Gold and silver nanoparticles effects to the earthworm Eisenia fetida - the importance of tissue over soil concentrations.

To address and to compare the respective impact of gold and silver nanoparticles (Au and Ag NPs) in soil invertebrate, the earthworm Eisenia fetida was exposed to soil containing 2, 10, and 50 mg/kg of Au and Ag in both nanoparticulate and ionic forms for 10 days. Both metal NPs were 2-15 times less bioavailable than their ionic forms, and displayed similar transfer coefficients from soil to earthworm tissues. Both metal NPs triggered the onset of an oxidative stress as illustrated by increased glutathione S-transferase levels, decreased catalase levels, and increased malondialdehyde concentrations. Protein carbonylation distinguished the nanoparticular from the ionic forms as its increase was observed only after exposure to the highest concentration of both metal NPs. Au and Ag NPs triggered DNA modifications even at the lowest concentration, and both repressed the expression of genes involved in the general defense and stress response at high concentrations as did their ionic counterparts. Despite the fact that both metal NPs were less bioavailable than their ionic forms, at equivalent concentrations accumulated within earthworms tissues they exerted equal or higher toxic potential than their ionic counterparts.Capsule: At equivalent concentrations accumulated within earthworm tissues Au and Ag NPs exert equal or higher toxic potential than their ionic forms.

Electromagnetic fields at a mobile phone frequency (900 MHz) trigger the onset of general stress response along with DNA modifications in Eisenia fetida earthworms.

Eisenia fetida earthworms were exposed to electromagnetic field (EMF) at a mobile phone frequency (900 MHz) and at field levels ranging from 10 to 120 V m-1 for a period of two hours (corresponding to specific absorption rates ranging from 0.13 to 9.33 mW kg-1). Potential effects of longer exposure (four hours), field modulation, and a recovery period of 24 h after two hours of exposure were addressed at the field level of 23 V m-1. All exposure treatments induced significant DNA modifications as assessed by a quantitative random amplified polymorphic DNA-PCR. Even after 24 h of recovery following a two hour-exposure, the number of probe hybridisation sites displayed a significant two-fold decrease as compared to untreated control earthworms, implying a loss of hybridisation sites and a persistent genotoxic effect of EMF. Expression of genes involved in the response to general stress (HSP70 encoding the 70 kDa heat shock protein, and MEKK1 involved in signal transduction), oxidative stress (CAT, encoding catalase), and chemical and immune defence (LYS, encoding lysenin, and MYD, encoding a myeloid differentiation factor) were up-regulated after exposure to 10 and modulated 23 V m-1 field levels. Western blots showing an increased quantity of HSP70 and MTCO1 proteins confirmed this stress response. HSP70 and LYS genes were up-regulated after 24 h of recovery following a two hour-exposure, meaning that the effect of EMF exposure lasted for hours.

Insights into the molecular phylogeny and historical biogeography of the white-clawed crayfish (Decapoda, Astacidae).

In this study, the evolutionary history of the white-clawed crayfish (WCC) was evaluated using large-scale datasets comprising >1350 specimens from the entire distribution range. Using species delimitation methods on mitochondrial DNA (mtDNA) sequences, we propose four primary species hypotheses for WCC. Sequences for several nuclear regions were screened but none showed significant variation within WCC. This result favours a single secondary species hypothesis and indicates the existence of a mito-nuclear discordance in WCC. Therefore, mtDNA groups were considered only as genetic units that carry information about ancient divergences within WCC and not as taxonomic units. The reconstruction of ancestral ranges and divergence time estimates were used to link the current genetic structure with paleogeographic processes. These results showed that the emergence of mtDNA groups in WCC could be related to the Messinian Salinity Crisis, the climate cooling during the Pliocene and Pleistocene, and (paleo)shifting of the Adriatic Sea coastline in the Padanovenezian Plain. The most recent common ancestor of the mtDNA groups most likely originated from Dalmatia (eastern Adriatic coast) as indicated by the reconstruction of ancestral ranges. This ecoregion, along with the Gulf of Venice Drainages, harbours a high genetic diversity and should be emphasised as an area of the highest conservation priority.

