[CANCER RESEARCH 38, 3509-3511, October 1978] 0008-5472/78/0038-0000$02.00 Increase of Urinary in 3,4-Benzopyrene Carcinogenesis and Its Inhibition by Putrescine

Keisuke Fujita,1 Toshiharu Nagatsu, Kan Shinpo, Kazuhiro Maruta, Hisahide Takahashi, and Atsushi Sekiya

Institute for Comprehensive Medical Science ¡K.F., K. S., K. M.], Research Center for Laboratory Animals ¡H.T.¡,and Department of Pharmacology ¡A.S.¡, Fujita-Gakuen University School of Medicine, Toyoake, Aichi 470-11, Japan, and Laboratory of Cell Physiology, Department of Life Chemistry, Graduate School at Nagatsuta, Tokyo Institute of Technology, Yokohama 227, Japan ¡T.N.¡

ABSTRACT were included. Thirty female BALB/c mice, 19 to 20 weeks old and 25 to 30 g body weight, were given s.c. injections of A significant increase in putrescine was noted in the 2.52 mg of 3,4-benzopyrene in 0.5 ml of tricaprylin (Group of mice with experimental s.c. tumors induced by a B) or of 2.52 mg of 3,4-benzopyrene plus 10 mg of putres single injection of 3,4-benzopyrene solution (2.52 mg of cine dissolved in 0.5 ml of tricaprylin (Group B + P). As 3,4-benzopyrene in 0.5 ml of tricaprylin). When 10 mg of controls, 30 mice were given injections of 0.5 ml of tricapry putrescine were added to the 3,4-benzopyrene solution, lin (Group C), and 15 mice received 10 mg of putrescine in the development of tumors was completely inhibited and 0.5 ml of tricaprylin (Group P). the increase of urinary putrescine in mice was suppressed The diameter of the tumor on the skin was measured by simultaneously. Animal weight data of a control group vernier calipers, for each mouse during carcinogenesis, receiving only putrescine indicated that the inhibitory and the surface area was calculated on the assumption that effect of putrescine is not due to its toxicity. tumors are round. When no tumor was detected the surface area was expressed as zero, and the mean surface area of INTRODUCTION the tumors of all mice was calculated. For example, at 8 weeks, 2 mice had 7-sq mm tumors, and the remaining 28 We reported that total polyamines (putrescine, spermi- mice had no tumor. Therefore, the mean surface area of the dine, and spermine) in urine of patients with blood and tumor of the 30 mice was calculated as follows: (2 x 7 + 28 solid cancers were significantly high and that putrescine, x 0)/30 = 0.5 (sq mm). which increased most significantly, could be of diagnostic Blood pressure was measured in the right carotid artery aid (1). Our results on the increase of urinary total polya with an electronic manometer (MPU-0.5 290; San-ei, Ja mines in blood and solid cancers agree with early reports pan). by Russell ef al. (5, 7), except for the pronounced increase Twenty-four-hr urine specimens were collected in a me in urinary putrescine (1). tabolism cage with a urine collector. Putrescine, spermi- We also reported a significant increase in urine not only dine, and spermine in 24-hr urine samples were determined of total polyamines but also of putrescine in rats with by our assay method (1). In principle polyamines were experimental stomach tumors induced by A/-methyl-/V-nitro- /V'-nitrosoguanidine; this increase in urinary putrescine was isolated with Dowex 50 column chromatography, separated by high-voltage paper electrophoresis, and finally measured observed earlier and was more pronounced (1). on a chromatogram with a Shimadzu CS-900 dual-wave On the other hand, Kallistratos ef al. (2-4) recently re length thin-layer chromatography scanner at 505 and 700 ported that 3,4-benzopyrene carcinogenesis can be delayed nm by the zigzag scanning method. Urinary polyamines or even completely inhibited by putrescine. Their results were expressed as ¿umol/mgcreatinine. prompted us to examine the effects of the injection of 3,4- benzopyrene together with putrescine to mice, and it was found that urinary putrescine increased during tumor de RESULTS velopment and that injected putrescine inhibited both 3,4- The experimental results of putrescine as inhibitor of 3,4- benzopyrene carcinogenesis and the increase in urinary putrescine. benzopyrene carcinogenesis are graphically summarized according to the method of Kallistratos (2) (Chart 1). Ani mals died within 23 weeks after a single s.c. injection of MATERIALS AND METHODS 3,4-benzopyrene due to the tumor development which was 3,4-Benzopyrene and putrescine were purchased from 100% (Group B). In contrast tumor development was com pletely inhibited in Group B + P (group treated with 3,4- Sigma Chemical Co., St. Louis, Mo., and tricaprylin was from Tokyo Kasei Kogyo Co., Tokyo, Japan. The design of benzopyrene plus putrescine). In this group 7 mice died 75 the experiments on 3,4-benzopyrene carcinogenesis and days after the injection, but none had developed a tumor on the effect of putrescine was essentially the same as that locally. In the control Group C, only 1 mouse died of reported by Kallistratos (2) except that putrescine controls pneumonia 90 days after the injection. In the control Group P, 3 mice died within 1 day, and a total of 7 mice died within 50 days, but none of the 7 had developed a tumor. In both 1To whom requests for reprints should be addressed. the B + P and P groups, while death occurring within 3 Received February 17, 1978; accepted July 18, 1978. days seemed to be due to acute toxicity of putrescine,

