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Sublethal toxicity of Roundup to immunological and molecular aspects of Biomphalaria alexandrina to Schistosoma mansoni infection
Ecotoxicology and Environmental Safety 74 (2011) 754–760
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Sublethal toxicity of Roundup to immunological and molecular aspects of Biomphalaria alexandrina to Schistosoma mansoni infection Azza H. Mohamed n Department of Zoology, Faculty of Science, Menoufia University, Shebin El-Kom, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 August 2010 Received in revised form 24 October 2010 Accepted 25 October 2010 Available online 3 December 2010
The present study was performed to elucidate the cellular mechanisms of Biomphalaria alexandrina snails hemocytes against sublethal concentration (10 mg/L) of herbicide Roundup (48% Glyphosate) and/or Schistosoma mansoni infection during 7 days of exposure. Obtained results indicated that herbicide treatment and/or infection led to significant increase (P o 0.05) in total hemocytes count during exposure period. Examination of hemocytes monolayers resulted in observation of 3 morphologically different cell types, round small, hyalinocytes and spreading hemocytes. Spreading hemocytes are the dominant, more responsive and highly phagocytic cell type in all experimental groups. Moreover, the exposure to herbicide, infection or both together led to a significant increase (P o0.05) of in vitro phagocytic activity against yeast cells during 7 days of exposure. In addition, flow cytometric analysis of cell cycle and comet assay, resulted in DNA damage in B. alexandrina hemocytes exposed to herbicide and/or S. mansoni infection when compared to control group. The immunological responses as well as molecular aspects in B. alexandrina snails have been proposed as biomarkers of exposure to environmental pollutants. & 2010 Elsevier Inc. All rights reserved.
Keywords: Roundup Biomphalaria alexandrina Schistosoma mansoni Hemocytes Phagocytosis Comet assay
1. Introduction Particular attention has been directed to immunological changes induced by environmental pollution in aquatic invertebrates (Livingstone et al., 2000). The defense mechanisms and immunological responses which consist of the immune system have been considered as biomarkers of pollution in aquatic invertebrates (Galloway and Depledge, 2001). Despite the lack of an adaptive immune system, invertebrates are able to survive among potential pathogens and respond to infection by activation of various defense mechanisms (Little et al., 2005). The immune system is likely to be one of the most sensitive physiological systems to pollutants (Fournier et al., 2000). Pollutants can interact with immune system components and interfere with protection function that induce immune suppression and decrease of disease resistance (Wong et al., 1992). Schistosomiasis is one of the major communicable diseases with socio-economic importance in the developing world. It was estimated that 800 million people in 74 countries are at risk while more than 200 million were infected (Steinmann et al., 2006). Biomphalaria snails have a great medical importance as intermediate hosts of S. mansoni (Paraense, 2001). In this respect, interactions between Biomphalaria snails and schistosomes have received much attention. Defense system of freshwater snails mainly depends on hemocytes, which are the
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circulating cells involved in mediating internal defense and immune functions (Yoshino et al., 1998). Phagocytosis is one of the various functions of hemocytes, which is a non-specific immune mechanism against non-self materials (Galloway and Depledge, 2001; NegraoCorrea et al., 2007). The response patterns, density and functions, of hemocytes can be affected by xenobiotics and parasites (Livingstone et al., 2000). Furthermore, the development of an infectious disease results from an imbalance between the host and the pathogen due to external factors, like pollutants, and/or internal factors, like susceptibility of the host (Snieszko, 1974). Herbicides are distinctive group of pesticides and are considered as selective weed killers. Roundup is a commercial herbicide with active compound glyphosate used in a broad spectrum in agricultural applications for weed control (USDA, 1984; Williams et al., 2000; Cavas and Konen, 2007). Due to its high water solubility and extensive usage, especially in shallow water systems, the exposure of non-target aquatic organisms to this herbicide is a concern (Tsui and Chu, 2003). Results of previous studies showed that glyphosate may have effect on plants, fishes, amphibians, arthropods and snails by causing physiological, immunological and biochemical alterations (Glusczak et al., 2006; Achiorno et al., 2008; Schneider et al., 2009; Benamu et al., 2010). Comet assay is becoming a major tool for environmental biomonitoring (Grazeffe et al., 2008) because of its sensitivity and determination of DNA strand breaks in individual cell (Lee and Steinert, 2003). Some studies reported that glyphosate treatment of human lymphocytes in vitro resulted in increased chromatid exchange (Bolognesi et al., 1997), chromosomal aberrations and
A.H. Mohamed / Ecotoxicology and Environmental Safety 74 (2011) 754–760
indicators of oxidative stress (Lioi et al., 1998). Furthermore, Roundup proved to increase DNA adducts in mice (Peluso et al., 1998). Recently, Mladinic et al. (2009) used comet assay to measure glyphosate impact on DNA of human lymphocytes and the results revealed that glyphosate increased tail intensity. It is hypothesized that immunological biomarkers in snails e.g. Biomphalaria would improve their use as diagnostic organisms in biomonitoring. Therefore, the present study was undertaken to evaluate the immunological and molecular responses of B. alexandrina snails to the effect of sublethal concentration of Roundup herbicide treatment and/or S. mansoni infection. 2. Materials and methods 2.1. Experimental animals and infection Laboratory bred Biomphalaria alexandrina snails and Schistosoma mansoni ova were obtained from The Schistosome Biological Supply Center (SBSC), Theodor Bilharz Research Institute, Giza, Egypt. The adult snails (8–10 mm shell diameter) were maintained in 5 L capacity plastic aquaria under lab conditions at a density of 50 individual per tank. Water temperature was maintained at 25 72 1C. The snails were fed dry lettuce leaves daily. For snail infection B. alexandrina snails were individually exposed to 7–9 freshly hatched miracidia from S. mansoni ova in glass test tubes for 2 h (Anderson et al., 1982). 2.2. Experimental material Roundup herbicide was used in the liquid commercial form produced by Monsanto Agricultural Company, USA. It consists of 48% (480 g/L active ingredient ‘‘a.i.’’) of glyphosate (N-phosphonomethyl glycine). Sublethal concentration of Roundup (10 mg/L), equivalent to 0.02 mg/L a.i. glyphosate was prepared from stock solution (1000 mg/L). Fifty snails were placed in 5 L aerated plastic aquaria containing the used concentration according to Osman et al. (2008). 2.3. Experimental bioassays Four treatments were employed in this study, (50 snails were used in each treatment): 1. 2. 3. 4.
Normal control group. S. mansoni-infected group. 10 mg/L Roundup-treated group. Treated-infected group.
The evaluation of sublethal effect of Roundup and/or S. mansoni was done by: (1) total and differential hemocyte counts, (2) in vitro phagocytosis, (3) comet assay and (4) flow cytometric analysis of cell cycle. Each ones are detailed below. 2.3.1. Total and differential hemocyte counts The hemolymph was collected from the four experimental groups as described by Sminia (1972). In brief, the headfoot was touched by a Pasteur pipette; as a result the snail was forced to retract deeply into its shell and extruded hemolymph. From each individual snail, ca.75 mL hemolymph was obtained and collected using a micropipette. Hemolymph samples were collected at intervals of 6 h, 1, 3 and 7 days post-exposure from the four experimental groups. The number of total hemocytes was counted in 10 snails individually from each experimental group using haemocytometer. For differential hemocytes count, hemolymph samples were placed onto glass slides and allowed to settle in moist chamber. The hemocytes
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monolayers were stained after methanol fixation with Giemsa’s stain. Data were expressed as mean values of a proportion of each cell population from the original 100 cells. 2.3.2. In vitro phagocytosis The suspension of freshly prepared yeast cells was diluted in PBS to get the ratio of 10,000 cell/mL for use and phagocytosis was carried out as described by AbdulSalam and Michelson (1980). Freshly collected hemolymph from 10 individual snails was overlaid with an equal volume of yeast suspension on a clean glass slide. Reaction mixtures were incubated in moist chamber for 60 min. The phagocytic reaction was stopped using absolute methanol after washing with PBS (pH 7.4). For each slide 100 cells was examined and phagocytic activity was expressed as the mean values 7 SE of positive phagocytized hemocyte during 7 days of exposure. 2.3.3. Comet assay Comet assay was performed for measuring DNA damage after 24 h postherbicide treatment and/or S. mansoni infection by single cell gel assay which permits the detection of single stranded DNA breaks (SSBs) in one cell according to Singh et al. (1988) and Grazeffe et al. (2008). For each experimental group, collected hemolymph from 5 individual snails was pooled in 1.5 Eppendorf tube. Three microscope slides were covered with 1.5% normal melting agarose dissolved in phosphate buffered saline (PBS) free of Ca2 + and Mg2 + and maintained overnight at room temperature (257 2 1C). A volume of 100 mL of pooled hemolymph was dissolved in 300 mL of 0.7% of low-melting agarose dissolved in PBS Ca2 + and Mg2 + free and placed on the first gel layer at 37 1C. After solidification at 4 1C for 10 min, the slides were immersed in a jar containing cold lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% sodium sarcozinate, 1% Triton X-100 and 10% dimethyl sulfoxide (DMSO)), pH 10 at 4 1C for 2 h. After lysis, the slides were placed in horizontal electrophoresis box. The unit was filled with a fresh alkaline electrophoresis buffer (300 mM NaOH and 1 mM EDTA, pH 13) to a level of 0.25 cm above the slides. The cells were exposed to alkali for 15 min to allow DNA unwinding and expression of alkali-labile sites. To electrophoresis the DNA, an electric current of 25 V (0.86 V/cm) and 300 mA was applied for 20 min. Alkali and electrophoresis treatments were performed in an ice bath. All these steps were conducted under dim light to prevent the occurrence of additional DNA damage. After electrophoresis, the slides were placed horizontally and Tris buffer (0.4 M Tris, pH 7.5) was added to neutralize the excess alkali. Finally, slides were fixed with absolute ethanol for 10 min and stained with 100 mL ethidium bromide (20 mg/mL). From each slide 100 cells were examined visually and the percentage of damaged hemocytes DNA was scored (Lee and Steinert, 2003). Examination of the slides was done under fluorescence microscope (Olympus BX 60, Japan) equipped with an excitation filter 510 nm and barrier filter of 590 nm (1000 magnification). All chemicals were purchased from Sigma. 2.3.4. Flow cytometric analysis of cell cycle Flow cytometric analysis was carried out for detecting apoptosis of B. alexandrina hemocytes isolated from pooled hemolymph samples from 15 snail in each experimental group 7 days post-herbicide treatment and/or S. mansoni infection. The flow cytometer used is FACS calibur flow cytometer (Becton Dickinson, Sunnyvale, CA, USA) equipped with a compact aircooked low power 15 mW argon ion laser beam (488 nm). The average number of evaluated nuclei per specimen was 20,000 and the number of nuclei scanned was 120 per second. DNA histogram derived from flow cytometry was obtained with a computer program for Dean and Jett mathematical analysis (Dean and Jett, 1974). Data analysis was conducted using DNA analysis program MODFIT (verity software house, ME 04086 USA, version, 2.0). Apoptosis was measured by using the sub G1 peak staining with propidium iodide (Cohen and Al-Rubeai, 1995). 2.3.5. Statistical analysis Total and differential hemocyte counts and phagocytosis data are presented as mean 7standard error. The significance of difference between the means was calculated according to the way analysis (ANOVA) followed by Student’s t-test (Sokal and Rohlf, 1981). Result was considered statistically significant at P o 0.05.
