Alternative Approaches for the Identification of Carcinogens are Closer than Usually Thought, but the Present Strategies and Regulations Need to be Updated
Published: November 29, 2011
Romualdo Benigni received his education in chemistry at the University of Rome “La Sapienza”. He then joined the Istituto Superiore di Sanità (Italian National Institute of Health) where he got a permanent position in 1977, and where he remained except for two sabbaticals, at the New York University in 1988 and at the Jawaharlal Nehru University in New Delhi in 2000. He worked experimentally in the field of molecular biology and environmental chemical mutagenesis. In the 1980’s, he turned his attention to the statistical analysis and modeling of toxicological data, and to the study of the relationships between the structure of organic compounds and their toxicological properties (mainly mutagenesis and carcinogenesis). Dr. Benigni has published over 160 journal articles and book chapters, applying a wide variety of quantitative analysis techniques, including (Q)SAR, to the examination of chemical toxicity information.
Dr. Romualdo Benigni
Istituto Superiore di Sanità
299 Viale Regina Elena
00161 – Roma (I)
Experimental long-term chemical carcinogenesis bioassays are designed and carried out to identify potential carcinogenic effects for humans. Carcinogenesis results in rodents, mainly rats and mice, have been shown to be a consistent and reliable indicator and predictor of human cancer risk (Haseman et al. 2001; Tomatis et al. 2001; Bucher 2000; Huff 1999a; Huff 1999b; Tomatis et al. 1997; Fung et al. 1993; Huff et al. 1991). All known human carcinogens that have been evaluated adequately in animal bioassays are also carcinogenic in animal bioassay studies. Of the nearly 100 recognized human carcinogens, about one-third were shown first to be carcinogenic in experimental animals (Huff 1999a; Huff 1999b). This includes both genotoxic and nongenotoxic carcinogens. In addition, a strong correlation between carcinogenic potencies estimated from epidemiological data and those estimated from animal carcinogenesis bioassays has been demonstrated (Allen et al. 1988; Goodman & Wilson, 1991). Hence, for chemicals discovered to be carcinogenic to laboratory animals, prudent public health policy suggests strongly that eliminating exposures to these agents would reduce or eliminate certain environmentally associated cancers (Tomatis et al. 1997).
The obvious negative side of the standard bioassay in rodents is that it is extremely time-consuming and costly, and requires the sacrifice of large numbers of animals. For these reasons, chemical carcinogenicity has been the target of numerous attempts to create alternative predictive models, ranging from short-term biological assays (e.g. the mutagenicity tests) to theoretical models. Among the theoretical models, the application of the science of Structure-Activity Relationships (SAR), both in its qualitative and quantitative (QSAR) form, has earned special prominence (Benigni et al. 2007; Hansch et al. 1989; Worth et al. 2007; Worth 2010).
The research for alternative methods now dates from the 1970’s, and is closely linked to the evolution of theories on the early stages of carcinogenesis. For decades, the prevailing paradigm for early-stage carcinogenesis has been the somatic mutation theory, which states that “cancer results from an accumulation of mutations and other heritable changes in susceptible cells” (Wunderlich 2007; Haber & Settleman 2007; Baker et al. 2010). The somatic mutation theory has changed over time, and multiple variations have occurred in recent years. These variations, which include epigenetic, chromosomal, and cancer stem-cell theories, differ in how the alteration occurs and in what types of cells are involved. A common feature is the underlying notion that cancer originates at the cellular level of biologic organization, and that carcinogens directly alter the DNA structure or function in cells in the tissue from which cancer arises (Baker et al. 2010).
However, several facts cannot be explained convincingly by the somatic mutation theory of cancer, thus an alternative paradigm, namely the tissue organization field theory, which proposes that cancer is a tissue-based disease, has arisen (Soto & Sonnenschein 2011; Potter 2007). Under the tissue organization field theory, cancer arises from disruption of tissue microarchitecture, driven by the collapse of the so-called morphostat gradients that maintain that integrity; mutations and genetic instability are viewed as consequence of the disruption of the morphostat gradient.
