j. Cosmet. Sci., 51, 169-182 (May/June 2000) Oxygen radicals from photoirradiated human hair: An ESR and fluorescence study LOUIS J. KIRSCHENBAUM, XINHUA QU, and EDWARD T. BORISH, Department of Chemistry, University of Rhode Island, Kingston, R102881. Accepted for publication March 15, 2000. Synopsis Exposure of hair to light causes degradation, the precise mechanism of which is not completely understood. Oxygen free radicals (superoxide and hydroxyl) are believed to be involved, and a variety of indirect evidence has supported this. However, to date, direct observation of oxyradicals from photoirradiated hair has been lacking. We have used complementary electron spin resonance and fluorescence techniques to assess oxyradical production when human hair is irradiated under UVA and visible light 0• -- 320 nm). ESR studies include direct observation of intrinsic melanin and keratin radicals as well as spin trapping with DMPO. The fluorescence technique is based on terephthalic acid dianion (TA) as a hydroxyl radical probe. Radical scavengers are used to compete with the probe (TA or DMPO). Our results indicate that (a) oxyradicals are produced during photoirradiation, (b) terephthalate is a convenient method to study hydroxyl production in hair, (c) radical production upon irradiation varies with hair type, and (d) the effects of radical scavengers suggest potential implications for hair care product development. INTRODUCTION Hair fibers consist primarily of fibrous proteins belonging to the keratin family. Mor- phologically, hair structure has three distinct components: the cuticle, the cortex, and the medulla. The shingle-like cuticles form the hair exterior and enclose the corticular mass. The cortex constitutes the bulk of the hair and contains tightly packed elongated cortical cells oriented parallel to the fiber axis. These cells contain alpha-helical micro- fibrils embedded in a cystine-rich amorphous protein matrix. The protein components comprising the hair fiber have differing amino acid compositions. The function of the medulla is not clear. It is composed of loosely attached spongy cells. The keratin in hair is accompanied by minor quantities of lipids. Pigmented hair contains melanin granules located within the cortex and medulla (1-3). Address correspondence to Louis J. Kirschenbaum. E-mail: Kirschenbaum@chm.uri.edu Edward T. Borish's present address is Bath and Body Works, 7 Limited Parkway East, Reynoldsburg, OH 43068. 169

170 JOURNAL OF COSMETIC SCIENCE Exposure of hair to light causes chemical and physical degradation of the fiber (4,5). Recent reports on the harmful effects of sunlight on human skin have raised awareness of the deleterious effects of sunlight on biological tissues in general. Because hair does not have the repair mechanism of skin (6), the effects of photodegradation must be dealt with through cosmetic means. The way that light affects hair is still not completely understood. The solar spectrum's UV range is known to lead to photooxidation of the protein structure through the oxidation of specific amino acids, in particular the oxi- dation of cystine to cysteic acid (7-9). The deleterious effects of solar irradiation are perceived by consumers as changes in texture and color, dryness, etc, and can be evalu- ated in terms of reduced elasticity, increased porosity or swelling properties, altered dye sorption characteristics, and photofading of natural or artificial hair color (10-12). The essential first event in hair photodamage, as in all photoprocesses, is light absorption by the fiber. Free radicals have been observed by electron spin resonance (ESR) spec- trometry during the light-induced degradation of keratin ('13-15). Carbon-centered radicals are formed with an action spectrum maximum of 285 nm, suggesting that they are primary photoproducts of aromatic amino acids (16). Kinetic measurements indicate that there are at least three types of radicals produced at this wavelength (17). Stable sulfur-centered radicals are formed from irradiation of dry keratin in the absence of oxygen, but their ESR signals disappear in the presence of moist air (18). The presence of small amounts of metal ion (trivalent iron or divalent copper) is especially conducive to the production of free radicals from irradiated keratin at wavelengths above 320 nm, apparently due to the formation of metal-protein complexes that absorb at higher wavelengths (19). Smith et aL (20) have suggested the presence of tetrahedral iron-sulfur centers that are easily oxidized upon irradiation, giving rise to carbon-centered radicals on neighboring groups. The most significant chromophores in proteins that absorb in the UVB region are the amino acids, tyrosine (Tyr, •max = 275 nm) and tryptophan (trp, •max = 280 nm). The irradiation of wool keratin under UVA and visible light does not cause the formation of tryptamine, which is the main photoproduct formed during irradiation of tryptophan itself in aqueous solution (21,22). Like wool photoyellowing and photodamage, a free-radical mechanism may be involved in the photodamage of hair at wavelengths above 320 nm. Melanin has an intrinsic ESR signal that increases significantly when irradiated with UV-visible light. In the presence of oxygen, superoxide is produced, which dismutates to hydrogen peroxide (23,24). This leads to the formation of hydroxyl radical in the presence of trace amounts of metal ions. We have recently extended the observations of earlier workers to the study of oxygen radical production in suspensions of melanin extracted from red and brown hair (25). By employing terephthalic acid dianion (TA) as a hydroxyl radical trap, we have introduced a convenient method for estimating relative amounts of oxyradicals formed under varying conditions. In the terephthalate analysis, TA reacts with hydroxyl radicals to form hydroxyterephthalate (HTA), as shown in Scheme I (26). HTA is highly fluorescent, with an excitation wavelength sufficiently high that there is little or no interference from tryptophan fluorescence. Our results on the melanin suspensions show that while red hair melanin (largely pheomelanin) has a smaller intrinsic ESR signal, and a smaller increase upon irradiation, compared to brown hair melanin (largely eumelanin), irradiation of red hair melanin produces a larger yield of oxygen radicals than either brown hair melanin or sepia, which is pure eumelanin. In this paper, we report on the combined use of ESR spectroscopy