in vivo formation of n-nitroso compounds and detection of ......mutagenic n-nitroso compounds formed...

[CANCER RESEARCH 37, 4572-4579, December 1977]

In Vivo Formation of N-Nitroso Compounds and Detection of TheirMutagenic 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 fÃ¼rKrebsforschung der Akademie der Wissenschaften der DDR, 1115 Berlin-Buch Â¡D.Z.], German Democratic Republic

SUMMARY

The formation of /V-nitroso compounds in mouse stomach from equimolar doses of sodium nitrite and secondaryamines or alkylurea derivatives given simultaneously by astomach tube was estimated by measuring the mutagenicactivity of the compounds in the i.p. host-mediated assaywith the use of Salmonella typhimurium TA1950 as geneticindicator system. A mutagenic response in the bacteriawas found after administration of the cyclic amines pipera-zine dihydrochloride, morpholine, and amitrole. The highest mutagenicity was exerted by piperazine dihydrochlorideplus nitrite, while amitrole plus nitrite was only weaklymutagenic. No mutagenic activity was observed for equimolar doses of sodium nitrite plus dimethylamine hydro-chloride, diphenylamine. methylbenzylamine hydrochlo-ride, and phenmetrazine hydrochloride. All /V-alkylurea derivatives tested were found to yield significant amounts of/V-nitroso compounds, which allowed detection of theirmutagenic activity in the host-mediated assay. The highestactivity was shown by nitrite plus ethylenebis(thiourea),while methylurea and ethylurea were found to be less activein combination with nitrite. Dose-response curves for themutagenic activity of /V-nitrosamines were used to estimatethe amounts of /V-nitroso derivatives formed in vivo fromthe precursors after acute treatment of the mice. In thecase of piperazine dihydrochloride, nitrosation of 50 to70% was estimated, while for morpholine nitrosationranged from 1 to 3%. The results are compared with thoseobtained in long-term carcinogenesis studies with sodiumnitrite plus amines.

INTRODUCTION

Since Druckrey ef al. (11) discussed the formation ofcarcinogenic /V-nitroso compounds from sodium nitrite andsecondary amines of /V-alkylureas under the acidic conditions of the mammalian stomach, much experimental workhas been done to explore this hypothesis from the point ofview of carcinogenic risk to humans. Besides the analysisof in vitro /V-nitrosation in buffer solutions (12-15, 23,41, 48, 52) and human gastric juice (57, 58), more than 30secondary and tertiary amines, alkylureas. and other nitros-able W-compounds have been tested in combination with

nitrite for carcinogenic activity in long-term animal studies(For reviews, see Refs. 28 and 35). In addition to thecarcinogenic hazards of exposure of humans to /V-nitrosocompounds formed intragastrally from nonactive precursors, a genetic risk cannot be excluded since most N-nitroso compounds are strong mutagens (for reviews, seeRefs. 29 and 31). The mutagenic and DMA-damaging activities of A/-nitroso derivatives from agricultural chemicals ofthe carbamate type are well documented (6, 15, 16, 44, 47).Soon after it was found that primary amines and nitritereact in vitro to form mutagenic products (22), host-mediated assay studies revealed the mutagenic activity ofcombined application of nitrite and the secondary amineDMA2 or the alkylurea derivatives Mil and EU (9, 38).

The present study was undertaken to determine therelationship between chemical structure and properties ofsecondary amines and alkylureas. It investigated the abilityof these compounds to undergo /V-nitrosation in the mousestomach, eventually resulting in the formation of mutagenicproducts detectable in the i.p. host-mediated assay withthe use of Salmonella typhimurium TA1950 as geneticindicator system. This system was introduced into mutagenicity testing by Garbridge and Legator (18) to bridgethe discrepancy between mammalian and microbial bio-transformation of xenobiotic compounds, and it has beenused successfully to detect the mutagenic potential ofnitrosamines and -amides (7, 32, 55, 56). The host-mediatedassay represents a useful method for the identification ofthe mutagenic activity of /V-nitroso compounds generatedin vivo from secondary amines and amides because, first,nitrosation takes place in the acidic environment of thehost animal's stomach, and second, the nitrosamines and

-amides preferentially induce gene mutations (49, 51) detectable in this system. The metabolic activation of thepromutagenic nitrosamines takes place in the biotransformation system of the host animals, while the ultimatemutagens are detected by the i.p.-growing indicator bacteria.

