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Submitted on 19 April 2013
The Effect of Acetaminophen on Neutrophil Recruitment in Danio rerio Embryos
Ryan J. Coon
Department of Molecular and Cellular Biology, Loras College, Dubuque, IA, USA
Abstract
Neutrophil migration is an important function of the immune system response to tissue injury or
infection. Certain drugs can affect neutrophil migration to the site of injury. Acetaminophen can
have detrimental effects on embryos and hepatotoxic levels of this drug can reduce neutrophil
migration. This study was designed to determine if a non-hepatotoxic concentration of
acetaminophen affects neutrophil migration to the injury site. The Danio rerio embryos were
placed into three groups: wounded and treated with acetaminophen, wounded lacking
acetaminophen, and unwounded and treated with acetaminophen. The corresponding Danio rerio
embryos were treated with acetaminophen, wounded and the neutrophil migration was observed
in all three groups. After statistical analysis there was a significant difference between the
wounded and treated with drug group and the unwounded and treated with drug group. There
was no significant difference between the wounded and lacking the drug group and wounded and
treated with drug group. There was no significant difference between the wounded and lacking
the drug group and the unwounded and treated with drug group. This suggests that
acetaminophen has no effect on neutrophil migration to the wound site. However, neutrophil
migration is due to whether or not the embryo is injured with an increased migration in injured
embryos.
Introduction
Organisms are composed of many cells that communicate with each other and move
throughout the organism. This is characteristic of the cells in our immune system that migrate in
order to fight off disease and infection to maintain homeostasis. A particular type of immune cell
is the neutrophil or better known as the white blood cell. These cells migrate towards a wound
and act as the first responders to the threat of infection, secreting chemicals to ward off infection
and recruit other neutrophils to the site of infection as well, causing inflammation at the site of
the wound.
Many drugs can affect neutrophil movement to the location of injury. However,
acetaminophen is an analgesic or pain reliever and also an antipyretic or fever reducer and does
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not have an effect on inflammation (McNicol 2008). Studies have shown that acetaminophen can
have detrimental effects on mouse embryos and fetuses (Laub et al., 2000). Such effects can
occur in development and have detrimental effects on overall functioning of the embryo by
inhibiting the Na+/K+ ATPase (Laub at al., 2000). Also, embryos that are not within the mother
are more sensitive to acetaminophen than embryos outside of the mother (Laub et al., 2000).
This may be due to maternal mechanisms that protect the embryo from toxic levels of
acetaminophen (Laub et al., 2000). Another study suggests that the inflammatory response
caused by acetaminophen hepatotoxicity limits the amount of pro-inflammatory mediators
present at the location of the trauma and is beneficial to the organism by stimulating tissue repair
(Jaeschke et al., 2012). This suggests that neutrophil migration which is triggered by proinflammatory mediators occurs less when cells are treated with high concentrations of
acetaminophen.
Thus, our experiment was performed to determine if a non-hepatotoxic concentration of
acetaminophen affects the migration of white blood cells in Danio rerio embryos. A nonhepatotoxic acetaminophen concentration in zebrafish embryos is 1mM (North et al., 2010).
Since the Danio rerio embryos are not developing within the mother the neutrophils may be more
sensitive to acetaminophen. Therefore, it was hypothesized that a non-hepatotoxic concentration
of acetaminophen will not affect the recruitment of neutrophils to the site of trauma in the Danio
rerio embryo.
Methods
600 μL of tricaine anesthetic was added to 15 mL of 1x E3 and 5 ml of the solution were
added to each of the three dishes (wound lacking drug, wound with drug, no wound with drug).
The concentration of tricaine that the fish were exposed to was 0.16 mg/ml. Four, three-day-old
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Danio rerio embryos were added to each dish and anesthetized for five minutes. Two of the three
groups were then treated with a 1 mM aqueous solution of acetaminophen. A syringe was used to
wound the fish on their fin, relatively the same location on each fish. This was only done to the
fish in the wound groups.
Two hours after the fish were wounded they were removed from their dishes and placed
in 1.5 mL eppendorf tubes. The E3 was removed from the eppendorf tubes and 250 μL of 4%
formaldehyde in PBS was added to each tube. The eppendorf tubes containing the fish embryos
were placed in the refrigerator for one week at 4˚C.
