Real-time visualization of photochemically induced fluorescence of 8-halogenated quinolones: lomefloxacin, clinafloxacin and Bay3118 in live human HaCaT keratinocytes.

Halogenoquinolones are potent and widely used antimicrobials blocking microbial DNA synthesis. However, they induce adverse photoresponses through the absorption of UV light, including phototoxicity and photocarcinogenicity. The phototoxic responses may be the result of photosensitization of singlet oxygen, production of free radicals and/or other reactive species resulting from photodehalogenation. Here, we report the use of laser scanning confocal microscopy to detect and to follow the fluorescence changes of one monohalogenated and three di-halogenated quinolones in live human epidermal keratinocyte cells during in situ irradiation by confocal laser in real time. Fluorescence image analysis and co-staining with the LysoTracker probe showed that lysosomes are a preferential site of drug localization and phototransformations. As the lysosomal environment is relatively acidic, we also determined how low pH may affect the dehalogenation and concomitant fluorescence. With continued UV irradiation, fluorescence increased in the photoproducts from BAY y3118 and clinafloxacin, whereas it decreased for lomefloxacin and moxifloxacin. Our images not only help to localize these phototoxic agents in the cell, but also provide means for dynamic monitoring of their phototransformations in the cellular environment.

Quenching of singlet oxygen by pyocyanin and related phenazines.

Pseudomonas aeruginosa is a human pathogen, which causes infections of various organs, including lung, skin and eye, particularly in individuals who are immunocompromised. Pyocyanin (1-hydroxy-5-methylphenazine), a cytotoxic pigment secreted by the bacterium, is among the factors that contribute to virulence of this pathogen. We have previously shown that rose bengal and riboflavin photosensitize oxidation of pyocyanin to a product(s) with diminished reactivity and toxicity. Singlet oxygen was suggested as the major oxidant, based on the inhibitory effect of sodium azide. In the present study, we used the time resolved technique to investigate direct interaction of pyocyanin and related phenazines (1-hydroxyphenazine [1-OH-Phen], 1-methoxy-5-methylphenazine [1-MeO-PCN] and phenazine methosulfate [PMS]) with (1)O(2). The rate constants for the (1)O(2) quenching (physical + chemical) by pyocyanin and 1-OH-Phen in D(2)O buffer (pD approximately 7.2) have been determined to be 4.8 x 10(8) and 6.8 x 10(8) M(-1) s(-1), respectively. 1-MeO-PCN and PMS were markedly less efficient (1)O(2) quenchers. Among the phenazines studied only phenazine methosulfate photogenerated (1)O(2) (Phi((1)O(2)) = 0.56 in acetonitrile). Interaction of (1)O(2) with pyocyanin and other related phenazines produced by the bacteria may be important in determining the potential utility of photochemical/pharmacological approaches to eradicate P. aeruginosa from infected tissues.

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.

Photochemical reactions involved in the phototoxicity of the anticonvulsant and antidepressant drug lamotrigine (Lamictal).

Lamotrigine (LTG) [3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine], an anticonvulsant and antidepressant drug Lamictal, produces a (photo)toxic response in some patients. LTG absorbs UV light, generating singlet oxygen (1O2) with a quantum yield of 0.22 in CH2Cl2, 0.11 in MeCN and 0.01 in D2O. A small production of superoxide radical anion was also detected in acetonitrile. Thus, LTG is a moderate photosensitizer producing phototoxicity and oxidizing linoleic acid. LTG is a weak 1O2 quencher (k(q) = 3.2 x 10(5) M(-1) s(-1) in MeCN), but its photodecomposition products in dimethyl sulfoxide (DMSO) quenched 1O2 very efficiently. Upon intense UV irradiation from a xenon lamp, LTG was photobleached rapidly in DMSO and slowly in acetonitrile, alcohol and water. The rate increased significantly when laser pulses at 266 nm were employed. The photobleaching products generated 1O2 twice as strongly as LTG. Photobleaching was usually accompanied by the release of chloride anions, which increased in the presence of ascorbic acid. This suggests the formation of aryl radicals via dechlorination, a process which may be responsible for the photoallergic response observed in some patients. Our results demonstrate that LTG is a moderate generator of 1O2 prone to photodechlorination, especially in a reducing environment, which can contribute to the reported phototoxicity of LTG.

