Toxicological Assays Research Articles

Chemical Exposure-Induced Developmental Neurotoxicity in Head-Regenerating Schmidtea mediterranea.

The growing number of commercially used chemicals that are under-evaluated for developmental neurotoxicity (DNT) combined with the difficulty in describing the etiology of exposure-related neurodevelopmental toxicity has created a reticent threat to human health. Current means of screening chemicals for DNT are limited to expensive, time-consuming, and labor-intensive traditional laboratory animal models. In this study, we hypothesize that exposed head-regenerating planarian flatworms can effectively and efficiently categorize DNT in known developmental neurotoxins (ethanol and bisphenol A [BPA]). Planarian flatworms are an established alternative animal model for neurodevelopmental studies and have remarkable regenerative abilities allowing neurodevelopment to be induced via head resection. Here, we observed changes in photophobic behavior and central nervous system (CNS) morphology to evaluate the impact of exposure to low concentrations of ethanol, BPA, and BPA industry alternatives bisphenol F, and bisguaiacol on neurodevelopment. Our studies show that exposure to 1% v/v ethanol during regeneration induces a recoverable 48-h delay in the development of proper CNS integrity, which aligns with behavioral assessments of cognitive ability. Exposure to BPA and its alternatives induced deviations to neurodevelopment in a range of severities, distinguished by suppressions, delays, or a combination of the 2. These results suggest that quick and inexpensive behavioral assessments are a viable surrogate for tedious and costly immunostaining studies, equipping more utility and resolution to the planarian model for neurodevelopmental toxicity in the future of mass chemical screening. These studies demonstrate that behavioral phenotypes observed following chemical exposure are classifiable and also temporally correlated to the anatomical development of the CNS in planaria. This will facilitate and accelerate toxicological screening assays with this alternative animal model.

Nanotechnology to Cure Breast Cancer and Obstacles in its Way

Nanomaterials owing to their remarkable pharmaceutical properties and potential to load large quantities of drugs are considered an ideal vector for carrying medicine to the target site. The treatment of breast cancer has been a pressing issue due the use of conventional methods of treatment resulting in many side effects. Nanotechnology offers a more targeted approach in breast cancer treatment. The present review examines the role of Carbon Nanotubes (CNTs) and nanoparticles in the treatment of breast cancer and the obstacles in the way of nanotechnology in becoming one of the best methods in breast cancer treatment and drug delivery. Use of monoclonal antibody trastuzumab against HER-2 biomarkers by attaching it with nanotubes or nanoparticles is considered the best-targeted therapy along with photo thermal ablation. The absence of human tumor models and lack of toxicological assays are the major factors contributing to differences between the preclinical and clinical trial results.

Large-Scale Transcriptional Profiling of Molecular Perturbations Reveals Cell Line Specific Responses and Implications for Environmental Screening

Cell-based bioassays represent nearly half of all high-throughput screens currently conducted for risk assessment of environmental chemicals. However, there has long been a concern about the sensitivity and heterogeneity among cell lines, which were explored only in a limited manner. Here, we address this question by conducting a large-scale transcriptome analysis of the responses of discrete cell lines to specific molecules. We report the collections of >223 300 gene expression profiles from a wide array of cell lines exposed to 2243 compounds. Our results demonstrate distinct responses among cell lines at both the gene and the pathway levels. Temporal variations for a very large proportion of compounds occur as well. High sensitivity and/or heterogeneity is either cell line-specific or universal depending on the modes of action of the compounds. Among 12 representative pathways analyzed, distinct cell-chemical interactions exist. On one hand, lung carcinoma cells are always best suited for glucocorticoid receptor agonist identification, while on the other hand, high sensitivity and heterogenic features are universal for histone deacetylase inhibitors and ATPase inhibitors. Our data provide novel insights into the understanding of cell-specific responses and interactions between cells and xenobiotics. The findings have substantial implications for the design, execution, and interpretation of high-throughput screening assays in (eco)toxicology.

