Damage to mitochondria of cultured human skin fibroblasts photosensitized by fluoroquinolones

The phototoxic fluoroquinolones ofloxacin, lomefloxacin, norfloxacin, ciprofloxacin and BAYy 3118 have ionizable groups with pKa values close to neutrality. Different ionic species of these fluoroquinolones, therefore, partition in various compartments and organelles of living cells according to their ionic equilibria. While all these fluoroquinolones accumulate in lysosomes, they more or less stain the rest of the cytoplasm of living HS 68 fibroblasts. As a result, photosensitized damage to other cytoplasmic sites than lysosomes can also be expected. Using microfluorometry and rhodamine 123 (Rh 123) as a specific fluorescent probe which is released from mitochondria by light absorption, we show that under ultraviolet A (UVA) irradiation norfloxacin and ciprofloxacin readily damage mitochondrial membranes. as evidenced by the UVA dose-dependent strongly accelerated release of Rh 123 from mitochondria in cells treated with norfloxacin and ciprofloxacin. Damages are already noticeable at UVA doses as low as 2 J/cm2. By contrast, no such photoinduced damage can be observed with ofloxacin, lomefloxacin and BAYy 3118, the latter being the most phototoxic derivative towards HS 68 fibroblasts. The initial photodamage induced by norfloxacin and ciprofloxacin can then propagate after the irradiation as shown by the strongly increased rate of release of Rh 123 from mitochondria of cells that have been incubated with these two fluoroquinolones and left in the dark after a pre-irradiation with 18 J/cm2 of UVA. Interestingly, the same pre-irradiation after cells have been treated with BAYy 3118 and lomefloxacin induces similar post-irradiation effects, although they have no apparent immediate photosensitizing action on mitochondria.     

Alteration of the endocytotic pathway by photosensitization with fluoroquinolones

The endocytotic pathway is profoundly altered by the UVA-induced photosensitization of HS 68 fibroblasts by the fluoroquinolone (FQ) antibiotics lomefloxacin, BAYy 3118, norfloxacin and ciprofloxacin, which preferentially localize in lysosomes. The endocytosis of low-density lipoproteins (LDL) loaded with two carbocyanine dyes compatible for effective Forster-type resonance energy transfer (FRET), namely 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) as the donor and 1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) as the acceptor, has been used as a model system. Binding of LDL to their cell surface receptors is impaired by irradiation with 10 J cm(-2) of UVA and/or treatment with 250 microM BAYy 3118 during 2 h. Perturbation of the plasma membrane by the FQ is revealed by the change in the rate of exchange of DiO from the LDL to the cell membrane as compared to untreated cells. The lysosomal degradation of LDL, demonstrated by the disappearance of FRET between DiO and DiI, is partly inhibited by the FQ. The actin filament network, involved in the fusion of mature endosomes with lysosomes, is readily destroyed upon photosensitization with the four FQ. However, actin depolymerization can be avoided by incubation of the cells with trans-epoxysuccinyl-1-leucylamido-(4-guanidino)butane, an inhibitor of lysosomal cathepsins prior to FQ photosensitization. All these data suggest that several components of the endocytotic pathway are impaired by photosensitization with these FQ.     

Lysosomes Are Sites of Fluoroquinolone Photosensitization in Human Skin Fibroblasts: A Microspectrofluorometric Approach*

The fluoroquinolone antibiotics are widely used despite their strong phototoxicity under solar UV irradiation. Although they are known as good photodynamic photosensitizers, other factors than production of activated oxygen species may play a role in the effectiveness of the phototoxic effect. Subcellular localization is one of the important parameters that may determine this strength. Using microspectrofluorometry, it is shown that norfloxacin, ofloxacin, lomefloxacin, ciproflaxin and BAYy3118 are readily incorporated into lysosomes of HS68 human skin fibroblasts although weak staining of the whole cytoplasm also occurs especially with norfloxacin. Consistent with their photoinstability in solutions, the fluoroquinolones under study are readily photobleached by UVA in the HS68 fibroblasts. The BAYy3118 derivative that has the fastest bleaching rate also shows the strongest phototoxicity toward HS68 fibroblasts. Photosensitization with these fluoroquinolones induces lysosomal membrane damage as shown by the increased rate of leakage of the lysosomal probe lucifer yellow as compared to that observed with untreated cells.     

