Adduct of the blistering warfare agent sesquimustard with human serum albumin and its mass spectrometric identification for biomedical verification of exposure.

Apart from the well-known sulfur mustard (SM), additional sulfur-containing blistering chemical warfare agents exist. Sesquimustard (Q) is one of them and five times more blistering than SM. It is a common impurity in mustard mixtures and regularly found in old munitions but can also be used in pure form. Compared to the extensive literature on SM, very little experimental data is available on Q and no protein biomarkers of exposure have been reported. We herein report for the first time the adduct of Q with the nucleophilic Cys34 residue of human serum albumin (HSA) formed in vitro and introduce two novel bioanalytical procedures for detection. After proteolysis of this HSA adduct catalyzed either by pronase or by proteinase K, two biomarkers were identified by high-resolution tandem mass spectrometry (MS/HR MS), namely a dipeptide and a tripeptide, both alkylated at their Cys residue, which we refer to as HETETE-CP and HETETE-CPF. HETETE represents the Q-derived thio-alkyl moiety bearing a terminal hydroxyl group: "hydroxyethylthioethylthioethyl." Targeting both peptide markers from plasma, a micro liquid chromatography-electrospray ionization tandem mass spectrometry method working in the selected reaction monitoring mode (µLC-ESI MS/MS SRM) was developed and validated as well suited for the verification of exposure to Q. Fulfilling the quality criteria defined by the Organisation for the Prohibition of Chemical Weapons, the novel methods enable the detection of exposure to Q alone or in mixtures with SM. We further report on the relative reactivity of Q compared to SM. Based on experiments making use of partially deuterated Q as the alkylating agent, we rule out a major role for six-membered ring sulfonium ions as relevant reactive species in the alkylation of Cys34. Furthermore, the results of molecular dynamics simulations are indicative that the protein environment around Cys34 allows adduct formation with elongated but not bulky molecules such as Q, and identify important hydrogen bonding interactions and hydrophobic contacts. Graphical abstract.

Toxic blister agents: Chemistry, mode of their action and effective treatment strategies.

Since their use during the First World War, Blister agents have posed a major threat to the individuals and have caused around two million casualties. Major incidents occurred not only due to their use as chemical warfare agents but also because of occupational hazards. Therefore, a clear understanding of these agents and their mode of action is essential to develop effective decontamination and therapeutic strategies. The blister agents have been categorised on the basis of their chemistry and the biological interactions that entail post contamination. These compounds have been known to majorly cause blisters/bullae along with alkylation of the contaminated DNA. However, due to the high toxicity and restricted use, very little research has been conducted and a lot remains to be clearly understood about these compounds. Various decontamination solutions and detection technologies have been developed, which have proven to be effective for their timely mitigation. But a major hurdle seems to be the lack of proper understanding of the toxicological mechanism of action of these compounds. Current review is about the detailed and updated information on physical, chemical and biological aspects of various blister agents. It also illustrates the mechanism of their action, toxicological effects, detection technologies and possible decontamination strategies.

Long-term Respiratory Effects of Mustard Vesicants.

Sulfur mustard and related vesicants are cytotoxic alkylating agents that cause severe damage to the respiratory tract. Injury is progressive leading, over time, to asthma, bronchitis, bronchiectasis, airway stenosis, and pulmonary fibrosis. As there are no specific therapeutics available for victims of mustard gas poisoning, current clinical treatments mostly provide only symptomatic relief. In this article, the long-term effects of mustards on the respiratory tract are described in humans and experimental animal models in an effort to define cellular and molecular mechanisms contributing to lung injury and disease pathogenesis. A better understanding of mechanisms underlying pulmonary toxicity induced by mustards may help in identifying potential targets for the development of effective clinical therapeutics aimed at mitigating their adverse effects.

Clinical management of casualties exposed to lung damaging agents: a critical review.

There is no specific antidote for the treatment of casualties exposed to chlorine, phosgene, or mustards; therefore, management is largely supportive. Corticosteroid treatment has been given to casualties accidentally exposed to chlorine. Clinical data on efficacy are inconclusive as the numbers given steroids have been small and the indications for administration unclear. There have been no clinical controlled studies. There is a stronger evidence base from animal studies, particularly from porcine and rodent models. Lung injury induced by phosgene and mustard appears to be mediated by glutathione depletion, lipid peroxidation, free radical generation, and subsequent cellular toxicity. There is limited evidence to suggest that repletion of glutathione reduces and/or prevents lung damage by these agents. This may provide an opportunity for therapeutic intervention.

