157
Mutation Research, 251 (1991) 157-161 © 1991 Elsevier Science Publishers B.V. All rights reserved 0027-5107/91/$03.50 ADONIS 002751079100225W
MUT 05021
A re-examination of the genotoxicity and carcinogenicity of azathioprine Herbert S. Rosenkranz and Gilles Klopman Departments of Environmental Health Sciences and Chemistry, Case Western Reserve University, Cleveland, OH 44106 (U.S.A.) (Received 29 May 1990) (Accepted 26 June 1990)
Keywords: Azathioprine, genotoxicity/carcinogenicity
Recently Voogd (1989) provided an informative as well as a thoughtful review of the genotoxic and carcinogenic potentials of azathioprine, a molecule composed of 6-mercaptopurine (6-MP) and 1-methyl-4-nitroimidazole (Fig. 1). Azathioprine is a rodent carcinogen which has also been identified as a human carcinogen (IARC, 1987a). In addition, azathioprine causes mutations in bacteria and induces cytogenetic effects in cultured mammalian cells (IARC, 1987b; Speck and Rosenkranz, 1976; Voogd, 1989). While we owe a debt of gratitude to Dr. Voogd for his erudite presentation, we are concerned by his extrapolation of risk to humans. Voogd (1989) suggests that the mutagenicity of azathioprine to bacteria is of minimal consequence because the required nitroreduction is characteristic of microbes and does not have a parallel in mammalian systems and he discounts the cytogenetic effects because they are achieved at elevated levels. The presence of chromosomal aberrations in patients treated with azathioprine he ascribes to the fact that given the immunosuppressive action of azathioprine, the aberrant cells accumulate because they cannot be eliminated as a result of a compromised immune system. Finally, regarding the induction of tumors, Voogd considers azathioprine
Correspondence: Prof. H.S. Rosenkranz, Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, 130 De Soto Street, Pittsburgh, PA 15261 (U.S.A.).
a 'promoter' and also suggests that the tumors are due to the immunosuppressive action of azathioprine in a manner similar to that of 'the non-mutagenic peptide cyclosporin A'. With respect to azathioprine, given its therapeutic effectiveness in life-threatening situations, obviously its benefits far outweigh its potential risks to humans. However, we feel that extending the same reasoning to other agents, which do not possess such a clear-cut benefit, could result in unnecessary human risks. We discuss herein some of our analyses regarding the properties of azathioprine and of other unrelated immunosuppressive agents. First, although nitroreduction is indeed enhanced in bacteria, both human and other mammals possess a constellation of enzymes with nitroreductase activity (xanthine oxidase, alcohol dehydrogenase, DT diaphorase, aldehyde oxidase, etc.). Indeed, the same bacterial enzymes which reduce azathioprine or its component niO
__N ~" ~
j
/! ~N
\N ---Y.-%
•
Fig. 1. Predicted carcinogenicity of azathioprine. The biophore is shown in bold. The dotted line separates 6-mercaptopurine (lower portion) from 4-methyl-l-nitroimidazole.
158
troimidazole, also reduce nitrofurans, nitropyrenes and niridazole which are recognized carcinogens and which derive their activity from a DNA-reactive mechanism. Voogd (1989) also argues that the in vitro cytogenetic effects can be disregarded because they occur at elevated levels of azathioprine; we disagree with this reasoning. Among the nitroreductases present in mammalian cells are some which exhibit optimal activity under anaerobic conditions. Obviously, the activity of these enzymes will be expressed only minimally in cultured cells. This activity, however, will be efficient in vivo in tissues that are normally anaerobic, such as the colon. Finally, the flora of humans and animals is composed of an enormous number of aerobic as well as anaerobic bacteria which contain potent nitroreductases and are, therefore, capable of activating nitro compounds to intermediates acting on human or animal tissues. Finally, although Voogd does not elaborate on his reasoning concerning the presumed lack of significance of chromosomal aberrations in azathioprine-treated individuals resulting from lack of immune clearance, it would seem that if chromosomal aberrations per se represent a risk, then the lack of removal of such cells is an increased risk. Additionally, because azathioprine clearly is a mutagen, it belongs to the class of 'genotoxic carcinogens' (Ashby and Tennant, 1988; Weisburger and Williams, 1981). Recent analyses of 'genotoxic carcinogens' have led to the conclusion that they represent a greater risk than 'nongenotoxic carcinogens' and it has even been suggested that genotoxic carcinogens should be regulated more stringently than non-genotoxic ones (Ashby and Purchase, 1988; Bartsch and Malaveille, 1989; Ennever et al., 1987; The Netherlands, 1980; Rosenkranz and Ennever, 1990; Shelby, 1988; Williams, 1987; Wilson, 1989). Recently, we developed CASE, a novel artificial intelligence structure-activity relational method (Klopman et al., 1990; Rosenkranz and Klopman, 1990a) of analysis which can be used to study the structural basis of carcinogenicity (Rosenkranz and Klopman, 1990a,b,c,d). We have applied CASE to an analysis of the structural
basis of the activity of azathioprine and compared it to that of cyclosporin A, an immunosuppressive agent which has also been identified as a human carcinogen (IARC, 1990).
