Ketalar

Mustard, a vesicant warfare agent, has cytotoxic, mutagenic, and cytostatic effects via alkylation of DNA and inhibition of DNA replication. Since symptoms appear following a latent period, it can cause some subacute and chronic effects to occur and delay in the treatment. Therefore, the main approach should be the use of protective preparation to reduce the skin toxicity. Thus, this study was conducted in guinea pigs (350-400 g) shaved in areas of 10 × 10 cm. Mechlorethamine HCl (100 mg), a nitrogen mustard derivative, in ethanol was applied by spraying on hairless regions where previously prepared pharmaceutical topical formulations were medicated before. The experimental regions of the animals were kept preserved from environmental factors. Forty-eight hours after the application of the protective ointments and mechlorethamine consecutively, skin-damaging effects were macroscopically evaluated in terms of erythema formation, ulceration, necrosis, and inflammation occurrences. Then, punch biopsy was performed from these damaged sites for histopathological evaluation. Although numerous topical formulations were prepared and tested for protection, according to microscopic examination of the pathologic sections, tissue specimen treated with the ointment containing the mixture of zinc oxide, zinc chloride, dimethylpolysiloxane in a base of petroleum jelly was determined as being the most effective protective against skin injury caused by the vesicant agent.

Introduction

Chemical warfare still remains a serious and realistic threat despite several international conventions and treaties signed to prevent its use. With regard to chemical warfare agents, chemicals capable of inducing blisters, known as blistering or vesicating agents, have been in use for more than 150 years.1 Among these agents, nitrogen mustard is a cytotoxic agent which is closely related chemically and toxicologically to classical blister agent sulfur mustard (2,2-di(chloroethyl) sulfide) and, as far as is known, sulfur mustard and nitrogen mustard behave in similar manners2 (Fig. 1). In 1935, it was discovered that the vesicant properties were completely preserved when the sulfur atom was substituted by a nitrogen atom.3 The nitrogen mustard bis(2-chloroethyl)methylamine known as HN^sub 2^, mechlorethamine, chloromethine, mustine, or 2-chloro-N-(2-chloroethyl)-N-methylethanamine, is also a common alkylating drug that has been used in the treatment of various types of malignancies such as Hodgkin's disease, lymphoma, and carcinoma of solid tumors for approximately 50 years. Its use is accompanied by severe systemic effects including bone marrow suppression, gastrointestinal disorders, teratogenesis, and carcinogenesis in case of dermal absorption.4 It has a potent vesicant action on human skin like ultraviolet radiation. Release and activation of proteases may also be predicted to cause damage of the dermo-epidermal junction and lead to separation of the injured skin region.5

Medical interest is primarily concerned with its action on the skin, eyes, and respiratory tract.6 Skin lesions appear generally within hours following exposure; therefore, first erythema and bullae develop, then gradually blisters follow ulceration and necrosis.7 They are simple to produce and cause many incapacitating, nonlethal or lethal injuries, so they can prevent the enemy from using a contaminated area. Mustard agent does not cause any immediate noticeable effects, but results in slowly appearing lesions.1 Because of its nature, mustard gas easily penetrates through skin and moves into the circulation within 15 to 20 minutes. Therefore, topical application of drugs will be useless if not applied within the duration of exposure. Skin lesions resulting from exposure to mustard vapor may progress to form large bullae that may require intensive medical management and surgical intervention. Thus, the use of a protective agent is required to protect the exposed tissues against its toxic action and to prevent further alkylation of cellular components. Therefore, the objective of this research was to test and evaluate some pharmaceutical topical preparations that have been reported and that are supposed to be protective against the effects of nitrogen mustard on skin.8-11

Therefore, the protective effects of seven different types of agent combinations were compared in an experimental model of HN^sub 2^ injury to guinea pig skin. Pathology observations at the gross and at the light microscopy level are also discussed.

Materials and Methods

Materials

Chemicals were used to prepare the pharmaceutical combinations that would be applied on the skin of animals. The formulations ready for use were stored in appropriate conditions until study. Mechlorethamine (HN^sub 2^), a nitrogen mustard derivative, was obtained from Sigma-Aldrich (M3878, St. Louis, Missouri). Other chemicals used in the content of formulation, including dimethylpolysiloxane (DMPS), niacinamide (N3376), α-tocopherol (T3251), aluminum silicate (AlSiO^sub 3^; K7375), were also Sigma-Aldrich originated. Additionally, zinc oxide (ZnO^sub 2^) and zinc chloride (ZnCl^sub 2^) were purchased from Merck (Darmstadt, Germany). Topical preparations of the mentioned drugs were prepared before application.

