Phenotypic screening in Organ-on-a-Chip systems: a 1537 kinase inhibitor library screen on a 3D angiogenesis assay.

Modern drug development increasingly requires comprehensive models that can be utilized in the earliest stages of compound and target discovery. Here we report a phenotypic screening exercise in a high-throughput Organ-on-a-Chip setup. We assessed the inhibitory effect of 1537 protein kinase inhibitors in an angiogenesis assay. Over 4000 micro-vessels were grown under perfusion flow in microfluidic chips, exposed to a cocktail of pro-angiogenic factors and subsequently exposed to the respective kinase inhibitors. Efficacy of compounds was evaluated by reduced angiogenic sprouting, whereas reduced integrity of the main micro-vessel was taken as a measure for toxicity. The screen yielded 53 hits with high anti-angiogenicity and low toxicity, of which 44 were previously unassociated with angiogenic pathways. This study demonstrates that Organ-on-a-Chip models can be screened in high numbers to identify novel compounds and targets. This will ultimately reduce bias in early-stage drug development and increases probability to identify first in class compounds and targets for today's intractable diseases.

Exposure Monitoring Strategies for Applying Low-Cost PM Sensors to Assess Flour Dust in Industrial Bakeries.

Low-cost particulate matter (PM) sensors provide new methods for monitoring occupational exposure to hazardous substances, such as flour dust. These devices have many possible benefits, but much remains unknown about their performance for different exposure monitoring strategies in the workplace. We explored the performance of PM sensors for four different monitoring strategies (time-weighted average and high time resolution, each quantitative and semi-quantitative) for assessing occupational exposure using low-cost PM sensors in a field study in the industrial bakery sector. Measurements were collected using four types of sensor (PATS+, Isensit, Airbeam2, and Munisense) and two reference devices (respirable gravimetric samplers and an established time-resolved device) at two large-scale bakeries, spread over 11 participants and 6 measurement days. Average PM2.5 concentrations of the low-cost sensors were compared with gravimetric respirable concentrations for 8-h shift periods and 1-min PM2.5 concentrations of the low-cost sensors were compared with time-resolved PM2.5 data from the reference device (quantitative monitoring strategy). Low-cost sensors were also ranked in terms of exposure for 8-h shifts and for 15-min periods with a shift (semi-quantitative monitoring strategy). Environmental factors and methodological variables, which can affect sensor performance, were investigated. Semi-quantitative monitoring strategies only showed more accurate results compared with quantitative strategies when these were based on shift-average exposures. The main factors that influenced sensor performance were the type of placement (positioning the devices stationary versus personal) and the company or workstation where measurements were collected. Together, these findings provide an overview of common strengths and drawbacks of low-cost sensors and different ways these can be applied in the workplace. This can be used as a starting point for further investigations and the development of guidance documents and data analysis methods.

In vitro grafting of hepatic spheroids and organoids on a microfluidic vascular bed.

With recent progress in modeling liver organogenesis and regeneration, the lack of vasculature is becoming the bottleneck in progressing our ability to model human hepatic tissues in vitro. Here, we introduce a platform for routine grafting of liver and other tissues on an in vitro grown microvascular bed. The platform consists of 64 microfluidic chips patterned underneath a 384-well microtiter plate. Each chip allows the formation of a microvascular bed between two main lateral vessels by inducing angiogenesis. Chips consist of an open-top microfluidic chamber, which enables addition of a target tissue by manual or robotic pipetting. Upon grafting a liver microtissue, the microvascular bed undergoes anastomosis, resulting in a stable, perfusable vascular network. Interactions with vasculature were found in spheroids and organoids upon 7 days of co-culture with space of Disse-like architecture in between hepatocytes and endothelium. Veno-occlusive disease was induced by azathioprine exposure, leading to impeded perfusion of the vascularized spheroid. The platform holds the potential to replace animals with an in vitro alternative for routine grafting of spheroids, organoids, or (patient-derived) explants.

Exploring Evaluation Variables for Low-Cost Particulate Matter Monitors to Assess Occupational Exposure.

(1) Background: Small, lightweight, low-cost optical particulate matter (PM) monitors are becoming popular in the field of occupational exposure monitoring, because these devices allow for real-time static measurements to be collected at multiple locations throughout a work site as well as being used as wearables providing personal exposure estimates. Prior to deployment, devices should be evaluated to optimize and quantify measurement accuracy. However, this can turn out to be difficult, as no standardized methods are yet available and different deployments may require different evaluation procedures. To gain insight in the relevance of different variables that may affect the monitor readings, six PM monitors were selected based on current availability and evaluated in the laboratory; (2) Methods: Existing strategies that were judged appropriate for the evaluation of PM monitors were reviewed and seven evaluation variables were selected, namely the type of dust, within- and between-device variations, nature of the power supply, temperature, relative humidity, and exposure pattern (peak and constant). Each variable was tested and analyzed individually and, if found to affect the readings significantly, included in a final correction model specific to each monitor. Finally, the accuracy for each monitor after correction was calculated; (3) Results: The reference materials and exposure patterns were found to be main factors needing correction for most monitors. One PM monitor was found to be sufficiently accurate at concentrations up to 2000 µg/m3 PM2.5, with other monitors appropriate at lower concentrations. The average accuracy increased by up to three-fold compared to when the correction model did not include evaluation variables; (4) Conclusions: Laboratory evaluation and readings correction can greatly increase the accuracy of PM monitors and set boundaries for appropriate use. However, this requires identifying the relevant evaluation variables, which are heavily reliant on how the monitors are used in the workplace. This, together with the lack of current consensus on standardized procedures, shows the need for harmonized PM monitor evaluation methods for occupational exposure monitoring.

