Comet Assay: Quantification of Damaged DNA in Catla catla Exposed to Endosulfan+Chlorpyrifos

Huma Naz1*, Sajid Abdullah2, Khalid Abbas2, Muhammad Rizwan Tariq3, Laiba Shafique4, Ghazala Nazeer5

1Department of Zoology, Cholistan University of Veterinary and Animal Sciences, Bahawalpur, Pakistan.

2Department of Zoology, Wildlife and Fisheries, University of Agriculture Faisalabad, Pakistan.

3Department of Diet and Nutritional Sciences, University of Lahore, Islamabad Campus, Pakistan.

4Animal Sciences and Technology, Guangxi University China.

5Department of Social and Allied Sciences, Cholistan University of Veterinary and Animal Sciences, Bahawalpur, Pakistan.

Abstract | In an acute toxicity trail, LC50 and LC100value (96 hr) of endosulfan (END)+chlorpyrifos (CPF) mixture for Catla catla was computed. The genotoxicity of END+CPF mixture in RBCs of fish was also evaluated by comet assay. To check the genotoxicity of END+CPF mixture, blood of fish was sampled after 24-hr of intervals. Some fishes were also kept in clean water known as negative control (NC) and for positive control (PC) cyclophosphamide was injected into fish. The tolerance limits of C. catla against END+CPF was computed as 1.35±0.01μgL-1(LC50) and 2.25±0.02μgL-1(LC100). Genotoxic results showed that END+CPF mixture caused significant damage to nuclei (22.58±5.37%) and GDI (0.73±0.15%) in RBCs of fish as compare to control. Aduration-specific damage to DNA was observed in RBCs of C. catla.

Article History

Received: September 04, 2018

Revised: June 03, 2019

Accepted: June 08, 2019

Published: June 21, 2019

Authors’ Contributions

HN performed the research and wrote the article. SA planned and supervised the study. KA provided lab facilities for research. MAT and LS helped in writing the article. GN helped in statistical anlyses.

Keywords

Fish, Acute effect, Blood, Insecticides mixture, Comet assay

Corresponding author: Huma Naz, dr.humanaz98@gmail.com, humanaz@cuvas.edu.pk

To cite this article: Naz, H., Abdullah, S., Abbas, K., Tariq, M.R., Shafique, L. and Nazeer, G., 2019. Comet Assay: quantification of damaged DNA in Catla catla exposed to endosulfan+chlorpyrifos. Punjab Univ. J. Zool., 34(1): 85-88. http://dx.doi.org/10.17582/journal.pujz/2019.34.1.85.88

Introduction

From last few years, pesticides have been extensively applied in agriculture to control the pest. In results of this natural aquatic system such as lakes, rivers, streams and wetlands have been contaminated all over the world (Ngidlo, 2013). Aquatic pollution by these pesticides cause the unwarranted mortalities of aquatic animals ingeneral and especially fish (Gupta et al., 2013). Fish is an important animal to examine the genotoxic effects of pollutants present in water bodies because fish can amass these toxicants in body organs (Bhatnagar et al., 2016).

Organochlorine (endosulfan) and organophosphate (chlorpyrifos) are indiscriminately used in agricultural to save crop from pests (Rusyniak and Nanagas, 2004 Kumari et al., 2007). These pesticides cause liver failure (Poet et al., 2003), genotoxicity (Mehta et al., 2008), neuro-chemical and neuro-behavioral alterations (Verma et al., 2009; Ojha et al., 2011).

Environmental contaminants induced genotoxicity can be assessed by using biomarkers such as comet assay. Comet assay is the sensitive methods to quantify the geno-toxic results of agrochemicals on integrity of DNA in different species of fish (Frenzilli et al., 2009; Nagarani et al., 2012). According to Kim et al. (2002), it is most reliable technique due to its efficiency for detecting damage at DNA level even in particular cell induced by physical and chemical toxicants. So, keeping in view above mentioned problems associated with insecticides, this work was carried out to assess the genotoxic effects of dual pesticides mixture on RBCs of Catla catla using comet assay.

