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The brominated flame retardant 3,3',5,5'-tetrabromobisphenol A (TBBPA) may accumulate in the environment, including surface waters, and degrade there to potentially toxic products. We have previously shown that singlet oxygen (1O2), produced by irradiation of rose bengal with visible light, oxidizes Triton X-100-solubilized TBBPA to yield the 2,6-dibromo-p-benzosemiquinone anion radical while consuming oxygen (Environ. Sci. Technol.42, 166, 2008). Here, we report that a similar 1O2-induced oxidation can be initiated in aqueous solutions by the irradiation of TBBPA dissolved in a humic acid (HA) solution. HA is a known weak 1O2 photosensitizer and we indeed detected the infrared 1O2 phosphorescence from HA preparations in D2O. When an aqueous preparation of HA was irradiated (lambda > 400 nm) in the presence of TBBPA, oxygen was consumed, and the 2,6-dibromo-p-benzosemiquinone anion radical was generated and detected using electron paramagnetic resonance. Radical formation and oxygen consumption were inhibited by sodium azide, a singlet oxygen quencher. Our results suggest that solar radiation, in the presence of HA, may play an important role in the photodegradation of TBBPA in the aquatic environment.

Wide use of flame retardants can pose an environmental hazard, and it is of interest to investigate how they may degrade. We report here that 3,3',5,5'-tetrabromobisphenol A (TBBPA) is subject to photosensitized oxidation involving singlet molecular oxygen ((1)O2). By using visible light and rose bengal or methylene blue as 102 photosensitizers, we have found that TBBPA is a 102 quencher. The quenching rate constant, k(q), depends on TBBPA ionization (pK = 7.4). In acetonitrile, where TBBPA is undissociated, the kq value is 6.1 x 10(5) M(-1) s(-1) for a TBBPA monomer and decreases to 2.9 x 10(4) M(-1) s(-1) for TBBPA dimers and/or aggregates. TBBPA dissociates in aqueous solutions, and its kq value is 1.44 x 10(9) M(-1) s(-1) in alkaline solution, decreasing to 3.9 x 10(8) M(-1) s(-1) at pH 7.2. The strong 102 quenching by TBBPA anion initiates an efficient oxidation of TBBPA, which results in oxygen consumption in aqueous micellar (e.g., Triton X-100) solutions containing photosensitizer. This oxygen consumption is mediated by transient radical species, which we detected by using EPR spectroscopy. We observed two major radicals and one minor radical generated from TBBPA by reaction with 102 at pH 10. One was identified as the 2,6-dibromo-p-benzosemiquinone radical (a2H = 2.36 G, g = 2.0056). A second radical (aH = 2.10 G, g = 2.0055) could not be identified butwas probably a 2,6-dibromo-p-benzosemiquinone radical containing an EPR-silent substituent at the 3-position. Spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DPMO) showed that other minor radicals (hydroxyl, carbon-centered) are also generated during the reaction of TBBPA with (1)O2. The photosensitized production of radicals and oxygen consumption were completely inhibited by the azide anion, an efficient physical (1)O2 quencher. Because TBBPA is a stable compound that at neutral pH does not absorb much of the atmosphere-filtered solar radiation, its photosensitized oxidation by (1)O2 may be the key reaction initiating or mediating TBBPA degradation in the natural environment.

The process of sight (photostasis) produces, as a by-product, a chromophore called 2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E, 5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E, 3E, 5E-hexatrienyl]-pyridinium (A2E), whose function in the eye has not been defined as yet. In youth and adulthood, A2E is removed from human retinal pigment epithelial (h-RPE) cells as it is made, and so it is present in very low concentrations, but with advanced age, it accumulates to concentrations reaching 20 microM. In the present study we have used photophysical techniques and in vitro cellular measurements to explore the role of A2E in h-RPE cells. We have found that A2E has both pro- and antioxidant properties. It generated singlet oxygen (phiso = 0.004) much less efficiently than its precursor trans-retinal (phiso = 0.24). It also quenched singlet oxygen at a rate (10(8) M(-1) s(-1)) equivalent to two other endogenous quenchers of reactive oxygen species in the eye: alpha-tocopherol (vitamin E) and ascorbic acid (vitamin C). The endogenous singlet oxygen quencher lutein, whose quenching rate is two orders of magnitude greater than that of A2E, completely prevented light damage in vitro, suggesting that singlet oxygen does indeed play a role in light-induced damage to aged human retinas. We have used multiphoton confocal microscopy and the comet assay to measure the toxic, phototoxic and protective capacity of A2E in h-RPE cells. At 1-5 microM, A2E protected these cells from UV-induced breaks in DNA; at 20 microM, A2E no longer exerted this protective effect. These results imply that the role of A2E is not simple and may change over the course of a lifetime. A2E itself may play a protective role in the young eye but a toxic role in older eyes.

The dried root or rhizome of Goldenseal (Hydrastis canadensis L.) contains several alkaloids including berberine, hydrastine, palmatine and lesser amounts of canadine and hydrastinine. Preparations derived from Goldenseal have been used to treat skin and eye ailments. Berberine, the major alkaloid in Goldenseal root powder, has been used in eye drops to treat trachoma, a disease characterized by keratoconjunctivitis. Berberine and palmatine are also present in extracts from Berberis amurensis Ruprecht (Berberidaceae) which are used to treat ocular disorders. We have previously shown that Goldenseal alkaloids are phototoxic to keratinocytes (Chem Res Toxicol. 14, 1529, 2001; ibid 19, 739, 2006) and now report their effect on human lens and retinal pigment epithelial cells. Human lens epithelial cells (HLE-B3) were severely damaged when incubated with berberine (25 microM) and exposed to UVA (5 J cm(-2)). Under the same conditions, palmatine was less phototoxic and hydrastine, canadine and hydrastinine were inactive. Moderate protection against berberine phototoxicity was afforded by the antioxidants ascorbate (2 mM) and N-acetylcysteine (5 mM). When exposed to UVA (5 J cm(-2)) both berberine (10 microM) and palmatine (10 microM) caused mild DNA damage as determined by the alkaline comet assay which measures single strand breaks. Berberine and palmatine are the only Goldenseal alkaloids with appreciable absorption above 400 nm. Because light at wavelengths below 400 nm is cut off by the anterior portion of the adult human eye only berberine and palmatine were tested for phototoxicity to human retinal pigment epithelial (hRPE) cells. Although berberine did damage hRPE cells when irradiated with visible light (lambda > 400 nm) approximately 10 times higher concentrations were required to produce the same amount of damage as seen in lens cells. Palmatine was not phototoxic to hRPE cells. Neither berberine nor palmatine photodamaged DNA in hRPE. Infusions of Goldenseal are estimated to contain approximately 1 mM berberine, while in tinctures the alkaloid concentration may be more than 10 times higher. Our findings show that eyewashes and lotions derived from Goldenseal or containing berberine must be used with caution when the eyes are exposed to bright sunlight but that oral preparations are not likely to cause ocular phototoxicity.

