scholarly journals Posttreatment Strategy Against Hypoxia and Ischemia Based on Selective Targeting of Nonnuclear Estrogen Receptors with PaPE-1

2021 ◽  
Author(s):  
A. Wnuk ◽  
K. Przepiórska ◽  
B. A. Pietrzak ◽  
M. Kajta
Keyword(s):  
Estrogen Receptors ◽  
Mitochondrial Membrane ◽  
Neutral Red ◽  
Apoptosis Inhibition ◽  
Selective Targeting ◽  
Cellular Metabolic Activity ◽  
Ros Formation ◽  
Neutral Red Uptake ◽  
Neuroprotective Action

AbstractNewly synthesized Pathway Preferential Estrogen-1 (PaPE-1) selectively activates membrane estrogen receptors (mERs), namely, mERα and mERβ, and has been shown to evoke neuroprotection; however, its effectiveness in protecting brain tissue against hypoxia and ischemia has not been verified in a posttreatment paradigm. This is the first study showing that a 6-h delayed posttreatment with PaPE-1 inhibited hypoxia/ischemia-induced neuronal death, as indicated by neutral red uptake in mouse primary cell cultures in vitro. The effect was accompanied by substantial decreases in neurotoxicity and neurodegeneration in terms of LDH release and Fluoro-Jade C staining of damaged cells, respectively. The mechanisms of the neuroprotective action of PaPE-1 also involved apoptosis inhibition demonstrated by normalization of both mitochondrial membrane potential and expression levels of apoptosis-related genes and proteins such as Fas, Fasl, Bcl2, FAS, FASL, BCL2, BAX, and GSK3β. Furthermore, PaPE-1-evoked neuroprotection was mediated through a reduction in ROS formation and restoration of cellular metabolic activity that had become dysregulated due to hypoxia and ischemia. These data provide evidence that targeting membrane non-GPER estrogen receptors with PaPE-1 is an effective therapy that protects brain neurons from hypoxic/ischemic damage, even when applied with a 6-h delay from injury onset.

The EYTEX™ System and the Neutral Red Uptake Inhibition Assay in Cultured Hep G2 Cells as Alternative Methods for In Vivo Eye Irritation Following the EEC Protocol

1990 ◽  
Vol 17 (4) ◽  
pp. 325-333
Author(s):  
Paul J. Dierickx ◽  
Virginia C. Gordon
Keyword(s):  
Neutral Red ◽  
Alternative Methods ◽  
Inhibition Assay ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Eye Irritation ◽  
Uptake Inhibition ◽  
In Vitro Methods ◽  
Neutral Red Uptake

The neutral red uptake inhibition assay and the EYTEX™ system were investigated as alternative methods for the assessment of eye irritation, determined according to the EEC protocol. The 17 test chemicals used were mainly organic solvents. The xenobiotics were applied to Hep G2 cells for 24 hours at different concentrations. Neutral red uptake inhibition was then measured. The results are expressed as the NI50 value, which is the concentration of test compound required to induce a 50% reduction in neutral red uptake. The same chemicals were also tested as coded samples by the EYTEX™ test according to the manufacturer's directions. A nearly identical quantitative correlation was found for both in vitro methods with corneal opacity scores: r = 0.84 for EYTEX™ scores and r = 0.83 for log NI50, expressed in μg/ml. Whilst these correlations are certainly not perfect, it is clear that both in vitro methods can be used as valuable prescreening methods.

scholarly journals Differential Effects of the Flavonolignans Silybin, Silychristin and 2,3-Dehydrosilybin on Mesocestoides vogae Larvae (Cestoda) under Hypoxic and Aerobic In Vitro Conditions

Molecules ◽  
2018 ◽  
Vol 23 (11) ◽  
pp. 2999 ◽  
Author(s):  
Gabriela Hrčková ◽  
Terézia Kubašková ◽  
Oldřich Benada ◽  
Olga Kofroňová ◽  
Lenka Tumová ◽  
...  
Keyword(s):  
Metabolic Activity ◽  
Neutral Lipids ◽  
Neutral Red ◽  
Suitable Model ◽  
Glucose Content ◽  
Morphological Alterations ◽  
Mitochondrial Functions ◽  
Neutral Red Uptake ◽  
Mesocestoides Vogae

