Immunotoxicity In Vitro Assays for Environmental Pollutants under Paradigm Shift in Toxicity Tests.

With the outbreak of COVID-19, increasingly more attention has been paid to the effects of environmental factors on the immune system of organisms, because environmental pollutants may act in synergy with viruses by affecting the immunity of organisms. The immune system is a developing defense system formed by all metazoans in the course of struggling with various internal and external factors, whose damage may lead to increased susceptibility to pathogens and diseases. Due to a greater vulnerability of the immune system, immunotoxicity has the potential to be the early event of other toxic effects, and should be incorporated into environmental risk assessment. However, compared with other toxicity endpoints, e.g., genotoxicity, endocrine toxicity, or developmental toxicity, there are many challenges for the immunotoxicity test of environmental pollutants; this is due to the lack of detailed mechanisms of action and reliable assay methods. In addition, with the strong appeal for animal-free experiments, there has been a significant shift in the toxicity test paradigm, from traditional animal experiments to high-throughput in vitro assays that rely on cell lines. Therefore, there is an urgent need to build high-though put immunotoxicity test methods to screen massive environmental pollutants. This paper reviews the common methods of immunotoxicity assays, including assays for direct immunotoxicity and skin sensitization. Direct immunotoxicity mainly refers to immunosuppression, for which the assays mostly use mixed immune cells or isolated single cells from animals with obvious problems, such as high cost, complex experimental operation, strong variability and so on. Meanwhile, there have been no stable and standard cell lines targeting immune functions developed for high-throughput tests. Compared with direct immunotoxicity, skin sensitizer screening has developed relatively mature in vitro assay methods based on an adverse outcome pathway (AOP), which points out the way forward for the paradigm shift in toxicity tests. According to the experience of skin sensitizer screening, this paper proposes that we also should seek appropriate nodes and establish more complete AOPs for immunosuppression and other immune-mediated diseases. Then, effective in vitro immunotoxicity assay methods can be developed targeting key events, simultaneously coordinating the studies of the chemical immunotoxicity mechanism, and further promoting the paradigm shift in the immunotoxicity test.

Integrated passive sampling and fugacity model to characterize fate and removal of organophosphate flame retardants in an anaerobic-anoxic-oxic municipal wastewater treatment system.

It is crucial to deeply understand the fate and removal mechanism of various organophosphate flame retardants (PFRs) in specified wastewater treatment processes. However, concentration fluctuation and matrix effect in wastewater challenge quantification of PFR flux for both field observation and model validation. We present measured seasonal distribution profiles of time-weighted average (TWA) concentrations by in situ hydrophobic and polar passive samplers and modeled mass transport and transformation by means of fugacity for 11 PFRs with varied structures in an anaerobic-anoxic-oxic (A-A-O) municipal wastewater treatment system, and provided a systematic approach to characterize fate and removal mechanism of PFRs in major compartments via various treatment processes. We find evidence that PFRs have a unique structural-dependent fate and removal in the A-A-O system. Hydrophilic chlorinated-PFRs present persistent in all major compartments and dominate in effluents with significant variations; alkyl-PFRs are majorly reduced by biodegradation; whereas hydrophobic aryl-PFRs have the highest removal percentage, contributed by both sorption on solids and biotransformation. Sensitive analysis shows the most influential operation parameters on removal efficiency varied among the PFRs with different properties. We also conclude passive sampling can be effectively applied to estimate TWA wastewater concentrations and to validate fugacity model prediction.

Water pollutant discharge permit allocation based on DEA and non-cooperative game theory.

Against the background of the ecological civilization system reform in the new era, the appropriate allocation of water pollutant discharge permits is an important policy for controlling the amount of wastewater discharge. Traditional allocation methods have disadvantages, such as high additional costs, an unfair allocation scheme, and market distortion. In the present study, a fixed-cost allocation model based on data envelopment analysis (DEA) and the Nash non-cooperative game theory is employed to allocate water pollutant discharge permits of totally 31 provinces in China from 2008 to 2017. The allocation scheme considers environmental efficiency. The results demonstrate regional differences in the allocation of water pollutant discharge permits. The eastern region has abundant allocations. The northeastern and central regions have insufficient allocations. Besides, the western region has a significant shortage of allocations. It indicates the higher the utilization efficiency of the water pollutant discharge permits, the higher the region's sustainable development is. Based on the analysis, we propose guidelines for industrial wastewater discharge reduction.

