handbook of toxicology of chemical warfare agents || immunotoxicity

C H A P T E R 40

Immunotoxicity

KAVITA GULATI AND ARUNABHA RAY

59

I. INTRODUCTION

Immunotoxicity is defined as adverse effects on the func-tioning of both local and systemic immune systems thatresult from exposure to toxic substances including chemicalwarfare agents. Observations in humans and animal studieshave clearly demonstrated that a number of environmentaland industrial chemicals can adversely affect the immunesystem. Alteration in the immune system may result ineither immunosuppression or exaggerated immune reaction.Immunosuppression may lead to the increased incidence orseverity of infectious diseases or cancer, since the immunesystem’s ability to respond adequately to invading agents issuppressed. Toxic agent-induced immunostimulation cancause autoimmune diseases, in which healthy tissue isattacked by an immune system that fails to differentiate self-antigens from foreign antigens. For example, the pesticidedieldrin induces an autoimmune response against red bloodcells, resulting in hemolytic anemia. Immunotoxicologydeals with the effects of toxic substances and explores themechanisms underlying these effects in a biological system.

Although immunotoxicology is a relatively new field,a considerable amount of data has accumulated during thepast few years on immunotoxicity of certain xenobiotics.The majority of the research thus far carried out has been onenvironmental contaminants. Thus, from the defense pointof view considerable work is required to investigate theimmunotoxicity of several chemicals and some bacterialand fungal toxins which may be potential chemical warfareagents. Furthermore, there are several chemicals used in thedefense industry to which the defense industrial workersmay be constantly exposed. These chemicals, followinglow-level exposure to humans and animals, may causeimmunological alterations. Thus immunotoxicity studies onsuch chemicals are being conducted to understand thepotential risks of such exposure on the host’s defense as wellas the cellular and molecular mechanism of such immuno-modulatory action.

A chemical warfare agent (CWA) is a substance which isintended for use in military operations to kill, seriouslyinjure, or incapacitate people because of its toxicologicaleffects. Although CWAs have been widely condemnedsince their first use on a massive scale during World War I,they have been used in many conflicts during the 20th

Handbook of Toxicology of Chemical Warfare Agents

century. As chemical weapons are cheap, relatively easy toproduce and can result in mass casualties, they will continueto be used in future wars and terrorist attacks.

Although most of the compounds of CWAs are notpersistent in the environment, repeated exposure andpersistence of some of the compounds result in immuno-toxicity. This chapter describes the immunotoxicity ofCWAs and gives an insight into the probable mechanisms ofsuch effects.

II. THE IMMUNE SYSTEM

The immune system is composed of several organs, cells,and noncellular components which act in an interrelatedmanner to protect the host against foreign organisms andchemical substances. The immune system participates in themechanisms responsible for the maintenance of homeostasisand an altered immune system reflects the adverse changesin both internal and external microenvironments. Theimmune system protects organisms against pathogens orother innocuous substances like pollens, chemicals, indoormolds, potential food allergens, and environmental agents,and acts as layered defenses of increasing specificity. Mostsimply, physical barriers (e.g. skin) prevent pathogens andxenobiotics from entering the organism. If they breach thesebarriers, the innate immune system provides an immediatebut nonspecific response. However, if pathogens success-fully evade the innate response, there is a third layer ofprotection, i.e. the adaptive immune system, which is acti-vated by the innate response. Here, the immune systemadapts during an infection to improve its recognition of thepathogen and its response is then retained after the pathogenor xenobiotic has been eliminated. This immunologicalmemory allows the adaptive immune system to respondfaster with a stronger attack each time the same insult isencountered (Kindt et al., 2007).

The immune system protects organisms from infectionwith layered defenses of increasing specificity. The layereddefense includes mechanical, chemical, and biologicalbarriers which protect organisms from toxic substances.Skin, a mechanical barrier, acts as the first line of defenseagainst infection. In the lungs, coughing and sneezingmechanically eject pathogens and other irritants from the

Copyright 2009, Elsevier Inc.All rights of reproduction in any form reserved.5

596 SECTION III $ Target Organ Toxicity

respiratory tract while mucus secreted by the respiratory andgastrointestinal tract traps and entangles microorganismsand other toxins (Boyton and Openshaw, 2002). Chemicalbarriers also protect against infection. The skin and respi-ratory tract secrete antimicrobial peptides such as the b-defensins. Enzymes such as lysozyme and phospholipaseA2 in saliva, tears, and breast milk are also antibacterials(Moreau et al., 2001; Hankiewicz and Swierczek, 1974). Inthe stomach, gastric acid and proteases serve as powerfulchemical defenses against ingested pathogens.

A. The Innate Immune System

The innate immune system defends the host from infectionand toxicants, in a nonspecific manner. This means that thecells of the innate system recognize, and respond ina generic way, but do not confer long-lasting or protectiveimmunity to the host. The innate immune response wasinitially dismissed by the immunologist as it was thought toprovide a temporary holding of the situation until a moreeffective and specific adaptative immune responsedevelops. But it has now been clear that it plays an impor-tant role as a dominant system of host defense in mostorganisms (Litman et al., 2005). The major function of theinnate immune system is to recruit immune cells to sites ofinfection and inflammation. Inflammation is one of the firstresponses of the immune system to infection or irritationthrough the production of cytokines. These cytokinesreleased by injured cells serve to establish a physical barrieragainst the spread of infection. Several chemical factors areproduced during inflammation, e.g. histamine, bradykinin,serotonin, leukotrienes, and prostaglandins, which sensitizepain receptors, cause vasodilation of the blood vessels, andattract phagocytes. The inflammatory response is charac-terized by the redness, heat, swelling, pain, and possibledysfunction of the organs or tissues involved. The fluidexudate contains the mediators for four proteolytic enzymecascades: the complement system, the coagulation system,the fibrinolytic system, and the kinin system. The exudate iscarried by lymphatics to lymphoid tissue, where the productof foreign organism can initiate an immune response.

The activation of the complement cascade helps toidentify the invading substance, activate cells, and promoteclearance of dead cells by specialized white blood cells. Thecascade is composed of nine major components, designatedC1 to C9, which are plasma proteins synthesized in the liver,primarily by hepatocytes. These proteins work together totrigger the recruitment of inflammatory cells. One of themain events is the splitting of the C3, which gives rise tovarious peptides. One of them, C3a (anaphylatoxin), canstimulate mast cells to secrete chemical mediators andanother, C3b (opsonin), can attach to the surface of a foreignbody and facilitates its ingestion by white blood cells. C5 isa powerful chemotactic of white cells and causes release ofmediators from mast cells. Later components from C5 to C9assemble in a sequence at the surface of bacteria/

xenobiotics and lead to their lysis, ridding the body ofneutralized antigen–antibody complexes. The main eventsof this system can also be directly initiated by the principalenzymes of the coagulation and fibrinolytic cascade,thrombin and plasmin, and by enzymes released from whiteblood cells. Further, an innate immune system leads to theactivation of an adaptive immune system.

B. The Adaptive Immune System

The adaptive immune system is composed of highlyspecialized, systemic cells and processes that eliminatepathogenic challenges and provide the ability to recognizeand mount stronger attacks each time the same pathogen isencountered. Antigen specificity requires the recognition ofspecific ‘‘nonself’’ antigens during a process called antigenpresentation. The ability to mount these immune responsesis maintained in the body by ‘‘memory cells’’. The cells ofthe adaptive immune system are special types of leukocytes,B cells and T cells, which constitue about 20–40% of whiteblood cells (WBCs). The peripheral blood contains 20–50%of circulating lymphocytes and the rest move within thelymphatic system (Kindt et al., 2007). B cells and T cells arederived from the same pluripotential hematopoietic stemcells in the bone marrow, and are indistinguishable from oneanother until after they are activated. B cells play a largerole in the humoral immune response, whereas T cells areintimately involved in cell-mediated immune responses. Bcells derive their name from the bursa of Fabricius, an organunique to birds, where the cells were first found to develop.However, in nearly all other vertebrates, B cells (and Tcells) are produced by stem cells in the bone marrow (Kindtet al., 2007). T cells are named after thymus where theydevelop and through which they pass. In humans, approxi-mately 1–2% of the lymphocyte pool recirculates each hourto optimize the opportunities for antigen-specific lympho-cytes to find their specific antigen within the secondarylymphoid tissues. Both B cells and T cells carry receptormolecules that recognize specific targets.

T cells express a unique antigen-binding molecule, the Tcell receptor (TCR), on their membrane. There are two well-defined subpopulations of T cell: T helper (TH) and Tcytotoxic (TC) cells. They can be distinguished from oneanother by the presence of either CD4 or CD8 membraneglycoproteins on their surfaces. T cells displaying CD4generally function as TH cells whereas those displayingCD8 function as TC cells. T cells recognize a ‘‘nonself’’target, such as a pathogen, only after antigens have beenprocessed and presented in combination with a ‘‘self’’receptor called a major histocompatibility complex (MHC)molecule. TC cells only recognize antigens coupled to classI MHC molecules, while TH cells only recognize antigenscoupled to class II MHC molecules.

B cells are the major cells involved in the creation ofantibodies that circulate in blood plasma and lymph, knownas humoral immunity. Like the T cell receptor, B cells

CHAPTER 40 $ Immunotoxicity 597

express a unique B cell receptor (BCR), in this case animmobilized antibody molecule. The BCR recognizes andbinds to only one particular antigen. A critical differencebetween B cells and T cells is how each cell ‘‘sees’’ anantigen. T cells recognize their cognate antigen in a pro-cessed form – as a peptide in the context of an MHCmolecule – while B cells recognize antigens in their nativeform. Once a B cell encounters its cognate (or specific)antigen [and receives additional signals from a helper T cell(predominantly Th2 type)], it further differentiates into aneffector cell, known as a plasma cell.

