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J. Biol. Chem., Vol. 278, Issue 36, 33928-33935, September 5, 2003
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¶
From the
Liver Unit, Institut de Malalties Digestives, Hospital Clínic y
Provincial, Instituto Investigaciones Biomédicas August Pi Suñer
and the Department of Experimental Pathology,
Instituto Investigaciones Biomédicas Barcelona, Consejo Superior de
Investigaciones Científicas, Barcelona 08036, Spain
Received for publication, October 25, 2002 , and in revised form, May 19, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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The MPT has been shown to function as a key device in the control of cell survival regardless of the phenotype of cell death (2, 6–8). Agents that induce or prevent MPT modulate cell survival (9–11). As a result of mitochondrial membrane permeabilization death-promoting factors are released from the intermembrane space, thereby stimulating the execution phase of the cell death program. Such factors include cytochrome c, which triggers the assembly of the cytochrome c/Apaf-1/procaspase-9 activation complex, called the apoptosome (12, 13); certain inactive caspases precursors (14, 15); heat shock protein 10, which favors apoptosome activation (16); AIF (17) and endonuclease G (18), both of which mediate the cleavage of DNA during caspase-independent cell death; Smac/DIABLO (19, 20) and Omi/HtrA2 (21) that ensure inactivation of caspase inhibitors called inhibitors of apoptosis (IAPs), and recently, pre-processed caspase-9 (22).
Although one of the consequences derived from MPT is the rupture of the outer membrane, some studies have pointed out that outer membrane permeabilization may occur in the absence of disrupted inner membrane (23–26). Indeed there is growing evidence supporting the MPT-independent release of cytochrome c during apoptosis primarily because of the rupture or permeabilization of the mitochondrial outer membrane. Although it remains unclear exactly how mitochondrial outer membrane permeabilization occurs by MPT, large pore opening, or even hyperpolarization subsequent to the closure of VDAC (27), it represents a key event allowing the release of intermembrane proteins, e.g. cytochrome c, that initiate the apoptosome activation leading to cell death.
Many different factors regulate MPT including Ca2+
and ADP levels (28), matrix pH
and 
m (29,
30), mitochondrial energetic
status (31,
32), lipid peroxidation
(33), oxidative stress because
of reactive oxygen species, and/or mitochondrial GSH depletion
(33–36).
In pathological conditions, one or several of these MPT-regulating factors may
contribute to altered susceptibility to MPT. For instance, liver mitochondria
from ethanol-fed rats showed enhanced susceptibility to MPT induced by
different stimuli (37,
38). The underlying mechanisms
for these observations are not completely understood and may include damage to
lipids or proteins, decrease of respiratory chain components, depletion of
mitochondrial GSH, and changes in the composition of mitochondrial lipids.
Recent studies have reported that induction of MPT in rat liver mitochondria was accompanied by an increase in mitochondrial membrane fluidity because of conformational change of pore forming protein(s) during the assembly of the pore (39). Therefore, because a key factor determining the physical properties of membranes is the cholesterol to phospholipid molar ratio, this study was undertaken to examine the influence of mitochondrial membrane dynamics on ANT-mediated MPT and subsequent release of intermembrane proapoptotic factors.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Mitochondria Preparation and Functional Integrity—Rat liver mitochondria were isolated from liver homogenates by differential centrifugation (40). Alternatively, highly purified mitochondria were prepared by rapid centrifugation through Percoll density gradient as described in detail previously (41). Enrichment and recovery of mitochondria were ascertained by the specific activity of succinic dehydrogenase. Mitochondrial purity was confirmed by estimating the contamination with other subcellular organelles such as sinusoidal and canalicular plasma membranes, microsomes and lysosomes assessed by the activity of Na+,K+-ATPase, Mg2+-ATPase, glucose 6-phosphatase, and acid phosphatase, respectively. Integrity was determined by the acceptor control ratio as the ADP-stimulated oxygen consumption over its absence using a Clark oxygen electrode with glutamate/malate or succinate as substrates for respiratory sites for complexes I or II.
