Chlorpyrifos induces apoptosis and DNA damage in Drosophila through generation of reactive oxygen species

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Abstract

The present study investigated the apoptosis and DNA damage inducing potential of chlorpyrifos (CP) in Drosophila melanogaster. Third instar larvae of Drosophila were treated with different concentrations of CP (0.015–15.0 μg/L) for 2–48 h. Reactive oxygen species (ROS) generation, oxidative stress markers, DNA damage and apoptotic cell death end points were measured in them. A significant increase in DNA damage was concomitant with apoptotic mode of cell death in 15.0 μg/L CP-treated organisms for 24 and 48 h. Depolarization in mitochondrial membrane potential and increased casapase-3 and caspase-9 activities in these organisms indicated both as potential targets of CP. A significant positive correlation was observed among ROS generation, apoptosis and DNA damage. The study suggests that (i) ROS may be involved in inducing apoptosis and DNA damage in the CP-exposed larvae of Drosophila and (ii) D. melanogaster may be used as an alternative in vivo animal model for xenobiotics hazard assessment.

Introduction

Aerobic organisms are exposed to ROS in a continuous and unavoidable manner. The ROS are generated as a part of the normal metabolism of a cell. Additionally, they can also be derived from exogenous sources following exposure of a cell/organism to environmental chemical insult including pesticides. Although ROS regulate various biological processes by regulating an array of signal transduction pathways when present in transient amounts, at high and/or sustained levels, they can cause severe damage to DNA, protein and lipids (Lau et al., 2008).

Various stressors present in the environment including pesticides are capable of reacting with DNA and causing DNA damage (Wilson et al., 2003). Given that stressors have the capability to generate ROS, one of the possible mechanisms for the induction of DNA damage may be through the generation of ROS. Excessive ROS production may lead to cellular dysfunction culminating in cell death. Apoptosis is one of the major forms of cell death, in which the cell designs and executes the program of its own demise. Recently, ROS produced during oxidative stress have been reported to initiate signaling cascades leading to apoptosis (Yuan et al., 2008). Although a mechanism for ROS-induced apoptosis has not been delineated, increasing amounts of research have indicated the involvement of mitochondria in this process (Ratha et al., 2007).

Chlorpyrifos (CP) is a broad spectrum organophosphorous pesticide that is widely used throughout the world in agriculture and non-agriculture applications. It has been reported to cause immunological abnormalities (Trasher et al., 2002) and to induce oxidative stress (Goel et al., 2005) and tissue damage (Jett and Navoa, 2000). The main toxicity of CP is neurotoxicity, which is caused by the inhibition of acetylcholinesterase (Yu et al., 2008). Nevertheless, the toxicity of CP may involve mechanisms other than the inhibition of cholinesterase. One such mechanism may be the inhibition of mitochondrial ATP production through the uncoupling of oxidative phosphorylation that could lead to the generation of ROS (Ishii et al., 2004).

Previous studies from this laboratory have shown that CP can induce toxicity in D. melanogaster (Gupta et al., 2007a, Nazir et al., 2001) wherein ROS have been considered to be a possible signaling molecule causing cellular toxicity. Therefore, the present study was conducted to examine the role of ROS in inducing DNA damage and apoptosis in the third instar larvae of D. melanogaster after CP exposure.

In recent years, one of the fundamental concerns for researchers has been toward reducing the number of higher laboratory animals for research and testing due to ethical issues with an emphasis on the use of alternative animal models. In the present study, Drosophila was used as a model for its well-defined genetics, molecular and developmental biology. During the last decade, the model has been used extensively for toxicological studies (Chowdhuri et al., 1999, Mukhopadhyay et al., 2003, Nazir et al., 2003, Siddique et al., 2005a), drug discovery and targeting pathway genes of human diseases (Chang et al., 2008, Ratnaparkhi et al., 2008). The model raises few ethical concerns and is exempted by the animal rights organizations (Benford et al., 2000).

Section snippets

Fly strain and culture

The wild type fly and larvae of D. melanogaster (Oregon R+) were reared at 22±1 °C on a standard Drosophila diet containing agar-agar, maize powder, sugar, yeast, nepagin (methyl-p-hydroxy benzoate) and propionic acid. For healthy growth of the organism, additional yeast suspension was provided.

Treatment schedule

Technical grade CP (97.15%) obtained from DE-NOCIL Crop Protection Ltd., Mumbai, India was used during the study. Four different concentrations (0.015, 0.15, 1.5 and 15.0 μg/L) of the test compound

Results

During the course of the study, no overt sign of toxicity was observed in test chemical treated organisms. Since DMSO itself was unable to induce any significant change in the end points examined as compared to control, only control was included for the comparison. The data presented are for a single significant concentration of CP (15.0 μg/L) to show time-course. Additionally, a single significant time point was also chosen to show the dose-response curve of significant values at that time.

