1H NMR-based metabolomics was used to measure the response of Eisenia fetida earthworms after exposure to sub-lethal concentrations of perfluorooctane sulfonate (PFOS) in soil. Earthworms were exposed to a range of PFOS concentrations (five, 10, 25, 50, 100 or 150 mg/kg) for two, seven and fourteen days. Earthworm tissues were extracted and analyzed by 1H NMR. Multivariate statistical analysis of the metabolic response of E. fetida to PFOS exposure identified time-dependent responses that were comprised of two separate modes of action: a non-polar narcosis type mechanism after two days of exposure and increased fatty acid oxidation after seven and fourteen days of exposure. Univariate statistical analysis revealed that 2-hexyl-5-ethyl-3-furansulfonate (HEFS), betaine, leucine, arginine, glutamate, maltose and ATP are potential indicators of PFOS exposure, as the concentrations of these metabolites fluctuated significantly. Overall, NMR-based metabolomic analysis suggests elevated fatty acid oxidation, disruption in energy metabolism and biological membrane structure and a possible interruption of ATP synthesis. These conclusions obtained from analysis of the metabolic profile in response to sub-lethal PFOS exposure indicates that NMR-based metabolomics is an excellent discovery tool when the mode of action (MOA) of contaminants is not clearly defined.
References
[1]
Kissa, E. Fluorinated Surfactants and Repellents, 2nd ed. ed.; Marcel Dekker: New York, NY, USA, 2001; pp. 349–352.
[2]
Lau, C.; Anitole, K.; Hodes, C.; Lai, D.; Pfahles-Hutchens, A.; Seed, J. Perfluoroalkyl acids: A review of monitoring and toxicological findings. Toxicol. Sci. 2007, 99, 366–394, doi:10.1093/toxsci/kfm128.
Renner, R. Growing concern over perfluorinated chemicals. Environ. Sci. Technol. 2001, 35, 154A–160A, doi:10.1021/es012317k.
[5]
Zhao, Y.G.; Wong, C.K.; Wong, M.H. Environmental contamination, human exposure and body loadings of perfluorooctane sulfonate (PFOS), focusing on Asian countrie. Chemosphere 2012, 89, 355–368, doi:10.1016/j.chemosphere.2012.05.043.
[6]
Guruge, K.S.; Taniyasu, S.; Yamashita, N.; Wijeratna, S.; Mohotti, K.M.; Seneviratne, H.R.; Kannan, K.; Yamanaka, N.; Miyazaki, S. Perfluorinated organic compounds in human blood serum and seminal plasma: A study of urban and rural tea worker populations in Sri Lanka. J. Environ. Monit. 2005, 7, 371–377, doi:10.1039/b412532k.
[7]
Karrman, A.; Ericson, I.; van Bavel, B.; Darnerud, P.O.; Aune, M.; Glynn, A.; Lignell, S.; Lindstrom, G. Exposure of perfluorinated chemicals through lactation: Levels of matched human milk and serum and a temporal trend, 1996–2004, in Sweden. Environ. Health Persp. 2007, 115, 226–230.
[8]
Sohlenius, A.K.; Lundgren, B.; Depierre, J.W. Perfluorooctanoic acid has persistent effects on peroxisome proliferation and related parameters in mouse-liver. J. Biochem. Toxicol. 1992, 7, 205–212, doi:10.1002/jbt.2570070403.
Seacat, A.M.; Thomford, P.J.; Hansen, K.J.; Olsen, G.W.; Case, M.T.; Butenhoff, J.L. Subchronic toxicity studies on perfluorooctanesulfonate potassium salt in cynomolgus monkeys. Toxicol. Sci. 2002, 68, 249–264, doi:10.1093/toxsci/68.1.249.
[11]
Governments Unite to Step-Up Reduction on Global DDT Reliance and Add Nine New Chemicals under inTernational Treaty; Stockholm Convention Secretariat: Geneva, Switzerland, 2009.
[12]
Joung, K.E.; Jo, E.H.; Kim, H.M.; Choi, K.; Yoon, J. Toxicological effects of PFOS and PFOA on earthwrom, Eisenia fetida. Environ. Health Toxicol. 2010, 25, 181.
