Phototoxic Substances Research Articles

MLtox, online phototoxicity prediction webpage

Phototoxicity, sometimes in the literature referred to as photo-irritation, is a chemically induced reaction requiring light. While it is generally accepted that phototoxicity testing can be performed in the majority of cases in vitro (i.e. without the use of experimental animals), these tests may sometimes provide contradictory predictions. Understanding the mechanisms of initiating events based on the molecule's structure and its ability to reach the excited state and consequently generate ROS enables the creation of predictive QSAR for this adverse outcome. The ability to predict the phototoxicity potential via a QSAR model is beneficial in reducing the number of mechanical in vitro/in chemico tests needed to demonstrate absence of phototoxicity and it is very helpful in the overall safety assessment process.The QSAR prediction model presented here focused on developing a robust platform freely available on the web via the link http://mltox.online to provide interpretable predictions of the phototoxicity of tested molecules. Great attention was devoted to interpretability and explainability of the prediction results. The web application allows the user to input a chemical by CAS number, SMILES code or trivial name. The user can choose between simple prediction or advanced tools options. These extended tools include the artificial intelligence explainability of model prediction using XSMILES (interactive visualization technique to support the interpretation of SMILES) and SHAP values (impact each element on the prediction). The comprehensive tools in question allow the user to explore the properties of phototoxic substances and to understand the prediction outcomes better.

Survey of cosmetologists’ knowledge of photoallergenic and phototoxic substances

Phototoxic and photoallergic reactions induced by ultraviolet A radiation belong to the group of exogenous photodermatoses. To induce changes in addition to sunlight, the presence of a photosensitizer is essential. These can be both externally applied and systemically ingested substances: drugs and supplements, plants, cosmetics, essential oils. The article aimed to assess cosmetologists’ knowledge of phototoxic and photoallergic substances. The study was carried out in a group of 103 cosmetologists based on an original questionnaire survey. Statistically significant differences were found in the level of knowledge between those with a master’s degree and a bachelor’s degree. The groups also differed in their willingness to expand their knowledge.

Validation of a Light Source for Phototoxicity in in vitro Conditions

Phototoxicity is a chemically induced skin irritation in the presence of sunlight. Phototoxic substances after irradiation with sunlight absorb and convert light energy into chemical energy, leading to the formation of highly reactive oxygen species with toxic effects. Recently LED technologies made possible their application for novel effective solar simulators, to be used in biology and medicine. We study the possibilities of application of LED sunlight simulator (Helios-iO, model LE-9ND55-H – 5500K) for in vitro testing of synthetic and natural products for phototoxicity. Cytotoxicity/phototoxicity was assessed by validated BALB/3T3 clone A31 Neutral Red Uptake Assay. The morphological alterations in BALB/3T3 cells induced by the Radachlorin® and irradiated with dose 2.4 J, were analyzed by fluorescent microscopy. The physical characteristics of the lamp were determined and the intensity distribution of the LED light over a model of a 96-well plate at a distance of 25 cm from the lamp diode matrix center was estimated. The obtained results show that the light emitted by the solar simulator used is similar to the natural sun light. The biological testing results reveal the high efficiency of the solar simulator in an in vitro experimental system for phototoxicity testing.

Reactive oxygen species (ROS) assay-based photosafety screening for complex ingredients: Modification of the ROS assay protocol.

A reactive oxygen species (ROS) assay has been widely used for photosafety assessment; however, the phototoxic potential of complex materials, including plant extracts, essential oils, and functional polymers, is unevaluable because of their undefined molecular weights. The present study was undertaken to modify the ROS assay protocol for evaluating phototoxic potentials of those materials with use of their apparent molecular weight (aMw). On preparing sample solutions for the ROS assay, aMw ranging from 150 to 350 was tentatively employed for test substances. The modified ROS assays were applied to 45 phototoxic and 19 non-phototoxic substances, including 44 chemicals and 20 complex materials (plant extracts) for clarification of the predictive performance. Generation of ROS from photo-irradiated samples tended to increase as aMW grew, resulting in the largest number of false-positive predictions at aMW of 350. Some false-negative predictions were also observed when aMW was set at 200 or less. At aMw of 250, all tested phototoxic substances could be correctly identified as photoreactive with no false-negative predictions. Based on these observations, aMw of 250 was found to be suitable for the ROS assay on complex materials, and the sensitivity, specificity, and positive and negative predictivity for the proposed ROS assay were calculated to be 100, 52.6, 83.3, and 100%, respectively. Thus, the proposed approach may be efficacious for predicting phototoxic potentials of complex materials and contribute to the development of new products with a wide photosafety margin.

