PURPOSE: The aim of this study was to investigate high molecular weight hyaluronan (HMW-HA) protection on human corneal epithelial (HCE) cells against ultraviolet B (UVB) radiation-induced toxic effects. METHODS: The HCE cell line was incubated with HMW-HA or phosphate-buffered salt solution (PBS), rinsed, and exposed to UVB radiation. Cell viability, reactive oxygen species (ROS) and glutathione (GSH) levels, 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) release, p53 phosphorylation, caspase-3,-8,-9 activation, and interleukin (IL)-6 and -8 production were assessed to evaluate and to compare UVB-induced toxicity between cells treated with HMW-HA and cells treated with PBS. RESULTS: Data indicate that HMW-HA had significant protective effects against UVB radiation. HMW-HA increased HCE cell viability, decreased IL-6 and -8 production, and decreased caspase-3 and -8 activation. However, HMW-HA had no significant effect on ROS and GSH levels, 8-oxo-dG release, and p53 phosphorylation. CONCLUSIONS: To our knowledge, we report for the first time the ability of HMW-HA to protect cells against UV irradiation. According to our results, HMW-HA provides anti-inflammatory and anti-apoptotic signals to cells exposed to UVB.

Tendinopathy and tendon rupture are the adverse effects observed with fluoroquinolone antibiotics in old patients. The aim of this study was to investigate the effect of anethole dithiolethione (5-[p-methoxyphenyl]3H-1,2-dithiole-3-thione) on the oxidative stress induced by three fluoroquinolones (pefloxacin, ofloxacin, ciprofloxacin) incubated with rabbit tenocyte cell line. Anethole dithiolethione is a well known antioxidant and glutathione inducer. Anethole dithiolethione is widely used in human therapy for its choleretic, sialogogic properties and recently proposed as cytoprotective agent in lung precancerous lesions prevention in smokers. In this purpose, protection against oxidative stress induced by fluoroquinolones has been assessed using cytofluorimetric probes to quantify cytotoxicity and reactive oxygen species production. Fluorescence signal was quantified in 96-well microplates, using cold light cytofluorometer. Significant reactive oxygen species production was detected after 45 minutes for all fluoroquinolones tested. Anethole dithiolethione has been evaluated on this parameter. Anethole dithiolethione significantly (*: P<0.05) reduces and normalizes reactive oxygen species induced by fluoroquinolones. So, anethole dithiolethione (Sulfarlem), well known for its antioxidant and glutathione inducing properties, good tissue diffusion and good tolerance in humans, could be beneficially associated to fluoroquinolones, and be proposed as a therapeutic adjuvant to prevent oxidative stress and tendinous adverse effects induced by xenobiotics and more precisely by fluoroquinolones.

It has been shown that some cytochrome P450-dependent enzyme activities could present daily fluctuations, particularly CYP3A isoenzymes which are enhanced during the dark period. The aim of this study was to investigate whether age and photoperiodic conditions at different circadian stages could influence these fluctuations. Young mature (10 weeks) and old (22 months) Wistar rats were initially exposed to light-dark cycles 12:12 during 4 weeks, and secondly 18:6 for either one week or six weeks. Erythromycin N-demethylase (CYP3A-dependent), 7-ethoxycoumarin O-deethylase (CYP1A-dependent) and aniline 4-hydroxylase (CYP2E-dependent) activities were determined in liver and kidney microsomes at different hours after darkness onset (HADO). In addition, liver and kidney GSH, GSHPx, ATP, TBARS were determined. During the LD 12:12 cycle, while no significant modification was observed in CYP1A- and 2E-dependent enzyme activities as functions of HADO, erythromycin N-demethylase activity (CYP3A-dependent) showed a significant increase during the second third of the dark period in both young and old rats. After switching to a LD 18:6 cycle, this variation was still observed during second third of the dark period, to a lesser but still significant degree, with no difference between one week and six weeks exposure to the new photoperiod. It can be noted that the old rats showed a significantly lower level of erythromycin N-demethylase activity than the young rats, in parallel to a decrease in GSH, GSHPx and ATP, and an increase in TBARS.These results confirm the lower resistance of old animals to oxidative stress. The observed variations in metabolism parameters underline the need for study designs in pharmaco-toxicology taking into account the possible risks induced by circadian changes, especially in aged subjects.

