J. Toxico1.-Cut. & Ocular Toxiad. 7(4), 255-261 (1988)

SKIN PENETRATION AND TISSUE DISTRIBUTION OF [ ‘4c]BUM 2-CHLoRoETHyL SULFIDE IN THE RAT GEORGE J . KLAIN, Ph.D. STEPHANIE J. BONNER, B. S. STANLEY T. OMAYE, Ph.D. Letterman Army Institute of Research Presidio of San Francisco San Francisco, California

Abstract In vivo percutaneous penetration and tissue distribution of radioactivity following a topical application of ‘%-labeled butyl 2-chloroethyl sulfide (BCS) were determined in the laboratory rat. Up to 70% of the topical BCS was lost by evaporation. Only a portion of BCS in the skin was extracted with ethanol. At 1 hr, approximately two-thirds of the applied BCS was ethanol-extractable. This level decreased to about 6% at 24 hr. Radioactiv- ity in blood was detected 5 min after application. Thereafter, a linear in- crease in blood radioactivity was observed throughout the entire 4 hr ex- perimental period. Topical BCS was absorbed into the circulation and was incorporated into various organs and tissues. The liver, kidneys, heart, lungs,

Address reprint requests to: George J. Klain, Ph.D., Division of Cutaneous Hazards, Letterman Army Institute of Research, Presidio of San Francisco, California 94129-6800.

In conducting this research, the investigators adhered to the “Guide for the Care and Use of Laboratory Animals,” as promulgated by the. Committee on Revision of the Guide for Laboratory Animal Facilities and Care of the In- stitute of Laboratory Resources, National Research Council. Citation of trade names in this report does not con- stitute an official endorsement or approval of the use of such items. The opinions and assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting views of the Department of the Army or the Department of Defense (AR 360-5).

255 Copyright 0 1988 by Marcel Dekker, Inc.

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256 KLAIN ET AL.

skeletal muscle, brain, eyes, and bone marrow were radioactive. On an equal weight basis, the kidneys and lungs contained the highest level of radioactivity. Following a subcutaneous injection of BCS, over 2% of the dose appeared in expired air. No radioactivity was detected in expired car- bon dioxide. In 24 hr, up to 70% of the applied radioactivity was excreted in the urine. Four urinary metabolites of BCS were found. This study demonstr-tes that skin contact with BCS forms an extensive reservoir of ethanol-extractable labeled BCS and/or its metabolites in the affected skin site.

Introduction

Butyl2-chloroethyl sulfide (BCS) is a volatile and highly reactive monofunctional analog of bis-2-chloroethyl sulfide (sulfur mustard). It readily reacts in vitro with purified proteins, including insulin, pepsin, and tobacco mosaic virus protein. Subsequent studies showed that BCS reacts with the nucleic acid and protein moiety of the virus.* In addition, BCS reacts with a variety of functional groups, including the amino, imidazolyl, sulfhydryl, and phenolic hydroxyl group^,^ and the mutagenic activity of BCS is similar to that of sulfur m ~ s t a r d . ~ Exposure to BCS vapor causes eye and skin irritation in humans and may lead to severe and slow- healing burns.s Exposure to liquid BCS produces histologic and metabolic altera- tions both in human skin grafted onto athymic nude mice and in mouse skin.6

No experimental data are available concerning the percutaneous penetration and systemic disposition of BCS. Because of the potential for exposure of laboratory per- sonnel, we examined in vivo penetration and tissue distribution of radioactive BCS applied topically or subcutaneously to the laboratory rat.

Materials and Methods

Nonradioactive BCS (Columbia Organic Company, Columbia, SC) was added to [14C]BCS (ICN Corporation, Irvine, CA) (sp. act. 1O.W mCi/mmol), labeled in both carbons of the chloroethyl group, to achieve the desired specific activity. A gas- liquid chromatogram of this compound showed one peak with a retention time coin- ciding with a standard nonradioactive sample.

Animals

Male Sprague-Dawley rats weighmg 280-300 g were used in all experiments. Sixteen hours prior to experimentation, the dorsal skin area was clipped with an electric hair clipper.

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PENETRATION AND DISTRIBUTION OF [ ‘‘CIBUTYL 2-CHLOROETHYL SULFIDE 257

Topical Administration

Ten rats were placed in an exhaust hood, and 5 p1 (6 pCi) of neat [14C]BCS was applied with a microliter syringe to a clipped 2 cm2 dorsal area of each rat. Blood samples were obtained periodically from the tail vein.’ The rats were sacrificed in groups of five 1 and 4 hr after application. The treated skin sites and selected tissues were excised, cooled in ice-cold saline, and weighed.

