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80
Environmental Toxicology and Chemistry, Vol. 26, No. 1, pp. 80–84, 2007
䉷
2007 SETAC
Printed in the USA
0730-7268/07 $12.00
⫹
.00
ATRAZINE IS AN IMMUNE DISRUPTOR IN ADULT NORTHERN LEOPARD FROGS
(
RANA PIPIENS
)
M
ARC
A. B
RODKIN
,* H
ARETH
M
ADHOUN
,M
UTHURAMANAN
R
AMESWARAN
, and I
TZICK
V
ATNICK
Department of Biology, Science Division, Widener University, One University Place, Chester, Pennsylvania 19013, USA
(
Received
18
January
2006;
Accepted
29
June
2006)
Abstract—Atrazine, the most widely used herbicide in the United States, has been shown in several studies to be an endocrine
disruptor in adult frogs. Results from this study indicate that atrazine also functions as an immune disruptor in frogs. Exposure to
atrazine (21 ppb for 8 d) affects the innate immune response of adult
Rana pipiens
in similar ways to acid exposure (pH 5.5), as
we have previously shown. Atrazine exposure suppressed the thioglycollate-stimulated recruitment of white blood cells to the
peritoneal cavity to background (Ringer exposed) levels and also decreased the phagocytic activity of these cells. Unlike acid
exposure, atrazine exposure did not cause mortality. Our results, from a dose–response study, indicate that atrazine acts as an
immune disruptor at the same effective doses that it disrupts the endocrine system.
Keywords—Atrazine Immune disruption Frog
Rana pipiens
INTRODUCTION
The global decline in amphibian populations has been under
close examination for more than 20 years; more recently, the
role played by environmental pollutants in this decline has
received increased attention. It is well documented that some
environmental pollutants disrupt the function of the endocrine
system in many animal species [1]. Therefore, these substances
are called endocrine disruptors and are defined by the Envi-
ronmental Protection Agency as ‘‘external agents that interfere
in some way with the role of natural hormones in the body’’
(http://www.epa.gov/scipoly/oscpendo/index.htm). Aspelin [2;
http://www.epa.gov/oppbead1/pestsales/] estimated that by
2000, there will be over 20,000 herbicides and pesticides used
in the United States and that these chemicals will have an
estimated 900 active ingredients.
A growing body of evidence shows that pesticides and her-
bicides reduce the ability of frogs to resist parasitic infection.
These compounds also suppress the humoral immune response
of
Rana pipiens
and may have species-specific effects on the
cellular immune response [3–5]. Atrazine is one of the most
heavily used agricultural herbicides in the United States and
can reach 40 ppb both in aquatic ecosystems and in precipi-
tation [6]. Atrazine is the most common pollutant found in
groundwater [7]. In
Xenopus laevis
and
Rana pipiens
, atrazine
exposure to concentrations of 0.1 ppb causes endocrine dis-
ruption manifested as gonadal abnormalities and hermaphro-
ditism in males [6,8] and thereby may disrupt their ability to
reproduce. Atrazine has also been shown to cause immune
deficiency in ranids during the larval stage of development
[5].
The Environmental Protection Agency’s maximum contam-
inant load for atrazine in drinking water is 3 ppb [9]. This
concentration is 30 times higher than the 0.1 ppb that Hayes
et al. [6,8] showed to cause feminization of male frogs in the
laboratory. We wanted to investigate the effects atrazine has
* To whom correspondence may be addressed
(mabrodkin@widener.edu).
on the innate immune response in adult
R. pipiens
at doses
similar to those that disrupt endocrine function. We hypoth-
esized that atrazine, even at as low a concentration as 0.1 ppb,
may act as an immune disruptor in similar ways to mild acid
exposure, that is, by disrupting the innate immune response
of adult
R. pipiens
(SETAC Globe, 2004, 5:48–49; [10]). To
assess the innate immune response, we stimulated frogs with
thioglycollate, a widely used inflammatory mediator [11–15].
Inflammatory response is characterized by an influx of leu-
kocytes to the site of infection or injury. We measured the
inflammatory response and phagocytic activity of white blood
cells (WBCs) as indicators of innate immune response. To
measure the phagocytic activity of WBCs, in vivo, the inoc-
ulation medium contained 1-micron beads impregnated with
fluorescein isothiocyanate.
