Subscribe Now Subscribe Today
Research Article
 

Malathion Induced DNA Damage in Freshwater Fish, Labeo rohita (Hamilton, 1822) Using Alkaline Single Cell Gel Electrophoresis



Sana Ullah, Maryam Begum, Kuldeep Dhama, Saeed Ahmad, Said Hassan and Ibrar Alam
 
Facebook Twitter Digg Reddit Linkedin StumbleUpon E-mail
ABSTRACT

The current study was aimed to investigate the genotoxic effect of an organophosphate pesticide malathion in the gills of a freshwater teleost rohu, Labeo rohita using alkaline Single Cell Gel Electrophoresis (SCGE)/comet assay. The 96 h LC50 of malathion was estimated for rohu in a semi-static system and was found to be 5 μg L–1. Specimens of rohu were exposed to LC50 of malathion. Gill tissues were sampled after 24, 48, 72 and 96 h of exposure. DNA damage was evaluated by studying different indices, including tail length (μm), percentage of DNA in tail, tail moment and olive tail moment using TriTek CometScoreTM. A linear relation was observed between exposure time and DNA damage in the gill cells. The current study revealed malathion as a potent inducer of DNA damage and comet assay as a reliable and sensitive assay for investigating and detecting DNA damage in vivo, induced in fish by genotoxic pesticides. In order to conserve the vanishing populations of rohu in natural aquatic bodies across the country, indiscriminate use of genotoxic pesticides such as malathion should be minimized.

Services
Related Articles in ASCI
Search in Google Scholar
View Citation
Report Citation

 
  How to cite this article:

Sana Ullah, Maryam Begum, Kuldeep Dhama, Saeed Ahmad, Said Hassan and Ibrar Alam, 2016. Malathion Induced DNA Damage in Freshwater Fish, Labeo rohita (Hamilton, 1822) Using Alkaline Single Cell Gel Electrophoresis. Asian Journal of Animal and Veterinary Advances, 11: 98-105.

DOI: 10.3923/ajava.2016.98.105

URL: https://scialert.net/abstract/?doi=ajava.2016.98.105

INTRODUCTION

Pesticides are used for preventing, repelling, destroying, mitigating and controlling agricultural and household pests (Ullah, 2015). These are also used for preventing infectious diseases. Although these chemicals are beneficial but pose a serious threat to humans as well as to the environment. Due to greater toxicity and higher persistence, organochlorine pesticides are banned worldwide or their use has been restricted, which established the second line pesticides, including pyrethroids and organophosphates as the most common and widely used pesticides (Ullah and Zorriehzahra, 2015). These pesticides might be beneficial, but their indiscriminate and inappropriate application can kill natural enemies of pests and can increase resistance of the pests. Some of these pesticides are highly persistent, thus contaminate the ground, soil and surface water. The toxic effects of these pesticides manifest in various ways, such as bioaccumulation and biomagnification, acute immune response, allergic reaction, chronic toxicity, teratogenic, carcinogenic and mutagenic effects (Ecobichon, 1996). These pesticides have led to serious environmental concerns, as some of these might not show instant effects in vivo but might lead to long term health hazards to humans. The ill environmental impacts of pesticides are often far greater as compared to the projected ones by those who use these pesticides. Approximately more than 98% employed insecticides and 95% herbicides lead to edible, non-target species, consequently reaching food and food chains, and eventually human beings (Damalas and Eleftherohorinos, 2011).

Malathion is one of the most widely employed organophosphate pesticides in agriculture, public health and for hygiene purpose in different countries around the globe (Ojha and Srivastava, 2014). Previous studies revealed organophosphates, including malathion, methyl parathion, chlorpyrifos and monchrotophos etc., as anticholinesterase agent and brought about different alterations in the level of neurotransmitters and neurobehavioral processes in different animals after exposure (Kamel and Hoppin, 2004; Satoh and Gupta, 2011). These pesticides also produce oxidative stress and extensive production of free radicals of oxygen, which is involved in pesticides induced toxicities in animals (Lukaszewicz-Hussain, 2010), in vitro experiments (Gultekin et al., 2001), pesticide manufacturing workers (Ranjbar et al., 2002) and pesticide sprayers (Lopez et al., 2007). Oxidative stress induced DNA damage has proposed mechanistic link of pesticide exposure to different health outcomes in previously conducted epidemiological studies (Olinski et al., 2002; Muniz et al., 2008). The diseases linked with the release of Reactive Oxygen Species (ROS), e.g., hydroxyl radical, superoxide anion radical, singlet oxygen, hydrogen peroxide, etc.) include atopic dermatitis, hepatitis, different autoimmune diseases (systemic lupus erythematosus and rheumatoid arthritis, etc.), male infertility and defective sperm function, aging, Alzheimer’s disease, Huntington’s disease and Parkinson’s disease (Shen and Ong, 2000; Olinski et al., 2002; Cooke et al., 2003; Tvrda et al., 2011).

Reactive oxygen species causes DNA oxidation, known as the most common kind of DNA damage. Hydrogen peroxides and superoxide anions induce base oxidation of intracellular DNA and strand breaks whereas, hydroxyl radicals induce DNA damage by direct reaction with DNA molecules (Friedberg et al., 1995). Despite being different tools for assessing DNA damage comet assay or Single Cell Gel Electrophoresis (SCGE) is the most commonly used measure for investigating induced damage in DNA. The SCGE is a well-established assay for the assessment and estimationof DNA damage both in vitro and in vivo, at the individual cell level due to its capability of detecting very low level oxidative damage induced in DNA (Singh et al., 1988).

