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HEW-3.0J‘IOL..I.$1JIfekm.rm.o.h-_tf1%_"~ 0 .0I II .n‘ 0. .0000. . . . 0 I 00 I ._ 0 0 . . . 1.....I0 . . .40. . v 0 09 l I 0 ll '0'! -0- cl 0 .l“ \ A.‘ 3'00.” O 0U)“. .— ol . . .00...‘ I. , . a. II .n .9 v.‘ 0. . .0“- ! I) .. 0.‘ (0. 55¢ Q .—.0 00....“ 0.4..” u. Tw‘afl w. ‘L'cfiait. .0 0.0.1.2. rsquY l 43!. ”uIfl’.,...0. t .va}.... .0‘0..5.4.. 00.‘ rift. h. fur. 0.. 00. b... . 0i 0.! 0.2.. Pint... ‘m ~030‘. ~0f10¢| 00.5’u’ 0" .- "JVP01IR...00 A0... 0.0? '0 Q 10 30"».A1’9‘ C...‘¥i’- u .. 0‘ _ . . 0 0 .0 .!. .. o THE-5‘s , fl - ,1 . ‘tflt. {ms-H! "a 1‘4" 5' towa- H . 4; Q‘J 1 37,3? ‘9 LIIL-xumz ! ’. 5 1 - .3 ‘ I»: ,7 in U My .2? BINDI‘NG IV. menu? 800K BINDERY mc. LISP-T ‘ZTY BINDERS unnulnl. 2?” // ‘/ / / / V‘-4N E ABSTRACT DNA SYNTHESIS IN REGENERATING LIVER I VITRO By Ralph Joel Rothenberg The adult rat liver is an organ in which almost all of the cells are in the non-proliferating (Go) state. Following partial hepatectomy, there is a compensatory hyperplasia and the liver will regenerate to it's original mass within a few days. Among the features of regenerating liver is a syn- chronous wave of hepatocyte DNA synthesis which occurs at about 20 to 30 hours post-hepatectomy. Work done by others i iv or i vitro has not yet succeeded in defining the —.—~ _ regulation of liver regeneration. This may be due, in part, to the complexity of the interactions between organs and within tissues that occur in an organism. This thesis tests the hypothesis that Slices of regenerating liver incubated 1 vitro have properties of liver regeneration similar to those 1 vivo, and therefore, this system could be a valuable tool in the study of the regulation of liver regeneration. The following methods were used in this study. At various times following 70% partial hepatectomy of male adult rats, Slices of regenerating liver were made and in— cubated in vitro. At specified times during the incubation (up to 24 hours) the slices were pulsed with radioactive DNA precursors and the resulting DNA specific activity of the Ralph Joel Rothenberg slices was determined and used to estimate rates of DNA syn- thesis in the slices. To begin the search for systemic fac- tors which may regulate liver cell division, the effects of including various serums in the incubation medium on slice DNA syntheses were determined. Prior to partial hepatectomy, some rats were fed a diet containing the hepatocarcinogen N-Z-fluorenylacetamide in order to see if exposure to this chemical would alter aspects of DNA synthesis. The results of this study indicate that slices of re- generating liver incubated in 11312 can be an important tool in studies of liver regeneration. When liver slices made from rats subjected to 70% partial hepatectomy l6 hours earlier are incubated in vitro, the times of the onset (16 hours) and peak (26 hours post-hepatectomy) DNA synthetic rates are Similar to those seen 1 vivo. When liver slices are made at earlier times after partial hepatectomy, they do not appear to progress through a wave of DNA synthesis. This suggests that prior to 16 hours post-hepatectomy there is a critical period i vivo during which exposure to a sys- temic factor(s) commits cells to enter the S phase of the cell cycle. This may be an important period in the control of liver regeneration. Calf serum, fetal calf serum, and serum from rats partially hepatectomized l8 hours earlier do not alter DNA synthesis in slices made at any time but dialyzed calf serum does appear to decrease this rate in slices prepared at l6 hours post-hepatectomy. Although esti- mates of DNA synthesis obtained with the use of ‘4C-formate Ralph Joel Rothenberg and 3H-thymidine are similar 1 vivo, this is not true in vitro. This may be becauSe the capacity of formate to be incorporated in lit£g_into the pool of proximate DNA precur- sors is a limiting factor, rather than DNA polymerase activity. DNA synthesis in liver slices made from rats fed a diet containing the hepatic carcinogen N-Z-fluorenylacetamide (0.05% w/w) for l4 days, followed by either 7 or 30 days on a control diet, and incubated beginning at 16 hours post- hepatectomy, is lower than normal and continually increases during a 25 hour incubation in vitro. This does not suggest a synchronous wave of DNA synthesis in these slices. DNA SYNTHESIS IN REGENERATING LIVER I VITRO By Ralph Joel Rothenberg A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pharmacology 1974 ACKNOWLEDGEMENTS The author thanks Dr. J. I. Goodman, his major advisor, for guidance in his graduate study program, and for his valuable suggestions and assistance in the preparation of this thesis. The same appreciation is extended to Dr. T. M. Brody, the Chairman of the Department of Pharmacology, for his interest and to Dr. T. Tobin and Dr. J. Trosko for their generous willingness to sit in on his guidance committee. ii TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES I. II. III. IV. INTRODUCTION l.l Objective l.2 Background l.3 Rationale l.4 Specific Aims METHODS AND MATERIALS 2.l Animals 2.2 Liver Slice Incubation 2.3 Experiments I Vivo 2.4 Experiments Using N-2-Fluorenyl- acetamide 2.5 Materials RESULTS 3.l Experiments I Vivo 3.2 Experiments Using 3H-Thymidine 3.3 Experiments Using 14C-Formate 3.4 Serum Effects on Slice DNA Synthesis 3.5 N-2-Fluorenylacetamide Effects on Slice DNA Synthesis DISCUSSION 4.l Slice DNA Synthesis 4.2 The 61 Phase of the Cell Cycle 4.3 14C-Formate DNA Synthesis iii 18 l8 20 20 23 23 24 25 25 31 38 43 49 52 56 4.4 Serum Effects on Slice DNA Synthesis 4.5 N-2-Fluorenylacetamide Effects on Slice DNA Synthesis SUMMARY LIST OF REFERENCES iv 60 63 66 LIST OF FIGURES Number Title Page l A comparison of 3H-dThd and ‘40- 26 formate incorporation into DNA of regenerating liver in vivo 2 A comparison of DNA specific activity 27 as a function of time in liver slices made and incubated starting at 14 or 16 hours post-hepatectomy 3 A comparison between liver DNA spe- 29 cific activity and the percentage of labeled nuclei at various times in slices made and incubated in vitro from one rat starting at 16 hours after 70% partial hepatectomy 4 A comparison of 3H-dThd and 14C- 33 formate incorporation into DNA at various times in liver slices made and incubated in vitro 18, 16. or 14 hours after—70% partial hepatectomy 5 The effect of formate oncentration on 35 She incorporation of 1 C-formate and H-dThd into DNA of liver slices made and incubated ig_vitro starting at 15 hours after 70% partial hepatectomy 6 A comparison of 14C-formate incorpora- 37 tion into DNA and into protein as a function of time in liver slices made and incubated in vitro starting at 18 hours after 70% partial hepatectomy 7 The effect of 20% calf serum on the 39 incorporation of 3H«dThd, 10'5M, 20 uCi/umole. into DNA at various times in liver slices made and incu- bated in vitro starting at 16 or 14 hours after 70% partial hepatectomy 8 The effect of 20% fetal calf serum on 40 the incorporation of 3H-dThd, 10'5M, 20 pCi/umole, into DNA at various times in liver slices made and incubated in vitro starting at 16 or 14 hours after partial hepatectomy Number 10 ll 12 l3 14 Title The effect of 5% serum from rats partially hepatectomized 18 hours earlier on the incorporation of 3H-dThd, 10'5M, 20 uCi/umole, into DNA of liver slices made and incubated in vitro starting at 16 or 14 hours post-hepatectomy The effect of 20% dialyzgd calf serum on ghe incorporation of H-dThd, 10' M, 20 pCi/pmole, into DNA at various times in liver slices made and incubated in vitro starting at 16 hours after 70% partial hepatectomy The effect of feeding a diet contain- ing 0.05% FAA for 14 days followed by a control diet Sor 7 days on the in- corporation of H-dThd, 10'5M, 20 pCi/ umole, into DNA at various times in liver slices made and incubated in vitro starting at 16 hours after 70% partial hepatectomy The effect of feeding a diet contain- ing 0.05% FAA for 14 days followed by a control diet for 30 days on the in- corporation of 3H-dThd, 10'5M, 20 uCi/ umole, into DNA at various times in liver slices made and incubated in vitro starting at 16 hours after-70% partial hepatectomy The effect of feeding a diet contain- ing 0.05% FAA for 14 days followed by a control diet for 7 days on the in- corporation of 3H-dThd, 10‘5M, 20 p01] Hmole, into DNA at various times in liver slices made and incubated in vitro starting at 18 hours after 70% partial hepatectomy Serum requirements of cells progressing through the G] phase of the cell cycle vi Page 41 42 45 46 48 LIST OF TABLES Number Title Page 1 The relationship between DNA 32 specific activity of half-slices and of slices made at different depths in the liver INTRODUCTION 1.1 Objective: The long range goal of this research is to develop an ifl_vi££g system to study the regulation of mam- malian cell division. This could then be used to study the alterations in growth regulation induced by exposure to chemical carcinogens. 1.2 Background: In order to understand the control of cell division, one approach now being used in many laboratories is to identify and evaluate the role(s) of endogenous fac- tors which alter rates of DNA synthesis and/or cell divi- sion in tissues. A lot of this work is being done in rat and mouse liver because the biochemistry of these species and this tissue have been extensively studied. In addition, the effect of partial hepatectomy, which has been carefully described by many authors,"2 is that liver cells normally not progressing through the cell cycle are converted into a group of cells which are synchronously going through the cell cycle. The ability to experimentally manipulate the liver so that this conversion occurs under defined conditions allows one to examine specific aspects of the cell cycle and its regulation. Since the liver, following partial hepatectomy, even— tually returns to the same percent of body weight as before the operation (which in the rat is about 4%‘), control of liver regeneration is exercised. Factors in the systemic circulation seem to influence this process because cross circulation experiments between a partially hepatectomized rat and a normal rat Show that DNA synthesis is stimulated not only in the partially hepatectomized rat but also in the normal rat.3 Also indicating that factors regulating liver regeneration are found in the blood is the observa- tion that cells in implanted subcutaneous bits of liver will proliferate in a manner similar to that of the liver remenant when the rat is subjected to partial hepatectomy.4 It is important to understand those aspects of regener- ating liver which form the basis, and some of the criti- cisms, of work in this area. The technique of partial hepatectomy, first quantified by Higgins and Anderson in l931.1 induces changes in the liver within one hour after the operation. Basophilic clumps (endoplasmic reticulum) in the hepatocyte cytoplasm disperse and fat globules soon accumulate.5’6 Glycogen stores decrease to a low point at 10 hours and then slowly increase.2 Approximately 12 hours after the partial hepatectomy, DNA synthesis begins, and it peaks at 20 to 30 hours after the operation.2 DNA synthe- sis,2 