Development of Sensitive In Vitro Protocols for the Biocompatibility Testing of Medical Devices and Pharmaceuticals Intended for Contact with the Eyes: Acute Irritation and Phototoxicity Assessment.

This study introduces a novel in vitro methodology that employs the 3-D reconstructed tissue model, EpiOcular, to assess the irritation and phototoxicity potential of medical devices and drugs in contact with the eye. Our study evaluated diverse test materials, including medical devices, ophthalmological solutions and an experimental drug (cemtirestat), for their potential to cause eye irritation and phototoxicity. The protocols used in this study with the EpiOcular tissue model were akin to those used in the ultra-mildness testing of cosmetic formulations, which is challenging to predict with standard in vivo rabbit tests. To design these protocols, we leveraged experience gained from the validation project on the EpiDerm skin irritation test for medical devices (ISO 10993-23:2021) and the OECD TG 498 method for photo-irritation testing. The predictions were based on the tissue viability and inflammatory response, as determined by IL-1α release. By developing and evaluating these protocols for medical devices, we aimed to expand the applicability domain of the tests referred to in ISO 10993-23. This will contribute to the standardisation and cost-effective safety evaluation of ophthalmic products, while reducing reliance on animal testing in this field. The findings obtained from the EpiOcular model in the photo-irritation test could support its implementation in the testing strategies outlined in OECD TG 498.

Tapinarof and its structure-activity relationship for redox chemistry and phototoxicity on human skin keratinocytes.

Tapinarof (3,5-dihydroxy-4-isopropylstilbene) is a therapeutic agent used in the treatment of psoriasis (VTAMA®). In this study, we examined the redox behaviour, (photo)stability, (photo)toxicity and (bio)transformation of tapinarof in the context of a structure-activity relationship study. Selected derivatives of the structurally related tapinarof were investigated, namely resveratrol, pterostilbene, pinosylvin and its methyl ether. Tapinarof undergoes electrochemical oxidation in a neutral aqueous medium at a potential of around +0.5 V (vs. Ag|AgCl|3M KCl). The anodic reaction of this substance is a proton-dependent irreversible and adsorption-driven process. The pKa value of tapinarof corresponds to 9.19 or 9.93, based on empirical and QM calculation approach, respectively. The oxidation potentials of tapinarof and its analogues correlate well with their HOMO (highest occupied molecular orbital) energy level. The ability to scavenge the DPPH radical decreased in the order trolox ≥ resveratrol > pterostilbene > tapinarof > pinosylvin â‰« pinosylvin methyl ether. It was also confirmed that tapinarof, being a moderate electron donor, is able to scavenge the ABTS radical and inhibit lipid peroxidation. The 4'-OH group plays a pivotal role in antioxidant action of stilbenols. During the stability studies, it was shown that tapinarof is subject to spontaneous degradation under aqueous conditions, and its degradation is accelerated at elevated temperatures and after exposure to UVA (315-399 nm) radiation. In aqueous media at pH 7.4, we observed an ∼50 % degradation of tapinarof after 48 h at laboratory temperature. The main UVA photodegradation processes include dihydroxylation and hydration. In conclusion, the phototoxic effect of tapinarof on a human keratinocytes cell line (HaCaT) was evaluated. Tapinarof exhibited a clear phototoxic effect, similar to phototoxic standard chlorpromazine. The IC50 values of the cytotoxicity and phototoxic effects of tapinarof correspond to 27.6 and 3.7 µM, respectively. The main HaCaT biotransformation products of tapinarof are sulfates and glucuronides.

In Silico Phototoxicity Prediction of Drugs and Chemicals by using Derek Nexus and QSAR Toolbox.

