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Carcinogenicity of N-nitrosoephedrine in rats
Cancer Le;~iers, 5 (1978) 103--106
~) Elsevier[North-Holland ,~lcientificPvlbl~shersLtd.
103
C~RCINOGENICITY OF N-NITROSOEPHEDR~krE IN R A T S G. EI~ISNBRAND, R. PREU.~$MANNaad D. SCHMAHL German Cancer Resea~r h C~n~er, Institute ~f Toxicology and Chemotherapy, Im Neuen$leimer Feld 280 6900Heidelberg. G.F.R.
(Received 6 April 1978]~ (Reverendversion receb~ed23 ~ y 1978)
(Accepted 31May 1978)
SLWII~i~,Y N-Nitmsoephedrb~e was adminis~tered orally to 32 male Sprague--Dawley rats at doses o f 120 mg]kg body wt. ~Lwiceweekly. Of the ~;reated animals, 50% died ~p~th prene~plastic and mal~,rnant lesions mainly in the liver, lung and forestomaeh. The median time o f death o f tumor beam~g ~Lnimals was 522 days after the beginning o f the experhnent. The observation o f hyperke:ratosis, papillomas, and I squamous cell cm~inoma o f ~he forestomach suggests that the compound not only exhibits sy.,~temic effects but is probably also a weak local caminogE~.
n~rRODUCTmN The formation of carcinogenic N-nitrosocompotmds from non-carcinogenic prectu'sors in vitro and in rive has received increasing attantion. C]iasses of compounds investigated chemically '.~d in animal experiments include nitrosatedL de.,2vatives of drugs, pasticide~, and many other biologically :,:e~evant N-nit~satable compoundb [3 ]. Ephedrine is a common][y used drag taken either as a single compotmd or, more oI~en, in combination with other: substances. Its N-ni'~roso dev::va£ive has been Lested for careinogel~[city in new!born mice b,y ~he int:mperitonea]: rout.e [4] ; 3 d,~sas of 200 mg]kg body w t , given on clays 1, 4 and 7 ~ffter b~rLh, induc~.
104
200 ml 1 N hydrochloric acid, and 1 M N a N O 2 was added. The N-nitroso compound quickly began to separate; it was redissolved by the addition of 200 rnl glacialaceticacid,and the mixture was stirredovernight.It w~s then poured into icewater,and theN-nitroso compoundwas extracted into diclflororne~ane~The organic phase was dried (Na2So+) and evaporated. The solid !~'esiduewas crystallized from dichlorornethane. It was unil'orrnby thin-laverchromatography (CH2Cl2--n-pentane--ether (5 : 5 : 3). Yield: 84%; rneltingpoint: 90--91°C; Elemental analysis(C~oHI+N20~): C: 61.74 (calc.61.85); H: 7.2~ (calc. 7.21); N: 14.48 (calc.14.43) ;U V spectrum (MeOH): max. 231.3 n m (]ge = 3.86); 208.1 n m (Ig e = 3.96); rnass spectrum (~0 eV): role 42 (C2H~q)+; role 39, 51, 77, 79, 91,107 (fragment seriestypicalfor substitutedhen~i,lalcohol) mZz 58 (C~HsN)+; role 87 (C3HsN20); role 194 (M)+; role 195 (M + I)+. The composition of the fragments at role 58, 87, 194, 195 was confirmed by high resolution mass spectrometry. N-nitrosoephedrine was given twice weekly by gavage, suspended in o'ii,to 32 male Sprague--Dawley rats at doses of 120 rng/kg body wt. ~he animals were about 100 days old at the beginning of the experimem~,.Tht~y received a pelleted diet (Altrornin®) and water ad libitum. Untreated controls (40 male Sprague--Dawiey ra~s) were kept under identicalstandard conditions. All animals were observed until spontaneous death. After complete autopsies, organs were fixed in 10% formaldehyde and examined histologically. PJ~.SULTSAND DISCUSSION The first animal 'with papi'llomas of ~he forestomach died on day 374 after
tl~ebeginning of the experknent~ At this time, 22 animals of ~he experirnen'tal group were stillalive.Of the remaining 21 animals, 13 died with tvrnors in the liver,lung and forestornach and 2 with tumors at other sites(see Table 1);the overalltumor yield thus was 50%. All animals that died with hepatocellular carcinomas of the liveralso showed lung metastases. In addition to 3 animals that died with papillomas of the forestornach,I died with hyperkeratosis of the forestomach (day 583);another one with a hepatoce~lularc~.rcinoma of the liver,also had hyperplasia of the glandv~ar stomach (day 588). The median time of death of tmnor bearing animals was 522 days afterthe beginning of the exper/ment. Interestingly,there were no cau;inornas in the oesophagu~, although the compound is a close analogue of the strong oesophageal careinogen methyl-phenetyl-nitrosarnine[11. The induction of hyperkeratosis,papillomas and a squamous celtcarcinoma in the fo:testornachgives some indicationthat,in addition to systemic effects,the cc~mpound seems to display localcareinogenicityactivity The present study shows that N-nitrosoephedrine is a carcinogen when given orally to rats,inducing preneoplastic and neoplasticlesions rn~inly in liver, lung and forestornach.A n earlierreport showed that t.hecompound induced hver con c~cinornas exclusivelywhen given intmperitoneally to n ~ w b o m mice [41.
