DNA damage induced by industrial solid waste leachates in Drosophila melanogaster: a mechanistic approach

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.     

DNA damage induced in human peripheral blood lymphocytes by industrial solid waste and municipal sludge leachates

Exposure of humans to toxic compounds occurs mostly in the form of complex mixtures. Leachates, consisting of mixtures of many chemicals, are a potential risk to human health. In the present study, leachates of solid wastes from a polyfiber factory (PFL), an aeronautical plant (AEL), and a municipal sludge leachate (MSL) were assessed for their ability to induce DNA damage in human peripheral blood lymphocytes using the alkaline Comet assay. The leachates also were examined for their physical and chemical properties. Lymphocytes were incubated with 0.5-15.0% concentrations (pH range 7.1-7.4) of the test leachates for 3 hr at 37 degrees C, and treatment with 1 mM ethyl methanesulfonate served as a positive control. All three leachates induced significant (P < 0.05), concentration-dependent increases in DNA damage compared with the negative control, as measured by increases in Olive tail moment (arbitrary units), tail DNA (%), and tail length (mum). A comparison of these variables among the treatment groups indicated that the MSL induced the most DNA damage. Inductively coupled plasma emission spectrometry analysis of the leachates indicated that they contained high concentrations of heavy metals, viz. iron, manganese, nickel, zinc, cadmium, chromium, and lead. The individual, synergistic, or antagonistic effects of these chemicals in the leachates may be responsible for the DNA damage. Our data indicate that the ever-increasing amounts of leachates from waste landfill sites have the potential to induce DNA damage and suggest that the exposure of human populations to these leachates may lead to adverse health effects.     

In vivo genotoxic effects of industrial waste leachates in mice following oral exposure

Contamination of ground water by industrial waste poses potential health hazards for man and his environment. The improper disposal of toxic wastes could allow genotoxic chemicals to percolate into ground waters, and these contaminated ground waters may produce toxicity, including mutation and eventually cancer, in exposed individuals. In the present study, we evaluated the in vivo genotoxic potential of leachates made from three different kinds of industrial waste (tannery waste, metal-based waste, and waste containing dyes and pigments) that are disposed of in areas adjoining human habitation. Three different doses of test leachates were administered by oral gavage for 15 consecutive days to Swiss albino mice; their bone marrow cells were examined for chromosome aberrations (CAs), micronucleated polychromatic erythrocytes (MNPCEs), and DNA damage using the alkaline Comet assay. Exposure to the leachates resulted in significant (P < 0.05 or P < 0.001) dose-dependent increases in chromosome and DNA damage. Fragmented chromosomes and chromatid breaks were the major CAs observed. Chemical analysis of the leachates indicated that chromium and nickel were elevated above the limits established by health organizations. The highest levels of genotoxicity were produced by the metal-based leachate and the tannery-waste leachate, while the dye-waste leachate produced weaker genotoxic responses. The cytogenetic abnormalities and DNA damage produced by the leachates indicate that humans consuming water contaminated with these materials are at increased risk of developing adverse health consequences.     

DNA damage in bone marrow and blood cells of mice exposed to municipal sludge leachates

Leachates of municipal solid waste from unsecured disposal sites contaminate sources of potable water and affect human health. In the present study, we have used the Comet assay to evaluate the DNA damage in mice exposed to municipal sludge leachates. Ten percent leachates were prepared from municipal sludge obtained from two different disposal drains. Male Swiss albino mice were treated daily with 0.1-0.4 ml of the leachates by oral gavage for 15 days, and the DNA damage was evaluated in bone marrow and blood using Olive tail moment, % tail DNA, and tail length as measures of DNA damage. Physicochemical and metal analysis of the leachates detected the presence of cadmium, chromium, copper, nickel, lead, and zinc, as well as elevated concentrations of sulfate and nitrate. Both of the leachates produced significant dose-responsive increases in DNA damage in both mouse tissues. There were no significant differences in the responses for any of the Comet endpoints between tissues (for the same leachate sample) or between leachate samples (for the same tissue). The results of this study indicate that municipal waste leachates produce DNA damage in vivo.     

