Planarian neoblast micronucleus assay for evaluating genotoxicity

Planarian neoblasts are somatic stem cells that have the potential to be used in genotoxicity assays due to their proliferative nature, sensitivity to genotoxic agents, and experimental accessibility. Two freshwater planarian species, Girardia tigrina and Girardia schubarti, were used to develop a neoblast-based micronucleus (MN) assay to assess genotoxicity. Intact or regenerating planarians were exposed to gamma-rays, methyl methanesulphonate (MMS), or cyclophosphamide (CP), and neoblast MN frequency was measured. Exposure to the clastogens had no detectable effect on the MN frequency of intact planarian neoblasts. However, for regenerating individuals, active neoblast proliferation was induced by decapitation, and gamma-ray doses as low as 0.5 Gy, and MMS and CP concentrations as low as 0.8 microM and 100 mM, respectively, induced a significant increase in MN frequency. Exposure to higher doses of gamma-rays consistently resulted in detectable increases in MN frequency. For MMS and CP, concentrations of up to 1.6 microM and 200 mM, respectively, resulted in significant increases in MN frequency, but exposures to higher concentrations led to a decrease to non-significant levels, possibly due to cytotoxic effects of MMS and CP. After completion of regeneration, the MN frequencies returned to those of non-exposed controls, indicating that the neoblast MN assay for regenerating G. tigrina or G. schubarti reflects chromosomal damage caused by acute exposure to clastogenic agents. Upon standardization, this assay may represent an interesting alternative that allows damage caused to freshwater organisms by potentially genotoxic environmental pollutants to be monitored.     

Improvement of Vicia-micronucleus test for assessment of soil quality: a proposal for international standardization

The Viciafaba root tip micronucleus test is one of the most employed plant genotoxicity assays, and has been used on various types of contaminated materials. This test has been standardized by AFNOR, the French member organization of ISO. However, this test is usually performed with a water extraction step but soil genotoxicity assessment would be more relevant when performed directly in the soil itself. In order to harmonize these protocols, an ISO standard for the V.faba micronucleus test in both liquid phase (exposure of plants to different liquid matrix, including soil water extracts) and solid phase (direct exposure of plants to the soil) would be very useful. In this context, we compared two exposure durations in the solid phase (48 h and 5 d) for the V.faba micronucleus test with two different well-known genotoxicants, maleic hydrazide and copper sulfate. We concluded that these two durations induced equivalent sensitivity: the micronucleus frequency was significantly increased with 5 μmol maleic hydrazide per kg dry soil and with 2 mmol copper sulfate per kg dry soil with both exposure durations. However, exposing roots to soil during 48 h is more practical. Moreover, organically and conventionally cultured seeds were employed to determine whether the seed provenance influenced the test sensitivity. Organic seeds were less sensitive to copper, possibly because copper-based treatments are permitted, and often applied, in organic farms. Therefore, in the absence of completely non-treated seeds, organically-cultured seeds did not appear to offer any advantages over conventional seeds.     

Evaluation of removal efficiency for acute toxicity and genotoxicity on zebrafish in anoxic–oxic process from selected municipal wastewater treatment plants

The anoxic–oxic (A/O) process has been extensively applied for simultaneous removal of organic contaminants and nitrogen in wastewater treatment. However, very little is known about its ability to remove toxic materials. Municipal wastewater contains various kinds of pollutants, some of which have recalcitrant genotoxicity and may cause potential threat to environment, and even can lead to extinction of many species. In this study, we have selected three municipal wastewater treatment plants (WWTPs) employing anoxic–oxic (A/O) process to evaluate their ability to remove acute toxicity and genotoxicity of wastewater. Mortality rate of zebrafish (Danio rerio) was used to evaluate acute toxicity, while micronucleus (MN) and comet assays were used to detect genotoxicity. Results showed that in this process the acute toxicity was completely removed as the treatment proceeded along with decrease in chemical oxygen demand (COD) (<50 mg L^?1) in the effluent. However, in these treatment processes the genotoxicity was not significantly reduced, but an increase in genotoxicity was observed. Both MN and comet assays showed similar results. The eliminated effluent may pose genotoxic threaten although its COD level has met the Chinese Sewage Discharge Standard. This study suggests that further treatment of the wastewater is required after the A/O process to remove the genotoxicity and minimize the ecotoxicological risk.     

