Cellular glutathione peroxidase (glutathione: H₂O₂ oxidoreductase, EC. 1.11.1.9, GPX1)⁵ was the first identified Se-dependent enzyme (Rotruck et al. 1973). Before the discovery of extracellular glutathione peroxidase (GPX3) (Takahashi and Cohen 1986), phospholipid hydroperoxide glutathione peroxidase (GPX4) (Ursini et al. 1985) and gastrointestinal glutathione peroxidase (GPX2) (Chu et al. 1993), selenoprotein P (Yang et al. 1987), type I iodothyronine deiodinase (Berry et al. 1991), mitochondrial capsule selenoprotein (Karimpour et al. 1992) and selenoprotein W (Vendeland et al. 1995), GPX1 had represented the only known biochemically functional form of body Se for many years. It has been assumed that GPX1 functions as an antioxidant enzyme, reducing hydrogen peroxide and organic hydroperoxides (Hoekstra 1975), and its activity has been used extensively to assess body Se status and nutritional requirements (Levander 1991).

The presumed antioxidative function of GPX1 has been questioned in light of recent advances in understanding of the regulation of GPX1 and other selenoproteins. Numerous studies have shown that expression of tissue GPX1 activity and mRNA is highly dependent on dietary Se level in rodents (Knight and Sunde 1987, Weitzel et al. 1990) and synthesis of tissue GPX1 competes for Se less efficiently in nutritional Se deficiency than does synthesis of other selenoproteins (Lei et al. 1995b). Although deprivation of Se causes GPX1 activity in liver to fall to extremely low levels, growth and well being in the first-generation animals remain apparently normal as long as they are fed adequate vitamin E (Sunde 1994). Moreover, Burk et al. (1980) demonstrated that Se-dependent protection against paraquat- and diquat-initiated oxidant injuries was independent of GPX1 activity in rats. Thus, it has been proposed that GPX1 may be a storage form of body Se, instead of an important antioxidative enzyme that serves a homeostatic function in Se metabolism (Burk 1991, Sunde 1994).

This "GPX1 buffer" theory, however, cannot explain the results from studies with cultured leukemia cells (Geiger et al. 1993) and cells transfected with the GPX1 gene (Mirault et al. 1991), in which GPX1 was shown to play an antioxidative role. Further, Mercurio and Combs (1986) showed that the nonmetabolized, synthetic compound, 2-phenyl-1,2-benzisoselenazol-3-(2H)on or PZ51, a GPX1-mimic, prevented the clinical signs of nutritional Se deficiency in vitamin E-deficient chicks. To clarify the metabolic role of GPX1, we used transgenic mice that overexpress the GPX1 gene to study the expression and physiological function of that enzyme. The objective of this experiment was to determine the effect of dietary Se on the GPX1 expression in these animals and the effect of the GPX1 overexpression on the expression of GPX3, GPX4 and glutathione S-transferase (EC 2.5.1.18, GST) and tissue lipid peroxidation.

MATERIALS AND METHODS

This 5.3-kb SacI fragment (Fig. 1) was used for microinjection into the fertilized mouse eggs from B6C3 mice. Briefly, female mice were superovulated with injections of pregnant mare's serum and human chorionic gonadotropin. They were killed the day after mating with stud males. Their eggs were harvested from the oviducts in sterile M2 medium (94.66 mmol/L NaCl, 4.78 mmol/L KCl, 1.71 mmol/L CaCl₂, 1.19 mmol/L KH₂PO₄, 1.19 mmol/L MgSO₄, 4.15 mmol/L NaHCO₃, 20.85 mmol/L HEPES, 23.28 mmol/L sodium lactate, 0.33 mmol/L sodium pyruvate, 5.56 mmol/L glucose, 4 g/L bovine serum albumin, 60 mg/L penicillin, 60 mg/L streptomycin and 10 mg/L phenol red), followed by incubation with 0.3 g/L hyaluronidase in M2 medium to remove the surrounding cumulus cells. The eggs were then washed in M2 medium and maintained at 37°C in sterile M16 medium that was identical to M2 medium except that it was buffered with 25 mmol/L NaHCO₃ without HEPES. Microinjection of mouse eggs was performed in sterile M2 medium according to the protocol described by Hogan et al. (1986). The eggs were then implanted into the oviducts of pseudopregnant dams for normal embryogenesis to proceed. The hemizygous transgenic and nontransgenic control littermates were identified by Southern blot analysis of genomic DNA isolated from mouse tails.

