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The effect of temperature on the formation of mutagens in heated beef stock and cooked ground beef
231
Mutation Research, 60 (1979) 231--237
© Elsevier/North-Holland Biomedical Press
THE E F F E C T OF TEMPERATURE ON THE FORMATION OF MUTAGENS IN HEATED BEEF STOCK AND COOKED GROUND BEEF
P. DOLARA, B. COMMONER, A. VITtIAYATHIL, G. CUCA, E. TULEY, P. MADYASTHA, S. NAIR and D. KRIEBEL Center for the Biology of Natural Systems, Washington University, St. Louis, MO 63130 (U.S.A.)
(Received 18 July 1978) (Revision received 8 November 1978) (Accepted 20 November 1978)
Summary The microsome-activatable mutagens {chromatographically distinguishable from benzo[a]pyrene and from the mutagens produced from pyrolysed amino acids and proteins) previously found in beef extract and in bacterial nutrients which contain beef extract axe produced when beef stock is heated. Reflux boiling of beef stock at 100°C results in a linear increase in mutagenic activity toward Salmonella strain TA1538. The rate of production of mutagenic activity at temperatures between 68°C and 98°C conforms closely to the Arrhenius equation, yielding an activation energy of 23 738 calories per mole. Extrapolation from these data predicts a sharp rise in the rate of mutagen formation between 140 and 180°C. This expectation is confirmed when ground beef patties (hamburgers) are prepared in various conventional electrically-heated appliances which operate at different cooking temperatures within this range. The mutagenic activity of hamburger cooked at high temperatures is limited to the surface layers; the temperature of the inside of the hamburger does not exceed 100°C during cooking. No mutagenic activity is found in comparable samples of uncooked meat. The results indicated that the mutagens may be formed as a result of the temperatures encountered in certain conventional cooking procedures.
We have previously shown that bacterial nutrients which contain beef extract, such as those c o m m o n l y used in the Ames bacterial mutagenesis tests, contain activatable mutagens [6]. We have also shown that these mutagens are preduced when beef stock is extensively boiled down to produce beef extract [2], or when raw beef is cooked in an electric home hamburger appliance at a temperature of about 200vC.
232 These observations suggested that cooking procedures might play a crucial role in the production of the beef-extract mutagens. As an initial step in investigating this subject we report here studies regarding the role of temperature in the production of mutagens in beef extract and in ground beef cooked by means of several procedures which differ in their temperatures. Materials Two batches of lean beef (round steak) were obtained from local markets, ground and used throughout the experiments. The microsome preparations and other materials used in the Ames test were prepared as described by Ames et al. [11. Methods
Cooking procedures Cooked beef patties. 100 g patties of lean ground beef were cooked with different, electrically heated, commercial appliances (see below). After being cooked the patties were homogenized in 2 vol. of distilled water in a Waxing blender. The homogenate was adjusted to pit 2.0 with concentrated HC1 and sufficient ammonium sulphate was added to achieve saturation. It was then filtered through glass wool to remove the beef residue and precipitated protein. The acidified filtrate was then extracted twice with equal volumes of methylene chloride and the latter discarded. The aqueous phase was then adjusted to pH 10 with ammonium hydroxide and extracted 3 times with methylene chloride. The methylene chloride extracts were dried with anhydrous sodium sulphate and evaporated to dryness under vacuum at a temperature of 40°C. The residue was taken up in a known volume of chloroform, from which various aliquots were taken for mutagenesis testing. Beef stock. Ground beef mixed with twice its volume of distilled water was heated for 20 min at a temperature of 40°C and then boiled for 10 min. The meat was removed by filtration through a muslin cloth, and the resultant beef stock kept in a 4°C cold room for 12 h. The solidified fat was then removed and the solution was filtered through glass wool and Whatman filter paper (No. 21). Cooking appliances. The following commercial cooking appliances were used: Hamilton Beach "Double Mac" electric hamburger cooker (rated at 750 W), Toastmaster electric toaster-broiler (1000W), Sunbeam electric frypan (1100 W), Amana microwave oven {1450 W), and a conventional laboratory hot plate which a caramic surface (600 W). Cooking temperatures were measured with a copper--constantan thermocouple by means of a Wheatstone bridge. Mutagenesis assay. Mutagenesis assays were carried out according to the conventional Ames procedure [1] using Salmonella strain TA1538. All samples were tested with and without PCB-induced rat-liver microsome preparation ($9). In keeping with our earlier ob,,~rvations, results obtained in the absence of the microsome preparation were uniformly negative and are not reported here.