Measurement of MPT—Large amplitude swelling was measured spectrophotometrically by recording absorbance at 540 nm. Isolated rat liver mitochondria (1 mg/ml) were suspended in a buffer consisting of 200 mM sucrose, 10 mM Tris-MOPS, 5 mM succinate, 1 mM potassium phosphate, 2 µM rotenone, 1 µg/ml oligomycin, 10 µM EGTA, pH 7.4, at 25 °C as described before (42). Opening of the pore was induced by the ANT ligand, ATR (100 µM), and prevented by preincubation with cyclosporin A (CSA) (5 µM).
Preparation of Mitoplasts—Mitoplasts were prepared as previously described (43). Briefly, to a suspension of 20 mg of mitochondrial protein/ml in 220 mM mannitol, 70 mM sucrose, 2 mM HEPES, and 0.05% BSA, pH 7.4, an equal volume of digitonin (2.4 mg/ml) in the same buffer without BSA was added. After incubation for 15 min at 4 °C, the sample was diluted three times, homogenized gently, and centrifuged for 10 min at 9,500 x g, and the pellet was resuspended in 3 volumes of buffer and centrifuged as before. The resulting supernatants were combined and centrifuged for 1 h at 144,000 x g to obtain the outer mitochondrial membrane. The efficiency of the procedure was verified by monitoring the MAO activity in the low speed pellet (mitoplasts) compared with the high speed pellet (outer membrane) or intact mitochondria from the oxidation of benzylamine (3.3 mM final concentration).
Mitochondrial Loading with Increasing Amounts of Cholesterol— Cholesterol-BSA complex was made by dissolving 50 mg of cholesterol in 5 ml of absolute ethanol as described (44). To the white solution obtained after addition of 5 ml of double distilled water, 2 g of BSA was added adjusting the pH to 7.3 and then centrifuged at 12,000 x g at 4 °C for 10 min. Ten to fifty µl of the cholesterol/BSA mixture was incubated at 4 °C with 50 mg of mitochondria protein for 1 min. Mitochondria were diluted 20 times with cold solution of 0.25 M sucrose, 1 mM EDTA, pH 7.3, and immediately recovered by centrifugation and washed three times, to eliminate excess cholesterol. Parallel control experiments were performed using only BSA. This procedure resulted in cholesterol loading from about 25 to 350% over basal levels.
Cholesterol Determination—The amount of cholesterol incorporated in mitochondria or mitoplasts was measured by high performance liquid chromatography (45). 10 mg of protein was saponified with alcoholic KOH in a 60 °C heating block for 30 min. No cholesterol was detectable in the remnant protein. After the mixture had cooled, 10 ml of hexane and 3 ml of distilled water were added and shaken to ensure complete mixing. Appropriate aliquots of the hexane layer were evaporated under nitrogen and used for cholesterol measurement. High performance liquid chromatography analyses were made using a Waters µBondapak C18 10-µm reversed-phase column (30 cm x 4 mm inner diameter), the mobile phase was 2-propanol/acetonitrile (50:50, v/v) at a flow rate of 1 ml/min.
Fluidization of Mitochondria—Mitochondrial suspension (2 mg/ml) was incubated in 10 mM Tris-HCl, pH 7.4, 150 mM KCl, 1 mM EDTA, with A2C (125 nmol/mg of protein) from a stock solution of 12 mg/ml as described in detail previously (43). The suspension was incubated at 37 °C for 30 min and then washed twice by spinning at 9,500 x g for 10 min.
Measurement of Fluorescence Anisotropy—Fluidity of mitochondrial membranes was evaluated by fluorescence anisotropy of mitochondria-bound dyes, TMA-DPH, DPH, or 9-(anthroyloxy)stearic acid, as described in detail previously (43). Briefly, TMA-DPH or 9-(anthroyloxy)stearic acid (final concentration 3 µM) were injected into stirred mitochondrial suspensions (0.5 mg/ml) and the mixture was incubated for 30 min at 37 °C, whereas DPH (20 mM in tetrahydrofuran) was diluted 100 times with 10 mM Tris-HCl, pH 7.4, 150 mM KCl, 1 mM EDTA. Fluorescence polarization was measured in a Hitachi spectrofluorometer at wavelengths of 366 nm for excitation and 425 nm for emission for TMA-DPH or DPH, and 366 nm excitation and 440 nm emission wavelengths for 9-(anthroyloxy)stearic acid (43). The results are expressed as anisotropy units (r), where r = (I0/I90)/(I0 + 2I90). I0 and I90 represent the intensities of light when polarizers were in parallel or perpendicular orientation, respectively. Correction for light scattering and intrinsic fluorescence were routinely made by subtracting the signal obtained from identical but unlabeled samples and the fluorescence of the buffer plus label alone.