The mice used as host animals were treated with theprecursor compounds plus equimolar amounts of sodiumnitrite, and the formation of /V-nitroso compounds wasmeasured via the mutagenic response of the indicator

1 Presented in part at the Sixth Annual Meeting of the European Environmental Mutagen Society. Gernrode. 1976 (36. 37).

Received March 24. 1977: accepted August 11. 1977.

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, methylbenzylamine hydrochloride: PZ, piperazine dihydrochloride; NPZ. 1-nitroso-piperazine: DiNPZ, 1,4-dinitrosopiperazine; NMBA, N-nitrosomethylben-zylamine: NMOR. /V-nitrosomorpholine; NPMZ, N-mtrosophenmetrazine.

4572 CANCER RESEARCH VOL. 37

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Mutagenic N-Nitroso Compounds Formed in Vivo

bacteria. Also, dose dependency of mutagenic activity ofthe nitrosamines thought to be formed was studied tocompare the mutagenic activity of the precursors plusnitrite with that of the final reaction products.

MATERIALS AND METHODS

Media. The bacterial cultures were maintained on nutrient agar slants at 4 and were grown for use in nutrientbroth (Sevac, Czechoslovakia) containing 7.5 g meat extract, 12.5 g peptone, and 5 g NaCI in a final volume of 1liter, his' revertants were scored on Spizizen's minimal

medium (52) supplemented with an excess of biotin (2).Bacteria. S. typhimurium TA1950 carries a missense

mutation within the first gene of the histidine biosynthesispathway and requires histidine for growth (4). Due to a deletion through the uvr-B locus, the bacteria are incapableof excision repair of DNA damage, which increases theirsensitivity to some chemical mutagens, as has been verifiedalso for nitrosamines in the host-mediated assay (7). Thereversion from auxotrophy to prototrophy was used asgenetic marker for mutation induction experiments. Thebacterial strain was kindly supplied by Professor B. N.Ames, Berkeley, Calif.

Animals. Male NMRI mice (outbred strain; Neuherberg,FRG), weighing 35 to 40 g and 12 to 14 weeks old, wereused for all experiments. Four to 7 animals were housed inplastic cages (500-sq cm basic area) on hardwood shavings.Water and standard Diet R from Mischfutterwerke Berlin(Alt Glienicke, Berlin, GDR) were given ad libitum.

Chemicals. AMT, MU, and EU (pure) were kindly providedby Dr. GÃ¼nther,VEB Chemiekombinat Bitterfeld, GDR. ETU(pure) was a gift from Dr. Srarh, Prague. DPA (pure) (Schering 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%GLC) (Merck-Schuchardt, Darmstadt, FRG), and sodiumnitrite (purest) (VEB Laborchemie Apolda, GDR) were purchased as commercial products and used without furtherpurification. DMA. MBA. and PZ were prepared from commercial distilled amines and hydrogen chloride in ethanoland recrystallized from ethanol. NPZ, DiNPZ, NMBA,NMOR, and NPMZ were prepared from the purified aminehydrochlorides by nitrosation with sodium nitrite and diluteaqueous hydrochloric acid. The compounds were distilledor recrystallized and were checked for purity by thin-layerchromatography on Kieselgel GF-,, (Merck-Schuchardt) inhexane:diethyl etherdichloromethane (4:3:2).

Host-mediated Assay. The test procedure is describedin detail in another publication (36). One ml of an overnightculture was inoculated into 10 ml fresh nutrient broth andincubated in a shaker at 37C in the dark. After 2 hr the

bacteria had reached the log phase of growth. This culturewas diluted with 0.9% NaCI solution to a titer of about 5 x107 cells/ml. Two ml of this culture were injected i.p. into

each mouse. The test compounds were dissolved in unbuffered 0.9% NaCI solution (pH 7.0) immediately before use.Each mouse received 0.2 ml by gavage. Nitrite and the A/compounds were mixed together prior to application toyield this volume. Injection of the bacteria and p.o. incubation of the compounds were performed simultaneously.