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106 Ephedrine is a secondary amine that might be N-nitrosated under conditions prevailing hi the stomach. In an in vitro system, maximum:yields of about 4--5% of the N-ni~r0s0 product were obtained When the nitt~sa~ion was carried out w~th rather high concen'~rations of nitrite(0.3 ]M N a N D ~ ) a n d ephedrine (0.025 M) [2]. Our o w n experiments, carried out at much lower concentrations (5 X I0 -s M N aNOn, I0 -3 M ephedrine), showed that above; 0.5% of the N-nitroso product was obtained under conditions simulating those in the stomach (pH 3, 37°C, i h). In human drug therapy, recommended single o~ml doses of ephedrine hydro. chloride are 50 m g with maxim-al total doses of 150 rag/day. These doses, in our view, do not necessarily represent a significant human health hazard with respect to in vivo formation of N-nitrosoephedlme, when the rehti'¢ely low yields of N-nitroso-product in the above reported in vitro exper~nents are taken into account. ACKNOWLEDGEMENTS We thank Dr. D. Komitowsk~ and Prof. P. Bannasch, Institute for Experimental Pathology, German Cancer Research Center, for histological e:~;aminations. REFERENCES
1 Dmckrey, H., Preussmann, R., Invaukovic, S. and Schmt/hl, D. (1967) Organotrope careinogene Wirkung bei 65 vemchiedenen N~Nitrosoverbindungenan BD-Ratten. Z. Krebsforsch., 69, 103--201. 2 Kinaw!, A. and Schu~er, T. (1978) Reaction l lneti~i."studies on formation of N-nitro. soephedrine in vitro and in vivo. Arzneim.-For~ch, 28, ~19--225. 3 Mindsh, S.S. (1975) Formation of N-nitroso comp0unds. Chemistry, kinetics, and in vivo occurrence. Toxieol. Appl. Pharmacol., 31, 325--351. 4 Wogan, G.N., Paglialunga, S., Archer, M.C. ~nd Tannenbaum, S.R. (1975) Catcinogenicity of nitTamtion product~ of ephedrb~e, sarcosine, folie acid, and creatinine. Cancer Res., 35, 1981--1984.
~) Elsevier[North-Holland ,~lcientificPvlbl~shersLtd.
103
C~RCINOGENICITY OF N-NITROSOEPHEDR~krE IN R A T S G. EI~ISNBRAND, R. PREU.~$MANNaad D. SCHMAHL German Cancer Resea~r h C~n~er, Institute ~f Toxicology and Chemotherapy, Im Neuen$leimer Feld 280 6900Heidelberg. G.F.R.
(Received 6 April 1978]~ (Reverendversion receb~ed23 ~ y 1978)
(Accepted 31May 1978)
SLWII~i~,Y N-Nitmsoephedrb~e was adminis~tered orally to 32 male Sprague--Dawley rats at doses o f 120 mg]kg body wt. ~Lwiceweekly. Of the ~;reated animals, 50% died ~p~th prene~plastic and mal~,rnant lesions mainly in the liver, lung and forestomaeh. The median time o f death o f tumor beam~g ~Lnimals was 522 days after the beginning o f the experhnent. The observation o f hyperke:ratosis, papillomas, and I squamous cell cm~inoma o f ~he forestomach suggests that the compound not only exhibits sy.,~temic effects but is probably also a weak local caminogE~.
n~rRODUCTmN The formation of carcinogenic N-nitrosocompotmds from non-carcinogenic prectu'sors in vitro and in rive has received increasing attantion. C]iasses of compounds investigated chemically '.~d in animal experiments include nitrosatedL de.,2vatives of drugs, pasticide~, and many other biologically :,:e~evant N-nit~satable compoundb [3 ]. Ephedrine is a common][y used drag taken either as a single compotmd or, more oI~en, in combination with other: substances. Its N-ni'~roso dev::va£ive has been Lested for careinogel~[city in new!born mice b,y ~he int:mperitonea]: rout.e [4] ; 3 d,~sas of 200 mg]kg body w t , given on clays 1, 4 and 7 ~ffter b~rLh, induc~.
