Mutagenesis vol 6 no.5 pp.381 -384, 1991
Omeprazole: an exploration of its reported genotoxicity
Herbert S.Rosenkranz and Gilles KJopman' Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261 and 'Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106, USA
Introduction The report of the induction of gastric tumors in rats by omeprazole following long-term administration (Ekman et al., 1985) has been attributed to an increased level of circulating gastrin (Hakanson et al., 1986). However, recently there has been considerable controversy regarding the significance of the reported genotoxicity of omeprazole (Figure 1) to the gastric mucosa (Anonymous, 1990; Burlinson etai, 1990,1991; De Rojas, 1990; Ekman et al., 1990; Misiewicz, 1990; Parry, 1990; Pounder, 1990; Poynter, 1990; Wright and Goodlad, 1990; Larsson et al., 1991). The controversy is of some importance as omeprazole has been shown to be an effective inhibitor of gastric acid secretion in humans. However, to some extent, the argument has dealt primarily with the physiology of the gastric mucosa and details of the unscheduled DNA synthesis (UDS) protocol employed. More recently the induction of UDS and of ornithine decarboxylase in the glandular stomach mucosa of rats was reported by another group of investigators (Furihata et al., 1991). Lost in the argument is the possible significance of genotoxicity in identifying a potential human hazard. In fact, the reason for determining genotoxic activity is primarily to identify a carcinogenic potential. It is, of course, recognized that genotoxicity is not a perfect measure; there are many agents which induce cancers in rodents by non-genotoxic mechanisms. However, the argument has been made that compared to nongenotoxic carcinogens, genotoxic carcinogens appear to represent an increased hazard (Netherlands, 1980; Williams, 1987, 1990; Wilson, 1989). As a class, such agents have been suggested as causing cancers in multiple species and at multiple sites in both genders while non-genotoxic carcinogens appear generally to be restricted to the single tissue of a single sex of a single species (Ashby and Tennant, 1988; Ashby etal., 1989; Gold etal., 1989). Moreover, genotoxic carcinogens seem to be more potent rodent carcinogens than non-genotoxic ones (Rosenkranz and Ennever, 1990). Finally, the vast majority of recognized human carcinogens are genotoxicants (Ennever etal., 1987; Shelby, 1988; Bartsch and Malaveille, 1989). This does not imply that © Oxford University Press
Methodology The CASE methodology has been described on a number of occasions (Rosenkranz and Klopman, 1989, 1990a,b; Klopman et al., 1900). CASE selects its own descriptors from a learning set composed of active as well as inactive molecules. These descriptors are readily recognized as continuous structural OMEPRAZOLE (A) 67% chance of being ACTIVE due to substructure (Conf. level = 50%): NH - C =N - C . = (B) 83% chance of being ACTIVE due to substructure (Conf. level = 100%); CH =CH - C =CH (O 95% chance of being ACTIVE due to substructure (Conf. level = 100%): CH =C -CH =C. (D) 97% chance of being ACTIVE due to substructure (Conf. level = 100%): O - C =CH (E) 70% chance of being ACTIVE due to substructure (Conf. level = 91%): NH - C . =CH (F) 91% chance of being ACTIVE due to substructure (Conf. level = 100%): O -CH3 *** OVERALL, the probability of Inducing Bone Marrow Nuclei is 100.0%«*
Fig. 1. CASE prediction of the ability of omeprazole to induce in vivo bone marrow nuclei. The biophores are indicated in bold. Biophore F is present twice. The biophores B, C, D and F are also present in the sulphenimide. C. indicates a carbon atom shared by two ring systems.
381
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Because of its reported ability to induce unscheduled DNA synthesis in the gastric mucosa, the safety of omeprazole, a potentially clinically useful anti-ulcer drug, has been the subject of debate. We have undertaken a detailed computerbased study of structural basis of the putative mutagenicity, genotoxicity and carcinogenidty in rodents of omeprazole and of its sulphenimide, and we conclude that omeprazole is a potential 'genotoxic' carcinogen. The analysis is consistent with the possibility that these activities are associated with the unstable sulphenimide metabolite.
genotoxic agents are to be shunned, especially if they possess therapeutic properties. Their adoption, however, needs to be evaluated carefully and subjected to a risk-benefit analysis. In this context, we have undertaken an in-depth analysis of the possible structural basis of the genotoxic and carcinogenic potential of omeprazole and some of its metabolites. Our study indicates that omeprazole has a potential for DNA reactivity, genotoxicity, as well as for inducing cancer in rodents. It is to be noted that these predictions are not based upon a single laboratory test but rather they are derived from the results of structural analyses involving over 2000 chemicals and a multiplicity of toxicological endpoints.
H.S.Rosenkranz and G.KJopman
fragments embedded in the molecule. The descriptors consist of either activating (biophore) or deactivating (biophobe) fragments. Each biophobe and biophore is characterized by its distribution among active and inactive molecules and the associated P value. Once biophores and biophobes have been identified, unknown molecules may be analyzed. Upon submission of such a molecule, the CASE program will generate all 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 and/or absence of these descriptors, CASE predicts activity or lack thereof.
