In Vivo Formation of N-Nitroso Compounds and Detection of Their Mutagenic Activity in the Host-Mediated Assay1

[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, JÃ¶rgSchÃ¶neich, and Dieter Ziebarth Zentralinstitut fÃ¼rGenetik und Kulturpflanzenforschung der Akademie der Wissenschaften der DDR. 4325 Gatersleben [R. B., J. S.], and Zentralinstitut fÃ¼r Krebsforschung der Akademie der Wissenschaften der DDR, 1115 Berlin-Buch Â¡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

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 exÃºdate 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 37Â°.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. GÃ¼nther,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 /Â¿moles/kg. hydrochlorides by nitrosation with sodium nitrite and dilute Mutagenicity of Combined Nitrite:Amine Application. aqueous hydrochloric acid. The compounds were distilled DPA (dissolved in DMSO), DMA, MBA and PMZ (dissolved or recrystallized and were checked for purity by thin-layer in NaCI solution) had no mutagenic activity when adminis chromatography on Kieselgel GF-,, (Merck-Schuchardt) in tered p.o. with equimolar amounts of sodium nitrite. Even hexane:diethyl etherdichloromethane (4:3:2). the very high DMA dose of 24,444 /nmoles/kg (2 g/kg) plus Host-mediated Assay. The test procedure is described nitrate, 2,900 /Â¿moles/kg (200 mg/kg), did not result in in detail in another publication (36). One ml of an overnight mutations. The cyclic compounds AMT, MOR, and PZ culture was inoculated into 10 ml fresh nutrient broth and showed dose-dependent mutagenic effectiveness. In com incubated in a shaker at 37C in the dark. After 2 hr the parable doses PZ plus nitrite proved to be the strongest bacteria had reached the log phase of growth. This culture mutagen, followed by MOR plus nitrite and AMT plus nitrite. was diluted with 0.9% NaCI solution to a titer of about 5 x The latter was found to be a weak mutagen at the highest 107 cells/ml. Two ml of this culture were injected i.p. into dose tested (Table 1). Dose-response studies of the indica each mouse. The test compounds were dissolved in unbuf tor bacteria revealed significant differences in mutagenic fered 0.9% NaCI solution (pH 7.0) immediately before use. response between combined PZ:nitrite and MOR:nitrite Each mouse received 0.2 ml by gavage. Nitrite and the A/ (Chart 1). The lowest effective dose of PZ plus nitrite was compounds were mixed together prior to application to found to be 145 ^moles/kg, while MOR plus nitrite was yield this volume. Injection of the bacteria and p.o. incuba mutagenic in doses higher than 363 /Â¿moles/kg. The in tion of the compounds were performed simultaneously. crease in induced mutant frequency in bacteria over a dose