Comparison of the mechanisms of estrogen disrupting effects between triphenyl phosphate (TPhP) and tris(1,3-dichloro-2-propyl) phosphate (TDCIPP).

As the typical aryl-organophosphate flame retardants (OPFRs), triphenyl phosphate (TPhP) and tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) were reported to be estrogen disruptors. However, estrogen receptor α (ERα) binding experiments could not explain their biological effects. In this study, their action on ERα, G protein-coupled estrogen receptor (GPER) and the synthesis of 17ß-estradiol (E2) were investigated using in vitro assays and molecular docking. The results showed that TPhP acted as an ERα agonist and recruited steroid receptor co-activator 1 (SRC1) and 3 (SRC3), which was found for the first time. Unlike TPhP, TDCIPP acted as an ERα antagonist. However, both TPhP and TDCIPP activated the estrogen pathway by GPER in SKBR3 cells which were lack of ERα. Although molecular docking results revealed that both TPhP and TDCIPP could dock into ERα and GPER, their substituent groups and combination mode might affect the receptor activation. In addition, by using estrogen biosynthesis assay in H295R cells, both of TPhP and TDCIPP were found to promote E2 synthesis and E2/T ratio involving their different alteration on levels of progesterone, testosterone and estrone, and expression of various key genes. Our data proposed estrogen-disrupting mechanism frameworks of TPhP and TDCIPP. Moreover, our results will contribute to future construction of adverse outcome pathway (AOP) framework of endocrine disruptors.

Fish forewarning of comprehensive toxicity in water environment based on Bayesian sequential method.

Environmental impact of pollutants can be analyzed effectively by acquiring fish behavioral signals in water with biological behavior sensors. However, a variety of factors, such as the complexity of biological organisms themselves, the device error and the environmental noise, may compromise the accuracy and timeliness of model predictions. The current methods lack prior knowledge about the fish behavioral signals corresponding to characteristic pollutants, and in the event of a pollutant invasion, the fish behavioral signals are poorly discriminated. Therefore, we propose a novel method based on Bayesian sequential, which utilizes multi-channel prior knowledge to calculate the outlier sequence based on wavelet feature followed by calculating the anomaly probability of observed values. Furthermore, the relationship between the anomaly probability and toxicity is analyzed in order to achieve forewarning effectively. At last, our algorithm for fish toxicity detection is verified by integrating the data on laboratory acceptance of characteristic pollutants. The results show that only one false positive occurred in the six experiments, the present algorithm is effective in suppressing false positives and negatives, which increases the reliability of toxicity detections, and thereby has certain applicability and universality in engineering applications.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-induced suppression of immunity in THP-1-derived macrophages and the possible mechanisms.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a well-known immunotoxic environmental pollutant. However, most immunotoxicology studies of TCDD were based on the animal models and the inner mechanisms have just focused on a few genes/proteins. In this study, the immune functions of THP-1-derived macrophages was measured with in-vitro bioassays after 24-h exposure of TCDD including environmentally relevant concentrations. RNA-seq and Weighted Gene Co-expression Network Analysis were used to characterize the immunotoxicity molecular mechanisms. Our study is the first report on the TCDD-induced effects of cell adhesion, morphology, and multiple cytokines/chemokines production on THP-1 macrophages. After TCDD treatment, we observed an inhibited cell adherence, probably attributed to the suppressed mRNA levels of adhesion molecules ICAM-1, VCAM-1 and CD11b, and a decrease in cell pseudopodia and expression of F-actin. The inflammatory cytokines TNF-α, IL-10 and other 8 cytokines/chemokines regulating granulocytes/T cells and angiogenesis were disrupted by TCDD. Alternative splicing event was found to be a sensitive target for TCDD. Using WGCNA, we identified 10 hub genes (TNF, SRC, FGF2, PTGS2, CDH2, GNG11, BDNF, WNT5A, CXCR5 and RUNX2) highly relevant to these observed phenotypes, suggesting AhR less important in the effects TCDD have on THP-1 macrophages than in other cells. Our findings broaden the understanding of TCDD immunotoxicity on macrophages and provide new potential targets for clarifying the molecular mechanisms.

