Cellular glutathione peroxidase (glutathione: H₂O₂ oxidoreductase, EC 1.11.1.9, GPX1)⁵ was discovered as an erythrocyte enzyme that protects against oxidative hemolysis (Mills 1957) and later found to be Se-dependent (Rotruck et al. 1973). For many years, GPX1 represented the only known biochemically functional form of body Se. Recent molecular approaches have helped unravel three other Se-dependent glutathione peroxidases: GPX2, gastrointestinal GPX (Chu et al. 1993); GPX3, plasma or extracellular GPX (Takahashi and Cohen 1986); and GPX4, phospholipid hydroperoxide GPX (Ursini et al. 1985). The interrelationship between the expression of GPX1 and these new members of the Se-dependent GPX enzyme family remains to be elucidated.

Because of its ability to reduce hydrogen peroxide and organic hydroperoxides in vitro, GPX1 has been assumed to be an in vivo antioxidative enzyme (Hoekstra 1975). However, questions have been raised concerning the linkage of GPX1 activity and growth, well being or health in animals (Sunde 1994). Furthermore, expression of tissue GPX1 mRNA and activity is affected by dietary Se deficiency to a much greater extent than are the other selenoproteins, and the incorporation of Se into GPX1 is fulfilled at dietary Se levels greater than those required for the other selenoproteins in rats and mice (Christensen et al. 1995, Vadhanavikit and Ganther 1993, Weitzel et al. 1990, Yang et al. 1989). Therefore GPX1 has been viewed as a storage form of body Se, rather than an important antioxidative enzyme that serves a homeostatic function in Se metabolism (Burk 1991, Sunde 1994). Although this "GPX1 buffer" hypothesis is not fully supported by the results of recent studies with cell models (Geiger et al. 1993, Kelner et al. 1995), it has raised concerns over the use of the maximal GPX1 activity in tissues to determine dietary Se needs for animals and humans (Levander and Whanger 1996). Thus it is critical to clarify the metabolic role of GPX1. Genetically intact animals are not ideal for this because of possible confounding effects of multiple selenoproteins that are all affected by dietary Se level.

We have used transgenic mice that either overexpress the GPX1 gene or do not express the GPX1 gene (knockout) to study the role of GPX1 in Se metabolism and function. Previously, we demonstrated that overexpression of GPX1 did not affect expression of GPX3, GPX4 or Se-independent glutathione S-transferase (GST) in Se-deficient or -adequate mice (Cheng et al. 1997). The objectives of this study were to determine the effects of the GPX1 knockout on the following: 1 ) the susceptibility of mice to dietary vitamin E and Se deficiency; 2 ) the expression of GPX3, GPX4 and GST, and 3 ) the total Se concentration in the liver of Se-adequate mice.

MATERIALS AND METHODS

The GPX1() mice derived from line 80 were used in this study. The knockout of GPX1 gene expression was confirmed by Northern analysis and did not affect activities of glutathione reductase, catalase, glucose 6-phosphate dehydrogenase, and Cu,Zn- and Mn-superoxide dismutases in various tissues (Ho, Y.-S., unpublished results). All mice were housed individually in hanging stainless steel cages in a temperature-controlled (22°C) animal room with a 12-h light:dark cycle and were given free access to distilled water and diets. The health condition of the mice was checked daily and body weights of individual mice were recorded weekly.

Table 1. Composition of Torula yeast basal diet1 [View Table]

Because GPX3 has been shown to be produced primarily by renal cells (Maser et al. 1994) and lung has relatively high GPX4 activity in mice, we determined the effect of the GPX1 knockout on kidney GPX3 and lung GPX4 mRNA expression in Experiment 1. Total RNA was isolated from the kidneys and lungs of three Se-deficient and two Se-adequate mice from both the control and GPX1() groups using TRIzol reagent (BRL). RNA (15 µg per lane) was loaded (10 lanes per gel) and separated by electrophoresis in a 1.5% agarose-formaldehyde gel, transferred and blotted to a hyblot membrane (National Labnet, Woodbridge, NJ). The Northern analysis was conducted as previously described (Cheng et al. 1997). Briefly, blots were prehybridized, hybridized and washed in a Hybaid Maxi 14 Hybridization Oven (Hybaid, Middlesex, UK). The GPX3 probe was a 277-bp Eco R I fragment of murine GPX3 (generously provided by J. P. Calvet, University of Kansas Medical Center, Maser et al. 1994). The GPX4 probe was a 637-bp Eco R I/Xho I fragment of rat GPX4, and the 18S rRNA probe was an 1.4-kb Bam H I fragment of human 18S rRNA (generously provided by R. A. Sunde, University of Missouri, Lei et al. 1995b). The probes were random-primed labeled with ³²P (dCTP, Du Pont, Boston, MA) using a DNA labeling kit (Pharmacia Biotech., Piscataway, NJ) followed by G-50 column purification (Pharmacia Biotech.). Levels of the GPX3 and the GPX4 mRNA were determined in two separate, but identical blots with the use of a FuJIX BAS100 Bio-imaging Analyzer (Fuji Photo Film, Kanagawa, Japan). The relative levels of mRNA were normalized by the levels of 18S rRNA detected on the same blots after removing the GPX3 or GPX4 probe.

