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Mutagenic activity in the stomach and small intestine of rats after oral administration of nitrosated beef extract
Cancer Letters, 25 (1984) 225-230 Elsevier Scientific Publishers Ireland Ltd.
hlUTAGENIC ACTIVITY IN THE STOMACH AND SMALL INTESTINE OF RATS AFTER ORAL ADMINISTRATION OF NITROSATED REEF EXTRACT RUTH ~ N Z N E R and JOACHIM WEVER Institute o f Biochemistry, Federal Research Centre for Nutrition, Engesserstrasse 20, D-7500 Karlsruhe (F.R.G.) (Received 17 September 1984) (Revised version received 31 October 1984) (Accepted 1 November 1984)
SUMMARY
The products formed by the reaction of beef extract with nitrite were assayed in the Salmonella/microsome mutagenicity test. In strain TA1538, TA98 and TAlOO a direct-acting mutagenic response was observed. The presence of liver-microsome preparation resulted in decreased mutagenicity. To study the absorption, distribution and excretion of mutagenic substances in nitrosated beef extract, the test material was given perorally to rats. Investigations of the stomach, bile fluid, urine, small intestine and blood samples were carried out, and mutagenicity was found in the contents of stomach and small intestine: It is supposed that unlike beef extract itself, its nitroso product is not excreted in the bile but passes directly from the stomach and small intestine.
INTRODUCTION
It is well documented that promutagens are formed in meat subjected to moderate cooking conditions and in commercially produced beef extract [2,3,9,10,12,14]. In previous investigations on the absorption, distribution and metabolism of ingested beef extract in the mammalian organism, we discovered mutagenic activity in the stomach contents, bile and urine after oral administration of beef extract to rats; using the Ames test, mutagenicity was only demonstrable after the addition of liver-microsome fraction (S9) derived from rats pretreated with Aroclor 1254 to the plates [Ill. It is known that mutagenic activity is present in nitrite-treated foods [7]. Many nitroso compounds are potent carcinogens in a wide range of animal species with marked organotropic effects [ 5 ] . It seemed interesting to investigate the absorption, distribution and excre0304-3835/84/$03.00 @ 1984 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
tion of the direct-acting mutagenic substances after oral administration of nitrite-treated beef extract to rats, and to compare the results with those previously obtained after administration of untreated beef extract. EXPERIMENTAL
Preparation o f the reaction product of beef extract with nitrite Beef extract (Difco) and half of its weight in sodium nitrite in aqueous solution was adjusted to pH 3.3 with 1N HC1 and left for 1 0 min at room temperature. The resulting red-brown precipitate was collected by centrifugation and washed twice with warm distilled water. Ten grams of beef extract and 5 g of sodium nitrite yielded about 1g (wet weight) of precipitate. This material is hereafter designated as nitrosated beef extract. Animal treatment and sampling Animals were treated and samples were taken as described [ l l ] . Briefly, male Sprague-Dawley rats from Charles River Wiga, Sulzfeld, F.R.G., were housed under standardized conditions. After fasting for 1 2 h the rats received 1 1 5 mg of nitrosated beef extract dissolved in 2 ml of dirnethylsulfoxide by gavage, For the investigation of the gut content the material was given in 2 ml of 1 % aqueous gelatine. Before surgical operation animals were anesthetized with 1g/kg of urethane i.p. Bile was collected by means of a cannula inserted into the common bile duct and the effluent bile fluid was investigated every hour for up t o 4 h after feeding. Urine was obtained by bladder puncture at the end of the experiment. Blood samples were drawn with a sterile syringe from the portal vein or the vena cava distal from the junction of the kidney. At the end of an experiment gastric and intestinal content, respectively, were obtained by gastrotomy and flushing the stomach or the small intestine with 5 ml of 0.9% NaCl solution. The supernatant of the centrifuged gastric or intestinal contents, respectively, as well as bile and urine, were filter sterilized (0.2 pm membrane filters) before being assayed in the Ames test. All assays were performed with at least 6 animals each. Mutagenicity assay The standard plate-incorporation test was carried out according to the procedure of Ames et al. [I], employing the his- tester strains TA1538, TA98 and TA100. The activation enzyme system consisted of 0.5 ml of S9 mix containing 50 pl of hepatic 9000 X g supernatant from Aroclor 1254-induced male Sprague-Dawley rats. Positive controls were performed by spot-test with 2-aminoanthracene. All plates were run in triplicate. RESULTS AND DISCUSSION
In preliminary investigations the mutagenic activity of the material formed by the reaction of beef extract with nitrite under acid conditions was
TABLE 1 blUTAGENIC ACTIVITY OF NITROSATED BEEF EXTRACT Dose (mgiplate)
his+ revertants per platea
. TA1538
a
TA9 8
T A l 00
Each vatue is the mean of 3 plates.
