Immunotoxicity

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Immunotoxicity KAVITA GULATI AND ARUNABHA RAY

century. As chemical weapons are cheap, relatively easy to produce and can result in mass casualties, they will continue to be used in future wars and terrorist attacks. Although most of the compounds of CWAs are not persistent in the environment, repeated exposure and persistence of some of the compounds result in immunotoxicity. This chapter describes the immunotoxicity of CWAs and gives an insight into the probable mechanisms of such effects.

I. INTRODUCTION Immunotoxicity is defined as adverse effects on the functioning of both local and systemic immune systems that result from exposure to toxic substances including chemical warfare agents. Observations in humans and animal studies have clearly demonstrated that a number of environmental and industrial chemicals can adversely affect the immune system. Alteration in the immune system may result in either immunosuppression or exaggerated immune reaction. Immunosuppression may lead to the increased incidence or severity of infectious diseases or cancer, since the immune system’s ability to respond adequately to invading agents is suppressed. Toxic agent-induced immunostimulation can cause autoimmune diseases, in which healthy tissue is attacked by an immune system that fails to differentiate selfantigens from foreign antigens. For example, the pesticide dieldrin induces an autoimmune response against red blood cells, resulting in hemolytic anemia. Immunotoxicology deals with the effects of toxic substances and explores the mechanisms underlying these effects in a biological system. Although immunotoxicology is a relatively new field, a considerable amount of data has accumulated during the past few years on immunotoxicity of certain xenobiotics. The majority of the research thus far carried out has been on environmental contaminants. Thus, from the defense point of view considerable work is required to investigate the immunotoxicity of several chemicals and some bacterial and fungal toxins which may be potential chemical warfare agents. Furthermore, there are several chemicals used in the defense industry to which the defense industrial workers may be constantly exposed. These chemicals, following low-level exposure to humans and animals, may cause immunological alterations. Thus immunotoxicity studies on such chemicals are being conducted to understand the potential risks of such exposure on the host’s defense as well as the cellular and molecular mechanism of such immunomodulatory action. A chemical warfare agent (CWA) is a substance which is intended for use in military operations to kill, seriously injure, or incapacitate people because of its toxicological effects. Although CWAs have been widely condemned since 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

II. THE IMMUNE SYSTEM The immune system is composed of several organs, cells, and noncellular components which act in an interrelated manner to protect the host against foreign organisms and chemical substances. The immune system participates in the mechanisms responsible for the maintenance of homeostasis and an altered immune system reflects the adverse changes in both internal and external microenvironments. The immune system protects organisms against pathogens or other innocuous substances like pollens, chemicals, indoor molds, potential food allergens, and environmental agents, and acts as layered defenses of increasing specificity. Most simply, physical barriers (e.g. skin) prevent pathogens and xenobiotics from entering the organism. If they breach these barriers, the innate immune system provides an immediate but nonspecific response. However, if pathogens successfully evade the innate response, there is a third layer of protection, i.e. the adaptive immune system, which is activated by the innate response. Here, the immune system adapts during an infection to improve its recognition of the pathogen and its response is then retained after the pathogen or xenobiotic has been eliminated. This immunological memory allows the adaptive immune system to respond faster with a stronger attack each time the same insult is encountered (Kindt et al., 2007). The immune system protects organisms from infection with layered defenses of increasing specificity. The layered defense includes mechanical, chemical, and biological barriers which protect organisms from toxic substances. Skin, a mechanical barrier, acts as the first line of defense against infection. In the lungs, coughing and sneezing mechanically eject pathogens and other irritants from the

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respiratory tract while mucus secreted by the respiratory and gastrointestinal tract traps and entangles microorganisms and other toxins (Boyton and Openshaw, 2002). Chemical barriers also protect against infection. The skin and respiratory tract secrete antimicrobial peptides such as the bdefensins. Enzymes such as lysozyme and phospholipase A2 in saliva, tears, and breast milk are also antibacterials (Moreau et al., 2001; Hankiewicz and Swierczek, 1974). In the stomach, gastric acid and proteases serve as powerful chemical defenses against ingested pathogens.

A. The Innate Immune System The innate immune system defends the host from infection and toxicants, in a nonspecific manner. This means that the cells of the innate system recognize, and respond in a generic way, but do not confer long-lasting or protective immunity to the host. The innate immune response was initially dismissed by the immunologist as it was thought to provide a temporary holding of the situation until a more effective and specific adaptative immune response develops. But it has now been clear that it plays an important role as a dominant system of host defense in most organisms (Litman et al., 2005). The major function of the innate immune system is to recruit immune cells to sites of infection and inflammation. Inflammation is one of the first responses of the immune system to infection or irritation through the production of cytokines. These cytokines released by injured cells serve to establish a physical barrier against the spread of infection. Several chemical factors are produced during inflammation, e.g. histamine, bradykinin, serotonin, leukotrienes, and prostaglandins, which sensitize pain receptors, cause vasodilation of the blood vessels, and attract phagocytes. The inflammatory response is characterized by the redness, heat, swelling, pain, and possible dysfunction of the organs or tissues involved. The fluid exudate contains the mediators for four proteolytic enzyme cascades: the complement system, the coagulation system, the fibrinolytic system, and the kinin system. The exudate is carried by lymphatics to lymphoid tissue, where the product of foreign organism can initiate an immune response. The activation of the complement cascade helps to identify the invading substance, activate cells, and promote clearance of dead cells by specialized white blood cells. The cascade is composed of nine major components, designated C1 to C9, which are plasma proteins synthesized in the liver, primarily by hepatocytes. These proteins work together to trigger the recruitment of inflammatory cells. One of the main events is the splitting of the C3, which gives rise to various peptides. One of them, C3a (anaphylatoxin), can stimulate mast cells to secrete chemical mediators and another, C3b (opsonin), can attach to the surface of a foreign body and facilitates its ingestion by white blood cells. C5 is a powerful chemotactic of white cells and causes release of mediators from mast cells. Later components from C5 to C9 assemble in a sequence at the surface of bacteria/

xenobiotics and lead to their lysis, ridding the body of neutralized antigen–antibody complexes. The main events of this system can also be directly initiated by the principal enzymes of the coagulation and fibrinolytic cascade, thrombin and plasmin, and by enzymes released from white blood cells. Further, an innate immune system leads to the activation of an adaptive immune system.