Evaluation of in vivo genotoxicity of cypermethrin in Drosophila melanogaster using the alkaline Comet assay

The single cell gel electrophoresis (SCGE) assay, also known as the Comet assay, is one of the most promising genotoxicity tests developed in recent years to measure and analyse DNA damage in single cells. The present study was undertaken to assess the in vivo genotoxicity of the synthetic pyrethroid cypermethrin in brain ganglia and anterior mid gut of Drosophila melanogaster. Freshly emerged first instar larvae (22 +/- 2 h) were placed in different concentrations of cypermethrin (0.0004, 0.0008, 0.002, 0.2 and 0.5 p.p.m.) mixed in standard Drosophila food and allowed to grow. At 96 +/- 2 h, brain ganglia and anterior midgut from control and treated larvae were dissected out, single cell suspensions were prepared and a Comet assay was performed. Our results revealed a significant dose-dependent increase in DNA damage in the cells of brain ganglia and anterior midgut of D.melanogaster exposed to cypermethrin as compared with controls (P < 0.05 at 0.002 p.p.m.; P < 0.001 at 0.2 and 0.5 p.p.m.). The present study shows in vivo genotoxicity of cypermethrin even at very low concentrations, which proves D.melanogaster as a model for in vivo genotoxicity assessment using the Comet assay.     

Genotoxicity of vegetable cooking oils in the Drosophila wing spot test

Seven vegetable oils consumed by humans were tested for genotoxic activity in the Drosophila somatic mutation and recombination test. The oils included five seed oils (sesame, sunflower, wheat germ, flax, and soy oil) and both first-class extra-virgin and low-grade (refined) olive oil. Larvae of the standard (STD) and highly bioactive (NORR) crosses of Drosophila melanogaster were fed medium containing 6% and 12% of each of the oils. Flax oil produced the strongest response, while sesame, wheat germ, and soy oil showed some genotoxic activity. Sunflower and the low-grade olive oil gave inconclusive results, and extra-virgin olive oil was clearly nongenotoxic. It is argued that the genotoxicity is probably due to the fatty acid composition of the oils, which after peroxidation can form specific DNA-adducts.     

Evaluation of micronucleus frequencies and DNA damage in glass workers exposed to arsenic

Arsenic (As) is a known human carcinogen; however, very little is known about the health consequences of occupational exposure to As. In the present study, we assessed the genotoxic damage in the blood cells and in the buccal cells of south Indian glass factory workers who are occupationally exposed to As. The As content in the whole blood of 200 workers and 165 controls was evaluated with inductively coupled plasma mass spectrometry. Blood leukocytes from the subjects were monitored for the level of DNA damage using the Comet assay (mean comet tail length); buccal cells were used to determine the frequency of micronuclei (MN). The mean As concentration was significantly higher in the workers (56.76 microg/L) than in the controls (11.74 microg/L) (P < 0.001). The workers also had increased frequencies of MN in the buccal cells and increased levels of DNA damage in leukocytes compared to the controls (P < 0.001). There were significant correlations between the genotoxicity endpoints that were evaluated and blood As concentration, smoking, age, and the duration of working in the factory. Also, a significant correlation was observed between the frequency of MN and comet tail-length for the worker samples. Our findings indicate that chronic occupational exposure to As is genotoxic and that the Comet assay and micronucleus test are useful assays for evaluating genotoxicity in humans occupationally exposed to As.     

