Electron Paramagnetic Resonance Oximetry Research Articles

How Do Xanthophylls Protect Lipid Membranes from Oxidative Damage?

Here, we address the problem of the antioxidant activity of carotenoids in biomembranes. The activity of lutein and zeaxanthin in the quenching of singlet oxygen generated by photosensitization was monitored in lipid vesicles using a singlet oxygen-sensitive fluorescent probe and with the application of fluorescence lifetime imaging microscopy. The antioxidant activity of xanthophylls was interpreted on the basis of electron paramagnetic resonance oximetry results showing that xanthophylls constitute a barrier to the penetration of molecular oxygen into lipid membranes: to a greater extent in the 13-cis configuration than in all-trans. These results are discussed in relation to the trans-cis photoisomerization of xanthophylls observed in the human retina. It can be concluded that photoisomerization of xanthophylls is a regulatory mechanism that is important for both the modulation of light filtration through the macula and photoprotection by quenching singlet oxygen and creating a barrier to oxygen permeation to membranes.

Murine Breast Cancer Radiosensitization Using Oxygen Microbubbles and Metformin: Vessels Are the Key.

Radiotherapy is a cornerstone of cancer treatment, but tumor hypoxia and resistance to radiation remain significant challenges. Vascular normalization has emerged as a strategy to improve oxygenation and enhance therapeutic outcomes. In this study, we examine the radiosensitization potential of vascular normalization using metformin, a widely used anti-diabetic drug, and oxygen microbubbles (OMBs). We investigated the synergistic action of metformin and OMBs and the impact of this therapeutic combination on the vasculature, oxygenation, invasiveness, and radiosensitivity of murine 4T1 breast cancer. We employed in vivo Doppler ultrasonographic imaging for vasculature analysis, electron paramagnetic resonance oximetry, and immunohistochemical assessment of microvessels, perfusion, and invasiveness markers. Our findings demonstrate that both two-week metformin therapy and oxygen microbubble treatment normalize abnormal cancer vasculature. The combination of metformin and OMB yielded more pronounced and sustained effects than either treatment alone. The investigated therapy protocols led to nearly twice the radiosensitivity of 4T1 tumors; however, no significant differences in radiosensitivity were observed between the various treatment groups. Despite these improvements, resistance to treatment inevitably emerged, leading to the recurrence of hypoxia and an increased incidence of metastasis.

Oxidative stability and oxygen permeability of oil-loaded capsules produced by spray-drying or electrospraying measured by electron spin resonance

The oxidative stability and the oxygen permeability of oil-loaded capsules were investigated by Electron Spin Resonance (ESR). The capsules were produced by spray-drying or electrospraying in the monoaxial or coaxial configuration using glucose syrup as the encapsulating agent. ESR-spin trapping results showed that electrosprayed capsules oxidized faster and during the early stages of incubation, irrespective of the emitter configuration (monoaxial or coaxial), when compared to those produced by spray-drying. Furthermore, ESR oximetry showed that oxygen inside the spray-dried capsules reached equilibrium with the surrounding atmosphere significantly slower than the monoaxially electrosprayed capsules (i.e., ∼2h and ∼10 min, respectively). These findings have been attributed to the larger particle size of the spray-dried capsules influencing the oxygen diffusion area (i.e., lower surface-to-volume ratio) and diffusion path (i.e., thicker encapsulating wall for a fixed oil load). Together, the lower oxygen uptake reported for the spray-dried capsules correlated well with their higher oxidative stability.

Photoreactivity and phototoxicity of experimentally photodegraded hair melanosomes from individuals of different skin phototypes.

