The Redox State of Glutathione,Cysteine and Homocysteine in the Extracellular Fluid in the Skin

Glutathione, the most abundant low-molecular weight thiol in the skin, has been shown to protect the skin from both photobiological and chemical injury. The thiols, glutathione in particular, have also been shown to be crucially involved in defence against contact allergens. Since the levels of extracellular thiol concentrations are important determinants of intracellular thiol status, we have compared the normal concentrations and the redox status of the main low-molecular weight thiol components in the extracellular fluid at the dermo-epidermal junction with the corresponding plasma levels. In their sulfhydryl form, all three thiols, i.e. glutathione, cysteine and homocysteine, were more abundant in experimental skin blister fluid than in plasma, as were the free disulfides of glutathione and homocysteine, whereas the free disulfides of cysteine were about the same in blister fluid and in plasma. Protein mixed disulfide levels were higher in plasma than in blister fluid. The present results provide information concerning the extracellular defence in the skin.     

The redox state of glutathione, cysteine and homocysteine in the extracellular fluid in the skin

Glutathione, the most abundant low-molecular weight thiol in the skin, has been shown to protect the skin from both photobiological and chemical injury. The thiols, glutathione in particular, have also been shown to be crucially involved in defence against contact allergens. Since the levels of extracellular thiol concentrations are important determinants of intracellular thiol status, we have compared the normal concentrations and the redox status of the main low-molecular weight thiol components in the extracellular fluid at the dermo-epidermal junction with the corresponding plasma levels. In their sulfhydryl form, all three thiols, i.e. glutathione, cysteine and homocysteine, were more abundant in experimental skin blister fluid than in plasma, as were the free disulfides of glutathione and homocysteine, whereas the free disulfides of cysteine were about the same in blister fluid and in plasma. Protein mixed disulfide levels were higher in plasma than in blister fluid. The present results provide information concerning the extracellular defence in the skin.     

An evaluation of the effects of photoactivation of bithionol, amiodarone and chlorpromazine on human keratinocytes in vitro

Human skin is a continual target for chemical toxicity, due to its constant exposure to xenobiotics. The skin possesses a number of protective antioxidant systems, including glutathione and enzymic pathways, which are capable of neutralising reactive oxygen species (ROS). In combination with certain chemicals, the presence of ROS might augment the levels of toxicity, due to photoactivation of the chemical or, alternatively, due to an oxidatively-stressed state in the skin which existed prior to exposure to the chemical. Bithionol is a phototoxic anti-parasitic compound. The mechanism of its toxicity and the possible methods of protection from its damaging effects have been explored. The capacity of keratinocytes to protect themselves from bithionol and other phototoxic chemicals has been investigated. In addition, the potential of endogenous antioxidants, such as vitamin C and E, to afford protection to the cells, has been evaluated. The intracellular glutathione stores of HaCaT keratinocytes were reduced following treatment with biothionol. Following photoactivation, both bithionol and chlorpromazine had similar effects, which suggests that glutathione is important in the detoxification pathway of these chemicals. This was confirmed by means of the visual identification of fluorescently-labelled glutathione. Endogenous antioxidants were unable to protect the HaCaT keratinocytes from bithionol toxicity or chlorpromazine phototoxicity. Amiodarone was shown to have no effect on cellular glutathione levels, which suggests that an alternative mechanism of detoxification was occurring in this case. This was supported by evidence of the protection of HaCaT cells from amiodarone phototoxicity via endogenous antioxidants. Thus, it appears that amiodarone toxicity is dependent on the levels of non-gluathione antioxidants present, whilst bithionol and chlorpromazine detoxification relies on the glutathione antioxidant system. This type of approach could indicate the likely mechanisms of phototoxicity of chemicals in vitro, with relevance to potential effects in vivo.     

Identification of a novel glutathione transferase in human skin homologous with class alpha glutathione transferase 2-2 in the rat.

