Delivery of Hydrophobic Drugs to the Posterior Ocular Region by Gel-in-Water Nanoemulsion.

Purpose: The aim of this study was to develop a nanogel emulsion as a minimally invasive, safe, and effective treatment alternative for posterior ocular diseases. Methods: A gel-in-water (G/W) nanoemulsion was developed by ultrasonication using beeswax as an organogelator. Different physicochemical properties were evaluated along with particle size analysis by dynamic light scattering. In vitro biocompatibility of G/W nanoemulsion using rat hepatocytes and human umbilical vein endothelial cells (HUVECs) and in vivo corneal permeability as eye drops were investigated. Results: The nanogel emulsion was monodispersed with a polydispersity index and particle diameter of approximately 0.2 and 200 nm, respectively. The zeta potential value of -8.1 mV suggested enhanced stability and improved retinal permeability of nanoparticles. The prepared nanoemulsion was found to be biocompatible with hepatocytes and HUVECs in vitro. Moreover, in vivo study demonstrated high permeability of G/W nanoemulsion to the retinal layer with no ocular irritation. Conclusions: G/W nanoemulsions have the potential for topical drug delivery in the posterior eye segment with maximum therapeutic efficacy. Translational Relevance: Organogel nanodispersion is a new concept to deliver hydrophobic drugs to the posterior segment of eyes as a novel drug delivery system.

Author Correction: Suppression of presbyopia progression with pirenoxine eye drops: experiments on rats and non-blinded, randomized clinical trial of efficacy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Development of New Pharmaceutical Candidates With Antioxidant Activity for the Treatment of Corneal Disorders.

The ocular surface is continuously exposed to physical and chemical factors in the environment. Oxidative stress, which strongly affects the ocular surface, is caused by several factors, including ultraviolet irradiation, fine particles, and tobacco smoke. Oxidative stress is one of the pathogeneses for corneal disorders. Thus, corneal epithelium and tear fluid contain antioxidants and antioxidative enzymes to protect the cornea against oxidative stress. Because autologous serum eye drops are useful for the treatment of corneal disorders caused by dry eye, these eye drops are globally used as a therapeutic intervention in patients with dry eye. We investigated the serum components that exert antioxidative effects to clarify the mechanism of action for serum antioxidants on corneal epithelial cells and to apply these components as drugs for the treatment of corneal disorders. We found that selenoprotein P, a known selenium-transfer plasma glycoprotein, was secreted into the tear fluid to supply the corneal epithelium with selenium. Selenium participates in the regulation of oxidative stress in many tissues, including the cornea. We subsequently developed selenium compounds for application in eye drops and successfully prepared Se-COMP as a new candidate for the treatment of corneal disorders.

Autologous Serum and Serum Components.

Dry eye syndrome is a multifactorial condition on the tear and ocular surface. Autologous serum eye drop is an effective method for treating dry eye. Autologous serum eye drops are now widely used by specialists since a first report in 1975. The results of a systematic study showed that the efficacy of autologous serum eye drops remains ambiguous because its preparation methods and clinical application have not been standardized. To elucidate the efficacy of autologous serum eye drops, well-designed, large-scale, high-quality randomized controlled trials need to be conducted with standardized treatment and use. Since serum components are partially similar to tear components, autologous serum eye drops improve dry eye by supplying tear components such as growth factors, proteins, and vitamins. Adding to the evidence based on the treatment of dry eye, we have found a new treatment candidate from serum: selenoprotein P (SeP). The efficacy of SeP as a treatment for dry eye was revealed by applying SeP eye drops to a dry eye rat model. Compared with phosphate-buffered saline treatment, SeP eye drops significantly reduced the fluorescein score of the cornea and suppressed the oxidative stress in the cornea, which is related to onset of dry eye, leading to improved corneal disorder. We have developed a new dry eye model caused by oxidative stress that will be used to screen candidate molecules for antioxidative activity.

Suppression of presbyopia progression with pirenoxine eye drops: experiments on rats and non-blinded, randomized clinical trial of efficacy.

