Modulatory effect of glutathione status and antioxidants on methylmercury-induced free radical formation in primary cultures of cerebral astrocytes

Molecular Brain Research 137 (2005) 11 – 22 www.elsevier.com/locate/molbrainres

Research report

Modulatory effect of glutathione status and antioxidants on methylmercury-induced free radical formation in primary cultures of cerebral astrocytes Gouri Shankera, Tore Syversene, Judy L. Aschner c,d, Michael Aschner a,b,c,d,T a

Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, NC 27109, USA b Interdisciplinary Program in Neuroscience, Wake Forest University School of Medicine, Winston-Salem, NC 27109, USA c Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN 37203, USA d Department of Pharmacology and The Kennedy Center, Vanderbilt University Medical Center, Nashville, TN 37203, USA e Department of Neuroscience, Norwegian University of Science and Technology, Trondheim, Norway Accepted 5 February 2005 Available online 17 March 2005

Abstract Excessive free radical formation has been implicated as one of the causative factors in neurotoxic damage associated with variety of metals, including methylmercury (MeHg). Although the mechanism(s) associated with MeHg-dependent neurotoxicity remains far from clear, overwhelming data give credence to a mediatory role for astrocytes, a major cell type that preferentially accumulates MeHg. To extend our recent findings of MeHg-induced increase in ROS formation (G. Shanker, J.L. Aschner, T. Syversen et al., Free radical formation in cerebral cortical astrocytes in culture induced by methylmercury, Mol. Brain Res. 128 (2004) 48 – 57), the present studies were designed to assess the effect of modulating intracellular glutathione (GSH) content, on ROS generation, in the absence and presence of MeHg. Intracellular GSH was reduced by treatment with 100 AM buthionine-l-sulfoxane (BSO) for 24 h, and increased by treatment with 1 mM l-2-oxothiazolidine-4-carboxylic acid (OTC) for 24 h. Additionally, the effects of the selective antioxidants, catalase (1000 U/ml for 1 h), an H2O2 scavenger, and n-propyl gallate (100 AM for 1 h), a superoxide radical (IO2 ) and possibly hydroxyl radical (IOH) scavenger on MeHg-induced ROS formation were examined. After these treatments, astrocytes were exposed to T10 AM MeHg for 30 min, following which the fluorescent probes, CM-H2DCFA and CM-H2XRos were added; 20 min later, laser scanning confocal microscopy (LSCM) images were obtained. Exposure of astrocytes for 24 h to 100 AM BSO, a GSH synthesis inhibitor, led to a significant increase in mitochondrial ROS (i.e., IO2 , INO, and ONOO ) formation, as assessed with CMH2XRos mitotracker red dye. Similarly, BSO increased ROS formation in various intracellular organelles, as assessed with CM-H2DCFDA. BSO in combination with MeHg increased fluorescence levels in astrocytes to levels above those noted with BSO or MeHg alone, but this effect was statistically indistinguishable from either of these groups (BSO or MeHg). Pretreatment of astrocytes for 24 h with 1 mM OTC abolished the MeHg-induced increase in ROS. Results similar to those obtained with OTC were observed with the free radical scavenger, n-propyl gallate (nPG). The latter had no significant effects on astrocytic fluorescence when administered alone. This IO2 and possibly IOH radical scavenger significantly attenuated MeHg-induced ROS formation. Catalase, an H2O2 scavenger, was less effective in reducing MeHg-induced ROS formation. Taken together, these studies point to the important protective effect of adequate intracellular GSH content as well as antioxidants against MeHg-triggered oxidative stress in primary astrocyte cultures. D 2005 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Neurotoxicology Keywords: Astrocytes; Methylmercury; Neurotoxicity; Laser scanning confocal microscopy; Reactive oxygen species; Glutathione; Redox status; Mitochondria; Catalase; n-Propyl gallate

T Corresponding author. Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN 37203, USA. Fax: +1 615 322 6541. E-mail address: [email protected] (M. Aschner). 0169-328X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2005.02.006

