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Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom;
* Disease Mechanisms and Therapeutics Research Group, Department of Biological Sciences, University of Essex, Essex CO4 3SQ, United Kingdom;
Centre for Tsukuba Advanced Research Alliance and Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba 305-8577, Japan
3To whom correspondence should be addressed. E-mail: john.hayes{at}cancer.org.uk.
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ABSTRACT |
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 TOP ABSTRACT Materials and methodsResultsDiscussionLITERATURE CITED |
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1.5-fold increase in NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase (GST) activities in stomach, small intestine, and liver of wild-type mice but not in mutant mice; increased transferase activity was associated with elevated levels of GSTA1/2, GSTA3, and GSTM1/2 subunits. These seeds also increased significantly the level of glutamate cysteine ligase catalytic (GCLC) subunit in the stomach and the small intestine of nrf2+/+ mice but not nrf2–/– mice. An aqueous broccoli seed extract was prepared for treatment of cultured cells that contained ITC at 600 µmol/L, composed of 61% 3-methylsulfinylpropyl ITC, 30% sulforaphane, 4% allyl ITC, and 4% 3-butenyl ITC. This extract induced GSTA1/2, GSTA3, NQO1, and GCLC between 3-fold and 10-fold in mouse Hepa-1c1c7 and rat liver RL-34 cells. The broccoli seed extract affected increases in GSTA3, GSTM1, and NQO1 proteins in nrf2+/+ mouse embryonic fibroblasts but not in nrf2–/– mouse embryonic fibroblasts. These experiments show that broccoli seeds are effective at inducing antioxidant and detoxication proteins, both in vivo and ex vivo, in an Nrf2-dependent manner.
KEY WORDS: • antioxidant response element • chemoprevention • glucosinolates • sulforaphane • myrosinase
Individuals who have a high dietary intake of fruit and vegetables appear to have a lower risk of cancer (1). Among vegetables with anticarcinogenic properties, members of the Cruciferae family have been reported to protect against neoplastic disease at a variety of sites, such as the gastrointestinal tract and the lungs (2–6).
The cancer chemopreventive effect of cruciferous vegetables has been attributed to the fact that they contain high levels of glucosinolates (7,8). During food preparation and eating, these glucosinolates are hydrolyzed by the plant enzyme myrosinase to yield a complex number of breakdown products, including isothiocyanates (ITC),4 thiocyanates, cyanides, nitriles, and epithio-containing compounds (7–9). Some of these breakdown products, and, in particular, ITCs can increase the levels of detoxication enzymes in rodent organs and in mouse, rat, and human cell lines (10–17). Inducible proteins include the drug-metabolizing enzymes aldo-keto reductase, NAD(P)H:quinone oxidoreductase 1 (NQO1), and glutathione S-transferase (GST). Increases in the levels of these detoxication enzymes would be expected to confer protection against chemical carcinogens such as benzo[a]pyrene, and, in experimental models, this prediction appears to hold true (14,18). Less well appreciated is the fact that glucosinolate breakdown products also induce antioxidant proteins, such as the glutamate cysteine ligase catalytic (GCLC) and glutamate cysteine ligase modifier (GCLM) subunits, that catalyze the rate-limiting step in the formation of reduced glutathione (19,20). They also induce glutathione reductase, ferritin, and glucose-6-phosphate dehydrogenase (20). Increases in the levels of detoxication enzymes and antioxidant proteins would be expected to protect against reactive oxygen species and the harmful metabolites they generate as a consequence of damaging cellular membranes, proteins, and nucleic acids (21).
Many genes encoding detoxication and antioxidant proteins are regulated by nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) (22). This basic-region leucine zipper transcription factor mediates the transcriptional activation of genes in response to oxidative and electrophile stress. Under normal homeostatic conditions, Nrf2 protein has a short half-life, being targeted for proteasomal degradation by Keap1 (23–26). Oxidants and electrophiles interfere with Keap1-facilitated degradation of Nrf2, causing it to become more stable. This process involves oxidation, modification, or both, of cysteine residues 273 and 288 in Keap1 by the inducing compounds (27). Induction of NQO1, GST, GCLC, and GCLM genes by Nrf2 occurs through it being recruited to antioxidant response elements (ARE) in their gene promoters (28); Nrf2 binds the ARE as a heterodimer with small Maf proteins (29,30). Mice in which the nrf2 gene has been disrupted by targeted homologous recombination have lower constitutive levels of NQO1 and GST proteins in liver and small intestine (19,31,32). Furthermore, nrf2–/– mice are either unable to respond or have a blunted response to the model cancer chemopreventive agent butylated hydroxyanisole (19,33,34).