Methods

Experimental Animals

Seventy male haired guinea pigs (600-700 g) were used in the experiments. They were depilated and shaved to provide a circular hairless skin area with the diameter of 10 cm on both sides of the ventral portion of the animals. They were maintained under great care and kept in stainless cages on pellet diet and tap water ad libitum.

Study Protocol

On the study day, mechlorethamine (HN^sub 2^) was freshly prepared. Mechlorethamine solution (1 g/100 mL ethyl alcohol) was put in a spraying device to be applied in aerosol form.

The experiment was performed in specific laboratory conditions where the necessary protective measures were taken. The prepared topical formulations listed in Table I were applied to the entire hairless skin area with the thickness of 0.5 cm 2 hours before the exposure of HN^sub 2^. The animals were anesthetized with an intramuscular injection of 12 mg/kg ketamine (Ketalar, Parke-Davis) and 6 mg/kg xylazine HCl (Rompun, Bayer, Wuppertal, Germany) in combination.

Following the topical application, freshly prepared HN^sub 2^ was sprayed onto the skin area on which the candidate topical creams were previously applied. Ten sprays were made for each skin area within 2 minutes, hence the total exposure dose of the agent was 0.38 mg/cm^sub 2^/minute for each skin sample. This phase of the experiment was performed in a laminar air flow hood with a fare velocity of 125 feet/minute to protect the researchers from any further contamination. Thirty minutes later, the exposed demarcated area of the skin was preserved with barrier disc to prevent damage from the external interfering factors. Then the anesthetized animals were placed in individual cages where they were kept until the end of the experiment.

Macroscopic and Histopathologic Examinations

After 48 hours from the time when HN^sub 2^ and the topical protective agent application were made, the exposed skin was examined with respect to macroscopic observations. Thus, skin macroscopic lesions were graded according to the severity of erythema, macroscopic ulceration, and necrosis which typically appeared following the exposure to the mustard. The skin was removed and 6-mm skin punches were taken from each of the exposure sites. The histopathologic examination of the skin area treated with the topical agents and HN^sub 2^ was also graded according to predetermined criteria given in Table II. The skin samples were fixed in 10% neutral-buffered Formalin and embedded paraffin. The histopathologic examinations were undertaken with thin sections of tissues at 4-µm thickness. Tissues were micromorphologically observed with respect to necrosis, ulceration, edema formation, epidermal inflammation, and dermoepidermal separation and evaluated. After skin biopsy, animals were euthanized with an overdose of ketamine HCl.

Statistical Evaluation

Statistical evaluation was carried out on SPSS software for Windows (SPSS, Chicago, Illinois). The Mann-Whitney U test was used for the comparison of the macroscopic and microscopic effects of the ointments along with controls when no protective agent was applied. Values of p

Results

Results revealed that according to macroscopic examination of the effects of candidate protective agents on the skin exposed to HN^sub 2^, the combination of ZnCl^sub 2^, ZnO^sub 2^, DMPS, and petroleum jelly was found to be the most effective topical formulation against dermal absorption of the vapor form of mechlorethamine, a nitrogen mustard derivative. Illustration of this effect compared with the topical agent-free control exposed to HN^sub 2^ is given in Figure 2. The resulting burns may be considered compatible in severity with the human skin lesions following exposure to mustard vapors under warfare conditions.

The cutaneous injury caused by HN^sub 2^ in the control group application reached a maximum level in all histopathologic indicators. When the chemicals formulated as in Table I were administered as protective agents before an HN^sub 2^ exposure, the injury was almost diminished. However, the protective action of the ointment (no. 4), consisting of the mixture of ZnCl^sub 2^, ZnO^sub 2^, and DMPS in a base of petroleum jelly, was shown to be significantly effective at a high level, especially against the formation of necrosis, ulceration, epidermal inflammation, and dermoepidermal separation throughout the study (p

The results showed that the use of compounds containing niacin, α-tocopherol, and AlSiO^sub 3^ when combined with ZnO^sub 2^ and ZnCl^sub 2^ did not provide a high level of skin protection as that provided by formulation 4 and caused deep skin lesions with the remaining histopathologic changes that were untreated at the injury site. However, formulations 1, 2, and 7 individually showed considerable protection against ulceration that occurred due to mustard exposure when compared with those of nonmedicated control animals (p = 0.001, p

In the histopathologic examination of skin pieces treated with ointment 4 before mustard application, the epidermis was intact and viable, and minimal intradermal mononuclear inflammatory cell infiltration and minimal vacuolar alterations in the basal layer of epidermis and in adnexial epithelial parts were the other pathologic findings observed with the preparations (Fig. 3A). However, histopathologic observations of the skin tissues of guinea pigs that were exposed only to HN^sub 2^, but had no protective treatment before, were found to be almost compatible with those found routinely in the victims of mustard exposure (Fig. 4).