RESULTS

Experiment 1

Experiment 2

DISCUSSION

In our previous study, we demonstrated that the overexpression of GPX1 in mice did not affect expression of either GPX3 or GPX4 in various tissues (Cheng et al. 1997). In the present study, we observed no effect of the GPX1 knockout on the expression of GPX3 mRNA in kidney, GPX3 activity in plasma, GPX4 mRNA in lung or GPX4 activity in any tissue except kidney, in mice fed both levels of dietary Se. Although renal GPX4 activity in Se-adequate GPX1() mice was significantly lower (P < 0.009) than that of controls, the 10% difference did not seem to be physiologically important. Therefore, we conclude that these selenoperoxidase genes are expressed independently in mice. Other researchers have also shown that overexpressions of GPX1 or GPX4 in cultured cells do not affect each other (Kelner et al. 1995, Lei et al. 1995a). Similarly, the GPX1 knockout, just as the GPX1 overexpression (Cheng et al. 1997), did not affect hepatic or renal GST activities in mice fed the Se-adequate or -deficient diets. In contrast, GST activities in these tissues were enhanced in both the control and GPX1() mice fed the Se-deficient diet. As a Se-independent enzyme, GST has been proposed to play an important role in the hydrolysis of bulky hydroperoxides in Se-deficient animals (Burk et al. 1978, Reiter and Wendel 1984). Our data clearly indicate that expressions of GPX1 and GST in mice are regulated independently, and that factors other than the fall of GPX1 activity in the Se-deficient mice are responsible for the elevated GST activity.

On the basis of the masses, Se contents and GPX1 activities of various tissues, Behne and Wolters (1983) estimated that 63% of liver total Se was bound to GPX1 in Se-adequate adult female rats. That estimate compares very favorably with the present result that the total liver Se concentration in the Se-adequate GPX1() mice was ~60% lower than that in the controls. As discussed above, the GPX1 knockout did not affect GPX3 or GPX4 expression in liver of the mice. Our previous work showed that the GPX1 overexpression did not affect plasma Se concentration (Cheng et al. 1997), 65% of which is bound to selenoprotein P (Read et al. 1990). Recently, we also found that the GPX1 knockout did not affect selenoprotein P mRNA levels in the liver of Se-adequate mice (Lei, X. G., unpublished results). Thus, the expression of selenoprotein P appears to be independent of that of GPX1. It is reasonable to conclude that mouse liver GPX1 represents ~60% of total liver Se, unless future work shows an effect of the GPX1 knockout on the expression of iodothyronine deiodinases (Berry et al. 1991, Croteau et al. 1996, Salvatore et al. 1995), selenoprotein W (Vendeland et al. 1995), or other selenoproteins (Karimpour et al. 1992) and/or on overall Se metabolism in the liver of mice.

Unexpectedly, no histopathology was detected in the liver of any of the mice in Experiment 1. Because all of the mice were fed diets without supplemental vitamin E for 13 wk, we had expected signs of liver necrosis in those fed the Se-deficient basal diet. Apparently, these mice remained healthy and had body weight gains comparable to those of vitamin E-fed mice. It is possible that this 129/SVJ × C57BL/6 mouse strain was more resistant to dietary Se and vitamin E deficiency than those used in the earlier studies (De Witt and Schwarz 1958). In addition, their dams were fed the diet supplemented with all-rac--tocopheryl acetate at 75 mg/kg and these mice were fed the basal diet supplemented with all-rac--tocopheryl acetate at 15 mg/kg for 2 wk before the start of the experiment. Thus, maternal endowment and tissue stores of vitamin E must have been sufficient to protect these Se-deficient mice from tissue damage. Still, it is clear that the GPX1 knockout did not make these mice more susceptible to dietary Se and vitamin E deficiency. Spector et al. (1996) also did not observe any significant change in the response of lens epithelial cells to medium hydrogen peroxide stress in the GPX1() mice compared with the controls. There was no effect of the GPX- overexpression on malondialdehyde concentrations in tissue homogenates of mice fed the diet deficient in both Se and vitamin E for 8 wk (Cheng et al. 1997). Furthermore, the GPX1() mice are fertile and show no apparent pathological changes. Therefore, it is premature to comment on the full physiological role of GPX1 from the results of the present study. More sensitive assays should be used in future studies, and oxidative stresses more direct than the dietary Se and vitamin E deprivation should also be employed in these mice to determine the metabolic role of GPX1.