Cecilia Bossa

Structure alerts for carcinogenicity, and the Salmonella assay system: a novel insight through the chemical relational databases technology.

In the past decades, chemical carcinogenicity has been the object of mechanistic studies that have been translated into valuable experimental (e.g., the Salmonella assays system) and theoretical (e.g., compilations of structure alerts for chemical carcinogenicity) models. These findings remain the basis of the science and regulation of mutagens and carcinogens. Recent advances in the organization and treatment of large databases consisting of both biological and chemical information nowadays allows for a much easier and more refined view of data. This paper reviews recent analyses on the predictive performance of various lists of structure alerts, including a new compilation of alerts that combines previous work in an optimized form for computer implementation. The revised compilation is part of the Toxtree 1.50 software (freely available from the European Chemicals Bureau website). The use of structural alerts for the chemical biological profiling of a large database of Salmonella mutagenicity results is also reported. Together with being a repository of the science on the chemical biological interactions at the basis of chemical carcinogenicity, the SAs have a crucial role in practical applications for risk assessment, for: (a) description of sets of chemicals; (b) preliminary hazard characterization; (c) formation of categories for e.g., regulatory purposes; (d) generation of subsets of congeneric chemicals to be analyzed subsequently with QSAR methods; (e) priority setting. An important aspect of SAs as predictive toxicity tools is that they derive directly from mechanistic knowledge. The crucial role of mechanistic knowledge in the process of applying (Q)SAR considerations to risk assessment should be strongly emphasized. Mechanistic knowledge provides a ground for interaction and dialogue between model developers, toxicologists and regulators, and permits the integration of the (Q)SAR results into a wider regulatory framework, where different types of evidence and data concur or complement each other as a basis for making decisions and taking actions.

Predictivity of QSAR.

A range of good quality, local QSARs for mutagenicity and carcinogenicity have been assessed and challenged for their predictivity in respect to real external test sets (i.e., chemicals never considered by the authors while developing their models). The QSARs for potency (applicable only to toxic chemicals) generated predictions 30-70% correct, whereas the QSARs for discriminating between active and inactive chemicals were 70-100% correct in their external predictions: thus the latter can be used with good reliability for applicative purposes. On the other hand internal, statistical validation methods, which are often assumed to be good diagnostics for predictivity, did not correlate well with the predictivity of the QSARs when challenged in external prediction tests. Nonlocal models for noncongeneric chemicals were considered as well, pointing to the critical role of an adequate definition of the applicability domain.

Alternatives to the carcinogenicity bioassay: in silico methods, and the in vitro and in vivo mutagenicity assays

Importance of the field: Carcinogenicity and mutagenicity are toxicological end points posing considerable concern for human health. Due to the cost in animal lives, time and money, alternative approaches to the rodent bioassay were designed based on: i) identification of mutations and ii) structure–activity relationships. Areas covered in this review: Evidence on i) and ii) is summarized, covering 4 decades (1971 – 2010). What the reader will gain: A comprehensive, state-of-the-art perspective on alternatives to the carcinogenicity bioassay. Take home message: Research to develop mutagenicity-based tests to predict carcinogenicity has generated useful results only for a limited area of the chemical space, that is, for the DNA-reactive chemicals (able to induce cancer, together with a wide spectrum of mutations). The most predictive mutagenicity-based assay is the Ames test. For non-DNA-reactive chemicals, that are Ames-negative and mutagenic in other in vitro assays (e.g., clastogenicity), no correlation with carcinogenicity is apparent. The knowledge on DNA reactivity permits the identification of genotoxic carcinogens with the same efficiency of the Ames test. Thus, a chemical mutagenic in Salmonella and/or with structural alerts should be seriously considered as a potential carcinogen. No reliable mutagenicity-based alternative tools are available to assess the risk of non-DNA-reactive chemicals.

