Synthetic Triterpenoid CDDO Derivatives Modulate Cytoprotective or Immunological Properties in Astrocytes, Neurons, and Microglia

David J. Graber & Paul J. Park & William F. Hickey & Brent T. Harris

Abstract

2-Cyano-3,12-dioxoolean-1, 9-dien-28-oic acid (CDDO) is a semisynthetic triterpenoid. CDDO derivatives with an amide, butyl ester (BE), imidazolide (IM), or trifluoroethyl amide (TFEA) group at position C-28 of CDDO were evaluated in glial and neuronal cells, in vitro. Changes in intracellular NADPH:quinone oxidoreductase (NQO1) levels, protection against oxidative toxicity, endotoxin-induced freeradical production, and the median lethal concentration (LC50) were assessed. All four CDDO derivatives at nanomolar concentrations increased NQO1 levels in astrocytes and moderately in neurons, but not in microglial cells. Pretreatment with 100 nM of CDDO-amide, CDDO-TFEA, or CDDO-IM protected astrocytes from hydrogen peroxide toxicity. Only CDDO-amide protected neuronal cells. Pretreatment of microglial cells with CDDO derivatives at nanomolar concentrations attenuated endotoxin-induced nitric oxide protection. The effectiveness for NQO1 induction, protection against oxidative toxicity, and attenuation of nitric oxide production, as well as cell viability at higher concentrations, varied among the derivatives withdifferentfunctional groups at C-28. CDDO-amide had comparable or even a greater effectiveness at altering cytoprotective and immunomodulatory properties while having higher LC50 values for each neural cell type examined. These results indicate that derivatives of CDDO modulate important pathways relevant to many neurological diseases that involve both chronic inflammation and free-radical damage with variable effects based on the functional group at C-28 and cell type.

Keywords Antioxidant . Astrocyte . Inflammation .Microglia . Motor neuron . Triterpenoid

Introduction

Extracts from plants, later recognized as containing triterpenoids, have been used as medicinal treatments for ailments such as fever, infection, and inflammation since being introduced in ancient Asian cultures. Triterpenoids are structurally similar to steroids and may, like steroids, diffuse freely through cell membranes to interact with intracellular molecular targets. The semisynthetic triterpenoid 2-cyano3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) had been developed along with chemically modified derivatives containing various functional groups on rings A and C (Honda et al. 1998, 2000b, a). These novel compounds are far more potent than natural triterpenoids and can affect signaling pathways in mammalian cells that are associated with detoxification (Liby et al. 2005), inflammation (Suh et al. 1998; Yore et al. 2006), and apoptosis (Ito et al. 2001).
In a number of cell types, nanomolar concentrations of various CDDO derivatives have been shown to induce the expression of genes encoding proteins associated with the NF-E2-related factor 2–antioxidant response element (Nrf2– ARE) pathway (Liby et al. 2005, 2007; Dinkova-Kostova et al. 2005; Thimmulappa et al. 2006). Increased NQO1 is one of several downstream detoxifying products of Nrf2 activation. An association between Nrf2 activation and attenuation of immunological activation has also been reported (Lin et al. 2008; Koh et al. 2009). Modification of functional groups on CDDO affects potencies with a linear correlation between the induction of NQO1 and inhibition of nitric oxide synthesis (Dinkova-Kostova et al. 2005).
Free-radical damage and inflammation in the central nervous system (CNS) are pathological features associated with many neurological diseases. The innate immunological response is thought to be involved in the degeneration of neurons and mediated by pattern recognition receptors (Takeuchi and Akira 2010; Zitvogel et al. 2010). Systemic administrationofseveral differentCDDOderivatives has been recently shown to induce neurological effects in mice that are potentially related to neuroprotection (Yang et al. 2009; Dumont et al. 2009; Yates et al. 2007; Stack et al. 2010). Astrocytes and microglia are important cell types for protection against free-radical damage and immunological challenges in the CNS. Attenuation of microglial activation in cell culture has been reported with CDDO and a derivative with a methyl ester at C-28 (Tran et al. 2008; Suh et al. 1999). Effects on astrocytes have not been elucidated.
To investigate the effects of semisynthetic triterpenoids in the CNS, four different derivatives of CDDO were tested using astrocytic, microglial, and neuronal cells, in vitro. The analogs, which have been previously demonstrated to induce NQO1 expression in CNS following systemic administration in mice (Yates et al. 2007), contained an amide, butyl ester, imidazolide, or trifluoroethyl amide group at C-28. Induction of NQO1, protection against oxidative toxicity, and concentration-dependent cytocidal activity were evaluated. Since lipopolysaccharide (LPS) acting through toll-like receptor 4 can activate microglia (Qin et al. 2005; Pei et al. 2007; Olson and Miller 2004), the effect of the CDDO derivatives on LPS-induced freeradical generation by microglia was also examined.

