Ecotoxicity of Single-Wall Carbon Nanotubes to Freshwater Snail Lymnaea luteola L.: Impacts on Oxidative Stress and Genotoxicity Daoud Ali,1 Mukhtar Ahmed,1 Saud Alarifi,1 Huma Ali2 1
Department of Zoology, College of Science, King Saud University, Riyadh, 11451, Saudi Arabia
2
Department of Chemistry, Maulana Azad National Institute of Technology, Bhopal, Madhya Pradesh, India
Received 2 November 2013; revised 19 December 2013; accepted 22 December 2013 ABSTRACT: Mammalian studies have raised concerns about the toxicity of carbon nanotubes, but there is very limited data on ecogenotoxicity to aquatic organisms. The aim of this study was to determine ecogeno toxic effects of single walled carbon nanotubes (SWCNTs) in fresh water snail, Lymnea luteola (L. luteola). A static test system was used to expose L. luteola to a freshwater control, 0.05, 0.15, 0.30, 0.46 mg/L SWCNTs for up to 4 days. SWCNTs changed a significant reduction in glutathione, glutathioneS-transferase, and glutathione peroxidase with in hepatopancreas of L. luteola. Lipid peroxidation (LPO) and catalase showed dose- and time-dependent and statistically significant increase in hepatopancreas during SWCNTs exposure compared with control. However, a significant (p < 0.01) induction in DNA damage was observed by the comet assay in hepatopancreas cells treated with SWCNTs. These results demonstrate that SWCNTs are ecogenotoxic to freshwater snail L. luteola. The oxidative stress and C 2014 Wiley comet assay can successfully be used as sensitive tools of aquatic pollution biomonitoring. V Periodicals, Inc. Environ Toxicol 00: 000–000, 2014.
Keywords: single walled carbon nanotubes; oxidative stress; DNA damage; comet assay; Lymnea luteola L
INTRODUCTION Carbon nanomaterials showing unique physicochemical, mechanical, and electrical properties are increasingly employed in nanotechnology and in several consumer products. Carbon nanotubes are currently used in desalination plants, artificial muscles, paints, solar cells, electronics, and industrial lubricants. Aquatic environments face potential Correspondence to: D. Ali; e-mail: [email protected] or [email protected] Contract grant sponsor: Deanship of Scientific Research at King Saud University. Contract grant number: RGP-VPP-300 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.21945
risks from nanomaterials through wastewater treatment plant spills, rain, and runoff (Glenn et al., 2012). Risk assessment of ecotoxicology of nanomaterials in the aquatic environment is essential as their use and manufacturing are rapidly expanding. Carbon nanotubes shown a significant impacts on survival or growth to several species of freshwater aquatic invertebrates including Hyallela azteca, Chironomus dilutes, Lumbriculus variegates, Villosa iris, and Daphnia magna (Petersen et al., 2009; Mwangi et al., 2012). A number of studies have reported potential impacts of nanoparticles on aquatic organisms and their toxicity can be related to dissolution, size, or surface properties (Blaise et al., 2008; KingHeiden et al., 2009; Jackson et al., 2013). Uptake of xenobiotics by endocytotic routes was identified as a probable major mechanism of entry into cells, particularly in
C 2014 Wiley Periodicals, Inc. V
1
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ALI ET AL.
suspension-feeding invertebrates, thus potentially lead to various types of toxic cell injury in many tissues, such as gills and digestive gland, as well as in immune cells which are endowed with high phagocytic capacity (Baun et al., 2008). Smith et al. (2007) reported that single-walled carbon nanotubes produced respiratory toxicity, organ pathologies, and other physiological effects in rainbow trout. Gastropod organisms are ubiquitous in the aquatic ecosystem and are considered a good bioindicators of contaminants in view of their wide geographic distribution, relatively sedentary life habit, and their easy availability. In mollusks, the organ which plays the most important role in the metabolism of endogenous and xenobiotic compounds is the digestive gland (hepatopancreas) (Wilbrink et al., 1990). In snails, pollutants are transferred by blood cells to the digestive gland, which is one of the major target tissues of accumulation. Reactive oxygen species (ROS) and the resulting oxidative stress to living organisms have been reported to be related with the pollution-mediated mechanism of toxicity, which can damage macromolecules, such as DNA, proteins, and membranes. Antioxidant biomarkers are able to eliminate the highly reactive intermediate ROS induced by pollutants to maintain cell homeostasis, and they include antioxidant enzymes and antioxidant nonenzymatic scavengers, e.g., superoxide dismutase (SOD), catalase, glutathione-S-transferases (GST), and reduced glutathione (Trachootham et al., 2008). Malondialdehyde (MDA) is an end point of lipid peroxidation; therefore, the formation of MDA is regarded as a general indicator of lipid peroxidation. SOD and catalase activities play important role in the antioxidant protection of invertebrates (Livingstone, 2001). GST is a family of phase II detoxification enzymes that catalyze the conjugation of glutathione to a wide variety of endogenous and exogenous electrophilic compounds, such as therapeutic drugs and environmental toxins (Hayes et al., 2005). Oxidative stress may manifest as damage to tissue macromolecules, including proteins and DNA (Di Giulio et al., 1989). In addition, DNA damage is considered to be a biomarker of genotoxicity induced by noxious substances, and the comet assay has recently been considered a robust genotoxicological tool with which to assess DNA damage at the individual cell level. In the present study, we designed to assess the acute toxicity of SWCNTs to freshwater snail L. luteola, which is an important component of river, ponds, and lakes of South East Asian freshwater ecosystems. Our results also provide critical information to regulatory agencies and industry to determine the need for monitoring and regulation regarding SWCNTs.
