[CANCER RESEARCH 37, 4572-4579, December 1977] In Vivo Formation of N-Nitroso Compounds and Detection of Their Mutagenic Activity in the Host-mediated Assay1 Rolf Braun, JörgSchöneich, and Dieter Ziebarth Zentralinstitut fürGenetik und Kulturpflanzenforschung der Akademie der Wissenschaften der DDR. 4325 Gatersleben [R. B., J. S.], and Zentralinstitut für Krebsforschung der Akademie der Wissenschaften der DDR, 1115 Berlin-Buch ¡D.Z.], German Democratic Republic SUMMARY nitrite for carcinogenic activity in long-term animal studies (For reviews, see Refs. 28 and 35). In addition to the The formation of /V-nitroso compounds in mouse stom carcinogenic hazards of exposure of humans to /V-nitroso ach from equimolar doses of sodium nitrite and secondary compounds formed intragastrally from nonactive precur amines or alkylurea derivatives given simultaneously by a sors, a genetic risk cannot be excluded since most N- stomach tube was estimated by measuring the mutagenic nitroso compounds are strong mutagens (for reviews, see activity of the compounds in the i.p. host-mediated assay Refs. 29 and 31). The mutagenic and DMA-damaging activi with the use of Salmonella typhimurium TA1950 as genetic ties of A/-nitroso derivatives from agricultural chemicals of indicator system. A mutagenic response in the bacteria the carbamate type are well documented (6, 15, 16, 44, 47). was found after administration of the cyclic amines pipera- Soon after it was found that primary amines and nitrite zine dihydrochloride, morpholine, and amitrole. The high react in vitro to form mutagenic products (22), host-me est mutagenicity was exerted by piperazine dihydrochloride diated assay studies revealed the mutagenic activity of plus nitrite, while amitrole plus nitrite was only weakly combined application of nitrite and the secondary amine mutagenic. No mutagenic activity was observed for equi DMA2 or the alkylurea derivatives Mil and EU (9, 38). molar doses of sodium nitrite plus dimethylamine hydro- The present study was undertaken to determine the chloride, diphenylamine. methylbenzylamine hydrochlo- relationship between chemical structure and properties of ride, and phenmetrazine hydrochloride. All /V-alkylurea de secondary amines and alkylureas. It investigated the ability rivatives tested were found to yield significant amounts of of these compounds to undergo /V-nitrosation in the mouse /V-nitroso compounds, which allowed detection of their stomach, eventually resulting in the formation of mutagenic mutagenic activity in the host-mediated assay. The highest products detectable in the i.p. host-mediated assay with activity was shown by nitrite plus ethylenebis(thiourea), the use of Salmonella typhimurium TA1950 as genetic while methylurea and ethylurea were found to be less active indicator system. This system was introduced into muta in combination with nitrite. Dose-response curves for the genicity testing by Garbridge and Legator (18) to bridge mutagenic activity of /V-nitrosamines were used to estimate the discrepancy between mammalian and microbial bio- the amounts of /V-nitroso derivatives formed in vivo from transformation of xenobiotic compounds, and it has been the precursors after acute treatment of the mice. In the used successfully to detect the mutagenic potential of case of piperazine dihydrochloride, nitrosation of 50 to nitrosamines and -amides (7, 32, 55, 56). The host-mediated 70% was estimated, while for morpholine nitrosation assay represents a useful method for the identification of ranged from 1 to 3%. The results are compared with those the mutagenic activity of /V-nitroso compounds generated obtained in long-term carcinogenesis studies with sodium in vivo from secondary amines and amides because, first, nitrite plus amines. nitrosation takes place in the acidic environment of the host animal's stomach, and second, the nitrosamines and -amides preferentially induce gene mutations (49, 51) de INTRODUCTION tectable in this system. The metabolic activation of the promutagenic nitrosamines takes place in the biotransfor Since Druckrey ef al. (11) discussed the formation of mation system of the host animals, while the ultimate carcinogenic /V-nitroso compounds from sodium nitrite and mutagens are detected by the i.p.-growing indicator bacte secondary amines of /V-alkylureas under the acidic condi ria. tions of the mammalian stomach, much experimental work The mice used as host animals were treated with the has been done to explore this hypothesis from the point of precursor compounds plus equimolar amounts of sodium view of carcinogenic risk to humans. Besides the analysis nitrite, and the formation of /V-nitroso compounds was of in vitro /V-nitrosation in buffer solutions (12-15, 23, measured via the mutagenic response of the indicator 41, 48, 52) and human gastric juice (57, 58), more than 30 secondary and tertiary amines, alkylureas. and other nitros- able W-compounds have been tested in combination with 1The abbreviations used are: DMA. dimethylamine hydrochloride; MU. methylurea; EU, ethylurea: AMT. amitrole; ETU, ethylenebis(thiourea); DPA. diphenylamine; MOR, morpholine; PMZ, phenmetrazine hydrochloride; DMSO. dimethyl sulfoxide; NDMA. W-nitrosodimethylamine; MBA, methyl 1 Presented in part at the Sixth Annual Meeting of the European Environ benzylamine hydrochloride: PZ, piperazine dihydrochloride; NPZ. 1-nitroso- mental Mutagen Society. Gernrode. 