Results and discussion A number of metabolites of omeprazole have been identified; these include omeprazole sulfone, hydroxyomeprazole, OMEPRAZOLE SULPHENAMIDE+ (A) 60% chance of being ACTIVE due to substructure (Conf. level = 75%): CH =CH - C =CH •*• downgraded from 75% because of incorrect Conformation (B) 87% chance of being ACTIVE due to substructure (Coof. level = 100%): C. =CH - C H =C (O 83% chance of being ACTIVE due to substructure (Conf. level = 94%): N" - C . =CH •*• OVERALL, the probability of being a Rodent Cardnogen is 98.0%***
Although the information contributed by the cytogenetic assays (chromosomal aberrations, sister chromatid exchange) is only Table I. Summary of CASE predictions Biological Activity
Fig. 2. CASE prediction of the carcinogenicity in rodents of omeprazole sulphenimide. This prediction is based upon the rodent carcinogenicity compilation of Gold et aL (1984, 1986, 1987). The biophores are shown in bold. The same biophores are also present in omeprazole.
382
Mutagenicity/Salmonella Structural alert Chromosomal aberrations Sister chromatid exchanges In vivo micronuclei Carcinogenicity: rodents (Gold etal., 1984, 1986, 1987) rat (NTP)
Sulphenimide Omeprazole overall probability (%) overall probability (%)
+ + +
0 0 70.9 80.0 100.0
+ + + +
80.0 83.3 68.2 8.8 100.0
+ +
98.0 60.3
+ +
98.0 61.4
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Data Bases The data bases used by CASE as 'learning sets' to identify the appropriate biophores and biophobes were generated primarily by the US National Toxicology Program. Mutagenicity in Salmonella, carcinogenicity in rats, induction of sister chromatid exchange and chromosomal aberrations (Galloway et al., 1985, 1987; Tennant et al., 1987; Zeiger, 1987; Zeiger et al., 1987; Ashby and Tennant, 1988; Ashby etal., 1989; Gulati etai, 1989; Loveday etal., 1989). In addition we also analyzed a rodent carcinogenicity database consisting of 290 chemicals derived from the compilation of Gold et al. (1984, 1986, 1987) and a data base of in vivo inducers of mammalian bone marrow micronuclei (Mavournin et al., 1990). The biophores and biophobes associated with individual endpoints have been described previously: carcinogenicity in rodents (rats) (Rosenkranz and Klopman, 1990a,c,d), mutagenicity in Salmonella (Rosenkranz and Klopman, 1990b), 'structural alerts' for genotoxicity (Rosenkranz and Klopman, 1990e), and the induction of chromosomal aberrations and of sister chromatid exchanges in CHO cells (Rosenkranz et al., 1990a).
omeprazole acid and the sulphenimide (Cederberg et al., 1989; Wallmark, 1989). However, the therapeutic effectiveness of omeprazole has been ascribed to its metabolic conversion to the corresponding sulphenimide (Cederberg etal., 1989). Additionally, it has been suggested that the in vivo genotoxicity of omeprazole, in contrast to its lack of in vitro mutagenicity and genotoxicity, is also due to the formation of the sulphenimide (Parry, 1990; Poynter, 1990). For these reasons, for the present analysis, only the data related to omeprazole and its sulphenimide (Figure 2) are presented. A systematic analysis using the various data bases revealed that while the parent molecule omeprazole is predicted to be nonmutagenic, the sulphenimide is predicted to be a mutagen (Table I). Similarly, omeprazole is devoid of a structural alert while its sulphenimide contains such a structure (Table I). On the other hand, both omeprazole and the sulphenimide are predicted to induce chromosomal aberrations in CHO cells while only omeprazole is a probable inducer of sister chromatid exchange and the sulphenimide is predicted to be devoid of such activity (Table I). The most significant portion of the study relates to the predictions of in vivo activity. It is noteworthy that while there is a dichotomy between the in vitro predictions for omeprazole and its sulphenimide, the in vivo results parallel one another (Table I). Thus both molecules are predicted to induce micronuclei in bone marrow due to the presence of multiple biophores (Figure 1; Table I). Additionally, by all of the criteria, using two different databases, both omeprazole and its sulphenimide are predicted to be rodent carcinogens (Figure 2; Table I). The present analyses which indicate that omeprazole is a potential rodent carcinogen are consistent with the reported experimental results (Ekman etal., 1985). Furthermore, the analyses are consistent with the possibility that this putative activity derives from the biotransformation of the parent molecule to the sulphenimide. If this is indeed the case, then in view of the fact that sulphenimide is predicted to be mutagenic as well as DNA-reactive, it can be hypothesized that omeprazole is a potential 'genotoxic carcinogen'. Obviously the present analyses only describe a potential for carcinogenicity in rodents; the actual realization of this risk is dependent upon many other variables including dose, genetically controlled homeostatic (e.g. DNA repair, metabolic activation) and immunological defense mechanisms as well as possible modifying effects introduced by lifestyle (cigarette smoking, diet, alcohol consumption). Certainly, in view of the present predictions, an in-depth analysis of the risks and benefits of omeprazole appears to be called for and this in turn may lead to further testing (Lave et al., 1988).
