Phototoxic Reactions Inducted by Hydrochlorothiazide and Furosemide in Normal Skin Cells-In Vitro Studies on Melanocytes and Fibroblasts.

Hypertension is known to be a multifactorial disease associated with abnormalities in neuroendocrine, metabolic, and hemodynamic systems. Poorly controlled hypertension causes more than one in eight premature deaths worldwide. Hydrochlorothiazide (HCT) and furosemide (FUR), being first-line drugs in the treatment of hypertension, are among others the most frequently prescribed drugs in the world. Currently, many pharmacoepidemiological data associate the use of these diuretics with an increased risk of adverse phototoxic reactions that may induce the development of melanoma and non-melanoma skin cancers. In this study, the cytotoxic and phototoxic potential of HCT and FUR against skin cells varied by melanin pigment content was assessed for the first time. The results showed that both drugs reduced the number of metabolically active normal skin cells in a dose-dependent manner. UVA irradiation significantly increased the cytotoxicity of HCT towards fibroblasts by approximately 40% and melanocytes by almost 20% compared to unirradiated cells. In the case of skin cells exposed to FUR and UVA radiation, an increase in cytotoxicity by approximately 30% for fibroblasts and 10% for melanocytes was observed. Simultaneous exposure of melanocytes and fibroblasts to HCT or FUR and UVAR caused a decrease in cell viability, and number, which was confirmed by microscopic assessment of morphology. The phototoxic effect of HCT and FUR was associated with the disturbance of redox homeostasis confirming the oxidative stress as a mechanism of phototoxic reaction. UVA-irradiated drugs increased the generation of ROS by 10-150%, and oxidized intracellular thiols. A reduction in mitochondrial potential of almost 80% in melanocytes exposed to HCT and UVAR and 60% in fibroblasts was found due to oxidative stress occurrence. In addition, HCT and FUR have been shown to disrupt the cell cycle of normal skin cells. Finally, it can be concluded that HCT is the drug with a stronger phototoxic effect, and fibroblasts turn out to be more sensitive cells to the phototoxic effect of tested drugs.

Involvement of type-1 pathway in phototoxicity of benzo[ghi]perylenean ingredient of tattoo ink at ambient exposure of UVR and sunlight.

Tattooing on different parts of the body is a very common fashion trend in all sections of society globally. Skin allergies and other related skin diseases are very common among tattoo users. Benzo[ghi]perylene (BP) is a PAH and an important component of tattoo ink that showed prominent absorption under ultraviolet radiation (UVR) region. Therefore, to provide safety to the skin, a thorough safety study of BP exposed under UVR and Sunlight is very essential to understand their hazardous impact on the skin. BP showed a strong absorption of UVA and UVB radiation of sunlight. It is photolabile and degraded under UVA, UVB, and Sunlight in progressing order of time (1-4 h) without generating any novel photoproducts. Further, BP showed a specific generation of O2.- and OH radicals via activation of type I photodynamic reaction under exposure to UVA, UVB and Sunlight. Photocytotoxicity results illustrated concentration-dependent cell viability reduction in all exposure conditions of UVA, UVB, and Sunlight, respectively. Fluorescent probes (2',7'-dichlorofluorescein diacetate and dihydroethidium) for intracellular reactive oxygen species (ROS) generation supported the involvement of ROS in the phototoxicity of BP in the HaCaT cell line. Hoechst staining showed significant genomic insult induced by BP under UVA and UVB. Photoexcited BP promoted cell cycle arrest in the G1 phase and induced apoptosis confirmed via acridine orange/ethidium bromide staining. The findings of gene expression also supported apoptotic cell death in photoexcited BP via an increase in the level of pro-apoptotic gene (Bax) and a decrease in the level of anti-apoptotic gene (Bcl-2). The aforementioned finding indicates that tattoo users should avoid using BP since it can cause skin damage/diseases if they are exposed to UVR or Sunlight while tattooing on the body.

