Inactivation of the Cytotoxic Activity of Repin, a Sesquiterpene Lactone

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Inactivation of the Cytotoxic Activity of Repin, a Sesquiterpene Lactone from Centaurea repens Francis F. Tukov,† S. Anand,‡ Rama Sarma V. S. Gadepalli,‡ A. A. Leslie Gunatilaka,§ John C. Matthews,† and John M. Rimoldi*,‡ Department of Pharmacology and Research Institute of Pharmaceutical Sciences and Department of Medicinal Chemistry and Laboratory for Applied Drug Design and Synthesis, The University of Mississippi, University, Mississippi 38677, and Southwest Center for Natural Products Research, The University of Arizona, Tucson, Arizona 85706 Received May 15, 2004

Prolonged ingestion of Yellow Starthistle (Centaurea solstitialis) and Russian Knapweed (Centaurea repens) by horses has been shown to result in a fatal neurodegenerative disorder called equine nigropallidal encephalomalacia (ENE). Bioassay-guided fractionation of extracts from Centaurea species using the PC12 cell line have led to the identification of one of several putative agents, which may contribute to ENE, namely, the sesquiterpene lactone (SQL) repin (1), previously linked to ENE due to its abundance in C. repens. To characterize the molecular basis of repin-induced neurotoxicity, the present study was designed to identify reactive functional groups that may contribute overall to its toxicity. The reaction of repin (1) with glutathione (GSH) led to the exclusive addition of GSH to the R-methylenebutyrolactone affording a GSH conjugate (3b) that lacked toxicity in the PC12 cell assay, while selective reduction of the R-methylenebutyrolactone double bond of 1 also resulted in an analogue (2) that was devoid of toxicity relative to the parent compound. Unlike repin, analogue 2 failed to decrease cellular dopamine levels in PC12 cells, further substantiating the requirement of the R-methylenebutyrolactone group. Results from this study are suggestive that GSH depletion by the SQL repin may be a primary event in the etiology of ENE, increasing the susceptibility to oxidative damage.

Introduction Prolonged ingestion of Yellow Starthistle (Centaurea solstitialis) and Russian Knapweed (Centaurea repens) perennial herbaceous plants, native to Arizona and California, by horses has been shown to result in a fatal neurodegenerative disorder called ENE.1 This Parkinsonlike disease typified by the degeneration and necrosis of the globus pallidus and substantia nigra has no known treatment (1, 2). However, repin, a highly reactive electrophile and the major SQL isolated from C. repens, is believed to be the neurotoxin due to its abundance relative to other secondary SQL metabolites found in these noxious weeds. It has been hypothesized that the toxicity of repin may be due to the presence of the electrophilic R-methylenebutyrolactone moiety (3, 4), albeit alternate reactive electrophilic groups are present on repin, which may also contribute to its overall toxicity (Figure 1). * To whom correspondence should be addressed. Tel: 1-662-9155119. Fax: 1-662-915-5638. E-mail: [email protected]. † Department of Pharmacology and Research Institute of Pharmaceutical Sciences, The University of Mississippi. ‡ Department of Medicinal Chemistry and Laboratory for Applied Drug Design and Synthesis, The University of Mississippi. § Southwest Center for Natural Products Research, The University of Arizona. 1 Abbreviations: ENE, equine nigropallidal encephalomalacia; SQL, sesquiterpene lactone; PC, pheochromocytoma; DMSO, dimethyl sulfoxide; MEK, methyl ethyl ketone; XTT, sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]bis(4-methoxy-6-nitro)benzenesulfonic acid; HRMS, high-resolution mass spectrometry; Q-TOF, quadrapole timeof-flight; ESI, electrospray ionization; NOESY, nuclear Overhauser effect spectroscopy; ROS, reactive oxygen species; DHBA, 3,4-dihydroxybenzylamine; PMS, phenazine methosulfate.

Figure 1. Structure of repin (1). The arrows denote electrophilic sites for nucleophile conjugation.

