Early transcriptional responses to uranium
The present study focused on a combination of classical statistical treatment and functional enrichment analysis followed by customized identification of potential protein-protein interactions, toxicity and canonical pathways on the basis of mapping to mammalian orthologs. The protein-protein interactions, toxicity and canonical pathway analyses were considered to be exploratory, as the mammalian pathways may not always represent the same functions as that in fish. However, the correspondence between the present data and existing knowledge on stress responses in fish after U exposure provide confidence that the approach chosen herein may serve as a platform to guide future investigations. The major findings will be summarized and discussed below.
Mitochondrion electron transport chain
The analysis of global transcriptional changes and associated functional interpretation in the present study suggested that the mitochondrion, which plays a central role in the production of adenosine-5’-triphosphate (ATP) in oxidative phosphorylation (OXPHOS), may be one of the main targets of U hepatotoxicity in Atlantic salmon after short-term exposure. This was first revealed by GO-based functional analysis of DEGs associated with the mitochondrion as a cellular target, and further confirmed by pathway analyses where mitochondrial dysfunction, increases depolarization of mitochondria and mitochondrial membrane, and swelling of mitochondria were found to be affected by all U concentrations. The mitochondrial dysfunction pathway (Additional file 2: Figure S1) showed that several encoding genes of major protein complexes in the mitochondrial electron transport chain (ETC), such as NADH dehydrogenase (ubiquinone) Fe-S protein 2 (NDUFS2, Complex I), succinate dehydrogenase complex subunit D (SDHD, complex II), ubiquinol-cytochrome c reductase (UQCR11, complex III) and cytochrome c oxidase subunit VI b polypeptide 1 (COX6B1, complex IV), were found to be up-regulated, indicating potentially elevated ETC activity after U exposure. The pathway analysis did not identify genes in the complex V being affected, probably due to the lack of successful mapping of salmon DEGs towards mammalian orthologs. Indeed, two unmapped salmon DEGs which have important functions in complex V were induced by U, including ATP synthase H+ transporting mitochondrial F0 complex subunit F6 (ATP5J), which was regulated by all U concentrations, and complex V subunit ATP synthase H+ transporting mitochondrial F1 complex beta polypeptide (ATP5B), which was regulated by medium U treatment. These findings supported the hypothesis that core components of the mitochondrial ETC were likely affected by U. The mitochondrion ETC activity and associated ATP synthesis may have been modulated by U after 48 h exposure. This was also supported by the result from GO-based functional analysis that generation of precursor metabolites and energy was found to be the top overrepresented GO biological process affected by U. In addition, the eukaryotic citric acid (TCA) cycle II as a main part of the nutrient metabolic processes in the aerobic respiration was identified to be one of the highly enriched canonical pathways, indicating potential effects of U on energy metabolism in fish. These findings were in agreement with that reported by a few previous fish studies. Work by Lerebours and co-workers  reported that genes involved in the mitochondrial metabolism such as cytochrome c oxidase subunit I (COX-I) and mitochondrial ATP synthase subunit b (ATP5f1) were induced by low concentrations (23 and 130 μg/L) of U during a 28 d exposure in zebrafish. Lerebours and colleagues  further measured the mitochondrial energetic metabolism in the skeletal muscles and brain of zebrafish exposed to 30 and 100 μg/L of U for 28 d and found that the basal respiration rate was increased in the brain at day 10 and in the muscles at day 28 with a few proteins involved in the ETC being up-regulated, such as COX-IV (brain) and COX-I (muscle).
