Environ Sci Pollut Res DOI 10.1007/s11356-014-3256-2
RESEARCH ARTICLE
Molecular and ultrastructural insights into the earthworm Eisenia fetida of the assessment of ecotoxicity during colistin exposure Ruizi Guo & Xueyao Ding & Xiaoxia Zhong & Shangji Gao & Yongxue Sun
Received: 25 January 2014 / Accepted: 24 June 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Colistin is a peptide antibiotic widely used as a feed additive in animal farming, especially in poultry and swine production, for treatment and prevention of gram-negative bacterial infections, as well as for growth promotion use. When orally ingested, colistin is poorly absorbed and is eliminated almost unaltered by the enteric canal into the environment. Thus, risk of environmental toxicity cannot be ignored. In the present study, we examined the effects of colistin on Heath Shock Protein (HSP) 70, metallothionein (MT) gene expressions, and the ultrastructure of intestinal cells, following treatment of the soil indicator earthworm Eisenia fetida with 10, 20, and 100 mg/kg colistin for 7, 14, and 21 days. The results showed that, compared with the control, the expressions of HSP70 and MT genes changed significantly. Colistin caused up-regulations of HSP70’s expression while inhibited the expression of MT gene. In addition, most mitochondria and endoplasmic reticulum were damaged in the group treated with high concentration. The investigation of gene expressions of HSP70 and MT, as well as pathological alterations in the intestinal cells, may provide important information in terms of ecotoxicity of colistin and can be used as early warning system.
Keywords Colistin . Feed supplement . Earthworm . Eisenia fetida . Ecotoxicity
Responsible editor: Philippe Garrigues R. Guo : X. Ding : X. Zhong : S. Gao : Y. Sun (*) National Laboratory of Safety Evaluation (Environmental Assessment) of Veterinary Drugs, South China Agricultural University, Guangzhou, China e-mail: [email protected]
Introduction Colistin (also called polymysin E) is widely used by farmers as a feed additive or medicated feed in animal farming, especially in poultry and swine production, both for treatment and control of gram-negative bacterial infections and growth promotion (Li et al. 2005; Casal, et al. 2007; Callens et al. 2012). When orally ingested, colistin is eliminated almost unaltered by the alimentary canal (Sarkar et al. 2007; Bressan et al. 2013) in soil in the proximity of animal farm facilities; therefore, a potential hazard from animals’ feces to the environment in active concentrations might become a reality. On the toxicity of colistin, its neurotoxicity to mice and chicks has been studied (Dai and Li 2012; and 2013), but its ecological toxicity research is still lacking. Earthworms, a key representative organism of soil, are essential in maintaining soil fertility through their normal physiological activities like burrowing, ingestion, and excretion (Edwards 2004). Earthworms are increasingly recognized as the indicators of agricultural ecosystem health (Pirooznia et al. 2007). Several earthworm species are suggested as sentinels to monitor pollution of terrestrial ecosystems (Simonsen and Scotte 2004; Spurgeon and Svendsen 2003), with testing on enzyme activities (Nadeau and Corneau 2001), structure observations (Svendsen and Spurgeon 2004; Sauve and Hendawi 2002), molecular genetics (Ricketts et al. 2004; Galay-Burgos and Spurgeon 2003), and behavioral responses (Hund-Rinke and Wiechering 2001). Heat shock proteins have been demonstrated to participate in multi-biochemical processes by promoting proper folding of newly synthesized polypeptides into functional proteins and repair or disposal of mis-folded and damaged proteins (Kiang and Tsokos 1998; Homa et al., 2007); while cysteinerich metallothionein participates in the homeostasis of essential metals and protection against metabolic regulation and
Environ Sci Pollut Res
oxidant damage (Dallinger 1996; Coyle et al. 2002; Dabrio et al. 2002). In the present study, we employed field soil as the exposure medium instead of artificial soil and filter paper (filter paper contact test) to provide more valuable information in relatively realistic conditions. To gain a better understanding of the events that occurred in Eisenia fetida cells during the stress imposed by colistin treatment, the intestinal epithelium ultrastructure of the earthworm was evaluated and analyzed. In addition, the stress-responsive-related genes of Heat Shock Protein (HSP70) and metallothionein (MT) were investigated using the real-time PCR approach.