In order to estimate the loss of BCS from the skin as a result of evaporation, 5 p1 (6 pCi) of neat [14C]BCS was applied to a 2 cm2 dorsal skin area of each of five rats. Each rat was placed in a metabolism cage, and air was drawn for 24 hr through a glass column containing 1 g Tenax@ absorbent. Preliminary experiments showed that BCS was retained on the Tenax column. At 24 hr the rats were sacrificed, and the treated skin sites were excised and treated as above.

Subcutaneous Administration

Before use, [14C]BCS was diluted with absolute ethanol (20 pUm1). Five rats were injected subcutaneously in the dorsal area with 0.2 ml (6 pCi) of the solution, and each rat was placed in a metabolism cage. Expired air was drawn through a glass column containing 1 g Tenax absorbent and through a 2 % solution of sodium hydrox- ide. Expired air and urine were collected for 24 hr. Then the rats were sacrificed, and selected organs and tissues were excised and treated as above.

Analytical Procedures

Aliquots of blood and portions of the excised tissues were combusted in a tissue oxidizer, and radioactivity in carbon dioxide was determined in a liquid scintillation spectrometer. Aliquots of urine were applied to a 60 x 0.9 cm column containing Dowex-50 ion exchange resin in H+ form. Radioactivity from the column was eluted with 3.4 N HC1. Four 5 ml fractions were collected per hour. Portions of the excised skin were homogenized in absolute ethanol and centrifuged at 600 X g for 20 min. The supernatant fluid was decanted and the extraction process was repeated three times. Tenax samples were placed into scintillation vials and the columns were flushed with 10 ml of aqueous counting solution. Radioactivity in all liquid portions was determined by liquid scintillation techniques.

Results

The data presented in Table 1 show that BCS rapidly penetrated the skin. At 5 min blood contained about 250 DPM/ml. Thereafter, a gradual increase in radio- activity was observed, and at 4 hr blood contained about 12,500 DPM/ml.

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258

Table 1 Radioactivity in Whole Blood after Topical Application of IWlBCS

KLAIN ET AL.

Minutes DPM/ml'

5 10 20 40 60

120 180 240

248 f 18 440f 53 750 f 61

1250 f 94 1530 f 128 58% f 327 7316 f 648

12510 f 852

'Values are mean f SE, n = 5 .

As shown in Table 2, l4C from BCS was widely distributed in the body and was detected in all the tissues and organs examined. At 4 hr the tissues contained a higher level of radioactivity than at 1 hr. Expressed per unit weight, the highest level of radioactivity was found in the kidneys, followed by the lungs, liver, and heart.

The level of radioactivity extracted with ethanol from the involved skin site following topical application is shown in Table 3. At 1 hr over 67 % of the applied radioactiv- ity was ethanol-extractable. The level decreased to about 26% at 4 hr. A further decrease to about 5 % was observed at 24 hr after application. The results obtained from the skin evaporation experiment show that 69.2 f 4.8% of the applied radio- activity evaporated in 24 hr.

Table 2 Distribution of Radioactivity in Rats 1 and 4 Hr after Tom4 ADDkXtiOn of I'*clBCS

DPM/100 mg' DPMlOrgatP

OrgadTissue l h r 4 h r l h r 4hr

Spleen 91 f 13 156 f 21 464 f 63 997 f 110 Adipose tissue 32 f 7 93 f 10 * * Kidneys 582 f 73 1,059 f 156 14,062 f 1,736 34,324 f 4,230 Diaphragm 4 O f 6 120 f 17 * * Lungs 205 f 27 583 f 71 2,665 f 218 9,949 f 1,148 Heart 127 f 15 204 f 27 1,080 f 134 2,711 f 314 Brain 76 f 9 123 f 8 1,462 f 173 2,337 f 298 Eyes 8 0 f 7 130 f 10 218 f 19 370 f 42 Skin, involved 13,170 f 980 4,810 f 530 167,650 f 18,320 63,190 f 8,630

Liver 173 f 23 332 f 47 19,547 f 2,056 48,097 f 3,958

'Values are mean f SE, n = 5 . *Not determined.

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PENETRATION AND DISTRIBUTION OF ['JCJBUTYL 2-CHLOROETHYL. SULFIDE 259

Table 3 Distribution of Radioactivity in the Skin after Topical Application of PCIBCS

Hours Ethanol Extract Skin Residue

1

4

24

67.7 f 8.4

26.4 f 5.4

5.4 f 1.9

30.7 f 5.1

65.3 f 4.3

89.7 f 6.8

Values are mean f SE 96 total radioactivity, three replicates from each of five rats.

The distribution of radioactivity in selected tissues after a subcutaneous administra- tion of [14C]BCS is summarized in Table 4. The highest level of radioactivity per unit weight was found in the kidneys, followed by the lungs, liver, and the heart. Blood and urine were highly radioactive. Radioactivity was detected in the muscle, bone marrow, brain, and the eyes.

