DA-9601 suppresses 2, 4-dinitrochlorobenzene and dust mite extract-induced atopic dermatitis-like skin lesions.

DA-9601 (Stillen™) is a novel anti-peptic formulation prepared from the ethanol extracts of Artemisia asiatica possessing anti-oxidative, anti-allergic and anti-inflammatory activities. However, their effect on atopic dermatitis (AD) has not been studied yet. In this study, we report that topical application of DA-9601 suppressed house dust mite extract (Dermatophagoides farinae extract, DFE) and 2, 4-dinitrochlorobenzene (DNCB)-induced AD-like skin lesions in BALB/c mice model. We established atopic dermatitis model in BALB/c mice by repeated local exposure of DFE/DNCB to the ears. Repeated alternative treatment of DFE/DNCB caused AD-like lesions. DA-9601 reduced AD-like skin lesions based on ear thickness and histopathological analysis, and serum IgE levels. DA-9601 inhibited mast cell infiltration into the ear and elevation of serum histamine in AD model. In addition, DA-9601 suppressed DFE/DNCB-induced expression of IL-4, IL-13, IL-31, and TNF-α in the ears. Taken together, our results showed that topical application of DA-9601 exerts beneficial effects in animal model of AD, suggesting that DA-9601 might be a candidate for the treatment of AD.

Role of early- or late-phase activation of p38 mitogen-activated protein kinase induced by tumour necrosis factor-alpha or 2,4-dinitrochlorobenzene during maturation of murine dendritic cells.

Dendritic cells (DCs) are maturated by a variety of stimuli. However, the precise mechanisms underlying the maturation of DCs are not fully understood. In the present study, we analysed the effects of tumour necrosis factor-alpha (TNF-alpha) and 2,4-dinitrochlorobenzene (DNCB) on phenotypic maturation and p38 mitogen activated protein kinase (MAPK) activity, using a murine DC line. TNF-alpha markedly increased the surface expression of major histocompatibility complex (MHC) and costimulatory molecules, CD86 and CD80, on DCs. DNCB more markedly enhanced the surface expression of costimulatory molecules, but showed less stimulatory capability on MHC molecules, compared with TNF-alpha. Simultaneous treatment of DCs with TNF-alpha and DNCB showed additive enhancement of costimulatory molecule expression. TNF-alpha activated p38 MAPK in DCs only at an early time-point (15 min). In contrast, DNCB activated p38 MAPK at later time-points (3-6 hr). SB203580, a specific inhibitor of p38 MAPK, partially or markedly inhibited the phenotypic changes of DCs induced by TNF-alpha or DNCB, respectively. In addition, N-acetyl-l-cysteine, a reducing supplier, completely inhibited the DNCB-induced expression of MHC and costimulatory molecules, but not those induced by TNF-alpha. These findings demonstrate that TNF-alpha and DNCB activate the p38 MAPK pathway at an early and a late phase, respectively, and thereby induce DC maturation through different signal pathways.

Contrasting effects of thiol-modulating agents on endothelial NO bioactivity.

The bioactivity of endothelium-derived nitric oxide (NO) is an important component of vascular homeostasis that is sensitive to intracellular redox status. Because glutathione (GSH) is a major determinant of intracellular redox state, we sought to define its role in modulating endothelial NO bioactivity. In porcine aortic endothelial cells (PAECs), we depleted intracellular GSH (>70%) using 1) buthionine-(S,R)-sulfoximine (BSO), which inhibits GSH synthesis; 2) diamide, which oxidizes thiols; or 3) 1-chloro-2,4-dinitrobenzene (CDNB), which putatively depletes GSH through glutathione S-transferase activity. Cellular GSH depletion with BSO had no effect on endothelial NO bioactivity measured as A-23187-induced cGMP accumulation. In contrast, oxidation of intracellular thiols with diamide inhibited both A-23187-induced cGMP accumulation and the cGMP response to exogenous NO. Diamide treatment of either PAECs, PAEC membrane fractions, or purified endothelial nitric oxide synthase (eNOS) resulted in significant inhibition (approximately 75%) of eNOS catalytic activity measured as L-[(3)H]arginine-to-L-[(3)H]citrulline conversion. This effect appeared related to oxidation of eNOS thiols as it was completely reversed by dithiothreitol. Glutathione depletion with CDNB inhibited A-23187-stimulated cGMP accumulation but not the cGMP response to exogenous NO. Rather, CDNB treatment impaired eNOS catalytic activity in intact PAECs, and this effect was reversed by excess NADPH in isolated purified eNOS assays. Consistent with these results, we found spectral evidence that CDNB reacts with NADPH and renders it inactive as a cofactor for either eNOS or glutathione reductase. Thus thiol-modulating agents exert pleiotropic effects on endothelial NO bioactivity, and these data may help to resolve a number of conflicting previous studies linking GSH status with endothelial cell NO bioactivity.

The role of glutathione S-transferases in the detoxification of some organophosphorus insecticides in larvae and pupae of the yellow mealworm, Tenebrio molitor (Coleoptera: Tenebrionidae).