MATERIALS AND METHODS
Experimental animals and conditions
We conducted two experiments. In both experiments,
R.
pipiens
of both sexes (average length
⬃
7 cm and average mass
⬃
30 g) were purchased from Amphibians of North America
(Nashville, TN, USA). Frogs were caught in the northeastern
United States by licensed collectors in September or October.
Since these frogs were caught in the wild, we cannot control
for previous exposure to environmental contaminants. The ex-
periments were conducted shortly after the animals arrived and
were acclimated to the laboratory for at least 5 d. The Widener
University Institutional Animal Care and Use Committee ap-
proved all protocols for the experiments described in this paper.
Prior to the experiments, frogs were allowed to acclimate
to the laboratory in 38-L tanks, filled 3 cm deep with aged
tap water, and supplied with a Styrofoam (Dow Chemical,
Midland, MI, USA) raft to allow the animals to climb out of
the water. Water was changed daily, and frogs were fed three
to four crickets (Fluker Farms, Port Allen, LA, USA) per frog
4 d a week. During the 8-d experiment individual frogs were
kept in autoclaved sterile Tupperware (Tupperware, Orlando,
FL, USA; 30
⫻
20
⫻
9 cm) containers with lids (treated with
Atrazine is an immune disruptor
Environ. Toxicol. Chem.
26, 2007 81
Fig. 1. Eight days of exposure to pH 5.5 or to atrazine (21 ppb) reduce
the mean number (
n
⫽
6) of white blood cells
⫾
standard error in
the peritoneal exudates of adult
Rana pipiens
stimulated with thio-
glycollate to background levels. Stimulated groups were injected with
thioglycollate to induce an inflammatory response. Control groups
were injected with an iso-osmotic Ringer solution. Acid exposuredata
are from a previous experiment [10]. Statistical significance (
p
⬍
0.05) is indicated by different letters.
70% ethanol) filled with 500 ml of the appropriate solution
(i.e., aged tap water or atrazine supplemented water). The so-
lutions were changed daily. During experimentation, the Tup-
perware containers were placed in an environmental chamber
at 25
⬚
C with 12:12-h light:dark cycle.
Experiment 1: Atrazine exposure at 21 ppb
Twenty-four adult
R. pipiens
were randomly allocated to
four experimental groups: an atrazine-exposed group at 21 ppb
followed by injection with Ringer solution (RA), an atrazine-
exposed group at 21 ppb followed by thioglycollate stimulation
(TA), a group exposed to aged tap water—no atrazine followed
by injection with Ringer solution (RN), and a group exposed
to aged tap water—no atrazine followed by stimulation with
thioglycollate (TN).
Experiment 2: Atrazine dose response
Thirty-six adult
R. pipiens
were randomly allocated into
six experimental groups with six frogs in each group: four
experimental groups exposed to atrazine and two control
groups exposed to aged tap water. The atrazine (ChemService,
West Chester, PA, USA) groups were exposed to nominal doses
of 10, 1, 0.1, and 0.01 ppb. All atrazine-exposed groups re-
ceived thioglycollate stimulation and therefore were labeled
TA groups. One control group exposed to aged tap water (no
atrazine) was labeled RN and received injection with Ringer
solution (Carolina Biological Supply, Burlington, NC, USA),
and another control group exposed to aged tap water (no at-
razine) received thioglycollate stimulation and was labeled the
TN group.
White blood cell counts
White blood cells (WBCs) from the lavage fluid were count-
ed using a Hausser hemocytometer (Fisher Scientific, Pitts-
burg, PA, USA). Aliquots of lavage fluid were added to a
hemocytometer, and all four fields from the upper chamber
were counted, and the average number was reported for each
animal.
Phagocytic activity
The frogs were injected on the seventh day with 2 ml of
either physiological Ringer solution or thioglycollate each con-
taining 1-micron fluorescent beads. The frogs were euthanized
on day 8 by ether asphyxiation, and a peritoneal lavage was
performed with 10 ml of physiological Ringer solution. Blood
was collected, and livers were excised and placed in
⫺
70
⬚
C
storage for later characterization. Fluorescein isothiocyanate–
labeled beads (Polysciences, Warrington, PA, USA) were di-
luted into the inoculation medium to a final concentration of
2.5
⫻
10
7
beads per ml and injected intraperitoneally on day
7. The number of beads used was carefully titrated, and cells
were washed to avoid the occurrence of background beads
associated with but not engulfed by peritoneal WBCs. The
number of beads phagocytosed by each WBC was counted
using a Leitz Diaplan fluorescence microscope (Leica Micro-
systems, Wetzlar, Germany). For both experiments, 100 or
more WBCs were counted per frog. Based on the number of
beads phagocytosed, cells were placed into the following cat-
egories: 0 beads (nonphagocytic cells), 1 to 3 beads, 4 to 6
beads, 7 to 9 beads, and
⬎
10 beads (highly phagocytic cells).
