Document RJ8EMkXrbyaRVLkJxk1nYp3JX

sb THE PATHOLOGICAL EFFECTS OF SUBCUTANEOUS INJECTIONS OF ASBESTOS FIBRES IN MICE: MIGRATION OF FIBRES TO SUBMESOTHELI^L TISSUES AND INDUCTION OF MESOTHELIOMATA by K J. C. Rol, R L Caiutr, M. A. Walters and J S. Harington Chester Beatty Research Institute, Institute o f Cancer Research: Royal Cancer Hospital, London, S W 3, and Cancer Research Vmt o f the National Cancer Association o f South Africa, South Afiican Institute fo r Medical Research, P .0 Box 1038, Johannesburg, South Africa Groups o f 20 fem ale CBA mice weie injected subcutaneously in two sites with suspensions o f croctdohte, amosite or chrysotde asbestos. Each injection consisted o f 10 mg fibre suspended tn 0 4 ml saline and each animal received 3 injections into each flank at sites well distant from the t/w iax. For comparison, two groups weie similarly treated with croctdohte and amosite from which natural and contaminant oils had been removed by solvent exit action. The most remaikable feature of the results were, first, the apparently specific transpot t o f asbestos fibres o f all thi ee types to the submesothehal tissues o f the thorax and abdomen, and secondly, the development o f extensive in flammatory and proliferative changes in these regions culminating, m JO instances, in the formation o f mesothehomata. Six animals developed sarcomas at the injection site but, m most mice, there was little cellular response to asbestos m the subcutaneous tissues in contrast to the vigorous reactions seen in serosal membranes. AH three types of asbestos induced both injection-siH tumours and distant mesothelial changes and the removal o f oils from amosite and crocidohte did not abolish-- though it may have redm ed--these manifestations o f carcinogenic activity. An association between pleural mesothelioma and a history of exposure to asbestos dust was first observed in man by Wagner et aL (i960); in an addendum to this paper, the authors also referred to one case o f pentoneal mesothelioma m a person exposed to asbestos. Since then, both peritoneal and pleural mesotheliomas have been reported in asbestos workers, their relatives, and in people living near asbestos mines and factories (for review, see Harington, 1967). The carcinogenic effects o f asbestos have also been studied in experimental animals. Schmahl (1958) reported the induction o f local sarcomas in rats injected subcutaneously with asbestos but did not mention the type o f fibre used. Subsequently, pleural mesotheliomas were induced in rats by intrapleural injections ot chrysotilc, crocidolile or amosite (Wagner, 1962, 1966) and in hamsters by intrapleural injections of chrysotilc or amosite (Smith et a l , 1965a, b). The mechanism of carcinogenesis by asbestos is unknown, but three important pieces of information have recently come to light. First, certain iorms o f asbestos (notably crocidolite and amosite) normally contain appreciable quantities ot mineral oils (Harington, 1962,1965). Secondly, asbestos is also readily contaminated with exogenous mineral oils such as oils from jute bags which are used to store and transport asbestos fibres (Harington and Roe, J965). Thirdly, natural and contaminating oils have been extracted from commercial amosite and crocidohte and shown to have tumour-promoting activity on mouse skin (Roe et al. 1966). Received; 3 July, 1967. Approved: 18 July, 1967. Despite the demonstrable activity ol these oils, ibeir significance is disputed at the present time (see Wagner, 1966) and further investigations b^vc now been carried out in an attempt to Jarify their role The experiments to be described bc*jow were devised to compare the carcinogenic clTcct on subcutaneous tissues of commercial asbestos with that o f fibres from which the oils had been extracted. Three types of asbestos were used--crocidohte, amosite and chrysotile. |u the event, the results were not conclusive but they proved to be o f considerable interest in another respect. It was observed that asbestos injected subcutaneously was subsequently found in conspicuous amounts in the mesothelium o f the pleural, pericardial and peritoneal spaces; striking reactions developed in these sites includ ing the appearance o f mesotheliomata. 