Structural analysis and predictive value of the rodent in vivo micronucleus assay results

In vivo genotoxicity studies-shortly followed by carcinogenicity-are posing high demand for test-related recourses in terms of animal lives and resources. Among those, the micronucleus test in rodents is the most widely used as a follow-up to positive in vitro mutagenicity results; therefore, the development and extensive use of estimation techniques based on the concept of Structure-Activity Relationships-such as (Quantitative) Structure-Activity Relationships, read-across and grouping of chemicals-might have a huge saving potential for this end point. In this paper, we present a newly derived compilation of Structural Alerts for the rodent in vivo micronucleus assay, thus providing a coarse-grain filter for preliminary screening of potentially in vivo mutagens. The compilation has been implemented as computerized rule of the expert system Toxtree and is freely available: http://ecb.jrc.ec.europa.eu/qsar/qsar-tools/index.php?c=TOXTREE. In addition, analyses on the performance of the micronucleus assay as pre-screening tool for carcinogenesis indicate that this assay is prone to give false-negative predictions and point to the need of improving the in vivo component of the present testing schemes.

Predictivity and Reliability of QSAR Models: The Case of Mutagens and Carcinogens

ABSTRACT This paper presents the results from an evaluation of the noncommercial, (quantitative) structure-activity relationship ([Q]SAR) models for the prediction of mutagenicity and carcinogenicity, carried out in a collaboration between the European Chemicals Bureau Group on Computational Toxicology and the Italian Istituto Superiore di Sanita’. Local QSAR models for congeneric chemical classes and structure alert (SA) models were investigated. The studied models can be interpreted mechanistically, agree with, and/or support the available scientific knowledge, and exhibit good statistics. These models were subjected to external prediction tests with chemicals not considered by the authors of the models. Local QSARs that estimated the potency of congeneric chemicals were 30% to 70% correct, whereas the models that discriminated active and inactive chemicals had considerably higher accuracy (70% to 100%). In addition, the commonly used statistical internal cross-validation procedures were poorly correlated with external validation statistics. The genotoxic-based SA models had an accuracy of about 65% for rodent carcinogens, and about 75% for Salmonella mutagens. However, the SA models did not discriminate well active and inactive chemicals within individual chemical classes, and are more suited for preliminary or large-scale screenings. Overall, the (Q)SAR-based predictions are able to significantly enrich the target of safer chemicals, contribute to the organization and rationalization of data, elucidate mechanisms of action, and complement data from other sources.

Alternative strategies for carcinogenicity assessment: an efficient and simplified approach based on in vitro mutagenicity and cell transformation assays

The need for tools able to predict chemical carcinogens in less time and at a lower cost in terms of animal lives and money is still a research priority, even after several decades of effort in that direction. Now, new regulatory requirements (e.g. the Registration, Evaluation, Authorisation and Restriction of Chemical substances recently implemented in Europe) have even increased the pressure to develop new tools in this field. Drawbacks of the present testing strategies have come to light again recently especially in view of new requirements in worldwide regulations. Among these are (i) the lack of assays able to identify non-genotoxic carcinogens, (ii) the exaggerated rate of misleading (false) positive results of the in vitro mammalian cell-based short-term mutagenicity tests and (iii) the extremely low sensitivity of in vivo short-term mutagenicity tests. Within this perspective, we analyse the contribution of cell transformation assays (CTAs), and we show that they are a valid complement to tools able to detect DNA-reactive carcinogens. We also show that a tiered strategy, with inexpensive and fast tests in Tier 1 (e.g. the Ames test or structural alerts) and the Syrian hamster embryo CTA in Tier 2, is able to identify up to 90% of carcinogens.

Functional analysis of MUTYH mutated proteins associated with familial adenomatous polyposis

The MUTYH DNA glycosylase specifically removes adenine misincorporated by replicative polymerases opposite the oxidized purine 8-oxo-7,8-dihydroguanine (8-oxoG). A defective protein activity results in the accumulation of G>T transversions because of unrepaired 8-oxoG:A mismatches. In humans, MUTYH germline mutations are associated with a recessive form of familial adenomatous polyposis and colorectal cancer predisposition (MUTYH-associated polyposis, MAP). Here we studied the repair capacity of the MUTYH variants R171W, E466del, 137insIW, Y165C and G382D, identified in MAP patients. Following expression and purification of human proteins from a bacterial system, we investigated MUTYH incision capacity on an 8-oxoG:A substrate by standard glycosylase assays. For the first time, we employed the surface plasmon resonance (SPR) technology for real-time recording of the association/dissociation of wild-type and MUTYH variants from an 8-oxoG:A DNA substrate. When compared to the wild-type protein, R171W, E466del and Y165C variants showed a severe reduction in the binding affinity towards the substrate, while 137insIW and G382D mutants manifested only a slight decrease mainly due to a slower rate of association. This reduced binding was always associated with impairment of glycosylase activity, with adenine removal being totally abrogated in R171W, E466del and Y165C and only partially reduced in 137insIW and G382D. Our findings demonstrate that SPR analysis is suitable to identify defective enzymatic behaviour even when mutant proteins display minor alterations in substrate recognition.

