IN VITRO Volume 15, No. 8, 1979 All rights reserved 9
CHANGES IN THE PROPERTIES OF FETAL RAT LIVER DURING ORGAN CULTURE CARL MONDER ANDALENA HATLE COUFALIK Research Institute for Skeletornuscular Diseases, Hospital.forJoint Diseases and Medical Center, 1919 Madison Avenue, New York, New York 10035
(Received November 30, 1977; accepted December 20, 1978)
SUMMARY
Explants of fetal rat liver maintained in organ culture lost about 40% of their mass in 42 hr of incubation as a result of decrease in blood cells and hepatocytes. Proteins from the cytosol and particulate elements of the tissue were found in the culture medium. About 60% of this protein was degraded to peptides during culture. The transfer of malate and lactate dehydrogenases from tissue to medium paralleled that of proteins. Glutamate dehydrogenase was lost from the mitochondria and in part leaked through the cell membrane into the medium. Net loss of activity of the three enzymes occurred, probably as a consequence of proteolytic degradation. Of 12 enzymes in liver tissue, the specific activities of eight--soluble malate dehydrogenase, glutamate dehydrogenase, succinate dehydrogenase, phosphopyruvate carboxylase, hexosediphosphatase, glucose-6-phosphatase, tyrosine aminotransferase, and alanine aminotransferase--were unchanged or increased. Glycogen synthetase, aspartate aminotransferase, pyruvate kinase, and lactate dehydrogenase decreased. Although changes in membrane permeability may have had some influence on the results reported, the predominant effect was due to loss of protein from tissue as a result of discharge of total contents of some of the cells into the medium. The residual explanted tissue retained its structural integrity. It is concluded that fetal rat liver in organ culture provides a suitable model system for controlled studies with this organ in vitro. Key words: fetal rat liver; organ culture; enzyme activity.
INTRODUCTION
The organ culture technique has been used for many years to study the biochemical and physiological behavior of a wide variety of tissues 11-7). It has been claimed by those who use organ culture that it has certain advantages over tissue culture in that the structural integrity of tissue fragments are retained to a much greater extent. The degree to which the viability of a tissue is maintained during organ culture is important to the interpretation of results using this technique. MacDonnell et al. {8~ demonstrated that certain biochemical changes take place during organ culture of fetal liver. It is possible that these changes adversely affect the use of this .procedure for studies on fetal liver function and development. As a result of the findings of MacDonnell et al.,
we have reexamined the stability of fetal liver explants under the culture conditions that have been used in this laboratory for the past several years. This paper describes the changes that occur in the activities and distribution of certain enzymes of fetal rat liver during organ culture. We report that the explant undergoes extensive spontaneous changes over an incubation period of 42 hr. During this interval, most of the enzymes do not decrease (and in some cases increase} in residual tissue.
MATERIALS AND M E T H O D S
Sprague-Dawley rats were purchased from Camm Research Institute, Wayne, New Jersey, and maintained in rooms with controlled tern579
580
MONDER AND COUFALIK
perature and humidity with a light-dark cycle of 12 hr each. Food and water were supplied ad lib. Males were allowed to impregnate the females (one female and one male per cage) during the 12hr dark period. The gestation period, therefore, can be estimated with an uncertainty of _+6 hr. At 20 days of gestation, fetuses were removed under sterile conditions after the mother was killed with ether. Liver fragments were established in culture as described by Wicks ~6) in a humidified desiccator with a mixture of 98% air-2% carbon dioxide. When indicated, the livers were washed after 42 hr of incubation in 0.14 M potassium chloride, transferred to new culture dishes with fresh medium, and incubated for an additional 22 hr. The pH of the medium was 7.4 _+ 0.2 and adjusted back, as necessary, to this pH with sterile dilute sodium hydroxide. After incubation, the livers were washed in 0.14 M potassium chloride and then homogenized at 4 ~ C in 0.15 M potassium chloride. The "soluble" and "particulate" fractions were prepared as described by MacDonnell et al. (8). Malate dehydrogenase IL-malate:NAD oxidoreductase EC 1.1.1.37) was measured as described by Ochoa {9); lactate dehydrogenase (Llactate:NAD oxMoreductase, EC 1.1.1.27) by the method of Kornberg ~10}; and glutamate dehydrogenase ~L-glutamate:NAD oxidoreductase EC 1.4.1.2 }by the method of Herzfeld (11 ). Succinate dehydrogenase [succinate: (acceptor} oxidoreductase, EC 1.3.99.1] was measured by a modification of the method of Bernath and Singer (12). Aliquots of particulate or soluble fractions, prepared as described by MacDonnell et al. (8), were incubated with 0.06 mM potassium phosphate, pH 7.6, and 0.0012 mM sodium cyanide for 7 rain at 37 ~ C. A mixture of 0.24 mM sodium succinate and 0.6 or 0.8 mg of phenazine methosulfate (both levels were used in separate runs) was added to the incubation mixture and oxygen
uptake was measured on an Oxygraph (Gilson Medical Electronics, Madison, Wisconsin} using a Clark electrode. Other enzymes were measured as described in our earlier papers (13,14). Protein was estimated by the method of Lowry et al. ~15). Blood content of liver was estimated according to Castagna (16}. To measure enzyme activity in blood cells: heparinized fetal blood was centrifuged at 600 x g; and the cells were washed twice with 0.9% sodium chloride, homogenized in 0.15 M potassium chloride, and separated into "soluble" and "particulate" fractions as described above.
RESULTS Fetal rat liver explants maintained in organ culture for periods up to 64 hr decreased in size and weight. Initially the fragments in each culture dish weighed a total of 57.9 _+ 1 mg Imean and S.D. of 11 determinations). After 42 hr, the explants weighed 30 _+ 2 mg and at 64 hr, 37.7 _+ 2 mg ~a value not significantly different from that at 42 hr}. The explanted tissue changed from deep red-brown to a light tan as a result of the loss of reticulocytes t6). The blood cell content of fetal liver accounted for 17 + 4% of the tissue weight at 20 days of gestation. This value is close to that reported by Greengard, Federman and Knox {17). By 42 hr of incubation the blood cells of the tissue decreased to 3.7 + 0.4% relative to the freshly isolated liver; at 64 hr, the reticulocyte content was unchanged at 2.5 _+ 1.5%. During the 42-hr incubation period, therefore, blood cells accounted for a decrease of 8 mg of tissue. Since the net weight loss was 27 mg, 30% of the total decrease was attributed to the blood cell elements; the remainder was derived from the liver tissue. Thus 33% of the hepatocyte content of the liver explant is released by 42 hr of culture.
TABLE 1 PROTEIN CONTENT OF FETAL LIVER EXPLANTS Incubation Time
Protein Contenta Supernate
Pellet
Medium
Total
mg per g o[ fresh liver 0 46.5 _+6.1 (11) 51.5 + 8.3 (11) 98.0 42 9.8 ---1.8 ~8) 11.3 _+1.6 (8) 11.7 ---7.9 {4) 21.1 64b 7.0 _+1.4 {8) 9.2 + 2.8 (8) 0.16 _+0.07 {2) 16.2 a Each value is the mean +_standard deviation from the mean. Number of litters studied is given in parentheses. b At 42 hr of incubation, the liver fragments were washed on the grid with 0.15 M KCI, transferred to fresh medium, and incubated for an additional 22 hr.
