Piotr Bilski

Symposium-in-Print Vitamin B6 (Pyridoxine) and Its Derivatives Are Efficient Singlet Oxygen Quenchers and Potential Fungal Antioxidants

Abstract Vitamin B6 (pyridoxine, 1) and its derivatives: pyridoxal (2), pyridoxal 5-phosphate (3) and pyridoxamine (4) are important natural compounds involved in numerous biological functions. Pyridoxine appears to play a role in the resistance of the filamentous fungus Cercospora nicotianae to its own abundantly produced strong photosensitizer of singlet molecular oxygen (1O2), cercosporin. We measured the rate constants (kq) for the quenching of 1O2 phosphorescence by 1–4 in D2O. The respective total (physical and chemical quenching) kq values are: 5.5 × 107 M−1 s−1 for 1; 7.5 × 107 M−1 s−1 for 2, 6.2 ×107 M−1 s−1 for 3 and 7.5 × 107 M−1 s−1 for 4, all measured at pD 6.2. The quenching efficacy increased up to five times in alkaline solutions and decreased ∼10 times in ethanol. Significant contribution to total quenching by chemical reaction(s) is suggested by the degradation of all the vitamin derivatives by 1O2, which was observed as declining absorption of the pyridoxine moiety upon aerobic irradiation of RB used to photosensitize 1O2. This photodegradation was completely stopped by azide, a known physical quencher of 1O2. The pyridoxine moiety can also function as a redox quencher for excited cercosporin by forming the cercosporin radical anion, as observed by electron paramagnetic resonance. All B6 vitamers fluoresce upon UV excitation. Compounds 1 and 4 emit fluorescence at 400 nm, compound 2 at 450 nm and compound 3 at 550 nm. The fluorescence intensity of 3 increased ∼10 times in organic solvents such as ethanol and 1,2-propanediol compared to aqueous solutions, suggesting that fluorescence may be used to image the distribution of 1–4 in Cercospora to understand better the interactions of pyridoxine and 1O2 in the living fungus.

Vitamin B6 (pyridoxine) and its derivatives are efficient singlet oxygen quenchers and potential fungal antioxidants.

Vitamin B6 (pyridoxine, 1) and its derivatives: pyridoxal (2), pyridoxal 5‐phosphate (3) and pyridoxamine (4) are important natural compounds involved in numerous biological functions. Pyridoxine appears to play a role in the resistance of the filamentous fungus Cercospora nicotianae to its own abundantly produced strong photosensitizer of singlet molecular oxygen (1O2), cercosporin. We measured the rate constants (kq) for the quenching of 1O2 phosphorescence by 1–4 in D2O. The respective total (physical and chemical quenching) kq values are: 5.5 × 107M−1 s−1 for 1; 7.5 × 107M−1 s−1 for 2, 6.2 ×107M−1 s−1 for 3 and 7.5 × 107M−1 s−1 for 4, all measured at pD 6.2. The quenching efficacy increased up to five times in alkaline solutions and decreased ∼10 times in ethanol. Significant contribution to total quenching by chemical reaction(s) is suggested by the degradation of all the vitamin derivatives by 1O2, which was observed as declining absorption of the pyridoxine moiety upon aerobic irradiation of RB used to photosensitize 1O2. This photodegradation was completely stopped by azide, a known physical quencher of 1O2. The pyridoxine moiety can also function as a redox quencher for excited cercosporin by forming the cercosporin radical anion, as observed by electron paramagnetic resonance. All B6 vitamers fluoresce upon UV excitation. Compounds 1 and 4 emit fluorescence at 400 nm, compound 2 at 450 nm and compound 3 at 550 nm. The fluorescence intensity of 3 increased ∼10 times in organic solvents such as ethanol and 1,2‐propanediol compared to aqueous solutions, suggesting that fluorescence may be used to image the distribution of 1–4 in Cercospora to understand better the interactions of pyridoxine and 1O2 in the living fungus.