Detection of environmental clastogens and aneugens in human fibroblasts by cytokinesis-blocked micronucleus assay associated with immunofluorescent staining of CENP-A in micronuclei

The cytokinesis-blocked micronucleus (CBMN) assay, in combination with fluorescent in situ hybridization (FISH) of human pan-centromeric DNA probes, or with CREST antibodies that specifically stain kinetochore proteins, is widely used on several cell types. It distinguishes micronuclei containing one or several whole chromosomes, which are positively labeled (centromere positive micronucleus, C+MN, due to aneugenic effect), or acentric chromosome fragments, which are unlabeled due to the absence of centromere (centromere negative micronucleus, C−MN, due to clastogenic effect). However, the very slight level of the centromeric signals obtained with the FISH technique on primary human fibroblasts, a cell type commonly used in environmental genetic toxicology, leads to great difficulties in distinguishing C+MN and C−MN. Furthermore, the CREST technique may lead to inappropriate results particularly with regards to variations in antibody composition between patient sera. Our results show that the in vitro CBMN, in combination with immunofluorescence staining of CENP-A (centromere protein A), efficiently screens genotoxicants for their ability to induce clastogenic and/or aneugenic effects. We propose the in vitro CBMN assay in combination with immunofluorescence staining of CENP-A as a suitable tool in environmental genotoxicity testing of primary human fibroblasts.     

Utilization of two invasive free-floating aquatic plants (Pistia stratiotes and Eichhornia crassipes) as sorbents for oil removal

Free-floating aquatic plants Pistia stratiotes and Eichhornia crassipes are well-known invasive species in the tropics and subtropics. The aim of this study was to utilize the plants as cost-effective and environmentally friendly oil sorbents. Multilevel wrinkle structure of P. stratiotes leaf (PL), rough surface of E. crassipes leaf (EL), and box structure of E. crassipes stalk (ES) were observed using the scanning electron microscope. The natural hydrophobic structures and capillary rise tests supported the idea to use P. stratiotes and E. crassipes as oil sorbents. Experiments indicated that the oil sorption by the plants was a fast process. The maximum sorption capacities for different oils reached 5.1–7.6, 3.1–4.8, and 10.6–11.7 g of oil per gram of sorbent for PL, EL, and ES, respectively. In the range of 5–35 °C, the sorption capacities of the plants were not significantly different. These results suggest that the plants can be used as efficient oil sorbents.     

In situ monitoring of the mutagenic effects of the gaseous emissions of a solid waste incinerator in metropolitan São Paulo, Brazil, using the Tradescantia stamen-hair assay

The present work was designed to determine the potential genotoxicity at the vicinity of a solid waste incinerator in the metropolitan area of S?o Paulo, using the Tradescantia stamen-hair bioassay. Experiments were carried out between December 1998 and April 1999 in four regions (40 pots of plants per site) selected on the basis of their pollution levels predicted by theoretical modeling of the dispersion of the incinerator's plume. The exposure sites were defined as follows: highest level (incinerator); a high level (museum) located 1.5 km from the emission point; a moderate level (school, at a distance of 3.5 km from the incinerator); and a control (at Jaguariúna countryside). The difference in genotoxicity among the groups was statistically significant (p < 0.001). The frequency of mutations observed in the countryside was significantly lower [2.25 +/- 1.55, mean +/- SD (standard deviation)] than that of the sites close to the incinerator. The frequency of mutations measured at the school (3.70 +/- 1.36) was significantly lower than that measured at both the museum (4.89 +/- 1.12) and the incinerator (5.69 +/- 1.34). In conclusion, we found a positive correlation between the spatial distribution of the emissions of the incinerator located in an urban area and the mutagenic events measured by the Tradescantia stamen-hair assay. The in situ approach employed in this study was simple, efficient, and of low cost. No air or chemical extraction of pollutants was necessary for genotoxicity testing as required by other assays.     

