Silvio Albertini

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

Discriminating different classes of toxicants by transcript profiling

Male rats were treated with various model compounds or the appropriate vehicle controls. Most substances were either well-known hepatotoxicants or showed hepatotoxicity during preclinical testing. The aim of the present study was to determine if biological samples from rats treated with various compounds can be classified based on gene expression profiles. In addition to gene expression analysis using microarrays, a complete serum chemistry profile and liver and kidney histopathology were performed. We analyzed hepatic gene expression profiles using a supervised learning method (support vector machines; SVMs) to generate classification rules and combined this with recursive feature elimination to improve classification performance and to identify a compact subset of probe sets with potential use as biomarkers. Two different SVM algorithms were tested, and the models obtained were validated with a compound-based external cross-validation approach. Our predictive models were able to discriminate between hepatotoxic and nonhepatotoxic compounds. Furthermore, they predicted the correct class of hepatotoxicant in most cases. We provide an example showing that a predictive model built on transcript profiles from one rat strain can successfully classify profiles from another rat strain. In addition, we demonstrate that the predictive models identify nonresponders and are able to discriminate between gene changes related to pharmacology and toxicity. This work confirms the hypothesis that compound classification based on gene expression data is feasible.

ECVAM retrospective validation of in vitro micronucleus test (MNT)

In the past decade several studies comparing the in vitro chromosome aberration test (CAT) and the in vitro micronucleus test (MNT) were performed. A high correlation was observed in each of the studies (>85%); however, no formal validation for the micronucleus in vitro assay had been carried out. Therefore, a working group was established by the European Centre for the Validation of Alternative Methods (ECVAM) to perform a retrospective validation of the existing data, in order to evaluate the validity of the in vitro MNT on the basis of the modular validation approach. The primary focus of this retrospective validation was on the evaluation of the potential of the in vitro MNT as alternative to the standard in vitro CAT. The working group evaluated, in a first step, the available published data and came to the conclusion that two studies [German ring trial, von der Hude, W., Kalweit, S., Engelhardt, G. et al. (2000) In-vitro micronucleus assay with Chinese hamster V79 cells: results of a collaborative study with 26 chemicals. Mutat. Res., 468, 137–163, and SFTG International Collaborative Study, Lorge, E., Thybaud, V., Aardema, M., Oliver, J., Wataka, A., Lorenzon, G. and Marzin, D. (2006) SFTG International Collaborative Study on in-vitro micronucleus test I. General conditions and overall conclusions of the study. Mutat. Res., 607, 13–36] met the criteria for a retrospective validation according to the criteria previously defined by the working group. These two studies were evaluated in depth (including the reanalysis of raw data) and provided the information required for assessing the reliability (reproducibility) of the test. For the assessment of the concordance between the in vitro MNT and the in vitro CAT, additional published data were considered. Based on this retrospective validation, the ECVAM Validation Management Team concluded that the in vitro MNT is reliable and relevant and can therefore be used as an alternative method to the in vitro CAT. Following peer review, these conclusions were formally endorsed by the ECVAM Scientific Advisory Committee.

Two-dimensional database of mouse liver proteins: Changes in hepatic protein levels following treatment with acetaminophen or its nontoxic regioisomer 3-acetamidophenol

Overdose of acetaminophen (APAP) causes acute hepatotoxicity in rodents and man. The mechanism underlying APAP‐induced liver injury remains unclear, but experimental evidence strongly suggests that activation of APAP and subsequent formation of protein adducts are involved in hepatotoxicity. Using proteomics technologies, we constructed a two‐dimensional protein database for mouse liver, comprising 256 different gene products and investigated the proteins affected after APAP‐induced hepatotoxicity. Adult male mice received a single dose of APAP (100 or 300 mg/kg) or its nontoxic regioisomer 3‐acetamidophenol (AMAP, 300 mg/kg). The extent of liver damage was assessed 8 h after administration by increased liver enzyme release and histopathology. Changes in the protein level were studied by comparison of the intensities of the corresponding spots on two‐dimensional (2‐D) gels. The expression level of about 35 of the identified proteins was modified due to treatment with APAP or AMAP. The observed changes were usually in the order of 10—50% of the control value and were more marked in the high‐ than in the low‐dose of APAP‐treated animals. Most of the changes caused by AMAP occurred in a subset of the proteins modified by APAP. Many of the proteins showing changed expression levels are either known targets for covalent modification by N‐acetyl‐p‐benzoquinoneimine (NAPQI) or involved in the regulation of mechanisms that are believed to drive APAP‐induced hepatotoxicity.

In vitro micronucleus assay with Chinese hamster V79 cells : results of a collaborative study with in situ exposure to 26 chemical substances

A collaborative study with 10 participating laboratories was conducted to evaluate a test protocol for the performance of the in vitro micronucleus (MN) test using the V79 cell line with one treatment and one sampling time only. A total of 26 coded substances were tested in this study for MN-inducing properties. Three substances were tested by all 10 laboratories and 23 substances were tested by three or four laboratories in parallel. Six aneugenic, 7 clastogenic and 6 non-genotoxic chemicals were uniformly recognised as such by all laboratories. Three chemicals were tested uniformly negative by three laboratories although also clastogenic properties have been reported for these substances. Another set of three clastogenic substances showed inconsistent results and one non-clastogenic substance was found to be positive by one out of three laboratories. Within the study, the applicability of the determination of a proliferation index (PI) as an internal cytotoxicity parameter in comparison with the determination of the mitotic index (MI) was also evaluated. Both parameters were found to be useful for the interpretation of the MN test result with regard to the control of cell cycle kinetics and the mode of action for MN induction. The MN test in vitro was found to be easy to perform and its results were mainly in accordance with results from chromosomal aberration tests in vitro.

