Singlet Molecular Oxygen Reactions with Nucleic Acids, Lipids, and Proteins.

Singlet oxygen (1O2) is a biologically relevant reactive oxygen species capable of efficiently reacting with cellular constituents. The resulting oxidatively generated damage to nucleic acids, membrane unsaturated lipids, and protein components has been shown to be implicated in several diseases, including arthritis, cataracts, and skin cancer. Singlet oxygen may be endogenously produced, among various possibilities, by myeloperoxidase, an enzyme implicated in inflammation processes, and also efficiently in skin by the UVA component of solar radiation through photosensitization reactions. Emphasis is placed in this Review on the description of the main oxidation reactions initiated by 1O2 and the resulting modifications within key cellular targets, including guanine for nucleic acids, unsaturated lipids, and targeted amino acids. Most of these reactions give rise to peroxides and dioxetanes, whose formation has been rationalized in terms of [4+2] cycloaddition and 1,2-cycloaddition with dienes + olefins, respectively. The use of [18O]-labeled thermolabile endoperoxides as a source of [18O]-labeled 1O2 has been applied to study mechanistic aspects and preferential targets of 1O2 in biological systems. A relevant major topic deals with the search for the molecular signature of the 1O2 formation in targeted biomolecules within cells. It may be anticipated that [18O]-labeled 1O2 and labeled peroxides in association with sensitive mass spectrometric methods should constitute powerful tools for this purpose.

Short-term high temperature treatment reduces viability and inhibits respiration and DNA repair enzymes in Araucaria angustifolia cells.

We evaluated the effect of global warming on Araucaria angustifolia (Bert.) O. Kuntze, a critically endangered native tree of Southern Brazil, by studying the effects of short-term high temperature treatment on cell viability, respiration and DNA repair of embryogenic cells. Compared with control cells grown at 25°C, cell viability was reduced by 40% after incubation at 30 and 37°C for 24 and 6 h, respectively, while 2 h at 40 and 42°C killed 95% of the cells. Cell respiration was unaffected at 30-37°C, but dramatically reduced after 2 h at 42°C. The in vitro activity of enzymes of the base excision repair (BER) pathway was determined. Apurinic/apyrimidine endonuclease, measured in extracts from cells incubated for 2 h at 42°C, was completely inactivated while lower temperatures had no effect. The activities of three enzymes of the mitochondrial BER pathway were measured after 30-min preincubation of isolated mitochondria at 25-40°C and one of them, uracil glycosylase, was completely inhibited at 40°C. We conclude that cell viability, respiration and DNA repair have different temperature sensitivities between 25 and 37°C, and that they are all very sensitive to 40 or 42°C. Thus, A. angustifolia will likely be vulnerable to the short-term high temperature events associated with global warming.

Singlet molecular oxygen: Düsseldorf - São Paulo, the Brazilian connection.

Inspired by Helmut Sies we continue the development of suitable chemical generators of (1)O2 based on the thermodissociation of naphthalene endoperoxide derivatives. The present manuscript focuses on how the use of [(18)O]-labeled endoperoxides and hydroperoxides can be applied to study mechanistic aspects related to the generation of singlet molecular oxygen and its reactions in biological systems. The peroxidation reactions of the main cellular targets including unsaturated lipids, proteins and nucleic acids have received major attention during the last three decades. Emphasis is placed in this manuscript on the description of the synthesis and the main use of [(18)O]-labeled compounds, and especially of peroxides and (1)O2, for tracer elucidation of reaction mechanisms.

Glutathione modifies the oxidation products of 2'-deoxyguanosine by singlet molecular oxygen.

The oxidation of the free nucleoside 2'-deoxyguanosine (dGuo) by singlet molecular oxygen ((1)O2) has been studied over the three last decades due to the major role of DNA oxidation products in process such as ageing, mutation and carcinogenesis. In the present work we investigated the dGuo oxidation by (1)O2 in the presence of the important low molecular antioxidant, glutathione, in its reduced (GSH) and oxidized (GSSG) forms. There were applied different conditions of concentration, pH, time of incubation, and the use of a [(18)O]-labeled thermolabile endoperoxide naphthalene derivative as a source of [(18)O]-labeled (1)O2. Data was obtained through high performance liquid chromatography (HPLC) and HPLC coupled to micrOTOF Q-II analysis of the main oxidation products: the diastereomers of spiroiminodihydantoin-2'-deoxyribonucleosides (dSp) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). An intriguing result was that 8-oxodGuo levels increased by 100 fold when dGuo was oxidized by (1)O2 in the presence of GSH and by 2 fold in the presence of GSSG, while dSp levels dropped to zero for both conditions. All data from dGuo, 8-oxodGuo and dSp quantification together with the analysis of residual GSH/GSSG content in each sample strongly suggest that glutathione modifies the mechanism of dGuo oxidation by (1)O2 by disfavoring the pathway of dSp formation.

