scholarly journals Potency ranking of per- and polyfluoroalkyl substances using high-throughput transcriptomic analysis of human liver spheroids

2021 ◽  
Author(s):  
A J F Reardon ◽  
A Rowan-Carroll ◽  
S S Ferguson ◽  
K Leingartner ◽  
R Gagne ◽  
...  
Keyword(s):  
Gene Expression ◽  
Risk Assessment ◽  
High Throughput ◽  
Human Liver ◽  
Human Health Risk ◽  
Carbon Chain Length ◽  
Human Liver Cell ◽  
Polyfluoroalkyl Substances ◽  
Potency Ranking

Abstract Per- and polyfluoroalkyl substances (PFAS) are some of the most prominent organic contaminants in human blood. Although the toxicological implications of human exposure to perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) are well established, data on lesser-understood PFAS are limited. New approach methodologies (NAMs) that apply bioinformatic tools to high-throughput data are being increasingly considered to inform risk assessment for data-poor chemicals. The aim of this study was to compare the potencies (i.e., benchmark concentrations: BMCs) of PFAS in primary human liver microtissues (3D spheroids) using high-throughput transcriptional profiling. Gene expression changes were measured using TempO-seq, a templated, multiplexed RNA-sequencing platform. Spheroids were exposed for 1 or 10 days to increasing concentrations of 23 PFAS in three subgroups: carboxylates (PFCAs), sulfonates (PFSAs), and fluorotelomers and sulfonamides. PFCAs and PFSAs exhibited trends toward increased transcriptional potency with carbon chain-length. Specifically, longer-chain compounds (7 to 10 carbons) were more likely to induce changes in gene expression and have lower transcriptional BMCs. The combined high-throughput transcriptomic and bioinformatic analyses support the capability of NAMs to efficiently assess the effects of PFAS in liver microtissues. The data enable potency ranking of PFAS for human liver cell spheroid cytotoxicity and transcriptional changes, and assessment of in vitro transcriptomic points of departure. These data improve our understanding of the possible health effects of PFAS and will be used to inform read-across for human health risk assessment.

scholarly journals High-throughput transcriptomics and benchmark concentration modeling for potency ranking of per- and polyfluoroalkyl substances (PFAS) in exposed human liver cell spheroids

2020 ◽  
Author(s):  
A.J.F. Reardon ◽  
A. Rowan-Carroll ◽  
S.S. Ferguson ◽  
K. Leingartner ◽  
R. Gagne ◽  
...  
Keyword(s):  
Gene Expression ◽  
Risk Assessment ◽  
High Throughput ◽  
Liver Cell ◽  
Human Liver ◽  
Human Health Risk ◽  
Human Liver Cell ◽  
Transcriptional Changes ◽  
Polyfluoroalkyl Substances ◽  
Potency Ranking

AbstractPer- and polyfluoroalkyl substances (PFAS) are some of the most prominent organic contaminants in human blood. Although the toxicological implications from human exposure to perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) are well established, data on lesser-understood PFAS are limited. New approach methodologies (NAMs) that apply bioinformatic tools to high-throughput data are being increasingly considered to inform risk assessment for data-poor chemicals. The aim of this investigation was to identify biological response potencies (i.e., benchmark concentrations: BMCs) following PFAS exposures to inform read-across for risk assessment of data-poor PFAS. Gene expression changes were measured in primary human liver cell microtissues (i.e., 3D spheroids) after 1-day and 10-day exposures to increasing concentrations of 23 PFAS. The cells were treated with four subgroups of PFAS: carboxylates (PFCAs), sulfonates (PFSAs), fluorotelomers, and sulfonamides. An established pipeline to identify differentially expressed genes and transcriptomic BMCs was applied. We found that both PFCAs and PFSAs exhibited a trend toward increased transcriptional changes with carbon chain-length. Specifically, longer-chain compounds (7 to 10 carbons) were more likely to induce changes in gene expression, and have lower transcriptional BMCs. The combined high-throughput transcriptomic and bioinformatic analyses supports the capability of NAMs to efficiently assess the effects of PFAS in liver microtissues. The data enable potency ranking of PFAS for human liver cell spheroid cytotoxicity and transcriptional changes, and assessment of in vitro transcriptomic points of departure. These data improve our understanding of the health effects of PFAS and will be used to inform read-across for human health risk assessment.