Six forms of glutathione transferase with pI values of 4.6, 5.9, 6.8, 7.1, 8.5 and 9.9 have been isolated from the cytosol fraction of normal skin from three human subjects. The three most abundant enzymes were an acidic Class Pi transferase (pI 4.6; apparent subunit Mr 23,000), a basic Class Alpha transferase (pI 8.5; apparent subunit Mr 24,000) and an even more basic glutathione transferase of Class Alpha (pI 9.9; apparent subunit Mr 26,500). The last enzyme, which was previously unknown, accounts for 10-20% of the glutathione transferase in human skin. The novel transferase showed greater similarities with rat glutathione transferase 2-2, another Class Alpha enzyme, than with any other known transferase irrespective of species. The most striking similarities included reactions with antibodies, amino acid compositions and identical N-terminal amino acid sequences (16 residues). The close relationship between the human most basic and the rat glutathione transferase 2-2 supports the classification of the transferases previously proposed and indicates that the similarities between enzymes isolated from different species are more extensive than had been assumed previously.     

UVR-induced oxidative stress in human skin in vivo: effects of oral vitamin C supplementation

Previous studies of cultured skin cells and murine skin in vivo have indicated that UVR-induced damage involves the generation of reactive oxygen species and depletion of endogenous antioxidant systems. In order to explore the relevance of this to UVR-induced damage to human skin, we have undertaken a detailed examination of the time-course of changes in markers of oxidative stress in human skin following exposure to physiological amounts of UVR in vivo. In addition, we have examined the skin bioavailability of a common nutritional antioxidant, vitamin C, and have assessed the effects of supplementation on markers of oxidative stress. Our hypothesis was that acute exposure of human skin to UVR in vivo would lead to oxidation of cellular biomolecules that could be prevented by prior vitamin C treatment. A UVR-challenge of 120 mJ/cm2 of broadband UVB (peak 310 nm, range 270-400 nm) was applied to buttock skin of 8 healthy volunteers. This caused a rapid and significant rise in activity of skin catalase at 1 h and an increase in the oxidized/total glutathione ratio at 6 h post-UVR. AP-1 DNA binding also peaked at 1-6 h post-UVR, then declined rapidly to baseline levels. No significant changes were seen in skin malonaldehyde content. Oral vitamin C supplements (500 mg/day) were taken by 12 volunteers for 8 weeks resulting in significant rises in plasma and skin vitamin C content. Supplementation had no effect on the UVR-induced erythemal response. The skin malonaldehyde content was reduced by vitamin C supplementation, but surprisingly, reductions in the skin content of total glutathione and protein thiols were also seen. We speculate that this apparently paradoxical effect could be due to regulation of total reductant capacity by skin cells, such that vitamin C may have been replacing other reductants in these cells. No evidence was obtained for an effect of the supplementary vitamin C on the mild oxidative stress seen in human skin following UVR exposure.     

Electrochemical determination of femtomole amounts of free reduced and oxidized glutathione: Application to human hair follicles

A simple and sensitive high-performance liquid chromatographic technique has been developed for the quantification of free reduced and free oxidized glutathione in biological samples. After acidic extraction and isocratic separation of the compounds of interest on a reversed-phase column, both forms of glutathione are quantified with a coulometric detector working in the oxidative mode. The limit of detection is 125 fmol for reduced glutathione and 400 fmol for the oxidized form (signal-to-noise ratio of 3). This sensitivity allows the measurement of the small amount of glutathione present in a single hair follicle. The technique is well adapted to microsamples, i.e. for non-invasive sampling technique (hair, skin, tears, etc.) and can be adapted to various cells or tissues.     

Glycyrrhizin exhibits potential chemopreventive activity on 12-O-tetradecanoyl phorbol-13-acetate-induced cutaneous oxidative stress and tumor promotion in Swiss albino mice

