Toxicity and detoxification of lipid-derived aldehydes in cultured retinal pigmented epithelial cells

Toxicology and Applied Pharmacology 204 (2005) 122 – 134 www.elsevier.com/locate/ytaap

Toxicity and detoxification of lipid-derived aldehydes in cultured retinal pigmented epithelial cells S. Choudharya, T. Xiaoa, S. Srivastavab, W. Zhanga, L.L. Chana, L.A. Vergarac, F.J.G.M. Van Kuijkd, N.H. Ansaria,* a

Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, TX 77555-0647, USA b Department of Medicine, Division of Cardiology, University of Louisville, Louisville, KY 40208, USA c Department of Physiology and Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0647, USA d Department of Ophthalmology and Visual Science, The University of Texas Medical Branch, Galveston, TX 77555-0647, USA Received 24 June 2004; accepted 30 August 2004 Available online 28 December 2004

Abstract Age-related macular degeneration (ARMD) is the leading cause of blindness in the developed world and yet its pathogenesis remains poorly understood. Retina has high levels of polyunsaturated fatty acids (PUFAs) and functions under conditions of oxidative stress. To investigate whether peroxidative products of PUFAs induce apoptosis in retinal pigmented epithelial (RPE) cells and possibly contribute to ARMD, human retinal pigmented epithelial cells (ARPE-19) were exposed to micromolar concentrations of H2O2, 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE). A concentration- and time-dependent increase in H2O2-, HNE-, and HHE-induced apoptosis was observed when monitored by quantifying DNA fragmentation as determined by ELISA, flow cytometry, and Hoechst staining. The broad-spectrum inhibitor of apoptosis Z-VAD inhibited apoptosis. Treatment of RPE cells with a thionein peptide prior to exposure to H2O2 or HNE reduced the formation of protein-HNE adducts as well as alteration in mitochondrial membrane potential and apoptosis. Using 3H-HNE, various metabolic pathways to detoxify HNE by ARPE-19 cells were studied. The metabolites were separated by HPLC and characterized by ElectroSpray Ionization-Mass Spectrometry (ESI-MS) and gas chromatography-MS. Three main metabolic routes of HNE detoxification were detected: (1) conjugation with glutathione (GSH) to form GS-HNE, catalyzed by glutathione-S-transferase (GST), (2) reduction of GS-HNE catalyzed by aldose reductase, and (3) oxidation of HNE catalyzed by aldehyde dehydrogenase (ALDH). Preventing HNE formation by a combined strategy of antioxidants, scavenging HNE by thionein peptide, and inhibiting apoptosis by caspase inhibitors may offer a potential therapy to limit retinal degeneration in ARMD. D 2004 Elsevier Inc. All rights reserved. Keywords: 4-Hydroxynonenal; 4-Hydroxyhexenal; Oxidative stress; Retinal pigmented; Epithelium; Apoptosis; Age-related macular degeneration

Introduction Age-related macular degeneration (ARMD) is the most common cause of blindness in elderly individuals over 55 Abbreviations: HNE, 4-hydroxynonenal; HHE, 4-hydroxyhexenal; LDAs, lipid-derived aldehydes; ARMD, Age-related macular degeneration; GC/CI-MS, gas chromatography/chemical ionization-mass spectrometry; ESI-MS, ElectroSpray Ionization-MS; AR, aldose reductase; ALDH, aldehyde dehydrogenase. * Corresponding author. Fax: +1 409 772 9679. E-mail address: [email protected] (N.H. Ansari). 0041-008X/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2004.08.023

years of age in developed nations (Klein et al., 1995). The exact etiology of ARMD is not known. Oxidative damage has been implicated in the pathogenesis of ARMD (Bressler et al., 1988), but the precise role of the damaging effects of the reactive oxygen species in the development and progression of this aging disease remains to be fully elucidated. Epidemiological studies of diet, environmental, and behavioral risk factors suggest that oxidative stress is a contributing factor of ARMD (Beatty et al., 2000). The retina is particularly susceptible

