Are Sugars Carcinogens? an Experimental Study

Are Sugars Carcinogens? An Experimental Study

W. C. HUEPER (National Cancer fnstitute, Bethesda, Marykind)

SUMMARY Injections (s.c.) of 25 % solutions of arabinose, dextrose, galactose, lactose, levulose, maltose, sorbose, and sucrose were given twice a week into the region of the nape of the neck to 480 rats and 480 mice (in groups of 60 animals) for periods up to 2 years. Only 2 rats given sorbose solutions developed sarcomas, after 21 months, at the site of injec tion. No tumors were found at this site in additional series of rats and mice injected similarly with water or traumatized by needle puncture. These observations do not support the claim that hypertonic sugar solutions paren terally introduced into rats and mice exert a carcinogenic effect. It is suggested that previously reported carcinogenic effects following repeated s.c. injection of sugar solu tions may be due to the presence of carcinogenic chemical impurities in the sugars used, possibly eluted from charcoal employed for the decoloration of sugar solutions.

In 1935, Nishiyama (11) first reported the carcinogenic compositions were represented, i.e., pentoses, hexoses, action of glucose solutions on the subcutaneous tissue of aldoses, ketoses, monosaccharides, and disaccharides. rats. Other investigatorsâ€”especially in Japan and Italy Solutions were prepared by dissolving 25 gm of a sugar â€”confirmed these observations and extended them to in 100 ml of distilled water in a volumetric flask, and auto other mono-, di-, and polysaccharides (levulose, maltose, claving in an Erlenmeyer flask (15 mm at 15 lb of steam fructose, galactose, lactose, sucrose, and glycogen (1â€”3,7, pressure and 250Â°F). The solutions of maltose assumed 9, 11â€”16,18â€”20,22). While rats were used in the majority a slightly brownish tint upon completion of sterilization. of the initial experiments, mice appeared subsequently to There occurred a small increase in viscosity of most sugar be equally responsive (2, 16, 18â€”20,22). In fact, Nagayo solutions after autoclaving, when measured by the Hellige (10) succeeded in transplanting a sugar sarcoma, obtained viscosimeter. The increases ranged from 0.1 for arabinose, in a mouse, to rats. Only 2 investigators (8, 26) failed sorbose, and levulose to 0.2 for dextrose, 0.4 for lactose, to corroborate the general claim that repeated s.c. injec and 0.8 for sucrose, suggesting that some polymerization tions of concentrated solutions of glucose into rats induced may have occurred in lactose and sucrose solutions. No the formation of sarcomas at the site of their adminis viscosity change from the average value of 2.0 occurred tration. in the solutions of galactose and maltose. New syringes Because of the distinct scientific and practical impor were used at all times. The needles were also new and tance of the apparent carcinogenic action of sugars and were used only once. Injections were given s.c. 3 times the various fundamental issues involved, it was considered a week for up to 2 years. necessary to test again a series of sugars under experi Rats were given 2 ml of the sugar solution into the sub mental conditions duplicating as closely as practicable cutaneous tissue of the nape of the neck at each injection; those employed by previous investigators, while using mice received 0.5 ml in the same area. Control groups of special precautions in their administration so as to pre rats and mice were given identical amounts of distilled dude the action of any carcinogenic impurities accident water. Additional control groups of rats and mice were ally introduced into the experimental agents and pro subjected to needle trauma only, while an untreated group cedures. of mice served as strain controls. Bethesda black rats and C57BL mice were used. The rats were 3 months MATERIALS AND METHODS old and the mice were 2 months old at the start of the The following sugars were tested : l-arabinose, anhy experiment. There were 60 rats or mice (30 male, 30 drous d-dextrose, d-galactose, lactose, d-levulose, l-sorbose female) in each test group, including the treated control (obtained from Mann Research Laboratories, Inc., New groups, while 125 mice served as strain controls. York), d-maltose, and d-sucrose (obtained from Nutri The maximal observation period was 2 years, at the end tional Biochemicals Corporation, Cleveland, Ohio). of which all surviving animals were killed. Autopsies Only those designated as chemically or chromatographi were performed on all animals and histologic examinations cally pure were used. Different chemical structures and were made of those tissues which showed any abnormali Received for publication September 28, 1964. ties. A total of 600 rats and 720 mice was used. 440