Smith-Lemli-Opitz syndrome (SLOS) is a severe developmental disorder caused by mutations in the DHCR7 gene coding for 7-dehydrocholesterol (7-DHC) reductase, the enzyme involved in the last step of cholesterol biosynthesis. SLOS homozygotes exhibit marked deficiency of cholesterol in plasma and tissues with concomitant increase in 7-DHC. Ultraviolet A (UVA) photosensitivity has been recognized as part of SLOS with maximal response occurring at 350 nm. 7-DHC itself has no UVA absorption and so cannot be the direct cause of SLOS photosensitivity. However, cholesta-5,7,9(11)-trien-3beta-ol (9-DDHC), a metabolite of 7-DHC, has been detected in plasma from SLOS patients. Because 9-DDHC has strong absorption in the UVA range (approximately 15,000 @ 324 nm), we have examined its photobiology to determine whether it could be involved in SLOS photosensitivity. High levels of 7-DHC (0.65 mg/100 g wet weight) and measurable amounts of 9-DDHC (0.042 mg/100 g wet weight) were found in skin lipids extracted from CD-1 mice treated with AY9944 (trans-1,4-bis(2-chlorobenzylaminomethyl)cyclohexane dihydrochloride), an inhibitor of 7-DHC reductase. Human HaCaT keratinocytes treated with 9-DDHC (10 microM) and then immediately exposed to UVA (15 J/cm2) exhibited an 88% decrease in viability (compared to dark controls). No damage was observed in cells exposed to 7-DHC/UVA or UVA alone. However, HaCaT keratinocytes treated with 7-DHC (5 microM) for 15 h and then exposed to UVA (30 J/cm2) were damaged. 9-DDHC was detected in keratinocytes incubated with 7-DHC. Reactive oxygen species were detected in 9-DDHC/UVA-exposed cells using the fluorescent probe 5-(and 6-)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester. Singlet oxygen was generated when 9-DDHC was UVA irradiated in CCl4. UVA irradiation of 9-DDHC in acetonitrile generated superoxide and carbon-centered and alkoxyl radicals which were trapped by 5,5-dimethyl-1-pyrroline N-oxide. These findings suggest that reactive oxygen species generated by 9-DDHC may play a role in the UVA skin photosensitivity of SLOS patients. Furthermore, several statin drugs inhibit 7-DHC reductase, in addition to hydroxymethylglutaryl-CoenzymeA reductase, so that 9-DDHC may also be responsible for statin-derived photosensitivity, dermatoses, and cataract formation. Finally, we have previously detected 9-DDHC in skin lipids from normal subjects, so this sterol may also be the skin chromophore responsible for skin photoaging and UV-induced skin cancer.

Recently an article about the new energy-saving compact fluorescent light (CFL) bulbs appeared in Parade magazine [Rosenfeld, I. (2008) Parade Feb 3, 22]. Under the heading "Bright Lights, Bad Headache?" the writer states that "new research suggests some dangers" involving these lights because they are fluorescent and "can aggravate skin rashes in people with lups, eczema, dermatitis or porphyria." We measured the emission spectrum of a 14 W compact fluorescent bulb (with the same luminous flux as a 60 W incandescent bulb) and compared it to 60 W soft white incandescent and cool white fluorescent (CWF) bulbs. Our results clearly show that the spectral irradiance of the compact fluorescent bulb is similar to that of the CWF bulb; both exhibit sharp Hg emission lines at 365 nm (very weak), 404 nm (weak), 435 nm (moderate) and 543 nm (strong). In contrast, the emission of the incandescent bulb begins at 375 nm and then increases monotonically to above 750 nm. From their respective absorption spectra we calculated the potential photosensitization indices of protoporphyrin IX (PPIX; a prototypic porphyria skin photosensitizer) and riboflavin (a putative lens photosensitizer) vs 14 W compact fluorescent, CWF and 60 W incandescent bulbs. A higher photosensitization index would indicate a greater chance that the light/photosensitizer combination would cause photosensitization of the skin or eyes. We found that for PPIX and riboflavin the photosensitization index of the compact fluorescent bulb is less than half that of the 60 W incandescent bulb. These results suggest that substitution of a compact fluorescent bulb for an incandescent bulb of the same luminous flux should not increase the phototoxicity of skin porphyrins or lens riboflavin.

Magnetite (Fe3O4) encapsulated in polystyrene microspheres dramatically decreased the time for 50% hemolysis (t1/2) of human erythrocytes irradiated (lambda > 300 nm) in the presence of ketoprofen (0.1 mM). The magnetic microspheres were present at a very low concentration (0.002%) such that on average there was only one particle per four erythrocytes. No such effect was seen when nonmagnetic microspheres were employed or when the equivalent concentration of soluble iron (FeCl3) was present. A decrease in t1/2 was also observed when the magnetic microspheres were added after UVA/ketoprofen treatment or when they were present during hemolysis initiated by thermolysis of 2,2'-azobis(2-amidinopropane). These findings may be attributed to an increase in the membrane concentration of lipid radicals as a result of a magnetic field-induced increase in radicals escaping from triplet radical pairs.

