A mutagen is defined as a chemical or substance that can induce heritable changes called mutations in the genotype of a particular cell resulting from aberrations or loss of genes, whole chromosomes, or regions of chromosomes. On the other hand, the term genotoxicity refers to the capability of a particular chemical or substance to interact with DNA material. This ability is widely believed to be due to electrophilic reactions between the target chemical or substance or any of its intermediates or metabolites, and DNA material which serves as a nucleophilic core (Combes et al., 2007).
Chemicals for industrial, commercial, home, and other uses need to undergo mutagenicity testing which is a significant part of regulatory hazard assessment of materials and substances. The objectives of this regulatory step are: first, to detect chemicals or substances that have the ability to induce genetic harm such as point mutations in germ cells causing an increased risk for the acquisition of heritable or genetic abnormalities in humans; and second, to detect substances or chemicals that may be classified as carcinogenic since mutagenesis is considered an important event in carcinogenesis (Combes, 1995).
There are many in vitro protocols that are available for genotoxicity testing. These detection methods are divided into two groups. The first group is called tier 1 or core assays. The second group is referred to as tier 2 or ancillary assays. Tier 1 or core assays are typically comprised of bacterial mutagenicity and cytogenetic screens including cultured mammalian cell gene mutation tests. Tier 2 or ancillary assays are taken to elucidate unexpected or suspicious results in tier 1 or core assays. This second strategy includes short-term in vivo studies such as cytogenetics assay using bone marrow. The objective is to establish whether any ability to induce genotoxicity at tier 1 in vitro level can be replicated in a living animal. Therefore, it is sufficient to claim that there is a lack of mutagenicity when tier 1 or core assays results are negative while positive results do not automatically merit mutagenic hazard classification (Zeiger 2001).
Tier 1 or core in vitro genotoxicity assays, as mentioned above, are anchored on the detection of mutagenicity in bacterial cells and chromosomal aberrations or injury in mammalian cells. These two methods are both used since particular chemicals can provide either effect and may result in inaccuracy in the tests if only one is performed (Combes, 1997).
Hypermutable indicator or tester strains of bacteria are used in bacterial gene mutation assays since these organisms have defective cell envelopes enhancing the absorption of test chemicals. In addition, hypermutable bacterial strains are characteristically deficient in one or more DNA repair functions. The Ames test is the most widely used assay wherein both forward and reverses mutation systems are available. The process involves picking Salmonella typhimurium which are histidine revertants on an agar medium that is supplemented minimally. Mutagenic potency, through a collection of concentration-response values, is derived by counting revertant colonies arising from mutated bacteria. Another assay involves trp reversion in WP2 strain of Eschirichia coli (Mitchell & Combes, 1997).
Chromosomal damage includes structural and chromosomal number aberrations. These abnormalities are generally detected in the metaphase stage of stained mammalian cells. Chinese hamster ovary, Chinese hamster lung, and human primary cell isolates are used to produce permanent cell lines which are exposed in containers or slides. The following changes are observed if present: chromosomal gaps, breaks, micronuclei exchanges, chromatid transfers, numerical abnormalities such as polyploidy and aneuploidy (Combes 2007).
A carcinogen is defined as a substance or chemical that induces cancer. Cancer, on the other hand, is defined as tumor formation with increased incidence. Carcinogenesis occurs during the transition of healthy cells into cancerous cells through the series of steps related to changes in cell growth rates, apoptosis or cell death, aberrations in cell differentiation, and the introduction and prevalence of cancer cells over healthy cells (Sato & Tomita, 2001).
Chemicals directly interacting with DNA material cause cancer and are referred to as genotoxic carcinogens are known to affect different species, sexes, and organs. On the other hand, there are non-genotoxic carcinogens, which indirectly interact with DNA material in so-called epigenetic mechanisms but are more specific to species, sexes, and organs affected than genotoxic carcinogens (Choy, 2001).
Carcinogenicity tests are dependent on the intended use of a particular chemical or compound and the level to which humans may be subjected to such chemicals. Most regulatory requirements indicate in vivo carcinogenicity testing which involves a rodent bioassay on its own or together with a transgenic mouse assay. The latter is utilized to satisfy risk assessment purposes and to establish of lack of carcinogenicity (Cronin, 1995).
Four hundred animals are used for each test. One dose or control group requires 50 animals for each sex. Two species are generally used in the rodent bioassay which is rats and mice since tumor induction, as mentioned above, is species-specific. Test substance introduction is performed through the following modes: oral, dermal, or inhalation based on the chemical and physical properties of the test substance although the oral introduction is the most commonly performed method (Combes, 2007).
The manner of substance administration to the test subject dictates the interval of the introduction although daily exposure is generally followed. This test is conducted for most of the life span of the test animal although for mice and hamsters termination occurs at 18 months and rats at 24 months. After termination, full necropsy and histopathology immediately follow (Sato & Tomita, 2001; Combes, 2007).
The primary alternative for a non-animal protocol for predicting carcinogenicity utilizes morphological cell transformation which is anchored on the induction of particular changes in the phenotype of cells in tissue culture behaving like tumorigenic cells. Genotoxic carcinogens and non-genotoxic carcinogens can be detected using cell transformation assays. Rodent cell lines, human origin fibroblast cell lines, primary cells, Syrian hamster embryo cell assays are cell transformation assays that have been developed (Combes, 2007).
Some of the above assay systems are employed to detect inducers of carcinogenesis through the mechanism called electrohilic interactions with DNA materials and other macromolecules. Other assays systems, on the other hand, are used to detect tumor promoters an example of which is the recently developed Bhas 42 cells which were derived from Balb/c 3T3 cells (Tsuchiya & Umeda, 1995).
Works Cited
Choy, W.N. (2001). Genotoxic and non-genotoxic mechanisms of carcinogenesis. In Genetic Toxicology and Cancer Risk Assessment (ed. W.N. Choy), pp. 47–72. New York, NY, USA: Marcel Dekker.
Combes, R., Grindon, C., Cronin, M., Roberts, D. and J. Garrod. (2007). Proposed integrated decision-tree testing strategies for mutagenicity and carcinogenicity in relation to the EU REACH legislation. ATLA 35, 267-287.
Combes, R.D. (1995). Regulatory genotoxicity testing: A critical appraisal. ATLA 23, 352–379.
Combes, R.D. (1997). Statistical analysis of doseresponse data from in vitro assays: an illustration using Salmonella mutagenicity data. Toxicology in Vitro 11, 683–687.
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Mitchell, I. de G. & Combes, R.D. (1997). In vitro genotoxicity and cell transformation assessment. In In Vitro Methods in Pharmaceutical Research (ed. J.V. Castell & M.J. Gómez-Lechón), pp. 318–352. London, UK & New York, NY, USA:Academic Press. London, UK: Springer-Verlag.
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Sato, S. and I. Tomita. (2001). Short-term screening method for the prediction of carcinogenicity of chemical substances: Current status and problems of an in vivo rodent micronucleus assay. Journal of Health Science, 47(1) 1–8.
Tsuchiya, T. & Umeda, M. (1995). Improvement in the efficiency of the in vitro transformation assay method using BALB/3T3 A31-1-1 cells. Carcinogenesis16, 1887–1894.
Zeiger, E. (2001). Genetic toxicity tests for predicting carcinogenicity. In Genetic Toxicology and Cancer Risk Assessment (ed. W.N. Choy), pp. 29–46. New York, NY, USA: Marcel Dekker.