B. The Adaptive Immune System The adaptive immune system is composed of highly specialized, systemic cells and processes that eliminate pathogenic challenges and provide the ability to recognize and mount stronger attacks each time the same pathogen is encountered. Antigen specificity requires the recognition of specific ‘‘nonself’’ antigens during a process called antigen presentation. The ability to mount these immune responses is maintained in the body by ‘‘memory cells’’. The cells of the adaptive immune system are special types of leukocytes, B cells and T cells, which constitue about 20–40% of white blood cells (WBCs). The peripheral blood contains 20–50% of circulating lymphocytes and the rest move within the lymphatic system (Kindt et al., 2007). B cells and T cells are derived from the same pluripotential hematopoietic stem cells in the bone marrow, and are indistinguishable from one another until after they are activated. B cells play a large role in the humoral immune response, whereas T cells are intimately involved in cell-mediated immune responses. B cells derive their name from the bursa of Fabricius, an organ unique to birds, where the cells were first found to develop. However, in nearly all other vertebrates, B cells (and T cells) are produced by stem cells in the bone marrow (Kindt et al., 2007). T cells are named after thymus where they develop and through which they pass. In humans, approximately 1–2% of the lymphocyte pool recirculates each hour to optimize the opportunities for antigen-specific lymphocytes to find their specific antigen within the secondary lymphoid tissues. Both B cells and T cells carry receptor molecules that recognize specific targets. T cells express a unique antigen-binding molecule, the T cell receptor (TCR), on their membrane. There are two welldefined subpopulations of T cell: T helper (TH) and T cytotoxic (TC) cells. They can be distinguished from one another by the presence of either CD4 or CD8 membrane glycoproteins on their surfaces. T cells displaying CD4 generally function as TH cells whereas those displaying CD8 function as TC cells. T cells recognize a ‘‘nonself’’ target, such as a pathogen, only after antigens have been processed and presented in combination with a ‘‘self’’ receptor called a major histocompatibility complex (MHC) molecule. TC cells only recognize antigens coupled to class I MHC molecules, while TH cells only recognize antigens coupled to class II MHC molecules. B cells are the major cells involved in the creation of antibodies that circulate in blood plasma and lymph, known as humoral immunity. Like the T cell receptor, B cells

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express a unique B cell receptor (BCR), in this case an immobilized antibody molecule. The BCR recognizes and binds to only one particular antigen. A critical difference between B cells and T cells is how each cell ‘‘sees’’ an antigen. T cells recognize their cognate antigen in a processed form – as a peptide in the context of an MHC molecule – while B cells recognize antigens in their native form. 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 an effector cell, known as a plasma cell. Plasma cells are short-lived cells (2–3 days) which secrete antibodies that circulate in blood plasma and lymph, and are responsible for humoral immunity. Antibodies (or immunoglobulin, Ig) are large Y-shaped proteins used by the immune system to identify and neutralize foreign objects. 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 easier targets for phagocytes, and trigger the complement cascade. About 10% of plasma cells will survive to become longlived antigen-specific memory B cells (Lu and Kacew, 2002). Already primed to produce specific antibodies, these cells can be called upon to respond quickly if the same foreign body reinfects the host. This is called ‘‘adaptive immunity’’ because it occurs during the lifetime of an individual as an adaptation to infection with that pathogen and prepares the immune system for future challenges. A number of animal models have been developed and validated to detect the chemical-induced direct immunotoxicity. Several compounds, including certain drugs, have been shown in this way to cause immunosuppression or skin allergic responses. In this chapter, the various mechanisms of immunotoxicity are discussed by which a compound affects different cell types and interferes with immune responses, ultimately leading to immunotoxicity as well as sensitizing capacity.

III. TARGETS OF IMMUNOTOXICITY A. Effects on Precursor Stem Cells The bone marrow is an organ with precursor stem cells that are responsible for synthesizing peripheral leukocytes. All leukocyte lineages originate from these stem cells, but once distinct subsets of leukocytes are established, their dependence on replenishment from the bone marrow differs vastly. The turnover of neutrophils is very rapid, i.e. more than 108 neutrophils enter and leave the circulation in a normal adult daily so there is dependence on new formation in the bone marrow. In contrast, macrophages are long-lived and have little dependence on new formation of precursor cells. The adaptive immune system, comprising antigen-specific T and B lymphocytes, is almost completely

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established around puberty and is therefore essentially bone marrow independent in the adult. As a consequence of their high proliferation rate, stem cells in the bone marrow are likely to be extremely vulnerable to cytostatic drugs and chemicals like CWAs. Lineages like neutrophils with rapid turnover will be most vulnerable and will be affected first by such treatments/ exposures. After prolonged exposure, macrophages and T or B cells of the adaptive immune system are also suppressed.

B. Effects on Maturation of Lymphocytes T lymphocytes mature in the thymus by a very complex selection process that takes place under the influence of the thymic microenvironment and ultimately generates an antigen-specific, host-tolerant population of mature T cells. This process involves cellular proliferation, gene rearrangement, apoptotic cell death, receptor up- and down-regulation and antigen-presentation processes, and is very vulnerable to a number of chemicals. Drugs may target different stages of T cell differentiation like naı¨ve T cells, proliferating and differentiating thymocytes, antigen-presenting thymic epithelial cells and dendritic cells, cell death processes, etc. (Vos et al., 1999). In general, immunosuppressive drugs may cause a depletion of peripheral T cells, particularly after prolonged treatment and during early stages of life when thymus activity is high and important in establishing a mature T cell population. In addition, suppression of T cells may result in suppression of the adaptive immune system by affecting the maturation of B cells and thus antibody level.

C. Effects on Initiation of Immune Responses The innate and adaptive immune systems act together to eliminate invading pathogens. Ideally, T cells tailor the responses to neutralise invaders with minimal damage to the host. The recognition of autoantigens is maintained by the two distinct signals that govern lymphocyte activation. One is the specific recognition of antigen via clonally distributed antigen receptors and the other is antigen nonspecific co-stimulation or ‘‘help’’ and involves interactions of various adhesive and signaling molecules expressed in response to tissue damage, linking initiation of immune responses to situations of acute ‘‘danger’’ for the host (Vos et al., 1999). This helps to aim immune responses at potentially dangerous microorganisms (nonself), while minimizing deleterious reactions to the host (self). Xenobiotics, however, can interfere with the initiation of immune responses if they act as antigens, by forming haptens or by releasing previously hidden self-antigens. They may also trigger an inflammatory response, or disturb T–B cell cooperation. CWAs with large molecular weight can function as antigens and become targets of specific immune responses themselves. This is particularly relevant for foreign protein

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pharmaceuticals, as these can activate both T and B lymphocytes. The resulting immune responses may lead to formation of antibodies, and induce specific memory which can lead to allergic responses to the drug. Immunotoxic effects may occur after repeated treatment with the same CWA. However, low molecular weight CWAs cannot function as antigens, because they are too small to be detected by T cells. Reactive chemicals that bind to proteins, however, can function as haptens and become immunogenic if epitopes derived from them prime T cells, which in turn provide co-stimulation for hapten-specific B cells. This effect is responsible for allergic responses to many new (neo) epitopes formed by chemical haptens. Modification of autoantigens can also lead to autoimmune responses to unmodified self-epitopes. Haptenated autoantigens can be recognized and internalized by antigen presenting cells. These cells subsequently present a mixture of neo- and self-epitopes complexed to distinct class II major histocompatibility (MHC-II) molecules on their surface and neospecific T cells. Th cells provide signals for the B cell. This leads to production of either antihapten or anti-self antibodies depending on the exact specificity of the B cell. Moreover, once these B cells are activated, they can stimulate autoreactive Th cells recognizing unmodified selfepitopes. This process is called epitope (determinant) spreading and causes the diversification of adaptive immune responses. For example, injection of mercury salts initially induces response directed only to unidentified chemically created neoepitopes, but after 3–4 weeks include reactivity to unmodified self-epitopes. Thus the allergic response may gradually culminate as autoimmune responses reflecting the relative antigenicity of the neo- and self-epitopes involved (Lu and Kacew, 2002).