 

Materials and Methods

Acute toxicity assay was accomplished in Toxicology laboratory at Fisheries Research Farms, UAF, Pakistan. Fish were acclimatized for 14 days in cemented tank. Commercially available feed (at 3% wet body weight) was used to fed fish during acclimatization; however fish were kept in fasting condition during toxicity trails. The test chemicals, endosulfan (END) and chlorpyrifos (CPF) were dissolved, separately, in methanol to prepare the stock-I solutions while to prepare the mixture solution of END+CPF further dilutions was made in the deionized water.

Toxicity trail

To conduct the acute toxicity tests, each group of C. catla (n=10) were placed in glass aquarium (70-L) and separately exposed to 14 different levels (0.15-2.10 μgL-1) of END+CPF for 96 hr. Each toxicity tests were carried out with three replicates. After 12 hr of interval fish mortality was observed and deceased fish were separated immediately.

Comet assay

Fish were exposed to LC50value of END+CPF for 96 hr. Some fishes were also placed same conditions in clean water known as negative control (NC) and for positive control (PC) cyclophosphamide was injected into fish. Blood from fish was sampled after 24-hr interval to see the genotoxic effects of CPF+END, PC and NC by comet assay.The blood was collected in eppendorf which contain anticoagulant (Kousar and Javed, 2015). Sing et al. (1988) procedure was followed to complete assay. Jose et al. (2011) criterion was followed to identify the different types of damaged cells.

Data analyses

Data collected from fish mortality was used to draw the concentration response curve by Probit analyses (Finney, 1971). Mann-Whitney U-test (non-parametric) was applied on data collected from DNA damaged (Steel et al., 1996) and graphs were draw in MS excel.

 

Results and Discussion

Toxicity trail

The tolerance limits in term of LC50 and LC100 value (96 hr) of C. catla against CPF+END was estimated as 1.35±0.01 and 2.25±0.03μgL-1, respectively. Fish mortality against different concentration was given in Figure 1. Several researchers also calculated the lethal concentrations of insecticides for different fish species (Verma and Saxena, 2013; Haloi et al., 2014; Ambreen and Javed, 2015; Naserabad et al., 2015; Naz et al., 2017; Naz et al., 2019).

 

 

Comet assay

Different types of DNA damage in RBCs of C. catla was as follow NC<PC<CPF+END (Figure 2). Regarding different treatments the damaged nuclei (DN) and genetic damage index (GDI) in RBCs of fish was higher in CPF+END mixture exposure than that of PC and NC. A duration-specific damage to DNA was observed in RBCs of C. catla (Figure 3-4). Ambreen et al. (2018) also confirmed the DN and GDI in RBCs of grass carp under chlorpyrifos+endosulfan exposure. Duration specific DNA damaged in RBCs of goldfish induced by monocrotophos was also noted by Zhao et al. (2015). Shukla et al. (2010) also noted the duration- dependent increase in DNA damage of Mystusvittatus exposed to dichlorvos. Malathion exposure also caused DNA damage in blood of common carp (Moradi et al., 2012). Blood of T. mossambica also demonstrated the DNA damage of monocrotophos (Banu et al., 2001). Genotoxic potential of some insecticides in RBCs of fish was also noted by some other authors (Simoniello et al., 2009; Gadhia et al., 2016).

 

 

Conclusion

In conclusion, insecticides mixture has potential to cause damage to DNA in blood of Catla catla. Comet assaycan be successfully applied as a good biomarker and screening approach for identifying the harmful impacts of these toxicants on DNA molecule.

 

References

Ambreen, F. and Javed, M., 2015. Assessment of acute toxicity of pesticides mixtures for Cyprinus carpio and Ctenopharyngodon idella. Pakistan J. Zool., 47: 133-139.

Ambreen, F., Javed, M., Abbas, S., Kousar, S., Ilyas, R. and Batool, M., 2018. DNA damage in peripheral erythrocytes of ctenopharyngodon idella during chronic exposure to pesticide mixture. Pakistan J. Zool., (13): 19-25.