Glycyrrhizin and its aglycone, glycyrrhetic acid has been found useful for various therapeutic purposes. Glycyrrhizin has been shown to possess many physiological functions like anti-inflammatory activity, detoxification and inhibition of carcinogenic promoters. 12-O-Tetradecanoyl phorbol-13-acetate (TPA), a well-known phorbal ester is known for its tumor promotion activity. The induction of inflammation in skin mediated by TPA is believed to be governed by cyclooxygenase (COX), lipoxygenase and ornithine decarboxylase (ODC). These markers of inflammatory responses are important for skin tumor promotion. In our present study, we studied the chemopreventive effect of glycyrrhizin on TPA (20 nmol/0.2 mL acetone/animal, topically)-induced oxidative stress and hyperproliferation markers in skin. TPA enhanced lipid peroxidation with reduction in the level of catalase, glutathione, glutathione peroxidase, glutathione reductase and glutathione-s-transferase. TPA treatment also enhanced ODC activity and [3H] thymidine incorporation into cutaneous DNA. Prophylactic treatment of mice with glycyrrhizin (2.0 & 4.0 mg/0.2 mL acetone/animal, topically) resulted in a significant decrease in cutaneous microsomal lipid peroxidation (P < 0.001) and recovery of cutaneous glutathione content (P < 0.001) and its dependent enzymes. A significant inhibition in ODC activity and DNA synthesis (P < 0.001) was also observed. Thus, the results demonstrate that pretreatment with glycyrrhizin is protective against TPA-induced oxidative stress and tumor promotion in Swiss albino mice.     

Changes in the levels of glutathione after cellular and cutaneous damage induced by squalene monohydroperoxide.

Squalene monohydroperoxide (Sq-OOH), the initial product of ultraviolet-peroxidated squalene, was used to investigate the effect of peroxidative challenge upon the glutathione contents in rabbit ear skin and primary-cultured fibroblasts derived from rabbit ear skin. The cellular reduced glutathione (GSH) contents decreased during 30-minute incubations in vitro with Sq-OOH, and oxidized glutathione (GSSG) was formed concomitantly, indicating that Sq-OOH had a potential for GSH-depleting activity in vitro. When Sq-OOH was applied topically to the skin in vivo, only GSSG contents increased significantly within 30 minutes. Moreover, pretreatment with the GSH depletors, DL-buthionine sulfoximine (BSO) and diethyl maleate (DEM), could potentiate the cytotoxicity and comedogenicity induced by Sq-OOH. These findings suggest that the endogenous antioxidant, glutathione, is quite sensitive to Sq-OOH and may be an important material for protecting cells and/or tissues against the oxidative stress induced by Sq-OOH treatment.     

Glycyrrhizin exhibits potential chemopreventive activity on 12-O-tetradecanoyl phorbol-13-acetate-induced cutaneous oxidative stress and tumor promotion in swiss albino mice

Glycyrrhizin and its aglycone, glycyrrhetic acid has been found useful for various therapeutic purposes. Glycyrrhizin has been shown to possess many physiological functions like anti-inflammatory activity, detoxification and inhibition of carcinogenic promoters. 12-O-Tetradecanoyl phorbol-13-acetate (TPA), a well-known phorbal ester is known for its tumor promotion activity. The induction of inflammation in skin mediated by TPA is believed to be governed by cyclooxygenase (COX), lipoxygenase and ornithine decarboxylase (ODC). These markers of inflammatory responses are important for skin tumor promotion. In our present study, we studied the chemopreventive effect of glycyrrhizin on TPA (20 nmol/0.2 mL acetone/animal, topically)-induced oxidative stress and hyperproliferation markers in skin. TPA enhanced lipid peroxidation with reduction in the level of catalase, glutathione, glutathione peroxidase, glutathione reductase and glutathione-s-transferase. TPA treatment also enhanced ODC activity and [³H] thymidine incorporation into cutaneous DNA. Prophylactic treatment of mice with glycyrrhizin (2.0 & 4.0 mg/0.2 mL acetone/animal, topically) resulted in a significant decrease in cutaneous microsomal lipid peroxidation (P < 0.001) and recovery of cutaneous glutathione content (P < 0.001) and its dependent enzymes. A significant inhibition in ODC activity and DNA synthesis (P < 0.001) was also observed. Thus, the results demonstrate that pretreatment with glycyrrhizin is protective against TPA-induced oxidative stress and tumor promotion in Swiss albino mice.     