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 CURCUMIN

Curcumin, bis(4‐hydroxy‐3‐methoxyphenyl)‐l,6‐diene‐3,5‐dione, is a yellow‐orange dye derived from the rhizome of the plant Curcuma longa. Curcumin has demonstrated phototoxicity to several species of bacteria under aerobic conditions (Dahl, T. A., et al., 1989, Arch. Microbiol. 151 183), denoting photodynamic inactivation. We have now found that curcumin is also phototoxic to mammalian cells, using a rat basophilic leukemia cell model, and that this phototoxicity again requires the presence of oxygen. The spectral and photochemical properties of curcumin vary with environment, resulting in the potential for multiple or alternate pathways for the exertion of photodynamic effects. For example, curcumin photogenerates singlet oxygen and reduced forms of molecular oxygen under several conditions relevant to cellular environments. In addition, we detected carbon‐centered radicals, which may lead to oxidation products (see accompanying paper). Such products may be important reactants in curcumins phototoxicity since singlet oxygen and reduced oxygen species alone could not explain the biological results, such as the relatively long lifetime (t12= 27 s) of the toxicant responsible for decreased cell viability.

Photogeneration of singlet oxygen and free radicals in dissolved organic matter isolated from the Mississippi and Atchafalaya River plumes

Abstract The photoreactivity to UV light of ultrafiltered dissolved organic matter (DOM) collected during cruises along salinity transects in the Mississippi and Atchafalaya River plumes was examined by measuring photogenerated free radicals and singlet molecular oxygen ( 1 O 2 ) photosensitization. Singlet oxygen was detected by its infrared phosphorescence at 1270 nm using both steady-state and time-resolved techniques. The 1 O 2 quantum yields were corrected for self-quenching of 1 O 2 by the DOM substrates. Photogenerated free radicals were monitored by electron paramagnetic resonance (EPR). Two size fractions of the dissolved organic matter were examined: material retained with a 3 kDa cut-off filter and material retained with a 1 kDa cut-off filter. The highest 1 O 2 quantum yields were found in the lower molecular mass material. There was little change in the 1 O 2 quantum yields with increasing salinity, indicating that the photosensitizing ability of the estuarine DOM does not decrease as terrestrial DOM is transported to sea and mixes with marine DOM. In contrast to 1 O 2 formation, the steady-state levels of photoproduced free radicals did not significantly differ between high and low molecular mass DOM, and the levels were substantially higher in riverine DOM than along plume salinity transects. This rapid transition in free radical level suggests that terrestrially-derived DOM experiences significant changes in this aspect of its photoreactivity in low (

Photosensitization by Norfloxacin is a Function of pH

Abstract— Norfloxacin is a fluoroquinolone (FQ) antibiotic that has been reported to cause cutaneous photosensitivity in animals and occasionally in humans. We have studied the fluorescence and singlet oxygen (1O2)‐generating properties of norfloxacin. Upon UV excitation the drug fluoresces in water, and the relative intensities of two major fluorescence bands at ca 420 and 450 nm are affected by pH. The overall quantum yield of fluorescence (φF) is also strongly pH dependent: φF is low in 0.2 N HC1 solution (0.2), increasing steeply to 0.12 at pH 4, then gradually decreasing to 0.01 at pH 10. The changes in φF are accompanied by changes in fluorescence lifetime from 0.6 ns at pH 1 to 1.8 ns at pH 4. Norfloxacin exhibits phosphorescence in low temperature glasses. The formation of a triplet state at room temperature is also suggested by 1O2 phosphorescence in aerobic D2O. This phosphorescence is “self‐quenched” by norfloxacin itself with an efficiency that is pH dependent: kq is 7.9 ×106M−1s−1 at pD 4, decreases to 1.9 × 106M−1 s−1 at pD 7.5 but then increases about 20‐fold in alkaline D2O solutions. This quenching causes the observed 1O2 production by norfloxacin (0.1 mM) to show a maximum at around pH 8–9. However, after correction for self‐quenching, the quantum yield of 1O2 production (φso), measured by using perinaphthenone as a standard, yielded the following values: φso is about 0.07 in 0.2 N DCl solution, 0.08 at pH 7.5 and then increases smoothly to ∼ 0.2 in 0.1 M NaOD solution. The relatively high, unquenched 1O2 production at physiological pH 7.4 (φso∼ 0.08) suggests that 1O2 reactions may play an important role in the cutaneous phototoxicity of norfloxacin and other FQ antibiotics.

