Biochemical and Pathological Effects of Methylazoxymethanol Acetate, a Potent Carcinogen1

[CANCER RESEARCH 30, 801-812, March 1970]

Morris S. Zedeck, Stephen S. Sternberg, Richard W. Poynter, and Jane McGowan Divisions of Pharmacology and Cytology, Sloan-Kettering Institute for Cancer Research, New York, New York 10021

SUMMARY vealed that the agent is cleaved by the intestinal flora which possess a 0-o-glucosidase, and that the free agly Methylazoxymethanol acetate is known to induce tu cone methylazoxymethanol is the active carcinogen (13- mors in rodents. Effects produced by a single nonlethal 15, 34). Matsumoto et al. (19) synthesized MAM acetate3 dose on nucleic acid and protein synthesis have been in (Chart 1), which is stable and not dependent upon vestigated in rat and mouse liver, small intestine, and ,0-glucosidase cleavage for activity. Single or only a few kidney, the 3 organs most susceptible to carcinogenesis. doses of cycasin, or of the aglycone methylazoxymetha The agent induced early inhibition of thymidine incor nol, are required to induce tumors in rats which are pri poration into DNA of rat tissues. Rat liver was most sen marily of the kidney, intestinal tract, and liver (11, 15). sitive to this effect and was the only organ to show marked Aqueous extracts of cycad nut have also been shown to inhibition of RNA and protein synthesis. In mice, liver induce tumors of liver and kidneys in mice (21). The was more sensitive than small intestine or kidney and ex present investigation was undertaken to study the acute hibited an inhibition of RNA and protein synthesis; there pathological and biochemical effects produced by MAM were no changes observed in small intestine or kidney. acetate in the kidney, small intestine, and liver of mice Pathological effects of methylazoxymethanol acetate have and rats. It was hoped that the information derived from been studied by light and electron microscopy. In rats, this study might help to elucidate the initial action of the 6 hr after treatment there was minimal focal necrosis in carcinogen which is requisite for tumor formation. liver; duodenum and colon, however, showed marked karyorrhexis in crypt epithelium. Intestinal recovery was MATERIALS AND METHODS evident by 1 week, whereas hepatic nuclear changes, MAM acetate was purchased from Mann Research consisting of enlarged and irregular nuclei, were detect Laboratories, Inc., New York, N. Y. Thymidine-2-14C able months later. Microsegregation in rat hepatic cell (53.8 mCi/mmole), uridine-6-'H (9.34 Ci/mmole), L-leu- nucleoli was evident within 1 hr after treatment, prior to cine-l-uC (25.6 mCi/mmole), cytidine-5-3H (26.2 Ci/ inhibition of nuclear RNA synthesis; the nucleolar change mmole), and orotic acid-6-HC hydrate (4.6 mCi/mmole) persisted for several months. Mouse liver showed necrosis were obtained from New England Nuclear Corporation, while duodenal changes were minimal. Kidneys of both Boston, Mass.; uridine-6-'H (10.4 Ci/mmole) was from species appeared normal at all times. The relationships Tracerlab, Waltham, Mass.; and o-phosphoric acid-t2P between inhibition of the synthesis of the various macro- (1 mCi/ml) was from E. R. Squibb and Sons, New York, molecules, the pathological effects, and carcinogenesis N. Y. Immature weanling (50- to 70-g) and young adult are discussed. (150-g) male Sprague-Dawley rats (CD line) and adult male CD1 mice (25 g) were obtained from Charles River INTRODUCTION Breeding Laboratories, Brookline, Mass., and given food and water ad libitum. In 1963 Laqueur et al. (16) reported that rats fed flour All solutions for injection were made with 0.9% NaCl prepared from the nuts of the plant Cycas circinalis de solution and administered i.v. either by tail vein or fem veloped benign and malignant tumors predominantly of oral vein, in a volume of 10 ml/kg. Solutions of MAM the liver and kidneys. Nishida et al. (20) and Riggs (27) acetate were prepared immediately before use. Animals had previously isolated from cycads a glucoside termed given injections of NaCl solution served as controls in all cycasin and had reported that the aglycone moiety experiments. The animals were killed by either cervical methylazoxymethanol is linked to glucose in the /3-Dcon dislocation (mice) or exsanguination from the abdominal figuration. Experiments in which cycasin was given by aorta while under ether anesthesia (rats). various routes to normal and to germ-free animals re- ' Aided by Grant CA 08748 from the National Cancer Institute, Precursor Incorporation Studies USPHS. Presented in part at the 60th Annual Meeting of the American The radiolabeled precursors used to measure the syn Association for Cancer Research, March 23 to 25, 1969. ' Present address: Department of Experimental Pathology and thesis of nucleic acids and protein were injected at vari- Cancer Research, University of Leeds, Leeds 2, England. 'The abbreviations used are: MAM acetate, methylazoxymethanol Received June 23, 1969; accepted August 25, 1969. acetate; TCA, trichloroacetic acid.

