Increased cytotoxic and genotoxic tolerance of Eisenia fetida (Oligochaeta) to cadmium after long-term exposure.
ABSTRACT Since life-cycle studies showed that the earthworm species Eisenia fetida can develop increased tolerance after long-term exposure to a sub-lethal concentration of Cd in the laboratory, we assessed both the cytotoxicity and genotoxicity of Cd in a long-term Cd-exposed population. We exposed E. fetida specimens from this population, from a laboratory control population and from a field population to various concentrations of CdSO(4) in artificial soil water. Toxic effects were measured using the MTT test and the comet assay. The group that had been exposed to Cd for more than a decade was found to be more tolerant to the deleterious effects of Cd at both cellular and molecular levels than the laboratory control population. The field population, which came from a severely metal polluted environment, displayed high tolerance at molecular level as well. The results provide novel biomarker evidence of increased Cd tolerance in E. fetida, but the mechanisms supporting the apparent tolerance, still need to be clarified.
- SourceAvailable from: Anna Lankoff[show abstract] [hide abstract]
ABSTRACT: The single-cell gel electrophoresis, also known as the comet assay, has gained wide-spread popularity as a simple and reliable method to measure genotoxic and cytotoxic effects of physical and chemical agents as well as kinetics of DNA repair. Cells are generally stained with fluorescent dyes. The analysis of comets--damaged cells which form a typical comet-shaped pattern--is greatly facilitated by the use of a computer image-analysis program. Although several image-analysis programs are available commercially, they are expensive and their source codes are not provided. For Macintosh computers a cost-free public domain macro is available on the Internet. No ready for use, cost-free program exists for the PC platform. We have, therefore, developed such a public domain program under the GNU license for PC computers. The program is called CASP and can be run on a variety of hardware and software platforms. Its practical merit was tested on human lymphocytes exposed to gamma-rays and found to yield reproducible results. The binaries for Windows 95 and Linux, together with the source code can be obtained from: http://www.casp.of.pl.Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 02/2003; 534(1-2):15-20. · 3.90 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The purpose of this study was to determine if metallothioneins are present in the aquatic oligochaete Limnodrilus udekemianus and to determine the interplay between the presence of these proteins, cadmium (Cd) exposure, and Cd toxicity. The latter was geared specifically towards evaluating the role of metallothionein as a homeostatic mechanism against Cd toxicity. These issues are important for evaluating the usefulness of the quantification of metallothioneins as a biomonitoring tool. Worms in sediment were exposed to Cd under static conditions, with Cd initially added to the aqueous phase. Survival was monitored while respiration (as a measure of sublethal Cd effects) was determined immediately following exposure. Metallothioneins were separated from the cytosol by gel permeation high performance liquid chromatography (HPLC) while Cd levels were quantified in whole worms, cytosol and cytosolic fractions. Also, a Cd-saturation assay was used to determine the amounts of Cd bound to metallothionein and the total Cd-binding capacity of the metallothionein. Limnodrilus udekemianus has a metallothionein-like protein (an inducible cytosolic protein with an apparent molecular weight of approximately 15 kD that binds high levels of Cd and shows a red shift upon Cd binding). Sediment Cd levels above 60 microg/g were lethal to the worms (in 8-day exposures). Respiration rates at 13 and 41 microg/g Cd were not significantly different from controls, though cytosolic Cd levels were substantially increased in the 41 microg/g exposure. In this latter cytosol, Cd levels were significantly elevated in the low molecular weight pool (which includes metallothioneins) but not in the other pools, while the Cd-saturation assay also showed that worms in this group had significantly elevated levels of metallothionein-bound Cd. However, in all treatments the metallothionein was far from saturated by Cd. These observations indicate that no 'spill-over' of Cd was evident as lethal levels of Cd were approached. The overall cytosolic Cd distribution, and the degree of metallothionein saturation in Limnodrilus udekemianus thus do not appear to be good predictors of Cd toxicity in this species.Environmental Pollution 10/1999; 106(3):381-9. · 3.73 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Earthworms have been shown to accumulate trace elements in general, and particularly high amounts of metal ions such as cadmium, copper and zinc. The earthworm's response to metal contamination has been linked to the induction and expression of metallothionein (MT) proteins, a detoxification strategy analogous to that found in other biological systems. The present study focuses on an inducible Cd-MT isolated from the compost-dwelling brandling worm Eisenia foetida (Savigny). A full characterization of the protein (including protein induction, MT cDNA, amino-acid sequence and metal stoichiometry) revealed a new dimension of knowledge to the molecular genetic information available to date. Whereas the elucidated cDNA codes for a putative protein which possesses 80 amino-acid residues, the characterized protein bears only 41 amino acids. The isolated product has evidently attained its size and shape by cleavage near the N-terminal site and at the linker region between the two putative metal-binding domains of the translated product, yielding a small MT moiety which contains 12 Cys residues (including one triple Cys-motif) binding four cadmium ions. It can be shown that the isolated MT molecule represents a self-sufficient one-domain MT which is stable in vitro. The isolation of the single-domain MT peptide raises the question about the method of formation and significance in vivo of such small MT moieties from tissues of E. foetida and possibly other terrestrial invertebrates. In this respect, two hypotheses are discussed: firstly, the possibility of formation of small MT peptides due to enzymatic cleavage of the intact protein during the process of preparation and isolation; and secondly, the possibility of deliberate post-translational processing of the translated gene product to yield functional one-domain MT moieties.European Journal of Biochemistry 02/2000; 267(2):573-82. · 3.58 Impact Factor