* Department of Biochemistry, Emory University, Atlanta, Georgia 30322, and Toxicology Program, Department of Environmental Health Sciences, 1420 Washington Heights, University of Michigan, Ann Arbor, Michigan 48109-2029
Received March 2, 2004; accepted April 13, 2004
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ABSTRACT |
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Key Words: glutamate-cysteine ligase; glutathione; visceral yolk sac; rat conceptus; -glutamylcysteine; rat embryo.
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INTRODUCTION |
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The proper redox environment is dependent on GSH status and is regulated through total concentrations, ratios of reduced and oxidized forms, and the capacity for GSH restoration via enzymatic reduction and de novo synthesis (Schafer and Buettner, 2001). Some tissues, such as intestinal epithelium and kidney proximal tubules, possess specific GSH transporters, which facilitate transport of intact GSH into cells (Hagen and Jones, 1987
; Lash and Jones, 1983
), but most cells require de novo synthesis to replenish GSH lost through oxidation, adduct formation, and routine catabolism. (Hiranruengchok and Harris, 1993
; Jones et al., 1986
).
Glutathione biosynthesis and turnover requires a series of six enzyme-catalyzed reactions, identified collectively as the -glutamyl cycle (Meister, 1974
, Meister and Anderson, 1983
). The glutathionase component of the
-glutamyl cycle may also be important as a means to facilitate transport of GSH precursors and other amino acids needed for new protein synthesis through the activity of
-glutamyl transpeptidase, which is found in plasma membranes (Meister, 1984
). Glutathione synthesis occurs via two consecutive enzymatic reactions (Meister, 1974
; Snoke and Bloch, 1954
). In the first step, glutamate is coupled with cysteine to form
-glutamylcysteine (GC) by a cytosolic enzyme, glutamate-cysteine ligase (EC 6.3.2.2, GCL), a process requiring ATP and Mg2+ as cofactors. Glycine is subsequently added to the GC cysteine by a second reaction that is catalyzed by glutathione synthetase (EC 6.3.2.3, GS), which also requires ATP and Mg2+. The first reaction is the rate-limiting step in GSH synthesis, and is primarily regulated by the availability of cysteine and through feedback inhibition by GSH (Kaplowitz et al., 1985
; Richman and Meister, 1975
). Although GSH de novo synthesis is well documented in rat liver, kidney, erythrocytes, and other tissues (Orlowski and Meister, 1971
; Seelig and Meister, 1985a
,b
), less is known about GSH synthesis in developing embryos in spite of the well-documented protective functions of GSH against embryotoxicity and teratogenicity produced by xenobiotics (Harris et al., 1995
).
The mouse GCL holoenzyme is a heterodimer constisting of a 72 kD, heavy subunit (GCLC) that contains the catalytic active site with corresponding glutamate and cysteine binding domains. The 27 kD, light subunit (GCLR) provides regulatory control for GSH synthesis through a thiol-sensitive sulfhydryl bridge that interacts with the intracellular GSH/redox environment to facilitate the feedback inhibition that is activated when GSH levels rise. GCLC and GCLR originate from separate genes and appear to be independently regulated (Diaz et al., 2002; Tsuchiya et al., 1995
). GCL is identified as the rate-limiting enzyme of GSH synthesis in biological systems and the rate limiting substrate is usually cysteine. Deficits in cysteine transport or availability have been shown to significantly slow the ability to restore GSH through de novo biosynthesis (Meister, 1974
).
Study of GCLC expression in developing mouse embryos has shown that expression is observed as early as gestational day 3 (blastocyst stage; Gardiner and Reed, 1995). By gestation day 10, expression is found in the neuroepithelium, spinal cord, branchial arches, and liver, and by gestational day (GD) 16, expression is detected in virtually every tissue (Diaz et al., 2002
). Mouse fetuses (GD 12/16) treated with methyl mercury showed oxidation of GSH in both fetuses and VYS, but only the VYS showed an increase in GCL activity (Thompson et al., 2000
). These experiments demonstrate the regulation and inducibility during development at later stages of development but do not address these responses in early organogenesis stage embryos (mouse: gestational day 8 and rat: gestational day 10).