[13]
Renner, R. EPA finds record PFOS, PFOA levels in Alabama grazing fields. Environ. Sci. Technol. 2009, 43, 1245–1246, doi:10.1021/es803520c.
[14]
Zareitalabad, P.; Siemens, J.; Hamer, M.; Amelung, W. Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) in surface waters, sediments, soils and wastewater - A review on concentrations and distribution coefficients. Chemosphere 2013, 91, 725–732, doi:10.1016/j.chemosphere.2013.02.024.
[15]
Cho, C.R.; Eom, I.; Kim, E.J.; Kim, S.; Choi, K.; Cho, H.; Yoon, J. Evaluation of the level of PFOS and PFOA in environmental media from industrial area and major river basin. J. Korea Soc. Environ. Anal. 2009, 12, 296–306.
[16]
Li, F.; Zhang, C.J.; Qu, Y.; Chen, J.; Chen, L.; Liu, Y.; Zhou, Q. Quantitative characterization of short- and long-chain perfluorinated acids in solid matrices in Shanghai, China. Sci. Total Environ. 2010, 408, 617–623, doi:10.1016/j.scitotenv.2009.10.032.
[17]
Sepulvado, J.G.; Blaine, A.C.; Hundal, L.S.; Higgins, C.P. Occurrence and fate of perfluorochemicals in soil following the land application of municipal biosolids. Environ. Sci. Technol. 2011, 45, 8106–8112, doi:10.1021/es103903d.
[18]
Das, P.; Megharaj, M.; Naidu, R. Perfluorooctane sulfonate release pattern from soils of fire training areas in Australia and its bioaccumulation potential in the earthworm Eisenia fetida. Environ. Sci. Pollut. Res. 2013, doi:10.1007/s11356-013-1782-y.
[19]
Edwards, C.A.; Bohlen, P.J. The effects of toxic-chemicals on earthworms. Rev. Environ. Contam. Toxicol. 1992, 125, 23–99, doi:10.1007/978-1-4612-2890-5_2.
[20]
Svendsen, C.; Spurgeon, D.J.; Hankard, P.K.; Weeks, J.M. A review of lysosomal membrane stability measured by neutral red retention: is it a workable earthworm biomarker? Ecotox. Environ. Saf. 2004, 57, 20–29, doi:10.1016/j.ecoenv.2003.08.009.
[21]
Sindermann, A.B.; Porch, J.R.; Krueger, H.O.; van Hoven, R.L. PFOS: An acute toxicity study with the earthworm in an artificial soil substrate. In Project No. 454–111. EPA Docket AR226-1106; Wildlife International Ltd.: Easton, MD, USA, 2002.
[22]
Mosleh, Y.Y.; Paris-Palacios, S.; Couderchet, M.; Vernet, G. Acute and sublethal effects of two insecticides on earthworms (Lumbricus terrestris L.) under laboratory conditions. Environ. Toxicol. 2003, 18, 1–8, doi:10.1002/tox.10095.
[23]
Neuhauser, E.F.; Callahan, C.A. Growth and reproduction of the earthworm Eisenia-Fetida exposed to sublethal concentrations of organic-chemicals. Soil Biol. Biochem. 1990, 22, 175–179, doi:10.1016/0038-0717(90)90083-C.
[24]
Stubberud, H. ？kotoksikologiske effecter av PFOS, PFOA og 6:2 FTS p？ meitemark (Eisenia fetida). (in Norwegian, English summary); Norwegian State Pollution Control Authority: Oslo, Norway, 2006.
[25]
Lankadurai, B.P.; Wolfe, D.M.; Simpson, A.J.; Simpson, M.J. 1H NMR-based metabolomic analysis of the time-dependent response of Eisenia fetida after sub-lethal phenanthrene exposure. Environ. Pollut. 2011, 159, 2845–2851, doi:10.1016/j.envpol.2011.04.044.
[26]
Beckwith-Hall, B.M.; Nicholson, J.K.; Nicholls, A.W.; Foxall, P.J.D.; Lindon, J.C.; Connor, S.C.; Abdi, M.; Connelly, J.; Holmes, E. Nuclear magnetic resonance spectroscopic and principal components analysis investigations into biochemical effects of three model hepatotoxins. Chem. Res. Toxicol. 1998, 11, 260–272, doi:10.1021/tx9700679.