In Situ Monitoring of the Mutagenic Effects of the Gaseous Emissions of a Solid Waste Incinerator in Metropolitan São Paulo,Brazil, Using the Tradescantia Stamen-Hair Assay

ABSTRACT

The present work was designed to determine the potential genotoxicity at the vicinity of a solid waste incinerator in the metropolitan area of Sâo Paulo, using the Tradescantia stamen-hair bioassay. Experiments were carried out between December 1998 and April 1999 in four regions (40 pots of plants per site) selected on the basis of their pollution levels predicted by theoretical modeling of the dispersion of the incinerator's plume. The exposure sites were defined as follows: highest level (incinerator); a high level (museum) located 1.5 km from the emission point; a moderate level (school, at a distance of 3.5 km from the incinerator); and a control (at Jaguariuna countryside). The difference in genotoxicity among the groups was statistically significant (p < 0.001). The frequency of mutations observed in the countryside was significantly lower [2.25 ± 1.55, mean ± SD (standard deviation)] than that of the sites close to the incinerator. The frequency of mutations measured at the school (3.70 ± 1.36) was significantly lower than that measured at both the museum (4.89 ± 1.12) and the incinerator (5.69 ± 1.34). In conclusion, we found a positive correlation between the spatial distribution of the emissions of the incinerator located in an urban area and the mutagenic events measured by the Tradescantia stamen-hair assay. The in situ approach employed in this study was simple, efficient, and of low cost. No air or chemical extraction of pollutants was necessary for genotoxicity testing as required by other assays.     

Aporrectodea caliginosa, a suitable earthworm species for field based genotoxicity assessment?

There is a growing interest for the application of biomakers to field-collected earthworms. Therefore we have evaluated the usability of native populations of endogeic, widely distributed earthworm Aporrectodea caliginosa in the assessment of soil genotoxicity using the Comet assay. Validation of the Comet assay on earthworm coelomocytes has been established using commercially available Eisenia fetida exposed to copper, cadmium, and pentachlorophenol, along with A. caliginosa exposed to copper in a filter paper contact test. Neutral red retention time (NRRT) assay was conducted on copper exposed and field-collected earthworms. Significant DNA and lysosomal damage was measured using Comet and NRRT assays in native populations of A. caliginosa sampled from the polluted soils in the urban area in comparison to the earthworms from the reference site. The results of this study confirm the employment of A. caliginosa as a suitable species for the in situ soil toxicity and genotoxicity field surveys.     

DNA damage in caged Gammarus fossarum amphipods: a tool for freshwater genotoxicity assessment

The aim of this study was to propose a tool for freshwater environmental genotoxicity assessment using Gammarus fossarum, a high ecologically relevant species. In a first part, gammarids were caged upstream and downstream wastewater treatment plant effluent output. The sensitivity of genotoxic responses of haemocytes, oocytes and spermatozoa was compared using the Comet assay. Spermatozoa appeared to be the most sensitive, suitable and relevant cell type for genotoxicity risk assessment. In a second part, a watershed-scale study was conducted over 2 years to evaluate the applicability of our caging procedure. The genotoxic impact of a contamination was followed, taking into account seasonal variability. DNA damage in spermatozoa exhibited low basal level and low variability in control upstream sites, providing a reliable discrimination of polluted sites. Finally, DNA damage in caged G. fossarum has been proved to be a sensitive and reproducible tool for freshwater genotoxicity assessment.     

Nitrogen dioxide inhalation induces genotoxicity in rats

Nitrogen dioxide (NO₂) is a ubiquitous reactive free-radical gas, which has been associated with momentary and chronic health effects. In the present study, comet, micronucleus (MN) and DNA–protein crosslinks (DPC) assays were used to investigate the genotoxicity following in vivo inhalation exposure of rats to NO₂. The results show that inhalation exposure of rats to NO₂ induced DNA strand breakage and the formation of DPC in the cells from various internal organs (brain, lung, liver, spleen, kidney and heart), as well as resulted in obvious increase of MN frequency in the bone marrow cells of rats. Furthermore, above genotoxic responses showed significant linear dose-dependent manners. These results implicate that NO₂ is a genotoxic agent and these observations are informative for understanding the mechanisms of adverse effects of nitrogen dioxide.     