OCTOBER 1978 3509

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1978 American Association for Cancer Research. K. Fujita et al. especially respiratory inhibition, death later in the experi injections of 3,4-benzopyrene alone was observed as early ment was due to pneumonia. as 9 weeks (0.305 ±0.193 /xmol/mg creatinine; p < 0.05), While the B group showed no early mortality, the 17 and became marked from 12 weeks (0.373 ±0.188 ¿tmol/mg 20% mortality rates of the B + P and P groups occurred creatinine; p < 0.01), and after 16 weeks reached a maxi relatively early in the experiment, and putrescine adminis mum (0.661 ±0.490 /¿mol/mg creatinine; p < 0.01). The tration might have been toxic. Therefore, the toxicity of decrease in urinary putrescine at Weeks 17 and 18 was due putrescine was examined in detail and its 50% lethal dose to death of mice that had been excreting higher levels of by s.c. injection for BALB/c mice was found to be 1770 mg/ urinary putrescine. On the other hand, the level of urinary kg (range, 1650 to 1894 mg/kg; p = 0.95); this toxicity was spermidine (Chart 4) in the 4 groups showed no significant acute and caused death by respiratory inhibition and a fall difference at 18 weeks. Urinary spermine also did not in blood pressure. From the weight data of the surviving increase or decrease significantly with the passage of animals (Chart 2), the inhibition of tumorigenesis does not weeks. seem to be due to poor general health of the animals, which might have occurred from the chronic toxic effects of pu DISCUSSION trescine. In agreement with the results of «alustraros ef al. (2-4), Changes in urinary polyamines (putrescine, spermidine, we found that putrescine completely inhibited 3,4-benzo- and spermine) were studied during the 3,4-benzopyrene carcinogenesis experiments; s.c. tumor was noticed to develop from 8 weeks after the 3,4-benzopyrene injection (Group B). The mean size of tumors increased markedly between 14 and 18 weeks. A significant increase in urinary •¿ Õ0.6 putrescine (Chart 3) of mice of Group B which were given U

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26MEAN OF MCE 30 30 27 121026 26 23 23 22 18 3 010.A$ 34OF SURFACE AREA 0 05 IB 39152410484359»17 29 THE TUMOR (sq mm) *•••**»11 B°0IN GFtOUP 123456789 10J 12 13 14 15 16 17 18 SO 100 150 TIME (weeks) DAYS OF SURVIVAL Chart 3. Effects of a single s.c. injection of 3,4-benzopyrene, 3,4-benzo Chart 1. The effect of putrescine on 3,4-benzopyrene carcinogenesis. pyrene plus putrescine, putrescine, or the solvent (tricaprylin) on the level of Group B, 30 mice treated with a single s.c. injection of 2.52 mg of 3,4- urinary putrescine. Samples of 24-hr urine were collected in benzopyrene; Group B + P, 30 mice treated with a single s.c. injection of cages. The concentrations of polyamines were determined by isolation of 2.52 mg of 3,4-benzopyrene with 10 mg of putrescine; Group P, 15 control the amine with Dowex 50 column chromatography, separation by high- mice treated with a single s.c. injection of 10 mg of putrescine; control voltage electrophoresis, and direct dual-wavelength densitometry, as de group, 30 control mice treated with a single s.c. injection of the solvent (0.5 scribed in "Materials and Methods." •¿,GroupB (30 mice treated with a ml tricaprylin) alone. The mortality rate of 30 mice due to 3,4-benzopyrene single s.c. injection of 2.52 mg of 3,4-benzopyrene); O, Group B + P (30 carcinogenesis is shown (Group B). Animals died within 23 weeks due to mice treated with a single s.c. injection of 2.52 mg of 3,4-benzopyrene plus tumor development which was 100%. Prolongation of life of mice treated 10 mg of putrescine added to 3,4-benzopyrene solution); A, Group C [30 with 3,4-benzopyrene plus putrescine and inhibition of tumor development mice treated with a single s.c. injection of the solvent (0.5 ml of tricaprylin) was observed in Group B + P. alone); D, Group P (15 mice treated with a single s.c. injection of 10 mg of putrescine).Tumor development was observedonly in Group B from 8 weeks after the injection, when mice began to die. Numbers of surviving mice and the mean surface area of the tumors (sq mm) in Group B are also shown.