Search for Alternative Methods: Genotoxic Carcinogens and Mutagenicity Tests
The somatic mutation theory of cancer has been by and large regarded as the predominant paradigm for the induction of cancer by chemicals, and has inspired the majority of studies. Two independent lines of research have been ongoing: a) the research by the Millers pointing to the carcinogenic properties of the electrophilic chemicals, potentially able to react with the DNA; b) the research of genetic toxicologists on the ability of chemicals to induce mutation, thus being potentially able to elicit heritable genetic damage. A highly productive cross-fertilization between the two fields gave rise to: a) the theory that electrophilic chemicals (per se, or after metabolic transformation) are able to induce both mutations and cancer; and b) the generation of mutagenicity short-term tests (STT) (e.g., the Salmonella typhimurium or Ames test, incorporating metabolic activation) for identifying mutagenic/genotoxic chemicals (hence potential carcinogens) (Zeiger 2004). Another important contribution came from John Ashby, who listed the chemical reactive groups present in carcinogens (Structural Alerts [SA]) (Ashby 1985).
Prompted by the above results, subsequent major research efforts focused on the hypothesis that “Mutation = Cancer” and more than 100 STTs were developed, based on different genetic endpoints and types of cells, as to (hopefully) complement the Salmonella assay (Zeiger 2004). The use of STTs as pre-screening of carcinogenicity was accepted by the scientific community and incorporated into regulatory schemes.
These regulatory schemes and strategies vary to a large extent, depending on the types of chemicals and intended use (e.g., whether the chemicals are industrial chemicals, pharmaceutical drugs, food additives or constituents); they also vary -for the same types of chemicals- from a regulatory authority to another. However, a dominant trend can be recognized. Most often, a 2-tiered integrated testing approach is used. Tier 1 includes in vitro assays. In this tier, bacterial mutation assays (such as the Ames test) are used first, followed by tests based on in vitro mammalian cells (detecting gene mutations or chromosomal aberrations). Tier 2 involves the use of short-term in vivo studies (usually a bone-marrow cytogenetics assay) to assess whether any potential for mutagenicity detected at the Tier 1 in vitro stage is actually expressed in the whole animal. Thus, negative results in vitro are usually considered sufficient to indicate lack of mutagenicity, whereas a positive result is not considered sufficient to indicate that the chemical represents a mutagenic hazard (i.e. it could be a misleading positive). The above approach to mutagenicity testing has a fundamental theoretical unity, and has been recommended internationally as part of the strategy for predicting and quantifying mutagenic and carcinogenic hazard.
Present Pre-screening Strategies are not Efficient in Identifying Genotoxic Carcinogens
Even though already noticed, recently pitfalls of the present testing strategies have come to the limelight again. Today it appears that the original hypothesis “mutation = cancer” is only valid within the limited area of the DNA-reactive chemicals: these induce cancer, together with a wide spectrum of mutations. For these chemicals, the best predictor of carcinogenicity is the Ames test. For the chemicals that are negative in Salmonella, but positive in other in vitro assays (e.g., clastogenicity) no correlation with, and predictive ability for carcinogenicity is apparent. In practice, the other in vitro assays generate an exaggerate rate of false positive results: thus, no efficient in vitro, mutagenicity-based STTs complementary to Salmonella for predicting carcinogenicity are available today (Zeiger 1998; Benigni et al. 2010b). The other working hypothesis was that in vitro positives should be confirmed through an in vivo genotoxicity assay; however it is demonstrated that existing in vivo tests are extremely insensitive and give a majority of false negative results for many clearly genotoxic carcinogens (Kirkland & Speit 2008; Benigni et al. 2011). It should be emphasized as well that overall, because of their intensive use worldwide, the in vivo mutagenicity tests involve the sacrifice of a much larger number of animals than the rodent bioassay itself (Pedersen et al. 2003; Van der Jagt et al. 2004).
