Applied In Vitro Toxicology最新文献

Due to the ∼86,000 chemicals registered under the Toxic Substances Control Act and increasing ethical concerns regarding animal testing, it is not economically or technically feasible to screen every registered chemical for toxicity using animal-based toxicity assays. To address this challenge, regulatory agencies are investigating high-throughput screening in vitro methods to increase speed of toxicity testing, while reducing the overall cost. One approach for rapid toxicity testing currently being investigated is monitoring of volatile emissions produced by cell lines in culture. Such a metabolomics approach would measure gaseous emissions from a cell line and determine if such gaseous metabolites are altered upon exposure to a xenobiotic. Herein, we describe the history and rationale of monitoring endogenously produced volatiles for identification of pathologic conditions, as well as emerging applications in toxicity testing for such an approach.

In vitro air-liquid interface (ALI) cell culture models can potentially be used to assess inhalation toxicology endpoints and are usually considered, in terms of relevancy, between classic (i.e., submerged) in vitro models and animal-based models. In some situations that need to be clearly defined, ALI methods may represent a complement or an alternative option to in vivo experimentations or classic in vitro methods. However, it is clear that many different approaches exist and that only very limited validation studies have been carried out to date. This means comparison of data from different methods is difficult and available methods are currently not suitable for use in regulatory assessments. This is despite inhalation toxicology being a priority area for many governmental organizations. In this setting, a 1-day workshop on ALI in vitro models for respiratory toxicology research was organized in Paris in March 2016 to assess the situation and to discuss what might be possible in terms of validation studies. The workshop was attended by major parties in Europe and brought together more than 60 representatives from various academic, commercial, and regulatory organizations. Following plenary, oral, and poster presentations, an expert panel was convened to lead a discussion on possible approaches to validation studies for ALI inhalation models. A series of recommendations were made and the outcomes of the workshop are reported.

Three-Dimensional (3D) liver microtissues, specifically prepared from primary human hepatocytes (PHH) in coculture with nonparenchymal cells (NPCs), have been shown to be a valuable tool for in vitro toxicology. However, a lack of thorough characterization on a functional, transcriptomic, and proteomic level of such models during long-term cultivation is evident. By integrating multiple omics technologies, we provide in this study an in-depth long-term characterization of 3D microtissues composed of PHH from three different donors cocultured with primary NPCs. The 3D human liver microtissues (hLiMTs) exhibited stable adenosine triphosphate (ATP) content and albumin secretion over 5 weeks. Histological analysis indicated a healthy liver tissue with polarized expression of bile salt export pump (BSEP) and multidrug resistance protein 2 (MRP2) in a structure reminiscent of bile canaliculi. The 3D microtissues exhibited stable basal and inducible cytochrome P450 activities up to 5 weeks in culture. Analysis of 40,716 transcripts using RNA arrays revealed distinct similarities to native human liver gene expression. Long-term culture showed a stable phenotype up to 5 weeks, with differences in liver gene expression primarily attributed to individual donors. Proteomic profiling of 2200 unique proteins by label-free LC-MS/MS revealed a relatively stable protein expression where only 7.3% were up- or downregulated more than twofold from day 7 to 35 in culture. Taken together, these results suggest that hLiMTs represent a responsive and physiologically relevant in vitro liver model that maintains stable function over 5 weeks and is therefore well suited for repeated-dose toxicity testing.

Introduction: Understanding biotransformation pathways in aquatic species is an integral part of ecological risk assessment with respect to the potential bioactivation of chemicals to more toxic metabolites. The long-range goal is to gain sufficient understanding of fish metabolic transformation reactions to be able to accurately predict fish xenobiotic metabolism. While some metabolism data exist, there are few fish in vivo exposure studies where metabolites have been identified and the metabolic pathways proposed. Previous biotransformation work has focused on in vitro studies which have the advantage of high throughput but may have limited metabolic capabilities, and in vivo studies which have full metabolic capacity but are low throughput. An aquatic model system with full metabolic capacity in which a large number of chemicals could be tested would be a valuable tool.

Materials and methods: The current study evaluated the ex vivo rainbow trout liver slice model, which has the advantages of high throughput as found in vitro models and non-dedifferentiated cells and cell to cell communication found in in vivo systems. The pesticide diazinon, which has been previously tested both in vitro and in vivo in a number of mammalian and aquatic species including rainbow trout, was used to evaluate the ex vivo slice model as a tool to study biotransformation pathways.