Omeprazole
ancillary for the present analysis, it should be noted that the predictivity of these endpoints for carcinogenicity in rodents has been questioned (Tennant et at., 1987; Rosenkranz et at., 1990b). Finally, it should be noted that detailed analyses of CASE predictions have shown that CASE can identify 'cryptic agents' as well, i.e. agents that possess intrinsic activity but which may not be expressed under the conditions of the assay (Rosenkranz and Klopman, 1990f). This certainly may represent the situation with the predictions, for example, of the induction of sister chromatid exchange. If the prediciton reflects the cytogenetic potential of the sulphenimide, this will be difficult to ascertain due to its existence only in an acidic milieu. This possibility has been mentioned previously (Parry, 1990). The present data exemplifies the utility of the prescreen for genotoxicity (UDS) and also lends support for the decision to reconsider the safety of omeprazole. Acknowledgements
References Anonymous (1990) Omeprazole and genotoxicity. Lancet, 335, 386. Ashby J. and Tennant,R.W. (1988) Chemical, structure, Salmonella mutagenicity and extent of carcinogenicity as indicators of genotoxic carcinogenesis among 222 chemicals tested in rodents by the US NCI/NTP. Mutat. ftn.,204, 17-115. AshbyJ., Tennant.R.W., Zeiger.E. and Stasiewicz.S. (1989) Classification according to chemical structure, mutagenicity to Salmonella and level of carcinogenicity of a further 42 chemicals tested for carcinogenicity by the US National Toxicology Program. Mutat. Res., 223, 73-103. Bartsch.H. and Malaveille.C. (1989) Prevalence of genotoxic chemicals among animal and human carcinogens evaluated in the IARC Monograph Series. Cell Biol. Toxic, 5, 115-127. Burlinson.B., Morriss.S.H., Gatehouse.D.G. and Tweats,D.J. (1990) Genotoxicity studies of gastric acid inhibiting drugs. Lancet, 335, 419. Burlinson.B., Morriss.S., Gatehouse,D.G., Tweats.D.J. and Jackson.M.R. (1991) Uptake of tritiated thymidine by cells of the rat gastric mucosa after exposure to loxtidine or omeprazole. Mutagenesis, 6, 11-18. Cederberg.C, Andersson.T. and Skanberg.I. (1989) Omeprazole: Pharmacokinetics and metabolism in man. Scent J. GastroenteroL, 24 , (Suppl. 166), 33-40. De Rojas.F.D. (1990) Omeprazole and genotoxicity. Lancet, 335, 910. Ekman.L., Hansson.E., Have.N., Carlsson.E. and Lundberg.C. (1985) Toxicological studies of omeprazole. Scand J. Gastroenterol., 20 (Suppl. 108), 53-69. Ekman.L., Bolcsfoldi.G., MacdonakU. and Nicols.W. (1990) Genotoxicity studies of gastric acid inhibiting drug. Lancet, 35, 419-420. Ennever.F.K., Noonan.T.J. and Rosenkranz.H.S. (1987) The predictivity of animal bioassays and short-term genotoxicity tests for carcinogenicity and noncarcinogenicity to humans. Mutagenesis, 2, 73-78. Furihata.C, Hirose.K. and Matsushima.T. (1991) Genotoxicity and cell proliferative activity of omeprazole in rat stomach mucosa. Mutat. Res., 162, 73-76. Galloway.S.M., Armstrong.MJ., Reuben.C, Colman.S., Brown,B., Carmon.C, Bloom,A.D., Nakamura.F., Ahmed,M., Duk.S., RimpoJ., Margolin,B.H., Resnick.M.A., Anderson,B. and Zeiger.E. (1987) Chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells. Evaluation of 108 chemicals. Environ. Mol. Mutagenesis, 10 (Suppl. 10), 1-175. Galloway.S.M., Bloom.A.D., Resnick.M., MargoIin.B.H., Nakamura.F., Archer.P. and Zeiger.E. (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. Mutagenesis, 7, 151. Gold.L.S., Sawyer.C.B., Magaw.R., Backman.G.M., de Veciana.M., Levinson.R., Hooper.N.K., Havender.W.R., Bemstein.L., Peto.R., Pike.M.C. and Ames.B.N. (1984) A carcinogenic potency database of the standardized results of animal bioassays. Environ. Health Perspea, 58, 9-319. Gold.L.S., de Veciana.M., Backman.G.M., Magaw.R., Lopipero.P., Smith,M., Blumenthal.M., Levinson.R., Bernstein,L. and Ames.B.N. (1986) Chronological supplement to the Carcinogenic Potency Database: Standardized results of animal bioassays published through December 1982. Environ. Health Perspea., 67, 161-200.
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This investigation was supported by the National Institute of Environmental Health Sciences (ES04659) and the US Environmental Protection Agency (R815488).
