DNA damage and transcriptional changes in the gills of mytilus galloprovincialis exposed to nanomolar doses of combined metal salts (Cd, Cu, Hg)

Abstract

Aiming at an integrated and mechanistic view of the early biological effects of selected metals in the marine sentinel organism Mytilus galloprovincialis, we exposed mussels for 48 hours to 50, 100 and 200 nM solutions of equimolar Cd, Cu and Hg salts and measured cytological and molecular biomarkers in parallel. Focusing on the mussel gills, first target of toxic water contaminants and actively proliferating tissue, we detected significant dose-related increases of cells with micronuclei and other nuclear abnormalities in the treated mussels, with differences in the bioconcentration of the three metals determined in the mussel flesh by atomic absorption spectrometry. Gene expression profiles, determined in the same individual gills in parallel, revealed some transcriptional changes at the 50 nM dose, and substantial increases of differentially expressed genes at the 100 and 200 nM doses, with roughly similar amounts of up- and down-regulated genes. The functional annotation of gill transcripts with consistent expression trends and significantly altered at least in one dose point disclosed the complexity of the induced cell response. The most evident transcriptional changes concerned protein synthesis and turnover, ion homeostasis, cell cycle regulation and apoptosis, and intracellular trafficking (transcript sequences denoting heat shock proteins, metal binding thioneins, sequestosome 1 and proteasome subunits, and GADD45 exemplify up-regulated genes while transcript sequences denoting actin, tubulins and the apoptosis inhibitor 1 exemplify down-regulated genes). Overall, nanomolar doses of co-occurring free metal ions have induced significant structural and functional changes in the mussel gills: the intensity of response to the stimulus measured in laboratory supports the additional validation of molecular markers of metal exposure to be used in Mussel Watch programs.

Figure 1. The mussel gill structure.

In the mussel shell, rows of interconnected gill filaments folded to form a W-shape create the trabecular structure of the gill lamellae on either sides of the visceral mass, with apical ciliated cells facilitating the flux of water, oxygen uptake and food collection (3 µm sections observed after haematoxylin and eosin staining in bright field microscopy, by courtesy of Tobia Pretto, Reference Natl. Centre on mollusc diseases, IZSVe, Italy).

Figure 2. Occurrence of gill cells with micronuclei (MN) or nuclear abnormalities (NA) in the mussels exposed for 48 h to the combined metals.

Panels A and B refer to offshore mussels collected in late spring and marketed winter mussels, respectively. Mean (N = 5) and standard deviation per dose point are reported (* and *** indicate significant increases at p<0.05 and 0.001 compared to the control mussels).

Figure 3. Number of genes differentially expressed in the mussel gills per metal dose in regard to control group.

Left: over- and under-expressed genes with related false discovery rate (FDR) and delta value (Δ). Right: Venn diagram of common and unique genes modulated at 50, 100 and 200 nM metal mixtures (right).

Figure 4. Unsupervised hierarchical clustering and relative expression trends of annotated genes differentially expressed in the mussel gills at least in one treatment dose.

Clustering of median values per dose point (left), individual trends (middle) and transcript description (right) have been reorganized by functional category.

Figure 5. Distribution of all differentially expressed genes in different functional categories.

Figure 6. Relative quantification levels (RQ) of selected mussel transcripts significantly modulated after exposure to the combined metals.

Pooled gill RNAs from both treated and control mussels were tested in triplicate according to the 2−ΔΔCt method and considering 18S rRNA as endogenous control gene.

Figure 7. Representation of the gill cell response at 48 hours from the mussel exposure to the combined metals.

