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Cytotoxicity, genotoxicity and oxidative stress of malachite green on the kidney and gill cell lines of freshwater air breathing fish Channa striata

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Abstract

The cytotoxicity, genotoxicity and oxidative stress of malachite green (MG) was investigated using the fish Channa striata kidney (CSK) and Channa striata gill (CSG) cell lines. Five concentrations ranging from 0.001 to 10 μg mL−1 were tested in three independent experiments. Cytotoxicity was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Rhodamine 123 and Alamar Blue. The mitochondrial changes and apoptosis of MG-exposed cells were observed by Rhodamine 123 and acridine orange/ethidium bromide (AO/EB) staining, respectively. In vitro potential DNA damaging effect of MG was tested using comet assay. Mitochondrial damage, apoptosis and DNA fragmentation increased in a concentration-dependent manner. Additionally, DNA electrophoretic mobility experiments were carried out to study the binding effect of MG to double-stranded DNA (dsDNA) of cells. DNA shift mobility experiments showed that MG is capable of strongly binding to linear dsDNA causing its degradation. Biochemical parameters such as lipid peroxidation (MDA), catalase (CAT) activity and reduced glutathione (GSH) levels were evaluated after exposure to MG. In CSK and CSG cell lines exposed to MG for 48 h, a significant increase in lipid peroxidation, which might be associated with decreased levels of reduced glutathione and catalase activity in these cell lines (p < 0.001), was observed.

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References

  • Abdul Majeed S, Nambi KSN, Taju G, Sundar Raj N, Madan N, Sahul Hameed AS (2013a) Establishment and characterization of permanent cell line from gill tissue of Labeo rohita (Hamilton) and its application in gene expression and toxicology. Cell Biol Toxicol 29:59–73

    Article  CAS  Google Scholar 

  • Abdul Majeed S, Nambi KSN, Taju G, Sahul Hameed AS (2013b) Development, characterization and application of a new fibroblastic-like cell line from kidney of a freshwater air breathing fish Channa striata (Bloch, 1793). Acta Trop 127:25–32

    Article  CAS  Google Scholar 

  • Abdul Majeed S, Nambi KSN, Taju G, Sarath Babu V, Farook MA, Sahul Hameed AS (2014) Development and characterization of a new gill cell line from air breathing fish Channa striata (Bloch 1793) and its application in toxicology: direct comparison to the acute fish toxicity. Chemosphere 96:89–98

    Article  CAS  Google Scholar 

  • Aebi H (1974) Catalase. In: Bergmeyer HU (ed) Methods Enzymatic Analysis. Academic Press, New York, pp 673–678

    Chapter  Google Scholar 

  • Afrati T, Dendrinou-Samara C, Raptopoulou C, Terzis A, Tangoulis V, Tsipis A, Kessissoglou DP (2008) Experimental and theoretical study of the antisymmetric magnetic behavior of copper inverse-9-metallacrown-3 compounds. Inorg Chem 47:7545–7555

    Article  CAS  Google Scholar 

  • Afrati T, Pantazaki AA, Dendrinou-Samara C, Raptopoulou C, Terzis A, Kessissoglou DP (2010) Copper inverse-9-metallacrown-3 compounds interacting with DNA. Dalton Trans 39:765–775

    Article  CAS  Google Scholar 

  • Aitcheson SJ, Arnett J, Murray KR, Zhang J (2000) Removal of aquaculture therapeutants by carbon adsorption. 1. Equilibrium adsorption behaviour of single components. Aquaculture 183(3–4):269–284

    Article  CAS  Google Scholar 

  • Albert A (1979) Selective toxicology, 6th edn. Chapman & Hall, London

    Google Scholar 

  • Alderman DJ (1985) Malachite green: a review. J Fish Dis 8:289–298

    Article  CAS  Google Scholar 

  • Alderman DJ (1992) Malachite green and alternatives as therapeutic agents. Eur Aquacult Soc Bredene 16:235–244

    Google Scholar 

  • Alderman DJ (2002) Trends in therapy and prophylaxis 1991–2001. Bul Eur Assoc Fish Pathol 22(2):117–125

    Google Scholar 

  • Azmi W, Saini RK, Banerjee UC (1998) Biodegradation of triphenylmethane dyes. Enzyme Microb Technol 22:185–191

    Article  CAS  Google Scholar 

  • Babich H, Borenfreund E (1991) Cytotoxicity and genotoxicity assays with cultured fish cells: a review. Toxicol In Vitro 5:91–100

    Article  CAS  Google Scholar 

  • Baskic D, Popovic S, Ristic P, Arsenijevic NN (2006) Analysis of cycloheximide-induced apoptosis in human leukocytes: fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide. Cell Bio Int 30(3):924–932

