Toxicology and Applied Pharmacology 194 (2004) 101–110 www.elsevier.com/locate/ytaap

Cocaine induces a mixed lysosomal lipidosis in cultured fibroblasts, by inactivation of and inhibition of A1$

Marie-Ce´cile Nassogne,a Chantal Lizarraga,a Francisca N’Kuli,a Francßoise Van Bambeke,b Roger Van Binst,c Pierre Wallemacq,c Paul M. Tulkens,b Marie-Paule Mingeot-Leclercq,b Thierry Levade,d and Pierre J. Courtoya,*

a Cell Biology Unit, Christian de Duve Institute of Cellular Pathology, Louvain University Medical School, B-1200 Brussels, Belgium b Unite´ de Pharmacologie cellulaire et mole´culaire, Universite´ catholique de Louvain, B-1200 Brussels, Belgium c Toxicology Laboratory, Clinical Biology Department, Saint-Luc University Hospital, B-1200 Brussels, Belgium d Biochemistry Laboratory, INSERM U 466, Louis Bugnard Institute, CHU Rangueil, Toulouse, France Received 16 July 2003; accepted 6 September 2003

Abstract

This paper reports that cocaine may induce a lysosomal storage disorder. Indeed, culture of Rat-1 fibroblasts with 250–500 AM cocaine induced after 2–3 days a major accumulation in lysosomes of electron-dense lamellar structures. By subcellular fractionation, this was reflected by a selective decrease of the buoyant density of several lysosomal , indicating lysosomal lipid overload. Biochemical analysis confirmed an increased cellular content of major phospholipids and sphingomyelin, but not of cholesterol. Cocaine, a membrane- permeant weak base, is concentrated by acidotropic sequestration, because its accumulation was abrogated by the proton ionophore, monensin and the vacuolar ATPase inhibitor, bafilomycin A1. At its estimated lysosomal concentration, cocaine almost completely inhibited phospholipase A1 activity on liposomes. Cell incubation with cocaine, but not with its inactive metabolite, benzoylecgonine, rapidly inactivated acid sphingomyelinase, as reflected by a 10-fold decrease in Vmax with identical Km. Acid sphingomyelinase inactivation was fully prevented by the thiol proteinases inhibitors, leupeptin and E64, indicating that cocaine induces selective sphingomyelinase proteolysis. Upon cocaine removal, acid sphingomyelinase activity was rapidly restored, pointing to its fast turnover. In contrast, the cellular content of several other lysosomal was increased up to 2-fold. Together, these data show that acidotropic accumulation of cocaine in lysosomes rapidly inhibits acid phospholipase A1 and inactivates acid sphingomyelinase, which can explain induction of a mixed lysosomal lipidosis. D 2003 Elsevier Inc. All rights reserved.

Keywords: Cocaine; Lysosomes; Phospholipidosis

Introduction variety of pharmacological agents, which include antipsy- chotic (e.g. chlorpromazine), antidepressant (imipramine Lysosomal phospholipidosis is a well-known cellular and desipramine), antiarrhythmic (amiodarone), antibacteri- disorder induced by an array of drugs belonging to a large al (aminoglycosides and macrolides), antimalarial (chloro- quine) and anorectic agents (chlorphentermine) (Kodavanti Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; FCS, and Mehendale, 1990). fetal calf serum. Most of these agents share a hydrophobic ring structure $ This work was supported by Grants from FNRS, IUAP, ARC and and a protonable nitrogen, with the notable exception of Re´gion Wallonne (Belgium). aminoglycosides, which are polybasic, hydrophilic com- * Corresponding author. Cell Biology Unit, ICP/UCL 7541, Christian pounds. Hydrophobic properties allow permeation of their de Duve Institute of Cellular Pathology, Louvain University Medical School, avenue Hippocrate, 75, B-1200 Brussels, Belgium. Fax: +32-2- non-protonated form across biological membranes, whereas 764-75-43. nitrogen protonation leads to sequestration into acid- E-mail address: [email protected] (P.J. Courtoy). bounded organelles, typically resulting in 2–3 logs higher

