The Journal of Nutrition Vol. 129 No. 1 January 1999, pp. 194-198

Aurothioglucose Inhibits Murine Thioredoxin Reductase Activity In Vivo¹

Allen D. Smith², Catherine A. Guidry, Virginia C. Morris, and Orville A. Levander

U.S. Department of Agriculture, Beltsville Human Nutrition Research Center, Nutrient Requirements and Functions Laboratory, Beltsville, MD 20705

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Gold (I)-containing compounds, including aurothioglucose (ATG), are potent in vitro inhibitors of several selenocysteine-containing enzymes. Gold compounds have also been shown to potentiate the virulence of several viruses in mice, including coxsackievirus, implicated as a possible infectious agent in Keshan disease. One possible mechanism by which gold compounds may be increasing the virulence of viral infections in mice is by acting as a selenium antagonist in vivo and inducing oxidative stress. To investigate the possible role of gold compounds in inducing oxidative stress in mice, we assessed the ability of ATG administered in vivo to inhibit the activity of the selenocysteine-containing enzymes thioredoxin reductase (TR) and glutathione peroxidase (GPX1). Doses as low as 0.025 mg ATG/g body weight caused significant and prolonged inhibition of TR activity in all tissues examined. No such inhibition of GPX1 activity was seen, indicating differential in vivo sensitivity of the enzymes to inhibition by ATG. In liver and heart, some recovery of TR activity was observed after a 7-d period, but no recovery was observed in pancreas or kidney. Because TR is involved in several important cellular redox functions, its inhibition most likely will affect multiple cellular processes. These results indicate that in vivo administration of ATG results in significant and long-lasting inhibition of TR activity. Such inhibition of TR could lead to increased levels of oxidative stress in vivo, thereby increasing the virulence of several viruses including the coxsackievirus.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Selenium plays an important role in host antioxidant defense by virtue of its incorporation into antioxidant enzymes as selenocysteine. The activities of the selenocysteine-containing enzymes, thioredoxin reductase (TR;³ NADPH-oxidized thioredoxin oxidoreductase, EC 1.6.4.5) and glutathione peroxidase (GPX1; H₂O₂:oxidoreductase, EC 1.11.1.9) can be altered by manipulating the selenium content of the diet (e.g., Hill et al. 1997a, Levander et al. 1983). In vitro, aurothioglucose (ATG) has been shown to inhibit TR (Hill et al. 1997a), GPX1 (Chaudiere and Tappel 1984), and some, but not all, selenodeiodinases (e.g., Berry et al. 1991, Croteau et al. 1995, Salvatore et al. 1996, St. Germain et al. 1994). It has also been demonstrated that gold (I) compounds can inhibit certain selenocysteine-containing enzymes in vivo. For example, Hu et al. (1988) reported that repeated administration of ATG to rats over an 8-wk period decreased platelet, kidney and liver GPX1 activity. Thus, administration of gold (I) compounds can have some of the same effects as selenium deficiency on selenocysteine-containing enzymes in vivo. Therefore, gold compounds may induce oxidative stress both in vitro and in vivo.

Gold compounds have also been shown to potentiate the virulence of several viruses, including members of the flavi- (Mehta and Webb 1987), toga- (Allner et al. 1974, Bradish et al. 1975), and picornavirus (Kabiri et al. 1978, Stebbing et al. 1978) families. The last-mentioned includes the coxsackieviruses, which have been implicated as possible infectious agents in the selenium-responsive cardiomyopathy Keshan disease (Levander and Beck 1997). Other studies have shown that induction of oxidative stress via dietary selenium or vitamin E deficiency in mice can lead to conversion of an avirulent strain of coxsackievirus B3 (CVB3/0) to virulence (Beck et al. 1994a, 1994b, 1994c and 1995). Therefore, the possibility exists that ATG, by acting as a selenium antagonist, may be inducing oxidative stress in mice that leads to changes in the virulence of RNA viruses similar to those observed when mice are rendered selenium- or vitamin E-deficient by dietary manipulation.