Methods

Primary glia and microglia-enriched cultures All animal procedures were approved by the Dartmouth Institutional Animal Care and Use Committee, under Association for Assessment and Accreditation of Laboratory Animal Care International-approved conditions, in accordance with National Institutes of Health guidelines. Cells were derived from trypsin-dissociated spinal cord (embryonic day 14) or brain (postnatal days 2–4) of Sasco Sprague Dawley rats (Charles River Laboratories International, Inc., Wilmington, MA) based on methods described previously (Giulian and Baker 1986; Nakajima et al. 1989). Dissociated cells were plated in polystyrene culture flasks at 107 cells/75 cm2 and incubated for 10–20 days. Loosely attached cells (microglia) were collected in media supernatant after shaking the flasks for 5 min. The adherent glial layer (astrocytes with some residual microglia) was then collected after treatment with trypsin–EDTA. Enriched microglia and glia were centrifuged at 300×g and resuspended separately in cell culture medium. Cell populations in glia cultures were approximately 90% astrocytes based on characteristic morphology and GFAP expression with the remaining cells being microglia. Cell populations in microglia-enriched primary cultures were typically greater than 95% Iba1 or ED1 positive with the remaining cells being astrocytes.

Primary spinal neuron cultures The primary neurons used in this experiment were isolated from the spinal cords of Sasco Sprague Dawley rat embryos at gestation day 14 as described previously (Stommel et al. 2007; Hanson et al. 1998). In brief, spinal cords were trypsinized and the dissociated tissues were spun over a density gradient of 6.8% metrizamide at 515×g at 4°C. Cellular pellets were used for generating embryonic spinal cord glia as described above. The larger, less dense cells collected above the metrizamide layer were further enriched for motor neurons by positive selection immunopanning using antibody against the low-affinity p75 NGF receptor (192 antibody was a gift of the E. M. Shooter laboratory, Stanford University School of Medicine, Palo Alto, CA, USA). After 1 h of immunopanning, unattached cells were collected and used as spinal neuron cultures. The antip75-attached cells were collected following trypsinization and used as enriched spinal motor neuron cultures. Plating medium was Neurobasal base media (Invitrogen, Carlsbad, CA, USA) supplemented with B27 (Invitrogen), 10 ng/ml each of brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and glial-derived neurotrophic factor (GDNF) (PeproTech, Rocky Hill, NJ, USA), 100 μg/ml transferrin, 60 ng/ml progesterone, 16 μg/ml putrescine, 40 ng/ml sodium selenite, 5 μg/ml insulin, 1 mM sodium pyruvate, 2 mM l-glutamine, 40 ng/ml triiodothyronine, 1 μg/ml laminin, 2.2 μg/ml isobutylmethylxanthine, 417 ng/ml forskolin, 5 μg/ml N-acetyl cysteine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Both spinal neurons and enriched spinal motor neurons were plated at 10,000 cells in 0.1 ml per well in 96-well polystyrene tissue culture trays previously coated with poly-d-lysine and then human placental laminin (Sigma, St. Louis, MO, USA). To limit astrocyte contamination, 100 nM of cytosine arabinoside was added to and maintained in culture medium from days 2 to 8 after plating. Growth medium components were limited to B27 minus antioxidants, insulin, laminin, and L-glutamine, penicillin, streptomycin, BDNF, CNTF, and GDNF on day 8. Wells were examined for any nonneuronal cells on day 8 with phase-contrast microscopy. Only seven out of 180 total wells contained cells with nonneuronal morphology and were excluded from analysis prior to adding treatments. Cells were treated with vehicle (0.1% dimethyl sulfoxide (DMSO); Sigma) or 100 nM CDDO-amide on day 8 and then analyzed after 20 h. Randomized differential interference contrast (DIC) images were captured before and after treatment using a PixeLINK (model PL-A662) digital camera mounted on a bright-field (Nikon Diaphot-TMD) microscope.