0041-008X/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2003.09.026 102 M.-C. Nassogne et al. / Toxicology and Applied Pharmacology 194 (2004) 101–110 concentration in lysosomes than in the extracellular medium ogies) under 8% CO2, at pH close to 7.2. For the experi- (de Duve et al., 1974). For non-permeable agents bearing ments, cells were seeded on 9.6 cm2 (biochemistry) or 78.5 several amino groups, such as aminoglycosides, drug uptake cm2 (electron microscopy) plastic Petri dishes (Lab-Tek, is secondary to absorptive endocytosis followed by vesicular Life Technologies, Nunc, Roskilde, Denmark). Cocaine, transfer to, and retention within, lysosomes (Just and Hab- benzoylecgonine and desipramine, freshly dissolved at ermann, 1977; Moestrup et al., 1995). Owing to their high 100–500 AM in medium, were added to cultures for 2 h to concentration in lysosomes, cationic drugs may reversibly 5 days, and the medium was replaced daily. inhibit or irreversibly inactivate acid and sphingomyelinases (Xia et al., 2000). In addition, binding Microscopy. Control or cocaine-treated fibroblasts were of drugs such as macrolides and aminoglycosides to phos- rapidly rinsed with PBS–Ca2+ (137 mM NaCl, 5.4 mM pholipids may shield the phospholipase substrate or nega- KCl, 0.34 mM Na2HPO4,0.44mMKH2PO4,3.6mM tively charged phospholipids required for optimal lysosomal CaCl2 and 0.74 mM MgSO4, pH 7.4), fixed in 2% (v/v) activity, resulting into inhibition by substrate depletion or glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 12 h at charge neutralization, respectively (Hostetler and Jellison, 4j C, and washed again thrice for 5 min each with 0.1 M 1990; Mingeot-Leclercq et al., 1988). sodium cacodylate buffer, pH 7.4. Cells were processed and In recent studies, we described that cocaine led to examined as previously described (Nassogne et al., 1997). neuronal apoptosis in a model of mouse embryonic cerebral cells co-culture after 2–3 days, without affecting survival of Lipid extraction and analysis. After scraping, cell pellets glial cells (Nassogne et al., 1995, 1997).Thedelayof were suspended in distilled water and lipids were extracted apoptosis suggested a metabolic process. In the course of according to Folch et al. (1957) with some modifications: these experiments, we noticed by electron microscopy of cell 800 Al of a lysate containing 0.5 mg/ml of cell protein was pellets the frequent occurrence of myelinic figures sugges- extracted with 3 ml of chloroform–methanol (2:1; v/v), tive of lysosomal lipid storage disorders. This prompted us vortex-mixed, and centrifuged at 1500 Â g for 10 min. The to study whether cocaine could be ranked among drugs organic phase was washed with 2 ml of 50 mM NaCl, then producing phospholipidosis, and to address the underlying with 4 ml of a 1:1 mixture of 360 mM CaCl2 in water and mechanisms. methanol. After addition of 50 Al of 10% Triton X-100 in For these investigations, we have selected cultured fibro- acetone (w/v), the liquid phase was extracted under nitrogen blasts as an experimental system where genetic lysosomal and the pellet was dissolved in 100 Al of water for total lipid disorders (Neufeld et al., 1977) and drug-induced phospho- phosphorus assay (Rouser et al., 1970). Individual phos- lipidosis (Aubert-Tulkens et al., 1979; Montenez et al., pholipids were separated by thin-layer chromatography, 1999) have been extensively studied, and for which frac- with one development with chloroform/methanol/ammo- tionation procedures to study lysosomal main properties and nia/water (24:16:2:1; v/v) for phosphatidylethanolamine functions are well established (Draye et al., 1987, 1988; and sphingomyelin and two developments with chloro- Tulkens et al., 1974). form/methanol/acetic acid/water (65:50:1:4; v/v) for phos- phatidylinositol, phosphatidylserine and phosophatidylcho- line. The plates were exposed to iodine vapour and the spots Methods identified by comparison with standards run in parallel. The silica gel was scraped off the plates and inorganic phos- Chemicals. Cocaine hydrochloride was obtained from St- phorus was assayed by the method of Bartlett (1959) after Luc University Hospital Central Pharmacy. Benzoylecgo- hydrolysis in 60% perchloric acid (v/v) at 210jC for 90 nine, a major cocaine metabolite, was a generous gift from min. the National Institute of Drug Abuse (NIDA, Bethesda, MD). Bafilomycin A1, monensin and desipramine were Subcellular fractionation. Cells were scraped with a rub- from by Sigma (St. Louis, MO); leupeptin and E64 were ber policeman, then homogenized in 4 ml of 250 mM from Boehringer (Mannheim, Germany). These inhibitors sucrose containing 1 mM EDTA and 3 mM imidazole, pH were all dissolved in DMSO at 1000-fold concentrated stock 7.0. Postnuclear particles were isolated by differential cen- solutions. None of these drugs significantly modified the trifugation as described (Draye et al., 1987), resuspended in total cell protein content. 1 ml and layered on the top of 11.5 ml linear sucrose gradients (from 1.1 to 1.3 g/ml in density), containing 1 mM Cell culture. All experiments were carried out using the EDTA and 3 mM imidazole/HCl, pH 7.0. After equilibration established cell line, Rat-1 fibroblasts (Veithen et al., 1996). at 70,000 Â g for 18 h in a SW40 swinging rotor (Beckman, Cells were cultured in Dulbecco’s modified Eagle’s medium Palo Alto, CA), 12 fractions of 1 ml were collected from the (DMEM; Life Technologies, Grand Island, NY) supple- tube bottom, weighed and analyzed for density, protein mented by 5 mM glucose, 4 mM glutamine, 10 mM content and marker enzymes. Postnuclear particles NaHCO3, 10 mM Hepes, 100 Ag/ml streptomycin, 66 Ag/ contained on the average 34% of total cell protein (Lowry ml penicillin and 10% fetal calf serum (FCS; Life Technol- assay, using BSA as standard; Lowry et al., 1951), together M.-C. Nassogne et al. / Toxicology and Applied Pharmacology 194 (2004) 101–110 103 with 79% of the homogenate for N-acetyl-h-hexosamini- choline (Amersham, Buckinghamshire, UK) incorporated dase, 77% of cathepsin B and 61% of acid as in small unilamellar liposomes (140 mCi/mol of phospha- marker for lysosomes (for assay conditions, see Cornillie et tidylcholine; total lipid concentration, 15.7 mM) and de- al., 1991), as well as 82% for alkaline I fined as the rate of substrate conversion into [14C]-h- and 94% of 5V- as marker of combined plasma as described (Mingeot-Leclercq et membrane and endosomes (assayed as described in Draye et al., 1988). al., 1987). There was no appreciable difference between control and cocaine-treated cells. Recoveries after differen- Determination of acid sphingomyelinase activity in tial sedimentation of the six analyzed constituents in two vitro. After culture, cell pellets were suspended in distilled control and two cocaine-treated cell samples were on the water and sonicated (3 Â 15 s). Cell lysates were incubated average 91 F 11% (mean F SD, n = 24). Frequency/density at 37j C for the indicated periods with a liposomal suspen- histograms are presented after normalization as described sion of 14C-choline-labelled sphingomyelin (Amersham) in previously (Leighton et al., 1968). 250 mM sodium acetate and 5 mM EDTA, pH 5.0, under previously reported conditions (Levade et al., 1983). The Cocaine assay. After incubation with 500 AM cocaine for liberated water-soluble product, 14C-phosphocholine, was 30 min, cells (f1 mg cell protein) were rapidly washed isolated by phase partitioning upon addition of chloroform with PBS (3 Â 10 s), lysed in 1.5 ml 0.01% Triton X-100 and methanol, and quantified by scintillation counting. and frozen at À20j C. One milliliter of cell lysate was extracted with 5 ml of dichloromethane/dichloroethane/ Statistical analysis. Individual comparisons in Figs. 5 and heptane/isopropanol (2.6:4.8:6.5:1; v/v). After extracts 6 were tested using Student’s t test. For multiple compar- were adjusted to pH 9.2 with 1 ml of 0.1 M carbonate/ isons in Table 1, Dunnett’s test for one-way analysis of bicarbonate buffer, cocaine was extracted by organic sol- variance (ANOVA) was used. Results were regarded as vents, then analyzed by gas chromatography coupled to statistically significant at P < 0.05, denoted *; NS, not mass spectrometry, using a quadripole GC-MS HP5890 significant. equipment, series 11 (Hewlett Packard, Palo Alto, CA). Cocaine content in the cell lysates was around 2 Ag/ml and normalized for each lysate to the protein content (about 0.8 Results mg/ml). This assay was validated for linearity, sensitivity and interference. Measurements of cocaine diluted in PBS Morphological evidence that cocaine induces a lysosomal or added to a lysate of untreated cells at 0.8 mg protein/ml storage disease were linear between 0.5 and 8 Ag cocaine/ml and were not affected by cellular protein. When cells were further Upon incubation of Rat-1 fibroblasts with 500 AM treated with monensin or bafilomycin A1, values in cell cocaine, numerous dark cytoplasmic granules started extracts were close to the lower limit of sensitivity of the appearing after a few days, as seen with the light micro- assay. scope. By electron microscopy, inclusions corresponded to distorted structures surrounded by a single membrane and Determination of phospholipase A1 activity. Phospholi- filled with heterogeneous, sometimes intensively osmio- pase A1 activity was assayed using purified lysosomal philic material (Fig. 1B). At higher magnification, electron- extracts toward 1-palmitoyl,2-[1-14C]-oleoylphosphatidyl- dense material revealed lamellar concentric structures, sug-