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1. Introduction Methylmercury (MeHg) is a significant environmental contaminant with well-known risks to human health. Excessive MeHg ingestion from a diet rich in fish has been linked to aberrant central nervous system (CNS) function. This ubiquitous environmental contaminant is capable of causing toxic effects as indicated by human poisoning epidemics following food-borne MeHg ingestion [11,65]. MeHg can easily pass placental and blood – brain barriers and cause CNS damage to both adult and developing brain [24,43]. Maternal mercury ingestion during pregnancy causes neurological as well as neuropsychological deficits in the offspring [25,34]. Another recent finding points to the damaging effects of MeHg on neurogenesis [29]. However, other studies found no definite connection between MeHg and neurodevelopmental deficits in children at 66 months of age [27,49]. Thus, there remains a clear need for additional studies on the basic mechanisms and consequences of MeHg exposure on brain function.

Numerous studies have established a prominent role for astrocytes in mediating MeHg neurotoxicity [22,33]. Astrocytes are a preferential cellular site for MeHg accumulation [6,21,31], MeHg induces inhibition of glutamate, cystine and cysteine uptake, thus adversely affecting intracellular GSH content and redox status in astrocytes [3,4,18,26,54,56,58], and MeHg also stimulates cytosolic phospholipase A2 (cPLA2) leading to arachidonic acid (AA) release from astrocytes, further inhibiting glutamate transporters [7,57]. Oxidative stress has been associated with a wide variety of neurodegenerative states as well as with metal-induced neurotoxicity [5,19,40,44,46]. In vivo as well as in vitro biochemical investigations using neuronal cultures, mixed neuronal/glial cultures, and recent studies with primary astrocytic cultures have demonstrated enhanced ROS formation with MeHg exposure [1,32,48,51 – 53,55,59,62,67]. One of the most effective and powerful techniques to examine oxidative stress is LSCM. With this technique, the approach to intracellular ROS detection is based on alterations in the fluorescence intensity of redox-sensitive

Fig. 1. (A) ROS production in rat primary cerebral astrocytes exposed to MeHg and BSO, assessed by changes in DCF fluorescence. Some of the culture dishes were pretreated for 24 h at 37 -C with 100 AM BSO, after which T10 AM MeHg was added for an additional 30 min. Twenty minutes after the addition of CMH2DCFDA (7 AM), fluorescent images were recorded by LSCM. The images demonstrate an enhancement of fluorescent intensity with MeHg, BSO, as well as with the combination of BSO and MeHg (the images shown are representative of three separate studies conducted in three different astrocytes cultures, A = Control; B = 10 AM MeHg; C = 100 AM BSO; D = 100 AM BSO plus 10 AM MeHg). Scale bar = 20 Am. (B) The images obtained in panel A were quantitatively analyzed for changes in fluorescence intensities within cells using the Zeiss LSM software. Data were collected from 3 different control and treated cultures (total number of cells analyzed = 12 – 18). The results indicate a significant increase in fluorescent intensity with both MeHg and BSO alone as well as in combination (*P < 0.05 versus control; **P < 0.01 versus control; mean T SEM).

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fluorescent probes selective for various reactive oxygen species/reactive nitrogen species (ROS/RNS) that are strongly associated with cellular oxidative stress [15,19, 28,38]. CM-H2DCFDA is known to react with free radical species in the cytosolic, nuclear, and mitochondrial compartments [39]. This dye passively diffuses into cells, where its acetate groups are cleaved by intracellular esterases and subsequent oxidation yields a fluorescent product, 2V, 7Vdichlorofluorescein (DCF) that is retained inside the cell. Various studies indicate H2O2 as the main ROS detected by this dye [20]. The second redox probe used in the present study is mitotracker red (CM-H2XRos), known to be mitochondrial specific [39,41]. The mitochondrial respiratory chain accounts for the majority of cellular oxygen uptake; a small percentage is converted to IO2 by complexes I, II, and III [50]. The IO2 radical can be further converted either to H2O2 by mitochondrial manganese superoxide dismutase (MnSOD) [30] or it can also react with INO produced by mitochondria [45] to form ONOO [66]. Thus, this probe may detect both ROS (predominantly