tested in Salmonella typhimurium strains TA1538, TA98 and TA100. As shown in Table 1 the test material exhibited mutagenic activity toward all tester strains with and without metabolic activation with rat-liver microsome preparation (89 mix). In the presence of S9 mix the number of revertants decreased. Because of the high revertant yield with TA1538 and the low histidine sensitivity the animal experiments were performed with this strain. Mutagenicity of the body fluids of the animals treated with the nitrosated beef extract was determined with and without S9 mix. In every case, the mutagenicity was reduced by the addition of S9 mix and, therefore, only the results of the Ames test without metabolic activation are shown in Tables 2 and 3. TABLE 2 MUTAGENICITY TESTING OF THE RINSES OF THE STOMACH OF RATS 4 H AFTER ORAL ADMINISTRATION O F NITROSATED BEEF EXTRACT Animal no.
Level of gastric filling
No. of his' revertants per platea
1 2
Completely filled Partialfy filled Partially filled Empty
6000 4100 2500 136 30
3 4 Control (fasted) a
Shown are mean values of his' revertants of strain TA1558 scored o n 3 plates (with 0.1 ml test material each) of 4 representative treated animals out of 1 0 and 1control out of 2.
TABLE 3 MUTAGENICITY TESTING OF THE RINSES OF THE SMALL INTESTINE OF RATS AFTER ORAL ADhIINISTRATION OF NITROSATED BEEF EXTRACT Animal No.
No. of his' revertants per platea Vol. of gut rinses per plate (ml)
1 2 3 Control (fasted) a
0.1
0.5
212 176 108 27
1050 560 460 34
Shown are mean values of his' revertants of strain TA1538 scored o n 3 plates o f 3 representative treated animals out of 6 and 1 control out o f 2.
Unlike nitrosamines most nitrosamides are direct-acting mutagens [6]. As the reaction products of beef extract and nitrite exert their mutagenic response without metabolic activation, it is assumed that they belong to this type of nitroso derivatives. Nitrosamides are predominantly contact carcinogens that tend to produce gastric lesions in animal studies [4,8,13,15]. In order to follow the routes of absorption, distribution and excretion of the ingested nitrosated beef extract in the animal body, the substance was fed t o the rats at a concentration higher than that considered appropriate for the animals. The gastric transit time of the test material dissolved in dimethylsulfoxide revealed considerable individual variation among the experimental rats. When the animals were killed 4 h after oral administration of the nitrosated beef extract the stomachs of some rats were still completely filled whereas the gastric content of other animals was small. In some rats the absorption of the test material was extremely slow. Thus even after 1 0 h in some cases the browncoloured substances was still present in the stomach in visible quantities. Depending on the extent t o which the stomach was filled, the gastric contents revealed varying degrees of mutagenicity. In Table 2 the number of revertants induced by the rinses from the stomach of 4 animals with different degrees of gastric filling is presented. The data show that at a higher level of gastric filling the mutagenicity was increased. When the stomach was empty, as determined by visual inspection, mutagenicity of the rinsing fluid was still detectable, though to a lesser extent. The control animals did not show mutagenicity of the stomach contents. The small intestine was investigated 1-2 h after oral administration of nitrosated beef extract given in gelatine. In most animals the test substance 1 was observed as a brown-coloured plug.in the small intestine near the caecum. In Table 3 the results of the Ames test of the gut contents of the small
