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Cellular lipid peroxidation end-products induce apoptosis in human lens epithelial cells
Free Radical Biology & Medicine, Vol. 32, No. 4, pp. 360 –369, 2002 Copyright © 2002 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/02/$–see front matter
PII S0891-5849(01)00810-3
Original Contribution CELLULAR LIPID PEROXIDATION END-PRODUCTS INDUCE APOPTOSIS IN HUMAN LENS EPITHELIAL CELLS S. CHOUDHARY,* W. ZHANG,* F. ZHOU,*1 G. A. CAMPBELL,† L. L. CHAN,* E. B. THOMPSON,* N. H. ANSARI*
and
*Department of Human Biological Chemistry & Genetics and †Department of Pathology, The University of Texas Medical Branch, Galveston, TX, USA (Received 12 July 2001; Accepted 5 December 2001)
Abstract—Hydrogen peroxide (H2O2), an oxidant present in high concentrations in the aqueous humor of the elderly eyes, is known to impart toxicity to the lens—apoptosis being one of the toxic events. Since H2O2 causes lipid peroxidation leading to the formation of reactive end-products, it is important to investigate whether the end-products of lipid peroxidation are involved in the oxidation-induced apoptosis in the lens. 4-Hydroxynonenal (HNE), a major cytotoxic end product of lipid peroxidation, has been shown to mediate oxidative stress-induced cell death in many cell types. It has been shown that HNE is cataractogenic in micromolar concentrations in vitro, however, the underlying mechanism is not yet clearly understood. In the present study we have demonstrated that H2O2 and the lipid derived aldehydes, HNE and 4-hydroxyhexenal (HHE), can induce dose- and time-dependent loss of cell viability and a simultaneous increase in apoptosis involving activation of caspases such as caspase-1, -2, -3, and -8 in the cultured human lens epithelial cells. Interestingly, we observed that Z-VAD, a broad range inhibitor of caspases, conferred protection against H2O2- and HNE-induced apoptosis, suggesting the involvement of caspases in this apoptotic system. Using the cationic dye JC-1, early apoptotic changes were assessed following 5 h of HNE and H2O2 insult. Though HNE exposure resulted in ⬃ 50% cells to undergo early apoptotic changes, no such changes were observed in H2O2 treated cells during this period. Furthermore, apoptosis, as determined by quantifying the DNA fragmentation, was apparent at a much earlier time period by HNE as opposed to H2O2. Taken together, the results demonstrate the apoptotic potential of the lipid peroxidation end-products and suggest that H2O2-induced apoptosis may be mediated by these end-products in the lens epithelium. © 2002 Elsevier Science Inc. Keywords— 4-Hydroxynonenal, Human lens epithelial cells, Apoptosis, Cataract, Oxidative damage, Hydrogen peroxide, Free radicals
INTRODUCTION
cataractous lens [3–5], the mechanisms by which oxidative stress in general, and lipid peroxidation in particular, lead to cataract formation, remain unclear. Lipid peroxidation generates high levels of alkoxyl radicals, which degenerate into saturated and unsaturated aldehydes. There is increasing evidence in nonlenticular tissues, suggesting that the unsaturated aldehydes, especially the 4-hydroxyalkenals formed by lipid peroxidative reactions, mediate the biological effects of free radicals by acting as toxic messengers and may be responsible for a significant portion of the tissue damage ascribed to their radical precursors (review [6]). The two most toxic 4-hydroxyalkenals are 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE) (review [6]). Peroxidation reactions of -6 polyunsaturated fatty acids (arachidonic, linoleic,
Due to its constant exposure to light, the ocular lens is continuously attacked by reactive oxygen species. Inadequate control of the redox reactions has been suggested to be the key trigger for the development of cataract under several etiologically unrelated conditions (reviews [1,2]). While significantly elevated concentrations of peroxides and other oxidants have been measured in the Address correspondence to: Naseem H. Ansari, Ph.D., Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, 6.642 Basic Science Bldg., Galveston, TX 7755-0647, USA; Tel: (409) 772-3905; Fax: (409) 772-9679; E-Mail: [email protected]. 1 Current address: National Flow Cytometry Resources, Bioscience Division, Los Alamos, NM 87545, USA. 360
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and ␥-linolenic acid) generate HNE and of -3 (docosohexanoeic acid) generate HHE. As a result of a conjugated double bond between the ␣ and  carbons, the ␥ carbon of these aldehydes is electron deficient and reacts readily with nucleophiles such as thiols and amines. Due to their high reactivity, these aldehydes display marked biological effects, which, depending upon their concentrations, cause selective alteration in cell signaling, protein and DNA damage, and cytotoxicity (review [6]) including induction of apoptosis [7–10]. Apoptosis is a form of cell death distinct from necrosis. Necrosis is the result of cellular “accidents” such as those occurring in tissues subjected to chemical trauma. Apoptosis, on the other hand, is programmed. Activation of programmed cell death in an individual cell is dependent on its own internal metabolism, environment, developmental background, and genetic information. Apoptosis is characterized by cell shrinkage, chromatin condensation, and DNA fragmentation, and can be induced by a variety of stimuli. The apoptotic process is thought to be under the control of caspases, a class of cysteine proteases, mainly discovered in Caenorhabditis elegans, with at least three families of death genes being involved termed ced-3, ced-4, and ced-9 [11–16]. Once caspases get activated, they cleave certain proteins like poly (ADP-ribose) polymerase (PARP) and lamin A involved in chromatin condensation as well as cytoskeleton regulation [16,17]. The expression of oncogenes, c-myc, and c-jun, which regulate cell proliferation and survival, has been shown to be modulated by HNE in K562 Erythroleukemic cells and HL-60 [18,19] human leukemic cells [10], suggesting a role of these aldehydes in the control of gene expression. The present study was undertaken to determine the induction of apoptosis in human lens epithelial cells (HLECs) by H2O2 and the 4-hydroxyalkenals, and to investigate the involvement of caspases in this event. EXPERIMENTAL PROCEDURES
Materials Cell culture media, fetal bovine serum, antibiotics, and all chemicals that are not otherwise specified were purchased from Sigma Chemical Co. (St. Louis, MO, USA). HNE was obtained from Caymen Chemicals (Ann Arbor, MI, USA). The substrates and inhibitors of caspases were purchased from Enzyme Systems (Dublin, CA, USA) and the Cell Death ELISA kit from Boehringer Mannheim (Mannheim, Germany). HHE was generously provided by Dr. F. M. J. van Kuijk at our institute.
