Understanding the impact of more realistic low-dose, prolonged engineered nanomaterial exposure on genotoxicity using 3D models of the human liver

Abstract Background With the continued integration of engineered nanomaterials (ENMs) into everyday applications, it is important to understand their potential for inducing adverse human health effects. However, standard in vitro hazard characterisation approaches suffer limitations for evaluating ENM and so it is imperative to determine these potential hazards under more physiologically relevant and realistic exposure scenarios in target organ systems, to minimise the necessity for in vivo testing. The aim of this study was to determine if acute (24 h) and prolonged (120 h) exposures to five ENMs (TiO2, ZnO, Ag, BaSO4 and CeO2) would have a significantly different toxicological outcome (cytotoxicity, (pro-)inflammatory and genotoxic response) upon 3D human HepG2 liver spheroids. In addition, this study evaluated whether a more realistic, prolonged fractionated and repeated ENM dosing regime induces a significantly different toxicity outcome in liver spheroids as compared to a single, bolus prolonged exposure. Results Whilst it was found that the five ENMs did not impede liver functionality (e.g. albumin and urea production), induce cytotoxicity or an IL-8 (pro-)inflammatory response, all were found to cause significant genotoxicity following acute exposure. Most statistically significant genotoxic responses were not dose-dependent, with the exception of TiO2. Interestingly, the DNA damage effects observed following acute exposures, were not mirrored in the prolonged exposures, where only 0.2–5.0 µg/mL of ZnO ENMs were found to elicit significant (p ≤ 0.05) genotoxicity. When fractionated, repeated exposure regimes were performed with the test ENMs, no significant (p ≥ 0.05) difference was observed when compared to the single, bolus exposure regime. There was < 5.0% cytotoxicity observed across all exposures, and the mean difference in IL-8 cytokine release and genotoxicity between exposure regimes was 3.425 pg/mL and 0.181%, respectively. Conclusion In conclusion, whilst there was no difference between a single, bolus or fractionated, repeated ENM prolonged exposure regimes upon the toxicological output of 3D HepG2 liver spheroids, there was a difference between acute and prolonged exposures. This study highlights the importance of evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

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Cytotoxic effect of Montivipera bornmuelleri’s venom on cancer cell lines: in vitro and in vivo studies

PeerJ ◽

10.7717/peerj.9909 ◽

2020 ◽

Vol 8 ◽

pp. e9909

Author(s):

Carol Haddoub ◽

Mohamad Rima ◽

Sandrine Heurtebise ◽

Myriam Lawand ◽

Dania Jundi ◽

...

Keyword(s):

Tumor Growth ◽

Cell Lines ◽

Cytotoxic Effect ◽

In Vivo Studies ◽

Dependent Manner ◽

In Vivo Testing ◽

Murine Fibrosarcoma ◽

Dose Dependent

Background Montivipera bornmuelleri’s venom has shown immunomodulation of cytokines release in mice and selective cytotoxicity on cancer cells in a dose-dependent manner, highlighting an anticancer potential. Here, we extend these findings by elucidating the sensitivity of murine B16 skin melanoma and 3-MCA-induced murine fibrosarcoma cell lines to M. bornmuelleri’s venom and its effect on tumor growth in vivo. Methods The toxicity of the venom on B16 and MCA cells was assessed using flow cytometry and xCELLigence assays. For in vivo testing, tumor growth was followed in mice after intratumoral venom injection. Results The venom toxicity showed a dose-dependent cell death on both B16 and MCA cells. Interestingly, overexpression of ovalbumin increased the sensitivity of the cells to the venom. However, the venom was not able to eradicate induced-tumor growth when injected at 100 µg/kg. Our study demonstrates a cytotoxic effect of M. bornmuelleri’s venom in vitro which, however, does not translate to an anticancer action in vivo.