In the present study, intrinsic GSH synthesis has been characterized in the rat embryo and VYS during early organogenesis using an in vitro GCL assay. Presence of active GCL has been demonstrated in the embryo and VYS by measuring the formation of -glutamylcysteine (GC) in tissue extracts. Relative activities of GCL and apparent, tissue-specific kinetic properties of GCL for the utilization of precursor amino acids have been determined in extracts of embryos and VYSs. Furthermore, GCL mRNA inducibility and protein expression will also be addressed to assess conceptal responses following exposure to substances that cause oxidative stress.
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MATERIALS AND METHODS |
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Animals. Primagravida Sprague-Dawley rats were obtained from the Reproductive Sciences Program Small Animal Core, University of Michigan or Charles River (Portage, MI) on GD 69. Day 0 was determined by a sperm-positive vaginal smear on the morning following copulation. Pregnant rats were maintained on a 12-h light/12-h dark cycle until explantation on GD 10. Food and water were given ad libitum. Conceptuses were explanted on day 10 of gestation and cultured for 2 h in a medium consisting of 33% (v/v) heat-inactivated rat serum in Hanks' balanced salt solution (HBSS, pH 7.4) and saturated with a gas mixture containing 20% O2/5% CO2/75% N2 (v/v/v). The conceptuses were withdrawn from the culture and rinsed (3X) with homogenization buffer (100 mM Tris.Cl, 50 mM KCl, 20 mM MgCl2 and 2 mM EDTA, pH 7.4 at 37°C). Embryos were dissected free of the VYSs and placed in separate microcentrifuge tubes containing buffer. Embryos and VYSs were homogenized by ultrasonic disruption and centrifuged at 14,900 x g for 10 min. The resulting supernatants were used for the determination of GCL activity.
Assay of GCL activity. GCL activity was determined in a reaction mixture containing 100 mM Tris.Cl, 50 mM KCl, 20 mM MgCl2, 2 mM EDTA, 6 mM ATP, 8 mM glutamic acid, 0.1 mM cysteine, and tissue extracts (equivalent to 200 µg of protein) in a 150 µl final volume (pH 8.2 at 37°C). The reaction was incubated at 37°C for 10 to 40 min, and terminated by adding 50 µl of 800 mM methanesulfonic acid on ice. The amount of GC produced in the reaction was determined by HPLC analysis following derivatization with monobromobimane as described in a previous study (Lee and Harris, 1995). GCL specific activity was expressed as pmol GC/mg protein/min.
Incorporation of [35S]-cysteine into GC and GSH. [35S]-Cysteine (1200.0 Ci/mmol) was diluted with deionized water to obtain specific activities of 240,000 dpm/pmol. Tissue extracts were prepared from GD 10 embryos and VYSs. The enzyme reaction was initiated by adding [35S]-cysteine into the reaction mixtures which does not contain unlabeled cysteine. After 20 min of incubation at 37°C, the reaction was terminated, and the products were resolved by HPLC. Fractions eluted from HPLC were further analyzed by scintillation counting to determine the amount of labeled GC and GSH produced by incorporation of [35S]-cysteine.
HPLC analysis. Glutathione and GC levels were determined by reverse phase HPLC analysis according to the method previously described (Fenton and Fahey, 1986). Samples were resolved on a Novapac C-18 4 µm column (Waters, Millipore Corporation, Milford, MA) with an isocratic mobile phase of 14.2% (v/v) methanol and 0.25% (v/v) acetic acid in water at a flow rate of 1.0 ml/min. Products were detected by a Waters Model 470 scanning fluorescence detector (l excitation 360 nm; l emission 455 nm). The column was washed with 90% (v/v) methyl alcohol and 0.25% (v/v) acetic acid in water to re-equilibrate before each run. Authentic standards (GC and GSH) were prepared for quantification of each sample.