ORGAN CULTURE OF FETAL RAT LIVER Table I shows that both soluble and particulate protein decreased to an equal extent in the explants during 42 hr of incubation. The net accumulation of protein in the medium, 11.7 _+ 7.9 mg per g of liver, was insufficient to account for the loss of protein from the tissue. The total combined increase in peptides and protein in the medium was 30.1 mg per g of liver. Two-thirds of the total, therefore, probably represented degraded protein. The elution pattern of the peptide components in the medium determined by gel filtration, and reproduced in Fig. 1, confirmed this conclusion. The figure shows that a complex pattern of peptides followed the elution of protein. The patterns for liver explants from two 20-dayold fetuses were similar. From the results we estimate that peptides (fractions 14 to 32) represent 0.700
0.600
0.500
0400 E c
0
0.300
0200 0.180
O. 140
o,oo~
o.o ol
TJ' 4
8
12
16 20 24 28 Froction
52
36 40
FIG, ]. Chromatographic separation of peptides in
incubation medium. The medium after 44 hr of culture with 60 mg of fetal liver was passed through a Bin-Gel P2 column (32 by 1.6-cml with 0.01 M sodium phosphate (pH 7.4) as developing solvent. Each fraction contained 2.0 ml effluent. Protein [determined by the method of Lowry et al. (15)] emerged between fractions 8 and 12; tyrosine emerged between fractions 34 and 38. The profile represents the difference between that of medium incubated with liver and one of identical composition incubated under the same conditions without tissue.
581
28% of the total polypeptides. Peptides and amino acids together represent at least 40% of the whole. The loss of both particulate and soluble components from the tissue was interpreted as evidence that the total cellular contents were discharged into the medium rather than through the selective leakage of protein through damaged cell membranes (8). Loss of protein from the tissue should be accompanied by a corresponding enzyme loss. Therefore the retention of enzyme activity by explants was studied. Four enzymes were measured in the medium and explant. These included soluble and particulate malate dehydrogenase, lactate dehydrogenase, glutamate dehydrogenase, and succinate dehydrogenase. The results are shown in Table 2. For each enzyme the total activity recovered after 42 hr of incubation was about one-half of the initial value. Part of the loss in activity is probably due to proteolysis of enzyme released into the medium as indicated above. Malate and lactate dehydrogenase of hematopoietic cells contributed less than 3% of the total activity of the liver tissue. Of the activity lost from the tissue and recovered in the medium, the blood elements contributed 7% to malate dehydrogenase and 18% to lactate dehydrogenase. The activities of these enzymes in the medium, therefore, are mainly due to loss from damaged liver cells. The decrease in glutamate dehydrogenase in the particulate fraction of the hepatocyte was far greater than could be accounted for by cell destruction. The loss was 87% of the initial amount. About one-half of the recoverable activity appeared in the medium. The total decrease of succinate dehydrogenase, which is a specific mitochondrial marker, was 85%. When explants were transferred after 42 hr of incubation to fresh medium for an additional 22 hr, further small decreases in lactate and malate dehydrogenase occurred. Glutamate dehydrogenase continued to redistribute itself into nonparticulate fractions. Total tissue mass, protein, and enzyme activity all decreased during incubation. The effect of organ culture on the specific activities of lactate, malate, glutamate and succinate dehydrogenase in the explant is shown in Table 2B. The specific activity of the soluble malate dehydrogenase was maintained at a constant level over the 64-hr period; the activity of the particulate fraction was halved during the first incubation interval and did not change thereafter.
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M O N D E R AND COUFALIK TABLE 2 EFFECT OF ORGAN CULTURE ON MALATE, LACTATE AND GLUTAMATE DEHYDROGENASES a
A. Total Enzyme Activity (~mol/min/g fresh tissue) Time After Explantation
Particulate
So|uble
Medium
Total
89.3 +_ 18.3 (5) 6.9 +_ 2.4 (41 5.8 +_ 1.3 (6)
101.3 +_26.6 {5) 20.7 +- 4.1 (4) 14.8 +_ 1.8 (6)
62.4+_113.8(4t 5.8+_ 1.1(6)
190.6 90.3 26.5
119.6 +_ 18.6 (7) 18.7 +_ 4.1 (5) 11.9 +_ 1.4 16)
0 66.0+_ 7.6+_
8.9(5) 2.5 (5)
129.2 85.9 20.6
0 3.8+_ 0.8(4) 1.1 +_ 0.6(3)
20.7 8.3 4.9
hr
Malate dehydrogenase 0 42 64 b Lactate dehydrogenase 0 42 64 b Glutamate dehydrogenase 0 42 64 b Succinate dehydrogenase 0 42
9.6(1) 1.2 (1) 1.1 (1) 19.8+_ 1.9(7) 2.3+_ 0.8(5) 1.1 +_ 0.1 (3) 23.2 +_ 4.3 (4) 3.5 +_ 0.8(4)
0.88 +_ 0.25(7) 2.2 +_ 0.8 t5) 2.7 +_ 0.8 (31 0 0
0 0
23.2 3.5
B. Enzyme Specific Activity (~mol/min / 100 mg of protein F T i m e After E xplantation
Particulate
Sohlble
Totat
hr
Malate dehydrogenase 0 176 +- 31 (5) 218 +_55 (5) 394 +- 31 42 68 +- 14 (4) 224 +_62 (4) 292 -+ 32 64 b 54 +- 10 (6) 214 +_42 (6) 268 +- 22 Lactate dehydrogenase 0 7.5(H 285+_55 (7) 293+-55 42 0(1) 199+ 15 (5) 199+_ 15 64b 0 (1) 179 +_40 (6) 179 +_40 Glutamate dehydrogenase 0 38.0 +_4.8 (7) 1.8 +_0.5 (7) 39.8 _+4.8 42 19.1 +- 6.6 (5) 20.9 +- 6.9 (5) 40.1 +- 4.8 6@ 15.7 +- 3.8 (31 38.5 +_7.2 (3t 54.1 +_4.1 Succinate dehydrogenase 0 48.8 +_6.9 (4) 0 48.8 +_6.9 42 35.7 +_9.5 (4) 0 35.7 +_9.5 a Values represent means + SD. Number of litters are given in parentheses. b Values shown were obtained after transferring washed explants to fresh medium at 42 hr and incubating for an additional 22 hr. c Values are determined on the basis of the protein remaining in the explant at the indicated time.
Succinate d e h y d r o g e n a s e specific activity was unchanged. T h e decrease in the specific activity of lactate d e h y d r o g e n a s e was small. T h e increase in specific activity of g l u t a m a t e d e h y d r o g e n a s e in the soluble f r a c t i o n occurred a t t h e expense of t h e activity in t h e p a r t i c u l a t e fraction. R e d i s t r i b u t i o n of g l u t a m a t e d e h y d r o g e n a s e activity was continuous over t h e entire 64 h r of i n c u b a t i o n . Other enzymes. C h a n g e s over a n i n c u b a t i o n period of 44 h r for eight a d d i t i o n a l enzymes (expressed as activity per g r a m wet weight of fresh tissue or p e r 100 m g of protein of i n c u b a t e d tissue) are p r e s e n t e d in T a b l e 3. T h e specific activities of
t h e e n z y m e s did n o t c h a n g e in a n y u n i f o r m direction. H e x o s e d i p h o s p h a t a s e (D-fructose-l,6-diphosphate 1-phosphate phosphohydrolase, EC 3.1.3.11) a n d g l u c o s e - 6 - p h o s p h a t a s e (D-glucose6 - p h o s p h a t e p h o s p h o h y d r o l a s e E C 3.1.3.9) were unchanged. Aspartate aminotransferase (Laspartate: 2-oxoglutarate a m i n o t r a n s f e r a s e E C 2.6.1.1), p y r u v a t e kinase ( A T P : pyruvate p h o s p h o t r a n s f e r a s e , E C 2.7.1.40) a n d glycogen synthetase ( U D P glucose: glucogen-a-4-glucosylt r a n s f e r a s e E C 2.4.1.11) decreased. T h e specific activities of several "soluble" e n z y m e s , phosphopyruvate carboxylase [(}riP: oxaloacetate
583
ORGAN CULTURE OF FETAL RAT LIVER TABLE 3 CHANGES IN ENZYME ACTIVITY DURING ORGAN CULTURE OF FETAL EXPLANTS a Time After Explantation
hr Phosphopyruvate carboxylase 0 44 Hexosediphosphatase 0 44 Glucose-6-phosphatase 0 44 Pyruvate kinase 0 44 Tyrosine aminotransferase 0 44 Alanine aminotransferase 0 44 Aspartate aminotransferase 0 44 Glycogen synthetase l a + b) 0 44
Enzyme Activity
#mol/min / g fresh tissue
nmol/min / 1O0 mg protein b
2.73 +_0.73 (7) 2.10_+0.49 ~6)
6.2 _+1.5 9.1 +2.0
25O 170_+ 30
{1 ) (2)
800 720_+ 10
1040 450_+ 30
( 1) (2) c
2350 1900_+ 100
4960 1180 _+10
( 1j (2) c
11200 5000 _+20 c
118_+45 163 _+54
(6) (48)
104_+40 270 _+10c
603 _+44 557-+ 71
(5) ~4)
1360 _+100 2350-+ 100 c
8507 -+ 1680 ~5) 2298 -+ 140 ~4)c 1150 _+400 (10) 73 _+7 (12)"
19200 _+3800 9700 _+600 c 2600 _+900 340 _+30 c
a Values represent means -+ S.D. Number of experiments is given in parentheses. b Protein of explant at time of measurement. '~44 Hr versus 0 hr. P < 0.01.
