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Designing of mouse model: A new approach for studying sulphur mustard-induced skin lesions
burns 37 (2011) 851–864
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Designing of mouse model: A new approach for studying sulphur mustard-induced skin lesions Vinay Lomash, Utsab Deb, Renuka Rai, Sunil E. Jadhav, R. Vijayaraghavan, S.C. Pant * Division of Pharmacology and Toxicology, Defence R & D Establishment, Jhansi Road, Gwalior 474 002, Madhya Pradesh, India
article info
abstract
Article history:
This study was planned to design a mouse model for studying sulphur mustard (SM)-
Received 15 July 2010
induced skin injury. SM was applied dermally at dose of 5 or 10 mg kg1 in polyethylene-
Received in revised form
glycol-300 (PEG-300) or dimethylsulphoxide (DMSO) or acetone once. The changes in body
20 October 2010
weight, organ body weight indices (OBWI) and haematological and oxidative stress param-
Accepted 10 December 2010
eters were investigated over a period of 3–7 days and supported by histopathological observations. Exposure to SM in PEG-300 or DMSO resulted in a significant depletion in body weight, OBWI, hepatic glutathione (GSH) and elevation in hepatic lipid peroxidation,
Keywords:
without affecting the blood GSH and hepatic oxidised glutathione (GSSG) levels. Interest-
Sulphur mustard
ingly, no aforesaid change was observed after dermal application of SM diluted in acetone.
PEG-300
These biochemical changes were supported by the histological observations, which
DMSO
revealed pronounced toxic effect and damage to liver, kidney and spleen after dermal
Acetone
application of SM diluted in PEG-300 or DMSO. The skin showed similar microscopic changes
Oxidative stress
after dermal application of SM in all the three diluents, however; the severity of lesions was
Skin lesions
found to be time and dose dependent. It can be concluded that dermal exposure of SM diluted in acetone can be used to mimic SM-induced skin toxicity without systemic toxicity in a mouse model. # 2010 Elsevier Ltd and ISBI. All rights reserved.
1.
Introduction
Sulphur mustard (SM) is bis-(2-chlorethyl) sulphide and is one of the blistering or vesicating agents among the various mustard agents. It is regarded as a significant chemical agent of historical and current interest because of its multitudinous acute and chronic adverse effects resulting from extensive use in previous armed conflicts [1]. The easy availability of precursors, the simple method of synthesis and the extremely stable nature of SM make it a chemical weapon of choice by military and terrorist groups [2]. SM has many debilitating effects including ocular and dermal injury, respiratory tract damage, reproductive and developmental toxicity and gastrointestinal and haematological effects [3]. SM forms sulphonium ion in the body and alkylates DNA and other cellular * Corresponding author. Tel.: +91 751 2233492; fax: +91 751 2341148. E-mail address: [email protected] (S.C. Pant). 0305-4179/$36.00 # 2010 Elsevier Ltd and ISBI. All rights reserved. doi:10.1016/j.burns.2010.12.010
macromolecules, which, in turn, leads to DNA strand breaks and cell death [4]. SM causes inflammation and extensive blistering of the skin, primarily because of the large surface area of exposure and the sensitivity of frequently dividing basal cells [4,5]. Skin response to SM manifests as erythema and oedema, which progress to blister formation, ulceration, necrosis and desquamation of the epidermis, and can lead to permanent residual effects in humans [6–11]. The formation of thinwalled blisters is preceded by an asymptomatic latent period of several hours, followed by itching, pain and erythema in humans. Progression of the lesions, the degree of blistering and necrosis are dependent on the quantity of SM delivered [12,13]. Histopathology of the skin following exposure to SM is characterised by oedema, dermal infiltration of inflammatory
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cells, premature death of basal-layer epidermal cells and dermo–epidermal separation [14–16]. Though a number of in vitro models have been used for antidote evaluation, their in vivo efficacy is yet to be established [17]. It appears that the in vitro models are appropriate for mechanistic studies but may not be suitable for protection studies. Many in vivo studies have used weanling pig, hairless guinea pig, rabbit or various mouse species as useful animal models for evaluating the efficacy of various antidotes against SM-induced skin toxicity [18–24]. But, there could be many variables such as dose of SM, physical and chemical nature of diluents and duration of SM exposure that influence the differential severity of pathogenesis of SM-induced lesions. Hairless mouse and hairless guinea pig with unknown genetic defect, and lack of polyclonal and monoclonal antibodies and lack of probes and primers to DNA and RNA sequences in guinea pig might limit the usefulness of these animals in the study of the pathophysiology of SM-induced skin lesions [15]. Our previous studies described the in vivo protection against systemic toxicity due to percutaneous exposure of SM diluted in PEG-300 [25–32], DMSO [29] and acetone [33]. However, for the screening of wound-healing efficacies of dermal formulations, there is need for development of an in vivo model, which should produce SMinduced skin lesions only without showing any signs of systemic toxicity. Thus, the goal of current study was to design a resourceful and consistent mouse model for studying SM-induced skin lesions by comparing in vivo toxicity of SM in various diluents (PEG-300, DMSO and acetone).
2.
Materials and methods
2.1.
Chemicals and reagents
SM was synthesised in the Synthetic Chemistry Division of the Establishment and was found to be above 99% pure by gas chromatographic analysis. Other chemicals of analytical grade were purchased from Sigma (USA) or Merck (India).
2.2.
Animals
All experiments were performed on randomly bred 25–30 g female Swiss albino mice. The animals were obtained from the Animal Facility of Defence Research and Development Establishment. The care and management of the animals were as per the approved guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA, India). The Institutional animal ethical committee approved the protocol for the experiment. The animals were maintained in polypropylene cages on dust-free and autoclaved paddy husk and fed standard pellet diet (Ashirwad Feeds, India) and water ad libitum. A day before the SM application, hairs from back region of all mice were closely clipped using a pair of scissors. Animals were randomly allocated to various groups and SM was applied once dermally at a dose of 5 or 10 mg kg1 body weight diluted in various solvents as mentioned below:
(1) group I: control (without any dermal application); (2) group II: control (single dermal application with acetone only); (3) group III: SM in acetone and sacrificed after 3 days; (4) group IV: SM in PEG-300 and sacrificed after 3 days; (5) group V: SM in DMSO and sacrificed after 3 days; (6) group VI: SM in acetone and sacrificed after 7 days; (7) group VII: SM in PEG-300 and sacrificed after 7 days; and (8) group VIII: SM in DMSO and sacrificed after 7 days. The experiment was performed initially with three mice per group and then repeated once, and the data were compiled for six mice as shown in the results. All the safety precautions were taken while handling SM during dermal application.
2.3.
Body weight and organ weight
The body weight of animals was recorded daily. After 3 or 7 days, the animals were sacrificed and liver, kidney and spleen were dissected out, weighed and preserved in Bouin’s fluid. A portion of skin exposed to the SM application site was also taken and preserved in Bouin’s fluid.
2.4.
Haematological and biochemical analysis
White blood cell (WBC) and red blood cell (RBC) count and haemoglobin, haematocrit (HCT) and mean cell haemoglobin concentration (MCHC) were measured on Sysmex Hematology Analyzer (model K4500). Blood GSH concentration was determined as per the method described by Ellman [34] and modified by Jollow et al. [35]. In brief, 0.2 ml of whole blood was added to 1.8 ml of distilled water and incubated for 10 min at 37 8C for complete haemolysis. After haemolysis, 3 ml of 4% sulphosalycylic acid was added, and the tubes were centrifuged at 2500 rpm for 15 min. The supernatant (0.2 ml) was mixed with 0.4 ml of 10 mM 5,50 dithiobis-(2-nitrobenzoic acid) (DTNB) and 1 ml phosphate buffer (0.1 M, pH 7.4). After 5 min, the absorbance was recorded at 412 nm. The fluorometric method of Hissin and Hilf [36] was used for the determination of hepatic GSH and GSSG concentration. A total of 250 mg of liver tissue was homogenised in 5 ml phosphate ethylene diamine tetraacetic acid (EDTA) buffer (pH 8.0) and metaphosphoric acid (25%). The samples were centrifuged and the supernatant was used for the estimation of GSH and GSSG using O-phthaldialdehyde as a fluorescent dye. Lipid peroxidation was determined by measuring the level of malondialdehyde (MDA), according to the modified method of Esterbauer and Cheeseman [37]. In brief, 200 mg of liver tissue was homogenised in 0.15 M KCl, and 30% trichloroacetic acid (TCA) and 0.8% thiobarbituric acid reagent (TBA) were added. The contents were boiled for 30 min, and the absorbance of the supernatant was measured at 535 nm. MDA was calculated using a molar extinction coefficient of 1.58 105 M1 cm1.
2.5.
Histopathology
Multiple tissue sections of 4–5-mm thickness were taken after dehydration in a graded ascending series of alcohol, clearing in toluene using an automatic tissue processor (Leica TP 1020) and embedded in paraffin wax. Tissue sections were further
burns 37 (2011) 851–864
[()TD$FIG]
853
stained with haemotoxylin and eosin (H&E) and analysed under light microscope (Leica, DMLB).
2.6.
Statistical analysis
The biochemical variables were analysed using one-way analysis of variance with Student–Newman Kewl’s multiple comparison. A probability of 0.05 and less was taken as statistically significant. The analyses were carried out using SigmaStat for Windows version 2.03 (SPSS Inc., USA).
3.
Results
3.1.
Effects on body weight and organ weight
Fig. 1 shows the percent change in body weight of control mice and SM (5 or 10 mg kg1) exposed mice at day 0, 3 and 7 post application. The animals exposed to SM (5 mg kg1) in PEG-300 showed a significant decrease in body weight on day 3 and 7 and animals applied with SM (5 mg kg1) in DMSO showed a significant decrease in body weight on day 7 compared with the control animals and SM (5 mg kg1) in acetone-exposed mice. The animals with SM (10 mg kg1) in PEG-300 and DMSO showed a significant decrease in body weight compared with the control mice and SM (10 mg kg1) in acetone-exposed mice both on day 3 and 7. There was no statistical difference in organ body weight indices (OBWIs) of liver and kidney of mice exposed to SM (5mg kg1) in acetone or PEG-300 or DMSO compared with the control mice on day 3 and 7 (Fig. 2). However, there was a significant decrease in OBWIs of liver on day 3 and 7 and of kidney on day 7 in mice to which SM (10 mg kg1) in PEG-300 and DMSO were applied compared with the liver and kidney of control (normal and with dermal acetone) mice and mice with dermal application of SM (10 mg kg1) diluted in acetone on the same days. A significant decrease in organ body weight indices was observed in spleen of mice applied with SM (5 or 10 mg kg1) in PEG-300 or DMSO on day 3 and 7 compared with spleen from control groups. In contrast to the above findings, a significant increase in spleen weight was noticed on day 3 and 7 in mice to which SM (10 mg kg1) in acetone was applied (Fig. 2).
3.2.