as determined by autoradiography with tritiated thy- midine, occurs in a wave starting in the periportal part of the hepatic lobule and moves inward as time passes, though with steadily diminishing activity. By 48 hours, this wave of hepatocyte DNA synthesis has subsided and only random cells are labeled. Six to eight hours after peak DNA synthesis. hepatocyte mitoses can be observed.2’7 Nonparenchymal cell DNA synthesis lags behind hepatocyte DNA synthesis by almost a whole day.2’7 and thus, experi- ments examining liver DNA synthesis which.are limited to the first 30 hours after partial hepatectomy essentially examine a homogeneous synchronized hepatocyte population. In addition to the events that were mentioned above, one can detect changes in the biochemistry of the liver such as increased RNA synthesis by 6 hours post-hepatectomy, in- creased protein synthesis by 12 hours, and increased acti- vities of enzymes which are needed for nucleotide synthe- sis and the assembly of DNA by 12 hours.2 Examples of these changes are increased activities of cytidine kinase, RNA and DNA polymerase, asparagine synthetase, and increased histone synthesis, all of which occur at or before DNA syn- thesis begins. If one injects tritiated thymidine at a time corresponding to peak DNA synthesis in partially hepa- tectomized rats, most of the mitotic figures that are seen in regenerating liver during the wave of hepatocyte mitoses contain labeled DNA as determined by autoradiography.7 This observation indicates that these cells synthesized DNA and progressed through the S phase of the cell cycle fol- lowing the operation. Based on this observation, it appears that almost all adult rat hepatocytes are arrested in their progression through the cell cycle somewhere in the 61 phase. _ As a first step in the identification of the factor(s) whichregulates liver cell DNA synthesis, LlanosB’9 injected mouse plasma i.p. isolated under various conditions, to intact and to partially hepatectomized mice. He noted that in intact mice, serum from partially hepatectomized mice stimulated DNA synthesis; that growth hormone also had this effect; and that there is a large release of growth hormone at a time corresponding to that when serum of partially hepatectomized mice was taken for use in the experiment. He noted that extracts of the pars distalis, which releases growth hormone, stimulated DNA synthesis in intact mouse liver only when the pars distalis was taken from partially hepatectomized mice and not from intact or sham operated mice.10 This effect was mimicked by injec- ting pure growth hormone into the mice. Growth hormone may, therefore, be one factor involved in liver regeneration. In contrast, growth hormone did not increase DNA syn- thesis in partially hepatectomized mice, and plasma from intact and partially hepatectomized mice inhibited DNA syn- thesis in partially hepatectomized mice.8 Llanos concluded that at least two factors affecting liver DNA synthesis are present in plasma and that qualities of the system used allow one or the other to be active. In intact mice, where liver growth is not normally present, factors which stimu- late DNA synthesis would be easiest to detect. In hepatec- tomized mice, where cell replication may already be maximal, the addition of stimulating factors would have no effect. Inhibitory factors, however, would be easily observed under these conditions. An alternative explanation, and one which is easily tested, is possible. The plasma that was injected may have contained toxic breakdown products as a result of storage or bacterial growth because it was kept at only 0°C. In intact mice, these toxic products could injure liver cells and cause a compensatory increase in DNA synthesis by uninjured cells. In hepatectomized mice where most cells are already synthesizing DNA, the toxic effects on liver cells will show up as decreased DNA synthesis since there are no reserve cells for DNA synthesis which can cover up the decrease. 0n the other hand, storage at 0°C itself may not result in the production of factors de- creasing DNA synthesis because none were observed in boiled serum in Morley's experiments,11 which will be discussed be- low. In summary, it would appear that Llanos has observed a stimulating effect of growth hormone on DNA synthesis but has not necessarily shown that plasma normally contains any inhibiting factors. Although growth hormone may influence DNA synthesis, it is not an obligatory factor because liver regeneration can occur in hypophysectomized and partially hepatectomized animals.12 A word of caution must be introduced at this point about any conclusions based on DNA synthesis rates. These rates can only be estimated by measuring the incorporation of radioactive precursors into DNA. Apparent differences in DNA synthesis rates may really be differences in the availability of the labeled precursor for incorporation into DNA. The use of the term DNA synthesis in this section and throughout this introduction is used loosely and really means DNA synthesis as estimated by the incorporation of the labeled precursor used into DNA. Despite problems in interpreting Llanos's results showing inhibition of DNA synthesis by serum factors, the factors may actually be present. In support of this possi- bility, 0nda13 has isolated an alpha-l-globulin fraction from serum which inhibits the appearance of mitotic figures in regenerating rat liver. He states that this factor is hepatocyte-specific because it does not act on nonparenchy- mal liver cells. However, he has not examined the effects of this factor on cell division in parenchymal cells of other organs. Because of this, the conclusion that this factor acts only in the liver is not necessarily correct. Because histological examination of regenerating liver shows that hepatocytes nearest portal blood areas are most likely to divide and are responsible for most of the liver regeneration,14 a factor in portal blood has been suspected of controling liver cell division. In support 15 of this belief, Fisher was able to block liver regenera- tion by partial intestinal resection. In contrast, Price16 eviscerated dogs and found a higher rate of DNA synthesis in these livers after partial hepatectomy than in dogs with partial hepatectomy alone. He noted that glucagon reduced the level of DNA synthesis of eviscerated and partially hepatectomized dogs to the level found in dogs only par- tially hepatectomized. In an important breakthrough in the study of liver growth, Short17 was able to induce liver DNA synthesis in intact rats by infusing a mixture into the tail vein for 3 hours which contained glucagon as well as triiodothyronine, amino acids, and heparin. Three consecutive days of this infusion induced a 40% increase in liver DNA content. A single infusion induced a pattern of incorporation of tri- tiated thymidine into DNA which was temporally similar to that following 70% partial hepatectomy. This mixture was chosen because the increased blood levels of amino acids and free fatty acids found in partially hepatectomized rats would be stimulated by administration of these compounds. Short17 hoped that levels of circulating amino acids and free fatty acids would be significant factors in the con- trol of liver cell division. One indication that the mix- ture of chemicals used results in a physiological stimula- tion of DNA synthesis is that this mixture causes a biphasic wave of increased cyclic-3',5'-adenosine monophosphate (cAMP) levels in rats which parallels the wave of cAMP changes ‘8 Short's mixture can, seen after partial hepatectomy. therefore, be used as an important tool to study the control of liver regeneration. The effect of hormones other than polypeptides such as growth hormone,tmjiodothyronine, and glucagon on liver cell division is also being investigated. Noting that cortico- steroids are known to inhibit the growth of a variety of normal and neoplastic tissues, Rizzo19 examined the effects of hydrocortisone on regenerating rat liver. He found that when the hydrocortisone was injected l9 hours after partial hepatectomy, the DNA synthesis normally seen at this time was inhibited and was detectable only after 30 hours, with a peak at 36 hours. Cortisone inhibits thymi- dine kinase and thymidylate kinase activity in regenerating liver, suggesting that cortisone may act by altering ac- tivities of enzymes important in DNA synthesis.20 The observation by Rizzo19 that orotic acid incorporation into DNA of partially hepatectomized and hydrocortisone—treated rats is decreased, as well as that of thymidine, supports this suggestion. Thus, adrenocorticosteroids as well as glucagon, growth hormone,and triiodothyronine may play a role in the regulation of liver DNA synthesis. The role is unclear, however, because both adrenalectomy and sham adrenalectomy result in higher than normal rates of DNA 2‘ In addition, synthesis in partially hepatectomized rats. the administration of growth hormone and cortisone to hypo- physectomized rats which are partially hepatectomized re- sults in a greater than normal mitotic activity while growth hormone or cortisone alone does not affect the mitotic response in partially hepatectomized rats which are hypophysectomized.2 The use of i.p. injections of various substances in order to evaluate their roles as physiological liver growth regulators is subject to misinterpretation. For example, any substance which causes an increase in the secretion of hydrocortisone may appear to inhibit liver regeneration following partial hepatectomy. By the same token, it has been observed that substances with irritating properties will induce DNA synthesis in liver.22 It is evident that work on factors affecting DNA synthesis 1 vivo is complica- ted by the interactions of organs and by inflammatory re- actions that may occur when serum, or fractions isolated from serum, are injected into the test animal. Various ifl_!i££2 systems to study liver growth regula- tion have been devised to avoid these problems. The organ perfusion system that Levi23 used is one of these techniques. He has found, when using this system, that normal liver in- creases its incorporation of tritiated thymidine into DNA when cross-circulated with livers removed 18 hours after partial hepatectomy but not when cross-circulated with liver removed 12 hours or earlier.23 Levi23 also observed that the perfusate from livers removed 18 hours after par- tial hepatectomy contains a substance which is not dialy- zable and is stable at -70°C and which causes an increase in apparent DNA synthesis in normal rat liver. He concluded that liver not only has receptors for this factor but also is "the source of its production“ since it could be collec- ted from isolated, perfused, partially hepatectomized livers. It seems possible that while a factor stimulating incorpora- tion of tritiated thymidine into liver DNA can be observed in a perfusate of an isolated, partially hepatectomized liver, no evidence in Levi's paper indicates that the liver 10 makes the factor. It is possible that the factor is syn- thesized somewhere else, is fixed to liver between 12 and 18 hours post-hepatectomy, and