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Genotoxicity Article:

Test Techniques

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The purpose of genotoxicity testing is to determine if a substrate will influence genetic material or may cause cancer. They can be performed in bacterial, yeast, and mammalian cells. With the knowledge from the conclusions of the tests, one can control early development of vulnerable organisms to genotoxic substances.

Bacterial Reverse Mutation Assay The Bacterial Reverse Mutation Assay, also known as the Ames Assay, is used in laboratories to test for gene mutation. The techniques uses many different bacterial strains in order to compare the different changes in the genetic material. The result of the test detects the majority of genotoxic carcinogens and genetic changes; the types of mutations detected are frame-shifts and base substitutions.

in vitro Testing The purpose of in vitro testing is to determine whether a substrate, product, or environmental factor induces genetic damages. One technique is cytogenetic assays in different mammalian cells. The types of aberrations detected in cells affected by a genotoxic material are chromatid and chromosome gaps; chromosome breaks; chromatid deletions; fragmentation; acentric fragments; translocation; triradial and quadradial; pulverized chromosomes and cells; complex rearrangements; ring, dicentric, and minute chromosomes; more than ten aberrations; polyploidy; and hyperdiploids. The clastogenic or aneugenic effects from the genotoxic damage will cause an increase in frequency of structural or numerical aberrations of the genetic material. This is similar to the micronucleus test and chromosome aberration assay, which detect structural and numerical chromosomal aberrations in mammalian cells. In a specific mammalian tissue, one can perform a mouse lymphoma TK+/- assay to test for changes in the genetic material. Gene mutations are commonly point mutations, altering only one base within the genetic sequence to alter the ensuing transcript and amino acid sequence; these point mutations include base substitutions, deletions, frame-shifts, and rearrangements. Also, the chromosomes' integrity may be altered through chromosome loss and clastogenic lesions causing multiple gene and multilocus deletions. The specific type of damage is determined by the size of the colonies, distinguishing between genetic mutations (mutagens) and chromosomal aberrations (clastogens). Lastly, the SOS/umu assay test evaluates the ability of the substance to induce DNA damage; it is based on the alterations in the induction of the SOS response due to DNA damage. The benefits of this technique are that it is a fast and simple method and convenient for numerous substances. Such technique is performed on water and wastewater in the environment.

in vivo Testing The purpose for in vivo testing is to determine the potential of DNA damage that can affect chromosomal structure or disturb the mitotic apparatus that changes chromosome number; the factors that could influence the genotoxicity are ADME and DNA repair. It can also detect genotoxic agents missed in in vitro tests. The positive result of induced chromosomal damage is an increase in frequency of micronucelated PCEs. A micronucleus is a small structure separate from the nucleus containing nuclear DNA arisen from DNA fragments or whole chromosomes that were not incorporated in the daughter cell during mitosis. Causes for this structure are mitotic loss of acentric chromosomal fragments (clastogenicity), mechanical problems from chromosomal breakage and exchange, mitotic loss of chromosomes (aneugenicity), and apoptosis. The micronucleus test in vivo is similar to the in vitro one because it tests for structural and numerical chromosomal aberrations in mammalian cells, especially in rats' blood cells.

Comet Assay Comet assays are one of the most common tests for genotoxicity. The technique involves lysing cells using detergents and salts. The DNA released from the lysed cell is electrophoresed in an agarose gel under neutral pH conditions. Cells containing DNA with an increased number of double stranded breaks will migrate more quickly to the anode. This technique is advantageous in that it detects low levels of DNA damage, only requires a very small number of cells, is cheaper than many techniques, easy to execute, and quickly displays results. However, it does not identify the mechanism underlying the genotoxic effect nor the exact chemical or chemical component causing the breaks.