Accumulation of cystine from glutathione-cysteine mixed disulfide in cystinotic fibroblasts; blockade by an inhibitor of gamma-glutamyl transpeptidase

Cystinotic and normal skin fibroblasts in tissue culture were treated with varying concentrations of reduced glutathione, oxidized glutathione and glutathione-cysteine mixed disulfide, substrates of gamma-glutamyl transpeptidase, the catabolic enzyme of the gamma-glutamyl cycle. Cystine accumulated more rapidly and to a greater extent from the glutathione-cysteine mixed disulfide in cystinotic than in normal cells. Inhibition of gamma-glutamyl transpeptidase activity by serine in a borate buffer partially blocked this accumulation of cystine. Reduced glutathione and oxidized glutathione have lesser effects on cystine accumulation. Stored cystine in cystinotic tissues may derive in part from glutathione-cysteine mixed disulfide via transpeptidation.     

Glutathione S-transferase, similar to sigma class, from skin secretions of Xenopus laevis

Using glutathione affinity chromatography followed by isoelectrofocusing, we purified from the skin secretion of Xenopus laevis an isoenzyme of glutathione S-transferase with an apparent subunit molecular mass of 22.5 kDa and an isoelectric point at pH 5.1. Its N-terminal amino acid sequence was highly similar to that of the sigma class glutathione S-transferase, which previously was demonstrated to have a glutathione-dependent prostaglandin D2 synthase activity. Immunohistochemistry analysis revealed that the isoenzyme was located in the cytoplasm of granular gland cells.     

Oxidative damage elicited by imbalance of free radical scavenging enzymes is associated with large-scale mtDNA deletions in aging human skin

Mitochondrial DNA (mtDNA) mutations and impaired respiratory function have been demonstrated in various tissues of aged individuals. We hypothesized that age-dependent increase of ROS and free radicals production in mitochondria is associated with the accumulation of large-scale mtDNA deletions. In this study, we first confirmed that the proportion of mtDNA with the 4977 bp deletion in human skin tissues increases with age. We then investigated the 8-hydroxy-2'-deoxyguanosine (8-OH-dG) content in skin tissues and lipid peroxides content of the skin fibroblasts from subjects of different ages. The results showed an age-dependent increase of 8-OH-dG level in the total DNA of skin tissues of the subjects above the age of 60 years. The specific content of malondialdehyde, an end product of lipid peroxidation, was also found to increase with age. On the other hand, we examined the enzyme activities of Cu, Zn-superoxide dismutase (Cu,Zn-SOD), Mn-superoxide dismutase (Mn-SOD), catalase, and glutathione peroxidase (GPx) in the skin fibroblasts. The activities of Cu,Zn-SOD, catalase and glutathione peroxidase were found to decrease with age. However, the activity of Mn-SOD was increased with age before 60 years but was decreased thereafter. Moreover, the activity ratios of Mn-SOD/catalase and Mn-SOD/GPx exhibited the same pattern of change with age. This indicates that free radical scavenging enzymes can effectively dispose of ROS and free radicals before 60 years of age. However, elevated oxidative stress caused by an imbalance between the production and removal of ROS and free radicals occurred in skin fibroblasts after 60 years of age. Taken together, we suggest that the functional decline of free radical scavenging enzymes and the elevation of oxidative stress may play an important role in eliciting oxidative damage and mutation of mtDNA during the human aging process.     

Chemomodulatory effect of Dolichos biflorus Linn. on skin and forestomach papillomagenesis in Swiss albino mice

Effect of consumption of three different doses (2%, 4% and 6%, w/w) of Dolichos biflorus Linn. seeds on hepatic drug metabolizing enzymes, antioxidant enzymes, reduced glutathione content, lactate dehydrogenase and lipid peroxidation in Swiss albino mice has been reported. Anti-carcinogenic effect has been studied by 7,12-dimethylbenzanthracene (DMBA)-induced skin and benzo(a)pyrene[B(a)P]-induced forestomach papillomagenesis models. D. biflorus consumption resulted in a significant increase in hepatic carcinogen metabolizing enzyme systems especially at 4% and 6% doses. Significant increase in reduced glutathione content (GSH) and specific activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione reductase (GR) in liver of mice, at 4% and 6% doses has been reported. Lactate dehydrogensae (LDH) activity and peroxidative damage has been significantly decreased at 4% and 6% doses. In skin papillomagenesis model, 4% and 6% dose in diet significantly reduced the tumor incidence (up to 25%), tumor multiplicity (up to 59%) and tumor volume per mouse (up to 70%) as compared to DMBA treated group. Importantly, significant reduction in tumor incidence (up to 33%) and tumor multiplicity (up to 61%) was evident for forestomach papillomagenesis model.     

