* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project
1040 Arsenic ecotoxicology and innate immunity Christopher R. Lage, Akshata Nayak, and Carol H. Kim* Department of Biochemistry, Microbiology and Molecular Biology, University of Maine, Orono, ME 04469, USA Synopsis Understanding the ecotoxicological effects of arsenic in the environment is paramount to mitigating its deleterious effects on ecological and human health, particularly on the immune response. Toxicological and long-term health effects of arsenic exposure have been well studied. Its specific effects on immune function, however, are less well understood. Eukaryotic immune function often includes both general (innate) as well as specific (adaptive) responses to pathogens. Innate immunity is thought to be the primary defense during early embryonic development, subsequently potentiating adaptive immunity in jawed vertebrates, whereas all other eukaryotes must rely solely on the innate immune response throughout their life cycle. Here, we review the known ecotoxicological effects of arsenic on general health, including immune function, and propose the adoption of zebrafish as a vertebrate model for studying such effects on innate immunity. Natural sources Arsenic is a naturally occurring metalloid element that is found in soil, air and water (Huang and others 2004; Duker and others 2005). Environmental arsenic exists in both organic and inorganic states. Organic arsenicals are generally considered nontoxic (Gochfield 1995), whereas inorganic forms are toxic. The most acutely toxic form is arsine gas (Leonard 1991). Inorganic arsenic exists predominantly in trivalent (As3þ) and pentavalent (As5þ) forms, where trivalent compounds are more toxic than pentavalent ones (Cervantes and others 1994; Smedley and others 1996; Duker and others 2005). Both trivalent and pentavalent arsenicals are soluble over a wide pH range (Bell 1998) and are routinely found in surface and groundwater (Feng and others 2001). Under aerobic conditions, pentavalent arsenic is more stable and predominates, whereas trivalent species predominate under anaerobic conditions (Duker and others 2005). Arsenic ranks 20th in abundance in relation to other elements in the earth’s crust and high concentrations are found in granite and in many minerals including copper, lead, zinc, silver and gold (NAS 1977). The geochemistry of arsenic in the environment was recently reviewed by Duker and others (2005). Arsenic naturally accumulates as both organic and inorganic forms in soil, surface and groundwater (Attrep and Anirudhan 1977; Lloyd-Smith and Wickens 2000), and seawater (Penrose and others 1977). The primary source of arsenic in soil is the parent rock (Smedley and Kinniburgh 2002). Additionally, volcanoes are a major natural source of arsenic released into the environment (Nriagu and Pacyna 1988; Nriagu 1989) that can generate high arsenic concentrations in natural waters (Smedley and Kinniburgh 2002). The chemistry of arsenic in aqueous environments has been reviewed by Ferguson and Gavis (1972). Arsenic concentrations in lakes are often less than in rivers, due to adsorption by iron oxides, although changes in water levels (Nimick and others 1998; Smedley and Kinniburgh 2002) and geothermal activity can enhance concentrations in some cases (Aggett and Kriegman 1988; Duker and others 2005). Groundwater from alluvial and deltaic watersheds generally has high arsenic concentration due to predominantly reducing conditions (Smedley and Kiniburgh 2002). Anthropogenic influences Human activities have intensified arsenic accumulation in the environment (Bell 1998) such as fossil fuel combustion and metal smelting, as well as the semiconductor and glass industries. Arsenic is also an ingredient in many commonly used materials including wood preservatives, pigments, insecticides, herbicides, rodenticides and fungicides (Hathaway and others 1991). Although most arsenic in soil is derived from the parent rock, the application of arsenic compounds in agriculture and forestry practices may lead to extreme soil contamination and subsequent groundwater From the symposium “Ecological Immunology: Recent Advances and Applications for Conservation and Public Health” presented at the annual meeting of the Society for Integrative and Comparative Biology, January 4–8, 2006, at Orlando, Florida. 1 E-mail: [email protected] Integrative and Comparative Biology, volume 46, number 6, pp. 1040–1054 doi:10.1093/icb/icl048 Advance Access publication October 11, 2006 Ó The Author 2006. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: [email protected] 1041 Effects of arsenic on innate immunity contamination, while the burning of coal and smelting of metals may be major sources of airborne arsenic. Mining activities may result in high levels of arsenic contamination in soil, surface water, groundwater and vegetation (Amasa 1975; Smedley and others 1996; Smedley and Kinniburgh 2002). Additionally, human modifications to the natural hydrograph, including the construction of dams (Armah and others 1998), wastewater recycling and irrigation practices (Siegel 2002), can potentiate arsenic accumulation in soil and in water supplies. The role of microbes Many microorganisms have adapted to arsenic-rich environments, including soils and waters (Nakahara and others 1977; De Vicente and others 1990; Cervantes and Chavez 1992; Ahmann and others 1994; Cervantes and others 1994; Laverman and others 1995; Saltikov and Olson 2002) and may be important factors in arsenic biotransformation (Shariatpanahi and others 1981) and mobilization (Cummings and others 1999) in the environment. Bacterial resistance to the toxic effects of arsenic may be a function of a specific arsenic-resistance operon, ars (Carlin and others 1995; Cai and others 1998), and may be facilitated by reduction in arsenic uptake and increased phosphate transport (Willsky and Malamy 1980). Homology across microbial taxa suggests that the ars operon is conserved in Gram-negative bacteria and that it has a functional role in arsenic detoxification (Diorio and others 1995). Bacterial populations have been shown to be associated with both oxidation and reduction of arsenic in soils (Macur and others 2004). Degradation of arsenic species has even been shown in bacterial symbionts of marine mussels (Jenkins and others 2003). Under anaerobic conditions, some microbes can reduce the less toxic arsenate to the more toxic arsenite (Andreae 1978; Nies and Silver 1995; Rensing and others 1999) through an energy-generating process (Ilyaletdinov and Abdrashitova 1981). Additionally, other microbes are able to methylate arsenic compounds (Gadd 1993), which may serve as a detoxification process. Seasonal variations in temperature and water levels can have strong effects on arsenic concentration and speciation in soil and water due to changes in microbial uptake (Andreae 1978, 1979). During warm, dry periods arsenic compounds are often oxidized (Maest and others 1992), potentially increasing toxicity (Savage and others 2000), while during wet periods oxidized arsenic is solubilized and distributed throughout the environment (McLaren and Kim 1995; Rodriguez and others 2004). Bioaccumulation and metabolism Arsenic accumulates across highly diverse environments within the soil, water and air where it is subsequently taken up and processed by microbes, plants and animals. Soluble arsenic taken up by plants rapidly accumulates in the food chain (Green and others 2001). Freshwater plants and peat moss have been shown to contain considerable amounts of arsenic (Reay 1972; Minkkinen and Yliruokanen 1978). Due to the high metal-binding affinity of their soils, wetlands may have elevated concentrations of arsenic (Beining and Ote 1996) when compared with uplands. High arsenic concentrations have been found in the tissues of wild birds (Fairbrother and others 1994) and in many marine organisms, including algae (Lunde 1972, 1973), crustaceans (Edmonds and others 1977), cetaceans, pinnipeds, sea turtles and sea birds (Kubota and others 2003). Ecotoxicants released into the environment, including arsenic, often accumulate most rapidly in aquatic habitats where they enter the biota and are subsequently transferred to higher trophic levels and, in many cases, eventually to humans. Extremely high levels of arsenic have been observed in many fish taxa (Bosnir and others 2003; Juresa and Blanusa 2003) and have been shown to be toxic (Suhendrayatna and others 2002; Tisler and Zagorc-Koncan 2002). Some species possess specific arsenic-binding proteins (Oladimeji 1985) that may increase bioaccumulation. Monitoring arsenic levels and their associated health effects in aquatic organisms, particularly in taxa at high trophic levels such as fish, may provide insight into overall ecosystem health (Zelikoff and others 2000) as well as into potential impacts on human health (Zelikoff 1998; Adams and Greeley 1999). Exposure from air and soil is usually minimal in humans. The major sources of exposure for humans are food and water (Bernstam and Nriagu 2000). Once ingested, arsenic that is not eliminated from the body may accumulate in the muscles, skin, hair and nails (Ishinishi and others 1986; Kitchin 2001). Food contains both organic and