Cell lines The CNS-1 astrocytoma cell line was previously developed in Lewis rat by repeated injections of the carcinogen N-nitroso-N-methylurea (Kruse et al. 1994). Tumorigenic BV-2 microglia were previously developed by infecting mouse primary microglia cultures with a v-raf/vmyc-oncogene-carrying retrovirus (Blasi et al. 1990). EOC.13 microglia is a colony-stimulating factor-1-dependent line previously developed from mouse microglial precursors (Walker et al. 1995). EOC.13 cells required the addition of conditioned medium (20%) from mouse bone marrow cell line LADMAC as a source of colony-stimulating factor 1. NSC-34 (Cedarlane Laboratories, Burlington, NC) is a hybrid cell line, produced by fusion of motor-neuronenriched, embryonic mouse spinal cord cells with mouse neuroblastoma (Cashman et al. 1992).

Cell culture medium DMEM/high-glucose base medium (HyClone Laboratories, Inc., Logan, Utah) with 10% fetal bovine serum (HyClone) and supplemented with L-glutamine, penicillin, and streptomycin was used for all cell types except for primary spinal neuron cultures. The fetal bovine serum was heat-inactivated and charcoal-stripped for cell cultures used to measure NQO1 activity. All cell cultures were incubated at 37°C and 6% CO2.
Treatments with CDDO derivatives CDDO derivatives were generously supplied by Drs. Michael Sporn and Karen Liby (Dartmouth Medical School). Each molecule had a different chemical functional group at carbon 28 (Fig. 1). Compounds were dissolved in DMSO and stored at −20°C. Cells were treated 1 day after being plated except for endotoxinstimulated microglia experiments which were treated 4 h after plating. Vehicle controls and all concentrations of CDDO treatments contained 0.1% DMSO in cell culture medium.

Cell viability determinations The reduction of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan is an indicative marker of viable cells (Mosmann 1983). Cells were plated at 12,500 cells per well in 96-well culture trays. After treatment with vehicle or CDDO derivatives, culture medium was replaced with 50 μl of 0.5 mg/ml MTT (Sigma, St. Louis, MO) in culture medium, and trays were incubated for 3 h. Formazan was solubilized by adding 50 μl of 10% SDS, 5% isopropanol, and 0.012 N HCl solution and incubating for 3 h. The absorbance was read at 570 nm in a microplate reader (MRX Revelation, Dynex Technologies, Chantilly, VA, USA), and values are expressed as an average percent of an untreated control group ± SEM. To confirm MTT reduction as a viable cell marker, cells were evaluated by phasecontrast microscopy and scored as normal (no different from untreated condition), partial death, and near-complete cell death for each treatment condition prior to addition of MTT. Normal and near-complete death were referenced with untreated (no vehicle) and supertoxic levels (1 mM) of hydrogen-peroxide-treated groups for qualitative assessments. Trends between qualitative microscopic observations and MTT reduction levels were similar.
The concentration at which 50% of the cells were lost (LC50) was calculated by probit analysis. Nonlinear regression was performed and a sigmoidal curve with variable slope was fitted to each of the data sets using GraphPad Prism 4. The equation used for the sigmoidal curve with variable slope was: Y ¼ bottom þðtop bottomÞ= 1 þ 10ðlogLC50XÞHillslope where X is the logarithm of concentration; Y is the percent viability; bottom is the Yvalue at the bottom plateau; top is the Y value at the top plateau; logLC50 is the X value of viability halfway between top and bottom; Hill slope is the Hill coefficient or slope factor (controls slope of curve). For curve fitting, only the mean value of each data point, without weighting, was considered. The LC50 was calculated from the x value of the response halfway between top and bottom plateau.