Materials and methods Colistin-contaminated modeling treatment of earthworms Soil pre-processing Soil was collected from the top 20-cm layer after removal of vegetation in South Agriculture University, air dried after homogenization, and sieved before use. Physical and chemical properties of the soil are shown in Table 1. Earthworm procuring Adult E. fetida were purchased from an earthworm farm in Nanjing, Jiangsu Province, China, and acclimatized under laboratory conditions without colistin treatment for a week. Treatment The reactions were carried in beakers. Concentration of the colistin was selected based on the recommended daily dose of colistin for animals. Different weights of colistin (Guangzhou Chemical Reagent Factory, Guangzhou, China) were dissolved in Milli-Q water mixed uniformly with 1 kg of preprocessed soil in each beaker, and then more Milli-Q water was added to ensure there was 25–35 % (w/w) water in each reactor (Table 2). It was allowed to balance for half an hour before 20 earthworms (about 300–400 mg) were introduced in each beaker. Reactors were placed in an artificial climate cabinet (Boxun, SPX-250I-C, Shanghai) and kept at 20± 1 °C with alternating 12 h in light and 12 h in darkness, and with the humidity being adjusted at 65–75 %. Food was supplied to the surface of all exposure soil in the form of dry cattle manure powder once a week. There were three copies of every type of reactor, and water was used as control treatment. Colistin exposure durations followed the scheme detailed in Table 2. After the colistin exposure, the earthworms were placed on wet filter paper for 48 h at room temperature to empty their guts of content at each sampling time point. Fresh
Table 1 Physical and chemical properties of soil used in the experiment Parameter (units)
Value (mean±standard error, n=3)
PH Organic carbon (g/kg) Total nitrogen (g/kg) Total phosphorus (g/kg)
6.2±0.05 35.89±3.05 2.38±0.19 0.51±0.044
Total potassium (g/kg) Available nitrogen (g/kg) Rapid available phosphorus (g/kg) Rapid available potassium (g/kg) Clay content (w/w, D
RESEARCH ARTICLE
Molecular and ultrastructural insights into the earthworm Eisenia fetida of the assessment of ecotoxicity during colistin exposure Ruizi Guo & Xueyao Ding & Xiaoxia Zhong & Shangji Gao & Yongxue Sun
Received: 25 January 2014 / Accepted: 24 June 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Colistin is a peptide antibiotic widely used as a feed additive in animal farming, especially in poultry and swine production, for treatment and prevention of gram-negative bacterial infections, as well as for growth promotion use. When orally ingested, colistin is poorly absorbed and is eliminated almost unaltered by the enteric canal into the environment. Thus, risk of environmental toxicity cannot be ignored. In the present study, we examined the effects of colistin on Heath Shock Protein (HSP) 70, metallothionein (MT) gene expressions, and the ultrastructure of intestinal cells, following treatment of the soil indicator earthworm Eisenia fetida with 10, 20, and 100 mg/kg colistin for 7, 14, and 21 days. The results showed that, compared with the control, the expressions of HSP70 and MT genes changed significantly. Colistin caused up-regulations of HSP70’s expression while inhibited the expression of MT gene. In addition, most mitochondria and endoplasmic reticulum were damaged in the group treated with high concentration. The investigation of gene expressions of HSP70 and MT, as well as pathological alterations in the intestinal cells, may provide important information in terms of ecotoxicity of colistin and can be used as early warning system.
