ToxSci Advance Access originally published online on August 29, 2007
Toxicological Sciences 2007 100(2):423-432; doi:10.1093/toxsci/kfm224
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Role of Docosahexaenoic Acid in Modulating Methylmercury-Induced Neurotoxicity
* Department of Neuroscience, Norwegian University of Science and Technology, N-7489 Trondheim, Norway
Departments of Pediatrics and Pharmacology, and the Kennedy Center, Vanderbilt University Medical Center, B-3307 Medical Center North, Nashville, Tennessee 37232-2495
1 To whom correspondence should be addressed at the Department of Neuroscience, Norwegian University of Science and Technology, Faculty of Medicine, Olav Kyrresgt, 3, N-7489 Trondheim, Norway. Fax: +47-73598655. E-mail: parvinder.kaur{at}ntnu.no.
Received July 11, 2007; accepted August 21, 2007
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ABSTRACT |
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The effect of docosahexaenoic acid (DHA) in modulating methylmercury (MeHg)-induced neurotoxicity was investigated in C6-glial and B35-neuronal cell lines. Gas chromatography measurements indicated increased DHA content in both the cell lines after 24 h supplementation. Mitochondrial activity evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide (MTT) reduction indicated that 10µM MeHg treatment for 50 min led to a significant (p < 0.001) and similar decrease in MTT activity in both the cell lines. However, DHA pretreatment led to more pronounced depletion (p < 0.05) in the MTT activity in C6 cells as compared to B35 cells. The depletion of glutathione (GSH) content measured with the fluorescent indicator monochlorobimane was more apparent (p < 0.001) in C6 cells treated with DHA and MeHg. The amount of reactive oxygen species (ROS) detected with the fluorescent indicator—chloromethyl derivative of dichloro dihydro fluorescein diacetate (CMH2DCFDA)—indicated a fourfold increase in C6 cells (p < 0.001) as compared to twofold increase in B35 cells (p < 0.001) upon DHA and MeHg exposure. However, the cell-associated MeHg measurement using 14C-labeled MeHg indicated a decrease (p < 0.05) in MeHg accumulation upon DHA exposure in both the cell lines. These findings provide experimental evidence that although pretreatment with DHA reduces cell-associated MeHg, it causes an increased ROS (p < 0.001) and GSH depletion (p < 0.05) in C6 cells.
Key Words: neurotoxicology; in vitro; cell culture; glutathione; reactive oxygen species.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Methylmercury (MeHg), a ubiquitous environmental poison from various sources is sustained in the marine ecosphere (Stokes and Wren, 1987
; Veiga et al., 1994) and after bioaccumulation reaches humans through fish consumption (Clarkson, 1997; Kamps et al., 1972; Spry and Wiener, 1991). Nearly all fish contain detectable amounts of MeHg which when consumed in surplus by humans can exert neurotoxic effects due to MeHg overload (Clarkson et al., 1988). Fish consumption also serves as a source of cis-4,7,10,13,16,19-docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid (PUFA). It is one of the most abundant PUFAs in phospholipid fractions of mammalian brain (Lopez et al., 1995; Okada et al., 1996). It is essential for the growth and development of the brain (Crawford et al., 2003; Green et al., 1999; Horrocks and Yeo, 1999; Innis and Elias, 2003). Besides fish consumption, transfer of MeHg (Franco et al., 2006; Manfroi et al., 2004) and DHA (Larque et al., 2002; Rodriguez et al., 1999) to the offspring can also take place by lactation. Therefore, a public health concern is whether PUFA intake from fish may modulate the MeHg-associated effects in central nervous system (CNS).
Dietary components can modulate MeHg-induced neurotoxicity (Beyrouty and Chan, 2006; Chapman and Chan, 2000; Egeland and Middaugh, 1997). DHA is one of the major beneficial PUFA derived from dietary fish intake (Mahaffey, 2004). It is considered to be important for the prenatal development of the CNS (Anderson et al., 1990) and plays a crucial role in diverse cellular functions ranging from controlling the cell body size (Ahmad et al., 2002) and outgrowth of neurites by promoting cell differentiation (Ikemoto et al., 1997) to being antiapoptotic (Akbar and Kim, 2002) and neuroprotective (Martin, 1998). However, owing to the multiplicity of double bonds, PUFAs are putative targets for free radical generation (Halliwell and Gutteridge, 1990; Leonardi et al., 2005). Elevating the concentration of PUFAs in brain lipids could make them susceptible to oxidative damage due to the high oxygen consumption and modest antioxidant defences in the CNS (Liu et al., 1997).