NQO1 measurement Induction of cytosolic NQO1 activity was used as an indicator of Nrf2–ARE activation as described previously (Prochaska and Santamaria 1988; Fahey et al. 2004). Cells were plated at 12,500 cells per well in 96-well culture trays. After treatment with vehicle or CDDO derivative for 20 h, culture medium was discarded and cells were rinsed with phosphate-buffered saline (PBS). Cells were then lysed in 45 μl of PBS by repeated freezing at 80°C and then thawing at 37°C three times. Reaction mixture (200 μl per well) of 25 mM Tris– HCl, 0.067% bovine serum albumin, 0.001% Tween-20, 2 U/ml glucose-6-phosphate dehydrogenase (Worthington Biochemical Corp., Lakewood, NJ), 0.28 mg/ml glucose-6-phosphate (Sigma) , 30 μM NADP (Sigma), 5 μM FAD (Alexis Biochemicals, San Diego, CA), 0.31 mg/ml MTT, and 50 μM menadione (MP Biochemicals, Solon, OH) was added. The reduction of MTT to formazan was measured at 570 nm in a microplate reader 5 min after adding reaction buffer. Specificity for NQO1 activity was confirmed with the lack of color change in conditions omitting menadione or containing the reaction inhibitor dicoumarol (MP Biochemicals) at 0.1 mg/ml. Values are expressed as the average absorbance of treatment groups divided by absorbance of vehicle group ± SEM.

Protection from oxidative toxicity Primary glial, CNS-1, or NSC-34 cells were plated at 15,000 cells per well in 96-well trays, and CDDO derivatives (100 nM) were added the following day. After 20 h, hydrogen peroxide (Fisher Scientific, Fair Lawn, NJ) or ethanol (Decon Labs, Inc., King of Prussia, PA) was added, and a viability assay (MTT reduction) was performed the next day. Concentrations of hydrogen peroxide (250 or 500 μM) were selected based on the level that induced greater than a 40% reduction in viable cells after 1 day. Serial dilutions of ethanol were made in DMEM base media and then added at one third of final cell culture volume. Values are expressed as the average percent of viable cells relative to the vehicle-pretreated group ± SEM following hydrogen peroxide toxicity.

Nitric oxide production Microglial cells were plated at 10,000 cells per well in 96-well culture trays or 50,000 cells per well in 24-well trays. After a 20-h treatment with vehicle or CDDO, microglia were stimulated with the endotoxin LPS (derived from Escherichia coli 055:B5, Sigma). An LPS concentration of 1 μg/ml was used to stimulate BV-2 and EOC.13 cells, whereas 0.1 μg/ml was used with primary microglia. Culture supernatant was assessed for nitrite levels using the Griess assay 2 days after adding endotoxin. Supernatant was mixed with equal volumes of 1% sulfanilamide (Sigma) in 5% phosphoric acid and 0.1% N-(1-naphthyl)-ethylenediamine (Sigma); the absorbance was measured at 530 nm. Values are expressed as average percent of nitrite levels relative to the endotoxin-stimulated vehicle group ± SEM.

Superoxide anion generation Intracellular superoxide anion production was measured based on the reduction of nitro blue tetrazolium (NBT; Sigma) to formazan with modifications to a previous protocol (Choi et al. 2006). In brief, BV-2 cells were plated at 10,000 cells per well in a 96-well tray and then treated with vehicle of CDDO derivative 4 h later. Media (unprimed) or 0.1 μg/ml LPS (primed) was added 20 h later and incubated overnight. Then, NBT (0.1%, mass/volume) in media with or without 1 μM phorbol-12-myristate-13-acetate (PMA; Sigma) was added and incubated at 37°C for 1 h. Media supernatant was discarded; cells were rinsed with PBS followed by methanol, air-dried for 5 min, and then solubilized in 48 μl 1 M KOH plus 56 μl DMSO. Absorbance was read at 630 nm after 10 min. Values are expressed as average percent of absorbance relative to vehicle-pretreated group plus SEM.

Statistics The statistical evaluation was performed using GraphPad Prism 4 on pooled data of two or more independent experiments using a one-way ANOVA followed by Dunnett’s test for comparison of multiple dose levels against vehicle. Unpaired Student’s T test was used to determine significant difference between two groups of data. A P value of less than 0.05 was considered significant.