Gold.L.S., Slone.T.H., Backman.G.M., Magaw.R., Da Costa.M., Lopipero.P., Blumenthal.M. and Ames.B.N. (1987) Second chronological supplement to the carcinogenic potency database: Standardized results of animal bioassays published through December 1984 and by the National Toxicology Program through May 1986. Environ. Health Perspea, 74, 237-329. Gold.L.S., Bemstein.L., Magaw.R. and Slone.T.H. (1989) Interspecies extrapolation in carcinogenesis: Prediction between rats and mice. Environ. Health Perspea., 81, 211-219. Gulati,D.K., Witt.K., Anderson.B., Zeiger.E. and Shelby.M.D. (1989) Chromosome aberration and sister chromatid exchange tests in Chinese hamster ovary cells in vitro. ID: Results with 27 chemicals. Environ. MoL Mutagenesis, 13, 133-193. Hakansson.R., Blom.H., Carlsson.E., Larsson.H., Ryberg.B. and Sundler.F. (1986) Hypergastrinaemia produces trophic effects in stomach but not in pancreas and intestine. Regulatory Peptides, 13, 225-233. Helander.H.F., Larsson.H. and Carlsson.E. (1990) Omeprazole and genotoxicity. Lancet, 335, 910-911. Klopman,G., Frierson.M.R. and Rosenkranz.H.S. (1990) The structural basis of the mutagenicity of chemicals in Salmonella typhimurium: The Genc-Tox Data Base. Mutat. Res., 228, 1-50. Larsson.H., FryklundJ., Helander.H.F. and Wallmark.B. (1991) Partial pronase digestion of rat gastric mucosa isolates cells undergoing replicative DNA synthesis. Mutagenesis, 6, 3 - 9 . Lave.L.B., Ennever.F.K., Rosenkranz.H.S. and Omenn.G.S. (1988) Information value of the rodent bioassay. Nature, 36, 631-633. Loveday.K.S., Lugo.M.H., Resnick.M.A., Anderson.B.E. and Zeiger.E. (1989) Chromosome aberration and sister chromatid exchange tests in Chinese hamster ovary cells in vitro: U. Results with 20 chemicals. Environ. Mol. Mutagenesis, 13, 60-94. Mavoumin.K.H., Blakey.D.H., Cimino.M.C, Salamone.M.F. and HeddleJ.A. (1990) The in vivo micronucleus assay in mammalian bone marrow and peripheral blood. A report of the US Environmental Protection Agency GeneTox Program. Mutat. Res., 239, 29-80. Misiewicz (1990) Omeprazole and genotoxicity. Lancet, 335, 610. Netherlands (1980) Health Council of the Netherlands Report of the Evaluation of the Carcinogenicity of Chemical Substances. Government Printing Office, The Hague. Parry.J.M. (1990) Omeprazole and genotoxicity. Lancet, 335, 611-612. Pounder.R. (1990) Omeprazole and genotoxicity. Lancet, 335, 612. Poynter.D. (1990) Omeprazole and genotoxicity. Lancet, 335, 611. Rosenltranz.H.S. and Ennever.F.K. (1990) An association between mutagenicity and carcinogenic potency. Mutat. Res., 244, 61-65. Rosenkranz.H.S. and Klopman.G. (1989) Structural Basis of the mutagenicity of phenylazoaniline dyes. Mutat. Res., 221, 217-239. Rosenkranz.H.S. and Klopman.G. (1990a) Stuctural basis of carcinogenicity in rodents of genotoxicants and non-genotoxicants. Mutat. Res., 228, 105 — 124. Rosenkranz,H.S. and Klopman.G. (1900b) The structural basis of the mutagenicity of chemicals in Salmonella typhimurium: The National Toxicology Program data base. Mutat. Res., 228, 51-80. Rosenkranz.H.S. and Klopman.G. (1900c) Identification of rodent carcinogens by an expert system. In Mendelsohn,M.L. and Albertini.E.J. (eds), Mutation and the Environment, Part B: Metabolism, Testing, Methods, and Chromosomes. Wiley-Liss, New York, pp. 2 3 - 4 8 . Rosenkranz.H.S. and Klopman.G. (1990d) Natural pesticides present in edible plants are predicted to be carcinogenic. Carcinogenesis, 11, 349—353. Rosenkranz.H.S. and Klopman.G. (1990e) Structural alerts to genotoxicity: The interaction of human and artificial intelligence. Mutagenesis, 5, 333-361. Rosenkranz,H.S. and Klopman.G. (1990f) 'Cryptic' mutagens and carcinogenicity. Mutagenesis, 5, 199-202. Rosenkranz.H.S., Ennever.F.K., Dimayuga.M. and Klopman.G. (1990a) Significant differences in the structural basis of the induction of sister chromatid exchanges and chromosomal aberrations in Chinese hamster ovary cells. Environ. Mol. Mutagenesis, 16, 149-177. Rosenkranz.H.S., Ennever.F.K. and Klopman.G. (1990b) Relationship between carcinogenicity in rodents and the induction of sister chromatid exchanges and chromosomal aberrations in Chinese Hamster Ovary Cells. Mutagenesis, 5, 559-571. Shelby.M.D. (1988) The genetic toxicity of human carcinogens and its implications. Mutation Res., 204, 3 - 1 5 . Tennant.R.W., Margolin,B.H., Shelby.M.D., Zeiger.E., HasemanJ.K., SpaldingJ., Caspary.W., Resnick.M., Stasiewicz.S., Anderson.B. and Minor.R. (1987) Prediction of chemical carcinogenicity in rodents from in vitro genotoxicity assays. Science, 236, 933-941. Wallmark.B. (1989) Omeprazole: Mode of action and effect on acid secretion in animals. Scand. J. Gastroenterol., 24 (Suppl. 166), 12-18. Williams.G.M. (1987) Definition of a human cancer hazard. In Butterworth,B.E. and Slaga.T.J. (eds), Nongenotoxic Mechanisms in Carcinogenesis, Banbury
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H.S.Rosenkranz and G.KIopman Report 25. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 367-380. Williams,G.M. (1990) Screening procedures for evaluating the potential carcinogenicity of food-packaging chemicals. Reg. Toxicol. Pharmacol., 12, 30-40. WilsonJ.D. (1989) Assessment of low-exposure risk from carcinogenesis: Implications of the Knudson—Moolgavkar Two-Critical Mutation Theory. In Travis.C.C. (ed.), Biologically-Based Methods for Cancer Risk Assessment. Plenum Press, New York, pp. 275-287. Wright.N.A. and Goodlad.R.A. (1990) Omeprazole and genotoxicity, Lancet, 335, 909-910. Zeiger,E. (1987) Carcinogenicity of mutagens: Predictive capability of the Salmonella mutagenesU assay for rodent carcinogenicity. Cancer Res., 47, 1287-1296. Zeiger.E., Anderson,B., Haworth.S., Lawlor.T., Mortelmans.K. and Speck.W. (1987) Salmonella mutagenicity tests: ID. Results from 255 chemicals. Environ. Mutagenesis, 9, (Suppl. 9), 1-109. Received on January 7, 1991; accepted on April 8, 1991