Plasma cells are short-lived cells (2–3 days) whichsecrete antibodies that circulate in blood plasma and lymph,and are responsible for humoral immunity. Antibodies (orimmunoglobulin, Ig) are large Y-shaped proteins used bythe immune system to identify and neutralize foreignobjects. In mammals there are five types of antibody: IgA,IgD, IgE, IgG, and IgM. Differing in biological properties,each has evolved to handle different kinds of antigens.These antibodies bind to antigens, making them easiertargets for phagocytes, and trigger the complement cascade.About 10% of plasma cells will survive to become long-lived antigen-specific memory B cells (Lu and Kacew,2002). Already primed to produce specific antibodies, thesecells can be called upon to respond quickly if the sameforeign body reinfects the host. This is called ‘‘adaptiveimmunity’’ because it occurs during the lifetime of anindividual as an adaptation to infection with that pathogenand prepares the immune system for future challenges.

A number of animal models have been developed andvalidated to detect the chemical-induced direct immuno-toxicity. Several compounds, including certain drugs, havebeen shown in this way to cause immunosuppression or skinallergic responses. In this chapter, the various mechanismsof immunotoxicity are discussed by which a compoundaffects different cell types and interferes with immuneresponses, ultimately leading to immunotoxicity as well assensitizing capacity.

III. TARGETS OF IMMUNOTOXICITY

A. Effects on Precursor Stem Cells

The bone marrow is an organ with precursor stem cells thatare responsible for synthesizing peripheral leukocytes. Allleukocyte lineages originate from these stem cells, but oncedistinct subsets of leukocytes are established, their depen-dence on replenishment from the bone marrow differsvastly. The turnover of neutrophils is very rapid, i.e. morethan 108 neutrophils enter and leave the circulation ina normal adult daily so there is dependence on newformation in the bone marrow. In contrast, macrophages arelong-lived and have little dependence on new formation ofprecursor cells. The adaptive immune system, comprisingantigen-specific T and B lymphocytes, is almost completely

established around puberty and is therefore essentially bonemarrow independent in the adult.

As a consequence of their high proliferation rate, stemcells in the bone marrow are likely to be extremelyvulnerable to cytostatic drugs and chemicals like CWAs.Lineages like neutrophils with rapid turnover will be mostvulnerable and will be affected first by such treatments/exposures. After prolonged exposure, macrophages and Tor B cells of the adaptive immune system are alsosuppressed.

B. Effects on Maturation of Lymphocytes

T lymphocytes mature in the thymus by a very complexselection process that takes place under the influence of thethymic microenvironment and ultimately generates anantigen-specific, host-tolerant population of mature T cells.This process involves cellular proliferation, gene rearrange-ment, apoptotic cell death, receptor up- and down-regulationand antigen-presentation processes, and is very vulnerable toa number of chemicals. Drugs may target different stagesof T cell differentiation like naıve T cells, proliferatingand differentiating thymocytes, antigen-presenting thymicepithelial cells and dendritic cells, cell death processes, etc.(Vos et al., 1999). In general, immunosuppressive drugs maycause a depletion of peripheral T cells, particularly afterprolonged treatment and during early stages of life whenthymus activity is high and important in establishinga mature T cell population. In addition, suppression of T cellsmay result in suppression of the adaptive immune system byaffecting the maturation of B cells and thus antibody level.

C. Effects on Initiation of Immune Responses

The innate and adaptive immune systems act together toeliminate invading pathogens. Ideally, T cells tailor theresponses to neutralise invaders with minimal damage tothe host. The recognition of autoantigens is maintained bythe two distinct signals that govern lymphocyte activation.One is the specific recognition of antigen via clonallydistributed antigen receptors and the other is antigennonspecific co-stimulation or ‘‘help’’ and involves interac-tions of various adhesive and signaling molecules expressedin response to tissue damage, linking initiation of immuneresponses to situations of acute ‘‘danger’’ for the host (Voset al., 1999). This helps to aim immune responses atpotentially dangerous microorganisms (nonself), whileminimizing deleterious reactions to the host (self). Xeno-biotics, however, can interfere with the initiation of immuneresponses if they act as antigens, by forming haptens or byreleasing previously hidden self-antigens. They may alsotrigger an inflammatory response, or disturb T–B cellcooperation.

CWAs with large molecular weight can function asantigens and become targets of specific immune responsesthemselves. This is particularly relevant for foreign protein


pharmaceuticals, as these can activate both T and Blymphocytes. The resulting immune responses may lead toformation of antibodies, and induce specific memory whichcan lead to allergic responses to the drug. Immunotoxiceffects may occur after repeated treatment with the sameCWA. However, low molecular weight CWAs cannotfunction as antigens, because they are too small to bedetected by T cells. Reactive chemicals that bind to proteins,however, can function as haptens and become immunogenicif epitopes derived from them prime T cells, which in turnprovide co-stimulation for hapten-specific B cells. Thiseffect is responsible for allergic responses to many new(neo) epitopes formed by chemical haptens.

Modification of autoantigens can also lead to autoim-mune responses to unmodified self-epitopes. Haptenatedautoantigens can be recognized and internalized by antigenpresenting cells. These cells subsequently present a mixtureof neo- and self-epitopes complexed to distinct class IImajor histocompatibility (MHC-II) molecules on theirsurface and neospecific T cells. Th cells provide signals forthe B cell. This leads to production of either antihapten oranti-self antibodies depending on the exact specificity of theB cell. Moreover, once these B cells are activated, they canstimulate autoreactive Th cells recognizing unmodified self-epitopes. This process is called epitope (determinant)spreading and causes the diversification of adaptive immuneresponses. For example, injection of mercury salts initiallyinduces response directed only to unidentified chemicallycreated neoepitopes, but after 3–4 weeks include reactivityto unmodified self-epitopes. Thus the allergic response maygradually culminate as autoimmune responses reflecting therelative antigenicity of the neo- and self-epitopes involved(Lu and Kacew, 2002).

IV. EXPOSITION OF AUTOANTIGENSAND INTERFERENCE WITHCO-STIMULATORY SIGNALS

Self-tolerance involves specific recognition of autoantigenleading to selective inactivation of autoreactive lympho-cytes at birth, but tolerance is not established for (epitopesof) autoantigens that are normally not available for immunerecognition. Pharmaceuticals can expose such sequesteredepitopes by disrupting barriers between the antigen and theimmune system (i.e. blood–brain barrier, blood–testisbarrier, cell membranes). Tissue damage, cell death, andprotein denaturation induced by chemicals can largelyincrease the chances of such (epitopes of) autoantigens forimmune recognition. Antigen recognition followed by co-stimulation of signaling molecules leads to activation oflymphocytes and initiation of immune responses. Manyxenobiotics have the inherent capacity to induce or inhibitthis co-stimulation due to their intrinsic adjuvant activity.

V. INDUCTION OF INFLAMMATIONAND NONCOGNATE T–B COOPERATION

Cytotoxic chemicals or their reactive metabolites can inducetissue damage which results in release of proinflammatorycytokines like tumor necrosis factor a (TNFa), interleukin-1(IL-1), and IL-6, and attracts inflammatory cells like gran-ulocytes and macrophages. Cytokines produced during thisinflammatory response activate antigen-presenting cells andaccumulation of tissue debris. The epitopes of antigens ondebris provide co-stimulation for Th cells, which lead to theinitiation of an adaptive immune response. Reactive xeno-biotics may also stimulate adaptive immune responses bydisturbing the normal cooperation of Th and B cells. Nor-mally, B cells receive stimulation from Th cells thatrecognize (epitopes of) the same antigen. However, whenTh cells respond to nonself-epitopes on B cells, such B cellsmay be noncognately stimulated by the Th cell. This occursduring graft-versus-host responses following bone marrowtransplantation, when Th cells of the host recognize nonself-epitopes on B cells of the graft and vice versa. This leads toT and B cell activation and results in production of auto-antibodies to distinct autoantigens like DNA, nucleoli,nuclear proteins, erythrocytes, and basal membranes. Drug/chemical-related lupus is characterized by a similar spec-trum of autoantibodies, and noncognate – graft-versus-host-like – T–B cooperation is therefore suggested to be one ofthe underlying mechanisms.

VI. REGULATION OF THE IMMUNERESPONSE

The type of immune response elicited in response toa foreign pathogen or allergen is the result of a complexinterplay of cytokines produced by macrophages, dendriticcells, mast cells, granulocytes, and lymphocytes. Immuno-toxic chemicals that somehow influence the immune systemcan lead to either immunosuppression or immune exagger-ation, i.e. hypersensitivity and autoimmunity. Hypersensi-tivity is an immune response that damages the body’s owntissues. Hypersensitivity reactions require a pre-sensitized(immune) state of the host. They are divided into four classes(Type I–IV) based on the mechanisms involved and the timecourse of the hypersensitive reaction. Type I hypersensitivityis an immediate or anaphylactic reaction, often associatedwith allergy. Symptoms can range from mild discomfort todeath. Type I hypersensitivity is mediated by IgE releasedfrom mast cells and basophils. Type II hypersensitivityoccurs when antibodies bind to antigens on the patient’s owncells, marking them for destruction. This is also calledantibody-dependent (or cytotoxic) hypersensitivity, and ismediated by IgG and IgM antibodies. Immune complexes(aggregations of antigens, complement proteins, and IgG andIgM antibodies) deposited in various tissues trigger Type IIIhypersensitivity reactions. Type IV hypersensitivity (also


known as cell-mediated or delayed type hypersensitivity)usually takes between 2 and 3 days to develop. Type IVreactions are involved in many autoimmune and infectiousdiseases, but may also involve contact dermatitis (poisonivy). These reactions are mediated by T cells, monocytes,and macrophages. Actual development of clinical symptomsis influenced by the route and duration of exposure, thedosage of the pharmaceutical, and by immunogenetic (MHChaplotype, Th1-type versus Th2-type responders) and phar-macogenetic (acetylator phenotype, sulfoxidizer, Ahreceptor, etc.) predisposition of the exposed individual.Moreover, atopic individuals that tend to mount Th2immune responses are more susceptible to anaphylaxistriggered by an IgE response to chemical haptens thantypical Th1 responders. Genetic variation in metabolism ofpharmaceuticals is important as it determines the formationand clearance of immunotoxic metabolites. The slow acet-ylating phenotype, for instance, predisposes for drug-relatedlupus because reactive intermediates of phase I metabolismhave an increased opportunity to bind proteins as they areonly slowly conjugated.

