In vitro phototoxic properties of new 6-desfluoro and 6-fluoro-8-methylquinolones

A representative set of potent antibacterial 6-desfluoro-8-methylquinolones, in which the C-6 fluorine atom is replaced by –NH₂ or –H, and their 6-fluoro counterparts, were investigated to evaluate their phototoxic potential and to explore the mechanism behind their phototoxicity. The capacity to photosensitize biological substrates (lipids, proteins, DNA) has been analyzed, as well as their photocytotoxicity on red blood cells and 3T3 murine fibroblasts. The results obtained show that the quinolones studied are able to photosensitize red blood cell lysis in an oxygen-dependent way and induce a high decrease in cell viability after UVA irradiation. A major correlation with phototoxicity lies in the structure of the individual antibacterials and their hydrophobicity; in particular, 6-amino derivatives are less phototoxic than corresponding unsubstituted and fluorinated compounds. Cellular phototoxicity was inhibited by the addition of free radical and hydroxyl radical scavengers (BHA, GSH and DMTU), suggesting the involvement of a radical mechanism in their cytotoxicity. A good correlation was observed between lipid peroxidation and phototoxicity, indicating that the test compounds exert their toxic effects mainly in the cellular membrane. Preliminary experiments on pBR322 DNA show that these derivatives do not photocleave DNA, differently from the two photogenotoxic fluoroquinolones, ciprofloxacin and lomefloxacin, used as reference compounds.     

In vitro method for prediction of the phototoxic potentials of fluoroquinolones

The phototoxic potential of eight fluoroquinolones (norfloxacin, ofloxacin, enoxacin, ciprofloxacin, lomefloxacin, tosufloxacin, sparfloxacin and gatifloxacin) was evaluated by using three in vitro methods of cytotoxicity against mammalian cells, erythrocyte lysis and DNA strand breakage. All fluoroquinolones tested with the exception of gatifloxacin, an 8-methoxy quinolone, showed DNA strand breaking activities under UV-A irradiation. Their cytotoxicity against HeLa cells was also enhanced by UV-A irradiation. In particular, the phototoxic potential of sparfloxacin, enoxacin and lomefloxacin was high in both methods. Ofloxacin is very photocytotoxic against HeLa cells, while it has low potential to cause DNA strand breakage. Norfloxacin, ciprofloxacin and enoxacin were very photohemolytic, but sparfloxacin was not, indicating that the in vivo phototoxic potencies of fluoroquinolones might not be predictable by the photohemolysis study. Gatifloxacin, a non-phototoxic quinolone, showed no phototoxic potential in any of these three in vitro tests. These results suggest that determination of DNA strand breaking activity, combined with cytotoxicity against mammalian cells, is available to predict the phototoxic potential of fluoroquinolones without laboratory animals.     

Ferritin enhances salsolinol-mediated DNA strand breakage: Protection by carnosine and related compounds

Salsolinol is an endogenous neurotoxin, known to be involved in the pathogenesis of neurodegenerative disorders. In this present study, we have attempted to characterize the oxidative damage of DNA induced by the reaction of salsolinol with ferritin. When DNA was incubated with salsolinol and ferritin, DNA strand breakage occurred. Hydroxyl radical scavengers and catalase reduced salsolinol/ferritin system-mediated DNA cleavage, whereas Cu,Zn-superoxide dismutase did not inhibit DNA cleavage. The reaction of salsolinol with ferritin resulted in a time-dependent increase in the release of free iron ions. A strong iron chelator, ferrozine, effectively inhibited the salsolinol/ferritin system-mediated DNA cleavage. Ferritin enhanced a mutation of the lacZ′ gene in the presence of salsolinol when measured as a loss of α-complementation. These results indicate that salsolinol/ferritin system-mediated DNA cleavage and mutation may be attributable to hydroxyl radical generation via the Fenton-like reaction of free iron ions released from oxidatively damaged ferritin. The endogenous dipeptides, carnosine and related compounds, are naturally occurring compounds with a multiplicity of neuroprotective properties. Carnosine, homocarnosine and anserine significantly inhibited salsolinol/ferritin system-mediated DNA strand breakage and mutation. These results indicate that carnosine and related compounds effectively suppressed the salsolinol/ferritin system-mediated DNA strand breakage via hydroxyl radical scavenging.     

