Photohemolysis Test Research Articles

524 In vitro evaluation of phototoxic properties of systemic antipsoriatics

524 In vitro evaluation of phototoxic properties of systemic antipsoriatics

Photosensitizing Properties of Compounds Related to Benzophenone

Benzophenone is a phototoxic compound with absorption maxima in the ultraviolet A (UVA) and ultraviolet B (UVB) range. Many benzophenone derivatives are known to be photosensitizing. On the other hand, 2-hydroxy-4-methoxybenzophenone is used as a photoprotective agent. The aim of the present study was to analyse a range of benzophenone derivatives and thus examine the effects of molecular changes in the benzophenone molecule on phototoxic behaviour. Phototoxicity was tested by an in vitro photohaemolysis test. The tested compounds were benzophenone itself and the derivatives 2-hydroxybenzophenone, 2-aminobenzophenone, 2-benzoylbenzoic acid, 3-hydroxybenzophenone, and 4-hydroxybenzo-phenone, as well as the structurally similar compounds 9-fluorenone, 9-fluorenone-2-carboxylic acid, cyclohexyl phenyl ketone, and 1,4-naphtho-quinone. It was shown that minor changes in molecular structure can result in highly different phototoxic characteristics.

Evaluation of Phototoxic Properties of Fragrances

Fragrances are widely used in topical formulations and can cause photoallergic or phototoxic reactions. To identify phototoxic effects, 43 fragrances were evaluated in vitro with a photohaemolysis test using suspensions of human erythrocytes exposed to radiation sources rich in ultraviolet (UV) A or B in the presence of the test compounds. Haemolysis was measured by reading the absorbance values, and photohaemolysis was calculated as a percentage of total haemolysis. Oakmoss caused photohaemolysis of up to 100% with radiation rich in UVA and up to 26% with radiation rich in UVB. Moderate UVA-induced haemolysis (5-11%) was found with benzyl alcohol, bergamot oil, costus root oil, lime oil, orange oil, alpha-amyl cinnamic aldehyde and laurel leaf oil. Moderate UVB-induced haemolysis was induced by hydroxy citronellal, cinnamic alcohol, cinnamic aldehyde, alpha-amyl cinnamic aldehyde and laurel leaf oil. The phototoxic effects depended on the concentration of the compounds and the UV doses administered. We conclude that some, but not all, fragrances exert phototoxic effects in vitro. Assessment of the correlation of the clinical effects of these findings could lead to improved protection of the skin from noxious compounds.

Evaluation of Phototoxic Properties of Antimicrobials Used in Topical Preparations by a Photohaemolysis Test

Antimicrobials are widely used in topical formulations as preservatives or as therapeutically active agents. Photosensitization by such compounds has not yet been studied systematically. To identify possible phototoxic properties, antimicrobials (benzyl alcohol, bronopol, chloracetamide, clioquinol, diazolidinyl urea, ethylenediamine dihydrochloride, formaldehyde, glutaraldehyde, imidazolidinyl urea, sodium benzoate, propylene glycol) were evaluated in vitro by means of a photohaemolysis test using suspensions of human erythrocytes. Irradiations were performed with UVA- and UVB-rich light sources. In the presence of bronopol or clioquinol, there was photohaemolysis up to 78.1% or 48.5% with UVA and up to 100% or 34.3% with UVB, respectively. The phototoxic effect depended on the concentration of the compounds and the UV doses administered. None of the other substances tested caused significant photohaemolysis. It is concluded that bronopol and clioquinol exert phototoxic effects in vitro and thus might also cause photosensitization when used on the skin. The clinical significance of this has to be established by further work.

Tobacco smoke is phototoxic.

Both cigarette smoke and ultraviolet (UV) radiation are known to cause changes of the skin which can be regarded as premature ageing. To assess the theory that the effects of these two exposures could be linked by a phototoxic action of cigarette smoke. A photohaemolysis test was used, in which human erythrocytes were incubated with cigarette smoke condensate, followed by UV irradiation and measurement of exposure-dependent haemolysis. Cigarette smoke condensate was clearly phototoxic. Photohaemolysis depended on the concentration of the condensate and UV dose and was more pronounced after exposure to UVA-rich than UVB-rich radiation. Phototoxicity may be a mechanism by which cigarette smoking causes premature skin ageing. An enhancing effect on photocarcinogenesis has also to be considered.