Methodology and data bases
The CASE methodology has been described on a number of occasions (Klopman, 1984; Klopman et al., 1990; Rosenkranz and Klopman, 1989). Basically, CASE selects its own descriptors automatically from a learning set composed of active and inactive molecules. The descriptors are easily recognizable single, continuous structural fragments that are embedded in the complete molecule (see Fig. 1). The descriptors consist of either activating (biophore) or inactivating (biophobe) fragments. Once the training set has been assimilated, CASE can be queried regarding the predicted activity of molecules of unknown activity. Thus, entry of an unknown chemical will result in the generation of all the possible fragments ranging from 2 to 10 heavy atoms accompanied by their hydrogens and these will be compared to the previously identified biophores and biophobes. On the basis of the presence a n d / o r absence of these descriptors, CASE predicts activity or lack thereof. The biological endpoints studied herein include carcinogenicity in rodents, mutagenicity in Salmonella typhimurium and the induction of sister-chromatid exchanges (SCE) and of chromosomal aberrations. In addition, 'structural alerts' for genotoxicity were also included in the analyses. The data were all derived from NTP-sponsored studies (Ashby and Tennant, 1988; Ashby et al., 1989; Galloway et al., 1985, 1987; Loveday et al., 1989; Tennant et al., 1987; Zeiger, 1987). These data bases have been analyzed by CASE and the major biophores and biophobes identified: mutagenicity in Salmonella (Rosenkranz and Klopman, 1990e), carcinogenicity in rodents (Rosenkranz and Klopman, 1990a,d), induction of SCEs and of chromosomal aberrations in CHO cells (Rosenkranz et al., 1990) and 'structural alerts' for genotoxicity (Rosenkranz and Klopman~ 1990g).
159 TABLE 1 CASE P R E D I C T I O N S OF M U T A G E N I C I T Y , G E N O T O X I C I T Y A N D R O D E N T C A R C I N O G E N I C I T Y Biological endpoint
Carcinogenicity in rodents Mutagenicity in Salmonella SCE Chromosomal aberrations Structural alert for genotoxicity
Azathioprine
6-Mercaptopurine
4-Methyl- 1-nitroimidazole
Overall
Percent
Overall
Percent
Overall
Percent
+ + + + +
80.0 67.0 86.7 71.9 67.0
m + + -
57.0 0 80.0 71.9 0
+ + + +
0 70.0 86.7 63.0 70.0
m = marginal.
Results and discussion
The various CASE predictions are summarized in Table 1. CASE predicted azathioprine to be a rodent carcinogen (Fig. 1), which is in concordance with the results of animal bioassays (IARC, 1987a). Moreover, it should be noted that the biophore associated with the carcinogenicity (C"S-C=, Fig. 1) spans the 6-MP and nitroimidazole moieties, thus suggesting that the intact parent molecule is required for carcinogenicity. Additionally, CASE predicted azathioprine to induce cytogenetic effects and to be mutagenic in Salmonella (Table 1) which is also in conformity with experimental results (IARC, 1987b). Finally, azathioprine was predicted to be DNA-reactive (i.e., to possess a 'structural alert' for genotoxicity). Thus, by all of the criteria, both experimental as well as structural, azathioprine is predicted to be a mutagen, thus fulfilling the criteria of a 'genotoxic carcinogen'. In addition to using biophores to predict carcinogenicity, CASE can also be used to determine the origin of carcinogenic biophores (Rosenkranz and Klopman, 1990f). When such an analysis was performed for azathioprine, it became apparent that the molecules which contain this biophore are primarily mutagenic ones (Table 2). Voogd (1989) suggests that the carcinogenicity of azathioprine derives from its bioconversion to 6-mercaptopurine (6-MP). The CASE analysis, however, suggests (a) that azathioprine is not split (see above) and (b) that 6-MP is only marginally carcinogenic. This is in accord with the animal bioassay results for 6-MP which were of such a nature as to preclude evaluation of the carcino-
genic properties of this chemical (IARC, 1987a). However, some genotoxic activities are predicted for 6-MP (Table 1), which again is in accordance with published data (IARC, 1987b). It is of further interest that 1-methyl-4-nitroimidazole, the other component of azathioprine, is also not predicted to be a rodent carcinogen, although clearly it possesses mutagenic potential (Table 1). Voogd (1989) suggests that azathioprine derives its carcinogenicity through a promotional mechanism resulting from immunosuppression. This would be a type of nonspecific, or indirect, mechanism which would not be predictable by TABLE 2 ORIGIN OF BIOPHORE ASSOCIATED WITH THE C A R C I N O G E N I C I T Y OF A Z A T H I O P I N E CAS No.