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In Vitro Glioblastoma Models: A Journey into the Third Dimension

Cancers ◽

10.3390/cancers13102449 ◽

2021 ◽

Vol 13 (10) ◽

pp. 2449

Author(s):

Mayra Paolillo ◽

Sergio Comincini ◽

Sergio Schinelli

Keyword(s):

Tumor Cells ◽

Single Cell Analysis ◽

3D Models ◽

Primary Brain Tumor ◽

Molecular Features ◽

Third Dimension ◽

One Year ◽

The Impact

Glioblastoma multiforme (GBM) is the most lethal primary brain tumor in adults, with an average survival time of about one year from initial diagnosis. In the attempt to overcome the complexity and drawbacks associated with in vivo GBM models, together with the need of developing systems dedicated to screen new potential drugs, considerable efforts have been devoted to the implementation of reliable and affordable in vitro GBM models. Recent findings on GBM molecular features, revealing a high heterogeneity between GBM cells and also between other non-tumor cells belonging to the tumoral niche, have stressed the limitations of the classical 2D cell culture systems. Recently, several novel and innovative 3D cell cultures models for GBM have been proposed and implemented. In this review, we first describe the different populations and their functional role of GBM and niche non-tumor cells that could be used in 3D models. An overview of the current available 3D in vitro systems for modeling GBM, together with their major weaknesses and strengths, is presented. Lastly, we discuss the impact of groundbreaking technologies, such as bioprinting and multi-omics single cell analysis, on the future implementation of 3D in vitro GBM models.

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Antimicrobial Agent Triclosan Inhibits Acetylcholinesterase Activity in Vitro in a Dose-Dependent Manner

10.1101/2021.06.11.448059 ◽

2021 ◽

Author(s):

Narasimha Pullaguri ◽

Andrea Kagoo ◽

Anamika Bhargava

Keyword(s):

Antimicrobial Agent ◽

Ache Activity ◽

Acetylcholinesterase Activity ◽

Consumer Products ◽

In Vivo Studies ◽

Dependent Manner ◽

Human Health Effects ◽

Dose Dependent

The antimicrobial agent, Triclosan, is widely used in many consumer products. It has been designated as a "contaminant of emerging concern (CEC)" because its exposure is known to cause adverse ecological and human health effects. Triclosan is not labelled as GRAS/GRAE (generally recognized as safe and effective), but its use is still prevailing. In vivo studies have revealed that exposure to triclosan results in a decreased acetylcholinesterase (AChE) activity. However mechanistic insights into AChE inhibition by triclosan are missing. Using in vitro AChE activity assay with purified AChE, we show that triclosan acts as a direct inhibitor of AChE and inhibits AChE activity in a dose-dependent manner. Given the function of AChE, any alteration in its activity can be neurotoxic. Our results provide important mechanistic insights into triclosan induced neurotoxicity with AChE as a target.

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Glial cells in the heart? Replicating the diversity of the myocardium with low-cost 3D models

10.1101/306233 ◽

2018 ◽

Author(s):

Jonathan R. Soucy ◽

Jody Askaryan ◽

David Diaz ◽

Abigail N. Koppes ◽

Nasim Annabi ◽

...

Keyword(s):

Low Cost ◽

Beat Frequency ◽

3D Models ◽

Cellular Heterogeneity ◽

Resting Potential ◽

Ec Coupling ◽

Electrical Pacing ◽

The Impact

Excitation-contraction (EC) coupling in the heart has, until recently, been solely accredited to cardiomyocytes. The inherent complexities of the heart make it difficult to examine nonmuscle contributions to contraction in vivo, and conventional in vitro models fail to capture multiple features and cellular heterogeneity of the myocardium. Here, we report on the development of a 3D cardiac μTissue towards recapitulating the architecture and composition of native myocardium in vitro. Cells are encapsulated within micropatterned gelatin-based hydrogels formed via visible light photocrosslinking. This system enables spatial control of cardiac microarchitecture, perturbation of the cellular composition, and functional measures of EC coupling via video microscopy and a custom algorithm to quantify beat frequency and degree of coordination. To demonstrate the robustness of these tools and evaluate the impact of altered cell population densities on cardiac μTissues, contractility and cell morphology were assessed with the inclusion of exogenous non-myelinating Schwann cells (SCs). Results demonstrate that the addition of exogenous SCs alter cardiomyocyte EC, profoundly inhibiting the response to electrical pacing. Computational modeling of connexin-mediated coupling suggests that SCs impact cardiomyocyte resting potential and rectification following depolarization. Cardiac μTissues hold potential for examining the role of cellular heterogeneity in heart health, pathologies, and cellular therapies.

