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TOXICOLOGY
Division of Basic Medical Sciences, Mercer University School of Medicine, Macon, Georgia
Received June 13, 2006; accepted August 21, 2006.
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Abstract |
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). Once in systemic circulation, each methylmercuric ion forms a strong bond with the reduced sulfur atom of one of several thiol-containing molecules, such as albumin, Cys, glutathione (GSH), homocysteine (Hcy), and N-acetylcysteine (NAC) (Fuhr and Rabenstein, 1973). Some of these mercuric conjugates serve as transportable substrates of certain carrier proteins in target organs such as the kidneys, liver, and brain (Bridges and Zalups, 2005a).
Within the kidney, the epithelial cells of the proximal tubule are the primary cells that take up CH3Hg+ and Hg2+ (Rodier and Kates, 1988; Rodier et al., 1988; Zalups, 1991, 2000). It has been shown that Hg2+ gains access to these cells, in the form of certain thiol S-conjugates, via sodium-dependent and -independent transporters present in the luminal plasma membrane (Cannon et al., 2000, 2001). In contrast, there are currently no data indicating specific mechanisms in the uptake of CH3Hg+ across the luminal plasma membrane of proximal tubular cells.
Because the chemical structures of CH3Hg-S-Cys and the Hcy S-conjugate of CH3Hg+ (CH3Hg-S-Hcy) are similar to that of the amino acid methionine, it has been postulated that these two conjugates may act as mimics of methionine to gain access to certain compartments of the body (Clarkson, 1993; Bridges and Zalups, 2005). In recent years, the theory of molecular mimicry has been put forth as a mechanism by which inorganic and methylated forms of mercury gain entry into target cells (Clarkson, 1993; Zalups, 2000; Ballatori, 2002; Zalups and Ahmad, 2003; Bridges and Zalups, 2005a). Indeed, several studies have provided indirect evidence for the involvement of molecular mimicry in the transport of CH3Hg-S-Cys (Aschner, 1989; Aschner and Clarkson, 1989; Kerper et al., 1992; Mokrzan et al., 1995). More direct evidence for this theory comes from a recent study in Xenopus laevis oocytes in which CH3Hg-S-Cys was transported by the Na+-independent amino acid transporter, system L (Simmons-Willis et al., 2002).
Because of these data and the structural similarities among CH3Hg-S-Cys, CH3Hg-S-Hcy, and methionine, we hypothesize that one or more transporters of methionine participate in the transport of these mercuric species. One possible candidate for this transport is the amino acid transporter, system B0,+. This carrier mediates the Na+/Cl–-dependent uptake of a variety of neutral and cationic amino acids, including methionine (Sloan and Mager, 1999; Nakanishi et al., 2001). In addition, system B0,+ is localized in the luminal plasma membrane of proximal tubular epithelial cells (Gonska et al., 2000), a location that corresponds to the site of CH3Hg+ accumulation and toxicity in the kidney.
Therefore, the purpose of the present study was to test the hypothesis that system B0,+ is capable of transporting methyl mercuric ions, as conjugates of Cys, Hcy, GSH, or NAC. This study focused exclusively on the Na+-dependent component of CH3Hg+ transport, although it should be noted the transport of this metal may also be mediated by one or more Na+-independent carriers. Analyses of time dependence, substrate specificity, and saturation kinetics of CH3Hg-S-Cys and CH3Hg-S-Hcy transport demonstrate that, in the present model, these two mercuric conjugates are transportable substrates of system B0,+.
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Materials and Methods |
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). In brief, connective tissue was digested with 1 mg/ml collagenase A (Roche Molecular Biochemicals, Indianapolis, IN), and follicles were removed by incubating oocytes in 100 mM K2HPO4. Oocytes were then placed in oocyte Ringer's 2 [82.5 mM NaCl, 2.5 mM KCl, 1 mM Na2HPO4, 3 mM NaOH, 1 mM CaCl2, 1 mM MgCl2, 1 mM sodium pyruvate, and 5 mM HEPES, pH 7.6] supplemented with 5% (v/v) horse serum (Atlanta Biological, Atlanta, GA) and 50 µg/ml gentamicin sulfate (Invitrogen, Carlsbad, CA) and stored in a shaking incubator at 18°C. The construct for mouse ATB0,+ (amino acid transporter B0,+), the cDNA-encoding system B0,+, was obtained from Dr. Vadivel Ganapathy (Medical College of Georgia, Augusta, GA). Capped RNA (cRNA) transcripts encoding ATB0,+ were generated using the mMessage mMachine RNA transcription kit (Ambion, Austin, TX). On the day following isolation, stage IV and V oocytes were microinjected with 28.5 ng (23 nl) of cRNA or an equal volume of water. Oocytes were stored in a shaking incubator at 18°C until the time of experimentation.