MARCH 1970 801

0 cally with diphenylamine and orcinol as reagents, respec t tively (30). Deoxyadenosine and adenosine were used as CH3-N=l\l-CH2-0-Acetyl standards for DNA and RNA determinations, respec tively. Protein content was determined according to the Methylazoxymethanolacetate method of Lowry et al. (17), with bovine serum albumin as the standard. The results are expressed as dpm/^mole deoxyribose, dpm/Mmole ribose, or dpm/mg protein MAIVIacetate for incorporation of thymidine-2-14C or orthophosphoric Chart 1. The structural formula of methylazoxymethanol acetate. acid-:i'P into DNA, uridine-6-:!H, orotic acid-6-uC hy drate, or cytidine-5-'H into RNA and leucine-l-uC into ous intervals of time after the administration of MAM protein, respectively. Since only the purine ribose or de acetate (see the tables and charts for details). After an oxyribose moieties give colorimetrie reactions, the results interval was allowed for precursor incorporation (the in have been adjusted to include the pyrimidine bases. corporation of each precursor was linear during the pulse time used), the organs were rapidly removed, placed in ice-cold water, where they were trimmed, and then Pathology Studies homogenized in iced water with a VirTis homogenizer, and a measured portion of homogenate was added to Light Microscopy. Weanling and young adult rats were TCA, to a final concentration of 10%. The acid-insoluble given a single injection of MAM acetate in a dose of 35 or pellet was washed several times with 5% TCA and de 50 mg/kg, respectively. Approximately equal numbers of fatted once with absolute ethanol and 3 times with eth- each age group were killed at varying intervals: a total of anol: ether (3:1). The pellet was heated twice at 90Â°for 4 at 1 hr, 10 at 6 hr, 8 at 24 hr, 6 at 1 week, 6 at 1 month, 15 min with 5% TCA, and the resulting supernatants, 4 at 2 months, and 4 at 6 to 7 months. Tissues were im mediately fixed in Bouin's solution and paraffin sections containing degraded nucleic acids, were combined for measurement of radioactivity and of nucleic acid content. were stained with hematoxylin and eosin. In the older When protein synthesis was measured with leucine-l-14C, rats given the higher dose all thoracic and abdominal the pellet which remained after the 2 acid-heat treatments viscera, salivary glands, sternum, thyroid, testes, and as above was dissolved in 0.5 N NaOH and the digest was skeletal muscle were examined microscopically, while in analyzed for protein content and radioactivity. The above the weanling rats only liver, duodenum, colon, kidney. procedure is similar to the method of Schneider (30). Nuclei from weanling rat liver and kidney and from 600r mouse liver were isolated by homogenizing the tissue in 4 volumes ice-cold 0.25 Msucrose with a Potter-Elvehjem TdR-2-C->DNA os smooth-walled glass tube fitted with a Teflon pestle. The Â£ 400 pestle was driven downward once (about 12,000 rpm) I into the tube and withdrawn (at 6,000 rpm); the homoge nate was then centrifuged at 1,000 X g for 10 min. The 200- precipitate was twice resuspended in 0.25 M sucrose and (6) recentrifuged at 600 X g for 10 min (4). To the final pellet W) I was added 10% TCA to precipitate the nucleic acids and o proteins. The pellet was washed with acid and defatted, 270 L-Leucine-l-'tâ€”Â»Protein and the DNA and RNA were separated according to the method of Schmidt-Thannhauser (29). DNA and RNA 180 determinations revealed a recovery of 50 to 60% of DNA Dl for liver and kidney with a RNA:DNA ratio of 0.28. 90 Occasionally, the nuclei were further purified by spinning (5)1'S(4)1er<Â»1IBI,/ them through 2.4 M sucrose containing 0.001 M MgCl2 1 481 " ,, 72 1 for 1 hr at 50,000 X g in a Spinco SW 40 rotor (38). 3 6 24 Samples of acid-containing fractions were added to Hours after MAM acetate liquid scintillator which consisted of 0.52 g POPOP, 26 g Chart 2. The effects of MAM acetate, 35 mg/kg, upon DNA and PPO, 416 g naphthalene, 1.2 liters methanol, 2 liters protein synthesis in weanling rat liver. At various times after treatment. toluene, and 2 liters dioxane (10). Samples of alkali- thymidine-2-'4C (TdR-2-"C) (21 â€žCi/5â€žmoles/kg)or leucine-l-MC digested protein were added to liquid scintillator which (25 ^Ci/50 Â¿imoles/kg)was injected i.v. After a 15- or 10-min pulse, consisted of 0.1 g POPOP, 8 g PPO, 0.6 liter absolute respectively, the animals were sacrificed and the DNA and protein spe cific activity was determined. The numbers in parentheses represent ethanol, and 1.4 liters toluene. The radioactivity was de the number of animals studied at that time. The shaded area represents termined in a Packard Tri-Carb liquid scintillation spec the mean Â±1 S.D. of all controls given injections of NaCl solution and trometer. is representative of the controls at any interval of time measured. The DNA and RNA content of the respective fractions Vertical bars, mean Â±1 S.D. of the treated animals for n values greater obtained as described above was determined colorimetri- than 2, and mean Â±range for n values of 2.

802 CANCER RESEARCH VOL. 30

and testes were examined. Fifteen control rats were each Orotic-6- C Acid Hydrate â€”RNA given 1 injection of NaCl solution, and 1 or more of these 10,000 were killed at each of the above indicated time intervals. Electron Microscopy. The ultrastructural studies re 8000 ported below were concerned primarily with nuclear changes in the hepatocytes of rats within the first 24 hr after treatment with MAM acetate, although a few pre

liminary observations were made at 1 and 2 months after 54000 (2) injection. As in the light microscopic studies both wean (2) ling and young adult male rats received a single injection of either 35 or 50 mg/kg, respectively. No differences E 2000 were observed with regard to age at time of injection or o1â€” to dose. Eight rats were examined at 6 hr and 6 rats at 24 hr after treatment. In addition, 4 rats were killed 2000r within the 1st hr; 1 each at 15 and 30 min and 2 rats at (2) 1 hr. Controls given injections of NaCl solution were also ÃŒS\j(6)i