Analysis of fluoroquinolone-mediated photosensitization of 2'-deoxyguanosine, calf thymus and cellular DNA: determination of type-I, type-II and triplet-triplet energy transfer mechanism contribution

Fluoroquinolone (FQ) antibacterials are known to exhibit photosensitization properties leading to the formation of oxidative damage to DNA. In addition, photoexcited lomefloxacin (Lome) was recently shown to induce the formation of cyclobutane pyrimidine dimers via triplet-triplet energy transfer. The present study is aimed at gaining further insights into the photosensitization mechanisms of several FQ including enoxacin (Enox), Lome, norfloxacin (Norflo) and ofloxacin (Oflo). This was achieved by monitoring the formation of DNA base degradation products upon UVA-mediated photosensitization of 2'-deoxyguanosine, isolated and cellular DNA. Oflo and Norflo act mainly via a Type-II mechanism whereas Lome and, to a lesser extent, Enox behave more like Type-I photosensitizers. However, the extent of oxidative damage was found to be relatively low. In contrast, it was found that cyclobutane thymine dimers represent the major class of damage induced by Enox, Lome and Norflo within isolated and cellular DNA upon UVA irradiation. This striking observation confirms that FQ are able to promote efficient triplet energy transfer to DNA. The levels of photosensitized formation of strand breaks, alkali-labile sites and oxidative damage to cellular DNA, as measured by the comet assay, were confirmed to be rather low. Therefore, we propose that the phototoxic effects of FQ are mostly accounted for energy transfer mechanism rather than by Type-I or -II photosensitization processes.     

Genotoxicity of visible light (400-800 nm) and photoprotection assessment of ectoin, L-ergothioneine and mannitol and four sunscreens

This study was designed to determine the genotoxic effects of visible (400-800nm) and ultraviolet A (UVA)/visible (315-800nm) lights on human keratinocytes and CHO cells. The alkaline comet assay was used to quantify DNA-damage. In addition, photo-dependent cytogenetic lesions were assessed in CHO cells by the micronucleus test. Three protective compounds [ectoin, l-ergothioneine (ERT) and mannitol] were tested with the comet assay for their effectiveness to reduce DNA single-strand breaks (SSB). Finally, the genomic photoprotections of two broad-band sunscreens and their tinted analogues were assessed by the comet assay. The WST-1 cytotoxicity assay revealed a decrease of the keratinocyte viability of 30% and 13% for the highest UVA/visible and visible irradiations (15 and 13.8J/cm(2), respectively). Visible as well as UVA/visible lights induced DNA SSB and micronuclei, in a dose-dependent manner. The level of DNA breakage induced by visible light was 50% of the one generated by UVA/visible irradiation. However, UVA radiations were 10 times more effective than visible radiations to produce SSB. The DNA lesions induced by visible and UVA/visible lights were reduced after a 1-h preincubation period with the three tested compounds. The maximal protective effects were 92.7%, 97.9% and 52.0% for ectoin (0.1mM), ERT (0.5mM) and mannitol (1.5mM), respectively, against visible light and 68.9%, 59.8% and 62.7% for ectoin (0.1mM), ERT (0.5mM) and mannitol (1.5mM), respectively, against UVA/visible light. Thus, visible light was genotoxic on human keratinocytes and CHO cells through oxidative stress mechanisms similar to the ones induced by UVA radiations. The four tested sunscreens efficiently prevented DNA lesions that were induced by both visible and UVA/visible irradiations. The tinted sunscreens were slightly more effective that their colorless analogues. There is a need to complement sunscreen formulations with additional molecules to obtain a complete internal and external photoprotection against both UVA and visible lights.     

The soluble eumelanin precursor 5,6-dihydroxyindole-2-carboxylic acid enhances oxidative damage in human keratinocyte DNA after UVA irradiation.

