The carcinoGENOMICS project - Semantic Scholar

Mutation Research 659 (2008) 202–210

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The carcinoGENOMICS project: Critical selection of model compounds for the development of omics-based in vitro carcinogenicity screening assays Mathieu Vinken a,1,*, Tatyana Doktorova a, Heidrun Ellinger-Ziegelbauer b, Hans-Ju¨rgen Ahr b, Edward Lock c, Paul Carmichael d, Erwin Roggen e, Joost van Delft f, Jos Kleinjans f, Jose´ Castell g, Roque Bort g, Teresa Donato g, Michael Ryan h, Raffaella Corvi i, Hector Keun j, Timothy Ebbels j, Toby Athersuch j, Susanna-Assunta Sansone k, Philippe Rocca-Serra k, Rob Stierum l, Paul Jennings m, Walter Pfaller m, Hans Gmuender n, Tamara Vanhaecke a,1, Vera Rogiers a a

Department of Toxicology, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, B-1090 Brussels, Belgium Bayer Healthcare AG, Department of Molecular and Genetic Toxicology, Aprather Weg 18a, 42096 Wuppertal, Germany c School of Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, United Kingdom d Safety & Environmental Assurance Center, Unilever Colworth, Sharnbrook, Bedford MK44 1LQ, United Kingdom e Novozymes A/S, Krogshoejvej 36, DK 2880 Bagsvaerd, Denmark f Department of Health Risk Analysis and Toxicology, Maastricht University PO Box 616, 6200 MD Maastricht, The Netherlands g Unidad de Hepatologıá Experimental, Centro de Investigacioń, University Hopital La Fe, Valencia, Spain h Department of Pharmacology, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland i European Centre for the Validation of Alternative Methods (ECVAM), Institute for Health and Consumer Protection (IHCP), Joint Research Centre of the European Commission (JRC), Ispra, Italy j Department of Biomolecular Medicine, Division of Surgery, Oncology, Reproductive Biology and Anaesthetics, Faculty of Medicine, Imperial College London, London SW7 2AZ, United Kingdom k The European Bioinformatics Institute – European Molecular Biology Laboratory Outstation (EMBL-EBI), Wellcome Trust Genome Campus CB10 1SD, Cambridge Hinxton, United Kingdom l Business Unit Biosciences, TNO Quality of Life, P.O. Box 360, 3700 AJ, Zeist, The Netherlands m Division of Physiology, Department of Physiology and Medical Physics, Innsbruck Medical University, Innsbruck, Austria n Genedata AG, CH-4016 Basel, Switzerland b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 30 January 2008 Received in revised form 2 April 2008 Accepted 21 April 2008 Available online 26 April 2008

Recent changes in the European legislation of chemical-related substances have forced the scientific community to speed up the search for alternative methods that could partly or fully replace animal experimentation. The Sixth Framework Program project carcinoGENOMICS was specifically raised to develop omics-based in vitro screens for testing the carcinogenic potential of chemical compounds in a pan-European context. This paper provides an in-depth analysis of the complexity of choosing suitable reference compounds used for creating and fine-tuning the in vitro carcinogenicity assays. First, a number of solid criteria for the selection of the model compounds are defined. Secondly, the strategy followed, including resources consulted, is described and the selected compounds are briefly illustrated. Finally, limitations and problems encountered during the selection procedure are discussed. Since selecting an appropriate set of chemicals is a frequent impediment in the

Keywords: carcinoGENOMICS FP6 project REACH Chemical carcinogenesis

* Corresponding author. Tel.: +32 2 4774587; fax: +32 2 4774582. E-mail address: [email protected] (M. Vinken). 1 These authors are postdoctoral research fellows of the Fund for Scientific Research Flanders (FWO-Vlaanderen), Belgium. Abbreviations: AhR, aryl hydrocarbon receptor; CA, chromosome aberration; CAS, Chemical Abstracts Service; ChEBI, Chemical Entities of Biological Interest; CYP, cytochrome P450; DCVC, S-(1,2-dichlorovinyl)-L-cysteine; ECOPA, European Consensus Platform on 3R-Alternatives; ECVAM, European Centre for the Validation of Alternative Methods; FP6, Sixth Framework Program; HPRT, hypoxanthine-guanine phosphoribosyltransferase; IARC, International Agency for Research on Cancer; Ki, kidney; Li, liver; Lu, lung; MLA, mouse lymphoma assay; MNT, micronucleus test; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NS, not specified; OECD, Organization for Economic Co-operation and Development; REACH, registration, evaluation and authorization of chemicals; UDS, unscheduled DNA synthesis; WP, work package. 1383-5742/$ – see front matter . Crown Copyright ß 2008 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.mrrev.2008.04.006

M. Vinken et al. / Mutation Research 659 (2008) 202–210 Genomics Metabonomics Metabolomics Model compounds Selection criteria Genotoxic carcinogen Non-genotoxic carcinogen.

