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Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/etap
Combined effects of oxytetracycline and Pb on earthworm Eisenia fetida Minling Gao ∗ , Qian Zhou, Wenhua Song, Xiaojun Ma School of Environmental and Chemical Engineering, Tianjin Polytechnic University, No. 399 Binshui Western Road, Xiqing District, Tianjin 300387, China
a r t i c l e
i n f o
a b s t r a c t
Article history:
Combined effects of oxytetracycline (OTC) and Pb on lysosomal membrane stability and
Received 14 August 2013
coelomocyte apoptosis of earthworm were studied in the paper. Compared with control,
Received in revised form
the lysosomal membrane stability decreased and coelomocyte apoptosis increased in the
25 January 2014
treatments of single OTC and Pb contamination. As for compound pollution, combined effect
Accepted 1 February 2014
of (5 mg/kg OTC + 50 mg/kg Pb) treatment on earthworm lysosomal was synergistic (except
Available online 8 February 2014
28 d). However, it was antagonistic at higher concentration of (10 mg/kg OTC + 50 mg/kg Pb) and (20 mg/kg OTC + 50 mg/kg Pb) treatment. In addition, coelomocyte apoptosis of
Keywords:
earthworm decreased significantly compared with single OTC, indicating an antagonistic
Oxytetracycline
reaction. And joint toxicity of OTC and Pb decreased significantly with the increasing OTC
Pb
concentration.
Combined effects
© 2014 Elsevier B.V. All rights reserved.
Lysosomal membrane stability Coelomocyte apoptosis
1.
Introduction
Among varieties of antibiotics, OTC is one of the most widely used veterinary antibiotics (Li et al., 2008), which is mainly used to treat disease, protect the health, enhance growth and feed efficiency in livestock industry (Sarmah et al., 2006). But most OTC could not be completely absorbed by animals and approximately 25 to 75% or even 70 to 90% of OTC administered to animals is excreted in an antimicrobially active form in urine or feces (Bao et al., 2013). The accumulation and residues of antibiotics occurred in soil environments when the animal dejecta are applied repeatedly into field as organic fertilizers. The concentration of the OTC was 5.172 mg kg−1 in the surface soils (0–20 cm) from agricultural fields treated with animal manures in the northern areas of Zhejiang province,
∗
Corresponding author. Tel.: +86 022 83956667; fax: +86 022 83956667. E-mail address: [email protected] (M. Gao). 1382-6689/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.etap.2014.02.004
China (Zhang et al., 2008). Bao (2008) found that the concentration of OTC was up to 200 mg kg−1 in the soil and 285 mg kg−1 in sediment in some places in China. Veterinary antibiotics can experience a series of changes in soil, such as sorption, translocation and degradation and then could make a harmful impact on soil organism (Li et al., 2008). It is found that OTC can inhibit the number of bacteria, actinomyces, and total microorganisms (Wang and Zhang, 2009), decrease activities of urease, sucrose phosphatase, and hydrogen peroxidase (Yao et al., 2010), inhibit root and shoot elongation of crops, wheat and Chinese cabbage (Jin et al., 2009), promote triticum elongation at low concentration (Boleas et al., 2005), induce genotoxicity of earthworm (Dong et al., 2012). Furthermore, questions have been raised over the potential impacts of antibiotics in environmental matrices on human health (Kong and Zhu, 2007).
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Pb is a typical heavy metal pollutant in agricultural land that is caused by irrigation of industrial wastewater and sewage slurry and precipitation of industrial and traffic waste gases (Li et al., 2006). The content of Pb is in range of 2–200 mg/kg in soil (Hou, 2007). It has been reported that the concentration of lead varies from 51.84 to 1185 mg kg−1 in polluted soil in China (Wu et al., 2008). Pb can be absorbed and accumulated by some plants. Many studies indicate that Pb is toxic to soil microorganism, plants and soil fauna (Hong et al., 2008; Wang et al., 2007). Moreover, excessive lead intake can inhibit the reproductive function and immunity of the human’s body; retard the intelligence development of children, resulting in various physical abnormalities (Chen and Zhu, 2004). Antibiotics and heavy metal are often detected to coexist in soil and organic fertilizer samples (Zhang et al., 2005). At present, little information is available on the potential concern of biochemical responses and genetic effects of combined pollution of antibiotics and metal on earthworm, though a few studies showed that interactive effect of antibiotics and heavy metals was potentially hazardous to bacteria and other organisms in the environment (Kong et al., 2006; Gao et al., 2013). Kong et al. (2006) reported the antibiotic (OTC) and Cu had significantly negative effects on soil microbial community function, particularly when both pollutants presented in the environment. However, the study about effect and mechanism of antibiotics and heavy metal on soil microorganisms is scarce. Thus it is significant for soil risk assessment to study the combined pollution of antibiotics and metal. Earthworm is one of the sensitive indicators of soil quality and widely used in terrestrial ecotoxicology. Eisenia fetida is considered a model organism for environmental surveillance and has been used in the standardization of acute and chronic ecotoxicological assays for industrial chemicals (ISO, 1998). To date, many kinds of biomarkers of earthworm including mortality, growth rate, reproduction rate, cell apoptosis, lysosomal membrane stability, total immune activity, metallothionein (MT) concentration, enzyme activities and DNA damage have been conducted to investigate the toxicological effects of chemicals (Wu et al., 2012; Sforzini et al., 2012). But most researches mainly focused on the mortality, growth rate or reproduction rate, which are often considered inappropriate to evaluate chemicals of lower toxicity and concentration. Qu et al. (2005) reported that OTC was not induced mortality of earthworm as the concentration of OTC was 500 mg/L. However, the comet assay indicated that OTC induced significant DNA damage in earthworm coelomocytes (P < 0.01). Of these biomarkers, lysosomal membrane stability has been identified to be a high sensitive biomarker and meet the needs of eco-toxicological soil risk assessment (Svendsen et al., 2004). Apoptosis is a controlled, regulated process and confers advantages during an organism’s life cycle. A series of changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation have occurred during apoptosis. The objectives of this study were (1) to investigate combined effects of OTC and Pb on lysosomal membrane stability of earthworms; (2) to investigate combined effects of OTC and Pb on coelomocyte apoptosis of earthworms.
