ABSTRACT
Earlier studies in our laboratory indicated the predominance of pentose cycle activity in early embryos (Jolley, Cheldelin & Newburgh, 1959; Coffey, 1963). In addition a correlation was demonstrated between enzymes generating reduced triphosphopyridine nucleotide and lipid synthesis (Bieber, Cheldelin & Newburgh, 1962; Baker & Newburgh, 1963). Since several of the biosynthetic pathways involve this compound, it is conceivable that in a rapidly growing system such as the early chick embryo enzymes generating TPNH may play a major röle. We have chosen to use explants of chick embryos in an attempt to gain insight into the relation of changes of these enzymes with other chemical changes which may more directly relate to differentiation. These latter changes include synthesis of such macromolecules as DNA, RNA, hemoglobin, collagen and myosin. This method permits the addition of certain chemicals to the media such as substrates or inhibitors (Hayashi et al., 1959), allowing easy manipulation of the environment. Prior to studying the above macromolecules it seemed appropriate to study the effect of certain inhibitors on glucose-6-phosphate, 6-phosphogluconic acid and isocitric dehydrogenase. It was hoped that such information might be useful for purposes of establishing base lines of biochemical activity. The inhibitors chosen were fluorouracil, chloramphenicol and acetyl pyridine, and it will be shown that these had several rather specific effects on the enzymes studied. The first two inhibitors were chosen because of their relation to RNA or protein biosynthesis and the latter since it is an analogue of the pyridine moiety of TPN.
In addition, Blackwood (1962) reported that chloramphenicol inhibited differentiation of the heart and ventral trunk if injected into eggs prior to Hamilton and Hamburger Stage 10 (1951). After this stage an inhibition of the rate of growth and development occurred without gross malformations with chick embryo explants. Herrmann (1963) showed a greater inhibition of protein increase and glycine-1-C14 incorporation in the central nervous system than the trunk tissue by acetyl pyridine.
METHODS
Explantation
The technique was similar to that reported by Spratt as modified by Klein (1963). The medium contained one part concentrated egg homogenate and one part chick Ringers containing 0 · 75 per cent, agar, 165 units/ml. of penicillin, 370 units/ml. of streptomycin, and inhibitors as indicated. The embryos were removed from the egg at an age of 11–13 somites (Hamilton and Hamburger Stage 6 (1951)) and incubated 24 or 48 hr. at 37°C. The gas atmosphere consisted of 40 per cent, air-60 per cent. O2. The levels of inhibitor added were 75 μg. chloramphenicol, 100 μg. acetyl pyridine, and 50 μg. of fluorouracil per ml. of medium.
Isolation of embryos and membranes
The embryos were removed from the medium and trimmed as described previously (Newburgh et al., 1962). They were separated from the extra-embryonic membrane and both the membrane and embryo were immediately frozen. They were then homogenized in a solution of 0·585 M sucrose, 0·03 M Tris and 0·004 M sodium ethylene diamine tetraacetic acid, pH 8·2. (Usually ten embryos or membranes were homogenized in 1·2 ml. of solution.) The homogenization and assay were completed within 24 hr. Optimum enzymic activity resulted with this procedure.
Analytical methods
The assay mixture for all enzymes contained 86 μM Tris buffer, pH 8·2, 0 ·1 μM TPN, 0·4 μM MgCl2, 0·4 μM of substrate as indicated, 0·05 ml. homogenate. Total volume 1·0 ml. Temperature 23–25°C.
Enzymic activity was determined by measuring the rate of TPN reduction using a Farrand fluorometer. Internal standards of 0·02 γ quinine sulfate were used. In addition, this standard was related to TPNH of known concentrations to permit calculation of activity on the basis of μmoles of TPNH reduced per unit time. One division on the fluorometer represented 3 × 10−4μM TPNH. Readings were taken at 1-min. intervals, and the enzymic activity was calculated from the linear portion of the curve. Activity depended on the amount of homogenate present in a linear fashion. In addition up to 10−3 M concentrations of the various inhibitors were added to homogenates of control explants and no inhibition of activity was noted. DNA was determined by the method of Burton (1956), protein was determined by nesslerization, (Lang, 1958) and RNA by the method of Mejbaum (1939).
RESULTS
Morphological effects
These effects are summarized in Table 1.
Chloramphenicol inhibited development only slightly while acetyl pyridine and fluorouracil were more effective. In the 48-hr. explants fluorouracil completely inhibited the development of the embryo. In fact, they were in worse condition than after 24 hr. growth. All the inhibitors affected the development of blood vessels in the extra embryonic membranes. The inhibition was complete in the presence of fluorouracil.
