Accepted Manuscript
Inactivating Hemocyanin from Oncomelania Hupensis With 4-(chloroace‐ tyl)catechol and Its Application in Snail Control
Daoyi Guo, Hong Pan, Dan Zeng, Yongdong Li, Xun Li, Xiaolin Fan
PII: S0048-3575(12)00007-7 DOI: 10.1016/j.pestbp.2012.01.006 Reference: YPEST 3427
To appear in: Pesticide Biochemistry and Physiology
Received Date: 11 July 2011 Accepted Date: 16 January 2012
Please cite this article as: D. Guo, H. Pan, D. Zeng, Y. Li, X. Li, X. Fan, Inactivating Hemocyanin from Oncomelania Hupensis With 4-(chloroacetyl)catechol and Its Application in Snail Control, Pesticide Biochemistry and Physiology (2012), doi: 10.1016/j.pestbp.2012.01.006
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1 Inactivating Hemocyanin from Oncomelania Hupensis With 4-(chloroacetyl)catechol and Its
2 Application in Snail Control
3
4 Daoyi Guo, Hong Pan, Dan Zeng, Yongdong Li , Xun Li*, Xiaolin Fan*
5 Key Laboratory of Organo-Pharmaceutical Chemistry, Jiangxi Province, Gannan Normal University,
6 Ganzhou 341000, People’s Republic of China
7 Corresponding author: Tel: +86-797-8393536; Fax: +86-797-8393536; E-mail address:
8 [email protected], [email protected]
9
10 Abstract: The hemocyanin of Oncomelania hupensis (OhH) is essential for the survival of
11 Oncomelania hupensis and may be an effective target for the development of new molluscicide.
12 4-(chloroacetyl)catechol is a substrate analogue of OhH. In this study, we evaluated the toxicity of
13 4-(chloroacetyl)catechol to O. hupensis and Kunming mice. 4-(chloroacetyl)catechol had strong
14 molluscicidal activities and the molluscicidal activities was time and dose-dependent. With the increase
15 of exposure time, the LC50 values of the 4-(chloroacetyl)catechol decreased from 6.5 mg/L (24 h) to
16 3.1 mg/L (72 h). The LC90 values decreased from 16.4 mg/L (24 h) to 4.9 mg/L (72 h). In the acute
17 toxicity test of mice, no evident poisoning symptoms and no animal death were detected after 14
18 days’continuous observation, which indicated that 4-(chloroacetyl)catechol was a low toxic substance
19 for Kunming mice. These results indicated that 4-(chloroacetyl)catechol is potent molluscicides.
20 Keywords: Oncomelania Hupensis, Hemocyanin, Schistosoma japonicum, 4-(chloroacetyl)catechol
21 Abbreviations: Oncomelania hupensis hemocyanin, OhH; Schistosoma japonicum, S. japonicum 22
23
24
25
Page 2 of 15
1
2 1. Introduction
3 Oncomelania hupensis, a gastropod mollusc, is a unique intermediate host of Schistosoma
4 japonicum, which causes schistosomiasis in China. One of the major preventive steps against
5 schistosomiasis is the control of the vector snail population. Sodium pentachlorophenate and
6 niclosamide are the most widely used molluscicides in China at present [1-3]. But they are highly toxic
7 towards non-target organisms and easily result in the destruction of the ecosystem. The r efore, advent of
8 an environmentally friendly molluscicide would be a significant addition to current methods of the
9 control of the vector snails population.
10 Hemocyanin is an oxygen transport protein in the hemolymph of O. hupensis. As an extracellular
11 oxygen carrier, it is responsible for the precise oxygen delivery from the respiratory organs to tissues.
12 Therefore, the hemocyanin of O. hupensis (OhH) is essential for vector snail survival. OhH is
13 biochemically distinct from the oxygen transport protein of mammal and may be an effective target for
14 the development of a new molluscicide [4-6].