Table 1 Effect of Roundup on total hemocytes count/ml in hemolymph of non-infected and S. mansoni-infected B. alexandrina snails during 7 days of exposure. Exposure period
6h 1 day 3 days 7 days
Experimental groups Control
Herbicide treated
Infected
Treated- infected
13.98 7 1.17 11.46 7 0.7 11.68 7 1.3 14.25 7 1.2
15.3 7 0.98 40.6 7 2.1n 39.3 7 2.2 n 39.8 7 2.7 n
3.6 7 0.5 n 15.9 7 1 n 8.26 7 0.5 4.7 7 0.4 n
8.2 7 0.3n 12.9 7 1 40.87 1.4 37.9 7 1.6
Data are presented as mean/104 7standard error (n¼ 10; 1 snail per replicate). n
Significant at Po 0.05.
n n
62.57 2.6 61.37 1.3 567 0.5 78.37 0.7n
Significant at Po 0.05.
44.25 7 2.3 26.37 0.02 387 0.7n 55.37 1.9 n
13.75 70.5 12.7 70.6 17 71.1 14.3 71.2n 49 72n 53.3 72.2 n 44.7 72.3n 28.3 70.7 n
31.7 7 1.4 33.4 7 1.7 33.7 7 1.8 51.3 7 1.8 58.7 7 0.4 567 0.8 547 1.5 44.7 7 1.5 19.8 7 0.9 19.4 7 1.1 237 0.8 29.7 7 0.4
21.5 7 0.7 24.6 7 0.7 237 0.6 25.6 7 0.9
19.5 7 1.2 15 7 1 21.7 7 0.7 20.3 7 0.2
Rs Hy Rs
Sp
Herbicide treated Control
Hy
n
Rs Sp
Infected
Hy
n
n
Using comet assay, the obtained results indicated that herbicide exposure and/or S. mansoni infection caused DNA single strand
Data are presented as mean 7standard error (n¼ 10; 1 snail per replicate). (Rs) Round small Hemocytes, (Hy) Hyalinocytes, (Sp) Granulocytes.
3.4. Comet assay
Experimental groups
The data illustrated in Table 3 indicated the effect of Roundup treatment and/or S. mansoni infection on phagocytic activity (in vitro) of B. alexandrina hemocytes against yeast cells during 7 days of exposure. The recorded data indicated that the exposure to herbicide, infection or both together led to a significant increase of phagocytic activity after 6 h post-exposure. The phagocytic activity was significantly higher after 3 days of exposure in snails exposed only to Roundup treatment. The mean values were 45.770.79, 30.77 0.5 and 40 70.9 for treated, infected and treated-infected groups, respectively, compared to 37.9870.8 for control group (Table 3). At the 7th day post-exposure, similar significant elevation level (Po0.05) was recorded in both treated and treated-infected groups while the elevation level in infected group was not significant.
Exposure period
3.3. Phagocytic activity
Table 2 Effect of Roundup on differential hemocytes count of non-infected and S. mansoni-infected B. alexandrina snails during 7 days of exposure.
Hemolymph samples from B. alexandrina snails contained three morphologically distinct types of hemocytes, designated as round small, hyalinocytes and spreading hemocytes. Round small hemocytes, measuring 8–10 mm in diameter; having a high cytoplasmnucleus ratio; few cytoplasmic granules and did not adhere to glass or emit pseudopodia. The spreading hemocytes, measuring 20–25 mm in diameter, had plentiful cytoplasm with numerous pseudopodia, an irregular nucleus and adhered to glass. Hyalinocytes were morphologically intermediate between round and spreading hemocytes, measuring about 12–15 mm in diameter; and can form few and short pseudopods. Spreading hemocytes are the dominant, more responsive and highly phagocytic cell type in all groups. It was significantly increased (Po0.05) in treated and infected groups after 6 h of exposure. After 1 and 3 days of exposure, the significant increase was continued in treated and infected groups when compared to control group. The mean number of spreading hemocytes was 53.3 72.2, 44.7 72.3 for treated group and 6170.4, 4570.5 for infected group compared to 24.6 70.7, 2370.6 for control group at 1 and 3 days postexposure. After 7 days of exposure, there was a significant decrease in spreading hemocytes of treated-infected group. The mean number was 10.370.9 and 25.6 70.9 for treated-infected and control groups (Table 2).
Sp
3.2. Differential hemocytes count
Rs
Treated infected
Hy
The effect of Roundup treatment and S. mansoni infection on total hemocytes count of B. alexandrina snails during 7 days of exposure is presented in Table 1. The presented data indicated that S. mansoni infection led to significant reduction (Po0.05) in total hemocytes count after 6 h of exposure. However, only herbicide and treatment combined with infection led to non-significant changes in counts at the same period. The total count in treated group was 40.672.1 compared to 11.4670.7, 15.9 71 and 12.971 for control, infected and treated-infected groups, respectively, one day post-exposure. The significant increase (P o0.05) continued till the end of the experiment (7 days) in both treated and treated-infected groups. However, in only infected group, there was a significant decrease by the 7th day post-exposure (Table 1).
14.25 7 1.5 17.7 7 1.7 24.3 7 0.9 11.3 7 0.6n
Sp
3.1. Total hemocytes count
45.75 7 3.1n 61 7 0.4n 45 7 0.5n 30.3 7 1.1
3. Results
23.25 7 1.1 217 1.2 197 0.98 10.37 0.9n
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6h 1 day 3 days 7 days
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Table 3 Effect of Roundup on phagocytic activity of non-infected and S. mansoni-infected B. alexandrina snails during 7 days of exposure. Exposure period
Experimental groups Control
6h 1 day 3 days 7 days
36.5 71.2 33 70.7 37.98 70.8 35.8 71
Herbicide treated 45.25 7 1.4 33.3 7 1 45.7 7 0.79 n 467 1.6 n n
Infected
Treated infected
53 71.3 51.7 71.28 30.7 70.5 35.7 71
n n
58.25 7 1.1 27.3 7 0.5 40 7 0.9 41 7 0.9
n
n
Data are presented as mean 7 standard error (n ¼10; 1 snail per replicate). n
Significant at Po 0.05.
3.5. Detection of apoptosis by flow cytometry The flow cytometric analysis showed that the percentage of apoptosis increased in all experimental groups when compared to control group post 7 days of herbicide exposure and/or S. mansoni infection. The percentage of apoptosis was 79.1%, 42.8% and 88.92% for treated, infected and treated-infected groups, respectively, compared with 2.5% for control group (Fig. 2).
4. Discussion
Fig. 1. Photomicrograph showing DNA single strand breaks (comet assay) in hemocytes of B. alexandrina snails exposed to Roundup (herbicide) for 24 h. (A) Undamaged, (B) moderately damaged and (C) strongly damaged.
breaks ‘‘SSBs’’ of B. alexandrina hemocytes (Fig. 1). Roundup treatment combined with the infection led to a higher DNA damage than treated with Roundup or infected group alone. The percentages of damaged spots were 21%, 12% and 25% for treated, infected and treated-infected group, respectively, compared to 4% for control group.