Quantitative fluorescence PCR. Embryos and VYS were collected, washed with HBSS and placed in Trizol (Invitrogen, Carlsbad, CA) for RNA extraction by the manufacturer's suggested protocols and followed by a clean up with the RNeasy kit (Qiagen, Valencia, CA). Reverse transcription (RT) of RNA and synthesis of cDNA were performed with M-MLV RT (Promega, Madison, WI) using 1 µg of isolated RNA, RT mix containing RT buffer (Promega), 40 mM dithiothreitol, 0.5 mM dNTPs, 10 U RNAase inhibitor (Promega), and 200 ng random primers (Promega). The reaction was incubated for 1 h at 37°C.
GCLC and GCLR primers were obtained from Integrated DNA Technologies (Coralville, IA) (GCLC forward primer: 5'-TGTGAATCCAGGGCAGCCTA-3'; GCLC reverse primer: 5'-ATCCTCAGTTCCTGCACAT-3'; GCLR forward primer: 5'-CCTCGGATCTAGACAAAAC-ACAGT-3'; GCLR reverse primer: ACATTGCCAAACCACCACATTCAC-3'). ß-actin polymerase chain reaction (PCR) was used as an internal control (forward primer: 5'-TCACCCACACTGTGCCCATGTACGA-3'; reverse primer 5'-CCTACGGTGTTCTAAGGTATGGGT-3').
Real-time PCR was performed using a LightCycler (Roche, Indianapolis, IN) and the LightCycler FastStart DNA Master SYBR Green I kit (Roche Applied Science, Indianapolis, IN). Reactions were carried out as described by the manufacturer's suggestions and protocols. Briefly, real-time fluorescence PCR analysis consisted of reactions using 50 ng of cDNA that included a preincubation period (95°C for 5 min), an amplification cycle repeated up to 45 times (cycle: 95°C for 1 sec, 60°C for 8 s, 72°C for 18 s) and melting curve analysis for verification of specific, desired product. Products were also run on a 2% agarose gel to verify product size. The ß-actin amplification cycle consisted of annealing at 60°C for 7 s and elongation at 72°C for 21 s.
GCLC protein detection and quantification by immunoblot. Following treatment and incubation in whole embryo culture, embryos and VYSs were collected in lysis buffer and snap frozen in liquid nitrogen. Protein amounts were determined by the Lowry method with the Dc protein assay kit (BioRad, Hercules, CA). Loading buffer was added to 25 µg of each sample and boiled for 5 min prior to loading on a 1020% gradient polyacrylamide ReadyGel (SDS-PAGE) (BioRad). Following separation samples were transferred to a nitrocellulose membrane and probed for GCLC with a primary anti-goat antibody (Santa Cruz Biotechnology, Santa Cruz, CA). An Alexafluor 680 nm donkey anti-goat secondary antibody (Molecular Probes, Eugene, OR) was used to reveal GCLC. Membranes were visualized on an Odyssey fluorescence detection system as described by the manufacturer (Li-Cor, Lincoln, NE). Densitometry was determined with the Odyssey software.
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RESULTS |
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Addition of the GSH depleting agents diethyl maleate (DEM, depletion through GSH conjugation) and diamide (depletion through chemical oxidation of GSH to GSSG) directly to the culture medium containing GD 10 conceptuses resulted in increased GCL activities on the order of 25125% in embryos and 65165% in VYS after 24 h (Fig. 4). Increased GCL specific activities were statistically significant for DEM and diamide in the VYS. Other known inducers of GSH synthesis such as PGA2 and BHT, exposed using the same protocol, had no effect on cultured rat embryo or VYS GCL activities (Fig. 4).
