DNA damage induced by industrial solid waste leachates inDrosophila melanogaster: A mechanistic approach

Embryotoxicology Section, Industrial Toxicology Research Centre, Lucknow, Uttar Pradesh, India.
Environmental and Molecular Mutagenesis (Impact Factor: 2.63). 04/2008; 49(3):206-16. DOI: 10.1002/em.20373
Source: PubMed


Genomic stability requires that error-free genetic information be transmitted from generation to generation, a process that is dependent upon efficient DNA repair. Industrial leachates which contain mixtures of diverse chemicals are a major environmental concern. The interaction between these chemicals may have synergistic, antagonistic, or simply additive effects on biological systems. In the present study, the Comet assay was used to measure the DNA damage produced by leachates of solid wastes from flashlight battery, pigment, and tanning factories in the midgut cells and brain ganglia of Drosophila melanogaster mutants deficient in DNA repair proteins. Larvae were allowed to feed for 48 or 72 hr on diets containing 0.1, 0.5, and 2.0% (v/v) of the leachates. Physicochemical analysis run on the solid wastes, leachates, and treated larvae detected elevated levels of heavy metals. Leachates produced significantly greater levels of DNA damage in mutant strains mei41 (deficient in cell cycle check point protein), mus201 (deficient in excision repair protein), mus308 (deficient in postreplication repair protein), and rad54 (deficient in double strand break repair protein) than in the OregonR(+) wild-type strain. Larvae of the ligaseIV mutant (deficient in double strand break repair protein) were hypersensitive only to the pigment plant waste leachate. Conversely, the dnase2 mutant (deficient in protein responsible for degrading fragmented DNA) was more sensitive to DNA damage induction from the flashlight battery and tannery waste leachates. Our data demonstrate that repair of DNA damage in organisms exposed to leachates is dependent upon several DNA repair proteins, indicative of the involvement of multiple overlapping repair pathways. The study further suggests the usefulness of the Comet assay for studying the mechanisms of DNA repair in Drosophila.