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to oxidative stress because of its high consumption of oxygen, its high proportion of polyunsaturated fatty acids (PUFAs), and its exposure to visible light (Bazan, 1989; Dargel, 1992; Sickel, 1972). Retinal pigmented epithelial (RPE) cells are postmitotic and a primary function is to phagocytize photoreceptor outer segment (Young, 1967); this process generates oxidants (Tate et al., 1995). Under oxidative stress, PUFAs are degraded and yield reactive a-h, unsaturated lipid aldehydes (LDAs) such as 4hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE). Since corresponding precursor PUFAs of HNE and HHE (arachidonic and docosohexanoeic acid, respectively) are present in abundance in the retina (Stone et al., 1979), it is likely that retinal degeneration associated with aging may be mediated via these LDAs. Because damage to the RPE is an early event in ARMD (Green et al., 1985; Spraul et al., 1996; Zarbin, 1998), it is important to delineate the role of LDAs in the degeneration of RPE. In vitro studies show that oxidant-treated RPE cells undergo apoptosis, a possible mechanism by which RPEs are lost during the early phase of ARMD (Cai et al., 1999). There is increasing evidence suggesting that LDAs formed by lipid peroxidative reactions mediate the biological effects of free radicals (Esterbauer et al., 1991). By acting as toxic messengers, LDAs may propagate oxidative stress and be responsible for a significant portion of the tissue damage ascribed to their radical precursors. As a result of a conjugated double bond between the a and h carbons, the g carbon of these aldehydes is electron deficient and reacts readily with nucleophiles such as thiols and amines, whereas the carbonyl group forms Schiff base with amino groups thereby forming adducts with proteins. Thus, these aldehydes are highly reactive and display marked biological effects, which, depending upon their concentration, can produce selective alterations in cell signaling, protein and DNA damage, and cytotoxicity (Esterbauer et al., 1993), including apoptosis (Choudhary et al., 2004; Herbst et al., 1999; Li et al., 1996; Malecki et al., 2000; Zhang et al., 2001). The extent and severity of damage produced by LDAs will depend, in part, upon how the particular cell type detoxifies these aldehydes. The metabolism and the injurious effects of LDAs have been extensively investigated mainly in tissues such as liver, heart, erythrocytes (Halliwell, 1991; Srivastava et al., 1998, 2000), and ocular lens (Choudhary et al., 2003). HNE can be detoxified by oxidation to 4-hydroxynonenoic acid (HNA) by aldehyde dehydrogenase (ALDH) and carbonyl reductases (Esterbauer et al., 1991) or reduced to dihydroxynonene (DHN) by aldose reductase (AR). HNE can also conjugate with glutathione (GSH) to form GS-HNE, catalyzed by glutathione-S-transferase (GST) (Choudhary et al., 2003; Halliwell, 1991; Srivastava et al., 1998, 2000), and then be reduced to GS-DHN by AR (Choudhary et al., 2003; Halliwell, 1991; He et al., 1998; Srivastava et al., 1998,

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2000). Toxic LDAs can be readily generated in the retina that normally functions under oxidative stress conditions. In the present study, we have investigated the metabolic pathways and the apoptotic potential of LDAs in ARPE19 cells.

Materials and methods Materials. Human RPE cell line ARPE-19 was purchased from ATCC. DMEM/F-12 media were obtained from Gibco. Fetal bovine serum (FBS), trypsin/EDTA, antibiotics, aldehyde dehydrogenase, cyanamide, reduced glutathione (GSH) fetal bovine serum (FBS), trypsin, and all chemicals that are not otherwise specified were purchased from Sigma (St. Louis, MO, USA). The substrates and inhibitors of caspases were purchased from Enzyme Systems (Dublin, CA) and the Cell Death ELISA kit from Boehringer Mannheim (Mannheim, Germany). Thionein hexapeptide (H-Lys-Cys-Thr-Cys-Cys-Ala-OH as a trifluoroacetate salt) was synthesized and HPLC purified at bThe Peptide Synthesis Core FacilityQ of University of Texas Medical Branch, Galveston. JC-1 dye, Vybrant Apoptosis Assay kit #5 (containing Hoechst 33342 and Propidium iodide) were purchased from Molecular Probes (Eugene, OR, USA). Anti-HNE Michael adduct antibodies were obtained from Calbiochem (San Diego, CA) and HNE from Cayman Chemicals (Ann Arbor, MI). HHE was generously provided by Dr. F.M.J. van Kuijk of University of Texas Medical Branch, Galveston. Trifluoroacetic acid (TFA) was obtained from Pierce (Rockford, IL, USA). Sorbinil was a gift from Pfizer (Groton, CT, USA). All other reagents were of the highest purity available. Cell line. ARPE-19 cells were cultured in a 1:1 mixture of Ham’s F medium with 2.5 mM l-glutamine and 10% fetal bovine serum at 37 8C. The medium was renewed every 3–4 days. Subculturing was done using trypsin/EDTA solution and the cells were subcultivated with a split ratio of 1:3 to 1:5. Before treating the cells with H2O2, HNE, or HHE, the cells were washed with PBS and the regular media were replaced by phenol red free media containing 5% FBS. In all the experiments where Z-VAD or thionein peptide were used, cells were preincubated with them for 1 h. Measurement of apoptosis. Apoptosis was determined by quantifying the cytosolic oligonucleosome-bound DNA using a bCell Death Detection ELISA KitQ (Boehringer Mannheim) as per the manufacture’s instructions and as previously described (Choudhary et al., 2003; Yang et al., 2002; Zhang et al., 2001). Measurement of caspase activity. Caspase activity was measured according to Sarin et al. (1996) and as previously described (Choudhary et al., 2004; Xiao et al., 2003; Yang et al., 2002). Briefly, cells (0.5  106 cells) were lysed

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with 100 Al of buffer containing 50 mM HEPES, pH 7.5, 10% sucrose, and 0.1% Triton-X-100. After 20 min on ice, the extracts were centrifuged at 10 000  g for 10 min at 4 8C. Dithiothreitol (DTT) was added to the supernatant fraction at a final concentration of 10 mM. The specific fluorogenic substrates (50 AM) for each caspase (1, 2, 3, and 8) were individually mixed with 10-Al aliquots of the cell extracts in 96-well plates and allowed to react for 1 h at room temperature. After dilution with 0.1 ml PBS, the fluorescence in the samples was measured using the Fluorocount Microplate Fluorometer (Packard Instruments, Meriden, CT) at an excitation of 400 nm and emission of 510 nm.