is slowly washed out during the perfusion. Morley'sn observations of the appearance of a DNA synthesis stimulating-protein in blood and Llanos's9 observations on the release of growth hormone at this time period support this possibility. However, Levi's observations of DNA synthesis stimulation in normal liver by perfusates of partially hepatectomized livers were not able to be duplicated in another laboratory.24 While organ perfusion experiments can yield important results, the liver contains several different cell types which make it difficult to pinpoint the site of action of any putative liver growth regulator. In addition, the technique of organ perfusion is technically more complicated and slower to use than some of the methods which are des- cribed below. Tissue culture is one method where the con- trol of growth of a homogeneous cell population derived from liver is studied. Rutzy25 reported that l0% serum from rats partially hepatectomized 48 hours earlier in- creased the rate of production of viable cells in the tis- sue culture. There are questions that can be raised about the significance of Rutzy'sz5 observations to the control of liver growth and about tissue culture work in general. Remembering that the onset of hepatocyte mitosis in par- tially hepatectomized rats occurs at about 30 to 32 hours post-hepatectomy, one would expect that any factor ll stimulating liver regeneration would be present at or be- fore this time period. Serum collected at 48 hours does not necessarily contain the factor acting to initiate liver parenchymal DNA synthesis. Rutzy's25 cells exhibited fibroblast-like qualities and littoral cells can differen- tiate into fibroblasts. Rutzy may be observing a littoral cell division stimulator. It, therefore, remains to be seen whether the factor stimulates hepatocyte cell division or whether more than one factor is active in the control of the various cell types in the liver during regeneration. In order to relate observations in this system to the con- trol of liver regeneration l vivo, one must assume that the tissue culture responSe to factors affecting cell divi- sion is similar to that of intact liver. However, young tissue cultures in general do not exhibit the arrest of cell division, despite an apparently adequate environment, that the liver does in 1112, The only things that stop the growth of many cells in culture are lack of space to expand or the presence of medium exposed to cells for several days. (Since new medium can increase DNA synthesis, it is possible that some nutritional factor has been ex- hausted in the serum or toxic factors added by the cells.) When either is rectified, the cells begin to divide again. Thus, tissue cultures may not be good models for the control of liver regeneration. One cannot dismiss Rutzy's25 work since factors affecting cells derived from the liver may in fact play a role in some of the processes of liver 12 26 can detect inhibition regeneration. For example, Aujard of DNA synthesis in synchronized cells in cultures by liver extracts made in a manner similar to those which have been reported to be inhibitors of DNA synthesis when using lg 1112 and other ifl,li££2 bioassay systems. As mentioned earlier, work 1g vivo is beset with many 13 problems. Morley was working on a factor stimulating liver DNA synthesis in lllg_but great variability between rats limited the conclusions he could draw from the ex- periments. In an attempt to avoid this problem, Morley13 developed a method to make mouse liver cell suspensions whose rate of DNA synthesis is higher when a 24 hour re- generating liver was used as the source of the cells than when an intact liver was used. This indicated that liver cell suspension DNA synthesis can reflect DNA synthesis rates 1 vivo. He found that the injection of growth hor- mone into an intact rat 24 hours before sacrifice resulted in cell suspensions made from the rat which had higher than normal rates of DNA synthesis. In addition, boiled serum (which inactivates any growth hormone in the serum) from partially hepatectomized rats also increased DNA synthesis. 8 but This is in contrast to observations made by Llanos, perhaps the differences in bioassay of the serum can account for the contradiction. In view of the ability of cell sus- pensions made from livers to show increases in apparent DNA synthesis due to growth hormone and also to boiled serum, Morley13 concluded that he observed two factors which may l3 stimulate l vivo liver DNA synthesis. Characterization of the boiled serum factor indicates that it is liver spe- cific and appears in the blood of partially hepatectomized rats after 12 hours. Since the factor appears just before stimulation of DNA synthesis 1 vivo, this factor may be important in liver regeneration. However, there is another possible explanation for the apparent stimulation of DNA synthesis. Enzymatic isolation of cells from a liver is a procedure which may have damaged the cells so that the suspension could not make DNA to its full capacity. Any factor which would somehow protect the cells during the isolation procedures would result in higher rates of DNA synthesis by suspensions and appear to be a stimulatory factor involved in the regeneration of the liver following partial hepatectomy. To support this possibility, it has been observed that liver plasma membranes have altered spectrofluorescence properties in growth hormone-treated rats.27 Hypophysectomized rat plasma membranes in the pre- sence of growth hormone have altered concentrations of various phospholipids and altered activities of Na-K-ATPase and 5'-nucleotidase.28 On a subcellular level, serum factors and heparin have been observed to increase DNA synthesis in isolated rat liver nuclei.29’30 This system lacks the interactions be- tween nucleus and cytoplasm and between cells, and as a result, the amount of information about the control of cell replication is limited. However, it certainly is not l4 irrelevant. The detection of heparin stimulation of DNA synthesis in nuclei for example, has also been reported in 1119 by Short.‘7 A better method to study the control of liver regenera- tion may be the liver slice incubation system. The in 11312 incubation avoids the complex 1 vivo interactions between organs and the indirect effects on liver by test substance actions on other organs. It is cheaper and less technically complicated than an organ perfusion system and allows many more tests to be performed in the same experiment since many slices can be incubated at the same time. It also avoids the problem of altered cell division properties seen in tissue and cell cultures. Furthermore, it has been ob- served that incorporation of tritiated thymidine into DNA of slices is qualitatively similar to what occurs in liver in vivo, suggesting that slices maintain at least some of the regenerative properties of liver i vivo.31 The first thorough investigation of the properties of liver slices i vitro was by Hecht and Potter in 1958.32 They noted that the initial rate of incorporation of precursors into DNA 13 vitro paralleled that seen 1 vivo in regenerating livers. However, they failed to observe changes in the rate of labeling of DNA with time as are seen 1 vivo. The presence of rat serum in the medium did not affect the incorporation of precursors into DNA. To explain the apparent lack of a wave of DNA synthesis in their slices, they suggested that the 4 hour incubation may have been too short to see any 15 changes in the rate of labeled precursor incorporation into DNA. In addition, possible other problems in their system were: 1) The incubation medium consisted only of Krebs- Ringer buffer and fructose; slices may have been deficient in nutrients needed to progress through the wave of DNA syn- thesis. 2) Labeled precursors were present throughout the 4 hour incubation and this might have allowed precursor con- centration or specific activity changes to occur. To the extent that liver slice DNA synthesis is due to hepatocyte DNA synthesis, as it is in the first 30 hours post-hepatec- tomy l vivo, a favorable aspect of studies of DNA synthesis in slices is that a single cell type can be studied. Using this type of system, Verly33 reported on a factor in rat liver homogenates which inhibited DNA synthesis in Slices from livers removed 24 hours after partial hepatec- tomy. He also showed that this homogenate, when injected into rats 21 hours after partial hepatectomy, decreased the incorporation of tritiated thymidine into DNA of liver slices prepared 24 hours post-hepatectomy. The factor ap- pears to be liver specific because it did not inhibit DNA synthesis in rat spleen or kidney slices. A criticism of this work is that slices were exposed to this factor at a time of peak DNA synthesis. For an inhibitory factor to be important in the regulation of liver growth, one would ex- pect it to prevent the onset of DNA synthesis, not to de- crease DNA synthesis already occuring. The inhibition of DNA synthesis by a liver homogenate may be due to toxic 16 34 However, Verly35 factors present in the homogenate. tested his factor for toxicity in liver-derived tissue and cell cultures and did not detect any increase in lethality over that normally seen in the cultures as judged by Trypan blue staining of dead cells. Chemicals known to cause cancer have been observed to interact covalently to macromolecules such as protein and nucleic acid.36 Alkylating agents such as uracil mustard can covalently bind without prior modification of their structures to nucleophilic centers on these macromolecules.37 In contrast, most other carcinogens appear to require metabolism to chemicals which can act as electrophiles be- fore they can react with the nucleophilic centers of cellu- lar macromolecules.38 Examples of carcinogens which re- quire metabolic activation are the aminoazodye 3'-methyl- 4'-dimethylaminoazobenzene, the aromatic amide N-Z-fluor- enylacetamide, and the nitrosamines. Chemicals such as N-2-fluorenylacetamide are not carcinogenic in animals or tissues which fail to metabolize them to a compound capable of binding to nucleophiles.36 An example is the lack of carcinogenicity of N-Z-fluorenylacetamide in the guinea pig.38 This suggests that binding of the carcinogen to macromolecules in a cell is an important step in transfor- mation to a malignant state. Alterations in cell biochemistry and morphology result from the administration of hepatocarcinogens to rats, per- haps as a result of carcinogen binding to proteins or to l7 DNA. For example,39 N-2-fluorenylacetamide induces hepatic nodular hyperplasia and it is believed that hepatomas can arise from these nodules. Besides changes in liver archi- tecture, there are also changes in certain liver enzyme activities. Areas of nodular hyperplasia in the liver have reduced activities of glucose-6-phosphatase and glycogen phosphorylase. In addition, glycogen levels change only Slightly following glucagon administration. In view of the widespread changes seen in carcinogen-treated liver, it seems reasonable to postulate that there may be changes in the levels of, or response to, factors controling