Hemolysate Thioredoxin Reductase and Glutathione ...

The Journal of Nutrition Nutrition and Disease

Hemolysate Thioredoxin Reductase and Glutathione Peroxidase Activities Correlate with Serum Selenium in a Group of New Zealand Men at High Prostate Cancer Risk1 Nishi Karunasinghe,2 Lynnette R. Ferguson,2* John Tuckey,3 and Jonathan Masters3 2 Discipline of Nutrition, University of Auckland, Auckland 1023, New Zealand; and 3Urology Department, Auckland Hospital, Auckland 1023, New Zealand

Abstract The study provides data relating serum selenium concentration to activities of 2 key selenoenzymes, hemolysate thioredoxin reductase (TR) and glutathione peroxidase (GPx), measured by spectrophotometry, in a group of men at high prostate cancer. Such individuals have a high risk of developing prostate cancer in the succeeding 5 y. In the men with baseline serum selenium concentrations ranging from 0.74–1.62 mmol/L (59–128 mg/L), hemolysate TR (r ¼ 0.359, P , 0.05) and GPx (r ¼ 0.341, P , 0.05) activities increased with increasing serum selenium. Furthermore, after a run-in period of 1 mo, men participated in a randomized, double-blind, placebo-controlled selenium supplementation trial for 6 mo and received a placebo, or 200 or 400 mg of Se per day, in the form of a seleno yeast. This study is a subsidiary of an ongoing Phase III cancer chemoprevention trial and, as such, randomization groups have not yet been revealed. After 6 mo of being on trial and with an estimated 66% of the group being supplemented with seleno yeast, the TR activity of the group increased by 80% relative to baseline. In contrast, 6 mo of selenium supplementation did not affect GPx activity. This study presents, to our knowledge for the first time, both measurements of human hemolysate TR activity and its relation to serum selenium. J. Nutr. 136: 2232–2235, 2006.

Introduction Selenium plays a critical role in human health. While frank Se deficiency leads to muscle wasting, low Se levels may also enhance the risk of degenerative diseases, including cancer (1,2). Glutathione peroxidase (GPx)4 enzymes are a group of selenoproteins that are necessary to regulate intracellular concentration of hydroperoxides and thought to play a role in antioxidant defense (3). Thioredoxin reductase (TR) is another key seleniumcontaining enzyme that, together with thioredoxin, forms a redox system with multiple roles, including redox regulation of transcription factors and provision of reducing equivalents for the synthesis of deoxyribonucleotides for DNA synthesis (4). TR also carries the function of reducing ubiquinone-10 to regenerate the antioxidant ubiquinol-10 (5–7), which is important in preventing peroxidation of lipids such as a-tocopherol, considered the primary lipid-soluble antioxidant in humans (8,9). TR also reverses selenium-induced inactivation of protein kinase C; 1

Supported by the Auckland Medical Research Foundation (Auckland, New Zealand) and National Cancer Center Grant RO1 CA 77789 from the NIH (Bethesda, MD). 4 Abbreviations used: Hb, hemoglobin; GPx, glutathione peroxidase; TR, thioredoxin reductase. * To whom correspondence should be addressed. E-mail: l.ferguson@auckland. ac.nz.

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this selenoenzyme serves as a safeguard against the toxicity induced by selenometabolites (10). The presence of TR has been reported from various tissues, including erythrocytes (11). Those world regions having soil Se levels ,0.6 mg/kg are considered Se deficient (12). Much of New Zealand falls into that category (13). The desirable Se level in humans, however, has been debated. The levels of selenium considered necessary to satisfy human physiological requirements have been calculated as the levels necessary to maximize activity of the functional protein GPx (14), and New Zealand data have been important in justifying this approach (15). However, selenium is a component of .30 enzymes, and it cannot be assumed that the same levels are required for each. The presence of polymorphisms in the genes for GPx suggests that human Se requirements may differ according to specific genotype (16,17). It is also possible that susceptibility to disease may play a role in individual Se requirement. A U.S.-based, double-blind, placebo-controlled, phase III clinical trial is investigating whether selenium supplementation will reduce the likelihood of prostate cancer development (18). The Negative Biopsy Trial enrolls patients with elevated prostatespecific antigen but negative biopsy for prostate cancer. Such individuals have a high risk of developing prostate cancer in the succeeding 5 y (19). This blind-coded trial supplements with 0,

0022-3166/06 $8.00 ª 2006 American Society for Nutrition. Manuscript received 12 March 2006. Initial review completed 29 March 2006. Revision accepted 6 June 2006.

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risk for prostate cancer. This trial enrolled 43 patients with elevated prostate-specific antigen but negative biopsy for

200, or 400 mg Se/d in the form of selenized yeast. The recruitment of patients for this trial at the Auckland Hospital in New Zealand allows us to investigate the relation between enzyme activities and serum selenium concentration in this low selenium/high cancer risk group (20). The present study aims at understanding the levels of activity of GPx and TR from hemolysates at trial entry and their relation with serum selenium levels. It also enables us to make preliminary observations on effects of 6 mo of selenium supplementation.

