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Biochemical, Molecular, and Genetic Mechanisms

Consuming Broccoli Does Not Induce Genes Associated with Xenobiotic Metabolism and Cell Cycle Control in Human Gastric Mucosa^1,2

³ Phytochemicals and Health Programme, Institute of Food Research, Colney, Norwich NR4 7UA, UK; ⁴ Centre for Analytical Bioscience, School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK; ⁵ Wolfson Digestive Diseases Centre, Queen's Medical Centre, Nottingham University NHS Trust, Nottingham NG7 2RD, UK; and ⁶ School of Biomedical Sciences, University of Nottingham, Nottingham NG7 2RD, UK

^* To whom correspondence should be addressed. E-mail: amy.gasper{at}bbsrc.ac.uk.

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Epidemiological studies suggest that a diet rich in broccoli can reduce the risk of cancer at several sites. The anticarcinogenic activity has been largely attributed to the biological activity of sulforaphane (SF), the isothiocyanate derived from 4-methylsulphinylbutyl glucosinolate, which accumulates in broccoli. SF induces xenobiotic metabolizing genes in both cell cultures and animal models and induces genes associated with cell cycle arrest and apoptosis. However, it is not known whether these genes are induced in humans after consumption of broccoli. Sixteen subjects were recruited into a randomized, 3-phase crossover dietary trial of standard broccoli, high glucosinolate broccoli, and water. Global changes in gene expression that occurred 6 h after consuming broccoli soups or water were quantified in gastric mucosal tissue, using Affymetrix whole genome microarrays (n = 4), and in selected genes by real-time RT-PCR in the other individuals. Consumption of high glucosinolate broccoli resulted in up-regulation of several xenobiotic metabolizing genes, including thioredoxin reductase, aldoketoreductases, and glutamate cysteine ligase modifier subunit, which have previously been reported to be induced in cell and animal models after exposure to SF. Only 1 such gene was significantly up-regulated after consumption of standard broccoli. The consequences of these results in relation to the potential anticarcinogenic action of broccoli are discussed.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

Many studies have demonstrated that sulforaphane, 1-isothiocyanato-4-methylsulphinyl butane, (SF) can induce changes in gene and protein expression in both cell cultures and animal models that are consistent with anticarcinogenic activity [reviewed in Juge et al. (13)]. One of the mechanisms by which SF affords this protection is modulation of the phase I/II biotransformation pathways. First, suppression of phase I activation enzymes, such as cytochrome p450s, has been reported (14–16). Additionally, SF induces Nrf2-regulated transcriptional activation of phase II detoxification enzymes, including quinone reductase (QR), thioredoxin reductase 1 (Tr1), and heme oxygensase 1 (HO-1) (17–21). This induction is mediated through binding of nuclear factor (erythroid-derived 2)–like 2 (Nrf2) to the antioxidant response element (ARE) present in the promoter of phase II genes. In addition, SF induces apoptosis both in vitro and in vivo by activating the mitochondrial/intrinsic and the death receptor/extrinsic apoptotic pathways (22–24). Moreover, SF arrests cell growth by inhibiting cell cycle progression at the G_o/G1 or G2/M phase, through regulation of cyclins (e.g., cyclins B and D) and associated cyclin-dependent kinase inhibitors (e.g., p21^waf1/cip1 and p27^kip1) (25–27). More recently, involvement of SF in inhibition of angiogenesis (28,29) and antiinflammatory activity (30–32) have been demonstrated, highlighting the complex mechanisms of potential anticarcinogenic action by SF.

Despite the epidemiological evidence and mechanistic studies with cell and animal models, surprisingly few intervention studies have investigated whether any of the phenomena described in model systems occurs in humans. Some have investigated whether consumption of Brassica vegetables induces enzymes that activate or detoxify xenobiotics, as predicted from cell culture studies. For example, a 6-d Brassica-rich diet increased cytochrome p450 1A2 (CYP1A2) activity, as extrapolated from the ratio of urinary caffeine metabolites (33), which is consistent with the induction of CYP1A2 in animal studies with degradation products of indolyl glucosinolates (34). The same diet also enhanced serum glutathione S-transferase (GST) concentration and GST activity (33), which is again consistent with animal and cell studies (18). However, contrary to expectation, a 2-wk intervention with broccoli sprouts did not reduce the excretion of aflatoxin-N(7)-guanine in a population in China consuming aflatoxin-contaminated foods, compared with a control (35). This would have been expected on the basis of animal studies, in which glucosinolate degradation products from broccoli sprouts are potent inducers of glutathione transferases (36).