Table 1 Effect of cocaine exposure on cell lipid content Lipids (nmol/mg protein) Treatment Control Cocaine Benzoylecgonine Desipramine Total phospholipids 218.2 F 1.7 253.7 F 11.7* 210.7 F 0.6 327.3 F 6.9* Phosphatidylcholine 81.5 F 9.5 96.6 F 4.7* 84.3 F 0.3 137.1 F 2.8* Phosphatidylserine 45.8 F 1.4 54.5 F 3.8* 43.9 F 2.1 53.4 F 1.7* Phosphatidylethanolamine 33.3 F 3.6 49.1 F 1.8* 35.4 F 3.6 62.5 F 2.0* Sphingomyelin 24.8 F 0.1 33.8 F 2.0* 24.4 F 1.6 41.0 F 0.7* Phosphatidylinositol 17.0 F 0.7 19.1 F 0.2 14.0 F 0.8* 34.4 F 1.5* Lysophosphatidylcholine 6.8 F 0.3 5.7 F 0.7 6.2 F 0.4 6.7 F 0.4 Cholesterol 86.0 F 16.0 70.0 F 4.0 NT NT Fibroblasts were cultured for 5 days in daily renewed DMEM supplemented by 10% FCS without any drug, or with 500 AM cocaine, benzoylecgonine or desipramine. Results are means F SD values (n = 3 dishes). This experiment was repeated twice with similar results. NT, not tested. Comparison of each treatment with control was performed by Dunnett’s test for one-way ANOVA, except for last row (comparison of two means by t test). *P < 0.05. 104 M.-C. Nassogne et al. / Toxicology and Applied Pharmacology 194 (2004) 101–110