In summary, the identification of genotoxic carcinogens has one strong arm, represented by the Ames test that detects efficiently the DNA-reactive chemicals: a chemical positive in this test has a very high probability (around 80%) of being also a carcinogen. It should be added that the SAs for DNA-reactive chemicals have a high predictivity for the Ames test and for the DNA-reactive carcinogens; thus, this fast and inexpensive computerized approach can replace the Salmonella assay in many instances (Benigni et al. 2010b). Unfortunately, the other in vitro STTs (e.g., chromosomal aberrations) and especially the in vivo ones do not have added value and rather impair the prediction ability of Salmonella alone.
In light of the evident failure of the above paradigm, different solutions have been proposed. One approach is based on the continued acceptance of the paradigm on the complementarity of the STTs in terms of genetic endpoint and phylogenetic position of the assay systems. Operational improvements are sought by trying to manipulate the assay systems to, for example, reduce the sensitivity of in vitro complements to Salmonella and to improve the in vivo systems (Kirkland et al. 2007; Kirkland et al. 2008). Other approaches explore completely new tools, such as tracing molecular perturbations related to specific biochemical pathways with the use of various omics technologies (in vitro high throughput assay systems). It appears that the latter approach is still in its infancy, and needs much work and refinement (Benigni et al. 2010a). Overall, both approaches have to be validated and are not yet available for improving the present day strategies.
Identification of Nongenotoxic Carcinogens: An (Almost) Forgotten Issue
The identification of nongenotoxic carcinogens is the other weak point of the present testing strategies adopted within regulatory schemes. Traditionally, nongenotoxic carcinogens have been detected in rodent 2-year cancer bioassays (Huff 1999a, 1999b), but the new regulatory policies (e.g., the EU REACH legislation) tend to drastically reduce the number of new cancer bioassays. The test strategy for carcinogenicity pre-screening of REACH is based on the pivotal role of genotoxic endpoints: the bioassay may be required by the Authorities in the case of high exposure plus genotoxicity evidence. However, nongenotoxic carcinogens are negative in all genotoxicity tests, and thus go undetected. Justification for the development of alternative methods for the detection of nongenotoxic carcinogens include their remarkable presence among the known human carcinogens (up to 25% in Class 1 human carcinogens as classified by the International Agency for Research on Cancer [IARC]), and the considerable potential risk associated with them (Hernandez et al. 2009). In addition, it should be emphasized that the proportion of nongenotoxic to genotoxic carcinogens in the environment is likely bound to increase in the near future, since the knowledge on DNA-reactivity is now widespread and solid enough as to permit to the industrial chemists to design new chemicals without overtly reactive moieties. The same does not hold for nongenotoxic carcinogens.
Is It Possible to Overcome the Pitfalls of the Present Strategies and, at the Same Time, to Reduce the Use of the Rodent Bioassay?
After several decades of investigations the need for tools able to predict chemical carcinogens in shorter times, and at a lower cost in terms of animal lives and money is still a research priority. In summary, the open issues are: a) the lack of alternative assays able to identify nongenotoxic carcinogens; b) the exaggerated rate of misleading (“false”) positive results of the in vitro mammalian cells-based mutagenicity short-tem tests; c) the extremely low sensitivity of in vivo mutagenicity short-term tests (which overall, because of their intensive use worldwide, employ more animals than the rodent bioassay). As a consequence, the present strategies for identifying carcinogens without the use of the rodent bioassay are not an efficient filter for protecting human health. The question is: are there alternative tools available today to deal with the above issues? Since the Ames test is an efficient tool to detect DNA-reactive carcinogens, the question can re-phrased as follows: are there non-mutagenicity in vitro assays for non-DNA-reactive carcinogens?
Recently, the contribution of the Cell Transformation Assays (CTA) has been re-explored. CTAs mimic some stages of in vivo multistep carcinogenesis. Cell transformation has been defined as the induction of certain phenotypic alterations in cultured cells that are characteristic of tumorigenic cells (Barrett & Ts’o 1978). These phenotypic alterations can be induced by exposing mammalian cells to carcinogens. Transformed cells that have acquired the characteristics of malignant cells have the ability to induce tumors in susceptible animals (Berwald & Sachs 1963, 1965). The CTAs have been proposed for assessing carcinogenic potential of chemicals for many years; however they have undergone different cycles of favor and disfavor among the scientific community, and have never been consistently included in regulatory testing schemes. Recently, the Organization for the Economic Cooperation and Development (OECD) has reconsidered the CTAs and published a report that includes both experimental results and data analyses (OECD 2007), while the European Centre for the Validation of Alternative Methods (ECVAM) has performed a pre-validation of CTAs (Corvi et al. 2008).