SQLs, although generally considered cytotoxic, possess diverse pharmacological and toxicological properties (57). Research from several laboratories has found that many of the toxic effects associated with SQL exposure are linked to the presence of the R-methylenebutyrolactone moiety in these molecules (8-10). Studies have shown that the R-methylenebutyrolactone group covalently binds in a Michael type reaction to various biological nucleophiles, especially those with cysteine sulfhydryl groups (8, 11). Thus, SQLs are believed to exert their numerous biological activities through the inhibition of enzymes and other functional proteins by forming covalent bonds with free cysteine residues in these macromolecules or by conjugation with GSH. Of the numerous SQLs isolated and characterized from the noxious weed C. repens, repin (1) exhibited the most pronounced cytotoxic activity (12-14). The activity of repin, thought to be mediated chemically by the

10.1021/tx049864e CCC: $27.50 © 2004 American Chemical Society Published on Web 07/31/2004

Inactivation of the Cytotoxicity of Repin

R-methylenebutyrolactone moiety, has been reported to conjugate GSH nonenzymatically at a ratio of 1:1 in vitro (3). However, the structure or biological activity of the repin-GSH conjugate has not been investigated. Although the etiology of neuronal death in neurodegenerative diseases remains puzzling, recent evidence suggests that disturbances in the GSH homeostatic system may have significant effects on the pathogenesis of several neurodegenerative disorders. Limited in vivo and in vitro studies have demonstrated that repin selectively reduces striatal and hippocampal GSH levels in rodents (14) and causes a dose-dependent depletion of GSH levels in cultured cells (3). Depletion of cellular GSH to about 20-30% of the total GSH levels can impair cellular defense against toxic actions of compounds and lead to cellular injury or death (15). Repin administration in mice induces a significant and consistent increase in DA content in striatal homogenates, while striatal extracellular DA declines to approximately 30% vs control (14). It has been postulated that these effects are due to a blockade of DA release. In addition to the R-methylenebutyrolactone moiety, epoxide groups present on repin have been suggested as possible contributors to repin-induced neurotoxicity (3, 13). In a study comparing the relative toxicity of different SQLs isolated from Russian Knapweed and Yellow Starthistle, repin was found to be the most cytotoxic, whereas other SQLs of similar structure, such as acroptilin, were much less active (13). Both repin and acroptilin have identical structures with the exception that the side chain 17,18-epoxide group in repin has been replaced with an epichlorohydrin group. GSH conjugation and collateral depletion with epoxides, specifically styrene oxide, have been reported in PC12 cells (16). To better understand the molecular mechanism of repin-induced neurotoxicity, the present study was designed to identify the functional groups on repin that are responsible for its toxicity by structural modification, followed by screening of the analogues for cytotoxic activity using the well-established PC12 cell line.

Experimental Procedures Materials. PC12 cells were obtained from the Culture Collection (ATCC; Rockville, MD). Matrigel was obtained from BD Biosciences (Bedford, MA). All other tissue culture materials were obtained from the ATCC (Mannasas, VA). Perchloric acid was obtained from Fischer Scientific (Pittsburgh, PA). HPLC grade methanol and all other solvents were obtained from Fischer Scientific Chemicals (Fair Lawn, NJ). XTT and all other chemicals were procured from Sigma-Aldrich Chemical Co. (St. Louis, MO). Collagen-coated 75 cm2 culture flasks were purchased from Becton Dickinson Labware (Franklin Lakes, NJ). Instrumentation and Methods. 1H NMR and 13C NMR were performed on a Bruker DRX 500 spectrometer, with either tetramethylsilane or solvent peak as an internal standard. Mass spectrometry was performed using a Waters ZQ-LC-MS (ESI+ or ESI- mode, where applicable), and HRMS was performed using a Micromass Q-TOF (ESI+ or ESI- mode, where applicable). Isolation of Repin. C. repens was collected in Riverside, CA, and the extraction/isolation protocols were conducted similar to the method of Stevens et al. (13). Briefly, the dried aerial portions of the plant were ground and subjected to exhaustive extraction with hexanes, MEK, methanol, and water. The MEK extract, comprising activity as demonstrated by the PC12 cell activity results, was subjected to column chromatography (silica gel) using a gradient of ethyl acetate:hexanes (30:70 to 100:0).

