RESUMEN

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.

Asunto(s)

Desinfectantes , Agua Potable , Contaminantes Químicos del Agua , Purificación del Agua , Femenino , Embarazo , Humanos , Desinfección , Desinfectantes/toxicidad , Desinfectantes/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Línea Celular Tumoral , Placenta/metabolismo , Agua Potable/análisis , Benzoquinonas/toxicidad , Fenoles/metabolismo , Estrógenos/metabolismo , Lípidos , Contaminantes Químicos del Agua/análisis , Halogenación

RESUMEN

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.

Asunto(s)

Hepatocitos , Hígado , Animales , Línea Celular , Técnicas de Cultivo de Célula/métodos , Peces

RESUMEN

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.

Asunto(s)

Nanopartículas , Pintura , Humanos , Tamaño de la Partícula , Aerosoles/análisis , Nanopartículas/toxicidad , Nanopartículas/química , Especies Reactivas de Oxígeno , Monitoreo del Ambiente/métodos

RESUMEN

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.

Asunto(s)

Desinfectantes , Contaminantes Químicos del Agua , Purificación del Agua , Embarazo , Humanos , Femenino , Placenta , Acetatos , Desinfección/métodos , Daño del ADN , Desinfectantes/toxicidad , Contaminantes Químicos del Agua/toxicidad , Purificación del Agua/métodos , Halogenación , Trihalometanos

RESUMEN

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.

Asunto(s)

Placenta , Espectrometría de Masas en Tándem , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Estrona , Femenino , Humanos , Embarazo , Espectrometría de Masas en Tándem/métodos

RESUMEN

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.

Asunto(s)

Compuestos de Bencidrilo/toxicidad , Fenoles/toxicidad , Contaminantes Químicos del Agua/toxicidad , Animales , Compuestos Epoxi/análisis , Éter , Éteres , Hepatocitos , Lipidómica , Hígado/química , Pez Cebra

RESUMEN

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.

Asunto(s)

Plásticos/toxicidad , Pruebas de Toxicidad/métodos , Contaminantes Químicos del Agua/toxicidad , Animales , Compuestos de Bencidrilo , Dibutil Ftalato , Compuestos Epoxi , Fundulidae , Hepatocitos , Hígado/efectos de los fármacos , Modelos Biológicos , Fenoles , Ácidos Ftálicos , Plastificantes/toxicidad

RESUMEN

Drospirenone (DRO) is one of the most commonly used progestins reaching the aquatic environment through wastewater treatment plant effluents. It is a progesterone receptor agonist, and as such, can act primarily in the brain and reproductive organs of fish. In order to better understand and predict its effects, this work evaluates the lipidomic changes induced in PLHC-1 cells after exposure to drospirenone at concentrations below the EC10 (1 and 10 µM) by direct injection of the lipid extracts into a ESI(+/-) Orbitrap mass spectrometer. A significant accumulation of triacylglycerides, particularly long chain ones with unsaturated fatty acid moieties (TGs 46:2, 56:4-7; 58:5-8) and a concomitant decrease of diacylglycerides (DGs 32:1, 34:1-2, 36:1-2, 38:2-4) was observed after 48 h exposure to 10 µM DRO, which corresponded to an intracellular concentration of 8.3 ng·mg-1 protein. No significant alteration of PLHC-1 cell lipids was observed following exposure to 1 µM DRO. EC50 for the cytotoxicity of DRO ranged from 105 to 119 µM (24 h exposure) to 51-58 µM (48 h exposure). The study evidences a dysregulation of neutral lipid metabolism and increased TG/DG ratio in fish hepatic cells exposed to DRO.

Asunto(s)

Androstenos/efectos adversos , Ciprinodontiformes/metabolismo , Glicéridos/metabolismo , Contaminantes Químicos del Agua/efectos adversos , Animales , Línea Celular Tumoral , Hepatocitos , Antagonistas de Receptores de Mineralocorticoides/efectos adversos

RESUMEN

BADGE (bisphenol A diglycidyl ether) is a synthesis product of bisphenol A (BPA), which, like other plasticizers, can cross the human placenta and reach the foetus. However, compared to BPA, there is almost no toxicological information. This work investigates the toxicity, endocrine and lipid disruption potential of BADGE and its hydrolysed and chlorinated derivatives (BADGE·H2O and BADGE·2HCl) in human placental JEG-3 cells. The analysis of culture medium by HPLC-ESI(+)-QqQ evidenced a good bioavailability of BADGE·2HCl and BADGE·H2O, but low stability of BADGE. Regardless, BADGE·2HCl and BADGE showed higher cytotoxicity than BADGE·H2O, which was the only compound that significantly inhibited CYP19 activity (IC50 49 ±â€¯5 µM). JEG-3 cells lipidome analyzed by FIA-ESI(+/-)-Orbitrap was significantly altered by exposure to BADGE·2HCl and BADGE at concentrations at the low µM range. BADGE·2HCl lead to a strong decrease of diacyl- and triacyl-glycerides (DGs,TGs) together with some membrane lipids, while BADGE lead to an accumulation of TGs. The results evidence the ability of BADGE and derivatives to affect placental lipid handling and to modulate placental CYP19 activity (BADGE·H2O) and highlights the need to monitor human exposure to these compounds, at least as intensely as BPA is monitored.