Inoculation with arbuscular mycorrhizal fungi and addition of composted olive-mill waste enhance plant establishment and soil properties in the regeneration of a heavy metal-polluted environment

A greenhouse experiment was carried out in order to investigate the effects of arbuscular mycorrhizal (AM) fungi inoculation and the use of composted olive waste (COW) in the establishment of Tetraclinis articulata and soil properties in a heavy metal-polluted soil. The treatments assayed were as follows: AM?+?0 % COW, AM?+?1 % COW, and AM?+?3 % COW. The higher doses of COW in combination with AM fungi increased shoot and root biomass production of T. articulata by 96 and 60 %, respectively. These treatments trended to improve the soil properties evaluated, highlighting the C compounds and N as well as the microbiological activities. In relation to the metal translocation in T. articulata, doses of COW applied decreased the Cr, Ni, and Pb contents in shoot, as well as Cr and As in root, although the most of them reached low levels and far from phytotoxic. The COW amendment aided Glomus mosseae-inoculated T. articulata plants to thrive in contaminated soil, mainly through an improvement in both nutrients uptake, mainly P and soil microbial function. In addition, the combined use of AM fungi plus COW could be a feasible strategy to be incorporated in phytoremediation programs because it promotes soil properties, a better performance of plants for supporting the stress in heavy metal-contaminated soils derived from the mining process, and also can be a good way for olive-mill waste disposal.     

Saprobe fungi decreased the sensitivity to the toxic effect of dry olive mill residue on arbuscular mycorrhizal plants

We studied the influence of olive mill dry residue (DOR) treated with saprobe fungi on growth of tomato and alfalfa colonized by Glomus deserticola. The application of 25g kg(-1) of dry DOR to soil decreased the shoot and root dry weight of tomato and alfalfa. Plants were more sensitive to the toxicity of DOR when colonized with the arbuscular mycorrhizal (AM) fungi. The sensitivity of both plants to the toxicity of DOR differed according to whether they were colonized by G. deserticola or by indigenous AM fungi. The phytotoxicity of DOR towards tomato and alfalfa was decreased by incubation the residue before planting with saprobe fungi for 20wk. The beneficial effects of AM fungi on plant growth added with DOR incubated with saprobe fungi depend of the type of the plant and AM fungi. The contribution of AM fungi to the beneficial effect of DOR incubated with saprobe fungi varied according to the type of the plant and AM fungi. G. deserticola increased the shoot and root dry weight of plants when they were grown in the presence of DOR incubated with saprobe fungi for 20wk. The beneficial effect of saprobe fungi on the dry weight and the level of AM mycorrhization of plants seem to be related to the decrease caused by these fungi in the phenol concentration in DOR. However, the toxicity of DOR due to substances other than phenols can not be ignored. The use of certain saprobe and AM fungi allows the possibility of using DOR as an organic fertilizer.     

Mutagenicity and genotoxicity assessment of industrial wastewaters

The genotoxicity of industrial wastewaters from Jajmau (Kanpur), was carried out by Ames Salmonella/microsome test, DNA repair-defective mutants, and Allium cepa anaphase–telophase test. Test samples showed maximum response with TA98 strain with and without metabolic activation. Amberlite resins concentrated wastewater samples were found to be more mutagenic as compared to those of liquid–liquid extracts (hexane and dichloromethane extracts). The damage in the DNA repair defective mutants in the presence of Amberlite resins concentrated water samples were found to be higher to that of liquid–liquid-extracted water samples at the dose level of 20 μl/ml culture. Among all the mutants, polA exhibited maximum decline with test samples. Mitotic index (MI) of root tip meristematic cells of A. cepa treated with 5, 10, 25, 50, and 100 % (v/v) wastewaters were significantly lower than the control. Complementary to the lower levels of MI, the wastewaters showed higher chromosomal aberration levels in all cases investigated.     