Quenching of Singlet Molecular Oxygen (1O2) by Azide Anion in Solvent Mixtures

The azide ion is a strong physical quencher of singlet molecular oxygen (1O2) and is frequently employed to show involvement of 1O2 in oxidation processes. Rate constants (kq) for the quenching of 1O2 by azide are routinely used as standards to calculate kq values for quenching by other substrates. We have measured kq for azide in solvent mixtures containing deuterium oxide (D2O), acetonitrile (MeCN), 1,4‐dioxane, ethanol (EtOH), propylene carbonate (PC), or ethylene carbonate (EC), mixtures commonly used for many experimental studies. The rate constants were calculated directly from 1O2 phosphorescence lifetimes observed after laser pulse excitation of rose bengal (RB), used to generate 1O2. In aqueous mixtures with MeCN and carbonates, the rate constant increased nonlinearly with increasing volume of organic solvent in the mixtures. kq was 4.78 × 108M−1 s−1 in D2O and increased to 26.7 × 108 and 27.7 × 108M−1 s−1 in 96% MeCN and 97.7% EC/PC, respectively. However, in EtOH/D2O mixtures, kq decreased with increasing alcohol concentration. This shows that a higher solvent polarity increases the quenching efficiency, which is unexpectedly decreased by the proticity of aqueous and alcohol solvent mixtures. The rate constant values increased with increasing temperature, yielding a quenching activation energy of 11.3 kJ mol−1 in D2O. Our results show that rate constants in most solvent mixtures cannot be derived reliably from kq values measured in pure solvents by using a simple additivity rule. We have measured the rate constants with high accuracy, and they may serve as a reliable reference to calculate unknown kq values.

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.

PHOTOCHEMICAL REACTIONS AND PHOTOTOXICITY OF STEROLS : NOVEL SELF-PERPETUATING MECHANISM FOR LIPID PHOTOOXIDATION

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

Influence of Solvent Polarity and Proticity on the Photochemical Properties of Norfloxacin

Abstract— The fluoroquinolone antibacterial norfloxacin (NF) is a moderate photosensitizer of singlet molecular oxygen (1O2). We have studied photosensitization by NF as a function of medium polarity and proticity in solvent mixtures. We have used 1,4‐dioxane and propylene carbonate mixtures to keep proticity constant while modulating polarity, and water/D2O and ethylene carbonate mixtures to alter proticity without large changes in polarity. The absorption spectrum of NF was little affected by solvent changes, as compared to the fluorescence spectrum that exhibited as much as a 50 nm blue‐shift, e.g. 1,4‐dioxane versus D2O. The quantum yield of NF fluorescence saturated at an almost 10 times higher value (?0.14) when proticity was increased by added water, up to 0.2 mol fraction, to ethylene carbonate. Less pronounced, the increasing polarity in 1,4‐dioxane/propylene carbonate mixtures affected the fluorescence yield much less. Norfloxacin produces 1O2 and is able to quench 1O2. The rate constant for 1O2 quenching is 4.5 × 107 M−1 s−1 in propylene carbonate but decreases ca four times in D2O. The quantum yield of 1O2 photogeneration was also up to five times higher in solvents that were both protic and polar than vice versa. Our data show that NF is more photochemically active in an environment that is both protic and polar. This suggests the involvement of polar excited state(s) and possible proton/hydrogen transfer during photoexcitation. Similar processes may initiate the phototoxic response reported in some patients treated with the fluoroquinolone drugs. The phototoxicity of NF and other fluoroquinolone antibiotics may strongly depend on their localization in hydrophilic or hydrophobic cell/tissue regions.