liver cell division. In support of this hypothesis are sugges- tions in the literature that factors inhibiting DNA syn- thesis in normal liver are less effective in chemical car- cinogen-induced hepatomas. For example, the inhibitory activity of one of Verly's extracts, which is marked on normal liver slices, is lower on slices of a rat hepatoma induced by oral administration of the hepatocarcinogen p- 35 Since cell division in hepa- dimethylaminoazobenzene. tomas is not inhibited to the degree that normal liver is, this decreased response to Verly's inhibitory factor is con- sistent with a physiological role for the factor in the control of liver growth. This relative lack of inhibition of DNA synthesis in hepatoma tissue by factors known to be inhibitory in normal tissue has been observed in other sys- tems by Terayama and Chany.26 18 1.3 Rationale: A good model for the study of the control of tissue growth is the liver since both a non-growing tissue, and by partial hepatectomy or perhaps by the use of Short's ‘7 a synchronously dividing cell population hormone mixture, can be studied in a controlled manner. This model can be related to the general case of regulation of cell division if one assumes that properties of the control of cell divi- sion in the liver are similar to those in all tissues; i.e., that there is in general a common basic mechanism(s) for the control of cell division in all mammalian tissues. A system to study this regulation of liver cell division is needed which avoids the complex interactions of factors and of organs which occurs lanlVO but which retains properties of liver cell division seen in animals following partial hepatectomy. Specifically, what is desired is a system which can resemble both the 50 state and the synchronized progression of cells through the cell cycle which can be observed 1 vivo. In this way, factors which influence liver cell division might be detected and studied in slices and the results might have important implications for the in vivo situation. 1.4 Specific Aims: One of the first steps in this study was to determine whether or not slices can progress through a wave of DNA synthesis in the absence of serum 13 vitro. A closely related experiment was to ask at what time the liver becomes committed to DNA synthesis following partial l9 hepatectomy. This was tested by determining how early after partial hepatectomy the liver remenant could be sliced, incubated, and still make DNA ifl_!i££2. To esti- mate the rate of DNA synthesis, DNA specific activity or percent labeled nuclei in an autoradiograph was measured following a pulse of 3H-thymidine at various times during an incubation. Another aspect of this project was to begin an exami- nation of the effects of blood-borne factors on liver slice DNA synthesis. This was done by incubating slices in me- dium containing various types of serum and searching for alterations in the normally observed pattern of DNA synthe- sis when incubations were performed in the absence of serum. METHODS AND MATERIALS 2.l Animals: Male Sprague-Dawley rats, 150 to 300 gm, were used in all experiments. Rats used in any given experiment were received on the same day and were within a 25 gm weight range. The rats were kept in a room with a lighting cycle set so that it was light from 7 p.m. to 7 a.m. The method 1 was used to perform 70% partial of Higgins and Anderson hepatectomies under ether anesthesia. The time of partial hepatectomy in all cases was 6 p.m. : 2 hours. Food and water were available to the rats at all times including the time after the partial hepatectomy and up to the time of sacrifice. 2.2 Liver Slice Incubation: At specified times after the partial hepatectomy the rats to be used in an experiment were decapitated and the livers perfused 1 situ with ice- cold incubation medium taken from the batch to be used in that day's experiment. The right lateral lobe of the liver was removed and sliced with a Stadie-Riggs microtome. Slices were placed in ice-cold incubation medium as they were cut and remained there until the incubation began. The first slice was normally discarded, except where noted. The incubation medium contained Swim's S-77 medium, and also final concentrations of 0.05 mM cystine, 1.8 mM CaClZ, 4 mM L-glutamine, 26 mM NaHCO3, 100 units/ml of penicillin, 100 ug/ml of streptomycin, and 0.25 ug/ml of 20 21 amphotericin 8. Into sterile 50 ml erlenmeyer flasks was placed 8 ml of incubation medium made up to 80% of its final volume. Then either 2 ml of a solution of 0.8% NaCl and 0.04% KCl or 2 ml of serum was added to the flask. Serums tested included fetal calf serum, calf serum, dialyzed calf serum, and serum from rats partially hepatectomized 18 hours earlier. To prepare rat serum, blood was obtained from a rat by cardiac puncture under ether anesthesia 18 hours after partial hepatectomy. After being allowed to clot for two hours in ice, the blood was centrifuged at 2000 x g x 10 minutes and the serum was then removed, pooled with serum from other rats similarly treated, and frozen at -20°C until used. Flasks containing the medium to be used in an experi— ment were tightly closed with sterile silicone stoppers and the medium was allowed to equilibrate for 20 minutes at 37°C in an atmosphere of 95% oxygen and 5% C02. The liver slices were cut in half and one piece was placed in each of the flasks, followed by replentishment of the atmosphere. In experiments testing the pattern of 3H-thymidine (3H-dThd) into DNA as a function of time, the half-slices were ran- domly placed in flasks but in the other experiments, slices were placed in flasks so that one half-slice served as a control for the other corresponding half-slice. All incu- bations were done at 37°C and the platform on which the flasks rested was rotated at 100 rpm in a New Brunswick gyrorotator incubator. 22 At various times during the incubation 10‘5M 3H-dThd, 20 uCi/umoie, and 5 x 10‘4M sodium l4c-formate, 0.8 uCi/umoie, as specified, were added to the flasks, the oxygen-C02 at- mosphere was replaced, and the flasks were allowed to incu- bate for one more hour. At the end of this time, the slice in each of the flasks pulsed with the labeled DNA precursors was immediately homogenized in 5% trichloracetic acid in a Potter-Elvejem homogenizer. The DNA was extracted by the method of Sneider and Potter40 after lipids were removed with the use of solutions of 95% ethanol and 10% potassium acetate,41 100% ethanol, chloroformzmethanol (1:2), and ether.42 To analyze protein labeling, the pellet remaining after the DNA extraction was dissolved in 0.5 ml of tetra- ethylammonium, diluted to 2 ml with water, and the radio- activity of this solution was measured. The amount of DNA recovered was determined by the method of Blobel and Potter.43 DNA radioactivity was determined by placing some of the extracted DNA in Multisol and analyzing it in a Packard model 3380 liquid scintillation counter. The calcu- lated DNA specific activity resulting from the pulses of labeled DNA precursors were then used as estimates of DNA synthesis during the pulse periods. DNA synthesis rates were not due to bacterial or fungal DNA synthesis because no colonies grew when incubation medium was plated on saubaurad or tryptose blood agar base. Nuclei were isolated at the end of a pulse of 3H-dThd by a modification of the method described by Goodman.42 The 23 slice was homogenized in 5 ml of a solution containing 0.05 M TRIS-HCl, pH 7.5 and 0.25 M sucrose, filtered through cheesecloth and mixed with Triton X-100 to give a final con- centration of 2% and centrifuged at 1000 x g x 10 minutes. The pellet was resuspended and recentrifuged twice in 0.25 M sucrose before suspending it in the TRIS-sucrose solution. This suspension was spread on microscope slides and allowed to air dry. Slides were then dipped in nuclear track emul- sion (Kodak NTB-3) and exposed for 23 days. They were de- veloped (Kodak D-19 developer), fixed (Kodak acid fixer), and were stained with Harris hematoxylin. The autoradio— graphs were analyzed by scoring the percent labeled nuclei (5 silver grains or more) in random fields throughout each slide. 2.3 Experiments In Vivo: IQ vivo DNA synthesis rates were estimated by injection of 29.8 nmoles, 200 uCi/kg 3H-dThd and 20 umoles, 40 uCi/kg sodium 14C-formate i.p. at various times after partial hepatectomy. The rats were sacrificed one hour later and the livers were perfused with a solution containing 0.05 M TRIS-HCl, pH 7.5, 0.025 M KCl, and 0.005 M MgClz. The right lateral lobe of the liver was then removed and the DNA specific activity was determined as described above. 2.4 Experiments Using N-2-Fluorenylacetamide: In experi- ments examining the effect of the hepatocarcinogen 24 N-2-fluorenylacetamide (FAA) on DNA synthesis in slices ig_yjtrg, rats were fed a powdered diet for 14 days which contained 0.05% FAA mixed in Farber basal carcinogenic diet44 supplemented with para-aminobenzoic acid, inositol, vitamin E acetate, and USP salt mix XIV. Following this period of time, rats were switched to a pelleted control diet for at least 7 days, as specified in the results. Procedures for 70% partial hepatectomy and for incubation were the same as described for normal rats. Rats weighed 150 to 175 gm when placed on the carcinogenic diet. 2.5 Materials: Sprague-Dawley rats were obtained from Spartan Farms, Haslett, Michigan. Swim's S-77 medium, calf serum, antibiotics, dialyzed calf serum, and fetal calf serum were purchased from Grand Island Biological Company, Grand Island, New York. Multisol was obtained from Isolab Incorporated, Akron, Ohio, and the 3H-thymidine (3H-methyl), 6.7 Ci/mmole and sodium 14C-formate, 3 mCi/mmole, was pur- chased from New England Nuclear, Boston, Massachusetts. The basal carcinogenic diet was obtained from General Biochemicals, Chagrin Falls, Ohio. RESULTS 3.1 Experiments In Vivo: The data in Figure 1 show the rate of incorporation of 3H-dThd and 14C-formate into DNA of the right lateral lobe of the liver 1 vivo following partial hepatectomy. Points are plotted at the time of sacrifice of the rats. The onset of a wave of apparent DNA synthesis in this liver lobe occurs at some time between 12 and 22 hours post-hepatectomy and peaks at 26 hours. These observations are consistent with the observations of others,2:7'45 who studied the total regenerating hepatic remnant. This sug- gests that the pattern of DNA synthesis of this liver lobe in experiments 1 vivo is a valid reflection of what occurs throughout the regenerating liver and that this lobe can be used to study the general aspects of liver regeneration lg vitro. 3.2 Experiments Using3H-thymidine: Figure 2 shows the typical pattern of incorporation of 3H-dThd into DNA as a function of time in liver slices made from the right lateral lobe when the rats were sacrificed at 14 or 16 hours after partial hepatectomy. Each point represents slices pulsed with 3H-dThd for one hour. The mean specific activity of DNA at the end of the pulse is plotted in this figure. At 14 hours there has never been a wave of 3H-dThd incorpora- tion into DNA during the incubation period. At 16 hours there usually was a wave of incorporation which peaked at 25 26 O m m we? .m o N h N M D D 081 m m .w I 1 W 8.: x x m M D m ~21 .._o mm CL u Oi. . . 