Comparative pharmacokinetics of 1-(2-chloroethyl)-3-(2,6-dioxo-1-piperidyl)-1-nitrosourea in rats and patients and extrapolation to clinical trials.

The plasma pharmacokinetics of 1-(2-chloroethyl)-3-(2,6-dioxo-1-piperidyl)1-nitrosourea (PCNU) were determined in ambulatory rats and in patients receiving PCNU chemotherapy in Phase 1 and II studies. After derivativization to the methyl carbamate, both rat and human PCNU plasma levels were measured by gas chromatography-mass spectrometry. Comparison of the tolerated dose levels and pharmacokinetics of PCNU to the values determined for 1,3-bis(2-chloroethyl)-1-nitrosourea in humans indicated that PCNU has a lower plasma drug area under the curve at equitoxic doses. We conclude that PCNU may show less clinical efficacy than 1,3-bis(2-chloroethyl)-1-nitrosourea in the treatment of solid tumors.

Full-thickness human skin explants for testing the toxicity of topically applied chemicals.

This report describes a model organ-culture system for testing the toxicity of chemical substances that are topically applied to human skin. In this system, the viable keratinocytes in the full-thickness skin explants are protected by the same keratinized layer as skin remaining on the donor, and toxicity can be assessed microscopically and/or biochemically. The human skin specimens were discards from a variety of surgical procedures. They were cut into full-thickness 1.0-cm2 explants, and briefly exposed to the military vesicant sulfur mustard (SM), which was used as a model toxicant. The explants were then organ cultured in small Petri dishes for 24 h at 36 degrees C. In the 0.03-1.0% dosage range, a straight-line dose-response relationship occurred between the concentration of SM applied and the number of paranuclear vacuoles seen histologically in the epidermis. Within the same SM dosage range, there was also a proportional decrease in 14C-leucine incorporation by the explants. Thus, the number of paranuclear vacuoles reflected decreases in protein synthesis by the injured epidermal cells. The epidermis of full-thickness untreated (control) human skin explants usually remained viable for 7 d when stored at 4 degrees C in culture medium. During storage, a relatively small number of paranuclear vacuoles developed within the epidermis, but the explants were still quite satisfactory for testing SM toxicity. Incubation (for 4 or 24 h at 36 degrees C) of such control skin explants reduced (often by 50%) the small number of paranuclear vacuoles produced during 4-7 d of storage. This reduction was probably caused by autolysis of many of the vacuolated cells. Two types of paranuclear vacuoles could be identified by both light and electron microscopy: a storage type and a toxicant type. The storage type seemed to be caused by autolysis of cell components. The toxicant type seemed to be caused by an invagination of the plasma membrane. Only toxicant-type vacuoles increased appreciably in number when skin explants were exposed to mustard, and to other toxicants.

Components of hypothalamic obesity: bipiperidyl-mustard lesions add hyperphagia to monosodium glutamate-induced hyperinsulinemia.

Rats with bilateral electrolytic lesions in the general region of the ventromedial hypothalamic (VMH) nucleus develop hyperinsulinemia, excessive food intake and obesity. Monosodium glutamate (MSG) destroys neurons of the arcuate hypothalamic (AH) nucleus and produces hyperinsulinemic but hypophagic obesity. Bipiperidyl mustard (BPM) primarily destroys VMH neurons, but has produced only a slight obesity even when rats were maintained on high-fat diets. In the present study, rats treated with MSG (AH lesion) were hyperinsulinemic, moderately obese and hypophagic; BPM rats (primarily VMH lesion) were not different from controls when fed standard chow diets. However, MSG/BPM rats (AH + VMH lesion) were hyperinsulinemic, massively obese and hyperphagic. Thus, two components of the electrolytic lesion syndrome previously attributed to VMH damage (hyperinsulinemia and obesity) were reproduced simply by MSG treatment alone. The third component (hyperphagia) occurred only when both AH and VMH were lesioned, suggesting that neurons in both nuclei may perform a satiety function and may be able to substitute for one another in this respect. Since MSG treatment is required for all components of both obesity syndromes described here, this underscores the importance of MSG-sensitive neurons in mechanisms of obesity. The combined treatment approach also represents the first rat model of hyperinsulinemic, hyperphagic obesity that can be entirely produced by systemic administration of neurotoxins.