Elucidation of xenoestrogen metabolism by non-targeted, stable isotope-assisted mass spectrometry in breast cancer cells

Environmental exposure to xenoestrogens, i.e., chemicals that imitate the hormone 17β-estradiol, has the potential to influence hormone homeostasis and action. Detailed knowledge of xenobiotic biotransformation processes in cell models is key when transferring knowledge learned from in vitro models to in vivo relevance. This study elucidated the metabolism of two naturally-occurring phyto- and mycoestrogens; namely genistein and zearalenone, in an estrogen receptor positive breast cancer cell line (MCF-7) with the aid of stable isotope-assisted metabolomics and the bioinformatic tool MetExtract II. Metabolism was studied in a time course experiment after 2 h, 6 h and 24 h incubation. Twelve and six biotransformation products of zearalenone and genistein were detected, respectively, clearly demonstrating the abundant xenobiotic biotransformation capability of the cells. Zearalenone underwent extensive phase-I metabolism resulting in α-zearalenol (α-ZEL), a molecule known to possess a significantly higher estrogenicity, and several phase-II metabolites (sulfo- and glycoconjugates) of the native compound and the major phase I metabolite α-ZEL. Moreover, potential adducts of zearalenone with a vitamin and several hydroxylated metabolites were annotated. Genistein metabolism resulted in sulfation, combined sulfation and hydroxylation, acetylation, glucuronidation and unexpectedly adduct formation with pentose- and hexose sugars. Kinetics of metabolite formation and subsequent excretion into the extracellular medium revealed a time-dependent increase in most biotransformation products. The untargeted elucidation of biotransformation products formed during cell culture experiments enables an improved and more meaningful interpretation of toxicological assays and has the potential to identify unexpected or unknown metabolites.

Feasibility assessment of oil sands process water treatment in a flow‐through microbial electrolysis cell

This study examines the feasibility of oil sands process water (OSPW) treatment in a gravitational flow-through membraneless microbial electrolysis cell (MEC) using crushed metallurgical coke as an electrode material. Synthetic OSPW was composed of the water soluble fraction (WSF) of diesel, naphthenic acids (NAs), and metals with a total chemical oxygen demand (COD) concentration of 200 mg L − 1 and total NA concentration of 9.4 mg L − 1. OSPW treatment under bioelectrochemical conditions (1.4 V applied voltage) was compared with a control system operated at 0 V. Furthermore, in an additional set of experiments, microaerobic conditions were applied to both MEC and control setups to facilitate NA biodegradation. Highest NA and COD removal rates were observed in the MEC with microaeration in electrode compartments, where total NA concentration decreased to 4.2 mg L − 1 at a hydraulic retention time of 4 days. Furthermore, microtox and microalgae toxicological assays showed substantial toxicity decrease in this MEC.

PH-Induced Modulation of Vibrio fischeri Population Life Cycle

Commonly used as biological chemosensors in toxicity assays, Vibrio fischeri bacteria were systematically characterized using complementary physicochemical and biological techniques to elucidate the evolution of their properties under varying environmental conditions. Changing the pH above or below the optimal pH 7 was used to model the long-term stress that would be experienced by V. fischeri in environmental toxicology assays. The spectral shape of bioluminescence and cell-surface charge during the exponential growth phase were largely unaffected by pH changes. The pH-induced modulation of V. fischeri growth, monitored via the optical density (OD), was moderate. In contrast, the concomitant changes in the time-profiles of their bioluminescence, which is used as the readout in assays, were more significant. Imaging at discrete timepoints by scanning electron microscopy (SEM) and helium-ion microscopy (HIM) revealed that mature V. fischeri cells maintained a rod-shaped morphology with the average length of 2.2 ± 1 µm and diameter of 0.6 ± 0.1 µm. Detailed morphological analysis revealed subpopulations of rods having aspect ratios significantly larger than those of average individuals, suggesting the use of such elongated rods as an indicator of the multigenerational environmental stress. The observed modulation of bioluminescence and morphology supports the suitability of V. fischeri as biological chemosensors for both rapid and long-term assays, including under environmental conditions that can modify the physicochemical properties of novel anthropogenic pollutants, such as nanomaterials and especially stimulus-responsive nanomaterials.

English

In recent years, essential oils (EOs) as alternatives to synthetic pesticides in managing pests have been assessed. The use of bioinsecticide in pest management is encouraged in agroecology for a sustainable agricultural system. Essential oils of Lantana camara L. leaves from different climatic zones of Kenya were extracted by steam distillation and analyzed through GC-MS to identify the compounds. The contact toxicity and repellent activity of EOs against the invasive tomato pest, Tuta absoluta, were tested. The toxicological assays were performed following the leaf-dip bioassay protocol, while the repellency activity was performed using the repellent response method for phytophagous pests and the data analyzed using the ANOVA test. It was found that L. camara EO has a good insecticidal activity with higher mortality (89%) on the 2nd instar larvae with a higher dosage (0.01 µl/µl). The repellence test also showed a higher average repellence (93.44%) effect with a higher dosage (0.01 µl/µl) of the EOs. According to these results, the EO of L. camara may well be a sustainable, eco-friendly alternative for synthetic insecticide in the T. absoluta management program. Key words: Bioinsecticide; essential oil, integrated pest management, invasive species, secondary metabolites.

