Modulation of Glutathione and Glutamate-L-cysteine Ligase by ...

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Borg, 1991; Guest et al., 1992; Hales and Brown, 1991; Harris et al., 1987 ... shown to adversely affect neonatal development (Calvin et al.,. 1986; Harris et al., ...
57, 141–146 (2000) Copyright © 2000 by the Society of Toxicology

TOXICOLOGICAL SCIENCES

Modulation of Glutathione and Glutamate-L-cysteine Ligase by Methylmercury during Mouse Development Sally A. Thompson,* ,† ,‡ ,1 Collin C. White,* ,‡ Cecile M. Krejsa,* ,‡ David L. Eaton,* ,‡ and Terrance J. Kavanagh* ,‡ ,2 Departments of *Environmental Health and †Comparative Medicine, and ‡NIEHS Center for Ecogenetics and Environmental Health, University of Washington, Seattle, Washington 98195 Received February 22, 2000; accepted May 3, 2000

The antioxidant tripeptide glutathione has been proposed to be important in defense against oxidative stress and heavy metal toxicity. We evaluated alterations in glutathione regulation and synthesis associated with low-level chronic methylmercury (MeHg) exposure in the developing mouse fetus. Female C57Bl/6 mice were given 0, 3, or 10 ppm MeHg in the drinking water for 2 weeks prior to breeding and throughout pregnancy. Fetuses were collected on gestational days (gd) 12 and 16. Total glutathione, reduced glutathione (GSH), oxidized glutathione (GSSR), and glutamate-L-cysteine ligase (Glcl) activity were assessed in yolk sacs and fetuses at gd 16. Western and Northern blots for Glclcatalytic (Glclc) and Glcl-regulatory (Glclr) subunits were performed on gd 12 and gd 16 fetuses. There were no changes in total glutathione in gd 16 mouse fetuses with exposure, but there were dose-related decreases in GSH and increases in GSSR. In contrast, visceral yolk sacs exhibited an increase in total glutathione in the low-dose groups, but no changes in the high-dose group. There were no changes in Glcl activity in fetuses, but there was a 2-fold increase in Glcl activity in yolk sacs from both low-dose and high-dose groups. There was a 2-fold induction in Glclc mRNA and protein in the gd 16 yolk sacs at both 3 and 10 ppm MeHg. No treatment-related changes in Glclr protein in either gd 12 or gd 16 yolk sacs or fetuses were found. Thus, the yolk sac is capable of up-regulating Glclc and GSH synthetic capacity in response to MeHg exposure. This increase appears to be sufficient to resist MeHg-induced GSH depletion in the yolk sac; however fetal glutathione redox status is compromised with exposure to 10 ppm MeHg. Key Words: glutamate-cysteine ligase; ␥-glutamylcysteine synthetase; glutathione; methylmercury; mouse development.

Glutathione (GSH) is important in free radical scavenging, xenobiotic detoxification, and maintaining protein thiol redox status. Several studies have examined whether GSH levels influence the susceptibility of the developing fetus towards well-known teratogens (Andrews et al., 1993; Eriksson and 1 Current address: Targeted Genetics Corporation, 1100 Olive Way, Suite 100, Seattle, WA 98101. 2 To whom correspondence should be addressed at University of Washington, Mail Box 354695, Seattle, Washington 98195. Fax: (206) 685-4696. E-mail: [email protected].