In vitro cell transformation assays for an integrated, alternative assessment of carcinogenicity: a data-based analysis

The study of the chemical carcinogenesis mechanisms and the design of efficient prevention strategies and measures are of crucial importance to protect human health. The long-term carcinogenesis bioassays have played a central role in protecting human health, but for ethical and practical reasons their use is dramatically diminishing, and the genotoxicity short-term tests have taken the pivotal role in the pre-screening of carcinogenicity. However, there is evidence that this strategy is not sensitive enough to detect all genotoxic carcinogens and it cannot detect nongenotoxic carcinogens. In a previous article, we have shown that an integrated strategy consisting of the in vitro Ames and Syrian Hamster Embryo cells transformation assays, combined with structure-activity relationships, is a valid alternative to the present pre-screening strategies. Here, we expand the previous investigation by (i) including results of cell transformation assays on inorganics, together with an additional assay (Bhas 42), and (ii) considering new structural alerts for nongenotoxic carcinogenicity. We also present a new analysis on global relationships between toxicological endpoints. The new results confirm that the previously proposed integrated, alternative strategy is an efficient tool to identify both genotoxic and nongenotoxic carcinogens, with an estimated 90-95% sensitivity.

DNA Damage and Repair in Human Cancer: Molecular Mechanisms and Contribution to Therapy-Related Leukemias

Most antitumour therapies damage tumour cell DNA either directly or indirectly. Without repair, damage can result in genetic instability and eventually cancer. The strong association between the lack of DNA damage repair, mutations and cancer is dramatically demonstrated by a number of cancer-prone human syndromes, such as xeroderma pigmentosum, ataxia-telangiectasia and Fanconi anemia. Notably, DNA damage responses, and particularly DNA repair, influence the outcome of therapy. Because DNA repair normally excises lethal DNA lesions, it is intuitive that efficient repair will contribute to intrinsic drug resistance. Unexpectedly, a paradoxical relationship between DNA mismatch repair and drug sensitivity has been revealed by model studies in cell lines. This suggests that connections between DNA repair mechanism efficiency and tumour therapy might be more complex. Here, we review the evidence for the contribution of carcinogenic properties of several drugs as well as of alterations in specific mechanisms involved in drug-induced DNA damage response and repair in the pathogenesis of therapy-related cancers.

The new ISSMIC database on in vivo micronucleus and its role in assessing genotoxicity testing strategies

This paper presents a new curated database on in vivo micronucleus mutagenicity results, called ISSMIC. It is freely available at: http://www.iss.it/ampp/dati/cont.php?id=233&lang=1&tipo=7. The experimental results were critically reviewed, and evidence on target cell exposure was considered as well. The inspection of ISSMIC demonstrates that a large proportion of reported negative results in the literature (231 out 566 ISSMIC chemicals) lack a clear-cut, direct demonstration of toxicity at the target cells. Using this updated database, the predictive value of a compilation of Structural Alerts (SA) for in vivo micronucleus recently implemented in the expert system Toxtree was investigated. Individually, most of the SA showed a high Positive Predictivity (∼80%), but the need for further expanding the list of alerts was pointed out as well. The role of in vivo micronucleus in strategies for carcinogenicity prediction was re-evaluated. In agreement with previous analyses, the data point to a low overall correlation with carcinogenicity. In addition, given the cost in animal lives and the time required for the experimentation, in many programs, the in vivo tests are used only to assess in vitro positive results. The ability of in vivo micronucleus to identify real positives (i.e. carcinogens) among chemicals positive in Salmonella or among chemicals inducing in vitro chromosomal aberrations was studied. It appears that the in vivo micronucleus test does not have added value and rather impairs the prediction ability of the in vitro tests alone. The overall evidence indicates that in vivo micronucleus--in its present form--cannot be considered an useful tool for routine genotoxicity testing but should be used in targeted mechanistic studies.