Immune dysregulation can also be in the form of immunesuppression and both innate and adaptive arms of theimmune systems play crucial roles. A wide variety ofphysiological, pharmacological, and environmental factorscan exert a negative influence on the immune system andsometimes result in immunotoxicity. Recent experimentaldata have shown that emotional and environmental stressorsinfluence the functioning of the immune system and this isreflected in the various markers of specific immunity (Rayet al., 1991; Koner et al., 1998). Such experimental stressorsconsistently suppressed both humoral and cell mediatedimmune responses in experimental animals. Both antibodyforming cell counts and antibody titer were lowered anda neuroendocrine–immune axis concept was proposed.Similar attenuations in cell mediated immune responseswere also seen after such stressors and DTH responses,leukocyte/macrophage migration indices and also cytokineprofiles (both Th1 and Th2 dependent). Further analysis ofthe mechanisms involved indicated that CNS mediatedchanges could have contributed to this immunotoxicity.Depletion or antagonism of brain dopamine aggravatedemotional stress-induced immune suppression, whereaspsychoactive drugs like benzodiazepines and opioids pre-vented this response (Ray et al., 1992; Puri et al., 1994). Inanother set of experiments, rats exposed to several envi-ronmental pollutants like DDT showed graded degrees ofimmune suppression and immunotoxicity, when the expo-sure lasted for a reasonably long period of time. Gradualaccumulation in the various body tissues resulted ina variety of untoward effects in the immune system, whichwas particularly susceptible to such xenobiotic-induceddamage (Banerji et al., 1996; Koner et al., 1998). Bothhumoral and cell mediated immune response were affecteddepending on the quantum and duration of exposure to thesexenobiotics. Further, a combination of emotional stress and

xenobiotic exposure had additive effects on the immuno-toxicity parameters studied (Banerjee et al., 1997). Recentstudies revealed that such emotional stress and xenobiotic-induced immunotoxicity was accompanied by derange-ments in oxidative stress parameters, such as enhancementsin MDA levels and lowering of GSH/SOD levels in theblood (Koner et al., 1997; Ray and Gulati, 2007; Gulatiet al., 2007).

VII. IMMUNOTOXICITY OF CHEMICALWARFARE AGENTS

A chemical warfare agent (CWA) is a substance which isintended for use in military operations to kill, seriouslyinjure, or incapacitate people because of the severe patho-physiological changes induced by them in various bodysystems. A United Nations report from 1969 defineschemical warfare agents as ‘‘chemical substances, whethergaseous, liquid or solid, which might be employed becauseof their direct toxic effects on man, animals and plants’’.However, the Chemical Weapons Convention defineschemical weapons as including not only toxic chemicals butalso ammunition and equipment for their dispersal. Toxicchemicals are stated to be ‘‘any chemical which, through itschemical effect on living processes, may cause death,temporary loss of performance, or permanent injury topeople and animals’’. Normally, they are either liquids orsolids.

Chemical agents have been used in war since timeimmemorial. In 600 BC Helleborus roots were usedsuccessfully by the Athenians to contaminate water suppliesduring the siege of Kirrha. Spartans ignited pitch and sulfurto create toxic fumes during the Peloponnesian War in 429BC. The uses of CWAs in battlefields reached a peak duringWorld War I and the French were the first to use ethyl-bromoacetate. It was followed by o-dianisidine chlor-osulphonate, chloroacetate, chlorine, phosgene, hydrogencyanide, diphenylchloroarsine, ethyl- and methyldi-chloroarsine, and sulfur mustard resulting in nearly 90,000deaths and over 1.3 million casualties (Eckert, 1991).CWAs were most brutally used by the Germans in the gaschambers for mass genocide of Jews during World War II,and have been used intermittently both in war, as in theIraq–Iran War, as well as in terrorist attacks in the Japanesesubway stations. It is estimated that nearly 100,000 UStroops may have been exposed to CWAs during operationDesert Storm (Chauhan et al., 2008).

CWAs have been widely condemned since they were firstused on a massive scale during World War I. However, theyare still stockpiled and used in many countries as they arecheap and relatively easy to produce, and can cause masscasualties. Although the blood agent CK is extremelyvolatile and undergoes rapid hydrolysis, the degradation ofthree types of vesicant CWAs, the sulfur mustards, nitrogenmustards, and Lewisite, results in persistent products. For


example, sulfonium ion aggregates formed during hydro-lysis may be persistent and may retain vesicant properties.The nerve agents include the V agent VX as well as three Gagents (tabun, sarin, and soman). VX gives rise to twohydrolysis products of possible concern: EA 4196, which ispersistent, and EA 2192, which is highly toxic and ispossibly persistent under certain limited conditions (Small,1984). Thus, their long-term persistence in the body maylead to alterations in the immune system of the exposedpopulation.

CWAs can be classified in many different ways. Thereare, for example, volatile substances, which mainlycontaminate the air, or persistent substances, which arenonvolatile and therefore mainly cover surfaces. CWAsmainly used against people may also be divided into lethaland incapacitating categories. A substance is classified as anincapacitating agent if less than 1/100 of the lethal dosecauses incapacitation, e.g. through nausea or visual prob-lems. The limit between lethal and incapacitatingsubstances is not absolute but refers to a statistical average.Chemical warfare agents are generally classified accordingto their principal target organs.

1. Organophosphate (OP) nerve agents. These agents areextremely toxic compounds that work by interfering withthe nervous system, and include soman, sarin, cyclosarin,tabun, and VX.

2. Blister agents/vesicants. These compounds severelyblister the eyes, respiratory tract and skin on exposure,and include nitrogen mustard, sulfur mustard, Lewisite,etc.

3. Choking agents. These agents cause severe irritationprimarily affecting the respiratory tract, and includephosgene, ammonia, methyl bromide, methyl isocyanate,etc.

4. Blood agents. These agents are absorbed into the bloodand interfere with the oxygen carrying capacity, e.g.arsine, cyanides, carbon monoxide, etc.

Very few studies have been conducted to explore theimmunomodulation and immunotoxic potential of CWAs,and there is little evidence that these drugs are associatedwith such undesirable, immunologically significant effects.The reason may be due to confounding factors such asstress, nutritional status, lifestyle, co-medication, andgenetics (Vos et al., 1999). The exposure to CWA can resultin immunodepressed conditions on the one hand and toallergic and autoimmune diseases on the other. Fewconventional compounds have been shown to induce unex-pected enhancement of immune competence. However,introduction of biotechnologically manufactured agents likecytokines has been shown to induce unwanted immunosti-mulation. Drug-induced hypersensitivity reactions andautoimmune disorders are a major concern, whereas some ofthese chemicals also result in immunosuppression. Inparticular, impaired activity of the first line of defense of thenatural immune system can have disastrous consequences.

These are generally not influenced by the genetic predis-position of the exposed individual, but on actual outbreak ofinfections and the general immune status prior to exposition.This explains why immunosupressive xenobiotics are mostlikely to have clinical consequences in immunocompro-mised individuals such as young children, the elderly, andtransplant recipients.

A. Nerve Agents

Nerve agents are highly toxic organophosphoruscompounds (OPs) which represent potential threats to bothmilitary and civilian populations, as evidenced in recentterroristic attacks in Japan (Ohtomi et al., 1996). Commonlyknown as nerve agents or nerve gases, these are the deadliestof CWAs. These agents have both chemical names as wellas two-letter NATO codes. These are categorized as G seriesagents: GA (tabun), GB (sarin), GD (soman), GF (cyclo-sarin), and V series agents: VE, VG, VM, and VX, the letter‘‘G’’ representing the country of origin ‘‘Germany’’ andletter ‘‘V’’ possibly denoting ‘‘Venomous’’. Their initialeffects occur within 1–10 min of exposure followed bydeath within 15–30 min for sarin, soman, and VX, andwithin 30–60 min for tabun. The ease and low cost ofproduction make sarin gas a tool of mass destruction in thehands of terrorist groups and rogue nations. While people inthe immediate vicinity of a sarin attack may receiveneurotoxic doses, people remote from the vicinity are likelyto receive subclinical exposures.

Short- and long-term health effects from exposure to OPnerve agents and insecticide nerve agents are compiled onthe basis of scientific literature published on health effects inhumans and animal studies. Four distinct health effects areidentified: acute cholinergic toxicity; OP-induced delayedneuropathy (OPIDN); subtle long-term neuropsychologicaland neurophysiological effects; and a reversible muscularweakness called ‘‘intermediate syndrome’’. Each effect hasdata suggesting threshold exposure levels below which it isunlikely to be clinically detectable. High-level exposureresults in definitive cholinergic poisoning; intermediate-level threshold cholinergic effects include miosis, rhinor-rhea or clinically measurable depression of cholinesterase;and low-level exposure results in no immediate clinicalsigns or symptoms. Threshold exposure levels for knownlong-term effects from OP nerve agent are at or aboveintermediate-level exposure (Brown and Brix, 1998).However, subclinical doses of sarin cause subtle changes inthe brain, and subclinical exposure to sarin has beenproposed as an etiology to the Gulf War Syndrome.

The wide use of cholinesterase inhibitors in variousspheres of human activities and the risk of acute and chronicintoxications associated with this process prompted inves-tigation of the role of acetylcholinesterase (AChE) andnonspecific esterases in the immunotropic effects of thesechemicals. They irreversibly bind to AChE that normallycatalyzes the hydrolysis of acetylcholine (ACh) at the


cholinergic synapses and neuromuscular junctions (NMJs).The inhibition of degradation results in accumulation ofACh in the cholinergic synapses, causes the overstimulationof peripheral as well as central cholinergic nervous systems,and is clinically manifested as acute cholinergic crisis(convulsions, respiratory failure, and/or death) (Marrs,1993; Taylor, 2006).