Even though melanin is commonly viewed as natural photoprotectant, the pigment demonstrates residual photoreactivity, which under certain conditions could contribute to UVA-dependent melanomagenesis. Skin melanin is constantly exposed to external stressors, including solar radiation, which could induce photodegradation of the pigment. Although photodegradation of melanin pigments was studied in synthetic models and RPE melanosomes, photochemical and photobiological effects of experimental photodegradation of human skin melanin of different chemical composition remain unknown. In this work, melanosomes isolated from hair of individuals of different skin phototypes (I-III, V) were exposed to high-intensity violet light and its impact on physical and chemical properties of the pigments were analyzed using electron paramagnetic resonance (EPR), spectrophotometry and dynamic light scattering (DLS). Photoreactivity of photodegraded melanins was examined by EPR oximetry, EPR spin-trapping and time-resolved singlet oxygen phosphorescence. Antioxidant potential of the pigments was measured using the EPR DPPH assay. Cellular effect of the exposure of melanosome-loaded HaCaT cells to UV-Vis light was determined by MTT assay, JC-10 assay, and iodometric assay. The data revealed that experimental photodegradation increased photoreactivity of natural melanins, while decreasing their antioxidant capacity. Photodegraded melanin was responsible for higher cell death, a decrease in mitochondrial membrane potential and elevated levels of lipid hydroperoxides.

Oxygen therapeutic window induced by myo-inositol trispyrophosphate (ITPP)-Local pO2 study in murine tumors.

Hypoxia, an inevitable feature of locally advanced solid tumors, has been known as an adverse prognostic factor, a driver of an aggressive phenotype, and an unfavorable factor in therapies. Myo-inositol trispyrophosphate (ITPP) is a hemoglobin modifier known to both increase O2 release and normalize microvasculature. Our goal was to measure the tumor oxygen partial pressure dynamic changes and timing of the therapeutic window after ITPP systemic administration. Two syngeneic tumor models in mice, B16 melanoma and 4T1 breast carcinoma, were used, with varying ITPP dose schedules. Tissue oxygenation level was measured over several days in situ in live animals by Electron Paramagnetic Resonance oximetry with implanted OxyChip used as a constant sensor of the local pO2 value. Both B16 and 4T1 tumors became more normoxic after ITPP treatment, with pO2 levels elevated by 10-20 mm Hg compared to the control. The increase in pO2 was either transient or sustained, and the underlying mechanism relied on shifting hypoxic tumor areas to normoxia. The effect depended on ITPP delivery intervals regarding the tumor type and growth rate. Moreover, hypoxic tumors before treatment responded better than normoxic ones. In conclusion, the ITPP-generated oxygen therapeutic window may be valuable for anti-tumor therapies requiring oxygen, such as radio-, photo- or immunotherapy. Furthermore, such a combinatory treatment can be especially beneficial for hypoxic tumors.

Quenching effect of cerium oxide nanoparticles on singlet oxygen: validation of the potential for reaction with multiple reactive oxygen species.

Here we studied cerium oxide nanoparticles (nanoceria) as an agent for the future treatment of oxidative damage by validating and evaluating its scavenging activity towards reactive oxygen species (ROS) in vitro. Nanoceria has been shown to mimic the activities of superoxide dismutase and catalase, degrading superoxide (O2•-) and hydrogen peroxide (H2O2). We examined the antioxidative activity of nanoceria, focusing on its ability to quench singlet oxygen (1O2) in an aqueous solution. Electron paramagnetic resonance (EPR) was used to determine the rates of second-order reactions between nanoceria and three ROS (1O2, O2•-, and H2O2) in aqueous solution, and its antioxidative abilities were demonstrated. Nanoceria shows a wide range of ultraviolet-light absorption bands and thus 1O2 was produced directly in a nanoceria suspension using high-frequency ultrasound. The quenching or scavenging abilities of nanoceria for 1O2 and hypoxanthine-xanthine oxidase reaction-derived O2•- were examined by EPR spin-trapping methods, and the consumption of H2O2 was estimated by the EPR oximetry method. Our results indicated that nanoceria interact not only with two previously reported ROS but also with 1O2. Nanoceria were shown to degrade O2•- and H2O2, and their ability to quench 1O2 may be one mechanism by which they protect against oxidative damage such as inflammation.

