Gene manipulation and genetic toxicology

Transgenic mice with recoverable target genes, with genes modifiedin expression or with ablated gene function provide importanttools for defining the biological effects of chemicals. In manycases studies can be conducted with fewer animals and in a shortertimeframe. They also provide important tools for studies ofcarcinogenesis mechanisms and processes, for early detectionof carcinogens and for identification of potential cancer therapies.     

Evaluating the genetic toxicology of DNA-based products using existing genetic toxicology assays

Unlike the development of drugs based on small chemical entities there are no conventional regulatory toxicity studies established for DNA-based products. As the potential for insertional mutagenesis is of particular concern for gene therapy, we have investigated the mutagenicity of model non-viral DNA-based products at the HPRT locus in Chinese hamster V79 cells. Cultures were transfected with a pL3112BSKS plasmid in combination with a number of non-viral transfection facilitators: Effectene, Lipofectamine 2000 and ExGen 500. The plasmid contains a green fluorescent protein gene, which was used as a reporter of transfection efficiency. Flow cytometry was used to analyse large numbers of cells. Small scale transient transfection efficiencies (7-90%) were obtained at low cytotoxicity, however, scaling up the process led to decreased transfection and increased cytotoxicity. Stable transfection (chromosomal) was observed, but only at very low levels (<1.5%). Two of the non-viral delivery facilitators (Effectene and ExGen 500) themselves induced mutation at the HPRT locus, although they were considerably less potent than the positive control ethyl methanesulphonate. In transfection experiments, neither of the non-viral delivery facilitators (Effectene and Lipofectamine 2000) had a mutagenic effect, whereas with ExGen 500 there was evidence of a mutagenic effect, consistent with the mutagenicity observed with the non-viral transfection facilitator alone. Moreover, treatment with the plasmid pL3112BSKS itself was able to induce a 3- to 7-fold increase in the mutation frequency at the HPRT locus. Our studies highlight some of the problems associated with using exisiting genetic toxicology testing procedures for the assessment of DNA-based products used in novel gene therapy approaches. However, given the limited level of sophistication of the current approach, the data suggest that non-viral gene therapy may present a detectable mutation risk and more appropriate testing strategies are needed to evaluate the nature of the risk.     

The genetic toxicology of some hydrocarban and oxygenated solvents

Two hydrocarbon solvents (heptane and Special Boiling PointSpirit 100/140) and eight oxygenated solvents [methyl ethylketone, methyl isobutyl ketone, diacetone alcohol, di-isobutylketone, isopropyl ether, hexylene glycol, secondary butyl alcoholand ME 6K (pentoxone)] have been tested for geno-toxic activity.The solvents were tested in bacterial mutation assays, a yeastassay for mitotic gene conversion and in cultured mammaliancells (either rat liver or Chinese hamster ovary) for structuralchromosome damage. All of the solvents gave a negative responsein the bacterial mutation assays and the yeast mitotic geneconversion assay. In the rat liver chromosome assay, diacetonealcohol evoked a weak positive response, the remaining solventsgave a negative response.     

Use of genetic toxicology information for risk assessment

Genetic toxicology data are used worldwide in regulatory decision-making. On the 25th anniversary of Environmental and Molecular Mutagenesis, we think it is important to provide a brief overview of the currently available genetic toxicity tests and to outline a framework for conducting weight-of-the-evidence (WOE) evaluations that optimize the utility of genetic toxicology information for risk assessment. There are two major types of regulatory decisions made by agencies such as the Environmental Protection Agency (EPA) and the Food and Drug Administration (FDA): (1) the approval and registration of pesticides, pharmaceuticals, medical devices, and medical-use products, and (2) the setting of standards for acceptable exposure levels in air, water, and food. Genetic toxicology data are utilized for both of these regulatory decisions. The current default assumption for regulatory decisions is that chemicals that are shown to be genotoxic in standard tests are, in fact, capable of causing mutations in humans (in somatic and/or germ cells) and that they contribute to adverse health outcomes via a "genotoxic/mutagenic" mode of action (MOA). The new EPA Guidelines for Carcinogen Risk Assessment [Guidelines for Carcinogen Risk Assessment, USEPA, 2005, EPA Publication No. EPA/630/P-03/001F] emphasize the use of MOA information in risk assessment and provide a framework to help identify a possible mutagenic and/or nonmutagenic MOA for potential adverse effects. An analysis of the available genetic toxicity data is now, more than ever, a key component to consider in the derivation of an MOA for characterizing observed adverse health outcomes such as cancer. We provide our perspective and a two-step strategy for evaluating genotoxicity data for optimal use in regulatory decision-making. The strategy includes integration of all available information and provides, first, for a WOE analysis as to whether a chemical is a mutagen, and second, whether an adverse health outcome is mediated via a mutagenic MOA.     