Ultraviolet irradiation (lambda > 300 nm) of the nonsteroidal anti-inflammatory agent ketoprofen (KP, 3-benzoyl-alpha-methylbenzoacetic acid) in aqueous solution, pH 7.4, results in heterolytic decarboxylation of the drug to give 3-ethylbenzophenone (EtBP). Ketoprofen caused the photohemolysis of human erythrocytes probably as a result of lipid peroxidation. Application of a static magnetic field (250-1500 G) during UV (> 300 nm) irradiation of KP and erythrocytes significantly decreased the time required for photohemolysis. This observation suggests that KP-induced photohemolysis involves the initial generation of a triplet radical pair derived from the reaction of triplet state KP (or 3-EtBP) with erythrocyte component(s) probably lipids. The magnetic field increases the concentration and/or lifetime of free radicals that escape from the radical pair so that the critical radical concentration needed to initiate membrane damage and cause cell lysis is reached sooner. Spin-trapping studies with 2,6-dibromo-1-nitrosobenzene-4-sulfonate confirmed that the application of an external static magnetic field increased the concentration of radicals released during the photolysis of either KP or 3-EtBP dissolved in organized media such as sodium dodecylsulfate micelles.

The fluoroquinolone antibacterial agents possess photosensitizing 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 (phi delta) by detection of the singlet oxygen (1O2) luminescence at 1270 nm for several fluoroquinolones, naphthyridines and other structurally related compounds. All the fluoroquinolones examined have low phi delta 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 10(6) M-1 s-1, except for lomefloxacin whose rate constant was 1.8 x 10(7) M-1 s-1. The phi delta values were significantly decreased in a solvent of lower polarity such as methanol (0.007 300 nm) of superoxide by these antibacterials in dimethylsulfoxide using electron paramagnetic resonance and the spin trap 5,5-dimethyl-1-pyrroline 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.

Sunlight has been implicated in the high incidence of skin cancer found in patients receiving 6-mercaptopurine (PSH) in the form of its pro-drug azathioprine. In this study we have used EPR spectroscopy in conjunction with the spin-trapping technique to determine whether PSH and its metabolic or photochemical oxidation products generate highly reactive free radicals upon UV irradiation. When an aqueous anaerobic solution (pH 5 or 9) of PSH (pKa = 7.7) and either 2-methyl-2-nitrosopropane (MNP) or nitromethane (NM) were irradiated (lambda > 300 nm) with a Xe arc lamp, the corresponding purine-6-thiyl (PS.) radical adduct and the reduced form of the spin trap (MNP/H. or CH3NO2.-) were observed. However, no radical adducts were detected when PSH and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were irradiated (lambda = 320 nm) in oxygen-free buffer. These findings suggest that PSH does not photoionize but that instead MNP and NM are reduced by direct electron transfer from excited state PSH, 1.3(PSH)*. In aerobic solution, oxygen can act as an electron acceptor and the O2.- and PS. radicals are formed and trapped by DMPO. 6-Mercaptopurine did photoionize when irradiated with a Nd:YAG laser at 355 nm as evidenced by the appearance of the DMPO/H.(eq- + H+) adduct, which decreased in intensity in the presence of N2O. 1.3(6-Mercaptopurine)* oxidized ascorbate, formate and reduced glutathione to the corresponding ascorbyl, CO2.- or glutathiyl radicals. The photochemical behavior of 6-thioxanthine and 6-thiouric acid was similar to PSH. However, the excited states of these metabolic oxidation products exhibited stronger reducing properties than 1.3(PSH)*.(ABSTRACT TRUNCATED AT 250 WORDS)

The photochemistry of 5,5-dimethyl-l-pyrroline N-oxide (DMPO) has been studied in benzene, cyclohexane and aqueous buffer solutions (pH 7.4) by means of electron paramagnetic resonance (EPR) and the spin trapping technique. Ultraviolet irradiation of DMPO in aqueous buffer with unfiltered UV radiation from a Xe arc lamp results in photoionization of the spin trap and the generation of the DMPO cation radical, DMPO+. The aqueous electron, eaq−, was trapped by DMPO and detected as the DMPO/H adduct. The DMPO+- reacted with the water to yield the DMPO/OH adduct. Ultraviolet irradiation of DMPO in nitrogen-saturated benzene gave an unidentified carbon-centered DMPO adduct that was replaced by hydroperoxyl and alkoxyl adducts of DMPO when oxygen was present. Experiments employing 17O2 gas indicated that the oxygen in the DMPO alkoxyl adduct was derived from molecular oxygen. However, UV irradiation of DMPO in cyclohexane yielded the cyclohexyl and cyclohexyloxyl adducts of DMPO in nitrogen-saturated and air-saturated solutions, respectively. These observations suggest that in aprotic solvents UV irradiation of DMPO generates a carbon-centered radical (R−), derived from the trap itself, which in benzene reacts with oxygen to yield an alkoxyl radical (RO−), possibly via a peroxyl radical (ROO−) intermediate. In cyclohexane R− abstracts a hydrogen atom from the solvent to yield the cyclohexyl radical in the absence of oxygen and the cyclohexyloxyl radical in the presence of oxygen. These findings indicate that when DMPO is used as a spin trap in studies employing short-wavelength UV radiation (λ < 300 nm) the photochemistry of DMPO cannot be ignored.

— The photochemistry, photophysics, and photosensitization (Type I and II) of indomethacin (IN) (N-[p-chlorobenzoyl]-5-methoxy-2-methylindole-3-acetic acid) has been studied in a variety of solvents using NMR, high performance liquid chromatography-mass spectroscopy, transient spectroscopy, electron paramagnetic resonance in conjunction with the spin trapping technique, and the direct detection of singlet molecular oxygen (l O2) luminescence. Photodecomposition of IN (λex > 330 nm) in degassed or air-saturated benzene proceeds rapidly to yield a major (2; N-[p-chlorobenzoyl]-5-methoxy-2-methyl-3-methylene-indoline) and a minor (3; N-[p-chlorobenzoyl]-5-methoxy-2, 3-dimethyl-indole) decarboxylated product and a minor indoline (5; 1-en-5-methoxy-2-methyl-3-methylene-in-doline), which is formed by loss of the p-chlorobenzoyl moiety. In air-saturated solvents two minor oxidized products 4 (N-[p-chlorobenzoyl]-5-methoxy-2-methylindol-3-aldehyde) and 6 (5-methoxy-2-methyl-indole-3-aldehyde) are also formed. When photolysis was carried out in 18O2-saturated benzene, the oxidized products 4 and 6 contained 18O, indicating that oxidation was mediated by dissolved oxygen in the solvent. In more polar solvents such as acetonitrile or ethanol, photodecomposition is extremely slow and inefficient. Phosphorescence of IN at 77 K shows strong solvent dependence and its emission is greatly reduced as polarity of solvent is increased. Flash excitation of IN in degassed ethanol or acetonitrile produces no transients. A weak transient is observed at 375 nm in degassed benzene, which is not quenched by oxygen. Irradiation of IN (λex > 325 nm) in N2-gassed C6H6 in the presence of 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) results in the trapping of two carbon-centered radicals by DMPO. One adduct was identified as DMPO/.COC6H4-p-CI, while the other was probably derived from a radical formed during IN decarboxylation. In air-saturated benzene, (hydro) peroxyl and alkoxyl radical adducts of DMPO are observed. A very weak luminescence signal from 1O2 at 1268 nm is observed initially upon irradiation (λex= 325 nm) of IN in air-saturated benzene or chloroform. The intensity of this 1O2 signal increases as irradiation is continued suggesting that the enhancement in 1O2 yield is due to photoproduct(s). Accordingly, when 2 and 3 were tested directly, 2 was found to be a much better sensitizer of 1O2 than IN. In air-saturated ethanol or acetonitrile no IN 1O2 luminescence is detected even on continuous irradiation. The inability of IN to cause phototoxicity may be related to its photo stability in polar solvents, coupled with the low yield of active oxygen species (1O2, O2−-) upon UV irradiation.

