RESUMEN
Among the factors determining the propensity of a chemical to induce skin allergy are the penetration into skin and the kinetics of ingress. Confocal Raman spectroscopy can provide such information as it enables direct, spatially resolved measurement of the skin and of any chemical uptake. Several chemicals can be monitored at once, and the method is non-destructive (light in, light out) so that the skin can be kept intact for repeated and continuous measurement. Raman spectroscopy was used to follow the penetration of 2.5 weight percent trans-cinnamaldehyde and its delivery vehicle into skin in vitro, up to 24 h after topical application. A custom-made Bronaugh-type diffusion cell that was suitable for the Raman experiment was used. Four different vehicles were tested: absolute ethanol, 50% aqueous ethanol, propylene glycol and acetone:olive oil (4:1); these gave different time scales for cinnamaldehyde penetration. The acetone:olive oil vehicle phase-separated on the skin surface and the cinnamaldehyde penetrated at different rates in the different phases, which may be of significance since this is the preferred solvent for the local lymph node assay (an in vivo animal test used to generate hazard information on skin sensitization). In conclusion, the Raman method gives valuable detailed information on chemical ingress, clearly differentiates between different delivery rates and allows solvent monitoring alongside the chemical of interest.
Asunto(s)
Acroleína/análogos & derivados , Antineoplásicos Fitogénicos/farmacocinética , Hipersensibilidad a las Drogas/fisiopatología , Vehículos Farmacéuticos/farmacocinética , Acroleína/administración & dosificación , Acroleína/farmacocinética , Acroleína/farmacología , Administración Cutánea , Animales , Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/farmacología , Oído , Excipientes , Humanos , Piel/efectos de los fármacos , Absorción Cutánea , Solventes , Espectrometría Raman , Porcinos , Factores de TiempoRESUMEN
Aqueous solutions (pH = 8) of both 3,3'-dimethyl and 4,4'-dimethyl substituted analogues of the photoallergen fentichlor (bis(2-hydroxy-5-chlorophenyl)sulphide) produced stable semiquinone radicals when irradiated with u.v. light (greater than 310 nm). These radicals have been characterised using electron spin resonance techniques: the results confirm the assignment of hyperfine coupling constants for the parent fentichlor radical. The binding of fentichlor to HSA was found to be partly oxygen dependent demonstrating a role for semiquinone type radicals in the binding mechanism. The stoichiometry and specificity of the binding of the dimethyl analogues to soluble proteins were found to be similar to that of fentichlor itself.
Asunto(s)
Antiinfecciosos Locales/efectos de la radiación , Clorofenoles/efectos de la radiación , Trastornos por Fotosensibilidad/inducido químicamente , Proteínas/metabolismo , Rayos Ultravioleta , Antiinfecciosos Locales/efectos adversos , Sitios de Unión , Clorofenoles/efectos adversos , Clorofenoles/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres , Humanos , Metilación , Estructura Molecular , Oxígeno/farmacología , Fotoquímica , Albúmina Sérica/metabolismoRESUMEN
The photochemical reactions of the bacteriocide bithionol (a known photoallergen in man) with soluble proteins and peptides, were investigated. Solutions of human serum albumin (HSA), human gamma-globulin, bovine insulin and poly-L-lysine were irradiated with ultraviolet light of wavelength 313 nm in the presence of [35S]-bithionol and the extent of photochemical (covalent) binding determined. HSA bound at least four molecules of bithionol per molecule of HSA. Bithionol was also found to bind to gamma-globulin to a similar extent; lower levels of binding were achieved with bovine insulin and poly-L-lysine. A bithionol-HSA photoadduct was treated with cyanogen bromide to determine the selectivity of binding. Fractionation after cyanogen bromide treatment showed that bithionol was bound to both major fragments of HSA, with a preference for the N-terminal region of the protein.
Asunto(s)
Bitionol/análisis , Péptidos/análisis , Fenoles/análisis , Proteínas/análisis , Bromuro de Cianógeno , Humanos , Fotoquímica , Unión Proteica , Albúmina Sérica/análisis , Solubilidad , Rayos Ultravioleta , gammaglobulinas/análisisRESUMEN
An in vitro screening procedure is described whereby potential photoallergens are irradiated with ultraviolet (UV) light in the presence of monomeric human serum albumin. UV spectroscopy before and after irradiation and after passage of the reaction mixture through Sephadex G-10, is used to determine whether or not the test compound has bound to the albumin. Using this method and others employing radiolabelled compounds, nine photoallergens have been shown to bind to protein under the influence of UV light. In contrast, a cinnamate sunscreen, which absorbs UV light but is not a photoallergen, does not bind under these conditions. The method is proposed as an in vitro screening procedure for potential photoallergens.
