Development of fish liver PLHC-1 spheroids and its applicability to investigate the toxicity of plastic additives.

Fish liver cell lines are valuable tools to understand the toxicity of chemicals in aquatic vertebrates. While conventional 2D cell cultures grown in monolayers are well established, they fail to emulate toxic gradients and cellular functions as in in-vivo conditions. To overcome these limitations, this work focuses on the development of Poeciliopsis lucida (PLHC-1) spheroids as a testing platform to evaluate the toxicity of a mixture of plastic additives. The growth of spheroids was monitored over a period of 30 days, and spheroids 2-8 days old and sized between 150 and 250 µm were considered optimal for conducting toxicity tests due to their excellent viability and metabolic activity. Eight-day-old spheroids were selected for lipidomic characterization. Compared to 2D-cells, the lipidome of spheroids was relatively enriched in highly unsaturated phosphatidylcholines (PCs), sphingosines (SPBs), sphingomyelins (SMs) and cholesterol esters (CEs). When exposed to a mixture of plastic additives, spheroids were less responsive in terms of decreased cell viability and generation of reactive oxygen species (ROS), but were more sensitive than cells growing in monolayers for lipidomic responses. The lipid profile of 3D-spheroids was similar to a liver-like phenotype and it was strongly modulated by exposure to plastic additives. The development of PLHC-1 spheroids represents an important step towards the application of more realistic in-vitro methods in aquatic toxicity studies.

Lipidomic analysis of mussel hemocytes exposed to polystyrene nanoplastics.

Plastics production and usage has exponentially increased in the last decades around the world. Due to the insufficient waste management, a significant amount of plastic ends up in the environment, where they tend to fragment into micro- and nano-plastics (NPs), and accumulate in aquatic organisms with still unknown effects. Although studies have indicated that lipid metabolism is a main target of NPs, this mechanism has not been extensively explored. In this study, we evaluated changes in the lipidome of mussel hemocytes after exposure to polystyrene (PS) NPs of 50 and 500 nm, at two different concentrations (106 and 109 particles/mL) for 24 h. The lipidome of hemocytes, analyzed by FIA-ESI (±) Orbitrap, was characterized by a relatively high abundance of cholesteryl esters (CEs) and phosphatidylcholine-plasmalogens (PC-Os/PC-Ps), involved in cell's defense against oxidative stress and membrane reorganization. In hemocytes exposed to PS NPs, a number of highly unsaturated membrane lipids were down-regulated, indicating a reorganization of the cell membranes after exposure to the particles and an oxidation of lipids with a high number of double bonds. This reduction was more evident after exposure to 50 nm NPs -both concentrations- and 500 nm NPs -high concentration-. The analysis of culture medium suggested increased release of vesicles enriched in triglycerides (TGs). The relevance of these responses to NP exposure on the immune function of hemocytes remains to be investigated.

PLHC-1 topminnow liver cells: An alternative model to investigate the toxicity of plastic additives in the aquatic environment.

Plasticizers are widespread environmental contaminants that have been described as obesogens in terrestrial vertebrates. However, its effects on fish lipids homeostasis are almost unknown. This work explores the use of PLHC-1 cells as an alternative model to assess the disruption of hepatic lipids by plastic additives and to gather information on the mode of action of these chemicals in fish. PLHC-1 lipid extracts were analyzed by flow injection coupled to high resolution mass spectrometry (FIA-ESI(+/-)-Orbitrap-Exactive) after 24 h exposure of the cells to the selected plasticizers: dibutyl phthalate (DBP), di-(2-ethylhexyl) phthalate (DEHP), bisphenol A (BPA), bisphenol F (BPF), and chlorinated bisphenol A diglycidyl ether (BADGE·2HCl). The analysis of the culture medium and the intracellular concentration of the chemicals revealed the highest bioconcentration of BADGE·2HCl, DBP and DEHP, which was in agreement with the strongest alteration of the cells lipidome. BADGE·2HCl induced a significant depletion of triacylglycerides (TGs), while DEHP and DBP stimulated the accumulation of TGs. Exposure to BPF induced the generation of reactive oxygen species in PLHC-1 cells and a significant depletion of phosphatidylcholine (PC)- and phosphatidylethanolamine (PE)-plasmalogens, and TGs (cell depots of polyunsaturated fatty acids). Overall, this study evidences different modes of action of plastic additives in topminnow liver cells, describes differential lipidomic signatures, and highlights the higher lipotoxicity of BADGE·2HCl and BPF compared to BPA.