The photochemistry of benzanthrone (7H-benz[de]-anthracene-7-one) has been studied using electron paramagnetic resonance (EPR) in conjunction with the spin trapping technique and the direct detection of singlet molecular oxygen luminescence. Irradiation (lambda ex = 394 nm) of benzanthrone (BA) in aerated ethanol, dimethylsulfoxide or benzene resulted in the generation of superoxide (O2-.) which was trapped by 5,5-dimethyl-1-pyrroline-N-oxide. The ethoxy radical was also detected in ethanol. Photolysis of BA in deaerated basic ethanol led to the formation of BA anion radical, BA-., which was detected directly by ESR. This radical anion decayed back to BA with a unimolecular rate constant of 1.5 x 10(-3) s-1. The 1O2 quantum yields (lambda ex greater than 345 nm) for BA in ethanol, 90% ethanol and basic ethanol (0.1N NaOH) were 0.89, 0.88 and 0.28 respectively relative to Rose Bengal. The lower yield of 1O2 in basic ethanol may be attributable to the reaction of oxygen with BA-. (which is generated in higher yield at alkaline pH) to give O2-.. These findings suggest that on exposure to light BA can generate active oxygen species which may be responsible for the photocontact dermatitis caused by BA in industrial workers exposed to this chemical.

Reactions of tryptophan residues in proteins with radical and other oxidative species frequently lead to cleavage of the indole ring, modifying tryptophan residues into N-formylkynurenine (NFK) and kynurenine. Tryptophan modification has been detected in physiologically important proteins and has been associated with a number of human disease conditions. Modified residues have been identified through various combinations of proteomic analyses, tryptic digestion, HPLC, and mass spectrometry. Here we present a novel, immunological approach using polyclonal antiserum for detection of NFK. The specificity of our antiserum is confirmed using photooxidation and radical-mediated oxidation of proteins with and without tryptophan residues. The sensitivity of our antiserum is validated through detection of NFK in photooxidized myoglobin (two tryptophan residues) and in carbonate radical-oxidized human SOD1, which contains a single tryptophan residue. Analysis of photooxidized milk also shows that our antiserum can detect NFK residues in a mixture of proteins. Results from mass spectrometric analysis of photooxidized myoglobin samples corroborate the immunological data, detecting an increase in NFK content as the extent of photooxidation increases.

The dried root or rhizome of Goldenseal (Hydrastis canadensis L.) contains several alkaloids including berberine, hydrastine, palmatine and lesser amounts of canadine and hydrastinine. Preparations derived from Goldenseal have been used to treat skin and eye ailments. Berberine, the major alkaloid in Goldenseal root powder, has been used in eye drops to treat trachoma, a disease characterized by keratoconjunctivitis. Berberine and palmatine are also present in extracts from Berberis amurensis Ruprecht (Berberidaceae) which are used to treat ocular disorders. We have previously shown that Goldenseal alkaloids are phototoxic to keratinocytes (Chem Res Toxicol. 14, 1529, 2001; ibid 19, 739, 2006) and now report their effect on human lens and retinal pigment epithelial cells. Human lens epithelial cells (HLE-B3) were severely damaged when incubated with berberine (25 microM) and exposed to UVA (5 J cm(-2)). Under the same conditions, palmatine was less phototoxic and hydrastine, canadine and hydrastinine were inactive. Moderate protection against berberine phototoxicity was afforded by the antioxidants ascorbate (2 mM) and N-acetylcysteine (5 mM). When exposed to UVA (5 J cm(-2)) both berberine (10 microM) and palmatine (10 microM) caused mild DNA damage as determined by the alkaline comet assay which measures single strand breaks. Berberine and palmatine are the only Goldenseal alkaloids with appreciable absorption above 400 nm. Because light at wavelengths below 400 nm is cut off by the anterior portion of the adult human eye only berberine and palmatine were tested for phototoxicity to human retinal pigment epithelial (hRPE) cells. Although berberine did damage hRPE cells when irradiated with visible light (lambda400 nm) approximately 10 times higher concentrations were required to produce the same amount of damage as seen in lens cells. Palmatine was not phototoxic to hRPE cells. Neither berberine nor palmatine photodamaged DNA in hRPE. Infusions of Goldenseal are estimated to contain approximately 1 mM berberine, while in tinctures the alkaloid concentration may be more than 10 times higher. Our findings show that eyewashes and lotions derived from Goldenseal or containing berberine must be used with caution when the eyes are exposed to bright sunlight but that oral preparations are not likely to cause ocular phototoxicity.