The obtained results demonstrated that short-term exposure of B. alexandrina snails to Roundup herbicide in sublethal concentration induced rapid changes in hemocytes responses. Changes in numbers, types and behavior of hemocytes in molluscs experimentally challenged by foreign materials are well documented (Amen et al., 1991; Russo and Lagadic, 2000, 2004). In the present work, the obtained results indicated that the B. alexandrina hemocytes are classified according to cell size and shape into three cell types, designated as small round, hyalinocytes and granular spreading hemocytes. The three reported circulating hemocytes are in agreement with the results of Matricon-Gondran and Letocart (1999), who identified three subpopulations of hemocytes in hemolymph of B. glabrata snails based on their size and ultrastructure aspects. Also, using optical microscopy, MartinsSouza et al. (2006) identified three circulating hemocytes subsets in Biomphalaria species. Recently, cytometric analysis carried out by Martins-Souza et al. (2009) revealed that B. glabrata snails and two strains of B. tenagophila have three major circulating hemocytes subsets, referred to as small, medium and large hemocytes. As reported previously, the round small cells have low immunological competence whereas spreading hemocytes displayed immunological activities (Van der Knaap et al., 1993). Exposure of B. alexandrina snails to Roundup herbicide in sublethal concentration led to significant increase in total hemocytes counts during 7 days of exposure; consequently, the spreading hemocytes were also increased during the whole experimental period. Similar results were obtained by Russo and Lagadic (2000), who stated that dramatic increase of the total number of hemocytes in L. palustris exposed to atrazin herbicide was recorded. In addition, Russo and Lagadic (2004) reported that atrazin was responsible for a significant increase in the number of L. stagnalis hemocytes, mainly granulocytes ‘‘spreading’’. The elevation of total hemocytes is the most commonly observed response in different molluscan species exposed to different stressors. The increase of hemocytes density in infected snails after 1 day of exposure may be resulted from resident and/or newly formed hemocytes from existing pools into connective tissue or in amoebocyts producing organ (Sminia and Van der Knaap, 1986). The obtained results in the present investigation also showed that S. mansoni infection led to
758
A.H. Mohamed / Ecotoxicology and Environmental Safety 74 (2011) 754–760
0
0
40
80 120 Channels
10 20 Channels
0
160
30
0
10
10
20 30 40 Channels
20 30 Channels
50
40
50
Fig. 2. Flow cytometric analysis of B. alexandrina hemocytes exposed to 10 mg/L of Roundup and/or S. mansoni infection 7 days post-exposure.
significant reduction in total hemocytes count after 6 h of exposure. This very rapid decrease may be due to migration of hemocytes out of hemolymph circulation to participate in the encapsulation of the parasite or take part in the repair of tissue damage caused by the parasite (Loker et al., 1982). Similar results were obtained by Martins-Souza et al. (2003), who reported that there was a significant reduction in the number of circulating cells in all studied Biomphalaria snails species after 5 h of infection by S. mansoni. Phagocytosis is a response helpful to assess the immunological impact of environmental pollutants (Galloway and Depledge, 2001). In the present investigation, the obtained results revealed that the phagocytic activity of B. alexandrina hemocytes was increased during 7 days of exposure. Sublethal concentration (10 mg/L) of herbicide Roundup may lead to cytotoxic effects. Matricon-Gondran and Letocart (1999) explained that these cytotoxic effects may be due to the changes in the sensitivity of intercellular adhesion molecules involved in the ingestion of foreign materials, resulting in increase of phagocytic activity. Phagocytic activity of mussel hemocytes was stimulated as a result of exposure to organic compounds at short-term exposure (Pipe et al., 1999; Fournier et al., 2002). Also, Burlando et al. (2002) explained that hemocyte intercellular membrane fusion events were increased, under the effect of experimental material, leading to activation of the bivalve hemocyte’s function. Moreover, Canesi et al. (2007) reported that phagocytic activity of Mytilius hemocytes was enhanced by estrogenic chemicals. In the present results, the decrease of phagocytic activity of S. mansoni-infected snails at 3 and 7 days post-exposure may be due to immunorecognition processes that prevent host immune attack (Hora´k and Van der Knaap, 1997). Noda and Loker (1989) suggested that active phagocytic cells had migrated out from circulation to infection site that led to statistically lower phagocytic activity of B. glabrata hemocytes. Russo and Lagadic (2000) reported that L. palustris is not able to eliminate the parasite by encapsulation or phagocytosis. Snail’s hemocytes were captured by the parasite to build an envelope. Martins-Souza et al. (2009) referred the decreased phagocytic activity of infected Biomphalaria species hemocytes to the significant decrease in the number of medium and large cell.
The obtained results indicated that using comet assay and flow cytometric analysis, either herbicide treatment or S. mansoni infection or both together led to hemocyte’s DNA damage. Some materials are known to be able to interact directly with molecular components of the cytoskeleton (Bellomo et al., 1990; Fagotti et al., 1996). Russo et al. (2007, 2009) stated that toxicants trigger a number of effects on Lymnea snails hemocytes, such as lysosomal fragility or apoptosis. Genotoxicity of glyphosate to human lymphocytes was previously evaluated by Mladinic et al. (2009) using comet assay, which is in agreement with the current results. Their results revealed that glyphosate increased tail length and intensity when compared to the control. On the other hand, the toxicity of Roundup may be attributed to its surfactant (Mann et al., 2009). Nevertheless, it has been demonstrated through several studies in invertebrates and humans that glyphosate formulation (Roundup) and the active ingredient alone caused similar deleterious effects (Gasnier et al., 2009; Benamu et al., 2010). Moreover, Achiorno et al. (2008) and Manas et al. (2009) evaluated the genotoxicity of formulation containing glyphosate using comet assay. They stated that the level of DNA damage in human lymphocytes showed a significant increment. Furthermore, an investigation by Poletta et al. (2009) confirmed that comet assay was useful tool in determining genotoxicity of Roundup on DNA of Caiman latirostris erythrocytes. Benachour and Seralini (2009) suggested that death of human cells as a result of Roundup treatment may be through inhibition of the mitochondrial succinate dehydrogenase and apoptosis may be via activation of enzymatic caspases. Binelli et al. (2009) reported that using comet assay it was found that antimicrobial trimethorim increased DNA damage in mussel hemocytes. It is clearly observed from the obtained data that B. alexandrina snails found to be more affected by Roundup treatment than S. mansoni infection. Thus, infected snails have effective immune responses but failed to express successful defense when interacted with Roundup sublethal toxicity. It can be concluded that herbicides as environmental pollutants affected B. alexandrina snails and its interactions with S. mansoni infection. Thus, such data provide information for aquatic management and for establishing limits of
A.H. Mohamed / Ecotoxicology and Environmental Safety 74 (2011) 754–760
use of herbicides near aquatic ecosystems. In addition, comet assay and flow cytometry were able to detect adverse environmental conditions, proving to be a very adequate short-term test and should be included as biomarkers to be used in the monitoring of aquatic environments.
References Abdul-Salam, J.M., Michelson, E.H., 1980. Biomphalaria glabrata amoebocytes, effect of Schistosoma mansoni infection on in vitro phagocytosis. J. Invertebr. Pathol. 35, 241–248. Achiorno, C.L., de Villalobos, C., Ferrari, L., 2008. Toxicity of the herbicide glyphosate to Chordodes nobilii (Gordiida, Nematomorpha). Chemosphere 71, 1817–1822. Amen, R.I., Tijnagel, J.M.G., Van der Knaap, W.P., Meuleman, E.A., de Lange-de Klerk, E.S.M., Sminia, T., 1991. Effects of Trichobilharzia ocellata on hemocytes of Lymnaea stagnalis. Dev. Comp. Immunol. 15, 105–115. Anderson, R.M., Mercer, J.G., Wilson, R.R., Carter, N.P., 1982. Transmission of Schistosoma mansoni from man to snail, experimental studies of miracidial survival and infectivity in relation to larval age, water temperature, host size and age. Parasitology 85, 339–360. Bellomo, G., Mirabelli, F., Richelmi, P., Malorni, W., Iosi, F., Orrenius, S., 1990. The cytoskeleton as in quinine toxicity. Free Rad. Res. Commun. 8, 391–399. Benachour, N., Seralini, G.E., 2009. Glyphosate formulations induce apoptosis and necrosis in human umbilical, embryonic, and placental cells. Chem. Res. Toxicol. 22, 97–105. Benamu, M.A., Schneider, M.I., Sa´nchez, N.E., 2010. Effects of the herbicide glyphosate on biological attributes of Alpaida veniliae (Araneae, Araneidae), in laboratory. Chemosphere 78, 871–876. Binelli, A., Parolini, M., Cogni, D., Pedriali, A., Provini, A., 2009. A multi-biomarker assessment of the impact of the antibacterial trimethoprim on the non-target organism Zebra mussel (Dreissena polymorpha). Comp. Biochem. Physiol. 150, 329–336. Bolognesi, C., Bonatti, S., Degan, P., Gallerani, E., Peluso, M., Rabboni, R., 1997. Genotoxicity activity of glyphosate and its technical formulation Roundup. J. Agric. Food Chem. 45, 1957–1962. Burlando, B., Marchi, B., Panfili, I., Viarengo, A., 2002. Essential role of Ca2 + dependent phospholipase A2 in estradiol-induced lysosome activation. Am. J. Physiol. Cell Physiol. 