Asunto(s)

Inhibidores de la Aromatasa/toxicidad , Aromatasa/metabolismo , Compuestos de Bencidrilo/toxicidad , Carcinógenos/toxicidad , Compuestos Epoxi/toxicidad , Fenoles/toxicidad , Placenta/citología , Plastificantes/toxicidad , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión/métodos , Diglicéridos/metabolismo , Femenino , Humanos , Lípidos de la Membrana/metabolismo , Placenta/efectos de los fármacos , Embarazo , Especies Reactivas de Oxígeno/metabolismo , Triglicéridos/metabolismo

RESUMEN

Alkylphenols (APs) are a diverse class of chemicals that can cross the placental barrier and interfere with embryonic and fetal development. This work investigates the comparative toxicity, ability to inhibit aromatase activity, and to alter the lipid composition of 10 alkylphenols in the human placenta choriocarcinoma cell line JEG-3. Among the selected APs, 4-dodecylphenol (DP), 4-heptylphenol (HP), and 4-cumylphenol (CP) showed the highest cytotoxicity (EC50: 18-65 µM). Aromatase inhibition was closely related to the hydrophobicity of APs. HP significantly induced the generation of reactive oxygen species (ROS) (43-fold), inhibited placental aromatase activity (IC50: 41 µM), and induced a general dose-dependent depletion of polyunsaturated lipids (10-20 µM), which were attributed to high levels of oxidative stress. In contrast, 2,4,6-tri-tert-butylphenol (TTBP) significantly induced the intracellular accumulation of triacylglycerides (TGs), whereas DP increased the synthesis of phosphatidylcholines (PCs) and TGs at the expense of diacylglycerides (DGs). Overall, this study evidences the different modes of action of alkylphenols in human placental JEG-3 cells, describes differential lipidomic fingerprints, and highlights DP, HP, CP, and TTBP as the ones that caused the most harmful effects.

Asunto(s)

Aromatasa/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Fenoles/toxicidad , Placenta/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Estructura Molecular , Estrés Oxidativo/efectos de los fármacos , Fenoles/química , Placenta/metabolismo , Placenta/patología , Embarazo , Relación Estructura-Actividad

RESUMEN

Industrial activity generates harmful substances which can travel via aerial or water currents thousands of kilometers away from the place they were used impacting the local biota where they deposit. The presence of harmful anthropogenic substances in the Antarctic is particularly surprising and striking due to its remoteness and the apparent geophysical isolation developed with the flows of the Antarctic Circumpolar current and the ring of westerly winds surrounding the continent. However, long-range atmospheric transport (LRAT) of pollutants has been detected in the Antarctic since the 70's along the Antarctic trophic food web from phytoplankton to birds. Still, no information exists on the presence of cytotoxic compounds in marine sediments neither at basin scales (thousands of kilometers) nor in water depths (hundreds of meters) beyond shallow coastal areas near research stations. Our results showed for the first time that there is cytotoxic activity in marine sediment extracts from water depths >1000 m and along thousands of kilometers of Antarctic continental shelf, in some cases comparable to that observed in Mediterranean areas. Ongoing anthropogenic pressure appears as a serious threat to the sessile benthic communities, which have evolved in near isolation for millions of years in these environments.

RESUMEN

A battery of cell-based bioassays, including PLHC-1 cells, zebrafish-Pxr-transfected COS-7 cells and estrogen receptor-recombinant yeast assay (ER-RYA), were applied to detect the presence of bioactive pollutants in sediments collected from Kastela Bay and Brac Channel (Croatia). Exposure of PLHC-1 cells to the sediment extracts evidenced significant cytotoxicity and presence of CYP1A inducers in sediments collected in Kastela Bay, near the industrial zone and cargo port of Split. Sediments from this area, which is highly contaminated with PCBs, HCB, DDTs and γ-HCH, also activated the zebrafish Pxr (zfPxr) reporter system. No evidence of estrogenicity was detected for any of the sediments extracts in the ER-RYA assay. Importantly, the battery of in vitro assays identified Kastela Bay as the area with the higher anthropogenic impact, where sediment-bound pollutants could pose a risk to aquatic organisms. In contrast, sediments from the Brac Channel showed rather low response in the different bioassays.