inorganic arsenic, whereas water primarily contains inorganic forms. Seafood may provide higher concentrations of arsenic when compared with terrestrial food products (Sakurai and others 2004), presumably due to increased bioaccumulation through generally longer trophic chains. As elemental arsenic is poorly absorbed, it is predominantly eliminated from the body unchanged (Duker and others 2005). Inorganic arsenic is absorbed through the gastrointestinal tract and is eliminated via renal function (Hindmarsh and McCurdy 1986); however, a small amount is biotransformed 1042 into "detoxified" forms via methylation and reduction in the liver (Winski and Carter 1995; Bernstam and Nriagu, 2000). Once thought to be a purely detoxification process, it has been shown that methylation of arsenic may, in some cases, actually increase arsenic toxicity in humans and rodents (Petrick and others 2000, 2001; Styblo and others 2000; Del Razo and others 2001). Variation in arsenic metabolism has been shown to occur in humans (Abernathy and others 1999). Interestingly, some mammals, including nonhuman primates, are deficient in arsenite methyltransferases necessary for effective methylation (Aposhian 1997). They may also show different tissuespecific expression (Abernathy and others 1999). The relationships between arsenical exposure, methylation and toxicity are paramount to understanding the risks posed to humans. Toxicology of arsenic Acute and chronic arsenic toxicities have been shown in a variety of organisms, and the data suggest that most inorganic arsenicals are more toxic than organic forms (Abernathy and others 1999; Duker and others 2005). Toxic effects of inorganic arsenic include denaturing of cellular enzymes through interaction with sulfhydryl groups (Graeme and Pollack 1998; Gebel 2000), causing cellular damage through increased reactive oxygen species (ROS) (Wang and others 1996; Ahmad and others 2000), and altering gene regulation (Rossman 1998; Abernathy and others 1999). Arsenic is known to inhibit more than 200 enzymes (Abernathy and others 1999) and has been implicated in multisystemic health effects via interference with enzymatic function and transcriptional regulation (NRC 1995). A variety of inhibitory effects on cellular metabolism have been shown, affecting mitochondrial respiration (Klaassen 1996; Abernathy and others 1999) and synthesis of adenosine triphosphate (ATP) (Winship 1984). Other effects of arsenic include activation of the estrogen receptor, inhibition of angiogenesis and tubulin polymerization, induction of heat-shock proteins, and oxidation of glutathione (Bernstam and Nriagu 2000). Due to its structural similarity to phosphate, arsenate may replace phosphorus in bone (Ellenhorn and Barceloux 1988). Among fish taxa, arsenic has been shown to induce apoptosis of fin cells (Wang and others 2004), to cause liver inflammation, hyperplasia and necrosis (Pedlar and others 2002), gall bladder inflammation, fibrosis and edema (Cockell and others 1991; Pedlar and others 2002;), kidney fibrosis (Kotsanis and IliopoulouGeorgudaki 1999), and the induction of various heatshock proteins (Kothary and Candido 1982). Arsenic C. R. Lage et al. has been shown to cause morphological changes, as well as to increase numbers of necrotic bodies, abnormal lysosomes and autophagic vacuoles in fish hepatocytes (Sorensen and others 1985). Additionally, effects on reproduction in fishes include disrupting ovarian cell cycles (Wang and others 2004), inhibiting ovarian follicle development (Shukla and Pandey 1984a), impairing spermatogenesis and changing testicular architecture (Shukla and Pandey 1984b). There is clear evidence that arsenic can disrupt gene expression, particularly through its effects on signal transduction (Abernathy and others 1999). Arsenic can interact directly with the glucocorticoid receptor (GR), selectively inhibiting GR-mediated transcription (Kaltreider and others 2001). It has been found to inhibit the Janus family of tyrosine kinase-signal transducers and activators of transcription (JAKSTATs) by interacting directly with JAK (Cheng and others 2004), to inhibit IkB-kinase (Roussel and Barchowsky 2000), as well as to inactivate protein tyrosine phosphatases, promote the activation of AP-1 and upregulate levels of MAPK (Cavigelli and others 1996). At low concentrations, arsenic has been shown to affect the DNA-binding capabilities of transcription factors NFkB and AP-1, leading to increased gene expression and stimulation of cell proliferation (Chen and others 2000; Wijeweera and others 2001). However, at high concentrations, arsenic may lower NFkB activation, inhibit cell proliferation and induce apoptosis (Shumilla and others 1998; Wei and others 2005). It has been suggested that arsenic can disrupt cell division by deranging the spindle apparatus (Abernathy and others 1999). Arsenic induces large deletion mutations (Hei and others 1998), chromosome damage and aneuploidy (Abernathy and others 1999) and causes micronucleus formation, DNA– protein cross-linking, and sister chromatid exchange (Huang and others 2004). It is known to inhibit DNA repair (Lynn and others 1997; Rossman 1998; Brochmoller and others 2000) and even to exacerbate the effects of other mutagenic agents (Abernathy and others 1999), thereby increasing susceptibility to multiple diseases (Duker and others 2005). Arsenic and the immune response Chronic exposure to arsenic, in addition to its general toxicity and its stimulation of many diseases, may affect lymphocyte, monocyte and macrophage activity in many mammals, resulting in immunosuppression (Blakley and others 1980; Gonsebatt and others 1994; Lantz and others 1994; Yang and Frenkel 2002; Wu and others 2003; Duker and others 2005; Sakurai and 1043 Effects of arsenic on innate immunity others 2006). Likewise, phagocytic activity of macrophages and other immune responses were found to be significantly reduced by arsenic exposure in birds (Fairbrother and others 1994; Vodela and others 1997). Generally, arsenic can disrupt glucocorticoid regulation of immune function (Kaltreider and others 2001) and arsenic-mediated apoptosis may lead to a diminished immune response in mice (Harrison and McCoy 2001), rats (Bustamante and others 1997) and humans (de la Fuente and others 2002; GonzalesRangel and others 2005). Additionally, arsenic exposure in mice has been shown to suppress the primary antibody response (Sikorski and others 1991), reduce macrophage and neutrophil abundance (Patterson and others 2004), increase susceptibility to infection (Aranyi and others 1985), increase mortality due to bacterial infection (Hatch and others 1985), decrease adhesion of macrophages, decrease nitric oxide (NO) production, and reduce chemotactic and phagocytotic indices (Sengupta and Bishayi 2002; Bishayi and Sengupta 2003). A field study of the effects of environmental arsenic exposure along a pollution gradient has also been shown to suppress immune function in wood mice (Tersago and others 2004). Arsenic has been shown to affect not only the immune response, but also behavior in rats (Schultz and others 2002). Dose-dependence of the immunotoxicological effects of arsenic is unclear. Dosedependent immunosuppressive relationships have been observed in mice (Burns and others 1991). However, in some studies the immunosuppressive effects of arsenic were most pronounced at low concentrations of exposure, compared to high concentrations (Blakley and others 1980; Savabieasfahani and others 1998), and it has even been proposed that in some situations arsenic may enhance certain immune responses (Yoshida and others 1987). Arsenic has been found to increase the expression of granulocyte macrophage-colony stimulating factor (GM-CSF), transforming growth factor-a (TGF-a) and TNFa in human keratinocytes (Germolec and others 1996), and IL-1a and IL-8 in murine keratinocytes (Yen and others 1996; Corsini and others 1999). While arsenic has been shown to induce expression of IL-1, IL-6 and IL-7 receptors, as well as inducible nitric oxidase (iNOS) in rat liver epithelial cells (Chen and others 2001), it has also been shown to downregulate expression of the IL-2 receptor (Yu and others 1998), IL-1a, IL-1b, IL-2, IL-8, IL-12A and B, IL-18, monocyte chemotactic protein-1 (MCP-1), TGF-b1 and b2 human osteoblasts (Yang and Frenkel 2002). Reactive oxygen species (ROS) generated during the innate respiratory-burst response destroy invading pathogens; however, they can also cause oxidative stress and tissue damage and affect signal transduction. The introduction of environmental arsenic may lead to the increased production of ROS through various pathways and has been shown in humans (Pineda-Zavaleta and others 2004). Arsenic can activate NADPH oxidase and induces the production of O2 and H2O2 (Chen and others 1998), thereby increasing the production of the intercellular messenger NO (Gurr and others 1998), and uncoupling mitochondrial oxidative phosphorylation (Klaassen 1996). Low levels of ROS can modulate gene expression and induce apoptosis (Chen and others 1998), whereas high levels of ROS can result in oxidative damage and cell death. DNA damage and micronucleus formation caused by ROS may also potentiate cancer by enhancing cell proliferation (Hei