Development of an albuminous reactive oxygen species assay for photosafety evaluation under experimental biomimetic conditions

Abstract

The generation of reactive oxygen species (ROS) from an ultraviolet (UV)-exposed chemical can be an experimental indicator of phototoxic potential. The aim of the present study was to develop a new ROS assay using serum albumin to provide photosafety assessment under experimental biomimetic conditions. To assess assay robustness, a validation study on an albuminous ROS (aROS) assay was conducted with a focus on intra- and inter-day precisions and Z'-factor reflecting both the assay signal-to-noise ratio and variation associated with signal measurements. In the aROS assay on quinine HCl (200 μM), a typical phototoxic drug, both intra- and inter-day precisions (coefficient of variation; CV) were found to be below 4%, and the Z'-factors for singlet oxygen and superoxide suggested a large separation band between samples and blank signals. To evaluate the prediction capacity, the aROS and ROS assays were applied to 21 phototoxins and 10 non-phototoxic chemicals. Upon aROS assay on these model chemicals, the individual specificity was 100%, and the positive and negative predictivities were found to be 100% and 81.8%, respectively. The aROS assay can be employed for poorly soluble chemicals for which the ROS assay is unavailable. Comparing the ROS assay data, there seemed to be a photochemical transition of some chemicals in albuminous solution. A molecular interaction between albumin and chemical was also assessed by UV and fluorescent spectroscopic analyses, and the results suggested the limited relationship between the albumin-chemical interaction and the photochemical change. The aROS assay may allow photosafety assessment of new drug entities with a wide range of applicability partly under experimental biomimetic conditions.

New Photosafety Assessment Strategy Based on the Photochemical and Pharmacokinetic Properties of Both Parent Chemicals and Metabolites

“…However, singlet oxygen generation from some irradiated samples was negligible in the ROS assay with S9 fractions, whereas potent singlet oxygen generation from irradiated FF and FA was observed in the mROS assay. Changes in photochemical reactions in the ROS assay were attributed to assay conditions, including the concentration of chemicals and additives (Onoue et al, 2008b(Onoue et al, , 2014b. Although further optimization of assay conditions is needed, the ROS assay employing drug-metabolizing enzymes might become a useful method for hazard identification of metabolite-mediated phototoxicity.…”

TG 495: Ros (Reactive Oxygen Species) Assay for Photoreactivity

“…The applicability domain of the ROS assay is currently restricted to only those chemicals that meet the solubility criteria outlined in the protocol (see paragraph 22). Insoluble chemicals in the reaction mixtures are not suitable for testing with the ROS assay using this protocol (DMSO or NaPB solvent) but might be tested in the ROS assay with addition of solubility enhancers in the reaction mixtures (22) (23) (24). However, further characterization and standardization of procedures using these alternative vehicles should be performed by testing proficiency chemicals before incorporation into routine use.…”

TG 495 : Essai de Photoréactivité DRO (Dérivés réactifs de l’oxygène)

“…Le domaine d'applicabilité de l'essai DRO est actuellement limité aux seuls produits chimiques qui répondent aux critères de solubilité énoncés dans le protocole (voir le paragraphe 22). Les produits chimiques insolubles dans les mélanges réactionnels ne sont pas adaptés aux essais par le test DRO selon le présent protocole (DMSO ou NaPB utilisés comme solvant), mais peuvent être testés moyennant l'addition d'agents solubilisants dans les mélanges réactionnels (22) (23) (24). Cependant, les procédures utilisant ces véhicules alternatifs doivent être mieux caractérisées et standardisées par des essais sur les produits chimiques indiqués pour les épreuves de compétence, avant d'être utilisées en routine.…”