2.
Materials and methods
2.1.
Chemicals
Oxytetracycline hydrochloride (95–105% purity) and EDTA, dimethyl sulfoxide (DMSO, >99% purity) was purchased from Amresco. Guaiacol glyceryl ether (>98% purity) was purchased from the Sigma Company in USA. Pb(NO3 )2 and other reagents were purchased from Beifang Tianyi Chemical Co. Ltd. (Tianjin, China).
2.2.
Earthworm
E. fetida were purchased from Tianjin Liming Jia Earthworm Farm Company, China. They maintained in the laboratory with controlled conditions (22 ± 2 ◦ C; 12 h:12 light: darkness) in natural soil for a week before experiment. Adult earthworms were selected for testing, which possessed clitellum and had an individual wet weight of 300–500 mg. Earthworm was kept on moist filter paper for 24 h, after which it was then washed again with distilled water and dried before use.
2.3.
Soil samples
The surface soils (0–30 cm) were collected from a suburban farmland of Tianjin, China. Which had never received any pesticide. The samples were air-dried, sieved at 2 mm, and then stored in containers at 4 ◦ C. Selected soil characteristics were analyzed according to the method descried in Lao (1988). The soil had a medium loam texture with density of 1.255 g/cm3 , a pH value of 8.24, the organic matter content of 1.61%, water holding capacity of 41.98%, sand of 10.58%, silt of 50.12%, clay of 29.30%, and cation exchange capacity of 10.86 cmol/kg. The soil background content of OTC and Pb was 0 mg/kg and 8.15 mg/kg, respectively.
2.4.
OTC and Pb exposure
Thirty portions of 500 g soils were weighed and put into each beaker. Different concentrations of OTC and Pb were added to each beaker. The concentrations were 0, 5, 10 and 20 mg/kg for OTC, 50, 200 and 800 mg/kg for Pb, 5 + 50, 10 + 50 and 20 + 50 mg/kg for OTC + Pb. The dosage of OTC and Pb toxicity experiments were designed according to exposure level of OTC and Pb in the soil. The soil moisture was adjusted to 60% of maximum water-holding capacity with aqueous solution. Each treatment was conducted in triplicates. The samples were mixed thoroughly in order to insure a uniform distribution of OTC and Pb applied. Twelve adult earthworms were set on each beaker, and the beaker was covered with plastic film with small ventilation holes. Tests were done in the light:dark (12:12) at 20 ± 2 ◦ C for 28 d. The humidity of an artificial climate chamber was 75 ± 5%. Two earthworms, one each for coelomocyte apoptosis and lysosomal membrane stability, were collected from each replicate beaker on the 7 d, 14 d and 28 d. During the experiment progress, the mortality of earthworm was 0%.
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2.5.
Harvesting of coelomocytes
Earthworms were allowed to depurate their guts for 18 h in Petri dishes on filter paper (moistened with distilled water) and were subsequently rinsed in distilled water and dried on filter paper. Coelomocytes were harvested using a modified version of the method described by Eyambe et al. (1991). Briefly, each earthworm was rinsed in saline (6.5 mg/mL NaCl, 4 ◦ C) and then was put into a 1.5 mL Eppendorf tube with 1 mL cold extrusion medium which contained 5% ethanol, 95% saline, 2.5 mg/mL EDTA, 10 mg/mL of the guaiacol glycerol ether. Subsequently, it was adjusted to pH 7.3 with 1 M NaOH. After 2 min, the earthworm was collected from the extrusion medium, and the cell suspension subsided naturally for 10 min. A part of the supernatant were transferred to another tube and washed once with calcium free Lumbricus balanced salt solution (LBSS) which consists of 71.5 mM NaCl, 4.8 mM KCl, 3.8 mM CaCl2 , 1.1 mM MgSO2 ·7H2 O, 0.4 mM KH2 PO4 , 0.3 mM Na2 HPO4 ·7H2 O, and 4.2 mM NaHCO3 . Then it was dissolved in distilled water and adjusted to pH 7.3 with NaOH (Goven et al., 1993), recovering cells by centrifugation at 100 g and 4 ◦ C for 3 min and resuspended in calcium free LBSS. Cell viability (trypan blue exclusion method) was assessed immediately after the extrusion and resulted about 90% in all cases.
2.6.
Neutral-red retention time
The neutral red retention time is applied to assess the lysosomal membrane stability coelomocyte membrane caused by environmental pollutants and this technique has already proven to be reliable and sensitive to use in earthworm (Svendsen et al., 1996). The method was described previously by Weeks and Svendsen (1996). Briefly, 20 L cell suspension was placed onto a microscope slide, allowed to adhere to the glass surface for 30 s. Subsequently, it was stained with an equal volume of neutral red working solution (80 mg/mL) which contained 2.5 mL lumbricus balanced salt solution (LBSS) and 10 L neutral red stock solution. The stock solution was obtained by dissolving 20 mg of neutral red powder in 1 mL DMSO. The working solution (80 mg/mL) was renewed every hour during the measuring process in order to avoid crystallization. After placing a coverslip, the slide was scanned every 2 min under a biological microscope at 400 times magnification. During these intervals, the slides were put into the artificial climate chamber. Observation was stopped when the ratio of cells with fully stained cytosols was over 50% of the total number of cells counted. This time was recorded as the neutral red retention time.