Results

NQO1 induction in primary glia and glial cell lines Glial cultures derived from perinatal rat brain and embryonic spinal cord were used to assess changes in intracellular NQO1 levels following a 20-h treatment with CDDOamide, CDDO-BE, CDDO-IM, or CDDO-TFEA. These cultures were comprised predominantly of astrocytes with a nanomolar concentrations of CDDO-amide, CDDO-BE, CDDO-IM, or CDDO-TFEA for 20 h. The absorbance of NQO1mediated MTT reduction was measured in cell lysates after 5 min with reaction mixture and expressed as the change from vehicle-treated cells. Viability was assessed after 20-h exposure to vehicle or CDDO derivatives in separate experiments, and values are expressed as the percent absorbance change of MTT reduction in vehicle-treated group. Significant difference (P<0.05) from vehicle group was determined using Dunnett’s multiple comparison test and represented by a (amide), b (BE), i (IM), or t (TFEA) either above (increase) or below (decrease) the data point (n>12 for NQO1; n>6 for viability) minor microglia population. Nanomolar levels of CDDO derivatives caused a concentration-dependent increase in NQO1 in glia derived from perinatal rat brain or embryonic spinal cord (Fig. 2). CDDO-IM was the most potent and effective, whereas CDDO-BE was the least. To determine whether NQO1 was induced in astrocytes and/or microglia, transformed cell lines of rat astrocytes (CNS-1 astrocytoma cells) and murine microglia (BV-2 and EOC.13) were also 
evaluated. CDDO derivatives increased NQO1 in CNS-1 astrocytes, but not in BV-2 and EOC.13 microglia (Fig. 2). In BV-2 microglia, a decrease in NQO1 relative to basal levels was observed with CDDO-amide and CDDO-BE. Viability of glial cells was not reduced between 1 and 100 nM except for those with CDDO-IM which caused a 9% decrease in glial cultures derived from embryonic spinal cord at 100 nM. Reductions in viability occurred with most derivatives and cell types at 250 nM. CDDO-amide and CDDO-BE actually resulted in a small increase in viable cells in EOC.13 and BV-2 cells compared to vehicle-treated controls, respectively.
NQO1 induction in a motor neuron cell line and primary spinal motor neurons Intracellular NQO1 levels were first measured in NSC-34 motor-neuron-like cells following exposure to CDDO derivatives for 20 h. Elevated NQO1 levels with maximum increases of 14–27% relative to basal levels were determined (Fig. 3a). CDDO-BE and CDDOIM at 250 nM reduced viability. CDDO-TFEA was no longer effective at 250 nM despite the fact that it did not cause a loss of cell viability. A small increase in viable cells was observed with CDDO-BE at 10 nM. Next, 100 nM of CDDO-amide was tested in 8-day-old primary spinal neuron cultures. After 20 h, a 39% and 30% increase of NQO1 relative to basal levels was observed in motorneuron-enriched and non-motor-neuron-enriched cultures, respectively (Fig. 3b, c). No change in neuron density was observed with 1-day exposure to 100 nM CDDO-amide relative to vehicle-treated cells.
Hydrogen peroxide toxicity in primary astrocytes and a motor neuron and astrocyte cell line Based on the concentrationdependent induction of NQO1 in astrocytic and neuronal cells, 100 nM of each CDDO derivative was selected to test for protection against hydrogen-peroxide-induced toxicity. Pretreatment with CDDO-amide, CDDO-IM, and CDDOTFEA for 20 h increased the overall cell viability in glial cultures observed 1 day following exposure to 500 μM of hydrogen peroxide (Fig. 4). All four CDDO derivatives were highly effective in protecting CNS-1 astrocytoma cells from hydrogen peroxide toxicity (Fig. 4). In NSC-34 motor neuron cells, only CDDO-amide pretreatment increased cell viability following exposure to 250 μM of hydrogen peroxide (Fig. 4). CDDO-BE pretreatment actually resulted in lower viability relative to pretreatment with the vehicle alone (Fig. 4).