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384
Omeprazole: an exploration of its reported genotoxicity
Herbert S.Rosenkranz and Gilles KJopman' Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261 and 'Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106, USA
Introduction The report of the induction of gastric tumors in rats by omeprazole following long-term administration (Ekman et al., 1985) has been attributed to an increased level of circulating gastrin (Hakanson et al., 1986). However, recently there has been considerable controversy regarding the significance of the reported genotoxicity of omeprazole (Figure 1) to the gastric mucosa (Anonymous, 1990; Burlinson etai, 1990,1991; De Rojas, 1990; Ekman et al., 1990; Misiewicz, 1990; Parry, 1990; Pounder, 1990; Poynter, 1990; Wright and Goodlad, 1990; Larsson et al., 1991). The controversy is of some importance as omeprazole has been shown to be an effective inhibitor of gastric acid secretion in humans. However, to some extent, the argument has dealt primarily with the physiology of the gastric mucosa and details of the unscheduled DNA synthesis (UDS) protocol employed. More recently the induction of UDS and of ornithine decarboxylase in the glandular stomach mucosa of rats was reported by another group of investigators (Furihata et al., 1991). Lost in the argument is the possible significance of genotoxicity in identifying a potential human hazard. In fact, the reason for determining genotoxic activity is primarily to identify a carcinogenic potential. It is, of course, recognized that genotoxicity is not a perfect measure; there are many agents which induce cancers in rodents by non-genotoxic mechanisms. However, the argument has been made that compared to nongenotoxic carcinogens, genotoxic carcinogens appear to represent an increased hazard (Netherlands, 1980; Williams, 1987, 1990; Wilson, 1989). As a class, such agents have been suggested as causing cancers in multiple species and at multiple sites in both genders while non-genotoxic carcinogens appear generally to be restricted to the single tissue of a single sex of a single species (Ashby and Tennant, 1988; Ashby etal., 1989; Gold etal., 1989). Moreover, genotoxic carcinogens seem to be more potent rodent carcinogens than non-genotoxic ones (Rosenkranz and Ennever, 1990). Finally, the vast majority of recognized human carcinogens are genotoxicants (Ennever etal., 1987; Shelby, 1988; Bartsch and Malaveille, 1989). This does not imply that © Oxford University Press
Methodology The CASE methodology has been described on a number of occasions (Rosenkranz and Klopman, 1989, 1990a,b; Klopman et al., 1900). CASE selects its own descriptors from a learning set composed of active as well as inactive molecules. These descriptors are readily recognized as continuous structural OMEPRAZOLE (A) 67% chance of being ACTIVE due to substructure (Conf. level = 50%): NH - C =N - C . = (B) 83% chance of being ACTIVE due to substructure (Conf. level = 100%); CH =CH - C =CH (O 95% chance of being ACTIVE due to substructure (Conf. level = 100%): CH =C -CH =C. (D) 97% chance of being ACTIVE due to substructure (Conf. level = 100%): O - C =CH (E) 70% chance of being ACTIVE due to substructure (Conf. level = 91%): NH - C . =CH (F) 91% chance of being ACTIVE due to substructure (Conf. level = 100%): O -CH3 *** OVERALL, the probability of Inducing Bone Marrow Nuclei is 100.0%«*
Fig. 1. CASE prediction of the ability of omeprazole to induce in vivo bone marrow nuclei. The biophores are indicated in bold. Biophore F is present twice. The biophores B, C, D and F are also present in the sulphenimide. C. indicates a carbon atom shared by two ring systems.
381
Downloaded from http://mutage.oxfordjournals.org/ at University of Sussex on January 5, 2013
Because of its reported ability to induce unscheduled DNA synthesis in the gastric mucosa, the safety of omeprazole, a potentially clinically useful anti-ulcer drug, has been the subject of debate. We have undertaken a detailed computerbased study of structural basis of the putative mutagenicity, genotoxicity and carcinogenidty in rodents of omeprazole and of its sulphenimide, and we conclude that omeprazole is a potential 'genotoxic' carcinogen. The analysis is consistent with the possibility that these activities are associated with the unstable sulphenimide metabolite.
genotoxic agents are to be shunned, especially if they possess therapeutic properties. Their adoption, however, needs to be evaluated carefully and subjected to a risk-benefit analysis. In this context, we have undertaken an in-depth analysis of the possible structural basis of the genotoxic and carcinogenic potential of omeprazole and some of its metabolites. Our study indicates that omeprazole has a potential for DNA reactivity, genotoxicity, as well as for inducing cancer in rodents. It is to be noted that these predictions are not based upon a single laboratory test but rather they are derived from the results of structural analyses involving over 2000 chemicals and a multiplicity of toxicological endpoints.
H.S.Rosenkranz and G.KJopman
fragments embedded in the molecule. The descriptors consist of either activating (biophore) or deactivating (biophobe) fragments. Each biophobe and biophore is characterized by its distribution among active and inactive molecules and the associated P value. Once biophores and biophobes have been identified, unknown molecules may be analyzed. Upon submission of such a molecule, the CASE program will generate all 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 and/or absence of these descriptors, CASE predicts activity or lack thereof.
