Photochemistry and Photobiology

Cover image for Vol. 90 Issue 6

Edited By: Jean Cadet

Impact Factor: 2.684

ISI Journal Citation Reports © Ranking: 2013: 36/74 (Biophysics); 161/291 (Biochemistry & Molecular Biology)

Online ISSN: 1751-1097

Virtual Issue: Type I and II Photosensitized Oxidation in Chemistry and Biology


Virtual Issue:  Type I and II Photosensitized Oxidation in Chemistry and Biology

Edited by: Ashwini Ghogare and Alexander Greer



Click here to read an introduction from the Editors.






Photoinduced superoxide radical anion and singlet oxygen generation in the presence of novel selenadiazoloquinolones (an EPR study)

Barbieriková, Z.; Bella, M.; Kučerák, J.; Milata, V.; Jantová, S.; Dvoranová, D.; Veselá, M.; Staško, A.; Brezová, V

Novel 7-substituted 6-oxo-6,9-dihydro[1,2,5]selenadiazolo[3,4-h]quinoline (SeQ(1–6)) and 8-substituted 9-oxo-6,9-dihydro[1,2,5]selenadiazolo[3,4-f ]quinoline derivatives (SeQN(1–5)) with R7, R8 = H, COOC2H5, COOCH3, COOH, COCH3 or CN were synthesized and their spectral characteristics were obtained by UV/Vis spectroscopy. Ultraviolet A photoexcitation of the selenadiazoloquinolones in dimethylsulfoxide or acetonitrile resulted in the formation of paramagnetic species coupled with molecular oxygen activation generating the superoxide radical anion or singlet oxygen, evidenced by electron paramagnetic resonance spectroscopy. The cytotoxic/photocytotoxic impact of selenadiazoloquinolones on murine and human cancer cell lines was demonstrated using the derivative SeQ5 (with R7 = COCH3).



Stability of flavonoids in the presence of riboflavin-photogenerated reactive oxygen species: a kinetic and mechanistic study on quercetin, morin and rutin


Montaña, M. P Massad, W. A Criado, S Biasutti, A García N.A.

 

Kinetic and mechanistic aspects on the stability of the flavones (FL) quercetin (Que), morin (Mor) and rutin (Rut), in methanolic solution and in the presence of reactive oxygen species (ROS) generated by visible light-promoted riboflavin (Rf, vitamin B2) photoirradiation were studied. The system was chosen as a model for the evaluation of the in vivo protective effect of biological targets by the flavones. The overall picture includes the vitamin as an endogenous natural photosensitizer. A systematic study on the effect of ROS on FL photostability shows that under work conditions Que is oxidized by singlet molecular oxygen (O2(1Δg)), superoxide radical anion (O2˙) and hydrogen peroxide; Mor is degraded by O2(1Δg) and O2˙ whereas Rut only reacts with O2(1Δg). Que and Rut, with an extremely poor overall rate constant, are mainly physical quenchers of O2(1Δg). Mor, with O2(1Δg)-interception ability slightly lower than the recognized synthetic antioxidant trolox (Tx), behaves as a typical sacrificial scavenger provided that ca 80% of the collisions with O2(1Δg) cause its own degradation, whereas this parameter reaches around 50% in the case of Tx.




Photosensitized oxidation of tetrabromobisphenol a by humic acid in aqueous solution


Han, S. K.; Sik, R. H.;Motten, A.G.; Chignell, C. F.;Bilski, P. J. 



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 (λ > 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.

Oxygen uptake in the vitamin B2-sensitized photo-oxidation of tyrosine and tryptophan in the presence of uracil: kinetics and mechanism


Montaña, M. P.; Blasich, N Haggi, E García, N. A 



Considering the significance of visible light-promoted reactions in complex biological media, the photo-oxidation of the amino acids (AAs) tyrosine (tyr) and tryptophan (trp) was studied in the presence of the naturally occurring oxidative scavenger uracil (ur). The involved photoprocesses, studied at pH 7 and 9, are driven through the reactive oxygen species (ROS) singlet molecular oxygen (O2(1Δg)), superoxide radical anion (O2) and hydrogen peroxide (H2O2). The effect on the effectiveness of the overall photo-oxidation process due to the presence of an added electron-donating substrate such as ur is not straightforwardly predictable. The addition of the pyrimidine compound, a much lesser photo-oxidizable substrate than the AAs themselves, produced different results: (1) antioxidative for tyr at pH 9, decreasing the overall rate of oxygen uptake; (2) synergistic for tyr at pH 7, increasing the oxidation rate more than the corresponding addition value of the respective individual rates and (3) no effect for trp at both pH values. The final result depends on the respective abilities of the substrates as quenchers of both the long-lived riboflavin triplet excited state and the generated ROS and the pH of the medium. An interpretation for the different cases is attempted through a kinetic and mechanistic analysis.