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DISCUSSION |
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The rate-limiting step in GSH synthesis involves the enzyme glutamate cysteine ligase (GCL), which is comprised of two subunits; a heavy, catalytic subunit (GCLC) and a light, regulatory subunit (GCLR), which facilitates the ligation of glutamate and cysteine. The two subunits are products of different genes, independently regulated, and may be distributed unequally between cells due to their selective expression. Our data show that, within the organogenesis-stage rat conceptus, constitutive specific activity for GSH synthesis differs significantly between the embryo proper and the associated VYS. Specific activities decrease as gestational age progresses, but the relative differences between embryo and higher activity VYS persist through mid-gestation.
Tissue-specific apparent kinetic values derived from cysteine and glutamate saturation curves in conceptal homogenates show high and equal affinities for cysteine in embryo and VYS and lower and tissue-specific affinities for glutamate, where the embryonic apparent Km value is twice that of the VYS Km value on gestational day 10. The observed high affinity of conceptal GCL for cysteine may be an embryonic adaptation to compensate for reported low levels of cysteine in the conceptus (Beckman et al., 1990; Hansen et al., 1999
). Differences for the apparent Km values for glutamate between embryo and VYS do not necessarily imply that distinct isoforms of the proteins exist. To our knowledge, there are no structural variations between embryonic and VYS GCLC that could account for these differences, suggesting that inherent differences in the tissue homogenates, possibly the presence of endogenous inhibitors or regulators, may be contributing to the differences. For both glutamate and cysteine, the VYS is capable of producing more GC than the embryo. Although the specifics for the observed disparities in the apparent catalytic binding affinity have not yet been characterized, it does suggest some significant implications for embryonic susceptibility and VYS resistance to chemical-induced oxidative stress and GSH depletion.
Previous studies with the electrophile, DEM, showed that considerable differences exist within the conceptus with regard to rates of GSH synthesis following DEM exposure and GSH depletion (Harris, 1993). Exposure of conceptuses to DEM in rats grown in whole embryo culture resulted in rapid and equivalent depletion of GSH in both embryo and VYS, but immediate replenishment occurred only in the VYS. Embryo GSH repletion lagged for several hours (Harris, 1993
). This observation agrees well with our determination that 23-fold higher basal GCL specific activities occur in the VYS when compared to the embryo (Table 1). The initial inability of embryos to rapidly respond to GSH depletion and resynthesize GSH was interpreted to be due to the lack of amino acid precursors in the embryo and a renewable supply of precursor in the VYS through constant degradation of maternal proteins. The current data show that basal activities and enzyme inducibility may also contribute to the differential behavior.
In this study, increases in GCL specific activity were observed after 24 h in both embryos and VYSs following DEM exposure, and to a lesser degree following oxidation of GSH by diamide. While these values correlate with changes in GCLC and GCLR mRNA and GCLC and GCLR protein levels in the embryo, no substantial induction was observed in the VYS. Quantitative PCR of embryos and VYS exposed to DEM and diamide showed significant increases in GCLC expression only in the embryo proper. Twenty-four hour GCLC protein concentrations were also proportional to the changes in gene expression in the embryo, suggesting that the induced expression of GCLC in the embryo was highest for DEM but that it did not exceed the basal expression levels seen constitutively in the VYS. This result implies that the increase in specific activity observed in the VYS is not due to induction (increased message and protein) per se, but may involve other regulatory events, possibly involving increased precursor availability, or feedback regulation and activation related to intracellular GSH status and redox environment. Cysteine, the rate limiting precursor to glutathione synthesis, is nearly 3-fold greater in the VYS (Hansen et al., 1999), again suggesting that the VYS may respond more completely and quickly than the embryo during periods of GSH depletion and resynthesis.
