Plasticity of animal genome architecture unmasked by rapid evolution of a pelagic tunicate

Genomes of animals as different as sponges and humans show conservation of global architecture. Here we show that multiple genomic features including transposon diversity, developmental gene repertoire, physical gene order, and intron-exon organization are shattered in the tunicate Oikopleura, belonging to the sister group of vertebrates and retaining chordate morphology. Ancestral architecture of animal genomes can be deeply modified and may therefore be largely nonadaptive. This rapidly evolving animal lineage thus offers unique perspectives on the level of genome plasticity. It also illuminates issues as fundamental as the mechanisms of intron gain.     

Membrane Dialysis Extraction (MDE): A Novel Approach for Extracting Toxicologically Relevant Hydrophobic Organic Compounds from Soils and Sediments for Assessment in Biotests

Goal, Scope and Background   Organic solvents are routinely used to extract toxicants from polluted soils and sediments prior to chemical analysis or bioassay. Conventional extraction methods often require the use of heated organic solvents, in some cases under high pressure. These conditions can result in loss of volatile compounds from the sample and the degradation of thermally labile target analytes. Moreover, extracts of soils and sediments also frequently contain substantial quantities of organic macromolecules which can act as sorbing phases for target analytes and in doing so interfere with both chemical analysis and bioassays. Membrane dialysis extraction (MDE) is described as a simple, passive extraction method for selectively extracting toxicologically relevant hydrophobic organic compounds (HOCs) from polluted soils and sediments and anaylzed for its applicability in ecotoxicological investigations. Methods   Toxicologically relevant hydrophobic organic compounds were extracted from wet and dry sediments by sealing replicate samples in individual lengths of pre-cleaned low-density polyethylene (LD-PE) tubing and then dialysing in n-hexane. The efficacy of the MDE method for use in ecotoxicological investigations was assessed by testing the concentrated extracts in the neutral red assay for acute cytotoxicity, in the EROD assay for the presence of dioxin-like compounds and in the Danio rerio fish egg assay for embryotoxic and teratogenic effects. Conditions of the sediment sample (with or without water content), dialysis membrane length and duration of dialysis were analyzed with respect to their impact on three endpoints. Results of the MDE investigations were compared to data obtained in samples prepared using conventional Soxhlet extraction. Results and Discussion   The membrane dialysis extraction was found to be at least as efficient as Soxhlet methodology to extract toxicologically relevant HOCs from sediment samples. In most cases, MDE-derived extracts showed a higher toxicological potential than the Soxhlet extracts. Lack of any significant effects in any MDE controls indicated these differences were not caused by contamination of the LD-PE membrane used. The elevated toxicological potential of MDE extracts is most likely the result of enhanced bioavailability of toxic compounds in consequence of lower amounts of organic macromolecules (i.e. sorbing phases) in the MDE extracts. This effect is probably the result of a size-selective restriction by the LD-PE membrane. Conclusion   Membrane dialysis extraction was found to be a simple, efficient and cost-effective method for the extraction of sediment samples. MDE can be used to extract toxicologically relevant hydrophobic organic compounds from both wet and dry sediments without the risk of loosing volatile and thermally labile target analytes. The size-selectivity of the LD-PE membrane also appears to have the capacity to increase the bioavailablity of potential target analytes in the resulting extracts by retaining much of the organic macromolecules present in the sample. Thus, results suggest that MDE may be particularly useful for the extraction of toxicologically relevant hydrophobic organic compounds from soils and sediments for bioassays and other ecotoxicological investigations. Recommendation and Perspective   Further validation of MDE has been initiated and the applicability of the methodology to other sample types will be investigated. Of particular interest is the potential application of MDE to recover hydrophobic target analytes from biological samples such as muscle, other soft tissues and blood.     

