Genotoxic evaluation of two mercury compounds in the Drosophila wing spot test

Few studies on the genotoxicity of mercury compounds have been carried out in Drosophila melanogaster, most of them focused in the effects on germinal cells, whereas studies in somatic cells are scarce. In the present study we have analyzed for the first time the genotoxic activity of mercury (II) chloride (MC) and methyl mercury (II) chloride (MMC) in the in vivo wing somatic mutation and recombination test in Drosophila, also known as the wing spot assay. This test is based on the principle that loss of heterozygosis and the corresponding expression of the suitable recessive markers, multiple wing hairs (mwh) and flare-3 (flr(3)), can lead to the formation of mutant clones in larval cells, which are then expressed as spots on the wings of adult flies. The mercury compounds were supplied to third instar larvae (72+/-2h old) at concentrations ranging from 1 to 50 microM for mercury chloride (MC) and from 0.5 to 5 microM for methyl mercury chloride (MMC). Both mercury compounds showed high toxicity; however, MMC was more toxic than MC. The results showed that none of the three categories of mutant spots recorded (small, large, and twin) increased significantly by the treatments, independently of the dose supplied, indicating that the mercury compounds tested exhibit a lack of genotoxic activity in the wing spot assay of D. melanogaster. These results contribute to increase the genotoxicity database on the in vivo evaluation of mercury compounds in Drosophila.     

Quantitative structure-activity relationships of nitroaromatics toxicity to the algae (Scenedesmus obliguus)

The DFT-B3LYP method, with the basis set 6-311G( * *), was employed to calculate the molecular geometries and electronic structures of 25 nitroaromatics. The acute toxicity (-lgEC(50)) of these compounds to the algae (Scenedesmus obliguus) along with hydrophobicity described by logK(OW), and two quantum chemical parameters-energy of the lowest unoccupied molecular orbital, E(LUMO), and the charge of the nitro group, [ForQ(NO2), were used to establish the quantitative structure-activity relationships (QSARs). For 18 mononitro derivatives, the hydrophobicity parameter logK(OW) could interpret the toxic mechanism successfully. Dinitro aromatic compounds were susceptible to be reduced to aniline for their electrophilic nature. Their toxicity was controlled mainly by electronic factors instead of hydrophobicity. The electronic parameters, E(LUMO) and Q(NO2), were used to yield the following model: -lg EC(50) = 3.746 - 25.053 E(LUMO) + 6.481 Q(NO2) (n=22, R=0.926, SE=0.206, F=56.854, P<0.001). The predicted toxic values using the above equation are in good agreement with the experimental values.     

Quantitative structure-activity relationships for polycyclic aromatic hydrocarbons: Correlation between molecular connectivity,physico-chemical properties,bioconcentration and toxicity in Daphnia pulex

High correlation coefficients (r = 0.9444 – 0.9996) were calculated for the linear one-parameter correlation between molecular weight, molecular connectivity indices, HPLC and TLC retention indices, partition coefficients between n-octanol or sediment and water, and bioconcentration factors or lethal concentrations in Daphnia Pulex of unsubstituted polycyclic aromatic hydrocarbons.     

Quantitative structure-activity relationships for the inhibition toxicity to root elongation of Cucumis sativus of selected phenols and interspecies correlation with Tetrahymena pyriformis