(Choudhary et al., 2002; Srivastava et al., 2000). Synthetic HNE metabolites were separated by HPLC using a Beckman reversed phase C18 column (particle size, 5 Am; dimensions 4.6  250 mm, Beckman Instruments, Inc., Fullerton, CA, USA) using a gradient consisting of solvent A (0.1% aqueous trifluoroacetic acid, TFA) and solvent B (100% acetonitrile) at a flow rate of 1 ml/min. The gradient was established such that B reached 24% in 15 min and held for 5 min. In an additional 10 min, B reached 26% and was held at this value for 5 min. Further, in the next 20 min, it reached 100%. Fractions (1 ml) were collected and the radioactivity was measured using a Beckman liquid scintillation radioactivity counter.

Detection of early stages of apoptosis by flow cytometry. Flow cytometry was performed using the cationic dye JC-1 (Molecular Probes) that exhibits potential-dependent accumulation in the mitochondria, indicated by a fluorescence emission shift from green (~525 nm) to red (~590 nm). Pellets of approximately 1  106 cells were suspended in 130 Al of warm 1:1 mixture of Ham’s F12 medium containing 5% FBS to which 250 Al of JC-1 solution (20 Ag/ml) was added. After incubation for 30 min, cells were washed with PBS, resuspended in 500 Al PBS, and subjected to flow cytometry.

Mass spectrometry. Since GS-HNE and GS-DHN were not separated on HPLC, to characterize the GS-conjugate mixture, ElectroSpray Ionization (ESI) mass spectra were acquired on a single quadrapole Micromass LCZ instrument. The ESI-Mass Spectrometry (ESI-MS) operating conditions were as follows: capillary voltage, 3.0 kV; cone voltage, 25 V; extractor voltage, 4 V; source block temperature, 80 8C; and dissolvation temperature, 200 8C. Nitrogen at 3 psi was used as nebulizer gas. Samples were reconstituted in 100 Al acetonitrile/water/acetic acid 50/50/ 0.1 (v/v/v) and then injected into the mass spectrometer using a Harvard Apparatus syringe pump at a rate of 10 Al/ min. Spectra were acquired at the rate of 200 atomic mass units/s over the range of 100–650 Da. Calculations for GSHNE and GS-DHN quantification were performed by using the arbitrary counts of the ions in the individual peaks of the ESI-MS spectra and the radioactivity counts of the GSconjugates obtained upon HPLC.

Hoechst staining. One microliter of each reagent of Vybrant apoptosis assay kit (V-13244), Hoechst 33342, and propidium iodide were added to 100 Al of the cell suspension (1  104 cells) and incubated for 20 min on ice. Following incubation, cells were cytospun (500 rpm, 2 min) on poly-l-Lysine-coated slides and visualized under a Nikon Eclipse 800 epifluorescence microscope equipped with a xenon arc lamp. A Texas red filter set (excitation 540–580 nm, dichroic mirror 595 nm, emission 600–660 nm) and a DAPI filter set (excitation 340–380 nm, dichroic mirror 400 nm, emission 435–485 nm) for red and blue fluorescence, respectively. Photographs were taken using a ROPER Scientific CoolSNAP Fx monochrome cooled CCD 12 bit digital camera. Formation of protein-HNE adducts in ARPE-19 cells exposed to H2O2/HNE. Cells were washed, cytospun on poly-l-lysine-coated slides, and dehydrated in cold acetone for 5 min. After thorough washing with TBS, the slides were incubated with normal goat serum (1:100 diluted) for 30 min at RT and then washed with TBS. The slides were incubated with anti-HNE antibodies (1:500 diluted) overnight at 4 8C. After several washes with TBS, the slides were incubated with FITC-conjugated goat anti-rabbit IgG (1:50 diluted) for 2 h at RT, washed with TBS, and photographed as described above using a FITC filter set. HPLC analysis. Tritiated HNE and its putative metabolites (DHN, HNA, GS-HNE, and GS-DHN) were synthesized and purified to homogeneity as previously described

GC/CI-MS. DHN, HNA, and HNE were characterized by gas chromatography/chemical ionization-mass spectrometry (GC/CI-MS). The samples were derivatized in 20 Al of acetonitrile with 20 Al of N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) for 1 h at 60 8C. The mixture was cooled to room temperature and 1-Al aliquots were used for analysis. The GC/CI-MS analysis was performed using a HP5890/HP5973 GC/CI-MS system (Hewlett Packard; Palo Alto, CA, USA) under 70 eV electron ionization conditions. The compounds were separated on a bonded phase capillary column (DB-5MS, 30 m  0.25 mm ID  0.25 Am film thickness, J7W Scientific Folsom, CA, USA). The GC injection port and interface temperature were set to 280 8C, with helium gas (carrier) maintained at 14 psi. Injections were made in the splitless mode with the inlet port purged for 1 min following injection. The GC oven temperature was held initially at 100 8C for 1 min, then increased at a rate of 10 8C min 1 to 280 8C, which was held for 5 min. Under these conditions, the retention time for HNA derivative was 9.94 min. Metabolic studies. To study the rate of detoxification of HNE in ARPE-19 cells, 0.8  106 cells were seeded in

