Cellular Injury and Carcinogenesis INHIBITION of METABOLISM of DIMETHYLNITROSAMINE by AMINOACETONITRILE

Biochem. J. (1970) 120, 601-605 601 Printed in Great Britain

Cellular Injury and Carcinogenesis INHIBITION OF METABOLISM OF DIMETHYLNITROSAMINE BY AMINOACETONITRILE

By L. FIUME,* GABRIELLA CAMPADELLI-FIUME,* P. N. MAGEE AND J. HOLSMAN Courtauld In8titute of Biochemi8try, Middle8ex Hospital Medical School, London W.1, U.K. (Received 15 June 1970)

1. Aminoacetonitrile, a lathyrogenic agent known to decrease the hepatotoxic action of dimethylnitrosamine, inhibited the metabolism of this compound by rats in vivo and by rat liver slices in vitro. 2. Methylation ofnucleic acids in rat liver and kidneybydimethylnitrosamineinvivowasinhibitedby treatment ofthe animalswith aminoacetonitrile. 3. These findings are discussed in relation to the hypothesis that dimethylnitrosamine requires metabolism to exert its hepatotoxic and carcinogenic action.

Aminoacetonitrile, a lathyrogenic compound tive action of aminoacetonitrile could be attributed (Wawzonek, Ponseti, Shepard & Wiedenman, to this mechanism. 1955), was shown to inhibit the production of cir- The experiments reported in the present paper rhosis of the liver in rats by chronic administration were undertaken to elucidate this point and to gain of carbon tetrachloride (Fiume & Favilli, 1961). further information on the inhibition ofmetabolism It prevents the necrosis of the liver induced by this of dimethylnitrosamine by aminoacetonitrile. hepatotoxin (Fiume, 1962a; Fiume & Laschi, 1963) and also the necrosis caused by allylamine (Fiume, 1962b), bromobenzene (Fiume, 1963), MATERIALS AND METHODS chloroform and dimethylnitrosamine (Fiume, Animal&. Female Wistar albino rats were maintained 1962c). on Rowett diet 81. The available evidence suggests that dimethyl- Chemical&. Dimethylnitrosamine was obtained from nitrosamine is not itself hepatotoxic but that it is British Drug Houses Ltd. (Poole, Dorset, U.K.) and purified by distillation (b.p. 1510C). Aminoacetonitrile metabolized into a methylating intermediate bisulphate was obtained from Koch-Light Laboratories compound which is the actual toxic agent (see Ltd. (Colnbrook, Bucks., U.K.). Magee & Barnes, 1967; Magee & Swann, 1969). Radioactive material. [14C]Dimethylnitrosamine was Among the earliest biochemical changes after a prepared by Dr D. F. Heath, by the method of Dutton & dose of dimethylnitrosamine sufficient to cause Heath (1956). The radioactive material (4.8mCi/mmol; liver necrosis is inhibition ofprotein synthesis in the 3.06 mg/ml ofwater) was diluted with unlabelled dimethyl- liver (Magee, 1958). Aminoacetonitrile adminis- nitrosamine before use. The labelled starting material tration was shown to prevent this early inhibition of was ['4C]methyl iodide (30mCi/mmol) purchased from hepatic protein synthesis (Fiume, 1964; Mager, The Radiochemical Centre, Amersham, Bucks., U.K. that Measurement of radioactivity. A Packard Tri-Carb Halbreich & Bornstein, 1965) suggesting its model 3320 liquid-scintillation spectrometer was used. protective action might be mediated by inhibition Corrections from c.p.m. to d.p.m. were made either by the of the metabolism of the nitrosamine. The finding addition of standard [14C]toluene (Packard Instrument by Fiume & Roffia (1965) that the concentration of Co., La Grange, Ill., U.S.A.) or by the channel-ratios dimethylnitrosamine in the livers of rats treated method (Baillie, 1960). Three scintillation solutions were with aminoacetonitrile was higher than that when used: 0.6% 2,5-diphenyloxazole in toluene for samples the animals received the nitrosamine alone gave that did not contain water; 1% 2,5-diphenyloxazole, support to the hypothesis that metabolism of 0.05% 1,4-bis-(4-methyl-5-phenyloxazol-2-yl)benzene and dimethylnitrosamine was inhibited. However, the 8% naphthalene dissolved in dioxan-Cellosolve-toluene of data on the extent of this inhibition made it (3:3:1, by vol.) (Bruno & Christian, 1961) was used for lack aqueous samples; this second scintillation solution plus impossible to ascertain whether the entire protec- 4% Cab-O-Sil (Packard Instrument Co.) was used to * On leave of absence from Department of General suspend Ba14CO3 precipitates. Nucleic acids and proteins Pathology, University ofBologna, Italy. Present address: were dissolved in Hyamine (Packard Instrument Co.). Max-Planck-Institut fur Medizinische Forschung, Abtei- Metaboli8m of dimethylnitrosamine in vivo. (a) With lung Chemie, Heidelberg, Germany. non-radioactive dimethylnitrosamine. Rats were given 602 L. FIUME, G. CAMPADELLI-FIUME, P. N. MAGEE AND J. HOLSMAN 1970 dimethylnitrosamine (30mg/kg body wt.) dissolved in Presentation of results. The results are presented as 0.9% NaCl in a volume of 5ml/kg body wt. by intraperi- means +S.D., with Student's t test for significance of toneal injection. At the time of dimethylnitrosamine difference between means where appropriate. administration some rats were injected subcutaneously with aminoacetonitrile, 200mg/kg body wt., as a 20% (w/v) solution of the bisulphate neutralized with NaOH. RESULTS At increasing time-intervals the rats were anaesthetized of aminoacetonitrile on the metabolism of with sodium pentobarbital and blood was withdrawn Effect from the abdominal aorta into a syringe moistened with dimethylnitrosamine. The rate of disappearance of heparin