Fig. 1. Subcellular alterations induced by cocaine. Rat-1 fibroblasts were cultured for 4 days in daily renewed DMEM supplemented by 10% FCS without (A) or with 500 AM cocaine (B, C), fixed, pelleted and processed for electron microscopy. Notice in cocaine-treated cells cytoplasmic inclusions (B) that correspond to enlarged lysosomes filled with osmiophilic material, usually organized in concentric lamellar structures (C). (A, B) Â10,000; (C) Â39,000. gesting membrane stacking, the so-called ‘‘myelinic bodies’’ Lipids accumulate in lysosomes (Fig. 1C). All other organelles had a normal appearance and there was no induction of apoptosis, in contrast to Subcellular fractionation by isopycnic centrifugation in embryonic mouse neurons that were treated similarly sucrose density gradients allows one to determine in which (Nassogne et al., 1997). This is not surprising because type of organelle lipids accumulate, due to a selective fibroblasts are much less prone to apoptosis than embry- decrease of equilibrium density, and is particularly suited onic neurons. These myelinic bodies were never seen in to evidence lysosomal lipid overload (Aubert-Tulkens et al., untreated cells (Fig. 1A) nor in cells treated with benzoy- 1979; Limet et al., 1985). Upon exposure to 500 AM cocaine lecgonine, a major inactive metabolite of cocaine (data not for 6 days, three lysosomal marker enzymes (N-acetyl-h- shown). hexosaminidase, cathepsin B and ) con- comitantly showed a major shift of median densities, from Cocaine produces cellular accumulation of phospholipids 1.21 g/ml in control cells to 1.16 g/ml in cocaine treated- and sphingomyelin cells (Fig. 2). In contrast, the median density of total protein, as well as that of 5V-nucleotidase and alkaline phosphodies- Because electron microscopy suggested a lysosomal lipid terase type I, two classical plasma membrane marker accumulation, phospholipid content was measured in fibro- enzymes, was not appreciably altered. The selective de- blasts exposed to 500 AM cocaine for 5 days. Table 1 shows crease of the buoyant density of lysosomes thus confirmed the comparison with the effects of 500 AM desipramine, an that lipids accumulated in these organelles. established phospholipidosis-inducing agent (Ruben et al., 1991). The pattern of biochemical alterations induced by Cocaine rapidly concentrates into cells by acidotropic cocaine was qualitatively comparable to that with desipra- sequestration mine, although less severe. Cocaine treatment produced a f20% increase of total cell phospholipid content, including Cocaine uptake was next measured by gas chromatog- phosphatidylcholine, phosphatidylserine and particularly raphy coupled to mass spectrometry (for validation, see phosphatidylethanolamine, as well as sphingomyelin. In Methods) and its mechanism of uptake was examined contrast, phosphatidylinosidol, lysophosphatidylcholine using agents known to abolish lysosomal acidification. and cholesterol content was similar in control and treated Monensin, a Na+/H+ ionophore, dissipates proton gradient cells. As a negative control, 500 AM benzoylecgonine across membranes (Tartakoff, 1983); bafilomycin A1 is a slightly decreased the phosphatidylinositol content and did specific inhibitor of vacuolar-type H+-ATPase (Bowman et not affect the other constituents tested. al., 1988). When control fibroblasts were incubated with M.-C. Nassogne et al. / Toxicology and Applied Pharmacology 194 (2004) 101–110 105