Most investigations into the ability of plant chemicals to increase antioxidant gene expression used highly purified compounds as inducing agents (11–20). Thus, ITCs such as sulforaphane have been shown to increase NQO1 enzyme activity in the mouse liver Hepa-1c1c7 cell line (35). Frequently, it is unclear whether the concentration of phytochemical used in cell culture experiments is physiologically relevant and whether, because of limitations caused by bioavailability or disposition, the dose of chemical used can be achieved in target tissues in vivo. The question of whether extracts of cruciferous plants are as effective as purified phytochemicals at stimulating gene expression is seldom addressed.
In this study, we investigated whether broccoli seeds, either in the diet or as aqueous extracts, can affect induction of antioxidant and detoxication genes in vivo, in transformed cells, and in nontransformed cells. We also tested the hypothesis that Nrf2, through stimulating ARE-driven gene transcription, is essential for gene induction by broccoli-derived phytochemicals.
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Materials and methods |
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TOPABSTRACTMaterials and methodsResultsDiscussionLITERATURE CITED |
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Allyl ITC (AITC) and sulforaphane were obtained from Aldrich and LKT Laboratories, respectively. All other chemicals used were of the highest purity that was available from commercial suppliers.
Broccoli seeds
Broccoli seeds were purchased from Thompson and Morgan.
Processing of broccoli seeds for induction experiments
The broccoli seeds were processed at room temperature (20°C). Extracts were prepared by crushing 10 g seeds (dry weight), by pestle and mortar, to a fine powder. For mice feeding experiments, crushed broccoli seeds were added directly to powdered RM1 laboratory animal feed (SDS) at 15% by weight. For cell culture experiments, the broccoli seed powder was suspended in 3 volumes of distilled water and was mixed vigorously for 5 min. The suspension was centrifuged at 800 x g for 10 min before being filtered through a 0.2-µm sterile filter. Aliquots (1 mL) of the aqueous filtered extract were snap-frozen in liquid nitrogen and were stored at –70°C before use; the entire process from crushing the broccoli seeds to snap-freezing the filtered aqueous extract was completed within 30 min. The frozen extracts were thawed rapidly and diluted 1/1000 in 6 mL of medium for cell culture experiments that were conducted in 60-mm dishes.
Analysis of glucosinolates and ITCs in broccoli seeds
Glucosinolates and corresponding ITCs were identified by liquid chromatography with triple quadrupole MS detection (LC-MS/MS). Standard reference glucosinolates were isolated and purified from Brassica seeds by modification of published methods (36), and the related ITCs were prepared by myrosinase-catalyzed hydrolysis (37) and purified by preparative reversed-phase HPLC. The following glucosinolates were analyzed by LC-MS/MS: sinigrin, gluconapin, progoitrin, glucoiberin, glucoraphanin, glucoalyssin, and gluconasturtiin and their related ITCs—AITC, 3-butenyl ITC, 5-vinyloxazolidine-2-thione, 3-methylsulfinylpropyl ITC, sulforaphane, 5-methylsulfinylpentyl ITC, and phenethyl ITC, respectively.
Glucosinolates and ITCs were determined in broccoli seeds by initial heating at 110°C for 2 h (to inactivate myrosinase). The seeds were then ground to a fine powder, lipid was removed by extraction with chloroform, and the residual solid was extracted twice with 75% methanol at 75°C. The combined methanol extracts were concentrated by removal of solvent under reduced pressure, filtered (0.2 µm), spiked with authentic standard analytes, and analyzed by LC-MS/MS. For detection of ITCs in samples of seed extract and in culture medium, ITCs were extracted into dichloromethane and derivatized with ammonia (1.33 mol/L, 24 h at 20°C). The derivatized extracts were then evaporated under reduced pressure, reconstituted in 50% methanol, filtered (0.2 µm), spiked with authentic standard analytes, and analyzed by LC-MS/MS.
