Aging Cell Culture - Genetic and Metabolic Effects of Passage Number on Zebrafish Z3 Cells.

BACKGROUND/AIMS: Since cell lines are cultured and extensively used in a variety of different research disciplines, we determined the effects of passage numbers on a commonly used embryonic zebrafish cell line (Z3). METHODS: Senescence markers, DNA damage, the redox state, gene expression, and metabolic parameters have been investigated in young (passage 5) up to very old (passage 40 and higher) cells. RESULTS: Besides increasing DNA damage, we also found elevated metabolic capacity and a shift to a more reduced cellular redox state in the cells. Interestingly, several parameters showed a non-linear course regarding the passage number or cell age, so that for example young and mid-aged cells appeared to cluster with very old rather than with old cells. CONCLUSION: This study illustrates the importance of passage number and suggests pre-testing specific parameters to assure the generation of accurate and reproducible data.

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.

Earthworms and cadmium - Heavy metal resistant gut bacteria as indicators for heavy metal pollution in soils?

Preservation of the soil resources stability is of high importance for ecosystems, particularly in the current era of environmental change, which presents a severe pollution burden (e.g. by heavy metals) to soil fauna. Gut microbiomes are becoming recognized as important players in organism health, with comprehension of their perturbations in the polluted environment offering new insights into the nature and extent of heavy metal effects on the health of soil biota. Our aim was to investigate the effect of environmentally relevant heavy metal concentrations of cadmium (Cd) on the earthworm (Lumbricus terrestris) gut microbiota. Our results revealed that Cd exposure led to perturbations of earthworm gut microbiota with an increase in bacteria previously described as heavy metal resistant or able to bind heavy metals, revealing the potential of the earthworm-gut microbiota system in overcoming human-caused heavy metal pollution. Furthermore, an 'indicator species analysis' linked the bacterial genera Paenibacillus, Flavobacterium and Pseudomonas, with Cd treatment, suggesting these bacterial taxa as biomarkers of exposure in earthworms inhabiting Cd-stressed soils. The results of this study help to understand the impact of anthropogenic disturbance on soil fauna health and will have implications for environmental monitoring and protection of soil resources.

Regulatory Plasticity of Earthworm wMT-2 Gene Expression.

Metallothioneins (MTs) are multifunctional proteins occurring throughout the animal kingdom. While the expression and transcriptional regulation of MTs is well-studied in vertebrates, the mechanism of MT activation is still unknown for most invertebrates. Therefore, we examined wMT-2 gene regulation and expression patterns in Lumbricus rubellus and L. terrestris. Transcription levels, the occupation of DNA binding sites, the expression of putative transcriptional regulators, and promotor DNA methylation were determined. We found that wMT-2 expression does not follow a circadian pattern. However, Cd-induced wMT-2 induction was observed, and was, interestingly, suppressed by physical injury. Moreover, the promotor region that is responsible for the wMT-2 gene regulation was elucidated. ATF, a putative transcriptional regulator, showed increased phosphorylation upon Cd exposure, suggesting that it plays a major role in wMT-2 gene activation. The promotor methylation of wMT-2, on the other hand, is probably not involved in transcriptional regulation. Elucidating the regulatory mechanism of the earthworm MT gene activation might provide insights into the molecular coordination of the environmental stress response in invertebrates, and might also reveal a link to wound repair and, in a broader sense, to immunity.

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.

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.

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.

Assessment of surface water in the vicinity of fertilizer factory using fish and plants.

The genotoxic and toxic potential of polluted surface water exposed to a fertilizer factory effluent was evaluated using assays with fish (Cyprinus carpio) and plant (Lemna minor) model organisms. Beside classical physicochemical parameters, the contents of fluorides, some heavy metals and polycyclic aromatic hydrocarbons were analyzed as well. Surface water caused inhibition of plant growth and decrease of photosynthetic pigment content. Regarding DNA damage and oxidative stress parameters, both fish and plants showed similar response to the surface water. In confirmation to biochemical markers, histopathological analysis of gill and liver tissues revealed a higher incidence of lesions in fish exposed to polluted surface water. Generally, results obtained by biological monitoring were mostly in agreement with chemical analysis of the surface water, although several discrepancies were observed which might be due to difference in sensitivity of model organisms or in experimental conditions (laboratory and field exposure). The results imply that conventional chemical analysis should be extended to genotoxicity/toxicity assays as measured biological effects and the potential health hazard cannot be predicted based on the physicochemical characteristics of water samples alone.

Marine Pollutant Tributyltin Affects DNA Methylation and Fitness of Banded Murex (Hexaplex trunculus) Populations.

