Richard J. Schmidt

Are free radicals and not quinones the haptenic species derived from urushiols and other contact allergenic mono- and dihydric alkylbenzenes? The significance of NADH, glutathione, and redox cycling in the skin

SummaryThe induction of allergic contact dermatitis to urushiols from poison ivy and related plants is generally believed to involve an initial oxidation event by which a protein-reactive quinone is formed. However, this does not readily account for the contact allergenicity of closely related mono- and dihydric alkylbenzenes such as the alkylphenols and alkylresorcinols which are not so easily oxidised to quinones in vitro. When the redox processes known to occur in living tissues are taken into consideration, a more plausible unifying mechanism involving the formation of protein-reactive radical species becomes apparent. Experiments described here examine the autoxidation of p-benzoquinone and various mono- and dihydric benzenes and alkylbenzenes, and their reactions with the diphenylpicrylhydrazyl radical, cysteine, glutathione, and NADH. We have also demonstrated that administration to mice of 2-oxo-4-thiazolidine carboxylate, a compound known to elevate intracellular glutathione levels, inhibits the irritancy and sensitising activity of 3-pentadecylphenol. This work suggests that redox cycling in the skin following penetration of allergenic mono- and dihydric alkylbenzenes initially depletes local levels of endogenous reducing equivalents such as glutathione and NADH; once depleted, further cycling results in the uncontrolled generation of radical species which may reasonably be expected to exhibit protein reactivity.

Biocompatibility of Wound Management Products: A Study of the Effects of Various Polysaccharides on Murine L929 Fibroblast Proliferation and Macrophage Respiratory Burst

Abstract— An in‐vitro screening method to examine the biocompatibility of materials used in wound management has been evaluated. This involved the use of a macrophage respiratory‐burst assay and a fibroblast proliferation assay to represent respectively the inflammatory and the granulation phases in wound healing. Standard polysaccharides (calcium and sodium alginates, i‐carrageenan, chitin, chitosan lactate, chondroitin sulphate and pectic acid) were used as test compounds. None of the polysaccharide samples caused a significant increase in L929 fibroblast cell numbers relative to control after 6 days incubation. The overall effect of exposure of the fibroblast cultures to the alginates, carrageenan and chondroitin sulphate was an extension of lag phase followed by an enhanced rate of cell proliferation in the logarithmic phase. Only calcium and sodium alginates and chondroitin sulphate enhanced the respiratory burst activity of murine macrophages; i‐carrageenan and chitosan lactate were markedly inhibitory. The results suggest that a macrophage activity assay should be included as part of an in‐vitro screening program to evaluate the biocompatibility of wound management materials and to detect intrinsic biological activity.

Skin irritant effects of esters of phorbol and related polyols

The skin irritant potencies of several naturally occurring and semisynthetic polyfunctional esters based on the tigliane hydrocarbon skeleton were studied using a mouse ear irritancy assay. The compounds used were esters of 12-deoxyphorbol, 12-deoxy-16-hydroxyphorbol, and phorbol. Observations were also made on the time interval between application of these compounds in acetone solution to the mouse ear, and the attainment of maximum response. Structure/activity relationships are discussed and compared with previous observations on the skin irritant potencies of esters based on the closely related daphnane hydrocarbon skeleton. The possible nature of the site of action in the target membrane is also discussed.

Point-wise and Whole-field Laser Speckle Intensity Fluctuation Measurements Applied to Botanical Specimens

Abstract Based on multi-scattering speckle theory, the speckle fields generated by plant specimens irradiated by laser light have been studied using a pointwise method. In addition, a whole-field method has been developed with which entire botanical specimens may be studied. Results are reported from measurements made on tomato and apple fruits, orange peel, leaves of tobacco seedlings, leaves of shihu seedlings (a Chinese medicinal herb), soy-bean sprouts, and leaves from an unidentified trailing houseplant. Although differences where observed in the temporal fluctuations of speckles that could be ascribed to differences in age and vitality, the growing tip of the bean sprout and the shihu seedling both generated virtually stationary speckles such as were observed from boiled orange peel and from localised heat-damaged regions on apple fruit. Our results suggest that both the identity of the botanical specimen and the site at which measurements are taken are likely to critically affect the observation or otherwise of temporal fluctuations of laser speckles.