Glucosinolates were detected by negative ion electrospray multiple reaction monitoring (MRM), where the fragment ion was hydrogen sulfate (38). Derivatized ITCs were detected by positive ion electrospray MRM, where fragmentation involved loss of ammonia. For LC-MS/MS, the HPLC column was a 100 x 2.1-mm octadecyl silica Symmetry column with a 10 x 2.1-mm guard column (Waters). The flow rate was 0.2 mL/min. The eluent was 0.1% (v:v) trifluoroacetic acid in water, with linear gradients of methanol (0–10% for glucosinolates and 0–80% for ITCs) over 30 min. Source and desolvation temperatures were 120 and 350°C, and the gas flows for cone and desolvation were 150 and 550 L/h, respectively. The capillary voltage was 2.50 kV, and the cone voltage was set at 50 V. Argon gas pressure in the collision cell was 2.9 x 10–3 mbar. Programmed molecular ions, fragment ions, and collision energies were optimized to ±0.1 Da and ±1 eV for MRM detection. Glucosinolate and ITC analytes were quantified by standard addition analysis. Samples analyzed were spiked with 1–100 pmol glucosinolate and 2–100 pmol ITC. The limits of detection for glucosinolates were 
0.4 pmol and, for ITCs, were 2 pmol. The interbatch coefficients of variation were <5%, and recoveries were 80–100%.
Mice feeding experiments
The Ethical Review Committee of the University of Dundee approved this program of work, and, throughout the study, mice were treated as advised by regulations contained in the Animals and Scientific Procedure Act (1986) of the United Kingdom. The nrf2+/+ and nrf2–/– mice were obtained as described previously (33). The mice used in this study have been backcrossed over 6 generations onto a C57BL/6 genetic background. Female mice of between 9 and 14 wk of age were used in all studies. Mice were fed on standard RM1 laboratory feed. Mice were given free access to RM1 feed with broccoli seeds at 15% (by weight) for 7 d immediately before being killed. During the administration of crushed broccoli seed, mice were monitored daily by measurement of body weight. Once the period of feeding these phytochemicals was complete, the mice were killed by exposure to a rising concentration of CO2. Organs were removed and snap-frozen immediately in liquid nitrogen before being stored at –70°C.
Cell culture
Mouse Hepa-1c1c7 cells (European Collection of Animal Cell Cultures) were maintained in minimal essential Eagle’s medium, with the Alpha modification (Sigma) supplemented with 10% (v:v) heat-inactivated fetal bovine serum, 50 U/mL penicillin-streptomycin mixture, and L-glutamine at 2 mmol/L. Rat liver RL-34 cells [Japanese Cancer Research Resources Bank (Setagaya-ku)] were grown in Dulbecco’s modified Eagle’s medium (Life Technologies) supplemented as described above. Wild-type and Nrf2-null mouse embryonic fibroblasts (MEF) were prepared from nrf2+/+ and nrf2–/– mouse lines as described by Tiemann and Deppert (39). These cells were maintained in tissue culture flasks coated with 0.1% (w:v) gelatin for 30 min before use and were grown in medium supplemented with 10 µg/L human recombinant epidermal growth factor, 1 x insulin-transferrin-selenium (Gibco), and 10% (v:v) fetal bovine serum. All cell lines were maintained at 37°C and 5% CO2.
The RL-34, Hepa-1c1c7, and MEF cells were cultured in monolayers and were allowed to grow to 80% confluence in 60-mm dishes before exposure for 24 h to phytochemicals. AITC and sulforaphane were both used to treat cells at a dose of 5 µmol/L. The aqueous broccoli seed extract used to treat cells contained several ITCs, with the total level in the culture media amounting to 0.6 µmol/L.
Enzyme assays and Western blotting
NQO1 enzyme activity was estimated by measuring the dicoumarol-inhibitable fraction of dichlorophenol indophenol reductase activity. GST enzyme activity was measured using 1-chloro-2,4-dinitrobenzene. Western blotting using antibodies against NQO1; class Apha, Mu, and Pi GST isoenzymes; and GCLC subunits was conducted as reported previously (12,19,31).
