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DRUG METABOLISM REVIEWS, 22(6-8), 863-869 (1990)
PROPOSED PLAN FOR A UNIFIED APPROACH FOR EVALUATING NITROFURAN RESlDUES* D. R. MCCALLA Department of Biochemistry McMaster University 1200 Main Street West Hamilton, Ontario, L8N 325 Canada
I.
INTRODUCTION ..........................................................
8~
11.
BACKGROUND ............................................................
8~
111.
SUGGESTIONS FOR ADDITIONAL RESEARCH .................865
IV.
CONCLUDING REMARKS .............................................
867
Acknowledgments .........................................................
.868
References. .................................................................. .868
*This paper was refereed by Suzanne C. Fitzpatrick, Ph.D., Division of Chemistry, HFV- 140, Center for Veterinary Medicine, FDA, 5600 Fishers Lane, Rockville, MD 20857. 863 Copyright 0 1991 by Marcel Dekker, Inc
864
MCCALLA
Drug Metabolism Reviews Downloaded from informahealthcare.com by Freie Universitaet Berlin on 07/08/15 For personal use only.
I. INTRODUCTION Nitrofuran derivatives have been used in human and veterinary medicine since the 1940s and are efficacious against a variety of bacterial and protozoal pathogens. Several hundred different derivatives have been prepared and subjected to some degree of biological testing. While early mechanism of action studies indicated that these agents specifically inhibited pyruvate oxidase (now pyruvate dehydrogenase), it later became clear that these compounds are mutagenic and that at least some of them are carcinogenic [ I ) . As a result of these findings and critiques, such as that by Nader and his associates [2], regulatory agencies began to subject these and related compounds to more intense scrutiny-requiring, first, development of analytical methods having low detection limits, and then asking for identification and characterization of all metabolites. The agencies also become concerned over the possible toxicity of the so-called bound residues (which are detected through the use of radiolabeled drugs). As many authors have pointed out, the task of characterizing bound residues is bedeviled by the low concentrations and low solubility of these materials. In part, at least, the concern of regulatory agencies over bound residues arose from experience with other material such as trichloroethyleneextracted soybean meal, which turned out to contain S-( I ,Zdichlorovinyl)L-cysteine residues in protein [3). In this situation the “dose” of trichloroethylene was immense, and the toxic product was stable in the meal but was converted to a reactive form when consumed by animals. This case, in a sense, establishes the paradigm for problem residues-they are stable where formed but activated when consumed. The soybean meal toxin was formed in a relatively inert material of plant origin and subjected to the full range of mammalian metabolism only when consumed. This situation is very different from that encountered with veterinary drugs. Bound residues derived from these materials are formed in mammalian cells and must persist there for days or weeks in spite of degradative reactions and protein turnover. It is conceivable that residues are formed in one type of cell or tissue and become active only when consumed, digested, and metabolized by another animal. However, it seems probable that the more similar the biochemistry of the organism in which the residues are formed is to that of the consuming organism, the less likelihood there is of the occurrence of toxic bound residues.
11. BACKGROUND
Nitrofurans are themselves relatively inert compounds that are activated in both bacteria and animal tissue by enzymatic reduction [ l , 41. At least in the case of furazolidone it appears that a reactive acrylonitrile deriv-
Drug Metabolism Reviews Downloaded from informahealthcare.com by Freie Universitaet Berlin on 07/08/15 For personal use only.