intestine rinsed with saline are shown. The data indicate that after the stomach passage there was a pronounced mutagenic effect (with a dose response relationship) demonstrable in the small intestine. In contrast to beef extract, where after oral administration to rats mutagenicity was excreted in the bile fluid and in the urine [ l l ] , the nitrossted beef extract exhibited no mutagenic activity in the bile during a study period of 1-4 h after oral feeding. There was also no mutagenic response in the urine, isolated 4 h after injection of the test material. The investigation of the blood samples of the portal vein and the vena cava also failed to show a mutagenic effect of the ingested nitrosation product of beef extract. (The negative results of the Ames test performed with bile fluid, urine and blood samples are not shown). Based on the lack of mutagenicity in the bile and in the urine of rats treated with the test substance it is assumed that unlike beef extract its nitrosation products are not, or only poorly, absorbed from the intestine and excreted in the bile, or that the nitrosated material is metabolized in the liver to products without mutagenic activity so that no mutagenicity is recovered in the bile fluid. It remains to be established by further investigations whether the contact of the gastro-intestinal mucosa with the direct-acting mutagenic material formed by the reaction of heat-treated beef with nitrite is of significance in potential chemical carcinogenesis. ACKNOWLEDGEMENTS
The authors wish to thank Mrs. G . Dubberke, Mrs. A. Fiirniss and Miss S. Schweikert for their skillful technical assistance. REFERENCES
1 Ames, B.N., hlccann, J. and Yamasaki, E. (1975)Methods for detecting carcinogens and mutagens with the Salmonella mammalian-microsome mutagenicity test. hlutat. Res., 31,347-364. 2 Commoner, B., Vithayathil, A., Dolara, P., Nair, S., Madyastha, P. and Cuca, G.C. (1978)Formation of mutagens in beef and beef extract during cooking. Science, 201, 913-916. 3 Dolara, P.,Commoner, B., Vithayathii, A., Cuca, G., Tuley, E., hladyastha, P., Nair, S. and Kriebel, D. (1979)The effect of temperature o n the formation of mutagens in heated beef stock and cooked ground beef. hlutat. Res., 60,231-237. 4 Druckrey, H., Ivankovic, S., Biicheler, J., Preussmann, R. and Thomas, C. (1968) Erzeugung von hlagen- und Pankreaskrebs beim llleerschweinchen durch hlethylnitrosoharnstoff und Urethan. Z. Krebsforsch., 71,167-182. 5 Druckrey, H.,Preussmann, R., Ivankovic, S. and Schmahl, D. (1967)Organotrope carcinogene Wirkungen bei 65 verschiedenen N-Nitroso-Verbindungen a n BD-Ratten. Z. Krebsforsch.. 69,103-201. 6 Lijinsky, W. and Andrews, A.W. (1979)T h e mutagenicity of nitrosamides in Salmonella typhimurium. hlutat. Res., 68,1-8.
7 hlarquardt, H.,Rufino, F. and Weisburger, J.H. (1977)hlutagenic activity of nitritetreated foods: human stomach cancer may be related t o dietary factors. Science, 196, 1000-1001. 8 hlirvish, S.S. (1983)T h e etiology of gastric cancer. Intragastric nitrosamide formation and other theories. J. Natl. Cancer Inst.. 71. 629-647. 9 hliinzner, R. (1981)hlutagenitatspriifung von Fleischextrakten. Fleischwirtschaft, 61, 1586-1588. 10 hliinzner, R. (1983)Untersuchungen zum mutagenen Potential von gebratenem Fleisch. Fleixhwirtschaft, 63,611-613. 11 hliinzner, R. and Wever, J. (1984)Investigations o n t h e detection of mutagenic activity of beef extract in rats after oral administration. Cancer Letters, 23, 109-114. 12 Pariza, hl.W., Ashoor, S.H. and Chu, F.S. (1979)hfutagens in heat-processed meat, bakery and cereal products. F o o d Cosrnet. Toxicol., 17,429-430. 13 Schoental, R. (1963)Induction of tumors of t h e stomach in rats and mice by N-nitroso-N-alkylurethanes. Nature, 199,190. 14 Spingarn, N.E. and Weisburger, J.H. (1979)Formation of mutagens in cooked food. I. Beef. Cancer Letters, 7, 259-264. 15 Sugirnura, T.,Fujimara, S. and Kogure, K. (1969)Production of adenocarcinoma in glandular stomach of rats by N-methyl-N-nitro-N-nitrosoguanidine. Nature, 216, 943-944.