Tendinitis and tendon rupture complicating fluoroquinolone therapy have been reported recently, especially affecting men over 60 years. These new quinolones are more potent antimicrobial agents than older nonfluorinated compounds like nalidixic acid. We compared the effects of one quinolone (nalidixic acid) and two fluoroquinolones (norfloxacin and pefloxacin) on cultured rabbit Achilles tendon cells. First, we examined their effects on cell viability, mitochondrial succinate dehydrogenase and global activity, mitochondrial activity using microtitration methods. Pefloxacin and norfloxacin were more cytotoxic than nalidixic acid according to IC50 values. These results confirm that mitochondria represent a biological target of fluoroquinolones. Moreover, the extracellular matrix was studied by molecular hybridization. After a 72 h treatment, the level of type I collagen transcripts was not modified with any of the three antimicrobial agents, whereas mRNA encoding decorin was decreased with 10(-4) mol/L pefloxacin only. The decrease of transcripts encoding decorin suggests that this matrix component is another target of pefloxacin and modification of decorin seems to be an early event (before mitochondrion alteration) which may contribute to the explanation of tendon rupture.

INTRODUCTION: The retina is located in a highly oxygenated environment and is therefore particularly susceptible to oxidative damage. Blueberries have a high antioxidant potential since they are rich in anthocyans. The aim of this study was to investigate the cytoprotective role of blueberries on human retinal cells. MATERIALS AND METHODS: Blueberry extract was incubated at 0.05% and 0.1% for 15 minutes or 24 hours on a human retinal cell line. Then oxidative stress was induced by 150 microM tert-butylhydroperoxide (tBHP) for 1 hour. Intracellular metabolism, reactive oxygen species, superoxide anion, and mitochondrial apoptosis were evaluated using Alamar blue, DCFDA, dihydroethidium, and nonylacridine orange dyes, respectively. Tests were performed using cytofluorometry adapted to microplates. RESULTS: Blueberry protected cells against tBHP-induced cytotoxicity. It increased cell viability, decreased oxidative stress and mitochondrial apoptosis. After a 24-hour preincubation time, blueberry totally inhibited tBHP-induced cytotoxicity. CONCLUSION: Blueberry seems to be a potent antioxidant and could be easily added to food complements to prevent or limit ocular pathologies induced by oxidative stress.

Purpose:To determine whether multipurpose solutions, widely used for contact lens disinfections, could be at the origin of ocular pathologies (contact lens intolerance and ocular infections).Methods:An observational cohort study (questionnaire analysis) was carried out to estimate the number of contact lens wearers, type of infection, and type of lens care regimen used by patients. Besides, multipurpose solutions cytotoxicity (necrosis and apoptosis) was evaluated on a conjunctival cell line using cytofluorometry.Results:In the general population, 59% of contact lens wearers use multipurpose solutions whereas 35% use oxidative products. Of the questioned contact lens wearers with ocular infections, 80% used multipurpose solutions. Multipurpose solutions are therefore not efficient enough against microorganisms, and cannot be considered as disinfectant solutions but only as preservatives. However, preservatives are known to be toxic to ocular surface, so apoptosis induced by multipurpose solutions could lead to ocular surface diseases. Our cytofluorometry study allowed us to demonstrate that contact lens multipurpose solutions containing preservatives are cytotoxic through caspase 3 induction, chromatin condensation and P2X7 cell-death receptor activation, in contrast with unpreserved sterile saline solutions that were found inert.Conclusions:Multipurpose solutions seem to be preservative but not disinfecting solutions. They are not adapted to the final rinse of contact lenses because of apoptosis induction. It could explain part of lens intolerance.Eye advance online publication, 16 May 2008; doi:10.1038/eye.2008.131.

The majority of chemical solar filters are cytotoxic, particularly on sensitive ocular cells (corneal and conjunctival cells). Consequently, a non-cytotoxic UV filter would be interesting in dermatology, but more especially in ophthalmology. In fact, light damage to the eye can be avoided thanks to a very efficient ocular antioxidant system; indeed, the chromophores absorb light and dissipate its energy. After middle age, a decrease in the production of antioxidants and antioxidative enzymes appears with accumulation of endogenous molecules that are phototoxic. UV radiations can induce reactive oxygen species formation, leading to various ocular diseases. Because most UV filters are cytotoxic for the eye, we investigated the anti-UV properties of Calophyllum inophyllum oil in order to propose it as a potential vehicle, free of toxicity, with a natural UV filter action in ophthalmic formulation. Calophyllum inophyllum oil, even at low concentration (1/10,000, v/v), exhibited significant UV absorption properties (maximum at 300nm) and was associated with an important sun protection factor (18-22). Oil concentrations up to 1% were not cytotoxic on human conjunctival epithelial cells, and Calophyllum inophyllum oil appeared to act as a cytoprotective agent against oxidative stress and DNA damage (85% of the DNA damage induced by UV radiations were inhibited with 1% Calophyllum oil) and did not induce in vivo ocular irritation (Draize test on New Zealand rabbits). Calophyllum inophyllum oil thus exhibited antioxidant and cytoprotective properties, and therefore might serve, for the first time, as a natural UV filter in ophthalmic preparations.