MATERIALS AND METHODS Chemicals Single-walled carbon nanotube (SWCNTs) (CAS Number 308068-56-6, Product No. 750522 and average diameter
Environmental Toxicology DOI 10.1002/tox
0.01) higher DNA damage in SWCNTs exposed cells than control groups (Fig. 5). The gradual nonlinear increase in DNA damage was observed in cells as concentration and time exposure of SWCNTs increased and the highest damage of DNA was recorded at 0.30 mg/L SWCNTs [Fig. 5(a,b,d)].
DISCUSSION Several studies accentuate gastropods as sentinel organisms for the assessment of pollution in aquatic ecosystems (Hoang and Rand, 2009). The application of nanotechnology has been recently extended in the areas of medicine, biotechnology,
Environmental Toxicology DOI 10.1002/tox
materials and energy and environment. However, SWCNTs are known toxic nanomaterial to fish; its toxicity to fresh water snail is under-investigated and poorly understood. SWCNTs may induce deleterious effects in aquatic life after release into aquatic environment. However, nanotubes may present special problems with respect to their retention in cells and tissues, since it is believed that there is a threshold for the length of tubes that is critical for induction of adverse biological effects (Hoet et al., 2004). Although some researchers reported that size of the nanomaterial itself cause toxicity and biodegradability may be a further significant factor in governing harmful biological effects (Brown et al., 2001; Howard, 2004). The most severe problem became evident in the hepatopancreas, which is the main metabolic organ in snails and also involved in detoxification and accumulation of xenobiotics (Dallinger and Wieser, 1984; Tanhan et al., 2005). Dallinger (1994) has reported that freshwater snails are sentinel model to monitor aquatic pollution, so it is important to study SWCNTs mechanisms of toxicity and the state of their defence systems by the snails. We have observed that SWCNTs induced oxidative stress and DNA damage in hepatopancreas and hemolymph of L. luteola as dose- and timedependent manner. The hepatopancreas (digestive gland) of gastropod molluscs is the key organ of metabolism and it is concerned with the production of digestive enzymes, absorption of nutrients, endocytosis of food substances, food storage, and excretion (Dallinger et al., 2002). It has been
OXIDATIVE STRESS AND GENOTOXICITY
7
Fig. 5. DNA damage in hepatopancreas cells of L. luteola after exposure to SWCNTs. a. % Tail DNA. b. Olive tail moment. c. Control. d. Exposed cells (at 0.30 mg/L of SWCNTs). Data are presented as the mean 6 SE of triplicate experiments. *p < 0.01 vs. control. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
reported as a major site of xenobiotic, oxy-radical generating and biotransformation enzymes (Livingstone et al., 1992). Pollutants accumulation through different routes are transported by blood cells (hemolymph) to the digestive gland, which also represents the main target organ for metabolic and detoxification processes (Beeby and Richmond, 2002; Regoli et al., 2005). So in the present study we have used the hepatopancreas tissue to investigate the biochemical responses to SWCNTs. A general pathway of toxicity for many environmental pollutants is mediated by the enhancement of intracellular ROS, which modulate the occurrence of cell damage (Regoli et al., 2002) via initiation and propagation of lipid peroxidation (Gutteridge, 1995). The estimation of malondialdehyde content (an index of lipid peroxidation) provides a relative measure of the potential for SWCNTs to cause oxidative injury. Nusetti et al. (2001) reported that LPO levels increased during exposure to metals in several organisms. In the present study, significantly elevations of lipid peroxidation levels in the digestive gland of SWCNTs exposed L. luteola indicate that some cell damage might have occurred. GSH is a tripeptide nonenzymatic antioxidant with a single cysteine residue and constitutes an important pathway of the antioxidant and detoxification defences. It is also a cofactor of many enzymes catalyzing the detoxification and excretion of several toxicants, which will be destroyed in the cytosolic and mitochondrial compartments by GPx in the presence of GSH (Doyotte et al.,