Evaluation of Transport and Cellular Disposition. Variability in oocyte quality may exist among groups of oocytes isolated from different frogs; therefore, each individual experiment used oocytes isolated from a single frog. Each experiment was repeated (using a different batch of oocytes) three times over the course of 3 consecutive weeks. Obvious seasonal variations in oocyte quality as determined by visual inspection were not noticed during the course of the study.
All experiments were carried out in both water- and ATB0,+-injected oocytes in a manner similar to that described previously (Bridges and Zalups, 2005b). In brief, 3 days following injection, the oocytes were separated into groups of five in 24-well plates and were washed with 1 ml of room temperature (25°C) uptake buffer (25 mM HEPES/Tris, 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, and 5 mM glucose, pH 7.5). Uptake was initiated by adding 350 µl of uptake buffer containing a radiolabeled substrate of interest. The incubation was allowed to proceed for 30 min at 25°C unless otherwise stated. Oocytes were then rinsed twice with 1 ml of ice-cold uptake buffer containing 1 mM sodium 2,3-dimercaptopropane-1-sulfonate (Sigma, St. Louis, MO), an effective chelator of mercuric ions. After washing, oocytes were lysed in 0.5 ml of 1% sodium dodecylsulfate/0.2 N NaOH, and each sample was added to 5 ml of Opti-Fluor scintillation cocktail (Perkin Elmer, Shelton, CT). The radioactivity contained therein was determined by standard isotopic techniques using a Beckman LS6500 scintillation counter (Beckman Instruments, Fullerton, CA).
Confirmation of X. laevis Oocytes as a Model for System B0,+-Mediated Transport. To confirm that a functional form of system B0,+ was being expressed in our Xenopus oocyte model, we measured the uptake of amino acids that have been characterized as substrates for this carrier: [3H]L-methionine (78 Ci/mmol; Amersham, Piscataway, NJ), [3H]L-phenylalanine (120 Ci/mmol; Amersham), [3H]L-valine (37 Ci/mmol; Amersham), [3H]L-isoleucine (92 Ci/mmol; Amersham), [3H]L-leucine (160 Ci/mmol; Amersham), [3H]L-alanine (85 Ci/mmol; Perkin Elmer), [3H]L-glycine (60 Ci/mmol; Perkin Elmer), [3H]L-serine (30 Ci/mmol; Amersham), [35S]L-cysteine (100 mCi/mmol; Amersham), [3H]L-threonine (16 Ci/mmol; Amersham), [3H]L-glutamine (52 Ci/mmol; Amersham), and [3H]L-lysine (98.5 Ci/mmol; Perkin Elmer). In addition, we measured the transport of [3H]L-aspartate (16.2 Ci/mmol; Amersham) and [35S]L-cystine (100 mCi/mmol; Amersham), neither of which has been identified as a substrate of system B0,+. The transport of each of the aforementioned amino acids (5 µM) was measured for 30 min at 25°C.
Analyses of CH3Hg+ Transport Using Biochemical Parameters. CH3Hg+ forms a strong bond with thiol-containing molecules. In plasma, the most common nonprotein thiol-containing molecules are Cys, Hcy, NAC, and GSH. Therefore, we studied the uptake of [14C]CH3HgCl (0.8 µCi/µg; American Radiolabeled Chemicals, St. Louis, MO) as an S-conjugate of each of these molecules. CH3Hg+ was presented to cells as CH3Hg-S-Cys, CH3Hg-S-Hcy, or as the GSH- or NAC-S-conjugate of CH3Hg+ (CH3Hg-S-GSH or CH3Hg-S-NAC, respectively). These complexes were formed by incubating 5 µM [14C]CH3HgCl with 6 µM Cys, Hcy, GSH, or NAC in uptake buffer for 5 min at 25°C. The 1:1.2 ratio of these compounds ensured that every molecule of CH3Hg+ bonded stably to a sulfhydryl group in a linear I coordinate manner.