Chemical and enzymic effects
The results of the effect of various inhibitors on DNA, RNA, and protein content of the embryos and extraembryonic membrane are recorded in Table 2.
The values for 24 hr. represent the average from 250 explants for the control and each inhibitor, while 100 explants are represented in the 48-hr. experiments. The standard error falls within a 5 per cent, deviation from the figures given. In the control there is a 4-fold increase in DNA and protein in 24 hr. and a 13-fold increase in 48 hr.; a 3-fold increase in RNA in 24 hr. and a 9-fold increase in 48 hr. In the presence of chloramphenicol inhibition of either DNA, protein and RNA accumulation is about the same, i.e. 30 per cent, in 24 hr. and 50 percent, in 48 hr. Acetyl pyridine inhibited the accumulation of these substances more extensively in the first 24 hr. (approximately 50 per cent), but in 48 hr. the inhibition is less than that obtained with chloramphenicol, or that occurring in the first 24 hr. This suggests a reversal in the second 24-hr. period. The addition of 5-fluorouracil results in no net accumulation of DNA, protein, or RNA in either the 24-or 48-hr. explants. A different picture emerges on examination of these substances in the extraembryonic membrane. In the control explants the increase of DNA and RNA is 1 · 5-fold in 24 hr., while in 48 hr. there is a three-fold increase in DNA and a two-fold increase in RNA. Protein nitrogen actually decreases in the 24-hr. explants and remains constant in the 48-hr. explants. Examination of the explants to which an inhibitor was added to the medium indicates little or no effect of either chloramphenicol or acetyl pyridine. Quite a different observation is evident on examination of the results with 5-fluorouracil. Neither DNA nor RNA increase, and the decrease in protein is somewhat greater than that found in the control.
The other part of these studies is the determination of the level of various enzymes in the explanted chick embryos. These enzymes are glucose-6-PO4, 6-phosphogluconic and isocitric dehydrogenase. It is evident from the data from the embryo (Table 3) with control explants that these three enzymes behave in quite a different manner. Regardless of the basis for the expression of activity, i.e., DNA, protein or RNA, the specific activity of glucose-6-PO4 dehydrogenase decreases in the 24- and 48-hr. periods; the activity of 6-phosphogluconic dehydrogenase increases slightly in 24 hr. and decreases to the zero time level in 48 hr.; the activity of isocitric dehydrogenase remains constant. When chloramphenicol is added to the medium, a significant change occurs in the level of glucose-6-phosphate dehydrogenase in both 24 and 48 hr. but not in the other enzymes, suggesting a specific effect. 5-Fluorouracil alters the level of all three enzymes in the 24-hr. period on the basis of DNA. Although drastic changes also occur in the 48-hr. period, the embryo is in a debilitated condition and one is more likely studying death.
A somewhat different pattern occurs in the membrane (Table 4). In the control experiments the activity of glucose-6-phosphate dehydrogenase decreases but to a much smaller extent while 6-phosphogluconic dehydrogenase behaves somewhat similarly in both the membrane and embryo. The dramatic difference is in the level of isocitric dehydrogenase. On the basis of DNA (or per cellular unit) we note an increase of 1-7-fold in 24 hr. and 2-4-fold in 48 hr. on the basis of DNA or RNA, while on the basis of protein the increase is 4- and 7 +-fold respectively. Contrary to the results found with the embryo, chloramphenicol has little or no effect while the addition of acetyl pyridine to the medium involves a rather dramatic increase in specific activity of all three enzymes in 48 hr. Similar results are found in the presence of 5-fluorouracil for glucose-6-phosphate and 6-phosphogluconic dehydrogenase.
DISCUSSION
The effect of the inhibitors on such gross parameters as DNA, RNA and protein varied. In the control explants the accumulation of DNA and protein paralleled one another, while the accumulation of RNA was somewhat less. Chloramphenicol seemed to be a general inhibitor in that the accumulation of DNA, RNA, and protein was inhibited to about the same extent. Acetyl pyridine inhibited the accumulation of these three compounds more than chloramphenicol in the first 24 hr. but less in the 48-hr. explants, suggesting a reversal. It is interesting to speculate that the point of inhibition is at the level of the coenzymes (TPN or TPNH) and that the organism adapts to the acetyl pyridine analogue. Of more interest is the comparison of the results with acetyl pyridine and 5-fluorouracil. In the presence of 5-fluorouracil, no net increase in DNA, protein and RNA occurs, in contrast to acetyl pyridine, yet the somite number in the presence of either one of these inhibitors was identical. This suggests that differentiation occurred without net growth. This occurred either at the expense of existing DNA, RNA and protein or the breakdown of these compounds exceeded their synthesis. For protein this is suggested by the results of Herrmann & Buckingham (1963).