Cocaine

Cocaine + monensin

Cocaine + bafilomycinA1

0.0 0.5 1.0 1.5 2.0 2.5 3.0 µg cocaine/mg cell protein

Fig. 3. Inhibition of lysosomal cocaine accumulation by monensin and bafilomycin A1. Rat-1 fibroblasts were pretreated or not with 25 AM monensin or 1 AM bafilomycin A1 for 30 min, then 500 AM cocaine was added. After 30 min, cells were rapidly washed three times with PBS– Ca2+, scraped and rapidly frozen in liquid nitrogen. After extraction, cell- associated cocaine was measured by gas chromatography coupled to mass spectrometry. Results are means F SD of three dishes. The experiment was repeated twice with similar results.

while that of acid sphingomyelinase was greatly decreased (see below). By contrast, no change of activity was observed for the two plasma membrane marker enzymes, 5V-nucleo- Fig. 2. Density shift of lysosomes upon cocaine exposure. After cells tidase and alkaline phosphodiesterase type I, nor for the were incubated for 6 days in daily renewed medium without (thin lines) cytosolic , phosphoglucomutase. or with 500 AM cocaine (thick lines), postnuclear particles were equilibrated in linear sucrose gradients; protein and the indicated marker enzymes were measured in the 14 collected fractions. Abscissa is the density span of the gradients between 1.05 and 1.27 g/ml divided in 14 100 sections of equal density increment (DRho). Ordinate is the relative frequency (DQ/DRho, where DQ is the enzymatic activity expressed as percentage of postnuclear particles, in each gradient section). Triangles represent median densities in the absence (open symbols) or presence of 75 cocaine (filled symbols). This experiment was repeated once with similar results.

50

500 AM cocaine for 30 min, cocaine concentration reached A1 activity

(%ofcontrol) 2.34 F 0.29 Ag/mg cell protein (mean F SD, n =3), suggesting a f100-fold concentration in lysosomes (see 25

Discussion). This accumulation was abrogated by either Phospho monensin or bafilomycin A1, demonstrating acidotropic sequestration of cocaine (Fig. 3). 0

Cocaine differentially affects the cellular content of 0 5 10 15 lysosomal hydrolases Cocaine (mM)

The activity of phospholipase A1, but also that of cathep- Fig. 4. Effect of cocaine on activity of phospholipase A1. The activity of sin B and N-acetyl-h-hexosaminidase, showed a time- and phospholipase A1 from purified lysosomes was assayed on 14C- concentration-dependent increase in cells treated with co- phosphatidylcholine incorporated into negatively charged liposomes in A the presence of the indicated cocaine concentrations. Results are expressed caine (reaching after 3 days of exposure to 500 M cocaine, as percentage of the activity measured in the absence of the drug and are 145%, 220% and 169% of untreated cells, respectively). The means F SD of three independent experiments. Error bars that are not activity of acid phosphatase was not significantly affected visible are included in the symbols. 106 M.-C. Nassogne et al. / Toxicology and Applied Pharmacology 194 (2004) 101–110

Cocaine inhibits phospholipase A1 activity Cocaine selectively inactivates acid sphingomyelinase