Effect on haematological and biochemical analysis
Mice exposed to SM (5 or 10 mg kg1 diluted in PEG-300 or DMSO or acetone) showed no significant influence on WBC and RBC count, haemoglobin, HCT and MCHC levels after day 3 compared with the mice of control groups (Table 1). SM (in PEG-300 or DMSO or acetone) had no effect on WBC and MCHC levels at day 7. After day 7, mice exposed to SM (10 mg kg1) diluted in PEG-300 or DMSO showed a significant increase in RBC count compared with control mice (Table 2). Haemoglobin concentration of mice exposed to SM (10 mg kg1) in PEG-300 or DMSO after day 7 was significantly higher compared with that in control mice and mice exposed to SM diluted in acetone at a similar dose. SM (10 mg kg1) in PEG-300 application resulted in significant increase in HCT than control mice and mice exposed to SM (10 mg kg1) in DMSO or acetone after day 7. Blood GSH in mice applied with SM (10 mg kg1) in acetone
Fig. 1 – Effect of SM (5 mg kgS1 or 10 mg kgS1, diluted in various vehicles) on body weight after single dermal application (A) control; (B) acetone only; (C) SM in acetone; (D) SM in PEG-300; (E) SM in DMSO. Mean W SE (n = 6). Same alphabets on bars indicate non-significant differences between groups at given dose on given day (P < 0.05).
was significantly higher as compared with control and PEG300 and DMSO groups (Fig. 3). However, it is difficult to explain this increase in blood GSH level. There was a significant decrease in liver GSH of mice applied with SM application (10 mg kg1 in PEG-300 or DMSO) on day 3 and 7, respectively, compared with the liver GSH of control (normal and with dermal acetone) mice on the same days and mice with dermal application of SM (10 mg kg1) diluted in acetone on day 7. However, no significant change was noted in liver GSH content in all groups with an SM dose of 5 mg kg1. MDA levels were significantly higher in mice with dermal application of SM (10 mg kg1) in PEG-300 or DMSO on day 3 and 7 as compared with control and SM (10 mg kg1) in acetone groups.
3.3.
Histopathology
The histopathological observations in liver, kidney and spleen following SM exposure are shown in Tables 3 and 4. Liver of control mice (with and without acetone) showed normal lobular architecture with hepatocytes arranged in cords encircling the central canal (Fig. 4). Similarly, liver sections of mice applied with percutaneous SM diluted in acetone did
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[()TD$FIG]
Fig. 2 – Effect of SM (5 mg kgS1 or 10 mg kgS1 diluted in various vehicles) on organ weight, 3 and 7 days after single dermal application. Mean W SE (n = 6). (A) Control; (B) acetone only; (C) SM in acetone; (D) SM in PEG-300; (E) SM in DMSO. Same alphabets on bars indicate non-significant differences between groups at given dose on given day (P < 0.05).
not reveal any significant histological changes at 5 mg kg1 and 10 mg kg1 doses compared with liver sections from control mice. The occasional occurrence of necrotic lesions and hepatocyte vacuolation of minimal severity in liver sections of control mice and mice applied with SM in acetone at 5 and 10 mg kg1 were restricted to individual animals, which were non-significant; and all these features suggest their spontaneous occurrence. However, liver sections from mice with percutaneously applied SM diluted in PEG-300 or DMSO showed significant pathological lesions in hepatic tissue as compared with control mice. The lesions were more pronounced in groups with SM in DMSO or PEG-300 at 10 mg kg1. The significant lesions were hepatocellular degeneration, hepatocyte vacuolation and hypertrophied Kupffer cells. Hepatocytes in groups with SM in PEG-300 or DMSO showed moderate-to-severe karyolysis and necrosis/apoptosis, mainly in the centrilobular area, which diffused margin-
ally in the midzonal area and rarely in the periportal area of the hepatic lobule. Focal accumulation of eosinophillic fibrinoid material entrapping inflammatory cells was also evident. A dose-dependent increase in severity of lesions was noticed in the liver of mice applied with SM (5 or 10 mg kg1) in PEG-300 or DMSO after day 3 and 7 (Fig. 4). The kidney of control mice showed the normal architecture (Fig. 5). It showed normal glomerulus, Bowman’s space and renal parenchyma. The occasional presence of lesions in kidney of control mice, acetone-treated mice and mice treated with SM in acetone at both the doses (5 or 10 mg kg1) showed minimal severity. These changes were common to all groups, which were considered spontaneous. Section of kidney of mice applied with SM in PEG-300 or DMSO (10 mg kg1) showed mild-to-moderate tubular degeneration, haemorrhage, necrosis and subsequent sloughing off of cuboidal cells of proximal and distal tubules into the lumen (Fig. 5).
Table 1 – Effect of SM (10 mg kgS1 and 5 mg kgS1) on hematological variables, 3 days after single percutaneous exposure. Groups
Control Acetone SM in Acetone SM in PEG-300 SM in DMSO
WBC (103 mm3)
RBC (106 mm3)
HGB (g%)
5 mg kg1
10 mg kg1
5 mg kg1
10 mg kg1
5 mg kg1
10 mg kg1
5 mg kg1
10 mg kg1
5 mg kg1
10 mg kg1
9.9 0.3 9.3 3.1 10.3 2.1 5.6 0.8 5.3 0.8
11.9 1.6 9.4 0.8 11.4 1.1 7.5 1.6 7.5 1.5
7.9 0.3 8.3 0.4 7.5 0.4 7.3 0.4 8.6 0.4
7.9 0.4 8.4 0.1 7.4 0.6 8.8 0.5 7.1 0.6
10.6 0.4 10.8 0.3 10.7 0.6 10.1 0.9 11.1 0.3
10.7 0.6 11.4 0.3 9.9 0.8 12.4 0.9 9.5 0.8
37.7 1.3 38.0 2.3 35.7 2.0 33.8 2.5 39.6 1.3
37.3 1.9 39.2 0.7 34.9 3.2 42.7 3.1 33.1 2.6
27.9 0.1 27.7 0.0 29.9 1.3 26.9 1.0 27.9 0.1
28.7 0.2 29.1 0.4 28.4 0.5 28.9 0.4 28.7 0.5
MCHC (g dl1)
HCT (%)
Mean SE (n = 6).
burns 37 (2011) 851–864
Table 2 – Effect of SM (10 mg kgS1 and 5 mg kgS1) on hematological variables, 7 days after single percutaneous exposure. Groups
Control Acetone SM in acetone SM in PEG-300 SM in DMSO
WBC (103/mm3)
RBC (106/mm3)
HGB (g%)
5 mg kg1
10 mg kg1
5 mg kg1
10 mg kg1
5 mg kg1
10 mg kg1
5 mg kg1
10 mg kg1
5 mg kg1
10 mg kg1
9.9 0.3 9.3 3.1 14.1 3.2 5.9 0.5 6.5 0.6
11.9 1.6 9.4 0.8 12.11.04 7.7 4.3 7.7 0.7
7.9 0.4 8.4 0.1 7.5 0.3 10.2 1.4 9.7 0.8
7.9 0.3 b 8.3 0.4 b 8.6 0.6 ab 10.2 0.8 a 10.4 0.5 a
10.7 0.6 bc 11.40.3 bc 10.2 0.7 b 14.3 1.2 a 13.5 1.0 ac
10.6 0.4 b 10.8 0.3 b 11.7 1.2 b 14.8 0.5 a 14.4 0.7 a
37.3 1.9 ab 39.2 0.7 ab 35.1 4.5 b 49.6 4.8 a 46.0 3.6 ab
37.7 1.3 b 38.0 2.3 b 41.3 2.6 b 53.5 2.9 a 41.3 6.3 b
27.9 0.1 27.7 0.0 29.0 0.4 29.1 0.2 29.2 1.5
28.7 0.2 29.1 0.4 29.1 0.6 28.9 0.5 29.4 0.3
MCHC (g dl1)
HCT (%)
Mean SE (n = 6; P < 0.05). Same alphabets in superscripts on values in the same column indicate non-significant differences between groups.
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[()TD$FIG]
Fig. 3 – Effect of SM (5 mg kgS1 or 10 mg kgS1 diluted in various vehicles) on GSH–blood, GSH–liver, GSSG–liver and MDA– liver, 3 and 7 days after single dermal application. Mean W SE (n = 6). (A) Control; (B) acetone only; (C) SM in acetone; (D) SM in PEG-300; (E) SM in DMSO. Same alphabets on bars indicate non-significant differences between groups at given dose on given day (P < 0.05).
Tubular cells showed swelling leading to narrowing of lumen along with the presence of eosinophillic debris. However, kidney sections of mice applied with SM in PEG-300 or DMSO at 5 mg kg1 showed a dose-dependent decrease in the severity of lesions. Control mice spleen showed normal splenic histology with germinal centre, red pulp and marginal zone of white pulp (Fig. 6). The severity of splenic lesions was diluent and dose dependent. There was a significant lymphoid depletion and loss of lymphoid follicle with the accumulation of fibrinoid material in the spleen of mice applied with SM in PEG-300 or DMSO. Lesions were pronounced with SM at 10 mg kg1 than with SM at 5 mg kg1 in PEG or DMSO. However, there was rare occurrence of lymphoid hyperplasia noticed in the spleen of mice with SM in acetone at both the doses (5 and 10 mg kg1).
