Technical evaluation and standardization of the human thyroid microtissue assay.

The success and sustainability of U.S. EPA efforts to reduce, refine, and replace in vivo animal testing depends on the ability to translate toxicokinetic and toxicodynamic data from in vitro and in silico new approach methods (NAMs) to human-relevant exposures and health outcomes. Organotypic culture models employing primary human cells enable consideration of human health effects and inter-individual variability but present significant challenges for test method standardization, transferability, and validation. Increasing confidence in the information provided by these in vitro NAMs requires setting appropriate performance standards and benchmarks, defined by the context of use, to consider human biology and mechanistic relevance without animal data. The human thyroid microtissue (hTMT) assay utilizes primary human thyrocytes to reproduce structural and functional features of the thyroid gland that enable testing for potential thyroid-disrupting chemicals. As a variable-donor assay platform, conventional principles for assay performance standardization need to be balanced with the ability to predict a range of human responses. The objectives of this study were to (1) define the technical parameters for optimal donor procurement, primary thyrocyte qualification, and performance in the hTMT assay, and (2) set benchmark ranges for reference chemical responses. Thyrocytes derived from a cohort of 32 demographically diverse euthyroid donors were characterized across a battery of endpoints to evaluate morphological and functional variability. Reference chemical responses were profiled to evaluate the range and chemical-specific variability of donor-dependent effects within the cohort. The data-informed minimum acceptance criteria for donor qualification and set benchmark parameters for method transfer proficiency testing and validation of assay performance.

Development of a bioprinter-based method for incorporating metabolic competence into high-throughput in vitro assays.

The acceptance and use of in vitro data for hazard identification, prioritization, and risk evaluation is partly limited by uncertainties associated with xenobiotic metabolism. The lack of biotransformation capabilities of many in vitro systems may under- or overestimate the hazard of compounds that are metabolized to more or less active metabolites in vivo. One approach to retrofitting existing bioassays with metabolic competence is the lid-based Alginate Immobilization of Metabolic Enzymes (AIME) method, which adds hepatic metabolism to conventional high-throughput screening platforms. Here, limitations of the lid-based AIME method were addressed by incorporating bioprinting, which involved depositing S9-encapsulated microspheres into standard 384-well plates with requisite cofactors for phase I and II hepatic metabolism. Objectives of this study included: 1) compare the lid-based and AIME bioprinting methods by assessing the enzymatic activity of a common cytochrome P450 (CYP) enzyme, 2) use biochemical assays with the bioprinting method to characterize additional measures of phase I and II metabolic activity, and 3) evaluate the bioprinting method by screening 25 chemicals of known metabolism-dependent bioactivity in the VM7Luc estrogen receptor transactivation (ERTA) assay. A comparison of the two methods revealed comparable precision and dynamic range. Activity of additional CYP enzymes and glucuronidation was observed using the AIME bioprinting method. The ERTA experiment identified 19/21 ER-active test chemicals, 14 of which were concordant with expected biotransformation effects (73.7%). Additional refinement of the AIME bioprinting method has the potential to expand high-throughput screening capabilities in a robust, accessible manner to incorporate in vitro metabolic competence.

The DevTox Germ Layer Reporter Platform: An Assay Adaptation of the Human Pluripotent Stem Cell Test.

Environmental chemical exposures are a contributing factor to birth defects affecting infant morbidity and mortality. The USA EPA is committed to developing new approach methods (NAMs) to detect chemical risks to susceptible populations, including pregnant women. NAM-based coverage for cellular mechanisms associated with early human development could enhance identification of potential developmental toxicants (DevTox) for new and existing data-poor chemicals. The human pluripotent stem cell test (hPST) is an in vitro test method for rapidly identifying potential human developmental toxicants that employs directed differentiation of embryonic stem cells to measure reductions in SOX17 biomarker expression and nuclear localization. The objective of this study was to expand on the hPST principles to develop a model platform (DevTox GLR) that utilizes the transgenic RUES2-GLR cell line expressing fluorescent reporter fusion protein biomarkers for SOX17 (endoderm marker), BRA (mesoderm marker), and SOX2 (ectoderm and pluripotency marker). Initial assay adaption to definitive endoderm (DevTox GLR-Endo) was performed to emulate the hPST SOX17 endpoint and enable comparative evaluation of concordant chemical effects. Assay duration was reduced to two days and screening throughput scaled to 384-well format for enhanced speed and efficiency. Assay performance for 66 chemicals derived from reference and training set data resulted in a balanced accuracy of 72% (79% sensitivity and 65% specificity). The DevTox GLR-Endo assay demonstrates successful adaptation of the hPST concept with increased throughput, shorter assay duration, and minimal endpoint processing. The DevTox GLR model platform expands the in vitro NAM toolbox to rapidly identify potential developmental hazards and mechanistically characterize toxicant effects on pathways and processes associated with early human development.

