Extragonadal 17β-hydroxysteroid dehydrogenase activity in rainbow trout

GENERAL AND COMPARATIVE ENDOCRINOLOGY 82, 197-205 (191)

Extragonadal 17P-Hydroxysteroid Dehydrogenase Activity in Rain bow Trout

RCJDIGERSCHULZ' AND VOLKERBLUM

Ruhr-Universit8t Bochum, Fakultiit Biologie, AG Vergleichrnde Endokrinologie, Universitiitsstr. 150, D-4630 Bochum I, Federal Republic of Germany

Accepted June 28, 1990

Blood cells of male and female rainbow trout showed 17B-hydroxysteroid dehydrogenase (17BHSD) activity in vitro, reducing 1 IB-hydroxyandrostenedione and 1 l-ketoandrostenedione (OA) to 1 lp-hydroxytestosterone and 1 I-ketotestosterone (OT), respectively. Enzyme activity did not vary with gonadal development in either sex. The conversion of tritiated precursors was partly inhibited in the presence of steroid-free serum or radioinert steroid, but inhibition was less strong when radioinert androgens were added to steroid-free serum or when the serum contained endogenous steroids. Treatment of male trout with salmon gonadotropin in vivo and/or incubation with a pituitary extract of mature salmon in vitro did not affect OA conversion when blood cells were incubated in the absence of serum, whereas it was slightly but significantly higher when they were incubated in the presence of serum and pituitary extract. In addition to blood cells and steroidogenic tissues, spleen, intestine, brain, liver, excretory kidney, and skin tissue also produced an OA metabolite isopolar to OT in vitro, so that 17BHSD appears to be present in a variety of trout issues. With respect to the biological significance of extragonadal steroid metabolism in vivo, the ligand binding characteristics of circulating steroid binding proteins may be of primary relevance in regulating substrate availability.

Steroid metabolism outside steroidogenic tissues in vertebrates often involves hormone inactivation and excretion. However, in brain, pituitary, fat, and blood platelets, for example (Pasmanik and Callard, 1988; Andersson et al., 1988; Deslypere et al., 1985; Milewich and Whisenant, 1982), the production of physiologically active steroids has also been observed. We have demonstrated in vitro 17B-hydroxysteroid dehydrogenase (17BHSD) activity in blood cells of seven fish species (Mayer et al., 1990a; Schulz, 1986a), catalyzing the reduction of 1 I-ketoandrostenedione (OA) to 1 l- ketotestosterone (OT), an important circulating androgen in teleost fish. Extrago-

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nadal 17BHSD activity also appears to be relevant in vivo, since after castration and implantation of OA-containing silastic cap- sules, high levels of OT but not OA were found in blood plasma of male salmon parr and stickleback (Mayer et al., 1990b,c). Furthermore, OA was the main androgen produced by mature stickleback testis in vitro (Borg et al., 1989) but OT predomi- nated in the circulation (Mayer et al., 1990~). In the trout, OA and II@-hydroxyandrostenedione (OHA) are present in plasma (Schulz, 1985) and are both converted by blood cell 17BHSD (Schulz, 1986a). Thus, steroid metabolism by blood cells may be relevant to the overall endocrine balance.

The present study examines, in rainbow trout, peripheral steroid metabolism of both sexes at different stages of gonadal development. 17BHSD activity was monitored in blood cells during the reproductive cycle,

197 0016~6480/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All right\ of reproduction in any form reserved.

198 SCHULZ AND BLijM

alongside an examination of some aspects of serum protein-steroid interactions, and a possible gonadotropic regulation of steroid conversion by blood cells was tested. Other tissues were also screened for 17PHSD activity.

MATERIAL AND METHODS

Fish. Two- to three-year-old rainbow trout were obtained from a commercial hatchery (Forellenzucht N. Niggemannn, Hattingen-Elfringhausen) where they were kept in open-air ponds under a natural tempera- ture and photoperiod. Further information about the fish, sampled during their first reproductive cycle, is given in Table 1. Except for fish treated with GTH, animals were transported to the University on the day of blood and/or tissue sampling.