Untargeted lipidomics reveals the toxicity of bisphenol A bis(3-chloro-2- hydroxypropyl) ether and bisphenols A and F in zebrafish liver cells.

Given the opposing responses reported for bisphenol A (BPA) in terms of induction of obesogenic effects and impaired lipid metabolism, the increasing use of bisphenol F (BPF), and the relatively low information available regarding the effects of bisphenol A bis(3-chloro-2- hydroxypropyl) ether (BADGE·2HCl) in aquatic organisms, this work aims to use the zebrafish liver cell line (ZFL) as an alternative model to characterize the toxicity and the lipid metabolism disruptive potential of the selected compounds in fish. All three bisphenols increased intracellular levels of dihydroceramides and ether-triacylglycerides (ether-TGs), suggestive of inhibited cell growth. However, while BPA and BADGE·2HCl caused an increase of saturated and lower unsaturated TGs, BPF caused oxidative stress and the decrease of TGs containing polyunsaturated fatty acids (PUFAs). Analysis by qPCR highlighted the up-regulation of the lipogenic genes scd and elovl6 by BPA and BPF in line with an increase of lipids containing saturated and monounsaturated FA and a decrease of lipids containing PUFAs. This study shows that BPA, BPF and BADGE·2HCl target lipid homeostasis in ZFL cells through different mechanisms, and highlights the higher lipotoxicity of BADGE·2HCl compared to BPA and BPF.

Skeletal Muscle Lipidomics as a New Tool to Determine Altered Lipid Homeostasis in Fish Exposed to Urban and Industrial Wastewaters.

This work applies ultrahigh performance liquid chromatography coupled with high-resolution mass spectrometry (UPLC-HRMS) to characterize for the first time the lipidome of the skeletal muscle of two fish species (Barbus meridionalis, Squalius laietanus) collected in a Mediterranean River affected by urban and industrial wastewater outflows. The untargeted analysis allowed a clear separation of the lipidome of fish from polluted and reference sites; phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), and their lyso and ether-linked forms were among the distinctive features. The targeted analysis consistently detected a decrease in PC-plasmalogens (36:4, 36:6, 38:6) and highly unsaturated PCs (36:5, 36:6, 38:6, 40:6, 40:7) and an increase in plasmanyl-PCs (36:5, 38:5), lyso-PCs (16:1, 18:1, 22:4) and cholesteryl esters (CEs) (16:0, 18:0, 20:4) in fish from polluted sites. These lipid profiles were indicative of oxidative stress and dysregulation of cholesterol homeostasis in fish from polluted sites. This methodology represents a promising tool for the development of novel noninvasive diagnostic methods based on muscle tissue biopsies to assess the effects of water pollution in wildlife.

The combined use of chemical and biochemical markers in Rutilus rutilus to assess the effect of dredging in the lower course of the Ebro River.

The lower course of the Ebro River is polluted with high concentrations of organochlorine compounds dumped by a chloro-alkali plant during the last century. A remediation plan, including building of a protective wall, removal and disposal of polluted sediments started in 2012. With the aim of assessing the effects of dredging of contaminated sediments and potential alterations of water quality, areas located upstream (RR) and downstream (BE, A) the chemical plant (FL) were monitored prior (October 2012) and during dredging (June 2013) using roach (Rutilus rutilus) as sentinel organisms. Concentrations of organochlorine compounds (OCs) in fish muscle and biliary levels of polycyclic aromatic hydrocarbons (PAHs), galaxolide (HHCB) and alkyphenols (APEs) were determined together with selected enzymatic activities (7-ethoxyresorufin-O-deethylase (EROD), 7-benzyloxy-4-trifluoromethyl-coumarin O-debenzyloxylase (BFCOD) and UDP-glucuronyltransferase (UGT)) in the liver. The obtained results proved the effectiveness of the wall retaining suspended particles and avoiding further contamination of downstream sites as fish sampled at downstream sites showed up to 9-fold higher concentrations of OCs in muscle during wall construction than during dredging. EROD and UGT activities were induced in fish from downstream sites; however, no clear response to the observed pollution gradient was detected.