1. IMMUNOTOXICITY

Kalra et al. (2002) suggested that low doses of sarin arehighly immunosuppressive, and suppress glucocorticoidproduction. The effects of sarin exposure on the immunesystem are attenuated by ganglionic blockers and decreasedglucocorticoid level may be a biomarker for cholinergictoxicity. In addition, nerve agents cause the activation ofmultiple noncholinergic neurotransmitter systems in thecentral nervous system (CNS) thus resulting in mutagenic,stressogenic, immunotoxic, hepatotoxic, membrane, andhematotoxic effects (Bajgar, 1992). The CNS and theimmune system communicate bidirectionally, and cholin-ergic agents modulate the immune system. The ability of OPcompounds to induce an alteration of the immune systemwas primarily demonstrated in animals or humans exposedto OP insecticides (OPIs). The results provide evidence that,especially neutrophil function, natural killer cell, cytotoxicT cell and humoral immune functions, and spontaneous aswell as mitogen-induced lymphocyte proliferation, arealtered in animals or humans exposed to OP compounds(Casale et al., 1984; Hermanowitz and Kossman, 1984; Liet al., 2002; Newcombe and Esa, 1992). In addition,a decreased number of cells in the spleen and thymus(Ladics et al., 1994), an inhibition of chemotaxis inneutrophils (Ward, 1968), inhibition of monocyte accessoryfunctions or inhibition of interleukin-2 production (Casaleet al., 1993; Pruett and Chambers, 1988) were reportedfollowing the exposure to OPs, at relatively high toxicdoses.

Lee et al. (1979) were the first to draw attention to thepossible effects of OPs on human leukocyte function. Theydemonstrated that lymphocyte proliferation to phytohema-glutinin in vitro was decreased in the presence of OPs.Although most of the studies described the results of OPIexposure, there are studies about the immunotoxic effects ofhighly toxic nerve agents and their by-products. Markedimpairment in neutrophil chemotaxis and neutrophil adhe-sion and a reduction in the natural killer cell and cytotoxic Tcell function were observed in workers exposed to OPIs andby-products of sarin (Hermanowitz and Kossman, 1984;Newcombe and Esa, 1992; Li et al., 2002). Kant et al.(1991) documented a decrease in the weight of thymus, animportant immune organ in severely affected soman survi-vors, but other tests of immune function did not showdifferences between control and soman-exposed rats. Sam-naliev et al. (1996) described a decrease in the number ofplaque forming cells in soman-exposed rats after theadministration of sheep red blood cells as an antigen.

However, Johnson et al. (2002) demonstrated that OP-induced modulation of immune functions can involve notonly their suppression but also their activation. Similaractivation of some immune functions involving ‘‘acutephase response’’ such as an increase in the synthesis of acutephase proteins, increase in release of histamine from baso-phile leukocytes and activation of macrophages wereobserved following the exposure to soman (Sevaljevic et al.,1992; Newball et al., 1986). Although most of the studiesdealt with exposure to high doses, Kassa et al. (2004)confirmed that not only symptomatic but also asymptomaticdoses of nerve agent sarin were able to modify variousimmune functions. The proportion of T lymphocytes wasfound to be decreased, while the B cell levels were raised.However, sarin significantly suppressed nonspecific in vitrostimulated proliferation of both T and B cells, whichsuggests that it can also block normal immune response toinfection. While the lymphocyte mediated immunity israther suppressed, the peritoneal as well as alveolarmacrophages and NK cells were activated after exposure toboth levels of sarin, which was explained to be the result ofcompensatory reactions of immune functions rather than theresult of direct effects of inhalation.

Immunosuppression may result from direct action ofacetylcholine upon the immune system or it may besecondary to the toxic chemical stress associated withcholinergic poisoning (Pruett et al., 1992). Further, immu-nomodulation at low levels seems to be very complex and itis suggested that there are probably other protein targetsvery sensitive to some anticholinesterases including nerveagents. However, the function of these protein targets is notyet known (Ray, 1998). Some immune functions are prob-ably stimulated due to the development of ‘‘acute phaseresponse’’ generally characterized for inflammatory reac-tion of OP-exposed organism (Sevaljevic et al., 1989,1992). Other immune functions are suppressed due toimmunotoxicity of OP compounds. Although these findingsare difficult to extrapolate directly to low-level exposures tonerve agents, they indicate that subtle alteration of immunesystem could also occur in humans at exposure levels whichdo not cause any clinical manifestation. Post-intoxicationimmunodeficiency can promote infectious complicationsand diseases.

It has been shown that T lymphocytes have AChElocated on the plasma membrane, while B cells are esterasenegative (Szelenyi et al., 1982). Thus, AChE inhibition bytoxic agents in sublethal doses may play an important role inimmunodeficiency following exposure to nerve gases.Zabrodskii et al. (2003) showed inhibition of AChE in Tcells and the decrease in the number of esterase-positive Tlymphocytes (and, to a certain extent, in monocytes andmacrophages) directly correlated with suppression of T cell-dependent antibody production and to the degree of DTHreduction, on exposure to dimethyl dichlorovinyl phosphate,sarin, VX, lewisite, tetraethyl lead, and dichloroethane. Thispresumably involves the loss of some functions by T


lymphocytes (e.g. by Th1 cells), which leads to attenuationof T-dependent immune reactions. This can be explained byexcessive acetylcholine (ACh) stimulation of muscarinicand nicotinic receptors present on T lymphocytes, as a resultof which the optimal cAMP to cGMP ratio in immunocytes,essential for their proliferation and differentiation, is dis-torted (Richman and Arnason, 1979). Thus, the anticholin-esterase effect of lewisite, TEL, and DCE may be one of theimportant mechanisms in the formation of T cell mediatedimmunodeficiency.

B. Blister or Vesicant Agents

These agents act on skin and other epithelial tissues andseverely blister the eyes, respiratory tract, and internalorgans, and also destroy different substances within cells ofliving tissue. The symptoms are variable depending uponthe compound and the sensitivity of the individual. Acutemortality is low; however, they can incapacitate the enemyand overload the already burdened health care servicesduring war time. Some of these agents are HD (sulfurmustard), HN (nitrogen mustard), L (Lewisite), and CX(phosgene oximine).

Sulfur mustard (SM) was the most widely used chemicalwarfare agent (CWA) in the Iran–Iraq War, resulting in over100,000 chemical casualties between 1980 and 1988. It actsas an alkylating agent with long-term toxic effects onseveral body organs, mainly the skin, eyes, and respiratorysystem (Willems, 1989). The extent of tissue injury dependson the duration and intensity of exposure. When absorbed inlarge amounts, SM can damage rapidly proliferating cells ofbone marrow and may cause severe suppression of theimmune system (Willems, 1989).

1. IMMUNOTOXICITY

Evidence that SM causes immunosuppression in humanshas emerged from several lines of investigation. The earliestevidence came from clinical observations of humansdirectly exposed to sulfur mustard during World War I, whoshowed significant changes (quantitative and qualitative) inthe circulating elements of the immune system. Stewart(1918) studied ten fatal cases of mustard poisoning andobserved striking depression of bone marrow production ofwhite blood cells. Among the sulfur mustard casualtiesduring the Iran–Iraq conflict leukopenia accompanied bytotal bone marrow aplasia and extensive losses of myeloidstem cells was the most common finding (Balali-Mood,1984; Eisenmenger et al., 1991). These findings providefurther evidence of an association between suppression ofimmunologic functions and an increased incidence ofinfectious disease.

SM has been widely used during Iran–Iraq conflict andthere are many reports of influence of SM on the respiratorysystem, gastrointestinal system, and endocrine system aswell as the immune system (Balali-Mood, 1984; Balali-Mood and Farhoodi 1990; Emad and Razaian 1997; Sasser

et al., 1996; Budiansky, 1984). The influence of SM on theimmune system has been the subject of many researcherssince 1919 (Krumbhaar and Krumbhaar, 1919; Hektoen andCorper 1921). Early investigations on SM casualties duringthe Iran–Iraq War showed decreased immunoresponsive-ness, expressed as leukopenia, lymphopenia, and neu-tropenia, as well as hypoplasia and atrophy of the bonemarrow (Willems, 1989; Tabarestani et al., 1990; Balali-Mood et al., 1991). Chronic exposure to SM has beenassociated with the impairment of NK cells among workersof poison gas factories in Japan (Yokogama, 1993). Simi-larly, cell mediated immunity was found to be suppressedfollowing mustard gas exposure (Zandieh et al., 1990).

Leukopenia has been the first manifestation to appearwithin the first days of post-exposure. Thrombocytopeniaand anemia followed later if the patients survived (whiteblood cells of some patients dropped to less than 1,000 percm3). Although most of these patients suffered skin burns,clinicians reported cases that had minor skin lesions and yetdeveloped leukopenia. Bone marrow biopsies revealedhypocellular marrow and cellular atrophy involving allelements (Willems, 1989). Studies on the status of immu-nocompetent cells in the blood of patients exposed to sulfurmustard showed that T cell and monocyte counts dropped in54% and 65% of the patients, respectively, from day 1 andup to 7th week post-exposure (Hassan and Ebtekar, 2002).Eosinophil counts dropped in 35% and neutrophil numbersin 60% of the patients. B lymphocyte counts were normal upto 7th week (Manesh, 1986). The majority of the patientsshowed increased levels of IgG and IgM during the 1stweek, but the percentage decreased over the next 6 months.The percentage of patients with increased levels of C3, C4,and CH50 was somewhat higher than of healthy controlsduring the 1st week and up to 6th month (Tabarestani et al.,1990) and remained higher 3 years post-exposure especiallyin the severely affected group. Eight years after exposurethere was a significant increase in the number of atypicalleukocytes (such as myelocytes). The severely affectedgroup presented with significantly lower CD56 NK as wellas CD4 and CD8 counts compared with healthy controls(Yokogama, 1993). Hassan and Ebtekar (2002) reported thatthere was no major difference between the severely affectedpatients and healthy controls concerning CD19 B cells,CD14 monocytes, and CD15 granulocytes. The moderatelyand mildly affected patients did not significantly differ intheir leukocyte subset counts from the control group 8 yearsafter exposure (Mahmoudi et al., 2005). Follow-up studieson the clinical conditions of exposed Iranian victims stillshow that they suffer from three major problems: recurrentinfection, septicemia and death, respiratory difficulties andlung fibrosis, as well as a high incidence of malignancies,septicemia, and death.

