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Originally published In Press as doi:10.1074/jbc.M600744200 on June 12, 2006

J. Biol. Chem., Vol. 281, Issue 33, 23643-23651, August 18, 2006
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Intracellular Dissemination of Peroxidative Stress

INTERNALIZATION, TRANSPORT, AND LETHAL TARGETING OF A CHOLESTEROL HYDROPEROXIDE SPECIES BY STEROL CARRIER PROTEIN-2-OVEREXPRESSING HEPATOMA CELLS*

Tamas Kriska{ddagger}, Vladislav V. Levchenko{ddagger}, Witold Korytowski{ddagger}§, Barbara P. Atshaves, Friedhelm Schroeder, and Albert W. Girotti{ddagger}1

From the {ddagger}Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, §Institute of Molecular Biology, Jagiellonian University, 31-120 Krakow, Poland, and Department of Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843

Received for publication, January 25, 2006 , and in revised form, May 30, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Sterol carrier protein-2 (SCP-2) plays a crucial role in the trafficking and metabolism of cholesterol and other lipids in mammalian cells. Lipid hydroperoxides generated under oxidative stress conditions are relatively long-lived intermediates that damage cell membranes and play an important role in redox signaling. We hypothesized that SCP-2-facilitated translocation of lipid hydroperoxides in oxidatively stressed cells might enhance cytolethality if highly sensitive sites are targeted and detoxification capacity is insufficient. We tested this using a clone (SC2A) of rat hepatoma cells that overexpress mature immunodetectable SCP-2. When challenged with liposomal cholesterol-7{alpha}-hydroperoxide (7{alpha}-OOH), SC2A cells were found to be much more sensitive to viability loss than vector control (VC) counterparts. Correspondingly, SC2A cells imported [14C]7{alpha}-OOH more rapidly. The clones were equally sensitive to tert-butyl hydroperoxide, suggesting that the 7{alpha}-OOH effect was SCP-2-specific. Fluorescence intensity of the probes 2',7'-dichlorofluorescein and C11-BODIPY increased more rapidly in SC2A than VC cells after 7{alpha}-OOH exposure, consistent with more rapid internalization and oxidative turnover in the former. [14C]7{alpha}-OOH radioactivity accumulated much faster in SC2A mitochondria than in VC, whereas other subcellular fractions showed little rate difference. In keeping with this, 7{alpha}-OOH-stressed SC2A cells exhibited a faster loss of mitochondrial membrane potential and development of apoptosis. This is the first reported evidence that peroxidative stress damage can be selectively targeted and exacerbated by an intracellular lipid transfer protein.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Non-esterified cholesterol (Ch)2 and various phospholipids are known to move from one membrane compartment to another in mammalian cells as needed for metabolic processing and membrane biogenesis/homeostasis (1–3). Desorption from the donor membrane is typically the rate-limiting step in a spontaneous lipid translocation process (1). Numerous studies with model systems have shown that natural lipids (particularly those in the phospholipid family) translocate relatively slowly on their own but that this is accelerated by specific lipid transfer proteins (4, 5). A well studied example is intracellular sterol carrier protein-2 (SCP-2), also known as nonspecific lipid transfer protein. SCP-2 not only facilitates the intermembrane translocation of Ch and other sterols but also various fatty acids and fatty acyl-CoAs (6–8). SCP-2 is a relatively small (13.2 kDa) translation product of a fusion gene encoded for 58-kDa SCP-x (~45 kDa of which represents a peroxisomal 3-ketoacyl-CoA thiolase) and 15-kDa pro-SCP-2 (7–10). Mature SCP-2 appears to arise mainly from post-translational cleavage of pro-SCP-2, although some direct formation by cleavage at the SCP-x level is also possible (7, 8). Examination of subcellular distribution by immunodetection methods has revealed that SCP-2 is at the highest concentration in peroxisomes, although significant levels are also found in mitochondria, lysosomes, and cytosol, presumably reflecting the wide-ranging trafficking activity of this protein (7). In mouse L-cells, for example, ~50% of the total immunoreactivity is extraperoxisomal (11). Numerous studies with model systems have shown that recombinant SCP-2, like the natural protein, can greatly accelerate the transfer of various sterols and phospholipids from donor to acceptor membranes (12–15). Various mechanisms for this have been proposed including one in which SCP-2 binds desorbed lipids in the aqueous compartment and another in which the protein binds resident lipids upon interacting with the donor membrane (7, 15). Support for the latter mechanism derives from evidence that the N-terminal {alpha}-helical segment of SCP-2 with several lysyl residues can interact with anionic phospholipids on donor/acceptor membrane surfaces (7, 16).

Oxidative stress-induced peroxidation of unsaturated lipids in cell membranes gives rise to a large number of reactive intermediates and end products (17–19). Highly prominent in the former category are lipid hydroperoxide (LOOH) species (19). Under redox-constrained conditions, LOOHs generated by singlet oxygen addition, for example, can accumulate and perturb membrane structure/function directly due to their increased hydrophilicity. However, in the presence of reductants and catalytic iron, LOOHs undergo one-electron reduction to oxyl radicals, which either directly or after conversion to epoxyallylic peroxyl radicals (20) can exacerbate damage by triggering chain peroxidation reactions (17–19). Opposing this is two-electron detoxification catalyzed by certain selenium- and non-selenium-dependent peroxidases (21, 22). Our recent studies have indicated that these reactions are not necessarily limited to a LOOH membrane of origin but can extend to other membranes via LOOH translocation through the aqueous phase (23–25). Using model membrane systems, we have shown that Ch- and phospholipid-derived hydroperoxide species translocate more rapidly than the respective parent lipids and that this is further accelerated by SCP-2 (25). Cytotoxic relevance was demonstrated by showing that SCP-2 accelerates transfer of 7{alpha}-OOH (a free-radical-derived ChOOH) from liposomes to isolated mitochondria, which enhances peroxide-induced loss of mitochondrial membrane potential (25).

