The current microarray analysis suggests that chemically dispersed oil has lower transcriptomic effect on larvae of Atlantic cod than mechanically dispersed oil, based on the number of significantly affected transcripts and fold changes of a few transcripts. In this work we link the magnitude of transcriptional response to toxicity, especially for well-established biomarkers of mode of action of hydrocarbons such as the cytochrome P450 genes, even though we have not examined higher-level toxicity endpoints. Increasing knowledge, for example publications included in the Comparative Toxicogenomic Database (http://ctdbase.org), suggests this to be a valid assumption for transcriptional responses. Earlier studies suggest similar toxicity of chemically and mechanically dispersed oil in invertebrates and fishes [5, 15, 16], or more toxic effects of mechanically dispersed oil than of chemically dispersed oil on copepods and fish . Clark et al.  showed for several organisms that the dispersants themselves did not alter the toxicity of oils, demonstrated by similar LC50 values for both chemically and mechanically dispersed crude oil. A similar finding was reported by Ramachandran et al. , who showed that the dispersant Corexit 9500 did not induce cyp1a in juvenile rainbow trout (Oncorhynchus mykiss). EPA has evaluated the contribution of dispersants on oil toxicity on shrimps and fish, including Corexit 9500A, which was used in the Gulf of Mexico 2010 incident, but were not able to see a universal trend . By reducing the size of the oil droplets and increasing the aromatic hydrocarbon concentration, one would suspect that the dispersed fraction is more bioavailable to fish for accumulation via the gills and oral uptake . However, conflicting evidence exists as to whether dispersed oil is more toxic than crude oil or untreated water-accommodated fraction of oil to fish. For example, Van Scoy et al.  showed that dispersant application significantly decreased hydrocarbon potency in Chinook salmon (Oncorhynchus tshawytscha) pre-smolts, whereas many studies suggest that the oil droplet fractions of oil dispersions increase the bioavailability and thereby the mechanism of toxicity of compounds of crude oil in fishes [18, 21–23] or have only moderate effects on fish [14, 24]. With a fold change cut-off of 1.5 and p<0.05, mechanically dispersed oil produced a much longer list of significantly affected transcripts than chemically dispersed oil. By comparing the significantly affected transcripts in larvae from the CDH and MDH exposure groups with the control in a PCA plot it also appears that mechanically dispersed oil is more toxic than chemically dispersed oil. One possible explanation for this finding is that the dispersant might have changed the characteristics of the oil droplets in a way that i) the dissolution rates of oil components into the water phase is lowered or ii) that the "stickiness" of oil droplet on fish larvae or rotifier (used as food for the larvae) surfaces is reduced. Since we obtained relatively comparable treatments in terms of oil concentrations, and the transcriptional effects are more pronounced for the mechanically dispersed oil than for the chemically dispersed oil, it is possible that the properties of the chemical dispersant decreases the exposure of cod larvae to oil components either through reduced dissolution of oil components, by reducing oil droplet fouling of cod larvae and/or reducing the uptake of oil droplets through food.
According to the microarray data, transcripts encoding cytochrome P450 system proteins were most strongly affected by the oil dispersions. Cyp1a1 (or cyp1a3, equally matched), the transcripts showing the highest induction, was most severely affected in larvae in the MDH treatment group. This result is in line with numerous previous studies showing that CYP1A is easily induced in fish via the aryl hydrocarbon receptor (AHR) by components in the oil [25, 26]. The induction of fish liver CYP1A has often been used as a molecular biomarker for exposure to petroleum hydrocarbons . Several components of the crude oil can induce CYP1A, which is largely responsible for metabolism of PAHs and a variety of other toxic compounds [27–29]. Significantly elevated levels of cyp1a following exposure to the two oil dispersions were also determined by the RT-qPCR analyses. However, the more specific RT-qPCR analyses did not confirm that mechanically dispersed oil was more toxic based on the transcriptional levels of cyp1a, neither in the low, medium or high-concentration exposure larval groups. Instead they suggested that cyp1a was about 60-fold up-regulated by both types of oil dispersions. In a recent study in which cod larvae were exposed to dispersed oil or to the water-soluble fraction of oil (WSF), we observed a stronger induction of cyp1a in terms of fold change . The relative levels of induction were greater following exposure to the dispersed oil, with a 300-fold up-regulation in the high-exposure group, compared to a 237-fold up-regulation in the high-exposure WSF group as suggested with the RT-qPCR data . The reason for the lower induction levels of cyp1a transcription observed in the current study is unknown.