Blood cell incubation. Blood was collected by punc- turing the caudal vein or the heart. Hematocrit values ranged from 38 to 47%; caudal vein samples were usu- ally higher. Blood (2 ml) was gently mixed with 6 ml of freshly prepared Alsever’s solution (0.1 M glucose, 30

TABLE I DATE OF BLOOD SAMPLING,%X,AND

GONADOSOMATIC INDEX (GSI)OF BLOODCELLDONORS

Date Sex GSI”

June 23 M 0.05 July 7 F 0.10 September 10 M 5.31 September 17 F 0.22 October 3 M 4.24 October 17 M 5.47 November 3 M (BSA*) 4.89

5 0.32 (GTHb) 4.63

2 0.47 November 4 F 10.62 December 4 M 5.33 January 26 M 3.72 February 2 M (BSAb) 3.87

2 0.31 (GTHb) 5.88

2 0.91 February 7 M 3.68 March 13 M 3.07 March 24 M 3.68 April 1 M 1.64

n Values are from individual fish or averaged from two fish at a similar stage of gonadal development.

b GSI values from males treated in viva with BSA or SGA-GTH, n = 4.

mM citric acid trisodium salt, 3 mM citric acid, 75 mM sodium chloride) and centrifuged (500 g, 10 min). The supematant was discarded and the pellet was sus- pended in trout balanced salt solution (TBSS) (Jalabert et al., 1973), followed by a second centrifugation. The final pellet was resuspended in TBSS such that 330 ~1 of the suspension contained 110 pl blood cells, where the TBSS volume necessary was calculated from the hematocrit value. The suspension (330 ~1) was added to glass vials to which radioactive steroid and other substances (see below) all dissolved in TBSS had been added previously. The final volume (550 pl in most cases) was attained with appropriate volumes of TBSS. The vials were placed in a rotator revolving 6 times/min. All procedures were carried out on ice or in walk-in cold-rooms (4”). To estimate the blood cell survival rate at the end of incubation, additional vials were prepared without radioactivity under otherwise identical conditions.

One or more of the following substances were added to the incubation vials as indicated: (1) 1 pg pituitary extract from mature Oncorhynchus sp., containing all water-soluble compounds (according to Syndel with a GTH content of 10.4%, w/w); (2) 5-250 ~1 blood serum from fish at the same stage of gonadal development as the blood cell donors in the specific experiment, containing or stripped of endogenous steroids by charcoal treatment; (3) 0.1-250 ng of radioinert androgen (all purchased from Sigma); (4) tritiated steroids (all purchased from NEN; sp act, 40-101 Ci/mmol)- 17a-hydroxyprogesterone (17P), testosterone (T), 17B-estradiol (E,), OT, OHA, or OA, the latter three androgens were synthesized from tritiated cortisol as described previously (Schulz, 1985). Unless otherwise indicated, 1 ng of radioactive steroid was added to blood cells.

At the end of incubation, aliquots from the vials without radioactivity were diluted l/10 with a trypan Blue solution (0.66 mg/ml TBSS). The total number of blood cells and those excluding the dye (viable cells) were counted. Except for one experiment, over 90% of the cells were viable at the end of incubation.

Incubations lasted between 1 and 24 hr and were terminated by centrifugation (500 g, 10 min). The su- pematants were mixed with appropriate nonradioactive carrier steroids (20 pg each) and extracted twice with 10 vol of diethyl ether. The samples were applied to thin-layer chromatography (TLC) plates. Following development in chloroform/methanol/H,0 = 188/12/l, radioactive fractions were localized by B-scanning. Radioactivity isopolar to starting material and product was eluted and aliquots were counted in a liquid scin- tillation counter. The aqueous residues remaining after ether extraction were also counted for radioactivity. The identity of OHA and OA conversion products with 1 lB-hydroxytestosterone (OHT) and OT, respectively (no other products were found), has been con- firmed previously by recrystallization to constant specific activity (Schulz, 1986a).

EXTRAGONADAL STEROID METABOLISM IN TROUT 199

GTH treatment. Male trout approaching the end of the spermatogenetic period (November 3) and in the first spawning season (February 2) were injected ip with a total dose of 10 pg of BSA or partially purified GTH from Oncorhynchus sp. (SGA-GTH, Syndel Ltd., Canada) split into two injections (10 and 90% of the total dose, respectively) administered 3 days apart. Twenty-four hours after the second injection, blood cells were prepared for in vitro incubation with 13H]OA.