In this study we have examined the effects of SCP-2 overexpression in rat hepatoma cells on uptake, distribution, and cytotoxicity of exogenous 7{alpha}-OOH delivered in liposomal form. Our findings are novel in that they are the first to implicate an intracellular lipid trafficking protein in the dissemination of lethal damage under peroxidative stress conditions.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Ch, cholesteryl linoleate, Ho258, JC-1, MTT, PI, Dulbecco's modified Eagle's medium, phenol red-free RPMI medium, fetal bovine serum, and other cell culture materials were from Sigma. Molecular Probes (Eugene, OR) supplied the C11-BODIPY581/591, DCFH-DA, and MitoTracker Deep Red 633. [4-14C]Ch (~50 mCi/ml) from Amersham Biosciences was HPLC-purified before use (23). Human recombinant SCP-2 was generated and isolated as described (25). The following rabbit-raised antibodies were kindly supplied as gifts: polyclonal anti-calnexin and monoclonal anti-lamin B receptor from Dr. Paula Traktman and polyclonal anti-cytochrome c oxidase from Dr. Margaret Wong-Riley. Peroxidase-conjugated anti-rabbit IgG was obtained from MP Biochemicals (Aurora, OH). Unlabeled or 14C-labeled 7{alpha}-OOH (~165 mCi/mmol) was prepared by dye-sensitized photoperoxidation of Ch or [14C]Ch, isolated to co-eluting sterol and peroxide homogeneity by normal and reverse-phase HPLC, and quantified by iodometric analysis (26, 27). Other Ch oxides, primarily used as TLC standards, were either prepared (26) or supplied by Steraloids (Wilton, NH).

Cell Culture—An SCP-2-overexpressing transfectant clone (SC2A) of rat McA-RH777 hepatoma cells (28) along with a vector control clone (VC) was grown in Dulbecco's modified Eagle's medium containing 10% serum, penicillin (100 units/ml), streptomycin (0.1 mg/ml), and Geneticin (G418, 0.35 mg/ml) using standard culture conditions (29). The SC2A cells had been transfected with a construct encoded for 15-kDa pro-SCP-2, which is post-translationally converted to 13.2 kDa SCP-2 (7, 28). Cells were taken off Geneticin 5 days before an experiment, reseeded 3 days later, and grown back to 50–60% confluence before beginning experimental manipulations.

Immunoblot Analysis—The level of SCP-2 protein expressed in SC2A and VC cells was determined by immunoblotting. Cells were recovered by trypsinization, washed with PBS and then hypotonic buffer, and lysed by sonication. Lysates were centrifuged at 100,000 x g for 1 h at 4°C. Recovered supernatant fractions were examined for protein content (30) and then subjected to Western analysis using a 15% polyacrylamide gel for electrophoresis and 0.45-µm polyvinylidene difluoride membrane for transblotting. Blots were blocked, treated with rabbit anti-mouse SCP-2 (10) and anti-actin antibodies and then with peroxidase-conjugated anti-rabbit IgG, and analyzed using enhanced chemiluminescence (31).

Cellular Enzymatic Activities and Glutathione Levels—GPx4 activity was determined by measuring the GSH-dependent decay kinetics of 7{alpha}-OOH using HPLC with electrochemical detection (26, 29). GPx1 was determined by coupled enzymatic assay using t-BuOOH as the peroxide substrate (29). Total glutathione (GSH + 2GSSG) was quantified by Tietze assay (32).

Determination of [14C]7{alpha}-OOH and [14C]Ch Uptake by Cells and Subcellular Distribution of Radioactivity—Two days before an experiment SC2A and VC cells were seeded into 10-cm dishes (5 x 105 cells each), and 12 h before the medium was changed to RPMI containing 5% lipoprotein-deficient serum to minimize Ch supply. This reduced Ch content of both cell types by ~30%, as determined by HPLC analysis (23). Small unilamellar liposomes (50 nm SUVs) consisting of 5.5 mM 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 2.5 mM [14C]7{alpha}-OOH (2.9 µCi/ml), 2.0 mM Ch, and 0.06 mM dicetyl phosphate in bulk phase PBS (pH 7.4) were prepared by an extrusion process as described (23). Identical composition SUVs containing [14C]Ch (2.5 µCi/ml) and unlabeled 7{alpha}-OOH were prepared alongside. Cells were first washed free of lipoprotein-deficient serum, then overlaid with [14C]7{alpha}-OOH- or [14C]Ch-containing SUVs in RPMI medium, with starting sterol concentrations being 25 and 20 µM, respectively. After various periods of incubation at 37 °C of up to 30 min, cells in each dish were washed extensively with cold PBS, recovered by scraping, centrifuged, and resuspended in 0.5 ml of PBS containing 0.1 mM EDTA. Samples were checked for protein content (30) and then extracted with 0.8 ml of cold chloroform/methanol (2:1, v/v) (27). Lipid-containing fractions were dried under N2, dissolved in hexane/isopropanol (97:3, v/v), applied to a TLC plate section in a hairline N2 stream, and subjected directly to phosphorimaging using a Storm 860 storage phosphor system (33). [14C]Ch and [14C]7{alpha}-OOH standards of known specific radioactivity were used for calibration.

