DNA Damage In Haemocytes Research Articles

Effects of cadmium on respiratory burst, intracellular Ca 2+ and DNA damage in the white shrimp Litopenaeus vannamei

Acute effects of heavy metal ions on shrimp have been an area of intense study worldwide. However, the molecular mechanism by which cadmium-induced injury occurs remains largely unclear, and methods for mitigating toxicity in vivo have rarely been reported. In this study, the changes in respiratory burst and intracellular free calcium in haemocytes of pacific white shrimp, Litopenaeus vannamei, after exposure to Cd 2+ (CdCl 2) were examined using flow cytometry. Meanwhile, DNA damage and repair in haemocytes and hepatopancreas cells were studied using the comet assay. Respiratory burst generation, intracellular Ca 2+ concentration ([Ca 2+]i) and DNA damage in haemocytes and hepatopancreas cells all exhibited a dose-dependent increase and a time-dependent change after treatment with Cd 2+ compared with controls. These results indicate that Cd can induce oxidative stress and DNA damage in the shrimp L. vannamei. Moreover, the results also demonstrate that these parameters can be used as sensitive indicators of exposure to this genotoxicant.

Detection of DNA damage in haemocytes of Mytilus galloprovincialis in the coastal ecosystems of Kaštela 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. Kaštela 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 Kaštela 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 Kaštela 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.

Protective effects of selenium on mercury-induced DNA damage in mussel haemocytes

Little is known of the antioxidant role of selenium (Se) in aquatic invertebrates. We investigated the effects of Se on mercury-induced DNA damage in haemocytes from Mytilus edulis using alkaline single cell gel electrophoresis, that is, the Comet assay. The basal percentage tail DNA value for mussel haemocytes was 9.8 ± 0.2% (mean ± S.E.M., n = 70). Exposing mussels to Hg 2+ (nominal concentration 20 μg L −1) for three days led to an increase in tail DNA to 61.1 ± 1.8% ( n = 10). With added Se (as selenite, nominal concentration 4 μg L −1), Hg-induced DNA damage was reduced to 39.5 ± 3.1% ( n = 10). Se pre-exposure also provided some protection against Hg-induced DNA damage (% tail DNA = 51.0 ± 2.9%, n = 10). Basal glutathione peroxidase (GPx) activity in cell-free haemolymph was 93.7 ± 3.5 nmol min −1 mg −1 (mean ± S.E.M., n = 70). Increases in GPx activity were seen when Se was added during and/or after exposure to Hg. For example, a 3–4-fold increase was seen after three days exposure to Hg in the presence of added Se. Interestingly GPx activity doubled after three days in the presence of added Se alone, but was unchanged after exposure to HgCl 2 alone. These results suggest that the availability of Se in the natural environment could affect the antioxidant status of mussels, and consequently could affect levels of DNA damage.

Effects of temperature on baseline and genotoxicant-induced DNA damage in haemocytes of Dreissena polymorpha

The potential application of the Comet assay for monitoring genotoxicity in the freshwater mussel Dreissena polymorpha was explored and a preliminary investigation was undertaken of the baseline levels of DNA damage in mussel haemocytes of animals kept at different temperatures. In addition, in vitro cell sensitivity against genotoxicants was assessed in relation to increasing temperatures. The mussels were kept at four different constant temperatures (4, 18, 28 and 37 °C) for 15 h. The haemocytes withdrawn were treated in vitro with melphalan, as a model genotoxic compound, or sodium hypochlorite, a common water disinfectant capable of producing mutagenic/carcinogenic by-products, at the established temperatures for 1 h. The data obtained in vivo, in cells directly withdrawn from the mussels showed a significant ( P<0.001, Student’s t test) inter-individual variability, probably due to genetic and epigenetic factors and an increasing amount of DNA damage at increasing temperature. Mussel haemocytes showed a clear dose–response effect after in vitro melphalan treatment. Hypochlorite treatment also significantly increased DNA migration: the damage was temperature dependent, with a similar increase at 4 and 28 °C and a minimum level at 18 °C. This study demonstrates the potential application of the Comet assay to haemocytes of D. polymorpha. However, these findings suggest that temperature could alter both DNA damage baseline levels in untreated animals and cell sensitivity towards environmental pollutants in in vitro conditions. Therefore, more information is needed about seasonal variations and the natural background levels of DNA damage in mussels living in the wild, before they are used for the monitoring of genotoxic effects in aquatic environments.