The mitochondrial membrane potential (MMP), a driving force for the operation of ATP synthase (complex V) to produce ATP, is dependent on the ETC activity . The present study identified two toxicity pathways involved in the depolarization of mitochondria and mitochondrial membrane. One of them was related to the increase in the MMP and the other related to the reduction of the MMP. These toxicity pathways were supported by different sets of DEGs, probably indicating the activation of multiple mechanisms related to potentially decreased MMP after U exposure. In fact, dissipation of MMP by U has been reported by a number of studies in mammals previously [22–26] and may also be a key mechanism of U chemical toxicity in fish. A study by Pourahmad and co-workers  found that the collapse of MMP was correlated with increased mitochondrial permeability transition (MPT) in rat hepatocyte due to the opening of mitochondrial permeability transition pore (MPTP) after U exposure. The opening of MPTP may be a consequence of the oxidation of thiol groups by U-induced ROS in the MPTP region in the mitochondrial inner membrane , or as a result of perturbed mitochondrial osmolarity, such as the alteration of intra-mitochondrial calcium level [12, 28, 29]. It has also been suggested that uranyl ions may form stable complexes with ATP by binding to the phosphate groups , thus influencing the synthesis of ATP and its cross-membrane translocation. Interestingly, several DEGs involved in these processes, such as the calcium channel voltage-dependent L type alpha 1C subunit (CACNA1C) and sodium/potassium transporting ATPase subunit beta-3 (ATP1B3) related to the calcium transportation, and adenosine diphosphate (ADP)/ATP translocase 2 (SLC25A5) related to the ADP/ATP cross-membrane transportation were found to be regulated following U exposure. The differential expression of these transporter genes may be potentially caused by disturbance of calcium and ATP homeostasis in the cells by U. Taken together, U may induce ROS, increase the ion concentration and/or interfere with ATP/ADP molecules in the mitochondria, thus causing an increase in the inner membrane MPT and dissipation of the proton gradient (i.e. MMP) by calcium overload and/or U ion accumulation on the mitochondrial inner membrane. The activation of ETC genes may therefore be a compensatory mechanism to re-establish the MMP, adjust the osmolarity in the mitochondria and overall secure the ATP production.
Oxidative stress response
The present study showed that genes encoding for antioxidant enzymes, such as peroxiredoxin 2 (PRDX2), PRDX3 and thioredoxin 2 (TXN2) were found to be up-regulated by U, indicating possible activation of antioxidant defense system. It has been commonly accepted that U may generate ROS and/or interfere with cellular redox reactions similar to other metals, such nickel, cadmium and copper , and subsequently cause oxidative stress as a key MoA [7, 15, 16, 19, 32]. Previous work by Song et al.  also documented the up-regulation of several genes involved in the antioxidant system in the liver of salmon after 48 h exposure to U, such as γ-glutamyl cysteine synthetase (GCS), glutathione reductase (GR) and glutathione peroxidase (GPx).
Since ROS can be formed following exposure to exogenous stressors such as U, or through normal biochemical processes in various cellular components such as cell membrane, mitochondria, peroxisomes and endoplasmic reticulum, clear identification of the actual source of ROS production may be challenging based on transcriptional responses. As the elevated level of mitochondrial ETC activity may also be a source of ROS production through normal physiological processes , it was likely that at least part of the oxidative stress response observed may occur in the mitochondria as a result of increased redox activities in the ETC Such hypothesis may also be supported by the identification of only limited number of DEGs related to antioxidant defense in this study. Furthermore, depolarization of mitochondrial membrane has been reported as a protective mechanism to avoid excessive ROS formation in the mitochondria in mammals [34, 35], although such causal relationship has not been confirmed in fish yet.
The swelling of mitochondria is frequently accompanied with apoptosis due to the release of cytochrome c as an initiator. Several DEGs found in this study, such as P53 and AIFM1 supporting the toxicity pathway of mitochondrial swelling, are also key regulators of apoptosis. The activation of apoptotic signaling (Additional file 2: Figure S2) supported by DEGs such as P53, AIFM1, bcl2-related myeloid cell leukemia sequence 1 (MCL1), B-cell lymphoma (Bcl) 2-associated X protein (BAX), apoptotic chromatin condensation inducer 1 (ACIN1) and spectrin alpha non-erythrocytic 1 (Fodrin) that were regulated by low and medium U, strengthened the hypothesis that apoptosis was potentially activated after short-term U exposure. Based on these DEGs, it seemed that three types of apoptotic signaling pathways may be affected, including the intrinsic apoptosis (BAX, P53), extrinsic apoptosis (MCL1) and caspase-independent apoptosis (AIFM1). However, genes regulating the outcomes of apoptotic signaling, such as ACIN1 promoted chromosome condensation and fodrin-regulated cell shrinkage and membrane blebbing were indeed repressed, probably indicating strict regulation of programmed cell death by other physiological processes as well. A previous study by the current research group  showed that caspase family genes such as BAX, Bcl-x and Caspase 6A were significantly up-regulated as an early sign of apoptosis in Atlantic salmon after 48 h exposure to U. Lerebours et al.  found that the BAX gene was 4-fold (23 μg U/L) and 10-fold (130 μg U/L) up-regulated in the liver of zebrafish after a 28 d exposure, suggesting that U exposure may cause apoptosis in fish at even lower concentrations than those tested herein.