In vivo and in vitro toxicity of newly synthesized monofunctional sulfur mustard derivatives.

The toxicity of two new monofunctional sulfur mustard derivatives was tested. The compound (4-carboxybutyl 2-chloroethyl sulfide, CBCS; 10-carboxydecyl 2-chloroethyl sulfide, CDCS) possess the 2-chloroethyl sulfide moiety present in mustard gas. Exposure of guinea pig skin to CBCS resulted in a dose-related ulcerative effect. CDCS exhibited similar pathological effects. Dimethylsulfoxide (DMSO) exacerbated CBCS toxicity. Regeneration and healing were prominent six days after application. Concentration-related effects were found in in vitro systems, using human SH-SY5Y neuroblastoma cells for acute toxicity and Y79 retinoblastoma cells for colony forming assay. CBCS or derivatives may serve as models compounds for investigating the mechanism of action of alkylating agents.

Response of mouse brain to a single subcutaneous injection of the monofunctional sulfur mustard, butyl 2-chloroethyl sulfide (BCS)*.

Exposure to mustard-type vesicants results in alkylation of DNA and vesication. However, the biochemical mechanism for vesicant injury and whether it is localized or diffuse are not clear. We postulated that vesicant damage is mediated by free radicals, resulting in oxidative stress. These free radicals-mediated reactions may propagate systemically distal to the site of exposure. To test this hypothesis, we examined the effects of a single subcutaneous injection of the monofunctional sulfur mustard, butyl 2-chloroethyl sulfide (BCS), on the brain. We injected 3 groups (6 mice/group) of 5-month-old male, athymic, nude mice, weighing 30-35 g, subcutaneously with neat (undiluted) BCS (5 microliters/mouse). After 1, 24, and 48 h, we sacrificed the treated mice along with an untreated control group and analyzed the brains for biochemical markers of oxidative stress. Compared to untreated controls, the activity of glutathione peroxidase increased by 76%, P less than 0.005 at 24 h, and that of glutathione S-transferases by 25-37%, P less than 0.05 over the entire period. Total glutathione content in the brain was significantly lower, 17%, after 1 h and 23% after 24 h. We found also, concomitant with decreased glutathione, almost a 3-fold increase in susceptibility to lipid peroxidation. Because these changes are consistent with oxidative stress, we conclude that the effect of BCS administered subcutaneously may be translocated, reaching mouse brain, and causing oxidative stress.

Chemical warfare agents: estimating oral reference doses.

