Protein binding and metabolism influence the relative skin sensitization potential of cinnamic compounds.

Skin protein modification (haptenation) is thought to be a key step in the manifestation of sensitization to low molecular mass chemicals (<500 g/mol). For sensitizing chemicals that are not protein reactive, it is hypothesised that metabolic activation can convert such chemicals into protein reactive toxins within the skin. trans-Cinnamaldehyde, alpha-amyl cinnamaldehyde, and trans-cinnamic alcohol are known sensitizers with differing potencies in man, where the former two are protein reactive and the latter is not. Here, we have used immunochemical methods to investigate the extent of protein-cinnamaldehyde binding in rat and human skin homogenates that have been incubated (for either 5, 15, 30, or 60 min) at 37 degrees C with cinnamaldehyde, alpha-amyl cinnamaldehyde (at concentrations of between 1 and 40 mM), and cinnamic alcohol (at higher concentrations of 200 or 400 mM). Cinnamaldehyde specific antiserum was raised specially. A broad range (in terms of molecular mass) of protein-cinnamaldehyde adducts was detected (as formed in a time- and concentration-dependent manner) in skin treated with cinnamaldehyde and cinnamic alcohol but not with alpha-amyl cinnamaldehyde. Mechanistic observations have been related to relative skin sensitization potential, as determined using the local lymph node assay (LLNA) as a biological read-out. The work presented here suggests that there is a common hapten involved in cinnamaldehyde and cinnamic alcohol sensitization and that metabolic activation (to cinnamaldehyde) is involved in the latter. Conversely, there does not appear to be a common hapten for cinnamaldehyde and alpha-amyl cinnamaldehyde. Such mechanistic work on protein modification is important in understanding the early mechanisms of skin sensitization. Such knowledge can then be used in order that effective and appropriate in vitro/in silico tools for predicting sensitization potential, with a high confidence, can be developed.

Cinnamic compound metabolism in human skin and the role metabolism may play in determining relative sensitisation potency.

BACKGROUND: trans-Cinnamaldehyde and trans-cinnamic alcohol cause allergic contact dermatitis (ACD) in humans; cinnamaldehyde is a more potent sensitiser than cinnamic alcohol. These two chemicals are principal constituents of the European Standard 'Fragrance Mix', as used in patch testing diagnostics of sensitisation to fragrances by clinical dermatologists. As contact sensitisers are usually protein reactive compounds, it is hypothesised that cinnamic alcohol (not protein-reactive) is a 'prohapten' that requires metabolic activation, presumably by cutaneous oxidoreductases, to the protein-reactive cinnamaldehyde (a 'hapten'). It is postulated that cinnamaldehyde can be detoxified by aldehyde dehydrogenase (ALDH) to cinnamic acid and/or by alcohol dehydrogenase (ADH) to cinnamic alcohol. Hence, a variety of metabolic pathways may contribute to the relative exposures and hence sensitising potencies of cinnamic alcohol and cinnamaldehyde. OBJECTIVE: To evaluate the extent of cinnamaldehyde and cinnamic alcohol metabolism in human skin and provide evidence for the role of cutaneous ADH and ALDH in such metabolism. METHODS: The extent of cinnamic alcohol and aldehyde metabolism was investigated in human skin homogenates and sub-cellular fractions. A high performance liquid chromatography method was used for analysis of skin sample extracts. Studies were conducted in the presence and absence of the ADH/cytochrome P450 inhibitor 4-methylpyrazole and the cytosolic ALDH inhibitor, disulfiram. RESULTS: Differential metabolism of cinnamic alcohol and cinnamaldehyde was observed in various subcellular fractions: skin cytosol was seen to be the major site of cinnamic compound metabolism. Significant metabolic inhibition was observed using 4-methylpyrazole and disulfiram in whole skin homogenates and cytosolic fractions only. CONCLUSIONS: This study has demonstrated that cutaneous ADH and ALDH activities, located within defined subcellular compartments, play important roles in the activation and detoxification of CAlc and CAld in skin. Such findings are important to the development of computational hazard prediction tools for sensitisation (e.g. the DEREK program) and also to dermatologists in understanding observed interindividual differences, cross-reactivities or co-sensitisation to different cinnamic compounds in the clinic.

