OVERVIEW ON DETECTION OF SPECIFIC BIOMARKERS OF LEWISITE EXPOSURE IN BIOMEDICAL SAMPLES

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1 226 Technical Sciences OVERVIEW ON DETECTION OF SPECIFIC BIOMARKERS OF LEWISITE EXPOSURE IN BIOMEDICAL SAMPLES Gabriel EPURE* Nicoleta GRIGORIU* D nu MO TEANU** * Scientific Research Center for CBRN Defence and Ecology, Bucharest, Romania ** Nicolae B lcescu Land Forces Academy, Sibiu, Romania ABSTRACT Identification and quantification of chemical warfare agents (CWA s) and their metabolic products in biomedical samples is an essential component of the complex measures applied to verification of the implementation of the Chemical Weapons Convention (CWC). Intact CWA s are metabolized fairy rapidly in the human body and, therefore, the objects of retrospective detection of exposure to CWA s are their more stable hydrolysis products and protein adducts. Arsenicals are considered a threat, not so much from large nation states but from smaller, less developed nations and/or by terrorist organizations. The relative ease of production coupled with their effectiveness against an unprotected population make organic arsenicals a continued threat in the 21 st century. The paper presents the metabolic products of Lewisite in biomedical samples and analytical methods to verify an exposure to Lewisite. KEYWORDS: lewisite, exposure, biomarkers, detection 1. Generalities Lewisite, an organoarsenic compound, was developed in an attempt to create a more effective blister agent than sulfur mustard. Its development is generally credited to Winford Lee Lewis at the Catholic University (Washington DC) and is based upon a thesis by Julius Arthur Nieuwland (1903) who described the synthesis of Lewisite from arsenic trichloride, acetylene, hydrochloric acid, and mercuric chloride. Early on, the compound was frequently referred to by the vividly descriptive term, Dew of Death. Like sulfur mustard, it is both a vesicant and systemic poison with target tissues not limited to the skin. A low-

2 Technical Sciences 227 dose exposure to skin (14 μg) can cause vesication, and higher doses (30-50 mg/kg) can lead to death. In an attempt to develop an antidote to lewisite, British Anti-lewisite (BAL; dimercaprol) was developed by Peters which later became invaluable in the treatment of arsenic poisoning. Late in World War I and into World War II, large quantities of lewisite were manufactured by Germany, the USA, Italy, the Soviet Union, and Japan. Large amounts of Lewisite were manufactured (up to 2 tons/day by Japan) and stored prior to and during World War II. Lewisite was frequently a component (often mixed with sulfur mustard) in artillery shells and aerial bombs. Like sulfur mustard, there are reports of large amounts of the compound being disposed of at sea. With the possible exception of its use against Iranian soldiers during the Iraq Iran conflict there has been little or no use of lewisite in battle situations. 2. Lewisite Mechanism Action The dominant element in the lewisite structure is arsenic, which is able to react with the sulfhydryl groups of various enzymes, disabling the enzyme in the process. Figure no. 1 depicts the structure of Lewisite and its toxic mechanism. Lipoic acid, a component of pyruvate oxidase, is particularly susceptible to this reaction and one of the most evident consequences is the inhibition of the enzyme pyruvate dehydrogenase (PDH), rapidly disabling the cell s metabolism of glucose and fatty acids. The resulting energy deficiency may lead to swift necrotic cell death. In comparison to sulfur mustard, the latency period (from exposure to first signs and symptoms) is significantly shorter and Lewisite injuries have been described as extremely painful from an early stage. Moreover, it is a much more lethal agent and has a large systemic toxicity; 0.5 ml of the agent may produce systemic effects, whereas 2 ml (approximately 3.6 g) can be fatal. Several other known enzymes are also inhibited alcohol dehydrogenase, succinic oxidase, hexokinase, and succinate dehydrogenase. Figure no. 2 summarizes the metabolism and reversal of adduct formation by BAL. Figure no. 1 Lewisite, its structure and mechanism of action. Lewisite forms covalent bonds with lipoic acid, inactivating the enzyme pyruvate dehydrogenase