In a previous publication, we reported the metabolism of SF in human subjects after consuming standard and high glucosinolate (HG) broccoli (37). Here, we investigated acute changes in gene expression in the human gastric mucosa after consumption of a single meal of SF-rich soup made from either standard or HG broccoli. The development of HG broccoli has been described elsewhere (38–40). We tested whether enhancing the glucosinolate content of broccoli leads to differences in gene expression profiles, compared with those previously reported to occur following exposure of cell cultures and animals to SF or broccoli extracts.

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Broccoli cultivation and preparation. The standard broccoli (cultivar Marathon) and the HG broccoli were grown at the ADAS experimental research station, Terrington, UK. Individual soup portions of the 2 cultivars were prepared by cooking 100 g of florets with 150 mL water for 90 s on high power in a 700 W microwave oven, followed by homogenization. Preliminary analysis had shown that with this cooking method, plant thioglucosidases remained active and glucosinolates were converted to ITC without nitrile formation (40). The broccoli soup was stored frozen at –18°C and portions were thawed at room temperature for 4 h before consumption, with no further processing, other than mixing. The mean SF concentration of the supernatants was 682.6 ± 113.30 µmol/L (n = 16) for standard broccoli and 2295.9 ± 217.53 µmol/L (n = 16) for HG broccoli, and was measured using a previously described method (41).

    Study protocol. Volunteers (7 male and 9 female) aged 18–46 y were recruited by research nurses at the Wolfson Digestive Diseases Center (WDDC), Queen's Medical Centre, Nottingham University Hospital NHS Trust. Ethical approval for the trial was obtained from the University of Nottingham Medical School Ethics Committee (reference E/10/2003). All participants gave written, informed consent and were screened for full blood count, liver function tests, urea, and electrolytes and fasted glucose. Volunteers were excluded if they were pregnant, smoked cigarettes, were diagnosed with a long-term medical condition, or were taking dietary supplements. Subjects were allocated into a randomized, 3-phase (HG broccoli, broccoli, and water), crossover design with 21 d of washout between each phase. One subject was unable to complete the study due to illness unrelated to the interventions. The trial was conducted from January 8 to May 19, 2004.

Volunteers avoided foods known to contain glucosinolates or ITC, spicy foods, and alcohol for 24 h prior to the start of the study. On the day before each study day, volunteers attended the center for a fasted baseline endoscopy. Four biopsies of the gastric mucosa were taken from the antrum and placed into RNAlater (Ambion) to preserve RNA. Samples were refrigerated overnight at 4°C to allow penetration of RNAlater and then snap frozen in liquid nitrogen and stored at –80°C until analysis. The following day, volunteers returned to the center and consumed a 150 mL test meal of HG broccoli soup, broccoli soup, or water. A sub-sample of the soup was taken immediately prior to consumption to enable the measurement of the precise concentration of SF and metabolites consumed by each individual. Blood and urine samples were collected for a metabolic study, the results of which were published separately (37). Four further biopsies were collected 6 h after consumption of the test meals. Volunteers were genotyped for GSTM1 as previously described (37).

    RNA extraction and array hybridization. Total RNA was isolated from biopsies using RNeasy mini kits, according the manufacturer's instructions (Qiagen). The quantity of resulting RNA was measured using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies). The RNA quality was determined using the 2100 Bioanalyzer (Agilent Technologies). A total of 24 RNA samples from all 3 interventions at baseline (t = 0) and study day (t = 6 h after intervention) from 4 subjects were hybridized onto Affymetrix Human U133 plus 2.0 microarrays, by the Arabidopsis Stock Centre, University of Nottingham, UK. These arrays allow for analysis of over 47,000 transcripts derived from >33,000 human genes. Double stranded cDNA synthesis and generation of biotin-labeled cRNA were performed according to the manufacturer's protocol (Affymetrix). The final cRNA was checked for quality before fragmentation and hybridization onto a total of 24 arrays.