Goldenseal is an herb that is widely used in dietary supplements, eye washes, and skin lotions. The presence of Goldenseal root powder in dietary supplements and the topical application of Goldenseal preparations raise the possibility that an adverse phototoxic reaction may result from an interaction between its constituent alkaloids and light in exposed tissues. We have previously shown that berberine, the major alkaloid in Goldenseal powder, in combination with UVA causes DNA damage and cell death in HaCaT keratinocytes [(2001) Chem. Res. Toxicol. 14, 1529]. We have studied the photochemical and photobiological properties of four minor alkaloids found in Goldenseal, namely, hydrastine, palmatine, canadine, and hydrastinine. UVA radiation of palmatine in aqueous solutions generated no (1)O(2), but in CH(2)Cl(2), copious amounts of (1)O(2) were detected (Phi = 0.2). Palmatine also photogenerated oxygen-centered radicals, (*)OH and O(2)(*)(-) in aerated aqueous buffer and acetonitrile, respectively, as detected by the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). In nitrogen-sparged acetonitrile containing DMPO, we observed the neutral palmatine radical formed by one-electron reduction. UVA irradiation (4 J/cm(2)) of HaCaT keratinocytes in the presence of palmatine (50 microM) resulted in a 50% decrease in cell viability but no DNA damage as measured by the comet assay. UVA irradiation of hydrastine, hydrastinine, or canadine (50 microM) did not cause DNA damage or cell death in keratinocytes. Although palmatine is photoactive, it is present in such small amounts in Goldenseal root powder that the phototoxicity of the herb is most likely due to berberine, the major constituent alkaloid.

Earlier studies have shown that on exposure to UVA, hydroperoxynaphthalene diimide (IA) generates hydroxyl radicals, induces DNA strand scission, and kills cells. Here we employed electron paramagnetic resonance (EPR) and spin trapping to investigate the free radical photochemistry of IA and that of related naphthalene diimides, which are devoid of the hydroperoxyl moiety (N,N'-bis[2-methyl]-1,4,5,8-naphthaldiimide [IB], N,N'-bis[2-thiomethyl-2-methoxyethyl]-1,4,5,8-naphthaldiimide [IC]) and therefore are unable to generate hydroxyl radicals. It is shown that on UV irradiation (300 nm) in air-free methanol or ethanol solutions all these naphthalene diimides undergo one-electron reduction to corresponding anion radicals, positively identified by EPR. With EPR and a spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), we found that the photogeneration of the naphthalene diimide radicals is concomitant with the formation of radicals from the solvents, presumably through electron/hydrogen atom abstraction by photoactivated diimides. Irradiation of IA, IB or IC in the presence of oxygen generates superoxide, which was detected as a DMPO adduct. The high photoreactivity of IB and IC supports the notion that hydroperoxide IA can induce oxidative damage via photoprocesses that are independent of *OH generation. These observations could be pertinent to the application of naphthalene diimides as selective photonucleases, PDT anticancer agents or both.

There have been several attempts to implicate reactive oxygen species in UVA-induced damage by loading cells with 2',7'-dichlorofluorescin (DCFH) and following the appearance of 2',7'-dichlorofluorescein (DCF), its highly fluorescent oxidation product. However, both DCF and DCFH have significant absorption in the 300-400 nm range so it is possible that photochemical reactions will occur in cells containing these dyes when they are irradiated with UVA. HaCaT keratinocytes loaded with DCFH were irradiated with 0, 1, 2, or 4 J/cm(2) UVA and DCF fluorescence was measured. A dose-dependent increase in DCF fluorescence was observed, with the cells exposed to 4 J/cm(2) UVA exhibiting an almost 10-fold increase over dark controls. However, there was no difference in cell viability, as measured by the MTS assay or LDH release, between the dark and the 4 J/cm(2) UVA-exposed groups. Furthermore, a large increase in DCF fluorescence was observed when a cell-free system containing DCF, DCFH, and horseradish peroxidase was UVA irradiated. As a control, keratinocytes loaded with DCFH were incubated in the dark with either exogenously added H(2)O(2) or 5-hydroxy-1,4-naphthoquinone (juglone), which redox cycles to generate superoxide (and H(2)O(2)). In both cases, the cells showed a concentration-dependent increase in DCF fluorescence and a concomitant decrease in viability. Our findings suggest that DCFH can not be used to detect the UVA-induced generation of reactive oxygen species in cells when the dye is present during exposure.

The process of sight (photostasis) produces, as a by-product, a chromophore called 2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E, 5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E, 3E, 5E-hexatrienyl]-pyridinium (A2E), whose function in the eye has not been defined as yet. In youth and adulthood, A2E is removed from human retinal pigment epithelial (h-RPE) cells as it is made, and so it is present in very low concentrations, but with advanced age, it accumulates to concentrations reaching 20 microM. In the present study we have used photophysical techniques and in vitro cellular measurements to explore the role of A2E in h-RPE cells. We have found that A2E has both pro- and antioxidant properties. It generated singlet oxygen (phiso = 0.004) much less efficiently than its precursor trans-retinal (phiso = 0.24). It also quenched singlet oxygen at a rate (10(8) M(-1) s(-1)) equivalent to two other endogenous quenchers of reactive oxygen species in the eye: alpha-tocopherol (vitamin E) and ascorbic acid (vitamin C). The endogenous singlet oxygen quencher lutein, whose quenching rate is two orders of magnitude greater than that of A2E, completely prevented light damage in vitro, suggesting that singlet oxygen does indeed play a role in light-induced damage to aged human retinas. We have used multiphoton confocal microscopy and the comet assay to measure the toxic, phototoxic and protective capacity of A2E in h-RPE cells. At 1-5 microM, A2E protected these cells from UV-induced breaks in DNA; at 20 microM, A2E no longer exerted this protective effect. These results imply that the role of A2E is not simple and may change over the course of a lifetime. A2E itself may play a protective role in the young eye but a toxic role in older eyes.

— Application of a static external magnetic field (3350 G) during UV-irradiation (>300 nm) reduced the time for 50% photohemolysis of human erythrocytes by the phototoxic drug ketoprofen (3-benzoyl-α-methylbenzoacetic acid) from 96 min to 78 min. This observation can be attributed to a magnetic field induced decrease in the rate of intersystem crossing (kISC) of the geminate triplet radical pair generated by the reduction of ketoprofen in its triplet excited state by erythrocyte membrane constituents, probably lipids. The decrease in kJSC results in an increase in the concentration and/or lifetime of free radicals that escape from the triplet radical pair. Thus the critical radical concentration needed to cause membrane damage and cell lysis is reached sooner in the presence of the magnetic field. In contrast, the photohemolysis induced by the photodynamic agent protoporphyrin IX was not affected by the magnetic field. Protoporphyrin IX photohemolysis, which is initiated by singlet oxygen, does not involve the initial generation of a triplet radical pair and so is not influenced by the magnetic field. The enhancement of ketoprofen-induced photohemolysis by an externally applied magnetic field is the first example of a magnetic field effect on a toxicological process involving free radicals.

