AMERICAN JOURNAL OF PHYSIOLOGY Vol. 231, No. 4, October 1976. Printed
in U.S.A.
Bile acid conjugation organ cultures
in fetal hepatic
R. C. DEBELLE, N. R. BLACKLOW, M. BAYLAN, J. M. LITTLE, AND R. LESTER Department of Pediatrics, McGill University Faculty of Medicine, Montreal, Canada; Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118; and Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
R. C., N. R. BLACKLOW, M. BAYLAN, J. M. LITR. LESTER. Bile acid conjugation in fetaZ hepatic organ cultures. Am. J. Physiol. 231(4): 1124-1128. 1976. -A new technique has been developed in which mammalian fetal liver can be maintained in organ culture for prolonged periods with intact structure and function. Near-term rat fetal liver explants were incubated in vitro for periods of up to 3 wk with preservation of normal cellular morphology and intercellular (organ) relationships. [ 14C]cholate was incorporated into tissue and medium conjugates at a constant rate during 21 days in vitro. During a 24-h incubation with radioactively labeled cholic acid, bile acid conjugates accumulated in tissues to a maximum value by 6 h and maintained this value through 24 h. During the same 24-h incubation with [‘“Clcholate, conjugates were secreted into the medium at a constant rate. Addition of 8 x lop4 M taurine to the medium during a 4-day incubation produced a threefold enhancement in the rate of conjugate formation in tissues and medium. Enhanced conjugation in the presence of additional taurine was due almost entirely to increased taurocholate formation and no significant difference was observed in the amount of glycocholate formed. Exposure of explants to 3.6 x lop4 M cycloheximide for prolonged periods resulted in inhibition of conjugate formation, but when this concentration of cycloheximide was maintained for only 24 h a significantly (P < 0.001) increased rate of conjugate formation was observed. The results indicate that metabolic processes in the organ-culture system are in a state of dynamic equilibrium and that morphologic integrity and specific hepatocytic function are maintained after 21 days in vitro. Preferential taurocholate formation was demonstrated in rat fetal liver, and the data suggest that glycine and taurine interact with separate enzymatic systems in bile acid conjugation. The possible mechanisms that mediate the effect of cycloheximide are discussed. DEBELLE, TLE, AND
liver explants; tissue culture
taurocholate;
glycocholate;
bile acid secretion;
CONJUGATION OF BILE ACIDS is important in the hepatic degradation of ch.olesterol (20) and in the formation and secretion of bile (26). Bile acid conjugates, due to their unusual “detergent” properties, play a central role in maintaining cholesterol in solution in bile (24); they also function in the intestine to solubilize the products of lipolysis and create an optimal environ .ment for lipid absorption (12). In adults, the synthesis, conju .gaTHE
tion, excretion, enterohepatic circulation, and physiologic function of bile acids have been studied extensively (17). Until recently, however, little was known about mammalian fetal bile acid metabolism except for information derived from limited observations on bile salts in perinatal gallbladder bile and meconium (3, 9, 19, 22). Recent studies in which in vivo techniques were used and in which fetal homeostasis was maintained have established that the near-term fetal dog has an efficient hepatic mechanism for the uptake, conjugation, and excretion of a bile acid load (13, 25). In the present study, a new model system has been developed in which organ cultures of rodent fetal liver can be maintained morphologically and metabolically intact for prolonged periods. This system permits a unique approach to the developing mammalian liver and has been used to characterize factors controlling fetal hepatic conjugation of primary bile acids. Preliminary reports of these studies have been presented at the Plenary Session, Forty-Fourth Annual Meeting of the Society for Pediatric Research, San Francisco (7), and the Twenty-Fifth Annual Meeting of the American Association for the Study of Liver Disease, Chicago (6). MATERIALS
AND
METHODS
Organ-culture system. Livers were dissected from fetal Sprague-Dawley rats at 20 days gestation (normal gestational period 22 days) and placed in sterile Leibovitz Ll5 medium (15) supplemented with a final concentration of 0.2% bovine serum albumin, 20 mM glutamine, and 250 U penicillin and streptomycin/ml; the pH of the medium was 7.6. The techniques for the preparation and maintenance of the organ cultures were patterned after the method employed by Dolin (8) in the establishment of fetal intestinal organ cultures. Five liver fragments, each l-2 mm diameter, were maintained in scored Falcon plastic petri dishes with 1.2 ml of supplemented L15 medium, which was renewed at 24 h and every 2nd day thereafter. Dishes with explants and media were placed in sealed plastic containers to prevent evaporation and then placed on a Bellco rocker platform that completed a full cycle once per minute. Incubations were carried out for periods up to 21 days at 36°C in air.