DECEMBER 1977 4573

Table 1 without mutagenic activity in the host-mediated assay (Ta Mutagenic activity ol several cyclic amines in combination with ble 2). equimolar doses of sodium nitrite after p.o. administration to mice Mutagenicity of Combined Nitrite:Alkylurea Application. in the host-mediated assay with S. typhimurium TA1950 as the The urea derivatives MU and EU (dissolved in 0.9% NaCI genetic indicator system solution) and the thiourea derivative ETU (dissolved in 10% Incubation of bacteria i.p. was limited to 5 hr in all cases. Under the experimental conditions, bacterial survival was 90 to 120%. DMSO) were mutagenic when administered p.o. in combi nation with equimolar amounts of sodium nitrite. The first Dose of Dose of Com amine nitrite statistically significant increase in mutant frequency in the pound (Â¿Â¿moles/kg)(Â¿tmoles/kg) indicator bacteria (lowest effective dose) was found for AMT 1450 1450 1.58 >0.05 ETU plus nitrite (7.25 /Â¿moles/kg), MU plus nitrite (1,450 2175 2175 2.05 >0.01 /Â¿moles/kg), and EU plus nitrite (3,650 /Â¿moles/kg). Also, 2900 2900 2.53 <0.01 combined nitrite:urea derivative mutagenicity, a linear dose dependency of mutagenic activity was found. Chart 2 MOR 1450 1450 7.87 <0.01 clearly indicates the very high mutagenic activity of ETU 2175 2175 12.00 <0.01 2900 2900 18.80 <0.01 plus nitrite compared with the lower activity of the other urea derivatives in combination with nitrite. PZ14502175290014502175290038.2248.7972.84<0.01<0.01<0.01 Mutagenic Activity of W-Nitrosamines. Among all nitrosamines tested, NMOR showed the strongest mutagenic activity in the host-mediated assay. The lowest effective dose, i.e., the first dose in a dose-response analysis that statistically significantly increased the mutant frequency of 50 the indicator bacteria, was 8.62 /Â¿moles/kg. The lowest effective doses of the other compounds were higher: 86.2 and 55.5 /Â¿moles/kg, respectively, for NPZ and DiNPZ. The dose-response curve for NPZ-induced mutagenesis shows 40 a faster increase in the C quotient with increasing dose than does that for DiNPZ. Both compounds were less active than NMOR. For all 3 compounds a linear dose dependency ~ 30 of mutagenic response in the indicator bacteria was re I vealed (Chart 3). The mutagenic activity of NDMA and O* NMBA was detectable only after p.o. administration of 0 20 336.7 and 2,166 /Â¿moles/kg, respectively. A linear increase with a dose of relative mutagenicity is evident from Table 3. NPMZ, up to a dose of 1,356 /Â¿moles/kg, was nonmutagenic in the host-mediated assay. 10- Estimation of the Rate of Nitrosation by Dose-Response Curves Obtained for Nitrite Plus Amine and for the Nitros- amines. The linear dose-response relationship for the mu

1000 2000 3000 tagenic activity of both PZ or MOR plus nitrite and NPZ or Dose morpholme(o)or piperazine(Â»J'NaNOj NMOR encouraged us to use these dose curves to estimate (yumoles/kg ) the amount of /V-nitrosamines formed from the precursors. Chart 1. Mutagenic effectiveness of nitrite plus either MOR or PZ appli On the assumption that the nitrosamines formed from the cation in the host-mediated assay with S. typhimurium TA1950 as the precursors have the same genetic activity as do the pure genetic indicator system. The compounds were given via stomach tube simultaneously with i.p. bacterial injection. Incubation time of bacteria was compounds applied in mutagenicity tests, we used the C limited to 5 hr in all cases. quotients for a given dose of nitrite plus amine and esti mated for the appropriate nitrosamine those amounts that resulted in similar genetic effects. The mathematical range up to 2,900 Â¿Â¿moles/kgwas faster for PZ than for expression for the linear dose-response regression was MOR in combination with nitrite. For both combinations a used for the calculation (Chart 4). For PZ plus nitrite, 1450 linear dose dependency of the C quotients was obtained Â¿Â¿moles/kg,an equimutagenic NPZ dose of 803.6 Â¿Â¿moles/ (correlation coefficient: PZmitrite, 0.988; MOR:nitrite, 0.995). kg was calculated, while for MOR plus nitrite only 45.5 Effects of Temporal Spacing of Nitrite Plus Amine Appli /xmoles/NMOR per kg were found to be equimutagenic. cation. The administration of PZ or MOR 10 min before Application of 2900 AmÃ³les precursors per kg, according nitrite did not result in changes of the interactive mutagen- to our calculations, probably results in the production of icity with nitrite. The C quotients estimated for temporal NPZ, 1357 /xmoles/kg, or NMOR, 104 Â¿Â¿moles/kg.For all spacing experiments did not differ statistically from those doses of nitrite plus amine tested, the resulting doses of N- obtained for the test variants with simultaneous intubation nitrosamine were found to be higher, by a factor of more of the precursors nitrite and amine. However, when the than 10, for PZ than for MOR. We used NPZ as a standard, administration of nitrite preceded that of amines, a reduc since the rate constant for the /V-nitrosation of PZ, resulting tion of about 80% of the mutagenic efficiency was observed in NPZ, is more than 10 times higher than the rate constant for PZ, while under these conditions MOR plus nitrite was for the /V-nitrosation of NPZ, resulting in DiNPZ. This means