Effect of oxygen concentration on photo-oxidation and photosensitizer bleaching in butter


Wold, J. P.; Veberg, D. A.; Lundby, F.; Nilsen, A. N.; Juzeniene, A.; Moan, J 



The effect of headspace oxygen concentration and color of light on photo-oxidation and degradation of photosensitizers in butter was studied. Butter samples were stored under 0%, 0.4%, 0.8%, 1.6%, 3.0%, 5.0%, 21% oxygen, and exposed to violet, green or red light. Storage time was 36 h. Degree of photo-oxidation was measured by a trained sensory panel. Photobleaching of six different photosensitizers was estimated based on front face fluorescence excitation and emission landscapes and spectral curve resolution (parallel factor analysis). The higher oxygen concentration, the more sensory degraded were the samples. Violet light resulted in slightly higher degrees of photo-oxidation than green and red light for low oxygen concentrations. Bleaching rate and course as function of O2 concentration differed between the photosensitizers. It is suggested that the rate of photobleaching is a balance between type I and type II photoreactions.



Time-resolved luminescence and singlet oxygen formation after illumination of the hypericin–low-density lipoprotein complex

Gbur, P.; Dedic, R.; Chorvat, D. Jr.; Miskovsky, P.; Hala, J.; Jancura, D 



Time-resolved fluorescence and phosphorescence study of hypericin (Hyp) in complex with low-density lipoproteins (LDL) as well as the evolution of singlet oxygen formation and annihilation after illumination of Hyp/LDL complexes at room temperature are presented in this work. The observed shortening of the fluorescence lifetime of Hyp at high Hyp/LDL molar ratios (>25:1) proves the self-quenching of the excited singlet state of monomeric Hyp at these concentration ratios. The very short lifetime (∼0.5 ns) of Hyp fluorescence at very high Hyp/LDL ratios (>150:1) suggests that at high local Hyp concentration inside LDL molecules fast and ultrafast nonradiative decay processes from excited singlet state of Hyp become more important. Contrary to the lifetime of the singlet excited state, the lifetime (its shorter component) of Hyp phosphorescence is not dependent on Hyp/LDL ratio in the studied concentration range. The amount of singlet oxygen produced as well as the integral intensity of Hyp phosphorescence after illumination of Hyp/LDL complexes resemble the dependence of the concentration of molecules of Hyp in monomeric state on Hyp/LDL until a concentration ratio of 60:1. This fact confirms that only monomeric Hyp is able to produce the excited triplet state of Hyp, which in aerobic conditions leads to singlet oxygen production. The value of singlet oxygen lifetime (∼8 μs) after its formation from the excited triplet state of Hyp in LDL proves that molecules of singlet oxygen remain for a certain period of time inside LDL particles and are not immediately released to the aqueous surrounding. That Hyp exists in the complex with LDL in the monodeprotonated state is also demonstrated.



Photosensitized oxidation of hypoxanthine and xanthine by aluminum phthalocyanine tetrasulfonate. Role of the alkylating quinone 2,5-dichloro-diaziridinyl-1,4-benzoquinone. 



Alegria, A. E.; Inostroza, Y.; Kumar, A. 