For determining subcellular distribution of radioactivity from incoming [14C]7{alpha}-OOH, cells were incubated with [14C]7{alpha}-OOH-containing SUVs for 3 h, then washed with PBS, resuspended in ice-cold hypoosmotic buffer (34) for 5 min, and Dounce-homogenized. Immediately thereafter, osmolarity was restored (34), and the lysate was layered onto a buffered 10-step sucrose gradient ranging from 12 to 83% (w/v) sucrose and centrifuged at 15,000 x g for 3 h to separate nuclear, mitochondrial, microsomal, and cytosolic fractions, each of which was analyzed for protein content. Other details were as specified previously (34). Purity of the microsomal, mitochondrial, and nuclear fractions was assessed by quantitative immunoblot analysis using antibodies against calnexin, cytochrome c oxidase, and lamin B receptor, respectively. Lipid material in each fraction was extracted and subjected to phosphorimaging analysis as described above for overall uptake. Protein-based specific radioactivity was corrected for any cross-contamination (e.g. microsomes with mitochondria), knowing the measured levels of the marker enzymes and also that microsomal fractions were devoid of nuclei and mitochondria.

Assessment of 7{alpha}-OOH Turnover in Cells—For monitoring the reactivity and fate of internalized hydroperoxide, we incubated SC2A and VC cells with 25 µM liposomal [14C]7{alpha}-OOH in RPMI medium for various time periods up to 4.5 h. Cell samples were washed, recovered, analyzed for protein, and extracted as described in the preceding section, after which lipid fractions were applied to a silica gel-60 HPTLC plate (EM Science, Gibbstown, NY), and chromatographed using benzene/ethyl acetate (1:1, v/v) as the mobile phase (27, 33). [14C]7{alpha}-OOH and resolved products thereof were detected and quantified by phosphorimaging (33).

Assessment of Peroxide-induced Cell Death and Mechanism Thereof—SC2A and VC cells at ~60% confluence in 12-well plates were overlaid with 7{alpha}-OOH-bearing SUVs in RPMI medium, giving a range of initial hydroperoxide concentrations up to 75 µM in bulk suspension (at the latter concentration, there was no significant cell detachment over at least a 4-h period). After a designated incubation time at 37 °C (typically 3 h), the cells were washed free of SUVs, overlaid with 1% serum-containing RPMI, and checked for viability by MTT assay (29, 35) after 20 h of additional incubation. Alternatively, cells were incubated with a fixed concentration of SUV 7{alpha}-OOH (typically 25 µM) for increasing periods up to 3 h, then washed, switched to 1% serum-containing RPMI, and tested for viability after an overnight incubation; the total time from peroxide introduction was 24 h in all cases. Cells were also challenged with t-BuOOH in increasing concentrations for 3 h and then checked by MTT assay after incubating for 20 h in the absence of peroxide. For evaluating death mechanism, cells were washed and treated with 5 µM Ho258 and 50 µM PI for 20 min, then examined by fluorescence microscopy using 4',6-diamidino-2-phenylindole and green filters for Ho258 and PI, respectively. Ho258 was used to detect any sustainable apoptosis and PI to confirm necrosis, which is not always discernable with Ho258 alone. Stained nuclei were counted (35, 36), and the percentage of apoptotic versus necrotic cells was determined.

Measurement of Mitochondrial Membrane Potential—At various times after exposure to liposomal 7{alpha}-OOH, cells were washed with PBS, overlaid with RPMI medium containing 2 µM JC-1, a probe of mitochondrial membrane potential ({Delta}{Psi}) (37), and incubated for 30 min at 37 °C. The cells then washed again with PBS, overlaid with RPMI alone, and checked for fluorescence emission intensity at 590 nm (red) versus 530 nm (green) using 488-nm excitation and a CytofluorTM (Bedford, MA) fluorescence plate reader. Strong 590 nm and weak 530 nm emission reflects a high {Delta}{Psi}, whereas the opposite reflects a low {Delta}{Psi} (37). Time course changes in the 590 nm/530 nm intensity ratio are represented.

Measurement of Overall Cellular Reactive Oxidants—DCFH-DA is taken up by cells and hydrolyzed to the more polar DCFH, which is trapped. DCFH in turn can be oxidized to DCF (the monitored fluorophore) by internally generated ROS (38). The following general protocol was used. Immediately after exposure to 7{alpha}-OOH, cells were washed with PBS, incubated with RPMI medium containing 10 µM DCFH-DA for 20 min at 37 °C, washed again, overlaid with RPMI alone, and examined by fluorescence microscopy using 488-nm excitation and 610-nm emission (36, 38). Fluorescence intensity of representative images was determined using MetaMorphTM software (36).

Imaging of 7{alpha}-OOH-induced Lipid Peroxidation—The lipophilic fluorophore C11-BODIPY581/591 localizes in subcellular membranes and "senses" lipid peroxidative damage in these compartments (39, 40). Cells were incubated with liposomal 7{alpha}-OOH for various periods at 37 °C, then washed and treated with 5 µM C11-BODIPY from a stock solution prepared as described (40). After 30 min of incubation, the cells were washed, overlaid with RPMI medium, and examined by fluorescence microscopy using 488 nm excitation and 590 nm (red) and 530 nm (green) emission filters for observing the unoxidized and oxidized probe, respectively (40). Fluorescence signals were quantified as described in the preceding section.

Confocal fluorescence microscopy was used for assessing whether mitochondria might be special targets of lipid peroxidation. SC2A cells grown on coverslips in a 12-well plate were exposed to liposomal 7{alpha}-OOH for a given period, then washed and overlaid with RPMI containing 2 µM C11-BODIPY and 0.5 µM MitoTracker Deep Red (from a 1 mM stock in dimethyl sulfoxide). After a 30-min dark incubation, the cells were washed with PBS, fixed with 4% paraformaldehyde, washed again, and mounted on a slide using Fluoromount-G medium. A Leica DMRE microscope equipped with TCS SP2 scanner (Leica Microsystems, Heidelberg GmbH) was used for confocal microscopy. The respective excitation and emission wavelengths were as follows: C11-BODIPY (488 nm, 507–535 nm); MitoTracker Deep Red (633 nm, 660–680 nm). Images were taken at 100-fold magnification using immersion oil.