Phototoxicity testing is crucial for evaluating the potential harmful effects of pharmaceuticals and chemicals on human skin when exposed to sunlight. Traditional in vivo models involving mice, rats, guinea pigs, as well as in vitro assays such as the 3T3 Neutral Red Uptake phototoxicity assay and methods based on the use of reconstructed human epidermis, have been established for phototoxicity testing. While these approaches are extremely valuable, they are costly in terms of both time and resources. Consequently, in silico approaches based on the use of predictive software tools can offer more rapid and cost-effective phototoxicity screening solutions. With this goal in mind, the current study evaluated two in silico tools - Derek Nexus 6.1.0/Derek Knowledge Base 2020 1.0 (Lhasa Limited, UK) and the QSAR Toolbox (v 4.5) developed by the Organisation for Economic Co-operation and Development (OECD) - for their capacity to predict the phototoxicity of several substances from diverse classes. Derek Nexus and the QSAR Toolbox were both found to be very useful for predicting the phototoxicity of drugs and other chemicals. Derek Nexus predicted phototoxicity of the compounds, with a sensitivity of 63%, specificity of 93%, Positive Predictive Values of 90% and Negative Predictive Value of 69%, overall accuracy of 77% and balanced accuracy of 78%. The QSAR Toolbox achieved sensitivity of 73%, specificity of 85%, Positive Predictive Value of 85% and Negative Predictive Value of 74%, overall accuracy of 79% and balanced accuracy of 79%. The results show that Derek Nexus and the QSAR Toolbox can be usefully incorporated in the workflow of phototoxicity testing for pharmaceuticals and chemicals.

Comparison of two biological systems used for phototoxicity testing: Cellular and tissue.

The OECD has approved two similar methods for testing the phototoxic potency of chemicals. The first method, OECD 432, is based on the cytotoxicity properties of materials to the mouse 3T3 (clone A31) cell line (fibroblasts) after exposure to light. The second method, OECD 498, is based on the same properties but using reconstructed human epidermis - EpiDerm (stratified keratinocytes). The aim of this study was to compare these two methods using statistical tests (specificity, sensitivity, negative predictive value, positive predictive value and accuracy) and non-statistical characteristics (e.g. price and experimental duration, amount of material, level of complications, cell type, irradiation dose). Both tests were performed according to the relevant guidelines using the same 11 control substances. Higher performance values were observed for OECD 432 in both phototoxic and non-phototoxic classifications. The accuracy of OECD 432 was 90.9%, while that of OECD 498 was 72.7%. OECD 432 was also shorter and less expensive. On the other hand, OECD 498 was less complicated, and used human cells with stratum corneum, which better reflects real skin. This method can also be used with oily substances that are poorly soluble in water. However, both methods are important for testing the phototoxic properties of materials, and can be used alone or in a tiered strategy.

Botanical Briefs: Fig Phytophotodermatitis (Ficus carica).

Patients presenting with a linear, erythematous, blistering eruption may experience a sudden painful sunburn that seems to get worse rather than better with time. In warm climates, exposure to the common fig tree (Ficus carica) may be the culprit. Dermatologists should recognize fig phytophotodermatitis as a possible cause and help the patient connect their symptoms with the inciting agent as well as administer proper treatment.

Effects of radical scavengers for reactive oxygen species on vitamin K-induced phototoxicity under UVA irradiation.

Vitamin K possesses efficacy as a topical dermatological agent. However, vitamin K is phototoxic and susceptible to photodegradation. Herein, we investigated the mechanisms underlying the phototoxicity of phylloquinone (PK, vitamin K1) and menaquinone-4 (MK-4, vitamin K2) under ultraviolet A (UVA) irradiation using various reactive oxygen species (ROS) scavengers. This resulted in the production of superoxide anion radicals via type I and singlet oxygen via type II photodynamic reactions, which were quenched by the ROS scavengers: superoxide dismutase and sodium azide (NaN3). In HaCaT cells, MK-4 and PK induced the production of intracellular ROS, particularly hydrogen peroxide, in response to UVA irradiation. Furthermore, the addition of catalase successfully decreased maximum ROS levels by approximately 30%. NaN3 and catalase decreased the maximum reduction in cell viability induced by UVA-irradiated PK and MK-4 in cell viability by approximately 2-7-fold. Additionally, ROS scavengers had no effect on the photodegradation of PK or MK-4 at 373 nm. Therefore, the phototoxicities of PK and MK-4 were attributed to the generation of singlet oxygen and hydrogen peroxide, underscoring the importance of photoshielding in circumventing phototoxicity.

