Earthworm Glutathione-Glutathione-S-Transferase: A Biomarker in Ecological Risk Assessment

Modernization of human society generates numerous chemical from different sources and stimulate serious hazard to the ecosystem. Consequently, people are exposed to thousands of chemicals on daily basis by food, air, water, soil, dust, or the products we use without knowing their harmful effect. It’s not easy for people to identify if those chemicals are risky to their wellbeing or not. These encountered problems require immediate scientific attention to find appropriate and cost effective solutions. Most ecological risk assessments are based on chemical residue analysis of environmental parameters, such as surface water, sediments and soils in addition to determination of species diversity and density of wildlife, though the biological significance of measured chemical concentrations are not so specific and quantitative [1]. Chemical analysis cannot predict actual bio available chemicals, assumed that it is 100% as obtained from analysis of environmental matrix.

Also the field assessment of individual survival and reproductive success cannot speculate the xenobiotics induced alteration in behavioural, biochemical, physiological or toxic responses in the organisms/ species, could affect the stability of a population. As a result, environmental modelling is unable to state real effect of the chemicals in people because of the large difference in predicted levels with respect to the actual levels of chemicals in the living organisms. To evaluate environmental health as well as effects of contaminants on organisms there is need of a biological approach as physicochemical analyses are unable to provide proper information on the biological status of ecosystems. Consequently, bio monitoring is most health relevant way of assessing exposure of environmental chemicals in organism to avoid errors in interpretation of environmental levels (measured in air, soil, water and food). In the course of bio-monitoring, biomarkers are the observable properties of an organism and are vital for the exposure assessment during direct measurement process. In true sense, Biologic markers or biomarkers may be used as the early warning indices of biologic effect and as quantitative measures of chemical exposures and biologically effective doses [2,3]. Biomarkers can provide us the evidence of exposure, the route and pathway of exposure, finally the effects of one or more chemical exposure. Biomarkers also have the ability to identifying priority exposures, recognizing time trends in exposure, identifying at risk populations (Large biomarker studies can distinguish exposure differences among racial, geographic or socio-economic groups), providing integrated dose measurements (biomarker analysis provides a direct assay of body burden that integrates exposure from all sources, even ones that hard to measure) and finally evaluating exposure prevention efforts [4]. For this solicitation, three specific types of biomarkers will be considered [5].

1. Biomarkers of exposure- Measure xenobiotics, metabolite(s) or the obtained products after interaction of a chemical with the cells or target molecules within an organism.
2. Biomarkers of effects- Assessable changes within an organism, which depends on the magnitude of exposure, can signify potential or recognized health impairment.
3. Biomarkers of susceptibility- Markers of inherent or acquired properties of an organism that make an individual more susceptible upon exposure to a specific chemical.

Continuous addition of foreign chemicals by urban communities and industries to the environment make people aware of the potential long-term adverse effects of these xenobiotics in general and their potential risks for aquatic and terrestrial ecosystems in particular. To maintain a healthy terrestrial food web, the sustainable use of soils is essential. The presence of a xenobiotic compound in a segment of a terrestrial ecosystem does not, by itself, indicate injurious effects. There should have established correlation between exposure levels, bio-accumulation of contaminants and early adverse effects.

In an environmental context, biomarkers offer promise as sensitive indicators demonstrating that toxicants have entered organisms, have been distributed between tissues, and are eliciting a toxic effect at critical targets [6]. Consequently, the development and subsequent application of sensitive laboratory bioassays, based on the biomarkers responses, such as the Glutathione (GSH), Glutathione-S-transferase (GST), Metallothionein (MT), Acetyl cholinesterase (AchE) responses [7-12] are important aspect of environmental research. Bioassays offer many advantages for comparing the relative toxicity of specific chemicals or specific effluents.

Earthworms have universally attracted considerable attention as one of the most suitable and representative animals to be used for ecotoxicity testing of industrial wastes disposed as land fillings and field applications of pesticides [13-18], by the European Economic Community (EEC), the Organization for Economic Cooperation and Development (OECD), the Food and Agriculture Organization of the United Nations (FAO), the American Society for Testing Materials [19,20], by many national pesticide registration authorities including the US EPA [21], and environmental pollution committees [22]. Earthworms have been regarded as an alternative to in vivo rodent bioassays [23,24]. Because they are readily available and easy to handle in laboratory and also in field condition. Earthworms are important component of terrestrial food web because of their contribution in enhancing overall soil productivity by changing soil properties like decomposition of organic litter, increasing soil porosity, water drainage, and aeration and enhancing microbial activities of soil. These features enhance their popularity to consider them as an excellent bio indicators of soil pollution [25,26]. Thus, earthworms provide the society a predictive tool of ecosystem quality as biomarker of chemical contamination [27,28]. This helps us to develop an experimental model that can assess ecotoxicological risk within an ecosystem. Pesticides can exert both direct toxicity against earthworms or produce latent effects on their growth, histology and fertility. In addition, metals and pesticide-contaminated earthworms are a source of contamination to higher members of the food web, e.g. birds or mammals.

