Rivers all over the world have a history of being heavily polluted by untreated waste waters from industrial and municipal discharges. Chemical analyses of priority pollutants are typically used to monitor contamination levels and are complemented by bioassay-based approaches and their modifications to serve as simple, rapid and sensitive screening systems to predict bioavailability [23
]. Changes in expression levels of single biomarkers (e.g. heat shock proteins, vitellogenins, or cytochromes P450) have been reported to reflect the toxic response and the underlying mode of action of few specific substances or environmental contaminants [24
]. The use of DNA microarrays attempts to link ecotoxicological effects with the changes in global gene expression patterns [4
]. This global approach is expected to cope more adequately with the complex matrices (such as soils and sediments) and to display the variety of potential modes of actions and strategies of the exposed organisms. Clearly, many species of invertebrates could be used to investigate transcriptional responsiveness in sediments, however two challenges need to be met, firstly few invertebrate species are omnipresent (and restricted to local habitats), and secondly none have, to date, been fully sequenced thus prohibiting the application of whole genome microarray technology. In contrast, the genome sequence of C. elegans
has been deciphered in fine detail, clearly representing an added value to comprehensive ecotoxicogenomics. Although C. elegans
is primarily a soil nematode, it has also been isolated from freshwater habitats (such as sediments) [25
]. Given that its natural habitat is the interstitial space between particles filled with water, the exposure to contaminants is similar in soils and sediments, thus making C. elegans
a suitable test organism for toxicity tests for both habitats. In the laboratory, the use of C. elegans
has been validated for single compounds, such as steroid hormones, PCB52, Cd, DEHP, as well as natural mixtures, such as humic substances [9
]. However, due to differences in experimental setups, e.g. the use of mixed populations vs. synchronized cultures or varying exposure times, the systematic and sound comparison between experiments is challenging and highlights, if anything, the need to streamline ecotoxicogenomic experiments. Of course, the use of C. elegans
in sediment toxicity is not a new concept introduced by us, but supported by numerous publications in classical sediment toxicity testing [6
], which have been instrumental for the implementation of the first standardized sediments toxicity test (ISO/DIS 10872 Draft).
The chemical analyses and three bioassays (reproduction, estrogenicity, genotoxicity) showed a clear pollution and toxicity gradient, with Elbe sediment being the most polluted characterized by elevated organic and heavy metal contamination. The YES test and comet assay identified the estrogenic and genotoxic potential of the Elbe sediment to be well above that of the Rhine and Danube sediment, respectively.
A laboratory media control (K-medium) was initially included, but deemed too artificial as subsequent PCA analysis identified the laboratory control exposures to cluster distinctly separate from the sediment exposures (see Additional file 9
) which masked the differences between the three sediments and thus diminished the robustness of GO and KEGG analyses and biologically meaningful data interpretation (data not shown). Sediments from the three geographically separated rivers are of course heterogeneous with distinct physico-chemical properties which may modulate effects (toxicities) of chemical contamination, a notion that may apply to any control sediment as well (such as the Danube). It could therefore be argued that observed effects may be a combination of chemical contamination and physio-chemical properties of sediments from different rivers. Nevertheless, for reasons of ecological relevance, and justified by the low chemical burden as well as the results from the bioassays, we selected the Danube sediment as reference for the DNA microarray study.
Of course, true DNA damaging or estrogen responsive effects of sediments are best assessed exploiting one of the many budding in vivo
techniques, including PCR based approaches to detect DNA damage in C. elegans
] or to assess changes of estrogen responsive genes in C. elegans
, such as vit-2 or vit-6 [29
]. However, the focus of this work was not to determine the definitive toxic status of the river sediments, but to observe, in a proof of principle experiment, if changes in global gene expression patterns can be used to study (and differentiate) the responses of exposure to complex sediments. Nematodes, as any other organism, exposed to contaminated environments will modulate their metabolic resources and available energy to combat the environmental insult [30
]. In line with this, we found that C. elegans
exposed to Elbe or Rhine sediments down-regulated higher-ranking GO categories which include catalytic activities, binding and metabolic processes. An analogous strategy of C. elegans
was observed by DNA microarray studies following single compound exposures to PCB52 [11
] and Cd [12
]. Interestingly, exposure to both sediments resulted in a down-regulation of several members of the starch and sucrose metabolism pathway, including the trehalase encoding genes tre-2
. The disaccharide trehalose is an invertebrate sugar transport and storage material [31
] that is present in C. elegans
at all life stages. The highest concentrations of trehalose (up to 2.3% of dry weight) are found in eggs and dauer larvae, two diapausing stages that are highly resistant to environmental stressors [32
], it is proposed to function as energy reserve and stress protectant [33
]. In contrast, the gene expression of α-amylase, whose yeast and human orthologs help to facilitate the breakdown of glycogen [34
], was found to have strongly increased. Although the Cd level of the Elbe sediment was found to be 5-fold higher than the Rhine sample, the sediment data presented here is remarkably divergent to the global gene expression pattern observed in laboratory exposures to Cadmium [12
]. This underlines how single compound exposures, though valuable in their own right, cannot model conditions of true environmental complexity. Observed effects are a sum of effects of chemical contamination and their bioavailability as well as further biotic and abiotic properties of sediments which may mask typical single compound transcript profiles. Indeed, the comparison of several gene lists resulted in only one obvious match, namely with PCB52 responsive genes, including two DNA damage checkpoint protein encoding genes (wrn-1
), which were found up-regulated in the Elbe derived sample, where the sediment was characterized by the highest measured genotoxicity and a high level amount of various POPs including different PCB compounds. In this context it is notable that the pathway involved in metabolism of xenobiotics by cytochrome P450 was induced in those nematodes exposed to the Elbe sediment, whereas the Rhine sediment failed to induce this pathway.
Besides well defined and annotated KEGG pathways, the Elbe sediment exposure resulted in the differential regulation of several related biological processes (GO categories), including development (organ, larval and genitalia development), aging (determination of adult life span) and a pool of 50 GO terms associated with reproduction. Detailed analysis of the latter GO terms will provide a detailed mechanistic and physiological/toxicological understanding of sediment toxicity and allow the linking of gene expression to higher level effects such as the observed reduction in brood size. Almost all members belonging to these categories are characterized by an abnormal developmental and/or aging phenotype (typically Age, Let, Emb, Ste, Gro, Egl, Lva; please find detailed information by referring to http://www.wormbase.org/db/misc/phenotype
), when silenced by RNAi. Given the significant estrogenicity of the Elbe sediment sample, as defined by the YES test, it is conceivable that at least some of the observed responses are due to this specific class of pollutants. Similarly, exposure to the Elbe sediment was shown to increase the aging process. This process is the determination of adult life span and provides a well-defined data record. First, genes deemed to be essential for embryogenesis and normal growth (bec-1
] or associated with an increased life-span (old-2
] were up-regulated. Second, the loss of daf-2
, found to be down-regulated, are known to promote a long life as well [39
]. Likewise, a gain of function of wrn-1
], found to be up-regulated in this experiment, has been shown to shortened life span. This pattern of regulation, and the results concerning starch and sucrose metabolism pathway, discussed above, are expected to increase nematode's life span and induce the dauer stadium, well known as a survival pathway to overcome unfavorable environmental conditions.