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IO 2

and possibly H2O2) and RNS (INO and ONOO ) [45,50,66]. Our recent study has shown increased MeHg-dependent oxidative stress in astrocytes [55]. One of the mechanisms of this increased oxidative stress may involve reduced intracellular GSH levels [23,42]. Depleted GSH levels are detrimental to cells as it leads to impairment of mitochondrial functions, including energy metabolism, increased free radical production, and ultimately cell death [14,23,47, 61,64]. Thus, oxidative stress associated with compromised intracellular GSH and mitochondrial dysfunction may represent key cellular events in MeHg-induced neurotoxic damage. Hence, studies examining alterations in intracellular GSH levels, and free radical scavengers and their effects on MeHg-induced ROS formation are of considerable importance. Therefore, the present studies were undertaken to examine the effects of agents which decrease or increase intracellular GSH levels, as well as of agents which are scavengers of reactive species on MeHg-dependent free radical formation.

Fig. 2. (A) Mitochondrial ROS production in rat primary cerebral astrocytes exposed to MeHg and BSO as assessed by changes in fluorescence of the oxidized form of the mitotracker red dye, CMXRos. Some culture dishes were treated with 10 AM MeHg (for 30 min); other dishes were pretreated for 24 h at 37 -C with 100 AM BSO, after which T10 AM MeHg was added for an additional 30 min. Twenty minutes following the addition of CM-H2XRos (150 nM), fluorescent images were recorded by LSCM. The images indicate an enhancement of fluorescent intensity with MeHg, BSO, as well as with the combination of BSO and MeHg (A = Control; B = 10 AM MeHg; C = 100 AM BSO; D = 100 AM BSO plus 10 AM MeHg). Scale bar = 20 Am. (B) The images obtained in panel A were quantified for changes in fluorescence intensities using the Zeiss LSM software. Data were collected from 3 different control and treated cultures (total number of cells = 12 – 18). The results indicate a significant increase in CMXRos fluorescent intensity with both MeHg and BSO alone as well as in combination (*P < 0.05 versus control; mean T SEM). (For interpretation of the references to colour in this and other figure legends, the reader is referred to the web version of this article.)

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2. Materials and methods 2.1. Materials Chloromethyl-X-rosamine (CM-H2XRos), and 5, 6-chloromethyl-2V, 7V-dichlorodihydrofluorescein diacetate (CMH2DCFDA), were obtained from molecular probes (Eugene, OR, USA). Poly-d-lysine-coated Corning 35-mm dishes were purchased from MatTek (Ashland, MA, USA). Methylmercuric chloride (MeHgCl) was obtained from ICN Biomedicals (Costa Mesa, CA, USA). Minimal essential medium (MEM) with Earle’s salts, heat-inactivated horse serum, streptomycin, penicillin, and Fungizone were purchased from Life Technologies (Gaithersburg, MD, USA). Buthionine-l-sulfoximine (BSO), l-2-oxothiazolidine-4-carboxylic acid (OTC), catalase, and n-propyl gallate (n-PG) were all purchased from Sigma (St. Louis, MO,

USA). All other reagents were either purchased from Sigma or from Fisher Scientific (Pittsburgh, PA, USA) and were of cell culture grade or higher. 2.2. Cell culture Primary cultures of astrocytes from cerebral cortices of newborn (1-day-old) Sprague –Dawley rats were established as previously described in detail [2]. Briefly, subsequent to removal of meninges, the cerebral cortices were digested with bacterial neutral protease (dispase) and astrocytes recovered by repeated aspiration of dissociated cells. Twenty-four hours subsequent to initial plating of cells in poly-d-lysine-coated Corning 35-mm dishes, the media were changed to preserve the adhering astrocytes, and remove the neurons and oligodendrocytes. The cultures were kept at 37 -C in a 5% CO2 incubator for 4– 5 weeks in