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Maternal Embryonic Leucine Zipper Kinase (MELK) Drives a High-Risk Gene Network and Represents an Attractive Novel Drug Target in Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.309.309 ◽

2016 ◽

Vol 128 (22) ◽

pp. 309-309 ◽

Cited By ~ 1

Author(s):

Arnold Bolomsky ◽

Roy Heusschen ◽

Karin Schlangen ◽

Kathrin Schönfelder ◽

Joséphine Muller ◽

...

Keyword(s):

Bone Marrow ◽

High Risk ◽

Leucine Zipper ◽

Myeloma Cell ◽

Spleen Weight ◽

Protein Levels ◽

The Impact ◽

Dose Dependent

Abstract Background: In spite of significant progress in the outcome of multiple myeloma (MM), effective treatment options are still missing for high-risk patients. A better understanding of key mechanisms in high-risk disease is therefore a necessary prerequisite for the development of novel therapeutic options for patients with poor prognosis. Here, we evaluated maternal embryonic leucine zipper kinase (MELK) for its implications in the prognosis of MM, its interactions with genes involved in high-risk MM and the impact of MELK inhibition in vitro and in vivo. Methods: Expression levels of genes associated with high-risk MM were analyzed in two large cohorts of publically available gene expression (GEP) datasets (GSE2658 and GSE9782; n=551 and n=264, respectively) and by quantitative PCR (qPCR) in human myeloma cell lines (HMCLs). The impact of MELK inhibition was studied using a selective inhibitor of MELK (OTSSP167). MELK and OTSSP167 target genes were analyzed by qPCR and Western Blot. MELK knockdown HMCLs were generated via lentiviral transduction of MELK specific shRNA. For the in vivo experiment, C57BL/KaLwRij mice were injected intravenously with 5x105 GFP-transfected 5TGM.1 cells. Starting the day after injection, two groups of mice were treated by different doses of OTSSP167 (15mg/kg/d or 7.5mg/kg/2d) and one group received the vehicle. Treatment was administered orally. Results: MELK expression was significantly elevated in the GEP-defined high-risk proliferation (PR) associated molecular subgroup compared to healthy donor bone marrow plasma cells, but barely detected in any other GEP defined MM subgroup. Consequently, overall survival was significantly shorter in patients with high compared to low MELK levels treated within the total therapy 2 (P=0.0003), total therapy 3 (P=0.04) and the APEX trial protocol (P=0.002). Of note, MELK expression was further elevated in relapsed patients compared to baseline (GSE31161) suggesting a potential role in drug resistance. In line with their proliferative character, MELK expression was detected in 8 of 8 HMCLs. Treatment with the MELK inhibitor OTSSP167 downregulated MELK protein levels and led to a dose-dependent reduction of viability in all primary MM samples and HMCLs tested (median IC50: 10.16 nM, range: 7.6 - 15.2 nM). We observed induction of apoptosis in all HMCLs investigated, verified by mitochondrial membrane depolarization, annexin V/7-AAD staining, detection of cleaved caspase 3, and cleaved PARP. This was accompanied by downregulation of gene expression levels of IRF4 and MCL-1. In addition, OTSSP167 induced a G2/M cell cycle arrest which was linked to downregulation of cyclin B1, aurora kinase A and PLK-1. Importantly, we also observed reduced clonogenic growth of MM cells treated with OTSSP167 and the anti-myeloma activity of OTSSP167 was upheld in the presence of bone marrow stromal cells. We next sought to clarify the impact of MELK on other genes implicated in high-risk myeloma. Strikingly, treatment of HMCLs with OTSSP167 reduced MELK and PLK-1 protein levels, but also those of FOXM1, EZH2 and DEPDC1. This relationship was confirmed in HMCLs with shRNA mediated MELK knockdown. All of these genes were significantly elevated in the GEP defined PR subgroup and associated with poor outcome. Finally, the activity of OTSSP167 was analyzed in the 5TGM.1 model of MM. In vivo, we observed a dose-dependent reduction of tumor parameters by OTSSP167. Low doses of OTSSP167 significantly reduced spleen weight and serum IgG2b levels but only tended to decrease BM infiltration, while high doses of OTSSP167 significantly decreased tumor infiltration analyzed by spleen weight (267mg vs 108mg) and bone marrow infiltration (14% vs 2%, P<0.05). Conclusion: The current study identified almost exclusive expression of MELK in the GEP defined PR subgroup of MM. Hence, MELK expression represents a novel poor prognostic marker. Inhibition of MELK with OTSSP167 impaired myeloma cell growth and survival in vitro and in vivo. Moreover, downregulation of MELK via shRNA or OTSSP167 treatment significantly reduced protein levels of several other genes of the GEP defined PR subgroup of myeloma, suggesting a central role for MELK in the signaling network of proliferation associated high-risk myeloma. These results place MELK in the center of a MM high-risk gene network and emphasize to initiate clinical testing of MELK inhibition in MM. Disclosures Ludwig: Amgen: Research Funding, Speakers Bureau; Janssen: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Takeda: Research Funding, Speakers Bureau.