and others 1998). In addition to increasing the amount of ROS produced, arsenic disturbs the natural oxidation and reduction equilibria of proteins, including many signal molecules such as AP-1, NFkB, IkB, p53 and p21ras (Simeonova and Luster 2000), by binding to cysteine-sulfhydryl groups. Arsenic and disease Arsenic compounds have been used directly on humans for treating many diseases including skin conditions, malaria, ulcers, syphilis, sleeping sickness and some forms of leukemia (Luh and others 1973; Nevens and others 1990; Zhang 1999; Miller and others 2002) although it is now rarely used medicinally (Azcue and Nriagu 1994). Organs most susceptible to arsenic toxicity are those involved with absorption, accumulation or excretion, including the skin, circulatory system, gastrointestinal tract, liver and kidney (Duker and others 2005). The primary symptom of arsenic exposure is dermal lesions (Zaloga and others 1985). Skin localizes and stores arsenic, presumably due to high levels of sulfhydryl-rich keratin (Kitchin 2001), potentially explaining this response. Arsenic is associated with multiple health effects, including Blackfoot disease (Abernathy and others 1999), diabetes (Longnecker and Daniels 2001), hypertension (Chen and others 1995), peripheral neuropathy and multiple vascular diseases (see Duker and others 2005). Other effects include anemia (ATSDR 2000), liver damage, portal cirrhosis, hematopoietic depression, anhydremia, sensory disturbance and weight loss (Webb 1966). It has been suggested that multiple factors, including genetics and nutrition, affect susceptibility to arsenic and disease manifestation (Mandal and others 1996; Hseuh and others 1998). 1044 In addition to acute toxicity, long-term exposure to inorganic arsenic is associated with certain forms of cancer of the skin, lung, colon, bladder, liver and breast (Nemery 1990; Abernathy and others 1999; Huang and others 2004; Duker and others 2005) although effects may not appear until more than 20 years after exposure (Jackson and Grainge 1975). It has been suggested that arsenic may act as a carcinogen through DNA hypomethylation and overexpression of protooncogenes (Zhao and others 1997). Cancer may induced by alteration of DNA-repair mechanisms, thus interfering with cell division, differentiation and tumor suppression (Chen and others 1996; Goering and others 1999). Additionally, arsenic may induce certain forms of cancer by enhancing the carcinogenic effects of other substances and by affecting metabolic pathways (Huang and others 2004). In summary, the effects of arsenic on health include various mechanisms of acute and chronic toxicity, enzymatic and genetic effects, and/or increasing susceptibility to multiple types of disease, both cancerous and noncancerous. Although it is clear that exposure to arsenic alters normal biological functions, resulting in the direct initiation of disease or, at least, predisposition of an organism to it, studying the impact of arsenic on the ability to fight viral and bacterial infections via specific immune responses is particularly important for full understanding of its overall effects on health. C. R. Lage et al. possibly other species, do not develop functional adaptive immunity until a distinct stage of development, until which time they rely entirely upon the innate immune response (Willett and others 1997). Because of its importance during development, generality of mechanisms of defense against pathogens and taxonomically conserved nature, innate immunity has particularly broad implications for overall health. Adaptive immune response The adaptive immune response is mediated primarily by B lymphocytes and T lymphocytes. Antigenpresenting cells transport and present antigenic molecules of pathogens to naı̈ve T lymphocytes, resulting in their differentiation into effector cells. By virtue of rearrangeable immune gene segments, this response can generate a repertoire of lymphocytes with receptors specific to antigenic components of any potential pathogen. The mature T cells either migrate to the site of infection to effect cell-mediated immunity, or circulate in the lymphoid organs to activate the B lymphocytes and participate in humoral immunity. B cells mature to form plasma cells and memory cells, which are responsible for producing antibodies against the pathogen and establishing immune memory, respectively. The adaptive immune response eradicates the infectious agent from the host body and provides an immunological memory against reinfection by the same pathogen. Overall immune response The immune system functions to protect a host from pathogenic infection. Its complex array of defense mechanisms relies mainly on the ability to distinguish between the host and foreign cells. To effectively manage this, the immune system has 2 separate responses that work synergistically to fight infection. The innate immune response uniquely recognizes molecular patterns, surface structures and cellular products conserved across a diversity of microbes. It is activated as the first line of defense, whereas the secondary adaptive immune response consists of a complex initiation process, followed by clonal selection and expansion of receptors specific to unique epitopes on the pathogen. They are mutually complementary, with the innate system acting as a prerequisite, potentiating factor for the adaptive immune response. All eukaryotic organisms, from amoebae to mammals, have mechanisms of innate immunity, whereas adaptive immunity, which appeared 450 million years ago, is present only in jawed vertebrates (Agrawal and others 1998). Some fish, and Innate immune response Innate immunity comprises a collection of defense mechanisms that protect an organism against infection without depending upon prior exposure and cell memory. As a rapid first response to assault by pathogens, the innate immune system halts infection or keeps it at bay until the adaptive immune response has sufficient time to develop. In addition to circulating cells, the innate immune system comprises epithelial barriers such as skin, scales and mucociliary membranes that separate the organism from the external environment. If this external barrier is compromised, receptors on circulating cells recognize specific molecular signatures and induce an inflammatory response, involving the recruitment and subsequent activation of leukocytes. Inflammation is considered to be the first sign of the wound-healing process and is characterized by localized elevation of temperature, pain, erythema and edema. Infectious agents are eliminated primarily by phagocytic cells, including macrophages, neutrophils and natural killer cells that migrate to the site of infection and respond Effects of arsenic on innate immunity by engulfing and destroying the pathogens. These cells also secrete effector molecules such as chemokines and cytokines that function as chemical messengers of the immune response and facilitate cell-to-cell communication. Detection of pathogens by the innate immune system is dependent on the recognition of invariant molecules of the microorganism by cell surfaceassociated receptors, the best characterized of which are the toll-like receptors (TLRs). TLR signaling was recently reviewed by Akira and Takeda (2004). Tolls were first discovered in Drosophila, associated with dorsoventral patterning during development (Hashimoto and others 1988). Further studies revealed the essential role of these transmembrane receptors in innate immunity (Lemaitre and others 1996); they detect conserved molecular patterns shared by large groups of microorganisms, such as lipopolysaccharide and double-stranded ribonucleic acid (RNA) (Akira and Takeda 2004). TLR family members are differentially expressed among immune cells and are characterized by the presence of a leucine-rich, extracellular domain and an intracellular toll/interleukin 1 receptor (TIR) domain. Ligand binding results in conformational changes in the TIR domains that facilitate interactions with downstream signaling molecules. This cascade results in the activation of cytoplasmic transcription factors, NF-kB or interferon regulatory factor 3, which subsequently translocate to the nucleus and begin transcription of proinflammatory cytokines (Medzhitov 2001). Chemokines are low molecular weight proteins that represent a superfamily of about 30 chemoattractants acting as vital initiators of the inflammatory process. They are secreted by a wide variety of cells including monocytes, neutrophils, epithelial cells, smooth muscle cells and T cells (Rollins 1997). Chemokines function mainly in leukocyte physiology by controlling inflammatory trafficking, but are also important for gene transcription, apoptosis and granule exocytosis (Thelen 2001). Some chemokines are inducible by physiological stress and recruit leukocytes to sites of injury, whereas others are constitutively active and are responsible for basal trafficking of leukocytes. All chemokines are tightly regulated by feedback mechanisms because of their potential for severely damaging host tissues by uncontrolled persistent expression. Cytokines are small, soluble, pleiotropic proteins that are secreted by virtually all cells in the body and possess both autocrine and paracrine