FS14.1 Safety of various fragrances: allergy and phototoxicity, joint study in Korea

The purpose of this study is to know the prevalence of allergic patch test responses in patients with suspected fragrance allergy in Korea and to know the photoxic potentials of limited fragrance substances. Nine dermatology departments of university hospitals and one research institute of cosmetic company were participated in this study for the past 1 year. To inquire the specific fragrance allergens in Korea, eighteen fragrance ingredients were added to Korean standard series and fragrance series (Chemotechnique Diagnosis, Sweden). Among 422 patients, 83% were women and the most common topographic site was face. Except the fragrance mix and Balsam of Peru, Cinnamic alcohol and Sandalwood oil showed high frequency of positive responses. Among the added specific fragrance ingredients, 3‐methyl‐5‐(2,2,3‐trimethyl‐3‐cyclopenen‐1‐yl) pent‐4‐en‐2‐ol, alpha‐isomethyl‐ionone(methyl ionone gamma), Lyral showed high positive responses. With eighteen added specific fragrance substances, we have also performed two in vitro phototoxicity tests, 3T3 NRU phototoxicity test and photohemolysis test. 3T3 NRU phototoxicity test is a screening method for DNA or cellular damage and Photohemolysis test is a useful screening method for phototoxic chemicals that cause oxygen‐dependent membrane damage. Only Lyral showed phototoxicity in photohemolysis test and no other fragrance ubstances showed phototoxicity in 3T3 NRU phtototoxicity test. We think our data are useful to know the current status of the frequent fragrance allergens in our society and the possible phototoxic potency of limited fragrance substances.

An In Vitro Phototoxicity Assay Battery (Photohaemolysis and 3T3 NRU PT test) to Assess Phototoxic Potential of Fragrances

The purpose of this study was to compare the in vivo and in vitro phototoxicity potentials of 13 fragrances. We used the 3T3 neutral red uptake phototoxicity (3T3 NRU PT) test and the photohaemolysis test as in vitro phototoxicity assays. In the 3T3 NRU PT test, all of the fragrances were non-phototoxic. Six fragrances were phototoxic in the photohaemolysis test. Three of the six photohaemolytic fragrances were phototoxic in the guinea-pig photoirritation test. These phototoxic fragrances did not cause cellular phototoxicity, but showed a photohaemolytic reaction. The photohaemolysis test was more sensitive than the 3T3 NRU PT test for screening for the phototoxicity of fragrances. The accuracy of this in vitro phototoxicity test battery was 82%. It is thought that the major phototoxic mechanism of fragrances is cell membrane damage. We suggest that a battery composed of the 3T3 NRU PT test and the photohaemolysis test is a simple and effective model for the in vitro phototoxicity assay of fragrances.

DETERMINATION OF THE PHOTOTOXICITY OF FENOFIBRIC ACID BY A SENSITIVE IN VITRO TEST ON POLYMORPHONUCLEAR CELLS

By means of a sensitive chemiluminescence's method, toxicity of fenofibric acid was demonstrated on respiratory burst of UVA-irradiated peripheral blood polymorphonuclear cells (PMNs). Through this method, it was possible detect such phototoxic effect at a minor concentration of fenofibric acid as compared with those previously reported in photohemolysis test. This effect was not related with losses of cell viability because PMNs did not lose its capabilities for excluding the trypan blue dye, when irradiated in presence of drug. In order to explain the results obtained in presence of scavengers of different reactive oxygen species, it was proposed a singlet oxygen-mediated (type II) mechanism of photosensitization and/or through formation of toxic photoproducts.

Increased phototoxicity of hydrochlorothiazide by photodegradation

The photodegradation products of hydrochlorothiazide produced by ultraviolet (UV) radiation were investigated for their phototoxicity utilizing the photohemolysis and Candida albicans tests. Hydrochlorothiazide was irradiated for 30, 60, 90 and 120 min with a 250 W xenon arc lamp using a WG295 cut‐off filter. Irradiation of hydrochlorothiazide resulted in the gradual decrease of all three absorption bands (225, 270 and 320 nm), the blue shift of the 225 nm band, and the appearance of a new band around 290 nm. Since previous results demonstrated that photosubstitution of chloride could occur, the main product of this photolysis most likely is ethoxyhydrochlorothiazide. The photohemolysis test revealed a significant increase in photohemolysis observed in the photodegradation products produced after 60, 90 and 120 min of UV irradiation. This increase in hemolysis value directly correlated with the UV‐irradiation time. However, there was no significant phototoxic killing of yeast in the Candida albicans test. This suggests photodegradation products of hydrochlorothiazide may play an important role in phototoxicity by acting on the cell membrane, but not on DNA. Considering the high in vitro phototoxicity observed in bendroflumethiazide and the data presented here, substitution of chloride seems to be responsible for the increased phototoxicity of hydrochlorothiazide.