Measurement of Mitochondrial (Dys)Function in Cellular Systems Using Electron Paramagnetic Resonance (EPR): Oxygen Consumption Rate and Superoxide Production.

The oxygen consumption rate (OCR) and superoxide production are crucial when assessing mitochondrial function and/or dysfunction. EPR spectroscopy allows the measurement of both components either independently or simultaneously in a same cellular or mitochondrial preparation. OCR determination using EPR oximetry is based on the change in EPR linewidth of a paramagnetic oxygen sensing probe (a perdeuterated nitroxide) in the presence of oxygen consuming cells in a closed system. Superoxide production can be monitored by the oxidation of cyclic hydroxylamines into nitroxides. The contribution of superoxide to the nitroxide formation is deduced from experiments in the presence and in the absence of SOD and PEG-SOD as appropriate controls.

OxyChip embedded with radio-opaque gold nanoparticles for anatomic registration and oximetry in tissues.

Electron paramagnetic resonance oximetry using the OxyChip as an implantable oxygen sensor can directly and repeatedly measure tissue oxygen levels. A phase I, first-in-human clinical study has established the safety and feasibility of using OxyChip for reliable and repeated measurements of oxygen levels in a variety of tumors and treatment regimens. A limitation in these studies is the inability to easily locate and identify the implanted probes in the tissue, particularly in the long term, thus limiting spatial/anatomical registration of the implant for proper interpretation of the oxygen data. In this study, we have developed and evaluated an enhanced oxygen-sensing probe embedded with gold nanoparticles (GNP), called the OxyChip-GNP, to enable visualization of the sensor using routine clinical imaging modalities. In vitro characterization, imaging, and histopathology studies were carried out using tissue phantoms, excised tissues, and in vivo animal models (mice and rats). The results demonstrated a substantial enhancement of ultrasound and CT contrast using the OxyChip-GNP without compromising its electron paramagnetic resonance and oxygen-sensing properties or biocompatibility. The OxyChips embedded with gold nanoparticles (OxyChip-GNP) can be readily identified in soft tissues using standard clinical imaging modalities such as CT, cone beam-CT, or ultrasound imaging while maintaining its capability to make repeated in vivo measurements of tissue oxygen levels over the long term. This unique capability of the OxyChip-GNP facilitates precisely localized in vivo oxygen measurements in the clinical setting.

First-In-Human Study in Cancer Patients Establishing the Feasibility of Oxygen Measurements in Tumors Using Electron Paramagnetic Resonance With the OxyChip.

ObjectiveThe overall objective of this clinical study was to validate an implantable oxygen sensor, called the ‘OxyChip’, as a clinically feasible technology that would allow individualized tumor-oxygen assessments in cancer patients prior to and during hypoxia-modification interventions such as hyperoxygen breathing.MethodsPatients with any solid tumor at ≤3-cm depth from the skin-surface scheduled to undergo surgical resection (with or without neoadjuvant therapy) were considered eligible for the study. The OxyChip was implanted in the tumor and subsequently removed during standard-of-care surgery. Partial pressure of oxygen (pO2) at the implant location was assessed using electron paramagnetic resonance (EPR) oximetry.ResultsTwenty-three cancer patients underwent OxyChip implantation in their tumors. Six patients received neoadjuvant therapy while the OxyChip was implanted. Median implant duration was 30 days (range 4–128 days). Forty-five successful oxygen measurements were made in 15 patients. Baseline pO2 values were variable with overall median 15.7 mmHg (range 0.6–73.1 mmHg); 33% of the values were below 10 mmHg. After hyperoxygenation, the overall median pO2 was 31.8 mmHg (range 1.5–144.6 mmHg). In 83% of the measurements, there was a statistically significant (p ≤ 0.05) response to hyperoxygenation.ConclusionsMeasurement of baseline pO2 and response to hyperoxygenation using EPR oximetry with the OxyChip is clinically feasible in a variety of tumor types. Tumor oxygen at baseline differed significantly among patients. Although most tumors responded to a hyperoxygenation intervention, some were non-responders. These data demonstrated the need for individualized assessment of tumor oxygenation in the context of planned hyperoxygenation interventions to optimize clinical outcomes.

