Toxicological Sciences vol. 81 no. 2 © Society of Toxicology 2004; all rights reserved.
TOXICOLOGICAL HIGHLIGHT |
Control of Glutathione Synthesis in Early Embryo Development
Indiana University School of Public and Environmental Affairs, Bloomington, Indiana 47405
Received July 12, 2004; accepted August 13, 2004
Hansen, Lee, and Harris' paper in this issue, entitled ‘Spatial Activities and Induction of Glutamate-Cysteine Ligase (GCL) in the Postimplantation Rat Embryo and Visceral Yolk Sac’ evaluates the molecular regulation of glutathione synthesis in embryos versus in the visceral yolk sac at a relatively early stage of embryonic development (rat gestational days 10 and 11). Glutathione is one of the principal antioxidant free radical scavengers in animal cells, and the most abundant low molecular weight thiol (Josephy, 1997
). Glutathione contributes to key antioxidant metabolic pathways by acting as a proton donor, or as the cofactor and nucleophilic conjugate. The first reaction, catalyzed by glutathione peroxidase, reduces hydrogen peroxide to water and yields oxidized glutathione. Similarly, glutathione can donate a proton directly to a free radical, including the very reactive hydroxyl radical. Glutathione also scavenges electrophiles, either by direct interaction (noncatalyzed) or in a reaction catalyzed by glutathione-S-transferase. It is by this last mode of action that glutathione acts as a key cofactor in metabolism (and usually detoxification) of many xenobiotics, as well as many endogenous compounds such as the steroid hormones. However, glutathione also plays a role in the antioxidant actions of other cellular protectants, helping maintain, for example, cellular ascorbate (vitamin C) levels (Josephy, 1997; Klaassen, 2001; Shaw, 1998).
Glutathione is a tripeptide made from glutamate, cysteine, and glycine, of which cysteine is usually considered to be the limiting resource (Meister, 1974). Glutathione synthesis, as discussed and evaluated in the paper by Hansen et al. (2004, this issue), is controlled by the first enzyme in the synthetic pathway, glutamate-cysteine ligase (GCL), otherwise known as 
-glutamylcysteine synthetase, which creates -L-glutamyl-L-cysteine (GC). Hansen et al. demonstrate that the GCL-mediated synthetic reaction is differentially controlled in the embryo versus in the visceral yolk sac. Overall, the synthesis of the GC dipeptide is higher in the visceral yolk sac than in the embryo, and this difference in enzyme activity increased between gestational days 10 and 11 (GD10 and GD11). Furthermore, the amount of change in GCL activity from GD10 to GD11 was greater in the embryo compared to the change in the visceral yolk sac. The basal activity of the GCL enzyme was somewhat reduced between GD10 and GD11 (118.9 pmol GC/mg/min to 71.3 pmol GC/mg/min) in the visceral yolk sac, but decreased to almost a third, from 60.5 pmol GC/mg/min in the GD10 embryo to 22.7 pmol GC/mg/min in the GD11 embryo. This is particularly interesting because, while heart formation, heart tube bending, and initial brain differentiation are ongoing throughout this period, GD11 marks the time when neural tube closure begins, and the initial outgrowths of the kidney (pronephros), inner ear, and thyroid are detectable (Altman and Bayer, 1988; Moorman and Christoffels, 2003). Both heart sculpting and neural tube closure require apoptosis (Abdelwahid et al., 2002; Weil, 1997).
Control experiments using a GCL inhibitor (buthionine-S-sulfoximine [BSO]) demonstrated that the embryonic tissues did contain active GCL, and that this GCL was the enzyme controlling the generation of the GC measured in the embryonic and visceral yolk sac tissues (Hansen et al., 2004). These control studies also demonstrated that there were some endogenous glutamate pools in the embryonic tissues, as trace levels of GC were detected even when no additional glutamate was added to the tissue incubation mixture.
Whereas earlier studies on the kinetics of the synthesis of glutathione have focused on cysteine as the rate-limiting controlled resource (Kaplowitz et al., 1985; Richman and Meister, 1975), Hansen et al. demonstrated that, at least in these early embryos, glutamate also played a role in the control of GCL activity. The affinity of cysteine for the GCL in the embryo was virtually identical in the embryonic and visceral yolk sac tissues (Km 0.030 mM), yet the affinity of glutamate for GCL was much lower in the embryonic versus the visceral yolk sac tissues. The higher Km of 1.38 mM in the embryonic tissues compared to the Km of 0.75 mM in the visceral yolk sac tissues indicate that a higher concentration of glutamate is needed in the embryonic tissues compared to the visceral yolk sac tissues in order to achieve a similar enzyme reaction rate. These results imply that the GCL in the embryonic tissues would be somewhat less sensitive to an equal concentration of glutamate in the embryonic and the visceral yolk sac tissues, and that, all other factors being equal, less glutathione would be produced in the embryo than in the visceral yolk sac. In both experiments evaluating the enzyme kinetics of GCL (i.e., one evaluating the glutamate influence on GCL activity, and the second evaluating cysteine influence) the maximal rate of the reaction (Vmax) was lower for the embryonic GCL compared to the visceral yolk sac GCL. As Hansen et al. point out, there is no evidence at this time that the GCL in the two tissues (embryonic vs. visceral yolk sac) represents two isozymes. An equally valid, but also unproven, hypothesis is simply that there are different concentrations of unidentified control factors in the two tissues.