Hassan and Ebtekar (2002) suggested that patients withmoderate clinical manifestations may be experiencinga shift from Th1 to Th2 cytokine patterns since leukocytecultures from this patient group showed a decrease in IFN-g


levels. When absorbed in large amounts, SM can damagerapidly proliferating cells of bone marrow and may causesevere suppression of the immune system (Sasser et al.,1996). Moreover, this alkylating agent has been reported toproduce short- and long-term suppression of antibodyproduction in both animals and humans. It also affectscomplement system factors C3 and C4. Incidences of acutemyelocytic and lymphocytic leukemia are reported to be 18and 12 times higher in patients exposed to SM, incomparison with the normal group (Zakeripanah, 1991).Willems (1989) reported that exposure to SM could result inthe impairment of human immune function, especially in thenumber of B and T lymphocytes. Hence, SM is stilla potential threat to the world and effective therapeuticmeasures must be taken for the relief of the victims of thisincapacitating agent. Ghotbi and Hassan (2002) showed thatthe percentage of NK cells, playing an important role incellular immunity, was significantly lower in severe patientsthan in the control group. Studies on animal models haveshown that alkylating agents such as SM mainly affect Bcells, which is why hypogammaglobulinemia is one of themain features in animal models, whereas studies on humancases, following a treatment with cytotoxic drugs, suggestthat low-dose exposure to alkylating agents impairs cellularimmunity and high-dose exposure to such agents impairsboth cellular and humoral responses (Marzban, 1989;Malaekeh et al., 1991). There are reports suggesting thatsulfur mustard can produce toxicity through the formationof reactive electrophobic intermediates, which in turncovalently modify nucleophilic groups in biomolecules suchas DNA, RNA, and protein (Malaekeh et al., 1991),resulting in disruption of cell function, especially celldivision (Crathorn and Robert, 1966). As a result, theseagents are particularly toxic to rapidly proliferating cellsincluding neoplastic, lymphoid, and bone marrow cells.Mahmoudi et al. (2005) reported higher IgM levels after 16to 20 years of exposure to SM, compared to the controlgroup. A significant decrease in the number of NK cells insevere patients is probably due to the destructive effect ofthis alkylating agent on NK cell precursors in bone marrow.However, activity of NK cells was found to be noticeablyabove normal which possibly compensates for the reductionin the number of these cells.

Recently, Korkmaz et al. (2006) explained the tox-icodynamics of sulfur mustards in three steps: (1) binding tocell surface receptors; (2) activation of ROS and RNSleading to peroxynitrite (OONO�) production, and (3)OONO�-induced damage to lipds, proteins, and DNA,leading to polyadenosine diphosphate ribose (PARP) acti-vation. This could provide a lead for devising strategies forprotection against/treatment of mustard toxicity.

In conclusion, the results suggest that exposure to SMcauses a higher risk of opportunistic infections, septicemia,and death following severe suppression of the immunesystem especially in the case of lesions and blistersproduced by these agents. As alkylating agents, they form

covalent linkages with biologically important molecules,resulting in disruption of cell function, especially celldivision. As a result, these agents are particularly toxic torapidly proliferating cells including neoplastic, lymphoid,and bone marrow cells. However, there is still a paucity ofinformation regarding the long-term immunosuppressiveproperties of SM in the setting of battlefield exposure to thisagent.

C. Choking Agents

Choking agents act on the pulmonary system causing severeirritation and swelling of the nose, throat, and lungs, e.g. CG(phosgene), DP (diphosgene), chlorine, and PS (chloro-picrin). These inhalational agents damage the respiratorytract and cause severe pulmonary edema in about 4 h,leading to death. The effects are variable, rapid, or delayeddepending on the specific agent (Gift et al., 2008).

Phosgene was first used as a chemical weapon in WorldWar I by Germany and later as offensive capability byFrench, American, and British forces. In this conflict,phosgene was often combined with chlorine in liquid-filledshells, so it was difficult to state the number of casualtiesand deaths attributable solely to phosgene. In militarypublications, it has been referred to as a choking agent,pulmonary agent, or irritant gas. Since World War I, phos-gene has rarely been used by traditional militaries, but theextremist cult Aum Shinrikyo used this agent in an attackagainst the Japanese journalist Shouko Egawa in 1994.Nowadays, phosgene is primarily used in the polyurethaneindustry for the production of polymeric isocyanates(USEPA, 1986). Phosgene is also used in the polycarbonateindustry and in the manufacture of carbamates and relatedpesticides, dyes, pharmaceuticals, and isocyanates.

As mentioned earlier, the primary exposure route forphosgene is by inhalation. Suspected sources of atmosphericphosgene are fugitive emissions, thermal decomposition ofchlorinated hydrocarbons, and photo-oxidation of chloro-ethylenes. Individuals are most likely to be exposed tophosgene in the workplace during its manufacture,handling, and use (USEPA, 1986). Phosgene is extremelytoxic by acute (short-term) inhalation exposure. Severerespiratory effects, including pulmonary edema, pulmonaryemphysema, and death have been reported in humans.Severe ocular irritation and dermal burns may resultfollowing eye or skin exposure. Chronic inhalation exposureto phosgene has been shown to result in some tolerance tothe acute effects noted in humans, but may also causeirreversible pulmonary changes of emphysema and fibrosis(US Department of Health and Human Services, 1993).

Primarily because of phosgene’s early use as a war gas,many exposure studies have been performed over the past100 years to examine the effects and mode of action ofphosgene following a single, acute (less than 24 h) exposure.Many studies have examined the effects of acute phosgene


exposure in animals but the human data are limited to casestudies following accidental exposures.

Most studies were performed in rodents and dogs, withexposure concentrations ranging between 0.5 and 40 ppm(2–160 mg/m3) and duration intervals ranging from 5 min to8 h. Acute exposure studies in animals suggest that rodentspecies may be more susceptible to the edematous effects ofphosgene acute exposure than larger species with lowerrespiratory volumes per body weight such as dogs andhumans (Pauluhn, 2006; Pauluhn et al., 2007).

Pauluhn et al. (2007) reported that acute phosgeneexposure results in increased lung lavage protein, phos-pholipid content, enzyme levels, number of inflammatorycells, and lethality (LC50). Rats seem to be able to surviveapproximately three-fold higher levels of lung edema thanhumans (100-fold versus 30-fold), thus rat responses inshort- and long-term assays may still be relevant to humanseven if it is ultimately shown that rats produce higher levelsof edema following acute phosgene exposure.

1. IMMUNOTOXICITY

Acute exposure to phosgene has been shown to result inimmunosuppression in animals, as evidenced by anincreased susceptibility to in vivo bacterial and tumor cellinfections (Selgrade et al., 1989) and viral infection (Ehr-lich and Burleson, 1991) as well as a decreased in vitrovirus-killing and T cell response (Burleson and Keyes,1989). Selgrade et al. (1989) reported that a single 4 hexposure to phosgene concentrations as low as 0.025 ppmsignificantly enhanced mortality due to streptococcalinfection in mice. Furthermore, when the exposure timewas increased from 4 to 8 h, a significant increase insusceptibility to streptococcus was seen at an exposureconcentration of 0.01 ppm.

Selgrade et al. (1995) administered Streptococcus zooe-pidemicus bacteria via an aerosol spray to the lungs of maleFischer 344 rats immediately after phosgene exposure andmeasured the subsequent clearance of bacteria. They alsoevaluated the immune response, as measured by an increasein the percentage of polymorphonuclear leukocytes (PMN),in lung lavage fluid of uninfected rats similarly exposed tophosgene. This experiment showed that all phosgeneconcentrations from 0.1 to 0.5 ppm impaired resistance tobacterial infection and that the immune response is stimu-lated by phosgene exposure. After 4 weeks followingexposure, bacterial resistance as well as immune responsereturned to normal.

Yang et al. (1995) also reported a decrease in bacterialclearance in the lungs at 24 h after infection followinga single 6 h exposure to phosgene concentrations of 0.1 and0.2 ppm. In comparison with single exposures, the multipledaily exposures extending to 4 and 12 weeks in the Selgradeet al. (1995) report showed a slight enhancement of effect inthe 0.1 ppm group at 24 h post-infection, but no ‘‘adapta-tion’’, or lessening of the effect. Yang et al. (1995) foundthat if the bacteria are administered 18 h after single

phosgene exposures rather than immediately, the clearanceis normal which indicates that recovery from the toxic effectof phosgene is rapid.

When inhaled, phosgene either is rapidly hydrolyzed toHCl and CO2 and exhaled (Schneider and Diller, 1989;Diller, 1985) or penetrates deep into the lungs and is elim-inated by rapid reactions with nucleophilic constituents ofthe alveolar region (Pauluhn et al., 2007). As phosgene iselectrophilic, it reacts with a wide variety of nucleophiles,including primary and secondary amines, hydroxy groups,and thiols. In addition, it also reacts with macromolecules,such as enzymes, proteins, or other polar phospholipids,resulting in a marked depletion of glutathione (Sciuto et al.,1996) and forms covalent adducts that can interfere withmolecular functions. Phosgene interacts with biologicalmolecules through two primary reactions: hydrolysis tohydrochloric acid and acylation reactions. Although thehydrolysis reaction does not contribute much to its clinicaleffects, the acylation reaction is mainly responsible for theirritant effects on mucous membranes. The acylation reac-tions occur between highly electrophilic carbon moleculesin phosgene and amino, hydroxyl, and sulfhydryl groups onbiological molecules. These reactions can result inmembrane structural changes, protein denaturation, anddepletion of lung glutathione. Acylation reactions withphosphatidylcholine are particularly important as it isa major constituent of pulmonary surfactant and lung tissuemembranes. Exposure to phosgene has been shown toincreases the alveolar leukotrienes, which are thought to beimportant mediators of phosgene toxicity to the alveolar–capillary interface. Phosgene exposure also increases lipidperoxidation and free radical formation. These processesmay lead to increased arachidonic acid release and leuko-triene production. Proinflammatory cytokines, such asinterleukin-6, are also found to be substantially higher 4–8 hafter phosgene exposure. In addition, studies have shownthat post-exposure phosphodiesterase activity increases,leading to decreased levels of cyclic AMP. Normal cAMPlevels are believed to be important for maintenance of tightjunctions between pulmonary endothelial cells and thus forprevention of vascular leakage into the interstitium.Oxygenation and ventilation both suffer, and breathing isdramatically increased.

