Marilyn J. Aardema

Toxicology and Genetic Toxicology in the New Era of “Toxicogenomics”: Impact of “-omics” Technologies

The unprecedented advances in molecular biology during the last two decades have resulted in a dramatic increase in knowledge about gene structure and function, an immense database of genetic sequence information, and an impressive set of efficient new technologies for monitoring genetic sequences, genetic variation, and global functional gene expression. These advances have led to a new sub-discipline of toxicology: “toxicogenomics”. We define toxicogenomics as “the study of the relationship between the structure and activity of the genome (the cellular complement of genes) and the adverse biological effects of exogenous agents.” This broad definition encompasses most of the variations in the current usage of this term, and in its broadest sense includes studies of the cellular products controlled by the genome (messenger RNAs, proteins, metabolites, etc.). The new “global” methods of measuring families of cellular molecules, such as RNA, proteins, and intermediary metabolites have been termed “-omic” technologies, based on their ability to characterize all, or most, members of a family of molecules in a single analysis. With these new tools, we can now obtain complete assessments of the functional activity of biochemical pathways, and of the structural genetic (sequence) differences among individuals and species, that were previously unattainable. These powerful new methods of high-throughput and multi-endpoint analysis, include gene expression arrays that will soon permit the simultaneous measurement of the expression of all human genes on a single “chip”. Likewise, there are powerful new methods for protein analysis (proteomics: the study of the complement of proteins in the cell) and for analysis of cellular small molecules (metabonomics: the study of the cellular This article has been reproduced from Mutation Research, Vol 499, 2002, pp 13–25, Aardema & MacGregor, by the permission of Elsevier Science, Ltd. metabolites formed and degraded under genetic control). This will likely be extended in the near future to other important classes of biomolecules such as lipids, carbohydrates, etc. These assays provide a general capability for global assessment of many classes of cellular molecules, providing new approaches to assessing functional cellular alterations. These new methods have already facilitated significant advances in our understanding of the molecular responses to cell and tissue damage, and of perturbations in functional cellular systems.

Report from the In Vitro Micronucleus Assay Working Group.

At the Washington International Workshop on Genotoxicity Test Procedures (March 25–26, 1999), the current methodologies and data for the in vitro micronucleus test were reviewed. From this, guidelines for the conduct of specific aspects of the protocol were developed. Because there are a number of important in vitro micronucleus validation studies in progress, it was not possible to design a definitive, internationally harmonized protocol at this time. Agreement was achieved on the following topics: Cells. The choice of cells is flexible, yet the choice of cell type should be justified and take into consideration doubling time, spontaneous frequency of micronuclei, and genetic background. Slide preparation. A fixation method that preserves the cytoplasm and cytoplasmic boundaries, and minimizes clumping should be used. Use of fluorescent DNA‐specific dyes is encouraged for better detection of small micronuclei. Analysis. Micronuclei should have a diameter less than one‐third of the main nucleus, and should be clearly distinguishable from the main nucleus. In the cytokinesis‐block method, binucleated cells selected for analysis should have two clearly distinguishable main nuclei. Cells where the main nucleus(ei) is undergoing apoptosis should not be scored for micronuclei because the assumed micronuclei may have been the result of nuclear fragmentation during the apoptotic process. Toxicity. Cytotoxicity can be measured by various methods including cell growth, cell counts, nucleation (i.e., percent binucleated), division/proliferation index, confluence. A majority of the group recommended that the highest concentration should induce at least 50% cytotoxicity (by whatever measure is selected). Cytochalasin B. There is much debate regarding the use of cytochalasin B. For human lymphocytes, the use of cytochalasin B (6 μg/ml [lymphocytes cultured from whole blood cells] and 3–6 μg/ml [isolated lymphocyte cultures]) is recommended. For cell lines, because there were no definitive data showing a clear advantage or disadvantage of the use of cytochalasin B for a variety of chemicals, the majority opinion of the group was that at this time, the use of cytochalasin B for cell lines is considered optional. Further studies (many chemicals of a variety of potencies, tested both with and without cytochalasin B) are clearly needed to resolve this issue. Number of doses. At least three concentrations should be scored for micronuclei. Treatment/harvest times. At this time, there are not enough data to define the most appropriate treatment/harvest times. Following the principles of the in vitro metaphase assay (with or without metabolic activation), it was agreed that there was a need for a short treatment followed by a recovery time in the absence of test chemical, there was a need for a long treatment (maybe with and without recovery time), and ideally, treatment should cover cells in different cell cycle stages. Environ. Mol. Mutagen. 35:167–172, 2000