Because the half-life of phosphatidylcholine depends Sphingomyelin accumulation in cells exposed to cocaine for two-thirds on lysosomal degradation by phospholipase could be directly ascribed to a loss of acid sphingomyelinase A1 (Fowler and de Duve, 1969; Laurent et al., 1982),it in cell lysates. Acid sphingomyelinase activity showed a may seem surprising that phosphatidylcholine would ac- concentration-dependent decrease, down to f20% of con- cumulate in lysosomes of cells where total activity (mea- trols after 24 h culture with 500 AM cocaine (Fig. 5A). The sured in diluted cell extracts) actually increased upon loss of acid sphingomyelinase activity was independent of cocaine treatment. This apparent paradox might be the dilution of the sample, excluding interference by cocaine explained if phospholipase A1 would be inhibited in situ, in the assay conditions, and demonstrating instead enzyme due to the high concentration of cocaine in lysosomes, but inactivation. This occurred remarkably fast, within 1 h of not in vitro, due to considerable dilution of the drug in incubation with cocaine (Fig. 5B); conversely, acid sphin- cell lysates (see Discussion). The inhibitory potency of gomyelinase activity was fully restored within 16 h follow- cocaine toward this enzyme was accordingly investigated ing cocaine withdrawal, suggesting both a fast efflux of in vitro, using small unilamellar, negatively charged lip- cocaine and a rapid turnover of the enzyme (t1/2 f8 h). osomes bearing radiolabelled phosphatidylcholine. As Inactivation was confirmed by the analysis of kinetic shown in Fig. 4, cocaine produced a concentration-depen- properties of the enzyme (Fig. 5C): Vmax was significantly dent inhibition of phospholipase A1 activity, approaching decreased by 10-fold, whereas Km was unaffected, at f1.4 100% inhibition above 5 mM, a value that is still about 1/ mM. Inactivation of acid sphingomyelinase was fully pre- 10 of the estimated drug concentration in lysosomes (see vented by pretreatment with the protease inhibitors, E64 and Discussion). leupeptin, indicating that cocaine accelerated acid sphingo-

Fig. 5. Time- and concentration-dependent reversible loss of acid sphingomyelinase activity induced by cocaine. Fibroblasts were cultured for 24 h in DMEM supplemented by 10% FCS with the indicated cocaine concentrations (A) or for the indicated times with 500 AM cocaine (filled circles at B). Alternatively, fibroblasts were cultured for 2 h in DMEM supplemented by 10% FCS with 500 AM cocaine, then in cocaine-free medium for indicated times (open squares in B). Sphingomyelinase activity was assessed in lysates and expressed as percentage of untreated cells. (C) The sphingomyelinase activity in lysates of untreated (open symbols) or cocaine-treated cells (filled symbols; 500 AM for 3 h) was assayed at increasing substrate concentrations and converted as Lineweaver–Burk plots. Data are averages of duplicates, with <5% variation. This experiment was reproduced thrice with similar results. M.-C. Nassogne et al. / Toxicology and Applied Pharmacology 194 (2004) 101–110 107

