TOXICOLOGICAL SCIENCES 98(1), 118124 (2007) doi:10.

1093/toxsci/kfm072 Advance Access publication March 30, 2007

Effects of Low Concentrations of Arsenic on the Innate Immune System of the Zebrash (Danio Rerio)
Akshata S. Nayak, Christopher R. Lage, and Carol H. Kim1
Department of Biochemistry, Microbiology and Molecular Biology, 5735 Hitchner Hall, University of Maine, Orono, Maine 04469 Received December 9, 2006; accepted March 6, 2007

Arsenic has been associated with a multitude of human health problems; however, its impact on host resistance to infection has not been extensively researched. In vertebrates, the innate immune response is vital for potentiating the adaptive immune response. Therefore, dampening of the innate immune response results in an immunocompromised host. In this present study, effects of low concentrations of arsenic on zebrash resistance to infection are evaluated. Exposure to 2 and 10 ppb arsenic, both considered safe levels in drinking water, resulted in a greater than 50-fold increase in viral load and at least a 17-fold increase in bacterial load in embryos. To determine the cause of this amplied pathogen load, important components of the innate immune system were analyzed. Presence of arsenic dampened the overall innate immune health of the sh as evidenced by reductions in respiratory burst activity. Viral infection, after arsenic exposure, showed decreases of up to 13- and 1.5-fold changes in interferon and Mx mRNA expression, respectively. Bacterial infection, post arsenic exposure, demonstrated at least 2.5- and 4-fold declines in interleukin-1b and tumor necrosis factor-a mRNA levels, respectively. Maximum expression of these essential cytokines was also delayed upon arsenic exposure. Our data indicate that arsenic exposure, at concentrations deemed safe in drinking water, suppresses the overall innate immune function in zebrash and present the zebrash as a unique model for studying immunotoxicity of environmental toxicants. To our knowledge, this is the rst report describing the effects of such low levels of arsenic on host resistance to infection. Key Words: arsenic; zebrash; innate immunity; cytokines; snakehead rhabdovirus; Edwardsiella tarda.

Arsenic is a naturally occurring element found in soil, air, and water (Duker et al., 2005; Huang et al., 2004); organic forms occurring in the environment are generally considered nontoxic, whereas inorganic forms are toxic (Cervantes et al., 1994; Duker et al., 2005). Arsenic accumulation in the environment has intensied due to human activities such as fossil fuel combustion, metal smelting and mining, semiconductor, and glass industries (Amasa, 1975; Smedley, 1996;
To whom correspondence should be addressed. Fax: (207) 581-2801. E-mail: carolkim@maine.edu.
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Smedley and Kinniburgh, 2002). Pollutants discharged into bodies of water are readily taken up by sh and subsequently move up the trophic levels, potentially even to humans. Fish have proven to be valuable model systems to study consequences of toxicant uptake and bioaccumulation on metabolic activities and immune function and therefore serve as important environmental sentinels. On the molecular level, there is evidence that arsenic can disrupt gene expression, particularly via signal transduction, and it has been implicated in the disruption of cell division (Abernathy et al., 1999), the inhibition of DNA repair (Andrew et al., 2006; Lynn et al., 1997), and in enhancing mutagenesis by other agents (Abernathy et al., 1999). Arsenic is associated with multiple health effects, including Blackfoot disease (Abernathy et al., 1999), diabetes (Longnecker and Daniels, 2001), hypertension (Chen et al., 1995), peripheral neuropathy, and vascular diseases (Duker et al., 2005). Although symptoms may be delayed for > 20 years, long-term exposure to inorganic arsenic is associated with skin, lung, colon, bladder, liver, and breast cancers (Huang et al., 2004). The innate immune response is activated as the rst line of defense by the immune system of the host and is a prerequisite for potentiating the adaptive immune response. Cytokines and chemokines, the effector molecules of the innate immune response, are essential for establishing a state of inammation critical in eradicating the pathogen from the host cells (Thelen, 2001; Zhang and Huang, 2006). These molecules also behave as chemoattractants for the cellular components of the innate immune system, namely macrophages and neutrophils (Thelen, 2001; Zhang and Huang, 2006). These immune cells destroy the pathogen by phagocytosis and production of reactive oxygen species (ROS), an essential mechanism known as the respiratory burst (Dewas et al., 2003; Thelen et al., 1990). The zebrash (Danio rerio) is a teleost that has become important as an animal model to study embryo development, genetics, and the immune system. Zebrash develop rapidly, are small in size, and easy to manage and breed compared to other vertebrate models. Optically clear embryos develop externally, allowing easy observation of cellular and organ development. A bioassay to measure the respiratory burst response has been developed (Hermann et al., 2004), and