To investigate the possible role of ATG in inducing oxidative stress in mice, we tested the ability of a single dose of ATG injected intraperitoneally to inhibit the selenocysteine-containing enzymes thioredoxin reductase (TR) and glutathione peroxidase (GPX1). In the experiments reported here, we found that the activity of TR was potently inhibited by a single intraperitoneal injection of ATG, whereas ATG had no such effect on GPX1 activity. These results suggest that TR activity may be more important then GPX1 activity in determining the effects of dietary selenium deficiency on viral virulence.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Materials.  Aurothioglucose (cat. # A 0632) was purchased from Sigma Chemical (St. Louis, MO). Dulbecco's PBS (D-PBS; cat. # 14040-133) was purchased from Gibco BRL (Grand Island, NY). Micro BCA Protein Assay Kits were obtained from Pierce (Rockford, IL).

Mice and diet.  Three-week-old weanling male C3H/HeJ mice were purchased from Jackson Laboratory (Bar Harbor, ME) and were given free access to water and a nonpurified diet. Mice were housed four or five to a cage at the USDA/ARS Beltsville Small Animal Facility, which is fully accredited by the American Association for Accreditation of Laboratory Animal Care. The protocols used in these experiments were approved by the Institutional Animal Care and Use Committee. Mice were killed by cervical dislocation. In anticipation of future experiments with virus, mice were housed after injection with ATG in Semi-Rigid Isolators purchased from Park Bioservices, (Lawrence, MA), operated under negative air pressure for the duration of the experiment.

The mice were fed nonpurified diet (Mouse Chow #7012, Harland TekLad, Madison, WI) containing 20% protein, 5.7% fat, 4.4% fiber and 6.5% ash. The diet was assayed for selenium content by isotope dilution gas chromatography/mass spectrometry (Reamer and Veillon 1981) with wheat flour SRM 1567a from NIST as a reference material; it was found to contain 210 ng Se/g (ambient moisture).

Injection of mice.  Solutions of ATG were prepared in D-PBS and sterile-filtered. Mice received a single injection of ATG. For Experiment 1, groups of eight mice were injected with 0 (D-PBS only), 0.2, 0.4, 0.6, 0.8 or 1.0 mg ATG/g body weight (BW). Body weight, mortality and morbidity were monitored for 6 d. On the basis of the results obtained from this pilot experiment, groups of 32 mice were injected with 0 (D-PBS only) or 0.2 mg ATG/g BW (Experiment 2). At 2, 4, 8, 24, 48, 72, 120 and 168 h postinjection, hearts, livers and pancreases were removed from groups of four mice, frozen in liquid nitrogen and stored at 80°C until the tissues were processed. To serve as a control, four mice were injected with D-PBS only and then their organs were removed and frozen. In Experiment 3, groups of 20 mice were injected with 0, 0.025, 0.05, 0.1 or 0.2 mg ATG/g BW. At 2, 24, 48 and 168 h after injection, hearts, livers, pancreases and kidneys were harvested from groups of five mice and frozen as described above. In addition, to serve as a zero time control, five mice were injected with D-PBS only and then immediately killed.

Tissue processing.  Tissues were thawed, weighed and homogenized in either 4 (heart, kidney, pancreas) or 9 (liver) volumes of 0.154 mol/L KCl solution. After low speed centrifugation to remove cellular debris, the samples were ultracentrifuged at 105,000 × g for 2 h to generate a postmicrosomal supernatant.

Protein and enzyme assays.  The protein concentration of the postmicrosomal supernatants was determined using the Micro BCA Protein Assay (Smith 1985) with bovine serum albumin as a standard. Thioredoxin reductase [adapted from Tamura and Stadtman (1996) as originally described by Holmgren (1977)] and GPX1 (McAdam et al. 1984) enzymatic activities were measured using assays modified to work in a 96-well microplate. Briefly, assays were initiated by the addition of 200 µL of assay mix to 50 µL of sample in a 96-well microplate; the change in optical density was measured with the use of the kinetics program of the SPECTRAmax PLUS microplate spectrophotometer (Molecular Devices, Sunnyvale, CA). Specific activities were calculated by using an assumed path length of 0.65 cm for a 0.25 mL assay volume. Contributions of nonspecific non-thioredoxin reductase activity were determined by running duplicate assays on a representative subset of samples from each tissue in the presence of 20 µmol/L ATG (Hill et al. 1997b). Background rates were consistent from sample to sample and were averaged; the average background reaction rate obtained in the presence of ATG was subtracted from that obtained in the absence of ATG to give the final corrected thioredoxin reductase rate. One unit of TR activity was defined as 1 µmol 5-thio-2-nitrobenzoic acid formed per minute. One enzyme unit of GPX1 activity was defined as 1 µmol NADPH oxidized per minute.

