To determine the optimal conditions that affect cadmium-responsive transcription, quantitative real-time PCR (qRT-PCR) was performed to assess the effects of different cadmium concentrations and exposure times on the expression of selected stress-responsive genes. The level of expression of three C. elegans cadmium-responsive genes, cdr-1, mtl-1 and mtl-2, significantly increased at all cadmium concentrations following a 24 h exposure (Figure 1a). However, the levels of expression of the two general stress-responsive genes, gst-38 and hsp-70, were induced only at concentrations greater than 50 μM (Figure 1a).

The time course of gene induction in response to 100 μM cadmium was also examined. The expression of cadmium-responsive genes was maximally induced after only 4 h; in contrast, the general stress-responsive gene gst-38 reached its highest level of expression after 24 h (Figure 1b). The C. elegans homolog of human jun, T24H10.7, did not respond significantly to cadmium exposure. Based on these results, subsequent microarray experiments were performed using C. elegans exposed to 100 μM cadmium for 4 and 24 h.

There were 37 and 95 genes significantly up-regulated after 4 h and 24 h exposures to cadmium, respectively; 6 genes were significantly down-regulated following a 24 h exposure (fold-change ≥2, p < 10^-6) (Table 1, Figure 2). The genes whose levels of expression significantly changed were clustered into three groups: group 1, early response genes; group 2, late response genes; and group 3, down-regulated genes. The early response group includes cdr-1, mtl-1, mtl-2, and phase I and phase II metabolism genes. The levels of expression from the microarray study of cdr-1, mtl-1, mtl-2 and gst-38 were consistent with the qRT-PCR results.

Microarray results were analyzed to identify the biological processes and molecular functions that are affected by cadmium exposure. To extend the scope of the analysis, 290 genes, whose expression levels were changed by cadmium by at least 1.5-fold (p < 0.001) following either 4 h or 24 h exposure, were used in the analysis (237 up-regulated and 53 down-regulated; Additional data file 2). Gene Ontology analysis indicated that C. elegans metabolic and localization pathways, which regulate establishment of localization and transportation of different chemical species (especially metal ions), were significantly enriched (p < 0.05) with up-regulated genes following the 4 h exposure (Figure 3, Additional data file 3). After a 24 h exposure, metabolic and localization pathways were enriched with both up- and down-regulated genes. Additional pathways that were overpopulated with down-regulated genes included fatty acid metabolism, cellular lipid metabolism and cell wall catabolism. Proteolysis pathways were enriched with up-regulated genes following a 24 h exposure, suggesting that increased protein degradation may occur after prolonged exposure to cadmium. The molecular functions enriched with over-expressed genes after both 4 h and 24 h exposures were catalytic activity and binding activities to many ion species, which agrees well with the results of the biological processes analysis (Figure 3, Additional data file 4).

Although GO analysis provides an overall understanding of the global transcription profile, current C. elegans GO data are not sufficient to predict the functions of all of the cadmium-responsive genes. Several C. elegans cadmium-regulated genes have been mapped to known metabolic pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database 18 (Table 2). Among these are four cytochrome P450 genes, which are involved in metabolism of both endogenous and exogenous compounds; gene W01A11.1, which is involved in degradation of tetrachloraethene; and gst-38, which is involved in phase II metabolism. The majority of the cadmium-responsive genes, however, are novel and have not been assigned GO categories or mapped to biochemical pathways. Of the 290 genes whose expression significantly changed following a 24 h cadmium exposure, only 83 (29%) have been assigned biological process GO terms. Similarly, only 109 (38%) of the genes following a 24 h exposure have been assigned molecular function GO terms (Additional data file 1).

Of the 53 down-regulated genes (≥1.5-fold), 8 have previously reported RNAi phenotypes, including embryonic lethality, slow growth, larval growth arrest, and sterility (Table 3). This suggests that the suppression of the expression of these genes by cadmium may adversely effect embryonic development, growth or reproduction. Several of the up-regulated genes also have previously reported phenotypes, such as F57B9.3 (embryonic lethal, larval arrest) and cyp-13A4 (locomotion abnormal, slow growth) 19. The biological consequence of changes in expression of the majority of the genes, and their roles in the defense against cadmium toxicity, however, are unknown. To investigate the relationship between cadmium-induced gene expression and resistance to cadmium toxicity, the effects of inhibiting the expression of the cadmium-responsive genes in the presence or absence of cadmium on C. elegans growth were determined.