The hypoxia-inducible factor (HIF) signaling pathway (Additional file 2: Figure S3) supported by DEGs such as P4HB, P53, HSP90AB1, NAD(P)H dehydrogenase quinone 1 (NQO1), ubiquitin-conjugating enzyme E2E 1 (UBE2E1) and SMT3 suppressor of mif two 3 homolog 1 (SUMO1) was found to be affected by all U concentrations. Key DEGs in this pathway promoting the hydroxylation (P4HB), ubiquitination (P53, HSP90AB1, NQO1, UBE2) and transcriptional regulation (SUMO1) of HIF-α were induced by U, suggesting that salmon may have suffered from U-caused hypoxia [36–42]. Although not assessed in the present study, the rationale for these observations may be several. Firstly, fish may have actually experienced hypoxia, as U may accumulate in the gill [6, 7, 43], alter gill structure and functions to reduce the gill oxygen uptake , influence the capacity of oxygen transport by red blood cells, or reduce the cardiac flow rate and oxygen supply . The hypoxic responses have been frequently observed in living organisms exposed to metals, such as chromium, nickel and cobalt . Secondly, dysfunction of the hematological system, which was indeed found to be one of the top gene networks regulated by all U concentrations, may potentially lead to hypoxia by affecting the transportation of oxygen. Hypoxic stress may cause further damage to the cardiovascular system and/or other physiological processes . Another potential mechanism may be that cross-talks between pathways such as P53 signaling and/or aryl hydrocarbon receptor (AhR) pathway may cause modulation of HIF-α signaling . In addition, U-induced ROS may also interfere with the iron availability and regulate the prolyl hydroxylase activity indirectly [48, 49]. Since 95% of the oxygen consumed by fish is used by the mitochondrial ETC to produce ATP, hypoxia may have considerable impact on the ETC activity. Several studies have proposed that mitochondrial ETC may regulate the cellular ROS level and HIF-1α expression , supporting the hypothesis that the induction of hypoxia signaling may also be a consequence of mitochondrial dysfunction.
DNA repair signaling
Uranium has been reported to cause DNA damage in fish [15, 19] either by causing oxidative DNA strand breaks through ROS, or by direct alteration of DNA structure through binding to the DNA molecules and forming uranium-DNA adducts [51, 52]. The top network found in this study showed that U regulated a group of DEGs associated with DNA replication, recombination and repair, representing transcriptional responses to potential DNA damage caused by U. Although no toxicity pathway directly associated with DNA damage was found based on the current data, the significant enrichment of a canonical pathway related to nucleotide excision repair (NER) may provide a link between U exposure and DNA damage. The NER pathway is usually responsible for repairing single strand DNA damage, either by global genome NER (GG-NER), or transcriptionally coupled NER (TC-NER). The present study identified up-regulated DEGs involved in both types of NER, such as DNA directed RNA polymerase II polypeptide I (POLR2I), DNA directed RNA polymerase II polypeptide F (POLR2F) and general transcription factor II H polypeptide 1 (GTF2H1) which are part of the TC-NER pathway. The nuclear excision repair protein RAD23 homolog B (RAD23B), which plays a central role in the GG-NER pathway, were also found to be affected by exposure to U. Song et al.  has previously observed that multiple genes involved in the cell cycle regulation and DNA repair processes, such as P53, cyclin-dependent kinase inhibitor 1 (P21), growth arrest and DNA damage-inducible gene gamma (GADD45G), proliferating cell nuclear antigen (PCNA) and RAD51, were up-regulated in the liver of Atlantic salmon after 48 h exposure to U. These evidences suggested that U may cause DNA damage after short-term exposure. But these damages may also be rapidly repaired by various DNA repair mechanisms.
Regulation of immune responses
Uranium has been documented to induce immune responses in fish. Cooley and co-workers  observed a wide range of histological changes, such as inflammation, tubules necrosis, haemorrhaging, glomerular lesions, pigmented macrophage proliferation in the liver of lake whitefish (Coregonus clupeaformis) after dietary exposure to U. A study on zebrafish  showed that hepatic IL-1b gene was 4-fold up-regulated by 23 μg /L U after 28 d waterborne exposure and 45-fold up-regulated by 130 μg /L U after 28 d exposure, suggesting that U may induce inflammatory responses. Gagnaire et al.  measured the immune biomarker enzyme phenoloxidase-like (PO) activity in zebrafish and found increased PO activity in adult fish after 48 h exposure to U, but significantly decreased PO activity in 96 h larvae after 4 d exposure to U. In mammals, Taulen and colleagues  found a group of genes associated with immune functions including tumor necrosis factor alpha-induced protein 1 (TNFAIP1) to be up-regulated in mouse kidney after a 48 h intraperitoneal injection exposure of 5 mg/kg uranyl nitrate. An in vitro gene expression study on murine macrophages and CD4+ T-cells showed that U induced multiple genes related to signal transduction, neurotrophic factors, chemokine and chemokine receptors, and interleukins, suggesting the immune modulation ability of U .
