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Cytotoxicity of the cocaine metabolite benzoylecgonine
BRAIN RESEARCH ELSEVIER
Brain Research 643 (1994) 108-114
Research Report
Cytotoxicity of the cocaine metabolite benzoylecgonine Ying Lin, Kenneth C. Leskawa * Department of Anatomical Sciences and Neurobiology, School of Medicine, University of Louist,ille, Louisville, KY 40292, USA (Accepted 21 December 1993)
Abstract
The NG108-15 and C6 cell lines were used in the present study as neuronal and glial models, respectively, to examine the cytotoxicity of the major metabolite of cocaine, benzoylecgonine (BE). Exposure of both cell types to varying concentrations of BE resulted in a loss of cells from the growth surface. Analysis of the unattached cells after such exposure, using a variety of techniques, revealed that these cells were not viable. Therefore, this effect could not be ascribed to BE interfering with cell-substratum interactions. The early events in BE cytotoxicity were examined by observing cells cultivated on the stage of an inverted microscope, using differential interference contrast (Nomarski) optics. Upon exposure of either cell type to 10/zM BE a retraction of cellular processes could be observed within 30 min. Within 6 h cell death was apparent. Similar analyses using 50 /xM BE in the growth medium resulted in similar results, except that process retraction could be observed as early as 15 min after exposure. These results demonstrate that the major metabolite of cocaine, benzoylecgonine, is cytotoxic to in vitro models of neuronal and glial cells. Key words: Cocaine; Benzoylecgonine; Neuroblastoma; Glioma; Neuron; Glia; Cytotoxicity; Toxicity; Morphology
I. Introduction
During the past decade cocaine use has increased dramatically among the childbearing population of the United States, resulting in a greater number of fetuses exposed to cocaine during critical stages of development [1,7]. Such children demonstrate abnormal development of the central nervous system resulting in neurobehavioral and learning deficits as well as increased incidence of sudden infant death syndrome [25], low birth weight, urogenital and visual abnormalities, decreased head size and poor cognitive ability [3], reviewed in [43]. Despite the fact that some of these observations have not been substantiated [5], such observations have attracted much attention to cocaine's possible teratogenic effects. Despite several studies of the teratogenic effects of cocaine, little is known regarding the mechanisms of action. It has been proposed to operate via indirect effects upon the fetus by maternal vasoconstriction, a lowering of placental blood flow or stimulation of
* Corresponding author. Fax: (1) (502) 852-6228. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved
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uterine contractility [23,32,44]. Conversely, it remains plausible that cocaine a n d / o r its metabolites pass through placental barriers and exert toxic effects directly upon fetal cells in the developing CNS. This latter concern may be important because both in vivo and in vitro studies demonstrate that cocaine is neurotoxic to the developing brain. It has been reported that cocaine inhibits D N A synthesis in all regions of the developing rat brain following administration in vivo [2]. This suggests that cocaine, or one of its metabolites, disrupts cell replication. In vitro studies of cultured whole rat and mouse embryos also demonstrate that cocaine inhibits mitochondrial N A D H oxidase activities [18], inhibits embryo development [17] and interferes with gliogenesis [21]. In addition, cocaine exposure results in fine structural changes in the nucleus and decreased cell viability of neuroblastoma cells (NG108-15) in culture [24]. All the above evidence suggests that cocaine has direct destructive a n d / o r teratogenic effects on differentiating brain cells. Cocaine has a very short circulatory half-life time (tl/2, approximately 0.8 h) and is metabolically converted as soon as 1 rain after entering the circulatory system [4,17,37]. The major metabolite, benzoylecgonine (BE), is the most stable of the metabolites [37].
Y Lin, K.C. Leskawa/Brain Research 643 (1994) 108-114
BE remains in the circulatory system of fetuses much longer than in the exposed mother [11] and accumulates in fetal brain in much higher concentrations [39]. These findings suggest either slower metabolism of cocaine or slower clearance from body systems of fetuses vs. adults. In a study of 25 cases of human fetal and newborn deaths associated with maternal cocaine use, the average fetal blood cocaine and BE levels were 0.26 and 1.73 ~ g / m l , respectively. In half of the cases, fetal blood cocaine was zero while BE remained at relatively high levels [31]. These findings suggest that BE may play an important role in fetal and newborn impairment after maternal cocaine use. In addition, a behavioral study using rats showed that BE caused seizures more frequently, and with significantly longer latencies, than those induced by equimolar amounts of cocaine [28]. This again, suggests that BE may be one of the causative agents in cocaine toxicity. For the present studies, we have used NG108-15 and C6 cultured cells to examine the effects of BE. NG108-15 neuroblastoma ceils are well-characterized as neuronal models and have many properties of neuronal cells [13,26,34]. C6 glioma cells are also wellcharacterized and have often been used as models of glial ceils [36,42]. At early passages (as used in the present studies), C6 cells are characterized as oligodendrocytes, while at late passages C6 ceils display characteristics of astrocytes [36]. For these reasons, NG108-15 and C6 ceils are used here as models of neuronal and glial cells, respectively, providing homogenous cell populations which are useful for detailed studies of teratogenic mechanisms in vivo.
2. Materials and methods 2.1. Cell culture NG108-15 cells, a mouse neuroblastoma-rat glioma hybrid, were cultivated in media composed of Dulbecco's Modified Eagle Medium (DMEM) containing 4500 m g / L glucose, 10% fetal bovine serum (FBS), 1% HAT (10 mM sodium hypoxanthine, 40 /~M aminopterin and 1.6 mM thymidine), and 1% antibiotic-antimycotic mixture (10,000 U / m l penicillin G, 10,000/~g/ml streptomycin and 25 tzg/ml amphotericin B). C6 rat glioma cells, were cultivated in media containing Ham's F-10 nutrient mixture, 10% horse serum, 2% FBS, and 1% antibiotic-antimycotic mixture. Both NG108-15 and C6 cells were routinely maintained in 75 cm 2 flasks (Corning Glass Works, Corning, NY) in a humidified atmosphere of 95% air and 5% CO 2 at 37°C. 2.2. Exposure o f cultured cells to BE BE (Sigma, St. Louis, MO) was dissolved in Hank's Balanced Salt Solution (HBSS) at a concentration of 1 m g / m l . Appropriate amounts of the BE stock solution were added directly to complete media to yield a final concentration of 2.5-200/zM. In some experi-
109
ments, BE concentrations were kept constant, but time of exposure was varied.
