TOXICOLOGICAL SCIENCES 98(1), 118–124 (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 Zebrafish (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 zebrafish 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 amplified pathogen load, important
components of the innate immune system were analyzed. Presence
of arsenic dampened the overall innate immune health of the fish as
evidenced by reductions in respiratory burst activity. Viral in-
fection, after arsenic exposure, showed decreases of up to 13- and
1.5-fold changes in interferon and Mx mRNA expression, re-
spectively. 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 zebrafish and present the zebrafish as a unique model
for studying immunotoxicity of environmental toxicants. To our
knowledge, this is the first report describing the effects of such low
levels of arsenic on host resistance to infection.
Key Words: arsenic; zebrafish; 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 intensified due to human activities such as
fossil fuel combustion, metal smelting and mining, semicon-
ductor, and glass industries (Amasa, 1975; Smedley, 1996;
1
To whom correspondence should be addressed. Fax: (207) 581-2801.
E-mail: carolkim@maine.edu.
Ó The Author 2007. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
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Smedley and Kinniburgh, 2002). Pollutants discharged into
bodies of water are readily taken up by fish and subsequently
move up the trophic levels, potentially even to humans. Fish
have proven to be valuable model systems to study consequen-
ces 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 dis-
ruption 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 first 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 inflammation
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 zebrafish (Danio rerio) is a teleost that has become
important as an animal model to study embryo development,
genetics, and the immune system. Zebrafish 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
 ARSENIC AND THE ZEBRAFISH INNATE IMMUNE RESPONSE
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 in-
dicate that knowledge gained from research in the zebrafish
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 zebrafish 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
host’s ability to fight viral and bacterial infection via specific
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
zebrafish. Effects of 2 and 10 ppb arsenic, concentrations that
fall within the range used previously in studies of fish 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 ex-
pression levels of essential antiviral, and antibacterial cyto-
kines were examined in zebrafish embryos.
MATERIALS AND METHODS
Zebrafish care and maintenance. AB strain zebrafish were maintained in
the Zebrafish Facility at The University of Maine, Orono, in recirculating
systems from Aquatic Habitat (Apopka, Florida). The water was maintained at
28°C with a flow 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 zebrafish facility was maintained according to Institutional Animal Care
and Use Committee standards.
Arsenic exposures. Zebrafish 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 28°C, 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 fish 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 fish
maintained as unexposed controls were handled in a similar manner to the
arsenic-exposed fish.
Determination of viral titer in arsenic-exposed embryos. Arsenic-
exposed 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 fish
were collected at 24 h post infection (hpi) for each treatment and homogenized
in minimum essential medium (GIBCO-Invitrogen, Carlsbad, CA) supple-
mented 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
119
(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. Arsenic-
exposed 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 fish
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
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zebrafish embryos by measuring oxidation of dihydrodichlorofluorescein
diacetate (H2DCFDA) to fluorescent dichlorofluorescein (Hermann et al.,
2004). Arsenic-exposed and unexposed embryos were used in the assay
between 3 and 10 dpf. At the designated time points, fish 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
fluorescence 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 fish 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 fish from each treatment per time point in
TRIzol reagent (Invitrogen, Carlsbad, CA) and stored at ÿ 80°C.
RNA was extracted according to the manufacturer’s instructions using
TRIzol reagent (Invitrogen), and reverse transcription reactions were per-
formed 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 94°C for 3 min to
activate the polymerase, followed by 40 cycles of 94°C for 30 s, 53°C for 30 s,
and 72°C for 30 s. The cytokines were amplified using similar conditions with
the exception that the annealing temperature was 56°C for TNF-a and 54°C 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 fish yielded the approximate difference in fold
reductions in cytokine expression upon arsenic exposure.
RESULTS
Exposure to Arsenic Inhibits the Ability of the Zebrafish
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 zebrafish after infection
were examined. At 24 hpi, the viral titer was determined to be
1.0 3 105 TCID50/ml in infected control fish, 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 signif-
icant increases of 57- and 87-fold in viral load ( p value < 0.006)
120 NAYAK, LAGE, AND KIM
FIG. 1. Exposure to arsenic inhibits the ability of the zebrafish to clear
viral and bacterial load. Viral (a) and bacterial (b) load were calculated in
arsenic-exposed and unexposed zebrafish. Each bar represents the average of
three experiments and the error bars are indicated by SEs of the mean
(significance 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. tarda–infected control
fish 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 significantly 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 fish.
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 measur-
ing the respiratory burst response (Fig. 2), using an assay
developed by Hermann et al. (2004). ROS production in PMA-
induced 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 fish 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 0.9-
fold. Fish exposed to 10 ppb arsenic showed a similar trend in
respiratory burst response, with a 1.5-fold induction observed
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FIG. 2. Arsenic exposure dampens the respiratory burst response. Res-
piratory burst was measured in arsenic-exposed and unexposed zebrafish by the
oxidation of H2DCFDA to dichlorofluorescein. Each bar represents the average
fluorescence from six embryos. Error bars are indicated by SEs of the mean.