2.7.
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Fig. 1 – Coelomocyte of earthworm at the beginning of dyeing.
samples were analyzed with a FACS calibur flow cytometer (BD Biosciences, America).
3.
Results
3.1.
Changes of coelomocyte
Lysosome, a kind of common organelle existing in eukaryocyte, could quickly absorb, hold and accumulate the neutral red dye after dyeing. The coelomocyte was colorless under the microscope at the beginning of dyeing (Fig. 1). After a period of time, the dye tended to leak out of the lysosomes into the cytosol, the coelomocyte was stained red (Fig. 2). The exposure to OTC, Pb and OTC-Pb could result in the lysosomal membrane damage and increase its permeability. The greater the membrane damage, the lower the retention time of the dye within the lysosomes. On the basis of this, the measure of NRRT could provide a rapid and sensitive indication of responses to altered environmental pollution.
Flow cytometer analysis
Samples of coelomocytes obtained previously were washed twice and then resuspended with 200 L 1× binding buffer. From the new cell suspension, 100 L cell solution was transferred to another 1.5 mL Eppendorf tube, mixed with 10 L Annexin-V-R-PE. Subsequently, it was placed into an ice-bath at the darkness for 20–30 min. After that, 380 L 1 × binding buffer and 10 L 7-AAD were added into the precious cell suspension and mixed uniformly. Finally, the obtained cell
Fig. 2 – Coelomocyte of earthworm with more than 50% stained red after dyeing.
Antagonism Antagonism 54.42 ± 10.74 65.82 ± 6.58 10 + 50 20 + 50 50 10 20 28
48.09 ± 5.80 67.09 ± 4.39
55.69 ± 4.38
Antagonism Antagonism Antagonism 74.56 ± 1.52 74.56 ± 4.02 82.27 ± 5.80 10 + 50 20 + 50 5 + 50 23.68 ± 3.73 10 20 5 14
50
4.04 1.37 3.88 1.86 ± ± ± ± 77.34 79.61 78.64 77.63 50 4.12 1.37 1.38 4.56 ± ± ± ± 34.95 54.37 83.50 52.63 5 10 20 5 7
NRRT inhibition rate (%) OTC concentration (mg/kg)
Table 1 – Joint effect of OTC and Pb on NRRT inhibition rate.
All data of NRRT are presented as means ± standard deviation (SD). According to the one-way analysis of variance (ANOVA) using SPSS 16.0 software, the NRRT of all exposure group decreased significantly compared with control (P < 0.05). The effect of single OTC on lysosomal was shown in Fig. 3. Although a downward trend of NRRT could be found with an increase of exposure time (except OTC 20 mg/kg), there was no obvious regularity for toxic effect. For example, at OTC 5 mg/kg treatment, the NRRT was (33.5 ± 2.1) min, (18.0 ± 3.5) min and (15.3 ± 2.5) min with an increase of exposure time. However, NRRT inhibition rate was (34.95 ± 4.12)%, (52.63 ± 4.56)%, (41.70 ± 9.57)%. A significant increase in the toxic effect of OTC on lysosomal was found with an increase of OTC concentrations (Fig. 3). The NRRT was (33.5 ± 2.1) min, (23.5 ± 0.7) min and (8.5 ± 0.7) min and NRRT inhibition rate was (34.95 ± 4.12)%, (54.37 ± 1.37)%, (83.50 ± 1.38)% at day 7 with increasing OTC concentrations. Meanwhile, there was a similar trend at day 14 and 28. Fig. 4 indicated that the toxicity of Pb on lysosomal increased with an increase of treatment concentrations. As the concentration of Pb was 800 mg/kg, lysosomal was damaged seriously, with a lower NRRT (10.3 ± 2.1) min and a higher NRRT inhibition rate (60.75 ± 7.91)% than that of other Pb concentrations. However, there was also no obvious regularity of toxicity to lysosomal considering the NRRT and NRRT inhibition rate comprehensively. As the concentration of Pb was 50 mg/kg, NRRT was (32.0 ± 2.7) min, (29.0 ± 1.4) min and (11.7 ± 1.2) min at day 7, 14 and 28 with a declining trend, while NRRT inhibition rate was (37.86 ± 5.14)%, (23.68 ± 3.73)% and (55.69 ± 4.38)%. Compared with the single toxicity of OTC and Pb, effects of combined pollution on lysosomal membrane stability were mainly related to the treatment concentrations (Table 1 and
Pb concentration (mg/kg)
(NRRTcontrol group −NRRTexposure group ) NRRTcontrol group
37.86 ± 5.14
NRRT inhibition rate (%)
accurately the toxic degree of pollutinhibition rate was calculated according following equation:NRRT inhibition rate =
Day
To respect ants, NRRT to the
59.65 ± 5.48 74.56 ± 5.48 41.70 ± 9.57
3.2. Combined effects of OTC and Pb on lysosomal membrane stability of earthworms
5 + 50 10 + 50 20 + 50 5 + 50
NRRT inhibition rate (%) OTC + Pb concentration (mg/kg)
Fig. 3 – Neutral red retention time of the earthworm exposed to single OTC.
Synergy Antagonism Antagonism Synergy
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Combined effects
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Fig. 4 – Neutral red retention time of the earthworm exposed to single Pb.
Fig. 5 – Neutral red retention time of the earthworm exposed to combined pollution of OTC and Pb.
