Role of PPAR-γ Ligands In Neuroprotection against Glutamate-Induced Cytotoxicity in Retinal Ganglion Cells

Abstract

purpose. The peroxisome proliferator-activated receptor-γ (PPAR-γ) is the target of the insulin sensitizing thiazolidinediones (TZDs), a class of drugs used in the treatment of type 2 diabetes mellitus. Glaucoma and other retinal disorders are some of the major complications in diabetes. In the present study, the role that PPAR-γ ligands play in protecting retinal ganglion cells (RGC-5) against glutamate insult was explored.

methods. Transformed rat RGC (RGC-5 cells) and two PPAR-γ agonists, 15-deoxy-d ^12,14-prostaglandin J2 (15d-PGJ2) and troglitazone were used. RGC-5 cells were incubated with either of the PPAR-γ ligands and were exposed to either l-glutamic acid or buthionine sulfoximine (BSO). Cell viability was determined with the neutral red dye uptake assay. Levels of PPAR-γ receptor proteins were monitored by immunoblot analysis.

results. Glutamate treatment resulted in RGC-5 cell death, and both 15d-PGJ2 and troglitazone protected the RGC-5 cells from glutamate cytotoxicity. The neuroprotective concentrations of both compounds ranged from approximately 1 to 5 μM. Troglitazone further protected against BSO toxicity, whereas 15d-PGJ2 did not. Glutamate treatment appears to exert its cytotoxicity through oxidative damage, because pretreatment of RGC-5 cells with the antioxidants N-acetyl cysteine (NAC) and thiourea resulted in the reversal of glutamate cytotoxicity. Furthermore, the glutamate effect was not reversed by pretreatment with MK801 or dl-threo-betabenzyloxyaspartate (dl-TBOA), suggesting that glutamate cytotoxicity is not mediated through the NMDA receptor and/or glutamate transporter, respectively. Levels of PPAR-γ receptor protein did not show any appreciable change in response to glutamate exposure, with or without 15d-PGJ2 or troglitazone.

conclusions. Two PPAR-γ ligands, 15d-PGJ2 and troglitazone, protect RGC-5, an established transformed rat retinal ganglion cell line, against glutamate cytotoxicity. The neuroprotective effects of the two compounds appear to be mediated through an antioxidant rather than a PPAR-γ–dependent pathway. These results suggest that PPAR-γ agonists, in addition to improving insulin sensitivity, may also provide a valuable antioxidant benefit that could prove valuable in targeting ocular complications including glaucoma.

Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear hormone receptor superfamily. Three different subtypes of PPAR (α, β/δ, and γ) coded by three separate genes have been identified in rodents and humans. ¹ PPAR-α is highly expressed in the liver and mediates the induction of enzymes of the peroxisomal fatty-acid oxidation pathways. ² PPAR-β or δ is ubiquitously expressed in a broad range of mammalian tissues ³ and in the adult rat. ¹ PPAR-γ is highly expressed in brown and white adipose tissues and, to a lesser extent, in large intestine, retina, and some parts of the immune system. ⁴ PPAR-γ regulates the process of adipogenesis ^{5 6} and is the target for the insulin-sensitizing thiazolidinediones (TZDs; i.e., ciglitazone and troglitazone) class of drug, ⁷ and PPAR-γ agonists are used for the treatment of type 2 diabetes mellitus. Rosiglitazone and pioglitazone are two TZD drugs that are used clinically for the treatment of type II diabetes. In addition to synthetic ligands, 15-deoxy-d ^12,14-PGJ2 (15d-PGJ2) appears to be a natural ligand for PPAR-γ with an EC₅₀ of 7.0 μM. ⁸  

Ocular complications from diabetes represent a significant public health problem. Because eye diseases represent an end-organ response to a generalized medical condition, all structures of the eye are susceptible. ⁹ Diabetic retinopathy alone represents a leading cause of new cases of blindness each year in the United States. ¹⁰ Diabetes is also a risk factor for open-angle glaucoma, ¹¹ which is characterized by elevated intraocular pressure, optic nerve degeneration, and visual field loss. ¹¹  