Report from working group on in vitro tests for chromosomal aberrations

The following summary represents a consensus of the working group except where noted. The items discussed are listed in the order in which they appear in the OECD guideline (473) for easy reference. Metabolic activation. S9 from animals induced either with Aroclor 1254 or with the combination of phenobarbital with beta-naphthoflavone is acceptable, and other systems could be used with suitable justification. Exposure concentrations. The upper limit of testing should be 10 mM (or 5 mg/ml where molecular weight is not known or mixtures are being tested), whichever is lower. Where this limit is inappropriate the investigator should give detailed justification of the choice of top concentration. Cytotoxicity should be measured not only in range-finding tests but also concurrently with the assay for chromosomal aberrations. Cytotoxicity should be assessed by measurements of cell growth such as cell counts or confluence estimation. Mitotic index data alone are not a sufficient measure of cytotoxicity, except in the case of blood cultures for which other methods are impractical. Cytotoxicity at the top dose should be greater than 50% of concurrent negative/solvent controls, if this can be achieved without exceeding a concentration limit of 10 mM or 5 mg/ml. There should be at least three concentrations scored for aberrations (each with and without S9), covering a toxicity range down to a concentration giving little or no cytotoxicity. This will usually mean that the concentrations scored will be quite closely spaced. It was not possible to reach a consensus on the issue of solubility limits. The group did not agree on whether (a) solubility rather than cytotoxicity should be the limiting factor, such that only one top dose with evident precipitate should be scored even if toxicity is not observed, or (b) several concentrations with evident precipitate should be scored for aberrations if this were necessary to obtain cytotoxicity. It was agreed that evidence of precipitation should be determined in the final culture medium. Controls. Concurrent positive controls are required but the working group thought it inappropriate to specify the control chemicals or the degree of response that should be obtained, leaving it up to the test laboratory to demonstrate that the system was working adequately based on historical data within the laboratory. It is not necessary to include both negative and solvent controls concurrently with the aberration test; solvent controls alone are acceptable provided that the laboratory has data to demonstrate that there is no effect of the solvent on baseline values. Preparation of cultures.(ABSTRACT TRUNCATED AT 400 WORDS)

Concepts of threshold in mutagenesis and carcinogenesis.

Although the existence of a threshold in the dose effect relationship is well documented for many, if not most, types of toxicological effects the existence of a threshold for the mutagenic effects of ionising radiation and of certain chemicals has been questioned since the middle of the century and only recently the question of thresholds for radiation and chemical carcinogenesis has been addressed. The essential facts for the interpretation of threshold dose-response curves are common to all type of effects and are: (i) the number and the identity of the target; (ii) the type and sensitivity of the endpoint used to quantify the effect. We therefore will first try to model the type of interactions which may be expected between a mutagen and its target and define from this whether a threshold dose-effect can be expected; in a second step the concept will be extended to heritable mutations and carcinogenesis.

Characterization of p53 in Chinese hamster cell lines CHO-K1, CHO-WBL, and CHL: implications for genotoxicity testing.

Since the p53 gene function is critical to how a cell responds to DNA damage, we investigated the p53 status in Chinese hamster cell lines commonly used in genotoxicity tests for cytogenetic damage around the world. These included: Chinese hamster ovary K1 (CHO-K1), Chinese hamster ovary WBL (CHO-WBL), and Chinese hamster lung (CHL) cells. The results of DNA sequencing, protein analysis, and cell cycle analysis demonstrate that the CHO-K1 and CHO-WBL cell lines have mutant p53 sequence [a mutation in codon 211 in exon 6 resulting in a change from Thr (ACA) to Lys (AAA)], mutant protein (high spontaneous levels that are non-inducible after X-irradiation), and mutant function (lack of G1 checkpoint). Interestingly, the CHL cell line has a completely wild-type p53 DNA sequence. However, the CHL cells have an abnormally high spontaneous level of wild-type p53 protein expression that is not inducible after X-irradiation, yet there is some evidence of G1 delay after irradiation. The protein data suggests that p53 in CHL cells is not being regulated normally, and thus is probably not functioning normally. The mechanism leading to this abnormal regulation of p53 in CHL cells clearly does not involve mutation in the p53 gene. Overall, the CHL cell line may be similar to the CHO cell lines, in that they all appear to have abnormal p53 function. Further work is needed to determine whether the presence of spontaneously high levels of wild-type p53 in CHL cells results in a difference in response to DNA damage (quantitatively or qualitatively) compared to the p53 mutant CHO cell lines.