Borg, 1991; Guest et al., 1992; Hales and Brown, 1991; Harris et al., 1987; McNutt and Harris, 1994; Peters et al., 1995; Reyes et al., 1995; Saillenfait et al., 1993; Slott and Hales, 1987; Ziegler et al., 1993). Most of these studies have evaluated the effects of GSH depletion by coadministration of buthionine sulfoximine (BSO), which inhibits glutamate-L-cysteine ligase (Glcl; also known as ␥-glutamylcysteine synthetase), the rate-limiting enzyme in GSH synthesis. Although BSO by itself does not appear to be teratogenic, it enhances the teratogenicity of many agents and has been shown to adversely affect neonatal development (Calvin et al., 1986; Harris et al., 1987; Harris et al., 1991; Little and Mirkes, 1990; Miranda et al., 1994; Reyes et al., 1995; Saillenfait et al., 1993). Other studies have found that GSH supplementation can decrease teratogenicity. Taken together, these studies indicate that GSH levels and the ability to up-regulate GSH synthesis are very important in fetal resistance to chemical exposures. GSH is synthesized in a two-step process. The first and rate-limiting step is the joining of glutamate and cysteine by Glcl to form ␥-glutamylcysteine (␥-GC). The second step is carried out by GSH synthetase which catalyzes the addition of glycine to ␥-GC to form GSH. Glcl is composed of two subunits, a catalytic subunit (Glclc) and a regulatory subunit (Glclr). We and others have shown an increase in Glcl in the brains and kidneys of rodents exposed to MeHg (Li et al., 1996a,b; Thompson et al., 1998; Thompson et al., 1999; Woods et al., 1992; Woods and Ellis, 1995). Glcl has been shown to be induced by a number of agents that induce oxidative stress (Griffith, 1999; Griffith and Mulcahy, 1999). MeHg binds to sulfhydryl groups on GSH and protein and interferes with several of the enzymes involved in detoxification of reactive oxygen species, leading to oxidative stress (Stohs and Bagchi, 1995), which can further deplete GSH levels. The goal of this study was to determine the effect of MeHg on fetal GSH status during in utero exposure. Our hypothesis was that because of its limited capacity to resynthesize GSH, the fetus is particularly susceptible to MeHg-induced GSH depletion, possibly explaining the sensitivity of the fetus to MeHg.

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MATERIALS AND METHODS Animals. Five-week-old C57Bl/6 female mice were housed in plastic cages (five mice/cage) with corn cob bedding and allowed to acclimate for 2 weeks prior to the beginning of the study. Food and water were provided ad libitum and animals were maintained on a 12-h light/dark cycle. This study was approved by the University of Washington Animal Care and Use Committee. At the end of the 2-week acclimation period, all females were weighed and arbitrarily placed into dose groups of control (15 mice), 3 ppm (20 mice), or 10 ppm (25 mice) mercury as methylmercury hydroxide (Alfa Aesar, Ward Hill, MA) in the drinking water. (Numbers of animals per dose group were intentionally skewed based on a preliminary study that showed decreased fecundity in the 10-ppm dose group.) Females were maintained on the MeHg for 2 weeks prior to breeding. Dosing solutions were evaluated for concentration of mercury and verified to be at the stated concentrations using cold vapor atomic absorption spectrometry by Dr. James Woods’ laboratory, Department of Environmental Health, University of Washington, Seattle, WA. Twice weekly, females were weighed, water was changed, and fluid consumption was recorded for the duration of the study. Breeding was conducted by placing one female with one male for the last hour of the dark cycle and the first hour of the light cycle. Presence of a coital plug in the morning was designated as gestational day (gd) 0. Females that had a coital plug were placed in separate cages and maintained on the respective dosing solution throughout pregnancy. Mice were rebred if not pregnant, as indicated by weight gain. Fetal collection. Pregnant females were sacrificed by cervical dislocation on gd 12 or gd 16. The uterus was removed with the fetuses intact and placed in ice-cold PBS. The uterine tissue was removed and fetuses with attached membranes were excised from the maternal tissue. Fetuses and visceral yolk sac were collected by incising Reichert’s membrane, removing the attached decidua, and dissecting free the intact fetus within the yolk sac. Visceral yolk sac was incised and removed from the fetus and the umbilicus was cut to separate the tissues from one another. Fetuses and yolk sacs were analyzed individually using the assays below. For the Western and Northern blots, a subset of fetuses and yolk sacs were snap-frozen on dry ice and stored at – 80° C until analysis. HPLC analysis of GSH:GSSR. Gestation day 16 fetuses and yolk sacs were weighed and placed in ice-cold 200 mM sulfosalycilic acid (SSA). Tissues were homogenized while on ice, then centrifuged at 10,000 ⫻ g for 10 min. The supernatants were collected and 50 ␮l was mixed with 80 ␮l of 20 mM ATP, 100 mM glutamic acid, 1.0 mM EDTA, 20 mM MgCl 2, 20 ␮l TES buffer (20 mM Tris, 250 mM sucrose, 1 mM EDTA, pH 7.4) and 200 ␮l of 0.2 M N-ethylmorpholine/0.02 M KOH (final pH 7– 8; all chemicals were supplied by Sigma, St. Louis, MO). Fifty microliters of either 20 mM dithiothreitol (DTT; for total GSH measurement), or distilled water (for reduced GSH measurement) was then added. Reduced GSH samples were derivitized immediately by addition of 20 ␮l of 10 mM monobromobimane (Calbiochem, CA). After an additional incubation for 30 min in the dark, these samples were then acidified with 200 ␮l of 200 mM SSA. For total glutathione measurements, samples were incubated for 2 h with DTT before derivitization and acidification. Samples were then analyzed by high-performance liquid chromatography (HPLC) as previously reported (Thompson et al., 1999) using reverse-phase HPLC with fluorescence detection. The amount of oxidized glutathione (GSSR; as both GSSG and mixed low molecular weight disulfides) was determined by halving the difference between total glutathione and reduced glutathione. All GSH and GSSR measurements were normalized to the wet weight of the tissues prior to homogenization. HPLC analysis of Glcl activity. Gestational day 16 fetuses and yolk sacs were placed in ice-cold TES buffer (20 mM Tris, 250 mM sucrose, 1 mM EDTA, pH 7.4), weighed, and homogenized. Fetuses and yolk sacs were evaluated for Glcl activity as previously described (Thompson et al., 1999). Briefly, triplicate samples of homogenate were incubated at 37 °C in Tris buffer containing L-glu, ATP, and L-cys for 10 min. Reactions were started by the addition of L-cys and stopped by the addition of ice-cold SSA. The samples