Each dose of a test substance or nitrite:compound mixturewas tested in a group of 6 animals against a control groupof 6 animals receiving only the solvent. All experimentswere performed at least twice. Changes in applicationvolume, time of treatment, or solvent used are indicated inthe appropriate tables and charts. Three or 5 hr afterbacterial injection, the animals were killed by cervicaldislocation, and the bacteria were recovered from theperitoneal cavity after injection of 2 ml 0.9% NaCI solution.Three-tenths ml exÃºdate of each mouse in each groupwere pooled together on nutrient agar plates for estimationof the viable bacteria (10 6 dilution, 0.1 ml per plate, 4plates per estimation). The bacterial titer within the peritoneal fluid was for all experiments approximately 1 to 2 x109 cells/ml after a 5-hr i.p. exposure. There was only asmall variation between the bacterial titers estimated foreach mouse separately, making it possible to determinethe titer for all animals of a group together. The remainingperitoneal fluid from each mouse was plated separately inminimal medium to establish the frequency of his' revert

ants (undiluted, 0.3 ml/plate). The spontaneous mutantfrequency for the tester strain TA1950 was found to be inthe range of 5 to 10 x 10 9. For estimation of survivors andprototrophs. the Petri dishes were incubated for 16 and 40hr, respectively, at 37Â°.The C quotient, i.e., the quotient

resulting from the mean mutant frequency of bacteria fromtreated animals (test group) and the mean mutant frequencyof bacteria from animals that received only the solvent(control group), was used as a measure of mutagenicity.The mutant frequencies of bacteria from all animals in thetest group were compared with those of the bacteria fromanimals in the control group (Wilcoxon signed-ranks test).An increase of the mutant frequency was to be statisticallysignificant when p < 0.01.

RESULTS

All secondary amines (DMA, DPA, MOR, MBA, PMZ,AMT, and PZ), urea derivatives (MU, EU, and ETU) andsodium nitrite were found to be without mutagenic activityin the host-mediated assay with S. tyhimurium TA1950 asgenetic indicator system when given in doses ranging from1,450 to 2,900 /Â¿moles/kg.

Mutagenicity of Combined Nitrite:Amine Application.DPA (dissolved in DMSO), DMA, MBA and PMZ (dissolvedin NaCI solution) had no mutagenic activity when administered p.o. with equimolar amounts of sodium nitrite. Eventhe very high DMA dose of 24,444 /nmoles/kg (2 g/kg) plusnitrate, 2,900 /Â¿moles/kg (200 mg/kg), did not result inmutations. The cyclic compounds AMT, MOR, and PZshowed dose-dependent mutagenic effectiveness. In comparable doses PZ plus nitrite proved to be the strongestmutagen, followed by MOR plus nitrite and AMT plus nitrite.The latter was found to be a weak mutagen at the highestdose tested (Table 1). Dose-response studies of the indicator bacteria revealed significant differences in mutagenicresponse between combined PZ:nitrite and MOR:nitrite(Chart 1). The lowest effective dose of PZ plus nitrite wasfound to be 145 ^moles/kg, while MOR plus nitrite wasmutagenic in doses higher than 363 /Â¿moles/kg. The increase in induced mutant frequency in bacteria over a dose

DECEMBER 1977 4573



fi. Braun et al.

Table 1Mutagenic activity ol several cyclic amines in combination with

equimolar doses of sodium nitrite after p.o. administration to micein the host-mediated assay with S. typhimurium TA1950 as the

genetic indicator systemIncubation of bacteria i.p. was limited to 5 hr in all cases. Under

the experimental conditions, bacterial survival was 90 to 120%.