carboxylase t t r a n s p h o s p h o r y l a t i n g ) E C 4.1.1.32], tyrosine a m i n o t r a n s f e r a s e lL-tyrosine: 2-oxoglutarate a m i n o t r a n s f e r a s e , E C 2.6.1.5) a n d alanine aminotransferase ~L-alanine: 2-oxoglutarate a m i n o t r a n s f e r a s e , E C 2.6.1.2), increased. H i s t o l o g y . T h e histological integrity of the explants w a s e x a m i n e d after 42 h r of i n c u b a t i o n in
organ culture. R e p r e s e n t a t i v e fields of sections of explants taken before i n c u b a t i o n a n d at 44 h r of incubation are shown in Fig. 2. D u r i n g 42 h r there was extensive decrease in reticulocytes. A decrease in cellular density w a s noted. I n other respects the tissue a p p e a r e d u n c h a n g e d w h e n comp a r e d with fresh tissue.
FIG. 2. Comparison of the structural integrity of fetal rat liver explants at 0 (A) and 44 {B) hr of incubation. H and E. x75.
584
MONDER AND COUFALIK DISCUSSION
During the first 42 hr of culture, a number of gross changes took place in the properties of the explants. The fragments decreased in mass, the color of the explanted tissue became pale, and red blood cells settled to the bottom of the dish. This was reflected on microscopic examination in a pronounced decrease in hematopoietic cells in the tissue. We have observed, as have others (6,8,18), that no other readily discernible disintegration of the tissue occurs in this time period. There was a loosening of the architecture of the explant over the period of incubation; the cells appeared to retain their integrity. Spaces freely permeable to medium and atmosphere developed. The remaining cells were now exposed to an environment in which nutrient and gas exchange necessary for maintenance of function was possible. In considering how the status of the long-term culture evolved, we have concluded that the destructive events we describe in this paper must be adaptive in nature. The life of the explant could not be sustained unless the individual ceils could nourish themselves and eliminate waste products in a way that was not damaging to the neighboring cells. This could only occur if sufficient cells were removed to provide to the remainder access to medium and air. As we have shown here, and as others have found (8), significant changes, which may profoundly affect tissue function, also were occurring on a molecular level. The decrease in tissue mass was due in large part to the loss of reticulocytes, degeneration of cells damaged during cutting of the tissue, and the loss of cells that died because of environmental deficiencies. We observed that both particulate and soluble proteins were lost from the tissue. We interpret this to mean that the total cell contents of the hepatocytes were discharged into the medium. Some cells, in addition, probably suffered membrane damage and selective loss of protein as MacDonnell et al. (8) have suggested. The data in this paper support the observations reported by MacDonnell et al. (8) that there is loss of cellular integrity during organ culture of fetal liver. Although they conclude that changes in membrane permeability account for most of the changes seen in culture, we conclude that destruction of whole or injured cells better account for our results. The loss of both particulate a~d soluble malate dehydrogenase from the explants during the initial 42 hr occurred in about the same proportion
as the loss of protein. Decrease of total enzyme activity in the explants during incubation was observed with all the enzymes listed in Tables 2 and 3 with the exception of phosphopyruvate carboxylase, tyrosine aminotransferase and alanine aminotransferase. If these losses are due to the sloughing off of damaged tissue into the medium, then the specific activities of the enzymes within the remaining tissue must be the same as the activities within the freshly isolated tissue, except for those in which specific selective changes have occurred. When the activities of 12 enzymes were expressed per 100 mg of protein, it was found that the intracellular levels of three (phosphopyrnvate carboxylase, tyrosine aminotransferase, and alanine aminotransferase} had increased; five ~hexosediphosphatase, glucose-6-phosphatase, lactate dehydrogenase, succinate dehydrogenase, soluble malate dehydrogenase) were unchanged; and four (pyruvate kinase, aspartate aminotransferase, and the two particulate enzymes, glycogen synthetase and particulate malate dehydrogenase) had decreased. The decrease in particulate malate dehydrogenase and glutamate dehydrogenase indicated that these enzymes were not yet firmly integrated into the mitochondrial superstructure. These enzymes are found in the matrix of the mature mitochondria. They might not be retained by the immature mitochondria. This conclusion is supported by the fact that the specific activity of sueeinate dehydrogenase, which is an integral part of the mitochondrial inner membrane, remained unchanged. Glutamate dehydrogenase is distributed in the cytosol in avian fetal tissue (19) and fungi (20) and, under certain conditions, in adult rat liver i21,22). Whether a portion of the activity is extramitochondrial in fetal liver remains to be investigated. The loss of particulate components from the explants in the experiments described in this paper most probably occurred as a resuh of the total destruction of the cell membrane. Changes in membrane permeability probably play an additional role in the transfer of macromolecules between the medium and explant. In suspension cultures, leakage of enzymes from cells into medium is a well known phenomenon. Leakage of several of the enzymes described in the paper, as well as of some others from dispersed liver cells (23-26) and ascites tumor suspensions (27,28), have been described. Changes in selective permeability of fetal and adult liver to glucose differ (29). The lag period of several hours, which is necessary for the
ORGANCULTUREOFFETALRATLIVER induction of tyrosine aminotransferase (6), glycogen synthesis (13,30), and mixed function oxidases {31,32} by steroids and other polycyclic hydrocarbon derivatives in organ culture, may be explained by assuming that increased selective permeability of the fetal cell membranes is needed in order for the inducers to gain entry into the cell. Net accumulation of polycyclic hydrocarbons and cortisol in fetal liver explants was always greater after preincubation (14,33D. Organ culture systems are useful as experimental models only if they remain viable during the period of study. The criteria of viability refer to the continuation of those functions that occur in the intact liver in vivo and the appearance of other functions that were absent in the intact fetus. Thus in explants the incorporation of orotate into R N A continues undiminished (25) although protein 134} and glycogen synthesis (13) continues at a slower rate than in the freshly isolated tissue. The histological appearance of the tissue remains unchanged. Gluconeogenesis increases from undetectable levels 135}. Tyrosine aminotransferase, phosphopyruvate carboxylase and alanine aminotransferase also increase. It is self-evident that fetal liver in organ culture, devoid of the stimuli and restraints to which it is subject in vivo, cannot develop in the way it does in situ. The changes that occur in culture were selective, sometimes following the path pursued during development and sometimes not. The reasons for this selectivity are poorly understood. A key issue to be explored is how the in vivo function of liver explants can be restored. Of those enzymes that decrease in activity in explants, but increase during development, most can be stimulated to increase in culture by appropriate hormones. Arginine synthetase t36} and glucose-6phosphatase are increased by cyclic AMP (37), glycogen phosphorylase by epinephrine and glucagon ~38}, and glycogen synthetase a by insulin ~39}. Pyruvate kinase, which decreases during fetal development, decreases in organ culture as well. Often the explant will respond to hormones whereas the indwelling tissue does not. It is remarkable, in the face of the extensive changes that we and n a c D o n n e l l et al. (8) have reported, that the fetal liver explant retains so great a responsiveness to hormones and other environmental stimuli. It has been argued that this is an aberrant response unrelated to fetal development ~8}. It seems to us, on the contrary, a matter to be explored for its relationship to physiological