Oxidative damage of DNA induced by methylglyoxal in vitro

Methylglyoxal is an endogenous metabolic by-product of glycolysis and has genotoxic effects. Previous studies suggested that the reaction of methylglyoxal with amino acid leads to the production of free radicals. In this study, oxidative damage of DNA by the reaction of methylglyoxal with amino acid was investigated. When plasmid DNA was incubated with methylglyoxal and lysine, DNA strand was cleaved. Cu(2+) enhanced DNA strand breakage induced by the reaction of methylglyoxal with lysine. The formation of superoxide anion was detected during the glycation reaction of methylglyoxal with lysine. Radical scavengers, catalase, and copper chelators inhibited the DNA breakage. The deoxyribose assay showed that hydroxyl radicals were generated during the reaction of methylglyoxal with lysine in the presence of Cu(2+). The generation of hydroxyl radicals was inhibited by radical scavenger, catalase, and copper chelator. These results suggest that superoxide anion and H2O2 may generate from the glycation reaction of methylglyoxal with lysine and then Cu(2+) likely participates in a Fenton's type reaction to produce hydroxyl radicals, which may cause DNA cleavage. This mechanism may be linked to several diverse biological processes including mutagenesis, aging, carcinogenesis, and diabetic complications.     

Reaction of chromium (VI) with hydrogen peroxide in the presence of glutathione: reactive intermediates and resulting DNA damage

The reaction of chromium(VI) with hydrogen peroxide was studied in the presence of glutathione. In vitro, reaction of chromium(VI) with hydrogen peroxide alone led to production of hydroxyl radical as the significant reactive intermediate, while reaction of chromium(VI) with glutathione led to formation of two chromium(V)-glutathione complexes and the glutathione thiyl radical. Incubation of chromium(VI) with glutathione prior to addition of hydrogen peroxide led to formation of peroxochromium(V) species and a dramatic increase in hydroxyl radical production over that detected in the reaction of chromium(VI) with hydrogen peroxide alone. In contrast, addition of chromium(VI) to a preincubated mixture of glutathione and hydrogen peroxide led to a decrease in hydroxyl radical production over that obtained in the reaction of chromium(VI) with hydrogen peroxide. When pBR322 DNA was added to the above reactions, the extent of chromium(VI)-induced DNA strand breakage correlated with the relative amount of hydroxyl radical formed. Reaction of chromium(VI) with calf thymus DNA in the presence of a preincubated mixture of glutathione and hydrogen peroxide led to detection of the 8-hydroxydeoxyguanosine adduct, whose formation correlated with that of hydroxyl radical production. No significant chromium-DNA adduct formation was detected. The results suggest that, in the cellular metabolism of chromium(VI), preformed chromium(V)-glutathione complexes may react with hydrogen peroxide in a Fenton-type manner to produce hydroxyl radical as the DNA-damaging agent. However, if glutathione reacts with hydrogen peroxide prior to exposure to chromium(VI), the amount of hydroxyl radical generated may not be sufficient to cause significant DNA damage.(ABSTRACT TRUNCATED AT 250 WORDS)     

姜黄素对长波紫外线所致DNA损伤的拮抗作用

目的　观察长波紫外线引起人表皮样癌细胞 DNA链断裂损伤的情况 ,以及姜黄素对这种损伤的拮抗作用。方法　用长波紫外线照射细胞 ,用单细胞凝胶电泳法检测 DNA损伤。结果　长波紫外线剂量依赖性地诱发表皮细胞的 DNA链断裂损伤 ,损伤的高峰出现在照射后 1h。而姜黄素可以拮抗长波紫外线引起的DNA链断裂 ,表现为彗星细胞百分比的降低和彗星尾巴长度的缩短 ,并有量效关系。结论　姜黄素可以预防长波紫外线照射引起的皮肤细胞 DNA链断裂损伤。     

Prooxidant action of furanone compounds: implication of reactive oxygen species in the metal-dependent strand breaks and the formation of 8-hydroxy-2'-deoxyguanosine in DNA.