RESUMEN
In the present study, the distribution and nature of specific xenobiotic-metabolizing enzymes has been studied. Immunocytochemistry revealed the specific isoenzyme profile of the different cell types in mouse, rat and human skin. Constitutive levels of cytochrome P-450 1A1/A2 (CYP1A1/A2), CYP2B1/B2 and glutathione S-transferase were concentrated in the epidermis and sebaceous glands of all three species. Enzymic digestion followed by density gradient centrifugation resulted in fractions enriched in sebaceous cells, basal cells and differentiated keratinocytes. The basal and sebaceous cells of mouse skin were found to contain high levels of CYP1A1/A2, which was induced approximately 10-fold following beta-naphthoflavone pretreatment. These findings suggest that xenobiotic metabolizing enzymes exhibit a similar qualitative distribution between the cell types of rodent and human skin and that these cell types may be isolated for use in mechanistic studies involving cutaneous metabolism and toxicity.
RESUMEN
Skin possesses metabolic capacity which may be significant for chemicals with direct skin effects. The conversion of some chemical contact allergens from an inactive to an active hapten has also been described. Eugenol and isoeugenol are contact allergens that fit this description, but despite close chemical similarity, they are not cross-reactive and this has led to the hypothesis that they may be activated by different mechanisms. There is evidence that eugenol follows a phenolic radical mechanism, while isoeugenol undergoes demethylation followed by oxidation to the orthoquinone. Since these reactions may be cytochrome P-450 mediated (the former being cytochrome P4501A driven) we investigated how modulation of this system affects skin sensitization to eugenol and isoeugenol. Using the local lymph node assay, which measures lymphocyte proliferation in draining lymph nodes, the effect of clotrimazole, an inhibitor of epidermal cytochrome P4501A, was assessed. Clotrimazole enhanced the response to eugenol and potassium dichromate (an irrelevant contact allergen) approximately two-fold and isoeugenol five-fold. It was anticipated that clotrimazole would have little effect on isoeugenol sensitization, but that through inhibition of cytochrome P4501A, eugenol sensitization would be reduced. However, the present data suggest that the presence of P4501A in skin may result in the metabolism of these chemicals to relatively harmless moieties.
RESUMEN
Iodide uptake (IU) by thyrocytes from the plasma against chemical and electrical gradients is by a specific iodide transporter or 'pump'. Perchlorate (ClO(4)(-)) and other univalent, symmetrical anions are competitive inhibitors of iodide uptake (IU), and apparent K(i) for individual anions can be correlated with ion size. This study uses cultured thyrocytes and a broad range of anion size, in particular a series of spherical hexafluoride ions, in order to understand more about the parameters governing the activity of competitive inhibitors of IU. (125)I uptake and organification by cultured porcine thyrocytes was combined with biochemical enzyme inhibition studies on thyroid peroxidase in order to identify specific effects on IU. Known inhibitors were used in a validation phase and demonstrated that a combination of the in vitro thyrocyte (125)I assay and thyroid peroxidase inhibition assay could be used to identify selective inhibitors that can be difficult to identify using thyrocytes alone. Anions of less than, or similar, volume to I(-) (35.0 A(3)) were weak inhibitors with potency increasing proportional to ion size up to an apparent maximum for AsF(6)(-) (94.45 A(3)); this correlation was strong (r = 0.96). PF(6)(-), AsF(6)(-) and SbF(6)(-) were identified as novel inhibitors of IU, showing that the size range of anions active in IU inhibition is greater than that previously identified. The biological significance in vivo of the inhibitory action of the hexafluoride ions is not known. Their potency in this study suggests that these anions may have the potential to affect thyroid function in vivo if they were available systemically.
RESUMEN
Ethanol is a major component of many aerosol sprays and consumer products that are designed to contact the skin. It is theoretically possible that small amounts of ethanol from alcohol-based sprays can be absorbed across the skin or inhaled during spraying. In order to assess the potential systemic dose, three parameters were measured: the evaporation of [14C]ethanol from the skin surface, the in vitro penetration of [14C]ethanol through excised pig skin and the ethanol concentration in the blood of human volunteers following simulated use of an alcohol based deodorant spray. The rate of evaporation from Benchkote and whole pig skin was similar (t(1/2)=13.6 sec and 11.7 sec, respectively) while that from glass was longer (t(1/2)=24.8 sec). Ethanol penetration through pig skin in vitro was greater in occluded cells than in non-occluded cells (2.19 mg/cm(2) and 0.10 mg/cm(2) in 24 hours, respectively). At the maximum flux seen in this experiment under occlusion, the amount of ethanol penetrating from a 1 m(2) area of skin would give a blood alcohol level of about 4 mg% in a 70-kg man. In the human use study, none of the blood samples taken from 16 human volunteers exhibited a detectable level of alcohol. These studies provide evidence that a systemic dose of ethanol is likely to be very low after the use of formulations delivering ethanol to the skin.