3 228 Technical Sciences Figure no. 2 Lewisite metabolism, adduct formation, and its reversal 3. Methods to Verify an Exposure to Lewisite Unlike other CWA s, Lewisite has not been a focus of researchers over the recent decades, even though tons of lewisite and mustard-lewisite mixtures are still buried in unknown places as old chemical weapons left after World War II [1]. Lewisite hydrolyses to form 2-chlorovinylarsonous acid (CVAA), which is the hydrated form of 2-chlorovinylarsine oxide and exists exclusively in aqueous solution. CVAA can be further oxidized to 2-chlorovinylarsonic acid (CVAOA). These two acids are lewisite markers, i.e., their detection in biomedical samples provides unequivocal evidence for exposure to lewisite. Both CVAA and CVAOA still retain the toxic properties of Lewisite, albeit at lower potency [2-5]. The literature concerning sensitive detection of CVAA and CVAOA in biomedical samples is fairly scarce. Specific biomarkers of lewisite exposure are currently based on a very limited number of in-vitro experiments and animal studies. Jakubowski et al. [6] developed a GC/MS method for CVAA spiked into guinea pig urine using 1,2-ethanedithiol for derivatization, with phenyl arsine oxide as the internal standard. The same group later expanded the method to include atomic emission detection (AED) [7]. CVAA was concentrated from urine (adjusted to ph 6 with 1M HCl) by SPE on C 18. After elution with methanol and concentration to dryness, the residue was reconstituted and derivatized with ethanolic 1,2- ethanedithiol. Detection was by GC combined with arsenic selective AED and by electron impact/mass spectrometry (EI/MS) using SIM. Ions monitored were the moderately intense M + ion at m/z 228, an intense ion [M C 2 H 4 ] +, m/z 200, and a base peak [AsS 2 C 2 H 4 ] +, m/z 167. The LOD was rather high at ~ 100 ng/ml. CVAA was detected in the urine of guinea pigs up to 24 h after exposure to lewisite (0.5 mg/kg s.c.). Excretion was rapid; a mean concentration of 3.5 ug/ml was detected in the 0-8 h urine, which had decreased to 100 ng/ml after h.

4 Technical Sciences 229 Wooten et al. [8] analyzed CVAA from urine after derivatization with propane-1,3-dithiol (PDT) and the resulting volatile cyclic disulfide was detected by solid phase microextraction (SPME) combined with GC-MS in the SIM mode, but the procedure was not tested on samples from in vivo experiments. The detection limit of CVAA was 7.4 pg/ml urine. In one study of four animals [7], guinea pigs were given a subcutaneous dose of Lewisite (0.5 mg/kg). Urine samples were analyzed for CVAA using both GC- MS and GC coupled with an atomic emission spectrometer set for elemental arsenic. The excretion profile indicated a very rapid elimination of CVAA in the urine. The mean concentrations detected were 3.5 g/ml, 250 ng/ml, and 50 ng/ml for the 0 to 8-hour, 8 to 16-hour, and 16 to 24-hour samples, respectively. Trace level concentrations (0-10 ng/ml) of CVAA were detected in the urine of the 24 to 32 hour and 32 to 40 hour samples. Following the incubation of human blood with radiolabeled lewisite, Fidder et al. [9] found that 90 % of the radioactivity was associated with the RBCs, and 25 % to 50% was found with the globin. Because of CVAA s reactive nature, derivatization using a thiol compound has generally been applied as part of the sample preparation process (Figure no. 3) [10]. Figure no. 3 Published analytical approaches for the analysis of chlorovinylarsonous acid in urine. (a) Reaction of lewisite (trans isomer shown) with water to form chlorovinylarsonous acid; (b) Reactions of chlorovinylarsonous acid with thiol compounds ethanedithiol, propanedithiol, and British anti-lewisite [7-9] (c) Derivatization using HFBI [9]