The generation of reactive oxygen species (ROS) and thiol depletion have been postulated as one of the major mechanisms behind MeHg toxicity (Chen et al., 2006; Gasso et al., 2001; Sarafian and Verity, 1990; Shanker et al., 2005; Thompson et al., 2000; Yasutake et al., 1997; Yee and Choi, 1996; Zalups, 2000). Therefore, the balance between oxidative and reductive cellular processes is critical for MeHg-induced neurotoxicity. Neuronal damage in response to MeHg exposure has been suggested to be mediated by astrocytes (Aschner et al., 2007; Morken et al., 2005). Therefore, both C6-glial (Benda et al., 1968; Schubert, 1974) and B35-neuronal (Otey et al., 2003; Schubert et al., 1974) cell lines from rat brain were selected to represent each cell type in the present study.
Hence, understanding the mechanistic basis of the effect of DHA and MeHg exposure in cell cultures may explain how fish consumption could modulate MeHg-induced neurotoxicity. This would help us in understanding the effect of potential natural modulators such as DHA in influencing the toxicity of fish-bound MeHg and assist in assessing risk/benefit from their exposure.
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MATERIALS AND METHODS |
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Materials.
Twenty-four–well plastic tissue culture plates were purchased from Falcon (Becton Dickinson Labware, USA). Fetal bovine serum (Cat. No. A15-151) was purchased from PAA Laboratories, Pasching (Austria). The medium for culturing C6 (F12 Kaighn's nutrient mixture, Cat. No. 21127) was purchased from Invitrogen (Norway). The Dulbecco's minimum essential medium media (Cat. No. E15-810) used for culturing B35 cells was purchased from PAA Laboratories. Fatty acid–free bovine serum albumin (FAF-BSA, Cat. No. A0281), DHA (Cat. No. D2534), monochlorobimane (MCB, Cat. No. 69899), MTT (Cat. No. M2128), and poly-D-lysine (Cat. No. P1024) were purchased from Sigma Aldrich (Norway). The fluorescent indicator, CMH2DCFDA (Cat. No. C6827) was purchased from Molecular Probes, Inc. (Eugene, OR). Radiolabeled 14C-MeHg (Cat. No. ARC-1302) was purchased from American Radiolabeled Chemicals Inc. (St Louis, MO). MeHgCl (Cat. No. 23308) was purchased from K&K Laboratories (Plainview, NY). All other chemicals were of analytical grade.
Cell lines.
The C6-glial and B35-neuronal cell lines were purchased from the ATCC-LGC Promochem (Sweden), and the stock samples were kept in liquid nitrogen. Both these cell lines are well established as a glial and neuronal cell model and were selected due to their suitability for live cell fluorescence measurements. Freshly thawed cells were used after three passages. At day 1, 60,000 cells per well for C6 cell line and 160,000 cells per well for B35 cell line were seeded in 24-well plates. The C6 cell line was cultured with 15% of heat-inactivated fetal bovine serum. For the B35 cells, 10% of non–heat-inactivated fetal bovine serum was used. Poly-D-lysine coating was used for plating the B35 cells. The different seeding concentrations were used due to the difference in cell division (C6 cells dividing at a much higher rate compared to B35 cells). On day 3, the media containing serum was removed, and the cells were washed twice with medium and then incubated with DHA-containing media. The cells were then used on day 4, that is, 24 h after incubation with DHA. The amount of protein present at the day of the experiment was between 0.5 and 0.6 mg protein per well for both C6 and B35 cell lines. The seeding density was chosen to facilitate the fluorescence measurements.
Cell culture and DHA modification.
