Antibiotics, frequently detected in aquatic ecosystems, can negatively impact the health of resident organisms. Although the study on the possible effects of antibiotics on these organisms has been increasing, there is still little information available on the molecular effects on exposed non-target organisms. In our study we used a label free proteomic approach and sea bream, Sparus aurata, to evaluate the effects of exposure to environmentally relevant concentrations of the antibiotic compounds ciprofloxacin (CIP), sulfadiazine (SULF) and trimethoprim (TRIM) produced at the protein level. Individuals of sea bream were exposed to single compounds at 5.2 ± 2.1 μg L− 1 of CIP, 3.8 ± 2.7 μg L− 1 of SULF and 25.7 ± 10.8 μg L− 1 of TRIM for 21 days. After exposure, the number of differentially expressed proteins in the liver was 39, 73 and 4 for CIP, SULF and TRIM respectively. In the brain, there was no alteration of proteins after CIP and TRIM treatment, while 9 proteins were impacted after SULF treatment. The differentially expressed proteins were involved in cellular biological, metabolic, developmental, growth and biological regulatory processes. Overall, our study evidences the vulnerability of Sparus aurata, after exposure to environmentally relevant concentrations of the major antibiotics CIP, SULF and TRIM and that their chronic exposure could lead to a stress situation, altering the proteomic profile of key organs such as brain and liver. | Ministerio de Economía y Competitividad (MINECO), Spain Universidad de Cádiz, Spain Asociación Universitaria Iberoamericana de Postgrado, Spain Ministerio de Asuntos Económicos y Transformación Digital, Gobierno de España, Spain Latin American Association of Postgraduates | 13 páginas

Silver nanomaterials (AgNMs) are broadly used and among the most studied nanomaterials. The underlying molecular mechanisms (e.g. protein and metabolite response) that precede phenotypical effects have been assessed to a much lesser extent. In this paper, we assess differentially expressed proteins (DEPs) and metabolites (DEMs) by high-throughput (HTP) techniques (HPLC-MS/MS with tandem mass tags, reversed-phase (RP) and hydrophilic interaction liquid chromatography (HILIC) with mass spectrometric detection). In a time series (0, 7, 14 days), the standard soil model Enchytraeus crypticus was exposed to AgNM300K and AgNO₃ at the reproduction EC20 and EC50. The impact on proteins/metabolites was clearly larger after 14 days. NM300K caused more upregulated DEPs/DEMs, more so at the EC20, whereas AgNO₃ caused a dose response increase of DEPs/DEMs. Similar pathways were activated, although often via opposite regulation (up vs down) of DEPs, hence, dissimilar mechanisms underlie the apical observed impact. Affected pathways included e.g. energy and lipid metabolism and oxidative stress. Uniquely affected by AgNO₃ was catalase, malate dehydrogenase and ATP-citrate synthase, and heat shock proteins (HSP70) and ferritin were affected by AgNM300K. The gene expression-based data in Adverse Outcome Pathway was confirmed and additional key events added, e.g. regulation of catalase and heat shock proteins were confirmed to be included. Finally, we observed (as we have seen before) that lower concentration of the NM caused higher biological impact. Data was deposited to ProteomeXchange, identifier PXD024444.

Microplastics (MPs) are plastic fragments less than 5 mm, which may have adverse impacts on organisms. In this study, we investigated the impacts and mechanisms of polystyrene MPs (10 μm and 100 μm) and nanoplastics (NPs, 100 nm) with different concentrations (10 mg/kg and 100 mg/kg) in soil on the uptake of metal Cd and semi-metal As in earthworms, Eisenia fetida. MPs facilitated the accumulation of (semi-)metals via damaging the integrity of earthworm intestine, and earthworms accumulated more (semi-)metals in MP treatment groups than NP treatment groups, especially in group of 100 mg/kg of 10 μm MP with concentrations of 1.13 mg/kg and 32.7 mg/kg of Cd and As, respectively. Higher genotoxicity to earthworms was observed for MPs than NPs. Antioxidant enzymes activity and their mRNA gene relative expression levels indicated that MPs with high concentration induced severer damage to earthworms, thus resulting in the increased accumulation of (semi-)metals by earthworms. In addition, proteomic and metabolomic analysis revealed that MPs (100 ppm of 10 μm) disturbed the earthworm immune and metabolic systems, resulting in the highest accumulation of (semi-)metals in earthworms. This study clarifies the influence mechanisms of MPs with different sizes and levels on the accumulation of (semi-)metals by terrestrial invertebrates.