Analysis of time dependence for the cellular uptake of CH3Hg-S-Cys and CH3Hg-S-Hcy was carried out by measuring the uptake of each compound for 5, 10, 15, 20, 30, 45, or 60 min. Saturation kinetics for the transport of these mercuric conjugates were assessed by measuring the transport of radiolabeled CH3Hg-S-Cys or CH3Hg-S-Hcy in the presence of increasing concentrations of unlabeled CH3Hg-S-Cys (1, 5, 10, 15, 25, 50, 75, and 100 µM) or CH3Hg-S-Hcy (5, 10, 25, 50, 75, 100 µM), respectively. Cells were not exposed to higher concentrations of these mercuric compounds because of concerns related to the effect of these compounds on cellular viability. The data were analyzed using the Michaelis-Menten equation: V = Vmax[S]/(Km + [S]), where Vmax is the maximal velocity of transport, [S] is the substrate concentration, and Km is the Michaelis-Menten constant. Substrate specificity analyses were also carried out, where transport of CH3Hg-S-Cys and CH3Hg-S-Hcy was measured in the presence of known substrates (3 mM) of system B0,+ (cysteine, methionine, alanine, glycine, leucine, isoleucine, phenylalanine, valine, lysine, glutamine, serine, tryptophan, asparagine, and arginine). In addition, the uptake of CH3Hg-S-Cys and CH3Hg-S-Hcy was measured in the presence of increasing concentrations of methionine (25, 50, 75, 100, 250, 500, 1000, and 2500 µM). Methionine is a high-affinity substrate for system B0,+ and is structurally similar to the mercuric conjugates being studied.
Determination of Oocyte Protein Content. Protein content of the oocytes was determined by the method of Bradford (1976). In brief, oocytes were solubilized in 1 ml of 1 N NaOH, and 100 µlofthe total solution was placed in a cuvette. Two milliliters of Bradford Reagent (Sigma) was added to the cuvette, and the sample was read in a Shimadzu UV-2101PC spectrophotometer at 595 nm.
Data Analyses. All experiments were performed in triplicate and were repeated at least twice (n = 6). Data for each parameter assessed were first analyzed using the Kolmogorov-Smirnov test for Normality and then with Levene's test for homogeneity of variances. Thereafter, the data were analyzed using either a two-tailed Student's t test (Fig. 1) or a two-way analysis of variance. Tukey's multiple comparison procedure was used to assess differences among the means. All data are presented as mean ± S.E. for an n of 3 to 4. p < 0.05 was considered statistically significant.
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Results |
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; Nakanishi et al., 2001). The transport of all of the amino acids except cystine and aspartate was significantly greater in oocytes expressing system B0,+ than in controls. When the uptake of cystine and aspartate was measured, there were no significant differences between water- and ATB0,+-injected oocytes. For many of the amino acids tested, the uptake in the oocytes injected with ATB0,+ was at least 3-fold greater than that in corresponding controls.
In addition, the saturation kinetics for the uptake of methionine (Fig. 2A) or arginine (Fig. 3A) confirmed that a functional form of system B0,+ was being expressed in the ATB0,+-injected oocytes. Because methionine is a high-affinity substrate of system B0,+ and a molecular homolog of CH3Hg-S-Cys, we chose to study the saturation kinetics of methionine transport as confirmation of the functional insertion of system B0,+ in the plasma membrane of the oocytes. Arginine was chosen for study because it is a positively charged substrate of system B0,+ at physiologic pH, and this amino acid is an effective substrate that can be used to help characterize the transporting features of this carrier protein. The uptake of both methionine and arginine was significantly greater in the oocytes injected with cRNA encoding system B0,+ than in the water-injected controls. To calculate the Vmax of transport and the Michaelis-Menten constant (Km) that are specific for the activity of system B0,+, the amount of uptake in the water-injected controls was subtracted from that in the ATB0,+-injected oocytes. For methionine, the Vmax was calculated to be 96.4 ± 7.3 pmol/mg protein/min and the Km was 257 ± 63.0 µM (r2 = 0.9887) (Fig. 2B). For arginine transport, the Vmax was estimated to be 163 ± 20.0 pmol/mg protein/min, and the Km was 1.84 ± 0.41 mM (r2 = 0.9937) (Fig. 3B).
