Desferal

The nitrogen mustard (HN^sub 2^), which contains chloroethyl groups, is a potent inhibitor of DNA synthesis and cell growth. It also has vesicant properties. Due to these damaging effects of HN^sub 2^ on the skin, zinc chloride (ZnCl^sub 2^) and desferrioxamine were used in the form of ointment for treatment and prevention of the histopathological destruction of skin of guinea pigs. According to results of the histopathological study, both ZnCl^sub 2^ and desferrioxamine had therapeutic and protective effects for HN^sub 2^-induced skin damage at moderate levels. Taking into consideration the alkylating effect of HN^sub 2^ on the skin, our results suggest that ZnCl^sub 2^ and desferrioxamine may be used in the prevention of nuclear damage and for handling the burns caused by the HN^sub 2^.

Introduction

The nitrogen mustard (HN^sub 2^) and a number of its derivatives are potent alkylating agents, and several of the nitrogen mustards are useful in cancer treatment.1 This compound has potent inhibitory effects on DNA synthesis and cell growth through the alkylation properties of its chloroethyl groups. Because of its toxic and vesicant properties, HN^sub 2^ has been used as a chemical weapon since 1935, giving rise to skin lesions being similar to those caused by sulfur mustard.2,3 If early decontamination is not performed, systemic toxicity may occur in addition to burns and tissue damage.4 Therefore, decontamination should be done as early as possible even if the local reaction has taken place. In this connection, several scientific studies have been performed using various decontaminants.5 It has been proposed that strong nucleophiles, oxidizing agents, and some others such as antihistamines and niacin might protect the tissue from the mustard gas.6-8 Povidone iodine ointment has also been used for the treatment of burns caused by mustard.3 This current study discusses the capabilities of easily accessible medications to induce a protective response to an alkylating agent HN^sub 2^. Besides, chemical burns are great concerns in war situations because they cause long-term incapacitation and blocking of treatment centers. Taking into consideration this negative situation, we strongly desired to treat and decontaminate the affected tissue exposed to HN^sub 2^, preparing different topical formulations including zinc and desferrioxamine separately.

In this study, we aimed to investigate the effects of ZnCl^sub 2^, an astringent and antiseptic agent, and desferrioxamine, a chelating agent, on the prevention of HN^sub 2^-induced skin damage in guinea pigs.

Materials and Methods

Materials

HN^sub 2^ (mechloroethamine) was purchased from Sigma-Aldrich (no. 12-256-4, St. Louis, MO), desferrioxamine B was obtained as the mesylate salt (desferal) from Ciba-Geigy (Basel, Switzerland), and zinc chloride (ZnCl^sub 2^) was purchased from Merck (no. 108816, Darmstadt, Germany). The desferrioxamine and ZnCl^sub 2^ ointments were prepared separately in accordance with pharmaceutical data (500 mg of desferal and 2.5 g of ZnCl^sub 2^ in Vaseline) at the Hebrew University-Israel.

In this study, 30 male guinea pigs treated with HN^sub 2^ were studied within a fume hood with a fare velocity of 125 feet per minute. At the end of the experiment, the animals were sacrificed, packed in three closed nylon sacks, and then immediately incinerated.

All contaminated glassware and disposable materials were neutralized for at least 24 hours in a solution containing 5:1 sodium hydroxyde (5 N)/ethanol (1/10v/v) during the study. All of the precautionary steps (dry and liquid decontamination means, laminair flow cabinet, appropriate isolation procedure) were fulfilled by investigators wearing protective suits and masks.3,9,10

