Transcript Limulus polyphemus - York College of Pennsylvania

Effects of heavy metals on blood coagulation of horseshoe crab,
Limulus polyphemus
Hanh Phung, Department of Biology, York College of Pennsylvania
Review literature
Project summary
Low level of metals were found in different
tissues of horseshoe crabs collected from Maine
to Florida (Burger et al. 2002).
• In the recent years populations of
horseshoe crabs have suffered a serious
decline due to overfishing, erosion and loss
of spawning habitat, and pollution.
•Pollution might cause potential problems to
the blood clotting system of horseshoe crabs
because the metals may interfere with the
defense molecules in the hemolymph and
inhibit their ability to clot and destroy the
bacteria upon exposure to endotoxins. Since
horseshoe crabs are taken from the wild,
which can be polluted with heavy metals, it is
essential to determine the effects of heavy
metals on the blood clotting of horseshoe
crab, and how the results of LAL could be
changed.
Experimental
Control
N= 10 hscs
No exposure to
metals
Bleed
&
LAL
Introduction
•To obtain the blood, horseshoe crabs are
collected from the wild, bled and released
back.
N= 250 hscs
10 hscs /replicate
5 Yrs in Captivity
The study is designed to test the effects of
heavy metals (Cd, Cu, Zn, and Hg) on the
blood clotting of horseshoe crabs and to
determine whether the results of Limulus
Amebocyte Lysate (LAL) could be changed.
In that case, the LAL test could fail to detect
the presence of bacteria in clinical settings,
which might cause adverse effects in patients
who receive treatments.
100
90
N = 240 hscs, divided into 4 groups
N1,…N4: each group/ treatment of a
metal
Cu
Cd
Zn
Hg
N=60hscs
N=60hscs
N=60hscs
N=60hscs
6 [Cu]
6 [Cd]
6 [Zn]
6[Hg]
70
(ppb)
60
Apodeme
Egg
Leg
50
40
LPS
1 μg/ ml
Color
change
30
20
*LPS: lipopolysaccharides
10
*hscs: horseshoe crabs
0
Cadmium Chromium Lead
Mercury
Horseshoe crab embryos exposed to heavy
metals using bioassay showed developmental
abnormalities (Itow et al. 1998).
• Segmented defective embryos
• Double embryos
• Embryos with abnormal eye areas
• Embryos with no posterior
Metals might cause a problem to blood clotting of
horseshoe crabs and the LAL test, which is not
known in literature. Metals might denature the
enzymes/proteins involved in blood coagulation.
In horseshoe crabs, 99% of all hemocytes (blood
cells) are granular hemocytes which contain the
defense molecules and release them upon
stimulation by lipopolysaccharides (LPS) (Ariki et
al. 2004).
*Coagulation cascade of horseshoe crab (Nagai
and Kawabata, 2002):
Expected results
*Lower absorbance normally indicates fewer bacteria. Since
the same amount of bacteria is added, lower absorbance
indicates reduced clotting ability.
Figure 4. The graph shows the expected results of
different concentrations of Hg in the chromogenic
substrate LAL test. There is a significant difference
between 0.5mg/L and 1mg/L, as shown in ANOVA test,
(p<0.05).
Conclusion
•High levels of metals (Cu, Zn, Cd, Hg) decrease color
change in the chromogenic substrate LAL test, which
means clotting intensity is reduced. In this case, the LAL
test could fail to detect the presence of bacteria in
clinical settings, which might cause adverse effects in
patients who receive treatments.
Literature Cited
Figure 1. The graph shows the expected results of different
concentrations of Cu in the chromogenic substrate LAL
test. There is a significant difference between 50mg/L and
100mg/L, as shown in ANOVA test, (p<0.05).
Hypothesis/Objectives
•Ho : There is no difference in the results of
a LAL test made from blood of a horseshoe
crab exposed to heavy metals as
comparison to a horseshoe crab with no
exposure.
•Metals of interests are: Copper, Cadmium,
Mercury, and Zinc.
Figure 3. The graph shows the expected results of
different concentrations of Cd in the chromogenic
substrate LAL test. There is a significant difference
between 5mg/L and 10mg/L, as shown in ANOVA test,
(p<0.05).
80
Level of metals
•Horseshoe crabs play an important role in
the biomedical industry because their blood
is used to make Limulus amebocyte lysate
(LAL), a method of detecting bacterial
endotoxins in clinical settings. A minute
amount of endotoxins can stimulate the
amebocytes, the blood cells of horseshoe
crabs, to form a clot. This method has
replaced the rabbit pyrogen test in non-oral
drugs, which saves thousands of rabbits.
Materials/Methods
Figure 2. The graph shows the expected results of different
concentrations of Zn in the chromogenic substrate LAL
test. There is a significant difference between 50mg/L and
100mg/L, as shown in ANOVA test, (p<0.05)
1. Ariki, S., Koori, K., Osaki, T., motoyama, K, Inamori, K. and Kawabata,
S. 2004. A serine protease zymogen functions as a pattern-recognition
receptor for lipopolysaccharides. Proceedings of the National Academy of
Sciences of the United States of America 101 (4) : 953-958
2. Botton, M.L. 2000. Toxicity of Cadmium and Mercury to Horseshoe crab
(Limulus polyphemus) embryos and larvae. Bulletin of Environmental
Contamination and Toxicology 64: 137-143
3. Botton, M. L., Johnson, K., and Helleby, L. 1998.Effects of copper and
zinc on embryos and larvae of the horseshoe crab, Limulus polyphemus.
Archives of Environmental Contamination and Toxicology 35 : 25-32
4. Burger, J., Dixon, C., Shukla, T., Tsipoura, N., and Gochfeld, M. 2002.
Metal levels in horseshoe crabs (Limulus polyphemus) from Maine to
Florida. Environmental Research 90: 227-236.
5. Itow, T., Loveland, R.E., and Botton, M.L. 1998. Developmental
abnormalities in horseshoe crab embryos caused by exposure to heavy
metals. Archives of Environmental Contamination and Toxicology 35 :3340
6. Nagai, T., and Kawataba, S. 2000. A link between blood coagulation and
prophenol oxidase activation in arthropod hose defense. The Journal of
Biological Chemistry 275 (38) :29264-29267
Acknowledgement
I would like to thank Dr Jessica Nolan, my mentor and also senior
thesis instructor, for her time, patience, expertise, and
willingness to guide me to the finish.