Malathion is a potent source of ROS and is also an alkylating agent that can cause DNA damage (Wild, 1975; Braun et al., 1982). The technical grade of malathion induced changes in chromosome such as chromosomal aberration and micronuclei in germ and somatic cells in mice (Flessel et al., 1993). The DNA might be damaged due to the metabolites of malathion. Commercially available malathion contains isomalathion and malaoxan, which can induce in vivo disturbances in DNA such as DNA breakage at the site of tumour suppressor genes or oncogenes. Thus play a distinct role in inducing malignancies in exposed individuals. Keeping in view malathion can be considered as a potent carcinogen/mutagen (Blasiak et al., 1999).

Malathion is widely used organophosphate pesticide. However, there is no report regarding its genotoxic potentials against commercially important indigenous fishes of Pakistan. Keeping in view the current scenario, the current study was designed to find out the LC50 of malathion and assess its genotoxic potential against a freshwater fish rohu, Labeo rohita, economically an important teleost.

MATERIALS AND METHODS

Experimental animal acclimatization: A total of 180 uniform sized and healthy specimens (8.17±0.794 cm and 6.55±1.01 g) of L. rohita were collected. The fish were acclimated for 10 days in aquaria (60×30×30 cm) and were fed with 35% protein to satiation at the rate of 5% body weight. The aquaria were siphoned off on a daily basis for avoiding stress to the fish and different water quality parameters were investigated regularly. Dead fish were removed as quickly as possible in order to maintain water quality.

Test chemical: The commercial formulation of malathion was purchased from local market and used during the study. A stock solution was prepared in acetone. During the experiment, required amount of dilutions was used.

Determination of Lethal Concentration (LC50): The LC50 of malathion against rohu was found through probit analysis. The fish were exposed to the different concentrations (2, 3, 4, 5, 6, 7 and 8 μg L–1) of malathion, using semi-static method. Fish mortality was noted from 24 through 96 h of exposure. Dead fish were removed to avoid water deterioration.

Alkaline Single Cell Gel Electrophoresis (SCGE)/comet assay: The alkaline Single Cell Gel Electrophoresis (SCGE)/comet assay was carried out as three layer procedure by following Singh et al. (1988) with minor modifications (Klaude et al., 1996) as performed by Pandey et al. (2011). The 15 μL cell suspension was mixed with Low Melting-Point Agarose (LMPA): 85 μL, 0.5% and was layered on a frosted slide, already coated with a layer of 1% 200 μL normal agarose. Then again, it was layered with LMPA (100 μL) after solidification of the gel. The slides were kept submerged in lysing solution overnight at 4°C. The slides were positioned side by side in horizontal unit of gel electrophoresis, dipped in fresh cold alkaline electrophoresis buffer, left at 4°C for 20 min in the same solution in order to unwind DNA and convert alkali-labile sites to single strand breaks. The same alkaline buffer was used for carrying out alkaline electrophoresis using 300 mA, 15 V (0.8 v/cm) for 20 min at 4°C. Then the slides were gently neutralized using 0.4 M tris buffer (pH 7.5). The slides were stained with acridine orange and were inspected under an epifluorescent microscope (400x, Nikon AFX-1 Optiphot). Digital images were captured for succeeding scoring with TriTek CometScoreTM, Freeware v1.5. A total of 100 randomly selected cells were scored for each specimen (50 cells from each of the two replicated slides). The parameters including Tail Length (TL), Percent DNA in tail (TDNA%), Tail Moment (TM) and Olive Tail Moment (OTM) were selected for quantifying the level of DNA damage.

Statistical analysis: Data obtained from the experiment were expressed as Mean±SE. The data were analysed by using one way analysis of variances (ANOVA) followed by HSDTukey test using Statistix Version 8.1. The value of p<0.05 was considered statistically significant.

RESULTS

Physico-chemical parameters of water: The temperature of the water was varying from 24.3-25.8°C. The dissolved oxygen ranged from 6.1-7.4 mg L–1, pH 6.9-7.5, conductivity 240-290 μM cm–1, total hardness 162-178 mg L–1 and Ammonia was lying under 0.25 ppm.

Toxicity evaluation of malathion: Acute toxicity bioassay of different concentrations, log concentrations, Percent mortality and values for probit kill is given in Table 1. A concentration dependent increase was observed in mortalities. Plotting percent mortality and probit kill against log concentration resulted in formation of curves shown in Fig. 1 and 2, respectively. Both the analyses revealed 50% mortality at log concentration 0.653, equal to 5 μg L–1 (Table 1).