Fig. 5). The joint toxic effects of (5 mg/kg OTC + 50 mg/kg Pb) treatment on lysosomal increased, indicating a synergistic reaction (except 28 d). However it was antagonistic reaction when combined concentration was (10 mg/kg OTC + 50 mg/kg Pb) and (20 mg/kg OTC + 50 mg/kg Pb).
pollution group (P < 0.05), indicating that both single and combined pollution induced toxicity to coelomocyte (Figs. 6–8). Furthermore, the exposure to single and combined pollution mainly caused late apoptosis of coelomocyte. When Pb treatment was 800 mg/kg, late apoptosis rate of coelomocyte was up to 52.81%, and early apoptosis only 5.70% (Fig. 6). The damage of coelomocyte apoptosis increased significantly with an increase of single OTC or Pb concentrations. As the Fig. 7 showed that the cell apoptosis rate of single OTC and Pb treatment increased from 35.75% to 67.37% (P < 0.05), and from 35.97% to 59.41% (P < 0.05) with increasing of treatment concentrations, respectively. However, coelomocyte apoptosis rate decreased with an increase of OTC concentration in combined pollution group, indicating an antagonism (Fig. 6). For example, the cell apoptosis rate decreased significantly from 45.89% to 33.14% with an increase of combined treatment concentration at day 7 (P < 0.05).
3.3. Combined effects of OTC and Pb on coelomocyte apoptosis of earthworms Presently, flow cytometry (FCM) has been widely used to characterize, identify and quantify apoptotic cells and considered sensitive and accurate in fast qualitative and quantitative analysis (Cossarizza et al., 2005). In this work, FCM can determine and demonstrate accurately earthworm coelomocyte apoptosis. Compared with control, coelomocyte apoptosis (containing early and late apoptosis) increased in single and combined
Fig. 6 – Coelomocyte apoptosis of the earthworm exposed to all the treatment after 7 days (In the histogram, x-coordinate represents the signal value of Annexin-V-R-PE fluorescence and y-coordinate represents the signal value of 7-AAD fluorescence. Meanwhile, each histogram is divided into four areas, namely upper left Q1: necrosis cells and impurities, upper right Q2: late apoptotic cells, lower left Q3: normal cells and lower right Q4: early apoptotic cells).
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Fig. 7 – Coelomocyte apoptosis of the earthworm exposed to all the treatment after 14 days.
Fig. 8 – Coelomocyte apoptosis of the earthworm exposed to all the treatment after 28 days.
Compared with the single pollution, combined toxicity of OTC and Pb on coelomocyte decreased, indicating an antagonistic effect. Furthermore, the toxicity of (20 mg/kg OTC + 50 mg/kg Pb) treatment was lower obviously than other treatment. At day 7, coelomocyte apoptosis rate of (20 mg/kg OTC + 50 mg/kg Pb) treatment was only 33.14%, which was lower than theoretical value 91.81%, indicating an antagonistic effect.
4.
Discussion
There is a general idea that toxic effects of a certain pollutant are firstly triggered at the molecular level, and then gradually revealed at cellular level, organismal level, individual
level, population level, community level and ecosystem level (NRC, 1987). The single and combined pollution of OTC and Pb did not induce mortality in the paper, however, lysosomal membrane stability was damaged and coelomocyte apoptosis of earthworms was observed. It indicated that biomarkers, such as lysosomal membrane stability and coelomocyte apoptosis are more sensitive and appropriate in assessing lower toxicity. Combined toxicity may be similar, stronger (synergistic), or weaker (antagonistic) than single toxicity (Zhu et al., 2006), which depended on various factors such as the constituents of the mixture, concentrations and exposure time. In this study, interaction of OTC and Pb on earthworm lysosomal was synergistic reaction at (10 mg/kg OTC + 50 mg/kg Pb) treatment, which might cause cellular lipid peroxidation
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of earthworm. Lin et al. (2012) reported that the antibiotic chlortetracycline (similar to OTC) might induce oxidative stress, cause cellular lipid peroxidation and react with critical cellular macromolecules, leading to DNA damage. According to the report by Wang et al. (2007), heavy metals like Pb could increase the formation of active oxygen radical, which incited oxidative stress-related responses and also caused cellular lipid peroxidation that could induce damages on the cell membrane permeability. The production of lipid peroxidation in organisms cans indirectly reflect the degree of intracellular damage. So it could be likely concluded that the combined toxicity of OTC and Pb was stronger than single effect at the lower combined concentration. With the increase of OTC concentration, antagonistic effect of combined toxicity on earthworm lysosomal and coelomocyte were found. This might be explained that complexation happened between OTC and Pb at higher combined concentration. Han et al. (2009) once reported that there was no complexation observed between OTC and Zn2+ at pH = 4. But with increasing of pH value, complexation was observed and might happen at the sites of O11, O12 or O12, O1. At pH 7.5, OTC aggregation and metal-OTC complexation were observed in solutions with Ca2+ and Mg2+ (Tongaree et al., 1999). Although there were few reports about whether complexation could happen between OTC and Pb2+ , it was probably deduced to the same from above metal-OTC studies. Moreover, in realistic soil environments, various factors could influence combined effects, such as absorption, settlement, degradation, interaction with other substance that should be further studied.
5.
Conclusions
This paper investigated the interactive effects of OTC and Pb on lysosomal membrane stability and coelomocyte apoptosis of earthworm. The results showed lysosomal membrane stability and coelomocyte apoptosis was damaged in single or combined pollution. Compared with single OTC pollution, interactive effect of OTC and Pb on lysosomal membrane stability was synergy or antagonism, which related mainly to combine concentration and targets it worked. However, joint effect of OTC and Pb on coelomocyte apoptosis was antagonism.
Conflict of interest The authors declare that there are no conflicts of interest.
Transparency document The Transparency document associated with this article can be found in the online version.
Acknowledgments We acknowledge the National Natural Science Foundation of China (No. 21007045) for financial support.
