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Chemical exposure in pathology labs

The Portuguese National Institute of Health (INSA), established in 1899, is a central department of the Ministry of Health with technical, administrative and financial autonomy. As the main Institution dealing with public health it includes several centres and laboratories with various locations in Lisbon and Porto, directing several well-integrated multidisciplinary programmes.

Comet Assay IV as a biomonitoring tool for DNA damage

An operative unit of INSA, the Centre of Occupational and Environmental Health’s (CSAO) primary function is to study the health impact of environmental factors, mainly in occupational environments. The overriding mission is to contribute to the knowledge of human exposure to environmental factors - chemical, physical and biological agents – and their effects on health, and to provide scientific and technical assistance in the field of prevention of occupational and environmental risks. The strategic goals are to promote healthy and safe environments (risk assessment) and contribute to the development of scientific and technical competences.

Dr. Joao Texeira and his colleagues at the CSAO began using the comet assay in 2005 primarily to assess the genotoxic potential of chemicals such as formaldehyde to which pathology and anatomy staff are exposed. The comet assay is widely used as a biomonitoring tool for DNA damage due to it’s versatility and relative simplicity. The technique is well suited to human monitoring studies because it is rapid, sensitive, requires a small number of cells and can be applied to proliferating and non proliferating cells, allowing the evaluation of DNA damage and repair at the single cell level.

Comet Assay IV in operation at the CSAO

Due to it’s extreme sensitivity the comet assay detects DNA damage at very low exposure levels. These characteristics make the assay suitable for analyzing cells such as leucocytes, exfoliated bladder cells, nasal and buccal epithelial cells leading to improved assessment of human exposure.

Methodology and protocol

The comet assay protocol used at the CSAO is based on that of Singh et al with some modifications. The cells used are lymphocytes harvested and isolated from heparinized venous blood. After mixed with low-melting-point agarose they were dropped onto a precoated slide with a 1% layer of normal-melting-point agarose. The cell membranes were removed by lysis. After lysis, the slides were placed in a horizontal electrophoresis tank with an alkali buffer. The DNA is denatured and electrophoresis is performed in the same buffer. During the electrophoresis the loops of DNA around strand breaks are more relaxed, and are pulled toward the anode, giving the appearance of a comet ‘tail’. Undamaged DNA remains tightly wound in the nucleoid, or comet ‘head’. After electrophoresis, the slides are rinsed, neutralized and stained with ethidium bromide. For each donor two slides are prepared and one hundred randomly selected cells (50 per replicate slide) are examined from each individual.

The slides are coded and examined using a Nikon Eclipse E400 microscope equipped with epi-fluorescence using a 40x objective. Analysis of the slides is performed using the Comet Assay IV scoring system with Olive Tail Moment and Tail Length used as the main indicators of DNA damage.

The group chose Comet Assay IV to score slides because of it’s ability to provide rapid scoring using one-click capture and analysis, automatic computation of the main parameters and storage of images and data. Statistical analysis of data is performed with the Spreadsheet Generator Excel macro supplied with Comet Assay IV.

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