0 4 ml saline and this procedure was lepcatcd twice at intervals of 5 weeks. The control group received saline only The animals were examined reguhriy lor the development o f tumours at the injection site or other lesions. Sick mice were kil'ed and the survivors sacrificed approximately 7 years after the beginning of the experiment. Except where precluded by post-mortem changes, a careful necropsy was carried out on all animals Tissues showing any macroscopic abnormality were fixed in Bourn's solution. Paraffin sections were prepared at 5 /t and stained with hacmatoxyUn and eosin, haematoxylin and Van Gieson, M asson's trichrome, Weigert's method for fibrin, and the modified Foot technique for reticuhn fibres. results MATERIAL AND METHODS Mice. One hundred and twenty CBA female mice were used, aged 6 to 10 weeks and weighing 30-35 g This strain has been maintained by brother-sister mating in the Chester Beatty Research Institute since 195) when it was intro duced from the University o f Minnesota. The jnimals were fed a cubed diet (Diet 86, Messrs. Dixon Limited, Ware, Herts., England) and water ad libitum and they were kept in metal cages, 10 mice in each. Asbestos. The three types o f asbestos used-- erocidolite, amosite and chrysotile--were derived from the same batch o f finely-divided fibres used in earlier experiments by Wagner (1965), Harington (1965) and Harington and Roe (1965). Natural and contaminating oils were extracted from erocidolite and amosite under reflux with 8 solvents of increasing polarity; these were, successively, petroleum ether (60-80), cyclo hexane, carbon tetrachloride, benzene, chloro form, ether, ethyl alcohol and methyl alcohol. Further details o f the process are described by Harington (1965). Five preparations were thus available for study: erocidolite, extracted crocidolite, amosite, extracted amosite and chrysotile. The mice were divided into 6 equal groups, 5 of which were treated with a preparation o f asbestos. The fibres were suspended in sterile physiological saline and injected through widebore needles (serum 2, 19 gauge) into the sub cutaneous tissues o f the flank. Each animal was injected in both flanks with 10 mg fibre/ Histological changes at injection sites The incidence o f tumours at the sites of injec tion is shown in Table I. in all animals, the tumours were single unilateral lesions, despite the fact that asbestos was injected into both flanks. Six o f the seven tumours were sarcomata; five o f these were spindle cell lesions (Fig. 1) while the sixth was a pleomorphic tumour with numerous multinucleate giant cells. 1n one mouse, a mesothelioma developed from the peritoneum o f the underlying body wall (Fig. 2) N o clear difference in the incidence of injection-site tumours was established in the five experimental groups: extracted erocidolite and amosite ap peared to be slightly less active than the corre sponding untreated fibres, but theshortersurviva! time o f mice which received extracted erocidolite and amosite may have been responsible for this difference. The local changes in animals which did not develop tumours were consistent and showed little variation according to the type of asbestos injected (Fig. 3). Asbestos fibres accumulated in the dermis, lying free or within macrophages, just deep to the pannieulus carnosus. The free-lying fibres were usually thin needle-like structures but, in some instances they appeared to be greatly thickened and resembled " asbestos bodies Apart from the presence of macro phages, other signs o f reaction were few: fibrosis was both slight and infrequent and inflammatory infiltrates were rare. Asbestos-laden macro phages showed some tendency to coalesce, K( >1 l I AI . IADLL I IN C ID I NCI- O l IN J rC T IO N -S lir TU M O U R S ItL.umom 1. Crocidohte No ol muc examined posi-inoium 40 or mine weeks aller Insi