A Standardized Protocol for Assessing Regulators of Pigmentation Victoria M. Virador, Nobuhiko Kobayashi, Jun Matsunaga, and Vincent J. Hearing 1 Laboratory of Cell Biology, National Institutes of Health, Bethesda, Maryland 20892 Received November 4, 1998 Varied effects of chemical or biological compounds on mammalian pigmentation have been reported by many groups, but to date, no standardized method has established necessary and/or optimal parameters for testing such agents. A standardized method has been developed to screen compounds with potential effects on pigmentation. The protocol comprises basic param- eters to analyze melanogenic effects and allows for further characterization of candidate compounds, providing important insights into their mechanism of action. In this protocol (termed STOPR, for standard- ized testing of pigmentation regulators), compounds are initially screened using purified tyrosinase and are then tested on melanocytes in culture. After treat- ment of melanocytes with potentially bioactive com- pounds, cell proliferation and viability, total melanin accumulated, and melanogenic potential are mea- sured. This protocol is an important first step in char- acterizing chemical regulation of effects on melano- genesis. When bioactive candidate compounds are identified, testing may proceed for pharmacological or otherwise commercial applications in coculture and/or organ culture models followed by in vivo test- ing. As an application of this method, results for com- pounds known to stimulate and/or inhibit melanogen- esis (including arbutin, hydroquinone, kojic acid, melanocyte-stimulating hormone, and thymidine dimers) as well as some commercial skin whiteners are reported. Key Words: melanin; pigmentation; assay; tyrosinase. Pigmentation in mammals is important to numerous distinct and critical processes, as illustrated by its ap- pearance in various tissues of the body. Despite recent breakthroughs in the characterization of genetic and biochemical determinants of pigmentation, much re- mains unknown about the role of melanin in embryonic development, its function(s) in certain parts of the brain, eyes, and ears, its photoprotective nature in the skin, and its determination of phenotypic appearance. Skin and hair color depends on the amount, size, and type of melanins produced by melanocytes, pigment- producing cells found at the epidermal– dermal junc- tion and in hair follicles (reviewed in 1–5). Starting with the enzyme tyrosinase, and in concert with other melanogenic enzymes, melanin is synthesized in mela- nosomes, is transferred from melanocytes to keratino- cytes, and eventually disappears with desquamation of the skin and/or hair growth. Melanin is also an impor- tant defense of human skin against the harmful effects of UV light due to its ability to absorb and reflect UV energy, and its ability to scavenge oxidative free radi- cals (6 –10). There is a dramatic inverse correlation between skin pigmentation and incidence of skin ma- lignancies and UV photodamage (11–15). Pigmenta- tion in humans also has an important cosmetic role which can be compromised in certain hyperpigmentary skin conditions and/or in lesions such as melasma, age spots, and postinflammatory hyperpigmentation (16 – 19). There has been great variation in studies on various bioactive compounds targeted at regulating melanin production; in part, this variation stems from diverse conditions of melanogenic assay, different cell lines (including melanoma cells) used as targets, and differ- ent end points used to establish efficacy (20 –23). In an attempt to standardize the assessment of novel bioac- tive agents for therapy of pigmentary lesions, a method has been developed which allows extensive testing of putative bioactives with minimal expense and effort, which renders outstanding reproducibility, and which accurately determines efficacy of action. In the 1 To whom correspondence should be addressed at Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Building 37, Room 1B25, Bethesda, MD 20892. Fax: (301) 402-8787. E-mail: hearingv@nih.gov. 0003-2697/99 207 Analytical Biochemistry 270, 207–219 (1999) Article ID abio.1999.4090, available online at http://www.idealibrary.com on