Data from experiment 1 are expressed as the total percentage
of phagocytic cells, and data from experiment 2 are expressed
as one of two categories: nonphagocytic or highly phagocytic.
Statistical analysis
Peritoneal WBC counts and phagocytic activity were an-
alyzed by a Kruskal–Wallis nonparametric analysis of variance
followed by a nonparametric multiple comparisons using the
Q stat test [16]. The percent of phagocytic activity was trans-
formed by taking the arcsine of the square roots to make a
normal distribution and then analyzed using a two-way anal-
ysis of variance with repetition with an alpha level of
p
⬍
0.05. The percentages of nonphagocytic cells and highly
phagocytic cells were analyzed using a Mann–Whitney
U
test.
RESULTS
Experiment 1: WBC counts
Frogs exposed to atrazine at 21 ppb followed by thiogly-
collate stimulation showed a statistically significant dimin-
ished innate immune response when compared to frogs that
had not been exposed to atrazine but injected with thiogly-
collate. These results are very similar to frogs that were ex-
posed to acid (Fig. 1); acid exposed results are from a previous
experiment [10] and are presented here only for comparison
between environmental contaminants. We further analyzed the
phagocytic activity by calculating the percentage of phagocytic
cells (all cells that contained beads) and compared this among
the groups (Fig. 2). The average percent
⫾
standard errors
(SE) of the thioglycollate-stimulated group with no atrazine
exposure (TN) was 22.4
⫾
4.8 compared to 3.3
⫾
0.8 in the
thioglycollate group exposed to atrazine (TA). The average
percent of the Ringer-stimulated group with no atrazine ex-
posure (RN) was 2.5
⫾
0.9 compared to 9.6
⫾
1.5 in the
Ringer-stimulated group exposed to atrazine (RA). The two-
way analysis of variance showed that the difference between
the treatments (thioglycollate vs Ringer) as well as between
the groups (atrazine exposure vs no atrazine exposure) were
significant (
p
⫽
0.006 and
p
⫽
0.014, respectively) as well
as the interaction term (
p
⫽
1.03
⫻
10
⫺
5
).
Experiment 2: Dose response
WBC count.
Thioglycollate-stimulated (TN) frogs exhibited
the greatest number of peritoneal exudate cells
⫾
SE (5.1
⫾
0.95,
⫻
10
5
; Fig. 3). Frogs exposed to atrazine at 10, 1, 0.1,
82
Environ. Toxicol. Chem.
26, 2007 M.A. Brodkin et al.
Fig. 2. Eight days of exposure to atrazine (21 ppb) reduced the mean
percent (
n
⫽
6) of peritoneal phagocytic cells
⫾
standard error in
thioglycollate-stimulated frogs. The experimental groups were no at-
razine exposure stimulated with thioglycollate (TN) and atrazine ex-
posed and thioglycollate-stimulated (TA). The control groups were
no atrazine exposure stimulated with Ringer solution (RN) and at-
razine-exposed and stimulated with Ringer solution (RA). Data were
analyzed using a two-way analysis of variance, and statistical signif-
icance (
p
⬍
0.05) is indicated by different letters.
Fig. 4. Atrazine exposure reduced the number of highly phagocytic
cells (cells that engulfed
⬎
10 beads) in a dose-dependent fashion.
The minimal effective dose was 0.01 ppb. TN represents the thiogly-
collate-stimulated group without atrazine exposure, and RN represents
the Ringer solution–stimulated group without atrazine exposure. Sta-
tistical significance (
p
⬍
0.05) is indicated by different letters.
