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Advances in Dermatological Sciences
By Robert Chilcott, Keith Brain The Royal Society of Chemistry
Copyright © 2014 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-1-84973-398-4
CHAPTER 1
SECTION EDITORIAL - CUTANEOUS BIOLOGY
F M Williams
Institute of Cellular Medicine and Medical Toxicology Centre, Medical School, Newcastle University, United Kingdom.
Understanding the biology of healthy skin and how it changes in disease is important for the development of new drugs to treat skin disease and for understanding and interpreting the effects of chemical exposure. Current developments in molecular medicine, use of in vitro models, in vivo imaging techniques integrated with clinical observation in dermatology are directed towards improving knowledge in this area. The chapters in this book section cover a wide range of such developing areas of biology and make a useful contribution to our current understanding of the biology of the skin.
The first two chapters in this section consider the nature of hydrolytic enzymes (carboxylesterases) and their role in the dermal metabolism of paraben esters. Esterases in the skin have traditionally received limited attention, as expression levels within the epidermis are relatively low compared to the liver and so they have been thought to have limited importance when compared to hepatic metabolism and subsequent initiation of systemic effects. However, low-level xenobiotic-metabolising enzymes such as cytochromes 450 (CYPs) and peroxidases have been shown to be important in development of DNA adducts which may lead to tumorigenesis. Also, the formation of activated protein adducts may result in hypersensitivity and inflammatory responses in the skin. A rate–limiting role for esterases has been dismissed by researchers as they are ubiquitous and highly expressed but, as indicated in the two presented papers, their variability in expression may influence the toxicity of esters during percutaneous penetration and carboxylesterase isoforms exhibit substrate specificity which influences metabolism of different chain-length parabens.
Both Williams et al. and Ford et al. addressed the local hydrolysis of paraben esters in skin using in vitro systems because of their potential oestrogenic effects and toxicity. Thus, these studies represent a positive contribution to the reduction, refinement and replacement of animals in research. The focus of the studies on parabens is highly relevant, as this class of compound is becoming ubiquitous in the environment and in the general population due to their application as anti-bacterial agents in a range of cosmetic, heath and household products. Esters are lipophilic and cross the stratum corneum but undergo hydrolysis in keratinocytes in the skin. Ford et al. also identified cross-esterification in whole skin ex vivo. This, in addition to the effects of inhibitors on the hydrolysis profile aid identification of the isoforms involved for specific molecules and so aid prediction of the hydrolysis profile for newly developed esters.
It is important to define the physiology and surface biology of healthy skin; how it varies between individuals and is influenced by age, skin treatments, sun exposure and chemical insults. Clearly, there may be a role for genetic polymorphisms in rate-limiting enzymes influencing skin biology as has recently been shown for fillagrin. The studies of paraben metabolism and carboxylesterases presented here are mainly in cultured skin cells and so metabolism profiles do not necessarily reflect those in vivo where the 3D architecture is intact and is more representative of a heterogenous genetic population. It is important to relate studies with cells in culture to 3D models such as skin equivalents and then to whole skin ex vivo and finally to the situation in humans, in vivo.
An area of cutaneous biology which tends to be overlooked is, perhaps surprisingly, the outer skin surface. Specifically, the material traditionally referred to as the acid mantle which arises from comeocyte debris and accumulated secretions of the sweat and sebaceous glands. In Chapter 4 by Shetage et al., preliminary work is presented which aims to characterise the nature of this superficial layer, more correctly termed residual skin surface material (RSSC). Using a traditional method (cigarette paper), samples of RSSC were acquired from the forehead of volunteers representative of a number of ethnic groups. Qualitative gas chromatography mass spectrophotometry (GCMS) indicated that RSSC contains at least 49 components from 5 lipid classes. Based on these data, Shetage et al. have proposed that RSSC may have utility as a bio-monitoring matrix to assess environmental exposure to chemicals or the secretion of specific endogenous biomarkers. The use of RSSC appears to provide several practical advantages as can be collected noninvasively and reproducibly from individuals. Moreover, the general composition was not significantly influenced by age, sex or ethnicity. Clearly, further investigations of the profile of RSSC in healthy individuals and patients with diseases will be an important aspect in developing the potential clinical applications of RSSC as a biomarker. The proposal for using RSSC as a medium for detecting chemical exposure deserves further investigation, but this approach will need considerable evaluation.
Non-invasive skin imaging techniques with good cellular resolution offer the potential to monitor changes in the skin in relation to chemical and drug use and in disease states. Indeed, the application of non-invasive visualising techniques to whole skin in vivo in healthy volunteers or patients and to whole ex vivo skin is evolving rapidly and provides important information about the 3D architecture of normal and diseased skin. Chapter 5 by Rolland et al. reports the application of Gabor Domain Optical Coherence Microscopy (GD OCM) as a technique for producing 3D images of skin in vivo at several anatomical locations. The resulting images clearly reveal the cutaneous microstructure and the array of epidermal keratinocytes and skin appendages. The technique was also used to compare normal and diseased (melanoma) skin in vitro. The work by Rolland et al. clearly demonstrates the potential utility of the technique: it will be interesting to see future studies where measurements in patients are compared directly with histological images of ex vivo samples.
Visualisation of the skin traditionally relies on the differential absorbance and reflectance of relatively low-energy photons. In contrast, synchrotron radiation (SR) can be used as a source of high energy photons with small-angle X ray scattering (SAXS) to characterise the structure of soft tissues such as skin. Its use is restricted by the need for a synchrotron (cyclic particle accelerator). In Chapter 6, Cocera et al. describe the advantages of the technique compared to Fourier transform infrared spectroscopy (FITR) for characterising pig skin in relation to collagen and lipid structure and also the influence of age and disease using ex vivo dermatomed and heat treated human skin. The use of SAXS, although not widely available, could provide useful 3D structural information on skin ex vivo but has no current in vivo application due to the potential adverse health effects of the high energy electromagnetic radiation. The continued development and improvement of such in vitro and ex vivo methodology will undoubtedly improve our understanding of skin biology in health and disease.