Discussion

Battlefield protection against blistering agent exposure requires wearing a protective mask and clothing which restricts the physical capability and cooperation, especially in extreme temperature and humidity conditions.12,13 To improve the functional abilities of soldiers in the Turkish Armed Forces in a mustard-contaminated environment, our department examined the efficacy of topical barrier compounds to protect the skin against dermal effects of mustard which could be easily obtained and have the potential to be used by terrorists and military units. A number of manually prepared compounds were applied as a thin layer 2 hours before to protect skin from exposure to mustard.

The protection of the skin is so essential in military action that the severity of possible lesions and postexposure rehabilitation and its overall economic burden could be reduced, as noted through the follow-up of the victims who survived after the Iran-Iraq conflict in the 1980s.14,15 In this respect, various chemicals were used in experiments. Topical creams containing combinations of chemicals that were already prepared were applied on the skin surfaces. Nitrogen mustard, a nitrogen analogue of mustard gas, was used as a model for chemical vesicant warfare agent, because it is available for clinical practice and chemical burns behave in almost the same way as those of the sulfur analogue of mustard.

The basis of the tissue injuries caused by vesicant agent still remains unclear. Like other alkylating agents, it affects DNA synthesis and inhibits cell proliferation. Mustard has also direct effects on collagen fibrils functioning in cell attachment to the basement membrane and causes enhanced protease synthesis and DNA damage.16 Reactions of HN^sub 2^ with other macromolecules may significantly contribute to the spectrum of cutaneous effects from the liquid form of exposure.2,16-18 Because of irreversible tissue damage from mustard gas, an effective protection is a must for a medical defense. Therefore, application of the protective drugs has been recently studied and investigated as an additional protection method other than individual protective equipment and shelters.

Results revealed that the application of the ointment containing ZnCl^sub 2^, ZnO^sub 2^, and DMPS in a base of petroleum jelly before nitrogen mustard exposure conferred a high degree of protection. The mechanism of action and the ability of ointment 4 to increase the protective index of HN^sub 2^ should also be studied. The possible mechanism might be the oxidative effects of ZnO^sub 2^ and ZnCl^sub 2^ on the nitrogen atoms existing in the toxic molecule. Additionally, another active ingredient, DMPS, a polymer silicone with a high molecular weight but a very low volatility, is supposed to act as an adsorbent barrier for the toxic agent.

Several studies were carried out to develop a number of protective agents against the skin effects of mustard agents. Vojvodic et al.19 examined the protective factors of some medications in injectable forms including sodium thiosulfate, vitamin E, dexamethosone, promethazine, heparin, and atropine; vitamin E (α-tocopherol) was found to be the most effective. Vitamin E was also used within this study in combination with other molecules in topical cream and found to be effective in diminishing the development of necrosis and ulcération visible by light microscopy.19 Fasth and Sörbo20 also found similar protective effects of sodium thiosulfate in rats poisoned by mustard. Since oxygen free radicals were postulated as causing at least part of the mustard gas toxicity, Superoxide dismutase, an antioxidant substance like vitamin E that can serve as scavenger of electrophilic compounds, was also used.16,21 Weltin el al.22 had also reported that antioxidant N-acetylcysteine would have been able to prevent apoplosis induced in lymphocytes by nitrogen mustard.

The experiment undertaken by Andrew et al.23 demonstrated significant protection in RPMI 2650 human upper respiratory cell lines treated with di-isopropyl esters of glutathione as cytoprotectants before mustard exposure. Esters of glutathione were reported to be effective at protecting against skin damage.

Wormser et al.11 found that the severity of chemical skin burns caused by nitrogen mustard was reduced by treatment with o-phenanthroline applied following the exposure. Histopathologic examination of the skin area treated with HN^sub 2^ followed by o-phenanthroline showed a slight diffuse acanthosis of the epidermal layer, whereas histology of the damaged tissue showed an area of epidermal ulceration associated with a covering layer of encrusted exudate.11

One of the protective agents used, as reported by Wormser et al., is povidone iodine, which has a lower degree of protection against mechlorethamine.4 The histopalhologic evaluation showed only moderate thickening of the epidermis with slight hyperkeratosis and only a small area of dermal leukocytic infiltration, but no ulceration was noted. However, areas without povidone iodine treatment showed deep epidermal ulceration associated with the involvement of the superficial dermis.4,10

Zinc chloride was also suggested to have a limited degree of protection against HN^sub 2^, with histopathologic findings on slightly damaged skin tissue. Karayylanoglu et al.24 found that edema and inflammation were histopathologically persistent following ZnCl^sub 2^ treatment; however, we observed that ointment 4, containing zinc compound, had a higher degree of protection on skin pathologies.25

Niacinamide, which was also applied in this study, was to restore the maintenance of the energy-providing system by intervening in the process of death of basal epidermal cells leading to blister formation. Therefore, therapy with niacinamide was reported to produce significant results following the exposure to mustard vapor.26

In conclusion, those initial positive results of the protection of those probably exposed to mustard obtained by the combinations discussed in the article may lead to a further improvement by testing other chemical combinations.