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Inasmuch as Cys, Hcy, NAC, or GSH are the principal nonprotein thiols present in blood, we chose to characterize the potential role of system B0,+ in the uptake of CH3Hg-S-Cys, CH3Hg-S-Hcy, CH3Hg-S-NAC, or CH3Hg-S-GSH in oocytes injected with either water or ATB0,+ (Fig. 4). The transport of CH3Hg-S-Cys was approximately 10-fold greater in the oocytes injected with ATB0,+ than in the controls. Likewise, the uptake of CH3Hg-S-Hcy was also significantly greater in oocytes expressing system B0,+. In contrast, the uptake of CH3Hg-S-NAC and CH3Hg-S-GSH was not significantly different between the oocytes expressing system B0,+ and the respective controls. Among the water-injected oocytes, there were no significant differences in the uptake of any species of CH3Hg+.
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The saturation kinetics for the uptake of CH3Hg-S-Cys (Fig. 7A) and CH3Hg-S-Hcy (Fig. 8A) were measured in oocytes injected with water or ATB0,+. The transport of each conjugate was significantly greater in oocytes expressing system B0,+ than in water-injected controls. As before, the amount of CH3Hg-S-Cys and CH3Hg-S-Hcy taken up by the water-injected oocytes was subtracted from that of the ATB0,+-injected oocytes. The Vmax and Km values for the transport of CH3Hg-S-Cys were estimated to be 42.7 ± 6.86 pmol/mg protein/min and 48.0 ± 16.4 µM, respectively (r2 = 0.9850) (Fig. 7B). The Vmax for the transport of CH3Hg-S-Hcy was 64.2 ± 25.6 pmol/mg protein/min, whereas the Km was calculated to be 164 ± 87.4 µM (r2 = 0.9963) (Fig. 8B).
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The ability of methionine to inhibit the transport of CH3Hg-S-Cys and CH3Hg-S-Hcy was measured in control and ATB0,+-injected oocytes. Methionine inhibited the uptake of CH3Hg-S-Cys (Fig. 11) and CH3Hg-S-Hcy (Fig. 12) in a dose-dependent manner. The ATB0,+-specific uptake of CH3Hg-S-Cys and CH3Hg-S-Hcy is shown in Figs. 11B and 12B, respectively.
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Discussion |
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; Rodier et al., 1988; Zalups and Barfuss, 1993); however, the mechanisms by which this accumulation occurs remain unclear. One of the logical candidates for the uptake of CH3Hg+ into proximal tubular cells is system B0,+, which is located in the luminal plasma membrane of these cells (Gonska et al., 2000). The expression of this carrier is probably greatest in the proximal tubule because 99% of filtered amino acids are absorbed by this region of the nephron (Giebisch and Windhager, 2003). Not surprisingly, the proximal tubular location of system B0,+ corresponds with the site of CH3Hg+ accumulation and toxicity. In addition, because methionine is a major substrate of system B0,+, and Cys S-conjugates of CH3Hg+ have been implicated as potential mimics of methionine, we sought to test the hypothesis that CH3Hg-S-Cys, as well as CH3Hg-S-Hcy, CH3Hg-S-NAC, and CH3Hg-S-GSH, are taken up from the tubular lumen by the Na+-dependent monomeric amino acid carrier, system B0,+.