The effect of mycorrhiza on the growth and elemental composition of Ni-hyperaccumulating plant Berkheya coddii Roessler

The effect of arbuscular mycorrhizal fungi (AMF) on growth and element uptake by Ni-hyperaccumulating plant, Berkheya coddii, was studied. Plants were grown under laboratory conditions on ultramafic soil without or with the AM fungi of different origin. The AM colonization, especially with the indigenous strain, significantly enhanced plants growth and their survival. AMF affected also the elemental concentrations that were studied with Particle-induced X-ray emission (PIXE). AMF (i) increased K and Fe in shoots, Zn and Mn in roots, P and Ca both, in roots and shoots; (ii) decreased Mn in shoots, Co and Ni both, in shoots and roots. Due to higher biomass of mycorrhizal plants, total Ni content was up to 20 times higher in mycorrhizal plants compared to the non-mycorrhizal ones. The AMF enhancement of Ni uptake may therefore provide an improvement of a presently used technique of nickel phytomining.     

DNA damage and biochemical toxicity of antibiotics in soil on the earthworm Eisenia fetida

DNA damage and changes in enzyme activities were used as biomarkers to evaluate the genotoxicity and oxidative stress of tetracycline and chlortetracycline on the earthworm Eisenia fetida. The results showed that both antibiotics induced significant genotoxicity on earthworms in a dose-dependent manner (p < 0.01) with chlortetracycline having a stronger effect than tetracycline in the short term. The tests on the activities of superoxide dismutase (SOD) and catalase (CAT) enzymes further indicated biochemical stresses induced by the antibiotics. An N-shaped activity pattern was noted with the enzyme activities being stimulated first, then inhibited, and stimulated again with increasing concentration. The induced activity of SOD or CAT could scavenge oxygen free radicals and protect the organisms against oxidative stress by alleviating the corresponding DNA damage. Compared to enzyme activities, DNA damage as a biomarker was more sensitive and is thus more suitable for detecting low concentration exposure and diagnosing the genotoxicity of contaminants in terrestrial environment.     

Effect of cadmium on cytogenetic toxicity in hairy roots of Wedelia trilobata L. and their alleviation by exogenous CaCl2

Effects of cadmium (Cd) alone and in combination with calcium on mitosis and chromosomal aberration in the hairy root tips of Wedelia trilobata were investigated. The results showed that Cd concentrations below 50 μmol/L had a lesser or even a promoting effect on the mitotic index (MI) and the rate of chromosomal aberration in hairy root tips, while those higher than 100 μmol/L significantly decreased the MI and gradually stimulated the rate of chromosomal aberrations with prolonged time and increasing concentrations of Cd. Concentrations of 50 μmol/L Cd mainly induced C-mitosis, while more than 100 μmol/L Cd mainly caused chromosome breakage and chromosome adhesion in hairy root tip cells. When cultured with 300 μmol/L Cd, micronuclei were only observed in the interphase, middle, and late phase of hairy root tip cells. Compared with untreated controls, exogenous calcium had an alleviating effect on Cd-induced cytotoxicity by effectively enhancing the MI and reducing the rate of chromosomal aberration in root tip cells. The results presented here provide evidence that W. trilobata hairy roots with rapid autonomous growth could be used as a sensitive tool for monitoring and evaluation of Cd pollution in the environment.     

Cytotoxicity and DNA damage of five organophosphorus pesticides mediated by oxidative stress in PC12 cells and protection by vitamin E

Previous studies have demonstrated that pesticides could induce cytotoxicity and genotoxicity in vivo and in vitro, and that oxidative stress may be an important factor involved. However, investigations comparing the capability of different organophosphorous (OP) compounds to induce cytotoxicity, genotoxicity and oxidative stress are limited. Hence, the aim of this paper was to access the cytotoxic and genotoxic effects of five OPs or metabolites, Acephate (ACE), Methamidophos (MET), Chloramidophos (CHL), Malathion (MAT) and Malaoxon (MAO), and to clarify the role of oxidative stress, using PC12 cells. The results demonstrated that MET, MAT and MAO caused significant inhibition of cell viability and increased DNA damage in PC12 cells at 40 mg L^?1. MAO was more toxic than the other OPs. ACE, MET, MAT and MAO increased the levels of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA), and decreased the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) at 20 mg L^?1 and 40 mg L^?1 to different degrees. Pre-treatment with vitamin E(600 μM)caused a significant attenuation in the cytotoxic and genotoxic effect; pre-treatment reversed subsequent OP-induced elevation of peroxidation products and the decline of anti-oxidant enzyme activities. These results indicate that oxidative damage is likely to be an initiating event that contributes to the OP-induced cytotoxicity.     