An elution pattern of radioactivity obtained when urine was chromatographed on a Dowex-50 column is shown in Figure 1. The pattern shows that BCS is metabo- lized into three major and one minor water-soluble compounds, which are excreted in the urine.

Table 4 Distribution of Radioactivity in Rats 24 Hr After Subcutaneous Administration of [14C]BCS

Organ/Tissue DPM/100 mp" DPM/Organa % Dose

Liver 542 f 63 58,790 f 7,240 0.45

Spleen 197 f 24 1,213 f 148 0.01

Adipose tissue 74 f 12 * Kidneys 839 f 69 23,760 f 3,470 0.18

Diaphragm

Lungs

Heart

Brain

180 f 17 * 657 f 73 8,278 f 945 0.06

394 f 51 4,474 f 672 0.03

85 f 11 1,745 f 156 0.01

Bone marrow 336 f 27

Eyes 137 f 9 401 f 23 0.003

Muscle, gastrocnemius 226 f 18 * Blood 374 f 27

*

*

'Values are mean f SE, n = 5 . *Not determined.

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260 KLAIN ET AL.

2 4 6 8 10 12 14 16 18 20

FRACTION

Figure 1. Radioactive components of rat urine after subcutaneous administration of [14C]BCS.

Discussion

The following events occur when BCS comes into contact with the skin: a substantial portion of the dose evaporates from the skin, and the remaining portion is absorbed by the skin. A fraction of the absorbed BCS may react with and be bound to specific components of the skin or be removed by the circulation and incorporated into various tissues. The uptake of BCS by the circulation appears to be somewhat slow and a constant process, as indicated by a steady increase in blood radioactivity and by the pool size of ethanol-extractable radioactivity in the treated skin sites. Even 24 hr after application, over 5% radioactivity was extracted with ethanol. We have not determined whether the radioactivity was associated with “free” BCS or with a com- plex of BCS with specific compounds in the skin.

Following absorption into the circulation and in the body, BCS is distributed throughout the various tissues and organs. Because of its size, the liver contains the highest level of the applied radioactive dose. However, on an equal weight basis the kidneys and the lungs contained more radioactivity than the liver. Other organs and tissues containing a high level of radioactivity include the bone marrow, brain, heart, and the spleen. In the tissues, presumably in the liver and the kidneys, BCS is

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PENETRATION AND DISTRIBUTION OF [WIBUTYL 2-CHLOROETHYL SULFIDE 261

metabolized into several water-soluble compounds or conjugates that are excreted in the urine. Four of these were separated and eluted from Dowex-50 columns. In our experiments all the radioactivity applied to the column was eluted with 3.4N HCl. The BCS degradation in the body is further evidenced by the appearance of a volatile compound(s) in the expired air following subcutaneous administration. Since BCS itself is a volatile compound, its presence in the expired air cannot be excluded. However, this possibility remains to be verified.

Our finding that topical BCS forms a reservoir in the skin is significant with respect to skin decontamination procedures. An effective decontaminant of skin exposed to BCS should neutralize the compound not only on the skin surface but also in the skin, to prevent or reduce systemic toxicity.

References

1. J. L. Wood, J. R. Rachele, C. M. Stevens, F. H. Carpenter, and V. du Vignaud, The reaction of some radioactive mustard-type vesicants with purified proteins, J. Am. Chem. Soc. 70:2547, 1948.

2. F. H. Carpenter, J. L. Wood, C. M. Stevens, and V. du Vignaud, Chemical studies on vesicant- treated proteins, J. Am. Chem. Soc. 702551, 1948.

3. V . du Vignaud, C. M. Stevens, H. F. McDuffir, Jr., J. L. Wood, and H. McKennis, Jr., Reac- tions with mustard-type vesicants with alpha-amino acids, J. Am. Chem. Soc. 7 0 1620, 1948.

4. C. M. Stevens and A. Mylroie, Mutagenic activity of compounds related to mustard gas, Biochim. Biophys. Acta 8:325, 1952.

5 . M. W . Goldblatt, Vesication and some vesicants, Br. J. Ind. Med. 2:183, 1945. 6. G. J. Main, S. J. Bonner, and W. G. Bell, Metabolic markers of chemically-induced cutaneous

irritationhjury. Letterman A m y Inst. of Research Rep. No. 223, Presidio of San Francisco, 1986. 7. S. T. Omaye, J . H. Skala, M. D. Gretz, E. E. Schaus, and C. E. Wade, Simple method for bleeding

the unanesthetized rat by venipuncture, Lab. Anim. 21:261, 1987.

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