Abstracts

2008 ◽  
Vol 27 (6) ◽  
pp. 405-405
Author(s):  
David J. Dix
Keyword(s):  
Risk Assessment ◽  
High Throughput ◽  
Environmental Protection Agency ◽  
High Throughput Screening ◽  
Human Health Risk ◽  
Environmental Chemicals ◽  
In Vitro Assays ◽  
The Impact ◽  
Toxicity Pathways

The U.S. Environmental Protection Agency (EPA), National Toxicology Program (NTP), and National Institutes of Health (NIH) Chemical Genomics Center (NCGC) have complementary research programs designed to improve chemical toxicity evaluations by developing high throughput screening (HTS) methods that evaluate the impact of environmental chemicals on key toxicity pathways. These federal partners are coordinating an extension of the EPA’s ToxCast program, the NTP’s HTS initiative, and the NCGC’s Molecular Libraries Initiative into a collaborative research program focused on identifying toxicity pathways and developing in vitro assays to characterize the ability of chemicals to perturb those pathways. The goal is to develop new paradigm for high throughput toxicity testing that collects mechanistic and quantitative data from in vitro assays measuring chemical modulation of biological processes involved in the progression to toxicity. As toxicity pathways are identified, the in vitro assays can be optimized for comparison to in vivo animal studies, and for predicting effects in humans. Subsequent computational modeling of toxicity pathway responses and appropriate chemical dosimetry will need to be developed to make these predictions relevant for human health risk assessment. This work was reviewed by EPA and approved for publication but does not necessarily reflect official Agency policy. Index Terms: Toxicogenomics, High Throughput Screening/Testing, EPA ToxCast, Chemical Risk Assessment

Human liver cell cultivation for long-term in vitro toxicity studies in a miniaturized multi-compartment 3D bioreactor system

2011 ◽  
Vol 49 (01) ◽  
Author(s):  
SA Hoffmann ◽  
M Lübberstedt ◽  
U Müller-Vieira ◽  
D Knobeloch ◽  
A Nüssler ◽  
...  
Keyword(s):  
Liver Cell ◽  
Human Liver ◽  
In Vitro Toxicity ◽  
Cell Cultivation ◽  
Toxicity Studies ◽  
Human Liver Cell ◽  
Bioreactor System

In Vitro Toxicology Methods in Risk Assessment

1996 ◽  
Vol 24 (3) ◽  
pp. 325-331
Author(s):  
Iain F. H. Purchase
Keyword(s):  
Risk Assessment ◽  
Hazard Assessment ◽  
Human Health Risk ◽  
In Vitro Test ◽  
In Vitro Methods ◽  
New Concepts ◽  
Vitro Test

The title of this paper is challenging, because the question of how in vitro methods and results contribute to human health risk assessment is rarely considered. The process of risk assessment usually begins with hazard assessment, which provides a description of the inherent toxicological properties of the chemical. The next step is to assess the relevance of this to humans, i.e. the human hazard assessment. Finally, information on exposure is examined, and risk can then be assessed. In vitro methods have a limited, but important, role to play in risk assessment. The results can be used for classification and labelling; these are methods of controlling exposure, analogous to risk assessment, but without considering exposure. The Ames Salmonella test is the only in vitro method which is incorporated into regulations and used widely. Data from this test can, at best, lead to classification of a chemical with regard to genotoxicity, but cannot be used for classification and labelling on their own. Several in vitro test systems which assess the topical irritancy and corrosivity of chemicals have been reasonably well validated, and the results from these tests can be used for classification. The future development of in vitro methods is likely to be slow, as it depends on the development of new concepts and ideas. The in vivo methods which currently have reasonably developed in vitro alternatives will be the easiest to replace. The remaining in vivo methods, which provide toxicological information from repeated chronic dosing, with varied endpoints and by mechanisms which are not understood, will be more difficult to replace.

scholarly journals Shortening and Improving the Embryonic Stem Cell Test through the Use of Gene Biomarkers of Differentiation

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Andrea C. Romero ◽  
Eugenio Vilanova ◽  
Miguel A. Sogorb
Keyword(s):  
Gene Expression ◽  
Risk Assessment ◽  
Stem Cell ◽  
Cell Viability ◽  
Embryonic Stem Cell ◽  
Total Duration ◽  
Embryonic Stem ◽  
End Points ◽  
Cell Test