In Vitro Immunotoxicity of Organophosphate Flame Retardants in Human THP-1-Derived Macrophages.

Scarce attention has been paid to the immunotoxicity of organophosphate flame retardants (PFRs), which poses a challenge to the systematic assessment of their health risks. In this study, a battery of in vitro immunotoxicity screening assays, including adhesion, phagocytosis, and 48 cytokine/chemokine production, was measured after exposing THP-1-derived macrophages to six selected common PFRs (TPHP, TDCPP, TNBP, TOCP, TCEP, and TBOEP) at a noncytotoxic concentration (≤50 µM). Our results showed that TPHP and TBOEP partially attenuated the adhesion and phagocytosis of the THP-1 mφs and that TDCPP caused a functional loss of phagocytosis, implying the potential immunosuppression. In contrast, TNBP and TOCP may cause an immunostimulation by significantly promoting cell adhesion and enhancing phagocytic efficiency. Additionally, the results from a cytokine/chemokine secretion analysis revealed the proinflammatory properties of TDCPP, TPHP, and TBOEP. TOCP was thought to disrupt the inflammatory balance by inhibiting both proinflammatory and antiinflammatory cytokines. TCEP showed no effect on adhesion or phagocytosis and little modulation of cytokine release at this experimental concentration. Overall, this study supports that PFRs can be immunotoxic to macrophages in different ways and provides evidence for developing more sensitive in vitro immunotoxicity bioassay methods.

Tricresyl phosphate isomers exert estrogenic effects via G protein-coupled estrogen receptor-mediated pathways.

Tricresyl phosphates (TCPs), as representative aromatic organophosphate flame retardants (OPFRs), have received much attention due to their potential neurotoxicity and endocrine-disrupting effects. However, the role of estrogen receptor α (ERα) and G protein-coupled estrogen receptor (GPER) in their estrogen disrupting effects remains poorly understood. Therefore, in this study, three TCP isomers, tri-o-cresyl phosphate (ToCP), tri-m-cresyl phosphate (TmCP) and tri-p-cresyl phosphate (TpCP), were examined for their activities on ERα by using two-hybrid yeast assay, and action on GPER by using Boyden chamber assay, cAMP production assay, calcium mobilization assay and molecular docking analysis. The results showed that three TCP isomers were found to act as ERα antagonists. Conversely, they had agonistic activity on GPER to promote GPER-mediated cell migration of MCF7 cells and SKBR3 cells. Both ToCP and TpCP activated GPER-mediated cAMP production and calcium mobilization, whereas TmCP had different mode of action, it only triggered GPER-mediated calcium mobilization, as evidenced by using the specific GPER inhibitor (G15) and GPER overexpressing experiments. Molecular docking further revealed that the way of interaction of TmCP and TpCP with GPER was different from that of ToCP with GPER, and higher activity of ToCP in activating GPER-mediated pathways might be associated with the alkyl substitution at the ortho position of the aromatic ring. Our results, for the first time, found a new target, GPER, for TCPs exerting their estrogen-disrupting effects, and demonstrated complex estrogen-disrupting effects of three TCP isomers involved their opposite activities toward ERα and GPER.

Distribution, sources and transport of organophosphorus flame retardants in the water and sediment of Ny-Ålesund, Svalbard, the Arctic.

Organophosphorus flame retardants (PFRs) are contaminants of emerging concern which have been detected globally. However, little information on PFRs in the Arctic freshwater environment is currently available. In this study, both hydrophilic and hydrophobic PFRs in the water and sediment of four areas (town, surroundings, coastal marine water, and glacier melt runoff) near Ny-Ålesund Svalbard were investigated by time-integrated passive sampling (water) and grab sampling (sediment). Seven kinds of PFRs were found in the Arctic waters with individual freely dissolved concentrations from 0.007 ng L-1 to 355 ng L-1, and the concentrations of chlorinated PFRs were 3-4 orders of magnitude higher than those of non-chlorinated PFRs. The distribution of different PFRs in freshwater showed significant spatial differences among the different areas, and the town was found to have most kinds of PFRs and highest PFRs concentrations. The sources and transport of different kinds of PFRs were explored based on a spatial overlay analysis of the contaminant distributions, environmental conditions, and PFR applications. As a result, human settlements, industrial activities, atmospheric deposition, and transportation in Ny-Ålesund were proposed to be related to the pollution of different PFRs at Ny-Ålesund. The PFRs in the inshore marine water were found to be affected by both local ship contamination and ocean current transport. Furthermore, nine PFRs were detected in the sediments of Ny-Ålesund. Accumulation of different PFRs in the Arctic sediment was found to be dominated by their physico-chemical properties (polarity).