GCLC and GCLR gene expression and protein synthesis were more responsive and robust in the embryo following DEM treatment than observed with diamide treatments. Although both chemicals effectively deplete GSH, depletion mechanisms are very different; DEM depletion occurs through covalent adduct formation and diamide occurs through chemical oxidation to GSSG. While oxidation of GSH to GSSG by diamide shifts the GSH:GSSG ratio and intracellular environment, the total amount of thiol does not necessarily change. Glutathoine disulfide reductase (GSSG-Rd) can convert GSSG back to GSH following oxidant exposures, such as with diamide, but not in cases of GSH adduct formation, such as with DEM. VYS GSSG-Rd activity is 23 fold greater than that measured in the embryo and may account for the VYS's superior ability to respond to chemically induced oxidation (Choe et al., 2001, Hiranruengchok and Harris, 1993
). Glutathione recovery from GSSG via GSSG-Rd is faster than de novo synthesis, the means by which GSH is restored in DEM treatment, yielding a shorter period of redox imbalance.
In response to conditions of GSH depletion and/or oxidative stress where a significant percentage of GSH becomes oxidized or lost, induction of GCLC and GCLR are possible by virtue of selective binding of redox-sensitive transcription factors such as AP-1 and NF-kB. AP-1 has been shown to be responsible for most changes in GCL expression. Ozolins et al. (2002) have determined that the changes in conditions that accompany the introduction of conceptuses into culture is sufficient to induce a shift in the GSSG/GSH ratio, inducing oxidative stress and activating AP-1 expression and DNA binding. Post-translational regulation of AP-1 activation and DNA binding differ in embryo and VYS and may contribute to observed organ specific differences in inducibility and activity related to the apparent constitutive production of GCLC in the VYS (Ozolins and Hales, 1997
, 1999a
,b
). Diamide may not be as effective an inducer of GCL due to the inherent cellular capacity to rapidly reduce GSSG and restore intracellular GSH without the need for de novo GSH biosynthesis.
The idea of redox regulation of transcription factors such as AP-1, Ref-1, NF-B, and nrf2 has been studied (Abate et al., 1990
; Erickson et al., 2002
; Ozolins and Hales, 1999a
,b
; Solis et al., 2002
). Our data in conceptuses suggest that redox-sensitive transcription factors may be more sensitive to overall thiol depletion (DEM) than conversion to an oxidized disulfide. Thus, the nature of GSH depletion, conditions of oxidative stress and the intrinsic abilities of conceptal tissues to restore GSH through new GSH synthesis may determine the overall susceptibility to chemical embryotoxicants and teratogens. Induction of GCLC and GCLR as well as differential modes of amino acid precursor supply are likely to contribute heavily to selective cell and tissue selectivity to toxicants an may act to regulate other redox-sensitive aspects of normal development.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: (734) 763-8095. E-mail: charris{at}umich.edu.
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REFERENCES |
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Beckman, D. A., Pubarelli, J. E., Jensen, M., Koszalka, T. R., Brent, R. L., and Lloyd, J. B. (1990). Sources of amino acids for protein synthesis during early organogenesis in the rat. I. Relative concentrations of free amino acids and proteins. Placenta 11, 109121.[ISI][Medline]
Berberian, R. M., Eurich, G. E., Rios, G. A., and Harris, C. (1996). Formation of glutathione adducts and 2-aminofluorene from 2-nitrosofluorene in postimplantation rat conceptuses in vitro. Reprod. Toxicol. 10, 273284.[CrossRef][ISI][Medline]
Choe, H., Hansen, J. M., and Harris, C. (2001). Spatial and temporal ontogenies of glutathione peroxidase and glutathione disulfide reductase during development of the prenatal rat. J. Biochem. Mol. Toxicol. 15, 197206.[CrossRef][ISI][Medline]
Cotgreave, I. A., and Gerdes, R. G. (1998). Recent trends in glutathione biochemistryglutathione-protein interactions: A molecular link between oxidative stress and cell proliferation. Biochem. Biophys Res. Commun. 242, 19.[CrossRef][ISI][Medline]
Diaz, D., Krejsa, C. M., and Kavanagh, T. J. (2002). Expression of glutamate-cysteine ligase during mouse development. Mol. Reprod. Dev. 62, 8391.[CrossRef][ISI][Medline]
Erickson, A. M., Nevarea, Z., Gipp, J. J., and Mulcahy, R. T. (2002). Identification of a variant antioxidant response element in the promoter of the human glutamate-cysteine ligase modifier subunit gene. Revision of the ARE consensus sequence. J. Biol. Chem. 277, 3073030737.