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Available online at www.sciencedirect.com
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Combined effects of oxytetracycline and Pb on earthworm Eisenia fetida Minling Gao ∗ , Qian Zhou, Wenhua Song, Xiaojun Ma School of Environmental and Chemical Engineering, Tianjin Polytechnic University, No. 399 Binshui Western Road, Xiqing District, Tianjin 300387, China
a r t i c l e
i n f o
a b s t r a c t
Article history:
Combined effects of oxytetracycline (OTC) and Pb on lysosomal membrane stability and
Received 14 August 2013
coelomocyte apoptosis of earthworm were studied in the paper. Compared with control,
Received in revised form
the lysosomal membrane stability decreased and coelomocyte apoptosis increased in the
25 January 2014
treatments of single OTC and Pb contamination. As for compound pollution, combined effect
Accepted 1 February 2014
of (5 mg/kg OTC + 50 mg/kg Pb) treatment on earthworm lysosomal was synergistic (except
Available online 8 February 2014
28 d). However, it was antagonistic at higher concentration of (10 mg/kg OTC + 50 mg/kg Pb) and (20 mg/kg OTC + 50 mg/kg Pb) treatment. In addition, coelomocyte apoptosis of
Keywords:
earthworm decreased significantly compared with single OTC, indicating an antagonistic
Oxytetracycline
reaction. And joint toxicity of OTC and Pb decreased significantly with the increasing OTC
Pb
concentration.
Combined effects
© 2014 Elsevier B.V. All rights reserved.
Lysosomal membrane stability Coelomocyte apoptosis
1.
Introduction
Among varieties of antibiotics, OTC is one of the most widely used veterinary antibiotics (Li et al., 2008), which is mainly used to treat disease, protect the health, enhance growth and feed efficiency in livestock industry (Sarmah et al., 2006). But most OTC could not be completely absorbed by animals and approximately 25 to 75% or even 70 to 90% of OTC administered to animals is excreted in an antimicrobially active form in urine or feces (Bao et al., 2013). The accumulation and residues of antibiotics occurred in soil environments when the animal dejecta are applied repeatedly into field as organic fertilizers. The concentration of the OTC was 5.172 mg kg−1 in the surface soils (0–20 cm) from agricultural fields treated with animal manures in the northern areas of Zhejiang province,
∗
Corresponding author. Tel.: +86 022 83956667; fax: +86 022 83956667. E-mail address: [email protected] (M. Gao). 1382-6689/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.etap.2014.02.004
China (Zhang et al., 2008). Bao (2008) found that the concentration of OTC was up to 200 mg kg−1 in the soil and 285 mg kg−1 in sediment in some places in China. Veterinary antibiotics can experience a series of changes in soil, such as sorption, translocation and degradation and then could make a harmful impact on soil organism (Li et al., 2008). It is found that OTC can inhibit the number of bacteria, actinomyces, and total microorganisms (Wang and Zhang, 2009), decrease activities of urease, sucrose phosphatase, and hydrogen peroxidase (Yao et al., 2010), inhibit root and shoot elongation of crops, wheat and Chinese cabbage (Jin et al., 2009), promote triticum elongation at low concentration (Boleas et al., 2005), induce genotoxicity of earthworm (Dong et al., 2012). Furthermore, questions have been raised over the potential impacts of antibiotics in environmental matrices on human health (Kong and Zhu, 2007).
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Pb is a typical heavy metal pollutant in agricultural land that is caused by irrigation of industrial wastewater and sewage slurry and precipitation of industrial and traffic waste gases (Li et al., 2006). The content of Pb is in range of 2–200 mg/kg in soil (Hou, 2007). It has been reported that the concentration of lead varies from 51.84 to 1185 mg kg−1 in polluted soil in China (Wu et al., 2008). Pb can be absorbed and accumulated by some plants. Many studies indicate that Pb is toxic to soil microorganism, plants and soil fauna (Hong et al., 2008; Wang et al., 2007). Moreover, excessive lead intake can inhibit the reproductive function and immunity of the human’s body; retard the intelligence development of children, resulting in various physical abnormalities (Chen and Zhu, 2004). Antibiotics and heavy metal are often detected to coexist in soil and organic fertilizer samples (Zhang et al., 2005). At present, little information is available on the potential concern of biochemical responses and genetic effects of combined pollution of antibiotics and metal on earthworm, though a few studies showed that interactive effect of antibiotics and heavy metals was potentially hazardous to bacteria and other organisms in the environment (Kong et al., 2006; Gao et al., 2013). Kong et al. (2006) reported the antibiotic (OTC) and Cu had significantly negative effects on soil microbial community function, particularly when both pollutants presented in the environment. However, the study about effect and mechanism of antibiotics and heavy metal on soil microorganisms is scarce. Thus it is significant for soil risk assessment to study the combined pollution of antibiotics and metal. Earthworm is one of the sensitive indicators of soil quality and widely used in terrestrial ecotoxicology. Eisenia fetida is considered a model organism for environmental surveillance and has been used in the standardization of acute and chronic ecotoxicological assays for industrial chemicals (ISO, 1998). To date, many kinds of biomarkers of earthworm including mortality, growth rate, reproduction rate, cell apoptosis, lysosomal membrane stability, total immune activity, metallothionein (MT) concentration, enzyme activities and DNA damage have been conducted to investigate the toxicological effects of chemicals (Wu et al., 2012; Sforzini et al., 2012). But most researches mainly focused on the mortality, growth rate or reproduction rate, which are often considered inappropriate to evaluate chemicals of lower toxicity and concentration. Qu et al. (2005) reported that OTC was not induced mortality of earthworm as the concentration of OTC was 500 mg/L. However, the comet assay indicated that OTC induced significant DNA damage in earthworm coelomocytes (P < 0.01). Of these biomarkers, lysosomal membrane stability has been identified to be a high sensitive biomarker and meet the needs of eco-toxicological soil risk assessment (Svendsen et al., 2004). Apoptosis is a controlled, regulated process and confers advantages during an organism’s life cycle. A series of changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation have occurred during apoptosis. The objectives of this study were (1) to investigate combined effects of OTC and Pb on lysosomal membrane stability of earthworms; (2) to investigate combined effects of OTC and Pb on coelomocyte apoptosis of earthworms.