Compound

Dose ofamine

(Â¿Â¿moles/kg)

Dose ofnitrite

(Â¿tmoles/kg)

AMT

MOR

145021752900

145021752900

145021752900

145021752900

1.582.052.53

7.8712.0018.80

>0.05>0.01<0.01

<0.01<0.01<0.01

PZ14502175290014502175290038.2248.7972.84<0.01<0.01<0.01

50

40

~ 30

I

O*

0 20

10-

1000 2000 3000Dose morpholme(o)or piperazine(Â»J'NaNOj

(yumoles/kg )

Chart 1. Mutagenic effectiveness of nitrite plus either MOR or PZ application in the host-mediated assay with S. typhimurium TA1950 as thegenetic indicator system. The compounds were given via stomach tubesimultaneously with i.p. bacterial injection. Incubation time of bacteria waslimited to 5 hr in all cases.

range up to 2,900 Â¿Â¿moles/kgwas faster for PZ than forMOR in combination with nitrite. For both combinations alinear dose dependency of the C quotients was obtained(correlation coefficient: PZmitrite, 0.988; MOR:nitrite, 0.995).

Effects of Temporal Spacing of Nitrite Plus Amine Application. The administration of PZ or MOR 10 min beforenitrite did not result in changes of the interactive mutagen-

icity with nitrite. The C quotients estimated for temporalspacing experiments did not differ statistically from thoseobtained for the test variants with simultaneous intubationof the precursors nitrite and amine. However, when theadministration of nitrite preceded that of amines, a reduction of about 80% of the mutagenic efficiency was observedfor PZ, while under these conditions MOR plus nitrite was

without mutagenic activity in the host-mediated assay (Ta

ble 2).Mutagenicity of Combined Nitrite:Alkylurea Application.

The urea derivatives MU and EU (dissolved in 0.9% NaCIsolution) and the thiourea derivative ETU (dissolved in 10%DMSO) were mutagenic when administered p.o. in combination with equimolar amounts of sodium nitrite. The firststatistically significant increase in mutant frequency in theindicator bacteria (lowest effective dose) was found forETU plus nitrite (7.25 /Â¿moles/kg), MU plus nitrite (1,450/Â¿moles/kg), and EU plus nitrite (3,650 /Â¿moles/kg). Also,combined nitrite:urea derivative mutagenicity, a linear dosedependency of mutagenic activity was found. Chart 2clearly indicates the very high mutagenic activity of ETUplus nitrite compared with the lower activity of the otherurea derivatives in combination with nitrite.

Mutagenic Activity of W-Nitrosamines. Among all nitros-

amines tested, NMOR showed the strongest mutagenicactivity in the host-mediated assay. The lowest effectivedose, i.e., the first dose in a dose-response analysis that

statistically significantly increased the mutant frequency ofthe indicator bacteria, was 8.62 /Â¿moles/kg. The lowesteffective doses of the other compounds were higher: 86.2and 55.5 /Â¿moles/kg, respectively, for NPZ and DiNPZ. Thedose-response curve for NPZ-induced mutagenesis shows

a faster increase in the C quotient with increasing dosethan does that for DiNPZ. Both compounds were less activethan NMOR. For all 3 compounds a linear dose dependencyof mutagenic response in the indicator bacteria was revealed (Chart 3). The mutagenic activity of NDMA andNMBA was detectable only after p.o. administration of336.7 and 2,166 /Â¿moles/kg, respectively. A linear increasewith a dose of relative mutagenicity is evident from Table3. NPMZ, up to a dose of 1,356 /Â¿moles/kg, was nonmuta-genic in the host-mediated assay.

Estimation of the Rate of Nitrosation by Dose-ResponseCurves Obtained for Nitrite Plus Amine and for the Nitros-amines. The linear dose-response relationship for the mu

tagenic activity of both PZ or MOR plus nitrite and NPZ orNMOR encouraged us to use these dose curves to estimatethe amount of /V-nitrosamines formed from the precursors.

On the assumption that the nitrosamines formed from theprecursors have the same genetic activity as do the purecompounds applied in mutagenicity tests, we used the Cquotients for a given dose of nitrite plus amine and estimated for the appropriate nitrosamine those amounts thatresulted in similar genetic effects. The mathematicalexpression for the linear dose-response regression was

used for the calculation (Chart 4). For PZ plus nitrite, 1450Â¿Â¿moles/kg,an equimutagenic NPZ dose of 803.6 Â¿Â¿moles/kg was calculated, while for MOR plus nitrite only 45.5/xmoles/NMOR per kg were found to be equimutagenic.Application of 2900 AmÃ³les precursors per kg, accordingto our calculations, probably results in the production ofNPZ, 1357 /xmoles/kg, or NMOR, 104 Â¿Â¿moles/kg.For alldoses of nitrite plus amine tested, the resulting doses of N-

nitrosamine were found to be higher, by a factor of morethan 10, for PZ than for MOR. We used NPZ as a standard,since the rate constant for the /V-nitrosation of PZ, resulting

in NPZ, is more than 10 times higher than the rate constantfor the /V-nitrosation of NPZ, resulting in DiNPZ. This means