585
events. The isolated explant has been profitably used to study the hormonal regulation of glycogen metabolism {40}, gluconeogenesis ~13,35}, and microsomal mixed function oxidases {31,32}. Parsa (41} and Parsa and Flancbaum (42} have shown that the morphological and biochemical development of fetal rat liver can be maintained in a synthetic medium enriched with hormones. Simkins, Eisen and Glinsmann (43) have recently described an improved technique for the maintenance of fetal rat liver in organ culture. Under their conditions, the explants generated albumin, a-feto protein and transferrin. These observations encourage us to believe that in a more completely defined environment the structural integrity of the fetal liver explant can be made to simulate that of the normal developing tissue. REFERENCES 1. Thomas, J.A. 1970. Organ Culture. Academic Press, New York. 2. Easty, G. C. 1970. Organ culture methods. In: H. Busch (Ed.}, Methods in Cancer Research. Vol. 5. Academic Press, New York, pp. 1-43. 3. Giannopoulos, G., S. Mulay, and S. Solomon. 1973. Glucocorticoid receptors in lung. II. Specific binding of glucocorticoids to nuclear components of rabbit fetal lung. J. Biol. Chem. 248: 5016-5023. 4. Calcagno, M., H. Goyena, E. Arrambide, and C. Arruti De Urse. 1970. Action of cortisone and cortisol upon biosynthesis of chondroitin sulfate in femur, in vitro cultures of chick embryo. Exp. Cell Res. 63: 131-137. 5. Corradino, R. A. 1973. Embryonic chick intestine in organ culture: Response to vitamin D3 and its metabolites. Science 179: 402-405. 6. Wicks, W. D. 1968. Induction of tyrosine transaminase in fetal rat liver. J. Biol. Chem. 243: 900-906. 7. Ballard, P. L., and R. A. Ballard. 1972. Glucocorticoid receptors and the role of glucocortieoids in fetal lung development. Proc. Nat. Acad. Sci. U.S.A. 69: 2668-2672. 8. MacDonnel, P. C., E. Ryder, J. A. Delvalle, and O. Greengard. 1975. Biochemical changes in cultured foetal rat liver explants. Bioehem. J. 150: 269-273. 9. Ochoa, S. 1955. Malic dehydrogenase from pig heart. Methods Enzymol. 1: 735-739. 10. Kornberg, A. 1955. Lactic dehydrogenase of muscle. Methods Enzymol. 1: 441-443. 11. Herzfeld, A. 1972. The distribution of glutamate dehydrogenase in rat tissues. Enzyme 13: 246-251. 12. P. Bernath, and T. P. Singer. 1962. Succinic dehydrogenase from heart. Methods in Enzymol. 5: 597-602. 13. Monder, C., and A. Coufalik. 1972. Influence of eortisoi on glycogen synthesis and gluconeogenesis in fetal rat liver in organ culture. J. Biol. Chem. 247: 3608-3617.
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14. Monder, C., and A. Coufalik. 1972. Separation of glycogen formation and aminotransferase activity induced by cortisol in fetal rat liver. Endocrinology 91 : 257-261. 15. Lowry, O. H., N. J. Rosenbrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. 16. Castagna, M. 1965. Blood content of ratliver homogenates. Nature 205: 905-907. 17. Greengard, O., M. Federman, and W. E. Knox. 1972. Cytomorphometry of developing rat liver and its application to enzymic differentiation. J. Cell Biol. 52: 261-272. 18. Kirby, L., and P. Hahn. 1973. Enzyme activities during culturing of fetal rat liver. Can. J. Biochem. 51: 476-481. 19. Solomon, J. B. 1959. Changes in the distribution of glutamic, lactic and malic dehydrogenase in liver cell fractions during development of the chick embryo. Dev. Biol. 1: 182-198. 20. Smith, E. L., B. M. Austen, K. M. Blumenthal, and J. F. Nyc. 1975. Glutamate dehydrogenases. In: P. D. Boyer tEd.L The Enzymes. Vol. llA. 3rd Edition. Academic Press, New York, pp. 305-306. 21. Hillar, M., and J. Reuben. 1976. Hormonal induction of glutamate dehydrogenase in rat liver. Experientia 32: 653-655. 22. Godinot, C., and H. A. Lardy. 1973. Biosynthesis of glutamate dehydrogenase in rat liver. Demonstration of its microsomal localization and hypothetical mechanism of transfer to mitochondria. Biochemistry 12: 21)51-2060. 23. Henley, K. S., H. S. Wiggins, H. M. Pollard, and E. Dullaert. 1958. Transaminase activity in suspensions of parenchymatous liver. Ann. N.Y. Acad. Sci. 75: 270-281. 24, Henley, K. S., O. Sorensen, and H. M. Pollard. 1959. Some enzymatic properties of suspensions of parenchymatous liver cells. Nature 184: 1400. 25. Henley, K. S., H. S. Wiggins, H. M. Pollard, and E. Dullaert. 1959. The transaminase content of parenchymatous liver cells. Gastroenterology 36: 1-5. 26. Takada, Y., A. Ichihara, H. Tanioka, and H. Inoue. 1963. The biochemistry of animal cells. 1. The effect of corticosteroids on leakage of enzymes from dispersed rat liver cells. J. Biol. Chem. 239: 3590-3596. 27. Holmberg, B. 1961. On the in vitro release of cytoplasmic enzymes from ascites tumor cells as compared with strain L cells. Cancer Res. 21: 1386-1393. 28. Monder, C., J. Ben-Ezzer, and A. White. 1962. Influence of steroids in vitro on transaminase activity and integrity of lymphocytes. Endocrinology 70: 123-130.
29. Dawkins, M. J. R. 1963. Glycogen synthesis and breakdown in fetal and newborn rat liver. Ann. N.Y. Acad. Sci. 111: 203-211. 30. Eisen, H. J., W. H. Glinsmann, and P. Sherline. 1973. Effect of insulin on glycogen synthesis in fetal rat liver in organ culture. Endocrinology 92: 584-588. 31. Cutroneo, K. R., R. A. Seibert, and E. Bresnick. 1972. Induction of benzpyrene hydroxylase by flavone and its derivatives in fetal rat liver explants. Biochem. Pharmacol. 21: 937-945. 32. Burki, K., R. A. Seibert, and E. Bresniek. 1971. Effects of 3-methylcholanthrene and some related compounds upon the benzpyrene hydroxylase activity in fetal rat liver explants. Biochem. Pharmacol. 20: 2947-2952. 33. Bresnick, E., and K. Burki. 1975. The fetal rat liver explant as a model system in the study of hormone action. Methods Enzymol. 39: 36-40. 34. Sereni, F., and L. P. Sereni. 1969. Spontaneous development of tyrosine aminotransferase activity in fetal liver cultures. Adv. Enzyme Regnl. 8: 253-267. 35. Coufalik, A., and C. Monder. 1976. Spontaneous development of gluconeogenesis in fetal rat livers during incubation in organ culture. Proc. Soc. Exp. Biol. Med. 152: 603-605. 36. Raiha, N. C. R., and A. L. Schwartz. 1973. Enzyme induction in human fetal liver in organ culture. Enzyme 15: 330-339. 37. Schwartz, A. L., N. C. R. Raiha, and T. W. Hall. 1974. Effect of dibutyryl cyclic AMP on glucose6-phosphatase activity in human fetal liver explants. Biochem. Biophys. Acta 343: 500-509. 38. Sherline, P., H. Eisen, and W. H. Glinsman. 1974. Acute hormonal regulation of cyclic AMP content and glycogen phosphorylase activity in fetal liver in organ culture. Endocrinology 94: 935-939. 39. Schwartz, A. L., and 3'. W. Rail. 1973. Hormonal regulation of glycogen metabolism in neonatal rat liver. Biochem. J. 134: 985-993. 40. Eisen, H . J . , I . D . Goldfine, and W. H. Glinsmann. 1973. Regulation of hepatic glycogen synthesis during fetal development: Roles of hydrocortisone, insulin, and insulin receptors. Proc. Nat. Acad. Sci. U.S.A. 70: 3454-3457. 41. Parsa, I. 1974 Liver cell differentiation in a chemically defined medium. Morphologic and enzymatic comparisons of in utero rat liver and liver rudiment grown in organ culture. Am. J. Pathol. 76: 107-116. 42. Parse, I., and L. Flancbaum. 1975. Long term organ culture of rat liver rudiment in a synthetic medium: Morphological and biochemical development. Dev. Biol. 46: 120-131. 43. Simkins, R. A., H . J . Eisen, and W. H. Glinsmann. 1978. Functional integrity of fetal rat liver explants cultured in a chemically defined medium. Dev. Biol. 66: 344-352.