The microscopic observation of control mice skin showed stratified epithelium that was united to the dermis by a thin basal lamina without showing any degenerative changes (Fig. 7). Following SM exposure, the skin showed severe coagulative necrosis of epidermal cells extending to the dermis and leading to erosions. There was extensive infiltration of inflammatory cells with oedema in the dermal region on day 3, which reduced after day 7. The severity of lesions, such as hyperaemia of blood vessels with dermo–epidermal separation and formation of vesicle, was increased from day 3 to day 7. Basal cells showed vacuolation and loss of connection from basement membrane (acantholysis) in the area surrounding the wound. The degenerated dermo–epidermal region was covered with the tissue comprising of necrosed leucocytes and RBCs in a network of fibrin. There was severe adenexal atrophy in all mice with SM exposure, regardless of
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Table 3 – Histopathology of liver, kidney, spleen and skin in mice 3 and 7 days after single percutaneous exposure of SM (5 mg kgS1). Number of animals showing lesions/total number of animals (severity). Organs
Group I
Group II
Group III
Group IV
Group V
Group VI
Group VII
Group VIII
Liver Necrosis/apoptosis Hepatocellular degeneration Hepatocellular vacuolation Infiltration of inflammatory cells Hypertrophy of Kupffer cells Karyolysis Congestion
0/6 0/6 0/6 0/6 0/6 0/6 0/6
0/6 0/6 0/6 0/6 0/6 0/6 0/6
0/6 0/6 0/6 0/6 2/6 (1) 0/6 0/6
2/6 1/6 3/6 3/6 4/6 2/6 2/6
3/6 1/6 3/6 3/6 4/6 2/6 2/6
0/6 0/6 0/6 0/6 2/6 (1) 0/6 0/6
3/6 2/6 3/6 2/6 4/6 3/6 2/6
(2) (2) (2) (2) (1) (1) (1)
2/6 1/6 3/6 3/6 4/6 3/6 2/6
(2) (2) (2) (2) (1) (1) (1)
Kidney Tubular degeneration Necrosis/apoptosis Glomerular/tubular congestion Hemorrhage Eosinophillic debris
0/6 1/6 (1) 1/6 (1) 0/6 0/6
0/6 2/6 (1) 0/6 0/6 0/6
0/6 2/6 (1) 0/6 0/6 0/6
0/6 0/6 0/6 0/6 0/6
0/6 0/6 0/6 0/6 0/6
0/6 2/6 (1) 1/6 (1) 0/6 0/6
4/6 3/6 2/6 2/6 2/6
(1) (1) (1) (1) (1)
4/6 4/6 2/6 2/6 2/6
(1) (1) (1) (1) (1)
Spleen Lymphoid depletion Lymphoid hyperplasia Lymphocyte necrosis/apoptosis Degeneration of germinal follicle Accumulation of fibrinoid material
0/6 0/6 0/6 0/6 0/6
0/6 0/6 0/6 0/6 0/6
0/6 3/6 (2) 0/6 0/6 0/6
4/6 (3) 0/6 4/6 (2) 3/6 (2) 0/6
5/6 0/6 5/6 3/6 2/6
4/6 (2) 0/6 4/6 (2) 4/6 (2) 0/6
4/6 0/6 4/6 3/6 2/6
(2)
(2) (2) (2)
0/6 0/6 0/6 0/6 0/6
(2) (2) (2)
Skin Inflammatory cell immigration Epidermal necrosis Vacuolar degeneration of basal cell Oedema Acantholysis Adenexal atrophy Vesicle formation
0/6 0/6 0/6 0/6 0/6 0/6 0/6
1/6 (1) 0/6 0/6 0/6 0/6 0/6 0/6
6/6 5/6 5/6 6/6 4/6 4/6 6/6
6/6 6/6 6/6 6/6 6/6 4/6 5/6
6/6 6/6 6/6 6/6 6/6 6/6 5/6
(4) (4) (4) (3) (3) (3) (3)
6/6 5/6 6/6 6/6 6/6 4/6 5/6
6/6 5/6 6/6 6/6 6/6 6/6 6/6
6/6 6/6 6/6 6/6 6/6 6/6 6/6
(4) (4) (4) (2) (4) (4) (4)
(3) (3) (3) (3) (2) (3) (2)
the diluents used. Dose-dependent increase was noticed in the severity of skin lesions of mice applied with SM at 5 mg kg1 than at 10 mg kg1. The details are presented in Tables 3 and 4.
4.
Discussion
The aim of our study was to design an in vivo model, which should produce SM-induced skin lesions without showing any signs of systemic toxicity; this required the use of suitable diluent for SM which will limit its action to the exposed part, that is, skin only. Acetone is more volatile and spreads evenly compared with other diluents used in this study. As a result, systemic absorption and resultant damage could be prevented and toxicity will only be restricted to skin. Thus, it was hypothesised that SM diluted in acetone may reduce systemic toxicity compared with other diluents, namely PEG-300 or DMSO, which have lower vapour pressure and are nonvolatile. In the present study, the topical application of SM (5 or 10 mg kg1) diluted in PEG-300 or DMSO caused progressive loss in body weight, and the animals appeared weak and emaciated. The OBWIs of liver and kidney (10 mg kg1) and spleen (5 or 10 mg kg1) were also reduced. Interestingly, there was no body weight loss and no change in the OBWI of animals with the dermal application of SM diluted in acetone. A similar pattern of decrease in body weight and OBWI has been reported earlier by us [1,29], after the dermal application of SM diluted in PEG-300 or DMSO. Increase in RBC count, haemo-
(2) (2) (2) (1) (1) (1) (1)
(4) (4) (4) (2) (3) (3) (3)
(2) (2) (2) (1) (1) (1) (2)
(3)
(4) (3) (3) (3) (3) (3) (3)
(4) (4) (4) (2) (3) (3) (4)
globin and HCT suggest haemoconcentration in animals with the dermal application of SM diluted in PEG-300 or DMSO [33,38]. There should be a decrease in WBC count as per the expected pathophysiology following the topical application of SM diluted in PEG-300 [38]. However, in the present study, due to haemoconcetration, decrease in WBC count was not evidenced following dermal application of 5 or 10 mg kg1 of SM. Haemoconcentration occurs due to the increase in the vascular permeability of the cutaneous blood vessels, which may also contribute to the increase in RBC count, haemoglobin concentration and the HCT. Median lethal dose (LD50) of SM diluted in acetone is 39.6 mg kg1, which was much higher than the reported LD50 of SM diluted in PEG-300 (8.1 mg kg1) or DMSO (7.2 mg kg1) [29]. The decrease in the LD50 of SM diluted in PEG-300 or DMSO may be due to the high lipophilic nature of the diluents [29]. GSH is an intracellular scavenger of SM. Thus, SM may cause GSH depletion and might be one of the leading causes of generation of reactive oxygen species (ROS) [23,25,26,29]. One of the major causative factors of SM-induced cell death could be accumulation of endogenous ROS leading to lipid peroxidation and irreversible membrane damage [39]. The present study shows that a single dermal application of 10 mg kg1 of SM diluted in PEG-300 or DMSO can cause depletion of GSH in liver and can induce hepatic lipid peroxidation, as evidenced by enhanced MDA levels [29]. However, no change was observed in hepatic GSH and lipid peroxidation following dermal application of 5 mg kg1 of SM in PEG-300 or DMSO and 5 or 10 mg kg1 of SM in acetone. Acetone, being volatile
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Table 4 – Histopathology of liver, kidney, spleen and skin in mice 3 and 7 days after single percutaneous exposure of SM (10 mg kgS1). Number of animals showing lesions/total number of animals (severity). Organs
Group I
Group II
Group III
Group IV
Group V
Group VI
Group VII
Group VIII
1/6 (1) 0/6 1/6 (1) 0/6 0/6 1/6 (1) 0/6
2/6 0/6 1/6 0/6 2/6 2/6 0/6
2/6 0/6 1/6 0/6 3/6 2/6 2/6
(2) (1) (2)
6/6 6/6 4/6 6/6 6/6 6/6 3/6
(3) (3) (4) (3) (2) (4) (2)
6/6 6/6 5/6 6/6 6/6 6/6 4/6
(4) (3) (3) (3) (2) (4) (3)
1/6 0/6 1/6 0/6 2/6 1/6 1/6
(1) (1) (1)
6/6 5/6 6/6 4/6 4/6 6/6 4/6
(3) (3) (3) (2) (2) (2) (2)
6/6 6/6 6/6 6/6 5/6 6/6 3/6
(3) (3) (3) (3) (2) (2) (2)
2/6 (1) 0/6 ( ) 2/6 (1) 0/6 0/6
2/6 (1) 0/6 2/6 (1) 0/6 0/6
2/6 (1) 0/6 3/6 (1) 0/6 0/6
6/6 5/6 5/6 4/6 4/6
(2) (2) (2) (2) (2)
6/6 5/6 4/6 5/6 5/6
(2) (2) (2) (2) (2)
1/6 (1) 0/6 2/6 (1) 0/6 0/6
6/6 6/6 5/6 5/6 5/6
(2) (3) (2) (2) (2)
6/6 6/6 4/6 5/6 5/6
(3) (3) (2) (2) (2)
Spleen Lymphoid depletion Lymphoid hyperplasia Lymphocyte necrosis/apoptosis Degeneration of germinal follicle Accumulation of fibrinoid material
0/6 0/6 0/6 0/6 0/6
0/6 0/6 0/6 0/6 0/6
0/6 2/6 (2) 0/6 0/6 0/6
6/6 0/6 5/6 5/6 4/6
(3)
(3)
6/6 0/6 6/6 6/6 5/6
(3) (3) (3)
6/6 0/6 6/6 6/6 4/6
(3)
(2) (3) (3)
0/6 2/6 (2) 0/6 0/6 0/6
(3)
(2) (2) (2)
6/6 0/6 6/6 5/6 3/6
Skin Inflammatory cell immigration Epidermal necrosis Vacuolar degeneration of basal cell Oedema Acantholysis Adenexal atrophy Vesicle formation
0/6 0/6 0/6 0/6 0/6 0/6 0/6
2/6 (1) 0/6 0/6 0/6 0/6 0/6 0/6
6/6 6/6 6/6 6/6 6/6 4/6 6/6
6/6 6/6 6/6 6/6 6/6 4/6 6/6
(4) (4) (3) (3) (3) (3) (2)
6/6 6/6 6/6 6/6 6/6 4/6 6/6
(3) (3) (4) (1) (3) (3) (4)
6/6 6/6 6/6 6/6 6/6 5/6 6/6
6/6 6/6 6/6 6/6 6/6 5/6 6/6
(4) (4) (4) (2) (3) (3) (4)
6/6 6/6 6/6 6/6 6/6 6/6 6/6
(3) (4) (4) (1) (3) (3) (4)
Liver Necrosis/apoptosis Hepatocellular degeneration Hepatocellular vacuolation Infiltration of inflammatory cells Hypertrophy of kupffer cells Karyolysis Congestion Kidney Tubular degeneration Necrosis/apoptosis Glomerular/tubular congestion Hemorrhage Eosinophillic debris
(1) (1) (1) (1)
(1) (1)
(4) (3) (3) (3) (3) (3) (2)
with better spreading effect, might have resulted in limited SM absorption, whereas the more lipophilic and deeper penetration properties of PEG-300 or DMSO might have acted as an internal carrier to contribute to the greater toxicity of SM. After the initial cutaneous exposure, it has been reported that skin reservoirs continue to distribute SM via circulation to the body tissues thereby increasing damage to several organs [23]. However, to elucidate if 5 or 10 mg kg1 of SM diluted in PEG-300 or DMSO or acetone induces oxidative stress, which may lead to organ damage, we supported our results with histopathological observations in the liver, kidney and spleen. Histomorphological alterations in the different structures in the various regions of these tissues were observed. Hepatocellular degeneration, hepatocyte vacuolation, necrosis/apoptosis, accumulation of fibrinoid material and hypertrophied Kupffer cells were observed in the liver sections of mice treated with SM (5 or 10 mg kg1) diluted in PEG-300 or DMSO in a dose-dependent manner. Sections of mice kidney showed tubular degenerative changes; haemorrhages, necrosis and sloughing off of cuboidal cells of tubules into lumen after percutaneous application with SM diluted in PEG-300 or DMSO. Similarly, spleen of mice with 5 or 10 mg kg1 of SM diluted in PEG-300 or DMSO showed atrophy of splenic tissue displaying lymphoid depletion, loss of lymphoid follicles and accumulation of fibrinoid material in a dose-dependent manner. However, sections of liver, kidney and spleen of mice exposed dermally with percutaneous 5 or 10 mg kg1 of SM diluted in acetone
(1) (1)
(4) (3) (3) (2) (3) (3) (3)
(3) (3) (2)
showed marginal variations in the structure and appeared normal. The results from histological changes revealed that percutaneous application of SM diluted in PEG-300 or DMSO shows more toxic effects and lesions to the visceral organs than SM diluted in acetone. These histopathological findings also support the results of biochemical observations and OBWIs. The appearance of subepidermal blister, basal cell vacuolation, ulceration and coagulative necrosis of epidermis, exudation of oedematous fluid, transcytosis of intramural inflammatory cells and adenexal atrophy in the dermis of mice skin after SM (in acetone, PEG-300 or DMSO) exposure at either dose in the present study is consistent with the observation in a variety of animal models including rabbit, guinea pig [14], pig skin [40], hairless guinea pig [15], hairless mouse [41] and Swiss albino mice [33]. However, we noticed that lesions with either dose of SM diluted in PEG-300 or DMSO showed lesser oedema, more acantholysis, more adexenal atrophy and severe necrosis as compared with the lesions induced by SM in acetone. This may be due to more loss of fluids in case of SM in PEG-300 or DMSO as compared with SM diluted in acetone. Healing is modified by both systemic and local host factors [42]. In the present study, damage to liver, kidney and spleen of mice on exposure to SM diluted in PEG-300 or DMSO was suggestive of impaired systemic host factors, which may retard wound healing and will interfere in the wound-healing efficacy of dermal formulations, affecting their proper evaluation. On the other hand, there was no noticeable
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[()TD$FIG]
859
Fig. 4 – Photomicrographs of control and SM exposed mice liver, H & E. (a) Control mice showing normal hepatic chord, hepatocytes, central canal and kupffer cells. (b) Section of mice liver after 7 days of exposure with 5 mg kgS1 SM diluted in PEG300 showing focal necrosis (arrow). (c) Mice liver after 7 days of exposure with 5 mg kgS1 SM diluted in DMSO showing mild vacuolation and ballooning of hepatocyte (arrow). (d) Section of mice liver 7 days post exposure with 10 mg kgS1 of SM diluted in acetone showing normal histology of liver. (e) Section of mice liver after 7 days of exposure with 10 mg kgS1 SM diluted in PEG-300 showing apoptosis (bold arrow), infiltration of inflammatory cells in hepatic parenchyma and necrotic hepatocyte (arrow). (f) Section of mice liver after 7 days of exposure with 10 mg kgS1 SM diluted in DMSO showing congestion in central vein infiltration of monocytoid cells, condensation of nucleus in hepatocytes, diffuse hepatocyte vacuolation (short arrow) and mid zonal eosinophillic focus (bold arrow) in hepatic parenchyma and necrosis of hepatocytes.