IV. EXPOSITION OF AUTOANTIGENS AND INTERFERENCE WITH CO-STIMULATORY SIGNALS Self-tolerance involves specific recognition of autoantigen leading to selective inactivation of autoreactive lymphocytes at birth, but tolerance is not established for (epitopes of) autoantigens that are normally not available for immune recognition. Pharmaceuticals can expose such sequestered epitopes by disrupting barriers between the antigen and the immune system (i.e. blood–brain barrier, blood–testis barrier, cell membranes). Tissue damage, cell death, and protein denaturation induced by chemicals can largely increase the chances of such (epitopes of) autoantigens for immune recognition. Antigen recognition followed by costimulation of signaling molecules leads to activation of lymphocytes and initiation of immune responses. Many xenobiotics have the inherent capacity to induce or inhibit this co-stimulation due to their intrinsic adjuvant activity.

V. INDUCTION OF INFLAMMATION AND NONCOGNATE T–B COOPERATION Cytotoxic chemicals or their reactive metabolites can induce tissue damage which results in release of proinflammatory cytokines like tumor necrosis factor a (TNFa), interleukin-1 (IL-1), and IL-6, and attracts inflammatory cells like granulocytes and macrophages. Cytokines produced during this inflammatory response activate antigen-presenting cells and accumulation of tissue debris. The epitopes of antigens on debris provide co-stimulation for Th cells, which lead to the initiation of an adaptive immune response. Reactive xenobiotics may also stimulate adaptive immune responses by disturbing the normal cooperation of Th and B cells. Normally, B cells receive stimulation from Th cells that recognize (epitopes of) the same antigen. However, when Th cells respond to nonself-epitopes on B cells, such B cells may be noncognately stimulated by the Th cell. This occurs during graft-versus-host responses following bone marrow transplantation, when Th cells of the host recognize nonselfepitopes on B cells of the graft and vice versa. This leads to T and B cell activation and results in production of autoantibodies to distinct autoantigens like DNA, nucleoli, nuclear proteins, erythrocytes, and basal membranes. Drug/ chemical-related lupus is characterized by a similar spectrum of autoantibodies, and noncognate – graft-versus-hostlike – T–B cooperation is therefore suggested to be one of the underlying mechanisms.

VI. REGULATION OF THE IMMUNE RESPONSE The type of immune response elicited in response to a foreign pathogen or allergen is the result of a complex interplay of cytokines produced by macrophages, dendritic cells, mast cells, granulocytes, and lymphocytes. Immunotoxic chemicals that somehow influence the immune system can lead to either immunosuppression or immune exaggeration, i.e. hypersensitivity and autoimmunity. Hypersensitivity is an immune response that damages the body’s own tissues. 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 time course of the hypersensitive reaction. Type I hypersensitivity is an immediate or anaphylactic reaction, often associated with allergy. Symptoms can range from mild discomfort to death. Type I hypersensitivity is mediated by IgE released from mast cells and basophils. Type II hypersensitivity occurs when antibodies bind to antigens on the patient’s own cells, marking them for destruction. This is also called antibody-dependent (or cytotoxic) hypersensitivity, and is mediated by IgG and IgM antibodies. Immune complexes (aggregations of antigens, complement proteins, and IgG and IgM antibodies) deposited in various tissues trigger Type III hypersensitivity reactions. Type IV hypersensitivity (also

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known as cell-mediated or delayed type hypersensitivity) usually takes between 2 and 3 days to develop. Type IV reactions are involved in many autoimmune and infectious diseases, but may also involve contact dermatitis (poison ivy). These reactions are mediated by T cells, monocytes, and macrophages. Actual development of clinical symptoms is influenced by the route and duration of exposure, the dosage of the pharmaceutical, and by immunogenetic (MHC haplotype, Th1-type versus Th2-type responders) and pharmacogenetic (acetylator phenotype, sulfoxidizer, Ah receptor, etc.) predisposition of the exposed individual. Moreover, atopic individuals that tend to mount Th2 immune responses are more susceptible to anaphylaxis triggered by an IgE response to chemical haptens than typical Th1 responders. Genetic variation in metabolism of pharmaceuticals is important as it determines the formation and clearance of immunotoxic metabolites. The slow acetylating phenotype, for instance, predisposes for drug-related lupus because reactive intermediates of phase I metabolism have an increased opportunity to bind proteins as they are only slowly conjugated. Immune dysregulation can also be in the form of immune suppression and both innate and adaptive arms of the immune systems play crucial roles. A wide variety of physiological, pharmacological, and environmental factors can exert a negative influence on the immune system and sometimes result in immunotoxicity. Recent experimental data have shown that emotional and environmental stressors influence the functioning of the immune system and this is reflected in the various markers of specific immunity (Ray et al., 1991; Koner et al., 1998). Such experimental stressors consistently suppressed both humoral and cell mediated immune responses in experimental animals. Both antibody forming cell counts and antibody titer were lowered and a neuroendocrine–immune axis concept was proposed. Similar attenuations in cell mediated immune responses were also seen after such stressors and DTH responses, leukocyte/macrophage migration indices and also cytokine profiles (both Th1 and Th2 dependent). Further analysis of the mechanisms involved indicated that CNS mediated changes could have contributed to this immunotoxicity. Depletion or antagonism of brain dopamine aggravated emotional stress-induced immune suppression, whereas psychoactive drugs like benzodiazepines and opioids prevented this response (Ray et al., 1992; Puri et al., 1994). In another set of experiments, rats exposed to several environmental pollutants like DDT showed graded degrees of immune suppression and immunotoxicity, when the exposure lasted for a reasonably long period of time. Gradual accumulation in the various body tissues resulted in a variety of untoward effects in the immune system, which was particularly susceptible to such xenobiotic-induced damage (Banerji et al., 1996; Koner et al., 1998). Both humoral and cell mediated immune response were affected depending on the quantum and duration of exposure to these xenobiotics. Further, a combination of emotional stress and

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xenobiotic exposure had additive effects on the immunotoxicity parameters studied (Banerjee et al., 1997). Recent studies revealed that such emotional stress and xenobioticinduced immunotoxicity was accompanied by derangements in oxidative stress parameters, such as enhancements in MDA levels and lowering of GSH/SOD levels in the blood (Koner et al., 1997; Ray and Gulati, 2007; Gulati et al., 2007).

VII. IMMUNOTOXICITY OF CHEMICAL WARFARE AGENTS A chemical warfare agent (CWA) is a substance which is intended for use in military operations to kill, seriously injure, or incapacitate people because of the severe pathophysiological changes induced by them in various body systems. A United Nations report from 1969 defines chemical warfare agents as ‘‘chemical substances, whether gaseous, liquid or solid, which might be employed because of their direct toxic effects on man, animals and plants’’. However, the Chemical Weapons Convention defines chemical weapons as including not only toxic chemicals but also ammunition and equipment for their dispersal. Toxic chemicals are stated to be ‘‘any chemical which, through its chemical effect on living processes, may cause death, temporary loss of performance, or permanent injury to people and animals’’. Normally, they are either liquids or solids. Chemical agents have been used in war since time immemorial. In 600 BC Helleborus roots were used successfully by the Athenians to contaminate water supplies during the siege of Kirrha. Spartans ignited pitch and sulfur to create toxic fumes during the Peloponnesian War in 429 BC. The uses of CWAs in battlefields reached a peak during World War I and the French were the first to use ethylbromoacetate. It was followed by o-dianisidine chlorosulphonate, chloroacetate, chlorine, phosgene, hydrogen cyanide, diphenylchloroarsine, ethyl- and methyldichloroarsine, and sulfur mustard resulting in nearly 90,000 deaths and over 1.3 million casualties (Eckert, 1991). CWAs were most brutally used by the Germans in the gas chambers for mass genocide of Jews during World War II, and have been used intermittently both in war, as in the Iraq–Iran War, as well as in terrorist attacks in the Japanese subway stations. It is estimated that nearly 100,000 US troops may have been exposed to CWAs during operation Desert Storm (Chauhan et al., 2008). CWAs have been widely condemned since they were first used on a massive scale during World War I. However, they are still stockpiled and used in many countries as they are cheap and relatively easy to produce, and can cause mass casualties. Although the blood agent CK is extremely volatile and undergoes rapid hydrolysis, the degradation of three types of vesicant CWAs, the sulfur mustards, nitrogen mustards, and Lewisite, results in persistent products. For