were derivatized with MBB, then separated and quantified by reverse-phase HPLC using fluorescence detection. Total ␥-GC was calculated as the sum of the ␥-GC and GSH peaks, minus baseline (nonincubated) GSH. ␥-GC was normalized to protein concentration in the homogenate. Activities were reported as nanomoles ␥-GC formed per minute per milligram protein. Northern blots. Tissues were homogenized in Trizol (Gibco), and RNA was isolated according to the manufacturer’s directions and resuspended in Formazol (Molecular Research Center, Inc., Cincinnati, OH). RNA was electrophoretically separated on a formaldehyde gel and transferred to a positively charged nylon membrane (Schleicher and Schuell, Keene, NH). The mouse cDNAs for Glclc (Reid et al., 1997a), Glclr (Reid et al., 1997b), and cyclophilin (kindly provided by Dr. Rick Finnell, Texas A&M) were labeled with 32 P-ATP (DuPont NEN, Boston, MA) using a random prime labeling kit (Stratagene, La Jolla, CA). The cDNA probes were purified using a Nuc-Trap column (Stratagene, La Jolla, CA), and labeling efficiency was determined on a Beckman scintillation counter. The membranes were prehybridized at 60° C for 2 h in a hybridization oven with 10% dextran sulfate, 1% SDS, 1M NaCl (all purchased from Sigma), and 100 mg/ml salmon sperm DNA (Stratagene, La Jolla, CA). They were then hybridized overnight with radiolabeled probe at a concentration of 400,000 cpm/ml. The membranes were washed with 2⫻ SSC/0.1% SDS for 15 min at room temperature, followed by a high-stringency wash with 0.1⫻ SSC/0.1% SDS for 50 min at 60° C, then exposed to Biomax film with a Biomax intensifying screen (Kodak, New Haven, CT) for the necessary exposure time. Membranes were stripped of bound probe by boiling twice in 0.1% SDS for 15 min. Western blots. Tissues were homogenized in TES buffer on ice. Proteins were measured by the Bradford method (Bio-Rad Laboratories, Hercules, CA), and 25 ␮g of sample per lane was separated by polyacrylamide gel electrophoresis. The proteins were transferred to PVDF membranes (Millipore, Bedford, MA) and stained with Glclc or Glclr antipeptide antisera (Thompson, et al., 1999). Autoradiographs were exposed with enhanced chemoluminescence (Amersham Life Sciences, Arlington Hts, Il), and protein levels were quantified by densitometry, or, alternatively, the blots were labeled with fluoresceinated goat anti-rabbit IgG (Amersham) and binding was quantified by Fluorimager analysis (Molecular Dynamics, Sunnyvale, CA). Statistical analysis. Data were analyzed by ANOVA using Systat (Systat, Inc., Evanston, IL). All statistical analyses were performed on raw data, which were then converted to percentage of control values for graphical representation. In some cases, because of small sample size, the Kruskal-Wallis Test was also performed. This test is insensitive to the magnitude of the differences between groups, so results for both the ANOVA and the Kruskal-Wallis Test are given for each significant result.