In vitro estrogen-disrupting effects of organophosphate flame retardants.

Organophosphate flame retardants (OPFRs), as substitutes for polybrominated diphenyl ethers (PBDEs), are frequently detected in the environment and biota due to their widespread use. Thus, there is a need to investigate their potential estrogen-disrupting effects and possible mechanisms of action in an effort to obtain a better risk assessment. In this study, we characterized the activities on estrogen receptor α (ERα) and the estrogen-disrupting potential of fourteen OPFRs, TMP, TEP, TPP, TnBP, TiBP, THP, TPhP, TCP, DPK, MDPP, IDPP, CDP, IPPDP and MPhP, using three in vitro assays representing different specific modes of action (MoAs). In the yeast two-hybrid assay, no OPFRs induced agonistic activity, but TiBP, DPK, TPhP, MDPP, CDP and IPPDP were shown to be hydrophobicity-dependent antagonists and to compete with E2 for binding to ERα. In the MVLN cell assay, TPhP was the only OPFR among the 14 tested that was able to activate ERα-estrogen responsive element (ERE) pathways. The results from the E-SCREEN assay showed that all tested OPFRs except TMP had estrogenic properties, and G protein-coupled receptor 30 (GPR30) was involved in the estrogenicity of eight OPFRs, TiBP, THP, TPhP, TCP, MDPP, IPPDP, CDP and MPhP. It was also found that in the E-SCREEN assay, the estrogenicity of alkyl-OPFRs but not aryl-OPFRs was closely correlated to hydrophobicity. Our research suggested that most OPFRs were estrogen disruptors, but their related mechanisms were complex and might involve ERα-mediated and/or ERα-independent pathways. Further in vitro studies concerning the estrogenic effects and involved mechanisms of OPFRs, as well as comprehensive evaluations of OPFRs including health and ecological assessments are needed to determine whether they are safe substitutes for PBDEs.

Tissue-specific bioaccumulation, metabolism and excretion of tris (2-ethylhexyl) phosphate (TEHP) in rare minnow (Gobiocyprisrarus).

Tris (2-ethylhexyl) phosphate (TEHP) is one of the most commonly used organophosphorus flame retardant (OPFR) analogues and is commonly detected in surface water and sediments. Limited information is available about the metabolic pathway or metabolite formation related to TEHP in fish. In this study, rare minnows (Gobiocyprisrarus) were exposed to TEHP in static water for 30 d to investigate the bioaccumulation and metabolite distribution in the fish muscle, liver, kidney, gill, GI-tract, ovary and testis. Based on the estimated kup,parent and kd,parent values, the bioconcentration factors (BCFparent) of TEHP in fish tissues were calculated in the order of kidney > ovary ≈ liver ≈ testis > gill ≈ GI-tract > muscle; this finding was consistent with the results of our previous study on other alkyl-substituted OPFRs. In addition, this study identified the metabolic profiles of TEHP in the liver. TEHP was oxidatively metabolized by the fish to a dealkylated metabolite (di 2-ethylhexyl phosphate; DEHP) and hydroxylated TEHP (OH-TEHP). OH-TEHP further underwent extensive phase II metabolism to yield glucuronic acid conjugates. DEHP was mainly distributed in rare minnow in the following order: liver > GI-tract > kidney â‰« other tissues. However, the metabolite showed lower accumulation potential in fish tissues than TEHP, with metabolite parent concentration factors (MPCFs) for DEHP of less than 0.1 in all the investigated tissues. The BCFparent values of TEHP in various fish tissues were only 9.0 × 10-3-7.2 × 10-4 times its estimated tissue-water partition coefficient (Ktissue-water) values based on tissue lipid, protein and water contents, which indicated the significance of biotransformation in reducing the bioaccumulation potential of TEHP in fish. The toxicokinetic data in the present study help in understanding the tissue-specific bioaccumulation and metabolism pathways of TEHP in fish and highlight the importance of toxicology research on TEHP metabolites in aquatic organisms.