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early stages of similar research projects, the information provided in this paper might be extremely valuable. Crown Copyright ß 2008 Published by Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The carcinoGENOMICS project at a glance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Selection of model compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Classification and definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Selection criteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Toxicological profile and gene expression profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. Diversity and selectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.3. Biochemical and biophysical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.4. Legal aspects and safety measures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Selected compounds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2. Genotoxic carcinogens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.3. Non-genotoxic carcinogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.4. Non-carcinogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Final considerations and perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction In December 2006, the European Parliament and Council adopted a novel legislation for chemicals, commonly known as REACH (registration, evaluation and authorization of chemicals), which has been ultimately implemented in June 2007. Basically, REACH demands the safety assessment of more than 30,000 manmade chemicals that are actually on the European market [1]. It is clear that such large-scale testing will require enormous amounts of laboratory animals. Varying numbers have been published in this respect, ranging from 1.2 million [2] to 45 million animals [3]. A more realistic estimate provided by ECOPA (European Consensus Platform on 3R-Alternatives) suggests that around 6.25 million experimental animals would be required, given that new testing approaches, such as read-across/grouping techniques and quantitative structure activity relationships, are accepted by the regulators. This estimate rises to around 12.2 million animals if only the existing alternative methods are optimally used [4]. For the safety evaluation of cosmetics, on the other hand, the European legislator expects a complete replacement of animal tests from 2013 onwards [5]. These legislative changes for chemical-related substances clearly illustrate that within the European Union, there is an urgent need for alternatives to animal testing. A major problem in this context is that the currently available and formally validated alternative methods can only be used to address acute or short-term toxicity [6]. Unfortunately, detection of chronic and systemic toxicity, including carcinogenicity, is not yet possible via alternative testing strategies and thus still requires large numbers of animals [7]. An additional problem is the fact that existing alternatives or those that are in the pipeline provide hazard identification and are not suitable for quantitative risk assessment [8,9]. Toxicological science has experienced tremendous advances in the last 2 decades. Undoubtedly, a milestone was the emergence of so-called toxicogenomics, resulting from the application of knowledge gained from genomics science into conventional

203 204 204 204 204 204 205 205 205 207 207 207 207 208 208 209 209

toxicology. This growing research field specifically tackles the complex interactions between toxic effects, elicited by exogenous stimuli (e.g. chemical compounds), and the structure and/or activity of the genome [10,11]. The basic tool in toxicogenomics, the DNA microarray, allows the simultaneous analysis of thousands of individual genes and thus permits the assessment of characteristic modifications in gene expression profiles induced by toxic compounds. Such ‘‘molecular signatures’’ are extremely useful for the elucidation of the mechanistic basis of toxic responses [10–15]. In this regard, several research groups have successfully applied DNA microarray technology to discriminate between direct-acting genotoxins and indirect-acting genotoxins [16–19]. Studies that have yet attempted to differentiate genotoxic carcinogens from their non-genotoxic counterparts have been conducted in vivo [20–22], used a very limited number of chemicals [23] or have only been focused on 1 specific target tissue [23,24]. Nevertheless, such studies frequently yield biomarkers that are relevant for both the ranking of carcinogenic compounds as well as for the prediction of chemically induced carcinogenicity. High-throughput screening of knockdown libraries using RNA interference may also lead to the identification of genes that are crucial for toxic effects provoked by different classes of carcinogenic compounds [25,26]. Furthermore, combining DNA microarray technology with other omics sciences, such as global protein expression profiling (‘‘proteomics’’) and/or metabolic profiling (‘‘metabonomics/metabolomics’’), may offer a solid and more rationalized basis for the large-scale estimation of adverse biological effects [11,27,28]. In recent years, a number of European initiatives have been launched to speed up the search for alternative methods, especially suitable for studying chronic toxicity induced by xenobiotics. Among those, the carcinoGENOMICS project was raised to develop omics-based in vitro screens for testing the carcinogenic potential of chemical compounds. In this paper, the carcinoGENOMICS project is presented. Particular attention is paid to the procedure of selecting chemicals for the project. Indeed, a critical selection of

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M. Vinken et al. / Mutation Research 659 (2008) 202–210

Table 1 Composition of the carcinoGENOMICS consortium Partner

Country

Maastricht University University Hospital La Fe Vrije Universiteit Brussel Cellartis Leiden University Medical Centre University College Dublin Novozymes Imperial College London AdvanCell European Bioinformatics Institute TNO Quality of Life Max Planck Institute for Molecular Genetics European Centre for the Validation of Alternative Methods European Consensus Platform for 3R-Alternatives Unilever Innsbruck Medical University Genedata Biopredic International John Moores University Future partner X (competitive call)

The Netherlands (Maastricht) Spain (Valencia) Belgium (Brussels) Sweden (Gothenburg) The Netherlands (Leiden) Ireland (Dublin) Denmark (Bagsvaerd) United Kingdom (London) Spain (Barcelona) Germany (Heidelberg) The Netherlands (Delft) Germany (Munich) Belgium (Brussels)

extensive bioinformatics, literature mining, and analysis of molecular expression datasets, differential genetic pathways will be identified that are capable of predicting mechanisms of chemical carcinogenesis in vivo. Furthermore, the integration of transcriptomic and metabonomic data will facilitate deeper understanding of the action of chemicals and the molecular mechanisms involved in carcinogenesis, which will aid in the generation of predictive and robust in silico models. Predictive genetic pathways will be subsequently used as the scientific basis to develop high-throughput technology for accelerating analysis of genomics responses in vitro. In the second phase of the project, the designed in vitro tests will go through the process of pre-validation, according to guidelines provided by the European Centre for the Validation of Alternative Methods (ECVAM) [30]. To this end, special attention will be paid to the robustness of the in vitro assays as well as to the further evaluation of their predictive power.

