Zebrafish on a Chip: A Novel Platform for Real-Time Monitoring of Drug-Induced Developmental Toxicity

Pharmaceutical safety testing requires a cheap, fast and highly efficient platform for real-time evaluation of drug toxicity and secondary effects. In this study, we have developed a microfluidic system for phenotype-based evaluation of toxic and teratogenic effects of drugs using zebrafish (Danio rerio) embryos and larvae as the model organism. The microfluidic chip is composed of two independent functional units, enabling the assessment of zebrafish embryos and larvae. Each unit consists of a fluidic concentration gradient generator and a row of seven culture chambers to accommodate zebrafish. To test the accuracy of this new chip platform, we examined the toxicity and teratogenicity of an anti-asthmatic agent-aminophylline (Apl) on 210 embryos and 210 larvae (10 individuals per chamber). The effect of Apl on zebrafish embryonic development was quantitatively assessed by recording a series of physiological indicators such as heart rate, survival rate, body length and hatch rate. Most importantly, a new index called clonic convulsion rate, combined with mortality was used to evaluate the toxicities of Apl on zebrafish larvae. We found that Apl can induce deformity and cardiovascular toxicity in both zebrafish embryos and larvae. This microdevice is a multiplexed testing apparatus that allows for the examination of indexes beyond toxicity and teratogenicity at the sub-organ and cellular levels and provides a potentially cost-effective and rapid pharmaceutical safety assessment tool.     

A prototype microfluidic chip using fluorescent yeast for detection of toxic compounds

A microfluidic chip has been developed to enable the screening of chemicals for environmental toxicity. The microfluidic approach offers several advantages over macro-scale systems for toxicity screening, including low cost and flexibility in design. This design flexibility means the chips can be produced with multiple channels or chambers which can be used to screen for different toxic compounds, or the same toxicant at different concentrations. Saccharomyces cerevisiae containing fluorescent markers are ideal candidates for the microfluidic screening system as fluorescence is emitted without the need of additional reagents. Microfluidic chips containing eight multi-parallel channels have been developed to retain yeast within the chip and allow exposure of them to toxic compounds. The recombinant yeast used was GreenScreentrade mark which expresses green fluorescent proteins when is exposed to genotoxins. After exposure of the yeast to target compounds, the fluorescence emission was detected using an inverted microscope. Qualitative and quantitative comparisons of the fluorescent emission were performed. Results indicated that fluorescent intensity per area significantly increases upon exposure to methyl-methanesulfonate, a well known genotoxic compound. The microfluidic approach reported here is an excellent tool for cell-based screening and detection of different toxicities. The device has the potential for use by industrial manufacturers to detect and reduce the production and discharge of toxic compounds, as well as to characterise already polluted environments.     

Comparative assessment of single and joint effects of diuron and Irgarol 1051 on Arctic and temperate microalgae using chlorophyll a fluorescence imaging

Ship groundings and ice-breakers can cause pollution of the polar environment with antifouling biocides such as diuron and Irgarol 1051. The present study used pulse amplitude modulated fluorometry to compare single and joint toxicities of diuron and Irgarol 1051 on two freshwater taxa of microalgae (Chlorella and Chlamydomonas) originating from Arctic and temperate regions. 30 min acute toxicity tests using chlorophyll a (Chl a) fluorescence revealed that Arctic strains of microalgae were more sensitive to herbicides than their temperate counterparts. Diuron and Irgarol 1051 had equal toxicities in the Arctic species, while Irgarol 1051 was more toxic (EC₅₀ = 5.55–14.70 μg L⁻¹) than diuron (EC₅₀ = 12.90–>40 μg L⁻¹) in the temperate species. Toxicity assessment of various mixtures of diuron and Irgarol 1051 revealed antagonistic, additive, and synergistic effects. Our data suggest that herbicides can adversely affect photosynthesis in Arctic microalgae at relatively low levels, and their impact can increase under complex mixture conditions.     