RESULTS

Treatment of dams with MeHg did not significantly change the average number of fetuses per litter or the average weight of the fetuses at gd 12 or gd 16. The percentage of copulatory plugs that resulted in pregnancy was 73, 67, and 57% for the 0-, 3-, and 10-ppm dose groups, respectively. Although no statistically significant change in fecundity was observed in the treated animals, several congenital malformations were common in the gd 16 animals from the 10-ppm dose group (see below). Results of the glutathione analyses are shown in Figure 1 and Table 1. In the fetuses, there was a significant decrease in the GSH with a corresponding significant increase in the amount of GSSR in the 10-ppm dose group, but total glutathione (GSH ⫹ GSSR) levels were not significantly altered. There was a significant decrease in the ratio of GSH to GSSR

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FIG. 1. High-performance liquid chromatography determination of reduced glutathione (GSH; Panel a); oxidized GSH (GSSR; Panel b); and total glutathione (GSH ⫹ GSSR; Panel c) in fetuses and yolk sacs after chronic methylmercury treatment. n ⫽ three litters/group. (Within each litter, three fetuses and their corresponding yolk sacs were analyzed in triplicate, and the mean of these three was then calculated as the litter mean.) Asterisks indicate significant difference from controls; p ⬍ 0.05 by ANOVA. Two asterisks indicate significant difference from controls; p ⬍ 0.01 by ANOVA and p ⱕ 0.05 by Kruskal-Wallis test.

in the 10-ppm dose group (as shown in Table 2), indicating that the thiol redox status was more oxidized with MeHg exposure. In contrast, in the yolk sac there was an increase in the GSH and GSSR in the 3-ppm dose group, which resulted in a significant increase in the total GSH of the yolk sac not seen in the 10-ppm dose group (Fig. 1). No treatment-related change in the ratio of GSH to GSSR was noted in yolk sacs (Table 2). Results of Glcl activity measurements are shown in Figure 2. We noted that basal Glcl activities were lower in the yolk sacs than in fetuses (0.27 ⫾ 0.17 and 0.47 ⫾ 0.04 nmole/min/mg protein, for mean ⫾ SD, respectively). Interestingly, there was no difference in the activity of Glcl in fetuses with exposure to MeHg, but there was a 2-fold increase in Glcl activity in the yolk sacs of both the 3-ppm and 10-ppm dose groups. Results of Northern blots are presented in Figure 3. There was an apparent increase in Glclc mRNA levels in the yolk sac of the gd 16 fetuses with both 3 and 10 ppm MeHg. Glclr mRNA was not detectable under the conditions used. TABLE 1 GSH and GSSR Levels in GD 16 Fetuses and Yolk Sacs from Mice Exposed to MeHg during Pregnancy Tissue Fetal Fetal Yolk Yolk

GSH GSSR sac GSH sac GSSR

0 ppm MeHg

3 ppm MeHg

10 ppm MeHg

920 ⫾ 83 172 ⫾ 15 575 ⫾ 60 172 ⫾ 23

853 ⫾ 57 185 ⫾ 13 714 ⫾ 46 238 ⫾ 12*

686 ⫾ 42* 237 ⫾ 31* 527 ⫾ 80 182 ⫾ 42

Note. GSH, reduced glutathione; GSSR, oxidized gluthathione. Numbers represent mean ⫾ SEM in nanomoles per gram. n ⫽ three litters/group. Three fetuses from each litter were examined in triplicate for each dose group. *Significantly different than controls, p ⬍ 0.05 by ANOVA.