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Cell culture and treatment Human lens epithelial cells (HLECs) with extended life span were generously provided by Dr. Usha Andley, Washington State University, St. Louis, MO, USA, and were used in this study as described previously [20]. Briefly, cells were grown in Eagle MEM (Sigma) containing 50 g/ml gentamicin and 20% fetal bovine serum in an atmosphere of 5% CO2 in air at 37°C. The pH of the medium was adjusted to 6.8 –7.0 prior to sterile filtration and addition of serum. The cells were fed twice a week. After attaining confluence, cells were passaged using Trypsin-EDTA. Cell survival was determined by trypan blue staining and counting with a hemocytometer. Cell viability is represented as the percentage of the number of live cells/number of total cells. Measurement of apoptosis Apoptosis was determined by quantifying the cytosolic oligonucleosomes-bound DNA by using a “Cell Death Detection ELISA Kit” according to the manufacturer’s instructions and described by us earlier [10,21]. Briefly, the cytosolic fraction (10,000 ⫻ g supernatant) of 5000 cells were used as the antigen source in a sandwich ELISA with a primary anti-histone antibody coated to the microliter plate and secondary anti-DNA antibody coupled to peroxidase. After adding the substrate, absorbance was recorded at 405 nm using the SpectroCount Microplate Spectrometer (Packard Instruments, Meriden, CT, USA). Apoptosis was also detected morphologically using Hoechst and propidium iodide stain. The Vybrant Apoptosis Assay Kit #5 (V-13244) from Molecular Probes (Eugene, OR, USA) containing both the dyes was used. One microliter of each dye was added to the cell suspension, mixed, and kept on ice for 20 min. Subsequently, the cells were plated on a glass slide using the cytospin (500 rpm, 2 min) and visualized under a Nikon Eclipse 800 upright epifluorescence microscope, equipped with a xenon arc lamp. Texas red filter (excitation 540 –580 nm band pass, Dichroic mirror 595 nm, emission 600 – 660 nm) and DAPI filter (excitation 340 –380 nm band pass, Dichroic mirror 400 nm, emission 435– 485 nm) for the red and blue fluorescence, respectively. Photographs were taken using a Dage-MTI (Michigan City, IN, USA) 300 T-RC monochrome single chip integrating cooled CCD video camera with a Flashbus (Integral Technologies, Indianopolis, IN, USA) video capture card. Detection of early stages of apoptosis by flow cytometer Flow cytometery was performed using the cationic dye, JC-1 (Molecular Probes) that exhibits potential de-
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pendent accumulation in mitochondria, indicated by a fluorescence emission shift from green (⬃ 525 nm) to red (⬃ 590 nm). Pellet of approximately 1 ⫻ 106 cells was suspended in 130 l of warm MEM ⫹ 5% FBS to which 250 l of JC-1 solution (20 g/ml) was added. After incubation for 30 min, cells were washed with PBS, re-suspended in 500 l PBS and subjected to flow cytometery. Measurement of caspase activity Caspase activity was measured essentially using the procedure described by Sarin et al. [22]. Cells (0.5 ⫻ 106 cells) were lysed with 100 l 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°C. Dithiothreitol (DTT) was added to the supernatant at a final concentration of 10 mM, then 50 M of the specific fluorogenic substrate for the various caspases (1, 2, 3, 8, and 9) were individually mixed with 10 l aliquots of the cell extracts in 96 well plates and the reaction was carried out 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) at ex 400 nm, em 510 nm. Measurement of oncogene expression The expression of c-myc was determined by Western blot, as described by us earlier for c-myc [10]. C-myc monoclonal antibody Myc-19E-10.2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), raised against a peptide corresponding to residues 408 to 439 within the C-terminal of the c-myc encoding protein, was generated as a culture supernatant of the hybridoma cell line CRL1729 (ATCC). Horseradish peroxidase conjugated secondary antibody catalyzed the oxidation of luminol, resulting in the emission of light-enhanced chemiluminescence, which was captured on autoradiography film. The densitometric analysis of c-myc protein was performed using a Lynx densitometer (Applied Imaging, Santa Cruz, CA, USA). RESULTS
Dose response The HLECs were exposed to various concentrations of H2O2 and after 24 h of exposure, cell viability was determined by trypan blue exclusion method. There was a dose-dependent decrease in the cell viability and 100% cell death was observed with 1 mM H2O2. LD50, defined as the concentration causing 50% cell death, for H2O2 in
Fig. 1. Dose-dependent effect of H2O2 on cell viability and apoptosis. HLECs (1 ⫻ 106 cells) cultured in MEM with 20% FBS and 50 g/ml gentamycin were treated with various concentrations of H2O2 and after 24 h of exposure, the (A) cell viability was determined by trypan blue dye exclusion and expressed as % survival of control without H2O2. (B) Apoptosis, expressed as ELISA absorbance/5000 cells, was determined as described in the text.
HLECs was found to be ⯝ 200 M (Fig. 1A). A dosedependent increase in apoptosis was observed in HLECs treated with H2O2 for 24 h as measured by quantifying cytosolic nucleosomal DNA fragments (Fig. 1B). A maximum of 16-fold increase in apoptosis as compared to that of control was observed with 200 M H2O2. However, when the cells were further exposed to higher concentrations of H2O2, a decrease in the apoptotic index was observed, which could be attributed to necrosis at higher concentrations of H2O2.
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Fig. 3. Time-dependent H2O2-, HNE-, and HHE-induced apoptosis in HLECs. 1 ⫻ 106 cells were incubated with 200 M H2O2 (‚-‚),30 M HNE (䊐-䊐) or 50 M HHE (x-x) for different time intervals. Cells were harvested at specific intervals and apoptosis was determined by ELISA as described in the text and expressed as absorbance at 405 nm/5000 cells for H2O2 and absorbance at 405 nm/2000 cells for HNE or HHE.
Time course
Fig. 2. Dose-dependent effect of HNE and HHE on cell viability and apoptosis. HLECs (1 ⫻ 106 cells) were incubated with various concentrations of HNE (⫹) and HHE (‚). After 24 h cells were harvested and (A) % cell viability and (B) apoptosis, expressed as ELISA absorbance/2000 cells, was determined as described in the text.
Similarly, when the cells were exposed to various concentrations of HNE or HHE, a dose-dependent decrease in the cell viability was observed (Fig. 2A). LD50 for HNE and HHE in HLECs cells were found to be approximately 30 and 50 M, respectively. HLECs exposed to different doses of HNE/HHE for 24 h resulted in a dose-dependent increase in apoptosis, which was more pronounced by HNE as compared to HHE (Fig. 2B). The difference was more distinct at higher concentrations. Cells exposed to 40 M HNE showed 50% higher apoptotic index as compared to the 40 M HHE-treated cells, suggesting that HNE is a more potent inducer of apoptosis as compared to HHE.