T h i s investigation was s u p p o r t e d by g r a n t s from the N a t i o n a l I n s t i t u t e of Child Health and Human Development (RO 1 HD09715L National Cancer I n s t i t u t e ICA 14194), a n d U n i t e d States P u b l i c H e a l t h Service G e n e r a l Research S u p p o r t G r a n t R R 5589.
CHANGES IN THE PROPERTIES OF FETAL RAT LIVER DURING ORGAN CULTURE CARL MONDER ANDALENA HATLE COUFALIK Research Institute for Skeletornuscular Diseases, Hospital.forJoint Diseases and Medical Center, 1919 Madison Avenue, New York, New York 10035
(Received November 30, 1977; accepted December 20, 1978)
SUMMARY
Explants of fetal rat liver maintained in organ culture lost about 40% of their mass in 42 hr of incubation as a result of decrease in blood cells and hepatocytes. Proteins from the cytosol and particulate elements of the tissue were found in the culture medium. About 60% of this protein was degraded to peptides during culture. The transfer of malate and lactate dehydrogenases from tissue to medium paralleled that of proteins. Glutamate dehydrogenase was lost from the mitochondria and in part leaked through the cell membrane into the medium. Net loss of activity of the three enzymes occurred, probably as a consequence of proteolytic degradation. Of 12 enzymes in liver tissue, the specific activities of eight--soluble malate dehydrogenase, glutamate dehydrogenase, succinate dehydrogenase, phosphopyruvate carboxylase, hexosediphosphatase, glucose-6-phosphatase, tyrosine aminotransferase, and alanine aminotransferase--were unchanged or increased. Glycogen synthetase, aspartate aminotransferase, pyruvate kinase, and lactate dehydrogenase decreased. Although changes in membrane permeability may have had some influence on the results reported, the predominant effect was due to loss of protein from tissue as a result of discharge of total contents of some of the cells into the medium. The residual explanted tissue retained its structural integrity. It is concluded that fetal rat liver in organ culture provides a suitable model system for controlled studies with this organ in vitro. Key words: fetal rat liver; organ culture; enzyme activity.
INTRODUCTION
The organ culture technique has been used for many years to study the biochemical and physiological behavior of a wide variety of tissues 11-7). It has been claimed by those who use organ culture that it has certain advantages over tissue culture in that the structural integrity of tissue fragments are retained to a much greater extent. The degree to which the viability of a tissue is maintained during organ culture is important to the interpretation of results using this technique. MacDonnell et al. {8~ demonstrated that certain biochemical changes take place during organ culture of fetal liver. It is possible that these changes adversely affect the use of this .procedure for studies on fetal liver function and development. As a result of the findings of MacDonnell et al.,
we have reexamined the stability of fetal liver explants under the culture conditions that have been used in this laboratory for the past several years. This paper describes the changes that occur in the activities and distribution of certain enzymes of fetal rat liver during organ culture. We report that the explant undergoes extensive spontaneous changes over an incubation period of 42 hr. During this interval, most of the enzymes do not decrease (and in some cases increase} in residual tissue.
MATERIALS AND M E T H O D S
Sprague-Dawley rats were purchased from Camm Research Institute, Wayne, New Jersey, and maintained in rooms with controlled tern579
580
MONDER AND COUFALIK
perature and humidity with a light-dark cycle of 12 hr each. Food and water were supplied ad lib. Males were allowed to impregnate the females (one female and one male per cage) during the 12hr dark period. The gestation period, therefore, can be estimated with an uncertainty of _+6 hr. At 20 days of gestation, fetuses were removed under sterile conditions after the mother was killed with ether. Liver fragments were established in culture as described by Wicks ~6) in a humidified desiccator with a mixture of 98% air-2% carbon dioxide. When indicated, the livers were washed after 42 hr of incubation in 0.14 M potassium chloride, transferred to new culture dishes with fresh medium, and incubated for an additional 22 hr. The pH of the medium was 7.4 _+ 0.2 and adjusted back, as necessary, to this pH with sterile dilute sodium hydroxide. After incubation, the livers were washed in 0.14 M potassium chloride and then homogenized at 4 ~ C in 0.15 M potassium chloride. The "soluble" and "particulate" fractions were prepared as described by MacDonnell et al. (8). Malate dehydrogenase IL-malate:NAD oxidoreductase EC 1.1.1.37) was measured as described by Ochoa {9); lactate dehydrogenase (Llactate:NAD oxMoreductase, EC 1.1.1.27) by the method of Kornberg ~10}; and glutamate dehydrogenase ~L-glutamate:NAD oxidoreductase EC 1.4.1.2 }by the method of Herzfeld (11 ). Succinate dehydrogenase [succinate: (acceptor} oxidoreductase, EC 1.3.99.1] was measured by a modification of the method of Bernath and Singer (12). Aliquots of particulate or soluble fractions, prepared as described by MacDonnell et al. (8), were incubated with 0.06 mM potassium phosphate, pH 7.6, and 0.0012 mM sodium cyanide for 7 rain at 37 ~ C. A mixture of 0.24 mM sodium succinate and 0.6 or 0.8 mg of phenazine methosulfate (both levels were used in separate runs) was added to the incubation mixture and oxygen
uptake was measured on an Oxygraph (Gilson Medical Electronics, Madison, Wisconsin} using a Clark electrode. Other enzymes were measured as described in our earlier papers (13,14). Protein was estimated by the method of Lowry et al. ~15). Blood content of liver was estimated according to Castagna (16}. To measure enzyme activity in blood cells: heparinized fetal blood was centrifuged at 600 x g; and the cells were washed twice with 0.9% sodium chloride, homogenized in 0.15 M potassium chloride, and separated into "soluble" and "particulate" fractions as described above.
RESULTS Fetal rat liver explants maintained in organ culture for periods up to 64 hr decreased in size and weight. Initially the fragments in each culture dish weighed a total of 57.9 _+ 1 mg Imean and S.D. of 11 determinations). After 42 hr, the explants weighed 30 _+ 2 mg and at 64 hr, 37.7 _+ 2 mg ~a value not significantly different from that at 42 hr}. The explanted tissue changed from deep red-brown to a light tan as a result of the loss of reticulocytes t6). The blood cell content of fetal liver accounted for 17 + 4% of the tissue weight at 20 days of gestation. This value is close to that reported by Greengard, Federman and Knox {17). By 42 hr of incubation the blood cells of the tissue decreased to 3.7 + 0.4% relative to the freshly isolated liver; at 64 hr, the reticulocyte content was unchanged at 2.5 _+ 1.5%. During the 42-hr incubation period, therefore, blood cells accounted for a decrease of 8 mg of tissue. Since the net weight loss was 27 mg, 30% of the total decrease was attributed to the blood cell elements; the remainder was derived from the liver tissue. Thus 33% of the hepatocyte content of the liver explant is released by 42 hr of culture.