The correlation between the natural levels of glutathione S-transferase (GST) and the tolerance to the organophosphorus insecticides parathion-methyl and paraoxon-methyl, as well as the interaction of affinity-purified enzyme and the insecticides were investigated in order to collect further information on the role of the glutathione S-transferase system as a mechanism of defence against insecticides in insects. The studies were carried out on the larvae and pupae of the coleopteran Tenebrio molitor L, which exhibit varying natural levels of GST activity. Stage-dependent susceptibility of the insect against insecticides was observed during the first 24 h. However, 48 h after treatment, the KD50 value increased significantly due to the recovery of some individuals. Simultaneous injection of insecticide with compounds which inhibit GST activity in vitro caused an alteration in susceptibility of insects 24 or 48 h post-treatment, depending on stage and insecticide used. Inhibition studies combined with competitive fluorescence spectroscopy revealed that the insecticides probably bind to the active site of the enzyme, thus inhibiting its activity towards 1-chloro-2,4-dinitrobenzene in a competitive manner. High-performance liquid chromatography and gas chromatography revealed that T molitor GST catalyses the conjugation of the insecticides studied to a reduced form of glutathione (GSH). From the above experimental results, it is considered that GST offers a protection against the organophosphorus insecticides studied by active site binding and subsequent conjugation with GSH.

Studies on the differential inhibition of glutathione conjugate formation of (+)-anti-benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide and 1-chloro-2,4-dinitrobenzene in V79 Chinese hamster cells.

V79 Chinese hamster cells have previously been shown to lack the capacity to detoxify the mutagenic and carcinogenic compound (+)-anti-benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide [(+)-anti-BPDE] by Pi class glutathione transferase (GSTPi)-catalysed conjugation with GSH, although these cells contain such an enzyme [Romert, Dock, Jenssen and Jernström (1989) Carcinogenesis 10, 1701-1707; Swedmark, Romert, Morgenstern and Jenssen (1992) Carcinogenesis 13, 1719-1723; Swedmark and Jenssen (1994) Gene 139, 251-256]. Previous findings also indicate that these results do not depend on an inactive GSTPi enzyme, since V79 cells conjugate 1-chloro-2, 4-dinitrobenzene (CDNB) with GSH, but more likely on (a) factor(s) that inhibit(s) V79 GSTPi selectively [Swedmark, Jernström and Jenssen (1996) Biochem. J. 318, 533-538]. The present study demonstrates that both human and V79 recombinant GSTPi enzymes are inhibited with respect to conjugating (+)-anti-BPDE, but not CDNB, after pre-incubation with V79-cell extract, but not with MCF-7-cell extract. In addition, it was found that the inhibition is dependent on the amount of cell extract present and that the factor(s) is heat-resistant and has a molecular mass of less than 10 kDa, suggesting that the factor(s) is (are) non-proteinaceous in nature.

Inhibition of irritation and contact hypersensitivity by phenoxyacetic acid methyl ester in mice.

New anti-irritant treatments are required to prevent irritation and sensitization reactions to consumer medicines and dermatological drugs. We report here that phenoxyacetic acid methyl ester (PAME) is an effective agent to prevent and treat irritant and allergic contact dermatitis. Balb/c mice skin-treated with 1% PAME do not lose weight relative to vehicle-treated mice, nor is it irritating to mouse skin. Topical PAME prevents skin irritation to a wide variety of irritants including: arachidonic acid, capsaicin, sodium lauryl sulfate (SLS), disodium laureth sulfosuccinate and tetradecanoylphorbol-13-acetate. Histological studies showed that 1% PAME greatly diminished dermal neutrophilic infiltration and dermal capillary vessel dilation, and prevented epidermal hyperproliferation and hyperkeratosis that accompanies detergent (SLS)-induced skin irritation. Topical PAME inhibited ear swelling following ear challenge during the elicitation phase of contact hypersensitivity in mice sensitized with 1-chloro-2, 4-dinitrochlorobenzene (DNCB), oxazolone and the hair coloring dye rho-phenylenediamine (PPD). Finally, topical administration of 1% PAME prior to PPD or DNCB sensitization prevented the induction phase of contact hypersensitivity. These results indicate that PAME represents a potential new category of potent topical anti-inflammatory agents.