examined: 1 at 30 min, 2 at 6 hr, 4 at 24 hr, and 1 each at I(2)lili31234â€”^('Â¿i151 1 and 2 months. Sections of the left lateral lobe of liver \\61 //124 were fixed by immersion at room temperature for 1 hr in 41 a mixture of 6% glutaraldehyde and 2% acrolein (28) and Hours after MAM ocelote postfixed for 1 hr in 2% osmium tetroxide (22) with added Chart 3. The effects of MAM acetate, 35 mg/kg. upon RNA synthe sucrose (3). Both solutions were buffered at pH 7.2 with sis in weanling rat liver (above) and kidney nuclei (below). At various Veronal acetate. Dehydration was carried out through a times after MAM acetate, orotic acid-6-"C hydrate (10 ^Ci/2.15 graded series of alcohols and propylene oxide. Specimens ^moles/kg) was injected, and, after a 20-min pulse, the nuclei were were embedded in Maraglas-Dow epoxy resin 732 (5). extracted and the specific activity of the RNA was determined as de Sections were stained with methanolic uranyl acetate (35) scribed. See Chart 2 for other details. and with lead citrate (25) and were examined in a Sie mens Elmiskop I electron microscope. appeared to follow more closely the inhibition of RNA synthesis in that it too began to recover 24 hr after treat ment. RESULTS The effects of MAM acetate upon nucleic acid and protein synthesis in small intestine and kidney were not Toxicity as marked as that in liver. Thymidine incorporation in weanling rat small intestine (Chart 4) and kidney (Chart 5) There was a narrow range between the dose of MAM was inhibited about 25 and 40%, respectively, although acetate when given i.v., which was lethal within 48 hr, the inhibitions appeared to last longer than that in liver. Inhibition of RNA synthesis in small intestine (Chart 4), and that which permitted normal survival. Whereas a approximately 30%, was detectable with uridine-6-:!H dose of 35 mg/kg in the mouse was lethal (5/5), 25 mg/kg while RNA synthesis in kidney nuclei using orotic acid- produced no deaths (0/9). Similar narrow ranges were 6-'4C hydrate was not affected even as late as 24 hr after observed with rats. The following doses, which produced no deaths, were used for both biochemical and patho MAM acetate (Chart 3). Protein synthesis was not af logical studies: adult rat, 50 mg/kg; weanling rat, 35 fected in either small intestine (Chart 4) or kidney (Chart 5). mg/kg; adult mouse, 25 mg/kg. To ascertain whether the inhibition of thymidine-2-14C incorporation into DNA was representative of de novo Precursor Incorporation Studies synthesis inhibition and not an artifact of thymidine metabolism, orthophosphoric acid-'"P incorporation into In weanling rat liver (Chart 2), thymidine-2-14C in DNA of weanling rat liver nuclei was determined. The corporation into DNA was inhibited 35% 1 hr after treat data in Table 1 indicate that DNA synthesis in liver was ment, and 65% from the 3rd to the 24th hr. The ability inhibited about 80% 1 to 3 hr after MAM acetate, a find to synthesize DNA recovered thereafter. RNA synthesis, ing in agreement with the data obtained using thymidine. however, measured by determining the incorporation of The lack of inhibition of RNA synthesis in kidney nu orotic acid-6-uC hydrate into nuclear RNA (Chart 3), clei as determined by orotic acid incorporation may be was not markedly affected until 3 hr after MAM acetate due to the poorer utilization of this precursor by this tis was given and was completely recovered by 24 hr (orotic sue as compared to liver (Chart 3). Therefore, nuclear acid was used as a precursor of RNA synthesis in liver RNA synthesis in kidney was measured with cytidine- and kidney because of the poor utilization of uridine for 5-'H. The data indicated that, as observed with orotic RNA synthesis by these organs). The inhibition of leu- acid, there was no inhibition of RNA synthesis 5 hr after cine-l-14C incorporation into protein, about 60% (Chart 2), MAM acetate, at a time when inhibition of DNA synthe-

MARCH 1970 803

S 400r sis was maximal. Cytidine incorporation into liver nuclear TdR-2-t â€”> DNA RNA was inhibited, however, a result in agreement with that obtained with orotic acid (Table 1). The incorporation of labeled precursors into their re 200 spective macromolecules was also determined in adult rats. The data show (Table 2) that MAM acetate exerts a similar effect in adult rats as in weanling rats. To ascer tain whether the adrenal gland played a role in the effects 140r L- Leuane-l-HC â€”Â»Protein produced by MAM acetate, thymidine-2-14C incorpora tion into DNA was measured in adrenalectomized female 2 weanling rats. (Animals were allowed NaCl solution and food ad libitum for 3 days prior to the experiment.) Six I 70 hr after MAM acetate, 50 mg/kg, thymidine incorpora tion into DNA of liver, small intestine, and kidney was inhibited 87, 45, and 66% respectively, results which are 3 6 24 48 72 similar to those obtained in intact animals. Hours after MAM acetate The effects of 25 mg/kg MAM acetate upon the syn Chart 5. The effect of MAM acetate, 35 mg/kg, upon DNA and thesis of nucleic acids and protein in mouse tissues were protein synthesis in weanling rat kidney. See Chart 2 for details. also investigated. There appears to be a slight inhibition of thymidine-2-14C incorporation into mouse liver DNA 24 hr after MAM acetate, whereas the effects upon uri- Pathology Studies dine-6-'H and leucine-l-14C incorporation appear earlier and have returned to normal at that time (Chart 6). Orotic Light Microscopy. Since the pathological effects ob acid-6-'4C hydrate incorporation into mouse liver nuclei served were similar in the weanling and young adult rats, was also determined. There was 52 and 18% inhibition at both groups are described together. No abnormalities 2 and 5 hr, respectively. The synthesis of nucleic acids were present in rats 1 hr after receiving MAM acetate. and protein in the small intestine and kidney of the mouse At 6 hr there was decreased cytoplasmic basophilia of the was not affected as late as 24 hr after the administration hepatocytes in all rats. Abnormal nuclei were observed in of MAM acetate. scattered hepatocytes in 9 of 10 rats. The abnormal nuclei contained irregular clear areas with marginated chromatin (Figs. 1 and 2). Only a minority of cells were involved. In addition, karyorrhectic material was present in sinusoids and Kupffer cells. The intestinal crypts in duodenum and colon contained numerous karyorrhectic cells (Fig. 6): the change was more marked in the duodenum. All other tissues were normal. At 24 hr all rats showed liver changes similar to those described at 6 hr. In addition, the liver of 2 of the 8 rats showed areas of congestion and hemorrhage; 1 of these contained focal collections of lymphocytes and polymorphonuclear leukocytes. The intestinal crypts in duo denum and colon contained only an occasional necrotic cell. The crypt nuclei, however, were no longer evenly aligned and many were enlarged and irregularly shaped. The colon was less affected than the duodenum. Seven days after treatment the hepatic cells showed greater variation in size of nuclei and density of chro matin pattern than did controls (Fig. 3). Many hepatic cells had enlarged clear areas in the cytoplasm principally in the perinuclear region (found to be glycogen by elec 0 1 3 6 24 48 72 Hours after MAM acetate tron microscopy). In 3 of the 6 rats many hepatic cells showed significant irregularities in size and shape with Chart 4. The effect of MAM acetate, 35 mg/kg, upon DNA, RNA. the result that the normal sinusoidal pattern was dis and protein synthesis in the weanling rat small intestine. At various times after treatment with MAM acetate. thymidine-2-MC (TdR-2-{'C) torted. The duodenum of 2 of the rats showed increased (21 MCi/5 â€žmoles/kg),uridine-o-'H (UR-6-'H) (500 MCi/25 â€žmoles/kg), numbers of inflammatory cells (mainly lymphocytes and or leucine-l-"C (25 ^Ci/50 ^moles/kg) was injected for 15-, 10-, or a few polymorphonuclear leukocytes) in the lamina 10-min pulses, respectively. The specific activities were determined as propria and ectasia of crypts. Similar changes were pres described under "Materials and Methods." See Chart 2 for other ent in the colon of 4 rats. In 2 animals there was a de details. crease in hematopoietic elements in the red pulp of the