Glutamate acts as a normal neurotransmitter in the retina, but its high levels may be neurotoxic both in vivo, when injected in vitreous, or in vitro when used as a treatment of cultured retinal neuronal cells, and results in apoptosis of retinal ganglion cells. ^{12 13 14 15} Although controversial, elevated glutamate levels may exist in the vitreous humor of patients with glaucoma, and the major causes of cell death from glutamate are the influx of calcium into cells and the generation of free radicals. ^{16 17 18 19 20 21}  

There is increasing interest in the role of oxidative stress and its potential role in diabetogenesis and development of the diabetic complications, atherosclerosis and associated cardiovascular disease. Lipid peroxidation is increased in patients with diabetes mellitus. ²² Reactive oxygen species modulate various biological functions by stimulating transduction signals, some of which are involved in diabetes pathogenesis and complications. ²³ Glutamate also stimulates the production of large quantities of nitric oxide (NO) and produces the free radical superoxide anion in mitochondria. ²⁴ Nitric oxide reacts with the superoxide anion, forming peroxynitrite, which triggers cell death by apoptosis. ²⁵  

In the present study, we investigated the effects of PPAR-γ ligands on a retinal ganglion cell line, RGC-5, in response to oxidative stress induced by glutamate treatment in an acute in vitro model of experimental glaucoma. Both 15d-PGJ2 and troglitazone protected RGC-5 cells against glutamate insults, and troglitazone further protected against buthionine sulfoximine (BSO) toxicity. 

Materials and Methods

Materials

15d-PGJ2 and troglitazone were purchased from Biomol (Plymouth Meeting, PA). (+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK801) and dl-threo-β-benzyloxyaspartate (dl-TBOA) were purchased from Tocris Cookson (Ballwin, MO). BSO, N-acetyl cysteine (NAC), and thiourea, were purchased from Sigma-Aldrich (St. Louis, MO). The antibody against PPAR-γ was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). 

Culture of the Retinal Ganglion Cell Line RGC-5

Cultures of the RGC-5 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum, 100 U/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich) in a humidified atmosphere of 95% air and 5% CO₂ at 37°C. The RGC-5 cells had a doubling time of approximately 18 to 20 hours and were passaged by trypsinization every 3 to 4 days. ²⁶  

Glutamate Cytotoxicity and Effect of Various Drugs on Retinal Ganglion Cells

The RGC-5 cells were seeded at 10,000 cells/well in 24-well plates. After 24 hours, cells were either subjected to troglitazone or 15d-PGJ2 at various concentrations and incubated for another 24 hours at 37°C in a humidified chamber incubator. After incubation, cells were treated with 5 mM l-glutamic acid or 10 μM BSO. The two PPAR-γ agonists were present during the glutamate treatment of the cells. Twenty-four hours after glutamate or BSO treatment, cell viability was determined using a neutral red dye uptake assay. ²⁶ To determine the effect of NAC and thiourea, an overnight culture of RGC-5 cells was simultaneously treated with either NAC or thiourea along with 5 mM glutamate for 24 hours. Non–drug- and glutamate-treated cells were also included as a control. Briefly, to conduct the neutral red dye assay, ²⁶ growth medium was washed from the cells with HEPES buffer (125 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 2 mM MgCl2, 0.5 mM NaH₂PO₄, 5 mM NaHCO3, 10 mM d-glucose, and 10 mM HEPES [pH 7.2]). Neutral red dye was added to a final concentration of 0.033% in HEPES buffer and incubated for 2 hours at room temperature. After the neutral red dye uptake by living cells, they were gently washed with 2 to 4 volumes of HEPES buffer to wash off the excess dye. Cells were allowed to air dry for 20 minutes and then treated with ice-cold solubilization buffer (1% acetic acid/50% ethanol; 300 μL) to extract the dye taken up by the cells. Twenty minutes later, 100-μL aliquots were transferred to wells of flat-bottomed 96-well plates, and optical densities of samples were read at 570 nm. 

