白癜风患者血清中谷胱甘肽及谷胱甘肽过氧化物酶的检测

为了探讨谷胱甘肽 (GSH)及谷胱甘肽过氧化物酶 (GSH-PX)在白癜风发病中的作用及相互关系,采用化学的方法,对 69例白癜风患者, 44名健康人血清进行了 GSH及 GSH-PX的测定,结果白癜风患者血清 GSH各个体间数值差距较大,但经统计学处理白癜风患者与健康对照无显著性差异 (P >0.05),患者各型别之间,进行期与稳定期之间 ,无显著性差别;白癜风患者血清中 GSH-PX与健康人血清对照有极显著性差异 (P< 0.01),但各型之间,进行期与稳定期均无显著性差异。本研究发现白癜风 GSH-PX显著低于健康人,提示 GSH-PX与白癜风的发病可能有一定的关系,而 GSH与白癜风未发现明显的相关性。     

Characterization of glutathione S-transferase in cultured human keratinocytes

The glutathione S-transferase activity and isozymic composition of cultured human keratinocytes were characterized. Keratinocytes were grown in culture and harvested at different stages of differentiation. Glutathione S-transferase activity was found in the soluble cell fraction but not in the microsomal cell fraction. The glutathione S-transferase specific activity of the soluble cell fraction was found to increase as the keratinocytes differentiated in culture. All of the enzymatic activity was found to reside with a single isozymic form that was concluded to be the pi form of the enzyme based on substrate specificity, sensitivity to inhibitors, molecular weight, and reactivity towards antibodies raised to alpha, mu, and pi forms of the enzyme. It is concluded that all of the isozymic forms of glutathione S-transferase noted in whole skin, with the exception of pi, are of extra-keratinocyte origin.     

Expression of glutathione S-transferase-pi in malignant skin tumors.

GST-pi has been known to be markedly increased in human (pre) neoplasms of several organs. In this paper, the significance of immunohistochemical detection of GST-pi in human malignant tumors of the skin was studied. In specimens from 40 patients with various skin cancers, malignant melanoma, Paget's disease and undifferentiated squamous cell carcinoma showed strong reactivity in GST-pi staining. The reactions were negative or weak in Bowen's disease, basal cell epithelioma and solar keratosis. In normal melanocytes, eccrine, apocrine, and breast gland cells stained positively but not in keratinocytes, sebaceus gland and fibroblasts. While immunohistochemical detection of GST-pi in the skin was not specific for malignancies, it contributed to aid the distinction of squamous cell carcinoma from other keratinocytic tumors. GST-pi might provide potentially useful information on chemosensitivity of skin cancer, and might serve as a biomarker of disease activity.     

E6* oncoprotein expression of human papillomavirus type-16 determines different ultraviolet sensitivity related to glutathione and glutathione peroxidase antioxidant defence

Clinical observations of non-melanoma skin cancer in immunocompromised patients, such as organ transplant recipients, suggest co-operative effects of human papillomavirus (HPV) and ultraviolet (UV) radiation. The aim of the present study is to evaluate UV sensitivity and DNA damage formation according to antioxidant status in HPV16-infected keratinocytes. We used SKv cell lines, infected with HPV16 and well characterized for their proliferative and tumorigenic capacities. We showed that SKv cell lines presented various E6* (a truncated form of E6) RNA levels. We demonstrated that the higher oncoprotein RNA expression level was associated with a higher resistance to solar-simulated radiation, more specifically to UVB radiation and to hydrogen peroxide. Moreover, this high resistance was associated with a low oxidative DNA damage formation after UV radiation and was related to high glutathione content and glutathione peroxidase activities. Therefore, the results of our study suggest that E6* levels could modulate the glutathione/glutathione peroxidase pathway providing a mechanism to protect HPV-infected keratinocytes against an environmental oxidative stress, such as UV radiation.     

Modulation of glutathione level in cultured human melanoma cells

The effects of buthionine sulphoximine (BSO) treatment on cellular glutathione (GSH) content and on the cytotoxic action of menadione were investigated in cultured IGRI human melanoma cells. Addition of BSO (10(-8)-0.5 X 10(-3) M) to the cultures resulted in a dose- and time-dependent depletion of cellular GSH. BSO (10(-5) and 10(-6) M) did not influence cell multiplication up to 48 h, as determined by trypan blue staining. Menadione (3 X 10(-5) M) treatment decreased the cellular GSH concentration and also reduced cell number after a 24 h exposure. Its cytotoxicity was increased by BSO (10(-5), 10(-6) M), though the potentiating effect was moderate.     