Animal Experiment

Thirty male guinea pigs (750-900 g) were used in this study. They were shaved and chemically depilated 24 hours before the experiment. The animals were randomly assigned into three groups: applied ZnCl^sub 2^ group, applied desferrioxamine group, and controls (N = 10 for each group). Animals were anesthetized with a combination of ketamine hydrochloride (30 mg/kg) and xylazine (6 mg/kg, intraperitoneally) before applying HN^sub 2^. A 100-[mu]L solution containing 5 mg of HN^sub 2^ was administered onto circular areas with a diameter of 1.5 cm, which were previously marked on each side of the waist. After HN^sub 2^ exposure, the animals were placed into individual cages, which remained in a fume hood for the duration of the experiment. At the end of 24 hours, a 0.2-mm layer of the ointments (ZnCl^sub 2^ and desferrioxamine) were applied separately with a steel spatula on the skin surface of each test group of 20 animals exposed to HN^sub 2^ previously. Another ointment without any effective chemical compounds was applied to the corresponding sides of the control group animals. All animals were kept in the holding rooms maintained at 21[degrees]C + or - 1[degrees]C with 50 + or - 10% relative humidity until histopathologic analysis.

Histopathologic Analysis

Skin punch biopsies were taken by members of the Department of Pathology from each exposed site 48 hours after the application of the ointments to observe whether therapeutic benefits occurred. Specimens were preserved in 10% neutral-buffered Formalin until examination of pathologic changes by light microscopy. Severe parameters were evaluated, including size and depth of the lesion (to determine tissue reactivity manifested by intracellular edema); epidermal ulceration, fibrosis, nuclear pyknosis, hyperkeratosis, and inflammation were investigated also. All animals were sacrificed by an overdose injection of anesthetic compound and were immediately incinerated.3

Statistical Evaluation

Statistical evaluation was performed with SPSS for Windows software. Fisher's exact [chi]^sup 2^ test was used for the comparison between groups, and values of p

Results

On histopathological examination, all skin tissues of the animals in the control group showed signs of chemical burn with deep epidermal ulceration associated with involvement of the superficial dermis as well as varying degrees of edema and inflammation (Fig. 1). Polymorphonuclear leukocytes (PMNL) were identified within dermis and epidermis where dense micro-abscesses full of PMNL existed. In this control group, extensive nuclear pyknosis in the lower epidermal cell layers and hyperkeratosis were also seen along with dermoepidermal separation and vesicle-bullous formation. Other two groups exposed to ZnCl^sub 2^ and desferrioxamine ointments were found to be affected at a lower degree. The findings included superficial epithelial degeneration associated with slight hyperkeratosis. In both study groups, few pyknotic cells, perinuclear vacuoles, and dermal leukocytic infiltration were noted with similar signs of edema and inflammation observed in the controls but no deep ulceration (Fig. 2). No difference was seen between the effects of ZnCl^sub 2^ and desferrioxamine on the skin exposed to HN^sub 2^ previously in regard to healing of the skin lesion. However, desferrioxamine was differentiated from ZnCl^sub 2^ in that it had an effect of reducing inflammation caused by itself (Fig. 3). A summary of the results is shown in Table I.

Discussion

HN^sub 2^ is a potent inhibitor of DNA synthesis because of its property as a powerful alkylating agent.1,11 It has been suggested that the alkylating activity of HN^sub 2^ is a result of its conversion to the ethylene immonium derivatives.12 In addition, its toxic and vesicant properties depend on chloroethyl groups located in its chemical structure.5,9,13 HN^sub 2^ has been recently used as a therapeutic agent.2,4 As a result of the damaging effects of HN^sub 2^ on living organisms, treatment and decontamination within a short time is essential. If the required intervention cannot be done immediately, irreversible tissue damage will occur and lead to DNA breaks.4 Therefore, removal and inactivation of HN^sub 2^ along with effective treatment are essential in terms of detoxification. For this reason, several studies have focused on this issue by using different approaches, including surgery, pharmacological antidotes, and topical therapy with ointments and creams and so forth.10,14-18 From this point of view, we aimed to develop a preparation that was able to remove and inactivate HN^sub 2^ and, at the same time, to provide beneficial effects in terms of healing of the tissue exposed to HN^sub 2^. Formulations containing the chemical compounds ZnCl^sub 2^ and desferrioxamine were applied on the skin of animals in each group. It was hypothesized that reactive skin ointments containing two different chemicals separately would be able to become efficient against the burning and damaging effects of HN^sub 2^ applied cutaneously. In view of the histopathological findings, it is thought that ZnCl^sub 2^ and desferrioxamine are effective against HN^sub 2^ and possibly other alkylating agents. Shackelford and Tobey19 tested zinc compound against HN^sub 2^ in vivo to determine the toxicity of HN^sub 2^. They suggested that the zinc compound provided a limited degree of protection against HN^sub 2^ toxicity in tumor-free female mice. Although suggesting the application of zinc compound against HN^sub 2^, we hypothesized that chlorine atoms on chloroethyl groups of HN^sub 2^ could be displaced by zinc elements. Therefore, the toxic and burning effects of HN^sub 2^ caused by the chloroethyl groups potentially could be reduced or removed. The slight histopathological damage seen in the group treated with ZnCl^sub 2^ supports this hypothesis. However, this finding should be supported by further advanced chemical methods.