Atomistic simulations modify interpretation of spin-label oximetry data. Part 1: intensified water-lipid interfacial resistances.

The role of membrane cholesterol in cellular function and dysfunction has been the subject of much inquiry. A few studies have suggested that cholesterol may slow oxygen diffusive transport, altering membrane physical properties and reducing oxygen permeability. The primary experimental technique used in recent years to study membrane oxygen transport is saturation-recovery electron paramagnetic resonance (EPR) oximetry, using spin-label probes targeted to specific regions of a lipid bilayer. The technique has been used, in particular, to assess the influence of cholesterol on oxygen transport and membrane permeability. The reliability of such EPR recordings at the water-lipid interface near the phospholipid headgroups has been challenged by all-atom molecular dynamics (MD) simulation data that show substantive agreement with spin-label probe measurements throughout much of the bilayer. This work uses further MD simulations, with an updated oxygen model, to determine the location of the maximum resistance to permeation and the rate-limiting barrier to oxygen permeation in 1-palmitoyl,2-oleoylphosphatidylcholine (POPC) and POPC/cholesterol bilayers at 25 and 35°C. The current simulations show a spike of resistance to permeation in the headgroup region that was not detected by EPR but was predicted in early theoretical work by Diamond and Katz. Published experimental nuclear magnetic resonance (NMR) oxygen measurements provide key validation of the MD models and indicate that the positions and relative magnitudes of the phosphatidylcholine resistance peaks are accurate. Consideration of the headgroup-region resistances predicts bilayer permeability coefficients lower than estimated in EPR studies, giving permeabilities lower than the permeability of unstirred water layers of the same thickness. Here, the permeability of POPC at 35°C is estimated to be 13 cm/s, compared with 10 cm/s for POPC/cholesterol and 118 cm/s for simulation water layers of similar thickness. The value for POPC is 12 times lower than estimated from EPR measurements, while the value for POPC/cholesterol is 5 times lower. These findings underscore the value of atomic resolution models for guiding the interpretation of experimental probe-based measurements.

Hypoxia Imaging As a Guide for Hypoxia-Modulated and Hypoxia-Activated Therapy.

Significance: Oxygen imaging techniques, which can probe the spatiotemporal heterogeneity of tumor oxygenation, could be of significant clinical utility in radiation treatment planning and in evaluating the effectiveness of hypoxia-activated prodrugs. To fulfill these goals, oxygen imaging techniques should be noninvasive, quantitative, and capable of serial imaging, as well as having sufficient temporal resolution to detect the dynamics of tumor oxygenation to distinguish regions of chronic and acute hypoxia. Recent Advances: No current technique meets all these requirements, although all have strengths in certain areas. The current status of positron emission tomography (PET)-based hypoxia imaging, oxygen-enhanced magnetic resonance imaging (MRI), 19F MRI, and electron paramagnetic resonance (EPR) oximetry are reviewed along with their strengths and weaknesses for planning hypoxia-guided, intensity-modulated radiation therapy and detecting treatment response for hypoxia-targeted prodrugs. Critical Issues: Spatial and temporal resolution emerges as a major concern for these areas along with specificity and quantitative response. Although multiple oxygen imaging techniques have reached the investigative stage, clinical trials to test the therapeutic effectiveness of hypoxia imaging have been limited. Future Directions: Imaging elements of the redox environment besides oxygen by EPR and hyperpolarized MRI may have a significant impact on our understanding of the basic biology of the reactive oxygen species response and may extend treatment possibilities.

Evaluation of a Refined Implantable Resonator for Deep-Tissue EPR Oximetry in the Clinic.