Prooxidant properties of furanone compounds including 2,5-furanone (furaneol, 4-hydroxy-2,5-dimethyl-furan-3-one), 4,5-furanone (4,5-dimethyl-3-hydroxy-2(5H)-furanone) (sotolone) and cyclotene (2-hydroxy-3-methyl-2-cyclopenten-1-one) were analyzed in relation to the metal-reducing activity. Only 2.5-furanone known as a "strawberry or pineapple furanone" inactivated aconitase the most sensitive enzyme to active oxygen in the presence of ferrous sulfate, suggesting the furaneol/iron-mediated generation of reactive oxygen species. 2,5-Furanone caused strand scission of pBR322 DNA in the presence of copper. Treatment of calf thymus DNA with 2,5-furanone plus copper produced 8-hydroxy-2'-deoxyguanosine in DNA. 2,5-Furanone showed a potent copper-reducing activity, and thus, DNA strand breaks and the formation of 8-hydroxy-2'-deoxyguanosine by 2,5-furanone can be initiated by the production of superoxide radical through the reduction of cupric ion to cuprous ion, resulting in the conversion to hydrogen peroxide and hydroxyl radical. However, an isomer and analog of 2,5-furanone, 4,5-furanone and cyclotene, respectively, did not show an inactivation of aconitase, DNA injuries including strand breakage and the formation of 8-hydroxy-2'-deoxyguanosine, and copper-reducing activity. Cytotoxic effect of 2,5-furanone with hydroxyketone structure can be explained by its prooxidant properties: furaneol/transition metal complex generates reactive oxygen species causing the inactivation of aconitase and the formation of DNA base damage by hydroxyl radical.     

Metal- and photo-induced cleavage of DNA by podophyllotoxin, etoposide, and their related compounds.

Podophyllotoxin (PD) and its derivative etoposide (VP-16), a clinically useful anticancer drug, exhibit different mechanisms of action. PD binds specifically to tubulin to prevent its polymerization, whereas VP-16 lacks this action. The DNA strand breakage caused by VP-16 is thought to be due to its interaction with topoisomerase II or to free radical formation by oxidation of its 4'-phenolic hydroxyl group to a semiquinone free radical. We have demonstrated that PD, VP-16, 4'-demethylepipodophyllotoxin (DEPD), and syringic acid (SA) exhibit no DNA-cleaving activity but, in the presence of metal ions such as Cu2+ and Fe3+, DEPD and SA form metal complexes, which in turn show high activity for DNA strand scission at pH 7.8 under air. Furthermore, it was found that DNA cleavage was greatly promoted by irradiation with UV light. The PD-Fe3+ system at pH 7.8 showed very low DNA-cleaving activity, but irradiation with UV light in the system induced almost complete DNA breakage. DNA cleavages were significantly inhibited in the presence of hydroxyl radical scavengers, such as sodium benzoate and dimethylurea, in the Cu(2+)-SA and Fe(3+)-PD systems, with or without UV irradiation. These reactions were investigated by optical and ESR spectra, coupled with ESR spin-trapping techniques, by which the formation of hydroxy radicals was clearly detected in all systems. These findings have led us to a new proposal of the metal- and photo-induced mechanism for understanding the antitumor action of PD, VP-16, and their related compounds.     

In vitro/in vivo phototoxic risk assessments of griseofulvin based on photobiochemical and pharmacokinetic behaviors

The present investigation aims to establish efficacious screening strategy to clarify the phototoxic potential of pharmaceutical substances and its possible pathways by characterizing both photobiochemical properties and pharmacokinetic profiles. Photochemical behavior of griseofulvin, as model compounds, was evaluated by reactive oxygen species (ROS) assay, and the photogenotoxic potential was also assessed by DNA binding assay, DNA photocleavage assay, and atomic force microscopy. Pharmacokinetic (PK) study was also carried out after dermal and oral administration of griseofulvin in rats. ROS assay suggested the phototoxic potential of griseofulvin via type II photochemical pathways, and the photogenotoxic risk of griseofulvin was also proposed as evidenced by high affinity toward DNA and potent DNA photocleaving activity. PK profiling and in vivo phototoxicity testing demonstrated that a highly concentrated griseofulvin in the skin might cause phototoxic skin reactions in rats, whereas oral administration of griseofulvin in single dosing regimen (20 mg/kg) resulted in 10³-fold less skin deposition than phototoxic skin concentration of griseofulvin. Upon these findings, the phototoxic potential of griseofulvin might not be severe at least in a single oral dosing regimen, whereas it might be phototoxic in dermal administration. The combination use of photobiochemical and pharmacokinetic data would be valuable to provide reliable prediction on phototoxic risk and possible toxic pathways of new drug entities in the early stage of drug discovery.     