µ-Opioid Receptors Expressed by Intrinsically Photosensitive Retinal Ganglion Cells Contribute to Morphine-Induced Behavioral Sensitization.

Opioid drugs are the most effective tools for treating moderate to severe pain. Despite their analgesic efficacy, long-term opioid use can lead to drug tolerance, addiction, and sleep/wake disturbances. While the link between opioids and sleep/wake problems is well-documented, the mechanism underlying opioid-related sleep/wake problems remains largely unresolved. Importantly, intrinsically photosensitive retinal ganglion cells (ipRGCs), the cells that transmit environmental light/dark information to the brain's sleep/circadian centers to regulate sleep/wake behavior, express µ-opioid receptors (MORs). In this study, we explored the potential contribution of ipRGCs to opioid-related sleep/circadian disruptions. Using implanted telemetry transmitters, we measured changes in horizontal locomotor activity and body temperature in mice over the course of a chronic morphine paradigm. Mice lacking MORs expressed by ipRGCs (McKO) exhibited reduced morphine-induced behavioral activation/sensitization compared with control littermates with normal patterns of MOR expression. Contrastingly, mice lacking MORs globally (MKO) did not acquire morphine-induced locomotor activation/sensitization. Control mice also showed morphine-induced hypothermia in both the light and dark phases, while McKO littermates only exhibited morphine-induced hypothermia in the dark. Interestingly, only control animals appeared to acquire tolerance to morphine's hypothermic effect. Morphine, however, did not acutely decrease the body temperature of MKO mice. These findings support the idea that MORs expressed by ipRGCs could contribute to opioid-related sleep/wake problems and thermoregulatory changes.

Pyrimethamine induces phototoxicity in human keratinocytes via lysosomal and mitochondrial dependent signaling pathways under environmental UVA and UVB exposure.

Pyrimethamine (PYR) is used to treat parasitic infections including toxoplasmosis, pneumonia and cystoisosporiasis in HIV patients. Various oral medicines have shown phototoxicity therefore, we aimed to study the phototoxicity of PYR and its molecular mechanism involving stress responsive lysosomal protein Lamp2 and mitochondrial mediated signaling pathway under normal UVA/B exposure. We found that photodegradation and subsequent photoproduct formation was evident through LCMS/MS analysis. Photosensitized PYR produces ROS that cause damage to DNA, cell membrane and membrane bound organelles in human keratinocytes. PYR triggered cytotoxicity and phototoxicity that was evident through MTT and NRU assay respectively. Intracellular ROS generation caused phosphatidyl serine (PS) translocation in cell membrane, lysosome membrane permeabilization (LMP) and mitochondrial membrane potential (MMP) collapse that was further validated through caspase3 activation. DNA damage was measured as tail DNA formation and cell cycle arrest in G1 phase. Photosensitized PYR induces oxidative stress in the form of overexpression of Lamp2 that ultimately led to cellular apoptosis. Moreover, the effects of UVB were higher than UVA, probably due to its direct interaction with various macromolecules. We propose that photoexcited PYR may be harmful to human health even at normal sunlight exposure. Therefore, protective procedures should be practiced during PYR medication.

Superoxide anion radical induced phototoxicity of 2,4,5,6-Tetraminopyrimidine sulfate via mitochondrial-mediated apoptosis in human skin keratinocytes at ambient UVR exposure.