The embryonic Stem cell Test (EST) is a validated assay for testing embryotoxicityin vitro. The total duration of this protocol is 10 days, and its main end-point is based on histological determinations. It is suggested that improvements on EST must be focused toward molecular end-points and, if possible, to reduce the total assay duration. Five days of exposure of D3 cells in monolayers under spontaneous differentiation to 50 ng/mL of the strong embryotoxic 5-fluorouracil or to 75 μg/mL of the weak embryotoxic 5,5-diphenylhydeantoin caused between 20 and 74% of reductions in the expression of the following genes:Pnpla6,Afp,Hdac7,Vegfa, andNes. The exposure to 1 mg/mL of nonembryotoxic saccharin only caused statistically significant reductions in the expression ofNes. These exposures reduced cell viability of D3 cells by 15, 28, and 34%. We applied these records to the mathematical discriminating function of the EST method to find that this approach is able to correctly predict the embryotoxicity of all three above-mentioned chemicals. Therefore, this work proposes the possibility of improve EST by reducing its total duration and by introducing gene expression as biomarker of differentiation, which might be very interesting forin vitrorisk assessment embryotoxicity.

scholarly journals Reversible Gene Expression Control inYersinia pestisby Using an Optimized CRISPR Interference System

2019 ◽  
Vol 85 (12) ◽  
Author(s):  
Tong Wang ◽  
Min Wang ◽  
Qingwen Zhang ◽  
Shiyang Cao ◽  
Xiang Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Throughput ◽  
Biofilm Formation ◽  
Prevention And Control ◽  
Target Gene ◽  
Gene Knockdown ◽  
Content Type ◽  
Short Palindromic Repeat ◽  
And Control

ABSTRACTMany genes in the bacterial pathogenYersinia pestis, the causative agent of three plague pandemics, remain uncharacterized, greatly hampering the development of measures for plague prevention and control. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been shown to be an effective tool for gene knockdown in model bacteria. In this system, a catalytically dead Cas9 (dCas9) and a small guide RNA (sgRNA) form a complex, binding to the specific DNA target through base pairing, thereby impeding RNA polymerase binding and causing target gene repression. Here, we introduce an optimized CRISPRi system usingStreptococcus pyogenesCas9-derived dCas9 for gene knockdown inY. pestis. Multiple genes harbored on either the chromosome or plasmids ofY. pestiswere efficiently knocked down (up to 380-fold) in a strictly anhydrotetracycline-inducible manner using this CRISPRi approach. Knockdown ofhmsH(responsible for biofilm formation) orcspB(encoding a cold shock protein) resulted in greatly decreased biofilm formation or impaired cold tolerance inin vitrophenotypic assays. Furthermore, silencing of the virulence-associated genesyscBorailusing this CRISPRi system resulted in attenuation of virulence in HeLa cells and mice similar to that previously reported foryscBandailnull mutants. Taken together, our results confirm that this optimized CRISPRi system can reversibly and efficiently repress the expression of target genes inY. pestis, providing an alternative to conventional gene knockdown techniques, as well as a strategy for high-throughput phenotypic screening ofY. pestisgenes with unknown functions.IMPORTANCEYersiniapestisis a lethal pathogen responsible for millions of human deaths in history. It has also attracted much attention for potential uses as a bioweapon or bioterrorism agent, against which new vaccines are desperately needed. However, manyY. pestisgenes remain uncharacterized, greatly hampering the development of measures for plague prevention and control. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been successfully used in a variety of bacteria in functional genomic studies, but no such genetic tool has been reported inY. pestis. Here, we systematically optimized the CRISPRi approach for use inY. pestis, which ultimately repressed target gene expression with high efficiency in a reversible manner. Knockdown of functional genes using this method produced phenotypes that were readily detected byin vitroassays, cell infection assays, and mouse infection experiments. This is a report of a CRISPRi approach inY. pestisand highlights the potential use of this approach in high-throughput functional genomics studies of this pathogen.

In vitro effects of estetrol on receptor binding, drug targets and human liver cell metabolism

Climacteric ◽  
2008 ◽  
Vol 11 (sup1) ◽  
pp. 64-68 ◽  
Author(s):  
M. Visser ◽  
J.-M. Foidart ◽  
H. J. T. Coelingh Bennink
Keyword(s):  
Receptor Binding ◽  
Liver Cell ◽  
Human Liver ◽  
Drug Targets ◽  
Cell Metabolism ◽  
Human Liver Cell ◽  
In Vitro Effects
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