Use of the Comet assay to assess DNA damage in hemocytes and digestive gland cells of mussels and clams exposed to water contaminated with petroleum hydrocarbons

Oil spills can result in the deposition of large quantities of petroleum hydrocarbons into intertidal and shallow waters seriously impacting bivalve populations. Petroleum hydrocarbons are enriched in polycyclic aromatic hydrocarbons (PAH) and PAH analogs many of which may have potential to damage DNA. The Comet assay is useful for assessing DNA damage and has been used to a limited degree with aquatic organisms, but mostly with studies in vitro. We have carried out studies with the Comet assay to assess the DNA damaging potential of complex mixtures of petroleum hydrocarbons for bivalves. Experiments were carried out with mussels (Mytilus edulis) and clams (Mya arenaria) with dispersions and water soluble fractions of an Arabian crude oil which was also chemically characterized in detail by GC–MS. Pilot studies were first conducted to evaluate test performance and reproducibility. An interindividual coefficient of variation ranging from 17 to 30% was established for the assay with hemocytes and digestive gland cells of both species. Exposure to hydrocarbon fractions had no significant impact on clams. However, an increase in DNA damage was observed at P<0.1 with digestive gland cells of mussels exposed to aqueous fractions of a light crude oil. These studies have demonstrated a potential for DNA damage in bivalves exposed to oil spills in inshore waters as well as potential for interspecies sensitivity.

Detection of DNA damage in haemocytes of zebra mussel using comet assay

The aim of the study was to use the comet assay on haemocytes of freshwater mussel, Dreissena polymorpha Pallas, for detection of possible DNA damage after exposure to pentachlorophenol (PCP) and to evaluate the potential application of the comet assay on mussel haemocytes for genotoxicity monitoring of freshwater environment. Zebra mussels were exposed for seven days to different concentrations (10, 80, 100, 150 μg/l) of PCP and in the river Sava downstream from Zagreb municipal wastewater outlet. Significant increase in DNA damage was observed after exposure to PCP at doses of 80 μg/l and higher and after in situ exposure in the river Sava as well. This study confirmed that the comet assay applied on zebra mussel haemocytes may be a useful tool in determining the potential genotoxicity of water pollutants.

Influence of sunlight on DNA damage in mussels exposed to polycyclic aromatic hydrocarbons

DNA single-strand break levels were compared in mussels deployed in San Diego Bay. Mussels were deployed at two locations, a reference site and at a location known to have high concentrations of PAHs. Two vertically oriented lines of mussels were deployed at each location for a period of 32 days. Mussels were attached to the lines at depths of 0.3, 1 and 3 m. One line was fully exposed to ambient sunlight, the other was shaded from sunlight to examine the role of PAH light activation on the expression of DNA damage in hemocytes and whole organism growth. Damage and growth measurements were made on days 0, 5, 14, and 32. Temperatures at the stations varied only by ≈0.5 °C. Hydrocarbon levels at the high PAH station were on average six times higher than the reference station. Slower growth and higher DNA damage was observed in cells from light exposed mussels at the PAH contaminated site. These effects decreased with depth. High levels of DNA damage could be induced with low intensity ultraviolet light exposure of cells from depurated or shaded mussels exposed to PAHs. DNA damage in mussels from the high impact site could be reduced to baseline levels after 24 h depuration in the dark. These results confirm previous findings showing increased DNA damage in PAH and sunlight exposed mussels, and demonstrate the significant impact of photoactivation on the health of these organisms.