Asunto(s)
Depresores del Sistema Nervioso Central/farmacocinética , Etanol/farmacocinética , Absorción Cutánea , Administración Tópica , Adulto , Aerosoles , Animales , Depresores del Sistema Nervioso Central/administración & dosificación , Depresores del Sistema Nervioso Central/sangre , Etanol/administración & dosificación , Etanol/sangre , Femenino , Humanos , Técnicas In Vitro , Masculino , Porcinos , VolatilizaciónRESUMEN
Conjugated linoleic acid (CLA) is reported as having several beneficial effects including anticarcinogenic, cholesterol-lowering and anti-atherogenic properties; however, CLA has also been reported as a putative peroxisome proliferator in mice. In this study the ability of CLA to cause peroxisome proliferation in the rat, as measured by accepted enzyme markers was investigated. Male Wistar rats were fed a semi-purified diet containing 0.0, 1.5 or 5.0 energy % CLA for 4 weeks. A positive control group were given 250 mg clofibrate/kg by gavage for 4 days. Hepatic cyanide-insensitive palmitoyl coenzyme A (PCoA) oxidase and carnitine acetyl transferase (CAT) activities and total cytochrome P450 (CYP) levels were measured. CLA had no effect on body weight or liver/body weight ratios, but clofibrate significantly increased mean liver/body weight ratio by 41.6%. Clofibrate-treated rats showed typical changes with increases in hepatic PCoA oxidase and CAT activity (5.8-fold and 22.8-fold) and in total CYP (1.66-fold) compared with control. There were no differences between the control group and the groups fed CLA for either the peroxisomal enzymes or total CYP. These results suggest that CLA does not act in the rat as a classical peroxisome proliferator and that there may be a species difference in the effects on rat and mice.
Asunto(s)
Ácidos Linoleicos/toxicidad , Proliferadores de Peroxisomas/toxicidad , Animales , Peso Corporal/efectos de los fármacos , Carnitina O-Acetiltransferasa/metabolismo , Clofibrato/toxicidad , Sistema Enzimático del Citocromo P-450/metabolismo , Ingestión de Líquidos/efectos de los fármacos , Ingestión de Alimentos/efectos de los fármacos , Isomerismo , Ácidos Linoleicos/química , Hígado/anatomía & histología , Hígado/efectos de los fármacos , Hígado/enzimología , Masculino , Tamaño de los Órganos/efectos de los fármacos , Oxidorreductasas/metabolismo , Proliferadores de Peroxisomas/química , Ratas , Ratas WistarRESUMEN
Synopsis Photocontact allergy, an acquired altered reactivity of the skin to light in the presence of a photosensitizer, has for many years been considered to be a delayed-type hypersensitivity. The response has been postulated as being mediated via the formation of a protein-photoallergen conjugate acting as a complete antigen. The purpose of this paper is to bring together evidence at the molecular level which supports this theory of photoallergy. All photoallergens studied so far have been shown to be able to bind to proteins under the influence of ultraviolet light. Photoallergen-protein binding in most cases is non-specific; the exception, that of tetrachlorosalicylanilide (T(4)CS), displays a high specificity towards serum albumin. The mechanism of protein-photoallergen binding is thought to proceed via the formation of highly reactive species such as free radicals. Free radicals have now been observed using electron spin resonance spectroscopy for at least five photoallergens. Macrophage inhibition and lymphocyte transformation experiments have indicated that protein-photoallergen conjugates act as complete antigens. Further evidence for this is provided by the observation that photoconjugates injected into guinea-pigs can induce a photoallergic response in the absence of irradiation. The response produced by T(4)CS-serum albumin conjugates is greater than that produced by any other combination of photoallergen and protein. The potency of the T(4)CS-serum albumin photoconjugate in inducing photoallergy, together with the binding specificity of T(4)CS, suggest that albumin may have a special role as a carrier protein in T(4)CS photoallergy.
RESUMEN
Guinea pig test methods are the most commonly used and reliable of predictive models for contact photoallergenicity of chemicals. The murine local lymph node assay (LLNA) has been developed recently as an alternative method for the identification of skin-sensitizing chemicals. Sensitization potential is measured from an assessment of the proliferation of lymphocytes in lymph nodes draining the site of exposure to the test chemical. This work investigates the activity of 6 widely reported photoactive chemicals in a modified LLNA (a photo-LLNA). The photoallergens tetrachlorosalicylanilide and fentichlor elicited positive ultraviolet radiation (UV)-dependent proliferative responses that were greater than their positive UV-independent responses, suggesting that they are both contact and photoallergic in the mouse. The lack of a proliferative response to 6-methylcoumarin and the absence of a reproducible response to musk ambrette suggest that the assay is insufficiently sensitive to identify weak photoallergic potential. Positive UV-dependent responses to acridine and anthracene, both photoirritants, cast doubt on the specificity of the photo-LLNA. Positive LLNA responses to these chemicals may be due to skin protein modification, based on evidence from the in vitro photo-chemical protein-binding assay. These results demonstrate that the photo-LLNA is able to detect at least moderate photoallergic potential.