Effects of heavy metals on blood coagulation of horseshoe crab, Limulus polyphemus

Hanh Phung, Department of Biology, York College of Pennsylvania

Introduction

•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.

•To obtain the blood, horseshoe crabs are collected from the wild, bled and released back.

• 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.

Project summary

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.

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.

Review literature

Low level of metals were found in different tissues of horseshoe crabs collected from Maine to Florida (Burger et al. 2002).

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):

Materials/Methods

N= 250 hscs10 hscs /replicate

Control

N= 10 hscsNo exposure to

metals

Experimental

N = 240 hscs, divided into 4 groupsN1,…N4: each group/ treatment of a metal

Cu Cd Zn Hg

N=60hscs N=60hscsN=60hscsN=60hscs

6 [Cu] 6 [Cd] 6 [Zn] 6[Hg]

5 Yrs in Captivity

Bleed & LAL

LPS

1 μg/ ml

Colorchange

Expected results

Literature Cited1. 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) : 953-9582. Botton, M.L. 2000. Toxicity of Cadmium and Mercury to Horseshoe crab (Limulus polyphemus) embryos and larvae. Bulletin of Environmental Contamination and Toxicology 64: 137-1433. 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 : 25-324. 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 :33-406. 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

AcknowledgementI 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.

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.

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).

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)

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).

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).

Chromium Mercury0

10

20

30

40

50

60

70

80

90

100

Cadmium Lead

Apodeme

Egg

Leg

Le

vel o

f m

eta

ls

(pp

b)

*LPS: lipopolysaccharides

*hscs: horseshoe crabs

*Lower absorbance normally indicates fewer bacteria. Since the same amount of bacteria is added, lower absorbance indicates reduced clotting ability.