In Vitro Methods to Test Materials for Ozone Protective Capabilities

As a sign of the times, in addition to the morning traffic and weather reports, many large news networks are also reporting ground level ozone measurements as an index of air pollution. Ozone has long been known to induce respiratory inflammation,1 and excessively high levels of ozone can put the elderly or individuals with compromised respiratory function at risk for injury. Currently 0.1 parts per million (ppm) is the maximum ozone level allowed in key European cities; however, most typical US cities have levels ranging between 0.1 and 0.5 ppm, with cities such as Los Angeles reaching highs of 2.0 ppm.2

At levels of 0.3 ppm the effects of extended ozone exposure become noticeable in healthy individuals, with predominate effects including headaches and throat irritation. As ozone levels rise symptoms can become more severe and include reduced lung function, reduced pulmonary immune function and an increased chance of mortality (see Ozone’s Effect on the Skin).3

In light of the effects of ozone pollution and the ever-increasing levels of ozone found in US cities, it should come as no surprise that 14 states have recently sued the US Environmental Protection Agency (EPA) in attempt to establish stricter laws regarding ozone pollution.4

While most research regarding ozone exposure has focused on the respiratory system, there has been increasing interest in the effects of ozone exposure on the skin. Early work by Thiele et al.5 reported two effects of ozone exposure in mice: depletion of α-tocopherol and ascorbic acid, two key antioxidants in the superficial levels of the epidermis; and induction of lipid peroxidation throughout the epidermis (see Ozone Levels and Effects). 

Ozone exposure also can result in the secondary generation of other types of intracellular reactive oxygen species (ROS).6 Finally, in epidermal keratinocytes, it can activate nuclear factor kappa beta-a transcription factor activated in response to oxidative stress-and induce the expression of heme oxygenase-I, an enzyme that protects cells from oxidative stress; and cyclooxygenase-2, an enzyme involved in the inflammation process.7 All of these markers can be used to indicate epidermal stress. Therefore, it becomes clear that the skin is also a sensitive target to ozone exposure and one that could greatly benefit from cosmetic or personal care materials designed to protect against ozone.

Overview of Ozone

Ozone is a highly reactive gas formed from three molecules of oxygen (O3). Ozone is naturally formed in the upper atmosphere (stratospheric ozone) via an interaction between normal O2 and UV light. However, ozone can also be formed at the ground level by an interaction between two types of air pollution: volatile organic compounds produced by gas pumps, some consumer products and certain types of chemical plants; and nitrogen oxides produced by combustion engines and industrial plants.8 When ozone is formed at the ground level it is referred to as tropospheric ozone and it is this type of ozone that is dangerous to human health.

Once formed, ozone can react with biological systems either directly or indirectly. As an example of a direct reaction, ozone can readily react with unsaturated fatty acids to form lipid peroxides. In this case, the ozone molecule modifies the unsaturated carbon bond via a Criegee mechanism to form a cyclo intermediate-a five-sided ring structure with three oxygens and two carbons forming the ring-within the lipids’ carbon backbone. This intermediate is unstable and will rapidly disintegrate to form an aldehyde and a ketone or zwitterion end product. If a zwitterion is the end product, it will break down again into a hydroxyl hydroperoxide and carbonyl end product. 

In addition to direct mechanisms, ozone can act indirectly by either breaking down into, or facilitating the generation of, other types of ROS. For example, in an aqueous environment, ozone combines with hydroxyl radicals to ultimately generate superoxide radicals, which can start a chain reaction because the superoxides in turn react with ozone to generate additional hydroxyl radicals and superoxide radicals. In fact, through either direct or indirect mechanisms, ozone can lead to the generation of hydroxyl radicals, hydrogen peroxide, peroxyl radicals, singlet oxygen, and superoxide radicals.9 These secondary free radicals are generated in proportion to the amount of original ozone present and are free to attack intracellular targets.

ROS-based Ozone Protection Assays

The formation of ROS serves as an excellent marker for screening cosmetic or personal care materials for ozone protection assays. ROS can be measured simply by using the dye 2′-7′-dichlorofluorescein (DCF). Initially it is relatively nonfluorescent; however, once it reacts with ROS, it becomes highly fluorescent. 

For assay purposes, DCF is loaded into cells or the viable cell layer of skin tissue equivalents before their exposure to ozone. The cells or tissues are subsequently treated with test materials, then exposed to ozone. After the ozone exposure, the fluorescence intensity of the cells or tissues is measured. If the cells or tissues treated with the test materials have a lower relative fluorescence than untreated cells or tissues, the tested materials may provide some protection against ozone exposure because the amount of ROS formed during the ozone exposure was reduced.6 

In addition to measuring the direct formation of ROS due to ozone, it is possible to measure the subsequent damage caused by the ROS on cellular targets. One of the most general targets to measure is the formation of protein carbonyls. Protein carbonyls are formed when ROS modify the amine side chains of arginine, lysine, proline and threonine to form aminoacyl carbonyls. When the skin is exposed to ozone, the level of protein carbonyls dramatically increases and this provides another useful  indicator to determine whether a material protects against ozone damage.10 

Ozone-induced protein carbonyl formation can be measured in cultured cells or in skin tissue equivalents through the use of 2,4-dinitrophenylhydrazine (DNPH). DNPH reacts with protein carbonyls to form a colored end product that can be measured spectrophotometrically. The greater the degree of color formation, the greater the extent of carbonyl formation and thus the greater the degree of ozone-induced damage. 