1124
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FETAL
HEPATIC
ORGAN
CULTURE
1125
Histology. Tissues were fixed in Bouin’s solution, dehydrated, and embedded in paraffin. Serial sections were cut at 6 pm and stained with hematoxylin and eosin for light microscopy. All sections were examined. Bile acid conjugation. During each incubation period 0.5 &i [14C]cholic acid (sp act 100 $.Xmg, New England Nuclear Corporation, Boston, Mass.) was added per milliliter of medium. The purity of the [14Clcholate was established by thin-layer chromatography (TLC) in two systems (11): system I, isooctane-ethyl acetate-acetic acid (551); system II, isooctane-isopropanol-acetic acid (3O:lO:l). At the end of each incubation with radioactively labeled cholic acid, tissues and media from three organ-culture dishes were pooled; tissues were homogenized in a Potter-Elvehjem apparatus in 0.5 ml absolute ethanol and dry weights were determined with a Cahn electrobalance on duplicate 25-J aliquots; from the remainder of the homogenate and from a l-ml aliquot of the pooled medium, bile acids were extracted in hot ethanol (23) and acid-butanol, respectively, then identified by TLC on Silica Gel-G in three solvent systems (10): system I, butanol-acetic acid-water (85:10:5); system 11, chloroform-methanol-acetic acid-water (65:20: 10:5); and system III, isoamyl acetate-propionic acid-npropanol-water (4:3:2:1). Bile acids were detected by spraying the chromatograms with phosphomolybdic acid solution and identified by comparison with simultaneously chromatographed standards. Gel bands corresponding to taurocholate and glycocholate were isolated, scraped into count vials, and quantitated in a FIG. 1. Rat fetal liver: magnification x500, hematoxylin and Packard Tri-Carb liquid scintillation spectrometer by Immaturity of hepatic lobular established liquid scintillation techniques with an ex- eosin stain. Top: prior to incubation. architecture is evidenced by paucity of central veins and portal areas ternal standard used for quench correction. and preponderance of double plates of hepatic parenchymal cells. Taurine and cycloheximide (Sigma Chemical Co.) Arrow A indicates hepatocyte; arrow B indicates sinusoid. Abunwere dissolved in the medium in concentrations of 8 x dance of hemopoietic tissue can be seen in the sinusoids. Bottom: cells show characteristic hepatocyte 10m4M and 3.6 x lop4 M, respectively, and the medium after 21 days in vitro. Surviving and intercellular relationships. Arrow A indicates hewith solute was filtered through a Swinnex 0.22-pm- morphology patic parenchymal cell; arrow B indicates sinusoid. Decrease in pore filter (Millipore Corporation). V4Clcholic acid was number of nucleated red blood cells in sinusoids is evident. added to a portion of the appropriate medium, which was then filtered as above. This labeled medium was pmol conjugated/mg dry wt per 24 h on day 20. In added to tissue samples for the final 24 h of a &day separate studies, when conjugate formation was meaincubation. sured at intervals of several hours on the 5th day of explant incubation, conjugates accumulated in the tisRESULTS sues to a maximum value of 90 pmol/mg dry wt 6 h after Figure 1 shows the appearance of rat fetal liver before addition of labeled cholate; this value was maintained and after 3 wk in vitro. During the incubation there was through 24 h (Fig. 2A 1. During the same 24-h incubation some tissue loss, especially in the central area of the with [‘4Clcholate, bile acid conjugates were secreted explant as evidenced by loss of cellular outline, cyto- into the medium at a constant rate of 70 pmol/mg dry wt plasmic coagulation, and nuclear pyknosis. Cells sur- per h (Fig. 2B). vived best at the periphery and were notable for their The effect of substrate on bile acid conjugation was increased granularity and basophilia. The hepatic par- determined by addition of 8 x 1O-4 M taurine to the enchyma maintained its fundamental structure as evi- medium during a 4-day incubation (Table 1). The rate of denced by preservation of interhepatocyte and, hepato- total conjugate formation was enhanced (P < O.OOl)l cyte-sinusoidal relationships. from 328 to 1,000 pmol conjugatedlmg dry wt per 24 h. Specific hepatocytic function (5) was determined by Corresponding increases in both tissue accumulation measuring the uptake, conjugation, and excretion of and secretion were observed. Examination of individual labeled cholic acid by the explants during a prolonged conjugates formed revealed that increased conjugation incubation and throughout a 24-h period after 4 days in was due almost exclusively to enhanced taurocholate vitro. During a 21-day incubation, the incorporation of V4Clcholate into tissue and medium conjugates equaled 1 All P values were determined with the Student t test for the 355 pmol conjugatedlmg dry wt per 24 h on day 5 and 360 differences of means.
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1126
ET
DEBELLE
AL.
3z 100 3 &
n
90
80 i
!I
11111
2
4
II
6
8 IO HOURS
I
12 14 I6 INCUBATION
18
I 20
I 22
I 24
2
8 IO 12 14 16 18 20 22 24 HOURS INCUBATION results of 3 pooled dishes; crosses indicate mean values. A: bile acid conjugate accumulation in tissues. B: values determined for conjugates in medium.