The Pearl River Estuary (PRE) is the largest estuary in southern China and under high metal stress. In the present study, we employed an integrated method of transcriptomics and proteomics to investigate the ecotoxicological effects of trace metals on the Hong Kong oyster Crassostrea hongkongensis. Three oyster populations with distinct spatial distributions of metals were sampled, including the Control (Station QA, the lowest metal levels), the High Cd (Station JZ, the highest Cd), and the High Zn–Cu–Cr–Ni (Station LFS, with the highest levels of zinc, copper, chromium, and nickel). Dominant metals in oysters were differentiated by principal component analysis (PCA), and theirgene and protein profiles were studied using RNA-seq and iTRAQ techniques. Of the 2250 proteins identified at both protein and RNA levels, 70 proteins exhibited differential expressions in response to metal stress in oysters from the two contaminated stations. There were 8 proteins altered at both stations, with the potential effects on mitochondria and endoplasmic reticulum by Ag. The genotoxicity, including impaired DNA replication and transcription, was specifically observed in the High Cd oysters with the dominating influence of Cd. The structural components (cytoskeleton and chromosome-associated proteins) were impaired by the over-accumulated Cu, Zn, Cr, and Ni at Station LFS. However, enhanced tRNA biogenesis and exosome activity might help the oysters to alleviate the toxicities resulting from their exposure to these metals. Our study provided comprehensive information on the molecular changes in oysters at both protein and RNA levels in responding to multi-levels of trace metal stress.

Here, we examined the combinational effect of ocean acidification (OA) and mercury (Hg) in the planktonic copepod Pseudodiaptomus annandalei in cross-factored response to different pCO₂ (400, 800 μatm) and Hg (control, 1.0 and 2.5 μg/L) exposures for three generations (F0–F2), followed by single-generation recovery (F3) under clean condition. Several phenotypic traits and Hg accumulation were analyzed for F0–F3. Furthermore, shotgun-based quantitative proteomics was performed for F0 and F2. Our results showed that OA insignificantly influenced the traits. During F0–F2, combined exposure reduced Hg accumulation as compared with the counterpart Hg treatment, supporting the mitigating effect of OA on Hg toxicity in copepods. Proteomics analysis indicated that the copepods probably increased energy production/storage and stress response to ensure physiological resilience against OA. However, Hg induced many toxic events (e.g., energy depletion and degenerated organomorphogenesis/embryogenesis for F0; cell cycle arrest and detrimental stress-defense for F2), which were translated to the population-level adverse outcome, i.e., compromised growth/reproduction. Particularly, compensatory proteome response was identified (e.g., increased immune defense for F0; energetic compensation and enhanced embryogenesis for F2), accounting for a negative interaction between OA and Hg. Together, this study provides the molecular mechanisms behind the effects of OA and Hg pollution in marine copepods.

Cadmium (Cd) is an important heavy metal pollutant in the Bohai Sea. Mitochondria are recognized as the key target for Cd toxicity. However, mitochondrial responses to Cd have not been fully investigated in marine fishes. In this study, the mitochondrial responses were characterized in gills of juvenile flounder Paralichthys olivaceus treated with two environmentally relevant concentrations (5 and 50 μg/L) of Cd for 14 days by determination of mitochondrial membrane potential (MMP), observation of mitochondrial morphology and quantitative proteomic analysis. Both Cd treatments significantly decreased MMPs of mitochondria from flounder gills. Mitochondrial morphologies were altered in Cd-treated flounder samples, indicated by more and smaller mitochondria. iTRAQ-based proteomic analysis indicated that a total of 128 proteins were differentially expressed in both Cd treatments. These proteins were basically involved in various biological processes in gill mitochondria, including mitochondrial morphology and import, tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), primary bile acid biosynthesis, stress resistance and apoptosis. These results indicated that dynamic regulations of energy homeostasis, cholesterol metabolism, stress resistance, apoptosis, and mitochondrial morphology in gill mitochondria might play significant roles in response to Cd toxicity. Overall, this study provided a global view on mitochondrial toxicity of Cd in flounder gills using iTRAQ-based proteomics.

Effects of contamination on aquatic organisms have been investigated and employed as biomarkers in environmental quality assessment for years. A commonly referenced aquatic organism, mollusks represent a group of major interest in toxicological studies. Both gastropods and bivalves have external mineral shells that protects their soft tissue from predation and desiccation. These structures are composed of an organic matrix and an inorganic matrix, both of which are affected by environmental changes, including exposure to hazardous chemicals. This literature review evaluates studies that propose mollusk shell alterations as biomarkers of aquatic system quality. The studies included herein show that changes to natural variables such as salinity, temperature, food availability, hydrodynamics, desiccation, predatory pressure, and substrate type may influence the form, structure, and composition of mollusk shells. However, in the spatial and temporal studies performed in coastal waters around the world, shells of organisms sampled from multi-impacted areas were found to differ in the form and composition of both organic and inorganic matrices relative to shells from less contaminated areas. Though these findings are useful, the toxicological studies were often performed in the field and were not able to attribute shell alterations to a specific molecule. It is known that the organic matrix of shells regulates the biomineralization process; proteomic analyses of shells may therefore elucidate how different contaminants affect shell biomineralization. Further research using approaches that allow a clearer distinction between shell alterations caused by natural variations and those caused by anthropogenic influence, as well as studies to identify which molecule is responsible for such alterations or to determine the ecological implications of shell alterations, are needed before any responses can be applied universally.