Use of agro-industrial organic sludge amendment to remediate degraded soil: chemical and eco(geno)toxicological differences between fresh and stabilized sludge and establishment of application rates

Soil degraded by coal mining activities can be remediated by amendment with agro-industrial organic sludge. However, the environmental impacts associated with this management practice must be properly addressed. In this context, the objective of this study was to evaluate the eco(geno)toxicity of a fresh and a stabilized sludge before use in a laboratory soil remediation test. Chemical analysis of the complex mixtures (degraded soil, fresh sludge, and stabilized sludge) was carried out, as well as a battery of eco(geno)toxicity tests on microbiological enzymes (fluorescein hydrolysis), earthworms, and higher plants (including Vicia faba genotoxicity test), according to published methodologies. The results of these tests showed that fresh sludge was more toxic than sludge stabilized over 6 months toward earthworms and higher plants (lettuce, corn, and wild cabbage), while phyto(geno)toxicity tests with V. faba indicated the same genotoxicity levels for the two types of sludge. In the soil remediation simulation using different mixtures of degraded soil and stabilized sludge, the proportions of 50:50 % (dry weight basis) provided the lowest phyto(geno)toxicity effects and this mixture can be used for the revegetation of the contaminated site.     

Assessing the genotoxicity of urban air pollutants using two in situ plant bioassays

Genotoxicity of urban air has been analysed almost exclusively in airborne particulates. We monitored the genotoxic effects of airborne pollutants in the urban air of Perugia (Central Italy). Two plant bioindicators with different genetic endpoints were used: micronuclei in meiotic pollen mother cells using Tradescantia-micronucleus bioassay (Trad-MCN) and DNA damage in nuclei of Nicotiana tabacum leaves using comet assay (Nicotiana-comet). Buds of Tradescantia clone # 4430 and young N. tabacum cv. Xanthi plants were exposed for 24 h at three sites with different pollution levels. One control site (indoor control) was also used. The two bioassays showed different sensitivities toward urban pollutants: Trad-MCN assay was the most sensitive, but DNA damage in N. tabacum showed a better correlation with the pollutant concentrations. In situ biomonitoring of airborne genotoxins using higher plants combined with chemical analysis is thus recommended for characterizing genotoxicity of urban air.     

Cytotoxicity and DNA damage of five organophosphorus pesticides mediated by oxidative stress in PC12 cells and protection by vitamin E

Previous studies have demonstrated that pesticides could induce cytotoxicity and genotoxicity in vivo and in vitro, and that oxidative stress may be an important factor involved. However, investigations comparing the capability of different organophosphorous (OP) compounds to induce cytotoxicity, genotoxicity and oxidative stress are limited. Hence, the aim of this paper was to access the cytotoxic and genotoxic effects of five OPs or metabolites, Acephate (ACE), Methamidophos (MET), Chloramidophos (CHL), Malathion (MAT) and Malaoxon (MAO), and to clarify the role of oxidative stress, using PC12 cells. The results demonstrated that MET, MAT and MAO caused significant inhibition of cell viability and increased DNA damage in PC12 cells at 40 mg L(-1). MAO was more toxic than the other OPs. ACE, MET, MAT and MAO increased the levels of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA), and decreased the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) at 20 mg L(-1) and 40 mg L(-1) to different degrees. Pre-treatment with vitamin E(600 μM)caused a significant attenuation in the cytotoxic and genotoxic effect; pre-treatment reversed subsequent OP-induced elevation of peroxidation products and the decline of anti-oxidant enzyme activities. These results indicate that oxidative damage is likely to be an initiating event that contributes to the OP-induced cytotoxicity.