Dual properties of hispidulin: antiproliferative effects on HepG2 cancer cells and selective inhibition of ABCG2 transport activity.

Hepatocellular carcinoma is the third most common cause of cancer-related deaths worldwide. Furthermore, the existing pharmacological-based treatments are insufficiently effective and generate many side effects. Hispidulin (6-methoxy-5,7,4'-trihydroxyflavone) is a flavonoid found in various medicinal herbs that present antineoplastic properties. Here we evaluated how modulation of reactive oxygen species (ROS) and alterations of antioxidant defenses could be associated to the antiproliferative effects of hispidulin in HepG2 cells. In addition, we studied the inhibitory activity of hispidulin on the efflux of drugs mediated by ABC transporters involved in multidrug resistance. In order to understand the increase of intracellular ROS promoted by hispidulin, we investigated the mRNA expression levels and activities of antioxidant enzymes, and the GSH/GSSG ratio. We showed that hispidulin significantly down-regulated the transcription levels of catalase, leading to reduction of enzyme activity and decrease of the GSH content. We also observed that, in the presence of N-acetylcysteine or exogenous catalase, the proliferation was lowered back to the control levels. These data clearly indicate a strong involvement of intracellular ROS levels for triggering the antiproliferative effects. We also demonstrated that the inhibition produced by hispidulin on drug efflux was specific for ABCG2, since no effects were observed with ABCB1 and ABCC1. Furthermore, HepG2 cells were more sensitive to hispidulin-mediated cell death than immortalized L929 fibroblasts, suggesting a differential toxicity of this compound between tumor and non-tumor cell lines. Our results suggest that hispidulin constitutes a promising candidate to sensitize chemoresistant cancer cells overexpressing ABCG2.

Melanogenesis stimulation in B16-F10 melanoma cells induces cell cycle alterations, increased ROS levels and a differential expression of proteins as revealed by proteomic analysis.

Considering that stimulation of melanogenesis may lead to alterations of cellular responses, besides melanin production, our main goal was to study the cellular effects of melanogenesis stimulation of B16-F10 melanoma cells. Our results show increased levels of the reactive oxygen species after 15 h of melanogenesis stimulation. Following 48 h of melanogenesis stimulation, proliferation was inhibited (by induction of cell cycle arrest in the G1 phase) and the expression levels of p21 mRNA were increased. In addition, melanogenesis stimulation did not induce cellular senescence. Proteomic analysis demonstrated the involvement of proteins from other pathways besides those related to the cell cycle, including protein disulfide isomerase A3, heat-shock protein 70, and fructose biphosphate aldolase A (all up-regulated), and lactate dehydrogenase (down-regulated). In RT-qPCR experiments, the levels of pyruvate kinase M2 mRNA dropped, whereas the levels of ATP synthase (beta-F1) mRNA increased. These data indicate that melanogenesis stimulation of B16-F10 cells leads to alterations in metabolism and cell cycle progression that may contribute to an induction of cell quiescence, which may provide a mechanism of resistance against cellular injury promoted by melanin synthesis.

Metabolism of the mesoionic compound (MI-D) by mouse liver microsome, detection of its metabolite in vivo, and acute toxicity in mice.