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P-60 dish a day before the experiment. Cells were washed twice with PBS to remove any trace of fetal bovine serum (FBS). Cells were incubated with 30 nmol of [3H]HNE (45 000 cpm) in 2.0 ml of Krebs–Hensleit (K–H) buffer containing 118 mM NaCl, 4.7 mM KCl, 1.25 mM MgCl2, 3.0 mM CaCl2, 1.25 mM KH2PO4, 0.5 mM EDTA, 25 mM NaHCO3, and 10 mM glucose (pH 7.4) at 37 8C for 0–90 min. For the time course, at the specified time (15, 30, 60, and 90 min), an aliquot of the incubation media was removed, ultrafiltered through Amicon microcon-10 (Millipore Corporation, Bedford, MA, USA). The filtrate thus obtained was used for separation by HPLC. The metabolites of [3H]HNE were quantified by determining the radioactivity in each fractions and the individual peaks were analyzed and characterized by ESI-MS or GC/MS. In order to elucidate the metabolic pathways involved in detoxifying HNE in these cells, RPE cells were preincubated with either 50 AM sorbinil (AR inhibitor) or 2.0 mM cyanamide (aldehyde dehydrogenase inhibitor) for 30 min at 37 8C in 2.0 ml K–H buffer. Subsequently, the cells were incubated with 30 nmol of [3H]HNE for 60 min at 37 8C. After incubation, the reaction mixture was ultrafiltered and the metabolites were separated by HPLC and analyzed. Statistical analysis. The results presented are of mean F SE. The data were analyzed by ANOVA using Turkey’s multiple comparison test or Student’s t test. Differences were considered significant at P b 0.05. Each experiment was performed at least two times.

Fig. 1. Concentration-dependent effect of H2O2 on ARPE-19 cell viability and apoptosis. 1  106 cells/2 ml cultured in 1:1 Ham’s F12 medium with 5% FBS and 1% penicillin/streptomycin were treated with various concentrations of H2O2, and after 24 h of exposure, the cell viability (A) and apoptosis (B) were measured. Cell viability was determined by trypan blue dye exclusion (D) and by MTT (x) and expressed as percent survival of control without H2O2. Values are mean F SEM of three experiments.

Results

of HNE or HHE for 24 h. A dose-dependent increase in apoptosis was observed with HNE as well as HHE. The HNE-induced apoptosis reached a maximum of 4-fold at 20 AM and declined thereafter. Similarly, HHE-induced apoptosis reached a maximum of approximately 2.5-fold and declined thereafter (Fig. 2B). The decline in apoptosis at higher concentrations of HNE and HHE may be attributed to necrosis. ARPE-19 cells incubated with 100 AM H2O2 showed 20% decrease in cell viability in the first 9 h of H2O2 treatment and by 24 h resulted in an additional 10% loss in cell viability. An exponential increase in apoptosis was observed with increase in time of exposure of cells to H2O2. A maximum of 13-fold increase in apoptosis was observed after 24 h (Fig. 3). Cells exposed to 50 AM HNE or HHE for various time intervals also resulted in a time-dependent increase in apoptosis, reaching a maximum of 4- or 3-fold, respectively, by 24 h (data not shown).

H2O2, HNE, or HHE decreases ARPE-19 cell viability and increases apoptosis LC50 for H2O2 in ARPE-19 was found to be approximately 100 and 60 AM by trypan blue exclusion and MTT, respectively, in phenol red free media (Fig. 1A). However, in media containing phenol red, LC50 was found to be approximately 700 and 500 AM by trypan blue exclusion and MTT, respectively (data not shown). A concentrationdependent (50–200 AM) increase in apoptosis (Fig. 1B) was observed in ARPE-19 cells incubated in H2O2 for 24 h. Similarly, when the cells were incubated with various concentrations of HNE or HHE for 24 h, a concentrationdependent decrease in cell viability was observed. LC50 for HNE and HHE in ARPE-19 cells as measured by trypan blue exclusion test was found to be approximately 40 and 60 AM, respectively (Fig. 2A), whereas by MTT test, it was approximately 30 and 40 AM for HNE and HHE, respectively, in phenol red free medium as well as in phenol red medium (data not shown). In order to elucidate the effect of HNE and HHE to induce apoptosis, ARPE-19 cells were exposed to different concentrations

Early apoptotic changes in ARPE-19 cells induced by oxidative stress In order to assess the effect of H2O2 and HNE on the early stages of apoptosis, RPE cells were first exposed to

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communications). Therefore, the effect of Z-VAD on H2O2- and HNE-induced apoptosis was investigated. A thiol, thionein peptide was also tested for efficacy in scavenging the generated HNE and thereby providing a protective effect. ARPE-19 cells (1  106) were incubated with either 20 AM Z-VAD or 50 AM thionein peptide (concentrations pretested to provide maximum inhibition of HNE-induced apoptosis and minimum loss of cell viability; data not shown) before addition of H2O2 or HNE. One hour later, H2O2 (200 AM) and HNE (50 AM) were added to the media and further incubated for 16 h. After incubation, cells were harvested and analyzed for apoptosis by ELISA. Fig. 5 shows that Z-VAD significantly attenuated H2O2- and HNE-induced apoptosis, suggesting the involvement of caspases. Similarly, thionein peptide at the concentration of 50 AM completely attenuated H2O2and HNE-induced apoptosis, supporting a protective efficacy. To visualize the protective effects of Z-VAD and thionein peptide on H2O2- and HNE-induced apoptosis, ARPE-19 cells were stained with nuclear dyes, Hoechst 33342, and propidium iodide. H2O2- and HNEtreated cells showed chromatin condensation; however, cells pretreated with Z-VAD or thionein peptide had a nuclear staining pattern similar to that of untreated cells (Fig. 6). The results of HHE-treated cells were similar to HNE-treated cells (data not shown). Fig. 2. Concentration-dependent effect of HNE and HHE on ARPE-19 cell viability and apoptosis. 1  106 cells/2 ml were incubated with various concentrations of HNE (D) and HHE (). After 24 h cells were harvested and cell viability was determined by trypan blue exclusion test (A) and apoptosis by ELISA as described in the text and expressed as absorbance at 405 nm (B). Values are mean of two separate experiments. The bars display the range of the value.