A time-dependent increase in apoptosis was observed when HLECs were treated with H2O2. Increase was exponential with respect to time until 16 h postexposure, and remained almost constant at 24 h of H2O2 exposure (Fig. 3). A similar trend was observed when the cells were treated with 30 M of HNE or 50 M of HHE (Fig. 3). Increased apoptosis was observed as early as 2 h in HNE-treated HLECs, which increases rapidly thereafter until 16 h postexposure. However, on the subsequent time intervals, a decrease in the apoptosis was observed. HNE-induced apoptosis was more pronounced as it can be seen from the figure, that at 16 h postexposure, HNE-treated cells showed 2.5-fold higher values of apoptosis as compared to HHE. In order to assess the effect of H2O2 and HNE on the early stages of apoptosis, the cells were first exposed to H2O2 or HNE for 5 h and then incubated with the dye JC-1 and subjected to flowcytometer. It was observed that at this time interval, very few cells undergo apoptotic changes following H2O2 exposure, however, in the HNE-treated cells, nearly 50% of cell population showed the early apoptotic change (change in the polarity of the mitochondrial membrane) (Fig. 4). Caspase activation To assess the involvement of caspases in H2O2- and HNE-induced apoptosis, activation of various caspases (1, 2, 3, 8, and 9) was determined at different time intervals using specific substrates of these caspases. A time-dependent increase in the activities of all the
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Fig. 4. Early apoptotic changes in HLECs treated with H2O2 and HNE. 0.5 ⫻ 106 cells were incubated with 200 M H2O2 or 50 M HNE for 5 h with and without preincubation with 20 M Z-VAD. Cells were harvested at the end of incubation and were further incubated with the dye JC-1 for 30 min as described in the text and were subjected to flowcytometry. (A) control; (B) H2O2-treated; (C) HNE-treated; (D) Z-VAD ⫹ HNE-treated.
caspases examined, except caspase-9, was observed following H2O2 treatment starting as early as 2 h (Fig. 5A). Caspase-1 and caspase-2 get activated almost simultaneously, however, after 6 h postexposure, activity of caspase-2 increases very rapidly, and after 24 h of H2O2 exposure caspase-2 activity increased by 80-fold as opposed to caspase-8 activity, which increases by 15-fold at
the same time interval. In the initial time periods, until 8 h postexposure, the caspase-8 activity was found to be higher than caspase-3 but thereafter, caspase-3 activity increased very rapidly and at 24 h postexposure, the caspase-3 activity was more pronounced than caspase-8 activity. As shown in Fig. 5, caspase-9 was not activated. The results of activation of caspases in HLECs ex-
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Fig. 5. Activation of caspases by H2O2 and HNE in HLECs. (A) Activity of caspases 1, 2, 3, 8, and 9 was determined in the cells treated with 200 M H2O2 at the indicated time intervals as described in the text. 䊐-䊐: caspase-1; E-E: caspase-2; x-x: caspase-3; ‚-‚: caspase-8; 〫-〫: caspase-9. (B) Activity of caspases in the cells treated with 30 M HNE at 16 h as described in the text. C ⫽ control; H ⫽ HNE.
posed to 40 M HNE for 16 h are given in Fig. 5B. Caspase-9 was not activated. A significant increase in both the initiator caspases 2 and 8 was observed. Caspase-3, the main executioner caspase, was activated approximately 6-fold. Prevention of apoptosis by Z-VAD The effect of Z-VAD-FMK, a broad-range inhibitor of the caspases, was studied on the H2O2- and HNE-
induced apoptosis a HLECs. The cells were treated with 20 M Z-VAD 1 h before the addition of H2O2 or HNE in order to ensure enough time for Z-VAD to enter the cells and inhibit the caspases. It was observed that ZVAD completely prevented the H2O2- and HNE-induced apoptosis (Fig. 6). Morphological assessment revealed similar results (Fig. 7, A–F). The effect of Z-VAD was also tested on the H2O2- and HNE-induced early apoptotic changes as measured by incubating the cells with the dye JC-1 and subjecting to flowcytometry. At this early
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the total cell population had undergone apoptosis after 5 h of HNE exposure, and Z-VAD had no protective effect on HNE-induced early apoptotic changes in the cells, demonstrating that mitochondrial membrane potential change is independent of caspase activation (Fig. 4, A–D). Induction of C-Myc by HNE and HHE
Fig. 6. Prevention of H2O2- and HNE-induced apoptosis by Z-VAD in HLECs. Cells were incubated with 20 M Z-VAD for 1 h before the addition of 200 M H2O2 or 30 M HNE for 16 h. Apoptosis was determined as described in the text and is expressed as ELISA absorbance/5000 cells for H2O2-treated cells and ELISA absorbance/2000 cells for HNE-treated cells. C ⫽ control; H ⫽ H2O2; HN ⫽ HNE; Z ⫽ Z-VAD. Values are mean ⫾ SE of three experiments and expressed as ELISA absorbance/5000 cells for H2O2-treated cells and ELISA absorbance/2000 cells for HNE-treated cells.
stage, H2O2 did not produce any apoptotic changes, hence no effect of Z-VAD was observed (data not shown). Interestingly, we observed that nearly 50% of
Figure 8 shows the induction of c-myc protein in HNE-treated cells. For this, the cells were incubated with HNE for 2 and 6 h with and without preincubation with 20 M Z-VAD for 1 h, followed by extraction of whole cell protein. C-Myc protein was estimated by Western blot using specific antibodies. It was observed that cells treated with HNE showed a 1.3- and 4-fold induction of c-myc protein at 2 and 6 h time points, respectively. The broad-spectrum caspase inhibitor Z-VAD prevented the induction of c-myc protein. Similar trend was observed with HHE (data not shown). DISCUSSION
4-Hydroxynonenal and 4-hydroxyhexenal, two highly reactive and cytotoxic end-products of lipid peroxidation
Fig. 7. Prevention of H2O2- and HNE-induced apoptosis by Z-VAD in HLECs. Cell were incubated with 20 M Z-VAD for 1 h before the addition of 200 M H2O2 or 30 M HNE for 16 h. Cells were stained using the Vybrant Apoptosis Kit and photographed (magnification, 40⫻) as described in the text. (A) Control; (B) H2O2-treated; (C) HNE-treated; (D) Z-VAD ⫹ control; (E) Z-VAD ⫹ H2O2-treated; (F) Z-VAD ⫹ HNE-treated.