Molecular characterization of corn glutathione S-transferase isozymes involved in herbicide detoxication

Corn ( Zea mays L.) glutathione S-transferases (EC 2.5.1.18) have attracted interest, in part, due to their involvement in the metabolism of several herbicides, including atrazine and alachlor. Three corn, glutathione S-transferases have been purified, and cDNA clones have been isolated and sequenced for two of these, GST I and GST III. In addition to showing some amino acid sequence similarity to each other, the two sequenced corn glutathione S-transferases also show some similarity to rat and human enzymes. The corn glutathione S-transferases responsible for atrazine tolerance have not yet been purified or cloned, but purification attempts indicate that corn has two glutathione S-transferases with activity towards atrazine. While many glutathione S-transferases from various organisms have been detected by using 1-chloro-2,4-dinitrobenzene as a substrate, the atrazine-specific glutathione S-transferases have very little or no activity with 1-chloro-2,4-dinitrobenzene. This shows the importance of assaying with a variety of substrates when characterizing glutathione S-transferases.     

Evaluation of chemopreventive action of Ginsenoside Rp1

We evaluated the chemopreventive properties of Ginsenoside Rp1 on 7,12-Dimethyl benz (a) anthracene (DMBA) skin papillomagenesis in Swiss albino mice. A significant reduction in values of tumor incidence, tumor burden, and cumulative number of papilloma was observed in mice treated orally with Ginsenoside Rp1 continuously at pre-, peri- and post-initiational stages of papillomagenesis as compared to the control group. Chemopreventive potential of Ginsenoside Rp1 was also observed on the skin metabolizing enzymes in Swiss albino mice. Ginsenoside Rp1 produced a significant elevation in the skin microsomal cytochrome p-450 and cytochrome b5, glutathione S-transferase (GST), reduced glutathione (GSH), glutathione peroxidase (GPX), glutathione reductase (GR), DT-diaphorase, superoxide dismutase (SOD) and catalase levels in the group of mice treated with Ginsenoside Rp1 for seven consecutive days. However, there was significant decrease in lipid peroxidation (LPO) level in Ginsenoside Rp1 treated group.     

Effects of antioxidants on glutathione levels and clinical recovery from the malnutrition syndrome kwashiorkor--a pilot study

Kwashiorkor is a severe edematous form of malnutrition with high prevalence and lethality in many African countries, and repeatedly has been reported to be associated with oxidative stress. The therapy of kwashiorkor is still ineffective. In this pilot study, we tested the hypothesis that oral application of thiol-containing antioxidants increases glutathione status and is beneficial for the clinical recovery of kwashiorkor patients. The longitudinal clinical intervention study was carried out at St Joseph's Hospital, Jirapa, Ghana. Children with severe kwashiorkor were randomly assigned to either a standard treatment (ST) receiving a therapeutic protocol based on the recommendations of the WHO or to one of three study groups receiving in addition 2 x 600 mg reduced glutathione or 2 x 50 mg alpha-lipoic acid or 2 x 100 mg N-acetylcysteine per day. Patients were followed up clinically and biochemically for 20 days and compared with 37 healthy controls. Both glutathione and alpha-lipoic acid supplementation had positive effects on survival. Also, the blood glutathione concentrations correlated positively with survival rates. Furthermore, the initial skin lesions, glutathione and total protein concentrations were found to be strong predictors of survival. The data strongly suggest that a therapy restoring the antioxidative capacity by applying cysteine equivalents in the form of glutathione and/or alpha-lipoic acid is beneficial for biochemical and clinical recovery of kwashiorkor patients.     