DNA transfection and luciferase reporter gene assays
Transfection and ARE-reporter gene assays were performed in Hepa-1c1c7 cells. The wild-type mouse nqo1 promoter reporter construct, containing the functional ARE (5'-TCACAGTGAGTCGGCAAAATT-3') in the pGL3-Basic luciferase reporter vector, was described previously and was designated –1016/nqo5'-luc (29). The mutant NQO1 reporter construct containing 1016 nucleotides of 5'-upstream nqo1 sequence but with the ARE scrambled (i.e., 5'-TTAGAGATACTAGACCACGTC-3', with mutated bases in italics) is called Mut1 (29). Transfection of –1016/nqo5'-luc and Mut1 into Hepa-1c1c7 cells was performed using Lipofectin Reagent (Life Technologies), and, in all experiments, the pRL-TK Renilla reporter vector (Promega) was used as an internal control. Renilla and firefly luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega).
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Results |
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TOPABSTRACTMaterials and methodsResultsDiscussionLITERATURE CITED |
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The glucosinolate content of broccoli seeds was examined before their ability to induce gene expression in mammalian cells was examined. Prior heating, to inactivate myrosinase, followed by LC-MS/MS analysis revealed that the seeds contained 38.8 mmol glucosinolates per kg. Glucoiberin accounted for 59% of the total glucosinolate recovered, whereas sinigrin and glucoraphanin accounted for 19 and 8% of the glucosinolates, respectively. Significant amounts of progoitrin, gluconapin, and gluconasturtiin were also detected (Table 1). The structures of these phytochemicals are shown in Figure 1.
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Feeding nrf2+/+ mice diets containing 15% (w:w) crushed broccoli seeds resulted in the induction of both NQO1 and GST enzyme activities in the stomach, the small intestine, and the liver, but no increase was observed in the large intestine.
In the wild-type mice, feeding the seeds increased NQO1 activity in the stomach from 155 ± 40 to 248 ± 50 nmol · min–1 · mg–1 protein; in the small intestine, the broccoli seed diet increased NQO1 activity from 106 ± 16 to 183 ± 8 nmol·min–1·mg–1 protein; and, in the liver, this diet increased NQO1 activity from 50 ± 7 to 72 ± 4 nmol ·min–1 · mg–1 protein. The NQO1 enzyme activity in the stomach, the small intestine, and the liver of nrf2–/– mice placed on a control diet was only 50 ± 16, 40 ± 20, and 7 ± 5 nmol · min–1 · mg–1 protein, respectively. The NQO1 enzyme activity did not appear to be increased in stomach, the small intestine, or the liver of nrf2–/– mice fed diet containing broccoli seeds.
In nrf2+/+ mice, feeding the broccoli seed diet for 7 d increased GST activity in the stomach from 1.55 ± 0.10 to 2.53 ± 0.47 µmol · min–1 · mg–1 protein, in the small intestine from 1.61 ± 0.11 to 2.02 ± 0.23 µmol · min–1 · mg–1 protein, and in the liver from 4.91 ± 0.52 to 7.7 ± 0.95 µmol ·min–1 · mg–1 protein. Not only was transferase activity substantially lower in nrf2–/– mice than in the wild-type mice, but also, it was not increased in the mutant mice fed broccoli seeds. In stomach, small intestine, and liver, GST activity in knockout mice on a control diet was 1.26 ± 0.11, 1.18 ± 0.12, and 1.72 ± 0.69 µmol · min–1 · mg–1 protein, respectively.
The levels of NQO1 protein in the tissues of mice fed broccoli seeds was examined by Western blotting to determine whether increases in oxidoreductase activity in stomach, small intestine, and liver reflected an increase in protein. Immunoblotting showed increases of 2-fold in the level of NQO1 in all 3 organs from nrf2+/+ mice administered broccoli seeds (Figs. 2and 3). Similar experiments were carried out using antisera against class Alpha, Mu, and Pi GST subunits. These revealed significant increases of class Alpha GSTA3 protein in stomach and small intestine and a modest increase in all organs of class Mu GSTM1. The level of the class Pi GSTP1 subunit did not appear to increase in mice after administration of broccoli seeds.