RESULTS appeared, which ulcerated unless the injection of the sugar The rats tolerated the injections of the various sugar solutions was stopped for some time. solutions without any untoward effects. The mice, on The principal pertinent observations made in rats and the other hand, not infrequently developed symptoms of mice composing test and control series are summarized in acute shock, especially during the early experimental Tables 1 and 2. period, soon after the introduction of the sugar solutions; Apart from some minor brownish discolorations of the some died with the anatomic reactions characterizing such connective tissue in the nape of the neck, rats did not a response to the hypertonic agent, i.e., hemorrhagic exhibit any abnormal local reactions. transudates in pleural and peritoneal cavities, severe The great majority of the benign and malignant tumors hyperemia, and hemorrhages in the lungs. In addition found in the test and control rats and mice were at sites to these acute general reactions, mice did not usually toler remote from the nape of the neck where the sugar solutions ate well the repeated local deposition of hypertonic sugar were administered. Only 2 fibrosarcomas, found in 2 solutions in the subcutaneous tissue of the nape of the rats which received sorbose solution, were near the in neck. In most of the mice, white, cheesy, necrotic masses jection site.

TABLE 1 DEATH DISTRIBUTION AND TUMOR YIELD IN RATS RECEIVING AQUEOUS SOLUTIONS OF DIFFERENT SUGARS 5.C. There were 60 rats, 30 of each sex, in each sugar (and control) group.

AT:â€˜@@â€œOtherArabinose TIME (months) oi TUMORS

SUGARS AND TUMORSSURVIVAL 0-6 7â€”9 10-12 13â€”15 16-18 19-21 22â€”24No.

f,g,i,i,e11DextroseTumors14Â° a,a112 a,c10 e,e32

e9GalactoseTumors213 a,a,a5 a6 r,b17 q26 e,

d,h,i,e,k11LactoseTumors5325 a8 i,i9 d,d,s28

d,i,l,l,m13LevuloseTumors2146 a11 a,a,b, e,i24 e, k12

d,e,h,i,l18MaltoseTumors83 a2 a,a6 a,d13 a,c,d,k10 c,d,i,k18

a,e,h,i14.SorboseTumors7134 a13 a,e,i14 a,a,e, k, k, i18

Tumors434 a4 a,d10 a,d11 e,i,o24 a,e,h,n, r213Sucrose 0, q,

i12Needle-controlTumors113 a,a15 a,b,g1010 a,a,e,h20 a,e,

e,i5Water-controlTumors13711 f,k7 C823

Tumors17 a,a,a,f397 a,b, b, a,e,e,e, q33 e,h,q,t16

a Key to tumor symbols : a, large round cell sarcoma of ileocecal lymph nodes ; b, mesothelioma of the pleura; c, mesothelioma of the peritoneum; d, reticulum cell sarcoma of the liver; e, carcinoma of the uterus; f, papilloma of the urinary bladder; g, lymphangiosarcoma of the subcutis; h, cholangioma; i, adenofibroma of the breast; k, carcinoma of the maxillary sinus or gingiva; 1, adenocarcinoma of the breast; m, adenocarcinoma of the pancreas; n, cholangiocarcinoma; o, fibrosarcoma of the subcutis of the back; q, leukemia; r, adenocarcinoma of the colon; s, teratoma of the testis; t, gelatinous adeno. carcinoma of the stomach.