solution. Dimethylnitrosamine was determined dimethylnitrosamine from the circulating blood of in blood samples by the polarographic method of Heath & rats to which it had been administered was greatly Jarvis (1955). decreased by treatment of the animals with amino- (b) With ['4C]dimethylnitrosamine. Rats received acetonitrile (Fig. 1). The rate of clearance of [14C]dimethylnitrosamine, (30mg and 20,uCi/kg body wt.) dimethylnitrosamine from the blood has been shown in 5 ml of 0.9% NaCl/kg body wt., by intraperitoneal injec- by Heath (1962) to correspond closely to the rate of tion. Aminoacetonitrile was administered subcutaneously its metabolism, which therefore appeared to be either as one dose (200mg/kg body wt.) simultaneously strongly inhibited. with the dimethylnitrosamine or as repeated doses The inhibition of metabolism of dimethylnitro- (100mg/kg body wt.) on the preceding 2 days and at the same time as the dimethylnitrosamine. Each rat was samine by aminoacetonitrile was confirmed by placed individually in a metabolism cage and air was measurement of the rate of exhalation of 14C02 by drawn through the cage and passed through three cylinders, rats treated with [14C]dimethylnitrosamine. each containing 400ml of 4M-NaOH, which was replaced Animals receiving aminoacetonitrile simultaneously every 8-9 h. Samples of the alkaline solution were taken with the nitrosamine, with or without treatment on at various times and the radioactive carbonate was the preceding 2 days, showed marked inhibition in precipitated with BaCl2 after the addition, when neces- their output of 14C02 (Fig. 2). sary, of carrier Na2CO3. The precipitate of BaCO3 was Dimethylnitrosamine is metabolized by rat liver washed repeatedly with water and assayed for radio- slices and, at a slower rate, by rat kidney slices activity. in vitro (Magee & Metabolism of dimethylnitrosamine in vitro. Liver and Vandekar, 1958; Montesano & kidney slices were cut with a hand microtome. About Magee, 1970). Addition of aminoacetonitrile to 150mg of slices was incubated at 37°C for 60min in 2.2 ml liver and kidney slices in vitro inhibited metabolism of Krebs-Ringer phosphate buffer (Umbreit, Burris & of [14C]dimethylnitrosamine, as shown by the lower Stauffer, 1964) containing 20,umol of glucose in a con- production of 14C02 (Table 1). ventional Warburg apparatus. ['4C]Dimethylnitrosamine Effect of aminoacetonitrile on methylation of liver (542 nmol, 0.01OtCi) dissolved in 0.2 ml of the phosphate buffer was added from the side arm after the flasks had been allowed to equilibrate at 37°C. The gas phase was 02 and '4C02 was trapped in 4 M-NaOH in the centre wells of the flasks. The radioactive carbonate, after addition of 0 carrier Na2CO3, was precipitated with BaCl2, washed 0 repeatedly with water, and assayed for radioactivity. .oz Methylation of nucleic acids and isolation of labelled protetns. Groups of eight rats were starved overnight and S ce injected intraperitoneally with [14C]dimethylnitrosamine CL (30mg and 20,uCi/kg body wt.) in 0.9% NaCl. Four rats o b4 received dimethylnitrosamine alone and the other four +' - rats were injected subcutaneously on the previous 2 days r. and at the time of dimethylnitrosamine administration Pa IR with aminoacetonitrile (100mg/kg body wt.). The animals were killed by exsanguination and the livers and kidneys were rapidly transferred to liquid N2. Total nucleic acids 0 16 24 32 40 48 were extracted from about 1 g of liver and from both Time (h) kidneys of each animal by the method of Schneider (1945). DNA and RNA were isolated separately from pooled livers Fig. 1. Effect of aminoacetonitrile on the disappearance of from each group of rats as described by Kidson, Kirby & injected dimethylnitrosamine from the blood. All rats Ralph (1963), as modified by Swann & Magee (1968). The received dimethylnitrosamine (30mg/kg body wt.) by nucleic acids were hydrolysed in m-HCI for 1 h at 100°C intraperitoneal injection. Those treated with amino- and chromatographed as described by Magee & Farber acetonitrile received 200mg/kg body wt. subcutaneously (1962). The extent of methylation was measured by the at the same time. Rats were killed after increasing time- amount of 7-methylguanine expressed as a percentage of intervals and the concentration of dimethylnitrosamine in the total guanine (Swann & Magee, 1968). Proteins were the blood was measured. *, Dimethylnitrosamine alone; isolated by the method of Rabinovitz, Olson & Greenberg o, dimethylnitrosamine+aminoacetonitrile. The calcu- (1954). lated regression lines are shown. Vol. 120 DIMETHYLNITROSAMINE AND AMINOACETONITRILE 603 50 and kidney nucleic acid8 by dimethylnitro8amin e in vivo. The marked inhibitory effect of aminoacetonitrile on metabolism of dimethylnitrosamine suggested that it might also inhibit methylation of 040- nucleic acids by the nitrosamine. Rats were given ['4C]dimethylnitrosamine at a dosage (30 mg/kg body wt.) that would induce necrosis of the liver in all and tumours of the liver in some (about 20%) of * 30 - the surviving treated animals. Nucleic acids and proteins were extracted from the livers and kidneys at increasing time-intervals after the injections and assayed for radioactivity. Aminoacetonitrile inhi- 20 - bited incorporation ofthe label into proteins of both 20 organs at 4h and 12h after injection ofthe dimethyl- 0 nitrosamine and the inhibition was still apparent in