Fig. 3. (A) ROS production in rat primary cerebral astrocytes exposed to MeHg and OTC, assessed by changes in DCF fluorescence. In addition to the pretreatment of some dishes with 10 AM MeHg (for 30 min), some additional dishes were pretreated for 24 h at 37 -C with 1 mM OTC, after which T10 AM MeHg was added for an additional 30 min. Twenty minutes after the addition of CM-H2DCFDA (7 AM), fluorescent images were collected by LSCM. The images, while indicating an enhancement of fluorescent intensity with MeHg, also indicate an attenuation with OTC both in the absence as well as presence of MeHg (A = Control; B = 10 AM MeHg; C = 1 mM OTC; D = 1 mM OTC plus 10 AM MeHg). Scale bar = 20 Am. (B) The effect of MeHg, OTC, and the combination of MeHg and OTC on ROS production was analyzed by DCF fluorescence. The images obtained in panel A were quantitatively analyzed for changes in fluorescence intensities with Zeiss LSM software. Data were collected from 3 different control and variously treated cultures (total number of cells analyzed = 12 – 18). The results demonstrate a significant increase in fluorescent intensity with MeHg (*P < 0.001 versus control). In addition, when compared to MeHg alone, addition of OTC alone or in combination with MeHg was found to significantly decrease the ROS formation (**P < 0.001 versus MeHg and indistinguishable from controls; mean T SEM).

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MEM with Earle’s salts supplemented with 10% heatinactivated horse serum, 100 U/ml penicillin, 100 Ag/ml streptomycin, and 0.25 Ag/ml Fungizone. The media were changed twice per week. These monolayer, surface-adhering cultures were >95% positive for the astrocytic marker, glial fibrillary acidic protein (GFAP). 2.3. Estimation of ROS formation To quantify the effect of MeHg on astrocyte oxidant species, two distinct oxidant-sensing fluorescent redox probes were utilized: CM-H2DCFDA and CM-H2XRos. The method used is as described in detail earlier [59]. Briefly, some astrocyte cultures were pretreated for 30 min at 37 -C with 10 AM MeHg. After 30 min, CM-H2DCFDA (7 AM) and CM-H2XRos (150 nM) were added and 20 min

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later fluorescence images were obtained by LSCM (LSM 510; Zeiss 63 water immersion objective; zoom factor 0.70). The cell selection for confocal imaging was nonbiased and random. For studies utilizing CM-H2DCFDA dye, an excitation wavelength of 488 nm (argon laser) and emission wavelength of 515 nm were used. For studies with the mitotracker red dye, CM-H2XRos, a rhodamine laser at 543 nm excitation wavelength and 570– 600 nm emission wavelength were used. The corresponding controls were run without MeHg and under identical microscopy settings (i.e., detector gain, pinhole, etc.). Additional astrocyte cultures were pre-incubated for 24 h at 37 -C with 100 AM BSO to reduce intracellular GSH levels, or with 1 mM OTC to increase intracellular GSH levels [9,35], or for 1 h at 37 -C with either 1000 U/ml catalase [42] or 100 AM n-PG [55]. Following these treatments, additional 30-min incubation