Western blots of both Glclc and Glclr for three fetuses each at gd 12 and gd 16 at each dose of MeHg are shown in Figure 4. There were no statistically significant changes in Glclc or Glclr protein levels in the gd 12 mouse fetuses or yolk sacs with MeHg exposure. In addition, it is interesting to note that in all dose groups, the protein levels of Glclc are much higher in yolk sacs than in the fetuses, whereas Glclr protein levels appear not to differ between these two tissues. On gd 16 there was a significant increase in Glclc protein in the yolk sac at both doses of MeHg exposure, but no changes were noted in Glclr protein levels in either the yolk sac or the fetus. DISCUSSION

Several outbreaks of MeHg poisoning have occurred that resulted in severe congenital malformations and neurological defects in the offspring of women who themselves showed no signs or symptoms of toxicity. Animal studies have further validated the susceptibility of the developing fetus to the toxic

TABLE 2 Ratio of GSH to GSSR in GD 16 Fetuses and Yolk Sacs from Mice Exposed to MeHg during Pregnancy Tissue Fetus Yolk sac

0 ppm MeHg

3 ppm MeHg

10 ppm MeHg

5.43 ⫾ 0.19 3.49 ⫾ 0.29

4.86 ⫾ 0.59 3.14 ⫾ 0.09

3.12 ⫾ 0.48* 3.64 ⫾ 0.81

Note. Numbers represent mean ⫾ SEM. n ⫽ three litters/group. Three fetuses from each litter were examined in triplicate for each dose group. *Significantly different than controls, p ⬍ 0.01 by ANOVA, (p ⫽ 0.07 by Kruskal-Wallis Test).

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FIG. 2. High-performance liquid chromatography determination of Glcl activity in fetuses and yolk sacs after chronic methylmercury treatment. Glcl activity was significantly increased in the yolk sac at both doses of MeHg exposure. n ⫽ three litters/group (Within each litter, three fetuses and their corresponding yolk sacs were analyzed in triplicate and the mean of these three was then calculated as the litter mean.) Asterisks indicate significant difference from controls; p ⬍ 0.01 by ANOVA and p ⱕ 0.05 by Kruskal-Wallis Test.

effects of MeHg. To determine what factors may contribute to fetal susceptibility to MeHg, we evaluated its effects on GSH regulation and biosynthesis in the developing mouse fetus. The yolk sac is particularly important in maintaining the supply of nutrients and energy to the fetus. It has also been shown that the yolk sac has a higher GSH synthesis capacity than the fetus, and that it up-regulates GSH synthesis in response to teratogenic insult (Harris, 1993; Serafini and Romeu, 1991). In contrast, the fetus itself does not respond as quickly and is not able to up-regulate GSH to the same degree as the

yolk sac. However, in a recent study of the effects of acute MeHg exposure on fetal brain and placental GSH content, Watanabe and colleagues (1999) found an increase in brain GSH on gd 17, which suggests that some fetal tissues may be able to increase GSH synthesis when treated in this fashion. In the present study we found that exposure to MeHg throughout gestation resulted in a depletion of GSH levels in the fetus and a significant decrease in the ratio of GSH to GSSR. However, there were no changes in the total GSH levels in the fetus. Interestingly, the ratios of GSH to GSSR observed in this study are much lower than the ratios reported for the ratio of GSH to GSSG in other tissues. This may be due to the contribution of low molecular weight mixed disulfides to the total GSH measurement. Alternatively, the fetus and yolk sac may have a more oxidized intracellular thiol redox status than adult tissues, leading to an increased potential for oxidantinduced injury. In contrast to the effects in the fetus, there was a significant increase in total GSH levels in the yolk sac of low-dose MeHg-exposed animals and no change in the ratio of oxidized to reduced glutathione at either of the doses tested. The activity of Glcl, the rate-limiting enzyme in GSH synthesis, was increased 2-fold in the yolk sac from both doses of MeHg, but was unchanged in the fetus. It is possible that there was tissue-specific induction of Glcl protein and activity in the fetus that was not detectable in the whole-fetus preparations used in this study. The apparent induction of Glclc mRNA and protein in the yolk sac suggests that the yolk sac, but not the fetus, is capable of providing GSH-dependent protection from oxidative stress induced by MeHg during fetal development. Western blots showed that levels of Glclc protein are much higher in the yolk sac than in the fetus, whereas levels of the Glclr protein were quite similar. Glclr protein was not induced by MeHg treat-