2,4,5,6-Tetraaminopyrimidine sulfate (TAPS) is worldwide the most commonly used developer in hair dyes. As skin is the major organ, which is directly exposed to these permanent hair dyes, a comprehensive dermal safety assessment is needed. Hereto, we studied the photosensitization potential and mechanism involved in dermal phototoxicity of TAPS exposed to the dark and UVA/UVB/Sunlight by using different in-chemico and mammalian (HaCaT) cells, as test systems. Our experimental outcomes illustrate that TAPS get photodegraded (LC-MS/MS) and specifically generated superoxide anion radical (O2•-) under UVA and UVB via type-I photodynamic reaction. The phototoxic potential of TAPS is measured through MTT, NRU, and LDH assays that depicted a significant cell viability reduction at 25 µg/ml concentration and higher. Different cellular stainings (PI uptake, AO/EB, JC-1, NR uptake) suggested the role of mitochondrial-mediated apoptosis. Further, the transcriptomics study revealed upregulation of Apaf-1, Bax, Cytochrome c, Caspase 3, Caspase 9 and downregulation of Catalase and Bcl-2 by TAPS treated cells that strengthen our findings. Thus, the above findings suggest that chronic application of TAPS may be hazardous for human skin and promote various skin diseases.

An approach to evaluate metabolite-related phototoxicity with combined use of photochemical properties and skin deposition.

Some chemicals have been reported to cause metabolite-related phototoxicity, and this study aimed to verify the applicability of photosafety assessment based on photochemical and pharmacokinetic properties to evaluate the metabolite-related phototoxicity risk. The phototoxic risk of imipramine (IMI) and its metabolite, desipramine (DMI), was evaluated by photochemical and pharmacokinetic analyses. IMI and DMI were found to have similar photoreactivities based on the generation of reactive oxygen species. The skin concentrations of IMI and DMI reached maximal levels at approximately 1 and 4 h, respectively, after oral administration of IMI (10 mg/kg), and DMI showed high skin deposition compared with IMI. According to the results, DMI was identified as a contributor to phototoxicity induced by orally-taken IMI. In in vivo phototoxicity testing, ultraviolet A irradiation from 3 to 6 h after oral administration of IMI (100 mg/kg) caused more potent phototoxic reactions compared with that from 0 to 3 h, and DMI yielded by metabolism of IMI would be associated with phototoxic reactions caused by orally-administered IMI. In addition to the data on IMI, a parent chemical, photochemical and pharmacokinetic profiling of its metabolite, DMI, led to reliable phototoxicity prediction of orally-administered IMI. Thus, characterization of the photosafety of metabolites would generate reliable information on the phototoxicity risk of parent chemicals, and the proposed strategy may facilitate comprehensive photosafety assessment of drug candidates in pharmaceutical development.

The possible neurobehavioral protective effects of natural antioxidant against phototoxicity attenuation of antimicrobial quinolone group in rats.

The fluoroquinolones absorb light in the 320 to 330 nm ultraviolet A (UV-A) wavelength and produce reactive oxygen species (ROS) such as superoxide anion, hydroxyl radical, and hydrogen peroxide; thus, the photodynamic generation of ROS may be the basis of phototoxicity of quinolones in human beings and animals. This study aimed to evaluate the damaging effects of UV-A radiation at different periods of exposure on rats' brains administered with ciprofloxacin. Ciprofloxacin administration in UV-A exposed animals exaggerated the brain-oxidative stress biomarkers and decreased the locomotor activity. Exposure of rats to UV-A for 60 minutes induced a significant increase of malondialdehyde (MDA), myeloperoxidase (MPO), and a decrease in the values of superoxide dismutase (SOD), glutathione (GSH) compared to a normal one; these changes were UV-A exposure time-dependent. However, the administration of vitamin C to the UV-60-treated group decreased the values of MDA, MPO, and shifted the values of SOD, GSH toward the normal values. Vitamin C, probably due to its strong antioxidant properties, could improve and partially counteract the toxic effect of UV-A on oxidative stress parameters and prevent the damage in rat's brain tissues.

Phototoxic risk assessment of dermally-applied chemicals with structural variety based on photoreactivity and skin deposition.

