Reduction of diphenyl diselenide and analogs by mammalian thioredoxin reductase is independent of their gluthathione peroxidase-like activity: a possible novel pathway for their antioxidant activity

Abstract

Since the successful use of the organoselenium drug ebselen in clinical trials for the treatment of neuropathological conditions associated with oxidative stress, there have been concerted efforts geared towards understanding the precise mechanism of action of ebselen and other organoselenium compounds, especially the diorganyl diselenides such as diphenyl diselenide, and its analogs. Although the mechanism of action of ebselen and other organoselenium compounds has been shown to be related to their ability to generally mimic native glutathione peroxidase (GPx), only ebselen however has been shown to serve as a substrate for the mammalian thioredoxin reductase (TrxR), demonstrating another component of its pharmacological mechanisms. In fact, there is a dearth of information on the ability of other organoselenium compounds, especially diphenyl diselenide and its analogs, to serve as substrates for the mammalian enzyme thioredoxin reductase. Interestingly, diphenyl diselenide shares several antioxidant and neuroprotective properties with ebselen. Hence in the present study, we tested the hypothesis that diphenyl diselenide and some of its analogs (4,4'-bistrifluoromethyldiphenyl diselenide, 4,4'-bismethoxy-diphenyl diselenide, 4.4'-biscarboxydiphenyl diselenide, 4,4'-bischlorodiphenyl diselenide, 2,4,6,2',4',6'-hexamethyldiphenyl diselenide) could also be substrates for rat hepatic TrxR. Here we show for the first time that diselenides are good substrates for mammalian TrxR, but not necessarily good mimetics of GPx, and vice versa. For instance, bis-methoxydiphenyl diselenide had no GPx activity, whereas it was a good substrate for reduction by TrxR. Our experimental observations indicate a possible dissociation between the two pathways for peroxide degradation (either via substrate for TrxR or as a mimic of GPx). Consequently, the antioxidant activity of diphenyl diselenide and analogs can be attributed to their capacity to be substrates for mammalian TrxR and we therefore conclude that subtle changes in the aryl moiety of diselenides can be used as tool for dissociation of GPx or TrxR pathways as mechanism triggering their antioxidant activities.

Figure 1

Structures of diselenide compounds.

Figure 2

Reduction of diselenide compounds I (A), II ( B), III (C), IV (D), V (E) e VI (F) by NADPH catalyzed by mammalian Thioredoxin Reductase (TrxR). Enzyme was mixed with a medium containing 50 mM Tris-HCl, 1 mM EDTA, pH 7,5 and then reaction was started by adding NADPH (final concentration 100 µM). 0 (□), 10(O), 15(∆), 20(∇) µM diselenide compounds. Statistical analysis were performed by three-way ANOVA (six diselenides × four concentrations × 11 sampling points). Data analysis yielded a significant diselenide × concentration × time interaction F(150, 1,440) = 42.5; p < 0.000001, which indicates that the consumption of NADPH was dependent on the concentration, on the type of compound and on the sampling time.

Figure 2

Reduction of diselenide compounds I (A), II ( B), III (C), IV (D), V (E) e VI (F) by NADPH catalyzed by mammalian Thioredoxin Reductase (TrxR). Enzyme was mixed with a medium containing 50 mM Tris-HCl, 1 mM EDTA, pH 7,5 and then reaction was started by adding NADPH (final concentration 100 µM). 0 (□), 10(O), 15(∆), 20(∇) µM diselenide compounds. Statistical analysis were performed by three-way ANOVA (six diselenides × four concentrations × 11 sampling points). Data analysis yielded a significant diselenide × concentration × time interaction F(150, 1,440) = 42.5; p < 0.000001, which indicates that the consumption of NADPH was dependent on the concentration, on the type of compound and on the sampling time.

Figure 3

Reduction of ebselen (0, 5, 7.5, 10, 15 or 20 µM) by NADPH catalyzed by mammalian TrxR. Enzyme was mixed with a medium containing 50 mM Tris-HCl, 1 mM EDTA, pH 7.5 and, then reaction was started by adding NADPH (final concentration 100 µM). Statistical analysis were performed by two-way ANOVA (six concentrations × 11 time points). Data analysis yielded a significant concentration × time interaction [F(50, 360) = 24.6; p < 0.000001], which indicates that the consumption of NADPH determined in the presence of ebselen depended both on its concentration and on time of sampling.

Figure 4

GPx like behavior of diselenide compounds: (I) diphenyl diselenide, (II) bistrifluoromethyldiphenyl diselenide, (III) bismethoxydiphenyl diselenide, (IV) biscarboxydiphenyl diselenide, (V) bischlorodiphenyl diselenide and (VI) hexamethyl-diphenyl diselenide. Two hundred µM of diselenide compounds was added. Statistical analysis were performed by two-way ANOVA (seven compounds × 13 sampling times). Data analysis yielded a significant compound type × time of sampling interaction [F(72, 504) = 57.9 p < 0.01), which indicates that PhSSPh production varied as a function of sampling time and the diselenide considered.

Figure 5

GPx like behavior of ebselen. Fifty µM of ebselen was tested. Statistical analysis were performed by two-way ANOVA (two concentrations × 13 times of sampling). Data analysis yielded a significant concentration × sampling time interaction [F(12,144) = 228,4; p < 0.000001], which indicates that ebselen caused an increase in PhSSPh formation that was time dependent.

Figure 6

Correlation analysis (by Sperman rank test) between the thiol oxidase activity with the effectiveness of diselenides and ebselen as substrate of hepatic mammalian TrxR (A) or with thiol peroxidase-like activity (B) or and between thiol-peroxidase like activity and the effectiveness of diselenides and ebselen as substrates for TrxR (C).