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Modified immunoslotblot assay to detect hemi and sulfur mustard DNA adducts
Chemico-Biological Interactions 206 (2013) 523–528
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Modified immunoslotblot assay to detect hemi and sulfur mustard DNA adducts Kai Kehe a,b,⇑, Verena Schrettl a,c, Horst Thiermann a, Dirk Steinritz a,b a
Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstr. 11, 80937 Munich, Germany Walther Straub Institute of Pharmacology and Toxicology, Ludwig Maximilians University of Munich, Goethestraße 33, 80336 Munich, Germany c Toxicological Department, Klinikum rechts der Isar, Technical University of Munich, Ismaninger Strasse 22, 81675 Munich, Germany b
a r t i c l e
i n f o
Article history: Available online 8 August 2013 Keywords: Sulfur mustard Nitrogen mustard Chemical warfare agents Vesicants
a b s t r a c t Sulfur mustard (SM) is an old chemical warfare agent causing blisters (vesicant). Skin toxicity is thought to be partly caused by SM induced DNA damage. SM and the hemi mustard 2-chloroethyl ethyl sulfide (CEES) are bi- and monofunctional DNA alkylating agents, respectively. Both chemicals react especially with N7 guanine. The most abundant adducts are 7-hydroxyethylthioethylguanine for SM (61%) and 7ethyl thioethylguanine for CEES. Thus, DNA alkylation should serve as a biomarker of SM exposure. A specific monoclonal antibody (2F8) was previously developed to detect SM and CEES adducts at N7 position by means of immunoslotblot (ISB) technique (van der Schans et al. (2004) [16]). Nitrogen mustards (HN1, HN-2, HN-3) are alkylating agents with structural similarities, which can form DNA adducts with N7 guanine. The aim of the presented work was to modify the van der Schans protocol for use in a field laboratory and to test the cross reactivity of the 2F8 antibody against nitrogen mustards. Briefly, human keratinocytes were exposed to SM and CEES (0–300 lM, 60 min) or HN-1, HN-2, HN-3 (120 min). After exposure, cells were scraped and DNA was isolated and normalized. 1 lg DNA was transferred to a nitrocellulose membrane using a slotblot technique. After incubation with 2F8 antibody, the DNA adducts were visualized with chromogen staining (3,30 -diaminobenzidine (DAB), SeramunGrün). Blots were photographed and signal intensity was quantified. In general, DAB was superior to SeramunGrün stain. A staining was seen from 30 nM to 300 lM of SM or CEES, respectively. However, statistically significant DNA adducts were detected after CEES and SM exposure above 30 lM which is below the vesicant threshold. No signal was observed after HN-1, HN-2, HN-3 exposure. The total hands-on time to complete the assay was about 36 h. Further studies are necessary to validate SM or CEES exposure in blister roofs of exposed patients. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Sulfur mustard was used as a chemical warfare agent since 1917. Due to its strong vesicating properties on skin, it is classified as a blistering agent [1]. Sulfur mustard, either as liquid or gas, is a lipophilic compound that can easily penetrate in the body. Exposure to sulfur mustard does not cause immediate signs of intoxication. A symptomless or -free period of several hours was frequently reported. The first symptoms are observed on the eyes, skin and in the bronchial tract [2]. SM is classified as a vesicant agent because of its strong capability to induce blistering on the skin. The epidermis detaches from the dermis. Keratinocyte cell death is detectable ⇑ Corresponding author at: Walther Straub Institute of Pharmacology and Toxicology, Ludwig Maximilians University of Munich, Goethestraße 33, 80336 Munich, Germany. Tel.: +49 89 1249 7950; fax: +49 89 3168 2333. E-mail addresses: [email protected], [email protected] (K. Kehe), [email protected] (V. Schrettl), [email protected] (H. Thiermann), [email protected] (D. Steinritz). 0009-2797/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbi.2013.08.001
within the stratum basale [3]. Eye symptoms involve redness of the eyes, swollen eyelids, tearing, pain, sensitivity to light and severe blepharospasm [4]. Inhalation of sulfur mustard vapor damages epithelial surfaces of the laryngeal and bronchial mucosa. Acute symptoms and signs are hoarseness, sneezing, lacrimation, hacking cough and in severe cases pseudomembrane formation in the bronchial tract [5,6]. Nevertheless, exposure to sulfur mustard results often in systemic poisoning with nausea, vomiting and leucopenia at a later stage. The clinical picture has been extensively reviewed in the past [6–8]. The continuous threat of poisoning has prompted a huge research program in the last century. Despite this effort, the pathophysiology of sulfur mustard poisoning is still not completely understood. It is widely accepted that the reaction of SM with the DNA is one if not the main pathophysiological event that triggers the cell death cascade. SM is a highly reactive bifunctional alkylating agent. It hydrolyzes in water to form an ethylene sulfonium ion intermediate. A highly reactive carbenium ion is formed,
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which is highly reactive. Cell constituents like DNA, RNA, proteins, and other molecules are rapidly alkylated [1]. SM reactions with DNA lead to mono- or bifunctional adducts with guanine and adenosine. Reactions with thymine or uracil were not detected under physiological conditions. SM preferentially reacts with ring nitrogen atoms of the DNA in decreasing order of reactivity with N7 of guanine, N3 of adenine, N1 of adenine and N1 of cytosine [9]. Thus, 61% of all alkylations occur at the N7 of guanine [10]. Nearly 17% of total alkylations produce intra- or interstrand-crosslinks [11]. The genotoxic properties of SM have been recognized to be the most important trigger of cell death [12]. The strong cytotoxic properties of SM against rapidly dividing cells were used against cancer cells. Several mustards were synthesized and screened for their usefulness as chemotherapeutic drugs. Initially, several N-mustards were introduced in anti-cancer therapy and used as starting substance for other chemotherapeutic drugs [13]. Nitrogen mustards (HN-1, HN-2, HN-3) were also synthesized as chemical warfare agents. Retrospective diagnosis and verification of SM poisoning may be difficult. The portion of SM that does not penetrate very rapidly through the skin evaporates quickly from the surface [14]. Off-gassing of SM vapor has been detected up to 24 h after exposure in pig models, and a reservoir of agent has been noted in the upper levels of the stratum corneum of pigs [15]. Rapid methods for reliable and fast detection of SM poisoning are needed to initiate appropriate clinical treatment. Sophisticated analytical methods may be useful to confirm a possible SM intoxication with high evidence. Recently, an immunochemical method was described to detect the monofunctional SM adduct at the N7 position of guanine [15]. A substantial portion of SM that has penetrated the skin is fixed at the DNA mainly within the epidermis. It is hypothesized that extracted DNA of blister roofs or affected skin contain sufficient amounts of reacted SM to be detectable with the above mentioned antibody. The aim of this study was to modify the immunoslotblot technique and to test the cross-reactivity of the 2F8 antibody against SM, semi mustard (CEES) and nitrogen mustards (HN-1, HN-2, HN-3). 