Glutatión S-transferasas en no-vertebrados y en mamíferos: su importancia en la detoxificación de insecticidas / The Glutathione S-transferases (GST) of non-vertebrate organisms and mammals

Las Glutatión S-transferasas (GST) de organismos no-vertebrados no han sido estudiadas con la misma intensidad que las de mamíferos. El interés en las GST en no-vertebrados radica en su importancia como protección bioquímica de los organismos expuestos a compuestos químicos ambientales. En efecto, se ha observado que niveles elevados de GST podrían estar asociados con la tolerancia a pesticidas. La intención de esta actualización es revisar el nivel de conocimiento actual sobre estas enzimas en no-vertebrados, comparándolas con las de mamíferos. Evaluar la contribución de estos estudios al conocimiento del rol de las glutinatión transferasas en general, e intentar discernir la dirección de las futuras investigaciones en este campo

Glutatión S-transferasas en no-vertebrados y en mamíferos: su importancia en la detoxificación de insecticidas / The Glutathione S-transferases (GST) of non-vertebrate organisms and mammals

Las Glutatión S-transferasas (GST) de organismos no-vertebrados no han sido estudiadas con la misma intensidad que las de mamíferos. El interés en las GST en no-vertebrados radica en su importancia como protección bioquímica de los organismos expuestos a compuestos químicos ambientales. En efecto, se ha observado que niveles elevados de GST podrían estar asociados con la tolerancia a pesticidas. La intención de esta actualización es revisar el nivel de conocimiento actual sobre estas enzimas en no-vertebrados, comparándolas con las de mamíferos. Evaluar la contribución de estos estudios al conocimiento del rol de las glutinatión transferasas en general, e intentar discernir la dirección de las futuras investigaciones en este campo (AU)

Prevention of UVB-induced immunosuppression in humans by a high sun protection factor sunscreen.

BACKGROUND AND DESIGN: To date, studies of the effectiveness of sunscreens in preventing UVB-induced suppression of contact hypersensitivity in humans have not been published. Several studies in mice of the protection afforded by sunscreens from UVB-induced suppression of contact hypersensitivity and rejection of transplanted skin cancers have yielded dissimilar results, ranging from "no" to "complete" protection. We sought to determine whether the effect of preapplication of a sun protection factor (SPF) 29 sunscreen (containing octyl methoxycinnamate, oxybenzone, and octyl salicylate) could prevent local UVB-induced suppression of contact hypersensitivity to dinitrochlorobenzene (DNCB). Nineteen subjects received either three minimal erythema doses of UVB daily on three consecutive days (UVB group) or sunscreen followed by this same dose of UVB irradiation (sunscreen plus UVB group) to a 16-cm2 area of the buttock. One day after completion of irradiation, DNCB was applied to this buttock site, and 2 weeks later, forearm challenge with four different concentrations of DNCB was performed. A control group of 10 subjects underwent DNCB testing as above, but with no prior exposure to UVB (no-UVB group). RESULTS: The UVB group had a reduced response rate to all challenge doses of DNCB (3.125, 6.25, and 8.8 micrograms), except for the highest dose (12.5 micrograms) compared with the no-UVB control group (Fisher's Exact test, P < or = .008), and compared with the sunscreen plus UVB group (P < or = .02). The no-UVB and sunscreen plus UVB groups showed no significant differences in response rates to any of the doses of DNCB tested (P > or = .53). CONCLUSIONS: These results indicate that application of a sunscreen with over ninefold greater protection than that needed to prevent erythema prior to localized UVB radiation prevents localized UVB-induced suppression of contact hypersensitivity. Further studies are needed to determine whether sunscreens providing less protection, yet still preventing erythema, adequately prevent suppression of contact hypersensitivity, a possible surrogate marker for skin cancer tumor antigen recognition and rejection.

Reduced glutathione esters--antidotes to toxicity. Cytotoxicity induced by hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and menadione in murine P388D1 macrophages in vitro.

Repletion of depleted cellular reduced glutathione (GSH) levels in oxidative stress and exposure to arylating agents is a strategy for the development of antidotes to chemical toxicity. The effect of GSH, reduced glutathione ethyl monoester (GSHEt), and reduced glutathione ethyl diester (GSHEt2) on the cytotoxicity of hydrogen peroxide, 1-chloro-2,4-dinitrobenzene (CDNB), and menadione to P388D1 macrophages in vitro was investigated. The median toxic concentration TC50 values of the toxicants were hydrogen peroxide 24 +/- 2 mM (N = 19), CDNB 63 +/- 6 microM (N = 18), and menadione 30 +/- 4 microM (N = 22). Reduced glutathione, GSHEt, and GSHEt2 were poor antidotes to hydrogen peroxide toxicity. Indeed, the observed antidote effects were attributed to the nonenzymatic reaction of the GSH derivatives with hydrogen peroxide in the extracellular medium. Reduced glutathione ethyl diester was a more potent antidote of CDNB- and menadione-mediated toxicity than GSHEt and GSH. For cell incubations with the approximate median toxic concentration TC50 values of hydrogen peroxide, CDNB, and menadione, the respective median effective antidote concentration EC50 values were GSHEt 23.8 +/- 4.1 mM (N = 9), 3.6 +/- 0.6 mM (N = 11), and 226 +/- 93 microM (N = 12); and GSHEt2 20.4 +/- 1.9 mM (N = 6), 603 +/- 2 microM (N = 9), and 7.6 +/- 2.3 microM (N = 12). Reduced glutathione ethyl diester was a potent antidote to CDNB- and menadione-induced toxicities but not to hydrogen peroxide-induced toxicity under acute intoxication conditions.