Sex steroids and metabolic responses in mussels Mytilus galloprovincialis exposed to drospirenone.

Drospirenone (DRO) is a synthetic progestin derived from 17α-spironolactone with a pharmacological mechanism of action similar to progesterone. Despite its wide use as pharmaceutical and consequent continuous release into the aquatic environment, DRO effects have been poorly investigated on aquatic biota. In order to unravel the toxicity mechanisms of DRO, mussels Mytilus galloprovincialis were exposed for 7 days to different concentrations of DRO, namely 20ng/L (Low; L), 200ng/L (Medium; M), 2000ng/L (High; H) and 10µg/L (Super High; SH) nominal doses. Following exposure, no significant effect was observed on gonad maturation of treated and untreated mussels. The levels of progesterone (P4) and testosterone (T) were measured in mantle/gonad tissues and no significant alteration detected after exposure. However, the application of a protonic nuclear magnetic resonance (1H NMR)-based metabolomics approach enabled a comprehensive assessment of DRO effects in mussels. Specifically, 1H NMR metabolic fingerprints of digestive glands of DRO treated mussel groups were clearly separated from each other and from controls through a principal component analysis (PCA). Moreover, a number of metabolites involved in different metabolic pathways were found to significantly change in DRO-exposed mussels compared to control, suggesting the occurrence of alterations in energy metabolism, amino acids metabolism, and glycerophospholipid metabolism. Overall, despite no changes in gonad maturation and steroids levels were recorded in mussels after DRO exposure, the metabolomics approach demonstrated its effectiveness and high sensitivity in elucidating DRO-induced metabolic disturbances in marine mussels, and thus its usefulness in the environmental risk assessment of pharmaceuticals.

Perfluorinated chemicals: differential toxicity, inhibition of aromatase activity and alteration of cellular lipids in human placental cells.

The cytotoxicity of eight perfluorinated chemicals (PFCs), namely, perfluorobutanoic acid (PFBA), perfluorohexanoic acid (PFHxA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorododecanoic acid (PFDoA), perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHxS) and perfluorooctanesulfonate (PFOS) was assessed in the human placental choriocarcinoma cell line JEG-3. Only the long chain PFCs--PFOS, PFDoA, PFNA, PFOA--showed significant cytotoxicity in JEG-3 cells with EC50 values in the range of 107 to 647 µM. The observed cytotoxicity was to some extent related to a higher uptake of the longer chain PFCs by cells (PFDoA>PFOS≫PFNA>PFOA>PFHxA). Moreover, this work evidences a high potential of PFOS, PFOA and PFBS to act as aromatase inhibitors in placental cells with IC50s in the range of 57-80 µM, the inhibitory effect of PFBS being particularly important despite the rather low uptake of the compound by cells. Finally, exposure of JEG-3 cells to a mixture of the eight PFCs (0.6 µM each) led to a relative increase (up to 3.4-fold) of several lipid classes, including phosphatidylcholines (PCs), plasmalogen PC and lyso plasmalogen PC, which suggests an interference of PFCs with membrane lipids. Overall, this work highlights the ability of the PFC mixture to alter cellular lipid pattern at concentrations well below those that generate toxicity, and the potential of the short chain PFBS, often considered a safe substitute of PFOS, to significantly inhibit aromatase activity in placental cells.

Protective role of fetal bovine serum on PLHC-1 spheroids exposed to a mixture of plastic additives: A lipidomic perspective.

The use of fetal bovine serum (FBS) in cell culture is being questioned for scientific and ethical reasons, prompting the exploration of alternative approaches. Nevertheless, the influence of FBS on cell functioning, especially in fish cells, has not been comprehensively examined. This study aims to evaluate the impact of FBS on the lipidome of PLHC-1 spheroids and investigate cellular and molecular responses to plastic additives in the presence/absence of FBS. Lipidomic analyses were conducted on PLHC-1 cell spheroids using liquid chromatography coupled with a high-resolution quadrupole time-of-flight mass spectrometer (HRMS-QToF). The removal of FBS from the culture medium for 24 h significantly changed the lipid profile of spheroids, resulting in a depletion of cholesterol esters (CEs), phosphatidylcholines (PCs) and lyso-phosphatidylcholines (LPCs), while ceramides and certain glycerophospholipids slightly increased. Additionally, the exclusion of FBS from the medium led to increased cytotoxicity caused by a mixture of plastic additives and increased lipidomic alterations, including an elevation of ceramides. This study emphasizes the protective role of serum components in fish liver spheroids against a mixture of plastic additives and underscores the importance of considering exposure conditions when studying metabolomic and lipidomic responses to toxicants.