Name
Carcino genicity in rodents a
Muta genicity b
00121-66-4 00149-30-4 00139-65-1 00055-38-9 00139-94-6
2-Amino-5-nit rot hiazole 2-Mercaptobenzothiazole 4,4'-Thiodianiline Fenthion Nithiazide
D B A E A
+ + + +
The classification of Ashby and Tennant (1988) was adopted. In that scheme, chemicals in Group A induce tumors in rats and mice. Group B includes chemicals which are carcinogenic to only one species but which include cance~s at two or more sites. Group C consists of chemicals which are carcinogenic at only a single site in both sexes of a single species. Group D contains chemicals carcinogenic at a single site in a single species. Group E, adequately studied chemicals for which only equivocal evidence of carcinogenicity was observed. Group NC, non-carcinogens. h Mutagenicity in Salmonella. a
160
Cyclosporin A 5'/% c h a n c e o f b e i n g N
ACTIVE due t o s u l ~ t r o e t o r e (Conf. level = 50%) :
-CO -CH -CH2-
*** d o w n g r e d e d f r o m 6T% because of d i f f e r e n t E n v i r o n m e n t
62% c h a n c e o f b e i n g INACTIVE due to s u b s t r u c t u r e (Conf. level = 75%) : CH3-N
-CH2-
*** d o w n g r e d e d f r o m T5% because of d i f f e r e n t E n v i r o n m e n t 64% c h a n n e o f b e i n g INACTIVE d u e t o s u l ~ t r u c t u r e (Conf. level = 8?%) : CH3-N
-CH -
*** d o w n g r a d e d f r o m 80% b e c a u s e of d i f f e r e n t E n v i r o n m e n t
69% c h a n c e o f b e i n g INACTIVE doe t o s u h s t r v e t n r e (Conf. level = 100%) : OH
-CH -
*** d o w n g r a d e d f r o m 91% b e c a u s e of d i f f e r e n t E n v i r o n m e n t *** O V E R A L L , t h e p r o b a b i l i t y of b e i n g a Rodent C a r c i n o g e n Is 17.0% ***
Fig. 2. CASE prediction of the lack of carcinogenicity of cyclosporin A.
FK506 is a result of the direct effect of these agents on the immune system (Harding et al., 1989; Siekierka et al., 1989; Thomson, 1989; White, 1981). Our analyses indicate that the carcinogenicity of azathioprine is associated with its genotoxicity. We do not wish to imply that our analyses suggest that these potential properties should be used as a counterindication to the therapeutic usage of azathioprine. Additionally, our disagreement with Voogd's specific interpretation of the risks posed by azathioprine in no way diminishes the value of his review concerning the genotoxic properties of azathioprine.
Acknowledgements CASE using the present data set since the agents contained therein are not considered to be immunosuppressors. To test this hypothesis, we investigated the potential carcinogenicity of cyclosporin A and FK506, two immunosuppressive agents which, although acting by apparently the same mechanism, are structurally very different. Neither of them is predicted to be carcinogenic (Figs. 2 and 3), thus confirming the expectation that the NTP carcinogenicity data base cannot be used to predict the carcinogenicity of immunosuppressive agents unless the latter act by way of a DNA-reactive mechanism (wherein the immunosuppressive action is secondary to their genotoxicity). It is known, however, that the immunosuppressive action of cyclosporin A and FK- 506
83% c h a n c e o f b e i n g
ACTIVE d u e t o s u b s t r u c t u r e (Conf. level = 87%) :
CH2-CH2-CH2-CH2-
*** d o w n g r a d e d f r o m 80% bemmse of Ineorroet C o n f o r m a t i o n 88% chance o f belnR CH2-CHI-CH2-CH
A C T I V E due to sel~trooturo (Conf. level : 97%) : -
*** d o w n g r a d e d f r o m 88% b e c a u s e of I n c o r r e c t C o n f o r m a t i o n 86% c h a n c e o f b e i n g INACTIVE d u e t o mlbetructuro (Conf. level : 97%) : OH*-CH
-CH2-
86% o h s n e e o f b e l q INACTIVE due to S u l ~ t r o c t u r e (Conf. level : 97%) : OH
-CH -CH -
71% c h a n c e o f b e i n g INACTIVE due t o Imbetruotore (Conf. l e ve l : 89%) : N -CH-
*** O V E R A L L , t h e probabllltay of b e i n g a R o d e n t C a r c i n o g e n Is 3.5% ***
Fig. 3. CASE prediction of the lack of carcinogenicity of the aminosuppressive agent FK-506.