Line Tg (GPX1) 41 transgenic mice that carried three copies of the transgene were used in this study. The elevation of GPX1 mRNA expression, confirmed by Northern analysis, was 2%, 24%, 6.0-, 5.8- and 2.6-fold in liver, kidney, heart, lung and muscle, respectively, of Se- and vitamin E-adequate GPX1(+) mice compared with the controls. This overexpression of the GPX1 gene did not involve changes in the activities of glutathione reductase, catalase, glucose 6-phosphate dehydrogenase, and Cu, Zn- and Mn-superoxide dismutases in various tissues (Ho et al. 1995).

The basal diet consisted of 40% Torula yeast and contained <0.02 mg Se/kg (by analysis, Table 1). Because our mice, plus their dams, were fed diets supplemented with all-rac--tocopheryl acetate (75 mg/kg) for 2 mo prior to this experiment, we expected these mice to have had appreciable stores of this vitamin. To show the maximal potential effect of GPX1 overexpression, we chose not to supplement vitamin E in the experimental diets. An experiment with a 2 × 2 factorial arrangement of treatments with 15 GPX1(+) and 15 control mice (2 mo old) was conducted for 8 wk. Ten mice within each group (half males and females) were fed the basal diet and five mice (3 males and 2 females) were fed the basal diet supplemented with 0.51 mg Se/kg as Na₂SeO₃. All mice were housed individually in hanging wire 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 food. Body weights of individual mice were recorded weekly.

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

RESULTS

Activities of GPX4 in all tissues except for heart and stomach were greatly affected by dietary Se concentrations (P < 0.0001), but not by the overexpression of the GPX1 gene in mice (Fig. 4). In contrast to other tissues, heart GPX4 activities in the Se-deficient GPX1(+) mice were greater than in Se-deficient controls (P < 0.05) and not different than that of the Se-adequate GPX1(+) mice. Stomach GPX4 activities were the lowest among the tissues assayed and were not different (P = 0.50) among the four treatment groups. Similarly, GPX3 activities in plasma were greatly affected (P < 0.0001) by dietary Se concentrations (Fig. 5), but not by the GPX1 overexpression.

DISCUSSION

The lack of effect on the expression of GPX3 activity in plasma and GPX4 activity and/or mRNA level in various tissues by the overexpression of GPX1 in mice fed both levels of dietary Se suggested that these selenoperoxidase genes were expressed independently. Although the effects of GPX1 overexpression on selenoprotein P and type I iodothyronine deiodinase expression were not determined in the present study, the lack of difference in plasma Se concentrations between the controls and the GPX1(+) mice implied that expression of selenoprotein P was likely unaffected. Similarly, overexpression of GPX1 did not alter the expression of GPX4 in NIH3T3 and MCF7 cells (Kelner et al. 1995) and overexpression of GPX4 did not alter the expression of GPX1 in FL5-12 cells (Lei et al. 1995a). In addition, the lack of differences in tissue GST activities due to overexpression of the GPX1 was consistent with the earlier suggestion that increases in GST activities in Se-deficient rats were not directly related to the fall in GPX1 activity (Hill et al. 1987).