Statistics—The two-tailed Student's t test was used for determining the significance of apparent differences between experimental values, with p ≥ 0.05 considered statistically insignificant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
SCP-2 Protein Expression in SC2A Versus VC Cells—The SCP-2 content of SC2A cells was found to be substantially higher than that of VC, consistent with previous findings (28). Actin-normalized band integration for the immunoblot shown in Fig. 1 indicated an ~10-fold elevation of SCP-2 in the overexpressing cells. No significant difference in the levels of other immunodetectable proteins, including SCP-x and an undefined ~32-kDa protein, was apparent, which also agrees with earlier results (28). Little if any pro-SCP-2 could be detected, indicating that proteolytic conversion to mature SCP-2 was highly efficient.


Figure 1
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  		FIGURE 1.
Immunoblot showing SCP-2 levels in transfectant SC2A and VC cells. Solubilized cell samples (each containing 60 µg of total cellular protein) were loaded onto a 4–15% SDS-PAGE gel, electrophoresed, transblotted to a polyvinylidene difluoride membrane, and probed with anti-SCP2 and anti-actin antibodies. A 10-ng sample of human recombinant SCP-2 was used as a standard (Std).

 
Glutathione Peroxidase Activities and Glutathione Contents—Cellular levels of selected antioxidants were determined, viz. glutathione and the GSH-dependent selenoperoxidases GPx1 and GPx4. Measured values (means ± S.D., n = 3) are as follows: GPx4 activity (units/mg of protein), 0.78 ± 0.12 (SC2A), 0.86 ± 0.09 (VC); GPx1 activity (units/mg of protein), 8.7 ± 1.1 (SC2A), 8.3 ± 0.6 (VC); GSH + 2GSSG (nmol/mg of protein), 29.2 ± 2.0 (SC2A), 26.3 ± 3.3 (VC). Thus, there was no significant difference between SC2A and VC cells with respect to these particular antioxidant levels, ruling this out as a possible contributing factor in any observed difference to a peroxidative challenge (see below).

Cytotoxic Effects of 7{alpha}-OOH and t-BuOOH—When challenged with liposomal 7{alpha}-OOH, SC2A cells were found to be substantially more sensitive to MTT-assessed killing than VC. This was observed in both an increasing [7{alpha}-OOH]/fixed time (Fig. 2A) and fixed [7{alpha}-OOH]/increasing time (Fig. 2B) format. The LC50 values for SC2A and VC cells were ~19 and ~75 µM, respectively (Fig. 2A). Control SUVs in which 7{alpha}-OOH was replaced by redox-inactive 7{alpha}-OH exhibited no significant cytotoxicity over the concentration range used (Fig. 2A). This indicates that the observed lethality was not merely a generalized oxysterol effect but required the OOH group specifically. In contrast to the 7{alpha}-OOH response, SC2A and VC cells were found to be equally sensitive to t-BuOOH (Fig. 2C), with the LC50 (~165 µM) significantly higher than those for the sterol hydroperoxide. Not seeing a toxicity difference with t-BuOOH (a non-lipid hydroperoxide) suggests that the effects observed with 7{alpha}-OOH were SCP-2-specific.

Comparative Kinetics of Sterol Uptake—Using [14C]7{alpha}-OOH-containing SUVs as donors and cells that had been deprived of Ch by growing in lipoprotein-deficient medium, we monitored uptake of radioactivity as a function of incubation time. As shown in Table 1, specific radioactivity of cellular lipid extracts increased progressively and nearly linearly over a 30-min period, the rate for SC2A cells being ~25% greater than that for VC. The same trend was observed when [14C]Ch uptake was tracked, the rate in this case being ~35% greater for SC2A cells (Table 1). In agreement with previous findings based on use of cholesterol oxidase for analysis (28), the [14C]Ch results probably reflect more rapid internalization by the SCP-2-overexpressing cells rather than mere surface association. A similar deduction is made for [14C]7{alpha}-OOH uptake (Table 1), and our data relating to the appearance of radioactivity from this hydroperoxide in subcellular compartments supports this (see below).


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  		TABLE 1
Time-dependent sterol uptake by SC2A and VC transfectants

[14C]Sterol in cell samples was extracted, deposited on a TLC plate, and determined by phosphorimaging. Each value is standardized to total cellular protein and represents the mean ± S.E. (n = 4).

 


Figure 2
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  		FIGURE 2.
Comparative sensitivity of SC2A and VC cells to peroxide-induced killing. SC2A ({triangleup}) and VC ({circ}) cells were incubated with SUV 7{alpha}-OOH in increasing concentrations for 3 h (A) or at a fixed concentration (25 µM) for increasing times (B), then washed and checked for viability by MTT assay 20 h later. Either clone (x) was also exposed to SUV 7{alpha}-OH in increasing concentrations for 3 h and checked 20 h later (A). The same time schedule was used for testing toxicity of t-BuOOH on SC2A ({triangleup}) and VC ({circ}) cells (C). Data points in each panel are the means ± S.E. (n = 6).

 
Subcellular Distribution of [14C]7{alpha}-OOH Radioactivity—In a separate experiment, cells prepared as described in Table 1 were exposed to liposomal [14C]7{alpha}-OOH for 3 h and then homogenized. Density gradient-separated fractions containing (i) cytosol, (ii) microsomes, (iii) mitochondria (with some microsomes), and (iv) mitochondria (with some microsomes and nuclei), based on immunodetection of marker enzymes, were analyzed for protein content and extracted. Phosphorimaging of recovered lipid material with correction for cross-contamination in fractions iii and iv showed no significant difference between SC2A and VC cells in the level of specific radioactivity associated with the nuclear, microsomal, and cytosolic fractions (Fig. 3). However, there was a striking difference in specific radioactivity of the mitochondrial fractions, the SC2A being ~2.5 times greater than the VC (Fig. 3). Therefore, SCP-2 overexpression resulted not only in more rapid internalization of 7{alpha}-OOH but also selectively greater localization of the hydroperoxide and/or metabolites thereof in the mitochondrial compartment.


