Methodology for Evaluation of Melanin Content and Production of Pigment Cells in Vitro†
This invited paper is part of the Symposium-in-Print: Melanins.
*email: firstname.lastname@example.org (Dan-Ning Hu)
Melanin has a photo-screening, a biophysical/biochemical and a cosmetic effect. Melanin content of cultured pigmented cells can be measured by spectrophotometry and expressed either as melanin content per cell or melanin content per culture (area). Melanin production can be calculated from melanin content and cell number at the beginning and at the end of a culture using various formulas and expressed as melanin production per cell per day or melanin production per culture per day. Melanin content or production per cell have been used widely to compare melanin content in various cell lines or to compare the melanin content during different stages in the culture (e.g. growing stage and senescent stage). For the evaluation of changes in melanin content and production in a given pigment cell line after treatment with a special chemical, physical or biological stimulator or inhibitor, different parameters used for the evaluation of experimental data can lead to conflicting results. Melanin content per area is determined by melanin content per cell and the number of cells in this area. The biological and cosmetic effects of melanin in vivo are determined mainly by melanin content per area, not melanin content per cell. For example, if melanin content per cell is the same, but the number of cells in a given area is increased after the treatment, then the melanin content per area is also increased. Under this circumstance, the color of skin turns darker and the total antioxidant activity provided by melanin in this area is increased even though the melanin content per cell measured remains the same; therefore, melanin content or production per culture is more important than melanin content or production per cell under this circumstance.
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Melanin plays an important role in the physiology, pathology and toxicology of the skin, the eye and the brain (1–10). Melanin is produced by various pigment cells and located in the melanosomes of these cells. Melanin has both a photo-screening effect and a biophysical/biochemical effect. Melanin absorbs near-infrared, visible light and UV radiation with absorption increasing in the shorter wavelengths, therefore melanin may protect the tissues from damage caused by visible light or UV radiation. Melanin is an antioxidant, a free radical scavenger and has an affinity for metals and other toxic chemical substances. Because of these properties, melanin efficiently filters toxic substances and protects tissues from oxidative and chemical stress. Unfortunately, with chronic exposure the properties of melanin change so that under severe oxidative stress and binding of excessive amount of toxic substances, melanin itself may induce damage to cells. Aging in pigment cells may also diminish the protective properties of melanin, leading to injury of tissues, resulting in diverse neural, ocular and dermal diseases.
The quantity and quality of melanin in the skin and iris are the most important factors that determine the color of the iris and skin. People from different ethnicities have different skin color preferences, for example, in the western countries, people prefer the tan color. On the contrary, in Asia, especially in China and Japan, people prefer lighter skin colors. Billions of dollars are spent on cosmetics each year that allow people to obtain their ideal skin color. Therefore, studies on the modulation of quantity and quality of melanin are important both in theoretical and practical aspects.
Dermal and ocular melanocytes and ocular pigment epithelial cells can be successfully cultured in vitro. Melanin content and melanin production in cultured pigmented cells correlate with those of corresponding cells in vivo (11–21). Therefore, cultured pigment cells have been used as an in vitro experimental model system for the study of the modulation of melanogenesis. Melanin content and melanin production of cultured pigmented cells have previously been measured and evaluated using a variety of methods and parameters (21–32). Because different parameters have been used for the evaluation of melanin content in cells, the results obtained from different in vitro studies can lead to conflicting opinions. How to evaluate the results obtained from these studies and which parameters should be selected to evaluate these results are the subject of the present study.
Methods for Measurement of Quantity of Melanin
Spectrophotometry, chemical degradation followed by HPLC and ESR are methods that have been used for the measurement of melanin content of specimens from pigmented tissues or from cultured pigment cells. Each method has its own advantages and disadvantages (21,32). Measurement of melanin content from cultured pigment cells is relatively easy compared with melanin analysis in pigmented whole tissues because there is interference from other tissue components not present in cells. Melanin in cultured cells is also relatively easy to dissolve. The spectrophotometric method, which is based on the absorption of light at a special wavelength by melanin, is the most popular and a relatively simple method used for the measurement of melanin contents in cultured pigment cells. If used properly, this method can provide reliable results, especially for the comparison of results before and after the treatment of cells with a specific reagent (21–32).
The spectrophotometric method for measuring melanin content has been previously reported. Briefly, cultured pigment cells are detached from the cultured flask by trypsin-EDTA solution and counted in a hemocytometer, the cell suspensions are centrifuged, and the pellet is dissolved in 1 N NaOH. Melanin concentration is determined by measurement of optic density at 475 nm and compare with a standard curve obtained using melanin isolated from sepia (SIGMA) (21,27,32).
Parameters Used For Evaluating The Results Of Measurement Of Melanin Content
There are two different parameters that have previously been widely used for the evaluation of melanin content: melanin content per cell (MC) and melanin content per culture (or per area, MT). Melanin content per cell is determined by the quantity of melanin in each pigment cell. Melanin content per area is determined both by melanin contents per cell and the density of cells in a given area (21–31).