Biocompatibility of wound management products: standardization of and determination of cell growth rate in L929 fibroblast cultures

Abstract— To facilitate the development of a bioassay procedure by which the biocompatibilities of materials used in wound management may be assessed and compared, those environmental factors affecting cell growth in mouse L929 fibroblast cultures have been identified. Standardization of the initial cell number and frequency of change of medium resulted in the virtual elimination of variation of growth curves of L929 cells cultured in flasks of specified surface area. In addition, three methods for assessing fibroblast growth rate in the presence of alginate products used in wound management were evaluated. These were the haemacytometer counting chamber method, the Coulter counting method, and a liquid scintillation counting method. The first two methods determine the number of cells in a given volume of a cell suspension, whereas the third method determines the rate of synthesis of deoxyribonucleic acid (DNA), and hence cell growth, by measuring the incorporation of [3H]thymidine. The haemacytometer method had significant advantages over the other two procedures in providing both qualitative and quantitative data on culture morphology and cell growth response.

The dermatitic properties of black bryony {Tamus communis L.)

The skin irritant properties of both the juice expressed from the ripe berries and the slimy mucilage present in the rhizomes of black bryony (Tamus communis L.) were investigated. The dermatitis produced on human skin after gently rubbing in either the berry juice or the rhizome mucilage was found in both cases to be a result of mechanical irritation, being produced by penetration of the skin by minute needle‐like crystals of calcium oxalate. Scanning electron micrographs supporting this conclusion are presented. Chemical investigation of the rhizome mucilage confirmed the presence of histamine. The rôle of histamine in the production of skin irritation following contact with the rhizome mucilage is discussed.

A study of hydrogen peroxide generation by, and antioxidant activity of, Granuflex™ (DuoDERM™) Hydrocolloid Granules and some other hydrogel/hydrocolloid wound management materials

Summary The effect of Granuflex™ Hydrocolloid Granules (0.0l–0.50% w/v) on the rate of proliferation of murine (L929) fibroblasts was examined. The dose–response curve showed a significant (P<0.02) pro‐proliferant effect at 0.05%, and a significant (P<0.02) antiproliferant effect at 0.50%, mirroring the dose–response curve produced by hydrogen peroxide in the concentration range 10−9–10−4 mol/l. The antiproliferant effect at 0.20% w/v was abolished by catalase, suggesting that the biological activity of Granuflex was mediated by the in situ generation of hydrogen peroxide. Formation of hydrogen peroxide by Granuflex was confirmed by performing the scopoletinhorseradish peroxidase assay in the presence and absence of catalase. The total concentration of hydrogen peroxide detected was about 8 × 10−6 mol/1 (using 0.5% w/v Granuflex) after 48 h at 37°C. In contrast, when hydrogen peroxide itself was added to L929 cultures, a similar antiproliferant activity was observed at concentrations between 10−4 and 10−5 mol/l. These results suggested that Granuflex was undergoing autoxidation in the culture medium, and hence that it might possess antioxidant activity. In assays for antioxidant activity using 1,l‐diphenyl‐2‐picrylhydrazyI (DPPH), Granuflex, and two other hydrocolloid dressings (Comfeel® Powder and Bard® Absorption Dressing) showed significant ability to reduce DPPH to DPPH2. These three dressings also displayed superoxide scavenging activity in a nitroblue tetrazolium reduction assay. We conclude that, in addition to providing a moist wound‐healing environment. Granuflex and certain other hydrocolloids might contribute to the establishment and maintenance of the reducing environment necessary for energy production and hence cell division. The release of hydrogen peroxide into the wound environment couid conceivably contribute both to the inflammation phase of wound healing and to fibroblast proliferation and hence the granulation phase.