17f3-HSD activity in trout tissues. The following tissues from two males at the beginning of the second reproductive cycle (June 29; GSI, 0.62 and 0.81) were prepared for in vitro incubation as described previously (Schulz, 1986b): testis, liver, kidney taken at the height of the spleen, rostrally situated head kidney containing interrenal tissue, fat, appendices pyloricae, spleen, muscle, skin, and brain. About 200 mg of each tissue was incubated with 1 ng [3H]OA in 1.7 ml TBSS for 24 hr at 10”. At the end of the incubation, medium was mixed with nonradioactive OA and OT, extracted with ether, subjected to TLC, and analyzed as before. The aqueous residues remaining after ether extraction were assayed for radioactivity. Aliquots of the product isopolar to OT were subjected to CrO, oxidation followed by another TLC run and P-scanning.

RESULTS

In a seasonal study with blood cells collected from trout at different stages of gonadal development (immature, during spermatogenesis, spawning; previtellogenesis, exogenous vitellogenesis), incubations were carried out in the absence or presence of a steroid-free serum volume imitating the blood cell to serum ratio in viva. The conversion of [3H]OA to [3H]OT showed no relation to the stage of gonadal development in either sex (Fig. la). Blood cells from females were slightly less active (P < 0.05, Student’s t test) in the presence of serum. Similar results were obtained when OA conversion was analyzed at different times after the beginning of incubation (Fig. lb and lc). Increasing volumes of steroid- free serum decreased OHA and OA reduction to OHT and OT, respectively (Fig. Id); 20-30% of the substrate was converted at the physiological ratio of blood cells to serum proteins. In two experiments with males at the beginning of the second reproductive cycle, blood cells were incubated with 0.1-5.5 ng [3H]OA (Fig. If). Substrate

saturation of 17BHSD was not reached but calculated with 5.5 ng OA and extrapolated to 1 ml blood, OT production was 1974 pg/ hr in the absence, and 124 pg/hr in the presence of steroid-free serum. When blood cells were incubated with 1 ng [3H]OA and increasing amounts of radioinert OA in the absence of serum (Fig. le), a significant reduction of [3H]OA conversion was observed in the presence of a lOO-fold excess of unlabeled OA. Under these conditions, OT production was calculated as 32 &ml blood x hr.

Tritiated T, E,, and 17P were not metabolized by blood cells from males at the end of the spawning season in the presence of 150 t.~l steroid-free serum (March 13 and 24; 110 ~1 blood cells, 24 hr at 4”), whereas OHA and OA were still converted to OHT and OT (21.5, 19, 27.3, 23.8%, respectively). The metabolism of different steroids was further analyzed in incubations of 110 pJ blood cells with [3H]OT, T, or OA with or without 100 ~1 steroid-free serum (i.e., a serum protein content one-third that in vivo), in the absence or presence of 250 ng of the respective radioinert androgen. After incubation with L3H]OA or OT, most radioactivity remained ether-soluble, whereas more than 80% was rendered water-soluble during incubation with 13H]T alone (Fig. 2a). The latter was completely inhibited by 100 ~1 steroid-free serum, whereas 250 ng radioinert T alone or in combination with serum was only partially effective. There was no ether-soluble radioactivity with TLC mobilities being different from the substrate in the case of OT (Fig. 2b). Radioactivity that remained ether- soluble after incubation with only [3H]T showed TLC mobilities lower than those of authentic T and was distributed in at least two fractions; i.e., the substrate had been completely metabolized. The only metabolite found after incubation with OA was isopolar to OT. T and more so OA conver- sions were relatively high in the presence of a 250-fold excess of radioinert androgen and were even higher when radioinert an-

SCHULZ AND SLUM

8 -

m.

. Jan 26, spawning

a’m OI, ais --I

0.2 0.25 mlsteroid-freeserum

1 N,rv A

exogenous . vilellogenesis

\

iif

C

\ sep 17 previtcllo- genesis

.