Soluble melanin precursors are present in serum and may act as skin chromophores contributing to UVR-induced oxidative damage. Our study aimed to determine whether the soluble eumelanin precursor 5,6-dihydroxy-indole-2-carboxylic acid (DHICA) photosensitizes DNA damage in human keratinocytes exposed to UVA irradiation. The HaCaT keratinocytes were incubated with and without DHICA, before irradiation with broadband UVA (320-400 nm). The DNA photodamage was assessed using the comet assay that detects frank single-strand breaks (SSB) and specific oxidative lesions with the addition of endonuclease III. Without DHICA incubation, there was no significant increase in SSB, compared to unirradiated cells, for doses up to 48.5 J/cm2 (< 1 minimum erythemal dose). Preincubation with 0.5 microM DHICA caused an increase in SSB at every UVA dose (significant from 12.1 to 48.5 J/cm2), while varying the DHICA concentration (0.125-2 microM) showed this effect to be concentration dependent such that SSB increased and endonuclease III-sensitive sites decreased with increasing DHICA concentration. The irradiation of cells in the presence of antioxidants (catalase, mannitol and histidine) suggests that DHICA-induced photosensitization is mediated via singlet oxygen and, to a lesser extent, hydroxyl radicals. These results indicate that DHICA can induce strand breaks with UVA at clinically relevant doses via a mechanism involving reactive oxygen species.     

Genotoxicity of visible light (400–800 nm) and photoprotection assessment of ectoin, l-ergothioneine and mannitol and four sunscreens

This study was designed to determine the genotoxic effects of visible (400–800 nm) and ultraviolet A (UVA)/visible (315–800 nm) lights on human keratinocytes and CHO cells. The alkaline comet assay was used to quantify DNA-damage. In addition, photo-dependent cytogenetic lesions were assessed in CHO cells by the micronucleus test. Three protective compounds [ectoin, l-ergothioneine (ERT) and mannitol] were tested with the comet assay for their effectiveness to reduce DNA single-strand breaks (SSB). Finally, the genomic photoprotections of two broad-band sunscreens and their tinted analogues were assessed by the comet assay. The WST-1 cytotoxicity assay revealed a decrease of the keratinocyte viability of 30% and 13% for the highest UVA/visible and visible irradiations (15 and 13.8 J/cm², respectively). Visible as well as UVA/visible lights induced DNA SSB and micronuclei, in a dose-dependent manner. The level of DNA breakage induced by visible light was 50% of the one generated by UVA/visible irradiation. However, UVA radiations were 10 times more effective than visible radiations to produce SSB. The DNA lesions induced by visible and UVA/visible lights were reduced after a 1-h preincubation period with the three tested compounds. The maximal protective effects were 92.7%, 97.9% and 52.0% for ectoin (0.1 mM), ERT (0.5 mM) and mannitol (1.5 mM), respectively, against visible light and 68.9%, 59.8% and 62.7% for ectoin (0.1 mM), ERT (0.5 mM) and mannitol (1.5 mM), respectively, against UVA/visible light. Thus, visible light was genotoxic on human keratinocytes and CHO cells through oxidative stress mechanisms similar to the ones induced by UVA radiations. The four tested sunscreens efficiently prevented DNA lesions that were induced by both visible and UVA/visible irradiations. The tinted sunscreens were slightly more effective that their colorless analogues. There is a need to complement sunscreen formulations with additional molecules to obtain a complete internal and external photoprotection against both UVA and visible lights.     

FORMATION OF SINGLE AND DOUBLE STRAND BREAKS IN DNA ULTRAVIOLET IRRADIATED AT HIGH INTENSITY

The induction of single-strand breaks (SSB) by two quantum processes in DNA is well established. We now report that biphotonic processes result in double-strand breaks (DSB) as well. pUC19 and bacteriophage M13 RF DNA were irradiated using an excimer laser (248 nm) at intensities of 10(7), 10(9), 10(10) and 10(11) W/m2 and doses up to 30 kJ/m2. The proportion of DNA as supercoil, open circular, linear and short fragments was determined by gel electrophoresis. Linear molecules were noted at fluences where supercoiled DNA was still present. The random occurrence of independent SSB in proximity to each other on opposite strands (producing linear DNA) implies introduction of numerous SSB per molecule in the sample. If so, supercoiled DNA that has sustained no SSB should not be observed. A model accounting for the amounts of supercoiled, open circular, linear and shorter fragments of DNA due to SSB, DSB and Scissions (opposition of two independently occurring SSB producing an apparent DSB) was developed, our experimental data and those of others were fit to the model, and quantum yields determined for SSB and DSB formation at each intensity. Results showed that high intensity laser radiation caused an increase in the quantum yields for both SSB and DSB formation. The mechanism of DSB formation is unknown, and may be due to simultaneous cleavage of both strands in one biphotonic event or the biased introduction of an SSB opposite a preexisting SSB, requiring two biphotonic events.     