Figure 3
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  		FIGURE 3.
Subcellular distribution of [14C]7{alpha}-OOH radioactivity in SC2A versus VC cells. Cells preincubated in lipoprotein-deficient serum-containing medium were exposed to SUV [14C]7{alpha}-OOH (25 µM; ~30 nCi/ml) for 3 h. After washing and homogenization, subcellular fractions were separated by sucrose density gradient centrifugation. Protein-based specific radioactivity of the lipid extract from each fraction was determined. Plotted values (corrected for cross-contamination where necessary) are the means ± S.E. (n = 4). *, significantly greater than corresponding VC value, p < 0.01. Mito, mitochondrial; Nucl, nuclear; Micro, microsomal; Cyto, cytosolic fractions.

 
Turnover and Fate of Internalized 7{alpha}-OOH—Like all other LOOHs, 7{alpha}-OOH would be expected to turn over in various ways upon entering cells, undergoing e.g. iron-catalyzed one-electron reduction, enzyme-catalyzed two-electron reduction (detoxification), or transesterification to give a peroxysterol ester (19, 41). To assess this in a comparative manner for [14C]7{alpha}-OOH-treated SC2A and VC cells, we analyzed lipid extracts at various time points by means of HPTLC with phosphorimaging detection (33). As shown by the chromatogram in Fig. 4, four prominent radiolabeled analytes were identified based on co-migration with authentic standards, 7{alpha}-OH, 7-one, 7{alpha}-OOH, and CE, in order of increasing relative mobility (the CE standard was cholesteryl linoleate; however, cellular CE could be any number of unresolved acyl-CoA:cholesterol acyltransferase-generated esters of 7{alpha}-OOH or species derived from it). Intensity of all analyte bands increased with time of cell incubation with [14C]7{alpha}-OOH. However, there was a striking difference in the rate of intensification between SC2A and VC cells, with 7{alpha}-OOH, 7-one, and CE increasing more rapidly in the latter but 7{alpha}-OH more slowly (Fig. 4). This is seen graphically in the time course plots for integrated 7{alpha}-OOH, 7{alpha}-OH, 7-one, and CE radioactivity (Fig. 4). The slower 7{alpha}-OOH and faster 7{alpha}-OH accumulation in SC2A cells is attributed to relatively rapid SCP-2-facilitated transfer of the hydroperoxide to redox-active subcellular sites. One-electron (free radical) reductive turnover at such sites would give rise to 7{alpha}-OH and other redox-inactive products (33, 41, 42). 7{alpha}-OH could also arise from two-electron (non-radical) reduction catalyzed by cytoprotective GPx4 (19, 41). No significant 7beta-OH was detected (Fig. 4), indicating that conversion of both 7{alpha}-OOH and 7{alpha}-OH to the more stable beta-epimers (43) was slow in this system. It is not clear why the buildup of 7-ketone, which should derive only from one-electron reduction of 7{alpha}-OOH (33, 42), was slower in SC2A cells. Interestingly, however, the [7-one]/[7{alpha}-OOH] ratio for these cells was significantly higher throughout than that for VC, i.e. ~1.6 versus 0.9 (Fig. 4), which is consistent with greater free radical activity in the former. In accordance with a previous study showing diminished CE synthesis in SCP-2-overexpressing cells (28), the observed CE slowdown in SC2A cells (Fig. 4) can be attributed to their more extensive dissemination of incoming peroxide, making less of it available at esterification sites.


Figure 4
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  		FIGURE 4.
Accumulation of [14C]7{alpha}-OOH turnover products as a function of incubation time with SC2A versus VC cells. Lipids extracted from cells incubated with [14C]7{alpha}-OOH-containing SUVs were analyzed by HPTLC with phosphorimaging detection. Each sample lane on the chromatogram represents ~1.3 x 107 cells. Incubation times for VC cells (lanes 1–3) and SC2A cells (lanes 4–6) were 1.5 h (lanes 1 and 4), 3.0 h (lanes 2 and 5), and 4.5 h (lanes 3 and 6). A mixture of non-radiolabeled 7{alpha}-OH, 7beta-OH, 7-one, 7{alpha}-OOH, and CE standards (Std) was run alongside and detected by spraying with 9 N H2SO4 with warming. O, origin; F, solvent front. Time courses for accumulation of radiolabeled cellular 7{alpha}-OOH, 7{alpha}-OH, 7-one, and CE (standardized to cell protein for each sample) are shown in the plots: {triangleup}, SC2A; {circ}, VC. Values are the means ± S.E. (n = 3) (at 4.5 h, 7{alpha}-OOH represents 14.6 and 4.5% of the total radioactivity in VC and SC2A, respectively).

 


Figure 5
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  		FIGURE 5.
Reactive oxidant generation in 7{alpha}-OOH-treated cells. SC2A and VC cells were washed after the indicated times of exposure to 25 µM SUV 7{alpha}-OOH, then treated with 10 µM DCFH-DA, washed again, and analyzed by fluorescence microscopy using 488-nm excitation and 610-nm emission. Images of representative cell populations are shown. Integrated fluorescence intensity is indicated below each panel. Values are the means ± S.E. (n = 4).

