A selection of corneal genes to this study.

<p>A selection of DEGs for at least one treatment or genes selected for RNA-seq validation (^) or time-related expression analysis (*). Where genes are DEG, fold change value and absolute ranks in the transcriptome was reported. FC, Fold Change.</p><p>A selection of corneal genes to this study.</p

DNA Damage and Transcriptional Changes in the Gills of <em>Mytilus galloprovincialis</em> Exposed to Nanomolar Doses of Combined Metal Salts (Cd, Cu, Hg)

<div><p>Aiming at an integrated and mechanistic view of the early biological effects of selected metals in the marine sentinel organism <em>Mytilus galloprovincialis</em>, 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.</p> </div

Expression levels of selected genes as measured by RNAseq and qPCR.

<p>The expression values generated by RNA-seq (red) or qPCR (blu) for eleven selected genes using the same corneal samples (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133173#pone.0133173.t001" target="_blank">Table 1</a>) are compared. Values detected for each gene were normalized to GAPDH expression and reported as a ratio between APCP-exposed samples and unexposed controls.</p

Detection of OGG1 in human corneas treated ex-vivo with APCP.

<p>Corneal tissues exposed for 2 min to APCP were analyzed by immunohistochemistry (a-d) and Western Blot (e-f). Frozen sections (5 μm) of corneas treated in the absence (a) or presence (b) of 10 mM NAC were incubated with polyclonal rabbit anti-OGG1 at 6 h post-treatment. Protein immunostaining (in red) was compared to that of untreated controls (c). Negative controls were prepared by omitting the primary antibody (d). For the Western Blot analysis, proteins were extracted at 6 and 24 h post-treatment: the OGG1 protein signal increased at 6 h, and was reduced in the presence of NAC, and returned to values comparable to that of controls within 24 h. Densitometric values of OGG1 autoradiographic bands were normalized to corresponding β-actin and expressed as percentage ± SE of the mean control value.</p

Differentially expressed corneal genes (DEGs) at 6 h after exposure to APCP.

<p>(a) over- and under-expressed DEGs are shown as common (overlapping area) or exclusive to the APCP (left) or APCP+NAC (right) treatments; (b) number of total (Baggerly’s test FDR p-value <0.01) and DEG, over- and under-expressed genes detected in HC1-HC6 samples, paired per condition.</p

Gene ID, Ensemble ID and related Forward and Reverse primers used in qPCR.

<p>GAPDH was used as housekeeping gene, other genes were selected for RNA-seq validation (underlined) or time-related expression analysis (*)</p><p>Gene ID, Ensemble ID and related Forward and Reverse primers used in qPCR.</p

qPCR analysis of selected genes at different time points.

<p>Two human corneas were used to evaluate the expression of specific genes by qPCR at selected time points from the exposure to APCP. To minimize the variability of response, both corneas, one used as negative control and the other exposed to 2 min APCP, were divided into three pieces, then collected at 3, 6 and 24 h post-treatment. Expression levels of the target genes, detected in the treated corneal sample relative to the untreated sample were normalized to GAPDH levels.</p

Concentration of selected elements in the mussels exposed for 48 h to the 200 nM metal dose.

<p>Triplicate analysis of three tissue pools, each one composed by the whole flesh of four mussels. Mean and standard deviation are reported in ww and dw; BCF, bioconcentration factor.</p

Gene Ontologies most represented in the corneal genes up-regulated by APCP.

<p>Gene Ontologies most represented in the corneal genes up-regulated by APCP in the absence (a) or presence (b) of NAC. Size and gray scale color of the circles reflect the importance of cell pathways, represented as functionally connected nodes.</p

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

<p>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: <i>M galloprovincialis</i> 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: <i>S. cerevisiae</i> 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.</p