Exacerbating effect of vitamin E supplementation on DNA damage induced in cultured human normal fibroblasts by UVA radiation

The effects of vitamin E supplementation were evaluated in cultured human normal fibroblasts exposed to ultraviolet A radiation (320-380 nm) (UVA). Cells were incubated in medium containing alpha-tocopherol, alpha-tocopherol acetate or the synthetic analog Trolox for 24 h prior to UVA exposure. DNA damage in the form of frank breaks and alkali-labile sites, collectively termed single-strand breaks (SSB), was assayed by the technique of single cell gel electrophoresis (comet assay), immediately following irradiation or after different repair periods. The generation of hydrogen peroxide (H2O2) and superoxide ion (O2.-) was measured by flow cytometry through the oxidation of indicators into fluorescent dyes. It was observed that pretreatment of cells with any form of vitamin E resulted in an increased susceptibility to the photoinduction of DNA SSB and in a longer persistence of damage, whereas no significant change was observed in the production of H2O2 and O2.- reactive oxygen species, compared to untreated controls. These findings indicate that in human normal fibroblasts, exogenously added vitamin E exerts a promoting activity on DNA damage upon UVA irradiation and might lead to increased cytotoxic and mutagenic risks.     

Induced and photoinduced DNA damage by quinolones: ciprofloxacin, ofloxacin and nalidixic acid determined by comet assay

Quinolones are degraded by light with loss of their antimicrobial activity, generating active species or radicals. Evidence exists that some fluoroquinolones (lomefloxacin, fleroxacin and enoxacin) induce damage to the cellular membrane and DNA cleavage by photosensitization. In this study, the genotoxic potential of the quinolones ofloxacin, nalidixic acid and ciprofloxacin (three antimicrobials frequently used in therapy) was evaluated upon irradiation with UV light by using the comet assay on cells of the Jurkat line. The results demonstrate that there are significant differences between the control groups (positive control with 50 microM H2O2, negative controls without drug and with and without irradiation) and the groups of irradiated quinolones (ofloxacin 2.76 x 10(-5) M, nalidixic acid 2.15 x 10(-4) M and ciprofloxacin 2.01 x 10(-5) M), indicating that, at the dose of irradiation employed (necessary to produce 50% photodegradation), the photodecomposition of the quinolones enhanced DNA damage. The unirradiated drugs also exhibited genotoxicity significantly different from that of the negative control.     

Evaluation of sunscreen protection in human melanocytes exposed to UVA or UVB irradiation using the alkaline comet assay

The in vivo assessment of sunscreen protection does not include the photogenotoxicity of UVA or UVB solar radiation. Using the comet assay we have developed a simple and rapid technique to quantify sunscreen efficacy against DNA damage induced by UV light. Cutaneous human melanocytes from primary cultures were embedded in low-melting point (LPM) agarose and exposed to UVA (0.8 J/cm2) or to UVB (0.06 J/cm2) through a quartz slide covered with 10 microL volumes of sunscreens. DNA single-strand breaks induced directly by UVA at 4 degrees C and indirectly through nucleotide excision repair by UVB following a 35 min incubation period at 37 degrees C were quantified using the comet assay. Tail moments (TM) (tail length x %tail DNA) of 100 cells/sample were determined by image analysis. DNA damage was evaluated with a nonlinear regression analysis on the normalized distribution frequencies of TM using a chi 2 function. The coefficients of genomic protection (CGP) were defined as the percentage of inhibition of DNA lesions caused by the sunscreens. Twenty-one sunscreens were evaluated, and the calculated CGP were compared with the in vivo sun protective factor (SPF) and with the protection factor UVA (PFA). Nonlinear relationships were found between SPF and CGPUVB and between PFA and CGPUVA.     

