Inactivation of Yeast Glutathione Reductase by O -Phthalaldehyde

It has been reported that the melanogenic activity can be inhibited by antioxidants, like a-tocopherol and hydrocoumarins, 1) and that reduced glutathione (GSH), an important biological reductant, plays a role in the modulation of the melanogenic process. 2)Melanogenesis, which is catalyzed enzymatically by the key enzyme, tyrosinase, is also influenced by other non-enzymatic factors such as ultraviolet rays and a-melanocytestimulating hormone (a-MSH).3) In the case of a-MSH-induced melanogenesis, the enhancement of tyrosinase activity, tyrosinase mRNA, and intracellular cyclic AMP (cAMP) were observed. 4)The requirement of cAMP for melanogenesis is quite clear for pigmentation. cAMP also increases tyrosinase mRNA and promotes increased microphthalmia transcription factor (MITF) expression. MITF is a melanocyte-specific transcription factor crucial for melanocyte survival, development and differentiation through the activation of protein kinase A (PKA).5) PKA is a serine/threonine kinase that is an inactive tetramer consisting of two regulatory subunits and two catalytic subunits, and its activation occurs by the binding of cAMP to its regulatory subunits and then releasing its catalytic subunits from the regulatory subunits. 6)Recently, we reported that 4,4Ј-dihydroxybiphenyl (44Ј-BP) has a melanogenesis inhibitory effect through the inhibition of tyrosinase and reduction of melanin content.7) However, the underlying process by which tyrosinase activity and melanogenesis were inhibited by 44Ј-BP was not elucidated. In this current study, we attempted to investigate the effect of 44Ј-BP on major cellular regulators involved in melanogenesis in B16 melanoma cells (B16 cells). The analysis was carried out by quantifying amounts of tyrosinase and MITF proteins, cAMP levels, phosphorylated PKA, and GSH and oxidized glutathione (GSSG) levels. Cell Culture System B16 cells (from Korean Cell Line Bank) were cultured in DMEM with 10% fetal bovine serum (FBS; Gibco) and penicillin/streptomycin (100 IU/50 mg/ml) in a humidified atmosphere containing 5% CO 2 in air at 37°C. B16 cells were cultured in 24-well plates for each assay. MATERIALS AND METHODS MaterialsAll the experiments were determined in triplicate and repeated three times to ensure reproducibility.GSH and GSSG Assay Assays for GSH and GSSG were carried out by the method of Pandey and Katiyar. 8)Twenty-five percent of the meta-phosphoric acid-added cell pellets were centrifuged at 12000 rpm for 10 min, and the supernatant was taken for assay. For GSH, 1 mM EDTA-50 mM phosphate buffer was added to the supernatant followed by ophthaladehyde. After 20 min at room temperature, the fluorescence was measured at excitation wavelength of 360 nm and emission wavelength of 460 nm. GSSG was assayed after preincubated with N-ethylmaleimide for 20 min and 0.1 M NaOH was substituted for 1 mM EDTA-50 mM phosphate buffer.Quantitation of cAMP To measure intracellular cAMP levels, an enzyme immunoassay kit was used. In brief, B16 cells (7ϫ10 4 ) were lyzed in 0.1 M of HCl to inh...

Section: Methodsmentioning

confidence: 99%

Inhibition of Melanogenic Activity by 4,4'-Dihydroxybiphenyl in Melanoma Cells

Kim²,

Lee

et al. 2006

“…19) Estimation of GSH and GSSG Levels in B16 Cells GSH levels were assayed according to the method of Pandey and Katiyar. 20) Twenty-five percent of the meta-phosphoric acid-added cell pellets were centrifuged at 12000×g for 10 min, and then the supernatant was taken for assay. One millimolar EDTA-50 mM phosphate buffer was added to the supernatant followed by o-phthalaldehyde.…”