Interactions between rhamnolipid biosurfactants and toxic chlorinated phenols enhance biodegradation of a model hydrocarbon-rich effluent

Surfactant-mediated treatment increases hydrocarbon solubilization and potentially facilitates biodegradation, unless toxic co-contaminants inhibiting microbial activity are present in the hydrocarbon mixture. We assessed the effect of rhamnolipids on the performance of a bacterial consortium degrading diesel fuel employed as a model hydrocarbon-rich effluent, co-contaminated with toxic phenol, 4-chlorophenol (4-CP) or 2,4-dichlorophenol (2,4-DCP). This approach led to the unexpected finding that rhamnolipids reduced toxicity of 4-CP and 2,4-DCP to the hydrocarbon-degrading cells. The facts that rhamnolipids decreased diesel fuel - water partition coefficient (K_FW) of 4-CP and 2,4-DCP and modified aggregate size distribution profiles of the dispersed diesel fuel - chlorinated phenols solutions, suggest the existence of specific interactions between rhamnolipids and the co-contaminants. Due to the polar nature of 4-CP and 2,4-DCP, possible explanations involve adsorption of 4-CP and 2,4-DCP on the surface of biosurfactant aggregates. This property of rhamnolipids is of interest to those using biosurfactants for microbial treatment of hydrocarbon-rich wastewaters co-contaminated with toxic compounds.     

Assessment of the genotoxic potential of Hoechst 33342, SYBR-14 and PI using the SOS ChromoTest™

Abstract

Fluorochromes in combination with flow cytometry can be used for laboratory assessment of semen quality in humans and domestic animals. Some studies have reported the potential toxicity of these fluorochromes toward the cells analyzed, but not toward the laboratory technician who operates the analytical instrument. We tested the genotoxic potential of three fluorochromes, SYBR-14, propidium iodide, and Hoechst 33342, using the colorimetric SOS ChromoTest^?. The test revealed no genotoxic potential for any of the three fluorochromes within the dilution ranges investigated. We conclude that occasional direct contact with these fluorescent probes does not necessarily pose a genotoxic hazard.     

Compound toxicity screening and structure-activity relationship modeling in Escherichia coli

Synthetic biology and metabolic engineering are used to develop new strategies for producing valuable compounds ranging from therapeutics to biofuels in engineered microorganisms. When developing methods for high-titer production cells, toxicity is an important element to consider. Indeed the production rate can be limited due to toxic intermediates or accumulation of byproducts of the heterologous biosynthetic pathway of interest. Conversely, highly toxic molecules are desired when designing antimicrobials. Compound toxicity in bacteria plays a major role in metabolic engineering as well as in the development of new antibacterial agents. Here, we screened a diversified chemical library of 166 compounds for toxicity in Escherichia coli. The dataset was built using a clustering algorithm maximizing the chemical diversity in the library. The resulting assay data was used to develop a toxicity predictor that we used to assess the toxicity of metabolites throughout the metabolome. This new tool for predicting toxicity can thus be used for fine-tuning heterologous expression and can be integrated in a computational-framework for metabolic pathway design. Many structure-activity relationship tools have been developed for toxicology studies in eukaryotes [Valerio (2009), Toxicol Appl Pharmacol, 241(3): 356-370], however, to the best of our knowledge we present here the first E. coli toxicity prediction web server based on QSAR models (EcoliTox server: http://www.issb.genopole.fr/～faulon/EcoliTox.php).     

Design and Validation of DNA Libraries for Multiplexing Proximity Ligation Assays

Here, we present an in silico, analytical procedure for designing and testing orthogonal DNA templates for multiplexing of the proximity ligation assay (PLA). PLA is a technology for the detection of protein interactions, post-translational modifications, and protein concentrations. To enable multiplexing of the PLA, the target information of antibodies was encoded within the DNA template of a PLA, where each template comprised four single-stranded DNA molecules. Our DNA design procedure followed the principles of minimizing the free energy of DNA cross-hybridization. To validate the functionality, orthogonality, and efficiency of the constructed template libraries, we developed a high-throughput solid-phase rolling-circle amplification assay and solid-phase PLA on a microfluidic platform. Upon integration on a microfluidic chip, 640 miniaturized pull-down assays for oligonucleotides or antibodies could be performed in parallel together with steps of DNA ligation, isothermal amplification, and detection under controlled microenvironments. From a large computed PLA template library, we randomly selected 10 template sets and tested all DNA combinations for cross-reactivity in the presence and absence of antibodies. By using the microfluidic chip application, we determined rapidly the false-positive rate of the design procedure, which was less than 1%. The combined theoretical and experimental procedure is applicable for high-throughput PLA studies on a microfluidic chip.     