 
Reactive Oxidant Generation and Lipid Peroxidation in 7{alpha}-OOH-challenged Cells—Overall ROS buildup in 7{alpha}-OOH-treated cells was assessed with the cell-permeating probe DCFH-DA. Internalized DCFH-DA is hydrolyzed and trapped as DCFH, which in turn is converted to DCF (the fluorophore reporter) by H2O2 and other strong oxidants (38). As shown in Fig. 5, barely any DCF fluorescence could be seen in either SC2A or VC cells 1.5 h after introducing peroxide. By 3 h the VC signal was greater but nevertheless quite weak compared with the very intense SC2A signal, which appeared uniform throughout most cell interiors. Internalized (priming) 7{alpha}-OOH was barely detectable in SC2A cells at this time (Fig. 4), suggesting that it itself was not responsible for the strong DCF response but, rather, downstream LOOHs and other oxidants arising from its free radical turnover. The higher ROS level attained by SC2A cells (Fig. 5) is consistent with the notion that their more rapid 7{alpha}-OOH turnover (Fig. 4) and loss of viability (Fig. 2) was due to more vigorous one-electron chemistry (19, 41). No significant DCF fluorescence was observed when cells were exposed to SUVs bearing 7{alpha}-OH instead of 7{alpha}-OOH (not shown).


Figure 6
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  		FIGURE 6.
Oxidation of endogenous lipids in 7{alpha}-OOH-treated cells. Cells were incubated with 25 µM SUV 7{alpha}-OOH for the indicated periods, washed, treated with 5 µM C11-BODIPY, washed again, and examined by fluorescence microscopy using 488-nm excitation and appropriate cutoff filters for detecting non-oxidized (red) and oxidized (green) probe. A, representative viewing fields. B, integrated fluorescence intensities plotted as percent oxidized C11-BODIPY versus contact time with 7{alpha}-OOH; means ± S.E. (n = 4). *, significantly greater than corresponding VC value, p < 0.001. C, confocal microscopy of SC2A cells stained with 2 µM C11-BODIPY and 0.5 µM MitroTracker Deep Red after 3 h of contact with 25 µM 7{alpha}-OOH. Images represent a single focal layer. The merged frame shows dye co-localization. Bar, 10 µm.

 
More direct evidence that 7{alpha}-OOH cytotoxicity is mediated by free radical lipid peroxidation was sought by using the fluorophore C11-BODIPY. This probe enters cells, intercalates with membrane lipids, and acts as an in situ "sensor" of peroxidative damage by changing from red emission in its non-oxidized form to green emission in its oxidized form (40). As shown by the fluorescence micrographs in Fig. 6A, C11-BODIPY-detected lipid peroxidation progressed more rapidly in SC2A cells than VC during 7{alpha}-OOH exposure, as indicated by faster disappearance of red fluorescence in the former with reciprocal appearance of green fluorescence. Thus, SC2A cells were more susceptible to this specific type of 7{alpha}-OOH-primed oxidative damage. In quantitative terms, the amount of probe in its oxidized form averaged ~3 times higher in the SCP-2-overexpressing cells after 3 h (Fig. 6B). Closer scrutiny revealed that the strongest signal from oxidized probe was concentrated in perinuclear zones (see SC2A at 3 h in Fig. 6A). To learn whether these might represent mitochondria, we examined the fluorescence pattern of individual cells co-stained with C11-BODIPY and MitoTracker Deep Red using confocal microscopy. As shown in Fig. 6C, both probes produced a punctuated perinuclear pattern in 3-h-stressed SC2A cells, the BODIPY green merging with the MitoTracker Red to give a yellow-orange composite. These findings, consistent with those shown in Fig. 3, indicate that mitochondrial membranes were major sites of 7{alpha}-OOH-provoked lipid peroxidation damage in SC2A cells.


Figure 7
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  		FIGURE 7.
Effect of 7{alpha}-OOH exposure on mitochondrial membrane potential. SC2A ({triangleup}) and VC ({circ}) cells were washed after the indicated times of incubation with 25 µM SUV 7{alpha}-OOH, then treated with 2 µM JC-1, washed again, and examined by fluorescence plate reader using 488-nm excitation and 590- or 530-nm emission. Time-dependent changes in RFI (fluorescence intensity at 590 nm relative to that at 530 nm) are plotted. Values are the means ± S.E. (n = 4).

 
Loss of Membrane Potential and Induction of Apoptosis in 7{alpha}-OOH-treated Cells—Evidence for mitochondrial functional damage in stressed cells was sought by using JC-1 to monitor {Delta}{Psi} changes. As shown in Fig. 7, {Delta}{Psi} remained high and nearly the same for VC and SC2A cells up to ~1 h after challenging with 25 µM 7{alpha}-OOH. The potentials decreased and diverged thereafter; that for VC cells dropped to ~80% of its starting value by 2 h, but that for SC2A dropped to <10% (Fig. 7). Thus, loss of {Delta}{Psi} after an initial lag was much more pronounced in the latter. The onset of this loss coincided with the strong surge of ROS development (Fig. 5) and lipid peroxidation (Fig. 6) in stressed SC2A cells. We found that under continuous 7{alpha}-OOH pressure, SC2A cells not only died faster than VC (Fig. 2), but death mechanism was dominated by apoptosis, at least at moderate lethality levels. As shown in Fig. 8, there was a progressive increase in the number of cells with apoptotic nuclei during the early stages of 7{alpha}-OOH treatment. At 4.5 h, the ~60% dead SC2A cells were ~65% apoptotic and 35% necrotic, whereas the ~10% dead VC cells were ~85% apoptotic and 15% necrotic. There were still far more dead SC2A cells than VC at 6 h, but by then the majority of the former were necrotic rather than apoptotic, possibly because of greater membrane damage and metabolic impairment (44). The relatively rapid entry of SC2A cells into apoptosis during 7{alpha}-OOH exposure may have been due to the greater susceptibility of their mitochondria to oxidative damage and dysfunction. Exemplifying this was their relatively rapid lipid peroxidation (Fig. 6) and decline of {Delta}{Psi} (Fig. 7), which preceded the earliest indication of any significant apoptosis (Fig. 8).