Image for - Malathion Induced DNA Damage in Freshwater Fish, Labeo rohita (Hamilton, 1822) Using Alkaline Single Cell Gel Electrophoresis
Fig. 1: Toxicity evaluation of malathion against rohu, Labeo rohita

Image for - Malathion Induced DNA Damage in Freshwater Fish, Labeo rohita (Hamilton, 1822) Using Alkaline Single Cell Gel Electrophoresis
Fig. 2: Toxicity evaluation of malathion against rohu, Labeo rohita

Table 1: Determination of LC50 value of malathion for 96 h against Lebeo rohita
Image for - Malathion Induced DNA Damage in Freshwater Fish, Labeo rohita (Hamilton, 1822) Using Alkaline Single Cell Gel Electrophoresis

DNA damage: Malathion induced DNA damage in gill tissues of Labeo rohita was quantified in terms of TL, TDNA%, TM and OTM. A significant (p<0.05) increasing trend was observed in TL of the comets, TDNA% and TM with exposure period in the treated group as shown in Table 2. The exposed fish specimens of treated group also exhibited significantly higher olive tail moment (p<0.05) in their gills as compared to fish specimens of control group (Fig. 3). The strand breaks in DNA of the fish in the treated group after malathion exposure is shown in Fig. 4.

With respect to duration of exposure, the lowest level of damage in DNA was observed at 24 h while, the highest after 96 h of exposure in the treated group in term of TL, TDNA%, TM and OTM.

Image for - Malathion Induced DNA Damage in Freshwater Fish, Labeo rohita (Hamilton, 1822) Using Alkaline Single Cell Gel Electrophoresis
Fig. 3:
Olive tail moment of comets in gills tissues of rohu at specific intervals after exposure to LC50 of malathion, data are presented as Mean±SE, (n = 6), Means with different superscripted letters are significantly different (p<0.05)

DISCUSSION

Previous studies revealed SCGE (Frenzilli et al., 2009; Galindo et al., 2010) as a handy assay for investigating the gentoxicological potential of different chemicals such as in birds (Baos et al., 2006), mammals (Park et al., 2007; Garaj-Vrhovac et al., 2009), amphibians (Cotelle and Ferard, 1999; Yin et al., 2009), reptiles (Bronikowski, 2008) and mollusks (Cotelle and Ferard, 1999; Canty et al., 2009). Aquatic organisms, including fish can serve as excellent sentries for assessing the carcinogenic, mutagenic and genotoxic potential of different toxicants (Banu et al., 2001; Ali et al., 2008). Different fish species from both marine and freshwater ecosystems have been employed for environmental biomonitoring such as Channa punctatus (Kushwaha et al., 2000; Pandey et al., 2006; Ali et al., 2008), Cyprinus carpio (Buschini et al., 2004; Gustavino et al., 2005), Ameiurus nebulosus (Pandrangi et al., 1995), Dreissena polymorpha (Pavlica et al., 2001), Mugil sp. and Netuma sp. (De Andrade et al., 2004).

Fish are used as a model organism in ecotoxicological studies on account of its economic value, crucial role in trophic web, sensitiveness even to lower concentration of toxicants as well as the capability of bioaccumulation of toxic substances (De Andrade et al., 2004; Jha, 2008). The fish is being used as an important bioindicator of pollution and earlier detection of problems in aquatic ecosystem (Cavas and Ergene-Gozukara, 2005; Steckert et al., 2009).

Image for - Malathion Induced DNA Damage in Freshwater Fish, Labeo rohita (Hamilton, 1822) Using Alkaline Single Cell Gel Electrophoresis
Fig. 4(a-b):
Fluorescent photomicrograph (40×) of cells in gills of rohu, after 96 h of exposure to LC50 of malathion using comet assay, Stain: Acridine orange, (a) Intact DNA in control fish and (b) Comets with tail formed in treated fish

Table 2:
Tail length, Tail DNA (%), tail moment observed in the gills of Lebeo rohita after exposure to malathion at different time intervals
Image for - Malathion Induced DNA Damage in Freshwater Fish, Labeo rohita (Hamilton, 1822) Using Alkaline Single Cell Gel Electrophoresis
Data are represented as Mean±SE (n = 6), means followed by different letters are significantly different at p<0.05. ANOVA followed by HSD Tukey test

Many tissues of fish, such as blood peripheral erythrocyte, gills, liver and brain can be used for conducting toxicological studies such as evaluating DNA damage, however, non-invasive method is highly preferred (Lee and Steinert, 2003; Zhou et al., 2006).

Pesticides are extensively used in modern era of the industrial revolution (Ullah, 2015; Ullah and Zorriehzahra, 2015). Therefore, researchers are actively involved in conducting experiments on exposed populations in order to investigate the risks associated with occupational exposure. As cancer is having a mutational base, therefore, monitoring of exposed animals to different mutagenic and genotoxic pesticides in polluted environments is of great importance in assuring protection to human health (Pandey et al., 2011). Several reports are available, demonstrating the genotoxic effects of different pesticides in various living organisms (Bhalli et al., 2006, 2009). Pesticides bioaccumulate in the tissues of living organisms, hence, the estimation of these chemicals in tissues might be more valuable for assessing natural or wild populations (Kumar and Chapman, 2001; Rao et al., 2003).

In the present study acute concentration of malathion causing 50% mortality after 96 h was found to be 5 μg L–1. This value was a bit higher than 4.5 μg L–1 (Patil and David, 2013) and much lower than 15 mg L–1 (Thenmozhi et al., 2011) for the same fish species. Changes in LC50 values of the same toxicant for the same fish species or different toxicants for various fish species might be due to the formulation, specification of the pesticides as well as the stereochemistry of their active molecules (Ullah, 2015). Toxicity of the pesticides mainly depends on stereochemistry of their molecules as isomers in formulations of pesticides vary in their precise toxicity (Ullah et al., 2014). Single isomer based pesticides are more toxic as compared to those having various combinations of isomers in their formulation (Bradbury and Coats, 1989).