To test our hypothesis, we used Xenopus oocytes microinjected with the cRNA for ATB0,+ (which encodes system B0,+). To ensure that this transporter was functioning properly in the current oocyte model, we measured the uptake of various amino acids in water- and ATB0,+-injected oocytes. All of the amino acids tested, except cystine and aspartate, have been identified as substrates of system B0,+ (Sloan and Mager, 1999; Nakanishi et al., 2001). The transport of these substrates was significantly greater in oocytes injected with ATB0,+ than in controls, indicating the presence of a functional system B0,+ transporter. To further confirm the functionality of system B0,+ in our model, we measured the saturation kinetics for two known substrates of this carrier, methionine and arginine. Given the number of endogenous amino acid transporters in X. laevis oocytes (i.e., systems B0,+, ASC, X–AG, asc, L, y+, b0,+, and xc–), it is possible that some of the measured uptake is mediated by transporters other than system B0,+ (Campa and Kilberg, 1989; Taylor et al., 1989, 1996; Van Winkle, 1993). However, when the uptake in water-injected oocytes was subtracted from that in ATB0,+-injected oocytes, it became obvious that system B0,+ mediates a large fraction of methionine and arginine transport. Collectively, the analyses of amino acid uptake and saturation kinetics support the validity of our in vitro model (i.e., X. laevis oocytes injected with ATB0,+) as an appropriate and useful model in which to study the activity of system B0,+.
In plasma, the most common nonprotein thiols that may come in contact with CH3Hg+ include Cys, Hcy, NAC, and GSH. Therefore, we evaluated the transport of CH3Hg+ as a conjugate of each of these compounds. Only CH3Hg-S-Cys and CH3Hg-S-Hcy seem to be transported by system B0,+. CH3Hg-S-NAC and CH3Hg-S-GSH were not taken up efficiently by this carrier; a phenomenon that may be related to the steric nature and negative charge of each complex. System B0,+ recognizes neutral and cationic, but not anionic, amino acids as substrates (Sloan and Mager, 1999; Nakanishi et al., 2001; Hatanaka et al., 2004); thus, it is not surprising that it does not accept the negatively charged molecules, CH3Hg-S-NAC and CH3Hg-S-GSH, as substrates. Likewise, these mercuric conjugates were not transported by the neutral amino acid carrier, system L, when it was expressed in Xenopus oocytes (Simmons-Willis et al., 2002). In addition, GSH and NAC conjugates of Hg2+ were not identified as substrates of the Na+-independent neutral and cationic amino acid transporter, system b0,+ (Bridges and Zalups, 2004; Bridges et al., 2004). Furthermore, the transport of NAC S-conjugates of Hg2+ and CH3Hg+ appears to occur almost exclusively at the basolateral plasma membrane of the proximal tubule by means of one or more multispecific carrier proteins (Zalups, 1998; Zalups and Barfuss, 1998, 2002; Koh et al., 2002; Zalups and Ahmad, 2005). The localization of system B0,+ on the luminal plasma membrane, therefore, eliminates this carrier as a potential mechanism for the transport of these conjugates.
The transport of CH3Hg-S-Cys and CH3Hg-S-Hcy via system B0,+ was further characterized by multiple biochemical analyses. Time course analyses of the transport of these conjugates demonstrated that uptake of each conjugate increased over 60 min of study without reaching a plateau. It seems that the transport of CH3Hg-S-Cys and CH3Hg-S-Hcy may plateau only after a much longer time period, possibly because of the large intracellular volume of the oocyte. The transport of the two mercuric species was not measured for time periods longer than 60 min because of potential toxicity issues. A similar trend in transport was observed in oocytes expressing system L where the transport of CH3Hg-S-Cys continued to increase after 180 min of uptake (Simmons-Willis et al., 2002).
Assessment of the saturation kinetics for the transport of CH3Hg-S-Cys and CH3Hg-S-Hcy also suggests that system B0,+ is capable of transporting these forms of mercury. CH3Hg-S-Cys and CH3Hg-S-Hcy transport was higher in the ATB0,+-injected oocytes than in the controls; thus, we can conclude that system B0,+ does indeed play a role in the transport of these conjugates. At the concentrations tested, we did not observe complete transporter saturation with either conjugate, possibly because the carriers responsible for this uptake have a low affinity for CH3Hg-S-Cys and CH3Hg-S-Hcy. It is likely that saturation may be reached at concentrations greater than those examined; however, they were not included in this study due to concerns related to toxicity. Regardless, these data provide strong support for the hypothesis that system B0,+ is involved in the transport of CH3Hg-S-Cys and CH3Hg-S-Hcy in oocytes injected with ATB0,+.