After one week the formaldehyde was removed and 500 μL of 1x PBS was added and the
fish were exposed to the solution for four minutes. This process was then repeated two more
times. After the third wash the 1x PBS was removed and 500 μL of Sudan Black working
solution was added to each tube. The stock solution was made of 0.18% Sudan Black B (Sigma
S2380) in 70% Ethanol, kept at room temperature. The working solution consisted of dilute stock
1:5 into 70% ethanol and 0.1% phenol was added at room temperature. The tubes were then
incubated on an orbital shaker for forty minutes at room temperature. After the incubation period
the Sudan Black was removed and 500 μL of 70% ethanol was added to wash the fish embryos.
This ethanol wash was repeated twice. After removing 250 μL of ethanol after the second wash
250 μL of 1x PBS+0.1% Tween was added and the fish were exposed to this solution for five
minutes. All of the liquid was removed and 500 μL of 1x PBS+0.1% Tween was added and let
sit for five minutes. All of the liquid was removed to clear the natural pigment of the fish
embryos and 500 μL of a solution of 1% KOH + 1% H2O2 (made up in water) was added. The
tubes were then placed on an orbital shaker for twenty minutes at room temperature. The fish
were then washed twice with 1xPBS+0.1% Tween. The zebrafish embryos were then placed in a
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dish containing PBS/Tween, one treatment group at a time, and observed under a microscope.
The neutrophils were counted near the site of the wound; see Figure 1 for location used for
counting neutrophils. Statistical analysis of neutrophil migration was done using a one-way
ANOVA with a post hoc tukey test in SPSS.
Wound site
Neutrophil
Figure 1. Observed neutrophil migration in wounded three-day-old Danio rerio embryos. This
depicts a zebrafish embryo in the wounded and treated with acetaminophen group. The zebrafish were
treated with 1 mM aqueous solution of acetaminophen and then wounded at the end of their tail fin with a
syringe. The embryos were stained with Sudan Black working solution and neutrophil migration was then
observed. If neutrophils fell within the circled region near the wound they were counted as migrated
neutrophils. The wound site and neutrophils observed are denoted by the respective arrows.
Results
Statistical analysis of neutrophil migration was done using a one-way ANOVA and
resulted in a p-value of 0.032 suggesting there was a significant difference between groups.
Additional statistical analysis using post hoc tukey testing in SPSS revealed there was a
significant difference (p-value=0.030) between the wounded and treated with drug group and the
unwounded and treated with drug group. There was no significant difference between the
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wounded and lacking the drug group and wounded and treated with drug group (p-value=0.631).
There was no significant difference between the wounded and lacking the drug group and the
unwounded and treated with drug group (p-value=0.127). These results are shown in Figure 2.
4
3.5
3
2.5
2
Average
number of
1.5
neutrophils
that migrated
1
0.5
0
-0.5
Wound lacking Drug Wound with drug
No wound with
drug
-1
Figure 2. The Average Number of Neutrophils that Migrated in four-day-old Danio rerio Embryos.
Four-day-old Danio rerio embryos were obtained. Four embryos were in each of the three treatment
groups. The three groups were: wounded embryos lacking acetaminophen, wounded embryos treated with
acetaminophen, and unwounded embryos treated with acetaminophen. The zebrafish embryos were
anesthetized and two of the three groups were then treated with a 1 mM aqueous solution of
acetaminophen. Two of the groups were wounded and let sit in 4% formaldehyde made in PBS and
placed in a 4˚C environment for one week. The zebrafish were washed three times and then stained with
Sudan Black at room temperature. Each treatment group was then analyzed under a microscope and the
migration of neutrophils towards the wound site was observed see Figure 1. Microsoft Excel was used to
determine the average number of neutrophils migrated for each treatment group (error bars indicate
SEM). Post hoc tukey analysis revealed a p-value < 0.05 between the wound with drug group and no
wound with drug group.
Discussion
The hypothesis for this experiment was that if Danio rerio embryos were treated with a
non-hepatotoxic concentration of acetaminophen it will not affect the recruitment of neutrophils
to the site of trauma. The data obtained though this experiment support our hypothesis. There
was no significant difference between the wounded zebrafish embryos treated with
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acetaminophen and the wounded group lacking acetaminophen (Figure 2). This suggests that the
presence of acetaminophen did not affect neutrophil migration in the wounded zebrafish
embryos. The group that was wounded and lacking the acetaminophen was not significantly
different than the unwounded and treated with acetaminophen group. However, there was a
significant difference between the wounded embryos treated with acetaminophen and the
unwounded group treated with acetaminophen. This reveals that neutrophil migration was
significantly different between those two groups if a wound was present or not.
