Calibration of the comet assay using ionising radiation.

Several trials have attempted to identify sources of inter-laboratory variability in comet assay results, aiming at achieving more equal responses. Ionising radiation induces a defined level of DNA single-strand breaks (per dose/base pairs) and is used as a reference when comparing comet results but relies on accurately determined radiation doses. In this ring test we studied the significance of dose calibrations and comet assay protocol differences, with the object of identifying causes of variability and how to deal with them. Eight participating laboratories, using either x-ray or gamma radiation units, measured dose rates using alanine pellet dosimeters that were subsequently sent to a specialised laboratory for analysis. We found substantial deviations between calibrated and nominal (uncalibrated) dose rates, with up to 46% difference comparing highest and lowest values. Three additional dosimetry systems were employed in some laboratories: thermoluminescence detectors and two aqueous chemical dosimeters. Fricke's and Benzoic Acid dosimetry solutions gave reliable quantitative dose estimations using local equipment. Mononuclear cells from fresh human blood or mammalian cell lines were irradiated locally with calibrated (alanine) radiation doses and analysed for DNA damage using a standardised comet assay protocol and a lab-specific protocol. The dose response of eight laboratories, calculated against calibrated radiation doses, was linear with slope variance CV= 29% with the lab-specific protocol, reduced to CV= 16% with the standard protocol. Variation between laboratories indicate post-irradiation repair differences. Intra-laboratory variation was very low judging from the dose response of 8 donors (CV=4%). Electrophoresis conditions were different in the lab-specific protocols explaining some dose response variations which were reduced by systematic corrections for electrophoresis conditions. The study shows that comet assay data obtained in different laboratories can be compared quantitatively using calibrated radiation doses and that systematic corrections for electrophoresis conditions are useful.

Interleukin-10 and PD150606 modulate expression of AMPA receptor GluA1 and GluA2 subunits under hypoxic conditions.

The goal of this study was to evaluate the effects of anti-inflammatory cytokine, interleukin-10 (IL-10), and calpain inhibitor, PD150606, on the expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits in rat hippocampal slices exposed to repeated brief hypoxic episodes. We studied both individual and combinatory effects of PD150606 and IL-10 on the expression of AMPA receptor subunits under hypoxic conditions for GluA1 and GluA2 as well as their phosphorylated forms - pSer831-GluA1 and pSer880-GluA2. Additionally, we studied whether brief hypoxic episodes and IL-10 may affect mRNA expression of transcriptional factors such as hypoxia-inducible factor-1α and nuclear factor κB (NF-κB). Western blotting analysis of hippocampal slice homogenates revealed that IL-10 and PD150606, both individually and in combination, ameliorate hypoxia-induced decrease in the expression of GluA1 and pSer831-GluA1, with different level of efficiency measured at 10, 50, and 90 min after hypoxia induction. Interestingly, brief hypoxic episodes did not induce any changes in the expression of GluA2 and pSer880-GluA2 subunits, whereas PD150606 showed biphasic effect, decreasing the expression of GluA2 and pSer880-GluA2 at 10 min and potentiating it at 90 min after hypoxia induction. IL-10 alone did not show any effect but was able to reverse PD150606 action on the expression of pSer880-GluA2 at 10 min and further potentiated it for GluA2 at 90 min after hypoxia. Finally, PCR analysis revealed that modulation of GluA1 and GluA2 expressions by hypoxia, and IL-10 was not associated with changes in the expression of hypoxia-inducible factor-1α and nuclear factor-κB (NF-κB) transcriptional factors.

Some causes of inter-laboratory variation in the results of comet assay.