2.3. Analysis of DNA synthesis DNA synthesis was analyzed by quantitating the incorporation of [3H]-thymidine into DNA. Cells were applied to 24-well dishes, allowed 24 h to attach and then changed to media containing varying concentrations of BE (0-200 /xM). During the last 6 h of 48 h BE exposure, each well received 2 tzCi of [3H]thymidine. Unattached and attached cells were harvested separately into 10% trichloroacetic acid (TCA) and TCA-insoluble material was collected by centrifugation. TCA pellets were suspended in 1N NaOH. Aliquots were neutralized with acetic acid and quantitated by scintillation spectrometry.
2.4. MTT-based cell growth determinations Cell growth was determined by mitochondrial dehydrogenase activity of viable cells. In this assay, the tetrazolium ring of MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide) is cleaved to yield purple MTT formazan chromogens [6,14,33,38], which were dissolved in isopropanol and measured spectrophotometrically. Control studies demonstrated that cell number correlated linearly with absorbance at 570 nm. Cells (2.5 x 104) were added to culture wells. After allowing 24 h to attach, cells were exposed to varying concentrations of BE (0-100/zM), and were maintained for an additional 24 h. During the last 4 h MTT assay reagents, in an amount equal to 10% of the culture media volume, were added and the chromophore was extracted. Sample absorbencies were converted into cell number by comparison to standard curves using linear regression and were compared by A N O V A and Tukey's protected t-test.
2.5. Photomicroscopy Cells (2.5 × 104) were seeded onto glass slide culture chambers (Nunc, Naperville, IL), previously coated with collagen type IV (Collagen Corp., Palo Alto, CA). After allowing 24 h to attach, cultures were transferred to an inverted microscope stage where they were maintained in an humidified atmosphere at 37°C. Growth media was removed and HEPES-buffered BE media (0-100/xM BE) was added. Morphology changes of the cells were monitored during time by differential interference contrast (Nomarski) optics and photomicroscopy. Control cells were treated in exactly the same manner except that non-BE, i.e. control, media was added.
3. Results
3.1. General NG108-15 and C6 cells were cultivated in 24-well culture vessels and allowed 24 h to attach. Cultures were then changed to media containing concentrations of BE which varied from 0-100 IzM. After 24 h of incubation a loss of cells from the substratum was apparent by visual observation. This was later confirmed by staining the remaining attached cells with Giemsa stain (not shown). In repeated experiments, the unattached cells were collected and analyzed. It
Y. Lin, K.C. Leskawa /Brain Research 643 (1994) 108-114
110 Table 1 Cytotoxicity of benzoylecgonine
Benzoylecgonine Concentration (/zM) 20 NG108-15 Cells Attached Unattached C6 Cells Attached Unattached
50
100
6.67 + 0.52 0.97+0.18 **
6.70 _+0.90 0.47_+0.03 **
6.43 + 0.46 1.23+0.68 **
67.7 +9.10 1.07+0.77 **
46.5 + 1.10 0.36+0.21 **
56.0 +1.90 0.17+0.03 **
NG108-15 and C6 cells were cultivated and exposed to BE at varying concentrations in the growth media as described in the text. After 24 h incubation, attached and unattached cells were collected and assayed for D N A synthesis using [3H]thymidine as a precursor. Values presented are picomole precursor incorporated per mg cellular protein, n = 3+S.E.M. * * P < 0.01. At least three replicate experiments gave similar results.
was found that virtually 100% of the unattached cells did not exclude Trypan Blue, indicating loss of viability (not shown). This was confirmed by collecting the unattached cells and determining D N A synthesis by the incorporation of [3H]thymidine. As shown in Table 1, the exposure of either cell type to either 20, 50 or 100 /xM BE resulted in a highly significant accumulation of unattached cells which were not viable. Unattached cells in control cultures were not detected. In additional experiments, viability of cells was analyzed by measuring the activity of mitochondrial dehydrogenase. Enzyme activity values were compared to cell number using standard curves derived .with control cells. Significant loss of cell viability could be observed at concentrations as low as 1 0 / , M in cultures of both cell types (Figs. 1 and 2). Attention then turned to early events during BE exposure. NG108-15 and C6 cells were cultivated on a stage of an inverted microscope and fields were exam-
ined during time following exposure to BE-containing media. When either cell type was exposed to media containing 10/zM BE a retraction of cellular processes was observed within 30 rain. By 6 h obvious cell death was observed, which was followed by an eventual loss of cells from the growth surface by 10 h (Figs. 3 and 4). Similar experiments were performed using media containing 50 /xM BE. A similar sequence of events was observed, except in this case the morphological changes occurred over a shorter length of time. Process withdrawal into the cell bodies could be observed as early as 15 min after exposure (not shown). Control cells were cultured and examined under the same exact conditions. In contrast, these cells were observed to extended processes, rather than withdraw them, and were observed to migrate on the growth surface (not shown).
4. Discussion
The use of cocaine during pregnancy has both direct and indirect effects on the developing fetal brain. It is difficult to distinguish between the two, especially direct toxic effects, by in vivo studies. In this regard, cell culture models are very useful because the extracellular environment can be carefully controlled, thus serving to detect direct toxic effects of cocaine on specific cell types (i.e., neuronal vs. glial cells). In addition, concentrations of cocaine can be regulated so that the minimum dosage required for toxic effects can be determined. Such detailed studies are impossible with whole animals because one must consider a multitude of variables. These include varying routes of administration, rates of metabolic conversion and transport across placental barriers and whether brain tissue esti-
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20
40
60
80
1O0
uM Benzoylecgonine
Fig. 1. Viability of NG108-15 cells following 24 h exposure to varying concentrations of BE. Cell culture and BE exposure conditions are described in the text. Viability was determined by assaying mitochondrial dehydrogenase activity, n = 3 + S.E.M. ** P < 0.01 A minimum of three replicate experiments gave similar results. In cases where error bars are not shown, the S.E.M. was smaller than the size of the point, as drawn.