accumulation. The cationic character of cocaine is probably responsible for the lysosomal accumulation of cocaine that leads to the induction of a mixed lipidosis. In contrast, the major zwitterionic cocaine metabolite, benzoylecgonine (pKa 10.82 and 3.49), was found to have lost by the same token lysosomal toxicity. Acidotropic sequestration results in considerable cocaine concentration in lysosomes. In our experiments, cocaine was used at 500 AM (160 Ag/l) and its measured cellular accumulation after 30 min reached 2.3 Ag/mg protein, that is, 1.5 mM, based on an estimated cellular protein concen- tration of 200 mg/ml. The lysosomal volume in fibroblasts was not detectably modified after 30 min of cocaine uptake and represents 3.3% of the cell volume (Draye et al., 1988). Thus, cocaine concentration in lysosomes is close to 50 mM, that is, f100-fold higher than the concentration introduced in the culture medium. This is entirely consistent with the estimated lysosomal pH of f5.2, that is a 100-fold higher Fig. 6. Effect of proteinase inhibitors on the cocaine-induced loss of acid proton concentration than in culture medium (Ohkuma and sphingomyelinase activity. After fibroblasts were incubated without or with Poole, 1978). In turn, high concentration of a weak base in 40 AM leupeptin or 10 Ag/ml E64 for 16–20 h, 500 AM cocaine was added lysosomes should not only compromise lysosomal acidifi- for the last 2 h, where indicated. Sphingomyelinase activity was assessed on cell lysates. For comparison, lack of effect of 500 AM benzoylecgonine is cation, thereby impairing in situ activity of acid hydrolases, obvious. Statistical significance: cocaine versus control. Other treatments but might also directly inhibit their catalytic site, or affect versus control: NS. their stability (Maxfield, 1982; Wibo and Poole, 1974). The accumulation of lipids upon cocaine exposure can be ascribed to at least two different effects, namely inhibition myelinase degradation by a thiol protease, presumably of acid phospholipase A1 in situ and inactivation of acid within lysosomes. Cell incubation with the inactive cocaine sphingomyelinase. In an in vitro phospholipase A1 assay on metabolite, benzoylecgonine, had no effect on sphingomye- liposomes, complete inhibition of lysosomal extracts was linase activity (Fig. 6). achieved at much lower cocaine concentrations than calcu- lated above for lysosomes. Phospholipase A1 has been shown to degrade both phosphatidylethanolamine and phos- Discussion phatidylcholine and therefore its inhibition may directly explain the concomitant accumulation of various phospho- The present study demonstrates that cocaine undergoes lipids, but not of phosphatidylinositol, which is well known acidotropic accumulation and may induce a typical lyso- as poor substrate for this enzyme. somal lipid overload, like several other cationic membrane- How do cationic drugs inhibit lysosomal phospholipase permeant drugs (Halliwell, 1997; Kodavanti and Mehen- activity remains debated (for a review, see Reasor and dale, 1990; Lu¨llmann-Rauch, 1979). Morphological and Kacew, 2001). Two mechanisms have been proposed: biochemical hallmarks of cocaine-induced lipidosis we binding of the drug to hydrophilic and/or hydrophobic evidenced in cultured fibroblasts include (i) accumulation moieties of phospholipids, resulting in complexes that are of typical electron-dense material and lamellar inclusions indigestible by phospholipases, or direct inhibition of phos- without structural alteration of other cytoplasmic organelles, pholipase by the drug. Consistent with the first mechanism, as shown by electron microscopy; (ii) cellular accumulation a non-specific weak binding of cocaine to lipids has been of phospholipids and sphingomyelin, as measured by bio- reported (Bailey, 1998). Second, the activity of lysosomal chemistry; and (iii) a major and selective shift of the phospholipase A1 toward phophatidylcholine critically equilibrium density of lysosomes toward more buoyant depends on the negative charge of the bilayer and increases fractions, as demonstrated by subcellular fractionation. Such with the content in acidic phospholipids (Mingeot-Leclercq a density shift has already been reported to reflect lysosomal et al., 1988). Third, local anesthetics may induce phospho- phospholipidosis induced by gentamicin in cultured fibro- lipidosis and this effect correlates with their differential blasts (Aubert-Tulkens et al., 1979) and proximal tubular affinities toward polar lipids (Ja¨gel and Lu¨llmann-Rauch, cells in vivo (Giurgea-Marion et al., 1986). 1984). Neutralization of acidic compartments by either dissipa- We noticed, however, that the total activity of phospho- tion of transmembrane proton gradients or inhibition of lipase A1 in cell lysates was actually increased upon vacuolar ATPase abrogated cocaine uptake by fibroblasts, prolonged cocaine incubation. Although this may seem showing that acidotropic sequestration accounts for cocaine paradoxical, such an increase has already been reported 108 M.