Combined use of photochemical and pharmacokinetic (PK) data for phototoxic risk assessment was previously proposed, and the system provided reliable phototoxic risk predictions of chemicals in same chemical series. This study aimed to verify the feasibility of the screening system for phototoxic risk assessment on dermally-applied chemicals with wide structural diversity, as a first attempt. Photochemical properties of test chemicals, 2-acetonaphthalene, 4'-methylbenzylidene camphor, 6-methylcoumarin, methyl N-methylanthranilate, and sulisobenzone, were evaluated in terms of UV absorption and reactive oxygen species (ROS) generation, and PK profiles of the test chemicals in rat skin were characterized after dermal co-application. All test chemicals showed strong UVA/B absorption with molar extinction coefficients of over 3000 M-1⋅cm-1, and irradiated 2-acetonaphthalene, 6-methylcoumarin, and methyl N-methylanthranilate exhibited significant ROS generation. Dermally-applied 2-acetonaphthalene and 4'-methylbenzylidene camphor indicated high and long-lasting skin deposition compared with the other test chemicals. Based on the photochemical and PK data, 2-acetonaphthalene was predicted to have potent phototoxic risk. The predicted phototoxic risk of the test chemicals by integration of obtained data was mostly consistent with their in vivo phototoxicity observed in rat skin. The screening strategy employing photochemical and PK data would have high prediction capacity and wide applicability for photosafety evaluation of chemicals.

A new photosafety screening strategy based on in chemico photoreactivity and in vitro skin exposure for dermally-applied chemicals.

This study involved an attempt to establish a new photosafety screening system for dermally-applied chemicals consisting of a reactive oxygen species (ROS) assay and an in vitro skin permeation test. The ROS assay was undertaken to evaluate photoreactivity of six test compounds, acridine (ACD), furosemide (FSM), hexachlorophene (HCP), 8-methoxypsoralen (MOP), norfloxacin (NFX), and promethazine (PMZ), and the in vitro skin permeation test was conducted to obtain steady-state concentration (Css) values of test compounds in removed rat skin. All test compounds were photoreactive based on ROS generation under simulated sunlight exposure. In particular, ROS generation from ACD was high compared with other test compounds, and photoreactivity of ACD was deduced to be potent. The Css values of ACD, HCP, MOP, and PMZ were over 50 µg/mL, and skin exposure to FSM and NFX was found to be extremely low. Upon these findings, ACD was judged to be highly phototoxic. The rank for phototoxic risk of test compounds based on photoreactivity and in vitro skin exposure was mostly in agreement with outcomes on their in vivo phototoxicity in rats. The proposed strategy, an alternative to animal testing, would be efficacious for photosafety evaluation of drug candidates in early stages of pharmaceutical development.

Porphyrin-Induced Protein Oxidation and Aggregation as a Mechanism of Porphyria-Associated Cell Injury.

Genetic porphyrias comprise eight diseases caused by defects in the heme biosynthetic pathway that lead to accumulation of heme precursors. Consequences of porphyria include photosensitivity, liver damage and increased risk of hepatocellular carcinoma, and neurovisceral involvement, including seizures. Fluorescent porphyrins that include protoporphyrin-IX, uroporphyrin and coproporphyrin, are photo-reactive; they absorb light energy and are excited to high-energy singlet and triplet states. Decay of the porphyrin excited to ground state releases energy and generates singlet oxygen. Porphyrin-induced oxidative stress is thought to be the major mechanism of porphyrin-mediated tissue damage. Although this explains the acute photosensitivity in most porphyrias, light-induced porphyrin-mediated oxidative stress does not account for the effect of porphyrins on internal organs. Recent findings demonstrate the unique role of fluorescent porphyrins in causing subcellular compartment-selective protein aggregation. Porphyrin-mediated protein aggregation associates with nuclear deformation, cytoplasmic vacuole formation and endoplasmic reticulum dilation. Porphyrin-triggered proteotoxicity is compounded by inhibition of the proteasome due to aggregation of some of its subunits. The ensuing disruption in proteostasis also manifests in cell cycle arrest coupled with aggregation of cell proliferation-related proteins, including PCNA, cdk4 and cyclin B1. Porphyrins bind to native proteins and, in presence of light and oxygen, oxidize several amino acids, particularly methionine. Noncovalent interaction of oxidized proteins with porphyrins leads to formation of protein aggregates. In internal organs, particularly the liver, light-independent porphyrin-mediated protein aggregation occurs after secondary triggers of oxidative stress. Thus, porphyrin-induced protein aggregation provides a novel mechanism for external and internal tissue damage in porphyrias that involve fluorescent porphyrin accumulation.