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multiple chemokines (David et al., 2002; Long et al., 2000) and cytokines (Altmann et al., 2003, 2004; Pressley et al., 2005) have been characterized. Genome sequence similarities indicate that knowledge gained from research in the zebrash model could be applicable to humans (Amemiya et al., 1999). The ease with which large numbers of embryos can be exposed to toxicants has contributed to making the zebrash a unique model for immunotoxicological studies involving cadmium, copper, mercury, and lead (Blechinger et al., 2002; Dave and Xiu, 1991; Fraysse et al., 2006). The use of whole embryos also allows study of holistic effects of toxicants on the host. It is clear that arsenic exposure alters normal biological functions resulting in the direct initiation or predisposition of an organism to disease. However, the impact of arsenic on the hosts ability to ght viral and bacterial infection via specic immune responses has not been extensively researched. In an effort to understand the overall effect of arsenic on the immune health of humans, our study characterizes the effects of low concentrations of arsenic on the innate immune response of the zebrash. Effects of 2 and 10 ppb arsenic, concentrations that fall within the range used previously in studies of sh and mammals (Cavigelli et al., 1996; Chen et al., 1998; Hermesz et al., 2002; Wang et al., 2004), on the ability to clear viral and bacterial infections, the respiratory burst response, mRNA expression levels of essential antiviral, and antibacterial cytokines were examined in zebrash embryos.

(CPE). At the end of 1 week, the number of wells with CPE was determined and the TCID50/ml of the virus calculated (Levine, 2001). The data were compared using ANOVA single factor analysis. Determination of bacterial load in arsenic-exposed embryos. Arsenicexposed and unexposed embryos were infected by static immersion at 7 dpe with 1 3 108 colony forming units (CFU)/ml Edwardsiella tarda (E. tarda), for 5 h or maintained as uninfected controls (Pressley et al., 2005). Twenty sh were collected at 4 hpi for each treatment and homogenized in Luria Bertani broth medium. Serial 10-fold dilutions were plated on Edwardsiella ictaluri medium plates. The number of colonies was counted and the corresponding CFU/ml calculated for each treatment. The data were compared using ANOVA single factor analysis. Respiratory burst assay. The respiratory burst assay was performed with zebrash embryos by measuring oxidation of dihydrodichlorouorescein diacetate (H2DCFDA) to uorescent dichlorouorescein (Hermann et al., 2004). Arsenic-exposed and unexposed embryos were used in the assay between 3 and 10 dpf. At the designated time points, sh were treated with either 0.2% dimethylsulfoxide or induced with 400 ng/ml phorbol myristate acetate (PMA) in the presence of 1 lg/ml of H2DCFDA. Intensity of uorescence was measured every 2.5 min for 150 min. The data were compared using ANOVA two factor with replication analysis. RNA extraction, cDNA synthesis, and quantitative real-time PCR. Total RNA was extracted from arsenic-exposed and unexposed sh after infection with viral or bacterial pathogens. Viral time points were collected at 12, 24, 48, 72, and 96 h post infection (hpi) and bacterial time points collected at 2, 4, 8, 12, and 24 hpi, by homogenizing 10 sh from each treatment per time point in TRIzol reagent (Invitrogen, Carlsbad, CA) and stored at 80C. RNA was extracted according to the manufacturers instructions using TRIzol reagent (Invitrogen), and reverse transcription reactions were performed to synthesize cDNA (Phelan et al., 2005a). Quantitation of type I interferon, Mx, tumor necrosis factor-a (TNF-a), and interleukin-1b (IL-1b) was carried out using an I-cycler IQ Detection System (Bio-Rad Laboratories, Hercules, CA). Reactions were performed as previously described (Phelan et al., 2005a). The cycling parameters for b-actin were 94C for 3 min to activate the polymerase, followed by 40 cycles of 94C for 30 s, 53C for 30 s, and 72C for 30 s. The cytokines were amplied using similar conditions with the exception that the annealing temperature was 56C for TNF-a and 54C for IFN, Mx, and IL-1b. Fluorescence measurements were taken at each cycle during the annealing step. The copy number was determined based on a standard curve by the iCycler software, and the value for each sample was normalized to the corresponding b-actin value to determine relative copy number. Fold inductions were calculated by dividing the relative copy number (RCN) of infected samples by the RCN of uninfected samples for the same treatment. Difference between the fold inductions of the arsenic-unexposed samples and the arsenic-exposed sh yielded the approximate difference in fold reductions in cytokine expression upon arsenic exposure.