functions. They act on their targets by binding specific membrane receptors and signaling through them to commence the transcription of certain genes, usually those involved in cellular activation or growth and 1045 differentiation. The central role of cytokines, however, is to modulate and direct the amplitude of immune responses. There are 2 major groups of cytokines: proinflammatory are secreted by activated macrophages, and anti-inflammatory are involved in the downregulation of inflammatory reactions. Important antiviral cytokines include certain interferons, whereas antibacterial cytokines include interleukins and tumor necrosis factors. Interferons are a multigene family of inducible cytokines that possess antiviral activity, broadly grouped into 2 categories, type I and type II, based on their biological, biochemical and immunological properties (Samuel 1991). Type I interferons are secreted upon viral infection and almost all virusinfected cells can synthesize them. Type II interferons are upregulated by antigenic stimuli and are secreted mainly by natural killer cells, CD4þ Th1 cells and CD8þ cytotoxic suppressor cells (Young 1996). All interferons act through membrane-associated receptor complexes. In response to ligand binding, JAK-STAT members become activated via a series of phosphorylation events, ultimately leading to the translocation of STATs to the nucleus, and upregulation of interferon-inducible genes (Stark and others 1998; Horvath 2000). Among the genes induced by interferons are proteins important in anti-RNA virus activity, including protein kinase (PKR), oligoadenylate synthetase (OAS) and interferon-induced Mx GTPases. PKR and OAS are essential for inhibition of mRNA translation and catalyzing RNA degradation, respectively (Jacobs and Langland 1996; Samuel 1998). Mx GTPases are key components for resisting a wide range of RNA viruses; produced for short periods of time, they function by sensing viral nucleocapsids. Their actions trap essential viral components and make them unavailable for viral replication, thus containing the infection (Haller and Kochs 2002). Mx has strong intrinsic antiviral properties, which makes it competent to induce an effective antiviral state independently (Hefti and others 1999). Interleukins (ILs) were initially considered to be cytokines essential only for the functioning of leukocytes, but research has shown that they affect nearly all cell types. The IL-1 gene family includes 3 members, interleukin-1a, interleukin-1b and an interleukin-1 receptor antagonist, IL-1Ra. IL-1b is considered to function as a hormone-like mediator, intended to be released by cells, whereas IL-1a is primarily a regulator of intracellular events and of localized inflammation. At low concentrations, IL-1b mediates inflammation by increasing synthesis of cell surface adhesion molecules, thus activating 1046 neutrophils and macrophages and stimulating their recruitment to the site of injury. At higher concentrations, it exerts systemic effects through the activation of NFkB, AP-1 and activating transcription factor (ATF) (Li and others 2001). Tumor necrosis factor (TNF)a, first discovered for its ability to induce necrosis of tumor cells, belongs to a superfamily of proteins that possess a wide range of proinflammatory functions. TNFa is secreted by activated macrophages and is critical for the normal functioning of T cells, natural killer (NK) cells, macrophages and dendritic cells. TNFa is also responsible for priming the respiratory-burst response in activated macrophages by increasing the production of NADPH-oxidase subunits. Phagocytosis by neutrophils and macrophages is an essential mechanism for the elimination of invading microorganisms. Upon initiation of phagocytosis, the phagosome fuses with lysosomes and destroys the ingested microbe through the production of reactive oxygen species and superoxides by a process known as the respiratory burst. The enzyme responsible for superoxide production is NADPH oxidase, which has membrane-bound and cytosolic components. The cytosolic subunits translocate to the membrane upon phosphorylation and assist in forming the functional enzyme complex that reduces molecular oxygen to produce superoxide (Smith and others 1996). Hydrogen peroxide, hydroxyl radicals and hypochlorites are reactive oxygen species formed from the superoxide via multiple enzymatic reactions (Hermann and others 2004). Zebrafish as a model for innate immunity Zebrafish or Danio rerio is a teleost that has become important as an animal model for studying the embryo development, genetics and immune system. It has several advantages over other animal models currently used. Zebrafish have rapid development, are small in size and easy to manage, and are highly fecund compared with other vertebrate models. Embryos develop externally as transparent larvae, allowing easy observation of cellular and organ development. Genomic libraries, combined with shotgun sequencing methods, have permitted the sequencing of the zebrafish genome, and analyses of genes reveal significant similarities to higher vertebrates (Amemiya and others 1999). Forward genetic screens have identified several mutants with phenotypes comparable to human genetic diseases (Karlovich and others 1998; Leimer and others 1999; Hostetter and others 2003) while reverse genetic approaches have also been C. R. Lage et al. designed, allowing knockdown of individual genes (Nasevicius and Ekker 2000). Zebrafish have been used as a model for understanding the genetic basis for both viral and bacterial infectious diseases. Pathogenic viral infections examined in a zebrafish model include the spring viremia carp virus (Sanders and others 2003), pancreatic necrosis virus and infectious hematopoietic necrosis virus (LaPatra and others 2000), and snakehead rhabdovirus (Phelan, Pressley and others 2005). In some viral infection models, zebrafish are able to rapidly clear the pathogen without increased mortality or clinical symptoms, whereas in other models infection results in increased mortality, hemorrhaging and increased expression of genes involved in the antiviral immune response. Differences in virulence and host immune response have been observed among different bacterial strains used for infection (Menudier and others 1996). In some cases, one strain of bacterium may cause systemic infection in all major organ systems (Neely and others 2002), whereas, in other cases, pathogenesis of a different strain may be restricted to certain tissues (Miller and Neely 2004). Bacterial infection may result in mortality corresponding with a dramatic increase in the amount of bacteria entering the bloodstream (van der Sar and others 2003). It has also been shown that bacterial infection by static immersion results in increased inflammatory cytokine production (Pressley and others 2005). Infections involving both Grampositive and Gram-negative bacteria in zebrafish have shown similar gene induction to that observed in mammalian systems, suggesting conserved immune mechanisms among fish and mammals (Lin and others 2006). Mycobacterial infection in the zebrafish resulted in granuloma-like lesions similar to those seen in mammals (Prouty and others 2003), while microarray analysis revealed upregulation of genes known to be associated with immune function (Meijer and others 2005). Functional adaptive immunity is not present in zebrafish until the 4th day of development (Willett and others 1997) such that, until this time, mechanisms of infectious defense rely entirely on the innate immune response. Early zebrafish macrophages develop and have chemotactic functions similar to their mammalian counterparts (Herbomel and others 1999). A bioassay to measure the respiratory-burst response has been developed in the zebrafish (Hermann and others 2004). Multiple chemokines (Long and others 2001; David and others 2002) and cytokines (Altmann and others 2003; Altmann and others 2004; Pressley and others 2005) have been characterized in the zebrafish. Additionally, over 1047 Effects of arsenic on innate immunity 20 putative variants of the toll-like receptors (Jault and others 2004; Meijer and others 2004; Phelan, Mellon and others 2005) as well as 4 adaptor proteins (Jault and others 2004) have been identified in the zebrafish. Thus, the zebrafish provides a unique model for studying vertebrate biology and, in particular, for investigating ecotoxicological effects on innate immunity. Arsenic and innate immunity in fish As many components of innate immunity are evolutionarily conserved (Hoffmann and others 1999; Ulevitch 2000), and as arsenic often accumulates most rapidly in aquatic habitats, monitoring arsenic levels and their associated health effects in fish may not only provide insight into overall ecosystem health (Zelikoff and others 2000) but may also act as a sentinel for potential impacts on human health (Zelikoff 1998; Adams and Greeley 1999). Because adaptive immunity is developmentally delayed in fish (Alexander and Ingram 1992), the effects of ecotoxicants on innate immunity may be more significant and thus easier to measure in fish than in mammals. The general effects of ecotoxicants (Bols and others 2001) and stress (Fletcher 1986) on innate immunity in fish have been