Photoirradn. of nitrogen-satd. aq. solns. contg. aluminum phthalocyanine tetrasulfonate (AlPcS4) at 675 nm in the presence of 2,5-dichloro-diaziridinyl-1,4-benzoquinone (AZDClQ) and hypoxanthine (HX) produces the oxidized HX derivs., xanthine (X) and uric acid (UA).  Concns. of the AZDClQ semiquinone, X and UA increase at the expense of HX with an increase in irradn. time.  Almost negligible decompn. of HX, as well as very low amts. of X, are detected if photolysis occurs under identical conditions but in the absence of AZDClQ.  Addn. of calf-thymus DNA produces quinone-DNA covalent adducts after photolysis of anaerobic samples contg. quinone, DNA and AlPcS4, in the presence or absence of HX and at pH 5.5.  However, larger amts. of quinone-DNA adducts are detected if HX is present.  The results presented here could have applications in the photodynamic treatment of hypoxic tissues such as solid tumors, under conditions of high HX concentration, where Type-I pathways could be more important than singlet oxygen generation.


Singlet oxygen reacts with 2',7'-dichlorodihydrofluorescein and contributes to the formation of 2',7'-dichlorofluorescein.



Daghastanli, N. A.;Itri, R.;Baptista, M. S. 



There are controversial reports in the literature concerning the reactivity ofsinglet oxygen((1)O(2)) with the redox probe 2',7'-dichlorodihydrofluorescein (DCFH). By carefully preparing solutions in which (1)O(2) is quantitatively generated in the presence of DCFH, we were able to show that the formation rate of the fluorescent molecule derived from DCFH oxidation, which is 2',7'-dichlorofluorescein (DCF), increases in D(2)O and decreases in sodium azide, proving the direct role of (1)O(2) in this process. We have also prepared solutions in which either (1)O(2) or dication (MB(2+)) and semi-reduced (MB) radicals of the sensitizer and subsequently super-oxide radical (O(2)(-)) are generated. The absence of any effect of SOD and catalase ruled out the DCFH oxidation by O(2)(-), indicating that both (1)O(2) and MB(2+) react with DCFH. Although the formation of DCF was 1 order of magnitude larger in the presence of MB(2+) than in the presence of (1)O(2), considering the rate of spontaneous decays of these species in aqueous solution, we were able to conclude that the reactivity of (1)O(2) with DCFH is actually larger than that of MB(2+). We conclude that DCFH can continue to be used as a probe to monitor general redox misbalance induced in biologic systems by oxidizing radicals and (1)O(2).


Chemiluminescence associated with singlet oxygen reactions with amino acids, peptides and proteins.


Alarcón, E.; Henríquez, C.; Aspée, A.; Lissi, E. A. 



Low level chemiluminescence (CL) is observed after protein oxidation mediated by singlet oxygen produced in Rose Bengal (RB) irradiation. This CL lasts for several minutes after the end of the photolysis. In this work, the mechanism of the process was assessed from the spectral characteristics of the CL and the effect of antioxidants (Trolox or ascorbate), Ebselen (a compound with peroxidase-like activity), azide (a singlet oxygen scavenger) and D2O, added prior to or after RB irradiation. It is concluded that most of the light emission is due to formation of excited states generated in the decomposition of peroxides and/or hydroperoxides accumulated during the photolysis. Experiments carried out in the presence of several amino acids (Cys, Met, His, Tyr and Trp) and di- and tripeptides suggest that peroxides (and/or hydroperoxides) of Trp residues are mainly responsible for the CL observed after singlet oxygen-mediated protein oxidation. The much weaker CL observed after the oxidation of proteins without Trp residues supports this conclusion. A comparison of the results obtained employing free Trp, Ala-Trp and Trp-Ala dipeptides, Ala-Trp-Ala tripeptide and Trp-containing proteins supports the conclusion that blocking the amino group of the Trp moiety strongly increases the efficiency of the chemiluminescent process, producing ≈2.5×10−8 photons per oxidized Trp group in Ala-Trp. A mechanism comprising two chemiluminescent oxidation pathways of Trp residues is proposed to explain the results.

A theoretical elucidation on the solvent-dependent photosensitive behaviors of C60

 

Shen, L.;Ji, H. F;Zhang, H. Y. 



In this paper, the solvent-dependent photosensitive behaviors of fullerene (C(60)) were investigated in polar and nonpolar solvents by time-dependent density functional theory (TD-DFT) calculation. Based on the calculated physicochemical parameters on triplet state, it is revealed that excited-state C(60) only generates (1)O(2) via energy transfer in benzene, but can give birth to O(2)(.-) and (1)O(2) in water via energy transfer and electron transfer, respectively. Considering the fact that electron transfer is more favorable compared with energy transfer in polar biological systems, especially with the presence of electron donors, the O(2)(.-)-generating process will get predominant in physiological systems. These results account well for the experimental observations that O(2)(.-) and (.)OH are primarily responsible for the photoinduced DNA cleavage by C(60) under physiological conditions, whereas (1)O(2) plays a critical role in nonpolar solvents.