Performance of a Single Layer of Clothing or Gloves to Prevent Dermal Exposure to Pesticides.

The suitability, availability, and use of protective clothing are critical factors determining the actual dermal exposure (ADE) of operators and workers to pesticides. A realistic assessment of occupational exposure to pesticides requires information about the performance of protective clothing during everyday use. In this study, the performance of clothing or gloves has been investigated based on available dermal exposure data in order to provide recommendations for default protection factors that can be used in regulatory exposure assessments. Suitable dermal exposure data from available exposure databases were collated and analysed. The data that met the selection criteria for the analysis of the performance of protective clothing comprised studies in which protective clothing like cotton coveralls, cotton clothing, polyester-cotton coveralls, Sontara coveralls, Tyvek coveralls, butyl/neoprene gloves, latex/PE/vinyl/PVC gloves, or nitrile gloves were worn. Based on available potential and ADE levels, the migration of pesticides through this protective clothing was estimated. Evaluation of exposure data showed that on average only 2.3-2.6% of the pesticides present on the outside of the clothing or gloves migrated through the garments, although there was a large variation with migration up to 99%. Forearms, legs, and chest areas of the clothing tended to have the greatest migration of pesticides. Caution is needed in the selection of the appropriate protection offered protective clothing for specific situations. This study gives valuable information on the performance of protective clothing, for use in exposure assessment and for default setting in exposure modelling, taking into account the type of clothing or gloves worn. As new data become available, it may be possible to further refine the protection factors offered by different types of clothing or gloves, particularly where a common protocol has been used.

Programmed Effects in Neurobehavior and Antioxidative Physiology in Zebrafish Embryonically Exposed to Cadmium: Observations and Hypothesized Adverse Outcome Pathway Framework.

Non-communicable diseases (NCDs) are a major cause of premature mortality. Recent studies show that predispositions for NCDs may arise from early-life exposure to low concentrations of environmental contaminants. This developmental origins of health and disease (DOHaD) paradigm suggests that programming of an embryo can be disrupted, changing the homeostatic set point of biological functions. Epigenetic alterations are a possible underlying mechanism. Here, we investigated the DOHaD paradigm by exposing zebrafish to subtoxic concentrations of the ubiquitous contaminant cadmium during embryogenesis, followed by growth under normal conditions. Prolonged behavioral responses to physical stress and altered antioxidative physiology were observed approximately ten weeks after termination of embryonal exposure, at concentrations that were 50-3200-fold below the direct embryotoxic concentration, and interpreted as altered developmental programming. Literature was explored for possible mechanistic pathways that link embryonic subtoxic cadmium to the observed apical phenotypes, more specifically, the probability of molecular mechanisms induced by cadmium exposure leading to altered DNA methylation and subsequently to the observed apical phenotypes. This was done using the adverse outcome pathway model framework, and assessing key event relationship plausibility by tailored Bradford-Hill analysis. Thus, cadmium interaction with thiols appeared to be the major contributor to late-life effects. Cadmium-thiol interactions may lead to depletion of the methyl donor S-adenosyl-methionine, resulting in methylome alterations, and may, additionally, result in oxidative stress, which may lead to DNA oxidation, and subsequently altered DNA methyltransferase activity. In this way, DNA methylation may be affected at a critical developmental stage, causing the observed apical phenotypes.

Zebrafish embryos as a screen for DNA methylation modifications after compound exposure.

Modified epigenetic programming early in life is proposed to underlie the development of an adverse adult phenotype, known as the Developmental Origins of Health and Disease (DOHaD) concept. Several environmental contaminants have been implicated as modifying factors of the developing epigenome. This underlines the need to investigate this newly recognized toxicological risk and systematically screen for the epigenome modifying potential of compounds. In this study, we examined the applicability of the zebrafish embryo as a screening model for DNA methylation modifications. Embryos were exposed from 0 to 72 h post fertilization (hpf) to bisphenol-A (BPA), diethylstilbestrol, 17α-ethynylestradiol, nickel, cadmium, tributyltin, arsenite, perfluoroctanoic acid, valproic acid, flusilazole, 5-azacytidine (5AC) in subtoxic concentrations. Both global and site-specific methylation was examined. Global methylation was only affected by 5AC. Genome wide locus-specific analysis was performed for BPA exposed embryos using Digital Restriction Enzyme Analysis of Methylation (DREAM), which showed minimal wide scale effects on the genome, whereas potential informative markers were not confirmed by pyrosequencing. Site-specific methylation was examined in the promoter regions of three selected genes vasa, vtg1 and cyp19a2, of which vasa (ddx4) was the most responsive. This analysis distinguished estrogenic compounds from metals by direction and sensitivity of the effect compared to embryotoxicity. In conclusion, the zebrafish embryo is a potential screening tool to examine DNA methylation modifications after xenobiotic exposure. The next step is to examine the adult phenotype of exposed embryos and to analyze molecular mechanisms that potentially link epigenetic effects and altered phenotypes, to support the DOHaD hypothesis.