The comparative toxicities of selected phenols to higher plants Cucumis sativus were measured and the negative logarithm molar concentration of the root elongation median inhibition (IRC50) were derived. Quantitative structure-activity relationships (QSARs) were developed to explore the toxicity influencing factors and for predictive purpose. The toxicity data, fell into two classes: polar narcosis and bio-reactive. For polar narcotic phenols, a highly significant two-parameter QSAR based on 1-octanol/water partition coefficient (logKow) and energy of the lowest unoccupied orbital (E(lumo)) was derived (IRC50 = 0.77 log Kow - 0.39E(lumo) + 2.36 n = 22 r2 = 0.89). The five bio-reactive chemicals proved to show elevated toxicity due to their typical substructure involved diverse reactive mechanisms. In an effort to model all chemicals, a robust multiple-variable QSAR combining logKow, E(lumo) and Qmax, the most negative net atomic charge, was developed (IRC50 = 0.65 logKow - 0.72E(lumo) + 0.23Qmax + 2.81 n = 27 r2 = 0.94), indicating that hydrophobicity, electrophilicity and hydrogen bond interaction contribute mainly to the phytotoxicity. The toxicological data was compared with Tetrahymena pyriformis 2-d population growth inhibition toxicity (IGC50) and excellent interspecies correlations were observed both for the polar narcotics and for five reactive chemicals (for polar narcotics: IRC50 = 0.95IGC50 + 1.07 n = 16 r2 = 0.89; for bio-reactive chemicals: IRC50 = 0.98IGC50 + 2.19 n = 5 r2 = 0.97; and for all: IRC50 = 0.93IGC50 + 1.63 n = 21 r2 = 0.87). This suggested that T pyriformis toxicity could serve as a surrogate of C. sativus toxicity for phenols and interspecies correlation also could be established for reactive chemicals.     

Studies of 3D-quantitative structure-activity relationships on a set of nitroaromatic compounds: CoMFA,advanced CoMFA and CoMSIA

By using comparative molecular field analysis (CoMFA), advanced CoMFA and comparative molecular similarity index analysis (CoMSIA) methods, the 3D relationships between the structures of 35 nitroaromatic compounds and their toxicities have been investigated to yield statistically reliable models of considerable predictive power. In contrast to CoMFA, CoMSIA produces better results for the correlation. Moreover, the obtained CoMSIA contour maps that interpret the correlations in terms of field contributions allow physicochemical properties relevant for binding to be easily mapped back onto molecular structures, and thus elucidate structural features among ligands that are responsible for toxicities. Besides, most of the highlighted regions in CoMSIA and CoMFA contour maps are mirrored by features in the surrounding environment. Thereby, CoMFA and CoMSIA both help to give explanations of the toxic mechanism of tested compounds.     

Bioremediation of halogenated compounds: comparison of dehalogenating bacteria and improvement of catalyst stability

Five bacterial strains were compared for halogenated compounds conversion in aqueous media. Depending on the strain, the optimal temperature for dehalogenase activity of resting cells varied from 30 to 45 degrees C, while optimal pH raised from 8.4 to 9.0. The most effective dehalogenase activity for 1-chlorobutane conversion was detected with Rhodococcus erythropolis NCIMB13064 and Escherichia coli BL21 (DE3) (DhaA). The presence of 2-chlorobutane or propanal in the aqueous media could inhibit the 1-chlorobutane transformation.     

Differential toxicity of silver and titanium dioxide nanoparticles on Drosophila melanogaster development, reproductive effort, and viability: size, coatings and antioxidants matter

Silver and titanium dioxide nanoparticles are known to induce oxidative stress in vitro and in vivo. Here we test if they impact development, mating success, and survivorship in Drosophila melanogaster, and if so, if these effects are reversible by antioxidants. Ingestion of nanotitanium dioxide during the larval stage of the life cycle showed no effects on development or survivorship, up to doses of 200 μg mL⁻¹. Conversely, ingestion of nanosilver had major dose, size, and coating-dependent effects on each of these aspects of life history. Each of these effects was partially or fully reversible by vitamin C. Larvae growing on nanosilver supplemented with vitamin C showed a greater than twofold increase in survivorship compared to flies reared on nanosilver alone, and a threefold increase in mating success. Vitamin C also rescued cuticular and pigmentation defects in nanosilver fed flies. Biochemical assays of superoxide dismutase and glutathione show these markers respond to nanotitanium dioxide and nanosilver induced oxidative stress, and this response is reduced by vitamin C. These results indicate that life history effects of nanosilver ingestion result from oxidative stress, and suggest antioxidants as a potential remediation for nanosilver toxicity. Conversely, the lack of nanotitanium dioxide life history toxicity shows that oxidative stress does not necessarily result in whole organism effects, and argues that nanoparticle toxicity needs to be examined at different levels of biological organization.     