Desferrioxamine (desferal) is a common drug used in the treatment of iron overload.20,21 In addition, it has been suggested that desferrioxamine has advantages in protecting against DNA single-strand breaks and becoming scavenger of different types of free radicals.22 In the light of this information, we believe that desferrioxamine may provide skin protection from HN^sub 2^ by functioning as a scavenger of free radicals generated by HN^sub 2^ action. Moreover, in accordance with the study performed by Ben-Zhan-Zhu et al.,22 desferrioxamine also has action to cause dechlorination via stimulation of the hydrolysis of halogenated substituent. Based on this report, we applied desferrioxamine in ointment onto the skin of animals contaminated with HN^sub 2^. Evaluating the histopathological findings for each group, both study groups had slight pathological changes when compared with those in controls. The control group was also found to have deep skin lesions and histologically extensive damage.

The pathology results strongly supported Papirmaister et al.'s5 biochemical hypothesis that skin injury caused by HN^sub 2^ is characterized by early nuclear damage progressing to basal cell necrosis and vesication. Although both drugs used in this study were found ineffective in treating erythema, they were very protective in preventing ongoing epidermal ulceration, fibrosis, nuclear pyknosis, and hyperkeratosis. Furthermore, desferrioxamine was found to be more effective than ZnCl^sub 2^ on inflammation. These findings imply that drugs tested in the study do not function based on erythema-induced mechanisms. As opposed to ZnCl^sub 2^, desferrioxamine may have an additional beneficial effect on inflammation-induced mechanisms.

In conclusion, our observations suggest that ZnCl^sub 2^ and desferrioxamine are both effective against the damaging effect of HN^sub 2^ on the skin, and these products may be used for the treatment of skin lesions occurring in individuals exposed to HN^sub 2^.

References

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17. Scheidegger D, Sparkers BG, Lusher N: Survival in major burn injuries treated by one bathing in cerium nitrate. Burns 1992; 18: 296-300.

18. Thomes WS, Hancock JR: L-Thiocitrulline: a potent protective agent against the toxicity of sulphur mustard in vitro. Toxicol Appl Pharmacol 1998; 151: 340-6.

19. Shackelford ME, Tobey 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.

20. Davies MJ, Donkor R, Dunster C: Desferrioxamine and superoxide radicals. Biochem J 1987; 246: 725-9.

21. Halliwell B: Protection against tissue damage in vivo by desferrioxamine. Free Radic Biol Med 1989; 7: 645-61.

22. Ben-Zhan-Zhu, Har-el R, Kitrossky N, Chevion M: New modes of desferrioxamine. Free Radic Biol Med 1998; 24: 360-9.

Guarantor: COL Turan Karayilanog lu

Contributors: COL Turan Karayilanog lu*; COL Omer Gunhan[dagger]; MAJ Levent Kenar*; LT Bulent Kurt[dagger]

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

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

This manuscript was received for review in August 2002. The revised manuscript was accepted for publication in November 2002.

Reprint & Copyright (C) by Association of Military Surgeons of U.S., 2003.

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