Simpson, M.J.; McKelvie, J.R. Environmental metabolomics: new insights into earthworm ecotoxicity and contaminant bioavailability in soil. Anal. Bioanal. Chem. 2009, 394, 137–149, doi:10.1007/s00216-009-2612-4.
[29]
Viant, M.R.; Bundy, J.G.; Pincetich, C.A.; de Ropp, J.S.; Tjeerdema, R.S. NMR-derived developmental metabolic trajectories: an approach for visualizing the toxic actions of trichloroethylene during embryogenesis. Metabolomics 2005, 1, 149–158, doi:10.1007/s11306-005-4429-2.
[30]
Bundy, J.G.; Sidhu, J.K.; Rana, F.; Spurgeon, D.J.; Svendsen, C.; Wren, J.F.; Sturzenbaum, S.R.; Morgan, A.J.; Kille, P. “Systems toxicology” approach identifies coordinated metabolic responses to copper in a terrestrial non-model invertebrate, the earthworm Lumbricus rubellus. BMC Biol. 2008, 6, 25–46, doi:10.1186/1741-7007-6-25.
[31]
Ekman, D.R.; Teng, Q.N.; Villeneuve, D.L.; Kahl, M.D.; Jensen, K.M.; Durhan, E.J.; Ankley, G.T.; Collette, T.W. Profiling lipid metabolites yields unique information on sex- and time-dependent responses of fathead minnows (Pimephales promelas) exposed to 17 alpha-ethynylestradiol. Metabolomics 2009, 5, 22–32, doi:10.1007/s11306-008-0138-y.
[32]
Lankadurai, B.P.; Simpson, A.J.; Simpson, M.J. H-1 NMR metabolomics of Eisenia fetida responses after sub-lethal exposure to perfluorooctanoic acid and perfluorooctane sulfonate. Environ. Chem. 2012, 9, 502–511, doi:10.1071/EN12112.
[33]
Brown, S.A.E.; McKelvie, J.R.; Simpson, A.J.; Simpson, M.J. H-1 NMR metabolomics of earthworm exposure to sub-lethal concentrations of phenanthrene in soil. Environ. Pollut. 2010, 158, 2117–2123, doi:10.1016/j.envpol.2010.02.023.
[34]
Whitfield ？slund, M.; Simpson, A.J.; Simpson, M.J. 1H NMR metabolomics of earthworm responses to polychlorinated biphenyl (PCB) exposure in soil. Ecotoxicology 2011, 20, 836–846, doi:10.1007/s10646-011-0638-9.
[35]
Test No. 207: Earthworm, Acute Toxicity Tests. OECD Guidelines for the Testing of Chemicals, Section 2: Effects on Biotic Systems; OECD Publishing: Paris, France, 1984.
[36]
Soil Screening Levels for Perfluorooctanoic Acid (PFOA) and Perfluorooctyl Sulfonate (PFOS). EPA Region 4; USEPA: USA, 2009.
Bundy, J.G.; Lenz, E.M.; Bailey, N.J.; Gavaghan, C.L.; Svendsen, C.; Spurgeon, D.; Hankard, P.K.; Osborn, D.; Weeks, J.A.; Trauger, S.A. Metabonomic assessment of toxicity of 4-fluoroaniline, 3,5-difluoroaniline and 2-fluoro-4-methylaniline to the earthworm Eisenia veneta (Rosa): Identification of new endogenous biomarkers. Environ. Toxicol. Chem. 2002, 21, 1966–1972.
[40]
Eriksson, L.; Johansson, E.; Kettaneh-Wold, N.; Trygg, J.; Wikstrom, C.; Wold, S. Multi- and Megavariate Data Analysis Part 1: Basic Principles and Applications, 2nd ed. ed.; Umetrics AB: Umea, Sweden, 2006; pp. 63–98.
[41]
Lankadurai, B.P.; Wolfe, D.M.; Whitfield ？slund, M.; Simpson, A.J.; Simpson, M.J. 1H NMR-based metabolomic analysis of polar and non-polar earthworm metabolites after sub-lethal exposure to phenanthrene. Metabolomics 2013, 9, 44–56.