233
Chromatographic fractionation. Aliquots of chloroform solutions prepared as indicated above were applied to Gelman (rrLC-SG) sheets, and separated with different solvent systems. 1-cm zones of the developed chromatograms were extracted with chloroform : methanol (90 : 10), dried, the residue taken up in DMSO, and tested on strain TA1538 in the presence of the microsome preparation. Results Given the foregoing observations, it was of interest to determine quantitatively the temperature dependence of the process which is responsible for the production of mutagens when beef stock is heated. Beef stok was prepared as described in the previous section. 1600 ml of beef stock was boiled for 13 h in a reflux flask and 100-ml samples were removed at intervals and analyzed for mutagenic activity in the usual way. The results, which are reported in Fig. 1, show that at 100°C mutagenic activity increases approximately linearly with time over a 13-h period. A similar experiment was carried out by refluxing 40-ml aliquots of beef stock at temperatures of 68 °, 78 °, 88 ° and 98°C. After a 24-h period the contents of each flask were analyzed for mutagenic activity in the usual way. The results are presented in the form of an Arrhenius plot in Fig. 2. It is evident from Fig. 2 that the data fit the Arrhenius equation quite well (In K = In A -E/RT; where K is the reaction rate at a given (absolute) temperature T, A is the "Arrhenius factor", E is the activation energy and R is the gas constant).
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From a least-square fit of a straight line to these data, an activation energy of 23 738 calories per mole was calculated. Based on this equation we can compute the expected rate of the reaction at more elevated temperatures than those actually used in the experiment. The resultant curve is shown in Fig. 3, from which it is evident that a sharp increase in the production of mutagens is expected to take place between 140 and 180°C. In view of these results, it was of interest to examine the relationship between the temperature-dependence of mutagen formation in beef stock and in beef itself. In particular, the data of Fig. 3 suggested that variations in temperature in the range of 100--200°C would considerably affect the rate of mutagen formation in cooked beef. This range of temperatures is spanned by several conventional cooking procedures. Thus in microwave cooking, in which the heat is generated by the absorption of microwave energy by the liquid water present in the meat, the cooking temperature is limited to 100°C. In contrast, meat which is grilled or fried is in contact with metal at temperatures of 200°C or more, and the surface temperature of the meat is likely to approach that temperature during cooking. Accordingly, we have compared the mutagen content of 100-g patties of a common batch of lean ground beef cooked in appliances which differ in their cooking temperatures. In each case the cooked meat was treated as described above, and dose--response curves were determined from increasing aliquots using Salmonella strain TA1538 in the presence of the microsome pre~,ar~tion. The results are shown in Fig. 4. When the ground beef patties we.,. cooked in an electrically heated home hamburger appliance {surface temperature 210°C) for 5.5 min or on an electric frypan for 3 min (surface temperature 190°C), substantial mutagenic activity
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Fig. 4. D o s e - - r e s p o n s e curves for m e t h y l e n e c h l o r i d e e x t r a c t s of h a m b u r g e r s c o o k e d w i t h d i f f e r e n t electrically h e a t e d appliances. Fig. 5. D o s e - - r e s p o n s e curves for m e t h y l e n e c h l o r i d e e x t r a c t s of the o u t s i d e l a y e r and the r e m a i n d e r of h a m b u r g e r s c o o k e d o n a h o t p l a t e (surface t e m p e r a t u r e 3 0 0 ° C ) for 10 m i n .