Biocompatibility of wound management products: the effect of various monosaccharides on L929 and 2002 fibroblast cells in culture

Abstract— The effects of various monosaccharides on the growth of human 2002 and mouse L929 fibroblast cultures have been investigated. Eleven monosaccharides having acidic, neutral, and basic characteristics were evaluated in a bioassay procedure developed for the investigation of biocompatibility of wound management materials. Rate of growth in both cell lines was inhibited by D‐galacturonic acid and by D‐glucuronic acid. Although most neutral sugars produced no significant change in the growth rate or in the morphology of the cells, galactose produced a significant increase in the growth rate of both cell lines whilst L‐fucose caused a significant decrease in growth of the L929 cells but did not significantly affect the growth of 2002 cells; xylose increased the growth rate of L929 but not 2002 cells. D‐Glucosamine, a basic sugar, produced inhibition of growth which followed a different pattern from that produced by the acidic sugars; N‐acetylglucosamine produced a species specific increase in cell growth of L929 cells. The results show that the effects produced by the monosaccharides on the cultured fibroblasts are related to their chemical structure and to cell line, and suggest that the use of galactose as a possible aid to wound healing should be investigated.

Biocompatibility of potential wound management products: Hydrogen peroxide generation by fungal chitin/chitosans and their effects on the proliferation of murine L929 fibroblasts in culture

Agaricus bisporus, Fusarium graminearum, Phycomyces blakesleeanus, unbleached and bleached, Rhizomucor miehei, and Rhizopus oryzae were examined as sources of fungal chitin/chitosan. The nitrogen content of the alkalitreated mycelia/sporangiophores obtained after optimization of culture conditions, and of similarly treated A. bisporus stipes, was 2.87, 1.29, 6.27, 6.50, 4.80, and 4.95% w/w, respectively, which relates to an estimated chitin content of 42, 19, 91, 94, 70, and 72%, respectively. The hydrogen peroxide (H2O2)-generating ability of the treated fungal materials after 8 h at pH 7.4 and 37 degrees C decreased in the order R. oryzae > P. blakesleeanus unbleached approximately R. miehi > F. graminearum > A. bisporus > P. blakesleeanus bleached. This did not correlate with estimated chitin content. The effect of these fungal materials on the rate of proliferation of murine L929 fibroblasts in culture also was examined. Both pro- and antiproliferant effects were observed. Significant (P < .05) proproliferant effects were observed on day 6 with R. miehei, R. oryzae, and P. blakesleeanus (unbleached and bleached) at 0.01% w/v. The greatest antiproliferant effect was observed with R. oryzae at 0.05% w/v on day 6 (-63% relative to the control, P < .05; cell viability, 95%). In contrast, A. bisporus failed to affect cell yield significantly at either 0.01 or 0.05% w/v. Addition of catalase to cultures containing R. oryzae or R. miehei at 0.05% w/v failed to abolish the antiproliferant effect on day 3, instead producing a small but significant (P < .05) increase in the effect. Catalase also failed to affect significantly the antiproliferant effect of F. graminearum at 0.05% w/v, but did abolish the proproliferant effect of P. blakesleeanus (unbleached and bleached) on day 3. Overall, our results suggest that the H2O2 being generated by the fungal materials modulates cell proliferation but that this effect is superimposed upon a H2O2-independent antiproliferant effect manifesting itself at the higher concentrations of fungal material. The antiproliferant effect was not attributable to Ca2+, Mg2+, or Fe2+ depletion although chelation of Fe2+ did correlate with H2O2-generating ability. Only P. blakesleeanus appears to lack this antiproliferant activity while retaining H2O2-generating activity. These results may aid the selection of fungal chitin/chitosan for further evaluation as a potential wound management material.

Contact dermatitis as an adverse reaction to some topically used European herbal medicinal products – part 1: Achillea millefolium–Curcuma longa

This review focuses on contact dermatitis as an adverse effect of a selection of topically used herbal medicinal products for which the European Medicines Agency has completed an evaluation up to the end of November 2013 and for which a Community herbal monograph has been produced. Part 1: Achillea millefolium L.–Curcuma longa L.