Comparison of Phototoxicity Sensitivity by the Neutral Red Uptake Method for BALB/c 3T3, HaCaT, and HDFa Cells In Vitro

ABSTRACT Introduction Phototoxicity is an acute photoinduced reaction. The 3T3 neutral red uptake (NRU) phototoxicity test has high sensitivity for the determination of phototoxic substances. To further optimize the method, this study mainly focused on comparing the phototoxicity sensitivity by using the NRU method for BALB/c 3T3, HaCaT, and HDFa cells in vitro. Methods The NRU method was used to evaluate the phototoxicity of chlorpromazine hydrochloride (CPZ), amiodarone hydrochloride (Amiodar), and L-histidine (L-His) on BALB/c 3T3 cells, HaCaT cells, and HDFa cells. The sensitivity of different cells to ultraviolet (UVA) irradiation in vitro was studied. Results L-His showed no phototoxicity, but the phototoxicity of CPZ and Amiodar showed different sensitivities among the three kinds of cells. The in vitro phototoxicity evaluation of HaCaT cells is closer to that of primary human fibroblasts. Conclusion This study provides a reference for cell line selection to optimize the existing in vitro evaluation method of 3T3 NRU phototoxicity.

Precision limit for simultaneous phase and transmittance estimation with phase-shifting interferometry

The use of phase-shifting interferometry (PSI) in the ultra-low-light region would open the way for important applications, such as imaging of phototoxic substances and sensing of ultrafast phenomena. In the ultra-low-light region, the statistical nature of photons can be a dominant source of noise compared with other noise caused by experimental instruments. Here we theoretically derive the precision limit determined by the statistical nature of photons for simultaneously measuring the transmittance and phase of a sample with PSI. We show that the precision of PSI depends on the phase and transmittance themselves. We also show a trade-off relation between transmittance and phase. Then we compare PSI with sequential optimal measurements in which the transmittance and phase of a sample are separately measured for each corresponding optimal measurement. We also discuss the case where the input number of photons fluctuates with a Poisson distribution and show that the fluctuation affects both the transmittance and phase precision for PSI.

Determination of phototoxic furanocoumarins in natural cosmetics using SPE with LC-MS

Furanocoumarins are a class of naturally occurring phototoxic substances widely spread throughout the Umbelliferae and Rutacea families of plants. Plant extract of these families (e.g. citrus extracts) are commonly used as ingredients in fragrances and cosmetic products. Due to their phototoxicity these substances are regulated within the European Union according to the European Regulation (EC) No. 1223/2009 on cosmetic products. The proposed analytical workflow aims to swiftly screen out and determine the amount of phototoxic furanocoumarins in natural cosmetics employing solid-phase extraction (SPE). Cosmetic samples were first extracted in methanol and subsequently diluted in 35% (v/v) methanol before being applied to solid-phase extraction. The extraction procedure allows extraction of the eight target compounds including the six marker-substances proposed by the International Fragrance Association (IFRA) from complex cosmetic samples with satisfactory recovery levels ranging from 84 to 116% with the exception of bergamottin (68–89%). HPLC-UV and ESI-MS were used for the analysis of the target compounds. Furanocoumarin quantification was performed by standard addition. The workflow was successfully validated according to the ISO 12787 international standard (“Cosmetics – Analytical methods- Validation criteria for analytical results using chromatographic techniques”). Results of the validation on standard solutions highlight a LOQ of 0.01 mg kg−1, acceptable linearity up to 1 mg/kg as well as good accuracy (Bias <15%) and precision (RSD <15%). Total furanocoumarin levels of the examined natural cosmetic products were in general in agreement with the European Regulation. However, it was shown that total levels of furanocoumarins might be significantly higher than assumed using the six proposed marker substances only.