Ethanol toxicity in a motor neuron cell line Exposure to ethanol at millimolar concentrations for 1 day reduced cell viability in NSC-34 neuronal cultures. This finding is similar to those noted using comparable ethanol concentrations with other neuron types (Ku et al. 2006; Sun et al. 1997; Li et al. 2001). Pretreatment with 100 nM CDDOamide attenuated cell loss seen with ethanol concentrations between 250 and 1,000 mM (Fig. 5).
Endotoxin-induced nitric oxide production in primary microglia and microglial cell lines The average nitrite levels in BV-2 and EOC.13 microglial culture supernatants 2 days following administration of 1 μg/ml LPS was 47 and 32 μM, respectively (Fig. 6a). A reduction in viable BV-2 (43%) and EOC.13 (28%) cells also occurred 2 days following LPS administration. Pretreatment with CDDO derivatives for 20 h prior to adding LPS caused a concentration-dependent reduction in nitric oxide production based on nitrite levels (Fig. 6b). More than an 80% reduction in nitrite levels occurred with 100 nM of CDDO derivates in BV-2 and EOC.13 microglia. A small decrease in viable cells was only observed with 100 nM of CDDOIM in EOC.13 cells. An increase in viable cells following LPS activation occurred at similar concentrations of CDDO derivative pretreatment that were highly effective at reducing nitric oxide production. Microglia-enriched cultures derived from perinatal brains and embryonic spinal cords were also examined. Pretreatment with CDDO derivatives decreased LPS-stimulated nitric oxide production (Fig. 6c). CDDO-IM resulted in the lowest nitrite levels but also caused a large reduction in viability in these primary microglia cultures at 100 nM. A small decrease in viability was also observed with 100 nM of CDDO-TFEA in microglia derived from brain tissue. CDDO-BE was the least effective in reducing LPS-stimulated nitric oxide production and also revealed a biphasic trend in EOC.13 and microglia derived from embryonic spinal cord.
Since attenuation of immunological activation occurred with some of the CDDO derivatives at the lowest nanomolar concentration tested and CDDO-BE revealed an unexpected biphasic pattern, picomolar concentrations were evaluated in the BV-2 and EOC.13 microglia. Only moderate reductions in LPS-stimulated nitric oxide production were observed with 100 pM (Fig. 7). Unexpectedly, CDDO-IM caused increased levels of nitrites at 1 and 10 pM in both cell lines. Enhanced nitric oxide synthesis was not observed with CDDO-amide or CDDO-BE at picomolar levels.
Superoxide anion generation in BV-2 microglial cell line Next, the effect of pretreatment with 100 nM CDDO-amide on NADPH oxidase-generated free-radical production was examined in LPS-stimulated BV-2 cells. PMA-induced superoxide anion increase was suppressed by CDDOamide (Fig. 8).
Primary and cell line astrocytic, neuronal, and microglial viability with high concentrations of synthetic triterpenoid derivatives Cell cultures were exposed to increasing concentrations of CDDO derivatives for 20 h and then viability was assessed. The LC50s were derived from fitting the change in viability relative to vehicle-treated cells to a variable slope sigmoidal dose–response curve. CDDO-amide had the highest LC50 value for astrocytic, neuronal, and microglial cells relative to the other three derivatives (Table 1). Reductions in viable microglia typically revealed steeper Hill slopes relative to astrocytic and neuronal cells, indicating a small concentration difference between no effect and large losses. NSC-34 motor neuron cells revealed the flattest (closer to zero) Hill slopes of the different cell types.

