Studies on α-Hexachlorocyclohexane Cytotoxicity, Genotoxicity and Cytochrome P450 Induction in Primary Hepatocytes and Hepatoma Cell Lines from Rodents and Humans
Margherita Ferro, Anna Maria Bassi, Daniela Adamo, Francesca Mattioli, Luigi Robbiano and Giorgio Nanni
α-Hexachlorocyclohexane (α-HCH) was examined for cytotoxicity, genotoxicity and cytochrome P450 induction in primary cultures of mouse, rat and human hepatocytes and in three hepatoma cell lines (Hepa 1c1c7, FaO and Hep G2, from mouse, rat and man, respectively). The cell lines were much more sensitive to the cytotoxicity of the classical inducers phenobarbital and 3-methylcholanthrene than that of α-HCH, whereas no cytotoxicity was observed in primary hepatocytes. Exposure for 24 hours to 0.32mM α-HCH produced a modest, but statistically significant, frequency of DNA breaks, as measured by the alkaline elution assay, in the mouse Hepa 1c1c7 cell line, and the human Hep G2 cell line, but not in the rat FaO cell line. In the Hep G2 cell line, the amount of DNA fragmentation was found to increase with the length of exposure. Compared with the results of previous observations on primary cultures, with regard to species specificity, only the human cell line gave a concordant positive response. Monooxygenase activity induction in primary hepatocytes, despite rather high initial levels of 7-ethoxycoumarin-O-deethylase activity, was low with the classical inducers phenobarbital and 3-methylcholanthrene. α-HCH caused no induction of monooxygenase. The rat FaO and human Hep G2 cell lines were sensitive to α-HCH, but only after long exposure. The results of this study support the hypothesis that α-HCH might act as a weak genotoxic agent in humans, but they also suggest caution in the extrapolation to the in vivo situation of the observations made in established cell lines.
Peter G. Odeigah, Jide Ijimakinwa, Bimbo Lawal and Rebecca Oyeniyi
Six solid wastes from the metal industry, from the chemical industry and from the production of pesticides were investigated for their toxic and genotoxic properties by using the Allium test. To simulate environmental conditions and better assess their environmental impact, the solid wastes were leached with water, and the water-extractable micropollutants were lyophilised before screening. The mean root lengths of onions exposed to different concentrations of the leachates were measured, and EC50 values were determined from the growth curves. The EC50 values of the wastes were ranked, in the order: organochlorine pesticide waste, settling tank sludge from a chemical company, paint sludge, lead slag, steel slag and aluminium slag. The leachates were also mitodepressive, and caused significant increases in the frequency of chromosome aberrations. These results demonstrate that the Allium test is a useful screening test for the evaluation and ranking of toxic industrial waste.
In Vitro Toxicity: Mechanisms, Alternatives and Validation A Report from the 19th Annual Scientific Meeting of the Scandinavian Society for Cell Toxicology
The Scandinavian Society for Cell Toxicology (SSCT) has arranged annual scientific meetings since 1983. These workshops were the forum for the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme. Along with the MEIC programme, which was completed in 1998, a wide range of topics relating to cytotoxicity have been discussed. The meetings have also given an opportunity for graduate students and young scientists to present their work to an international audience. At the same time, experts in the fields of in vitro toxicity have been invited as speakers. The 19th SSCT scientific meeting, which was held in 2001 at Sørup Manor in Ringsted, Denmark, was no exception. The meeting consisted of four sessions: mechanisms of toxicity; environmental toxicological testing; alternatives to animal experiments; and validation of in vitro tests.
The Comet Assay with MCL-5 Cells as an Indicator of Genotoxic Treatment with Chemicals and Cigarette Smoke Condensate
Lucie Wolz, Günter Krause and Gerhard Scherer
The metabolically competent human lymphoblastoid cell line MCL-5 was treated with a panel of mutagens to assess the induction of DNA damage. Treatment effects were observed by monitoring cell proliferation and by single-cell gel electrophoresis (SCGE). The direct-acting mutagens benzo[a]pyrene-7,8-diol 9,10-epoxide (BPDE) and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), as well as pro-mutagens requiring metabolic activation, i.e. benzo[a]pyrene (BaP), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 4-N-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and cigarette-smoke condensate (CSC), were assayed by SCGE. Assay schemes were adapted for the MCL-5 cell line and for low levels of strand break induction, by inclusion of the DNA synthesis inhibitors cytosine arabinoside and hydroxyurea, and by extending the electrophoresis time. For all mutagens tested, dose-dependent increases of median and average tail moment values among 50 nucleoids per slide were observed. The determining factors for selecting the treatment doses for mutation-induction experiments were the solubility of BaP and PhIP in the exposure medium, and the cytotoxicity exhibited by BPDE, MNNG and CSC. Induction of DNA strand breaks was obtained at mutagen concentrations permitting sufficient cell proliferation, except in the case of MNNG.