References

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2. Smith KJ, Smith WJ, Hamilton T, et al: Histopathologic and immunohistochcmical features in human skin after exposure to nitrogen and sulfur mustard. Am J Dermatopathol 1998; 20: 22-8.

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7. Vogt RF Jr, Dannenberg AM Jr, Schofield BH, Hyncs NA, Papirmcister B: Pathogenesis of skin lesions caused by sulfur mustard. Fundam Appl Toxicol 1984; 4: S71-83.

8. Kumar O, Sugendran K. Vijayaraghavan 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.

9. Koper O, Lucas E, Klabunda KJ: Development of reactive topical skin protectants against sulfur mustard and nerve agents. J Appl Toxicol. 1999; Suppl 1: S59-70.

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11. Wormser U, Nyska A: Protective effect of o-phenanthroline against mechlorelhamine toxicity in the rat liver slice system and in the guinea pig skin. Arch Toxicol 1991:65:666-70.

12. Laird IS, Goldsmith R, Pack RJ, Vitalis A: The effect on heart rale and facial skin temperature of wearing respiratory protection at work. Ann Occup Hyg 2002; 46: 143-8.

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15. Momeni AZ, Aminjavaheri M: Skin manifestations of mustard gas in a group of 14 children and teenagers: a clinical study. Int J Dermatol 1994; 33: 184-7.

16. Naghii MR: Sulfur mustard intoxication, oxidative stress, and antioxidants. Milit Med 2002; 167: 573-5.

17. Mol MAE, de Vries R, Kluivers AW: Effects of induced by sulfur mustard in human skin organ cultures. Toxicol Appl Pharmacol 1991; 107: 439-49.

18. Sidell FR, Takafuji ET, Franz DR: Vesicants, Medical Aspects of Chemical and Biological Warfare, pp 198-217. Belhesda, MD. Office of the Surgeon General, 1997.

19. Vojvodie V, Milosavljevic Z, Boskovic B, Bojanic N: The protective effect of different drugs in rats poisoned by sulfur and nitrogen mustards, Fundam Appl Toxicol 1985; 5: S160-8.

20. Fasth AI, Sörbo B: Protective effect of thiosulfate and metabolic thiosulfate precursors against toxicity of nitrogen mustard (HN^sub ^2). Biochem Pharmacol 1973; 22: 1337-51.

21. Eldad A, Ben Meir P, Breiterman S, Chaouat M, Shafran A, Ben-Bassat H: Superoxide dismutase (SOD) for mustard gas burns. Burns 1998; 24: 114-9.

22. Weltin D, Aupeix K. Iltis C, et al: N-acetylcysteine protects lymphocytes from nitrogen mustard-induced apoptosis. Biochem Pharmacol 1996: 51: 1123-9.

23. DJ Andrew, Lindsay CD: Protection of upper respiratory tract cell lines against sulphur mustard toxicity by glutathione esters. Hum Exp Toxicol 1998; 17: 387-95.

24. Karayylanoglu T, Gunhan Ö, Kenar L, Kurt B: The protective and therapeutic effects of zinc chloride and desferrioxamine on skin exposed to nitrogen mustard. Milit Med 2003; 168: 614-17.

25. Shackelford ME, Tobay RA: Attempted use of Zn in vivo to protect against nitrogen mustard toxicity in tumour-free female B6D2F^sub 1^ mice. J Appl Toxicol 1992; 12: 295-300.

26. Mol MAE, DeVries R, Kluivers AW: Effects of nicotinamide on biochemical changes and mlcroblistering induced by sulfur mustard in human skin organ cultures. Toxicol Appl Pharmacol 1991; 107: 439-49.

Guarantor: COL Turan Karayilanoglu

Contributors: MAJ Levent Kenar*; COL Turan Karayilanoglu*; Altan Yuksel, PharmPhD[dagger]; COL Omer Gunhan[double dagger]; Songul Kose*; 1st Lieut Bulent Kurt[double dagger]

* Department of NBC Defense, Gulhane Military Medical Academy, 06018 Ankara, Turkey.

[dagger] Department of Pharmaceutical Technology, Faculty of Pharmacy, Mersin, Turkey.

Department of Pathology, Gulhane Military Medical Academy, 06018 Ankara, Turkey.

This manuscript was received for review in November 2003. The revised manuscript was accepted for publication in March 2004.

Reprint & Copyright © by Association of Military Surgeons of U.S., 2005.

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