Substrate specificity experiments provide additional support for the notion that CH3Hg-S-Cys and CH3Hg-S-Hcy are potential transportable substrates of system B0,+. The uptake of these mercuric species into oocytes injected with ATB0,+ was inhibited by neutral and cationic amino acids. Interestingly, in the water-injected oocytes, some amino acids were able to significantly inhibit the uptake of CH3Hg-S-Cys. This reduction in transport is most likely due to the inhibition of endogenous amino acid transporters. The presence of these transporters, however, does not alter the significance of the current findings. Our initial experiments indicate that the uptake of individual amino acids is much greater in ATB0,+-injected oocytes than in water-injected controls. Therefore, the activity of system B0,+ can be studied easily by comparing the transport of various substrates in both sets of oocytes.
In contrast to the uptake of CH3Hg-S-Cys, the transport of CH3Hg-S-Hcy in the water-injected oocytes was not affected by the presence of additional amino acids. Given this, we can postulate that CH3Hg-S-Cys is a preferred substrate for one or more of the endogenous transporters. These results are similar to those obtained from studies on the transport of Cys and Hcy S-conjugates of Hg2+ in MDCK cells transfected stably with the Na+-independent amino acid transporter, system b0,+, wherein the transporter affinity for the Cys conjugate was greater than that of the Hcy conjugate (Bridges and Zalups, 2004; Bridges et al., 2004).
In the present study, the uptake of CH3Hg-S-Cys by system B0,+ was significantly greater than the uptake of CH3Hg-S-Hcy in all of the experiments carried out. Likewise, in oocytes expressing system L, the uptake of CH3Hg-S-Cys appeared to be greater than the uptake of CH3Hg-S-Hcy (Simmons-Willis et al., 2002). These differences in transport may be due, in part, to the size of the molecule. CH3Hg-S-Hcy possesses an additional methyl group not present on CH3Hg-S-Cys. Therefore, the differences in transport may be due partially to the differences in size between the two mercuric species.
Transport of CH3Hg-S-Cys and CH3Hg-S-Hcy was also measured in the presence of increasing concentrations of methionine. In addition to being a high-affinity substrate of system B0,+, methionine is chemically and structurally similar to CH3Hg-S-Cys and CH3Hg-S-Hcy. Our data demonstrate that in oocytes expressing system B0,+, methionine is able to inhibit the uptake of Cys and Hcy S-conjugates of CH3Hg+ in a concentration-dependent manner. These data indicate that CH3Hg-S-Cys, CH3Hg-S-Hcy, and methionine share a common transport mechanism, i.e., system B0,+. It is important to note that the implications for this finding may spread beyond the scope of system B0,+. Because methionine is a neutral amino acid, it may be transported by a number of amino acid carriers, including systems L, b0,+, B0, y+L, and A (Ganapathy et al., 2004). These transporters are found in numerous organ systems and may serve as mechanisms for the entry of CH3Hg-S-Cys and CH3Hg-S-Hcy. It is possible that the distribution of these transporters in the various organs contribute to the detrimental effects observed following exposure to CH3Hg+.
In conclusion, the identification of system B0,+ as a mechanism for the transport of CH3Hg-S-Cys and CH3Hg-S-Hcy is an important step in understanding the way in which CH3Hg+ is handled by the human body. More specifically, because of its location in the luminal plasma membrane of proximal tubular epithelial cells, system B0,+ may play a role in the uptake of thiol conjugates of CH3Hg+ from the tubular fluid. To our knowledge, this study represents the first time that a Na+-dependent amino acid transporter has been implicated in the transport of any form of mercury.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: Hg2+, inorganic mercury; CH3Hg+, methylmercury; GSH, glutathione; Hcy, homocysteine; NAC, N-acetylcysteine; CH3Hg-S-Cys, cysteine S-conjugate of methylmercury; CH3Hg-S-Hcy, homocysteine S-conjugate of methylmercury; cRNA, capped RNA; CH3Hg-S-GSH, glutathione S-conjugate of methylmercury; CH3Hg-S-NAC, N-acetylcysteine S-conjugate of methylmercury.
Address correspondence to: Dr. Christy C. Bridges, Mercer University School of Medicine, 1550 College Street, Macon, GA 31207. E-mail: bridges_cc{at}mercer.edu
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