Elsevier

Toxicology in Vitro

Volume 27, Issue 6, September 2013, Pages 1838-1846
Toxicology in Vitro

Development of micellar reactive oxygen species assay for photosafety evaluation of poorly water-soluble chemicals

https://doi.org/10.1016/j.tiv.2013.05.008Get rights and content

Highlights

  • Reactive oxygen species (ROS) assay has solubility issues.

  • Micellar ROS (mROS) assay was developed for photosafety testing on poorly soluble chemicals.

  • The mROS assay exhibited high robustness, reproducibility, and enhanced applicability.

  • Complementary use of mROS assay might strengthen assay performance of ROS assay.

Abstract

A reactive oxygen species (ROS) assay was previously developed for photosafety assessment; however, the phototoxic potential of some chemicals cannot be evaluated because of their limited aqueous solubility. The present study was undertaken to develop a new micellar ROS (mROS) assay system for poorly water-soluble chemicals using a micellar solution of 0.5% (v/v) Tween 20 for solubility enhancement. In repeated mROS assay, intra- and inter-day precisions (coefficient of variation) were found to be below 11%, and the Z′-factors for singlet oxygen and superoxide suggested a large separation band between positive and negative standards. The ROS and mROS assays were applied to 65 phototoxins and 18 non-phototoxic compounds for comparative purposes. Of all 83 chemicals, 25 were unevaluable in the ROS assay due to poor solubility, but only 2 were in the mROS assay. Upon mROS assay on these model chemicals, the individual specificity was 76.5%, and the positive and negative predictivities were found to be 93.9% and 86.7%, respectively. The mROS assay provided 2 false negative predictions, although negative predictivity for the ROS assay was found to be 100%. Considering the pros and cons of these assays, strategic combined use of the ROS and mROS assays might be efficacious for reliable photosafety assessment with high applicability and predictivity.

Introduction

Several classes of pharmaceutics, cosmetics and food ingredients can be excited by sunlight, consisting of partial ultraviolet (UV) B (290–320 nm), UVA (320–400 nm) and visible light (400–700 nm); then, these photo-excited agents can elicit phototoxic reactions in skin and eyes (Epstein, 1983, Moore, 2002, Onoue et al., 2009). For photosafety evaluation, a number of effective in vitro methodologies have been proposed within the past few decades (Seto et al., 2012), and, notably, a UV absorption system (Henry et al., 2009) and a 3T3 neutral red uptake phototoxicity test (Spielmann et al., 1994) were recommended in the Organisation for Economic Co-operation and Development (OECD) guideline (OECD, 2004). Considering the implementation of the 3Rs principle (replacement, reduction and refinement), interest in the development of in vitro assessments based on photochemical and photobiological mechanisms should be increasing in photosafety assessments. A reactive oxygen species (ROS) assay was designed for the in vitro photoreactivity assessment of pharmaceuticals on the basis of ROS generation from photoirradiated chemicals, including singlet oxygen and superoxide (Onoue and Tsuda, 2006). The experimental conditions of the ROS assay were optimized (Onoue et al., 2008a, Onoue et al., 2008b) and validated (Onoue et al., in press), offering high assay productivity and prediction capacity.

Although the ROS assay demonstrated high prediction capacity for photosafety assessment, there appeared to be at least two assay limitations in a multi-laboratory validation study: (i) false positive predictions and (ii) solubility issues (Onoue et al., in press). Since the ROS assay is carried out in early phases of photosafety assessments, false positives would be re-evaluated by appropriate follow-up assessments. In this context, the former assay limitation might not be a severe problem. In contrast, the solubility issues would be a serious problem for reliable photosafety assessment. In the validation study (Onoue et al., in press), 43% of tested chemicals could not be dissolved in reaction mixtures at 200 μM owing to their poor water solubility, and, additional experiments on these chemicals had to be performed at lower concentrations (20 or 2 μM). The ROS data on some phototoxins at lower concentrations led to different observations among three laboratories, and ROS data from chemicals at lower concentrations might not be suitable for photosafety assessment. Hence, appropriate modifications to the ROS assay system for enhanced applicability would be required for reliable photosafety assessment on poorly water-soluble chemicals.