283, C1461–C1468. Canesi, L., Lorusso, L.C., Ciacci, C., Betti, M., Rocchi, M., Pojana, G., Marcomini, A., 2007. Immunomodulation of Mytilus hemocytes by individual estrogenic chemicals and environmentally relevant mixtures of estrogens, In vitro and in vivo studies. Aquat. Toxicol. 81, 36–44. Cavas, T., Konen, S., 2007. Detection of cytogenetic and DNA damage in peripheral erythrocytes of goldfish (Carassius auratus) exposed to a glyphosate formulation using the micronucleus test and the comet assay. Mutagenesis 22, 263–268. Cohen, J.J., Al- Rubeai, M., 1995. Apoptosis-targeted therapies the next big thing in biotechnology? Trends Biotechnol. 13, 281–283. Dean, P.N., Jett, J.H., 1974. Mathematical analysis of DNA distributions derived from flow microfluorometry. J. Cell. Biol. 60, 523. Fagotti, A., Di Rosa, I., Simoncelli, F., Pipe, R., 1996. The effects of copper on actin and fibronectin organization in Mytilus galloprovincialis haemocytes. Dev. Comp. Immunol. 20, 383–391. Fournier, M., Cyr, D., Blakley, B., Boermans, H., Brousseau, P., 2000. Phagocytosis as a biomarker of immunotoxicity in wildlife species exposed to environmental xenobiotics. Am. Zool. 40, 412–420. Fournier, M., Pellerin, J., Lebeuf, M., Brousseau, P., Morin, Y., Cyr, D., 2002. Effects of exposure of Mya arenaria and Mactromeris polynyma to contaminated marine sediments on phagocytic activity of hemocytes. Aquat. Toxicol. 59, 83–92. Galloway, T.S., Depledge, M.H., 2001. Immunotoxicity in invertebrates, Measurement and ecotoxicological relevance. Ecotoxicology 10, 5–23. Gasnier, C., Dumont, C., Benachour, N., Clair, E.J., Chagnon, M.C., Se´ralini, G.E., 2009. Glyphosate-based herbicides are toxic and endocrine disruptors in human cell lines. Toxicology 262, 184–189. Grazeffe, V.S., Tallarico, L.F., Pinheiro, A., Kawano, T., Suzuki, M.F., Okazaki, K., Pereira, C.A.B., Nakano, E., 2008. Establishment of the comet assay in the freshwater snail Biomphalaria glabrata (Say, 1818). Mutat. Res. 654, 58–63. Glusczak, L., Miron, D.S., Crestani, M., Fonseca, M.B., Pedron, F.A., Duarte, M.F., Vieira, V.L.P., 2006. Effect of glyphosate herbicide on acetylcholinesterase activity and metabolic and hematological parameters in piava (Leporinus obtusidens). Ecotoxicol. Environ. Saf. 65, 237–241. Hora´k, P., Van der Knaap, W.P.W., 1997. Lectins in snails-trematode immune interaction, a review. Folia Parasitol. (Praha) 44, 161–172. Lee, R.F., Steinert, S., 2003. Use of the single cell gel electrophoresis/comet assay for detecting DNA damage in aquatic (marine and freshwater) animals. Mutat. Res. 544, 43–64. Lioi, M.B., Scarfi, M.R., Santoro, R., Barbieri, O.S., Salvemini, F., Di Berardino, D., Ursini, M.V., 1998. Cytogenetic damage and induction of pro-oxidant state in human lymphocytes exposed in vitro to glyphosate, vinclozolin, atrazine and DPX-E9636. Environ. Mol. Mutagen. 32, 39–46. Livingstone, D.R., Chipman, J.K., Lowe, D.M., Minier, C., Mitchelmore, C.L., Moore, M.N., Peters, L.D., Pipe, R.K., 2000. Development of biomarkers to detect the effects of organic pollution on aquatic invertebrates: recent molecular,
759
genotoxic, cellular and immunological studies on the common mussel (Mytilus edulis L.) and other mytilids. Int. J. Environ. Pollut. 13, 56–91. Little, T.J., Hultmark, D., Read, A.F., 2005. Invertebrate immunity and the limits of mechanistic immunology. Nat. Immunol. 6, 651–654. Loker, E.S., Bayn, C.J., Buckley, P.M., Kruse, K.T., 1982. Ultrastructure of encapsulation of Schistosoma mansoni mother sporocystes by hemocytes of juneniles of the 10-R2 strain of Biomphalaria glabrate. J. Parasitol. 68, 84–94. Manas, F., Peralta, L., Raviolo, J., Garcia Ovando, H., Weyers, A., Ugnia, L., Gonzalez, Cid, M, Larripa, I., Gorla, N., 2009. Genotoxicity of AMPA, the environmental metabolite of glyphosate, assessed by the comet assay and cytogenetic tests. Ecotoxicol. Environ. Saf. 72, 834–837. Mann, R.M., Hyne, R.V., Choung, C.B., Wilson, S.P., 2009. Amphibians and agricultural chemicals, review of the risks in a complex environment. Environ. Pollut. 157, 2903–2927. Matricon-Gondran, M., Letocart, M., 1999. Internal defenses of the snail Biomphalaria glabrata I. Characterization of hemocytes and fixed phagocytosis. J. Invertebr. Pathol. 74, 224–234. Martins-Souza, R.L., Pereira, C.A., Coelho, P.M.Z., Negrao-Correa, D., 2003. Silica treatment increases the susceptibility of the Cabo Frio strain of Biomphalaria tenagophila to Schistosoma mansoni infection but does alter the natural resistance of the Taim strain. Parasitol. Res. 91, 500–507. Martins-Souza, R.L., Pereira, C.A., Coelho, P.M.Z., Martins Filho, O.A., Negrao-Correa, D., 2009. Flow cytometry analysis of the circulating haemocytes from Biomphalaria glabrata and Biomphalaria tenagophila following Schistosoma mansoni infection. Parasitology 136, 67–76. Martins-Souza, R.L., Pereira, C.A., Martins Filho, O.A., Coelho, P.M.Z., Correa Jr., A., Negrao-Correa, D., 2006. Differential lectin labeling of circulating hemocytes from Biomphalaria glabrata and Biomphalaria tenagophila resistant or susceptible to Schistosoma mansoni infection. Mem. Inst. Oswaldo Cruz 101, 185–192. Mladinic, M., Berend, S., Vrdoljak, A.L., Kopjar, N., Radic, B., Zeljezic, D., 2009. Evaluation of genome damage and its relation to oxidative stress induced by glyphosate in human lymphocytes in vitro. Environ. Mol. Mutagen. 50, 800–807. Negrao-Correa, D., Pereira, C.A., Rosa, F.M., Martins-Souza, R.L., Andrade, Z.A., Coelho, P.M.Z., 2007. Molluscan response to parasite, Biomphalaria and Schistosoma mansoni interaction. Invertebr. Sur. J. 4, 101–111. Noda, S., Loker, E.S., 1989. Effects of infection with Echinostoma paraensei on the circulating haemocytes population of the host snail Biomphalaria glabrata. Parasitology 98, 1–10. Osman, G.Y., Mohamed, A.H., Mohamed, A.M., Al-Qormuti, S.A., 2008. Effect of Roundup herbicide on biological activity of Biomphalaria alexandrina snails infected with Schistosoma mansoni. Mansoura J. Biol. 35, 147–167. Paraense, W.L., 2001. The schistosome vectors in the Americas. Mem. Inst. Oswaldo Cruz 96, 7–16. Pipe, R.K., Coles, J.A., Carissan, F.M., Ramanathan, K., 1999. Copper induced immunomodulation in the marine mussel Mytilus edulis. Aquat. Toxicol. 46, 43–54. Peluso, M., Munnia, A., Bolognesi, C., Parodi, S., 1998. 32p-Postlabeling detection of DNA adducts in mice treated with the herbicide Roundup. Environ. Mol. Mutagen. 31, 55–59. Poletta, G.L., Larriera, A., Kleinsorge, E., Mudry, M.D., 2009. Genotoxicity of the herbicide formulation Roundup (glyphosate) in broad-snouted caiman (Caiman latirostris) evidenced by the comet assay and the micronucleus test. Mutat. Res. 672, 95–102. Russo, J., Lagadic, L., 2000. Effects of parasitism and pesticide exposure on characteristics and functions of hemocyte populations in the freshwater snail Lymnaea palustris (Gastropoda, Pulmonata). Cell Biol. Toxicol. 16, 15–30. Russo, J., Lagadic, L., 2004. Effects of environmental concentrations of atrazine on hemocyte density and phagocytic activity in the pond snail Lymnaea stagnalis (Gastropoda, Pulmonata). Environ. Pollut. 127, 303–311. Russo, J., Lefeuvre-Orfila, L., Lagadic, L., 2007. Hemocyte-specific responses to the peroxidizing herbicide fomesafen in the pond snail Lymnaea stagnalis (Gastropoda, Pulmonata). Environ. Pollut. 146, 420–427. Russo, J., Madec, L., Brehelin, M., 2009. Haemocyte lysosomal fragility facing an environmental reality, A toxicological perspective with atrazine and Lymnaea stagnalis (Gastropoda, Pulmonata) as a test case. Ecotoxicol. Environ. Safe. 72, 1719–1726. Schneider, M., Sanchez, N., Pineda, S., Chi, H., Ronco, A., 2009. Impact of glyphosate on the development, fertility and demography of Chrysoperla externa (Neuroptera: Chrysopidae): ecological approach. Chemosphere 76, 1451–1455. Singh, N.P., Mc Coy, M.T., Tice, R.R., Schnider, E.L., 1988. A simple technique for quantification of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184–191. Sminia, T., 1972. Structure and function of blood and connective tissue cells of the fresh water pulmonate Lymnaea stagnalis studied by electron microscopy and enzyme histochemistry. Z. Zellforsch. 130, 497–526. Sminia, T., Van der Knaap, W.P.W., 1986. Immunorecognition in invertebrates with special reference to molluscs. In, Immunity in Invertebrates. In: Brehelin, M. (Ed.), Springer, Berlin/Heidelberg 112–124. Snieszko, S.F., 1974. The effects of environmental stress on outbreaks of infectious diseases of fishes. J. Fish Biol. 6, 197–208. Steinmann, P., Keiser, J., Bos, R., Tanner, M., Utzinger, J., 2006. Schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk. Lancet Infect. Dis. 6, 411–425. Sokal, R.R., Rohlf, F.J. (Eds.), 1981. Biometry, 2nd ed. W. H. Freeman and Co., San Francisco.
760
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Tsui, M.T.K., Chu, L.M., 2003. Aquatic toxicity of glyphosate-based formulations, comparison between different organisms and the effects of environmental factors. Chemosphere 52, 1189–1197. USDA 1984. Herbicide background statement, Glyphosate. In: ‘‘Pesticide Background Statement’’. 1, Herbicides USDA forest Service, Agricultural Handbook No 633, G-1-72. Van der Knaap, W..P.W., Adema, C.M., Sminia, T., 1993. Invertebrate blood cells, morphological and functional aspects of the haemocytes in the pond snail Lymnaea stagnalis. Comp. Haematol. Int. 3, 20–26.
Williams, G.M., Kroes, R., Monrot, I.C., 2000. Safety evaluation and risk assessment of the herbicide Roundup and its active ingredient, glyphosate, for humans. Regul. Toxicol. Pharmacol. 31, 117–165. Wong, S., Fournier, M., Coderre, D., Banska, W., Krzstyniak, K., 1992. Environmental immunotoxicology. In: Peakall, D. (Ed.), Animals Biomarkers as Pollution Indicators. Chapman and Hall, London, pp. 167–189. Yoshino, T.P., Davids, B.J., Castillo, M.G., Johnston, L.A., 1998. Invertebrate cellular adhesion receptors, their structure and function as primordial recognition molecules in the immune system. Trends Comp. Biochem. Physiol. 5, 49–65.