mjeelion 17 Average survival mile ol mice * examined post-mortem (weeks) 75.3 (Range = 62 to 105) No o f mice wiih injcction-sue tumours 2 Spindle cell sarcoma 1me of appearance of tumour luicj m previuto column 4 (weeks) 65, 105 11. Extracted crocidohte 14 6 4 .6 (Range -- 41 to 104) 0 - 111. Amosiie 13 96 4 3 ife, 118 (Range -- 41 to 118) Spindle cell sarcoma (2) Local mesothelioma close to injection 62 site (1) IV. Exti acted amosite 15 79 7 1 109 (Range - - 41 to 109) Spindle cell sarcoma V. Chrysotilc 12 75 5 1 108 (Range == 41 to 108) Pleomorphic sarcoma VI. Saline controls 15 104 (Range = 71 to 108) 0 - 1 i c Hint when tumour first evident on macroscopic examm.it ion o f living animal v s m S l O S - l M H J C l I ) Ml S O I 111 I IOM \ I N M K I lotmmg mulunudcatc giant culls, and m animals surviving for long pciiods ol time loci ol necrosis and calcification appealed m the injection sites, these changes were more prominent in mice treated with chrysotilc Disti ibution oj asbestos at distant sites and the associated tissue i factions HOURb 3 Injection site showing abundant asbestos, lying free and within macrophages. Note the paucity ol ihc tissue icsponse Haematoxylin and cosin. x!6 0 The distribution of asbestos and the reactions evoked by this material are summarized b e lo w the principal histological findings in the thorax and in the abdomen are shown in Table 11 Although no clear-cut differences were established between the various types of asbestos which were studied, several important features emerged. First, it is clear that asbestos fibres are not only disseminated widely from the sites ot injection but are also distributed in a highly selective fashion. In the chest, the main sites in which asbestos accumulated were the visceral and parietal pleura and pericardium with in volvement of the adjacent parts of the lungs, chest wall, diaphragm and heart In the abdomen, asbestos was seen on the serosal surfaces of the stomach, intestine, liver and spleen, in the [T'>T<sjwr, F igure l An early injection site sarcoma, surrounded by asbestos fibres Hacnutoxyiin and eosin. 160. F igure 2 Mesothelioma developing in abdominal wall beneath injection site. Haematoxylin and eosin. x 160, TABLE II HISTOLOGICAL REACTIONS IN THE THORAX A N D ABDOMEN ASSOCIATED WITH ASBESTOS Treatment No o f mice examined post-mortem 40 or more weeks after first injection Mactoscopic deposits of asbestos on serosal surfaces Thorax Abdomen Inflammatory changes in serosal surfaces Thorax Abdomen Non-malign.ml mcsoiheliat proliferation in scrostil membranes and adhesions Thorax Abdomen Mesothelioma 1 Thorax Thorax Abdomen and Abdomen 1. Crocidohte 17 10 8 1 2 9 9 <2 l (7 i,~ 8 2 ) (65) l (71) 11. Extracted crocidohte 111. Amosite 14 a 14 12 48 2 2 1 8 11 l 0 (41) 0 1 4 8 13 0 (1 0 4 ) (59, 68, 109) IV. Extracted amosite V. Chrysotile VI. Saline 15 12 15 7 8 I 2 7 70 0 0 3 5 10 3 50 00 (73) 000 o 0 00 0 0 u ni 1 1 ai nroscmeiy, and also in retroperitoneal structuiex, paitiLulaily around the paneleus and kidneys Secondly, in contiast to the relatively incon spicuous response which was described in the subcutaneous tissues, the serosal elements usually reacted vigorously to the presence o f asbestos (ibres. A wide range o f histological changes was seen although, in some circumstances, it was noticeable that the tissue reactions developed at a small distance away from the deposited fibres (Fig 4) The most common response was the formation o f adhesions, together with thickening o f pre-existing fibrous capsules (Fig. 5 to 7) Such changes were usually confined to the surfaces o f the organs concerned and were frequently unevenly distributed, forming discrete fibrous nodules, asbestos fibres, cither lying free or within macrophages, were readily scon within them In two sites--the heart and the liver-- these fibrotic zones sometimes extended inwards producing considerable local destruction (Fig. 6, 7) Not infrequently, dilated lymphatics were observed within adhesions; some o f them, particularly those in the hepatic capsule