1976 (36. 37). piperazine: DiNPZ, 1,4-dinitrosopiperazine; NMBA, N-nitrosomethylben- Received March 24. 1977: accepted August 11. 1977. zylamine: NMOR. /V-nitrosomorpholine; NPMZ, N-mtrosophenmetrazine. 4572 CANCER RESEARCH VOL. 37 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1977 American Association for Cancer Research. Mutagenic N-Nitroso Compounds Formed in Vivo bacteria. Also, dose dependency of mutagenic activity of Each dose of a test substance or nitrite:compound mixture the nitrosamines thought to be formed was studied to was tested in a group of 6 animals against a control group compare the mutagenic activity of the precursors plus of 6 animals receiving only the solvent. All experiments nitrite with that of the final reaction products. were performed at least twice. Changes in application volume, time of treatment, or solvent used are indicated in the appropriate tables and charts. Three or 5 hr after MATERIALS AND METHODS bacterial injection, the animals were killed by cervical dislocation, and the bacteria were recovered from the Media. The bacterial cultures were maintained on nutri peritoneal cavity after injection of 2 ml 0.9% NaCI solution. ent agar slants at 4 and were grown for use in nutrient Three-tenths ml exúdate of each mouse in each group broth (Sevac, Czechoslovakia) containing 7.5 g meat ex were pooled together on nutrient agar plates for estimation tract, 12.5 g peptone, and 5 g NaCI in a final volume of 1 of the viable bacteria (10 6 dilution, 0.1 ml per plate, 4 liter, his' revertants were scored on Spizizen's minimal plates per estimation). The bacterial titer within the perito medium (52) supplemented with an excess of biotin (2). neal fluid was for all experiments approximately 1 to 2 x Bacteria. S. typhimurium TA1950 carries a missense 109 cells/ml after a 5-hr i.p. exposure. There was only a mutation within the first gene of the histidine biosynthesis small variation between the bacterial titers estimated for pathway and requires histidine for growth (4). Due to a dele each mouse separately, making it possible to determine tion through the uvr-B locus, the bacteria are incapable the titer for all animals of a group together. The remaining of excision repair of DNA damage, which increases their peritoneal fluid from each mouse was plated separately in sensitivity to some chemical mutagens, as has been verified minimal medium to establish the frequency of his' revert also for nitrosamines in the host-mediated assay (7). The ants (undiluted, 0.3 ml/plate). The spontaneous mutant reversion from auxotrophy to prototrophy was used as frequency for the tester strain TA1950 was found to be in genetic marker for mutation induction experiments. The the range of 5 to 10 x 10 9. For estimation of survivors and bacterial strain was kindly supplied by Professor B. N. prototrophs. the Petri dishes were incubated for 16 and 40 Ames, Berkeley, Calif. hr, respectively, at 37°.The C quotient, i.e., the quotient Animals. Male NMRI mice (outbred strain; Neuherberg, resulting from the mean mutant frequency of bacteria from FRG), weighing 35 to 40 g and 12 to 14 weeks old, were treated animals (test group) and the mean mutant frequency used for all experiments. Four to 7 animals were housed in of bacteria from animals that received only the solvent plastic cages (500-sq cm basic area) on hardwood shavings. (control group), was used as a measure of mutagenicity. Water and standard Diet R from Mischfutterwerke Berlin The mutant frequencies of bacteria from all animals in the (Alt Glienicke, Berlin, GDR) were given ad libitum. test group were compared with those of the bacteria from Chemicals. AMT, MU, and EU (pure) were kindly provided animals in the control group (Wilcoxon signed-ranks test). by Dr. Günther,VEBChemiekombinat Bitterfeld, GDR. ETU An increase of the mutant frequency was to be statistically (pure) was a gift from Dr. Srarh, Prague. DPA (pure) (Scher significant when p < 0.01. ing A. G., West Berlin), MOR (99% GLC) (Riedel-DeHaen A. G., Hannover, FRG), PMZ (C. H. Boehringer, Mannheim, FRG). DMSO (purest) (Ferak, West Berlin), NDMA (99% RESULTS GLC) (Merck-Schuchardt, Darmstadt, FRG), and sodium nitrite (purest) (VEB Laborchemie Apolda, GDR) were pur All secondary amines (DMA, DPA, MOR, MBA, PMZ, chased as commercial products and used without further AMT, and PZ), urea derivatives (MU, EU, and ETU) and purification. DMA. MBA. and PZ were prepared from com sodium nitrite were found to be without mutagenic activity mercial distilled amines and hydrogen chloride in ethanol in the host-mediated assay with S. tyhimurium TA1950 as and recrystallized from ethanol. NPZ, DiNPZ, NMBA, genetic indicator system when given in doses ranging from NMOR, and NPMZ were prepared from the purified amine 1,450 to 2,900 /¿moles/kg.