ARTICLE
Oxidative stress is a condition of pro-oxidant/ antioxidant disequilibrium,
in which the generation of potentially harmful reactive oxygen species
(ROS) exceeds the ability of the tissue's antioxidant defence mechanisms
to quench them. Damage to cell membranes by way of lipid peroxidation,
and damage to DNA, sulphur-containing enzymes and proteins, and carbohydrates
are amongst the major resultant effects [1, 2].
A number of skin diseases are believed to be associated with oxidative
stress, including psoriasis, atopic dermatitis, erytheme multiforme and
cutaneous vasculitis [3]. There is also evidence that ROS are involved
in allergic contact dermatitis, both during the early pre-immunological
phase following exposure to contact allergens which readily auto-oxidise,
such as paraphenylene diamine, and during the later stages of inflammatory
cell infiltration [4, 5]. Chemical irritants may also generate free radicals,
the most well known from a dermatological point of view being the anti-psoriatic
agent, dithranol, which undergoes rapid light-catalysed auto-oxidation
in aqueous solution forming ROS (singlet oxygen and superoxide anion radical)
as reaction intermediates [6, 7].
Defence against ROS in the skin has evolved by way of a variety of antioxidant
enzymes, free radical quenchers and inducible responses, the activities
and tissue levels of which serve as important indicators of the skin's
response to oxidative stress [1, 2]. In this study, our aims were two-fold;
firstly to examine whether the generation of free radicals by dithranol
following topical application in human volunteers is detectable by changes
in the epidermal levels of Cu,Zn-superoxide dismutase (Cu,Zn-SOD), an
enzyme which catalyzes the conversion of the superoxide anion to hydrogen
peroxide and oxygen [2] and which would therefore be expected to be affected
by dithranol, and secondly to investigate whether such changes also take
place following exposure to an irritant which is not traditionally regarded
as generating ROS, namely the anionic detergent, sodium lauryl sulphate
(SLS). Detection and quantification of the antioxidant enzyme within the
epidermis were conducted using image analysis techniques applied to immunocytochemically
labelled tissue sections.
Materials and methods
Subjects
Eighteen healthy, non-atopic, male volunteers (age range 18-58 years,
mean 32 years) participated in the study. Approval was given by the Wycombe
Local Research Ethics Committee and all subjects gave written, informed
consent.
Irritants
Sodium lauryl sulphate (purity > 99%, Sigma Chemical Co., Poole,
Dorset, UK) was freshly prepared in distilled water at a concentration
of 5% (w/v). Dithranol was prepared at a concentration of 0.2% (w/w) in
white soft paraffin (wsp), with 0.25% (w/w) salicylic acid added.
Patch testing
Half of the volunteers received patch tests containing SLS, the remaining
half being patch tested with dithranol. Each individual received a total
of four, 8 mm Finn Chambers (Epitest Ltd Oy, Rannankoukku, Tuusula, Finland),
two on the mid-volar area of each forearm. One chamber on each arm was
filled with the irritant (15 µl of SLS/25 mg of dithranol), whilst
the other contained a similar quantity of the appropriate vehicle control.
The patch tests were left in contact with the skin for either 5 h or 47
h, depending upon the designated biopsy time. Immediately before biopsying,
the intensity of the irritant reactions was visually assessed for erythema,
according to the following grading system: 0, no visible reaction; 0.5,
faint, patchy erythema; 1, weak erythema; 2, moderate erythema; 3, marked
erythema; 4, intense erythema.
Biopsy procedure
Five elliptical biopsies (4 mm in diameter) were taken from each subject
following injection of lignocaine. Two were taken from the irritant patch
test sites, two from the vehicle control sites, and one from an area of
untreated, normal skin adjacent to the patch test sites on one arm. Two
time periods were selected for each subject from the following three sampling
times; 6 h (Finn chamber application time, 5 h), 48 h or 96 h (chamber
application time for both, 47 h). Vehicle control sites were always biopsied
at the same time as the irritant test sites and the normal skin sample
was taken during the first biopsy session. A total of five biopsies per
irritant and control time point were obtained. All biopsies were immediately
embedded in OCT compound, snap frozen stored in liquid nitrogen.
Immunocytochemistry
Serial sections of 4 µm thickness were cut from each biopsy and
mounted onto Vectabond subbed (Vector Laboratories, Peterborough, UK),
Teflon coated, multi-well slides (ICN, Thame, Oxon., UK). They were air
dried overnight and stored at 35° C until required. Following
10 minutes fixation in acetone at room temperature, serial sections were
incubated for 30 minutes in the following primary monoclonal antibody:
Anti-Cu,Zn-SOD 1/500 (Clone SD-G6, Sigma BioSciences, St Louis,
MO, USA).