Analysis of phototoxin taste closely correlates nucleophilicity to type 1 phototoxicity.

Pigments often inflict tissue-damaging and proaging toxicity on light illumination by generating free radicals and reactive oxygen species (ROS). However, the molecular mechanism by which organisms sense phototoxic pigments is unknown. Here, we discover that Transient Receptor Potential Ankyrin 1-A isoform [TRPA1(A)], previously shown to serve as a receptor for free radicals and ROS induced by photochemical reactions, enables Drosophila melanogaster to aphotically sense phototoxic pigments for feeding deterrence. Thus, TRPA1(A) detects both cause (phototoxins) and effect (free radicals and ROS) of photochemical reactions. A group of pigment molecules not only activates TRPA1(A) in darkness but also generates free radicals on light illumination. Such aphotic detection of phototoxins harboring the type 1 (radical-generating) photochemical potential requires the nucleophile-sensing ability of TRPA1. In addition, agTRPA1(A) from malaria-transmitting mosquitoes Anopheles gambiae heterologously produces larger current responses to phototoxins than Drosophila TRPA1(A), similar to their disparate nucleophile responsiveness. Along with TRPA1(A)-stimulating capabilities, type 1 phototoxins exhibit relatively strong photo-absorbance and low energy gaps between the highest occupied molecular orbital and the lowest unoccupied molecular orbital. However, TRPA1(A) activation is more highly concordant to type 1 phototoxicity than are those photochemical parameters. Collectively, nucleophile sensitivity of TRPA1(A) allows flies to taste potential phototoxins for feeding deterrence, preventing postingestive photo-injury. Conversely, pigments need to bear high nucleophilicity (electron-donating propensity) to act as type 1 phototoxins, which is consistent with the fact that transferring photoexcited electrons from phototoxins to other molecules causes free radicals. Thus, identification of a sensory mechanism in Drosophila reveals a property fundamental to type 1 phototoxins.

Photocatalytic production of hydroxyl radicals by commercial TiO2 nanoparticles and phototoxic hazard identification.

This study identifies the phototoxic potential of commercial titanium dioxide nanoparticles (TiO2 NPs) used in sunscreens and consumer products by employing a tiered testing approach comprising physicochemical, in vitro and ex vivo tests. Our results revealed that all the test samples of TiO2 NPs, varied in surface coating, crystallinity and primary particle size, produced hydroxyl radicals upon UVA photoexcitation as determined by electron spin resonance (ESR) spectroscopy. Their phototoxic potentials were assessed first by combining the validated 3T3 neutral red uptake phototoxicity test and red blood cell phototoxicity test and subsequently in ex vivo models of chick chorioallantoic membrane (CAM) and reconstructed human 3D skin model (H3D). Crystalline structure and particle size of TiO2 NPs were found to exert a major influence on the photocatalytic activity and the associated phototoxic effects. Besides, a medium-sized sample with silica/alumina also exhibited high phototoxic potency with no obvious relevance to the enhanced hydroxyl radicals and lipidperoxidation. This effect might be taken place through the interaction of harmful metal ions released from the oxide coating. However, no phototoxicity was observed on a H3D skin model probably due to the lack of efficient percutaneous absorption of TiO2 NPs. This study demonstrates the efficacy of a tiered testing strategy for identifying phototoxic hazards of TiO2 NPs and suggests the need for a comprehensive assessment that takes account of the effects of different coating materials and potential interactions between multiple mechanisms.

JAK/STAT and TGF-ß activation as potential adverse outcome pathway of TiO2NPs phototoxicity in Caenorhabditis elegans.