All living organisms contain enzymes and bio molecules with antioxidant capabilities to protect them against the adverse effects of reactive oxygen species (ROS) and xenobiotics [29]. GSH and GST of the phase II biotransformation system have been considered as biomarkers of environmental pollution because of their major role in cellular defence mechanisms [30]. GSTs and glutathione interact perfectly in protecting cells from electrophilic xenobiotics and endogenous toxic compounds. They are also involved in hormone metabolism and intercellular signal cascades [31]. The enzyme class shows a high flexibility and represents the starting point of well-developed metabolic pathways in the degradation and excretion of xenobiotic conjugates [32].

The cytosolic as well as the microsomal GSTs of most species are dimeric enzymes (consisting of subunits of the same or similar size between MW 23 and 30kDa) that have extensive ligand binding properties in addition to their catalytic role in detoxification [33,34]. The determination of subunit size is in most cases deduced from SDS-PAGE separation and migration [31]. The ubiquitous distribution of GSTs and glutathione in animals and plants assume their joint co-evolution. However, so far, little is known about the oxidative stress and antioxidant defences in earthworms. [35,36], reported the existence of glutathione (GSH)-glutathione-S-transferase (GST) involved operating system in six species of Lumbricidae. Maity et al. [8] recorded a significant perturbation in GSH-GST and other enzymes (glutathione peroxidise and glutathione reductase) involved in antioxidant defence systems in Lampito mauritii against Pb and Zn treatment. A recent report of [37] noted that a combined effect of different trace elements induces GST activity and reduces cellular GSH level in earthworm Aporrectodea caliginosa, sampled from different heavy metal polluted sites of eastern Slovakia. According to the earlier investigations [38,39], the coupled reaction of GSH consumption and GST activation is essential to retain homeostasis of the cellular internal environment. Also reported that GST-GST responses depend on the dose and duration of metal exposure and the species of earthworm. In response to organic and inorganic pollutant perturbations of glutathione concentration, activity of glutathione-S-transferases, glutathione reductase and glutathione peroxidase have been reported in earthworm [40-45], recorded induction in GST activity of earthworm (Pheretima posthuma) exposed to three insecticides (aldrin, endosulphan or lindane) with subsequent decline to control level due to the biotransformation and elimination of pesticides [46]. Also reported induction in GST activity of Apporectodea caliginosa exposed to organophosphate pesticides (chlorpyrifos and diazinon) [47]. The biochemical/ molecular biomarker like GST responses in earthworms could have an ecological meaning when they can be related directly to behavioural responses with significant ecological impact. However, very little research has demonstrated such a relationship. In light of the above discussion we can conclude that at the present time we need extensive research associating earthworm biomarkers as the biomarker is increasingly becoming a suitable tool for soil contamination survey for assessing the progress of remediation actions in contaminated soils.