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Fig. 8. Induction of c-myc by HNE in HLECs. The cells were incubated with 30 M HNE with or without preincubation with 20 M Z-VAD as described in the text for 2 and 6 h. Total proteins were extracted and assayed for the expression of c-myc 1: control; 2: HNE-treated (2 h); 3: HNE-treated (6 h); 4: Z-VAD ⫹ HNE-treated (6 h); 5: Z-VAD ⫹ HNE-treated (2 h). (A) c-myc protein as detected by immunoblot analysis using specific antibodies described in text. (B) quantified data of the c-myc bands shown in Panel A.
have been shown to produce oxidative damage to various tissues under the oxidative stressed conditions. Earlier, we have shown that HNE is cataractogenic in vitro [23] and that high levels of protein-HNE adducts are present in the human diabetic cataractous lenses [24]. Induction of apoptosis in the various cell types is one of the major cytotoxic effects of HNE [7–9]. In the present study, we studied the effect of HNE and HHE as well as H2O2, an oxidant, which is known to be produced in high concentrations under oxidative stress, in the lens. Our study shows LD50 for HNE and HHE in HLECs to be 30 M and 50 M, respectively, suggesting HNE to be more cytotoxic as compared to HHE; LD50 for H2O2 in HLECs was found to be 200 M. The toxic concentrations of HNE and HHE appear to be higher than those reported in the tissues under certain pathological conditions [6]. It has been argued that levels of HNE may build up locally near or within the peroxidizing membranes because of their high lipophilicity. Therefore, the effective cytotoxic concentrations may be much higher than the levels of HNE/HHE expressed as per milligram
tissue [6]. The single layer of lens epithelial cells is important for maintaining metabolic homeostasis and the transparency of the entire lens [24]. Normally, these cells have a long life span under physiological conditions. However, if such conditions are altered or disturbed by factors such as oxidative stress, aging, and other pathological conditions, the viability of these cells can be effected, which leads to the opacification of the lens. In this context, it has been reported that human cataract subjects have a higher percentage of apoptotic lens epithelial cells as compared to normal human lenses of the comparable age group [24]. This indicates a role of oxidative stress-induced apoptosis of lens epithelial cells in cataractogenesis. Contrary to this finding, Harocopos et al. [25] were unable to demonstrate any difference in the number of dead cells in the lens epithelia from the cataractous and normal lenses obtained from the eye bank. It is not clear whether the discrepancy in the two reports could be due to some trivial but crucial difference in obtaining the samples or involvement of other factors. Nevertheless, studies showing that oxidants, such as
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H2O2, induce apoptosis in lens cells [26,27] and that LDAs, such as HNE, induce apoptosis in various cell types [7–9] suggests that the oxidants may impart their toxicity via their peroxidative end-products. When the effect of H2O2 and HNE on the early apoptotic changes (depolarization of the mitochondrial membrane) was studied, we did not observe any significant change in the H2O2-treated cells, whereas ⬃ 50% of the HNE-treated cells were found to undergo early apoptotic changes. This indicates that H2O2-induced apoptosis is a delayed event as opposed to a relatively instantaneous induction of apoptosis by HNE. It may be possible that exposing the cells to H2O2 may lead to lipid peroxidation resulting in the excessive production of lipid-derived aldehydes, such as HNE and HHE, which may then induce apoptosis in these cells. Hence, HNE and HHE could be a mediator of H2O2-induced apoptosis in HLECs. Alternatively, it is possible that HNE could be modifying the mitochondrial membrane proteins leading to the changes in the mitochondrial membrane potential. Since proteolysis is a requirement for the induction of apoptosis in most of the apoptotic systems, we investigated the activation of various caspases in our apoptotic systems. Activation of caspases 1, 2, 3, and 8 by H2O2/ HNE suggests the involvement of these caspases in the induction of H2O2- and HNE-induced apoptosis. While there was activation of initiator caspases (caspase 1 and 8; Fig. 5), we did not observe activation of the initiator caspase 9. Since Z-VAD did not prevent the HNEinduced change in mitochondrial membrane potential (Fig. 4) but did prevent apoptosis (Fig. 6), our studies suggest that the HNE-induced activation of all the caspases and execution of apoptosis in this system are independent of mitochondrial changes. A systematic study to establish the caspase-cascade is in progress. The proto-oncogene product, c-myc has been shown to encourage cell proliferation and apoptosis, since it has both the positive as well as negative factor to support apoptosis. Many studies have shown the induction of apoptosis following overexpression of c-myc in various cell types [28,29]. However, there are reports suggesting that a reduction of c-myc expression also induces apoptosis [30 –32]. In fact, previously we have reported that HNE exposure to CEMC7 human leukemic cell line results in the downregulation of c-myc prior to activation of caspases [10]. However, in the present study, we observed an increase in the expression of c-myc protein, which was inhibited by Z-VAD, demonstrating the importance of caspase activation in this system. Induction of c-myc has been found to lead into the processing/ activation of caspases [33,34]. Furthermore, it has been reported that several targets of caspases (PARP, fodrin, lamins, MDM2, etc.) are cleaved during c-myc-induced apoptosis in Rat-1 MycER cells [35]. Our results (Fig. 8)
showing that caspase inhibitor Z-VAD prevents the HNE-induced induction of c-myc suggest that c-myc gets induced downstream of, at least, the intiator caspases. Whether and how c-myc is indeed involved in this apoptotic system is yet to be established by our work under progress. In conclusion, our study demonstrates that H2O2, HNE, and HHE are cytotoxic to HLECs at micromolar concentrations and they kill the cells by inducing apoptosis. Furthermore, our results suggest that apoptosis of lens epithelial cells, following oxidative insult, may be mediated by HNE and HHE. Acknowledgements — This work was supported by the National Institutes of Health EY 13014 and Lions Eye Bank Foundation.