Chemical Screening in an Estrogen Receptor Transactivation Assay With Metabolic Competence.

The U.S. EPA continues to utilize high-throughput screening data to evaluate potential biological effects of endocrine active substances without the use of animal testing. Determining the scope and need for in vitro metabolism in high-throughput assays requires the generation of larger data sets that assess the impact of xenobiotic transformations on toxicity-related endpoints. The objective of the current study was to screen a set of 768 ToxCast chemicals in the VM7Luc estrogen receptor transactivation assay (ERTA) using the Alginate Immobilization of Metabolic Enzymes hepatic metabolism method. Chemicals were screened with or without metabolism to identify estrogenic effects and metabolism-dependent changes in bioactivity. Based on estrogenic hit calls, 85 chemicals were active in both assay modes, 16 chemicals were only active without metabolism, and 27 chemicals were only active with metabolism. Using a novel metabolism curve shift method that evaluates the shift in concentration-response curves, 29 of these estrogenic chemicals were identified as bioactivated and 59 were bioinactivated. Human biotransformation routes and associated metabolites were predicted in silico across the chemicals to mechanistically characterize possible transformation-related ERTA effects. Overall, the study profiled novel chemicals associated with metabolism-dependent changes in ERTA bioactivity, and suggested routes of biotransformation and putative metabolites responsible for the observed estrogenic effects. The data demonstrate a range of metabolism-dependent effects across a diverse chemical library and highlight the need to evaluate the role of intrinsic xenobiotic metabolism for endocrine and other toxicity-related health effects.

Landscape-level variation in Bt crops predict Helicoverpa zea (Lepidoptera: Noctuidae) resistance in cotton agroecosystems.

BACKGROUND: Helicoverpa zea (Boddie) damage to Bt cotton and maize has increased as a result of widespread Bt resistance across the USA Cotton Belt. Our objective was to link Bt crop production patterns to cotton damage through a series of spatial and temporal surveys of commercial fields to understand how Bt crop production relates to greater than expected H. zea damage to Bt cotton. To do this, we assembled longitudinal cotton damage data that spanned the Bt adoption period, collected cotton damage data since Bt resistance has been detected, and estimated local population susceptibility using replicated on-farm studies that included all Bt pyramids marketed in cotton. RESULTS: Significant year effects of H. zea damage frequency in commercial cotton were observed throughout the Bt adoption period, with a recent damage increase after 2012. Landscape-level Bt crop production intensity over time was positively associated with the risk of H. zea damage in two- and three-toxin pyramided Bt cotton. Helicoverpa zea damage also varied across Bt toxin types in spatially replicated on-farm studies. CONCLUSIONS: Landscape-level predictors of H. zea damage in Bt cotton can be used to identify heightened Bt resistance risk areas and serves as a model to understand factors that drive pest resistance evolution to Bt toxins in the southeastern United States. These results provide a framework for more effective insect resistance management strategies to be used in combination with conventional pest management practices that improve Bt trait durability while minimizing the environmental footprint of row crop agriculture. © 2021 Society of Chemical Industry. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Characterization of Novel Human Immortalized Thyroid Follicular Epithelial Cell Lines.