reviewed. Arsenic has been shown to induce metallothionein (MT), part of the oxidative stress response (Schlenk and others 1997; Hermesz and others 2002; Pedlar and others 2002) in various species of fish. The duration of arsenic exposure may affect the generation of a stress response in some fish, as it has been shown in the snakehead that after initial arsenic exposure and reduction in ROS scavenging enzyme production, an extended exposure upregulated enzymatic activity, resulting in increased arsenic resistance over time (Allen and Rana 2004). Arsenic has been shown to regulate transcriptionfactor activation in zebrafish cell cultures (Carvan and others 2000). Effects of arsenic on the innate immune system, including the ability to mount an adequate respiratory-burst response, express essential antiviral genes and produce sufficient levels of TNFa, within the concentration range of arsenic found in contaminated groundwater (Clark and Raven 2004), have been evaluated in the zebrafish model system (Hermann and Kim 2005). The ability to mount an appropriate respiratory burst is an indicator of the general immune health of an organism and reductions in respiratory-burst activity were found in zebrafish on exposure to even low concentrations of arsenic. In an effort to elucidate the possible mechanism for this inhibition, examination of TNFa levels revealed decreased expression upon exposure, supporting the possibility that arsenic hinders respiratory-burst activity by reducing the expression of TNFa, which is essential for priming this response. The antiviral response of the fish was examined upon arsenic exposure before and after infection with snakehead rhabdovirus. Gradual increases in interferon expression were observed over time in arsenic-exposed zebrafish. However, Mx levels failed to mirror this trend. The disruption of Mx induction by interferon indicates arsenic inhibition of interferon signaling. Upon viral exposure, expression levels of both antiviral genes were downregulated when compared with arsenic-unexposed controls. These data suggest that the ability of the zebrafish to mount an effective innate immune response is reduced by arsenic exposure, indicating that the presence of this metalloid in water has potentially adverse effects on the components of innate immunity essential for an antiviral response in fish. Concluding remarks Mounting evidence of links to human disease underscores the need for in-depth investigations into the health effects of arsenic. Furthermore, from our studies and those of others, it is becoming increasingly evident that fish, and in particular zebrafish, serve as an ideal animal model for studying the effects of arsenic. It is now clear that arsenic disrupts the immune response, potentially playing an important role in the outcome of infection and of host resistance to infectious diseases. Conflict of interest: None declared. References Abernathy CO, Liu Y, Longfellow D, Aposhia HV, Beck B, Fowler B, Goyer R, Menzer R, Rossman T, Thompson C, Waalkes M. 1999. Arsenic: health effects, mechanisms of actions, and research issues. Environ Health Perspect 107:593–7. Adams SM, Greeley MS. 1999. Establishing possible links between aquatic ecosystem health and human health: an integrated approach. In: Di Giulo RT, Monosson E, editors. Interconnections between human and ecosystem health. London: Chapman and Hall. p 91–102. Agency for Toxic Substances and Disease Registry, 2000. Toxicological profile for arsenic: health effects chapter; [cited 2006 April]. Available at: http://www.atsdr.cdc.gov/ toxpro2.html#Final. Aggett J, Kriegman MR. 1988. The extent of formation of arsenic (III) in sediment interstitial waters and its release to hypolimnetic waters in Lake Ohakuri. Water Res 22:407–11. 1048 Agrawal A, Eastman QM, Schatz DG. 1998. Transposition mediated by RAG1 and RAG2 and its implications for the evolution of the immune system. Nature 394:744–51. Ahmad S, Kitchin KT, Cullen WR. 2000. Arsenic species that cause release ofiron from ferritin and generation of activated oxygen. Arch Biochem Biophys 382:195–202. Ahmann D, Roberts AL, Krumholz LR, Morel FMM. 1994. Microbe grows by reducing arsenic. Nature 371:750. Akira S, Takeda K. 2004. Toll-like receptor signalling. Nat Rev Immunol 4:499–511. Alexander JB, Ingram GA. 1992. Noncellular nonspecific defence mechanisms of fish. Annu Rev Fish Dis 2:249–79. Allen T, Rana SV. 2004. Effect of arsenic (AsIII) on glutathionedependent enzymes in liver and kidney of the freshwater fish Channa punctatus. Biol Trace Elem Res 100:39–48. Altmann SM, Mellon MT, Distel DL, Kim CH. 2003. Molecular and functional analysis of an interferon gene from the zebrafish, Danio rerio. J Virol 77:1992–2002. Altmann SM, Mellon MT, Johnson MC, Paw BH, Trede NS, Zon LI, Kim CH. 2004. Cloning and characterization of an Mx gene and its corresponding promoter from the zebrafish, Danio rerio. Dev Comp Immunol 28:295–306. Amasa SK. 1975. Arsenic pollution at Obuasi goldmine, town and surrounding countryside. Environ Health Perspect 12:131–5. Amemiya CT, Zhong TP, Silverman GA, Fishman MC, Zon LI. 1999. Zebrafish YAC, BAC, and PAC genomic libraries. Methods Cell Biol 60:235–58. Andreae MO. 1978. Distribution and speciation of arsenic in natural waters and some marine algae. Deep Sea Res 25:391–402. Andreae MO. 1979. Arsenic speciation in seawater and interstitial waters: the biological, chemical interactions on the chemistry of a trace element. Limnol Oceanol 24:440–52. C. R. Lage et al. Bernstam L, Nriagu J. 2000. Molecular aspects of arsenic stress. J Toxicol Environ Health B Crit Rev 3:293–322. Bishayi B, Sengupta M. 2003. Intracellular survival of Staphylococcus aureus due to alteration of cellular activity in arsenic and lead intoxicated mature Swiss albino mice. Toxicology 184:31–9. Blakley BR, Sisodia CS, Mukkur TK. 1980. The effect of methylmercury, tetraethyl lead, and sodum arsenite on the humoral immune response in mice. Toxiol Appl Pharmacol 52:245–54. Bols NC, Brubacher JL, Ganassin RC, Lee LEJ. 2001. Ecotoxicology and innate immunity in fish. Dev Comp Immunol 25:853–73. Bosnir J, Puntaric D, Skes I, Klaric M, Simic S, Zoric I, Galic R. 2003. Toxic metals in freshwater fish from the Zagreb area as indicators of environmental pollution. Coll Antropol 27 (Suppl 1):31–9. Brochmoller J, Cascorbi I, Henning S, Meisel C, Roots I. 2000. Molecular genetics of cancer susceptibility. Pharmacology 61:212–27. Burns LA, Sikorski EE, Saady JJ, Munson AE. 1991. Evidence for arsenic as the immunosuppressive component of gallium arsenide. Toxicol Appl Pharmacol 110:157–69. Bustamante J, Dock L, Vahter M, Fowler B, Orrenius S. 1997. The semiconductor elements arenic and indium induce apoptosis in rat thymocytes. Toxicology 118:129–36. Cai J, Salmon K, DuBow MS. 1998. A chromosomal ars operon homologue of Psuedomonas aeruginosa confers increased resistance to arsenic and antimony in Esherichia coli. Microbiology 144:2705–13. Carlin A, Shi W, Dey S, Rosen BP. 1995. The ars operon of Escherichia coli confers arsenical and antimonial resistance. J Bacteriol 177:981–6. Aposhian HV. 1997. Enzymatic methylation of arsenic species and other approaches to arsenic toxicity. Annu Rev Pharmacol Toxicol 37:397–419. Carvan MJ III, Solis WA, Gedamu L, Nebert DW. 2000. Activation of transcription factors in zebrafish cell cultures by environmental pollutants. Arch Biochem Biophys 376:320–7. Aranyi C, Bradof JN, O’Shea WJ, Graham JA, Miller FJ. 1985. Effects of arsenic trioxide inhalation exposure on pulmonary antibacterial defenses in mice. J Toxicol Environ Health 15:163–72. Cavigelli M, Li WW, Lin A, Su B, Yoshioka K, Karin M. 1996. The tumor promoter arsenite stimulates AP-1 activity by inhibiting a JNK phosphatase. EMBO J 15:6269–79. Armah AK, Darpaah GA, Carboo D. 1998. Heavy metal levels and physical parameters of drainage ways and wells in three mining areas in Ghana. J Ghana Sci Assoc 1:113–7. Cervantes C, Ji G, Ramirez JL, Silver S. 1994. Resistance to arsenic compounds in microorganisms. FEMS Microbiol Rev 15:355–67. Attrep M Jr, Anirudhan M. 1977. Atmospheric inorganic and organic arsenic. Trace Subst Environ Health 11:365–9. Cervantes C, Chavez J. 1992. Plasmid-determined resistance to arsenic and antimony in Psuedomonas aeriginosa. Antonie Van Leeuwenhoek 61:333–7. Azcue JM, Nriagu JO. 1994. Arsenic: historical perspectives. In: Nriagu JO, editor. Arsenic in the environment. Vol. 26. New York: John Wiley & Sons. p 1–15. Chen CJ, Hsueh YM, Lai MS, Shyu MP, Chen SY, Wu MM. 1995. Increased prevalence of hypertension and long-term arsenic exposure. Hypertension 25:53–60. Beining BA, Ote MI. 1996. Retention of metals originating from an abandoned lead-zinc mine by a wetland at Glenalough, Co Wicklow. Biol Environm Proc R Ir Acad Sci 96(B2):117–26. Chen GQ, Zhu J, Shi XG, Ni JH, Zhong HJ, Si GY, Jin XL, Tang W, Li XS, Xong SM. and others. 1996. In vitro studies on cellular and molecular mechanisms of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia: As2O3 induces NB4 cell apoptosis with downregulation of Bcl-2 expression and modulation of PML-RAR alpha/PML proteins. Blood 88:1052–61. Bell FG. 1998. Environmental geology and health. Environmental geology: principles and practice. London: Blackwell Science. p 487–500. 