2. Materials and methods 2.1. Chemicals SM was obtained from TNO, Rijswijk, The Netherlands. All other chemicals used were reagent grade products obtained from Sigma (Deisenhofen, Germany). SeramunGrün was from Seramun Diagnostica, Heidesee, Germany. The tested sulfur or nitrogen mustards are listed in Table 1. 2.2. Cell cultures HaCaT cells, a spontaneously transformed human keratinocyte cell line [16,17], was kindly provided by Prof. Dr. N. Fusenig (German Cancer Research Center, Heidelberg, FRG). HaCaT cells were cultured in Dulbecco’s Modified Eagle Medium/Ham’s F12 (DMEM/F12) supplemented with 2.45 mmol/l glutamine, 10% fetal calf serum (Life Technologies, Eggenstein, FRG) using standard cell culture flasks (75 cm2, Falcon, Heidelberg, FRG). Cultures were maintained at 37 °C in a humidified atmosphere with 5% CO2. Cell doubling was 22 h. Cells were seeded with 105 cells/cm2 and experiments were performed with subconfluent cultures on the 1st day after seeding. 2.3. Exposure protocol HaCaT cells were washed with PBS and incubated at 25 °C for 60 min with various concentrations of SM or CEES, or for 120 min with various concentrations of HN-1, HN-2, or HN-3 in
Table 1 Structures of tested chemical compounds. chemical compound
structure
CEES CAS-Nr. 693-07-2 sulfur mustard CAS-Nr. 505-60-2 HN-1 (Chlormethine) CAS-Nr. 51-75-2
HN-2 (Mustine, Mustargen) CAS-Nr. 107-99-3
HN-3 (Trimustine) CAS-Nr. 817-09-4
Modified Eagle’s Medium (MEM). SM was dissolved first in ethanol. The final concentration of the solvent in the exposure fluids was below 1%. Control cultures were exposed to the same amount of ethanol. The obtained stock solutions of SM, CEES and nitrogen mustards in MEM were used immediately after dilution to minimize hydrolysis. After exposure, HaCaT cells were washed with PBS and subjected to analysis. Every experiment was repeated three times and each immunoslotblot consists of three replicate experiments (n = 9). 2.4. Cell isolation HaCaT cells were treated with 2 ml of 0.05%-Trypsin–EDTA (GIBCOÒ invitrogen) and incubated at 37 °C until trypsination was complete. The HaCaT cells were diluted with 13 ml of Dulbecco’s Modified Eagle Medium (DMEM) containing FKS and transferred to 50 ml tubes, from which 100 ll samples were taken for cell count (CASY Modell TTC, Innovatis Systems, Reutlingen). The tubes were centrifuged for 5 min at 1350 g (ROTINA35 R, Hettich Zentrifugen, Tübingen) and the supernatant removed. The remaining HaCaT cells were re-suspended in DMEM to a concentration of 5–10 106 cells/ml. From each sample 200 ll were transferred to 1.5 ml sample tubes and centrifuged for 5 s at 16.1 g (Centrifuge 5415 R, Eppendorf, Hamburg). The supernatant medium was removed and the sample tubes were vortexed. 2.5. DNA isolation DNA was isolated from the HaCaT cells using the Puregene Core Kit A (QIAGEN, Hilden, Germany). Cell lysis solution (300 ll) was added to the isolated cells and vortexed for 10 s. 1.5 ll RNase A (50 mg/ml) was added, the mixture was incubated for 15 min at 37 °C and cooled at 0 °C for 1 min. Protein precipitation solution (100 ll) was added to the mixture, vortexed for 20 s and centrifuged for 5 min at 16.1 g. The supernatant was carefully decanted from theformed pellet into isopropanol (300 ll). The obtained solution was mixed by mild shaking, and centrifuged for 5 min at 16.1 g. The supernatant was decanted from the formed pellet, ethanol (300 ll, 70%) was added to the pellet and again centrifuged for 5 min at 16.1 g. After decantation of the supernatant the samples were dried for 15 min at room temperature, DNA Hydration Solution (50 ll) was added, the mixture was incubated for 1 h at
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65 °C, and mixed on a shaker (2/4 digital, IKAÒ MTS, Staufen) over night at moderate speed. The DNA concentration was determined with the NanoDropÒ by using 1 ll of the obtained DNA solutions (measurements at 280 nm and 260 nm). 2.6. DNA denaturation DNA-denaturation-buffer-solution was prepared from formamide (8 ll), formaldehyde (164 lL; 50%), and 1828 lL Tris/acetic acid/EDTA buffer (Bio-Rad Laboratories GmbH, Munich, Germany) and added to the DNA samples to give a final DNA concentration of 50 lg/ml. These samples were incubated at 52 °C for 20 min, cooled at 0 °C for 1 min, frozen at 80 °C, and defrosted at room temperature. 2.7. Immunoslot blot technique Denaturated DNA from the previous step was diluted in PBS to a final concentration of 5 lg/ml and 200 ll of each sample were pipetted into a slot of the blotting apparatus (Blotting Unit SHM48) equipped with a nitrocellulose membrane (Nitrocellulose Transfer Membrane: Protran BA 79, Pore size 0.1 lm; Whatman, Dassel, Germany) and a blotting paper. The DNA was blotted with a vacuum of 350 mbar onto the membrane after which each slot was washed with PBS (300 ll). The membrane was air-dried for 15 min and subsequently the DNA was fixated onto the membrane for 2 h at 80 °C. Thereafter, the membrane was washed twice with a 1:100 SCC buffer (SCC buffer 20 (3 M NaCl, 0.3 M Na-Citrat, pH 7.0)) for 15 min on a horizontal shaker at moderate speed and room temperature. After washing, the membrane was incubated for 20 min with Protein block (Protein Block Serum-free, Dako, Hamburg, Germany). 2.8. Incubation with the 2F8 Antibody The 2F8 antibody (2F8 Antibody purified Ab 246, 1.05 mg/ml, BBI Dundee) was diluted with Antibody Diluent (Dako) to give a concentration of 50.25 ng/ml 2F8. The membrane was covered with this antibody solution and incubated over night at 4 °C under continued pivoting. On the next day, the membrane was washed using PBS with 0.1% Tween 20 (Tween 20 Sigma) at room temperature on a shaker according to the following procedure: the membrane was washed four times for 15 min, incubated with the HRPLink (Dako, Hamburg, Germany) for 30 min, washed again four times for 10 min, incubated for 30 min with the HRP-Enzyme DakoÒ ADVANCE™, and finally washed four times for 10 min. For staining, the membrane was treated with the corresponding dye solutions. DAB stock solution (50 ll) or SeramunGrünÒ working solution (5 ml; ready to use) was filtered (0.4 lm pore size) and added to the membrane, respectively. Then the membrane was washed three times for 10 min with distilled water and air-dried.