Impact of treated wastewater on organismic biosensors at various levels of biological organization.

Relating the treated wastewater quality and its impact on organismic biosensors (Prussian carp, Carassius gibelio and earthworm, Eisenia fetida) was the main objective of the study. The impact on health status of fish living downstream, microbiological contamination and antimicrobial resistance, fish tissue structure, blood biochemistry, oxidative stress, genotoxic effects, as well as multixenobiotic resistance mechanism (MXR) was assessed. Treated wastewater discharged from the WWTP modified the environmental parameters and xenobiotic concentrations of the receiving surface waters. Potential bacterial pathogens from fish and respective waters were found in relatively low numbers, although they comprised aeromonads with a zoonotic potential. High resistance profiles were determined towards the tested antimicrobial compounds, mostly sulfamethoxazole and erythromycin. Histopathology primarily revealed gill lamellar fusion and reduction of interlamellar spaces of effluent fish. A significant increase in plasma values of urea, total proteins, albumins and triglycerides and a significant decrease in the activity of plasma superoxide dismutase were noted in carp from the effluent-receiving canal. Micronucleus test did not reveal significant differences between the examined groups, but a higher frequency of erythrocyte nuclear abnormalities was found in fish sampled from the effluent-receiving canal. Earthworms indicated to the presence of MXR inhibitors in water and sludge samples, thus proving as a sensitive sentinel organism for environmental pollutants. The integrative approach of this study could serve as a guiding principle in conducting evaluations of the aquatic habitat health in complex bio-monitoring studies.

Zebrafish genome instability after exposure to model genotoxicants.

Sublethal exposure to environmental genotoxicants may impact genome integrity in affected organisms. It is therefore necessary to develop tools to measure the extent and longevity of genotoxicant-induced DNA damage, and choose appropriate model organisms for biomonitoring. To this end, markers of DNA damage were measured in zebrafish larvae and adults following exposure to model genotoxicants (benzo[a]pyrene and ethyl methanesulfonate). Specifically, we assessed primary DNA damage and the existence of potentially persistent genomic alterations through application of the comet assay, quantitative random amplified polymorphic DNA (qRAPD) and amplified fragment length polymorphism (AFLP) assays. Furthermore, expression of genes involved in DNA repair, oxidative stress response and xenobiotic metabolism was evaluated as well. Additionally, the AFLP method was applied to adult specimens 1 year after larval exposure to the genotoxicants to evaluate the longevity of the observed DNA alterations. Large numbers of DNA alterations were detected in larval DNA using the comet assay, qRAPD and AFLP, demonstrating that zebrafish larvae are a sensitive model for revealing genotoxic effects. Furthermore, some of these genomic alterations persisted into adulthood, indicating the formation of stable genomic modifications. qRAPD and AFLP methods proved to be highly sensitive to genotoxic effects, even in cases when the comet assay indicated a lack of significant damage. These results thus support the use of zebrafish larvae as a sensitive model for monitoring the impact of genotoxic insult and give evidence of the longevity of genomic modifications induced by genotoxic agents.

Genomic and gene expression responses to genotoxic stress in PAC2 zebrafish embryonic cell line.

PAC2 cell line is, along most of the developed zebrafish cell lines, poorly characterized concerning its response to genotoxicants. To define the PAC2 cell line response to different forms of genotoxic stress, we exposed the cells to model genotoxic agents (benzo[a]pyrene, B[a]P, and ethyl methanesulfonate) and subsequently monitored DNA damage and alterations by using the battery of tests, including the Comet assay, quantitative random-amplified polymorphic DNA and amplified fragment length polymorphism. The expression of several DNA repair (xpc, xpd, hr23b, rad51, msh2) and oxidative stress response (sod (Cu/Zn)) genes was monitored as well. To obtain an indication of the PAC2 cell line metabolizing capacity, the expression of genes belonging to cyp1, cyp2 and cyp3 families was assessed upon exposure to B[a]P. Genotoxic responses were observed in all the used methods, and quantitative random-amplified polymorphic DNA and amplified fragment length polymorphism proved to be more sensitive by revealing DNA alterations even when the Comet assay indicated lack of significant damage. The PAC2 cell line demonstrated basal and B[a]P-induced expression of several cyp genes, suggesting its ability to metabolize indirect acting xenobiotics to a certain point. Based on these results, PAC2 cells seem to be sensitive zebrafish in vitro model in the genotoxicity assessment of the direct acting genotoxicant; however, they are less sensitive toward the indirect acting genotoxicant due to their limited metabolizing properties.