FIG. 2. Bile acid conjugation during a 24-h incubation after 4 days in vitro. Medium with radioactively labeled cholic acid was added to all dishes at beginning of incubation period. Each point represents
TABLE
I. Effect of taurine
and cycloheximide
4
6
on bile acid conjugation Taurine
Cycloheximide
Sample Taurine
Concn
n
Total
M
Tissue
Medium
Tissue
+ medium
0
7 7
0
4 4
0 8 x lO-4
Values
are means
pmol
8 x 1O-4
8 x 1O-4
t SE; n is number
Conjugates
4 4
Taurocholate conjugatedlmg
Glycocholate
Cycloheximide
dry wt per 24 h
Concn
n
M
Total
Conjugates
pmol conjugatedlmg dry wt per 24 h
531 + 6 109 * 11
0 x 1O-4
7
3.6
* 35 890 2 35
3.6
0 x lO-4
7 6
580 1,050
* 43 + 77
3.6
0 x 1O-4
7 6
600 1,062
+ 46 + 78
270
328
1,000
+ 51 + 53
309 + 44 823 + 120
44 38
+ 5 t 6
6
26 + 3 7kl
of determinations.
formation and that glycocholate formation was not significantly altered in the presence of 8 x low4 M taurine. The effect of an inhibitor of protein synthesis on the in vitro rate of bile acid conjugation was investigated by incubating explants for 24 h in medium containing 3.6 x lo-" M cycloheximide (Table 1). Although conjugate formation in tissues decreased from 26 to 7 pmol conjugated/mg dry wt per 24 h, surprisingly, the rate of secretion of conjugates into the medium increased significantly (P < 0.001) from 580 to 1,050 pmol conjugated/ mg dry wt per 24 h, so that total conjugate formation was significantly (P < 0.001) enhanced in the presence of cycloheximide. In other experiments it was determined that incubation with cycloheximide for 48 h or longer produced up to 95% inhibition of conjugate formation, but alteration of explant viability could not be excluded. DISCUSSION
The analysis of metabolic processesin isolated tissues provides the advantage of complete environmental control and eliminates variables encountered in studying intact organisms. The techniques of organ perfusion and tissue slice incubation have been used extensively in the
past, but unfortunately limit experiments to short-term observations. Recently, tissue-culture methods have been applied to maintaining liver cells for prolonged periods in vitro; however, the derivation of hepatocytes for these systems requires subjecting intact liver to mechanical (18) or chemical (2) separation techniques. The disadvantages of such systems are the potential injury to the cells during the isolation procedures, the elimination of organ architecture and intercellular relationships, and the possibility of gross or subtle dedifferentiation of the cells in culture. Organ culture is a technique in which whole fragments of organs are maintained in vitro. These systems have the distinct advantages of the elimination of preparative cellular injury, the maintenance of organ architecture, and extreme simplicity (27). Recent attempts to establish adult and fetal liver in organ culture have been limited by the short survival of the explants in vitro (1). The results presented here document prolonged survival of the explanted fragments of rat fetal liver by morphologic and metabolic criteria. Unlike standard tissue-culture techniques, normal cellular morphology and normal intercellular relationships were preserved throughout the in vitro period. The constant rate of formation of bile acid conjugates in explant tis-
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FETAL
HEPATIC
ORGAN
1127
CULTURE
sues and medium throughout 3 wk in vitro establishes that specific hepatocyt,ic function (5) was maintained and supports the histologic evidence that cellular dedifferentiation did not occur. During the 5th day in vitro, the conjugates accumulated in tissues until a plateau was reached at 6 h. Thereafter, the plateau was maintained through 24 h, which suggests that a dynamic state of equilibrium between uptake, conjugation, and secretion of bile acids was attained in the tissues at this time. Furthermore, the constant rate of secretion of conjugates into the medium throughout 24 h establishes metabolic stability of the system after 5 days in vitro. Since bile acid conjugates accumulated in the medium in concentrations exceeding those of the tissues, an active secretory mechanism for cholic acid conjugates has been demonstrated in rat fetal liver. Preliminary evidence indicates that chenodeoxycholate conjugates are also actively secreted. The addition of taurine to the medium resulted in a greatly enhanced rate of conjugate formation in tissues and medium. As significant bile acid conjugation could be demonstrated in explants of rat fetal liver prior to increasing medium taurine concentration (unpublished observations), it is unlikely that this effect was due to enzyme induction; more probably it represents a massaction phenomenon: increased product formation concomitant with increased substrate concentration. Analysis of individual bile acid conjugates revealed that increased conjugation in the presence of additional taurine was due solely to enhanced taurocholate formation; the proportion of total taurocholate formed increased from 88% to 95%. As the final concentration of taurine in the medium was equimolar with that of glycine, these results establish preferential taurocholate formation in rat fetal liver. In addition, failure to demonstrate competitive inhibition of glycocholate formation with increased taurine availability suggests that glycine and