The mesoionic derivative 4-phenyl-5-[4-nitrocinnamoyl]-1,3,4-thiadiazolyl-2-phenylamine chloride (MI-D) has antitumoral and anti-inflammatory effects. In this study, we present aspects of its metabolism and toxicity in mice. MI-D was metabolized in vitro by liver microsome, generating a main product with a much shorter retention time than MI-D in high-performance liquid chromatography (HPLC) analysis but with a spectrum similar to that of the original molecule. Mass spectrometry with electrospray ionization in positive mode analysis of the purified compound by HPLC indicated that the product of metabolism has four additional hydroxyl groups (m/z = 465) compared with MI-D (m/z = 401). The HPLC analyses of plasma and urine samples from mice treated with MI-D showed the presence of the metabolite product. The kinetic parameters K(m) (19.5 +/- 4.5 microM) and V(max) [1.5 +/- 0.4 units of fluorescence/(100 microg of microsomal protein/mL/s)] were estimated, confirming the metabolism of MI-D and indicating that the reaction follows Michaelis-Menten kinetics. Acute toxicity was established on the basis of an estimation of mean lethal dose (LD-50; 181.2 mg/kg) and histopathological analysis of animals that survived the LD-50 test. Abdominal adhesions, inflammatory foci, and formation of granulomas were observed. Altogether, the results contribute to the advancement of research in support of MI-D as a future chemotherapeutic drug.

Novel rhythms of N1-acetyl-N2-formyl-5-methoxykynuramine and its precursor melatonin in water hyacinth: importance for phytoremediation.

N1-acetyl-N2-formyl-5-methoxykynuramine (AMFK) is a major metabolite of melatonin in mammals. To investigate whether AFMK exists in plants, an aquatic plant, water hyacinth, was used. To achieve this, LC/MS/MS with a deuterated standard was employed. AFMK was identified in any plant for the first time. Both it and its precursor, melatonin, were rhythmic with peaks during the late light phase. These novel rhythms indicate that these molecules do not serve as the chemical signal of darkness as in animals but may relate to processes of photosynthesis or photoprotection. These possibilities are supported by higher production of melatonin and AFMK in plants grown in sunlight (10,000-15,000 microW/cm2) compared to those grown under artificial light (400-450 microW/cm2). Melatonin and AFMK, as potent free radical scavengers, may assist plants in coping with harsh environmental insults, including soil and water pollutants. High levels of melatonin and AFMK in water hyacinth may explain why this plant more easily tolerates environmental pollutants, including toxic chemicals and heavy metals and is successfully used in phytoremediation. These novel findings could lead to improvements in the phytoremediative capacity of plants by either stimulating endogenous melatonin synthesis or by adding melatonin to water/soil in which they are grown.

Eupafolin: Effect on mitochondrial energetic metabolism.

This study evaluated the effects of flavone eupafolin (6-methoxy 5,7,3',4'-tetrahydroxyflavone), extracted from dry leaves of Eupatorium litoralle. Eupafolin (25-200microM) promoted inhibition of the respiratory rate in state 3, in the presence of glutamate or succinate. During succinate oxidation, it was found that only state 4 respiratory rate was stimulated approximately 30% by eupafolin (100microM) and ADP/O ratio and RCC were reduced with all doses. When glutamate was used as substrate, RCC was similarly reduced. Eupafolin caused a reduction of enzymatic activities between complexes I and III of the respiratory chain. Cytochrome c oxidase and ATPase activities were not affected. Using voltammetry cyclic analysis, eupafolin give rise to irreversible oxidation with an anodic peak potential at +0.08V (SHE). We also observed that eupafolin can undergo oxidation catalyzed by EDTA-Fe, promoting cytochrome c reduction in the presence of NADH, resulting in the production of the superoxide radical and hydrogen peroxide. All together, the results could explain the cytotoxic effects observed previously with the eupafolin.

Cytotoxicity of η6-areneruthenium-based molecules to glioblastoma cells and their recognition by multidrug ABC transporters.