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2.10. Analysis of data Statistical differences were assessed with the Mann–WhitneyU-Test (PASW Statistics Version 18). All results from each concentration of each staining agent were compared to the negative control (C). The value of p was determined with p < 0.05 and p > 0.05 indicating statistically significant difference. 3. Results 3.1. Sulfur mustard (SM) N7-DNA-adducts of SM were detectable with DAB and SeramunGrünÓ. Fig. 1 shows a typical membrane after immunoslotblot and staining with DAB. A concentration dependent increase of signal intensity could be detected visually. With densitometry of DAB stained membranes, significant different signals were detected with concentrations down to 0.3 lM SM in comparison to sham treated controls (p < 0.05) (Fig. 2). The dye SeramunGrünÓ showed a similar intensity compared to DAB (median, 147.0 AU vs. 156.9 AU) at 100 lM SM (Fig. 2). With densitometry, the SeramunGrünÓ stained membranes gave significant signals down to 1 lM SM when compared to sham treated controls (p < 0.05). 3.2. CEES N7-DNA-adducts of CEES were detectable with both staining dyes, DAB and SeramunGrünÓ, from 30 lM to 300 lM (Fig. 3). CEES concentrations equal to and below 10 lM of CEES showed only a faint staining. DAB staining was more intense and easier to identify than SeramunGrünÓ staining. This observation was underlined by densitometry. When CEES was incubated with SeramunGrünÓ, the signal intensity was 2-fold lower than after DAB incubation with 300 lM of CEES (median, 68.3 AU vs. 123.8 AU). DAB stained blots showed a better signal to noise ratio compared to SeramunGrünÓ. 3.3. Nitrogen mustards (HN-1, HN-2, HN-3) N7-DNA-adducts of HN-1, HN-2 or HN-3 were not detectable with both staining dyes, DAB and SeramunGrünÓ, up to 300 lM in our experimental setup. A slight but not significant increase of
2.9. Analysis of the membrane The membrane was scanned with a Image Scanner III (GE Healthcare; LabScan™ 6.0 Powered by Melanie™, Swiss Institute of Bioinformatics). The settings were 150 dpi for the resolution. The scanned image was inverted and transformed into a Tif file with Image J 1.42 (Wayne Rasband, NIH). The resulting image was analyzed densitometrically with AndorIQ 1.9.1 (Bioimaging, UK). After background subtraction, gray values of each pixel were summed and the resulting value was referred to the area of measurement (mm2). For comparison, the slots on the negative image and an area of the background with as little staining as possible were marked with a fixed rectangle. The results were given in arbitrary units.
Fig. 1. Immunoslotblot of SM induced DNA adducts detected with 2F8 antibody in HaCaT cells. Following exposure with SM (0.1–100 lM, 60 min) cells were washed and DNA was extracted, blotted, immunostained with 2F8 antibody and visualized with DAB.
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Fig. 2. Monofunctional DNA adducts detected with 2F8 antibody in HaCaT cells after SM exposure. Following exposure with SM (0.1–100 lM, 60 min) cells were washed and DNA was extracted, blotted, immunostained with 2F8 antibody and visualized either with DAB or SeramunGrün followed by densitometry. ⁄p < 0.05; Mann–Whitney-U-Test.
Fig. 3. Monofunctional DNA adducts detected with 2F8 antibody in HaCaT cells after CEES exposure. Following exposure with CEES (0.3–300 lM, 60 min) cells were washed and DNA was extracted, blotted, immunostained with 2F8 antibody and visualized either with DAB or SeramunGrün followed by densitometry. ⁄p < 0.05; Mann–Whitney-UTest.
signal intensity was observed for HN-3 after SeramunGrünÓ staining (data not shown). 3.4. Hands-on time of the immunoslotblot procedure The total hands-on time to complete the assay was about 36 h. The two overnight steps were used to denaturate the DNA and for incubation with the first antibody (2F8). 4. Discussion Blister agents or vesicants are chemical warfare agents that produce pronounced skin symptoms ranging from erythema to large bullae. Within the group of blister agents three subgroups are listed: mustard agents, lewisite, phosgene oxime. The last produces more likely urticaria than vesicle. Blister agents are still a threat
until today and have indeed been used in various military conflicts starting from 1917. SM is the only large scale used blister agent until today [2]. After World War I, several programs were initiated to identify substances similar to SM with blister inducing properties. SM has two reactive chlorethyl groups. An analogue with one reactive chlorethyl group is CEES (half mustard) which produces solely monofunctional DNA adducts and has been used as a SM analogue in various studies [18–21]. HN-1, HN-2 and HN-3 are nitrogen mustards, which are listed as chemical warfare agents [22]. However, HN-1 was used as a wart remover, HN-2 is also known as Mustine, a pharmaceutical compound to treat cancer [23]. HN-3 has no medical use so far. Taken together, SM related substances have one or more chlorethyl groups attached to nitrogen (nitrogen mustards) and are less toxic than SM [24,25]. Thus, it is likely that they share some similarities in their properties to damage DNA. In fact, sulfur and nitrogen
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mustards attack predominately guanine at the N7-position [26– 28]. This alkylation site was used to develop an antibody against this specific adduct. The antigen 7-hydroxyethylthioethyl-guanine (HETEG) was synthesized and a monoclonal antibody (2F8) was developed and recently characterized [29]. The 2F8 antibody was successfully applied in toxicokinetic studies [30] and for verification of SM exposure in blood of Iranian patients by means of immunoslotblot technique [31]. More recently, the immunoslotblot technique was optimized and a standard operating procedure was published [32]. However, the published techniques used fluorescent methods to visualize and quantify the signals. The advantage of fluorescent detection is a good signal to noise ratio. The disadvantage is the need for a more complex imaging system. Generally, detection using fluorescent substrates is 2–10-fold more sensitive than chromogenic detection [16]. In a military setting or at rural laboratories less complicated methods are needed. To achieve this goal, we have replaced the fluorescent label by a horseradish peroxidase system, which allows staining techniques with chromogenic dyes. In the presented study, we used the two chromogenic dyes DAB and SeramunGrün. DAB precipitates in the presence of hydrogen peroxide that is produced by peroxidase. DAB is widely used in various immunochemical protocols, which produces a brown color. SeramunGrün comprises is a ready-to-use substrate solutions for horseradish peroxidase. During the catalytic decomposition of hydrogen peroxide electrons are transferred from 3,30 5,50 -Tetramethylbenzidine to o-Dianisidine (o-Dia) by horseradish peroxidase. Oxidation of o-Dia leads to formation of dimers with deep green color. SeramunGrün is a substrate optimized for use in blotting systems with visual assessment. Both reagents proved to be suitable for the detection of SM or CEES DNA adducts. SeramunGrün showed a less intense signal in our hands compared to DAB and consecutively leading to a decreased sensitivity. Taken together, DAB is superior to SeramunGrün in the presented immunoslotblot assay. DNA adducts induced by nitrogen mustards (HN-1, HN-2, HN-3) were not detected in the immunoslotblot assay. This may be due to a different antigen structure, which is not detectable by the 2F8 antibody. On the other hand, nitrogen mustards react more slowly with cellular DNA than SM [27,33]. Thus, it is possible that DNA adduct formation has not reached a detectable amount of mono-adducts. This hypothesis needs further investigations, which was beyond the scope of our study. Only a small but not significant signal increase was seen after blotting DNA of HN-3 treated cells and SeramunGrünÓ staining, which may be a hint to some DNA alkylation. On the other hand, SeramunGrünÓ was not as sensitive as DAB. Therefore, this signal was considered as an artifact. Taken together, skin contact with nitrogen mustards less than 2 h will not result in detectable DNA adducts in the presented immunoslotblot assay. The detection level of SM DNA adducts was 0.3 lM in our experimental setup. In a previous study, SM poisoned Iranian soldiers showed levels of alkylation in leukocytes comparable to 0.9 lM SM [35]. Thus, the achieved level of detection was sensitive enough to detect SM induced DNA alkylation even in the blood of severely SM poisoned victims. As SM concentrations at the site of body entry are likely to be much higher, the presented method is suitable to verify skin exposure to SM. Skin blistering is the main clinical symptom of SM poisoning [2]. Histopathologically, this macroscopic picture corresponds to apoptotic cell death in the epidermal stratum basale [34]. In vitro, keratinocyte apoptosis is observed after 100 lM SM exposure [35–38]. Thus, for meaningful studies 100 lM SM can be considered as an equivalent blister inducing SM concentration in vitro [38,39]. The presented immunoslotblot assay has a detection limit for SM exposure that is more than 300-fold below the vesicant dose. The hands-on time was
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36 h. Further studies are needed to reduce the time. However, the presented method is simple and does not need sophisticated equipment (GC–MS, LC–MS) compared to the detection of SM metabolites in blister fluid and urine, or adducts on proteins found in the blood [39]. Our study showed a simple method to detect full and hemimustard induced DNA adducts in human keratinocytes with high sensitivity and specificity in vitro. Further studies are necessary to validate this method for skin samples and to confirm the relevance for in vivo exposure. Source of funding H.T. received fund from German Ministry of Defense. Conflict of interest statement None declared. References [1] B. Papirmeister, C.L. Gross, H.L. Meier, J.P. Petrali, J.B. Johnson, Molecular basis for mustard-induced vesication, Fundam. Appl. Toxicol. 5 (1985) S134–49. [2] K. Kehe, L. Szinicz, Medical aspects of sulphur mustard poisoning, Toxicology 214 (2005) 198–209. [3] B. Papirmeister, C.L. Gross, J.P. Petrali, C.J. Hixson, Pathology produced by sulfur mustard in human skin grafts on athymic nude mice. I. Gross and light microscopic changes, J. Toxicol. Cutaneous Ocul. Toxicol. 3 (1984) 371–391. [4] Y. Solberg, M. Alcalay, M. Belkin, Ocular injury by mustard gas, Surv. Ophthalmol. 41 (1997) 461–466. [5] A. Emad, G.H. Rezaian, The diversity of the effects of sulfur mustard gas inhalation on respiratory system 10 years after a single, heavy exposure: analysis of 197 cases, Chest 112 (1997) 734–738. [6] M. Balali-Mood, M. Hefazi, Comparison of early and late toxic effects of sulfur mustard in Iranian veterans, Basic Clin. Pharmacol. Toxicol. 99 (2006) 273– 282. [7] K. Kehe, H. Thiermann, F. Balszuweit, F. Eyer, D. Steinritz, T. Zilker, Acute effects of sulfur mustard injury-Munich experiences, Toxicology 263 (2009) 3– 8. [8] M. Balali-Mood, A. Navaeian, B. Heyndrickx. Clinical and paraclinical findings in 233 patients with sulfur mustard poisoning. In: Proceedings of the Second World Congress on New Compounds in Biological and Chemical Warfare (1986) 464–473. [9] P.D. Lawley, P. Brookes, Further studies on the alkylation of nucleic acids and their constituent nucleotides, Biochem. J. 89 (1963) 127–138. [10] D.B. Ludlum, W.P. Tong, J.R. Mehta, M. Kirk, B. Papirmeister, Formation of O6ethylthioethyldeoxyguanosine from the reaction of chloroethyl ethyl sulfide with deoxyguanosine, Cancer Res. 44 (1984) 5698–5701. [11] D.B. Ludlum, B. Papirmeister, DNA modification by sulfur mustards and nitrosoureas and repair of these lesions, Basic Life Sci. 38 (1986) 119–125. [12] I.J. Lodhi, J.F. Sweeney, R.E. Clift, D.B. Hinshaw, Nuclear dependence of sulfur mustard-mediated cell death, Toxicol. Appl. Pharmacol. 170 (2001) 69–77. [13] L.S. Goodman, M.M. Wintrobe, W. Dameshek, M.J. Goodman, A. Gilman, M.T. McLennan, Landmark article Sept. 21, 1946: nitrogen mustard therapy. Use of methyl-bis(beta-chloroethyl)amine hydrochloride and tris(betachloroethyl)amine hydrochloride for Hodgkin’s disease, lymphosarcoma, leukemia and certain allied and miscellaneous disorders. By Louis S. Goodman, Maxwell M. Wintrobe, William Dameshek, Morton J. Goodman, Alfred Gilman and Margaret T. McLennan, JAMA 251 (1984) 2255–2261. [14] R.P. Chilcott, J. Jenner, W. Carrick, S.A. Hotchkiss, P. Rice, Human skin absorption of bis-2-(chloroethyl)sulphide (sulphur mustard) in vitro, J. Appl. Toxicol. 20 (2000) 349–355. [15] I.J. Hattersley, J. Jenner, C. Dalton, R.P. Chilcott, J.S. Graham, The skin reservoir of sulphur mustard, Toxicol. In Vitro 22 (2008) 1539–1546. [16] G.P. van der Schans, A.G. Scheffer, R.H. Mars-Groenendijk, A. Fidder, H.P. Benschop, R.A. Baan, Standard operating procedure for immunuslotblot assay for analysis of DNA/sulfur mustard adducts in human blood and skin, J. Anal. Toxicol. 28 (2004) 316–319. [17] P. Boukamp, R.T. Petrussevska, D. Breitkreutz, J. Hornung, A. Markham, N.E. Fusenig, Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line, J. Cell Biol. 106 (1988) 761–771. [18] N.E. Fusenig, D. Breitkreutz, R.T. Dzarlieva, P. Boukamp, A. Bohnert, W. Tilgen, Growth and differentiation characteristics of transformed keratinocytes from mouse and human skin in vitro and in vivo, J. Invest. Dermatol. 81 (1983) 168s–175s. [19] M. Blaha, W. Bowers, J. Kohl, D. DuBose, J. Walker, A. Alkhyyat, G. Wong, Effects of CEES on inflammatory mediators, heat shock protein 70A, histology and ultrastructure in two skin models, J. Appl. Toxicol. 20 (2000) S101–S108.