Cd, Cu and Hg ions can enter the cytosol across various transporters or channels, also by endocytosis, with potential inhibition of physiological membrane import processes. Contaminant metal ions can bind small molecules (e.g. GSH), transporters and chaperons (e.g. ferritin, MTs), and apoproteins (e.g. superoxide dismutase, cytochrome c oxidase). Hence, they may compete with endogenous cations, substitute their natural ligands, disturb Ca⁺⁺ homeostasis, and accumulate also in metal-rich or mineralized granules. Redox reactions with sulfur groups and direct/indirect formation of ROS/RNS can trigger multiple signalling pathways, disrupt the regulated expression of many genes and deplete the antioxidant cell defences. The unbalance towards the oxidative stress associated to extensive damage to organelles such as mytochondria and lysosomes, macromolecules and their precursors may lead to cell death either by necrosis or apoptosis. Deregulation of genes involved in the cell cycle homeostasis might also lead to uncontrolled replication and tumour development. Based on the experimental data, we have exemplified genes up-regulated (red), down-regulated (green) and contrasting (brown) expression trends, altogether outlining the enhancement or depression of specific cellular processes. Transcripts represented in the MytArray are reported in bold. Related abbreviations are the following. α: 26S proteasome subunit α (α1: Myt01-003G03; α2: Myt01-007D11, Myt01-013D06). ABCB: ATP binding cassette p-glycoprotein (Myt01-004E12, Myt01-018G06). APAF: Apoptotic Peptidase Activating Factor. ATP: Adenosine-5'-TriPhosphate. ATOX: AnTiOXdant protein AVEN: Caspase Activation Inhibitor (Myt01-018H01). β: proteasome subunit beta type (β1: Myt01-002G08; β5: Myt01-007H02; β7: Myt01-016F07). BAT2: HLA-B-associated Transcript 2 (BAT2 domain containing 1-like: Myt01-012B10). Bcl-xS: B cell lymphoma X apoptosis regulator. Bcl2: B cell lymphoma 2-like protein 1. CAL: Calmodulin (Myt01-003H01). CASP: Caspase (CASP3/7: Myt01-011F10; CASP1: Myt01-014F12). cMyc: cellular Myelocytomatosis proto-oncogene (Myt01-003C05). COX: Cytochrome c Oxidase (subunit I: Myt01-006G10, Myt01-019B06; subunit II: Myt01-019B11; subunit III: Myt01-004F10, Myt01-019B03, Myt01-016F03; subunit IV: Myt01-007E11). CREB: C-amp-Responsive Element-Binding (Myt01-009E09). cPLA2: cytoplasmic Ca2+-dependent PhosphoLipase A2 (Myt01-014H05). CTR: Cell surface Transporter. Cytb: Cytochrome b (Myt01-019B12, Myt01-011C05). Cytc: Cytochrome c (Myt01-005A03). DAD1: Defender Against apoptotic cell Death 1 (Myt01-017B11). DCT1: Divalent Cation Transporter 1. DEAD box: DEAD (Asp-Glu-Ala-Asp) box polypeptide 17, Myt01-007F09; polypeptide 42 (Myt01-017E11). DUSP7: Dual Specificity protein Phosphatase 7 (Myt01-016G07). ENaC; Epithelial Na Channel. EPN1: Epsin1 (Myt01-014H03). Fk506-BP: FK506-binding protein (Myt01-012A04). FT: Ferritin (Myt01-013D11). GSH: Glutathione. GST: Glutathione S-Transferase (GST3: Myt01-012G04; GSTpi1: Myt01-010C12). IAP1: Inhibitor of APoptosis 1 (Myt01-007H08). INO80: INO80 complex subunit C-like (Myt01-003G12). MAPBPIP: mitogen-activated protein-binding protein-interacting protein (Myt01-015D08). MEKK5: Mitogen activated protein Kinase Kinase Kinase 5 (Myt01-006H07). MePCs: Metal Protein Complexes. MgC1q: M galloprovincialis C1q domain containing protein (MgC1q8: Myt01-015F11; MgC1q4: Myt01-015C12; MgC1q48: Myt01-018E07; MgC1q89: Myt01-015H10). MMgT: Membrane Magnesium Transporter (Myt01-002C12). MRAS: Ras-related protein M-Ras (Myt01-005E12). MRE: Metal Response Element. MRP: Multidrug Resistance-associated Protein (Myt01-010D05). MSMB: MicroSeMinoprotein Beta (Myt01-016C09). MT: Metallothionein (MT10: Myt01-016C08). MTF: MRE-binding Transcription Factor. NELL1: protein kinase C-binding protein, Neural Epidermal growth factor-Like 1 (Myt01-015F09). NOLC1: Nucleolar and Coiled-body phosphoprotein 1 (Myt01-015B10). P-ATPase: P-type ATPase. PCNA: Proliferating Cell Nuclear Antigen (Myt01-016A01). PPIase: PeptidylProlyl Isomerase (cyclophilin-like) (Myt01-009D06). RAB: Ras-related GTP-binding protein (Rab6 subfamily protein: Myt01-002B09; RAB27: Myt01-018A11). RACK: Receptor for Activated C-Kinase (Myt01-007H10). RalGEF: Ral Guanine nucleotide Exchange Factor (Myt01-009C10). RNS : Reactive Nitrogen Species. ROS: Reactive Oxygen Species. SEC: S. cerevisiae endoplasmic reticulum membrane protein translocator (SEC61: Myt01-011C12; SEC63: Myt01-018G07). SCO: Synthesis of Cytochrome c Oxidase SOCS2: Suppressor Of Cytokine Signaling 2 (Myt01-012D01). SOD: SuperOxide Dismutase. TCTP: Translationally Controlled Tumor Protein (Myt01-007H05, Myt01-010H05). TF: TransFerrin. TFR: TransFerrin Receptor. VDAC: mitochondrial Voltage-Dependent Anion membrane Channel. ZRT: Zinc-Regulated Transporter.