Enhanced photodynamic efficacy towards melanoma cells by encapsulation of Pc4 in silica nanoparticles.

Nanoparticles have been explored recently as an efficient means of delivering photosensitizers for cancer diagnosis and photodynamic therapy (PDT). Silicon phthalocyanine 4 (Pc4) is currently being clinically tested as a photosensitizer for PDT. Unfortunately, Pc4 aggregates in aqueous solutions, which dramatically reduces its PDT efficacy and therefore limits its clinical application. We have encapsulated Pc4 using silica nanoparticles (Pc4SNP), which not only improved the aqueous solubility, stability, and delivery of the photodynamic drug but also increased its photodynamic efficacy compared to free Pc4 molecules. Pc4SNP generated photo-induced singlet oxygen more efficiently than free Pc4 as measured by chemical probe and EPR trapping techniques. Transmission electron microscopy and dynamic light scattering measurements showed that the size of the particles is in the range of 25-30 nm. Cell viability measurements demonstrated that Pc4SNP was more phototoxic to A375 or B16-F10 melanoma cells than free Pc4. Pc4SNP photodamaged melanoma cells primarily through apoptosis. Irradiation of A375 cells in the presence of Pc4SNP resulted in a significant increase in intracellular protein-derived peroxides, suggesting a Type II (singlet oxygen) mechanism for phototoxicity. More Pc4SNP than free Pc4 was localized in the mitochondria and lysosomes. Our results show that these stable, monodispersed silica nanoparticles may be an effective new formulation for Pc4 in its preclinical and clinical studies. We expect that modifying the surface of silicon nanoparticles encapsulating the photosensitizers with antibodies specific to melanoma cells will lead to even better early diagnosis and targeted treatment of melanoma in the future.

Photocytotoxicity of the fluorescent nonsteroidal androgen receptor ligand TDPQ.

1,2,3,4-tetrahydro-2,2-dimethyl-6-(trifluoromethyl)-8-pyridono[5,6-g]quinoline (TDPQ), a selective nonsteroidal androgen receptor (AR) ligand, is a fluorescent compound. We characterized its spectral properties in comparison with the structural precursor carbostyril 151 (C151) and with its racemic structural isomer 4-ethyl-1,2,3,4-tetrahydro-6-(trifluoromethyl)-8-pyridino[5,6-g]quinoline (ETPQ). The absorption maximum in CH3CN of either TDPQ or ETPQ is 400 nm whereas that of C151 is 350 nm. The fluorescence lifetimes (tau) and quantum yields (phif) in CH3CN are typical of fluorescent dyes: TDPQ (4.2 ns, 0.8) and ETPQ (4.6 ns, 0.76). C151 showed lower tau and phif of 0.2 ns and 0.02, respectively. TDPQ can function as a fluorescent label at (sub)micromolar concentrations. We detected TDPQ fluorescence in human breast tumor cells using confocal microscopy. While the fluorescence maxima of the compounds were solvent insensitive, the phif for ETPQ decreased in aqueous solutions regardless of the presence of albumin or DNA. The phif of TDPQ was less affected. The quantum yield of singlet oxygen (1O2) photosensitization (phiso) by TDPQ and ETPQ was about 7% in CH3CN, sufficient to induce photocytotoxicity. TDPQ was photocytotoxic in AR-positive MDA-MB-453 breast cancer cells but not in AR-negative MDA-MB-231 cells. The combination of AR selectivity with photocytotoxicity makes TDPQ a promising candidate for selective targeting of AR-positive cells during photodynamic therapy.

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.