This investigation was supported by the National Institute of Environmental Health Sciences (ES04659) and the U.S. Environmental Protection Agency (R815488).
References Ashby, J., and I.F.H. Purchase (1987) Reflections on the declining ability of the Salmonella assay to detect rodent carcinogens as positive, Mutation Res., 205, 51-58. Ashby, J., and R.W. Tennant (1988) Chemical structure, Salmonella mutagenicity and extent of carcinogenicity as indicators of genotoxic carcinogenesis among 222 chemicals tested in rodents by the U.S. NCI/NTP, Mutation Res., 204, 17-115. Ashby, J., R.W. Tennant, E. Zeiger and S. Stasiewicz (1989) Classification according to chemical structure, mutagenicity to Salmonella and level of carcinogenicity of a further 42 chemicals tested for carcinogenicity by the U.S. National Toxicology Program, Mutation Res., 223, 73-103. Bartsch, H., and C. Malaveille (1989) Prevalence of genotoxic chemicals among animal and human carcinogens evaluated in the IARC Monograph Series, Cell Biol. Toxicol., 5, 115-127. Ennever, F.K., T.J. Noonan and H.S. Rosenkranz (1987) The predictivity of animal bioassays and short-term genotoxicity tests for carcinogenicity and non-carcinogenicity to humans, Mutagenesis, 2, 73-78. Galloway S.M., A.D. Bloom, M. Resnick, B.H. Margolin, F. Nakamura, P. Archer, and E. Zeiger (1985) Development of a standard protocol for in vitro cytogenetic testing with Chinese hamster ovary cells: Comparison of results for 22 compounds in two laboratories, Environ Mutagen., 7, 1-51. Galloway, S.M., M.J. Armstrong, C. Reuben, S. Colman, B. Brown, C. Cannon, A.D. Bloom, F. Nakamura, M. Ahmed, S. Duk, J. Rimpo, B.H. Margolin, M.A. Resnick, B. Anderson and E. Zeiger (1987) Chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells:
161 Evaluation of 108 chemicals, Environ. Mol. Mutagen., 10, Suppl. 10, 1-175. Gulati, D.K., K. Witt, B. Anderson, E. Zeiger and M.D. Shelby (1989) Chromosome aberration and sister chromatid exchange tests in Chinese hamster ovary cells in vitro. III: Results with 27 chemicals, Environ. Mol. Mutagen., 13, 133-193. Harding, M.W., A. Galat, D.E. Uehling and S.L. Schreiber (1989) A receptor for the immunosuppressant FK-506 is a cis-trans peptidyl-propyl isomerase, Nature (London), 341, 758-760. IARC (1987a) Monographs on the Evaluation of Carcinogenic Risks to Humans: Genetic and Related Effects, Suppl. 7, Overall Evaluation of Carcinogenicity: An Updating of IARC Monographs Vols. 1-42, International Agency for Research on Cancer, Lyon, France. IARC (1987b) Monographs on the Evaluation of Carcinogenic Risks to Humans: Genetic and Related Effects, Suppl. 6, Genetic and Related Effects, International Agency for Research on Cancer, Lyon, France. IARC (1990) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 50, International Agency for Research on Cancer, Lyon, France. Klopman, G. (1984) Artificial intelligence approach to structure-activity studies, Computer automated structure evaluation of biological activity of organic molecules, J. Am. Chem. Soc., 106, 7315-7321. Klopman, G., M.R. Frierson and H.S. Rosenkranz (1990) The structural basis of the mutagenicity of chemicals in Salmonella typhimurium: The Gene-Tox Data Base, Mutation Res., 228, 1-50. Loveday, K.S., M.H. Lugo, M.A. Resnick, B.E. Anderson and E. Zeiger (1989) Chromosome aberration and sister chromatid exchange tests in Chinese hamster ovary cells in vitro II. Results with 20 chemicals, Environ. Mol. Mutagen., 13, 60-94. The Netherlands (1980) Health Council of The Netherlands, Report on the Evaluation of the Carcinogenicity of Chemical Substances, Government Printing Office, The Hague. Rosenkranz, H.S., and F.K. Ennever (1990) An association between mutagenicity and carcinogenic potency, Mutation Res., 244, 61-65. Rosenkranz, H.S., and G. Klopman (1989) Structural basis of the mutagenicity of phenylazoaniline dyes, Mutation Res., 221, 217-239. Rosenkranz, H.S., and G. Klopman (1990a) Structural basis of carcinogenicity in rodents of genotoxicants and nongenotoxicants, Mutation Res., 228, 105-124. Rosenkranz, H.S., and G. Klopman (1990b) Identification of rodent carcinogens by an expert system, in: M.L. Mendelsohn and R.J. Albertini (Eds.), Mutation and the Environment, Part B. Metabolism, Testing Methods and Chromosomes, Wiley-Liss, New York, pp. 23-48. Rosenkranz, H.S., and G. Klopman (1990c) New structural concepts for predicting carcinogenicity in rodents: An artificial intelligence approach, Teratogen. Carcinogen. Mutagen., 10, 73-79.
