in Vitro 1 l (1997)
Morphological, Biochemical and Molecular Effects of Cocaine on Mouse Neuroblastoma Cells Culture In Vitro G. REPETTO*t,
A. del PESO?, M. SALGUERO&
A. GARFIAt, M. J. GONZALEZ-MUfiOZS, P. SANZt and M. REPETTOt
tNational Institute of Toxicology, PO Box 863, 41080, Seville SDepartamento de Nutrition y Bromatologia, Facultad de Farmacia, Universidad de Alcala de Henares, Madrid and @Departamento de Citologia e Histologia Normal y Patolbgica, Facultad de Medicina, Universidad de Sevilla, Seville, Spain Abstract-In order to compare the effects of cocaine at morphological, basal cytotoxicity, biochemical and molecular levels, cultured mouse neuroblastoma cells (Neuro-2a) were exposed to a range of concentrations of cocaine hydrochloride. Neuroblastoma cell proliferation, evaluated by quantification of total protein content, was very sensitive to cocaine, being increasingly inhibited from I2 to 72 hr of exposure (ECs = 3.1 mM at 24 hr). Cytoplasmic membrane permeability to lactate dehydrogenase was not particularly increased and lysosomal function was stimulated from 0.05 to I.5 mM, and inhibited from 2.5 mM. A shift to anaerobiosis was detected as intracellular lactate dehydrogenase (LDH) activity was mcreased and mitochondrial succinate dehydrogenase (SDH) activity decreased. Hexosaminidase (HEX), a lysosomal enzyme involved in sphingolipid degradation, was stimulated only at I mM and neural acetylcholinesterase (AChE) activity was stimulated from 2.5 mM. Morphological examination of exposed cultures revealed that most cells became bipolar and multipolar neurons by extension of neurites, but also suffered cytoplasmic vacuolization, hydropic degeneration and nuclear pyknosis. Although cells developing apoptosis were observed, no DNA oligonucleosomal fragmentation was detected by agarose gel electrophoresis of DNA from cells exposed to cocaine. In conclusion, in addition to predominance of anaerobiosis, little disruption of membranes and severe morphologic injury, biochemical and morphological differentiation-like effects were the most prominent alterations produced by cocaine on mouse neuroblastoma cells. 8 1997 Published by Elseoier Science Ltd Abbreviations: AChE = acetylcholinesterase; HEX = hexosaminidase; RNRU = relative neutral red uptake; SDH = succinate dehydrogenase. Keywords:
Cocaine presents both sympathomimetic (inhibition of neuronal reuptake of norepinephrine and dopamine) and local anaesthetic (Na + channels blockade) properties. However, no single mechanism may explain a given alteration; among the possible mechanisms of acute or chronic neurotoxicity are: alteration of sodium channel and monoamine transporter; release of epinephrine from the adrenal medulla with subsequent hyperglycaemia; vasoconstriction with subsequent hypoxia and decrease of nutrient supply; calcium ion quelation; superoxide formation; enzyme inhibition; reduced neurotrophic activity; altered gene expression; programmed cell death; and plasma membrane changes (Cascales et al., 1994; Olsen, 1995).
0887-2333/97/%17.00 + 0.00 0 SSDI 0887-2333(97)00066-O
in oitro; differentiation
*Author for correspondence.
LDH = lactate
The aim of this study was to compare the effects of cocaine at morphological, basal cytotoxicity, biochemical and molecular levels. Neuro-2a mouse neuroblastoma cells were exposed in vitro to different concentrations of cocaine hydrochloride. Toxicity indicators assessed in the in oitro test system were: cell culture morphology; cell proliferation by quantification of total protein content of the cell culture; cytoplasmic membrane integrity to cytosolic lactate dehydrogenase (LDH) leakage; LDH activity; mitochondrial succinate dehydrogenase (SDH) activity in the citric acid cycle; lysosomal function evaluated by the relative uptake of neutral red (RNRU) (Repetto and Sanz, 1993); lysosomal hexosaminidase (HEX) sphingolipid degradation activity; neuronal acetylcholinesterase (AChE) activity; and DNA fragmentation by agarose gel electrophoresis.
by Elsevier Science
Ltd. All rights
in Great Britain
G. Repetto et 01.
:, .5 g
; n -25
Cocaine 2.5 mM
= $ E 0 z w
Fig. 2. Comparative modification of the different biomarkers produced by 2.5 mM cocaine in Neuro-2a cells. Toxicity indicators assessed in the in vitrotest system were: cell proliferation (PROL), lysosomal function as relative neutral red uptake (RNRU), cytoplasmic membrane integrity to cytosolic lactate dehydrogenase leakage (LDHL), lactate dehydrogenase (LDH) intracellular activity, mitochondrial succinate dehydrogenase (SDH) activity, lysosomal hexosaminidase (HEX) intracellular activity and neuronal acetylcholinesterase (AChE) activity. Data expressed relative to mean value in respective unexposed controls. *Indicates significant difference from control value (P < 0.01).