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MATERIALS AND METHODS

Zebrash care and maintenance. AB strain zebrash were maintained in the Zebrash Facility at The University of Maine, Orono, in recirculating systems from Aquatic Habitat (Apopka, Florida). The water was maintained at 28C with a ow rate of 150 l/min. Adults were bred and all embryos were collected in petri dishes at the one-cell stage before the start of the experiment. The zebrash facility was maintained according to Institutional Animal Care and Use Committee standards. Arsenic exposures. Zebrash embryos, at the one-cell stage, were exposed to 2 or 10 ppb sodium arsenite (Sigma, St Louis, MO) in egg water (60 lg/ml Instant Ocean, Aquarium Systems, Mentor, OH) or retained as unexposed controls. One hundred embryos per treatment were maintained in each petri dish at 28C, and the arsenic was changed daily by replacing all the arsenic egg water in the dish with freshly made arsenic solutions at the appropriate concentrations. From 4 days post fertilization (dpf), the sh were transferred to 2-l tanks, were fed rotifers, and the tanks were cleaned twice daily. Exposure to 2 and 10 ppb arsenic revealed no visible developmental defects. All sh maintained as unexposed controls were handled in a similar manner to the arsenic-exposed sh. Determination of viral titer in arsenic-exposed embryos. Arsenicexposed and unexposed embryos were infected by static immersion at 7 days post exposure (dpe) with 1 3 106 TCID50/ml snakehead rhabdovirus (SHRV), for 5 h or maintained as uninfected controls (Phelan et al., 2005b). Twenty sh were collected at 24 h post infection (hpi) for each treatment and homogenized in minimum essential medium (GIBCO-Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO-Invitrogen) and 50 lg/ml each of penicillin, streptomycin, and ampicillin. The supernatants were used in TCID50/ml experiments and monitored daily for cytopathic effect

RESULTS

Exposure to Arsenic Inhibits the Ability of the Zebrash to Clear Viral and Bacterial Load To determine the effects of arsenic on the ability of the host to resist infection, the viral (Fig. 1a) and bacterial (Fig. 1b) loads in arsenic-exposed and unexposed zebrash after infection were examined. At 24 hpi, the viral titer was determined to be 1.0 3 105 TCID50/ml in infected control sh, 5.73 3 106 TCID50/ml upon exposure to 2 ppb arsenic, and 8.77 3 106 TCID50/ml upon exposure to 10 ppb corresponding to significant increases of 57- and 87-fold in viral load ( p value < 0.006)

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FIG. 2. Arsenic exposure dampens the respiratory burst response. Respiratory burst was measured in arsenic-exposed and unexposed zebrash by the oxidation of H2DCFDA to dichlorouorescein. Each bar represents the average uorescence from six embryos. Error bars are indicated by SEs of the mean. Results are representative of three independent experiments (signicance was determined using ANOVA two factor with replication analysis; p value < 0.04).

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FIG. 1. Exposure to arsenic inhibits the ability of the zebrash to clear viral and bacterial load. Viral (a) and bacterial (b) load were calculated in arsenic-exposed and unexposed zebrash. Each bar represents the average of three experiments and the error bars are indicated by SEs of the mean (signicance was determined using ANOVA single factor analysis; viral load p value < 0.006 and bacterial load p value < 0.007).