1997). The decrease in GSH concentrations in our study might be attributed to the intensification of turnover between reduced and oxidized glutathione under the conditions, which cause increased consumption of this peptide for the synthesis of heavy metal-binding proteins, like metallothioneins. A decrease in GSH content in the digestive gland appears to be a common response of molluscs to metal exposure (Regoli and Principato, 1995). In this study, we observed a decrease in GSH content accompanied by the elevation of LPO levels. Through catalyzation by glutathione peroxidase, GSH can eliminate H2O2 and lipid hydro peroxide (Ahmed, 2005); therefore, a negative relationship between MDA and GSH was a reasonable result. GSH is one of the most important factors protecting from oxidative attacks by reactive oxygen species such as lipid peroxidation, because GSH acts as a reducing agent and free-radical trapper and is known to be a cofactor substrate and/or GSH-related enzymes (Verma et al., 2007). Catalase converts H2O2 to H2O and O2 to prevent oxidative stress and maintain cell homeostasis. Increased catalase activity indicated that O22 and H2O2 were formed during the freshwater snail exposure to contaminant (Zheng et al., 2013). The antioxidant catalase is an extremely important component of intracellular and antioxidant defences of organisms (Jamil, 2001). It reduces the H2O2 into water and oxygen to prevent oxidative stress and for maintaining cell homeostasis. Many studies have found varying responses of
Environmental Toxicology DOI 10.1002/tox
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catalase to increased metal exposures, with some organisms exhibiting increased activity, others exhibiting decreased activity, and still others showing no catalase response at all (Regoli et al., 1998). In the present study, we observed that catalase activity was significantly increased; this data suggests that the increase in antioxidant defences would be due to enhanced oxygen free radicals production, which could stimulate antioxidant activities (Torres et al., 2002) to cope with this increased oxidative stress and protect the cells from damage. The obtained results are in accordance with the findings of Almeida et al. (2004), who found that catalase activity was increased in mussels after exposure to lead. GPx is the most important peroxidase for the detoxification of hydro peroxides (Orbea et al., 2000). However, the decreased activities of GPx might be due to over production of ROS, especially O22, by the SWCNTs and depletion of its substrate level (GSH). It is also reported that under high rates of free radicals input, enzyme inactivation prevails and the enzymatic activities are reduced, leading to autocatalysis of oxidative damage process (Escobar et al., 1996). The role of GST is to conjugate tripeptide glutathione with electrophilic and other xenobiotic. Inhibition of GST activity may be occurred either through direct action of the metal on the enzyme or indirectly via the production of ROS that interact directly with the enzyme, depletion of its substrate (GSH), and/or down regulation of GST genes through different mechanisms (Roling and Baldwin, 2006). This explanation might be the reason for the GST activity decrease that was caused in the present study in the case of snails exposed to SWCNTs. Changes in the levels of antioxidants have been proposed as biomarkers of a contaminantmediated pro oxidant challenge in a variety of invertebrates (Regoli et al., 2002). Genotoxicity is also one of the most important toxic endpoints in most chemical toxicity testing and risk assessment; however, little is known about the genotoxicity of SWCNTs, especially towards aquatic organisms L. luteola. The results of the comet assay suggested that SWCNTs may provoke DNA damage in L. luteola. It is also observed DNA damaging effects, in which oxidative stress may be attributed as one of the probable cause. Reactive oxygen species is known to react with DNA molecule causing damage to purine and pyrimidine bases as well as DNA backbone. Another important outcome of reactive oxygen species generation, DNA damage resulting from any of these probable mechanisms may trigger signal transduction pathways leading to apoptosis or cause interferences with normal cellular processes thereby causing cell death. The results of this investigation may demonstrate ecological implications of SWCNTs release in aquatic ecosystems. These biomarkers have also opened a wide perspective in aquatic toxicology, as freshwater snail L. luteola is being exposed to environmental pollutants. Our results provide critical information to regulatory agencies and industry to determine the need for monitoring and regulation regarding SWCNTs.