In vitro studies of the phototoxic potential of the antidepressant drugs amitriptyline and imipramine

Amitriptyline and imipramine, two tricyclic antidepressant drugs, have been studied to evaluate their phototoxic potential using various models. Reactive oxygen species production was investigated. A negligible production of singlet oxygen was observed for both compounds whereas a significant production of superoxide anion was noted for amitriptyline in particular. Moderate red blood cell lysis under UVA light (365 nm) was induced in the presence of the two drugs at a concentration of 50 microM. Cellular phototoxicity was investigated on a murine fibroblast cell line (3T3). The two drugs were phototoxic causing cell death at a concentration of 100 microM and a UVA dose in the range of 3.3-6.6 J/cm2. Furthermore, the two drugs photosensitized the peroxidation of linoleic acid, as monitored by the formation of dienic hydroperoxides. The presence of BHA and GSH, two free radical scavengers, significantly reduced the lipid oxidation photoinduced by the drugs, suggesting a predominant involvement of radical species. Finally, the involvement of nucleic acids in the phototoxicity mechanism was also investigated using a pBR322 plasmid DNA as a model.     

Myricetin affords protection against peroxynitrite-mediated DNA damage and hydroxyl radical formation

Peroxynitrite has been extensively implicated in the pathogenesis of various forms of neurodegenerative disorders via its cytotoxic effects, this study was undertaken to investigate whether the neuroprotective effect of myricetin is associated with inhibition of peroxynitrite-mediated DNA damage, a critical event leading to peroxynitrite elicited cytotoxicity. We observed that peroxynitrite can cause DNA damage in ?X-174 plasmid DNA and rat primary astrocytes. The presence of myricetin at physiological concentration was found to significantly inhibit DNA strand breakage induced by both peroxynitrite and its generator 3-morpholinosydnonimine (SIN-1). Moreover, the consumption of oxygen caused by SIN-1 was found to be decreased in the presence of myricetin, indicating that myricetin might affect the auto-oxidation of SIN-1. Furthermore, EPR spectroscopy demonstrated that the formation of DMPO-hydroxyl radical adduct (DMPO-OH) from peroxynitrite, and that myricetin inhibited the adduct signal in a concentration-dependent manner. Taken together, these results demonstrate for the first time that myricetin can inhibit peroxynitrite-mediated DNA damage and hydroxyl radical formation.     

Novel Methodology for Predicting Photogenotoxic Risk of Pharmaceutical Substances Based on Reactive Oxygen Species (ROS) and DNA-Binding Assay

Drug-induced phototoxic skin responses involves photoirritation, photoallergy, and photogenotoxicity. In the present study, we attempted to develop effective analytical tools to predict the photogenotoxic potential of pharmaceutical substances on the basis of their photobiological and DNA-binding properties. Photocleavage of plasmid DNA was assessed by capillary gel electrophoresis (CGE), and generation of reactive oxygen species (ROS) from photoirradiated photosensitizers was also assessed by spectrophotometrical determination. The affinity of drugs with DNA was determined by fluorescent titration or competitive binding assay using ethidium bromide. Structural transition from supercoiled form to open circular form of plasmid DNA were observed in the presence of nine irradiated-photosensitizers, however DNA damage by six phototoxic and five nonphototoxic drugs were negligible or weak even though some chemicals exhibited significant generation of ROS. DNA photocleavers tended to interact with DNA, binding constants of which were found to be less than 1 µM, whereas most nonphotogenotoxic chemicals failed to bind with DNA. The differences in DNA-binding properties might be attributed to a data discrepancy between ROS-generation and DNA photocleaving activity. The combination use of ROS assay and DNA-binding assay could be fast and reliable prediction for the photogenotoxic potential of a large number of drug candidates at the drug-discovery stage. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3647–3658, 2009     

High-throughput screening strategy for photogenotoxic potential of pharmaceutical substances using fluorescent intercalating dye