How to obtain large amounts of location- and time-specific PM2.5 with homogeneous mass and composition? A possible approach, from particulate collection to chemical characterization

A suitable protocol for the collection, extraction, and characterization of a large quantity of a local and current fine particulate matter (PM2.5) was developed, to provide homogeneous PM2.5 amounts for use in in vitro and in vivo toxicological assays rather than a generic and obsolete standard reference material (SRM). PM2.5 samples were collected at an outdoor urban background site in Como, Northern Italy, using a high-volume air sampler (500 L/min) equipped with polytetrafluoroethylene (PTFE)-coated glass fiber filters (eight sampling campaigns of 24–96 h each, from February to April 2019). PM2.5 was extracted from the filters by sequential sonication steps and the final pooled extract was filtered to remove the glass fibers released during extraction. Several PM2.5 aliquots (n = 55) were then prepared from the PM2.5 dry residue for i) a chemical characterization of the elements, inorganic ions, and the carbonaceous fraction and ii) in vivo and in vitro studies. The proposed protocol allowed a large amount of ambient PM2.5 (>300 mg) to be collected within a reasonably short time. An average extraction efficiency of 75% was achieved, and the chemical analyses conducted on a set of PM2.5-replicates revealed a satisfactory level of precision, ranging from 3% (anions and OC) to 10% (trace metals and EC), thereby confirming the possibility of obtaining aliquots with homogeneous mass and composition. Finally, the emerging differences with the SRM 1648a elemental composition suggest the importance of location- and time-specific particulate testing, which is truly representative of the actual exposure scenario for specific populations.

Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse.

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells entering meiotic prophase I are eliminated during Fetal Oocyte Attrition (FOA) and the first week of postnatal life. Three major mechanisms regulate the number of oocytes that survive during development and establish the ovarian reserve in females entering puberty. In the first wave of oocyte loss, 30-50% of the oocytes are eliminated during early FOA, a phenomenon that is attributed to high Long interspersed nuclear element-1 (LINE-1) expression. The second wave of oocyte loss is the elimination of oocytes with meiotic defects by a meiotic quality checkpoint. The third wave of oocyte loss occurs perinatally during primordial follicle formation when some oocytes fail to form follicles. It remains unclear what regulates each of these three waves of oocyte loss and how they shape the ovarian reserve in either mice or humans. Immunofluorescence and 3D visualization have opened a new avenue to image and analyze oocyte development in the context of the whole ovary rather than in less informative 2D sections. This article provides a comprehensive protocol for whole ovary immunostaining and optical clearing, yielding preparations for imaging using multiphoton microscopy and 3D modeling using commercially available software. It shows how this method can be used to show the dynamics of oocyte attrition during ovarian development in C57BL/6J mice and quantify oocyte loss during the three waves of oocyte elimination. This protocol can be applied to prenatal and early postnatal ovaries for oocyte visualization and quantification, as well as other quantitative approaches. Importantly, the protocol was strategically developed to accommodate high-throughput, reliable, and repeatable processing that can meet the needs in toxicology, clinical diagnostics, and genomic assays of ovarian function.

A geospatial modeling approach to quantifying risk of exposure to environmental chemical mixtures via a common molecular initiating event

BACKGROUND AND AIM: The exposome, a measure of all chemical exposures experienced through a lifetime, plays an important role in human health outcomes. Thus, identifying and quantifying lifetime exposure to chemicals present in the environment is important for risk assessment. Linking health outcomes to environmental chemicals exposures is challenging, as real-world exposures involve multiple chemicals and unresolved spatial and temporal distributions. To address these challenges, we propose a geospatially resolved biological target-based risk assessment for exposure to chemical mixtures. This approach models the geospatial distribution of environmental chemicals based on common activation of toxicological adverse outcome pathways (AOPs) as indicated by Tox21 high throughput assays. METHODS: We exemplify this with a USA-wide proof-of-concept that quantifies the predicted geospatial likelihood of thyroid peroxidase (TPO) inhibition. This enzyme is necessary for producing the thyroid hormone T3 and T4, and inhibition has been associated with decreased cognitive function. First, environmental chemicals were identified that both 1) inhibit TPO based on Tox21 data, and 2) were measured by the USGS and EPA in environmental samples (surface water). Next, the geospatial chemical distributions were modeled with combined land-use regression and Gaussian process models. The individual chemical exposure surfaces can be combined into an aggregate risk surface using an unweighted approach or weighted by chemical potency from the toxicological assay concentration-response data. RESULTS:Maps of the aggregate risk surface will identify regions where thyroid peroxidase inhibition is more likely based on combined environmental chemical exposures and chemical potency. CONCLUSIONS:This work will help advance risk assessment methods by providing a method for assessing the geospatial risk of exposure to environmental chemical mixtures and identifying regions with a higher cumulative risk of activating the selected molecular pathways, which may lead to adverse health outcomes. KEYWORDS: Mixtures, External exposome, Exposure assessment-general, risk assessment, Spatial statistics, big data