Pesticide toxicity is also correlated with the carriers of contaminants and active and inert ingredients (Ullah et al., 2014). Moreover, the toxicity of pesticides against fish also depends on certain factors, including temperature, age of the fish, the size of the fish and health condition of the fish (Farah et al., 2004). The temperature during the study was ranging from 24.3 and 25.8°C. No association between temperature and the value of LC50 was observed while, an inverse relation between temperature and LC50 was observed by Kumaragura and Beamish (Kumaraguru and Beamish, 1981). Studies have shown that pesticides are less toxic in summer as compare to winter. Tenfold difference was observed at 10, 15 and 20°C in the value of LC50 for 96 h (Singh et al., 2010), while an inverse association between pesticide toxicity and body weight do exist (WHO., 1992).

The results of the current study revealed a significant DNA damage in the gills of rohu. An increase was observed in DNA damage with the time period of exposure. The higher DNA damage in gills might be due to the fact that the gills are constantly, directly and continuously exposed tissue to the toxicant (Pandey et al., 2006). Gill has been shown as the suitable tissues for conducting genotoxicological studies in goldfish, rainbow trout and shellfish (Liepelt et al., 1995; Sasaki et al., 1997; Masuda et al., 2004; Pandey et al., 2011). Some previous studies on various species of fish showed higher sensitivity of gills to DNA damage than other cells, including lymphocyte, erythrocytes, liver and kidney (Wilson et al., 1998; Kilemade et al., 2004; Ali et al., 2008, 2009).

The basic principle of electrophoresis is that damaged DNA migrate faster toward anode as compared to intact DNA, thus result in tail formation. The fragments of DNA freely move during electrophoresis, while tranquilloops of DNA drag out of the head/nucleus. Thus the tail length determines the extent of DNA migrated out of the nuclear head. As smaller fragments of DNA migrate farthermost, hence the tail length of the comets is primarily showed by the size of the fragments of DNA, produced during the alkaline unwinding step of SCGE (Kumaravel and Jha, 2006). Percent tail DNA shows the percentage of DNA moved out of the head and is also considered as a suitable parameter (Kilemade et al., 2004). Olive tail moment shows the distance from the head centre to tail centre and is used for the assessment of degree of DNA damage (Singh et al., 1988).

The biotransformation of xenobiotic leads to an increase in the production of Reactive Oxygen Species (ROS) highly toxic to fish. It can directly break DNA through OH- and H2O2 resulting in oxidized bases of DNA (Akcha et al., 2003). Fish have an antioxidant defence system against ROS but when higher production of ROS surpasses the defence systems of fish, cellular lesions and DNA damage occur (Cadet et al., 2003; Cavalcante et al., 2008; Jha, 2008). Oxidative DNA damage due to production of ROS is attributing for variable and higher DNA damage in the cells of the gills (Wilson et al., 1998; Pavlica et al., 2001).

Our results are in congruence with the previous studies conducted on mutagenic and genotoxic potentials of different organophosphates pesticides in different organisms (Fahmy and Abdalla, 1998; Das et al., 2006; Rao et al., 2006; Ganguly et al., 2010; Li et al., 2010). The result of the present study demonstrated malathion as genotoxicity inducer at acute concentration. The findings, display a serious concern regarding the potential threats to aquatic organisms from malathion. Thus, it should be carefully and judiciously employed in domestic, industrial and agricultural practices. The current study has a broad perspective regarding aquatic toxicology as gills of the fish species are constantly exposed to different type of toxicants.

CONCLUSION

Our results clearly concluded that malathion possesses mutagenic and genotoxic potential as well as showed SCGE as a potent assay for bio-monitoring genotoxicity in aquatic environments. This study provides a biomarker for malathion induced mutagenicity and genotoxicity, which might be useful for evaluating the toxicants impacts in acute concentrations on freshwater fish species. This system might also work as a sensitive and useful tool for investigating the exposure of different fish species to genotoxins and assessing the mutagenic hazards in surface water.

REFERENCES

1:  Akcha, F., F.V. Hubert and A. Pfhol-Leszkowicz, 2003. Potential value of the comet assay and DNA adduct measurement in dab (Limanda limanda) for assessment of in situ exposure to genotoxic compounds. Mutat. Res./Genet. Toxicol. Environ. Mutagen., 534: 21-32.
CrossRef  |  Direct Link  |  

2:  Ali, D., N.S. Nagpure, S. Kumar, R. Kumar and B. Kushwaha, 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.
CrossRef  |  Direct Link  |  

3:  Ali, D., N.S. Nagpure, S. Kumar, R. Kumar, B. Kushwaha and W.S. Lakra, 2009. Assessment of genotoxic and mutagenic effects of chlorpyrifos in freshwater fish channa punctatus (bloch) using micronucleus assay and alkaline single-cell gel electrophoresis. Food Chem. Toxicol., 47: 650-656.
CrossRef  |  Direct Link  |  

4:  Banu, B.S., K. Danadevi, M.F. Rahman, Y.R. Ahuja and J. Kaiser, 2001. Genotoxic effect of monocrotophos to sentinel species using comet assay. Food Chem. Toxicol., 39: 361-366.
CrossRef  |  Direct Link  |  