Fig. 4. (A) ROS production in rat primary cerebral astrocytes exposed to MeHg and OTC, assessed by changes in CM-XRos fluorescence. In addition to the pretreatment of some dishes with 10 AM MeHg (for 30 min), some additional dishes were pretreated for 24 h at 37 -C with 1 mM OTC, after which T10 AM MeHg was added for an additional 30 min. Twenty minutes after the addition of CM-H2XRos (150 nM), fluorescent images were collected by LSCM. The images, while indicating an enhancement of fluorescent intensity with MeHg, also indicate an attenuation with OTC both in the absence as well as presence of MeHg (A = Control; B = 10 AM MeHg; C = 1 mM OTC; D = 1 mM OTC plus 10 AM MeHg). Scale bar = 20 Am. (B) The effect of MeHg, OTC, and the combination of MeHg and OTC on ROS production was analyzed by CM-XRos fluorescence. The images obtained in panel A were quantitatively analyzed for changes in fluorescence intensities with Zeiss LSM software. Data were collected from 3 different control and treated cultures (total number of cells analyzed = 12 – 18). The results demonstrate a significant increase in fluorescent intensity with MeHg (*P < 0.001 versus control). In addition, when compared to MeHg alone, addition of OTC alone or in combination with MeHg was found to significantly decrease the ROS formation (**P < 0.001 versus MeHg and indistinguishable from controls; mean T SEM).

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(with or without 10 AM MeHg) was carried out. Fluorescent measurements were recorded 20 min after loading the cultures with dye. The resultant confocal data were quantitatively analyzed using the Zeiss LSM software.

resulted in rejection of the null hypothesis (P < 0.05), the source of variance was determined with the Tukey – Kramer test. All analyses were carried out with GraphPad Instat 3.02 for Windows (GraphPad Software, San Diego, CA, USA).

2.4. Statistical analysis 3. Results To assess the effects of MeHg and various other agents on ROS formation, the confocal images were quantitatively analyzed for changes in fluorescence intensities within defined regions of interest by circumscribing boxes over cell somata using the Zeiss LSM software. For each treatment, cellular fluorescence was analyzed from 2 to 4 culture dishes obtained from different culture batches (for each dish, the total number of cells used for fluorescence analysis was between 4 and 6). All results are presented as mean T SEM and represent treatment-induced changes in fluorescent intensity normalized to the corresponding cross-sectional area for each cell/region of interest. For multiple treatment paradigms (i.e., control, MeHg, BSO, BSO plus MeHg), the statistical analysis was carried out by one-way analysis of variance (ANOVA) and when the overall significance

3.1. Effect of BSO and MeHg on ROS formation Using the CM-H2DCFDA redox probe, intracellular H2O2 species production in astrocytes treated with MeHg (30 min), BSO (24 h), and BSO plus MeHg (24 h followed by 30 min, respectively) was examined by changes in DCF fluorescence (Fig. 1A). The images shown in Fig. 1A, taken 20 min after CM-H2DCFDA addition, are representative of 3 separate studies done on 3 different astrocyte cultures. Quantification of the fluorescent signal indicates an enhancement in fluorescent intensity in MeHg-treated and BSO-treated astrocytes when compared to control (Fig. 1B). Furthermore, depletion of GSH by the addition of BSO for 24 h exacerbates the ROS formation in MeHg-exposed

Fig. 5. (A) ROS formation in rat primary cerebral astrocytes exposed to MeHg and catalase, as assessed by changes in DCF fluorescence. In addition to the pretreatment of some dishes with 10 AM MeHg (for 30 min), some additional dishes were also pre-incubated with 1000 U/ml of catalase for 1 h at 37 -C, following which 10 AM MeHg was added for an additional 30 min. Twenty minutes after the addition of CM-H2DCFDA (7 AM), fluorescent images were recorded by LSCM (A = Control; B = 10 AM MeHg; C = 1,000 U/ml catalase plus 10 AM MeHg). Scale bar = 20 Am. (B) The images obtained in panel A were analyzed for changes in fluorescence intensities using the Zeiss LSM software. Data were obtained from 3 different control and treated cultures (total number of cells = 12 – 18). Although MeHg caused a significant increase in DCF fluorescence (*P < 0.01 versus control; mean T SEM), the observed decrease with catalase did not reach statistical significance.