20
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I
I
40 60 80 uM Benzoylecgonine
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Fig. 2. Viability of C6 cells following 24 h exposure to varying concentrations of BE. Cell culture and BE exposure conditions are described in the text. Viability was determined by assaying mitochondrial dehydrogenase activity, n = 3 + S.E.M. ** P < 0.01 A minimum of three replicate experiments gave similar results. In cases where error bars are not shown, the S.E.M. was smaller than the size of the point, as drawn.
Y. Lin, K.C. Leskawa /Brain Research 643 (1994) 108-114
111
cles [13]. Biochemically, they form functional cholinergic synapses with muscle cells, with requisite mechanisms for transmitter synthesis, storage and depolarization initiated release. In addition, these cells have large numbers of high-affinity opiate receptors and were used to characterize the enkephalin receptor [26,34]. Physiologically, NG108-15 cells are very excitable electrically and generate action potentials [34]. In regard to this project it is important to note that NG108-15 cells
mates of cocaine or BE concentration are truly those presented to brain cells or whether they reflect compartmentalization in the vasculature. NG108-15 neuroblastoma cells have well-characterized neuronal properties and have often been used as models of neuronal cells [13,26]. Morphologically, they exhibit an obvious neuronal phenotype including neurite-like processes containing both microtubules and filaments as well as many dense-core vesi-
w
y~
Fig. 3. Time-lapse photomicroscopic observations of NG108-15 cells exposed to 10/~M BE using differential interference contrast (Nomarski) optics.
112
Y. Lin, K.C. Leskawa /Brain Research 643 (1994) 108-114
contain complex gangliosides of the 'A' pathway, rather than just the simpler ganglioside structures (GM3, GM2 and GM1) seen in most other neuroblastoma cells [12]. C6 glioma cells are also well characterized and have often been used as models of glial cells [36,42]. At early passages (as used in the present studies), C6 cells are characterized as oligodendrocytes, while at late passages C6 cells assume properties of astrocytes [36]. For
the above reasons, NG108-15 and C6 cells are useful as models of neuronal and glial cells. The present studies demonstrate that BE is cytotoxic and causes dose-dependent reductions in cell viability of both NG108-15 and C6 cells in culture. Release of cells from the growth surface was observed at media BE concentrations as low as 10 /xM. This cytotoxicity is not due to an impairment of cell-sub-
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v
k 7
Fig. 4. Time-lapse photomicroscopic observations of C6 cells exposed to 10 /zM BE using differential interference contrast (Nomarski) optics,
Y. Lin, K.C. Leskawa /Brain Research 643 (1994) 108-114
stratum interactions, because unattached cells were nonviable, as determined by lack of D N A synthesis, loss of mitochondrial dehydrogenase activity and cellular inclusion of Trypan blue. Others have reported BE concentrations in fetal plasma and brain following maternal cocaine use which range from 0.4 to 11.1 /xM [28,39]. It is important to note that the concentrations used in the present in vitro studies were within, or near to, this range of values. Although this is the first report of BE cytotoxicity in vitro, others have reported BE causing seizures in rats [27,28]. It was found that BE induced seizures more frequently and with significantly longer latencies, than those due to equimolar amounts of cocaine [28]. In addition, the seizures could be prevented by antiepileptic drugs [27], suggesting that BE may be one of the causative agents in cocaine toxicity. There are many reports of cocaine's toxicity and teratogenic effects [8-11,15,16,22,31,35]. However, a recent report shows no evidence of irreversible neuronal damage in the rat brain after cocaine administration using immunocytochemical methods [20]. Other reports show that cocaine disrupts cell replication in all regions of the developing rat brain [2]. In vitro studies have also demonstrated that cocaine inhibits mitochondrial N A D H oxidase activities in cultured rat embryos [18], increases rat neuronal and astrocytic vulnerability to neurotoxic injury [40], interferes with the microtubule-associated tau protein metabolism and eventually causes cell death in cultured SH-SY5Y human neuroblastoma cells [30]. These same researchers have shown that cocaine causes a decrease in tau protein in cytoplasmic as well as membrane fractions, possibly disrupting microtubule stability and assembly. Cocaine has a very short circulatory half-life time (plasma levels peak between 20 min and 1 h) and is converted to its metabolites soon after it enters the circulatory system [4,17,37]. Benzoylecgonine (BE) is the major metabolite and is the most stable. BE remains in the fetal circulatory system much longer [11] and accumulates in fetal brain in much higher concentrations [39] than in the mother. This, and related studies [28,31] suggest that BE may play an important role in fetal and newborn impairment after maternal cocaine use. Although this is the first report of neuronal and glial cytotoxicity of BE in vitro, it opens a wide range of questions regarding cellular responses. The mechanism by which cocaine is converted to BE is still unclear. It has been proposed to be spontaneous or due to serum or liver enzymes [19,29,41]. It remains possible that the previous reports of cocaine toxicity to cells in culture may be due to its conversion to BE either by enzymes present in the serum in the culture media or by the cells themselves. This remains to be examined. In addi-
113
tion, very early morphological responses to BE exposure, such as possible alterations in cytoskeletal organization, warrant further detailed examination.