Fenton, S. S., and Fahey, R. C. (1986). Analysis of biological thiols: Determination of thiol components of disulfides and thioesters. Anal. Biochem. 154, 3442.[ISI][Medline]
Gardiner, C. S., and Reed, D. J. (1995). Synthesis of glutathione in the preimplantation mouse embryo. Arch. Biochem. Biophys. 318, 3036.[CrossRef][ISI][Medline]
Hagen, T. W., and Jones, D. P. (1987). Transepithelial transport of glutathione in vascularly perfused small intestine of rat. Am. J. Physiol. 252, G607G613.[ISI][Medline]
Hansen, J. M., Carney, E. W., and Harris, C. (1999). Differential alteration by thalidomide of the glutathione content of rat vs. rabbit conceptuses in vitro. Reprod. Toxicol. 13, 547554.[CrossRef][ISI][Medline]
Harris, C. (1993). Glutathione biosynthesis in the postimplantation rat conceptus in vitro. Toxicol. Appl. Pharmacol. 120, 247256.[CrossRef][ISI][Medline]
Harris, C., Hiranruengchok, R., Lee, E., Berberian, R. M., and Eurich, G. E. (1995). Glutathione status in chemical embryotoxicity: Synthesis, turnover and adduct formation. Toxicol. in vitro 9, 623631.[CrossRef][ISI]
Harris, C., Stark, K. L., and Juchau, M. R. (1988). Glutathione status and the incidence of neural tube defects elicited by direct acting teratogens in vitro. Teratology 37, 577590.[ISI][Medline]
Hiranruengchok, R., and Harris, C. (1993) Glutathione oxidation and embryotoxicity elicited by diamide in the developing rat conceptus in vitro. Toxicol. Appl. Pharmacol. 120, 6271.[CrossRef][ISI][Medline]
Hiranruengchok, R., and Harris, C. (1995a). Diamide-induced alterations of intracellular thiol status and the regulation of glucose metabolism in the developing rat conceptus in vitro. Teratology 52, 205214.[ISI][Medline]
Hiranruengchok, R., and Harris, C. (1995b). Formation of protein-glutathione mixed disulfides in the developing rat conceptus following diamide treatment in vitro. Teratology 52, 196204.[ISI][Medline]
Jones, T. W., Thor, H., and Orrenius, S. (1986). Cellular defense mechanisms against toxic substances. Arch. Toxicol. Suppl. 9, 259271.[Medline]
Kaplowitz, N., Aw, T. Y., and Ookhtens, M. (1985). The regulation of hepatic GSH. Annu. Rev. Pharmacol. Toxicol. 25, 714744.
Kosower, N. S., and Kosower, E. M. (1978). The glutathione status of cells. Int. Rev. Cytol. 54, 109160.[Medline]
Lash, L. H., and Jones, D. P. (1983). Transport of glutathione by renal basal-lateral membrane vesicles. Biochem. Biophys. Res. Comm. 112, 5560.[ISI][Medline]
Lee, E., and Harris, C. (1995). Differential chemical modulation of glutathione and cysteine status in rat embryo and visceral yolk sac in vitro: Microinjection and addition to the culture medium. In Vitro Toxicol. 8, 129138.
Meister, A. (1974). Glutathione synthesis. In The Enzymes (P. D. Boyer, Ed.), pp. 671697. Academic Press, New York.