DHA was used to modify the fatty acid content of the cell lines. A range of concentrations were tested, and the 30 and 90µM doses were selected for further study. The highest dose resulted in DHA constituting 19–24% of the total fatty acids in the cultures which is similar to what has been found in the gray matter of brain (Salem et al., 1986) and marine fish (Kainz et al., 2006; Wang et al., 1990). DHA was added to the cell cultures as a DHA-FAF-BSA complex in order to facilitate the uptake of the fatty acid. For making the DHA-FAF-BSA complex, the DHA is taken in a test tube and converted to K+ salts by using 0.05M KOH in the ratio of 1:1.5. To this solution, the media containing FAF-BSA is added in a ratio of 2.5:1. The freshly prepared DHA-FAF-BSA complex was added directly to the wells 24 h prior to MeHg exposure.
Treatments.
A stock solution of 1mM MeHgCl was prepared in 5mM Na2CO3. From this stock, a working solution was prepared in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer (122mM NaCl, 3.3mM KCl, 0.4mM MgSO4.7H2O, 1.3mM CaCl2, 1.2mM KH2PO4, 10mM glucose, and 25mM HEPES adjusted to pH 7.4 with 10N NaOH). On the day of the experiment, the cells were washed once with HEPES buffer and incubated with 10µM MeHg for 50 min. For the last 20 min, the cells were incubated with either fluorescent probes CMH2DCFDA (7µM) or MCB (40µM) or with colorimetric reagent MTT (2.4mM). We selected a relatively high MeHg concentration in the media for a short exposure time on the basis of MTT activity timeline study (Fig. 4). The exposure time was selected on the basis of previous mercury uptake studies (data not shown) showing a stable cell-associated mercury level at 50 min of exposure.
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Determination of fatty acid content in media and cells.
Fatty acid composition of total lipids was analyzed by using a Perkin Elmer AutoSystem XL gas chromatograph (PerkinElmer Life And Analytical Sciences, Inc, Waltham, Massachusetts, USA) with an "on column" injector. The gradient temperature program started at 60°C for 1 min
before rising to 160°C for 25 min (at 25°C/min) then rising to 190°C for 17 min (at 25°C/min) 220°C for 10 min (at 5°C /min), equipped with a 50-m CP-sil 88 (Chromopack)–fused silica capillary column (internal diameter 0.32 mm). Lipids from the samples were extracted and then saponified and methylated using NaOH and BF3 (both in methanol) under heat, respectively. Fatty acids were detected by flame ionization detector (FID) and identified by retention time using standard mixtures of methyl esters (Nu-Chek, Elyian) and then quantified using Totalchrom software (version 6.2, Perkin Elmer). The amount of fatty acid per weight of the tissue or media was calculated using 19:0 methyl ester as an internal standard.
Determination of free thiol levels.
The content of intracellular free thiols was determined by using the fluorescent indicator, MCB (40µM). After 20 min of MCB incubation, the cells were washed twice with cold HEPES buffer. The resulting fluorescence was detected on a GENios Plus multilabel reader (Tecan, Austria) by scanning the whole well using an excitation wavelength of 360 nm and an emission wavelength of 465 nm, respectively. For each well, MCB fluorescence was calculated as described previously (Kaur et al., 2006, 2007). The final values of fluorescence were corrected for intracellular protein in each well and expressed as a percent of fluorescence in control wells.
Detection of intracellular ROS accumulation.
Intracellular ROS accumulation was monitored using CMH2DCFDA (7µM) for 20 min. The cells were then rinsed twice with HEPES buffer and scanned by using excitation and emission wavelengths of 485 and 535 nm at a GENios plus multilabel counter. The final values were corrected for intracellular protein in each well and expressed as a percent of fluorescence in control wells. In the absence of cells, incubation of MeHg with any of the fluorescent compounds did not induce any change in the signal produced by the reagent blank.
Cytotoxicity activity.
Cytotoxicity was determined by colorimetric MTT (2.4mM) reduction assay (Carmichael et al., 1987; Dahlin et al., 1999). An MTT timeline study was also performed to evaluate the effect of three different MeHg concentrations and exposure time intervals. The absorbance obtained was measured at 570 nm using a Sunrise absorbance reader (Tecan, Austria). Cytotoxicity was expressed as percent of MTT activity in control wells.