Various methods can correct presbyopia, but all require devices or surgeries. Recently, supplements or warming devices to relieve presbyopic symptoms have been developed, but no eye drops have been developed. We screened certain compounds possibly related to lens degeneration and identified pirenoxine, which has been used for cataracts, as a possible new pharmacologic treatment for presbyopia. We first researched the anti-presbyopic activity of pirenoxine in rats. The lens elasticity significantly (p = 0.028) increased with exposure to tobacco smoke for 12 days, and pirenoxine eye drops significantly (p < 0.001) suppressed lens hardening, which causes presbyopia in humans. In a parallel randomized controlled clinical study of the subjects in their fifth decade of life, the objective accommodative amplitude (AA) decreased significantly (p < 0.01) by 0.16 diopter (D) in the control group, and there was no detectable change in the treatment group after a 6-month treatment period, suggesting that pirenoxine eye drops might prevent progression of presbyopia. Subjects in their sixth decade of life, in whom the AA was already nearly 0 D, did not show similar results. Pirenoxine eye drops might be a new and the first pharmacologic treatment for preventing progression of presbyopia.

Selenium-binding lactoferrin is taken into corneal epithelial cells by a receptor and prevents corneal damage in dry eye model animals.

The ocular surface is strongly affected by oxidative stress, which causes many ocular diseases including dry eye. Previously, we showed that selenium compounds, e.g., selenoprotein P and Se-lactoferrin, were candidates for treatment of dry eye. This paper shows the efficacy of Se-lactoferrin for the treatment of dry eye compared with Diquas as a control drug using two dry eye models and incorporation of lactoferrin into corneal epithelial cells via lactoferrin receptors. We show the efficacy of Se-lactoferrin eye drops in the tobacco smoke exposure rat dry eye model and short-term rabbit dry eye model, although Diquas eye drops were only effective in the short-term rabbit dry eye model. These results indicate that Se-lactoferrin was useful in the oxidative stress-causing dry eye model. Se-lactoferrin was taken into corneal epithelium cells via lactoferrin receptors. We identified LRP1 as the lactoferrin receptor in the corneal epithelium involved in lactoferrin uptake. Se-lactoferrin eye drops did not irritate the ocular surface of rabbits. Se-lactoferrin was an excellent candidate for treatment of dry eye, reducing oxidative stress by a novel mechanism.

Evaluation of treatment for dry eye with 2-hydroxyestradiol using a dry eye rat model.

PURPOSE: 2-hydroxy estradiol (2-OHE2) is a catechol derivative of 17ß -Estradiol (E2) and it is synthesized from E2 catalyzed by cytochrome P4501A1. Previous studies reported that 2-OHE2 is a physiologic antioxidant in lipoproteins, liver microsomes, and the brain. Catechol derivatives show an anti-inflammatory effect through the inhibition of prostaglandin endoperoxide synthase (PGS) activity. Corneal erosion caused by dry eye is related to an increase in oxidative stress and inflammation in ocular surface cells. We investigated the therapeutic effects of 2-OHE2 on corneal damage caused by dry eye. METHODS: Steroidal radical scavenging activity was confirmed through the electron spin resonance (ESR) method. PGS activity was measured using the COX Fluorescent Activity Assay Kit. To evaluate the effect of 2-OHE2 on the treatment for dry eye, 2-OHE2 was applied as an eye drop experiment using dry eye model rats. RESULTS: 2-OHE2 scavenged tyrosyl radical and possibly suppressed oxidative stress in corneal epithelial cells. In addition, 2-OHE2 inhibited PGS activity, and 2-OHE2 is probably a competitive inhibitor of PGS. Corneal PGS activity was upregulated in the dry eye group. Therefore, 2-OHE2 eye drops improved corneal erosion in dry eye model rats. CONCLUSIONS: 2-OHE2 is a candidate for the treatment of dry eye through the suppression of inflammation and oxidative stress in the cornea.

The effect of Nrf2 knockout on ocular surface protection from acute tobacco smoke exposure: evidence from Nrf2 knockout mice.