(Fig. 1a). The CFU/ml isolated from E. tardainfected control sh at 4 hpi was calculated as 1.06 3 105 CFU/ml and 2.10 3 106 CFU/ml and 1.83 106 CFU/ml when exposed to 2 and 10 ppb arsenic (Fig. 1b). These values are signicantly higher, by 17- and 19-fold, respectively, when compared to infected controls ( p value < 0.007). These data suggest that exposure to arsenic blunts the ability of the embryo to clear an infection as effectively as unexposed control sh. Arsenic Exposure Dampens the Respiratory Burst Response To test our hypothesis that arsenic exposure dampens host resistance to infection, the overall immune health of the embryos upon exposure to arsenic was determined by measuring the respiratory burst response (Fig. 2), using an assay developed by Hermann et al. (2004). ROS production in PMAinduced control embryos peaked at 4 dpf with a twofold induction (Fig. 2) compared to uninduced controls. A gradual decrease in respiratory burst activity was then observed until 9 dpf when ROS induction was reduced to 1.2-fold. When sh were exposed to 2 ppb arsenic, a peak of 1.7-fold induction of ROS was observed at 4 dpf (Fig. 2). This gradually decreased until 9 dpf when ROS induction was reduced to 0.9fold. Fish exposed to 10 ppb arsenic showed a similar trend in respiratory burst response, with a 1.5-fold induction observed

at 4 dpf, which gradually decreased until 9 dpf, when a 0.9-fold induction was observed. Overall, presence of 2 or 10 ppb in the water resulted in small, but signicant, differences in respiratory burst activity in arsenic-exposed embryos when compared to unexposed controls ( p value < 0.04). Variations in fold induction were observed in different experiments, but the overall trend remained constant. Our data indicate that the ability of embryos to produce an effective respiratory burst response is dampened by arsenic exposure. Exposure to Arsenic Diminishes Induction of Essential Antiviral Cytokines The induction of an immune response to a viral pathogen was examined upon arsenic exposure to assess the effects of arsenic on immunocompetence. The mRNA expression patterns of type I interferon (Fig. 3a) and the interferon-inducible gene, Mx (Fig. 3b), were examined in arsenic-exposed and unexposed zebrash after infection with SHRV. Control sh, not exposed to arsenic and infected with SHRV, showed steady increases in interferon expression from 12 hpi (1.04 3 103 RCN), reaching a maximum expression level at 48 hpi (1.63 3 104 RCN) (Fig. 3a). A decrease was then observed until 96 hpi (3.12 3 103 RCN). As an interferon-inducible gene, expression levels of Mx followed a similar pattern with an increase from 12 hpi (2.80 3 103 RCN), reaching maximum induction at 48 hpi (4.39 3 104 RCN), followed by a decrease until 96 hpi (2.92 3 103 RCN) (Fig. 3b). Fish exposed to 2 ppb arsenic and infected with SHRV, demonstrated an increase in expression of interferon levels from 12 hpi (1.14 3 103 RCN), reaching a maximum expression level at 72 hpi (1.18 3 104 RCN) (Fig. 3a). The expression level of interferon in these sh decreased at 96 hpi

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observed at 72 hpi (1.72 3 104 RCN) (Fig. 3b). The RCN did not vary signicantly between 72 and 96 hpi. The Mx peak was delayed by 24 h and the difference in the fold induction was lower, by approximately 0.5-fold, than the maximum Mx expression measured in infected control sh. Variations in RCN were noted between experiments, but the overall trend remained constant. These data suggest that presence of arsenic in the water diminished the ability of the sh to mount an effective antiviral immune response. Exposure to Arsenic Diminishes Induction of Essential Antibacterial Cytokines To characterize the expression pattern of essential antibacterial cytokines in response to bacterial infection, arsenicexposed and unexposed zebrash were infected with E. tarda, and mRNA expression patterns for IL-1b (Fig. 4a) and TNF-a (Fig. 4b) were examined. Infected control sh demonstrated a decrease in IL-1b expression from 2 hpi (9.42 3 102 RCN) to 4 hpi (4.84 3 102 RCN) (Fig. 4a). The level of IL-1b increased at 8 hpi (1.17 3 103 RCN) but showed a decrease again at 12 hpi (5.12 3 102 RCN) nally reaching maximum induction at 24 hpi (3.97 3 103 RCN). The expression pattern of TNF-a followed an identical trend, where mRNA levels of the cytokine decreased from 2 hpi (4.50 3 10 RCN) to 4 hpi (3.83 3 10 RCN) (Fig. 4b). RCN of TNF-a then increased at