Modulatory influence of Brassica compestris Linn var sarson on phase-II carcinogen metabolizing enzymes and glutathione levels in mice

The present study reports the modulatory influence of 95% ethanolic extract from the seeds of B. compestris on the activity of phase-II enzymes such as glutathione S-transferase (GST), DT-diaphorase (DTD) and reduced glutathione (GSH) level in the skin, lung, kidney and forestomach of the mouse. Oral treatment with the seed extract at 800 mg/kg body wt. for 15 days significantly elevated GST in lung and forestomach and DT-diaphorase in forestomach and skin and GSH level in lung, kidney forestomach and skin. The lower dose 400 mg/kg body wt was effective only in inducing GST and DT-diaphorase activity in forestomach and reduced glutathione level in lung. The findings suggest that B. compestris seed extract may block or suppress the events associated with chemical carcinogenesis at least in part, by inducing metabolic detoxification of the carcinogen.     

Chemomodulation of carcinogen metabolising enzymes,antioxidant profiles and skin and fore-stomach papillomagenesis by Spirulina platensis

Numerous reports have revealed an inverse association between consumption of some selective natural products and risk of developing cancer. In the present study the effect of 250 and 500 mg/kg body weight of Spirulina was examined on drug metabolising phase I and phase II enzymes, antioxidant enzymes, glutathione content, lactate dehydrogenase and lipid peroxidation in the liver of 7-week-old Swiss albino mice. The implications of these biochemical alterations have been further evaluated adopting the protocol of benzo(a)pyrene induced forestomach and 7,12 dimethylbenz(a)anthracene (DMBA) initiated and croton oil promoted skin papillomagenesis. Our primary findings reveal the Monofunctional nature of Spirulina as deduced from its potential to induce only the phase II enzyme activities associated mainly with carcinogen detoxification. The glutathione S-transferase and DT-diaphorase specific activities were induced in hepatic and all the extrahepatic organs examined (lung, kidney and forestomach) by Spirulina pretreatment (significance level being from p < 0.05 to p < 0.005) except for the low dose treatment in forestomach. With reference to antioxidant enzymes viz., superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase and reduced glutathione were increased significantly by both the chosen doses of Spirulina from p < 0.01 to p < 0.005. Chemopreventive response was quantitated by the average number of papillomas per effective mouse (tumor burden) as well as percentage of tumor bearing animals. There was a significant inhibition of tumor burden as well as tumor incidence in both the tumor model systems studied. In the skin tumor studies tumor burden was reduced from 4.86 to 1.20 and 1.15 by the low and high dose treatment respectively. In stomach tumor studies tumor burden was 2.05 and 1.73 by the low and high doses of Spirulina treatment against 3.73 that of control.     

Inhibitory effect of 18beta-glycyrrhetinic acid on 12-O-tetradecanoyl phorbol-13-acetate-induced cutaneous oxidative stress and tumor promotion in mice

Glycyrrhetinic acid is an aglycone of glycyrrhizic acid, another major active component of licorice roots. Licorice root extract has been used for a long time as a medicine and a natural sweetening additive. In the present study, we found that glycyrrhetinic acid inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA) mediated oxidative stress and tumor promotion in murine skin. Topical application of TPA alone in mouse skin enhances ornithine decarboxylase activity and also increases [3H]-thymidine incorporation in DNA. Topical application of TPA also resulted in the depletion of glutathione, activities of glutathione metabolizing and antioxidant enzymes. Application of glycyrrhetinic acid prior to TPA treatment reduces this enhanced ODC activity, [3H]-thymidine incorporation in DNA and oxidative stress. Glycyrrhetinic acid was also found to inhibit DMBA/TPA-induced skin tumor formation at doses of 1.25 and 2.5 mg by reducing the number of tumors per mouse by 24% (P < 0.05) and 62% (P < 0.05), respectively. These results suggest that glycyrrhetinic acid, an antioxidant, is a potential chemopreventive agent that can inhibit DMBA/TPA-induced cutaneous oxidative stress and tumor promotion.