    Real-time RT-PCR. We selected 3 genes for further analysis by RT-PCR. These genes have been reported to be up-regulated in cell cultures and have frequently been included in mechanistic explanations for the anticarcinogenic activity of broccoli (12,13). Two of these, Tr1 and glutamate cysteine ligase modifier (GCLM), were shown to be up-regulated by 1.58-fold (P < 0.05) and 1.78-fold (P < 0.05), respectively, in the microarray data. The third gene, p21^waf1/cip1, had previously been shown to be up-regulated by SF in cell culture data, but was not changed by either broccoli treatment in this study. Primers (Sigma-Genosys) and probes (Applied Biosystems) for Tr1 and p21^waf1/cip1 were designed using ABI PRISM Primer Express software v1.5 (Applied Biosystems). The probes were labeled with a 5' reporter dye (FAM, 6-carboxyfluorescein) and a 3' quencher dye (TAMRA, 6-carboxytetramethylrhodamine). GCLM was quantified using a TaqMan gene expression assay kit Hs00157694_m1 (Applied Biosystems).

RT-PCR were performed using an Applied Biosystems AB7700 real-time PCR system on an optical 96-well plate in a total volume of 25 µL/well, consisting of TaqMan 1-step RT-PCR master mix reagent kit (Applied Biosystems), 20 ng total RNA, and the following optimized concentrations of primers and probes: Tr1 forward sequence [5'-CCACTGGTGAAAGACCACGTT-3' 200 nmol/L, reverse sequence 5'-AGGAGAAAAGATCATCACTGCTGAT-3' 300 nmol/L, probe 5'-CAGTATTCTTTGTCACCAGGGATGCCCA-3' 100 nmol/L] and p21^waf1/cip1 forward sequence [5'-CTGGAGACTCTCAGGGTCGAA-3' 200 nmol/L, reverse sequence 5'-CGGCGTTTGGAGTGGTAGA-3' 400 nmol/L, probe 5'-ACGGCGGCAGACCAGCATGAC-3' 100 nmol/L]. Reverse transcription was performed for 30 min at 48°C, AmpliTaq gold activation for 10 min at 95°C, followed by 40 PCR cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min. Reactions were carried out in triplicate and were normalized against the expression of an invariant endogenous control, 18S ribosomal RNA, according to methods previously described (42).

    Statistical analysis. Fluorescence intensity for each microarray chip was captured with an Affymetrix HP GeneArray laser confocal scanner (Agilent Technologies). Affymetrix Microarray Suite version 5.0 was used to quantitate each chip. The 24 raw CEL files generated were loaded into the DNA-Chip Analyzer software, (version dChip2006) for normalization, generation of expression values, and statistical analysis. For normalization, dChip uses the invariant set normalization method, which chooses a subset of perfect match (PM) probes with small within-subset rank difference in the 2 arrays (baseline and target array), to serve as the basis for fitting a normalization curve. This is used to generate new normalized values for each probe on the chip. Probe expression values were subsequently calculated using the perfect match (PM-only) model, which was shown previously to produce consistently less variable results than the PM-mismatch difference model (43).

Microarray data were analyzed using dChip2006 software (44). Probe lists were created using combined comparisons between baseline and 6 h after consumption of each treatment. Paired t tests, adjusted to compensate for false discovery rate (FDR) with 100 permutations, were conducted applying the following criteria: 1) fold change cut-off (1.0 and 1.5); 2) probability (P < 0.05, P < 0.01, and P < 0.001); and 3) absolute difference between the 2 group means >100. To interpret the biological consequences of the differences in gene expression, Onto-Express software (45) was used to calculate significant differences in gene ontology. The gene lists input for classification were as follows: no fold cut-off, P < 0.05, and group means >100 for each test meal, after removal of multiple probes using dChip (Table 2). Hypergeomatric distribution was used to calculate statistically significant (P < 0.0001) functional gene ontology categories with FDR-correction for each test meal.