Thresholds in genotoxicity responses

It has been commonly accepted that risk assessments of genotoxic chemicals are based on linear extrapolation methods. However, there is substantial evidence that some chemicals may be genotoxic only at high doses by mechanisms that do not occur at low doses, or only under specific conditions in genotoxicity assays, but are inactive at concentrations within the range of human exposure levels. There are a variety of possible mechanisms of thresholded genotoxicity, including disruption of cell division and chromosome segregation, inhibition of DNA synthesis, overloading of oxidative defence mechanisms, metabolism or plasma binding capacity, disturbances of metal homeostasis, cytotoxicity and physiological perturbations in in vivo assays. The degrees of evidence supporting the proposed mechanisms are variable and not all are sufficiently robust to be universally accepted as yet by the scientific community. However, a survey of industrial companies indicated that data have been accepted by some regulatory authorities indicating thresholds contributing to genotoxicity responses.

Photomutagenesis test development: I. 8-methoxypsoralen, chlorpromazine and sunscreen compounds in bacterial and yeast assays

Two in vitro genotoxicity tests have been adapted to the evaluation of photomutagenic activity of test compounds. The study was initiated to obtain an experimental basis relating to newly proposed guidelines of the EC which request the screening of UV-absorbing compounds, for example, those employed in sunscreen preparations, for their photomutagenic potential. The well established photomutagens 8-methoxypsoralen and chlorpromazine were used to define relevant test protocols. The compounds were evaluated with the Ames test and the Saccharomyces cerevisiae D7 test for gene conversion. The influence of various parameters such as UV light sources, spectral composition, UV sensitivity of the test systems, absorbance by test materials and different exposure conditions is indicated. Two exemplary screening experiments with cosmetic ingredients are presented. Both test systems can be employed for the evaluation of compounds for photomutagenic activity although the standard excision-deficient strains of S. typhimurium pose problems because of their high UV sensitivity. The present experience in this complex field suggests that rigid test protocols and a restrictive test battery would be inadequate.

Effect of two 5-HT6 receptor antagonists on the rat liver: a molecular approach.

ABSTRACTSerotonin is involved in disorders of the central nervous system; thus, specific 5-HT6 receptor antagonists have therapeutic potential. Nevertheless, preclinical tests showed that Ro 65-7199 caused hepatic steatosis. Here, we investigated the hepatic effects of Ro 65-7199 and Ro 66-0074 using toxicogenomics. The profiles obtained after exposure of rats to both compounds clearly show that two pharmacologically closely related compounds with different toxicological profiles can be distinguished based on gene expression profiles. Moreover, side effects can be detected earlier with toxicogenomics than with conventional end points. A possible link between the sterol metabolic pathway, the induction of CYP2B, and the hepatic fat accumulation was also established. Summarizing, gene expression profiles allow both compounds to be distinguished according to their toxicity and provide mechanistic insights. The results clearly show the power of toxicogenomics as a tool for obtaining characteristic fingerprints at early time-points and for generating mechanistic hypotheses.

The detection of chemically induced chromosomal malsegregation in Saccharomyces cerevisiae D61.M: A literature survey (1984–1990)

Our objective is to summarize the published data obtained with a recently developed tester strain suitable for the detection of chromosomal malsegregation in yeast. Results from 25 papers were reviewed in which numerical data for 111 chemicals tested in Saccharomyces cerevisiae D61.M are reported (a total of 316 independent tests; 279 acceptable, 37 not meeting our criteria). Of the 111 compounds analyzed 43 compounds are positive for chromosomal malsegregation, 56 compounds are negative and 12 compounds do not meet our criteria for acceptance (inconclusive). Of the 43 compounds judged positive 5 (acetone, acetonitrile, benzonitrile, ethylacetate and propionitrile) were only positive using a cold interruption protocol. Recommendations are made for standardization of methods and protocols for screening purposes. Finally, a comparison with in vitro tubulin assembly data using mammalian tubulin is presented.

Evaluation of the GreenScreen GADD45α-GFP indicator assay with non-proprietary and proprietary compounds

The GreenScreen GADD45alpha indicator assay has been assessed for its concordance with in vitro genotoxicity and rodent carcinogenicity bioassay data. To test robustness, sensitivity, and specificity of the assay, 91 compounds with known genotoxicity results were screened in a blinded manner. Fifty seven of the compounds were classified as in vitro genotoxic whereas 34 were non-genotoxic. Out of the 91 compounds, 50 had been tested in 2-year carcinogenicity assays, with 33 identified to be rodent carcinogens and 17 non-carcinogens. Gadd45alpha assay sensitivity and specificity for genotoxicity was 30% and 97%, respectively (17/57 and 33/34), whereas its sensitivity and specificity for rodent carcinogenicity was 30% and 88%, respectively (10/33 and 15/17). Gadd45alpha assay genotoxicity results from this validation study exhibited a high concordance with previously published results as well as for compound test results generated at two different sites (91%, 19/21), indicating that the assay is both robust and reproducible. In conclusion, results from this blinded and independent validation study indicate that the GreenScreen GADD45 indicator assay is reproducible and reliable with low sensitivity and high specificity for identifying genotoxic and carcinogenic compounds.