| Objective: The fluoroquinolones have well pharmacodynamic nature(metabolic rate in human being and animals < 25%), used in treatment of human diseases, livestock and aquaculture activities, so the majority of fluoroquinolones, as the primary form in medical waste water, sanitary sewage and animal waste enter into the environment. After proliferating rapidly from aquatic environment to soils, it can be adsorbed by mineral substance and organic matter and accumulates in the form of hydrochloride. This ecotoxicological effect has been a serious environmental problem that Chinese people even all human beings have to face. Our objective is to investigate the exposed toxicity and the potential exposure biomarkers of Ciprofloxacin by studying acute and chronic toxic effect and the protein expression profile in earthworm Eisenia fetida exposed to Ciprofloxacin hydrochloride. This can provide experimental method and referential data for early diagnosing and evaluating the risk of ecological contamination by Ciprofloxacin in environment.Method: The acute toxicity test includes the contact filter paper method(≤96 h) and the artificial soil method(≤14 d) and the chronic toxicity test use the artificial soil method(>14 d) based on OECD standard. We measured the LD50 of earthworm in filter paper contact after 24 h’s, 48 h’s and 96 h’s exposure and the antioxidant enzymes(SOD, POD, CAT) of earthworm in the artificial soil test after 12 h, 1 d, 2 d, 7 d, 14 d, 28 d and 56 d. According to the result of enzyme assay, we selected earthworm of four key points in time(2 d, 7 d, 28 d and 56 d) to make histopathologic slides stained by HE. Meanwhile, the total protein of these groups was extracted, separated by 2-DE and identified by MALDI-TOF-MS and the relative protein databases, for searching the differential expression proteins at each time, which were verified by Western blot.Result: After 24 h’s, 48 h’s and 96 h’s exposure in filter paper contact, LD50 was respectively 2.20×10-1 mg·cm-2, 1.60×10-1 mg·cm-2 and 1.12×10-1 mg·cm-2. In the artificial soil test, no individual died, so LD50 exceeded 5 000 mg?kg-1. Under the conditions of the test, the activity of POD in 1 000 mg?kg-1 group was 2.48 times than that of the control group at the 2th day; the activity of SOD in 5 000 mg?kg-1 group was 1.83 times than that of the control group at the 56 th day. Through HE staining,we could detect the muscle fiber was slightly damaged and arranged loosely at the 2th day and the 28 th day. In 7 days, the muscle fiber ruptured and was damaged seriously. However, there were no obvious injuries in the muscle fiber after 56 days. In proteomics research, approximate 1100 protein spots were found in each gel by the gel analysis software PDQuset 8.0. In total, the expression quantity of 110 protein points was significantly(P<0.05) changed(up-regulation 1.50 times or down-regulation 0.70 times), of which 36 protein spots were successfully identified and belonged to 15 kinds of proteins. In these 36 points, there were 21 points up-regulation and 6 points down-regulation after 2 days exposure(200 mg/kg) to Ciprofloxacin hydrochloride; 10 points up-regulation and 7 points down-regulation after 7 days; 6 points up-regulation and 12 points down-regulation after 28 days; 2 points up-regulation and 5 points down-regulation after 56 days. Actin protein accounting for 30.6%, Myosin protein accounting for 22.2%, these points significantly up regulated in two days and significantly down regulated in 28 days; other proteins associated with metabolic pathways or were hypothetical proteins. At one moment, the alone differentially most expressed proteins were Actin(2 d, 3.52 times), Intermediate filament protein(7 d, 3.55 times) and Regucalcin(28 d, 0.45 times). Myosin was checked by Western blot. Statistical analysis showed that exposing Eisenia fetida to Ciprofloxacin hydrochloride(200 mg/kg), Myosin protein expression was significantly higher compared to the control group(P<0.05) in two days, significantly reduced in 7 days, and did not change in 28 days and 56 days, which was in keeping with the trend of proteomics research results.Conclusion: Ciprofloxacin is certain lethal toxic to earthworms through filter paper contact method. The result of the antioxidant enzymes assay showed that Ciprofloxacin was toxic to Eisenia fetida. POD is more sensitive in acute phase(≤14 d) while SOD is more sensitive in chronic phase(>14 d). POD and SOD could be used as the effective biomarkers for toxicity assessment of Ciprofloxacin to Eisenia fetida. By exposing 200 mg/kg Ciprofloxacin hydrochloride to Eisenia fetida in 2 days, 7 days, 28 days and 56 days, the proteomics study found 36 differentially expressed protein spots, mainly containing cytoskeletal proteins(Actin and Myosin), which were also consistent with the results of HE staining and Western blot validation results. Meanwhile, Extracellular globin also was found, which associated with the oxygen transport protein. These proteins could, to some extent, reflect the oxidative stress damage to the cytoskeleton and contractile activities of muscles and serve as the potential exposed molecular biomarkers in antibiotics contaminated soil. |
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