was detected. In both cases the cooking time was sufficient to produce a "welld o n e " hamburger -- i.e., one cooked throughout. In contrast, as shown in Fig. 4, no significant amounts of mutagens were formed when the meat was cooked for 10 min in an electric broiler under the heating element. In this case, although the hamburger was also "well
236 Discussion The results reported above, together with our earlier findings, show that mutagens which are very active in the Ames test are formed when beef stock or ground beef is heated. From the evidence reported in Fig. 2, it is evident that the reaction which converts beef-tissue constituents into mutagens can occur in an aqueous medium at temperatures as low as 68°C. The Arrhenius plot obtained from data derived from such a system (Fig. 3) predicts a sharp increase in the rate of mutagen production at temperatures in excess of about 150°C. The mutagen content of ground beef cooked in different appliances confirms this expectation. Thus, hamburgers cooked at 100°C ill a microwave oven contain no mutagens. The same is true of hamburgers cooked in an electric broiler by radiant heat from a heating element situated above the meat. In this case, although the air temperature is about 350°C, because of the low heat conductivity of air, the temperature of the upper surface of the hamburger rises slowly from room temperature, never exceeding 150 ° during the 10-min cooking period. In contrast, comparable hamburgers cooked in contact with the metal surface of an electric frying pan (190°C) or an electric hamburger cooking appliance (210°C) for 3--5.5 min contain considerable amounts of mutagens. In these appliances the hight heat conductivity of the metal quickly brings the surface of the meat to the temperature of the cooking surface. The evidence that the mutagen content of hamburgers cooked in this way is restricted to the surface layers also confirms this conclusion, since the interior temperature does not exceed 100°C during cooking. Thus, the mutagen content of ground beef cooked in various ways is consistent with the influence of temperature on the rate of mutagen formation, as determined from mutagen formation in beef stock. It is relevant to note that the range of temperatures achieved in these conventional cooking procedures results in very considerable differences in mutagen content, for this allows practical options in cooking meat which can essentially eliminate whatever risk may be associated with the presence of the mutagens in cooked beef. It is relevant to take note, here, of the current information regarding the identity of these mutagens. To begin with, chromatographic comparisons show that they are neither benzo[a]pyrene nor the mutagens reported by Matsumoto and others [3--5] in the charred surface of broiled meat and fish, which are produced by the pyrolysis of amino acids and proteins at temperatures in excess of 300°C [2]. From our present studies of the production of the beefextract mutagens we can infer the following properties. They are (a) basic substances, soluble in water and extractable from alkaline solutions into organic solvents such as methylene chloride; (b) stable to refluxing in 6 N tICl for 6 h; and (c) converted to inherently active mutagens (i.e. mutagenic in the absence of a microsome preparation) by treatment with nitrous acid. Separate analysis of the fat and non-fat parts of cooked beef hamburgers show that the mutagens are chiefly formed in the non-fat constituents but are soluble in the liquid fat formed during cooking. The foregoing results give rise to a number of problems which require further investigation. Extension of such studies to tissues other than beef would help to delineate the conditions which foster heat-induced formation of the muta-
237