Section: Evaluation Of • O 2 Levels In B16 Cellsmentioning

confidence: 99%

Hyperin and Quercetin Modulate Oxidative Stress-Induced Melanogenesis

Kim

2012

Hyperin and quercetin are phenolic compounds present in fruits and vegetables that have been reported to possess strong anti-oxidative properties. Although increasing evidence strongly suggests that antioxidants suppress the melanin synthesis that is causally associated with oxidative stress, the protective actions of hyperin and quercetin against oxidative stress-induced melanogenesis have not been fully explored. To elucidate the suppressive effects of hyperin and quercetin on oxidative stress and melanin synthesis, peroxynitrite (ONOO ) scavenging activity was measured in vitro as were quantifications of melanin content, intracellular total RS, ONOO , superoxide (• O 2 ), nitric oxide (NO • ), catalase activity and the reduced glutathione (GSH)/ oxidized glutathione (GSSG) ratio. Results showed that in vitro, hyperin and quercetin reduced ONOO . Additionally, hyperin and quercetin suppressed total RS, ONOO ,• O 2 , and NO• , catalase activity, and melanin synthesis, while they boosted the GSH/GSSG ratio in B16F10 melanoma cells (B16 cells). Therefore, I propose that hyperin and quercetin have a powerful capacity to modulate oxidative stress-induced melanogenesis.Key words hyperin; quercetin; antioxidant; oxidative stress; melanogenesis Melanogenesis is the process by which pigment forms and melanin is synthesized in special organelles called melanosomes.1) Melanin plays a key role in the protection of the skin against deleterious sunlight under normal conditions, but also can increase the generation and excessive accumulation of melanin that lead to clinical and cosmetic concerns such as freckles, age spots, sites of actinic damage, and melasma. Chronic inflammation, UV ray radiation, and the release of abnormal α-melanocyte stimulating hormone (α-MSH) also are well-known triggering factors for abnormal melanogenesis and inflammatory pigmentation. 1,2)Oxidative stress caused by excessive reactive species (RS), including reactive oxygen species (ROS) and reactive nitrogen species (RNS), is known to be associated with skin aging and hyperpigmentation. Also, a deficient anti-oxidative defense system, such as a disturbed redox balance and weak anti-oxidant enzyme activity, exacerbate skin aging and hyperpigmentaion.3-5) Therefore, searching for natural antioxidants that inhibit melanogenesis has been an important strategy in the development of skin medications and cosmetics. 6) We have reported on the significance of maintaining cellular redox through the down-regulation of RS and the boosting of antioxidant defenses in the inhibition of melanogenesis. 5,7,8) Various phenolic compounds from natural sources have been reported as melanogenic inhibitors. 5,7-11) Hyperin and quercetin are two phenolic flavonoids reported to have antioxidative 12) and anti-viral 13) properties. However, the effects of hyperin and quercetin on oxidative stress-induced melanogenesis have not been fully explored to date.To estimate the effects of hyperin and quercetin on oxidative insult in vitro, peroxynitrite (ONOO − ) scavenging act...

“…21) Twenty-five percent of the meta-phosphoric acid-added cell pellets were centrifuged at 12000ϫg for 10 min, and the supernatant was taken for assay. For GSH, 1 mM EDTA-50 mM phosphate buffer was added to the supernatant followed by ophthaladehyde.…”

Section: Estimation Of Melanogenesis In B16 Cellsmentioning

confidence: 99%

Antimelanogenic and Antioxidant Properties of Gallic Acid

Kim¹

2007

295

175

To find novel skin-whitening agents, the melanogenesis inhibitory action of gallic acid (GA) was investigated. In this current study, the effects of GA on mushroom tyrosinase, tyrosinase inhibitory activity, and melanin content were assessed in B16 melanoma cells (B16 cells). Results indicated that GA has a strong antityrosinase activity (IC 50 ‫01؋95.3؍‬ ؊6 M). Furthermore, data on murine tyrosinase activity and melanin biosynthesis revealed that GA effectively suppressed murine tyrosinase action and the amount of melanin. To investigate the relation between GA's inhibition of melanogenesis and antioxidant activity, the effect of GA on reactive species (RS) generation and the reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio in were determined in B16 cells. Results indicated that GA effectively down-regulated the RS generation and enhanced the GSH/GSSG ratio. Based on these results, I propose that GA exerts antimelanogenic activity coupled with antioxidant properties by suppressing RS generation and maintaining a higher GSH/GSSG ratio.