Mesocestoides vogae larvae represent a suitable model for evaluating the larvicidal potential of various compounds. In this study we investigated the in vitro effects of three natural flavonolignans—silybin (SB), 2,3-dehydrosilybin (DHSB) and silychristin (SCH)—on M. vogae larvae at concentrations of 5 and 50 μM under aerobic and hypoxic conditions for 72 h. With both kinds of treatment, the viability and motility of larvae remained unchanged, metabolic activity, neutral red uptake and concentrations of neutral lipids were reduced, in contrast with a significantly elevated glucose content. Incubation conditions modified the effects of individual FLs depending on their concentration. Under both sets of conditions, SB and SCH suppressed metabolic activity, the concentration of glucose, lipids and partially motility more at 50 μM, but neutral red uptake was elevated. DHSB exerted larvicidal activity and affected motility and neutral lipid concentrations differently depending on the cultivation conditions, whereas it decreased glucose concentration. DHSB at the 50 μM concentration caused irreversible morphological alterations along with damage to the microvillus surface of larvae, which was accompanied by unregulated neutral red uptake. In conclusion, SB and SCH suppressed mitochondrial functions and energy stores, inducing a physiological misbalance, whereas DHSB exhibited a direct larvicidal effect due to damage to the tegument and complete disruption of larval physiology and metabolism.

Neutral Red Uptake, Cellular Growth and Lysosomal Function: In Vitro Effects of 24 Metals

1993 ◽  
Vol 21 (4) ◽  
pp. 501-507 ◽  
Author(s):  
Guillermo Repetto ◽  
Pilar Sanz
Keyword(s):  
Neuroblastoma Cells ◽  
Neutral Red ◽  
Cellular Growth ◽  
Metal Compounds ◽  
Lysosomal Activity ◽  
Neutral Red Assay ◽  
Neutral Red Uptake ◽  
Group B ◽  
Relative Uptake

Specific toxic interference in lysosomal activity was evaluated by the relative uptake of neutral red by lysosomes. In this adaptation of the standard neutral red cytotoxicity assay, the results are expressed relative to cell culture protein content to avoid misinterpretation due to the influence on cell proliferation of the chemicals tested. Neuro-2a mouse neuroblastoma cells were exposed in vitro to 24 metal compounds and the lysosomal activity was quantified. Five different patterns of alterations were identified. Group A (ZnCl2, NaAsO2, Na2HAsO4, CdCl2, SnCl2, HgCl2, HgCH3Cl and TlCOOCH3) produced an inhibition of the relative uptake of the dye, in marked contrast to the greater inhibition shown by the neutral red uptake assay. Group B (MgCl2, A1C13, KMnO4) NiCl2, BaCl2 and Pb[NO3]2) showed less inhibition with strong parallelism with the neutral red assay. In Group C (CrCl3, Na2Cr2O7, FeCl3, CoCl2, CuCl2 and Sn[C2H5]4), low-dose stimulation, and inhibition at higher concentrations were found. Group D (LiCl and CrCl2) stimulated uptake, and Group E (MnCl2 and Sr[NO3]2) produced no significant modifications. The relative uptake of neutral red can be a convenient tool for the study of specific toxic alterations of lysosomes.

scholarly journals Selective Targeting of Non-nuclear Estrogen Receptors with PaPE-1 as a New Treatment Strategy for Alzheimer’s Disease

2020 ◽  
Vol 38 (4) ◽  
pp. 957-966
Author(s):  
Agnieszka Wnuk ◽  
Karolina Przepiórska ◽  
Joanna Rzemieniec ◽  
Bernadeta Pietrzak ◽  
Małgorzata Kajta
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Membrane Potential ◽  
Estrogen Receptors ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Neurodegenerative Disorder ◽  
Apoptotic Pathway ◽  
Selective Targeting ◽  
Nuclear Estrogen Receptors

Abstract Alzheimer’s disease (AD) is a multifactorial and severe neurodegenerative disorder characterized by progressive memory decline, the presence of Aβ plaques and tau tangles, brain atrophy, and neuronal loss. Available therapies provide moderate symptomatic relief but do not alter disease progression. This study demonstrated that PaPE-1, which has been designed to selectively activate non-nuclear estrogen receptors (ERs), has anti-AD capacity, as evidenced in a cellular model of the disease. In this model, the treatment of mouse neocortical neurons with Aβ (5 and 10 μM) induced apoptosis (loss of mitochondrial membrane potential, activation of caspase-3, induction of apoptosis-related genes and proteins) accompanied by increases in levels of reactive oxygen species (ROS) and lactate dehydrogenase (LDH) as well as reduced cell viability. Following 24 h of exposure, PaPE-1 inhibited Aβ-evoked effects, as shown by reduced parameters of neurotoxicity, oxidative stress, and apoptosis. Because PaPE-1 downregulated Aβ-induced Fas/FAS expression but upregulated that of Aβ-induced FasL, the role of PaPE-1 in controlling the external apoptotic pathway is controversial. However, PaPE-1 normalized Aβ-induced loss of mitochondrial membrane potential and restored the BAX/BCL2 ratio, suggesting that the anti-AD capacity of PaPE-1 particularly relies on inhibition of the mitochondrial apoptotic pathway. These data provide new evidence for an anti-AD strategy that utilizes the selective targeting of non-nuclear ERs with PaPE-1.