In vitro oxidative stress, mitochondrial impairment and G1 phase cell cycle arrest induced by alkyl-phosphorus-containing flame retardants.

Phosphorus-containing flame retardants (PFRs) have been frequently detected in various environmental samples at relatively high concentrations and are considered emerging environmental pollutants. However, their biological effects and the underlying mechanism remain unclear, especially alkyl-PFRs. In this study, a battery of in vitro bioassays was conducted to analyze the cytotoxicity, oxidative stress, mitochondrial impairment, DNA damage and the involved molecular mechanisms of several selected alkyl-PFRs. Results showed that alkyl-PFRs induced structural related toxicity, where alkyl-PFRs with higher logKow values induced higher cytotoxicity. Long-chain alkyl-PFRs caused mitochondrial and DNA damage, resulting from intracellular reactive oxygen species (ROS) and mitochondrial superoxide overproduction; while short-chain alkyl-PFRs displayed adverse outcomes by significantly impairing mitochondria without obvious ROS generation. In addition, alkyl-PFRs caused DNA damage-induced cell cycle arrest, as determined by flow cytometry, and transcriptionally upregulated key transcription factors in p53/p21-mediated cell cycle pathways. Moreover, compared to the control condition, triisobutyl phosphate and trimethyl phosphate exposure increased the sub-G1 apoptotic peak and upregulated the p53/bax apoptosis pathway, indicating potential cell apoptosis at the cellular and molecular levels. These results provide insight into PFR toxicity and the involved mode of action and indicate the mitochondria is an important target for some alkyl-PFRs.

The steroid receptor coactivator 1 (SRC1) and 3 (SRC3) recruitment as a novel molecular initiating event of 4-n-nonylphenol in estrogen receptor α-mediated pathways.

Recently, the action of steroid receptor coactivators (SRCs) has been recognized to be an important molecular initiating event (MIE) in estrogenic adverse outcome pathways (AOPs). However, the role of SRCs in the molecular mechanisms of many highly concerned environmental estrogens remains poorly understood. In this study, the widely studied environmental estrogen, 4-n-nonylphenol (4-n-NP), was used as a typical pollutant to study SRCs recruitment in its estrogenic effects. In MCF7 cell proliferation (E-SCREEN) assay and MVLN cell assay, 4-n-NP showed significant estrogenic potency that involved an increase in estrogen receptor α (ERα), SRC1 and SRC3 transcript levels. Moreover, 4-n-NP was found to induce estrogen response element (ERE)-mediated activity via ERα in MVLN cells. To investigate the mechanism by which SRCs recruitment is induced by 4-n-NP-ERα, a coactivators recruitment assay was performed, and the results showed that 4-n-NP-ERα recruited both SRC1 and SRC3, whereas it failed to recruit SRC2. Similarly, it had no interaction with SRC2 in the ERα-SRC2 two-hybrid yeast assay. This is the first report to investigate the novel MIE of SRCs recruitment in 4-n-NP-ERα-induced estrogenicity. Overall, our results suggest that the action of 4-n-NP on estrogenic effects involves the following MIEs: the activation of ERα, the recruitment of SRC1 and SRC3, and the induction of ERE-mediated activity. The findings also provide valuable insights into the MIE associated with the different SRCs that are recruited in the adverse outcome pathways of environmental estrogens.

Organophosphorus flame retardants and persistent, bioaccumulative, and toxic contaminants in Arctic seawaters: On-board passive sampling coupled with target and non-target analysis.

