• Intrinsic metabolism

During aerobic energy generation about 2% of all the oxygen burned ends up as reactive oxygen species (ROS). These damage DNA most frequently by oxidizing the guanine base to form 8-oxo-guanine (8oGua), which is often misread by the DNA replication machinery causing a change in the sequence of the progeny strands, resulting in mutations. This is particularly serious for mitochondrial DNA, which is closest to the source of the short-lived ROS. An unfolding area of research is the role of mitochondrial DNA in chronic diseases of aging.

• Environmental insult

By far the most serious damage to skin DNA is from the sun. Solar ultraviolet radiation (UV) also causes the formation of ROS which result in 8oGua. In mitochondria, repeated doses of UVA result in the accumulation of a characteristic deletion mutation in mitochondrial DNA. The frequency of this characteristic mutation in human skin increases with sun exposure, suggesting that it is an internal dosimeter for cumulative sun exposure. But 10-times more frequent than 8oGua is the cyclobutane pyrimidine dimer (CPD), which is DNA damage caused by direct absorption of UV photons without any ROS intermediate. UV causes two adjacent DNA bases to fuse together in a cyclobutane ring, in a photochemical reaction that takes about 15 picoseconds; antioxidants cannot stop it because no free radicals are involved. A second type of base fusion, called a 6–4 photoproduct, is similarly formed about one-sixth as frequently as CPD. Because these fused bases distort the DNA much more than 8oGua, they are much more mutagenic than 8oGua, and consequently much more carcinogenic. CPD causes a characteristic type of DNA mutation, and these signature mutations are frequently found in key cancer genes in squamous and basal cell carcinomas. This is the smoking gun that connects CPDs to skin cancer.

Alkylation is a third type of DNA damage in which an alkyl group is added to DNA. The most prevalent additions are at the 7 position of guanine (N⁷meGua) and to the phosphates of the DNA backbone, but a much less common form of damage—alkylation of the 6 position of guanine (O⁶meGua)—is the most mutagenic and hence the most dangerous. This type of damage is usually caused by toxins, such as from some of the chemicals in cigarette smoke, and by drugs. The premature signs of aging in the skin induced by cigarette smoking may be associated with the alkylation damage from alkylating agents in cigarette smoke transported through the circulation to the skin. Additionally, exposure to other chemicals via exposure to industrial pollution and drugs with DNA alkylating potential may have some role in what has been considered normal skin aging.

• The comet assay

Cultured mammalian cells are seeded onto glass slides and placed in an electric field. The DNA, which is ordinarily tightly wound, cannot leave the nucleus. But if the DNA contains breaks in the double helix, then the stands can unwind and stream out of the nucleus toward the cathode (positive charged pole). When the DNA is stained and examined under the microscope, the nuclei appear to be ‘comets’ with a ball-shaped head and strands trailing out into a tail Fig. 34.1.

Fig. 34.1 The Comet assay for DNA damage. An example of a cell containing single-stranded breaks which was plated in agarose, lysed in situ, and subjected to electrophoresis. The DNA was then stained with ethidium bromide and examined by fluorescence microscopy. The ‘comet’ streaking out toward the left from the nucleus on the right represents DNA migrating to the cathode

What causes the breaks in DNA? UV itself does not directly cut DNA, so the comets that form after UV reflect the ongoing DNA repair, which has as its first step the introduction of a break in the double strand. Cosmeceutical ingredients that reduce comets after UV may be detrimental by inhibiting DNA repair. Ionizing radiation and free radicals, on the other hand, do directly result in DNA breaks, so cosmeceutical ingredients that reduce comets after these treatments may be beneficial. Therefore, the comet assay alone does not prove that DNA repair is enhanced or inhibited.

• Antibody-based assays

Several antibodies raised against specific types of DNA damage (e.g. anti-CPD, anti-8oGua or anti-O⁶meGua) have been developed to directly measure the appearance and removal of DNA lesions. Cultured cells may be stained with these antibodies, and counterstained for the DNA of the nucleus, and then examined under the microscope for the amount of antibody binding as a measure of the extent of DNA damage. Figure 34.2A shows UV-irradiated cells stained for the nucleus (blue); Figure 34.2B shows the same cells also stained for CPD (red). In other assays the DNA is purified from the cells, or from skin, and bound to a filter membrane, which is then probed with the damage-specific antibodies. A reduction in the amount of antibody binding after treating the cells, artificial or natural skin with a cosmeceutical ingredient (always compared to an untreated control) is a direct measure of DNA repair.

Fig. 34.2 Antibody-based assay for DNA damage. UV-irradiated cells were stained with antibodies against CPD and counterstained for DNA with DAPI, then examined by fluorescence microscopy. (A) Nuclei appear blue. (B) CPD appear red

• Apoptosis and sunburn cells

Cells that have sustained too much DNA damage enter a suicide mode called apoptosis. In skin, these apoptotic cells are recognized as small, rounded-up, darkly staining cells called ‘sunburn cells’; not surprisingly, they were first described in sunburned skin! Although they represent excessive damage, apoptosis is considered a process beneficial to the skin, because it removes cells loaded with DNA lesions that have a high potential of turning into mutated or even cancerous cells. Tests that show cosmeceutical ingredients inhibiting apoptosis and sunburn cells are often interpreted to mean the ingredient is desirable, but this is not necessarily the case. An ingredient may reduce apoptosis by inhibiting DNA damage, increasing DNA repair, but it may also reduce apoptosis by blocking the suicide mode, and thus preserving heavily damaged cells that can cause cancer.

The best tests for DNA damage are those that directly follow the loss of the altered bases from DNA. These data, when combined with evidence from the Comet assay or the sunburn cell assay, can give a full picture of the effects of cosmeceutical ingredients.