Statistical analysis.  Results were analyzed statistically using a one-way ANOVA and a Student-Newman-Keuls multiple range test or a paired Student's t test using the SigmaStat program (SPSS, Chicago, IL). Probability values <0.05 were considered significant. Data in the figures are reported as means ± SEM.

	RESULTS

Abstract Introduction Methods Results Discussion References

The purpose of Experiment 1 was to identify the highest dose of ATG that could be administered to C3H/HeJ mice without causing mortality or excessive weight loss under our specific set of housing and dietary conditions, so as to avoid complicating our analysis. This dose ranging study showed that ATG doses >0.4 mg/g BW led to significant mortality and morbidity (data not shown). Over a 6-d period after injection, control mice receiving D-PBS only (no ATG) had a group mean weight change of +13%. On the other hand, mice from groups receiving 0.2 or 0.4 mg ATG/g BW had group mean weight changes of +2 and 9%, respectively.

On the basis of the toxicity data from Experiment 1, a second experiment was conducted to investigate the effect of 0.2 mg ATG/g BW on TR and GPX1 activity. TR activity was inhibited by >90% within 2 h of ATG administration in heart, liver and pancreas (Fig. 1A), whereas GPX1 activity was largely unaffected by ATG treatment (Fig. 1B). A tendency of TR activity to recover was observed in heart and liver over a 7-d period, and the TR activity observed at 7 d post-ATG administration was significantly greater than the activity at 2 h postinjection (P < 0.05). In contrast, TR activity remained completely inhibited in pancreas during this time. However, it was also noted that transient weight loss still occurred at the ATG dose of 0.2 mg/g BW (data not shown), and potential complicating effects due to toxicity could not be completely ruled out.

View larger version (23K): [in this window] [in a new window]   Fig 1. Effect of intraperitoneal injection of 0.2 mg aurothioglucose (ATG)/g body weight on the activities of thioredoxin reductase (TR; panel A) and glutathione peroxidase (GPX1; panel B) in heart, liver and pancreas of C3H/HeJ mice. Mice were killed at various time points after injection, tissues harvested, processed and the postmicrosomal supernatant assayed for TR and GPX1 activity. Values are means ± SEM, n = 4. Asterisk indicates that the values at 168 h are significantly different from the 2-h time point by paired Student's t test (P < 0.05).

To identify a dosage of ATG that minimized toxicity and still resulted in significant inhibition of TR activity, the dosage of ATG administered was decreased from 0.2 to 0.025 mg/g BW in 50% decrements (Experiment 3). Dosages of ATG >0.05 mg/g BW led to less weight gain and transient weight loss over the first 48 h post-ATG administration (data not shown). These results indicate that, under our conditions, administration of ATG 0.05 mg/g BW should not result in mortality or weight loss in C3H/HeJ mice.

The inhibitory effect of ATG against TR activity was strong, long-lasting and consistent, even at the lowest dosage tested (0.025 mg/g BW) (Fig. 2). Significant inhibition was observed within 2 h of ATG administration at all dosages tested (P < 0.05). As noted in Experiment 2, there was a pronounced difference in the way the various tissues responded to the ATG treatment. TR activity in the pancreas and kidney was blocked completely by the ATG over the entire 7-d test period and showed no signs of recovering. However, a significant recovery of TR activity was observed at d 7 compared with 2 h post-ATG administration in heart for all four ATG doses tested and in liver for the three lowest ATG doses. In the heart at d 7, the recovery of TR activity was greater with lower gold doses, but the differences in recovery among the ATG doses were not significant. In liver, there was an overall dosage-dependent trend toward greater recovery of TR activity at lower ATG doses.