H2O2 (200 AM), HNE (50 AM), or HHE (50 AM) for 5 h, followed by incubation with JC-1 dye, and then subjected to flow cytometry. As shown in Fig. 4, approximately 30% of the cells underwent early apoptotic change following H2O2 treatment (4D); however, in HNE-treated cells, ~80% of the cells showed a change in polarity of the mitochondrial membrane (4C), and in HHE-treated cells, ~65% of the cells underwent early apoptotic changes (data not shown). Treating the cells with thionein peptide (50 AM) for 30 min prior to the addition of HNE or H2O2 resulted in a significant restoration of the mitochondrial membrane potential (4G and H). Exposure of cells to the broadspectrum caspase inhibitor Z-VAD did not provide any protection against HNE- and H2O2-induced alteration of the mitochondrial membrane potential (4E and 4F). Prevention of apoptosis by caspase inhibitor and thionein peptide Other results indicate that HNE, HHE, and H2O2 activate caspases in ARPE-19 cells (N.H. Ansari, personal

Formation of protein-HNE adducts The formation of protein-HNE adducts was assessed by fluorescence microscope and observed to be significant in ARPE-19 cells exposed to HNE alone (Fig. 7). Cells treated with 200 AM H2O2 alone also showed a substantial increase in the level of protein-HNE adduct as compared to untreated cells. Pretreatment of cells with thionine peptide ameliorated the formation of protein-HNE adducts in both HNE- and H2O2-exposed cells.

Fig. 3. Time-dependent H2O2-induced apoptosis in ARPE-19 cells. 1  106 cells/2 ml were incubated with 100 AM H2O2 for different time intervals. Cells were harvested at specific intervals and cell survival (D) was determined by trypan blue exclusion and expressed as percent survival of control without H2O2, and apoptosis (E) was determined by quantifying the cytosolic oligonucleosome-bound DNA as described in the text. Values are mean F SEM of three experiments.

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Fig. 4. Early apoptotic changes in ARPE-19 cells treated with H2O2 and HNE: 0.5  106 cells/ml were incubated with 200 AM H2O2 or 50 AM HNE for 5 h. At the end of incubation, the cells were harvested and further incubated with JC-1 dye for 30 min. The cells were washed three times with PBS and then subjected to flow cytometry. In order to see the protective effect of Z-VAD and thionein peptide on H2O2- and HNE-induced early apoptosis, cells were incubated with 20 AM Z-VAD or 50 AM thionein peptide for 1 h before addition of H2O2 or HNE. Panels represent (A) control, (B) thionein peptide alone, (C) HNE alone, (D) H2O2 alone, (E) HNE + Z-VAD, (F) H2O2 + Z-VAD, (G) HNE + thionein peptide, and (H) H2O2 + thionein peptide.

HNE metabolism in ARPE-19 cells HNE metabolism in ARPE-19 cells was evaluated by HPLC (Choudhary et al., 2003). Fig. 8A shows the separation profile of the synthetic standards of HNE and the metabolites of HNE obtained by incubation of ARPE-19 cells with [3H]HNE for various time intervals. The s R of the GS-conjugates, DHN, HNA, and HNE were 23, 26, 29, and

34 min, respectively. It is shown in Fig. 8B that the amount of free HNE decreased approximately 20%, 30%, 55%, and 75% in 15, 30, 60, and 90 min, respectively, with a concomitant increase in the formation of HNE metabolites, viz., GS-conjugates and HNA. The formation of GS conjugates was undetectable at the 15-min time point. Radioactive peaks obtained upon HPLC of the incubation media were tentatively assigned to HNE metabolites on the

Fig. 5. Protective effect of Z-VAD and thionein peptide on H2O2- and HNE-induced apoptosis in ARPE-19 cells. Cells (1  106) were incubated with 20 AM ZVAD or 50 AM of thionein peptide for 1 h before addition of 200 AM H2O2 or 50 AM HNE. The cells were harvested after 16 h and apoptosis was measured by ELISA as described in the text. C: Control; HN: HNE; HO: H2O2; Z: Z-VAD; and P: thionein peptide. Data represent the mean F SEM of three experiments; P b 0.005.