Mutagenic N-N/troso Compounds Formed in Vivo

Table 2Effects of temporal spacing of nitrite and amino application on interactive mutagenic activity in

the host-mediated assayIncubation of S. typhimurium TA1950 i.p. was limited to 5 hr.

CompoundMORPZDose

ofamine

(/Â¿moles/kg)14501450Dose

ofnitrite

(AmÃ³les/kg)14501450Administration

ofnitriteSimultaneouslywith

amine10minbeforeamine10

minafteramineSimultaneouslywith

amine10minbeforeamine10

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 bacteriafrom animals treated with nitrite plus amine with the mutant frequencies of the bacteria fromanimals that received only the solvent.

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

400

rA

io-Ã³

100*

O

f

10 T

100Dose ETU [Â°

200

NaNO2 ( AJmoles/kg )

1000 2000 3000

Dose MU (Â°)or EU H*NaNO2 (umoles/kg)

Chart 2. Mutagenic effectiveness of combined nitrite plus alkylurea application in the host-mediated assay. The compounds were given via stomachtube simultaneously with i.p. bacteial injection. Incubation time of bacteriawas limited to 3 hr in all cases. Asterisk, lowest effective dose.

j

U

io

250 500 750

Dose nitrosomorpholine (o),1-nitrosopiperazine (â€¢).or 1,4-dinitrosopiperazine (â€¢) (yumoles/kg)

Chart 3. Linear dose dependence of mutagenic activity of W-nitrosaminesin the host-mediated assay. The compounds were given via stomach tubesimultaneously with i.p. bacterial injection. Incubation time of bacteria waslimited to 5 hr in all cases.

that the formation of NPZ will be faster than its conversioninto the binitrosated state (28). For in vivo studies thisfinding is of great importance, since nitrite eliminationfrom the stomach is very rapid (17). Secondly, a completeformation of DiNPZ will be possible only in the presence ofexceas nitrite. In vitro studies with low concentrations ofnitrite revealed a high yield of NPZ, whereas only traces ofDiNPZ could be estimated (40). Furthermore, under ourconditions, the mononitrosated PZ was a stronger mutagenthan was DiNPZ (Chart 3).

The percentages of theoretical yield of nitrosamine forvarious equimolar doses of nitrite plus amine are presentedin Charts 5 and 6. For MOR plus nitrite, the relative nitrosa-tion seemed to increase with an increase in precursor

concentration. In doses ranging from 725 to 1450 /xmoles/kg, the relative nitrosation increased from 1.7 to 3.1%. Fornitrite plus MOR, 2900 Â¿Â¿moles/kg,the relative nitrosationwas 3.6%. With increasing nitrite plus PZ doses, the relativenitrosation of PZ decreased (Chart 6). Although for a doseof 145 /nmoles/kg, 74% amine was found to be nitrosated,only 55% relative nitrosation could be demonstrated afterprecursor application of 1450 /Â¿moles/kg.

DISCUSSION

This paper describes the possibility of detection of invivo formation of A/-nitroso compounds by estimating their

DECEMBER 1977 4575



R. Braun et al.

a u

Table 3Mutagenic activity of several p.o.-administered N-nitrosamines in the host-mediated

assay

Incubation time of bacteria was 5 hr in all cases.

W-Nitros-

amineNDMA

DiNPZNPZNMBANMORNPMZLowest

effectivedose"

(mg/kg)30

810

3601

--300C2-dose"Co

LOED2.76

2.983.332.643.310,r0.6520.263

0.4770.0341.303r"0.9840.988

0.9960.9690.995mg/kg29

108

3230.05

â€¢300^moles/kg392

6867

21360.45

â€¢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 studieswith 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 thehost-mediated assay.