Titanium dioxide nanoparticles (TiO2NPs) are widely used nanoparticles, whose catalytic activity is mainly due to photoactivation. In this study, the toxicity of TiO2NPs was investigated on the nematode Caenorhabditis elegans, with and without UV activation. Comparative analyses across the four treatments revealed that UV-activated TiO2NPs led to significant reproductive toxicity through oxidative stress. To understand the underlying molecular mechanism, transcriptomics and metabolomics analyses were conducted, followed by whole-genome network-based pathway analyses. Differential expression analysis from microarray data revealed only 4 DEGs by exposure to TiO2NPs alone, compared to 3,625 and 3,286 DEGs by UV alone and UV-activated TiO2NPs, respectively. Pathway analyses suggested the possible involvement of the JAK/STAT and TGF-ß pathways in the phototoxicity of TiO2NPs, which correlated with the observation of increased gene expression of those pathways. Comparative analysis of C. elegans response across UV activation and TiO2NPs exposure was performed using loss-of-function mutants of genes in these pathways. Results indicated that the JAK/STAT pathway was specific to TiO2NPs, whereas the TGF-ß pathway was specific to UV. Interestingly, crosstalk between these pathways was confirmed by further mutant analysis. We consider that these findings will contribute to understand the molecular mechanisms of toxicity of TiO2NPs in the natural environment.

Enzymatic reactive oxygen species assay to evaluate phototoxic risk of metabolites.

The present study aimed to verify the feasibility of an enzymatic reactive oxygen species (eROS) assay to evaluate the phototoxic risk of compounds after their metabolization. The eROS assay was designed based on the combined use of an in vitro drug metabolism system and a ROS assay. The incubation time of compounds with human hepatic S9 fractions was optimized with the use of fenofibrate (FF), a typical phototoxicant with metabolite-related phototoxicity, and the reproducibility and robustness of the eROS assay were examined using FF. The eROS assay was applied to 12 phototoxic compounds, including 7 phototoxicants with metabolite-related phototoxicity, to clarify the assay performance. According to the eROS data on singlet oxygen generation from FF and metabolic conversion profiles of FF and fenofibric acid, the incubation time of chemicals with human hepatic S9-mix was determined to be 4min. The singlet oxygen-based evaluation system in the eROS assay was found to be acceptable as a high-throughput assay because of its favorable intra-/inter-day reproducibility (coefficient of variation: ca. 8%) and robustness (Z'-factor: 0.23). Singlet oxygen data on phototoxicants with phototoxic metabolites tended to exceed 120% of control, suggesting the feasibility of the eROS assay to evaluate metabolite-related phototoxic potentials. However, further data accumulation is still needed to improve the assay performance because the eROS assay provided false predictions for some compounds. The present eROS assay may be applicable in part for evaluating the phototoxic risk of drug candidates after their metabolization in the early stage of drug discovery.

Ultraviolet A photosensitivity profile of dexchlorpheniramine maleate and promethazine-based creams: Anti-inflammatory, antihistaminic, and skin barrier protection properties.

BACKGROUND: Unwanted side effects such as dryness, hypersensitivity, and cutaneous photosensitivity are challenge for adherence and therapeutical success for patients using treatments for inflammatory and allergic skin response. AIMS: In this study, we compared the effects of two dermatological formulations, which are used in inflammatory and/or allergic skin conditions: dexchlorpheniramine maleate (DCP; 10 mg/g) and promethazine (PTZ; 20 mg/g). METHODS: We evaluated both formulations for phototoxicity potential, skin irritation, anti-inflammatory and antihistaminic abilities, and skin barrier repair in vitro and ex vivo using the standard OECD test guideline n° 432, the ECVAM protocol n° 78, and cultured skin explants from a healthy patient. Ultraviolet A was chosen as exogenous agent to induce allergic and inflammatory response. RESULTS: Both PTZ and DCP promoted increases in interleukin-1 (IL-1) synthesis in response to ultraviolet A (UVA) radiation compared to control. However, the increase observed with PTZ was significantly greater than the DCP, indicating that the latter has a lower irritant potential. DCP also demonstrated a protective effect on UVA-induced leukotriene B4 and nuclear factor kappa B (NF-κB) synthesis. Conversely, PTZ demonstrates more robust UVA antihistaminic activity. Likewise, PTZ promoted a significantly greater increase in the production of involucrin and keratin 14, both associated with protective skin barrier property. CONCLUSION: In conclusion, these data suggest possible diverging UVA response mechanisms of DCP and PTZ, which gives greater insight into the contrasting photosensitizing potential between DCP and PTZ observed in the patients.