Asunto(s)

Bioensayo , Monitoreo del Ambiente , Sedimentos Geológicos/química , Contaminantes Químicos del Agua/análisis , Animales , Línea Celular , Croacia , Peces , Pez Cebra

RESUMEN

The present study characterizes the quality of sediments from the Paranaguá Estuarine Complex (South Brazil). Polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and organochlorine pesticides (OCPs) were determined in sediment samples together with a series of different in vitro bioassays. The fish hepatoma cell line (PLHC-1) was used to determine the presence of cytotoxic compounds and CYP1A- and oxidative stress-inducing agents in sediment extracts. Ovarian microsomal fractions from sea bass (Dicentrarchus labrax) were used to detect the presence of endocrine disrupters that interfered with the synthesis of estrogens (ovarian CYP19). Despite the relatively low levels of pollutants and no evidence of negative effects based on guideline levels, sediments collected close to harbors were enriched with CYP1A-inducing agents and they showed higher cytotoxicity. In contrast, sediments from internal areas inhibited CYP19 activity, which suggests the presence of endocrine disrupters at these sites. Overall, the selected bioassays and the chemistry data led to the identification of potentially impacted areas along the Paranaguá Estuarine Complex that would require further action to improve their environmental quality. Environ Toxicol Chem 2017;36:1811-1819. © 2016 SETAC.

Asunto(s)

Bioensayo , Cromatografía de Gases y Espectrometría de Masas , Sedimentos Geológicos/análisis , Contaminantes Químicos del Agua/análisis , Animales , Aromatasa/metabolismo , Hidrocarburo de Aril Hidroxilasas/metabolismo , Lubina/metabolismo , Bahías , Brasil , Línea Celular , Disruptores Endocrinos/química , Disruptores Endocrinos/toxicidad , Monitoreo del Ambiente , Estrógenos/biosíntesis , Femenino , Sedimentos Geológicos/química , Microsomas/efectos de los fármacos , Microsomas/enzimología , Ovario/metabolismo , Bifenilos Policlorados/análisis , Bifenilos Policlorados/química , Hidrocarburos Policíclicos Aromáticos/análisis , Hidrocarburos Policíclicos Aromáticos/química , Análisis de Componente Principal , Especies Reactivas de Oxígeno/metabolismo , Contaminantes Químicos del Agua/química , Contaminantes Químicos del Agua/toxicidad

RESUMEN

Plasticizers are suspected to be toxic and/or to modulate or disrupt the endocrine system of humans and to cross the placental barrier, being embryonic and fetal development a particularly vulnerable period. This work investigates the comparative toxicity and ability to interfere with the synthesis of steroids and to generate reactive oxygen species (ROS) of a selected number of plasticizers, including bisphenol A (BPA), nonyl- (NP) and octylphenol (OP), benzyl butyl phthalate (BBP), dibutyl phthalate (DBP), di(2-ethylhexyl)phthalate (DEHP) and dimethyl phthalate (DMP), in the human placenta JEG-3 cells. Moreover, the bioavailability of chemicals in culture medium has been investigated. After 24h exposure, OP and NP showed the highest cytotoxicity (EC50: 36-40µM) followed by BPA (138-219µM), whereas no significant toxicity was observed for phthalates. Notwithstanding, BBP and DBP significantly decreased P450 aromatase activity (experimental IC50: 14-15µM), while NP and OP (20µM) increased the activity. Overall, this study evidences the differential toxicity and ability to modulate placental aromatase activity of some of the compounds nowadays used as plasticizers, and highlights the need of an accurate determination of the bioavailability of chemicals to improve the sensitivity of in-vitro tests.

Asunto(s)

Disruptores Endocrinos/toxicidad , Fenoles/toxicidad , Ácidos Ftálicos/toxicidad , Plastificantes/toxicidad , Aromatasa/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Estrés Oxidativo/efectos de los fármacos , Placenta/citología , Embarazo , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

The environmental quality of marine sediments collected in the area of influence of the Po and Danube Rivers was assessed by using a battery of bioassays based on the use of PLHC-1 cells, zebrafish-Pxr-transfected COS-7 cells, and sea bass ovarian subcellular fractions. This allowed the determination of multiple endpoints, namely, cytotoxicity, oxidative stress, induction of CYP1A, activation of zebrafish Pxr and inhibition of ovarian aromatase. Organic extracts of sediments influenced by the Danube River and collected near harbors and urban discharges showed significant cytotoxicity, CYP1A induction and inhibition of aromatase activity. An analogous response of CYP1A induction and zfPxr activation was observed, which suggests the existence of common ligands of AhR and PXR in the sediment extracts. The study highlights the usefulness of the selected bioassays to identify those sediments that could pose a risk to aquatic organisms and that require further action in order to improve their environmental quality.