View larger version (28K): [in this window] [in a new window]   Fig 2. Effect of aurothioglucose (ATG) dose on thioredoxin reductase (TR) activity in heart (panel A), liver (panel B), pancreas (panel C) and kidney (panel D) of C3H/HeJ mice. Mice were killed at 2, 24, 48 and 168 h after administration of different doses of ATG, tissues harvested, processed and the postmicrosomal supernatant assayed for TR activity. Values are means ± SEM, n = 5. At any given time, means with different letters were significantly different (Student-Newman-Keuls Multiple range test; P < 0.05). For clarity, significance indicated only where appropriate at 2 and 168 h. Asterisk indicates that the values at 168 h are significantly different from the 2-h time point by paired Student's t test (P < 0.05).

In contrast to the results obtained with TR, the inhibitory effects of graded doses of ATG on GPX1 activity were transient and inconsistent (Fig. 3). For example, ATG had no significant effect whatsoever on GPX1 activity in the heart. ATG, however, did cause a significant dose-dependent decrease in GPX1 activity in the pancreas and kidney 2 h after treatment, but this inhibitory effect was lost by 24 h. ATG even caused a paradoxical increase in GPX1 activity in the liver at 2 h, which disappeared after 24 h. This ATG-induced increase in hepatic GPX1 activity 2 h after treatment was not observed in Experiment 2 and the reason for this inconsistency is not known.

View larger version (31K): [in this window] [in a new window]   Fig 3. Effect of aurothioglucose (ATG) dose on glutathione peroxidase (GPX1) activity in heart (panel A), liver (panel B), pancreas (panel C) and kidney (panel D) of C3H/HeJ mice. Mice were killed at 2, 24, 48 and 168 h after administration of different doses of ATG, tissues harvested, processed and the postmicrosomal supernatant assayed for GPX1 activity. Values are means ± SEM, n = 5. At any given time, means with different letters were significantly different (Student-Newman-Keuls Multiple range test; P < 0.05). For clarity, significance indicated only where appropriate at 2 and 168 h.

Because ATG is a potent inhibitor of certain selenocysteine-containing enzymes, it was of interest to determine the effect of ATG on GR, an enzyme that relies on cysteine moieties for activity but contains no selenocysteine. No significant inhibitory effect of ATG on GR activity was noted in the heart, liver or pancreas (data not shown). A transient dose-dependent inhibition of GR activity seen in kidney 2 h after ATG treatment (67% maximum inhibition) was not observed at later time points (data not shown).

	DISCUSSION

Abstract Introduction Methods Results Discussion References

The results presented here demonstrate that treatment of mice with ATG in vivo causes significant and long-term inhibition of TR activity, but not of GPX1 or GR activity. The TR inhibition occurred at ATG doses that did not adversely affect the weight gain of the animals, indicating that feed refusal was not responsible for the observed changes in enzyme activity.

Several factors are known to influence ATG toxicity. For example, ATG causes variable mortality depending on the dose and route of administration (Brecher and Walker 1949, Larsson 1967), the age and strain of the mice (Liebelt et al. 1960), the environmental temperature (De Laey et al. 1974) and the nutritional state (Edelman et al. 1965, Larsson 1967, Soyka et al. 1969). Furthermore, mice injected with ATG can go through an initial phase of weight loss followed by excessive weight gain (De Laey et al. 1974). Because food deprivation can lead to decreases in tissue glutathione content and antioxidant enzyme activities, we sought to identify doses of ATG that did not significantly alter the weight gain of the mice, thus avoiding an additional confounding factor. Our results indicate that ATG at doses 0.4 mg/g BW caused significant mortality and were in agreement with those reported by De Laey et al. (1974) for CD-1 mice. We also noted that injection of ATG led to transient initial weight loss and lower overall total weight gain 7 d after administration of doses >0.05 mg ATG/g BW. Thus, to eliminate any potential confounding factors due to weight loss, doses 0.05 mg ATG/g BW should be employed.

