INTRODUCTION

Skin is the largest and outermost organ of the human body. The skin is frequently and directly exposed to a pro-oxidative environment, including ultraviolet radia­tion (UV), drugs, and air pollutants. Be­sides external inducers of oxidative at­tack, the skin has to cope with endogen­ous gen­eration of reactive oxygen spe­cies (ROS) and other free radicals, which are con­tinuously produced during physi­ological cellular metabolism.

Balance between oxidants and antioxi­dants is needed to minimize molecular, cellular, and tissue damage. However, if the balance is upset in favour of the oxi­dants, oxidative stress could occur and results in oxidative damage. Reactive oxygen species (ROS) are known to cause oxidative modification of DNA, proteins, lipids and small cellular mole­cules (Kang et al. 2005). To counteract the harmful effects of ROS, the various compartments of the skin (epidermis, dermis, subcutis) are equipped with lay­erspecific antioxidant systems to main­tain an equilibrium between ROS and antioxi­dants and thus prevent oxida­tive stress (Thiele and Ekanayake-Mudiyanselage 2007).

Skin aging is defined as structural changes of skin as the age increase. Ag­ing is indicated as inability to balance the important functions of skin leading to cell death. The free radical theory of ag­ing explains the importance of antioxi­dants in reducing pigmentation in older people (Chandrosoma & Taylor 2001).

The human pigmentary system is de­pendent on the production of the light ab­sorbing biopolymer, melanin, within epi­dermal, ocular and follicular melano­cytes (Nordlund et al. 1998). Melano­cytes within the skin are situated on the basal layer between the dermis and epi­dermis and have a number of dendritic processes that interdigitate with the sur­rounding keratino­cytes. While pigment synthesis occurs within the melanocytes, the majority of pigment within the skin is found in melanin laden vesicles known as melanosomes located within the kerati­nocytes (Sturm et al. 2001).

Melanin synthesis is also known as melanogenesis where the pigment mela­nin is formed. The melanin pigments are of no fixed molecular weight but are all derived by enzymatic oxidation of the amino acid tyrosine and eventually pro­duce two types of melanin in mammalian skin which are feomelanin and eumelanin (Prota 1992; Nordlund et al. 1998). Tyro­sinases catalyze two distinct reactions in melanin synthesis: the hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and the oxidation of DOPA to dopaquinone (Tripathi et al. 1992). The eumelanins are derived from the metabo­lites of DOPAchrome, whereas the pheo-melanins are derived from metabo­lites of 5-S-cysteinylDOPA. The isomeri­zation of DOPAchrome to 5,6-dihydroxy­indole-2-carboxylic acid (DHICA) is cata­lysed by DOPAchrome tautomerase and the oxi­dation of DHICA is performed by a DHICA-oxidase enzyme (Sturm et al. 2001).

Important genes involved in melanin synthesis pathway are TYR, TYRP1 and TYRP2. Tyrosinase is encoded by the TYR or c-locus that maps to chromo­some 11q14–21 in humans (Barton et al. 1988) and chromosome 7 in mice, re­spectively. It is composed of five exons and four in­trons (Jimbow et al. 2000). The gene for TYRP1 is 37 kb long and contains eight exons separated by seven introns (Nordlund et al. 1998). The gene for TYRP2 is 60 kb long and contains eight exons and seven introns; all eight exons encode the final protein (Budd and Jackson 1995). Gene and protein struc­tures of tyrosinase, TYRP1 and TYRP2 consist of NH2- terminal domain of tyrosi­nase which comprises of the NH2-terminal signal peptide that is important for intra­cellular trafficking and process­ing, the EGF-like/cysteine-rich domain, two histi­dine-rich regions binding copper with a cysteine-rich region between them (the important catalytic domain), as well as the COOH-terminal hydrophobic trans­membrane segment and cytoplas­mic tail (Kwon 1993). The transmem­brane and cyto­plasmic domains are nec­essary for tar­geting the enzyme to the melanosome (Jimbow et al. 2000; Selaturi 2000)while the NH2 terminus cysteine-rich region may serve as a pro­tein binding/regulatory do­main unrelated to enzymatic function.