Species variations in cutaneous alcohol dehydrogenases and aldehyde dehydrogenases may impact on toxicological assessments of alcohols and aldehydes.

Alcohol dehydrogenase (ADH; EC. 1.1.1.1) and aldehyde dehydrogenase (ALDH; EC 1.2.1.3) play important roles in the metabolism of both endogenous and exogenous alcohols and aldehydes. The expression and localisation patterns of ADH (1-3) and ALDH (1-3) were investigated in the skin and liver of the mouse (BALB/c and CBA/ca), rat (F344) and guinea-pig (Dunkin-Hartley), using Western blot analysis and immunohistochemistry with class-specific antisera. ALDH2 expression and localisation was also determined in human skin, while ethanol oxidation, catalysed by ADH, was investigated in the mouse, guinea-pig and human skin cytosol. Western blot analysis revealed that ADH1, ADH3, ALDH1 and ALDH2 were expressed, constitutively, in the skin and liver of the mouse, rat and guinea-pig. ADH2 was not detected in the skin of any rodent species/strain, but was present in all rodent livers. ALDH3 was expressed, constitutively, in the skin of both strains of mouse and rat, but was not detected in guinea-pig skin and was absent in all livers. Immunohistochemistry showed similar patterns of expression for ADH and ALDH in both strains of mouse, rat, guinea-pig and human skin sections, with localisation predominantly in the epidermis, sebaceous glands and hair follicles. ADH activity (apparent V(max), nmoles/mg protein/min) was higher in liver (6.02-16.67) compared to skin (0.32-1.21) and lower in human skin (0.32-0.41) compared to mouse skin (1.07-1.21). The ADH inhibitor 4-methyl pyrazole (4-MP) reduced ethanol oxidation in the skin and liver in a concentration dependent manner: activity was reduced to approximately 30-40% and approximately 2-10% of the control activity, in the skin and liver, respectively, using 1 mM 4-MP. The class-specific expression of ADH and ALDH enzymes, in the skin and liver and their variation between species, may have toxicological significance, with respect to the metabolism of endogenous and xenobiotic alcohols and aldehydes.

Evaluation of barrier creams against sulphur mustard. I. In vitro studies using human skin.

The purpose of this study was to assess the effectiveness of a range of passive and reactive barrier cream formulations against the chemical warfare agent sulphur mustard (SM) using an in vitro diffusion cell system containing human skin. In general, proprietary formulations were relatively effective under occluded conditions, but ineffective under unoccluded conditions. For example, SM skin absorption rates through occluded control and Stokoderm pre-treated skin were 538 +/- 193 and 200 +/- 51 microg x cm(-2) x h(-1), respectively (p < 0.05). Under unoccluded conditions, control and Stokoderm pre-treated skin absorption rates were 4.41 +/- 1.90 and 36.84 +/- 15.19 microg x cm(-2) x h(-1) (p < 0.05). Novel (perfluorinated) barrier creams were generally more effective under unoccluded conditions; pre-treatment with one formulation led to an 18-fold reduction in skin absorption rate and reduced the total amount of SM penetrated by 95% of the applied dose. Several proprietary formulations also had adverse effects on the effectiveness of the skin decontaminant fuller's earth. The rate (Jss) and total amount (percentage of dose) of SM absorbed through the skin were deemed to be independent parameters of barrier cream performance. These data indicate that (1) perceived conditions of use, (2) compatibility with existing protective equipment and (3) the rate and extent of SM skin absorption must all be taken into account when evaluating barrier creams in vitro.