Validation of Drosophila melanogaster as an in vivo model for genotoxicity assessment using modified alkaline Comet assay

The single cell gel electrophoresis or Comet assay is one of the most popular techniques for genotoxicity assessment. The present study was undertaken to validate our previously modified version of the Comet assay for genotoxicity assessment in Drosophila melanogaster (Oregon R(+)) with four well-known mutagenic and carcinogenic alkylating agents, i.e. ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-ethyl-N-nitrosourea (ENU) and cyclophosphamide (CP). Third instar larvae (74 +/- 2 h) of D.melanogaster were fed different concentrations of EMS, MMS, ENU and CP (0.05, 0.5 and 1.0 mM) mixed standard Drosophila food for 24 h. At 98 +/- 2 h, the anterior midgut from control and treated larvae were dissected out, single-cell suspensions were prepared and Comet assay was performed. Our results show a dose-dependent increase in DNA damage with all the four alkylating agents, in comparison to control. The lower concentration (0.05 mM) of the test chemicals, except MMS, did not induce any DNA damage in the gut cells of the exposed larvae. When comparison of Comet parameters was made among the chemicals, MMS was found to be the most potent genotoxicant and ENU the least. The present study validated our previous observation and shows that D.melanogaster is a sensitive and suitable model for the in vivo assessment of genotoxicity using our modified alkaline Comet assay.     

Metabolism of arsenic in Drosophila melanogaster and the genotoxicity of dimethylarsinic acid in the Drosophila wing spot test

Inorganic arsenic is nongenotoxic in the Drosophila melanogaster wing somatic mutation and recombination test (SMART). Recent evidence in mammalian systems indicates that methylated metabolites of arsenic are more genotoxic than inorganic arsenic. Thus, we hypothesized that inorganic arsenic is nongenotoxic in Drosophila because they are unable to biotransform arsenic to methylated forms. In the present study, we fed trivalent and pentavalent inorganic arsenic to Drosophila larvae and adults and measured the production of methylated derivatives. No biomethylated arsenic species were found in the organisms or in the growth medium, which suggests that Drosophila are unable to biomethylate inorganic arsenic. Exposure of Drosophila to the methylated arsenic derivative dimethylarsinic acid (DMA(V)) resulted in incorporation of this organoarsenic compound without demethylation. In addition, we used the SMART wing spot assay, which measures loss of heterozygosity (LOH) resulting from gene mutation, chromosomal rearrangement, chromosome breakage, and chromosome loss, to evaluate the genotoxicity of DMA. DMA by itself induced significant increases in the frequency of total spots, small spots, and large single spots. These results are consistent with the important role of arsenic biomethylation as a determinant of the genotoxicity of arsenic compounds. The absence of biomethylation in Drosophila could explain the lack of genotoxicity for inorganic arsenic and the genotoxicity of methylated arsenic species in the SMART wing spot assay.     

Recombinagenic activity of four compounds in the standard and high bioactivation crosses of Drosophila melanogaster in the wing spot test

The wing somatic mutation and recombination test (SMART) using Drosophila melanogaster was employed to determine the recombinagenic and mutagenic activity of four chemicals in an in vivo eukaryotic system. Two different crosses involving the wing cell markers mwh and flr(3) were used: the standard cross and a high bioactivation cross. The high bioactivation cross is characterized by a high constitutive level of cytochromes P450 which leads to an increased sensitivity to a number of promutagens and procarcinogens. Three-day-old larvae derived from both crosses were treated chronically with the oxidizing agent potassium chromate and with the three procarcinogens cyclophosphamide, p-dimethylaminoazobenzene and 9,10-dimethylanthracene. From both crosses two types of progeny were obtained: marker-heterozygous and balancer-heterozygous. The wings of both genotypes were analysed for the occurrence of single and twin spots expressing the mwh and/or flr(3) mutant phenotypes. In the marker-heterozygous genotype the spots can be due either to mitotic recombination or to mutation. In contrast, in the balancer-heterozygous genotype only mutational events lead to spot formation, all recombination events being eliminated. The oxidizing agent potassium chromate was equally and highly genotoxic in both crosses. Surprisingly, the promutagen cyclophosphamide also showed equal genotoxicity in both crosses, whereas p-dimethylaminoazobenzene was negative in the standard cross, but clearly genotoxic in the high bioactivation cross. 9,10-Dimethylanthracene showed a rather weak genotoxicity in the high bioactivation cross. Analyses of the dose-response relationships for mwh clones recorded in the two wing genotypes demonstrated that all four compounds are recombinagenic. The fraction of all genotoxic events which are due to mitotic recombination ranged from 83% (9,10-dimethylanthracene) to 99% (p-dimethylaminoazobenzene). These results demonstrate that the wing spot test in Drosophila is most suited to the detection of recombinagenic activity of genotoxic chemicals.     