KoCVAM-led development of phototoxicity alternative test method using reconstructed human epidermis model (KeraSkin™).

Phototoxicity is an acute toxic reaction induced by topical skin exposure to photoreactive chemicals followed by exposure to environmental light and thus chemicals that absorb UV are recommended to be evaluated for phototoxic potential. There are currently three internationally harmonized alternative test methods for phototoxicity. One of them is the in vitro Phototoxicity: RhE Phototoxicity test method (OECD TG498). Korean center for the Validation of Alternative Methods (KoCVAM) developed an in vitro phototoxicity test method using a KeraSkin™ reconstructed human epidermis model (KeraSkin™ Phototoxicity Assay) as a 'me-too' test method of OECD TG498. For the development and optimization of KeraSkin™ Phototoxicity Assay, the following test chemicals were used: 6 proficiency chemicals in OECD TG498 (3 phototoxic and 3 non-phototoxic), 6 reference chemicals in OECD Performance Standard No. 356 (excluding the proficiency test chemicals, 3 phototoxic and 3 non-phototoxic) and 13 additional chemicals (7 phototoxic and 6 non-phototoxic). Based on the test results generated from the test chemicals above, the overall predictive capacity of KeraSkin™ Phototoxicity Assay was calculated. In particular, the assay exhibited 100 % accuracy, 100 % sensitivity, and 100 % specificity. Therefore, it fulfills the requirements to be included as a 'me-too' test method in OECD TG498.

Low-Toxicity Self-Photosensitized Biohybrid Systems for Enhanced Light-Driven H2 Production.

Nanoparticles (NPs) represent a potential optoelectronic source capable of significantly boosting hydrogen production; however, their inevitable cytotoxicity may lead to oxidative damage of bacterial cell membranes. In this study, we employed non-photosynthetic Escherichia coli K-12 as a model organism and utilized self-assembled cadmium sulfide (CdS) nanoparticles to construct a low-toxicity and hydrogen-production-enhancing self-photosensitive hybrid system. To mitigate the cytotoxicity of CdS NPs and synthesize biocompatible CdS NPs on the cell surface, we employed engineered E. coli (efeB/OE) for bioremediation, achieving this goal through the overexpression of the peroxidase enzyme (EfeB). A comparative analysis with E. coli-CdS revealed a significant downregulation of genes encoding oxidative stress proteins in efeB/OE-CdS post-irradiation. Atomic force microscopy (AFM) confirmed the stability of bacterial cell membranes. Due to the enhanced stability of the cell membrane, the hydrogen yield of the efeB/OE-CdS system increased by 1.3 times compared to the control, accompanied by a 49.1% reduction in malondialdehyde (MDA) content. This study proposes an effective strategy to alleviate the toxicity of mixed biological nanoparticle systems and efficiently harness optoelectronic electrons, thereby achieving higher hydrogen production in bioremediation.

Lime Dermatitis.

Phytophotodermatitis are skin eruptions caused by contact with a plant substance and exaggerated by exposure to sunlight. They are due to the presence of furocoumarins (psoralens, xanthotoxins, and bergaptenes) in the plant and involve 2 mechanisms of skin photosensitivity: phototoxicity or photoallergy.

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.

Photosensitizing antihypertensive medication and risk of skin cancer among postmenopausal women.