Banu, B.S., Danadevi, K., Rahman, M.F., Ahuja, Y.R. and Kaiser, J., 2001. Genotoxic effect of monocrotophos to sentinel species using comet assay. Food Chem. Toxicol., 39: 361-366. https://doi.org/10.1016/S0278-6915(00)00141-1

Bhatnagar, A., Yadav, A.S. and Cheema, N., 2016. Genotoxic effects of chlorpyrifos in freshwater fish Cirrhinus mrigala using micronucleus assay. Adv. Biol., pp.1-6. https://doi.org/10.1155/2016/9276963

Finney, D.J., 1971. Probit Analysis, 3rd Ed. Cambridge Univ. Press, London, 318pp.

Frenzilli, G., Nigro, M. and Lyons, B.P., 2009. The comet assay for the evaluation of genotoxic impact in aquatic environments. Mut. Res.,681: 80-92. https://doi.org/10.1016/j.mrrev.2008.03.001

Gadhia, M., Prajapati, R. and Gadhia, P., 2016. Cypermethrin induced DNA damage in Labeorohita assessed by comet assay. Int. J. Environ. Sci., 6: 1113- 1116.

Gupta, S.K., Pal, A.K., Sahu, N.P., Jha, A.K., Akhtar, M.S., Mandal, S.C., Das, P. and Prusty, A.K., 2013. Supplementation of microbial levan in the diet of Cyprinus carpio fry (Linnaeus, 1758) exposed to sublethal toxicity of fipronil: effect on growth and metabolic responses. Fish Physiol. Biochem., 39: 1513-1524. https://doi.org/10.1007/s10695-013-9805-7

Haloi, K., Kalita, M. and Borkotoki, A., 2014. Acute toxicity of endosulfan on Channa punctatus (Bloch.). Int. J. Res. Environ. Sci. Technol.,4: 88-92.

Jose, S., Jayesh, P., Mohandas, A., Philip, R. and Singh, I.S.B., 2011. Application of primary haemocyte culture of Penaeus monodon in the assessment of cytotoxicity and genotoxicity of heavy metals and pesticides. Marine environ. Res.,71:169-177. https://doi.org/10.1016/j.marenvres.2010.12.008

Kim, B., Park, J.J., Edlaer, L., Fournier, D., Haase, W., Sautter-Bihl, M., Gotzes, F. and Thielmann, H.W., 2002. New measurement of DNA repairs in the single-cell gel electrophoresis (comet) assay. Environ. Mol. Mut., 40: 50-56. https://doi.org/10.1002/em.10090

Kousar, S. and Javed, M., 2015. Diagnosis of metals induced DNA damage in fish using comet assay. Pak. Vet. J., 35: 168-175.

Kumari, B., Madan,V.K. and Kathpal, T.S., 2007. Pesticide residues in rain water from Hisar, Idia. Environ. Monit. Assess., 133: 467-71. https://doi.org/10.1007/s10661-006-9601-2

Mehta, A., Verma, R.S. and Srivastava, N., 2008. Oxidative DNA damage induced by chlorpyrifos in rat tissues. Environ. Mol. Mut., 49: 426-433. https://doi.org/10.1002/em.20397

Moradi, A.M., Mozdarani, H., Salimi, P.A. and Salimi. M.A., 2012. Evaluation of DNA damage in Cyprinus carpio (L. 1758) exposed to malathion using single cell gel electrophoresis. Int. J. Marine Sci. Eng., 2: 185-188, 2012.

Nagarani, N., Devi, V.J. and Kumaraguru, A.K., 2012. Identification of DNA damage in marine fish Theraponjarbuaby comet assay technique. J. Environ. Biol., 33: 699-703.

Naserabad, S.S., Mirvaghefi, Gerami, M.H. and Farsani, H.G., 2015. Acute toxicity and behavioral changes of the gold fish (Carassius Auratus) exposed to malathion and hinosan. Iranian J. Toxicol., 8: 1203-1208.

