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.


Available from: Hifzur Siddique
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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.
    Frontiers in Genetics 08/2014; 5:304. DOI:10.3389/fgene.2014.00304
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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.
    Mutation Research/Genetic Toxicology and Environmental Mutagenesis 05/2014; 766. DOI:10.1016/j.mrgentox.2014.02.004 · 2.42 Impact Factor
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    • "Therefore, recent biological research has emphasized alternative animal models that follow the principles of the 3R's: reduction, replacement and refinement [38]. Drosophila melanogaster is a model organism for both genetic and developmental biology studies, as well as an established insect model for human disease and toxicological research [27] [39] [40]. Moreover, the European Centre for the Validation of Alternative Methods (ECVAM) for research and testing has advocated studies in this organism [41]. "
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    ABSTRACT: Modifications to the alkaline Comet assay by using lesion-specific endonucleases, such as formamidopyrimidine-DNA glycosylase (FPG) and endonuclease III (ENDOIII, also known as Nth), can detect DNA bases with oxidative damage. This modified assay can be used to assess the genotoxic/carcinogenic potential of environmental chemicals. The goal of this study was to validate the ability of this modified assay to detect oxidative stress-induced genotoxicity in Drosophila melanogaster (Oregon R(+)). In this study, we used three well known chemical oxidative stress inducers: hydrogen peroxide (H(2)O(2)), cadmium chloride (CdCl(2)) and copper sulfate (CuSO(4)). Third instar larvae of D. melanogaster were fed various concentrations of the test chemicals (50-200μM) mixed with a standard Drosophila food for 24h. Alkaline Comet assays with and without the FPG and ENDOIII enzymes were performed with midgut cells that were isolated from the control and treated larvae. Our results show a concentration-dependent increase (p<0.05-0.001) in the migration of DNA from the treated larvae. ENDOIII treatment detected more oxidative DNA damage (specifically pyrimidine damage) in the H(2)O(2) exposed larvae compared to FPG or no enzyme treatment (buffer only). In contrast, FPG treatment detected more oxidative DNA damage (specifically purine damage) in CuSO(4) exposed larvae compared to ENDOIII. Although previously reported to be a potent genotoxic agent, CdCl(2) did not induce more oxidative DNA damage than the other test chemicals. Our results show that the modified alkaline Comet assay can be used to detect oxidative stress-induced DNA damage in D. melanogaster and thus may be applicable for in vivo genotoxic assessments of environmental chemicals.
    Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 12/2011; 726(2):222-6. DOI:10.1016/j.mrgentox.2011.09.017 · 3.68 Impact Factor
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