Toxicity and underlying lipidomic alterations generated by a mixture of water disinfection byproducts in human lung cells.

Complex mixtures of disinfection by-products (DBPs) are present in disinfected waters, but their mixture toxicity has been rarely described. Apart from ingestion, DBP exposure can occur through inhalation, which may lead to respiratory effects in highly exposed individuals. However, the underlying biological mechanisms have yet to be elucidated. This study aimed to investigate the toxicity of a mixture of 10 DBPs, including haloacetic acids and haloaromatics, on human alveolar A549 cells by assessing their cytotoxicity, genotoxicity, and impact on the cell lipidome. A DBP mixture up to 50 µM slightly reduced cell viability, induced the generation of reactive oxygen species (ROS) up to 3.5-fold, and increased the frequency of micronuclei formation. Exposure to 50 µM DBP mixture led to a significant accumulation of triacylglycerides and a decrease of diacylglycerides and phosphatidylcholines in A549 cells. Lipidomic profiling of extracellular vesicles (EVs) released in the culture medium revealed a marked increase in cholesterol esters, sphingomyelins, and other membrane lipids. Overall, these alterations in the lipidome of cells and EVs may indicate a disruption of lipid homeostasis, and thus, potentially contribute to the respiratory effects associated with DBP exposure.

Non-regulated haloaromatic water disinfection byproducts act as endocrine and lipid disrupters in human placental cells.

The disinfection of drinking water generates hundreds of disinfection byproducts (DBPs), including haloaromatic DBPs. These haloaromatic DBPs are suspected to be more toxic than haloaliphatic ones, and they are currently not regulated. This work investigates their toxicity and ability to interfere with estrogen synthesis in human placental JEG-3 cells, and their genotoxic potential in human alveolar A549 cells. Among the haloaromatic DBPs studied, halobenzoquinones (2,6-dichloro-1,4-benzoquinone (DCBQ) and 2,6-dibromo-1,4-benzoquinone (DBBQ)) showed the highest cytotoxicity (EC50: 18-26 µg/mL). They induced the generation of very high levels of reactive oxygen species (ROS) and up-regulated the expression of genes involved in estrogen synthesis (cyp19a1, hsd17b1). Increased ROS was linked to significant depletion of polyunsaturated lipid species from inner cell membranes. The other DBPs tested showed low or no significant cytotoxicity (EC50 ≥ 100 µg/mL), while 2,4,6-trichloro-phenol (TCP), 2,4,6-tribromo-phenol (TBP) and 3,5-dibromo-4-hydroxybenzaldehyde (DCHB) induced the formation of micronuclei at concentrations much higher than those typically found in water (100 µg/mL). This study reveals the different modes of action of haloaromatic DBPs, and highlights the toxic potential of halobenzoquinones, which had a significant impact on the expression of placenta steroid metabolism related genes and induce oxidative stress, implying potential adverse health effects.

Comparative toxicity of beach mesoplastics from South Spain: An in vitro approach.

Plastics, particularly mesoplastics, dominate beach debris and act as carriers of hazardous chemicals, either initially present in plastics or absorbed from the surrounding environment. In this study, mesoplastics were collected from five beaches in the southern region of Spain to investigate their potential impact on marine life. In vitro assays employing fish liver cells (PLHC-1) were conducted to evaluate the toxicity of methanolic extracts derived from intact mesoplastics and after simulated photodegradation. LC-MS analysis of the methanolic extracts revealed the presence of organophosphate esters, phthalates, and phthalate alternatives. The extracts from photodegraded plastics generally showed higher cytotoxicity, ability to generate reactive oxygen species (ROS), and genotoxicity (micronuclei formation) than those from intact mesoplastics. All the extracts induced EROD activity in PLHC-1 cells, indicating the presence of significant amounts of CYP1A inducers in beach mesoplastics. Thus, mesoplastics contain chemicals able to induce cytotoxicity and genotoxicity in PLHC-1 cells, and further photodegradation of mesoplastics facilitates the release of additional chemicals, increasing the overall toxicity. This work also highlights the usefulness of cell-based assays to better define the risks of plastic pollution.