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example, sulfonium ion aggregates formed during hydrolysis may be persistent and may retain vesicant properties. The nerve agents include the V agent VX as well as three G agents (tabun, sarin, and soman). VX gives rise to two hydrolysis products of possible concern: EA 4196, which is persistent, and EA 2192, which is highly toxic and is possibly persistent under certain limited conditions (Small, 1984). Thus, their long-term persistence in the body may lead to alterations in the immune system of the exposed population. CWAs can be classified in many different ways. There are, for example, volatile substances, which mainly contaminate the air, or persistent substances, which are nonvolatile and therefore mainly cover surfaces. CWAs mainly used against people may also be divided into lethal and incapacitating categories. A substance is classified as an incapacitating agent if less than 1/100 of the lethal dose causes incapacitation, e.g. through nausea or visual problems. The limit between lethal and incapacitating substances is not absolute but refers to a statistical average. Chemical warfare agents are generally classified according to their principal target organs. 1. Organophosphate (OP) nerve agents. These agents are extremely toxic compounds that work by interfering with the nervous system, and include soman, sarin, cyclosarin, tabun, and VX. 2. Blister agents/vesicants. These compounds severely blister the eyes, respiratory tract and skin on exposure, and include nitrogen mustard, sulfur mustard, Lewisite, etc. 3. Choking agents. These agents cause severe irritation primarily affecting the respiratory tract, and include phosgene, ammonia, methyl bromide, methyl isocyanate, etc. 4. Blood agents. These agents are absorbed into the blood and interfere with the oxygen carrying capacity, e.g. arsine, cyanides, carbon monoxide, etc. Very few studies have been conducted to explore the immunomodulation and immunotoxic potential of CWAs, and there is little evidence that these drugs are associated with such undesirable, immunologically significant effects. The reason may be due to confounding factors such as stress, nutritional status, lifestyle, co-medication, and genetics (Vos et al., 1999). The exposure to CWA can result in immunodepressed conditions on the one hand and to allergic and autoimmune diseases on the other. Few conventional compounds have been shown to induce unexpected enhancement of immune competence. However, introduction of biotechnologically manufactured agents like cytokines has been shown to induce unwanted immunostimulation. Drug-induced hypersensitivity reactions and autoimmune disorders are a major concern, whereas some of these chemicals also result in immunosuppression. In particular, impaired activity of the first line of defense of the natural immune system can have disastrous consequences.

These are generally not influenced by the genetic predisposition of the exposed individual, but on actual outbreak of infections and the general immune status prior to exposition. This explains why immunosupressive xenobiotics are most likely to have clinical consequences in immunocompromised individuals such as young children, the elderly, and transplant recipients.

A. Nerve Agents Nerve agents are highly toxic organophosphorus compounds (OPs) which represent potential threats to both military and civilian populations, as evidenced in recent terroristic attacks in Japan (Ohtomi et al., 1996). Commonly known as nerve agents or nerve gases, these are the deadliest of CWAs. These agents have both chemical names as well as two-letter NATO codes. These are categorized as G series agents: GA (tabun), GB (sarin), GD (soman), GF (cyclosarin), and V series agents: VE, VG, VM, and VX, the letter ‘‘G’’ representing the country of origin ‘‘Germany’’ and letter ‘‘V’’ possibly denoting ‘‘Venomous’’. Their initial effects occur within 1–10 min of exposure followed by death within 15–30 min for sarin, soman, and VX, and within 30–60 min for tabun. The ease and low cost of production make sarin gas a tool of mass destruction in the hands of terrorist groups and rogue nations. While people in the immediate vicinity of a sarin attack may receive neurotoxic doses, people remote from the vicinity are likely to receive subclinical exposures. Short- and long-term health effects from exposure to OP nerve agents and insecticide nerve agents are compiled on the basis of scientific literature published on health effects in humans and animal studies. Four distinct health effects are identified: acute cholinergic toxicity; OP-induced delayed neuropathy (OPIDN); subtle long-term neuropsychological and neurophysiological effects; and a reversible muscular weakness called ‘‘intermediate syndrome’’. Each effect has data suggesting threshold exposure levels below which it is unlikely to be clinically detectable. High-level exposure results in definitive cholinergic poisoning; intermediatelevel threshold cholinergic effects include miosis, rhinorrhea or clinically measurable depression of cholinesterase; and low-level exposure results in no immediate clinical signs or symptoms. Threshold exposure levels for known long-term effects from OP nerve agent are at or above intermediate-level exposure (Brown and Brix, 1998). However, subclinical doses of sarin cause subtle changes in the brain, and subclinical exposure to sarin has been proposed as an etiology to the Gulf War Syndrome. The wide use of cholinesterase inhibitors in various spheres of human activities and the risk of acute and chronic intoxications associated with this process prompted investigation of the role of acetylcholinesterase (AChE) and nonspecific esterases in the immunotropic effects of these chemicals. They irreversibly bind to AChE that normally catalyzes the hydrolysis of acetylcholine (ACh) at the

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cholinergic synapses and neuromuscular junctions (NMJs). The inhibition of degradation results in accumulation of ACh in the cholinergic synapses, causes the overstimulation of 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 are highly immunosuppressive, and suppress glucocorticoid production. The effects of sarin exposure on the immune system are attenuated by ganglionic blockers and decreased glucocorticoid level may be a biomarker for cholinergic toxicity. In addition, nerve agents cause the activation of multiple noncholinergic neurotransmitter systems in the central nervous system (CNS) thus resulting in mutagenic, stressogenic, immunotoxic, hepatotoxic, membrane, and hematotoxic effects (Bajgar, 1992). The CNS and the immune system communicate bidirectionally, and cholinergic agents modulate the immune system. The ability of OP compounds to induce an alteration of the immune system was primarily demonstrated in animals or humans exposed to OP insecticides (OPIs). The results provide evidence that, especially neutrophil function, natural killer cell, cytotoxic T cell and humoral immune functions, and spontaneous as well as mitogen-induced lymphocyte proliferation, are altered in animals or humans exposed to OP compounds (Casale et al., 1984; Hermanowitz and Kossman, 1984; Li et 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 in neutrophils (Ward, 1968), inhibition of monocyte accessory functions or inhibition of interleukin-2 production (Casale et al., 1993; Pruett and Chambers, 1988) were reported following the exposure to OPs, at relatively high toxic doses. Lee et al. (1979) were the first to draw attention to the possible effects of OPs on human leukocyte function. They demonstrated that lymphocyte proliferation to phytohemaglutinin in vitro was decreased in the presence of OPs. Although most of the studies described the results of OPI exposure, there are studies about the immunotoxic effects of highly toxic nerve agents and their by-products. Marked impairment in neutrophil chemotaxis and neutrophil adhesion and a reduction in the natural killer cell and cytotoxic T cell function were observed in workers exposed to OPIs and by-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, an important immune organ in severely affected soman survivors, but other tests of immune function did not show differences between control and soman-exposed rats. Samnaliev et al. (1996) described a decrease in the number of plaque forming cells in soman-exposed rats after the administration of sheep red blood cells as an antigen.