胎盘型谷胱甘肽转换酶在皮肤角朊细胞肿瘤中的表达

为了探讨谷胱甘肽转换酶π（ＧＳＴπ）与皮肤角朊细胞肿瘤发生的关系，采用免疫组化技术检测了４５例皮肤角朊细胞肿瘤和５例正常皮肤。结果∶ＧＳＴπ在正常皮肤主要表达于颗粒细胞层、大部分棘细胞层和外毛根鞘、小汗腺。在日光性角化病、Ｂｏｗｅｎ病、基底鳞状细胞癌和鳞状细胞癌中胞浆和部分胞核ＧＳＴπ阳性，基底细胞癌胞浆弱阳性，部分脂溢性角化症和角化棘皮瘤胞浆呈弱阳性。本研究提示ＧＳＴπ主要存在于表皮上层，并在皮肤角朊细胞肿瘤发生中起一定作用。     

Serum selenium levels and blood glutathione peroxidase activities in vitiligo

It has recently been shown that patients with vitiligo can accumulate epidermal hydrogen peroxide (H2O2) in association with low catalase levels. This study examined serum selenium levels and blood glutathione peroxidase activities in 61 patients and controls. The results showed high serum selenium levels in 56% of the patients. As at least one isoform of glutathione peroxidase requires selenium for its activity, enzyme activities were also evaluated. The overall results were not significantly different compared with controls, but further age-related analysis of the data indicated significantly lower activities in patients up to 46 years. As glutathione peroxidase can also efficiently degrade H2O2, the results of this study could indicate an additional impaired H2O2 metabolism in vitiligo.     

Decrease in glutathione may be involved in pathogenesis of acne vulgaris

Background Some past studies reported that oxidative stress components such as reactive oxygen species (ROS) or lipid peroxide (LPO) are involved in the pathogenesis and progression of acne vulgaris. In this study, we hypothesized that the pathogenesis of acne vulgaris may depend on the differences in antioxidative activity among antioxidants in our body. We collected samples of stratum corneum from acne patients and healthy subjects and compared the quantity of gluthathione (GSH), one of many antioxidative components in our body, for comparison. Methods Samples of stratum corneum were collected from facial acne‐involved lesion, facial uninvolved area, and the medial side of the upper arm in acne vulgaris patients. Similarly, samples were collected from a facial uninvolved area and the medial side of the upper arm in healthy subjects. The quantity of GSH was measured in each area. In vitro effects of alpha‐melanocyte stimulating hormone (α‐MSH) on GSH synthesis‐related gene were also examined. Results The quantity of GSH in stratum corneum from each area was significantly lower in acne vulgaris patients than that of healthy subjects. There was no significant difference in quantity of GSH between the acne‐involved lesion and uninvolved area in acne patients. In vitro studies showed that the expression level of Glutamate‐cysteine ligase catalytic subunit (GCLC), one of the GSH synthesis‐related genes, was significantly decreased by the additional use of α‐MSH. Conclusions We conclude that a decline in antioxidative activity led by a decrease in GSH quantity may play an important role in pathogenesis of acne vulgaris. The use of α‐MSH may further decrease the GSH level.     

Effect of glutathione depletion on sunburn cell formation in the hairless mouse

Cutaneous protection against ultraviolet B (UVB) radiation damage by endogenous glutathione (GSH) was evaluated in the epidermis of the hairless mouse by measuring the influence of GSH depletion on sunburn cell (SBC) formation. Cellular GSH exerts antioxidant effects and recent studies have suggested a role for oxygen radicals in the production of SBC. Hairless mice (Skh/h 1) received oral treatment with buthionine S,R-sulfoximine (BSO), an irreversible inhibitor of gamma-glutamylcysteine synthetase, to deplete cutaneous GSH; 4 d later their ears were exposed to UVB radiation. BSO treatment significantly reduced GSH levels in the epidermis to 10-15% of control levels. Twenty-four hours after UVB exposure, SBC counts in the ears of animals with and without BSO treatment were measured, and those exposed to UVB were found to have increased. Greater numbers of SBC were found in the ears of BSO-treated mice exposed to 15 or 20 mJ/cm2 UVB, than in non-BSO-treated mice exposed to the same UVB doses. At higher UVB doses, there were no statistically significant differences between the groups. The results show that endogenous GSH provides the epidermis with measurable protection against injury by low or moderate UVB doses.     