gens and help to evaluate their practical significance relative to the dietary origin of potential carcinogens. It is also of considerable importance to determine the molecular structure of the mutagens that occur in beef extract and in beef cooked above the critical temperature range. Identification of the beefextract mutagens will facilitate the elucidation of the processes which produce them and the determination of their toxicological and carcinogenic properties in test animals. Investigations designed to accomplish to those aims are in progress. References 1 A m e s , B.N., J. M c C a n n a n d E. Y a m a s a k i , M e t h o d s f o r d e t e c t i n g c a r c i n o g e n s a n d m u t a g e n s w i t h t h e S a l o m o n e U a / m a m m a l i a n m i c r o s o m e s t e s t , M u t a t i o n Res., 3 1 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 2 C o m m o n e r , B., A. V i t h a y a t h i l , P. D o l a r a , S. N a i r , P. M a d y a s t h a a n d G. C u c a , F o r m a t i o n o f m u t a g e n s in b e e f a n d b e e f e x t r a c t d u r i n g c o o k i n g , S c i e n c e , 201 ( 1 9 7 8 ) 9 1 3 - - 9 1 6 . 3 M a t s u m o t o , T., D. Y o s h i d a , S. M i z u s a k i a n d H. O k a m o t o , M u t a g e n i c i t y o f t h e p y r o l y s a t e s o f peptides a n d p r o t e i n s , M u t a t i o n Res., 56 ( 1 9 7 8 ) 2 8 1 - - 2 8 8 . 4 N a g a o , M., M. H o n d a , Y. S e i n o , T. Y a h a g i a n d T. S u g i m u r a , M u t a g e n i c i t y o f s m o k e e o n d e n s a t e s a n d t h e c h a r r e d s u r f a c e o f fish a n d m e a t , C a n c e r L e t t . , 2 ( 1 9 7 7 ) 2 2 1 - - 2 2 6 . 5 S u g i m u r a , T., M. N a g a o , T. K a w a c h i , M. H o n d a , T. Y a h a g i , Y. S e i n o , T. M a t . ~ l s h i m a , A. Shirai, M. S a w a m u r a a n d H. M a t s u m o t o , M u t a g e n ~ c a r c i n o g e n s in f o o d w i t h special reference to h i g h l y m u t a g e n i c p y r o l y t i c p r o d u c t s in b r o i l e d f o o d , in: O r i g i n s o f H u m a n C a n c e r , C o l d S p r i n g H a r b o r L a b o r a t o r y Ed., 1977, pp. 1561--1578. 6 V i t h a y a t h i l , A . J . , B. C o m m o n e r , S. N a i r a n d P. M a d y a s t h a , I s o l a t i o n o f m u t a g e n s f r o m b a c t e r i a l n u t r i e n t s c o n t a i n i n g b e e f e x t r a c t , J. T o x i c o l . E n v i r o n . H e a l t h , 4 ( 1 9 7 8 ) 1 8 9 - - 2 0 2 .
Mutation Research, 60 (1979) 231--237
© Elsevier/North-Holland Biomedical Press
THE E F F E C T OF TEMPERATURE ON THE FORMATION OF MUTAGENS IN HEATED BEEF STOCK AND COOKED GROUND BEEF
P. DOLARA, B. COMMONER, A. VITtIAYATHIL, G. CUCA, E. TULEY, P. MADYASTHA, S. NAIR and D. KRIEBEL Center for the Biology of Natural Systems, Washington University, St. Louis, MO 63130 (U.S.A.)
(Received 18 July 1978) (Revision received 8 November 1978) (Accepted 20 November 1978)
Summary The microsome-activatable mutagens {chromatographically distinguishable from benzo[a]pyrene and from the mutagens produced from pyrolysed amino acids and proteins) previously found in beef extract and in bacterial nutrients which contain beef extract axe produced when beef stock is heated. Reflux boiling of beef stock at 100°C results in a linear increase in mutagenic activity toward Salmonella strain TA1538. The rate of production of mutagenic activity at temperatures between 68°C and 98°C conforms closely to the Arrhenius equation, yielding an activation energy of 23 738 calories per mole. Extrapolation from these data predicts a sharp rise in the rate of mutagen formation between 140 and 180°C. This expectation is confirmed when ground beef patties (hamburgers) are prepared in various conventional electrically-heated appliances which operate at different cooking temperatures within this range. The mutagenic activity of hamburger cooked at high temperatures is limited to the surface layers; the temperature of the inside of the hamburger does not exceed 100°C during cooking. No mutagenic activity is found in comparable samples of uncooked meat. The results indicated that the mutagens may be formed as a result of the temperatures encountered in certain conventional cooking procedures.
We have previously shown that bacterial nutrients which contain beef extract, such as those c o m m o n l y used in the Ames bacterial mutagenesis tests, contain activatable mutagens [6]. We have also shown that these mutagens are preduced when beef stock is extensively boiled down to produce beef extract [2], or when raw beef is cooked in an electric home hamburger appliance at a temperature of about 200vC.