Modulation of cell surface molecules involved in immune recognition and cellular interactions (class I major histocompatibility complex or MHC-I, B7.1 or CD80, integrin alpha4 or CD49d, tetraspanins CD9, CD81) was examined in modified B16 melanoma cells displaying either inhibited IGF-I expression or transfected OVA encoding gene. It was shown that inhibiting IGF-I expression or inserting OVA encoding gene did not lead to modification relevant to the presence of MHC-I or B7.1. However downregulation of tetraspanin CD9 was observed in modified IGF-I but not in OVA encoding gene inserted melanoma cells. Expression of tetraspanin CD81 and integrin alpha4/CD49d remained unchanged. Inoculated into syngeneic recipients, the modified melanoma cells exhibited significant delayed outgrowth with a reduction in the percentage of lethal tumors observed essentially in hosts injected with inhibited IGF-I expression cells.

Introduction: Following material vigilance cases encountered with the hydrophilic acrylic intraocular lens, ACR6D SE preloaded in the Premier(R) shooter, we studied the cytotoxicity of the intraocular lens and its conditioning to identify the cytotoxic element. We proposed medical device modification to improve its biocompatibility. Materials and Methods: Biocompatibility-cytotoxicity assays were carried out according to ISO 10993-5 recommendations. Tests were performed on the SRA 01/04 human lens epithelial cell line. Neutral red, Hoechst 33342, and YO-PRO-1 fluorescent probes were used to assess membrane integrity, total DNA, and membrane fluidity, respectively. Materials samples were prepared in culture medium according to the ISO 10993-5 elution procedure. Pure saline solutions and conditioning liquids were tested directly on cells. RESULTS: The intraocular lens and injector were not cytotoxic. Conditioning liquids induced membrane fluidity perturbation characteristic of apoptosis. Tests performed on new versions of the medical device identified a better adapted conditioning liquid. CONCLUSION: The results suggest that the cytotoxicity of the conditioning liquid could explain the postoperative complication rate. When we changed the conditioning liquid with sterile irrigating solution (i.e., rich divalent cation marine solution), we eliminated cellular stress. Fluorescent probes are well adapted to assess medical device biocompatibility-cytotoxicity.

The age-related difference in fluoroquinolone-induced tendon toxicity was investigated. In vitro tendon cells from juvenile and young adult rabbits, respectively, were incubated with quinolone (nalidixic acid, NA) or fluoroquinolone (ofloxacin, OFX or pefloxacin, PEF) at 0.01 muM to 1 mM for 72 h. Redox status, glutathione (GSH), reactive oxygen species (ROS), and mitochondrial activity were assessed using intracellular fluorescent probes. Fluorescence signal was detected on living adherent tenocytes in microplates using cold-light cytofluorometry. Tendon toxicity differed significantly between the two cell groups and the difference was greatest with highest dose (1 mM). For 72 h, significant (p<0.001) differences between immature and young adult primary tenocytes were observed for redox status decrease, GSH decrease, and ROS production increase. Mitochondrial activity remained unaltered in immature tenocytes. We confirm two groups of intrinsic tendon toxicity (OFX/NA vs. PEF) associated to oxidative stress (GSH decrease). Our in vitro experimental model confirms the clinical observations of age dependent tenotoxicity. First group (NA, OFX) showed greater intrinsic tenotoxicity for young adult than immature tenocytes, second group (PEF) was highly toxic for immature and young adult cells. The three quinolones do not altered mitochondrial activity in immature tenocytes whereas alteration was observed in young adult tenocytes.

Introduction: Cyclosporine is a molecule used in ophthalmology for the prevention of corneal graft rejection. The systemic use of this product can lead to serious adverse side effects that can be avoided using the topical formulation of cyclosporine. However, cyclosporine application can induce ocular irritation. MATERIAL: and methods: The aim of this study is to evaluate the cytotoxicity of four formulations of 2% cyclosporine eye drops: Sandimmum(R) intravenous solution diluted with NaCl 0.9%, Sandimmun(R) oral solution diluted in castor oil or corn oil after ethanol evaporation, and Sandimmun(R) oral solution diluted in castor oil without previous ethanol evaporation. Two tests - the Draize test and the evaluation of cytotoxicity of adherent alive cells with cold light cytofluorimetry on microplates - were used in this study. RESULTS: These tests demonstrated that the aqueous solution shows more toxicity than the other formulations, and the type of oil and ethanol concentration influence cell viability. CONCLUSION: These results helped the Pharmacy unit choose the vehicles for a safe cyclosporine eye drop formulation and thus decrease the side effects of cyclosporine eye drop instillation with a decrease in ethanol concentration compared to published formulations.

Tendinitis and tendon ruptures induced by fluoroquinolones, while uncommon, have been documented in the literature since 1983. We report five cases of tendinitis induced by fluoroquinolones, three caused by levofloxacin and two by ciprofloxacin. We revise actual knowledge of this association and we insist on how important is an early detection to prevent tendon rupture.