5. References [1] Abelson, H.I. and Miller, J.D., A decade of trends in cocaine use in the household population. In N.J. Kozel and E.H. Adams (Eds.), Cocaine Use In America: Epidemiologic and Clinical Perspectives, Office of Science of National Institute on Drug Abuse, Rockville, MD, 1985, pp. 35-49. [2] Anderson-Brown, T., Slotkin, T.A. and Seidler, F.J., Cocaine acutely inhibits DNA synthesis in developing rat brain regions: evidence for direct actions, Brain Res., 537 (1990) 197-202. [3] Azuma, S.D. and Chasnoff, I.J., Outcome of children prenatally exposed to cocaine and other drugs--a path analysis of 3-year data, Pediatrics, 92 (1993) 396-402. [4] Barnett, G., Hawks, R. and Resnick, R., Cocaine pharmacokinetics in humans, J. Ethnopharmacol., 3 (1981) 353-366. [5] Bauchner, H., Zuckerman, B., McClain, M., Frank, D., Fried, L.E. and Kayne, H., Risk of sudden infant death syndrome among infants with in utero exposure to cocaine, J. Pediatr., 113 (1988) 831-834. [6] Carmichael, J., DeGraff, W.G., Gazdar, A.F., Minna, J.D. and Mitchell, J.B., Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing, Cancer Res., 47 (1987) 936-942. [7] Chasnoff, I.J., Drug use and women: establishing a standard of care, Ann. NYAcad. Sci., 562 (1989) 208-210. [8] Chasnoff, I.J., Burns, K.A. and Burns, W.J., Cocaine use in pregnancy: perinatal morbidity and mortality, Neurotoxicol. Teratol., 9 (1987) 291-293. [9] Chasnoff, I.J., Burns, W.J., Schnoll, S.H. and Burns, K.A., Cocaine use in pregnancy, N. Engl. J. Med., 313 (1985) 666-669. [10] Chasnoff, I.J., Chisum, G.M. and Kaplan, W.E., Maternal cocaine use and genitourinary tract malformations, Teratology, 37 (1988) 201-204. [11] Chasnoff, I.J., Lewis, D.E., Griffith, D.R. and Willey, S., Cocaine and pregnancy: clinical and toxicological implications for the neonate, Clin. Chem., 35 (1989) 1276-1278. [12] Dahms, N.M. and Schnaar, R.L., Ganglioside composition is regulated during differentiation in the neuroblastomaxglioma hybrid cell line NG108-15, J. Neurosci., 3 (1983) 806-817. [13] Daniels, M.P. and Hamprecht, B., The ultrastructure of neuroblastoma glioma somatic cell hybrids, J. Cell Biol., 63 (1974) 691-699. [14] Denizot, F. and Lang, R., Rapid colorimetric assay for cell growth and survival modifications to the tetrazolium dye procedure giving improved sensitivity and reliability, J. Immunol. Meth., 89 (1986) 271-277. [15] Dixon, S.D., Coen.R, W. and Crutchfield, S., Visual disfunction in cocaine exposed infants, Pediatr. Res., 21 (1987) 359A. [16] Dow-Edwards, D.L., Freed, L.A. and Fico, T.A., Structural and functional effects of prenatal cocaine exposure in adult rat brain, Dev. Brain Res., 57 (1990) 263-268. [17] E1-Bizri, H., Guest, I. and Varma, D.R., Effects of cocaine on rat embryo development in vivo and in cultures, Pediatr. Res., 29 (1991) 187-190. [18] Fantel, A.G., Person, R.E., Burroughs-Gleim, C.J. and Mackler, B., Direct embryotoxicity of cocaine in rats: effects on mitochondrial activity, cardiac function, and growth and development in vitro, Teratol., 42 (1990) 35-43. [19] Fleming, J.A., Byck, R. and Barash, P.G., Pharmacology and therapeutic applications of cocaine, Anesthesiology, 73 (1990) 518-531.
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[20] Goodman, J.H. and Sloviter, R.S., Cocaine neurotoxicity and altered neuropeptide-Y immunoreactivity in the rat hippocampus--a silver degeneration and immunocytochemical study, Brain Res, 616 (1993) 263-272. [21] Gressens, P., Gofflot, F., Maele-Fabry, G.V., Misson, J.P., Gadisseux, J.F., Evrard, P. and Picard, J.J., Early neurogenesis and teratogenesis in whole mouse embryo cultures. Histochemical, immunocytological and ultrastructural study of the premigratory neuronal-glial units in normal mouse embryo and in mouse embryos influenced by cocaine and retinoic acid, J. NeuropathoL Exp. Neurol., 51 (1992) 206-219. [22] Hadeed, A.J. and Siegel, S.R., Maternal cocaine use during pregnancy--effect on the newborn infant, Pediatrics, 84 (1989) 205-210. [23] Hurd, W.W., Smith, A.J., Gauvin, J.M. and Hayashi, R.H., Cocaine blocks extraneuronal uptake of norepinephrine by the pregnant human uterus, Obstet. Gynecol., 78 (1991) 249-253. [24] Johnson Jr., J.E. and Weissman, A.D., Cocaine produces fine structural nuclear alterations in cultured neuroglioblastoma cells, Brain Res. Bull., 20 (1988) 39-47. [25] Kandall, S.R., Gaines, J., Habel, L., Davidson, G. and Jessop, D., Relationship of maternal substance abuse to subsequent sudden infant death syndrome in offspring, J. Pediatr., 123 (1993) 120-126. [26] Klee, W.A. and Nirenberg, M., A neuroblastomax glioma hybrid cell line with morphine receptors, Proc. Natl. Acad. Sci. USA, 71 (1974) 3474-3477. [27] Konkol, R.J., Doerr, J.K. and Madden, J.A., Effects of benzoylecgonine on the behavior of suckling rats: a preliminary report, J. Child NeuroL, 7 (1992) 87-92. [28] Konkol, R.J., Erickson, B.A., Doerr, J.K., Hoffman, R.G. and Madden, J.A., Seizures induced by the cocaine metabolite benzoylecgonine in rats, Epilepsia, 33 (1992) 420-427. [29] Leighty, E.G. and Fentiman Jr., A.F., Metabolism of cocaine to norcocaine and benzoylecgonine by an in vitro microsomal enzyme system, Res. Commun. Chem. Path. PharmacoL, 8 (1974) 65-74. [30] Lew, G.M., Microtubnlar tan protein after cocaine in cultured SH-SY5Y human neuroblastoma, Gen. PharmacoL, 23 (1992) 1111-1113. [31] Meeker, J.E. and Reynolds, P.C., Fetal and newborn death associated with maternal cocaine use, J. Anal ToxicoL, 14 (1990) 379-382.