Genotoxicity of tamoxifen citrate and 4-nitroquinoline-1-oxide in the wing spot test of Drosophila melanogaster

Tamoxifen (TAM) is an anti-oestrogen used for treatment and prevention of human breast cancer, but it is also related to human endometrial and uterine cancer. The wing spot test in Drosophila melanogaster was employed to determine the genotoxic effects of TAM and 4-nitroquinoline-1-oxide (4-NQO), a carcinogen that produces adducts similar to TAM-DNA adducts detected in rodent liver and human liver microsomes. As Drosophila spp. have no oestrogen receptor, no effects can result in binding of TAM to a receptor. Chronic treatments with TAM citrate were performed with 3-day-old larvae of the standard (ST) and high bioactivation (HB) crosses of the wing spot test at concentrations of 0.66, 1.66 and 3.33 mM. In addition, the carcinogen 4-NQO was administered at 2.5 and 5.0 mM. Somatic spots on normal wings from marker-heterozygous flies and on serrate wings from balancer-heterozygous flies were scored to determine mutation and recombination events in somatic cells for each compound. The results showed genotoxic effects of TAM at 1.66 and 3.33 mM in the ST cross only and without a clear dose-response effect. This suggests a weak genotoxicity of this anti-oestrogen. The negative results obtained with TAM in the HB cross may indicate efficient detoxification of the compound by the increased xenobiotic metabolism present in this cross. As reported before, 4-NQO showed genotoxic effects in the ST cross with a clear dose-response effect. For the first time, we report enhanced effects of this compound in the HB cross. It is concluded that the genotoxicity of TAM in the Drosophila wing spot test is different from that of 4-NQO.     

Genotoxicity of three mouthwash products, Cepacol, Periogard, and Plax, in the Drosophila wing-spot test

Antiseptic mouthwashes used in biofilm control are widely available in the marketplace, despite inconsistent data concerning their genetic and cellular toxicity. In the present study, we investigated the genotoxic potential of three antiseptics currently used for odontologic treatment, Cepacol (containing cetylpyridinium chloride), Periogard (chlorhexidine digluconate), and Plax (triclosan). Genotoxicity was evaluated using the Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster, employing flies having normal bioactivation (the standard cross) and flies with increased cytochrome P450-dependent biotransformation capacity (the high bioactivation cross). Periogard and Plax produced negative responses in both types of flies; however, Cepacol (75 and 100%) produced positive responses in both the standard and high bioactivation assays, with the genotoxic responses mainly due to the induction of mitotic recombination. Assays performed with ethanol and cetylpirydinium chloride, two major ingredients of Cepacol, indicated that the genotoxicity of the mouthwash is likely to be due to ethanol.     