2.
Materials and methods
2.1.
Chemicals
Oxytetracycline hydrochloride (95–105% purity) and EDTA, dimethyl sulfoxide (DMSO, >99% purity) was purchased from Amresco. Guaiacol glyceryl ether (>98% purity) was purchased from the Sigma Company in USA. Pb(NO3 )2 and other reagents were purchased from Beifang Tianyi Chemical Co. Ltd. (Tianjin, China).
2.2.
Earthworm
E. fetida were purchased from Tianjin Liming Jia Earthworm Farm Company, China. They maintained in the laboratory with controlled conditions (22 ± 2 ◦ C; 12 h:12 light: darkness) in natural soil for a week before experiment. Adult earthworms were selected for testing, which possessed clitellum and had an individual wet weight of 300–500 mg. Earthworm was kept on moist filter paper for 24 h, after which it was then washed again with distilled water and dried before use.
2.3.
Soil samples
The surface soils (0–30 cm) were collected from a suburban farmland of Tianjin, China. Which had never received any pesticide. The samples were air-dried, sieved at 2 mm, and then stored in containers at 4 ◦ C. Selected soil characteristics were analyzed according to the method descried in Lao (1988). The soil had a medium loam texture with density of 1.255 g/cm3 , a pH value of 8.24, the organic matter content of 1.61%, water holding capacity of 41.98%, sand of 10.58%, silt of 50.12%, clay of 29.30%, and cation exchange capacity of 10.86 cmol/kg. The soil background content of OTC and Pb was 0 mg/kg and 8.15 mg/kg, respectively.
2.4.
OTC and Pb exposure
Thirty portions of 500 g soils were weighed and put into each beaker. Different concentrations of OTC and Pb were added to each beaker. The concentrations were 0, 5, 10 and 20 mg/kg for OTC, 50, 200 and 800 mg/kg for Pb, 5 + 50, 10 + 50 and 20 + 50 mg/kg for OTC + Pb. The dosage of OTC and Pb toxicity experiments were designed according to exposure level of OTC and Pb in the soil. The soil moisture was adjusted to 60% of maximum water-holding capacity with aqueous solution. Each treatment was conducted in triplicates. The samples were mixed thoroughly in order to insure a uniform distribution of OTC and Pb applied. Twelve adult earthworms were set on each beaker, and the beaker was covered with plastic film with small ventilation holes. Tests were done in the light:dark (12:12) at 20 ± 2 ◦ C for 28 d. The humidity of an artificial climate chamber was 75 ± 5%. Two earthworms, one each for coelomocyte apoptosis and lysosomal membrane stability, were collected from each replicate beaker on the 7 d, 14 d and 28 d. During the experiment progress, the mortality of earthworm was 0%.
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2.5.
Harvesting of coelomocytes
Earthworms were allowed to depurate their guts for 18 h in Petri dishes on filter paper (moistened with distilled water) and were subsequently rinsed in distilled water and dried on filter paper. Coelomocytes were harvested using a modified version of the method described by Eyambe et al. (1991). Briefly, each earthworm was rinsed in saline (6.5 mg/mL NaCl, 4 ◦ C) and then was put into a 1.5 mL Eppendorf tube with 1 mL cold extrusion medium which contained 5% ethanol, 95% saline, 2.5 mg/mL EDTA, 10 mg/mL of the guaiacol glycerol ether. Subsequently, it was adjusted to pH 7.3 with 1 M NaOH. After 2 min, the earthworm was collected from the extrusion medium, and the cell suspension subsided naturally for 10 min. A part of the supernatant were transferred to another tube and washed once with calcium free Lumbricus balanced salt solution (LBSS) which consists of 71.5 mM NaCl, 4.8 mM KCl, 3.8 mM CaCl2 , 1.1 mM MgSO2 ·7H2 O, 0.4 mM KH2 PO4 , 0.3 mM Na2 HPO4 ·7H2 O, and 4.2 mM NaHCO3 . Then it was dissolved in distilled water and adjusted to pH 7.3 with NaOH (Goven et al., 1993), recovering cells by centrifugation at 100 g and 4 ◦ C for 3 min and resuspended in calcium free LBSS. Cell viability (trypan blue exclusion method) was assessed immediately after the extrusion and resulted about 90% in all cases.
2.6.
Neutral-red retention time
The neutral red retention time is applied to assess the lysosomal membrane stability coelomocyte membrane caused by environmental pollutants and this technique has already proven to be reliable and sensitive to use in earthworm (Svendsen et al., 1996). The method was described previously by Weeks and Svendsen (1996). Briefly, 20 L cell suspension was placed onto a microscope slide, allowed to adhere to the glass surface for 30 s. Subsequently, it was stained with an equal volume of neutral red working solution (80 mg/mL) which contained 2.5 mL lumbricus balanced salt solution (LBSS) and 10 L neutral red stock solution. The stock solution was obtained by dissolving 20 mg of neutral red powder in 1 mL DMSO. The working solution (80 mg/mL) was renewed every hour during the measuring process in order to avoid crystallization. After placing a coverslip, the slide was scanned every 2 min under a biological microscope at 400 times magnification. During these intervals, the slides were put into the artificial climate chamber. Observation was stopped when the ratio of cells with fully stained cytosols was over 50% of the total number of cells counted. This time was recorded as the neutral red retention time.
