Photosensitized oxidation of tetrabromobisphenol a by humic acid in aqueous solution.

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.

Immunological detection of N-formylkynurenine in oxidized proteins.

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 photosensitizing potential of compact fluorescent vs incandescent light bulbs.

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.

Photochemistry and photocytotoxicity of alkaloids from Goldenseal (Hydrastis canadensis L.) 3: effect on human lens and retinal pigment epithelial cells.

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.

Ultraviolet A sensitivity in Smith-Lemli-Opitz syndrome: Possible involvement of cholesta-5,7,9(11)-trien-3 beta-ol.

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.

Photochemistry of naphthalene diimides: EPR study of free radical formation via photoredox process.

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.

Expression profiling of human keratinocyte response to ultraviolet A: implications in apoptosis.

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.

A photochemical study of cells loaded with 2',7'-dichlorofluorescin: implications for the detection of reactive oxygen species generated during UVA irradiation.

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 role of A2E in prevention or enhancement of light damage in human retinal pigment epithelial cells.

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.