Biliary PAH and alkylphenol metabolites, biomarker enzyme activities, and gene expression levels in the deep-sea fish Alepocephalus rostratus.

Biliary polycyclic aromatic hydrocarbon (PAH) and alkylphenol (AP) metabolites, hepatic gene expression, and corresponding enzyme activities were determined in the deep-sea fish Alepocephalus rostratus from two sites within the Mediterranean. Biliary metabolites included the hydroxylated PAH metabolites (OH-PAHs) 1-naphthol, 2-naphthol, 9-fluorenol, 9-phenanthrol, and 1-pyrenol and the APs 4-nonylphenol (NP) and 4-tert-octylphenol (OP). Five biomarker genes, namely, cytochrome P450 1A (CYP1A), vitellogenin (Vtg), catalase (CAT), Cu/Zn-superoxide-dismutase (Cu/Zn-SOD), and glutathione reductase (GR), were quantified using qRT-PCR. Moreover, corresponding enzyme activities (ethoxyresorufin-O-deethylase (EROD), CAT, SOD, and GR) were also determined. The ΣOH-PAHs detected ranged from 21.1 to 300.3 ng/g bile and were mainly composed of 1-naphthol. Both NP and OP metabolites were detected in all samples with concentrations ranging from 17.4 to 107.2 ng/g bile and 4.9 to 17.3 ng/g bile, respectively, and levels were significantly higher in samples from the western Mediterranean (WM) compared to those from the Catalan slope (CS). Accordingly, gene expression was significantly induced in male fish from the WM; however, these results were not reflected in enzyme activity levels. In particular, males caught at 2000 m in the WM exhibited 35-times higher Vtg levels compared to those from the CS, suggesting that endocrine-disrupting effects may potentially be occurring in such remote environments as the deep-sea.

Genotoxicity and endocrine disruption potential of haloacetic acids in human placental and lung cells.

Chlorination of water results in the formation of haloacetic acids (HAAs) as major disinfection byproducts (DBPs). Previous studies have reported some HAAs species to act as cytotoxic, genotoxic, and carcinogenic. This work aimed at further exploring the toxicity potential of the most investigated HAAs (chloroacetic (CAA), bromoacetic (BAA), iodoacetic (IAA) acid) and HAAs species with high content of bromine (tribromoacetic acid (TBAA)), and iodine in their structures (chloroiodoacetic (CIAA) and diiodoacetic acid (DIAA)) to human cells. Novel knowledge was generated regarding cytotoxicity, oxidative stress, endocrine disrupting potential, and genotoxicity of these HAAs by using human placental and lung cells as in vitro models, not previously used for DBP assessment. IAA showed the highest cytotoxicity (EC50: 7.5 µM) and ability to generate ROS (up to 3-fold) in placental cells, followed by BAA (EC50: 20-25 µM and 2.1-fold). TBAA, CAA, DIAA, and CIAA showed no significant cytotoxicity (EC50 > 250 µM). All tested HAAs decreased the expression of the steroidogenic gene hsd17b1 up to 40 % in placental cells, and IAA and BAA (0.01-1 µM) slightly inhibited the aromatase activity. HAAs also induced the formation of micronuclei in A549 lung cells after 48 h of exposure. IAA and BAA showed a non-significant increase in micronuclei formation at low concentrations (1 µM), while BAA, CAA, CIAA and TBAA were genotoxic at exposure concentrations above 10 µM (100 µM in the case of DIAA). These results point to genotoxic and endocrine disruption effects associated with HAA exposure at low concentrations (0.01-1 µM), and the usefulness of the selected bioassays to provide fast and sensitive responses to HAA exposure, particularly in terms of genotoxicity and endocrine disruption effects. Further studies are needed to define thresholds that better protect public health.

Comparative toxicity of conventional versus compostable plastic consumer products: An in-vitro assessment.