Mechanisms

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The genotoxic substances induce damage to the genetic material in the cells through interactions with the DNA sequence and structure. For example, the transition metal chromium interacts with DNA in its high-valent oxidation state so to incur DNA lesions leading to carcinogenesis. The metastable oxidation state Cr(V) is achieved through reductive activation. Sugden et al. performed an experiment to study the interaction between DNA with the carcinogenic chromium by using a Cr(V)-Salen complex at the specific oxidation state. The interaction was specific to the guanine nucleotide in the genetic sequence. In order to narrow the interaction between the Cr(V)-Salen complex with the guanine base, the researchers modified the bases to 8-oxo-G so to have site specific oxidation. The reaction between the two molecules caused DNA lesions; the two lesions observed at the modified base site were guanidinohydantoin and spiroiminodihydantoin. To further analyze the site of lesion, it was observed that polymerase stopped at the site and adenine was inappropriately incorporated into the DNA sequence opposite of the 8-oxo-G base. Therefore, these lesions predominately contain G-->T transversions. In the end, "the mechanism of damage and base oxidation products for the interaction between high-valent chromium and DNA... are relevant to in vivo formation of DNA damage leading to cancer in chromate-exposed human populations." Consequently, it shows how high-valent chromium can act as a carcinogen with 8-oxo-G forming xenobiotics. Another example of a genotoxic substance causing DNA damage are pyrrolizidine alkaloids. These substances are found mainly in plant species and are poisonous to animals, including humans; about half of them have been identified as genotoxic and many as tumorigenic. "Upon metabolic activation, PAs produce DNA adducts, DNA cross-linking, DNA breaks, sister chromatid exchange, micronuclei, chromosomal aberrations, gene mutations, and chromosome mutations in vivo and in vitro." The most common mutation within the genes ares G:C --> T:A tranversions and tandem base substitution. The pyrrolizidine alkaloids are mutagenic in vivo and in vitro and, therefore, responsible for the carcinogenesis prominently in the liver. Comfrey is an example of a plant species that contains fourteen different PAs. The active metabolites interact with DNA to cause DNA damage, mutation induction, and cancer development in liver endothelial cells and hepatocytes. "Overall, comfrey is mutagenic in liver, and PA contained in comfrey appear to be responsible for comfrey-induced toxicity and tumor induction."

Cancer

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Genotoxic effects such as deletions, breaks and/or rearrangements can lead to cancer if the damage does not immediately lead to cell death. Regions sensitive to breakage, called fragile sites, may result from genotoxic agents (such as pesticides). Some chemicals have the ability to induce fragile sites in regions of the chromosome where oncogenes are present which could lead to carcinogenic effects. In keeping with this finding, some occupational exposure to mixtures of pesticides are positively correlated with increased genotoxic damage in the exposed individuals. Individuals vary in their ability to activate or detoxify genotoxic substances, which leads to variability in the incidence of cancer among individuals. The difference in ability to detoxify certain compounds in due to individuals’ inherited polymorphisms of genes involved in the metabolism of the chemical. Differences may also be attributed to individual variation in efficiency of DNA repair mechanisms. The metabolism of some chemicals results in the production of reactive oxygen species which is a possible mechanism of genotoxicity. This is seen in the metabolism of arsenic which produces hydroxyl radicals, which are known to cause genotoxic effects. Similarly, ROS have been implicated in genotoxicity caused by particles and fibers. Genotoxicity of nonfibrous and fibrous particles is characterized by high production of ROS from inflammatory cells.


References

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  • Bolognesi, Claudia (2003): Genotoxicity of Pesticides: A review of human biomonitoring studies. Mutation Research 543(3): 251-272. doi:10.1016/S1383-5742(03)00015-2
  • Furman, Grace M. (2008): Genotoxicity Testing for Pharmaceuticals Current and Emerging Practices. UPTEK Seminar Series (PDF)
  • Liu, Su X.; Athar, M.; Lippai, I.; Waldren, C.; Hei, T. (2000): Induction of oxyradicals by arsenic: Implication for mechanism of genotoxicity. Proceedings of the National Academy of Sciences of the United States of America 98(4): 1643-1648.
  • Sugden, Kent D.;Campo, Christina K. & Martin, Brooke D. (2001): Direct Oxidation of Guanine and 7,8-Dihydro-8-oxoguanine in DNA by a High-Valent Chromium Complex:  A Possible Mechanism for Chromate Genotoxicity. Chemical Research in Toxicology 14(9): 1315–1322. doi:10.1021/tx010088+ (HTML fulltext)
  • Tice, R.R.; Agurell, E.; Anderson, D.; Burlinson, B.; Hartmann, A.; Kobayashi, H.; Miyamae, Y.; Rojas, E.; Ryu, J.-C. & Sasaki, Y.F. (2000): Single Cell Gel/Comet Assay: Guidelines for In Vitro and In Vivo Genetic Toxicology Testing. Environmental and Molecular Mutagenesis 35(3): 206-221. doi:10.1002/(SICI)10982280
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