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Sublethal toxicity of Roundup to immunological and molecular aspects of Biomphalaria alexandrina to Schistosoma mansoni infection Azza H. Mohamed n Department of Zoology, Faculty of Science, Menoufia University, Shebin El-Kom, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 August 2010 Received in revised form 24 October 2010 Accepted 25 October 2010 Available online 3 December 2010
The present study was performed to elucidate the cellular mechanisms of Biomphalaria alexandrina snails hemocytes against sublethal concentration (10 mg/L) of herbicide Roundup (48% Glyphosate) and/or Schistosoma mansoni infection during 7 days of exposure. Obtained results indicated that herbicide treatment and/or infection led to significant increase (P o 0.05) in total hemocytes count during exposure period. Examination of hemocytes monolayers resulted in observation of 3 morphologically different cell types, round small, hyalinocytes and spreading hemocytes. Spreading hemocytes are the dominant, more responsive and highly phagocytic cell type in all experimental groups. Moreover, the exposure to herbicide, infection or both together led to a significant increase (P o0.05) of in vitro phagocytic activity against yeast cells during 7 days of exposure. In addition, flow cytometric analysis of cell cycle and comet assay, resulted in DNA damage in B. alexandrina hemocytes exposed to herbicide and/or S. mansoni infection when compared to control group. The immunological responses as well as molecular aspects in B. alexandrina snails have been proposed as biomarkers of exposure to environmental pollutants. & 2010 Elsevier Inc. All rights reserved.
Keywords: Roundup Biomphalaria alexandrina Schistosoma mansoni Hemocytes Phagocytosis Comet assay
1. Introduction Particular attention has been directed to immunological changes induced by environmental pollution in aquatic invertebrates (Livingstone et al., 2000). The defense mechanisms and immunological responses which consist of the immune system have been considered as biomarkers of pollution in aquatic invertebrates (Galloway and Depledge, 2001). Despite the lack of an adaptive immune system, invertebrates are able to survive among potential pathogens and respond to infection by activation of various defense mechanisms (Little et al., 2005). The immune system is likely to be one of the most sensitive physiological systems to pollutants (Fournier et al., 2000). Pollutants can interact with immune system components and interfere with protection function that induce immune suppression and decrease of disease resistance (Wong et al., 1992). Schistosomiasis is one of the major communicable diseases with socio-economic importance in the developing world. It was estimated that 800 million people in 74 countries are at risk while more than 200 million were infected (Steinmann et al., 2006). Biomphalaria snails have a great medical importance as intermediate hosts of S. mansoni (Paraense, 2001). In this respect, interactions between Biomphalaria snails and schistosomes have received much attention. Defense system of freshwater snails mainly depends on hemocytes, which are the
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circulating cells involved in mediating internal defense and immune functions (Yoshino et al., 1998). Phagocytosis is one of the various functions of hemocytes, which is a non-specific immune mechanism against non-self materials (Galloway and Depledge, 2001; NegraoCorrea et al., 2007). The response patterns, density and functions, of hemocytes can be affected by xenobiotics and parasites (Livingstone et al., 2000). Furthermore, the development of an infectious disease results from an imbalance between the host and the pathogen due to external factors, like pollutants, and/or internal factors, like susceptibility of the host (Snieszko, 1974). Herbicides are distinctive group of pesticides and are considered as selective weed killers. Roundup is a commercial herbicide with active compound glyphosate used in a broad spectrum in agricultural applications for weed control (USDA, 1984; Williams et al., 2000; Cavas and Konen, 2007). Due to its high water solubility and extensive usage, especially in shallow water systems, the exposure of non-target aquatic organisms to this herbicide is a concern (Tsui and Chu, 2003). Results of previous studies showed that glyphosate may have effect on plants, fishes, amphibians, arthropods and snails by causing physiological, immunological and biochemical alterations (Glusczak et al., 2006; Achiorno et al., 2008; Schneider et al., 2009; Benamu et al., 2010). Comet assay is becoming a major tool for environmental biomonitoring (Grazeffe et al., 2008) because of its sensitivity and determination of DNA strand breaks in individual cell (Lee and Steinert, 2003). Some studies reported that glyphosate treatment of human lymphocytes in vitro resulted in increased chromatid exchange (Bolognesi et al., 1997), chromosomal aberrations and
A.H. Mohamed / Ecotoxicology and Environmental Safety 74 (2011) 754–760
indicators of oxidative stress (Lioi et al., 1998). Furthermore, Roundup proved to increase DNA adducts in mice (Peluso et al., 1998). Recently, Mladinic et al. (2009) used comet assay to measure glyphosate impact on DNA of human lymphocytes and the results revealed that glyphosate increased tail intensity. It is hypothesized that immunological biomarkers in snails e.g. Biomphalaria would improve their use as diagnostic organisms in biomonitoring. Therefore, the present study was undertaken to evaluate the immunological and molecular responses of B. alexandrina snails to the effect of sublethal concentration of Roundup herbicide treatment and/or S. mansoni infection. 2. Materials and methods 2.1. Experimental animals and infection Laboratory bred Biomphalaria alexandrina snails and Schistosoma mansoni ova were obtained from The Schistosome Biological Supply Center (SBSC), Theodor Bilharz Research Institute, Giza, Egypt. The adult snails (8–10 mm shell diameter) were maintained in 5 L capacity plastic aquaria under lab conditions at a density of 50 individual per tank. Water temperature was maintained at 25 72 1C. The snails were fed dry lettuce leaves daily. For snail infection B. alexandrina snails were individually exposed to 7–9 freshly hatched miracidia from S. mansoni ova in glass test tubes for 2 h (Anderson et al., 1982). 2.2. Experimental material Roundup herbicide was used in the liquid commercial form produced by Monsanto Agricultural Company, USA. It consists of 48% (480 g/L active ingredient ‘‘a.i.’’) of glyphosate (N-phosphonomethyl glycine). Sublethal concentration of Roundup (10 mg/L), equivalent to 0.02 mg/L a.i. glyphosate was prepared from stock solution (1000 mg/L). Fifty snails were placed in 5 L aerated plastic aquaria containing the used concentration according to Osman et al. (2008). 2.3. Experimental bioassays Four treatments were employed in this study, (50 snails were used in each treatment): 1. 2. 3. 4.
Normal control group. S. mansoni-infected group. 10 mg/L Roundup-treated group. Treated-infected group.
The evaluation of sublethal effect of Roundup and/or S. mansoni was done by: (1) total and differential hemocyte counts, (2) in vitro phagocytosis, (3) comet assay and (4) flow cytometric analysis of cell cycle. Each ones are detailed below. 2.3.1. Total and differential hemocyte counts The hemolymph was collected from the four experimental groups as described by Sminia (1972). In brief, the headfoot was touched by a Pasteur pipette; as a result the snail was forced to retract deeply into its shell and extruded hemolymph. From each individual snail, ca.75 mL hemolymph was obtained and collected using a micropipette. Hemolymph samples were collected at intervals of 6 h, 1, 3 and 7 days post-exposure from the four experimental groups. The number of total hemocytes was counted in 10 snails individually from each experimental group using haemocytometer. For differential hemocytes count, hemolymph samples were placed onto glass slides and allowed to settle in moist chamber. The hemocytes
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monolayers were stained after methanol fixation with Giemsa’s stain. Data were expressed as mean values of a proportion of each cell population from the original 100 cells. 2.3.2. In vitro phagocytosis The suspension of freshly prepared yeast cells was diluted in PBS to get the ratio of 10,000 cell/mL for use and phagocytosis was carried out as described by AbdulSalam and Michelson (1980). Freshly collected hemolymph from 10 individual snails was overlaid with an equal volume of yeast suspension on a clean glass slide. Reaction mixtures were incubated in moist chamber for 60 min. The phagocytic reaction was stopped using absolute methanol after washing with PBS (pH 7.4). For each slide 100 cells was examined and phagocytic activity was expressed as the mean values 7 SE of positive phagocytized hemocyte during 7 days of exposure. 2.3.3. Comet assay Comet assay was performed for measuring DNA damage after 24 h postherbicide treatment and/or S. mansoni infection by single cell gel assay which permits the detection of single stranded DNA breaks (SSBs) in one cell according to Singh et al. (1988) and Grazeffe et al. (2008). For each experimental group, collected hemolymph from 5 individual snails was pooled in 1.5 Eppendorf tube. Three microscope slides were covered with 1.5% normal melting agarose dissolved in phosphate buffered saline (PBS) free of Ca2 + and Mg2 + and maintained overnight at room temperature (257 2 1C). A volume of 100 mL of pooled hemolymph was dissolved in 300 mL of 0.7% of low-melting agarose dissolved in PBS Ca2 + and Mg2 + free and placed on the first gel layer at 37 1C. After solidification at 4 1C for 10 min, the slides were immersed in a jar containing cold lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% sodium sarcozinate, 1% Triton X-100 and 10% dimethyl sulfoxide (DMSO)), pH 10 at 4 1C for 2 h. After lysis, the slides were placed in horizontal electrophoresis box. The unit was filled with a fresh alkaline electrophoresis buffer (300 mM NaOH and 1 mM EDTA, pH 13) to a level of 0.25 cm above the slides. The cells were exposed to alkali for 15 min to allow DNA unwinding and expression of alkali-labile sites. To electrophoresis the DNA, an electric current of 25 V (0.86 V/cm) and 300 mA was applied for 20 min. Alkali and electrophoresis treatments were performed in an ice bath. All these steps were conducted under dim light to prevent the occurrence of additional DNA damage. After electrophoresis, the slides were placed horizontally and Tris buffer (0.4 M Tris, pH 7.5) was added to neutralize the excess alkali. Finally, slides were fixed with absolute ethanol for 10 min and stained with 100 mL ethidium bromide (20 mg/mL). From each slide 100 cells were examined visually and the percentage of damaged hemocytes DNA was scored (Lee and Steinert, 2003). Examination of the slides was done under fluorescence microscope (Olympus BX 60, Japan) equipped with an excitation filter 510 nm and barrier filter of 590 nm (1000 magnification). All chemicals were purchased from Sigma. 2.3.4. Flow cytometric analysis of cell cycle Flow cytometric analysis was carried out for detecting apoptosis of B. alexandrina hemocytes isolated from pooled hemolymph samples from 15 snail in each experimental group 7 days post-herbicide treatment and/or S. mansoni infection. The flow cytometer used is FACS calibur flow cytometer (Becton Dickinson, Sunnyvale, CA, USA) equipped with a compact aircooked low power 15 mW argon ion laser beam (488 nm). The average number of evaluated nuclei per specimen was 20,000 and the number of nuclei scanned was 120 per second. DNA histogram derived from flow cytometry was obtained with a computer program for Dean and Jett mathematical analysis (Dean and Jett, 1974). Data analysis was conducted using DNA analysis program MODFIT (verity software house, ME 04086 USA, version, 2.0). Apoptosis was measured by using the sub G1 peak staining with propidium iodide (Cohen and Al-Rubeai, 1995). 2.3.5. Statistical analysis Total and differential hemocyte counts and phagocytosis data are presented as mean 7standard error. The significance of difference between the means was calculated according to the way analysis (ANOVA) followed by Student’s t-test (Sokal and Rohlf, 1981). Result was considered statistically significant at P o 0.05.