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Fig. 6. Effect of thionein peptide and Z-VAD on H2O2- or HNE-induced apoptosis in ARPE-19 cells. After exposure to H2O2 or HNE alone or in presence of 50 AM thionein peptide or 20 AM Z-VAD, the cells were co-stained with Hoechst 33342 and propidium iodide as described in the text. Panel represents (A) untreated cells, (B) Z-VAD, (C) H2O2, (D) HNE, (E) H2O2 + Z-VAD, (F) HNE + Z-VAD, (G) H2O2 + thionein peptide, and (H) HNE + thionein peptide treated cells.

basis of the s R of the synthesized standards. In order to confirm the identity of various metabolites, the peak samples were individually spiked with known amounts of synthetic-radiolabeled HNE and its putative metabolite. The co-elution of synthetic metabolites with the putative assigned peaks confirmed our assignments. Furthermore, both the HNA and HNE peaks were identified by GC/CIMS as described below. Since we did not observe any crosscontamination of metabolites in different peaks, all the values given in Table 1 were calculated based on the radioactivity determination in each peak. The peak fraction obtained upon HPLC analysis of the ARPE-19 cell incubation media, tentatively assigned to be HNA (based upon the s R and spiking the peak with synthetic HNA), was derivatized with BSTFA and upon GC/CI-MS yielded a prominent peak at 9.94 min (Fig. 9A), a retention time identical to synthetic HNA (Choudhary et al., 2003; Srivastava et al., 1995, 1998, 2000). The fragmentation pattern of the species eluted at 9.94 min showed fragments at m/z 73, 83, 147, and 245 (Fig. 9B), similar to those of synthetic HNA (Choudhary et al., 2003; Srivastava et al., 1998). The HPLC profile of the incubation mixture of the cells did not show any detectable peak at the s R of synthetic DHN. HNE metabolism in ARPE-19 cells involves conjugation with GSH to form GS-HNE and oxidation of HNE to HNA

and possibly reduction of GS-HNE to GS-DHN, as suggested by the results in Fig. 8. In order to identify the biochemical pathways involved in the generation of these metabolites, 0.8  106 cells were preincubated either with or without inhibitors of AR (50 AM sorbinil) or aldehyde dehydrogenase (2 mM cyanamide) in 2.0 ml of KH buffer at 37 8C for 30 min. Subsequently, the cells were incubated with 30 nmol of [4-3H] HNE for 60 min. We have previously reported (Choudhary et al., 2003; Srivastava et al., 1995, 1998, 2000) that ESI-MS of synthesized GS-DHN and GS-HNE each exhibit a single pseudo-molecular ion [MH]+ with a m/z of 466.2 and 464.2, respectively. When ARPE-19 cells were incubated with 30 nmol [4-3H] HNE, 46% of the recovered metabolites were GS-conjugates and 36% were HNA (Table 1). The relative abundance of GS-HNE and GSDHN in the peak corresponding to synthetic GS conjugates was determined by ESI-MS (Fig. 10). No detectable signal at m/z of 464.2 was observed in the absence of sorbnil, suggesting that most of the GS-HNE formed was quickly reduced to GS-DHN. In the presence of sorbinil, the relative apparent abundance of GS-DHN decreased from 100% to 52%, with a concomitant increase (48%) in GS-HNE (Fig. 10). These results implicate a sorbinil-inhibitable AR in ARPE-19 cells in the reduction of GS-HNE to GS-DHN.

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Fig. 7. Protective effect of thionein peptide in HNE and H2O2 induced protein-HNE adduct formation in ARPE-19 cells. Cells were given various treatments as described in the text. Protein-HNE adduct was probed by using anti-HNE as the primary antibodies and FITC-conjugated secondary antibody. CTL: control; HO: H2O2; HN: HNE; and TP: thionein peptide.

The effect of the aldehyde dehydrogenase inhibitor cyanamide on HNE metabolism in ARPE-19 cells was also investigated. Cyanamide decreased the oxidation of HNE to HNA significantly (Table 1), supporting the possible involvement of aldehyde dehydrogenase in the oxidation of HNE in these cells.

Discussion Peroxidation of PUFAs leads to the generation of cytotoxic and metastable LDAs that have been shown to be mediators of oxidative stress-induced cell injury (Esterbauer et al., 1991). The RPE cells phagocytize the PUFArich tips of photoreceptor outer segment (Young, 1967), it is therefore likely that under oxidative stress LDAs produced may contribute in the degeneration of RPE cells. In the present study, we have shown that H2O2 and the lipid peroxidation end products HNE and HHE are cytotoxic in micromolar concentrations as evident by MTT assay and induce apoptosis in ARPE-19 cells as evident by trypan blue exclusion assay and ELISA (Fig. 1). It is noteworthy that the apparent LC50 of H2O2, but not HNE/HHE, is higher in the presence of phenol red. This could be attributed to the

reduction of H2O2 by phenol red; HNE/HHE being unable to perform the oxidation–reduction reaction with phenol red, showing no change in their LC50. At higher concentrations of HNE/HHE, a relative decrease in apoptosis indicates that some cells underwent primary/secondary necrosis, a phenomenon also displayed in other cell types by these aldehydes (Zhang et al., 2001). The toxic concentrations of HNE and HHE appear to be higher than the concentrations reported in the tissues of certain pathological conditions (Esterbauer et al., 1991). It has been argued that levels of LDAs may build up locally near or within the peroxidizing membranes because of their high lipophilicity (Esterbauer, 1993). Therefore, the effective cytotoxic concentrations may be much higher than the levels of HNE/ HHE expressed per milligram tissue. Both HNE and HHE induced apoptosis in ARPE-19 cells in a concentration- and time-dependent fashion. It therefore appears that with aging as the production of LDAs increases and their detoxification decreases, RPE may become more vulnerable to damage by these LDAs. Our results show that H2O2 also induces apoptosis in these cells in a concentration-dependent and time-dependent manner. Barak et al. (2001) have also reported H2O2induced apoptosis in ARPE-19 cells. It is possible that