Just as ozone-induced ROS modify proteins, they also modify lipids, resulting in the formation of lipid peroxides. Traditionally, lipid peroxides have been measured using the method of thiobarbituric acid-reactive substances (TBARS). In cultured cells or tissues, lipid peroxides are unstable and can decompose, resulting in the formation of malondialdehyde. Malondialdehyde, in return, reacts with thiobarbituric acid to form a colored end product that also can be measured spectrophotometrically. 

AGE-based Ozone Protection Assays

Advanced glycation end products (AGE) can be formed by the covalent bonding of aldehyde groups in sugars with proteins that contain free amine groups.11 Exemplary sugars are glucose or ribose. Among the proteins are the amino acids lysine and arginine. The AGE-modified proteins are dangerous to the cell because they may diminish the proteins’ function, and may also serve to cross-link adjacent proteins. 

AGE-modified proteins in the skin can serve to cross-link type I collagen, leading to a decrease in the elasticity of the skin.12 AGE modification of proteins occurs via non-enzymatic reactions, and the rate of AGE formation can be substantially increased by the presence of ROS.11

Due to the variability in the types of sugars and the types of amino acids with free amine groups there are a number of possible types of AGE. Interestingly, quite a few of these products have fluorescent properties and can be measured noninvasively in the skin.13 In the author’s lab, ozone exposure of skin tissue equivalents was shown to increase the tissue’s autofluorescence using fluorescent wavelengths associated with pentosidine, an AGE formed by cross-linking arginine and lysine side chains with a ribose sugar.12 

AGE formation can also be assayed by measuring the formation of specific AGE species using immunological-based techniques. For example, carboxymethyl lysine (CML) is an AGE that is commonly formed.11 Several companies have developed antibodies that recognize CML. Again, in the author’s lab, ozone exposure of skin tissue equivalents was shown to increase the amount of CML within the tissues. 

Autofluorescence and CML measurements can be used to determine whether a material could protect against ozone exposure. Because skin tissue equivalents are used for these measurements, the tested materials can be applied topically to the tissues prior to the ozone exposure. After the exposure, the tissue autofluorescence can be measured quickly using a fluorometer and compared to the tissue’s fluorescence prior to the ozone exposure. Then the tissue can be homogenized to measure CML formation via methods based on Western blotting or slot blotting. If the change in autofluorescence and CML content is less in the tissues treated with the test materials than in the untreated tissues exposed to ozone, the material could provide ozone protective properties, warranting further investigations into endpoints associated with ozone-induced damage.

Concluding Remarks

The methods described in this article highlight some of the markers to assess the ability of a test material to protect against ozone damage to the skin. It should be noted that because ozone is capable of generating a variety of ROS and AGE, a test material may be proficient at protecting against some effects yet not others. Thus the effectiveness of the material will depend upon the mechanism by which the material protects against ozone damage. 


1.HK Yoon, HY Cho and SR Kleeberger, Protective role of MMP-9 in ozone induced airway inflammation, Environ Health Perspect 115 1557–1563 (2007)

2.Ozone levels and their effects, (Accessed Aug 3, 2008)

3.JW Hollingsworth, SR Kleeberger and WM Foster, Ozone and pulmonary innate immunity, Proc Am Thorac Soc 4(3) 240–246 (2007)

4.States sue EPA over ozone pollution standards, Reuters News Service (May 28, 2008) (Accessed Aug 3, 2008) 

5.JJ Thiele, MJ Traber, K Tsang, CE Cross and L Packer, In vivo exposure to ozone depletes vitamins C and E and induces lipid peroxidation in murine skin, Free Radic Bio Med 23(3) 385–391 (1997)

6.J Cotovio, L Onno, P Justine, S Lamure and P Catroux, Generation of oxidative stress in human cutaneous models following in vitro ozone exposure, Toxicol in vitro 15(4–5) 357–362 (2001)

7.G Valacchi, E Pagnin, AM Corbacho, E Olano, PA Davis, L Packer and CE Cross, In vivo ozone exposure induces antioxidant/stress-related responses in murine lung and skin, Free Radic Bio Med 36(5) 673–681 (2004)

8.What is ozone?, An air quality awareness statement from the EPA, (Accessed Aug 3, 2008)

9.BS Berlett, RL Levine and ER Stadtman, Comparison of the effects of ozone on the modification of amino acid residues in glutamine synthetase and bovine serum albumin, J Biol Chem 271(8) 4177–4182 (1996) 

10.G Valacchi, A Van Der Vliet, BC Schock, T Okamoto, U Obermuller-Jevic, CE Cross and L Packer, Ozone exposure activates oxidative stress response in murine skin, Toxicol 179 163–170 (2002)

11.KF Hanssen, Making sense of advanced glycation endproducts (AGE), Internat Diab Mon 10(4) 1–5 (1998)

12.JL Wautier and PJ Guillausseau, Advanced glycation end products, their receptors and diabetic angiopathy, Diabet Metab (Paris) 27 535–542 (2001)

13.JW Hartog, AP de Vries,  HL Lutgers, R Meerwaldt, RM Huisman, WJ van Son, PE de Jong and AJ Smit, Accumulation of advanced glycation end products, measured as skin autofluorescence, in renal disease, Ann NY Acad Sci 1043 299–307 (2005)              

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