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Microarray analysis of gene expression in the gastric mucosa. Totals of 133 and 165 probes were differentially expressed 6 h after consumption of HG broccoli and standard broccoli, respectively, compared with 25 probes following consumption of water (Table 1). The relatively high median FDR makes biological interpretation of the probe list complex, but this was overcome using Onto-Express analysis to link expression profiles with biological processes, molecular functions, and cellular components for each test meal. The ontological analysis for HG broccoli found that oxidoreductase activity was a highly significant (10 genes at P < 0.05) molecular function in this gene list. For the standard broccoli, transcription factor activity was the most significant molecular function (9 genes at P < 0.05), which was also significant in the HG broccoli list (6 genes at P < 0.05). There were fewer changes in gene expression and a far higher median FDR with the water intervention (Table 1), which was reflected in the functional ontology analysis.

View larger version (5K): [in this window] [in a new window]   FIGURE 1  Confirmation of microarray results for expression of Tr1 (A), GCLM (B), and p21 (C), by real time RT-PCR in a subjects 6 h after consumption of HG broccoli, standard broccoli (S), and water (W). Quantitative data are expressed as log2 fold change and are means ± SEM, n = 10 (A and C) or 6 (B). Asterisks indicate different from preintervention: * P 0.05; ** P 0.001.

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

Despite the 3-fold difference in SF concentration between HG broccoli and standard broccoli soups, similar numbers of probes were significantly altered in expression after the intervention (Table 1). Of the 133 probes altered in expression following HG broccoli, only 18 of these changed after consumption of standard broccoli. Thus, most of the changes in gene expression were specific to the 2 broccoli cultivars. When grouped by function, following the intervention with the HG broccoli soup, there was induction of several xenobiotic metabolising genes with oxidoreductase activity (Table 2). Changes in expression of these genes were previously observed following exposure of Caco-2 cells to SF (42). Four of these [GCLM; aldo-keto reductase family 1, member C1 (AKR1C1); aldo-keto reductase family 1, member C2 (AKR1C2); and Tr1] have also been found by several other studies (36,47) to be modified in expression by SF in both cell cultures and animal models. These genes all contain an ARE in their promoter region, so they are likely to be up-regulated by SF via the Nrf-2 transcription factor, as previously described (47). The enhanced expression of GCLM is likely to be related to glutathione depletion after conjugation to SF, as it is the rate limiting enzyme in glutathione biosynthesis.

Solute carrier family 7, member 11, was also induced following HG broccoli consumption. This gene is specifically associated with the import of cysteine into cells for glutathione biosynthesis. In addition, heat shock proteins HSPA1A and HSPH1 were found to be induced. HSPA1A is known for its cytoprotective role and HSPH1 has been associated with apoptosis (48). Both were also up-regulated by 50 µmol/L SF in Caco-2 cells (42). Two other genes coding for oxidoreductases, not previously reported to be induced by SF, were also enhanced in expression. They were carbonyl reductase, an NADPH-dependent oxidoreductase having wide specificity for carbonyl compounds, including prostaglandins (49), and leukotriene B4 12-hydroxydehydrogenase, which is involved in prostaglandin catabolism (50,51).

Consumption of standard broccoli soup resulted in a significant change in expression of only 1 gene with oxidoreductase activity, hydroxysteroid (11-ß dehydrogenase 2) (Table 3). The simplest explanation is that the SF concentration was insufficient to induce expression of ARE-mediated genes, but it is also conceivable that induction may have been transitory, and thus was not detected through expression profiling of biopsy tissue. Expression of 4 genes with transcription factor activity was suppressed by both standard and HG broccoli. They were the nuclear receptor subfamily 1 group D2, thyrotrophic embryonic factor, MAX dimerization protein 1, and basic helix-loop-helix domain class B2, but they were not affected by the water treatment (Tables 2–4). In addition, pleckstrin homology like domain A2, involved in apoptosis, was suppressed by both interventions, and not by water. As the extent of suppression was similar with both broccoli soups, factors other than SF may be responsible, and these genes may have additional importance for the biological activity of broccoli.