Chem. Res. Toxicol., Vol. 17, No. 9, 2004 1171 Bioassay-guided fractionation of extracts from C. repens using the PC12 cell line led to the identification and consequent isolation of repin as the main active constituent. PC12 cells were exposed to repin dissolved in DMSO and diluted in growth medium, such that the final DMSO concentration per well was less than 0.1% (v/v). Chemistry. Reduction of the Exocyclic Methylene (2). To a solution of repin (20 mg, 0.06 mmol) in 800 µL of methanol were added, with stirring, anhydrous CdCl2 (150 mg, 0.082 mmol) and Mg powder (118.56 mg, 4.94 mmol). Water (300 µL, 16.66 mmol) was added dropwise to this reaction mixture over a period of 5 min, accompanied by an instantaneous exothermic reaction, with the liberation of hydrogen. After 15 min, the reaction mixture was diluted with 400 µL of CH2Cl2, washed with water, and dried over sodium sulfate. Evaporation of the solvent under reduced pressure, followed by purification by preparative TLC (Si gel, 90:10; ethyl acetate:hexanes) furnished product 2 (16 mg, 80% yield). 1H NMR (500 MHz, CDCl3): δ 1.27 (3H, H-13, d, J ) 7 Hz), 1.61 (3H, H-19, s), 1.89 (1H, H-2β, ddd, J ) 5, 9, 14 Hz), 2.15 (1H, H-5, dd, J ) 9, 10.5 Hz) 2.19 (1H, H-7, m), 2.27 (1H, H-9β, dd, J ) 6.5, 13.5 Hz), 2.45 (1H, H-2R, ddd, J ) 7, 9, 14 Hz), 2.50 (1H, H-12, dq, J ) 7, 11 Hz), 2.77 (1H, H-9R, dd, J ) 4, 13.5 Hz), 2.81 (1H, H-18R, d, J ) 6 Hz), 3.07 (1H, H-15β, d, J ) 4 Hz), 3.15 (1H, H-18β, d, J ) 6 Hz), 3.25 (1H, H-1, t, J ) 9 Hz), 3.28 (1H, H-15R, d, J ) 4 Hz), 4.011 (1H, H-3, t, J ) 6 Hz), 4.45 (1H, H-6, t, J ) 10.5 Hz), 5.02 (1H, H-8, ddd, J ) 4,6,10 Hz), 5.08 (1H, H-14A, s), 5.20 (1H, H-14B, s). 13C NMR (125 MHz, CDCl3): δ 177.02 (C-11), 169.63 (C-16), 141.16 (C-10), 117.90 (C-14), 77.17 (C-6), 77.09 (C-8), 75.63 (C-3), 68.56 (C-4), 54.03 (C-7), 53.94 (C-17), 53.12 (C-18), 51.57 (C-5), 48.58 (C-15), 44.54 (C-1), 41.35 (C-12), 39.57 (C-9), 37.03 (C-2), 17.79 (C-19), 15.84 (C-13). HRMS (ESI+) m/z calcd for C19H24O7Na, 387.1420; found, 387.1406. Formation of the GSH-Repin Conjugate (3a; NMR Analysis). Repin (5.0 mg; 0.0138 mmol) was dissolved in a minimum quantity of acetone-d6, 200 µL, and to this