EVALUATING NITROFURAN RESIDUES
865
ative formed by rearrangement of the hydroxylamino metabolite is probably the “ultimate” active species [5]. This agent reacts with a variety of cellular constituents including nucleic acid [6] and protein [7]. Similar nucleic acid [8] and protein binding [9] has been observed with other nitrofuran derivatives. Among the compounds formed from activated furazolidone are conjugates with GSH [7], mercaptoethanol [5], and protein thiol groups [6]. These products, too, are reactive. Indeed, the GSH conjugate is so reactive that it cannot be recovered from microsomal incubation mixtures unless the mixture is subjected to ultrafiltration [7]. Also, when the mercaptoethanol conjugate reacts with GSH to form the GSH conjugate, approximately 50% of the material is found in unknown products [7]. The mercaptoethanol conjugate is apparently mutagenic in the Salmonella assay. We know from published data on nitrofurans that a portion of the “bound residue” formed from furazolidone and other nitrofurans consists of I4C, which, through metabolism, has become incorporated into normal cellular constituents and thence into protein and other macromolecules. (It is of interest that while Tatsumi [lo] has shown that the aldehyde carbon of furazolidone is found in a-keto glutaric acid, from which it enters metabolic pathways leading to a number of amino acids and thus to protein, the methylene carbons of the oxizolidone ring apparently do not. Available data also show that additional radioactivity in the residue results from the reaction of activated nitrofurans with proteins, nucleic acids, and possibly other cellular constituents [6-91. Nevertheless, as noted above, regulatory agencies remain suspicious that a portion of the bound residue may be reactive and toxic. While in special circumstances relay toxicology may provide useful data, with furazolidone this approach is pointless. The concentration of bound residue from furazolidone is of the order of I ppm or O.OOOl%, whereas the concentration of nitrofurans used in typical rodent carcinogenicity tests with these agents has been 0.2% of the diet!
111. SUGGESTIONS FOR ADDITIONAL RESEARCH
In considering the question of what additional data might help to clarify the risks involved, it should be noted that the thiol conjugates described above have been observed at short times and may have little relevance to the situation after the drug has been withdrawn for a period of days or weeks. In general, it is thermodynamically unlikely that compounds that are themselves reactive enough to pose a toxic hazard will survive for long periods in a warm, moist environment containing a wide array of chemicals. Unless there are special features involved (e.g., halogen-containing
Drug Metabolism Reviews Downloaded from informahealthcare.com by Freie Universitaet Berlin on 07/08/15 For personal use only.
866
MCCALLA
conjugates susceptible to P-lyase action), it is much more likely that the reactivity will decrease as the initial species react progressively with cellular constituents to form more stable adducts. At the present time the reactions in which the acrylonitrile moiety is transferred from one thiol to another seem to be well established. However, these reactions are not quantitative-some 50% of the material ends up in unidentified compounds. Experiments should be undertaken to try to ascertain whether or not these unidentified products represent unreactive “dead ends.” It would also be useful to have additional information on the reactivity of the mercaptoethanol and GSH conjugates. Specifically, we need to know whether or not these materials react (however slowly) with typical cellular constituents to form stable products. It is of interest that acrylonitrile itself is known to react with unprotonated amino groups and the imidazole ring of histidine as well as with thiols [ I 1 1. Since the concentrations of such unprotonated groups are low, the reactions will be slow but nevertheless may be important in determining the nature of the long-term residue. We also need more information on the stability of the conjugates themselves both at 37°C and under conditions typically encountered during food preparation. Such information will provide a basis of chemical evidence which could be used to judge whether or not the thiol conjugates are likely to be persistent or whether the activated furazolidone winds up in stable compounds. Another line of experimentation should be directed to determining whether or not reactive thiol conjugates persist in the bound residue. If protein thiol conjugates do persist, it should be possible to determine their presence, since treatment of the residue with mercaptoethanol would be expected to result in formation of the soluble mercaptoethanol conjugate. It is essential that these experiments be performed on material obtained after an appropriate withdrawal period, otherwise short-term reactive products will inevitably be present and may predominate. To facilitate such experiments, consideration should be given to the use of furazolidone having considerably higher specific activity than has been used in the past [6]. If expense limits the amount available, it would be possible to use rodents rather than pigs to examine whether or not the residue (both before and after enzymatic hydrolysis) can react with mercaptoethanol to regenerate the mutagenic conjugate. If (as is the case with pigs) 1 ppm of residue were to be formed from furazolidone having specific activity of 10 mCi/mmol, each g of tissue would contain some 800,OOO dpm. Even if only 0.1% of this material were reactive, 800 dpm would be solublized. Thus, if thiol conjugates do persist they should be easy to detect. Further, it should be possible, using HPLC, to show whether or not the soluble product found is actually the mercaptoethanol conjugate.
Drug Metabolism Reviews Downloaded from informahealthcare.com by Freie Universitaet Berlin on 07/08/15 For personal use only.