7 O o m 3 .m ~ 3 we am .225 >m._.mm 3:352. Imv>dm0fi0§< =5:an mdocxm .. > noaooxsmo: om wziaqza mag donimoxamnm Annex- uowwndo: dawo 02> ow «momsmxmnszm fim m: fizm «flora dmfimsmd doam 2mm amamxaszma. man: coda”. ndoaama ma asm man om fiym cam :ocs ucdmm. «muxmmmzfim cam «on. 27 1001 1" S O a: 2 2: C) a! E E? 9. X if O. C) 0 a I Q r o 5 1'0 1% 20 TIME (hours) Figure 2. A comparison of DNA specific activity as a function of time in liver slices made and incubated starting at 14 or 16 hours post-hepatectomy. Following 70% partial hepatectomy, slices incubated beginning at 14 hours G-O , and at 16 hgurs H post-hepatectomy were pulsed for one hour with H-dThd, 10’5M, 20 uCi/umole, at various times during the incubation. Each point, plotted at the end of the pulse, represents the mean of 4 slices except for the last point which represents the average of 2 slices. Each curve represents liver slices pooled from 3 rats. 28 about 10 hours after the start of the incubation; i.e., at 26 hours after partial hepatectomy. Those slices which failed to show this wave had a pattern Similar to that shown at 14 hours post-hepatectomy, as did slices from rats not subjected to partial hepatectomy. One can note that the peak effect is at the same time as that seen in 1112 in the right lateral lobe of the rat liver (Figure 1). At 18 hours after partial hepatectomy (not shown) a wave of incorpora- tion of labeled thymidine into DNA has always been observed. This suggests that liver slices prepared from the right lateral lobe of rats sacrificed at 16 hours post-hepatectomy can progress through the S phase of the cell cycle 13 11319 independent of systemic influences and in a manner similar to that of the whole liver lobe i vivo. Slices from rats made 14 hours post-hepatectomy do not appear to synthesize DNA ig_yit£g (Figure 2). No major differences in these patterns were observed when rat weight ranged from 150 to 300 gm. Presented in Figure 3 are the results of an experiment which compared DNA specific activities and percent labeled nuclei in slices from a liver removed 16 hours post-hepatec- tomy. Figure 3a Shows the pattern of incorporation of 3H- dThd into DNA in slices from one rat which showed a wave of incorporation while Figure 3b is of a rat whose liver slices failed to progress through this wave. (While slices made 14 hours post-hepatectomy or earlier always failed to progress through a wave of 3H-dThd incorporation into DNA, this also 29 A. 30+ '3 § zo~ ~2 i in Z d D 101 -1 D a: :2 E C) e?“ u: 53 .1 EE .1 C) 33 o 5 1o 15 2'0 TIME (hours) Figure 3. A comparison between liver DNA specific activity and the percentage of labeled nuclei at vari- ous times in slices made and incubated lg vitro from one rat starting at 16 hours after 70% partial hepa- tectomy. Slices were cut in half and at indicated imes each half was separately pulsed for one hour with H-dThd, 10'5M, 20 pCi/umole. In one half H, DNA specific activity was determined, while in the other half 0—0, the nuclei were isolated and the percentage of labeled nuclei was determined by autoradiography. A and 8 represent the incorporation of 3H-dThd into DNA in Slices made from different rats. 30 occasionally occurred at 16 hours.) It is important to remember that the number of grains over a nucleus had no influence on the decision that it was labeled (as long as there are at least 5 grains present). Therefore, changing rates of DNA synthesis in the slice are not reflected in the resulting plot of the percentage of labeled cells as a function of time. Because the plot of DNA specific activity in the slice as a function of time is similar to that of the percentage of labeled cells, this suggests that changes in DNA specific activity reflect changes in numbers of cells making DNA. At 6 hours after the start of the incubation, the percentage of labeled cells is near its peak while DNA specific activity at this time is only slightly increased (Figure 3a). While the cause for this discrepancy has not been determined, a similar high percentage of labeled cells and a low DNA specific activity a few hours before peak levels of incorporation of 3H-dThd into DNA are reached has been observed in experiments studying liver regeneration i vivo.45 In addition to what is shown in Figure 3, one slice from the rat liver represented in Figure 3a was incubated in the presence of 3H-dThd throughout the 23 hour incubation period. The percentage of labeled nuclei in this slice, an indication of the percentage of cells which progressed through the wave of 3H-dThd incorporation into DNA seen in Figure 3a was 5.70% (95% confidence interval = 4.33 to 7.07%). 31 The assumption made in these experiments is that the liver is homogeneous; that is, that Slices taken from various depths in the liver, as well as sections from the same slice, are comparable to each other. Table 1 shows an experiment in which the depth of the slices and specific activities in the two halves of a slice were examined. All the values represent slices made from a single rat liver 16 hours post-hepatectomy and incubated 9 hours plus a one hour pulse period. This time for the pulse was chosen be- cause the incorporation of 3H-dThd into DNA of the slices should be at a peak, as indicated by Figure 2. There was no significant (analysis of variance, F > 0.05) difference be- tween corresponding halves of a slice or between slices made throughout the liver lobe. However, inspection of the table indicates that a given half-slice specific activity may vary by a factor of two from other slices or from its correspon- ding half-slice. This variability accounts for the large standard errors shown in experiments in this report. 3.3 Experiments Using‘4C-formate: Although the incorpora- tion of 14C-formate into liver DNA 10.1112 paralleled that of 3H-dThd (Figure 1), this was not the case in slices in liELQ- Figures 4a, 4b, and 4c represent experiments where the incorporation of labeled precursors into DNA in slices was measured when the livers were sliced 18, 16, and 14 hours post-hepatectomy, respectively. As a result of the different times of slice preparation, three different 32 Table l. The relationship between DNA specific activity of half-slices and of slices made at different depths in the liver. One rat was sacrificed 16 hours post-hepatectomy. Consecutive slices were made from the right lateral liver lobe. Each slice was cut in half, and each piece was then separately incubated 11 vitro for 9 hours. Following a subsequent one hour pulse 6T_3H-dThd, io-SM, 20 uCi/umole, the DNA specific activity of the tissue was determined. Slice #1 is normally discarded. DNA Specific Activity (DPMXlO3/mg DNA/hour) Slice # Half-slice Half-slice l 29.69 21.50 2 29.31 22.22 3 24.81 24.36 4 21.45 40.19 5 20.86 42.44 6 51.39 24.72 7 45.74 27.93 8 58.33 16.40 9 4.28 38.38 33 B 100‘ A 5 § 150 3 ‘ < 50- 2 z T Z O \ o 5’ °’ 100- E o . 1 ”5 ‘3 o 10 20 g '; TIME (hours) x 504 , e E 40 c E \. o D 20 0 r i O 0 10 20 0 10 20 TIME (hours) TIME (hours) Figure 4. A comparison of 3H-dThd and 14C-formate incor- poration into DNA at various times in liver slices made and incubated lg vitro l8, 16, or 14 hours after 70% par- tial hepatectomy. Slices were pulsed for one hour with 3H-dThd, 10'5M, 20 uCi/umole 0—0, and 14C-formate, 5 x 10'4M, 0.8 uCi/umole o—o. Points are plotted at the end of the pu se. A. Slices made at 18 hours post-hepatectomy; slices from 2 rat livers were pooled and were distributed so that each point represents the mean of 4 slices except for the first point which is the average of 2 slices. 8. Slices made at 16 hours post-hepatectomy; slices from 3 rat livers were pooled and distributed so that each point represents the mean of 5 slices. C. Slices made 14 hours post-hepatectomy; slices from 3 rat livers were pooled and distributed as described in Figure 2. 34 patterns of 3H-dThd incorporation into DNA are displayed but only a single pattern of 14C-formate incorporation occurred at these times. The incorporation of 14C-formate (5 x 10‘4M. 0.8 uCi/pmole) into DNA, therefore, appears to be independent of the time of sacrifice of the rats used in the experiment and dependent only on the length of the incubation. No wave of incorporation of 14C-formate is apparent at the times tested. The question immediately arose as to whether the 14C- formate incorporation into DNA affects the conclusions about liver Slice properties as determined by 3H-dThd incorpora- tion since many of the experiments used both 3H-dThd and 14C-formate in the pulses. This question was approached by examining the effects of formate concentration on the in- corporation of 3H-dThd into DNA as shown in Figure 5. In this experiment, the amount of labeled formate was kept constant while cold formate was added to obtain the specific final concentrations. The activities at the three concen- trations of formate tested, 2, 6.5, and 10 x 10’4M, are 2, 0.62, and 0.4 uCi/umole, respectively. In Figure 5a, one can see that DNA specific activity decreases as the formate concentration is increased in slices incubated beginning at 15 hours post-hepatectomy and pulsed at either the start of the incubation or 12 hours after the start of the incubation. One can note that while the specific activity of DNA in the slices decreases by a factor of five, the incorporation of 14C-formate into DNA decreases by only a factor of two at 35 804 A 60- s 40- l 0 =5 ‘2‘ o 20- D e 67‘ E! 0 . . i i x o 2 4 6 8 10 E FORMATE CONCENTRATION (x WW) 0 20 B n- L o E i a o 2 4i 6 e 10 FORMATE CONCENTRATION (x 104M) Figure 5. The effect of formate concentration on the in- corporation of 4C-formate and 3H-dThd into DNA of liver slices made and incubated jfl_vitro starting at 15 hours after 70% partial hepatectomy. The 3H-dThd concentration in the medium in all cases was 10'5M, 20 uCi/pmole. 14C- formate specific activity at formate concentrations of 2, 6.5, and 10 x 10‘4M are 2, 0.62, and 0.4 Ci/ mole respec- tively. Slices from 2 rat livers were pooled and distribu- ted so that each point represents the mean of 5 slices. Points are plotted at the end of a one hour pulse which be- gan at the start of the incubation I—O, or at 12 hours af- ter the start of the incubation, 0—0. A. The incorporation of C-formate into DNA as a function of formate concentration. B. The incorporation of 3H-dThd into DNA as a function of formate concentration. 