Changes in toxicity and genotoxicity of industrial sewage sludge samples containing nitro- and amino-aromatic compounds following treatment in bioreactors with different oxygen regimes

Goals, Scope and Background

From 2005, deposition of organic waste will be banned in Sweden. Likewise, in Germany and Austria, similar bans are being planned, and further countries will probably follow. Thus, there is a need to develop new methods and to refine established techniques for sludge management in the whole of the European Union. For this end, there is also an urgent need for appropriate ecotoxicological approaches to elucidate and assess the hazard potential of sewage sludge. Therefore, the present study was designed to assess the capacity of various established sludge treatment methods using different oxygen regimes to degrade recalcitrant nitro-substituted organic compounds and reduce their toxicity. Sewage sludge samples from a wastewater treatment plant in Sweden (Cambrex Karlskoga AB, industrial area Björkborn) receiving wastewater from industries manufacturing pharmaceutical substances, chemical intermediates and explosives were processed with different sludge treatment methods. Among other treatment methods, bioreactors (for anaerobic and aerobic sludge treatment) were used. In the present investigation, a battery ofin vitro bioassays was employed to compare the cytotoxic and genotoxic potentials of different fractions of sludge samples in order to elucidate whether the treatments were suitable to reduce the toxicity of the sludge.

Methods

In order to investigate the cytotoxicity of the extracts of treated and untreated sludge samples, the acute cytotoxicity test with the permanent cell line RTL-W1 was used. Genotoxicity was tested by means of the comet assay (single cell gel electro-phoresis) with RTL-W1cells, and mutagenicity was assessed with the Ames test using the Salmonella typhimurium strains TA98, TA98NR and TA100. Sludge toxicity was tested in different fractions of organic extracts produced by acetone and hexane extractions. The subsequent clean-up procedure (silica gel chro-matography and elution with hexane and dichloromethane) resulted in two fractions, a lipophilic hexane-fraction and a semi-lipophilic dichloromethane-fraction. For the genotoxicity and mutagenicity tests, these fractions were reunited at equal ratios.

Results and Discussion

The acute cytotoxicity test with RTL-Wl cells revealed a high cytotoxic potential for the semi-li-pophilic DM-fractions of all sludge samples with NR₅₀ values (= effective concentration for 50% cell death in the neutral red test) from 8.9 up to 20 mg sludge d.w./ml medium. A low cytotoxic potential for the hexane fractions of the untreated sludge samples (NR₅₀ 400 to < 400 mg sludge d.w./ml medium) was observed, whereas the hexane fractions of the treated sludge samples showed elevated cytotoxicity increasing further with treatment in the bioreactors. The comet assay indicated that three out of eight of the reunited fractions had a significant genotoxic potential. Whereas the genotoxic potential of one sample treated anaerobically was very high with an induction factor of 11.6, a similar sample (taken from the same anaerobic reactor four months later) and one untreated sample showed lower potentials. The samples treated in another anaerobic bioreactor as well as the samples treated aerobically showed no genotoxic potential. Results indicate that aerobic treatment was basically adequate for reducing the genotoxicity of the sludge, whereas anaerobic treatment was only partly useful for reduction of genotoxicity. The Ames test revealed a very high mutagenic potential for the reunited fractions of the untreated sludge samples with strain TA98 (maximum induction factors (IF_max up to 45) and a relatively high potential for one of the samples treated aerobically (S2, IF_max = 18 (TA98, S9-)), thus documenting the suitability of both anaerobic and aerobic treatments to reduce the mutagenicity of the samples, however, with the aerobic treatment being less effective. Conclusions. Overall, none of the microbiological treatments for wastewater sludge in bioreactors was found to be ideal for general toxicity reduction of the sludge samples. Whereas cytotoxicity of the sludge increased or levelled off in most cases following either treatment, genotoxicity both increased or decreased after anaerobic treatment, depending on the specific sample. However, mutagenicity could generally be reduced by anaerobic treatment and, to a lesser degree, by aerobic treatment. Recommendationsand Perspectives. The complex modification of the diverse damage potentials of sludge sample extracts by use of anin vitro biotest battery following treatment for toxicity reduction in bioreactors showed that considerations of different toxicological endpoints is essential for an adequate hazard assessment. Whereas in the case of cytotoxicity reduction, the reactors proved ineffective, mutagenicity could be reduced significantly at least in some cases in this case study.