EVALUATING NITROFUR AN RESIDUES
867
Finally, the mutagenicity of the mercaptoethanol conjugate needs further exploration since, obviously, the mutagenic potential of the conjugate (and hence, by inference, of the protein conjugates as well) is a principal cause for concern. Furazolidone can adsorb to glassware and therefore be present unexpectedly [ 121. Although the chances of contamination seem somewhat remote, a small amount of furazolidone (which is 30 times more mutagenic than the conjugate) might be responsible for the activity observed. The simplest approach to clarifying this point would be to use test strains of bacteria such as Salmonella ryphimurium TA IOONR [ 131 or Escherichia coli NFR 502 [ 141 that have lost the nitroreductase necessary to activate nitrofurans and are therefore insensitive to nitrofurans themselves but should remain sensitive to the conjugate. Further, as noted by Zeiger earlier in this symposium, thiols themselves can give positive results in mutagenicity assays because, in the presence of certain metal ions, they lead to the generation of hydrogen peroxide. While it again seems unlikely, it is not beyond the realm of possibility that the conjugate’s activity could be a consequence of the thiol moiety rather than the acrylonitrile. If this mechanism were operative, the mutagenic activity should be suppressed when catalase is added to the test plates.
IV. CONCLUDING REMARKS It will be obvious that while the additional experiments suggested in the preceding section will yield relevant and useful data, they will not provide absolute assurance that the bound residues are harmless. Indeed, there exists no practical way of obtaining such assurance. The problem is not confined to the three classes of chemicals being discussed in this symposium but applies to all veterinary drugs and feed additives whose bound residue levels exceed the tolerance allowed for the parent compound. What, then, is to be done? Logically, we need to find some way of dealing with the uncertainty or abandon the use of such agents. Regulatory agencies have become adept at dealing with other forms of uncertainty such as extrapolations from high to low doses and from rodents to humans. The problems posed by bound residues, while different, do not pose inherently greater difficulty. Since, as pointed out by Frazier (p. 821), toxic residues really represent a “second-order” problem in toxicology, one approach would be to make reasonable estimates of the maximum hazard that might exist and compare that value to other, accepted hazards. Vroomen has shown that ‘‘C-labeled residues in the tissues of pigs after a 14-day withdrawal period are present in amounts of about 1 ppm (61. It is
MCCALLA
Drug Metabolism Reviews Downloaded from informahealthcare.com by Freie Universitaet Berlin on 07/08/15 For personal use only.
868
likely that much of this material consists of stable adducts formed with insoluble cellular constituents. Let us assume, however, that as much as 10% of the long-term bound residue may consist of mutagenic material having the same potency as the mercaptoethanol conjugate and that this material persists through processing and food preparation. From Vroomen’s data [7] a 100-g portion of meat would then contribute some 4OOO revertants if Salmonella typhimurium strain TA 100 were used in the assay. To put this value in perspective, cooking of that meat could produce another 5000 revertants (TA 1538) from protein pyrolysis products 1151. These later compounds are aromatic amines which are known to be powerful multiorgan and multispecies carcinogens [16]. Further, a cup of instant coffee contains mutagenic material sufficient to induce about 50,000 revertants (TA 100) [17]. While no claim is made that the hazard to humans is necessarily proportional to the levels of mutagenicity of the materials, the comparison indicates that even if long-term bound residues are mutagenic, the hazards they pose will be small relative to that of other mutagens present in food.
Acknowledgments Support for research on nitrofurans in the author’s laboratory has been provided by the Natural Sciences and Engineering Research Council of Canada and the National Cancer Institute of Canada. The collaboration over many years of colleagues, especially Dr. Douglas W. Bryant and Mrs. Christel Kaiser-Farrell, is gratefully acknowledged.
REFERENCES [ I ] D. R. McCalla, Environ. Mutagenesis, 5 , 745 (1983). [2] H. Wellford, The Nader Report; Sowing the Wind,Grossman, New York, 1972. [3] M. 0. Schultze, I? Klubes, V. Perman. N. S. Mizuno, F. W. Bates, J. H. Sautter, Blood, 14, 1015 (1959). [4] J. J. Gavin, F. F. Ebetino, R. Freedman, and W. E. Waterbury, Arch. Biochem. Biophys., 113, 399 (1966). [ 5 ] L. H. M. Vroomen. J. F? Groten, K. van Muiswinkel, A. van Veldhuizen. and F? J. van Bladeren, in L. H. M. Vroomen’s Ph.D. thesis, Agricultural University of Wageningen, 1987, p. 105.