Preparation of Cytoplasmic and Nuclear Extracts

The RGC-5 cells were plated in 100-mm dishes, and the next day, 15d-PGJ2 or troglitazone were administered to the RGC-5 cultures. After a 24-hour incubation period with the PPAR-γ agonists, glutamate (5 mM) was administered and incubated for an additional 24 hours. After this, the nuclear and cytoplasmic extracts were prepared. ²⁷ Briefly, the cells were suspended in 100 μL of buffer C (10 mM HEPES [pH 7.9], 1.5 mM MgCl₂, 10 mM KCl, 10% glycerol, 1 mM dithiothreitol [DTT], and 0.5 mM phenylmethylsulfonyl fluoride [PMSF]) and incubated on ice for 15 minutes. Then, 3 μL of 10% NP-40 was added to the suspension and then vortexed briefly and the nuclei were pelleted by centrifugation at low speed. The supernatant (cytoplasmic extract) was collected and stored at −80°C. The nuclear pellet was resuspended in 70 μL of buffer D (20 mM HEPES [pH 7.9], 400 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 20% glycerol, 1 mM DTT, and 0.5 mM PMSF) and incubated for 20 minutes at 4°C before being centrifuged at 8000g for 5 minutes. The resultant supernatant containing extracted nuclear proteins was transferred to a fresh microfuge tube and stored at −80°C until used for immunoblot analysis. 

Immunoblot Analysis for PPAR-γ Receptor

Equal amounts of cytoplasmic and nuclear proteins, as determined by the Bradford method, were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA) in an electrophoresis apparatus (Trans-Blot; Bio-Rad, Hercules, CA) at 100 V for 2 hours, using Towbin’s buffer (25 mM Tris, [pH 8.3], 192 mM glycine, and 20% methanol). The membranes containing immobilized proteins were blocked with 5% skim milk in assay buffer (20 mM Tris, [pH 7.5], and 0.5 M NaCl). A polyclonal PPAR-γ antibody (Santa Cruz Biotechnology) that cross-reacts with rat PPAR-γ was prepared in TS buffer and added to the transblots for overnight incubation. After the membranes were washed, they were incubated with a goat anti-rabbit IgG horseradish peroxidase (HRP)–conjugated antibody as the secondary antibody in assay buffer. Immunoreactive bands were visualized by a standard enhanced chemiluminescence (ECL) procedure. 

Statistical Analysis

Statistical significance was determined by one-way analysis of variance (ANOVA) followed by a Tukey multiple-comparison test. P < 0.05 was considered significant for all experiments. The results are reported as the mean ± SEM. 

Results

Effects of Glutamate on RGC-5 Cell Viability

To determine the effect of glutamate on RGC-5 cell viability, RGC-5 cells were treated with various concentrations of l-glutamate for a period of 24 hours. This treatment resulted in a dose-dependent decrease in cell viability (Fig. 1) . We investigated whether the cytotoxicity of glutamate in RGC-5 cells was mediated through either the glutamate transporter or the NMDA receptor. dl-TBOA was used to inhibit the sodium-dependent glutamate/aspartate (excitatory amino acid) transporter. ²⁸ MK801 was used to block NMDA receptor ion channels. ²⁹ At concentrations above their K _i values, neither dl-TBOA nor MK801 inhibited glutamate cytotoxicity (Fig. 2) . 

Mediation of Glutamate Cytotoxic Effects in RGC-5 Cells

To assess the possibility that glutamate treatment results in oxidative damage, we studied the effects of inclusion of two antioxidants, NAC and thiourea, in the glutamate cytotoxicity assay. Figure 3 shows that NAC reversed and thiourea attenuated the glutamate-induced cytotoxicity in RGC-5 cells, suggesting that, at a concentration of 5 mM, glutamate causes oxidative damage to RGC-5 cells and that this cytotoxicity can be reversed by antioxidants. 

Protective Effects of 15d-PGJ2 and Troglitazone on RGC-5 Cell Viability

To determine the neuroprotective effect of PPAR-γ agonists against glutamate-induced cytotoxicity, RGC-5 cells were preincubated with 15d-PGJ2 or troglitazone for 24 hours. The next day, glutamate was added to the cells containing either the 15d-PGJ2 or troglitazone. Cell viability was determined 24 hours later, and the cells were photomicrographed for morphologic changes. 