Several canonical pathways were found to be associated with U-mediated immune responses in this study. The antigen presentation pathway, which plays an important role in the development of both innate and adaptive immunity, may be one of the key pathways linking U exposure to the initiation of immune responses. It is well-known that T helper (CD4+) cells are vital in assisting other white blood cells in immunologic processes, whereas cytotoxic (CD8+) T-cells are eliminators of problematic cells. Through the antigen presentation pathway, cell types such as macrophages and dendritic cells are able to capture antigens and recognized by CD4+ and CD8+ T-cells. A few central DEGs in this pathway, such as major histocompatibility complex class II DQ beta 2 (HLA-DQB2), major histocompatibility complex class I C (HLA-C) and ATP-binding cassette sub-family B transporter 1 (TAP1) were found to be up-regulated following U exposure. The major histocompatibility complexes (MHCs) are often considered to be the main functioning molecules for immune recognition. The TAP1 gene encodes protein for transporting fragmented peptides during antigen presentation. The effect of U on antigen presentation has not been well studied yet, but the toxicological mechanisms may be similar to that proposed for nickel , including indirect actions to self-proteins which are then processed and presented by MHC, or directly binding to MHC/peptide complexes. Other immune-related canonical pathways, such as the IL-22 and HMGB1 signaling, which are involved in inflammation as an innate immune response , were found to be commonly regulated by all U concentrations. Another canonical pathway, the IL-8 signaling, which is central to inflammation, angiogenesis and tumor growth processes, was found to be regulated by low and medium U treatments. However, as the key regulator genes in these pathways such as IL-22, HMGB1 and IL-8 were not identified as DEGs in the current study, it was not clear how these pathways were affected by U.
Other potential mechanisms
Besides the major MoAs identified, the global transcriptional analysis performed herein may also provide some insight into other potential toxicological mechanisms of U which have not been well-studied. Although not as clearly supported by either experimental evidences or previous studies in fish, interference with nuclear receptors (NRs), interaction with peripheral nervous system (PNS) and disturbance of blood coagulation may also be important biological processes that were affected by U in the present study.
Pathways related to the glucocorticoid receptor signaling and retinoid X receptors (RXR) functions such as lipopolysaccharide (LPS)/IL-1 mediated inhibition of RXR function and farnesoid X receptor (FXR)/RXR activation were found to be affected by U. These pathways are important in the transcriptional regulation through NRs and may influence many downstream processes such as regulation of endocrine system, transportation, enzyme metabolism and biosynthetic processes. As DEGs identified in this study only supported the downstream parts of these pathways, it was not clear whether the NR signaling was directly or indirectly affected by U.
Another pathway of potential toxicological interest may be the γ-aminobutyric acid (GABA) receptor signaling regulated by all U concentrations. Supporting DEGs, including two main neurotransmitter receptors in this pathway, GABA A receptor rho 1 (GABRR1) and GABA A receptor beta 2 (GABRB4), were up-regulated after 48 h exposure. The GABA signaling is usually present in the central nervous system (CNS), but also widely found in peripheral tissues, including fish liver . The non-CNS roles of GABA in fish have been considered to participate in the regulation of reproduction, such as enhancing the release of gonadotropins from the pituitary or altering the plasma luteinizing hormone level . It has been proposed for mammals that the activation of GABA receptors may reduce the oxidative damage to the liver . Since no fish studies have been performed so far on U-induced GABA signaling in peripheral tissues, it may only be speculated that the induction of GABA signaling may be a secondary effect of oxidative stress or altered calcium level, as these primary effects may interfere with the neurotransmitter receptor signaling and/or inter-cellular signal transductions.
Uranium may also affect the blood coagulation in fish, as the canonical pathway related to the coagulation system was found to be among the most affected pathways by all U concentrations. Key DEGs such as coagulation factor VII (F7), IX (F9) and X (F10) were found to be up-regulated following short-term U exposure. The coagulation system may cross-talk with the immune system, as by forming blood clots, coagulation may physically trap the invading substances. It was also reported that metal-induced hydroxyl radicals may affect the blood coagulation in human . Whether this is also the case in fish still needs to be further investigated.