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Hydroxyalkenal-induced apoptosis in human lens epithelial cells [16] Thornberry, N. A.; Lazebnik, Y. Caspases: enemies within [Review] [50 refs]. Science 281:1312–1316; 1998. [17] Villa, P.; Kaufmann, S. H.; Earnshaw, W. C. Caspases and caspase inhibitors [Review] [78 refs.]. Trends Biochem. Sci. 22: 388 –393; 1997. [18] Fazio, V. M.; Barrera, G.; Martinotti, S.; Farace, M. G.; Giglioni, B.; Frati, L.; Manzari, V.; Dianzani, M. U. 4-Hydroxynonenal, a product of cellular lipid peroxidation, which modulates c-myc and globin gene expression in K62 erythroleukemic cells. Cancer Res. 52:4866 – 4871; 1992. [19] Barrera, G.; Muraca, R.; Pizzimenti, S.; Serra, A.; Rosso, C.; Saglio, G.; Farace, M. G.; Fazio, V. M.; Dianzani, M. U. Inhibition of c-myc expression induced by 4-hydroxynonenenal, a product of lipid peroxidation, in the HL-60 human leukemic cell line. Biochem. Biophys. Res. Commun. 203:553–561; 1994. [20] Andley, U. P.; Rhim, J. S.; Chylack, L. T. Jr.; Fleming, T. P. Propagation and immortalization of human lens epithelial cells in culture. Invest. Ophthalmol. Vis. Sci. 35:3094 –3102; 1994. [21] Zhang, W.; Khanna, P.; Chan, L. L.; Campbell, G.; Ansari, N. H. Diabetes-induced apoptosis in rat kidney. Biochem. Mol. Med. 61:58 – 62; 1997. [22] Sarin, A.; Wu, M.-L.; Henkart, P. A. Different interleukin-1converting enzyme (ICE) family protease requirements for the apoptotic death of T lymphocytes triggered by diverse stimuli. J. Exp. Med. 184:2445–2450; 1996. [23] Ansari, N. H.; Wang, L.; Srivastava, S. K. Role of lipid aldehydes in cataractogenesis: 4-hydroxynonenal-induced cataract. Biochem. Mol. Med. 58:2–30; 1996. [24] Ansari, N. H.; Khanna, P.; Bhatnagar, A.; Bhalla, P.; Szweda, L. Protein-HNE adducts in human diabetic cataractous lenses. Invest. Ophthalmol. Vis. Sci. 38:S1171; 1997. [25] Harocopos, G. J.; Alvares, K. M.; Kolker, A. E.; Beebe, D. C. Human age-related cataract and lens epithelial cell death. Invest. Ophthalmol. Vis. Sci. 39:2696 –2706; 1998. [26] Li, W.-C.; Kuszak, J. R.; Dunn, K.; Wang, R.-R.; Ma, W.; Wang, G.-M.; Spector, A.; Leib, M.; Cotliar, A. M.; Weiss, M.; Espy, J.; Howard, G.; Farris, R. L.; Auran, J.; Donn, A.; Hofeldt, A.; Mackay, C.; Merriam, J.; Mittl, R.; Smith, T. R. Lens epithelial cell apoptosis appears to be a common cellular basis for noncongenital cataract development in humans and animals. J. Cell Biol. 130:169 –181; 1995. [27] Li, D. W.; Spector, A. Hydrogen peroxide-induced expression of
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DTT— dithiothreitol HLECs— human lens epithelial cells HNE— 4-hydroxynonenal HHE— 4-hydroxyhexenal H2O2— hydrogen peroxide
PII S0891-5849(01)00810-3
Original Contribution CELLULAR LIPID PEROXIDATION END-PRODUCTS INDUCE APOPTOSIS IN HUMAN LENS EPITHELIAL CELLS S. CHOUDHARY,* W. ZHANG,* F. ZHOU,*1 G. A. CAMPBELL,† L. L. CHAN,* E. B. THOMPSON,* N. H. ANSARI*
and
*Department of Human Biological Chemistry & Genetics and †Department of Pathology, The University of Texas Medical Branch, Galveston, TX, USA (Received 12 July 2001; Accepted 5 December 2001)
Abstract—Hydrogen peroxide (H2O2), an oxidant present in high concentrations in the aqueous humor of the elderly eyes, is known to impart toxicity to the lens—apoptosis being one of the toxic events. Since H2O2 causes lipid peroxidation leading to the formation of reactive end-products, it is important to investigate whether the end-products of lipid peroxidation are involved in the oxidation-induced apoptosis in the lens. 4-Hydroxynonenal (HNE), a major cytotoxic end product of lipid peroxidation, has been shown to mediate oxidative stress-induced cell death in many cell types. It has been shown that HNE is cataractogenic in micromolar concentrations in vitro, however, the underlying mechanism is not yet clearly understood. In the present study we have demonstrated that H2O2 and the lipid derived aldehydes, HNE and 4-hydroxyhexenal (HHE), can induce dose- and time-dependent loss of cell viability and a simultaneous increase in apoptosis involving activation of caspases such as caspase-1, -2, -3, and -8 in the cultured human lens epithelial cells. Interestingly, we observed that Z-VAD, a broad range inhibitor of caspases, conferred protection against H2O2- and HNE-induced apoptosis, suggesting the involvement of caspases in this apoptotic system. Using the cationic dye JC-1, early apoptotic changes were assessed following 5 h of HNE and H2O2 insult. Though HNE exposure resulted in ⬃ 50% cells to undergo early apoptotic changes, no such changes were observed in H2O2 treated cells during this period. Furthermore, apoptosis, as determined by quantifying the DNA fragmentation, was apparent at a much earlier time period by HNE as opposed to H2O2. Taken together, the results demonstrate the apoptotic potential of the lipid peroxidation end-products and suggest that H2O2-induced apoptosis may be mediated by these end-products in the lens epithelium. © 2002 Elsevier Science Inc. Keywords— 4-Hydroxynonenal, Human lens epithelial cells, Apoptosis, Cataract, Oxidative damage, Hydrogen peroxide, Free radicals
INTRODUCTION
cataractous lens [3–5], the mechanisms by which oxidative stress in general, and lipid peroxidation in particular, lead to cataract formation, remain unclear. Lipid peroxidation generates high levels of alkoxyl radicals, which degenerate into saturated and unsaturated aldehydes. There is increasing evidence in nonlenticular tissues, suggesting that the unsaturated aldehydes, especially the 4-hydroxyalkenals formed by lipid peroxidative reactions, mediate the biological effects of free radicals by acting as toxic messengers and may be responsible for a significant portion of the tissue damage ascribed to their radical precursors (review [6]). The two most toxic 4-hydroxyalkenals are 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE) (review [6]). Peroxidation reactions of -6 polyunsaturated fatty acids (arachidonic, linoleic,
Due to its constant exposure to light, the ocular lens is continuously attacked by reactive oxygen species. Inadequate control of the redox reactions has been suggested to be the key trigger for the development of cataract under several etiologically unrelated conditions (reviews [1,2]). While significantly elevated concentrations of peroxides and other oxidants have been measured in the Address correspondence to: Naseem H. Ansari, Ph.D., Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, 6.642 Basic Science Bldg., Galveston, TX 7755-0647, USA; Tel: (409) 772-3905; Fax: (409) 772-9679; E-Mail: [email protected]. 1 Current address: National Flow Cytometry Resources, Bioscience Division, Los Alamos, NM 87545, USA. 360
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and ␥-linolenic acid) generate HNE and of -3 (docosohexanoeic acid) generate HHE. As a result of a conjugated double bond between the ␣ and  carbons, the ␥ carbon of these aldehydes is electron deficient and reacts readily with nucleophiles such as thiols and amines. Due to their high reactivity, these aldehydes display marked biological effects, which, depending upon their concentrations, cause selective alteration in cell signaling, protein and DNA damage, and cytotoxicity (review [6]) including induction of apoptosis [7–10]. Apoptosis is a form of cell death distinct from necrosis. Necrosis is the result of cellular “accidents” such as those occurring in tissues subjected to chemical trauma. Apoptosis, on the other hand, is programmed. Activation of programmed cell death in an individual cell is dependent on its own internal metabolism, environment, developmental background, and genetic information. Apoptosis is characterized by cell shrinkage, chromatin condensation, and DNA fragmentation, and can be induced by a variety of stimuli. The apoptotic process is thought to be under the control of caspases, a class of cysteine proteases, mainly discovered in Caenorhabditis elegans, with at least three families of death genes being involved termed ced-3, ced-4, and ced-9 [11–16]. Once caspases get activated, they cleave certain proteins like poly (ADP-ribose) polymerase (PARP) and lamin A involved in chromatin condensation as well as cytoskeleton regulation [16,17]. The expression of oncogenes, c-myc, and c-jun, which regulate cell proliferation and survival, has been shown to be modulated by HNE in K562 Erythroleukemic cells and HL-60 [18,19] human leukemic cells [10], suggesting a role of these aldehydes in the control of gene expression. The present study was undertaken to determine the induction of apoptosis in human lens epithelial cells (HLECs) by H2O2 and the 4-hydroxyalkenals, and to investigate the involvement of caspases in this event. EXPERIMENTAL PROCEDURES
Materials Cell culture media, fetal bovine serum, antibiotics, and all chemicals that are not otherwise specified were purchased from Sigma Chemical Co. (St. Louis, MO, USA). HNE was obtained from Caymen Chemicals (Ann Arbor, MI, USA). The substrates and inhibitors of caspases were purchased from Enzyme Systems (Dublin, CA, USA) and the Cell Death ELISA kit from Boehringer Mannheim (Mannheim, Germany). HHE was generously provided by Dr. F. M. J. van Kuijk at our institute.