Table 1 Effect of Roundup on total hemocytes count/ml in hemolymph of non-infected and S. mansoni-infected B. alexandrina snails during 7 days of exposure. Exposure period
6h 1 day 3 days 7 days
Experimental groups Control
Herbicide treated
Infected
Treated- infected
13.98 7 1.17 11.46 7 0.7 11.68 7 1.3 14.25 7 1.2
15.3 7 0.98 40.6 7 2.1n 39.3 7 2.2 n 39.8 7 2.7 n
3.6 7 0.5 n 15.9 7 1 n 8.26 7 0.5 4.7 7 0.4 n
8.2 7 0.3n 12.9 7 1 40.87 1.4 37.9 7 1.6
Data are presented as mean/104 7standard error (n¼ 10; 1 snail per replicate). n
Significant at Po 0.05.
n n
62.57 2.6 61.37 1.3 567 0.5 78.37 0.7n
Significant at Po 0.05.
44.25 7 2.3 26.37 0.02 387 0.7n 55.37 1.9 n
13.75 70.5 12.7 70.6 17 71.1 14.3 71.2n 49 72n 53.3 72.2 n 44.7 72.3n 28.3 70.7 n
31.7 7 1.4 33.4 7 1.7 33.7 7 1.8 51.3 7 1.8 58.7 7 0.4 567 0.8 547 1.5 44.7 7 1.5 19.8 7 0.9 19.4 7 1.1 237 0.8 29.7 7 0.4
21.5 7 0.7 24.6 7 0.7 237 0.6 25.6 7 0.9
19.5 7 1.2 15 7 1 21.7 7 0.7 20.3 7 0.2
Rs Hy Rs
Sp
Herbicide treated Control
Hy
n
Rs Sp
Infected
Hy
n
n
Using comet assay, the obtained results indicated that herbicide exposure and/or S. mansoni infection caused DNA single strand
Data are presented as mean 7standard error (n¼ 10; 1 snail per replicate). (Rs) Round small Hemocytes, (Hy) Hyalinocytes, (Sp) Granulocytes.
3.4. Comet assay
Experimental groups
The data illustrated in Table 3 indicated the effect of Roundup treatment and/or S. mansoni infection on phagocytic activity (in vitro) of B. alexandrina hemocytes against yeast cells during 7 days of exposure. The recorded data indicated that the exposure to herbicide, infection or both together led to a significant increase of phagocytic activity after 6 h post-exposure. The phagocytic activity was significantly higher after 3 days of exposure in snails exposed only to Roundup treatment. The mean values were 45.770.79, 30.77 0.5 and 40 70.9 for treated, infected and treated-infected groups, respectively, compared to 37.9870.8 for control group (Table 3). At the 7th day post-exposure, similar significant elevation level (Po0.05) was recorded in both treated and treated-infected groups while the elevation level in infected group was not significant.
Exposure period
3.3. Phagocytic activity
Table 2 Effect of Roundup on differential hemocytes count of non-infected and S. mansoni-infected B. alexandrina snails during 7 days of exposure.
Hemolymph samples from B. alexandrina snails contained three morphologically distinct types of hemocytes, designated as round small, hyalinocytes and spreading hemocytes. Round small hemocytes, measuring 8–10 mm in diameter; having a high cytoplasmnucleus ratio; few cytoplasmic granules and did not adhere to glass or emit pseudopodia. The spreading hemocytes, measuring 20–25 mm in diameter, had plentiful cytoplasm with numerous pseudopodia, an irregular nucleus and adhered to glass. Hyalinocytes were morphologically intermediate between round and spreading hemocytes, measuring about 12–15 mm in diameter; and can form few and short pseudopods. Spreading hemocytes are the dominant, more responsive and highly phagocytic cell type in all groups. It was significantly increased (Po0.05) in treated and infected groups after 6 h of exposure. After 1 and 3 days of exposure, the significant increase was continued in treated and infected groups when compared to control group. The mean number of spreading hemocytes was 53.3 72.2, 44.7 72.3 for treated group and 6170.4, 4570.5 for infected group compared to 24.6 70.7, 2370.6 for control group at 1 and 3 days postexposure. After 7 days of exposure, there was a significant decrease in spreading hemocytes of treated-infected group. The mean number was 10.370.9 and 25.6 70.9 for treated-infected and control groups (Table 2).
Sp
3.2. Differential hemocytes count
Rs
Treated infected
Hy
The effect of Roundup treatment and S. mansoni infection on total hemocytes count of B. alexandrina snails during 7 days of exposure is presented in Table 1. The presented data indicated that S. mansoni infection led to significant reduction (Po0.05) in total hemocytes count after 6 h of exposure. However, only herbicide and treatment combined with infection led to non-significant changes in counts at the same period. The total count in treated group was 40.672.1 compared to 11.4670.7, 15.9 71 and 12.971 for control, infected and treated-infected groups, respectively, one day post-exposure. The significant increase (P o0.05) continued till the end of the experiment (7 days) in both treated and treated-infected groups. However, in only infected group, there was a significant decrease by the 7th day post-exposure (Table 1).
14.25 7 1.5 17.7 7 1.7 24.3 7 0.9 11.3 7 0.6n
Sp
3.1. Total hemocytes count
45.75 7 3.1n 61 7 0.4n 45 7 0.5n 30.3 7 1.1
3. Results
23.25 7 1.1 217 1.2 197 0.98 10.37 0.9n
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6h 1 day 3 days 7 days
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Table 3 Effect of Roundup on phagocytic activity of non-infected and S. mansoni-infected B. alexandrina snails during 7 days of exposure. Exposure period
Experimental groups Control
6h 1 day 3 days 7 days
36.5 71.2 33 70.7 37.98 70.8 35.8 71
Herbicide treated 45.25 7 1.4 33.3 7 1 45.7 7 0.79 n 467 1.6 n n
Infected
Treated infected
53 71.3 51.7 71.28 30.7 70.5 35.7 71
n n
58.25 7 1.1 27.3 7 0.5 40 7 0.9 41 7 0.9
n
n
Data are presented as mean 7 standard error (n ¼10; 1 snail per replicate). n
Significant at Po 0.05.
3.5. Detection of apoptosis by flow cytometry The flow cytometric analysis showed that the percentage of apoptosis increased in all experimental groups when compared to control group post 7 days of herbicide exposure and/or S. mansoni infection. The percentage of apoptosis was 79.1%, 42.8% and 88.92% for treated, infected and treated-infected groups, respectively, compared with 2.5% for control group (Fig. 2).
4. Discussion
Fig. 1. Photomicrograph showing DNA single strand breaks (comet assay) in hemocytes of B. alexandrina snails exposed to Roundup (herbicide) for 24 h. (A) Undamaged, (B) moderately damaged and (C) strongly damaged.
breaks ‘‘SSBs’’ of B. alexandrina hemocytes (Fig. 1). Roundup treatment combined with the infection led to a higher DNA damage than treated with Roundup or infected group alone. The percentages of damaged spots were 21%, 12% and 25% for treated, infected and treated-infected group, respectively, compared to 4% for control group.