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FIG. 3. Exposure to arsenic diminishes induction of essential antiviral cytokines. cDNA samples from arsenic-exposed and unexposed zebrash, infected with SHRV, were used to quantitate the essential antiviral cytokines, type I interferon (a), and Mx (b). For each treatment, reading from left to right, the bars correspond to RNA time points collected at 12, 24, 48, 72, and 96 hpi. Error bars are indicated by SEs. The results are representative of three experiments.

(3.32 3 103 RCN). Analysis of the difference in fold induction between infected and uninfected sh exposed to arsenic or unexposed controls revealed that the interferon peak observed in these sh was lower, by approximately 9-fold, than the maximum induction measured in the infected controls and was also delayed by 24 h. As with the control sh, the Mx expression pattern followed the interferon expression model for each time point (Fig. 3b). Mx levels increased from 12 hpi (1.63 3 103 RCN) peaking at 72 hpi (2.04 3 104 RCN). Mx expression then gradually decreased until 96 hpi (1.36 3 104 RCN). The difference in the fold induction for the Mx peak observed was lower, by approximately 1.5-fold, than the maximum Mx expression observed in infected controls and was also induced 24 h later. Fish exposed to 10 ppb arsenic and infected with SHRV demonstrated a slight decrease in interferon expression levels between 12 hpi (2.48 3 103 RCN) and 24 hpi (2.08 3 103 RCN) (Fig. 3a). Maximum induction was observed at 72 hpi (3.60 3 104 RCN), with the RCN not varying signicantly between 72 and 96 hpi. As with the sh exposed to 2 ppb arsenic, the interferon peak appeared 24 h later and the difference in the fold induction was dramatically lower, by approximately 13-fold, than the maximum expression observed in infected control sh. RCN of Mx followed this interferon pattern at each time point, with maximum expression level

FIG. 4. Exposure to arsenic diminishes induction of essential antibacterial cytokines. cDNA samples from arsenic-exposed and unexposed zebrash, infected with E. tarda, were used to quantitate the essential antibacterial cytokines, IL-1b (a), and TNF-a (b). For each treatment, reading from left to right, the bars correspond to RNA time points collected at 2, 4, 8, 12, and 24 hpi. Error bars are indicated by SEs. The results are representative of three experiments.