Introduction: Investigation of normal human thyroid function using in vitro culture systems is dependent on cells that recapitulate physiology of differentiated thyrocytes. Primary thyrocytes retain features of the native organ but have limited lifespan in culture. Immortalized thyrocytes offer an alternative if challenges maintaining phenotypic stability can be overcome to retain functional features of primary cells. Materials and Methods: CI-SCREEN immortalization technology was applied to normal human thyroid tissue to generate four cell line variants. The lines were characterized for transgene integration, biomarker expression, genomic stability, and proliferation rates. Thyroid Stimulating Hormone (TSH)-dependent morphology, thyroglobulin production, thyroxine hormone synthesis, and viability were assessed using conventional 2D monolayer and 3D microtissue culture formats in huThyrEC or h7H medium. Results: Despite differential transgene profiles, the lines had similar biomarker expression patterns and proliferation rates. In 2D culture there was no thyroxine synthesis or changes in viability, but TSH-dependent thyroglobulin production was more significant for several lines in h7H than huThyrEC medium. Comparatively, in 3D microtissues, TSH-dependent thyroglobulin induction was greater for cell lines in h7H medium. Synthesis of thyroxine in one cell line was higher than background with TSH exposure, but not significantly different than control. Discussion: Immortalization of primary human thyrocytes yielded transgenic lines of epithelial origin. When evaluated in 2D or 3D culture formats, h7H medium supported thyroglobulin production to a greater magnitude than huThyrEC medium. One cell line cultured in 3D microtissue format marginally recapitulated T4 synthesis under continuous TSH exposure. Conclusion: Select human thyroid cell lines exhibited morphological and functional features of primary thyrocytes and are a novel resource for in vitro disease modeling and toxicity testing that will enable reproducible culture models more representative of normal human thyroid function.

Estimations of Fine-Scale Species Distributions of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in Eastern Florida.

Many species distribution maps indicate the ranges of Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse) overlap in Florida despite the well-documented range reduction of Ae. aegypti. Within the last 30 yr, competitive displacement of Ae. aegypti by Ae. albopictus has resulted in partial spatial segregation of the two species, with Ae. aegypti persisting primarily in urban refugia. We modeled fine-scale distributions of both species, with the goal of capturing the outcome of interspecific competition across space by building habitat suitability maps. We empirically parameterized models by sampling 59 sites in south and central Florida over time and incorporated climatic, landscape, and human population data to identify predictors of habitat suitability for both species. Our results show human density, precipitation, and urban land cover drive Ae. aegypti habitat suitability, compared with exclusively climatic variables driving Ae. albopictus habitat suitability. Remotely sensed variables (macrohabitat) were more predictive than locally collected metrics (microhabitat), although recorded minimum daily temperature showed significant, inverse relationships with both species. We detected minor Aedes habitat segregation; some periurban areas that were highly suitable for Ae. albopictus were unsuitable for Ae. aegypti. Fine-scale empirical models like those presented here have the potential for precise risk assessment and the improvement of operational applications to control container-breeding Aedes mosquitoes.

Sunshine versus gold: The effect of population age on genetic structure of an invasive mosquito.

The genetic diversity and structure of invasive species are affected by the time since invasion, but it is not well understood how. We compare likely the oldest populations of Aedes aegypti in continental North America with some of the newest to illuminate the range of genetic diversity and structure that can be found within the invasive range of this important disease vector. Aedes aegypti populations in Florida have probably persisted since the 1600-1700s, while populations in southern California derive from new invasions that occurred in the last 10 years. For this comparison, we genotyped 1,193 individuals from 28 sites at 12 highly variable microsatellites and a subset of these individuals at 23,961 single nucleotide polymorphisms (SNPs). This is the largest sample analyzed for genetic structure for either region, and it doubles the number of southern California populations previously analyzed. As predicted, the older populations (Florida) showed fewer indicators of recent founder effect and bottlenecks; in particular, these populations have dramatically higher genetic diversity and lower genetic structure. Geographic distance and driving distance were not good predictors of genetic distance in either region, especially southern California. Additionally, southern California had higher levels of genetic differentiation than any comparably sized documented region throughout the worldwide distribution of the species. Although population age and demographic history are likely driving these differences, differences in climate and transportation practices could also play a role.