Discussion

In the present study, exposure of third instar larvae of D. melanogaster to CP was found to induce oxidative stress, apoptosis and DNA damage in the exposed organisms.

Chlorpyrifos has been in extensive use for various purposes and therefore, environmental presence and subsequently effect on biota is of concern. In a field study, 0.12 μg/L CP was detected in Royal lake, Washington (Gruber and Munn, 1998). A compilation of >60 studies in a review by Schulz (2004) showed a maximum of 3.8 μg/L CP in

Conclusion

In conclusion, the present study provides evidence of CP-induced negative impact (viz. DNA damage and apoptosis) on Drosophila larvae which may be attributed due to generation of ROS. The study further demonstrates that mitochondria and caspases are potential targets of CP in the exposed organism. However, further studies to understand the mechanism involved in ROS-induced changes in the mitochondrial membrane potential and activation of caspases following CP exposure to Drosophila larvae is

Acknowledgments

The authors are thankful to the Director of the Indian Institute of Toxicology Research, Lucknow for his interest throughout the study. We thank Bloomington Stock Centre, USA for D. melanogaster (Oregon R+) stock, Dr. A. Dhawan, Scientist, Indian Institute of Toxicology Research, and Dr. M. Dixit, Scientist, Central Drug Research Institute, Lucknow for providing Comet assay and flow cytometry facilities, respectively, and Miss Barbara Heck, The Ohio State University, USA, for editorial

References (51)

  • A.P.K. Jantunen et al.

    Bioaccumulation of atrazin and chlorpyrifos to Lumbriculus variegates from lake sediments

    Ecotoxicol. Environ. Saf.

    (2008)
  • P. Jaruga et al.

    Oxidative DNA base damage and antioxidant enzyme activities in human lung cancer

    FEBS Lett.

    (1994)
  • R.L. Levine et al.

    Determination of carbonyl content in oxidatively modified proteins

    Methods Enzymol.

    (1990)
  • O.H. Lowry et al.

    Protein measurement with the Folin Phenol reagent

    J. Biol. Chem.

    (1951)
  • A. Nakadai et al.

    Chlorpyrifos induces apoptosis in human monocyte cell line U937

    Toxicology

    (2006)
  • M. Nishikimi et al.

    The occurrence of superoxide anion in the reaction of reduced phenazine methosulphate and molecular oxygen

    Biochem. Biophys. Res. Commun.

    (1972)
  • H. Ohkawa et al.

    Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction

    Anal. Biochem.

    (1979)
  • P.L. Olive et al.

    Factors influencing DNA migration from individual cells subjected to gel electrophoresis

    Exp. Cell Res.

    (1992)
  • M.D. Saulsbury et al.

    Characterization of chlorpyrifos-induced apoptosis in placental cells

    Toxicology

    (2008)
  • H.R. Siddique et al.

    Induction of biochemical stress markers and apoptosis in transgenic Drosophila melanogaster against complex chemical mixtures: role of reactive oxygen species

    Chem. Biol. Interact.

    (2007)
  • N.P. Singh et al.

    A simple technique for quantification of low levels of DNA damage in individual cells

    Exp. Cell Res.

    (1988)
  • A.K. Sinha

    Colorimetric assay of catalase

    Anal. Biochem.

    (1972)
  • M. Valko et al.

    Free radicals and antioxidants in normal physiological functions and human disease

    Int. J. Biochem. Cell Biol.

    (2007)
  • F. Yu et al.

    Apoptotic effect of organophosphorous insecticide chlorpyrifos on mouse retina in vivo via oxidative stress and protection of combination of vitamins C and E

    Exp. Toxicol. Pathol.

    (2008)
  • B. Yuan et al.

    Imbalance between ROS production and elimination results in apoptosis induction in primary smooth chorion trophoblast cells prepared from human fetal membrane tissues

    Life Sci.

    (2008)
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    Funding sources: (1) Council of Scientific and Industrial Research, New Delhi, India and (2) Indian Council of Medical Research, New Delhi, India.

    1

    Present address: Department of Experimental Therapeutics, MD Anderson Cancer Center, TX 77030, USA.

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    Present address: Laboratory for Membrane Trafficking, Center for Human Genetics and Department of Molecular and Developmental Genetics, Gasthuisberg, Leuven, Belgium.

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