hlUTAGENIC ACTIVITY IN THE STOMACH AND SMALL INTESTINE OF RATS AFTER ORAL ADMINISTRATION OF NITROSATED REEF EXTRACT RUTH ~ N Z N E R and JOACHIM WEVER Institute o f Biochemistry, Federal Research Centre for Nutrition, Engesserstrasse 20, D-7500 Karlsruhe (F.R.G.) (Received 17 September 1984) (Revised version received 31 October 1984) (Accepted 1 November 1984)
SUMMARY
The products formed by the reaction of beef extract with nitrite were assayed in the Salmonella/microsome mutagenicity test. In strain TA1538, TA98 and TAlOO a direct-acting mutagenic response was observed. The presence of liver-microsome preparation resulted in decreased mutagenicity. To study the absorption, distribution and excretion of mutagenic substances in nitrosated beef extract, the test material was given perorally to rats. Investigations of the stomach, bile fluid, urine, small intestine and blood samples were carried out, and mutagenicity was found in the contents of stomach and small intestine: It is supposed that unlike beef extract itself, its nitroso product is not excreted in the bile but passes directly from the stomach and small intestine.
INTRODUCTION
It is well documented that promutagens are formed in meat subjected to moderate cooking conditions and in commercially produced beef extract [2,3,9,10,12,14]. In previous investigations on the absorption, distribution and metabolism of ingested beef extract in the mammalian organism, we discovered mutagenic activity in the stomach contents, bile and urine after oral administration of beef extract to rats; using the Ames test, mutagenicity was only demonstrable after the addition of liver-microsome fraction (S9) derived from rats pretreated with Aroclor 1254 to the plates [Ill. It is known that mutagenic activity is present in nitrite-treated foods [7]. Many nitroso compounds are potent carcinogens in a wide range of animal species with marked organotropic effects [ 5 ] . It seemed interesting to investigate the absorption, distribution and excre0304-3835/84/$03.00 @ 1984 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
tion of the direct-acting mutagenic substances after oral administration of nitrite-treated beef extract to rats, and to compare the results with those previously obtained after administration of untreated beef extract. EXPERIMENTAL
Preparation o f the reaction product of beef extract with nitrite Beef extract (Difco) and half of its weight in sodium nitrite in aqueous solution was adjusted to pH 3.3 with 1N HC1 and left for 1 0 min at room temperature. The resulting red-brown precipitate was collected by centrifugation and washed twice with warm distilled water. Ten grams of beef extract and 5 g of sodium nitrite yielded about 1g (wet weight) of precipitate. This material is hereafter designated as nitrosated beef extract. Animal treatment and sampling Animals were treated and samples were taken as described [ l l ] . Briefly, male Sprague-Dawley rats from Charles River Wiga, Sulzfeld, F.R.G., were housed under standardized conditions. After fasting for 1 2 h the rats received 1 1 5 mg of nitrosated beef extract dissolved in 2 ml of dirnethylsulfoxide by gavage, For the investigation of the gut content the material was given in 2 ml of 1 % aqueous gelatine. Before surgical operation animals were anesthetized with 1g/kg of urethane i.p. Bile was collected by means of a cannula inserted into the common bile duct and the effluent bile fluid was investigated every hour for up t o 4 h after feeding. Urine was obtained by bladder puncture at the end of the experiment. Blood samples were drawn with a sterile syringe from the portal vein or the vena cava distal from the junction of the kidney. At the end of an experiment gastric and intestinal content, respectively, were obtained by gastrotomy and flushing the stomach or the small intestine with 5 ml of 0.9% NaCl solution. The supernatant of the centrifuged gastric or intestinal contents, respectively, as well as bile and urine, were filter sterilized (0.2 pm membrane filters) before being assayed in the Ames test. All assays were performed with at least 6 animals each. Mutagenicity assay The standard plate-incorporation test was carried out according to the procedure of Ames et al. [I], employing the his- tester strains TA1538, TA98 and TA100. The activation enzyme system consisted of 0.5 ml of S9 mix containing 50 pl of hepatic 9000 X g supernatant from Aroclor 1254-induced male Sprague-Dawley rats. Positive controls were performed by spot-test with 2-aminoanthracene. All plates were run in triplicate. RESULTS AND DISCUSSION
In preliminary investigations the mutagenic activity of the material formed by the reaction of beef extract with nitrite under acid conditions was
TABLE 1 blUTAGENIC ACTIVITY OF NITROSATED BEEF EXTRACT Dose (mgiplate)
his+ revertants per platea
. TA1538
a
TA9 8
T A l 00
Each vatue is the mean of 3 plates.