804 CANCER RESEARCH VOL. 30

spleen and a slight loss of corticomedullary demarcation In a limited study with mice, selected tissues were in the thymus. studied at 6 and 24 hr following a single administration At 1 month, all treated rats had nuclear and cytoplas- of 25 mg/kg MAM acetate. In mice killed at 6 hr, di mic alterations in the liver like those noted at 7 days. minished cytoplasmic basophilia was present in central There was, however, greater variation in nuclear size and zone hepatocytes; in addition, a few necrotic hepatocytes an increase in the number of enlarged nuclei. Cells with were seen immediately adjacent to central veins in increased glycogen deposits occurred in clusters. In 1 nearly all central zones. In the duodenum, a few karyor- rat some of the hepatic cells were also enlarged. Two rats rhectic crypt cells were present in a minority of crypts. had testicular damage consisting of a few enlarged and The kidneys were normal. Of 4 mice killed at 24 hr, 2 multinucleated germinal epithelial cells in some seminif had central necrosis in liver, involving one-fourth to one- erous tubules. The livers of the 8 rats killed at 2, 6, or 7 half of each lobule. In the other 2 mice, only a few ne months had changes in hepatocytes like those described crotic cells were present around central veins in all above at 1 month, but with variations in severity that lobules. In the duodenum of the 24-hr mice a few kary- appeared in this small series to be independent of time. orrhectic cells were present in the majority of crypts. For example, the most abnormal hepatocytes were ob The kidneys were normal. served in 2 rats at 2 months (Fig. 4). At 6 and 7 months Electron Microscopy. While this study was in progress, after receiving MAM acetate, 1 rat had hyperplastic GanÃ³te and Rosenthal (7) described hepatic changes in nodules of the liver and an infiltrating adenocarcinoma young male Osborne-Mendel rats that had received 60 to of the duodenum, another had an in situ adenocarcinoma 110 mg/kg MAM acetate i.p. and had been killed at 2, of the duodenum, and a 3rd had hyperplastic nodules 6, 12, and 24 hr. Our studies, confirming and extending of the liver (Fig. 5). theirs, are summarized below. The kidney did not show any pathological changes or As early as 15 min, and persisting until 24 hr, most preneoplastic alterations in any of the 42 rats examined. hepatocyte nucleoli contained nucleolar plaques. These were round discrete, electron-dense bodies composed of Table 1 fibrillar or granular material (Figs. 7, 8, and 9). Similar The effect of MAM acetate upon the synthesis of DNA structures have been described as "spotted" nucleoli in and RNA in the weanling raÃliver Orthophosphoric acid-12? (1 mCi/kg), or cytidine-5-r'H (1 mCi/25 tumor cells (2) and as nucleolar plaques (26) or micro- ^mole/kg) was injected i.v. into weanling rats at various intervals of spherules (37) following administration of actinomycin D. time after receiving either 0.9% NaCl solution or MAM acetate, 35 mg/kg. At the end of the pulse time the animals were sacrificed and At 15 and 30 min in the present studies, the presence their livers were removed. The nuclei were separated and the DNA of plaques was the only nucleolar change. At 1 hr there and RNA were extracted as discussed in "Materials and Methods." was, in addition, an irregular separation of nucleolar Aliquots of the extracts were taken for radioactivity determination and components with loss of skein-like arrangement (Fig. 8). the results are expressed as dpm/fimole deoxyribose or ribose for At 6 hr, segregation of nucleolar components appeared DNA and RNA. respectively. The values represent the mean of data obtained from at least 2 animals for each treatment. (Fig. 9). Some nucleoli at this time had a reticulated pat tern composed of fibrillar and granular components (Fig. Time after Pulse time Control Treated % of control 10). At 24 hr some nucleoli consisted of a central granular MAM (hr) (hr) mass with peripheral extensions of fibrillar material (Fig. Orthophosphoric acid-'2P incorporation into DNA 11). At 1 hr and thereafter, the nucleolar alterations were present in less than one-half of the hepatocytes. 0.5 1 407 151 37 1.0 2 1054 130 12 Occasional necrotic cells were present at 6 and 24 hr 1.0 2 1040 256 25 (see "Results, Light Microscopy"). These were char Cytidine-5-'H incorporation into RNA (specific activity X 10 ') acterized by finely granular homogenous nucleoplasm 5 0.25 142 45 32 with a few varied electron-dense structures, not other-