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However, Johnson et al. (2002) demonstrated that OPinduced modulation of immune functions can involve not only their suppression but also their activation. Similar activation of some immune functions involving ‘‘acute phase response’’ such as an increase in the synthesis of acute phase proteins, increase in release of histamine from basophile leukocytes and activation of macrophages were observed following the exposure to soman (Sevaljevic et al., 1992; Newball et al., 1986). Although most of the studies dealt with exposure to high doses, Kassa et al. (2004) confirmed that not only symptomatic but also asymptomatic doses of nerve agent sarin were able to modify various immune functions. The proportion of T lymphocytes was found to be decreased, while the B cell levels were raised. However, sarin significantly suppressed nonspecific in vitro stimulated proliferation of both T and B cells, which suggests that it can also block normal immune response to infection. While the lymphocyte mediated immunity is rather suppressed, the peritoneal as well as alveolar macrophages and NK cells were activated after exposure to both levels of sarin, which was explained to be the result of compensatory reactions of immune functions rather than the result of direct effects of inhalation. Immunosuppression may result from direct action of acetylcholine upon the immune system or it may be secondary to the toxic chemical stress associated with cholinergic poisoning (Pruett et al., 1992). Further, immunomodulation at low levels seems to be very complex and it is suggested that there are probably other protein targets very sensitive to some anticholinesterases including nerve agents. However, the function of these protein targets is not yet known (Ray, 1998). Some immune functions are probably stimulated due to the development of ‘‘acute phase response’’ generally characterized for inflammatory reaction of OP-exposed organism (Sevaljevic et al., 1989, 1992). Other immune functions are suppressed due to immunotoxicity of OP compounds. Although these findings are difficult to extrapolate directly to low-level exposures to nerve agents, they indicate that subtle alteration of immune system could also occur in humans at exposure levels which do not cause any clinical manifestation. Post-intoxication immunodeficiency can promote infectious complications and diseases. It has been shown that T lymphocytes have AChE located on the plasma membrane, while B cells are esterase negative (Szelenyi et al., 1982). Thus, AChE inhibition by toxic agents in sublethal doses may play an important role in immunodeficiency following exposure to nerve gases. Zabrodskii et al. (2003) showed inhibition of AChE in T cells and the decrease in the number of esterase-positive T lymphocytes (and, to a certain extent, in monocytes and macrophages) directly correlated with suppression of T celldependent antibody production and to the degree of DTH reduction, on exposure to dimethyl dichlorovinyl phosphate, sarin, VX, lewisite, tetraethyl lead, and dichloroethane. This presumably involves the loss of some functions by T

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lymphocytes (e.g. by Th1 cells), which leads to attenuation of T-dependent immune reactions. This can be explained by excessive acetylcholine (ACh) stimulation of muscarinic and nicotinic receptors present on T lymphocytes, as a result of which the optimal cAMP to cGMP ratio in immunocytes, essential for their proliferation and differentiation, is distorted (Richman and Arnason, 1979). Thus, the anticholinesterase effect of lewisite, TEL, and DCE may be one of the important mechanisms in the formation of T cell mediated immunodeficiency.

B. Blister or Vesicant Agents These agents act on skin and other epithelial tissues and severely blister the eyes, respiratory tract, and internal organs, and also destroy different substances within cells of living tissue. The symptoms are variable depending upon the compound and the sensitivity of the individual. Acute mortality is low; however, they can incapacitate the enemy and overload the already burdened health care services during war time. Some of these agents are HD (sulfur mustard), HN (nitrogen mustard), L (Lewisite), and CX (phosgene oximine). Sulfur mustard (SM) was the most widely used chemical warfare agent (CWA) in the Iran–Iraq War, resulting in over 100,000 chemical casualties between 1980 and 1988. It acts as an alkylating agent with long-term toxic effects on several body organs, mainly the skin, eyes, and respiratory system (Willems, 1989). The extent of tissue injury depends on the duration and intensity of exposure. When absorbed in large amounts, SM can damage rapidly proliferating cells of bone marrow and may cause severe suppression of the immune system (Willems, 1989). 1. IMMUNOTOXICITY Evidence that SM causes immunosuppression in humans has emerged from several lines of investigation. The earliest evidence came from clinical observations of humans directly exposed to sulfur mustard during World War I, who showed significant changes (quantitative and qualitative) in the circulating elements of the immune system. Stewart (1918) studied ten fatal cases of mustard poisoning and observed striking depression of bone marrow production of white blood cells. Among the sulfur mustard casualties during the Iran–Iraq conflict leukopenia accompanied by total bone marrow aplasia and extensive losses of myeloid stem cells was the most common finding (Balali-Mood, 1984; Eisenmenger et al., 1991). These findings provide further evidence of an association between suppression of immunologic functions and an increased incidence of infectious disease. SM has been widely used during Iran–Iraq conflict and there are many reports of influence of SM on the respiratory system, gastrointestinal system, and endocrine system as well as the immune system (Balali-Mood, 1984; BalaliMood and Farhoodi 1990; Emad and Razaian 1997; Sasser

et al., 1996; Budiansky, 1984). The influence of SM on the immune system has been the subject of many researchers since 1919 (Krumbhaar and Krumbhaar, 1919; Hektoen and Corper 1921). Early investigations on SM casualties during the Iran–Iraq War showed decreased immunoresponsiveness, expressed as leukopenia, lymphopenia, and neutropenia, as well as hypoplasia and atrophy of the bone marrow (Willems, 1989; Tabarestani et al., 1990; BalaliMood et al., 1991). Chronic exposure to SM has been associated with the impairment of NK cells among workers of poison gas factories in Japan (Yokogama, 1993). Similarly, cell mediated immunity was found to be suppressed following mustard gas exposure (Zandieh et al., 1990). Leukopenia has been the first manifestation to appear within the first days of post-exposure. Thrombocytopenia and anemia followed later if the patients survived (white blood cells of some patients dropped to less than 1,000 per cm3). Although most of these patients suffered skin burns, clinicians reported cases that had minor skin lesions and yet developed leukopenia. Bone marrow biopsies revealed hypocellular marrow and cellular atrophy involving all elements (Willems, 1989). Studies on the status of immunocompetent cells in the blood of patients exposed to sulfur mustard showed that T cell and monocyte counts dropped in 54% and 65% of the patients, respectively, from day 1 and up to 7th week post-exposure (Hassan and Ebtekar, 2002). Eosinophil counts dropped in 35% and neutrophil numbers in 60% of the patients. B lymphocyte counts were normal up to 7th week (Manesh, 1986). The majority of the patients showed increased levels of IgG and IgM during the 1st week, 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 controls during the 1st week and up to 6th month (Tabarestani et al., 1990) and remained higher 3 years post-exposure especially in the severely affected group. Eight years after exposure there was a significant increase in the number of atypical leukocytes (such as myelocytes). The severely affected group presented with significantly lower CD56 NK as well as CD4 and CD8 counts compared with healthy controls (Yokogama, 1993). Hassan and Ebtekar (2002) reported that there was no major difference between the severely affected patients and healthy controls concerning CD19 B cells, CD14 monocytes, and CD15 granulocytes. The moderately and mildly affected patients did not significantly differ in their leukocyte subset counts from the control group 8 years after exposure (Mahmoudi et al., 2005). Follow-up studies on the clinical conditions of exposed Iranian victims still show that they suffer from three major problems: recurrent infection, septicemia and death, respiratory difficulties and lung fibrosis, as well as a high incidence of malignancies, septicemia, and death. Hassan and Ebtekar (2002) suggested that patients with moderate clinical manifestations may be experiencing a shift from Th1 to Th2 cytokine patterns since leukocyte cultures from this patient group showed a decrease in IFN-g