solution a freshly prepared solution of GSH (6.36 mg; 0.0207 mmol in D2O, 200 µL) was added. The contents were transferred into a NMR sample tube, and the 1H NMR data (500 MHz) were recorded periodically from the time of mixing, and every hour thereafter, up to 24 h. Formation of the repin-GSH conjugate (3a) was confirmed by changes in the 1H NMR spectra relative to repin. Formation of the GSH-Repin Conjugate (3b). Repin (10.0 mg; 0.0276 mmol) was dissolved in a minimum quantity of acetone (400 µL), and to this solution, a freshly prepared solution of GSH (12.7 mg; 0.0414 mmol in H2O, 400 µL) was added. The reaction mixture was stirred at room temperature for 48 h, and the progress of the reaction was monitored by LCMS. Excess repin was removed by extraction with diethyl ether, and the aqueous solvent was removed by vacuum evaporation. The residue was reconstituted in methanol, and the conjugate was slowly precipitated with diethyl ether. 1H NMR (500 MHz, D2O): δ 1.52 (3H, H-19, s), 1.80 (1H, H-2β, m), 2.10 (2H, H-27, t, J ) 7 Hz), 2.2-2.33 (2H, H-9β, H-2R, m), 2.40 (1H, H-5, m), 2.47 (2H, H-26, t, J ) 7 Hz), 2.70 (1H, H-7, m), 2.78 (1H, H-9R, m), 2.8-2.9 (2H, H-13 and H-20, m), 2.91 (1H, H-18A, d, J ) 5 Hz), 3.02 (2H, H-13 and H-20, m) 3.07 (1H, H-15B, d, J ) 4 Hz), 3.10 (1H, H-18B, d, J ) 5 Hz), 3.18 (1H, H-1, m), 3.21 (1H, H-15A, d, J ) 4 Hz), 3.73 (1H, H-28, m), 3.88 (2H, H-23, s), 4.10 (1H, H-3, t, J ) 7.5 Hz), 4.51 (2H, H-6 and H-21, m), 5.00 (1H, H-8, m), 5.05 (1H, H-14A, s), 5.15 (1H, H-14B, s). 13C NMR (125 MHz, D2O): δ 178.5 (C-11), 174.4,a 173.3,a 173.3,a 172.0,a 171.1 (C-16), 141.06 (C-10), 117.53 (C-14), 78.6 (C-6), 77.68 (C-8), 72.41 (C-3), 69.43 (C-4), 55.81 (C-7), 55.22 (C-17), 54.08 (C-28), 54.00 (C-18), 53.40 (C-21), 52.8 (C-5), 48.58 (C-15), 48.13 (C-1), 46.4 (C-12), 42.08 (C-23), 39.12 (C-2), 38.98 (C-9), 35.66 (C-13), 34.81 (C-20), 31.64 (C-26), 26.48 (C-27), 16.91 (C-19). HRMS (ESI-) m/z calcd for C29H38N3O13S1, 668.2125; found, 668.2139. The superscript letter a means that the GSH carbonyl peaks were not assigned. Biological Activity Assessment. Cell Culture. For experiments evaluating changes in mitochondrial function/cell viability