-C. Nassogne et al. / Toxicology and Applied Pharmacology 194 (2004) 101–110 for related conditions. For example, phospholipidosis in- myelinase activity was also decreased and inhibitors of duced by the acidotropic accumulation of the macrolide cysteine proteinases exerted a protective effect (Montenez antibiotic, azithromycin, was accompanied by a selective et al., 1994a,b). Similarly, cell treatment with the antide- increase of total phospholipase A1 activity (Gerbaux et al., pressant, desipramine, induced a loss of sphingomyelinase 1996). The explanation proposed was that (i) the drug activity that was prevented by leupeptin (Hurwitz et al., reversibly inhibits the activity of phospholipases in the cells 1994). Several mechanisms were proposed in the latter (presumably by binding to phospholipids, as discussed report to explain the selective proteolytic degradation of hereunder for cocaine); (ii) as an adaptation, cells would acid sphingomyelinase induced by this drug. These include increase the synthesis of acid phospholipase, but the enzyme structural alteration of the enzyme itself due to high wouldremaininactiveaslongasthedrugremainsin concentrations of the drug, unknown signal transduction lysosomes; and (iii) upon dilution of cell lysates for enzyme processes (e.g. phosphorylation events) or modification of activity in vitro, full enzyme activity would be recovered the lysosomal membrane permeability, all resulting in a and even appear enhanced due to increased synthesis. The more pronounced susceptibility to degradation. Whether same mechanism was also proposed to account for the these or additional mechanisms apply to cocaine-induced increased activity of cathepsin B in lysates of fibroblasts proteolysis of sphingomyelinase would require further treated with leupeptin, a reversible cathepsin B inhibitor extensive work. (Montenez et al., 1994a,b), although a mere protection The clinical relevance of our observations remains to be against cathepsin B degradation by itself could not be evaluated. First, we do not know to what extent cocaine excluded. accumulates in lysosomes in vivo and remains intact in Similarly, the concomitant increase of total activity of target tissues long enough to produce significant phospho- other lysosomal enzymes could either reflect increased lipid accumulation. Indeed, to produce phospholipidosis in synthesis as an adaptation to lysosomal overload, or vivo, drug tissue retention is at least as important deter- decreased degradation due to impaired activity of cathe- minant as binding to phospholipids (Joshi et al., 1989). psins in situ. These mechanisms, alone or combined, Second, it is clear that cocaine concentrations used in the could account for the increase in the total activity of present in vitro study are well above those recorded in several lysosomal enzymes in inborn or acquired lyso- recreational drug users, although some addicted humans somal storage disorders. In Hunter and Sanfilippo type B may take much higher doses during the so-called diseases, the total activity of several non-genetically ‘‘binges’’. Cocaine concentration used in this study also affected enzymes, including N-acetyl-h-hexosaminidase, largely exceeded the level required to induce behavioural is greatly enhanced (Kint et al., 1973). Similarly, upon changes and addiction in rats [f1.5–3 AM (Lau and Sun, lysosomal overload induced in fibroblasts by azithromycin 2002); this concentration corresponds to the Cmax observed (Gerbaux et al., 1996) and in proximal kidney tubular in rats receiving 2 mg cocaine/kg i.v. (Sun and Lau, cells by the accumulation of the indigestible compound, 2001)]. In dogs, convulsion is induced by an acute poly-D-glutamic acid (Kishore et al., 1996), the activity of injection of 12 mg/kg and death at 21 mg/kg (Catravas N-acetyl-h-hexosaminidase and cathepsin B are both in- et al., 1978). However, we have noticed that prolonged creased. In the latter case, co-precipitation and/or stable incubation of fibroblasts with concentrations much lower binding of selected hydrolases to the insoluble matrix was than 500 AM still resulted in distinct ultrastructural evi- proposed to confer protection against proteolysis by lyso- dence for lipidosis in vitro (our unpublished results). somal cathepsins. Several drugs, including cocaine and gentamicin, induce Sphingomyelin accumulation in cocaine-treated cells both a mixed lysosomal lipidosis and apoptosis (El Moued- can be ascribed to a completely different mechanism, den et al., 2000a). For cocaine, apoptosis has been reported namely induction at high cocaine concentration of sphin- in sensitive cells such as neurons in the developing brain gomyelinase proteolysis, by a lysosomal cysteine protein- (Nassogne et al., 1995, 1997), coronary endothelial cells ase that is not sensitive to this drug. Indeed, incubation of (He et al., 2000) and hepatocytes (Zaragoza et al., 2000). All cells with cocaine resulted in a rapid disappearance of acid these studies were conducted at high cocaine concentrations sphingomyelinase in cell extracts, that was reflected by a (typically 50–100 AM range or higher). After in vivo 10-fold decrease in Vmax without change in Km, indicating administration of cocaine, apoptosis has also been observed that the affinity of the residual enzyme was not affected. in testis (Li et al., 1999) and thymocytes (Wu et al., 1997), Moreover, the loss of acid sphingomyelinase was only but fairly high doses were required (15–50 mg/kg). In observed when cells were incubated with cocaine, but not contrast, gentamicin induces both phospholipidosis and with benzoylecgonine. Finally, loss of sphingomyelinase apoptosis at intracellular concentrations similar to those activity was fully prevented by two inhibitors of cysteine observed in vivo in kidney proximal tubular cells of rats, proteinases, leupeptin and E64. The rapid recovery of acid where apoptosis could also be demonstrated under clinically sphingomyelinase upon cocaine withdrawal points to a fast relevant conditions (El-Mouedden et al., 2000b). Apoptosis turnover of acid sphingomyelinase in the cells. Interesting- is a common finding in the brain of human patients who ly, in gentamicin-induced lipid storage disorders, sphingo- died of lysosomal lipids storage disorders, such as Tay- M.-C. Nassogne et al. / Toxicology and Applied Pharmacology 194 (2004) 101–110 109

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