Effects of freshwater pollution on the genetics of zebra mussels (Dreissena polymorpha) at the molecular and population level.

Revealing long-term effects of contaminants on the genetic structure of organisms inhabiting polluted environments should encompass analyses at the population, molecular, and cellular level. Following this concept, we studied the genetic constitution of zebra mussel populations from a polluted (Dp) and reference sites (Cl) at the river Drava, Croatia, and applied microsatellite and DNA damage analyses (Comet assay, micronucleus test (MNT)). Additionally, mussels from both populations were exposed to polluted wastewater in the laboratory for three days, and DNA damage was analyzed to evaluate acclimatization and genetic adaptation of the investigated populations to the polluted environment. The two populations differed in their genetic constitution. Microsatellite analysis suggested that Dp had undergone a genetic bottleneck. Comet assay did not indicate any difference in DNA damage between the two populations, but MNT revealed that Dp had an increased percentage of micronuclei in hemocytes in comparison to Cl. The laboratory experiment revealed that Dp had a lower percentage of tail DNA and a higher percentage of micronuclei than Cl. These differences between populations were possibly caused by an overall decreased fitness of Dp due to genetic drift and by an enhanced DNA repair mechanism due to acclimatization to pollution in the source habitat.

Gene flow vs. pollution pressure: genetic diversity of Mytilus galloprovincialis in eastern Adriatic.

Environmental pollution may modify all the evolutionary processes involved in shaping the genetic patterns of exposed populations. In order to evaluate the pollution impact on the genetic diversity of Mediterranean mussel Mytilus galloprovincialis ten populations inhabiting differently polluted sites along the eastern Adriatic coast, from pristine bays to heavily trafficked harbours, were studied. Pollution pressure was assessed through an integrated study of biological effects and responses across different levels of biological organization. Eight microsatellite markers were analysed to assess genetic diversity of investigated populations. Both the principal component analysis (PCA) of the biomarker data set as well as the biomarker response index (BRI) confirmed substantial pollution pressure at the highly polluted sites, and very low pollution exposure at the three reference sites. Very shallow genetic differentiation was found in respect to maritime distances or pollution status, and this was attributed to a high gene flow among the populations. However, populations inhabiting polluted sites exhibited higher levels of genetic diversity and evolutionary mechanisms underlying this phenomenon are discussed.

What is Comet assay not telling us: AFLP reveals wider aspects of genotoxicity.

DNA damage detected by genotoxicity biomarkers such as the Comet assay is not always a reliable indicator of the consequences that genotoxic agents can have on the genome integrity of the exposed organisms. Therefore, to reveal the existence of more permanent alterations of DNA structure after genotoxic stress, the RTG-2 rainbow trout cell line was exposed for 3 days to benzo[a]pyrene (B[a]P, 0.1-10 µM) and ethyl methanesulfonate (EMS, 0.1-1mM) followed by 3 days of recovery period. Primary DNA damage was evaluated by the Comet assay and DNA alterations were assessed using AFLP (amplified fragment length polymorphism). Qualitative and quantitative modifications in AFLP profiles were analyzed in order to detect genetic alterations arising from mutation events and/or DNA damage. Significant induction in DNA damage measured by the Comet assay was noticed after B[a]P treatment at all concentrations but values returned to the control level after recovery. Exposure to EMS induced significant DNA damage only at the highest concentration and damage persisted after the recovery period. AFLP profiles detected DNA alterations even when Comet assay indicated complete DNA repair, revealing more persistent damage. Since such DNA damage can impair its structure and function, Comet assay results should preferably be supplemented with other methods in order to predict the consequences of genotoxic insult more accurately.