5 230 Technical Sciences The developed procedure of Koryagina et al. [11] was based on the researches of Wooten, but applied to the analysis of urine samples obtained in experiments in vivo and also adapted to the analysis of blood plasma and red blood cells (RBCs). Koryagina et al. [12] explored in their research the possibility of determination of CVAA in the blood plasma, red blood cells and urine of animals after exposure to Lewisite with and without antidotal therapy. The procedure that involved derivatization of the CVAA with propane-1,3-dithiol and head-space SPME of the derivative on 100- μm polydimethylsiloxane fiber followed by GC-MS was applied for the first time of in vivo samples. The detection limits of CVAA in urine, plasma and red blood cells were 0.1, 1.0, and 10 ng/ml, respectively. Upon exposure to lewisite at a dose of 1.6 mg/kg, CVAA could be detected in rat urine for about 3 months, and in red blood cells 7 days after. Study of the effect of a single injection of the antidote unithiol - 2,3-dimercapto-1-propanesulfonate on the CVAA excretion profile revealed more active CVAA excretion during the first 2 days after the injection, compared to that observed in the absence of antidotal therapy. 4. Conclusions The toxicological experiments showed that urine is a universal and preferential matrix for the determination of lewisite biomarkers. Blood (red blood cells) could be a perspective matrix for confirmation of the results of urine analysis. Conversely, blood plasma is considered a non-perspective matrix in terms of establishment of exposure to Lewisite, since the blood plasma levels of the biomarker are relatively low. 2-chlorovinylarsonic acid is the key marker of exposure to high doses of lewisite, whereas 2-chlorovinylarsonous acid is primarily detected in biomedical samples on low-dose exposure. In terms of detection of exposure of a human to lewisite, combined determination of CVAOA and CVAA in biomedical samples treated with a reducer is a reasonable approach, since both these compounds are absolute markers of lewisite. However, separate determination of CVAOA and CVAA is important for understanding the metabolism of Lewisite and for developing schemes of antidotal therapy. REFERENCES 1. N.L. Koryagina, E.S. Ukolova, E.I. Savel eva, N.G. Voitenko, O.I. Orlova, R.O. Jenkins and N.V. Goncharov, High-Sensitivity Determination of 2-Chlorovinylarsonous Acid in Biomedical Samples for Retrospective Detection of Exposure to Lewisite Upon Antidotal Therapy, Spectroscopy 26, Hindawi Publishing Corporation, Egypt, (2011): S.L. Bartlet-Hunt, D.R.U. Knappe and M.A. Barlaz, A Review of Chemical Warfare Agent Simulants for the Study of Environmental Behavior, Critical Reviews Environmental Science and Technology, 38, Taylor & Francis Group, (2008): B.A. Tomkins, G.A. Sega and C.-H. Ho, Determination of Lewisite Oxide in Soil Using Solid Phase Microextraction Followed by Gas Chromatography With Flame Photometric or Mass Spectrometric Detection, Journal of Chromatography A, 909, Elsevier, USA, (2001): N.B. Munro, S.S. Talmage, G.D. Griffin, L.C. Waters, A.P. Watson, J.F. King and V. Hauschild, The Sources, Fate, and Toxicity of Chemical Warfare Agent Degradation Products, Environmental Health Perspectives, 107, National Institute of Environmental Health Sciences, USA, (1999):