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astrocytes (Fig. 1B); however, statistically, this effect is indistinguishable from the effects of MeHg or BSO treatments alone. Using the mitotracker red dye, CM-H2XRos, production of mitochondrial ROS/RNS in astrocytes was examined by changes in CM-XRos fluorescence (Fig. 2A). As depicted in Fig. 2B, there was a significant increase in fluorescence intensity in MeHg-exposed and BSO-exposed cells when compared to control. Additionally, depletion of GSH by the addition of BSO for 24 h exacerbated this free radical formation in MeHg-treated astrocytes (Fig. 2B); however, statistically, this effect is indistinguishable from the effects of MeHg or BSO treatments alone.

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fluorescence intensity in MeHg-exposed astrocytes when compared to control. However, augmentation of intracellular GSH levels by the addition of OTC for 24 h alone as well as in combination with MeHg attenuated ROS to levels statistically indistinguishable from control (Fig. 3B). Using mitotracker red dye, CM-H2XRos, production of mitochondrial ROS/RNS was assessed by changes in CMXRos fluorescence (Figs. 4A and B). As shown in Fig. 4B, there was a significant increase in fluorescence intensity in MeHg-exposed astrocytes when compared to control (Fig. 4B). However, increasing intracellular GSH levels by treatment with OTC for 24 h alone as well as in combination with MeHg attenuated ROS to levels statistically indistinguishable from control (Fig. 4B).

3.2. Effect of OTC and MeHg on ROS formation 3.3. Effect of catalase and MeHg on ROS formation Using the CM-H2DCFDA probe, production of intracellular H2O2 species in MeHg-exposed (30 min), OTCexposed (24 h), and OTC plus MeHg-exposed (24 h followed by 30 min, respectively) astrocytes was assessed by changes in DCF fluorescence (Figs. 3A and B). As shown in Fig. 3B, there was a significant increase in

Using the CM-H2DCFDA probe, production of intracellular H2O2 species in astrocytes treated with MeHg (30 min) or catalase plus MeHg (1 h followed by 30 min, respectively) was assessed by changes in DCF fluorescence (Figs. 5A and B). There was a significant increase in

Fig. 6. (A) ROS formation in rat primary cerebral astrocytes exposed to MeHg and catalase, as assessed by changes in CM-H2XRos fluorescence. In addition to the pretreatment of some dishes with 10 AM MeHg (for 30 min), some additional dishes were also pre-incubated with 1000 U/ml of catalase for 1 h at 37 -C, following which 10 AM MeHg was added for an additional 30 min. Twenty minutes after the addition of CM-H2XRos (150 nM), fluorescent images were recorded by LSCM (A = Control; B = 10 AM MeHg; C = 1000 U/ml catalase plus 10 AM MeHg). Scale bar = 20 Am. (B) The images obtained in panel A were analyzed for changes in fluorescence intensities using the Zeiss LSM software. Data were obtained from 3 different control and treated cultures (total number of cells = 12 – 18). Although MeHg caused a significant increase in CM-XRos fluorescence (*P < 0.001 versus control; mean T SEM), the observed decrease with catalase did not reach statistical significance.

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fluorescent intensity in MeHg-exposed astrocytes, when compared to control (Fig. 5B). Pre-incubation with catalase, an H2O2 scavenger, did not significantly reduce MeHginduced ROS production. Using the CM-H2XRos probe, formation of intracellular H2O2 species was examined by alterations in DCF fluorescence (Figs. 6A and B). There was a significant enhancement in fluorescent intensity in MeHg-treated astrocytes, when compared to control (Fig. 6B). Pre-incubation with catalase did not significantly reduce MeHg-induced ROS production. 3.4. Effect of n-PG and MeHg on ROS formation Using the CM-H2DCFDA probe and LSCM, production of intracellular H2O2 species in MeHg-exposed (30 min), nPG exposed (1 h), and n-PG plus MeHg-exposed (1 h followed by 30 min, respectively) astrocytes was assessed by changes in DCF fluorescence (Figs. 7A and B). There was a significant increase in fluorescence intensity in MeHg-exposed astrocytes when compared to control (Fig.