Environmental Toxicology DOI 10.1002/tox
REFERENCES Ahmed RG. 2005. Is there a balance between oxidative stress and antioxidant defence system during development? Med J Islam Acad Sci 15:255–263. Ali D, Nagpure NS, Kumar S, Kumar R, Kushwaha B. 2008. Genotoxicity assessment of acute exposure of chlorpyrifos to freshwater fish Channa punctatus (Bloch) using micronucleus assay and alkaline single-cell gel electrophoresis. Chemosphere 71:1823–1831. Almeida EA, Miyamoto S, Bainy ACD, Medeiros MH, Mascio P. 2004. Protective effect of phosphor lipid hydroperoxide glutathione peroxidase (PHGPx) against lipid peroxidation in mussels Perna perna exposed to different metals. Mar Pollut Bull 49:386–392. Anderson D, Yu TW, Phillips BJ, Schmerzer P. 1994. The effect of various antioxidants and other modifying agents on oxygenradical generated DNA damage inhuman lymphocytes in the comet assay. Mutat Res 307:261–271. APHA, AWWA, WPCF. 2005. Standard Methods for the Examination of Water and Wastewater, 21st ed. Washington, DC: American Publication of Health Association. Baun A, Hartmann NB, Grieger K, Kisk KO. 2008. Ecotoxicity of engineerednanoparticles to aquatic invertebrates: A brief review and recommendations for future toxicity testing. Ecotoxicology 17:387–395. Beeby A, Richmond L. 2002. Evaluating Helix aspersa as a sentinel for mapping metal pollution. Ecol Indicators 1:261–270. Beers RF Jr, Sizer IW. 1952. Spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem 195:133–140. Blaise C, Gagne F, Ferard JF, Eullaffroy P. 2008. Ecotoxicity of selected nanomaterialsto aquatic organisms. Environ Toxicol 223:591–598. Bradford MM. 1976. A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. Brown DM, Wilson MR, MacNee W, Stone V, Donaldson K. 2001. Size-dependent proinflammatory effects of ultrafine polystyrene particles: A role for surface area and oxidative stress in the enhanced activity of ultrafines. Toxicol Appl Pharmacol 175:191–199. Chiu DTY, Stults FH, Tappal AL. 1976. Purification and prosperities of rat lung soluble glutathione peroxidase. Biochem Biophys Acta 445:558–566. Dallinger R, Wieser W. 1984. Patterns of an accumulation, distribution and liberation of Zn, Cu, Cd, and Pb in different organs of the land snail Helix pomatia L. Comp Biochem Physiol C 79:117–124. Dallinger R. 1994. Invertebrate organisms as biological indicators of heavy metal pollution. Appl Biochem Biotechnol 48:27–31. Dallinger R, Berger B, Triebskorn R, Kohler H. 2002. Soil biology and ecotoxicology. In: Barker GM, editor. The Biology of Terrestrial Molluscs. Oxon, UK: CAB International. pp 489–525. Di-Giulio RT, Washburn PC, Wenning RJ, Winston GW, Jewell CS. 1989. Biochemical responses in aquatic animals: A review of determinants of oxidative stress. Environ Toxicol Chem 8: 1103–1123.
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Doyotte A, Cossu C, Jacquin MC, Babut M, Vasseur P. 1997. Antioxidant enzymes, glutathione and lipid peroxidation as relevant biomarkers of experimental or field exposure in the gills and the digestive gland of the freshwater bivalve Unio tumidus. Aquat Toxicol 39:93–110. Finney DJ. 1971. Probit Analysis, 3rd ed. London: Cambridge University Press. pp 318. Glenn JB, White SA, Klaine SJ. 2012. Interactions of gold nanoparticles with freshwater aquatic macrophytes are size and species dependent. Environ Toxicol Chem 31:194–201. Gutteridge MC. 1995. Lipid peroxidation and antioxidants as biomarker of tissue damage. Clin Chem 41:1819–1828. Hayes JD, Flanagan JU, Jowsey IR. 2005. Glutathione transferases. Annu Rev Pharmacol Toxicol 45:51–88. Hoang TC, Rand GM. 2009. Exposure routes of copper: Short term effects on survival, weight, and uptake in Florida apple snail (Pamacea paludosa). Chemosphere 76:407–414. Hoet PHM, Br€uske-Hohlfeld I, Salata OV. 2004. Nanoparticles known and unknown health risks. J Nanobiotechnol 2:12. Howard CV. 2004. Small particles big problems. Int Lab News 34:28–29. Jackson P, Jacobsen NR, Baun A, Birkedal R, Kuhnel D, Jensen KA, Vogel U, Wallin H. 2013. Bioaccumulation and ecotoxicity of carbon nanotubes. Chem Central J 7:154.