Health risk assessments for sites contaminated with chemical warfare agents require a comparison of the potential levels of exposure with a characterization of the toxic potency of each chemical. For noncancer health effects, toxic potency is expressed in terms of Reference Doses (RfD). A RfD is a daily exposure level or dose (usually expressed in units of milligrams of chemical per kilogram body weight per day) for the human population, including sensitive subpopulations, that is likely to be without an appreciable risk of deleterious effects. A daily exposure at or below the RfD is not likely to be associated with health risks, but as the amount of chemical that an individual is exposed to increases above the RfD, the probability that an adverse effect will occur also increases. A RfD is derived by first examining the available human or animal toxicity data to identify a dose or exposure that corresponds to a no-observed-adverse-effect level (NOAEL) or a lowest-observed-adverse-effect level (LOAEL). The NOAEL is the exposure level at which there are no statistically or biologically significant increases in frequency or severity of adverse effects between the exposed population and its appropriate control. Effects may be produced at this level, but they are not considered to be adverse if they do not result in functional impairment or pathological lesions that affect the performance of the whole organism or which reduce an organism's ability to cope with additional challenge. The LOAEL is the lowest exposure level at which there are statistically or biologically significant increases in frequency or severity of adverse effects between the exposed population and its appropriate control. If only a LOAEL is identified by the toxicity data, a NOAEL is estimated by dividing the LOAEL by a factor no greater than 10. This extrapolation factor of 10 or less is termed the LOAEL-to-NOAEL Uncertainty Factor (UFL). The NOAEL is also adjusted by the application of other Uncertainty Factors, including (1) a UFH < or = 10 to ensure that the resulting RfD protects segments of the human population that may be more sensitive to the chemical than the average person; (2) a UFA < or = 10 to extrapolate from the experimental animal species to humans; (3) a UFS < or = 10 to extrapolate from an experimental subchronic exposure study to a potential chronic exposure; and (4) a UFD < or = 10 to ensure that the resulting RfD is protective for all possible adverse effects, particularly those that may not have been adequately evaluated in the available studies. A Modifying Factor (MF), based on a qualitative professional assessment of the data, may also be used to account for other factors (e.g., deficiencies in the critical study) that are not adequately covered by the standard Uncertainty Factors. 1. Agent HD (Sulfur Mustard). RfDe = 7 x 10(-6) mg kg-1 d-1. A LOAEL was identified in a two-generation reproductive toxicity study conducted in rats. A total uncertainty factor of 3000 was applied to account for protection of sensitive subpopulations (10), animal-to-human extrapolation (10), LOAEL-to-NOAEL extrapolation (3), and extrapolation from a subchronic to chronic exposure (10). A LOAEL-to-NOAEL UF of 3, instead of the default value of 10, was used because the critical effect (stomach lesions) was considered to be "mild" in severity and may have been enhanced by the vehicle used (sesame oil in which sulfur mustard is fully soluble) and the route of administration (gavage), which is more likely to result in localized irritant effects. The key study did identify a toxic effect that is consistent with the vesicant properties of sulfur mustard. In none of the other available studies was there any indication of a different effect occurring at a lower exposure level.

Chemical weapons: documented use and compounds on the horizon.

Man's inhumanity to man is expressed through a plethora of tools of modern warfare and terror. The use of chemical and biological weapons with the goals of assault, demoralisation and lethality has been documented in recent history, both on the battlefield and in urban terror against civilians. A general review of a few of the currently employed chemical weapons and biological toxins, along with a look at potential chemical weapons and tools of counter-terrorism, follows. While these weapons are fearsome elements, the dangers should be viewed in the context of the widespread availability and efficacy of conventional weapons.

2,2'-Dichlorodiethyl sulfide (sulfur mustard) decreases NAD+ levels in human leukocytes.

2,2'-Dichlorodiethyl sulfide (sulfur mustard, HD) extensively alkylates DNA in a concentration-dependent manner in many cell types. We have proposed a biochemical hypothesis that explains HD-induced injury by linking DNA alkylation and DNA breaks with activation of poly(ADP-ribose) polymerase, resulting in depletion of cellular NAD+. This hypothesis was tested by treating human leukocytes with HD to determine whether NAD+ depletion occurred as predicted. These cells demonstrated a decrease in NAD+ levels which was dependent on both concentration of HD and time after exposure. Inhibitors of poly(ADP-ribose) polymerase or substrates for NAD+ synthesis were able to prevent the HD-induced NAD+ decrease.

Molecular dosimetry for sister-chromatid exchange induction and cytotoxicity by monofunctional and bifunctional alkylating agents.

The induction of sister-chromatid exchanges (SCEs) and cytotoxicity in 9L cells treated with monofunctional and bifunctional alkylating agents has been investigated. Three classes of monofunctional and bifunctional agents were studied: nitrosoureas, mustards and epoxides. Independent of class the bifunctional agents were 55-630-fold more effective at inducing SCEs and 300-2400-fold more effective at inducing cellular cytotoxicity than the corresponding monofunctional agents. Comparing the induction of SCEs and cytotoxicity by these agents showed that these two cellular responses to DNA damage are highly correlated. The extent of DNA alkylation in cells treated with 1-ethyl-1-nitrosourea (ENU) or 1-(2-chloro-ethyl)-1-nitrosourea (CNU) was similar indicating that the increased effectiveness of CNU to induce SCEs and cytotoxicity was not due to increased DNA alkylation. Molecular dosimetry calculations indicate that for CNU and ENU treatment of 9L cells there are 116 and 8500 alkylations per SCE induced and 2.6 x 10(4) and 4.6 x 10(6) alkylations at the dose required to reduce survival of 9L cells by 90%. Comparison of the DNA alkylation products produced by CNU and ENU treatment of 9L cells suggests that the formation of the intrastrand crosslink N7-bis(guanyl)ethane and the interstrand crosslink 1-(3-deoxycytidyl)-2-(1-deoxyguanosinyl)ethane by CNU is responsible for the increased effectiveness of CNU treatment at both induction of SCEs and cytotoxicity.