TABLE 2 tion of an unknown carcinogenic factor into the â€œnormalâ€• DEATH DISTRIBUTION AND TUMOR YIEu IN MICE environment of these animals. Such an agent may be RECEIVING AQUEOUS SOLUTIONS OF DIFFERENT contained, for instance, in the commercial feed. Since SUGARS S.C. urinary calculi and cystitis were noted in test and control There were 60 mice, 30 of each sex, in each sugar (and control) animals, it cannot justly be concluded that the sugar group, except for 125 in the strain-control group. administered played an essential role in eliciting the ab normal reactions in the bladder wall. For the same rca TIME (months)No.oF son, the administration of sugars was not responsible for TUMORSâ€”â€”0â€”67â€”910â€”1213â€”1316-1819â€”2122â€”24Arabinose401064Dextrose31214Â°4Tumora1Galactose40146Lactose1618121031Levulose18181392Maltose1318236TumorbiSorbose117122361Sucrose26195352Needle-control26108745Water-control218114547Strain-control512212167710Tumorc,da,b4SUGARSANDTUMORSSURVIVAL the production of the bladder stones, although Vermeulen et at. (23) reported that rats developed urinary concre tions when kept on a milk or lactose diet. The essentially negative outcome of the present experi ments on the alleged carcinogenic action of subcutaneously introduced sugar solutions in rats and mice supports the similar observations previously reported by Meier (8), who failed to induce sarcoma formation in 2 series of about 50 rats each given s.c. solutions of 50 % and 5% glucose, respectively, for up to 539 days. The main local changes found by Meier (8) in the rats receiving the hypertonic glucose solutions were of a degenerative, necrotizing, and granulomatous character and thus re sembled those seen in the mice similarly treated in the present experiment. The results obtained in this investigation differ sharply aKeytotumorsymbols:a,adenomaoflung;b,leukemiaand from those previously reported by Japanese and Italian lymphoma; C, adenoma of liver; d, spindle-cell sarcoma of groin. workers who obtained, in mice and rats receiving hyper tonic sugar solutions s.c., fibrosarcomas and spindle-cell Chronic hemorrhagic and purulent cystitis, associated sarcomas at the site of injection in a significant percentage in about one-third of the cases with bladder stones, was of animals surviving for more than 250 days : 10 rats with observed in some rats and mice of both the test and con sarcomas among 47 survivors (14) ; 3 rats with sarcomas trol groups (18 rats, 7 mice). There was no histologic among 4 survivors (15) ; 4 mice with sarcomas among 8 evidence of an injurious effect of the injected sugars upon survivors receiving galactose; 5 sarcomas among 18 sur any internal organ, especially the liver and kidneys, vivors receiving glucose (18â€”20); 5 mice with sarcomas although nephrotic changes of varying degrees were noted among 20 mice receiving lactose; and 4 mice with in many animals, including controls. Rather extensive sarcomas among 20 mice receiving glucose and fructose amyloidosis of the liver, spleen, and kidneys often oc (2). curred in the mice. The only point of apparent agreement between the find DISCUSSION ings of the Japanese and Italian investigators and those of the present experiment, is the occurrence of 2 sarcomas in Almost all of the benign and malignant neoplasms seen rats at the site of sorbose injections. Because of the in mice and rats of the test series occurred at sites and in absence of any similar neoplastic reactions in the 7 other numbers similar to those at which such tumors were ob groups of rats and in all of the mice receiving the 8 sugars, served in the controls, and have been seen in many previous this evidence tends to support the view that sugars are experiments (4â€”6). It is, therefore, unlikely that the not carcinogenic. The exception to the rule seems to development of most of them was related causally to the suggest that the carcinogenic reactions attributed to sugars administration of the sugar solutions. They may properly by previous investigators may be due to a carcinogenic be regarded as â€œenvironmental background tumorsâ€• impurity present in the sugars used. Such carcinogenic elicited by undetermined endogenous as well as exogenous impurities may be introduced into sugars, for instance, factors. It is uncertain, however, whether this explana tion safely accounts for the presence of an adenocarcinoma when concentrated sugar solutions are filtered for de of the pancrease in a lactose rat, of adenocarcinomas of colorizing purposes through improperly prepared char the colon in a dextrose and a sorbose rat, and of a chol coal, containing polycyclic hydrocarbons. Chemicals of the dibenzanthracene type are eluted from charcoal by angiocarcinoma in a sorbose rat. This uncertainty is concentrated sugar solutions, according to Druckrey based, in part, on the fact that such cancers have never (1963,personalcommunication); traces may be introduced before been seen in normal and carcinogenically tested animals of this strain of rat, of which many thousands in this manner and may remain in apparently chemically have been employed in various types of carcinogenic pure sugars. experiments; in part, it is related to the occurrence of It is unlikely that the different responses obtained by hemorrhagic and purulent cystitis in a significant number Japanese workers and by a Swiss investigator, as well as of rats and mice of both test and control series, and of in this experiment, may be attributable to an extraordinary papillomas of this viscus in 1 rat each of the arabinose, carcinogenic susceptibility of Japanese rats to sugar solu needle-control, and water-control series. These tumors tions introduced s.c., a property lacking in Swiss and may, therefore, be due to the recent, inadvertent introduc American rats and mice (8).