Ocular surface mucosa is the first-line ocular tissue to be exposed to environmental stress. We evaluated tear functions and keratoconjunctival epithelial alterations after sidestream cigarette smoke (SCS) exposure and tried to clarify the role of the transcription factor nuclear factor erythroid 2-related factor 2 (Nfe2l2, also known as Nrf2), on the ocular surface. In wild-type and Nrf2(-/-) mice, tear volume did not change after SCS exposure. Tear film breakup time (tear stability) in Nrf2(-/-) mice was significantly shorter than that in wild-type mice after SCS exposure. Vital staining scores, including fluorescein and Rose Bengal staining, showed significantly higher values in Nrf2(-/-) mice than in wild-type mice after SCS exposure. Excessive oxidative stress accumulation was detected in Nrf2(-/-) mice after SCS exposure using immunohistochemical analysis. Immunohistochemical analysis also revealed decreased mucin 1 (Muc1) and Muc5ac staining in Nrf2(-/-) mice after SCS exposure. mRNA expression levels of Muc1, Muc4, and Muc5ac and of SAM-pointed domain epithelial-specific transcription factor in Nrf2(-/-) mice were lower than those in wild-type mice after SCS exposure. Mean tear IL-6 concentrations increased significantly in Nrf2(-/-) mice after SCS exposure. In conclusion, SCS exposure induced decreased tear stability, ocular surface damage, and altered conjunctival phenotype in Nrf2(-/-) mice. Nrf2 could play an important role in protection of the ocular surface against SCS exposure.

Diclofenac protects cultured human corneal epithelial cells against hyperosmolarity and ameliorates corneal surface damage in a rat model of dry eye.

PURPOSE: Dry eye syndrome (DES) is characterized by an increase in tear osmolarity and induction of the expression and nuclear localization of an osmoprotective transcription factor (nuclear factor of activated T-cells 5 [NFAT5]) that plays an important role in providing protection against hyperosmotic tears. In this study, we screened medicines already in clinical use with a view of finding compounds that protect cultured human corneal epithelial cells against hyperosmolarity-induced cell damage. METHODS: Viable cell number was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and cellular NFAT5 level was measured by immunoblotting. The rat model for DES was developed by removal of the lacrimal glands, with an assessment of corneal surface damage based on levels of fluorescein staining and epithelial apoptosis. RESULTS: Some nonsteroidal anti-inflammatory drugs (NSAIDs), including diclofenac sodium (diclofenac), were identified during the screening procedure. These NSAIDs were able to suppress hyperosmolarity-induced apoptosis and cell growth arrest. In contrast, other NSAIDs, including bromfenac sodium (bromfenac), did not exert such a protective action. Treatment of cells with diclofenac, but not bromfenac, stimulated both the nuclear localization and expression of NFAT5 under hyperosmotic conditions. In the rat model for DES, topical administration of diclofenac (but not bromfenac) to eyes reduced corneal surface damage without affecting the volume of tear fluid. CONCLUSIONS: Diclofenac appears to protect cells against hyperosmolarity-induced cell damage and NFAT5 would play an important role in this protective action. The findings reported here may also indicate that the topical administration of diclofenac to eyes may be therapeutically beneficial for DES patients.

Selenium compound protects corneal epithelium against oxidative stress.

The ocular surface is strongly affected by oxidative stress, and anti-oxidative systems are maintained in corneal epithelial cells and tear fluid. Dry eye is recognized as an oxidative stress-induced disease. Selenium compound eye drops are expected to be a candidate for the treatment of dry eye. We estimated the efficacy of several selenium compounds in the treatment of dry eye using a dry eye rat model. All of the studied selenium compounds were uptaken into corneal epithelial cells in vitro. However, when the selenium compounds were administered as eye drops in the dry eye rat model, most of the selenium compounds did not show effectiveness except for Se-lactoferrin. Se-lactoferrin is a lactoferrin that we prepared that binds selenium instead of iron. Se-lactoferrin eye drops suppressed the up-regulated expression of heme oxygenase-1, cyclooxygenase-2, matrix metallopeptidase-9, and interleukin-6 and also suppressed 8-OHdG production in the cornea induced by surgical removal of the lacrimal glands. Compared with Se-lactoferrin, apolactoferrin eye drops weakly improved dry eye in high dose. The effect of Se-lactoferrin eye drops on dry eye is possibly due to the effect of selenium and also the effect of apolactoferrin. Se-lactoferrin is a candidate for the treatment of dry eye via regulation of oxidative stress in the corneal epithelium.

The antiaging approach for the treatment of dry eye.

