Single cell gel electrophoresis assay: methodology and applications.

Human biomonitoring using the single cell gel electrophoresis (SCGE) or comet assay is a novel approach for the assessment of genetic damage in exposed populations. This assay enables the detection of various forms of DNA damage in individual cells with ease and speed and is, therefore, well suited to the analysis of a large group in a population. Here, application of SCGE assay in the identification of dietary protective factors, in clinical studies and in monitoring the risk of DNA damage resulting from occupational, environmental or lifestyle exposures is reviewed. Also, the comparative sensitivity of SCGE assay and conventional cytogenetic tests to detect genetic damage is discussed. Finally, strengths and shortcomings of the SCGE assay are addressed.

Single cell gel electrophoresis assay: A new technique for human biomonitoring studies

Age-related changes in the immune system may contribute to morbidity and mortality due to decreased resistance to infection and, possibly, certain cancers in the aged Many studies mostly performed in mice, rats and man but also including monkeys and dogs have established that age-associated immune decline is characterized by decreases in both humoral and cellular responses The former may be largely a result of the latter, because observed changes both in the B cell germline-encoded repertoire and the age-associated decrease in somatic hypermutation of the B cell antigen receptors are now known to be critically affected by helper T cell aging As antigen presenting cell (APC) function appears to be well-maintained in the elderly, this review will focus on the T cell Factors contributing to T cell immunosenescence may include a) altered production of T cell progenitors (stem cell defects, stromal cell defects), b) decreased levels of newly-generated mature T cells (thymic involution), c) aging of resting immune cells, d) disrupted activation pathways in immune cells (stimulation via the T cell receptor for antigen, costimulation, apoptosis control), e) replicative senescence of clonally expanding cells This review aims to consider the current state of knowledge on the scientific basis for and potential clinical relevance of those factors in immunosenescence in humans Experiments in other species will be touched upon with the proviso that there are clearly differences between them, especially between humans and rodents, but exactly what those differences are is not completely clear Given its potential importance and the increasing proportion of elderly people the world over, coupled with the realisation that whereas mortality is decreasing, morbidity may not be decreasing in parallel (1), a better understanding of the causes and impact of immunosenescence may offer the possibility of identifying where prevention or delay of onset, as well as therapeutic intervention, might be beneficial Amelioration of the effects of dysregulated immune responses in the elderly by replacement therapy, supplementation therapy or other approaches may result in an enhancement of their quality of life, and significant reductions in the cost of medical care in old age

T cells and aging, January 2002 update.

The hazards of surgical smoke.

Over 200 agents/factors have been examined in the single cell gel electrophoresis assay, more commonly known as the Comet assay, performed either in vitro or in vivo in a variety of species. Unequivocal carcinogenicity data are available for 119 of them, amongst which unequivocal Comet assay data exist for 95 agents. Of these 95 agents the prevalence of carcinogens was 88% (84/95). The carcinogens that were Comet positive (sensitivity) formed 88% (74/84), the non-carcinogens that were Comet negative (specificity) formed 64% (7/11). This simple analysis of the Comet assay has not taken account of the difference between in vitro and in vivo responses, species differences or organ and tissue differences. Also, limitations as to the conduct of the assay have not been examined in any depth. Thus, at the present time the Comet assay has high sensitivity for carcinogens, but its specificity is uncertain because few non-carcinogens have been tested.

/pdf/comet-assay-responses-as-indicators-of-carcinogen-exposure-3vuknq91i0.pdf

Comet assay responses as indicators of carcinogen exposure

Laser therapy has gained wide acceptance and application in many medical disciplines. Nevertheless, during surgical procedures, the thermal destruction of tissue creates a smoke plume. Recent research data indicate that pyrolysates liberated during vaporisation of tissue induce DNA damage. However, assessing potential health hazards during medical laser treatment requires comprehensive insight into the cytotoxic, genotoxic, clastogenic and mutagenic capacity of laser pyrolysis products (LPP). Therefore, the aim of this study was to evaluate the cytotoxic, genotoxic, clastogenic and mutagenic potential of substances resulting from laser irradiation. Four different types of porcine tissues were irradiated with a surgical CO2 laser, the aerosols were sampled under defined conditions and subjected to the SCE test, micronucleus test and the HPRT test. The results showed that the pyrolysis products are strong inducers of cytotoxic effects. The pyrolysis products induced positive effects in the SCE test, micronucleus test and the HPRT test. The ability and extent to induce genotoxic and mutagenic effects turned out to be dependent on the type of tissue that had been irradiated. In general, the effects were most pronounced with liver pyrolysate. In all test systems, a clear dose relationship could be established. In conclusion, we were able to prove that the particulate fraction of laser pyrolysis aerosols originating from biological tissues undoubtedly have to be classified as cytotoxic, genotoxic, clastogenic and mutagenic. Therefore, they could be potential health hazards for humans.