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[30] A. Fidder, G.W. Moes, A.G. Scheffer, G.P. van der Schans, R.A. Baan, L.P. De Jong, H.P. Benschop, N7-(2-hydroxyethylthioethyl)-guanine: a novel urinary metabolite following exposure to sulphur mustard, Chem. Res. Toxicol. 7 (1994) 199–204. [31] J.P. Langenberg, G.P. van der Schans, H.E. Spruit, W.C. Kuijpers, R.H. MarsGroenendijk, H.C. van Dijk-Knijnenburg, H.C. Trap, H.P. van Helden, H.P. Benschop, Toxicokinetics of sulfur mustard and its DNA-adducts in the hairless guinea pig, Drug Chem. Toxicol. 21 (1998) 131–147. [32] H.P. Benschop, G.P. van der Schans, D. Noort, A. Fidder, R.H. Mars-Groenendijk, L.P. de Jong, Verification of exposure to sulfur mustard in two casualties of the Iran-Iraq conflict, J. Anal. Toxicol. 21 (1997) 249–251. [33] T. Porstmann, S.T. Kiessig, Enzyme immunoassay techniques, An overview. J. Immunol. Methods 150 (1992) 5–21. [34] J.R. Hansson, R. Lewensohn, U. Ringborg, B. Nilsson, Standard operating procedure for immunuslotblot assay for analysis of DNA/sulfur mustard adducts in human blood and skin, Cancer Res. 47 (1987) 2631–2637. [35] K.J. Smith, R.P. Casillas, J.S. Graham, H.G. Skelton, F.W. Stemler, B.E. Hackley, Histopathologic features seen with different animal models following cutaneous sulfur mustard exposure, J. Dermatol. Sci. 14 (1997) 126–135. [36] K. Kehe, H. Reisinger, L. Szinicz, Sulfur mustard induces apoptosis and necrosis in SCL II cells in vitro, J. Appl. Toxicol. (2000) S81–S86. [37] K. Kehe, K. Raithel, H. Kreppel, M. Jochum, F. Worek, H. Thiermann, Inhibition of poly(ADP-ribose) polymerase (PARP) influences the mode of sulfur mustard (SM)-induced cell death in HaCaT cells, Arch. Toxicol. (2007) 1–10. [38] C.M. Simbulan-Rosenthal, R. Ray, B. Benton, E. Soeda, A. Daher, D. Anderson, W.J. Smith, D.S. Rosenthal, Calmodulin mediates sulfur mustard toxicity in human keratinocytes, Toxicology 227 (2006) 21–35. [39] B.R. Capacio, J.R. Smith, M.T. DeLion, D.R. Anderson, J.S. Graham, G.E. Platoff, W.D. Korte, Monitoring sulfur mustard exposure by gas chromatography-mass spectrometry analysis of thiodiglycol cleaved from blood proteins, J. Anal. Toxicol. 28 (2005) 306–310.
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Modified immunoslotblot assay to detect hemi and sulfur mustard DNA adducts Kai Kehe a,b,⇑, Verena Schrettl a,c, Horst Thiermann a, Dirk Steinritz a,b a
Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstr. 11, 80937 Munich, Germany Walther Straub Institute of Pharmacology and Toxicology, Ludwig Maximilians University of Munich, Goethestraße 33, 80336 Munich, Germany c Toxicological Department, Klinikum rechts der Isar, Technical University of Munich, Ismaninger Strasse 22, 81675 Munich, Germany b
a r t i c l e
i n f o
Article history: Available online 8 August 2013 Keywords: Sulfur mustard Nitrogen mustard Chemical warfare agents Vesicants
a b s t r a c t Sulfur mustard (SM) is an old chemical warfare agent causing blisters (vesicant). Skin toxicity is thought to be partly caused by SM induced DNA damage. SM and the hemi mustard 2-chloroethyl ethyl sulfide (CEES) are bi- and monofunctional DNA alkylating agents, respectively. Both chemicals react especially with N7 guanine. The most abundant adducts are 7-hydroxyethylthioethylguanine for SM (61%) and 7ethyl thioethylguanine for CEES. Thus, DNA alkylation should serve as a biomarker of SM exposure. A specific monoclonal antibody (2F8) was previously developed to detect SM and CEES adducts at N7 position by means of immunoslotblot (ISB) technique (van der Schans et al. (2004) [16]). Nitrogen mustards (HN1, HN-2, HN-3) are alkylating agents with structural similarities, which can form DNA adducts with N7 guanine. The aim of the presented work was to modify the van der Schans protocol for use in a field laboratory and to test the cross reactivity of the 2F8 antibody against nitrogen mustards. Briefly, human keratinocytes were exposed to SM and CEES (0–300 lM, 60 min) or HN-1, HN-2, HN-3 (120 min). After exposure, cells were scraped and DNA was isolated and normalized. 1 lg DNA was transferred to a nitrocellulose membrane using a slotblot technique. After incubation with 2F8 antibody, the DNA adducts were visualized with chromogen staining (3,30 -diaminobenzidine (DAB), SeramunGrün). Blots were photographed and signal intensity was quantified. In general, DAB was superior to SeramunGrün stain. A staining was seen from 30 nM to 300 lM of SM or CEES, respectively. However, statistically significant DNA adducts were detected after CEES and SM exposure above 30 lM which is below the vesicant threshold. No signal was observed after HN-1, HN-2, HN-3 exposure. The total hands-on time to complete the assay was about 36 h. Further studies are necessary to validate SM or CEES exposure in blister roofs of exposed patients. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Sulfur mustard was used as a chemical warfare agent since 1917. Due to its strong vesicating properties on skin, it is classified as a blistering agent [1]. Sulfur mustard, either as liquid or gas, is a lipophilic compound that can easily penetrate in the body. Exposure to sulfur mustard does not cause immediate signs of intoxication. A symptomless or -free period of several hours was frequently reported. The first symptoms are observed on the eyes, skin and in the bronchial tract [2]. SM is classified as a vesicant agent because of its strong capability to induce blistering on the skin. The epidermis detaches from the dermis. Keratinocyte cell death is detectable ⇑ Corresponding author at: Walther Straub Institute of Pharmacology and Toxicology, Ludwig Maximilians University of Munich, Goethestraße 33, 80336 Munich, Germany. Tel.: +49 89 1249 7950; fax: +49 89 3168 2333. E-mail addresses: [email protected], [email protected] (K. Kehe), [email protected] (V. Schrettl), [email protected] (H. Thiermann), [email protected] (D. Steinritz). 0009-2797/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbi.2013.08.001
within the stratum basale [3]. Eye symptoms involve redness of the eyes, swollen eyelids, tearing, pain, sensitivity to light and severe blepharospasm [4]. Inhalation of sulfur mustard vapor damages epithelial surfaces of the laryngeal and bronchial mucosa. Acute symptoms and signs are hoarseness, sneezing, lacrimation, hacking cough and in severe cases pseudomembrane formation in the bronchial tract [5,6]. Nevertheless, exposure to sulfur mustard results often in systemic poisoning with nausea, vomiting and leucopenia at a later stage. The clinical picture has been extensively reviewed in the past [6–8]. The continuous threat of poisoning has prompted a huge research program in the last century. Despite this effort, the pathophysiology of sulfur mustard poisoning is still not completely understood. It is widely accepted that the reaction of SM with the DNA is one if not the main pathophysiological event that triggers the cell death cascade. SM is a highly reactive bifunctional alkylating agent. It hydrolyzes in water to form an ethylene sulfonium ion intermediate. A highly reactive carbenium ion is formed,