Effect and mechanism of a new photodynamic therapy with glycoconjugated fullerene


Otake, E.; Sakuma, S.; Torii, K.; Maeda, A.; Ohi, H.; Yano, S.; Morita, A. 



When irradiated, fullerene efficiently generates reactive oxygen species (ROS) and is an attractive photosensitizer for photodynamic therapy (PDT). Ideally, photosensitizers for PDT should be water-sol. and tumor-specific. Because cancer cells end-ocytose glucose more effectively than normal cells, the characteristics of fullerene as a photosensitizer were improved by combining it with glucose. The cytotoxicity of PDT was studied in several cancer cell lines cultured with C60-(Glc)1 (D-glucose residue pendant fullerene) and C60-(6Glc)1 (a maltohexaose residue pendant fullerene) subsequently irradiated with UVA1. PDT alone induced significant cytotoxicity. In contrast, PDT with the glycoconjugated fullerene exhibited no significant cytotoxicity against normal fibroblasts, indicating that PDT with these compds. targeted cancer cells. To investigate whether the effects of PDT with glycoconjugated fullerene were because of the generation of singlet oxygen 1O2), NaN3 was added to cancer cells during irradn. NaN3 extensively blocked PDT-induced apoptosis, suggesting that PDT-induced cell death was a result of the generation of 1O2. Finally, to investigate the effect of PDT in vivo, melanoma-bearing mice were injected intratumorally with C60-(Glc)1 and irradiated with UVA1. PDT with C60-(Glc)1 suppressed tumor growth. These findings indicate that PDT with glycoconjugated fullerene exhibits tumor-specific cytotoxicity both in vivo and in vitro via the induction of 1O2.



Photochemistry of bacteriochlorophylls in human blood plasma: 2. Reaction mechanism investigated by product analysis and deuterium isotope effect

Dandler, J.; Wilhelm, B.; Scheer, H. 



Transmetalated (Pd) bacteriochlorophyll derivs. are currently being clin. tested as sensitizers for photodynamic therapy. Protocols using short delay times between injection and irradn. generate interest in the photochem. of these pigments in the blood. Using near-IR irradn. where these pigments absorb strongly, we have studied the mechanism of photo-oxidn. In two lipoprotein fractions, low- and high-d. lipoproteins, derived from human blood plasma that preferentially accumulate these pigments. Using quenchers of reactive oxygen species, and chem. reporters, in particular peroxides generated from cholesterol as an inherent component of the lipoproteins, a Type II mechanism generating singlet oxygen has been demonstrated for Pd- and Zn-bacteriopheophorbides. In homogeneous systems, accelerated bleaching in D2O, compared with H2O, supports this mechanism. An unusual deuterium isotope effect was obsd., by contrast, in heterogeneous amphiphilic-water systems. In the early phase, and under high oxygen concns., again a pos. D-isotope effect is obsd. which later, in a second phase, is reversed to a neg. D-isotope effect. The latter cannot be explained by heterogeneous pigment populations in the amphiphilic system; we, therefore, conclude a mechanistic switch, and discuss a possible mechanism.

Monitoring singlet oxygen and hydroxyl radical formation with fluorescent probes during photodynamic therapy

Reiners, J. J.; Santiago, A. M.; Kessel, D. 