TABLE 1 PROTEIN CONTENT OF FETAL LIVER EXPLANTS Incubation Time
Protein Contenta Supernate
Pellet
Medium
Total
mg per g o[ fresh liver 0 46.5 _+6.1 (11) 51.5 + 8.3 (11) 98.0 42 9.8 ---1.8 ~8) 11.3 _+1.6 (8) 11.7 ---7.9 {4) 21.1 64b 7.0 _+1.4 {8) 9.2 + 2.8 (8) 0.16 _+0.07 {2) 16.2 a Each value is the mean +_standard deviation from the mean. Number of litters studied is given in parentheses. b At 42 hr of incubation, the liver fragments were washed on the grid with 0.15 M KCI, transferred to fresh medium, and incubated for an additional 22 hr.
ORGAN CULTURE OF FETAL RAT LIVER Table I shows that both soluble and particulate protein decreased to an equal extent in the explants during 42 hr of incubation. The net accumulation of protein in the medium, 11.7 _+ 7.9 mg per g of liver, was insufficient to account for the loss of protein from the tissue. The total combined increase in peptides and protein in the medium was 30.1 mg per g of liver. Two-thirds of the total, therefore, probably represented degraded protein. The elution pattern of the peptide components in the medium determined by gel filtration, and reproduced in Fig. 1, confirmed this conclusion. The figure shows that a complex pattern of peptides followed the elution of protein. The patterns for liver explants from two 20-dayold fetuses were similar. From the results we estimate that peptides (fractions 14 to 32) represent 0.700
0.600
0.500
0400 E c
0
0.300
0200 0.180
O. 140
o,oo~
o.o ol
TJ' 4
8
12
16 20 24 28 Froction
52
36 40
FIG, ]. Chromatographic separation of peptides in
incubation medium. The medium after 44 hr of culture with 60 mg of fetal liver was passed through a Bin-Gel P2 column (32 by 1.6-cml with 0.01 M sodium phosphate (pH 7.4) as developing solvent. Each fraction contained 2.0 ml effluent. Protein [determined by the method of Lowry et al. (15)] emerged between fractions 8 and 12; tyrosine emerged between fractions 34 and 38. The profile represents the difference between that of medium incubated with liver and one of identical composition incubated under the same conditions without tissue.
581
28% of the total polypeptides. Peptides and amino acids together represent at least 40% of the whole. The loss of both particulate and soluble components from the tissue was interpreted as evidence that the total cellular contents were discharged into the medium rather than through the selective leakage of protein through damaged cell membranes (8). Loss of protein from the tissue should be accompanied by a corresponding enzyme loss. Therefore the retention of enzyme activity by explants was studied. Four enzymes were measured in the medium and explant. These included soluble and particulate malate dehydrogenase, lactate dehydrogenase, glutamate dehydrogenase, and succinate dehydrogenase. The results are shown in Table 2. For each enzyme the total activity recovered after 42 hr of incubation was about one-half of the initial value. Part of the loss in activity is probably due to proteolysis of enzyme released into the medium as indicated above. Malate and lactate dehydrogenase of hematopoietic cells contributed less than 3% of the total activity of the liver tissue. Of the activity lost from the tissue and recovered in the medium, the blood elements contributed 7% to malate dehydrogenase and 18% to lactate dehydrogenase. The activities of these enzymes in the medium, therefore, are mainly due to loss from damaged liver cells. The decrease in glutamate dehydrogenase in the particulate fraction of the hepatocyte was far greater than could be accounted for by cell destruction. The loss was 87% of the initial amount. About one-half of the recoverable activity appeared in the medium. The total decrease of succinate dehydrogenase, which is a specific mitochondrial marker, was 85%. When explants were transferred after 42 hr of incubation to fresh medium for an additional 22 hr, further small decreases in lactate and malate dehydrogenase occurred. Glutamate dehydrogenase continued to redistribute itself into nonparticulate fractions. Total tissue mass, protein, and enzyme activity all decreased during incubation. The effect of organ culture on the specific activities of lactate, malate, glutamate and succinate dehydrogenase in the explant is shown in Table 2B. The specific activity of the soluble malate dehydrogenase was maintained at a constant level over the 64-hr period; the activity of the particulate fraction was halved during the first incubation interval and did not change thereafter.
582
M O N D E R AND COUFALIK TABLE 2 EFFECT OF ORGAN CULTURE ON MALATE, LACTATE AND GLUTAMATE DEHYDROGENASES a
A. Total Enzyme Activity (~mol/min/g fresh tissue) Time After Explantation
Particulate
So|uble
Medium
Total
89.3 +_ 18.3 (5) 6.9 +_ 2.4 (41 5.8 +_ 1.3 (6)
101.3 +_26.6 {5) 20.7 +- 4.1 (4) 14.8 +_ 1.8 (6)
62.4+_113.8(4t 5.8+_ 1.1(6)
190.6 90.3 26.5
119.6 +_ 18.6 (7) 18.7 +_ 4.1 (5) 11.9 +_ 1.4 16)
0 66.0+_ 7.6+_
8.9(5) 2.5 (5)
129.2 85.9 20.6
0 3.8+_ 0.8(4) 1.1 +_ 0.6(3)
20.7 8.3 4.9
hr
Malate dehydrogenase 0 42 64 b Lactate dehydrogenase 0 42 64 b Glutamate dehydrogenase 0 42 64 b Succinate dehydrogenase 0 42
9.6(1) 1.2 (1) 1.1 (1) 19.8+_ 1.9(7) 2.3+_ 0.8(5) 1.1 +_ 0.1 (3) 23.2 +_ 4.3 (4) 3.5 +_ 0.8(4)
0.88 +_ 0.25(7) 2.2 +_ 0.8 t5) 2.7 +_ 0.8 (31 0 0
0 0
23.2 3.5
B. Enzyme Specific Activity (~mol/min / 100 mg of protein F T i m e After E xplantation
Particulate
Sohlble
Totat
hr
Malate dehydrogenase 0 176 +- 31 (5) 218 +_55 (5) 394 +- 31 42 68 +- 14 (4) 224 +_62 (4) 292 -+ 32 64 b 54 +- 10 (6) 214 +_42 (6) 268 +- 22 Lactate dehydrogenase 0 7.5(H 285+_55 (7) 293+-55 42 0(1) 199+ 15 (5) 199+_ 15 64b 0 (1) 179 +_40 (6) 179 +_40 Glutamate dehydrogenase 0 38.0 +_4.8 (7) 1.8 +_0.5 (7) 39.8 _+4.8 42 19.1 +- 6.6 (5) 20.9 +- 6.9 (5) 40.1 +- 4.8 6@ 15.7 +- 3.8 (31 38.5 +_7.2 (3t 54.1 +_4.1 Succinate dehydrogenase 0 48.8 +_6.9 (4) 0 48.8 +_6.9 42 35.7 +_9.5 (4) 0 35.7 +_9.5 a Values represent means + SD. Number of litters are given in parentheses. b Values shown were obtained after transferring washed explants to fresh medium at 42 hr and incubating for an additional 22 hr. c Values are determined on the basis of the protein remaining in the explant at the indicated time.