1049 Effects of arsenic on innate immunity Chen H, Liu J, Merrick BA, Waalkes MP. 2001. Genetic events associated with arsenic-induced malignant transformation: applications of cDNA microarray technology. Mol Carcinog 30:79–87. Chen NY, Ma WY, Huang C, Ding M, Dong Z. 2000. Activation of PKC is required for arsenite-induced signal transduction. J Environ Pathol Toxicol Oncol 19:297–305. Chen YC, Lin-Shiau SY, Lin JK. 1998. Involvement of reactive oxygen species and caspase 3 activation in arsenite-induced apoptosis. J Cell Physiol 177:324–33. Cheng HY, Li P, David M, Smithgall TE, Feng L, Lieberman MW. 2004. Arsenic inhibition of the JAK-STAT pathway. Oncogene 23:3603–12. Clark ID, Raven KG. 2004. Sources and circulation of water and arsenic in the Giant Mine, Yellowknife, NWT, Canada. Isotopes Environ Health Studies 40:115–28. Cockell KA, Hilton JW, Bettger WJ. 1991. Chronic toxicity of dietary disodium arsenate heptahydrate to juvenile rainbow trout (Oncorhynchus mykiss). Arch Environ Contam Toxicol 21:518–27. Corsini E, Asti L, Viviani B, Marinovich M, Galli CL. 1999. Sodium arsenate induces overproduction of interleukin1alpha in murine keratinocytes: role of mitochondria. J Invest Dermatol 113:760–5. Cummings DE, Caccavo F Jr, Fendorf SE, Rosenzweig RF. 1999. Arsenic mobilization by the dissimilatory Fe(III)-reducing bacterium Shewanella alga BrY. Environ Sci Technol 33:723–9. David NB, Sapede D, Saint-Etienne L, Thisse C, Thisse B, Dambly-Chaudiere C, Rosa FM, Ghysen A. 2002. Molecular basis of cell migration in the fish lateral line: role of chemokine receptor CXCR4 and of its ligand, SDF1. Proc Natl Acad Sci USA 99:16297–302. de la Fuente H, Portales-Perez D, Baranda L, Diaz-Barriga F, Saavedre-Alanis V. 2002. Effect of arsenic, cadmium and lead on the induction of apoptosis of normal human mononuclear cells. Clin Exp Immunol 129:69–77. Del Razo LM, Styblo M, Cullen WR, Thomas DJ. 2001. Determination of trivalent methylated arsenicals in biological matrices. Toxicol Appl Pharmacol 174(3):282–93. De Vicente A, Aviles M, Codina JC, Borrego JJ, Romero P. 1990. Resistance to antibiotics and heavy metals of Pseudomonas aeruginosa isolated from natural waters. J Appl Bacteriol 68:625–32. Diorio C, Cai J, Mormor J, Shinder R, DuBow MS. 1995. An Escherichia coli chromosomal ars operon homolog is functional in arsenic detoxification and is conserved in gram-negative bacteria. J Bacteriol 177(8):2050–6. Duker AA, Carranza EJM, Hale M. 2005. Arsenic geochemistry and health. Environ Int 31:631–41. Edmonds JS, Francesoni KA, Cannon JR, Raston CL, Sketon BW, White AH. 1977. Isolation, crystal structure and synthesis of arsenobetaine, the arsenical constituent of the western rock lobster Panulirus longipes cygnus George. Tetrahedron Lett 18:1543–6. Ellenhorn MJ, Barceloux DG. 1988. Arsenic in medical toxicology: diagnosis and treatment of human poisoning. New York: Elsevier. p 1012–6. Fairbrother A, Fix M, O’Hara T, Ribic CA. 1994. Impairment of growth and immune function of avocet chicks from sites with elevated selenium, arsenic, and boron. J Wildl Dis. 30:222–33. Feng Z, Xia Y, Tian D, Wu K, Schmitt M, Kwok RK, Mumford JL. 2001. DNA damage in buccal epithelial cells from individuals chronically exposed to arsenic via drinking water in Inner Mongolia, China. Anticancer Res 21:51–8. Ferguson JF, Gavis J. 1972. A review of the arsenic cycle in natural waters. Water Res 6:1259–74. Fletcher TC. 1986. Modulation of non specific host defenses in fish. Vet Immunol Immunopathol 12:59–67. Gadd GM. 1993. Microbial formation and transformation of organometallic and organometalloid compounds. FEMS Microbiol Rev 11:297–316. Gebel T. 2000. Confounding variables in the environmental toxicology of arsenic. Toxicology 144:155–62. Germolec DR, Yoshida T, Gaido K, Wilmer JL, Simeonova PP, Kayama F, Burleson F, Dong W, Lange RW, Luster MI. 1996. Arsenic induces overexpression of growth factors in human keratinocytes. Toxicol Appl Pharmacol 141:308–18. Gochfeld M. 1995. Chemical agents. In: Brooks S, Gochfeld M, Herzstein J, Schenker M, editors. Environmental medicine. St. Louis: Mosby. p 592–614. Goering PL, Aposhian HV, Mass MJ, Cebrian M, Beck BD, Waalkes MP. 1999. The enigma of arsenic carcinogenesis: role of metabolism. Toxicol Sci 49:5–14. Gonzales-Rangel Y, Escudero-Lourdes sensitizes CD3þ arsenite-mediated 48:89–91. Portales-Perez DP, Galicia-Cruz O, C. 2005. Chronic exposure to arsenic and CD56þ human cells to sodium apoptosis. Proc West Pharmacol Soc Gosenbatt ME, Vega L, Montero R, Garcia-Vargas G, DelRazo LM, Albores A, Cebrian ME, OstroskyWegman P. 1994. Lymphocute replicating ability in individuals exposed to arsenic via drinking water. Mutat Res 313:293–9. Graeme HM, Pollack JVC. 1998. Selected topics: toxicology: Part I Arsenic and mercury. J Emerg Med 16:45–56. Green K, Broome L, Heinze D, Johnston S. 2001. Long distance transport of arsenic by migrating Bogon Moth from agricultural lowlands to mountain ecosystem. Victorian Nat 118(4):112–6. Gurr JR, Liu F, Lynn S, Jan KY. 1998. Calcium-dependent nitric oxide production is involved in arsenite-induced micronuclei. Mutat Res 416:137–48. Haller O, Kochs G. 2002. Interferon induced Mx proteins: dynamin like GTPases with antiviral activity. Traffic 3:710–7. Harrison MT, McCoy KL. 2001. Immunosuppression by arsenic: a comparison of cathepsin L inhibition and apoptosis. Int Immunopharmacol 1:647–56. 1050 Hashimoto C, Hudson KL, Anderson KV. 1988. The Toll gene of Drosophila, required for dorsal-ventral embryonic polarity, appears to encode a transmembrane protein. Cell 52:269–79. Hatch GE, Boykin E, Graham JA, Lewtas J, Pott F, Loud K, Mumford JL. 1985. Inhalable particles and pulmonary host defense: in vivo and in vitro effects of ambient air and combustion particles. Environ Res 36:67–80. Hathaway GJ, Proctor NH, Hughes JP, Fischman ML. 1991. Arsenic and arsine. In: Proctor NH, Hughes JP, editors. Chemical hazards of the workplace. Third edition. New York: Van Nostrand Reinhold. p 92–6. Hefti HP, Frese M, Landis H, Di Paolo C, Aguzzi A, Haller O, Pavlovic J. 1999. Human MxA protein protects mice lacking a functional alpha/beta interferon system against La Crosse virus and other lethal viral infections. J Virol 73:6984–91. Hei TK, Liu SX, Waldren C. 1998. Mutagenicity of arsenic in mammalian cells: role of reactive oxygen species. Proc Natl Acad Sci USA 95:8103–7. Herbomel P, Thisse B, Thisse C. 1999. Ontogeny and behaviour of early macrophages in the zebrafish embryo. Development 126:3735–45. Hermann AC, Kim CH. 2005. Effects of arsenic on zebrafish innate immune system. Marine Biotechnol 7(5):494–505. Hermann AC, Millard PJ, Blake SH, Kim CH. 2004. Development of a respiratory burst assay using zebrafish kidneys and whole embryos. J Immunol Methods 292(1–2):119–29. Hermesz E, Gazdag AP, Ali KS, Nemcsok J, Abraham M. 2002. Differential regulation of the two metallothionein genes in common carp. Acta Biol Hung 53:343–50. Hindmarsh JT, McCurdy RF. 1986. Clinical and environmental aspects of arsenic toxicity. CRC Crit Rev Clin Lab Sci 23:315–47. Hoffmann JA, Kafatos FC, Janeway CA, Ezekowitz RA. 1999. Phylogenetic perspectives in innate immunity. Science 284:1313–8. Horvath CM. 2000. STAT proteins and transcriptional responses to extracellular signals. Trends Biochem Sci 25:496–502. C. R. Lage et al. the toxicology of metals. 2nd edition. Amsterdam: Elsevier Science Publishers. p 43–83. Jackson R, Grainge JW. 1975. Arsenic and cancer. Can Med Assoc J 113:396–401. Jacobs BL, Langland JO. 1996. When two strands are better than one: the mediators and modulators of the cellular responses to double-stranded RNA. Virology 219:339–49. Jault C, Pichon L, Chluba J. 2004. Toll-like receptor gene family and TIR-domain adaptors in Danio rerio. Mol Immunol 40:759–71. Jenkins RO, Ritchie AW, Edmonds JS, Goessler W, Molenat N, Kuehnelt D, Harrington CF, Sutton PG. 2003. Bacterial degradation of arsenobetaine via dimethylarsinoylacetate. Arch Microbiol 180(2):142–50. Juresa D, Blanusa M. 2003. Mercury, arsenic, lead and cadmium in fish and shellfish from the Adriatic Sea. Food Addit Contam 20:241–6. Kaltreider RC, Davis AM, Lariviere JP, Hamilton JW. 2001. Arsenic alters the function of the glucocorticoid receptor as a transcription factor. Environ Health Perspect 109:245–51. Karlovich CA, John RM, Ramirez L, Stainier DY, Myers RM. 1998. Characterization of the Huntington’s disease gene homologue in the zebrafish Danio rerio. Gene 217:117–25. Kitchin KT. 2001. Recent advances in arsenic carcinogenesis: modes of action, animal model systems, and methylated arsenic metabolites. Toxicol Appl Pharmacol 172:249–61. Klaassen CD. 1996. Heavy metals and heavy-metal antagonists. In: Hardman JG, Limbird LE, Molinoff PB, Ruddon RW, Gilman AG, editors. Goodman & Gilman’s The pharmacological basis of therapeutics. New York: McGraw-Hill. p 1649–72. Kothary RK, Candido EP. 1982. Induction of a novel set of polypeptides by heat shock or sodium arsenite in cultured cells of rainbow trout, Salmo gairdnerii. Can J Biochem 60:347–55. Kotsanis N, Iliopoulou-Georgudaki J. 1999. Arsenic induced liver hyperplasia and kidney fibrosis in rainbow trout (Oncorhynchus mykiss) by microinjection technique: a sensitive animal bioassay for environmental metal-toxicity. Bull