Organic pollutants in the Arctic seas have been of concern to many researchers; however, the vast dynamic marine water poses challenges to their comprehensive monitoring within appropriate spatial and temporal scales in the Arctic. In this study, on-board passive sampling of organic pollutants using a self-developed device coupled with triolein-embedded cellulose acetate membranes (TECAMs) was performed during an Arctic cruise. The TECAM extracts were used for target analysis of organophosphorus flame retardants (PFRs), and non-target screening of persistent, bioaccumulative, and toxic (PBT) contaminants using two-dimensional gas chromatography with time-of-flight mass spectrometry (GC × GC-TOFMS). Sixteen chemicals were screened out as PBT contaminants from the 1500 features in the non-target analysis and further identified. Consequently, two chlorinated PFRs (tris(chloroisopropyl)phosphate and tris(1,3-dichloroisopropyl)phosphate) and four PBT contaminants (4-tert-butylphenol, 2-isopropylnaphthalene, 1,1,3-trimethyl-3-phenylindane, and 1-phenylnonan-1-one) were accurately quantified, with the temporally and spatially integrated concentrations ranging from 0.83 ng L-1 to 20.82 ng L-1 in the seawaters. Sources and transport of the contaminants were studied, and ocean current transport (West Spitsbergen Current, WSC) and local sources (human settlement, Arctic oil exploitation, and petroleum fuel emissions) were found to contribute to the presence of the different contaminants. Finally, annual transport fluxes of the contaminants from the North Atlantic to the Arctic Ocean by WSC were estimated, and the results indicate that their hazard to the Arctic should be concerned.

Comparative toxicity study of a novel non-ionic surfactant, vanillin ethoxylates, and nonylphenol ethoxylates in Chinese hamster ovary cells in vitro.

Surfactants such as alkylphenol polyethoxylates (APEOs) and nonylphenol ethoxylates (NPEOs) are commonly used worldwide, but the majority of these compounds, together with their metabolites, have been reported to induce severe biological toxicity. Here, we evaluated for the first time the cytotoxicity, genotoxicity and mitochondrial damage in Chinese hamster ovary (CHO-K1) cells caused by a novel non-ionic surfactant, vanillin ethoxylates (VAEOs), an alternative to APEOs. In parallel, the same in vitro bioassays were conducted on NPEOs along with their metabolic byproducts 4-nonylphenol (4-NP) and vanillin. The results showed that the cytotoxic potency order was NPEOs > 4-NP > VAEOs>vanillin using CCK-8 assays. Also, 4-NP showed potential direct DNA damage in SOS/umu tests, whereas NPEOs, VAEOs and vanillin showed no positive result with and without S9 addition. In addition, none of the test compounds showed obvious genotoxic effects with low olive tail moment value using comet assays. However, all test compounds were shown to cause mitochondrial impairment by increasing mitochondrial mass and decreasing mitochondrial membrane potential in a concentration-dependent manner. And further analysis of reactive oxygen species (ROS) and mitochondrial superoxide (MNSOD) measurement showed that mitochondrial impairment was induced by oxidative stress with intracellular ROS and MNSOD overproduction. It's worth noting that VAEOs and vanillin cause relative lower cytotoxic, genotoxic and mitochondrial damage effects than NPEOs and 4-NP, indicating that VAEOs have the potential to substitute NPEOs as suitable surfactants. Take together, this study elucidates the toxicity profiles of VAEOs and NPEOs relatively comprehensively, and further toxicity analyses are suggested in the population, community and ecosystem.

Insight into the generation of toxic products during chloramination of carbamazepine: Kinetics, transformation pathway and toxicity.

As a widely used antiepileptic drug, carbamazepine (CBZ) has been frequently detected in aquatic environments, even in drinking water. Chloramine is a widely used alternative disinfectant due to its low-level formation of regulated disinfection byproducts (DBPs). However, there is previous evidence linking product mixtures of chloraminated CBZ to stronger DNA damage effects than those caused by CBZ itself. The present study further investigated the reaction rate, transformation mechanism and multi-endpoint toxicity of transformation products (TPs) of CBZ treated with NH2Cl under different pH conditions. The results showed that the reaction between CBZ and NH2Cl at pH 8.5, where NH2Cl is stable, is a second-order reaction with a rate of 4.2 M-1 h-1. Compared to both alkaline and acidic conditions, CBZ was quickly degraded at pH 7. This indicated that HOCl produced from NH2Cl hydrolysis is more effective in degrading CBZ than NH2Cl and NHCl2. Furthermore, the concentration variation of four TPs formed during the chloramination of CBZ under different pH conditions was investigate by quantitative analysis, and the transformation pathway from CBZ to 9(10H)-acridone was confirmed. Three of the detected TPs showed cytotoxicity, DNA damage effects or chromosome damage effects. Acridine and 9(10H)-acridone, which accumulated with increasing time, showed higher cytotoxic or genotoxic effects than CBZ itself. In addition, a similar transformation mechanism was observed in real ambient water during simulated chloramination with a low level of CBZ. These results suggested that despite the chloramination of CBZ being slower than chlorination, TPs with higher cytotoxicity or genotoxicity may lead to greater toxic risks.