Melanin content per cell can be calculated by dividing the amount of melanin by the number of melanin containing cells (ng per cell). Melanin content per culture can be calculated by multiplying melanin per cell by the total number of cells in the flask (ng per well). For example, melanocytes in a confluent culture flask are detached by trypsin-EDTA and counted, the number of cells in this flask is 1 × 106. One fifth of the cells (2 × 105) are centrifuged, the cell pellet is dissolved in 1N NaOH. The amount of melanin as determined by measurement of optic density at 475 nm and compared with a standard curve is 4400 ng melanin. The final melanin content per cell would be 4,400 ng divided by 2 × 105 and equal to 0.022 ng per cell. Melanin content per culture is 0.022 ng × 1 × 106 and equal to 22 000 ng, or 22 μg melanin per flask (21).
These two different parameters have previously been used for the description and evaluation of melanin content in a given pigment cell line at a certain growth period or for the evaluation of the changes in melanin content in a pigment cell line after treatment with a special chemical, physical or biological stimulator or inhibitor (21–31).
For the description of melanin content in a given pigment cell line at a certain growth period, melanin per cell has been widely used to compare melanin content in various cell lines or to compare the melanin content during different stages in the culture (e.g. growing stage and senescent stage) (15,16,21).
For the evaluation of the changes in melanin content in a given pigment cell line after treatment with a special chemical, physical or biological stimulator or inhibitor, melanin content per culture is more important than melanin content per cell, because the biological and cosmetic effects of melanin in vivo are determined mainly by melanin content per area, not melanin content per cell. Melanin content per area is determined by the melanin per cell and the number of cells in a given area. If melanin content per cell is the same, but the number of cells in a given area is increased after the treatment, then the melanin content per area is also increased. Under this circumstance, the color of skin turns darker and antioxidant activity by melanin in this area is increased even though the melanin content per cell measured remains the same. Therefore, melanin content per area (or per culture) is a better parameter than melanin content per cell for the evaluation of changes in melanin content after cells have been with a stimulator or inhibitor in vitro (25,27,29,31).
Methods And Parameters For Measurement Of Melanin Production
There are mainly two different parameters for the measurement of melanin production—the measurement of the changes of melanin content per culture in a given time period (MPT) or the melanin production by each cell in a given period (MPC).
In the first method, the calculation is relatively simple and can be calculated using Eq. (1):
where MPT is the production of melanin in a culture dish per day (ng per culture per day), t is the time period (day), MTo and MTt represent melanin content per dish at the beginning and at the end of the culture, respectively. For example, 2 × 105 pigment cells with melanin content per cell at 0.025 ng are seeded into a flask. After culture for 6 days, the cell number increases to 1 × 106 cells and melanin content per cell measured is 0.030 ng, then MTt is 0.030 × 1 × 106 = 30 μg and MTo is 0.025 × 2 × 105 = 5 μg. The total melanin produced by cells in this flask is 25 μg and equals 4.17 μg per culture per day.
In the second method, melanin production by each cell in a given period can be calculated using Eq. (2) (21):
where MPC is melanin production per cell per day during time t (ng per cell per day); MCt and MCo are the melanin content per cell (ng per cell) at the end and beginning of culture, respectively; t is the time period (day); P is the population increase during time t; and D is the doubling time of the cells, which can be calculated using Eq. (3) (12):
where Nt and No represent the cell number at the end and at the beginning of culture during time t (day), respectively.
For example, 2 × 105 pigment cells (No) with melanin content per cell at 0.025 ng (MCo) are seeded into a flask. After culture for 6 days, 1 × 106 cells (Nt) are collected and the melanin content per cell measured is 0.030 ng (MCt), then, P is 1 × 106/2 × 105 = 5. D as calculated by Eq. (3) is 2.58. The melanin content production by each cell per day (MPC) as calculated by Eq. (2) is 0.00932 ng per cell per day.
Based on the same reason previously mentioned for melanin content, for the description of melanin production in a given pigment cell line at a certain growth period, melanin production per cell has been previously used to compare melanin production in various cell lines or to compare the melanin production during different stages in the culture (e.g. growing stage and senescent stage) (15,21).
For the evaluation of the changes in melanin production in a given pigment cell line after treatment with a special chemical, physical or biological stimulator or inhibitor, melanin production per culture is more important than melanin production per cell.
Besides the measurement of melanin, measurement of tyrosinase activity can also be used to estimate melanogenesis of cultured pigment cells (33). Tyrosinase holds a central position in the biosynthesis of melanin because of its ability to catalyze the first two rate-limiting synthesis reactions, namely the hydroxylation of tyrosine to DOPA and its subsequent oxidation to dopaquinone. It was previously thought that the subsequent steps proceeded more or less spontaneously, ending with the formation of melanin. However, more recently, it has been found that many other factors regulate melanogenesis, such as the activity of auxiliary enzymes (dopachrome tautomerase and peroxidase, etc.), and certain metal ions, especially copper and iron (34–36). An additional level of genetic control is involved in the synthesis of melanin even prior to tyrosinase (37,38). Therefore, tyrosinase activity is an important factor, but not the sole factor for determining the rate of melanin production.
