PracticeUpdate: Dermatology - Winter 2018

IID 2018 27

Vitamin C and Its Derivatives Suppress Melanogenesis Vitamin C and its derivatives have been shown to inhibit tyrosinase activity and melanogenesis, and constitute a promising strategy for screening depigmenting agents. In addition, a new model has shown usefulness in evaluating effects of skin-lightening agents on melanogenesis, results of two in vitro studies show.

Suppression of melanogenesis using vitamin C and its derivatives T iechi Lei, MD, of Wuhan University in China, explained that a melanosome is an acidic organelle enveloped by the monolayer plasma membrane. The intraluminal pH of melanosomes is approximately 4–5. The optimal pH for tyrosinase, however, has been shown at 6.8. Modulation of intraluminal pH in mel- anosomes seems helpful for screening reversible skin-lightening agents. Dr. Lei and colleagues set out to deter- mine whether vitamin C and its two derivatives, magnesium ascorbyl phos- phate and 3-o-ethyl-L-ascorbic acid, would acidify melanosomes, thereby inhibiting melanogenesis. Melan-a cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 200 nM phorbol 12-myristate 13-acetate, and 100 μM 2-mercaptoetha- nol, at 37°C in 5% CO2. L-ascorbic acid, magnesium ascorbyl phosphate, and 3-O-ethyl-L-ascorbic acid were synthe- sized in Dr. Lei’s laboratory. The above compounds were prepared immediately before use and buffered to PH 7.0 using sodium hydroxide. Melan-a cells were treated with 1 mM, 1 mM magnesium ascorbyl phosphate, and 1 mM 3-o-ethyl-L- ascorbic acid for 48 h. Concanamycin A, a selective vacuolar type H ATPase inhibitor; and ammonium chloride were used to change the acidic intracellular condition. Viabilities of cells treated or untreated with compounds were analyzed using cell counting kit-8 assay. Melan-a cells were assayed for tyrosinase activity and stained to visualize cell labeling using a confocal microscope. Fluorescence was examined using a con- focal microscope. Fluorescence microscopy imaging anal- ysis revealed that the pH in melanocytes treated with vitamin C and its two deriva- tives was substantially lower than that in the untreated control. Concanamycin A and ammonium chlo- ride were able to reverse the inhibition of melanogenesis induced by vitamin C and its derivatives by further alkalizing melanosomes.

In parallel to measurements taken with the chromometer, total melanin content of tis- sues was also quantified. Over time, cultures became increasingly pigmented with retention of normal epi- thelial morphology, with the expected pigmentation level of donor tissue, that is, black, Asian, or Caucasian, when cultured in media containing α melanocyte-stimulat- ing hormone and β fibroblast growth factor. Several over-the-counter skin lightening products were also evaluated in cultures containing normal human melanocytes from black donors. Over the 2–3 week treatment period, control cultures became increasingly pig- mented while tissues treated topically with cosmetic skin-lightening agents containing tyrosinase inhibitors such as kojic acid and magnesium ascorbyl phosphate remained distinctly lighter than control cultures. After 14 days in culture, total melanin con- tent was found to correlate inversely with surface reflectance. Dr. Bachelor concluded that the results suggested that the model is useful for evaluating effects on melanogenesis, skin lightening, and other pigmentation phe- nomena of skin in vitro. In particular, the results highlight two dis- tinct endpoints, total melanin content and skin color measurement, that can be used to evaluate skin pigmentation in vitro. www.practiceupdate.com/c/68653

Dr. Lei concluded that vitamin C and its derivatives were found to inhibit tyrosinase activity and melanogenesis. This inhibi- tion was likely due to the acidification of melanosomes. The finding provides a promising strat- egy for screening of depigmenting agents through acidification of melanosomes and reversible inhibition of tyrosinase. A chromometer in a 3D epidermal model containing functional melanocytes M ichael Bachelor, PhD, of MatTek Corporation in Ashland, Massachu- setts, explained that considerable interest has arisen in evaluating effects on skin pigmentation using treatment with new materials, skin care formulations, and as side effects induced by medication. A convenient way to screen such effects employs the MelanoDerm tissue model, a highly differentiated, three-dimensional tissue culture model of human epidermis. The model contains normal human melano- cytes and keratinocytes. Use of this in vitro model can provide valu- able in vitro data as an early screening tool prior to costly clinical trials. Dr. Bachelor and colleagues evaluated pigmentation over 2–3 weeks using a tristimulus chromometer to measure bright- ness in MelanoDerm tissue produced with normal human melanocytes from black, Asian, or Caucasian donors.

Courtesy of IID 2018

VOL. 2 • NO. 3 • 2018