Banded murex, Hexaplex trunculus, is a marine gastropod whose reproductive fitness can be severely affected by very low concentrations of antifouling compound tributyltin (TBT). TBT has strong xenoandrogen impacts on snails, causing the development of imposex (e.g., the superimposition of male sexual characteristic in females), thereby affecting the fitness of entire populations. TBT is also known as a DNA-demethylating agent and an obesogenic factor. The aim of this study was to unravel the interactions between TBT bioaccumulation, phenotypic responses, and epigenetic and genetic endpoints in native populations of H. trunculus. Seven populations inhabiting environments along the pollution gradient were sampled in the coastal eastern Adriatic. These included sites of intense marine traffic and boat maintenance activity and sites with low anthropogenic impact. Populations inhabiting intermediately and highly polluted sites exhibited higher TBT burdens, higher incidences of imposex, and higher wet masses of snails than populations in lowly polluted sites. Other morphometric traits and cellular biomarker responses did not show clear differentiation among populations in relation to marine traffic/pollution intensity. An analysis of methylation sensitive amplification polymorphism (MSAP) revealed environmentally driven population differentiation and higher epigenetics than genetic within-population diversity. Moreover, decreases in genome-wide DNA methylation coincided with the imposex level and snail mass, suggesting an epigenetic background of the animal phenotypic response.

Environmental Epigenetics in Soil Ecosystems: Earthworms as Model Organisms.

One of the major emerging concerns within ecotoxicology is the effect of environmental pollutants on epigenetic changes, including DNA methylation, histone modifications, and non-coding RNAs. Epigenetic mechanisms regulate gene expression, meaning that the alterations of epigenetic marks can induce long-term physiological effects that can even be inherited across generations. Many invertebrate species have been used as models in environmental epigenetics, with a special focus on DNA methylation changes caused by environmental perturbations (e.g., pollution). Among soil organisms, earthworms are considered the most relevant sentinel organisms for anthropogenic stress assessment and are widely used as standard models in ecotoxicological testing of soil toxicity. In the last decade, several research groups have focused on assessing the impact of environmental stress on earthworm epigenetic mechanisms and tried to link these mechanisms to the physiological effects. The aim of this review is to give an overview and to critically examine the available literature covering this topic. The high level of earthworm genome methylation for an invertebrate species, responsiveness of epigenome to environmental stimuli, availability of molecular resources, and the possibility to study epigenetic inheritance make earthworms adequate models in environmental epigenomics. However, there are still many knowledge gaps that need to be filled in, before we can fully explore earthworms as models in this field. These include detailed characterization of the methylome using next-generation sequencing tools, exploration of multigenerational and transgenerational effects of pollutants, and information about other epigenetic mechanisms apart from DNA methylation. Moreover, the connection between epigenetic effects and phenotype has to be further explored.

DNA Methylation and Detoxification in the Earthworm Lumbricus terrestris Exposed to Cadmium and the DNA Demethylation Agent 5-aza-2'-deoxycytidine.

Earthworms are well-established model organisms for testing the effects of heavy metal pollution. How DNA methylation affects cadmium (Cd) detoxification processes such as the expression of metallothionein 2 (MT2), however, is largely unknown. We therefore exposed Lumbricus terrestris to 200 mg concentrations of Cd and 5-aza-2'-deoxycytidine (Aza), a demethylating agent, and sampled tissue and coelomocytes, cells of the innate immune system, for 48 h. MT2 transcription significantly increased in the Cd- and Cd-Aza-treated groups. In tissue samples, a significant decrease in MT2 in the Aza-treated group was detected, showing that Aza treatment inhibits basal MT2 gene activity but has no effect on Cd-induced MT2 levels. Although Cd repressed the gene expression of DNA-(cytosine-5)-methyltransferase-1 (DNMT1), which is responsible for maintaining DNA methylation, DNMT activity was unchanged, meaning that methylation maintenance was not affected in coelomocytes. The treatment did not influence DNMT3, which mediates de novo methylation, TET gene expression, which orchestrates demethylation, and global levels of hydroxymethylcytosine (5hmC), a product of the demethylation process. Taken together, this study indicates that Aza inhibits basal gene activity, in contrast to Cd-induced MT2 gene expression, but does not affect global DNA methylation. We therefore conclude that Cd detoxification based on the induction of MT2 does not relate to DNA methylation changes.

Common mechanisms cannot explain time- and dose-dependent DNA methylation changes in earthworms exposed to cadmium.