Since Japanese investigators have also reported that 9. Murayama, H. Experimental Induction of Sarcoma in Mice hypertonic saline solutions given s.c. to mice and rats with a Concentrated Solution of Maltose as a Reducing Sac charide, and a Comparative Experiment with a Solution of induce the development of sarcomas (21), whereas physio Mannitol, as a Non-Reducing Saccharide. Trans. Soc. Pathol. logic saline solution was said to lack such properties when Japon. 48: 1345-52, 1960. similarly used (2, 14, 19), the old concept of carcinogenesis 10. Nagayo, M. On Heteroplastic Transplantation of Mouse Sar on the basis of chronic irritation and regeneration has been coma. Gann, 85: 323â€”45,1941. advanced as an explanation of sugar and salt carcinogene 11. Nishiyama, Y. tjber die Sarkombildung durch wiederholte Injektion der hockhonzentrierten Glukoselosung bein den mit sis (8, 21). It is remarkable that Tokoro (19) failed, o-Amidoazotoluol gefutterten Ratten. Ibid. , @9:1â€”9,1935. however, to induce sarcoma formation in mice receiving 12. Nishiyama, Y. Experimentelle Erzeugung der Sarkome bei solutions of 5 % and 25 % sodium chloride, and that he Ratten durch chemische Substanzen. Ibid., SO: 419â€”20,1936. was successful in this respect only when a 15 % saline 13. Nishiyama, Y. Experimentelle Erzeugung der Sarkome bei Ratten durch chemische Substanzen. II. Mitteilung. Ibid., 31: solution was used. Moreover, it is noteworthy that, 223â€”25,1937. according to several investigators, sarcomas could be 14. Nishiyama, Y. Experimentelle Erzeugung des Sarkomas bei produced in the subcutaneous tissue of mice by repeated Ratten durch wiederholte Injektionen von Glukoselosung. s.c. injections of hot water, cold water, and water at room mid., 3@:85-99, 1938. temperature (24, 25). Before such extraordinary claims 15. Nonaka, T. The Occurrence of Subcutaneous Sarcomas in the Rat, after Repeated Injections of Glucose Solution. Ibid., 39: (10, 17) can be accepted as valid, these experiments should 234â€”35,1938. be repeated with water and saline of unquestionable chemi 16. Seki, M. Experimental Study of Freezing in Laevulose Sar cal purity, and should be performed under conditions coma. Trans. Soc. Pathol. Japan., 48: 1353â€”74,1960. which exclude secondary contamination of the injected 17. Tagashira, U. Studies on the Interruptions of the Ferment System of Glucose Metabolism in the Sarcoma Producing material by any extraneous carcinogenic chemical. These Tissues. Second Report. Supplementary Evidence of the 511- precautions should be observed since the present experi Conjugation Hypothesis in Carcinogenesis. Gann, 45: 601â€”15, ments have clearly shown that distified water given in this 1954. manner does not induce in rats and mice the development 18. Takizawa, N. tYber die Erzeugung des Maussarkoms durch die of subcutaneous sarcomas and that chronic mechanical subcutane