    Article  CAS  Google Scholar 

  • Bergwerff AA, Kuiper RV, Scherpenisse P (2004) Persistence of residues of malachite green in juvenile eels (Anguilla anguilla). Aquaculture 233:55–63

    Article  CAS  Google Scholar 

  • Bloch ME (1793) Naturgeschichte der Ausländischen fische, 7: Berlin, Germany, Morino & Co., p 144

  • Borenfreund E, Babich H, Martin-Alguacil N (1988) Comparision of two in vitro cytotoxicity assays: the neutral red and tetrazolium (MTT) tests. Toxicol In Vitro 2:1–6

    Article  CAS  Google Scholar 

  • Caminada D, Escher C, Fent K (2006) Cytotoxicity of pharmaceuticals found in aquatic systems: comparison of PLHC-1 and RTG-2 fish cell lines. Aquat Toxicol 79:114–123

    Article  CAS  Google Scholar 

  • Campbell RE, Lilley JH, Taukhid, Panyawachira V, Kanchanakhan S (2001) In vitro screening of novel treatments for Aphanomyces invadans. Aquacult Res 32(3):223–233

  • Castano A, Bols N, Braunbeck T, Dierickx P, Halder M, Isomaa B, Kawahara K, Lee LEJ, Mothersill C, Part P, Repetto G, Sintes JR, Rufli H, Smith R, Wood C, Segner H (2003) The use of fish cells in ecotoxicology. ATLA 31(3):317–351

    CAS  Google Scholar 

  • Chang CF, Yang CH, Shu YO, Chen TI, Shu MS, Liao IC (2001) Effects of temperature, salinity and chemical drugs on the in vitro propagation of the Dinoflagellate parasite, Amylodinium ocellatum. Asian Fish Soc P31

  • Chowdhury S, Saha P (2010) Sea shell powder as a new adsorbent to remove Basic Green 4 (Malachite Green) from aqueous solutions: equilibrium, kinetic and thermodynamic studies. Chem Eng J 164:168–177

    Article  CAS  Google Scholar 

  • Chowdhury S, Mishra R, Saha P, Kushwaha P (2011) Adsorption thermodynamics, kinetics and isosteric heat of adsorption of malachite green onto chemically modified rice husk. Desalination 265:159–168

    Article  CAS  Google Scholar 

  • Culp SJ, Beland FA (1996) Malachite green: a toxicological review. Int J Toxicol 15(3):219–238

    Article  Google Scholar 

  • Dayeh VR, Bols NC, Schirmer K, Lee LEJ (2003) The use of fish derived cell lines for investigation of environmental contaminants. Current protocols in toxicology. Wiley, New York, pp 1–17

    Google Scholar 

  • Ellman G (1959) Tissue sulphydryl groups. Arch Biochem Biophys 82:70–77

    Article  CAS  Google Scholar 

  • Fent K (2001) Fish cell lines as versatile tools in ecotoxicology: assessment of cytotoxicity, cytochrome P4501A induction potential and estrogenic activity of chemicals and environmental samples. Toxicol In Vitro 15:477–488

    Article  CAS  Google Scholar 

  • Fessard V, Godard T, Huet S, Mourot A, Poul JM (1999) Mutagenicity of malachite green and leucomalachite green in in vitro tests. J Appl Toxicol 19:421–430

    Article  CAS  Google Scholar 

  • Foster FJ, Woodbury L (1936) The use of malachite green as a fish fungicide and antiseptic. Prog Fish Cult 18:7–9

    Article  Google Scholar 

  • Freshney RI (1994) Culture of animal cells: a manual of basic technique. Wiley-Liss, New York

    Google Scholar 

  • Gerundo N, Alderman DJ, Clifton-Hadely RS, Feist SW (1991) Pathological effects of repeated doses of malachite green: a preliminary study. J Fish Dis 14:521–532

    Article  CAS  Google Scholar 

  • Hecht T, Endemann F (1998) The impact of parasites, infections and disease on the development of aquaculture in sub-Saharan Africa. J Appl Ichth 14(3–4):213–221

    Article  Google Scholar 

  • Hoffman GL, Meyer FP (1974) Parasites of freshwater fishes. TFH Publications, Neptune

    Google Scholar 

  • Hussein MMA, Wada S, Hatai K, Yamamoto A (1999) Antimicotic activity of eugenol against some water molts. In: Symposium on Diseases in Asian Aquaculture, Philippines

  • Johnson LV, Walsh ML, Bockus BJ, Chen LB (1981) Monitoring of relative mitochondrial membrane potential in living cells by fluorescence microscopy. J Cell Biol 88:526–535

    Article  CAS  Google Scholar 

  • Kasuga Y, Hishida M, Tanahashi N, Arai M (1992) Studies on disappearance of malachite green in cultured rainbow trout. J Food Hygienic Soc Jpn 33(6):539–542