2.7.
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Fig. 1 – Coelomocyte of earthworm at the beginning of dyeing.
samples were analyzed with a FACS calibur flow cytometer (BD Biosciences, America).
3.
Results
3.1.
Changes of coelomocyte
Lysosome, a kind of common organelle existing in eukaryocyte, could quickly absorb, hold and accumulate the neutral red dye after dyeing. The coelomocyte was colorless under the microscope at the beginning of dyeing (Fig. 1). After a period of time, the dye tended to leak out of the lysosomes into the cytosol, the coelomocyte was stained red (Fig. 2). The exposure to OTC, Pb and OTC-Pb could result in the lysosomal membrane damage and increase its permeability. The greater the membrane damage, the lower the retention time of the dye within the lysosomes. On the basis of this, the measure of NRRT could provide a rapid and sensitive indication of responses to altered environmental pollution.
Flow cytometer analysis
Samples of coelomocytes obtained previously were washed twice and then resuspended with 200 L 1× binding buffer. From the new cell suspension, 100 L cell solution was transferred to another 1.5 mL Eppendorf tube, mixed with 10 L Annexin-V-R-PE. Subsequently, it was placed into an ice-bath at the darkness for 20–30 min. After that, 380 L 1 × binding buffer and 10 L 7-AAD were added into the precious cell suspension and mixed uniformly. Finally, the obtained cell
Fig. 2 – Coelomocyte of earthworm with more than 50% stained red after dyeing.
Antagonism Antagonism 54.42 ± 10.74 65.82 ± 6.58 10 + 50 20 + 50 50 10 20 28
48.09 ± 5.80 67.09 ± 4.39
55.69 ± 4.38
Antagonism Antagonism Antagonism 74.56 ± 1.52 74.56 ± 4.02 82.27 ± 5.80 10 + 50 20 + 50 5 + 50 23.68 ± 3.73 10 20 5 14
50
4.04 1.37 3.88 1.86 ± ± ± ± 77.34 79.61 78.64 77.63 50 4.12 1.37 1.38 4.56 ± ± ± ± 34.95 54.37 83.50 52.63 5 10 20 5 7
NRRT inhibition rate (%) OTC concentration (mg/kg)
Table 1 – Joint effect of OTC and Pb on NRRT inhibition rate.
All data of NRRT are presented as means ± standard deviation (SD). According to the one-way analysis of variance (ANOVA) using SPSS 16.0 software, the NRRT of all exposure group decreased significantly compared with control (P < 0.05). The effect of single OTC on lysosomal was shown in Fig. 3. Although a downward trend of NRRT could be found with an increase of exposure time (except OTC 20 mg/kg), there was no obvious regularity for toxic effect. For example, at OTC 5 mg/kg treatment, the NRRT was (33.5 ± 2.1) min, (18.0 ± 3.5) min and (15.3 ± 2.5) min with an increase of exposure time. However, NRRT inhibition rate was (34.95 ± 4.12)%, (52.63 ± 4.56)%, (41.70 ± 9.57)%. A significant increase in the toxic effect of OTC on lysosomal was found with an increase of OTC concentrations (Fig. 3). The NRRT was (33.5 ± 2.1) min, (23.5 ± 0.7) min and (8.5 ± 0.7) min and NRRT inhibition rate was (34.95 ± 4.12)%, (54.37 ± 1.37)%, (83.50 ± 1.38)% at day 7 with increasing OTC concentrations. Meanwhile, there was a similar trend at day 14 and 28. Fig. 4 indicated that the toxicity of Pb on lysosomal increased with an increase of treatment concentrations. As the concentration of Pb was 800 mg/kg, lysosomal was damaged seriously, with a lower NRRT (10.3 ± 2.1) min and a higher NRRT inhibition rate (60.75 ± 7.91)% than that of other Pb concentrations. However, there was also no obvious regularity of toxicity to lysosomal considering the NRRT and NRRT inhibition rate comprehensively. As the concentration of Pb was 50 mg/kg, NRRT was (32.0 ± 2.7) min, (29.0 ± 1.4) min and (11.7 ± 1.2) min at day 7, 14 and 28 with a declining trend, while NRRT inhibition rate was (37.86 ± 5.14)%, (23.68 ± 3.73)% and (55.69 ± 4.38)%. Compared with the single toxicity of OTC and Pb, effects of combined pollution on lysosomal membrane stability were mainly related to the treatment concentrations (Table 1 and
Pb concentration (mg/kg)
(NRRTcontrol group −NRRTexposure group ) NRRTcontrol group
37.86 ± 5.14
NRRT inhibition rate (%)
accurately the toxic degree of pollutinhibition rate was calculated according following equation:NRRT inhibition rate =
Day
To respect ants, NRRT to the
59.65 ± 5.48 74.56 ± 5.48 41.70 ± 9.57
3.2. Combined effects of OTC and Pb on lysosomal membrane stability of earthworms
5 + 50 10 + 50 20 + 50 5 + 50
NRRT inhibition rate (%) OTC + Pb concentration (mg/kg)
Fig. 3 – Neutral red retention time of the earthworm exposed to single OTC.
Synergy Antagonism Antagonism Synergy
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Combined effects
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Fig. 4 – Neutral red retention time of the earthworm exposed to single Pb.
Fig. 5 – Neutral red retention time of the earthworm exposed to combined pollution of OTC and Pb.