tested in Salmonella typhimurium strains TA1538, TA98 and TA100. As shown in Table 1 the test material exhibited mutagenic activity toward all tester strains with and without metabolic activation with rat-liver microsome preparation (89 mix). In the presence of S9 mix the number of revertants decreased. Because of the high revertant yield with TA1538 and the low histidine sensitivity the animal experiments were performed with this strain. Mutagenicity of the body fluids of the animals treated with the nitrosated beef extract was determined with and without S9 mix. In every case, the mutagenicity was reduced by the addition of S9 mix and, therefore, only the results of the Ames test without metabolic activation are shown in Tables 2 and 3. TABLE 2 MUTAGENICITY TESTING OF THE RINSES OF THE STOMACH OF RATS 4 H AFTER ORAL ADMINISTRATION O F NITROSATED BEEF EXTRACT Animal no.
Level of gastric filling
No. of his' revertants per platea
1 2
Completely filled Partialfy filled Partially filled Empty
6000 4100 2500 136 30
3 4 Control (fasted) a
Shown are mean values of his' revertants of strain TA1558 scored o n 3 plates (with 0.1 ml test material each) of 4 representative treated animals out of 1 0 and 1control out of 2.
TABLE 3 MUTAGENICITY TESTING OF THE RINSES OF THE SMALL INTESTINE OF RATS AFTER ORAL ADhIINISTRATION OF NITROSATED BEEF EXTRACT Animal No.
No. of his' revertants per platea Vol. of gut rinses per plate (ml)
1 2 3 Control (fasted) a
0.1
0.5
212 176 108 27
1050 560 460 34
Shown are mean values of his' revertants of strain TA1538 scored o n 3 plates o f 3 representative treated animals out of 6 and 1 control out o f 2.
Unlike nitrosamines most nitrosamides are direct-acting mutagens [6]. As the reaction products of beef extract and nitrite exert their mutagenic response without metabolic activation, it is assumed that they belong to this type of nitroso derivatives. Nitrosamides are predominantly contact carcinogens that tend to produce gastric lesions in animal studies [4,8,13,15]. In order to follow the routes of absorption, distribution and excretion of the ingested nitrosated beef extract in the animal body, the substance was fed t o the rats at a concentration higher than that considered appropriate for the animals. The gastric transit time of the test material dissolved in dimethylsulfoxide revealed considerable individual variation among the experimental rats. When the animals were killed 4 h after oral administration of the nitrosated beef extract the stomachs of some rats were still completely filled whereas the gastric content of other animals was small. In some rats the absorption of the test material was extremely slow. Thus even after 1 0 h in some cases the browncoloured substances was still present in the stomach in visible quantities. Depending on the extent t o which the stomach was filled, the gastric contents revealed varying degrees of mutagenicity. In Table 2 the number of revertants induced by the rinses from the stomach of 4 animals with different degrees of gastric filling is presented. The data show that at a higher level of gastric filling the mutagenicity was increased. When the stomach was empty, as determined by visual inspection, mutagenicity of the rinsing fluid was still detectable, though to a lesser extent. The control animals did not show mutagenicity of the stomach contents. The small intestine was investigated 1-2 h after oral administration of nitrosated beef extract given in gelatine. In most animals the test substance 1 was observed as a brown-coloured plug.in the small intestine near the caecum. In Table 3 the results of the Ames test of the gut contents of the small