4574 CANCER RESEARCH VOL. 37

Table 2 Effects of temporal spacing of nitrite and amino application on interactive mutagenic activity in the host-mediated assay Incubation of S. typhimurium TA1950 i.p. was limited to 5 hr. of of amine nitrite Com (/Â¿moles/ (AmÃ³les/ of poundMORPZDosekg)14501450Dosekg)14501450AdministrationnitriteSimultaneouslywith

amine10 minbeforeamine10

minafteramineSimultaneouslywith

amine10 minbeforeamine10

minafteramineC7.871.506.4038.418.1542.01P"<0.01>0.05<0.01<0.01<0.01<0.01p"<0.01>0.05<0.01>0.05

" The statistical calculations were done by comparing the mutant frequencies of the bacteria from animals treated with nitrite plus amine with the mutant frequencies of the bacteria from animals that received only the solvent. '' The statistical calculations were done by comparing the mutant frequencies of bacteria from animals treated with nitrite plus amine simultaneously with the mutant frequencies of bacteria from animals that received the precursors at different times.

400

r j iA o- 100* 10 T Ã³ U io

100 200 Dose ETU [Â° NaNO2 ( AJmoles/kg ) 250 500 750 1000 2000 3000 Dose nitrosomorpholine (o),1-nitrosopiperazine (â€¢). Dose MU (Â°)or EU H*NaNO2 (umoles/kg) or 1,4-dinitrosopiperazine (â€¢)(yumoles/kg) Chart 2. Mutagenic effectiveness of combined nitrite plus alkylurea appli Chart 3. Linear dose dependence of mutagenic activity of W-nitrosamines cation in the host-mediated assay. The compounds were given via stomach in the host-mediated assay. The compounds were given via stomach tube tube simultaneously with i.p. bacteial injection. Incubation time of bacteria simultaneously with i.p. bacterial injection. Incubation time of bacteria was was limited to 3 hr in all cases. Asterisk, lowest effective dose. limited to 5 hr in all cases. that the formation of NPZ will be faster than its conversion concentration. In doses ranging from 725 to 1450 /xmoles/ into the binitrosated state (28). For in vivo studies this kg, the relative nitrosation increased from 1.7 to 3.1%. For finding is of great importance, since nitrite elimination nitrite plus MOR, 2900 Â¿Â¿moles/kg,therelative nitrosation from the stomach is very rapid (17). Secondly, a complete was 3.6%. With increasing nitrite plus PZ doses, the relative formation of DiNPZ will be possible only in the presence of nitrosation of PZ decreased (Chart 6). Although for a dose exceas nitrite. In vitro studies with low concentrations of of 145 /nmoles/kg, 74% amine was found to be nitrosated, nitrite revealed a high yield of NPZ, whereas only traces of only 55% relative nitrosation could be demonstrated after DiNPZ could be estimated (40). Furthermore, under our precursor application of 1450 /Â¿moles/kg. conditions, the mononitrosated PZ was a stronger mutagen than was DiNPZ (Chart 3). The percentages of theoretical yield of nitrosamine for DISCUSSION various equimolar doses of nitrite plus amine are presented in Charts 5 and 6. For MOR plus nitrite, the relative nitrosa- This paper describes the possibility of detection of in tion seemed to increase with an increase in precursor vivo formation of A/-nitroso compounds by estimating their

DECEMBER 1977 4575

Table 3 Mutagenic activity of several p.o.-administered N-nitrosamines in the host-mediated assay Incubation time of bacteria was 5 hr in all cases.