36 both pulse times. This indicates that the concentrations of formate are not saturating for DNA synthesis since the decrease Should be a factor of 5 if it was. It seems that factors other than formate concentration alone play a role in determining the incorporation of 14C-formate into DNA, especially in view of the lack of saturation of 14C-formate incorporation into DNA at the high formate concentrations used in this experiment. Figure 5b shows a relatively con- stant incorporation of 3H-dThd into DNA despite increases in formate concentration in the medium. The Slices in this experiment were incubated beginning at 15 hours post-hepa- tectomy and were pulsed at the start or at 12 hours after the start of the incubation. It is, therefore, evident that the concentrations of formate tested in this experiment do not alter the incorporation of 3H-dThd into DNA. This sug— gests that the apparent lack of a relationship between 14C— formate incorporation into DNA of slices from regenerating liver incubated ig'vitgp and the time at which rats were sac- rificed probably do not affect the conclusions drawn through the use of 3H-dThd to estimate DNA synthesis. One explanation for the pattern of formate incorporation into DNA is that some of the labeled protein made during the incubation is extracted in the DNA fraction of the procedure for DNA isolation.40 Figure 6 compares the total incorpora- tion at various times during the incubation. Since the rates of incorporation appear to be similar, protein hydroly- sis could account for some of the radioactivity since the 37 10‘ f3. 5 (D J: a? 6- S .. >< E 4- C3 :2- O i I l O 2 4 6 TIME (hours) Figure 6. A comparison of 14C-formate incorporation into DNA and into protein as a function of time in liver slices made and incubated lg vitro starting at 18 hours after 70% partial hepatectomy. Slices were pulsed for one hour with 4C-formate, 2 x 10‘4M, 2 uCi/umole. Total radioactivity incorporation into DNA 0-0, and into protein H, in a slice is plotted at the end of the one hour pulse. Slices from 2 rat livers were pooled and distributed so that the middle point represents 4 slices while the other points represent 5 slices. 38 DNA fraction in a Slice contains 30 to 40% of the total radioactivity measured in the DNA and protein fractions, as can be calculated from the data in Figure 6. 3.4 Serum Effects on Slice DNA Synthesis: Figure 7 Shows the apparent lack of effect (paired Student's t-test, p > 0.05) of 20% calf serum on slices incubated beginning at 16 (Figure 7a) and 14 (Figure 7b) hours post-hepatectomy. Al- though points are displayed as grouped means, each half- slice incubated in medium plus serum was compared to its other half of the slice which was in control medium and was pulsed at the same time as the test half-slice. There was a similar lack Of effect when 20% fetal calf serum (Figure 8), or 5% serum from rats partially hepatectomized 18 hours earlier (Figure 9) was tested. Because of these observations, we must conclude that factors affecting DNA synthesis are not present in the serums tested or that this liver slice incubation system was not able to detect the presence of these factors. It still seemed possible that stimulatory factors were present but that unlabeled thymidine in the serum diluted the 3H-dThd specific activity and caused the stimulation to be obscured. Accordingly, dialyzed calf serum was tested to see if it stimulated DNA synthesis in 16 hour post- hepatectomy liver slices. To our surprise, the dialyzed serum decreased the incorporation of 3H—dThd into slice DNA, as Shown in Figure 10. One explanation of this observation 39- 20% CALF SERUM 100‘ A T 80« 601 '5 . O 5 . < . Z 40' . 4‘ C) D x e. , ° &T‘ 20- 53 x E O I l 1 T Q o 5 1o 15 20 25 B 20« ‘2 0 a . t . o 5 1o 15 20 25 TIME (hours) Figure 7. The effect of 20% calf serum on the incorpora- tion of 3H-dThd, 10-5M, 20 uCi/umole, into DNA at various times in liver slices made and incubated ip_vitro starting at 16 or 14 hours after 70% partial hepatectomy. Slices per point are as described in Figure 2. Points are plot- ted at the end of the one hour pulse. o—o- No serum pre- sent in the incubation medimn. QrC-20% calf serum pre- sent in the incubation medium. A. Slices made from 3 rat livers were pooled and were in- cubated beginning at 16 hours post-hepatectomy. B. Slices made from 2 rat livers were pooled and were in- cubated beginning at 14 hours post-hepatectomy. 4O 20% FETAL CALF SERUM 7 170: 1201 \V A. 100. J. 40 L 20- DPM x 103/mg DNA/hour o a 16 1T5 20 TIME (hours) 201 B o 5 10 is 20 TIME (hours) Figure 8. The effect of 20% fetal calf serum on the in- corporation of 3H-dThd, 10'5M, 20 uCi/umole, into DNA at various times in liver slices made and incubated lg vitro starting at 16 or 14 hours after 70% partial hepatectomy. In each case, Slices from 3 rat livers were pooled and dis- tributed as described in Figure 2. Points are plotted at the end of the one hour pulse. 0~O-No serum present in the incubation medium. H- 20% fetal calf serum present in the incubation medium. A. Slices incubated beginning at 16 hours post-hepatectomy. B. Slices incubated beginning at 14 hours post-hepatectomy. 41 5% RAT SERUM A 40- 20a 5 O £5 < 0 l I i i E 0 5 10 15 20 5, TIME (hours) E a} S X z 40* B a. C3 20- O l I I I O 5 10 15 20 TIME (hours) Figure 9. The effect of 5% serum from rats partially hepa- tectomized 18 hours earlier on the incorporation of H- dThd, 10'5M , 20 uCi/umole, into DNA of liver slices made and in- cubated in vitro starting at 16 or 14 hours post- hepatectomy. Slices were pooled from 4 rat livers and distributed as des- cribed in Figure 2. Points are plotted at the end of the one hour pulse. OFO- No serum present in the incubation medium. 0—0-20% fetal calf serum present in the incuba- tion medium. 42 20% DIALYZED 3 80- CALF SERUM 2 2 Each U, E 2’. 40- X 2 3 20- 0 I I I T o 5 10 15 20 TIME (hours) Figure 10. The effect of 20% dialyzed calf serum on the incorporation of 3H-dThd, 10'5M, 20 uCi/umoie, into DNA at various times in liver slices made and incubated lg vitro starting at 16 hours after 70% partial hepatectomy. Slices were pooled from 4 rat livers and were distributed as described in Figure 2. Points are plotted at the end of the one hour pulse. <><>- No serum present in the incu- bation medium. 04- 20% dialyzed calf serum present in the incubation medium. 43 is that serum contains low molecular weight stimulators of DNA synthesis”,29 and high molecular weight inhibitors]3 whose effects are equal in whole serum. However, another explanation is that inhibitory factors are produced during dialysis. 3.5 N-2-Fluorenylacetamide Effects on Slice DNA Synthesis: Factors important in the normal control of liver growth might be altered by the effects of a hepatocarcinogen such as FAA. It seems reasonable that an alteration in the con- trol of liver growth should occur before the gross appear- ance of liver cancer. (At later times, secondary changes might occur such as when chromosomes are lost from tumor cells.) Therefore, a test of the importance of a change in the properties of regenerating liver at 16 hours post- hepatectomy (as evaluated by the onset of a wave of DNA syn- thesis in liver slices incubated beginning at this time), and of the importance of the synchronous wave of DNA synthe- sis, to the control of normal liver cell division would be to see if FAA alters the ability of regenerating liver Slices to make DNA from that seen normally at a time before the gross appearance of liver cancer. After 4 weeks on a diet containing 0.05% FAA, rats were observed to have gross evidence of hepatic cirrhosis. In addition, 2 out of 21 rats had an abnormal mass, in one rat in the lower thoracic cavity and in the other a subcutaneous abdominal mass. Two weeks of feeding on a diet containing FAA was chosen as a starting time to examine precancerous 44 effects of FAA on Slice DNA synthesis ip_yit£g. Rats ex- posed to FAA for 14 days, followed by seven days on a con- trol diet (to decrease or eliminate the acute toxic effects of FAA in this study) were partially hepatectomized and their livers used in an experiment 16 hours later. Figure 11 shows that there was an almost continual increase in DNA specific activities with time rather than the wave of incorporation that was seen in normal rats. This suggests asynchronous DNA synthesis in the slices. To determine if the alteration in DNA synthetic rate is due to acute or to Chronic effects of FAA, rats were kept on the control diet for 30 days after 14 days eating FAA. In this experiment (Figure 12), the rats were again sacrificed 16 hours post-hepatectomy but the pattern of the incorpora- tion Of 3H-dThd into DNA with time did not duplicate that Shown in Figure 11. Instead, it resembled that of 14 hour post-hepatectomy slices; namely the pattern of DNA specific activity seen when a liver is removed from a rat before the critical period has passed in the animal (Figure 2). This suggests that the presence of a critical period in rats exposed to FAA and allowed to recover from FAA acute effects is unchanged from that seen in normal rats or that there is a decreased response to partial hepatectomy in these rats. Unfortunately, no conclusions can be drawn from this experi- ment about the stability of changes in incorporation of 3H—dThd into DNA that were observed in Figure 11. Since slices from livers removed before the critical period sometimes show a small gradual increase in DNA 45 14 DAYS 0.05% FAA 7 DAYS CONTROL DIET 00 CD 1 DPM x 103/mg DNA/hour -d DO CD CD 1 l o 5 1O 1'5 211 25 TIME (hours) Figure 11. The effect of feeding a diet containing 0.05% FAA for 14 days followed by a control diet for 7 days on the incorporation of 3H-dThd, 10'5M, 20 pCi/umole, into DNA at various times in liver slices made and incubated 13 vitro starting at 16 hours after 70% partial hepatec- tomy. Slices were pooled from 2 rat livers and each point, .plotted at the end of the one hour pulse, represents 5 Slices except for the last point where 4 slices were used. 45 no CD 3 O> mo: in amkm moddozma ck m nozflwod asmfl mow wo amkm o: fizm dznosuoxm- ”so: 54 mI-e«:a. do-mz. No eom\eaoso. mafia oz» 5a < E C) () l l l 1 r o 2 4 5 8 10 TIME (hours) Figure 13. The effect of feeding a diet containing 0.05% FAA for 14 days followed by a control diet for 7 days on the incorporation of 3H-dThd, 10-5M, 20 uCi/umole, into DNA at various times in liver slices made and incubated in vitro starting at 18 hours after 70% partial hepatec- tomy. Slices from one rat were used in this experiment and are distributed as described in Figure 2. DISCUSSION 4.1 Slice DNA Synthesis: These studies are the first, to our knowledge, to show that slices of rat liver from the right lateral lobe are capable of progressing through a wave of incorporation of 3H-dThd into DNA 1Q 11339 when the liver is removed 16 hours or later after partial hepatectomy. The observation that the peak rate of 3H—dThd incorporation into DNA 1p vitro is seen at the same time as is seen i vivo sug- gests that this wave represents progression of liver slice cells through the S phase in vitro in a manner reflecting that seen 1 vivo. This, in turn, indicates that liver slice cells 13 11313, when taken from livers after 16 hours post-hepatectomy, are capable of maintaining a physiological regulation of progression through the S phase independent of any putative systemic factors which may be required to con- trol liver cell division. Autoradiography of slices made 16 hours post-hepatec- tomy (Figure 3) resulted in a peak of 2.65% labeled nuclei. Since this number represents the percentage of all nuclei in the slices, the labeling of hepatocytes (which make up 60% of liver cells) was probably about 4.3%. (Only hepatocytes make DNA during the first 26 hours post-hepatectomy 1 vivo and assuming that this also holds true for the