Injektion der konzentrierten Zuckerlosung. Ibid., 33:193â€”95,1939. trauma resulting from the repeated s.c. introduction of a 19. Takizawa, N. t@ber die Erzeugung des Sarkoms der Maus und needle was equally ineffective in this respect. The cvi Ratte durch wiederholte subkutane Injectionen der konzen dence reported here and elsewhere (5) militates against trierten Zuckerlosungen. Thid., 32: 236â€”37,1936. the acceptance of the concept that nonspecific chronic 20. Takizawa, N. Experimentelle Erzeugung des Sarkoms bei der irritation represents a carcinogenic mechanism. Maus durch die Injektion von Glucose, Fructose and Galac tose. Em Beitrag zur Frage der Histogenese des fibroplasti REFERENCES schen Sarkoms. Ibid., 34: 1â€”5,1940. 1. Amano, S., and Ito, S. Glykogen und Geschwulstbildung. 21. Tokoro, Y. Uber die artificielle Erzeugung des Sarkoms bei den Gann, 57: 200-03, 1943. weissen Ratten mittels konzentrierter Kochsalzlosung. Ibid., 2. Arakawa, S. Experimental Production of Sarcoma of Mice 34: 149â€”55,1940. with the Subcutaneous High Concentrated Sugar Solutions, 22. Uekusa, T. Influence of Triphenyl-Tetrazolium Chloride on Especially Lactose, and Mixture of Laevulose and Glucose. the Production of Levulose Sarcoma in Mice. Ibid., 45: 393â€” Ibid.,48:363â€”64,1956. 95,1954. 3. Cappellato, M. Sui sarcomi sperimentali da gluosio nd ratto 23. Vermeulen, C. W., Proctor, D. L., and Seibutis, L. Urinary bianco. Tumori, 16: 38â€”54,1942. Calculi on Milk or Lactose Diet. J. Lab. Clin. Med., .57:883â€”93, 4. Hueper, W. C. Environmental and Occupational Cancer Haz 1961. ards and Cancer. Clin. Pharmacol. Therap., 3: 776â€”813,1962. 24. Warabioka, K. Experimental Carcinogenesis with Aqua Des 5. Hueper, W. C., and Conway, W. D. Chemical Carcinogenesis tillata. Third Report. On the influence of Temperature of and Cancers, p. 592. Springfield, Ill. : Charles C Thomas, 1965. Aqua Destillata Injected Subcutaneously into Rats. Proc. 6. Hueper, W. C., and Payne, W. W. Carcinogenic Studies on Polyoxyethylene(8)Stearate. Arch. Environ. Health, 6: 484â€”94, Japan. Cancer Assoc., 1958. Suppl. to Gann, 49: 147â€”48,1959. 25. Watanabe, F., and Azuma, M. Six New Rat Sarcomas, Pro 1963. 7. Ito, S. Experimentelle Sarkomerzeugung bei Ratten durch duced by the Subcutaneous Injection of Hot Water. J. Naga wiederholte Injektion von Glyogenemulsion. Gann, 38: 103â€”30, saki Med. Assoc. 34: 834, 1959. 1944. 26. Zarattini, A. Sulla produzione sperimentale del sarcoma nei 8. Meier, A. Gibt es einen Zuckerkrebs? Schweiz. Z. Allgen. ratti mediante somministrazione parenterale di glucosio. Pathol. Bakteriol., 5: 226â€”37,1942. Tumori, 14: 77â€”84,1941.

W. C. Hueper

Cancer Res 1965;25:440-443.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/25/4_Part_1/440

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/25/4_Part_1/440. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.