0 1 the kidney at 18h, but the incorporation in the liver oA0 was not significantly different from that in the control at this time (Table 2). Incorporation into '4 total plasma proteins was also measured at 12h, when the mean specific radioactivity in four rats 0 8 16 24 30 afterdimethylnitrosamine alone was 332± 31 d.p.m./ Time (h) mg and that in four rats that also received amino- Fig. 2. Effect of aminoacetonitrile on the exhalation of acetonitrile was 147±34d.p.m./mg, indicating an 14C02 after injection of [14C]dimethylnitrosamine. All inhibition comparable with that observed in the rats received [14C]dimethylnitrosamine (30mg, 201ACi/ liver kg body wt.). Each point represents the mean of values and kidney proteins. Since these inhibitions from two rats. 0, [14C]Dimethylnitrosamine alone; A, might have been due to an effect of aminoaceto- [14C]dimethylnitrosamine+aminoacetonitrile (200mg/kg nitrile on the distribution of the dimethylnitro- body wt.) simultaneously; A, [14C]dimethylnitrosamine+ samine in the bodies of the animals the radioactivi- aminoacetonitrile (100mg/kg body wt.) on the preceding ties of the acid-soluble fractions of the liver and 2 days and at the same time as the nitrosamine. kidney were measured at 12h. The mean acid-soluble

Table 1. Effect of aminoacetonitrile added in vitro on the metabolim of [14C]dimethylnitro8amine by liver and kidney 8lice8 The metabolism of [14C]dimethylnitrosamine was measured by measuring the amounts of 14C02 produced during incubation with tissue slices in a conventional Warburg apparatus as described in the Materials and Methods section. The number of flasks used are given in parentheses. Production of 14C02 (d.p.m./min per g wet wt. of tissue) Liver Kidney Control 44.8± 8.8 (8) 11.7 1.6 (5) Aminoacetonitrile (0.5mM) 15.3±3.0 (8) (66% inhibition) 8.1 t 0.6 (P < 0.01) (5) (31% inhibition) Aminoacetonitrile (5mM) 6.1 ± 1.06 (6) (86% inhibition)