This study investigates the toxicity of methanolic extracts obtained from compostable plastics (BPs) and conventional plastics (both virgin and recycled). Additionally, it explores the potential influence of plastic photodegradation and composting on toxic responses using a battery of in vitro assays conducted in PLHC-1 cells. The extracts of BPs, but not those of conventional plastics, induced a significant decrease in cell viability (<70%) in PLHC-1 cells after 24 h of exposure. Toxicity was enhanced by either photodegradation or composting of BPs. Extracts of conventional plastics, and particularly those of recycled plastics, induced 7-ethoxyresorufin-O-deethylase (EROD) activity and micronucleus formation in exposed cells, indicating the presence of significant amounts of CYP1A inducers and genotoxic compounds in the extracts, which was enhanced by photodegradation. These findings highlight the importance of investigating the effects of degradation mechanisms such as sunlight and composting on the toxicity of BPs. It is also crucial to investigate the composition of newly developed formulations for BPs, as they may be more harmful than conventional ones.

Size-resolved chemical composition and toxicity of particles released from refit operations in shipyards.

Ship refit and repair operations in shipyards generate aerosol emissions with high potential for environmental impacts. Metal-bearing nano-, fine and coarse particles are incidentally formed and can be released to indoor and ambient air and the aquatic environment. This work aimed to further the understanding of these impacts by characterising particle size-resolved chemical composition (15 nm - 10 µm), organophosphate esters (OPEs) content (e.g., plasticisers) and cytotoxic and genotoxic potential. Results showed that nanoparticle emissions (20-110 nm) took place in bursts, coinciding with the use of mechanical abraders and spray-painting guns. Tracers of these activities were Sc, V, Cr, Co, Ni, Cu, Rb, Nb, and Cs. Key components were V and Cu, probably sourcing from nanoadditives in the coatings. Abrasion of coatings also emitted OPEs, especially from old paints. Toxicity assessments consistently evidenced hazardous potential for the different endpoints assessed, for a number of samples. Exposures to spray-painting aerosols were linked with reduced cell viability (cytotoxicity), significant generation of reactive oxygen species (ROS), and increases in micronuclei frequency (genotoxicity). Even though spray-painting did not contribute significantly to aerosol mass or number concentrations, it was a major driver of potential health effects. Results suggest that aerosol chemical composition (e.g., content in nano-sized Cu or V) may have a larger impact on toxicity than aerosol concentration. While direct human exposures may be prevented using personal and collective protective equipment and environmental release can be minimised by enclosures and filtration systems, impacts on ambient air and the aquatic environment cannot be fully prevented. The continued use of good practices (exhaust, dilution, general ventilation systems, PPE, already in place) is encouraged to reduce inhalation exposures inside the tents. Understanding the size-resolved chemical and toxicological properties of aerosols is key to reducing human health and environmental impacts of ship refit operations in shipyards.

Integrative omics-analysis of lipid metabolism regulation by peroxisome proliferator-activated receptor a and b agonists in male Atlantic cod.

Lipid metabolism is essential in maintaining energy homeostasis in multicellular organisms. In vertebrates, the peroxisome proliferator-activated receptors (PPARs, NR1C) regulate the expression of many genes involved in these processes. Atlantic cod (Gadus morhua) is an important fish species in the North Atlantic ecosystem and in human nutrition, with a highly fatty liver. Here we study the involvement of Atlantic cod Ppar a and b subtypes in systemic regulation of lipid metabolism using two model agonists after in vivo exposure. WY-14,643, a specific PPARA ligand in mammals, activated cod Ppara1 and Ppara2 in vitro. In vivo, WY-14,643 caused a shift in lipid transport both at transcriptional and translational level in cod. However, WY-14,643 induced fewer genes in the fatty acid beta-oxidation pathway compared to that observed in rodents. Although GW501516 serves as a specific PPARB/D ligand in mammals, this compound activated cod Ppara1 and Ppara2 as well as Pparb in vitro. In vivo, it further induced transcription of Ppar target genes and caused changes in lipid composition of liver and plasma. The integrative approach provide a foundation for understanding how Ppars are engaged in regulating lipid metabolism in Atlantic cod physiology. We have shown that WY-14,643 and GW501516 activate Atlantic cod Ppara and Pparb, affect genes in lipid metabolism pathways, and induce changes in the lipid composition in plasma and liver microsomal membranes. Particularly, the combined transcriptomic, proteomics and lipidomics analyses revealed that effects of WY-14,643 on lipid metabolism are similar to what is known in mammalian studies, suggesting conservation of Ppara functions in mediating lipid metabolic processes in fish. The alterations in the lipid profiles observed after Ppar agonist exposure suggest that other chemicals with similar Ppar receptor affinities may cause disturbances in the lipid regulation of fish. Model organism: Atlantic cod (Gadus morhua). LSID: urn:lsid:zoobank.org:act:389BE401-2718-4CF2-BBAE-2E13A97A5E7B. COL Identifier: 6K72F.