Comparative genotoxicity testing of rhine river sediment extracts using the comet assay with permanent fish cell lines (rtg-2 and rtl-w1) and the ames test*

Goals, Scope and Background

Improved quality of surface waters and sediments requires advanced strategies for ecotoxicological assessment. Whilst at least in Germany assessment strategies on the basis of chemical analysis and acute toxicity data dominated the last decades, the development of more specific biological endpoints and biomarkers in ecotoxicology is required in order to arrive at a good ecological potential and good chemical status of surface waters in the European river basins until the year 2015, as required by the European Water Framework Directive. Since sediments have for long been known to function both as a sink and as a source of pollutants in aquatic systems, and since part of the particle-associated substances have frequently been demonstrated to cause mutagenic and carcinogenic effects in aquatic organisms, particularly in fish, there is, among other requirements, an urgent need to develop, standardize and implement integrated vertebrate-based test systems addressing genotoxicity into recent sediment investigation strategies. Thus, the present study was designed to compare the suitability of two commonly used test systems, the comet assay and the Ames test, for the evaluation of the ecotoxicological burden of surface and core sediment samples from the river Rhine.

Methods (or Main Features)

In order to determine the importance of inherent enzymatic activities, two permanent fish cell lines with different biotransformation capacities, RTL-W1 and RTG-2, were compared with respect to their capability of detecting genotoxic effects in 18 surface and core sediment samples from 9 locations along the River Rhine in the comet assay with and without exogenous bioactivation. For further comparison, as a prokaryotic mutagenicity assay, theSalmonella plate incorporation assay (Ames test) with the test strains TA98 and TA 100 with and without exogenous metabolic activation was used.

Results and Discussion

Whereas all sediment extracts induced genotoxic effects in the comet assay with RTL-W1 cells, only 12 out of 18 sediment extracts revealed significant genotoxicity in the tests with the less biotransformation-competent RTG-2 cells. Exogenous bioactivation by addition of ß-naphthoflavone /phenobarbital-induced S9 from rat liver resulted in both reduction or increase of genotoxicity in samples from different sites, however, without consistent reaction patterns. In general, the responses of RTL-W1 cells indicated higher biotransformation capacity than in RTG-2 cells without S9 complementation. In Ames tests using TA98 with S9, 16 out of 18 extracts induced significant mutagenicity with induction factors up to 4. Compared to TA98, the strain TA100 proved less sensitive, with maximum induction factors of 1.3, indicating the potential presence of substances inducing frarneshift mutations, which can only be detected in the strain TA98. Chemical analyses revealed particularly high levels of hexachlorbenzene (up to 860 µg/kg) and priority PAHs (up to 4.8 mg/kg); so far, however, no correlation could be found between compounds analyzed and the corresponding biotests.

Conclusions

Results document that both comet assay and Ames test are capable of detecting xenobiotic interaction with DNA in consequence of exposure to complex environmental samples. Whereas the alkaline version of the comet assay detects a broad range of interactions with the DNA, however without information about their eventual importance, the Ames test only reveals established mutations, but fails to detect transient (reparable) DNA alterations. However, even transient primary changes in the DNA structure might result in carcinogenic processes and, eventually, in implications at the population level. As a consequence, for hazard assessment purposes, a combination of both assays is required to avoid false negatives in genotoxicity evaluation. Poor correlation between data obtained by chemical analysis and results in bioassays is indicative of our limited understanding of the sources of genotoxicity. In fact, numerous studies combining chemical and biological approaches for hazard assessment of complex environmental mixtures indicate that priority pollutant concentrations are a poor indicator of toxicity.If compared to the cell line RTG-2, RTL-W1 proved more effective in detecting genotoxicity in surface sediment samples and, thus, indicated the importance of bioactivation of at least part of the compounds in superficial layers of sediments. Results further document that the common assumption may be wrong that, in comparison to deeper strata, surface layers carry a lower toxic burden in consequence of the current decrease in water pollution. This might at least in part be due to remobilization of more heavily polluted sediments from deeper layers during severe flood events followed by re-sedimentation in flood plains or upstream weirs, where they might cover less polluted younger sediment layers.

Recommendations and Perspectives

For a comprehensive assessment of genotoxicity in surface and core sediments, a combination of eukaryotic (comet assay) and prokaryotic assays (Ames test) with and without exogenous bioactivation is recommended. Since studies with organic sediments extracts simulate a worst-case scenario and fail to take into account bioavailability, there is broad consensus that whole-sediment exposure protocols represent the most realistic scenarios. Whereas more realistic solid phase exposure has frequently been applied in both microbial and invertebrate acute toxicity testing, there is an urgent need to develop corresponding whole sediment fish-based genotoxicity tests.