’ OA -0T . Feb 7, spawning

150 dpm ~HI-oT x lo4

lrn P

e

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ll ng radioinert OA dpm (%fJ-OA x 10’

FIG. 1. Substrate ([3H]OA or OHA) conversion to [‘HIOT or I lp-hydroxytestosterone (OHT) by trout blood cells in percentage of radioactivity added. Vertical lines on columns indicate SEM. (a) 110 t.tI blood cells collected from trout at different stages of gonadal development were incubated for 24 hr with 1 ng [3H]OA at 4” in the absence (open columns) or presence (hatched columns) of 150 p,l steroid-free serum, prepared from fish at a similar stage of gonadal development. The bars indicate the high/low range. Since no difference was found with respect to the stage of gonadal development, data from all experiments were pooled. (b,c) Experimental conditions as in a, except that substrate conversion was analyzed at different times after the beginning of incubation, in the absence (black lines) or presence (open lines) of 150 ~1 steroid-free serum. (d) Substrate conversion by blood cells (110 p,I, 24 hr, 4”) of male trout in the presence of increasing volumes of steroid-free serum. The bar indicates the physiological blood cell to serum volume ratio. (e) Percentage substrate conversion (1 ng [3H]OA, 4 hr, 4”) in the presence of a l- to lOO-fold excess of radioinert OA by 110 pl blood cells of mature male trout (February 7). (f) Production of [3H]OT by 110 pl blood cells of male trout at the beginning of the second cycle (April 1) incubated for 4-24 hr with different amounts of [3H]OA in the absence or presence of 150 pl steroid-free serum. Data points show the mean of duplicates (b-d and f).


loo-

OT

b

I

OT T

OA

OA

0 TBSS

q 0.1 ml serum

n 250 ng OT, T, or OA

H serum and 250 ng of the respective androgen

0 0.15 ml serum and O-100 ng ‘I

C

0 0.1 1 10 100 FIG. 2. Incubation of I10 ~1 blood cells from trout during spermatogenesis (a and b, October 17) or

during the spawning season (c, February 7), with 1 ng [3H]OT, T, or OA alone or in the presence of 100 (a and b) or 150 (c) (~1 steroid-free serum, radioinert androgen, or a combination of serum and radioinert androgen. (a) Percentage ether-soluble radioactivity in the medium of total radioactivity added. (b) Percentage of the ether-soluble radioactivity with TLC mobihties different from the respective substrates. (c) Percentage substrate (1 ng [‘H]OA) conversion to OT in the presence of O-100 ng radioinert T. All values are means of duplicates.

drogen and serum were concomitantly Blood cells from in vivo BSA- or SGA- present. When [3H]OA was incubated with GTH-treated males at the end of spermato- blood cells and 150 ~1 steroid-free serum, genesis were incubated with r3H]OA in the addition of radioinert T led to an increase of absence of serum (Fig. 3a). A significantly OA conversion (Fig. 2~). In an experimen- higher substrate conversion was found with tal setup more akin to in vivo circum- blood cells from SGA-GTH-injected fish. stances, blood cells of mature males were However, cells from BSA-treated males incubated with serum containing endoge- showed an unusually low survival rate nous steroids, and [3H]OA conversion was (BSA, 78.8%; SGA-GTH, 97.3%), and nor- found to be significantly higher in the pres- malization to the number of viable cells ence than in the absence of endogenous ste- eliminated the differences. Blood cells col- roids (32.5 f 1.6% vs 20.4 2 2.9%; n = 4, lected from spawning males after BSA or P < 0.01; Student’s t test). SGA-GTH treatment were incubated with

- PE

Pn rsirsi a

SCHULZ AND BLijM

serumwith b steroid-free endogenous

-m

PE ’ - PE ’ - PE FIG. 3. Percentage substrate (1 ng [3H]OA) conversion to [3H]OT by blood cells (110 ~1, 4”) col-

lected from male trout at the end of spermatogenesis (a, November 3) or in the first spawning season (b, February 2), after BSA (open columns) or SGA-GTH treatment (hatched columns) in viva. Blood cells were incubated with or without 1 kg of a pituitary extract (PE) from mature salmon, in the absence (5 hr) or presence (24 hr) of 150 pl serum before or after charcoal treatment. S, significantly different (P < 0.05, Student’s t test, n = 4; vertical lines on columns indicate SEM). Blood cells of BSA-treated fish at the end of spermatogenesis (a) showed an unusually low survival rate (BSA, 78.8%; SGA-GTH, 97.3%).