Results/discussion: While somewhat limited by the analytical chemistry method employed, results of the liver slice model, mainly that hydroxypyrimidine was the major diazinon metabolite, are in line with the results of previous rainbow trout in vivo studies.

Conclusion: Therefore, the rainbow trout liver slice model is a useful tool for the study of metabolism in aquatic species.

Arsenic, a naturally occurring environmental contaminant, is harmful to humans at elevated concentrations. Increased levels of arsenic in the environment occur as a result of human activities and from natural geologically sourced leaching into ground and surface water. These sources pose an exposure risk above the USEPA standard to individuals whose food and water sources become contaminated. Arsenic exposure negatively impacts organ function and increases the risk for developing pathologies, including cancer. Some of the effects of arsenic on cancer translate to normal cell function in wound healing. To evaluate whether arsenic influences wound healing, an in vitro scratch assay was employed to study the effects of arsenic on cellular migration, which is a key component in the normal wound-healing process. In this study, skin cells were exposed to environmentally relevant concentrations of arsenic, and wound closure was evaluated. Results indicated that arsenic significantly decreased the rate of cellular migration in the scratch assay when compared with controls. In addition, estradiol, which has been shown to positively influence cellular and tissue processes involved in wound healing, reversed the slowing effects of arsenic on wound closure. These results suggest that arsenic contamination may inhibit, and estrogen may provide a therapeutic benefit for individuals with arsenic-contaminated wounds.

In vitro substrate depletion methods developed by the pharmaceutical industry are being used with increasing frequency to support chemical bioaccumulation assessments for fish. However, the application of these methods to high log K _ow chemicals poses special challenges. Biotransformation of three polycyclic aromatic hydrocarbons (PAHs) was measured using trout liver S9 fractions. Measured activity declined with incubation time and was reduced by acetone (used as a spiking solvent) at concentrations greater than 0.5%. Addition of alamethicin, a pore-forming peptide used to support UDP-glucuronosyltransferase activity, also reduced activity in a concentration-dependent manner. The substrate concentration dependence of activity was evaluated to estimate K _M and V _max values for each compound. Derived kinetic constants suggested that all three PAHs are transformed by the same reaction pathway and indicated an inverse correlation between K _M and chemical log K _ow. Binding effects on activity were evaluated by measuring unbound chemical concentrations across a range of S9 protein levels. Reaction rates were proportional to the unbound concentration except when these concentrations approached saturating levels, providing a direct demonstration of the free chemical hypothesis. These findings suggest that previous in vitro work with high log K _ow compounds was conducted at inappropriately high substrate concentrations resulting in underestimation of true in vivo activity. Preliminary calculations also indicate that PAH metabolism in fish may approach saturation during standardized in vivo testing efforts, potentially resulting in concentration-dependent accumulation and/or steady-state levels of accumulation greater than those which occur in a natural setting.

Introduction: The Adverse Outcome Pathway framework is increasingly used to integrate data generated based on traditional and emerging toxicity testing paradigms. As the number of AOP descriptions has increased, so has the need to define the AOP in computable terms.

Materials and methods: Herein, we present a comprehensive annotation of 172 AOPs housed in the AOP-Wiki as of December 4, 2016 using terms from existing biological ontologies.

Results: AOP Key Events (KEs) were assigned ontology terms using a concept called the Event Component, which consists of a Process, an Object, and an Action term, with each term originating from ontologies and other controlled vocabularies. Annotation of KEs with ontology classes from fourteen ontologies and controlled vocabularies resulted in a total of 685 KEs being annotated with a total of 809 Event Components. A set of seven conventions resulted, defining the annotation of KEs via Event Components.

Discussion: This expanded annotation of AOPs allows computational reasoners to aid in both AOP development and applications. In addition, the incorporation of explicit biological objects will reduce the time required for converting a qualitative AOP description into a conceptual model that can support computational modeling. As high throughput genomics becomes a more important part of the high throughput toxicity testing landscape, the new approaches described here for annotating key events will also promote the visualization and analysis of genomics data in an AOP context.

Chemical-induced liver injury can be manifested in a number of ways, such as cholestasis, steatosis, fibrosis, and cancer. The mechanisms driving these toxicological processes have been well characterized and have been embedded in adverse outcome pathway frameworks in recent years. This article provides a concise overview of these constructs.