A new series of amphiphilic η6-areneruthenium(II) compounds containing phenylazo ligands (group I: compounds 1a, 1b, 2a and 2b) and phenyloxadiazole ligands (group II: compounds 3a, 3b, 4a and 4b) were synthesized and characterized for their anti-glioblastoma activity. The effects of the amphiphilic η6-areneruthenium(II) complexes on the viability of three human glioblastoma cell lines, U251, U87MG and T98G, were evaluated. The azo-derivative ruthenium complexes (group I) showed high cytotoxicity to all cell lines, whilst most oxadiazole-derivative complexes (group II) were less cytotoxic, except for compound 4a. The cationic complexes 2a, 2b and 4b were more cytotoxic than the neutral complexes. Compounds 2a and 2b caused a significant reduction in the percentage of cells in the G0/G1 phase, with concomitant increases in the G2/M phase and fragmented DNA in the T98G cell line. The η6-areneruthenium(II) compounds were also tested in cell lines that overexpress the multidrug ABC transporters P-gp, MRP1 and ABCG2. Compounds 2b and 4a were substrates for the P-gp protein, with resistance indexes of 8.6 and 1.9, respectively. Compound 2b was also a substrate for ABCG2 and MRP1 proteins, with lower resistance indexes (1.8 and 1.6, respectively). The contribution of multidrug ABC transporters to the cytotoxicity of compound 2b in T98G cells was evidenced, since verapamil (a characteristic inhibitor of MRP1) increased the cytotoxicity of compound 2b at concentrations up to 20 µmol L-1, whilst GF120918 and Ko143 (specific inhibitors of P-gp and ABCG2, respectively) had no significant effect. In addition, we showed that compound 2b interacts with glutathione (GSH), which could explain its cellular efflux by MRP1. Our results showed that the amphiphilic η6-areneruthenium(II) complexes are promising anti-glioblastoma compounds, especially compound 2b, which was cytotoxic for all three cell lines, although it is transported by the three main multidrug ABC transporters.

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

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 photogenerated 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 spiroiminodihy-dantoin 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.

Synthesis of 8-oxo-7,8-dihydro-2'-deoxyguanosine from 2'-deoxyguanosine using Cu(II)/H2O2/ascorbate: A new strategy for an improved yield.

The free nucleoside 2'-deoxyguanosine (dGuo) is the most susceptible to oxidation by reactive oxygen species (ROS) compared to the other free nucleosides, and its oxidation product 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) has been used as a biomarker to quantify oxidative stress damage. We investigated different reactions using Fe(2+) or Cu(2+) and H2O2 in order to identify the reaction with the best yield. HPLC coupled with a UV detector and micrOTOF mass spectrometry were used to detect and confirm the identity of 8-oxodGuo. The optimized reaction synthesized 8-oxodGuo with a yield of 72.0%, much higher than that previously described in the literature. Our improved method for 8-oxodGuo synthesis could be extremely useful for assays that require the synthesis of internal standards labeled with stable isotopes.

Identification of the main oxidation products of 8-methoxy-2'-deoxyguanosine by singlet molecular oxygen.

It is now well established that oxidation of 2'-deoxyguanosine (dGuo) in DNA by singlet molecular oxygen [O2 (1Delta(g))] produces 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), whereas the main degradation products of free dGuo in aqueous solution have been identified as the two diastereomers of spiroiminodihydantoin nucleoside. Interestingly, O2 (1Delta(g))-mediated oxidation of free 8-oxodGuo gives rise to a pattern of degradation products that is different from that observed when the nucleoside is inserted into DNA. The reasons for these differences and the mechanisms involved in the oxidation reactions are not yet completely understood for either dGuo or 8-oxodGuo, either free or within DNA. In the present work, we report a study of the reaction of O2 (1Delta(g)) toward a modified nucleoside, 8-methoxy-2'-deoxyguanosine (8-MeOdGuo), either free or incorporated into an oligonucleotide. The reason for the choice of 8-MeOdGuo as a chemical model to study in more detail the oxidation pathways of 8-oxodGuo or, more precisely, of the tautomeric 8-hydroxy-2'-deoxyguanosine was dictated by the fact that only the 7,8-enolic tautomer is present in the molecule. The thermolysis of an endoperoxide of a naphthalene derivative as a clean chemical source of 18O-labeled O2 (1Delta(g)) was used to oxidize 8-MeOdGuo. The main O2 (1Delta(g)) oxidation products that were separated and analyzed by HPLC coupled to tandem mass spectrometry were identified as the 2'-deoxyribonucleoside derivatives of 2,2,4-triamino-5-(2H)oxazolone, 2,5-diamino-4H-imidazol-4-one together with the methyl-substituted derivatives of spiroiminodihydantoin, oxidized iminoallantoin and urea. On the other hand, O2 (1Delta(g)) oxidation of 8-MeOdGuo-containing oligonucleotide generated imidazolone as the predominant degradation product. These results provided new mechanistic insights into the reactions of O2 (1Delta(g)) with purine nucleosides.

Oxidative and alkylating damage in DNA.