15 Hemocyanins and phenoloxidases serve different physiological functions as oxygen transporters
16 and as enzymes involved in defense response, respectively. However, they are equipped with a
17 structurally similar oxygen-binding active site. The active site contains two copper atoms. Either of
18 them, CuA and CuB, is coordinated by three histidines. The binuclear Cu active site in hemocyanin
19 reversibly binds O2, whereas the binuclear Cu active site in phenoloxidase activates O2 for substrate
20 hydroxylation or oxidation [7-10]. Recent results indicate that several hemocyanins can also exhibit
21 phenoloxidase activity[11-15]. We also found that the OhH exhibited phenoloxidase activity which can
22 activate O2 for oxidation of catechol [16].
23 Because catechol is a substrate of OhH, which can bind to the oxygen-binding active site of OhH
24 and then be oxidated by the activated O2, so it is possible that a specific inactivator of OhH can be
25 designed and synthesized based on the chemical structure of catechol, and the structure of the
Page 3 of 15
1 oxygen-binding active site. This specific inactivator can bind to the oxygen-binding active site and
2 form a covalent bond with the protein, thus irreversibly inactivating the snail oxygen transporters and
3 could be used in snail control. In this study, we found that the bands at 340nm of OhH were observably
4 descended with the addition of 4-(Chloroacetyl)catechol, which indicates that 4-(chloroacetyl)catechol
5 can inactivate oxygen-binding active site of OhH. In addition, we evaluated the toxicity of
6 4-(chloroacetyl)catechol to O. hupensis and Kunming mice.
7 2. Materials and methods
8 2.1 Mice and snails and reagents
9 Adult O. hupensis snails (9-11 mm in length) were caught from the Poyang Lake, Jiangxi Province,
10 China. The snails were kept in a laboratory at 20°C for a week before experiment. Kunming mice,
11 six-eight weeks old, were purchased from the Center of Experimental Animals, Jiangxi University of
12 Traditional Chinese Medicine. 4-(chloroacetyl)catechol were purchased from Alfa Aesar (CAS:
13 99-40-1).
14 2.2 Preparation of OhH protein
15 The OhH was prepared as previously described by our group [17]. Briefly, Hemolymph of O.
16 hupensis was collected from the foot muscles of the snails. Hemocytes and other cells were removed by
17 centrifugation at 8000g for 20 min at 4°C. The hemocyanin was pelleted in an ultracentrifuge at
18 180,000g for 3 h at 4 . The blue pellet was resuspended in “stabilizing buffer” consisting of 0.05 M
19 Tris, 5 mM CaCl2, 5 mM MgCl2 and 1 mM phenylmethylsulfonyl fluoride, pH 7.0 and stored at 4 .
20 2.3 Effects of 4-(chloroacetyl)catechol on oxygen binding
21 In spectroscopic analysis, OhH shows a peak at 340 nm, which is typical for oxygenated
22 hemocyanins and reflects the formation of the copper-oxygen complex. The degree of oxygenation of
23 the protein can be determined from the absorbance at 340nm [18]. Inactivating oxygen-binding active
24 site will result in the decrease of the absorbance at 340nm (A340). The changes in A340 at a protein
25 concentration of 2 mg/ml of OhH will be recorded with a spectrophotometer in the presence of 50ppm
Page 4 of 15
1 4-(chloroacetyl)catechol.
2 2.4. Molluscicidal assay
3 The molluscicidal effects of 4-(chloroacetyl)catechol against O. hupensis were determined by
4 Mao’s method [19]. The 4-(chloroacetyl)catechol was first dissolved in ethanol at 100mg/ml, then
5 diluted by dechlorinated water to 20mg/L, 10mg/L, 5mg/L and 2.5mg/L, respectively. Snails were
6 collected in a nylon mesh bag (30 snails per bag) and immersed into 1000mL dechlorinated water
7 solution of a known concentration of 4-(chloroacetyl)catechol and exposed for 2 4, 48 and 72,
8 respectively. After exposure and 24 h recovery period in only dechlorinated tap water, mortality was
9 checked. No response to a needle probe under dissecting microscope was the evidence of death. The
10 data were subjected to the probit analysis and the half and nearly full-lethal doses LC50 and LC90 were
11 calculated. Ethanol (200µl /L) controls were added to the test.