Schneider and Diller (1989) and Diller (1985) reportedthat inhalation of phosgene at high concentrations results ina sequence of events, including an initial bioprotectivephase, a symptom-free latent period, and a terminal phasecharacterized by pulmonary edema. The first is an imme-diate irritant reaction likely caused by the hydrolysis ofphosgene to hydrochloric acid on mucous membranes,which results in conjunctivitis, lacrimation, and oropha-ryngeal burning sensations. This symptom complex occursonly in the presence of high-concentration (>3–4 ppm)exposures but does not have any prognostic value for thetiming and severity of later respiratory symptoms. The mostimportant finding to identify during this stage is a laryngeal


irritant reaction causing laryngospasm, which may lead tosudden death. The irritant symptoms last only a few minutesand then resolve as long as further exposure to phosgeneceases.

The second phase, when clinical signs and symptoms aregenerally lacking, may last for several hours after phosgeneexposure. The duration of the latent phase is an extremelyimportant prognostic factor for the severity of the ensuingpulmonary edema. Patients with a latent phase of less than4 h have a poor prognosis. Increased physical activity mayshorten the duration of the latent phase and worsen theoverall clinical course. Unfortunately, there are no reliablephysical examination findings during the latent phase topredict its duration. However, histologic examinationreveals the beginnings of an edematous swelling, withexudation of blood plasma into the pulmonary interstitiumand alveoli. This may result in damage to the alveolar type Icells and a rise in hematocrit. The length of this phase variesinversely with the inhaled dose. The third clinical phasepeaks approximately 24 h after an acute exposure and iflethality does not occur, recedes over the next 3–5 days. Inthe third clinical phase of phosgene toxicity, the accumu-lating fluid in the lung results in edema. Oxygenation andventilation both suffer, and the breathing is dramaticallyincreased. Often positive end expiratory pressure (PEEP) isrequired to stent open alveoli that would otherwise collapseand result in significant ventilation/perfusion (V/Q)mismatch. This hyperventilation causes the protein-richfluid to take on a frothy consistency. A severe edema mayresult in an increased concentration of hemoglobin in theblood and congestion of the alveolar capillaries.

Increased levels of protein in bronchoalveolar lavagehave been shown to be among the most sensitive endpointscharacterizing the early, acute effects of phosgene exposure,and are rapidly reduced after the cessation of exposure(Sciuto, 1998; Schiuto et al., 2003). With continuous,chronic, low-level phosgene exposure, there may be tran-sition of edema to persistent cellular inflammation leadingto the synthesis of abnormal Type I collagen and pulmonaryfibrosis. An increased synthesis of Type I relative to TypeIII collagen can lead to chronic fibrosis (Pauluhn et al.,2007). Surfactant lipids are important for maintainingalveolar stability and for preventing pulmonary edema.Pauluhn et al. (2007) reported that the induction of surfac-tant abnormalities following phosgene exposures is a keypathophysiological event leading to pulmonary edema andchronic cellular inflammation, leading to the stimulation offibroblasts and the synthesis of ‘‘abnormal’’ collagen inpulmonary fibrosis. As discussed earlier, a breach in thechemical layer of defense followed by pulmonary edemamay lead to a cascade of other immunological responses/reactions. There are limited studies, in both humans andexperimental animals, to evaluate immunotoxicity ofchronic low-level environmental exposures to phosgene.The lack of studies examining the effects in humans orlaboratory animals from chronic exposure to phosgene is

a concern and the sequela of effects leading to phosgene-induced pulmonary fibrosis is not well understood.

D. Blood Agents

Agents like SA (arsine), cyanide, and carbon monoxide areabsorbed into the blood and affect its oxygen carryingcapacity, and are thus termed blood agents. They are highlyvolatile and rapidly acting, and produce seizures, respiratoryfailure, and cardiac arrest. Hydrogen cyanide has beenknown as a potent toxicant for over 200 years. It was used asa chemical warfare agent during World War I by France.Although it is highly volatile (and was later considered‘‘militarily useless’’ because of its volatility), no deathsfrom its military use during World War I were ever reported.After World War II, the importance of hydrogen cyanide asa chemical warfare agent diminished rapidly, primarily asa result of the rise of nerve agents. Although reduced inimportance, there are some reports of hydrogen cyanidebeing used as a war gas by Vietnamese forces in Thailandterritories and during the Iran–Iraq War in the 1980s (Sidell,1992).

Hydrogen cyanide can be detoxified rapidly by humans.It is very volatile and massive amounts of the gas are neededfor it to be effective as a chemical warfare agent. Cyanide isprimarily an environmental contaminant of industrialprocesses. It is used in the metal-processing industry forelectroplating, heat treating, and metal polishing and can befound in waste waters from many mining operations that usecyanide compounds in the extraction of metals, such as goldand silver, from ore.

The acute toxicity of cyanide has been well documentedin humans and experimental animals. Symptoms of toxicityin humans include headache, breathlessness, weakness,palpitations, nausea, giddiness, and tremors (Gupta et al.,1979). Depending on the degree of intoxication, symptomsmay include ‘‘metallic’’ taste, anxiety and/or confusion,headache, vertigo, hyperpnea followed by dyspnea,convulsions, cyanosis, respiratory arrest, bradycardia, andcardiac arrest. Death results from respiratory arrest (Berlin,1977). Onset is usually rapid. Effects on inhalation of lethalamounts may be observed within 15 s, with death occurringin less than 10 min. Hydrogen cyanide should be suspectedin terrorist incidents involving prompt fatalities, especiallywhen the characteristic symptoms of nerve agent intoxica-tion are absent. Chronic exposure to low-level cyanide canresult in neuropathies, goiter, and diabetes. Cyanide andderivatives prevent the cells of the body from using oxygen.Cyanide acts by binding to mitochondrial cytochromeoxidase, blocking electron transport, thus inhibitingenzymes in the cytochrome oxidase chain and in turnblocking oxygen use in metabolizing cells and preventingthe use of oxygen in cellular metabolism. These chemicalsare highly toxic to cells and in high doses may result indeath. Cyanide is more harmful to the heart and brain asthese organs require large amounts of oxygen.


1. IMMUNOTOXICITY

There are very few reports on immunotoxicity of thecompound; however, acrylonitrile (vinyl cyanide, VCN), anenvironmental pollutant which is metabolized to cyanide,has been shown to be an animal and human carcinogenparticularly for the gastrointestinal tract (Mostafa et al.,1999; National Toxicology Program Technical ReportSeries, 2001). Earlier Hamada et al. (1998) evaluated thesystemic and/or local immunotoxic potential of VCN anddemonstrated that VCN induces immunosuppression asevident by a decrease in the plaque forming cell (PFC)response to SRBCs (sheep red blood cells), a markeddepletion of spleen lymphocyte subsets, as well as bacterialtranslocation of the normal flora leading to brachial lymphnode abscess. These results suggested that VCN hasa profound immunosuppressive effect which could bea contributing factor in its gastrointestinal tractcarcinogenicity.

VIII. CONCLUDING REMARKSAND FUTURE DIRECTION

The immune system is extremely vulnerable to the action ofxenobiotics for several reasons. The immune response isassociated with rapidly multiplying cells and synthesis ofregulatory/effector molecules and the immune systemworks as an amplifier for this integrated informationnetwork. Immunologic tissue damage can result from acti-vation of the cellular and biochemical systems of the host.The interactions of an antigen with a specific antibody orwith effector lymphocytes trigger the sequence of humoraland cellular events to produce the pathophysiologic effectsthat lead to tissue injury or disease. Stem cells often appearto be sensitive targets for therapeutic and environmentaltoxicants, most likely because of their rapid proliferation.Xenobiotics or various drugs that are toxic to the myelo-cytes of the bone marrow can cause profound immunosup-pression due to loss of stem cells.

Humans are now under sustained and increasing pressureof xenobiotics exposure. Xenobiotics can stimulate theimmune system as antigens by provoking a substantialimmune response. Even mild disturbances of this networkcould result in detrimental health effects. The influence ofthe xenobiotics on the immune system is either suppressiveor enhancing. The former leads into immunosuppressionwith consequent increased susceptibility to infection andcancer. The latter is associated with the development ofautoimmune reactivity such as delayed hypersensitivity,atopy, systemic or organ-specific immunopathology, andgranulomas formation. It is likely that overall immunosup-pressive effects of xenobiotics are caused by the interfer-ence with cellular proliferation and differentiation, down-regulation of the cytokine signaling, and enhanced apoptosisof immune cells. In contrast, autoimmune reactions areinduced by abnormal activation of immune cells followed

by dysregulated production of cytokines resulting in harm-ful inflammatory response.

The field of immunotoxicology is new but developingrapidly. Attempts must be made to conduct basic research toaddress the cellular and molecular mechanism of immuno-modulatory action of various xenobiotics. The newlyemerging technologies such as genomics, proteomics, andbioinformatics will be certainly helpful to investigate theinteractions between the immune system and xenobiotics intheir full complexities. Toxic compounds may be antigenicor act as haptens and can evoke an antibody response. Ifthese antibodies bind to the determinant on the parentmolecule which is responsible for causing toxicity, then itcan lead to the biological inactivation of the parent moleculeand thereby prevent toxicity. This may constitute animmunological antidote approach to neutralize the toxicityof certain compounds. Thus, passive administration of theantibodies may be used to prevent the toxic effects of thespecific compound and this approach may be useful inbiological or chemical warfare to protect against the toxicityof known chemicals or toxins. The antibodies can also beused to protect industrial workers against the toxic effects ofknown chemicals or gases during accidental exposure.Although this assumption seems logical, it will involveelaborate and time-consuming research to identify the siteof the parent molecule responsible for causing toxicity, tochemically link the molecule with a large protein moleculewhich should be immunogenic but not toxic, and to screenvarious antibodies raised for their capacity to prevent thetoxicity of the compound.