Discussion

Directtreatmentofprimarygliaandneuronsandcorresponding representative cell lines with semisynthetic triterpenoids was employed to delineate effects based on cell type and chemical modifications of CDDO. Both cytoprotective and antiinflammatory properties were observed. Chemical modifications at C-28 of CDDO changed the effectiveness and toxicity. CDDO and several of its derivatives have been reported to activate the Nrf2–ARE pathway, most probably by dissociating Nrf2 from Kelch-like ECH-associated protein 1 (KEAP1) in the cytoplasm (Liby et al. 2005; DinkovaKostova et al. 2002, 2005; Kobayashi et al. 2006). Transcription of ARE results in the synthesis of many antioxidant and phase II detoxifying enzymes including NQO1, catalase, SOD1, heme oxygenase 1, and enzymes involved in glutathione production (Chen and Kunsch 2004). The CDDO derivatives evaluated in this study induced NQO1 expression in astrocytes and spinal neurons. A 190% maximal increase above basal levels was achieved in astrocytes, whereas only a 39% increase occurred in neurons. The effects in primary spinal neurons and the spinal motor-neuron-like cell line, NSC-34, were very similar. This differential effect between astrocytic and neuronal cells was not unexpected. Other known inducers of the Nrf2–ARE pathway have also shown a disparity between astrocytes and neurons (Murphy et al. 2001; Johnson et al. 2002), and neurons have been reported to have lower basal levels of Nrf2 relative to astrocytes (Shih et al. 2003). Nrf2 activation has also been demonstrated in SH-SY5Y cells, resulted in elevations in nitrites and reductions in the level of viable cells relative to nonstimulated conditions. Significant difference (P<0.05) from vehicle group was determined using Student’s T test and represented by asterisks (n>56 for nitrite; n>46 for viability). b Changes in nitric oxide production and level of viable cells were determined with 20-h pretreatment with vehicle (0.1% DMSO) and nanomolar concentrations of CDDO-amide, CDDO-BE, CDDO-IM, or CDDO-TFEA prior to LPS stimulation. c Changes in nitric oxide production and level of viable cells were also determined in microglia-enriched primary cultures derived from rat brain or spinal cord that were stimulated with 0.1 μg/ml LPS. Values are expressed as the percent absorbance change in vehicle-pretreated group. Significant difference (P<0.05) from vehicle group was determined using Dunnett’s multiple comparison test and represented by a (amide), b (BE), i (IM), or t (TFEA) either above (increase) or below (decrease) the data point (n>11 for nitric oxide; n>7 for viability) a cell line derived from a human neuroblastoma, by CDDO with a methyl amide group at C-28 (Yang et al. 2009). CDDO with IM at C-28 was the most potent and effective inducer of NQO1 in primary astrocytic cell cultures, whereas CDDO-BE was the least. NQO1 was not elevated in BV-2 and EOC.13 microglial cell lines at CDDO derivative concentrations that induced effects in astrocytes and neurons. The lack of NQO1 induction in microglial cells was unexpected, but this does not necessary indicate that the Nrf2–ARE pathway was not activated. Other known inducers of the Nrf2–ARE pathway have increased other downstream products such as heme oxygenase 1 (Chen et al. 2005; Jeong et al. 2009; Ishii et al. 2000; Innamorato et al. 2008), but evidence of NQO1 induction in microglia in the literature is lacking.
Activation of the Nrf2–ARE pathway is known to protect against oxidative damage such as with toxic levels of hydrogen peroxide (Shih et al. 2003; Kraft et al. 2004; Kwak et al. 2007). Since NQO1 was induced in astrocytic and neuronal cells by each CDDO derivatives at 100 nM, this concentration was selected to pretreat cells before exposure to toxic levels of hydrogen peroxide. An increased number of viable cells in primary glial cultures occurred with CDDO-amide, CDDO-IM, and CDDO-TFEA. CDDO-BE was the least effective in inducing NQO1 and was not effective in protecting cells from peroxide toxicity. All four derivatives were protective in the CNS-1 astrocytoma cells to an even greater degree than in primary cultures of astrocytes. A greater fold change in NQO1 production relative to basal levels also occurred in CNS-1 cells, indicating a stronger response to CDDO derivatives by these neoplastic astrocytes. The primary glial cultures were predominantly astrocytes, but the presence of some microglia might explain the lower degree in NQO1 induction in addition to CNS-1 being a transformed neoplastic cell line. All four derivatives induced a small increase in NQO1 in NSC-34 neurons; however, only pretreatment with CDDOamide increased viability in the NSC-34 motor-neuron-like cells following toxic levels of hydrogen peroxide. Ethanol toxicity in neuronal cultures involves oxidative damage (Sun et al. 1997; Mitchell et al. 1999). CDDO-amide also attenuated ethanol-induced toxicity in NSC-34 cells. It is important to note that activation of the Nrf2 pathway in astrocytes is also relevant for protecting against neuronal loss by proxy as demonstrated with transgenic overexpression of Nrf2 in astrocytes and containing the mutant human superoxide dismutase enzyme, a murine model of motor neuron disease (Vargas et al. 2008).