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Fig. 8. (A) HNE metabolism in ARPE-19 cells. (A-A) HPLC profile of the mixture of synthesized putative metabolites of [3H]HNE; GC conjugates (GS-HNE and GS-DHN), designated as (I), eluted with a s R of 23 min, whereas DHN (II), HNA (III), and HNE (IV) were eluted at s R of 26, 29 and 34 min, respectively. Panels A-B, A-C, and A-D show the HPLC profile of the metabolites obtained by incubating ARPE-19 cells with [3H]HNE for 15, 60, and 90 min, respectively. (B) Time course of HNE metabolism in ARPE-19 cells. Each metabolite separated upon HPLC (A) was quantified by taking the radioactive counts under respective peaks. The identity of the metabolites was confirmed as described in the text. Radioactive counts of each metabolite was plotted against the time of incubation of ARPE-19 cells with [3H]HNE. Values represent F SEM of three sets of experiments.

apoptosis induced by H2O2 could be mediated via these LDAs. This contention is supported by our experiments shown in Fig. 7 and discussed later in this section. Since mitochondria are believed to be a central control center of apoptosis (Kroemer and Reed, 2000; Rego and Oliveira, 2003; Wallace, 1999), which regulates the release of cytochrome c and other pro-apoptotic signals, we investigated the effect of H2O2 and HNE on mitochondrial membrane potential change as an early event in the process of apoptosis. Exposure to 200 AM H2O2 or 50 AM HNE for 5 h resulted in changed mitochondrial membrane potential in ~30% and 80% of cell population, respectively, suggesting that H2O2 does not induce apoptosis instanta-

neously. However, exposing the cells to H2O2 would lead to lipid peroxidation resulting in the production of LDAs like HNE and HHE, which may then induce apoptosis in cells. Inability of Z-VAD to protect H2O2- and HNEinduced mitochondrial depolarization suggests that this event is independent of caspase activation. Metallothionein is an acute phase stress protein that participates in the detoxification of heavy metals and scavenges hydroxyl radicals (Sato and Bremner, 1993; Webb, 1987). Its presence has been demonstrated in human RPE cells, where it is in lower concentration in the macular region than in the periphery (Sato and Bremner, 1993). Further, there is an age-related decline

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Table 1 Metabolic transformations of HNE in ARPE-19 cells

Control Sorbinil Cyanamide

GS conjugates (nmol)

4-Hydroxynonenoic acid (nmol)

4-Hydroxynonenal (nmol)

Total recovery (nmol)

8.9 F 0.36 8.5 F 0.43 8.6 F 0.7

6.9 F 0.53 6.6 F 0.21 5.3 F 0.36*

3.5 F 0.01 3.3 F 0.34 4.1 F 1.0

19.3 (83.9%) 18.4 (82.8%) 18.0 (81%)

RPEs (0.8  106 cells) were first incubated with either 50 AM sorbinil or 2 mM cyanamide followed by incubation with 30 nmol of [3H]HNE for 60 min. Incubation media were filtered through centriprep-10 and the HNE metabolites (equivalent to 23 nmol of [3H]HNE) were separated by HPLC. Fractions corresponding to the respective peaks were pooled and quantified by their radioactive counts. Values are mean F SD of three separate experiments. * P V 0.05 significantly different (Student’s t test; two-tailed distribution) from control group (without inhibitors).

in the macular RPE content of metallothionein (Tate et al., 1993). Suppression of metallothionein synthesis was also reported in cynomolgus monkeys that showed early onset of macular degeneration (Fujimura et al., 1996). These reports suggest a protective role of metallothionein in ARMD. Since these proteins contain unusually high content of cysteine (Buhler and Kagi, 1974; Kagi et al., 1974) and the fact that g-carbon of HNE readily reacts with thiols, these proteins may have a function to scavenge the lipid-derived aldehydes such as HNE or HHE. We synthesized the ester of a hexapeptide H-Lys-Cys-Thr-CysCys-Ala-OH, which is a portion of mouse liver thionein- I (Huang et al., 1977; Yoshida et al., 1979). This hexapeptide provided significant protection to H2O2- and HNEinduced mitochondrial depolarization and ameliorated H2O2- and HNE-induced apoptosis. Protein adducts of HNE, which have been used as markers of oxidative stress, have also been shown to be involved in the pathogenesis of various diseases initiated by oxidative stress (French et al., 1993; Newcombe et al., 1994; Waeg et al., 1996; Yoritaka et al., 1996). ProteinHNE adduct formation has also been associated with the induction of apoptosis in other systems (Kirichenko et al., 1996; Vieira et al., 2000). Our studies show that proteinHNE adduct formation increases in ARPE-19 cells when exposed to H2O2 or HNE. Formation of excessive amount of protein-HNE adduct in H2O2-exposed cells supports the contention that H2O2-induced ARPE-19 degeneration is mediated via production of HNE. Pretreatment of cells with thionein peptide effectively reduces the H2O2- and