232 These observations suggested that cooking procedures might play a crucial role in the production of the beef-extract mutagens. As an initial step in investigating this subject we report here studies regarding the role of temperature in the production of mutagens in beef extract and in ground beef cooked by means of several procedures which differ in their temperatures. Materials Two batches of lean beef (round steak) were obtained from local markets, ground and used throughout the experiments. The microsome preparations and other materials used in the Ames test were prepared as described by Ames et al. [11. Methods
Cooking procedures Cooked beef patties. 100 g patties of lean ground beef were cooked with different, electrically heated, commercial appliances (see below). After being cooked the patties were homogenized in 2 vol. of distilled water in a Waxing blender. The homogenate was adjusted to pit 2.0 with concentrated HC1 and sufficient ammonium sulphate was added to achieve saturation. It was then filtered through glass wool to remove the beef residue and precipitated protein. The acidified filtrate was then extracted twice with equal volumes of methylene chloride and the latter discarded. The aqueous phase was then adjusted to pH 10 with ammonium hydroxide and extracted 3 times with methylene chloride. The methylene chloride extracts were dried with anhydrous sodium sulphate and evaporated to dryness under vacuum at a temperature of 40°C. The residue was taken up in a known volume of chloroform, from which various aliquots were taken for mutagenesis testing. Beef stock. Ground beef mixed with twice its volume of distilled water was heated for 20 min at a temperature of 40°C and then boiled for 10 min. The meat was removed by filtration through a muslin cloth, and the resultant beef stock kept in a 4°C cold room for 12 h. The solidified fat was then removed and the solution was filtered through glass wool and Whatman filter paper (No. 21). Cooking appliances. The following commercial cooking appliances were used: Hamilton Beach "Double Mac" electric hamburger cooker (rated at 750 W), Toastmaster electric toaster-broiler (1000W), Sunbeam electric frypan (1100 W), Amana microwave oven {1450 W), and a conventional laboratory hot plate which a caramic surface (600 W). Cooking temperatures were measured with a copper--constantan thermocouple by means of a Wheatstone bridge. Mutagenesis assay. Mutagenesis assays were carried out according to the conventional Ames procedure [1] using Salmonella strain TA1538. All samples were tested with and without PCB-induced rat-liver microsome preparation ($9). In keeping with our earlier ob,,~rvations, results obtained in the absence of the microsome preparation were uniformly negative and are not reported here.
233
Chromatographic fractionation. Aliquots of chloroform solutions prepared as indicated above were applied to Gelman (rrLC-SG) sheets, and separated with different solvent systems. 1-cm zones of the developed chromatograms were extracted with chloroform : methanol (90 : 10), dried, the residue taken up in DMSO, and tested on strain TA1538 in the presence of the microsome preparation. Results Given the foregoing observations, it was of interest to determine quantitatively the temperature dependence of the process which is responsible for the production of mutagens when beef stock is heated. Beef stok was prepared as described in the previous section. 1600 ml of beef stock was boiled for 13 h in a reflux flask and 100-ml samples were removed at intervals and analyzed for mutagenic activity in the usual way. The results, which are reported in Fig. 1, show that at 100°C mutagenic activity increases approximately linearly with time over a 13-h period. A similar experiment was carried out by refluxing 40-ml aliquots of beef stock at temperatures of 68 °, 78 °, 88 ° and 98°C. After a 24-h period the contents of each flask were analyzed for mutagenic activity in the usual way. The results are presented in the form of an Arrhenius plot in Fig. 2. It is evident from Fig. 2 that the data fit the Arrhenius equation quite well (In K = In A -E/RT; where K is the reaction rate at a given (absolute) temperature T, A is the "Arrhenius factor", E is the activation energy and R is the gas constant).
~-7 . . . . .With Microsome Preparat~n 360"f Strain TA 1558i ! Sample. Beef Stock
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Fig. 2. A r r h e n i u s p l o t o f t h e m u t a g e n i c a c t i v i t y r e f l u x e d in b e e f s t o c k as a f u n c t i o n o f t e m p e r a t u r e . T h e r a t e o f m u t a g e n f o r m a t i o n ° K, is e x p r e s s e d as t h e n u m b e r o f r e v e r t a n t c o l o n i e s p e r plate o b t a i n e d f r o m a 7 0 - m l s a m p l e o f b e e f s t o c k ( 0 . 9 8 g d r y w e i g h t ) a f t e r 24 h o f r e f l u x b o i l i n g . T h e line r e p r e s e n t s the leasts q u a r e fit to t h e p o i n t s a n d is d e s c r i b e d b y the e q u a t i o n : In K = 3 6 . 4 4 -- 11 5 7 0 × 1/T. Fig. 3. T h e o r e t i c a l r a t e o f f o r m a t i o n o f m u t a g e n s in b e e f s t o c k at d i f f e r e n t t e m p e r a t u r e s , c o m p u t e d f r o m A r r h e n i u s e q u a t i o n s h o w n in Fig. 2.