Comparative genotoxic evaluation of 2-furylethylenes and 5-nitrofurans by using the comet assay in TK6 cells

The genotoxicity of three 2-furylethylene derivatives and four 5-nitrofurans was evaluated by using the comet assay in human lymphoblastoid cultured TK6 cells. The 2-furylethylene derivatives were 2-furyl-1-nitroethene, 1-(5-bromofur-2-yl)-2-nitroethene and 1-(5-bromofur-2-yl)-2-bromo-2-nitroethene, while the 5-nitrofurans were nitrofurantoin, nitrofurazone, furazolidone and 5-nitro-2-furanacrolein. The treatments lasted for 3 h in the absence of metabolic activation. No genotoxic effects were observed for two of the 2-furylethylene compounds, while the derivative 1-(5-bromofur-2-yl)-2-nitroethene showed a statistically significant response mainly at the highest concentration tested; this effect was considered biologically relevant and the compound was classified as slightly genotoxic. On the other hand, for the classical 5-nitrofurans tested there is a tendency towards a dose-related increase of the DNA damage in the comet assay and the observed increases for the parameters analysed (Olive tail moment, tail % DNA and tail length) were significant for all compounds. Then, the four 5-nitrofurans tested were considered genotoxic. These results show that the position of the nitro group influences the genotoxicity of the assayed compounds. Thus, in this comet assay, the 2-furylethylene derivatives having the nitro group attached outside the furan ring appear to be much less genotoxic than the 5-nitrofurans.     

Genotoxicity of vesicular fluid and saline extract of Taenia solium metacestodes in somatic cells of Drosophila melanogaster

Neurocysticercosis, the most common parasitic disease of the central nervous system, is caused by cysticerci of the helminth Taenia solium, which is prevalent in developing countries and is reemerging in affluent societies. This helminth is associated with brain tumors and hematological malignancies in humans. In the present study, we analyzed the genotoxicity of vesicular fluid (VF) and a saline extract (SE) of T. solium metacestodes in the Drosophila melanogaster wing somatic mutation and recombination test (SMART). Third-instar larvae derived from standard and high bioactivation crosses were treated for approximately 48 hr with 12.5, 25.0, and 50.0 microg/ml of VF and SE of T. solium metacestodes. Negative (phosphate buffered saline) and positive (10 mM urethane) controls were also included. The results showed that the two test compounds were genotoxic in both crosses of Drosophila. Nevertheless, further research is needed to determine the genotoxic potential of specific compounds present in VF and SE and their role in the development of cancer.     

Comparison of camptothecin derivatives presently in clinical trials: genotoxic potency and mitotic recombination

The genotoxicity of camptothecin (CPT) and its clinical antineoplastic analogues irinotecan (CPT-11) and topotecan (TPT) were evaluated using the wing somatic mutation and recombination test (SMART) in Drosophila melanogaster. These compounds stabilize and trap the topoisomerase I-DNA complex, preventing the religation step of the breakage/rejoining reaction mediated by the enzyme. The standard version of the wing SMART was used to evaluate the three compounds and to compare the wing spots induced in marker-heterozygous and balancer-heterozygous flies. The results demonstrate that all compounds tested have a significant genotoxic effect in both genotypes analysed. At the same time, a comparison of the clone induction frequencies in marker-heterozygous and balancer-heterozygous flies shows that mitotic recombination is the prevalent mechanism through which the three compounds induce all categories of wing spots (78-93% recombination). TPT was the most genotoxic compound, probably because substitutions of amino groups for the 9-carbon of the CPT A ring leads to compounds with greater in vivo activity. CPT and CPT-11 induced, respectively, about 7 and 28 times fewer mutant clones per millimolar exposure unit than TPT.     

Use of the microscreen phage-induction assay to assess the genotoxicity of 14 hazardous industrial wastes

The Microscreen phage-induction assay, which quantitatively measures the induction of prophage lambda in Escherichia coli WP2s(lambda), was used to test 14 crude (unfractionated) hazardous industrial waste samples for genotoxic activity in the presence and absence of metabolic activation. Eleven of the 14 wastes induced prophage, and induction was observed at concentrations as low as 0.4 pg per ml. Comparisons between the ability of these waste samples to induce prophage and their mutagenicity in the Salmonella reverse mutation assay indicate that the phage-induction assay detected genotoxic activity in all but one of the wastes that were mutagenic in Salmonella. Moreover, the Microscreen assay detected as genotoxic five additional wastes that were not detected in the Salmonella assay. The applicability of the Microscreen phage-induction assay for screening hazardous wastes for genotoxic activity is discussed, as are some of the problems associated with screening highly toxic wastes containing toxic volatile compounds.     