Dry eye is one of the most common eye disorders affecting millions of people. It causes ocular irritation or discomfort, and decreases functional vision, causing a dramatic deterioration in the quality of life. Although new treatments such as the P2Y2 agonist or cyclosporine eye drops have been developed and a certain level of patient satisfaction can now be obtained, no fundamental treatment has been developed. Currently, there is no therapy available to recover lacrimal function to its normal status. Recent progress in the understanding of aging has laid the foundations for a new way of thinking about intervention of the aging process. Because dry eye is accelerated by aging, a useful approach for the prevention or treatment of dry eye may be to interfere with the aging process. In the scientific community, there is a global consensus that calorie restriction can extend the life span of various kinds of animals, establishing an intervention to aging. Another important hypothesis believed to be involved in aging is the free radical theory. According to these theories, the aging process may be managed by controlling levels of calories or reactive oxygen species. In this review, these 2 important aging theories, calorie restriction and free radical aging, are examined, and we discuss how to apply these theories to the prevention and treatment of dry eye.

IL-6 induction in desiccated corneal epithelium in vitro and in vivo.

PURPOSE: To investigate the effect of desiccation on secretion of inflammatory cytokines in corneal epithelial cells and in the rat desiccation model. METHODS: A human corneal epithelial cell line (CEPI) was grown in keratinocyte growth medium 2 (KGM2) to approximately 80% confluence. The medium was aspirated and dishes were left for 0 to 30 min with the cover left open to dry the cells (short-term desiccation). After desiccation, KGM2 was added to the dishes and collected from the dishes 15 min later to measure the concentrations of cytokines in the medium by sandwich enzyme immunoassay (ELISA). Viability of the cells was estimated with alamer blue. To study the effect of long-term desiccation, cultivated cells on transwells were used. After dessiccation for up to 8 h, the viability of the cells and levels of cytokines in the culture medium were examined. The expression of cytokines in the cornea of the dry eye model rat was measured by real-time PCR. RESULTS: Short-term dessication of CEPI cells significantly increased the interleukin (IL)-6 level and slightly increased the tumor necrosis factor (TNF)-α level. Anti-IL-6 antibody partially suppressed cell death caused by desiccation. Upon long-term desiccation, IL-6 and IL-8 levels were increased. In the dry eye model rats, the IL-6 mRNA level in the cornea significantly increased, whereas TNF-α mRNA level slightly increased. CONCLUSIONS: Desiccation induced IL-6 expression in corneal epithelial cells, suggesting that IL-6 participates in desiccation-induced cell death.

Corneal damage and lacrimal gland dysfunction in a smoking rat model.

Smoking is a serious public health problem around the world and causes many diseases such as chronic obstructive pulmonary disease, lung cancer, and some eye diseases. Cytochrome P450s (CYPs) are xenobiotic-metabolizing enzymes and are distributed in the corneas, protecting the ocular surface against chemical compounds in the environment. Although CYPs are principally detoxification enzymes, CYP1A1 and CYP2A6 are known to participate in the induction of lung cancer by smoking. We studied the participation of CYPs in corneal dysfunction caused by exposure to mainstream cigarette smoke (MCS) in a smoking rat model. Six-week-old male Sprague-Dawley rats were exposed to MCS. Exposure to MCS caused corneal damage and lacrimal gland dysfunction. Immunohistochemical analysis revealed that CYP1A1 expression was upregulated in the corneal epithelium and ducts of the lacrimal glands, accompanied by an increase in production of reactive oxygen species (ROS). An increase in 8-hydroxy-2'-deoxyguanosine, which is a marker of oxidative DNA damage, was detected only in areas where CYP1A1 was expressed, whereas the level of hexanoyl-lysine adduct, which is an initial marker of oxidative damage of phospholipids, did not increase. Exposure to MCS damaged the corneas and lacrimal glands probably through DNA oxidation by ROS produced by CYP1A1. Although the influence of other components in MCS remains unclear, CYPs, especially CYP1A1, probably participate in corneal damage and lacrimal gland dysfunction induced by smoking.

The era of antiaging ophthalmology comes of age: antiaging approach for dry eye treatment.

Recent advances in the understanding of aging have paved a new way of thinking about intervening with the aging process. There is a global agreement in the scientific community that calorie restriction (CR) can actually extend the life span of various kinds of animals so that this has become a real intervention in aging. In addition to the CR theory, the free radical theory is another important hypothesis, which is believed to be involved in aging. According to this theory, we can manage the aging process by controlling calories or reactive oxygen species. In this paper, these two important aging theories, CR and free radical aging, are reviewed, and it is discussed how to apply these theories to the prevention and treatment of eye diseases. Finally, we share the preliminary results of our animal study on dry eye, and I report my personal experience as a dry eye patient, which has been alleviated by the antiaging approach.