The aim of the present study was to provide an intercalator-based photogenotoxicity (IBP) assay as a high-throughput in vitro screening system for predicting the photogenotoxic potential of pharmaceutical substances. The conditions of the high-throughput IBP assay using thiazole orange (TO), a fluorescent intercalating dye, were optimized and validated by a fluorescence titration experiment and reproducibility/robustness test. The IBP assay was applied to 27 phototoxic and 5 weak/non-phototoxic commercially available compounds, and other phototoxicity screenings were also carried out for comparison; these included the reactive oxygen species (ROS) assay for overall phototoxic potential and the DNA-photocleavage assay for photogenotoxic risk. According to the results from the comparative experiments, a decreased level of intercalated TO in the IBP assay could theoretically be related to the DNA-photocleaving behaviors of phototoxic drugs, but not to their ROS-generating abilities. The IBP assay could predict the photodynamic DNA impairment caused by irradiated drugs with a prediction accuracy of 78%. These findings suggest that the IBP assay could be a fast and reliable tool for predicting the photogenotoxic potential of a large number of drug candidates at early stages of drug discovery.     

Nickel induces oxidative stress and genotoxicity in human lymphocytes

In order to elucidate the oxidative effects and genotoxicity of nickel on human lymphocytes in vitro, we report the level of intracellular reactive oxygen species (ROS), lipid peroxidation, hydroxyl radical ((*)OH), and DNA damage in human lymphocytes after acute exposure to inorganic nickel. NiCl(2) appeared to increase the formation of the fluorescent oxidized compound dichlorofluorescein (DCF). Lipid peroxidation in lymphocytes significantly increased compared to control. 2,3- and 2,5-DHB increased markedly in a concentration-dependent manner. Single-strand DNA breakage induced by Ni in lymphocytes was evaluated by Comet assay. Significant increase in DNA damage score (arbitrary units) showed a dose-related elevation after treatment with NiCl(2). NiCl(2) induced lipid peroxidation at 0.5 mM but had no effect on DNA strand breakage. These results support the emerging concept that NiCl(2)-induced oxidative stress and genotoxicity may be caused by oxygen radical intermediates. NiCl(2)-induced DNA strand breakage is related to the generation of the (*)OH radical.     

Effects of UVA and visible light on the photogenotoxicity of benzo[a]pyrene and pyrene

This study investigated the role of UVA/visible light (U, 320–800 nm) and visible light (V, 400–800 nm) in the phototoxicity and photogenotoxicity of two ubiquitous polycyclic aromatic hydrocarbons (PAH): benzo[a]pyrene (BaP) and Pyrene (Pyr). These mechanisms were evaluated by the WST‐1 test and the comet assay on normal human keratinocytes (NHK) and by the micronucleus test on CHO cells. The production of reactive oxygen species (ROS) was assessed through the induction of 8‐oxodeoxyguanine (8‐oxodG) lesions by immunofluorescence staining in NHK. Results of the WST‐1 test revealed the phototoxic properties of BaP and Pyr after irradiation with U and V lights. BaP presented the highest phototoxic properties. Results of the comet assay showed that U‐ and V‐irradiated BaP and Pyr induced increasing rates of DNA single‐strand breaks in NHK, in a dose dependent manner. The tested PAH could also induce increased levels of micronuclei in CHO cells after U and V irradiations. Increasing 8‐oxodG levels were detected after U and V irradiations in BaP‐ and Pyr‐treated keratinocytes and confirmed the involvement of ROS in the photogenotoxicity of PAH. Overall, this study highlighted the existence of an alternative pathway of PAH genotoxicity that is induced by UVA and/or visible light. Visible light is suggested to photoactivate PAH by a mechanism which is mainly based on oxidative reactions. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2009.     

