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The comet assay and the earthworm

Wildlife can be exposed to ionising radiation both from natural and man-made sources. In the majority of cases of routine radiation release, the exposure is long-term and dose rates are generally low, but after an accidental release, the levels can be significantly elevated. Methods for analysing oxidised DNA lesions in the immune and reproductive cells (coelomocytes and spermatogenic cells, respectively) from the earthworm Eisenia fetida were established by associates at The Norwegian Radiation Protection Authority and The Norwegian Institute of Public Health.

The Comet Assay IV scoring system, supplied by Perceptive Instruments Ltd, was used for the scoring of comets.

Here, the comet assay was used as a tool for measuring the DNA integrity in cells from E. fetida continuously exposed to (60)Co gamma-radiation. The DNA integrity refers to the strand breaks plus alkali labile sites and formamidopyrimidine glycosylase (Fpg)-sensitive sites. This integrity was measured and related to effects on reproduction end points which had previously been reported. Here, both the standard comet assay method, and a method for determining Fpg-sensitive sites (the Fpg-modified comet assay) were used. For further information on the experimental details please follow the link below this article (subject to access permissions). The Comet Assay IV scoring system, supplied by Perceptive Instruments Ltd, was used for the scoring of comets. Cells were selected randomly and ‘hedgehogs’ (cells with small or non-existent head and large diffused tails containing >90% of the DNA) were not excluded from the dataset.

In vivo DNA repair was studied by measuring the decrease in damage (strand breaks and Fpg-sensitive sites) in coelomocytes and spermatogenic cells isolated from worms at different times. These data indicated that strand breaks were repaired in coelomocytes. This repair followed a biphasic kinetic pattern, which means that a fast and a slow half-life (36 min (95%) and 6.7 hour (5%)) were both observed. The Fpg-sensitive sites were repaired at considerably lower rates (between 4 and 5 hours). In general, a reduced repair of Fpg-sensitive sites suggests a higher potential for accumulation of oxidised lesions, compared to DNA strand breaks, in earthworms exposed to radiation and other environmental contaminants. This is the first study comparing DNA damage with reproduction in earthworms exposed to ionising radiation.

This case study is based on:
Induction and repair of DNA strand breaks and oxidised bases in somatic and spermatogenic cells from the earthworm Eisenia fetida after exposure to ionising radiation.
Hertel-Aas T, Oughton DH, Jaworska A, Brunborg G.
Mutagenesis. 2011 Nov;26(6):783-93.