12.28 f 5.65 5.6 4.4

3.59 f 0.25 2.90 f 0.378.b

1.75 f 0.20 24.28 f 1.24 40.31 f 1.61

8.55 (13/152)"

0.66

5.92 2.63

-

1.32 f 0.27a,b 21.75 f 2.83a.b 34.84 f 6.13a,b 43.02 (37/86)"'

11.62 23.25 22.09 39.53 22.09 23.25

"Significantly different from control group. b . Significantly different from 150 mgkg group.

one with open brain folds, nine with somite alterations and four with reduction of the telencephalic vesicles.

Rats at Term When we examined female rats at term we recorded a significantly higher inci-

dence of resorptions only at the high dose level associated with a reduced fetal weight (Table V).

The incidence of major external malformations was very low and there were no neural tube defects. A significantly higher incidence of cardiovascular and urogenital malformations was recorded only in the fetuses of the high dose group (Table VI).

A characteristic pattern of malformations was observed at the level of the axial skeleton of fetuses treated with 300 mg/kg b.w., namely extra cervical vertebrae in some cases associated with extra cervical ribs (Fig. 2a). A lack of one thoracic verte- bra with its ribs was observed in a high number of fetuses, so only six pairs of ribs were inserted on the sternum which had only five sternebrae (Fig. 2b). Only spo- radic malformations were recorded in fetuses exposed to the low dose.

DISCUSSION

The treatment regimen used in these experiments was chosen on the basis of preliminary experiments carried out in order to identify comparable stages of devel- opment in mouse and rat embryos. The first treatment corresponds to the presomite stage 10 of Fujinaga et al. [25]. With the other treatments the main phases of early somitogenesis and neurulation of these species were ongoing when repeated drug treatments were given. These in vivo VPA exposure times correspond to the period of development amenable to in vitro whole embryo culture and could permit a com- parison with the result obtained in such studies. The pattern of malformations ob-

104 Menegola et al.

TABLE V. Reproductive and Litter Parameters Near Term in Rats Treated with Sodium Valproate

Mated Females Pregnant females Totally bed litters Implantation (mean f SD) Live fetuses (mean f SD) Postimplantation loss (%) Fetal weight (g) (mean f SD) Placental weight (8) (mean f SD)

Control

17 12

15.25 f 2.26 14.83 f 2.33

2.73 3.82 f 0.20 0.48 k 0.06

-

150 mgkg

16 16

15.31 k 2.36 14.81 k 2.54

3.26 3.67 f 0.31 0.46 f 0.04

-

300 mgkg

17 16 -

16.44 f 3.78 12.87 f 4.05

2 1 .70".' 3.27 f 0.3Ia.' 0.43 f 0.14

"Significantly different from control group. bSignificantly different from 150 mgkg group.

TABLE VI. Malformations in Fetuses From Rats Treated with Sodium Valproate

Control 150 mgkg 300 mgkg

External examination Malformed fetuses (%)

Acaudia Micrognathia Palatoschisis

Visceral examination Malformed fetuses (%)

Cardiovascular malformations Urogenital malformations

Skeletal examination

Cervical rib Extra cervical vertebra Fused cervical vertebrae Lack 7th cervical vertebra Asymmetric sternum Sternum cleft Agenesis 13th rib Fused ribs Fused thoracic vertebrae Lack of 1 thoracic

Thickened ribs Wavy ribs Extra lumbar vertebra Fused lumbar vertebrae Lack 1st lumbar vertebra Lumbar ribs Agenesis sacrum

Malformed fetuses (%)

vertebra + rib + sternebra

- (0/236) -

-

-

2.46 (3/122)" 1.64 0.82

17.54 (20/114)" -

-

-

0.88

5.26 -

-

- -

0.88

-

2.63 -

-

-

1.75

1.46 (3/206) 0.97 0.48 0.48

22.85 (24/105)".' 5.71"

I 8.0Y'

50.49 (5 I/lOl)a,h 22.77".' 30.69",'

2.97 0.99 3.96 4.95 1.98 3.96 5.94?

28.7 1

0.99

1.98 0.99 1.98

10.89".'

-

v 6.14" 4.95

"Significantly different from control group. hSignificantly different from 150 mgkg group.