Increased phototoxicity of hydrochlorothiazide by photodegradation.

The photodegradation products of hydrochlorothiazide produced by ultraviolet (UV) radiation were investigated for their phototoxicity utilizing the photohemolysis and Candida albicans tests. Hydrochlorothiazide was irradiated for 30, 60, 90 and 120 min with a 250 W xenon arc lamp using a WG295 cut-off filter. Irradiation of hydrochlorothiazide resulted in the gradual decrease of all three absorption bands (225, 270 and 320 nm), the blue shift of the 225 nm band, and the appearance of a new band around 290 nm. Since previous results demonstrated that photosubstitution of chloride could occur, the main product of this photolysis most likely is ethoxyhydrochlorothiazide. The photohemolysis test revealed a significant increase in photohemolysis observed in the photodegradation products produced after 60, 90 and 120 min of UV irradiation. This increase in hemolysis value directly correlated with the UV-irradiation time. However, there was no significant phototoxic killing of yeast in the Candida albicans test. This suggests photodegradation products of hydrochlorothiazide may play an important role in phototoxicity by acting on the cell membrane, but not on DNA. Considering the high in vitro phototoxicity observed in bendroflumethiazide and the data presented here, substitution of chloride seems to be responsible for the increased phototoxicity of hydrochlorothiazide.

Photodegradation and phototoxicity studies of furosemide. Involvement of singlet oxygen in the photoinduced hemolysis and lipid peroxidation

The phototoxic diuretic drug furosemide ( 1), a 5-(aminosulfonyl)-4-chloro-2-[(2-furanylmethyl)-amino] benzoic acid is photolabile under aerobic and anaerobic conditions. Irradiation of a methanol solution of 1 under oxygen produces photoproducts 2, 3, 4 and singlet oxygen, while under argon the photoproducts 2 and 4 were isolated. A peroxidic unstable photoproduct was detected during the photolysis under oxygen atmosphere. The formation of singlet oxygen by photolysis of 1 was evidenced by trapping with 2,5-dimethylfuran (GC-mass), furfuryl alcohol and 1,3-cyclohexadiene-1,4-diethanoate (HPLC) as 1O 2 scavengers and by the histidine test. Furosemide was screened in vitro at different concentrations for UV-Vis-induced phototoxic effects in a photohemolysis test, in the presence and absence of different radical scavengers, singlet oxygen and hydroxyl radical quenchers. However, furosemide photosensitized the peroxidation of linoleic acid, as monitored by the UV-detection of dienic hydroperoxides and it also photosensitized the oxidation of histidine. The photodegradation was catalyzed in the presence of human serum albumin. Studies on peripheral blood mononuclear and polymorphonuclear cells (lymphocytes and neutrophils) demonstrated no phototoxicity on these cell lines.

Phototoxic properties of neuroleptic drugs.

Photo-induced eruptions are well-known adverse effects of some neuroleptic drugs, particularly chlorpromazine. By a photohemolysis test we assessed in vitro the phototoxic properties of 12 phenothiazines (chlorpromazine, dixyrazine, fluphenazine, levomepromazine, perazine, perphenazine, promazine, promethazine, prothipendyl, thioridazine, trifluoperazine, triflupromazine) and 5 thioxanthenes (chlorprothixene, clopenthixol, flupenthixol, thiothixene, zuclopenthixol). Human erythrocytes from 3 donors were incubated with the compounds and irradiated with light sources rich in UVA or UVB, respectively. Doses were up to 100 J/cm2 UVA or up to 1,600 mJ/cm2 UVB. Photo-induced hemolysis was calculated as percentage of complete hemolysis. Photo-induced hemolysis >10% due to radiation rich in UVA was found with chlorpromazine (maximal median: 98%), dixyrazine (100%), fluphenazine (84%), perazine (100%), perphenazine (100%), promazine (16%), promethazine (25%), prothipendyl (96%), trifluoperazine (100%), triflupromazine (76%), chlorprothixene (100%) and thiothixene (31%). UVB-rich radiation induced hemolysis only with chlorpromazine (73%), dixyrazine (45%) and perazine (60%). Most neuroleptics are strongly phototoxic in vitro indicating a potential risk for photo-induced reactions also to occur in patients treated with these drugs.

