The Assessment of Meloxicam Phototoxicity in Human Normal Skin Cells: In Vitro Studies on Dermal Fibroblasts and Epidermal Melanocytes

Meloxicam (MLX), which belongs to the oxicam nonsteroidal anti-inflammatory drug derivatives, is an inhibitor of the cyclooxygenase-2 (COX-2) enzyme. Cutaneous adverse effects caused by interaction between UVA radiation and exogenous factors can manifest as phototoxic reactions. Phototoxicity may be a reason for the accumulation of genetic and molecular changes in long-lived cells with low proliferation potential, leading to tumor development. There are several potentially phototoxic drugs, the active component of which is meloxicam. The research aimed to evaluate the influence of MLX and UVAR on skin cells—fibroblasts and melanocytes homeostasis. The obtained results indicated that co-treatment with MLX and UVAR inhibited skin cell proliferation, proportionally to the drug concentration. The observation was confirmed by cytometric analysis of the cell number and viability. The phototoxic effect of MLX was revealed in morphological changes. It was stated that MLX with UVAR lowered the mitochondrial transmembrane potential and changed the cell cycle profile. Additionally, MLX and UVAR caused the disruption of redox homeostasis by lowering the intracellular level of reduced thiols. The presented study revealed that the phototoxic activity of MLX is associated with oxidative stress induction and disruptions in cell homeostasis. The differences in the phototoxic effects of MLX at the cellular level may be related to the different content of melanin pigments.     

Visible-light Photochemistry and Phototoxicity of Thiarubrines

Abstract. Thiarubrines, a group of intensely red, sulfur-containing pigments produced by asteraceous plants, are photola-bile, yielding thiophenes and elemental sulfur upon exposure to UV or visible light. The mechanism of this light-induced conversion for thiarubrines A (la), B (lb) and D (lc), isolated from the roots of Ambrosia chamis-sonis (Asteraceae), was investigated. Visible-light irradiation of thiarubrines resulted in the formation of novel 2,6-dithiabicyclo[3.1.0]hex-3-ene polyyne photointerme-diates (photosulfides) that rapidly undergo desulfuriza-tion to yield thiophenes. Six photosulfides, photosulfides 3a and 3a' from thiarubrine A (la), photosulfides 3b and 3b' from thiarubrine B (lb) and photosulfides 3c and 3c' from thiarubrine D (lc) were characterized. Thiarubrine photointermediates are short-lived and unstable, with the photosulfides formed from thiarubrine A having a half-life of 12.3 min at room temperature. While the immediate fate of the extruded sulfur is unknown, we identified cyclooctasulfur (S₈) in photolysis solutions of thiarubrine A using electron impact mass spectrometry. Visible-light irradiation of Candida albicans cell suspensions treated with thiarubrine A led to a 99% decrease in cell viability, suggesting that the photosulfides, or other molecules generated by the exposure of thiarubrines to light, have significant toxicity.     

Phototoxicity testing by online irradiation and HPLC

A high-performance liquid chromatography (HPLC) system was developed for the determination of drug photostability and phototoxicity based on an automated column-switching system with aqueous online UV-A irradiation and hyphenated organic separation of the drug and its photoproducts. The photoreactor is built with an poly(ethylene-co-tetrafluoroethylene) (ETFE) reaction coil knitted around a UV-A light source. The chromatographic separation was performed with two special C₁₈ columns, which are also suitable for using with pure water as eluent. Degradation of chlorpromazine (CPZ) by ultraviolet light was investigated at pH 7 and pH 3. Furthermore chlorpromazine was irradiated in the presence of guanosine-5-monophosphate (GMP) in pH 7 buffered solution, leading to a new photoproduct. In the pH 3 irradiation studies of CPZ and GMP, no reaction was detected between the molecules.     

A CE Method for Measuring Phototoxicity in Vitro

This paper presents an experimental setup which employs capillary electrophoresis with electrochemical and UV detection to test phototoxicity of plant extracts and components in terms of oxygen consumption and generation of reactive oxygen species upon irradiation with visible light. The experimental setup was used to test the phototoxicity of different buckwheat extracts and individual plant derived substances. The buckwheat extracts showed differences in their phototoxic behavior which might be due to different phytochemical composition. Screening of individual components revealed that rutin and quercetin alone were not phototoxic, but quercetin in combination with hypericin and chlorophyll caused considerable oxygen consumption. It has been demonstrated that the apparatus is a valuable tool to screen in vitro potential phototoxic reactions of plant extracts and individual constituents.     