The pH 6.7 Syrian hamster embryo cell transformation assay for assessing the carcinogenic potential of chemicals

Cell transformation models have been established for studying the cellular and molecular basis of the neoplastic process. Transformation models have also been utilized extensively for studying mechanisms of chemical carcinogenesis and, to a lesser degree, screening chemicals for their carcinogenic potential. Complexities associated with the conduct of cell transformation assays have been a significant factor in discouraging broad use of this approach despite their reported good predictivity for carcinogenicity. We previously reported that many of the experimental difficulties with the Syrian hamster embryo (SHE) cell transformation assay could be reduced or eliminated by culturing these cells at pH 6.7 culture conditions compared to the historically used pH 7.1-7.3. We and others have shown that morphological transformation (MT), the earliest recognizable phenotype in the multi-step transformation process and the endpoint used in the standard assay to indicate a chemicals transforming activity, represents a pre-neoplastic stage in this model system. In the collaborative study reported here, in which approx. 50% of the chemicals were tested under code in one laboratory (Hazelton) and the other 50% evaluated by several investigators in the second laboratory (P & G), we have evaluated 56 chemicals (30 carcinogens, 18 non-carcinogens, 8 of inconclusive carcinogenic activity) in the SHE cell transformation assay conducted at pH 6.7 culture conditions with a standardized, Good Laboratory Practices-quality protocol. An overall concordance of 85% (41/48) between SHE cell transformation and rodent bioassay results was observed with assay sensitivity of 87% (26/30) and specificity of 83% (15/18), respectively. The assay exhibited a sensitivity of 78% (14/18) for Salmonella assay negative carcinogens, supporting its value for detecting non-mutagenic carcinogens. For maximum assay sensitivity, two exposure durations were required, namely a 24-h exposure and a 7-day exposure assay. Depending on the duration of chemical treatment required to induce transformation, insight into the mechanism of transformation induction may also be gained. Based on the data reported here, as well as the larger historical dataset reviewed by Isfort et al. (1996), we conclude that the SHE cell transformation assay provides an improved method for screening chemicals for carcinogenicity relative to current standard genotoxicity assays.

In vitro genotoxicity testing using the micronucleus assay in cell lines, human lymphocytes and 3D human skin models

The toxicological relevance of the micronucleus (MN) test is well defined: it is a multi-target genotoxic endpoint, assessing not only clastogenic and aneugenic events but also some epigenetic effects, which is simple to score, accurate, applicable in different cell types. In addition, it is predictive for cancer, amenable for automation and allows good extrapolation for potential limits of exposure or thresholds and it is easily measured in experimental both in vitro and in vivo systems. Implementation of in vitro micronucleus (IVMN) assays in the battery of tests for hazard and risk assessment of potential mutagens/carcinogens is therefore fully justified. Moreover, the final draft of an OECD guideline became recently available for this test. In this review, we discuss the prerequisites for an acceptable MN assay, including the cell as unit of observation, importance of cell membranes, the requirement of a mitotic or meiotic division and the assessment of cell division in the presence of the test substance. Furthermore, the importance of adequate design of protocols is highlighted and new developments, in particular the in vitro 3D human skin models, are discussed. Finally, we address future research perspectives including the possibility of a combined primary 3D human skin and primary human whole blood culture system, and the need for adaptation of the IVMN assays to assess the genotoxic potential of new materials, in particular nanomaterials.

Xenobiotic metabolism gene expression in the EpiDerm™ in vitro 3D human epidermis model compared to human skin

There is an urgent need to validate in vitro human skin models for use in safety testing. An important component of validation is characterizing the metabolizing capacity of these models. We report comparison of the expression of 139 genes encoding xenobiotic metabolizing enzymes in the EpiDerm model and human skin. In microarray analysis, the expression of 87% of the genes was consistent between the EpiDerm model and human skin indicating the presence of similar metabolic pathways suggesting commonality in function. Analysis of EpiDerm models constructed from four donors showed highly comparable expression of xenobiotic metabolizing genes demonstrating reproducibility of the model. Overall, the expression of Phase II enzymes appeared to be more pronounced in human skin and the EpiDerm model than that of Phase I enzymes, consistent with the role of skin in detoxification of xenobiotics. Though the basal expression of CYPs in particular was low in EpiDerm, significant induction of CYP1A1/1B1 activity was observed following treatment with 3-methylcholanthrene. These results indicate that the xenobiotic metabolizing capacity of the EpiDerm model appears to be representative of human skin. Models such as EpiDerm provide a valuable in vitro approach for evaluation of metabolism and toxicity of cutaneous exposures to xenobiotics.