CHAPTER 40 $ Immunotoxicity

levels. When absorbed in large amounts, SM can damage rapidly proliferating cells of bone marrow and may cause severe suppression of the immune system (Sasser et al., 1996). Moreover, this alkylating agent has been reported to produce short- and long-term suppression of antibody production in both animals and humans. It also affects complement system factors C3 and C4. Incidences of acute myelocytic and lymphocytic leukemia are reported to be 18 and 12 times higher in patients exposed to SM, in comparison with the normal group (Zakeripanah, 1991). Willems (1989) reported that exposure to SM could result in the impairment of human immune function, especially in the number of B and T lymphocytes. Hence, SM is still a potential threat to the world and effective therapeutic measures must be taken for the relief of the victims of this incapacitating agent. Ghotbi and Hassan (2002) showed that the percentage of NK cells, playing an important role in cellular immunity, was significantly lower in severe patients than in the control group. Studies on animal models have shown that alkylating agents such as SM mainly affect B cells, which is why hypogammaglobulinemia is one of the main features in animal models, whereas studies on human cases, following a treatment with cytotoxic drugs, suggest that low-dose exposure to alkylating agents impairs cellular immunity and high-dose exposure to such agents impairs both cellular and humoral responses (Marzban, 1989; Malaekeh et al., 1991). There are reports suggesting that sulfur mustard can produce toxicity through the formation of reactive electrophobic intermediates, which in turn covalently modify nucleophilic groups in biomolecules such as DNA, RNA, and protein (Malaekeh et al., 1991), resulting in disruption of cell function, especially cell division (Crathorn and Robert, 1966). As a result, these agents are particularly toxic to rapidly proliferating cells including neoplastic, lymphoid, and bone marrow cells. Mahmoudi et al. (2005) reported higher IgM levels after 16 to 20 years of exposure to SM, compared to the control group. A significant decrease in the number of NK cells in severe patients is probably due to the destructive effect of this alkylating agent on NK cell precursors in bone marrow. However, activity of NK cells was found to be noticeably above normal which possibly compensates for the reduction in the number of these cells. Recently, Korkmaz et al. (2006) explained the toxicodynamics of sulfur mustards in three steps: (1) binding to cell surface receptors; (2) activation of ROS and RNS leading to peroxynitrite (OONO ) production, and (3) OONO -induced damage to lipds, proteins, and DNA, leading to polyadenosine diphosphate ribose (PARP) activation. This could provide a lead for devising strategies for protection against/treatment of mustard toxicity. In conclusion, the results suggest that exposure to SM causes a higher risk of opportunistic infections, septicemia, and death following severe suppression of the immune system especially in the case of lesions and blisters produced by these agents. As alkylating agents, they form

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covalent linkages with biologically important molecules, resulting in disruption of cell function, especially cell division. As a result, these agents are particularly toxic to rapidly proliferating cells including neoplastic, lymphoid, and bone marrow cells. However, there is still a paucity of information regarding the long-term immunosuppressive properties of SM in the setting of battlefield exposure to this agent.

C. Choking Agents Choking agents act on the pulmonary system causing severe irritation and swelling of the nose, throat, and lungs, e.g. CG (phosgene), DP (diphosgene), chlorine, and PS (chloropicrin). These inhalational agents damage the respiratory tract and cause severe pulmonary edema in about 4 h, leading to death. The effects are variable, rapid, or delayed depending on the specific agent (Gift et al., 2008). Phosgene was first used as a chemical weapon in World War I by Germany and later as offensive capability by French, American, and British forces. In this conflict, phosgene was often combined with chlorine in liquid-filled shells, so it was difficult to state the number of casualties and deaths attributable solely to phosgene. In military publications, it has been referred to as a choking agent, pulmonary agent, or irritant gas. Since World War I, phosgene has rarely been used by traditional militaries, but the extremist cult Aum Shinrikyo used this agent in an attack against the Japanese journalist Shouko Egawa in 1994. Nowadays, phosgene is primarily used in the polyurethane industry for the production of polymeric isocyanates (USEPA, 1986). Phosgene is also used in the polycarbonate industry and in the manufacture of carbamates and related pesticides, dyes, pharmaceuticals, and isocyanates. As mentioned earlier, the primary exposure route for phosgene is by inhalation. Suspected sources of atmospheric phosgene are fugitive emissions, thermal decomposition of chlorinated hydrocarbons, and photo-oxidation of chloroethylenes. Individuals are most likely to be exposed to phosgene in the workplace during its manufacture, handling, and use (USEPA, 1986). Phosgene is extremely toxic by acute (short-term) inhalation exposure. Severe respiratory effects, including pulmonary edema, pulmonary emphysema, and death have been reported in humans. Severe ocular irritation and dermal burns may result following eye or skin exposure. Chronic inhalation exposure to phosgene has been shown to result in some tolerance to the acute effects noted in humans, but may also cause irreversible 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 past 100 years to examine the effects and mode of action of phosgene following a single, acute (less than 24 h) exposure. Many studies have examined the effects of acute phosgene

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exposure in animals but the human data are limited to case studies following accidental exposures. Most studies were performed in rodents and dogs, with exposure concentrations ranging between 0.5 and 40 ppm (2–160 mg/m3) and duration intervals ranging from 5 min to 8 h. Acute exposure studies in animals suggest that rodent species may be more susceptible to the edematous effects of phosgene acute exposure than larger species with lower respiratory volumes per body weight such as dogs and humans (Pauluhn, 2006; Pauluhn et al., 2007). Pauluhn et al. (2007) reported that acute phosgene exposure results in increased lung lavage protein, phospholipid content, enzyme levels, number of inflammatory cells, and lethality (LC50). Rats seem to be able to survive approximately three-fold higher levels of lung edema than humans (100-fold versus 30-fold), thus rat responses in short- and long-term assays may still be relevant to humans even if it is ultimately shown that rats produce higher levels of edema following acute phosgene exposure. 1. IMMUNOTOXICITY Acute exposure to phosgene has been shown to result in immunosuppression in animals, as evidenced by an increased susceptibility to in vivo bacterial and tumor cell infections (Selgrade et al., 1989) and viral infection (Ehrlich and Burleson, 1991) as well as a decreased in vitro virus-killing and T cell response (Burleson and Keyes, 1989). Selgrade et al. (1989) reported that a single 4 h exposure to phosgene concentrations as low as 0.025 ppm significantly enhanced mortality due to streptococcal infection in mice. Furthermore, when the exposure time was increased from 4 to 8 h, a significant increase in susceptibility to streptococcus was seen at an exposure concentration of 0.01 ppm. Selgrade et al. (1995) administered Streptococcus zooepidemicus bacteria via an aerosol spray to the lungs of male Fischer 344 rats immediately after phosgene exposure and measured the subsequent clearance of bacteria. They also evaluated the immune response, as measured by an increase in the percentage of polymorphonuclear leukocytes (PMN), in lung lavage fluid of uninfected rats similarly exposed to phosgene. This experiment showed that all phosgene concentrations from 0.1 to 0.5 ppm impaired resistance to bacterial infection and that the immune response is stimulated by phosgene exposure. After 4 weeks following exposure, bacterial resistance as well as immune response returned to normal. Yang et al. (1995) also reported a decrease in bacterial clearance in the lungs at 24 h after infection following a single 6 h exposure to phosgene concentrations of 0.1 and 0.2 ppm. In comparison with single exposures, the multiple daily exposures extending to 4 and 12 weeks in the Selgrade et al. (1995) report showed a slight enhancement of effect in the 0.1 ppm group at 24 h post-infection, but no ‘‘adaptation’’, or lessening of the effect. Yang et al. (1995) found that if the bacteria are administered 18 h after single