1000 2000 3000

Dose morpholine or piperazine-NaNOj( Aimoles/kg ]

Chart 4. Regression lines for the equimutagenic doses of N-nitrosaminesdetermined by means of the dose-response curves for nitrite plus amine andN-nitrosamine mutagenesis in the host-mediated assay.

mutagenic properties in the host-mediated assay. This system for mutagenicity testing seems to be very useful forthis purpose since /V-nitrosation is an acid-catalyzedprocess taking place in mammalian stomach (28) and theA/-nitroso compounds formed preferentially induce genemutations (29, 49, 51). In the case of nitrosamine synthesis,these promutagens become activated by drug-metabolizingenzymes localized in the microsomes of host animal tissues(5, 19, 25, 30).

In the host-mediated assay, all alkylureas administeredp.o. to mice in combination with equimolar doses of sodiumnitrite were found to increase the mutant frequencies ofthe indicator bacteria. Neither the alkylurea derivatives MU,EU, and ETU nor nitrite were mutagenic alone. Therefore,these results are consistent with the theory that mutagenic

3-

ez$S 1- 0

NO

1000 2000 3000

Dose NaNOj* morpholine (umoles/kg)

Chart 5. Relative rate of nitrosation for MOR in mouse stomach as afunction of the nitrite plus MOR dose administered to the animals.

75-

Ã®

No1000 2000 3000

Dose NaNOjÂ» piperazine (jiimoles|kg)

Chart 6. Relative rate of nitrosation for PZ in mouse stomach as afunction of the nitrite plus PZ dose administered to the animals.

A/-mtroso compounds are synthesized when these compounds are in combination. MU possessed a higher mutagenic activity than did EU when administered in similardoses and under the same conditions; this is attributed, inpart at least, to the more rapid nitrosation of MU under





acidic conditions (27, 28). These findings confirm the earlierstudies of Couch and Friedman (9), who also demonstratedhigher mutagenic activity of MU plus nitrite. Also, in long-term studies, MU showed stronger tumorigenic potential incombination with nitrite than did EU plus nitrite (35). Although at the present time no data concerning the muta-genicity of both nitroso alkylureas in the host-mediatedassay with the repair-defective strain TA1950 S. typhimu-rium are available, studies with the corresponding repair-effective strain revealed a higher activity of nitrosomethylu-rea in this test system than of nitrosoethylurea after p.o.application to mice (32). Therefore, the finding of strongermutagenic activity of nitrite plus MU in our study, as wellas in that of Couch and Friedman (9), seems to be theresult of both a higher rate constant for the nitrosation andthe stronger mutagenic activity of nitrosomethylurea produced within the stomach of host animal. The alkylureasMU and EU are not considered to be significant environmental contaminants. However, ETU is widely distributedin our environment as the decomposition and metabolicbreakdown product of fungicides belonging to the ethyl-enebis(dithiocarbamate) group (39). Studies dealing withthe nitrosation kinetics of this thiourea derivative are notavailable. In the present host-mediated assay experiments,a very low effective dose for ETU:nitrite was demonstrated,this lowest effective dose is in fact 200-fold lower than thelowest effective dose for MU:nitrite and 500-fold lower thanthat of EU:nitrite. Although /V-nitrosoethylenebis(thiourea)was found to be a very potent mutagen in the host-mediatedassay (R. Braun, unpublished observation), these resultsindicate a high turnover and a high rate constant for thenitrosation reaction in vivo.