The results showed that glutamate treatment caused substantial loss of cells (Fig. 4A) . The inclusion of either 15d-PGJ2 or troglitazone protected the RGC-5 cells from glutamate toxicity (Fig. 4A) . To further quantify the neuroprotective effects of the two compounds, cell viability was assessed by neutral red assay (Fig. 4B) . At concentrations ranging from 1 to 5 μM, both 15d- PGJ2 and troglitazone significantly reduced the cytotoxicity of glutamate (Fig. 4B) . 

Effect of Glutamate, 15d-PGJ2, and Troglitazone on the Expression of PPAR-γ Receptor in RGC-5 Cells

We investigated the effects of glutamate, with or without 15d-PGJ2 or troglitazone, on the expression of PPAR-γ receptor protein in cytoplasmic and nuclear extracts of RGC-5 cells. Immunoblot analysis showed that there was no appreciable change in the expression levels of the PPAR-γ receptor in the various treatment groups compared with the control RGC-5 cells (Fig. 5) . 

Effects of PPAR-γ Agonists on RGC-5 Cell Viability from BSO Toxicity

BSO is a specific and irreversible inhibitor of γ-glutamylcysteine synthetase (γ-GCS), the rate-limiting enzyme in glutathione (GSH) biosynthesis. We used BSO to assess the effects of PPAR-γ agonists against this pro-oxidant in RGC-5 cells. Troglitazone protected RGC-5 cells against BSO cytotoxicity (Fig. 6) , whereas, 15d-PGJ2 did not (data not shown). Near maximal protection was conferred at the 5-μM concentration of troglitazone. 

Discussion

In the present study, 15d-PGJ2 and troglitazone protected retinal ganglion cells, RGC-5, from glutamate cytotoxicity. Troglitazone further protected RGC-5 cells from another pro-oxidant, BSO. Levels of PPAR-γ receptor protein did not show any appreciable change in response to glutamate exposure, with or without 15d-PGJ2 or troglitazone. The cytotoxic effects of glutamate were not reversed by pretreatment with MK801 or dl-TBOA, suggesting that glutamate cytotoxicity is not mediated through an NMDA receptor and/or glutamate transporter. Glutamate treatment exerted its cytotoxicity through oxidative damage; pretreatment of RGC-5 cells with two antioxidants, NAC and thiourea, reversed glutamate cytotoxicity. Thus, these findings suggest an antioxidant neuroprotective property of PPAR-γ ligands in retinal ganglion cells. 

The beneficial effects of PPAR-γ ligands on the ocular system have been supported by various reports. PPAR-γ ligands inhibits the vascular endothelial growth factor–induced choroidal angiogenesis in human retinal pigment epithelial cells and bovine choroidal endothelial cells. ³⁰ They also inhibit choroidal neovascularization (CNV) in vivo, ³⁰ suggesting the potential benefit of these agents in patients with age-related macular degeneration complicated by CNV. ³⁰ Ershov and Bazan ³¹ argue that the selective activation of PPAR-γ may play an important role in regulating the expression of target genes that are involved in lipid and fatty acid metabolism in the photoreceptor renewal process. 

Glutamate causes excitotoxicity by overstimulation of the NMDA glutamate receptor and generation of a cascade of events resulting in an overload of intracellular calcium and release of NO, which has free radical properties. NO combines with reactive oxygen species, causing oxidative damage, which may lead to apoptosis. Glutamate is essential as a normal neurotransmitter in the retina. However, high levels may be neurotoxic through an apoptotic mechanism, both in vivo when injected in vitreous or in vitro in cultured retinal neuronal cells. ^{12 13 14 15} The occurrence of neurotoxic glutamate levels in the vitreous body of patients with glaucoma, as well as in various animal models, is controversial, with some, but not all, studies showing an increase with elevated intraocular pressure. ^{16 17 18 19 20 21} Nevertheless, treatment of retinal ganglion cells with glutamate results in a glaucomatous phenotype (i.e., the apoptosis of retinal ganglion cells), ^{13 14 15 32 33 34} both in vivo and in vitro. In the present studies, glutamate was used to induce apoptosis in cultured rat retinal ganglion cells (RGC-5) as an in vitro model of glaucoma. RGC-5 cells are an established transformed rat retinal ganglion cell line that have been shown to be retinal ganglion cells based on the positive expression of Thy-1, Brn-3C, neuritin, NMDA receptor, GABA-B2 receptor, and synaptophysin and the negative expression of glial fibrillary acidic protein (GFAP), HPC-1, and 8A1. ²⁶  