1. Mc Carthy JF, Halbrook RS, Shugart LR (1991) Conceptual strategy for design, implementation, and validation of a biomarker-based biomonitoring capability. Environmental Sciences Division, Oak Ridge National Laboratory, Tennessee, USA.
2. NRC (1987) Committe on biological markers. Environ Health Perspect 74: 3-9.
3. Depledge MH, Aagard A, Györkös P (1995) Assessment of trace metal toxicity using molecular, physiological and behavioural biomarkers. Mar Pollut Bull 31(3): 19-27.
4. Sexton K, Needham LL, Pirkle JL (2004) Human bio monitoring of environmental chemicals. Measuring chemicals in human tissue is the ‘gold standard’ for assessing people’s exposure to pollution. American Scientist 92(1): 38-45.
5. Bearer CF (1998) Biomarkers in pediatric environmental health: a cross-cutting issue. Environ Health Perspect 106(3): 813-816.
6. Mc Carthy JF, Shugart LR (1990) Biological markers of environmental contamination. Biomarkers of environmental contamination. Lewis Publishers, Boca Raton, USA, pp. 3-16.
7. Saint DM, Narbonne JF, Arnaud C, Ribera D (2001) Biochemical responses of the earthworm Eisenia fetida andrei exposed to contaminated artificial soil: effects of lead acetate. Soil Biology & Biochemistry 33(3): 395-404.
8. Maity S, Roy S, Chaudhury S, Bhattacharya S (2008) Antioxidant responses of the earthworm Lampito mauritii exposed to Pb and Zn contaminated soil. Environ Pollut 151(1): 1-7.
9. Maity S, Bhattacharya S, Chaudhury S (2009) Metallothionein response in earthworms Lampito mauritii (Kinberg) exposed to fly ash. Chemosphere 77(3): 319-324.
10. Maity S, Roy S, Bhattacharya S, Chaudhury S (2010) Accumulation of lead and zinc in earthworm Lampito mauritii (Kinberg): effect on survival, growth and acetylcholinesterase activity. Recent Research in Science and Technology 2(4): 46-53.
11. Maity S, Roy S, Bhattacharya S, Chaudhury S (2010) Metallothionein responses in the earthworm Lampito mauritii (Kinberg) exposed to lead and zinc: a biomarker of metal contamination. European Journal of Soil Biology 47(1): 69-71.
12. Maity S, Poráčová J, Dey P, Vašková J, Vaško L, et al. (2018) Antioxidant responses in the earthworm Aporrectodea caliginosa of eastern Slovakia: application of principal component analysis as a tool to identify metal contaminated areas. Environmental Monitoring and Assessment 190(1): 21.
13. Viswanathan R (1997) Physiological basis in the assessment of ecotoxicity of pesticides to soil organisms. Chemosphere 35 (1-2): 323- 334.
14. Leland JE, Mullis DE, Berry DF (2001) Evaluating environmental hazards of land applying composted diazinon using earthworm bioassays. J Environ Sci Health 36(6): 831-834.
15. Booth LH, Heeppelthwaite V, Eason CT (1998) Choline esterase and glutathione S-transferase in the earthworm Aporrectodea caliginosa as biomarkers of organophosphate exposure. Proc 51st New Zeeland Plant Protection Conf, New Zealand, pp. 138-142.
16. Booth LH, Ohalloran K (2001) A comparison of biomarker responses in the earthworms Aporrectodea caliginosa to the organophosphorus insecticides diazinon and chlropyrifos. Environ Toxicol Chem 20(11): 2498-2502.
17. American Society for Testing and Materials (1995) Standard guide for conducting a laboratory soil toxicity test with lumbricid earthworm. In: Eisenia foetida E (Ed.), Annual Book of ASTM Standards ASTM, Philidelphia, USA, pp. 1123-1139.
18. OECD (1984) Guidelines for testing of chemicals, Test 207, Earthworms acute toxicity test. OECD Paris, pp. 1-11.
19. Greene JC, Bartels CL, Warren HW, Parkhurst BR, Linder GL, et al. (1989) Protocols for short term toxicity screening of hazardous waste sites. Environmental Protection Agency, USA.
20. Goats GC, Edwards CA (1988) The prediction of field toxicology of chemicals to earthworms by laboratory methods. In: Earthworms in Waste and Environmental Management. In Edwards CA, Neuhauser EF (Eds.), SPB Academic Publishing, The Hague, Belgium, pp. 283-294.
21. Römbke J, Förster B, Jänsch S, Scheffczyk A, Garcia M (2005) Terrestrische ökotoxikologische testmethoden für die Tropen - teil 1: labortests mit regenwürmern und arthropoden. UWSF - Umwelt Chem Ökotox 17(1): 20-27.
22. Cortet J, Gomot DV, Poinsot BN, Gomot L, Texier C, et al. (1999) The use of invertebrate soil fauna in monitoring pollutant effects. European Journal of Soil Biology 35: 115-134.
23. Lanno RP, Wells J, Conder K, Bradham, Basta N (2004) The bioavailability of chemicals in soil for earthworms. Ecotoxicology and Environmental Safety 57(1) 39- 47.
