Journal of Nutritional Biochemistry 14 (2003) 173–179 Induction of hepatic thioredoxin reductase activity by sulforaphane, both in Hepa1c1c7 cells and in male Fisher 344 rats Korry J. Hintzea, Anna-Sigrid Keckb, John W. Finleyc, Elizabeth H. Jefferyb,* aDepartment of Animal and Range Science, North Dakota State University, Fargo, ND 58105 bDepartment of Food Science and Human Nutrition, University of Illinois, Urbana, IL 61801 cUnited States Department of Agriculture, Agricultural Research Service, Grand Forks Human Nutrition Research Center, Grand Forks, ND 58202-9034 Received 13 May 2002; received in revised form 6 November 2002; accepted 10 December 2002 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 ϭ 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 ␮M) plus SF (2.0 ␮M) 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. Published by Elsevier Inc. All rights reserved. Keywords: Thioredoxin reductase; Sulforaphane; Selenium; Glutathione peroxidase 1. Introduction Mammalian TR is an antioxidant that it is able to reduce various substrates including lipoic acid, lipid hydroperox- Mammalian TR is a 110 kDa homodimeric selenoprotein ides, NK-lysin, vitamin K3, dehydroascorbic acid, ascorbyl that catalyzes the NADPH-dependent reduction of thiore- free radical, and the tumor suppressor protein p53 [for doxin. Electrons are transferred from NADPH via FAD to a review see 2]. It is hypothesized that the presence of the conserved redox site. Each monomer is comprised of a selenocysteine redox site in mammalian TR allows for this prosthetic FAD group, a redox active disulfide site and a broad reducing substrate specificity [2]. pentultimate selenocysteine amino acid residue [1]. Thioredoxin provides reducing equivalents for ribonu- cleotide reductase, which is essential for DNA synthesis [3]. ϩ ϩ Also, it regulates the action of several gene transcription * Corresponding author. Tel.: 217-333-3820; fax: 217-265-0925 ␬ (E.H. Jeffery). factors including NF- B [4], AP-1 [5] and the glucocorti- The U.S. Department of Agriculture, Agricultural Research Service, coid receptor [6] through a redox mechanism by providing Northern Plains Area, is an equal opportunity/affirmative action employer reducing equivalents to facilitate binding to DNA [7]. Thi- and all agency services are available without discrimination. Names are oredoxin must be in a reduced form to be active and TR is necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the the primary means of reduction. Ultimately, all of the func- name by USDA implies no approval of the product to the exclusion of tions of thioredoxin are tied to the presence of TR. others that may also be suitable. In animals, TR activity is regulated by dietary Se, and 0955-2863/03/$ – see front matter Published by Elsevier Inc. All rights reserved. doi:10.1016/S0955-2863(02)00282-6 174 K.J. Hintze et al. / Journal of Nutritional Biochemistry 14 (2003) 173–179 activity drops to less than 5% of normal values in selenium 2.3. Animals and housing deficient rat livers [8,9]. In cell culture, TR activity is increased in a dose dependent manner by 1-10 ␮M supple- Animal usage was approved by the University of Illinois mental Se, and falls to below 5% of controls when cultures Animal Care and Use Committee. Male F344 rats (Harlan, are depleted of Se [10]. Indianapolis, IN), 4 weeks old (40–60 g), were housed Both TR activity and protein levels are induced by t- individually, in shoebox cages with corncob bedding in a butylhydroquinone (BHQ) in cultured cortical astrocytes controlled environment with a 12-hr light/dark cycle under [11]. Because BHQ is known to upregulate a number of uniform temperature and humidity. The animals were accli- detoxification enzymes via the ARE, these researchers sug- mated over six days; during the first two days they received gested that TR may be regulated in part by the ARE which rat chow followed by four or more days of powdered semi- is known to mediate the induction of QR and other enzymes purified control diet (AIN76 B-40, ICN Biochemicals, such as ␥-glutamylcysteine synthetase and glutathione S- Cleveland, OH). Animals were allowed ad libitum access to transferase [for review see 12]. The objective of the present food and water throughout the acclimation period. 