Arsenic (As) is a metalloid element that is ubiquitous in the marine environment and its contamination has received worldwide attention due to its potential toxicity. Arsenic can induce multiple adverse effects, such as lipid metabolism disorder, immune system dysfunction, oxidative stress and carcinogenesis, in animals. Inorganic arsenic includes two chemical forms, arsenite (As (III)) and arsenate (As (V)), in natural environment. As (V) is the dominant form in natural waters. In the present study, metabolomic and proteomic alterations were investigated in juvenile rockfish Sebastes schlegelii exposed to environmentally relevant concentrations of As (V) for 14 d. The analysis of iTRAQ-based proteomics combined with untargeted NMR-based metabolomics indicated apparent toxicological effects induced by As (V) in juvenile rockfish. In details, the metabolites, including lactate, alanine, ATP, inosine and phosphocholine were significantly altered in As-treated groups. Proteomic responses suggested that As (V) could not only affected energy and primary metabolisms and signal transduction, but also influenced cytoskeleton structure in juvenile rockfish. This work suggested that the combined proteomic and metabolomic approach could shed light on the toxicological effects of pollutants in rockfish S. schlegelii.

Hydrogen gas (H₂) has been shown as an important factor in plant tolerance to abiotic stresses, but the underlying mechanisms remain unclear. In the present study, the effects of H₂ and its interaction with nitric oxide (NO) on alleviating cadmium (Cd) stress in Brassica campestris seedlings were investigated. NO donor (SNP) or hydrogen-rich water (HRW) treatment showed a significant improvement in growth of Cd-stressed seedlings. Cd treatment upregulated both endogenous NO and H₂ (36% and 66%, respectively), and the increase of H₂ was prior to NO increase. When treated with NO scavenger (PTIO) or NO biosynthesis enzyme inhibitors (L-NAME and Gln), HRW-induced alleviation under Cd stress was prevented. Under Cd stress, HRW pretreatment significantly enhanced the NO accumulation, and together up-regulated the activity of NR (nitrate reductase) and expression of NR. HRW induced lower reactive oxygen species (ROS), higher AsA content, enhanced activity of POD (peroxidase) and SOD (superoxide dismutase) in seedling roots were inhibited by PTIO, L-NAME and Gln. Through proteomic analysis, the level of 29 proteins were changed in response to H₂ and NO-induced amelioration of Cd stress. Nearly half of them were involved in oxidation-reduction processes (about 20%) or antioxidant enzymes (approximately 20%). These results strongly indicate that in Cd-stressed seedlings, pretreatment with HRW induces the accumulation of H₂ (biosynthesized or permeated), which further stimulates the biosynthesis of NO through the NR pathway. Finally, H₂ and NO together enhance the antioxidant capabilities of seedlings in response to Cd toxicity.

Dibenzofuran (DBF) derivatives have caused serious environmental problems, especially those produced by paper pulp bleaching and incineration processes. Prominent for its resilient mutagenicity and toxicity, DBF poses a major challenge to human health. In the present study, a new strain of Pseudomonas aeruginosa, FA-HZ1, with high DBF-degrading activity was isolated and identified. The determined optimum conditions for cell growth of strain FA-HZ1 were a temperature of 30 °C, pH 5.0, rotation rate of 200 rpm and 0.1 mM DBF as a carbon source. The biochemical and physiological features as well as usage of different carbon sources by FA-HZ1 were studied. The new strain was positive for arginine double hydrolase, gelatinase and citric acid, while it was negative for urease and lysine decarboxylase. It could utilize citric acid as its sole carbon source, but was negative for indole and H2S production. Intermediates of DBF 1,2-dihydroxy-1,2-dihydrodibenzofuran, 1,2-dihydroxydibenzofuran, 2-hydroxy-4-(3′-oxo-3′H-benzofuran-2′-yliden)but-2-enoic acid, 2,3-dihydroxybenzofuran, 2-oxo-2-(2′-hydrophenyl)lactic acid, and 2-hydroxy-2-(2′-hydroxyphenyl)acetic acid were detected and identified through liquid chromatography-mass analyses. FA-HZ1 metabolizes DBF by both the angular and lateral dioxygenation pathways. The genomic study identified 158 genes that were involved in the catabolism of aromatic compounds. To identify the key genes responsible for DBF degradation, a proteomic study was performed. A total of 1459 proteins were identified in strain FA-HZ1, of which 100 were up-regulated and 104 were down-regulated. A novel enzyme “HZ6359 dioxygenase”, was amplified and expressed in pET-28a in E. coli BL21(DE3). The recombinant plasmid was successfully constructed, and was used for further experiments to verify its function. In addition, the strain FA-HZ1 can also degrade halogenated analogues such as 2, 8-dibromo dibenzofuran and 4-(4-bromophenyl) dibenzofuran. Undoubtedly, the isolation and characterization of new strain and the designed pathways is significant, as it could lead to the development of cost-effective and alternative remediation strategies. The degradation pathway of DBF by P. aeruginosa FA-HZ1 is a promising tool of biotechnological and environmental significance.