75 so l LDH
Cl300 cell line, Neuro-2a clone (ATCC CCL 131, Flow, Irvine, UK) was grown in modified Eagle’s medium supplemented with 10% foetal calf serum (Flow Laboratories, Helsinki, Finland). Cells were plated at a density of 10,000 cells/well in 96-well tissue culture plates (Costar). After 24 hr, the culture medium was replaced with 0.2 ml medium containing the test chemical in solution and incubated for another 24 hr (also for 12, 48 and 72 hr to evaluate effects on cell growth). Assessment of cytotoxicity and metabolic markers
l LDH 0 SOH
1 . . ..I
. . ..I
All the determinations were carried out in the same 96-well tissue culture plates where exposure took place, as previously stated (Repetto et al., 1993). Absorbance was read on a Titertek Multiscan plate
Fig. I. In vitro effects of exposure to different concentrations of cocaine on Neuro-2a (a) cell proliferation at I2 (m), 24 (0). 48 (A) and 72 (+) hr; (b) lactate dehydrogenase leakage at 24 hr (m) and lysosomal function (a); in order to avoid interference of chemicals in enzyme activities, the percentage of enzyme release in a culture was calculated as extracellular activity x 100 divided by total activity (extracellular + intracellular activity) of the culture; (c) lactate dehydrogenase activity (a) and succinate dehydrogenase activity (a); (d) hexosaminidase activity (w) and acetylcholinesterase activity (0). Each point represents the mean value of at least three experiments using six replicate cultures per concentration, expressed as the arithmetical mean percentage of control f SEM. *Indicates significant difference from control value (P < 0.01).
Plate 1. Morphology of Nemo-2a cell cultures stained with Giemsa: (A) control culture ( x 400) showing a epitheliod-like proliferative cell culture, with predominance of non-differentiated cells; (B) culture exposed for 24 hr to 2.5 mM cocaine ( x 400) showing a fine cytoplasmic vacuolization. Fine filopodia were emitted, changing the morphological pattern to a more differentiated culture composed by plaques of bipolar and multipolar cells (t); (C) culture exposed for 24 hr to 5 mM cocaine ( x 400) showing loss of neurites and cells; confluent cytoplasmic vacuoles, pyknosis (-) and cells showing nuclear fragmentation (-==); (D ) culture exposed for 72 hr to 0.1 mM cocaine ( x 200) showing the formation of pseudovascular spaces; (E) culture exposed for 72 hr to 5 mM cocaine ( x 400) showing pyknotic and dead cells surrounded by rests of nuclear material (-z). Note the presence of long beaded cytoplasmic processes interconnecting cells, as expression of neuronal differentiation (-). Insert ( x 1000) shows a detail of the varicose processes; (F) culture exposed for 72 hr to 5 mM cocaine ( x 1000) showing large confluent cytoplasmic vacuoles (t) with intense hydropic degeneration.
Plate 2. Agarose gel electrophoresis (at 50 V for 3 hr) of ethidium bromi’ de stained DNA from new0 lblastoma cells: UInexposed cultures (lane 2). or exposed to 2.5 mM cocaine for 24 (lane 3) or 72 hr (lane 4). to 7.5 rnM for 24 hr (lane 5.) or 72 hr (lane 6) and to JO mM for 72 hr (lane 7). Lanes I and 8 correspond to DNA Molecular Weight Marker VI.