For solubilizing poorly water-soluble drugs in oral and injectable solution forms, micelle systems are widely used in commercially available formulations (Strickley, 2004). In addition, a previous study demonstrated that the use of micellar solution systems, such as Tween 20, sodium laurate and sodium dodecyl sulfate (SDS), would be effective for monitoring singlet oxygen generation from poorly water-soluble chemicals because of the intense solubilizing potency and production of the biomembrane-mimetic environment (Onoue et al., 2008c). Thus, the present study attempted to develop a micellar ROS (mROS) assay with the aim of overcoming solubility issues of ROS assay, and thus a micellar solution of Tween 20 (polyoxyethylene sorbitan monolaurate), a non-ionic detergent, was applied to the ROS assay system. The precision and robustness of the mROS assay were evaluated by repeated measurement and calculation of Z′-factor, a parameter reflecting the quality of the assay. To verify the utility of the mROS assay, the number of evaluable compounds and the predictability for photosafety were compared between the ROS and mROS assays using 65 phototoxins and 18 non-phototoxic compounds.

Section snippets

Chemicals

Amlodipine besylate (>98%; 5), chlorpromazine HCl (>99%; 12), ciprofloxacin (>98%; 14), fenofibrate (>98%; 19), fluvastatin Na (>98%; 21), glibenclamide (>98%; 23), gliclazide (>98%; 24), griseofulvin (>95%; 25), hydrochlorothiazide (>98%; 26), ibuprofen (>98.5%; 27), indomethacin (>98%; 28), ketoprofen (>98%; 29), lomefloxacin HCl (>98%; 31), lovastatin (>95%; 33), meloxicam (>98%; 35), methotrexate (>98%; 36), 6-methylcoumarin (>99%; 38), mequitazine (>98%; 39), nicardipine HCl (>99%; 42),

Selection of detergent for mROS assay

On the basis of a previous study (Onoue et al., 2008c), the micellar solution system was applied to the ROS assay system with the aim of solubilizing poorly water-soluble chemicals. In general, superoxide can be detected by the conversion of NBT to monoformazan; however, a previous study demonstrated that the weak acidity at pH 6.2 attenuated the conversion from tetrazolium salt to formazan (Johno et al., 2010). Since the use of acidic detergents, including sodium laurate and SDS, might affect

Conclusion

The mROS assay was developed with the use of 0.5% (v/v) Tween 20 for photosafety assessment on poorly water-soluble chemicals. This mROS assay exhibited high robustness and reproducibility, and the addition of micellar solution significantly enhanced the applicability of the ROS assay system to poorly water-soluble chemicals. Despite some false negative predictions in the mROS assay, complementary use of mROS assay might strengthen the assay performance of the ROS assay on wide range of new

Conflict of Interest

None of the authors have any conflicts of interest associated with this study.

Acknowledgements

This work was supported in part by a Health Labour Sciences Research Grant from The Ministry of Health, Labour and Welfare, Japan, and a Grant-in-Aid for Scientific Research (C) (No. 24590200; S. Onoue) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

References (39)

  • N. Canudas et al.

    Photodegradation of bezafibrate in aqueous media. Studies of its in vitro phototoxicity

    Arzneimittelforschung

    (1996)
  • E. Durbize et al.

    Spectrum of cross-photosensitization in 18 consecutive patients with contact photoallergy to ketoprofen: associated photoallergies to non-benzophenone-containing molecules

    Contact Dermatitis

    (2003)
  • E. Gebhardt et al.

    In vitro evaluation of phototoxic properties of proton pump inhibitors, H2-receptor antagonists and statins

    Acta Derm. Venereol.

    (2012)
  • S.A. Grabczynska et al.

    Amlodipine induced-photosensitivity presenting as telangiectasia

    Br. J. Dermatol.

    (2000)
  • N. Hayashi et al.

    New findings on the structure-phototoxicity relationship and photostability of fluoroquinolones with various substituents at position 1

    Antimicrob. Agents Chemother.

    (2004)
  • H. Johno et al.

    Influences of acidic conditions on formazan assay: a cautionary note

    Appl. Biochem. Biotechnol.

    (2010)
  • Y. Kurumaji

    Photo Koebner phenomenon in erythema-multiforme-like eruption induced by contact dermatitis due to bufexamac

    Dermatology

    (1998)
  • B.A. Lipsky et al.

    Fluoroquinolone toxicity profiles: a review focusing on newer agents

    Clin. Infect Dis.

    (1999)
  • G. Miolo et al.

    In vitro phototoxicity of phenothiazines: involvement of stable UVA photolysis products formed in aqueous medium

    Chem. Res. Toxicol.

    (2006)
  • Cited by (0)

    1

    Current address: Pharmacokinetics and Safety Research Department, Central Research Laboratories, Kaken Pharmaceutical Co. Ltd., 301 Gensuke, Fujieda, Shizuoka 426-8646, Japan.

    View full text