Selenoprotein P controls oxidative stress in cornea.

The ocular surface is always attacked by oxidative stress, and cornea epithelial cells are supposed to have their own recovery system against oxidative stress. Therefore we hypothesized that tears supply key molecules for preventing oxidative stress in cornea. The potential target key molecule we focused is selenoprotein P (SeP). SeP is a carrier of selenium, which is an essential trace element for many animals, for oxidative stress metabolism in the organism, and was extremely expressed in lacrimal gland. An experiment was performed with SeP eye drops in a rat dry eye model, prepared by removing the lacrimal glands. The anticipated improvement in corneal dry eye index and the suppression of oxidative stress markers were observed in SeP eye drop group. Furthermore, the concentration of SeP was significantly higher in dry eye patients compared with normal volunteers. Collectively, we concluded that tear SeP is a key molecule to protect the ocular surface cells against environmental oxidative stress.

The role of fractalkine as an accelerating factor on the autoimmune exocrinopathy in mice.

PURPOSE: Sjögren's syndrome (SS) is an organ-specific autoimmune disease caused by the progressive loss of exocrine glands and is associated with several autoimmune phenomena. Various research studies have been performed, and many molecules have been suggested as responsible for the pathogenesis of SS. Here the authors show the increased expression of fractalkine (CX(3)CL1) in lacrimal glands of SS model mice. Among more than 50 known chemokines, fractalkine is the sole member of the CX(3)C family and has unique structural and functional attributes. The purpose of this study was to analyze the role of fractalkine in exocrine glands. METHODS: The expression of fractalkine in the lacrimal glands of thymectomized NFS/sld mice was investigated by immunohistochemistry and RT-PCR. To confirm the effects of fractalkine in exocrine glands, tissue-specific fractalkine transgenic mice were generated using the salivary amylase promoter. RESULTS: The results demonstrated the upregulated fractalkine expression in thymectomized NFS/sld mice. Furthermore, the lacrimal and salivary gland-specific fractalkine transgenic mice showed the expression of fragmented fractalkine and lymphocytic infiltration in their lacrimal and submandibular glands. Interestingly, the dominant population was B cells in the lacrimal glands, whereas B cells and CD4(+) T cells were infiltrated in the submandibular glands. These mice also demonstrated slightly decreased tear and salivary secretion compared with wild-type mice. CONCLUSIONS: Based on these results, it may be that fractalkine contributes to the development of SS, especially in lymphocyte migration to exocrine glands, and that it accelerates the disease in association with other molecules.

Autologous serum eye drops for the treatment of dry eye diseases.

Conventional treatment of dry eye mainly consists of the use of preservative-free artificial eye drops and punctal occlusion. None of the commercially available artificial tear preparations include essential tear components such as epidermal growth factor, hepatocyte growth factor, fibronectin, neurotrophic growth factor, and vitamin A-all of which have been shown to play important roles in the maintenance of a healthy ocular surface epithelial milieu. We reported previously that autologous serum (AS) eye drops contain these essential factors and that AS eye drops are beneficial in the treatment of ocular surface diseases such as persistent epithelial defects, superior limbic keratoconjunctivitis, keratoconjunctivitis sicca, and neurotrophic keratopathy. However, there is some controversy regarding the efficacy of AS treatment. We demonstrated that this modality is more effective than artificial tears in a randomized control study. In in vivo and in vitro experiments, AS eye drops showed marked suppression of apoptosis in the conjunctival and corneal epithelium. Albumin, the major protein in serum, improved ocular surface damage in vivo and rescued apoptosis after serum deprivation in vitro. The biological background of AS eye drops and previous clinical studies of these medications for the treatment of dry eye are discussed.

Albumin rescues ocular epithelial cells from cell death in dry eye.