The Role of Singlet Oxygen in Photoreactivity and Phototoxicity of Curcumin

Curcumin is a plant-derived yellow-orange compound widely used as a spice, dye and food additive. It is also believed to have therapeutic effects against different disorders. On the other hand, there are data showing its phototoxicity against bacteria, fungi and various mammalian cells. Since the mechanism of its phototoxic action is not fully understood, we investigated here the phototoxic potential of curcumin in liposomal model membranes and in HaCaT cells. First, detection of singlet oxygen (¹O₂) luminescence proved that curcumin generates ¹O₂ upon blue light irradiation in organic solvent and in liposomes. Then, HPLC-EC(Hg) measurements revealed that liposomal and cellular cholesterol is oxidized by ¹O₂ photogenerated by curcumin. Enrichment of liposome membranes with curcumin significantly increased the oxygen photo-consumption rate compared to the control liposomes as determined by EPR oximetry. Cytotoxicity measurements, mitochondrial membrane potential analyses and protein hydroperoxides detection confirmed strong phototoxic effects of curcumin in irradiated HaCaT cells. These data show that since curcumin is advertised as a valuable dietary supplement, or a component of cosmetics for topical use, caution should be recommended especially when skin is exposed to light.     

Phototoxicity of a core-modified porphyrin and induction of apoptosis

A core-modified porphyrin, 5-phenyl-10,15-bis(carboxylatomethoxyphenyl)-20-(2-thienyl)-21,23-dithiaporphyrin (IY69) was studied in vitro for photodynamic activity under a variety of experimental protocols. Variables included the cell line (the rodent mammary tumor cell line R3230AC or the human breast cancer cell line MCF-7), light fluence, time of exposure of the cell cultures to IY69, and the time post-irradiation for cell counting. The length of time cell cultures were exposed to IY69 impacted cellular accumulation and cellular localization, phototoxicity, and the apparent mode of cell death - apoptosis vs. necrosis.     

Fluoroquinolone Antimicrobials: Singlet Oxygen, Superoxide and Phototoxicity

The fluoroquinolone antibacterial agents possess photo-sensitizing properties that lead to phototoxic responses in both human and animal subjects. The phototoxicity order reported in humans is: fleroxacin > lomefloxacin, pefloxacin > ciprofloxacin ? enoxacin, norfloxacin and ofloxacin. Studies both in vivo and in vitro have related this phototoxicity to the generation of reactive oxygen species including hydrogen peroxide and the hydroxyl radical. We determined the quantum yields of singlet oxygen generation (φ_Δ,) by detection of the singlet oxygen (¹O₂) luminescence at 1270 tun for several fluoroquinolones, naphthyridines and other structurally related compounds. All the fluoroquinolones examined have low φ_Δ values ranging from 0.06 to 0.09 in phosphate buffer at pD 7.5. We also determined the ¹O₂ quenching constants for these compounds and their values were on the order of 10⁶M^?1 s¹, except for lomefloxacin whose rate constant was 1.8 × 10⁷M^?1 s^?1. The φ_Δ values were significantly decreased in a solvent of lower polarity such as methanol (0.007 ≤φ_Δ≤ 0.02). The production of ¹O₂ by these antibiotics did not correlate with the order reported for their phototoxicity. We also measured the photogeneration (λ > 300 nm) of superoxide by these antibacterials in dimethylsulfoxide using electron paramagnetic resonance and the spin trap 5,5-dimethyl-l-pyrroiine N-oxide. Although there is not a one-to-one correspondence between the relative rates of superoxide generation and the phototoxicity ranking of the fluoroquinolones, the more phototoxic compounds tended to produce superoxide at a faster rate. Nevertheless, the magnitudes of the observed differences do not appear sufficient to explain the range of fluoroquinolone phototoxicity potencies in human and animal subjects in general and the high activity of fleroxacin and lomefloxacin in particular. For these latter drugs the photoinduced loss of the F₈ atom as fluoride and the concomitant generation of a highly reactive carbene at C-8 provide a more plausible mechanism for their potent phototoxic and photocarcinogenic properties.     