5:  Baos, R., R. Jovani, N. Pastor, J.L. Tella and B. Jimenez et al., 2006. Evaluation of genotoxic effects of heavy metals and arsenic in wild nestling white storks (Ciconia ciconia) and black kites (Milvus migrans) from southwestern spain after a mining accident. Environ. Toxicol. Chem., 25: 2794-2803.
CrossRef  |  PubMed  |  Direct Link  |  

6:  Bhalli, J.A., T. Ali, M.R. Asi, Z.M. Khalid, M. Ceppi and Q.M. Khan, 2009. DNA damage in Pakistani agricultural workers exposed to mixture of pesticides. Environ. Mol. Mutagen., 50: 37-45.
CrossRef  |  PubMed  |  Direct Link  |  

7:  Bhalli, J.A., Q.M. Khan and A. Nasim, 2006. DNA damage in pakistani pesticide-manufacturing workers assayed using the comet assay. Environ. Mol. Mutagen., 47: 587-593.
CrossRef  |  Direct Link  |  

8:  Blasiak, J., P. Jaloszynski, A. Trzeciak and K. Szyfter, 1999. In vitro studies on the genotoxicity of the organophosphorus insecticide malathion and its two analogues. Mutation Res./Genet. Toxicol. Environ. Mutagen., 445: 275-283.
CrossRef  |  Direct Link  |  

9:  Bradbury, S.P. and J.R. Coats, 1989. Comparative Toxicology of the Pyrethroid Insecticides: Reiews of Environmental Contamination and Toxicology, Vol. 108, Springer-Verlag, New York, pp: 132-177

10:  Braun, R., J. Schoneich, L. Weissflog and W. Dedek, 1982. Activity of organophosphorus insecticides in bacterial tests for mutagenicity and DNA repair-direct alkylation vs. Metabolic activation and breakdown. I. Butonate, vinylbutonate, trichlorfon, dichlorvos, demethyl dichlorvos and demethyl vinylbutonate. Chemico-Biol. Interact., 39: 339-350.
CrossRef  |  Direct Link  |  

11:  Bronikowski, A.M., 2008. The evolution of aging phenotypes in snakes: A review and synthesis with new data. Age, 30: 169-176.
CrossRef  |  Direct Link  |  

12:  Buschini, A., A. Martino, B. Gustavino, M. Monfrinotti and P. Poli et al., 2004. Comet assay and micronucleus test in circulating erythrocytes of Cyprinus carpio specimens exposed in situ to lake waters treated with disinfectants for potabilization. Mutat. Res./Genet. Toxicol. Environ. Mutagen., 557: 119-129.
CrossRef  |  Direct Link  |  

13:  Cadet, J., T. Douki, D. Gasparutto and J.L. Ravanat, 2003. Oxidative damage to DNA: Formation, measurement and biochemical features. Mutation Res./Fundam. Mol. Mech. Mutagen., 531: 5-23.
CrossRef  |  PubMed  |  Direct Link  |  

14:  Canty, M.N., T.H. Hutchinson, R.J. Brown, M.B. Jones and A.N. Jha, 2009. Linking genotoxic responses with cytotoxic and behavioural or physiological consequences: Differential sensitivity of echinoderms (Asterias rubens) and marine molluscs (Mytilus edulis). Aquatic Toxicol., 94: 68-76.
CrossRef  |  Direct Link  |  

15:  Cavalcante, D.G.S.M., C.B.R. Martinez and S.H. Sofia, 2008. Genotoxic effects of Roundup® on the fish Prochilodus lineatus. Mutat. Res./Genet. Toxicol. Environ. Mutagenesis, 655: 41-46.
CrossRef  |  Direct Link  |  

16:  Cavas, T. and S. Ergene-Gozukara, 2005. Micronucleus test in fish cells: A bioassay for in situ monitoring of genotoxic pollution in the marine environment. Environ. Mol. Mutagen., 46: 64-70.
CrossRef  |  Direct Link  |  

17:  Cooke, M.S., M.D. Evans, M. Dizdaroglu and J. Lunec, 2003. Oxidative DNA damage: Mechanisms, mutation and disease. FASEB J., 17: 1195-1214.
CrossRef  |  PubMed  |  Direct Link  |  

18:  Cotelle, S. and J.F. Ferard, 1999. Comet assay in genetic ecotoxicology: A review. Environ. Mol. Mutagen., 34: 246-255.
CrossRef  |  PubMed  |  3.0.CO;2-V/abstract? target='_blank' class='botlinks'>Direct Link  |  

19:  Damalas, C.A. and I.G. Eleftherohorinos, 2011. Pesticide exposure, safety issues and risk assessment indicators. Int. J. Environ. Res. Public Health, 8: 1402-1419.
CrossRef  |  Direct Link  |  

20:  De Andrade, V.M., T.R.O. de Freitas and J. da Silva, 2004. Comet assay using mullet (Mugil sp.) and sea catfish (Netuma sp.) erythrocytes for the detection of genotoxic pollutants in aquatic environment. Mutat. Res./Genet. Toxicol. Environ. Mutagen., 560: 57-67.
CrossRef  |  Direct Link  |  