Drug Metabolism Reviews Downloaded from informahealthcare.com by Freie Universitaet Berlin on 07/08/15 For personal use only.
EVALUATING NITROFUR AN RESIDUES
869
[6] L. H. M. Vroomen, M. C. J. Berghmans, F? van Leeuwen, T. D. B. van der Struijs, F? H. U. De Vries, and H. A. Kuiper, Food Add. & Contam., 3 , 331 (1986). [7] L. H. M. Vroomen, M. C. J. Berghmans, J. F? Groten, J. H. Koeman, and F? J. van Bladeren. in L. M. H. Vroomen’s Ph.D. thesis, Agricultural University of Wageningen, 1987, p. 123. [8] B. Wentzell and D. R . McCalla, Chem. Biol. Interactions, 31, 151 (1980). [9] D. R. McCalla, A. Reuvers, and C. Kaiser-Farrell, Biochem Pharmacol., 20, 3532 (1971). [lo] K. Tatsumi, H. Nakabeppu, H. Takahashi, and S. Kitamura, Arch. Biochem. Biophys., 234, 112 (1984). [Ill L. Weil and T. S. Seibles, Arch. Biochem. Biophys., 95, 470 (1961). [12] J. I? Heotis, unpublished results. [13] H. S. Rosehkranz and W. T. Speck, Biochem. Biophys. Res. Commun., 66, 520 (1975). [I41 D. R. McCalla, C. Kaiser-Farrell, and M. H. L. Green, J. Bacreriol., 133, 10 (1978). [I51 L. F. Bjeldanes, M. M. Morris, J. S. Felton, S. Heally, D. Stuermu, F? Berry, H. Timourian, and F. T. Hatch, Fd. Chem. Toxicol,20, 357 (1982). [I61 M. Nagao, Y. Takahashi, H. Yamakawa, and T. Sugimura, Mutat. Res., 68, 101 (1979). [17] H. Ohgaki, L. Hasegawa, T. Kato, M. Suenaga, M. Ubukata, S. Sato, S. Takayama, and T. Sugimura. Environ. Health Persp., 67, 129 (1986).
DRUG METABOLISM REVIEWS, 22(6-8), 863-869 (1990)
PROPOSED PLAN FOR A UNIFIED APPROACH FOR EVALUATING NITROFURAN RESlDUES* D. R. MCCALLA Department of Biochemistry McMaster University 1200 Main Street West Hamilton, Ontario, L8N 325 Canada
I.
INTRODUCTION ..........................................................
8~
11.
BACKGROUND ............................................................
8~
111.
SUGGESTIONS FOR ADDITIONAL RESEARCH .................865
IV.
CONCLUDING REMARKS .............................................
867
Acknowledgments .........................................................
.868
References. .................................................................. .868
*This paper was refereed by Suzanne C. Fitzpatrick, Ph.D., Division of Chemistry, HFV- 140, Center for Veterinary Medicine, FDA, 5600 Fishers Lane, Rockville, MD 20857. 863 Copyright 0 1991 by Marcel Dekker, Inc
864
MCCALLA
Drug Metabolism Reviews Downloaded from informahealthcare.com by Freie Universitaet Berlin on 07/08/15 For personal use only.
I. INTRODUCTION Nitrofuran derivatives have been used in human and veterinary medicine since the 1940s and are efficacious against a variety of bacterial and protozoal pathogens. Several hundred different derivatives have been prepared and subjected to some degree of biological testing. While early mechanism of action studies indicated that these agents specifically inhibited pyruvate oxidase (now pyruvate dehydrogenase), it later became clear that these compounds are mutagenic and that at least some of them are carcinogenic [ I ) . As a result of these findings and critiques, such as that by Nader and his associates [2], regulatory agencies began to subject these and related compounds to more intense scrutiny-requiring, first, development of analytical methods having low detection limits, and then asking for identification and characterization of all metabolites. The agencies also become concerned over the possible toxicity of the so-called bound residues (which are detected through the use of radiolabeled drugs). As many authors have pointed out, the task of characterizing bound residues is bedeviled by the low concentrations and low solubility of these materials. In part, at least, the concern of regulatory agencies over bound residues arose from experience with other material such as trichloroethyleneextracted soybean meal, which turned out to contain S-( I ,Zdichlorovinyl)L-cysteine residues in protein [3). In this situation the “dose” of trichloroethylene was immense, and the toxic product was stable in the meal but was converted to a reactive form when consumed by animals. This case, in a sense, establishes the paradigm for problem residues-they are stable where formed but activated when consumed. The soybean meal toxin was formed in a relatively inert material of plant origin and subjected to the full range of mammalian metabolism only when consumed. This situation is very different from that encountered with veterinary drugs. Bound residues derived from these materials are formed in mammalian cells and must persist there for days or weeks in spite of degradative reactions and protein turnover. It is conceivable that residues are formed in one type of cell or tissue and become active only when consumed, digested, and metabolized by another animal. However, it seems probable that the more similar the biochemistry of the organism in which the residues are formed is to that of the consuming organism, the less likelihood there is of the occurrence of toxic bound residues.