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Potentials of medicinal nanostructured diamond particles and coatings

10.31219/osf.io/h68xz ◽

2021 ◽

Author(s):

Moataz Dowaidar

Keyword(s):

Chemical Stability ◽

Clinical Medicine ◽

The Body ◽

Specific Cell ◽

Diamond Surfaces ◽

Diamond Particles ◽

In Vivo Testing ◽

The Impact

Particles and coatings can be manufactured utilizing low-cost, scalable processes. In both in vitro and in vivo experiments, unmodified diamond materials displayed good cell biocompatibility. Most current nanostructured diamond research focuses on diamond surfaces for specific cell and tissue interactions. Although the chemical stability of functionalized diamond surfaces has been explored for relatively short periods of time, the functionalized surface's chemical stability while its use in the body deserves additional exploration. In addition, the impact of chemistry (e.g., pH), enzymes, and microorganisms on the functionalized diamond surface's chemical stability must be considered. Moreover, the goals of studies evaluating the biological functionality of nanostructured diamond materials must be better aligned with regulatory requirements (e.g., ASTM International and the International Organization for Standardization) and in vitro and in vivo testing requirements of government agencies (e.g. the U.S. Food & Drug Administration). Because national regulatory authorities generally review medical devices rather than biomaterials, the parameters of in vitro and in vivo research should be closer to the implantation site's biological, chemical and mechanical features. Research on the breakdown of nanostructured diamond devices and the presence of degradation products in surrounding and remote tissues is needed. Thinking about how the sterilizing technique impacts the surface properties and biocompatibility of nanostructured diamond materials is equally significant. As the stability and fate of functionalized diamond particles and surfaces is better known via more in-vitro and in-vivo testing, nanostructured diamond materials are likely to play a greater role in clinical medicine.

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In vitro and in vivo biological approach to validate folkloric claims of Trianthema triquetra Rottler & Willd

Bangladesh Journal of Botany ◽

10.3329/bjb.v50i1.52664 ◽

2021 ◽

Vol 50 (1) ◽

pp. 21-28

Author(s):

Fatima Saqib ◽

Mehwish Shaukat ◽

Sana Javad ◽

Muhammad Riaz ◽

Zahid Khan ◽

...

Keyword(s):

Calcium Channels ◽

Analgesic Activity ◽

Rabbit Aorta ◽

Lipoxygenase Activity ◽

Traditional Practices ◽

Response Curves ◽

In Vivo Testing ◽

Dose Dependent

Trianthema triquetra Rottler & Willd (Tt.Cr) is used in traditional practices as a remedy for various ailments. Hence current research was commenced to authenticate the folkloric uses. To discover spasmolytic potential, Tt.Cr was applied to isolate jejunum, while isolated tracheal and aorta tissues were used to determine the tissue relaxing properties of the extract. Anti-lipoxygenase activity was determined in vitro using Baicalein as standard. In vivo testing was carried to examine the potentiality of the herb to treat pyrexia and pain. Tt.Cr showed dose-dependent (0.01 - 3.0 mg/ml) spasmolytic effects in jejunum tissues and relaxed K+ (80 mM)-induced spasm and triggered rightwards shift of Ca+2 concentration-response curves. Carbachol (1μM)- together with K+ (80 mM) - induced tracheal spasm was also relaxed by Tt.Cr (0.01 to 1.0 mg/ml). Additionally, Tt.Cr (0.01 - 1.0 mg/ml) relaxed phenylephrine (1 μM) and K+ (80 mM) - treated constricted rabbit aorta. Tt.Cr (0.5 mM) inhibited lipoxygenase enzyme. Tt.Cr (80 mg/kg) settled pyrexia in rabbits comparable to aspirin and prolonged tail deflection time in mice (100 mg/kg) hence proving analgesic activity. The Tt.Cr demonstrated antispasmodic, bronchodilation and vasodilation properties probably by blocking calcium channels. These outcomes generate logic behind ancient application of herb for numerous ailments such as asthma, cough, heart problems and spasm.