HNE-induced protein-HNE adduct formation and apoptosis. A positive correlation between the amounts of proteinHNE adduct formation and the induction of apoptosis in response to H2O2 or HNE alone, or in presence of thionein peptide, suggests that protein modification is likely to play a direct causative role in the induction of apoptosis. Modification of thiol proteins in the mitochondria, such as the permeability transition pore, has been proposed as a trigger mechanism that initiates apoptosis (Costantini et al., 1996; Petronilli et al., 1994; Takeyama et al., 2002; Zamzami et al., 1998). Modification of key membrane proteins may compromise their physiological function and lead to cellular degeneration. Thus, in view of the toxic potential of the LDAs to RPE cells, it is important to investigate how these LDAs, such as HNE, are detoxified in this cell type. Our results suggest that in ARPE-19 cells, the major pathways of HNE metabolism are the conjugation of HNE with GSH and oxidation to its corresponding acid HNA. Approximately 46% of the recovered HNE metabolites were GS conjugates. HNE can conjugate with GSH to form a Michael adduct, a reaction facilitated by glutathione-Stransferase (GST) (Alin et al., 1985). GS-HNE can be efficiently reduced to GS-DHN by AR (Km GS-HNE ~30 AM) in the presence of NADPH (Srivastava et al., 1995). As shown by the ESI-MS profile (Fig. 10), the glutathione conjugate of HNE in ARPE-19 cells appears to be predominantly in the reduced form. GS-DHN could arise either from the reduction of GS-HNE or by conjugation of DHN with GSH. Since DHN is not electrophilic, it is

Fig. 9. GC/CI-MS analysis of peak II obtained from HPLC separation of the incubation media of ARPE-19 cells incubated with [4-3H] HNE. (A) GC chromatogram of peak II in Fig. 8A, panel B, C, or D. Peak eluting at a retention time of 9.94 corresponds to BSTFA-derivatized synthetic HNA. (B) Positive ionization mass spectrum of the peak at s R of 9.94 in A showing indicated fragments at m/z 73, 83, 147, and 245.

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Fig. 10. ElectroSpray Mass Spectra of GS-HNE and GS-DHN. ESI-MS of peak I obtained upon HPLC of ARPE-19 cells incubated with [3H]HNE without (A) and with 50 AM sorbinil (B), as described in the text. The peaks at m/z 464.2 and 466.4 were assigned to GS-HNE and GS-DHN, respectively.

unlikely that it could spontaneously react with GSH. Therefore, the most probable route of GS-DHN formation appears to be via the enzymatic reduction of GS-HNE rather than spontaneous conjugation of DHN to GSH. The role of AR in reducing the glutathione conjugate of HNE in ARPE-19 cells is also supported by the observation that the formation of GS-DHN decreased in the presence of the AR inhibitor sorbinil. As shown in Fig. 10, sorbinil produced a decrease in the relative abundance of GS-DHN to 52% with a concomitant increase in GS-HNE to 48%. Since GS-HNE can spontaneously dissociate at neutral pH to form GSH and HNE (Esterbauer et al., 1975), if the conjugate is not reduced then it can produce transcellular and transorgan toxicity, therefore reduction of GS-HNE to GS-DHN by AR appears to be a physiologically relevant detoxification step. Although AR can efficiently reduce free HNE to DHN under in vitro condition (Srivastava et al., 1995; Vander Jagt et al., 1995), the present study shows that conversion of HNE to DHN is not a significant metabolic route for the

detoxification of HNE in ARPE-19 cells. This could be due to the high conjugation rate of HNE to GSH such that AR encounters mostly the glutathione conjugate and not the free aldehyde. In addition to conjugation, HNE is also metabolized to its corresponding acid HNA by aldehyde dehydrogenase. HNA accounted for 36% of the recovered metabolites. The role of aldehyde dehydrogense in HNE metabolism was strengthened by the observation that in the presence of the aldehyde dehydrogenase inhibitor cyanamide, the formation of HNA was significantly decreased (Table 1). In summary, we have shown the presence of conjugation, reduction, and oxidation pathways to detoxify HNE in ARPE-19 cells. If one of the pathway is blocked, the other pathway would take over. Therefore, under normal and mild oxidizing conditions, a small amount of HNE formed, partitions in the cytosol, and is detoxified where GSH and other cofactors such as NADPH, NAD+, and the HNE metabolizing enzymes are present. Thus, under mild oxidative stress, the LDA-induced damage could be none or minimal. However, under severe oxidative stress, when the formation of ROS overwhelms the reduction capacity of the tissue, excessive amounts of LDAs could be formed in the membrane due to oxidation of membrane lipids. Increased amounts of LDAs would then produce membrane damage by forming membrane protein adducts as well as LDA-protein crosslinks, which would hamper the structural and functional integrity of the cell, leading to cell degeneration via apoptosis. It appears that antioxidants + scavengers of HNE + apoptotic inhibitors may be a potential therapeutic combination to prevent the degeneration of aging RPE.

Acknowledgments This study was supported in part by NIH grants EY 13014 (NHA) and HL61618 (SS) and the Lions Eye Bank Foundation (NHA).

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