[42]
Sverdrup, L.E.; Nielsen, T.; Krogh, P.H. Soil ecotoxicity of polycyclic aromatic hydrocarbons in relation to soil sorption, lipophilicity, and water solubility. Environ. Sci. Technol. 2002, 36, 2429–2435, doi:10.1021/es010180s.
[43]
Ekman, D.R.; Teng, Q.; Villeneuve, D.L.; Kahl, M.D.; Jensen, K.M.; Durhan, E.J.; Ankley, G.T.; Collette, T.W. Investigating compensation and recovery of fathead minnow (Pimephales promelas) exposed to 17 alpha-ethynylestradiol with metabolite profiling. Environ. Sci. Technol. 2008, 42, 4188–4194, doi:10.1021/es8000618.
[44]
Yuk, J.; Simpson, A.J.; Simpson, M.J. 1D and 2D NMR metabolomics of earthworm responses to sub-lethal trifluralin and endosulfan exposure. Environ. Chem. 2011, 8, 281–294, doi:10.1071/EN11033.
[45]
Lankadurai, B.P.; Wolfe, D.M.; Simpson, A.J.; Simpson, M.J. 1H NMR-based metabolomic observation of a two-phased toxic mode of action in Eisenia fetida after sub-lethal phenanthrene exposure. Environ. Chem. 2011, 8, 105–114, doi:10.1071/EN10094.
[46]
Vanden Heuvel, J.P.; Thompson, J.T.; Frame, S.R.; Gillies, P.J. Differential activation of nuclear receptors by perfluorinated fatty acid analogs and natural fatty acids: A comparison of human, mouse, and rat peroxisome proliferator-activated receptor-alpha, -beta, and -gamma, liver X receptor-beta, and retinoid X receptor-alpha. Toxicol Sci. 2006, 92, 476–489, doi:10.1093/toxsci/kfl014.
[47]
Haughom, B.; Spydevold, O. The mechanism underlying the hypolipemic effect of perfluorooctanoic acid (PFOA), perfluorooctane sulfonic-acid (Pfosa) and clofibric acid. Biochim. Biophys. Acta 1992, 1128, 65–72, doi:10.1016/0005-2760(92)90258-W.
[48]
Van Gilst, M.R.; Hadjivassiliou, H.; Jolly, A.; Yamamoto, K.R. Nuclear hormone receptor NHR-49 controls fat consumption and fatty acid composition in C. elegans. PLoS Biol. 2005, 3, e53, doi:10.1371/journal.pbio.0030053.
[49]
Atherton, H.J.; Jones, O.A.H.; Malik, S.; Miska, E.A.; Griffin, J.L. A comparative metabolomic study of NHR-49 in Caenorhabditis elegans and PPAR-alpha in the mouse. FEBS Lett. 2008, 582, 1661–1666, doi:10.1016/j.febslet.2008.04.020.
[50]
McKelvie, J.R.; Wolfe, D.M.; Celejewski, M.; Simpson, A.J.; Simpson, M.J. Correlations of Eisenia fetida metabolic responses to extractable phenanthrene concentrations through time. Environ. Pollut. 2010, 158, 2150–2157, doi:10.1016/j.envpol.2010.02.022.
[51]
Vanwezel, A.P.; Opperhuizen, A. Narcosis due to environmental-pollutants in aquatic organisms—Residue-based toxicity, mechanisms, and membrane burdens. Crit. Rev. Toxicol. 1995, 25, 255–279.
[52]
Jones, O.A.H.; Spurgeon, D.J.; Svendsen, C.; Griffin, J.L. A metabolomics based approach to assessing the toxicity of the polyaromatic hydrocarbon pyrene to the earthworm Lumbricus rubellus. Chemosphere 2008, 71, 601–609, doi:10.1016/j.chemosphere.2007.08.056.
Cheng, W.; Yu, Z.; Feng, L.; Wang, Y. Perfluorooctane sulfonate (PFOS) induced embryotoxicity and disruption of cardiogenesis. Toxicol. in Vitro 2013, 27, 1503–1512, doi:10.1016/j.tiv.2013.03.014.
[57]
Panaretakis, T.; Shabalina, I.G.; Grander, D.; Shoshan, M.C.; DePierre, J.W. Reactive oxygen species and mitochondria mediate the induction of apoptosis in human hepatoma HepG2 cells by the rodent peroxisome proliferator and hepatocarcinogen, perfluorooctanoic acid. Toxicol. Appl. Pharm. 2001, 173, 56–64, doi:10.1006/taap.2001.9159.