In vitro toxicity
reader (Flow Laboratories). Cell proliferation according to total cellular protein was quantified in situ by a modification of the method of Bradford (1976), as previously stated (Repetto and Sanz, 1993). LDH (EC 22.214.171.124) activity both intracellular and in the culture medium, was determined by the method of Korzeniewski and Callewaert (I983). HEX (N-acetylP-D-hexosaminidase, EC 126.96.36.199) activity in cells and in medium was quantified by the method of Landegren (I 984). Lysosomal function was evaluated by the relative uptake of neutral red (Borenfreund et al., 1988; Repetto and Sanz, 1993). The determination of SDH (EC I .3.99.1) activity on intact cells was performed by the method of Mosmann (1983) modified by Borenfreund et al. (1988) and Repetto et al. (1994). AChE (acetylcholine acetylhydrolase, EC 188.8.131.52) activity on intact cells was measured using quinidine sulfate as a butyryl cholinesterase inhibitor by a previous adaptation (Repetto et a/., 1994) of the method of Ellman et al. (1961). Morphological study
Cells for the morphological study were seeded in Lab-Tek” tissue culture chamber slides (Nunc, Inc., Naperville, IL, USA), fixed in 70% ethanol and stained with Giemsa. DNA fragmentation
The extracted DNA from 10 million cells was loaded in 1.8% agarose gels for electrophoresis at 50 V for 3 hr. Gels were stained with ethidium bromide and visualized by UV (McCabe ef al., 1993). Calculations and statistical analysis
Values for enzyme activities and relative neutral red uptake were corrected for cell culture total protein content to avoid misinterpretation due to the influence on cell proliferation and cell detachment of the chemical tested. All experiments were performed at least three times using six wells per concentration and statistical analysis was carried out using analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. RESULTS AND DISCUSSION
The results of this study demonstrate a dose-dependent toxicity of cocaine to cultured neuroblastoma cells, in which cell proliferation was inhibited. Cell growth, evaluated by quantification of total protein content, was increasingly inhibited from 12 to 72 hr of exposure (EC,, = 4.5, 3.1, 1.5 and 1.2 mM at 12, 24,48 and 72 hr, respectively) (Fig. 1A). The stability of membranes was not markedly altered (Fig. 1B) and cytoplasmic membrane permeability evaluated according to LDH leakage was only significantly increased from 2.5 mM (EC% = 7.5 mM). The fact that effects on membrane disruption were much less than those on cell proliferation may be attributed to
the membrane stabilizing activity of cocaine, which gives rise to its local anaesthetic properties (Yuan and Acosta, 1996). Lysosomal function was evaluated according to the relative uptake of neutral red (Repetto and Sanz, 1993). In this modification of the standard neutral red cytotoxicity assay (Borenfreund et al., 1988), the results of lysosomal uptake of the faintly cationic supravital dye by the cells are expressed relative to cell culture protein content to avoid misinterpretation due to the influence on cell proliferation and cell detachment of the chemical tested. This marker of lysosomal function was stimulated by cocaine from 0.05 to 1.5 mM (EC,, = 0.3 mM) and a subtle inhibition was found at concentrations higher than 2.5 tTIM (Fig. 1B). This effect of cocaine may be related to the reduction of intracellular pH produced by cocaine (Altura and Gupta, 1992; Lei et al., 1985; Repetto and Sanz, 1993). Metabolic markers were altered much more than cytotoxicity indicators (Fig. IC). LDH intracellular activity was stimulated with increasing cocaine concentrations, being the most sensitive change observed (EC,, = 0.2 mM). At the tricarboxylic acid cycle level, mitochondrial SDH activity on intact Neuro-2a cells was reduced at I mM (EC,, = 1.7 mM). This stimulation of anaerobic glycolysis is concordant with the severe lactic acidosis reported in rat brain in vivo (Altura and Gupta, 1992) and the decrease in hepatocyte glycogen previously described in vitro (Ponsoda et al., 1992). HEX, a lysosomal enzyme involved in sphingolipid degradation, was previously found to be a sensitive marker of toxicity (Repetto et a/., 1994). HEX activity was stimulated at 1 mM and preceeding the cytoplasmic membrane disruption (Fig. lD), in a similar way to the previously reported reaction to thallium and trivalent and pentavalent arsenic (Repetto et al., 1994). It has been reported that cocaine produces free radicals, leading to hpoperoxidation of membrane phospholipids in different organs (Goldlin and Boelsterli, 1994) and the altered molecules may be metabolized by different enzymes including HEX, after proteolysis of the precursor form and the lysosomal redistribution of the enzyme (Lei et al., 1985). Neural AChE activity elicited a triphasic curve with a subtle stimulation at 0.01 mM, reduction between 0.1 and 1 mM and stimulation beyond this concentration (EC0 = 1 mM). The inhibition can be related to the binding of cocaine to the enzyme (Schwarz et al., 1995). To compare in vivo human toxicity and in vitro results, toxicokinetic factors should be considered. The minimum human lethal blood concentration reported for cocaine is 0.003 mM (Repetto and Repetto, 1997). However, due to tolerance, very high doses can be used by addicts. in addition, the brain-blood ratio for cocaine ranges from 4 to IO, and cocaine brain levels are also 60-70% higher after several administrations compared with a single dose