Certain 9-anthrone derivatives are useful in treating psoriasis and are also known to be tumor promoters in mouse skin. Their therapeutic use is accompanied by side effects of severe skin inflammation, irritation, and staining. The precise biochemical mechanisms of therapeutic action, tumor promotion, and side effects are presently uncertain, although the corresponding 9-anthron-10-yl radicals have been proposed as important intermediates. In order to gain insight into the possible role of anthrone-derived radicals in mediating the biological effects of these compounds, in the present study free radicals from a number of anthrone derivatives were generated by thermolysis in nonpolar solvents. Hyperfine splitting constants (hfsc) of the radicals were determined by electron paramagnetic resonance (EPR) spectroscopy. The experimentally determined hfsc's were also compared with spin densities obtained by molecular calculations (MOPAC 6.0). The experimental and theoretical data were found to be consistent in all cases. The formation of 9-anthron-10-yl radicals appears to be a general phenomenon among 9-anthrones regardless of therapeutic or tumor-promoting effectiveness, although there is a trend toward easier radical formation for the more active compounds.

Curcumin, bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, is a natural yellow-orange dye derived from the rhizome of Curcuma longa, an East Indian plant. In order to understand the photobiology of curcumin better we have studied the spectral and photochemical properties of both curcumin and 4-(4-hydroxy-3-methoxy-phenyl)-3-buten-2-one (hC, half curcumin) in different solvents. In toluene, the absorption spectrum of curcumin contains some structure, which disappears in more polar solvents, e.g. ethanol, acetonitrile. Curcumin fluorescence is a broad band in acetonitrile (lambda max = 524 nm), ethanol (lambda max = 549 nm) or micellar solution (lambda max = 557 nm) but has some structure in toluene (lambda max = 460, 488 nm). The fluorescence quantum yield of curcumin is low in sodium dodecyl sulfate (SDS) solution (phi = 0.011) but higher in acetonitrile (phi = 0.104). Curcumin produced singlet oxygen upon irradiation (lambda > 400 nm) in toluene or acetonitrile (phi = 0.11 for 50 microM curcumin); in acetonitrile curcumin also quenched 1O2 (kq = 7 x 10(6) M-1 s-1). Singlet oxygen production was about 10 times lower in alcohols and was hardly detectable when curcumin was solubilized in a D2O micellar solution of Triton X-100. In SDS micelles containing curcumin no singlet oxygen phosphorescence could be observed. Curcumin photogenerates superoxide in toluene and ethanol, which was detected using the electron paramagnetic resonance/spin-trapping technique with 5,5-dimethyl-pyrroline-N-oxide as a trapping agent. Unidentified carbon-centered radicals were also detected.(ABSTRACT TRUNCATED AT 250 WORDS)

The UV or visible irradiation of pigments such as curcumin, anthralin, benzanthrone, 1,8-dihydroxyanthraquinone, and rose bengal- or eosine-complexes with cationic surfactants in aerated aprotic solvents, such as benzene, toluene, acetone, n-heptane, cyclohexane, in the presence of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) generates EPR spectra with hyperfine splitting constants (hfsc's) aN = 12.75 G, a beta H = 10.50 G, a gamma H = 1.26 G in toluene, 12.83 G, 10.64 G, 1.24 G in benzene, 12.75 G, 10.19 G, 1.35 G in acetone and 12.54 G, 10.46 G, 1.38 G in n-heptane and cyclohexane. These spectra are similar to those observed when DMPO reacts with 18-crown-6 ether-solubilized KO2 in the respective solvents and suggests that the photoinduced EPR spectra can be safely assigned to the DMPO/superoxide radical adduct (1). A correlation between the hfsc's of 1 and solvent parameters, the solvent acceptor number AN and the Kosower Z value, has been evaluated in terms of its usefulness for the identification of the DMPO/superoxide adduct in organic media.

— The photochemistry (Type I and II) of anthralin and its photo-oxidation product 1,8-dihydroxyanthraquinone (1,8-DHAQ) has been studied in ethanol, acetonitrile and dimethylsulfoxide using spin-trapping and direct detection of singlet oxygen (1O2) luminescence techniques. In ethanol, where it exists in its neutral form (AN), anthralin does not undergo either Type I or II reactions upon UV-irradiation. In contrast, irradiation of anthralin in acetonitrile, a solvent in which anthralin is partially converted to its corresponding mono-anion (AN), generates both superoxide and singlet oxygen. Irradiation of anthralin in dimethylsulfoxide, where the AN− form is present in substantial quantity, generates superoxide and solvent derived radicals but no detectable singlet oxygen. UV-irradiation of 1,8-DHAQ in ethanol and acetonitrile produces both superoxide and singlet oxygen in significant yields. In dimethylsulfoxide, on the other hand, only superoxide and solvent derived radicals are observed. The 1O2 quantum yield for AN− and 1,8-DHAQ in acetonitrile were determined to be 0.14 and 0.88 relative to rose bengal in the same solvent. These findings suggest that the AN photosensitization occurs via Type I and II pathways, is solvent dependent and involves AN− as well as its oxidation product 1,8-DHAQ, which is a more potent generator of both singlet oxygen and superoxide.

The Tg.AC mouse is a good predictor of carcinogenic potential when the test article is administered by dorsal painting (Tennant et al. (1995) Environ. Health Perspect. 103, 942). We have used lomefloxacin (LOME) and 8-methoxypsoralen (8-MOP) in combination with UVA to determine whether the Tg.AC transgenic mouse also responds to parenterally administered photocarcinogens. Female Tg.AC mice were given LOME (25 mg/kg intraperitoneal in normal saline) followed by UVA (25 J/cm2) 1–2 h later, five times every 2 weeks on a repetitive schedule. Other groups received LOME, UVA or vehicle alone. After 16 weeks, the mean numbers of papillomas/mouse ± SD (% responding) were: saline, 0.3 ± 0.5 (33%); UVA + saline, 1.3 ± 0.6 (100%); LOME, 1.9 ± 1.6 (86%) and LOME–UVA, 1.5 ± 1.9 (64%). Only the 100% incidence of tumors in the UVA group and the maximum tumor yields in the LOME and UVA groups are significant (P < 0.05) when compared with the control. In a second study, Tg.AC mice were administered the classical ...

