Anamaria Štambuk

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.

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.

Persistence of DNA Damage in the Freshwater Mussel Unio pictorum Upon Exposure to Ethyl Methanesulphonate and Hydrogen Peroxide

An important endpoint in assessing pollution‐related toxicity is genotoxicity. To obtain insight into the time‐course of oxidative‐ and alkylation‐induced DNA damage in the freshwater mussel, Unio pictorum, mussels were exposed for 24 hr to concentration gradients of pro‐oxidant hydrogen peroxide (H2O2) and a mono‐functional alkylating agent, ethyl methanesulfonate (EMS). DNA damage was assessed in haemocytes immediately upon exposure and over the recovery period of up to 72 days by means of comet and micronucleus assays. Following exposure to H2O2, DNA damage as detected by the comet assay returned to control values after one day, except for the mussels exposed to the highest dose when damage was detectable for the next 3 days. In contrast, alkylation‐induced DNA damage was detectable even after 72 days of recovery in de‐chlorinated water, with a dose‐response relationship observable throughout the whole recovery period. Micronucleus frequency was the highest on Day 3 after exposure to EMS; it decreased considerably by Day 7 and returned almost to the control levels 19 days after exposure, while no significant induction of micronuclei was observed in mussels exposed to H2O2. Although the comet assay is considered a biomarker of recent genotoxic exposure, detecting DNA damage of shorter longevity than with the micronucleus assay, results presented here show that in the case of alkylation damage the comet assay reveals genotoxic exposure of U. pictorum in a dose‐dependent manner even after 2 months. Environ. Mol. Mutagen. 2008.

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.

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.

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.

Prospects and challenges for the conservation of farm animal genomic resources, 2015-2025

Livestock conservation practice is changing rapidly in light of policy developments, climate change and diversifying market demands. The last decade has seen a step change in technology and analytical approaches available to define, manage and conserve Farm Animal Genomic Resources (FAnGR). However, these rapid changes pose challenges for FAnGR conservation in terms of technological continuity, analytical capacity and integrative methodologies needed to fully exploit new, multidimensional data. The final conference of the ESF Genomic Resources program aimed to address these interdisciplinary problems in an attempt to contribute to the agenda for research and policy development directions during the coming decade. By 2020, according to the Convention on Biodiversitys Aichi Target 13, signatories should ensure that “…the genetic diversity of …farmed and domesticated animals and of wild relatives …is maintained, and strategies have been developed and implemented for minimizing genetic erosion and safeguarding their genetic diversity.” However, the real extent of genetic erosion is very difficult to measure using current data. Therefore, this challenging target demands better coverage, understanding and utilization of genomic and environmental data, the development of optimized ways to integrate these data with social and other sciences and policy analysis to enable more flexible, evidence-based models to underpin FAnGR conservation. At the conference, we attempted to identify the most important problems for effective livestock genomic resource conservation during the next decade. Twenty priority questions were identified that could be broadly categorized into challenges related to methodology, analytical approaches, data management and conservation. It should be acknowledged here that while the focus of our meeting was predominantly around genetics, genomics and animal science, many of the practical challenges facing conservation of genomic resources are societal in origin and are predicated on the value (e.g., socio-economic and cultural) of these resources to farmers, rural communities and society as a whole. The overall conclusion is that despite the fact that the livestock sector has been relatively well-organized in the application of genetic methodologies to date, there is still a large gap between the current state-of-the-art in the use of tools to characterize genomic resources and its application to many non-commercial and local breeds, hampering the consistent utilization of genetic and genomic data as indicators of genetic erosion and diversity. The livestock genomic sector therefore needs to make a concerted effort in the coming decade to enable to the democratization of the powerful tools that are now at its disposal, and to ensure that they are applied in the context of breed conservation as well as development.

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 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 Mrežnica) 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.

Two distinct evolutionary lineages of the Astacus leptodactylus species-complex (Decapoda : Astacidae) inferred by phylogenetic analyses

Abstract. Narrow-clawed crayfish (Astacus leptodactylus Eschscholtz, 1823 species-complex) is one of five European freshwater crayfish species. Even though widely distributed, it hasn’t been frequently studied and its taxonomy and systematics are unresolved. The results of a recent comparative morphometric character study revealed that morphometry of Asian and European populations differ significantly. In this research, for the first time, mitochondrial molecular markers (16S rRNA and COI) were used with the aim of elucidating the phylogenetic relationship between European and Asian populations of the narrow-clawed crayfish. Analyses included crayfish from Croatia, Bulgaria, Armenia, Russia, Poland and Turkey, and three different optimality criteria were applied. Phylogenetic relationships were reconstructed using the COI dataset, as well as the concatenated one (COI + 16S rRNA). For both datasets, congruent topologies were obtained and trees were characterised by the existence of two well supported phylogroups, one that included European populations, and the other Asian. Results indicate the presence of distinct evolutionary lineages within the A. leptodactylus species-complex, and corroborate previous results obtained using morphometric analyses.