Tocopherols and tocotrienols are sub­families of vitamin E (Brigelius-Flohe & Traber 1999). Both tocopherols and to­cotrienols have isomers, designated as -α, -β,-δ and -γ which differ by the number and position of the methyl groups on the chromanol ring (Machlin 1991). Tocotrie­nol rich fractions are vitamin E that con­sists of 75% alpha-tocotrienol and 25% tocopherol (Mutalib et al. 2003). Toco­trie­nol rich fractions are found in barley, oats, palm, and commercial rice brans (Qureshi & Qureshi 1993).

In this study, the anti pigmentation prop­erties of palm tocotrienol rich fraction was evaluated. Primary culture of skin melanocytes was treated with tocotrienol rich fraction to evaluate the effects of tocotrienol rich fraction on melanin syn­thesis by determining melanin content and tyro­sinase enzyme activity. Determi­nation of gene expression involved in the regulation of melanin synthesis such as TYR, TYRP1 and TYRP2 was also car­ried out.

MATERIALS AND METHODS

Cell culture

Human melanocytes obtained from the fore­skin of nine to 12 year-old boys were grown in Medium 254, which is a basal medium containing essential and non essential amino acids, vitamins, organic com­pounds, trace minerals, and inor­ganic salts (Cascade Biologics, USA). The me­dium was supplemented with Human Melanocyte Growth Supplement-2 (HMGS-2), which contains 0.5% fetal bo­vine serum, 3 ng/ml basic fibroblast growth factor (human recombinant), 0.2% bovine pituitary extract, 3 µg/ml heparin, 0.18 µg/ml hydrocortisone, 5 µg/ml insu­lin, 5 µg/ml transferrin, and 10 ng/ml phorbol 12- myristate 13-acetate. Written consent was obtained from parents or guardians of all subjects.

Cell viability assay

Cell viability was assessed with CellTiter 96* Aqueous Non-Radioactive Cell Pro­liferation Assay (MTS, Promega, USA). The MTS assay em­ploys 3-(4,5-di­me­thyl­thiazol-2-yl)-5-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium (MTS) and the electron coupling agent phenazine methosul­phate (PMS). The MTS com­pound is reduced by the dehy­droge­nase en­zymes found in metaboli­cally ac­tive cells into a formazan product that is solu­ble in the medium. The amount of colored formazan product is propor­tional to the number of viable cells. Briefly, 20 µl MTS solution was added to each well and incu­bated in a humidi­fied incubator at 370C in 5% CO2 for 2 - 4 hours. The quantity of formazan product present was determined by measuring the absor­b­ance at 490 nm with a micro­titer plate reader (VeraMax Molecular Devices, USA).

Determination of melanin content

Melanin content was determined ac­cording to Huang et al. (2008) with slight modification. The cells (105) were treated with 500µg/ml TRF (Sime Darby Bhd, Malaysia) for 24 hours. Cell pellets were dissolved in 1 ml of 1 N NaOH at 37 °C overnight and centrifuged for 10 minutes at 10,000 x g. The optical density (OD) of the supernatants was measured at 450nm using the µQuant microplate reader. Melanin concentration was calculated by comparing the OD at 450 nm of unknown samples with a standard curve (Figure 2).

Determination of cellular tyrosinase activity

Cellular tyrosinase activity was measured according to Lin et al. (2007), with slight modification. Melanocytes (105) were cultured in 24-well plates for 24 hours followed by 24 hours treatment with 500µg/ml TRF (Sime Darby Bhd, Malay­sia). Cells were then washed and lysed with potassium phosphate-buffered sa­line (PBS) pH 6.8 containing 1% Triton X-100 and ruptured by freezing at -80°C and thawing in a water bath. Cell lysates were then clarified by centrifugation at 10,000 x g for 10 minutes. Protein con­tent was determined using Bradford as­say. Protein concentrations were ad­justed with lysis buffer until each lysate contained the same amount of protein (40 µg). The final reaction mixture in each well contained the cell lysate, 10 µl 2.5 mM L-dopa, and 0.1 M PBS pH 6.8. Absorbance was then measured at 475 nm using the µQuant microplate reader after incubation at 37°C for 1 hour.