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which is highly reactive. Cell constituents like DNA, RNA, proteins, and other molecules are rapidly alkylated [1]. SM reactions with DNA lead to mono- or bifunctional adducts with guanine and adenosine. Reactions with thymine or uracil were not detected under physiological conditions. SM preferentially reacts with ring nitrogen atoms of the DNA in decreasing order of reactivity with N7 of guanine, N3 of adenine, N1 of adenine and N1 of cytosine [9]. Thus, 61% of all alkylations occur at the N7 of guanine [10]. Nearly 17% of total alkylations produce intra- or interstrand-crosslinks [11]. The genotoxic properties of SM have been recognized to be the most important trigger of cell death [12]. The strong cytotoxic properties of SM against rapidly dividing cells were used against cancer cells. Several mustards were synthesized and screened for their usefulness as chemotherapeutic drugs. Initially, several N-mustards were introduced in anti-cancer therapy and used as starting substance for other chemotherapeutic drugs [13]. Nitrogen mustards (HN-1, HN-2, HN-3) were also synthesized as chemical warfare agents. Retrospective diagnosis and verification of SM poisoning may be difficult. The portion of SM that does not penetrate very rapidly through the skin evaporates quickly from the surface [14]. Off-gassing of SM vapor has been detected up to 24 h after exposure in pig models, and a reservoir of agent has been noted in the upper levels of the stratum corneum of pigs [15]. Rapid methods for reliable and fast detection of SM poisoning are needed to initiate appropriate clinical treatment. Sophisticated analytical methods may be useful to confirm a possible SM intoxication with high evidence. Recently, an immunochemical method was described to detect the monofunctional SM adduct at the N7 position of guanine [15]. A substantial portion of SM that has penetrated the skin is fixed at the DNA mainly within the epidermis. It is hypothesized that extracted DNA of blister roofs or affected skin contain sufficient amounts of reacted SM to be detectable with the above mentioned antibody. The aim of this study was to modify the immunoslotblot technique and to test the cross-reactivity of the 2F8 antibody against SM, semi mustard (CEES) and nitrogen mustards (HN-1, HN-2, HN-3). 2. Materials and methods 2.1. Chemicals SM was obtained from TNO, Rijswijk, The Netherlands. All other chemicals used were reagent grade products obtained from Sigma (Deisenhofen, Germany). SeramunGrün was from Seramun Diagnostica, Heidesee, Germany. The tested sulfur or nitrogen mustards are listed in Table 1. 2.2. Cell cultures HaCaT cells, a spontaneously transformed human keratinocyte cell line [16,17], was kindly provided by Prof. Dr. N. Fusenig (German Cancer Research Center, Heidelberg, FRG). HaCaT cells were cultured in Dulbecco’s Modified Eagle Medium/Ham’s F12 (DMEM/F12) supplemented with 2.45 mmol/l glutamine, 10% fetal calf serum (Life Technologies, Eggenstein, FRG) using standard cell culture flasks (75 cm2, Falcon, Heidelberg, FRG). Cultures were maintained at 37 °C in a humidified atmosphere with 5% CO2. Cell doubling was 22 h. Cells were seeded with 105 cells/cm2 and experiments were performed with subconfluent cultures on the 1st day after seeding. 2.3. Exposure protocol HaCaT cells were washed with PBS and incubated at 25 °C for 60 min with various concentrations of SM or CEES, or for 120 min with various concentrations of HN-1, HN-2, or HN-3 in
Table 1 Structures of tested chemical compounds. chemical compound
structure
CEES CAS-Nr. 693-07-2 sulfur mustard CAS-Nr. 505-60-2 HN-1 (Chlormethine) CAS-Nr. 51-75-2
HN-2 (Mustine, Mustargen) CAS-Nr. 107-99-3
HN-3 (Trimustine) CAS-Nr. 817-09-4
Modified Eagle’s Medium (MEM). SM was dissolved first in ethanol. The final concentration of the solvent in the exposure fluids was below 1%. Control cultures were exposed to the same amount of ethanol. The obtained stock solutions of SM, CEES and nitrogen mustards in MEM were used immediately after dilution to minimize hydrolysis. After exposure, HaCaT cells were washed with PBS and subjected to analysis. Every experiment was repeated three times and each immunoslotblot consists of three replicate experiments (n = 9). 2.4. Cell isolation HaCaT cells were treated with 2 ml of 0.05%-Trypsin–EDTA (GIBCOÒ invitrogen) and incubated at 37 °C until trypsination was complete. The HaCaT cells were diluted with 13 ml of Dulbecco’s Modified Eagle Medium (DMEM) containing FKS and transferred to 50 ml tubes, from which 100 ll samples were taken for cell count (CASY Modell TTC, Innovatis Systems, Reutlingen). The tubes were centrifuged for 5 min at 1350 g (ROTINA35 R, Hettich Zentrifugen, Tübingen) and the supernatant removed. The remaining HaCaT cells were re-suspended in DMEM to a concentration of 5–10 106 cells/ml. From each sample 200 ll were transferred to 1.5 ml sample tubes and centrifuged for 5 s at 16.1 g (Centrifuge 5415 R, Eppendorf, Hamburg). The supernatant medium was removed and the sample tubes were vortexed. 2.5. DNA isolation DNA was isolated from the HaCaT cells using the Puregene Core Kit A (QIAGEN, Hilden, Germany). Cell lysis solution (300 ll) was added to the isolated cells and vortexed for 10 s. 1.5 ll RNase A (50 mg/ml) was added, the mixture was incubated for 15 min at 37 °C and cooled at 0 °C for 1 min. Protein precipitation solution (100 ll) was added to the mixture, vortexed for 20 s and centrifuged for 5 min at 16.1 g. The supernatant was carefully decanted from theformed pellet into isopropanol (300 ll). The obtained solution was mixed by mild shaking, and centrifuged for 5 min at 16.1 g. The supernatant was decanted from the formed pellet, ethanol (300 ll, 70%) was added to the pellet and again centrifuged for 5 min at 16.1 g. After decantation of the supernatant the samples were dried for 15 min at room temperature, DNA Hydration Solution (50 ll) was added, the mixture was incubated for 1 h at
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65 °C, and mixed on a shaker (2/4 digital, IKAÒ MTS, Staufen) over night at moderate speed. The DNA concentration was determined with the NanoDropÒ by using 1 ll of the obtained DNA solutions (measurements at 280 nm and 260 nm). 2.6. DNA denaturation DNA-denaturation-buffer-solution was prepared from formamide (8 ll), formaldehyde (164 lL; 50%), and 1828 lL Tris/acetic acid/EDTA buffer (Bio-Rad Laboratories GmbH, Munich, Germany) and added to the DNA samples to give a final DNA concentration of 50 lg/ml. These samples were incubated at 52 °C for 20 min, cooled at 0 °C for 1 min, frozen at 80 °C, and defrosted at room temperature. 2.7. Immunoslot blot technique Denaturated DNA from the previous step was diluted in PBS to a final concentration of 5 lg/ml and 200 ll of each sample were pipetted into a slot of the blotting apparatus (Blotting Unit SHM48) equipped with a nitrocellulose membrane (Nitrocellulose Transfer Membrane: Protran BA 79, Pore size 0.1 lm; Whatman, Dassel, Germany) and a blotting paper. The DNA was blotted with a vacuum of 350 mbar onto the membrane after which each slot was washed with PBS (300 ll). The membrane was air-dried for 15 min and subsequently the DNA was fixated onto the membrane for 2 h at 80 °C. Thereafter, the membrane was washed twice with a 1:100 SCC buffer (SCC buffer 20 (3 M NaCl, 0.3 M Na-Citrat, pH 7.0)) for 15 min on a horizontal shaker at moderate speed and room temperature. After washing, the membrane was incubated for 20 min with Protein block (Protein Block Serum-free, Dako, Hamburg, Germany). 2.8. Incubation with the 2F8 Antibody The 2F8 antibody (2F8 Antibody purified Ab 246, 1.05 mg/ml, BBI Dundee) was diluted with Antibody Diluent (Dako) to give a concentration of 50.25 ng/ml 2F8. The membrane was covered with this antibody solution and incubated over night at 4 °C under continued pivoting. On the next day, the membrane was washed using PBS with 0.1% Tween 20 (Tween 20 Sigma) at room temperature on a shaker according to the following procedure: the membrane was washed four times for 15 min, incubated with the HRPLink (Dako, Hamburg, Germany) for 30 min, washed again four times for 10 min, incubated for 30 min with the HRP-Enzyme DakoÒ ADVANCE™, and finally washed four times for 10 min. For staining, the membrane was treated with the corresponding dye solutions. DAB stock solution (50 ll) or SeramunGrünÒ working solution (5 ml; ready to use) was filtered (0.4 lm pore size) and added to the membrane, respectively. Then the membrane was washed three times for 10 min with distilled water and air-dried.
