Figure 2. LDA of an independent prostate microarray data set. Linear discriminant analysis (LDA) using the benign (B) and malignant (M) TURP prostate tissue for this study as training samples to classify the laser-capture microdissected (LCD) epithelial prostate cell samples (GEO Accession:GDS1439), consisting of benign (Be), primary cancer (PCa) and metastatic cancer (MCa) samples. LDA was performed on a gene list that distinguished the benign and malignant TURP samples as described in Methods. Here, the first linear discriminant (LD1) is shown. doi:10.1371/journal.pone.0002568.g002 positive and men who had been on the broccoli diet for six months, but few, if any, differences in gene expression between GSTM1 positive and men who had been on the pea diet (Figure 3a, Table 3b). To investigate the potential consequences of the differences in gene expression between GSTM1 genotypes following the broccoli-rich diet, we analyzed these data via GenMapp. Three pathways, EGF receptor, adipogenesis and TGFb receptor, were identified in which genes occurred at a higher frequency than they would by chance (Table 2b). The relative expression of genes between the GSTM1 positive and men in the EGF receptor and TGFb receptor pathways are given as supporting information (Table S2 and S3). Changes in gene expression before and after the dietary intervention We used paired t-tests to identify genes that had changed in expression between 0 and 6 months and 0 and 12 months in biopsy samples from individuals within each arm of the intervention to quantify changes in expression with time. Within Broccoli & Signalling Pathways the broccoli arm, we restricted analyses to GSTM1 positive individuals. We found after both 6 months and 12 months there were more changes in expression within the broccoli-rich arm than the pea-rich arm (Figure 3b, Table 3c). Pathway analyses with genes that changed in expression between 0 and 12 months identified changes only in the androgen receptor pathway in the pea-rich arm, while in the broccoli-rich arm androgen receptor pathway was identified, along with several other signalling pathways, including insulin signalling, TGFb and EGF receptor pathways (Table 2c, Table S4 and S5). Analyses with genes that changed in expression between 0 and 6 months in the broccoli arm also identified changes in TGFb receptor pathway (adjusted P = 0.001), insulin signalling (adjusted P = 0.035) and EGF receptor signalling (adjusted P = 0.068). Thus, evidence for the effect of broccoli consumption on modulation of TGFb and EGF signalling has been obtained in two independent analyses: Firstly, the comparison of gene expression profiles of GSTM1 positive and individuals who had consumed the broccoli-rich diet for six months, and, secondly, the paired analyses of gene expression profiles from biopsies obtained at 0 and 12 months from GSTM1 positive individuals who had consumed the broccoli-rich diet. It is important to note that these analyses do not share any array data sets. Chemical interactions between TGFb1, insulin and EGF peptides and broccoli isothiocyanates Having demonstrated that broccoli consumption modulates several cell signalling pathways, we sought a mechanistic explanation. Incubation of insulin, EGF and TGFb1 peptides with the isothiocyanates SF or IB in PBS pH 7.4 at 37uC for a period of 0.5 to 24 hours gave consistent evidence of the formation of a covalently bound conjugate of the respective peptide and the ITC. This was further investigated for physiological relevance by performing the same incubations in human plasma depleted of high MW proteins. LC-MS/MS analysis showed the appearance of one or more additional LC-MS peaks when SF or IB were incubated with the peptides. For example, in Figure 4 an extracted ion chromatogram (m/z 1183.9, corresponding to insulin- SF MH55+) shows the appearance of two insulin-SF conjugates compared with the control incubation. MS2 analysis of these peaks (Figure 5) confirmed the presence of two diagnostic fragment ions at m/z 235 and m/z 325 corresponding to the addition of SF to the two N-terminal amino acids of insulin Gly-SF and Phe-SF. Figure 3. Effect of dietary intervention on gene transcription. a, Number of probes that differ between GSTM1 positive and genotypes (P#0.005, Welch modified two-sample t-test) in TURP tissue from benign (Ben) and malignant (Mal) prostates, and TRUS-guided biopsy tissue from volunteers at pre-intervention (Pre), post 6 months broccoli-rich diet (Broc) and post 6 months pea-rich diet (Peas). b, Number of probes that differ between pre-intervention TRUS-guided biopsy samples and after 6 months broccoli (6B)-, 6 month pea (6P)-, 12 month broccoli (12B)- and 12 month pea (12 P)-rich diets (P#0.005, Welch modified two-sample paired t-test). Shading correspond to different fold cutoffs applied. See Table 2 for full details of probe numbers, P-values and median false discovery rates. doi:10.1371/journal.pone.0002568.g003 PLoS ONE | www.plosone.org 7 July 2008 | Volume 3 | Issue 7 | e2568