Results and discussion A number of metabolites of omeprazole have been identified; these include omeprazole sulfone, hydroxyomeprazole, OMEPRAZOLE SULPHENAMIDE+ (A) 60% chance of being ACTIVE due to substructure (Conf. level = 75%): CH =CH - C =CH •*• downgraded from 75% because of incorrect Conformation (B) 87% chance of being ACTIVE due to substructure (Coof. level = 100%): C. =CH - C H =C (O 83% chance of being ACTIVE due to substructure (Conf. level = 94%): N" - C . =CH •*• OVERALL, the probability of being a Rodent Cardnogen is 98.0%***
Although the information contributed by the cytogenetic assays (chromosomal aberrations, sister chromatid exchange) is only Table I. Summary of CASE predictions Biological Activity
Fig. 2. CASE prediction of the carcinogenicity in rodents of omeprazole sulphenimide. This prediction is based upon the rodent carcinogenicity compilation of Gold et aL (1984, 1986, 1987). The biophores are shown in bold. The same biophores are also present in omeprazole.
382
Mutagenicity/Salmonella Structural alert Chromosomal aberrations Sister chromatid exchanges In vivo micronuclei Carcinogenicity: rodents (Gold etal., 1984, 1986, 1987) rat (NTP)
Sulphenimide Omeprazole overall probability (%) overall probability (%)
+ + +
0 0 70.9 80.0 100.0
+ + + +
80.0 83.3 68.2 8.8 100.0
+ +
98.0 60.3
+ +
98.0 61.4
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Data Bases The data bases used by CASE as 'learning sets' to identify the appropriate biophores and biophobes were generated primarily by the US National Toxicology Program. Mutagenicity in Salmonella, carcinogenicity in rats, induction of sister chromatid exchange and chromosomal aberrations (Galloway et al., 1985, 1987; Tennant et al., 1987; Zeiger, 1987; Zeiger et al., 1987; Ashby and Tennant, 1988; Ashby etal., 1989; Gulati etai, 1989; Loveday etal., 1989). In addition we also analyzed a rodent carcinogenicity database consisting of 290 chemicals derived from the compilation of Gold et al. (1984, 1986, 1987) and a data base of in vivo inducers of mammalian bone marrow micronuclei (Mavournin et al., 1990). The biophores and biophobes associated with individual endpoints have been described previously: carcinogenicity in rodents (rats) (Rosenkranz and Klopman, 1990a,c,d), mutagenicity in Salmonella (Rosenkranz and Klopman, 1990b), 'structural alerts' for genotoxicity (Rosenkranz and Klopman, 1990e), and the induction of chromosomal aberrations and of sister chromatid exchanges in CHO cells (Rosenkranz et al., 1990a).
omeprazole acid and the sulphenimide (Cederberg et al., 1989; Wallmark, 1989). However, the therapeutic effectiveness of omeprazole has been ascribed to its metabolic conversion to the corresponding sulphenimide (Cederberg etal., 1989). Additionally, it has been suggested that the in vivo genotoxicity of omeprazole, in contrast to its lack of in vitro mutagenicity and genotoxicity, is also due to the formation of the sulphenimide (Parry, 1990; Poynter, 1990). For these reasons, for the present analysis, only the data related to omeprazole and its sulphenimide (Figure 2) are presented. A systematic analysis using the various data bases revealed that while the parent molecule omeprazole is predicted to be nonmutagenic, the sulphenimide is predicted to be a mutagen (Table I). Similarly, omeprazole is devoid of a structural alert while its sulphenimide contains such a structure (Table I). On the other hand, both omeprazole and the sulphenimide are predicted to induce chromosomal aberrations in CHO cells while only omeprazole is a probable inducer of sister chromatid exchange and the sulphenimide is predicted to be devoid of such activity (Table I). The most significant portion of the study relates to the predictions of in vivo activity. It is noteworthy that while there is a dichotomy between the in vitro predictions for omeprazole and its sulphenimide, the in vivo results parallel one another (Table I). Thus both molecules are predicted to induce micronuclei in bone marrow due to the presence of multiple biophores (Figure 1; Table I). Additionally, by all of the criteria, using two different databases, both omeprazole and its sulphenimide are predicted to be rodent carcinogens (Figure 2; Table I). The present analyses which indicate that omeprazole is a potential rodent carcinogen are consistent with the reported experimental results (Ekman etal., 1985). Furthermore, the analyses are consistent with the possibility that this putative activity derives from the biotransformation of the parent molecule to the sulphenimide. If this is indeed the case, then in view of the fact that sulphenimide is predicted to be mutagenic as well as DNA-reactive, it can be hypothesized that omeprazole is a potential 'genotoxic carcinogen'. Obviously the present analyses only describe a potential for carcinogenicity in rodents; the actual realization of this risk is dependent upon many other variables including dose, genetically controlled homeostatic (e.g. DNA repair, metabolic activation) and immunological defense mechanisms as well as possible modifying effects introduced by lifestyle (cigarette smoking, diet, alcohol consumption). Certainly, in view of the present predictions, an in-depth analysis of the risks and benefits of omeprazole appears to be called for and this in turn may lead to further testing (Lave et al., 1988).