Results are representative of three independent experiments (significance was
determined using ANOVA two factor with replication analysis; p value < 0.04).
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 significant, differences in re-
spiratory 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 pat-
terns of type I interferon (Fig. 3a) and the interferon-inducible
gene, Mx (Fig. 3b), were examined in arsenic-exposed and
unexposed zebrafish after infection with SHRV.
Control fish, 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 ex-
pression level at 72 hpi (1.18 3 104 RCN) (Fig. 3a). The ex-
pression level of interferon in these fish decreased at 96 hpi
 ARSENIC AND THE ZEBRAFISH INNATE IMMUNE RESPONSE
FIG. 3. Exposure to arsenic diminishes induction of essential antiviral
cytokines. cDNA samples from arsenic-exposed and unexposed zebrafish,
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 fish exposed to arsenic or
unexposed controls revealed that the interferon peak observed
in these fish 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 fish, the Mx ex-
pression 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 significantly
between 72 and 96 hpi. As with the fish 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 fish. RCN of Mx followed this interferon
pattern at each time point, with maximum expression level
121
observed at 72 hpi (1.72 3 104 RCN) (Fig. 3b). The RCN did
not vary significantly 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 fish. 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 fish to mount an
effective antiviral immune response.
Exposure to Arsenic Diminishes Induction of Essential
Antibacterial Cytokines
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To characterize the expression pattern of essential antibac-
terial cytokines in response to bacterial infection, arsenic-
exposed and unexposed zebrafish were infected with E. tarda,
and mRNA expression patterns for IL-1b (Fig. 4a) and TNF-a
(Fig. 4b) were examined. Infected control fish 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) finally 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
FIG. 4. Exposure to arsenic diminishes induction of essential antibacterial
cytokines. cDNA samples from arsenic-exposed and unexposed zebrafish,
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.
122 NAYAK, LAGE, AND KIM
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 zebrafish (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 fish at all time points (Fig. 4a). At 24 hpi, fish
exposed to 2 ppb showed a higher IL-1b RCN (6.40 3 103
RCN) when compared to the infected control fish 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 fish
for each treatment, at 24 hpi, demonstrated a 5-fold induction
in infected control fish and a 2.6-fold induction in fish 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 fish 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 final
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 fish, whereas 0.6-, 0.3-, and 2.7-
fold 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 fish.
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 de-
crease 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
fish. Also, the typical bimodal expression pattern observed in
infected control fish 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 fish to mount an effective antibacterial
immune response upon infection.
DISCUSSION
This is the first 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. Zebrafish embryos were used in this study because
the innate immune system of the zebrafish is active from birth
and is the only means of defense during the first 3 days of
development. This system is functionally as efficient 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 fish, zebrafish embryos can be
genetically manipulated to knock down genes of interest, which
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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 zebrafish 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 zebrafish in a dose-
dependent manner (data not shown), but even these slight
increases in total arsenic correlate with dramatic declines in
essential innate immune functions. Exposure to arsenic in-
hibited the host’s 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 specific 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 profiles of the zebrafish, upon
infection (Phelan et al., 2005b; Pressley et al., 2005). The
concentrations of pathogens used in our challenge experiments
were based on our previous findings, which established the
infective doses that resulted in 50% mortality and characteristic
changes in cytokine profiles in the zebrafish embryos (Phelan
et al., 2005b; Pressley et al., 2005). Data from these earlier
experiments served as an internal control for our present
experiments, specifically 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 zebrafish. It
is possible that the use of a high starting density of bacteria,
1 3 10 8 CFU/ml, could have overwhelmed the immune sys-
tem, thereby masking a significant difference between the fish
exposed to 2 and 10 ppb arsenic. Arsenic-exposed and control
embryos could be challenged with lower numbers of bacteria;
however, cytokine profiles and mortality curves in response to
the lower bacterial dose would need to be established in
zebrafish 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.
 ARSENIC AND THE ZEBRAFISH INNATE IMMUNE RESPONSE
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, macro-
phages, and dendritic cells (So et al., 2006). TNF-a also primes
the phagocytic cells for protein kinase C–dependent 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 cyto-
kines 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 in-
volved in cytokine signaling pathways in the zebrafish. Higher
pathogen load, coupled with delays or reductions in important
antiviral and antibacterial cytokine profiles, confirms that
presence of arsenic in the water blunts the ability of the fish
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 adap-
tive 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 demon-
strated 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 findings 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 contrib-
utes to further concentration at higher trophic levels. Arsenic
has also been shown to have deleterious effects on fish species
other than the zebrafish, 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 short-
term arsenic exposure, in addition to compromising overall
innate immune health, could leave individuals more susceptible
to opportunistic pathogen infections. Arsenic could potentially
123
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
Downloaded from https://academic.oup.com/toxsci/article/98/1/118/1659888 by guest on 21 February 2022
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
zebrafish as an excellent model for further immunotoxicolog-
ical studies. Therefore, since human health complications
associated with arsenic, including various forms of cancer,
aberrant inflammation, 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.
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 Environmen-
tal 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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