21:  Ecobichon, D.J., 1996. Toxic Effects of Pesticides. In: Casarett and Doull's Toxicology: The Basic Science of Poisons, Casarett, L.J., C.D. Klaassen, M.O. Amdur and J. Doull (Eds.). 5th Edn., MacMillan, New York, USA., ISBN-13: 9780071054768, pp: 643-689

22:  Fahmy, M.A. and E.F. Abdalla, 1998. Genotoxicity evaluation of buprofezin, petroleum oil and profenofos in somatic and germ cells of male mice. J. Applied Toxicol., 18: 301-305.
CrossRef  |  Direct Link  |  

23:  Farah, M.A., B. Ateeq, M.N. Ali, R. Sabir and W. Ahmad, 2004. Studies on lethal concentrations and toxicity stress of some xenobiotics on aquatic organisms. Chemosphere, 55: 257-265.
CrossRef  |  Direct Link  |  

24:  Flessel, P., P.J.E. Quintana and K. Hooper, 1993. Genetic toxicity of malathion: A review. Environ. Mol. Mutagen., 22: 7-17.
CrossRef  |  Direct Link  |  

25:  Frenzilli, G., M. Nigro and B.P. Lyons, 2009. The comet assay for the evaluation of genotoxic impact in aquatic environments. Mutat. Res./Rev. Mutat. Res., 681: 80-92.
CrossRef  |  Direct Link  |  

26:  Friedberg, E.C., G.C. Walker and W. Siede, 1995. DNA Repair and Mutagenesis. 2nd Edn., ASM Press, Washington, DC., USA., ISBN-13: 9781555810887, Pages: 698

27:  Galindo, B.A., G. Troilo, I.M.S. Colus, C.B.R. Martinez and S.H. Sofia, 2010. Genotoxic effects of aluminum on the neotropical fish Prochilodus lineatus. Water Air Soil Pollut., 212: 419-428.
CrossRef  |  Direct Link  |  

28:  Ganguly, S., S. Bhattacharya, S. Mandi and J. Tarafdar, 2010. Biological detection and analysis of toxicity of organophosphate- and azadirachtin-based insecticides in Lathyrus sativus L. Ecotoxicology, 19: 85-95.
CrossRef  |  Direct Link  |  

29:  Garaj-Vrhovac, V., G. Gajski, I. Trosic and I. Pavicic, 2009. Retracted: Evaluation of basal DNA damage and oxidative stress in wistar rat leukocytes after exposure to microwave radiation. Toxicology, 259: 107-112.
CrossRef  |  Direct Link  |  

30:  Gultekin, F., N. Delibas, S. Yasar and I. Kilinc, 2001. In vivo changes in antioxidant systems and protective role of melatonin and a combination of vitamin C and vitamin E on oxidative damage in erythrocytes induced by chlorpyrifos-ethyl in rats. Arch. Toxicol., 75: 88-96.
CrossRef  |  PubMed  |  Direct Link  |  

31:  Gustavino, B., A. Buschini, M. Monfrinotti, M. Rizzoni, L. Tancioni, P. Poli and C. Rossi, 2005. Modulating effects of humic acids on genotoxicity induced by water disinfectants in Cyprinus carpio. Mutat. Res./Genet. Toxicol. Environ. Mutagen., 587: 103-113.
CrossRef  |  Direct Link  |  

32:  Jha, A.N., 2008. Ecotoxicological applications and significance of the comet assay. Mutagenesis, 23: 207-221.
CrossRef  |  Direct Link  |  

33:  Kamel, F. and J.A. Hoppin, 2004. Association of pesticide exposure with neurologic dysfunction and disease. Environ. Health Perspect., 112: 950-958.
CrossRef  |  Direct Link  |  

34:  Kilemade, M.F., M.G.J. Hartl, D. Sheehan, C. Mothersill, F.N.A.M. van Pelt, J. O'Halloran and N.M. O'Brien, 2004. Genotoxicity of field-collected inter-tidal sediments from Cork Harbor, Ireland, to juvenile turbot (Scophthalmus maximus L.) as measured by the Comet assay. Environ. Mol. Mutagen., 44: 56-64.
CrossRef  |  Direct Link  |  

35:  Klaude, M., S. Eriksson, J. Nygren and G. Ahnstrom, 1996. The comet assay: Mechanisms and technical considerations. Mutat. Res./DNA Repair, 363: 89-96.
CrossRef  |  Direct Link  |  

36:  Kumar, A. and J.C. Chapman, 2001. Profenofos residues in wild fish from cotton-growing areas of New South Wales, Australia. J. Environ. Qual., 30: 740-750.
CrossRef  |  PubMed  |  Direct Link  |  

37:  Kumaraguru, A.K. and F.W.H. Beamish, 1981. Lethal toxicity of permethrin (NRDC-143) to rainbow trout, Salmo gairdneri, in relation to body weight and water temperature. Water Res., 15: 503-505.
CrossRef  |  Direct Link  |  

38:  Kumaravel, T.S. and A.N. Jha, 2006. Reliable comet assay measurements for detecting DNA damage induced by ionising radiation and chemicals. Mutat. Res./Genet. Toxicol. Environ. Mutagen., 605: 7-16.
CrossRef  |  Direct Link  |  

39:  Kushwaha, B., S.K. Srivastava, B. Singh, N.S. Nagpure and A.G. Ponniah, 2000. Evaluation of comet assay and micronuclei test as genotoxic assays in channa punctatus. Natl. Acad. Sci. Lett., 23: 177-179.
Direct Link  |  