Omeprazole
ancillary for the present analysis, it should be noted that the predictivity of these endpoints for carcinogenicity in rodents has been questioned (Tennant et at., 1987; Rosenkranz et at., 1990b). Finally, it should be noted that detailed analyses of CASE predictions have shown that CASE can identify 'cryptic agents' as well, i.e. agents that possess intrinsic activity but which may not be expressed under the conditions of the assay (Rosenkranz and Klopman, 1990f). This certainly may represent the situation with the predictions, for example, of the induction of sister chromatid exchange. If the prediciton reflects the cytogenetic potential of the sulphenimide, this will be difficult to ascertain due to its existence only in an acidic milieu. This possibility has been mentioned previously (Parry, 1990). The present data exemplifies the utility of the prescreen for genotoxicity (UDS) and also lends support for the decision to reconsider the safety of omeprazole. Acknowledgements
References Anonymous (1990) Omeprazole and genotoxicity. Lancet, 335, 386. Ashby J. and Tennant,R.W. (1988) Chemical, structure, Salmonella mutagenicity and extent of carcinogenicity as indicators of genotoxic carcinogenesis among 222 chemicals tested in rodents by the US NCI/NTP. Mutat. ftn.,204, 17-115. AshbyJ., Tennant.R.W., Zeiger.E. and Stasiewicz.S. (1989) Classification according to chemical structure, mutagenicity to Salmonella and level of carcinogenicity of a further 42 chemicals tested for carcinogenicity by the US National Toxicology Program. Mutat. Res., 223, 73-103. Bartsch.H. and Malaveille.C. (1989) Prevalence of genotoxic chemicals among animal and human carcinogens evaluated in the IARC Monograph Series. Cell Biol. Toxic, 5, 115-127. Burlinson.B., Morriss.S.H., Gatehouse.D.G. and Tweats,D.J. (1990) Genotoxicity studies of gastric acid inhibiting drugs. Lancet, 335, 419. Burlinson.B., Morriss.S., Gatehouse,D.G., Tweats.D.J. and Jackson.M.R. (1991) Uptake of tritiated thymidine by cells of the rat gastric mucosa after exposure to loxtidine or omeprazole. Mutagenesis, 6, 11-18. Cederberg.C, Andersson.T. and Skanberg.I. (1989) Omeprazole: Pharmacokinetics and metabolism in man. Scent J. GastroenteroL, 24 , (Suppl. 166), 33-40. De Rojas.F.D. (1990) Omeprazole and genotoxicity. Lancet, 335, 910. Ekman.L., Hansson.E., Have.N., Carlsson.E. and Lundberg.C. (1985) Toxicological studies of omeprazole. Scand J. Gastroenterol., 20 (Suppl. 108), 53-69. Ekman.L., Bolcsfoldi.G., MacdonakU. and Nicols.W. (1990) Genotoxicity studies of gastric acid inhibiting drug. Lancet, 35, 419-420. Ennever.F.K., Noonan.T.J. and Rosenkranz.H.S. (1987) The predictivity of animal bioassays and short-term genotoxicity tests for carcinogenicity and noncarcinogenicity to humans. Mutagenesis, 2, 73-78. Furihata.C, Hirose.K. and Matsushima.T. (1991) Genotoxicity and cell proliferative activity of omeprazole in rat stomach mucosa. Mutat. Res., 162, 73-76. Galloway.S.M., Armstrong.MJ., Reuben.C, Colman.S., Brown,B., Carmon.C, Bloom,A.D., Nakamura.F., Ahmed,M., Duk.S., RimpoJ., Margolin,B.H., Resnick.M.A., Anderson,B. and Zeiger.E. (1987) Chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells. Evaluation of 108 chemicals. Environ. Mol. Mutagenesis, 10 (Suppl. 10), 1-175. Galloway.S.M., Bloom.A.D., Resnick.M., MargoIin.B.H., Nakamura.F., Archer.P. and Zeiger.E. (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. Mutagenesis, 7, 151. Gold.L.S., Sawyer.C.B., Magaw.R., Backman.G.M., de Veciana.M., Levinson.R., Hooper.N.K., Havender.W.R., Bemstein.L., Peto.R., Pike.M.C. and Ames.B.N. (1984) A carcinogenic potency database of the standardized results of animal bioassays. Environ. Health Perspea, 58, 9-319. Gold.L.S., de Veciana.M., Backman.G.M., Magaw.R., Lopipero.P., Smith,M., Blumenthal.M., Levinson.R., Bernstein,L. and Ames.B.N. (1986) Chronological supplement to the Carcinogenic Potency Database: Standardized results of animal bioassays published through December 1982. Environ. Health Perspea., 67, 161-200.
Downloaded from http://mutage.oxfordjournals.org/ at University of Sussex on January 5, 2013
This investigation was supported by the National Institute of Environmental Health Sciences (ES04659) and the US Environmental Protection Agency (R815488).