Potentiation of sulphur mustard or cisplatin-induced toxicity by caffeine in Chinese hamster cells correlates with formation of DNA double-strand breaks during replication on a damaged template.

Caffeine inhibited the elongation of nascent DNA and induced breaks in the template DNA of sulphur mustard-treated Chinese hamster cells. The sizes of template and nascent DNAs, as indicated by alkaline sucrose gradient sedimentation, were similar suggesting that incision of template DNA occurred opposite gaps formed in nascent DNA by the action of caffeine, forming, effectively, double-strand breaks in DNA. Double-strand break formation was demonstrated, by means of elution of labelled DNA through polycarbonate filters at neutral pH, in both sulphur mustard- and cisplatin-treated cells when they were incubated in the presence of caffeine for 24 h. Double-strand breaks were only formed in that DNA which had been replicated in the presence of caffeine after treatment with sulphur mustard or cisplatin. Non-toxic concentrations of cycloheximide abolished the potentiation by caffeine of sulphur mustard-induced toxicity to Chinese hamster cells and at the same time abolished the formation of the low molecular weight nascent DNA, and as a consequence of its inhibitory effect on DNA synthesis, and the formation of double-strand breaks in DNA. Potentiation of the lethal and clastogenic effects of genotoxic agents by caffeine is therefore due to effects on the rate and mode of DNA synthesis which lead finally to double-strand breaks in DNA.

[Diastereomers of nonionic analogs of nucleic acids. V. Alkylation of nucleic acids in the living cells by ethylated derivatives of oligonucleotides containing the residue of nitrous yperite. The effect of the phosphotriester fragment configuration]. / Issledovnie diasteromerov neionnykh analogov oligonukleotidov. V. Alkilirovanie nukleinovykh kislot v zhivykh kletkakh étilirovannymi proizvodnymi oligonukleotidov, soderzhashchikh ostatok azotistogo iprita. Vliianie konfiguratsii fosfotriéfirnogo fragmenta.

The alkylation of the cell biopolymers (RNA, DNA, proteins) by reagents Tp'(Et)Tp'(Et)Tp'(Et)TpU(CHRCl) (1) Tp'(Et)Tp''(Et)Tp'(Et)TpU(CHRCl) (2) Tp''(Et)Tp'(Et)Tp''(Et)TpU(CHRCl) (3) Tp''(Et)Tp''(Et)Tp''(Et)TpU(CHRCl) (4) Tp(Et)Tp(Et)Tp(Et)TpU(CHRCl) (5) Tp(Et)Tp(Et)Tp(Et)Tp(Et)U(CHRCl) (6) TpTpTpTpU(CHRCl) (7) where (CHRCl) is the residue of 2',3'-O-[4-N-(2-chloroethyl)-N-methylamino]-benzylidene has been investigated in the case of the ascite carcinoma Krebs-2. p' and p" designate the enantiomeric configurations at the internucleotide phosphorus atoms of the triester fragment--Tp(Et)T--, and p designates the racemic mixture. Completely and partly ethylated reagents (1)-(6) have been found to bind to the cells 4-15 fold more effectively than the diester derivative. The concentration of reagents (1)-(6) in the cells is 2-7 fold higher than in the external medium. Among the diastereomers (1)-(4) reagent (4) with the p"-configuration is the most efficient in binding with the cells 2-3 fold more efficient than reagents (1)-(3). The main targents of modifications performed in the cells by means of reagents (1)-(7) have been established. These are RNA, DNA and proteins. The share of the reagents which react with nucleic acids increases from 45% [reagent (1)] to 80% [reagent (4)], and that reacting with proteins decreases from 50 to 20% correspondingly. Reagent (4) with the p" configurations at phosphotriester fragments alkylates nucleic acids most effectively among the phosphotriester diastereomers (1)-(4): 11-fold more efficient than reagent (1) with configuration p'. The extent of modification of poly(A)+-tracts of m-RNA by reagent (4) in comparison with reagent (1) is 50-fold higher.