effective W-Nitros- dose"

amineNDMA (mg/kg)30 LOED2.76

DiNPZ 810 2.98 108 68 NPZ 3.33 0.477 0.996 67 NMBA 360 2.64 0.034 0.969 323 2136 NMOR 1 3.310,r0.6520.2631.303r"0.9840.9880.995mg/kg29 0.05 0.45 NPMZLowest --300C2-dose"Co â€¢300^moles/kg392â€¢1356 " Lowest effective dose of a compound in dose-response studies in the host-mediated assay analogous to the lowest effective concentration estimated in mutagenicity studies with Drosophila (57). '' C quotient found for the lowest effective dose. ' Ascent of the regression line for the dose-effect curve according to the formula y yâ€ž+b,x. '' Correlation coefficient for the linear dose response. ' Dose estimated by means of regression analysis of the dose-response studies that result in a doubling of the spontaneous mutant frequency of the indicator bacteria in the host-mediated assay.

e z $ S 1- 0 NO

1000 2000 3000 Dose NaNOj* morpholine (umoles/kg) Chart 5. Relative rate of nitrosation for MOR in mouse stomach as a function of the nitrite plus MOR dose administered to the animals.

a u 1000 2000 3000 75- Dose morpholine or piperazine-NaNOj ( Aimoles/kg ] Chart 4. Regression lines for the equimutagenic doses of N-nitrosamines determined by means of the dose-response curves for nitrite plus amine and N-nitrosamine mutagenesis in the host-mediated assay.

mutagenic properties in the host-mediated assay. This sys N tem for mutagenicity testing seems to be very useful for this purpose since /V-nitrosation is an acid-catalyzed o Ã® process taking place in mammalian stomach (28) and the A/-nitroso compounds formed preferentially induce gene mutations (29, 49, 51). In the case of nitrosamine synthesis, 1000 2000 3000 these promutagens become activated by drug-metabolizing Dose NaNOjÂ» piperazine (jiimoles|kg) enzymes localized in the microsomes of host animal tissues Chart 6. Relative rate of nitrosation for PZ in mouse stomach as a (5, 19, 25, 30). function of the nitrite plus PZ dose administered to the animals. In the host-mediated assay, all alkylureas administered p.o. to mice in combination with equimolar doses of sodium A/-mtroso compounds are synthesized when these com nitrite were found to increase the mutant frequencies of pounds are in combination. MU possessed a higher muta the indicator bacteria. Neither the alkylurea derivatives MU, genic activity than did EU when administered in similar EU, and ETU nor nitrite were mutagenic alone. Therefore, doses and under the same conditions; this is attributed, in these results are consistent with the theory that mutagenic part at least, to the more rapid nitrosation of MU under