Oxidative and genotoxic effects of 900 MHz electromagnetic fields in the earthworm Eisenia fetida.

Accumulating evidence suggests that exposure to radiofrequency electromagnetic field (RF-EMF) can have various biological effects. In this study the oxidative and genotoxic effects were investigated in earthworms Eisenia fetida exposed in vivo to RF-EMF at the mobile phone frequency (900 MHz). Earthworms were exposed to the homogeneous RF-EMF at field levels of 10, 23, 41 and 120 V m(-1) for a period of 2h using a Gigahertz Transversal Electromagnetic (GTEM) cell. At the field level of 23 V m(-1) the effect of longer exposure (4h) and field modulation (80% AM 1 kHz sinusoidal) was investigated as well. All exposure treatments induced significant genotoxic effect in earthworms coelomocytes detected by the Comet assay, demonstrating DNA damaging capacity of 900 MHz electromagnetic radiation. Field modulation additionally increased the genotoxic effect. Moreover, our results indicated the induction of antioxidant stress response in terms of enhanced catalase and glutathione reductase activity as a result of the RF-EMF exposure, and demonstrated the generation of lipid and protein oxidative damage. Antioxidant responses and the potential of RF-EMF to induce damage to lipids, proteins and DNA differed depending on the field level applied, modulation of the field and duration of E. fetida exposure to 900 MHz electromagnetic radiation. Nature of detected DNA lesions and oxidative stress as the mechanism of action for the induction of DNA damage are discussed.

Genotoxicity monitoring of freshwater environments using caged crayfish (Astacus leptodactylus).

Genotoxicity of freshwater pollution was assessed by measuring DNA damage in haemocytes of caged freshwater crayfish Astacus leptodactylus by the means of Comet assay and micronucleus test, integrated with the measurements of physiological (total protein concentration) and immunological (total haemocyte count) haemolymph parameters as biomarkers of undergone stress. Crayfish were collected at the reference site (River Mreznica) and exposed in cages for 1 week at three polluted sites along the Sava River (Zagreb, Sisak, Krapje). The long term pollution status of these locations was confirmed by chemical analyses of sediments. Statistically significant increase in DNA damage measured by the Comet assay was observed at all three polluted sites comparing to the crayfish from reference site. In addition, native crayfish from the mildly polluted site (Krapje) cage-exposed on another polluted site (Zagreb) showed lower DNA damage than crayfish from the reference site exposed at the same location indicating adaptation and acclimatisation of crayfish to lower levels of pollution. Micronuclei induction showed similar gradient of DNA damage as Comet assay, but did not reach the statistical significance. Observed increase in total haemocyte count and total protein content in crayfish from polluted environments in the Sava River also confirmed stress caused by exposure to pollution. The results of this study have proved the applicability of caging exposure of freshwater crayfish A. leptodactylus in environmental genotoxicity monitoring using Comet assay and micronucleus test.

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.

Genotoxicity of marine sediments in the fish hepatoma cell line PLHC-1 as assessed by the Comet assay.

The main goal of this study was to test the usefulness of the Comet assay in the PLHC-1 hepatoma fish cell line as a tool for detecting the presence of genotoxic compounds in contaminated marine sediments. The system has been tested using both model chemicals (benzo[a]pyrene (B[a]P) and ethyl methanesulfonate (EMS)) and extracts of sediment samples obtained with solvent dichloromethane/methanol. For all of the analysed sediment extracts as well as for the model chemicals a concentration dependent genotoxic effect was observed. The sediment with the highest observed genotoxic potential was additionally extracted using various solvents in order to test which class of compounds, according to their polarity, is most responsible for the observed genotoxic effect. Non-polar solvents (cyclohexane and dichloromethane) yielded stronger genotoxic effect but the highest level of DNA damage was determined after exposure to sediment extract obtained with the solvent mixture dichloromethane/methanol which extracts a wide range of contaminants. Our results indicate that the PLHC-1 cell line is a suitable in vitro model in sediment genotoxicity assessment and encourage the use of fish cell lines as versatile tools in ecogenotoxicology.