taurine interact with two separate enzyme systems. This proposed mechanism of bile acid conjugation is in agreement with that postulated by Bremer (4) on the basis of comparative studies in different animal species. As expected, the addition of cycloheximide to the medium for periods of 48 h or longer inhibited conjugate formation and diminished explant viability. To our surprise, however, conjugate formation was nearly doubled when tissues were incubated with cycloheximide and substrate for 24 h. The fact that at the end of 24 h decreased conjugate was present in tissues, while increased quantities had accumulated in the medium, is compatible with an initial stimulatory effect followed by inhibition later during the 24-h period. Cycloheximideinduced stimulation of metabolic processes is not unprecedented. The inhibition of synthesis of a protein that is itself an inhibitor is postulated to explain cycloheximide-induced stimulation of paramylum degradation inEugLena (21) and may explain the observed effect on conjugation. It is of interest that an inhibitor of phenobarbital-induced hepatic microsomal function is said to be extractable from fetal liver (14). Alternatively, nonspecific effects of cycloheximide on cell metabolism (16) may have changed substrate availability, cell permeability, or enzyme activity and secondarily provided the stimulus for conjugate synthesis. Further evaluation of this phenomenon may provide a lever for clarifying the mechanism of development of fetal hepatic bile acid conjugation. This study was supported in part by the Medical Research Council of Canada, National Institutes of Health Grants AMHD17847 and HD08954, and U. S. Army Medical Research and Development Command Contract No. 17-72-C-2071. Address reprint requests to: Dr. Roger Lester, 1000G Scaife Hall, University of Pittsburgh School of Medicine, Pittsburgh, Pa. 15261. Received
for publication
17 February
1976.
REFERENCES 1. BAINES, P. M., P. E. TAYLOR, AND A.. J. ZUCKERMAN. Establishment of organ cultures of human embryonic liver for growth of viruses. Arch. Ges. Virusforsch. 37: 230-235, 1972. 2. BISSELL, D. M., AND J. G. TILLES. Morphology and function of cells of human embryonic liver in monolayer culture. J. Cell Biol. 50: 222-231, 1971, 3. BONGIOVANNI, A. M. Bile acid content of gall bladder of infants, children and adults. J. Clin. Endocrinol. Metab. 25: 678-685, 1965. 4. BREMER, J. Thesis. Oslo: Akadem Trykningssentral-Blindern, 1956. 5. CAREY, J. B., JR. Bile salt metabolism in man. In: The BiZe Acids. Physiology and Metabolism, edited by P. P. Nair and D. Kritchevsky. New York: Plenum, 1973, vol. 2, p. 63. 6. DEBELLE, R. C., N. R. BLACKLOW, J. M. LITTLE, R. WHALEN, AND R. LESTER. Organ culture of fetal liver. GastroenteroZogy 67: 788, 1974. 7. DEBELLE, R. C., A. S. BROWN, N. R. BLACKLOW, R. M. DONALDSON, JR., AND R. LESTER. Organ culture of fetal liver: a new model system. Pediat. Res. 7: 292, 1973. 8. DOLIN, R., N. R. BLACKLOW, R. A. MALMGREN, AND R. M. CHANOCK. Establishment of human fetal intestinal organ cultures for growth of viruses. J. Infect. Diseases 122: 227-231, 1970. 9. ENCRANTZ, J. C., AND J. SJOVALL. On the bile acids in duodenal contents on infants and children. Bile acids and steroids 71. CZin. Chim. Acta 4: 793-799, 1959. 10. ENEROTH, P. Thin layer chromatography of bile alcohols and bile
11,
12.
13.
14.
15.
16.
17.
acids. In: Lipid Chromatographic Analysis, edited by G. V. Marinetti. New York: Dekker, 1969, vol. 2, p. 154-161. HOFMANN, A. F. Thin-layer chromatography of bile acids and their derivatives. In: New Biochemical Separations, edited by A. T. James and H. J. Morris. London: Van Nostrand, 1964, p. 270279. HOFMANN, A. F., AND B. J. BERGSTROM. The intraluminal phase of fat digestion in man: the lipid content of micellar and oil phases of intestinal content obtained during fat digestion and absorption. J. Clin. Invest. 43: 247-257, 1964. JACKSON, B. T., R. A. SMALLWOOD, G. J. PIASECKI, A. S. BROWN, H. F. J. RAUSCHECKER, AND R. LESTER. Fetal bile salt metabolism 1. The metabolism of sodium cholate-‘“C in the fetal dog. J. Clin. Invest. 50: 1286-1299, 1971. JACKSON, S. F. The molecular organization of cells and tissues in culture. In: Cells and Tissues in CuZture, edited by E. N. Willmer. New York: Academic, 1966, vol. I, p. l-62. KLINGER, W., G. ZWACKA, AND H. ANKERMANN. Untersuchungen zum Mechanisms der Enzyminduktion. XIII. Die Ubertragung eines zytoplasmatischen Hemmfaktors der Entwicklung mikrosomaler Leberenzyme aus der Leber von Rattenfoeten auf infantile Ratten. Acta BioZ. Med. Germ. 20: 137-145, 1968. LEIBOVITZ, A. The growth and maintenance of tissue-cell cultures in free gas exchange with the atmosphere. Am. J. Hyg. 78: 173-180, 1963. MCMAHON, D. Cycloheximide is not a specific inhibitor of protein synthesis in vivo. PZant Physiol. 55: 815-821, 1975.