6 Technical Sciences W.K. Fowler, D.C. Stewart and D.S. Weinberg, Gas Chromatographic Determination of the Lewisite Hydrolysate, 2-Chlorovinylarsonous Acid, After Derivatization with 1,2-Ethanedithiol, Journal of Chromatography A, 558, Elsevier, USA, (1991): E.M. Jakubowski, J.R. Smith, T.P. Logan, N.D. Wiltshire, C.L. Woodard, R.A. Evans and T.W. Dolzine, Verification of Lewisite Exposure: Quantification of Chlorovinyl Arsonous Acid in Biological Samples, Proceedings of the 1993 Medical Defense Bioscience Review, May, 1993, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, (1993): T.P. Logan, J.R. Smith, E.M. Jakubowski and R.E. Nielson, Verification of Lewisite Exposure by the Analysis of 2-Chlorovinyl Arsonous Acid in Urine, Toxicology Mechanisms and Methods, 9, United Kingdom, (1999): J.V. Wooten, D.L. Ashley, and A.M. Calafat, Quantification of 2- Chlorovinylarsonous Acid in Human Urine by Automated Solid Phase Microextraction Gas Chromatography Mass Spectrometry, Journal of Chromatography B, 772, Elsevier, USA, (2002): A. Fidder, D. Noort, A.G. Hulst, L.P.A. de Jong and H.P. Benschop, Biomonitoring of Exposure to Lewisite Based on Adducts to Haemoglobin, Archives of Toxicology, 74, Springer Berlin Heidelberg, (2000): B.R. Capacio, J.R. Smith, R.K. Gordon, J. R. Haigh, J.R. Barr and G.E. Platoff. Medical diagnostics, Medical aspects of chemical warfare, USA, (2012): N.L. Koryagina, E.S. Ukolova, E.I. Savel eva, N.G. Voitenko, O.I. Orlova, R.O. Jenkins and N.V. Goncharov, cit. ed. 12. Ibidem. BIBLIOGRAPHY Bartlet-Hunt, S.L., D.R.U. Knappe, and M.A. Barlaz. A review of chemical warfare agent simulants for the study of environmental behavior. Critical Reviews Environmental Science and Technology, 38, Taylor & Francis Group, (2008): Capacio, B.R., J.R. Smith, R.K. Gordon, J.R. Haigh, J.R. Barr, and G.E. Platoff. Medical diagnostics. Medical aspects of chemical warfare, USA, (2012): Fidder, A., D. Noort, A.G. Hulst, L.P.A. de Jong, and H.P. Benschop. Biomonitoring of Exposure to Lewisite Based on Adducts to Haemoglobin. Archives of Toxicology, 74, Springer Berlin Heidelberg, (2000): Fowler, W.K., D.C. Stewart, and D.S. Weinberg. Gas chromatographic determination of the Lewisite Hydrolysate, 2-Chlorovinylarsonous Acid, After Derivatization with 1,2- Ethanedithiol. Journal of Chromatography A, 558, Elsevier, USA, (1991); Jakubowski, E.M., J.R. Smith, T.P. Logan, N.D. Wiltshire, C.L. Woodard, R.A. Evans, and T.W. Dolzine. Verification of Lewisite Exposure: Quantification of Chlorovinyl Arsonous Acid in Biological Samples. Proceedings of the 1993 Medical Defense Bioscience Review, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, (10-13 May, 1993): Koryagina, N.L., E.S. Ukolova, E.I. Savel eva, N.G. Voitenko, O.I. Orlova, R.O. Jenkins, and N.V. Goncharov. High-sensitivity Determination of 2-Chlorovinylarsonous Acid in Biomedical Samples for Retrospective Detection of Exposure to Lewisite Upon Antidotal Therapy. Spectroscopy 26, Hindawi Publishing Corporation, Egypt, (2011): 1-10.

7 232 Technical Sciences Logan, T.P., J.R. Smith, E.M. Jakubowski, and R.E. Nielson. Verification of Lewisite Exposure by the Analysis of 2-Chlorovinyl Arsonous Acid in Urine. Toxicology Mechanisms and Methods, 9, United Kingdom, (1999): Munro, N.B., S.S. Talmage, G.D. Griffin, L.C. Waters, A.P. Watson, J.F. King, and V. Hauschild. The Sources, Fate, and Toxicity of Chemical Warfare Agent Degradation Products. Environmental Health Perspectives, 107, National Institute of Environmental Health Sciences, USA, (1999): Tomkins, B.A., G.A. Sega, and C.-H. Ho. Determination of Lewisite Oxide in Soil Using Solid Phase Microextraction Followed by Gas Chromatography with Flame Photometric or Mass Spectrometric Detection. Journal of Chromatography A, 909, Elsevier, USA, (2001): Wooten, J.V., D.L. Ashley, and A.M. Calafat. Quantification of 2-Chlorovinylarsonous Acid in Human Urine by Automated Solid-Phase Microextraction Gas Chromatography Mass Spectrometry. Journal of Chromatography B, 772, Elsevier, USA, (2002):