11. BACKGROUND
Nitrofurans are themselves relatively inert compounds that are activated in both bacteria and animal tissue by enzymatic reduction [ l , 41. At least in the case of furazolidone it appears that a reactive acrylonitrile deriv-
Drug Metabolism Reviews Downloaded from informahealthcare.com by Freie Universitaet Berlin on 07/08/15 For personal use only.
EVALUATING NITROFURAN RESIDUES
865
ative formed by rearrangement of the hydroxylamino metabolite is probably the “ultimate” active species [5]. This agent reacts with a variety of cellular constituents including nucleic acid [6] and protein [7]. Similar nucleic acid [8] and protein binding [9] has been observed with other nitrofuran derivatives. Among the compounds formed from activated furazolidone are conjugates with GSH [7], mercaptoethanol [5], and protein thiol groups [6]. These products, too, are reactive. Indeed, the GSH conjugate is so reactive that it cannot be recovered from microsomal incubation mixtures unless the mixture is subjected to ultrafiltration [7]. Also, when the mercaptoethanol conjugate reacts with GSH to form the GSH conjugate, approximately 50% of the material is found in unknown products [7]. The mercaptoethanol conjugate is apparently mutagenic in the Salmonella assay. We know from published data on nitrofurans that a portion of the “bound residue” formed from furazolidone and other nitrofurans consists of I4C, which, through metabolism, has become incorporated into normal cellular constituents and thence into protein and other macromolecules. (It is of interest that while Tatsumi [lo] has shown that the aldehyde carbon of furazolidone is found in a-keto glutaric acid, from which it enters metabolic pathways leading to a number of amino acids and thus to protein, the methylene carbons of the oxizolidone ring apparently do not. Available data also show that additional radioactivity in the residue results from the reaction of activated nitrofurans with proteins, nucleic acids, and possibly other cellular constituents [6-91. Nevertheless, as noted above, regulatory agencies remain suspicious that a portion of the bound residue may be reactive and toxic. While in special circumstances relay toxicology may provide useful data, with furazolidone this approach is pointless. The concentration of bound residue from furazolidone is of the order of I ppm or O.OOOl%, whereas the concentration of nitrofurans used in typical rodent carcinogenicity tests with these agents has been 0.2% of the diet!
111. SUGGESTIONS FOR ADDITIONAL RESEARCH
In considering the question of what additional data might help to clarify the risks involved, it should be noted that the thiol conjugates described above have been observed at short times and may have little relevance to the situation after the drug has been withdrawn for a period of days or weeks. In general, it is thermodynamically unlikely that compounds that are themselves reactive enough to pose a toxic hazard will survive for long periods in a warm, moist environment containing a wide array of chemicals. Unless there are special features involved (e.g., halogen-containing
Drug Metabolism Reviews Downloaded from informahealthcare.com by Freie Universitaet Berlin on 07/08/15 For personal use only.