Materials and Methods

Statistical methods. We analyzed the data statistically using 2 approaches. One was a cross-sectional examination of the relation between Se levels and enzyme activity using the baseline data of the study. We used plots of linear curves and estimated Pearson’s correlation coefficients between hemolysate GPx activity and serum selenium concentration and

Results At baseline, the men’s serum selenium concentration ranged from 0.74–1.62 mmol/L (59–128 mg/L) with a mean of 1.24 6 0.21 mmol/L (97.8 6 16.6 mg/L). The serum selenium concentration of all individuals at baseline and at randomization at 1 mo were correlated (r ¼ 0.62, P , 0.0001). All patients from the trial had detectable hemolysate GPx activity that ranged from 3.273–10.726 mU/mg Hb. At baseline, the GPx activity increased linearly with increasing serum selenium (r ¼ 0.341, P , 0.05; Fig. 1). The GPx activity at randomization and at 6 mo did not differ from baseline (Table 1). The GPx activity at baseline was correlated with that after 6 mo of supplementation (r ¼ 0.52, P , 0.001). Hemolysate TR activity ranged from 0.05–1.212 mU/mg Hb at baseline and 0.06–1.98 mU/mg Hb at 6 mo. The hemolysate TR activity in 6 of 43 patients at baseline and 6 of 38 at 6 mo was below the detectable limit. At baseline, TR activity increased exponentially with increasing serum selenium (r ¼ 0.359, P , 0.05; Fig. 2). Hemolysate TR activity increased ;80% between baseline and 6 mo but did not change between baseline and randomization at 1 mo (Table 1).

Discussion This study provides, to our knowledge, the first data relating serum selenium to activity of TR in a group of men at high risk

Figure 1 GPx activity in hemolysates and serum Se concentration of men at high risk for prostate cancer at baseline (r ¼ 0.341, P , 0.05). Equation for the regression line is GPx activity ¼ 3.35 1 2.41 Se level. Open circle, an outlier that was eliminated from the regression analysis.

Selenium and selenoenzyme activity

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Cohort. Forty-three men, between the ages of 50 and 75 y, were recruited from records at the Auckland Hospital Urology Clinic. Recruitment criteria were a prostate-specific antigen .4 and prostate biopsy negative for cancer or high-grade prostatic intraepithelial neoplasia, ,80 y of age, 5-y life expectancy, no history of prior malignancy except for basal cell or squamous cell carcinoma of the skin, and not taking .50 mg of Se a day as a dietary supplement. Patient characteristics are summarized in Karunasinghe et al. (20). After a run-in period of 1 mo, subjects were randomized to receive a placebo, or 200 mg or 400 mg Se/d, in the form of seleno yeast (Seleno Excell tablets A2603 Lot 1086ZR5A, A2601 Lot 1086ZR6A, and A2602 Lot 1086ZR7A, respectively) manufactured for Nutrilite. We estimate that this protocol will supplement ;66% of the study subjects with 200 or 400 mg Se/d, whereas the remaining ;33% will receive a placebo. Nonfasting venous blood samples were collected into sodium heparin tubes for selenium and selenoenzyme assays at the initial visit, randomization at 1 mo, and 6 mo after this point. This study was approved by the Auckland Ethics Committee (2000/045). Serum selenium was analyzed using hydride generation atomic absorption spectrometry (Model 951 dual-channel atomic absorption spectrometry equipped with a single-slot burner head, Instrumentation Laboratories) using protocols of Hershey and Osstdyk (21) as described (20). These assays were done at Alpha Scientific, a branch of Gribbles Veterinary Pathology. Another sample of 100 mL of blood was used to prepare the hemolysates as described by Calbiochem (cellular glutathione peroxidase assay kit, Catalog No. 354104). The protocol of Wendel (22) was followed for the measurement of hemoglobin (Hb) using Drabkin’s solution. The conversion of the hemolysates to cyanomethemoglobin using transformation solution was also carried out according to the same protocol before the enzyme activity for both GPx and TR was measured. GPx was assayed using the protocol of Wendel (22) modified to suit a 96well plate format, and the samples were assayed in duplicate. All spectrophotometric measurements were carried out for 4 min at 366 nm using the kinetic protocol on Spectra Max Plus spectrophotometer (Molecular Devices). Each well contained an equivalent of 250 mg Hb. One unit of GPx activity is defined as 1 mmol NADPH oxidized/min at 37C. The intraassay %CV of this assay was 4.01, whereas the interassay %CV was 4.14. TR was assayed with a minor modification to the protocol of Smith and Levander (23), whereby 20 mmol of aurothiomalate instead of 20 mmol aurothioglucose was used as the suppressor of TR for the measurement of non-TR activities. Data collection was started 1 min after the initiation of the reaction to allow nonenzymatic reduction of 5,5#dithiobis(2-nitrobenzoic acid) to go to completion. All spectrophotometric measurements were carried out for 4 min at 412 nm using the kinetic protocol on Spectra Max Plus spectrophotometer (Molecular Devices). Each well contained an equivalent of 125 mg Hb. One unit of TR activity is defined as 1 mmol 5-thio-2-nitrobenzoic acid formed/min at 37C. Both untreated and aurothiomalate-treated samples were assayed in duplicate. The intraassay %CV was 10.05, whereas the interassay %CV was 8.11.