Among the different CTAS, it appears that the Syrian Hamster Embryo cells transformation assay (with pH=7 protocol) (SHE_7) performs best in predicting rodent carcinogenicity, with high sensitivity and specificity. It is quite remarkable that SHE_7 has a high performance in respect to both the DNA-reactive and non-DNA-reactive/nongenotoxic carcinogens. Based on this, the following tiered approach to the prediction of carcinogens was tested: Ames or SAs in Tier 1, and SHE_7 applied in Tier 2 to the chemicals negative in Tier 1. The result was quite exciting, and pointed to the tiered approach as a powerful screening/priority setting approach: only 5-10 % of carcinogens remained undetected (Benigni & Bossa 2011a). It should be noticed that the tiered approach reduces the use of the more time- and skill-requiring tests (SHE_7, Tier 2) only to the chemicals that are negative in the more economical and quick approaches of Tier 1 (SAs or Salmonella).
A Mechanistic Consideration on Alternatives to the Bioassay
It is interesting to comment on the mechanistic relevance of the two in vitro assays whose combination appears to represent an optimal strategy for the detection of carcinogens. The cell transformation assay SHE_7 detects phenotypic alterations which are characteristic of tumorigenic cells. It should be emphasized that in vitro cell transformation can be produced by a plethora of different molecular mechanisms that do not include the induction of mutations (Bignami et al. 1984). Among other mechanisms, these transformation assays are models of cell-cell and cell-stroma communication phenomena typical of cancer. Cancer cannot be seen as a ‘single cell’ condition, but is linked to modifications of the relations among cells in tissues (Kalluri & Zeisberg 2006). This interpretation is favored by the growing evidence on tumor microenvironment (Whiteside 2008) and cell adhesion mechanisms (Alcaraz et al. 2008), and is exemplified by the tissue organization field theory (Soto & Sonnenschein 2011; Potter 2007). On the other hand, the Ames test is sensitive to a very large family of carcinogens that are able to interact with DNA according to various molecular mechanisms (e.g., direct or indirect alkylation, acylation, intercalation, formation of aminoaryl DNA-adducts), and is the typical example of the somatic mutation theory of cancer.
The successful combination of two assays that exemplify theories often presented as antagonistic may indicate that the distinction between genotoxic and nongenotoxic carcinogens is not so sharp, and that the theories on the early stages of carcinogenesis are not mutually exclusive; on the contrary, different pathways in the carcinogenesis process may co-exist and should be taken into account in testing strategies.
The contribution of SAR concepts in the identification and coding of the action mechanisms, and in the development of alternative strategies should be remarked as well. In this commentary, the use of SAs for the rapid and inexpensive identification of DNA-reactive carcinogens has been presented. The availability of free, user-friendly implementations, like e.g., the expert system Toxtree (Worth 2010; Benigni & Bossa 2011b), allows every scientist to easily apply the SAs to the query chemicals of interest (free download: http://ihcp.jrc.ec.europa.eu/our_labs/computational_toxicology/qsar_tools/toxtree). It should be emphasized as well that the SAR concepts have a much wider application, and are also at the basis of the Read-Across and Regulatory Category approaches aimed at filling gaps in experimental data by similarity with other, already tested chemicals (Van Leeuwen et al. 2009; Worth 2010).
The good news is that the goal of identifying carcinogens with alternative methods is closer than usually thought. The Ames test and the SAs for the DNA-reactive carcinogens, and the CTAs for the nongenotoxic carcinogens permit -in combination- the identification of a very large proportion of carcinogens. So, they constitute a solid ground for refinements by future research. However, the present, commonly accepted strategies for the pre-screening of carcinogenicity are quite inefficient, do not adequately protect public health and so need to be updated urgently.
©2011 Romualdo Benigni
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