Interestingly, the three CYP1 transcripts quantified with RT-qPCR in the current study showed a different level of induction, with cyp1a1, cyp1b1 and cyp1c1 being 65, 12 and 8-fold up-regulated in larvae from the CDH group and 61, 10, and 8-fold up-regulated in larvae from the MDH group. Based on the microarray sequences used to design our PCR primers, the cyp1a1 assay matched equally well against cyp1a3 with BlastX searches, while the cyp1c1 assay matched almost equally against cyp1c2, suggesting that more research are needed into the transcription of the different CYP1 genes and organ-specific function of their encoded proteins in cod.
In addition to the CYP1 genes, the aryl hydrocarbon receptor repressor (ahrr) transcript was also up-regulated in cod larvae for the high-exposure groups. The protein encoded by the ahrr transcript participates in the AHR signaling cascade, and is involved in regulation of cell growth and differentiation (GeneCards). AHRR represses the transcription of CYP1A1 by binding to the xenobiotic response element (XRE) sequence present in the promoter regulatory region of variety of genes. AHRR acts by recruiting ankyrin repeat, family A (RFXANK-like), 2 (ANKRA2), HDAC4 and/or HDAC5 to repress CYP1A1 in mammals (GeneCards). Several transcripts annotated to ankyrin genes were also up-regulated in cod larvae from the high-exposure groups, among them ankyrin repeat and btb domain containing 1 (abtb1). Histone deacetylase 1 (hdac1) was significantly down-regulated in larvae from both the CDH and MDH groups, while histone deacetylase 5 (hdac5) was significantly up-regulated in larvae from the MDH exposure group. These results suggest that both cyp1a1 and ahrr mRNA inducibility is part of a mechanistic basis for resistance of fish larvae against compounds in dispersed oil, explaining the simultaneous induction of cyp1a1 and ahrr mRNA. A similar finding has been reported for Atlantic tomcod (Microgadus tomcod), with a positive correlation between ahrr and cyp1a1 mRNA levels in fish exposed to AH-responsive compounds . Another explanation for this finding could also be that the dispersed oil mediated different effects in different organs, e.g. strong induction of cyp1a1 transcription via AHR activation by aromatic hydrocarbons in liver, and effects via other mechanisms on ahrr transcription in other tissues. Organ-specific mechanisms cannot be studied in pooled whole larvae, representing a methodological limitation of using RNA from whole fish larvae for microarray examinations.
Mechanistic effects of contaminants can be studied with a number of tools. In this study we chose to use gene set enrichment analysis (GSEA) and pathway analysis with the Ingenuity Pathways Analysis (IPA) system. The GSEA data suggest that the two oil dispersions partly affected different cellular mechanisms, with several gene sets suggesting an effect on the proteasome complex. As part of the ubiquitin protein degradation system, the ubiquitin-protein ligases target specific proteins for ubiquitin-mediated proteolysis, and some of these genes potentially have a role in regulation of cell proliferation or differentiation (GeneCards). Components in the oil dispersions may therefore affect protein folding, and thereby activating ubiquitin-mediated proteolysis of misfolded proteins . Comparing the two high-exposure groups CDH and MDH, in addition to the mentioned effect on the proteasome complex, the main difference between them seems to be that chemically dispersed oil specifically affected nucleosome assembly and DNA methylation by up-regulation of transcripts involved in these mechanisms, while mechanically dispersed oil mediated a down-regulation of the same gene sets. The mechanistic basis for this response is unclear, but this finding suggests that compounds in oil dispersions may affect epigenetic mechanisms in the developing cod larvae. Chromatin remodeling and altered DNA methyltransferase activity are key components of epigenetic regulation of gene expression, and these effects of dispersed oil should be studied more closely in follow-up investigations.
According to the IPA-Tox data, it appears that the oil dispersions have affected many well-known toxicological mechanisms, including aryl hydrocarbon receptor signaling, acute phase response proteins, oxidative stress, cell proliferation and nuclear receptor mediated responses. All of these represent well-known effects of toxic compounds in crude oil such as PAHs. Using a broader approach as shown in Additional files 4 and 5, the IPA Core Analyses suggest that chemically and mechanically dispersed oil share many of the top networks. Even when looking at transcripts that were uniquely affected in larvae from the different exposure groups, the data suggest a relatively similar mode of action in both exposure groups. As shown in Figures 4 and 5, transcripts common for the CDH and MDH groups, suggest that the dispersed oil mainly affected genes involved in DNA replication, recombination, and repair.