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.ALPHA.-Lipoic Acid Inhibits Melanogenesis in Three-dimensional Human Skin Model


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The overproduction of pigmentary melanin in the skin causes an undesirable cosmetic appearance. The present study was undertaken to evaluate the inhibitory effect of α-Iipoic acid (LA) on melanogenesis using a three-dimensional human skin model with ultraviolet (UV) irradiation. It is evident that melanin biosynthesis in human skin cells was decreased by the treatment with LA under the concentration range from 0.01 to 0.1% (w/v) in a concentration-dependent fashion, reflecting no effect on cell viability.
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Food Sci. Technol. Res., 15 (5), 537540, 2009
-Lipoic Acid Inhibits Melanogenesis in Three-dimensional Human Skin Model
Tomonori unno
, Fumihiro ito and Yuko M. SageSaka
Central Research Institute, ITO EN, Ltd., 21 Mekami, Makinohara-shi, Shizuoka 421-0516, Japan
Present afliation: Faculty of Home Economics, Tokyo Kasei-Gakuin University, 2600 Aihara-machi, Machida-shi, Tokyo 194-0292,
Received November 20, 2008; Accepted May 8, 2009
The overproduction of pigmentary melanin in the skin causes an undesirable cosmetic appearance.
The present study was undertaken to evaluate the inhibitory effect of
-lipoic acid (LA) on melanogenesis
using a three-dimensional human skin model with ultraviolet (UV) irradiation. It is evident that melanin
biosynthesis in human skin cells was decreased by the treatment with LA under the concentration range
from 0.01 to 0.1% (w/v) in a concentration-dependent fashion, reecting no effect on cell viability.
-lipoic acid, melanin, tyrosinase, three-dimensional human skin model
*To whom correspondence should be addressed.
-Lipoic acid (LA), chemically designated 1,2-dithio-
lane-3-pentanoic acid, is a natural substance found in plant
and animal foods. It is known that LA functions as a mito-
chondrial cofactor in multienzyme complexes that catalyze
the oxidative decarboxylation of
-keto acids (Bunik, 2003).
Apart from its biological role in the body, LA has an impres-
sive list of beneficial health effects, such as powerful anti-
oxidant activity (Biewenga et al., 1997), removal of several
toxic metals from the body (Patrick, 2002), suppression of
diabetes complications (Packer et al., 2001) and prevention
of obesity by suppressing hypothalamic AMP-activated pro-
tein kinase activity (Kim et al., 2004).
Topical application of LA to the skin to protect against
a variety of skin damage, including photoaging and hyper-
pigmentation, has recently drawn considerable interest. Ex
vivo pig skin models have demonstrated that LA decreases
UVB radiation-induced cell death or apoptosis (Rijnkels et
al., 2003). Animal studies using dark-skinned Yucatan swine
have also shown that topical treatments with LA for 8 wk
suppressed hyperpigmentation of skin (Lin et al., 2002).
However, there is little evidence of a link to a melanin-
suppressing effect by LA in human skin cells. The present
study aimed to assess the inhibitory effects of LA on mela-
nin biosynthesis in a cultured epidermal human skin model.
Three-dimensional human skin equivalent is a useful tool to
investigate cell-cell interactions in the regulation of in vivo
melanogenesis (Monteiro-Riviere et al., 1997), and the ef-
fects of LA have not been examined using this model to date.
Materials and Methods
-lipoic acid and kojic acid were pur-
chased from Wako Pure Chemical Industry, Ltd. (Osaka, Ja-
pan). Proteinase K, synthetic melanin and thiazoyl blue tetra-
zolium bromide (MTT) were obtained from Sigma Chemical
Co. (St. Louis, MO, USA). All other chemicals and organic
solvents were of reagent grade.