systemic injury observed in mice exposed to SM diluted in acetone, thus diminishing the systemic host factors that influence wound healing. Using current model in efficacy evaluation of various wound healants after dermal SM
exposure, skin lesions can be scored on the basis of histomorphologic scale as described by Graham et al. [3]. Thus, the developed mouse model in the present investigation is a suitable model for studying SM-induced skin lesions.
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[()TD$FIG]
Fig. 5 – Photomicrographs of control and SM exposed mice kidney (H & E). (a) Control mice kidney showing normal glomerulus, bowman’s space and renal parenchyma. (b) Mice kidney after 7 days of exposure with 5 mg kgS1 SM diluted in PEG-300 showing mild swelling in convoluted tubules, degenerative changes, necrosis (arrow) with eosinophillic cast in tubules (bold arrow). (c) Mice kidney after 7 days of exposure with 5 mg kgS1 SM diluted in DMSO showing necrosis (arrow), infiltration of inflammatory cells and degenerative changes in tubular cells (bold arrow). (d) Mice kidney 7 days post exposure with 10 mg kgS1 of SM diluted in acetone showing normal renal architecture. (e) Mice kidney after 7 days of exposure with 10 mg kgS1 SM diluted in PEG-300 showing extensive cellular necrosis leading to degeneration of convoluted tubules (arrow) lobulated glomerulus. (f) Mice kidney after 7 days of exposure with 10 mg kgS1 SM diluted in DMSO showing necrosed tubules (arrow) with vacuolation in glomeruli and degenerative changes in tubules (bold arrow).
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[()TD$FIG]
861
Fig. 6 – Photomicrographs of control and SM exposed mice spleen (H & E). (a) Control mice spleen showing normal splenic histology with germinal center, red pulp and marginal zone of white pulp. (b) Mice spleen after 7 days of exposure with 5 mg kgS1 SM diluted in PEG-300 showing degeneration of lymphocyte in germinal lymphocyte (arrow). (c) Mice spleen after 7 days of exposure with 5 mg kgS1 SM diluted in DMSO showing lymphoid depletion. (d) Mice spleen 7 days post exposure with 10 mg kgS1 of SM diluted in acetone showing similarity with the control spleen. (e) Mice spleen after 7 days of exposure with 10 mg kgS1 SM diluted in PEG-300 showing hypocellularity of white pulp and accumulation of fibrinoid material (arrow). (f) Mice spleen after 7 days of exposure with 10 mg kgS1 SM diluted in DMSO showing necrosis (arrow) of lymphocyte, dead necrosed cells showing hypocellularity of white pulp.
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[()TD$FIG]
Fig. 7 – Photomicrographs of control and SM exposed mice skin (H&E). (a) Control mice skin section showing normal arrangement of epidermis, dermis and adenexa. (b) 3 day post acetone exposure showing normal arrangement of all the layers of skin along with intact adenexal tissue. (c) Mice skin 3 days after dermal application of 5 mg kgS1 of SM in PEG-300 showing mild inflammatory reaction and changes in epidermis and dermis. (d) Mice skin 3 days after dermal application of
burns 37 (2011) 851–864
Conflict of interest The authors declare no conflict of interest either financially or personally with other people or organisations that could influence their work.
references
[1] Vijayaraghavan R, Gautam A, Sharma M. Medical countermeasures and other therapeutic strategies for sulfur mustard toxicity. In: Gupta RC, editor. Handbook of toxicology of chemical warfare agents. New York: Academic Press; 2009. p. 897–918. [2] Balali-Mood M, Hefazi M. The pharmacology toxicology and medical treatment of sulphur mustard poisoning. Fund Clin Pharmacol 2005;19:297–315. [3] Graham JS, Stevenson RS, Mitcheltree LW, Hamilton TA, Deckert RR, Lee RB, et al. Medical management of cutaneous sulfur mustard injuries. Toxicology 2009;263:47– 58. [4] Dacre JC, Goldman M. Toxicology and pharmacology of the chemical warfare agent sulphur mustard. Pharmacol Rev 1996;48:289–326. [5] Graham JS, Chilcott RP, Rice P, Milner SM, Hurst CG, Maliner BI. Wound healing of cutaneous sulfur mustard injuries: strategies for the development of improved therapies. J Burns Wounds 2005;4:1–45. [6] Balali-Mood M, Hefazi M. Comparison of early and late toxic effects of sulfur mustard in Iranian veterans. Basic Clin Pharmacol Toxicol 2006;99:273–82. [7] Greenberg S, Kamath P, Petrali J, Hamilton T, Garfield J, Garlick JA. Characterization of the initial response of engineered human skin to sulfur mustard. Toxicol Sci 2006;90:549–57. [8] Sabourin CL, Danne MM, Buxton KL, Casillas RP, Schlager JJ. Cytokine, chemokine, and matrix metalloproteinase response after sulfur mustard injury to weanling pig skin. J Biochem Mol Toxicol 2002;16:263–72. [9] Smith KJ, Skelton HG, Hobson DW, Reid FM, Blank JA, Hurst CG. Cutaneous histopathologic features in weanling pigs after exposure to three different doses of liquid sulfur mustard. Am J Dermatopathol 1996;18: 515–20. [10] Smith KJ, Casillas R, Graham J, Skelton HG, Stemler F, Hackley Jr BE. Histopathologic features seen with different animal models following cutaneous sulfur mustard exposure. J Dermatol Sci 1997;14:126–35. [11] Wormser U, Brodsky B, Proscura E, Foley JF, Jones T, Nyska A. Involvement of tumor necrosis factor-alpha in sulfur mustard-induced skin lesion; effect of topical iodine. Arch Toxicol 2005;79:660–70. [12] Evison D, Hinsley D, Rice P. Chemical weapons. BMJ 2002;324:332–5. [13] Ruff AL, Dillman JF. Signaling molecules in sulfur mustardinduced cutaneous injury. Eplasty 2007;8:e2.
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[14] Vogt RF, Dannenberg AM, Schofield BH, Hynes NA, Papirmeister B. Pathogenesis of skin lesions caused by sulfur mustard. Fundam Appl Toxicol 1984;4:S71–83. [15] Smith KJ, Graham JS, Moeller RB, Okerberg CV, Skelton H, Hurst CG. Histopathologic features seen in sulfur mustard induced cutaneous lesions in hairless guinea pigs. J Cutan Pathol 1995;22:260–8. [16] Smith KJ, Smith WJ, Hamilton T, Skelton HG, Graham JS, Okerberg C, et al. Histopathologic and immunohistochemical features in human skin after exposure to nitrogen and sulfur mustard. Am J Dermatopathol 1998;20:22–8. [17] Sawyer TW, Risk D. Effects of selected arginine analogues on sulphur mustard toxicity in human and hairless guinea pig skin keratinocytes. Toxicol Appl Pharmacol 2000;163:75–85. [18] Blaha M, Bowers WJ, Kohl J, DuBose D, Walker J. Il-1-related cytokine responses of nonimmune skin cells subjected to CEES exposure with and without potential vesicant antagonists. In Vitro Mol Toxicol 2000;13:99–111. [19] Brodsky B, Trivedi S, Peddada S, Flagler N, Wormser U, Nyska A. Early effects of iodine on DNA synthesis in sulfur mustard-induced skin lesions. Arch Toxicol 2006;80:212–6. [20] Cowan FM, Broomfield CA, Lenz DE, Smith WJ. Putative role of proteolysis and inflammatory response in the toxicity of nerve and blister chemical warfare agents: implications for multi-threat medical countermeasures. J Appl Toxicol 2003;23:177–86. [21] Gross CL, Nealley EW, Nipwoda MT, Smith WJ. Pretreatment of human epidermal keratinocytes with DLsulforaphane protects against sulfur mustard cytotoxicity. Cutan Ocul Toxicol 2006;25:155–63. [22] Isidore MA, Castagna MP, Steele KE, Gordon RK, Nambiar MP. A dorsal model for cutaneous vesicant injury by 2chloroethyl ethyl sulfide using C57BL/6 mice. Cutan Ocul Toxicol 2007;26:265–76. [23] Paromov V, Suntres Z, Smith M, Stone WL. Sulfur mustard toxicity following dermal exposure: role of oxidative stress, and antioxidant therapy. J Burns Wounds 2007;7:e7. [24] Simpson R, Lindsay C. D. Effect of sulphur mustard on human skin cell lines with differential agent sensitivity. J Appl Toxicol 2005;25:115–28. [25] Pant SC, Vijayaraghavan R, Kannan GM, Ganeshan K. Sulphur mustard induced oxidative stress and its prevention by sodium 2,3-dimercapto propane sulponic acid (DMPS) in mice. Biomed Environ Sci 2000;13:225–32. [26] Kumar O, Sugendran K, Vljayaraghavan R. Protective effect of various antioxidants on the toxicity of sulphur mustard administered to mice by inhalation or percutaneous routes. Chem Biol Interact 2001;134:1–12. [27] Kulkarni AS, Vijayaraghavan R, Pathak U, Raza SK, Pant SC, Satish HT, et al. Evaluation of analogues of DRDE-07 as prophylactic agents against the lethality and toxicity of sulphur mustard administered through percutaneous routes. J Appl Toxicol 2006;26:115–25. [28] Vijayaraghavan R, Kumar P, Joshi U, Raja SK, Rao PVL, Malhotra RC, et al. Prophylactic efficacy of amifostine and its analoges against sulphur mustard toxicity. Toxicology 2001;163:83–91.