866
MCCALLA
conjugates susceptible to P-lyase action), it is much more likely that the reactivity will decrease as the initial species react progressively with cellular constituents to form more stable adducts. At the present time the reactions in which the acrylonitrile moiety is transferred from one thiol to another seem to be well established. However, these reactions are not quantitative-some 50% of the material ends up in unidentified compounds. Experiments should be undertaken to try to ascertain whether or not these unidentified products represent unreactive “dead ends.” It would also be useful to have additional information on the reactivity of the mercaptoethanol and GSH conjugates. Specifically, we need to know whether or not these materials react (however slowly) with typical cellular constituents to form stable products. It is of interest that acrylonitrile itself is known to react with unprotonated amino groups and the imidazole ring of histidine as well as with thiols [ I 1 1. Since the concentrations of such unprotonated groups are low, the reactions will be slow but nevertheless may be important in determining the nature of the long-term residue. We also need more information on the stability of the conjugates themselves both at 37°C and under conditions typically encountered during food preparation. Such information will provide a basis of chemical evidence which could be used to judge whether or not the thiol conjugates are likely to be persistent or whether the activated furazolidone winds up in stable compounds. Another line of experimentation should be directed to determining whether or not reactive thiol conjugates persist in the bound residue. If protein thiol conjugates do persist, it should be possible to determine their presence, since treatment of the residue with mercaptoethanol would be expected to result in formation of the soluble mercaptoethanol conjugate. It is essential that these experiments be performed on material obtained after an appropriate withdrawal period, otherwise short-term reactive products will inevitably be present and may predominate. To facilitate such experiments, consideration should be given to the use of furazolidone having considerably higher specific activity than has been used in the past [6]. If expense limits the amount available, it would be possible to use rodents rather than pigs to examine whether or not the residue (both before and after enzymatic hydrolysis) can react with mercaptoethanol to regenerate the mutagenic conjugate. If (as is the case with pigs) 1 ppm of residue were to be formed from furazolidone having specific activity of 10 mCi/mmol, each g of tissue would contain some 800,OOO dpm. Even if only 0.1% of this material were reactive, 800 dpm would be solublized. Thus, if thiol conjugates do persist they should be easy to detect. Further, it should be possible, using HPLC, to show whether or not the soluble product found is actually the mercaptoethanol conjugate.
Drug Metabolism Reviews Downloaded from informahealthcare.com by Freie Universitaet Berlin on 07/08/15 For personal use only.
EVALUATING NITROFUR AN RESIDUES
867
Finally, the mutagenicity of the mercaptoethanol conjugate needs further exploration since, obviously, the mutagenic potential of the conjugate (and hence, by inference, of the protein conjugates as well) is a principal cause for concern. Furazolidone can adsorb to glassware and therefore be present unexpectedly [ 121. Although the chances of contamination seem somewhat remote, a small amount of furazolidone (which is 30 times more mutagenic than the conjugate) might be responsible for the activity observed. The simplest approach to clarifying this point would be to use test strains of bacteria such as Salmonella ryphimurium TA IOONR [ 131 or Escherichia coli NFR 502 [ 141 that have lost the nitroreductase necessary to activate nitrofurans and are therefore insensitive to nitrofurans themselves but should remain sensitive to the conjugate. Further, as noted by Zeiger earlier in this symposium, thiols themselves can give positive results in mutagenicity assays because, in the presence of certain metal ions, they lead to the generation of hydrogen peroxide. While it again seems unlikely, it is not beyond the realm of possibility that the conjugate’s activity could be a consequence of the thiol moiety rather than the acrylonitrile. If this mechanism were operative, the mutagenic activity should be suppressed when catalase is added to the test plates.
IV. CONCLUDING REMARKS It will be obvious that while the additional experiments suggested in the preceding section will yield relevant and useful data, they will not provide absolute assurance that the bound residues are harmless. Indeed, there exists no practical way of obtaining such assurance. The problem is not confined to the three classes of chemicals being discussed in this symposium but applies to all veterinary drugs and feed additives whose bound residue levels exceed the tolerance allowed for the parent compound. What, then, is to be done? Logically, we need to find some way of dealing with the uncertainty or abandon the use of such agents. Regulatory agencies have become adept at dealing with other forms of uncertainty such as extrapolations from high to low doses and from rodents to humans. The problems posed by bound residues, while different, do not pose inherently greater difficulty. Since, as pointed out by Frazier (p. 821), toxic residues really represent a “second-order” problem in toxicology, one approach would be to make reasonable estimates of the maximum hazard that might exist and compare that value to other, accepted hazards. Vroomen has shown that ‘‘C-labeled residues in the tissues of pigs after a 14-day withdrawal period are present in amounts of about 1 ppm (61. It is
MCCALLA
Drug Metabolism Reviews Downloaded from informahealthcare.com by Freie Universitaet Berlin on 07/08/15 For personal use only.