Belgium (Brussels) United Kingdom (London) Austria (Innsbruck) Switzerland (Basel) France (Rennes) United Kingdom (Liverpool)

The project is coordinated by Maastricht University and involves 20 participating groups, including academia, research institutions, industry and regulatory bodies.

compounds in the initial stage of carcinoGENOMICS is of crucial importance, as this set of chemicals will be used as a reference during the development and optimization of the in vitro carcinogenicity assays. The information provided in this paper might be very helpful during preliminary stages of analogous research projects that aim at the prediction of in vivo chemically induced carcinogenicity from in vitro testing. 2. The carcinoGENOMICS project at a glance The carcinoGENOMICS project is an integrated project within the European Sixth Framework Program (FP6) and comprises 20 participating groups, including academia, research institutions, industry and regulatory bodies (Table 1) [29]. It is operational since November 2006 and set to run over a 5-year period. Its overall goal is to create in vitro tests for assessing the carcinogenic potential of chemical compounds as an alternative to existing chronic rodent bioassays for carcinogenicity. In addition, it hopes to deliver more reliable in vitro tools for predicting genotoxicity as current in vitro tests are hampered by the risk of reporting false positives, thereby falsely suggesting the necessity of performing in vivo carcinogenicity studies. In particular, a battery of mechanism-based in vitro tests representative for various modes of carcinogenic action in 3 major target organs, namely the liver, the kidney and the lung, will be designed. These novel assays will be based on the application of omics technologies (i.e. transcriptomics and metabonomics) in robust in vitro systems. The latter are of both human and rodent origin and include organotypical primary cell culture systems, tumor-derived cell lines, as well as embryonic stem cell-derived models. As such, carcinoGENOMICS is organized in 12 work packages (WPs) and consists of 2 phases (Fig. 1) [29]. In the first stage, genomic and metabonomic responses from a set of model compounds of genotoxicity and carcinogenicity will be generated. Phenotypic markers will be assessed for the purpose of anchoring gene expression modulations, metabolic profiles and mechanistic pathways. Such markers include cytogenetical endpoints for chromosome stability, cellular proliferation, apoptosis and necrosis, and the production of reactive oxygen species. Through

3. Selection of model compounds 3.1. Classification and definitions Within existing carcinogen ranking systems worldwide, such as proposed by the International Agency for Research on Cancer (IARC) [31], agents are usually classified according to their carcinogenic hazard to humans [32–34]. In view of the project’s objectives, however, a more mechanistically-based categorization for carcinogens is addressed, at least in the first stage of the project. Thus, a distinction is made between genotoxic carcinogens, nongenotoxic carcinogens and non-carcinogens. In the context of carcinoGENOMICS, a carcinogen is defined as any chemical of which the carcinogenic potential has been unequivocally demonstrated on the basis of a 2-year combined rodent in vivo chronic toxicity/carcinogenicity test, thereby considering either ingestion (liver and kidney) or inhalation (lung) as the route of exposure. To be designated a genotoxic carcinogen, compounds must have been proven positive in the classical bacterial reverse mutation assay (Ames test) combined with at least 1 of the other commonly used in vitro genotoxicity tests (e.g. in vitro chromosome mammalian aberration test, in vitro mammalian cell gene mutation test or unscheduled DNA synthesis in mammalian cells in vitro), as well as with at least 1 in vivo genotoxicity test (e.g. mammalian erythrocyte micronucleus test, mammalian bone marrow chromosome aberration test or unscheduled DNA synthesis test with mammalian liver cells in vivo). By contrast, carcinogenic compounds for which a non-genotoxic mechanism is considered to be the primary cause of carcinogenicity are judged as being nongenotoxic carcinogens. Chemical substances that are negative in the Ames test as well as in in vivo cytogenetics testing, and that clearly lack carcinogenicity potential in rodent bioassays are considered as non-carcinogens (i.e. negative controls). 3.2. Selection criteria 3.2.1. Toxicological profile and gene expression profile Chemicals are selected on the basis of already existing toxicological information, namely data derived from in vitro and in vivo genotoxicity testing as well as from 2-year bioassays, as described above. In view of the scope of carcinoGENOMICS, chemicals for which transcriptomics data (i.e. DNA microarray data) in the specific target organ are available are being prioritized. It is evident that in vivo transcriptomics data are preferred over in vitro transcriptomics data. This kind of information is found in scientific literature as well as in public and/or in-house databases. One could, however, expect major heterogeneity in terms of types of DNA microarrays, experimental set-up and expression of the


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Fig. 1. Organization of carcinoGENOMICS: interrelationships of the respective work packages. The project is organized in 12 work packages (WPs) and consists of 2 phases. WP1 involves the selection of model compounds for the project. In WP2, WP3 and WP4, a number of robust liver-, kidney-and lung-based in vitro assays, respectively, are developed that can detect the carcinogenic actions of the selected compounds. Transcriptomic and metabonomic responses induced by the model compounds are processed in WP5 and WP6, respectively. WP7 is fully devoted to data warehousing, statistical data analysis, pathway analysis and in silico modelling. WP8 is expected to add highthroughput features in order to facilitate the analysis of specific genomic responses to human carcinogens at high reliability and speed, and at low cost. In WP9, the designed in vitro tests are pre-validated according to ECVAM’s internationally accepted criteria [30]. WP10 foresees training and educational activities among carcinoGENOMICS researchers by transfer of knowledge and scientific skills, exchange of researchers between laboratories, networking of existing training and research, and practical training on the best available technologies. WP11 aims at the organization of the flow of scientific information within the carcinoGENOMICS network and dissemination of information regarding the results of the research performed in the individual work packages, as well as relevant scientific findings from the wider scientific community. Finally, the global management of carcinoGENOMICS is established in WP12.