5 mg kgS1 of SM in acetone showing coagulative necrosis of epidermal cells (arrow) with severe infiltration of inflammatory cells in dermis. (e) Mice skin 7 days after dermal application of 5 mg kgS1 of SM in DMSO showing loss of epidermis with ulcer formation (arrow). (f) Mice skin 3 days after dermal application of 10 mg kgS1 of SM in acetone showing intensive oedema, transcytosis of inflammatory cells and dermo–epidermal separation (arrow). (g) Mice skin 7 days after dermal application of 10 mg kgS1 of SM in PEG-300 showing necrosis and loss of epidermal cells, oedema, transmigrated inflammatory cells and ulcer formation (arrow). (h) Mice skin 7 days after dermal application of 10 mg kgS1 of SM in DMSO showing coagulative necrosis of epidermal cells (arrow) along with severe inflammatory reaction in dermis.
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[29] Vijayaraghavan R, Kulkarni A, Pant SC, Kumar P, Rao PVL, Gupta N, et al. Differential toxicity of sulfur mustard administered through percutaneous, subcutaneous, and oral routes. Toxicol Appl Pharmacol 2005;202:180–8. [30] Gautam A, Vijayaraghavan R, Pant SC, Kumar O, Singh S, Satish kumar HT. Protective effect of quercetin against sulphur mustard-induced oxidative stress in mice. Def Sci J 2007;57:707–20. [31] Gautam A, Vijayaraghavan R. Prophylactic effect of gossypin against percutaneously administered sulphur mustards in mice. Biomed Environ Sci 2007;20:250–9. [32] Sharma M, Vijayaraghavan R, Gautam A. DRDE07 and its analogues as promising cytoprotectants to nitrogen mustard (HN-2)—an alkylating anti-cancer and chemical warfare agent. Toxicol Lett 2009;188:243–50. [33] Gautam A, Singh S, Kulkarni AS, Pant SC, Vijayaraghavan R. Protective effect of Aloe vera L. gel against sulphur mustardinduced systemic toxicity and skin lesions. Ind J Pharmacol 2005;87:103–10. [34] Ellman GL. Tissue sulfhydryl groups. Arch Biochem 1959;82:70–7. [35] Jollow DJ, Mitchell JR, Zamppaglione Z, Gillette JR. Bromobenzene induced liver necrosis: protective role of glutathione and evidence for 3,4-bromobenzene oxide as the hepatotoxic metabolites. Pharmacology 1974;11:151–7.
[36] Hissin PJ, Hilf RA. Fluorometric method for determination of oxidized and reduced glutathione in tissue. Anal Biochem 1976;74:214–26. [37] Esterbauer H, Cheeseman KH. Determination of aldehydic lipid peroxidation products: malonaldehyde and 4hydroxyl nonenal. Methods Enzymol 1990;186:407. [38] Sugendran K, Jeevaratnam K, Bhaskar ASB, Pant SC. Effect of topically applied sulphur mustard on hematological, biochemical and histological parameters in mice. Def Sci J 1996;46:149–54. [39] Papirmeister B, Feister AF, Robinson SI, Ford RD. Medical defense against mustard gas: toxic mechanisms pharmacological implications. Boca Raton, FL: Press CRC; 1991. [40] Lindsay CD, Rice P. Changes in connective tissue macromolecular components of Yucatan mini-pig following application of sulphur mustard vapour. Hum Exp Toxicol 1995;14:341–8. [41] Tewari-Singh N, Rana S, Gu M, Pal A, Orlicky DJ, White CW, et al. Inflammatory biomarkers of sulphur mustard analog 2-chloro ethyl sulphide-induced skin injury in SKH-1 hairless mice. Toxicol Sci 2009;108:194–206. [42] Kumar V, Abbas AK, Fausto N. Robbins and Cotran pathologic basis of disease. Philadelphia: Saunders (Elsevier); 2004.
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Designing of mouse model: A new approach for studying sulphur mustard-induced skin lesions Vinay Lomash, Utsab Deb, Renuka Rai, Sunil E. Jadhav, R. Vijayaraghavan, S.C. Pant * Division of Pharmacology and Toxicology, Defence R & D Establishment, Jhansi Road, Gwalior 474 002, Madhya Pradesh, India
article info
abstract
Article history:
This study was planned to design a mouse model for studying sulphur mustard (SM)-
Received 15 July 2010
induced skin injury. SM was applied dermally at dose of 5 or 10 mg kg1 in polyethylene-
Received in revised form
glycol-300 (PEG-300) or dimethylsulphoxide (DMSO) or acetone once. The changes in body
20 October 2010
weight, organ body weight indices (OBWI) and haematological and oxidative stress param-
Accepted 10 December 2010
eters were investigated over a period of 3–7 days and supported by histopathological observations. Exposure to SM in PEG-300 or DMSO resulted in a significant depletion in body weight, OBWI, hepatic glutathione (GSH) and elevation in hepatic lipid peroxidation,
Keywords:
without affecting the blood GSH and hepatic oxidised glutathione (GSSG) levels. Interest-
Sulphur mustard
ingly, no aforesaid change was observed after dermal application of SM diluted in acetone.
PEG-300
These biochemical changes were supported by the histological observations, which
DMSO
revealed pronounced toxic effect and damage to liver, kidney and spleen after dermal
Acetone
application of SM diluted in PEG-300 or DMSO. The skin showed similar microscopic changes
Oxidative stress
after dermal application of SM in all the three diluents, however; the severity of lesions was
Skin lesions
found to be time and dose dependent. It can be concluded that dermal exposure of SM diluted in acetone can be used to mimic SM-induced skin toxicity without systemic toxicity in a mouse model. # 2010 Elsevier Ltd and ISBI. All rights reserved.
1.
Introduction
Sulphur mustard (SM) is bis-(2-chlorethyl) sulphide and is one of the blistering or vesicating agents among the various mustard agents. It is regarded as a significant chemical agent of historical and current interest because of its multitudinous acute and chronic adverse effects resulting from extensive use in previous armed conflicts [1]. The easy availability of precursors, the simple method of synthesis and the extremely stable nature of SM make it a chemical weapon of choice by military and terrorist groups [2]. SM has many debilitating effects including ocular and dermal injury, respiratory tract damage, reproductive and developmental toxicity and gastrointestinal and haematological effects [3]. SM forms sulphonium ion in the body and alkylates DNA and other cellular * Corresponding author. Tel.: +91 751 2233492; fax: +91 751 2341148. E-mail address: [email protected] (S.C. Pant). 0305-4179/$36.00 # 2010 Elsevier Ltd and ISBI. All rights reserved. doi:10.1016/j.burns.2010.12.010
macromolecules, which, in turn, leads to DNA strand breaks and cell death [4]. SM causes inflammation and extensive blistering of the skin, primarily because of the large surface area of exposure and the sensitivity of frequently dividing basal cells [4,5]. Skin response to SM manifests as erythema and oedema, which progress to blister formation, ulceration, necrosis and desquamation of the epidermis, and can lead to permanent residual effects in humans [6–11]. The formation of thinwalled blisters is preceded by an asymptomatic latent period of several hours, followed by itching, pain and erythema in humans. Progression of the lesions, the degree of blistering and necrosis are dependent on the quantity of SM delivered [12,13]. Histopathology of the skin following exposure to SM is characterised by oedema, dermal infiltration of inflammatory
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cells, premature death of basal-layer epidermal cells and dermo–epidermal separation [14–16]. Though a number of in vitro models have been used for antidote evaluation, their in vivo efficacy is yet to be established [17]. It appears that the in vitro models are appropriate for mechanistic studies but may not be suitable for protection studies. Many in vivo studies have used weanling pig, hairless guinea pig, rabbit or various mouse species as useful animal models for evaluating the efficacy of various antidotes against SM-induced skin toxicity [18–24]. But, there could be many variables such as dose of SM, physical and chemical nature of diluents and duration of SM exposure that influence the differential severity of pathogenesis of SM-induced lesions. Hairless mouse and hairless guinea pig with unknown genetic defect, and lack of polyclonal and monoclonal antibodies and lack of probes and primers to DNA and RNA sequences in guinea pig might limit the usefulness of these animals in the study of the pathophysiology of SM-induced skin lesions [15]. Our previous studies described the in vivo protection against systemic toxicity due to percutaneous exposure of SM diluted in PEG-300 [25–32], DMSO [29] and acetone [33]. However, for the screening of wound-healing efficacies of dermal formulations, there is need for development of an in vivo model, which should produce SMinduced skin lesions only without showing any signs of systemic toxicity. Thus, the goal of current study was to design a resourceful and consistent mouse model for studying SM-induced skin lesions by comparing in vivo toxicity of SM in various diluents (PEG-300, DMSO and acetone).
2.
Materials and methods
2.1.
Chemicals and reagents
SM was synthesised in the Synthetic Chemistry Division of the Establishment and was found to be above 99% pure by gas chromatographic analysis. Other chemicals of analytical grade were purchased from Sigma (USA) or Merck (India).
2.2.
Animals
All experiments were performed on randomly bred 25–30 g female Swiss albino mice. The animals were obtained from the Animal Facility of Defence Research and Development Establishment. The care and management of the animals were as per the approved guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA, India). The Institutional animal ethical committee approved the protocol for the experiment. The animals were maintained in polypropylene cages on dust-free and autoclaved paddy husk and fed standard pellet diet (Ashirwad Feeds, India) and water ad libitum. A day before the SM application, hairs from back region of all mice were closely clipped using a pair of scissors. Animals were randomly allocated to various groups and SM was applied once dermally at a dose of 5 or 10 mg kg1 body weight diluted in various solvents as mentioned below:
(1) group I: control (without any dermal application); (2) group II: control (single dermal application with acetone only); (3) group III: SM in acetone and sacrificed after 3 days; (4) group IV: SM in PEG-300 and sacrificed after 3 days; (5) group V: SM in DMSO and sacrificed after 3 days; (6) group VI: SM in acetone and sacrificed after 7 days; (7) group VII: SM in PEG-300 and sacrificed after 7 days; and (8) group VIII: SM in DMSO and sacrificed after 7 days. The experiment was performed initially with three mice per group and then repeated once, and the data were compiled for six mice as shown in the results. All the safety precautions were taken while handling SM during dermal application.