Succinate d e h y d r o g e n a s e specific activity was unchanged. T h e decrease in the specific activity of lactate d e h y d r o g e n a s e was small. T h e increase in specific activity of g l u t a m a t e d e h y d r o g e n a s e in the soluble f r a c t i o n occurred a t t h e expense of t h e activity in t h e p a r t i c u l a t e fraction. R e d i s t r i b u t i o n of g l u t a m a t e d e h y d r o g e n a s e activity was continuous over t h e entire 64 h r of i n c u b a t i o n . Other enzymes. C h a n g e s over a n i n c u b a t i o n period of 44 h r for eight a d d i t i o n a l enzymes (expressed as activity per g r a m wet weight of fresh tissue or p e r 100 m g of protein of i n c u b a t e d tissue) are p r e s e n t e d in T a b l e 3. T h e specific activities of
t h e e n z y m e s did n o t c h a n g e in a n y u n i f o r m direction. H e x o s e d i p h o s p h a t a s e (D-fructose-l,6-diphosphate 1-phosphate phosphohydrolase, EC 3.1.3.11) a n d g l u c o s e - 6 - p h o s p h a t a s e (D-glucose6 - p h o s p h a t e p h o s p h o h y d r o l a s e E C 3.1.3.9) were unchanged. Aspartate aminotransferase (Laspartate: 2-oxoglutarate a m i n o t r a n s f e r a s e E C 2.6.1.1), p y r u v a t e kinase ( A T P : pyruvate p h o s p h o t r a n s f e r a s e , E C 2.7.1.40) a n d glycogen synthetase ( U D P glucose: glucogen-a-4-glucosylt r a n s f e r a s e E C 2.4.1.11) decreased. T h e specific activities of several "soluble" e n z y m e s , phosphopyruvate carboxylase [(}riP: oxaloacetate
583
ORGAN CULTURE OF FETAL RAT LIVER TABLE 3 CHANGES IN ENZYME ACTIVITY DURING ORGAN CULTURE OF FETAL EXPLANTS a Time After Explantation
hr Phosphopyruvate carboxylase 0 44 Hexosediphosphatase 0 44 Glucose-6-phosphatase 0 44 Pyruvate kinase 0 44 Tyrosine aminotransferase 0 44 Alanine aminotransferase 0 44 Aspartate aminotransferase 0 44 Glycogen synthetase l a + b) 0 44
Enzyme Activity
#mol/min / g fresh tissue
nmol/min / 1O0 mg protein b
2.73 +_0.73 (7) 2.10_+0.49 ~6)
6.2 _+1.5 9.1 +2.0
25O 170_+ 30
{1 ) (2)
800 720_+ 10
1040 450_+ 30
( 1) (2) c
2350 1900_+ 100
4960 1180 _+10
( 1j (2) c
11200 5000 _+20 c
118_+45 163 _+54
(6) (48)
104_+40 270 _+10c
603 _+44 557-+ 71
(5) ~4)
1360 _+100 2350-+ 100 c
8507 -+ 1680 ~5) 2298 -+ 140 ~4)c 1150 _+400 (10) 73 _+7 (12)"
19200 _+3800 9700 _+600 c 2600 _+900 340 _+30 c
a Values represent means -+ S.D. Number of experiments is given in parentheses. b Protein of explant at time of measurement. '~44 Hr versus 0 hr. P < 0.01.
carboxylase t t r a n s p h o s p h o r y l a t i n g ) E C 4.1.1.32], tyrosine a m i n o t r a n s f e r a s e lL-tyrosine: 2-oxoglutarate a m i n o t r a n s f e r a s e , E C 2.6.1.5) a n d alanine aminotransferase ~L-alanine: 2-oxoglutarate a m i n o t r a n s f e r a s e , E C 2.6.1.2), increased. H i s t o l o g y . T h e histological integrity of the explants w a s e x a m i n e d after 42 h r of i n c u b a t i o n in
organ culture. R e p r e s e n t a t i v e fields of sections of explants taken before i n c u b a t i o n a n d at 44 h r of incubation are shown in Fig. 2. D u r i n g 42 h r there was extensive decrease in reticulocytes. A decrease in cellular density w a s noted. I n other respects the tissue a p p e a r e d u n c h a n g e d w h e n comp a r e d with fresh tissue.
FIG. 2. Comparison of the structural integrity of fetal rat liver explants at 0 (A) and 44 {B) hr of incubation. H and E. x75.
584
MONDER AND COUFALIK DISCUSSION
During the first 42 hr of culture, a number of gross changes took place in the properties of the explants. The fragments decreased in mass, the color of the explanted tissue became pale, and red blood cells settled to the bottom of the dish. This was reflected on microscopic examination in a pronounced decrease in hematopoietic cells in the tissue. We have observed, as have others (6,8,18), that no other readily discernible disintegration of the tissue occurs in this time period. There was a loosening of the architecture of the explant over the period of incubation; the cells appeared to retain their integrity. Spaces freely permeable to medium and atmosphere developed. The remaining cells were now exposed to an environment in which nutrient and gas exchange necessary for maintenance of function was possible. In considering how the status of the long-term culture evolved, we have concluded that the destructive events we describe in this paper must be adaptive in nature. The life of the explant could not be sustained unless the individual ceils could nourish themselves and eliminate waste products in a way that was not damaging to the neighboring cells. This could only occur if sufficient cells were removed to provide to the remainder access to medium and air. As we have shown here, and as others have found (8), significant changes, which may profoundly affect tissue function, also were occurring on a molecular level. The decrease in tissue mass was due in large part to the loss of reticulocytes, degeneration of cells damaged during cutting of the tissue, and the loss of cells that died because of environmental deficiencies. We observed that both particulate and soluble proteins were lost from the tissue. We interpret this to mean that the total cell contents of the hepatocytes were discharged into the medium. Some cells, in addition, probably suffered membrane damage and selective loss of protein as MacDonnell et al. (8) have suggested. The data in this paper support the observations reported by MacDonnell et al. (8) that there is loss of cellular integrity during organ culture of fetal liver. Although they conclude that changes in membrane permeability account for most of the changes seen in culture, we conclude that destruction of whole or injured cells better account for our results. The loss of both particulate a~d soluble malate dehydrogenase from the explants during the initial 42 hr occurred in about the same proportion
as the loss of protein. Decrease of total enzyme activity in the explants during incubation was observed with all the enzymes listed in Tables 2 and 3 with the exception of phosphopyruvate carboxylase, tyrosine aminotransferase and alanine aminotransferase. If these losses are due to the sloughing off of damaged tissue into the medium, then the specific activities of the enzymes within the remaining tissue must be the same as the activities within the freshly isolated tissue, except for those in which specific selective changes have occurred. When the activities of 12 enzymes were expressed per 100 mg of protein, it was found that the intracellular levels of three (phosphopyrnvate carboxylase, tyrosine aminotransferase, and alanine aminotransferase} had increased; five ~hexosediphosphatase, glucose-6-phosphatase, lactate dehydrogenase, succinate dehydrogenase, soluble malate dehydrogenase) were unchanged; and four (pyruvate kinase, aspartate aminotransferase, and the two particulate enzymes, glycogen synthetase and particulate malate dehydrogenase) had decreased. The decrease in particulate malate dehydrogenase and glutamate dehydrogenase indicated that these enzymes were not yet firmly integrated into the mitochondrial superstructure. These enzymes are found in the matrix of the mature mitochondria. They might not be retained by the immature mitochondria. This conclusion is supported by the fact that the specific activity of sueeinate dehydrogenase, which is an integral part of the mitochondrial inner membrane, remained unchanged. Glutamate dehydrogenase is distributed in the cytosol in avian fetal tissue (19) and fungi (20) and, under certain conditions, in adult rat liver i21,22). Whether a portion of the activity is extramitochondrial in fetal liver remains to be investigated. The loss of particulate components from the explants in the experiments described in this paper most probably occurred as a resuh of the total destruction of the cell membrane. Changes in membrane permeability probably play an additional role in the transfer of macromolecules between the medium and explant. In suspension cultures, leakage of enzymes from cells into medium is a well known phenomenon. Leakage of several of the enzymes described in the paper, as well as of some others from dispersed liver cells (23-26) and ascites tumor suspensions (27,28), have been described. Changes in selective permeability of fetal and adult liver to glucose differ (29). The lag period of several hours, which is necessary for the