RNA extraction

Total RNA from fibroblast cells in differ­ent groups were extracted using TRI Re­agent (Molecular Research Centre, Cin­cinnati OH) according to the manu­fac­turer’s instruction. Polyacryl Carrier was added in each extraction to precipi­tate the total RNA. Extracted RNA pellet was then washed with 75% ethanol and dried before being dissolved in RNase and DNase free distilled water (Gibco Invitro­gen Corp.). Total RNA was stored at -80°C immediately after extraction. Yield and purity of the extracted RNA was de­termined by Nanodrop (Thermo Scien­tific).

Primer design

Primers for human GADPH and gene of interest were designed with Primer 3 software and blasted with Genebank da­tabase sequences. The sequence of the primers is shown in Table 1.

Real time RT-PCR

Gene expression of TYR, TYRP1, TYRP2 was quantitatively analysed with real time RT-PCR technique. The expression level of each targeted gene was normalized to GAPDH. Primers for hu­man GAPDH, TYR, TYRP1, TYRP2 were designed with Primer 3 software and blasted with GeneBank database se­quences in order to obtain primers with high specificity. The efficiency and speci­ficity of each primer set were confirmed with standard curve (Ct value versus se­rial dilution of total RNA) and melting profile evaluation. Real time RT-PCR re­action was performed with 100 ng of total RNA, 400 nM of each primer and iScript One-Step RT-PCR kit with SYBR Green (Biorad) according to the manu­facturer’s instruction. Reactions were run using Bio-Rad iCycler with reaction pro­file as follows; cDNA synthesis for 30 min at 50°C; pre-denaturation for 2 min at 94°C; PCR amplification for 38 cycles with 30 sec at 94°C, 30 sec at 60°C and 30 sec at 72°C. This was followed by a melt curve analysis to determine the reaction specificity. Agarose gel electro­phoresis was performed for confirmation of the PCR product. Expression level of each targeted gene was normalized to GAPDH.

Statistical analysis

Each experiment was carried out in tripli­cates with at least 3 independent cultures with comparable results. Data are re­ported as mean + SD of at least three experiments. Comparison between groups was made by Student’s t-test (two-tailed). P<0.05 was considered sta­tistically significant.

RESULTS

Effects of Tocotrienol Rich Fraction on cell viability

Incubation of melanocytes with different concentrations of tocotrienol rich fraction (100, 200, 300, 400, 500 ug/ml) for 24 hours caused an increase in the number of viable cells (Figure 1). The percentage of the viable cells was increased with in­creased concentration of tocotrienol rich fraction. Tocotrienol rich fraction at 500 ug/ml was used in this study to determine its effects on melanin synthesis.

Effects of Tocotrienol Rich Fraction on melanin content and tyrosinase activity.

Melanin concentration was calculated by comparing the OD at 450 nm of unknown samples with a standard curve (Figure 2). Results showed that treatment with toco­trienol rich fraction caused a signifi­cant reduction in melanin content (Figure 3) (p<0.05) as compared to the untreated control. Tyrosinase activity was signifi- cantly lower (p<0.05) in melanocytes treated with tocotrienol rich fraction (Fig­ure 4) compared to the untreated control.

Effects of tocotrienol rich fraction on the expression of TYR, TYRP1 and TYRP2 genes

The agarose gel electrophoresis showed a single band of PCR product indicating that the primers were specific (Figure 5a). The melting curve analysis showed single and narrow peak as an indication of primer specificity (Figure 5b-e)

Results showed that treatment with to­cotrienol rich fraction caused a significant downregulation of TYRP2 gene (p<0.05) compared to the untreated control (Fig­ure 6). Expression of TYR and TYRP1 genes was not significant from the un­treated control.