Figure 8
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  		FIGURE 8.
Relative cell susceptibility to 7{alpha}-OOH-induced apoptosis. Immediately after incubating with 25 µM liposomal 7{alpha}-OOH for the indicated times, SC2A and VC cells were treated with 5 µM Ho258/50 µM PI and examined for apoptotic (bright blue) versus necrotic (red) nuclei by fluorescence microscopy. Images of representative cell populations after various peroxide exposure times are shown. Percentages of apoptotic (A) and necrotic (N) cells are indicated below each panel. Values are the means ± S.E. (n = 8).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Free radical-mediated oxidation of unsaturated lipids in cell membranes and lipoproteins gives rise to numerous oxidized intermediates and products (18, 19). In the case of Ch oxidation, these are collectively referred to as Ch oxides. Among the latter, the epimeric hydroperoxides 7{alpha}-OOH and 7beta-OOH are prominent intermediates whose lifetimes are significantly greater than those of free radical precursors or products (19, 41). We predicted earlier (23, 45) that this attribute along with greater hydrophilicity compared with parent lipids would allow 7-OOHs to depart membranes of origin relatively rapidly and translocate to other membranes. Similar to parental lipid translocation (1–3), this could be fostered by favorable intermembrane concentration gradients or by larger acceptor compartments serving as sinks. Initial supporting evidence came from model membrane studies (23, 24). These showed that a group of photochemically generated ChOOHs translocated spontaneously with a rate constant at least 60 times greater than that of parent Ch, the rate-limiting step being desorption from the donor bilayer. As in the case of Ch itself (1), spontaneous ChOOH transfer was found to occur mainly via aqueous diffusion rather than membrane collision (23). We subsequently showed that three individual ChOOH isomers, including 7{alpha}-OOH, translocate at different rates and that these increase proportionately with degree of hydrophilicity (24). Experiments with a GPx4-deficient cell line (24) revealed that the time-dependent degree of cell killing by SUV-borne 7{alpha}-OOH and two other ChOOHs increased in parallel with their rates of spontaneous transfer uptake, thus demonstrating that transfer-limited cytotoxicity is possible. More recent in vitro studies (25) showed that intermembrane ChOOH transfer could be further accelerated by a recombinant SCP-2, raising the possibility that this protein plays a role not only in the intracellular trafficking of Ch but also potentially damaging ChOOH species. Similarly, SCP-2 further enhanced the intermembrane shuttling of several phospholipid hydroperoxide families (25), suggesting broad range LOOH applicability. We postulated on the basis of these findings that transfer protein-facilitated dissemination of stress-generated LOOHs would greatly expand their range of prooxidant cytotoxic and signaling activity (23–25, 45). In initial testing of direct relevance to the present work, we showed that transfer of SUV-borne 7{alpha}-OOH to isolated mitochondria with the resultant loss of membrane potential was significantly enhanced by SCP-2 (25).

This study is a natural extension of our work with isolated SCP-2 (25) and provides the first known evidence that LOOH cytotoxicity can be enhanced by an endogenous lipid transfer protein. SCP-2-enriched SC2A cells exhibited no obvious difference in morphology or growth rate compared with VC counterparts nor were their levels of protective antioxidants such as GSH, GPx1, and GPx4 any different. It was important to check at least some key antioxidants to be certain that interpretation of our cytotoxicity results would not be complicated by any down-regulation of these. We found that SC2A cells internalized 7{alpha}-OOH more rapidly than VC and were substantially more sensitive to 7{alpha}-OOH-induced killing. On the other hand, SC2A and VC cells were equally sensitive to the non-lipid hydroperoxide t-BuOOH (Fig. 2), suggesting that enhanced cytotoxicity of 7{alpha}-OOH depended on its ability to interact with and be transported by SCP-2 similarly to parent Ch (5–7). It is reasonable to assume that delivery of internalized 7{alpha}-OOH to vital redox-active membrane sites played a key role in the underlying mechanism of SC2A hypersensitivity. At such sites the hydroperoxide could undergo iron-catalyzed one-electron reduction with generation of oxyl and/or epoxyallylic peroxyl radicals (19, 20), which trigger damaging chain peroxidation. In support of this, we showed that 7{alpha}-OOH-challenged SC2A cells (i) accumulated DCFH-detectable ROS more rapidly than VC, (ii) oxidized the lipid-like reporter C11-BODIPY faster, and (iii) effected a faster turnover of internalized [14C]7{alpha}-OOH, as monitored by HPTLC with phosphorimaging detection. The latter was manifested by accumulation rates of 7{alpha}-OOH and its reduction product 7{alpha}-OH, which were substantially lower and higher, respectively, than those for VC (Fig. 4). 7{alpha}-OH derives not only from one-electron but also two-electron reduction of 7{alpha}-OOH (19, 41); therefore, some of it observed in Fig. 4 may have arisen from detoxification rather than toxicity-expanding free radical reactions. How these reactions were proportioned in our system is not known. However, identification of 7-one provides unambiguous evidence for free radical activity, and the higher level of 7-one relative to 7{alpha}-OOH observed in stressed SC2A versus VC cells (Fig. 4) is consistent with more vigorous radical activity in the former. It should be stressed that the Fig. 4 data represent only the turnover fate of internalized [14C]7{alpha}-OOH, the "priming" hydroperoxide. One-electron reduction of this ChOOH in an acceptor compartment with ensuing chain peroxidation would give rise to various endogenous LOOHs, including phospholipid-derived species and Ch-derived 7{alpha}-OOH. These downstream LOOHs might also be translocated by SCP-2, thereby increasing the possibility of peroxidative membrane damage at key subcellular sites. It is noteworthy in this regard that t-BuOOH, which would be expected to give rise to cellular LOOHs via its own one-electron decomposition (46), was not hypertoxic toward SC2A cells. One possible explanation is that t-BuOOH-induced lipid peroxidation was slow compared with its diffusion to vital subcellular sites.

