Neutral Red Uptake Cytotoxicity Tests for Estimating Starting Doses for Acute Oral Toxicity Tests

Stokes, William S.; Casati, Silvana; Strickland, Judy; Paris, Michaël

doi:10.1002/0471140856.tx2004s36

Cited by 35 publications

(28 citation statements)

References 4 publications

(4 reference statements)

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“…According to Stokes et al [32], the evaluation of neutral red uptake is directly proportional to living cell number [33]. In our study, in all tested concentration, the extracts and the mixed sample reduced HaCat cell viability to less than 50% (Figure 6).…”

Section: Resultssupporting

confidence: 53%

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Evaluation of In Vitro Solar Protection Factor (SPF), Antioxidant Activity, and Cell Viability of Mixed Vegetable Extracts from Dirmophandra mollis Benth, Ginkgo biloba L., Ruta graveolens L., and Vitis vinífera L.

Cefali

Ataide

Fernandes

et al. 2019

Plants

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The aim of this study was to validate a HPLC method for the assay of flavonoids in extracts obtained from natural sources, i.e., from Dirmophandra mollis Benth, Ginkgo biloba L., Ruta graveolens L., and Vitis vinífera L. The potential sun protecting effect, antioxidant activity, and cell viability of the extracts were also determined. Individual extracts (obtained from each individual species) and a mixed extract (containing the four extracts) were analyzed by the validated HPLC method for the identification of flavonoids and quantification of rutin and quercetin. An in vitro cell viability study was carried out using the neutral red method. The in vitro sun protection factor was determined by spectral transmittance and in vitro antioxidant efficacy was evaluated against DPPH, ABTS, and AAPH radicals. The HPLC method used for the identification and quantification of flavonoids in extracts exhibited linearity, precision, accuracy, and robustness. Detection and quantification limits were, respectively, 2.881 ± 0.9 μg·mL−1 and 0.864 ± 0.9 μg·mL−1 for quercetin, and 30.09 ± 1 μg·mL−1 and 9.027 ± 1.1 μg·mL−1 for rutin. All extracts did not affect cell viability at the evaluated concentration range and exhibited a sun protection effect and antioxidant activity. Among the evaluated extracts, Ginkgo biloba L. and the mixed extract depicted the most expressive antioxidant activity. The mixed extract exhibited sunscreen protection against ultraviolet A (UVA) and ultraviolet B (UVB) and a critical wavelength of 372.7 ± 0.1. Our results translate the enhanced flavonoids’ composition of the mixed extract, which may be a potential alternative over sunscreens and antioxidants in pharmaceutic/cosmetic formulations.

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Section: Resultssupporting

confidence: 53%

“…The in vitro cell viability assay was performed as described by Stokes et al [32] and OECD [33]. The HaCaT (4 × 104 cells·mL −1 ), in 96-well plates (100 µL cells·well −1 ), were exposed to the samples’ final concentrations, in triplicate, for 48 h. The final DMSO concentration (≤0.25%) did not affect cell viability.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of In Vitro Solar Protection Factor (SPF), Antioxidant Activity, and Cell Viability of Mixed Vegetable Extracts from Dirmophandra mollis Benth, Ginkgo biloba L., Ruta graveolens L., and Vitis vinífera L.

Cefali

Ataide

Fernandes

et al. 2019

Plants

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“…Cells were washed with PBS then distained using (50% EtOH and 5% glacial acetic acid in distilled water). The absorbance was measured at 540 nm using spectrophotometer (Hewlett Packard, USA) [11]. All determinations were performed in triplicates.…”

Section: Evaluation Of Cytotoxicity Using Neutral Red Uptake Assaymentioning

confidence: 99%

Ganoderma applanatum secondary metabolites induced apoptosis through different pathways: In vivo and in vitro anticancer studies

Elkhateeb

Zaghlol

El-Garawani

et al. 2018

Biomedicine & Pharmacotherapy

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Ganoderma applanatum is a widely distributed saprobic or parasitic mushroom, it was found at the bases of decaying logs in Hakozaki Higashi-ku Fukuoka-shi. Japan. The mushroom was extracted with 80% methanol, and LC-HRMS analysis was conducted to illustrate the bioactive ingredients. The cytotoxicity of the total metabolite extract was evaluated against human colon cancer cell line (Caco-2) which showed IC value of 160 ± 4.08 μg/ml. G. applanatum methanolic extract caused different morphological alterations and increased glutathione level in the treated cells. Interestingly, G. applanatum increased Bax/Bcl-2 ratio significantly (P ˂ 0.05) at concentrations of 80 and 160 μg/ml on Caco-2 undergoing apoptotic p53-independent pathway with lake expression of p53 protein and up-regulated Cas-3 mRNA. The in vivo study on solid Ehrlich tumor (SEC) revealed a decrease in the volume of the developed tumor mass after five days of G. applanatum (200 μg/ml) treatment. The apoptotic p53-dependant pathway was confirmed by mRNA Bax/Bcl-2 increased ratio in addition to p53 and Cas-3 up-regulation. In conclusion, G. applanatum could exert apoptotic antitumor properties in Caco-2 by p53-independent pathway and p53-dependant in SEC. The findings proved that G. applanatum can be a promising candidate as alternative or co-anticancer medications.