Overexpression of GPX1 was also shown to be tissue specific. In nutritional Se adequacy, GPX1 activity, just as its mRNA, was not greater in liver and was only marginally greater in kidney in GPX1(+) mice than in control mice. This contrasted to the well-documented, large fluctuation of liver GPX1 expression caused by changes in dietary Se levels (Lei et al. 1995b, Weitzel et al. 1990). Because our Se-supplemented diet contained an amount of Se (0.51 mg Se/kg) that should be sufficient to saturate Se metabolic systems, factors other than Se (Moriarty et al. 1995) must have limited the overexpression of GPX1 in liver and to some extent in kidney in the present study. Because liver expresses the greatest GPX1 activity among all of the tissues, it is likely that under conditions of Se adequacy the selenoprotein synthetic system might have reached a maximal rate such that further increase in GPX1 expression was not possible. Christensen et al. (1995) reported that dietary Se in excess of the nutritional needs did not increase GPX1 mRNA in liver of rats. Alternatively, the 5.3-kb mouse GPX1 genomic fragment used herein might not contain sufficient genetic information to direct a specific overexpression of GPX1 in liver.

The tissue-specific overexpression of GPX1 was also demonstrated in lung, in which there was sixfold greater GPX1 activity in Se-deficient mice compared to the controls. In contrast, there was only ~100% greater expression in other tissues of Se-deficient mice or in lung of Se-adequate mice. It remained unclear to us whether overexpression of GPX1 in the lungs of the GPX1(+) mice was affected by dietary Se deficiency differently from that in other tissues. Hawker et al. (1993) reported that Se deficiency augmented the pulmonary toxic effects of oxygen exposure. An oxygen-responsive element in the flanking region of the GPX1 gene has been identified, and oxidative stress may directly regulate GPX1 transcription (Cowan et al. 1993). In addition, in Se-deficient mice, it is unclear why there exists greater GPX4 activity in heart of the GPX1(+) mice compared with controls, except for the fact that GPX4 activity comprises a substantial portion of total heart GPX activity of the Se-deficient mice (Weitzel et al. 1990). Stomach GPX4 might be too low for us to detect any differences.

The present results were somewhat unexpected in that they indicated a significantly higher expression of GPX1 activities in various tissues and GPX1 mRNA levels in kidney and lung of the Se-deficient GPX1(+) mice than in the controls. Compared with their Se-adequate counterparts, the Se-deficient GPX1(+) mice retained greater amounts of the residual GPX1 activity in tissues than the Se-deficient controls. Because numerous researchers have shown rapid declines of GPX1 expression in tissues or cells to minimal levels by Se depletion, GPX1 mRNA has been presumed to be so unstable that expression of GPX1 is either minimized or precluded (Bermano et al. 1995). Subsequently, the limited Se would be channeled for preferential maintenance of some critical metabolic forms of Se (Sunde 1994). However, the present observations did not fully support this view. Although Se-deficient control mice had only 3% of the residual GPX1 activity in liver of the Se-adequate control mice, several-fold higher GPX1 activities and/or mRNA levels were still expressed in various tissues of the Se-deficient GPX1(+) mice than in those of the control mice.

Because GPX1 has been assumed to reduce hydrogen peroxide and organic hydroperoxides in vivo (Hoekstra 1975), we tested whether the substantially greater (up to sixfold) GPX1 activity expression in Se-deficient GPX1(+) mice than in the controls had any effect on the status of lipid peroxidation. Because we did not see any differences in TBARS concentrations in tissue homogenates of these two groups of mice, the physiological implication of the overexpressed GPX1 in Se-deficient mice in the present study apparently was not closely related to lipid peroxidation. Because our mice were fed diets that were not supplemented with vitamin E, a clearly potent antioxidant that prevents lipid peroxidation (Lee and Csallany 1994), we should have had a better chance to demonstrate an antioxidative role of GPX1 if there was one. It might be possible that our measure of lipid peroxidation was not sensitive enough to show such a role of GPX1. In addition, transgenic mice that overexpressed human GPX1 or GPX3 had lower concentrations of peroxides in the brain than the control mice (Mirochnitchenko et al. 1995). Thus, we are using more sensitive measures than TBARS to clarify the role of GPX1 in both GPX1 overexpressing and knockout mice.

ACKNOWLEDGMENTS