Broccoli & Signalling Pathways Table 1. Volunteer characteristics and plasma PSA levels. Age BMI GSTM1 PSA (ng/ml) Broccoli intervention Pre-intervention 6-month 12-month Mean (sd) 4.9 (3.25) 4.3 (2.50) 3.84 (2.44) 68 68 64 63 66 57 66 69 62 59 68 64 63 70 65 59 61 57 70 61 57 29 27 26 31 28 27 30 27 23 29 25 32 27 28 24 24 35 26 23 29 30 positive positive positive positive positive positive positive positive positive positive positive 4.6 3.1 9.4 6.5 0.9 5.5 13.6 6.9 2.2 10.8 9.7 6.4 7.9 4.1 7.5 9.3 1.1 5.5 8.9 2.3 3.5 after broccoli consumption (i.e. ,2 mM for less than one hour). At these physiological concentrations, it is likely that any SF entering cells would be immediately conjugated with glutathione due to the relatively high intracellular glutathione concentration, with no effect on, for example, the Keap1-Nrf2 complex [16,17]. Consistent with this hypothesis, there was no evidence for induction of phase 2 gene transcription in the gastric mucosa after an acute broccoli intervention [18]. In this study, we initially adopt an empirical approach in humans to elucidate the mechanisms that underlie the beneficial effects of a broccoli-rich diet, and explore the interaction with GSTM1. We compare and interpret global gene expression profiles in human prostate biopsy tissue before, during and after a 12 month broccoli-rich diet and a 12-month pea-rich diet. Subse- quently, we provide a mechanistic explanation for how the observed changes in gene expression may be induced by normal dietary broccoli consumption. Methods Subjects and study design Full details of the study protocol and the supporting CONSORT checklist and flow diagram are available as supporting information see Protocol S1, Checklist S1 and Figure S1. As stated in the protocol, it was intended to recruit forty men with a previous diagnosis of high-grade prostatic intraepithelial neoplasia (HGPIN), the pre-invasive in situ stage of prostatic adenocarcinoma, into a dietary intervention trial to study the effects of a diet rich in broccoli and a diet rich in peas on prostate gene expression. However, due to changes in clinical practice during the recruitment phase of the study there was marked reduction in the numbers of men diagnosed with HGPIN and it was not possible to recruit 40 men within the time constraints of the research programme. Thus, twenty-two male volunteers aged 57–70 years (Table 1) were recruited by a consultant urologist at Norfolk and Norwich University Hospital NHS Trust. Histological diagnosis was made by two consultant histopathologists, who had a special interest in prostate pathology. Ethical approval for the trial was obtained from the Norfolk Research Ethics Committee (reference 05/Q0101/9, see Text S1). All participants gave written, informed consent. Volunteers were excluded if they were undergoing chemopreventive therapy, were receiving testosterone replacement medication or 5 alpha reductase inhibitor, had active infection requiring treatment, had a body mass index (BMI) ,18.5 or .35, or were diabetic. Subjects were allocated into a 12-month, parallel dietary intervention trial consisting of two dietary intervention groups: (i) consuming 400 g broccoli per week or (ii) consuming 400 g peas per week, in addition to their normal diet. The trial was conducted from April 2005–April 2007. Plasma prostate specific antigen (PSA) levels were quantified prior to the intervention study and after six and 12 months at the Norfolk and Norwich University Hospital with the use of a total PSA immunoassay. Volunteers avoided foods known to contain glucosinolates for 48 hours prior to each biopsy appointment to avoid acute effects. In addition to the transrectal ultrasound scan (TRUS)-guided needle biopsies of the prostate obtained from the volunteers immediately prior to the intervention study, and after six and twelve months, 18 benign and 14 malignant transurethral resection of the prostate (TURP) tissues were also obtained from the Norfolk & Norwich University Hospital Partners in Cancer Research Human Tissue Bank. Dietary intervention Vegetables were delivered to the volunteers on a monthly basis. They were provided with a steamer and the volunteers were given PLoS ONE | www.plosone.org 3 July 2008 | Volume 3 | Issue 7 | e2568 Mean (sd) 7.9 (3.5) 8.3 (4.85) 7.56 (3.95) Peas intervention Mean (sd) 5.5 (3.15) 6.2 (3.92) 5.4 (2.92) Mean (sd) 4.9 (3.52) 4.3 (1.82) 3.7 (1.70) One of the 22 volunteers was diagnosed with prostatic adenocarcinoma at the study baseline biopsy (pre-intervention) and was removed from the study. *This volunteer developed prostatic adenocarcinoma six months into the intervention and was removed from the study. doi:10.1371/journal.pone.0002568.t001 a demonstration by the diet cooks at the Institute of Food Research of how to cook the vegetables. Portions of broccoli were steamed for 4–5 minutes and portions of peas were steamed for 2– 3 minutes. Frozen peas (Birds Eye Garden Peas, http://www. birdseye.co.uk/) were purchased from a local retail outlet. To ensure consistency in glucosinolate content in frozen broccoli provided to the volunteers, the broccoli required for the intervention study was grown in one batch at an ADAS experimental farm at Terrington, near King’s Lynn, UK (http://www.adas.co.uk/) and processed by Christian Salvesen (Bourne, Lincolnshire, UK, http://www.salvesen.co.uk/). It was blanched at 90.1uC for 74 s, frozen at 230uC and packaged into 100 g portions, then stored at 218uC until steamed by the volunteer. The broccoli was a high glucosinolate variety [19,20]. The levels, mean (SD), of 4-methylsulphinylbutyl and 3-methyl- sulphinylpropyl glucosinolates (the precursors of SF and IB, respectively) were 10.6 (0.38) and 3.6 (0.14) mmolesg21 dry weight, respectively, compared to 4.4 (0.12) and 0.6 (0.01) mmolesg21 dry weight in broccoli purchased from local retail outlets. Although the level of glucosinolates were higher than standard broccoli, 5.4 3.2 3.5 7.9 1.3 5.5 16.8 3.8 2.2 10.4 12.5 6.1 9 4.2 9.3 10.8 1.1 5.4 5.2 2.2 5.5 5.3 2.9 2.9 7.2 0.9 5.7 13.4 3.7 1.9 11.2 7.2 6.6 10.8 4.4 8.2 8 1.1 5.2 N/A* 2.5 4.9