BACKGROUND: Few prospective studies exist with an evaluation of a dose-response relationship between use of some photosensitizing antihypertensive medications and skin cancer. PATIENT AND METHODS: We used prospective data from the Women's Health Initiative Observational Study to investigate the association between antihypertensive use and risk of non-melanoma skin cancer (NMSC) and melanoma in postmenopausal women aged 50-79 years at baseline (n  =  64,918). Multivariable Cox proportional hazards regression models were used and hazard ratios (HRs) and 95 confidence intervals (CIs) were calculated. RESULTS: 8,777 NMSC and 1,227 melanoma cases were observed. Use of antihypertensives (HR [95% CI]: 1.12 [1.07-1.18]), ACE inhibitors (1.09 [1.01-1.18]), calcium channel blockers (1.13 [1.05-1.22]), diuretics (1.20 [1.12-1.27]), loop diuretics (1.17 [1.07-1.28]), and thiazides (1.17 [1.03-1.33]) were each associated with higher NMSC risk. NMSC risk linearly increased with use of multiple antihypertensives (p-trend  =  0.02) and with longer duration of use (p-trend < 0.01). Antihypertensives (1.15 [1.00-1.31]), angiotensin-II receptor blockers (1.82 [1.05-3.15]), and diuretics (1.34 [1.13-1.59]) were each associated with elevated melanoma risk. Effect modification by solar radiation exposure was found between antihypertensive use and incidence of melanoma (p-interaction  =  0.02). CONCLUSIONS: Use of antihypertensives overall, and several individual classes thereof, were associated with higher incidence of NMSC and melanoma with dose-response relationship.

Surfactants for stabilization of dermal emulsions and their skin compatibility under UVA irradiation: Diacyl phospholipids and polysorbate 80 result in high viability rates of primary human skin cells.

Phospholipids are versatile formulation compounds with high biocompatibility. However, no data on their effect on skin in combination with UVA radiation exist. Thus, it was the aim of this work to (i) develop o/w nanoemulsions (NEs) differing in surfactant type and to investigate their physicochemical stability at different storage temperatures, (ii) establish a standardized protocol for in vitro phototoxicity testing using primary human skin cells and (iii) investigate the phototoxicity of amphoteric phospholipids (S45, S75, E80, S100, LPC80), sodium lauryl ether sulfate (SLES) and polysorbate 80 (PS80). Satisfying systems were developed with all surfactants except S100 due to low zeta potential (-21.4 mV ± 4.69). SLES and PS80-type NEs showed the highest stability after eight weeks; temperature-dependent variations in storage stability were most noticeable for phospholipid surfactants. For phospholipid-based NEs, higher phosphatidylcholine content led to unstable formulations. Phototoxicity assays with primary skin fibroblasts confirmed the lack of UVA-related phototoxicity but revealed cytotoxic effects of LPC80 and SLES, resulting in cell viability as low as 2.7 % ±0.78 and 1.9 % ±1.57 compared to the control. Our findings suggest that surfactants S45, S75 and PS80 are the most promising candidates for skin-friendly emulsifiers in sensitive applications involving exposure to UV light.

Potential therapeutic efficacy of photodynamic therapy on female hormonal-dependent cancers in a hormonal simulated microenvironment.

BACKGROUND: Photodynamic Therapy (PDT) is a clinically approved cancer treatment. Sex hormones, the key drivers for the development of female hormonal dependent cancers, might affect cancer treatment. There are seldom studies to evaluate the effect of sex hormones mimicked the menstrual cycle on the PDT mediated by prodrug 5-aminolevulinic acid (ALA) and its ester derivatives to the hormonal dependent cancers. AIMS: To evaluate the efficacy of sex hormones on Hexyl-ALA-PDT in hormonal dependent cancers and the effect of the sex hormones on heme biosynthetic pathway. METHODS: Cell culture system that mimicked the fluctuation of sex hormones 17ß-estradiol (E2) and progesterone (PG) in the menstrual cycle was developed. Two pairs of hormonal-independent and hormonal dependent uterine sarcoma and breast cancer cell lines were used as cell models. Hexyl-ALA induced PpIX production and intracellular localization were examined. Key enzymes for PpIX synthesis were analysed. Hexyl-ALA-PDT mediated phototoxicity was evaluated. RESULTS: The PpIX generation was increased in the hormonal-dependent cells (28-50 %) when cultured in the hormonal microenvironment with long incubation of Hexyl-ALA for 15 and 24 h compared to that cultured without hormones; whereas only slight difference in PpIX generation in their hormonal-independent counterpart. The PpIX generation was in a time-dependent manner. The CPOX, PPOX and FECH expressions were significantly enhanced by Hexyl-ALA-PDT in uterine sarcoma cells in hormonal microenvironment. Hexyl-ALA-PDT triggered significant increase of PPOX expression in breast cancer cells in hormonal microenvironment. The Hexyl-ALA-PDT phototoxicity was enhanced by 18-40 % in cells cultured in the hormonal system in a dose-dependent manner. CONCLUSION: The PpIX generation and the efficacy of Hexyl-ALA-PDT in both uterine sarcoma and breast cancer cells was significantly enhanced by the sex hormones via cultured in the hormonal microenvironment.