G. Repetto PI N/.
(Cass and Zahniser, 1993). The lowest statistically significant critical cellular neurotoxic concentration (Walum rt al., 1993) that we found on neuroblastoma cells was 0.1 nlM cocaine. Moreover, the results were slightly less sensitive than those of neurotransmitter re-uptake inhibition and membrane stabilization, that occurred from 0.03 mM (Liu 611al.. 1996). Figure 2 compares the extent of variation of each cytotoxicity and biochemical biomarker studied after exposure of Neuro-2a cells for 24 hr to 2.5 mM cocaine. Non-statistically significant increases were observed in cytoplasmic membrane permeability and in HEX activity. A shift to anaerobiosis is suggested as intracellular lactate dehydrogenase activity was increased and mitochondrial succinate dehydrogenase activity decreased. Lysosomal function was also reduced at this concentration. However, the most marked effects were the stimulation of AChE activity and the inhibition of cell proliferation, biochemically confirming the differentiation of neuroblastoma cells. Morphological changes were a sensitive indication of the effects of cocaine. When neuroblastoma cell cultures exposed for 24 hr to cocaine were compared with their respective controls (Plate IA), a fine cytoplasmic vacuolization was increasingly observed at concentrations higher than 0.001 mM. Fine filopodia were emitted (Plate IB), changing the pattern of the epitheliod-like proliferative cell culture. with predominance of mitotic and post-mitotic cells, to a more differentiated culture (HafTke and Seeds, 1975). composed by plaques of bipolar and multipolar cells with large nuclei. disposed in rosettes and pseudoganglia (Plate IB). Changes were even more evident at 5 mM with confluent cytoplasmic vacuoles, pyknosis, chromatin fragmentation and loss of neurites and cells (Plate IC). More marked injury was detected at 72 hr of exposure to 0.1 ITIM. causing the formation of pseudovascular spaces; some ceils had a pseudoepithelial aspect, and others showed apoptotic compatible morphology. At 5 mM (Plate IE), pyknotic and dead cells were surrounded by remains of nuclear material. Very long varicose processes interconnected several cells. Large confluent cytoplasmic vacuoles, hydropic degeneration and vacuolization of nucleoli showed the severe degree of the injury (Plate IF). In contrast to our results. Zachor <‘I (11. (1994) showed that cocaine differentially inhibited neuronal differentiation and proliferation in PC-12 cells in vitro. However, they did not assay concentrations of cocaine beyond 0.03 mM. The increase in both number and length of neurites induced by cocaine in neuroblastoma cells, producing a change from a proliferative undifferentiated pattern towards a multipolar neuronal culture, may be interpreted as a defence mechanism. In fact, it has been reported that decreased butyrylcholinesterase activity enhances cocaine toxicity not only because of poor metabolization, but also because of its non-catalytic role as a scavenger of toxins protecting AChE (Schwarz et al.,
1995). As butyrylcholinesterase may be increased in parallel with AChE during differentiation of neural cells (Ellman et al., 1961), differentiated neuronal cells can be more resistant to cocaine than naive cells. As cocaine crosses the placenta to interact with foetal tissues (Olsen, l995), the relevance of the possible interference in cell differentiation on the teratogenic potency of cocaine should be further explored. Cascales et al. (1994) reported the induction of programmed hepatocyte death in cocaine-treated mice. As we have observed apoptotic cells in the morphological study, the possible DNA degradation was investigated by molecular biology methods (Plate 2). Although some DNA degradation occurred. the typical apoptotic DNA ladder pattern was not detected by agarose gel electrophoresis of the DNA from cells exposed for 24 and 72 hr to cocaine 2.5, 7.5 and IO mM. Furthermore, the capacity of Neuro-2a cells to undergo DNA internucleosomal fragmentation after morphological apoptosis is under current investigation. In conclusion, in addition to predominance of anaerobiosis, little disruption of membranes and severe morphologic injury, biochemical and morphological differentiation-like effects were the most prominent alterations produced by cocaine on mouse neuroblastoma cells.
il[,h-rlo,l./r~l~rrnPnr.v-The authors thank S. Jimenez. F. Repetto and D. Osuna for their technical assistance, A. M. Eguino and S. A. Owens. MRPharmS, for their assistance in the preparation of this manuscript.