PURPOSE: Because autologous serum is useful for the treatment of severe dry eye, serum components may be a potential candidate for the treatment of dry eye. Serum albumin is abundantly contained in human serum and plays many physiologic roles. We investigated the efficacy of serum albumin in a dry eye animal model. METHODS: Sprague-Dawley rats were used to make dry eye model rats according to a previous study. The central region of the corneal epithelium was scraped mechanically, and the rats were placed in a desiccation room (temperature, 23 +/- 2 degrees C; humidity, 28 +/- 2%; air flow, 2-4 m/s) for 12 hr. During desiccation, one eye of each rat was treated with human serum albumin eye drops, and the other eye was given a drop of phosphate buffered saline (PBS). Human corneal and conjunctival cell lines were used to investigate suppression effect of albumin on apoptosis induced by addition of apoptosis inducers or serum deprivation, respectively. RESULTS: The erosion area was increased by 12 hr of desiccation. Albumin treatment decreased the area of erosion compared with PBS treatment. Apoptosis suppression assay using cell lines revealed that caspase-3 activation induced by serum deprivation and DNA fragmentation induced by addition of apoptosis inducers were dose-dependently suppressed by albumin. CONCLUSIONS: Albumin showed a therapeutic effect in dry eye model rats. This efficacy may be related to the suppression of apoptosis by albumin.

Protective effect of D-beta-hydroxybutyrate on corneal epithelia in dry eye conditions through suppression of apoptosis.

PURPOSE: To investigate the effect of D-beta-hydroxybutyrate (HBA) on ocular surface epithelial disorders induced by tear fluid deficiency, the potency of HBA and serum, the efficacy of which has been well documented in clinical application, were compared. METHODS: Rat corneal epithelial erosion was induced by exposure of rat eyes to continuous low-humidity airflow, which accelerated the tear evaporation. During desiccation, one eye of each rat was treated with HBA (20, 40, or 80 mM) or rat serum (5%, 20%, or 100%), and in the other eye a drop of phosphate-buffered saline (PBS) was instilled as the control. Histopathologic examination and quantification of the epithelial defect area were performed. The apoptosis in the epithelia was determined by chromatin condensation using the Hoechst 33342 fluorescein probe. RESULTS: In PBS-treated eyes, thinning in the cell layer was seen on the periphery of the initial wound after 6 hours, and it progressed to defects after 12 hours. In the 80-mM HBA and 20% serum applications, the pathologic change in the epithelia was moderate, and the structure was maintained in an almost normal state in the 100% serum application. Significant decreases in the defect areas were observed in the 5%, 20%, and 100% serum and 40- and 80-mM HBA treatment groups compared with the PBS-treated eyes (n=12). A significant suppression of chromatin condensation was observed with HBA and serum treatment. CONCLUSIONS: These results suggest the potential clinical application of HBA for ocular surface epithelial disorders to maintain epithelial cell viability in patients with dry eye.

Inhibitory activity of epigallocatechin gallate (EGCg) in paraquat-induced microsomal lipid peroxidation--a mechanism of protective effects of EGCg against paraquat toxicity.

Recently we have reported that epigallocatechin gallate (EGCg), a major component of Japanese green tea, significantly increased the survival rate of paraquat (Pq) poisoned mice. This paper describes two biochemical activities of EGCg, which relate to its protective effects against Pq toxicity. EGCg inhibited Pq-induced microsomal malondialdehyde (MDA) productions in rat liver microsome system containing 40 microM FeSO(4). Forty micromolar EGCg inhibited MDA production significantly. EGCg may inhibit the Pq-induced MDA production by at least two mechanisms. One may be iron-chelating activity as the inhibition disappeared when excess amounts of FeSO(4) were added to the reaction mixture, which indicated that EGCg reduced iron driven lipid peroxidation by pulling out available irons in the reaction mixture. The other is radical scavenging activity. EGCg scavenged DMPO-OOH spin adducts generated by the microsome-Pq system. The dose response curve of EGCg was similar to that obtained by ascorbic acid which is a typical water-soluble radical scavenger. Although ascorbic acid had a potential activity of scavenging superoxide radicals, it can not be recommended to use for the treatment of Pq poisoning, because ascorbic acid acts as a pro-oxidant in the presence of free transition metal ions by accelerating the Fenton reaction (Fe(2+)+H(2)O(2)-->Fe(3+)+OH(-)+OH*), which is responsible for lipid peroxidation. On the contrary, EGCg inhibited iron-driven lipid peroxidation presumably not only by chelating to Fe ions but also by scavenging superoxide radicals, which are responsible for the reduction of ferric (Fe(3+)) to ferrous (Fe(2+)) that catalyzes the Fenton reaction. Chelating and radical scavenging activity of EGCg can be expected simultaneously in the occurrence of Pq toxicity, which may explain the protective effects of EGCg against Pq toxicity.