Exploring toxicity of perfluorinated compounds through complex network and pathway modeling

Abstract

Perfluorinated compounds (PFCs) have serious impacts on human health, which could interfere with the body's signal pathways and affect the normal hormone balance of humans. PFCs were reported to bind to many proteins causing a series of biological effects. It was quite possible that the in vivo action of PFCs was not a single target or a single pathway, suggesting the toxic effect was due to the disturbance of protein or gene network, not limited to the modification of a single target protein or gene. Thus, a PFCs-targets interaction network was constructed and the significant differences in the characteristics of complex networks between the branched PFCs and linear PFCs were observed. A molecular dynamics simulation proved that binding ability of the branched PFCs to the target protein was much weaker than that of the linear PFCs, explaining why the branched PFCs presented significantly difference from the linear PFCs in terms of complex network characteristics. In addition, four target genes were identified as the central node genes of the network. The four target genes were proved to present certain influences on some diseases, which suggested a high correlation between PFCs to these diseases, including obesity, hepatocellular carcinoma and diabetes. The present work was helpful to develop new approaches to identify the key toxic targets of compounds and to explore the toxicity effects on pathways. Abbreviations AR androgen receptor

BPA bisphenol A

ESR1 estrogen receptor 1

ESR2 estrogen receptor 2

GLTP glycolipid transfer protein

HbF the fetal hemoglobin

HBG1 hemoglobin subunit γ-1

hERα human ERα

HSD17B1 hydroxysteroid 17-β dehydrogenase 1

KEGG Kenya encyclopedia of genes and genomes

MD molecular dynamics simulation

PFCs perfluorinated compounds

PFOA perfluorooctanoic acid

PFOS perfluorooctane sulfonate

POPs persistent organic pollutants

RMSD root-mean-square deviation

SHBG sex hormone binding globulin

SPC/E extended simple point charge model

TR thyroid hormone receptor

Communicated by Ramaswamy H. Sarma     

Enhanced CO2 biofixation and protein production by microalgae biofilm attached on modified surface of nickel foam

In this work, a photobioreactor with microalgae biofilm was proposed to enhance CO₂ biofixation and protein production using nickel foam with the modified surface as the carrier for immobilizing microalgae cells. The results demonstrated that, compared with microalgae suspension, microalgae biofilm lowered mass transfer resistance and promoted mass transfer efficiency of CO₂ from the bubbles into the immobilized microalgae cells, enhancing CO₂ biofixation and protein production. Moreover, parametric studies on the performance of the photobioreactor with microalgae biofilm were also conducted. The results showed that the photobioreactor with microalgae biofilm yielded a good performance with the CO₂ biofixation rate of 4465.6 µmol m⁻³ s⁻¹, the protein concentration of effluent liquid of 0.892 g L⁻¹, and the protein synthesis rate of 43.11 g m⁻³ h⁻¹. This work will be conducive to the optimization design of microalgae culture system for improving the performance of the photobioreactor.

     

Mutation of microalgae from antifouling sensitivity to antifouling resistance allows phytoplankton dispersal through ships’ biofouling

Marine ecosystems are affected by introduced species including microalgae. We propose that biofouling on ships’ hulls is a potentially important mechanism for microalgae dispersal worldwide. Biofouling samples, for phytoplankton composition analysis, were collected in Spanish Mediterranean ports from the hulls of ships that had completed oceanic journeys from other Mediterranean ports, and long journeys from the Atlantic and Indian Oceans. Samples representing the local population of phytoplankton either in the water column or attached to the biofouling of locally-based ship-hulls were used as controls. A broad variety of microalgae species (including toxic dinoflagellates), which were not present in the local phytoplankton populations were found on the biofouling film of the ships that had been on distant journeys. In spite of the presence of the antifouling paints containing toxic compounds, microalgae were able to rapidly adapt to these non-favourable conditions. Consequently, our study shows that ships’ biofouling seems to be a powerful vector for microalgae dispersal at a global scale due to the capacity of microalgae to attach to the biofouling film and to cope by adaptation mechanisms with antifouling compounds.     

