Quantification of Hypopigmentation Activity In Vitro

Summary

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

Abstract

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.

In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

Introduction

Melanin plays a critical role in the physiology, pathology, and toxicology of several organs including the skin, eyes, and brain¹. Major functions of melanin are photo-screening and biochemical effects. Melanin absorbs near-infrared and visible light as well as ultraviolet (UV) radiation, with increased absorption rates at shorter wavelengths of light; thus, melanin protects tissues from damage caused by visible light or UV radiation². Melanin is an antioxidant and has an affinity for metals and other toxic chemicals; therefore, it can protect tissues from oxidative and chemical stress³. However, ....

Protocol

1. Preparation of Medium, Compounds, and Reagents

1. Prepare B16F10 growth complete medium. Supplement Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PEST).
   1. To make 500 mL of complete medium, mix 445 mL of Dulbecco’s modified Eagle’s medium (DMEM) with 50 mL of FBS and 5 mL of PEST in a 1 L sterilized glass bottle. After mixing gently, filter the medium through a 0.2-µm bottle-top filter to a new sterilized glass bottle and then store at 4 °C.
      CAUTION: All cell culture techniques should be undertaken in microbiological safety cabinet using a....

Results

Representative results for the hypopigmentation activity of arbutin, an anti-melanogenic compound, in B16F10 melanocytes are shown below. Figure 1A shows that arbutin significantly suppressed the cellular tyrosinase activity compared with the vehicle-treated control. Similarly, the melanin content of cells stimulated with arbutin was significantly reduced compared with the controls (Figure 1B). Microscopic images of cells stained.......

Discussion

We presented protocols for evaluating the hypopigmentation activity of test compounds using cultured melanocytes. The representative results showed the hypopigmentation effect of arbutin, a tyrosinase inhibitor that inhibited tyrosinase activity and cellular melanin content. These methods are widely used in anti-melanogenic activity research. Using these assays, we have also successfully identified several bioactive compounds that have melanogenesis inhibitory effects on B16F10 cells in the past decade^9.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3,4-Dihydroxy-l-phenylalanine	Sigma	D9628
Ammonium hydroxide solution	Sigma	#320145
Arbutin	Fluka	#10960
DMEM	HyClone	SH30243.01
FBS	HyClone	SH30084.03
Formalin	Yakuri Pure Chemicals	#16223	37%
Gold chloride	American MasterTech	AHG0226	0.10%
HCl	Samchun chemical	H0255
KCl	Bio basic Canada Inc.	PB0440
KH2PO4	Sigma	#60218
Na2HPO4	J.T.Baker	#3817-01
NaCl	Duksan pure chemicals	#81
NaOH	Sigma	655104
Nuclear fast red	Merck	100121	Nuclear fast red 0.1% in 5% aluminum sulfate.
Penicillin and streptomycin solution	HyClone	SV30010
Silver nitrate	Duksan Pure Chemicals	#900
Sodium phosphate dibasic (Na2HPO4)	J.T. Baker	#3817-01
Sodium phosphate monobasic (Na2HPO4)	Sigma	S5011
Sodium thiosulfate pentahydrate	Duksan Pure Chemicals	#2163
Tris(hydroxymethyl)aminomethane	Bio Basic Canada	TB0196
Triton X-100	Union Carbide	T8787
Tyrosinase from mushroom	Sigma	T3824	25 KU