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Glucosinolate breakdown products identified in broccoli seed extracts
The total amount of glucosinolate in the broccoli seed extract was <3.6 µmol/L, whereas the total amount of ITC in the extract was 596 µmol/L. Table 2 shows that 3-methylsulfinylpropyl ITC and sulforaphane account for 61 and 30%, respectively, of the ITCs present in the seed extract. Significant amounts of AITC and 3-butenyl ITC were also obtained.
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Aqueous broccoli seed extracts were used to treat cells at an estimated concentration of total ITC of 0.6 µmol/L in the media. The transformed mouse Hepa-1c1c7 liver cell line and the nontransformed rat liver RL-34 epithelial cells were used in these experiments. The broccoli seed extract increased NQO1 enzyme activity 3-fold and 5-fold in the Hepa-1c1c7 and RL-34 cells, respectively. Treatment with AITC at 5 µmol/L induced NQO1 catalytic activity 2-fold in both Hepa-1c1c7 and RL-34 cells. Treatment with sulforaphane at 5 µmol/L induced NQO1 catalytic activity 4.5-fold and 5.2-fold in Hepa-1c1c7 and RL-34 cells, respectively. By contrast, GST activity was not increased to the same extent in either cell line.
Western blotting showed that the level of NQO1 protein in Hepa-1c1c7 and RL-34 cells (Fig. 4A and B, respectively) grown in normal cell culture medium without the addition of phytochemicals was barely detectable. Treatment of both cell lines with the broccoli seed extract containing a mixture of ITCs substantially increased NQO1 protein. This increase was comparable to the induction of NQO1 protein affected by sulforaphane at 5 µmol/L.
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In both Hepa-1c1c7 and RL-34 cells, the broccoli seed extract induced large increases in GCLC protein (Fig. 4).
Broccoli seed extracts stimulate ARE-driven gene expression
To determine whether broccoli seed extracts can activate gene expression controlled through an ARE enhancer, RL-34 cells were transfected with the mouse –1016/nqo5'-luc reporter construct. Treatment of transfected cells with the standard dose of broccoli seed extract produced a 4.6-fold increase in luciferase activity compared with transfected cells treated with vehicle alone (Fig. 5). By contrast, AITC and sulforaphane, each at 5 µmol/L, produced 1.9-fold and 3.3-fold increases, respectively, in luciferase activity. Similar experiments using a reporter construct driven by the mouse nqo1 promoter that contained a mutant ARE (i.e., Mut1) proved to be unresponsive to broccoli seed extract, AITC, and sulforaphane.
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To explore whether GST subunits and NQO1 can be induced by broccoli in an Nrf2-dependent fashion, wild-type and mutant MEFs were treated with the seed extract. In the nrf2+/+ MEFs, treatment with the standard dose of broccoli seed extract caused a significant increase in GSTM1, GSTA3, and NQO1. This increase was similar to that seen in wild-type MEFs treated with sulforaphane at 5 µmol/L. In the nrf2–/– MEFs, the levels of GSTM1, GSTA3, and NQO1 were lower than in the wild-type cells, and the seed extract failed to induce these proteins (Fig. 6).
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Discussion |
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TOPABSTRACTMaterials and methodsResultsDiscussionLITERATURE CITED |
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Most of the studies into the cellular effects of glucosinolate-derived compounds have used purified phytochemicals such as sulforaphane, benzyl ITC, and phenethyl ITC (2,14–18). How relevant the doses of pure phytochemical used in such gene induction and cell cycle arrest experiments are to the in vivo situation is unclear. This issue is complicated, because the yield of ITCs from different parental glucosinolates varies substantially and can be influenced significantly by the presence of epithiospecifier protein present in certain crucifers (9,42). In the present paper, broccoli seeds were used as the source of plant glucosinolates because we wished to avoid variations in the content of these chemicals that arise from postgermination metabolism. Furthermore, Fahey et al. (43) reported that the ability of broccoli to induce NQO1 in Hepa-1c1c7 cells diminishes with the age of the plant. Therefore, in this study, we used crushed broccoli seeds in the mice feeding experiments and aqueous seed extracts in the cell culture experiments. Analysis of the glucosinolates revealed that the seeds used in this study contained primarily glucoiberin and sinigrin, with lesser amounts of glucoraphanin and progoitrin (Table 1). In the aqueous broccoli seed extracts, LC-MS/MS revealed the presence of large amounts of ITCs, primarily 3-methylsulfinylpropyl ITC, and sulforaphane (Table 2). The low recovery of AITC in the extracts is noteworthy given the large amount of sinigrin in the broccoli seeds.