Photoprotective Effect of Black Tea Extracts Against UVB-induced Phototoxicity in Skin

In previous studies, we showed that green tea and black tea extracts and their major polyphenolic constituents protect against UVB light-induced carcinogenesis in murine skin. All of these studies required chronic administration of tea extracts or specific constituents either topically or orally. However, it is not known whether acute or subchronic administration of black tea extracts or constituents can ameliorate UVB-induced early effects in skin. In the present study, cultured keratinocytes and mouse and human skin were employed to assess the effect of both oral and topical administration of standardized black tea extract (SBTE) and its two major polyphenolic subfractions namely BTF1 and BTF2 against UVB-induced photodamage. In SKH-1 hairless mice, topical application of SBTE (0.2 mg/cm2) prior to UVB exposure (180 mJ/cm2) resulted in 40% reduced incidence and 64% reduced severity of erythema and 50% reduction in skinfold thickness by day 6 when compared to nontreated UVB-exposed animals. The SBTE was also effective in protecting against UVB-induced erythema in human volunteers. Administration of SBTE 5 min after UVB irradiation was similarly effective in reducing UVB-induced inflammation in both murine and human skin. The major polyphenolic subfractions, BTF1 and BTF2, were also effective in protecting in mouse skin. The SBTE subfractions inhibited UVB-induced tyrosine phosphorylation of epidermal growth factor receptor (EGFR). The UVB irradiation of human epidermoid carcinoma cells resulted in 3.3-fold induction of tyrosine phosphorylation of EGFR. Pretreatment with BTF1 and BTF2 reduced tyrosine phosphorylation of EGFR by 53% and 31%, respectively. The UVB-mediated enhanced expression of the early response genes, c-fos and c-jun in human epidermal keratinocytes was reduced in a dose-dependent manner by SBTE. Topical application of SBTE was also effective in reducing accumulation of c-fos and p53 proteins by 82% and 78%, respectively, in UVB-exposed mouse skin. These data provide evidence that constituents of black tea can abrogate UVB-induced erythema and associated early events in murine and human skin.     

Phototoxicity in human lens epithelial cells promoted by St. John's Wort

St. John's Wort (SJW), an over-the-counter antidepressant, contains hypericin, which absorbs light in the UV and visible ranges and is phototoxic to skin. To determine if it also could be phototoxic to the eye, we exposed human lens epithelial cells to 0.1-10 microM hypericin and irradiated them with 4 J/cm2 UV-A or 0.9 J/cm2 visible light. Neither hypericin exposure alone nor light exposure alone reduced cell viability. In contrast, cells exposed to hypericin in combination with UV-A or visible light underwent necrosis and apoptosis. The ocular antioxidants lutein and N-acetyl cysteine did not prevent damage. Thus, ingested SJW is potentially phototoxic to the eye and could contribute to early cataractogenesis. Precautions should be taken to protect the eye from intense sunlight while taking SJW.     

In Vitro Evaluation of the Photoreactivity and Phototoxicity of Natural Polyphenol Antioxidants

Polyphenols are a large family of natural compounds widely used in cosmetic products due to their antioxidant and anti-inflammatory beneficial properties and their ability to prevent UV radiation-induced oxidative stress. Since these compounds present chromophores and are applied directly to the skin, they can react with sunlight and exert phototoxic effects. The available scientific information on the phototoxic potential of these natural compounds is scarce, and thus the aim of this study was to evaluate the photoreactivity and phototoxicity of five phenolic antioxidants with documented use in cosmetic products. A standard ROS assay was validated and applied to screen the photoreactivity of the natural phenolic antioxidants caffeic acid, ferulic acid, p-coumaric acid, 3,4-dihydroxyphenylacetic acid (DOPAC), and rutin. The phototoxicity potential was determined by using a human keratinocyte cell line (HaCaT), based on the 3T3 Neutral Red Uptake phototoxicity test. Although all studied phenolic antioxidants absorbed UV/Vis radiation in the range of 290 to 700 nm, only DOPAC was able to generate singlet oxygen. The generation of reactive oxygen species is an early-stage chemical reaction as part of the phototoxicity mechanism. Yet, none of the studied compounds decreased the viability of keratinocytes after irradiation, leading to the conclusion that they do not have phototoxic potential. The data obtained with this work suggests that these compounds are safe when incorporated in cosmetic products.     