CHAPTER 2
FACTORS INFLUENCING EXPRESSION AND SPECIFICITY OF CARBOXYLESTERASES IN SKIN: IMPLICATIONS FOR LOCAL AND SYSTEMIC PARABEN ESTER TOXICITY
F M Williams, V Ravi, D McGarry, S J Boulton, E Mutch, C Jewell and S C Wilkinson
Institute of Cellular Medicine and Medical Toxicology Centre, Medical School, Newcastle University, United Kingdom.
1 INTRODUCTION
Carboxylesterases (CES) are present in a number of skin cells and have known physiological and exogenous substrates including drugs, chemicals, pro-drugs, carbamate and pyrethroid insecticides, cosmetic chemicals and environmental toxicants. A typical reaction scheme for CES is presented in Figure 1. During drug discovery and design, an ester linkage is frequently included to selectively target a pro-drug to a tissue or to improve dermal absorption and deliver a novel compound. Skin is an important tissue regulating uptake of environmental chemicals as well as a delivery site for drugs targeted locally to the skin and systemically.
Satoh and Hosokawa classified five groups of CES enzymes (CES 1–5) on the basis of amino acid homology and substrate specificity, with the majority of identified CES enzymes belonging to either the CES-1 or CES-2 sub-families. Amino acid sequence homology between human carboxylesterase 1 (hCE-1; a member of CES-1 family) and human carboxylesterase 2 (hCE-2, CES-2 family) is 48%. However, the substrate selectivity of these two enzymes is different. The hCE-1 enzyme mainly hydrolyses substrates with low molecular weight alcohol groups and higher molecular weight acyl groups such as cocaine (methyl ester), meperidine, and delapril. In contrast to hCE-1, the hCE-2 enzyme efficiently hydrolyses compounds with high molecular weight alcohol groups and relatively smaller carboxylate groups such as 4-methylumbelliferyl acetate, heroin, and 6-acetylmorphine CPT-11 or SN38. The hCE-1 and hCE-2 genes encode the two major forms of human liver microsomal carboxylesterase enzymes. Carboxylesterase activity has been found both in the microsomal and cytosolic fractions of the skin.
There are differences in the distribution of CES isoforms between extra-hepatic tissues (such as the small intestine, lung, skin) and the liver which results in differing hydrolysis profiles. Staudinger et al. summarised the differences in distribution between CES-1 and CES-2, but did not refer to skin. Hydrolysis profiles have been defined using isozyme specific substrates such as CPT-11 ironotecan or procaine for CES-2 and methyl phenidate for CES-1. Several nuclear receptor (NR) family members regulate drug-inducible expression and activity of CES-1 and 2 in mammalian liver and intestine but these have not been investigated in the skin.
Parabens (4-hydroxy benzoic acid esters) are substrates for CES enzymes. Parabens are widely used in topically applied cosmetic products and are known to penetrate the skin and have some oestrogenic effects in vitro. Paraben esters have been used as model substrates to identify carboxylesterase activity in human liver and skin. Previous studies have shown that parabens are rapidly hydrolysed after oral intake through first pass metabolism by the liver. The capacity for hydrolysis of the parabens in skin varies with the sidechain of the substrate. Lobemeier et al. showed that in human skin and subcutaneous fat tissue, paraben esters could be hydrolysed by 4 different carboxylesterase enzymes classified by their isoelectric points. Paraben esters are hydrolysed to 4-OH benzoic acid by carboxylesterases in both skin microsomes and in cytosol by CES-1 and CES-2 differentially relating to the leaving group. Inhibition by loperamide (specific for CES-2) indicated that butyl paraben hydrolysis was more inhibited than methyl paraben suggesting involvement of CES-2 with higher molecular weight leaving groups.
In the liver, greater hydrolysis of methyl paraben is consistent with higher expression of CES-1. The profile is less easy to explain for skin where the hydrolysis rate for methyl paraben is greater than for butyl paraben, although previous studies had suggested lower expression of CES-1 in the skin. The degree of hydrolysis during absorption through the skin could potentially influence the efficacy and toxicity of esters and thus be influenced by variability in esterase expression in the skin. Absolute variability in activity of carboxylesterases in skin versus other enzymes (e.g. balance between hydrolysis and conjugation, oxidation or transport) may be important. CES are localised in keratinocytes so the absorbed ester needs access to enzymes and so higher lipid solubility might promote uptake into keratinocytes. We had postulated that transporters might be required for efflux of the metabolite from cells but found no evidence of this (unpublished data). It was previously shown for fluazifop butyl ester pesticides that inhibition of all serine esterases in skin by BNPP reduced absorption through skin in flow-through diffusion cell system.
The aims of the present study were to further define the activity and expression profile of carboxylesterases isoforms in human skin and to determine whether expression of carboxylesterases or hydrolysis of paraben esters could be induced by the steroid dexamethasone or 8-methoxypsoralen (8-MOP) which are applied to skin. Dexamethasone is used orally and applied topically to the skin in preparations for inflammation to produce a local effect, whilst 8-MOP is used orally for psoriasis and distributes to the skin from the systemic compartment. Studies were conducted in vitro using keratinocytes and human skin in culture compared with hepatocytes. Also the interaction between UV light and paraben ester local hydrolysis and toxicity in skin cells was examined.
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Excerpted from Advances in Dermatological Sciences by Robert Chilcott, Keith Brain. Copyright © 2014 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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