Cysteine-dependent 5-S-cysteinyldopa formation and its regulation by glutathione in normal epidermal melanocytes

Recent evidence suggests that the melanogenesis intermediate 5-S-cysteinyldopa (5-S-CD) could display antioxidative activity. In the present study, the synthesis of 5-S-CD was examined in human epidermal melanocytes isolated from dark skin type VI (MT) and from white skin type III (GT). The MT melanocytes showed the higher melanin content and dopa oxidase activity. In addition, they produced eumelanin as shown by their ultrastructure, and the solubility and UV/visible absorption of the isolated pigment. Both MT and GT cells showed high levels of 5-S-CD (5.5–6.9 nmol/mg protein). 5-S-CD was also detected in culture supernatants from MT cells; the secretion rate was estimated to be 2.5 nmol/mg protein per 24 h. The role of cysteine and glutathione in 5-S-CD formation was investigated by exposing the melanocytes to theγ-glutamylcysteine synthetase inhibitorl-buthionine sulfoximine (BSO). A strong reduction in glutathione levels (4–8% of the untreated controls) associated with an increase in cysteine levels (152–154%) was observed. In addition, BSO induced a moderate increase in the cellular levels of 5-S-CD (114–129%) and a decrease in dopa oxidase activity (75–83%). Our results indicate that the direct addition of cysteine to dopaquinone is the main source of 5-S-CD in human epidermal melanocytes. It is proposed that the synthesis of 5-S-CD is a mechanism regulating dopaquinone levels during pigment formation and/or a defence mechanism against oxidative stress.     

Dimethylfumarate induces immunosuppression via glutathione depletion and subsequent induction of heme oxygenase 1

A mixture of different fumaric acid esters (FAE) is established for systemic therapy of psoriasis, a frequent inflammatory skin disease. The main active compound of FAE, however, has not been identified so far, and the mechanisms of activity are only partially understood. We analyzed the impact of FAE on in vitro immune function and aimed to gain knowledge about the mode of action. Dimethylfumarate (DMF) and diethylfumarate (DEF), but not fumaric acid, methylhydrogenfumarate and ethylhydrogenfumarate, exhibited potent depression of inflammatory cytokine secretion (e.g., tumor necrosis factoralpha, IL-12, and IFNgamma) in activated human peripheral blood mononuclear cells. Moreover, solely DMF and DEF inhibited alloreactive T-cell proliferation in mixed leukocyte reaction. Interestingly, these immunosuppressive effects were accompanied by the strong induction of the anti-inflammatory stress protein heme oxygenase 1 (HO-1). Supplementation with exogenous glutathione (GSH), which is known to bind DMF, prevented both HO-1 induction as well as the anti-inflammatory effects of DMF. Moreover, inhibition of HO-1 activity restored the diminished IL-12 and IFNgamma production after FAE treatment. These results suggest that DMF acts as active compound within the FAE mixture and at least partially mediates its immunomodulatory activity by the induction of the anti-inflammatory stress protein HO-1 ascribed to the functional depletion of reduced GSH.     

Erythrocyte glutathione peroxidase activity and plasma vitamin E status in patients with psoriasis

Erythrocyte selenium dependent glutathione peroxidase (GSH-Px) activity and plasma vitamin E levels were measured in 20 psoriatics. Psoriatics had significantly lower GSH-Px and plasma vitamin E levels than did controls. Psoriatics were divided into three grades by the rule of nine: Grade I—less than 33% skin involvement, Grade II—33-66%, and Grade III—over 66% skin involvement. Grade III psoriatics showed much lower values of GSH-Px and plasma vitamin E, showing a definite correlation with the percentage of surface area involved.     