We postulate that binding to SCP-2 sheltered 7{alpha}-OOH from redox degradation, presumably because catalytic iron or detoxifying enzyme access to the peroxide was restricted. Although this remains to be investigated, a recent report indicated that C11-BODIPY is protected against oxidative attack by Cu2+/H2O2-generated hydroxyl radical when bound to SCP-2 (47). Similar redox protection of bound 7{alpha}-OOH or other LOOHs would prolong their lifetimes in transit and also minimize free radical damage to the protein.

The data in Fig. 4 represent a composite of overall internalized [14C]7{alpha}-OOH turnover. We have not yet examined its turnover profile in separated subcellular compartments, e.g. nuclear, cytosolic, and mitochondrial fractions. However, a key observation with clear implications for this is that SC2A mitochondria incorporated [14C]7{alpha}-OOH radioactivity much more rapidly than VC (Fig. 3). The uptake rate in other compartments was the same, suggesting that mitochondria were special targets for SCP-2-mediated hydroperoxide transfer. This is consistent with previous evidence (i) that SCP-2 can enhance 7{alpha}-OOH transfer to isolated mitochondria with damaging consequences and (ii) that cellular mitochondria can harbor significant amounts of SCP-2, presumably for internal lipid trafficking (7, 48). Because mitochondria are iron-rich and highly redox-active, more rapid peroxide delivery to them would explain their greater extent of lipid peroxidation damage and loss of membrane potential in SC2A cells (Figs. 6 and 7). Significant mitochondrial targeting in these cells may account for fact that they showed early signs of dying apoptotically, supposedly via the intrinsic mitochondrial pathway (49).

Immunohistochemical studies have revealed that steady state levels of SCP-2 vary in different tissues, liver, and adrenal cortex, for example, exhibiting much higher amounts than lung and kidney (7, 50, 51). In adrenocortical cells the protein has been reported to play a crucial role in delivering Ch from endoplasmic reticulum and lipid droplet depots to mitochondria for steroid hormone synthesis (48, 52). If oxidatively stressed, such cells might be susceptible to mitochondrial injury due to SCP-2-mediated transfer of ChOOHs along with Ch to mitochondria. Similar reasoning could apply to other cell types expressing relatively high constitutive levels of SCP-2.

In view of the present findings, we have focused up to this point on the prooxidant cytotoxic potential of SCP-2-dependent LOOH translocation. However, one can imagine situations in which LOOH transfer by constitutive SCP-2 could have the opposite effect, i.e. act cytoprotectively by easing peroxidative stress. This might occur, for example, if LOOHs are translocated to compartments whose content of redox-active metal ions is low, but content of antioxidants such as GSH and GPx4 is relatively high, leading to more efficient LOOH detoxification than toxicity enhancement (19). One can predict that this situation would require relatively modest peroxidative pressure and antioxidant-rich compartments that already contain SCP-2 or are readily accessed by it. The existence of SCP-2-facilitated LOOH detoxification and its importance relative to toxicity enhancement under various stress conditions awaits critical examination.

In summary, we have shown for the first time that up-regulation of the low specificity lipid transfer protein SCP-2 puts affected cells at greater risk of lethal injury from LOOHs that can interact with and be translocated by this protein. Mitochondria were found to be special targets of priming hydroperoxide (7{alpha}-OOH) delivery and damaging turnover in SCP-2-overexpressing cells. These findings provide further support for the idea (25, 45) that under stress conditions, which overwhelm detoxification capacity, the cytotoxic and signaling ranges of LOOHs can be greatly expanded by SCP-2-mediated transfer. Whether similar effects can be seen with other intracellular lipid transfer proteins, e.g. those with greater specificity for phospholipid ligands (4, 5), remains to be explored.


   FOOTNOTES
 
* This work was supported by United States Public Health Service, National Institutes of Health Grants CA72630 (to A. W. G.) and GM31651 (to F. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 414-456-8432; Fax: 414-456-6510; E-mail: agirotti{at}mcw.edu.

2 The abbreviations used are: Ch, cholesterol; ChOOH, cholesterol hydroperoxide; CE, cholesteryl ester; 7{alpha}(beta)-OH, cholest-5-ene-3beta, 7{alpha}(beta)-diol; 7{alpha}-OOH, 3beta-hydroxycholest-5-ene-7{alpha}-hydroperoxide; 7-one, 3beta-hydroxycholest-5-ene-7-one; C11-BODIPY, 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a, 4a-diaza-s-inda-cene-3-undecanoic acid; DCF, 2',7'-dichlorofluorescein; DCFH-DA, 2',7'-dichlorofluorescin diacetate; GPx1 and GPx4, glutathione peroxidase isotype 1 and 4; Ho258, Hoechst 33258; HPLC, high performance liquid chromatography; HPTLC, high performance thin layer chromatography; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine iodide; LOOH, lipid hydroperoxide; MTT, 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide; PBS, Chelex-treated phosphate-buffered saline (25 mM sodium phosphate/125 mM NaCl (pH 7.4)); PI, propidium iodide; ROS, reactive oxygen species; SCP-2, sterol carrier protein-2; SUV, small unilamellar vesicle; t-BuOOH, tert-butyl hydroperoxide; VC, vector control. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Charles Baum for kindly providing the clones of SCP-2-overexpressing and vector control hepatoma cells. We also thank Dr. Alexandra Lerch-Gaggl of the Bryant Imaging Core Facility, Department of Cell Biology, Neurobiology, and Anatomy for assisting with the confocal microscopy.



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 DISCUSSION
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