and in subpleural zones, contained aggregates o f lym phoid cells but asbestos fibres were not identified within them with certainty. Asbes tos fibres were, however, often seen in lymph nodes, particularly in mediastinal, mesen teric and pararenal glands. In some animals, the adhesions were infiltrated with granulation tissue and chronic inflammatory cells, but the latter were never seen in large numbers. More frequently, the adhesions contained cells which were regarded as proliferating mcsothelial Cle men's. These appeared somewhat swollen and were usually arranged as plaques on serous membranes (Fig. 8). The individual cells varied in shape, ranging from flattened to columnar forms; the nuclei were usually vesicular with an open chromatin arrangement and the cytoplasm was diffuse and faintly eosinophilic. In some animals, a blood-stained ascites was found at autopsy. The most striking response to asbestos fibres in these sites was, however, the development of mesotheliomata. Ten examples were seen; 4 were confined to the chest, 4 to the abdomen, and 2 involved both body cavities. The tumours were usually multifocal: in the chest, for example, nodules were commonly scattered in the sub pleural parts o f the lung, the chest wall and the F igure 5 Dense adhesions around the pancreas which are infiltrated with asbestos fibres and macrophages. Haematoxylin and eosin. x 160. Hcure 6 Large deposit of asbestos in pericardium with fibrosis and inward extension involving the myo cardium. Haematoxylin and cosin. x5.5. F igure 8 Subpleural zone showing an early mesothelioma which has developed on top of a layer containing granulation tissue and non-malignant mcsothelial elements. Haematoxylin and eosin xt6 0 diaphragm. N o tumours were encountered in the pericardial cavity, but oesophageal involvement was observed in one mouse. In the abdomen, multiple tumours were commonly seen, partic ularly in the mesentery, in adhesions binding together the liver, stomach, spleen and intestine, and near the pancreas and kidneys (Fig. 9), These lesions were usually arranged either as solid plaques or in more complex configurations with pedunculated or papilliferous patterns. The cells were relatively homogeneous in appearance and, in most instances, their origin from m so thlial cells was readily apparent (Fig. 10, 11). Early neoplastic lesions presented considerable 4 F igure 7 Massive fibrosis of liver capsule extending into the parenchyma. There are numerous asbestos fibres. Haematoxylin and eosin. x 160. V I. 1 l i' i----wiP'W mb. mmmmmmmmmm 1 - ',, % a *"' i s . * ' > ' . 'V " 4 s * - i u , "fe munitili Fio u r e II 1 tu m -o f lesion at its centre. H aem aiotylin mesothelioma which contains a granulomatous type ol les F igure 9 Mesothelioma arising from the mesentery, showing a characteristic papilhferous arrangement. Haenutoxyhn and eosin, x200. TABLE Itl SARCOMAS AND MESOTHELIOMAS INCIDENCE OF TUMOURS OTHER THAN INJECTION-SITE Treatment 1 Crocidohtc II Extracted c r o c c o lile III A m osite . . IV. Extracted am osite V. C hrysotile VI. Saline No o f mice examined poxt-moricm 40 or more wuAs alter firsl m iction Mammary tumours 17 Malignant lymphoma (74, 76) 14 0 0 _ 7T 5 2 (41, 118) IT 2 2 (65, 109) (41, 109) I T (713)_____ ((36) "IT 0 (108) Hepatoma Pulmonary adenoma Other tumours 0 0 ! Granulosu-cell tumour of ovary (76) 00 00 0 5 1 (109) 1 (109) 0 ' (108) 22 (108, 108) (108, 108) 0 "o 0 ,,me, measured m weeks, at whtcl, the tumours were observed NOT E Figures in parentheses refer FlGURr 10 Peritoneal mesothelioma which is infiltrated with lymphoid cells. Huematoxylin and eosin. x200. problems in diagnosis, particularly when in flammatory and librotic reactions to asbestos were also well developed in the same region f (.Fig. 8). In such circumstances foci of msothlial I pioliferation were regarded as neoplastic if the 1 cells concerned were present in reasonably large numbers, showed any tendency to form clu m ps or to invade underlying tissues, or exhibited the usual cytological tcatures associated with