Table2 The effect of MAM acetate upon nucleic acid and protein svnlhesis in the adult rat At various intervals of time after MAM acetate, 50 mg/kg, the rats were given injections of either thymidine-2-l4C (10 ^Ci/2 ^moles/kg), orotic acid-6-MC hydrate (10 MCi/2.15 ^moles/kg), or leucine-l-'4C (25 MCi/50 Mmoles/kg), and after 15-, 20-, and 10-min pulses, respec tively, the animals were sacrificed. The tissues were removed and treated as described under "Materials and Methods." DNA and protein syn thesis were measured in whole cell homogenates; RNA synthesis was determined in nuclear preparations. The results are expressed as dpm/^mole deoxyribose. dpm/^mole ribose, or dpm/mg protein for DNA, RNA, and protein, respectively. The values represent the mean of data obtained from at least 2 animals for each treatment. LiverPrecursor intestine% MAMporationincor- Time after ofcontrol104453135Control747837298Treated653490235%ofcontrol875979KidneyControl7971112Treated8832112%nfcontrol11145100 (hr)ThvmidineOrotic acetate

DNA-.

acidLeucineRNAprotein1656Control95994554164Treated9945141757Small â€”â€¢-.

MARCH 1970 805

acetate to induce tumors in rats has been reported by others (14, 15), and we have observed the development of adenocarcinomas of the rat duodenum and hyperplastic nodules of the rat liver 6 to 7 months after a sin gle administration of this agent. In mice, however, we found no inhibition of DNA synthesis, and there have been no reports of tumors in this species following MAM acetate. In our laboratory, we have not observed tumors in mice as late as 1 year after a single i.v. administra tion of the agent. The results suggest, therefore, that the initial actions of MAM acetate which result in early inhibition of DNA synthesis may also be responsible for nuclear changes in rat liver which persisted for several months after treatment, and for the subsequent develop ment of tumors. Whether the cells showing late nuclear changes are those which were originally affected or cells in which the response was delayed is difficult to say at this time. DNA synthesis in rat kidney was less sensitive to the inhibitory effects of MAM acetate than was that of liver I and did not show any pathological effects throughout 3 6 the course of this study. Tumors which have been ob Hours after MAM acetate served in rat kidney (15) were the result of multiple in Chart 6. The effect of MAM acetate. 25 mg/kg, upon DNA. RNA, jections of MAM acetate. and protein synthesis in mouse liver. At various intervals after MAM acetate. thymidine-2-"C (TdK-2-l'C) (100 â€žCi/20.6 ^moles/kg), The inhibition of DNA and RNA synthesis in rat liver uridine-6-:'H (UR-6-'H) (260 ^Ci/100 ,/moles/kg), or leucine-l-14C may be the result of a change in DNA template activity (50 fiCi/100 ^moles/kg) was injected for 15-, 10-, or 10-min pulses, because of methylation of the guanine moieties in DNA respectively. The specific activity of each macromolecule was deter by MAM acetate (18, 33) with the subsequent loss of mined as described. See Chart 2 for other details. both DNA and RNA polymerase activity. It is also pos sible that there exist separate mechanisms of action upon wise identifiable. Chromatin was marginateci at the nu the synthesis of each macromolecule. clear envelop (Fig. 12). These changes correlate well The lesser effects of MAM acetate upon the synthesis with the light microscopic observations of clear nuclei of DNA in rat small intestine and kidney as compared described above (Fig. 2). Whorls of smooth endoplasmic to liver and the finding that similar degrees of inhibition reticulum were noted in hepatocytes 24 hr after treat of the synthesis of DNA in the 2 former tissues are as ment; these changes were similar to those described by sociated with different effects upon RNA synthesis may GanÃ³teand Rosenthal (7). be a reflection of less uptake of the carcinogen by these Preliminary observations of 4 rats, 2 killed at 1 and 2 tissues, lower amounts of methylation of the nucleic acids, at 2 months after treatment, confirmed the findings noted or different sensitivities of the polymerases to the methy by light microscopy, i.e., nuclear enlargement, irregular lated DNA (18, 33). It has been observed that RNA of chromatin patterns, and increased amounts of glycogen liver is methylated to a greater extent after cycasin than deposits in cytoplasm in some cells. In the 2 rats killed is that of kidney or ileum (33) and the same may be true at 2 months hepatocytes were noted which contained for DNA. The different rates of proliferation between structures considered to be abnormal nucleoli. Some of the small intestine and kidney may also be a factor. Al the nuclei containing these bodies also had in addition though the inhibition of thymidine incorporation is less a normal or an enlarged nucleolus. The ring-like abnor pronounced in small intestine and kidney than that pro mal structures were composed of granular material con duced in liver, it is of a longer duration. taining vacuoles (Figs. 13, 14, and 15). In one instance The liver, which is less proliferative than small intes a fibrillar mass formed a cap around the periphery (Fig. tine, was more sensitive to inhibition of DNA synthesis 13). by MAM acetate. We have explored this phenomenon somewhat further and have found that 3 hr after MAM acetate, at a time when liver DNA synthesis was in DISCUSSION hibited 70%, the inhibition of DNA synthesis in thymus, spleen, and testis was 50%. These last-mentioned tissues In the present studies, inhibition of DNA synthesis showed no pathological effects. (as measured by changes in thymidine and orthophos- In both small intestine and kidney, however, in con phate incorporation) was observed in rat liver, small in trast to liver, no effect upon leucine incorporation into testine, and kidney within the first few hr following the protein could be observed. Shank and Magee (33) have administration of MAM acetate. The ability of MAM reported that cycasin inhibits leucine incorporation into