40:  Lee, R.F. and S. Steinert, 2003. Use of the single cell gel electrophoresis/comet assay for detecting DNA damage in aquatic (marine and freshwater) animals. Mutat. Res./Rev. Mutat. Res., 544: 43-64.
CrossRef  |  Direct Link  |  

41:  Li, X., S. Li, S. Liu and G. Zhu, 2010. Lethal effect and in vivo genotoxicity of profenofos to chinese native amphibian (Rana spinosa) tadpoles. Arch. Environ. Contaminat. Toxicol., 59: 478-483.
CrossRef  |  Direct Link  |  

42:  Liepelt, A., L. Karbe and J. Westendorf, 1995. Induction of DNA strand breaks in rainbow trout Oncorhynchus mykiss under hypoxic and hyperoxic conditions. Aquat. Toxicol., 33: 177-181.
CrossRef  |  Direct Link  |  

43:  Lopez, O., A.F. Hernandez, L. Rodrigo, F. Gil and G. Pena et al., 2007. Changes in antioxidant enzymes in humans with long-term exposure to pesticides. Toxicol. Lett., 171: 146-153.
CrossRef  |  Direct Link  |  

44:  Lukaszewicz-Hussain, A., 2010. Role of oxidative stress in organophosphate insecticide toxicity-short review. Pestic. Biochem. Physiol., 98: 145-150.
CrossRef  |  Direct Link  |  

45:  Masuda, S., Y. Deguchi, Y. Masuda, T. Watanabe and H. Nukaya et al., 2004. Genotoxicity of 2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) and 4-amino-3,3'-dichloro-5,4'-dinitro-biphenyl (ADDB) in goldfish (Carassius auratus) using the micronucleus test and the comet assay. Mutat. Res./Genet. Toxicol. Environ. Mutagen., 560: 33-40.
CrossRef  |  PubMed  |  Direct Link  |  

46:  Muniz, J.F., L. McCauley, J. Scherer, M. Lasarev and M. Koshy et al., 2008. Biomarkers of oxidative stress and DNA damage in agricultural workers: A pilot study. Toxicol. Applied Pharmacol., 227: 97-107.
CrossRef  |  Direct Link  |  

47:  Ojha, A. and N. Srivastava, 2014. In vitro studies on organophosphate pesticides induced oxidative DNA damage in rat lymphocytes. Mutat. Res./Genet. Toxicol. Environ. Mutagen., 761: 10-17.
CrossRef  |  Direct Link  |  

48:  Olinski, R., D. Gackowski, M. Foksinski, R. Rozalski, K. Roszkowski and P. Jaruga, 2002. Oxidative DNA damage: Assessment of the role in carcinogenesis, atherosclerosis and acquired immunodeficiency syndrome. Free Radic. Biol. Med., 33: 192-200.
CrossRef  |  Direct Link  |  

49:  Pandey, A.K., N.S. Nagpure, S.P. Trivedi, R. Kumar and B. Kushwaha, 2011. Profenofos induced DNA damage in freshwater fish, Channa punctatus (Bloch) using alkaline single cell gel electrophoresis. Mutat. Res./Genet. Toxicol. Environ. Mutagen., 726: 209-214.
CrossRef  |  Direct Link  |  

50:  Pandey, S., N.S. Nagpure, R. Kumar, S. Sharma, S.K. Srivastava and M.S. Verma, 2006. Genotoxicity evaluation of acute doses of endosulfan to freshwater teleost Channa punctatus (Bloch) by alkaline single-cell gel electrophoresis. Ecotoxicol. Environ. Saf., 65: 56-61.
CrossRef  |  Direct Link  |  

51:  Pandrangi, R., M. Petras, S. Ralph and M. Vrzoc, 1995. Alkaline single cell gel (comet) assay and genotoxicity monitoring using bullheads and carp. Environ. Mol. Mutagen., 26: 345-356.
CrossRef  |  Direct Link  |  

52:  Park, E., M. Glei, Y. Knobel and B.L. Pool-Zobel, 2007. Blood mononucleocytes are sensitive to the DNA damaging effects of iron overload-in vitro and ex vivo results with human and rat cells. Mutat. Res./Fundam. Mol. Mech. Mutagen., 619: 59-67.
CrossRef  |  Direct Link  |  

53:  Patil, V.K. and M. David, 2013. Oxidative stress in freshwater fish, Labeo rohita as a biomarker of malathion exposure. Environ. Monit. Assess., 185: 10191-10199.
CrossRef  |  Direct Link  |  

54:  Pavlica, M., G.I.V. Klobucar, N. Mojas, R. Erben and D. Papes, 2001. Detection of DNA damage in haemocytes of zebra mussel using comet assay. Mutat. Res./Genet. Toxicol. Environ. Mutagen., 490: 209-214.
CrossRef  |  Direct Link  |  

55:  Das, G.P., A.P. Shaik and K. Jamil, 2006. Cytotoxicity and genotoxicity induced by the pesticide profenofos on cultured human peripheral blood lymphocytes. Drug Chem. Toxicol., 29: 313-322.
CrossRef  |  PubMed  |  Direct Link  |  

56:  Ranjbar, A., P. Pasalar and M. Abdollahi, 2002. Induction of oxidative stress and acetylcholinesterase inhibition in organophosphorous pesticide manufacturing workers. Hum. Exp. Toxicol., 21: 179-182.
CrossRef  |  Direct Link  |  