We performed an inter-laboratory study to determine the variation of comet assay results and to identify its possible reasons. An exchange of slides between Labs in different stages of the comet assay protocol was performed. Because identical slides, durations of alkali treatment and electrophoresis, and similar electric field strengths (2.0 V/cm and 2.14 V/cm) were used, we concluded that the observed inter-laboratory difference in the results is directly associated with the electrophoresis step. In Lab 1, mouse bone marrow cells were exposed to methyl methanesulfonate at concentrations of 10, 25 and 50 µM for 3 h at 37 °C. In Lab 2, cells the same as in Lab 1 were immobilized in LMA on slides and exposed to X-rays at doses of 3-8 Gy. We found that the transportation of slides after lysis or electrophoresis step, as well as different dyes used for scoring did not produce any significant effect on the results. No substantial difference in the data was also revealed when various software packages were used for image analysis. The temperature of the alkaline solution was shown to increase during electrophoresis and, besides, the temperature heterogeneity of the solution took place in the area of the platform, with a maximum in the middle of the chamber. The temperature heterogeneity could affect the rate of conversion of alkali labile sites into single stranded breaks. Thus, it was clearly indicated that real temperature variations during the alkali treatment and electrophoresis were an essential factor in the variability of the results between our Labs.

The quality and fertility of sperm collected from European common frog (Rana temporaria) carcasses refrigerated for up to 7 days.

There is a catastrophic decrease in the biodiversity of amphibians coupled with the loss of genetic variation. The perpetuation of amphibian biodiversity demands a multifaceted approach, including the use of reproduction technologies (RTs), to enable efficient reproduction in captivity and to prevent the loss of genetic variation. Reproduction technologies for the storage of amphibian sperm for days to weeks, when refrigerated at 4°C, or for millennia when cryopreserved have recently undergone rapid development. Sperm from amphibians may be obtained through excision and maceration of testes; however, this is sometimes not possible with rare or endangered species. Alternate methods of obtaining sperm are through hormonal induction, or as spermatozoa from the carcasses of recently dead amphibians. The use of sperm from carcasses of recently dead amphibians is particularly valuable when sampled from genetically important founders in conservation breeding programs, or where catastrophic mortality is occurring in natural population. Sperm harvested over a period of 7 days from the testes of European common frog (Rana temporaria) carcasses stored in a refrigerator were assessed for percentage and progressive motility, cell membrane integrity, nuclear DNA fragmentation, and fertilizing ability. In addition, the survival of resulting embryos to hatch was recorded. Results indicated that some sperm of R. temporaria remain motile and fertile when harvested from frog carcasses refrigerated up to 7 days post-mortem, and resulting embryos can develop to hatch.

The increase in mitochondrial DNA copy number in the tissues of gamma-irradiated mice.

Changes in the number of mitochondrial DNA (mtDNA) copies in the brain and spleen tissues of gamma-irradiated (3 Gy) mice were studied by comparative analysis of the long-extension PCR products of mtDNA (15.9 kb) and a fragment of the cluster nuclear beta-globin gene (8.7 kb) amplified simultaneously in one and the same test-tube within total DNA. The analysis showed that, compared to the nuclear beta-globin gene, an increase in mtDNA copy number (polyploidization) took place in the brain and spleen cells of mice exposed to gamma-radiation. This data led to the suggestion that the major mechanism for maintenance of the mitochondrial genome, which is constantly damaged by endogenous ROS and easily affected by ionizing radiation or other exogenous factors, is the induction of synthesis of new mtDNA copies on intact or little affected mtDNA templates because the repair systems in the mitochondria function at a low level of efficiency.

DNA damage in frog erythrocytes after in vitro exposure to a high peak-power pulsed electromagnetic field.

Till the present time, the genotoxic effects of high peak-power pulsed electromagnetic fields (HPPP EMF) on cultured cells have not been studied. We investigated possible genotoxic effects of HPPP EMF (8.8 GHz, 180 ns pulse width, peak power 65 kW, repetition rate 50 Hz) on erythrocytes of the frog Xenopus laevis. We used the alkaline comet assay, which is a highly sensitive method to assess DNA single-strand breaks and alkali-labile lesions. Blood samples were exposed to HPPP EMF for 40 min in rectangular wave guide. The specific absorption rate (SAR) calculated from temperature kinetics was about 1.6 kW/kg (peak SAR was about 300 MW/kg). The temperature rise in the blood samples at steady state was 3.5 +/- 0.1 degrees C. The data show that the increase in DNA damage after exposure of erythrocytes to HPPP EMF was induced by the rise in temperature in the exposed cell suspension. This was confirmed in experiments in which cells were incubated for 40 min under the corresponding temperature conditions. The results allow us to conclude that HPPP EMF-exposure at the given modality did not cause any a-thermal genotoxic effect on frog erythrocytes in vitro.