Improvement of in vivo genotoxicity assessment: Combination of acute tests and integration into standard toxicity testing

A working group convened at the 2009 5th IWGT to discuss possibilities for improving in vivo genotoxicity assessment by investigating possible links to standard toxicity testing. The working group considered: (1) combination of acute micronucleus (MN) and Comet assays into a single study, (2) integration of MN assays into repeated-dose toxicity (RDT) studies, (3) integration of Comet assays into RDT studies, and (4) requirements for the top dose when integrating genotoxicity measurements into RDT studies. The working group reviewed current requirements for in vivo genotoxicity testing of different chemical product classes and identified opportunities for combination and integration of genotoxicity endpoints for each class. The combination of the acute in vivo MN and Comet assays was considered by the working group to represent a technically feasible and scientifically acceptable alternative to conducting independent assays. Two combination protocols, consisting of either a 3- or a 4-treament protocol, were considered equally acceptable. As the integration of MN assays into RDT studies had already been discussed in detail in previous IWGT meetings, the working group focussed on factors that could affect the results of the integrated MN assay, such as the possible effects of repeated bleeding and the need for early harvests. The working group reached the consensus that repeated bleeding at reasonable volumes is not a critical confounding factor for the MN assay in rats older than 9 weeks of age and that rats bled for toxicokinetic investigations or for other routine toxicological purposes can be used for MN analysis. The working group considered the available data as insufficient to conclude that there is a need for an early sampling point for MN analysis in RDT studies, in addition to the routine determination at terminal sacrifice. Specific scenarios were identified where an additional early sampling can have advantages, e.g., for compounds that exert toxic effects on hematopoiesis, including some aneugens. For the integration of Comet assays into RDT studies, the working group reached the consensus that, based upon the limited amount of data available, integration is scientifically acceptable and that the liver Comet assay can complement the MN assay in blood or bone marrow in detecting in vivo genotoxins. Practical issues need to be considered when conducting an integrated Comet assay study. Freezing of tissue samples for later Comet assay analysis could alleviate logistical problems. However, the working group concluded that freezing of tissue samples can presently not be recommended for routine use, although it was noted that results from some laboratories look promising. Another discussion topic centred around the question as to whether tissue toxicity, which is more likely observed in RDT than in acute toxicity studies, would affect the results of the Comet assay. Based on the available data from in vivo studies, the working group concluded that there are no clear examples where cytotoxicity, by itself, generates increases or decreases in DNA migration. The working group identified the need for a refined guidance on the use and interpretation of cytotoxicity methods used in the Comet assay, as the different methods used generally lead to inconsistent conclusions. Since top doses in RDT studies often are limited by toxicity that occurs only after several doses, the working group discussed whether the sensitivity of integrated genotoxicity studies is reduced under these circumstances. For compounds for which in vitro genotoxicity studies yielded negative results, the working group reached the consensus that integration of in vivo genotoxicity endpoints (typically the MN assay) into RDT studies is generally acceptable. If in vitro genotoxicity results are unavailable or positive, consensus was reached that the maximum tolerated dose (MTD) is acceptable as the top dose in RDT studies in many cases, such as when the RDT study MTD or exposure is close (50% or greater) to an acute study MTD or exposure. Finally, the group agreed that exceptions to this general rule might be acceptable, for example when human exposure is lower than the preclinical exposure by a large margin.

SFTG international collaborative study on in vitro micronucleus test: III. Using CHO cells

In this report, results are presented from an international study of the in vitro micronucleus assay using mouse lymphoma L5178Y cells. This study was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Test chemicals included mannitol, bleomycin, 5-fluorouracil, colchicine and griseofulvin. Mitomycin C was used as a positive control. Each chemical was evaluated in at least two laboratories following a variety of different protocols (short and long exposures, varying recovery times, with and without cytochalasin B) in order to help determine a standard protocol for routine testing in mouse lymphoma L5178Y cells. Mannitol was the only exception, being tested in only one laboratory. Mannitol was negative, while bleomycin induced a concentration-dependent increase in micronucleated cells. Equivocal results were obtained for 5-fluorouracil, colchicine and griseofulvin. High levels of cytotoxicity interfered with the assessment of aneuploidy for colchicine and griseofulvin, preventing the ability to obtain clear results in all the treatment schedules. Experiments with 5-fluorouracil, colchicine and griseofulvin showed that both short and long treatment times are required as each compound was detected using one or more treatment protocol. No clear differences were seen in the sensitivity or accuracy of the responses in the presence of absence of cytochalasin B. It was also found that a recovery period may help to detect compounds which induce a genotoxicity associated to a reduction in cell number or cell proliferation. Overall, the results of the present study show that mouse lymphoma L5178Y cells are suitable for the in vitro micronucleus assay.