Modification of cellular DNA upon exposure to reactive oxygen and nitrogen species is the likely initial event involved in the induction of the mutagenic and lethal effects of various oxidative stress agents. Evidence has been accumulated for the significant implication of singlet oxygen (1O(2)), generated as the result of UVA activation of endogenous photosensitizers as porphyrins and flavins. 7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxodGuo) has been shown to be the exclusive product of the reaction of 1O(2) with the guanine moiety of cellular DNA, in contrast to the hydroxyl radical, which reacts almost indifferently with all the nucleobases and the sugar moiety of DNA. Furthermore 8-oxodGuo is also produced by other oxidants and can be used as an ubiquitous biomarker of DNA oxidation but can not be a specific marker of any particular species. The role of DNA etheno adducts in mutagenic and carcinogenic processes triggered by known occupational and environmental carcinogens has also been studied. Much interest in etheno adducts resulted from the detection of increased levels of 1,N(6)-etheno-2'-deoxyadenosine and 3,N(4)-etheno-2'-deoxycytidine in DNA from human, rat and mouse tissues under pathophysiological conditions associated with oxidative stress. A method involving on-line HPLC with electrospray tandem mass spectrometry detection has been developed for the analysis of 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-epsilondGuo) in DNA. This methodology permits direct quantification of 20 fmol (7.4 adducts/10(8) dGuo) of the etheno adduct from approximately 350 microg of crude DNA hydrolysates. This method provides the first evidence of the occurrence of 1,N(2)-epsilondGuo as a basal endogenous lesion and may be utilized to better assess the biological consequences of etheno DNA damage under normal and pathological conditions. This work addresses the importance of isotope labeling associated with mass spectrometry technique for biomolecule damage studies.

18O-Labeled lipid hydroperoxides and HPLC coupled to mass spectrometry as valuable tools for studying the generation of singlet oxygen in biological system.

Decomposition of lipid hydroperoxides (LOOH) is known to generate toxic products capable to induce tissue injury. We have recently confirmed that decomposition of LOOH into peroxyl radicals is a potential source of singlet oxygen ((1)O(2) in biological system. Using (18)O-labeled linoleic acid hydroperoxide (LA(18)O(18)OH) in the presence of Ce(4+) or Fe(2+), we observed the formation of (18)O-labeled (1)O(2) ((18)[(1)O(2)]) by chemical trapping of (1)O(2) with 9,10-diphenylanthracene (DPA) and detecting the corresponding (18)O-labeled DPA endoperoxide (DPA(18)O(18)O) by HPLC coupled to tandem mass spectrometry (HPLC-MS/MS). (18)O-Labeled alcohol and ketone were also detected providing further evidence for the generation of (1)O(2) by the Russell mechanism. Similarly the reaction of LA(18)O(18)OH with peroxynitrite also generated (18)[(1)O(2)].In conclusion, these results indicates that the use of (18)O-labeled LOOH associated with HPLC-MS/MS can be an useful tool to clarify mechanistic features involved in the reaction of LOOH in biological media.

Singlet molecular oxygen generated by biological hydroperoxides.

The chemistry behind the phenomenon of ultra-weak photon emission has been subject of considerable interest for decades. Great progress has been made on the understanding of the chemical generation of electronically excited states that are involved in these processes. Proposed mechanisms implicated the production of excited carbonyl species and singlet molecular oxygen in the mechanism of generation of chemiluminescence in biological system. In particular, attention has been focused on the potential generation of singlet molecular oxygen in the recombination reaction of peroxyl radicals by the Russell mechanism. In the last ten years, our group has demonstrated the generation of singlet molecular oxygen from reactions involving the decomposition of biologically relevant hydroperoxides, especially from lipid hydroperoxides in the presence of metal ions, peroxynitrite, HOCl and cytochrome c. In this review we will discuss details on the chemical aspects related to the mechanism of singlet molecular oxygen generation from different biological hydroperoxides.

Excited singlet molecular O2(¹Δg) is generated enzymatically from excited carbonyls in the dark.