Short communication: A potential role of tannins in the control of American foulbrood

Aim of study: To evaluate the use of tannins extracts in the control of the American foulbrood pathology and to investigate if these extracts present levels of toxicity on Apis mellifera.Area of study: Paenibacillus larvae strains C1 and C2 were from Balcarce, province of Buenos Aires, strain C6 from Rio Cuarto, in Cordoba province and strain C9 from Concordia in Entre Rios province. Bees larvae used for toxicological assays were collected in Santa Paula experimental apiary, Mar del Plata (belonging to the Centro de Investigación en Abejas Sociales (CIAS-IIPROSAM) from UNMdP.Material and methods: The minimal inhibitory concentration (MIC) of five different tannin extracts were obtained by agar diffusion method on four P. larvae strains; using the MIC value, the toxicity test on A. mellifera larvae was performed afterwards.Main results: The MIC value was in the range of 6.9 to 898.6 µg/mL. Three tannin extracts did not show toxicity against bee larvae; however, those that were fed with the latter showed a significant increase in weight.Research highlights: Three tanninS extracts showed a good antimicrobial activity against P. larvae and they did not show toxicity against bee larvae.

Speciation of platinum nanoparticles in different cell culture media by HPLC-ICP-TQ-MS and complementary techniques: A contribution to toxicological assays

Toxicological studies of nanoparticles (NPs) are highly demanded nowadays but they are very challenging. In the in vitro assays, the understanding of the role of cell culture media is crucial to derive a proper interpretation of the toxicological results and to do so, new analytical tools are necessary. In this context, an analytical strategy based on reversed-phase liquid chromatography hyphenated to inductively coupled plasma-triple quadrupole mass spectrometry (HPLC-ICP-TQ-MS) has been developed for the first time for the detection and characterization of both 5 and 30 nm PtNPs, as well as ionic platinum species, in commonly used cell culture media. For this purpose, Dulbecco's Modified Eagle Medium, DMEM-high glucose, DMEM-F12, DMEM 31053–028, and Roswell Park Memorial Institute, RPMI-1640 (supplemented with 10% fetal bovine serum (FBS) and antibiotics) at several incubation times (24, 48, and 96 h at 37 °C) were tested. After a careful optimization and analytical performance, the developed method allows to simultaneously study the oxidation process, leading to the release of ionic species, and the increase in the hydrodynamic volume of PtNPs, probably related to the formation of new biological entities (protein corona). The magnitude of both processes was found to be dependent on the tested cell culture media and incubation times. Dynamic light scattering (DLS) and high-resolution scanning electron microscopy (HR-SEM) were used as complementary techniques to study the important process of both soft and hard protein corona formation. The feasibility of the HPLC-ICP-TQ-MS to get relevant information for toxicological studies has been demonstrated and in light of our results, the influence of the cell culture media on the behavior of PtNPs should not be underestimated.

Zebrafish as a new model for rhododendrol-induced leukoderma.

Idiopathic leukoderma is a skin disorder characterized by patchy loss of skin pigmentation due to melanocyte dysfunction or deficiency. Rhododendrol (RD) was approved as a cosmetic ingredient in Japan in 2008. However, it was shown to induce leukoderma in approximately 20,000 customers. The prediction of cytotoxicity, especially to melanocytes in vivo, is required to avoid such adverse effects. Since the use of higher vertebrates is prohibited for medicinal and toxicological assays, we used zebrafish, whose melanocytes were regulated by mechanisms similar to mammals. Zebrafish larvae were treated with RD in breeding water for 3days, which caused body lightening accompanied by a decrease in the number of melanophores. Interestingly, black particles were found at the bottom of culture dishes, suggesting that the melanophores peeled off from the body. In addition, RT-PCR analysis suggested that the mRNA levels of melanophore-specific genes were significantly low. An increase in the production of reactive oxygen species was found in larvae treated with RD. The treatments of the fish with other phenol compounds, which have been reported to cause leukoderma, also induced depigmentation and melanophore loss. These results suggest that zebrafish larvae could be used for the evaluation of leukoderma caused by chemicals, including RD.