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8 hpi (2.39 3 102 RCN), followed by a decrease again at 12 hpi (5.65 3 10 RCN) and maximum induction noted at 24 hpi (3.53 3 102 RCN). This bimodal expression pattern is typical for both IL-1b and TNF-a induction in response to bacterial infection in zebrash (Pressley et al., 2005). Fish exposed to 2 ppb arsenic and infected with E. tarda demonstrated an IL-1b expression pattern similar to that of infected control sh at all time points (Fig. 4a). At 24 hpi, sh exposed to 2 ppb showed a higher IL-1b RCN (6.40 3 103 RCN) when compared to the infected control sh at the same time point. However, the presence of arsenic in the water alone appeared to increase the level of IL-1b in all treatments. An analysis of fold induction between uninfected and infected sh for each treatment, at 24 hpi, demonstrated a 5-fold induction in infected control sh and a 2.6-fold induction in sh exposed to 2 ppb arsenic. This resulted in approximately a 2.5-fold difference in the fold induction (suppression) of IL-1b in the infected sh exposed to 2 ppb arsenic when compared to the infected controls. The bimodal induction pattern of IL-1b was also abrogated on exposure to 2 ppb arsenic. TNF-a expression decreased from 2 hpi (1.03 3 102 RCN) to 4 hpi (5.71 3 10 RCN) with an increase at 8 hpi (2.89 3 102 RCN), which was followed by a decrease at 12 hpi (7.27 3 10 RCN) and a nal maximum induction at 24 hpi (5.32 3 102 RCN) (Fig. 4b). Analyses at 2, 4, and 24 hpi revealed 1.2-, 0.4-, and 6.8-fold inductions in infected control sh, whereas 0.6-, 0.3-, and 2.7fold inductions were observed with 2 ppb arsenic. These resulted in approximately 0.6-, 0.1-, and 4.1-fold differences in fold induction (suppressions) with 2 ppb arsenic at the respective time points when compared to infected control sh. Fish exposed to 10 ppb and infected with E. tarda demonstrated reduced RCN for both IL-1b and TNF-a when compared to infected controls. Expression of IL-1b decreased between 2 hpi (8.90 3 102 RCN) and 8 hpi (6.33 3 102 RCN), followed by an increase at 12 hpi (1.05 3 103 RCN). A decrease in TNF-a levels was observed from 2 hpi (1.23 3 102 RCN) to 4 hpi (4.78 3 101 RCN). An increase in expression was then noted at 8 hpi (5.22 3 101 RCN). The maximum inductions observed at 24 hpi for IL-1b (2.66 3 103 RCN) and TNF-a (2.34 3 102 RCN) showed an approximate 4-fold difference in induction (suppression) than in infected control sh. Also, the typical bimodal expression pattern observed in infected control sh for IL-1b and TNF-a was abrogated upon exposure to 10 ppb arsenic. Variations in RCN were noted between experiments, but the overall trend was conserved. Our data indicate that presence of arsenic in the water diminished the ability of the sh to mount an effective antibacterial immune response upon infection.

DISCUSSION

This is the rst report describing the effects of low levels of arsenic, considered safe in drinking water, on important

components of the innate immune system and host resistance to infection. Zebrash embryos were used in this study because the innate immune system of the zebrash is active from birth and is the only means of defense during the rst 3 days of development. This system is functionally as efcient in embryos as it is in adults, and the adaptive immune system is not completely functional until the fourth to sixth week of development (Trede et al., 2004). Therefore, the use of embryos allows monitoring of the innate immune system alone and without interference from the adaptive immune response. Also, unlike adult or juvenile sh, zebrash embryos can be genetically manipulated to knock down genes of interest, which will be especially useful in future studies, directed toward determining mechanisms by which arsenic exerts deleterious effects on innate immunity. The use of embryos also provides a longer time span over which measurement of the effects of arsenic on zebrash mortality after pathogen infection can be carried out. The presence of 2 and 10 ppb arsenic resulted in slight increases in total arsenic content in the zebrash in a dosedependent manner (data not shown), but even these slight increases in total arsenic correlate with dramatic declines in essential innate immune functions. Exposure to arsenic inhibited the hosts ability to clear both viral and bacterial infection, caused reduction in the respiratory burst response, and delayed or abrogated the typical induction of antiviral and antibacterial cytokines when compared to unexposed controls. The specic time points, 12 hpi and 4 hpi, were chosen to determine viral and bacterial loads, respectively, because these time points preceded the peaks expected in important antiviral and antibacterial cytokine proles of the zebrash, upon infection (Phelan et al., 2005b; Pressley et al., 2005). The concentrations of pathogens used in our challenge experiments were based on our previous ndings, which established the infective doses that resulted in 50% mortality and characteristic changes in cytokine proles in the zebrash embryos (Phelan et al., 2005b; Pressley et al., 2005). Data from these earlier experiments served as an internal control for our present experiments, specically with respect to the embryos that were not exposed to arsenic. However, we did not observe a dose dependency of the bacterial load on arsenic level in zebrash. It is possible that the use of a high starting density of bacteria, 1 3 10 8 CFU/ml, could have overwhelmed the immune system, thereby masking a signicant difference between the sh exposed to 2 and 10 ppb arsenic. Arsenic-exposed and control embryos could be challenged with lower numbers of bacteria; however, cytokine proles and mortality curves in response to the lower bacterial dose would need to be established in zebrash to quantify any changes that might be brought about by arsenic exposure under these conditions. Phagocytosis and production of ROS by neutrophils and macrophages is an essential mechanism for the elimination of invading microorganisms. Cytokines secreted by immune cells are vital in modulating the amplitude of an immune response.