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Cell culture and treatment Human lens epithelial cells (HLECs) with extended life span were generously provided by Dr. Usha Andley, Washington State University, St. Louis, MO, USA, and were used in this study as described previously [20]. Briefly, cells were grown in Eagle MEM (Sigma) containing 50 g/ml gentamicin and 20% fetal bovine serum in an atmosphere of 5% CO2 in air at 37°C. The pH of the medium was adjusted to 6.8 –7.0 prior to sterile filtration and addition of serum. The cells were fed twice a week. After attaining confluence, cells were passaged using Trypsin-EDTA. Cell survival was determined by trypan blue staining and counting with a hemocytometer. Cell viability is represented as the percentage of the number of live cells/number of total cells. Measurement of apoptosis Apoptosis was determined by quantifying the cytosolic oligonucleosomes-bound DNA by using a “Cell Death Detection ELISA Kit” according to the manufacturer’s instructions and described by us earlier [10,21]. Briefly, the cytosolic fraction (10,000 ⫻ g supernatant) of 5000 cells were used as the antigen source in a sandwich ELISA with a primary anti-histone antibody coated to the microliter plate and secondary anti-DNA antibody coupled to peroxidase. After adding the substrate, absorbance was recorded at 405 nm using the SpectroCount Microplate Spectrometer (Packard Instruments, Meriden, CT, USA). Apoptosis was also detected morphologically using Hoechst and propidium iodide stain. The Vybrant Apoptosis Assay Kit #5 (V-13244) from Molecular Probes (Eugene, OR, USA) containing both the dyes was used. One microliter of each dye was added to the cell suspension, mixed, and kept on ice for 20 min. Subsequently, the cells were plated on a glass slide using the cytospin (500 rpm, 2 min) and visualized under a Nikon Eclipse 800 upright epifluorescence microscope, equipped with a xenon arc lamp. Texas red filter (excitation 540 –580 nm band pass, Dichroic mirror 595 nm, emission 600 – 660 nm) and DAPI filter (excitation 340 –380 nm band pass, Dichroic mirror 400 nm, emission 435– 485 nm) for the red and blue fluorescence, respectively. Photographs were taken using a Dage-MTI (Michigan City, IN, USA) 300 T-RC monochrome single chip integrating cooled CCD video camera with a Flashbus (Integral Technologies, Indianopolis, IN, USA) video capture card. Detection of early stages of apoptosis by flow cytometer Flow cytometery was performed using the cationic dye, JC-1 (Molecular Probes) that exhibits potential de-
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pendent accumulation in mitochondria, indicated by a fluorescence emission shift from green (⬃ 525 nm) to red (⬃ 590 nm). Pellet of approximately 1 ⫻ 106 cells was suspended in 130 l of warm MEM ⫹ 5% FBS to which 250 l of JC-1 solution (20 g/ml) was added. After incubation for 30 min, cells were washed with PBS, re-suspended in 500 l PBS and subjected to flow cytometery. Measurement of caspase activity Caspase activity was measured essentially using the procedure described by Sarin et al. [22]. Cells (0.5 ⫻ 106 cells) were lysed with 100 l 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°C. Dithiothreitol (DTT) was added to the supernatant at a final concentration of 10 mM, then 50 M of the specific fluorogenic substrate for the various caspases (1, 2, 3, 8, and 9) were individually mixed with 10 l aliquots of the cell extracts in 96 well plates and the reaction was carried out 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) at ex 400 nm, em 510 nm. Measurement of oncogene expression The expression of c-myc was determined by Western blot, as described by us earlier for c-myc [10]. C-myc monoclonal antibody Myc-19E-10.2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), raised against a peptide corresponding to residues 408 to 439 within the C-terminal of the c-myc encoding protein, was generated as a culture supernatant of the hybridoma cell line CRL1729 (ATCC). Horseradish peroxidase conjugated secondary antibody catalyzed the oxidation of luminol, resulting in the emission of light-enhanced chemiluminescence, which was captured on autoradiography film. The densitometric analysis of c-myc protein was performed using a Lynx densitometer (Applied Imaging, Santa Cruz, CA, USA). RESULTS
Dose response The HLECs were exposed to various concentrations of H2O2 and after 24 h of exposure, cell viability was determined by trypan blue exclusion method. There was a dose-dependent decrease in the cell viability and 100% cell death was observed with 1 mM H2O2. LD50, defined as the concentration causing 50% cell death, for H2O2 in
Fig. 1. Dose-dependent effect of H2O2 on cell viability and apoptosis. HLECs (1 ⫻ 106 cells) cultured in MEM with 20% FBS and 50 g/ml gentamycin were treated with various concentrations of H2O2 and after 24 h of exposure, the (A) cell viability was determined by trypan blue dye exclusion and expressed as % survival of control without H2O2. (B) Apoptosis, expressed as ELISA absorbance/5000 cells, was determined as described in the text.
HLECs was found to be ⯝ 200 M (Fig. 1A). A dosedependent increase in apoptosis was observed in HLECs treated with H2O2 for 24 h as measured by quantifying cytosolic nucleosomal DNA fragments (Fig. 1B). A maximum of 16-fold increase in apoptosis as compared to that of control was observed with 200 M H2O2. However, when the cells were further exposed to higher concentrations of H2O2, a decrease in the apoptotic index was observed, which could be attributed to necrosis at higher concentrations of H2O2.
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Fig. 3. Time-dependent H2O2-, HNE-, and HHE-induced apoptosis in HLECs. 1 ⫻ 106 cells were incubated with 200 M H2O2 (‚-‚),30 M HNE (䊐-䊐) or 50 M HHE (x-x) for different time intervals. Cells were harvested at specific intervals and apoptosis was determined by ELISA as described in the text and expressed as absorbance at 405 nm/5000 cells for H2O2 and absorbance at 405 nm/2000 cells for HNE or HHE.