Single-cell gel/comet assay applied to the analysis of UV radiation-induced DNA damage in Rhodomonas sp. (Cryptophyta).

The single-cell gel/comet assay is an electrophoretic technique used to detect single-strand breaks in DNA. Damage is assessed examining individual cells under an epifluorescent microscope. UV-induced DNA damage consists mostly of the formation of pyrimidine dimers; therefore, most of the damage cannot be detected using a standard comet assay. The enzyme T4 endonuclease V breaks DNA strands at sites of pyrimidine dimers. The main objective of this work is to evaluate the comet assay to detect UV-induced damage in DNA after an initial treatment of cells with T4 endonuclease V. This work was conducted on Rhodomonas sp. (Cryptophyta), a marine unicellular flagellate. Cells of Rhodomonas sp. were exposed to 12 h visible + ultraviolet-A + ultraviolet-B (VIS + UVA + UVB) and VIS (control), with and without T4 endonuclease V. Cells exposed to VIS + UVA + UVB showed approximately 200% more damage than control if these were treated with T4 endonuclease V. Rhodomonas sp. were exposed to 3, 6, 9 and 12 h of VIS, VIS + UVA and VIS + UVA + UVB. Damage induced by VIS + UVA + UVB as detected by the comet assay increased along with exposure time. However, damage caused by VIS and VIS + UVA remained relatively constant at all times. Results of this study indicate that the comet assay is more sensitive to UV radiation damage when used in conjunction with T4 endonuclease V. This modification of the comet assay can be used as an alternative technique to detect DNA damage in single cells caused by UV radiation.     

CORRELATION BETWEEN CELL SURVIVAL AND DNA SINGLE-STRAND BREAK REPAIR PROFICIENCY IN THE CHINESE HAMSTER OVARY CELL LINES AA8 AND EM9 IRRADIATED WITH 365-nm ULTRAVIOLET-A RADIATION

Cell survival parameters and the induction and repair of DNA single-strand breaks were measured in two Chinese hamster ovary cell lines after irradiation with monochromatic UVA radiation of wavelength 365 nm. The radiosensitive mutant cell line EM9 is known to repair ionizing-radiation-induced single-strand breaks (SSB) more slowly than the parent line AA8. EM9 was determined to be 1.7-fold more sensitive to killing by 365-nm radiation than AA8 at the 10% survival level, and EM9 had a smaller shoulder region on the survival curve (alpha = 1.76) than AA8 (alpha = 0.62). No significant differences were found between the cell lines in the initial yields of SSB induced either by gamma-radiation (as determined by alkaline sucrose gradient sedimentation) or by 365-nm UVA (as determined by alkaline elution). For measurement of initial SSB, cells were irradiated at 0.5 degrees C to minimize DNA repair processes. Rejoining of 365-nm induced SSB was measured by irradiating cells at 0.5 degrees C, allowing them to repair at 37 degrees C in full culture medium, and then quantitating the remaining SSB by alkaline elution. The repair of these breaks followed biphasic kinetics in both cell lines. EM9 repaired the breaks more slowly (t1/2 values of 1.3 and 61.3 min) than did AA8 (t1/2 values of 0.9 and 53.3 min), and EM9 also left more breaks unrepaired 90 min after irradiation (24% vs 8% for AA8). Thus, the sensitivity of EM9 to 365-nm radiation correlated with its deficiency in repairing DNA lesions revealed as SSB in alkaline elution.(ABSTRACT TRUNCATED AT 250 WORDS)     

LIGHT ACTIVATION OF A COMPLEX MIXTURE: EFFECTS OF UV EXCISION REPAIR ON THE MODULATION OF GENOTOXIC AND MOLECULAR EVENTS IN CHINESE HAMSTER CELLS

