Metabolism of Dimethylnitrosamine to Mutagenic Intermediates by Kidney Microsomal Enzymes and Correlation With Reported Host Susceptibility to Kidney Tumors

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Magee and Barnes (1) first demonstrated the oncogenicity of dimethylnitrosamine (DMNA) in rats. Many nitrosamines including DMNA have subsequently been shown to be potent carcinogens in many rodent species (2, 3). Substantial evidence indicates that DMNA and other aliphatic nitrosamines are tumorigenic only after metabolic activation to proximal carcinogens by the mixed-function oxidase enzyme system present in microsomes of the liver and other organs (1, 4-10). Comparative carcinogenicity tests in rodents demonstrated distinct differences in strain and organ specificities for the neoplastic activities of DMNA (9, 11-13). Takayama and Oota (14) reported that DMNA induced tumors of the liver, lung, kidney, and testes and a low incidence of leukemia in mouse strains ICR, ddN, and C3HjAHe. Clapp et al. (15) showed that DMNA caused lung adenomas and liver hemangiosarcomas in RF jUn and BALB j c strains though no kidney tumors were reported (16). In addition to the results with ddN, ICR, and C3HjHe mice (9, 14), DMNA-induced kidney tumors were also observed in Swiss mice (13). These results indicate that organs refractile to tumor induction may be unable, or have reduced capacity, to metabolize DMNA (15). The mutagenic properties of DMNA were also studied extensively in several organisms (17-21) and indicate that the parental compound is not active initially but requires conversion in vivo or in vitro to mutagenic metabolites. Such findings suggest an association between the induction of neoplastic and mutagenic events. The presence of a correlation between the formation of the proximal carcinogen and the proximal mutagen in specific strains and organs of differentially susceptible miee enhances the proposal that the two phenomena are related and that a single metabolic intermediate may be involved in the induction of both events. In this report the capability of kidney microsomal enzymes from HajlCR, RF jJ, BALBjcJ, and C3HjHeJ mice to activate DMNA to a mutagen in vitro is compared with susceptibility of the four strains to DMNAinduced kidney tumors in vivo. MATERIALS AND METHODS

The preparation of purified microsomes and of reaction mixture, and the in vitro microsomal enzyme assay

were described in (22). DMNA (Eastman Kodak Co., Rochester, N.Y.) was used at a final concentration of 100 p.molesjml. Salmonella typhimurium(his-, G-46), a missense mutation, was obtained from Dr. Bruce Ames, University of California at Berkeley, Berkeley, California (23). Yeast tryptone broth was used for bacteria growth (22). Yeast tryptone agar and Spizizen's minimal agar (24) were used for the determination of the total population and his: revertants, respectively. Control tests were done with components of the in vitro assay minus DMNA, as well as chemical controls consisting of the assay components plus DMNA but minus microsomal enzymes. The average spontaneous frequency observed in the control tests was one revertant/ l O" surviving cells. RFjJ, BALBjcJ, and C3HjHeJ male mice, 8-9 weeks old, were obtained from The Jackson Laboratory, Bar Harbor, Maine. Outbred HajlCR albino Swiss male mice, 8-9 weeks old, were from Sprague-Dawley, Madison, Wisconsin. Kidneys removed from 15-20 mice were pooled to obtain a single microsomal enzyme preparation. Replicate activation tests were conducted to ensure that microsome activity would not be lost over the period the preparation was used. RESULTS AND DISCUSSION

The metabolism of DMNA involves an initial oxidative dealkylation catalyzed by N-demethylase activity (6, 8, 10). Further, the oxidative demethylation of DMNA by isolated mouse liver microsomes and the activation of DMNA to a bacterial mutagen followed similar kinetics (25); thus the induction of biologically active intermediates of DMNA as measured by mutation induction frequencies in bacterial indicator cells in vitro can accurately reflect the rate and degree of the enzymatic activation of this compound. Purified kidney microsomal enzymes obtained from HajlCR and C3HjHeJ mice exhibited an unusually high capability to generate mutagenic intermediates from DMNA when compared with those from BALBjcJ and RF /J strains (text-fig. 1). Table 1 demonstrates the reproducibility of this effect by the comparison of HajlCR and BALBjcJ mutation induction tests. In each instance, kidney microsomes from Ha j ICR mice produced considerably higher mutation frequencies than did BALBjcJ kidney microsomal preparations. The levels and rates of DMNA activation by the HajlCR and C3HjHeJ kidney microsomal enzymes proReceived April 21, 1975; accepted July 16, 1975. Supported by grant IROI FD00521 from the Food &: Drug Administration, Public Health Service grant IPOI CA14718 from the National Cancer Institute, and Howard University General Research Support grant 470K-9. 8 Department of Microbiology, College of Medicine, Howard University, Washington, D.C. 20001. Present address: Department of Genetics, Litton Bionetics, Inc., 5516 Nicholson Lane, Kensington, Md. 20795. 4 I am grateful to Dr. David Brusick for his valuable suggestions and discussions during the preparation of this manuscript. 1

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JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 55, NO.5, NOVEMBER 1975

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SUMMARY-Purified kidney microsomal enzymes from C3H/HeJ and Ha/lCR mice exhibited an unusually high capacity to generate mutagenic metabolites from dimethylnitrosamine (DMNA) when compared with similar enzyme preparations from BALB/cJ and RF/J mice. These results suggested that the DMNA-activating enzymes involved in mutagen formation were either present at higher levels or were more active in the kidneys of C3H/HeJ and HallCR mice than in those of the other two strains. This strain difference correlated with the established susceptibility of these four strains to the neoplastic activity of DMNA.-J Nat! Cancer Inst 55: 1199-1201, 1975.