Rosenkranz, H.S., and G. Klopman (1990d) Natural pesticides present in edible plants are predicted to be carcinogenic, Carcinogenesis, 11,349-353. Rosenkranz, H.S., and G. Klopman (1990e) The structural basis of the mutagenicity of chemicals in Salmonella typhimurium: The National Toxicology Program data base, Mutation Res., 228, 51-80. Rosenkranz, H.S., and G. Klopman (19900 Novel structural concepts in elucidating the potential genotoxicity and carcinogenicity of tetrandrine, a traditional herbal drug, Mutation Res., 244, 265-271. Rosenkranz, H.S., and G. Klopman (1990g) Structural alerts to genotoxicity: The interaction of human and artificial intelligence, Mutagenesis, 5, 333-361. Rosenkranz, H.S., F.K. Ennever, M. Dimayuga and G. Klopman (1990) Significant differences in the structural basis of the induction of sister chromatid exchanges and chromosomal aberrations in Chinese hamster ovary cells, Environ. Mol. Mutagen., 5, 16, 149-177. Shelby, M.D. (1988) The genetic toxicity of human carcinogens and its implications, Mutation Res., 204, 3-15. Siekierka, J.J., S.H.Y. Hung, M. Poe, C.S. Lin and N.H. Sigal (1989) A cytosolic binding protein for the immunosuppressant FK-506 has peptidyl-prolyl isomerase activity but is distinct from cyclophilin, Nature (London), 341 755-757. Speck, William T., and Herbert S. Rosenkranz (1976) Mutagenicity of azathioprine, Cancer Res., 36, 108-109. Tennant, R.W., B.H. Margolin, M.D. Shelby, E. Zeiger, J.K. Haseman, J. Spalding, W. Caspary, M. Resnick, S. Stasiewicz, B. Anderson and R. Minor (1987) Prediction of chemical carcinogenicity in rodents from in vitro genotoxicity assays, Science, 236, 933-941. Thomson, A.W. (1989) FK-506 - - How much potential? Immunology Today, 10, 6-9. Voogd, C.E. (1989) Azathioprine, a genotoxic agent to be considered non-genotoxic in man, Mutation Res., 221, 133-152. Weisburger, J.H., and G.M. Williams (1981) Carcinogen testing: Current problems and new approaches, Science, 214, 401-407. White, P.C.G. (1982) Cyclosporin A: Proceedings of an International Symposium on Cyclosporin A, Elsevier, Amsterdam, 578 pp. Williams, G.M. (1987) Definition of a human cancer hazard, in: B.E. Butterworth and T.J. Slaga (Eds.), Nongenotoxic Mechanisms in Carcinogenesis, Banbury Report 25 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 367-380. Wilson, J.D. (1989) Assessment of low-exposure risk from carcinogenesis: Implications of the Knudson-Moolgavkar Two-Critical Mutation Theory, in: C.C. Travis (Ed.), Biologically-Based Methods for Cancer Risk Assessment, Plenum, New York, pp. 275-287. Zeiger, E. (1987) Carcinogenicity of mutagens: Predictive capability of the Salmonella mutagenesis assay for rodent carcinogenicity, Cancer Res., 47 1287-1296.