The obtained results demonstrated that short-term exposure of B. alexandrina snails to Roundup herbicide in sublethal concentration induced rapid changes in hemocytes responses. Changes in numbers, types and behavior of hemocytes in molluscs experimentally challenged by foreign materials are well documented (Amen et al., 1991; Russo and Lagadic, 2000, 2004). In the present work, the obtained results indicated that the B. alexandrina hemocytes are classified according to cell size and shape into three cell types, designated as small round, hyalinocytes and granular spreading hemocytes. The three reported circulating hemocytes are in agreement with the results of Matricon-Gondran and Letocart (1999), who identified three subpopulations of hemocytes in hemolymph of B. glabrata snails based on their size and ultrastructure aspects. Also, using optical microscopy, MartinsSouza et al. (2006) identified three circulating hemocytes subsets in Biomphalaria species. Recently, cytometric analysis carried out by Martins-Souza et al. (2009) revealed that B. glabrata snails and two strains of B. tenagophila have three major circulating hemocytes subsets, referred to as small, medium and large hemocytes. As reported previously, the round small cells have low immunological competence whereas spreading hemocytes displayed immunological activities (Van der Knaap et al., 1993). Exposure of B. alexandrina snails to Roundup herbicide in sublethal concentration led to significant increase in total hemocytes counts during 7 days of exposure; consequently, the spreading hemocytes were also increased during the whole experimental period. Similar results were obtained by Russo and Lagadic (2000), who stated that dramatic increase of the total number of hemocytes in L. palustris exposed to atrazin herbicide was recorded. In addition, Russo and Lagadic (2004) reported that atrazin was responsible for a significant increase in the number of L. stagnalis hemocytes, mainly granulocytes ‘‘spreading’’. The elevation of total hemocytes is the most commonly observed response in different molluscan species exposed to different stressors. The increase of hemocytes density in infected snails after 1 day of exposure may be resulted from resident and/or newly formed hemocytes from existing pools into connective tissue or in amoebocyts producing organ (Sminia and Van der Knaap, 1986). The obtained results in the present investigation also showed that S. mansoni infection led to
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0
0
40
80 120 Channels
10 20 Channels
0
160
30
0
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10
20 30 40 Channels
20 30 Channels
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Fig. 2. Flow cytometric analysis of B. alexandrina hemocytes exposed to 10 mg/L of Roundup and/or S. mansoni infection 7 days post-exposure.
significant reduction in total hemocytes count after 6 h of exposure. This very rapid decrease may be due to migration of hemocytes out of hemolymph circulation to participate in the encapsulation of the parasite or take part in the repair of tissue damage caused by the parasite (Loker et al., 1982). Similar results were obtained by Martins-Souza et al. (2003), who reported that there was a significant reduction in the number of circulating cells in all studied Biomphalaria snails species after 5 h of infection by S. mansoni. Phagocytosis is a response helpful to assess the immunological impact of environmental pollutants (Galloway and Depledge, 2001). In the present investigation, the obtained results revealed that the phagocytic activity of B. alexandrina hemocytes was increased during 7 days of exposure. Sublethal concentration (10 mg/L) of herbicide Roundup may lead to cytotoxic effects. Matricon-Gondran and Letocart (1999) explained that these cytotoxic effects may be due to the changes in the sensitivity of intercellular adhesion molecules involved in the ingestion of foreign materials, resulting in increase of phagocytic activity. Phagocytic activity of mussel hemocytes was stimulated as a result of exposure to organic compounds at short-term exposure (Pipe et al., 1999; Fournier et al., 2002). Also, Burlando et al. (2002) explained that hemocyte intercellular membrane fusion events were increased, under the effect of experimental material, leading to activation of the bivalve hemocyte’s function. Moreover, Canesi et al. (2007) reported that phagocytic activity of Mytilius hemocytes was enhanced by estrogenic chemicals. In the present results, the decrease of phagocytic activity of S. mansoni-infected snails at 3 and 7 days post-exposure may be due to immunorecognition processes that prevent host immune attack (Hora´k and Van der Knaap, 1997). Noda and Loker (1989) suggested that active phagocytic cells had migrated out from circulation to infection site that led to statistically lower phagocytic activity of B. glabrata hemocytes. Russo and Lagadic (2000) reported that L. palustris is not able to eliminate the parasite by encapsulation or phagocytosis. Snail’s hemocytes were captured by the parasite to build an envelope. Martins-Souza et al. (2009) referred the decreased phagocytic activity of infected Biomphalaria species hemocytes to the significant decrease in the number of medium and large cell.
The obtained results indicated that using comet assay and flow cytometric analysis, either herbicide treatment or S. mansoni infection or both together led to hemocyte’s DNA damage. Some materials are known to be able to interact directly with molecular components of the cytoskeleton (Bellomo et al., 1990; Fagotti et al., 1996). Russo et al. (2007, 2009) stated that toxicants trigger a number of effects on Lymnea snails hemocytes, such as lysosomal fragility or apoptosis. Genotoxicity of glyphosate to human lymphocytes was previously evaluated by Mladinic et al. (2009) using comet assay, which is in agreement with the current results. Their results revealed that glyphosate increased tail length and intensity when compared to the control. On the other hand, the toxicity of Roundup may be attributed to its surfactant (Mann et al., 2009). Nevertheless, it has been demonstrated through several studies in invertebrates and humans that glyphosate formulation (Roundup) and the active ingredient alone caused similar deleterious effects (Gasnier et al., 2009; Benamu et al., 2010). Moreover, Achiorno et al. (2008) and Manas et al. (2009) evaluated the genotoxicity of formulation containing glyphosate using comet assay. They stated that the level of DNA damage in human lymphocytes showed a significant increment. Furthermore, an investigation by Poletta et al. (2009) confirmed that comet assay was useful tool in determining genotoxicity of Roundup on DNA of Caiman latirostris erythrocytes. Benachour and Seralini (2009) suggested that death of human cells as a result of Roundup treatment may be through inhibition of the mitochondrial succinate dehydrogenase and apoptosis may be via activation of enzymatic caspases. Binelli et al. (2009) reported that using comet assay it was found that antimicrobial trimethorim increased DNA damage in mussel hemocytes. It is clearly observed from the obtained data that B. alexandrina snails found to be more affected by Roundup treatment than S. mansoni infection. Thus, infected snails have effective immune responses but failed to express successful defense when interacted with Roundup sublethal toxicity. It can be concluded that herbicides as environmental pollutants affected B. alexandrina snails and its interactions with S. mansoni infection. Thus, such data provide information for aquatic management and for establishing limits of
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use of herbicides near aquatic ecosystems. In addition, comet assay and flow cytometry were able to detect adverse environmental conditions, proving to be a very adequate short-term test and should be included as biomarkers to be used in the monitoring of aquatic environments.
References Abdul-Salam, J.M., Michelson, E.H., 1980. Biomphalaria glabrata amoebocytes, effect of Schistosoma mansoni infection on in vitro phagocytosis. J. Invertebr. Pathol. 35, 241–248. Achiorno, C.L., de Villalobos, C., Ferrari, L., 2008. Toxicity of the herbicide glyphosate to Chordodes nobilii (Gordiida, Nematomorpha). Chemosphere 71, 1817–1822. Amen, R.I., Tijnagel, J.M.G., Van der Knaap, W.P., Meuleman, E.A., de Lange-de Klerk, E.S.M., Sminia, T., 1991. Effects of Trichobilharzia ocellata on hemocytes of Lymnaea stagnalis. Dev. Comp. Immunol. 15, 105–115. Anderson, R.M., Mercer, J.G., Wilson, R.R., Carter, N.P., 1982. Transmission of Schistosoma mansoni from man to snail, experimental studies of miracidial survival and infectivity in relation to larval age, water temperature, host size and age. Parasitology 85, 339–360. Bellomo, G., Mirabelli, F., Richelmi, P., Malorni, W., Iosi, F., Orrenius, S., 1990. The cytoskeleton as in quinine toxicity. Free Rad. Res. Commun. 8, 391–399. Benachour, N., Seralini, G.E., 2009. Glyphosate formulations induce apoptosis and necrosis in human umbilical, embryonic, and placental cells. Chem. Res. Toxicol. 22, 97–105. Benamu, M.A., Schneider, M.I., Sa´nchez, N.E., 2010. Effects of the herbicide glyphosate on biological attributes of Alpaida veniliae (Araneae, Araneidae), in laboratory. Chemosphere 78, 871–876. Binelli, A., Parolini, M., Cogni, D., Pedriali, A., Provini, A., 2009. A multi-biomarker assessment of the impact of the antibacterial trimethoprim on the non-target organism Zebra mussel (Dreissena polymorpha). Comp. Biochem. Physiol. 150, 329–336. Bolognesi, C., Bonatti, S., Degan, P., Gallerani, E., Peluso, M., Rabboni, R., 1997. Genotoxicity activity of glyphosate and its technical formulation Roundup. J. Agric. Food Chem. 45, 1957–1962. Burlando, B., Marchi, B., Panfili, I., Viarengo, A., 2002. Essential role of Ca2 + dependent phospholipase A2 in estradiol-induced lysosome activation. Am. J. Physiol. Cell Physiol. 