Cellular MeHg accumulation.
For the cell-associated MeHg studies, cells were washed once with warm buffer and then incubated with 14C-labeled 10µM MeHg (82 nCi/µg Hg) for 50 min. After incubation, the cells were washed with ice-cold buffer and treated with 1 N NaOH for 90 min. Samples were neutralized with 10 N HCl, and 100 µl aliquots were added to 500 µl of Ultima Gold (Packard, Norway) scintillation cocktail and then counted in a 1450 Micro Beta Trilux Liquid scintillation counter (Wallac, Perkin Elmer Life Sciences, Norway). For each well, radioactivity was corrected for cellular protein.
Estimation of protein.
Protein concentration was determined by the folin reagent with BSA as a standard (Lowry et al., 1951).
Data analysis.
All results are given as mean ± SD. Differences between groups were analyzed statistically with one-way ANOVA followed by the Turkey Honestly significant difference (HSD) post hoc test for multiple comparisons. In addition, a three-way ANOVA was done to evaluate the interactive effects between different parameters. Cell type (C6 or B35), MeHg treatment, and DHA pretreatment were considered as three fixed factors for the dependent variables, DCF, MCB, and MTT. For the dependent variable, fatty acids, MeHg treatment was replaced with type of fatty acid, and for the dependent variable, MTT timeline, DHA pretreatment was replaced with time. For the dependent variable MeHg Uptake, cell type and DHA pretreatment were the two fixed factors, and p < 0.05 was considered statistically significant.
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RESULTS |
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The interactive effect between cell type, MeHg treatment, and DHA pretreatment is shown in Table 1. For both the cell types, a significant interactive effect between all three parameters was obtained only for the dependent variable ROS. For the dependent variable GSH, a significant interactive effect between MeHg treatment and DHA pretreatment was obtained.
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Modification of Fatty Acids by Treatment with DHA
The fatty acid composition in the incubation media (Table 2) and cells (Table 3) from C6 and B35 cell lines were analyzed with gas chromatography flame ionization detector (GC-FID). The significant interaction between the type of fatty acid x DHA pretreatment in the incubation media and cells (Table 1) indicated that pretreatment with DHA for 24 h resulted in a concentration-dependent cellular uptake of DHA from the incubation media. The incubation media (Table 2) contained the majority of DHA with small amounts of other PUFAs, such as arachidonic acid (AA), eicosapentaenoic acid (EPA), and linoleic acid (LA). The levels of AA, EPA, and LA in the incubation media did not vary significantly between the 30 and 90µM DHA–treated groups. However, the cells (Table 3) incubated with this incubation media varied significantly (p < 0.05) in the levels of AA, EPA, and LA. With respect to the fatty acid uptake, the control cultures from C6 and B35 cells did not vary significantly from each other (Table 3). However, in the 90µM DHA–pretreated group, the C6 cells differed significantly from the B35 cells in the amount of DHA (p < 0.05) and AA (p < 0.001) and LA (p < 0.05) present.
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Modification of Mitochondrial Activity by Treatment with MeHg and DHA
Exposure to MeHg (10µM) resulted in a comparable reduction of MTT activity in C6 and B35 cells compared to controls (Fig. 1). For both the cell lines, the group treated with DHA varied significantly (p < 0.001) from the DHA and MeHg–treated group. For the dependent variable, MTT, nonsignificant interaction between the cell type x MeHg x pretreatment (Table 1) was observed. However, significant interactions between the cell type x pretreatment (Table 1) indicated that C6 cells differed significantly from the B35 cells in the MTT activity upon DHA treatment with C6 cells exhibiting a greater reduction in MTT activity. In addition, the 90µM DHA and MeHg–treated C6 cells varied significantly (p < 0.001) from the B35 cells with C6 cells exhibiting a greater reduction in MTT activity.
