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Induction of hepatic thioredoxin reductase activity by sulforaphane, both in Hepa1c1c7 cells and in male Fisher 344 rats

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Abstract

Sulforaphane (SF), a glucosinolate-derived isothiocyanate found in cruciferous vegetables, is considered an anticarcinogenic component in broccoli. Sulforaphane induces a battery of detoxification enzymes, including quinone reductase (QR). Induction is thought to be mediated through a common regulatory region termed the antioxidant response element (ARE). To test the hypothesis that the antioxidant selenoprotein thioredoxin reductase (TR) may be induced as part of this coordinated host-defense response to dietary anticarcinogenic compounds, TR activity was measured in livers of rats pair-fed diets containing SF and/or broccoli (n = 6/group). At the doses used, neither SF nor broccoli alone significantly elevated TR activity, whereas treatments containing both broccoli and SF caused a significant increase in TR activity. Glutathione peroxidase (GSH-Px), a second selenium-dependant enzyme with antioxidant activity, was downregulated in rats fed both SF and broccoli, compared to the control diet.

A second experiment, using mouse hepatoma Hepa1c1c7 cells, tested whether an interaction exists between selenium (Se) and SF in TR inducibility, since Se is known to induce TR activity. Selenium (2.5 μM) plus SF (2.0 μM) caused significantly greater TR activity than either treatment alone. All treatments with added Se or SF caused significantly greater TR activities than no Se or SF treatment. Glutathione peroxidase activity was elevated by Se, but not by SF. These data suggest that TR, known to be regulated by Se, is also upregulated as part of a host response to the dietary anticarcinogen SF, a trait not shared by another Se-dependent enzyme, GSH-Px.

Introduction

Mammalian TR is a 110 kDa homodimeric selenoprotein that catalyzes the NADPH-dependent reduction of thioredoxin. Electrons are transferred from NADPH via FAD to a conserved redox site. Each monomer is comprised of a prosthetic FAD group, a redox active disulfide site and a pentultimate selenocysteine amino acid residue [1].

Mammalian TR is an antioxidant that it is able to reduce various substrates including lipoic acid, lipid hydroperoxides, NK-lysin, vitamin K3, dehydroascorbic acid, ascorbyl free radical, and the tumor suppressor protein p53 [for review see 2]. It is hypothesized that the presence of the selenocysteine redox site in mammalian TR allows for this broad reducing substrate specificity [2].

Thioredoxin provides reducing equivalents for ribonucleotide reductase, which is essential for DNA synthesis [3]. Also, it regulates the action of several gene transcription factors including NF-κB [4], AP-1 [5] and the glucocorticoid receptor [6] through a redox mechanism by providing reducing equivalents to facilitate binding to DNA [7]. Thioredoxin must be in a reduced form to be active and TR is the primary means of reduction. Ultimately, all of the functions of thioredoxin are tied to the presence of TR.

In animals, TR activity is regulated by dietary Se, and activity drops to less than 5% of normal values in selenium deficient rat livers [8], [9]. In cell culture, TR activity is increased in a dose dependent manner by 1-10 μM supplemental Se, and falls to below 5% of controls when cultures are depleted of Se [10].

Both TR activity and protein levels are induced by t-butylhydroquinone (BHQ) in cultured cortical astrocytes [11]. Because BHQ is known to upregulate a number of detoxification enzymes via the ARE, these researchers suggested that TR may be regulated in part by the ARE which is known to mediate the induction of QR and other enzymes such as γ-glutamylcysteine synthetase and glutathione S-transferase [for review see 12]. The objective of the present study was to determine if the antioxidant selenoprotein TR can be induced by SF, a compound from broccoli known to induce detoxification enzymes via the ARE [12].

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Materials

Solvents used for extraction and purification were of reagent-grade, and solvents used for gas chromatography (GC) or high performance liquid chromatography (HPLC) analysis were of HPLC-grade. All solvents were purchased from Fisher Scientific (Fair Lawn, NJ). Sulforaphane was purified from broccoli seed by the method of Matusheski et al., [13]. Purity was determined to be >99% by GC analysis. Unless otherwise noted, all other materials used in this study were obtained from Sigma (St. Louis,

Animal experiment

Food intake for the highest treatment group, [20% broccoli + 5mmol SF/kg diet] was significantly lower than intake for the group receiving control diet. However, it was not different from any other diet containing 20% broccoli. Weight gain was not different among groups (data not shown).

Diets containing both 20% broccoli and purified SF increased hepatic TR activity compared to the control diet (P < 0.05, Fig. 1). In contrast, hepatic GSH-Px activity was significantly depressed in animals fed

Discussion

The present study demonstrates that in livers of rats fed adequate Se, TR activity and protein can be increased by dietary SF + broccoli. Conversely, we found that hepatic GSH-Px activity and protein were decreased in rats by dietary SF + broccoli. The animals employed in this study were also used in a study relating dietary SF to the induction of hepatic and colonic QR.2 In that study, hepatic QR induction was found to correlate closely with urinary SF mercapturate (r = 0.73, P = 0.0006).

Notes

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