Singlet oxygen (1O2) is the primary oxidant generated in photodynamic therapy (PDT) protocols involving sensitizers resulting in type II reactions. 1O2 can give rise to additional reactive oxygen species (ROS) such as the hydroxyl radical (*OH). The current study was designed to assess 3'-p-(aminophenyl) fluorescein (APF) and 3'-p-(hydroxyphenyl) fluorescein (HPF) as probes for the detection of 1O2 and *OH under conditions relevant to PDT. Cell-free studies indicated that both APF and HPF were converted to fluorescent products following exposure to 1O2 generated by irradiation of a water-soluble photosensitizing agent (TPPS) and that APF was 35-fold more sensitive than HPF. Using the 1O2 probe singlet oxygen sensor green (SOSG) we confirmed that 1 mm NaN3 quenched 1O2-induced APF/HPF fluorescence, while 1% DMSO had no effect. APF and HPF also yielded a fluorescent product upon interacting with *OH generated from H2O2 via the Fenton reaction in a cell-free system. DMSO quenched the fluorogenic interaction between APF/HPF and *OH at doses as low as 0.02%. Although NaN3 was expected to quench *OH-induced APF/HPF fluorescence, co-incubating NaN3 with APF or HPF in the presence of *OH markedly enhanced fluorescence. Cultured L1210 cells that had been photosensitized with benzoporphyhrin derivative exhibited APF fluorescence immediately following irradiation. Approximately 50% of the cellular fluorescence could be suppressed by inclusion of either DMSO or the iron-chelator desferroxamine. Combining the latter two agents did not enhance suppression. We conclude that APF can be used to monitor the formation of both 1O2 and *OH in cells subjected to PDT if studies are performed in the presence and absence of DMSO, respectively. That portion of the fluorescence quenched by DMSO will represent the contribution of *OH. This procedure could represent a useful means for evaluating formation of both ROS in the context of PDT.


Advanced glycation endproducts induce photocrosslinking and oxidation of bovine lens proteins through type-I mechanism

Denis, F.; Bertrand, F.; Eduardo, S. 



Advanced glycation endproducts (AGEs) have been suggested as photosensitizers that are capable of mediating eye lens photo-damage during aging. In the present work, we investigate the photo-crosslinking and oxidation of bovine lens proteins sensitized by AGEs, with special regard to low oxygen conditions. A mechanistic study was conducted using different oxygen concentrations and specific additives with the aim either to scavenge or enhance Type-I or Type-II photoprocesses. Quantum yields for Trp decomposition were determined at 5%, 20% and 100% O2, in the presence of ferricyanide and D2O to elucidate the mechanism of action of AGEs. Type-I mechanism proved to be the most efficient pathway for AGE-sensitized Trp decomposition at low oxygen concentration. Photocrosslinking of lens proteins and crystallin fractions due to Type-I interaction was observed. The influence of the oxygen concentration and additives was also studied. The results show that both Type-I mechanism and oxygen-mediated reactions contribute to protein crosslinking. Carbonyl group formation due to protein photo-oxidation was detected with Oxyblot technique. The generation of high levels of hydrogen peroxide during the irradiations was detected and attributed mainly to Type-I reactions. The results support that AGEs act preferentially as Type-I sensitizers at the low oxygen concentration found in the lens and are capable of inducing protein crosslinking, oxidation and peroxide formation.

Genotoxicity of combined exposure to polycyclic aromatic hydrocarbons and UVA-A mechanistic study 

Douki, T.; Ksoury, Z.; Marie, C.; Favier, A.; Ravanat, J.L.; Maitre, A. 



Solar UV radiation and benzo[a]pyrene (BaP) are two carcinogenic agents.  When combined, their deleterious properties are synergistic.  In order to get insights into the underlying processes, we carried out a mechanistic study within isolated DNA photosensitized to UVA radiation by either BaP, its diol epoxide metabolite (BPDE) or the tetrol arising from the hydrolysis of this last mol.  Measurement of the level of the oxidized base 8-oxo-7,8-dihydroguanine revealed that BaP is a poor sensitizer while BPDE and tetrol are more potent ones.  None of these compds. was found to photosensitize formation of cyclobutane pyrimidine dimers through triplet energy transfer.  On the basis of the distribution of oxidized DNA bases, we could show that photosensitization of DNA by BPDE involves electron abstraction (Type I) while tetrol acts mainly through singlet oxygen prodn. (Type II).  Under our exptl. conditions, Type I was the major photosensitization process, which shows the lack of involvement of tetrol in the obsd. photo-oxidn. reaction.  Finally, we could show that the adducts, resulting from the alkylation of DNA by BPDE, are very potent sensitizers.  Indeed, they are located in the close vicinity of the double helix and thus perfectly placed to induce oxidn. reactions.