104
200 ml 1 N hydrochloric acid, and 1 M N a N O 2 was added. The N-nitroso compound quickly began to separate; it was redissolved by the addition of 200 rnl glacialaceticacid,and the mixture was stirredovernight.It w~s then poured into icewater,and theN-nitroso compoundwas extracted into diclflororne~ane~The organic phase was dried (Na2So+) and evaporated. The solid !~'esiduewas crystallized from dichlorornethane. It was unil'orrnby thin-laverchromatography (CH2Cl2--n-pentane--ether (5 : 5 : 3). Yield: 84%; rneltingpoint: 90--91°C; Elemental analysis(C~oHI+N20~): C: 61.74 (calc.61.85); H: 7.2~ (calc. 7.21); N: 14.48 (calc.14.43) ;U V spectrum (MeOH): max. 231.3 n m (]ge = 3.86); 208.1 n m (Ig e = 3.96); rnass spectrum (~0 eV): role 42 (C2H~q)+; role 39, 51, 77, 79, 91,107 (fragment seriestypicalfor substitutedhen~i,lalcohol) mZz 58 (C~HsN)+; role 87 (C3HsN20); role 194 (M)+; role 195 (M + I)+. The composition of the fragments at role 58, 87, 194, 195 was confirmed by high resolution mass spectrometry. N-nitrosoephedrine was given twice weekly by gavage, suspended in o'ii,to 32 male Sprague--Dawley rats at doses of 120 rng/kg body wt. ~he animals were about 100 days old at the beginning of the experimem~,.Tht~y received a pelleted diet (Altrornin®) and water ad libitum. Untreated controls (40 male Sprague--Dawiey ra~s) were kept under identicalstandard conditions. All animals were observed until spontaneous death. After complete autopsies, organs were fixed in 10% formaldehyde and examined histologically. PJ~.SULTSAND DISCUSSION The first animal 'with papi'llomas of ~he forestomach died on day 374 after
tl~ebeginning of the experknent~ At this time, 22 animals of ~he experirnen'tal group were stillalive.Of the remaining 21 animals, 13 died with tvrnors in the liver,lung and forestornach and 2 with tumors at other sites(see Table 1);the overalltumor yield thus was 50%. All animals that died with hepatocellular carcinomas of the liveralso showed lung metastases. In addition to 3 animals that died with papillomas of the forestornach,I died with hyperkeratosis of the forestomach (day 583);another one with a hepatoce~lularc~.rcinoma of the liver,also had hyperplasia of the glandv~ar stomach (day 588). The median time of death of tmnor bearing animals was 522 days afterthe beginning of the exper/ment. Interestingly,there were no cau;inornas in the oesophagu~, although the compound is a close analogue of the strong oesophageal careinogen methyl-phenetyl-nitrosarnine[11. The induction of hyperkeratosis,papillomas and a squamous celtcarcinoma in the fo:testornachgives some indicationthat,in addition to systemic effects,the cc~mpound seems to display localcareinogenicityactivity The present study shows that N-nitrosoephedrine is a carcinogen when given orally to rats,inducing preneoplastic and neoplasticlesions rn~inly in liver, lung and forestornach.A n earlierreport showed that t.hecompound induced hver con c~cinornas exclusivelywhen given intmperitoneally to n ~ w b o m mice [41.
. . . . . . .
105
~
. . . .
.I
Io1,~
~
.~
I~.
~
~,~
~1~:
X
~.~.
r.r,l
= ~
~
.~
r~
Io Z
~S
o
~.~
ii
0
8
~
106 Ephedrine is a secondary amine that might be N-nitrosated under conditions prevailing hi the stomach. In an in vitro system, maximum:yields of about 4--5% of the N-ni~r0s0 product were obtained When the nitt~sa~ion was carried out w~th rather high concen'~rations of nitrite(0.3 ]M N a N D ~ ) a n d ephedrine (0.025 M) [2]. Our o w n experiments, carried out at much lower concentrations (5 X I0 -s M N aNOn, I0 -3 M ephedrine), showed that above; 0.5% of the N-nitroso product was obtained under conditions simulating those in the stomach (pH 3, 37°C, i h). In human drug therapy, recommended single o~ml doses of ephedrine hydro. chloride are 50 m g with maxim-al total doses of 150 rag/day. These doses, in our view, do not necessarily represent a significant human health hazard with respect to in vivo formation of N-nitrosoephedlme, when the rehti'¢ely low yields of N-nitroso-product in the above reported in vitro exper~nents are taken into account. ACKNOWLEDGEMENTS We thank Dr. D. Komitowsk~ and Prof. P. Bannasch, Institute for Experimental Pathology, German Cancer Research Center, for histological e:~;aminations. REFERENCES
1 Dmckrey, H., Preussmann, R., Invaukovic, S. and Schmt/hl, D. (1967) Organotrope careinogene Wirkung bei 65 vemchiedenen N~Nitrosoverbindungenan BD-Ratten. Z. Krebsforsch., 69, 103--201. 2 Kinaw!, A. and Schu~er, T. (1978) Reaction l lneti~i."studies on formation of N-nitro. soephedrine in vitro and in vivo. Arzneim.-For~ch, 28, ~19--225. 3 Mindsh, S.S. (1975) Formation of N-nitroso comp0unds. Chemistry, kinetics, and in vivo occurrence. Toxieol. Appl. Pharmacol., 31, 325--351. 4 Wogan, G.N., Paglialunga, S., Archer, M.C. ~nd Tannenbaum, S.R. (1975) Catcinogenicity of nitTamtion product~ of ephedrb~e, sarcosine, folie acid, and creatinine. Cancer Res., 35, 1981--1984.