Application of toxicogenomics to genetic toxicology risk assessment

Based on the assumption that compounds having similar toxic modes of action induce specific gene expression changes, the toxicity of unknown compounds can be predicted after comparison of their molecular fingerprints with those obtained with compounds of known toxicity. These predictive models will therefore rely on the characterization of marker genes. Toxicogenomics (TGX) also provides mechanistic insight into the mode of toxicity, and can therefore be used as an adjunct to the standard battery of genotoxicity tests. Promising results, highlighting the ability of TGX to differentiate genotoxic from non-genotoxic carcinogens, as well as DNA-reactive from non-DNA reactive genotoxins, have been reported. Additional data suggested the possibility of ranking genotoxins according to the nature of their interactions with DNA. This new approach could contribute to the improvement of risk assessment. TGX could be applied as a follow-up testing strategy in case of positive in vitro genotoxicity findings, and could contribute to improve our ability to identify the molecular mechanism of action and to possibly better assess dose-response curves. TGX has been found to be less sensitive than the standard genotoxicity end-points, probably because it measures the whole cell population response, when compared with standard tests designed to detect rare events in a small number of cells. Further validation will be needed (1) to better link the profiles obtained with TGX to the established genotoxicity end-points, (2) to improve the gene annotation tools, and (3) to standardise study design and data analysis and to better evaluate the impact of variability between platforms and bioinformatics approaches.     

Theory and practice of genetic toxicology tests. Tests on eukaryotes

The search for a mutation prone activity on bacteria must be completed by tests on eucaryotes, in vitro as well as in vivo. Tests on eucaryotes enable to study agents inducing gene mutations [on yeats, cultures of mammal cells (V 79, L 5278 Y), lethal recessive mutation linked to the sex chromosome, on drosophils...], chromosomal mutations (metaphases analysis in vitro and in vivo, micronucleus, lethal dominance), the involvement of repair mechanisms of DNA (non-programmed DNA synthesis, exchange of sister chromatides...). One must well differentiate the tests usable routinely for a screening from the tests currently validated and routine tests. It is only at the completion of a set of tests on bacteria and eucaryotes cells that the potential mutation risk of a product for human health, may be ascertained.     

Predictive genetic testing: mediators and moderators of anxiety

Mediators and moderators of anxiety following predictive genetic testing were investigated in a cross-sectional study of 208 individuals at risk for familial adenomatous polyposis (FAP). Receiving a positive test result was associated with increased anxiety. The relationship between test result and anxiety was mediated by how threatened individuals felt by their test results. The impact of a positive test result was greater for those who felt distressed about FAP in their families, perceived FAP to be more serious, and perceived the genetic test to be more accurate. The results suggest that assessing, and possibly modifying, people’s appraisals of the condition and of its impact on the family and of the threat of the genetic test may help to reduce subsequent anxiety. This has implications for the practice of genetic counseling.     

Predictive genetic testing and beyond: a theory of engagement

This article presents a tentative grounded theory, which can provide some explanation of variation in behaviour around predictive genetic testing (PGT) for Hereditary Non-Polyposis Colorectal Cancer (HNPCC), based on interviews with individuals (n = 55) from families with a clinical diagnosis of HNPCC, 12 of whom were followed through the PGT protocol. The theory is built around a core category of engagement, a newly constructed concept reflecting the degree of cognitive and emotional involvement with cancer risk in individuals from these families, and models the psychosocial process of engaging with cancer risk. The degree of engagement at the time of testing can explain variations in approaches and reactions to PGT. A series of social factors, many related to the experiences of family life, emerged as either facilitating or blocking the process of engaging with cancer risk; a series of psychological factors emerged as interacting in a recursive, dynamic manner with each other and with engagement status. The degree of engagement can change with the unfolding of time and events in family life. The theory of engagement (TE) provides an explanatory framework for understanding behaviour related to PGT for HNPCC, and can potentially be applied to research on risk perception in the social sciences more generally. In addition, the theory may have potential uses in the genetics clinic, in identifying individuals at risk of adverse reactions to PGT for cancer, thus enabling better targeting of genetic counselling resources.     

Gastric cancer: genetic and molecular abnormalities

In this work, the genetic and molecular alterations involved in gastric carcinogenesis are shown; the affected genes are those that encoded for matrix metalloproteinases, cell growth factors and receptors, oncogenes, tumor suppressors, DNA repair and cell adhesion molecule genes, cell cycle regulators and others related to metastasis, as well as lost of heterozygosity in chromosomal regions.     