2.3.
Body weight and organ weight
The body weight of animals was recorded daily. After 3 or 7 days, the animals were sacrificed and liver, kidney and spleen were dissected out, weighed and preserved in Bouin’s fluid. A portion of skin exposed to the SM application site was also taken and preserved in Bouin’s fluid.
2.4.
Haematological and biochemical analysis
White blood cell (WBC) and red blood cell (RBC) count and haemoglobin, haematocrit (HCT) and mean cell haemoglobin concentration (MCHC) were measured on Sysmex Hematology Analyzer (model K4500). Blood GSH concentration was determined as per the method described by Ellman [34] and modified by Jollow et al. [35]. In brief, 0.2 ml of whole blood was added to 1.8 ml of distilled water and incubated for 10 min at 37 8C for complete haemolysis. After haemolysis, 3 ml of 4% sulphosalycylic acid was added, and the tubes were centrifuged at 2500 rpm for 15 min. The supernatant (0.2 ml) was mixed with 0.4 ml of 10 mM 5,50 dithiobis-(2-nitrobenzoic acid) (DTNB) and 1 ml phosphate buffer (0.1 M, pH 7.4). After 5 min, the absorbance was recorded at 412 nm. The fluorometric method of Hissin and Hilf [36] was used for the determination of hepatic GSH and GSSG concentration. A total of 250 mg of liver tissue was homogenised in 5 ml phosphate ethylene diamine tetraacetic acid (EDTA) buffer (pH 8.0) and metaphosphoric acid (25%). The samples were centrifuged and the supernatant was used for the estimation of GSH and GSSG using O-phthaldialdehyde as a fluorescent dye. Lipid peroxidation was determined by measuring the level of malondialdehyde (MDA), according to the modified method of Esterbauer and Cheeseman [37]. In brief, 200 mg of liver tissue was homogenised in 0.15 M KCl, and 30% trichloroacetic acid (TCA) and 0.8% thiobarbituric acid reagent (TBA) were added. The contents were boiled for 30 min, and the absorbance of the supernatant was measured at 535 nm. MDA was calculated using a molar extinction coefficient of 1.58 105 M1 cm1.
2.5.
Histopathology
Multiple tissue sections of 4–5-mm thickness were taken after dehydration in a graded ascending series of alcohol, clearing in toluene using an automatic tissue processor (Leica TP 1020) and embedded in paraffin wax. Tissue sections were further
burns 37 (2011) 851–864
[()TD$FIG]
853
stained with haemotoxylin and eosin (H&E) and analysed under light microscope (Leica, DMLB).
2.6.
Statistical analysis
The biochemical variables were analysed using one-way analysis of variance with Student–Newman Kewl’s multiple comparison. A probability of 0.05 and less was taken as statistically significant. The analyses were carried out using SigmaStat for Windows version 2.03 (SPSS Inc., USA).
3.
Results
3.1.
Effects on body weight and organ weight
Fig. 1 shows the percent change in body weight of control mice and SM (5 or 10 mg kg1) exposed mice at day 0, 3 and 7 post application. The animals exposed to SM (5 mg kg1) in PEG-300 showed a significant decrease in body weight on day 3 and 7 and animals applied with SM (5 mg kg1) in DMSO showed a significant decrease in body weight on day 7 compared with the control animals and SM (5 mg kg1) in acetone-exposed mice. The animals with SM (10 mg kg1) in PEG-300 and DMSO showed a significant decrease in body weight compared with the control mice and SM (10 mg kg1) in acetone-exposed mice both on day 3 and 7. There was no statistical difference in organ body weight indices (OBWIs) of liver and kidney of mice exposed to SM (5mg kg1) in acetone or PEG-300 or DMSO compared with the control mice on day 3 and 7 (Fig. 2). However, there was a significant decrease in OBWIs of liver on day 3 and 7 and of kidney on day 7 in mice to which SM (10 mg kg1) in PEG-300 and DMSO were applied compared with the liver and kidney of control (normal and with dermal acetone) mice and mice with dermal application of SM (10 mg kg1) diluted in acetone on the same days. A significant decrease in organ body weight indices was observed in spleen of mice applied with SM (5 or 10 mg kg1) in PEG-300 or DMSO on day 3 and 7 compared with spleen from control groups. In contrast to the above findings, a significant increase in spleen weight was noticed on day 3 and 7 in mice to which SM (10 mg kg1) in acetone was applied (Fig. 2).
3.2.
Effect on haematological and biochemical analysis
Mice exposed to SM (5 or 10 mg kg1 diluted in PEG-300 or DMSO or acetone) showed no significant influence on WBC and RBC count, haemoglobin, HCT and MCHC levels after day 3 compared with the mice of control groups (Table 1). SM (in PEG-300 or DMSO or acetone) had no effect on WBC and MCHC levels at day 7. After day 7, mice exposed to SM (10 mg kg1) diluted in PEG-300 or DMSO showed a significant increase in RBC count compared with control mice (Table 2). Haemoglobin concentration of mice exposed to SM (10 mg kg1) in PEG-300 or DMSO after day 7 was significantly higher compared with that in control mice and mice exposed to SM diluted in acetone at a similar dose. SM (10 mg kg1) in PEG-300 application resulted in significant increase in HCT than control mice and mice exposed to SM (10 mg kg1) in DMSO or acetone after day 7. Blood GSH in mice applied with SM (10 mg kg1) in acetone
Fig. 1 – Effect of SM (5 mg kgS1 or 10 mg kgS1, diluted in various vehicles) on body weight after single dermal application (A) control; (B) acetone only; (C) SM in acetone; (D) SM in PEG-300; (E) SM in DMSO. Mean W SE (n = 6). Same alphabets on bars indicate non-significant differences between groups at given dose on given day (P < 0.05).
was significantly higher as compared with control and PEG300 and DMSO groups (Fig. 3). However, it is difficult to explain this increase in blood GSH level. There was a significant decrease in liver GSH of mice applied with SM application (10 mg kg1 in PEG-300 or DMSO) on day 3 and 7, respectively, compared with the liver GSH of control (normal and with dermal acetone) mice on the same days and mice with dermal application of SM (10 mg kg1) diluted in acetone on day 7. However, no significant change was noted in liver GSH content in all groups with an SM dose of 5 mg kg1. MDA levels were significantly higher in mice with dermal application of SM (10 mg kg1) in PEG-300 or DMSO on day 3 and 7 as compared with control and SM (10 mg kg1) in acetone groups.
3.3.
Histopathology
The histopathological observations in liver, kidney and spleen following SM exposure are shown in Tables 3 and 4. Liver of control mice (with and without acetone) showed normal lobular architecture with hepatocytes arranged in cords encircling the central canal (Fig. 4). Similarly, liver sections of mice applied with percutaneous SM diluted in acetone did
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Fig. 2 – Effect of SM (5 mg kgS1 or 10 mg kgS1 diluted in various vehicles) on organ weight, 3 and 7 days after single dermal application. Mean W SE (n = 6). (A) Control; (B) acetone only; (C) SM in acetone; (D) SM in PEG-300; (E) SM in DMSO. Same alphabets on bars indicate non-significant differences between groups at given dose on given day (P < 0.05).
not reveal any significant histological changes at 5 mg kg1 and 10 mg kg1 doses compared with liver sections from control mice. The occasional occurrence of necrotic lesions and hepatocyte vacuolation of minimal severity in liver sections of control mice and mice applied with SM in acetone at 5 and 10 mg kg1 were restricted to individual animals, which were non-significant; and all these features suggest their spontaneous occurrence. However, liver sections from mice with percutaneously applied SM diluted in PEG-300 or DMSO showed significant pathological lesions in hepatic tissue as compared with control mice. The lesions were more pronounced in groups with SM in DMSO or PEG-300 at 10 mg kg1. The significant lesions were hepatocellular degeneration, hepatocyte vacuolation and hypertrophied Kupffer cells. Hepatocytes in groups with SM in PEG-300 or DMSO showed moderate-to-severe karyolysis and necrosis/apoptosis, mainly in the centrilobular area, which diffused margin-
ally in the midzonal area and rarely in the periportal area of the hepatic lobule. Focal accumulation of eosinophillic fibrinoid material entrapping inflammatory cells was also evident. A dose-dependent increase in severity of lesions was noticed in the liver of mice applied with SM (5 or 10 mg kg1) in PEG-300 or DMSO after day 3 and 7 (Fig. 4). The kidney of control mice showed the normal architecture (Fig. 5). It showed normal glomerulus, Bowman’s space and renal parenchyma. The occasional presence of lesions in kidney of control mice, acetone-treated mice and mice treated with SM in acetone at both the doses (5 or 10 mg kg1) showed minimal severity. These changes were common to all groups, which were considered spontaneous. Section of kidney of mice applied with SM in PEG-300 or DMSO (10 mg kg1) showed mild-to-moderate tubular degeneration, haemorrhage, necrosis and subsequent sloughing off of cuboidal cells of proximal and distal tubules into the lumen (Fig. 5).
Table 1 – Effect of SM (10 mg kgS1 and 5 mg kgS1) on hematological variables, 3 days after single percutaneous exposure. Groups
Control Acetone SM in Acetone SM in PEG-300 SM in DMSO
WBC (103 mm3)
RBC (106 mm3)
HGB (g%)
5 mg kg1
10 mg kg1
5 mg kg1
10 mg kg1
5 mg kg1
10 mg kg1
5 mg kg1
10 mg kg1
5 mg kg1
10 mg kg1
9.9 0.3 9.3 3.1 10.3 2.1 5.6 0.8 5.3 0.8
11.9 1.6 9.4 0.8 11.4 1.1 7.5 1.6 7.5 1.5
7.9 0.3 8.3 0.4 7.5 0.4 7.3 0.4 8.6 0.4
7.9 0.4 8.4 0.1 7.4 0.6 8.8 0.5 7.1 0.6
10.6 0.4 10.8 0.3 10.7 0.6 10.1 0.9 11.1 0.3
10.7 0.6 11.4 0.3 9.9 0.8 12.4 0.9 9.5 0.8
37.7 1.3 38.0 2.3 35.7 2.0 33.8 2.5 39.6 1.3
37.3 1.9 39.2 0.7 34.9 3.2 42.7 3.1 33.1 2.6
27.9 0.1 27.7 0.0 29.9 1.3 26.9 1.0 27.9 0.1
28.7 0.2 29.1 0.4 28.4 0.5 28.9 0.4 28.7 0.5
MCHC (g dl1)
HCT (%)
Mean SE (n = 6).