Table 2. Effect of aminoacetonitrile on the 8pecific radioactivity ofprotein8from liver8 and kidney8 of rats given [14C]dimethylnitro8amine All rats received [14C]dimethylnitrosamine (30mg, 20 &Ci/kg body wt.) by intraperitoneal injection and those treated with aminoacetonitrile received 100mg/kg body wt. ofthis compound subcutaneously at the same time as the injection of the nitrosamine and on the previous 2 days. There were four animals per group. Time after injection of Liver proteins (d.p.m./mg) Kidney proteins (d.p.m./mg) [14C]dimethylnitrosamine (h) -Aminoacetonitrile +Aminoacetonitrile -Aminoacetonitrile +Aminoacetonitrile 4 120±10 48± 7 97± 13 24± 2 12 298±19 112±26 196±29 62±16 18 117±28 112±18 107±11 74± 8 604 L. FIUME, G. CAMPADELLI-FIUME, P. N. MAGEE AND J. HOLSMAN 1970 radioactivities in the livers and kidneys of the rats receiving dimethylnitrosamine alone were 20 830 ± .9 m 3683d.p.m./g wet wt. of tissue and 10200+ '4 2180d.p.m./g respectively and in the animals t0 o Caa co receiving dimethylnitrosamine plus aminoaceto- 404 ~44- nitrile the corresponding values were 39500 ± 0 4124d.p.m./g and 40620 ± 3277 d.p.m./g respectively. These results indicate that there was prob- ° J -HZ ably no failure of the nitrosamine to reach the two 4 organs. The higher amounts of total acid-soluble ;> 0 radioactivity in the organs of the rats receiving d4+I aminoacetonitrile is consistent with the greatly 'H lowered rate of metabolism of the nitrosamine 'P o I O 454. 0 (Table 1; Figs. 1 and 2). Ccda2 d The incorporation of radioactivity from labelled C) C; dimethylnitrosamine into cellular components s4 ((D4 0 ° probably follows two different pathways (Magee & 0 O Farber, 1962). After initial demethylation form- s eas IS aldehyde is produced, which enters the normal c4$4 metabolic pool of Cl intermediates and becomes 0 incorporated into nucleic acids and proteins by normal metabolic processes. In addition to this Q 4a M pathway the active methylating intermediate '4OQ that is formed metabolically reacts with proteins, nucleic acids and probably other cellular components. In the nucleic acids of organs of rats e 'e t dS '4 treated with dimethylnitrosamine the main site of -, I-. Z-t methylation is on the 7-position of guanine (Magee X00 0 so lOco & Farber, 1962; Lawley, Brookes, Magee, Craddock - l X @ A-> & 4-3 0 -H -H Swann, 1968) and the extent of methylation has Tl been expressed as the percentage of the nucleic acid V: guanines converted into 7-methylguanine (Swann & l o Magee, 1968). Methylation of nucleic acids of liver and kidney was measured in groups of rats killed at 4a a 4Q Ca intervals after injection with [14C]dimethylnitrosamine with or without aminoacetonitrile. In some 4, cases DNA plus RNA was extracted with hot trichloroacetic acid by the Schneider (1945) method

'4 -4D and in others DNA and RNA were prepared o * ~ 01 separately. Aminoacetonitrile clearly inhibited *t _ methylation of both nucleic acids at all times z4 01 studied (Table 3). ca S5 -.! 00 E"u3d-4* X z4 DISCUSSION 4cD 98 00 The experiments reported above clearly show Ce p'4m that aminoacetonitrile inhibits the metabolism of vivo in seems dimethylnitrosamine in and vitro. It 01X probable that the inhibition of metabolism of *44 d g g dimethylnitrosamine in vivo is strong enough to 0- E> d _ account for the protective action of aminoaceto- 0 nitrile against the liver lesions induced by the nitro- e Em- samine. The metabolic inhibition may also explain the ability of aminoacetonitrile to prevent d the appearance of liver tumours in rats fed on C a diet containing a carcinogenic concentration * ^ of dimethylnitrosamine (E. Bonetti & L. Fiume, unpublished work) since the available evidence Vol. 120 DIMETHYLNITROSAMINE AND AMINOACETONITRILE 605 indicates that dimethylnitrosamine requires meta- Fellowship arranged between the European Science bolic activation for its carcinogenic as well as for its Exchange Programme and the Accademia Nazionale dei hepatotoxic activity (see Magee & Barnes, 1967; Lincei of Rome. Magee & Swann, 1969). If the protective action of aminoacetonitrile REFERENCES against the toxicity of dimethylnitrosamine is due Baillie, L. A. (1960). Int. J. appl. Radiat. I8otope8, 8, 1. to inhibition of metabolism of the latter, it is Bruno, G. A. & Christian, J. E. (1961). Analyt. Chem. 33, possible that a similar mechanism may operate 1216. when aminoacetonitrile modifies the action of other Dutton, A. H. & Heath, D. F. (1956). J. chem. Soc. p. 1892. hepatotoxins. There is evidence largely from the Farber, E., Shull, K. H., Villa-Treviino, S., Lombardi, B. work of & Thomas, M. (1964). 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