Abstract— An aqueous effluent produced during the retorting of oil shale has been shown to induce a significant genotoxic response in cultured Chinese hamster (CHO) cells following activation by near ultraviolet light (UVA). In this report the light-activated responses induced by this complex mixture were compared between two DNA excision repair deficient mutants and their parental strain, CHO-AA8-4. The mutants, UV-5 and UV-135, were hypersensitive to both the cytotoxic and mutagenic effects of concurrent exposures to the retort process water and UVA. Repair proficiency appeared to render AA8-4 relatively insensitive to low doses of UVA treatment, whereas in the two mutants a linear dose-response in the induction of 6-thioguanine resistant (6TG^®) mutations was observed even at the lower UVA doses examined. Filter DNA alkaline elution methods were utilized to demonstrate that both single-strand breaks and DNA-DNA interstrand crosslinks were induced in CHO DNA following process water and UVA treatment. Results were also obtained which indicated that the inability to repair DNA-DNA crosslinks contributed significantly to the hypersensitive response seen in the excision repair mutants following the photoactivation of this complex mixture.     

Single,double, and multiple double strand breaks induced in DNA by 3-100 eV electrons

Nonthermal secondary electrons with initial kinetic energies below 100 eV are an abundant transient species created in irradiated cells and thermalize within picoseconds through successive multiple energy loss events. Here we show that below 15 eV such low-energy electrons induce single (SSB) and double (DSB) strand breaks in plasmid DNA exclusively via formation and decay of molecular resonances involving DNA components (base, sugar, hydration water, etc.). Furthermore, the strand break quantum yields (per incident electron) due to resonances occur with intensities similar to those that appear between 25 and 100 eV electron energy, where nonresonant mechanisms related to excitation/ionizations/dissociations are shown to dominate the yields, although with some contribution from multiple scattering electron energy loss events. We also present the first measurements of the electron energy dependence of multiple double strand breaks (MDSB) induced in DNA by electrons with energies below 100 eV. Unlike the SSB and DSB yields, which remain relatively constant above 25 eV, the MDSB yields show a strong monotonic increase above 30 eV, however with intensities at least 1 order of magnitude smaller than the combined SSB and DSB yields. The observation of MDSB above 30 eV is attributed to strand break clusters (nano-tracks) involving multiple successive interactions of one single electron at sites that are distant in primary sequence along the DNA double strand, but are in close contact; such regions exist in supercoiled DNA (as well as cellular DNA) where the double helix crosses itself or is in close proximity to another part of the same DNA molecule.     

EFFECTS OF INTENSITY AND FLUENCE UPON DNA SINGLE-STRAND BREAKS INDUCED BY EXCIMER LASER RADIATION

Abstract— A Xenon-chloride excimer laser emitting energy at 308 nm was used to induce single-strand breaks (SSBs, frank breaks plus alkali-labile lesions as assayed by alkaline sucrose sedimentation techniques) in purified DNA from Bacillus subtilis . A fluence response study and a peak pulse intensity study were performed. At a pulse energy of 0.1 mJ/pulse, the radiation induced SSBs in a linear fashion (91 SSB/10⁸ Da per MJ/m²) to a maximum exprimental fluence of 1.28 MJ/m². The pulse intensity study showed that there were no significant changes in DNA breakage (105 SSB/10⁸ Da) between 2.93 times 10⁹ and 5.86 times 10¹¹ W/m² (0.11 and 22.0 mJ/pulse) at a constant total fluence of 1.1 MJ/m² (27000 mJ dose). This study has verified and extended previous work by quantifying the yield of SSBs induced in DNA by this laser radiation.     