Fig. 5). The joint toxic effects of (5 mg/kg OTC + 50 mg/kg Pb) treatment on lysosomal increased, indicating a synergistic reaction (except 28 d). However it was antagonistic reaction when combined concentration was (10 mg/kg OTC + 50 mg/kg Pb) and (20 mg/kg OTC + 50 mg/kg Pb).
pollution group (P < 0.05), indicating that both single and combined pollution induced toxicity to coelomocyte (Figs. 6–8). Furthermore, the exposure to single and combined pollution mainly caused late apoptosis of coelomocyte. When Pb treatment was 800 mg/kg, late apoptosis rate of coelomocyte was up to 52.81%, and early apoptosis only 5.70% (Fig. 6). The damage of coelomocyte apoptosis increased significantly with an increase of single OTC or Pb concentrations. As the Fig. 7 showed that the cell apoptosis rate of single OTC and Pb treatment increased from 35.75% to 67.37% (P < 0.05), and from 35.97% to 59.41% (P < 0.05) with increasing of treatment concentrations, respectively. However, coelomocyte apoptosis rate decreased with an increase of OTC concentration in combined pollution group, indicating an antagonism (Fig. 6). For example, the cell apoptosis rate decreased significantly from 45.89% to 33.14% with an increase of combined treatment concentration at day 7 (P < 0.05).
3.3. Combined effects of OTC and Pb on coelomocyte apoptosis of earthworms Presently, flow cytometry (FCM) has been widely used to characterize, identify and quantify apoptotic cells and considered sensitive and accurate in fast qualitative and quantitative analysis (Cossarizza et al., 2005). In this work, FCM can determine and demonstrate accurately earthworm coelomocyte apoptosis. Compared with control, coelomocyte apoptosis (containing early and late apoptosis) increased in single and combined
Fig. 6 – Coelomocyte apoptosis of the earthworm exposed to all the treatment after 7 days (In the histogram, x-coordinate represents the signal value of Annexin-V-R-PE fluorescence and y-coordinate represents the signal value of 7-AAD fluorescence. Meanwhile, each histogram is divided into four areas, namely upper left Q1: necrosis cells and impurities, upper right Q2: late apoptotic cells, lower left Q3: normal cells and lower right Q4: early apoptotic cells).
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Fig. 7 – Coelomocyte apoptosis of the earthworm exposed to all the treatment after 14 days.
Fig. 8 – Coelomocyte apoptosis of the earthworm exposed to all the treatment after 28 days.
Compared with the single pollution, combined toxicity of OTC and Pb on coelomocyte decreased, indicating an antagonistic effect. Furthermore, the toxicity of (20 mg/kg OTC + 50 mg/kg Pb) treatment was lower obviously than other treatment. At day 7, coelomocyte apoptosis rate of (20 mg/kg OTC + 50 mg/kg Pb) treatment was only 33.14%, which was lower than theoretical value 91.81%, indicating an antagonistic effect.
4.
Discussion
There is a general idea that toxic effects of a certain pollutant are firstly triggered at the molecular level, and then gradually revealed at cellular level, organismal level, individual
level, population level, community level and ecosystem level (NRC, 1987). The single and combined pollution of OTC and Pb did not induce mortality in the paper, however, lysosomal membrane stability was damaged and coelomocyte apoptosis of earthworms was observed. It indicated that biomarkers, such as lysosomal membrane stability and coelomocyte apoptosis are more sensitive and appropriate in assessing lower toxicity. Combined toxicity may be similar, stronger (synergistic), or weaker (antagonistic) than single toxicity (Zhu et al., 2006), which depended on various factors such as the constituents of the mixture, concentrations and exposure time. In this study, interaction of OTC and Pb on earthworm lysosomal was synergistic reaction at (10 mg/kg OTC + 50 mg/kg Pb) treatment, which might cause cellular lipid peroxidation
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of earthworm. Lin et al. (2012) reported that the antibiotic chlortetracycline (similar to OTC) might induce oxidative stress, cause cellular lipid peroxidation and react with critical cellular macromolecules, leading to DNA damage. According to the report by Wang et al. (2007), heavy metals like Pb could increase the formation of active oxygen radical, which incited oxidative stress-related responses and also caused cellular lipid peroxidation that could induce damages on the cell membrane permeability. The production of lipid peroxidation in organisms cans indirectly reflect the degree of intracellular damage. So it could be likely concluded that the combined toxicity of OTC and Pb was stronger than single effect at the lower combined concentration. With the increase of OTC concentration, antagonistic effect of combined toxicity on earthworm lysosomal and coelomocyte were found. This might be explained that complexation happened between OTC and Pb at higher combined concentration. Han et al. (2009) once reported that there was no complexation observed between OTC and Zn2+ at pH = 4. But with increasing of pH value, complexation was observed and might happen at the sites of O11, O12 or O12, O1. At pH 7.5, OTC aggregation and metal-OTC complexation were observed in solutions with Ca2+ and Mg2+ (Tongaree et al., 1999). Although there were few reports about whether complexation could happen between OTC and Pb2+ , it was probably deduced to the same from above metal-OTC studies. Moreover, in realistic soil environments, various factors could influence combined effects, such as absorption, settlement, degradation, interaction with other substance that should be further studied.
5.
Conclusions
This paper investigated the interactive effects of OTC and Pb on lysosomal membrane stability and coelomocyte apoptosis of earthworm. The results showed lysosomal membrane stability and coelomocyte apoptosis was damaged in single or combined pollution. Compared with single OTC pollution, interactive effect of OTC and Pb on lysosomal membrane stability was synergy or antagonism, which related mainly to combine concentration and targets it worked. However, joint effect of OTC and Pb on coelomocyte apoptosis was antagonism.
Conflict of interest The authors declare that there are no conflicts of interest.
Transparency document The Transparency document associated with this article can be found in the online version.
Acknowledgments We acknowledge the National Natural Science Foundation of China (No. 21007045) for financial support.
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