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Available from: Hifzur Siddique
    • "Drosophila melanogaster EMS, MMS, CP, ENU Alkaline Midgut cells [33] Drosophila melanogaster Cypermethrin Alkaline Midgut cells [34] Drosophila melanogaster MMS, EMS, ENU, CP Alkaline Midgut cells [35] Drosophila melanogaster Industrial wastes Alkaline Midgut cells [36] Drosophila melanogaster SNAP, paraquat Alkaline S2 cell culture [37] Drosophila melanogaster Cisplatin Alkaline Neuroblasts [38] Drosophila melanogaster Industrial wastes Alkaline Midgut and brain cells [39] Drosophila melanogaster Chlorpyrifos Alkaline Midgut cells [40] Drosophila melanogaster K 2 Cr 2 O 7 , CrCl 3 Alkaline Midgut cells [44] Drosophila melanogaster H 2 O 2 , CdCl 2 , CuSO 4 Alkaline Comet assay with lesion-specific endonucleases Midgut cells [48] "
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    ABSTRACT: The Comet assay has been recently adapted to investigate DNA damage in insects. The first reports of its use in Drosophila melanogaster appeared in 2002. Since then, the interest in the application of the Comet assay to studies of insects has been rapidly increasing. Many authors see substantial potential in the use of the Comet assay in D. melanogaster for medical toxicology studies. This application could allow the testing of drugs and result in an understanding of the mechanisms of action of toxins, which could significantly influence the limited research that has been performed on vertebrates. The possible perspectives and benefits for science are considered in this review.In the last decade, the use of the Comet assay has been described in insects other than D. melanogaster. Specifically, methods to prepare a cell suspension from insect tissues, which is a difficult task, were analyzed and compared in detail. Furthermore, attention was paid to any differences and modifications in the research protocols, such as the buffer composition and electrophoresis conditions.Various scientific fields in addition to toxicological and ecotoxicological research were considered. We expect the Comet assay to be used in environmental risk assessments and to improve our understanding of many important phenomena of insect life, such as metamorphosis, molting, diapause and quiescence. The use of this method to study species that are of key importance to humans, such as pests and beneficial insects, appears to be highly probable and very promising. The use of the Comet assay for DNA stability testing in insects will most likely rapidly increase in the future.
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    • "Midgut cells, with or without brain cells, have been used to study oxidative DNA damage, using incubations with FPG and Endo III enzymes (Shukla et al., 2011; Sharma et al., 2012), and to demonstrate the genotoxicity of chromium salts (Mishra et al., 2011, 2013; Sharma et al., 2011), pesticides like cypermethrin (Mukhopadhyay et al., 2004), endosulfan (Sharma et al., 2012), and dichlorvos (Mishra et al., 2014), contaminants as industrial waste leachates (Siddique et al., 2005b, 2008), and nanomaterials like graphene copper nanocomposite (Siddique et al., 2013). In addition, some of these genotoxic agents, like chromium salts, dichlorvos, and industrial waste leachates, were analyzed in different repair conditions, with the in vivo comet repair assay (Siddique et al., 2008; Mishra et al., 2011, 2013, 2014), checking the influence of pre-and post-replication DNA repair pathways on their genotoxicity. Other genotoxic agents, like endosulfan and graphene copper nanocomposite, were analyzed in transgenic strains for genes encoding heat shock proteins (hsp), to check responses to xenobiotic stress, and influence of xenobiotic metabolism (Sharma et al., 2012; Siddique et al., 2013). "
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    ABSTRACT: The comet assay, a very useful tool in genotoxicity and DNA repair testing, is being applied to Drosophila melanogaster since around 15 years ago, by several research groups. This organism is a valuable model for all kind of processes related to human health, including DNA damage response. The assay has been performed mainly in vivo using different larvae cell types (from brain, midgut, hemolymph, and imaginal disk), but also in vitro with the S2 cell line. Since its first application, it has been used to analyze the genotoxicity and action mechanisms of different chemicals, demonstrating good sensitivity and proving its usefulness. Moreover, it is the only assay that can be used to analyze DNA repair in somatic cells in vivo, comparing the effects of chemicals in different repair strains, and to quantitate repair activities in vitro. Additionally, the comet assay in Drosophila, in vivo and in vitro, has been applied to study the influence of protein overexpression on genome integrity and degradation. Although the assay is well established, it could benefit from some research to determine optimal experimental design to standardize it, and then to allow comparisons among laboratories independently of the chosen cell type.
    Full-text · Article · Aug 2014 · Frontiers in Genetics
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    • "For understanding the mechanisms of DNA damage and repair, chemical-induced mutagenesis and subsequent generation of mutants have been immensely useful. A number of DNA repair deficient mutants have been characterized in Drosophila based on the observed defects in repairing damaged DNA [19] [20] [21] [22] and are also useful in the screening of chemicals for their potential to induce DNA damage [23] [24] [25]. In Drosophila and in most of the eukaryotes , DNA damage repair pathways are predominantly categorized under pre-and post-replication repair pathways [26] [27]. "
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    ABSTRACT: Dichlorvos (DDVP), an organophosphate pesticide, is reported to induce genotoxicity in in vitro and in certain in vivo models at its higher concentrations. However, studies on the affected DNA damage repair pathways are elusive. Hypersensitivity of mutagen sensitive strains (mus) in Drosophila to mutagens and chemicals are known and accordingly, these mutants were grouped in pre- and post-replication repair pathways. For examining chemical-induced genotoxicity, mutants belonging to one particular repair pathway may not be adequate when other repair pathways may neutralize its effect. To test whether both pre- and post-replication pathways are affected by DDVP-induced genotoxicity in exposed Drosophila, we examined changes in DNA migration of Comet assay in the midgut cells of Oregon R+ larvae and selected mutants of pre- (mei-9, mus201 and mus207) and post-replication (mei-41 and mus209) repair pathways exposed to increasing yet environmentally relevant concentrations of DDVP (0.0, 0.15, 1.5 and 15.0 ng/ml) for 48 h. We observed greater DNA damage in pre-replication repair mutants after exposure to 0.15 ng/ml DDVP, while effects on Oregon R+ and post-replication repair mutants were insignificant. On the other, significant DNA damage was observed in the post-replication repair mutants after their exposure to 1.5 and 15.0 ng/ml DDVP. Among the mutants, pre-replication repair mutant, mus207, exhibited maximum sensitivity to DDVP which is suggestive of alkylation damage to DNA apart from its role in DSB repair. We also examined mutants (SOD- and urate-null) that are sensitive to oxidative stress and observed significant oxidative DNA damage in DDVP exposed mutants as compared to Oregon R+. The present study suggests requirement of both pre- and post-replication repair pathways against DDVP induced DNA damage in Drosophila with increased oxidative DNA damage leading to increased genotoxicity.
    Full-text · Article · May 2014 · Mutation Research/Genetic Toxicology and Environmental Mutagenesis
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