Coexposure to benzo[a]pyrene plus ultraviolet A induces 8-oxo-7,8-dihydro-2'-deoxyguanosine formation in human skin fibroblasts: preventive effects of anti-oxidant agents

The influence of environmental benzo[a]pyrene (BaP) contamination under ultraviolet A (UVA) on normal human skin fibroblasts was examined. Treatment of human skin fibroblasts with UVA in the presence of BaP induced cytotoxicity in a UVA- and BaP-dose-dependent manner, involving oxidative DNA damage (formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG)). Singlet oxygen quenchers significantly inhibited the formation of 8-oxo-dG, whereas hydroxyl radical and superoixide anion radical scavengers showed no effect. N-Acetyl-l-cysteine prevented the formation of 8-oxo-dG. These findings suggested the possibility of increased carcinogenesis in the skin via singlet oxygen produced by sunlight plus environmental BaP contamination and the efficiency of anti-oxidant agents for its prevention.     

Prooxidant action of chebulinic acid and tellimagrandin I: Causing copper-dependent DNA strand breaks

The prooxidant activity of two hydrolysable tannins, chebulinic acid and tellimagrandin I, on plasmid DNA and genomic DNA of cultured MRC-5 human embryo lung fibroblasts was assessed. The results revealed that both hydrolysable tannins in combination with Cu(II) induced DNA strand breaks in pBR322 plasmid DNA in a concentration-dependent manner. Chebulinic acid and tellimagrandin I also induced genomic DNA strand breaks of MRC-5 human embryo lung fibroblasts in the presence of Cu(II). After treatment with chebulinic acid or tellimagrandin I alone, the pBR322 plasmid DNA and genomic DNA in MRC-5 cells kept intact. In addition, addition of Cu(I) reagent bathocuproinedisulfonic acid or catalase markedly inhibited the copper-dependent DNA strand breaks by both tannins. However, three typical hydroxyl radical scavengers, DMSO, ethanol and mannitol, did not inhibit the DNA strand breaks. Both tannins were able to reduce Cu(II) to Cu(I). These results indicated that chebulinic acid and tellimagrandin I induced the copper-dependent strand breaks of pBR322 plasmid DNA and MRC-5 genomic DNA with prooxidant action, in which Cu(II)/Cu(I) redox cycle and H₂O₂ were involved and hydroxyl radical formation is important in the hypothetical mechanism by which DNA strand breaks are formed.     

Shock, inflammation and PARP.

The activation of poly(ADP-ribose) polymerase (PARP) is well considered to play an important role in various patho-physiological conditions like inflammation and shock. A vast amount of circumstantial evidence implicates oxygen-derived free radicals (especially, superoxide and hydroxyl radical) and high-energy oxidants (such as peroxynitrite) as mediators of inflammation and shock. ROS (e.g., superoxide, peroxynitrite, hydroxyl radical and hydrogen peroxide) are all potential reactants capable of initiating DNA single strand breakage, with subsequent activation of the nuclear enzyme poly(ADP-ribose) synthetase (PARS), leading to eventual severe energy depletion of the cells, and necrotic-type cell death. During the last years, numerous experimental studies have clearly demonstrated the beneficial effects of PARP inhibition in cell cultures through rodent models and more recently in pre-clinical large animal models of acute and chronic inflammation. The aim of this review is to describe recent experimental evidence implicating PARP as a pathophysiological modulator of acute and chronic inflammation.     

DNA damage induced in cells by gamma and UVA radiation as measured by HPLC/GC-MS and HPLC-EC and Comet assay

The aim of the work was to measure DNA damage induced within tumoral human monocytes by gamma rays, UVA radiation, and exogenous photosensitizers. The accurate HPLC-EC assay was used to determine the level of 8-oxodGuo. The formation of FapyGua and FapyAde was monitored by HPLC/GC-MS analyses after formic acid hydrolysis at room temperature. For this purpose, cells were exposed to relatively high doses of gamma rays and UVA radiation. The extent of formation of FapyGua in the DNA of cells exposed to gamma rays was estimated to be more than 2-fold higher than that of 8-oxodGuo, i.e., about 0. 027 lesion per 10(6) bases per Gy. The yield of FapyAde was estimated to be 1 order of magnitude lower. The latter results were used to calibrate the alkaline comet assay associated with DNA N-glycosylases. The latter approach allowed the determination of the background level (0.11-0.16 Fpg-sensitive site/10(6) bases) and the yields of strand breaks and DNA base damage upon low irradiation doses. Insights into the mechanism of radiation-induced DNA damage were gained from these measurements. A major involvement of (1)O(2) with respect to hydroxyl radicals and type I photosensitization was thus observed within cells exposed to UVA radiation.