ORGANCULTUREOFFETALRATLIVER induction of tyrosine aminotransferase (6), glycogen synthesis (13,30), and mixed function oxidases {31,32} by steroids and other polycyclic hydrocarbon derivatives in organ culture, may be explained by assuming that increased selective permeability of the fetal cell membranes is needed in order for the inducers to gain entry into the cell. Net accumulation of polycyclic hydrocarbons and cortisol in fetal liver explants was always greater after preincubation (14,33D. Organ culture systems are useful as experimental models only if they remain viable during the period of study. The criteria of viability refer to the continuation of those functions that occur in the intact liver in vivo and the appearance of other functions that were absent in the intact fetus. Thus in explants the incorporation of orotate into R N A continues undiminished (25) although protein 134} and glycogen synthesis (13) continues at a slower rate than in the freshly isolated tissue. The histological appearance of the tissue remains unchanged. Gluconeogenesis increases from undetectable levels 135}. Tyrosine aminotransferase, phosphopyruvate carboxylase and alanine aminotransferase also increase. It is self-evident that fetal liver in organ culture, devoid of the stimuli and restraints to which it is subject in vivo, cannot develop in the way it does in situ. The changes that occur in culture were selective, sometimes following the path pursued during development and sometimes not. The reasons for this selectivity are poorly understood. A key issue to be explored is how the in vivo function of liver explants can be restored. Of those enzymes that decrease in activity in explants, but increase during development, most can be stimulated to increase in culture by appropriate hormones. Arginine synthetase t36} and glucose-6phosphatase are increased by cyclic AMP (37), glycogen phosphorylase by epinephrine and glucagon ~38}, and glycogen synthetase a by insulin ~39}. Pyruvate kinase, which decreases during fetal development, decreases in organ culture as well. Often the explant will respond to hormones whereas the indwelling tissue does not. It is remarkable, in the face of the extensive changes that we and n a c D o n n e l l et al. (8) have reported, that the fetal liver explant retains so great a responsiveness to hormones and other environmental stimuli. It has been argued that this is an aberrant response unrelated to fetal development ~8}. It seems to us, on the contrary, a matter to be explored for its relationship to physiological
585
events. The isolated explant has been profitably used to study the hormonal regulation of glycogen metabolism {40}, gluconeogenesis ~13,35}, and microsomal mixed function oxidases {31,32}. Parsa (41} and Parsa and Flancbaum (42} have shown that the morphological and biochemical development of fetal rat liver can be maintained in a synthetic medium enriched with hormones. Simkins, Eisen and Glinsmann (43) have recently described an improved technique for the maintenance of fetal rat liver in organ culture. Under their conditions, the explants generated albumin, a-feto protein and transferrin. These observations encourage us to believe that in a more completely defined environment the structural integrity of the fetal liver explant can be made to simulate that of the normal developing tissue. REFERENCES 1. Thomas, J.A. 1970. Organ Culture. Academic Press, New York. 2. Easty, G. C. 1970. Organ culture methods. In: H. Busch (Ed.}, Methods in Cancer Research. Vol. 5. Academic Press, New York, pp. 1-43. 3. Giannopoulos, G., S. Mulay, and S. Solomon. 1973. Glucocorticoid receptors in lung. II. Specific binding of glucocorticoids to nuclear components of rabbit fetal lung. J. Biol. Chem. 248: 5016-5023. 4. Calcagno, M., H. Goyena, E. Arrambide, and C. Arruti De Urse. 1970. Action of cortisone and cortisol upon biosynthesis of chondroitin sulfate in femur, in vitro cultures of chick embryo. Exp. Cell Res. 63: 131-137. 5. Corradino, R. A. 1973. Embryonic chick intestine in organ culture: Response to vitamin D3 and its metabolites. Science 179: 402-405. 6. Wicks, W. D. 1968. Induction of tyrosine transaminase in fetal rat liver. J. Biol. Chem. 243: 900-906. 7. Ballard, P. L., and R. A. Ballard. 1972. Glucocorticoid receptors and the role of glucocortieoids in fetal lung development. Proc. Nat. Acad. Sci. U.S.A. 69: 2668-2672. 8. MacDonnel, P. C., E. Ryder, J. A. Delvalle, and O. Greengard. 1975. Biochemical changes in cultured foetal rat liver explants. Bioehem. J. 150: 269-273. 9. Ochoa, S. 1955. Malic dehydrogenase from pig heart. Methods Enzymol. 1: 735-739. 10. Kornberg, A. 1955. Lactic dehydrogenase of muscle. Methods Enzymol. 1: 441-443. 11. Herzfeld, A. 1972. The distribution of glutamate dehydrogenase in rat tissues. Enzyme 13: 246-251. 12. P. Bernath, and T. P. Singer. 1962. Succinic dehydrogenase from heart. Methods in Enzymol. 5: 597-602. 13. Monder, C., and A. Coufalik. 1972. Influence of eortisoi on glycogen synthesis and gluconeogenesis in fetal rat liver in organ culture. J. Biol. Chem. 247: 3608-3617.
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29. Dawkins, M. J. R. 1963. Glycogen synthesis and breakdown in fetal and newborn rat liver. Ann. N.Y. Acad. Sci. 111: 203-211. 30. Eisen, H. J., W. H. Glinsmann, and P. Sherline. 1973. Effect of insulin on glycogen synthesis in fetal rat liver in organ culture. Endocrinology 92: 584-588. 31. Cutroneo, K. R., R. A. Seibert, and E. Bresnick. 1972. Induction of benzpyrene hydroxylase by flavone and its derivatives in fetal rat liver explants. Biochem. Pharmacol. 21: 937-945. 32. Burki, K., R. A. Seibert, and E. Bresniek. 1971. Effects of 3-methylcholanthrene and some related compounds upon the benzpyrene hydroxylase activity in fetal rat liver explants. Biochem. Pharmacol. 20: 2947-2952. 33. Bresnick, E., and K. Burki. 1975. The fetal rat liver explant as a model system in the study of hormone action. Methods Enzymol. 39: 36-40. 34. Sereni, F., and L. P. Sereni. 1969. Spontaneous development of tyrosine aminotransferase activity in fetal liver cultures. Adv. Enzyme Regnl. 8: 253-267. 35. Coufalik, A., and C. Monder. 1976. Spontaneous development of gluconeogenesis in fetal rat livers during incubation in organ culture. Proc. Soc. Exp. Biol. Med. 152: 603-605. 36. Raiha, N. C. R., and A. L. Schwartz. 1973. Enzyme induction in human fetal liver in organ culture. Enzyme 15: 330-339. 37. Schwartz, A. L., N. C. R. Raiha, and T. W. Hall. 1974. Effect of dibutyryl cyclic AMP on glucose6-phosphatase activity in human fetal liver explants. Biochem. Biophys. Acta 343: 500-509. 38. Sherline, P., H. Eisen, and W. H. Glinsman. 1974. Acute hormonal regulation of cyclic AMP content and glycogen phosphorylase activity in fetal liver in organ culture. Endocrinology 94: 935-939. 39. Schwartz, A. L., and 3'. W. Rail. 1973. Hormonal regulation of glycogen metabolism in neonatal rat liver. Biochem. J. 134: 985-993. 40. Eisen, H . J . , I . D . Goldfine, and W. H. Glinsmann. 1973. Regulation of hepatic glycogen synthesis during fetal development: Roles of hydrocortisone, insulin, and insulin receptors. Proc. Nat. Acad. Sci. U.S.A. 70: 3454-3457. 41. Parsa, I. 1974 Liver cell differentiation in a chemically defined medium. Morphologic and enzymatic comparisons of in utero rat liver and liver rudiment grown in organ culture. Am. J. Pathol. 76: 107-116. 42. Parse, I., and L. Flancbaum. 1975. Long term organ culture of rat liver rudiment in a synthetic medium: Morphological and biochemical development. Dev. Biol. 46: 120-131. 43. Simkins, R. A., H . J . Eisen, and W. H. Glinsmann. 1978. Functional integrity of fetal rat liver explants cultured in a chemically defined medium. Dev. Biol. 66: 344-352.
T h i s investigation was s u p p o r t e d by g r a n t s from the N a t i o n a l I n s t i t u t e of Child Health and Human Development (RO 1 HD09715L National Cancer I n s t i t u t e ICA 14194), a n d U n i t e d States P u b l i c H e a l t h Service G e n e r a l Research S u p p o r t G r a n t R R 5589.