Mutation Research, 251 (1991) 157-161 © 1991 Elsevier Science Publishers B.V. All rights reserved 0027-5107/91/$03.50 ADONIS 002751079100225W
MUT 05021
A re-examination of the genotoxicity and carcinogenicity of azathioprine Herbert S. Rosenkranz and Gilles Klopman Departments of Environmental Health Sciences and Chemistry, Case Western Reserve University, Cleveland, OH 44106 (U.S.A.) (Received 29 May 1990) (Accepted 26 June 1990)
Keywords: Azathioprine, genotoxicity/carcinogenicity
Recently Voogd (1989) provided an informative as well as a thoughtful review of the genotoxic and carcinogenic potentials of azathioprine, a molecule composed of 6-mercaptopurine (6-MP) and 1-methyl-4-nitroimidazole (Fig. 1). Azathioprine is a rodent carcinogen which has also been identified as a human carcinogen (IARC, 1987a). In addition, azathioprine causes mutations in bacteria and induces cytogenetic effects in cultured mammalian cells (IARC, 1987b; Speck and Rosenkranz, 1976; Voogd, 1989). While we owe a debt of gratitude to Dr. Voogd for his erudite presentation, we are concerned by his extrapolation of risk to humans. Voogd (1989) suggests that the mutagenicity of azathioprine to bacteria is of minimal consequence because the required nitroreduction is characteristic of microbes and does not have a parallel in mammalian systems and he discounts the cytogenetic effects because they are achieved at elevated levels. The presence of chromosomal aberrations in patients treated with azathioprine he ascribes to the fact that given the immunosuppressive action of azathioprine, the aberrant cells accumulate because they cannot be eliminated as a result of a compromised immune system. Finally, regarding the induction of tumors, Voogd considers azathioprine
Correspondence: Prof. H.S. Rosenkranz, Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, 130 De Soto Street, Pittsburgh, PA 15261 (U.S.A.).
a 'promoter' and also suggests that the tumors are due to the immunosuppressive action of azathioprine in a manner similar to that of 'the non-mutagenic peptide cyclosporin A'. With respect to azathioprine, given its therapeutic effectiveness in life-threatening situations, obviously its benefits far outweigh its potential risks to humans. However, we feel that extending the same reasoning to other agents, which do not possess such a clear-cut benefit, could result in unnecessary human risks. We discuss herein some of our analyses regarding the properties of azathioprine and of other unrelated immunosuppressive agents. First, although nitroreduction is indeed enhanced in bacteria, both human and other mammals possess a constellation of enzymes with nitroreductase activity (xanthine oxidase, alcohol dehydrogenase, DT diaphorase, aldehyde oxidase, etc.). Indeed, the same bacterial enzymes which reduce azathioprine or its component niO
__N ~" ~
j
/! ~N
\N ---Y.-%
•
Fig. 1. Predicted carcinogenicity of azathioprine. The biophore is shown in bold. The dotted line separates 6-mercaptopurine (lower portion) from 4-methyl-l-nitroimidazole.
158
troimidazole, also reduce nitrofurans, nitropyrenes and niridazole which are recognized carcinogens and which derive their activity from a DNA-reactive mechanism. Voogd (1989) also argues that the in vitro cytogenetic effects can be disregarded because they occur at elevated levels of azathioprine; we disagree with this reasoning. Among the nitroreductases present in mammalian cells are some which exhibit optimal activity under anaerobic conditions. Obviously, the activity of these enzymes will be expressed only minimally in cultured cells. This activity, however, will be efficient in vivo in tissues that are normally anaerobic, such as the colon. Finally, the flora of humans and animals is composed of an enormous number of aerobic as well as anaerobic bacteria which contain potent nitroreductases and are, therefore, capable of activating nitro compounds to intermediates acting on human or animal tissues. Finally, although Voogd does not elaborate on his reasoning concerning the presumed lack of significance of chromosomal aberrations in azathioprine-treated individuals resulting from lack of immune clearance, it would seem that if chromosomal aberrations per se represent a risk, then the lack of removal of such cells is an increased risk. Additionally, because azathioprine clearly is a mutagen, it belongs to the class of 'genotoxic carcinogens' (Ashby and Tennant, 1988; Weisburger and Williams, 1981). Recent analyses of 'genotoxic carcinogens' have led to the conclusion that they represent a greater risk than 'nongenotoxic carcinogens' and it has even been suggested that genotoxic carcinogens should be regulated more stringently than non-genotoxic ones (Ashby and Purchase, 1988; Bartsch and Malaveille, 1989; Ennever et al., 1987; The Netherlands, 1980; Rosenkranz and Ennever, 1990; Shelby, 1988; Williams, 1987; Wilson, 1989). Recently, we developed CASE, a novel artificial intelligence structure-activity relational method (Klopman et al., 1990; Rosenkranz and Klopman, 1990a) of analysis which can be used to study the structural basis of carcinogenicity (Rosenkranz and Klopman, 1990a,b,c,d). We have applied CASE to an analysis of the structural
basis of the activity of azathioprine and compared it to that of cyclosporin A, an immunosuppressive agent which has also been identified as a human carcinogen (IARC, 1990).