[32] Moore, T.R., Sorg, J., Miller, L., Key, T.C. and Resnik, R., Hemodynamic effects of intravenous cocaine on the pregnant ewe and fetus, Am. J. Obstet. Gynecol., 155 (1986) 833-888. [33] Mosmann, T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. ImmunoL Meth., 65 (1983) 55-63. [34] Nelson, P.G., Neuronal cell lines. In S. Fedoroff and L. Hertz (Eds.), Cell, Tissue, and Organ Cultures in Neurobiology, Academic Press, Inc., New York, 1977, pp, 348-365, [35] Oro, A.S, and Dixon, S.D., Perinatal cocaine and methamphetamine exposure: maternal and neonatal correlates, J. Pediatr., 111 (1987) 571-578. [36] Parker, K.K., Norenberg, M.D. and Vernadakis, A., Transdifferentiation of C6 glial cells in culture, Science, 208 (1980) 179-181. [37] Sandberg, J.A. and Olsen, G.D., Cocaine pharmacokinetics in the pregnant guinea pig, J. Pharmaeol. Exp. Ther., 258 (1991) 477-482. [38] Slater, T.F., Sawyer, B. and Strauli, U., Studies on succinate-tetrazolium reductase systems III. Points of coupling of four different tetrazolium salts, Biochim. Biophys. Acta, 77 (1963) 383-393. [39] Spear, L.P., Kirstein, C.L. and Frambes, N.A., Cocaine effects on the developing central nervous system: behavioral, psychopharmacological and neurochemical studies, Ann. NYAcad. Sci., 562 (1989) 290-307. [40] Stadlin, A., Tsang, D., Macdonall, J.S., Mahadik, S.P. and Karpiak, S.E., An in vitro study on increased neuronal and astrocytic vulnerability to neurotoxic injury after in utero cocaine exposure: the reversal effects of GM1 treatment. In A.C.H. Yu, L. Hertz, M.D. Norenberg, E. Sykova and S.G. Waxman (Eds.), Progress in Brain Research, Elsevier, New York, 1992, pp. 339-350. [41] Stewart, D.J., lnaba, T., Lucassen, M. and Kalow, W., Cocaine metabolism: cocaine and norcocaine hydrolysis by liver and serum esterases, Clin. PharmacoL Ther., 25 (1979) 464-468. [42] Vernadakis, A. and Culver, B., Neural tissue culture: a biochemical tool. In S. Kumar (Ed.), Biochemistry of Brain, Pergamon Press, New York, 1980, pp. 407-452. [43] Volpe, J.J., Mechanisms of disease--effect of cocaine use on the fetus, N. Eng. ]. Med., 327 (1992) 399-407. [44] Woods, J.R., Plessinger, M.A. and Clark, K.E., Effect of cocaine on uterine blood flow and fetal oxygenation, J. Am. Med. Assoc., 257 (1987) 957-961.
Brain Research 643 (1994) 108-114
Research Report
Cytotoxicity of the cocaine metabolite benzoylecgonine Ying Lin, Kenneth C. Leskawa * Department of Anatomical Sciences and Neurobiology, School of Medicine, University of Louist,ille, Louisville, KY 40292, USA (Accepted 21 December 1993)
Abstract
The NG108-15 and C6 cell lines were used in the present study as neuronal and glial models, respectively, to examine the cytotoxicity of the major metabolite of cocaine, benzoylecgonine (BE). Exposure of both cell types to varying concentrations of BE resulted in a loss of cells from the growth surface. Analysis of the unattached cells after such exposure, using a variety of techniques, revealed that these cells were not viable. Therefore, this effect could not be ascribed to BE interfering with cell-substratum interactions. The early events in BE cytotoxicity were examined by observing cells cultivated on the stage of an inverted microscope, using differential interference contrast (Nomarski) optics. Upon exposure of either cell type to 10/zM BE a retraction of cellular processes could be observed within 30 min. Within 6 h cell death was apparent. Similar analyses using 50 /xM BE in the growth medium resulted in similar results, except that process retraction could be observed as early as 15 min after exposure. These results demonstrate that the major metabolite of cocaine, benzoylecgonine, is cytotoxic to in vitro models of neuronal and glial cells. Key words: Cocaine; Benzoylecgonine; Neuroblastoma; Glioma; Neuron; Glia; Cytotoxicity; Toxicity; Morphology
I. Introduction
During the past decade cocaine use has increased dramatically among the childbearing population of the United States, resulting in a greater number of fetuses exposed to cocaine during critical stages of development [1,7]. Such children demonstrate abnormal development of the central nervous system resulting in neurobehavioral and learning deficits as well as increased incidence of sudden infant death syndrome [25], low birth weight, urogenital and visual abnormalities, decreased head size and poor cognitive ability [3], reviewed in [43]. Despite the fact that some of these observations have not been substantiated [5], such observations have attracted much attention to cocaine's possible teratogenic effects. Despite several studies of the teratogenic effects of cocaine, little is known regarding the mechanisms of action. It has been proposed to operate via indirect effects upon the fetus by maternal vasoconstriction, a lowering of placental blood flow or stimulation of
* Corresponding author. Fax: (1) (502) 852-6228. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved
SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 0 4 0 - J
uterine contractility [23,32,44]. Conversely, it remains plausible that cocaine a n d / o r its metabolites pass through placental barriers and exert toxic effects directly upon fetal cells in the developing CNS. This latter concern may be important because both in vivo and in vitro studies demonstrate that cocaine is neurotoxic to the developing brain. It has been reported that cocaine inhibits D N A synthesis in all regions of the developing rat brain following administration in vivo [2]. This suggests that cocaine, or one of its metabolites, disrupts cell replication. In vitro studies of cultured whole rat and mouse embryos also demonstrate that cocaine inhibits mitochondrial N A D H oxidase activities [18], inhibits embryo development [17] and interferes with gliogenesis [21]. In addition, cocaine exposure results in fine structural changes in the nucleus and decreased cell viability of neuroblastoma cells (NG108-15) in culture [24]. All the above evidence suggests that cocaine has direct destructive a n d / o r teratogenic effects on differentiating brain cells. Cocaine has a very short circulatory half-life time (tl/2, approximately 0.8 h) and is metabolically converted as soon as 1 rain after entering the circulatory system [4,17,37]. The major metabolite, benzoylecgonine (BE), is the most stable of the metabolites [37].