Topical fig sap induced phytophotodermatitis to eradicate molluscum contagiosum and flat warts

Folk medicine describes the use of figs to treat skin lesions. This therapeutic use of fig sap is even mentioned in the Bible. Fig sap can cause irritant or phototoxic reactions when combined with sun exposure termed Phytophotodermatitis. Contact with plant-derived phototoxic substances (furocoumarins or psoralens) followed by sunlight exposure produces clinical lesions that have therapeutic activity for small skin lesions. In children, conventional topical treatment of flat warts or Molluscum Contagiosum may be difficult because of poor compliance. Here, we present two cases of induced phytophotodermatitis by using fig sap over a period of 4-6 weeks to treat multiple flat warts in a 12-year-old boy, and a case of Molluscum Contagiosum in a 6-year-old girl. These cases suggest an alternative treatment for skin lesions.

Photomicronucleus assay of phototoxic and pseudophotoclastogenic chemicals in human keratinocyte NCTC2544 cells

Photochemical genotoxicity was evaluated in human keratinocyte NCTC2544 cells. The cells were pre-treated with photogenotoxic or pseudophotoclastogenic chemicals and irradiated with a solar-simulator for 50 min at a total UV dose of 5 J/cm 2 or placed in the dark for the same period. After washing, the cells were cultured for 1.5–2 cell cycles with fresh culture medium. At the end of culturing, slide specimens were prepared and examined for micronucleus formation. 8-Methoxypsoralen, a photogenotoxic chemical, strongly induced micronucleated cells with UV irradiation but not under non-irradiation conditions. Therefore, NCTC2544 cells were subjected to further investigation to evaluate the possible photogenotoxicity of other chemicals. 6-Methylcoumarin, 3,3′,4′,5-tetrachlorosalicylanilide and protoporphyrin IX disodium salt, which are all known phototoxic substances, induced micronucleated cells with irradiation but not in the non-irradiation state. These phototoxic substances were confirmed to be photogenotoxic. Tetrabenzoporphine and 5-aminolevulinic acid, which are used for photodynamic therapy, showed phototoxicity. However, these chemicals did not induce micronucleated cells in the irradiated or non-irradiated state, suggesting a lack of photogenotoxicity. Among 3 pseudophotoclastogenic chemicals having no light absorbance at 290–700 nm, neither cycloheximide nor disulfoton induced micronucleated cells with or without irradiation; zinc oxide induced micronucleated cells with irradiation and, to a lesser extent, without irradiation. Based on the results of the photogenotoxicity assays of these 9 chemicals, NCTC2544 cells are considered to be a suitable test system to evaluate the photogenotoxic potential of chemicals.

Five Cases of Berlocq Dermatitis and Analytical Results of Phototoxic Substances Included in Responsible Products

Five Cases of Berlocq Dermatitis and Analytical Results of Phototoxic Substances Included in Responsible Products

Analytical Studies on Photochemical Behavior of Phototoxic Substances; Effect of Detergent Additives on Singlet Oxygen Generation

Monitoring of reactive oxygen species (ROS) generation from photoirradiated compounds would be effective for the prediction of the phototoxic potential. The aim of this investigation was to clarify the possible role of biomimetic vehicle systems on the photochemical properties of phototoxic compounds, focusing on the singlet oxygen generation. Nine phototoxic and one non-phototoxic compounds (200 microM), dissolved in Tween 20, sodium laurate, or sodium dodecyl sulfate (SDS) micellar solution, were exposed to UVA/B light (250 W/m2), and singlet oxygen generation was monitored by RNO bleaching methodology. Photochemical properties of photosensitizers were also evaluated by UV measurement, and the interaction of photosensitizers with surfactant micelles was assessed by Z-potential and NMR spectroscopic analyses. All phototoxic compounds tended to generate singlet oxygen under light exposure in the all micellar solutions tested. There appeared to be some differences in photoreactivity of both cationic and anionic photosensitizers among the micelles tested, whereas ROS data on anthracene, dissolved in three micellar solutions, were found to be quite similar. Photosensitizers exhibited no significant changes in UV spectral patterns among the dissolving micellar solutions. Addition of cationic photosensitizer at the final concentration of 100 microM into 100 mM SDS solution resulted in the 20 mV increase of zeta potential and transition of NMR spectral pattern, which would reflect the electrostatic interaction with anionic micelles. Based on the data obtained, the photoreactivity of photosensitizing molecules, especially cationic and anionic photosensitizers, strongly depends on the physicochemical properties of the microenvironment.