Gold.L.S., Slone.T.H., Backman.G.M., Magaw.R., Da Costa.M., Lopipero.P., Blumenthal.M. and Ames.B.N. (1987) Second chronological supplement to the carcinogenic potency database: Standardized results of animal bioassays published through December 1984 and by the National Toxicology Program through May 1986. Environ. Health Perspea, 74, 237-329. Gold.L.S., Bemstein.L., Magaw.R. and Slone.T.H. (1989) Interspecies extrapolation in carcinogenesis: Prediction between rats and mice. Environ. Health Perspea., 81, 211-219. Gulati,D.K., Witt.K., Anderson.B., Zeiger.E. and Shelby.M.D. (1989) Chromosome aberration and sister chromatid exchange tests in Chinese hamster ovary cells in vitro. ID: Results with 27 chemicals. Environ. MoL Mutagenesis, 13, 133-193. Hakansson.R., Blom.H., Carlsson.E., Larsson.H., Ryberg.B. and Sundler.F. (1986) Hypergastrinaemia produces trophic effects in stomach but not in pancreas and intestine. Regulatory Peptides, 13, 225-233. Helander.H.F., Larsson.H. and Carlsson.E. (1990) Omeprazole and genotoxicity. Lancet, 335, 910-911. Klopman,G., Frierson.M.R. and Rosenkranz.H.S. (1990) The structural basis of the mutagenicity of chemicals in Salmonella typhimurium: The Genc-Tox Data Base. Mutat. Res., 228, 1-50. Larsson.H., FryklundJ., Helander.H.F. and Wallmark.B. (1991) Partial pronase digestion of rat gastric mucosa isolates cells undergoing replicative DNA synthesis. Mutagenesis, 6, 3 - 9 . Lave.L.B., Ennever.F.K., Rosenkranz.H.S. and Omenn.G.S. (1988) Information value of the rodent bioassay. Nature, 36, 631-633. Loveday.K.S., Lugo.M.H., Resnick.M.A., Anderson.B.E. and Zeiger.E. (1989) Chromosome aberration and sister chromatid exchange tests in Chinese hamster ovary cells in vitro: U. Results with 20 chemicals. Environ. Mol. Mutagenesis, 13, 60-94. Mavoumin.K.H., Blakey.D.H., Cimino.M.C, Salamone.M.F. and HeddleJ.A. (1990) The in vivo micronucleus assay in mammalian bone marrow and peripheral blood. A report of the US Environmental Protection Agency GeneTox Program. Mutat. Res., 239, 29-80. Misiewicz (1990) Omeprazole and genotoxicity. Lancet, 335, 610. Netherlands (1980) Health Council of the Netherlands Report of the Evaluation of the Carcinogenicity of Chemical Substances. Government Printing Office, The Hague. Parry.J.M. (1990) Omeprazole and genotoxicity. Lancet, 335, 611-612. Pounder.R. (1990) Omeprazole and genotoxicity. Lancet, 335, 612. Poynter.D. (1990) Omeprazole and genotoxicity. Lancet, 335, 611. Rosenltranz.H.S. and Ennever.F.K. (1990) An association between mutagenicity and carcinogenic potency. Mutat. Res., 244, 61-65. Rosenkranz.H.S. and Klopman.G. (1989) Structural Basis of the mutagenicity of phenylazoaniline dyes. Mutat. Res., 221, 217-239. Rosenkranz.H.S. and Klopman.G. (1990a) Stuctural basis of carcinogenicity in rodents of genotoxicants and non-genotoxicants. Mutat. Res., 228, 105 — 124. Rosenkranz,H.S. and Klopman.G. (1900b) The structural basis of the mutagenicity of chemicals in Salmonella typhimurium: The National Toxicology Program data base. Mutat. Res., 228, 51-80. Rosenkranz.H.S. and Klopman.G. (1900c) Identification of rodent carcinogens by an expert system. In Mendelsohn,M.L. and Albertini.E.J. (eds), Mutation and the Environment, Part B: Metabolism, Testing, Methods, and Chromosomes. Wiley-Liss, New York, pp. 2 3 - 4 8 . Rosenkranz.H.S. and Klopman.G. (1990d) Natural pesticides present in edible plants are predicted to be carcinogenic. Carcinogenesis, 11, 349—353. Rosenkranz.H.S. and Klopman.G. (1990e) Structural alerts to genotoxicity: The interaction of human and artificial intelligence. Mutagenesis, 5, 333-361. Rosenkranz,H.S. and Klopman.G. (1990f) 'Cryptic' mutagens and carcinogenicity. Mutagenesis, 5, 199-202. Rosenkranz.H.S., Ennever.F.K., Dimayuga.M. and Klopman.G. (1990a) Significant differences in the structural basis of the induction of sister chromatid exchanges and chromosomal aberrations in Chinese hamster ovary cells. Environ. Mol. Mutagenesis, 16, 149-177. Rosenkranz.H.S., Ennever.F.K. and Klopman.G. (1990b) Relationship between carcinogenicity in rodents and the induction of sister chromatid exchanges and chromosomal aberrations in Chinese Hamster Ovary Cells. Mutagenesis, 5, 559-571. Shelby.M.D. (1988) The genetic toxicity of human carcinogens and its implications. Mutation Res., 204, 3 - 1 5 . Tennant.R.W., Margolin,B.H., Shelby.M.D., Zeiger.E., HasemanJ.K., SpaldingJ., Caspary.W., Resnick.M., Stasiewicz.S., Anderson.B. and Minor.R. (1987) Prediction of chemical carcinogenicity in rodents from in vitro genotoxicity assays. Science, 236, 933-941. Wallmark.B. (1989) Omeprazole: Mode of action and effect on acid secretion in animals. Scand. J. Gastroenterol., 24 (Suppl. 166), 12-18. Williams.G.M. (1987) Definition of a human cancer hazard. In Butterworth,B.E. and Slaga.T.J. (eds), Nongenotoxic Mechanisms in Carcinogenesis, Banbury
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H.S.Rosenkranz and G.KIopman Report 25. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 367-380. Williams,G.M. (1990) Screening procedures for evaluating the potential carcinogenicity of food-packaging chemicals. Reg. Toxicol. Pharmacol., 12, 30-40. WilsonJ.D. (1989) Assessment of low-exposure risk from carcinogenesis: Implications of the Knudson—Moolgavkar Two-Critical Mutation Theory. In Travis.C.C. (ed.), Biologically-Based Methods for Cancer Risk Assessment. Plenum Press, New York, pp. 275-287. Wright.N.A. and Goodlad.R.A. (1990) Omeprazole and genotoxicity, Lancet, 335, 909-910. Zeiger,E. (1987) Carcinogenicity of mutagens: Predictive capability of the Salmonella mutagenesU assay for rodent carcinogenicity. Cancer Res., 47, 1287-1296. Zeiger.E., Anderson,B., Haworth.S., Lawlor.T., Mortelmans.K. and Speck.W. (1987) Salmonella mutagenicity tests: ID. Results from 255 chemicals. Environ. Mutagenesis, 9, (Suppl. 9), 1-109. Received on January 7, 1991; accepted on April 8, 1991
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