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2.10. Analysis of data Statistical differences were assessed with the Mann–WhitneyU-Test (PASW Statistics Version 18). All results from each concentration of each staining agent were compared to the negative control (C). The value of p was determined with p < 0.05 and p > 0.05 indicating statistically significant difference. 3. Results 3.1. Sulfur mustard (SM) N7-DNA-adducts of SM were detectable with DAB and SeramunGrünÓ. Fig. 1 shows a typical membrane after immunoslotblot and staining with DAB. A concentration dependent increase of signal intensity could be detected visually. With densitometry of DAB stained membranes, significant different signals were detected with concentrations down to 0.3 lM SM in comparison to sham treated controls (p < 0.05) (Fig. 2). The dye SeramunGrünÓ showed a similar intensity compared to DAB (median, 147.0 AU vs. 156.9 AU) at 100 lM SM (Fig. 2). With densitometry, the SeramunGrünÓ stained membranes gave significant signals down to 1 lM SM when compared to sham treated controls (p < 0.05). 3.2. CEES N7-DNA-adducts of CEES were detectable with both staining dyes, DAB and SeramunGrünÓ, from 30 lM to 300 lM (Fig. 3). CEES concentrations equal to and below 10 lM of CEES showed only a faint staining. DAB staining was more intense and easier to identify than SeramunGrünÓ staining. This observation was underlined by densitometry. When CEES was incubated with SeramunGrünÓ, the signal intensity was 2-fold lower than after DAB incubation with 300 lM of CEES (median, 68.3 AU vs. 123.8 AU). DAB stained blots showed a better signal to noise ratio compared to SeramunGrünÓ. 3.3. Nitrogen mustards (HN-1, HN-2, HN-3) N7-DNA-adducts of HN-1, HN-2 or HN-3 were not detectable with both staining dyes, DAB and SeramunGrünÓ, up to 300 lM in our experimental setup. A slight but not significant increase of
2.9. Analysis of the membrane The membrane was scanned with a Image Scanner III (GE Healthcare; LabScan™ 6.0 Powered by Melanie™, Swiss Institute of Bioinformatics). The settings were 150 dpi for the resolution. The scanned image was inverted and transformed into a Tif file with Image J 1.42 (Wayne Rasband, NIH). The resulting image was analyzed densitometrically with AndorIQ 1.9.1 (Bioimaging, UK). After background subtraction, gray values of each pixel were summed and the resulting value was referred to the area of measurement (mm2). For comparison, the slots on the negative image and an area of the background with as little staining as possible were marked with a fixed rectangle. The results were given in arbitrary units.
Fig. 1. Immunoslotblot of SM induced DNA adducts detected with 2F8 antibody in HaCaT cells. Following exposure with SM (0.1–100 lM, 60 min) cells were washed and DNA was extracted, blotted, immunostained with 2F8 antibody and visualized with DAB.
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Fig. 2. Monofunctional DNA adducts detected with 2F8 antibody in HaCaT cells after SM exposure. Following exposure with SM (0.1–100 lM, 60 min) cells were washed and DNA was extracted, blotted, immunostained with 2F8 antibody and visualized either with DAB or SeramunGrün followed by densitometry. ⁄p < 0.05; Mann–Whitney-U-Test.
Fig. 3. Monofunctional DNA adducts detected with 2F8 antibody in HaCaT cells after CEES exposure. Following exposure with CEES (0.3–300 lM, 60 min) cells were washed and DNA was extracted, blotted, immunostained with 2F8 antibody and visualized either with DAB or SeramunGrün followed by densitometry. ⁄p < 0.05; Mann–Whitney-UTest.
signal intensity was observed for HN-3 after SeramunGrünÓ staining (data not shown). 3.4. Hands-on time of the immunoslotblot procedure The total hands-on time to complete the assay was about 36 h. The two overnight steps were used to denaturate the DNA and for incubation with the first antibody (2F8). 4. Discussion Blister agents or vesicants are chemical warfare agents that produce pronounced skin symptoms ranging from erythema to large bullae. Within the group of blister agents three subgroups are listed: mustard agents, lewisite, phosgene oxime. The last produces more likely urticaria than vesicle. Blister agents are still a threat
until today and have indeed been used in various military conflicts starting from 1917. SM is the only large scale used blister agent until today [2]. After World War I, several programs were initiated to identify substances similar to SM with blister inducing properties. SM has two reactive chlorethyl groups. An analogue with one reactive chlorethyl group is CEES (half mustard) which produces solely monofunctional DNA adducts and has been used as a SM analogue in various studies [18–21]. HN-1, HN-2 and HN-3 are nitrogen mustards, which are listed as chemical warfare agents [22]. However, HN-1 was used as a wart remover, HN-2 is also known as Mustine, a pharmaceutical compound to treat cancer [23]. HN-3 has no medical use so far. Taken together, SM related substances have one or more chlorethyl groups attached to nitrogen (nitrogen mustards) and are less toxic than SM [24,25]. Thus, it is likely that they share some similarities in their properties to damage DNA. In fact, sulfur and nitrogen
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mustards attack predominately guanine at the N7-position [26– 28]. This alkylation site was used to develop an antibody against this specific adduct. The antigen 7-hydroxyethylthioethyl-guanine (HETEG) was synthesized and a monoclonal antibody (2F8) was developed and recently characterized [29]. The 2F8 antibody was successfully applied in toxicokinetic studies [30] and for verification of SM exposure in blood of Iranian patients by means of immunoslotblot technique [31]. More recently, the immunoslotblot technique was optimized and a standard operating procedure was published [32]. However, the published techniques used fluorescent methods to visualize and quantify the signals. The advantage of fluorescent detection is a good signal to noise ratio. The disadvantage is the need for a more complex imaging system. Generally, detection using fluorescent substrates is 2–10-fold more sensitive than chromogenic detection [16]. In a military setting or at rural laboratories less complicated methods are needed. To achieve this goal, we have replaced the fluorescent label by a horseradish peroxidase system, which allows staining techniques with chromogenic dyes. In the presented study, we used the two chromogenic dyes DAB and SeramunGrün. DAB precipitates in the presence of hydrogen peroxide that is produced by peroxidase. DAB is widely used in various immunochemical protocols, which produces a brown color. SeramunGrün comprises is a ready-to-use substrate solutions for horseradish peroxidase. During the catalytic decomposition of hydrogen peroxide electrons are transferred from 3,30 5,50 -Tetramethylbenzidine to o-Dianisidine (o-Dia) by horseradish