Cultured skin model Three-dimensional tissue culture
model of human epidermis from an Asian donor (MEL-
300A, Lot. 5765) was purchased from Kurabo Industries,
Ltd. (Osaka, Japan). Skin cultures were kept at 4
until use,
usually within 24 h of arrival. Before treatment, skin cultures
were transferred to 6-well plastic culture plates containing
0.9 mL of pre-warmed long-life maintenance medium, which
is supplied with the MEL-300A kit, and were equilibrated in
a 5% CO
incubator at 37
for 1 h. After the 1-h pre-equil-
ibration period, the medium was aspirated off, and the skin
model was exposed to 31.5 mJ/cm
UVB for 30 s using a UV
irradiator (Model DT-20MP, ATTO, Co., Tokyo, Japan). Five
milliliters of fresh medium was replaced beneath the skin
ultures, and then a 100L aliquot of tested solution was
applied directly onto the corneum of the skin tissue. LA was
rst dissolved in dimethyl sulfoxide (DMSO), and was then
diluted with phosphate-buffered saline (PBS), with the nal
concentration of DMSO being 0.5% (v/v). The skin cultures
were again maintained in the 5% CO
incubator in a humidi-
fied atmosphere at 37
for 7 d. In the meantime, sample
solutions were changed on the rst and fourth day.
Melanin extraction Skin tissue was homogenized in 0.45
mL of 10 m
M Tris-HCl buffer (pH 6.8) containing 1% so-
dium dodecyl sulfate and 50 μ
M ethylenediaminetetraacetic
acid. To each homogenate, 20 μL of 205 units/mL Proteinase
K was added, and digestion proceeded overnight at 45
. An
dditional 20-μL aliquot of Proteinase K was then added, and
further incubation was performed for 4 h. Samples were then
with 50 μL of 500 m
M sodium carbonate and 10 μL of
30% hydrogen peroxide, and were maintained at 80
for 30
min. After cooling to room temperature, the mixture was ex-
with 100 μL of chloroform:methanol (2:1, v/v). After
centrifuging at 10,000 × g and room temperature for 10 min,
the optical density of the upper layer was read at 405 nm.
Using a commercial melanin reagent, a standard curve was
constructed so that the melanin contents of unknowns could
be determined.
MTT assay Skin tissue was washed with 0.01
three times, Skin models were transferred to well plates
lled with 300 µL of 1 mg/mL MTT solution. After 3-h in-
cubation at 37
in a humidied 5% CO
atmosphere, they
were washed with 0.01
M PBS. Extraction was carried out
by adding 2 mL of isopropanol containing 0.04
n hydrogen
chloride. After leaving to stand for 2 h at room temperature,
absorbance of the extract was determined using a spectro-
photometer at 560 nm. The absorbance at 655 nm was used
as a reference.
Statistical analysis Results are represented as means ±
SeM. One-way analysis of variance (ANOVA) followed by
Tukey’s multiple comparison test was used to assess the sta-
tistical signicance of differences, with a p value of < 0.05
used as a measure of significance. Data calculations were
performed using GraphPad Prism for Windows version 4.0
(GraphPad Software, San Diego, CA, USA).
Inhibition of melanogenesis Figure 1 shows typical
photographs of cultured skin samples 7 d after UV irradia-
tion. The apparent darkening was veried in the control skin
model (Fig. 1a). Supplementation with 0.1% (w/v) LA in the
culture media reduced the biosynthesis of melanin in human
skin cells (Fig. 1b). Quantitative evaluation of its reducing
effects was performed by spectrophotometric analysis of
the melanin extracted from the cultured skin model. When
compared with phosphate-buffered saline, exposure of hu-
man epidermal skin cells to 0.5% (v/v) DMSO tended to
stimulate the melanin biosynthesis, although not to a signi-
cant level. Treatment with LA resulted in the reduction of the
melanin biosynthesis in a dose-dependent manner (Fig. 2).
Tukey’s multiple comparison test applied to the data showed
signicant differences with exposure to LA solutions at nal
concentrations of 0.1% (4.8 m
M) versus the control (0.5%
DMSO only). Meanwhile, kojic acid at 0.1% (7.0 m
M) was
less active than LA.
Cell viability To exclude the possibility that LA inu-
ences cell damage to a significant degree, MTT assay of
cultured human epidermis was performed 7 d after treatment
with LA. The results demonstrated that LA did not show de-
tectable cytotoxicity in the concentration range from 0.01 to
0.1% (w/v) (data not shown).
The three-dimensional cultured skin model is valuable
for dermatological research as an in vitro model for assess-
ing the pharmacological potency, toxicity and metabolism of
Fig. 1. Microscopic observation of three-dimensional reconstructed
skin tissue after treatment without (A) and with 0.1% LA (B). The
skin model was irradiated with UVB at 31.5 mJ/cm
for 30 min, and
was incubated for 7 d with test agents. Magnication: ×100.