In vitro alternatives and phototoxicity testing. I. Evaluation of in vitro phototoxicity assays.

Three in vitro phototoxicity assays -- the photohaemolysis assay, the haemoglobin (Hb) photo-oxidation assay, and the 3T3 cell neutral red uptake phototoxicity (3T3 NRU PT) assay -- were evaluated for use as screening methods in predicting the phototoxicity of test substances. Twenty-seven test substances, including cosmetic ingredients and drugs, were assessed in this study. The phototoxicity predicted in each assay was compared with that in guinea-pigs. In total, nine phototoxic substances used in this study could be detected by some of the in vitro phototoxicity assays. Eleven test substances regarded as non-phototoxic in vivo were predicted as non-phototoxic in the in vitro assays. Of the eight false positives revealed in some of the in vitro assays, five are believed to be photo-allergens. This suggests that in vitro phototoxicity assays might not be able to discriminate clearly between in vivo phototoxicity and in vivo photo-allergy, because the mechanisms in both processes are based on photodynamic reactions. The predictability of the three in vitro assays was comparatively good, but positive predictive values were low. The equivalence values of the photohaemolysis assay, the Hb photo-oxidation assay and the 3T3 NRU PT assay were 81%, 70% and 70%, respectively. From these results, we suggest that the three in vitro phototoxicity assays could be used as screens in predicting phototoxicity.

A modified murine local lymph node assay for the differentiation of contact photoallergy from phototoxicity by analysis of cytokine expression in skin-draining lymph node cells

Since predictive differentiation of photoallergenic from phototoxic reactions, induced by low molecular weight compounds, represents a current problem, we tried to improve the differentiation between the two reactions by using a modified protocol of the local lymph node assay (LLNA). Briefly, groups of female BALB/c mice received compound solution or vehicle alone on the dorsum of both ears on 3 consecutive days. Immediately after compound application indicated groups of mice were exposed to a UVA light-dose of 10 J/cm2. Auricular lymph nodes draining the ear tissue were excised 24 h following the last exposure. Evaluation consisted of assessing lymph node weights and cell counts to monitor organ hyperplasia and in vivo-proliferative events following substance application. Furthermore, we analysed cytokine gene transcription in freshly prepared lymph node cells (LNC) and the cytokine release in vitro by restimulated CD4+ T-cells and antigen presenting cells (APC), both purified from the skin-draining lymph nodes. Both contact (photo) allergenic (oxazolone and tetrachlorosalicylanilide) and phototoxic substances (8-methoxypsoralen and acridine) caused a dose dependent increase in lymph node weights and cell counts pointing to an inflammatory process in the lymph nodes. Analysis of cytokine gene transcription ex vivo and cytokine release in vitro revealed that during the induction phase of contact (photo) allergy CD4+ T-cells produced IL-2 and IFN-γ as well as IL-4 and IL-10, whereas IL-6 was derived from APC. In contrast, phototoxic reactions caused only an upregulation of IL-2 and IFN-γ. Furthermore, we demonstrate that the release of IL-4 and IL-10 by CD4+ T-cells was clearly increased, whereas IL-6 and IFN-γ expression was reduced or not changed following a challenge with contact (photo) allergens revealing an allergy-indicative shift in cytokine expression. In conclusion, our results show that contact photoallergenic reactions could be differentiated from phototoxic events by analysis of LNC cytokine expression patterns.

Photo hen's egg test: a model for phototoxicity

The aim of this investigation was to establish a new model for phototoxicity which is more advanced than the widely used cultures of yeasts, bacteria or cells of various origin, and at the same time to avoid animal testing. We studied the extraembryonal vasculature of the incubated hen's egg. This model was originally introduced by toxicologists as an alternative to the rabbit's eye irritation test (Draize test). In the photo hen's egg test, substances are applied to the embryo's yolk-sac blood vessel system at a non-toxic concentration and are irradiated with 5 J/cm2 ultraviolet A (UVA) (320-400 nm). Promethazine, haematoporphyrin, ciprofloxacin and 8-methoxypsoralen were tested in this system. Death of the embryo, membrane discoloration and haemorrhage are parameters for phototoxic damage, which were recorded during an observation period of 24 h. These well-known phototoxic substances induced pronounced damage of the yolk-sac membrane and blood vessels which was not found in the controls (test substance alone, UVA alone or untreated) using a 2 x 2 factorial test design. The photo hen's egg test serves as a valid screening model for substances supposed to be photosensitizers owing to a phototoxic mechanism.