In mammalian tissues, ultraweak chemiluminescence arising from biomolecule oxidation has been attributed to the radiative deactivation of singlet molecular oxygen [O2 ((1)Δg)] and electronically excited triplet carbonyl products involving dioxetane intermediates. Herein, we describe evidence of the generation of O2 ((1)Δg) in aqueous solution via energy transfer from excited triplet acetone. This involves thermolysis of 3,3,4,4-tetramethyl-1,2-dioxetane, a chemical source, and horseradish peroxidase-catalyzed oxidation of 2-methylpropanal, as an enzymatic source. Both sources of excited carbonyls showed characteristic light emission at 1,270 nm, directly indicative of the monomolecular decay of O2 ((1)Δg). Indirect analysis of O2 ((1)Δg) by electron paramagnetic resonance using the chemical trap 2,2,6,6-tetramethylpiperidine showed the formation of 2,2,6,6-tetramethylpiperidine-1-oxyl. Using [(18)O]-labeled triplet, ground state molecular oxygen [(18)O2 ((3)Σg(-))], chemical trapping of (18)O2 ((1)Δg) with disodium salt of anthracene-9,10-diyldiethane-2,1-diyl disulfate yielding the corresponding double-[(18)O]-labeled 9,10-endoperoxide, was detected through mass spectrometry. This corroborates formation of O2 ((1)Δg). Altogether, photoemission and chemical trapping studies clearly demonstrate that chemically and enzymatically nascent excited carbonyl generates (18)O2 ((1)Δg) by triplet-triplet energy transfer to ground state oxygen O2 ((3)Σg(-)), and supports the long formulated hypothesis of O2 ((1)Δg) involvement in physiological and pathophysiological events that might take place in tissues in the absence of light.

Flavone induces cell death in human hepatoma HepG2 cells.

Flavones have received considerable attention because of their antiproliferative properties and selective effects on cancer cells, making them good candidates for use in cancer therapy. In contrast to other flavones, little is known about the effects of the flavone core structure (2-phenyl-4H-1-benzopyran-4one) on cancer cells. Here, we report that flavone induces cell death in human hepatoma HepG2 cells. Furthermore, annexin-V+/PI- and SubG1 populations of HepG2 cells increased after flavone treatment. Exposure of HepG2 to flavone did not result in either cytochrome c release into the cytosol or changes in the mitochondrial membrane potential. Treatment of HepG2 cells with flavone for 24 h reduced the accumulation of intracellular ROS, which correlated with upregulation of Gred, CuZnSOD and MnSOD mRNA levels. Taken together, our results provided useful insights into the mechanism of cell death caused by flavones, in order to evaluate their future application in hepatocarcinoma therapy.

Melanin photosensitization and the effect of visible light on epithelial cells.

Protecting human skin from sun exposure is a complex issue that involves unclear aspects of the interaction between light and tissue. A persistent misconception is that visible light is safe for the skin, although several lines of evidence suggest otherwise. Here, we show that visible light can damage melanocytes through melanin photosensitization and singlet oxygen (1O2) generation, thus decreasing cell viability, increasing membrane permeability, and causing both DNA photo-oxidation and necro-apoptotic cell death. UVA (355 nm) and visible (532 nm) light photosensitize 1O2 with similar yields, and pheomelanin is more efficient than eumelanin at generating 1O2 and resisting photobleaching. Although melanin can protect against the cellular damage induced by UVB, exposure to visible light leads to pre-mutagenic DNA lesions (i.e., Fpg- and Endo III-sensitive modifications); these DNA lesions may be mutagenic and may cause photoaging, as well as other health problems, such as skin cancer.

DNA damage by singlet oxygen and cellular protective mechanisms.

Reactive oxygen species, as singlet oxygen ((1)O(2)) and hydrogen peroxide, are continuously generated by aerobic organisms, and react actively with biomolecules. At excessive amounts, (1)O(2) induces oxidative stress and shows carcinogenic and toxic effects due to oxidation of lipids, proteins and nucleic acids. Singlet oxygen is able to react with DNA molecule and may induce G to T transversions due to 8-oxodG generation. The nucleotide excision repair, base excision repair and mismatch repair have been implicated in the correction of DNA lesions induced by (1)O(2) both in prokaryotic and in eukaryotic cells. (1)O(2) is also able to induce the expression of genes involved with the cellular responses to oxidative stress, such as NF-κB, c-fos and c-jun, and genes involved with tissue damage and inflammation, as ICAM-1, interleukins 1 and 6. The studies outlined in this review reinforce the idea that (1)O(2) is one of the more dangerous reactive oxygen species to the cells, and deserves our attention.