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(XTT reduction), PC12 cells were maintained in Kaighn’s modification of Ham’s F12 medium (F12K) containing 2 mM L-glutamine and supplemented with 15% horse serum, 2.5% fetal bovine serum, 10 000 IU/mL penicillin, and 10 mg/mL streptomycin in collagen-coated 75 cm2 culture flasks. Prior to the experiments, the cells were seeded at a density of 1.0 × 105 cells/well in 96 well culture plates coated with matrigel (5 mg/ mL) and allowed to attach overnight at 37 °C. For all experiments measuring changes in cellular DA levels, PC12 cells were maintained in 75 cm2 culture flasks at 37 °C in a 95% O2/5% CO2 humidified incubator. The cells were fed a complete growth medium (10% RPMI complete media) consisting of RPMI 1640 medium supplemented with 5% (v/v) fetal bovine serum and 10% (v/v) heat-inactivated horse serum, 10 000 IU/mL penicillin, and 10 mg/mL streptomycin. Inactivation of the horse serum was achieved by heating at 56 °C for 30 min. The growth medium was changed every 2-3 days. Prior to the experiment, these cells seeded at a density of 5 × 105 cells/well in 24 well culture plates (Corning, NY) coated with matrigel (5 mg/mL) were allowed to grow for 7 days in growth medium containing 50 ng/mL nerve growth factor (NGF). The growth medium containing NGF was changed every 2-3 days. Mitochondrial Function/Cell Viability. Mitochondrial function/cell viability was assessed using the XTT reduction assay. This colorimetric assay measures the reduction of XTT, a tetrazolium salt, by the mitochondrial “succinate-tetrazolium reductase” system of metabolically active cells, once XTT enters the cells (17). A decrease in XTT reduction is a sign of mitochondrial dysfunction or reduced cell viability. Because the XTT reduced product is soluble in aqueous solutions, it can be quantified directly using a spectrophotometer without the need of a solubilization step as with the MTT assay, which has received wide use as an index of cell viability and mitochondrial function (18-20). Following attachment to the plate, the cells were exposed to either the vehicle (culture media) or varying concentrations of 1, 2, or 3b and allowed to incubate for 24 h at 37 °C in a humidified incubator. Postincubation, the media from each well was poured off the plate and 100 µL of lukewarm (25-30 °C) PBS without calcium or magnesium was added to each well. This was followed by the addition of 25 µL of XTT/PMS solution to each well. The plate was then incubated for 4 h at 37 °C. The absorbance of the solution (PBS and XTT/PMS) in each well was then measured at a wavelength of 450 nm, using a spectrophotometric plate reader (Bio-TEK Instruments Inc., United States). All experiments were performed in triplicate, and the average absorbance at each concentration level was calculated and expressed as a percentage of control absorbance [i.e., % of control ) [(absorbance of treated cells/absorbance of untreated control) × 100]. Alteration of Cellular Concentrations of DA in NGFDifferentiated PC12 Cells. A modified HPLC method combined with electrochemical detection previously described by Ali and colleagues (21) was used to determine cellular concentrations of DA in differentiated PC12 cells exposed to 1 or 2. After the cells were differentiated by culturing for 7 days with NGF, the media was changed and the cells were treated with vehicle (media) or various concentrations of 1 (0.5-20 µM) or 2 (0.520 µM) in vehicle for 24 h at 37 °C in the humidified incubator. After this incubation, 1 or 2-treated PC12 cells were harvested by trypsinization with 0.1% trypsin/EDTA and washed by centrifugation (4 °C; 5000g) with PBS. To the washed cells was added 400 µL of 0.2 M perchloric acid containing internal standard DHBA, 100 ng/mL, and then sonicated by ultrasonication and centrifuged at 4 °C (14000g) for 10 min. Postcentrifugation, the supernatants were removed and filtered through Nylon-66 microfilters (pore size, 0.2 µm; MF-1 centrifugal filter; Bioanalytical Systems, West Lafayette, IN). Aliquots (25 µL) of the filtrate were injected directly onto the HPLC with an electrochemical detection system for separation and detection of analytes. The mobile phase consisted of 92% of a solution of 0.07 M KH2PO4, 0.107 mM EDTA, and 1.09 mM heptanesulfonic