A versatile microfluidic tool for the 3D culture of HepaRG cells seeded at various stages of differentiation

The development of livers-on-a-chip aims to provide pharmaceutical companies with reliable systems to perform drug screening and toxicological studies. To that end, microfluidic systems are engineered to mimic the functions and architecture of this organ. In this context we have designed a device that reproduces series of liver microarchitectures, each permitting the 3D culture of hepatocytes by confining them to a chamber that is separated from the medium conveying channel by very thin slits. We modified the structure to ensure its compatibility with the culture of hepatocytes from different sources. Our device was adapted to the migratory and adhesion properties of the human HepaRG cell line at various stages of differentiation. Using this device, it was possible to keep the cells alive for more than 14 days, during which they achieved a 3D organisation and acquired or maintained their differentiation into hepatocytes. Albumin secretion as well as functional bile canaliculi were confirmed on the liver-on-a-chip. Finally, an acetaminophen toxicological assay was performed. With its multiple micro-chambers for hepatocyte culture, this microfluidic device architecture offers a promising opportunity to provide new tools for drug screening applications.

Assessing Experimental Uncertainty in Defined Approaches: Borderline Ranges for In Chemico and In Vitro Skin Sensitization Methods Determined from Ring Trial Data

Introduction: Prediction models of most toxicological assays translate continuous data to binary classifications (“positive” or “negative”) using cutoff values. Mostly these cutoffs do not consider...

Biologically Derived, Three-Dimensional, Embryonic Scaffolds for Long-Term Cardiomyocyte Culture.

The ability to maintain viable cultures of mature, primary cardiomyocytes is challenging. The lack of viable cardiomyocyte cultures severely limits in vitro biochemical assays, toxicology assays, drug screening assays, and other analyses. Here, we describe a novel three-dimensional (3D) embryonic scaffold, which supports the culture of postnatal day 7 murine cardiomyocytes within the embryonic heart for, at least, 28 days. We have observed that these cardiomyocytes display normal differentiation, protein expression, and function after extended culture. This novel culture system will allow for prolonged treatment of cardiomyocytes in a natural 3D orientation and has the potential for providing a superior tool for the screening of therapeutic compounds.

Studies of Melody Efficacy Vaccine Against SARS-CoV-2 are Closing COVID-19 Pandemic Doors? Single Dose is Recommended as Therapeutic Drug and Two Doses Application as Preventive Vaccine

In vitro studies provide data that is important for proof-of-concept determination, function validation, and peer review manuscript preparation, FDA applications and clinical trials. Our in vitro studies of Melody efficacy as a vaccine against SARS-CoV-2 were made in five cell cultures (BEAS-2B, HUVEC, HMEC-1, HEK293 and WM266), where the dead cell number monitored by using flow cytometry. Melody’s efficacy was tested by incubating melody RpA (RNA-peptide A) and melody RpAB (RNA-peptide AB). Both incubations were pending 16 hours with melody Lethal Concentration 50 % (LC50 µg/µL), which was determinated in a toxicological assay in the five cell cultures as previously described. Our conclusion was focused to understand the statistical difference in LC50 in order to estimate the melody preventive efficacity as vaccine in “In vitro” test. In the present study, the preventive efficacy values obtained in the five cell lines differ statistically between the value of two doses when compared with a single or three doses, p=5.321. These results suggested that the application of two doses is more effective. We used the Student’s t-test with n=6 to calculate p-value according to mRNA avian coronavirus action after one, two and three doses incubations of mRNA-peptide-A (melody therapeutic) of 16 hours. Our conclusion was focused to understand the statistical difference in LC50 in order to estimate the melody therapeutic efficacity as vaccine in “In vitro” test in cell lines (BEAS-2B, HUVEC, HMEC-1, HEK293 and WM-266). Results: Melody Therapeutic Efficacity was found to be 93.82% with using a single dose. Meanwhile, Melody Preventive Efficacity was found as 92.53% with using two doses. The therapeutic efficacy values obtained in the five cell lines in this study did not vary statistically significantly between the three doses used, p**=0.004. As a result, the Melody application of a single dose is recommended as therapeutic drug and two doses application as preventive vaccine.