    Article  CAS  Google Scholar 

  • Lakra WS, Raja Swaminathan T, Joy KP (2011) Development, characterization, conservation and storage of fish cell lines: a review. Fish Physiol Biochem 37:1–20

    Article  CAS  Google Scholar 

  • Lee LEJ, Dayeh VR, Schirmer K, Bols NC (2009) Applications and potential uses of fish gill cell lines: examples with RTgill-W1. Vitro Cell Dev Biol-Animal 45(3–4):127–134

    Article  CAS  Google Scholar 

  • Lilley JH, Callinan RB, Chinabut S, Kanchanakhan S, Macrae IH, Phillips MJ (1998) Epizootic ulcerative syndrome (EUS) technical handbook. Aquatic Animal Health Research Institute, Bangkok, Thailand

  • Lo CF, Leu JH, Ho CH, Chen CH, Peng SE, Chen YT, Yeh PY, Huang CJ, Wang CH, Kou GH (1996) Detection of baculovirus associated with white spot syndrome (WSSV) in penaeid shrimps using polymerase chain reaction. Dis Aquat Org 25:133–41

    Article  CAS  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with folin phenol reagent. J Biol Chem 193:265–275

    CAS  Google Scholar 

  • Martinez RM, Morales AE, Sanz A (2005) Antioxidant defenses in fish: biotic and abiotic factors. Rev Fish Biol Fisher 15:75–88

    Article  Google Scholar 

  • Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63

    Article  CAS  Google Scholar 

  • Mpountoukas P, Pantazaki A, Kostareli E, Christodoulou P, Kareli D, Poliliou S, Mourelatos C, Lambropoulou V, Lialiaris T (2010) Cytogenetic evaluation and DNA interaction studies of the food colorants amaranth, erythrosine and tartrazine. Food Chem Toxicol 48:2934–2944

    Article  CAS  Google Scholar 

  • Nordén B, Tjerneld F, Palm E (1978) Linear dichroism studies of binding site structures in solution. Complexes between DNA and basic arylmethane dyes. Biophys Chem 8:1–15

    Article  Google Scholar 

  • Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95:351–358

    Article  CAS  Google Scholar 

  • Onal Y (2006) Kinetics of adsorption of dyes from aqueous solution using activated carbon prepared from waste apricot. J Hazard Mater B 137:1719–1728

    Article  Google Scholar 

  • Panandiker A, Fernandes C, Rao KVK (1992) The cytotoxic properties of malachite green are associated with the increased demethylase, aryl hydrocarbon hydroxylase and lipid peroxidation in primary cultures of Syrian hamster embryo cells. Cancer Lett 67:93–101

    Article  CAS  Google Scholar 

  • Poe WE, Wilson RP (1983) Absorption of malachite green by channel catfish. Prog Fish Cult 45:228–229

    Article  CAS  Google Scholar 

  • Pointing SB, Vrijmoed LLP (2000) Decolorisation of azo and triphenylmethane dyes by Pycnoporus sanguineus producing laccase as the sole phenol oxidase. World J Microbiol Biotechnol 16:317–318

    Article  CAS  Google Scholar 

  • Qureshi TA, Chauhan R, Prasad Y, Mastan SA (1998) Effects of certain drugs on pathogenic fungi isolated from EUS affected fishes. J Ecotoxicol Environ Mont 8(1):2–15

    Google Scholar 

  • Radko L, Minta M, Stypula-Trebas S (2011) Cellular toxicity of malachite green and leucomalachite green evaluated on two rat cell lines by MTT, NRU, LDH and protein assays. Bull Vet Inst Pulawy 55:347–353

    Google Scholar 

  • Ross LG, Ward KMH, Ross B (1985) The effects of formaline, malachite green and suspended solids on the respiratory activity of rainbow trout, Salmo gairdneri Richandson. Aquacult Fish Manage 16:129–138

    CAS  Google Scholar 

  • Saito H, Iwami S, Shigeoka T (1991) In vitro cytotoxicity of 45 pesticides to goldfish GF-Scale (GFS) cells. Chemosphere 23(4):525–537

    Article  CAS  Google Scholar 

  • Schirmer K (2006) Proposal to improve vertebrate cell cultures to establish them as substitutes for the regulatory testing of chemicals and effluents using fish. Toxicol 224:163–183

    Article  CAS  Google Scholar 

  • Schnick RA (1988) The impetus to register new therapeutants for aquaculture. Prog Fish Cult 50:190–196

    Article  Google Scholar 

  • Segner H (1998) Fish cell lines as a tool in aquatic toxicology. EXS 86:1–38

    CAS  Google Scholar 

  • Singh NP, McCoy MT, Tice RR, Scheider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191

    Article  CAS  Google Scholar 

  • Srivastava S, Sinha R, Roy D (2004) Toxicological effects of malachite green. Aquat Toxicol 66:319–329