malignant cells. Established tumours were not 67 5 MWI W iW ' mlicquently infiltrated with chiom e inflammatory periods of time. Conversely, mcsothelionuu \ xTk (}-ig (0), lymphocytes and macrophages appeared cat her and in a higher incidence, often accumulated in clusters withm tumours despite the fact that the concentration of usbestot and, in a few instances, ill-delined granulomatous m serous mcmbianes must be far less than n zones were observed (Fig II) Asbestos ftbies injection sites. were larely seen within tumours Lymphatic channels adjacent to tumour-bearing areas were sometimes dilated and putative tumour cells were occasionally seen within them The histological reactions to asbestos in nj around serous membranes o f the chest ami abdomen have already been described in detail, but certain general points may be considered It must be stressed that the subcutaneous in Incidence of tumours other than injection site jections o f asbestos were given with great care s(ii comas and mesotheliomas to ensure that no fibres were introduced into the peritoneal cavity. Routine histological The incidence o f neoplasms at other sites, such as malignant lymphoma, hepatoma and pulmonary adenoma, is shown in Table IIL A low incidence was observed m all groups and no significant differences were established. examination o f the injection sites indicated that the asbestos deposits were invariably localized to the body waif, just deep to the panmeulus carnosus muscle; in no instance was there any indication of a needle-track leading into the peritoneal cavity. Accidental injection into the pleura! or pericardial spaces is impossible since DISCUSSION all ir tchons were made low down in the Hanks, at least l cm distant to the lowest part of the twelfth rib. There can be no doubt, therefore, Two main findings emerge from the present that asbestos fibres are transported from the work. First, asbestos, injected subcutaneously subcutaneous tissues to submesothelial regions; into female CBA mice, is disseminated widely on histological grounds, this distribution appears but selectively and accumulates in serosal to be specific. Certain questions inevitably arise-- membranes in the thorax and abdomen. Secondly, in particular, how are the fibres transported? the presence o f asbestos in these sites evokes a Is this tropism to the mesothelium a peculiarity varied and vigorous cellular response, not of asbestos or does it occur with other, less infrequently culminating in the formation of readily identified, materials? Such evidence mesotheliomata. These observations are equally as is available at the present time suggests that applicable to crocidohte, amosite and chrysotile tropism to mesothehal tissues may indeed be a and although there is a suggestion that removal of special attribute o f asbestos fibres but the mode mineral oils reduces the carcinogenic properties o f transport is unknown. The pattern of o f the fibres, it has already been stressed that the distribution, their occurrence within lymph difference in activity may be distorted by the nodes, and the presence o f dilated lymphatics in shorter survival time o f mice treated with ex serosal membranes, however, favour the view tracted fibres. The reasons for the poorer survival that the fibres may be disseminated by lymphatic of mice in these two experimental groups arc channels. obscure, but they effectively preclude any firm comparison between the effects of extracted and untreated asbestos. Finally, the mesotheliomas themselves present a number of noteworthy features. The tumours were relatively homogeneous in histological The distant changes seen in serous membranes appearance, in contrast to the pleomorphism are all the more remarkable when contrasted which has been described in mesotheliomas from with the relatively feeble response which the other experimental animals (Wagner et al., 1962; various types o f asbestos evoke in the sub Smith et a!., 19656) and also in man (McCaughey, cutaneous tissues. Although a few sarcomas 1958, 1965; Churg et a l 