海洋微藻溶血毒素研究进展

对海洋微藻溶血毒素的类型、理化性质、生物合成和毒性作用进行了综述,分析了存在的问题和应用前景的展望。     

Microplates as a microreactor platform for microalgae research

High‐throughput platforms for microalgae screening are not yet commercially available. In this study, the feasibility of 96‐well microplates was analyzed for microalgae research. Equivalence among wells, as culture microreactors, was investigated in controlled high CO₂ conditions. Specific growth rates of two microalgae species, Scenedesmus sp. UTEX1589 and an environmental isolate, were significantly higher in border wells than in internal positions. Furthermore, growth rate gradients analyzed as contours throughout the platform were observed for Scenedesmus sp. However, the output variable exhibited high precision associated with a low coefficient of variation (CV), between 6.8 and 7.8%. In a demonstrative experiment to determine the effect of culture media dilution on six microalgae species, treatments were randomized in the central subset of a microplate. Results were consistent and statistically sound (CV 9.4–12.9%), and showed that microalgae species could grow with no detrimental effect in 50% (v/v) dilution of the culture medium. Provided border wells exclusion and a randomized design, 96‐well microplates are a practical and statistical robust platform for microalgae research. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:638–644, 2013     

Challenges of including human exposure to chemicals in food packaging as a new exposure pathway in life cycle impact assessment

Purpose

Limiting exposure to potentially toxic chemicals in food packaging can lead to environmental impact trade-offs. No available tool, however, considers trade-offs between environmental impacts of packaging systems and exposure to potentially toxic chemicals in food packaging. This study therefore explores the research needs for extending life cycle impact assessment (LCIA) to include exposure to chemicals in food packaging.

Methods

The LCIA framework for human toxicity was extended for the first time to include consumer exposure to chemicals in food packaging through the product intake fraction (PiF) metric. The related exposure pathway was added to LCIA without other modifications to the existing toxicity characterization framework used by USEtox®, i.e., effect factor derivation. The developed method was applied to a high impact polystyrene (HIPS) container case study with the functional unit of providing 1 kg of yogurt in single servings. Various exposure scenarios were considered, including an evidence-based scenario using concentration data and a migration model. Human toxicity impact scores in comparative toxic units (CTU_h) for the use stage were evaluated and then compared to human toxicity impact scores from a conventional LCIA methodology.

Results and discussion

Data allowed toxicity characterization of use stage exposure to only seven chemicals in HIPS out of fourty-four identified. Data required were the initial concentration of chemicals in food packaging, chemical mass transfer from packaging into food, and relevant toxicity information. Toxicity characterization demonstrated that the combined CTU_h for HIPS material acquisition, manufacturing, and disposal stages exceeded the toxicity scores related to consumer exposure to previously estimated concentrations of the seven characterizable chemicals in HIPS, by about two orders of magnitude. The CTU_h associated with consumer exposure became relevant when migration was above 0.1% of the European regulatory levels. Results emphasize missing data for chemical concentrations in food contact materials and a need to expand the current USEtox method for effect factor derivation (e.g., to consider endocrine disruption, mixture toxicity, background exposure, and thresholds when relevant).

Conclusions

An LCIA method was developed to include consumer exposure to chemicals in food packaging. Further study is required to assess realistic scenarios to inform decisions and policies, such as circular economy, which can lead to trade-offs between environmental impacts and potentially toxic chemicals in packaging. To apply the developed method, data regarding occurrence, concentration, and toxicity of chemicals in food packaging are needed. Revisiting the derivation of effect factors in future work could improve the interpretation of human toxicity impact scores.