    Article  CAS  Google Scholar 

  • Sudova E, Machova J, Svobodova Z, Vesely T (2007) Negative effects of malachite green and possibilities of its replacement in the treatment of fish eggs and fish: a review. Vet Med 52(12):527–539

    CAS  Google Scholar 

  • Tacal O, Ozer I (2004) Adduct-forming tendencies of cationic triarylmethane dyes with proteins: metabolic and toxicological implications. J Biochem Mol Toxicol 18:253–256

    Article  CAS  Google Scholar 

  • Taju G, Abdul Majeed S, Nambi KSN, Sarath Babu V, Vimal S, Kamatchiammal S, Sahul Hameed AS (2012) Comparison of in vitro and in vivo acute toxicity assays in Etroplus suratensis (Bloch, 1790) and its three cell lines in relation to tannery effluent. Chemosphere 87:55–61

    Article  CAS  Google Scholar 

  • Taju G, Abdul Majeed S, Nambi KSN, Sahul Hameed AS (2013) Development and characterization of cell line from the gill tissue of Catla catla (Hamilton, 1822) for toxicological studies. Chemosphere 90:2172–2180

    Article  CAS  Google Scholar 

  • Taju G, Abdul Majeed S, Nambi KSN, Sahul Hameed AS (2014) In vitro assay for the toxicity of silver nanoparticles using heart and gill cell lines of Catla catla and gill cell line of Labeo rohita. Comp Biochem Physiol C 161:41–52

    CAS  Google Scholar 

  • Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF (2000) Single cell gel/ Comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35:206–221

    Article  CAS  Google Scholar 

  • Trenzado C, Carmen HM, Gallego MG, Morales AE, Furne M, Domezain A, Domezain J, Sanz A (2006) Antioxidant enzymes and lipid peroxidation in sturgeon Acipencer naccarii and trout Oncorhynchus mykiss. A comparative study. Aquaculture 254:758–767

    Article  CAS  Google Scholar 

  • Werth G (1958) Die erzeugung von storungen im erbgefuge und von tumoren durch experimentelle gewebsanoxie. Arzn Forsch 8:725–744

    Google Scholar 

  • Werth G, Boiteaux A (1967) The toxicity of the triphenylmethane dyestuff malachite green, as an uncoupler of oxidative phosphorylation in vivo and in vitro. Arch Fur Toxicol 23:82–103

    Google Scholar 

  • Werth G, Boiteaux A (1968) Zur biologischen wirkung von malachitgrun. Arzn Forsch 18:39

    CAS  Google Scholar 

  • Wolf K, Quimby MC (1962) Established eurythermic line of fish cells in vitro. Science 135:1065–1066

    Article  CAS  Google Scholar 

  • Wu J, Yi W, Jin L, Hu D, Song B (2012) Antiproliferative and cell apoptosis-inducing activities of compounds from Buddleja davidii in Mgc-803 cells. Cell Div 7:20

    Article  CAS  Google Scholar 

  • Yang MC, Fang JM, Kuo TF, Wang DM, Hunag YL, Liu LY, Chen PH, Chang TH (2007) Production of antibodies for selective detection of malachite green and the related triphenylmethane dyes in fish and fishpond water. J Agric Food Chem 55:8851–8856

    Article  CAS  Google Scholar 

  • Yilmaz S, Atessahin A, Sahna E, Karahan I, Ozer S (2006) Protective effect of lycopene on adriamycin-induced cardiotoxicity and nephrotoxicity. Toxicology 218:164–171

    Article  CAS  Google Scholar 

  • Yonar ME, Yonar SE (2010) Changes in selected immunological parameters and antioxidant status of rainbow trout exposed to malachite green (Oncorhynchus mykiss, Walbaum, 1792). Pestic Biochem Phys 97:19–23

    Article  CAS  Google Scholar 

  • Zahn T, Braunbeck T (1995) Cytotoxic effects of sublethal concentrations of malachite green in isolated hepatocytes from rainbow trout (Oncorhynchus mykiss). Toxicol In Vitro 9(5):729–741

    Article  CAS  Google Scholar 

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Acknowledgments

The first author is a recipient of INSPIRE Fellowship from the Department of Science and Technology, Government of India, New Delhi, India. The authors thank the management of C. Abdul Hakeem College, Melvisharam, India, for providing the facilities to carry out this work. This work was funded by the Department of Biotechnology, Government of India, New Delhi, India.

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Correspondence to A. S. Sahul Hameed.

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Majeed, S.A., Nambi, K.S.N., Taju, G. et al. Cytotoxicity, genotoxicity and oxidative stress of malachite green on the kidney and gill cell lines of freshwater air breathing fish Channa striata . Environ Sci Pollut Res 21, 13539–13550 (2014). https://doi.org/10.1007/s11356-014-3279-8

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