The concentration at which this antibody was applied was optimized during
preliminary studies so as to produce very slight background staining of
the dermis. This ensured that maximum specific labelling of the enzyme
was achieved. During image analysis, the background dermal values were
subtracted from those of the specific epidermal staining.
For each biopsy, the antibody was applied to sections taken from three
different areas. Negative controls, using an irrelevant antibody of the
same isotype (anti-follicle stimulating hormone, Clone 1038, ICN, Thame,
Oxon, UK), were included. Visualization of the antibody/antigen reaction
was performed using the Vectastain ABC Elite peroxidase kit as
directed (Vector Laboratories), with 3',3 diaminobenzidine employed as
the chromogen. No counterstaining was carried out in order to avoid wavelength
interference during image analysis. All incubations were conducted at
room temperature, and, throughout the study, test and control samples
were prepared and stained in parallel, so as to maintain the consistency
required for quantitative image analysis.
Additional sections from each biopsy were also routinely stained using
haematoxylin and eosin for assessment of histopathological changes.
Image analysis
Microscopy and analysis were performed on a blinded basis, using a Zeiss
Axioplan microscope linked by means of a Neotech Image Grabber to a Power
Macintosh 8100 computer, loaded with Optilab Pro 2.6 software. For analysis
of superoxide dismutase levels, a dedicated software programme was employed
which measures the total quantity of stain present on an area basis, and
expresses the levels of antigen in terms of the total number of grey levels/µm2
epidermis (GEMStain, ME Electronics, Reading, UK). So as to avoid any
investigator bias, the entire epidermal areas of all three sections from
each biopsy were analysed, and the mean value calculated. Areas where
obvious sectioning artefacts were present were omitted from the analysis,
as were intrafollicular, parakeratotic and stratum corneal regions of
the epidermis.
Statistics
The mean and standard deviation (SD) of each of the sample groups were
calculated. Test and vehicle control values were compared using the Wilcoxon
matched pairs signed ranks test. Results were considered significant at
p < 0.05.
Results
Patch test reactions
SLS induced mild inflammation in most individuals after 6 h (mean visual
score 1.4, SD 0.7). After 48 h, the responses were considerably more intense
(mean score 3.0, SD 0.4), diminishing again slightly after 96 h (mean
2.3, SD 0.3). The water vehicle control produced a slight reaction in
some individuals after 48 h (mean 0.5, SD 0.4), the skin appearing clinically
normal at the other two time periods investigated. Reactions to dithranol
were also mild after 6 h (mean 1.0, SD 0.7), with, again, the peak response
in visual terms occurring at 48 h (mean 3.4, SD 0.4). A mean visual score
of 3.2 (SD 0.4) was obtained at 96 h. The white soft paraffin vehicle
control produced a mild reaction of 0.5 in only one individual after 48
h.
Histopathology
After 6 h of exposure to SLS, some biopsies exhibited small areas of
mild spongiosis. By 48 h, spongiosis was present in most samples, with
marked parakeratosis also being evident (Fig.
3B). 96 h samples were characterized by parakeratosis, spongiosis
and, in some cases, acanthosis.
Patch testing with dithranol resulted in little change to the appearance
of the epidermal cells after 6 h. Spongiosis within the lower and mid
epidermis, and some swelling of keratinocytes in the upper epidermal regions
were seen at the 48 h and 96 h time points. One biopsy (4+
reaction) contained regions of marked cellular damage (Fig.
2B).
Vehicle controls showed little or no evidence of pathological change.
Distribution of Cu,Zn-SOD labelling
In the epidermis of normal skin, intense cytoplasmic labelling of the
basal and epibasal regions was present, the staining gradually reducing
in intensity towards the upper cell layers (Figs.
1A, 2A, 3A). Some biopsies possessed positive staining which extended
throughout the stratum spinosum and stratum granulosum, in others labelling
gradually diminished through the stratum spinosum. Within the dermis,
Cu,Zn-SOD was detected in the eccrine sweat glands and ducts, and in the
basal cells of the outer root sheath of hair follicles.
The majority of the irritant-treated skin samples, with the exception
of the 6 h SLS samples, showed reduced staining intensity in the basal
and epibasal layers of the epidermis, the stratum spinosum and stratum
granulosum being either palely stained or negative (Figs.
1B, 1C, 2B, 3B). Most patch test reactions were moderate (3+)
in intensity by 48 h and it was therefore not possible to distinguish
any relationship between intensity of reaction and the density of epidermal
Cu,Zn-SOD staining. The distribution of Cu,Zn-SOD within the dermal structures
was unchanged from that of normal skin, whilst the majority of the infiltrating
inflammatory cells were positively stained. Vehicle control samples were
similar to those of untreated skin controls, in terms of epidermal staining.