In view of the current global scenario, it appears thatCWAs are likely to be used in different types of warfare, andit is unlikely their usage will cease in the near future. CWAsfor warfare and other related activities are here to stay.These agents are not only inexpensive but easy to dissem-inate with the help of unsophisticated devices. Hence themedical profession should assemble on a common platformthrough globally recognized organizations like the WHOand put in efforts to monitor, research, and study thescientific and medical aspects of CWAs in the interest ofhumankind. Guidelines should be regularly updated on theprevention and management of CWA-induced insults andthereby aim to reduce morbidity and mortality. Nationsworldwide should ensure that adequate supplies of antidotes(wherever available), protective equipment, and decon-tamination devices are available in adequate quantities andat all times. The need of the hour is a multisectorialapproach involving health, defense, agriculture, and envi-ronmental specialists, with clearly defined roles of each, forestablishing and maintaining effective, robust, and sustain-able strategies to countermeasure this threatening situation.

References

Bajgar, J. (1992). Biological monitoring of exposure to nerveagents. Br. J. Ind. Med. 49: 648–53.


Balali-Mood, M. (1984). Clinical and laboratory findings inIranian fighters with chemical gas poisoning. In Proceeding ofthe First World Congress on Biological and ChemicalWarfare, Toxicological Evaluation (A. Heyndricks, ed.), 254,Ghent University Press, Ghent.

Balali-Mood, M., Farhoodi, M. (1990). Hematological findings ofsulfur mustard poisoning in Iranian combatants. Med. J. Islam.Repub. Iran 413: 185–96.

Balali-Mood, M., Tabarestani, M., Farhoodi, M., Panjvani, F.K.(1991). Study of clinical and laboratory findings of sulfurmustard in 329 war victims. Med. J. Islam. Repub. Iran 34:7–15.

Banerjee, B.D., Koner, B.C., Ray, A. (1996). Immunotoxicity ofpesticides: pespectives and trends. Indian J. Exp. Biol. 34:723–33.

Banerjee, B.D., Koner, B.C., Ray, A. (1997). Influence of stress onDDT-induced modulation of the humoral immune respon-siveness in mice. Environ. Res. 74: 43–7.

Berlin, C. (1977). Cyanide poisoning – a challenge. Arch. Intern.Med. 137: 993–4.

Boyton, R., Openshaw, P. (2002). Pulmonary defenses to acuterespiratory infection. Br. Med. Bull. 61: 1–12.

Brown, M.A., Brix, K.A. (1998). Review of health consequencesfrom high-, intermediate- and low-level exposure to organo-phosphorus nerve agents. J. Appl. Toxicol. 18: 393–408.

Budiansky, S. (1984). Chemical weapons: United Nations accusesIraq of military use. Nature 308: 483.

Burleson, G.R., Keyes, L.L. (1989). Natural killer activity inFischer-344 rat lungs as a method to assess pulmonaryimmunocompetence: immunosuppression by phosgene inha-lation. Immunopharm. Immunother. 11: 421–43.

Casale, G.P., Cohen, S.D., Di Capua, R.A. (1984). Parathion-induced suppression of humoral immunity in inbred mice.Toxicol. Lett. 23: 239–47.

Casale, G.P., Vennerstrom, J.L., Bavari, S., Wang, T.L. (1993).Inhibition of interleukin-2 driven proliferation of mouseCTLL2 cells, by selected carbamate and organophosphateinsecticides and congeners of carbaryl. Immunopharmacol.Immunotoxicol. 15: 199–215.

Chauhan, S. Chauhan, S., D’Cruzf, R., Faruqic, S., Singhd, K.K.,Varmae, S., Singha, M., Karthike, V. (2008). Chemical warfareagents. Environ. Toxicol. Pharmacol. 26: 113–22.

Crathorn, A.R., Robert, J.J. (1966). Mechanism of cytotoxicaction of alkylating agents in mammalian cells and evidencefor the removal of alkylated groups from deoxyribonuclei acid.Nature 211: 150–3.

Diller, W.F. (1985). Pathogenesis of phosgene poisoning. Toxicol.Ind. Health 1: 7–15.

Eckert, W.G. (1991). Mass death by gas or chemical poisoning.Am. J. Forensic Med. Pathol. 12: 119.

Ehrlich, J.P., Burleson, G.R. (1991). Enhanced and prolongedpulmonary influenza virus infection following phosgene inha-lation. J. Toxicol. Environ. Health 34: 259–73.

Eisenmenger, W., Drasch, G., von Clarmann, M., Kretschmer,E., Roider, G. (1991). Clinical and morphological findings onmustard gas [bis(2-chloroethyl)sulfide] poisoning. J. ForensicSci. 36: 1688–98.

Emad, A., Razaian, G.R. (1997). The diversity of the effect ofsulfur mustard gas inhalation on respiratory system 10 yearsafter a single heavy exposure; analysis of 197 cases. Chest112(3): 734–8.

Ghotbi, L., Hassan, Z. (2002). The immunostatus of natural killercells in people exposed to sulfur mustard. Int. Immuno-pharmacol. 2: 981–5.

Gift, J.S., McGaughy, R., Singh, D.V., Sonawane, B. (2008).Health assessment of phosgene: approaches for derivation ofreference concentration. Regul. Toxicol. Pharmacol. 51:98–107.

Gulati, K., Ray, A., Debnath, P.K., Bhattacharya, S.K. (2002).Immunomodulatory Indian medicinal plants. J. Nat. Remed. 2:121–31.

Gulati, K., Chakraborti, A., Ray, A. (2007). Stress-inducedmodulation of the neuro-immune axis and its regulation bynitric oxide (NO) in rats. In Proceedings of 40th AnnualConference of the Indian Pharmacological Society, NIPER,Mohali, p. 76.

Gupta, B.N., Clerk, S.H., Chandra, H., Bhargava, S.K., Mahendra,P.N. (1979). Clinical studies on workers chronically exposed tocyanide. Indian J. Occup. Health 22: 103–12.

Hamada, F.M., Abdel-Aziz, A.H., Abd-allah, A.R., Ahmed, A.E.(1998). Possible functional immunotoxicity of acrylonitrile(VCN). Pharmacol. Res. 37: 123–9.

Hankiewicz, J., Swierczek, E. (1974). Lysozyme in human bodyfluids. Clin. Chim. Acta 57: 205–9.

Hassan, Z.M., Ebtekar, M. (2002). Immunological consequence ofsulfur mustard exposure. Immunol. Lett. 83: 151–2.

Hektoen, H., Corper, H.J. (1921). The effect of mustard gas onantibody formation. J. Infect. Dis. 192: 279.

Hermanowitz, A., Kossman, S. (1984). Neutrophil function andinfectious disease occupationally exposed to phosphoorganicpesticides: role of mononuclear-derived chemotactic factor forneutrophils. Clin. Immunol. Immunopathol. 33: 13.

Johnson, V.J., Rosenberg, A.M., Lee, K., Blakley, B.R. (2002).Increased T-lymphocyte dependent antibody production infemale SJL/J mice following exposure to commercial grademalathion. Toxicology 170: 119–29.

Kalra, R., Singh, S.P., Razani-Boroujerdi, S., Langley, R.J.,Blackwell, W.B., Henderson, R.F., Sopori, M.L. (2002).Subclinical doses of the nerve gas sarin impair T cell responsesthrough the autonomic nervous system. Toxicol. Appl. Phar-macol. 184: 82–7.

Kant, G.J., Shih, T.M., Bernton, E.W., Fein, H.G., Smallridge,R.C., Mougey, E.H. (1991). Effects of soman on neuroen-docrine and immune function. Neurotoxicol. Teratol. 13:223–8.

Kassa, J., Krocova, Z., Sevelova, L., Sheshko, V., Kasalova, I.,Neubauerova, V. (2004). The influence of single or repeatedlow-level sarin exposure on immune functions of inbredBALB/c mice. Basic Clin. Pharmacol. Toxicol. 94: 139–43.

Kindt, T.J., Goldsby, R.A., Osborne, B.A. (2007). Cells andorgans of immune system. In Kuby Immunology, pp. 23–49.W.H. Freeman and Co., New York.

Koner, B.C., Banerjee, B.D., Ray, A. (1997). Effect of oxygenfree radicals on immune responsiveness in rabbits: an in vivostudy. Immunol. Lett. 59: 127–31.

Koner, B.C., Banerjee, B.D., Ray, A. (1998). Organochlorineinduced oxidative stress and immune suppression in rats.Indian J. Exp. Biol. 36: 395–8.

Korkmaz, A., Yaren, H., Topal, T., Oter, S. (2006). Moleculartargets against mustard toxicity: implication of cell surfacereceptors, peroxynitrite production and PARP activation. Arch.Toxicol. 80: 662–70.


Krumbhaar, E.B., Krumbhaar, H.D. (1919). The blood and bonemarrow in yellow cross gas (mustard gas) poisoning. J. Med.Res. 40: 497–506.

Ladics, G.S., Smith, C., Heaps, K., Loveless, S.E. (1994). Eval-uation of the humoral immune response of CD rats followinga 2-week exposure to the pesticide carbaryl by the oral, dermalor inhalation routes. J. Toxicol. Environ. Health 42: 143–56.

Lee, T.P., Moscati, R., Park, B.H. (1979). Effects of pesticides onhuman leucocyte functions. Res. Commun. Chem. Pathol.Pharmacol. 23: 597–605.

Li, Q., Nagahara, N., Takahashi, H., Takeda, K., Okumura, K.(2002). Organophosphorus pesticides markedly inhibit theactivities of natural killer, cytotoxic T lymphocyte andlymphocyte-activated killer: a proposed inhibiting mechanismvia granzyme inhibition. Toxicology 172: 181–90.

Litman, G., Cannon, J., Dishaw, L. (2005). Reconstructingimmune phylogeny: new perspectives. Nat. Rev. Immunol. 5:866–79.

Lu, F.C., Kacew, S. (2002). Toxicolology of the immune system. InLu’s Basic Toxicology, pp. 155–67. Taylor and Francis, London.

Mahmoudi, M., Hefazi, M., Rastin, M., Balali-Mood, M. (2005).Long-term hematological and immunological complications ofsulfur mustard poisoning in Iranian veterans. Int. Immuno-pharmacol. 5: 1479–85.