Western Blot Analysis of Cytochrome c, Smac/Diablo, and AIF— Mitochondrial supernatants and pellets were collected by centrifugation at 10,000 x g for 5 min at 4 °C. 20–25 µg of proteins were separated by SDS-PAGE (15% gel) and transferred to nitrocellulose filters. Blots were probed with anti-cytochrome c (mouse monoclonal antibody; clone 7H8.2C12, dilution 1:2000), anti-AIF (rabbit antiserum, dilution 1:2000), and anti-Smac/Diablo (rabbit polyclonal antibody, dilution 1:1 000). After a 1-h incubation with the primary antibody, bound antibodies were visualized using horseradish peroxidase-coupled secondary antibodies and ECL developing kit (Amersham Biosciences). Parallel aliquots were analyzed by immunoblotting for the release of cytochrome oxidase using monoclonal antibody anticytochrome oxidase subunit II to confirm the specificity of mitochondrial proteins release.
Adenine Nucleotide Transport Assay—The adenine nucleotide transport was measured in mitochondria by the atractyloside stop method. The initial rate of [14C]ADP (55 mCi/mmol, Amersham Biosciences) transport (2.5–40 µM) into mitochondria was performed in exchange for endogenous mitochondrial ATP. The assay was initiated by the addition of mitochondrial suspension (30 mg/ml) in a final volume of 766 µl and terminated by addition of 60 nmol of atractyloside followed by addition of excess cold transport buffer (120 mM KCl, 20 mM Tris-HCl, pH 7.4, 1.1 mM MgCl2, and 25 mM sucrose) and subjected to vacuum filtration (Millipore, 0.45 µm) to separate medium from mitochondria. Retained mitochondria in filters were washed twice with 2.5 ml of ice-cold buffer. The filters were dried and placed in 10 ml of Aquasol for scintillation counting.
Electron Microscopy—Two milligrams of mitochondria or mitoplasts were fixed in 3% glutaraldehyde, 10 mM sodium phosphate buffer, pH 7.2, postfixed in 1% osmium tetroxide, dehydrated in graded steps of ethanol through propylene oxide, and embedded in SPURR. Finally, ultrathin sections were stained with uranyl acetate and lead citrate and photographed in a JEOL JEM 1010 electron microscope.
Statistical Analyses—Statistical analyses for comparison of mean values for multiple comparisons between mitochondrial preparations were made by one-way analysis of variance followed by Fisher's test.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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We next examined the effect of cholesterol enrichment on the ability of ATR to cause mitochondrial swelling in relation to membrane fluidity. Cholesterol was incorporated into mitochondria by incubation with a BSA/cholesterol mixture as previously characterized (44). This approach increased cholesterol content over control levels (Figs. 2A and 3). Furthermore, the recovery of total mitochondrial protein matched the initial protein concentration of the mitochondrial suspension before the addition of the cholesterol-BSA complex, indicating that BSA was not bound to mitochondria and that it functions just as a carrier. Control experiments with BSA alone confirmed that BSA did not interfere with the different parameters analyzed (data not shown). To examine the dynamic properties of mitochondrial membranes after cholesterol enrichment, we analyzed the changes in the steady-state fluorescence anisotropy of mitochondria-bound TMA-DPH and DPH. The measurement of fluorescence polarization has been used as a common approach to determine the rotational diffusion freedom of the reported probes with respect to both the rate and the range or extent of the rotational motion (39, 46). The extent of depolarization of the exciting polarized light is a measure of the degree to which a population of photoselected probes lose their original orientation during the lifetime of the excited state (53). Mitochondria with increased cholesterol levels showed higher fluorescence polarization of TMA-DPH, a fluorescence dye incorporated in the membrane surface, compared with control mitochondria (Fig. 2B). Similar results were obtained using DPH, a fluorescence probe that is incorporated underneath the polar region of the bilayer (Fig. 2B). Interestingly, when mitochondria were incubated with the isomer of stearic acid labeled with the 9-anthroyloxy group to monitor fluidity in the core of the lipid bilayer, no changes were observed in the steady-state fluorescence anisotropy from cholesterol-enriched mitochondria compared with control (data not shown). Cholesterol-enriched mitochondria incubated with ATR did not exhibit any significant change in optical density over time (Fig. 2C).