868
likely that much of this material consists of stable adducts formed with insoluble cellular constituents. Let us assume, however, that as much as 10% of the long-term bound residue may consist of mutagenic material having the same potency as the mercaptoethanol conjugate and that this material persists through processing and food preparation. From Vroomen’s data [7] a 100-g portion of meat would then contribute some 4OOO revertants if Salmonella typhimurium strain TA 100 were used in the assay. To put this value in perspective, cooking of that meat could produce another 5000 revertants (TA 1538) from protein pyrolysis products 1151. These later compounds are aromatic amines which are known to be powerful multiorgan and multispecies carcinogens [16]. Further, a cup of instant coffee contains mutagenic material sufficient to induce about 50,000 revertants (TA 100) [17]. While no claim is made that the hazard to humans is necessarily proportional to the levels of mutagenicity of the materials, the comparison indicates that even if long-term bound residues are mutagenic, the hazards they pose will be small relative to that of other mutagens present in food.
Acknowledgments Support for research on nitrofurans in the author’s laboratory has been provided by the Natural Sciences and Engineering Research Council of Canada and the National Cancer Institute of Canada. The collaboration over many years of colleagues, especially Dr. Douglas W. Bryant and Mrs. Christel Kaiser-Farrell, is gratefully acknowledged.
REFERENCES [ I ] D. R. McCalla, Environ. Mutagenesis, 5 , 745 (1983). [2] H. Wellford, The Nader Report; Sowing the Wind,Grossman, New York, 1972. [3] M. 0. Schultze, I? Klubes, V. Perman. N. S. Mizuno, F. W. Bates, J. H. Sautter, Blood, 14, 1015 (1959). [4] J. J. Gavin, F. F. Ebetino, R. Freedman, and W. E. Waterbury, Arch. Biochem. Biophys., 113, 399 (1966). [ 5 ] L. H. M. Vroomen. J. F? Groten, K. van Muiswinkel, A. van Veldhuizen. and F? J. van Bladeren, in L. H. M. Vroomen’s Ph.D. thesis, Agricultural University of Wageningen, 1987, p. 105.
Drug Metabolism Reviews Downloaded from informahealthcare.com by Freie Universitaet Berlin on 07/08/15 For personal use only.
EVALUATING NITROFUR AN RESIDUES
869
[6] L. H. M. Vroomen, M. C. J. Berghmans, F? van Leeuwen, T. D. B. van der Struijs, F? H. U. De Vries, and H. A. Kuiper, Food Add. & Contam., 3 , 331 (1986). [7] L. H. M. Vroomen, M. C. J. Berghmans, J. F? Groten, J. H. Koeman, and F? J. van Bladeren. in L. M. H. Vroomen’s Ph.D. thesis, Agricultural University of Wageningen, 1987, p. 123. [8] B. Wentzell and D. R . McCalla, Chem. Biol. Interactions, 31, 151 (1980). [9] D. R. McCalla, A. Reuvers, and C. Kaiser-Farrell, Biochem Pharmacol., 20, 3532 (1971). [lo] K. Tatsumi, H. Nakabeppu, H. Takahashi, and S. Kitamura, Arch. Biochem. Biophys., 234, 112 (1984). [Ill L. Weil and T. S. Seibles, Arch. Biochem. Biophys., 95, 470 (1961). [12] J. I? Heotis, unpublished results. [13] H. S. Rosehkranz and W. T. Speck, Biochem. Biophys. Res. Commun., 66, 520 (1975). [I41 D. R. McCalla, C. Kaiser-Farrell, and M. H. L. Green, J. Bacreriol., 133, 10 (1978). [I51 L. F. Bjeldanes, M. M. Morris, J. S. Felton, S. Heally, D. Stuermu, F? Berry, H. Timourian, and F. T. Hatch, Fd. Chem. Toxicol,20, 357 (1982). [I61 M. Nagao, Y. Takahashi, H. Yamakawa, and T. Sugimura, Mutat. Res., 68, 101 (1979). [17] H. Ohgaki, L. Hasegawa, T. Kato, M. Suenaga, M. Ubukata, S. Sato, S. Takayama, and T. Sugimura. Environ. Health Persp., 67, 129 (1986).