Malaekeh, M., Baradaran, H., Balali, M. (1991). Evaluation ofglobulin and immunoglobulins in serum of victims of chemicalwarfare in late phase of intoxication with sulfur mustard. In 2ndCongress of Toxicology, University of Tabrize Press, Tabrize.

Manesh, M. (1986). Evaluation of the immune system of patientexposed to sulfur mustard. Dissertation, University of Tehran,Teheran.

Marrs, T.C. (1993). Organophosphate poisoning. Pharmacol.Ther. 58: 51–66.

Marzban, R.S. (1989). Treatment of Chemical Warfare Victims.Jahad Daneshgahi Press, Teheran.

Moreau, J., Girgis, D., Hume, E., Dajcs, J., Austin, M., O’Call-aghan, R. (2001). Phospholipase A2 in rabbit tears: a hostdefense against Staphylococcus aureus. Invest. Ophthalmol.Vis. Sci. 42: 2347–54.

Mostafa, A.M., Abdel-Naim, A.B., Abo-Salem, O., Abdel-Aziz,A.H., Hamada, F.M. (1999). Renal metabolism of acrylonitrileto cyanide: in vitro studies. Pharmacol. Res. 40: 195–200.

National Toxicology Program Technical Report Series (2001).Toxicology and carcinogenesis studies of acrylonitrile (CASNo. 107–13-1) in B6C3F1 mice (gavage studies), 506: 1–201.

Newball, H.H., Donlon, M.A., Procell, L.R., Helgeson, E.A.,Franz, D.R. (1986). Organophosphate-induced histaminerelease from mast cells. J. Pharmacol. Exp. Ther. 238: 839–46.

Newcombe, D.S., Esa, A.H. (1992). Immunotoxicity of organo-phosphorus compounds. In Clinical Immunotoxicology (D.S.Newcombe, N.R. Rose, J.C. Bloom, eds), pp. 349–63. RavenPress, New York.

Ohtomi, S., Takase, M., Kumagai, F. (1996). Sarin poisoning inJapan. A clinical experience in Japan Self Defense Force(JSDF) Central Hospital. Int. Rev. Arm. For. Med. Ser. 69: 97.

Parrish, J.S., Bradshaw, D.A. (2004). Toxic inhalational injury:gas, vapor and vesicant exposure. Respir. Care Clin. North Am.10: 43–58.

Pauluhn, J. (2006). Acute head-only exposure of dogs to phos-gene. Part III: Comparison of indicators of lung injury in dogsand rats. Inhal. Toxicol. 18: 609–21.

Pauluhn, J., Carson, A., Costa, D.L. et al. (2007). Workshopsummary: phosgene-induced pulmonary toxicity revisited:appraisal of early and late markers of pulmonary injury fromanimal models with emphasis on human significance. Inhal.Toxicol. 19: 789–810.

Pruett, S.B., Chambers, J.E. (1988). Effects of paraoxon,p-nitrophenol, phenyl saligenin cyclic phosphate and phenolon the rat inteleukin-2 system. Toxicol. Lett. 40: 11–20.

Pruett, S.B., Han, Y., Munson, A.E., Fuchs, B.A. (1992). Assess-ment of cholinergic influences on a primary humoral immuneresponse. Immunology 77: 428–35.

Puri, S., Ray, A., Chakravarti, A.K., Sen, P. (1994). Role ofdopaminergic mechanisms in the regulation of stress responsesin rats. Pharmacol. Biochem. Behav. 48: 53–6.

Ray, A. Gulati, K. (2007). CNS-immune interactions duringstress: possible role for free radicals. In Proceedings of 2ndWorld Conference of Stress, Budapest, p. 432.

Ray, A., Mediratta, P.K., Puri, S., Sen, P. (1991). Effects of stresson immune responsiveness, gastric ulcerogenesis and plasmacorticosterone: modulation by diazepam and naltrexone.Indian J. Exp. Biol. 29: 233–6.

Ray, A., Mediratta, P.K., Sen, P. (1992). Modulation bynaltrexone of stress induced changes in humoral immuneresponsiveness and gastric mucosal integrity in rats. Physiol.Behav. 51: 293–6.

Ray, D.E. (1998). Chronic effects of low level exposure to anti-cholinesterases – a mechanistic review. Toxicol. Lett. 102:527–33.

Richman, D.P., Arnason, B.G.W. (1979). Nicotinic acetylcholinereceptor: evidence for a functionally distinct receptor onhuman lymphocytes. Proc. Natl Acad. Sci. USA 76: 4632–5.

Samnaliev, I., Mladenov, K., Padechky, P. (1996). Investigationsinto the influence of multiple doses of a potent cholinesteraseinhibitor on the host resistance and humoral mediated immu-nity in rats. Voj. zdrav. Listy 65: 143.

Sasser, L.B., Miller, R.A., Kalkwarf, D.R. et al. (1996). Sub-chronic toxicity evaluation of sulfur mustard in rats. J. Appl.Toxicol. 6(1): 5–13.

Schneider, W., Diller, W. (1989). Phosgene. In Ullmann’s Ency-clopedia of Industrial Chemistry, Vol. A, pp. 411–20. VCHVerlag, Weinheim, Germany.

Sciuto, A.M. (1998). Assessment of early acute lung injury inrodents exposed in phosgene. Arch. Toxicol. 72: 283–8.

Sciuto, A.M., Strickland, P.T., Kennedy, T.P. et al. (1996).Intratracheal administration of DBcAMP attenuates edemaformation in phosgene-induced acute pulmonary injury.J. Appl. Physiol. 80: 149–57.

Sciuto, A.M., Clapp, D.L., Hess, Z.A. (2003). The temporal profile ofcytokines in the bronchoalveolar lavage fluid in mice exposed tothe industrial gas phosgene. Inhal. Toxicol. 15: 687–700.

Selgrade, M.K., Starnes, D.M., Illing, J.W. et al. (1989). Effects ofphosgene exposure on bacterial, viral, and neoplastic lungdisease susceptibility in mice. Inhal. Toxicol. 1: 243–59.

Selgrade, M.K., Gilmore, M.T., Yang, Y.G. et al. (1995).Pulmonary host defenses and resistance to infection followingsubchronic exposure to phosgene. Inhal. Toxicol. 7: 1257–68.

Sevaljevic, L., Marinkovic, S., Bogojevic, D., Matic, S., Boskovic,B. (1989). Soman intoxication-induced changes in serum acutephase protein levels, corticosterone concentration and immu-nosuppressive potency of the serum. Arch. Toxicol. 63:406–11.


Sevaljevic, L., Poznakovic, G., Ivanovic-Matic, S. (1992). Theacute phase of rats to soman intoxication. Toxicology 75: 1–12.

Sidell, F.R. (1992). Civil emergencies involving chemical warfareagents: medical considerations. In Chemical Warfare Agents(S.M. Somani, ed.), pp. 341–56. Academic Press, San Diego, CA.

Small, M.J. (1984). Compounds formed from the chemicaldecontamination of HD, GB, and VX and their environmentalfate. Tech. Rpt 8304; AD A149515. US Army MedicalBioengineering Research and Development Laboratory, FortDetrick, MD.

Stewart, G.W. (1918). The Americal association for the advance-ment of science. Science 47: 569–70.

Szelenyi, J.G., Bartha, E., Hollan S.R. (1982) Acetylcholines-terase activity of lymphocytes: an enzyme characteristic ofT-cells. Br. J. Haematol. 50: 241–5.

Tabarestani, M., Balali-Mood, M., Farhoodi, M. (1990). Hema-tological findings of sulfur mustard poisoning in Iraniancombatants. Med. J. Islam. Repub. Iran 4: 185–90.

Taylor, P. (2006). Anticholinesterase agents. In The Pharmaco-logical Basis of Therapeutics (L.L. Brunton, J.S. Lazo, K.L.Parker, eds), pp. 201–216. McGraw-Hill, New York.

US Department of Health and Human Services (1993). HazardousSubstances Data Bank (HSDB, online database). NationalLibrary of Medicine, National Toxicology InformationProgram, Bethesda, MD.

US Environmental Protection Agency (1986). Health AssessmentDocument for Phosgene (External Review Draft). EPA/600/8-86/022A. Environmental Criteria and Assessment Office,

Office of Health and Environmental Assessment, Office ofResearch and Development, Research Triangle Park, NC.

Vos, J.G., De Waal, E.J., Van Loveren, H., Albers, R., Pieters,R.H.H. (1999). Immunotoxicology. In Principles of Immuno-pharmacology (F.P. Nijkamp, M.J. Parnham, eds), pp. 407–41.Birkhauser Verlag, Basel.

Ward, P.A. (1968). Chemotaxis of mononuclear cells. J. Exp.Med. 128: 1201–21.

WHO (World Health Organization) (1997). Environmental HealthCriteria Monograph on Phosgene, Monograph 193. Interna-tional Programme on Chemical Safety, Geneva, Switzerland.

Willems, J.L. (1989). Clinical management of mustard gas casu-alties. Ann. Med. Mil. Belg. 3: 1–59.

Yang, Y.G., Gilmore, M.T., Lang, R. et al. (1995). Effect of acuteexposure to phosgene on pulmonary host defense and resis-tance infection. Inhal. Toxicol. 7: 393–404.

Yokogama, M.W. (1993). Recognition of natural killer cells.Curr. Opin. Immunol. 5: 67–73.

Zabrodskii, P.F., Germanchuk, V.G., Kirichuk, V.F., Nodel, M.L.,Aredakov, A.N. (2003). Anticholinesterase mechanism asa factor of immunotoxicity of various chemical compounds.Bull. Exp. Biol. Med. 2: 176–8.

Zakeripanah, M. (1991). Hematological malignancies in chemicalwar victims. In 5th Seminar on Study of Chronic Effect ofChemical War Gases, Tehran University Press, Teheran.

Zandieh, T., Marzaban, S., Tarabadi, F., Ansari, H. (1990).Defects of cell mediated immunity in mustard gas injury afteryears. Scand. J. Immunol. 32: 423.

handbook of toxicology of chemical warfare agents || immunotoxicity

Documents