Development of a modified 3T3 Neutral Red Uptake Phototoxicity Test protocol for evaluation of poorly water-soluble substances

Toyoda, Akemi; Sugiyama, Maki; Furihata, Seiichiro; Nishizumi, Keiji; Omori, Takashi; Itagaki, Hiroshi

doi:10.2131/jts.42.569

Cited by 5 publications

(4 citation statements)

References 15 publications

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“…However, it may be limited by compounds that are volatile, unstable in water, or with low solubility. 103…”

Section: Neutral Red Assaymentioning

confidence: 99%

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Cell viability and cytotoxicity assays: Biochemical elements and cellular compartments

Khalef,

Lydia,

Filicia

et al. 2024

Cell Biochemistry & Function

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Cell viability and cytotoxicity assays play a crucial role in drug screening and evaluating the cytotoxic effects of various chemicals. The quantification of cell viability and proliferation serves as the cornerstone for numerous in vitro assays that assess cellular responses to external factors. In the last decade, several studies have developed guidelines for defining and interpreting cell viability and cytotoxicity based on morphological, biochemical, and functional perspectives. As this domain continues to experience ongoing growth, revealing new mechanisms orchestrating diverse cell cytotoxicity pathways, we suggest a revised classification for multiple assays employed in evaluating cell viability and cell death. This classification is rooted in the cellular compartment and/or biochemical element involved, with a specific focus on mechanistic and essential aspects of the process. The assays are founded on diverse cell functions, encompassing metabolic activity, enzyme activity, cell membrane permeability and integrity, adenosine 5′‐triphosphate content, cell adherence, reduction equivalents, dye inclusion or exclusion, constitutive protease activity, colony formation, DNA fragmentation and nuclear splitting. These assays present straightforward, reliable, sensitive, reproducible, cost‐effective, and high‐throughput approaches for appraising the effects of newly formulated chemotherapeutic biomolecules on the cell survival during the drug development process.

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“…However, it may be limited by compounds that are volatile, unstable in water, or with low solubility. 103…”

Section: Neutral Red Assaymentioning

confidence: 99%

“…It requires minimal equipment and is compatible with determining protein content. However, it may be limited by compounds that are volatile, unstable in water, or with low solubility 103 …”

Section: Cell Viability Assaysmentioning

confidence: 99%

Cell viability and cytotoxicity assays: Biochemical elements and cellular compartments

Khalef,

Lydia,

Filicia

et al. 2024

Cell Biochemistry & Function

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“…However, some difficulty may occur in the measurement process due to precipitate formation in the case of highly hydrophobic chemicals. Therefore, some modifications, such as vehicle selection, have been attempted to broaden the applicability of these tests to hydrophobic chemicals (Seto, Kato, Yamada, & Onoue, 2013; Toyoda et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Development of a new photosafety test method based on singlet oxygen generation detected using electron spin resonance

Hinoshita

Abe

Sato

et al. 2020

J of Applied Toxicology

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Photosafety evaluations of chemicals used in consumer products, such as pharmaceuticals and cosmetics, are very important. Currently, two non-animal tests for photosafety evaluations, the in vitro 3T3 neutral red uptake phototoxicity test (NRU PT) and the reactive oxygen species (ROS) assay, are used to detect photoreactive chemicals. However, these two tests are difficult to apply to hydrophobic chemicals. In the present study, we attempted to develop a new photosafety test method, named the electron spin resonance-based photosafety test (ESR-PT), that would be applicable even to hydrophobic chemicals based on the detection of singlet oxygen generation after irradiation using ESR spectroscopy with 4-hydroxy-2,-2,6,6-tetramethyl-piperidine as a spin trap reagent. To achieve a quantitative evaluation, the singlet oxygen formation (SOF) value, which can be calculated as the increment in relative intensity after irradiation of the test mixture normalized by the increment in relative intensity after irradiation of the vehicle control solution, was calculated. The performance of the ESR-PT was evaluated by testing all the proficiency chemicals of the ROS assay plus additional chemicals, including hydrophobic chemicals and chemicals that tested false negative in the 3T3-NRU PT and ROS assay. SOF values were successfully calculated for all the chemicals tested including the hydrophobic chemicals, and the accuracy of the ESR-PT using a tentative cutoff value of 2.8 against the photosafety information was 100%. Therefore, the SOF value could be an effective parameter for photosafety evaluations, suggesting that the newly developed ESR-PT is a promising non-animal test applicable even to hydrophobic chemicals.

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“…Numerous methods have been introduced for photosafety testing, but priority has been attributed to animal-free test methods, in particular in vitro assays, such as the 3T3 neutral red uptake assay (3T3 NRU), − the photohemolysis assays based on erythrocytes, or advanced human reconstructed epidermis models including a corneal barrier, and mechanistic assays. Those latter include the ROS assay or suntest in solution which probes for photochemical degradation using a commercial sun simulator .…”

Section: Introductionmentioning

confidence: 99%

Computational Investigation of Drug Phototoxicity: Photosafety Assessment, Photo-Toxophore Identification, and Machine Learning

Schmidt

Wenzel

Halland

et al. 2019

Chem. Res. Toxicol.

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One of the most appreciated capabilities of computational toxicology is to support the design of pharmaceuticals with reduced toxicological hazard. To this end, we have strengthened our drug photosafety assessments by applying novel computer models for the anticipation of in vitro phototoxicity and human photosensitization. These models are typically used in pharmaceutical discovery projects as part of the compound toxicity assessments and compound optimization methods. To ensure good data quality and aiming at models with global applicability we separately compiled and curated highly chemically diverse data sets from 3T3 NRU phototoxicity reports (450 compounds) and clinical photosensitization alerts (1419 compounds) which are provided as supplements. The latter data gives rise to a comprehensive list of explanatory fragments for visual guidance, termed phototoxophores, by application of a Bayesian statistics approach. To extend beyond the domain of well sampled fragments we applied machine learning techniques based on explanatory descriptors such as pharmacophoric fingerprints or, more important, accurate electronic energy descriptors. Electronic descriptors were extracted from quantum chemical computations at the density functional theory (DFT) level. Accurate UV/vis spectral absorption descriptors and pharmacophoric fingerprints turned out to be necessary for predictive computer models, which were both derived from Deep Neural Networks but also the simpler Random Decision Forests approach. Model accuracies of 83–85% could typically be reached for diverse test data sets and other company in-house data, while model sensitivity (the capability of correctly detecting toxicants) was even better, reaching 86%–90%. Importantly, a computer model-triggered response-map allowed for graphical/chemical interpretability also in the case of previously unknown phototoxophores. The photosafety models described here are currently applied in a prospective manner for the hazard identification, prioritization, and optimization of newly designed molecules.

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Development of a modified 3T3 Neutral Red Uptake Phototoxicity Test protocol for evaluation of poorly water-soluble substances

Cited by 5 publications

References 15 publications

Cell viability and cytotoxicity assays: Biochemical elements and cellular compartments

Cell viability and cytotoxicity assays: Biochemical elements and cellular compartments

Development of a new photosafety test method based on singlet oxygen generation detected using electron spin resonance

Computational Investigation of Drug Phototoxicity: Photosafety Assessment, Photo-Toxophore Identification, and Machine Learning

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