Enzyme assay and Western blotting showed that addition of broccoli seeds at 15% (w:w) in the RM1 diet induced NQO1 about 2-fold in stomach, small intestine, and liver of wild-type mice. No induction was observed in the nrf2–/– mice. Similar results were observed by treating the nrf2+/+ and nrf2–/– mouse embryonic fibroblasts with broccoli seed extracts. Because the promoter of mouse nqo1 contains a functional ARE that recruits Nrf2 after treatment with sulforaphane (29), it is highly likely that transcriptional activation of mouse nqo1 caused by preparations of broccoli seed is a direct consequence of ITCs stimulating the basic-region leucine zipper protein to transactivate directly the oxidoreductase gene.
Among GSTs, modest increases of GSTM1 protein were observed in the stomach and the small intestine of wild-type but not of nrf2–/– mice after feeding with broccoli seeds. This diet also produced significant increases of the GSTA3 subunit in the stomach and large increases in the small intestine of wild-type mice. However, no such increases were observed in mutant mice. In MEFs from the wild-type and knockout mice, the Nrf2 dependency of induction of GSTM1 and GSTA3 by broccoli was clearly observed. Both the GSTM1 and GSTA3 subunit genes have been reported to contain an ARE (30,44), and it is likely that Nrf2 mediates induction directly through this enhancer. Chromatin immunoprecipitation experiments are required to confirm this hypothesis.
In the stomach and the small intestine of wild-type mice, substantial increases in GCLC were observed after treatment with the broccoli seed preparations. It is likely that Nrf2 mediates the increase in mouse GCLC and requires the existence of a functional ARE in the gene promoter, because this occurs in the human gene (45). However, the presence of a functional ARE in mouse gclc remains to be established.
Cellular models for screening the cancer chemopreventive properties of phytochemicals have frequently used induction of NQO1 enzyme activity in Hepa-1c1c7 cells (46). Our study revealed that besides NQO1 induction, GSTA1/2, GSTA3, and GCLC are also increased significantly in this transformed cell line by broccoli seed extract and by sulforaphane. Importantly, we also found that in nontransformed RL-34 cells, the seed extract and sulforaphane cause large increases in NQO1 and GCLC proteins. Modest increases in GSTA3 were also observed. Nakamura et al. (13) suggested that measurement of GST activity in RL-34 cells provides a useful assay for identifying potential inducing agents. However, our data suggest that induction of NQO1 in these cells may provide the most sensitive assay to identify chemopreventive phytochemicals, because the Western blots in Figure 4 suggest that a 10-fold increase of the protein can be readily achieved.
Significant variations in the amounts and the types of glucosinolates in different broccoli strains appear to exist (7–9). Because this will result in distinct ITCs being generated by myrosinase from different broccoli strains, these differences in glucosinolate content will also influence the level of induction that can be achieved in the host and also possibly the sensitivity to cell-cycle arrest. The significance of variation in the glucosinolate content of cruciferous vegetables in terms of antioxidant and detoxication gene induction and stimulation of cell-cycle arrest and apoptosis warrants further study.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by grant 2000/11 from the World Cancer Research Fund and by grant G0000268 from the Medical Research Council of the UK (L.G.H.).
4 Abbreviations used: AITC, allyl isothiocyanate; ARE, antioxidant response element; GCLC, glutamate cysteine ligase catalytic; GCLM, glutamate cysteine ligase modifier; GST, glutathione S-transferase; ITC, isothiocyanate; LC-MS/MS, liquid chromatography with triple quadrupole mass spectrometric detection; MEF, mouse embryonic fibroblast; MRM, multiple reaction monitoring; NQO1, NAD(P)H:quinone oxidoreductase 1; Nrf2, nuclear factor-erythroid 2 p45-related factor 2.
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LITERATURE CITED |
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