results. Such critical parameters therefore need to be carefully taken into consideration while interpreting gene profiling experiments. 3.2.2. Diversity and selectivity By definition, genotoxic carcinogens act by interfering with genetic material. In in vitro experimental settings, they can be further subdivided into direct-acting (DNA-reactive) and indirect-acting genotoxins. The former may target single genes as well as whole chromosomes. Genotoxicity triggered by DNA non-reactive genotoxic agents may indirectly result from a number of actions, including disturbance of nucleotide synthesis, inhibition of topoisomerase activity and reduction of microtubule formation [18,32,33]. An even greater variety exists when considering mechanisms that underlie non-genotoxic carcinogenicity, such as inhibition of apoptosis, downregulation of gap junctional intercellular communication, enhancement of cell proliferation and peroxisome proliferation as well as induction of xenobiotic biotransformation capacity [35,36]. Accordingly, the selection of compounds for carcinoGENOMICS must be as diverse as possible in terms of mode of carcinogenic action. This can be accomplished, at least in part, by avoiding structurally related carcinogenic compounds, which typically act via a common mechanism. To allow comparison of carcinogenic actions at the molecular level in different tissues, compounds, and the genotoxins in particular, should preferably affect all of the 3 target organs. In addition, they have to be effective in rodents (i.e. rat and mouse), and, whenever such data are available, in humans. Both male and female subjects should

be affected by the selected chemicals, a criterion that specifically holds for non-genotoxic carcinogens. Since primary cell systems will be used in the project and because such in vitro models are typically derived from male animals, only male-specific carcinogens are to be taken into consideration in case of gender specificity. 3.2.3. Biochemical and biophysical properties A great majority of chemical carcinogens, particularly genotoxins, rely on biotransformation enzymes, typically belonging to the cytochrome P450 (CYP) superfamily, to become effective. Thus, pro-carcinogens are converted into their ultimate counterparts that actually elicit deleterious effects in the organism [37]. To closely mimic this in vivo reality, chemicals that require metabolic activation are preferred over non-metabolized compounds. Furthermore, in order to ensure maximal uptake of the compounds into the cells, and consequently to maximize the toxicological response, chemicals should be handled in their most lipophilic analytical form. This practically implies that the use of salts is discouraged, if technically possible with respect to solubility. In addition, to allow well-controlled incubation in the liver- and kidney-based in vitro systems, volatile compounds should be avoided. As inhalation and thus local contact is a more realistic exposure route in the occurrence of pulmonary cancer, this principle does not apply to lung carcinogens involved. 3.2.4. Legal aspects and safety measures It is clear that carcinoGENOMICS implies the use of very toxic compounds. These chemicals may be subjected to specific national

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Table 2 Model compounds selected for the first phase of carcinoGENOMICS Compound

Genotoxic carcinogens Aflatoxin B1 4-(Methylnitrosamino)-1-(3-pyridyl)1-butanone Dimethylnitrosamine 2-Nitrofluorene Benzo[a]pyrene

Non-genotoxic carcinogens Wy-14,643 MethapyrileneHCl Piperonyl butoxide Sodium phenobarbital Tetradecanoyl phorbol acetate Ochratoxin A Monuron Chlorothalonil Bromodichloromethane S-(1,2-dichlorovinyl)-L-cysteine 2,3,7,8-Tetrachlorodibenzo-para-dioxin Cadmium dichloride Sodium arsenate Asbestos Chloroprene Non-carcinogens Nifedipine Tolbutamide Clonidine Sodium diclofenac D-mannitol Ethylene Beclomethasone dipropionate Ipratropium bromide monohydrate

ChEBI accession numbers [85,86]

In vitro genotoxicity

In vivo genotoxicity

Carcinogenicity

IARC

Organ involved

1162-65-8 64091-91-4

2504 32692

+Ames, +MLA, +UDS, +MNT, +CA [39,41,42,74] +Ames, +UDS, +HPRT [41,42]

+CA [42] +MNT [42]

+Rats, +mice [38,40,41] +Rats, +mice [38,40,41]

1 2B

Li [38,40,41] Li [38,40,41]

62-75-9

35807

+CA, +MNT [41,42]

+Rats, +mice [38,40,41]

2A

Li, Ki, Lu [38,40,41]

607-57-8 50-32-8

1224 29865

+CA, +MNT [41,42]

+Rats [38,41] +Rats, +mice [38,40,41]

2B 2A

Li, Ki [38,40,41] Li, Ki, Lu [40,41]

7758-01-2 18883-66-4 106-99-0 75-01-4 10588-01-9 542-56-3

38211 9288 39478 28509 39483 46643

+Ames, +UDS, +HPRT, +MLA, +MNT, +CA [39,41,42,74] +Ames, +MLA, +CA, +MNT, +UDS [37,38] +Ames, +HPRT, +MLA, +MNT, +CA, +UDS [39,41,42,74] +Ames, +MNT, +CA [39,41,74] +Ames, +CA, +HPRT, +MLA [38,41,74] +Ames, +HPRT, MLA [38,41,74] +Ames, +CA [39,41,42,74] +Ames, +MLA, +CA [39,41,42,74] +Ames, + MLA, +MNT, +CA [40–42,74]