Time course
Fig. 2. Dose-dependent effect of HNE and HHE on cell viability and apoptosis. HLECs (1 ⫻ 106 cells) were incubated with various concentrations of HNE (⫹) and HHE (‚). After 24 h cells were harvested and (A) % cell viability and (B) apoptosis, expressed as ELISA absorbance/2000 cells, was determined as described in the text.
Similarly, when the cells were exposed to various concentrations of HNE or HHE, a dose-dependent decrease in the cell viability was observed (Fig. 2A). LD50 for HNE and HHE in HLECs cells were found to be approximately 30 and 50 M, respectively. HLECs exposed to different doses of HNE/HHE for 24 h resulted in a dose-dependent increase in apoptosis, which was more pronounced by HNE as compared to HHE (Fig. 2B). The difference was more distinct at higher concentrations. Cells exposed to 40 M HNE showed 50% higher apoptotic index as compared to the 40 M HHE-treated cells, suggesting that HNE is a more potent inducer of apoptosis as compared to HHE.
A time-dependent increase in apoptosis was observed when HLECs were treated with H2O2. Increase was exponential with respect to time until 16 h postexposure, and remained almost constant at 24 h of H2O2 exposure (Fig. 3). A similar trend was observed when the cells were treated with 30 M of HNE or 50 M of HHE (Fig. 3). Increased apoptosis was observed as early as 2 h in HNE-treated HLECs, which increases rapidly thereafter until 16 h postexposure. However, on the subsequent time intervals, a decrease in the apoptosis was observed. HNE-induced apoptosis was more pronounced as it can be seen from the figure, that at 16 h postexposure, HNE-treated cells showed 2.5-fold higher values of apoptosis as compared to HHE. In order to assess the effect of H2O2 and HNE on the early stages of apoptosis, the cells were first exposed to H2O2 or HNE for 5 h and then incubated with the dye JC-1 and subjected to flowcytometer. It was observed that at this time interval, very few cells undergo apoptotic changes following H2O2 exposure, however, in the HNE-treated cells, nearly 50% of cell population showed the early apoptotic change (change in the polarity of the mitochondrial membrane) (Fig. 4). Caspase activation To assess the involvement of caspases in H2O2- and HNE-induced apoptosis, activation of various caspases (1, 2, 3, 8, and 9) was determined at different time intervals using specific substrates of these caspases. A time-dependent increase in the activities of all the
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Fig. 4. Early apoptotic changes in HLECs treated with H2O2 and HNE. 0.5 ⫻ 106 cells were incubated with 200 M H2O2 or 50 M HNE for 5 h with and without preincubation with 20 M Z-VAD. Cells were harvested at the end of incubation and were further incubated with the dye JC-1 for 30 min as described in the text and were subjected to flowcytometry. (A) control; (B) H2O2-treated; (C) HNE-treated; (D) Z-VAD ⫹ HNE-treated.
caspases examined, except caspase-9, was observed following H2O2 treatment starting as early as 2 h (Fig. 5A). Caspase-1 and caspase-2 get activated almost simultaneously, however, after 6 h postexposure, activity of caspase-2 increases very rapidly, and after 24 h of H2O2 exposure caspase-2 activity increased by 80-fold as opposed to caspase-8 activity, which increases by 15-fold at
the same time interval. In the initial time periods, until 8 h postexposure, the caspase-8 activity was found to be higher than caspase-3 but thereafter, caspase-3 activity increased very rapidly and at 24 h postexposure, the caspase-3 activity was more pronounced than caspase-8 activity. As shown in Fig. 5, caspase-9 was not activated. The results of activation of caspases in HLECs ex-
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Fig. 5. Activation of caspases by H2O2 and HNE in HLECs. (A) Activity of caspases 1, 2, 3, 8, and 9 was determined in the cells treated with 200 M H2O2 at the indicated time intervals as described in the text. 䊐-䊐: caspase-1; E-E: caspase-2; x-x: caspase-3; ‚-‚: caspase-8; 〫-〫: caspase-9. (B) Activity of caspases in the cells treated with 30 M HNE at 16 h as described in the text. C ⫽ control; H ⫽ HNE.
posed to 40 M HNE for 16 h are given in Fig. 5B. Caspase-9 was not activated. A significant increase in both the initiator caspases 2 and 8 was observed. Caspase-3, the main executioner caspase, was activated approximately 6-fold. Prevention of apoptosis by Z-VAD The effect of Z-VAD-FMK, a broad-range inhibitor of the caspases, was studied on the H2O2- and HNE-
induced apoptosis a HLECs. The cells were treated with 20 M Z-VAD 1 h before the addition of H2O2 or HNE in order to ensure enough time for Z-VAD to enter the cells and inhibit the caspases. It was observed that ZVAD completely prevented the H2O2- and HNE-induced apoptosis (Fig. 6). Morphological assessment revealed similar results (Fig. 7, A–F). The effect of Z-VAD was also tested on the H2O2- and HNE-induced early apoptotic changes as measured by incubating the cells with the dye JC-1 and subjecting to flowcytometry. At this early
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the total cell population had undergone apoptosis after 5 h of HNE exposure, and Z-VAD had no protective effect on HNE-induced early apoptotic changes in the cells, demonstrating that mitochondrial membrane potential change is independent of caspase activation (Fig. 4, A–D). Induction of C-Myc by HNE and HHE
Fig. 6. Prevention of H2O2- and HNE-induced apoptosis by Z-VAD in HLECs. Cells were incubated with 20 M Z-VAD for 1 h before the addition of 200 M H2O2 or 30 M HNE for 16 h. Apoptosis was determined as described in the text and is expressed as ELISA absorbance/5000 cells for H2O2-treated cells and ELISA absorbance/2000 cells for HNE-treated cells. C ⫽ control; H ⫽ H2O2; HN ⫽ HNE; Z ⫽ Z-VAD. Values are mean ⫾ SE of three experiments and expressed as ELISA absorbance/5000 cells for H2O2-treated cells and ELISA absorbance/2000 cells for HNE-treated cells.
stage, H2O2 did not produce any apoptotic changes, hence no effect of Z-VAD was observed (data not shown). Interestingly, we observed that nearly 50% of
Figure 8 shows the induction of c-myc protein in HNE-treated cells. For this, the cells were incubated with HNE for 2 and 6 h with and without preincubation with 20 M Z-VAD for 1 h, followed by extraction of whole cell protein. C-Myc protein was estimated by Western blot using specific antibodies. It was observed that cells treated with HNE showed a 1.3- and 4-fold induction of c-myc protein at 2 and 6 h time points, respectively. The broad-spectrum caspase inhibitor Z-VAD prevented the induction of c-myc protein. Similar trend was observed with HHE (data not shown). DISCUSSION
4-Hydroxynonenal and 4-hydroxyhexenal, two highly reactive and cytotoxic end-products of lipid peroxidation
Fig. 7. Prevention of H2O2- and HNE-induced apoptosis by Z-VAD in HLECs. Cell were incubated with 20 M Z-VAD for 1 h before the addition of 200 M H2O2 or 30 M HNE for 16 h. Cells were stained using the Vybrant Apoptosis Kit and photographed (magnification, 40⫻) as described in the text. (A) Control; (B) H2O2-treated; (C) HNE-treated; (D) Z-VAD ⫹ control; (E) Z-VAD ⫹ H2O2-treated; (F) Z-VAD ⫹ HNE-treated.