logarithmically converted TR (ln TR) activity and serum selenium concentration, using linear regression analysis. Correlations were considered significant at P , 0.05. The other was a comparison of enzyme activities at baseline, randomization at 1 mo, and after 6 mo of Se supplementation. As the randomization allotments are still not available, we used a pairwise comparison across subjects at different time points rather than a comparison between the placebo and supplemented groups separately. Of the 43 men that were recruited at baseline, there were 5 dropouts by 6 mo. Data collected at baseline, randomization, and after 6 mo of Se supplementation were compared by paired t tests. Differences were considered significant at P , 0.05. Only a sample of 7 men was available for the enzyme study at randomization due to physical constraints. However, this subset of data was used to support our conclusion that enzyme activity changes between baseline and 6 mo were not due to regression to the mean. Values in the text are means 6 SD.

TABLE 1

GPX and TR activities in hemolysates of men at high risk for prostate cancer at baseline, randomization at 1 mo, and after 6 mo of Se supplementation1,2 Baseline

Randomization

6 mo

mU/mg Hb GPX TR

6.46 6 0.244 (43) 0.390 6 0.05 (43)

6.11 6 0.472 (7) 0.476 6 0.09 (7)

6.49 6 0.261 (38) 0.705 6 0.191* (38)

1

Values are means 6 SD (n). * Different from baseline, P , 0.05. We estimate that ;66% of the study subjects received 200 or 400 mg Se/d; the remaining ;33% received a placebo.

2

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7. Figure 2 TR activity in hemolysates and serum Se concentration of men at high risk for prostate cancer at baseline (r ¼ 0.359, P , 0.05). Equation for the regression line is ln TR activity ¼ 22.873 1 1.41 Se level.

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for prostate cancer. It provides evidence that, at least over this range of serum selenium concentrations, TR activity increases with increasing serum selenium and this has not reached saturation even at concentrations up to 1.62 mmol/L (128 mg/L). The data also indicate that, over the serum selenium concentrations studied in this Auckland population, there is an increase in GPx activity in relation to increased serum selenium. Furthermore, after 6 mo, hemolysate TR activity, but not that of GPx, increased (Table 1). However, an important limitation of this study is that we have not yet revealed the randomization groups. Therefore, it is not possible to fully attribute the increase in TR activity at 6 mo to Se supplementation. The GPx data are consistent with the Berggren et al. (24) study on rat tissue and Thomson et al. (25) study on erythrocyte GPx activity on New Zealand women. Previous studies with human colon cancer cell lines and cancer cells of epithelial origin supplemented with selenite have shown a dose-dependent increase in TR activity (26,27). A 150–210% increase in TR activity in lung, liver, and kidney tissues of rats fed a moderately high level of Se as selenite (1 mg/kg diet) compared with rats fed normal levels of Se (0.1 mg/kg diet) was reported by Berggren and co-workers (24). The observations from our study might be consistent with the above data. Although baseline hemolysate GPx activity increased with the serum selenium concentration, selenium supplementation to an estimated 66% of this group for 6 mo has not significantly affected GPx activity. This suggests an individual carrying capacity for the selenoenzyme GPx, as previously suggested (25). As this trial has not been completed, we cannot determine whether there is a correlation between the 6-mo GPx or TR

activities and the corresponding serum selenium concentration. However, it is possible that individuals who have a higher serum selenium concentration have higher hemolysate GPx and TR activities as well. A 4-wk selenium supplementation study of women between the ages of 20 and 40 y awaiting cholecystectomy had results similar to those in our study, with erythrocyte GPx activities remaining the same as presupplementation levels (25). The authors suggested that erythrocyte GPx measures only give long-term indications of selenium status. We cannot be certain whether our observations extend to normal individuals. The GPx activities recorded in the present study are toward the lower end of the activities recorded for whole blood for New Zealanders using a similar assay protocol (28–30). There are reports that serum Se concentration and GPx activity in cancer patients are significantly lower than those in the control groups, although the serum selenium concentrations are correlated with GPx activity in both groups (31). As this study group is at high risk for prostate cancer, their selenium levels and GPx activities may be lower than those of normal individuals. The GPx family of genes (GPX-1–GPX-4) is crucial for cell detoxification (32), and it appears that the function of GPX-4 in particular is dependent on Se availability (33). It has been hypothesized that, during periods of Se deficiency, synthesis of GPx has priority over other selenoproteins and that this is mediated through the higher affinity of selenocysteine-tRNA for GPX-4 and/or differential changes in the stability of GPX mRNAs (33). Ganther (4) extensively discussed the possible effects of supranutritional levels of selenium on TR activity decline and suggested this could be related to the usefulness of Se as a chemopreventive agent (1). Given that TR activity is high in some tumor tissues, the question has been raised as to whether increasing TR is necessarily beneficial (34). It seems essential that we gather sufficient information on changes in a wide range of biomarkers with selenium supplementation and depletion in humans before we can set a rationally developed recommended daily intake or, more specifically, an individually tailored dosage for humans.

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