7B). One-hour prereatment of astrocytes with n-PG alone led to fluorescent intensity that was statistically indistinguishable from control astrocytes. However, 1-h pretreatment with n-PG, a scavenger of mainly IO2 , both alone as well as in combination with MeHg, attenuated ROS to levels to statistically indistinguishable levels from control astrocytes (Fig. 7B). Using the CM-H2XRos probe and LSCM, production of intracellular H2O2 species in astrocytes was examined by changes in CM-XRos fluorescence (Figs. 8A and B). There was a significant enhancement in fluorescence intensity in MeHg-treated astrocytes when compared to control (Fig. 8B). However, 1-h pretreatment with n-PG alone or in combination with MeHg attenuated ROS to levels statistically indistinguishable from control (Fig. 8B).

4. Discussion The present LSCM study employing two novel oxidant-sensing fluorescent probes examined systemati-

Fig. 7. (A) ROS production in rat primary cerebral astrocytes exposed to MeHg and n-PG, assessed by changes in DCF fluorescence. Some dishes were pretreated for 1 h at 37 -C with 100 AM n-PG, following which T10 AM MeHg was added for an additional 30 min. Twenty minutes after the addition of CMH2DCFDA (7 AM), fluorescent images were recorded by LSCM. The images, while indicating an enhancement of fluorescent intensity with MeHg, also indicate an attenuation with n-PG, both in the absence and presence of MeHg (A = Control; B = 10 AM MeHg; C = 100 AM n-PG; D = 100 AM n-PG plus 10 AM MeHg). Scale bar = 20 Am. (B) The effect of MeHg, n-PG, and the combination of MeHg and n-PG on ROS formation was analyzed by DCF fluorescence. The images obtained in panel A were analyzed for changes in fluorescence intensities with Zeiss LSM software. Data were collected from 3 different control and treated cultures (total number of cells analyzed = 12 – 18). The results demonstrate a significant increase in fluorescent intensity with MeHg (*P < 0.01 versus control). When compared to MeHg alone, addition of n-PG alone or in combination with MeHg was found to significantly decrease the ROS formation (*P < 0.05 versus control; **P < 0.05 versus MeHg and indistinguishable from control; mean T SEM).

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Fig. 8. (A) ROS production in rat primary cerebral astrocytes exposed to MeHg and n-PG, assessed by changes in CM-XRos fluorescence. Some dishes were pretreated for 1 h at 37 -C with 100 AM n-PG, after which T10 AM MeHg was added for an additional 30 min. Twenty minutes after the addition of CM-H2XRos (150 nM), fluorescent images were collected by LSCM. The images, while indicating an enhancement of fluorescent intensity with MeHg, also indicate an attenuation with n-PG both in the absence as well as presence of MeHg (A = Control; B = 10 AM MeHg; C = 100 AM n-PG; D = 100 AM n-PG plus 10 AM MeHg). Scale bar = 20 Am. (B) The effect of MeHg, n-PG, and the combination of MeHg and n-PG on ROS production was analyzed by CM-XRos fluorescence. The images obtained in panel A were quantitatively analyzed for changes in fluorescence intensities with Zeiss LSM software. Data were collected from 3 different control and treated cultures (total number of cells analyzed = 12 – 18). The results demonstrate a significant increase in fluorescent intensity with MeHg (*P < 0.01 versus control). In addition to this, when compared to MeHg-induced increase, addition of n-PG alone or in combination with MeHg was found to significantly decrease the ROS formation (**P < 0.01 versus MeHg alone and indistinguishable from control; mean T SEM).

