Balázs Kakasi

Mitochondrial toxicity of triclosan on mammalian cells

Highlights • We show (sub)cellular toxicity of triclosan (TCS) on six types of mammalian cells.• 1–5 μg ml−1 TCS induced metabolic acidification and uncoupled respiration.• TCS ceased progressive boar sperm motility at 1 μg ml−1.• TCS uncouples ATP synthetase complex V in mitochondrion.• TCS caused regression of pancreatic islets to pycnotic cells.

Flow cytometric detection of oxidative DNA damage in fish spermatozoa exposed to cadmium - Short communication

The aim of the present pilot study was to apply a flow cytometric assay, the so-called OxyDNA test, to determine the level of oxidative DNA damage in fish spermatozoa exposed to different concentrations (0.01-10,000 mg/L) of cadmium. Milt was collected from three randomly selected Prussian carp (Carassius auratus gibelio) males. Oxidative DNA damage was assessed with the OxyDNA kit and using flow cytometry. The ratio of OxyDNA-positive events increased significantly at higher cadmium concentrations. The results indicate that direct contact of fish spermatozoa with cadmium-polluted water initiates genotoxic damage.

Assessment of the genotoxic potential of Hoechst 33342, SYBR-14 and PI using the SOS ChromoTest™

Abstract Fluorochromes in combination with flow cytometry can be used for laboratory assessment of semen quality in humans and domestic animals. Some studies have reported the potential toxicity of these fluorochromes toward the cells analyzed, but not toward the laboratory technician who operates the analytical instrument. We tested the genotoxic potential of three fluorochromes, SYBR-14, propidium iodide, and Hoechst 33342, using the colorimetric SOS ChromoTest™. The test revealed no genotoxic potential for any of the three fluorochromes within the dilution ranges investigated. We conclude that occasional direct contact with these fluorescent probes does not necessarily pose a genotoxic hazard.

Ecotoxicological characterisation of sedimentation in the Kis-Balaton Water Protection System.

The main function of the Kis-Balaton Water Protection System is to retain nutrients and total suspended solids, thus protecting the water quality of Lake Balaton. In this paper, the toxic nature of the sediment in the 2nd reservoir of the KBWPS has been characterised, using a battery of tests: Vibrio fischeri acute bioassay on whole sediment samples, and V. fischeri bioassay on pore water and elutriate samples. The latest version of the V. fischeri bioluminescence inhibition was applied, the Flash assay which uses a kinetic mode and is able to detect the toxicity of solid, turbid/coloured samples. Whole sediment toxicity showed a clear spatial distribution of toxicity, in parallel with elutriate toxicity. However, no pore water toxicity was detected, leading to the conclusion that contaminants are not water soluble.

Eco- and genotoxicity profiling of a rapeseed biodiesel using a battery of bioassays

Biodiesel is considered an important renewable energy source but still there is some controversy about its environmental toxicity, especially to aquatic life. In our study, the toxicity of water soluble fraction of biodiesel was evaluated in relatively low concentrations using a battery of bioassays: Vibrio fischeri bioluminescence inhibition, Sinapis alba root growth inhibition, Daphnia magna immobilization, boar semen live/dead ratio and DNA fragmentation and Unio pictorum micronucleus test. While the S. alba test indicated nutritive (stimulating) effect of the sample, the biodiesel exerted toxic effect in the aquatic tests. D. magna was the most sensitive with EC50 value of 0.0226%. For genotoxicity assessment, the mussel micronucleus test (MNT) was applied, detecting considerable genotoxic potential of the biodiesel sample: it elucidated micronuclei formation already at low concentration of 3.3%. Although this test has never been employed in biodiesel eco/genotoxicity assessments, it seems a promising tool, based on its appropriate sensitivity, and representativity.

Effects of high ambient temperature on fish sperm plasma membrane integrity and mitochondrial activity — A flow cytometric study

Local extreme climatic conditions occurring as a result of global climate change may interfere with the reproduction of animals. In the present study fish spermatozoa were incubated at different temperatures (20, 25, 30 and 40 °C) for 10 and 30 minutes, respectively and plasma membrane integrity and mitochondrial membrane potential changes were evaluated with flow cytometry using SYBR-14/PI and Mitotracker Deep Red FM fluorescent dyes. No significant differences were found in plasma membrane integrity at either incubation temperatures or time points. Mitotracker Deep Red FM histogram profiles indicating mitochondrial activity showed significant (p < 0.001) alterations in all cases of higher (25, 30 and 40 °C) temperature treatments as compared to the samples incubated at 20 °C. Our results indicate that fish spermatozoa exposed to high temperatures suffer sublethal damage that cannot be detected with conventional, vital staining techniques.

A comparison of alternative assays to measure DNA damage in stallion spermatozoa: TUNEL test versus ‘Nicoletti assay’

The aberrations of sperm DNA may cause various problems and have negative consequences on fertility. These influence embryonic development or might lead to early embryo loss. Sperm Chromatin Structure Assay (SCSA) is the flow cytometric method most often used for the detection of DNA lesions; however, some studies using that method reached confusing conclusions. The aim of this pilot study was to adjust and compare two alternative tests, namely the TUNEL test and the Nicoletti assay. The above-mentioned two flow cytometric methods capable of detecting the fragmented DNA of sperm were tested on 12 frozen-thawed stallion semen samples. The TUNEL test demonstrated much higher DNA fragmentation ratio than the Nicoletti assay (mean ± SD: 30.77 ± 13.03% vs. 1.93 ± 0.89%, respectively). A fluorescent microscopic check of the samples showed that TUNEL labelled the plasma membrane and the mitochondria in a nonspecific way, rather than detecting only the fragmented DNA, thus eventually resulting in a false positive sign. The Nicoletti assay is simpler, quicker and does not detect nonspecific binding; however, further analyses are required to determine its diagnostic value.

Geno- and cytotoxicologic assessment of wastewater effluents with mussel micronucleus assay and with flow cytometric sperm toxicity assay: a comparison

Several pharmaceutical drugs have potential harmful effect on wildlife such as aquatic toxicity, genotoxicity, or endocrine disruption effect. Removal rates of pharmaceuticals from municipal sewage during waste water treatment is questionable, several studied drugs are insufficiently or not removed while passing through the sewage treatment plants (STP).The analytical monitoring of potentially harmful drugs and especially drug residues in influent and effluent of STP are rather costly and not always possible on a day to day basis. Toxicity bioassays, on the other hand, are relatively cost-effective short-term tests, estimating the aggregate genotoxicity of the samples on different taxonomic levels. In our study the cytoand genotoxicity of the pre-treated potentially pharmaceutical containing influent and the effluent sample of a Hungarian STP were estimated with mussel (Unio pictorum) micronucleus (MN) assay and flow cytometric boar spermatozoa assay. The influent induced in the flow cytometric assay significant changes in mitochondrial transmembrane potential, oxidative DNA lesions and DNA fragmentation, but no significant genotoxic effect was detected by the MN assay. These results point to deficiencies of present wastewater treatment systems and remind us to choose carefully among available toxicity assays. Keywords— genotoxicity, cytotoxicity, pharmaceutical sewage, micronucleus assay, flow cytometric spermatozoa assay