806 CANCER RESEARCH VOL. 30

protein of liver but not of kidney or ileum, data which and MAM acetate, as well as noncarcinogenic actinomy- are similar to those obtained in these experiments with cin D, bind to DNA has suggested that the alteration MAM acetate. Since the inhibition by cycasin began 5 of nucleolar structure and inhibition of RNA synthesis hr after administration, they postulated that metabolism are related to an effect upon the DNA template (8). Be of cycasin to MAM was responsible for the delay in on cause of the temporal differences in onset and in re set. The earlier effect by MAM acetate on leucine in covery following MAM acetate, it may be possible to de corporation into liver protein in these experiments is con termine which of the 2 phenomena is more sensitive to sistent with their hypothesis. alteration of the DNA template. Furthermore, the pres The inhibition of protein synthesis in rat liver may be ence of nuclear abnormalities in liver which persist for due to methylation of RNA (18, 33), resulting in a tem months after a single injection of MAM acetate may per plate which is less efficient to direct the synthesis of mit the study of the process of carcinogenesis within protein or because of disaggregation of polysomes and single cells from the time of administration of the car depletion of rough endoplasmic reticulum which have cinogen to the eventual appearance of neoplasia. Such been observed ultrastructurally by GanÃ³te and Rosenthal studies are currently under investigation. It is also of in (7) and in our laboratory as well. Furthermore, polysome terest that retrorsine, a senecio alkaloid, is capable of dissociation into monomer and dirtier units has been re producing carcinogenic and nuclear effects similar to those ported following the administration of MAM and produced by MAM acetate after a single injection (1). cycasin (32). The administration of aqueous extracts of Many of the present findings with MAM acetate have Macrozamia communis, a plant which contains the same also been observed in studies with dimethylnitrosamine, aglycone as present in cycasin, produces an inhibition of another potent carcinogen which is chemically similar to leucine incorporation in vitro in rat liver microsomal prep MAM acetate and has been postulated to react similarly arations (9). The addition of synthetic polyribonucleotide in vivo (33). Other carcinogens which are closely related to these microsomal preparations produced a stimulation to MAM acetate because of the presence of nitroso- or of amino acid incorporation into acid-insoluble material azoxy-constituents are streptozotocin (23) and elaiomycin (9). This suggests that fewer polysome units were pres (31), respectively, and each of these are naturally occur ent in treated animals and allowed for greater binding ring and capable of producing tumors upon single or few of synthetic template in vitro. administrations. The azoxy- or nitroso-containing agents The RNA:DNA ratio of weanling rat liver nuclei de are of great interest because of their potential hazard to creases 35% 5 hr after MAM acetate, at a time when man. Their marked potency, relatively simple chemical orotate incorporation into RNA appears to be maximally structures, and ease of administration make them unique inhibited. The ratio of RNA: DNA in kidney nuclei is tools for the study of carcinogenesis. not decreased. The lowered ratio in liver reflects either less RNA per nucleus, more DNA per nucleus, or a com bination of both. Hoch-Ligeti et al. (12) have found ADDENDUM similar data in rats 4 hr after feeding l g fresh cycad husk (equivalent to 2 mg cycasin); in agreement, we have found the lowered ratio to be due to less total RNA Since the acceptance of this manuscript, we have found that rats per nucleus. which receive a single i.v. injection of MAM acetate, 35 mg/kg, de Alterations in nucleolar structure have been observed velop tumors of the colon and kidney 6 to 9 months later. These tu with other hepatocarcinogens. Aflatoxin, 3'-methyldi- mors are in addition to the duodenal tumors and hyperplastic nodules mentioned in the text. The findings are consistent with the suggestion, methylaminoazobenzene, lasiocarpine, and tannic acid, as discussed above, that the rapidly developing, although transient, which produce macrosegregation (24, 36); and dimethyl- effects of MAM acetate which produce inhibition of DNA synthesis in nitrosamine, which produces microsegregation (36) of these organs may also be responsible for subsequent tumor formation. In nucleolar components, also inhibit RNA synthesis (6, 24, accord with this relationship is the recent finding of Hirono el al. (Can 39). The effects on RNA synthesis were studied, how cer Res, 29: 1658-1662, 1969) that cycasin-treated newborn mice de ever, at a time when nucleolar changes were already velop hepatomas predominantly, but few tumors of the intestine and present and the temporal relationship between inhibition kidney. In our studies (see text), mouse liver, but not duodenum and of synthesis and nucleolar segregation was not defined. kidney, was sensitive to induction of early biochemical and pathological Actinomycin D, which is not a hepatocarcinogen, in changes by MAM acetate. hibits RNA synthesis and simultaneously induces changes in nucleolar structure such as macrosegregation [nu cleolar capping (26)] in all the hepatocytes (8). The nu ACKNOWLEDGMENTS cleolar effects observed with MAM acetate occurred earlier than the decrease in RNA synthesis and per sisted for longer than 24 hr, by which time the synthesis We wish to thank Dr. Frederick S. Philips for his advice and co operation during the course of this study. Miss Maggie Byler for prepa of RNA had already recovered. In view of these findings, ration of the samples for electron microscopy, and Mr. John Hlinka and it would appear that the phenomena of RNA synthesis Mr. William Matz for the photomicrographs and electron micrographs, inhibition and nucleolar segregation are independent. respectively. During the course of this study. Miss Jane Sodergren also The fact that the hepatocarcinogens discussed above was very helpful.