Meister, A. (1984). New aspects of glutathione biochemistry and transport: Selective alteration of glutathione metaboism. Fed. Proc. 43, 30313042.[ISI][Medline]
Meister, A., and Anderson, M. E. (1983). Glutathione. Ann. Rev. Biochem. 52, 711760.[CrossRef][ISI][Medline]
Orlowski, M., and Meister, A. (1971). Isolation of highly purified -glutamylcysteine synthetase from rat kidney. Biochemistry 10, 372380.[ISI][Medline]
Ozolins, R. S., and Hales, B. F. (1997) Oxidative stress regulates the expression and activity of transcription factor activator protein-1 in rat conceptus. J. Pharmacol. Exp. Ther. 280, 10851093.
Ozolins, R. S., and Hales, B. F. (1999a). Post-translational regulation of AP-1 transcription factor DNA-binding activity in the rat conceptus. Mol. Pharmacol. 56, 537544.
Ozolins, R. S., and Hales, B. F. (1999b). Tissue-specific regulation of glutathione homeostasis and the activator protein-1 (AP-1) response in the rat conceptus. Biochem. Pharmacol. 57, 11651175.[CrossRef][ISI][Medline]
Ozolins, T. R, Harrouk, W., Doerksen, T., Trasler, J. M., and Hales, B. F. (2002). Buthionine sulfoximine embryotoxicity is associated with prolonged AP-1 activation. Teratology 66, 192200.[CrossRef][ISI][Medline]
Richman, P., and Meister, A. (1975). Regulation of -glutamyl-cysteine synthetase by nonallosteric feedback inhibition by glutathione. J. Biol. Chem. 250, 14221426.[Abstract]
Schafer, F. Q., and Buettner, G. R. (2001). Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Rad. Biol. Med. 30, 11911212.[CrossRef][ISI][Medline]
Seelig, G. F., and Meister, A. (1985a). Glutathione biosynthesis; -glutamylcysteine synthetase from rat kidney. Methods Enzymol. 113, 379390.[ISI][Medline]
Seelig, G. F., and Meister, A. (1985b). -Glutamylcysteine synthetase from erythrocytes. Methods Enzymol. 113, 390392.[ISI][Medline]
Slott, V. L., and Hales, B. F. (1987). Protection of rat embryos in culture against the embryotoxicity of acrolein using exogenous glutathione. Biochem. Pharmacol. 36, 21872194.[CrossRef][ISI][Medline]
Snoke, J. E., and Bloch, K. (1954). The biosynthesis of glutathione. In Glutathionea symposium (S. Colowick, A. Lazarow, E. Racker, D. R. Schwarz, E. Stadtman, and H. Waelsch, Eds.), pp. 12941. Academic Press, New York.
Solis, W. A., Dalton, T. P., Dieter, M. Z., Freshwater, S., Harrer, J. M., He, L., Shertzer, H. G., and Nebert, D. W. (2002). Glutamate-cysteine ligase modifier subunit: Mouse Gclm gene structure and regulation by agents that cause oxidative stress. Biochem. Pharmacol. 63, 17391754.[CrossRef][ISI][Medline]
Stark, K. L., Harris, C., and Juchau, M. R. (1987). Embryotoxicity elicited by inhibition of -glutamyltransferase by acivicin and transferase antibodies in cultured rat embryos. Toxicol. Appl. Pharmacol. 89, 8896.[ISI][Medline]
Thompson, S. A., White, C. C., Krejsa, C. M., Eaton, D. L., and Kavanagh, T. J. (2000). Modulation of glutathione and glutamate-L-cysteine ligase by methylmercury during mouse development. Toxicol. Sci. 57, 141146.
Tsuchiya, K., Mulcahy, R. T., Reid, L. L., Disteche, C. M., and Kavanagh, T. J. (1995). Mapping of the glutamate-cysteine ligase catalytic subunit gene (GLCLC) to human chromosome 6p12 and mouse chromosome 9D-E and of the regulatory subunit gene (GLCLR) to human chromosome 1p21-p22 and mouse chromosome 3H1-3. Genomics 30, 630632.[CrossRef][ISI][Medline]