Sediment from lake with missing egg bank is toxic to hatchlings of model zooplankton: A reason to consider obligate dormancy in toxicological assessment.

Over the last 60 years, valuable progress was made in the standardization of environmental monitoring with model zooplankton. However, obligate dormancy in zooplankton life cycles is not yet considered in standardized toxicology methods. Most zooplankton from coastal and inland waters use dormancy as a critical ecological strategy, and exposure to toxicants during dormancy or resurrection from dormancy alters developmental patterning and hatching success. The present study accounts for this by using both standardized and novel toxicology assays to assess the impacts of coal ash contaminated sediments and water on development, hatching, and survivorship of model zooplankton. The results demonstrate that standardized assays with rotifer and cladoceran models detect no toxicity in surface water and sediment pore water from Lake Sutton, North Carolina, USA. By contrast, novel toxicity assays with cladoceran and anostracan models demonstrate that development and larval survivorship are negatively impacted by Lake Sutton water and sediment. Embryos of Artemia franciscana display developmental patterning and hatching aberrations that match those observed in previous studies with metals when hatched in filtered surface water or pore water after a period of anoxia-induced dormancy. Larval survivorship in Daphnia magna and A. franciscana also decreases when post-diapause embryos are hatched in the presence of sediment. The effects of whole sediment on larval survivorship are not explained by coal ash impacts on water pH. These data provide an explanation for the missing egg bank and historic community restructure in Lake Sutton. The data also demonstrate a need for standardized assays that include dormant life stages.

Environmental Toxicology Assays Using Organ-on-Chip.

Adverse effects of environmental toxicants to human health have traditionally been assayed using in vitro assays. Organ-on-chip (OOC) is a new platform that can bridge the gaps between in vitro assays (or 3D cell culture) and animal tests. Microenvironments, physical and biochemical stimuli, and adequate sensing and biosensing systems can be integrated into OOC devices to better recapitulate the in vivo tissue and organ behavior and metabolism. While OOCs have extensively been studied for drug toxicity screening, their implementation in environmental toxicology assays is minimal and has limitations. In this review, recent attempts of environmental toxicology assays using OOCs, including multiple-organs-on-chip, are summarized and compared with OOC-based drug toxicity screening. Requirements for further improvements are identified and potential solutions are suggested.

Three-Dimensional in vitro Models of Healthy and Tumor Brain Microvasculature for Drug and Toxicity Screening.

Tissue vascularization is essential for its oxygenation and the homogenous diffusion of nutrients. Cutting-edge studies are focusing on the vascularization of three-dimensional (3D) in vitro models of human tissues. The reproduction of the brain vasculature is particularly challenging as numerous cell types are involved. Moreover, the blood-brain barrier, which acts as a selective filter between the vascular system and the brain, is a complex structure to replicate. Nevertheless, tremendous advances have been made in recent years, and several works have proposed promising 3D in vitro models of the brain microvasculature. They incorporate cell co-cultures organized in 3D scaffolds, often consisting of components of the native extracellular matrix (ECM), to obtain a micro-environment similar to the in vivo physiological state. These models are particularly useful for studying adverse effects on the healthy brain vasculature. They provide insights into the molecular and cellular events involved in the pathological evolutions of this vasculature, such as those supporting the appearance of brain cancers. Glioblastoma multiform (GBM) is the most common form of brain cancer and one of the most vascularized solid tumors. It is characterized by a high aggressiveness and therapy resistance. Current conventional therapies are unable to prevent the high risk of recurrence of the disease. Most of the new drug candidates fail to pass clinical trials, despite the promising results shown in vitro. The conventional in vitro models are unable to efficiently reproduce the specific features of GBM tumors. Recent studies have indeed suggested a high heterogeneity of the tumor brain vasculature, with the coexistence of intact and leaky regions resulting from the constant remodeling of the ECM by glioma cells. In this review paper, after summarizing the advances in 3D in vitro brain vasculature models, we focus on the latest achievements in vascularized GBM modeling, and the potential applications for both healthy and pathological models as platforms for drug screening and toxicological assays. Particular attention will be paid to discuss the relevance of these models in terms of cell-cell, cell-ECM interactions, vascularization and permeability properties, which are crucial parameters for improving in vitro testing accuracy.