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) on the horizontal bar indicates p < 0.001 where DHA versus DHA and MeHg–treated group. Values are presented as a percentage of activity in control cells.Modification of Cellular Thiols by Treatment with MeHg and DHA
The C6-glial and B35-neuronal cultures expressing blue fluorescent bimane-GSH adduct were observed under a fluorescence microscope, and the GSH levels were quantified by the plate reader from the fluorescence intensity of the bimane-GSH adduct (Fig. 2). The significant interactions for the dependent variable GSH (Table 1) indicated that C6 cells varied significantly from B35 cells after MeHg treatment (cell type x MeHg; p = 0.000), DHA pretreatment (cell type x pretreatment; p = 0.000), and MeHg and DHA pretreatment (MeHg x pretreatment; p = 0.008). A slight increase in GSH content (p < 0.001) in B35 cells was observed after MeHg treatment when compared to controls. However, the DHA supplementation was not able to increase the GSH content in B35 cells. The 90µM DHA–treated C6 cells varied significantly (p < 0.001) from the 90µM DHA and MeHg–treated cells.
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Depletion of GSH Content Induces ROS Generation
ROS levels quantified in both C6-glial and B35-neuronal cells by the fluorescence intensity of the oxidized product, DCF, are shown in Figure 3. A significant interaction between cell type x MeHg x pretreatment (Table 1) indicated that the amount of ROS produced in the C6 cells varied significantly from the B35 cells. The C6 cells exhibited increased production of ROS for all type of treatments (p < 0.001) when compared to B35 cells. An increased production of ROS was indicated with DHA, MeHg, or a combined treatment of the two in C6 cells (p < 0.001) with respect to the control groups. In contrast, only a combination of DHA and MeHg resulted in increased production of ROS in B35 cells (p < 0.001) with respect to the control. The combination of DHA and MeHg resulted in twofold increase in ROS in B35 cells as compared to fourfold increase in C6 cells. For both cell lines, the DHA-treated group varied significantly (p < 0.001) from the DHA and MeHg–treated group.
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DHA Modulation Affects Cell-Associated MeHg
Treatment with DHA led to decreased MeHg accumulation in both C6 and B35 cell lines (Table 4). There was a significant (p < 0.05) decrease in cell-associated MeHg in B35 cells at both 30 and 90µM DHA dose when compared to MeHg alone. However, in C6 cells a significant decrease (p < 0.001) was observed only at 90µM DHA concentration. For the dependent variable MeHg uptake, the nonsignificant interaction between cell type x pretreatment indicated that the C6 cells did not vary significantly from the B35 cells for this parameter. Within the C6 cells, the two DHA-treated groups varied significantly (p < 0.05) from each other.
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MTT Timeline Studies
A significant effect of time and MeHg concentration (p = 0.000) indicated that an increase in MeHg concentration and exposure time results in reduced mitochondrial activity (Fig. 4). The significant interaction between cell type x MeHg (Table 1) indicated that at 60 min, the control group varied significantly from 10 to 25µM MeHg–treated group for both the cell types. In addition, the significant interaction between MeHg x time (Table 1) indicated that for a particular MeHg concentration, the MTT activity at different time intervals varied significantly from each other. The C6 cell line was significantly (p < 0.05) different from the B35 cell line only at 25µM MeHg dose at 30-min interval.
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DISCUSSION |
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Previous studies at Faroe Islands (Grandjean et al., 1997
) and New Zealand (Kjellstrom et al., 1989) have indicated association between low-level MeHg exposure and neurological deficits. However, this outcome is not replicated by other studies at the Seychelles (Davidson et al., 1998; Myers et al., 1997) and Peru (Marsh et al., 1995). In a U.S. cohort, gestational maternal fish intake was positively associated with, but mercury levels in hair were negatively associated with, visual recognition memory scores in infancy (Mozaffarian and Rimm, 2006; Oken et al., 2005), indicating possible opposing effects of overall fish consumption (i.e., providing DHA) and MeHg exposure. Few studies have been performed using rat as a model to study the interaction between MeHg and DHA. Jin et al. (2007) have shown a beneficial effect of DHA diet against MeHg-induced decreased serum albumin levels, whereas no protection against MeHg-induced behavioral defects was reported by other studies (Day et al., 2005; Paletz et al., 2007). The susceptibility of neural cells from MeHg and DHA exposure has not been investigated. The present study investigates whether DHA supplementation can modulate the susceptibility of the neural cells from MeHg exposure.