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Table 2 – Effect of SM (10 mg kgS1 and 5 mg kgS1) on hematological variables, 7 days after single percutaneous exposure. Groups
Control Acetone SM in acetone SM in PEG-300 SM in DMSO
WBC (103/mm3)
RBC (106/mm3)
HGB (g%)
5 mg kg1
10 mg kg1
5 mg kg1
10 mg kg1
5 mg kg1
10 mg kg1
5 mg kg1
10 mg kg1
5 mg kg1
10 mg kg1
9.9 0.3 9.3 3.1 14.1 3.2 5.9 0.5 6.5 0.6
11.9 1.6 9.4 0.8 12.11.04 7.7 4.3 7.7 0.7
7.9 0.4 8.4 0.1 7.5 0.3 10.2 1.4 9.7 0.8
7.9 0.3 b 8.3 0.4 b 8.6 0.6 ab 10.2 0.8 a 10.4 0.5 a
10.7 0.6 bc 11.40.3 bc 10.2 0.7 b 14.3 1.2 a 13.5 1.0 ac
10.6 0.4 b 10.8 0.3 b 11.7 1.2 b 14.8 0.5 a 14.4 0.7 a
37.3 1.9 ab 39.2 0.7 ab 35.1 4.5 b 49.6 4.8 a 46.0 3.6 ab
37.7 1.3 b 38.0 2.3 b 41.3 2.6 b 53.5 2.9 a 41.3 6.3 b
27.9 0.1 27.7 0.0 29.0 0.4 29.1 0.2 29.2 1.5
28.7 0.2 29.1 0.4 29.1 0.6 28.9 0.5 29.4 0.3
MCHC (g dl1)
HCT (%)
Mean SE (n = 6; P < 0.05). Same alphabets in superscripts on values in the same column indicate non-significant differences between groups.
855
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Fig. 3 – Effect of SM (5 mg kgS1 or 10 mg kgS1 diluted in various vehicles) on GSH–blood, GSH–liver, GSSG–liver and MDA– liver, 3 and 7 days after single dermal application. Mean W SE (n = 6). (A) Control; (B) acetone only; (C) SM in acetone; (D) SM in PEG-300; (E) SM in DMSO. Same alphabets on bars indicate non-significant differences between groups at given dose on given day (P < 0.05).
Tubular cells showed swelling leading to narrowing of lumen along with the presence of eosinophillic debris. However, kidney sections of mice applied with SM in PEG-300 or DMSO at 5 mg kg1 showed a dose-dependent decrease in the severity of lesions. Control mice spleen showed normal splenic histology with germinal centre, red pulp and marginal zone of white pulp (Fig. 6). The severity of splenic lesions was diluent and dose dependent. There was a significant lymphoid depletion and loss of lymphoid follicle with the accumulation of fibrinoid material in the spleen of mice applied with SM in PEG-300 or DMSO. Lesions were pronounced with SM at 10 mg kg1 than with SM at 5 mg kg1 in PEG or DMSO. However, there was rare occurrence of lymphoid hyperplasia noticed in the spleen of mice with SM in acetone at both the doses (5 and 10 mg kg1).
The microscopic observation of control mice skin showed stratified epithelium that was united to the dermis by a thin basal lamina without showing any degenerative changes (Fig. 7). Following SM exposure, the skin showed severe coagulative necrosis of epidermal cells extending to the dermis and leading to erosions. There was extensive infiltration of inflammatory cells with oedema in the dermal region on day 3, which reduced after day 7. The severity of lesions, such as hyperaemia of blood vessels with dermo–epidermal separation and formation of vesicle, was increased from day 3 to day 7. Basal cells showed vacuolation and loss of connection from basement membrane (acantholysis) in the area surrounding the wound. The degenerated dermo–epidermal region was covered with the tissue comprising of necrosed leucocytes and RBCs in a network of fibrin. There was severe adenexal atrophy in all mice with SM exposure, regardless of
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Table 3 – Histopathology of liver, kidney, spleen and skin in mice 3 and 7 days after single percutaneous exposure of SM (5 mg kgS1). Number of animals showing lesions/total number of animals (severity). Organs
Group I
Group II
Group III
Group IV
Group V
Group VI
Group VII
Group VIII
Liver Necrosis/apoptosis Hepatocellular degeneration Hepatocellular vacuolation Infiltration of inflammatory cells Hypertrophy of Kupffer cells Karyolysis Congestion
0/6 0/6 0/6 0/6 0/6 0/6 0/6
0/6 0/6 0/6 0/6 0/6 0/6 0/6
0/6 0/6 0/6 0/6 2/6 (1) 0/6 0/6
2/6 1/6 3/6 3/6 4/6 2/6 2/6
3/6 1/6 3/6 3/6 4/6 2/6 2/6
0/6 0/6 0/6 0/6 2/6 (1) 0/6 0/6
3/6 2/6 3/6 2/6 4/6 3/6 2/6
(2) (2) (2) (2) (1) (1) (1)
2/6 1/6 3/6 3/6 4/6 3/6 2/6
(2) (2) (2) (2) (1) (1) (1)
Kidney Tubular degeneration Necrosis/apoptosis Glomerular/tubular congestion Hemorrhage Eosinophillic debris
0/6 1/6 (1) 1/6 (1) 0/6 0/6
0/6 2/6 (1) 0/6 0/6 0/6
0/6 2/6 (1) 0/6 0/6 0/6
0/6 0/6 0/6 0/6 0/6
0/6 0/6 0/6 0/6 0/6
0/6 2/6 (1) 1/6 (1) 0/6 0/6
4/6 3/6 2/6 2/6 2/6
(1) (1) (1) (1) (1)
4/6 4/6 2/6 2/6 2/6
(1) (1) (1) (1) (1)
Spleen Lymphoid depletion Lymphoid hyperplasia Lymphocyte necrosis/apoptosis Degeneration of germinal follicle Accumulation of fibrinoid material
0/6 0/6 0/6 0/6 0/6
0/6 0/6 0/6 0/6 0/6
0/6 3/6 (2) 0/6 0/6 0/6
4/6 (3) 0/6 4/6 (2) 3/6 (2) 0/6
5/6 0/6 5/6 3/6 2/6
4/6 (2) 0/6 4/6 (2) 4/6 (2) 0/6
4/6 0/6 4/6 3/6 2/6
(2)
(2) (2) (2)
0/6 0/6 0/6 0/6 0/6
(2) (2) (2)
Skin Inflammatory cell immigration Epidermal necrosis Vacuolar degeneration of basal cell Oedema Acantholysis Adenexal atrophy Vesicle formation
0/6 0/6 0/6 0/6 0/6 0/6 0/6
1/6 (1) 0/6 0/6 0/6 0/6 0/6 0/6
6/6 5/6 5/6 6/6 4/6 4/6 6/6
6/6 6/6 6/6 6/6 6/6 4/6 5/6
6/6 6/6 6/6 6/6 6/6 6/6 5/6
(4) (4) (4) (3) (3) (3) (3)
6/6 5/6 6/6 6/6 6/6 4/6 5/6
6/6 5/6 6/6 6/6 6/6 6/6 6/6
6/6 6/6 6/6 6/6 6/6 6/6 6/6
(4) (4) (4) (2) (4) (4) (4)
(3) (3) (3) (3) (2) (3) (2)
the diluents used. Dose-dependent increase was noticed in the severity of skin lesions of mice applied with SM at 5 mg kg1 than at 10 mg kg1. The details are presented in Tables 3 and 4.
4.
Discussion
The aim of our study was to design an in vivo model, which should produce SM-induced skin lesions without showing any signs of systemic toxicity; this required the use of suitable diluent for SM which will limit its action to the exposed part, that is, skin only. Acetone is more volatile and spreads evenly compared with other diluents used in this study. As a result, systemic absorption and resultant damage could be prevented and toxicity will only be restricted to skin. Thus, it was hypothesised that SM diluted in acetone may reduce systemic toxicity compared with other diluents, namely PEG-300 or DMSO, which have lower vapour pressure and are nonvolatile. In the present study, the topical application of SM (5 or 10 mg kg1) diluted in PEG-300 or DMSO caused progressive loss in body weight, and the animals appeared weak and emaciated. The OBWIs of liver and kidney (10 mg kg1) and spleen (5 or 10 mg kg1) were also reduced. Interestingly, there was no body weight loss and no change in the OBWI of animals with the dermal application of SM diluted in acetone. A similar pattern of decrease in body weight and OBWI has been reported earlier by us [1,29], after the dermal application of SM diluted in PEG-300 or DMSO. Increase in RBC count, haemo-
(2) (2) (2) (1) (1) (1) (1)
(4) (4) (4) (2) (3) (3) (3)
(2) (2) (2) (1) (1) (1) (2)
(3)
(4) (3) (3) (3) (3) (3) (3)
(4) (4) (4) (2) (3) (3) (4)
globin and HCT suggest haemoconcentration in animals with the dermal application of SM diluted in PEG-300 or DMSO [33,38]. There should be a decrease in WBC count as per the expected pathophysiology following the topical application of SM diluted in PEG-300 [38]. However, in the present study, due to haemoconcetration, decrease in WBC count was not evidenced following dermal application of 5 or 10 mg kg1 of SM. Haemoconcentration occurs due to the increase in the vascular permeability of the cutaneous blood vessels, which may also contribute to the increase in RBC count, haemoglobin concentration and the HCT. Median lethal dose (LD50) of SM diluted in acetone is 39.6 mg kg1, which was much higher than the reported LD50 of SM diluted in PEG-300 (8.1 mg kg1) or DMSO (7.2 mg kg1) [29]. The decrease in the LD50 of SM diluted in PEG-300 or DMSO may be due to the high lipophilic nature of the diluents [29]. GSH is an intracellular scavenger of SM. Thus, SM may cause GSH depletion and might be one of the leading causes of generation of reactive oxygen species (ROS) [23,25,26,29]. One of the major causative factors of SM-induced cell death could be accumulation of endogenous ROS leading to lipid peroxidation and irreversible membrane damage [39]. The present study shows that a single dermal application of 10 mg kg1 of SM diluted in PEG-300 or DMSO can cause depletion of GSH in liver and can induce hepatic lipid peroxidation, as evidenced by enhanced MDA levels [29]. However, no change was observed in hepatic GSH and lipid peroxidation following dermal application of 5 mg kg1 of SM in PEG-300 or DMSO and 5 or 10 mg kg1 of SM in acetone. Acetone, being volatile
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Table 4 – Histopathology of liver, kidney, spleen and skin in mice 3 and 7 days after single percutaneous exposure of SM (10 mg kgS1). Number of animals showing lesions/total number of animals (severity). Organs
Group I
Group II
Group III
Group IV
Group V
Group VI
Group VII
Group VIII
1/6 (1) 0/6 1/6 (1) 0/6 0/6 1/6 (1) 0/6
2/6 0/6 1/6 0/6 2/6 2/6 0/6
2/6 0/6 1/6 0/6 3/6 2/6 2/6
(2) (1) (2)
6/6 6/6 4/6 6/6 6/6 6/6 3/6
(3) (3) (4) (3) (2) (4) (2)
6/6 6/6 5/6 6/6 6/6 6/6 4/6
(4) (3) (3) (3) (2) (4) (3)
1/6 0/6 1/6 0/6 2/6 1/6 1/6
(1) (1) (1)
6/6 5/6 6/6 4/6 4/6 6/6 4/6
(3) (3) (3) (2) (2) (2) (2)
6/6 6/6 6/6 6/6 5/6 6/6 3/6
(3) (3) (3) (3) (2) (2) (2)
2/6 (1) 0/6 ( ) 2/6 (1) 0/6 0/6
2/6 (1) 0/6 2/6 (1) 0/6 0/6
2/6 (1) 0/6 3/6 (1) 0/6 0/6
6/6 5/6 5/6 4/6 4/6
(2) (2) (2) (2) (2)
6/6 5/6 4/6 5/6 5/6
(2) (2) (2) (2) (2)
1/6 (1) 0/6 2/6 (1) 0/6 0/6
6/6 6/6 5/6 5/6 5/6
(2) (3) (2) (2) (2)
6/6 6/6 4/6 5/6 5/6
(3) (3) (2) (2) (2)
Spleen Lymphoid depletion Lymphoid hyperplasia Lymphocyte necrosis/apoptosis Degeneration of germinal follicle Accumulation of fibrinoid material
0/6 0/6 0/6 0/6 0/6
0/6 0/6 0/6 0/6 0/6