Evaluation of Sparfloxacin Distribution by Mass Spectrometry Imaging in a Phototoxicity Model

Mass spectrometry imaging (MSI) was applied to samples from mouse skin and from a human in vitro 3D skin model in order to assess its suitability in the context of photosafety evaluation. MSI proved to be a suitable method for the detection of the model compound sparfloxacin in biological tissues following systemic administration (oral gavage, 100 mg/kg) and subsequent exposure to simulated sunlight. In the human in vitro 3D skin model, a concentration-dependent increase as well as an irradiation-dependent decrease of sparfloxacin was observed. The MSI data on samples from mouse skin showed high signals of sparfloxacin 8 h after dosing. In contrast, animals irradiated with simulated sunlight showed significantly lower signals for sparfloxacin starting already at 1 h postirradiation, with no measurable intensity at the later time points (3 h and 6 h), suggesting a time- and irradiation-dependent degradation of sparfloxacin. The acquisition resolution of 100 μm proved to be adequate for the visualization of the distribution of sparfloxacin in the gross ear tissue samples, but distinct skin compartments were unable to be resolved. The label-free detection of intact sparfloxacin was only the first step in an attempt to gain a deeper understanding of the phototoxic processes. Further work is needed to identify the degradation products of sparfloxacin implicated in the observed inflammatory processes in order to better understand the origin and the mechanism of the phototoxic reaction. Figure

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Phototoxicity and cytotoxicity of chlorophyll a/cyclodextrins complexes on Jurkat cells

The aggregation status of chlorophyll a (Chl a) and the ability of four cyclodextrins, hydroxypropyl-beta-cyclodextrin (HP-beta-CD), hydroxypropyl-gamma-cyclodextrin (HP-gamma-CD), heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DIMEB), and heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin (TRIMEB), to solubilize the pigment in the complete cellular medium RPMI 1640 was estimated by means of UV-Vis absorption and static resonance light scattering (RLS) measurements. The results indicate that the pigment interacts with cyclodextrins in the cellular medium differently to that observed in water. The cytotoxic and phototoxic activity of these complexes towards human leukemia T-lymphocytes (Jurkat cells) was tested by means of experiments aimed to discriminate between the intrinsic toxicity and the toxicity induced by light. The overall data indicate that the HP-beta-CD is the cyclodextrins having the best characteristics to form with Chl a a potential supramolecular system for the photodynamic therapy.     

重稀碱金属Cs~+跨人红细胞膜行为的研究(英文)

采用原子吸收光谱法检测体外人红细胞摄取Cs+的含量,系统讨论了胞外Cs+浓度,温育时间、温育温度、介质pH值对人红细胞摄取Cs+过程的影响。选用不同离子通道或离子载体的特异性抑制剂进一步探讨Cs+的跨膜途径和机理。结果显示,各实验参数对人红细胞摄取Cs+均有一定的促进作用。Cs+主要借助Na+/K+-泵的主动运输方式跨膜;少量的Cs+能"漏入"细胞,微量的Cs+可以模拟Na+/Li+-反向协同运输的方式跨膜;在允许HCO3-存在的pH环境下,少量Cs+以Cl-/CsCO3-交换的形式通过膜上带3蛋白进入人红细胞;Ca2+通道对Cs+没有通透作用。     

High-Performance Liquid Chromatographic Assay of D-Glucose in Erythrocytes

Abstract

A procedure is presented after several attempts with different modes of chromatography for measuring high concentrations of d-glucose in erythrocytes. The procedure utilizes rapid deproteinization of hemolysate by mixing with acetonitrile. The supernatant is analyzed by strong cation exchange chromatography, using an Organic Analysis Column. Separation conditions are: eluent = 0.01 N H₂SO₄, flow rate = 0.6 ml/min, detection = 195nm at 0.05 AUFS, sample size = 20 μl and temperature = ambient. The coefficients of variation for 5 mg/ml samples were (within-run) 6.7%, and (day-to-day) 7.1%. This study shows the presence of a high concentration (1900 mg/dl) of d-glucose within the erythrocytes as a result of a high external d-glucose concentration (2000 mg/dl) in plasma, and suggests that d-glucose is rapidly transported into the cell.     