Asunto(s)

Monitoreo del Ambiente , Sedimentos Geológicos , Animales , Bioensayo , Mar Negro , Italia , Ríos , Rumanía , Contaminantes Químicos del Agua

RESUMEN

The aim of this study was to develop a method based on ultra high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS) for lipid profiling in human placental choriocarcinoma (JEG-3) cells. Lipids were solid-liquid extracted from JEG-3 cells using a solution of chloroform/methanol (2:1, v/v) in a simple procedure requiring minimal sample alteration. Simultaneous separation of complex lipid mixtures in their major classes was achieved with a reversed-phase (C8) UHPLC column and a mobile phase containing methanol with 1 mM ammonium formate and 0.2 % formic acid (A)/water with 2 mM ammonium formate and 0.2 % formic acid (B). Lipids were characterized using time-of-flight (TOF) and Orbitrap under full scan and positive electrospray ionization mode with both analyzers. A total of 178 species of lipids, including 37 phosphatidylcholines (PC), 32 plasmalogen PC, 9 lyso PC, 4 lyso plasmalogen PC, 30 triacylglycerols, 22 diacylglycerols, 7 cholesterol esters, 25 phosphatidylethanolamines, and 12 sphingomyelins, were identified using TOF and Orbitrap. The identification of all lipid classes was based on exact mass characterization with an error < 5 ppm. The developed methodology was applied to study lipid alterations in human placental cells against the exposure to perfluorinated chemicals (PFCs) and tributyltin (TBT).

Asunto(s)

Cromatografía Líquida de Alta Presión/métodos , Fluorocarburos/toxicidad , Lípidos/análisis , Espectrometría de Masas/métodos , Compuestos de Trialquiltina/toxicidad , Línea Celular Tumoral , Humanos , Lípidos/aislamiento & purificación , Metaboloma/efectos de los fármacos

RESUMEN

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.

Asunto(s)

Inhibidores de la Aromatasa/toxicidad , Aromatasa/metabolismo , Disruptores Endocrinos/toxicidad , Fluorocarburos/toxicidad , Metabolismo de los Lípidos/efectos de los fármacos , Placenta/efectos de los fármacos , Inhibidores de la Aromatasa/química , Inhibidores de la Aromatasa/metabolismo , Transporte Biológico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Disruptores Endocrinos/química , Disruptores Endocrinos/metabolismo , Femenino , Fluorocarburos/química , Fluorocarburos/metabolismo , Humanos , Concentración 50 Inhibidora , Estructura Molecular , Placenta/enzimología , Placenta/patología , Embarazo , Medición de Riesgo , Relación Estructura-Actividad , Factores de Tiempo

RESUMEN

This study characterizes the environmental quality of sediments from the Arade and Guadiana estuaries using different in-vitro bioassays: a) fish hepatoma cell line (PLHC-1) to determine cytotoxicity and presence of CYP1A and oxidative stress inducing agents; b) gonad subcellular fractions from sea bass (Dicentrarchus labrax) to detect compounds that are likely to act as endocrine disrupters by interfering with the synthesis of androgens (CYP17, CYP11ß) and estrogens (CYP19). Approximately 60% of extracts from the Arade estuary were cytotoxic when tested at 60 mg eQsed/mL, while only one sample from Guadiana showed cytotoxicity. Sediments from Arade collected close to harbours and waste water effluents were enriched with CYP1A inducing agents, while those from the upper Guadiana induced oxidative stress in PLHC-1 cells. On the other hand, several extracts from both estuaries were able to significantly inhibit CYP17, CYP11ß and CYP19 activities in gonad subcellular fractions of sea bass, which indicates the presence of endocrine disrupters, particularly in several sites from the Arade estuary. Overall, the study highlights the usefulness of in-vitro bioassays to identify those sediments that could pose risk to aquatic organisms and that require further action to improve their environmental quality.

Asunto(s)

Disruptores Endocrinos/toxicidad , Monitoreo del Ambiente , Sedimentos Geológicos/análisis , Estrés Oxidativo/efectos de los fármacos , Contaminantes Químicos del Agua/análisis , Contaminantes Químicos del Agua/toxicidad , Andrógenos/metabolismo , Animales , Lubina/metabolismo , Línea Celular Tumoral , Ciprinodontiformes , Sistema Enzimático del Citocromo P-450/metabolismo , Exposición a Riesgos Ambientales , Estrógenos/metabolismo , Estuarios , Femenino , Masculino , Ovario/efectos de los fármacos , Portugal , Especies Reactivas de Oxígeno/metabolismo , Testículo/efectos de los fármacos