1965). Secondly, many were induced at the sites o f injection, these o f them were infiltrated with chronic inflamma appeared late and, in the majority o f animals, tory cells. The significance o f this finding is there was negligible cellular response to asbestos uncertain but the most likely explanation is even though this material persisted for long that such cells are survivors of an earlier i jojlammatoiy response. Thirdly, like olher mcaodicliomas reported m the literature, the tumours {rammed in the present work hardly ever con fined recognizable asbestos fibres i-urlhei studies based on the piesent findings, particularly with respect to the mode o! transport of asbestos and its apparent predilection for the inesothehum, are now in progress ACKNOWLEDGEMENTS VVc are grateful to Miss J. Hotham for help ifi the preparation o f the manuscript and to the Photographic Dcpaitment o f the Institute for their help in the preparation o f the illustrations. This investigation has been supported by grants to the Chester Beatty Research Institute (institute o f Cancer Research: Royal Cancer Hospital) from the Medical Research Council and the British Empire Cancer Campaign for Research, and by the Public Health Service Research Grant No. CA - 03188 from the National Cancer Institute, US Public Health Sei vice. EFFETS PATHOLOGIQUES DE L'INJECTION SOUSCUTANE DE FIBRES D 'AM IANTE CHEZ LA SOURIS: M IGRATION DES FIBRES VERS LES TISSUS SUBM SOTHLIAUX ET IND U C TIO N DE MSOTHLIOMES Des groupes de 20 souris CBA femelles ont reu en deux points, par injection sous-cutane, des suspensions d'amiante ( crocidoiite, amosite ou d u y s o tile ). Vinjection tait faite avec 10 mg de fibre en suspension dans 0,4 ml d'eau physiologique; chaque animal a reu 3 injections dans les deux flancs, en des points loigns du thorax. Aux fins de comparaison, deux groupes ont t traits de la mme manire avec de la crocidoUte et de Vamosite dont on avait limin des huiles naturelles contaminantes par extrac tion au moyen de solvants. Les deux fa its les plus remarquables que l'on ait observs sont, premirement, la migration appai emment spcifique des fibres d'amiante des trois sortes vers les tissus submsothliaux du thorax et de l'abdomen; deuximement, dans ces rgions, des modifications tendues de caractre inflammatoire et prolifrant, abou tissant dans JO cas la formation de msothliomes. Six animaux ont eu des sarcomes au point d'injection mais, chez la plupart des souris, on n'a observ qu'une faible raction cellulaire /'amiante dans les tissus sous-cutans alors que la raction tait trs vive dans les membranes sreuses. Les trois types d'amiante ont provoqu la fols des tumeurs au pom t d'injection e t des modifications msothliales distance; l'limination des huiles de Vamosite et de la crocidoiite n'a pas supprim, mais elle a peut-tre diminu, ces manifestations d'activit carcinogne. REFERENCES Churg, J., R osen, S. H., and Moolten, S., Histo logical characteristics of mesothelioma associated with asbestos. Aim. N. Y. Acad. Sci., 132, 614-622 (1965). Harington, J. S., Natural occurrence of oils contain ing 3,4-benzpyrene and related substances in asbestos. Nature (Loud.), 193, 43-45 (1962). Harington, J. S., Chemical studies o f asbestos. Ann. N.Y. Acad. Set., 132, 31-47 (1965). H arjncton, J. S,, Mesothelioma, in R. \V. Raven and F. J. C. Roe (ed.), The Prevention of Cancer p. 207, Butterworth, London (1967). H arington, J. S., and Roe, F. J. C,, Studies of car cinogenesis o f asbestos fibers and their natural oils. Ann. N. Y. Acad. Sci., 132, 439-450 (1965). M c Caughey, W. T. E., Primary tumours of the pleura. J. Path. Bact., 76, 517-529 (1958). KOI 1 1 AI If,. J. Cancel. 