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]. 2.4. Experimental design The experiment used a completely randomized design. 2. Materials and methods Animals were stratified by weight and randomly allotted to one of six dietary treatments. Treatment duration and doses were based on previous work [15]. 2.1. Materials Solvents used for extraction and purification were of 2.5. Experimental diets reagent-grade, and solvents used for gas chromatography (GC) or high performance liquid chromatography (HPLC) Experimental diets were provided for 5 days; all diets analysis were of HPLC-grade. All solvents were purchased were provided fresh daily. During the 5-day experimental from Fisher Scientific (Fair Lawn, NJ). Sulforaphane was period, groups of 5 rats received one of 6 diets: 1) control purified from broccoli seed by the method of Matusheski et diet (modified AIN 76 B40); 2) 20% broccoli, 3) 0.32 mmol al., [13]. Purity was determined to be Ͼ99% by GC analy- SF/kg diet, 4) 5.16 mmol SF/kg diet, 5) 20% broccoli ϩ sis. Unless otherwise noted, all other materials used in this 0.16 mmol SF/kg diet, or 6) 20% broccoli ϩ 5 mmol SF/kg study were obtained from Sigma (St. Louis, MO). diet. The broccoli used in diets contained 11 ␮mol glucora- phanin/g dry weight broccoli or 2.2 mmol SF/kg diet. Glu- 2.2. Broccoli processing coraphanin is hydrolyzed by the plant enzyme myrosinase to form SF. Based on preliminary work, under laboratory Broccoli (Brassica oleracea var. Italica cv. Majestic) conditions the conversion of glucoraphanin to SF was esti- plants were grown at the University of Illinois, Urbana, IL. mated to be 6%.1 Therefore, rats consuming 20% broccoli At harvest, heads were cut from the mature vegetables, and diets would be supplied with approximately 0.16 mmol held on ice for Ͻ4 hr. Florets (2 inches long) were cut, SF/kg diet. Food intake was recorded daily and animals plunged into liquid nitrogen, and stored at 80°C until being were pair-fed to the mean of the group with the lowest freeze-dried. Freeze-dried broccoli samples were finely average food intake on the previous day. ground in a water-cooled Tekmar A-10 analytic mill (IKA௡ Labortechnik, Staufen, Germany) for 15 s and stored at 2.6. Tissue collection and preparation 20°C until used. Control diets (AIN 76-B40) were formulated to contain Rats were anesthetized with ketamine (87 mg/ml)/xyla- the same amount of fiber as the 20% broccoli diets. Exper- imental diets containing broccoli (20%) were adjusted to zine (13 mg/ml) dosed at 0.1 ml/100 g BW, and killed by balance nutrients and maintain a constant caloric value. cervical dislocation. Perfused livers were blotted dry, Nutritive values for broccoli were from the USDA Nutrient weighed, cut into 1 g pieces, and snap-frozen in liquid Ϫ Database for standard reference (Release 13, Nov. 1999). nitrogen. Samples were stored at 80°C. Defrosted livers Purified SF was premixed with corn oil for 15 min before were homogenized in four volumes of buffer (0.15 M KCl, mixing with the rest of the diet. All experimental diets were 0.25 M K2HPO4/KH2PO4, pH 7.25) and centrifuged mixed the same day and stored in the dark for fewer than 10 (10,000 ϫ g for 20 min). The supernatants were centrifuged days at 4°C in airtight plastic bags. Diets containing iso- at 105,000 ϫ g for 1 hr and the cytosolic fraction was thiocyanates may be stored for at least 2 weeks and remain collected, snap-frozen in liquid nitrogen and stored at stable [14]. Ϫ80°C. K.J. Hintze et al. / Journal of Nutritional Biochemistry 14 (2003) 173–179 175 2.7. Tissue collection and preparation Western blots of rat liver cytosolic TR and GSH-Px were performed using NuPage (Carlsbad, CA) protocols and re- Rats were anesthetized with ketamine (87 mg/ml)/xyla- agents: Bis-Tris, 12% polyacrylamide gels, MOPS running zine (13 mg/ml) dosed at 0.1 ml/100 g BW, and killed by buffer and NuPage sample buffer.