NHIK 3025 Cell Culture: A New in Vitro Test for Phototoxicity Screening

In this study, we used NHIK (Norsk Hydro's Institutt for Kreftforskning) 3025 cells, derived from a human carcinoma of the cervix, to screen for phototoxicity. Previously, NHIK 3025 cells have been used in porphyrin research. A number of sulphonamide-derived oral anti-diabetics and diuretics were incubated together with the NHIK cells for one hour and irradiated with broad band UV and UVA radiation. Cell death was observed in the presence of two oral anti-diabetic drugs and ten diurectic drugs, and was dependent on UVA exposure and the concentration of the test substance. This model proved valuable for the detection of known phototoxic substances, and, in addition, phototoxic properties were demonstrated for a number of substances not previously shown to exert these effects. Electron microscopy of the cells, in the presence of the diuretic bemetizide, showed that biomembranes are the main target for phototoxicity, although nuclear damage was also demonstrated. The addition of antioxidants reduced the amount of phototoxic cell death, indicating the participation of reactive oxygen species in the process. These procedures show that this model can be useful both in screening procedures and in mechanistic investigations.

Combined in vitro assay for photohaemolysis and haemoglobin oxidation as part of a phototoxicity test system assessed with various phototoxic substances

12 well-known phototoxicants used in the EC/COLIPA (The European Cosmetic, Toiletry and Perfumery Association) interlaboratory validation trial on ‘Photoirritation in vitro’ have been analysed in view of their ability to induce photohaemolysis. As well as, or instead of, this effect, oxidative reactions involving oxyhaemoglobin have been monitored, by assessing intra- and extracellular methaemoglobin formation, as an additional parameter in the screening of deleterious photoeffects on the cellular protein of red blood cells. A combination of both photohaemolysis and photo-oxidation of haemoglobin is proposed for the investigation of light-induced toxicity in an in vitro test system. Piroxicam and 8-methoxypsoralen did not produce any marked phototoxic alterations in this system. Rose bengal and amiodarone demonstrated predominantly membrane effects, whereas the antibiotics tetra- and doxycycline damaged exclusively the intracellular haemoglobin. All other phototoxins caused both photohaemolysis and photo-oxidation of haemoglobin.

Experimental studies on the mechanisms of tiaprofenic acid photosensitization

Red blood cell lysis and histidine degradation, photosensitized by tiaprofenic acid (TIA), were investigated. Photohaemolysis was markedly enhanced in oxygenated solutions, but was also intense in the presence of nitrogen. Photohaemolysis was inhibited by butylated hydroxyanisole and reduced glutathione, but was unaffected by sodium azide, superoxide dismutase and mannitol. The TIA-induced photo-oxidation of histidine was greatly enhanced in the presence of oxygen and almost completely inhibited in solutions bubbled with nitrogen. Sodium azide, butylated hydroxyanisole and reduced glutathione inhibited the photodegradation of histidine. Phototoxicity to histidine was unaffected by mannitol and superoxide dismutase. The overall results suggest that molecular mechanisms involving free radicals and singlet oxygen are responsible for TIA-photosensitized reactions. These two in vitro models (photohaemolysis and histidine degradation represent different mechanisms of phototoxicity, but complement one another in the investigation of potential phototoxic substances.