phosgene exposures rather than immediately, the clearance is normal which indicates that recovery from the toxic effect of phosgene is rapid. When inhaled, phosgene either is rapidly hydrolyzed to HCl and CO2 and exhaled (Schneider and Diller, 1989; Diller, 1985) or penetrates deep into the lungs and is eliminated by rapid reactions with nucleophilic constituents of the alveolar region (Pauluhn et al., 2007). As phosgene is electrophilic, 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 with molecular functions. Phosgene interacts with biological molecules through two primary reactions: hydrolysis to hydrochloric acid and acylation reactions. Although the hydrolysis reaction does not contribute much to its clinical effects, the acylation reaction is mainly responsible for the irritant effects on mucous membranes. The acylation reactions occur between highly electrophilic carbon molecules in phosgene and amino, hydroxyl, and sulfhydryl groups on biological molecules. These reactions can result in membrane structural changes, protein denaturation, and depletion of lung glutathione. Acylation reactions with phosphatidylcholine are particularly important as it is a major constituent of pulmonary surfactant and lung tissue membranes. Exposure to phosgene has been shown to increases the alveolar leukotrienes, which are thought to be important mediators of phosgene toxicity to the alveolar– capillary interface. Phosgene exposure also increases lipid peroxidation and free radical formation. These processes may lead to increased arachidonic acid release and leukotriene production. Proinflammatory cytokines, such as interleukin-6, are also found to be substantially higher 4–8 h after phosgene exposure. In addition, studies have shown that post-exposure phosphodiesterase activity increases, leading to decreased levels of cyclic AMP. Normal cAMP levels are believed to be important for maintenance of tight junctions between pulmonary endothelial cells and thus for prevention of vascular leakage into the interstitium. Oxygenation and ventilation both suffer, and breathing is dramatically increased. Schneider and Diller (1989) and Diller (1985) reported that inhalation of phosgene at high concentrations results in a sequence of events, including an initial bioprotective phase, a symptom-free latent period, and a terminal phase characterized by pulmonary edema. The first is an immediate irritant reaction likely caused by the hydrolysis of phosgene to hydrochloric acid on mucous membranes, which results in conjunctivitis, lacrimation, and oropharyngeal burning sensations. This symptom complex occurs only in the presence of high-concentration (>3–4 ppm) exposures but does not have any prognostic value for the timing and severity of later respiratory symptoms. The most important finding to identify during this stage is a laryngeal

CHAPTER 40 $ Immunotoxicity

irritant reaction causing laryngospasm, which may lead to sudden death. The irritant symptoms last only a few minutes and then resolve as long as further exposure to phosgene ceases. The second phase, when clinical signs and symptoms are generally lacking, may last for several hours after phosgene exposure. The duration of the latent phase is an extremely important prognostic factor for the severity of the ensuing pulmonary edema. Patients with a latent phase of less than 4 h have a poor prognosis. Increased physical activity may shorten the duration of the latent phase and worsen the overall clinical course. Unfortunately, there are no reliable physical examination findings during the latent phase to predict its duration. However, histologic examination reveals the beginnings of an edematous swelling, with exudation of blood plasma into the pulmonary interstitium and alveoli. This may result in damage to the alveolar type I cells and a rise in hematocrit. The length of this phase varies inversely with the inhaled dose. The third clinical phase peaks approximately 24 h after an acute exposure and if lethality does not occur, recedes over the next 3–5 days. In the third clinical phase of phosgene toxicity, the accumulating fluid in the lung results in edema. Oxygenation and ventilation both suffer, and the breathing is dramatically increased. Often positive end expiratory pressure (PEEP) is required to stent open alveoli that would otherwise collapse and result in significant ventilation/perfusion (V/Q) mismatch. This hyperventilation causes the protein-rich fluid to take on a frothy consistency. A severe edema may result in an increased concentration of hemoglobin in the blood and congestion of the alveolar capillaries. Increased levels of protein in bronchoalveolar lavage have been shown to be among the most sensitive endpoints characterizing 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 transition of edema to persistent cellular inflammation leading to the synthesis of abnormal Type I collagen and pulmonary fibrosis. An increased synthesis of Type I relative to Type III collagen can lead to chronic fibrosis (Pauluhn et al., 2007). Surfactant lipids are important for maintaining alveolar stability and for preventing pulmonary edema. Pauluhn et al. (2007) reported that the induction of surfactant abnormalities following phosgene exposures is a key pathophysiological event leading to pulmonary edema and chronic cellular inflammation, leading to the stimulation of fibroblasts and the synthesis of ‘‘abnormal’’ collagen in pulmonary fibrosis. As discussed earlier, a breach in the chemical layer of defense followed by pulmonary edema may lead to a cascade of other immunological responses/ reactions. There are limited studies, in both humans and experimental animals, to evaluate immunotoxicity of chronic low-level environmental exposures to phosgene. The lack of studies examining the effects in humans or laboratory animals from chronic exposure to phosgene is

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

D. Blood Agents Agents like SA (arsine), cyanide, and carbon monoxide are absorbed into the blood and affect its oxygen carrying capacity, and are thus termed blood agents. They are highly volatile and rapidly acting, and produce seizures, respiratory failure, and cardiac arrest. Hydrogen cyanide has been known as a potent toxicant for over 200 years. It was used as a 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 deaths from its military use during World War I were ever reported. After World War II, the importance of hydrogen cyanide as a chemical warfare agent diminished rapidly, primarily as a result of the rise of nerve agents. Although reduced in importance, there are some reports of hydrogen cyanide being used as a war gas by Vietnamese forces in Thailand territories 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 needed for it to be effective as a chemical warfare agent. Cyanide is primarily an environmental contaminant of industrial processes. It is used in the metal-processing industry for electroplating, heat treating, and metal polishing and can be found in waste waters from many mining operations that use cyanide compounds in the extraction of metals, such as gold and silver, from ore. The acute toxicity of cyanide has been well documented in humans and experimental animals. Symptoms of toxicity in humans include headache, breathlessness, weakness, palpitations, nausea, giddiness, and tremors (Gupta et al., 1979). Depending on the degree of intoxication, symptoms may include ‘‘metallic’’ taste, anxiety and/or confusion, headache, vertigo, hyperpnea followed by dyspnea, convulsions, cyanosis, respiratory arrest, bradycardia, and cardiac arrest. Death results from respiratory arrest (Berlin, 1977). Onset is usually rapid. Effects on inhalation of lethal amounts may be observed within 15 s, with death occurring in less than 10 min. Hydrogen cyanide should be suspected in terrorist incidents involving prompt fatalities, especially when the characteristic symptoms of nerve agent intoxication are absent. Chronic exposure to low-level cyanide can result in neuropathies, goiter, and diabetes. Cyanide and derivatives prevent the cells of the body from using oxygen. Cyanide acts by binding to mitochondrial cytochrome oxidase, blocking electron transport, thus inhibiting enzymes in the cytochrome oxidase chain and in turn blocking oxygen use in metabolizing cells and preventing the use of oxygen in cellular metabolism. These chemicals are highly toxic to cells and in high doses may result in death. Cyanide is more harmful to the heart and brain as these organs require large amounts of oxygen.