L3H]OA under different conditions (Fig. 3b). In the absence of serum, there were no differences with respect to BSA or to SGA- GTH injection in vivo, nor with respect to the absence or presence of pituitary proteins in vitro. In the presence of serum, however, substrate conversion of cells from BSA-injected fish was slightly but significantly higher when incubated with 1 Kg of a pituitary extract from mature salmon. Furthermore, cells from GTH-injected fish showed a higher activity than cells from BSA-injected fish in the presence of the pituitary extract. As observed previously, OA conversion to OT was higher when the serum contained endogenous steroids, compared to incubations with steroid-free serum.

Of the various tissues of male trout screened for [3H10A conversion activity, only muscle tissue was inactive (Table 2). Between 14.4 and 40.4% of the ether- soluble radioactivity in medium of nonsteroidogenic tissues was isopolar to OT and isopolar to OA after CrO, oxidation.

TLC-analysis often revealed the presence of further products and most tissues produced water-soluble products, indicative of steroid-conjugating enzyme activities.

DISCUSSION

17PHSD activity is present in blood cells of both sexes and in a variety of other tissue types in male trout. With respect to blood cells, the seasonal study, the incubations with or without serum protein in the absence or presence of nonradioactive steroid, and the effect of SGA-GTH in vivo and/or a pituitary extract of mature salmon in vitro all suggest that substrate availability probably is the most important factor with regard to the biological significance of 17PHSD and steroid conjugation activities; the same may apply to steroid metabolism in other nonsteroidogenic tissue. In trout, eel, and gold&h blood, a sex steroid binding plasma protein (SBP) with a relatively high capacity was described (KD = 2-4 nM, B max = 0.1-2.4 +‘U; determined using T as


TABLE 2 IN VITRO CONVERSION OF I ng 13H]OA BYTROUT

TISSUES INCUBATED FOR 24 HR AT 10”

Tissue (mg)

Testis (217) Spleen (208) Intestine (222) Muscle (208) Brain (208) Liver (208) Head kidney

% Substrate conversion: total

conv.“iconv. to OT’

d60.2151 .6 38.8i32.8 48.0114.4

None 47.4140.4 646123.2

% Water soluble’

13.3 2.5

10.9 0.7 8.7

23.1

changes in the hematocrit were not seen in the present study possibly because blood was collected either from the heart or cau- da1 vein.

(205) 27.7121.2 6.7 Kidney (214) 57.5125.2 6.0 Skin (202) 27.7118.0’ 4.6

a Percentage of ether-soluble radioactivity with TLC mobilities different from OA.

b Percentage of ether-soluble radioactivity isopolar to OT and to OA after Cr03 oxidation.

c Percentage of water-soluble radioactivity from total radioactivity added.

d All values are the means of duplicates. ’ Part of the radioactivity was not isopolar to OA

after CrO, oxidation.

In the presence of serum and pituitary extract in vitro, blood cells of SGA- GTH-injected fish produced more OT than blood cells from BSA-injected fish, and the pituitary extract stimulated blood cell OT production from BSA-injected fish in the presence of serum protein. This may be indicative of a direct effect of the pituitary proteins. Except for GTH, we have no information on the pituitary extract’s exact hormone composition, but in view of the relatively small effect of SGA-GTH treatment in viva or of PE in vitro, and in view of the absence of seasonal changes of OA conversion in both sexes despite fluctuations in GTH levels, a direct regulation of steroid metabolism in blood cells by GTH is probably of minor relevance. Indirect effects of pituitary hormones via steroid hormones however, for example through a modula- tion of erythropoiesis, cannot be excluded.