12 2.5 Study on Toxicity and Safety Evaluation of 4-(chloroacetyl)catechol on Kunming Mice
13 The acute toxicities of 4-(chloroacetyl)catechol on Kunming mice were determined according to the
14 Regulatory Guide on the Techniques for Drug Research [20]. Kunming mice were randomly assigned
15 to groups and weighed. 4-(chloroacetyl)catechol are slightly soluble in water, so suspensions were used
16 for their administration. Study substance was administered intragastrically to mice via a stomach tube.
17 Control mice received the same amount of water. After administration of the study substance, the
18 animals were closely observed during the first 8h, and occasionally thereafter for 14 days, for the onset
19 of convulsions, toxic signs and symptoms, and death.
20 2.6 Data analysis
21 The effect of 4-(chloroacetyl)catechol on O. hupensis was expressed by LC50 and LC90 and their
22 95% confidence limit.
23 3. Results
24 3.1 Effects of 4-(chloroacetyl)catechol on oxygen binding
25 The absorption peak of OhH was observed at 275 nm and 340 nm, which corresponded to aromatic
Page 5 of 15
1 residues and Cu2+-O2+ respectively (Fig.1.I). The bands at 340nm were observably descended by the
2 addition of 4-(chloroacetyl)catechol (Fig.1.II), which indicates the degree of oxygenation of the protein
3 decline. The bands at 275nm were also slightly descended by the addition of 4-(chloroacetyl)catechol.
4 In the spectroscopic analysis, 4-(chloroacetyl)catechol shows a peak at 275 nm, which reflects phenyl.
5 It is speculated that 4-(chloroacetyl)catechol can bind to oxygen-binding active site and form a covalent
6 bond with the OhH, then the 4-(chloroacetyl)catechol will be warped by the OhH which results in the
7 slight decline of the absorption peak at 275 nm.
8 3.2 Molluscicidal activities
9 Molluscicidal activities of 4-(chloroacetyl)catechol against the snail O. hupensis was shown in
10 Table 1. It shows time- and dose-dependent effects. The dead numbers of snails increased with the
11 increase of 4-(chloroacetyl)catechol concentration and exposure time. With the increase of exposure
12 time, the LC50 values of the 4-(chloroacetyl)catechol decreased from 6.5 mg/L (24 h) to 3.1 mg/L (72
13 h) against O. hupensis. The LC90 values of the 4-(chloroacetyl)catechol decreased from 16.4 mg/L (24
14 h) to 4.9 mg/L (72 h).
15 3.3 Study on Toxicity and Safety Evaluation of 4-(chloroacetyl)catechol on Kunming Mice
16 Mice were filled into the stomach with three doses of 500mg/kg, 1000mg/kg and 2000mg/kg of
17 4-(chloroacetyl)catechol. Water was used as the control group. The result showed that no evident
18 poisoning symptoms and no animal death were detected after 14 days’continuous observation.
19 4. Discussion
20 The control of snails is currently a main strategy in the Chinese National Schistosomiasis Control
21 Programme, which covers an area of 0.92 billion m2 annually. The search for environmentally friendly
22 synthetic or naturally occurring molluscicides is still ongoing[21-24]. In recent years, a large number of
23 plant extracts with molluscicidal activity have been identified[25-30], but due to low yields extracted
24 from raw plant material, these compounds have not been applied on a wider scale in the field. Synthetic
25 molluscicides are still considered the most effective measures for the control of schistosomiasis[31-37].