The final sets of experiments that Hansen et al. discuss compared the differential expression of the two subunits of the GCL enzyme, the catalytic GCLC subunit and the regulatory GCLR subunit, under basal and glutathione-depleted conditions. Hansen et al. used two methods to limit the concentration of the glutathione end product in the two tissues: diethyl maleate (DEM), which conjugates the glutathione directly, and diamide, which oxidizes reduced glutathione (the antioxidizing form) to oxidized glutathione. As expected from the enzyme kinetics results, constitutive expression of both GCL subunits was higher in the visceral yolk sac tissues than in the embryonic tissues. Both methods of depleting reduced glutathione in the tissues increased GCLC expression significantly in the embryonic tissues, but produced no corresponding significant increase in the visceral yolk sac tissues. However, only the direct conjugation of glutathione by DEM, and not the oxidation of glutathione by diamide, produced a significant increase in the expression of the regulatory GCLR subunit in the embryonic tissues. Again, there was no significant change in the expression of the GCLR in the visceral yolk sac tissues after either glutathione depletion treatment.
These findings are very intriguing. The obvious question is what reason would there be for (such) differential control over glutathione production in the early embryo versus in the visceral yolk sac? Theoretically, if glutathione was functioning solely as a metabolic cofactor and/or antioxidant scavenger, it would be logical for higher concentrations to be in the very sensitive early embryo rather than the visceral yolk sac membranes. It is, after all, the embryo that is being produced here, and the visceral yolk sac is in many ways just a supportive component of the system.
GD10 is at the point where the nervous system is just forming, and the very critical and toxicologically sensitive presumptive cortical neuroblasts are just about to start proliferating. By GD11, the brain is starting to differentiate into subregions, and the neural tube is about to start closing. After the presumptive neuroblasts have undergone final cell division, they will emigrate from the internal ventricular zone toward the external surface of the brain to become the cortical plate, the very early future cortex of the brain. Initial differentiation of cortical neuroblasts starts in rats on GD12, and the cortical plate first appears on GD15. Early control of neuronal process outgrowth and activity is controlled in part by neurotransmitter activity. Glutamate, one of the key precursors in the control of GCL activity in the GD10 and GD11 embryonic tissues, also plays a role in apoptotically controlled developmental modeling of the embryonic nervous system, including neural tube closure, acting through the N-methyl-d-aspartate (NMDA) receptor.
One role for glutathione in the nervous system is to help control glutamate neurotoxicity. Among other actions, glutathione acts as a free radical scavenger, binding and neutralizing the reactive oxygen species generated by glutamate-induced oxidative stress (Han et al., 1997; Shaw, 1998; Tan et al., 1998). Increasing glutathione concentrations by intracellular synthesis also results in incorporation of a portion of the available pool of free glutamate into glutathione, thus reducing the amount of glutamate available to act on the NMDA receptors. Glutathione synthesis also helps control cysteine availability in the nervous system, and cysteine has been shown to have agonist activity at the NMDA receptor (Shaw, 1998; Zaman and Ratan, 1998). One possibility is that glutathione synthesis is in some way helping control the availability of glutamate and/or cysteine, which may be differentially controlled in the embryo versus the visceral yolk sac membranes.
No NMDA-controlled glutamate responses can be elicited from the neuroblasts until they migrate out from the ventricular zone (LoTurco et al., 1991). Dubin et al. (1999), however, have shown that even neuroblasts proliferating in the ventricular zone prior to emigration have distinctive physiological responses to at least one endogenous chemical, lysophosphatidic acid (LPA). Further, these responses take two forms, mimicking responses stimulated by the excitatory neurotransmitter (and amino acid) L-glutamate and the inhibitory neurotransmitter -aminobutyric acid (GABA). Whereas the ventricular zone cells do not appear to have altered conductances in response to GABA and glutamate at the same time as they are responsive to LPA, and the responsiveness to LPA precedes the responsiveness to GABA and glutamate, Dubin's studies clearly demonstrate that, even before the presumptive neuroblasts have left the ventricular zone, they are already physiologically active and responsive to endogenously produced biochemicals.
Interestingly, glutathione by itself may have neurotransmitter activity. A glutathione receptor and clearly defined physiological responses to glutathione exist in some invertebrates (e.g., hydra), and 3H-glutathione binds selectively within subregions of the rat retina and brain (layer 4 of the visual cortex, for example). Glutathione may also stimulate adenylate cyclase (thereby increasing intracellular concentrations of the second messenger, cAMP) through a yet unidentified mechanism (Shaw, 1998).
Thus, Hansen et al.'s evidence that glutathione synthesis is controlled differently in the embryo may imply that glutathione is a developmental modulator of early embryonic development in some way. This might be via the glutathione antioxidative effects on developmentally controlled programmed cell death, or via a more direct effect on embryonic tissues, such as via a receptor in the developing nervous system. Such a modulatory mechanism remains to be elucidated.
NOTES
1 To whom correspondence should be addressed at Indiana University, School of Public and Environmental Affairs, Spea Building, Bloomington, IN 47405. Fax: (812) 855-4556. E-mail: dhenshel{at}indiana.edu.
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