MARCH 1970 807

REFERENCES Methylazoxymethanol, the Aglycone of Cycasin: A Synthesis of Methylazoxymethyl Acetate. Biochem. J., 95: 13c-14c, 1965. 20. Nishida, K., Kobayashi, A., and Nagahama, T. Studies on Cy casin, a New Toxic Glycoside, of Creas revoluta Thunb. Part I. 1. Afzelius, B. A., and Schoental, R. The Ultrastructure of the En Isolation and the Structure of Cycasin. Bull. Agr. Chem. Soc. Japan, larged Hepatocytes Induced in Rats with a Single Oral Dose of 19: 77-83, 1955. Retrorsine, a Pyrrolizidine (Senecio) Alkaloid. J. Ultrastruct. Res., 21. O'Gara, R. W., Brown, J. M., and Whiting, M. G. Induction of 20: 328-345, 1967. Hepatic and Renal Tumors by Topical Application of Aqueous Ex 2. Bernhard, W. Some Problems of Fine Structure in Tumor Cells. tract of Cycad Nut to Artificial Skin Ulcers in Mice. Federation Progr. Exptl. Tumor Res., 3: 1 34, 1963. Proc., 23: 1383, 1964. 3. Caulfield, J. B. Effects of Varying the Vehicle for OsO, in Tissue 22. Palade, G. E. A Study of Fixation for Electron Microscopy. J. Fixation. J. Biophys. Biochem. Cytol., 3: 827-830, 1957. Exptl. Med., 95: 285-298, 1952. 4. de Duve, C, Pressman, B. C., Gianetto. R., Wattiaux, R., and 23. Rakieten, N., Gordon, B. S., Cooney, D. A., Davis, R. D., and Appelmans, F. Tissue Fractionation Studies. 6. Intracellular Dis Schein, P. S. Renal Tumorigenic Action of Streptozotocin (NSC- tribution Patterns of Enzymes in Rat Liver Tissue. Biochem. J., 85998) in Rats. Cancer Chemotherapy Rept., 52: 563-567, 1968. 60: 604 617, 1955. 24. Reddy, J., and Svoboda, D. The Relationship of Nucleolar Segre 5. Erlandson, R. A. A New Maraglas. D.E.R. 732, Embedment for gation to Ribonucleic Acid Synthesis following the Administration Electron Microscopy. J. Cell Biol., 22: 704-709, 1964. of Selected Hepatocarcinogens. Lab. Invest., 19: 132-145, 1968. 6. Floyd, L. R., Unuma, T., and Busch, H. Effects of Aflatoxin B, and 25. Reynolds, E. S. The Use of Lead Citrate at High pH as an Elec Other Carcinogens upon Nucleolar RNA of Various Tissues in the tron-Opaque Stain in Electron Microscopy. J. Cell Biol., 17: 208 Rat. Exptl. Celt Res., 5/. 423 438, 1968. 212, 1963. 7. GanÃ³te, C. E., and Rosenthal, A. S. Characteristic Lesions of 26. Reynolds, R. C., Montgomery, P. O'B., and Hughes, B. Nucleolar Methylazoxymethanol-induced Liver Damage. A Comparative Ul- "Caps" Produced by Actinomycin D. Cancer Res., 24: 1269 trastructural Study with Dimethylnitrosamine, Hydrazine Sulfate, 1277, 1964. and Carbon Tetrachloride. Lab. Invest.. 19: 382 398, 1968. 27. Riggs. N. V. Glucosyloxyazoxymethane, a Constituent of the Seeds 8. Goldblatt, P. J., Sullivan, R. J., and Farber, E. Morphologic and of Cecas cirdnalis L. Chem. Ind. (London), 8: 926, 1956. Metabolic Alterations in Hepatic Cell Nucleoli Induced by 28. Sandborn, E., Koen, P. F., McNabb, J. D., and Moore, G. Cyto- Varying Doses of Actinomycin D. Cancer Res., 29: 124-135, 1969. plasmic Microtubules in Mammalian Cells. J. Ultrastruct. Res., 9. Healy, P. J. Studies on Poisoning by Macrozamia communisâ€”I. //: 123-138, 1964. Biochemical Disturbances in the Liver. Biochem. Pharmacol., 18: 29. Schmidt, G., and Thannhauser, S. J. A Method for the Determina 85 92, 1969. tion of Deoxyribonucleic Acid, Ribonucleic Acid, and Phospho- 10. Herberg, R. J. Determination of Carbon-14 and Tritium in Blood proteins in Animal Tissues. J. Biol. Chem., 161: 83-89, 1945. and Other Whole Tissues. Liquid Scintillation Counting of Tissues. 30. Schneider, W. C. Determination of Nucleic Acids in Tissues by Anal. Chem., 32: 42-46, 1960. Pentose Analysis. In: S. P. Colowick, and N. O. Kaplan, (eds.), 11. Hirono, I., Laqueur, G. L., and Spatz, M. Tumor Induction in Methods in Enzymology, Vol. 3, pp. 680 684. New York: Academic Fischer and Osborne-Mendel Rats by a Single Administration of Press, 1957. Cycasin. J. Nati. Cancer Inst., 40: 1003-1010, 1968. 31. Schoental, R. Carcinogenic Action of Elaiomycin in Rats. Nature, 12. Hoch-Ligeti, C., Stutzman, E., and Arvin, J. M. Cellular Com 221: 765 766, 1969. position during Tumor Induction in Rats by Cycad Husk. J. Nati. 32. Shank, R. C. Effect of Cycasin on Protein Synthesis. Biochim. Bio Cancer Inst., 41: 605 611, 1968. phys. Acta, 166: 578 580, 1968. 13. Kobayashi, A., and Matsumoto, H. Studies on Methylazoxy- 33. Shank, R. C., and Magee, P. N. Similarities between the Bio methanol, the Aglycone of Cycasin. Isolation. Biological and Chemi chemical Actions of Cycasin and Dimethylnitrosamine. Biochem. cal Properties. Arch. Biochem. Biophys.. i 10: 373 380, 1965. J., 105: 521-527, 1967. 14. Laqueur, G. L., and Matsumoto, H. Neoplasms in Female Fischer 34. Spatz, M., Smith, D. W. E., McDaniel, E. G., and Laqueur, G. L. Rats following Intraperitoneal Injection of Methylazoxymethanol. Role of Intestinal Microorganisms in Determining Cycasin Toxicity. J. Nati. Cancer Inst., 37: 217-232, 1966. Proc. Soc. Exptl. Biol. Med., 124: 691-697, 1967. 15. Laqueur, G. L., McDaniel, E. G., and Matsumoto, H. Tumor In 35. Stempack, J. G., and Ward, R. T. An Improved Staining Method duction in Germfree Rats with Methylazoxymethanol (MAM) and for Electron Microscopy. J. Cell. Biol., 22: 697-701, 1964. Synthetic MAM Acetate. J. Nati. Cancer Inst., 39: 355-361, 1967. 36. Svoboda, D., RÃ¡cela,A., and Higginson, J. Variations in Ultra- 16. Laqueur. G. L., Mickelsen, O., Whiting, M. G., and Kurland, structural Nuclear Changes in Hepatocarcinogenesis. Biochem. L. T. Carcinogenic Properties of Nuts from Cyeas cirdnalis L In Pharmacol., 16: 651 657, 1967. digenous to Guam. J. Nati. Cancer Inst., 31: 919-933, 1963. 37. Unuma, T., and Busch, H. Formation of Microspherules in Nu 17. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. cleoli of Tumor Cells Treated with High Doses of Actinomycin Protein Measurement with the Folin Phenol Reagent. J. Biol. D. Cancer Res., 27:1232-1242, 1967. Chem., 193: 265-275, 1951. 38. Widnell, C. C., and Tata, J. R. A Procedure for the Isolation of 18. Matsumoto, H., and Higa, H. H. Studies on Methylazoxymethanol, Enzymically Active Rat-Liver Nuclei. Biochem. J., 92: 313-317, the Aglycone of Cycasin: Methylation of Nucleic Acids in Vitro. 1964. Biochem. J., 98: 20c-22c, 1966. 39. Wogan, G. N. Biochemical Responses to Aflatoxins. Cancer Res., 19. Matsumoto, H., Nagahama, T., and Larson, H. O. Studies on 25:2282-2287, 1968.