Fig. 3. Atrazine exposure reduced the total number of peritoneal ex-
udate white blood cells (WBCs) following thioglycollate stimulation
in a dose-dependent fashion. The minimal effective dose was 0.01
ppb. TN represents the thioglycollate-stimulated group without atra-
zine exposure, and RN represents the Ringer solution–stimulated
group without atrazine exposure. Statistical significance (
p
⬍
0.05)
is indicated by different letters.
and 0.01 ppb showed a significantly reduced number of peri-
toneal exudate cells compared to the TN group: 0.2
⫾
0.1,
0.71
⫾
0.15, 0.5
⫾
0.05, and 3.1
⫾
0.91,
⫻
10
5
, respectively
(
p
⬍
0.05 for all). The number of peritoneal exudate cells at
0.01, 0.1, 1, and 10 ppb atrazine are significantly different
from each other (
p
⬍
0.05). However, the number of peritoneal
exudate cells at 10 ppb and RN are not significantly different
from each other (
p
⬎
0.2).
Experiment 2: Dose response
WBC phagocytosis.
Thioglycollate-stimulated (TN) frogs
had the highest percentage of highly phagocytic cells
⫾
SE
(cells that engulfed
⬎
10 beads, 9.8%
⫾
1.5%). Peritoneal
WBCs from frogs stimulated by thioglycollate and exposed to
atrazine showed suppressed levels of phagocytic activity at
doses of 1 ppb (5.1%
⫾
1.0%), 0.1 (3.96%
⫾
1.69%), and
0.01 ppb (6.5%
⫾
1.7%) when compared to the TN group.
Cells from frogs exposed to 0.1 ppb had phagocytic activity
(3.96%
⫾
1.69%) significantly different from that of nonstim-
ulated (RN) resident peritoneal cells. However, peritoneal
WBC from frogs stimulated by thioglycollate and exposed to
10 ppb had the same phagocytic activity (1.0%
⫾
0.8%) as
nonstimulated (RN) resident peritoneal cells (1.3%
⫾
0.6%)
but were significantly different from the TN-stimulated group
(Fig. 4).
The thioglycollate-stimulated group (TN, no atrazine ex-
posure) had the lowest level of nonphagocytic cells
⫾
SE
(70.7%
⫾
1.45%). At 10 ppb atrazine exposure with thiogly-
collate stimulation, the percent of nonphagocytic cells (94.7%
⫾
0.71%) was the same as the nonstimulated, background (RN)
group (95.4%
⫾
0.61%). The percent of nonphagocytic cells
in the thioglycollate-stimulated group (TA) at 1 ppb atrazine
(84.2%
⫾
1.0%), 0.1 ppb atrazine (84.3%
⫾
1.69%), and 0.01
ppb atrazine (84.6%
⫾
1.73%) was reduced when compared
to the 10 ppb atrazine-exposed group and the RN groups;
however, they still appear to be greater than the TN group.
The 10 ppb atrazine-exposed group (TA) was significantly
different than the thioglycollate-stimulated group without
atrazine exposure (TN; Fig. 5).
DISCUSSION
Our study indicates that atrazine acts as an innate immune
system disruptor in addition to its endocrine disruption action
as shown by Hayes et al. [6,8]. Atrazine (21 ppb) affects the
innate immune response of adult
R. pipiens
in similar ways
to acid (Fig. 1). That is, atrazine exposure suppressed the
experimentally induced (thioglycollate-stimulated) recruit-
ment of WBCs to the peritoneal cavity to background (Ringer
control) levels. Unlike mild acid exposure, however, atrazine
does not cause mortality during an 8-d exposure period. There-
fore, it appears that both acid and atrazine function as immune
disruptors. Immune disruptors may act on both innate and
acquired immunity. A disruptor may suppress innate immunity
by decreasing the inflammatory response, reducing phagocytic
activity, and disrupting cytokine networks.
Thioglycollate-induced peritonitis is a commonly used
model to study inflammation [17]. Previous studies of ours
[10] support findings that phagocytosis of fluorescein isothio-
cyanate microspheres can be reliably used to assess the activity
Atrazine is an immune disruptor
Environ. Toxicol. Chem.
26, 2007 83
Fig. 5. Atrazine exposure increased the number of nonphagocytic cells
(cells that did not engulf any beads). The minimal effective dose was
0.01 ppb. TN represents the thioglycollate-stimulated group without
atrazine exposure, and RN represents the Ringer solution–stimulated
group without atrazine exposure. Statistical significance (
p
⬍
0.05)
is indicated by different letters.
of phagocytic WBCs. The inflammatory response elicited by
intraperitoneal injection of thioglycollate varies among species
[18] and among strains (of mice) used. Furthermore, the mech-
anisms by which this inflammatory response is elicited are
complex, vary among species and strains, and are sensitive to
a variety of endogenous and exogenous factors. However, this
widely used model is a useful tool to study the innate immune
response of vertebrate phyla.