Effect of red dyes on blue light phototoxicity against VSC producing bacteria in an experimental oral biofilm.

Oral malodour is considered to be caused mainly by the production of volatile sulfide compounds (VSC) by anaerobic Gram-negative oral bacteria. Previous study showed that these bacteria were susceptible to blue light (wavelengths of 400-500 nm). In the present study, we tested the effect of blue light in the presence of red dyes on malodour production in an experimental oral biofilm. Biofilms were exposed to a plasma-arc light source for 30, 60, and 120 s (i.e. fluences of 41, 82, and 164 J cm-2, respectively) with the addition of erythrosine, natural red and rose bengal (0.01, 0.1 and 1% w/v). Following light exposure biofilm samples were examined for malodour production (Odour judge), VSC production (Halimeter™), VSC producing bacteria quantification using microscopy sulfide assay (MSA) and reactive oxygen species (ROS) production. Results showed that the exposure of experimental oral biofilm to blue light in the presence of rose bengal caused an increased reduction in VSC and malodour production concomitant with an increase in ROS production. These results suggest that rose bengal might be effective as a blue light photosensitizer against VSC producing bacteria.

Interplay Between Membrane Lipid Peroxidation and Photoproduct Formation in the Ultraviolet A-Induced Phototoxicity of Vemurafenib in Skin Keratinocytes.

According to some authors, the phototoxic response to ultraviolet A (UVA) of patients treated with vemurafenib (VB) may involve VB metabolites. However, the production of singlet oxygen and free radicals and photoproduct formation upon UVA light absorption by the lipophilic VB have been demonstrated. This work is aimed at determining the contribution of reactive oxygen species (ROS), lipid photoperoxidation, and VB photochemistry in the UVA-induced photocytotoxicity in NCTC 2544 keratinocytes. The potent membrane lipid peroxidation effectiveness of VB-photosensitization has been proved by the observation of an effective photohemolysis accompanied by thiobarbituric reactive substances (TBARS) formation in 2% red blood cell (RBC) suspensions. Photohemolysis is inhibited by human serum albumin (HSA) that binds VB and by the antioxidants 2,6-di-tert-butyl-4-methylphenol and Trolox. These data on RBC suggest that VB is readily incorporated in cell membranes and provide clues for understanding the UVA-induced VB-photosensitization of keratinocytes. In keratinocytes, ROS and TBARS formation with 10 µM VB is inhibited by approximately 40% and 50% by 30 µM Trolox and 50 µM vitamin E, respectively, but the light dose-dependent cell survival is unaffected. Whereas cell photokilling depends on the VB concentration, much smaller changes in the lethal doses (LD) than theoretically expected are observed for 25% or 50% cell photokilling when changing absorbed UVA doses and irradiation wavelengths. The lack of antioxidant effect on cell survival and the unexpectedly small LD dependence on absorbed UVA light doses and on irradiation wavelengths strongly suggest that, instead of metabolites, membrane photosensitization and photoproduct formation contribute to the cell photocytotoxicity.

Development of novel in vitro photosafety assays focused on the Keap1-Nrf2-ARE pathway.