cally the effects of depleting or augmenting intracellular GSH levels, as well as the effects of selective free radical scavengers on MeHg-induced ROS formation in astrocytes. Both MeHg and the GSH synthesis inhibitor BSO, alone and in combination, caused a significant enhancement in DCF fluorescence, which primarily measures H2O2, and in CM-XRos fluorescence, which primarily measures mitochondrial specific ROS (predominantly IO2 and possibly H2O2) and RNS (INO and ONOO ) [30,45,50,66]. With both redox probes, significant increase in ROS was observed after exposure to MeHg or BSO. The effect of combined exposure of MeHg and BSO was more pronounced than after separate exposure (but statistically indistinguishable from MeHg or BSO alone), and especially so for the CM-XRos probe. Our findings of MeHg-induced increase in ROS formation is in agreement with our earlier observations [55] as well as with the observations of others [14]. Preincubation with OTC, an agent known to increase intracellular GSH levels [9], both in the absence as well

as presence of MeHg, attenuated ROS to levels statistically indistinguishable from controls. These results allude to the importance of adequate intracellular GSH concentrations for protection of cells against MeHg-induced oxidative damage. These effects reflect the ability of MeHg to bid to – SH groups, thus most likely reducing MeHg binding to sensitive target sites. In contrast, pretreatment with the H2O2 scavenger, catalase, caused only a partial and statistically insignificant decrease in H2O2 species formation and mitochondrial reactive species. The antioxidant, n-PG, when administered alone to astrocytes (1 h pretreatment) led to no change in fluorescence intensity (Fig. 7). However, n-PG significantly attenuated the MeHg-induced increase in ROS formation in mitochondria (as monitored by CM-XRos) as well as in various subcellular organelles (as monitored by DCF fluorescence). Free radicals are known to be involved in toxicanttriggered processes leading to induction of apoptosis as well as necrosis [10,61]. Multiple mechanisms have been

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proffered to explain the protective effects of free radical scavengers, such as n-PG and catalase, including: scavenging and removal of free radicals [32,42], reversal of excitatory amino acid (EAA) uptake inhibition [3], and inhibition of cytochrome C release and caspase activation [10]. Our results with n-PG demonstrating significant attenuation of MeHg-induced ROS formation are in agreement with the reported observations [32]. Protective effects of catalase in glioma cells as well as in renal epithelial cells against various toxic insults have also been demonstrated [10,42]. We observed only partial reduction in MeHg-induced ROS formation with catalase in the presence of CM-H2DCFDA as well as CM-H2XRos probes. One possible explanation is that H2O2 may not be a major contributor towards total cellular ROS or that endogenous levels of catalase are sufficient to handle the increased H2O2 load, rendering the addition of exogenous catalase only marginally effective. A number of studies including those employing pharmacological as well as knockout/transgenic approaches suggest a key role for ROS/RNS in observed neurotoxicity in response to various insults [37,60]. The mitochondrial electron transport chain has been suggested as one of the most susceptible sites for toxic radical species-induced damage [36]. One of the postulated mechanisms of MeHg-associated neurotoxicity is enhanced glutamate release [8] through the mediation of free radical species like INO or H2O2 [3,12]. Increased levels of glutamate in extracellular space are not only toxic to astrocytes but can also stimulate neuronal N-methyl-daspartate (NMDA) receptors of juxtaposed neurons leading to neurotoxicity [7,12]. Depleted GSH levels following MeHg exposure may also contribute to neurotoxic damage [17,23]. Several toxic reactive species (i.e., IO2 , INO, and ONOO ) have been found to be damaging to the activities of various mitochondrial complexes in GSH-depleted astrocytes or in neurons with relative scarcity of GSH [13,16,17,36,63]. In summary, the present study examined in detail the effects of selective antioxidants as well as agents that manipulate intracellular GSH levels on MeHg-induced cytotoxicity, by utilizing the technique of LSCM. Taken together, our results suggest that, in addition to elevated extracellular glutamate concentrations, mitochondrial dysfunction and impaired antioxidant status represent downstream mechanisms contributing to ROS/RNS-mediated neurotoxicity upon MeHg exposure.

Acknowledgments The authors gratefully acknowledge the technical expertise of Lysette A. Mutkus and Qi Wu in setting up the astrocytic cultures. This study was supported by Public Health Service Grant ES07331 to M.A.

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