first 10 hours in an 13 vitro incubation of 16 hour post-hepatectomy Slices.) Reports in the literature indicate that peak per- centages of labeled cells seen in regenerating liver i vivo 49 50 range from 15% to 44%.7’13’45’46 One must note that the peak Specific activity observed in Figure 3 was lower than that seen in other experiments in this report so that in those experiments the percentage of hepatocytes participa- ting in DNA synthesis might have been higher. In addition, some cell death without nucleus breakdown might occur either in the preparation of the slices or during the incu- bation itself. Therefore, the percentage of hepatocytes in liver slices that make DNA during the incubation could be a fairly good reflection of the percentage of hepatocytes that participate in DNA synthesis in regenerating liver 1 vivo. Slices made from livers removed 14 hours or earlier after partial hepatectomy failed to progress through a wave Of incorporation of 3H-dThd into DNA. This indicates that there is a critical period at around 16 hours after partial hepatectomy when certain properties of the liver change so that Slices made after this time can progress through a wave of 3H-dlhd incorporation into DNA 13 11353. It is possible that this critical period, corresponding to a time near the beginning of DNA synthesis 1 vivo, may represent a control point in the regulation of cell division in the liver. This may be a time when blood-borne factors act to initiate a change from the normal GO state of liver cells to a state of active progression through the cell cycle beginning near the end of G]. An alternative is that this critical period is one of stabilization of intracellular factors needed for DNA synthesis and that these factors are labile and sensitive to 51 disturbances such as the preparation of slices before 16 hours post-hepatectomy. This situation would be similar to models of heat shock synchronization of cell division in Tetrahymena,47 although in the case of Tetrahymena, cell division, but not DNA synthesis, is delayed. If this possi- bility is correct, then the time at which specific steps are initiated which lead to DNA synthesis may be earlier than 16 hours post-hepatectomy. Evidence suggesting this transition of cells in GO to the early G] phase includes the increased levels of ribosomes and RNA seen by 6 hours post- .hepatectomy and increased protein content and activities of enzymes important in DNA synthesis by 12 hours post-hepa- tectomy. A factor might act before 6 hours post-hepatectomy and a sequence of events would be initiated which affect levels of protein and RNA and eventually DNA synthesis. As an alternative, there may be several control points, inclu- ding ones for increased protein and ribosome levels in a cell, one for increased activities of enzymes needed for DNA synthesis, and one for the critical phase observed in this report which occurs just before the onset of DNA syn- thesis. It has been pointed out that the onset of DNA syn— thesis in regenerating liver 1 vivo is dependent on the time of day at which the partial hepatectomy is performed.45 In this study, the time of operation was relatively constant (1 2 hours) and rats sacrificed at 14 or 16 hours post- hepatectomy were operated on randomly during the time period at which all operations were done. No relationship between 52 time of operation and the ability of liver slices to pro- gress through a wave of 3H-dThd incorporation into DNA was observed within the limited time period evaluated. Further- more, Figure 3 is an example of an experiment where the par- tial hepatectomy of one rat (Figure 3b) was completed 17 minutes after the first rat (Figure 3a) and yet a wave of incorporation of 3H-dThd into DNA of liver slices occurred in one rat (Figure 3a) but not in the other (Figure 3b), indicating that the onset of a wave of DNA synthesis in liver slices is not dependent on time of partial hepatectomy within the period tested. In summary, there are three possible explanations for the presence of the critical phase in regenerating liver. The first is that Go is located in late G]. A factor acts during the critical phase to cause the Go cell to reenter the cell cycle and to begin to make DNA. To explain the changes in protein and RNA observed before the criiical period, one need only to hypothesize that this represents a nonspecific response to hepatic insuf- ficiency without a commitment to DNA synthesis. The second possibility is that the critical phase is an artifact, due to the preparation of slices before 16 hours post-hepatec- tomy. The third possibility is that the critical phase is only one of several control points which occur in early G] and throughout the G] phase and which must be passed before DNA synthesis can begin. 4.2 The G] Phase of the Cell gycle: In an attempt to under- stand the regulation of cell division in chicken fibroblasts, 53 Temin examined the serum requirements of these cells.48 He found that the G1 phase could be divided into three seg- ments. In the absence of serum, cells stop progressing through the cell cycle in a phase Temin named Gla‘ Serum was required (Glb) for these cells to enter Glc’ a phase where serum is no longer required and where cells are com— mitted to DNA synthesis but have not yet begun to make DNA. In view of the requirement for serum in most cell and tissue cultures, one can suspect that liver also requires serum for cell division despite the apparent lack, in this system, of whole serum effects on liver slice incorporation of 3H-dThd into DNA. This lack of effect of serum might be explained by postulating that the slices are made after the serum re- quirement has passed, or that stimulating and inhibiting factors in the serum are exactly balanced. Slices made from livers 16 hours post-hepatectomy or later appear to be in late 61 (Figure 3a) or in early S phase (Figures 2.4.5.7-10) as judged by the apparent initial rates of DNA synthesis in these experiments. Since slices made earlier than 16 hours post-hepatectomy do not appear to enter the S phase even in the presence of serum, something more than a generalized serum requirement may be needed for liver to enter the S phase. (Temin48 showed that bovine serum could substitute for chicken serum in the chicken fibroblast system so the requirement of his tissue culture for serum was not specific.) The time during which this factor would act is represented by the phase Gld in Figure 14. This phase would be the 54 @ daughter cells f) (32 Gia Gic s ’ Gid Figure 14. Serum requirements of cells progressing through the 6la Gib Glc GI phase of the cell cycle. Cells stop progressing through the cell cycle here in the absence of serum. A period of time when serum is required for cells to enter Glc- Serum is no longer required in order to enter the S phase. Cells are committed to make DNA but have not yet begun to do so. The critical phase observed in the liver slices. Cer- tain properties of the liver change so that slices will make DNA in vitro only after the liver progresses through this phase in vivo. DNA synthetic phase Premitotic phase Mitotic phase 55 critical phase observed in the liver slices. Aside from 48 are in a state the species difference, Temin's fibroblasts of constant growth except when serum is removed from the medium or when serum becomes aged and has a decreased ability to support cell division due to the presence of the cells over a period of time. The plateau of cell division in the cultures may be due to a nutritional deficiency which de- velops as the serum ages. This nondividing state of the fibroblasts is not the same as is found in the liver 1 vivo, where cells are in a Go state despite apparently adequate nutrition. Thus, the conversion of the G0 state to active progression through the cell cycle in the liver may not be comparable to the serum requirement seen in tissue or cell culture. It may be that the Go conversion of liver to ac- tive cell division does not require a generalized serum pre- sence at all but rather something else or perhaps something in addition to serum. In view of the lack of DNA synthesis in slices made be- fore the critical period, one can conclude that there pro- bably is a positive factor acting at this time to initiate DNA synthesis. Should an inhibitory factor have been present which prevented liver progression through the cell cycle and which disappeared at a time corresponding to the onset of DNA synthesis, one would have expected all slices to progress through a wave of DNA synthesis 11 11319 since this factor would not be present in the medium. This does not eliminate the possibility that inhibitory factors control DNA 56 synthesis at different times in the regeneration of liver than those examined here. 4.3 ‘4C-Formate DNA Synthesis: The use of l4C-formate as a precursor for DNA in slices was intended as a way to examine whether the g; 9919 pathway for DNA synthesis was under similar control as the salvage pathway to DNA synthesis as determined by 3H-dThd. While the experiments comparing 3H-dlhd incorporation into DNA and the autoradiography of nuclei provide strong evidence that 3H-dThd incorporation into DNA reflects the true rate of DNA synthesis, it is still possible that real DNA synthetic rates may vary from the rate estimated by the use of 3H-dThd. This would occur if the dg pgvp DNA synthesis rate varied with time from the rate of 3H-dThd incorporation into DNA via the salvage pathway. The use of a fig pgvg precursor of DNA might have ruled out this possibility. It was, therefore, disappointing to find that ‘4C-formate was not a good precursor for slice DNA synthesis. Although 14C-formate incorporation paralleled 3H-dThd incorporation 1 vivo, it was much less sensitive than 3H-dThd. In Figure l, the peak incorporation of 14C-formate into DNA was only 4 times as high as at early times during the incubation while the incorporation of 3H-dThd into DNA increased 60-fold. LE 1135p, 14C-formate incorporation did not parallel 3H-dThd incorporation into DNA (Figure 4), al- though it did parallel formate incorporation into protein 57 (Figure 6). During the extraction of DNA, small amounts of protein are hydrolyzed and appear in the DNA extraction as oligopeptides and amino acids.40 However, this hydroly- sis is not sufficient to explain most of the radioactivity in the DNA fraction since 30 to 40% of the total radioac- tivity measured in the DNA and protein fractions appears in the DNA fraction. The data in Figure 5 indicate that the rate of incorporation of MC-formate into DNA is not satur- ated at concentrations as high as 10'3M. This suggests that the capacity of formate to be incorporated into the pool of proximate DNA precursors is a limiting factor in the in- corporation of 14C-formate into DNA, rather than DNA poly- merase activity. There are several possible steps in DNA precursor synthesis where this might occur. Formate is used to make N‘O-formyltetrahydrofolate in an ATP requiring re- action. Two steps later this compound has been converted to N5,N‘O-methylenetetrahydrofolate. This second step is coupled with the oxidation of NADPH. Purine and thymidine synthesis require these tetrahydrofolate compounds so that the limiting step could be either in the steps of the tetra- hydrofolate pathway just described, in nucleotide synthesis, or in the phosphorylation of the nucleotides to triphos- phates. One possible explanation for the lack of saturation of l4C-formate incorporation into DNA is that the amino acids serine, histidine, tryptophane, and glycine can donate one carbon units to the tetrahydrofolate pathway and thus com- pete with formate for incorporation into nucleotides. This 58 competitive inhibition of formate incorporation could be overcome at high formate concentrations and this would mean that 14C-formate incorporation into DNA would increase at high concentrations rather than appear to be saturating as it might if the amino acid competition for incorporation in- to proximate DNA precursors was not present. This hypothe- sis could be tested by adding adenosine, guanosine, and cytidine to the medium and by removing the amino acids named above from the incubation medium to see if formate incorporation into DNA increases. 