The Use of a Chemistry-based Profiler for Covalent DNA Binding in the Development of Chemical Categories for Read-across for Genotoxicity
Steven J. Enoch, Mark T.D. Cronin and Claire M. Ellison
An important molecular initiating event for genotoxicity is the ability of a compound to bind covalently with DNA. However, not all compounds that can undergo covalent binding mechanisms will result in genotoxicity. One approach to solving this problem, when in silico prediction techniques are being used, is to develop tools that allow chemicals to be grouped into categories based on their ability to bind covalently to DNA. For this analysis to take place, compounds need to be placed within categories where the trend in toxicity can be explained by simple descriptors, such as hydrophobicity. However, this can occur only when the compounds within a category are structurally and mechanistically similar. Chemistrybased profilers have the ability to screen compounds and highlight those with similar structures to a target compound, and are thus likely to act via a similar mechanism of action. Here, examples are reported to highlight how structure-based profilers can be used to form categories and hence fill data gaps. The importance of developing a well-defined and robust category is discussed in terms of both mechanisms of action and structural similarity.
Damien Breheny, Oluwatobiloba Oke and Stephen P. Faux
Carcinogenesis is a highly complex, multi-stage process that can occur over a relatively long period before its clinical manifestation. While the sequence in which a cancer cell acquires the necessary traits for tumour formation can vary, there are a number of mechanisms that are common to most, if not all, cancers across the spectrum of possible causes. Many aspects of carcinogenesis can be modelled in vitro. This has led to the development of a number of mechanistically driven, cell-based assays to assess the pro-carcinogenic and anti-carcinogenic potential of chemicals. A review is presented of the current in vitro models that can be used to study carcinogenesis, with examples of cigarette smoke testing in some of these models, in order to illustrate their potential applications. We present an overview of the assays used in regulatory genotoxicity testing, as well as those designed to model other aspects that are considered to be hallmarks of cancer. The latter assays are described with a view to demonstrating the recent advances in these areas, to a point where they should now be considered for inclusion in an overall testing strategy for chemical carcinogens.
Robert D. Combes
Published in silico, in vitro, in vivo laboratory animal and human data, together with information on biotransformation and data from structure–activity analyses with two decision-tree systems (ACToR and Toxtree), have been used in a weight-of-evidence (WoE) assessment to determine whether phenylbutazone (PBZ) is a genotoxic or a non-genotoxic carcinogen. This was undertaken to facilitate the risk assessment of human exposure to this veterinary drug via the consumption of horsemeat from treated animals. Despite problems with data interpretation at all tiers of the database, it was concluded that PBZ behaves like a genotoxic carcinogen with a threshold dose. This conclusion is based mainly on the results of a definitive rodent bioassay, and on the following observations: a) that PBZ has weak in vitro activity only at high concentrations in some genotoxicity assays, accompanied by high levels of cytotoxicity; b) that it (and a major metabolite) is able to cause sister chromatid exchanges in vivo in rodents; and c) that it can induce cytogenetic effects in vivo in humans. It also takes into account the known and predicted activities of the parent drug, some of its metabolites and two structural analogues, and, importantly, several of the drug’s other biochemical effects that are unrelated to toxicity. However, this conclusion is not fully supported by all the evidence, and much of the information is based on old papers. Therefore, more studies are required to establish whether the concentration thresholds seen in vitro would translate to dose thresholds for carcinogenicity, such that a safe dose-level could be defined for the purposes of assessing risk. It was disappointing that a WoE approach to evaluating all of the available hazard data, as is increasingly being advocated to improve the hazard identification paradigm, was unable to provide definitive answers in this case, particularly in view of the large numbers of animals that had been used to provide much of the information.