Environ Contam Toxicol 62:169–78. Hostetter CL, Sullivan-Brown JL, Burdine RD. 2003. Zebrafish pronephros: a model for studying cystic kidney disease. Dev Dyn 22:514–22. Kubota R, Kunito T, Tanabe S. 2003. Occurrence of several arsenic compounds in the liver of birds, cetaceans, pinnipeds, and sea turtles. Environ Toxicol Chem 22(6):1200–7. Hseuh YM, We WL, Huang YL, Chiou HY, Chiou CH, Chen CJ. 1998. Low serum carotene level and increased risk of ischemic heart disease related to long-term arsenic exposure. Atherosclerosis 141:249–57. Lantz RC, Parliman G, Chen GJ, Carter DE. 1994. Effect of arsenic exposure on alveolar marcrphage function. I. Effect of soluble As(III) and As(V). Environ Res 67:183–95. Huang C, Ke Q, Costa M, Shi X. 2004. Molecular mechanisms of arsenic carcinogenesis. Mol Cell Biochem 255:57–66. Ilyaletdinov AN, Abdrashitova SA. 1981. Autotrophic oxidation of arsenic by a culture of Pseudomonas arsenitoxidans. Microbiologiya 50:197–204. Ishinishi N, Tsuchiya K, Vahter M, Fowler BA. 1986. Arsenic. In: Friberg L, Nordberg GF, Vouk V, editors. Handbook on LaPatra SE, Barone L, Jones GR, Zon LI. 2000. Effects of infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus infection on hematopoietic precursors of the zebrafish. Blood Cells Mol Dis 26:445–52. Laverman AM, Blum JS, Schaeffer JK, Philips EJP, Lovley DR, Oremland RS. 1995. Growth of strain SES-3 with arsenate and other diverse electron acceptors. Appl Environ Microbiol 61(10):3556–61. 1051 Effects of arsenic on innate immunity Leimer U, Lun K, Romig H, Waleter J, Grunberg J, Brand M, Haass C. 1999. Zebrafish (Danio rerio) presenilin promotes aberrant amyloid beta-peptide production and requires a critical aspartate residue for its function in amyloidogenesis. Biochemistry 38:13602–9. Lemaitre B, Nicolas E, Michaut L, Reichhart J-M, Hoffmann JA. 1996. The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86:973–83. Leonard A. 1991. Arsenic. In: Meriam E, editor. Metals and their compounds in the environment. VCH: Weinheim. p 751–72. Li X, Commane M, Jiang Z, Stark GR. 2001. IL-1 induced NFkB and c-Jun N-terminal kinase activation diverge at IL-1 receptor associated kinase (IRAK). Proc Natl Acad Sci USA 98(8):4461–5. Lin B, Chen S, Cao Z, Lin Y, Mo D, Zhang H, Gu J, Dong M, Liu Z, Xu A. 2006. Acute phase response in zebrafish upon Aeromonas salmonicida and Staphylococcus aureus infection: striking similarities and obvious differences with mammals. Mol Immunol 44(4):295–301. Lloyd-Smith M, Wickens J. 2000. Mapping the hotspots: DDT-contaminated dipsites in Australia. Glob Pestic Camp 10(1) [April] [cited 2006 April]. Available at: www.panna. org/resources/gpc/gpc_200004.10.1.02.dv.html. Long Q, Quint E, Lin S, Ekker M. 2001. The zebrafish scyba gene encodes a novel CXC-type chemokine with distinctive expression patterns in the vestibulo-acoustic system during embryogenesis. Mech Dev 97(1–2):183–6. Longnecker MP, Daniels JL. 2001. Environmental contaminants as etiologic factors for diabetes. Environ Health Perpect 109:871–6. Luh MD, Baker RA, Henley DE. 1973. Arsenic analysis and toxicity—a review. Sci Total Environ 2:1–12. Lunde G. 1972. Analysis of arsenic and bromine in marine and terrestrial oils. J Am Oil Chem Soc 49:44–7. Lunde G. 1973. Separation and analysis of organic-bound and inorganic arsenic in marine organisms. J Sci Food Agric 24:1021–7. Lynn S, Lai HT, Gurr JR, Jan KY. 1997. Arsenite retards DNA break rejoining by inhibiting DNA ligation. Mutagenesis 12:353–8. Macur RE, Jackson CR, Botero LM, McDermott TR, Inskeep WP. 2004. Bacterial populations associated with the oxidation and reduction of arsenic in an unsaturated soil. Environ Sci Technol 38(1):104–11. Maest AS, Pasilis SP, Miller LG, Nordstrom DK. 1992. Redox geochemistry of arsenic and iron in Mono Lake, California, USA. In: Kharaka YK, Maest AS, editors. Proceedings of the 7th international symposium water–rock interactions. Rotterdam: A.A. Balkema. p 507–11. Mandal BK, Chowdhury TR, Samanta G, Basu GK, Chowdhury PP, Chandra CR, Losh D, Karan NK, Dhar RK, Tamili DK. 1996. Arsenic in groundwater in seven districts of West Bengal, India–the biggest arsenic calamity in the world. Curr Sci 70:976–86. McLaren SJ, Kim ND. 1995. Evidence for a seasonal fluctuations of arsenic in New Zealand’s longest river and the effect of treatment on concentrations in drinking water. Environ Pollut 90:67–73. Medzhitov R. 2001. Toll-like receptors and innate immunity. Nat Rev Immunol 1:135–45. Meijer AH, Gabby Krens SF, Medina Rodriguez IA, He S, Bitter W, Ewa Snaar-Jagalska B, Spaink HP. 2004. Expression analysis of the Toll-like receptor and TIR domain adaptor families of zebrafish. Mol Immunol 40:773–83. Meijer AH, Verbeek FJ, Salas-Vidal E, Corredor-Adamez M, Bussman J, van der Sar AM, Otto GW, Geisler R, Spaink HP. 2005. Transcriptome profiling of adult zebrafish at the late stage of chronic tuberculosis due to Mycobacterium marinum infection. Mol Immunol 42(10):1185–203. Menudier A, Rougier FP, Bosgiraud C. 1996. Comparative virulence between different strains of Listeria in zebrafish (Brachydanio rerio) and mice. Pathol Biol (Paris) 44(9):783–9. Miller JD, Neely MN. 2004. Zebrafish as a model host for streptococcal pathogenesis. Acta Trop 91(1):53–68. Miller WH Jr, Schipper HM, Lee JS, Singer J, Waxman S. 2002. Mechanisms of action of arsenic trioxide. Cancer Res 62:3893–903. Minkkinen P, Yliruokanen I. 1978. The arsenic distribution in Finnish peat bogs. Kemia-Kemi 7–8:331–5. Nakahara H, Ishikawa T, Sarai Y, Kondo I. 1977. Frequency of heavy-metal resistance in bacteria from in-patients in Japan. Nature 266:165–7. NAS (National Academy of Sciences), 1977. Medical and biologic effects of environmental pollutants: Arsenic. Washington, DC: National Academy of Sciences. Nasevicius A, Ekker SC. 2000. Effective target “knockdown” in zebrafish. Nat Genet 26:216–20. gene Neely MN, Pfeifer JD, Caparon M. 2002. Streptococcuszebrafish model of bacterial pathogenesis. Infect Immun 70(7):3904–14. Nemery B. 1990. Metal toxicity and the respiratory tract. Eur Respir J 3:202–19. Nevens F, Fevery J, van Steenbergen W, Sciot R, Desmet V, de-Groot J. 1990. Arsenic and cirrhotic portal hypertension: a report of 8 cases. J Hepatol 1:80–5. Nies DH, Silver S. 1995. Ion efflux systems involved in bacterial metal resistances. J Ind Microbiol 14:186–99. Nimick DA, Moore JN, Dalby CE, Savka MW. 1998. The fate of geothermal arsenic in the Madison and Missouri Rivers, Montana and Wyoming. Water Resour Res 34:3051–67. NRC (National Research Council), 1995. Arsenic in drinking water. Washington, DC: National Academy Press. Nriagu JO. 1989. A global assessment of natural sources of atmospheric trace metals. Nature 338:47–9. Nriagu JO, Pacyna JM. 1988. Quantitative assessment of worldwide contamination of air, water and soils by trace metals. Nature 333:134–9. 1052 Oladimeji AA. 1985. An arsenic-binding protein in rainbow trout. Ecotoxicol Environ Safety 9:1–5. Patterson R, Vega L, Trouba K, Bortner C, Germolec D. 2004. Arsenic-induced alterations in the contact hypersensitivity response in Balb/c mice. Toxicol Appl Pharmacol 198:434–43. Pedlar RM, Ptashynski MD, Evans R, Klaverkamp JF. 2002. Toxicological effects of dietary arsenic exposure in lake whitefish (Coregonus clupeaformis). Aquat Toxicol 57:167–89. Penrose WR, Conacher HBS, Black R, Meranger JC, Miles W, Cunningham HM, Squires WR. 1977. Implications of inorganic/organic interconversion on fluxes of arsenic in marine food webs. Environ Health Perspect 19:53–9. Petrick JS, Ayala-Fierro F, Cullen WR, Carter DE, Aposhian HV. 2000. Monomethylarsonous acid (MMAIII) is more toxic than arsenite in Chang human hepatocytes. Toxicol Appl Pharmacol 163:203–7. Petrick JS, Bhumasamudram J, Mash EA, Aposhian HV. 2001. Monomethylarsonous acid (MMAIII) and arsenite: LD50 in hamsters and in vitro inhibition of pyruvate dehydrogenase. Chem Res Toxicol 14:651–6. C. R. Lage et al. Roussel RR, Barchowsky A. 2000. Arsenic inhibits NF-kappaBmediated gene transcription by blocking IkappaB kinase activity and IkappaBalpha phosphorylation and degradation. Arch Biochem Biophys 377:204–12. Sakurai T, Jojima C, Ochiai M, Ohta T, Fujiwara K. 2004. Evaluation of in vivo acute immunotoxicity of a major organic arsenic compound arsenobetaine in seafood. Int Immunopharmacol 4(2):179–84. Sakurai T, Ohta T, Tomita N, Kojima C, Hariya Y, Mizukami A, Fujiwara K. 2006. Evaluation of immunotoxic and immunodisruptive effects of inorganic arsenite on human monocytes/macrophages. Int Immunopharmacol 6:304–15. Saltikov CW, Olson BH. 2002. Homology of Escherichia coli R773 arsA, arsB, and arsC genes in arsenic-resistant bacteria isolated from raw sewage and arsenic-enriched creek waters. Appl Environ Microbiol 68(1):280–8. Samuel CE. 1991. Antiviral actions of interferon. Interferonregulated cellular proteins and their surprisingly selective antiviral activities. Virology 183:1–11. Samuel CE. 1998. Protein-nucleic acid interactions and cellular responses to interferon. Methods J 15:161–5. Phelan PE, Mellon MT, Kim CH. 2005. Functional characterization of full-length TLR3, IRAK-4, and TRAF6 in zebrafish (Danio rerio). Mol Immunol 42:1057–71. Sanders GE, Batts WN, Winton JR. 2003. Susceptibility of zebrafish (Danio rerio) to a