Naz, H., Abdullah, S., Abbas, K. and Zia, M.A., 2017. Pesticides mixture toxicity; effects on superoxide dismutase activity in indian major carps. Pak. J. Agri. Sci., 54: 607-611. https://doi.org/10.21162/PAKJAS/17.5939

Naz, H., Abdullah, S., Abbas, K., Hassan, W., Batool, M., Perveen, S., Maalik, S. and Mushtaq, S., 2019. Toxic effect of insecticides mixtures on antioxidant enzymes in different organs of fish, Labeorohita. Pakistan J. Zool., 51: 1355-1361. https://doi.org/10.17582/journal.pjz/2019.51.4.1355.1361

Ngidlo, R.T., 2013. Impacts of pesticides and fertilizers on soil, tail water and groundwater in three vegetable producing areas in the cordillera region, Northern Philippines. American J. Exp. Agri., 3: 780-793. https://doi.org/10.9734/ajea/2013/4696 

Ojha, A., Yaduvanshik, S. and Srivastava, N., 2011. Effect of combined exposure of commonly used organophosphate pesticides on lipid peroxidation and antioxidant enzymes in rat tissues. Pestic. Biochem. Physiol., 99: 148-156. https://doi.org/10.1016/j.pestbp.2010.11.011

Poet, T.S., Wu, H., Kousba, A.A. and Timchalk, C., 2003. In vitro rat hepatic and intestinal metabolism of the organophsophate pesticides chlorpyrifos and diazinon. Toxicol. Sci.,72: 193-200. https://doi.org/10.1093/toxsci/kfg035

Rusyniak, D.E. and Nanagas, K.A., 2004. Organophosphate poisoning, Seminars in Neurology, 24: 197-204. https://doi.org/10.1055/s-2004-830907

Shukla, D., Nagpure, N.S., Kumar, R. and Singh, P.J., 2010. Assessement of genotoxicity of dichlorvos to Mystusvittatus (Bloch)by comet assay. Indian J. Fish., 57:39-44.

Simoniello, M.F., Gigena, F., Poletta, G., Loteste, A., Kleinsorge, E., Campana, M., Scagnetti, J. and Parma, M.J., 2009. Alkaline comet assay for genotoxic effect detection in Neotropical fish Prochilodus lineatus (Pisces, Curimatidae). Bullet. Environ. Contam. Toxicol., 83: 155-158. https://doi.org/10.1007/s00128-009-9771-z

Singh, N.P., Mccoy, M.T., Tice, R.R. and Schneider, E.L., 1988. A simple technique for quantization of low levels of DNA damage in individual cells. Exp. Cell Res., 175: 184-191. https://doi.org/10.1016/0014-4827(88)90265-0

Steel, R.G.D., Torrie, J.H. and Dinkkey, D.A., 1996. Principles and Procedures of Statisticas (3rd Ed.). McGraw Hill Book Co, Singapore, 627p.

Verma, R.S., Mehta, A. and Srivastava, N., 2009. Comparative studies on chlorpyrifos and methyl parathion induced oxidative stress in different parts of rat brain: attenuation by antioxidant vitamins. Pestic. Biochem. Physiol., 95: 152-158. https://doi.org/10.1016/j.pestbp.2009.08.004

Verma, V.K. and Saxena, A., 2013. Investigations on the acute toxicity and behavioural alterations induced by the organophosphate pesticide, chlorpyrifos on Puntius chola (Hamilton-Buchanan). Indian J. Fish., 60:141-145.

Zhao, F., Wang, B., Zhang, X., Tian, H., Wang, W. and Ru, S., 2015. Induction of DNA base damage and strand breaks in peripheral erythrocytes and the underlying mechanism in goldfish (Carassius auratus) exposed to monocrotophos. Fish Physiol. Biochem., 41:613-624. https://doi.org/10.1007/s10695-015-0032-2