+CA, +MNT [41] +CA, +MNT [42] +CA [42] +CA [40]

+Rats, +mice [38,41] +Rats, +mice [38,40,41] +Rats, +mice [38,40] +rats, +mice [38,40] +Rats [38,40] +Rats, +mice [38,40]

2B 2B 2A 1 1 1

Ki [38,41] Ki [38,40,41] Lu [38,40] Lu [38,40,41] Lu [38,40] Lu [38,40]

50892-23-4 135-23-9 51-03-6 57-30-7 16561-29-8 303-47-9 150-68-5 1897-45-6 75-27-4 627-72-5 1746-01-6 10108-64-2

32509 38213 32687 8070 37537 7719 38214 3639 34591 46650 28119 35456

+Rats, +mice [38,40] +Rats [38,40] +Rats, +mice [38,40] +Rats, +mice [38] +Mice [41] +Rats, +mice [38,40,41] +Rats, mice [38,40,41] +Rats, mice [38,40,41] +Rats, +mice [38,40,41] +Rats* [40] +Rats, +mice [38,40] +Rats, mice [38,40,41]

– – 3 – – 2B 3 2B 2B – 1 1

Li [38,40] Li [5,6] Li [38,40] Li [38] Li [41] Ki [38,40,41] Ki [38,40,41] Ki [38,40,41] Ki [38,40,41] Ki* [40] Li, Ki, Lu [38,40] Lu [38,40,41]

7784-46-5 1332-21-4 126-99-8

29678 46661 39481

rats, mice [38] +Rats [40] +Rats, +mice [38,40,41]

1 1 2B

Lu [40] Lu [40] Lu [38,40,41]

21829-25-4 64-77-7 4205-90-7 15307-79-6 69-65-8 74-85-1 5534-09-8 66985-17-9

7565 27999 46631 4507 16899 18153 3002 5957

– – – – – 3 – –

Li, Ki Li, Ki Li, Ki Li, Ki Li, Ki Lu Lu Lu

Ames, MNT, UDS [39–42] Ames, MLA, +CA [41,42,74] Ames, UDS, HPRT +MLA, CA [41,42,74] Ames [38,41,42,74] Ames, HPRT, MNT, CA [39,41,42,74] Ames, UDS, HPRT, MLA, CA [41,42,74] Ames, MLA, CA [39,41,42] Ames, MLA, +CA [39–42,74] Ames, MLA, UDS, CA [39,41,74] Ames, UDS [41] Ames, MLA, CA [39,41,42,74] Ames, UDS, +MNT, +MLA, HPRT, CA [39,41,42,74] Ames, CA, +MNT, +MLA [41,42] Ames, +CA [39,74] Ames [41]

Ames [41,72] Ames, MLA, CA [41,74] Ames, UDS [41,72] Ames, CA, MLA, HPRT [41,72] Ames, MLA, CA [41,42,74] Ames, CA [40,42] Ames, CA, HPRT [72] Ames, CA [72]

CA [41] CA, +MNT [42] MNT [42] MNT [38] CA [42] MNT [42] MNT [41,42] MNT, +CA [41] MNT, CA [41]

NS [72] NS [72] NS [72] CA, MNT [42] MNT [42] NS [72]

Rats [72] Rats, mice Rats [72] Rats, mice Rats, mice Rats, mice Rats, mice Rats, mice

[38,40] [72] [41] [40] [72] [72]

Compounds were selected according to the established criteria (see text) and cover a wide range of chemical substances, including industrial chemicals, biocidal products as well as pharmaceuticals. For both kidney and liver carcinogens, ingestion is considered as the route of exposure, whereas inhalation is regarded as the main way of contact for lung carcinogens. Chemicals presented in italics are part of the learning set of compounds ((+) positive outcome; ( ) negative outcome; (*) trichloroethylene-associated carcinogenicity; Ames, bacterial reverse mutation assay; CA, chromosome aberration test; CAS, Chemical Abstracts Service; ChEBI, Chemical Entities of Biological Interest; HPRT, hypoxanthine-guanine phosphoribosyltransferase mutation test; IARC, International Agency for Research on Cancer; Ki, kidney; Li, liver; Lu, lung; MLA, mouse lymphoma assay; MNT, micronucleus test; NS, not specified; UDS, unscheduled DNA synthesis test).


Potassium bromate Streptozotocin 1,3-Butadiene Vinyl chloride Sodium dichromate Isobutyl nitrite

