Carcinogen and Mutagen Testing

CARCINOGEN AND MUTAGEN TESTING

A carcinogen is a substance that causes living tissues to become carcinomatous, i.e. to produce a malignant tumour. A mutagen is a chemical (or physical) agent that induces mutation in a human (or other) cell.

Mutagenicity tests are used to screen a wide variety of chemicals for their ability to cause a mutation in the DNA of a cell. Such mutations can occur at either the gene level (a point mutation) at individual chromosomes, or at the level of a chromosome set, i.e. a change in the number of chromosomes (aneuploidy). Some compounds are only mutagenic or carcinogenic after metabolism (often in the liver). This aspect must, therefore, be considered in designing a suitable test method for such agents.

A)     Mutations At The Gene Level

Forward mutation refers to mutation of the natural (‘wild-type’) organism to a more stringent organism. By contrast, reverse (backward) mutation is the return of a mutant strain to the wild-type form, i.e. it is a heritable change in a previously mutated gene that restores the original function of that gene.

There are two types of reverse mutation:

•                                                 Frame-shift.

 In these mutants, the gene is altered by the addition or deletion of one or more bases so that the triplex reading frame for RNA is modified;

•                                                 Base-pair.

In these mutants, a single base is altered so that the triplex reading frame is again modified.

These principles of reverse mutation are utilized in one important method, the Ames test, which is used to detect compounds that act as mutagens or carcinogens (most carcinogens are mutagens).

B)   The Ames Test

The Ames test is used to screen a wide variety of chemicals for potential carcinogenicity or conversely for their potential as cancer chemotherapeutic agents. The test enables a large number of compounds to be screened rapidly by examining their ability to induce mutagenesis in several specially constructed bacterial mutants derived from Salmonella enterica serovar Typhi. The test strains contain mutations in the histidine operon such that they cannot synthesize the amino acid histidine. Two additional mutations increase further the sensitivity of the system. The first is a defect in their lipopolysaccharide structure such that they are in fact deep rough mutants possessing only 2-keto-3-deoxyoctonate (KDO) linked to lipid A. This mutation increases the permeability of the mutants to large hydrophobic molecules. The second mutation concerns a DNA excision repair system, which prevents the organism repairing its damaged DNA following exposure to a mutagen.

The assay method involves treatment of a large population of these mutant tester strains with the test compound. Histidine-requiring mutants are used to detect mutagens capable of causing base-pair substitutions (in some strains) or frame-shift mutations (other strains). This can be carried out by incorporating both the test strain and test compound in molten agar (at 45 °C), which is then poured on to a minimal glucose agar plate. Alternatively, the suspected mutagens can be applied to the surface of the top agar as a liquid or as a few crystals. The medium used for the top agar contains a limited concentration of histidine, which permits the bacteria on the plate to undergo several divisions, since for many mutagens some growth is a necessary prerequisite for mutagenesis to occur. After incubation for 2 days at 37 °C the number of ‘revertant’ colonies can be counted and compared with control plates from which the test compound has been omitted. Each revertant colony is assumed to be derived from a cell that has mutated back to the wild-type and thus can now synthesize its own histidine: see Figure 26.9 for a summary.

A further refinement to the Ames test permits screening of agents that require metabolic activation before their mutagenicity or carcinogenicity is apparent. This is achieved by incorporating into the top agar layer, along with the bacteria, homogenates of liver (commonly rat or human) whose activating enzyme systems have been induced by exposure to polychlorinated biphenyl mixtures. This test is sometimes referred to as the Salmonella/ microsome assay because the fraction of liver homogenate used, called the S9 fraction, contains predominantly liver microsomes.

It is important to realize that this test is flexible and is still undergoing modification and development. Almost all the known human carcinogens have been tested and shown to be positive. These include agents such as βnaphthylamine, cigarette smoke condensates, aflatoxin B and vinyl chloride, as well as drugs used in cancer treatment such as adriamycin, daunomycin and mitomycin C. Although the test is not perfect for the prediction of mammalian carcinogenicity or mutagenicity and for making definitive conclusions about potential toxicity or lack of toxicity in humans, it nevertheless provides useful screening information rapidly and cheaply. The Ames test remains an important part of a battery of tests, the others of which are non-microbial in nature, for detecting mutagenicity or carcinogenicity.