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.

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.

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.

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.

Drospirenone induces the accumulation of triacylglycerides in the fish hepatoma cell line, PLHC-1.

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.

Differential Toxicity of Alkylphenols in JEG-3 Human Placental Cells: Alteration of P450 Aromatase and Cell Lipid Composition.

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.

Toxic effects of bisphenol A diglycidyl ether and derivatives in human placental cells.

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.

Assessing the environmental quality of sediments from Split coastal area (Croatia) with a battery of cell-based bioassays.

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.

Characterization of quality of sediments from Paranaguá Bay (Brazil) by combined in vitro bioassays and chemical analyses.

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.

Comparative toxicity, oxidative stress and endocrine disruption potential of plasticizers in JEG-3 human placental cells.

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.

Characterization of complex lipid mixtures in contaminant exposed JEG-3 cells using liquid chromatography and high-resolution mass spectrometry.

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).

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.

Characterization of the environmental quality of sediments from two estuarine systems based on different in-vitro bioassays.

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.