4576 CANCER RESEARCH VOL. 37

acidic conditions (27, 28). These findings confirm the earlier reaction (28), and the relatively high lowest effective dose studies of Couch and Friedman (9), who also demonstrated of NDMA in the host-mediated assay (Table 3) might explain higher mutagenic activity of MU plus nitrite. Also, in long- the absence of genetic activity of combined nitrite plus term studies, MU showed stronger tumorigenic potential in DMA administraiton. MBA is a weaker base and has a combination with nitrite than did EU plus nitrite (35). Al higher rate constant for nitrosation (28). In both mice and though at the present time no data concerning the muta- rats, tumor development was demonstrated after adminis genicity of both nitroso alkylureas in the host-mediated tration of nitrite and MBA-containing food (35). On the assay with the repair-defective strain TA1950 S. typhimu- other hand, a combined MBA plus nitrite intubation was rium are available, studies with the corresponding repair- without mutagenic activity in our experiments up to the effective strain revealed a higher activity of nitrosomethylu- highest dose tested (2900 /^moles/kg). We attribute these rea in this test system than of nitrosoethylurea after p.o. negative findings to the very weak mutagenic activity of the application to mice (32). Therefore, the finding of stronger resulting NMBA in the host-mediated assay. The lowest mutagenic activity of nitrite plus MU in our study, as well effective dose for this nitrosamine was the highest of all as in that of Couch and Friedman (9), seems to be the compounds tested (Table 3). In addition, tumor induction result of both a higher rate constant for the nitrosation and in long-term studies is the result of repeated treatment the stronger mutagenic activity of nitrosomethylurea pro over weeks (or months), while under our test conditions duced within the stomach of host animal. The alkylureas the mutagenicity of the amount of nitrosamine produced MU and EU are not considered to be significant environ after single acute treatment was estimated. The absence of mental contaminants. However, ETU is widely distributed mutagenic activity of DPA and PMZ in combination with in our environment as the decomposition and metabolic sodium nitrite is due to the nonmutagenicity of the respec breakdown product of fungicides belonging to the ethyl- tive nitrosamines. NPMZ was tested in the host-mediated enebis(dithiocarbamate) group (39). Studies dealing with assay with S. typhimurium TA1950 as the genetic indicator the nitrosation kinetics of this thiourea derivative are not system (Table 3), while /V-nitrosodiphenylamine was with available. In the present host-mediated assay experiments, out mutagenic activity in the Sa/mone//a/microsome test in a very low effective dose for ETU:nitrite was demonstrated, which TA1535 or TA100 were used as the genetic indicators this lowest effective dose is in fact 200-fold lower than the (26, 54). Both compounds were also negative in carcinoge- lowest effective dose for MU:nitrite and 500-fold lower than nicity studies with rats (10, 24). The cyclic amines AMT, MOR, that of EU:nitrite. Although /V-nitrosoethylenebis(thiourea) and PZ possessed mutagenic properties in experiments was found to be a very potent mutagen in the host-mediated with simultaneous application of equimolar amounts of assay (R. Braun, unpublished observation), these results aminÃ©plus sodium nitrite. The nitrosation kinetics of AMT indicate a high turnover and a high rate constant for the is not known, and the same is true for the structure and nitrosation reaction in vivo. chemical properties of the A/-nitroso derivative formed. Our Of the secondary amines tested in combination with results demonstrate the formation of a mutagenic product nitrite, only the cyclic compounds MOR, PZ, and AMT in mouse stomach. Since AMT is a pesticide widely distrib increased the mutant frequencies of the indicator bacteria. uted in the human environment, further studies are needed Contrary to the observation on the interactive mutagenicity to elucidate whether this compound is carcinogenic and of DMA plus nitrite in the host-mediated assay in which ICR thus a health hazard for humans. The mutagenic activity of mice were used, published recently by Couch and Friedman combined PZ or MOR plus nitrite application is detectable (9), our studies with NMRI mice showed no increase in the in much lower doses than in AMT:nitrite (Table 1). Although mutant frequencies of the repair-defective indicator bacte the nitroso derivative of MOR showed stronger mutagenic ria. Even the very high dose of DMA, 24,444 Â¿Â¿moles/kg,effectiveness than did the /V-nitrosopiperazines, the oppo plus nitrite, 1450 /Â¿moles/kg,was without mutagenic effec- site was true for the genetic effectiveness of precursors tivity (the same dose was found to be mutagenic in the (Charts 1 and 3). The only explanation for this phenomenon paper cited above). In addition to differences between host is the production of a higher yield of nitrosamine from PZ animal strains, differences in sensitivity of the bacterial than from MOR. Equimutagenic doses of the respective tester strains G46 and TA1950 may account for the discrep nitrosamines inferred from the linear regression lines for ancy between the results obtained by Couch and Friedman the dose-response studies of both nitrite plus amine and and those of the present study. The repair-effective strain nitrite plus nitrosamine support this assumption (Chart 4). G46 used by those authors was found to be superior to its This interpretation gains further support from experiments derivatives lacking excision repair for NDMA and some on the effects of temporal spacing of precursor administra other methylatmg agents (3). The absence of interactive tion (Table 2). Since sodium nitrite absorption from gastric mutagenicity of DMA plus nitrite in our experiments is in juice is very fast (17, 34), the application of this compound conformity with studies on their carcinogenic potential 10 min before amine resulted in an 80% decrease in PZ (20). The negative results for DMA and the absence of plus nitrite mutagenicity. If the administration of nitrite carcinogenic effects of diethylamine (10, 42) and other preceded MOR treatment, no mutagenic response was strongly basic amines in combination with nitrite (35) led to induced. These results demonstrate that although nitrosa the conclusion that the carcinogenic risk exerted by a mine synthesis was depressed, in the case of PZ, it was secondary amine in the presence of nitrite depends on the still high enough to be detected in the host-mediated assay. basicity of the amines: the weaker the base, the faster the The higher amounts of nitrosamine formed in mouse stom rate constant for nitrosation (28). The strongly basic char ach from PZ relative to MOR and the significant mutagen acter (pKa, 10.7), the low rate constant for the nitrosation icity of nitrite plus PZ in experiments with temporal spacing