     

Automatic bio-sampling chips integrated with micro-pumps and micro-valves for disease detection

The present study reports a microfluidic system using the concept of membrane-movement to design and fabricate micro-pneumatic valves and pumps to form a bio-sensing diagnostic chip. The automatic bio-sampling system includes a micro-diagnostic chip fabricated by using MEMS (micro-electro-mechanical systems) technology and an automatic platform comprising of a control circuit, a compressed air source and several electromagnetic valve switches. The control circuit is used to regulate the electromagnetic valve switches, causing thin PDMS membranes to deflect pneumatically by the compressed air and generate valving and pumping effects. The micro-diagnostic chip allows for the quick detection of diseases. Compared to large-scale systems, the new microfluidic system uses smaller amounts of samples and reagents and performs fast diagnosis in an automated format. Instead of using traditional pneumatic micro-pumps, the current study adopts a new design called "spider-web" micro-pumps to increase the pumping rate, and more importantly, improve the uniformity of flow rates inside multiple micro-channels. Experimental data show that for disease diagnosis, the bio-sensing chips integrated with the micro-pneumatic valves and the peristaltic micro-pumps could successfully perform diagnosis tests. Small amounts of samples and reagents could be injected into the diagnosis chips using the micro-pumps and the micro-pneumatic valves could effectively control the movement of the samples and reagents. In order to demonstrate the functionality of the developed device, detection of hepatitis C virus (HCV) and syphilis has been performed using the bio-sampling chips. Experimental data show that fluorescence signals from the microfluidic system were comparable to the ones using conventional testing methods. The developed chip could be easily extended for multiple disease detection. The automatic bio-sensing chips could provide a useful tool for fast disease detection and be crucial for a micro-total-analysis system.     

光照对光生物反应器中微藻高密度光自养培养的影响

光生物反应器是实现微藻高密度培养的重要装置,其设计的关键技术之一是选择合适的光照方式。根据国内外近十年来的相关研究成果,重点介绍了入射光性质(光源、光强、光质和光暗循环)和光能分布对微藻生长的影响,评述了用于微藻高密度培养的光照技术,展望了进一步的研究方向,为高效光生物反应器的设计和优化提供参考。     

Biokinetic and toxicodynamic modelling and its role in toxicological research and risk assessment

Toxicological risk assessment for chemicals is still mainly based on highly standardised protocols for animal experimentation and exposure assessment. However, developments in our knowledge of general physiology, in chemicobiological interactions and in (computer-supported) modelling, have resulted in a tremendous change in our understanding of the molecular mechanisms underlying the toxicity of chemicals. This permits the development of biologically based models, in which the biokinetics as well as the toxicodynamics of compounds can be described. In this paper, the possibilities are discussed of developing systems in which the systemic (acute and chronic) toxicities of chemicals can be quantified without the heavy reliance on animal experiments. By integrating data derived from different sources, predictions of toxicity can be made. Key elements in this integrated approach are the evaluation of chemical functionalities representing structural alerts for toxic actions, the construction of biokinetic models on the basis of non-animal data (for example, tissue-blood partition coefficients, in vitro biotransformation parameters), tests or batteries of tests for determining basal cytotoxicity, and more-specific tests for evaluating tissue or organ toxicity. It is concluded that this approach is a useful tool for various steps in toxicological hazard and risk assessment, especially for those forms of toxicity for which validated in vitro and other non-animal tests have already been developed.     

Key issues for the development and application of the species sensitivity distribution (SSD) model for ecological risk assessment