REFERENCES Altura B. M. and Gupta R. K. (1992) Cocaine induces intracellular free Mg deficits, ischemia and stroke as observed by in vivo 31P-NMR of the brain. Biochimiccz c/ B/ophy~i(.a Acra 1111, 27 I-274. Borenfreund E., Babich H. and Martin-Alguacil N. (1988) Comparisons of two in Iirro cytotoxicity assays-the neutral red (NR) and tetrazolium MTT tests. To.ricolog~ irt Vitro 2 l-6. Bradford M: (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analyfical Eiochemisr,:1, 72, 248-254.
Cascales M.. Alvarez A., Gasco P., Fernandez-Simon L., Sanz N. and Bosca L. (1994) Cocaine-induced liver injury in mice elicits specific changes in DNA ploidy and induces programmed death of hepatocytes. Hepatology 20, 992-1001.
Cass W. A. and Zahniser N. R. (1993) Cocaine levels in striatum and nucleus accumbent: augmentation following challenge injection in rats withdrawn from repeated cocaine administration. Neuroscience Lerters 152, 177180. Ellman G. L.. Courtney K. D.. Andres V. and Featherstone R. M. (1961) A new and rapid calorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology i, 88-95. Goldlin C. and Boelsterli U. A. (19941 Dissociation of covalent protein adduct formation from oxidative injury in cultured hepatocytes exposed to cocaine. Xenobiorico
In rifro toxicity of cocaine HatIke S. C. and Seeds N. W. (1975) Neuroblastoma: the E. coli of neurobiology? Lije Sciences 16, 164991658. Korzeniewski C. and Callewaert M. (1983) An enzyme-release assay for natural cytotoxicity. Journal qf Immunological
64, 3 13-320.
Landegren 0. (1984) Measurement of cell numbers by means of the endogenous enzyme hexosaminidase. Applications to detection of lymphocytes and cell surface antigens. Journal of Immunological Method.7 67, 379-388.
Lei H.. Carroll K.. Au L. and Krag S. S. (1985) An in vitro screen for potential inflammatory agents using cultured fibroblasts. In Alternurire Methods in To.tico1og.v. Vol. 3: In Vi/r0 Toxicolog!. Edited by A. M. Goldberg. pp. 73..85. Mary Ann Liebert Inc., New York. Liu D.. Hariman R. J. and Bauman J. L. (1996) Cocaine concentration-effect relationship in the presence and absence of lidocaine: evidence of competitive binding between cocaine and lidocaine. Journal a/ Pharmacologic and Experimental
McCabe M. J., Jiang S. A. and Orrenius S. (1993) Chelation of intracellular zinc triggers apoptosis in mature thymocytes. LaborarorJ, Incestigarion 69, 101-l IO. Mosmann T. (1983) Rapid calorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal a/Immunological Methods 65, 55-63.
Olsen G. D. (1995) Potential mechanisms of cocaineinduced developmental neurotoxicity: a minireview. ,Veeuroro.x~cologr16, 159-167. Ponsoda X.. Jover R.. Caste11V. and Gomez-Lechon M. J.
(1992) Potentiation of cocaine hepatotoxicity in human hepatocytes by ethanol. Toxicology in Virro 2, 155-158. Repetto G. and Sanz P. (1993) Neutral red uptake, cellular growth and lysosomal function: in vitro effects of 24 metals. ATLA 21, 501-507. Repetto G., Sanz P. and Repetto M. (1993) In vitro effects of mercuric chloride and methylmercury chloride on neuroblastoma cells. Toxicology in Virro 7, 353-357. Repetto G., Sanz P. and Repetto M. (1994) Comparative in cirro effects of sodium arsenite and sodium arsenate on neuroblastoma cells. To.xicol0g.r 92, 143-I 53. Repetto R. and Repetto M. (1997) Habitual, toxic and lethal concentrations of 103 drugs of abuse in humans. Journal
Schwarz M., Glick D., Loewenstein Y. and Soreq H. (1995) Engineering of human cholinesterase explains and predicts diverse consequences of administration of various drugs and poisons. Pharmacology and Therapeurics 67, 283-322.
Walum E.. Nordin M.. Beckman M. and Odland L. (1993) Cellular methods for identification of neurotoxic chemicals and estimation of neurotoxicological risk. Tosicologj in Vitro
Yuan C. and Acosta D. (1996) Dissociation of the cytotoxicity of cocaine from its local anaesthetic effect: a comparison with lidocaine. To.ricology in Vitro 10, 195-204.
&chor D.. Cherkes J. K.. Fay C. T. and Ocrant I. (1994) Cocaine differentially inhibits neuronal differentiation and proliferation in rirro. Journal o/Clinical Incesrigarion 93, II79