Fig. 2. Effects of LA on melanin production in cultured human
skin cells. The amount of melanin in the cells was equivalent for
commercial synthetic melanin. Results are means ±
SeM of triplicate
samples. *p < 0.05 vs. control.
nno et al.
PBS Control 0.01% 0.025% 0.05% 0.1% 0.1%
Melanin (µg/culture
α-Lipoic acid
tested compounds because of its similarity to actual human
skin having the interaction between dermis and epidermis
(Bernerd and Asselineau, 2008, Duval et al., 2003, Shigeta
et al., 2004). UVB radiation can increase melanin biosyn-
thesis and proliferation of melanocytes in the epidermis by
acting on melanocytes directly (Friedmann and Gilchrest,
1987). The present study aimed to evaluate the suppressive
effects of LA on the magnitude of melanogenesis using the
three-dimensional cultured human skin cell. As compared to
controls (0.5% DMSO), treatment of this cultured cell model
with LA resulted in a dose-dependent reduction of melanin
biosynthesis in a concentration range from 0.01% to 0.1%,
not reecting the relevant marker of cytotoxic activity.
It has been documented that the mechanism underly-
ing skin protection by LA may be specic to its antioxidant
potential to scavenge free radicals generated (Podda et al.,
2001). Melanocytes in the epidermis contain melanosomes,
the activity of which is a major determinant of the skin color
to produce melanin. Melanosomes acquire tyrosinase, which
is the key enzyme catalyzing the conversion of
to dopa and dopaquinone, as well as the subsequent auto-
polymerization of melanin. The microphthalmia-associated
transcription factor is implicated in melanocyte development
and also upregulates melanogenesis via transactivation of
tyrosinase gene expression. Lin et al. (2002) showed that LA
reduced the microphthalmia-associated transcription factor
and tyrosinase promoter activities, resulting in depigmenta-
tion. Attention should be focused on the regulative mecha-
nisms of LA, not only a reduction of intracellular tyrosinase
expression but also direct tyrosinase inhibition. The diverse
mechanisms responsible for reducing melanin biosynthesis
in human skin cells are matters for further consideration.
LA can be readily absorbed from after oral dosage and
appears to be readily converted into its reduced form, dihy-
drolipoic acid, in mammalian cells (Takaishi et al., 2007).
Dihydrolipoic acid is capable of enhancing antioxidant
potency by regenerating
L-ascorbic acid vitamin E (Kagan
et al., 1992), and reportedly impacts both the regulation of
tyrosinase gene expression and direct inhibition of tyrosinase
(Lin et al., 2002). Moreover, dihydrolipoic acid is considered
to have superior inhibitory activity in melanin biosynthesis
when compared to parent LA (Tsuji-Naito et al., 2006). To
better understand the detailed mechanisms of orally or topi-
cally administered LA on the inhibition of melanogenesis,
further investigations into the involvement of such LA me-
tabolites and endogenous antioxidants are warranted.
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icity. Altern. Med. Rev., 7, 456-471.
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unno et al.540
... On one hand, it inhibits the expression of microphthalmia-associated transcription factor (MITF), thus inhibiting the expression of the melanogenic enzymes (Tyrosinase, TYRP-1, TYRP-2). On the other hand, it has antioxidant properties in its oxidized form, although it is more antioxidant in its reduced form (dihydropholic acid) (17)(18)(19)(20). In this work, some widely used depigmenting cosmetic agents, which are arbutin, kojic acid, azelaic acid, and α-lipoic acid (see Figure 1), will be evaluated individually and in different combinations to look for synergistic effects between them. ...
... A cytoprotective and antioxidant activity of arbutin has been reported by Seyfi zadeh et al. and Takebayashi et al. (25,26), although they used liver cells and fi broblasts, respectively, instead of melanocytes. Antioxidant effect of kojic acid, azelaic acid, and α-lipoic acid has also been reported (10,12,19,20). So, it might explain this proliferative effect (25). ...
Full-text available
Melanin plays a key role in our skin, protecting us against ultraviolet radiation, but there are situations in which its anomalous accumulation can lead to either aesthetic problems or diseases like melasma. For this reason, it is important to find agents that are able to decrease the skin pigmentation. It has been demonstrated that the melanin synthesis pathway can be inhibited at different levels by different mechanisms of action. The aim of this project is to combine some of these agents with different mechanisms of action on this pathway in order to find synergistic effects in the inhibition of tyrosinase and melanin synthesis. Kojic acid + α-lipoic acid combination are the only ones that have shown a synergistic effect over mushroom tyrosinase. However, this effect is not seen in melanin synthesis inhibition, although this combination is the most effective one. A potentiation effect is seen in arbutin + α-lipoic acid and kojic acid + azelaic acid combination. Kojic acid and α-lipoic acid combination might prove a good approach as treatment for hyperpigmentation disorders.