     

Evaluation of solid sorbents for sampling aliphatic and aromatic nitro compounds in work-room air

Amberlite XAD-2 and XAD-7 porous polymers were evaluated for the solid sampling of aliphatic and aromatic nitro compounds in work-room air. With the use of solvent desorption, XAD-2 gave 80–100% recovery of nitroaromatics, and XAD-7 85–100% recovery of nitroaliphatics. The compounds studied were nitromethane, nitroethane, 2-nitropropane, nitrobenzene, $\text{m}$-dinitrobenzene and 2,4,6-trinitrotoluene. All six compounds gave very poor recoveries from activated charcoal.     

Kinetic assessment of persistent halogenated xenobiotics in cell culture models: comparison of mono- and poly-halogenated compounds

We evaluated the suitability of single and multiple cell type cultures as model systems to characterise cellular kinetics of highly lipophilic compounds with potential ecotoxicological impact. Confluent mono-layers of human skin fibroblasts, rat astrocytoma C6 cells, non-differentiated and differentiated mouse 3T3 cells were kept in culture medium supplemented with 10% foetal calf serum. For competitive uptake experiments up to four different cell types, grown on glass sectors, were exposed for 3 h to ¹⁴C-labelled model compounds, dissolved either in organic solvents or incorporated into unilamellar lecithin liposomes. Bromo-, or chloro-benzenes, decabromodiphenylether (DBP), and dichlorodiphenyl ethylene (DDE) were tested in rather high concentration of 20 μM. Cellular toxicity was low. Compound levels were related to protein, DNA, and triglyceride contents. Cellular uptake was fast and dependent on physico-chemical properties of the compounds (lipophilicity, molecular size), formulation, and cell type. Mono-halogenated benzenes showed low and similar uptake levels (= low accumulation compounds). DBP and DDE showed much higher cellular accumulations (= high accumulation compounds) except for DBP in 3T3 cells. Uptake from liposomal formulations was mostly higher than if compounds were dissolved in organic solvents. The extent of uptake correlated with the cellular content of triglycerides, except for DBP. Uptake competition between different cell types was studied in a sectorial multi-cell culture model. For low accumulation compounds negligible differences were found among C6 cells and fibroblasts. Uptake of DDE was slightly and that of DBP highly increased in fibroblasts. Well-defined cell culture systems, especially the sectorial model, are appropriate to screen for bioaccumulation and cytotoxicity of (unknown) chemical entities in vitro.     

Adaptation of the Daphnia sp. acute toxicity test: miniaturization and prolongation for the testing of nanomaterials

Manufacturing of nanomaterials (NMs) is often complex and expensive, and their environmental risks are poorly understood or even unknown. An economization of testing NMs is therefore desirable, which can be achieved by miniaturizing test systems. However, the downsizing of test vessels and volumes can enlarge the surface/volume ratio (SVR) which in turn can affect the bioavailable concentration of adsorbing substances like NMs. The present study focused on the miniaturization of the acute toxicity test with Daphnia magna. The adaptations were verified with three reference substances, the non-adsorbing potassium dichromate (K₂Cr₂O₇) and as potentially highly-adsorbing substances silver nanoparticles (AgNPs) and silver nitrate (AgNO₃). The miniaturized test was conducted in 24-well microtiter plates (MT) and simultaneously compared to the OECD standard test (ST). Furthermore, the test duration was prolonged from 48 to 96 h since NMs tend to show effects only after extended exposure. The toxicity of K₂Cr₂O₇ and AgNPs continued to increase within the prolonged test span. The test comparisons with K₂Cr₂O₇ did not reveal any significant differences between ST and MT. AgNO₃ toxicity was significantly decreased in MT compared to ST due to the enlarged SVR. The toxicity of AgNPs in MT after 24 h was equal to ST. Contrary to our expectations an exposure longer than 24 h resulted in an increase of AgNP toxicity in MT, possibly due to enhanced dissolution of silver. Microtiter plates are appropriate alternative test vessels for the Daphnia sp. acute toxicity test; thus, its miniaturization is feasible. The enlarged SVR has to be taken into account since it can affect the toxicity of potentially adsorbing substances. Furthermore, the standard test duration of 48 h might underestimate the toxicity of many substances, especially of NMs.