CDDO and several of its derivatives have been reported to attenuate macrophage immunological activation with changes in pathways related to NF-κB (Yore et al. 2006), PPAR-γ (Wang et al. 2000), STAT3 (Liby et al. 2006; Ahmad et al. 2008), and Nrf2 (Dinkova-Kostova et al. 2005; Thimmulappa et al. 2006). Nrf2 deficiency has also been shown to exacerbate LPS-induced inflammation while Nrf2 activation attenuates it (Thimmulappa et al. 2006; Lin et al. 2008; Koh et al. 2009; Innamorato et al. 2008; Nagai et al. 2009; Brandenburg et al. 2010), implicating this transcription factor as a promising target for immunomodulation. Although NQO1 was not induced in the microglial cell lines, each CDDO derivative tested attenuated LPS-stimulated nitric oxide production in microglia. CDDO-BE was the least effective for reducing nitric oxide synthesis, which was a similar trend observed for NQO1 induction in astrocytes. CDDO-amide and CDDO-IM had similar dose–responses in both the BV-2 and EOC.13 microglial cell lines at nanomolar concentrations, but CDDO-IM had an opposite effect on LPS-induced nitric oxide production at picomolar levels. Although small, this effect at picomolar concentrations was unexpected. Each derivative was also examined in primary microglia cultures at nanomolar concentrations. CDDO-IM appeared to be highly effective in reducing nitric oxide relative to CDDOamide but reduced cell viability likely contributed to these lower levels of nitrite accumulation. Nitric oxide can react with other molecules such as with superoxide anion to form peroxynitrite, which is highly reactive and is considered a prominent damage-causing product involved in neuroinflammation (Beckman et al. 1990; Ebadi and Sharma 2003; Boje 2004). CDDO-amide pretreatment prevented PMAinduced superoxide anion generation in LPS-stimulated BV-2 microglia. Although reduced de novo synthesis of inducible nitric oxide synthase induced by triterpenoids has been demonstrated (Suh et al. 1998), the mechanism involved in reduced levels of superoxide anion requires further investigation. Additionally, pretreatment of CDDO with a methyl ester at C-28 has been shown to attenuate cytokine production in murine microglia (Tran et al. 2008).
Semisynthetic triterpenoids have been shown to be well tolerated in rodents and nonhuman primates (Kral et al. 2005). However, very high doses are known to induce cell death in vitro via a mechanism reported to involve mitochondrial cytochrome c release and caspase-3,8,9 activation (Ito et al. 2001; Ikeda et al. 2003; Konopleva et al. 2004; Deeb et al. 2009). Mechanisms of cell death were not evaluated in the current study, but LC50 values were determined using the MTT reduction assay that determines the level of viable cells based on the presence of functional mitochondria. For most cell types and CDDO derivatives, the metabolically effective concentration of 100 nM did not reduce cell viability. For each neural cell type analyzed, CDDO-amide revealed LC50 values that were typically twice the concentration as compared to the LC50 values for the other three derivatives. Interestingly, the Hill slope for loss of viability as a function of concentration was typically steeper in microglia compared to astrocytic and neuronal cells. This indicates a smaller range of concentration tolerance between no loss of viable cells and large losses. It is unknown why microglia were more sensitive, but targeting cell death in cells of the immune system is of pharmacological relevance in diseases involving autoimmunity or overactive immunological responses.
The different functional groups at C-28 of CDDO altered the pharmacodynamics in neurons and glia. A proposed molecular mechanism for semisynthetic triterpenoids is the binding of electrophilic Michael addition sites (α, β unsaturated carbonyl groups) on rings A and C of CDDO and its derivatives to one or more critical cysteines on protein targets (Couch et al. 2005). Sulfhydryl groups on KEAP1 are reportedly targets of several inducers of Nrf2– ARE (Dinkova-Kostova et al. 2005). CDDO-IM was highly effective and potent for the induction of NQO1 in astrocytic cells, whereas CDDO-BE was the least effective. This suggests that the IM group at C-28, relative to BE, resulted in CDDO having a greater ability to activate Nrf2, thus resulting in increased transcription at the ARE. CDDO-BE was also the least effective for attenuation of nitric oxide production in activated microglia, indicating a possible similar molecular target as for Nrf2 activation in astrocytes. Despite being less effective for inducing NQO1 synthesis and attenuating nitric oxide production, CDDO-BE had similar effects at higher concentrations on cell viability relative to CDDO-IM. CDDO-amide was comparatively effective in inducing cytoprotective and anti-inflammatory properties as CDDO-IM but had twofold to sevenfold higher LC50 values. Taken together, this suggests that the molecular target involved in reduced cell viability at higher CDDO concentrations is different than that for Nrf2 activation. The complexity of the potential molecular targets involved in various downstream events needs to be explored further, but these results support the importance and capabilities of chemical modifications of CDDO.
Oxidative/nitrosative damage and chronic innate immunological activations are promising therapeutic targets for many inflammatory and neurological disorders. A single reagent that can reduce free-radical generation in activated microglia and protect against free-radical damage would be ideal. Further understanding of the structural changes that improve the pharmacodynamics of semisynthetic triterpenoids could lead to newly designed drugs that are effective for cellular protection and reducing inflammation in neurological disease states. Their ability to access the CNS tissue following systemic administration needs to be explored further and may also be controlled by chemical modifications at C-28.
Overall, this study reveals that CDDO-amide is an attractive compound to test further in animal models of neurological disorders based on its effectiveness in cytoprotection and immunomodulation at nanomolar concentrations in cells of both neuronal and glial lineages.

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