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1128 18. NAIR, P. P., AND D. KRITCHEVSKY (Editors). The Bile Acids: Chemistry, PhysioZogy and Metabolism. New York: Plenum, 1971, 1973, ~01s. 1 and 2. 19. NOYES, W. F. Culture of human fetal liver. Proc. Sot. Exptl. Biol. Med. 144: 245-248, 1973. 20. POLEY, J. R., J. C. DOWER, C. A. OWEN, JR., AND G. B. STICKLER. Bile acids in infants and children. J. Lab. C&z. Med. 63: 838-846, 1964. 21. SCHERSTEN, T. The synthesis of cholic acid conjugates in human liver. Acta Chir. &and. Suppl. 373: l-38, 1967. 22. SCHWARTZBACK, S. D., J. A. SCHIFF, AND N. H. GOLDSTEIN. Events surrounding the early development of Euglena chloroplasts. 5. Control of paramylum degradation. PZant PhysioZ. 56: 313-321, 1975. 23. SHARP, H. L., J. PELLER, J. B. CAREY, JR., AND W. KRIVIT.
DEBELLE
ET
AL.
Primary 274-279,
24.
25.
26.
27.
and secondary bile acids in meconium. Pediat. Res. 5: 1971. SJOVALL, J. Separation and determination of bile acids. In: Methods of Biochemical Analysis, edited by D. Glick. New York: Interscience, 1964, vol. 12, p. 97-142. SMALL, D. M. The physical chemistry of cholanic acids. In: The BiZe Acids. Chemistry, edited by P. P. Nair and D. Kritchevsky. New York: Plenum, 1971, vol. 1, p. 249-356. SMALLWOOD, R. A., R. LESTER, G. J. PIASECKI, P. D. KLEIN, R. GRECO, AND B. T. JACKSON. Fetal bile salt metabolism II. Hepatic excretion of endogenous bile salt and of a taurocholate load. J. Clin. Invest. 51: 1388-1397, 1972. WHEELER, H. 0. Secretion of bile acids by the liver and their role in the formation of hepatic bile. Arch. Internal Med. 130: 533541, 1972.
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in U.S.A.
Bile acid conjugation organ cultures
in fetal hepatic
R. C. DEBELLE, N. R. BLACKLOW, M. BAYLAN, J. M. LITTLE, AND R. LESTER Department of Pediatrics, McGill University Faculty of Medicine, Montreal, Canada; Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118; and Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
R. C., N. R. BLACKLOW, M. BAYLAN, J. M. LITR. LESTER. Bile acid conjugation in fetaZ hepatic organ cultures. Am. J. Physiol. 231(4): 1124-1128. 1976. -A new technique has been developed in which mammalian fetal liver can be maintained in organ culture for prolonged periods with intact structure and function. Near-term rat fetal liver explants were incubated in vitro for periods of up to 3 wk with preservation of normal cellular morphology and intercellular (organ) relationships. [ 14C]cholate was incorporated into tissue and medium conjugates at a constant rate during 21 days in vitro. During a 24-h incubation with radioactively labeled cholic acid, bile acid conjugates accumulated in tissues to a maximum value by 6 h and maintained this value through 24 h. During the same 24-h incubation with [‘“Clcholate, conjugates were secreted into the medium at a constant rate. Addition of 8 x lop4 M taurine to the medium during a 4-day incubation produced a threefold enhancement in the rate of conjugate formation in tissues and medium. Enhanced conjugation in the presence of additional taurine was due almost entirely to increased taurocholate formation and no significant difference was observed in the amount of glycocholate formed. Exposure of explants to 3.6 x lop4 M cycloheximide for prolonged periods resulted in inhibition of conjugate formation, but when this concentration of cycloheximide was maintained for only 24 h a significantly (P < 0.001) increased rate of conjugate formation was observed. The results indicate that metabolic processes in the organ-culture system are in a state of dynamic equilibrium and that morphologic integrity and specific hepatocytic function are maintained after 21 days in vitro. Preferential taurocholate formation was demonstrated in rat fetal liver, and the data suggest that glycine and taurine interact with separate enzymatic systems in bile acid conjugation. The possible mechanisms that mediate the effect of cycloheximide are discussed. DEBELLE, TLE, AND
liver explants; tissue culture
taurocholate;
glycocholate;
bile acid secretion;
CONJUGATION OF BILE ACIDS is important in the hepatic degradation of ch.olesterol (20) and in the formation and secretion of bile (26). Bile acid conjugates, due to their unusual “detergent” properties, play a central role in maintaining cholesterol in solution in bile (24); they also function in the intestine to solubilize the products of lipolysis and create an optimal environ .ment for lipid absorption (12). In adults, the synthesis, conju .gaTHE