The Neutral Red Uptake Assay: Comments on the Results of the Istituto Superiore di Sanità in the EC/HO Validation Study

1998 ◽  
Vol 26 (1) ◽  
pp. 61-68
Author(s):  
Annalaura Stammati ◽  
Franco Zampaglioni ◽  
Cristiana Zanetti
Keyword(s):  
Validation Study ◽  
Rank Correlation ◽  
Neutral Red ◽  
Home Office ◽  
Uptake Assay ◽  
Neutral Red Uptake ◽  
British Home ◽  
Chemical Groups

The neutral red uptake (NRU) assay was included, among others, in a validation study sponsored by the European Commission/British Home Office (EC/HO) study, for its reliability as an in vitro alternative to the Draize eye irritancy test. The test was performed in parallel by four laboratories (Istituto Superiore di Sanità [ISS], Microbiological Associates, Hatano Research Institute and Kurabo Industries) on 60 selected chemicals. The results obtained by the ISS are reported in this paper. A poor rank correlation was obtained between the in vivo endpoint and the ISS in vitro results for the full set of chemicals and for the subsets, with the exception of surfactants, by an independent statistics group. The same unsatisfactory results were obtained by the ISS group when the rank correlation was calculated for compounds divided into chemical groups. The performance of the NRU assay, as an alternative to the Draize eye irritancy test, is discussed.

scholarly journals In VitroToxicity of Epigallocatechin Gallate in Rat Liver Mitochondria and Hepatocytes

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Otto Kucera ◽  
Vojtech Mezera ◽  
Alena Moravcova ◽  
Rene Endlicher ◽  
Halka Lotkova ◽  
...  
Keyword(s):  
Rat Liver ◽  
Mitochondrial Membrane ◽  
Epigallocatechin Gallate ◽  
Rat Hepatocytes ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Main Compound ◽  
Ros Formation ◽  
High Doses

Epigallocatechin-3-gallate (EGCG) is the main compound of green tea with well-described antioxidant, anti-inflammatory, and tumor-suppressing properties. However, EGCG at high doses was reported to cause liver injury. In this study, we evaluated the effect of EGCG on primary culture of rat hepatocytes and on rat liver mitochondria in permeabilized hepatocytes. The 24-hour incubation with EGCG in concentrations of 10 μmol/L and higher led to signs of cellular injury and to a decrease in hepatocyte functions. The effect of EGCG on the formation of reactive oxygen species (ROS) was biphasic. While low doses of EGCG decreased ROS production, the highest tested dose induced a significant increase in ROS formation. Furthermore, we observed a decline in mitochondrial membrane potential in cells exposed to EGCG when compared to control cells. In permeabilized hepatocytes, EGCG caused damage of the outer mitochondrial membrane and an uncoupling of oxidative phosphorylation. EGCG in concentrations lower than 10 μmol/L was recognized as safe for hepatocytesin vitro.

A Multicentre Study of Acute In Vitro Cytotoxicity in Rat Hepatocytes: Tentative Correlation Between In Vitro Toxicities and In Vivo Data

1993 ◽  
Vol 21 (2) ◽  
pp. 281-284
Author(s):  
Alain Fautrel ◽  
Christophe Chesné ◽  
André Guillouzo ◽  
Georges De Sousa ◽  
Michel Placidi ◽  
...  
Keyword(s):  
Rat Hepatocytes ◽  
Neutral Red ◽  
Model System ◽  
In Vitro Cytotoxicity ◽  
Rat Hepatocyte ◽  
Ic50 Value ◽  
Neutral Red Uptake ◽  
Tentative Correlation

A multicentre validation study of the acute in vitro cytotoxicities of 31 liquid or solid chemicals was carried out by six laboratories, using primary rat hepatocyte cultures as a model system. We report here a comparison of neutral red uptake IC50 and LD50 values. Oral, i.p. and i.v. LD50 values were available for 27, 24 and 18 chemicals, respectively, and an IC50 value was obtained for 15, 14 and 11 of these compounds, respectively. A significant correlation was found only between IC50 and i.v. LD50 values.

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