Most drugs used in the treatment of malaria produce phototoxic side effects in both the skin and the eye. Cutaneous and ocular effects that may be caused by light include changes in skin pigmentation, corneal opacity, cataract formation and other visual disturbances including irreversible retinal damage (retinopathy) leading to blindness. The mechanism for these reactions in humans is unknown. We irradiated a number of antimalarial drugs (amodiaquine, chloroquine, hydroxychloroquine, mefloquine, primaquine and quinacrine) with light (lambda > 300 nm) and conducted electron paramagnetic resonance (EPR) and laser flash photolysis studies to determine the possible active intermediates produced. Each antimalarial drug produced at least one EPR adduct with the spin-trap 5,5-dimethyl-1-pyrroline N-oxide in benzene: superoxide/hydroperoxyl adducts (chloroquine, mefloquine, quinacrine, amodiaquine and quinine), carbon-centered radical adducts (all but primaquine), or a nitrogen-centered radical adduct only (primaquine). In ethanol all drugs except primaquine produced some superoxide/hydroperoxyl adduct, with quinine, quinacrine, and hydroxychloroquine also producing the ethoxyl adduct. As detected with flash photolysis and steady-state techniques, mefloquine, quinine, amodiquine and a photoproduct of quinacrine produced singlet oxygen ([symbol: see text]delta = 0.38; [symbol: see text]delta = 0.36; [symbol: see text]delta = 0.011; [symbol: see text]delta = 0.013 in D2O, pD7), but only primaquine quenched singlet oxygen efficiently (2.6 x 10(8) M-1 s-1 in D2O, pD7). Because malaria is a disease most prevalent in regions of high light intensity, protective measures (clothing, sunblock, sunglasses or eye wraps) should be recommended when administering antimalarial drugs.

— The effect of three different fluorescent lighting systems on the growth, reproduction, and lifespan of the C3H/HeN(MTV) mouse has been investigated. The mice were born, weaned, and paired under luminaires containing daylight-simulating (DS; 425 μW/cm2), cool-white (CW; 425μW/cm2) or pink (PK; 234 μW/cm2) fluorescent lights. At the end of the study (19 months), 97% of the females in the DS and CW groups and 100% of females in the PK group had developed mammary tumors. The median times for mammary tumor development were 51 weeks (DS), 47 weeks (CW) and 42 weeks (PK) The median age of the dam at first litter and the median time between the first and second litters were not significantly different for the three lighting conditions. However, there was some evidence that the first litter was significantly delayed in both the CW and PK groups compared to the DS group. There were no significant differences in the total number of litters/dam, the total number of pups/dam, the neonatal mortality of the pups (at birth or weaning), the weights of the pups at weaning or the sex ratios among the three lighting groups. The growth of the male mice was the same under all three lighting conditions. The observed differences in tumor latency and reproduction, between female mice exposed to the DS and CW fluorescent lights, may be related to the different spectral energy distributions of these lighting systems since their irradiances were the same.

— The photodecomposition of sulfanilamide, 4-aminobenzoic acid and other related analogs has been studied with the aid of the spin trap 2-methyl-2-nitrosopropane. UV photolysis of an aqueous solution of sulfanilamide yielded the following radicals C˙6H4SO2NH2, C6H5SO2˙, S˙O2NH2 and SO-#˙ (or SO-2dot;). Under the same conditions 4-aminobenzoic acid gave C˙6H4COOH. In addition both sulfanilamide and 4-aminobenzoic acid, but not 4-dimethylaminobenzoic acid, generated e-aq or hydrogen atoms during UV irradiation. The C6H4SO2NH2 radical was also produced by photolysis of 4-iodobenzenesul-fonamide and 4-nitrobenzenesulfonamide. The C6H4COOH radical was generated by photolysis of 4-nitrobenzoic acid and 4-iodobenzoic acid. Finally the C6H4NO2 radical was formed during the irradiation of 4-nitroaniline, 1,4-dinitrobenzene and 4-iodonitrobenzene. The free radicals generated by sulfanilamide and 4-aminobenzoic acid may play an important role in the phototoxic and photoallergic responses elicited by these drugs in certain individuals.

— The photodecomposition of sulfanilamide, 4-aminobenzoic acid and related analogs in aqueous solution has been studied with the aid of spin traps 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and CH3NO2 as well as by direct electron spin resonance techniques. The NH2 radical was trapped by DMPO during the photolysis of aqueous solutions of sulfanilamide with a Xe arc lamp. Studies with [15N1]-sulfanilamide indicated that the NH2 radical was generated by homolytic fission of the sulfur-nitrogen bond. Under the same conditions DMPO trapped the H and SO−3 radicals during photolysis of sulfanic acid. Direct photolysis of sulfanilamide, sulfanilic acid and Na2SO3 in the absence of any spin trap yielded the SO−3 radical. Photolysis of 4-aminobenzoic acid at pH 7 gave the H radical which was trapped by DMPO. At low pH values OH and C6H4COOH radicals were generated during the photolysis of 4-aminobenzoic acid. No e−aq were trapped by CH3NO2 when acid (pH 4) and neutral aqueous solutions of sulfanilamide or 4-aminobenzoic acid were photoirradiated. The mechanism of formation of known photoproducts from the free radicals generated by sulfanilamide and 4-aminobenzoic acid during irradiation are discussed. The free radicals generated by these agents may play an important role in their phototoxic and photoallergic effects.

Ultraviolet A radiation from sunlight is a major human health concern, as it is not absorbed by the ozone layer and can deeply penetrate into the skin causing skin damage. To study the molecular mechanism involved in the ultraviolet A effect, human HaCaT keratinocytes were exposed to ultraviolet A at doses of 10 J per cm2 and 30 J per cm2. Ultraviolet A irradiation caused dose- and time-dependent apoptotic cell death, as evidenced by DNA fragmentation, flow cytometry, and the activation of caspase-3. To study the genes altered by ultraviolet A at an apoptosis-inducing dose (30 J per cm2), cells were harvested immediately after ultraviolet A treatment (0 h), and 6 h and 24 h after ultraviolet A exposure. Total RNA was extracted for microarray and real-time RT-PCR analysis, and cellular proteins were extracted for western blot analysis. Of the selected critical genes/proteins, the induction of c-Jun, c-myc, and p33ING1, and the repression of epidermal growth factor receptor, inhibitor of apoptosis protein, and survivin pathways, could be involved in ultraviolet-A-induced apoptosis. On the other hand, the late induction of cyclin D1 and cyclin-dependent kinase 4 was indicative of possible cell cycle recovery in surviving cells. Real-time RT-PCR analysis confirmed these results and a majority of the protein levels paralleled their corresponding RNA levels. In addition, ultraviolet A treatment altered the expression of genes involved in signal transduction, RNA processing, structural proteins, and metabolism in a time-dependent manner. This initial microarray analysis could advance our understanding of cellular responses to ultraviolet A exposure, and provide a platform from which to further study ultraviolet-A-induced apoptosis and carcinogenesis.