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Figs. 1 to 6. Paraffin sections stained with hematoxylin and eosin. Figs. 7 to 15. Thin sections from rat liver fixed in glutaraldehyde. postfixed in osmium, embedded in Maraglas, and stained with uranyl acetate followed by lead citrate. All rats received a single i.v. injection of MAM acetate at the indicated dosage. Fig. 1. Liver of adult rat given an injection of NaCI solution and killed 1 month after injection. Hepatocyte nuclei vary only slightly in size and configuration and have a uniform chromatin distribution, x 340. Fig. 2. Liver of rat killed 6 hr after receiving MAM acetate, 35 mg/kg. Note that several nuclei have relatively clear nucleoplasm and prominent nuclear membranes. X 340. Fig. 3. Liver of rat killed 7 days after MAM acetate, 50 mg/kg. Hepatocyte nuclei vary in size and have irregular chromatin pattern. Enlarged clear areas around nuclei represent glycogen. X 340. Fig. 4. Liver of rat killed 2 months after MAM acetate, 50 mg/kg. This is representative of most marked nuclear abnormalities. X 340. Fig. 5. Liver of rat killed 7 months after MAM acetate, 50 mg/kg. One of several hyperplastic nodules. X 130. Fig. 6. Duodenum of rat killed 6 hr after MAM acetate. 35 mg/kg showing karyorrhexis in the crypts. X 340. Fig. 7. Liver of rat killed 15 min after MAM acetate, 35 mg/kg. Dense nucleolar plaques were present in the nucleolus which otherwise appeared normal. Similar plaques were present at later times (see Figs. 8 and 9). X 31,000. Fig. 8. Liver of rat killed I hr after MAM acetate, 50 mg/kg. The nucleolus shows a loss of skein-like arrangement, separation of granular and fibrillar components, and plaques. X 43,000. Fig. 9. Liver from a rat killed 6 hr after MAM acetate, 35 mg/kg. Segregation of nucleolar components is apparent with fibrillar material in the upper part of the nucleolus and granular component below. Nucleolar plaques are also present. X 17,500. Fig. 10. Liver from a rat killed 6 hr after MAM acetate, 50 mg/kg. This nucleolus has a reticulated configuration (see Ref. 7, Figs. 5 and 6). X 5800. Fig. II. Liver from a rat killed 24 hr after MAM acetate, 35 mg/kg. In this nucleolus, fibrillar material forms peripheral strands around a central granular mass (see Ref. 7, Fig. 7). X 21,000. Fig. 12. Liver from a rat killed 24 hr after MAM acetate, 35 mg/kg. Example of necrotic cell (near center) seen at 6 and 24 hr after treatment (see Fig. 2). Relatively homogenous nucleoplasm with condensed chromatin at nuclear envelope. Lipid and dense bodies are present in cytoplasm. Adjacent cells show little change. X 3500. Fig. 13. Liver from a rat killed 2 months after MAM acetate, 50 mg/kg. One enlarged nucleolus is below. At upper right is an abnormal nucleolar-like accumulation of granular material with vacuole, and semicircular cap of fibrillar material. X 19,000. Fig. 14. Liver from same rat as in Fig. 13. Granular mass with vacuoles. X 6600. â€¢ Fig. 15. Liver of rat given MAM acetate, 50 mg/kg, and killed 2 months later. Nucleolus at upper left appears normal. At right is an abnormal nucleolar-like mass with vacuolated granular material. X 10,500.

MARCH 1970 809

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1970 American Association for Cancer Research. Biochemical and Pathological Effects of Methylazoxymethanol Acetate, a Potent Carcinogen

Morris S. Zedeck, Stephen S. Sternberg, Richard W. Poynter, et al.

Cancer Res 1970;30:801-812.

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