Inhibition of TR activity varied, depending on the tissue examined. Two hours after administration of ATG, complete inhibition of TR activity was observed in heart, pancreas and kidney, whereas ~9-17% of the original TR activity remained in liver, depending on the dose of ATG administered. The loss in TR activity within 2 h of injection agrees with the reported peak serum gold concentrations that occur ~2 h after intramuscular injection of rheumatoid arthritis patients with another gold (I) compound, aurothiomalate (Blocka et al. 1986). Recovery of TR activity with time also varied in a tissue-specific manner, with recovery of activity occurring in heart and liver but not in pancreas or kidney. Pancreas has been reported to be more sensitive to oxidative stress than other tissues (e.g., Malaisse 1982, Tiedge et al. 1997); these results suggest that the role of TR in the oxidative defenses of this organ should be investigated further.

The lack of inhibition of GPX1 by ATG in vivo beyond 2 h postinjection may be related to the differential sensitivity of GPX1 and TR to ATG in vitro. Only one tenth as much ATG was needed to inhibit TR to the same degree as GPX1 in rat liver supernatants (Hill et al. 1997a). The transient inhibition of GPX1 activity observed at 2 h (in pancreas and kidney) may be explained by the pharmacokinetics and localization of gold (I) compounds after their administration. Peak serum gold concentrations have been reported to occur ~2 h after intramuscular administration of aurothiomalate (Blocka et al. 1986), which corresponds to the time at which we observed transient inhibition of GPX1. In addition, gold is concentrated in the kidneys; this may help to account for the increased inhibition observed in this tissue (Atkins et al. 1975, Baker et al. 1985). Thus, the lack of long-term inhibition of GPX1 by ATG, as well as the tissue-dependent pattern of inhibition, may simply be a reflection of the different tissue concentrations of gold and the lower sensitivity of the enzyme to inhibition by ATG compared with TR. Alternatively, Chaudiere and Tappel (1984) reported that inhibition of GPX1 by ATG is reversible upon dilution and further demonstrated inhibition of GPX1 in rats receiving multiple injections of ATG with the use of highly concentrated rat liver supernatants that were minimally diluted for analysis (Hu et al. 1988). Thus, the protocols described herein cannot completely rule out the possibility that some inhibition of GPX1, in addition to that observed at 2 h, was occurring in vivo.

The results reported here indicate that the antioxidant defenses of mice treated with ATG may be impaired via inhibition of TR and possibly other antioxidant enzyme activities as well. In addition to its well-known role in ribonucleotide reduction, TR is also important in the redox control of certain enzymes, receptors and transcription factors (reviewed in Björnstedt et al. 1997). Thus, inhibition of TR may affect multiple cellular processes. Furthermore, TR has been shown to reduce directly lipid hydroperoxides (Björnstedt et al. 1995), dehydroascorbate (May et al. 1997), lipoamide and lipoic acid (Arner et al. 1996) and may act as an alternative pathway to glutathione peroxidases for the detoxification of hydroperoxides. These studies suggest that inhibition of TR could result in increased oxidative stress via several mechanisms. Reglinski et al. (1997) recently provided evidence that oxidative stress in rheumatoid arthritis patients may be increased by the gold(I) compound aurothiomalate. Therefore, it seems reasonable that ATG treatment of mice, which results in significant and long-lasting inhibition of TR activity, also leads to increased oxidative stress. Dillard and Tappel (1986) proposed that ATG can act as a selenium antagonist, thus mimicking the effects of a selenium deficiency, which then may cause the increase in virulence observed with a number of RNA viruses passaged through gold I-treated mice. To understand further the role of ATG treatment in antioxidant defense and its role in viral pathogenesis, we are currently investigating the effects of ATG treatment on the pathogenesis of an avirulent strain of coxsackievirus B3 (CVB3/0).

	FOOTNOTES

Manuscript received 10 July 1998. Initial reviews completed 23 July 1998. Revision accepted 21 October 1998.

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