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Regoli F, Gorbi S, Frenzilli G, Nigro M, Corsi I, Forcardi S, Winston GW. 2002. Oxidative stress in ecotoxicology: From the analysis of individual antioxidants to a more integrated approach. Mar Environ Res 54:419–423. Regoli F, Gorbi S, Machella N, Tedesco S, Benedetti M, Bocchetti R, Notti A, Fattorini D, Piva F, Principato G. 2005. Prooxidant effects of extremely low frequency electromagnetic fields (ELFEM) in the land snail Helix aspersa. Free Radic Biol Med 39:1620–1628. Regoli F, Hummel H, Amiard-Triquet C, Larroux C, Sukhotin A. 1998. Trace metals and variations of antioxidant enzymes in Arctic bivalve populations. Arch Environ Contam Toxicol 35: 594–601. Regoli F, Principato G. 1995. Glutathione, glutathione-dependent and antioxidant enzymes in mussel, Mytilus galloprovincialis, exposed to metals under field and laboratory conditions: Implications for the use of biochemical biomarkers. Aquat Toxicol 31:143–164. Roling JA, Baldwin WS. 2006. Alterations in hepatic gene expression by trivalent chromium in Fundulus heteroclitus. Mar Environ Res 62:122–127. Singh NP, McCoy MT, Tice RR, Schneider EL. 1988. A simple technique for quantization of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191.
Jamil, K., 2001. Bioindicators and Biomarkers of Environmental Pollution and Risk Assessment. Enfield, NH & Plymouth, UK: Science Publishers, Inc.
Smith CJ, Shaw BJ, Handy RD. 2007. Toxicity of single walled carbon nanotubes to rainbow trout, (Oncorhynchus mykiss): Respiratory toxicity, organ pathologies, and other physiological effects. Aquat Toxicol 82:94–109.
King-Heiden TC, Wiecinski PN, Mangham AN, Metz KM, Nesbit D, Pedersen JA, Hamers RJ, Heideman W, Peterson RE. 2009. Quantum dot nanotoxicityassessment using the zebrafish embryo. Environ Sci Technol 43:1605–1611.
Tanhan P, Sretarugsa P, Pokethitiyook P, Kruatrachue M, Upatham ES. 2005. Histopathological alterations in the edible snail, Babylonia areolata (spotted babylon), in acute and subchronic cadmium poisoning. Environ Toxicol 20:142–149.
Livingstone DR. 2001. Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms. Mar Pollut Bull 42:656–666.
Trachootham D, Weiqin LU, Marcia AO, Valle N, Huang P. 2008. Redox regulation of cell survival. Antioxid Redox Signal 10:1343–1374.
Livingstone DR, Lips F, Garcia MP, Pipe RK. 1992. Antioxidant enzymes in the digestive gland of the common mussel Mytilus edulis. Mar Biol 112:265–276. Mwangi JN, Wang N, Ingersoll CG, Hardesty DK, Brunson EL, Li H, Deng B. 2012. Toxicity of carbon nanotubes to freshwater aquatic invertebrates. Environ Toxicol Chem 31:1823–1830. Nair V, Turner GE. 1984.The thiobarbituric acid test for lipid peroxidation structure of the adduct with malondialdehyde. Lipids 19:84–95. Nusetti O, Esclapes M, Salazar G, Nusetti S, Pulido S. 2001. Biomarkers of oxidative stress in the polychaete Eurthoecom planata (Amphinomidae) under short-term copper exposure. Bull Environ Contam Toxicol 66:76–81. Orbea A, Fahimi HD, Cajaraville MP. 2000. Immunolocalization of four antioxidant enzymes in digestive glands of mollusks and crustaceans and fish liver. Histochem Cell Biol 114:393–404. Owens WI, Belcher RV. 1965. A colorimetric micro-method for the determination of glutathione. Biochem J 94:705–711. Petersen EJ, Akkanen J, Kukkonen JV, Weber WJ. 2009. Biological uptake and depuration of carbon nanotubes by Daphnia magna. Environ Sci Technol 43:2969–2975.
Torres MA, Testa CP, Gaspari C, Masutti MB, Panitz CMN, CuriPedrosa R, Almeida EA, Di Mascio P, Wilhelm FD. 2002. Oxidative stress in the mussel Mytella guyanensis from polluted mangroves on Santa Catarina Island, Brazil. Mar Pollut Bull 44:923–932. Verma RS, Mehta A, Srivastava N. 2007. In vivo chlorpyrifos induced oxidative stress: Attenuation by antioxidant vitamins. Pestic Biochem Physiol 88:191–196. Vessey DA, Boyer TD. 1984. Differential activation and inhibition of different forms of rat liver glutathione-S-transferase by the herbicides 2, 4-dichloro phenoxy acetate (2,4-D) and 2,4, strichloro phenoxy acetate (2,4,S-T). Toxicol Appl Pharmacol 73:492–499. Wilbrink M, Treskes M, De Vlieger TA, Vermeulen NPE. 1990. Comparative toxicokinetics of 2,2’- and 4,4’-dichlorobiphenyls in the pond snail Lymnaea stagnalis (L.). Arch Environ Contam Toxicol 9:565–571. Zheng S, Wang Y, Zhou Q, Chen C. 2013. Responses of oxidative stress biomarkers and DNA damage on a freshwater snail (Bellamya aeruginosa) stressed by ethylbenzene. Arch Environ Contam Toxicol 65:251–259.