model pathogen, spring viremia of carp virus. Comp Methods 53:514–21. Phelan PE, Pressley ME, Witten PE, Mellon MT, Blake SL, Kim CH. 2005. Characterization of infection with snakehead rhabdovirus in zebrafish (Danio rerio). J Virol 79(3):1842–52. Savabieasfahani M, Lochmiller RL, Rafferty DP, Sincleair JA. 1998. Sensitivity of wild cotton rats to the immunotoxic effects of low-level arsenic exposure. Arch Environ Contam Toxicol 34:289–96. Pineda-Zavaleta AP, Garcia-Vargas C, Borja-Aburto VH, Acosta-Saavedra LC, Vera-Aguilar E, Gomez-Munoz A, Cebrian ME, Caldeon-Aranda ES. 2004. Nitric oxide and superoxide anion production in monocytes from children exposed to arsenic and lead in region Lagunera, Mexico. Toxicol Appl Pharmacol 198:283–90. Pressley ME, Phelan PE, Witten PE, Mellon MT, Kim CH. 2005. Pathogenesis and inflammatory response to Edwardsiella tarda infection in the zebrafish. Dev Comp Immunol 29(6):501–13. Prouty MG, Correa NE, Barker LP, Jagadeeswaran P, Klose KE. 2003. Zebrafish-Mycobacterium marinum model for mycobacterial pathogenesis. FEMS Microbiol Lett 225:177–82. Reay PF. 1972. The accumulation of arsenic from arsenic-rich natural waters by aquatic plants. J Appl Ecol 9:557–65. Rensing C, Ghosh M, Rosen B. 1999. Families of soft-metalion-transporting ATPases. Bacteriol 181:5891–7. Rodriguez R, Ramos JA, Armienta A. 2004. Groundwater arsenic variations: the role of local geology and rainfall. Appl Geochem 19:245–50. Rollins BJ. 1997. Chemokines. Blood 90:909–28. Rossman TG. 1998. Molecular and genetic toxicology of arsenic. In: Rose J, editor. Environmental toxicology: current developments. Amsterdam: Gordon and Breach Publishers. p 171–87. Savage KS, Bird DK, Ashley RP. 2000. Legacy of the California gold rush: environmental geochemistry of arsenic in southern mother lode gold district. Int Geol Rev 42(5):385–415. Schlenk D, Wolford L, Chelius M, Steevens J, Chan KM. 1997. Effect of arsenite, arsenate, and the herbicide monosodium methyl arsonate (MSMA) on hepatic metallothionein expression and lipid peroxidation in channel catfish. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 118:177–83. Schultz H, Nagymajtenyi L, Institoris L, Papp A, Siroki O. 2002. A study on hebavioral, neurotoxicological, and immunotoxicological effects of subchronic arsenic treatment in rats. J Toxicol Environ Health A 65:1181–93. Sengupta M, Bishayi B. 2002. Effect of lead and arsenic on murine macrophage response. Drug Chem Toxicol 25:459–72. Shariatpanahi M, Anderson AC, Abdelghani AA, Englande AJ, Hughes J, Wilkinson RF. 1981. Biotransformation of the pesticide sodium arsenate. J Environ Sci Health B 16(1):35–47. Shukla JP, Pandey K. 1984a. Impaired ovarian functions in arsenic-treated freshwater fish, Colisa fasciatus (Bl. and Sch.). Toxicol Lett 20:1–3. Shukla JP, Pandey K. 1984b. Impaired spermatogenesis in arsenic treated freshwater fish, Colisa fasciatus (Bl. and Sch.). Toxicol Lett 21:191–5. Effects of arsenic on innate immunity Shumilla JA, Wetterhahn KE, Barchowsky A. 1998. Inhibition of NF-kappa B binding to DNA by chromium, cadmium, mercury, zinc, and arsenite in vitro: evidence of a thiol mechanism. Arch Biochem Biophys 349:356–62. Siegel FR. 2002. Environmental geochemistry of potentially toxic metals. Berlin: Springer-Verlag. Sikorski EE, Burns LA, Stern ML, Luster MI, Munson AE. 1991. Splenic cell targets in gallium arsenide-induced suppression of the primary antibody response. Toxicol Appl Pharmacol 110:129–42. Simeonova PP, Luster MI. 2000. Mechanisms of arsenic carcinogenicity: genetic or epigenetic mechanisms? J Environ Pathol Toxicol Oncol 19:281–6. Smedley PL, Edmunds WM, Pelig-Ba KB. 1996. Mobility of arsenic in groundwater in the Obuasi gold-mining area of Ghana: some implications for human health. In: Appleton JD, Fuge R, McCall GJH, editors. Environmental geochemistry and health. Geological Society special publication. New York: Chapman and Hall. p 163–81. Smedley PL, Kinniburgh DG. 2002. A review of the source, behaviour and distribution of arsenic in natural waters. Appl Geochem 17:517–68. Smith RM, Connor JA, Chen LM, Babior BM. 1996. The cytosolic subunit p67phox contains NADPH-binding site that participates in the catalysis by the leukocyte NADPH oxidase. J Clin Invest 98:977–83. Sorensen EM, Ramirez-Mitchell R, Pradzynski A, Bayer TL, Wenz LL. 1985. Stereological analyses of hepatocyte changes parallel arsenic accumulation in the livers of green sunfish. J Environ Pathol Toxicol Oncol 6:195–210. Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD. 1998. How cells respond to interferons. Annu Rev Biochem 67:227–64. Styblo M, Del Razo LM, Vega L, Germolec DR, LeCluyse EL, Hamilton GA, Reed W, Wang C, Cullen WR, Thomas DJ. 2000. Comparative toxicity of trivalent and pentavalent inorganic and methylated arsenicals in rat and human cells. Arch Toxicol 74(6):289–99. Suhendrayatna Ohki A, Nakajima T, Maeda S. 2002. Studies on the accumulation and transformation of arsenic in freshwater organisms I. Accumulation, transformation and toxicity of arsenic compounds on the Japanese medaka, Oryzias latipes. Chemosphere 46:319–24. Tersago K, DeCoen W, Scheirs J, Vermeulen K, Blust R, VanBockstaele D, Verhagen R. 2004. Immunotoxicology in wood mice along a heavy metal pollution gradient. Environ Pollut 132:385–94. Thelen M. 2001. Dancing to the tune of chemokines. Nat Immunol 2(2):129–34. Tisler T, Zagorc-Koncan J. 2002. Acute and chronic toxicity of arsenic to some aquatic organisms. Bull Environ Contam Toxicol 69:421–9. 1053 embryos as a model host for the real time analysis of Salmonella typhimurium infections. Cell Microbiol 5(9):601–11. Vodela JK, Renden JA, Lenz SD, McElhenney WH, Kemppainen BW. 1997. Drinking water contaminants (arsenic, cadmium, lead, benzene, and trichloroethylene). 1. Interaction of contaminants with nutritional status on general performance and immune function in broiler chickens. Poult Sci 76:1474–92. Wang TS, Kuo CF, Jan KY, Huang H. 1996. Arsenite induces apoptosis in Chinese hamster ovary cells by generation of reactive oxygen species. J Cell Physiol 169:256–68. Wang YC, Chaung RH, Tung LC. 2004. Comparison of the cytotoxicity induced by different exposure to sodium arsenite in two fish cell lines. Aquat Toxicol 69:67–79. Webb JL. 1966. Enzymes and metabolic inhibitors. Vol. 3. New York: Academic Press. p 595–793. Wei LH, Lai KP, Chen CA, Cheng CH, Huang YJ, Chou CH, Kuo ML, Hsieh CY. 2005. Arsenic trioxide prevent radiatioenhanced tumor invasiveness and inhibits matrix metalloproteinase-9 through downregulation on nuclear factor kB. Oncogene 24:390–8. Wijeweera JB, Gandolfi AJ, Parrish A, Lantz RC. 2001. Sodium arsenite enhances AP-1 and NFkappaB DNA binding and induces stress protein expression in precision-cut rat lung slices. Toxicol Sci 61:283–94. Willett CE, Cherry JJ, Steiner LA. 1997. Characterization and expression of the recombination activating genes (RAG1 and RAG2) of zebrafish. Immunogenetics 45:394–404. Willsky GR, Malamy MH. 1980. Effects of arsenate on inorganic phosphate transport in Escherichia coli. J Bacteriol 144:366–74. Winship KA. 1984. Toxicity of inorganic arsenic salts. Adverse Drug React Acute Poisoning Rev 3:129–60. Winski SL, Carter DE. 1995. Interaction of the rat blood cell sulphydryls with arsenate and arsenite. J Toxicol Environ Health 46:379–97. Wu MM, Chiou HY, Ho IC, Chen CJ, Lee TC. 2003. Gene expression of inflammatory molecules in circulating lymphocytes from arsenic-exposed human subjects. Environ Health Perspect 111:1429–38. Yang C, Frenkel K. 2002. Arsenic-mediated cellular signal transduction, transcription factor activation, and aberrant gene expression: implications in carcinogenesis. J Environ Pathol Toxicol Oncol 21:331–42. Yen HT, Chiang LC, Wen KH, Chang SF, Tsai CC, Yu CL, Yu HS. 1996. Arsenic induces interleukin-8 expression in cultured keratinocytes. Arch Dermatol Res 288:716–7. Ulevitch RJ. 2000. Molecular mechanisms of innate immunity. Immunol Res 21:49–54. Yoshida T, Shimamura T, Shigeta S. 1987. Enhancement of the immune response in vitro by arsenic. Int J Immunopharmacol 9:411–5. van der Sar AM, Musters RJ, van Eeden FJ, Appelmelk BJ, Vandenbroucke-Grauls CM, Bitter W. 2003. Zebrafish Young HA. 1996. Regulation of interferon-g gene expression. J Interferon Cytokine Res 16:563–8. 1054 Yu HS, Change KL, Yu CL, We CS, Chen GS, Jo JC. 1998. Defective IL-2 receptor expression in lymphocytes of patients with arsenic-induced Bowen’s disease. Arch Dermatol Res 290:681–7. Zaloga GP, Deal J, Spurling T, Richter J, Chernow B. 1985. Case report: unusual manifestations of arsenic intoxication. Am J Med Sci 289:210–4. Zelikoff JT. 1998. Biomarkers of immunotoxicity in fish and other non-mammalian sentinel species: predictive value for mammals? Toxicology 129:63–71. C. R. Lage et al. Zelikoff JT, Caymond A, Carlson E, Li Y, Beanan JR, Anderson M. 2000. Biomarkers of immunotoxicity in fish: from the lab to the ocean. Toxicol Lett 112/113:325–31. Zhang P. 1999. The use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia. J Biol Regul Homeost Agents 13:195–200. Zhao CW, Young MR, Biwan BA, Coogan TP, Waalkes MP. 1997. Association of arsenic-induced malignant transformation with DNA hypomethylation and aberrant gene expression. Proc Natl Acad Sci USA 94:10907–12.