the
From a least-square fit of a straight line to these data, an activation energy of 23 738 calories per mole was calculated. Based on this equation we can compute the expected rate of the reaction at more elevated temperatures than those actually used in the experiment. The resultant curve is shown in Fig. 3, from which it is evident that a sharp increase in the production of mutagens is expected to take place between 140 and 180°C. In view of these results, it was of interest to examine the relationship between the temperature-dependence of mutagen formation in beef stock and in beef itself. In particular, the data of Fig. 3 suggested that variations in temperature in the range of 100--200°C would considerably affect the rate of mutagen formation in cooked beef. This range of temperatures is spanned by several conventional cooking procedures. Thus in microwave cooking, in which the heat is generated by the absorption of microwave energy by the liquid water present in the meat, the cooking temperature is limited to 100°C. In contrast, meat which is grilled or fried is in contact with metal at temperatures of 200°C or more, and the surface temperature of the meat is likely to approach that temperature during cooking. Accordingly, we have compared the mutagen content of 100-g patties of a common batch of lean ground beef cooked in appliances which differ in their cooking temperatures. In each case the cooked meat was treated as described above, and dose--response curves were determined from increasing aliquots using Salmonella strain TA1538 in the presence of the microsome pre~,ar~tion. The results are shown in Fig. 4. When the ground beef patties we.,. cooked in an electrically heated home hamburger appliance {surface temperature 210°C) for 5.5 min or on an electric frypan for 3 min (surface temperature 190°C), substantial mutagenic activity
235 1800" 1600Q}
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Fig. 4. D o s e - - r e s p o n s e curves for m e t h y l e n e c h l o r i d e e x t r a c t s of h a m b u r g e r s c o o k e d w i t h d i f f e r e n t electrically h e a t e d appliances. Fig. 5. D o s e - - r e s p o n s e curves for m e t h y l e n e c h l o r i d e e x t r a c t s of the o u t s i d e l a y e r and the r e m a i n d e r of h a m b u r g e r s c o o k e d o n a h o t p l a t e (surface t e m p e r a t u r e 3 0 0 ° C ) for 10 m i n .
was detected. In both cases the cooking time was sufficient to produce a "welld o n e " hamburger -- i.e., one cooked throughout. In contrast, as shown in Fig. 4, no significant amounts of mutagens were formed when the meat was cooked for 10 min in an electric broiler under the heating element. In this case, although the hamburger was also "well
236 Discussion The results reported above, together with our earlier findings, show that mutagens which are very active in the Ames test are formed when beef stock or ground beef is heated. From the evidence reported in Fig. 2, it is evident that the reaction which converts beef-tissue constituents into mutagens can occur in an aqueous medium at temperatures as low as 68°C. The Arrhenius plot obtained from data derived from such a system (Fig. 3) predicts a sharp increase in the rate of mutagen production at temperatures in excess of about 150°C. The mutagen content of ground beef cooked in different appliances confirms this expectation. Thus, hamburgers cooked at 100°C ill a microwave oven contain no mutagens. The same is true of hamburgers cooked in an electric broiler by radiant heat from a heating element situated above the meat. In this case, although the air temperature is about 350°C, because of the low heat conductivity of air, the temperature of the upper surface of the hamburger rises slowly from room temperature, never exceeding 150 ° during the 10-min cooking period. In contrast, comparable hamburgers cooked in contact with the metal surface of an electric frying pan (190°C) or an electric hamburger cooking appliance (210°C) for 3--5.5 min contain considerable amounts of mutagens. In these appliances the hight heat conductivity of the metal quickly brings the surface of the meat to the temperature of the cooking surface. The evidence that the mutagen content of hamburgers cooked in this way is restricted to the surface layers also confirms