DNA integrity of chub erythrocytes (Squalius cephalus L.) as an indicator of pollution-related genotoxicity in the River Sava.

An alkaline comet assay and a micronucleus test were carried out on erythrocytes of the European chub, Squalius cephalus L., collected in spring and autumn in 2005 and 2006 at three sampling sites in River Sava, near Zagreb, Croatia. The results of comet assay showed the lowest genotoxic influence at the least polluted site, while higher DNA damage was observed at the polluted sites. Although the basal levels of DNA damage were elevated, a clear gradation of DNA damage was found due to pollution intensity in all sampling periods. The lowest cytogenetic damage as revealed by the micronucleus test (MNT) was observed as well at the least polluted site. High variations in MN frequency were observed between sampling periods, although the number of micronucleated erythrocytes was consistently the highest one at the polluted site. The comet assay as a biomarker of genotoxic effect exhibited higher sensitivity in discriminating the genotoxic capacity of studied polluted sites while the MNT was less sensitive. However, both tests should be used together in biomonitoring studies because they can reveal different aspects of DNA damage; comet assay, the early event of genotoxic exposure, and MNT, its final result as a mutagenic potential.

Genotoxic, physiological and immunological effects caused by temperature increase, air exposure or food deprivation in freshwater crayfish Astacus leptodactylus.

The aim of this research was to investigate influence of different environmental stressors, such as temperature increase, air exposure and food deprivation on DNA integrity of a bioindicator species, freshwater crayfish Astacus leptodactylus. DNA damage was measured in crayfish haemocytes using Comet assay and micronucleus test. Crayfish haemolymph was subsequentially sampled during their 7 days of exposure to increased temperatures (25 and 30 degrees C) and during 24 h of air exposure. Both groups were also monitored through the following 7 days of recovery period. Food deprived crayfish were monitored over a period of 2 weeks. Alterations of measured physiological and immunological haemolymph parameters (THC, lactate, glucose and protein concentration) indicated stress response in exposed crayfish. However, only the stress induced by increased temperature significantly increased DNA damage in freshwater crayfish while food deprivation or air exposure did not cause a significant genotoxic effect.

Cage exposure of European sea bass (Dicentrarchus labrax) for in situ assessment of pollution-related genotoxicity.

Genotoxic effects are often the earliest signs of pollution-related environmental disturbance. In this study, we used the comet assay and micronucleus test to assess DNA damage in the erythrocytes of the European sea bass (Dicentrarchus labrax) exposed to environmental pollution in situ. Fish were collected from a fish farm in the Trogir Bay and their cages placed at an unpolluted reference site Solta (Necujam Bay) and a polluted site Vranjic (Kastela Bay) for four weeks. A group of fish which remained at the fish farm Trogir Bay were used as the second control group. Fish exposed at the Vranjic site showed a significantly higher erythrocyte DNA damage, measured by the comet assay, than either control group. Micronucleus induction showed a similar gradient of DNA damage, but did not reach statistical significance. Our results show that cage exposure of a marine fish D. labrax can be useful in environmental biomonitoring and confirm the comet assay as a suitable tool for detecting pollution-related genotoxicity.

Genotoxicity monitoring of freshwater environments using caged carp (Cyprinus carpio).