Genotoxicity of the anti-juvenile hormone agent precocene II as revealed by the Drosophila wing spot test

The anti-juvenile hormone agent, precocene II, designated as a prototype of potential fourth-generation insecticides, was subjected to genotoxicity screening by means of the somatic mutation and recombination test in Drosophila melanogaster. Larvae heterozygous for recessive wing trichome mutations, mwh and flr3, were exposed to sublethal concentrations of precocene II, and wings of emerged adult females were inspected for the presence of phenotypically mutant mosaic spots. The compound significantly increased the frequency of mosaic spots in mwh/flr3 wings, but revealed only a slight effect in mwh/TM2 wings. The results suggest that the main sources of genotoxic activity of precocene II are due to chromosome-breakage phenomena resulting from mitotic recombination. The possible mechanism of this effect is discussed.     

Genotoxicity testing of antiparasitic nitrofurans in the Drosophila wing somatic mutation and recombination test

Nifurtimox and eight structurally related 5-nitrofuran compounds active against Trypanosoma cruzi were tested for genotoxicity in the wing somatic mutation and recombination test in Drosophila melanogaster. Nifurtimox, compound ada and compound 1B were clearly mutagenic and recombinogenic whereas the remaining six compounds were negative. In contrast to the situation in bacterial mutagenicity tests, nitroreductase activity is probably not decisive for the genotoxicity of these compounds in Drosophila. The three non-genotoxic nitrofurans with high antiparasitic activity are promising candidates for the replacement of nifurtimox. However, these compounds require further genotoxicity testing in eukaryotic assay systems for a final evaluation.     

Use of the Drosophila wing spot test in the genotoxicity testing of different herbicides

Four herbicides, namely propanil, maleic hydrazide, glyphosate, and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), were investigated for genotoxicity in the wing spot test of Drosophila melanogaster. The herbicides were administered by chronic feeding to 3-day-old larvae. Two different crosses, a standard (ST) and a high-bioactivation (HB) cross, involving the flare-3 (flr(3)) and the multiple wing hairs (mwh) markers, were used. The HB cross uses flies characterized by an increased cytochrome P-450-dependent bioactivation capacity, which permits a more efficient biotransformation of promutagens and procarcinogens. In both crosses, the wings of the two types of progeny, which are inversion-free marker heterozygotes and balancer heterozygotes, were analyzed. Maleic hydrazide and glyphosate proved to be more genotoxic in the ST cross, whereas propanil appeared to be slightly more genotoxic in the HB cross. On the other hand, the herbicide 2,4,5-T increased the mutation frequency for only the small single spots in the ST cross.     

Sodium thiosulfate inhibits cisplatin-induced mutagenesis in somatic tissue of Drosophila

The antitumor agent cisplatin was evaluated for genotoxicity in the somatic tissue of Drosophila melanogaster in combination with the nucleophilic compound sodium thiosulfate (STS). Third instar larvae transheterozygous for mwh and flr3 were grown on media containing (1) water, (2) 200 mM STS, (3) 0.05 mM cisplatin in 200 mM STS, or (4) 0.05 mM cisplatin. Wings of surviving adults were scored for the presence of twin spots and both small and large single spots; 200 mM STS was nongenotoxic in the assay. Whereas 0.05 mM cisplatin significantly induced all three endpoints, 0.05 mM cisplatin in combination with 200 mM STS was found to have no genotoxic activity. Hence, STS possessed antimutagenic activity in the wing-spot assay and completely inhibited cisplatin-induced mutagenesis and mitotic recombination.