Mortality of Metalloporphyrin-Treated Neonatal Rats after Light Exposure

Tin protoporphyrin (SnPP) has been used to suppress hyperbilirubinemia in human neonates through inhibition of heme oxygenase. Some of the subjects exhibited mild erythema upon receiving phototherapy. SnPP and three proposed alternatives, tin mesoporphyrin (SnMP), zinc protoporphyrin (ZnPP) and zinc mesoporphyrin (ZnMP) are potential photosensitizers. We therefore studied the phototoxic effects of these compounds in the neonatal rat model. Fed Wistar rats (24-36 h old) were injected intraperitoneally with up to 40 mumol SnPP/kg body weight, 30 mumol SnMP/kg body weight, 60 mumol ZnPP/kg body weight, or 45 mumol ZnMP/kg body weight. The animals were placed over cool white light (20 microW/cm2/nm) for 12 h. Light exposure resulted in SnPP dose-dependent mortality, and the LD50 was determined to be 11.7 mumol/kg body weight. No deaths were observed in pups treated with up to 20 mumol SnMP/kg; treatment with 30 mumol SnMP/kg resulted in a 40% mortality rate. No fatalities were observed among the light-exposed ZnPP- or ZnMP-treated pups. No deaths were observed among control pups treated with the highest metalloporphyrin doses and kept in the dark; similarly, no mortality was observed in untreated light-exposed control animals. We conclude that (1) SnPP and SnMP are potentially fatal phototoxic substances in the neonatal rat; (2) ZnPP and ZnMP may be safer drugs for neonatal rats receiving light exposure, and (3) further studies are needed to fully assess the photobiological hazards of metalloporphyrin administration to humans.

Demonstration of Quinolone Phototoxicity in vitro

The 'first-generation' quinolones cinoxacin, nalidixic acid, oxolinic acid, pipemidic acid and rosoxacin and the newly developed quinolones ciprofloxacin, enoxacin, fleroxacin, norfloxacin and ofloxacin were screened in vitro at 10(-5), 10(-4) and 10(-3) M concentrations for ultraviolet (UV)-induced phototoxic effects in a photohemolysis test. At 10(-3) M, photodependent effects of norfloxacin could not be evaluated, as hemolysis occurred without irradiation. All other compounds with the exception of ciprofloxacin were found to be phototoxic at 10(-3) M and some also at 10(-4) M. Hemolysis was UV dose dependent and for all compounds most prominent after exposure to UVA-rich radiation except for fleroxacin which was most active in the UVB range. Besides fleroxacin, also oxolinic acid, pipemidic acid and rosoxacin induced hemolysis with irradiations rich in UVB. It is concluded that quinolones have to be regarded as potentially phototoxic substances. Therefore intense light exposure should be avoided during treatment with these agents.

Role of complement and polymorphonuclear cells in demethylchlortetracycline-induced phototoxicity in guinea pigs. Inhibition by decomplementation in vivo.

In this study, demethylchlortetracycline was used as a prototype of exogenous phototoxic substances. In vitro, exposure of serum containing demethylchlortetracycline to ultraviolet-A irradiation resulted in the diminution of total complement hemolytic activity and C4, C2, C3, and C5 activities. In addition, chemotactic activity for human polymorphonuclear cells was generated, which was thermostable and antigenically related to human C5 but not human C3. In vivo, phototoxic lesions were induced in guinea pigs upon intradermal injections of demethylchlortetracycline solution, followed by ultraviolet-A irradiation. On a scale of 0-3+, the animals developed a maximal response of 2.5 at 20 h. This clinical response was associated with cellular infiltrate in the dermis, consisting of 29 +/- 2% of neutrophils at 24 h. The participation of the polymorphonuclear cells was evaluated in guinea pigs rendered neutropenic by treatment with cyclophosphamide. In these guinea pigs, demethylchlortetracycline and ultraviolet-A induced a maximal response of 0.75 +/- 0.5, which was associated histologically with 1.2 +/- 0.5% neutrophils in the dermis. The role of complement in this process was studied in guinea pigs congenitally deficient in C4, and in guinea pigs decomplemented by treatment with cobra venom factor. In contrast to normal guinea pigs, C4-deficient animals exhibited a maximal reaction of 0.83 +/- 0.16 at 6 h, which subsided within 24 h. Cobra venom factor-treated guinea pigs developed a maximal response of 0.5 at 0.5 and at 6 h. These clinical changes were associated with the development of an increased vascular permeability, as demonstrated by studies using guinea pigs injected intravenously with Evans blue solution. In animals with a normal complement system, there was intense localized bluing at the sites of phototoxic lesion. In contrast, only minimal bluing was observed in decomplemented guinea pigs. These data indicate that a normal number of polymorphonuclear cells and an intact complement system are required for the full development of demethylchlortetracycline-induced phototoxic lesions.