Of the secondary amines tested in combination withnitrite, only the cyclic compounds MOR, PZ, and AMTincreased the mutant frequencies of the indicator bacteria.Contrary to the observation on the interactive mutagenicityof DMA plus nitrite in the host-mediated assay in which ICRmice were used, published recently by Couch and Friedman(9), our studies with NMRI mice showed no increase in themutant frequencies of the repair-defective indicator bacteria. Even the very high dose of DMA, 24,444 Â¿Â¿moles/kg,plus nitrite, 1450 /Â¿moles/kg,was without mutagenic effec-tivity (the same dose was found to be mutagenic in thepaper cited above). In addition to differences between hostanimal strains, differences in sensitivity of the bacterialtester strains G46 and TA1950 may account for the discrepancy between the results obtained by Couch and Friedmanand those of the present study. The repair-effective strainG46 used by those authors was found to be superior to itsderivatives lacking excision repair for NDMA and someother methylatmg agents (3). The absence of interactivemutagenicity of DMA plus nitrite in our experiments is inconformity with studies on their carcinogenic potential(20). The negative results for DMA and the absence ofcarcinogenic effects of diethylamine (10, 42) and otherstrongly basic amines in combination with nitrite (35) led tothe conclusion that the carcinogenic risk exerted by asecondary amine in the presence of nitrite depends on thebasicity of the amines: the weaker the base, the faster therate constant for nitrosation (28). The strongly basic character (pKa, 10.7), the low rate constant for the nitrosation

reaction (28), and the relatively high lowest effective doseof NDMA in the host-mediated assay (Table 3) might explainthe absence of genetic activity of combined nitrite plusDMA administraiton. MBA is a weaker base and has ahigher rate constant for nitrosation (28). In both mice andrats, tumor development was demonstrated after administration of nitrite and MBA-containing food (35). On theother hand, a combined MBA plus nitrite intubation waswithout mutagenic activity in our experiments up to thehighest dose tested (2900 /^moles/kg). We attribute thesenegative findings to the very weak mutagenic activity of theresulting NMBA in the host-mediated assay. The lowesteffective dose for this nitrosamine was the highest of allcompounds tested (Table 3). In addition, tumor inductionin long-term studies is the result of repeated treatmentover weeks (or months), while under our test conditionsthe mutagenicity of the amount of nitrosamine producedafter single acute treatment was estimated. The absence ofmutagenic activity of DPA and PMZ in combination withsodium nitrite is due to the nonmutagenicity of the respective nitrosamines. NPMZ was tested in the host-mediatedassay with S. typhimurium TA1950 as the genetic indicatorsystem (Table 3), while /V-nitrosodiphenylamine was without mutagenic activity in the Sa/mone//a/microsome test inwhich TA1535 or TA100 were used as the genetic indicators(26, 54). Both compounds were also negative in carcinoge-nicity studies with rats (10, 24). The cyclic amines AMT, MOR,and PZ possessed mutagenic properties in experimentswith simultaneous application of equimolar amounts ofaminÃ©plus sodium nitrite. The nitrosation kinetics of AMTis not known, and the same is true for the structure andchemical properties of the A/-nitroso derivative formed. Ourresults demonstrate the formation of a mutagenic productin mouse stomach. Since AMT is a pesticide widely distributed in the human environment, further studies are neededto elucidate whether this compound is carcinogenic andthus a health hazard for humans. The mutagenic activity ofcombined PZ or MOR plus nitrite application is detectablein much lower doses than in AMT:nitrite (Table 1). Althoughthe nitroso derivative of MOR showed stronger mutageniceffectiveness than did the /V-nitrosopiperazines, the opposite was true for the genetic effectiveness of precursors(Charts 1 and 3). The only explanation for this phenomenonis the production of a higher yield of nitrosamine from PZthan from MOR. Equimutagenic doses of the respectivenitrosamines inferred from the linear regression lines forthe dose-response studies of both nitrite plus amine andnitrite plus nitrosamine support this assumption (Chart 4).This interpretation gains further support from experimentson the effects of temporal spacing of precursor administration (Table 2). Since sodium nitrite absorption from gastricjuice is very fast (17, 34), the application of this compound10 min before amine resulted in an 80% decrease in PZplus nitrite mutagenicity. If the administration of nitritepreceded MOR treatment, no mutagenic response wasinduced. These results demonstrate that although nitrosamine synthesis was depressed, in the case of PZ, it wasstill high enough to be detected in the host-mediated assay.The higher amounts of nitrosamine formed in mouse stomach from PZ relative to MOR and the significant mutagenicity of nitrite plus PZ in experiments with temporal spacing