Evaluation of the phototoxic properties of some hypolipidemics in vitro: fenofibrate exhibits a prominent phototoxic potential in the UVA and UVB region

As some fibric acid derivatives have been reported to exhibit photosensitizing effects in vivo, the antilipemic drugs fenofibrate, bezafibrate, clofibrate, and gemfibrozil were tested for their phototoxic potential in vitro by a photohemolysis test using human erythrocytes and different irradiation sources. In this system only fenofibrate revealed strong phototoxic properties, which were dependent both on the drug concentration and the irradiation doses. Above a surface dose of 40 J/cm 2 UVA of an UVA (320–400 nm)-rich irradiation source or 1.6 J/cm 2 UVB / 0.8 J/cm 2 UVA of an UVB (280–320 nm)-rich irradiation source human red blood cells were completely photohemolysed in the presence of fenofibrate. Bezafibrate- and gemfibrozil-induced photohemolysis remained beneath 10%, and clofibrate showed no phototoxicity at all. As the phototoxic potential of fenofibrate lies in the UVB and UVA range, this might be of relevance with regard to clinical photosensitivity.

Inhibition of thiazide photohemolysis in vitro by antioxidants and a nitrogen atmosphere

The diuretics acetazolamide, bemetizide, bendroflumethiazide, benzthiazide, benzylhydrochlorothiazide, bumetanide, butizide, chlorazanile, chlorothiazide, chlortalidone, clopamide, cyclopenthiazide, cyclothiazide, diazoxide, etozoline, furosemide, hydrochlorothiazide, hydroflumethiazide, indapamide, mefruside, metolazone, piretanide, polythiazide, trichlormethiazide, and xipamide were screened in vitro for phototoxic effects by means of a photohemolysis test. In all, 19 out of the 25 test substances revealed phototoxic hemolytic properties after irradiation with either solar stimulating irradiation, UVA and/or visible light. Addition of the antioxidants ascorbic acid, alpha-tocopherole or superoxide dismutase significantly inhibited the phototoxic hemolysis, as well as did investigations carried out in a nitrogen rich atmosphere, findings which indicate the involvement of reactive oxygen species in the phototoxic process.

Phototoxicity of non-steroidal anti-inflammatory drugs: in vitro studies with visible light.

Photosensitivity reactions due to non-steroidal anti-inflammatory drugs (NSAID) are well-known side-effects of these agents and are usually attributed to ultraviolet (UV) radiation. Eight NSAID (carprofen, diclofenac, ibuprofen, ketoprofen, naproxen, piroxicam, phenylbutazone, tiaprofenic acid) were assessed in a photohemolysis test for phototoxic effects in the visible light range. For the studies an experimental visible light lamp was used, emitting 5% UVA at most. To control for UVA-induced effects, additional samples were exposed to appropriate UVA doses from an UVA source and as well to the visible light lamp while being covered by a GG 400 or GG 420 filter. Photo-induced hemolysis was found after exposure to the visible light lamp with carprofen, ketoprofen, naproxen, piroxicam, phenylbutazone or tiaprofenic acid; diclofenac or ibuprofen did not cause photohemolysis. Control irradiation with UVA induced minor hemolysis only with carprofen, ketoprofen and tiaprofenic acid. When the samples were exposed to the filtered visible light lamp, using the GG 400 filter, photohemolysis was found with carprofen, naproxen and phenylbutazone and, using the GG 420 filter, with carprofen and phenylbutazone. Phototoxic effects induced by visible light may be of importance with regard to clinical photosensitization.

Photosensitization by fenofibrate. II. In vitro phototoxicity of the major metabolites.

Fenofibric acid, the major metabolite of fenofibrate, was found to be photolabile. Its irradiation in aqueous solution gave rise to two photoproducts, whose formation involves photodecarboxylation of the dissociated acid to an aryloxy-substituted carbanion, which is directly protonated or, alternatively, undergoes a Wittig rearrangement. A comparative in vitro phototoxicity study has been carried out on the anti-hyperlipoproteinemic drug fenofibrate, its metabolites and the photoproducts of fenofibric acid. Fenofibrate, fenofibric acid and its two photoproducts were found to be active when examined by the photohemolysis test and were able to photosensitize peroxidation of linoleic acid, as evidenced by the UV monitoring of dienic hydroperoxides. In summary, the major metabolite of fenofibrate (fenofibric acid), as well as its photoproducts, are phototoxic in vitro. This behavior can be attributed to the fact that the four compounds retain the benzophenone chromophore present in fenofibrate and is indicative of free radical-mediated photosensitization. In agreement with this rationalization, the metabolites with a reduced ketone functionality exhibit no detectable in vitro phototoxicity.