DETECTION OF UVR-INDUCED DNA DAMAGE IN MOUSE EPIDERMIS in vivo USING ALKALINE ELUTION

Abstract— Alkaline elution has been used to detect ultraviolet radiation (UVR)-induced DNA damage in the epidermis of C3H/Tif hr/hr mice. This technique detects DNA damage in the form of single-strand breaks and alkali-labile sites (SSB) formed directly by UVA (320–400 nm) or indirectly by UVB (280–320 nm). The latter induces DNA damage such as cyclobutane pyrimidine dimers and pyrimidine-pyrimidone (6–4)-photoproducts, which are then converted into transient SSB by cellular endonucleases, during nucleotide excision repair (NER). The irradiation system used had a spectral output similar in effect to solar UVR, with the UVB component inducing 94% of the edema response observed in mice. Consequently, the majority of SSB detected were those formed via NER of UVB-induced photoadducts. The number of SSB detected immediately after 8 kj/m² (2.7 minimum erythema doses determined at 48 h post-UVR [MED]) was low, indicating the formation of only small numbers of transient SSB. When DNA repair inhibitors hydroxyurea and 1 -β-D-arabinofuranosylcytosine were administered (intraperi-toneally) to mice 30 min before UVR, they prevented sealing of the DNA SSB formed during NER. A four-fold increase in the number of SSB detected resulted, which was found to be linearly related to the UVR dose. The SSB induced by 2 kj/m² (less than an MED) were readily detected, with the ear showing lower numbers of SSB than the dorsum. When repair inhibitors were added post-UVR, the rate of formation of SSB declined rapidly with time of administration, reflecting repair of DNA lesions. After a UVR dose of 6 kj/m² (2 MED), 50% of the initial repair-dependent SSB had been removed after approximately 2 h in the ear and 4 h in the dorsum; no more SSB appeared to be incised by 24 h post-UVR. The technique described is an efficient and highly sensitive one for the quantification of SSB induced in UV-irradiated skin samples in vivo.     

Evaluation of photolyase (Photosome) repair activity in human keratinocytes after a single dose of ultraviolet B irradiation using the comet assay

Photosome is constituted of photolyases included in liposomes. Photolyase is a bacterial enzyme that can repair ultraviolet B (UVB)-induced cyclobutane pyrimidine dimers (CPD) in eukaryotic cells. A modified version of the alkaline comet assay has been set up to evaluate the repair activity of this enzyme after a single dose of UVB (312 nm, 0.06 J/cm2) in human keratinocytes. The formation of single strand breaks (SSB) induced by the UVA photoactivation of the enzyme (1.2 J/cm2) was inhibited by the pretreatment of the cells with 4 mM L-ergothioneine (ERT) during 30 min at 37 degrees C. To increase the sensitivity of the comet assay, an additional lysis was used with a buffer containing sodium dodecyl sulfate (0.5%) and proteinase K (0.1 mg/ml) for 60 min at 37 degrees C. Unrepaired CPD by photolyase were revealed by a second enzymatic treatment with T4 endonuclease V, a CPD specific glycosylase. UVB irradiation increased the SSB level in keratinocytes and additional T4NV treatment enhanced this SSB level by 1.5-2.0-fold confirming that CPD were the major base modifications generated by UVB irradiation. UVA-photoactivated Photosome repaired CPD lesions and decreased the SSB levels by 2.6-3.3-fold. Photosome could be an additional component of sunscreens to reduce the development of skin cancer.     

Detection and Prevention of Ocular Phototoxicity of Ciprofloxacin and Other Fluoroquinolone Antibiotics†

Fluoroquinolone (FLQ) drugs are a potent family of antibiotics used to treat infections including ocular infections. To determine if these antibiotics may be phototoxic to the eye, we exposed human lens epithelial cells to 0.125–1 mm FLQs (ciprofloxacin [Cipro], lomefloxacin [Lome], norfloxacin [Nor] and ofloxacin [Ofl]), the precursor quinolone nalidixic acid (Nalid) and UVA radiation (2.5 J cm⁻²). Based on fluorescence confocal microscopy, FLQs are diffused throughout the cytoplasm and preferentially located in the lysosomes of lens epithelial cells. Neither FLQ exposure alone nor UVA exposure alone reduced cell viability. However, with exposure to UVA radiation the FLQs studied (Cipro, Nor, Lome and Ofl) induced a phototoxic reaction that included necrosis, apoptosis, loss of cell viability as measured by MTS, and membrane damage as determined by the lactate dehydrogenase assay. Both Nalid and all FLQs studied (Cipro, Nor, Lome and Ofl) photopolymerized the lens protein α-crystallin. Phototoxic damage to lens epithelial cells and/or α-crystallin will lead to a loss of transparency of the human lens. However, if precautions are taken to filter all UV radiation from the eye while taking these antibiotics, eye damage may be prevented.