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TABLE 2.-Qrgan weight and microsomal protein content of

Ha/ICR, BALB/cJ, C3H/HeJ, and RF /J kidneys

Strain TEXT-FIGURE I.-Comparison of the general activity of DMNA metabolites " prod uced by kidney ~ microsomal enzymes from S,oil four strains of mice. Each" curve represents the number of his+ revertan ts of ~ G-46/lO s surviving cells i corrected for spontaneous frequency, "

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TABLE I.-Activation of DMNA to genetically active intermediates by kidney microsomes from BALB/cJ and Ha/ICR male mice a Reversion frequency X 10- 8 c Experiments - - - - - - - - - - - - - - - - - - - - - - Control BALB/cJ Ha/ICR 0.40 0.08 0.23

7882.35 7253.52 6903.23

1405.60 1040.20 1051.80

• Jagannath DR: Unpublished results. • Each set of data represents an independent expt with fresh activating enzymes from the respective mouse strains. , Duration of exposure 'to DMNA was 60 min.

duced mutagenic effects unlike those generated by enzymes from either of the other strains tested, but were more like the kinetics exhibited by hepatic microsomal enzymes (text-fig. 2). This indicated a high level of activity for the de alkylation enzyme in the kidneys of these two strains when compared to that of BALB/cJ or RF/J mice. However, the capacity for metabolic activation of DMNA by three other organs of the Ha/lCR mice examined (liver, lung, testes) was similar to that of the BALB/cJ and RF /J strains (26). Studies of liver and lung microsomes of C3H/HeJ mice also showed similarities to those of BALB/cJ and RF /J mice. These results

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TEXT-FIGURE 2.-o:>mparison of the genetic activity of DMNA metabolites "I produced by liver microsomal enzymes from four ,. strains of mice. Each ;; curve represents the number of his+ revertan ts of ! G-46/108 surviving cells corrected for spontaneous frequency.

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2.418 2.074 2.071 2.720

• Based on averages from 15-18 mice. • Relative protein concentrations of dispersed 100,00 Xu microsome pellets were measured at 280 nm with a Gilford 2400 spectrophotometer. Samples were compared to a standard of 0.25 M sucrose.

indicate that in the strains studied, two levels of DMNA activation were observed in microsome preparations from the kidneys, whereas in the microsome preparations from other organ comparisons, only a single level of activity was seen. The kidneys of C3H/HeJ and Ha/lCR animals were not abnormally large, though total protein content of the Ha/lCR and C3H/HeJ preparations as measured by UV spectroscopy did show a slight increase compared to RF /J and BALB/cJ mice (table 2). However, it is not known if the higher protein content was related to the increased metabolic activity of the kidney microsomes from these strains. Studies to determine cytochrome P450 content of the microsome preparations are in progress. The high level of DMNA activation by C3H/HeJ and Ha/lCR kidney enzymes can be demonstrated by tumor production in vivo as well as by mutagen production in vitro. Clapp and Toya (16) observed that no kidney tumors were induced in BALB/c or RF mice after DMNA administration by feeding or in drinking water, whereas Takayama and Oota (14) studied tumor induction in C3H and ICR mice after similar exposure to DMNA and observed kidney tumors in 16.0 and 5.7% of the treated mice, respectively. High incidences of liver and lung tumors were induced in all four strains. There were no spontaneous kidney tumor frequencies reported for any strains in either study. The demonstration of a correlation between the degrees of susceptibility to DMNA-induced tumors of certain organs in the mouse strains examined in this study and the differential ability of these organs to activate DMNA to mutagenic metabolites in vitro provide additional support for proposals linking mutagenicity and carcinogenicity. These findings also demonstrate the importance of genetic factors involved in tumor etiology in mammals; additional genetic studies are in progress to determine the number of loci controlling the DMNAdemethylase enzyme in mice. REFERENCES

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(1) MAGEE PN, BARNES JM: The production of malignant primary hepatic tumors in the rat by feeding dimethylnitrosamine. Br J Cancer 10:114-121, 1956 (2) ARGUS MF, HOCH-LIGETI C: Induction of malignant tumors in the guinea pig by oral administration of diethylnitrosamine. J Nat! Cancer Inst 30:533-551, 1963 (3) SCHMAHL D, PREUSSMANN R: Cancerogene Wirkung von Nitrosodimethylamin bei Ratten. Naturwissenschaften 46: 175, 1959 (4) BROUWERS JA, EMMELOT P: Microsomal N-demethylation and the effect of the hepatic carcinogen dimethylnitrosamine on amino acid incorporation into the proteins of rat livers and hepatomas. Exp Cell Res 19:467-474, 1960