The microenvironment of DNA switches the activity of singlet oxygen generation photosensitized by berberine and palmatine

Hirakawa, K.; Hirano, T. 



The effect of the interaction between DNA and the photosensitizer on photosensitized singlet oxygen (1O2) generation was investigated using DNA-binding alkaloids, berberine and palmatine. These photosensitizers were bound to DNA by electrostatic force. Near-infrared luminescence measurement demonstrated that the photoexcited alkaloids can generate 1O2 only when the photosensitizers are bound to DNA. A fluorescence decay study showed significant enhancement of the lifetime of their photoexcited state with the DNA binding. A calculation study suggested that the electrostatic interaction with DNA inhibits the quenching of the photoexcited state of these alkaloids via intramolecular electron transfer, leading to the prolongation of the lifetime of their excited state. This effect should enhance their intersystem crossing and the yield of energy transfer to molecular oxygen. The results show that the electrostatic interaction with DNA significantly affects the 1O2 generation activity of a photosensitizer. In addition, this interaction may be applied to the control and the design of photosensitizers for medical applications such as photodynamic therapy.

Photodynamic action of benzo[a]pyrene in K562 cells


Filgueira, D. M.;Salomão de Freitas, D. P.;de Souza Votto, A.P.;Fillmann, G.;Monserrat, J.M.;Geracitano, L. A.;Santos, T. G 



Benzo[a]pyrene (BaP) is ubiquitously distributed in the environment, being considered the most phototoxic element among polycyclic aromatic hydrocarbon (PAHs). In presence of oxygen, PAHs can act as a photosensitizer by means of promoting photo-oxidation of biological molecules (photodynamic action, PDA). Thus, the present study analyzed the photodynamic action of BaP under UVA irradiation (BaP + UVA) and its oxidative effects on K562 cells. The evaluation of BaP kinetics showed that the highest intracellular concentration occurred after 18 h of incubation. Cell viability, reactive oxygen species (ROS) generation, lipid peroxidation, DNA damage (breaks and DNA-protein cross-link [DNAPC]) were assessed after exposure to BaP, UVA and BaP plus UVA irradiation (BaP + UVA). Cell viability was decreased just after exposure to BaP + UVA. Lipid peroxidation and DNA breaks increased after BaP + UVA exposure, while the DNAPC increased after BaP, UVA and BaP + UVA exposure, suggesting that BaP + UVA effects were a consequence of both type II (producing mainly singlet oxygen) and type I (producing others ROS) mechanisms of PDA.

Photosensitizing potential of ciprofloxacin at ambient level of UV radiation


Agrawal, N.; Ray, R.S.; Farooq, M.; Pant, A. B.; Hans, R. K 



Ciprofloxacin is a widely used fluoroquinolone drug with broad spectrum antibacterial activities. Clinical experience has shown incidences of adverse effects related to skin, hepatic, central nervous system, gastrointestinal and phototoxicity. India is a tropical country and sunlight is abundant throughout the day. In this scenario exposure to ambient levels of ultraviolet radiation (UV-R) in sunlight may lead to harmful effects in ciprofloxacin users. Phototoxicity assessment of ciprofloxacin was studied by two mouse fibroblast cell lines L-929 and NIH-3T3. Generation of reactive oxygen species (ROS) like singlet oxygen (1O2), superoxide anion radical (O2*-) and hydroxyl radical (*OH) was studied under the exposure of ambient intensities of UV-A (1.14, 1.6 and 2.2 mW cm(-2)), UV-B (0.6, 0.9 and 1.2 mW cm(-2)) and sunlight (60 min). The drug was generating 1O2, O2*- and *OH in a concentration and dose-dependent manner. Sodium azide (NaN3) and 1,4-diazabicyclo 2-2-2-octane (DABCO) inhibited the generation of 1O2. Superoxide dismutase (SOD) inhibited 90-95% O2*- generation. The drug (5-40 microg mL(-1)) was responsible for linoleic acid peroxidation. Quenching study of linoleic acid peroxidation with SOD (25 and 50 U mL(-1)) confirms the involvement of ROS in drug-induced lipid peroxidation. The generation of *OH radical was further confirmed by using specific quenchers of *OH such as mannitol (0.5 M) and sodium benzoate (0.5 M). 2'-deoxyguanosine (2'-dGuO) assay and linoleic acid peroxidation showed that ROS were mainly responsible for ciprofloxacin-sensitized photo-degradation of guanine base. L-929 cell line showed 29%, 34% and 54% reduced cell viability at higher drug concentration (300 microg mL(-1)) under UV-A, UV-B and sunlight, respectively. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in NIH-3T3 cell line at higher drug concentration (300 microg mL(-1)) showed a decrease in cell viability by 54%, 56% and 59% under UV-A, UV-B and sunlight, respectively. Results of neutral red uptake assay (NRU) in L-929 cell line were in accordance with MTT assay. The NIH-3T3 cell line showed a higher photosensitizing potential than L-929. The phototoxicity end point shows a time- and concentration-dependent statistically significant (P<0.001) damage. Ciprofloxacin produced ROS by Type I and Type II photodynamic reactions, interacted with nucleic acid moiety and inhibited cell viability. Further, UV-induced photo-peroxidation of linoleic acid accorded the involvement of ROS in the manifestation of drug phototoxicity. Appearance of ciprofloxacin-induced phototoxicity at the ambient level of sunlight is a real risk for the people of India and for those of other tropical countries. We suggest that sunlight exposure should be avoided (especially peak hours) during ciprofloxacin treatment.