(1) Summarize revisions made to the implantable resonator (IR) design and results of testing to characterize biocompatibility;(2) Demonstrate safety of implantation and feasibility of deep tissue oxygenation measurement using electron paramagnetic resonance (EPR) oximetry. In vitro testing of the revised IR and in vivo implantation in rabbit brain and leg tissues. Revised IRs were fabricated with 1-4 OxyChips with a thin wire encapsulated with two biocompatible coatings. Biocompatibility and chemical characterization tests were performed. Rabbits were implanted with either an IR with 2 oxygen sensors or a biocompatible-control sample in both the brain and hind leg. The rabbits were implanted with IRs using a catheter-based, minimally invasive surgical procedure. EPR oximetry was performed for rabbits with IRs. Cohorts of rabbits were euthanized and tissues were obtained at 1 week, 3 months, and 9 months after implantation and examined for tissue reaction. Biocompatibility and toxicity testing of the revised IRs demonstrated no abnormal reactions. EPR oximetry from brain and leg tissues were successfully executed. Blood work and histopathological evaluations showed no significant difference between the IR and control groups. IRs were functional for up to 9 months after implantation and provided deep tissue oxygen measurements using EPR oximetry. Tissues surrounding the IRs showed no more tissue reaction than tissues surrounding the control samples. This pre-clinical study demonstrates that the IRs can be safely implanted in brain and leg tissues and that repeated, non-invasive, deep-tissue oxygen measurements can be obtained using in vivo EPR oximetry.

Oxygenation Status in Normal Tissues, Pathological Tissues and Malignant Tumors: A pO2 Database Based on Electron Paramagnetic Resonance (EPR) Oximetry Measurements

Oxygenation Status in Normal Tissues, Pathological Tissues and Malignant Tumors: A pO2 Database Based on Electron Paramagnetic Resonance (EPR) Oximetry Measurements

Estimation of pO2 distribution in EPR oximetry

Electron paramagnetic resonance (EPR) oximetry, using oxygen-sensing implant such as OxyChip, is capable of measuring oxygen concentration in vivo – a critical tissue information required for successful medical treatment such as cancer, wound healing and diabetes. Typically, EPR oximetry produces one value of the oxygen concentration, expressed as pO2 at the site of implant. However, it is well recognized that in vivo one deals with a distribution of oxygen concentration and therefore reporting just one number is not representative_a long-standing critique of EPR oximetry. Indeed, when it comes to the assessment of radiation efficacy one should be guided not by the mean or median but the proportion of oxygenated cancer cells which can be estimated only when the whole oxygen distribution in the tumor is known. Although there is a handful of papers attempting estimation of the oxygen distribution they suffer from the problem of negative frequencies and no theoretical justification and no biomedical interpretation. The goal of this work is to suggest a novel method using the empirical Bayesian approach realized via nonlinear mixed modeling with a priori distribution of oxygen following a two-parameter lognormal distribution with parameters estimated from the multi-implant single component EPR scan. Unlike previous work, the result of our estimation is the distribution with positive values for the frequency and the associated pO2 value. Our algorithm based on nonlinear regression is illustrated with EPR measurements on OxyChips equilibrated with gas mixtures containing four values of pO2 and computation of the proportion of volume with pO2 greater than any given threshold. This approach may become crucial for application of the EPR oximetry in clinical setting when the sucsess of the treatment depends of the proportion of tissue oxygenated.

Photoreactivity of Hair Melanin from Different Skin Phototypes-Contribution of Melanin Subunits to the Pigments Photoreactive Properties.