this conclusion, since the interior temperature does not exceed 100°C during cooking. Thus, the mutagen content of ground beef cooked in various ways is consistent with the influence of temperature on the rate of mutagen formation, as determined from mutagen formation in beef stock. It is relevant to note that the range of temperatures achieved in these conventional cooking procedures results in very considerable differences in mutagen content, for this allows practical options in cooking meat which can essentially eliminate whatever risk may be associated with the presence of the mutagens in cooked beef. It is relevant to take note, here, of the current information regarding the identity of these mutagens. To begin with, chromatographic comparisons show that they are neither benzo[a]pyrene nor the mutagens reported by Matsumoto and others [3--5] in the charred surface of broiled meat and fish, which are produced by the pyrolysis of amino acids and proteins at temperatures in excess of 300°C [2]. From our present studies of the production of the beefextract mutagens we can infer the following properties. They are (a) basic substances, soluble in water and extractable from alkaline solutions into organic solvents such as methylene chloride; (b) stable to refluxing in 6 N tICl for 6 h; and (c) converted to inherently active mutagens (i.e. mutagenic in the absence of a microsome preparation) by treatment with nitrous acid. Separate analysis of the fat and non-fat parts of cooked beef hamburgers show that the mutagens are chiefly formed in the non-fat constituents but are soluble in the liquid fat formed during cooking. The foregoing results give rise to a number of problems which require further investigation. Extension of such studies to tissues other than beef would help to delineate the conditions which foster heat-induced formation of the muta-
237
gens and help to evaluate their practical significance relative to the dietary origin of potential carcinogens. It is also of considerable importance to determine the molecular structure of the mutagens that occur in beef extract and in beef cooked above the critical temperature range. Identification of the beefextract mutagens will facilitate the elucidation of the processes which produce them and the determination of their toxicological and carcinogenic properties in test animals. Investigations designed to accomplish to those aims are in progress. References 1 A m e s , B.N., J. M c C a n n a n d E. Y a m a s a k i , M e t h o d s f o r d e t e c t i n g c a r c i n o g e n s a n d m u t a g e n s w i t h t h e S a l o m o n e U a / m a m m a l i a n m i c r o s o m e s t e s t , M u t a t i o n Res., 3 1 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 2 C o m m o n e r , B., A. V i t h a y a t h i l , P. D o l a r a , S. N a i r , P. M a d y a s t h a a n d G. C u c a , F o r m a t i o n o f m u t a g e n s in b e e f a n d b e e f e x t r a c t d u r i n g c o o k i n g , S c i e n c e , 201 ( 1 9 7 8 ) 9 1 3 - - 9 1 6 . 3 M a t s u m o t o , T., D. Y o s h i d a , S. M i z u s a k i a n d H. O k a m o t o , M u t a g e n i c i t y o f t h e p y r o l y s a t e s o f peptides a n d p r o t e i n s , M u t a t i o n Res., 56 ( 1 9 7 8 ) 2 8 1 - - 2 8 8 . 4 N a g a o , M., M. H o n d a , Y. S e i n o , T. Y a h a g i a n d T. S u g i m u r a , M u t a g e n i c i t y o f s m o k e e o n d e n s a t e s a n d t h e c h a r r e d s u r f a c e o f fish a n d m e a t , C a n c e r L e t t . , 2 ( 1 9 7 7 ) 2 2 1 - - 2 2 6 . 5 S u g i m u r a , T., M. N a g a o , T. K a w a c h i , M. H o n d a , T. Y a h a g i , Y. S e i n o , T. M a t . ~ l s h i m a , A. Shirai, M. S a w a m u r a a n d H. M a t s u m o t o , M u t a g e n ~ c a r c i n o g e n s in f o o d w i t h special reference to h i g h l y m u t a g e n i c p y r o l y t i c p r o d u c t s in b r o i l e d f o o d , in: O r i g i n s o f H u m a n C a n c e r , C o l d S p r i n g H a r b o r L a b o r a t o r y Ed., 1977, pp. 1561--1578. 6 V i t h a y a t h i l , A . J . , B. C o m m o n e r , S. N a i r a n d P. M a d y a s t h a , I s o l a t i o n o f m u t a g e n s f r o m b a c t e r i a l n u t r i e n t s c o n t a i n i n g b e e f e x t r a c t , J. T o x i c o l . E n v i r o n . H e a l t h , 4 ( 1 9 7 8 ) 1 8 9 - - 2 0 2 .