Keywords Colistin . Feed supplement . Earthworm . Eisenia fetida . Ecotoxicity
Responsible editor: Philippe Garrigues R. Guo : X. Ding : X. Zhong : S. Gao : Y. Sun (*) National Laboratory of Safety Evaluation (Environmental Assessment) of Veterinary Drugs, South China Agricultural University, Guangzhou, China e-mail: [email protected]
Introduction Colistin (also called polymysin E) is widely used by farmers as a feed additive or medicated feed in animal farming, especially in poultry and swine production, both for treatment and control of gram-negative bacterial infections and growth promotion (Li et al. 2005; Casal, et al. 2007; Callens et al. 2012). When orally ingested, colistin is eliminated almost unaltered by the alimentary canal (Sarkar et al. 2007; Bressan et al. 2013) in soil in the proximity of animal farm facilities; therefore, a potential hazard from animals’ feces to the environment in active concentrations might become a reality. On the toxicity of colistin, its neurotoxicity to mice and chicks has been studied (Dai and Li 2012; and 2013), but its ecological toxicity research is still lacking. Earthworms, a key representative organism of soil, are essential in maintaining soil fertility through their normal physiological activities like burrowing, ingestion, and excretion (Edwards 2004). Earthworms are increasingly recognized as the indicators of agricultural ecosystem health (Pirooznia et al. 2007). Several earthworm species are suggested as sentinels to monitor pollution of terrestrial ecosystems (Simonsen and Scotte 2004; Spurgeon and Svendsen 2003), with testing on enzyme activities (Nadeau and Corneau 2001), structure observations (Svendsen and Spurgeon 2004; Sauve and Hendawi 2002), molecular genetics (Ricketts et al. 2004; Galay-Burgos and Spurgeon 2003), and behavioral responses (Hund-Rinke and Wiechering 2001). Heat shock proteins have been demonstrated to participate in multi-biochemical processes by promoting proper folding of newly synthesized polypeptides into functional proteins and repair or disposal of mis-folded and damaged proteins (Kiang and Tsokos 1998; Homa et al., 2007); while cysteinerich metallothionein participates in the homeostasis of essential metals and protection against metabolic regulation and
Environ Sci Pollut Res
oxidant damage (Dallinger 1996; Coyle et al. 2002; Dabrio et al. 2002). In the present study, we employed field soil as the exposure medium instead of artificial soil and filter paper (filter paper contact test) to provide more valuable information in relatively realistic conditions. To gain a better understanding of the events that occurred in Eisenia fetida cells during the stress imposed by colistin treatment, the intestinal epithelium ultrastructure of the earthworm was evaluated and analyzed. In addition, the stress-responsive-related genes of Heat Shock Protein (HSP70) and metallothionein (MT) were investigated using the real-time PCR approach.
Materials and methods Colistin-contaminated modeling treatment of earthworms Soil pre-processing Soil was collected from the top 20-cm layer after removal of vegetation in South Agriculture University, air dried after homogenization, and sieved before use. Physical and chemical properties of the soil are shown in Table 1. Earthworm procuring Adult E. fetida were purchased from an earthworm farm in Nanjing, Jiangsu Province, China, and acclimatized under laboratory conditions without colistin treatment for a week. Treatment The reactions were carried in beakers. Concentration of the colistin was selected based on the recommended daily dose of colistin for animals. Different weights of colistin (Guangzhou Chemical Reagent Factory, Guangzhou, China) were dissolved in Milli-Q water mixed uniformly with 1 kg of preprocessed soil in each beaker, and then more Milli-Q water was added to ensure there was 25–35 % (w/w) water in each reactor (Table 2). It was allowed to balance for half an hour before 20 earthworms (about 300–400 mg) were introduced in each beaker. Reactors were placed in an artificial climate cabinet (Boxun, SPX-250I-C, Shanghai) and kept at 20± 1 °C with alternating 12 h in light and 12 h in darkness, and with the humidity being adjusted at 65–75 %. Food was supplied to the surface of all exposure soil in the form of dry cattle manure powder once a week. There were three copies of every type of reactor, and water was used as control treatment. Colistin exposure durations followed the scheme detailed in Table 2. After the colistin exposure, the earthworms were placed on wet filter paper for 48 h at room temperature to empty their guts of content at each sampling time point. Fresh
Table 1 Physical and chemical properties of soil used in the experiment Parameter (units)
Value (mean±standard error, n=3)
PH Organic carbon (g/kg) Total nitrogen (g/kg) Total phosphorus (g/kg)
6.2±0.05 35.89±3.05 2.38±0.19 0.51±0.044
Total potassium (g/kg) Available nitrogen (g/kg) Rapid available phosphorus (g/kg) Rapid available potassium (g/kg) Clay content (w/w, D