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We next loaded mitochondria with increasing amounts of cholesterol to determine the dependence between cholesterol content, membrane fluidity, and MPT induced by atractyloside. As shown (Fig. 3), fluorescence anisotropy of mitochondria labeled with DPH increased as a function of the amount of cholesterol loaded, whereas the ability of ATR to induce MPT was impaired as reflected in absorbance changes over time after atractyloside exposure. The impairment of mitochondrial swelling in cholesterol-enriched mitochondria was accompanied with a decreased release of the apoptogenic factors, cytochrome c, Smac/DIABLO, and AIF induced by ATR (Fig. 4).
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Because the cholesterol levels between the inner and outer mitochondrial membranes differ, and because ANT is located within the inner membrane (51), we next examined the relative distribution of cholesterol within mitochondrial membranes. Mitoplasts were prepared by selective solubilization of the outer membrane and used to determine the cholesterol levels and the ability of ATR to induce MPT. The removal of the outer membrane was assessed by monitoring MAO activity, an enzyme that localizes specifically in the outer membrane, and by electron microscopy. The MAO activity in low speed pellets containing mitoplasts was 10–15% of that found in intact mitochondria, with an outer/inner membrane ratio around 8.5 for both control and cholesterol-enriched organelles (Fig. 5A). Furthermore, electron microscopy confirmed the almost absence of outer membrane in both control and cholesterol-enriched mitoplasts compared with intact mitochondria (Fig. 5B). As shown, cholesterol levels in mitoplasts were lower than in corresponding intact mitochondria (Fig. 5C). However, the cholesterol content of mitoplasts from cholesterol-enriched mitochondria was higher than in mitoplasts from control mitochondria (Fig. 5C). Interestingly, cholesterol-enriched mitoplasts behaved in a similar fashion as intact mitochondria showing the resistance to ATR-induced swelling (Fig. 5D). Thus, although the main bulk of cholesterol is found in the outer membrane, the cholesterol incorporated in the inner membrane is enough to blunt ATR-induced MPT.
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Measurement of fluorescence anisotropy of membranes labeled with fluorescent probes, e.g. DPH, has been widely used to monitor membrane fluidity changes that corresponds to the transition of membranes from gel to liquid crystalline state induced by cholesterol (46–50). In these prior studies, the range of change in fluorescence anisotropy using DPH was similar to our present findings. For instance mitochondria from ischemic pig hearts exhibited higher mitochondrial cholesterol content (2-fold) and increased DPH fluorescence polarization (16% increase) (47). Moreover, membranes from HEK-OTR and HEK-CCKR cells loaded with cholesterol showed increased (13–16%) fluorescence anisotropy values for DPH (49). Fluorescence polarization of large multilamellar liposomes labeled with DPH increased gradually with increasing amounts of cholesterol (46). Thus taken together these findings indicate that cholesterol loading decreases mitochondrial membrane fluidity and that the function of ANT in MPT requires an appropriate membrane fluidity range.
Moreover, in addition to the mobility of fluorescence probes within membranes the lifetime of the excited state of the probe is another critical factor that can influence membrane fluidity measurements (46, 53). Previous studies using multilamellar liposomes loaded with increasing amounts of cholesterol reported no significant changes in the lifetime of the excited state of DPH (46). Thus, the differences in the steady-state fluorescence anisotropy of cholesterol-enriched mitochondria represent changes in the degree of static orientation constraint of the probe rather than changes in factors that modulate membrane fluidity such as the lifetime of the probe or the diffusion rates of the label.