57:  Rao, J.V., D. Shilpanjali, P. Kavitha and S.S. Madhavendra, 2003. Toxic effects of profenofos on tissue acetylcholinesterase and gill morphology in a euryhaline fish, Oreochromis mossambicus. Arch. Toxicol., 77: 227-232.
Direct Link  |  

58:  Sasaki, Y.F., F. Izumiyama, E. Nishidate, S. Ishibashi and S. Tsuda et al., 1997. Detection of genotoxicity of polluted sea water using shellfish and the alkaline single-cell gel electrophoresis (SCE) assay: A preliminary study. Mutat. Res./Genet. Toxicol. Environ. Mutagen., 393: 133-139.
CrossRef  |  Direct Link  |  

59:  Satoh, T. and R.C. Gupta, 2011. Anticholinesterase Pesticides: Metabolism, Neurotoxicity and Epidemiology. John Wiley & Sons, New York, USA., ISBN: 9780470410301, Pages: 625

60:  Shen, H.M. and C.N. Ong, 2000. Detection of oxidative DNA damage in human sperm and its association with sperm function and male infertility. Free Radic. Biol. Med., 28: 529-536.
CrossRef  |  Direct Link  |  

61:  Singh, N.P., M.T. McCoy, R.R. Tice and E.L. Schneider, 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res., 175: 184-191.
CrossRef  |  PubMed  |  Direct Link  |  

62:  Singh, S.K., S.K. Singh and R.P. Yadav, 2010. Toxicological and biochemical alterations of cypermethrin (synthetic pyrethroids) against freshwater teleost fish Colisa fasciatus at different season. World J. Zool., 5: 25-32.
Direct Link  |  

63:  Steckert, A.V., C.E. Schnack, J. Silvano, F. Dal-Pizzol and V.M. Andrade, 2009. Markers of pesticide exposure in irrigated rice cultures. J. Agric. Food Chem., 57: 11441-11445.
CrossRef  |  Direct Link  |  

64:  Thenmozhi, C., V. Vignesh, R. Thirumurugan and S. Arun, 2011. Impacts of malathion on mortality and biochemical changes of freshwater fish Labeo rohita. Iran. J. Environ. Health Sci. Eng., 8: 393-400.
Direct Link  |  

65:  Tvrda, E., Z. Knazicka, L. Bardos, P. Massanyi and N. Lukac, 2011. Impact of oxidative stress on male fertility-A review. Acta Veterinaria Hungarica, 59: 465-484.
CrossRef  |  Direct Link  |  

66:  Ullah, R., A. Zuberi, S. Ullah, I. Ullah and F.U. Dawar, 2014. Cypermethrin induced behavioral and biochemical changes in mahseer, Tor putitora. J. Toxicol. Sci., 39: 829-836.
CrossRef  |  Direct Link  |  

67:  Ullah, S., 2015. Protective role of vitamin C against Cypermethrin induced toxicity in Labeo rohita (Ham.): Biochemical aspects. M.Phil Thesis, Department of Animal Sciences, Quaid-i-Azam University, Islamabad, Pakistan.

68:  Ullah, S. and M.J. Zorriehzahra, 2015. Ecotoxicology: A review of pesticides induced toxicity in fish. Adv. Anim. Vet. Sci., 3: 40-57.
CrossRef  |  Direct Link  |  

69:  Rao, J.V., G. Begum, N.M. Jakka, K. Srikanth and R.N. Rao, 2006. Sublethal effects of profenofos on locomotor behavior and gill architecture of the mosquito fish, Gambusia affinis. Drug Chem. Toxicol., 29: 255-267.
CrossRef  |  Direct Link  |  

70:  WHO., 1992. Alpha-cypermethrin. Environmental Health Criteria No. 142, World Health Organization/United Nation Geneva, Switzerland.

71:  Wild, D., 1975. Mutagenicity studies on organophosphorus insecticides. Mutat. Res./Rev. Genet. Toxicol., 32: 133-149.
CrossRef  |  Direct Link  |  

72:  Wilson, J.T., P.L. Pascoe, J.M. Parry and D.R. Dixon, 1998. Evaluation of the comet assay as a method for the detection of DNA damage in the cells of a marine invertebrate, Mytilus edulis L. (Mollusca: Pelecypoda). Mutat. Res./Fundam. Mol. Mechan. Mutagen., 399: 87-95.
CrossRef  |  PubMed  |  Direct Link  |  

73:  Yin, X., G. Zhu, X.B. Li and S. Liu, 2009. Genotoxicity evaluation of chlorpyrifos to amphibian Chinese toad (Amphibian: Anura) by Comet assay and Micronucleus test. Mutat. Res./Genet. Toxicol. Environ. Mutagen., 680: 2-6.
CrossRef  |  PubMed  |  Direct Link  |  

74:  Zhou, B., W. Liu, W.H. Siu, D. O'Toole, P.K. Lam and R.S. Wu, 2006. Exposure of spermatozoa to duroquinone may impair reproduction of the common carp (Cyprinus carpio) through oxidative stress. Aquat. Toxicol., 77: 136-142.
CrossRef  |  PubMed  |  Direct Link  |  

©  2021 Science Alert. All Rights Reserved