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Soluble Thrombomodulin Purified from Human Urine Exhibits a Potent Anticoagulant Effect In Vitro and In Vivo

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653872 ◽

1995 ◽

Vol 73 (05) ◽

pp. 805-811 ◽

Cited By ~ 12

Author(s):

Yasuo Takahashi ◽

Yoshitaka Hosaka ◽

Hiromi Niina ◽

Katsuaki Nagasawa ◽

Masaaki Naotsuka ◽

...

Keyword(s):

Human Urine ◽

Specific Activity ◽

Anticoagulant Activity ◽

Rabbit Lung ◽

Soluble Thrombomodulin ◽

Molecular Weights ◽

Dose Dependent Fashion ◽

Dose Dependent

SummaryWe examined the anticoagulant activity of two major molecules of soluble thrombomodulin purified from human urine. The apparent molecular weights of these urinary thrombomodulins (UTMs) were 72,000 and 79,000, respectively. Both UTMs showed more potent cofactor activity for protein C activation [specific activity >5,000 thrombomodulin units (TMU)/mg] than human placental thrombomodulin (2,180 TMU/mg) and rabbit lung thrombomodulin (1,980 TMU/mg). The UTMs prolonged thrombin-induced fibrinogen clotting time (>1 TMU/ml), APTT (>5 TMU/ml), TT (>5 TMU/ml) and PT (>40 TMU/ml) in a dose-dependent fashion. These effects appeared in the concentration range of soluble thrombomodulins present in human plasma and urine. In the rat DIC model induced by thromboplastin, administration of UTMs by infusion (300-3,000 TMU/kg) restored the hematological abnormalities derived from DIC in a dose-dependent fashion. These results demonstrate that UTMs exhibit potent anticoagulant and antithrombotic activities, and could play a physiologically important role in microcirculation.

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Vasorelaxant and Antihypertensive Effects of Mentha pulegium L. in Rats: An in vitro and in vivo Approach

Endocrine Metabolic & Immune Disorders - Drug Targets ◽

10.2174/1871530320666200909093908 ◽

2020 ◽

Vol 20 ◽

Author(s):

Mohammed Ajebli ◽

Mohamed Eddouks

Keyword(s):

Blood Pressure ◽

Acute Experiment ◽

Antihypertensive Activity ◽

Mentha Pulegium ◽

In Vitro Experiment ◽

Arterial Blood ◽

Dose Dependent ◽

Ca2 Channels

Aims and objective: The aim of the study was to investigate the effect of aqueous aerial part extract of Mentha pulegium L. (Pennyrile) (MPAE) on arterial pressure parameters in rats. Background: Mentha pulegium is a medicinal plant used to treat hypertension in Morocco. Material and methods: In the current study, MPAE was prepared and its antihypertensive activity was pharmacologically investigated. L-NAME-hypertensive and normotensive rats have received orally MPAE (180 and 300 mg/kg) during six hours for the acute experiment and during seven days for the sub-chronic treatment. Thereafter, systolic, diastolic, mean arterial blood pressure and heart rate were evaluated. While, in the in vitro experiment, isolated denuded and intact thoracic aortic rings were suspended in a tissue bath system and the tension changes were recorded. Results: A fall in blood pressure was observed in L-NAME-induced hypertensive treated with MPAE. The extract also produced a dose-dependent relaxation of aorta pre-contracted with NE and KCl. The study showed that the vasorelaxant ability of MPAE seems to be exerted through the blockage of extracellular Ca2+ entry. Conclusion: The results demonstrate that the extract of pennyrile exhibits antihypertensive activity. In addition, the effect may be, at least in part, due to dilation of blood vessels via blockage of Ca2+ channels.

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