The short-term colony transformation assay employing Syrian hamster embryonic (SHE) cells has been widely used as a simple method for detection of chemical and physical carcinogens. However, little investigation has been done on the biological properties of the early transformed colony (ETC: colony characterized by piling up and criss-cross pattern of growth) itself. This study was performed to examine the properties of these colonies. Secondary or tertiary cultures of SHE cells were treated with benzo[a]pyrene or N-methyl-N’-nitro-N-nitrosoguanidine. In total, 37 ETCs and 17 normal colonies (NCs) were cloned and analyzed. Obtained results were as follows: (1) Stability of transformed morphology; immediately after cloning, the cells from 3/37 of the ETCs maintained their transformed phenotype, but all cells from other ETCs (34/37) showed flat or well-oriented morphology. Thus, the “transformed” morphology of more than 90% of the ETCs was reversible. (2) Chromosome abnormality; 3/15 of the clones from ETCs were hypo diploid or tetraploid, while the others (12/15) were normal diploid immediately after cloning. (3) Immortalization; up to about one month after cloning, most of the clones (from transformed or normal colonies) could be subcultured at 1:2 or 1:4 split ratio per week, but thereafter all the clones ceased growing. After about a one month or longer latency, 6/37 of the clones from ETCs and 4/17 of the clones from NCs restarted growing and acquired immortality. That is, there was no significant difference in the frequency of immortalization between ETCs and NCs. Thus, from the present experiment, there was no direct evidence that ETC correlates to acquisition of immortality or tumorigenesis. Further experiments (e.g. comparison of gene expression profiles between cells from transformed and normal colonies using microarray) would be required to give a logical meaning to this short-term transformation assay.
Has mutation research contributed sufficiently to the genetic risk assessment of chemicals? The answer may be “No”. Although it has contributed much to predict carcinogenicity, how much benefit has exposure prevention of those mutagens brought to public health? It is suggested that the proportion of human carcinogenesis related to environmental chemicals is less than 10%. The main causes for cancer are tobacco smoking, diet, and aging. As genetic toxicologists, we have become too satisfied with a qualitative evaluation of mutation assay results and try to detect as many “carcinogens” as possible. This approach ignores the quantitative evaluation, which is more important in risk assessment. Many “carcinogens”, especially socalled non-genotoxic carcinogens, do not cause cancer at the human exposure level. The “genotoxic non-carcinogens”, if they exist, may be more important because cancer is not the only toxicological outcome of genotoxicity. We should pay more attention to heritable genetic effects of chemicals. An increasing incidence of smoking among young women alerts us to investigate the genetic effects of smoking in their progeny. The paradigm shift from hazard identification to risk assessment is important in mutation research. In this regard, quantitative, mechanism-based, and humanized mutation assays are required.
Over ten thousands kinds of chemical substances have been widely used for manufacture, agriculture, household and other purposes in industrialised countries. Various unintended by-products are also generated in manufacturing processes and in combustion of fossil fuel. Some of these chemicals released to the environment, e.g. into air and water, are likely to be hazardous and pose a public health concern. Regulation by Chemical Substances Control Low and PRTR systems are important frame works for managing release of hazardous chemicals to the environment in Japan. Biological monitoring is a novel methodology for assessing the health risk of exposure to environmental chemicals. Mutation is one practical endpoint for biological monitoring, whereby exposure to mutagenic chemicals may result in DNA mutations, e.g. base substitutions. Therefore, our studies have been focused on development of a bio-assay method for estimating in vivo mutagenicity of environmental chemicals, especially in ambient air. The potency of urban air for DNA adduct formation was estimated using rats maintained in a small animal facility beside a road with heavy traffic. The amount of DNA adducts in the lung was significantly elevated after a 4-week exposure, indicating that mutagenicity of urban air should be further evaluated. Diesel exhaust, a major source of mutagens in urban areas, was evaluated for in vivo mutagenicity using transgenic animals (Big Blue rat and gpt delta mouse) developed for detecting mutagens. Mutant frequency in lung of Big Blue rat was significantly increased by exposure to diesel exhaust for 4 weeks at the concentration of 6 mg SPM/m3. For detecting mutagens in water, we have developed a transgenic zebrafish line; this project is also reviewed.
Safety assessment of chemicals was previously based on qualitative determination of hazard. Principles and methods of safety evaluation have been developed through experience in international collaboration of experts, initially by introducing the notion of ADI (acceptable daily intake) while applying safety factors to compensate for uncertainties in safety evaluations. Recently, risk assessment based on quantitative data from animal experiments or from human observations with critical examination of experimental design and data processing has obtained more importance owing to progress in toxicological sciences and quality assurance systems of data management. In 1993, the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) jointly proposed a new paradigm of risk analysis in food safety in the framework of Codex Alimentarius. Risk analysis is composed of risk assessment, risk communication, and risk management which should be integrated in the process of securing food safety at every step from “farm to fork”. More input of science into uncertainty analysis and integration of various study data, and also more involvement of every stakeholder in the risk analysis process is now requested.