Suppressive effect of caffeic acid and its derivatives on the generation of UVA-induced reactive oxygen species in the skin of hairless mice and pharmacokinetic analysis on organ distribution of caffeic acid in ddY mice


Yamada, Y.Yasui, HSakurai, 



Caffeic acid (CA) and its analogues such as rosmarinic acid are well known as antioxidative agents. Exposure to UVA is known to generate reactive oxygen species (ROS) such as singlet oxygen (1O2) and superoxide anion radical (*O2-) in the skin of animals, which in turn induces skin photodamage and photoaging. Because CA and its analogues quench 1O2, these compounds were topically applied to the abdominal skin of live hairless mice and were found to suppress ROS generation upon UVA exposure. Furthermore, the generation of UVA-induced ROS was also suppressed in the skin of mice that were orally given CA. In order to understand the mechanism by which CA blocks ROS production in UVA-exposed skin, the pharmacokinetics of CA upon oral administration to mice was followed and CA was found to efficiently distribute in the skin. These results suggest that skin damage by UVA-induced ROS generation is reduced by oral supplementation of CA, which has a scavenging and quenching activity against ROS.



Singlet oxygen oxidation of isolated and cellular DNA: product formation and mechanistic insights


Cadet, J.; Ravanat, J.-L., Martinez, G. R.; Medeiros, M. H. G.; Di Mascio, P. 



This survey focuses on recent aspects of the singlet oxygen oxidation of the guanine moiety of nucleosides, oligonucleotides, isolated and cellular DNA that has been shown to be the exclusive DNA target for this biologically relevant photo-generated oxidant. A large body of mechanistic data is now available from studies performed on nucleosides in both aprotic solvents and aqueous solutions. A common process to both reaction conditions is the formation of 8-oxo-7,8-dihydroguanine by reduction of 8-hydroperoxyguanine that arises from the rearrangement of initially formed endoperoxide across the 4,8-bond of the purine moiety. However, in organic solvent the hydroperoxide is converted as a major degradation pathway into a dioxirane that subsequently decomposes into a complex pattern of oxidation products. A different reaction that involved the formation of a highly reactive quinonoid intermediate consecutively to the loss of a water molecule from the 8-hydroperoxide has been shown to occur in aqueous solution. Subsequent addition of a water molecule at C5 leads to the generation of a spiroiminodihydantoin compound via a rearrangement that involves an acyl shift. However, in both isolated and cellular DNA the latter decomposition pathway is at the best a minor process, because only 8-oxo-7,8-dihydroguanine has been found to be generated. It is interesting to point out that singlet oxygen has been shown to contribute predominantly to the formation of 8-oxo-7,8-dihydroguanine in the DNA of bacterial and human cells upon exposure to UVA radiation. It may be added that the formation of secondary singlet-oxygen oxidation products of 8-oxo-7,8-dihydroguanine, including spiroiminodihydantoin and oxaluric acid that were characterized in nucleosides and oligonucleotide, respectively, have not yet been found in cellular DNA.

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