0/6 2/6 (2) 0/6 0/6 0/6
6/6 0/6 5/6 5/6 4/6
(3)
(3)
6/6 0/6 6/6 6/6 5/6
(3) (3) (3)
6/6 0/6 6/6 6/6 4/6
(3)
(2) (3) (3)
0/6 2/6 (2) 0/6 0/6 0/6
(3)
(2) (2) (2)
6/6 0/6 6/6 5/6 3/6
Skin Inflammatory cell immigration Epidermal necrosis Vacuolar degeneration of basal cell Oedema Acantholysis Adenexal atrophy Vesicle formation
0/6 0/6 0/6 0/6 0/6 0/6 0/6
2/6 (1) 0/6 0/6 0/6 0/6 0/6 0/6
6/6 6/6 6/6 6/6 6/6 4/6 6/6
6/6 6/6 6/6 6/6 6/6 4/6 6/6
(4) (4) (3) (3) (3) (3) (2)
6/6 6/6 6/6 6/6 6/6 4/6 6/6
(3) (3) (4) (1) (3) (3) (4)
6/6 6/6 6/6 6/6 6/6 5/6 6/6
6/6 6/6 6/6 6/6 6/6 5/6 6/6
(4) (4) (4) (2) (3) (3) (4)
6/6 6/6 6/6 6/6 6/6 6/6 6/6
(3) (4) (4) (1) (3) (3) (4)
Liver Necrosis/apoptosis Hepatocellular degeneration Hepatocellular vacuolation Infiltration of inflammatory cells Hypertrophy of kupffer cells Karyolysis Congestion Kidney Tubular degeneration Necrosis/apoptosis Glomerular/tubular congestion Hemorrhage Eosinophillic debris
(1) (1) (1) (1)
(1) (1)
(4) (3) (3) (3) (3) (3) (2)
with better spreading effect, might have resulted in limited SM absorption, whereas the more lipophilic and deeper penetration properties of PEG-300 or DMSO might have acted as an internal carrier to contribute to the greater toxicity of SM. After the initial cutaneous exposure, it has been reported that skin reservoirs continue to distribute SM via circulation to the body tissues thereby increasing damage to several organs [23]. However, to elucidate if 5 or 10 mg kg1 of SM diluted in PEG-300 or DMSO or acetone induces oxidative stress, which may lead to organ damage, we supported our results with histopathological observations in the liver, kidney and spleen. Histomorphological alterations in the different structures in the various regions of these tissues were observed. Hepatocellular degeneration, hepatocyte vacuolation, necrosis/apoptosis, accumulation of fibrinoid material and hypertrophied Kupffer cells were observed in the liver sections of mice treated with SM (5 or 10 mg kg1) diluted in PEG-300 or DMSO in a dose-dependent manner. Sections of mice kidney showed tubular degenerative changes; haemorrhages, necrosis and sloughing off of cuboidal cells of tubules into lumen after percutaneous application with SM diluted in PEG-300 or DMSO. Similarly, spleen of mice with 5 or 10 mg kg1 of SM diluted in PEG-300 or DMSO showed atrophy of splenic tissue displaying lymphoid depletion, loss of lymphoid follicles and accumulation of fibrinoid material in a dose-dependent manner. However, sections of liver, kidney and spleen of mice exposed dermally with percutaneous 5 or 10 mg kg1 of SM diluted in acetone
(1) (1)
(4) (3) (3) (2) (3) (3) (3)
(3) (3) (2)
showed marginal variations in the structure and appeared normal. The results from histological changes revealed that percutaneous application of SM diluted in PEG-300 or DMSO shows more toxic effects and lesions to the visceral organs than SM diluted in acetone. These histopathological findings also support the results of biochemical observations and OBWIs. The appearance of subepidermal blister, basal cell vacuolation, ulceration and coagulative necrosis of epidermis, exudation of oedematous fluid, transcytosis of intramural inflammatory cells and adenexal atrophy in the dermis of mice skin after SM (in acetone, PEG-300 or DMSO) exposure at either dose in the present study is consistent with the observation in a variety of animal models including rabbit, guinea pig [14], pig skin [40], hairless guinea pig [15], hairless mouse [41] and Swiss albino mice [33]. However, we noticed that lesions with either dose of SM diluted in PEG-300 or DMSO showed lesser oedema, more acantholysis, more adexenal atrophy and severe necrosis as compared with the lesions induced by SM in acetone. This may be due to more loss of fluids in case of SM in PEG-300 or DMSO as compared with SM diluted in acetone. Healing is modified by both systemic and local host factors [42]. In the present study, damage to liver, kidney and spleen of mice on exposure to SM diluted in PEG-300 or DMSO was suggestive of impaired systemic host factors, which may retard wound healing and will interfere in the wound-healing efficacy of dermal formulations, affecting their proper evaluation. On the other hand, there was no noticeable
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[()TD$FIG]
859
Fig. 4 – Photomicrographs of control and SM exposed mice liver, H & E. (a) Control mice showing normal hepatic chord, hepatocytes, central canal and kupffer cells. (b) Section of mice liver after 7 days of exposure with 5 mg kgS1 SM diluted in PEG300 showing focal necrosis (arrow). (c) Mice liver after 7 days of exposure with 5 mg kgS1 SM diluted in DMSO showing mild vacuolation and ballooning of hepatocyte (arrow). (d) Section of mice liver 7 days post exposure with 10 mg kgS1 of SM diluted in acetone showing normal histology of liver. (e) Section of mice liver after 7 days of exposure with 10 mg kgS1 SM diluted in PEG-300 showing apoptosis (bold arrow), infiltration of inflammatory cells in hepatic parenchyma and necrotic hepatocyte (arrow). (f) Section of mice liver after 7 days of exposure with 10 mg kgS1 SM diluted in DMSO showing congestion in central vein infiltration of monocytoid cells, condensation of nucleus in hepatocytes, diffuse hepatocyte vacuolation (short arrow) and mid zonal eosinophillic focus (bold arrow) in hepatic parenchyma and necrosis of hepatocytes.
systemic injury observed in mice exposed to SM diluted in acetone, thus diminishing the systemic host factors that influence wound healing. Using current model in efficacy evaluation of various wound healants after dermal SM
exposure, skin lesions can be scored on the basis of histomorphologic scale as described by Graham et al. [3]. Thus, the developed mouse model in the present investigation is a suitable model for studying SM-induced skin lesions.
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Fig. 5 – Photomicrographs of control and SM exposed mice kidney (H & E). (a) Control mice kidney showing normal glomerulus, bowman’s space and renal parenchyma. (b) Mice kidney after 7 days of exposure with 5 mg kgS1 SM diluted in PEG-300 showing mild swelling in convoluted tubules, degenerative changes, necrosis (arrow) with eosinophillic cast in tubules (bold arrow). (c) Mice kidney after 7 days of exposure with 5 mg kgS1 SM diluted in DMSO showing necrosis (arrow), infiltration of inflammatory cells and degenerative changes in tubular cells (bold arrow). (d) Mice kidney 7 days post exposure with 10 mg kgS1 of SM diluted in acetone showing normal renal architecture. (e) Mice kidney after 7 days of exposure with 10 mg kgS1 SM diluted in PEG-300 showing extensive cellular necrosis leading to degeneration of convoluted tubules (arrow) lobulated glomerulus. (f) Mice kidney after 7 days of exposure with 10 mg kgS1 SM diluted in DMSO showing necrosed tubules (arrow) with vacuolation in glomeruli and degenerative changes in tubules (bold arrow).
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[()TD$FIG]
861
Fig. 6 – Photomicrographs of control and SM exposed mice spleen (H & E). (a) Control mice spleen showing normal splenic histology with germinal center, red pulp and marginal zone of white pulp. (b) Mice spleen after 7 days of exposure with 5 mg kgS1 SM diluted in PEG-300 showing degeneration of lymphocyte in germinal lymphocyte (arrow). (c) Mice spleen after 7 days of exposure with 5 mg kgS1 SM diluted in DMSO showing lymphoid depletion. (d) Mice spleen 7 days post exposure with 10 mg kgS1 of SM diluted in acetone showing similarity with the control spleen. (e) Mice spleen after 7 days of exposure with 10 mg kgS1 SM diluted in PEG-300 showing hypocellularity of white pulp and accumulation of fibrinoid material (arrow). (f) Mice spleen after 7 days of exposure with 10 mg kgS1 SM diluted in DMSO showing necrosis (arrow) of lymphocyte, dead necrosed cells showing hypocellularity of white pulp.
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[()TD$FIG]
Fig. 7 – Photomicrographs of control and SM exposed mice skin (H&E). (a) Control mice skin section showing normal arrangement of epidermis, dermis and adenexa. (b) 3 day post acetone exposure showing normal arrangement of all the layers of skin along with intact adenexal tissue. (c) Mice skin 3 days after dermal application of 5 mg kgS1 of SM in PEG-300 showing mild inflammatory reaction and changes in epidermis and dermis. (d) Mice skin 3 days after dermal application of
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Conflict of interest The authors declare no conflict of interest either financially or personally with other people or organisations that could influence their work.
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5 mg kgS1 of SM in acetone showing coagulative necrosis of epidermal cells (arrow) with severe infiltration of inflammatory cells in dermis. (e) Mice skin 7 days after dermal application of 5 mg kgS1 of SM in DMSO showing loss of epidermis with ulcer formation (arrow). (f) Mice skin 3 days after dermal application of 10 mg kgS1 of SM in acetone showing intensive oedema, transcytosis of inflammatory cells and dermo–epidermal separation (arrow). (g) Mice skin 7 days after dermal application of 10 mg kgS1 of SM in PEG-300 showing necrosis and loss of epidermal cells, oedema, transmigrated inflammatory cells and ulcer formation (arrow). (h) Mice skin 7 days after dermal application of 10 mg kgS1 of SM in DMSO showing coagulative necrosis of epidermal cells (arrow) along with severe inflammatory reaction in dermis.
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