... It is also a cosmetic ingredient classified as an AOX. 117 Various studies have suggested applications in anti-fibrosis, 171 wound healing, 172 skin lightening, 173 UVinduced pigmentation control, 174 protection from skin damage induced by smoke, 175 as well as anti-aging, 176,177 photoaging 178 and photoprotection, 179 although this is controversial. 180 Among its various mechanisms of action, lipoic acid is an Nrf2-dependent inducer of phase II detoxification enzymes. ...
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The skin is the organ that is most susceptible to the impact of the exposome. Located at the interface with the external environment, it protects internal organs through the barrier function of the epidermis. It must adapt to the consequences of the harmful effects of solar radiation, the various chemical constituents of atmospheric pollution, and wounds associated with mechanical damage: oxidation, cytotoxicity, inflammation, and so forth. In this biological context, a capacity to adapt to the various stresses caused by the exposome is essential; otherwise, more or less serious conditions may develop accelerated aging, pigmentation disorders, atopy, psoriasis, and skin cancers. Nrf2-controlled pathways play a key role at this level. Nrf2 is a transcription factor that controls genes involved in oxidative stress protection and detoxification of chemicals. Its involvement in UV protection, reduction of inflammation in processes associated with healing, epidermal differentiation for barrier function, and hair regrowth, has been demonstrated. The modulation of Nrf2 in the skin may therefore constitute a skin protection or care strategy for certain dermatological stresses and disorders initiated or aggravated by the exposome. Nrf2 inducers can act through different modes of action. Keap1-dependent mechanisms include modification of the cysteine residues of Keap1 by (pro)electrophiles or prooxidants, and disruption of the Keap1-Nrf2 complex. Indirect mechanisms are suggested for numerous phytochemicals, acting on upstream pathways, or via hormesis. While developing novel and safe Nrf2 modulators for skin care may be challenging, new avenues can arise from natural compounds-based molecular modeling and emerging concepts such as epigenetic regulation.
... Lipoic Acid. In a three-dimensional human skin model, alpha-lipoic acid was reported to inhibit melanogenesis [90]. Alpha-lipoic acid shows great efficacy in improving UV-induced pigmentation and epidermal thickening in a mouse model [91]. ...
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Oxidative stress represents an imbalance between the generation of reactive oxygen and nitrogen species and the ability of antioxidant systems to decompose those products. Oxidative stress is implicated in the pathogenesis of hyperpigmentation, hypopigmentation, melanoma, and other skin diseases. Regulatory networks involving oxidative stress and related pathways are widely represented in hypopigmentation diseases, particularly vitiligo. However, there is no complete review into the role of oxidative stress in the pathogenesis of hyperpigmentation disorders, especially regarding associations involving oxidative stress and cellular signaling pathways. Here, we review oxidative and antioxidant systems, oxidative stress-induced signal transduction mechanisms, and effects of antioxidant drugs used in preclinical and clinical settings in hyperpigmentation disorders.
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Mercury exposure is the second-most common cause of toxic metal poisoning. Public health concern over mercury exposure, due to contamination of fish with methylmercury and the elemental mercury content of dental amalgams, has long been a topic of political and medical debate. Although the toxicology of mercury is complex, there is evidence for antioxidant protection in the prevention of neurological and renal damage caused by mercury toxicity. Alpha-lipoic acid, a coenzyme of pyruvate and alpha-ketoglutarate dehydrogenase, has been used in Germany as an antioxidant and approved treatment for diabetic polyneuropathy for 40 years. Research has attempted to identify the role of antioxidants, glutathione and alpha-lipoic acid specifically, in both mitigation of heavy metal toxicity and direct chelation of heavy metals. This review of the literature will assess the role of glutathione and alpha-lipoic acid in the treatment of mercury toxicity.