A somewhat different picture emerges when the equivalent data from the membrane are considered. The rate of accumulation of DNA, protein and RNA is less than in the embryo. The only inhibitor that had any effect was 5-fluorouracil.
One group of chemical compounds whose changes probably represent early processes of differentiation are the enzymes. Comparison of the results of the control experiments for both the embryo and membrane reveal some interesting facts. Of particular interest is the different behavior of glucose-6-phosphate and isocitric dehydrogenase. In the embryo and membrane glucose-6-phosphate dehydrogenase decreases in specific activity when based either on DNA, RNA or protein. This is suggestive of the formation of new cell types with a lower content of this enzyme. This, of course, does not imply the same step in differentiation in both the embryo and membrane. Isocitric dehydrogenase is quite different in that the activity remains relatively constant in the embryo but increases drastically in the membrane. The increase in the membrane is much greater on the basis of protein than either DNA or RNA. This is also true of 6-phosphogluconic dehydrogenase. Since the change occurring in the membrane is the formation of blood vessels and blood, it is interesting to propose that these enzyme changes are related to this process. The inhibitors also behave differently in that chloramphenicol alters the content of glucose-6-phosphate dehydrogenase in the embryo while only a slight effect is found in the membrane. On the contrary acetyl pyridine affects all these enzymes in the membrane but none in the embryo. On the basis of measurement of these enzymes, in 48 hr. the addition of this inhibitor results in cells containing much higher amounts of the three enzymes. The effect is more dramatic on the basis of DNA than either RNA or protein and is most likely the result of decreased cellular proliferation. Since the protein is greater in the acetyl pyridine explants than the controls it appears that these enzymes are synthesized more extensively than other proteins found in the extra-embryonic membranes of the controls. Since blood vessels and blood do not occur in the 5-fluorouracil treated explants and the activity of the three enzymes is similar to that in acetyl pyridine explants a relation to this aspect of differentiation is suggested. It is apparent that these inhibitors have variable effects on the explants. This variation is probably indicative of interference with different phases of development of the chick embryo. In the extra-embryonic membrane this effect is probably related to the formation of a circulatory system. Prior to assigning a relation between the enzyme changes and morphological events it is necessary to examine individual parts of the embryo.
SUMMARY
Chick embryos were explanted at the 11-13 somite stage and grown in vitro for 24- and 48-hr. periods in the presence of various inhibitors.
In the control embryos DNA and protein accumulation paralleled one another while RNA accumulation was somewhat less. Chloramphenicol was a general inhibitor of these parameters. In the presence of 5-fluorouracil no net increase in DNA, protein and RNA occurs, in contrast to acetyl pyridine, yet the somite number in the presence of either one of these inhibitors was identical.
In the membrane only 5-fluorouracil inhibited accumulation of DNA, protein and RNA.
The three enzymes studied, glucose-6-phosphate, 6-phosphogluconate, and isocitrate dehydrogenase varied considerably during development, and the relations between these variations and development are discussed.
RÉSUMÉ
L’effet de différentes substances inhibitrices sur plusieurs enzymes et sur d’autres substances biochimiques dans des explants provenants d’embryon de poulet
On explante des embryons de poulet au stade 11-13 somites et on les cultive in vitro pendant des périodes de 24 et 48 heures en présence d’inhibiteurs variés.
Chez les embryons témoins, le DNA et les protéines augmentent paralèlle-ment, tandis que l’accumulation de RNA est un peu moindre. Le Chloramphenicol est un inhibiteur général de ces phénomènes. En présence de 5-fluoro-uracil, il n’y a pas d’augmentation nette de DNA, de protéines, ni de RNA.
Ce résultat contraste avec celui qu’on obtient avec l’acétylepyridine, bien que le nombre de somites soit identique en présence de l’un ou l’autre de ces inhibiteurs.
Seul le 5-fluorouracile inhibe l’accumulation de DNA, de protéines et de RNA dans la membrane.
La distribution de 3 enzymes, glucose-6-phosphate, 6-phosphogluconate, et l’isocitrate déhydrogénase varie beaucoup pendant le développement, et les relations entre ces variations et le développement sont discutées dans cet article.
ACKNOWLEDGEMENTS
This investigation was supported in part by a Public Health Service research career program award 3-K3-GM-14, 336 from the Division of General Medical Sciences and grants-in-aid from the National Science Foundation, Division of Research Grants, USPHS (H-2967) and the Oregon Division of the American Cancer Society. Published with the approval of the Monographs Publication Committee, Research Paper No. 462, School of Science, Department of Chemistry.