Table 4. Dietary analysis of average daily intakes of nutrients. Baseline 6 months P-value* 9081.88 (2480.80) 9506.00 (2405.38) Pea-rich diet (n = 7) Variable Fat (g) Protein (g) CHO (g) Energy (KJ) Alcohol (g) Cholesterol (mg) Vitamin C (mg) Vitamin E (mg) Vitamin D (mg) b-Carotene (mg) Folate (mg) Iron (mg) Selenium (mg) Peas (g) Broccoli (g) Fat (g) Protein (g) CHO (g) Energy (KJ) Alcohol (g) Cholesterol (mg) Vitamin C (mg) Vitamin E (mg) Vitamin D (mg) Beta Carotene (mg) Folate (mg) Iron (mg) Selenium (mg) Peas (g) Broccoli (g) Estimated GSL mmol 93.76 (32.97) 87.50 (15.15) 240.04 (79.85) 12.65 (9.87) 353.57 (74.43) 81.00 (66.05) 8.24 (5.64) 3.77 (3.25) 1.94 (1.57) 261.00 (89.36) 11.73 (4.36) 50.14 (13.01) 8.57 (10.23) 18.49 (30.89) 90.93 (29.27) 96.99 (20.02) 276.28 (76.03) 9.75 (7.20) 337.27 (168.29) 262.55 (175.83) 11.31 (5.73) 5.22 (3.17) 4.07 (3.01) 14.29 (2.09) 77.09 (29.89) 4.16 (5.51) 25.89 (24.49) 13.10 (12.39) 90.99 (25.83) 95.77 (33.31) 242.94 (72.97) 25.07 (35.42) 345.29 (201.06) 79.43 (62.58) 7.15 (3.60) 3.84 (1.76) 3.22 (2.29) 332.86 (214.50) 13.32 (4.92) 49.14 (23.08) 57.41 (18.86) 9.90 (13.61) 5.01 (6.88) 91.57 (33.70) 96.97 (21.26) 296.18 (72.99) 10.48 (9.70) 298.46 (123.99) 303.00 (188.52) 11.14 (4.82) 3.65 (1.08) 3.63 (2.53) 14.09 (2.25) 68.82 (26.22) 7.60 (8.46) 55.84 (7.71) 79.30 (10.94) 0.831 0.526 0.872 0.738 0.415 0.930 0.846 0.395 0.949 0.184 0.489 0.555 0.923 0.001 0.431 0.431 0.929 0.996 0.305 0.488 0.841 0.211 0.590 0.924 0.076 0.667 0.762 0.916 0.304 0.227 0.002 ,0.0001 9633.45 (2311.35) 9980.73 (2286.62) 477.82 (188.20) 491.36 (193.47) Estimated GSL (mmol) 9.36 (15.63) Broccoli-rich diet (n = 11) Variables shown are given in mean (sd) units per day. GSL refers to the glucosinolate precursors of sulforaphane and iberin (ie 4-methylsulphinylbutyl and 3-methylsulphinylpropyl glucosinolate) respectively. Similar analysis between GSTM1 positive and individuals showed no difference in dietary intakes after 6 months within either broccoli-rich or pea-rich intervention. *P-values were calculated in Minitab using a paired t-test. doi:10.1371/journal.pone.0002568.t004 Similar results were obtained to identify Gly-IB (m/z 221) and Phe-IB (m/z 311) from the incubation (data not shown). Comparable evidence was obtained for the formation of EGF conjugates with SF in human plasma corresponding to the addition of SF to the N-terminal asparagines residue (m/z 309) of EGF. To provide additional information of modifications to TGFb1, we adopted a complementary approach. 1 mg aliquots of the protein were incubated with either DMSO or 1.2 mmoles of SF for 30 minutes at 37uC and separated by SDS-PAGE electrophoresis. Bands were excised and digested with trypsin before analysis by LC-MS/MS. TGFb1 was robustly identified in bands of 25 kDa Broccoli & Signalling Pathways corresponding to the active dimer. The N-terminal peptide ALDTNYCFSSTEK was identified from parent ion m/z 768.5 in both DMSO (control) and SF-treated samples (Figure 6). A precursor ion m/z 877.2 was observed only in SF treated samples. MS/MS analysis of both precursor ions revealed a strong series of fragment peaks that were common to both (Figure 7) precursor ions. These fragmentation patterns are consistent with the unmodified y ion series for the peptide ALDTNYCFSSTEK (including carbamidomethyl cysteine +57) and a b ion series shifted by 217.460.8 Da in the SF-treated sample. These results strongly support an N-terminal modification to TGFb1 by SF. Addition of SF would result in a mass addition of 177, as observed with LC-MS analyses of intact TGFb1, as described above. It is highly likely that the addition of 217, as opposed to 177, is due to subsequent reaction of the thiourea with iodoacetamide, added to the reaction mixture to alkylate reduced disulphide linkages, to result in a mixture of isomeric carbamimidoylsulfanylacetamides, which undergo cyclisation and loss of NH3 to give the corresponding iminothiazolidinones (Figure S2). Enhancement of TGFb1 signalling after pre-incubation with sulforaphane As thiourea derivatives of proteins by isothiocyanates have been shown to modify physicochemical and enzymatic properties [26,27], we sought to assess whether SF modification of extracellular signalling proteins had functional consequences. We focussed on TGFb1 signalling due to its profound role in maintaining tissue homoeostasis through controlling cell prolifer- ation and behaviour [28,29]. TGFb1-induced Smad-mediated transcription was quantified in NIH3T3 cells stably transfected with a CAGA12-luc plasmid, in which luciferase activity can be measured upon activation of Smad proteins [30]. Exposure of cells to TGFb1 induced luciferase activity as expected. When cells were exposed to TGFb1 that had been pre-incubated with physiolog- ically appropriate concentrations of SF (2 mM) for 30 minutes followed by dialysis, to simulate SF plasma pharmacokinetics [14], there was an increase in Smad-mediated transcription compared to exposure to TGFb1 alone (Figure 8). Exposure of cells to the residual SF (34 nM) did not result in enhanced transcription suggesting that SF induces Smad activation indirectly, consistent with our previous observation that SF binds to the ligand itself. It is also conceivable that SF may interact with the extracellular domain of the receptor to alter downstream signalling. Discussion To our knowledge, this is the first dietary intervention study to analyse global gene expression profiles within a target tissue before and after a 12 month intervention, and to stratify gene expression profiles by genotype. While we do not observe any consistent changes in plasma PSA levels over the 12 month period of the intervention, we were able to quantify extensive changes in gene expression. We find little evidence to support potential mecha- nisms derived from animal and cell models to explain the observational data that consuming broccoli may reduce risk of cancer, but considerable evidence for the perturbation of several signalling pathways that are associated with carcinogenesis and inflammation (Table 2b and c). It is possible that the net effect of perturbation of these pathways may reduce the risk of cell proliferation, and maintain cell and tissue homoeostasis. However, whilst quantification of gene expression and pathway analyses provides information concerning which pathways may be modified by time or diet, it can provide little information about the precise nature of how these pathways are perturbed. This requires further PLoS ONE | www.plosone.org 8 July 2008 | Volume 3 | Issue 7 | e2568