The concentration of DHA is particularly high in gray matter of cerebral cortex in human adults (Skinner et al., 1993) as well as infants (Farquharson et al., 1992). The DHA constitutes > 17% of weight of the total fatty acids in the brain of adult rats (Hamano et al., 1996). The 30 and 90µM doses of DHA used in the present study increased the %DHA of the total fatty acids content from 2 to 2.5% in control to 13–24% in DHA-treated cells. These levels are in correlation with the previous study in C6 glial cells (Leonardi et al., 2005).
We have used a relatively high concentration of MeHg for a short time period and then measured the cell-associated MeHg as an indicator of dose. This may raise the question if such a dose is physiologically relevant. The MeHg levels found in the cat cerebellum was 0.225µg Hg/mg protein (Eto et al., 2001) which is 14 times less than the content observed in the cell lines used in the present study. A study by Sakaue et al. (2006) have reported 0.54 µg MeHg/mg protein content in cerebellar granule cells after in vitro exposure to 0.1µM MeHg for 24 h. Thus the MeHg content observed in the present study is 10–20 times that of in vivo studies or in extended in vitro exposure studies. For a range of toxic substances, the effective dose during in vitro exposure must be considerable higher than the in vivo situation when the target concentration is used as a dose indicator.
In the present study, alterations in the lipid content of the cells exposed to 90µM DHA was seen with a preferential accumulation of the DHA. It has been reported by Salem et al. (1986) that most of the DHA is accumulated in the phospholipids of the cellular membrane. At 90µM DHA dose, significant changes in the content of AA, EPA, and LA were also observed. Previous studies have shown either increase in AA levels in endothelial cells (Alexander-North et al., 1994) or decrease in AA levels in neurons (Kan et al., 2007) and rat brain (Wainwright et al., 1999) after DHA supplementation. However, in this context the exposure period and biological matrix differs between these studies. Neural tissue characteristically contains high proportions of DHA and AA (Svennerholm, 1968), which are the elongation-desaturation products of the parent fatty acids, ALA, and LA (Sprecher, 2000). The increased LA can be desaturated and elongated to AA (Salem et al., 1999) and this is likely to occur in C6 and B35 cells. In addition, Moore (2001) reported that DHA cannot be synthesized by neurons and needs to be supplied by the cerebrovascular endothelium and astrocytes. In the present study, the control cultures from both the cell lines did not differ in the content of DHA; however, after DHA supplementation they varied significantly from each other in the content of DHA, AA, and LA with C6 cells exhibiting higher fatty acid content.
The present study reports reduced-mitochondrial activity after 90µM DHA exposure in C6 cells but not in B35 cells. DHA has been shown to be cytotoxic in Caco-2 cells (Roig-Perez et al., 2004) while being protective in hippocampal cultures (Wang et al., 2003). The present study reports that both C6 and B35 cells had comparable sensitivity toward MeHg-induced cytotoxicity. However, the combined exposure to MeHg and DHA or DHA alone resulted in a more pronounced reduction in mitochondrial activity in C6 cells as compared to B35 cells. Moreover, the DHA and MeHg–treated group caused a greater reduction in MTT activity when compared to the DHA group, suggesting that combined exposure is able to inhibit mitochondrial enzymes more significantly in both the cell types.
MeHg in fish is consistently bound to sulfhydryl groups of proteins (Harris et al., 2003), and it is well established that it can deplete the intracellular GSH pool, thus priming oxidative damage (Carty and Malone, 1979; Hughes, 1957; Mokrzan et al., 1995). This effect is corroborated in C6 cells in the present study. However in contrast, the B35 cells exhibited an increase in GSH content after MeHg exposure for 50 min. This might indicate an upregulation of protective mechanisms in this cell type. However, this is contrary to the primary neurons that are not able to upregulate the synthesis of GSH on their own as they depend on astrocytes for this purpose (Wang and Cynader, 2000). The B35 cells have been extensively used as a neuronal model (Diestel et al., 2005; Otey et al., 2003; Schmid et al., 2000), and they express a variety of neuronal markers including neurotransmitters (Schubert et al., 1974). It has been reported by Sakaue et al. (2005) that B35 cells express increased viability toward MeHg as compared to cerebellar granule cells. Our present observation of GSH upregulation may thus explain their findings. The ability of B35 cells to increase their GSH content has, to our knowledge, not been reported before and should be further exploited together with other neuronal cell lines. Treatment with MeHg, DHA, or a combined exposure was associated with greater depletion of GSH in C6 cells, indicating that B35 cells were more resistant to MeHg-induced depletion of GSH. These differences in the C6 and B35 cells cannot be explained with respect to the cell-associated MeHg in these cell types. The significant interactions between cell type x MeHg, cell type x DHA pretreatment, and MeHg x DHA pretreatment for GSH also indicated that C6 cells varied significantly from B35 cells for all the tested treatments.
