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1.
Environ Sci Technol ; 58(1): 143-149, 2024 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-38154793

RESUMO

Human exposure to perfluorinated alkylate substances (PFASs) is usually assessed from the concentrations in serum or plasma, assuming one-compartment toxicokinetics. To characterize body distributions of major PFASs, we obtained and extracted tissue samples from 19 forensic autopsies of healthy adult subjects who had died suddenly and were not known to have elevated levels of PFAS exposure. As target organs of toxicological importance, we selected the liver, kidneys, lungs, spleen, and brain, as well as whole blood. Samples weighing about 0.1 g were analyzed by liquid chromatography coupled to triple mass spectrometers. Minor variations in PFAS concentrations were found between the kidney cortex and medulla and between lung lobes. Organ concentrations of perfluorooctanoic sulfonate (PFOS) and perfluorononanoate (PFNA) correlated well with blood concentrations, while perfluorooctanoate (PFOA) and perfluorohexanoic sulfonate (PFHxS) showed more variable associations. Likewise, the liver concentrations correlated well with those of other organs. Calculations of relative distributions were carried out to assess the interdependence of organ retentions. Equilibrium model predictions largely explained the observed PFAS distributions, except for the brain. Although the samples were small and affected by a possible lack of homogeneity, these findings support the use of blood-PFAS concentrations as a measure of PFAS exposure, with the liver possibly acting as the main organ of retention.


Assuntos
Ácidos Alcanossulfônicos , Poluentes Ambientais , Fluorocarbonos , Adulto , Humanos , Alcanossulfonatos , Plasma , Fluorocarbonos/farmacocinética
2.
Environ Sci Technol ; 57(40): 14817-14826, 2023 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-37756184

RESUMO

Animal studies have pointed at the liver as a hotspot for per- and polyfluoroalkyl substances (PFAS) accumulation and toxicity; however, these findings have not been replicated in human populations. We measured concentrations of seven PFAS in matched liver and plasma samples collected at the time of bariatric surgery from 64 adolescents in the Teen-Longitudinal Assessment of Bariatric Surgery (Teen-LABS) study. Liver:plasma concentration ratios were perfectly explained (r2 > 0.99) in a multilinear regression (MLR) model based on toxicokinetic (TK) descriptors consisting of binding to tissue constituents and membrane permeabilities. Of the seven matched plasma and liver PFAS concentrations compared in this study, the liver:plasma concentration ratio of perfluoroheptanoic acid (PFHpA) was considerably higher than the liver:plasma concentration ratio of other PFAS congeners. Comparing the MLR model with an equilibrium mass balance model (MBM) suggested that complex kinetic transport processes are driving the unexpectedly high liver:plasma concentration ratio of PFHpA. Intratissue MBM modeling pointed to membrane lipids as the tissue constituents that drive the liver accumulation of long-chain, hydrophobic PFAS, whereas albumin binding of hydrophobic PFAS dominated PFAS distribution in plasma. The liver:plasma concentration data set, empirical MLR model, and mechanistic MBM modeling allow the prediction of liver from plasma concentrations measured in human cohort studies. Our study demonstrates that combining biomonitoring data with mechanistic modeling can identify underlying mechanisms of internal distribution and specific target organ toxicity of PFAS in humans.


Assuntos
Ácidos Alcanossulfônicos , Cirurgia Bariátrica , Poluentes Ambientais , Fluorocarbonos , Animais , Humanos , Adolescente , Estudos de Coortes , Fígado , Fluorocarbonos/análise
3.
Environ Toxicol Chem ; 2022 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-36039972