The present study deals with genotoxicity assessment of freshwaters using caged carp (Cyprinus carpio). Carps were transplanted from a fish-farm to three differently polluted sites in eastern Croatia. Two polluted sites were situated in the river Drava, downstream from the cities of Belisce and Osijek, while the reference site was in the Nature Park Kopacki rit, a preserved wetland area with limited anthropogenic influence. Exposure lasted for 3 weeks and was repeated for 3 years (2002-2004). DNA damage was assessed in erythrocytes of the exposed animals by the Comet assay and micronucleus test (MNT). In order to evaluate possible differences in stress responses to polluted water in situ and in aquaria a laboratory exposure was performed with water from the studied location in the second year of the study. Carp from the sites with high anthropogenic influence (Belisce and Osijek) had higher average DNA damage as expressed in both the MNT and Comet assay. Of the two, the Comet assay appeared to be more sensitive following both caging and aquaria exposures. The results from this study suggest that 3 weeks caging exposure of C. carpio may be a useful strategy to monitor for genotoxic agents in freshwater ecosystems.

Assessment of genotoxicity in polluted freshwaters using caged painter's mussel, Unio pictorum.

This study was undertaken to evaluate the applicability of caged painter's mussel, Unio pictorum for freshwater environmental genotoxicity assessment. Mussels in cages were exposed for 3 weeks in 2002-2004 to polluted sites in two large rivers in the Croatia, the Sava and Drava, and on the respective reference sites. DNA damage was assessed in haemocytes of the exposed mussels by the comet and micronucleus assays. Both assays provided good discriminative power between polluted and control sites and showed the same gradation of sites according to their genotoxic properties, with high concordance between investigated years. Background levels of the DNA damage in haemocytes of painter's mussels are defined for both assays for easier detection of contamination-related genotoxicity. U. pictorum is found to be a very suitable sentinel species, sufficiently sensitive to the impact of pollution but at the same time unsusceptible to stress caused by translocation or cage exposure.

Detection of DNA damage in haemocytes of Mytilus galloprovincialis in the coastal ecosystems of Kastela and Trogir bays, Croatia.

Coastal waters are burdened with different contaminants of anthropogenic origin due to intensive urbanisation and economical development. Bays, semi-enclosed areas with limited water renewal ability, are particularly endangered by contaminant inputs. Kastela Bay (Dalmatia, Eastern Adriatic) has earlier been identified as an area loaded with diffuse sources of pollution, including genotoxic agents. However, there is lack of data on the effects of these contaminants on the local marine fauna. The aim of this study was to assess genotoxic impacts in Kastela Bay and the neighbouring Trogir Bay using the micronucleus test and Comet assay with mussel (Mytilus galloprovincialis) haemocytes. Native and caged mussels were included in the studies. Our results confirmed that mussels in Kastela and Trogir Bays are affected by genotoxic contaminants. In addition to mussels from the most known polluted site (Vranjic), there was evidence for genotoxic effects in mussels collected at other locations. The response in the micronucleus test and the Comet assay differed somewhat between sites, the latter apparently being more sensitive, but the two methods complement each other and it is therefore desirable to use them both in monitoring the impacts of genotoxic pollution in coastal waters.

Haemolymph as compartment for efficient and non-destructive determination of P-glycoprotein (Pgp) mediated MXR activity in bivalves.

Measurement of the modulation of accumulation rate of model P-glycoprotein (Pgp) substrates has been a well established methodology for determination of the presence and activity of the multixenobiotic resistance (MXR) defence mechanism in aquatic invertebrates. Most studies have been focused on the gill tissue of various bivalves as a primary compartment for this type of measurements. In this study, we evaluated the potential of measuring the accumulation rate of a fluorescent model Pgp substrate rhodamine B (RB) in haemolymph, plasma and haemocytes of the freshwater painter's mussel (Unio pictorum) as additional potentially useful compartments. The obtained results demonstrated several important advantages of the determination of Pgp mediated MXR transport activity in haemolymph over determinations in gill tissue. The overall MXR response correlated well with the level of Pgp activity simultaneously determined in gills. The method is more sensitive, the procedure is easier and less laborious, and repeated use of same individuals is possible. Finally--the approach is non-destructive, offering a potentially powerful biomarker and research tool for studies directed to the evaluation of ecotoxicological importance of MXR defence and the presence of MXR inhibitors in the environment.