A normal healthy cell will attempt to prevent expression of the genotoxic mutation by either enzymatic DNA repair or by promoting innate programmed cell death termed apoptosis. Apoptosis deletes the cell so healthy functional tissue is maintained. If damage is not corrected, it may lead to mutagenesis. As noted, the alteration of the genetic material can have direct or indirect effects on the DNA; i.e., the induction of mutations, mistimed event activation and direct DNA damage leading to mutations. Since the surface of the skin, the stratum corneum, is composed of non-living material (corneocytes and lipids), and since genotoxicity is only applicable to living cells, a genotoxic effect of a topically applied compound will be imparted should the compound penetrate the outer barrier and is able to interact with living skin layers or communicate an effect to the living layers. Therefore, it might be of interest to run a skin absorption study (as described in my March 2015 column).
A Key Component of Safety
Genotoxicity should not be confused with mutagenicity. Compounds that are mutagens are genotoxic because they generate genetic changes. However, not all genotoxic compounds are mutagenic. Meaning, a compound can alter the genetic information but not cause mutations.
The analysis of potential genotoxicity is of key importance in the determination of the relevancy of the assay as part of a tier approach in safety assessment. If the compound’s suggested efficacy mode of action relates to changes in cell genes or proteins, the genotoxicity test may be incorporated in the earlier set of studies and a more comprehensive study package should be generated to exclude such potential toxicity. For example, compounds that are tested and marketed with claims to “repair DNA,” “attenuate cellular death” or “enhance cell viability” should be studied with extra care for this safety endpoint. Changes to the genome at various levels can be a lengthy process and usually are not a result of short time exposure. As noted, since cosmetic products are regularly applied to skin for months and even years, they should be tested for genotoxicity. The genotoxic effect can be to the skin as the exposed organ; and if penetrates through the skin to the circulation – to internal organs as well.
Moreover, skin and other organs contain metabolic mechanisms known as Cytochrome P-450. The most intense P-450 in the body is present in the liver, the main metabolic organ; however, skin contains metabolic enzymes as well. This is an orchestrated cassette of proteins, primarily membrane-associated, that metabolize endogenous and exogenous chemicals with the ultimate goal to enable neutralization and excretion out of the body. While the tested compound may not exhibit genotoxic effect, its metabolites may—therefore a valid genotoxicity assay should include evaluating effects from potential metabolites.
A column about skin metabolism will be published later this year.
Numerous assays can determine whether a chemical has the potential to induce genetic damage; only a few will be included here. The assays differ in the model utilized, means and times of exposure, tested endpoints and translated implications.
Endpoints established can include the following:
- Single-gene mutations.
- Multilocus mutations, which comprise structural changes to chromosomes such as breaks, deletion, and rearrangement or numerical changes in the genome. The numerical changes to the genome can be aneuploidy (presence of one or a few chromosomes above or below the normal number) and polyploidy (effect on one or more extra sets of chromosomes).
- Changes to the DNA; determination of DNA adducts or DNA strand breakages, or measurement of unscheduled DNA synthesis as a cellular response to DNA damage.
The most widely employed and acknowledged assay to evaluate genotoxicity is the Ames assay, which detects mutagenicity in a bacterial reverse gene mutation test. This test has been shown to detect relevant genetic changes and the majority of genotoxic rodent and human carcinogens. If an Ames test is conducted, the second study in the tier should evaluate the hazard potential to mammalian cells. The Ames test was detailed in the early 1970s by Bruce Ames at the University of California Berkley. It is designed to detect potential mutations in bacteria and serves as a rapid, relatively inexpensive and convenient way to evaluate the mutagenic potential of a compound. The test is performed by incubating a mutated bacterium with the compound of interest, with or without an exogenous metabolic system. The metabolic system, generally a rat liver homogenate (termed “S9 fraction”), is added to evaluate the possible genotoxic effect of metabolites in the system. As previously explained, metabolites are compounds that are formed in the culture as a result of enzymatic conversion. They can potentially be inert; exert activity that is similar to the substrate (which is the tested compound) or toxic. Five or more concentrations of the substance are usually tested and include negative (vehicle) and positive controls with and without metabolic activation. Mutated bacteria, which are capable of producing histidine, will form colonies in the histidine-free media.