RESUMO

Aquatic toxicity tests with benthic organisms are used to predict the toxicity of hydrophobic organic chemicals (HOCs) in sediments, assuming that the freely dissolved concentration (Cfree ) is a good surrogate of bioavailability in the exposure system. However, Cfree of HOCs is difficult to control in water-only setups. Moreover, the role of dissolved organic carbon (DOC) in the occurrence of toxicity needs clarification because DOC concentrations in sediment porewater can be substantially higher than in typical test water. We introduced biocompatible polyethylene meshes with high sorptive capacities and fast release kinetics as a novel passive dosing phase, which maintained Cfree and Cwater (i.e., free + DOC-bound) in Hyalella azteca water-only tests. Adding the supernatant fraction of peat to test water as a DOC source increased Cwater to an extent comparable to sediment porewater and significantly increased and decreased the observed toxicity of permethrin and benzo[a]pyrene, respectively, to H. azteca. This result indicates that DOC can both benefit and harm test species likely due to the increased health after ingestion of DOC and to the uptake of DOC-bound HOCs, respectively. Passive dosing in combination with the addition of sediment DOC surrogates may better reflect exposure and habitat conditions in sediment porewater than conventional aquatic tests. Environ Toxicol Chem 2022;00:1-10. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

4.
Toxicol In Vitro ; 73: 105133, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33662518

RESUMO

Nominal effect concentrations from in vitro toxicity assays may lead to inaccurate estimations of in vivo toxic doses because the nominal concentration poorly reflects the concentration at the molecular target in cells in vitro, which is responsible for initiating effects and can be referred to as the biologically effective dose. Chemicals can differentially distribute between in vitro assay compartments, including serum constituents in exposure medium, microtitre plate plastic, headspace and extracellular matrices. The partitioning of test chemicals to these extracellular compartments reduces the concentration at the molecular target. Free concentrations in medium and cell-associated concentrations are considered better proxies of the biologically effective dose. This paper reviews the mechanisms by which test chemicals distribute between in vitro assay compartments, and also lists the physicochemical properties driving the extent of this distribution. The mechanisms and physicochemical properties driving the distribution of test chemical in vitro help explain the makeup of mass balance models that estimate free concentrations and cell-associated concentrations in in vitro toxicity assays. A thorough understanding of the distribution processes and assumptions underlying these mass balance models helps define chemical and biological applicability domains of individual models, as well as provide a perspective on how to improve model predictivity and quantitative in vitro-in vivo extrapolations.


Assuntos
Bioensaio , Modelos Biológicos , Testes de Toxicidade , Animais , Simulação por Computador , Humanos , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismo
5.
Environ Health Perspect ; 128(7): 77007, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32700975

RESUMO

BACKGROUND: High-throughput screening of chemicals with in vitro reporter gene assays in Tox21 has produced a large database on cytotoxicity and specific modes of action. However, the validity of some of the reported activities is questionable due to the "cytotoxicity burst," which refers to the supposition that many stress responses are activated in a nonspecific way at concentrations close to cell death. OBJECTIVES: We propose a pragmatic method to identify whether reporter gene activation is specific or cytotoxicity-triggered by comparing the measured effects with baseline toxicity. METHODS: Baseline toxicity, also termed narcosis, is the minimal toxicity any chemical causes. Quantitative structure-activity relationships (QSARs) developed for baseline toxicity in mammalian reporter gene cell lines served as anchors to define the chemical-specific threshold for the cytotoxicity burst and to evaluate the degree of specificity of the reporter gene activation. Measured 10% effect concentrations were related to measured or QSAR-predicted 10% cytotoxicity concentrations yielding specificity ratios (SR). We applied this approach to our own experimental data and to ∼8,000 chemicals that were tested in six of the high-throughput Tox21 reporter gene assays. RESULTS: Confirmed baseline toxicants activated reporter gene activity around cytotoxic concentrations triggered by the cytotoxicity burst. In six Tox21 assays, 37%-87% of the active hits were presumably caused by the cytotoxicity burst (SR<1) and only 2%-14% were specific with SR≥10 against experimental cytotoxicity but 75%-97% were specific against baseline toxicity. This difference was caused by a large fraction of chemicals showing excess cytotoxicity. CONCLUSIONS: The specificity analysis for measured in vitro effects identified whether a cytotoxicity burst had likely occurred. The SR-analysis not only prevented false positives, but it may also serve as measure for relative effect potency and can be used for quantitative in vitro-in vivo extrapolation and risk assessment of chemicals. https://doi.org/10.1289/EHP6664.