DECEMBER 1977 4577



R. Braun et al.

of compound administration correspond to the higher rateconstant for the nitrosation of PZ (28). The second aminogroup within the cyclic structure causes PZ to become aweaker base than is MOR and to participate readily innitrosation. By estimating the relative rates of nitrosationfor these amines, significant differences became clear(Charts 5 and 6). According to our observations in thehost-mediated assay, for MOR a fraction of 1 to 3% of the

theoretical yield must be taken into consideration. Therelative rate of nitrosation increased as a function of precursor dose. PZ nitrosation was, under the same experimental conditions, in the range of 50 to 70% of the theoretical yield, but in contrast to MOR these rates decreasedwith increasing precursor doses. Similar observations havebeen made for chemical estimation of in vivo formation ofnitrosopiperazines (34). These findings are difficult to explain, but changes of the pH from optimal to less-optimal

levels by high PZ concentrations in the gastric juice mightbe the cause of both findings. The relative rates of nitrosation shown by host-mediated assay techniques represent

only approximations, since biological activity, which inturn might be influenced by several factors, especially bythe presence of the precursors nitrite and amine, is beingmeasured. Couch and Friedman observed a decreasinginfluence of sodium nitrite, even at low doses, on themutagenic activity of NDMA in the host-mediated assay

and attributed this to an inhibition of the biotransformationof the promutagenic compound into ultimate forms (8). Ifthe same enzyme system is responsible for metabolism ofNMOR and the /V-nitrosopiperazines, the precursor nitrite

will reduce the mutagenic activity of the nitrosaminesformed from them and will therefore contribute to a decrease in the actual relative rate of nitrosation. Both PZand MOR proved to be tumorigenic in long-term animalstudies after administration with nitrite in food (35, 43) oradministration with nitrite in drinking water (1). Thesestudies revealed a higher carcinogenicity for PZ (35) thanfor MOR.

In agreement with the carcinogenicity studies performedby Sander and Schweinsberg (35), our results with the host-mediated assay clearly demonstrate that only easily nitros-

able compounds become sufficiently nitrosated in vivo forestimation of their mutagenic properties. These chemicalsare essentially the same as those described for long-term

studies of carcinogenesis and represent secondary amineswith weakly basic properties. A second aspect that must betaken into consideration is the mutagenic effectiveness of theresulting N-nitroso derivative. Mutagenic activity of combined nitrite plus amine or amide intubation will be detectable only for those compounds for which nitroso derivativesare strong mutagens. This is true for the nitrosopiperazines,NMOR, and nitrosoethylenebis(thiourea), the synthesis fromthe precursors of which has been estimated for a widedose range.

Our observations, along with those of other authors onthe significance of mutagenic activity of /V-nitroso com

pounds formed in vivo from nonmutagenic precursors, adda further dimension to the extensively studied carcinogenicpotential of such chemical interactions, but we should becautious in attempting to extrapolate experimental data tothe human situation. Human exposure to the precursors.

especially to nitrite, is much lower than simulated in thisstudy. In addition to nitrite-containing food (21, 33), nitrate

reduction in saliva contributes to a low, but significant,nitrite ingestion (46), which has been calculated at 12.8mg/day for the average United States resident (53). Theother precursors, the amines or amides, are present inminute amounts also, but much higher doses are ingestedas drugs; the dose for PZ ascariasis therapy may reach5000 mg within 1 day. In this special case, the likelihood ofthe formation of mutagenic and carcinogenic nitrosopiperazines seems to be rather high, since the therapy regimensresult in a close and continuous contact for days of bothnitrite and PZ under the acidic conditions of the humanstomach. However, it is difficult to determine from thepresent results whether these interactions might be relatedto the induction of mutation in human germ cells; only suchmutations will be hazardous from the point of view of agenetic risk for humans. In addition to differences in exposure and dosage, differences in the sensitivity of targetcells must be taken into account. Otherwise, it would beunrealistic to ignore the mutagenic potential of /V-nitroso

compounds synthesized in vivo from nonmutagenic chemicals and to delegate the human health hazards into thefield of chemical carcinogenesis.

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DECEMBER 1977 4579



1977;37:4572-4579. Cancer Res Rolf Braun, Jörg Schöneich and Dieter Ziebarth Their Mutagenic Activity in the Host-mediated Assay

-Nitroso Compounds and Detection ofN Formation of In Vivo

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