Y Lin, K.C. Leskawa/Brain Research 643 (1994) 108-114
BE remains in the circulatory system of fetuses much longer than in the exposed mother [11] and accumulates in fetal brain in much higher concentrations [39]. These findings suggest either slower metabolism of cocaine or slower clearance from body systems of fetuses vs. adults. In a study of 25 cases of human fetal and newborn deaths associated with maternal cocaine use, the average fetal blood cocaine and BE levels were 0.26 and 1.73 ~ g / m l , respectively. In half of the cases, fetal blood cocaine was zero while BE remained at relatively high levels [31]. These findings suggest that BE may play an important role in fetal and newborn impairment after maternal cocaine use. In addition, a behavioral study using rats showed that BE caused seizures more frequently, and with significantly longer latencies, than those induced by equimolar amounts of cocaine [28]. This again, suggests that BE may be one of the causative agents in cocaine toxicity. For the present studies, we have used NG108-15 and C6 cultured cells to examine the effects of BE. NG108-15 neuroblastoma ceils are well-characterized as neuronal models and have many properties of neuronal cells [13,26,34]. C6 glioma cells are also wellcharacterized and have often been used as models of glial ceils [36,42]. At early passages (as used in the present studies), C6 cells are characterized as oligodendrocytes, while at late passages C6 ceils display characteristics of astrocytes [36]. For these reasons, NG108-15 and C6 ceils are used here as models of neuronal and glial cells, respectively, providing homogenous cell populations which are useful for detailed studies of teratogenic mechanisms in vivo.
2. Materials and methods 2.1. Cell culture NG108-15 cells, a mouse neuroblastoma-rat glioma hybrid, were cultivated in media composed of Dulbecco's Modified Eagle Medium (DMEM) containing 4500 m g / L glucose, 10% fetal bovine serum (FBS), 1% HAT (10 mM sodium hypoxanthine, 40 /~M aminopterin and 1.6 mM thymidine), and 1% antibiotic-antimycotic mixture (10,000 U / m l penicillin G, 10,000/~g/ml streptomycin and 25 tzg/ml amphotericin B). C6 rat glioma cells, were cultivated in media containing Ham's F-10 nutrient mixture, 10% horse serum, 2% FBS, and 1% antibiotic-antimycotic mixture. Both NG108-15 and C6 cells were routinely maintained in 75 cm 2 flasks (Corning Glass Works, Corning, NY) in a humidified atmosphere of 95% air and 5% CO 2 at 37°C. 2.2. Exposure o f cultured cells to BE BE (Sigma, St. Louis, MO) was dissolved in Hank's Balanced Salt Solution (HBSS) at a concentration of 1 m g / m l . Appropriate amounts of the BE stock solution were added directly to complete media to yield a final concentration of 2.5-200/zM. In some experi-
109
ments, BE concentrations were kept constant, but time of exposure was varied.
2.3. Analysis of DNA synthesis DNA synthesis was analyzed by quantitating the incorporation of [3H]-thymidine into DNA. Cells were applied to 24-well dishes, allowed 24 h to attach and then changed to media containing varying concentrations of BE (0-200 /xM). During the last 6 h of 48 h BE exposure, each well received 2 tzCi of [3H]thymidine. Unattached and attached cells were harvested separately into 10% trichloroacetic acid (TCA) and TCA-insoluble material was collected by centrifugation. TCA pellets were suspended in 1N NaOH. Aliquots were neutralized with acetic acid and quantitated by scintillation spectrometry.
2.4. MTT-based cell growth determinations Cell growth was determined by mitochondrial dehydrogenase activity of viable cells. In this assay, the tetrazolium ring of MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide) is cleaved to yield purple MTT formazan chromogens [6,14,33,38], which were dissolved in isopropanol and measured spectrophotometrically. Control studies demonstrated that cell number correlated linearly with absorbance at 570 nm. Cells (2.5 x 104) were added to culture wells. After allowing 24 h to attach, cells were exposed to varying concentrations of BE (0-100/zM), and were maintained for an additional 24 h. During the last 4 h MTT assay reagents, in an amount equal to 10% of the culture media volume, were added and the chromophore was extracted. Sample absorbencies were converted into cell number by comparison to standard curves using linear regression and were compared by A N O V A and Tukey's protected t-test.
2.5. Photomicroscopy Cells (2.5 × 104) were seeded onto glass slide culture chambers (Nunc, Naperville, IL), previously coated with collagen type IV (Collagen Corp., Palo Alto, CA). After allowing 24 h to attach, cultures were transferred to an inverted microscope stage where they were maintained in an humidified atmosphere at 37°C. Growth media was removed and HEPES-buffered BE media (0-100/xM BE) was added. Morphology changes of the cells were monitored during time by differential interference contrast (Nomarski) optics and photomicroscopy. Control cells were treated in exactly the same manner except that non-BE, i.e. control, media was added.