Theoretical and experimental studies of intermediate species of photolysis, and phototoxicity of anti-hyperlipoproteinemic drugs (fibrates)

Gemfibrozil (1), bezafibrate (2), fenofibrate (3), and clofibric acid (4) were phototoxicin vitro, when examined by the photohemolysis test using UV-A + UV-B (290–400 nm) under aerobic conditions. No photohemolysis was observed for clofibrate (5). This behavior can be explained through the involvement of free radicals. A satisfactory correlation between the theoretical parameters (heat of formation and HOMO energy) of the postulated radical species and the observed phototoxic effects was found.

In vitro phototoxicity of clofibrate. Photochemical and photohemolytic studies on its metabolite clofibric acid

Aqueous or methanolic solutions of clofibrate and clofibric acid are photolabile towards UVB light under aerobic as well as anaerobic conditions. Nine photoproducts have been identified; their formation involves primary cleavage of the carbon-halogen bond or of the aryloxy-carbon bond, followed by hydrogen abstraction and/or radical recombination. Clofibric acid is phototoxic in vitro as indicated by the photohemolysis test, under both oxygen and argon atmospheres, although the photohemolysis rate is markedly higher under aerobic conditions. Partial inhibition of this process on addition of butylated hydroxyanisole (BHA), reduced glutathione (GSH), sodium azide (NaN 3) or 1,4-diazabicyclo[2.2.2]octane (DABCO) suggests the involvement of type I as well as type II mechanisms.

Evaluation of phototoxic properties of some food additives: sulfites exhibit prominent phototoxicity.

Additives are used widely to enhance the quality of food products. To identify possible phototoxic properties, 13 food additives (benzoic acid, sodium benzoate, 4-hydroxybenzoic acid, 4-hydroxybenzoic acid methyl ester, 4-hydroxybenzoic ethyl ester, 4-hydroxybenzoic acid propyl ester, p-hydroxybenzoic acid n-butyl ester, benzyl alcohol, sorbic acid, potassium sorbate, propionic acid, sodium disulfite and sodium sulfite) were evaluated in vitro by means of a photohemolysis test using suspensions of human erythrocytes. Irradiation was performed with various light sources differing with regard to their spectral irradiance. Sodium sulfite and sodium disulfite induced photohemolysis up to almost 100%, the effect depending on the concentration of the compounds and UV dose administered. Radiation rich in UVB was most effective; a sunlight-simulating lamp induced photohemolysis to a lesser degree. All other substances tested did not cause significant photohemolysis. As sulfites are frequently encountered, they may contribute to UVB sensitivity. The clinical significance of these findings has to be established by further work.

PHOTODEGRADATION AND in vitro PHOTOTOXICITY OF FENOFIBRATE, A PHOTOSENSITIZING ANTI‐HYPEIUIPOPROTEINEMIC DRUG

The phototoxic anti-hyperlipoproteinemic drug fenofibrate was found to be photolabile under aerobic and anaerobic conditions. Irradiation under argon of a methanol solution of this drug produced the photoproducts isopropyl 4-(1-[4-chlorophenyl]-1,2-dihydroxy)ethylphenoxyisobutyrate, 1,2-bis(4-chlorophenyl)-1,2bis (4-[isopropoxycarbonylisopropoxy]phenyl)ethane-1,2-diol and 4-(4-chlorobenzoyl)phenol, while under oxygen the photoproducts were 4-chloroperbenzoic acid, methyl 4-chlorobenzoate, 4-chlorobenzoic acid and singlet oxygen, as evidenced by trapping with 2,5-dimethylfuran. These results can be rationalized through hydrogen abstraction by excited fenofibrate, to afford a free radical as key intermediate. Biologically active antioxidants such as glutathione and cysteine efficiently reduced 4-chloroperbenzoic acid to 4-chlorobenzoic acid. The involvement of an electron transfer mechanism is suggested by detection (UV-vis spectrophotometry) of the radical cation TMP+. during the oxidation of tetramethylphenylenediamine (TMP) with 4-chloroperbenzoic acid. Fenofibrate was phototoxic in vitro when examined by the photohemolysis test, both under oxygen and argon atmosphere, although the photohemolysis rate was markedly lower under anaerobic conditions. The photoproducts 4-(1-[4-chlorophenyl]-1,2-dihydroxy)ethylphenoxyisobutyrate and 4-chloroperbenzoic acid induced hemolysis in the dark; however, this effect was quantitatively less important than photohemolysis by fenofibrate. On the other hand, fenofibrate photosensitized peroxidation of linoleic acid, monitored by the UV detection of dienic hydroperoxides. Based on the inhibition of this process upon addition of butylated hydroxyanisole, a radical chain (type I) mechanism appears to operate. In summary, fenofibrate is phototoxic in vitro. This behavior can be explained through the involvement of free radicals, singlet oxygen and stable photoproducts.