Methodology and data bases
The CASE methodology has been described on a number of occasions (Klopman, 1984; Klopman et al., 1990; Rosenkranz and Klopman, 1989). Basically, CASE selects its own descriptors automatically from a learning set composed of active and inactive molecules. The descriptors are easily recognizable single, continuous structural fragments that are embedded in the complete molecule (see Fig. 1). The descriptors consist of either activating (biophore) or inactivating (biophobe) fragments. Once the training set has been assimilated, CASE can be queried regarding the predicted activity of molecules of unknown activity. Thus, entry of an unknown chemical will result in the generation of all the possible fragments ranging from 2 to 10 heavy atoms accompanied by their hydrogens and these will be compared to the previously identified biophores and biophobes. On the basis of the presence a n d / o r absence of these descriptors, CASE predicts activity or lack thereof. The biological endpoints studied herein include carcinogenicity in rodents, mutagenicity in Salmonella typhimurium and the induction of sister-chromatid exchanges (SCE) and of chromosomal aberrations. In addition, 'structural alerts' for genotoxicity were also included in the analyses. The data were all derived from NTP-sponsored studies (Ashby and Tennant, 1988; Ashby et al., 1989; Galloway et al., 1985, 1987; Loveday et al., 1989; Tennant et al., 1987; Zeiger, 1987). These data bases have been analyzed by CASE and the major biophores and biophobes identified: mutagenicity in Salmonella (Rosenkranz and Klopman, 1990e), carcinogenicity in rodents (Rosenkranz and Klopman, 1990a,d), induction of SCEs and of chromosomal aberrations in CHO cells (Rosenkranz et al., 1990) and 'structural alerts' for genotoxicity (Rosenkranz and Klopman~ 1990g).
159 TABLE 1 CASE P R E D I C T I O N S OF M U T A G E N I C I T Y , G E N O T O X I C I T Y A N D R O D E N T C A R C I N O G E N I C I T Y Biological endpoint
Carcinogenicity in rodents Mutagenicity in Salmonella SCE Chromosomal aberrations Structural alert for genotoxicity
Azathioprine
6-Mercaptopurine
4-Methyl- 1-nitroimidazole
Overall
Percent
Overall
Percent
Overall
Percent
+ + + + +
80.0 67.0 86.7 71.9 67.0
m + + -
57.0 0 80.0 71.9 0
+ + + +
0 70.0 86.7 63.0 70.0
m = marginal.
Results and discussion
The various CASE predictions are summarized in Table 1. CASE predicted azathioprine to be a rodent carcinogen (Fig. 1), which is in concordance with the results of animal bioassays (IARC, 1987a). Moreover, it should be noted that the biophore associated with the carcinogenicity (C"S-C=, Fig. 1) spans the 6-MP and nitroimidazole moieties, thus suggesting that the intact parent molecule is required for carcinogenicity. Additionally, CASE predicted azathioprine to induce cytogenetic effects and to be mutagenic in Salmonella (Table 1) which is also in conformity with experimental results (IARC, 1987b). Finally, azathioprine was predicted to be DNA-reactive (i.e., to possess a 'structural alert' for genotoxicity). Thus, by all of the criteria, both experimental as well as structural, azathioprine is predicted to be a mutagen, thus fulfilling the criteria of a 'genotoxic carcinogen'. In addition to using biophores to predict carcinogenicity, CASE can also be used to determine the origin of carcinogenic biophores (Rosenkranz and Klopman, 1990f). When such an analysis was performed for azathioprine, it became apparent that the molecules which contain this biophore are primarily mutagenic ones (Table 2). Voogd (1989) suggests that the carcinogenicity of azathioprine derives from its bioconversion to 6-mercaptopurine (6-MP). The CASE analysis, however, suggests (a) that azathioprine is not split (see above) and (b) that 6-MP is only marginally carcinogenic. This is in accord with the animal bioassay results for 6-MP which were of such a nature as to preclude evaluation of the carcino-
genic properties of this chemical (IARC, 1987a). However, some genotoxic activities are predicted for 6-MP (Table 1), which again is in accordance with published data (IARC, 1987b). It is of further interest that 1-methyl-4-nitroimidazole, the other component of azathioprine, is also not predicted to be a rodent carcinogen, although clearly it possesses mutagenic potential (Table 1). Voogd (1989) suggests that azathioprine derives its carcinogenicity through a promotional mechanism resulting from immunosuppression. This would be a type of nonspecific, or indirect, mechanism which would not be predictable by TABLE 2 ORIGIN OF BIOPHORE ASSOCIATED WITH THE C A R C I N O G E N I C I T Y OF A Z A T H I O P I N E CAS No.