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KIDNEY METABOLISM OF DMNA TO MUTAGENIC INTERMEDIATES

(17) (18) (19) (20)

(21) (22)

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rate on dimethylnitrosamine oncogenesis in RF mice. J Natl Cancer Inst 45:495-498, 1970 GABRIDGE MG, LEGATOR MS: A host-mediated microbial assay for the detection of mutagenic compounds. Proc Soc Exp BioI Med 130:831-834, 1969 GEISSLER E: Uber Die Wirkung von Nitrosaminen auf Mikroorganismen. Naturwissenschaften 49:380-381, 1962 MALLING HV: Mutagenicity of two potent carcinogens, dimethylnitrosamine and diethylnitrosamine in Neurospora crassa, Mutat Res 3:537-540, 1966 MARQUARDT H, ZIMMERMAN FK, SCHWAIER R: Die Wirkung Krebsauslosender Nitrosamine and Nitrosamide auf das Adenin-6-45-ruckmutationssystem von Saccharomyces cereuisiae. Naturwissenschaften 98:82-96, 1964 PASTERNAK L: Mutagene Wirkung von Dimethylnitrosamine bei Drosophila melanogaster. Naturwissenschaften 49:381, 1962 GLETTEN F, WEEKES U, BRUSICK D: In vitro metabolic activation of chemical mutagens. I. Development of an in vitro mutagenicity assay using liver enzymes for activation of dimethylnitrosamine. Mutat Res 28: 113--122, 1975 AMES BN: The detection of chemical mutagens with enteric bacteria. In Chemical Mutagens: Principles and Methods for Their Detection (Hollaender A, ed.) . New York, Plenum Press, 1971, pp 267-282 SPIZIZEN J: Transformation in biochemically deficient strains of Bacillus subtilis by deoxyribonucleic acid. Proc Natl Acad Sci USA 44:1072-1078, 1958 CZYGAN P, GREIM H, GARRO AJ, et al: Microsomal metabolism of dimethylnitrosamine and the cytochrome P-450 dependency of its activation to a mutagen. Cancer Res 33:29832986, 1973 WEEKES U, BRUSICK D: In vitro metabolic activation of chemical mutagens. II. The relationships among mutagen formation, metabolism and carcinogenicity for dimethyl- and diethylnitrosamine in the livers, kidneys and lungs of BALB/cJ, C57BL/6J and RF IJ mice. Mutat Res 31:175-184, 1975

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(5) DONTENWILL W, MOHR U, ZAGEL M: Uber die Unterschiedliche Lungencarcinogene Wirkung des Diathylnitrosamin bei Hamster und Ratte. Z Krebsforsch 64:499-502, 1962 (6) GILLETTE JR: Metabolism of drugs and other foreign compounds by enzymatic mechanism. Progr Drug Res 6:13--73, 1963 (7) HULTIN T, ARRHENIUS EA, Low H, et al: Toxic liver injury, inhibition by dimethylnitrosamine of incorporation of labeled amino acids into proteins of rat-liver preparations in vitro. Biochem J 76:109-Il6, 1960 (8) MANNERING GJ: Significance of stimulation and inhibition of drug metabolism in pharmacological testing methods. In Selected Pharmacological Testing Methods (Burger A, 00.) . New York, Marcel Dekker, 1968, pp 51-IlO (9) TAKAYAMA S, OOTA K: Malignant tumors in mice fed with N-nitrosodimethylamine. Gann 54:465-472, 1963 (10) UEHLEKE H: N-Hydroxylation of carcinogenic amines in vivo and in vitro with liver microsomes, Biochem Pharmacol 12:219-221, 1963 (II) MAGEE PN, BARNES JM: Induction of kidney tumors in the rat with dimethylnitrosamine (N-nitrosodimethylamine). J ~athol Bacteriol 84:19-31, 1962 (12) ZAK FG, HOLZNER JH, SINGER EJ, et al: Renal and pulmonary tumors in rats fed dimethylnitrosamine. Cancer Res 20: 96-99, 1960 (13) TERRACINI B, PALESTRO G, GIGLIARD MR, et al: Carcinogenicity of dimethylnitrosamine in Swiss mice. Br J Cancer 20: 871-876, 1966 (14) TAKAYAMA S, OOTA K: Induction of malignant tumors in various strains of mice by oral administration of N-nitrosodimethylamine and N-nitrosodiethylamine. Gann 56: 189-199, 1965 (15) CLAPP NK, TYNDALL RL, OTTEN JA: Differences in tumor types and organ susceptibility in BALB/c and RF mice following dimethylnitrosamine and diethylnitrosamine. Cancer Res 31:196,1971 (16) CLAPP NK, TOYA RE SR: Effect of cumulative dose and dose

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Metabolism of dimethylnitrosamine to mutagenic intermediates by kidney microsomal enzymes and correlation with reported host susceptibility to kidney tumors.

Purified kidney microsomal enzymes from C3H/HeJ and Ha/1CR mice exhibited an unusually high capacity to generate mutagenic metabolites from dimethylni...
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