The core principal of the Ames test is the use of amino acid-dependent strains of Salmonella typhimurium and Escherichia coli. Each carries different mutations in various genes in the histidine operon. This means that they cannot produce histidine and will not grow on a histidine deficient media unless they mutate. These mutations act as cores for mutagens that cause DNA damage through various mechanisms. In the absence of an external histidine source, the bacteria cannot grow and form colonies. Therefore only bacteria that have mutated to revert to histidine independence will form colonies. The number of spontaneously induced reverting colonies per plate is relatively constant. When a mutagen is added to the plate, the number of reverting colonies per plate is increased, usually in a dose-related manner. The Ames test cannot be utilized in cases when the test substance is toxic to bacteria such as antibiotics or preservatives. In such case it is recommended that a tier of two tests in mammalian cells is conducted. In addition the fact that this assay uses prokaryotes means that it may not represent human cell behavior. To identify false positive mutagens further tests are required. For example, selected nitrate-containing substances (such as nitroglycerin) can produce false positive results by generating nitric oxide. But not all carcinogens (such as asbestos for example) are mutagens. As a result, the Ames assay may not identify some carcinogens.
Conducting Ames Assays
Ames assays are usually run in two tiers. The initial assay includes five strains of Salmonella typhimurium and one strain of Escherichia coli. Eight to ten dose levels may be tested, as well as vehicle controls, positive mutagens and S9 metabolic activation systems. Triplicate culture per strain or dose is conducted for both test systems to allow statistical generation. The second tier in the assay is perceived as a confirmatory one. Here, the same five strains are tested for at least five dose levels that have been adjusted as determined in the initial assay as well as metabolic activation. The scholars that highlight Ames test limitations point to the fact that it is utilizing bacteria that is very different from human cell and therefore advocate for models using human or mammalian cells.
One established in vitro mammalian cell system for testing mutagenicity is the micronucleus assay. In this assay the presence of chromosomal fragments that appear as small round additions to the nucleus (therefore termed “micronuclei”) are determined in erythrocytes following exposure to the test substance. Mature erythrocytes normally expel their nuclei during development, and thus do not contain DNA. If chromosomal fragments are formed during cell division, they may be retained in the cell and are considered to be a measure of chromosomal damage. The in vitro micronucleus assay has undergone retrospective validation by ECVAM and is described in details in a publication from 2008. ECVAM’s management team concluded that the in vitro micronucleus test is reliable and relevant and can be used as an alternative to the in vitro chromosome aberration test.
This test detects chromosomal structural aberrations as well as numerical aberrations (both polyploidy and aneuploidy; see prior explanation). A more recent modification of the in vitro micronucleus test is the use of 3D human reconstructed skin models for the assessment of genotoxicity of dermally applied materials. An example for such model, EpiDerm is described in the column on skin irritation published previously (see my September 2015 column).
In brief, the protocol utilizes various topical doses of the test material exposed for 48 to 72 hours with repeat exposure every 24 hours in the presence of 3µg/ml of Cytochalasin-B in the medium. Cells are harvested from the tissue 24 hours after the last dose as follow:
- Tissues are washed with DPBS (Dulbecco’s Phosphate-Buffered Saline), followed by EDTA exposure, and then exposed to trypsin-EDTA;
- Tissues are separated from the membrane and again exposed to trypsin-EDTA;
- Trypsin-EDTA neutralizing solution is added to the cell suspension before centrifuging;
- After centrifugation, KCl solution is slowly added to the cells followed by 3ml of cold;
- MeOH/acetic acid (3:1);
- The cell suspension is re-centrifuged, the medium removed;
- 4ml of MeOH/acetic acid is added. The cell suspension is again centrifuged, and the medium removed;
- The cells are gently placed on the slide immediately; and
- Slides are stained in acridine orange (AO) solution, rinsed, and then scored using a fluorescent microscope.
Mutagenesis vol. 23 no. 4 pp. 271–283, 2008 doi:10.1093/mutage/gen010
Advance Access Publication 7 March 2008 ECVAM retrospective validation of in vitro micronucleus
Dr. Nava Dayan LLC
Nava Dayan Ph.D. is the owner of Dr. Nava Dayan L.L.C, a skin science and research consultancy and serving the pharmaceutical, cosmetic, and personal care industries. She has 25 years of experience in the skin care segment, and more than 150 publication credits.