tion, excretion, enterohepatic circulation, and physiologic function of bile acids have been studied extensively (17). Until recently, however, little was known about mammalian fetal bile acid metabolism except for information derived from limited observations on bile salts in perinatal gallbladder bile and meconium (3, 9, 19, 22). Recent studies in which in vivo techniques were used and in which fetal homeostasis was maintained have established that the near-term fetal dog has an efficient hepatic mechanism for the uptake, conjugation, and excretion of a bile acid load (13, 25). In the present study, a new model system has been developed in which organ cultures of rodent fetal liver can be maintained morphologically and metabolically intact for prolonged periods. This system permits a unique approach to the developing mammalian liver and has been used to characterize factors controlling fetal hepatic conjugation of primary bile acids. Preliminary reports of these studies have been presented at the Plenary Session, Forty-Fourth Annual Meeting of the Society for Pediatric Research, San Francisco (7), and the Twenty-Fifth Annual Meeting of the American Association for the Study of Liver Disease, Chicago (6). MATERIALS
AND
METHODS
Organ-culture system. Livers were dissected from fetal Sprague-Dawley rats at 20 days gestation (normal gestational period 22 days) and placed in sterile Leibovitz Ll5 medium (15) supplemented with a final concentration of 0.2% bovine serum albumin, 20 mM glutamine, and 250 U penicillin and streptomycin/ml; the pH of the medium was 7.6. The techniques for the preparation and maintenance of the organ cultures were patterned after the method employed by Dolin (8) in the establishment of fetal intestinal organ cultures. Five liver fragments, each l-2 mm diameter, were maintained in scored Falcon plastic petri dishes with 1.2 ml of supplemented L15 medium, which was renewed at 24 h and every 2nd day thereafter. Dishes with explants and media were placed in sealed plastic containers to prevent evaporation and then placed on a Bellco rocker platform that completed a full cycle once per minute. Incubations were carried out for periods up to 21 days at 36°C in air.
1124
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FETAL
HEPATIC
ORGAN
CULTURE
1125
Histology. Tissues were fixed in Bouin’s solution, dehydrated, and embedded in paraffin. Serial sections were cut at 6 pm and stained with hematoxylin and eosin for light microscopy. All sections were examined. Bile acid conjugation. During each incubation period 0.5 &i [14C]cholic acid (sp act 100 $.Xmg, New England Nuclear Corporation, Boston, Mass.) was added per milliliter of medium. The purity of the [14Clcholate was established by thin-layer chromatography (TLC) in two systems (11): system I, isooctane-ethyl acetate-acetic acid (551); system II, isooctane-isopropanol-acetic acid (3O:lO:l). At the end of each incubation with radioactively labeled cholic acid, tissues and media from three organ-culture dishes were pooled; tissues were homogenized in a Potter-Elvehjem apparatus in 0.5 ml absolute ethanol and dry weights were determined with a Cahn electrobalance on duplicate 25-J aliquots; from the remainder of the homogenate and from a l-ml aliquot of the pooled medium, bile acids were extracted in hot ethanol (23) and acid-butanol, respectively, then identified by TLC on Silica Gel-G in three solvent systems (10): system I, butanol-acetic acid-water (85:10:5); system 11, chloroform-methanol-acetic acid-water (65:20: 10:5); and system III, isoamyl acetate-propionic acid-npropanol-water (4:3:2:1). Bile acids were detected by spraying the chromatograms with phosphomolybdic acid solution and identified by comparison with simultaneously chromatographed standards. Gel bands corresponding to taurocholate and glycocholate were isolated, scraped into count vials, and quantitated in a FIG. 1. Rat fetal liver: magnification x500, hematoxylin and Packard Tri-Carb liquid scintillation spectrometer by Immaturity of hepatic lobular established liquid scintillation techniques with an ex- eosin stain. Top: prior to incubation. architecture is evidenced by paucity of central veins and portal areas ternal standard used for quench correction. and preponderance of double plates of hepatic parenchymal cells. Taurine and cycloheximide (Sigma Chemical Co.) Arrow A indicates hepatocyte; arrow B indicates sinusoid. Abunwere dissolved in the medium in concentrations of 8 x dance of hemopoietic tissue can be seen in the sinusoids. Bottom: cells show characteristic hepatocyte 10m4M and 3.6 x lop4 M, respectively, and the medium after 21 days in vitro. Surviving and intercellular relationships. Arrow A indicates hewith solute was filtered through a Swinnex 0.22-pm- morphology patic parenchymal cell; arrow B indicates sinusoid. Decrease in pore filter (Millipore Corporation). V4Clcholic acid was number of nucleated red blood cells in sinusoids is evident. added to a portion of the appropriate medium, which was then filtered as above. This labeled medium was pmol conjugated/mg dry wt per 24 h on day 20. In added to tissue samples for the final 24 h of a &day separate studies, when conjugate formation was meaincubation. sured at intervals of several hours on the 5th day of explant incubation, conjugates accumulated in the tisRESULTS sues to a maximum value of 90 pmol/mg dry wt 6 h after Figure 1 shows the appearance of rat fetal liver before addition of labeled cholate; this value was maintained and after 3 wk in vitro. During