Quantification of Cu,Zn-SOD labelling
The results for dithranol-treated subjects are given in Fig.
4. Statistically significant reductions in the overall epidermal
levels of Cu,Zn-SOD were present at all three time points investigated.
Inter-individual variation was relatively high. Patch testing with SLS
also led to reductions in the epidermal levels of Cu,Zn-SOD, but, with
this irritant, only after 48 h and 96 h, not as early as the 6 h time
point (Fig. 5).
In order to ensure that any acanthosis induced in response to the irritants,
particularly at the 96 h time point, was not responsible for the reduction
in overall staining density, measurements were also taken of the basal
and epibasal areas alone. Similar results were obtained (data not shown).
Discussion
The demonstration that levels of Cu,Zn-SOD within the epidermis are
significantly reduced following topical exposure to irritants strongly
suggests that oxidative stress is involved in the inflammatory process.
Importantly, as regards our understanding of the mechanisms involved in
ICD, this change appears not to be restricted to dithranol, which is known
to generate ROS during auto-oxidation, but also extends to chemicals such
as SLS, which are not normally directly associated with ROS generation.
Oxidative stress occurs when there is a disequilibrium between pro-oxidants
and antioxidants within a tissue, in favour of the former. Pro-oxidants
in the form of ROS and free radicals may be introduced exogenously through
exposure to sources such as tobacco smoke and UV radiation, and/or endogenously,
as metabolites of normal biochemical pathways and as an adjunct to defence
mechanisms against micro-organisms and xenobiotics [9, 10]. In the case
of dithranol-induced irritation, it is likely that both routes of ROS
generation co-exist, with the reaction intermediates of auto-oxidation
perhaps contributing significantly to the early 6 h reduction in Cu,Zn-SOD
levels. The later time course for Cu,Zn-SOD reduction observed in SLS
reactions may reflect endogenous ROS generation alone. Infiltrating neutrophils
and macrophages, which can generate and release an array of reactive oxidants,
are likely to be rich sources of ROS in ICD [9, 10].
Of possible relevance also to this study, is the observation that reduced
Cu,Zn-SOD activity occurs in hyperproliferative skin disorders, such as
psoriasis, and in experimentally-induced epidermal hyperproliferation
[15-17]. This is thought to be due to increased oxidative metabolism,
which leads to enhanced production of superoxide anion and accumulation
of hydrogen peroxide [18]. Immunolabelling with the monoclonal antibody,
Ki-67, which was conducted as part of a separate study on this series
of biopsies [19], revealed that there was, in fact, a significant increase
in the density of dividing keratinocytes in the 48 h and 96 h reactions
to SLS, in accordance with earlier observations [20].
The observation in man that irritants induce oxidative stress demonstrable
by changes in pro-oxidant enzymes, is in keeping with the results of a
number of recent studies conducted in animal models. Following topical
exposure to the chemical irritants/carcinogens, sulphur mustard and 12-O-tetradecanoylphorbol-13-acetate,
rodent skin exhibits reductions in the specific activities not only of
superoxide dismutase, but also of catalase and glutathione peroxidase
[11, 12]. Interestingly, in situations of more chronic exposure to pro-oxidant
sources, the skin appears to respond by producing enhanced levels of intracellular
antioxidants such as Cu,Zn-SOD, thereby increasing its protective capabilities
[13, 14].
That oxidative stress is involved in chemically-induced cutaneous reactions
is further supported by evidence of inhibition of inflammation following
the application of antioxidant therapy. Superoxide dismutase, itself,
has been shown to be effective at reducing the erythema induced by both
dithranol and another irritant, lauroylsarcosine, as have other naturally
occurring antioxidants, such as catalase and R lipoate [21,22]. The use
of these and other antioxidants [23] may well represent an effective therapeutic
avenue for acute irritant contact dermatitis.
CONCLUSION
This study has shown not only that oxidative stress may be demonstrated
immunocytochemically in skin exposed to a known ROS generating irritant,
but also that similar enzyme changes take place following the application
of an irritant with a different oxidative profile, suggesting that oxidative
stress plays a broader and more general role in the pathogenesis of acute
irritant contact dermatitis than was perhaps previously thought.
Acknowledgements
The authors would like to thank all the subjects who participated in
the study and Sally Barth and Maria Nicholson for providing expert nursing
assistance. Financial support was given by the British Occupational Health
Research Foundation and the Erasmus Wilson Dermatological Research Fund.
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