Heritability and molecular genetic studies of endometriosis

Endometriosis is a common disease defined as the growth of endometrial tissue outside the uterine cavity that often results in a vast array of gynaecological problems including dyspareunia, dysmenorrhoea, pelvic pain and infertility. Despite the increasing evidence that supports a genetic component to this common gynaecological condition, the basic aetiology and pathogenesis of endometriosis remain unknown. It is likely that endometriosis is a common polygenic/multifactorial disease caused by an interaction between multiple genes as well as the environment. Such conditions do not have a clear Mendelian pattern of inheritance. Recent molecular cytogenetic studies on endometriotic tissue and an established endometriosis-derived cell line provide novel evidence that acquired chromosome-specific alterations may be involved in endometriosis, possibly reflecting clonal expansion of chromosomally abnormal cells. Molecular DNA studies examining the role of loss of heterozygosity in endometriotic lesions has identified candidate tumour suppressor gene loci, including 5q, 6q, 9p, 11q and 22q, that may play a role in the malignant transformation of endometriotic implants to endometrioid ovarian cancers. Evidence of mutations in the tumour suppressor PTEN gene in the endometrioid subtype of epithelial ovarian cancer further suggests that somatic genetic alterations represent early events in the transformation of benign endometriotic cells. Genetic factors are also likely to influence individual susceptibility to endometriosis. There is now evidence that heritable allelic differences in drug-metabolizing enzymes play an important role in the development of endometriosis. Further studies are warranted to identify major susceptibility gene(s) and the mechanism involved in endometriosis to assist in the development of better methods for early detection, diagnosis and prevention.     

Use of transgenic mouse lacl/Z mutation assays in genetic toxicology

The Big Blue^TM (lacl) and the Muta^TMMouse (lacZ/GalE) assaysoffer a practical means to generate mutation data from a widerange of tissues of treated animals. Our experience with theseassays has so far been encouraging, but several fundamentalquestions require addressing. Chief among these is whether theseassays are to be regarded as replacements for standard rodentgenotoxicity assays, or if they are to be regarded as assaysfor the prediction of potential carcinogenic organotropy usinga multiple-dose protocol. The interplay between the independentchemical properties of mitogenesis and DNA adduction requirealso to be studied. The value and sensitivity of these assayscan only be assessed when these questions have been internationallyaddressed and concluded.     

Comparative overview of current international strategies and guidelines for genetic toxicology testing for regulatory purposes

National and international regulatory agencies historically have used genotoxicity information as part of a weight-of-evidence approach to evaluate potential human carcinogenicity. Additionally, some agencies consider heritable mutation a regulatory endpoint. Furthermore, genotoxicity has the potential to contribute to other adverse health conditions. This article provides a comparative overview of the testing strategies used by regulatory agencies throughout the world. Despite minor variations in details, the genotoxicity test schemes for most regulatory entities generally comprise three tests: a bacterial gene mutation assay, an in vitro mammalian cell assay for gene mutation and/or chromosome aberrations, and often an in vivo assay for chromosomal effects. In some cases, fewer than these three tests are required. In other cases, when exposure data, structure-activity considerations, or other factors warrant, even chemicals negative in the three baseline tests may be subject to additional testing. If genotoxicity is identified by the baseline screening tests, assessment of the ability of the chemical to interact with DNA in the gonad may be required. This may apply regardless of whether or not a cancer bioassay has been triggered. Mutagens positive in second stage gonadal assay(s) may be tested in third stage in vivo rodent tests to provide data for a quantitative risk assessment. In all testing, theutilization of internationally-recognized protocols, where they exist, is advisable, although not in all instances required. When testing for regulatory purposes, it is advisable to verify the testing program with the specific regulatory body or bodies responsible forregulatory oversight before beginning testing.     

3Rs-friendly approach to exogenous metabolic activation that supports high-throughput genetic toxicology testing

MultiFlow® DNA Damage—p53, γH2AX, Phospho-Histone H3 is a miniaturized, flow cytometry-based assay that provides genotoxic mode of action information by distinguishing clastogens, aneugens, and nongenotoxicants. Work to date has focused on the p53-competent human cell line TK6. While mammalian cell genotoxicity assays typically supply exogenous metabolic activation in the form of concentrated rat liver S9, this is a less-than-ideal approach for several reasons, including 3Rs considerations. Here, we describe our experiences with low concentration S9 and saturating co-factors which were allowed to remain in contact with cells and test chemicals for 24 continuous hours. We exposed TK6 cells in 96-well plates to each of 15 reference chemicals over a range of concentrations, both in the presence and absence of 0.25% v/v phenobarbital/β-naphthoflavone-induced rat liver S9. After 4 and 24 hr of treatment cell aliquots were added to wells of a microtiter plate containing the working detergent/stain/antibody cocktail. After a brief incubation robotic sampling was employed for walk-away flow cytometric data acquisition. PROAST benchmark dose (BMD) modeling was used to characterize the resulting dose–response curves. For each of the 8 reference pro-genotoxicants studied, relative nuclei count, γH2AX, and/or p53 biomarker BMD values were order(s) of magnitude lower for 0.25% S9 conditions compared to 0% S9. Conversely, several of the direct-acting reference chemicals exhibited appreciably lower cytotoxicity and/or genotoxicity BMD values in the presence of S9 (eg, resorcinol). These results prove the efficacy of the low concentration S9 system, and indicate that an efficient and highly scalable multiplexed assay can effectively identify chemicals that require bioactivation to exert their genotoxic effects.