The expression of 92 cadmium-responsive genes, which were induced by cadmium (≥1.5-fold), was inhibited by RNAi in the presence of four different cadmium concentrations in an mtl-2 null background. In RNAi control animals, slow growth and uncoordinated movement were observed after cadmium exposure. Morphological changes (protruding vulva, multivulva) were occasionally observed at higher cadmium concentrations (100 and 200 μM). Lethality was not observed under any experimental condition. RNAi-mediated inhibition of 50 of the 92 genes tested resulted in slower growth in the presence of cadmium, compared to the RNAi control in the same treatment group (visual observation under microscope; Additional data file 5). The only gene that exhibited a morphological phenotype when inhibited by RNAi in the absence of cadmium was F57B9.3, which encodes a translation initiation related protein. As described previously 1920, inhibition of F57B9.3 caused embryonic lethality and L1 larval arrest.

To confirm and quantify the effect of the 50 cadmium-responsive genes that affected nematode growth in the presence of cadmium, we repeated the RNAi-mediated inhibition of these genes in the presence of 100 μM cadmium and measured nematode body length (as a measure of growth/development) using the COPAS Biosort 21. Inhibiting the expression of these genes resulted in different degrees of slow growth in the presence of cadmium, compared to the RNAi control in the same cadmium treatment group (Figure 4, Additional data file 6). Based on the changes in cadmium sensitivity caused by RNAi, the genes were grouped into three classes, strong, medium and weak protective effects against cadmium toxicity (Additional data file 6). Several of the genes in the strong and medium category (cdr-1, ttm-1, mtl-2, and mtl-1) have been previously reported to be involved in cadmium detoxification 172223. However, the majority of the genes have not been shown to be involved in resistance to metal toxicity. GO molecular function analysis indicated that many of these genes have metal ion binding and catalytic activities (Table 4).

The genes that had the strongest protective effects against cadmium toxicity in the growth assay were also examined for an effect on C. elegans reproduction. RNAi of cyp-13A4 or thn-1 resulted in a significant decrease in the number of C. elegans offspring when nematodes were exposed to cadmium, compared to the RNAi control in the same cadmium treatment group. RNAi did not significantly affect reproduction in the remainder of the tested genes (Table 5).

In order to further define the molecular mechanisms of the cadmium defense response, the program Cytoscape was used in protein interaction analysis to identify potential regulatory pathways 24. C. elegans has a relatively small interaction database (approximately 3,000 proteins and approximately 5,000 interactions) 25. A larger data set of predicted interactions in C. elegans, based on data from Drosophila and Saccharomyces interlogs, was recently released 26. We merged the two data sets into an interaction network designated WI_combined. Of the 290 cadmium-responsive genes, 49 were mapped to the interaction network, including 6 genes that were functionally important in cadmium defense response, as identified in the RNAi analysis (Figure 5a). Among these functional local networks, Y46G5A.24, which encodes a β,β-carotene 15,15'-dioxygenase like protein, was highly cadmium-inducible and inhibition of this gene by RNAi resulted in hypersensitivity to cadmium (Table 1, Figure 4). Two proteins that interact with Y46G5A.24, KEL-8 and BRP-1, are themselves centers of other interactions.

KEL-8 can interact with several proteins, including MEK-1, PMK-1, MPK-1 and MKK-4, which are components of the mitogen-activated protein kinase (MAPK) pathway (Figure 5b). The MAPK pathway is involved in the C. elegans heavy metal response 2728. Inhibiting the expression of Y46G5A.24 or kel-8 by RNAi resulted in enhanced sensitivity to cadmium exposure in wild-type and mtl-2 mutant C. elegans (Figure 6a). This suggests that both Y46G5A.24 and kel-8 can protect C. elegans from cadmium toxicity. The mek-1 mutant alone was slightly more sensitive to cadmium than wild-type nematodes. However, inhibition of kel-8 in mek-1 null background did not cause hypersensitivity to cadmium compared to mek-1 mutant alone, suggesting that the protective function of kel-8 against cadmium toxicity depends on the normal function of mek-1 (Figure 6a).

We also tested the function of brp-1, the other gene that was shown to interact with Y46G5A.24 brp-1 mutant nematodes showed a similar response to cadmium as wild-type nematodes, implying that brp-1 is not involved in the response to cadmium (Figure 6b).

Microarray technology has been used to examine the effects of cadmium exposure in a variety of organisms 29303132. Although cadmium-regulated gene expression has been documented, in <