Pristine (C60) and hydroxylated [C60(OH)24] fullerene phototoxicity towards HaCaT keratinocytes: type I vs type II mechanisms.

The increasing use of fullerene nanomaterials has prompted widespread concern over their biological effects. Herein, we have studied the phototoxicity of gamma-cyclodextrin bicapped pristine C 60 [(gamma-CyD) 2/C 60] and its water-soluble derivative C 60(OH) 24 toward human keratinocytes. Our results demonstrated that irradiation of (gamma-CyD) 2/C 60 or C 60(OH) 24 in D 2O generated singlet oxygen with quantum yields of 0.76 and 0.08, respectively. Irradiation (>400 nm) of C 60(OH) 24 generated superoxide as detected by the EPR spin trapping technique; superoxide generation was enhanced by addition of the electron donor nicotinamide adenine dinucleotide (reduced) (NADH). During the irradiation of (gamma-CyD) 2/C 60, superoxide was generated only in the presence of NADH. Cell viability measurements demonstrated that (gamma-CyD) 2/C 60 was about 60 times more phototoxic to human keratinocytes than C 60(OH) 24. UVA irradiation of human keratinocytes in the presence of (gamma-CyD) 2/C 60 resulted in a significant rise in intracellular protein-derived peroxides, suggesting a type II mechanism for phototoxicity. UVA irradiation of human keratinocytes in the presence of C 60(OH) 24 produced diffuse intracellular fluorescence when the hydrogen peroxide probe Peroxyfluor-1 was present, suggesting a type I mechanism. Our results clearly show that the phototoxicity induced by (gamma-CyD) 2/C 60 is mainly mediated by singlet oxygen with a minor contribution from superoxide, while C 60(OH) 24 phototoxicity is mainly due to superoxide.

Photo-induced reactive oxygen species generation by different water-soluble fullerenes (C) and their cytotoxicity in human keratinocytes.

In this study we report the phototoxicity toward HaCaT keratinocytes that results from the photogeneration of superoxide and singlet oxygen ((1)O(2)) by four different "water-soluble" fullerene (C(60)) preparations-monomeric (gamma-CyD)(2)/C(60) (gamma-cyclodextrin bicapped C(60)) and three aggregated forms-THF/nC(60) (prepared by solvent exchange from THF solution); Son/nC(60) (prepared by sonication of a toluene/water mixture); and gamma-CyD/nC(60) (prepared by heating the [gamma-CyD](2)/C(60) aqueous solution). Our results demonstrate that all four C(60) preparations photogenerate (1)O(2) efficiently. However, the properties of C(60)-generated (1)O(2), including its availability for reactions in solution, are markedly different for the monomeric and aggregated forms. (1)O(2) produced by monomeric (gamma-CyD)(2)/C(60) is quenchable by NaN(3) and its quantum yield in D(2)O, which is only weakly dependent on oxygen concentration, is as high as C(60) in toluene. In contrast, (1)O(2) generated from aggregated C(60) is not quenchable by NaN(3), exhibits a solvent-independent short-lived lifetime (ca 2.9 micros), is highly sensitive to oxygen concentration while its phosphorescence is redshifted. All these features indicate that (1)O(2) is sequestered inside the C(60) aggregates, which may explain why these preparations were not phototoxic toward HaCaT cells. Electron paramagnetic resonance studies demonstrated the generation of the C(60) anion radical (C(60)) when (gamma-CyD)(2)/C(60) was irradiated (lambda > 300 nm) in the presence of a reducing agent (NADH); spin trapping experiments (lambda > 400 nm) with 5,5-dimethyl-1-pyrroline N-oxide clearly showed the generation of superoxide resulting from the reaction of C(60) with oxygen. In vitro tests with HaCaT keratinocytes provided evidence that (gamma-CyD)(2)/C(60) phototoxicity is mainly mediated by (1)O(2) (Type II mechanism) with only a minor contribution from free radicals (Type I mechanism).

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.