In the present study, we exposed transformed rat retinal ganglion cells to high concentrations of glutamate (5 mM), which resulted in more than a 50% cell death after overnight incubation. This concentration of glutamate used in our studies is higher than the physiological concentrations to which the retinal ganglion cells are exposed in vivo and higher than that needed to kill primary retinal ganglion cells, as well as succinyl Concanavalin A [(s)ConA]-differentiated RGC-5 cells. ^{13 26 32 35} At this concentration, glutamate appears to induce a pro-oxidant–mediated cell death in RGC-5 cells, as suggested by the reversal of glutamate cytotoxicity by NAC and thiourea. In this model, we demonstrated, for the first time, that two PPAR-γ ligands, troglitazone and 15d-PGJ2, could protect retinal ganglion cells from the pro-oxidant toxicity induced by exposure to glutamate. These effects were robust, although complete protection was not observed over the 1- to 5-μM doses tested. 

Troglitazone, but not 15d-PGJ2, was also effective against BSO toxicity in RGC-5 cells. This apparent paradox may be due to distinctly different neuroprotective actions of the two compounds. Although both compounds are effective PPAR-γ agonists, studies in various cell models showed receptor-dependent, as well as, receptor-independent activities of various PPAR-γ ligands. Uryu et al. ³⁶ showed PPAR independent neuroprotective effects of troglitazone against both postglutamate neurotoxicity and low-potassium–induced apoptosis in cerebellar granule neurons. In an HT-22 mouse hippocampal cell model, we have shown that 15d-PGJ2 and troglitazone display different properties in achieving their neuroprotective effects. ³⁷  

Potential targets for PPAR-γ ligands are NF-κB, ^{38 39} other cell signaling pathways, ^{40 41} and the regulation of genes involved in neurodegeneration. ^{42 43}  

In the present study, we showed that neuroprotection by either troglitazone or 15d-PGJ2 in RGC-5 cells against glutamate cytotoxicity is likely to be independent of the PPAR-γ receptor. This conclusion is supported by the observation that there was no change in the PPAR-γ receptor protein levels with exposure to either troglitazone or 15d-PGJ2 in the presence of glutamate. 

The toxic effects of glutamate to mammalian retinal ganglion cells have been well documented. ^{12 13 14 15 16 17 18 19 20 21} The RGC-5 cells have been shown to express functional NMDA receptors. ²⁶ Furthermore, in sConA-treated RGC-5 cells, MK801 has been shown to reverse the toxic effects of l-glutamate (Ref. ²⁶ and unpublished observations). Thus, sConA treatment may result in differentiation of RGC-5 cells with respect to increased expression of NMDA receptors. In the present studies, RGC-5 cells were not pretreated with sConA, and thus they may not be as sensitive to MK801 as cells treated with sConA. Glutamate may be directly toxic to cultured RGC-5 cells through two different processes, both of which result in the production of free radicals. ⁴⁴ The classic pathway, known as excitotoxicity, occurs through the activation of NMDA and non-NMDA glutamatergic receptors. ⁴⁵ The oxidative glutamate toxicity pathway occurs through the inhibition of a cystine/glutamate antiporter. ⁴⁶ The competition by glutamate for the cystine/glutamate antiporter decreases the availability of intracellular cystine, the precursor of glutathione. 

In the current studies, glutamate cytotoxicity in RGC-5 cells was not reversed by the NMDA receptor antagonist MK801. Oxidative glutamate pathway is more likely to be involved in glutamate-mediated RGC-5 cell death. Earlier, and relevant to the present report, primary cultures of retinal ganglion cells were shown to be susceptible to lipid peroxidation induced by chemical hypoxia, hypoglycemia, and inhibition of glycolysis. ⁴⁷ Taken together, the present study suggests that PPAR-γ agonists, in addition to improving insulin sensitivity, may also provide a valuable antioxidant benefit that could be valuable in targeting ocular complications, including glaucoma. 