There was no evidence for changes in expression of the many other genes that have been reported to be modified in expression following exposure of cell cultures to SF, including the cell cycle regulator p21^waf1/cip1 (52), glutathione transferases, and UDP-glucuronosyl transferase. There are several explanations for this. First, the induction of these genes may be largely restricted to cell and animal model systems, in which the SF concentration and time of exposure is considerably greater than that which occurs following broccoli consumption. Notably, aldo-keto reductases and Tr1 were among the few genes induced by the lowest concentrations of SF (1 and 5 µmol/L) in the report by Traka and colleagues (42). Second, the tissue obtained in this study was healthy mucosal tissue comprising a mixture of cell types, whereas cell lines such as Caco-2 are derived from cancerous epithelial cells, which have faster growth rates and metabolism. Third, cell cultures may undergo changes during culturing that render them more or less sensitive to external stimuli, compared with in vivo tissue. Real-time RT-PCR confirmed the quantification of expression of 3 genes, GCLM, Tr1, and p21^waf1/cip1 in further individuals. The results were similar to the arrays in terms of the direction of fold change, although there was a tendency for the arrays to underestimate the magnitude of the change in gene expression as observed by others (42).

In this study, we found that a SF-rich soup can induce a small number of xenobiotic metabolizing genes (Table 2) in the human gastric mucosa that have also been shown to be induced in cell culture. The genes that were induced were previously found to be those most sensitive to induction by SF (42). However, this induction was evident following consumption of a soup prepared to maximize glucosinolate conversion to SF from a broccoli that contains 3-fold the concentration of glucosinolates than standard broccoli. Thus, although it is notable that the soup prepared from HG broccoli induced more genes involved in xenobiotic metabolism than standard broccoli soup, the latter resulted in changes in expression of many genes that were not significantly altered by HG broccoli soup, particularly those involved in transcription and metal ion binding.

This human intervention study quantified changes in gene expression in the gastric mucosa following a single broccoli meal. The broccoli soup was prepared in a manner to maximize SF concentration. Habitual consumption of broccoli and other cruciferous vegetables may result in long term changes in gene (and protein) expression that are likely to be distinct to those that are reported in this study. These may be important in understanding the mechanistic basis for the likely reduction in cancer risk resulting from consuming these vegetables. It is important that further dietary intervention studies are designed to investigate these types of changes.

In conclusion, a small number of genes involved in xenobiotic metabolism and other metabolic processes that are commonly associated with the putative anticarcinogenicity of broccoli were induced in human gastric mucosa following consumption of a SF-rich preparation of a high glucosinolate broccoli, but not following consumption of a SF-rich preparation from standard broccoli. Given that domestic preparation of standard broccoli is likely to result in <100% conversion of glucosinolates to ITC, caution should be exercised in extrapolation from cell and animal models in which concentrations of SF exceed those habitually consumed within the diet.

	ACKNOWLEDGMENTS

 
We thank research nurses Clare Atherton and Rosie Bayliss and Drs. Paul Fortun, Tony Shonde, Steve Foley, and Martin James for their hard work and endoscopy support. Thanks to Zoe Emmerson and Henrik Townsend at the Arabidopsis Stock Centre, University of Nottingham, for hybridization of the RNA to Affymetrix microarrays. Additionally, we thank Dr. Yongping Bao (University of East Anglia, UK) and Andrew Wilkinson (IFR, UK) for designing RT-PCR primers for Tr1 and p21, respectively. The sponsors of the study had no role in the design of the study, data collection, analysis, interpretation, or in writing the report.

	FOOTNOTES

 
¹ Supported by the Biotechnology and Biological Sciences Research Council and the University of Nottingham.

² Author disclosures: A. V. Gasper, M. Traka, J. R. Bacon, J. A. Smith, M. A. Taylor, C. J. Hawkey, D. A. Barrett, and R. F. Mithen, no conflicts of interest.

⁷ Abbreviations used: AKR, aldoketoreductase; ARE, antioxidant response element; CYP, cytochrome p450, FAM, 6-carboxyfluorescein; FDR, false discovery rate; GCLM, glutamate cysteine ligase modifier; GST, glutathione S-transferase, HG, high glucosinolate; HO-1, heme oxygenase; ITC, isothiocyanate; (Nrf2), nuclear factor (erythroid-derived 2)–like 2; PM, perfect match; QR, quinone reductase; SF, sulforaphane; TAMRA, 6-carboxytetramethylrhodamine; Tr1, thioredoxin reductase; WDDC, Wolfson Digestive Diseases Center.

Manuscript received 29 January 2007. Initial review completed 6 March 2007. Revision accepted 11 May 2007.

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TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 

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