Our data indicate that atrazine at 21 ppb with Ringer stim-
ulation (without thioglycollate) stimulates phagocytic activity
of resident peritoneal cells but suppresses phagocytic activity
of thioglycollate-recruited cells (Fig. 2). This difference may
be an example of a nonmonotonic dose response to atrazine
that has been reported in the literature [19].
We also used thioglycollate-induced peritonitis to study the
dose response of atrazine’s effects on the innate immune re-
sponse in our frogs. Hayes et al. [6] showed that atrazine-
induced endocrine disruption occurs at 0.1 ppb, a dose 30 times
lower than the Environmental Protection Agency’s maximum
contaminant level. Therefore, we designed our dose–response
experiment with similar atrazine doses to those used by Hayes
et al. [8]. Our results are similar to those published by Hayes
et al. [6,8] and indicate that immune disruption, like endocrine
disruption, occurred at 0.1 ppb (Figs. 3 to 5). At 10 ppb atrazine
exposure, both recruitment and phagocytic activity were re-
duced to the level of nonexposed control frogs, that is, to
background levels (RN; Figs. 3 and 4). At doses between 0.1
and 1.0 ppb atrazine exposure, WBC recruitment to the in-
flammatory site was still reduced but to a lesser degree. How-
ever, recruited cells appear to have high phagocytic activity
although less than stimulated nonexposed frogs (TN; Fig. 4).
Therefore, atrazine appears to still function as an immune dis-
ruptor even at these low doses. Although not tested for, this
result suggests that the mechanism underlying recruitment is
more sensitive to atrazine than the mechanism regulating
phagocytosis. This may be due to a differential effect of
atrazine on different cytokine networks.
Gilbertson et al. [3] studied the effects of exposure to a
mixture of pesticides on the innate, humoral, and cell-mediated
immune response of adult
R. pipiens
. Overall, the results of
that study suggest that pesticides can stimulate or suppress
different aspects of the immune response. The activation of
R. pipiens
phagocytic cells in the Gilbertson et al. [3] study
was measured by whole blood chemiluminescence assay, de-
scribed in detail in Marnilla et al. [20]. Frogs exposed to pes-
ticides had a lower chemiluminescence value (indicating lower
phagocytic activity) than frogs from a pesticide-free popula-
tion. Our results provide a measure of phagocytic activity in
vivo using a single herbicide and also demonstrated a herbi-
cide-induced suppression of the innate immune response.
Christin et al. [4] examined the in vitro phagocytic activity of
isolated splenocytes of adult
R. pipiens
exposed to a mixture
of six pesticides and herbicides (including atrazine) at an eco-
logically relevant dose. They found that pesticide exposure did
not have suppressive effects on phagocytosis and splenocyte
numbers. Therefore, phagocytic cells present in the spleen may
be less sensitive than phagocytic cells recruited to an inflam-
matory site. Recruited cells have responded to chemical signals
and cell surface receptors, and therefore they are in a physi-
ologically activated state. However, at the highest dose, ani-
mals that were exposed to the pesticides had the lowest re-
sistance to infection by the lung worm,
Rhabdias ranae
, sug-
gesting that agricultural herbicides and pesticides affect frogs’
ability to deal with parasitic infection. In a field study, Kie-
secker [5] found a synergistic relationship between trematode
infection leading to limb deformities and pesticide exposure.
Furthermore, in a laboratory study, Kiesecker [5] found sup-
porting evidence to suggest an association between pesticide
exposure and increased trematode infection. These results sug-
gested a pesticide-induced decrease in immunocompetence.
A growing body of literature indicates that exposure to
ecologically relevant mixtures of pesticides and herbicides
cause immunosuppression [3,4,20]. Our study indicates that
exposure to very low doses of atrazine alone also act as an
immunosuppressor of the innate immune response. Immuno-
suppression affects host–pathogen interactions, and because
all recently documented amphibian extinctions in Australia,
Britain, North America, and Central America [21] are asso-
ciated with pathogens, immunosuppression may play a role in
the global amphibian decline.
Acknowledgement
—This work was supported partially by Widener
University Provost’s and Faculty Development Grants, Sigma Xi
Grants in Aid of Research, and Tribeta Foundation Research Schol-
arship. We greatly appreciate the support of our former Provost Larry
Buck and our former Dean Larry Panek for their support of our work.
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