 Supported by the American Health Assistance Foundation-National Glaucoma Program (NA), and Grant AG10485 from the National Institute on Aging (JWS).

 Submitted for publication October 15, 2002; revised February 5, 2003; accepted February 24, 2003.

 Disclosure: P. Aoun, None; J.W. Simpkins, None; N. Agarwal, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Effects of l-glutamate on RGC-5 cell viability. RGC-5 cells were plated, incubated overnight, and treated with various concentrations of glutamate. Twenty-four hours later, cell viability was determined and expressed as the percentage of cells surviving compared with the control (non–glutamate-treated cultures). Data are the mean ± SEM, n = 4. *P < 0.05 vs. respective control.

Figure 2.

 

Effects of the NMDA receptor antagonist MK801 and glutamate transporter inhibitor dl-TBOA on RGC-5 cell survival during glutamate exposure. RGC-5 cells were plated, and 24 hours later, cells were preincubated with MK801 (100 mM) or dl-TBOA (100 mM). A day later, glutamate (5 mM) was added. Cell viability was determined after a 24-hour glutamate insult and expressed as the percentage of cells surviving compared with the control (non–glutamate-treated cultures). Data are the mean ± SEM; n = 4. *P < 0.05 versus respective control.

Figure 3.

 

Effects of NAC and thiourea on RGC-5 cell viability during glutamate exposure. RGC-5 cells were plated, and the next day, NAC (2 mM) or thiourea (7 mM), was added along with glutamate (5 mM). Twenty-four hours later, cell viability was determined and expressed as the percentage of cells surviving compared with the control (non–glutamate-treated cultures). Data are the mean ± SEM; n = 4. *P < 0.05 versus respective control; +P < 0.05 versus glutamate-treated cultures.

Figure 4.

 

(A) Effects of PPAR-γ agonists on RGC-5 cells viability during glutamate exposure. RGC-5 cells of an early passage were plated, and the next day, either 15d-PGJ2 or troglitazone was administered, along with an untreated control. Twenty-four hours later, glutamate (5 mM) was administered without the removal of either of the PPAR-γ agonists from the treated wells. Cells were observed microscopically for morphologic changes and photomicrographed 24 hours after glutamate insult. Glutamate cytotoxicity caused profound cell loss, with rounding up of the cells (G; arrowheads). Preincubation of RGC-5 cells with either 15d-PGJ2 or troglitazone reversed the cytotoxic effects of glutamate (P and T, respectively), and the cells appeared to be morphologically similar to untreated control RGC-5 cells (C). (B) Quantification of the effects of PPAR-γ agonists on RGC-5 cell viability during glutamate exposure. RGC-5 cells of an early passage were plated, and the next day, either 15d-PGJ2 or troglitazone was administered, along with an untreated control. Twenty-four hours later, glutamate (5 mM) was administered without the removal of either of the PPAR-γ agonists from the treated wells. Cell viability was determined approximately 1 day after the glutamate insult and expressed as the percentage of cells surviving compared with the control (non–glutamate-treated cultures). Data are the mean ± SEM; n = 4. *P < 0.05 versus respective control; +P < 0.05 versus glutamate-treated cultures.

Figure 5.

 

Immunoblot analysis of PPAR-γ receptor. RGC-5 cells of an early passage were plated, and the next day 15d-PGJ2 or troglitazone was administered. After a 24-hour incubation period, glutamate (5 mM) was administered without the removal of either of the PPAR-γ agonists from the treated dishes. Cytoplasmic (C) and nuclear (N) extracts of RGC-5 cells were subjected to immunoblot analysis, using an antibody to the PPAR-γ receptor. A 48-kDa band of PPAR-γ receptor was observed.

Figure 6.

 

Effects of troglitazone on RGC-5 cell viability during BSO exposure. RGC-5 cells were plated, and the next day, troglitazone was administered. 24 hours later, BSO (10 μM) was added. One day after BSO insult, cell viability was determined and expressed as the percentage of cells surviving compared with the control (non–BSO-treated cultures). Data are the mean ± SEM; n = 6. *P < 0.05 versus respective control (dimethyl sulfoxide).