CAS number


legislation, which in turn could impede their availability. When a problem arises in this context, such compounds should be omitted. Likewise, the safety measures that must be taken during handling of these chemicals should be carefully addressed. In addition, to ensure maximal safety, appropriate training of investigators should be foreseen. 3.3. Selected compounds 3.3.1. Methodology For the purpose of finding chemicals that meet the established selection criteria, we first mined public electronic databases. These included the Carcinogenic Potency Database [38], the Carcinogenicity and Genotoxicity Experience Database [39], the National Toxicology Program database [40] and the Chemical Carcinogenesis Research Information System database [41]. In addition, documents provided by regulatory bodies, such as the IARC [31] and the Organization for Economic Co-operation and Development (OECD) [42], were also consulted. Based on the information available, a preliminary set of common non-carcinogens, nongenotoxic carcinogens and genotoxic carcinogens was appointed. In-depth identification of the pre-selected compounds was subsequently performed using the commercial (Chemical Abstracts Service) factual database SciFinder [43]. Scientific literature was screened in parallel, thereby using PubMed [44] as a bibliographical resource. All information obtained was then compiled into datasheets presenting compound-specific properties relevant to the selection criteria. These compound profiles eventually allowed final selection of the chemicals. The ultimate set of selected compounds for the first stage of carcinoGENOMICS is shown in Table 2. In the following paragraphs, a brief description of each of these chemicals is provided. 3.3.2. Genotoxic carcinogens Aflatoxin B1 is a fungal metabolite, produced by the genus Aspergillus, which is known to contaminate agricultural products, such as cereal grains [45]. Administered orally or by intraperitoneal injection, it was repeatedly found to cause hepatocellular or cholangiocellular liver tumors in experimental animals [40]. Aflatoxin B1 is a well-known genotoxic substance and biotransformation mediated by the cytochrome P450 enzyme system is crucial for its genotoxicity. Indeed, aflatoxin B1 is converted into an epoxide metabolite that actually forms DNA adducts [45]. 4(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a tobaccospecific nitrosamine that causes tumors in liver and lung of rat when administered by injection or via drinking water [38,40]. NNK is a pro-carcinogen, whereby bioactivation to its DNA-interacting metabolites is controlled by several cytochrome P450 isoenzymes [46]. Dimethylnitrosamine (N-nitrosodimethylamine) is an environmental contaminant, produced in industrial chemistry, which causes tumors at multiple sites (e.g. liver, kidney and lung) in laboratory animals independent of the route of exposure [40]. Both in vivo and in vitro, dimethylnitrosamine displays potent genotoxic properties, which are in fact attributed to its N-nitroso-hydroxymethyl-methylamine metabolite [47]. 2-Nitrofluorene, a byproduct of combustion processes, produces tumors in liver and kidney of rat when added to the diet [40,41]. The 2-nitrofluorene parent molecule as such is mutagenic, but DNA adduct formation also occurs following conversion (mediated by intestinal microflora and/or cytochrome P450 enzymes) to DNA-binding metabolites, including 2-aminofluorene and 2-acetylaminofluorene [48]. Benzo[a]pyrene is a prototypic polycyclic aromatic hydrocarbon that forms as a result of incomplete combustion of organic compounds. It becomes active upon cytochrome P450-mediated biotransformation to a genotoxic epoxide metabolite [49]. When adminis-

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tered to experimental animals via several ways (inhalation, ingestion, dermal application, . . .), benzo[a]pyrene induces tumor growth in a number of organs, including liver, kidney and lung [40]. Potassium bromate has been used for a long time as a chemical oxidizing agent in food and cosmetic industries [50]. When added to the drinking water, it induces tumorigenesis in the kidney of rats and mice [38,41]. At the molecular level, potassium bromate causes DNA damage due to oxidative stress [50]. Streptozotocin is a fungal metabolite, produced by Streptomyces Achromogenes, that displays antibiotic and antineoplastic properties. Steptozotocin triggers the formation of both DNA adducts and DNA strand breaks [51]. By doing so, streptozotocin was found to increase the incidence of renal tumors in rodents [38,40]. 1,3-Butadiene, produced in rubber industry, is a gaseous substance that causes lung tumors in rodents [38,40,41]. It induces DNA adduct formation following cytochrome P450-mediated biotransformation [52]. Vinyl chloride is a colorless gas that is used in plastic industry [52]. Mice and rats exposed to vinyl chloride develop tumors at multiple sites, including lung [38,40,41]. In the organism, vinyl chloride is converted into a DNA-binding metabolite [52]. Sodium dichromate is a widely used oxidizing agent in chemical industry that undergoes reduction from hexavalent to DNA-interactive trivalent chromium after cellular uptake [53]. Sodium dichromate administration via inhalation causes lung tumors in rat [38,40]. Isobutyl nitrite is a volatile liquid that acts as a vasodilator [54]. It is a genotoxic substance that induces alveolar and bronchiolar adenomas and carcinomas in both rat and mice [38,40]. 3.3.3. Non-genotoxic carcinogens Wy-14,643 (pirinixic acid) is a selective peroxisome proliferator-activated receptor a agonist, that causes hepatocellular carcinomas and adenomas in (male) rats and mice following dietary uptake [38,41]. Its carcinogenic mode of action not only involves induction of peroxisome proliferation [55], but also reduction of gap junctional intercellular communication [56]. MethapyrileneHCl is a H1 histamine receptor blocking agent [57] that induces liver tumor growth when administered to rats by gavage or via diet [38,41]. The mechanisms that underlie its carcinogenicity are unclear, but could involve induction of oxidative stress and lipid peroxidation, modulation of biotransformation capacity [56], as well as reduction of gap junctional intercellular communication [57]. Piperonyl butoxide is an insecticide synergist that causes liver tumors in rats and mice from both sexes after oral uptake [38,41]. It acts through induction of oxidative stress, modulation of cytochrome P450-mediated biotransformation capacity and downregulation of gap junctional intercellular communication [58]. Sodium phenobarbital is an antiepileptic drug that is frequently used as a model tumor promoter in rodent liver. It is a typical inducer of cytochrome P450 isoenzymes and also blocks gap junctional intercellular communication [59]. Tetradecanoyl phorbol acetate, being a phorbol ester present in the oil of the Croton tiglium plant, is a known activator of protein kinase C [60]. It is a commonly used compound for tumor promotion studies, whereby the liver is a frequent target in both male and female mice [41]. Ochratoxin A is a food-contaminating mycotoxin from Aspergillus ochraceus and Penicillium verrucosum that interferes with a number of intracellular signalling pathways [61]. Gavage or dietary administration of ochratoxin A to rats and mice burgeons in the occurrence of renal cell tumors [39–41]. Monuron is a herbicide that induces tumors in the kidney, and to a lesser extent in the liver, of male rats, but not of female rats nor mice, following oral uptake. Its exact mode of action, however, remains elusive, and does not necessarily exclude mutagenic effects [39–41]. Chlorothalonil, a pesticide, specifically causes