Page 6 of 15
1 The OhH is essential for vector snail survival and may be an effective target for the development of
2 new molluscicide. In spectroscopic analysis, the bands at 340nm and 275 nm of OhH were observably
3 or slight descended respectively with the addition of 4-(chloroacetyl)catechol. We speculated that
4 4-(chloroacetyl)catechol can bind to the oxygen-binding active site of OhH in a substrate-like manner,
5 but, instead of being hydroxylated or oxidated, it probably alkylates a histidine residue in the active site
6 of the OhH, thus irreversibly inactivating the snail oxygen transporters, which could be used in snail
7 control. In addition, we evaluated the toxicity of 4-(chloroacetyl)catechol to O. hupens i s and Kunming
8 mice. 4-(chloroacetyl)catechol had strong molluscicidal activities against the snail O. hupensis and the
9 molluscicidal activities were time and dose-dependent. The time-dependent effect of
10 4-(chloroacetyl)catechol might be due to the uptake of the active moiety which progressively increased
11 the amount of active components in the snail body with an increase in the exposure period. In the acute
12 toxicity test of Kunming mice, the result showed that no evident poisoning symptoms and no animal
13 death were detected after 14 days’continuous observation, which indicates that 4-(chloroacetyl)catechol
14 was a low toxic substance for Kunming mice. The toxicity of 4-(chloroacetyl)catechol to other
15 non-target organisms such as fish and invertebrates need further evaluation in the future study. These
16 results indicated that 4-(chloroacetyl)catechol is potent molluscide.
17 Acknowledgments
18 This work was supported by National Science Foundation of China (50968002, 50727803,
19 30860073), National Key Technologies R&D Program (2009BAI78B01, 2009BAI78B02), National
20 Science Foundation of Jiangxi province (2008GQY0102), and Foundation of Jiangxi Educational
21 Committee (GJJ10236).
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1
2
3
4
5 6 Figure legends: 7 8 9 Fig.1 Absorption spectra of total hemocyanin from Oncomelania hupensis. Maximum peaks were at
10 275 and 340 nm ( ), the bands at 340 nm observably descended by the addition of
11 4-(Chloroacetyl)catechol ( ).
Page 12 of 15
1 Highlights 2 3 1. Hemocyanin was isolated and purified from Oncomelania hupensis 4 5 2. 4-(chloroacetyl)catechol can inactivate oxygen-binding active site of Hemocyanin from Oncomelania hupensis. 6 7 3. 4-(chloroacetyl)catechol had strong molluscicidal activities and the molluscicidal activities were time and 8 dose-dependent. 9
Page 13 of 15
1 Graphical Abstract 2 1. 4-(chloroacetyl)catechol can inactivate oxygen-binding active site of Hemocyanin from Oncomelania 3 hupensis. 4
5
6 Fig.1 Absorption spectra of total Hemocyanin from Oncomelania hupensis. Maximum peaks were at 275 and 340 nm
7 ( ), the bands at 340 nm observably descended by the addition of 4-(Chloroacetyl)catechol ( ¡ ). 8 9 2. 4-(chloroacetyl)catechol had strong molluscicidal activities and the molluscicidal activities were time and 10 dose-dependent. 11 Table 1 Concentration- and time-dependent effects of 4-(chloroacetyl)catechol on O. hupensis after different time 12 exposure under laboratory condition
NO.of death £ Concentration ¢ mg/L 24h 48h 72h Ethanol control 0 0 0 2.5 3 7 8 5 11 21 27 10 20 25 30 20 29 30 30 LC50 6.5(5.3-8.0) 4.0(2.7-4.5) 3.1(2.6-3.6) LC90 16.4(12.5-25.7) 10.2(7.9-15.9) 4.9(4.2-6.8) 13
Page 14 of 15
1 Table 1 Concentration- and time-dependent effects of 4-(chloroacetyl)catechol
2 on O. hupensis after different time exposures under laboratory condition
Concentration No. of death
mg/L 24h 48h 72h
Ethanol control 0 0 0
2.5 3 7 8
5 11 21 27
10 20 25 30
20 29 30 30
LC50 6.5(5.3-8.0) 4.0(2.7-4.5) 3.1(2.6-3.6)
LC90 16.4(12.5-25.7) 10.2(7.9-15.9) 4.9(4.2-6.8)
3
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1