Environmental Toxicology DOI 10.1002/tox
Department of Zoology, College of Science, King Saud University, Riyadh, 11451, Saudi Arabia
2
Department of Chemistry, Maulana Azad National Institute of Technology, Bhopal, Madhya Pradesh, India
Received 2 November 2013; revised 19 December 2013; accepted 22 December 2013 ABSTRACT: Mammalian studies have raised concerns about the toxicity of carbon nanotubes, but there is very limited data on ecogenotoxicity to aquatic organisms. The aim of this study was to determine ecogeno toxic effects of single walled carbon nanotubes (SWCNTs) in fresh water snail, Lymnea luteola (L. luteola). A static test system was used to expose L. luteola to a freshwater control, 0.05, 0.15, 0.30, 0.46 mg/L SWCNTs for up to 4 days. SWCNTs changed a significant reduction in glutathione, glutathioneS-transferase, and glutathione peroxidase with in hepatopancreas of L. luteola. Lipid peroxidation (LPO) and catalase showed dose- and time-dependent and statistically significant increase in hepatopancreas during SWCNTs exposure compared with control. However, a significant (p < 0.01) induction in DNA damage was observed by the comet assay in hepatopancreas cells treated with SWCNTs. These results demonstrate that SWCNTs are ecogenotoxic to freshwater snail L. luteola. The oxidative stress and C 2014 Wiley comet assay can successfully be used as sensitive tools of aquatic pollution biomonitoring. V Periodicals, Inc. Environ Toxicol 00: 000–000, 2014.
Keywords: single walled carbon nanotubes; oxidative stress; DNA damage; comet assay; Lymnea luteola L
INTRODUCTION Carbon nanomaterials showing unique physicochemical, mechanical, and electrical properties are increasingly employed in nanotechnology and in several consumer products. Carbon nanotubes are currently used in desalination plants, artificial muscles, paints, solar cells, electronics, and industrial lubricants. Aquatic environments face potential Correspondence to: D. Ali; e-mail: [email protected] or [email protected] Contract grant sponsor: Deanship of Scientific Research at King Saud University. Contract grant number: RGP-VPP-300 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.21945
risks from nanomaterials through wastewater treatment plant spills, rain, and runoff (Glenn et al., 2012). Risk assessment of ecotoxicology of nanomaterials in the aquatic environment is essential as their use and manufacturing are rapidly expanding. Carbon nanotubes shown a significant impacts on survival or growth to several species of freshwater aquatic invertebrates including Hyallela azteca, Chironomus dilutes, Lumbriculus variegates, Villosa iris, and Daphnia magna (Petersen et al., 2009; Mwangi et al., 2012). A number of studies have reported potential impacts of nanoparticles on aquatic organisms and their toxicity can be related to dissolution, size, or surface properties (Blaise et al., 2008; KingHeiden et al., 2009; Jackson et al., 2013). Uptake of xenobiotics by endocytotic routes was identified as a probable major mechanism of entry into cells, particularly in
C 2014 Wiley Periodicals, Inc. V
1
2
ALI ET AL.