Cholesterol Impairment of ATR-induced MPT Is Because of Increased Membrane Microviscosity—Having established that cholesterol hampers the ATR-induced MPT, we next examined whether this effect on ANT is mediated by cholesterol per se or through changes in the dynamic properties of mitochondrial membranes. To this end, we used A2C, a fatty acid derivative that intercalates into the lipid bilayer resulting in its fluidization (54). The incubation of cholesterol-enriched mitochondria with this agent fluidized mitochondrial membrane to control values (Fig. 6A). Expectedly this strategy did not affect the cholesterol levels (Fig. 6B). Interestingly, the fluidization of cholesterol-enriched mitochondria by A2C treatment restored the sensitivity of ANT to ATR as shown by the time-dependent optical density loss (Fig. 6C). To examine whether fluidization per se modulates the response to ATR, we analyzed the effect of A2C on control mitochondria. The treatment of control mitochondria with A2C resulted in further fluidization of mitochondria (Fig. 7A), but it did not affect ATR-induced mitochondrial swelling (Fig. 7B). These data establish that the modulation of cholesterol on ATR-induced mitochondrial swelling through ANT is exerted through alteration of membrane fluidity and that a critical fluid environment is necessary for optimal MPT induction by ATR.
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ATR acts on ANT from the cytosolic face of the protein inducing the so
called c-conformation. Because the affinity of ATR for ANT is in the nanomolar
range (51), we would expect a
saturation of the binding sites of ANT at the concentration of ATR used in our
conditions (100 µM), thus minimizing the possibility that the
impairment of ATR by cholesterol was because of perturbation of binding of the
ligand. In addition, the fact that A2C normalizes
cholesterol-enriched mitochondria membrane fluidity and restores the response
to ATR indicate that cholesterol does not play a direct disturbing effect on
ANT. Moreover, the reversing effect of A2C provides additional
evidence to discard changes in the physical parameters of the probes
(e.g. lifetime of excited state) by cholesterol. Other examples for
the regulation of membrane function by appropriate membrane fluidity have been
reported (49,
50). Treatment of HEK cells
with methyl-
-cyclodextrin, a cholesterol-extracting agent, fluidized the
plasma membrane, as probed by DPH fluorescence anisotropy measurements, and
enhanced the activity of 
-secretase ADAM 10
(50).
Effect of Cholesterol on the Adenine Nucleotide Exchange Function of ANT—ANT constitutes the most abundant protein of the mitochondrial inner membrane and in physiological conditions ANT catalyzes the import of cytosolic ADP and the export of matrix ATP (51, 55). Therefore, we next examined the regulation by cholesterol of the adenine nucleotide transport. Freshly isolated rat liver mitochondria contain a physiological ATP concentration of 4–6 nmol/mg of protein, which did not change upon cholesterol loading, and hence we monitored the initial rate of ADP uptake into mitochondria. The uptake of ADP (in exchange for ATP) was linear for up to 1 min (not shown) and thus we examined the rate of ADP transport at three different concentrations of ADP (2.5–40 µM). The initial rate of ADP transport in control mitochondria increased with increasing concentrations of the substrate being similar to the reported Vmax for ADP transport in rat liver mitochondria by ANT (56–59). Interestingly, the rate of ADP transport was not altered in mitochondria loaded with different amounts of cholesterol (Fig. 8) despite the fact that cholesterol dose dependently decreased membrane fluidity monitored by DPH fluorescence anisotropy (Fig. 3). Thus in contrast to the effect on mitochondrial swelling induced by ATR cholesterol loading does not perturb the ADT/ATP exchange function of ANT, thus establishing a divergent regulation of ANT by cholesterol-mediated changes in membrane fluidity.
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The insensitivity of the adenine nucleotide exchange to conditions that alter mitochondrial membrane fluidity has been shown previously. Mitochondria from chronic alcohol-fed rat livers have decreased membrane fluidity because of altered mitochondrial lipid composition and cholesterol deposition (43, 60–62); the DPH fluorescence anisotropy values reported in these membranes were similar to the range we observed in our present observations. Consistent with the current findings the adenine nucleotide exchange in mitochondria from chronic alcohol-fed rats was unaltered (43, 63, 64), confirming that this transport function of mitochondria is not regulated by membrane fluidity.