283, C1461–C1468. Canesi, L., Lorusso, L.C., Ciacci, C., Betti, M., Rocchi, M., Pojana, G., Marcomini, A., 2007. Immunomodulation of Mytilus hemocytes by individual estrogenic chemicals and environmentally relevant mixtures of estrogens, In vitro and in vivo studies. Aquat. Toxicol. 81, 36–44. Cavas, T., Konen, S., 2007. Detection of cytogenetic and DNA damage in peripheral erythrocytes of goldfish (Carassius auratus) exposed to a glyphosate formulation using the micronucleus test and the comet assay. Mutagenesis 22, 263–268. Cohen, J.J., Al- Rubeai, M., 1995. Apoptosis-targeted therapies the next big thing in biotechnology? Trends Biotechnol. 13, 281–283. Dean, P.N., Jett, J.H., 1974. Mathematical analysis of DNA distributions derived from flow microfluorometry. J. Cell. Biol. 60, 523. Fagotti, A., Di Rosa, I., Simoncelli, F., Pipe, R., 1996. The effects of copper on actin and fibronectin organization in Mytilus galloprovincialis haemocytes. Dev. Comp. Immunol. 20, 383–391. Fournier, M., Cyr, D., Blakley, B., Boermans, H., Brousseau, P., 2000. Phagocytosis as a biomarker of immunotoxicity in wildlife species exposed to environmental xenobiotics. Am. Zool. 40, 412–420. Fournier, M., Pellerin, J., Lebeuf, M., Brousseau, P., Morin, Y., Cyr, D., 2002. Effects of exposure of Mya arenaria and Mactromeris polynyma to contaminated marine sediments on phagocytic activity of hemocytes. Aquat. Toxicol. 59, 83–92. Galloway, T.S., Depledge, M.H., 2001. Immunotoxicity in invertebrates, Measurement and ecotoxicological relevance. Ecotoxicology 10, 5–23. Gasnier, C., Dumont, C., Benachour, N., Clair, E.J., Chagnon, M.C., Se´ralini, G.E., 2009. Glyphosate-based herbicides are toxic and endocrine disruptors in human cell lines. Toxicology 262, 184–189. Grazeffe, V.S., Tallarico, L.F., Pinheiro, A., Kawano, T., Suzuki, M.F., Okazaki, K., Pereira, C.A.B., Nakano, E., 2008. Establishment of the comet assay in the freshwater snail Biomphalaria glabrata (Say, 1818). Mutat. Res. 654, 58–63. Glusczak, L., Miron, D.S., Crestani, M., Fonseca, M.B., Pedron, F.A., Duarte, M.F., Vieira, V.L.P., 2006. Effect of glyphosate herbicide on acetylcholinesterase activity and metabolic and hematological parameters in piava (Leporinus obtusidens). Ecotoxicol. Environ. Saf. 65, 237–241. Hora´k, P., Van der Knaap, W.P.W., 1997. Lectins in snails-trematode immune interaction, a review. Folia Parasitol. (Praha) 44, 161–172. Lee, R.F., Steinert, S., 2003. Use of the single cell gel electrophoresis/comet assay for detecting DNA damage in aquatic (marine and freshwater) animals. Mutat. Res. 544, 43–64. Lioi, M.B., Scarfi, M.R., Santoro, R., Barbieri, O.S., Salvemini, F., Di Berardino, D., Ursini, M.V., 1998. Cytogenetic damage and induction of pro-oxidant state in human lymphocytes exposed in vitro to glyphosate, vinclozolin, atrazine and DPX-E9636. Environ. Mol. Mutagen. 32, 39–46. Livingstone, D.R., Chipman, J.K., Lowe, D.M., Minier, C., Mitchelmore, C.L., Moore, M.N., Peters, L.D., Pipe, R.K., 2000. Development of biomarkers to detect the effects of organic pollution on aquatic invertebrates: recent molecular,
759
genotoxic, cellular and immunological studies on the common mussel (Mytilus edulis L.) and other mytilids. Int. J. Environ. Pollut. 13, 56–91. Little, T.J., Hultmark, D., Read, A.F., 2005. Invertebrate immunity and the limits of mechanistic immunology. Nat. Immunol. 6, 651–654. Loker, E.S., Bayn, C.J., Buckley, P.M., Kruse, K.T., 1982. Ultrastructure of encapsulation of Schistosoma mansoni mother sporocystes by hemocytes of juneniles of the 10-R2 strain of Biomphalaria glabrate. J. Parasitol. 68, 84–94. Manas, F., Peralta, L., Raviolo, J., Garcia Ovando, H., Weyers, A., Ugnia, L., Gonzalez, Cid, M, Larripa, I., Gorla, N., 2009. Genotoxicity of AMPA, the environmental metabolite of glyphosate, assessed by the comet assay and cytogenetic tests. Ecotoxicol. Environ. Saf. 72, 834–837. Mann, R.M., Hyne, R.V., Choung, C.B., Wilson, S.P., 2009. Amphibians and agricultural chemicals, review of the risks in a complex environment. Environ. Pollut. 157, 2903–2927. Matricon-Gondran, M., Letocart, M., 1999. Internal defenses of the snail Biomphalaria glabrata I. Characterization of hemocytes and fixed phagocytosis. J. Invertebr. Pathol. 74, 224–234. Martins-Souza, R.L., Pereira, C.A., Coelho, P.M.Z., Negrao-Correa, D., 2003. Silica treatment increases the susceptibility of the Cabo Frio strain of Biomphalaria tenagophila to Schistosoma mansoni infection but does alter the natural resistance of the Taim strain. Parasitol. Res. 91, 500–507. Martins-Souza, R.L., Pereira, C.A., Coelho, P.M.Z., Martins Filho, O.A., Negrao-Correa, D., 2009. Flow cytometry analysis of the circulating haemocytes from Biomphalaria glabrata and Biomphalaria tenagophila following Schistosoma mansoni infection. Parasitology 136, 67–76. Martins-Souza, R.L., Pereira, C.A., Martins Filho, O.A., Coelho, P.M.Z., Correa Jr., A., Negrao-Correa, D., 2006. Differential lectin labeling of circulating hemocytes from Biomphalaria glabrata and Biomphalaria tenagophila resistant or susceptible to Schistosoma mansoni infection. Mem. Inst. Oswaldo Cruz 101, 185–192. Mladinic, M., Berend, S., Vrdoljak, A.L., Kopjar, N., Radic, B., Zeljezic, D., 2009. Evaluation of genome damage and its relation to oxidative stress induced by glyphosate in human lymphocytes in vitro. Environ. Mol. Mutagen. 50, 800–807. Negrao-Correa, D., Pereira, C.A., Rosa, F.M., Martins-Souza, R.L., Andrade, Z.A., Coelho, P.M.Z., 2007. Molluscan response to parasite, Biomphalaria and Schistosoma mansoni interaction. Invertebr. Sur. J. 4, 101–111. Noda, S., Loker, E.S., 1989. Effects of infection with Echinostoma paraensei on the circulating haemocytes population of the host snail Biomphalaria glabrata. Parasitology 98, 1–10. Osman, G.Y., Mohamed, A.H., Mohamed, A.M., Al-Qormuti, S.A., 2008. Effect of Roundup herbicide on biological activity of Biomphalaria alexandrina snails infected with Schistosoma mansoni. Mansoura J. Biol. 35, 147–167. Paraense, W.L., 2001. The schistosome vectors in the Americas. Mem. Inst. Oswaldo Cruz 96, 7–16. Pipe, R.K., Coles, J.A., Carissan, F.M., Ramanathan, K., 1999. Copper induced immunomodulation in the marine mussel Mytilus edulis. Aquat. Toxicol. 46, 43–54. Peluso, M., Munnia, A., Bolognesi, C., Parodi, S., 1998. 32p-Postlabeling detection of DNA adducts in mice treated with the herbicide Roundup. Environ. Mol. Mutagen. 31, 55–59. Poletta, G.L., Larriera, A., Kleinsorge, E., Mudry, M.D., 2009. Genotoxicity of the herbicide formulation Roundup (glyphosate) in broad-snouted caiman (Caiman latirostris) evidenced by the comet assay and the micronucleus test. Mutat. Res. 672, 95–102. Russo, J., Lagadic, L., 2000. Effects of parasitism and pesticide exposure on characteristics and functions of hemocyte populations in the freshwater snail Lymnaea palustris (Gastropoda, Pulmonata). Cell Biol. Toxicol. 16, 15–30. Russo, J., Lagadic, L., 2004. Effects of environmental concentrations of atrazine on hemocyte density and phagocytic activity in the pond snail Lymnaea stagnalis (Gastropoda, Pulmonata). Environ. Pollut. 127, 303–311. Russo, J., Lefeuvre-Orfila, L., Lagadic, L., 2007. Hemocyte-specific responses to the peroxidizing herbicide fomesafen in the pond snail Lymnaea stagnalis (Gastropoda, Pulmonata). Environ. Pollut. 146, 420–427. Russo, J., Madec, L., Brehelin, M., 2009. Haemocyte lysosomal fragility facing an environmental reality, A toxicological perspective with atrazine and Lymnaea stagnalis (Gastropoda, Pulmonata) as a test case. Ecotoxicol. Environ. Safe. 72, 1719–1726. Schneider, M., Sanchez, N., Pineda, S., Chi, H., Ronco, A., 2009. Impact of glyphosate on the development, fertility and demography of Chrysoperla externa (Neuroptera: Chrysopidae): ecological approach. Chemosphere 76, 1451–1455. Singh, N.P., Mc Coy, M.T., Tice, R.R., Schnider, E.L., 1988. A simple technique for quantification of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184–191. Sminia, T., 1972. Structure and function of blood and connective tissue cells of the fresh water pulmonate Lymnaea stagnalis studied by electron microscopy and enzyme histochemistry. Z. Zellforsch. 130, 497–526. Sminia, T., Van der Knaap, W.P.W., 1986. Immunorecognition in invertebrates with special reference to molluscs. In, Immunity in Invertebrates. In: Brehelin, M. (Ed.), Springer, Berlin/Heidelberg 112–124. Snieszko, S.F., 1974. The effects of environmental stress on outbreaks of infectious diseases of fishes. J. Fish Biol. 6, 197–208. Steinmann, P., Keiser, J., Bos, R., Tanner, M., Utzinger, J., 2006. Schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk. Lancet Infect. Dis. 6, 411–425. Sokal, R.R., Rohlf, F.J. (Eds.), 1981. Biometry, 2nd ed. W. H. Freeman and Co., San Francisco.
760
A.H. Mohamed / Ecotoxicology and Environmental Safety 74 (2011) 754–760
Tsui, M.T.K., Chu, L.M., 2003. Aquatic toxicity of glyphosate-based formulations, comparison between different organisms and the effects of environmental factors. Chemosphere 52, 1189–1197. USDA 1984. Herbicide background statement, Glyphosate. In: ‘‘Pesticide Background Statement’’. 1, Herbicides USDA forest Service, Agricultural Handbook No 633, G-1-72. Van der Knaap, W..P.W., Adema, C.M., Sminia, T., 1993. Invertebrate blood cells, morphological and functional aspects of the haemocytes in the pond snail Lymnaea stagnalis. Comp. Haematol. Int. 3, 20–26.
Williams, G.M., Kroes, R., Monrot, I.C., 2000. Safety evaluation and risk assessment of the herbicide Roundup and its active ingredient, glyphosate, for humans. Regul. Toxicol. Pharmacol. 31, 117–165. Wong, S., Fournier, M., Coderre, D., Banska, W., Krzstyniak, K., 1992. Environmental immunotoxicology. In: Peakall, D. (Ed.), Animals Biomarkers as Pollution Indicators. Chapman and Hall, London, pp. 167–189. Yoshino, T.P., Davids, B.J., Castillo, M.G., Johnston, L.A., 1998. Invertebrate cellular adhesion receptors, their structure and function as primordial recognition molecules in the immune system. Trends Comp. Biochem. Physiol. 5, 49–65.