suspension-feeding invertebrates, thus potentially lead to various types of toxic cell injury in many tissues, such as gills and digestive gland, as well as in immune cells which are endowed with high phagocytic capacity (Baun et al., 2008). Smith et al. (2007) reported that single-walled carbon nanotubes produced respiratory toxicity, organ pathologies, and other physiological effects in rainbow trout. Gastropod organisms are ubiquitous in the aquatic ecosystem and are considered a good bioindicators of contaminants in view of their wide geographic distribution, relatively sedentary life habit, and their easy availability. In mollusks, the organ which plays the most important role in the metabolism of endogenous and xenobiotic compounds is the digestive gland (hepatopancreas) (Wilbrink et al., 1990). In snails, pollutants are transferred by blood cells to the digestive gland, which is one of the major target tissues of accumulation. Reactive oxygen species (ROS) and the resulting oxidative stress to living organisms have been reported to be related with the pollution-mediated mechanism of toxicity, which can damage macromolecules, such as DNA, proteins, and membranes. Antioxidant biomarkers are able to eliminate the highly reactive intermediate ROS induced by pollutants to maintain cell homeostasis, and they include antioxidant enzymes and antioxidant nonenzymatic scavengers, e.g., superoxide dismutase (SOD), catalase, glutathione-S-transferases (GST), and reduced glutathione (Trachootham et al., 2008). Malondialdehyde (MDA) is an end point of lipid peroxidation; therefore, the formation of MDA is regarded as a general indicator of lipid peroxidation. SOD and catalase activities play important role in the antioxidant protection of invertebrates (Livingstone, 2001). GST is a family of phase II detoxification enzymes that catalyze the conjugation of glutathione to a wide variety of endogenous and exogenous electrophilic compounds, such as therapeutic drugs and environmental toxins (Hayes et al., 2005). Oxidative stress may manifest as damage to tissue macromolecules, including proteins and DNA (Di Giulio et al., 1989). In addition, DNA damage is considered to be a biomarker of genotoxicity induced by noxious substances, and the comet assay has recently been considered a robust genotoxicological tool with which to assess DNA damage at the individual cell level. In the present study, we designed to assess the acute toxicity of SWCNTs to freshwater snail L. luteola, which is an important component of river, ponds, and lakes of South East Asian freshwater ecosystems. Our results also provide critical information to regulatory agencies and industry to determine the need for monitoring and regulation regarding SWCNTs.
MATERIALS AND METHODS Chemicals Single-walled carbon nanotube (SWCNTs) (CAS Number 308068-56-6, Product No. 750522 and average diameter
Environmental Toxicology DOI 10.1002/tox
0.01) higher DNA damage in SWCNTs exposed cells than control groups (Fig. 5). The gradual nonlinear increase in DNA damage was observed in cells as concentration and time exposure of SWCNTs increased and the highest damage of DNA was recorded at 0.30 mg/L SWCNTs [Fig. 5(a,b,d)].
DISCUSSION Several studies accentuate gastropods as sentinel organisms for the assessment of pollution in aquatic ecosystems (Hoang and Rand, 2009). The application of nanotechnology has been recently extended in the areas of medicine, biotechnology,
Environmental Toxicology DOI 10.1002/tox
materials and energy and environment. However, SWCNTs are known toxic nanomaterial to fish; its toxicity to fresh water snail is under-investigated and poorly understood. SWCNTs may induce deleterious effects in aquatic life after release into aquatic environment. However, nanotubes may present special problems with respect to their retention in cells and tissues, since it is believed that there is a threshold for the length of tubes that is critical for induction of adverse biological effects (Hoet et al., 2004). Although some researchers reported that size of the nanomaterial itself cause toxicity and biodegradability may be a further significant factor in governing harmful biological effects (Brown et al., 2001; Howard, 2004). The most severe problem became evident in the hepatopancreas, which is the main metabolic organ in snails and also involved in detoxification and accumulation of xenobiotics (Dallinger and Wieser, 1984; Tanhan et al., 2005). Dallinger (1994) has reported that freshwater snails are sentinel model to monitor aquatic pollution, so it is important to study SWCNTs mechanisms of toxicity and the state of their defence systems by the snails. We have observed that SWCNTs induced oxidative stress and DNA damage in hepatopancreas and hemolymph of L. luteola as dose- and timedependent manner. The hepatopancreas (digestive gland) of gastropod molluscs is the key organ of metabolism and it is concerned with the production of digestive enzymes, absorption of nutrients, endocytosis of food substances, food storage, and excretion (Dallinger et al., 2002). It has been
OXIDATIVE STRESS AND GENOTOXICITY
7
Fig. 5. DNA damage in hepatopancreas cells of L. luteola after exposure to SWCNTs. a. % Tail DNA. b. Olive tail moment. c. Control. d. Exposed cells (at 0.30 mg/L of SWCNTs). Data are presented as the mean 6 SE of triplicate experiments. *p < 0.01 vs. control. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