intestine rinsed with saline are shown. The data indicate that after the stomach passage there was a pronounced mutagenic effect (with a dose response relationship) demonstrable in the small intestine. In contrast to beef extract, where after oral administration to rats mutagenicity was excreted in the bile fluid and in the urine [ l l ] , the nitrossted beef extract exhibited no mutagenic activity in the bile during a study period of 1-4 h after oral feeding. There was also no mutagenic response in the urine, isolated 4 h after injection of the test material. The investigation of the blood samples of the portal vein and the vena cava also failed to show a mutagenic effect of the ingested nitrosation product of beef extract. (The negative results of the Ames test performed with bile fluid, urine and blood samples are not shown). Based on the lack of mutagenicity in the bile and in the urine of rats treated with the test substance it is assumed that unlike beef extract its nitrosation products are not, or only poorly, absorbed from the intestine and excreted in the bile, or that the nitrosated material is metabolized in the liver to products without mutagenic activity so that no mutagenicity is recovered in the bile fluid. It remains to be established by further investigations whether the contact of the gastro-intestinal mucosa with the direct-acting mutagenic material formed by the reaction of heat-treated beef with nitrite is of significance in potential chemical carcinogenesis. ACKNOWLEDGEMENTS
The authors wish to thank Mrs. G . Dubberke, Mrs. A. Fiirniss and Miss S. Schweikert for their skillful technical assistance. REFERENCES
1 Ames, B.N., hlccann, J. and Yamasaki, E. (1975)Methods for detecting carcinogens and mutagens with the Salmonella mammalian-microsome mutagenicity test. hlutat. Res., 31,347-364. 2 Commoner, B., Vithayathil, A., Dolara, P., Nair, S., Madyastha, P. and Cuca, G.C. (1978)Formation of mutagens in beef and beef extract during cooking. Science, 201, 913-916. 3 Dolara, P.,Commoner, B., Vithayathii, A., Cuca, G., Tuley, E., hladyastha, P., Nair, S. and Kriebel, D. (1979)The effect of temperature o n the formation of mutagens in heated beef stock and cooked ground beef. hlutat. Res., 60,231-237. 4 Druckrey, H., Ivankovic, S., Biicheler, J., Preussmann, R. and Thomas, C. (1968) Erzeugung von hlagen- und Pankreaskrebs beim llleerschweinchen durch hlethylnitrosoharnstoff und Urethan. Z. Krebsforsch., 71,167-182. 5 Druckrey, H.,Preussmann, R., Ivankovic, S. and Schmahl, D. (1967)Organotrope carcinogene Wirkungen bei 65 verschiedenen N-Nitroso-Verbindungen a n BD-Ratten. Z. Krebsforsch.. 69,103-201. 6 Lijinsky, W. and Andrews, A.W. (1979)T h e mutagenicity of nitrosamides in Salmonella typhimurium. hlutat. Res., 68,1-8.
7 hlarquardt, H.,Rufino, F. and Weisburger, J.H. (1977)hlutagenic activity of nitritetreated foods: human stomach cancer may be related t o dietary factors. Science, 196, 1000-1001. 8 hlirvish, S.S. (1983)T h e etiology of gastric cancer. Intragastric nitrosamide formation and other theories. J. Natl. Cancer Inst.. 71. 629-647. 9 hliinzner, R. (1981)hlutagenitatspriifung von Fleischextrakten. Fleischwirtschaft, 61, 1586-1588. 10 hliinzner, R. (1983)Untersuchungen zum mutagenen Potential von gebratenem Fleisch. Fleixhwirtschaft, 63,611-613. 11 hliinzner, R. and Wever, J. (1984)Investigations o n t h e detection of mutagenic activity of beef extract in rats after oral administration. Cancer Letters, 23, 109-114. 12 Pariza, hl.W., Ashoor, S.H. and Chu, F.S. (1979)hfutagens in heat-processed meat, bakery and cereal products. F o o d Cosrnet. Toxicol., 17,429-430. 13 Schoental, R. (1963)Induction of tumors of t h e stomach in rats and mice by N-nitroso-N-alkylurethanes. Nature, 199,190. 14 Spingarn, N.E. and Weisburger, J.H. (1979)Formation of mutagens in cooked food. I. Beef. Cancer Letters, 7, 259-264. 15 Sugirnura, T.,Fujimara, S. and Kogure, K. (1969)Production of adenocarcinoma in glandular stomach of rats by N-methyl-N-nitro-N-nitrosoguanidine. Nature, 216, 943-944.