Opposite regulation of tyrosinase and glutathione peroxidase by intracellular thiols in human melanoma cells

Received: 16 July 1996     

Antimelanogenic activity of hydrocoumarins in cultured normal human melanocytes by stimulating intracellular glutathione synthesis

The antimelanogenic activity of six hydrocoumarins and alpha-tocopherol (alpha-Toc) in normal human melanocytes was evaluated in both cell culture systems and cell homogenates. The inhibitory effects of hydrocoumarins depended upon their substituent groups. alpha-Toc and some of the hydrocoumarins inhibited melanogenesis in cultured normal human melanocytes, although they did not influence melanin synthesis in enzyme solution prepared as cell homogenates. In addition, alpha-Toc and the hydrocoumarins stimulated intracellular glutathione (GSH) synthesis. In particular, 7-allyl-6-hydroxy-4,4,5,8-tetramethylhydrocoumarin strongly inhibited melanogenesis and intracellular GSH synthesis in normal human melanocytes, more so than alpha-Toc. Furthermore, hydrocoumarins exhibited higher scavenging and quenching activities against with tert-butyl peroxyl radicals and singlet oxygen species. These results suggest that 7-allyl-6-hydroxy-4,4,5,8-tetramethyl hydrocoumarin would be useful as an antimelanogenic agent for the prevention or improvement of skin pigmentation induced by reactive oxygen compounds and free radicals, and may inhibit melanogenesis, including tyrosinase transfer and melanosome differentiation, by interrupting melanization by increasing the intracellular GSH content.     

Analyses in blood of dermatological patients. I. Glutathione and glutathione reductase.

Glutathione was estimated in 98 blood samples from dermatological patients; in only two cases, both of contact eczema, a value considerably below normal was found. Glutathione reductase was assayed in blood samples from 139 different patients and 21 normal controls. The activity was significantly higher in atopic dermatitis (17 patients). A significantly greater variable was found among patients with non methotrexate-treated psoriasis (44), light sensitivity (12) and scleroderma (5). In the methotrexate-treated psoriatic group (24) and mean and variability did not differ significantly from normal. In most hospitalized patients a low glutathione reductase activity rose within a few weeks, but in a case of dermatitis herpetiformis a very low level persisted for 3 months. Blood samples with very low glutathione reductase activity, taken from a case of psoriasis and from a patient on griseofulvin treatment, gave a positive peroxide test and tended to hemolyze; these returned to normal together with the glutathione reductase activity.     

Analysis of initial melanogenesis including tyrosinase transfer and melanosome differentiation through interrupted melanization by glutathione

Because glycosylation-dependent melanization inhibition induced in cultured B-16 melanoma cells by glucosamine is reversible, producing synchronized initiation of melanogenesis after its removal, we have analyzed the possible dynamics of initial melanogenesis through their interruption by glutathione. The addition of glutathione at 0.2% concentration to the theophylline-stimulated recovery process completely interrupts the initiation of melanization for at least 72 h. At the electron microscopic level, theophylline-treated cells have many vacuolar melanosomes with distinct pigmentation which contain some vesicles (64% of total premelanosomes) or amorphous, filamentous, or granular materials within the interior which are suggestive of pheomelanotic melanosomes. The addition of glutathione induces a complete absence of melanization in the premelanosomes, within which a filamentous interior with periodicity is generally re-formed with almost complete disappearance of internal vesicles, providing dramatic changes to the size and shape characteristic of eumelanotic melanosome. Electron microscopic dopa reaction of glutathione-treated cells shows a predominant localization of tyrosinase activity in the Golgi-associated endoplasmic reticulum-lysosome and coated vesicles, but not in premelanosomes, in contrast to their dispersed distribution in all melanogenic organelles in the theophylline-treated control, suggesting a lack of tyrosinase translocation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of tyrosinase in the large granule fraction shows that in analogy with electron microscopic observations, glutathione blocks the reappearance of membrane-bound T3 tyrosinase which occurs in the theophylline-treated control during the recovery process, whereas the dynamics of T1 tyrosinase is almost the same as that of the control. These findings suggest that glutathione provides a new situation of interrupted melanogenesis in which melanization cannot proceed despite complete formation of melanosome matrix structure and a lack of inhibition of cellular metabolisms including protein glycosylation.