4.4 Serum Effects on Slice DNA Synthesis: Calf serum, fetal calf serum, and serum frOm rats subjected to partial hepa- tectomy 18 hours earlier did not alter the pattern of in- corporation of 3H-dThd into DNA at any time tested (Fig- ures 7-9). Bovine serums were tested because of reports using other systems and other tissues that factors in serums affecting DNA synthesis were not species specific.33’46 The rat serum was tested because of reports that factors stimulating liver DNA synthesis appear 12 to 18 hours post- hepatectomy“:23 The apparent lack of serum effects sug- gests that factors regulating rat liver DNA synthesis are never present in the blood, that factors with equal and op- posite effects may be present, or that the slice incubation system is not sensitive to the serums tested. Another pos- sibility to explain the lack of stimulatory effects of serum on 14 hour post-hepatectomy Slices is that the control point S9 for DNA synthesis may occur earlier in time than 16 hours post-hepatectomy and that liver is sensitive to factors only at this earlier time. If serum is required for DNA synthesis to occur and does act at 14 hours post-hepatectomy, it may be that slice preparation had destabilized other factors needed for DNA synthesis and thus, serum would ap- pear not to have a role in stimulating DNA synthesis at this time as judged by 13 vltgg assay of serum effects on slice DNA synthesis. Of course, one may also theorize that fac- tors controlling liver DNA synthesis are present in plasma but are removed or inactivated during blood clotting. This would explain the lack of effect of the serums on DNA syn- thesis. To test this,one would like to use plasma in the incubation but problems exist with this approach. To prevent clotting, either calcium must be removed from the medium, a condition judged not to be physiological and thus not ac- ceptable, or heparin would have to be added to the medium. Heparin, however, is known to stimulate DNA synthesis both 1 vivo and in isolated nuclei.]7’3O The inhibitory effect of dialyzed calf serum was puz- zling in view of the lack of effect of calf serum in 3H-dThd incorporation into DNA. Perhaps low molecular weight stimu- latory factorsn’29 were removed from the serum by the dialysis, allowing inhibitory factors of high molecular weight13 to decrease the incorporation of 3H-dThd into DNA. It is also possible that the inhibition was due to factors introduced during the dialysis. 60 Verly33 has reported that a factor in a liver homogenate can inhibit incorporation of 3H-dThd into DNA in a two hour incubation of liver slices from rats partially hepatecto- mized 24 hours earlier. He believes that this factor circu— lates in the blood and inhibits liver cell DNA synthesis when it binds to the liver. One wonders whether his inhi- bitory factor is a physiological regulator of hepatic DNA synthesis because liver cells in Go appear to be blocked before the S phase rather than in its middle since DNA con- 2 Since most of tent is always an even multiple of two. the cells committed to DNA synthesis have begun to make DNA before 24 hours, it seems that this inhibitor isolated by Verly is acting at a phase that is not normally inhi- bited lg 1112. Unless this factor can block the onset of DNA synthesis in liver cells, one must question the bio- logical significance of Verly's33 observations. 4.5 N-2-Fluorenylacetamide Effects on Slice DNA Synthesis: Among the changes in liver resulting from the administration Of FAA is an altered control of growth in the liver. Two components of growth in normal slices that have been observed in this study are the pattern of incorporation of 3H—dThd into DNA and the critical period seen at about 16 hours post- hepatectomy. An alteration in either of these aspects of DNA synthesis in livers of rats exposed to FAA might indi- cate that that aspect of DNA synthesis is important in the control of cell division and would suggest that a change in 61 that factor is important in the mechanism of chemical car- cinogenesis due to FAA. Although the study of FAA effects on liver slice DNA synthesis is not complete, the results depicted in Figures 11 and 12 suggest that the change in certain liver properties at around 16 hours post-hepatectomy that allows a wave of DNA synthesis to occur 1p 11352 still exists and occurs at the same time as in normal slices, or that there is a decreased response to partial hepatectomy in FAA-treated rats.49 The pattern of incorporation of 3H-dThd into DNA indicates that an asynchronous wave of DNA synthesis occurs in Figure 11. Of course, it is always possible that the use of 3H-dThd as a precursor for DNA in FAA treated liver does nOt reflect the real rate of DNA synthesis. It may also be possible that the alteration of the pattern of DNA synthesis in FAA treated liver slices is not due to the carcinogenic actions of FAA but rather to the hepatotoxic effects of this chemical.50 If so, then hepa- totoxic chemicals such as chloroform5i should be able to reproduce this pattern. One piece of evidence suggesting that the altered pattern of 3H-dThd incorporation into DNA in liver slices may be a significant change is that female rats treated with FAA and partially hepatectomized Show a similar pattern of incorporation of 3H-dThd into DNA 1__vivo 49 to that seen in Figure 11. Caution in interpreting these results is needed because female rats are less sensitive to the hepatocarcinogenic effects of FAA than the male rats used here to make the liver slices.52 62 4.6 Significance: It seems possible that the liver slice incubation system will be an important tool in the study of the control of cell division. It can be used as a bioassay in the search for factors active in various phases of liver regeneration. Because the control of liver growth is al- tered by the administration of hepatic carcinogens, it is reasonable that alterations in the control of cell division induced by these chemicals may be detected in this system before the gross appearance of cancer. Should this be the case, then the liver slice incubation system would have po- tential as a screen for hepatic carcinogens and as a tool to aid in the understanding of the mechanism(s) of action of hepatic carcinogens. SUMMARY Liver DNA synthesis in the right lateral lobe, as esti- mated by the incorporation of 3H-dThd into DNA, began at some time between 12 and 22 hours after 70% partial hepa- tectomy and peaked at about 26 hours. When regenerating slices were made from this lobe and incubated in 11313 be- ginning at 16 hours post-hepatectomy, there was a wave of DNA synthesis which peaked 10 hours after the start of the incubation; i.e., at 26 hours after partial hepatectomy. Autoradiography of slices in one experiment at this time indicated a peak of 2.65% labeled nuclei and thus, an esti- mated 4.3% of hepatocytes, a fairly good reflection of the percentage of labeled hepatocytes that participate in DNA synthesis in regenerating liver 1 vivo. These Observations suggest that liver slices prepared from the right lateral lobe of rats sacrificed at 16 hours post-hepatectomy can progress through the S phase of the cell cycle ifl.li££2 in- dependent of systemic influences and in a manner similar to that of the whole liver lobe i vivo. Slices made at 14 hours post-hepatectomy failed to pro- gress through a wave of DNA synthesis. This indicates that —l n vivo there is a critical period at around 16 hours after partial hepatectomy when certain properties of the liver Change so that slices made after this time can progress through a wave of DNA synthesis in_vitro. It is possible that this critical period, corresponding to a time near the 63 64 beginning of DNA synthesis In vivo, may represent a control point in the regulation of cell division in the liver. Although estimates of DNA synthesis obtained with the use of 14C-formate and 3H-thymidine were Similar 1 vivo, this was not true in 11359. In contrast to a pattern of in- corporation of 3H-dThd into slice DNA which was related to the time of sacrifice of a rat, 14C-formate incorporation into DNA was similar in slices made from rats sacrificed at any time tested and appeared to be dependent only on the length of the incubation. This may be because the capacity of formate to be incorporated into the pool of proximate DNA precursors is a limiting factor in the incorporation of 14C-formate into DNA Of regenerating liver slices, rather than DNA polymerase activity. To begin an examination of the effects of blood-borne factors on liver DNA synthesis, the effects of various serums on regenerating liver slice DNA synthesis were evaluated. Calf serum, fetal calf serum, and serum from rats subjected to partial hepatectomy 18 hours earlier did not appear to alter DNA synthesis in regenerating liver slices at any time tested. In contrast, dialyzed calf serum decreased slice DNA synthesis. One possibility suggested to explain these ob- servations was that various opposing factors were present in whole serum and that dialyzed serum contained predominantly inhibitory ones. Aspects of regenerating liver slice DNA synthesis im- portant in the control of normal liver cell division were 65 altered by prior exposure of the liver to FAA i vivo. Rats were fed a diet containing 0.05% FAA for 14 days followed by a control diet for 7 days or 30 days. In one experiment, DNA synthesis in regenerating liver slices made from rats treated in this manner and incubated beginning 16 hours post-hepatectomy was somewhat lower than normal and con- tinually increased. This suggests that an asychronous DNA synthesis occurred in those slices. In another experiment, the rate of DNA synthesis appeared to be very low and simi— lar to that seen in normal liver slices incubated beginning at 14 hours post-hepatectomy. This suggests that the change in certain liver properties at around 16 hours post-hepa- tectomy that allows a wave of DNA synthesis to occur lg 111:9 still exists in livers treated with FAA or that these livers have a decreased response to partial hepatectomy. 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