Thioctic (lipoic) acid is used as a therapeutic agent in a variety of diseases in which enhanced free radical peroxidation of membrane phospholipids has been shown to be a characteristic feature. It was suggested that the antioxidant properties of thioctic acid and its reduced form, dihydrolipoic acid, are at least in part responsible for the therapeutic potential. The reported results on the antioxidant efficiency of thioctic and dihydrolipoic acids obtained in oxidation models with complex multicomponent initiation systems are controversial. In the present work we used relatively simple oxidation systems to study the antioxidant effects of dihydrolipoic and thioctic acids based on their interactions with: (1) peroxyl radicals which are essential for the initiation of lipid peroxidation, (2) chromanoxyl radicals of vitamin E, and (3) ascorbyl radicals of vitamin C, the two major lipid- and water-soluble antioxidants, respectively. We demonstrated that: (1) dihydrolipoic acid (but not thioctic acid) was an efficient direct scavenger of peroxyl radicals generated in the aqueous phase by the water-soluble azoinitiator 2,2'-azobis(2-amidinopropane)-dihydrochloride, and in liposomes or in microsomal membranes by the lipid-soluble azoinitiator 2,2'-azobis(2,4-dimethylvaleronitrile); (2) both dihydrolipoic acid and thioctic acid did not interact directly with chromanoxyl radicals of vitamin E (or its synthetic homologues) generated in liposomes or in the membranes by three different ways: UV-irradiation, peroxyl radicals of 2,2'-azobis(2,4-dimethylvaleronitrile), or peroxyl radicals of linolenic acid formed by the lipoxygenase-catalyzed oxidation; and (3) dihydrolipoic acid (but not thioctic acid) reduced ascorbyl radicals (and dehydroascorbate) generated in the course of ascorbate oxidation by chromanoxyl radicals. This interaction resulted in ascorbate-mediated dihydrolipoic acid-dependent reduction of the vitamin E chromanoxyl radicals, i.e. vitamin E recycling. We conclude that dihydrolipoic acid may act as a strong direct chain-breaking antioxidant and may enhance the antioxidant potency of other antioxidants (ascorbate and vitamin E) in both the aqueous and the hydrophobic membraneous phases.
In humans the major stimulus for cutaneous pigmentation is ultraviolet radiation (UVR). Little is known about the mechanism underlying this response, in part because of the complexity of interactions in whole epidermis. Using a recently developed culture system, human melanocytes were exposed daily to a physiologic range of UVR doses from a solar simulator. Responses were determined 24 hours after the last exposure. There was a dose-related increase in melanin content per cell and uptake of 14C-DOPA, accompanied by growth inhibition. Cells from donors of different racial origin gave proportionately similar increases in melanin, although there were approximately tenfold differences in basal values. Light and electron microscopy revealed UVR-stimulated increases in dendricity as well as melanosome number and degree of melanization, analogous to the well-recognized melanocyte changes following sun exposure of intact skin. Similar responses were seen with Cloudman S91 melanoma cells, although this murine cell line required lower UVR dosages and fewer exposures for maximal stimulation. These data establish that UVR is capable of directly stimulating melanogenesis. Because cyclic AMP elevation has been associated in some settings with increased pigment production by cultured melanocytes, preliminary experiments were conducted to see if the effects of UVR were mediated by cAMP. Both alpha-MSH and isobutylmethylxanthine (IBMX), as positive controls, caused a fourfold increase in cAMP level in human melanocytes and/or S91 cells, but following a dose of UVR sufficient to stimulate pigment production there was no change in cAMP level up to 4 hours after exposure. Thus it appears that the UVR-induced melanogenesis is mediated by cAMP-independent mechanisms.
EpiDerm, an in vitro human skin equivalent (HSE), was compared to normal human breast skin (NHS) to morphologically and biochemically assess its feasibility for dermatological research. Intralot and interlot variability was studied in day 0, 1, 2, and 3 in vitro cultures and in day 0, 3, 5, and 7 NHS. For NHS, light microscopy (LM) at day 0 showed stratified epidermis which exhibited an increase in vacuoles and dark basal cells as storage increased to 3, 5, and 7 days. Transmission electron microscopy (TEM) revealed typical organelles in the epidermis and a convoluted basement membrane at day 0. With increased storage, vacuoles and paranuclear clefts became numerous, necrosis increased, tonofilaments became less organized, and overall cellular integrity decreased. Biochemical data showed consistent MTT and glucose utilization (GU) through day 5, while lactate production decreased to 75% by day 3. By LM, day 0 HSE consisted of a thick, compact, stratum corneum that sent projections between the stratum granulosum cells. By TEM, the configuration organization, differentiation, distribution, and frequency of the organelles differed slightly from NHS. In addition, the basement membrane of the HSE was not completely differentiated, and the dermis was thin and acellular. Although day 1 and 2 cultures showed little change, day 3 exhibited an overall degeneration. Biochemical analysis showed GU and the lactate production decreased through day 3. In conclusion, the EpiDerm HSE, although exhibiting slight differences, was morphologically and biochemically similar to normal human epidermis and may be a valuable model in assessing the toxicology, metabolism, or pharmacology of nonvesicating compounds.