Laser pyrolysis products-genotoxic, clastogenic and mutagenic effects of the particulate aerosol fractions.

The use of lasers in medical applications has grown enormously in the last few years. Recent chemical analysis of the laser pyrolysis products revealed that aerosols generated by pyrolytic decomposition of tissue could be health hazards. Therefore we analysed the genotoxic and mutagenic effects of laser pyrolysis products from different types of porcine tissue. The tissues were irradiated with a surgical CO2 laser and the generated aerosols were sampled as particulate fractions as well as low and highly volatile fractions. Then human leukocytes were incubated with the pyrolysis products and subjected to the comet assay. The results of the comet assay indicated the pyrolysis products being inducers of DNA damage. The ability to induce genotoxic effects turned out to be strongly dependent on the type of tissue that had been irradiated during laser treatment. To check whether the pyrolysis products also have mutagenic properties the Salmonella mutagenicity assay was performed. The particulate aerosol fractions of skin, muscle tissue and liver tissue clearly proved to be mutagenic in TA98 in the presence of S9 mix. There was no mutagenic effect detectable without metabolic activation. In conclusion, our experiments showed that the laser pyrolysis products originating from porcine tissues induced very potent genotoxic as well as mutagenic effects and therefore they could be potential health hazards for humans.

Laser pyrolysis products: sampling procedures, cytotoxic and genotoxic effects

Different types of porcine tissue were irradiated with a surgical CO2 laser. The generated aerosols were sampled on glass fiber filters and incubated with human peripheral blood cells. Afterwards, these exposed cells were subjected to the comet assay. The single cell gel electrophoresis or comet assay represents a powerful technique for the detection of DNA strand breaks in eukaryotic cells. In short, the electrophoretic mobility of DNA fragments is proportional to the quantity of DNA damage caused by the genotoxic agent in question. By investigating porcine tissue laser pyrolysis products, it is demonstrated that the comet assay is an appropriate tool to assess the genotoxic capacity of even a heterogeneously composed class of substances with unknown modes of action and interaction.© (1996) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Genotoxity of pyrolysis products originating from laser-treated porcine tissues

The purpose of this study was to examine whether changes in the repair capacity of blood cells could be used as a valuable biomarker for radiation exposure. To characterize the repair kinetics in nonirradiated and irradiated cells we first performed in vitro split dose experiments. DNA damage and DNA repair capacity were analysed using the comet assay. Our results showed that the first in vitro irradiation affects the repair system of the cells, resulting in a decreased repair capacity after the second irradiation. Furthermore, the second irradiation results in a large amount of DNA damage in the blood cells. To test whether the analysis of the DNA repair capacity after in vitro irradiation is also a valuable method for in vivo studies of donors exposed to radiation, we analysed the repair capacity of blood cells of two exposed groups: patients subjected to a radioiodine therapy and chronically irradiated volunteers from the Chernobyl region. Both groups also showed a significantly impaired repair capacity indicating a stress on the hematopoietic system. In addition, in the group of the Ukrainians DNA damage after in vitro irradiation was significantly higher than in a control group. These results lead to the presumption that the repair capacity and the DNA damage after in vitro irradiation might be a very useful biological marker for radiation exposure in population monitoring.

Changes in the Repair Capacity of Blood Cells as a Biomarker for Chronic Low-Dose Exposure to Ionizing Radiation

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Laser pyrolysis products-genotoxic, clastogenic and mutagenic effects of the particulate aerosol fractions.

Laser pyrolysis products: sampling procedures, cytotoxic and genotoxic effects

Genotoxity of pyrolysis products originating from laser-treated porcine tissues

Changes in the Repair Capacity of Blood Cells as a Biomarker for Chronic Low-Dose Exposure to Ionizing Radiation