A Review of Phototoxic Plants, Their Phototoxic Metabolites, and Possible Developments as Photosensitizers.

This study provides a comprehensive overview of the current knowledge regarding phototoxic terrestrial plants and their phototoxic and photosensitizing metabolites. Within the 435,000 land plant species, only around 250 vascular plants have been documented as phototoxic or implicated in phototoxic occurrences in humans and animals. This work compiles a comprehensive catalog of these phototoxic plant species, organized alphabetically based on their taxonomic family. The dataset encompasses meticulous details including taxonomy, geographical distribution, vernacular names, and information on the nature and structure of their phototoxic and photosensitizing molecule(s). Subsequently, this study undertook an in-depth investigation into phototoxic molecules, resulting in the compilation of a comprehensive and up-to-date list of phytochemicals exhibiting phototoxic or photosensitizing activity synthesized by terrestrial plants. For each identified molecule, an extensive review was conducted, encompassing discussions on its phototoxic activity, chemical family, occurrence in plant families or species, distribution within different plant tissues and organs, as well as the biogeographical locations of the producer species worldwide. The analysis also includes a thorough discussion on the potential use of these molecules for the development of new photosensitizers that could be used in topical or injectable formulations for antimicrobial and anticancer phototherapy as well as manufacturing of photoactive devices.

Phototoxic action of meloxicam contributes to dysregulation of redox homeostasis in normal human skin cells - Molecular and biochemical analysis of antioxidant enzymes in melanocytes and fibroblasts.

The phototoxic effect of meloxicam (MLX) raises the question of the effect of the drug on the redox homeostasis of normal human skin cells. The main objective of the study was to analyze the effect of MLX and/or UVA radiation (UVAR) on the redox homeostasis of human normal skin cells - melanocytes and fibroblasts. MLX was found to affect the activity and expression of enzymes of the antioxidant system differently depending on the cell line used. The drug decreased the activity and expression of superoxide dismutase type 1 and 2 (SOD1 and SOD2), catalase (CAT) and glutathione peroxidase (GPx) in fibroblasts, while increasing the activity of these enzymes in melanocytes. UVA radiation enhanced the effects of the drug. In conclusion, MLX in combination with UVAR induces oxidative stress in melanocytes and fibroblasts, however, the analyses showed that the drug's effect the activity and expression of SOD, CAT and GPx differently, depending on the cell line. The observed dissimilarity between tested cell lines may result from the presence of melanin pigments.

Modified ESR-based photosafety test (ESR-PT) detecting singlet oxygen and free radical formation.

The electron spin resonance-based photosafety test (ESR-PT) was modified using a new parameter, photoreactivity index (PRI), to detect singlet oxygen and free radical photoproducts simultaneously. With this modification, the modified ESR-PT is expected to reduce the number of false negative results due to chemicals producing free radical photoproducts other than singlet oxygen. The assay performance of the modified ESR-PT was evaluated using 56 chemicals, including hydrophobic chemicals. When using the PRI cutoff value of 2.0 in the modified ESR-PT, the accuracy relative to photosafety reference data was 91.1%, and the applicability (100%) was better than the other non-animal photosafety test. Among the chemicals producing positive results, bithionol, fenticlor, and doxycycline HCl were considered positive based on the detection of free radical photoproducts, suggesting that these three chemicals may have phototoxic or photoallergic potential via radical reactions. Additionally, this finding demonstrated the fundamental advantage of the modified ESR-PT using ESR spectroscopy, which can detect radicals selectively and quantitatively. Accordingly, the new parameter PRI is effective for photosafety evaluations based on not only singlet oxygen but also free radical photoproducts generated from chemicals. Therefore, the modified ESR-PT has a great potential for a photosafety test method applicable to various chemicals.