DNA hypermethylation caused by environmental pollutants like cadmium (Cd) has already been demonstrated in many invertebrates, including earthworms. However, the exact epigenetic mechanisms that drive this hypermethylation are largely unknown and even basic DNA methylation and demethylation processes are hardly characterized. Therefore, we used an important bioindicator, the earthworm Lumbricus terrestris, as a model organism to determine time- and dose-dependent effects of Cd on global and gene-specific DNA methylation and its underlying mechanisms. We revealed Cd-induced adenine and cytosine hypermethylation using specific antibodies in dot blots and found that the methylation level of adenine compared to cytosine changed even to a bigger extent. However, the levels of hydroxymethylated cytosine did not differ between treatment groups. General methylation and demethylation components like methyltransferases (DNMT1 and 3), and ten-eleven translocation (TET) genes were confirmed in L. terrestris by quantitative RealTime PCR. However, neither gene expression, nor DNMT and TET enzyme activity showed significant differences in the Cd exposure groups. Using bisulfite conversion and sequencing, gene body methylation (gbm) of metallothionein 2 (MT2), one of the most important detoxification proteins, was characterized. Cd-dependent changes in MT2 gbm could, however, not be correlated to MT2 gene activity evaluated by quantitative RealTime PCR. Future directions as well as missing links are discussed in the present study hinting towards the importance of studying epigenetic marks and mechanistic insights in a broad variety of species to deepen our knowledge on the effects of changing environmental conditions.

Ecotoxicological epigenetics in invertebrates: Emerging tool for the evaluation of present and past pollution burden.

The effect of environmental pollution on epigenetic changes and their heredity in affected organisms is of major concern as such changes can play a significant role in adaptation to changing environmental conditions. Changes of epigenetic marks including DNA methylation, histone modifications, and non-coding RNA's can induce changes in gene transcription leading to physiological long-term changes or even transgenerational inheritance. Such mechanisms have until recently been scarcely studied in invertebrate organisms, mainly focusing on model species including Caenorhabditis elegans and Daphnia magna. However, more data are becoming available, particularly focused on DNA methylation changes caused by anthropogenic pollutants in a wide range of invertebrates. This review examines the literature from field and laboratory studies utilising invertebrate species exposed to environmental pollutants and their effect on DNA methylation. Possible mechanisms of epigenetic modifications and their role on physiology and adaptation as well as the incidence of intergenerational and transgenerational inheritance are discussed. Furthermore, critical research challenges are defined and the way forward is proposed. Future studies should focus on the use of next generation sequencing tools to define invertebrate methylomes under environmental stress in higher resolution, those data should further be linked to gene expression patterns and phenotypes and detailed studies focusing on transgenerational effects are encouraged. Moreover, studies of other epigenetic mechanisms in various invertebrate species, apart from DNA methylation would provide better understanding of interconnected cross-talk between epigenetic marks. Taken together incorporating epigenetic studies in ecotoxicology context presents a promising tool for development of sensitive biomarkers for environmental stress assessment.

Biomarker response of Mediterranean mussels Mytilus galloprovincialis regarding environmental conditions, pollution impact and seasonal effects.

The complexity of seasonally and spatially variable environments, coupled with complex biological interactions, makes it difficult to pinpoint biological responses to specific environmental stressors, including chemical pollution. To disentangle causative factors and reveal biomarker responses, we applied biomarker-based multivariate approaches to 15 native populations of Mediterranean mussel Mytilus galloprovincialis in spring and autumn. In addition, we used a subset of these populations in transplant experiments between clean and polluted environments in nature and in lab mesocosms. The extent of biomarker responses in native populations is affected by season, and significantly lower variability across seasons was observed among mussels from clean than from polluted sites. Results of paired block designed transplant experiment demonstrated both regional and pollution effect, with mussels uniformly exhibiting higher responses on more impacted sites in each of the Adriatic regions. Biomarker status of mussels varied among Adriatic regions in dependence on the set of environmental variables, and between clean and polluted sites in dependence on measured concentrations of metals in mussels' tissue. Results of the mesocosm experiment revealed distinctive biomarker responses of two populations of different origin when exposed to common conditions. Multivariate description of biomarker activity and application of specific experiments allowed us to link environmental condition, exposure to pollution and seasonality to mussels' biomarker responses.

Low levels of Cd induce persisting epigenetic modifications and acclimation mechanisms in the earthworm Lumbricus terrestris.

Toxic effects of cadmium (Cd), a common soil pollutant, are still not very well understood, particularly in regard to its epigenetic impact. Therefore, the aim of this study was to assess DNA methylation changes and their persistence in the earthworm Lumbricus terrestris upon chronic low dose Cd exposure using methylation sensitive amplification polymorphism (MSAP). Moreover, the biomarker response and fitness of the earthworms, as well as the expression of detoxification-related genes (metallothionein (MT) and phytochelatin synthase (PCS)) was evaluated. Low levels of Cd caused an increase in genome-wide DNA methylation, which remained partly modified, even after several months of recovery in unpolluted soil. Increased cellular stress seemed to decrease after two weeks of exposure whereas fitness parameters remained unaffected by Cd, probably as a result from the activation of detoxification mechanisms like the expression of MTs. Interestingly, even though the level of Cd exposure was very low, MT expression levels indicate the development of acclimation mechanisms. Taken together, this study demonstrates that acclimation, as well as epigenetic modifications can occur already in moderately polluted environments. In addition, these effects can have long-lasting impacts on key species of soil invertebrates and might persist long after the actual heavy metal challenge has passed.

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.

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.

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.

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.

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.