DECEMBER 1977 4577

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1977 American Association for Cancer Research. R. Braun et al. of compound administration correspond to the higher rate especially to nitrite, is much lower than simulated in this constant for the nitrosation of PZ (28). The second amino study. In addition to nitrite-containing food (21, 33), nitrate group within the cyclic structure causes PZ to become a reduction in saliva contributes to a low, but significant, weaker base than is MOR and to participate readily in nitrite ingestion (46), which has been calculated at 12.8 nitrosation. By estimating the relative rates of nitrosation mg/day for the average United States resident (53). The for these amines, significant differences became clear other precursors, the amines or amides, are present in (Charts 5 and 6). According to our observations in the minute amounts also, but much higher doses are ingested host-mediated assay, for MOR a fraction of 1 to 3% of the as drugs; the dose for PZ ascariasis therapy may reach theoretical yield must be taken into consideration. The 5000 mg within 1 day. In this special case, the likelihood of relative rate of nitrosation increased as a function of pre the formation of mutagenic and carcinogenic nitrosopiper cursor dose. PZ nitrosation was, under the same experi azines seems to be rather high, since the therapy regimens mental conditions, in the range of 50 to 70% of the theoret result in a close and continuous contact for days of both ical yield, but in contrast to MOR these rates decreased nitrite and PZ under the acidic conditions of the human with increasing precursor doses. Similar observations have stomach. However, it is difficult to determine from the been made for chemical estimation of in vivo formation of present results whether these interactions might be related nitrosopiperazines (34). These findings are difficult to ex to the induction of mutation in human germ cells; only such plain, but changes of the pH from optimal to less-optimal mutations will be hazardous from the point of view of a levels by high PZ concentrations in the gastric juice might genetic risk for humans. In addition to differences in expo be the cause of both findings. The relative rates of nitrosa sure and dosage, differences in the sensitivity of target tion shown by host-mediated assay techniques represent cells must be taken into account. Otherwise, it would be only approximations, since biological activity, which in unrealistic to ignore the mutagenic potential of /V-nitroso turn might be influenced by several factors, especially by compounds synthesized in vivo from nonmutagenic chemi the presence of the precursors nitrite and amine, is being cals and to delegate the human health hazards into the measured. Couch and Friedman observed a decreasing field of chemical carcinogenesis. influence of sodium nitrite, even at low doses, on the mutagenic activity of NDMA in the host-mediated assay and attributed this to an inhibition of the biotransformation REFERENCES of the promutagenic compound into ultimate forms (8). If 1. Akin, F. J., and Wasserman, A. E. Effect on Guinea Pigs of Feeding the same enzyme system is responsible for metabolism of Nitrosomorpholine and It Precursors in Combination with Ascorbic NMOR and the /V-nitrosopiperazines, the precursor nitrite Acid. Food Cosmet. Toxicol., 13: 239-242. 1975. 2. Ames, B. 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Rolf Braun, Jörg Schöneich and Dieter Ziebarth

Cancer Res 1977;37:4572-4579.

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