Simultaneous passive sampling of hydrophilic and hydrophobic emerging organic contaminants in water.

Passive sampling techniques have been considered robust tools for monitoring freely dissolved concentrations of contaminants in aquatic systems. However, few passive samplers are currently available for the simultaneous sampling of both hydrophilic and hydrophobic chemicals. In this study, we developed a novel passive sampler (a hydrophilic-lipophilic balance sorbent-embedded cellulose acetate membrane (HECAM)) for estimating the time-weighted average (TWA) concentrations of both hydrophilic and hydrophobic organic contaminants in water. In our laboratorial controlled dynamic experiments, the accumulation results of thirty-seven target chemicals (including organophosphorus flame retardants, phenols, estrogens, organophosphorus pesticides, and triazine herbicides) with a wide polarity range (1.44 < log Kow < 9.49) in the HECAM followed first-order kinetics well, and the passive sampling parameters were estimated successfully. The estimated sampling rates for the target chemicals in the HECAM ranged from 0.14 to 6.90 L d-1 in the laboratory experiment, and the log Ksw (equilibrium partition coefficient between the sampler and water) values ranged from 2.75 to 6.00. The HECAM exhibited high sampling rate for moderately hydrophilic and moderately hydrophobic chemicals. The field validation study in an urban river resulted in the detection of four target chemicals (tris(chloroisopropyl)phosphate, tris(1,3-dichloroisopropyl)phosphate, prometryn, and 4-tert-octylphenol) by the HECAM at estimated TWA concentrations of 10.9-179.5 ng L-1, which were in agreement with the measured levels found in traditional grab samples by solid-phase extraction. In summary, both the laboratory tests and field deployment showed practicable results for the HECAM passive sampling, which suggests that it is an efficient approach for simultaneous monitoring of hydrophilic and hydrophobic organic contaminants in water.

Aryl-phosphorus-containing flame retardants induce oxidative stress, the p53-dependent DNA damage response and mitochondrial impairment in A549 cells.

Aryl phosphorus-containing flame retardants (aryl-PFRs) have been frequently detected with increasingly used worldwide as one of alternatives for brominated flame retardants. However, information on their adverse effects on human health and ecosystem is insufficient, with limited study on their molecular mode of action in vitro. In this study, the cytotoxicity, DNA damage, mitochondrial impairment and the involved molecular mechanisms of certain frequently detectable aryl-PFRs, including 2-ethylhexyldiphenyl phosphate (EHDPP), methyl diphenyl phosphate (MDPP), bisphenol-A bis (diphenyl phosphate) (BDP), isodecyl diphenyl phosphate (IDPP), cresyl diphenyl phosphate (CDP) and the structurally similar and widely used organophosphorus pesticide chlorpyrifos (CPF), were evaluated in A549 cells using high-content screening (HCS) system. Aryl-PFRs showed different lethal concentration 50 (LC50) values ranging from 97.94 to 546.85 µM in A549 cells using CCK-8 assay. EHDPP, IDPP, CDP, MDPP and CPF demonstrated an ability to induce DNA damage, evidenced by increased DNA content and S phase-reducing cell cycle arrest effect using fluorophore dye cocktail assay. Additionally, the selected aryl-PFRs induced mitochondrial impairment by the increasing mitochondrial mass and decreasing mitochondrial membrane potential. Moreover, BDP, MDPP, and CDP, which contain short alkyl chains showed their potential oxidative stress with intracellular ROS and mitochondrial superoxide overproduction from an initially relatively low concentration. Additionally, based on the promotion of firefly luminescence in p53-transfected A549 cells, p53 activation was found to be involved in aryl-PFRs-induced DNA damage. Further real-time PCR results showed that all selected aryl-PFRs triggered p53/p21/gadd45ß-, and p53/p21/mdm2-mediated cell cycle pathways, and the p53/bax mediated apoptosis pathway to induce DNA damage and cytotoxic effects. These results suggest that aryl-PFRs (e.g., BDP, MDPP, CDP) cause oxidative stress-mediated DNA damage and mitochondrial impairment, and p53-dependent pathway was involved in the aryl-PFRs-induced DNA damage and cell cycle arrest. In conclusion, this study improves the understanding of PFRs-induced adverse outcomes and the involved molecular mechanism.