ABSTRACT: Fluoroquinolone-associated tendinopathy is well described. This adverse effect however does not appear to be widely known among medical practitioners. We hereby described a case of ciprofloxacin-associated tendinopathy for which the adverse drug reaction was not suspected initially and the patient was inappropriately reassured and incorrectly advised to complete the antibiotic course. Given the frequent use of fluoroquinolones in clinical practice and the potential for severe disability from tendon rupture, we consider it important to remind your readers of this uncommon but potentially devastating adverse drug reaction.

With the expanded use of fluoroquinolones and increasing number of reports of tendon injury linked to these agents, clinicians must be aware of the frequency and strength of this association. In the past, pefloxacin and ciprofloxacin were most frequently implicated, but tendon injury is reported with most fluoroquinolones. As many as half of patients with fluoroquinolone-associated tendinopathy experience tendon rupture, and almost one third have received long-term corticosteroids. Tendon injury is mostly reported in the lower extremities, but injury in the upper extremities, including the hand, has also been reported. Management is similar regardless of the location of the injury. Use of fluoroquinolones requires careful patient assessment and follow-up in view of this complication with potential for sequelae.

Antimicrobial therapy with fluoroquinolones can be associated with tendinitis and other tendon disorders as an adverse reaction associated with this class of antimicrobials. Here we investigated aspects of the mechanism of quinolone-induced tendotoxicity in human tenocytes focussing mainly on the question whether fluoroquinolones may induce apoptosis. Monolayers of human tenocytes were incubated with ciprofloxacin or levofloxacin at different concentrations (0, 3, 10, 30 and 100mg/L medium) for up to 4 days. Ultrastructural changes were studied by electron microscopy, and alterations in synthesis of specific proteins were determined using immunoblotting. At concentrations, which are achievable during quinolone therapy, 3mg ciprofloxacin/L medium significantly decreased type I collagen; similar changes were observed with 3mg ciprofloxacin or 10mg levofloxacin/L medium for the beta(1)- integrin receptors. Effects were intensified at higher concentrations and longer incubation periods. Cytoskeletal and signalling proteins, such as activated shc or erk 1/2, were significantly reduced by both fluoroquinolones already at 3mg/L. Furthermore, time- and concentration-dependent increases of matrix metalloproteinases as well as of the apoptosis marker activated caspase-3 were found. Apoptotic changes were confirmed by electron microscopy: both fluoroquinolones caused typical alterations like condensed material in the nucleus, swollen cell organelles, apoptotic bodies and bleb formation at the cell membrane. Our results provide evidence that besides changes in receptor and signalling proteins apoptosis has to be considered as a final event in the pathogenesis of fluoroquinolone-induced tendopathies.

Fluoroquinolones are the most potent oral antibiotics in clinical use today. Increasingly, these drugs are being prescribed for relatively benign infections and for new categories of patients, including paediatric patients. As their use becomes more frequent, so will the adverse events. This review focuses on a rare but debilitating adverse reaction, the fluoroquinolone-associated tendinopathy. Despite many published case reports and approximately 3500 cases reported to the World Health Organization Collaborating Centre for Drug Monitoring, little is known about the mechanisms behind this fluoroquinolone-specific toxicity. Data on chemical properties, mode of action, pharmacokinetic features, clinical presentation and risk factors in relation to tendon toxicity are discussed and the literature reviewed. As long as the musculoskeletal toxicity cannot be predicted by in vitro or in vivo models and this class of antibiotics is one of the most commonly linked to selection of resistant bacteria, a more prudent use of fluoroquinolones is warranted.

Low doses, chronic exposure to mercurial organic compounds is a worldwide health concern and could be pathogenetically relevant as co-factor in several neurodegenerative diseases. In this in vitro study we wanted to further improve our knowledge on the mechanisms of toxicity of methylmercury hydroxide (MeHgOH) in the unprimed PC12 cell line. Cell viability, mitochondrial function, redox state, and cell morphology were recorded at different time points to sequence the events leading to cell death. The lowest cytotoxic concentration and EC50 were 0.3 and 1.3 microM, respectively. 5 microM MeHgOH was fatal for 80% of the cell population after 24 h; within 1 h it caused glutathione (GSH) depletion and a partial dissipation of Deltapsim. At this concentration, reactive oxygen species (ROS) generation was only slight and delayed. After 6h more than 50% of ATP was available and caspase 3 was active. Time-lapse confocal microscopy showed that only a fraction of the cells completed apoptosis while others turned toward necrosis (necrapoptosis). Pre-incubation with N-acetylcysteine (NAC) and GSH but not Cyclosporin A rescued over 80% of the cells. These results provide experimental evidence that, in this cell model, MeHgOH triggers cell death via a primary depletion of GSH but in the absence of ROS overproduction.