24. Paoletti MG (1999) The role of earthworms for the assessment of sustainability and as bio indicators. Agric Ecosystem & Environ 74(1-3): 137-155.
25. Hund RK, Achazi R, Römbke J, Warnecke D (2003) Avoidance test with Eisenia fetida as indicator for the habitat function of soils: Results of a laboratory comparison test. J Soils Sediments 3(1): 7-12.
26. Saint DM, Labrot F, Narbonne JF, Ribera D (1998) Glutathione, glutathione related enzymes and catalase activities in the earthworm Eisenia fetida andrei. Arch Environ Contam Toxicol 35(4): 602-614.
27. Muthukaruppan G, Janardhanan S, Vijayalakshmi GS (2005) Sublethal toxicity of the herbicide butachlor on the earthworm Perionyx sansibaricus and its histological changes. J Soils Sediments 5(2): 82-86.
28. Cole D (1994) Detoxification and activation of agrochemicals in plants. Pest Sci 42: 209-222.
29. Listowsky I, Abramovitz M, Homma H, Niitsu Y (1988) Intracellular binding and transport of hormones and xenobiotics by glutathione S-transferase. Drug Metab Rev 19(4): 305-318.
30. Schröder P (2001) The role of glutathione amd glutathione s-transferase in plant reaction and adaptation to xenobiotics. In Grill D, Tausz M, DeKok LJ (Eds.), Significance of glutathione in plant adaptation to the environment. Kluwer Academic Publ, Dordrecht, Netherlands, pp. 155- 218.
31. Ketley JN, Habig WH, Jakoby WB (1976) Binding of non substrate ligands to the glutathione S-transferases. J Biol Chem 250: 8670-8673.
32. Barycki JJ, Colman RF (1997) Identification of the nonsubstrate steroid binding site of the rat liver glutathione S-transferase, isoenzyme 1-1, by the steroid affinity label, 3b-(iodoacetoxy) dehydroisoandrosterone. Arch Biochem Biophys 345:16-31.
33. Stenersen J, Guthenberg C, Mannervik B (1979) Glutathione S-transferase in earthworms (Lumbricidae). Biochem J 181(1): 47-50.
34. Stenersen J, Oien N (1981) Glutathione S-transferase in earthworms (Lumbricidae). Substrate specificity, tissue and species distribution and molecular weight. Comp Biochem Physiol C 69(2): 243-252.
35. Stenersen J (1984) Detoxification of xenobiotic by earthworms. Comp Biochem Physiol C 78(2): 249-252.
36. Stenersen J, Brekke E, Engelstad F (1992) Earthworms for toxicity testing: species difference in response towards choline esterase inhibiting insecticides. Soil Biol Biochem 24(12): 1761-1764.
37. Zhang X, Lua Y, Shia Y, Chena C, Yang Z, et al. (2009) Antioxidant and metabolic responses induced by cadmium and pyrene in the earthworm Eisenia fetida in two different systems: contact and soil tests. Chem Ecol 25(3): 205-215.
38. Maity S, Banerjee R, Goswami P, Chakrabarti M, Mukherjee A (2018) Oxidative stress responses of two different ecophysiological species of earthworms (Eutyphoeus waltoni and Eisenia fetida) exposed to Cdcontaminated soil. Chemosphere 203: 307-317.
39. Panzarino O, Hyr P, Dobe P, Vojtek L, Vernile P, et al. (2016) Rank-based biomarker index to assess cadmium ecotoxicity on the earthworm Eisenia andrei. Chemosphere 145: 480-486.
40. Saint DM, Narbonne JF, Arnaud C, Thybaud E, Ribera D (1999) Biochemical responses of the earthworm Eisenia fetida andrei exposed to contaminated artificial soil: effects of benzo(a) pyrene. Soil Biology & Biochemistry 31(13): 1837-1846.
41. Saint DM, Narbonne JF, Arnaud C, Thybaud E, Ribera D (1998) Glutathione, glutathione related enzymes, and catalase activities in the earthworm Eisenia fetida andrei. Archives of Environmental Contamination and Toxicology 35(4): 602-614.
42. Łaszczyca P, Augustyniak M, Babczynska A, Bednarska K, Kafel A, et al. (2004) Profiles of enzymatic activity in earthworms from zinc, lead and cadmium polluted areas near Olkusz (Poland). Environment International 30(7): 901-910.
43. Hans (RK), Khan MA, Farooq M, Beg MU (1993) Glutathione-Stransferase activity in an earthworm (Pheretima posthuma) exposed to three insecticides. Soil Biology and Biochemistry 25(4): 509-511.
44. Borgeraas J, Nilsen K, Strensen J (1996) Methods for purification of glutathione transferase in the earthworm genus Eisenia and their characterization. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 114(2): 129-140.
45. Farenhorstn A, Bowman BT (2000) Sorption of atrazine and metolachlor by earthworm surface castings and soil. J Environ Sci Health B 35(2): 157-173.
46. Furst A (2002) My saga with earthworms. Food Chem Toxicol 40(6): 789-791.