3. Results
3.1. General NG108-15 and C6 cells were cultivated in 24-well culture vessels and allowed 24 h to attach. Cultures were then changed to media containing concentrations of BE which varied from 0-100 IzM. After 24 h of incubation a loss of cells from the substratum was apparent by visual observation. This was later confirmed by staining the remaining attached cells with Giemsa stain (not shown). In repeated experiments, the unattached cells were collected and analyzed. It
Y. Lin, K.C. Leskawa /Brain Research 643 (1994) 108-114
110 Table 1 Cytotoxicity of benzoylecgonine
Benzoylecgonine Concentration (/zM) 20 NG108-15 Cells Attached Unattached C6 Cells Attached Unattached
50
100
6.67 + 0.52 0.97+0.18 **
6.70 _+0.90 0.47_+0.03 **
6.43 + 0.46 1.23+0.68 **
67.7 +9.10 1.07+0.77 **
46.5 + 1.10 0.36+0.21 **
56.0 +1.90 0.17+0.03 **
NG108-15 and C6 cells were cultivated and exposed to BE at varying concentrations in the growth media as described in the text. After 24 h incubation, attached and unattached cells were collected and assayed for D N A synthesis using [3H]thymidine as a precursor. Values presented are picomole precursor incorporated per mg cellular protein, n = 3+S.E.M. * * P < 0.01. At least three replicate experiments gave similar results.
was found that virtually 100% of the unattached cells did not exclude Trypan Blue, indicating loss of viability (not shown). This was confirmed by collecting the unattached cells and determining D N A synthesis by the incorporation of [3H]thymidine. As shown in Table 1, the exposure of either cell type to either 20, 50 or 100 /xM BE resulted in a highly significant accumulation of unattached cells which were not viable. Unattached cells in control cultures were not detected. In additional experiments, viability of cells was analyzed by measuring the activity of mitochondrial dehydrogenase. Enzyme activity values were compared to cell number using standard curves derived .with control cells. Significant loss of cell viability could be observed at concentrations as low as 1 0 / , M in cultures of both cell types (Figs. 1 and 2). Attention then turned to early events during BE exposure. NG108-15 and C6 cells were cultivated on a stage of an inverted microscope and fields were exam-
ined during time following exposure to BE-containing media. When either cell type was exposed to media containing 10/zM BE a retraction of cellular processes was observed within 30 rain. By 6 h obvious cell death was observed, which was followed by an eventual loss of cells from the growth surface by 10 h (Figs. 3 and 4). Similar experiments were performed using media containing 50 /xM BE. A similar sequence of events was observed, except in this case the morphological changes occurred over a shorter length of time. Process withdrawal into the cell bodies could be observed as early as 15 min after exposure (not shown). Control cells were cultured and examined under the same exact conditions. In contrast, these cells were observed to extended processes, rather than withdraw them, and were observed to migrate on the growth surface (not shown).
4. Discussion
The use of cocaine during pregnancy has both direct and indirect effects on the developing fetal brain. It is difficult to distinguish between the two, especially direct toxic effects, by in vivo studies. In this regard, cell culture models are very useful because the extracellular environment can be carefully controlled, thus serving to detect direct toxic effects of cocaine on specific cell types (i.e., neuronal vs. glial cells). In addition, concentrations of cocaine can be regulated so that the minimum dosage required for toxic effects can be determined. Such detailed studies are impossible with whole animals because one must consider a multitude of variables. These include varying routes of administration, rates of metabolic conversion and transport across placental barriers and whether brain tissue esti-
,dO
×
0
4
2-
x
4
~n
o\..
•~
2
T
T
**
.. I
20
40
60
80
1O0
uM Benzoylecgonine
Fig. 1. Viability of NG108-15 cells following 24 h exposure to varying concentrations of BE. Cell culture and BE exposure conditions are described in the text. Viability was determined by assaying mitochondrial dehydrogenase activity, n = 3 + S.E.M. ** P < 0.01 A minimum of three replicate experiments gave similar results. In cases where error bars are not shown, the S.E.M. was smaller than the size of the point, as drawn.
20
I
I
I
40 60 80 uM Benzoylecgonine
I
1O0
Fig. 2. Viability of C6 cells following 24 h exposure to varying concentrations of BE. Cell culture and BE exposure conditions are described in the text. Viability was determined by assaying mitochondrial dehydrogenase activity, n = 3 + S.E.M. ** P < 0.01 A minimum of three replicate experiments gave similar results. In cases where error bars are not shown, the S.E.M. was smaller than the size of the point, as drawn.
Y. Lin, K.C. Leskawa /Brain Research 643 (1994) 108-114
111
cles [13]. Biochemically, they form functional cholinergic synapses with muscle cells, with requisite mechanisms for transmitter synthesis, storage and depolarization initiated release. In addition, these cells have large numbers of high-affinity opiate receptors and were used to characterize the enkephalin receptor [26,34]. Physiologically, NG108-15 cells are very excitable electrically and generate action potentials [34]. In regard to this project it is important to note that NG108-15 cells
mates of cocaine or BE concentration are truly those presented to brain cells or whether they reflect compartmentalization in the vasculature. NG108-15 neuroblastoma cells have well-characterized neuronal properties and have often been used as models of neuronal cells [13,26]. Morphologically, they exhibit an obvious neuronal phenotype including neurite-like processes containing both microtubules and filaments as well as many dense-core vesi-
w
y~
Fig. 3. Time-lapse photomicroscopic observations of NG108-15 cells exposed to 10/~M BE using differential interference contrast (Nomarski) optics.