2, 639-646 (1967) M i C a u g i i i v , W. T E,, Cruet,j fo, dmgnos.s of clitlusc mosoUicfial tumois. Ann N Y Acad Sei 132, 603-622 (1965) ' R ur, F J. C , Wa i. i ik s , M A , and K a r im , io n , J S,, Jimioui iniiuition by natural and contaminating asbestos oils. nt. J Cancer, /, 491-495 (1966). Schmahl, D., Cancerogene Wirkung von Asbcsl bei Implantation an Ratten Z Krebsforsch 62 561-567 (1958). ' S mith, W. E., M iller, L,, E lssser, R. e ., and H uhlrt, D. D,, Tests for carcinogenicity of asbestos Ann N Y Acad Sei , J32, 456-488 (1965(7). S mith, W. E., M iller, L,, C h urg, J., and S ilikoet, I. J., Mesotheliomas in hamsters following intra pleural injections of asbestos. J Ml Swat Hasp 32,1-8 (19656). Wagner, J. C,, S leggs, C. A., and M a rchand, P Diffuse pleural mesotheliomas and asbestos expo- sure in North Western Cape Province Ben j iildmlr. M ed. 21, 260-271 (I960). ' W agner, J C , M unim y , D . E,, and H aringios J. S,, Histochemical demonstration of hyalurouc acid in pleural mesotheliomas. J. Bath Baa tu 7T .T S llQ A -n 1w IV. ' W agner, J. C., Experimental production of mso thlial tumours of the pleura by implantation of dusts in laboratory animals. Naiure (Loud I ig* 180-181 (1962). '' W agner, J. C , in d iscu ssion o f paper by Peacock P. R , and Peacock, A , A sbestos-induced tumors in white leghorn fow ls. Am. N .Y . Acad Set 132, 505-506(1965). '' W agner, J. C., The induction of tumours by die aainnstbirmeasaptlloses.urAPaielsrtu,ignhioai cL(uJ.l9a6Sti6eo)vn.en o(efd.)v, aLruionugs tutmypoeusn omf BIOCHEMICAL MECHANISMS OF DRUG RESISTANCE DEVELOPMENT OF RESISTANCE TO 5-AZACYT1DINE AND SIMULTANEOUS DEPRESSION OF PYRIMIDINE METABOLISM IN LEUKEMIC MICE by J. V esely, A. Cihak and F. Sorm Institute o f Organic Chemistry and Biochemistry, Czechoslovak Academy o f Sciences, Prague The development o f resistance o f leukemic A K R mice to 5-azacytidme administered in small doses was investigated. The activity o f uridine kinase dect eased simultaneously with the development o f resistance. The gradual shortening o f the survival time o f treated animals and the depression o f enzyme activity had a continuous character. The application o f 5-azacytidine along with hydrocortisone or actmomycin D did not appreciably affect the development o f resistance, lit leukemic cells resistant to 5-azacyUdine the incorporation o f uridine, cytidine and 5-azacytidine itself into ribonucleic acids was depressed in accordance with the impaired anabolic transformations o f 5-azacytidme in vivo as well as with the soluble fraction in vitro. 5-Azacytidine which has been synthesized tn";i'I The purpose of the present report was to these laboratories (Piskala and orm, 1964) investigate the pyrimidine metabolism of mouse has potent biological effects; its antileukemic leukemic cells made resistant to 5-azacytidine properties in mouse leukemia are particularly It was of interest to find out whether a correlation pronounced (Sormand Vesely, 1964; Cihak and existed between the development ol resistance Sorm, 1965; Vesety and Sorm, 1965). Preliminary and the uridine kinase activity which is respon clinical trials revealed that 5-azacytidine might sible for the phosphorylation of 5-azacytidine be suitable for therapeutic application. (Jurovclk et al., 1965). Like 5-fluorouracil (Chaudhuri et al.. 1958; Reichard et al., 1961), 6-azauridtne (Habermann ind Sorm, 1958; Pasternak et at., 1961), I-(1-D-arabinofuranosyl -cytosine (Chu and Fischer, 1965) and other pyrimidine analogues (Welch, 1963), 5-azacytidine is phosphorylated in animal tissues and incorporated into ribo nucleic acids (Juroviik et al., 1965). The low liability of incorporated 5-azacytidine (Pithovd et al., 1965) is probably responsible for its striking biological activity. Furthermore, 5-azacytidine S'-phosphate inhibits orotidylic acid decarboxyl ase (Vesely et al., 1967). MATERIAL AND .METHODS Reagents. 5-Azacytidine was synthesized by Dr. A. Piskala o f this Institute. Sodium hydro cortisone succinate was supplied by Spofa (Prague, Czechoslovakia). Lyovac Cosmegen Dactinomycin was obtained from Merck, Sharp and Dohme (West Point, N.J., USA). Sodium adenosine 5'-triphosphate was obtained from Calbiochem (Lucerne, Switzerland). 5-Azacytidine-4-u C (4.1 pel,iniol) was prepared by Dr. J. Mordvek of this Institute. 6-Azauridine-4,5-u C (72 /<c///mol), cytidine-U -11C (30 /ic/junol) and Received: 5 July, 1967.