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“…The neutral red uptake (NRU) assay was performed according to the standard protocol of Stokes [27] modified by NICEATM-ECVAM validation study [28]. The NRU cytotoxicity assay procedure was based on the ability of viable cells to incorporate and bind neutral red, which is a supravital dye.…”

Section: Methodsmentioning

confidence: 99%

Contribution of Molecular Structure to Self-Assembling and Biological Properties of Bifunctional Lipid-Like 4-(N-Alkylpyridinium)-1,4-Dihydropyridines

et al. 2019

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The design of nanoparticle delivery materials possessing biological activities is an attractive strategy for the development of various therapies. In this study, 11 cationic amphiphilic 4-(N-alkylpyridinium)-1,4-dihydropyridine (1,4-DHP) derivatives differing in alkyl chain length and propargyl moiety/ties number and position were selected for the study of their self-assembling properties, evaluation of their cytotoxicity in vitro and toxicity on microorganisms, and the characterisation of their interaction with phospholipids. These lipid-like 1,4-DHPs have been earlier proposed as promising nanocarriers for DNA delivery. We have revealed that the mean diameter of freshly prepared nanoparticles varied from 58 to 513 nm, depending upon the 4-(N-alkylpyridinium)-1,4-DHP structure. Additionally, we have confirmed that only nanoparticles formed by 4-(N-dodecylpyridinium)-1,4-DHP derivatives 3 and 6, and by 4-(N-hexadecylpyridinium)-1,4-DHP derivatives 10 and 11 were stable after two weeks of storage. The nanoparticles of these compounds were found to be homogenous in size distribution, ranging from 124 to 221 nm. The polydispersity index (PDI) values of 1,4-DHPs samples 3, 6, 10, and 11 were in the range of 0.10 to 0.37. We also demonstrated that the nanoparticles formed by 4-(N-dodecylpyridinium)-1,4-DHP derivatives 3, 6, and 9, and 4-(N-hexadecylpyridinium)-1,4-DHP derivatives 10 and 11 had zeta-potentials from +26.07 mV (compound 6) to +62.80 mV (compound 11), indicating a strongly positive surface charge and confirming the relative electrostatic stability of these nanoparticle solutions. Transmission electron microscopy (TEM) images of nanoaggregates formed by 1,4-DHPs 3 and 11 confirmed liposome-like structures with diameters around 70 to 170 nm. The critical aggregation concentration (CAC) value interval for 4-(N-alkylpyridinium)-1,4-DHP was from 7.6 µM (compound 11) to 43.3 µM (compound 6). The tested 4-(N-alkylpyridinium)-1,4-DHP derivatives were able to quench the fluorescence of the binary 1,6-diphenyl-1,3,5-hexatriene (DPH)—1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) system, demonstrating hydrophobic interactions of 1,4-DHPs with phospholipids. Thus, 4-(N-dodecylpyridinium)-1,4-DHP derivative 3 quenched the fluorescence of the DPH–DPPC system more efficiently than the other 4-(N-alkylpyridinium)-1,4-DHP derivatives. Likewise the compound 3, also 4-(N-dodecylpyridinium)-1,4-DHP derivative 9 interacted with the phospholipids. Moreover, we have established that increasing the length of the alkyl chain at the quaternised nitrogen of the 4-(N-alkylpyridinium)-1,4-DHP molecule or the introduction of propargyl moieties in the 1,4-DHP molecule significantly influences the cytotoxicity on HT-1080 (human fibrosarcoma) and MH-22A (mouse hepatocarcinoma) cell lines, as well as the estimated basal cytotoxicity. Additionally, it was demonstrated that the toxicity of the 4-(N-alkylpyridinium)-1,4-DHP derivatives on the Gram-positive and Gram-negative bacteria species and eukaryotic microorganism depended on the presence of the alkyl chain length at the N-alkyl pyridinium moiety, as well as the number of propargyl groups. These lipid-like compounds may be proposed for the further development of drug formulations to be used in cancer treatment.

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Neutral Red Uptake Cytotoxicity Tests for Estimating Starting Doses for Acute Oral Toxicity Tests

Cited by 35 publications

References 4 publications

Evaluation of In Vitro Solar Protection Factor (SPF), Antioxidant Activity, and Cell Viability of Mixed Vegetable Extracts from Dirmophandra mollis Benth, Ginkgo biloba L., Ruta graveolens L., and Vitis vinífera L.

Evaluation of In Vitro Solar Protection Factor (SPF), Antioxidant Activity, and Cell Viability of Mixed Vegetable Extracts from Dirmophandra mollis Benth, Ginkgo biloba L., Ruta graveolens L., and Vitis vinífera L.

Ganoderma applanatum secondary metabolites induced apoptosis through different pathways: In vivo and in vitro anticancer studies

Contribution of Molecular Structure to Self-Assembling and Biological Properties of Bifunctional Lipid-Like 4-(N-Alkylpyridinium)-1,4-Dihydropyridines

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