Photoreactivity of melanin has become a major focus of research due to the postulated involvement of the pigment in UVA-induced melanoma. However, most of the hitherto studies were carried out using synthetic melanin models. Thus, photoreactivity of natural melanins is yet to be systematically analyzed. Here, we examined the photoreactive properties of natural melanins isolated from hair samples obtained from donors of different skin phototypes (I, II, III, and V). X-band and W-band electron paramagnetic resonance (EPR) spectroscopy was used to examine the paramagnetic properties of the pigments. Alkaline hydrogen peroxide degradation and hydroiodic acid hydrolysis were used to determine the chemical composition of the melanins. EPR oximetry and spin trapping were used to examine the oxygen photoconsumption and photo-induced formation of superoxide anion, and time-resolved near infrared phosphorescence was employed to determine the singlet oxygen photogeneration by the melanins. The efficiency of superoxide and singlet oxygen photogeneration was related to the chemical composition of the studied melanins. Melanins from blond and chestnut hair (phototypes II and III) exhibited highest photoreactivity of all examined pigments. Moreover, melanins of these phototypes showed highest quantum efficiency of singlet oxygen photogeneration at 332 nm and 365 nm supporting the postulate of the pigment contribution in UVA-induced melanoma.

Oxygen-Sensing Paramagnetic Probes for Clinical Oximetry.

Electron paramagnetic resonance (EPR) oximetry based on lithium naphthalocyanine paramagnetic crystals as oxygen sensors enables direct measurement of the partial pressure of oxygen (pO2) in tissues. The method uses topical or implantable forms of these oxygen-sensing crystals embedded in a biocompatible siloxane elastomer. This article presents a summary of these sensors for EPR oximetry and their applicability for tissue oxygen measurement in the clinic.

Discerning Membrane Steady-State Oxygen Flux by Monte Carlo Markov Chain Modeling.

Molecular oxygen (O2) permeability coefficients for lipid bilayers have previously been estimated using both electron paramagnetic resonance (EPR) oximetry and molecular dynamics simulation data. Yet, neither technique captures the fluxes that exist physiologically. Here, the dynamic steady state is modeled using a stochastic approach built on atomic resolution molecular dynamics simulation data. A Monte Carlo Markov chain technique is used to examine membrane-level fluxes of oxygen in lipid-water systems. At steady state, the concentration of oxygen is found to be higher inside the model membranes than in surrounding water, consistent with the known favorable partitioning of O2 toward the lipid phase. Pure phospholipid 1-palmitoyl,2-oleoyl-phosphatidylcholine (POPC) bilayers accrue ~40% more O2 molecules at steady state than POPC/cholesterol bilayers (1:1 molecular ratio) mimicking the red blood cell membrane. Steady-state levels of oxygen were reached inside both bilayer types within the same timeframe, but depletion of oxygen from the bilayer interior occurred 17% faster for POPC than for POPC/cholesterol. Likewise, first-order rate constants estimated for accrual to steady state were the same for POPC and POPC/cholesterol, at 190μs-1, while first-order rate constants for depletion of the accrued O2 from the bilayers differed, at 95μs-1 for POPC and 81μs-1 for POPC/cholesterol (lower by 15%). These results are consistent with prior experiments in red blood cells (RBCs) with varying membrane cholesterol content, in which additional cholesterol slowed oxygen uptake and release. Further work is needed to understand whether differences in RBC membrane cholesterol content would affect the delivery of oxygen to tissues.

OxyChip Implantation and Subsequent Electron Paramagnetic Resonance Oximetry in Human Tumors Is Safe and Feasible: First Experience in 24 Patients.