Toxicokinetic patterns, metabolites formation and distribution in various tissues of the Chinese rare minnow (Gobiocypris rarus) exposed to tri(2­butoxyethyl) phosphate (TBOEP) and tri-n-butyl phosphate (TNBP).

Alkylated organophosphate esters (alkyl-OPEs) are widely used and extensively detected in aquatic organisms. This work investigated the tissue-specific toxicokinetics of two common alkyl-OPEs, tri(2­butoxyethyl) phosphate (TBOEP) and tri­n­butyl phosphate (TNBP) in Chinese rare minnow (Gobiocypris rarus) through a 50 day uptake and depuration experiment. The tissue-specific bioconcentration factor (BCF) values for the two alkyl-OPEs ranged from 1 to 30 L/kg wet weight (ww), with the kidney and ovary as the tissues with the highest accumulation. The tissue BCFs only exhibited a significant correlation with lipid contents only in storage tissues (i.e., muscle, brain, ovary and testis), indicating that lipids might not be the major contributor to tissue distribution of TBOEP and TNBP. However, the contribution of blood perfusion and active transport to tissue-specific OPE accumulation needs to be further investigated. Lower accumulation of metabolites than parent chemicals was observed, with metabolite parent concentration factors (MPCFs) <1. Di-alkyl phosphate (DAP), bis(2­butoxyethyl) phosphate (BBOEP) and di(n-butyl) phosphate (DNBP) were the most abundantly formed metabolites of TBOEP and TNBP in various tissues, followed by the monohydroxylated OPEs (OH-OPEs). However, bis(2­butoxyethyl) hydroxyethyl phosphate (BBOEHEP), was detected at much lower levels in the tissues. All the investigated metabolites showed high production rates (kprod,metabolites) in the fish liver, followed by the GI tract and the kidney, indicating the importance of the hepatobiliary and urinary systems in eliminating the metabolites. Our study suggested that metabolism plays an important role in eliminating these two alkyl-OPEs in rare minnow and results in different tissue distribution mechanisms for metabolites and their compounds.

A comparison of endocrine disruption potential of nonylphenol ethoxylate, vanillin ethoxylate, 4-n-nonylphenol and vanillin in vitro.

The widely used surfactant nonylphenol ethoxylate (NPEO) and its raw material 4-n-nonylphenol (4-n-NP), as well as its degradation products, are recognized as endocrine disrupting chemicals. The USA Environmental Protection Agency (EPA) released an assessment that looked for safe alternatives to NPEO. Vanillin ethoxylate (VAEO) is a novel substitute for NPEO and is quite similar to NPEO in structure; there is a risk that it has similar endocrine disrupting effects to NPEO. However, their effects on various nuclear hormone receptors have not been thoroughly examined. In this study, the effects of NPEO, VAEO, 4-n-NP and Vanillin on the estrogen receptor α (ERα), androgen receptor (AR), thyroid hormone receptor (TR), retinoic X receptor ß (RXRß) and estrogen-related receptor γ (ERRγ) were determined and compared using a battery of recombined yeast strains expressing ß-galactosidase. The results showed that NPEO and 4-n-NP acted as significant antagonists of ER, AR, TR and ERRγ. In addition, 4-n-NP also had antagonistic activity toward RXRß. Moreover, VAEO was shown to be a very weak antagonist of TR and ERRγ, and Vanillin had no interaction with any nuclear receptors. For the first time, it was found that NPEO had AR, TR and ERRγ antagonistic effects and that 4-n-NP was an antagonist of RXRß. The in vitro data indicated that NPEO, 4-n-NP and VAEO have the potential to act as endocrine disruptors involving more than one nuclear hormone receptor, but VAEO has much lower endocrine disrupting potential than NPEO. Thus, it is critical to find safe substitutes for NPEO and a substitute of NPEO with structural analogues should be carried out with caution. Furthermore, to look for preferable alternatives for NPEO, more in vivo and in vitro studies of the alternatives concerning endocrine disruption are needed, especially in vitro studies need to involve various target points, not only focus on their effects on ER but also take other nuclear hormone receptor pathways into consideration.