112
Y. Lin, K.C. Leskawa /Brain Research 643 (1994) 108-114
contain complex gangliosides of the 'A' pathway, rather than just the simpler ganglioside structures (GM3, GM2 and GM1) seen in most other neuroblastoma cells [12]. C6 glioma cells are also well characterized and have often been used as models of glial cells [36,42]. At early passages (as used in the present studies), C6 cells are characterized as oligodendrocytes, while at late passages C6 cells assume properties of astrocytes [36]. For
the above reasons, NG108-15 and C6 cells are useful as models of neuronal and glial cells. The present studies demonstrate that BE is cytotoxic and causes dose-dependent reductions in cell viability of both NG108-15 and C6 cells in culture. Release of cells from the growth surface was observed at media BE concentrations as low as 10 /xM. This cytotoxicity is not due to an impairment of cell-sub-
~v
v
k 7
Fig. 4. Time-lapse photomicroscopic observations of C6 cells exposed to 10 /zM BE using differential interference contrast (Nomarski) optics,
Y. Lin, K.C. Leskawa /Brain Research 643 (1994) 108-114
stratum interactions, because unattached cells were nonviable, as determined by lack of D N A synthesis, loss of mitochondrial dehydrogenase activity and cellular inclusion of Trypan blue. Others have reported BE concentrations in fetal plasma and brain following maternal cocaine use which range from 0.4 to 11.1 /xM [28,39]. It is important to note that the concentrations used in the present in vitro studies were within, or near to, this range of values. Although this is the first report of BE cytotoxicity in vitro, others have reported BE causing seizures in rats [27,28]. It was found that BE induced seizures more frequently and with significantly longer latencies, than those due to equimolar amounts of cocaine [28]. In addition, the seizures could be prevented by antiepileptic drugs [27], suggesting that BE may be one of the causative agents in cocaine toxicity. There are many reports of cocaine's toxicity and teratogenic effects [8-11,15,16,22,31,35]. However, a recent report shows no evidence of irreversible neuronal damage in the rat brain after cocaine administration using immunocytochemical methods [20]. Other reports show that cocaine disrupts cell replication in all regions of the developing rat brain [2]. In vitro studies have also demonstrated that cocaine inhibits mitochondrial N A D H oxidase activities in cultured rat embryos [18], increases rat neuronal and astrocytic vulnerability to neurotoxic injury [40], interferes with the microtubule-associated tau protein metabolism and eventually causes cell death in cultured SH-SY5Y human neuroblastoma cells [30]. These same researchers have shown that cocaine causes a decrease in tau protein in cytoplasmic as well as membrane fractions, possibly disrupting microtubule stability and assembly. Cocaine has a very short circulatory half-life time (plasma levels peak between 20 min and 1 h) and is converted to its metabolites soon after it enters the circulatory system [4,17,37]. Benzoylecgonine (BE) is the major metabolite and is the most stable. BE remains in the fetal circulatory system much longer [11] and accumulates in fetal brain in much higher concentrations [39] than in the mother. This, and related studies [28,31] suggest that BE may play an important role in fetal and newborn impairment after maternal cocaine use. Although this is the first report of neuronal and glial cytotoxicity of BE in vitro, it opens a wide range of questions regarding cellular responses. The mechanism by which cocaine is converted to BE is still unclear. It has been proposed to be spontaneous or due to serum or liver enzymes [19,29,41]. It remains possible that the previous reports of cocaine toxicity to cells in culture may be due to its conversion to BE either by enzymes present in the serum in the culture media or by the cells themselves. This remains to be examined. In addi-
113
tion, very early morphological responses to BE exposure, such as possible alterations in cytoskeletal organization, warrant further detailed examination.
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[32] Moore, T.R., Sorg, J., Miller, L., Key, T.C. and Resnik, R., Hemodynamic effects of intravenous cocaine on the pregnant ewe and fetus, Am. J. Obstet. Gynecol., 155 (1986) 833-888. [33] Mosmann, T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. ImmunoL Meth., 65 (1983) 55-63. [34] Nelson, P.G., Neuronal cell lines. In S. Fedoroff and L. Hertz (Eds.), Cell, Tissue, and Organ Cultures in Neurobiology, Academic Press, Inc., New York, 1977, pp, 348-365, [35] Oro, A.S, and Dixon, S.D., Perinatal cocaine and methamphetamine exposure: maternal and neonatal correlates, J. Pediatr., 111 (1987) 571-578. [36] Parker, K.K., Norenberg, M.D. and Vernadakis, A., Transdifferentiation of C6 glial cells in culture, Science, 208 (1980) 179-181. [37] Sandberg, J.A. and Olsen, G.D., Cocaine pharmacokinetics in the pregnant guinea pig, J. Pharmaeol. Exp. Ther., 258 (1991) 477-482. [38] Slater, T.F., Sawyer, B. and Strauli, U., Studies on succinate-tetrazolium reductase systems III. Points of coupling of four different tetrazolium salts, Biochim. Biophys. Acta, 77 (1963) 383-393. [39] Spear, L.P., Kirstein, C.L. and Frambes, N.A., Cocaine effects on the developing central nervous system: behavioral, psychopharmacological and neurochemical studies, Ann. NYAcad. Sci., 562 (1989) 290-307. [40] Stadlin, A., Tsang, D., Macdonall, J.S., Mahadik, S.P. and Karpiak, S.E., An in vitro study on increased neuronal and astrocytic vulnerability to neurotoxic injury after in utero cocaine exposure: the reversal effects of GM1 treatment. In A.C.H. Yu, L. Hertz, M.D. Norenberg, E. Sykova and S.G. Waxman (Eds.), Progress in Brain Research, Elsevier, New York, 1992, pp. 339-350. [41] Stewart, D.J., lnaba, T., Lucassen, M. and Kalow, W., Cocaine metabolism: cocaine and norcocaine hydrolysis by liver and serum esterases, Clin. PharmacoL Ther., 25 (1979) 464-468. [42] Vernadakis, A. and Culver, B., Neural tissue culture: a biochemical tool. In S. Kumar (Ed.), Biochemistry of Brain, Pergamon Press, New York, 1980, pp. 407-452. [43] Volpe, J.J., Mechanisms of disease--effect of cocaine use on the fetus, N. Eng. ]. Med., 327 (1992) 399-407. [44] Woods, J.R., Plessinger, M.A. and Clark, K.E., Effect of cocaine on uterine blood flow and fetal oxygenation, J. Am. Med. Assoc., 257 (1987) 957-961.