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1. IMMUNOTOXICITY There are very few reports on immunotoxicity of the compound; however, acrylonitrile (vinyl cyanide, VCN), an environmental pollutant which is metabolized to cyanide, has been shown to be an animal and human carcinogen particularly for the gastrointestinal tract (Mostafa et al., 1999; National Toxicology Program Technical Report Series, 2001). Earlier Hamada et al. (1998) evaluated the systemic and/or local immunotoxic potential of VCN and demonstrated that VCN induces immunosuppression as evident by a decrease in the plaque forming cell (PFC) response to SRBCs (sheep red blood cells), a marked depletion of spleen lymphocyte subsets, as well as bacterial translocation of the normal flora leading to brachial lymph node abscess. These results suggested that VCN has a profound immunosuppressive effect which could be a contributing factor in its gastrointestinal tract carcinogenicity.

VIII. CONCLUDING REMARKS AND FUTURE DIRECTION The immune system is extremely vulnerable to the action of xenobiotics for several reasons. The immune response is associated with rapidly multiplying cells and synthesis of regulatory/effector molecules and the immune system works as an amplifier for this integrated information network. Immunologic tissue damage can result from activation of the cellular and biochemical systems of the host. The interactions of an antigen with a specific antibody or with effector lymphocytes trigger the sequence of humoral and cellular events to produce the pathophysiologic effects that lead to tissue injury or disease. Stem cells often appear to be sensitive targets for therapeutic and environmental toxicants, most likely because of their rapid proliferation. Xenobiotics or various drugs that are toxic to the myelocytes of the bone marrow can cause profound immunosuppression due to loss of stem cells. Humans are now under sustained and increasing pressure of xenobiotics exposure. Xenobiotics can stimulate the immune system as antigens by provoking a substantial immune response. Even mild disturbances of this network could result in detrimental health effects. The influence of the xenobiotics on the immune system is either suppressive or enhancing. The former leads into immunosuppression with consequent increased susceptibility to infection and cancer. The latter is associated with the development of autoimmune reactivity such as delayed hypersensitivity, atopy, systemic or organ-specific immunopathology, and granulomas formation. It is likely that overall immunosuppressive effects of xenobiotics are caused by the interference with cellular proliferation and differentiation, downregulation of the cytokine signaling, and enhanced apoptosis of immune cells. In contrast, autoimmune reactions are induced by abnormal activation of immune cells followed

by dysregulated production of cytokines resulting in harmful inflammatory response. The field of immunotoxicology is new but developing rapidly. Attempts must be made to conduct basic research to address the cellular and molecular mechanism of immunomodulatory action of various xenobiotics. The newly emerging technologies such as genomics, proteomics, and bioinformatics will be certainly helpful to investigate the interactions between the immune system and xenobiotics in their full complexities. Toxic compounds may be antigenic or act as haptens and can evoke an antibody response. If these antibodies bind to the determinant on the parent molecule which is responsible for causing toxicity, then it can lead to the biological inactivation of the parent molecule and thereby prevent toxicity. This may constitute an immunological antidote approach to neutralize the toxicity of certain compounds. Thus, passive administration of the antibodies may be used to prevent the toxic effects of the specific compound and this approach may be useful in biological or chemical warfare to protect against the toxicity of known chemicals or toxins. The antibodies can also be used to protect industrial workers against the toxic effects of known chemicals or gases during accidental exposure. Although this assumption seems logical, it will involve elaborate and time-consuming research to identify the site of the parent molecule responsible for causing toxicity, to chemically link the molecule with a large protein molecule which should be immunogenic but not toxic, and to screen various antibodies raised for their capacity to prevent the toxicity of the compound. In view of the current global scenario, it appears that CWAs are likely to be used in different types of warfare, and it is unlikely their usage will cease in the near future. CWAs for warfare and other related activities are here to stay. These agents are not only inexpensive but easy to disseminate with the help of unsophisticated devices. Hence the medical profession should assemble on a common platform through globally recognized organizations like the WHO and put in efforts to monitor, research, and study the scientific and medical aspects of CWAs in the interest of humankind. Guidelines should be regularly updated on the prevention and management of CWA-induced insults and thereby aim to reduce morbidity and mortality. Nations worldwide should ensure that adequate supplies of antidotes (wherever available), protective equipment, and decontamination devices are available in adequate quantities and at all times. The need of the hour is a multisectorial approach involving health, defense, agriculture, and environmental specialists, with clearly defined roles of each, for establishing and maintaining effective, robust, and sustainable strategies to countermeasure this threatening situation. References Bajgar, J. (1992). Biological monitoring of exposure to nerve agents. Br. J. Ind. Med. 49: 648–53.

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Ghotbi, L., Hassan, Z. (2002). The immunostatus of natural killer cells in people exposed to sulfur mustard. Int. Immunopharmacol. 2: 981–5. Gift, J.S., McGaughy, R., Singh, D.V., Sonawane, B. (2008). Health assessment of phosgene: approaches for derivation of reference 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-induced modulation of the neuro-immune axis and its regulation by nitric oxide (NO) in rats. In Proceedings of 40th Annual Conference 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 to cyanide. 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 body fluids. Clin. Chim. Acta 57: 205–9. Hassan, Z.M., Ebtekar, M. (2002). Immunological consequence of sulfur mustard exposure. Immunol. Lett. 83: 151–2. Hektoen, H., Corper, H.J. (1921). The effect of mustard gas on antibody formation. J. Infect. Dis. 192: 279. Hermanowitz, A., Kossman, S. (1984). Neutrophil function and infectious disease occupationally exposed to phosphoorganic pesticides: role of mononuclear-derived chemotactic factor for neutrophils. Clin. Immunol. Immunopathol. 33: 13. Johnson, V.J., Rosenberg, A.M., Lee, K., Blakley, B.R. (2002). Increased T-lymphocyte dependent antibody production in female SJL/J mice following exposure to commercial grade malathion. 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 responses through the autonomic nervous system. Toxicol. Appl. Pharmacol. 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 neuroendocrine 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 repeated low-level sarin exposure on immune functions of inbred BALB/c mice. Basic Clin. Pharmacol. Toxicol. 94: 139–43. Kindt, T.J., Goldsby, R.A., Osborne, B.A. (2007). Cells and organs 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 oxygen free radicals on immune responsiveness in rabbits: an in vivo study. Immunol. Lett. 59: 127–31. Koner, B.C., Banerjee, B.D., Ray, A. (1998). Organochlorine induced oxidative stress and immune suppression in rats. Indian J. Exp. Biol. 36: 395–8. Korkmaz, A., Yaren, H., Topal, T., Oter, S. (2006). Molecular targets against mustard toxicity: implication of cell surface receptors, peroxynitrite production and PARP activation. Arch. Toxicol. 80: 662–70.

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