SummaryUltraviolet irradiation of the nitroimidazole derivatives metronidazole, misonidazole, azathioprine and 1-methyl-4-nitroimidazole in aqueous solution with various reductants produced the respective nitro radical anions, as detected by electron spin resonance spectroscopy. The most effective reductant, yielding high concentrations of the radical anions, was EDTA at pH 10. NADH, NADPH, formaldehyde glutathione and methanol were also tested but were less efficient as reductants.

— Several antibacterial halogenated salicylanilides, including 3,3′,4′,5-tetrachlorosalicylanilide (TCSA) and 3,4′,5-tribromosalicylanilide (TBSA) are known to cause photoallergy. We have carried out photochemical and spin trapping studies to determine whether free radicals may be involved in the photoallergic response. Irradiation (λ > 300 nm) of TCSA in buffered (pH 7.4) 50% ethanol resulted in the rapid loss of the 3-chloro atom, followed by the much slower release of 5- and then the 4′-chloro atoms to give 3′-chlorosalicylanilide as a stable photoproduct. Under the same conditions TBSA successively lost the 3-, 5- and 4′-bromine atoms to give salicylanilide. When TCSA or TBSA were irradiated (λ= 356 nm) in buffered (pH 7.4) 50% ethanol containing 2-methyl-2-nitrosopropane (MNP) only solvent-derived free radicals were detected. However, irradiation (λ= 356 nm) of TCSA and MNP in 0.1 N NaOH generated an ESR spectrum consisting of a broad triplet (aN= 15.6 G).This spectrum was attributed to the adduct formed by the reaction of MNP with the aryl radical generated by the loss of a chlorine atom from the sterically hindered 3-(or 4′-)-position. Under the same conditions TBSA initially generated a broad triplet (aN= 15.5 G) similar to that observed for TCSA. However, upon further irradiation a 21-line spectrum (aN= 14.4 G, a2H= 2.0 G and a2H= 0.9 G) appeared. Since the same ESR spectrum was obtained by irradiating 4′-bromosalicylanilide, the spectrum must be generated by an adduct formed by reaction of MNP with an aryl radical derived from loss of the 4′-bromine atom of TBSA. Irradiation (λ= 356 nm) of TBSA, glutathione and the spin trap 5,5-dirrrethyl-l-pyrroline-N-oxide (DMPO) yielded the glutathione thiyl adduct of DMPO. Irradiation of (λ= 356 nm) TCSA, MNP and glycylglycine resulted in the trapping of a carbon-centered radical derived from hydrogen abstraction from the backbone methylene of the C-terminal glycine residue. These studies demonstrate that photo-irradiation of TCSA or TBSA generates highly reactive aryl radicals, which can react with peptides to form secondary radicals. The in vivo reaction of secondary protein radicals with the primary aryl radicals from TCSA or TBSA would result in the covalent binding of the salicylanilides to skin proteins to give molecules with antigenic potential.

Compound 48/80, a potent liberator of histamine from mast cells, is a polymer prepared by heating 4-methoxy-N-methylphenethylamine with an equimolar amount of formaldehyde in acid solution. We have attempted to elucidate the structure of 48/80 with the aid of carbon-13 nuclear magnetic resonance (NMR). The NMR spectrum of 48/80 suggests that it is a polymer in which methylene groups link 4-methoxy-N-methylphenethylamine moieties via their 3 and 39 positions. Some of the polymer chains of 48/80 are terminated by -CH2OH groups. In accord with the findings of Read and co-workers (J. Med. Chem. 15: 320, 1972; ibid 16: 1292, 1973), the most active high molecular weight polymers appear to be hexamers; however, contrary to the suggestion of these authors, there was no evidence for the presence of tetrahydroisoquinoline moieties in 48/80. Light scattering and fluorescence probe measurements with 1-anilino-naphthalene-8-sulfonic acid indicate that at concentrations above 2 mg/ml (0.01 M based on the monomer molecular weight) compound 48/80 formed micelles. This is reflected in the NMR spectrum of 48/80 by the sharpness of the N-CH3 resonance.

— Benoxaprofen [2-(4-chlorophenyl)-α-methyl-5-benzoxazoleacetic acid] is a phototoxic non-steroidal anti-inflammatory agent. Irradiation of human erythrocytes in the presence of benoxaprofen (8 μM) and oxygen resulted in rapid cell lysis which began after 10 min and was complete within 30 min. While photohemolysis was also observed under anerobic conditions, its onset was delayed for more than 20 min and it took nearly 100 min for complete lysis to occur. Photohemolysis was also delayed by butylated hydroxyanisole but was unaffected by reduced glutathione. 1,4-diazabicyclo[2.2.2]octane, D2O. β-carotene, or superoxide dismutase. The main photoproduct of benoxaprofen, 2-(4-chlorophenyl)-5-ethylbenzoxazole, was almost as effective in causing photohemolysis as benoxaprofen itself. In the presence of UV irradiation, benoxaprofen (10 (μM) caused the degranulation of rat peritoneal mast cells and the release of histamine. The release of mast cell histamine may provide a reasonable explanation for the urticarial response to benoxaprofen and irradiation seen in human subjects.

The effect of high pressure sodium vapor lamps and daylight-simulating fluorescent lights on the growth, hematology, and behavior of Sprague-Dawley rats has been investigated. Rats weaned under the sodium vapor lamps had slightly heavier adrenals than those exposed to the daylight lamps when the two lighting systems were equalized either for total irradiance or for scotopic illuminance. However, no differences were observed in the tailflick response, hot plate response, or swimming endurance of rats housed under the two lighting conditions. No consistent differences were seen in the hemoglobin, red and white cell count, hematocrit, and mean cell volume between rats weaned under the high pressure sodium vapor lamps and daylight-simulating fluorescent lights.