FIG. 3. Northern blot analysis of Glclc mRNA levels in gestational day-16 fetuses and yolk sacs after chronic methylmercury treatment. Shown are representative Northern blots. Northern blotting was repeated twice with samples from different fetuses from different litters. Results were consistent among the groups and showed an increase in the Glclc mRNA in GD 16 mouse fetuses exposed to 3 or 10 ppm MeHg.

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FIG. 4. Western blot analysis of Glclc and Glclr protein levels in gestational day-16 fetuses and yolk sacs after chronic methylmercury treatment. Mean values for densitometric analyses of bands corresponding to Glclc and Glclr are shown. There was a statistically significant increase in the protein for Glclc in the yolk sac from fetuses at both low-dose and high-dose MeHg (p ⬍ 0.01 by ANOVA and p ⱕ 0.05 by Kruskal-Wallis Test).

ment in the fetus or the yolk sac. The lack of coordinate regulation of Glcl subunits in MeHg-exposed animals may be due to an abundance of Glclr relative to that of Glclc. Alternatively, Glclr could serve another function in addition to regulating the activity of Glclc. Differential tissue expression of the two enzyme subunits in adult tissues has been described previously (Gipp et al., 1995). Interestingly, whereas Glclc protein levels were clearly higher in the yolk sac than the fetus, these differences did not translate into higher Glcl activity. Basal activity of the fetuses was about twice that of the yolk sacs from untreated animals. Treatment with MeHg led to an increase of Glcl activity in the yolk sac to about the same level of activity seen in the fetus. This suggests that other factors besides absolute Glcl protein levels, i.e., post-translational modifications, may be important for determining tissue-specific control of Glcl activity. It is important to note that although the induction of Glcl activity occurred in the 3-ppm dose group, this was not associated with any obvious congenital malformations. However, we did record some severe malformations at the 10-ppm dose (congenital hydrops, subcutaneous hemorrhage, anophthalmia, hydrocephalus, and limb malformations [Thompson et al., in preparation]). Additionally, there was an increase in total GSH in the yolk sac at 3 ppm MeHg that was not present at the high dose. This may be due to the exhaustion of GSH pools at the high dose of MeHg. Whereas 3 ppm MeHg exposure may stimulate an “overshoot” in GSH production, the 10-ppm dose might create an excess demand on cellular reducing equivalents and result in a net GSH depletion. The fact that malformations were seen at the high dose but not at the lower dose suggests that adaptive mechanisms were inadequate at 10 ppm MeHg. Alterations in GSH metabolism, including increased

total GSH, Glclc mRNA, and protein expression, occurred at MeHg levels that resulted in no overt signs of toxicity. These changes likely reflect adaptive responses of the fetus to oxidative stress induced by MeHg, and thus should be considered (in addition to teratogenicity data) when determining what levels of MeHg might present a risk to the developing fetus. In conclusion, we have shown that 10 ppm MeHg exposure throughout gestation produces alterations in glutathione redox status of the fetus and induces the expression of Glcl in the yolk sac. Exposure to 3 ppm MeHg caused no changes in fetal or yolk sac glutathione redox status. However, 3 ppm MeHg did cause an increase in total glutathione and Glcl activity and an increased expression of the catalytic subunit of Glcl in the yolk sac. These findings point to the possible importance of yolk sac Glcl activity in protecting the fetus from MeHg toxicity and suggest that Glclc induction may be a useful oxidative stress-related biomarker during fetal development. ACKNOWLEDGMENT This work was supported by National Institutes of Health grants P30ES04696, P50-ES07033, and T32-ES07032.

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