Teratogenic Effects of VPA in Mice and Rats 105

Fig. 2. a: Cervical region of a rat fetus at term showing the presence of eight cervical vertebrae (I- VIII c.v.). b: The sterum of a rat fetus at t e m with only six ribs (I-VI) inserted and, as a consequence, only five sternebrae. Both of these fetuses come from females treated with 300 mg/kg VPA.

served in mouse embryos on day 9 of gestation was comparable to that described by Naruse et al. [30] after in vitro culture of mouse embryos exposed to 1.2-1.6 mM VPA. Also, the concentrations inducing the observed abnormalities are quite similar to those determined in the present study (serum levels of 2 and 3.3 mM in mice 30 min after treatments with 150 or 300 mg/kg b.w., VPA, respectively). In rats the serum levels 30 min after treatments were lower than in mice (1.7 and 2.7 mM) but similar to those obtained by Klug et al. [21] after administration of 330 mg/kg b.w. VPA. However, the incidence of abnormal embryos (68%) and of neural tube defects

106 Menegola et al.

(52%) obtained by Klug et al. [21] was higher than that observed in our experiment. This could be due to the different schedules of treatment used in their experiment where pregnant rats were treated with 2 x 330 mgkg b.w. on day 10 of gestation at 08:OO A.M. and 12:OO A.M., producing two peaks of VPA in a narrow period of time.

A comparison of the embryotoxic effects induced by VPA in rats and mice con- firms the higher sensitivity of mice both in terms of embryolethality and in terms of teratogenicity. In rats it is interesting to note that we were unable to obtain neural tube defects in fetuses at term in spite of the relatively high incidence of embryos with open brain folds observed at midgestation. This result may mean that the neural alterations observed in the rat embryos have to be interpreted more as a delay in the closure of the neural tube rather than a real malformation. This interpretation is sup- ported by the observation that the open brain folds in rat embryos exposed to VPA never showed eversion of the neuroepithelium, while in mouse embryos this was frequently observed. Similar results were obtained by Piersma et al. [31] after ad- ministration of retinyl palmitate to pregnant rats, suggesting that in some cases de- layed cranial neural tube closure may be completed later in gestation. Alternatively, it is possible that the rat embryos with neural tube defects die during later stages of pregnancy as shown by the increased frequency of resorptions at term in comparison to midgestation. In contrast to the rat, there was a very high incidence of mouse fetuses with exencephaly; furthermore, the resorption rate near term almost tripled in comparison to that observed at midgestation. This means that the open neural folds observed in mouse embryos at midgestation result in exencephaly at term, but a high frequency of affected embryos dies before term. These different sensitivities at the level of the neural tube in these species are difficult to explain. The more severe effects observed in the mouse could be due to a greater accumulation of VPA in embryonic tissues or to a greater intrinsic sensitivity of mouse embryos to VPA.

A good correlation was observed both in rats and in mice between the irregu- larities of somites at midgestation and malformations near term of the axial skeleton. This region seems to be the common target of VPA teratogenesis in both species. Vertebral development can be disturbed by a variety of insults including chemicals such as mitomycin C [32], irradiation [33], or hyperthermia [34]. Recently, Kessel [35] and Kessel and Gruss [36] described homeotic transformations of vertebrae in mouse fetuses, including abnormalities of cervical vertebrae and extra vertebroster- nal ribs after exposure to teratogenic doses of retinoic acid. This latter malformation, which induces an extra sternebra, was explained as an anterior transformation of the 15th segment, i.e., the 8th thoracic somite. In our mouse fetuses there was clear evidence of a duplication of one thoracic segment leading also to the induction of an extra sternebra so the sternal appearance was similar to that reported by Kessel and Gruss [36]; however, our observations do not indicate which metamere had been duplicated. In contrast, in rat fetuses the most typical alteration observed at the level of the thoracic skeleton was the absence of a thoracic segment. No explanation is available at the moment for this difference in the two species. One possibility is that VPA produces different patterns of malformations because in mouse em- bryos the exposure covered more advanced stages of development in comparison to rat embryos due to the faster development of mice. In any case, it is an in- triguing hypothesis that VPA can interfere in some way with the expression of the “hox code” identifying the vertebral segments leading to the observed skel- etal alterations.

Teratogenic Effects of VPA in Mice and Rats 107

ACKNOWLEDGMENTS

This research was supported by EC grant BI02-CT93-0107. The authors thank Dr. N.A. Brown and Prof. J.J. Picard for helpful discussion.

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