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kidney tumor growth in rats of both sexes upon dietary uptake [39–41]. The carcinogenicity evoked by chlorothalonil is attributed to 1 of its nephrotoxic metabolites, that is generated via b lyasemediated catalyzation [62]. Bromodichloromethane is a reactant in chemical industry, that increases the incidence of renal cell tumors in rats and mice following oral uptake [39–41]. Potential mechanisms underlying bromodichloromethane carcinogenicity include glutathione S-transferase-mediated formation of reactive metabolites [63] as well as induction of DNA hypomethylation [64]. S-(1,2-dichlorovinyl)-L-cysteine (DCVC) is a nephrotoxic glutathione metabolite of trichloroethylene [65]. The latter is a volatile industrial solvent that causes both malignant and benign tumors at multiple tissue sites in several species of experimental animals [40]. 2,3,7,8-Tetrachlorodibenzo-para-dioxin (‘‘dioxin’’), a member of the chlorinated dibenzo-p-dioxins, is a well-known industrial and environmental contaminant that leads to the development of tumors in multiple organs in rats and mice from both sexes when administered via multiple routes [40]. Dioxin does not show genotoxic potential, both in vivo and in vitro [42]. Dioxin is known to interact with the cytosolic aryl hydrocarbon receptor (AhR). This results in the modulation of gene expression of AhR-responsive targets, such as biotransformation enzymes [66]. Dioxin carcinogenicity may also rely on reduction of gap junctional intercellular communication [56]. Cadmium dichloride is a widely used substance in chemical industry. It serves many purposes, including stabilization of plastics [40]. Cadmium dichloride clearly causes lung tumors in rats and mice when administered through inhalation [39–41]. Mechanisms of cadmium dichloride carcinogenicity that have been proposed include induction of oxidative stress, upregulation of mitogenic signalling and suppression of apoptotic activity [67]. Sodium arsenite (inorganic arsenic), released from both natural and man-made sources, is widely present in the environment, whereby exposure mainly occurs through oral and inhalation routes. The latter was found to correlate with the occurrence of lung tumors [40]. However, rats and mice that are exposed to sodium arsenite via the drinking water do not display tumorigenesis [39–41]. Modes of nongenotoxic sodium arsenite carcinogenicity could imply the induction of oxidative stress, upregulation of mitogenic signalling as well as the alteration of DNA methylation patterns [68,69]. Asbestos is a generic name for a group of 6 naturally occurring fibrous silicate minerals, including chrysotile, crocidolite, amosite, anthophyllite, actinolite and tremolite. Asbestos minerals are used for a number of applications, such as heat stabilization. All commercial forms of asbestos have been shown to cause cancer (e.g. in lung) in multiple species by various exposure routes, including inhalation [40]. Its exact mode of carcinogenic action is yet unclear but may involve both induction of oxidative stress and upregulation of mitogenic signalling [70]. Chloroprene is primarily used in the production of rubber [40]. When administered to rats and mice via inhalation, chloroprene triggers tumor growth at multiple sites, including lung [39–41]. The underlying molecular mechanisms are unknown, but do not include genotoxic actions [40]. 3.3.4. Non-carcinogens Nifedipine is an L-type Ca2+ channel blocking agent that is currently on the European market as an antihypertensive drug [71]. No tumor growth is observed in rats that have been administered nifedipine. Nifedipine also lacks genotoxicity potential, both in vitro and in vivo [72]. Tolbutamide is a hypoglemic drug that is used to treat type II diabetes [73]. It was found negative in carcinogenicity evaluation [38,41] as well as in genotoxicity testing [41,74]. Clonidine, an a adrenoceptor activating agent, is nowadays prescribed as an antihypertensive drug [75]. It does not

