Comet Assay measurements: a perspective.

The Comet Assay or single cell gel electrophoresis assay is one of the very widely used assays to microscopically detect DNA damage at the level of a single cell. The determination of damage is carried out either through visual scoring of cells (after classification into different categories on the basis of tail length and shape) or by using different commercially available or public domain software (which automatically recognise the extent of damage). In this assay, the shape, size and amount of DNA within the 'comet' play important roles in the determination of the level of damage. The use of a software in particular also provides a range of different parameters, many of which might not be relevant in determining the extent of DNA damage. As a large number of factors could influence the shape, size, identification and determination of induced damage, which includes the scoring criteria, staining techniques, selection of parameters (whilst using the software packages) and appearance of 'hedgehog' or 'clouds', this article aims (a) to provide an overview of evolution of measurements of DNA damage using the Comet Assay and (b) to summarise and critically analyse the advantages and disadvantages of different approaches currently being adopted whilst using this assay. It is suggested that judicious selection of different parameters, staining methods along with inter-laboratory validation and harmonisation of methodologies will further help in making this assay more robust and widely acceptable for scientific as well as regulatory studies.

Epithelial thickness and lipid droplets in the hepatopancreas of Porcellio scaber (Crustacea: Isopoda) in different physiological conditions.

We investigated the morphometric characteristics of the hepatopancreatic epithelium in the terrestrial isopod Porcellio scaber during acclimatization to laboratory conditions, during the daily cycle, the molt cycle, and fasting. The hepatopancreatic epithelium was analyzed using computer-assisted microscopy of serial sections of the hepatopancreatic tubes. In addition, the abundance, the distribution, and the size of lipid droplets in the hepatopancreatic epithelium were recorded. The experimental animals were collected in the field and transferred to the laboratory. The hepatopancreatic epithelium was thinner and lipid droplets reduced after 2 months of acclimatization to laboratory conditions. The daily cycle and the molt cycle affected neither the epithelial thickness nor the abundance of lipid droplets. But in animals fasted for 2 weeks, these two parameters were significantly reduced. Based on both the epithelial thickness and the abundance of lipid droplets in B cells, we propose criteria for estimating the stress status of the animals. With the possibility to determine the stress status, many studies on isopods gain in relevance.

Natural revegetation in the vicinity of the former lead smelter in Zerjav, Slovenia.

The response of plant communities to pollution associated with the lead smelter in Zerjav, Slovenia, was investigated on spatial and temporal scales. In 2001, the total concentrations of contaminating metals in the soil measured at the most polluted plot were 59000 mg kg(-1) Pb, 180 mg kg(-1) Cd, and 3300 mg kg(-1) Zn. A negative correlation between the concentration of metals in the soil and plant biodiversity parameters along the gradient of pollution in 2001 was detected. Plant species lists were compiled in 2001 for plots located at different distances from the emission source and compared to that of 1981. In the period from 1981 to 2001, smelter emissions were reduced, and plant species richness increased at all examined plots. Among the successful survivals were some metal hyperaccumulators (Minuartia gerardii, Thlaspi praecox, and Biscutella laevigata). Of special interest were plants that survived the period of highest pollution. We believe that these species can be used in metal-degraded environments for natural revegetation to immobilize heavy metals. The ecosystem in the surroundings of the former smelter is presently recovering. Our results suggest that high metal concentrations in soil are a potential limiting factor for revegetation.