Assuntos
Bioensaio , Testes de Toxicidade/métodos , Genes Reporter , Humanos , Relação Quantitativa Estrutura-Atividade
6.
Chem Res Toxicol ; 33(7): 1770-1779, 2020 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-32227843

RESUMO

High-throughput in vitro reporter gene assays are increasingly applied to assess the potency of chemicals to alter specific cellular signaling pathways. Genetically modified reporter gene cell lines provide stable readouts of the activation of cellular receptors or transcription factors of interest, but such reporter gene assays have been criticized for not capturing cellular metabolism. We characterized the metabolic activity of the widely applied AREc32 (human breast cancer MCF-7), ARE-bla (human liver cancer HepG2), and GR-bla (human embryonic kidney HEK293) reporter gene cells in the absence and in the presence of benzo[a]pyrene (BaP), an AhR ligand known to upregulate cytochrome P450 in vitro and in vivo. We combined fluorescence microscopy with chemical analysis, real-time PCR, and ethoxyresorufin-O-deethylase activity measurements to track temporal changes in BaP and its metabolites in the cells and surrounding medium over time in relation to the expression and activity of metabolic enzymes. Decreasing BaP concentrations and formation of metabolites agreed with the high basal CYP1 activity of ARE-bla and the strong CYP1A1 mRNA induction in AREc32, whereas BaP concentrations were constant in GR-bla, in which neither metabolites nor CYP1 induction was detected. The study emphasizes that differences in sensitivity between reporter gene assays may be caused not only by different reporter constructs but also by a varying biotransformation rate of the evaluated parent chemical. The basal metabolic capacity of reporter gene cells in the absence of chemicals is not a clear indication because we demonstrated that the metabolic activity can be upregulated by AhR ligands during the assay. The combination of methods presented here is suitable to characterize the metabolic activity of cells in vitro and can improve the interpretation of in vitro reporter gene effect data and extrapolation to in vivo human exposure.


Assuntos
Benzopirenos/farmacologia , Bioensaio , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Modelos Biológicos , Genes Reporter , Células HEK293 , Células Hep G2 , Humanos , Células MCF-7 , RNA Mensageiro/metabolismo
7.
Chem Res Toxicol ; 32(8): 1462-1468, 2019 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-31328914

RESUMO

Controlling the exposure of chemicals in in vitro mammalian cell assays is an important prerequisite for the application of in vitro methods in risk and hazard assessment of chemicals. Existing models require numerous physicochemical and system parameters to quantify the effective concentration in the assay. Synthesizing these studies, this article briefly communicates how the protein-rich supplement in the medium can be utilized to adjust constant and quantifiable exposure concentrations without the need for measurements and complex modeling. We present a simplified mass balance equation based on chemical properties and system parameters from openly accessible databases, which can be used to adjust the dose of chemicals in the exposure medium, leading to defined and stable freely dissolved concentrations (Cfree). The proposed framework prevents experimental artifacts associated with the use of cosolvents and medium oversaturation and enables the conversion of in vitro effect data to freely dissolved effect concentrations (ECfree), which can directly be applied in quantitative in vitro to in vivo extrapolation models and compared to other exposure scenarios.


Assuntos
Bioensaio , Meios de Cultura/análise , Ensaios de Triagem em Larga Escala , Animais , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura/farmacologia , Bases de Dados de Compostos Químicos , Relação Dose-Resposta a Droga , Humanos , Modelos Biológicos
8.
Arch Toxicol ; 93(8): 2295-2305, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31230094

RESUMO

Improved understanding of chemical exposure in in vitro bioassays is required for quantitative in vitro-in vivo extrapolation (QIVIVE). In this study, we quantified freely dissolved concentrations in medium sampled from in vitro cell-based bioassays (Cfree,medium) for nine chemicals with different hydrophobicity and speciation at the time point of dosing and after an incubation period of 24 h using solid-phase microextraction. The chemicals were tested in two reporter gene assays, the AREc32 assay indicative of the oxidative stress response and the PPARγ-GeneBLAzer assay that responds to chemicals which bind to the peroxisome proliferator-activated receptor gamma. For seven of the nine chemicals, Cfree,medium did not change significantly over time in both assays and the experimentally determined Cfree,medium generally agreed well with predictions of a mass balance model that describes the partitioning between proteinaceous and lipidous medium constituents, cells and the aqueous phase. Two chemicals showed a decrease of Cfree,medium in the AREc32 assay over time that was probably caused by cellular metabolism. Furthermore, Cfree,medium of the acidic chemical diclofenac deviated from the model predictions by more than a factor of 10 at higher concentrations, which indicates nonlinear binding and saturation of the medium proteins. Bioassay results are typically reported as nominal effect concentrations (ECnom), although it is established that freely dissolved effect concentrations (ECfree) are a better measure for the bioavailable dose and the method developed here provides a simple experimental approach to measure and model ECfree in in vitro bioassay for improved QIVIVE models.