display genotoxic or carcinogenic properties [41,72]. Sodium diclofenac, a widely used analgesic and anti-inflammatory drug [76], was repeatedly shown to lack genotoxicity [41,72]. In addition, no tumorigenesis was observed in rats and mice that received sodium diclofenac via diet [72]. D-Mannitol, a sugar alcohol, is applied as an osmotic diuretic agent [77]. It is frequently addressed as a negative control in genotoxicity and carcinogenicity testing [41,42,74]. Ethylene is petrochemical that is not mutagenic and that does not cause carcinogenicity in rats following chronic exposure via inhalation [40,42]. Beclomethasone dipropionate, a glucorticoid, is a potent inflammatory drug that is used in aerosols for the treatment of asthma [78]. It was found negative in a panel of genotoxicity assays and does not evoke cancer in laboratory animals [72]. Ipratropium bromide monohydrate is a M2 muscarinic receptor blocking agent that is currently marketed as an antiasthmatic drug [79]. It lacks genotoxic potential and does not cause tumor growth in rodents [72]. 4. Final considerations and perspectives Historically, the carcinogenic potential of chemical-related substances has been routinely tested in laboratory animals. At present, a conventional rodent lifetime carcinogenicity study takes up to 5 years to design, conduct and analyze, and consumes as many as 800 mice and rats at a total cost of s1.5–3 million per chemical tested [80]. In view of the recent legislative changes for chemical-related substances in Europe, such as envisaged under the REACH policy [1], these figures will substantially increase in near future. Although a number of non-animal carcinogenicity testing protocols are currently available [80–82], there is an urgent need for validated alternatives to the rodent bioassay. The FP6 project carcinoGENOMICS was raised in response to this ubiquitous shortage and is specifically targeted towards organ specificity (i.e. liver, kidney and lung) of genotoxic and non-genotoxic carcinogens. Its innovative character lies in the combination of optimized organotypical cell culture systems, including stem cellderived models, with both transcriptomics and metabonomics as well as with phenotypic anchoring. A key step in the first phase of carcinoGENOMICS implies the establishment of a collection of data that can serve as a platform to predict the carcinogenic potential of chemicals in lung, liver and kidney. This is achieved by including a set of chemicals that display well-characterized carcinogenic properties during the optimization of the in vitro assays. In fact, 3 classes of chemicals have been considered and defined, namely (i) genotoxic carcinogens, (ii) nongenotoxic carcinogens, and (iii) non-carcinogens. It is clear that this mechanistically-based categorization system is not exclusive, as some carcinogens (e.g. monuron) can be categorized into more than 1 class. One could also consider subdivision of the current classes of (non-)carcinogenic chemicals. In this regard, several acknowledged non-carcinogens, such as sodium chloride, caffeine and nitrofurantoin, are known to have a positive outcome in genotoxicity testing [41]. These alternative classes of chemicals will be included in the second stage of carcinoGENOMICS. For the purpose of appropriate selection of compounds for the first phase of the project, a number of theoretical criteria have been proposed, including (i) toxicological profile and gene expression profile (ii) diversity and selectivity, (iii) biochemical and biophysical properties, and (iv) legal aspects and safety measures. While initiating the search for chemicals that meet these ‘‘golden standards’’, it became immediately evident that the availability of such compounds is limited. Compounds that were ultimately selected are those that have been extensively described in scientific literature and/or that have already been well characterized in terms of carcinogenicity and genotoxicity by participating


partners. These compounds fulfilled most of the requirements postulated and covered a broad range of chemical-related substances, including industrial chemicals, biocidal products as well as pharmaceuticals. Subsequently, the final set of chemicals was submitted for critical review to a panel of external experts. Following approval, a restricted number of ‘‘priority chemicals’’ were appointed within the consensus compound set (Table 2). These prioritized chemicals are used as a learning set of compounds during the early stages of development of the in vitro carcinogenicity assays. A major challenge within the carcinoGENOMICS project now lies ahead in creating in vitro systems that can cope with the selected carcinogenic substances. Several properties inherent to these systems have to be thoroughly evaluated and optimized whenever necessary. The metabolic capacity of the in vitro models, for instance, is of the utmost importance. In vivo, a considerable number of carcinogens require biotransformation to become effective in the target organ [37]. The metabolic competence of the in vitro systems must therefore be kept at a level that reflects the in vivo situation. Strategies that will be followed to achieve this goal include the stable transfection with tissue-specific transcription factors, known to trigger biotransformation capacity [83], and the stabilization of the (functional) differentiated phenotype by using differentiation-promoting culture medium additives [84] in the case of hepatic cell lines and primary hepatocytes, respectively. The generation of hepatocyte-like cells from human embryonic stem cells will also be explored. For the lung-based in vitro models, an even greater test remains to be overcome, namely mimicking inhalation as an exposure route for carcinogens in experimental in vitro settings. In the second phase of carcinoGENOMICS, the designed in vitro toxicogenomic assays will enter the process of pre-validation, according to ECVAM’s guidelines [30]. Multi-laboratory validation is foreseen upon adding high-throughput features to the developed assays. Special attention is paid to the robustness of the assays as well as to the further evaluation of their predictive power. An additional and more tricky set of chemicals will be used for this fine-tuning of the in vitro tests, including compounds with less matched (non-)carcinogenic and (non-)genotoxic properties (e.g. nitrofurantoin) and structurally related pairs of chemicals of which only 1 isomer is carcinogenic (e.g. 2-aminoacetylfluorene and 4aminoacetylfluorene). It is expected that eventually in vitro assays will be produced that are able to forecast carcinogenic actions of yet uncharacterized compounds. In this way, carcinoGENOMICS will assist in meeting the ever increasing safety measures for chemical substances whilst reducing animal experimentation. Conflict of interest statement None. Acknowledgement The carcinoGENOMICS FP6 project is sponsored by the European Union (PL037712). References [1] Regulation (EC) No. 1907/2006 of the European Parliament and of the Council of 18 December 2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), establishing a European Chemicals Agency, amending Directive 1999/45/EC and repealing Council Regulation (EEC) No. 793/ 93 and Commission Regulation (EC) No. 1488/94 as well as Council Directive 76/ 769/EEC and Commission Directives 91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC, Official Journal L396, 30/12/2006 p.1; Corrected Official Journal L136, 29/05/2007 p.3.

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