Photochemical Reactions and Phototoxicity of Sterols: Novel Self-perpetuating Mechanism for Lipid Photooxidation

Abstract— Sterols are important lipid components that may contribute to phototoxicity. We have found that phototoxic response in earthworms is related to sterols extractable with lipophilic solvents. The photochemically active compounds in worm lipids are 5,7,9(11),22-ergostatetraen-3bT-ol (9-DHE) and 5,7,9(11)-cholestatrien-3bT-ol (9-DDHC), respectively. Human skin lipids are known to contain 9-DHE. We have also found 9-DDHC in human skin, which is reported here for the first time. In the presence of an excess of the corresponding 5,7-dienes (ergosterol or 7-dehydrocholesterol), these photoactive sterols constitute a self-regenerating source of singlet molecular oxygen (₁O₂) during irradiation in vivo or in vitro with UVA bT15-400 nm). The quantum yield for photosensitization of ₁O₂ by 9-DHE was estimated to be 0.09. The ₁O₂ is scavenged by the dienes and the rate constant for ₁O₂ quenching by ergosterol was found to be 1.2 times 10₇ M -₁ s_-1 in methyl t-butyl ether (MTBE). This scavenging ultimately leads to the production of 5,8-endo-peroxide and hydrogen peroxide. Photochemically induced superoxide radical was also produced on irradiation of sterol 5,7,9-trienes and trapped with the spin trap 5,5-dimeth-yl-1-pyrroline W-oxide (DMPO). The production of singlet oxygen, peroxides and radicals by the sterols may be significant in the cell damaging and tumor promoting action of UVA light on skin.     

Phototoxicity of non-steroidal anti-inflammatory drugs: in vitro testing of the photoproducts of Butibufen and Flurbiprofen.

In this work, the phototoxicity of two non-steroidal anti-inflammatory drugs, Butibufen and Flurbiprofen, was examined. Both were unstable to light, to give several photoproducts which were isolated and identified. The different photoproducts were formed by a primary photochemical mechanism which involves an initial cleavage of the C-C bond alpha to the carbonyl group, followed by several secondary processes. The cytotoxic effects of the xenobiotics were evaluated using two well-established biological in vitro tests: (a) enzyme leakage lactate dehydrogenase and glutamate-oxaloacetate transaminase from cultured fibroblasts and (b) lysis of red blood cells. The benzylic alcohols caused extensive leakage from cultured fibroblasts at the different concentrations assayed. The alcohol obtained from Butibufen was a potent lytic agent for human red blood cells. The other photoproducts, Butibufen and Flurbiprofen did not produce observable toxic effects on cells.     

HPLC Separation of Fluorescent Products of Lipid Peroxidation in Erythrocytes and Mitochondria

Polyunsaturated fatty acids found in membrane phospholipids are readily oxidized by free radicals producing highly reactive aldehydes such as malondialdehyde. These aldehydes react with biological material to form fluorescent lipid peroxidation end products known as lipofuscin-like pigments. We studied fluorescent pigments from beef heart mitochondria incubated with malondialdehyde in vitro and from erythrocytes of patients with Alzheimer’s disease to evaluate their potential as markers of oxidative stress and to develop HPLC methods for their qualitative analysis. We used tridimensional fluorescence spectral arrays and synchronous fluorescence spectra in connection with HPLC separation with fluorescence detection. Stable fluorophores were found in both models and were successfully resolved into several distinctive fractions. This creates the basis for further characterization of this relatively less studied group of products of oxidative damage.     

LC Determination of Nosiheptide in Swine Kidney and Liver

A liquid chromatographic method was developed for determination of nosiheptide in swine kidney and liver. The tissue samples were extracted with acetonitrile and defatted with hexane. The analytes were determined at a fluorescence excitation/emission wavelength of 357/515 nm and with gradient elution program. The limits of detection and limits of quantification were 5 and 20 ng g^?1, respectively, for swine kidney and liver. The mean recoveries for nosiheptide in swine kidney and liver ranged from 70.3 to 97.4% with coefficients of variation below 11.3% at 20–100 ng g^?1 fortification levels.