reported as a major site of xenobiotic, oxy-radical generating and biotransformation enzymes (Livingstone et al., 1992). Pollutants accumulation through different routes are transported by blood cells (hemolymph) to the digestive gland, which also represents the main target organ for metabolic and detoxification processes (Beeby and Richmond, 2002; Regoli et al., 2005). So in the present study we have used the hepatopancreas tissue to investigate the biochemical responses to SWCNTs. A general pathway of toxicity for many environmental pollutants is mediated by the enhancement of intracellular ROS, which modulate the occurrence of cell damage (Regoli et al., 2002) via initiation and propagation of lipid peroxidation (Gutteridge, 1995). The estimation of malondialdehyde content (an index of lipid peroxidation) provides a relative measure of the potential for SWCNTs to cause oxidative injury. Nusetti et al. (2001) reported that LPO levels increased during exposure to metals in several organisms. In the present study, significantly elevations of lipid peroxidation levels in the digestive gland of SWCNTs exposed L. luteola indicate that some cell damage might have occurred. GSH is a tripeptide nonenzymatic antioxidant with a single cysteine residue and constitutes an important pathway of the antioxidant and detoxification defences. It is also a cofactor of many enzymes catalyzing the detoxification and excretion of several toxicants, which will be destroyed in the cytosolic and mitochondrial compartments by GPx in the presence of GSH (Doyotte et al.,
1997). The decrease in GSH concentrations in our study might be attributed to the intensification of turnover between reduced and oxidized glutathione under the conditions, which cause increased consumption of this peptide for the synthesis of heavy metal-binding proteins, like metallothioneins. A decrease in GSH content in the digestive gland appears to be a common response of molluscs to metal exposure (Regoli and Principato, 1995). In this study, we observed a decrease in GSH content accompanied by the elevation of LPO levels. Through catalyzation by glutathione peroxidase, GSH can eliminate H2O2 and lipid hydro peroxide (Ahmed, 2005); therefore, a negative relationship between MDA and GSH was a reasonable result. GSH is one of the most important factors protecting from oxidative attacks by reactive oxygen species such as lipid peroxidation, because GSH acts as a reducing agent and free-radical trapper and is known to be a cofactor substrate and/or GSH-related enzymes (Verma et al., 2007). Catalase converts H2O2 to H2O and O2 to prevent oxidative stress and maintain cell homeostasis. Increased catalase activity indicated that O22 and H2O2 were formed during the freshwater snail exposure to contaminant (Zheng et al., 2013). The antioxidant catalase is an extremely important component of intracellular and antioxidant defences of organisms (Jamil, 2001). It reduces the H2O2 into water and oxygen to prevent oxidative stress and for maintaining cell homeostasis. Many studies have found varying responses of
Environmental Toxicology DOI 10.1002/tox
8
ALI ET AL.
catalase to increased metal exposures, with some organisms exhibiting increased activity, others exhibiting decreased activity, and still others showing no catalase response at all (Regoli et al., 1998). In the present study, we observed that catalase activity was significantly increased; this data suggests that the increase in antioxidant defences would be due to enhanced oxygen free radicals production, which could stimulate antioxidant activities (Torres et al., 2002) to cope with this increased oxidative stress and protect the cells from damage. The obtained results are in accordance with the findings of Almeida et al. (2004), who found that catalase activity was increased in mussels after exposure to lead. GPx is the most important peroxidase for the detoxification of hydro peroxides (Orbea et al., 2000). However, the decreased activities of GPx might be due to over production of ROS, especially O22, by the SWCNTs and depletion of its substrate level (GSH). It is also reported that under high rates of free radicals input, enzyme inactivation prevails and the enzymatic activities are reduced, leading to autocatalysis of oxidative damage process (Escobar et al., 1996). The role of GST is to conjugate tripeptide glutathione with electrophilic and other xenobiotic. Inhibition of GST activity may be occurred either through direct action of the metal on the enzyme or indirectly via the production of ROS that interact directly with the enzyme, depletion of its substrate (GSH), and/or down regulation of GST genes through different mechanisms (Roling and Baldwin, 2006). This explanation might be the reason for the GST activity decrease that was caused in the present study in the case of snails exposed to SWCNTs. Changes in the levels of antioxidants have been proposed as biomarkers of a contaminantmediated pro oxidant challenge in a variety of invertebrates (Regoli et al., 2002). Genotoxicity is also one of the most important toxic endpoints in most chemical toxicity testing and risk assessment; however, little is known about the genotoxicity of SWCNTs, especially towards aquatic organisms L. luteola. The results of the comet assay suggested that SWCNTs may provoke DNA damage in L. luteola. It is also observed DNA damaging effects, in which oxidative stress may be attributed as one of the probable cause. Reactive oxygen species is known to react with DNA molecule causing damage to purine and pyrimidine bases as well as DNA backbone. Another important outcome of reactive oxygen species generation, DNA damage resulting from any of these probable mechanisms may trigger signal transduction pathways leading to apoptosis or cause interferences with normal cellular processes thereby causing cell death. The results of this investigation may demonstrate ecological implications of SWCNTs release in aquatic ecosystems. These biomarkers have also opened a wide perspective in aquatic toxicology, as freshwater snail L. luteola is being exposed to environmental pollutants. Our results provide critical information to regulatory agencies and industry to determine the need for monitoring and regulation regarding SWCNTs.
Environmental Toxicology DOI 10.1002/tox
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