There is also a race/ethnicity relationship involved with melanin synthesis. In our previous studies, uveal melanocytes from eyes from Black donors contained higher tyrosinase activity than those from Caucasian donor eyes. However, when the tyrosinase activity of cultured uveal melanocytes from eyes with light irides were compared to that from dark irides from only Caucasian donor eyes, approximately equivalent levels of tyrosinase activity were present in these two groups. Furthermore, in uveal melanocytes from Caucasians, the tyrosinase activity did not correlate with melanin content and melanin production in vitro, indicating that although a racial difference of tyrosinase activity was present in the cultured uveal melanocytes, within the same race, the tyrosinase activity in vitro did not correlate well with the iris pigmentation in vivo (21). Measurement of tyrosinase activity has its limitation in the evaluation of melanogenesis. Furthermore, measurement of melanin content is more direct and meaningful than that of tyrosinase activity. Therefore, our discussion on the measurement of melanogenesis of cultured pigment cells will be restricted to the studies of melanin production calculated by melanin content and cell growth capacity.
Relationship Between Melanin Content, Melanin Production And Cell Growth
Melanin content per cell in cultured pigment cells is not only determined by the production of melanin, but also by the cell growth rate. In stationary or near-stationary cells (for example, in senescent cells), the melanin produced accumulates within the cell and results in a rapid increase in melanin content per cell (accumulation effect) (Table 1). In growing cells, the melanin is diluted in daughter cells during division (dilution effect). If the melanin production rate equals the rate of dilution, the melanin content per cell will remain unchanged, as seen in the growing stage of melanocytes (Table 1). In rapidly growing cells, if the dilution rate is greater than that of melanin production, melanin content per cell will decrease, as in the first few passages of melanocytes. In pigment cells that do not produce melanin in vitro (for example, in human adult retinal pigment epithelium), during growth stage, melanin content per cell decreases rapidly and in proportion to cell division (Table 2), while melanin content per cell remains unchanged if the cell does not grow (as in stationary cells) (21,27,31).
Table 1. Melanin content and production and cell growth in cultured uveal melanocytes.
| No||2 × 105||2 × 105||2 × 105|
| Nt||15 × 105||16 × 105||2.4 × 105|
| MTt||40 500||41 600||36 000|
Table 2. Melanin content and production and cell growth in cultured human retinal pigment cells.
| No||3 × 105||2.5 × 105||2.3 × 105|
| Nt||15 × 105||13 × 105||12 × 105|
In the study of the effects of various stimulators or inhibitors on melanogenesis of cultured pigment cells, cell growth rate also plays a role in the results of melanin content per cell. A growth stimulator that has no effect on the production of melanin per cell causes an increase in cell number and a resultant reduction in melanin per cell due to the dilution effect compared with the control (cultured without stimulator). Based on the melanin content per cell, the effect of this stimulator on melanin content could be evaluated as an inhibitor. On the contrary, the melanin content per culture is increased compared with the control because the number of cells increases over the proportion of decrease in melanin per cell (Table 3). Because the biological and cosmetic effects of melanin are determined by melanin content per area, the use of this stimulator may cause an increase in the darkness of skin color and an increase in total antioxidant ability of melanin in this area (if the in vivo effect of this stimulator is similar to the in vitro effect). Therefore, this stimulator can be evaluated as a growth stimulator and a mild melanin content elevating agent (e.g. effects of 12-O-tetradecanoyl-phorbol-13-acetate on cultured uveal melanocytes). Only if the melanin content per culture and melanin production per culture in the treated group remain similar to that in the control group, this stimulator can be evaluated as a simple growth stimulator without effects on melanin content (e.g. effects of basic fibroblast growth factor on cultured uveal melanocytes) (Table 4).
Table 3. Effects of a growth stimulator on melanin content and production and cell growth in cultured uveal melanocytes.
| No||2 × 104||2 × 104|
| Nt||16 × 104||8 × 104|
Table 4. Effects of a growth stimulator on melanin content and production and cell growth in cultured uveal melanocytes.
| No||2 × 104||2 × 104|
| Nt||16 × 104||2.4 × 104|
In conclusion, the determination of melanin content in either pigment containing tissues or cultured cells can be complex. Often the evaluation of changes in melanin content and production in a given pigment cell line after treatment with a special chemical, physical or biological stimulator or inhibitor lead to conflicting results because different parameters are used for the evaluation of experimental data. It is hoped that this review has at least clarified some of the reasons for the differing reported results.