Patterns of genetic divergence among populations of Aedes aegypti L. (Diptera: Culicidae) in the southeastern USA.

BACKGROUND: The yellow fever mosquito, Aedes aegypti is a public health concern in the USA, especially in the wake of emergent diseases such as Zika and chikungunya. Aedes aegypti populations dwindled after the invasion of Aedes albopictus in the 1980s and many populations were extirpated. However, in some areas Ae. aegypti persisted in small populations and there are reports of recent resurgences of Ae. aegypti in Florida, Louisiana, Nevada and California. We assessed the population genetic structure of Ae. aegypti in Florida and Georgia, which has concomitant consequences related to mosquito dispersal, pesticide resistance and vectorial capacity. METHODS: We collected Ae. aegypti across Florida and in Georgia using ovitraps. We hatched the eggs and reared them to adults, and after sacrifice we extracted their DNA. We then probed each individual for variation in 6 microsatellite markers, which we used to address population genetic characteristics. RESULTS: We collected Ae. aegypti and genotyped seven Florida populations and one Georgia population using microsatellite markers. We found evidence of isolation by distance model of gene flow supported by driving distance among cities within Florida and two theoretic genetic clusters. CONCLUSIONS: Significant genetic structure between some populations with substantial gene flow between geographically distant cities suggests regional genetic structuring of Ae. aegypti in Florida. This study provides information on the genetic exchange between populations of Ae. aegypti in the southeastern USA and suggests potential routes of spread of this species.

Simplification of vector communities during suburban succession.

Suburbanization is happening rapidly on a global scale, resulting in changes to the species assemblages present in previously undeveloped areas of land. Community-level changes after anthropogenic land-use change have been studied in a variety of organisms, but the effects on arthropods of medical and veterinary importance remain poorly characterized. Shifts in diversity, abundance, and community composition of such arthropods, like mosquitoes, can significantly impact vector-borne disease dynamics due to varying vectorial capacity between different species. In light of these potential implications for vector-borne diseases, we investigated changes in mosquito species assemblage after suburbanization by sampling mosquitoes in neighborhoods of different ages in Wake County, North Carolina, US. We found that independent of housing density and socioeconomic status, mosquito diversity measures decreased as suburban neighborhoods aged. In the oldest neighborhoods, the mosquito assemblage reached a distinct suburban climax community dominated by the invasive, peridomestic container-breeding Aedes albopictus, the Asian tiger mosquito. Aedes albopictus is a competent vector of many pathogens of human concern, and its dominance in suburban areas places it in close proximity with humans, allowing for heightened potential of host-vector interactions. While further research is necessary to explicitly characterize the effects of mosquito community simplification on vector-borne disease transmission in highly suburbanized areas, the current study demonstrates that suburbanization is disrupting mosquito communities so severely that they do not recover their diversity even 100 years after the initial disturbance. Our understanding of the community-level effects of anthropogenic land-use change on arthropod vectors will become increasingly important as we look to mitigate disease spread in a global landscape that is continually developed and altered by humans.

Surveillance for Immature Mosquitoes in Windshield Wash Basins at Gas Stations.

Gas stations often provide windshield wash basins (WWBs) that customers may use to clean their windshields. Motivated by casual observations, we conducted a survey of WWBs in and around Raleigh, NC, to determine whether these WWBs also serve as larval habitats for mosquitoes. We found that 27.7% (95% CI: 12.4-43.14%) of the 36 surveyed gas stations had mosquito larvae in their WWBs, and 22.4% (95% CI: 15.07-29.1%) of the 152 WWBs surveyed were positive for mosquito larvae. Two species were identified inhabiting these containers: Aedes albopictus (Skuse) and Culex quinquefasciatus Say. Aedes albopictus was associated with clear, unturbid water, whereas Cx. quinquefasciatus did not have any significant association with water characteristics. Pupae of both species were observed, suggesting these habitats could be sources of pest mosquitoes. Gas stations may be a convenient surveillance target for vector control specialists and may provide insight into human-aided mosquito dispersal.