In a previous study Jaeschke et al. (2012) treated liver cells in mice with hepatotoxic
levels of acetaminophen (~10mM) which resulted in a reduction in the presence of neutrophils at
the wound site. However, a non-hepatotoxic acetaminophen concentration in zebrafish embryos
is 1mM (North et al., 2010). When non-hepatotoxic levels of acetaminophen were used in this
current experiment, there was no significant difference in neutrophil migration in wounded
embryos that were treated with the drug and wounded embryos lacking acetaminophen
treatment. This suggests that the presence of acetaminophen in non-hepatotoxic levels does not
affect neutrophil migration, but at hepatotoxic levels of acetaminophen neutrophil migration is
reduced. Another study performed by Laub et al. (2000) states that acetaminophen can have
detrimental effects on embryo development and functioning. This effect is seen especially if the
embryo is not within the mother because they do not receive maternal mechanisms to protect it
from the acetaminophen. Even though both groups in the current experiment used zebrafish
embryos that develop outside of the mother, there was no significant difference in neutrophil
migration between the wounded group that were treated with acetaminophen and the wounded
group that was not treated with acetaminophen. Thus, the acetaminophen treatment did not have
an effect on neutrophil migration and did not affect this aspect of embryo functioning.
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Acetaminophen should not have an effect on neutrophil migration because it is an analgesic or
pain reliever and also an antipyretic or fever reducer, not an anti-inflammatory medication
(McNicol 2008). This also was shown in data collected from the two wounded groups previously
discussed because neutrophil migration was not significantly different.
There was a significant difference between the wounded group treated with
acetaminophen and the unwounded group treated with acetaminophen. This agrees with a study
performed by Mathias et al., (2006) which indicated that neutrophil migration is an essential
aspect of the immune system response to tissue wounding or infection. This study supports our
findings and suggests that neutrophil migration is affected by the presence of tissue injury and
not by the presence of acetaminophen.
There was no significant difference between the wounded zebrafish embryos lacking
acetaminophen and the unwounded group treated with acetaminophen. This could have been
caused if the wounded group did not receive a deep enough injury. Thus, neutrophil migration
may have not been affected in the wounded group because the organism did not stimulate a
physiological immune response. The presence of acetaminophen in one group and the lack of
acetaminophen in the other will not affect neutrophil migration as stated previously in the
discussion.
Future studies can be done to determine what signaling pathways enable the immune
system to trigger neutrophil migration. This would enable researchers to develop a drug that can
reduce the inflammatory response which is triggered by wound infliction.
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Works cited
Jaeschke H., Williams C.D., Ramachandran A., and Bajt M.L. (2012). Acetaminophen
hepatotoxicity and repair: the role of sterile inflammation and innate immunity. Liver Int. 32, 820.
Laub D.N., Elmagbari N.O., Hausburg M.A., and Gardiner C.S. (2000). Effects of
acetaminophen on preimplantation embryo glutathione concentration and development in vivo
and in vitro. Toxicological Sciences 56, 150-155.
Mathias J.R., Perrin B.J., Liu T.X., Kanki J., Look A.T., and Huttenlocher A. (2006). Resolution
of inflammation by retrograde chemotaxis of neutrophils in transgenic zebrafish. J. Leuok. Biol.
8, 1281-1288.
McNicol E. (2008). How does acetaminophen relieve pain? I’m always amazed that it can take
my headache away—and reduce a fever—so quickly. Tufts Journal. Available at:
http://tuftsjournal.tufts.edu/2008/04/professor/01/. Accessed April 16, 2013.
North T.E., Babu I.R., Vedder L.M., Lord A.M., Wishnok J.S., Tannenbaum S.R., Zon L.I., and
Goessling W. (2010). PGE2-regulated wnt signaling and N-acetylcysteine are synergistically
hepatoprotective in zebrafish acetaminophen injury. Proc. Natl. Acad. Sci. 107, 17315-17320.