DISCUSSION

Previous study reported that com­pounds with redox properties or (antioxidants) may have anti pigmentation effects by interacting with o-quinones or interacting with copper at the tyrosinase active site, thus avoiding the oxidative polymeriza­tion of melanin intermediates. Redox agents could inhibit second messengers which were able to stimulate epidermal melanogenesis either directly or indirectly by scavenging reactive oxygen species generated in the skin following UV expo­sure (Karg et al. 1993). Our findings showed that melanin content and tyrosi­nase activity were significantly reduced in melanocytes treated with 500 µg/ml to­cotrienol rich fraction.

It has been reported that α-tocopherol derivatives could inhibit tyrosinase in vitro (Shimizu et al. 2001) and inhibit melano­genesis in epidermal melanocytes (Ichihashi et al. 1999). The antioxidant properties of a-tocopherol (25% in to­cotrienol rich fraction), which interfere lipid peroxidation of melanocyte mem­branes and increased intracellular glu­tathione content, could explain its depig­menting effect (Marmol et al. 1993). Other antioxidants such as ascorbic acid interfere with the different steps of melanization by interacting with copper ion at the tyrosinase active site (Gukasyan 2002) or/and reducing dopaquinone as well as blocking DHICA oxidation (Ros et al. 1993) thus causing reduction in pigmentation.

Tyrosinase activity was an important and significant parameter to measure melanogenesis in pigment cell culture (Hu 2008). The significant reduction in tyrosinase activity and melanin content in the tocotrienol rich fraction-treated melano-cytes in this study showed inhibi­tion of melanogenesis in these cells. Similar findings were reported in previous studies that used active constituents from formosan apple (Lin et al. 2007), aloesin (Jones et al. 2002), picnogenol (Kim et al. 2008) and anemonin (Huang et al. 2008).

Melanin synthesis was affected by many factors such as the presence of TRP-1 and TRP-2 proteins, UV expo­sure, prostaglandin, vitamins, growth factors, interleukin, interferon and hor­mones such as alpha-melanocyte stimu­lating hormone, adrenocortropic hormone and endothelin-1 (Fang et al. 2002). Ge­netic control was also involved in melanin synthesis (Rinchik et al. 1993).

Our results showed that TYRP2 geneexpression was significantly reduced in tocotrienol rich fraction-treated melano­cytes while TYR and TYRP1 genesde­creased insignificantly. These findings showed TYRP2 expression was affected by tocotrienol rich fraction treatment but not for TYR and TYRP1 genes expres­sion. A previous study reported that anti­oxidants caused reduction in tyrosinase activity but did not affect the tyrosinase related protein itself (Khatib et al. 2005; Kim et al. 2006).

Huang et al. (2008) reported that anemonin, a natural bioactive compound, could regulate tyrosinase-related proteins and mRNA in human melanocytes. Eberle et al. (2001) showed that catechol could inhibit gene expression encoded for TRP-2 protein which was TYRP2.

TYRis controlled by a quantitative rheostat-like switch which allows con­tinuous transcription of TYR meanwhile TYRP1 is controlled by binary switch that would allow transcription of genes only at certain time. Therefore TYR has variable control mechanisms such as regulation at transcription level, and post translational mechanism as well as affected by mRNA stability (Hazily et al. 2002).

TYR, TYRP1and TYRP genes have been cloned and extrinsic factors regu­lating their expression were recently identified. It was reported that TYRP1 and TYRP2 genes may act together to modulate TYR activity (Manga et al. 2000). This may explained the down regulation of TYRP2 gene in this study that affect the activity of tyrosinase en­zyme.

CONCLUSION

In conclusion, the significant reduction in tyrosinase activity and melanin content in melanocytes treated with tocotrienol rich fraction and down regulation of TYRP2 gene confirmed the anti pigmentation property of palm tocotrienol rich fraction, an invaluable finding in the cosmetic in­dustry.

ACKNOWLEDGEMENT

This study was funded by Sime Darby Bhd, Malaysia grant JJ-001-2008 and Universiti Kebangsaan Malaysia.