Assuntos
Bioensaio/métodos , Microextração em Fase Sólida/métodos , Sobrevivência Celular/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Técnicas In Vitro , Estresse Oxidativo/efeitos dos fármacos , PPAR gama/metabolismo , Solubilidade
9.
Chem Res Toxicol ; 32(1): 168-178, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30585484

RESUMO

The effects measured with in vitro cell-based bioassays are typically reported as nominal effect concentrations ( Cnom), but the freely dissolved concentration in the exposure medium ( Cw) and the total cellular concentration ( Ccell) are considered more quantitative dose metrics that allow extrapolation to the whole-organism level. To predict Cw and Ccell, the partitioning of the test chemicals to medium proteins and lipids and cells has to be known. In this study, we developed a solid-phase microextraction (SPME) method based on C18-coated fibers to quantify the partitioning of diclofenac, 2,4-dichlorophenoxyacetic acid (2,4-D), ibuprofen, naproxen, torasemide, warfarin, and genistein to bovine serum albumin (BSA), phospholipid liposomes, fetal bovine serum (FBS), and cells. For ibuprofen, 2,4-D, naproxen, and warfarin, the partitioning to the SPME fibers was found to be concentration dependent, which had to be considered for the calculation of distribution ratios to biological materials. The sorption isotherms to FBS were nonlinear for diclofenac, 2,4-D, ibuprofen, naproxen, and warfarin. The FBS isotherms could be described by assuming that the total amount of chemical bound to FBS is the sum of the amount specifically bound to the binding sites of albumin and nonspecifically bound to all medium proteins and lipids. The determined cell-water distribution ratios ( Dcell/w) differed considerably between four different cell lines (up to 1.83 log-units) and also between different batches of the same cell line (up to 0.48 log-units). The relative importance of protein and lipid content for Dcell/w was evaluated with a mass balance model and different types of cellular proteins and lipids as input parameters. Existing in vitro mass balance models may underestimate Cw because they do not account for saturable protein binding and overestimate Ccell for organic acids, if BSA is used as surrogate for cellular proteins.


Assuntos
Fosfolipídeos/química , Soroalbumina Bovina/química , Microextração em Fase Sólida , Ácido 2,4-Diclorofenoxiacético/análise , Animais , Bovinos , Células Cultivadas , Diclofenaco/análise , Genisteína/análise , Células HEK293 , Humanos , Ibuprofeno/análise , Cinética , Lipossomos/química , Naproxeno/análise , Torasemida/análise , Varfarina/análise
10.
Environ Sci Technol ; 52(22): 13511-13522, 2018 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-30298728

RESUMO

Sorption to the polystyrene (PS) of multiwell plates can affect the exposure to organic chemicals over time in in vitro and in vivo bioassays. Experimentally determined diffusion coefficients in PS ( DPS) were in a narrow range of 1.25 to 8.0 · 10-16 m2 s-1 and PS-water partition constants ( KPS/w) ranged from 0.04 to 5.10 log-units for 22 neutral organic chemicals. A kinetic model, which explicitly accounts for diffusion in the plastic, was applied to predict the depletion of neutral organic chemicals from different bioassay media by sorption to various multiwell plate formats. For chemicals with log Kow > 3, the medium concentrations decreased rapidly and considerably in the fish embryo toxicity assay but medium concentrations remained relatively constant in the cell-based bioassays with medium containing 10% fetal bovine serum (FBS), emphasizing the ability of the protein- and lipid-rich medium to compensate for losses by multiwell plate sorption. The PS sorption data may serve not only for exposure assessment in bioassays but also to model the contaminant uptake by and release from plastic packaging material and the chemical transport by PS particles in the environment.