The species sensitivity distribution (SSD) model is one of the most commonly used methods for ecological risk assessment based on the potentially affected fraction (PAF) of and the combined PAF (msPAF) as quantitative indicators. There are usually four steps for the development of SSD models and their applications: (1) obtain the toxicity data of the pollutants; (2) fit the SSD curves; (3) calculate the potentially affected fractions (PAFs) of the individual pollutants for the ecological risk assessment of an individual pollutant; and (4) calculate the accumulated multi-substance potentially affected fractions (msPAFs) for the joint ecological risk assessment of multiple pollutants. Among the above mentioned four steps, the first two steps are paramount. In the present study, the following six key issues are discussed: (1) how to select the appropriate species, (2) how to preprocess the toxicity data collected from the ecotoxicity database, (3) how to transform the acute toxicity data into chronic data, (4) how to best fit the toxicity data, (5) how to calculate the msPAF of multiple pollutants, and (6) how to determine the uncertainty of the SSD model”. In response to these questions, several principles were proposed to select appropriate species; three data processing methods, including the geometric mean, weight assigning and using all raw data without processing, were compared to determine the appropriate method for the DDT (dichloro diphenyl trichloroethane) toxicity data preprocessing. The method of acute to chronic ratio (ACR) and binary correlation analysis were contrasted using the zinc toxicity data for the transformation of the acute toxicity data into chronic data. The Burr III, Loglogistic and Lognormal models were compared to determine the best fit model using the DDT toxicity data for invertebrates. The concentration addition or response addition were discussed to calculate msPAF according to the toxic model of action (TMoA). The uncertainties of the SSD models for five heavy metals and for eight polycyclic aromatic hydrocarbons (PAHs) were performed. The comparison of the coefficients of variation (CVs) for the toxicity data and exposure levels in Lake Chaohu for eight polycyclic aromatic hydrocarbons (PAHs) were also presented to demonstrate the uncertainties of the ecological risks assessed by the SSD model based on 5000 Monte Carlo simulations.     

微藻趋向运动及其靶向运输应用研究进展

微藻主要是指一类能进行光合自养的微生物,且很多藻株还兼具运动特性.因而将微藻与微流控芯片结合,实现精确靶向,或用于分子药物递送,在生物医学治疗和药效学分析等领域有重要的潜在应用价值,也是当今的研究热点之一.然而,目前关于微藻趋向运动的研究及潜在的应用的综述报道却相对较少.本文主要以模式微藻莱茵衣藻为例,概述基于微藻细胞...     

A method for real-time mechanical characterisation of microcapsules

Characterising the mechanical properties of flowing microcapsules is important from both fundamental and applied points of view. In the present study, we develop a novel multilayer perceptron (MLP)-based machine learning (ML) approach, for real-time simultaneous predictions of the membrane mechanical law type, shear and area-dilatation moduli of microcapsules, from their camera-recorded steady profiles in tube flow. By MLP, we mean a neural network where many perceptrons are organised into layers. A perceptron is a basic element that conducts input–output mapping operation. We test the performance of the present approach using both simulation and experimental data. We find that with a reasonably high prediction accuracy, our method can reach an unprecedented low prediction latency of less than 1 millisecond on a personal computer. That is the overall computational time, without using parallel computing, from a single experimental image to multiple capsule mechanical parameters. It is faster than a recently proposed convolutional neural network-based approach by two orders of magnitude, for it only deals with the one-dimensional capsule boundary instead of the entire two-dimensional capsule image. Our new approach may serve as the foundation of a promising tool for real-time mechanical characterisation and online active sorting of deformable microcapsules and biological cells in microfluidic devices.

     

Ferrowax microvalves for fully automated serial dilution on centrifugal microfluidic platforms

We herein describe a centrifugal microfluidic system to accomplish a fully automated serial dilution. The liquid flow on the disc was regulated by utilizing ferrowax microvalves systematically integrated into the channels within specially designed metering structures. By opening the differently positioned microvalves through irradiation of IR laser to allow metering, the same amount of diluent was serially eluted to the dilution chamber from the same diluent chamber. After dilution, the diluted samples were automatically delivered to the respective final product chambers by appropriately opening or closing the microvalves in the connecting channels, followed by rotating the disc. Based on this unique design principle, six consecutive two-fold and 10-fold dilutions were successfully achieved, yielding excellent accuracy in a wide dynamic range up to six orders of magnitude. Very importantly, the overall serial dilution process, including the diluent addition, mixing, and product transfer steps, was completed very rapidly within 5 min, due to the minimized procedures enabled by the automated actuation of the ferrowax microvalves at the rationally designed positions. We expect our centrifugal microfluidic system would serve as a powerful elemental tool to realize fully automated diagnostic microsystems involving the serial dilution process.