High-throughput analysis of the steroid profile in placental cell cultures to evaluate endocrine disrupting effects of contaminant exposure.

Human placental JEG-3 cells conserve a high P450 aromatase activity and are therefore suitable to evaluate how contaminants may interfere with the routes involved in estrogen synthesis during pregnancy. This has been traditionally assessed by measuring aromatase activity through the amount of tritiated water (3H2O) formed during the aromatization of 1ß-3H-androst-4-ene-3,17-dione (3H-AD). This work presents a greener and safer analytical approach for this purpose, which consists of the determination of the trace amounts of the steroids (estradiol, estrone, testosterone, and androstenedione) present in the culture medium. Turbulent flow chromatography coupled to liquid chromatography-tandem mass spectrometry (TFC-HPLC-MS/MS) delivered the high selectivity and sensitivity (limits of detection between 2 and 5 pg/mL) required for these measurements. Moreover, its automation allows high-throughput of samples with minimum sample handling and achieves high precision in the analysis (relative standard deviation values <6%). As a proof of concept, the method was applied to evaluate the effect of monohaloacetic acid exposure on the steroid profile of JEG-3 cells. Iodoacetic acid showed an estrogenic effect (statistically significant increase of estradiol levels compared to unexposed cells) at the highest concentration level tested (0.5 µM) that deserves further evaluation.

Precision cut tissue slices to investigate the effects of triclosan exposure in Mytilus galloprovincialis.

Precision-cut tissue slices (PCTS) are frequently used in mammalian research, but its application in the area of aquatic toxicology is still humble. This work proposes the use of PCTS to investigate the effects of the antimicrobial triclosan (TCS) in the mussel Mytilus galloprovincialis. PCTS sectioned from the digestive gland (400 µm) were exposed to 10, 100, and 500 nM TCS for 24 h, and the expression of selected genes, together with the biomarkers, carboxylesterases (CbE) and glutathione S-transferases (GST), and the analysis of lipids in PCTS and culture medium, were used to investigate the molecular initiating events of triclosan in the digestive gland of mussels. Significant dysregulation in the expression of phenylalanine-4-hydroxylase (PAH), glutamate dehydrogenase (GDH), fatty acid synthase (FASN), and 7-dehydrocholesterol reductase (DHCR7), involved in energy, phenylalanine and lipid metabolism, were detected. The analysis of lipids evidenced significant changes in cholesteryl esters (CEs) and membrane lipids in the culture medium of exposed PCTS, suggesting dysregulation of energy and lipid metabolism that can affect lipid dynamics in mussels exposed to triclosan.

Dysregulation of lipid metabolism in PLHC-1 and ZFL cells exposed to tributyltin an all-trans retinoic acid.

There is increasing awareness that exposure to endocrine disrupters interferes with lipid homeostasis in vertebrates, including fish. Many of these compounds exert their action by binding to nuclear receptors, such as peroxisome proliferator-activated receptors and retinoid X receptor. This work investigates the use of fish liver cells (PLHC-1 and ZFL cells) for the screening of metabolic and lipid disrupters in the aquatic environment by assessing changes in the cell's lipidome after exposure to the model compounds, tributyltin chloride and all-trans retinoic acid. Lipid extracts, analyzed by FIA-ESI (+/-) Orbitrap, evidenced the intracellular accumulation of triglycerides and diglycerides in both cell models after exposure to 100 and 200 nM tributyltin chloride for 24 h. Exposure to 1 µM all-trans retinoic acid led to a significant accumulation of triglycerides in PLHC-1 cells, while few triglycerides were accumulated in ZFL cells. Retinoic acid (cyp26b1, cyp3a65, lrata) and lipid metabolism (fasn, scd, elovl6) related genes were up-regulated by tributyltin chloride and all-trans retinoic acid, while only all-trans retinoic acid down-regulated the expression of dgat1a. The two cell models show sensitivity and responses to tributyltin chloride and all-trans retinoic acid comparable to those previously reported in mammalian cells. These results support the use of fish liver cells as alternative models for the detection of contaminants that act as lipid disrupters in the aquatic environment.