[58]
Horton, H.R.; Moran, L.A.; Scrimgeour, K.G.; Perry, M.D.; Rawn, J.D. Principles of Biochemistry, 4th ed. ed.; Pearson Prentice Hall: Upper Saddle River, NJ, USA, 2006.
[59]
Rylander, C.; Dumeaux, V.; Olsen, K.S.; Waaseth, M.; Sandanger, T.M.; Lund, E. Usingdy blood gene signatures for assessing effects of exposure to perfluoroalkyl acids (PFAAs) in humans: The NOWAC postgenome study. Int. J. Mol. Epidemiol. Genet. 2011, 2, 207–216.
[60]
Guruge, K.S.; Yeung, L.W.Y.; Yamanaka, N.; Miyazaki, S.; Lam, P.K.S.; Giesy, J.P.; Jones, P.D.; Yamashita, N. Gene expression profiles in rat liver treated with perfluorooctanoic acid (PFOA). Toxicol. Sci. 2006, 89, 93–107.
[61]
Zhang, H.X.; Ding, L.N.; Fang, X.M.; Shi, Z.M.; Zhang, Y.T.; Chen, H.B.; Yan, X.Z.; Dai, J.Y. Biological responses to perfluorododecanoic acid exposure in rat kidneys as determined by integrated proteomic and metabonomic studies. PLoS One 2011, 6, e20862.
[62]
Strange, K.; Morrison, R.; Heilig, C.W.; Dipietro, S.; Gullans, S.R. Up-regulation of inositol transport mediates inositol accumulation in hyperosmolar brain-cells. Am. J. Physiol. 1991, 260, C784–C790.
R Development Core Team, R: A Language and Environment for Statistical Computing, R Foundation for Statistical Computing, Vienna, Austria, 2009.
[73]
Filzmoser, P.; Varmuza, K. Chemometrics: Multivariate Statistical Analysis in Chemometrics, R package Version 0:8, 2010.
[74]
Varmuza, K.; Filzmoser, P. Introduction to Multivariate Statistical Analysis in Chemometrics; CRC Press: Boca Raton, FL, USA, 2009.
[75]
Westerhuis, J.A.; Hoefsloot, H.C.J.; Smit, S.; Vis, D.J.; Smilde, A.K.; van Velzen, E.J.J.; van Duijnhoven, J.P.M.; van Dorsten, F.A. Assessment of PLSDA cross validation. Metabolomics 2008, 4, 81–89, doi:10.1007/s11306-007-0099-6.
[76]
Alam, T.M.; Alam, N.K.; Neerathilingam, M.; Volk, D.E.; Sarkar, S.; Shakeel Ansari, G.A.; Luxon, B.A. 1H NMR metabonomic study of rat response to tri-phenyl phosphate and tri-butyl phosphate exposure. Metabolomics 2010, 6, 386–394, doi:10.1007/s11306-010-0205-z.
[77]
Eriksson, L.; Jaworska, J.; Worth, A.P.; Cronin, M.T.; McDowell, R.M.; Gramatica, P. Methods for reliability and uncertainty assessment and for applicability evaluations of classification- and regression-based QSARs. Environ. Health Persp. 2003, 111, 1361–1375, doi:10.1289/ehp.5758.
[78]
Rochfort, S.J.; Ezernieks, V.; Yen, A.L. NMR-based metabolomics using earthworms as potential indicators for soil health. Metabolomics 2009, 5, 95–107, doi:10.1007/s11306-008-0140-4.
[79]
Higgins, C.P.; Luthy, R.G. Sorption of perfluorinated surfactants on sediments. Environ. Sci. Technol. 2006, 40, 7251–7256, doi:10.1021/es061000n.
[80]
Sohlenius, A.K.; Eriksson, A.M.; Hogstrom, C.; Kimland, M.; Depierre, J.W. Perfluorooctane sulfonic-acid Is a potent inducer of peroxisomal fatty-acid beta-oxidation and other activities known to be affected by peroxisome proliferators in mouse-liver. Pharmacol. Toxicol. 1993, 72, 90–93, doi:10.1111/j.1600-0773.1993.tb00296.x.