Introduction: Tumor hypoxia confers both a poor prognosis and increased resistance to oncologic therapies, and therefore, hypoxia modification with reliable oxygen profiling during anticancer treatment is desirable. The OxyChip is an implantable oxygen sensor that can detect tumor oxygen levels using electron paramagnetic resonance (EPR) oximetry. We report initial safety and feasibility outcomes after OxyChip implantation in a first-in-humans clinical trial (NCT02706197, www.clinicaltrials.gov).Materials and Methods: Twenty-four patients were enrolled. Eligible patients had a tumor ≤ 3 cm from the skin surface with planned surgical resection as part of standard-of-care therapy. Most patients had a squamous cell carcinoma of the skin (33%) or a breast malignancy (33%). After an initial cohort of six patients who received surgery alone, eligibility was expanded to patients receiving either chemotherapy or radiotherapy prior to surgical resection. The OxyChip was implanted into the tumor using an 18-G needle; a subset of patients had ultrasound-guided implantation. Electron paramagnetic resonance oximetry was carried out using a custom-built clinical EPR scanner. Patients were evaluated for associated toxicity using the Common Terminology Criteria for Adverse Events (CTCAE); evaluations started immediately after OxyChip placement, occurred during every EPR oximetry measurement, and continued periodically after removal. The OxyChip was removed during standard-of-care surgery, and pathologic analysis of the tissue surrounding the OxyChip was performed.Results: Eighteen patients received surgery alone, while five underwent chemotherapy and one underwent radiotherapy prior to surgery. No unanticipated serious adverse device events occurred. The maximum severity of any adverse event as graded by the CTCAE was 1 (least severe), and all were related to events typically associated with implantation. After surgical resection, 45% of the patients had no histopathologic findings specifically associated with the OxyChip. All tissue pathology was “anticipated” excepting a patient with greater than expected inflammatory findings, which was assessed to be related to the tumor as opposed to the OxyChip.Conclusion: This report of the first-in-humans trial of OxyChip implantation and EPR oximetry demonstrated no significant clinical pathology or unanticipated serious adverse device events. Use of the OxyChip in the clinic was thus safe and feasible.

The Effect of Antioxidants on Photoreactivity and Phototoxic Potential of RPE Melanolipofuscin Granules from Human Donors of Different Age.

One of the most prominent age-related changes of retinal pigment epithelium (RPE) is the accumulation of melanolipofuscin granules, which could contribute to oxidative stress in the retina. The purpose of this study was to determine the ability of melanolipofuscin granules from younger and older donors to photogenerate reactive oxygen species, and to examine if natural antioxidants could modify the phototoxic potential of this age pigment. Electron paramagnetic resonance (EPR) oximetry, EPR-spin trapping, and time-resolved detection of near-infrared phosphorescence were employed for measuring photogeneration of superoxide anion and singlet oxygen by melanolipofuscin isolated from younger and older human donors. Phototoxicity mediated by internalized melanolipofuscin granules with and without supplementation with zeaxanthin and α-tocopherol was analyzed in ARPE-19 cells by determining cell survival, oxidation of cellular proteins, organization of the cell cytoskeleton, and the cell specific phagocytic activity. Supplementation with antioxidants reduced aerobic photoreactivity and phototoxicity of melanolipofuscin granules. The effect was particularly noticeable for melanolipofuscin mediated inhibition of the cell phagocytic activity. Antioxidants decreased the extent of melanolipofuscin-dependent oxidation of cellular proteins and disruption of the cell cytoskeleton. Although melanolipofuscin might be involved in chronic phototoxicity of the aging RPE, natural antioxidants could partially ameliorate these harmful effects.

The effect of aging and antioxidants on photoreactivity and phototoxicity of human melanosomes: An in vitro study.

Aging may significantly modify antioxidant and photoprotective properties of melanin in retinal pigment epithelium (RPE). Here, photoreactivity of melanosomes (MS), isolated from younger and older human donors with and without added zeaxanthin and α-tocopherol, was analyzed by electron paramagnetic resonance oximetry, time-resolved singlet oxygen phosphorescence, and protein oxidation assay. The phototoxic potential of ingested melanosomes was examined in ARPE-19 cells exposed to blue light. Phagocytosis of FITC-labeled photoreceptor outer segments (POS) isolated from bovine retinas was determined by flow cytometry. Irradiation of cells fed MS induced significant inhibition of the specific phagocytosis with the effect being stronger for melanosomes from older than from younger human cohorts, and enrichment of the melanosomes with antioxidants reduced the inhibitory effect. Cellular protein photooxidation was more pronounced in samples containing older melanosomes, and it was diminished by antioxidants. This study suggests that blue light irradiated RPE melanosomes could induce substantial inhibition of the key function of the cells-their specific phagocytosis. The data indicate that while photoreactivity of MS and their phototoxic potential increase with age, they could be reduced by selected natural antioxidants.