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Figure 2. Selective reduction of the exocyclic methylene group. acid (pH 3.0) and 8% methanol. The flow rate was 0.8 mL/min. The amount of DA was calculated using standard curves generated by determining in triplicate the ratio between three different known amounts of the amine and a constant amount of internal standard. With each experiment, we ran a series of standards containing known levels of DHBA, DA, and DOPAC to ensure that the instrument was running linearly and properly. Statistics. Statistical analyses were performed by unpaired Student’s t-test. The statistical significance was accepted at the conventional P e 0.05 level by two-tailed evaluation. The results were expressed as means ( SEM from three experiments.

Results Chemistry. We chose to focus our initial efforts on the chemistry of the R-methylenebutyrolactone group of repin. Selective reduction of the exocyclic methylene group proved to be more difficult than anticipated, due to the inherent reactivity of the ester and epoxide groups. Attempts were made to selectively reduce this group using a variety of standard methods; all failed to afford the requisite product, with multiple products forming as a result of concomitant ester hydrolysis and/or epoxide ring opening. Selective reduction was finally achieved by using a metal salt-metal reduction system, namely, cadmium chloride-magnesium in methanol/water (22), which afforded a single diastereomeric product. Analysis by ESI(+)-LC-MS confirmed the addition of 2 mass units, and reaction at the R-methylenebutyrolactone group was established by extensive analysis of the 1H and 13C NMR spectra. Disappearance of the 1H NMR doublets at 5.57 and 6.21 ppm with collateral appearance of a doublet at 1.27 ppm indicated selective reduction of the methylene lactone olefin (C12-C13), affording compound 2. The stereochemistry of the methyl group about C-12 was established by analysis of the NOESY spectra; correlations between C-12H and C-6H/C-8H and C-13-CH3 and C-7H were observed, denoting an R-configuration of the C13 methyl group (Figure 2). It has been previously reported that incubation of repin with GSH affords a conjugate in 1:1 ratio, as initially described by Robles (3). Initially, we monitored the conjugation reaction between repin and GSH (acetone/ argon degassed water) by LC-MS, utilizing varying concentrations of GSH relative to repin. The spectra were recorded every 4 h, over a 48 h period in both ESI(+) and ESI(-) mode; the positive ion mode allowed for monitoring of the depletion of repin, while the negative ion mode allowed for monitoring of the consumption of GSH and the formation of the GSH-repin conjugate. Addition of either 1.0, 1.5, or 2.0 equiv of GSH relative to repin afforded an adduct with a molecular mass of m/z 668 (ESI negative mode). Increasing the GSH concentration increased the rate of reaction but did not lead to the formation of additional conjugates (i.e., 2× GSH: 1). This

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Figure 3. Reaction of repin (1) with GSH.

is not an unusual finding, since a previous report had demonstrated that incubation of 1.0 equiv GSH with a related SQL, chamissonolide (containing an R-methylenebutyrolactone group), had failed to produce an adduct after 20 h (23). Similarly, conjugation rates of GSH addition to the SQL helenalin (containing two Michael type acceptors, cyclopentenone and R-methylenebutyrolactone) were found to be approximately 10 times faster to the cyclopentenone group vs the R-methylenebutyrolactone group (23). It is apparent that the relative reactivity of the Michael acceptor dictates the addition reaction of GSH. We then chose to monitor the conjugate formation by 1H NMR analysis, to confirm that GSH was reacting exclusively with a single electrophilic group. 1H NMR spectra were recorded in acetone-d6:D2O, and spectral data were collected prior to the addition of 1.5 equiv of GSH and recorded every hour for 24 h. The NMR analysis indicated that GSH added exclusively to the R-methylenebutyrolactone group, yielding 3a, as demonstrated by the slow disappearance of the olefinic (C13H) doublets at 5.57 and 6.21 ppm. No effect of GSH addition to the epoxides was observed, since the diagnostic epoxide proton signals (C15-Ha/Hb; C18-Ha/Hb) experienced modest changes in terms of their chemical shift values, while their multiplicity remained unchanged. LC-MS analysis of the NMR reaction mixture further confirmed the presence of a GSH-repin conjugate (3a). To ascertain the stereochemistry of GSH addition at C-13 and to provide a sample for biological evaluation, repin was reacted with 1.5 equiv of GSH in acetone/H2O (instead of D2O). After 48 h, conjugate formation was nearly complete, with less than 10% repin remaining, as measured by LC-MS analysis. The conjugate (3b) was isolated by extracting residual repin with diethyl ether and concentrating the aqueous solution under highvacuum, followed by slow precipitation of reconstituted residue with methanol/ether. One-dimensional (1D) and two-dimensional (2D) NMR analysis allowed for the assignment of a majority of the proton and carbon signals; however, the stereochemistry about C-12 proved difficult to ascertain, due to overlapping peaks (C-13/ C-20) in the NOESY spectra, as well as clustering of overlapping signals in the 2.5-3.0 ppm range. Biological Assessment. The biological activity of the analogues was assessed using PC12 cells (1 × 105 cells per well) plated on matrigel-coated 96 well flat-bottom plates, incubated overnight, and exposed to varying concentrations of 1, 2, and 3b. The mitochondrial function/cell viability was assessed using the XTT reduction assay. While repin treatment caused a concentrationdependent decrease in cell viability/mitochondrial activity, no significant change in cell viability and/or mitochondrial activity was evident in cells treated with 2 or the repin-GSH conjugate 3b (Figure 4). Although the diminished toxicity of GSH conjugate 3b may be partially explained by the lack of cell penetration due to the

Figure 4. Effects of varying concentrations of repin (1), 2, and repin-GSH conjugate (3b) on mitochondrial activity of PC12 cells. The results are expressed as means ( SEM of three different experiments performed in triplicate. *P < 0.05 in comparison with corresponding control (0 µM repin group) (unpaired Student’s t-test, two-tail analysis).