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Fig. 8. Induction of c-myc by HNE in HLECs. The cells were incubated with 30 M HNE with or without preincubation with 20 M Z-VAD as described in the text for 2 and 6 h. Total proteins were extracted and assayed for the expression of c-myc 1: control; 2: HNE-treated (2 h); 3: HNE-treated (6 h); 4: Z-VAD ⫹ HNE-treated (6 h); 5: Z-VAD ⫹ HNE-treated (2 h). (A) c-myc protein as detected by immunoblot analysis using specific antibodies described in text. (B) quantified data of the c-myc bands shown in Panel A.
have been shown to produce oxidative damage to various tissues under the oxidative stressed conditions. Earlier, we have shown that HNE is cataractogenic in vitro [23] and that high levels of protein-HNE adducts are present in the human diabetic cataractous lenses [24]. Induction of apoptosis in the various cell types is one of the major cytotoxic effects of HNE [7–9]. In the present study, we studied the effect of HNE and HHE as well as H2O2, an oxidant, which is known to be produced in high concentrations under oxidative stress, in the lens. Our study shows LD50 for HNE and HHE in HLECs to be 30 M and 50 M, respectively, suggesting HNE to be more cytotoxic as compared to HHE; LD50 for H2O2 in HLECs was found to be 200 M. The toxic concentrations of HNE and HHE appear to be higher than those reported in the tissues under certain pathological conditions [6]. It has been argued that levels of HNE may build up locally near or within the peroxidizing membranes because of their high lipophilicity. Therefore, the effective cytotoxic concentrations may be much higher than the levels of HNE/HHE expressed as per milligram
tissue [6]. The single layer of lens epithelial cells is important for maintaining metabolic homeostasis and the transparency of the entire lens [24]. Normally, these cells have a long life span under physiological conditions. However, if such conditions are altered or disturbed by factors such as oxidative stress, aging, and other pathological conditions, the viability of these cells can be effected, which leads to the opacification of the lens. In this context, it has been reported that human cataract subjects have a higher percentage of apoptotic lens epithelial cells as compared to normal human lenses of the comparable age group [24]. This indicates a role of oxidative stress-induced apoptosis of lens epithelial cells in cataractogenesis. Contrary to this finding, Harocopos et al. [25] were unable to demonstrate any difference in the number of dead cells in the lens epithelia from the cataractous and normal lenses obtained from the eye bank. It is not clear whether the discrepancy in the two reports could be due to some trivial but crucial difference in obtaining the samples or involvement of other factors. Nevertheless, studies showing that oxidants, such as
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H2O2, induce apoptosis in lens cells [26,27] and that LDAs, such as HNE, induce apoptosis in various cell types [7–9] suggests that the oxidants may impart their toxicity via their peroxidative end-products. When the effect of H2O2 and HNE on the early apoptotic changes (depolarization of the mitochondrial membrane) was studied, we did not observe any significant change in the H2O2-treated cells, whereas ⬃ 50% of the HNE-treated cells were found to undergo early apoptotic changes. This indicates that H2O2-induced apoptosis is a delayed event as opposed to a relatively instantaneous induction of apoptosis by HNE. It may be possible that exposing the cells to H2O2 may lead to lipid peroxidation resulting in the excessive production of lipid-derived aldehydes, such as HNE and HHE, which may then induce apoptosis in these cells. Hence, HNE and HHE could be a mediator of H2O2-induced apoptosis in HLECs. Alternatively, it is possible that HNE could be modifying the mitochondrial membrane proteins leading to the changes in the mitochondrial membrane potential. Since proteolysis is a requirement for the induction of apoptosis in most of the apoptotic systems, we investigated the activation of various caspases in our apoptotic systems. Activation of caspases 1, 2, 3, and 8 by H2O2/ HNE suggests the involvement of these caspases in the induction of H2O2- and HNE-induced apoptosis. While there was activation of initiator caspases (caspase 1 and 8; Fig. 5), we did not observe activation of the initiator caspase 9. Since Z-VAD did not prevent the HNEinduced change in mitochondrial membrane potential (Fig. 4) but did prevent apoptosis (Fig. 6), our studies suggest that the HNE-induced activation of all the caspases and execution of apoptosis in this system are independent of mitochondrial changes. A systematic study to establish the caspase-cascade is in progress. The proto-oncogene product, c-myc has been shown to encourage cell proliferation and apoptosis, since it has both the positive as well as negative factor to support apoptosis. Many studies have shown the induction of apoptosis following overexpression of c-myc in various cell types [28,29]. However, there are reports suggesting that a reduction of c-myc expression also induces apoptosis [30 –32]. In fact, previously we have reported that HNE exposure to CEMC7 human leukemic cell line results in the downregulation of c-myc prior to activation of caspases [10]. However, in the present study, we observed an increase in the expression of c-myc protein, which was inhibited by Z-VAD, demonstrating the importance of caspase activation in this system. Induction of c-myc has been found to lead into the processing/ activation of caspases [33,34]. Furthermore, it has been reported that several targets of caspases (PARP, fodrin, lamins, MDM2, etc.) are cleaved during c-myc-induced apoptosis in Rat-1 MycER cells [35]. Our results (Fig. 8)
showing that caspase inhibitor Z-VAD prevents the HNE-induced induction of c-myc suggest that c-myc gets induced downstream of, at least, the intiator caspases. Whether and how c-myc is indeed involved in this apoptotic system is yet to be established by our work under progress. In conclusion, our study demonstrates that H2O2, HNE, and HHE are cytotoxic to HLECs at micromolar concentrations and they kill the cells by inducing apoptosis. Furthermore, our results suggest that apoptosis of lens epithelial cells, following oxidative insult, may be mediated by HNE and HHE. Acknowledgements — This work was supported by the National Institutes of Health EY 13014 and Lions Eye Bank Foundation.
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DTT— dithiothreitol HLECs— human lens epithelial cells HNE— 4-hydroxynonenal HHE— 4-hydroxyhexenal H2O2— hydrogen peroxide