An alternative possibility to the divergent regulation of ANT by cholesterol loading would imply that the impairment seen in the ATR-induced MPT could have been mediated by the effect of cholesterol on other putative MPT components that interact with ANT. There is considerable evidence supporting a role of ANT as a permeability transition pore component, although the precise molecular composition and regulation of permeability transition pore remain poorly understood (65). Current models indicate the interaction and association of ANT with outer mitochondrial membrane proteins including the VDAC and Bax (3–5, 66). In this regard, we observed that the release from mitochondria of cytochrome c, AIF, and Smac/Diablo induced by xanthine/xanthine oxidase-mediated superoxide anion generation, which has been shown to target VDAC (67), was independent of cholesterol loading and prevented by anti-VDAC antibody (25).2 Thus, our data indicate a divergent sensitivity of ANT to membrane dynamics depending on its mode of action.
Concluding Remarks—The present study has relied on signature changes of MPT. Onset of MPT has a major consequence in the permeabilization of the inner mitochondrial membrane to small solutes. Thus, in addition to the dissipation of ion or metabolite gradient across the inner membrane, MPT results in a colloidal osmotic pressure that causes massive swelling, outer membrane rupture, and release of intermembrane proteins. The sensitivity to CSA stands as another important feature of regulated MPT (65). By monitoring these features we provide evidence that cholesterol deposition in mitochondria impairs the ATR-induced mitochondrial swelling and release of proapoptotic proteins that occur in control organelles. These dramatic changes induced by cholesterol are mediated by perturbation in mitochondrial membrane fluidity and not by cholesterol per se suggesting that an appropriate membrane fluidity range is needed for ATR to induce the conformational change in ANT that mediates MPT. An intriguing finding is that the ADP/ATP exchange function of ANT is insensitive to decreased mitochondrial membrane fluidity induced by cholesterol deposition. Accordingly these separated functions of ANT can be regulated independently and respond to cholesterol deposition in divergent ways. Consistent with these observations similar suggestions have been described for Bax-mediated regulation of ANT (68). The Bax-mediated inhibition of the ADP/ATP exchange was dissociated from Bax-stimulated formation of pores by ANT.
Being that MPT is an important gateway to cell death pathways, these observations may have important consequences in the control of cell death. According to the present findings mitochondrial cholesterol impairs the proapoptotic function of ANT without compromising the ADP/ATP exchange. Because stimulated ADP/ATP exchange has been shown to contribute to cell survival (69), our results imply that the divergent regulation of the two functions of ANT by cholesterol-mediated alteration in membrane fluidity may provide an advantage for cell survival. We would speculate that these conditions existing in certain tumor cells, e.g. Yoshida ascites and Morris hepatoma cells whose mitochondrial cholesterol levels are greater than in normal tissue (70, 71), may determine resistance to apoptosis stimuli that work through ANT.
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FOOTNOTES |
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Contributed equally to the results of this work.
¶ To whom correspondence should be addressed: Liver Unit, Institut de Malalties Digestives, Hospital Clínic i Provincial. C/Villarroel 170., 08036 Barcelona, Spain. Tel.: 34-93-227-5709; Fax: 34-93-451-5272; E-mail: checa229{at}yahoo.com.
1 The abbreviations used are: MPT, mitochondrial permeability transition;
A2C,
2-(2-methoxyethoxy)ethyl-8-(cis-2-n-octylcyclopropyl)octane; ANT,
adenine nucleotide translocator; AIF, apoptosis inducing factor; ATR,
atractyloside; CSA, cyclosporin A; DPH, 1,6-diphenyl-1,3,5-hexatriene; MAO,
monoamine oxidase; PBR, peripheral benzodiazepine receptor; TMA-DPH,
trimethylammonium 1,6-diphenyl-1,3,5-hexatriene; VDAC, voltage-dependent anion
channel; BSA, bovine serum albumin; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MOPS,
4-morpholinepropanesulfonic acid.
2 A. Colell, C. García-Ruiz, and J. C. Fernández-Checa,
unpublished observations.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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