Figure 5. Effects of repin (1) and 2 on cellular concentrations of DA in NGF-differentiated PC12 cells. After treatment with varying concentrations of 1, 2, or vehicle (culture media), the intracellular concentration of DA was measured using a modified HPLC-ECD method as described under the Experimental Procedures. The results are expressed as means ( SEM (N ) 4-6). *P < 0.05 in comparison with the corresponding control (CON) group (unpaired Student’s t-test, two-tail analysis).

enhanced hydrophilicity of the conjugate, the lack of cytotoxicity of 2 relative to repin demonstrates the necessity for an R-methylenebutyrolactone, since compound 2 is similar to 1 in terms of lipophilicity, as predicted by the ClogP estimations. To confirm that the C13-R-methylenebutyrolactone group of repin contributes substantially to repin-induced cytotoxicity in vitro, PC12 cells induced to differentiate into neuronlike cells by NGF treatment were exposed to either the vehicle (culture media) or varying concentrations of 1 or 2 for 24 h, and changes in cellular DA concentration were measured. The results obtained show that repin caused a concentration-dependent decrease in intracellular DA levels, whereas no significant change in cellular DA concentration was observed in cells treated with 2 (Figure 5). To determine whether any cross-reactivity exists in vitro between 1 and 2, NGF-differentiated PC12 cells

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Figure 6. Effects of 2 on repin-induced depletion of cellular DA level in NGF-differentiated PC12 cells. Cells pretreated with 2 at 5 or 10 µM for 1 h were incubated with 5 or 10 µM repin (1) for 24 h. The intracellular DA level was measured as described under the Experimental Procedures. The results are expressed as means ( SEM of three experiments performed in duplicate. *P < 0.05 vs control, and **P < 0.05 vs the corresponding compound 2-treated cells.

were pretreated for 1 h in the presence or absence of 5 and 10 µM 2 prior to incubation with either the vehicle control (culture media) or different concentrations of 1 for 24 h and changes in cellular DA concentration were measured. Pretreatment of these cells with either 5 or 10 µM 2 did not alter the effects of repin on these cells and had no effect on cellular DA levels in cells not treated with repin (Figure 6).

Discussion Cell culture techniques are finding increasing use in assessing drug-induced toxicity (24, 25) and have proved to be particularly useful in assessing the neurotoxic potential and mechanism of action of MPTP and related compounds on dopaminergic neurons (26, 27). In the present study, PC12 cells were used to assess the biological activity of repin (1) and related analogues (2 and 3b). These cells are preferred to primary cultures for studying specific biochemical mechanisms (28) of a variety of neurotoxicants and/or potential neurotoxicants because they are known to synthesize, store, release, and metabolize DA and norepinephrine (NE) in a manner analogous to that observed in vivo in the mammalian central nervous system (29-32). When exposed to NGF, PC12 cells cease to proliferate and gradually develop many of the characteristics of mature sympathetic neurons, including the outgrowth of long, branching neurites (33), thus making these cells a useful model for the determination of the neurotoxic mechanism of various neurotoxins in vitro. Oxidative stress and GSH depletion have been suggested as crucial factors in the degeneration of nigrostriatal dopaminergic neurons in horses affected by ENE (3, 14). The assay results that were obtained in our laboratory revealed that GSH conjugation by repin produced a conjugate that lacks toxicity, as demonstrated by the unaltered mitochondrial activity of PC12 cells. This result implicates the importance of the R-methylenebutyrolactone group as a site essential for GSH conjugation and collateral GSH depletion. This finding is in agreement with several other studies, which have