radioligand), which may account for most 13H]T and OA metabolism was rather of the serum’s steroid binding capacity high when serum and the respective radio- (Pasmanik and Callard, 1986; Q&rat et al., inert androgen, or when [3H]OA and radio- 1983; Fostier and Breton, 1975). It there- inert T were present together. Because fore appears reasonable to assume that sub- 17BHSD in 110 ~1 blood cells was not sat- strate availability is related to the capacity urated by 5.5 ng [“HJOA, and because re- and affinity with which serum proteins bind duction of 1 ng [‘H]OA (or conjugation of 1 circulating steroid hormones. The extent of ng L3H]T) was not or only partly inhibited in OA or T conversion also depended on the the presence of up to 250 ng radioinert an- number of blood cells (Schulz, 1986a). drogen, the enzymes’ catalytic capacities Snieszko (1961) described seasonal and do not appear to be a limiting factor at high sex-related changes of hematocrit values in steroid concentrations. Under these condi- rainbow trout with maximum values in ma- tions, radioactive substrate displaced from ture males. Pottinger and Pickering (1987) serum proteins by unlabeled steroid may recorded the erythrocyte number and sex have become accessible for the blood cell steroid levels in brown trout and suggested enzymes. The quantitative differences be- that the maturity-related erythropoiesis in tween T conjugation and OA reduction may males is stimulated by OT. In context with reflect the serum proteins’ different binding the present results, such an increase in characteristics toward the two androgens erythrocyte number may itself be of rele- and/or differences in the effectiveness with vance for attaining the high OT plasma lev- which 17BHSD and steroid glucuronyl- els recorded in mature male rainbow trout transferase metabolize OA and T, respec- (Scott and Sumpter, 1989). However, tively .

204 SCHULZ AND BLijM

Fostier and Breton (1975) showed that OT is a poor competitor for T binding to SBP. Similar results were obtained in brown trout (Pottinger, 1988) and goldfish (Pasmanik and Callard, 1986). OHA and OHT are also poor competitors for T binding in eel plasma (Qt.&at et al., 1983), so that 1 l-oxygenated androgens in general may be bound with low affinity and would be readily displaced by T. A competition between OA and T for serum protein binding sites is also suggested by the stimula- tory effect of radioinert T on [3H]OA conversion in the presence of steroid-free serum and by a higher conversion of [3H]OA in the presence of serum-containing endogenous steroids. The fact that OA conversion by blood cells collected from females was significantly lower than that in males in the presence of serum may also be understood in this context. If female serum showed a higher steroid binding capacity and/or affinity, a lower conversion would reflect differences in substrate availability instead of differences in 17PHSD activity.

If SBP was important for peripheral steroid metabolism, in blood cells or in other tissues, the regulation of SBP levels would become a crucial parameter. In mammals, sex steroids were initially considered as the main regulators of hepatic SBP synthesis, but evidence is not accumulating that growth hormone (GH) is more important while steroids may only play a modulatory role (von Schoultz and Carlstrom, 1989). Pasmanik and Callard (1986) found no sex differences in plasma SBP levels in goldfish at different stages of gonadal development, which would support the notion of sex steroids’ minor role. However, such changes were observed in brown trout plasma (Pot- tinger, 1988). Ng and Idler (1980) reported the disappearance of steroid binding activity from the blood following hypophysec- tomy of male flounder. The binding capacity reappeared after treatment with matura- tional GTH II or other pituitary proteins. It is interesting to note that salmon gonado-

tropin releasing hormone also stimulated GH release from the goldfish pituitary in vitro (Marchant and Peter, 1989), which may represent a link between reproductive hormones, GH, and SBP synthesis.

In summary, extragonadal 17PHSD activity may favor the production of OHT and OT from OHA and OA in vivo. With 40 ml blood/kg body wt (Nichols, 1987) and calculated with the data in the presence of steroid-free serum under nonsaturating conditions (i.e., minimum values), about 5 ng OT could be produced by blood cells per hour and kilogram body weight; steroids com- peting with OA for SBP binding sites most likely would increase OT production. The 1 l-oxygenated androgens show a low affinity to SBP whereas T appears to be either protein bound or is rapidly metabolized, so that its hormonal function at extragonadal sites, if not the displacement of Il-oxygenated androgens from serum proteins, must be understood in the context that protein-bound sex steroids may develop biological activity, viz, the internalization of SBP-steroid complexes by target cells (Si- iteri et al., 1982; Avvakumov et al., 1986).

ACKNOWLEDGMENTS

This study was supported by Grant Bl 62/15 from the Deutsche Forschungsgemeinschaft. The authors thank Renate Oberstebrink-Scholl for technical assis- tance

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Extragonadal 17β-hydroxysteroid dehydrogenase activity in rainbow trout

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