1. Lipoic acid is an example of an existing drug whose therapeutic effect has been related to its antioxidant activity. 2. Antioxidant activity is a relative concept: it depends on the kind of oxidative stress and the kind of oxidizable substrate (e.g., DNA, lipid, protein). 3. In vitro, the final antioxidant activity of lipoic acid is determined by its concentration and by its antioxidant properties. Four antioxidant properties of lipoic acid have been studied: its metal chelating capacity, its ability to scavenge reactive oxygen species (ROS), its ability to regenerate endogenous antioxidants and its ability to repair oxidative damage. 4. Dihydrolipoic acid (DHLA), formed by reduction of lipoic acid, has more antioxidant properties than does lipoic acid. Both DHLA and lipoic acid have metal chelating capacity and scavenge ROS, whereas only DHLA is able to regenerate endogenous antioxidants and to repair oxidative damage. 5. As a metal chelator, lipoic acid was shown to provide antioxidant activity by chelating Fe2+ and CU2+; DHLA can do so by chelating Cd2+. 6. As scavengers of ROS, lipoic acid and DHLA display antioxidant activity in most experiments, whereas, in particular cases, pro oxidant activity has been observed. However, lipoic acid can act as an antioxidant against the pro oxidant activity produced by DHLA. 7. DHLA has the capacity to regenerate the endogenous antioxidants vitamin E, vitamin C and glutathione. 8. DHLA can provide peptide methionine sulfoxide reductase with reducing equivalents. This enhances the repair of oxidatively damaged proteins such as α-1 antiprotease. 9. Through the lipoamide dehydrogenase-dependent reduction of lipoic acid, the cell can draw on its NADH pool for antioxidant activity additionally to its NADPH pool, which is usually consumed during oxidative stress. 10. Within drug-related antioxidant pharmacology, lipoic acid is a model compound that enhances understanding of the mode of action of antioxidants in drug therapy.
Alpha-lipoic acid (LA) and its reduced form, dihydrolipoic acid, are powerful antioxidants. LA scavenges hydroxyl radicals, hypochlorous acid, peroxynitrite, and singlet oxygen. Dihydrolipoic acid also scavenges superoxide and peroxyl radicals and can regenerate thioredoxin, vitamin C, and glutathione, which in turn can recycle vitamin E. There are several possible sources of oxidative stress in diabetes including glycation reactions, decompartmentalization of transition metals, and a shift in the reduced-oxygen status of the diabetic cells. Diabetics have increased levels of lipid hydroperoxides, DNA adducts, and protein carbonyls. Available data strongly suggest that LA, because of its antioxidant properties, is particularly suited to the prevention and/or treatment of diabetic complications that arise from an overproduction of reactive oxygen and nitrogen species. In addition to its antioxidant properties, LA increases glucose uptake through recruitment of the glucose transporter-4 to plasma membranes, a mechanism that is shared with insulin-stimulated glucose uptake. Further, recent trials have demonstrated that LA improves glucose disposal in patients with type II diabetes. In experimental and clinical studies, LA markedly reduced the symptoms of diabetic pathologies, including cataract formation, vascular damage, and polyneuropathy. To develop a better understanding of the preventative and therapeutic potentials of LA, much of the current interest is focused on elucidating its molecular mechanisms in redox dependent gene expression.