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shown that the R-methylenebutyrolactone moiety plays a critical role in the activity and/or toxicity of most active SQLs. GSH can be depleted directly by conjugation with electrophiles and indirectly by the addition of inhibitors of GSH biosynthesis and regeneration (15, 34). GSH depletion has been associated with increased ROS generation and cell death. Studies using cerebellar granule neurons and PC12 cells have shown that direct depletion of mitochondrial and cytoplasmic GSH resulted in increased generation of ROS, disruption of the mitochondrial transmembrane potential, and rapid loss of mitochondrial function (35, 36). In this present study, analogue 2 or 3b failed to induce a concentration-dependent decrease in cellular mitochondrial activity at even high concentrations. It is not premature to suggest that the inactivity of either 2 or 3b is due to the fact that there no longer exists an available reactive electrophilic site on repin to react with cellular GSH; hence, the GSH protective status of the cells is maintained leading to unaltered mitochondrial activity. However, one cannot rule out the decreased cell permeability of 3b relative to the parent compound, which may also explain the diminished cell cytotoxicity. We have also demonstrated in a related study that depletion of GSH with either (i) a potent depletor (ethacrynic acid) or (ii) a GSH synthesis inhibitor (buthionine sulfoximine) prior to repin treatment enhanced the repininduced mitochondrial change (37). Furthermore, increases in intracellular GSH levels induced by pretreatment with reducing agents (N-acetyl-L-cysteine or reduced GSH) completely protected the cells from repin-induced mitochondrial and dopaminergic toxicity, while the antioxidants, coenzyme-Q and ascorbic acid, completely blocked repin-induced dopaminergic toxicity (37). Degeneration of dopaminergic neurons in the substantia nigra is a predominant neuropathologic feature in ENE (1, 2). Studies have linked the neurotoxic effects of repin, the putative neurotoxin responsible for ENE development, to altered dopaminergic function (3, 14). In a previous study, repin was found to inhibit DA release without affecting DA uptake in PC12 cells (14), and it was hypothesized that inhibition of DA release represents one of the earliest pathogenetic events in ENE, ultimately leading to striatal extracellular DA denervation, oxidative stress, and degeneration of nigrostriatal pathways. However, in this study, analogue 2 had no significant effects on reducing the levels of cellular DA, suggesting that the R-methylenebutyrolactone moiety appears to be an essential site through which repin manifests its dopaminergic toxicity and may be a result of compromised GSH homeostasis. Alterations in GSH levels in the substantia nigra and subsequent mitochondrial dysfunction have been correlated with neuronal degeneration accompanying Parkinson’s disease (38). The depletion of GSH in the substantia nigra in presymptomatic Parkinson’s disease is the earliest known indicator of oxidative stress preceding decreases in both mitochondrial complex 1 activity and DA levels (39, 40). It has also been observed that blocking GSH synthesis in PC12 cells leads to a significantly decreased accumulation of [3H]DA, suggesting that GSH is involved in the granular storage of DA, and it is likely that GSH is used to protect susceptible parts of this system against (possibly DA-induced) oxidative damage (41).

Inactivation of the Cytotoxicity of Repin

In summary, these results suggest that the cytotoxic potential of repin appears to reside on its C13-R-methylenebutyrolactone moiety. The results also support the suggestion that intracellular GSH depletion by repin may represent an early pathogenic event in ENE, leading to oxidative stress, DA depletion, and ultimate disruption of nigrostriatal pathways in C. repens intoxicated horses. These findings could pave way for the selection and/or development of an antidote strategy to combat C. repens intoxication.

Acknowledgment. We thank the USDA NRI Competitive Grant Program (2001-35208-09969) and the Department of Medicinal Chemistry, University of Mississippi, for financial support. We thank Frank Wiggers, NMR specialist with the National Center for Natural Products Research, for help acquiring the 1D and 2D NMR data. Supporting Information Available: NMR spectra (1D and 2D proton/carbon) for compounds 2 and 3b. This material is available free of charge via the Internet at http://pubs.acs.org.

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