Assuntos
Compostos Orgânicos , Poliestirenos , Animais , Bioensaio , Cinética , Plásticos
11.
Chem Res Toxicol ; 31(8): 646-657, 2018 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-29939727

RESUMO

Cellular uptake kinetics are key for understanding time-dependent chemical exposure in in vitro cell assays. Slow cellular uptake kinetics in relation to the total exposure time can considerably reduce the biologically effective dose. In this study, fluorescence microscopy combined with automated image analysis was applied for time-resolved quantification of cellular uptake of 10 neutral, anionic, cationic, and zwitterionic fluorophores in two reporter gene assays. The chemical fluorescence in the medium remained relatively constant during the 24-h assay duration, emphasizing that the proteins and lipids in the fetal bovine serum (FBS) supplemented to the assay medium represent a large reservoir of reversibly bound chemicals with the potential to compensate for chemical depletion by cell uptake, growth, and sorption to well materials. Hence FBS plays a role in stabilizing the cellular dose in a similar way as polymer-based passive dosing, here we term this process as serum-mediated passive dosing (SMPD). Neutral chemicals accumulated in the cells up to 12 times faster than charged chemicals. Increasing medium FBS concentrations accelerated uptake due to FBS-facilitated transport but led to lower cellular concentrations as a result of increased sorption to medium proteins and lipids. In vitro cell exposure results from the interaction of several extra- and intracellular processes, leading to variable and time-dependent exposure between different chemicals and assay setups. The medium FBS plays a crucial role for the thermodynamic equilibria as well as for the cellular uptake kinetics, hence influencing exposure. However, quantification of cellular exposure by an area under the curve (AUC) analysis illustrated that, for the evaluated bioassay setup, current in vitro exposure models that assume instantaneous equilibrium between medium and cells still reflect a realistic exposure because the AUC was typically reduced less than 20% compared to the cellular dose that would result from instantaneous equilibrium.


Assuntos
Substâncias Perigosas/farmacocinética , Microscopia de Fluorescência/métodos , Animais , Área Sob a Curva , Linhagem Celular , Linhagem Celular Tumoral , Meios de Cultura , Genes Reporter , Humanos , Técnicas In Vitro , Termodinâmica
12.
Chem Res Toxicol ; 30(5): 1197-1208, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28316234

RESUMO

High-throughput in vitro bioassays are becoming increasingly important in the risk characterization of anthropogenic chemicals. Large databases gather nominal effect concentrations (Cnom) for diverse modes of action. However, the biologically effective concentration can substantially deviate due to differences in chemical partitioning. In this study, we modeled freely dissolved (Cfree), cellular (Ccell), and membrane concentrations (Cmem) in the Tox21 GeneBLAzer bioassays for a set of neutral and ionogenic organic chemicals covering a large physicochemical space. Cells and medium constituents were experimentally characterized for their lipid and protein content, and partition constants were either collected from the literature or predicted by mechanistic models. The chemicals exhibited multifaceted partitioning to proteins and lipids with distribution ratios spanning over 8 orders of magnitude. Modeled Cfree deviated over 5 orders of magnitude from Cnom and can be compared to in vivo effect data, environmental concentrations, and the unbound fraction in plasma, which is needed for the in vitro to in vivo extrapolation. Ccell was relatively constant for chemicals with membrane lipid-water distribution ratios of 1000 or higher and proportional to Cnom. Representing a sum parameter for exposure that integrates the entire dose from intracellular partitioning, Ccell is particularly suitable for the effect characterization of chemicals with multiple target sites and the calculation of their relative effect potencies. Effective membrane concentrations indicated that the specific effects of very hydrophobic chemicals in multiple bioassays are occurring at concentrations close to baseline toxicity. The equilibrium partitioning model including all relevant system parameters and a generic bioassay setup is attached as an excel workbook to this paper and can readily be applied to diverse in vitro bioassays.


Assuntos
Bioensaio , Exposição Ambiental , Modelos Teóricos , Testes de Toxicidade , Genes Reporter , Células HEK293 , Células Hep G2 , Ensaios de Triagem em Larga Escala , Humanos , Técnicas In Vitro , Relação Quantitativa Estrutura-Atividade
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