The microphthalmia-associated transcription factor is implicated in melanocyte development and in the regulation of melanogenesis. Microphthalmia-associated transcription factor is thought to bind to the M-box promoter elements of tyrosinase, tyrosinase-related protein-1 and dopachrome tautomerase/tyrosinase-related protein-2 and transactivate these genes, resulting in increased pigmentation. Using a luciferase reporter construct driven by the microphthalmia-associated transcription factor promoter, we identified agents that modulate microphthalmia-associated transcription factor promoter activity. Changes in endogenous microphthalmia-associated transcription factor expression levels upon treatment with these agents were confirmed using northern and western blots, and their pigmentary modulating activities were demonstrated. Ultraviolet B irradiation and traditional Chinese medicine-1, a natural extract used in traditional Chinese medicine, upregulated microphthalmia-associated transcription factor gene expression and enhanced tyrosinase activity in vitro. Dihydrolipoic acid, lipoic acid, and resveratrol reduced microphthalmia-associated transcription factor and tyrosinase promoter activities. These agents also inhibited the forskolin- and ultraviolet B-stimulated promoter activities of these genes and significantly reduced tyrosinase activity in melanocyte cultures, resulting in depigmentation. Overexpressed microphthalmia-associated transcription factor was capable of rescuing the repressive effects of these compounds on the cotransfected tyrosinase promoter. Dark-skinned Yucatan swine treated with these agents showed visible skin lightening, which was confirmed histologically, whereas ultraviolet B-induced tanning of light-skinned swine was inhibited using these agents. Our findings suggest that modulation of microphthalmia-associated transcription factor expression can alter skin pigmentation and further confirm the central role of microphthalmia-associated transcription factor in melanogenesis.
Topically applied antioxidants constitute an important group of protective agents against skin damage induced by ultraviolet radiation. The current study was performed to investigate whether a recently developed ex vivo pig skin model was suitable for short-term studies of the mechanism(s) of UVB-radiation-induced skin damage; the protective effect of topical application of alpha-tocopherol, l-ascorbic acid, alpha-lipoic acid, glutathione ethylester and N-acetylcysteine was tested. Increasing doses of the antioxidants were applied topically on ex vivo pig skin explants and allowed to penetrate for 60 min. Epidermal antioxidant bioavailability was measured before and 60 min after exposure to an ultraviolet B (UVB) radiation of 7.5 kJ/m2. Cell viability (trypan blue dye exclusion) and apoptosis were measured 48 h later in isolated keratinocytes. UVB-radiation-induced epidermal lipid peroxidation was determined immediately after exposure of the skin to a UVB dose of 28 kJ/m2. All antioxidants tested became bioavailable in pig skin epidermis, and none of them were depleted after UVB-radiation exposure. Increasing doses of the antioxidants tested decreased UVB-radiation-induced cell death and apoptosis. The highest doses of antioxidants prevented UVB-radiation-induced lipid peroxidation; alpha-lipoic acid only tended to decrease lipid peroxidation. In conclusion, a single topical dose of the above antioxidants on ex vivo pig skin can reduce UVB-radiation-induced oxidative stress and lipid peroxidation and thereby reduce apoptotic stimuli and cell death. Furthermore, the ex vivo pig skin model was a useful tool for testing compounds for their antioxidant activity.
A number of cellular systems cooperate in redox regulation, providing metabolic responses according to changes in the oxidation (or reduction) of the redox active components of a cell. Key systems of central metabolism, such as the 2-oxo acid dehydrogenase complexes, are important participants in redox regulation, because their function is controlled by the NADH/NAD+ ratio and the complex-bound dihydrolipoate/lipoate ratio. Redox state of the complex-bound lipoate is an indicator of the availability of the reaction substrates (2-oxo acid, CoA and NAD+) and thiol-disulfide status of the medium. Accumulation of the dihydrolipoate intermediate causes inactivation of the first enzyme of the complexes. With the mammalian pyruvate dehydrogenase, the phosphorylation system is involved in the lipoate-dependent regulation, whereas mammalian 2-oxoglutarate dehydrogenase exhibits a higher sensitivity to direct regulation by the complex-bound dihydrolipoate/lipoate and external SH/S-S, including mitochondrial thioredoxin. Thioredoxin efficiently protects the complexes from self-inactivation during catalysis at low NAD+. As a result, 2-oxoglutarate dehydrogenase complex may provide succinyl-CoA for phosphorylation of GDP and ADP under conditions of restricted NAD+ availability. This may be essential upon accumulation of NADH and exhaustion of the pyridine nucleotide pool. Concomitantly, thioredoxin stimulates the complex-bound dihydrolipoate-dependent production of reactive oxygen species. It is suggested that this side-effect of the 2-oxo acid oxidation at low NAD+in vivo would be overcome by cooperation of mitochondrial thioredoxin and the thioredoxin-dependent peroxidase, SP-22.