peroxidase. Oxidation of o-Dia leads to formation of dimers with deep green color. SeramunGrün is a substrate optimized for use in blotting systems with visual assessment. Both reagents proved to be suitable for the detection of SM or CEES DNA adducts. SeramunGrün showed a less intense signal in our hands compared to DAB and consecutively leading to a decreased sensitivity. Taken together, DAB is superior to SeramunGrün in the presented immunoslotblot assay. DNA adducts induced by nitrogen mustards (HN-1, HN-2, HN-3) were not detected in the immunoslotblot assay. This may be due to a different antigen structure, which is not detectable by the 2F8 antibody. On the other hand, nitrogen mustards react more slowly with cellular DNA than SM [27,33]. Thus, it is possible that DNA adduct formation has not reached a detectable amount of mono-adducts. This hypothesis needs further investigations, which was beyond the scope of our study. Only a small but not significant signal increase was seen after blotting DNA of HN-3 treated cells and SeramunGrünÓ staining, which may be a hint to some DNA alkylation. On the other hand, SeramunGrünÓ was not as sensitive as DAB. Therefore, this signal was considered as an artifact. Taken together, skin contact with nitrogen mustards less than 2 h will not result in detectable DNA adducts in the presented immunoslotblot assay. The detection level of SM DNA adducts was 0.3 lM in our experimental setup. In a previous study, SM poisoned Iranian soldiers showed levels of alkylation in leukocytes comparable to 0.9 lM SM [35]. Thus, the achieved level of detection was sensitive enough to detect SM induced DNA alkylation even in the blood of severely SM poisoned victims. As SM concentrations at the site of body entry are likely to be much higher, the presented method is suitable to verify skin exposure to SM. Skin blistering is the main clinical symptom of SM poisoning [2]. Histopathologically, this macroscopic picture corresponds to apoptotic cell death in the epidermal stratum basale [34]. In vitro, keratinocyte apoptosis is observed after 100 lM SM exposure [35–38]. Thus, for meaningful studies 100 lM SM can be considered as an equivalent blister inducing SM concentration in vitro [38,39]. The presented immunoslotblot assay has a detection limit for SM exposure that is more than 300-fold below the vesicant dose. The hands-on time was
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36 h. Further studies are needed to reduce the time. However, the presented method is simple and does not need sophisticated equipment (GC–MS, LC–MS) compared to the detection of SM metabolites in blister fluid and urine, or adducts on proteins found in the blood [39]. Our study showed a simple method to detect full and hemimustard induced DNA adducts in human keratinocytes with high sensitivity and specificity in vitro. Further studies are necessary to validate this method for skin samples and to confirm the relevance for in vivo exposure. Source of funding H.T. received fund from German Ministry of Defense. Conflict of interest statement None declared. References [1] B. Papirmeister, C.L. Gross, H.L. Meier, J.P. Petrali, J.B. Johnson, Molecular basis for mustard-induced vesication, Fundam. Appl. Toxicol. 5 (1985) S134–49. [2] K. Kehe, L. Szinicz, Medical aspects of sulphur mustard poisoning, Toxicology 214 (2005) 198–209. [3] B. Papirmeister, C.L. Gross, J.P. Petrali, C.J. Hixson, Pathology produced by sulfur mustard in human skin grafts on athymic nude mice. I. Gross and light microscopic changes, J. Toxicol. Cutaneous Ocul. Toxicol. 3 (1984) 371–391. [4] Y. Solberg, M. Alcalay, M. Belkin, Ocular injury by mustard gas, Surv. Ophthalmol. 41 (1997) 461–466. [5] A. Emad, G.H. Rezaian, The diversity of the effects of sulfur mustard gas inhalation on respiratory system 10 years after a single, heavy exposure: analysis of 197 cases, Chest 112 (1997) 734–738. [6] M. Balali-Mood, M. Hefazi, Comparison of early and late toxic effects of sulfur mustard in Iranian veterans, Basic Clin. Pharmacol. Toxicol. 99 (2006) 273– 282. [7] K. Kehe, H. Thiermann, F. Balszuweit, F. Eyer, D. Steinritz, T. Zilker, Acute effects of sulfur mustard injury-Munich experiences, Toxicology 263 (2009) 3– 8. [8] M. Balali-Mood, A. Navaeian, B. Heyndrickx. Clinical and paraclinical findings in 233 patients with sulfur mustard poisoning. In: Proceedings of the Second World Congress on New Compounds in Biological and Chemical Warfare (1986) 464–473. [9] P.D. Lawley, P. Brookes, Further studies on the alkylation of nucleic acids and their constituent nucleotides, Biochem. J. 89 (1963) 127–138. [10] D.B. Ludlum, W.P. Tong, J.R. Mehta, M. Kirk, B. Papirmeister, Formation of O6ethylthioethyldeoxyguanosine from the reaction of chloroethyl ethyl sulfide with deoxyguanosine, Cancer Res. 44 (1984) 5698–5701. [11] D.B. Ludlum, B. Papirmeister, DNA modification by sulfur mustards and nitrosoureas and repair of these lesions, Basic Life Sci. 38 (1986) 119–125. [12] I.J. Lodhi, J.F. Sweeney, R.E. Clift, D.B. Hinshaw, Nuclear dependence of sulfur mustard-mediated cell death, Toxicol. Appl. Pharmacol. 170 (2001) 69–77. [13] L.S. Goodman, M.M. Wintrobe, W. Dameshek, M.J. Goodman, A. Gilman, M.T. McLennan, Landmark article Sept. 21, 1946: nitrogen mustard therapy. Use of methyl-bis(beta-chloroethyl)amine hydrochloride and tris(betachloroethyl)amine hydrochloride for Hodgkin’s disease, lymphosarcoma, leukemia and certain allied and miscellaneous disorders. By Louis S. Goodman, Maxwell M. Wintrobe, William Dameshek, Morton J. Goodman, Alfred Gilman and Margaret T. McLennan, JAMA 251 (1984) 2255–2261. [14] R.P. Chilcott, J. Jenner, W. Carrick, S.A. Hotchkiss, P. Rice, Human skin absorption of bis-2-(chloroethyl)sulphide (sulphur mustard) in vitro, J. Appl. Toxicol. 20 (2000) 349–355. [15] I.J. Hattersley, J. Jenner, C. Dalton, R.P. Chilcott, J.S. Graham, The skin reservoir of sulphur mustard, Toxicol. In Vitro 22 (2008) 1539–1546. [16] G.P. van der Schans, A.G. Scheffer, R.H. Mars-Groenendijk, A. Fidder, H.P. Benschop, R.A. Baan, Standard operating procedure for immunuslotblot assay for analysis of DNA/sulfur mustard adducts in human blood and skin, J. Anal. Toxicol. 28 (2004) 316–319. [17] P. Boukamp, R.T. Petrussevska, D. Breitkreutz, J. Hornung, A. Markham, N.E. Fusenig, Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line, J. Cell Biol. 106 (1988) 761–771. [18] N.E. Fusenig, D. Breitkreutz, R.T. Dzarlieva, P. Boukamp, A. Bohnert, W. Tilgen, Growth and differentiation characteristics of transformed keratinocytes from mouse and human skin in vitro and in vivo, J. Invest. Dermatol. 81 (1983) 168s–175s. [19] M. Blaha, W. Bowers, J. Kohl, D. DuBose, J. Walker, A. Alkhyyat, G. Wong, Effects of CEES on inflammatory mediators, heat shock protein 70A, histology and ultrastructure in two skin models, J. Appl. Toxicol. 20 (2000) S101–S108.
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