the incubation there was through 24 h (Fig. 2A 1. During the same 24-h incubation some tissue loss, especially in the central area of the with [‘4Clcholate, bile acid conjugates were secreted explant as evidenced by loss of cellular outline, cyto- into the medium at a constant rate of 70 pmol/mg dry wt plasmic coagulation, and nuclear pyknosis. Cells sur- per h (Fig. 2B). vived best at the periphery and were notable for their The effect of substrate on bile acid conjugation was increased granularity and basophilia. The hepatic par- determined by addition of 8 x 1O-4 M taurine to the enchyma maintained its fundamental structure as evi- medium during a 4-day incubation (Table 1). The rate of denced by preservation of interhepatocyte and, hepato- total conjugate formation was enhanced (P < O.OOl)l cyte-sinusoidal relationships. from 328 to 1,000 pmol conjugatedlmg dry wt per 24 h. Specific hepatocytic function (5) was determined by Corresponding increases in both tissue accumulation measuring the uptake, conjugation, and excretion of and secretion were observed. Examination of individual labeled cholic acid by the explants during a prolonged conjugates formed revealed that increased conjugation incubation and throughout a 24-h period after 4 days in was due almost exclusively to enhanced taurocholate vitro. During a 21-day incubation, the incorporation of V4Clcholate into tissue and medium conjugates equaled 1 All P values were determined with the Student t test for the 355 pmol conjugatedlmg dry wt per 24 h on day 5 and 360 differences of means.
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1126
ET
DEBELLE
AL.
3z 100 3 &
n
90
80 i
!I
11111
2
4
II
6
8 IO HOURS
I
12 14 I6 INCUBATION
18
I 20
I 22
I 24
2
8 IO 12 14 16 18 20 22 24 HOURS INCUBATION results of 3 pooled dishes; crosses indicate mean values. A: bile acid conjugate accumulation in tissues. B: values determined for conjugates in medium.
FIG. 2. Bile acid conjugation during a 24-h incubation after 4 days in vitro. Medium with radioactively labeled cholic acid was added to all dishes at beginning of incubation period. Each point represents
TABLE
I. Effect of taurine
and cycloheximide
4
6
on bile acid conjugation Taurine
Cycloheximide
Sample Taurine
Concn
n
Total
M
Tissue
Medium
Tissue
+ medium
0
7 7
0
4 4
0 8 x lO-4
Values
are means
pmol
8 x 1O-4
8 x 1O-4
t SE; n is number
Conjugates
4 4
Taurocholate conjugatedlmg
Glycocholate
Cycloheximide
dry wt per 24 h
Concn
n
M
Total
Conjugates
pmol conjugatedlmg dry wt per 24 h
531 + 6 109 * 11
0 x 1O-4
7
3.6
* 35 890 2 35
3.6
0 x lO-4
7 6
580 1,050
* 43 + 77
3.6
0 x 1O-4
7 6
600 1,062
+ 46 + 78
270
328
1,000
+ 51 + 53
309 + 44 823 + 120
44 38
+ 5 t 6
6
26 + 3 7kl
of determinations.
formation and that glycocholate formation was not significantly altered in the presence of 8 x low4 M taurine. The effect of an inhibitor of protein synthesis on the in vitro rate of bile acid conjugation was investigated by incubating explants for 24 h in medium containing 3.6 x lo-" M cycloheximide (Table 1). Although conjugate formation in tissues decreased from 26 to 7 pmol conjugated/mg dry wt per 24 h, surprisingly, the rate of secretion of conjugates into the medium increased significantly (P < 0.001) from 580 to 1,050 pmol conjugated/ mg dry wt per 24 h, so that total conjugate formation was significantly (P < 0.001) enhanced in the presence of cycloheximide. In other experiments it was determined that incubation with cycloheximide for 48 h or longer produced up to 95% inhibition of conjugate formation, but alteration of explant viability could not be excluded. DISCUSSION
The analysis of metabolic processesin isolated tissues provides the advantage of complete environmental control and eliminates variables encountered in studying intact organisms. The techniques of organ perfusion and tissue slice incubation have been used extensively in the
past, but unfortunately limit experiments to short-term observations. Recently, tissue-culture methods have been applied to maintaining liver cells for prolonged periods in vitro; however, the derivation of hepatocytes for these systems requires subjecting intact liver to mechanical (18) or chemical (2) separation techniques. The disadvantages of such systems are the potential injury to the cells during the isolation procedures, the elimination of organ architecture and intercellular relationships, and the possibility of gross or subtle dedifferentiation of the cells in culture. Organ culture is a technique in which whole fragments of organs are maintained in vitro. These systems have the distinct advantages of the elimination of preparative cellular injury, the maintenance of organ architecture, and extreme simplicity (27). Recent attempts to establish adult and fetal liver in organ culture have been limited by the short survival of the explants in vitro (1). The results presented here document prolonged survival of the explanted fragments of rat fetal liver by morphologic and metabolic criteria. Unlike standard tissue-culture techniques, normal cellular morphology and normal intercellular relationships were preserved throughout the in vitro period. The constant rate of formation of bile acid conjugates in explant tis-