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1 2 34 56 7 8 9 10 11 12 13 14 15 16 17 18 01 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 TIME (weeks) TIME (weeks) Chart 2. Effects of a single s.c. injection of 3,4-benzopyrene, 3,4-benzo Chart 4. Effects of a single s.c. injection of 3,4-benzopyrene, 3,4-benzo pyrene plus putrescine, putrescine, or the solvent (tricaprylin) on the body pyrene plus putrescine, putrescine, or the solvent (tricaprylin)on the level of weight of mice. A, control; •¿,3,4-benzopyrene;O, 3,4-benzopyrene plus urinaryspermidine. Experimental conditions and symbolsare the same as in putrescine; D, putrescine. Charts.

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pyrene carcinogenesis. In this study we first noticed a to the interaction of benzopyrene and putrescine at the significant and specific increase of urinary putrescine in injection site and/or an effect of benzopyrene metabolism. mice with experimental tumors induced by a single s.c. The effects of the injection of putrescine at a different site injection of 3,4-benzopyrene. This increase of urinary pu and at different times relative to the carcinogen remain to trescine in mice agrees with our previous findings that be investigated. putrescine increased significantly in urine of patients with Russell has recently proposed that polyamines could be blood and solid cancers and in rats with experimental biochemical markers of tumor kinetics (6). Our previous stomach tumors induced by A/-methyl-/V-nitro-A/'-nitroso- report (1) and the present results suggest that of the guanidine (1). When compared to control rats (0.29 ±0.065 polyamines putrescine may be the biochemical marker of /¿mol/mgcreatinine), a significant increase in urinary pu tumor kinetics. trescine in experimental rats with stomach tumors after administration of A/-methyl-A/-nitro-/V'-nitrosoguanidine REFERENCES was observed as early as 20 weeks (0.32 ±0.082 ¿imol/mg creatinine; p < 0.01) and became marked after 36 weeks 1. Fujita, K., Nagatsu, T., Maruta, K., Ito, M., Senba, H., and Miki, K. Urinary Putrescine, Spermidine, and Spermine in Human Blood and (0.52 ±0.343 /¿mol/mgcreatinine; p < 0.01) (1). Therefore, Solid Cancers and in an Experimental Gastric Tumor of Rats. Cancer the present results further support the concept that putres Res.,36. 1320-1324, 1976. 2. Kailistratos, G. Putrescine as Inhibitor of 3,4-Benzopyrene Canceroge- cine concentrations in urine can be of diagnostic aid in nesis. European J. Cancer, 77: 717-719, 1975. cancers, as proposed in our previous study (1). 3. Kailistratos, G., and Fasske, E. Prevention of 3,4-Benzopyrene Carcino Interestingly, this increase of urinary putrescine in 3,4- genesis in Presence of Putrescine. Z. Krebsforsch. Klin. Onkol., 87: 81- 92,1976. benzopyrene carcinogenesis was completely inhibited by 4. Kailistratos, G., and Kailistratos, U. 3,4-Benzopyrene Carcinogenesis the injection of putrescine with 3,4-benzopyrene. and Its Inhibition by Natural and Synthetic Compounds. Folia Biochim From the animal weight data of the control Group P Biol. Graeca, 13: 1-10, 1976. 5. Russell, D. H. Increased Polyamine Concentrations in the Urine of receiving only putrescine (Chart 2), changes in tumor yield Human Cancer Patients. Nature New Biol.. 233: 144-145. 1971. and urinary polyamines may not be due to poor caloric 6. Russell, D. H. Clinical Relevance of Polyamines as Biochemical Markers of Tumor Kinetics. Clin. Chem., 23: 22-27,1977. intake caused by illness of the animals. Hence, it seems 7. Russell, D. H., Levy, C. C., Schimpf, S. C., and Hawk, I. A. Urinary likely that the inhibition of tumorigenesis seen here is due Polyamines in Cancer Patients. Cancer Res., 37: 1555-1558, 1971.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1978 American Association for Cancer Research. Increase of Urinary Putrescine in 3,4-Benzopyrene Carcinogenesis and Its Inhibition by Putrescine

Keisuke Fujita, Toshiharu Nagatsu, Kan Shinpo, et al.

Cancer Res 1978;38:3509-3511.

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