Name
Carcino genicity in rodents a
Muta genicity b
00121-66-4 00149-30-4 00139-65-1 00055-38-9 00139-94-6
2-Amino-5-nit rot hiazole 2-Mercaptobenzothiazole 4,4'-Thiodianiline Fenthion Nithiazide
D B A E A
+ + + +
The classification of Ashby and Tennant (1988) was adopted. In that scheme, chemicals in Group A induce tumors in rats and mice. Group B includes chemicals which are carcinogenic to only one species but which include cance~s at two or more sites. Group C consists of chemicals which are carcinogenic at only a single site in both sexes of a single species. Group D contains chemicals carcinogenic at a single site in a single species. Group E, adequately studied chemicals for which only equivocal evidence of carcinogenicity was observed. Group NC, non-carcinogens. h Mutagenicity in Salmonella. a
160
Cyclosporin A 5'/% c h a n c e o f b e i n g N
ACTIVE due t o s u l ~ t r o e t o r e (Conf. level = 50%) :
-CO -CH -CH2-
*** d o w n g r e d e d f r o m 6T% because of d i f f e r e n t E n v i r o n m e n t
62% c h a n c e o f b e i n g INACTIVE due to s u b s t r u c t u r e (Conf. level = 75%) : CH3-N
-CH2-
*** d o w n g r e d e d f r o m T5% because of d i f f e r e n t E n v i r o n m e n t 64% c h a n n e o f b e i n g INACTIVE d u e t o s u l ~ t r u c t u r e (Conf. level = 8?%) : CH3-N
-CH -
*** d o w n g r a d e d f r o m 80% b e c a u s e of d i f f e r e n t E n v i r o n m e n t
69% c h a n c e o f b e i n g INACTIVE doe t o s u h s t r v e t n r e (Conf. level = 100%) : OH
-CH -
*** d o w n g r a d e d f r o m 91% b e c a u s e of d i f f e r e n t E n v i r o n m e n t *** O V E R A L L , t h e p r o b a b i l i t y of b e i n g a Rodent C a r c i n o g e n Is 17.0% ***
Fig. 2. CASE prediction of the lack of carcinogenicity of cyclosporin A.
FK506 is a result of the direct effect of these agents on the immune system (Harding et al., 1989; Siekierka et al., 1989; Thomson, 1989; White, 1981). Our analyses indicate that the carcinogenicity of azathioprine is associated with its genotoxicity. We do not wish to imply that our analyses suggest that these potential properties should be used as a counterindication to the therapeutic usage of azathioprine. Additionally, our disagreement with Voogd's specific interpretation of the risks posed by azathioprine in no way diminishes the value of his review concerning the genotoxic properties of azathioprine.
Acknowledgements CASE using the present data set since the agents contained therein are not considered to be immunosuppressors. To test this hypothesis, we investigated the potential carcinogenicity of cyclosporin A and FK506, two immunosuppressive agents which, although acting by apparently the same mechanism, are structurally very different. Neither of them is predicted to be carcinogenic (Figs. 2 and 3), thus confirming the expectation that the NTP carcinogenicity data base cannot be used to predict the carcinogenicity of immunosuppressive agents unless the latter act by way of a DNA-reactive mechanism (wherein the immunosuppressive action is secondary to their genotoxicity). It is known, however, that the immunosuppressive action of cyclosporin A and FK- 506
83% c h a n c e o f b e i n g
ACTIVE d u e t o s u b s t r u c t u r e (Conf. level = 87%) :
CH2-CH2-CH2-CH2-
*** d o w n g r a d e d f r o m 80% bemmse of Ineorroet C o n f o r m a t i o n 88% chance o f belnR CH2-CHI-CH2-CH
A C T I V E due to sel~trooturo (Conf. level : 97%) : -
*** d o w n g r a d e d f r o m 88% b e c a u s e of I n c o r r e c t C o n f o r m a t i o n 86% c h a n c e o f b e i n g INACTIVE d u e t o mlbetructuro (Conf. level : 97%) : OH*-CH
-CH2-
86% o h s n e e o f b e l q INACTIVE due to S u l ~ t r o c t u r e (Conf. level : 97%) : OH
-CH -CH -
71% c h a n c e o f b e i n g INACTIVE due t o Imbetruotore (Conf. l e ve l : 89%) : N -CH-
*** O V E R A L L , t h e probabllltay of b e i n g a R o d e n t C a r c i n o g e n Is 3.5% ***
Fig. 3. CASE prediction of the lack of carcinogenicity of the aminosuppressive agent FK-506.
This investigation was supported by the National Institute of Environmental Health Sciences (ES04659) and the U.S. Environmental Protection Agency (R815488).
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