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FETAL
HEPATIC
ORGAN
1127
CULTURE
sues and medium throughout 3 wk in vitro establishes that specific hepatocyt,ic function (5) was maintained and supports the histologic evidence that cellular dedifferentiation did not occur. During the 5th day in vitro, the conjugates accumulated in tissues until a plateau was reached at 6 h. Thereafter, the plateau was maintained through 24 h, which suggests that a dynamic state of equilibrium between uptake, conjugation, and secretion of bile acids was attained in the tissues at this time. Furthermore, the constant rate of secretion of conjugates into the medium throughout 24 h establishes metabolic stability of the system after 5 days in vitro. Since bile acid conjugates accumulated in the medium in concentrations exceeding those of the tissues, an active secretory mechanism for cholic acid conjugates has been demonstrated in rat fetal liver. Preliminary evidence indicates that chenodeoxycholate conjugates are also actively secreted. The addition of taurine to the medium resulted in a greatly enhanced rate of conjugate formation in tissues and medium. As significant bile acid conjugation could be demonstrated in explants of rat fetal liver prior to increasing medium taurine concentration (unpublished observations), it is unlikely that this effect was due to enzyme induction; more probably it represents a massaction phenomenon: increased product formation concomitant with increased substrate concentration. Analysis of individual bile acid conjugates revealed that increased conjugation in the presence of additional taurine was due solely to enhanced taurocholate formation; the proportion of total taurocholate formed increased from 88% to 95%. As the final concentration of taurine in the medium was equimolar with that of glycine, these results establish preferential taurocholate formation in rat fetal liver. In addition, failure to demonstrate competitive inhibition of glycocholate formation with increased taurine availability suggests that glycine and
taurine interact with two separate enzyme systems. This proposed mechanism of bile acid conjugation is in agreement with that postulated by Bremer (4) on the basis of comparative studies in different animal species. As expected, the addition of cycloheximide to the medium for periods of 48 h or longer inhibited conjugate formation and diminished explant viability. To our surprise, however, conjugate formation was nearly doubled when tissues were incubated with cycloheximide and substrate for 24 h. The fact that at the end of 24 h decreased conjugate was present in tissues, while increased quantities had accumulated in the medium, is compatible with an initial stimulatory effect followed by inhibition later during the 24-h period. Cycloheximideinduced stimulation of metabolic processes is not unprecedented. The inhibition of synthesis of a protein that is itself an inhibitor is postulated to explain cycloheximide-induced stimulation of paramylum degradation inEugLena (21) and may explain the observed effect on conjugation. It is of interest that an inhibitor of phenobarbital-induced hepatic microsomal function is said to be extractable from fetal liver (14). Alternatively, nonspecific effects of cycloheximide on cell metabolism (16) may have changed substrate availability, cell permeability, or enzyme activity and secondarily provided the stimulus for conjugate synthesis. Further evaluation of this phenomenon may provide a lever for clarifying the mechanism of development of fetal hepatic bile acid conjugation. This study was supported in part by the Medical Research Council of Canada, National Institutes of Health Grants AMHD17847 and HD08954, and U. S. Army Medical Research and Development Command Contract No. 17-72-C-2071. Address reprint requests to: Dr. Roger Lester, 1000G Scaife Hall, University of Pittsburgh School of Medicine, Pittsburgh, Pa. 15261. Received
for publication
17 February
1976.
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1128 18. NAIR, P. P., AND D. KRITCHEVSKY (Editors). The Bile Acids: Chemistry, PhysioZogy and Metabolism. New York: Plenum, 1971, 1973, ~01s. 1 and 2. 19. NOYES, W. F. Culture of human fetal liver. Proc. Sot. Exptl. Biol. Med. 144: 245-248, 1973. 20. POLEY, J. R., J. C. DOWER, C. A. OWEN, JR., AND G. B. STICKLER. Bile acids in infants and children. J. Lab. C&z. Med. 63: 838-846, 1964. 21. SCHERSTEN, T. The synthesis of cholic acid conjugates in human liver. Acta Chir. &and. Suppl. 373: l-38, 1967. 22. SCHWARTZBACK, S. D., J. A. SCHIFF, AND N. H. GOLDSTEIN. Events surrounding the early development of Euglena chloroplasts. 5. Control of paramylum degradation. PZant PhysioZ. 56: 313-321, 1975. 23. SHARP, H. L., J. PELLER, J. B. CAREY, JR., AND W. KRIVIT.
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