Previous studies in different cell cultures have tested either the oxidative effects of MeHg (Sanfeliu et al., 2001; Sorg et al., 1998) or DHA (Leonardi et al., 2005) but not their combined effects. The present study demonstrates that treatment with MeHg, DHA, or combined exposure augments ROS generation in C6 cells, which correlates with GSH depletion in this cell type. The B35 cells on the other hand behave differently from the C6 cells when exposed to MeHg or DHA. They upregulate their GSH content, which causes nonsignificant changes in ROS content when compared to control in this cell type. The differential status of GSH in C6 and B35 cells for each type of treatment provides an explanation for the increased susceptibility of C6 cells to MeHg-induced ROS.
Previous study by Frenkel et al. (1988) has shown that nuclei isolated from B50 rat neuroblastoma cell line have decreased sensitivity to MeHg-induced stimulation of alpha-amantin–sensitive RNA synthesis. However, they suggested that nuclei isolated from B35 cells exhibited similar levels of toxicity to MeHg as HeLa nuclei or human neuroblastoma cell line. The MeHg-induced oxidative effects on B35 cells have not been previously investigated. This novel ability of B35 cells to resist MeHg-induced oxidative stress is quite intriguing and needs further investigation.
In the present study, DHA supplementation led to reduced cell-associated MeHg in both the cell cultures. Previous study by Berntssen et al. (2004) has also shown a significant lower degree of MeHg in the brains of rats fed naturally contaminated fish as compared to rats fed with chemically added MeHg to the same matrix. The cell-associated MeHg content did not vary between the C6 and B35 cells, indicating that this parameter did not reflect the differences observed otherwise with biomarkers of oxidative stress. Moreover, the differential and selective vulnerability of cells is not simply due to the preferential accumulation of MeHg since the resistant Purkinje cells accumulate more MeHg than the sensitive granule cells (Leyshon-Sorland et al., 1994). Further studies on differences in cell-specific MeHg accumulation may improve our understanding of the differential sensitivity of cell populations to MeHg.
In summary, the DHA affects the peroxidative machinery of the CNS and augments the C6 cells response to MeHg challenge. This indicates that combined effect of DHA and MeHg are more efficient in generating oxidative effects than exposure to MeHg alone. The B35 cells exhibit decreased sensitivity to MeHg-induced oxidative stress, which is likely explained by the upregulated GSH content after MeHg exposure in this cell line. However, there is much to learn concerning the effects of different PUFAs, their combinations, and their derivatives. Another exciting question is whether the primary cell cultures would respond differently to DHA exposure. Identifying these effects may improve the basis for risk/benefit assessment of a MeHg-containing fish diet.
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FUNDING |
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Norwegian Research Council through National Institute of Nutrition and Seafood Research (NIFES, Norway-173389/110 to P.K.).
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ACKNOWLEDGMENTS |
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The authors gratefully acknowledge Chris Glover (SCION, New Zealand) for his guidance and Keith M. Erikson (University of North Carolina at Greensboro, NC) for the statistical help. The technical assistance from Bente Urfjell and Lars Evje (NTNU, Norway) is highly appreciated. The authors also thank the project collaborators Anne-Katrine Lundebye (NIFES, Norway) and Christer Hogstrand (King's College London, UK) for their cooperation and valuable guidance. The work was assisted by the technical support from Jan Idar Hjelle and Bente Torstensen at NIFES. Conflict of interest statement: The authors confirm that they do not have any financial, personal, or institutional interests which may be in conflict with the content or use of the present study.
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