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Type I interferons interfere with viral replication and Mx traps viral components essential for replication, thus containing the infection (Haller and Kochs, 2002; Samuel, 2001). The bimodal induction pattern for both IL-1b and TNF-a is essential for the recruitment of phagocytes to the site of infection. IL-1b activates neutrophils and macrophages and stimulates their recruitment to the site of injury (Dinarello, 1996), whereas, TNF-a is secreted by activated macrophages and is critical for the normal functioning of T cells, natural killer cells, macrophages, and dendritic cells (So et al., 2006). TNF-a also primes the phagocytic cells for protein kinase Cdependent processes, including the respiratory burst response (Dewas et al., 2003; Phillips et al., 1992a; Phillips et al., 1992b). Therefore, alterations in expression patterns of these cytokines indicate an immunosuppressed host. It has been previously demonstrated that arsenic can directly inhibit phosphorylation events involved in interferon signaling (Cheng et al., 2004), and could have similar inhibitory effects on phosphorylation involved in cytokine signaling pathways in the zebrash. Higher pathogen load, coupled with delays or reductions in important antiviral and antibacterial cytokine proles, conrms that presence of arsenic in the water blunts the ability of the sh to mount a competent immune response vital for eradicating infection. To effectively protect the host from infection, the immune system has two countermeasures, the innate and the adaptive response, that work synergistically to eradicate pathogens. These complement each other, with the former working to halt infection until the latter develops, with the consequence that a vigorous innate immune response translates into a robust adaptive immune response. Immune surveillance by the cellular components of the innate immune system consists of germ line encoded receptors that can sense molecular signatures of abnormal cells. Evidence of these receptors being utilized as targets for drugs against cancer demonstrates the key role played by this system in destruction of cancerous cells (Romagne, 2007). Chronic, long-term exposure to arsenic has been demonstrated to be carcinogenic in humans (Huang et al., 2004) and causal of vascular diseases, diabetes, and hypertension (Abernathy et al., 1999; Chen et al., 1995; Duker et al., 2005). The implications of our ndings are broad, with potential impact on both the environment and human health. This is true, because the concentrations of arsenic used in our study are presently considered safe in drinking water, and the main sources of exposure to arsenic are food and water. Plants absorb arsenic easily from their surroundings, which in turn contributes to further concentration at higher trophic levels. Arsenic has also been shown to have deleterious effects on sh species other than the zebrash, birds, and mammals, including mice, rats, and humans. This has been reviewed recently by Lage et al. (2006). The results presented here demonstrate that even shortterm arsenic exposure, in addition to compromising overall innate immune health, could leave individuals more susceptible to opportunistic pathogen infections. Arsenic could potentially

have similar effects on the innate immunity of other animals as well, affecting their disease resistance, growth, and termination of oncogenic cells. The generation of immunocompromised organisms could lead to a disruption of the ecological balance, with negative impact on industry and research. The production of arsenicals has increased as a consequence of human activity, which is further exacerbated by the inability of arsenic to be destroyed once it has entered the environment. The net result is the spread of this toxin through the ecosystem, potentially interfering with the immune systems of organisms at all trophic levels. Selective proliferation of arsenic-resistant organisms could thus give rise to widespread ecological imbalance. Where the carcinogenic properties of arsenic have been the focus of most studies, our data provide another perspective in evaluating its toxicological effects. Our experiments also establish the zebrash as an excellent model for further immunotoxicological studies. Therefore, since human health complications associated with arsenic, including various forms of cancer, aberrant inammation, and cell apoptosis, can be attributed to interference with immune system function, investigation of the impairment of the innate immune system is likely to be a key to understanding the mechanisms of overall arsenic toxicity and will provide new information that may be important for establishing future guidelines for safe water standards.

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ACKNOWLEDGMENTS
The project described was supported in part by Grant Numbers R15AI049237-02 and R21ES014028-02 to C.H.K. from the National Institute for Allergy and Infectious Disease and the National Institute for Environmental Health Science. The authors would also like to thank Kristin Bodwell and Lauren Fournier for their technical assistance and Paul Millard for his review of the manuscript.

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