Although photoallergens require UV energy for antigen formation, the subsequent immune response is considered to be the same as in ordinary skin sensitization. Therefore, in vitro tests for skin sensitization should also be applicable for photoallergy testing. In this study, we examined whether activation of the Keap1 (Kelch-like ECH-associated protein 1)-Nrf2 (nuclear factor-erythroid 2-related factor 2)-ARE (antioxidant response element) pathway could be used to assess the photoallergenic potential of chemicals, using the reporter cell line AREc32 or KeratinoSens(TM) . First, we identified an appropriate UVA irradiation dose [5 J cm(-2) irradiation in phosphate-buffered saline (PBS)] by investigating the effect of UV irradiation on ARE-dependent gene induction using untreated or 6-methylcoumarin (6-MC)-treated cells. Irradiation of well-known photoallergens under this condition increased ARE-dependent gene expression by more than 50% compared with both vehicle and non-irradiated controls. When the cut-off value for detecting photoallergens was set at 50% induction, the accuracy of predicting photoallergenic/phototoxic chemicals was 70% in AREc32 cells and 67% in KeratinoSens(TM) cells, and the specificity was 100% in each case. We designate these assays as a photo-ARE assay and photo-KeratinoSens(TM) , respectively. Our results suggest that activation of the Keap1-Nrf2-ARE pathway is an effective biomarker for evaluating both photoallergenic and phototoxic potentials. Either of the above tests might be a useful component of a battery of in vitro tests/in silico methods for predicting the photoallergenicity and phototoxicity of chemicals. Copyright © 2015 John Wiley & Sons, Ltd.

Phototoxic Risk Assessments on Benzophenone Derivatives: Photobiochemical Assessments and Dermal Cassette-Dosing Pharmacokinetic Study.

This study aimed to qualify photosafety screening on the basis of photochemical and pharmacokinetic (PK) data on dermally applied chemicals. Six benzophenone derivatives (BZPs) were selected as model compounds, and in vitro photochemical/phototoxic characterization and dermal cassette-dosing PK study were carried out. For comparison, an in vivo phototoxicity test was also conducted. All of the BZPs exhibited strong UVA/UVB absorption with molar extinction coefficients of over 2000 M(-1) × cm(-1), and benzophenone and ketoprofen exhibited significant reactive oxygen species (ROS) generation upon exposure to simulated sunlight (about 2.0 mW/cm(2)); however, ROS generation from sulisobenzone and dioxybenzone was negligible. To verify in vitro phototoxicity, a 3T3 neutral red uptake phototoxicity test was carried out, and benzophenone and ketoprofen were categorized to be phototoxic chemicals. The dermal PK parameters of ketoprofen were indicative of the highest dermal distribution of all BZPs tested. On the basis of its in vitro photochemical/phototoxic and PK data, ketoprofen was deduced to be highly phototoxic. The rank of predicted phototoxic risk of BZPs on the basis of the proposed screening strategy was almost in agreement with the results from the in vivo phototoxicity test. The combined use of photochemical and cassette-dosing PK data would provide reliable predictions of phototoxic risk for candidates with high productivity.

Evaluation of chemical phototoxicity, focusing on phosphorylated histone H2AX.

Histone H2AX is a minor component of nuclear histone H2A. The phosphorylation of histone H2AX at Ser 139, termed γ-H2AX, was originally identified as an early event after the direct formation of DNA double-strand breaks (DSBs) by ionizing radiation. Now, the generation of γ-H2AX is also considered to occur in association with secondarily formed DSBs by cellular processing such as DNA replication and repair at the site of the initial damage, including DNA adducts, crosslinks, and UV-induced photolesions. Therefore, γ-H2AX is currently attracting attention as a new biomarker for detecting various genotoxic insults. We have determined the toxic impact of various environmental stresses such as chemicals, light and/or their coexposure using γ-H2AX, and found that the γ-H2AX assay exhibited high sensitivity and a low false-positive rate as a detection system of genotoxic potential. In this review, we introduced our recent findings concerning the evaluation of chemical phototoxicity, focusing on γ-H2AX.