defect by heterologous expression of FADS2, evidence of novel substrate specificity for FADS1, and competition between FADS1 and FADS2 for the same substrates, leading to the production of unusual LCPUFA which are not substrates for eicosanoid production. Results and Discussion FADS2 and FADS1 transiently transfected MCF7 cells were probed for evidence of bioactivity toward 18:2n26 and 18:3n23. FADS2 transfected cells showed activity towards both the substrates; gas chromatography-covalent adduct chemical ioniza- tion tandem mass spectrometry (GC-CACI-MS/MS) confirmed the new peaks to be 18:3n26 and 18:4n23 respectively (Figure 1). As expected, no products were observed when either 18:2n26 or 18:3n23 were incubated with the FADS1 and the empty vector controls. These data provide the first unambiguous molecular evidence that the metabolic defect in these cells can be restored by replacing the rate limiting D6-desaturase enzyme encoded by FADS2. When FADS1-transfected cells were incubated with 20:2n26 and 20:3n23, CACI-MS/MS shows gain of synthetic function to generate two new butylene-interrupted PUFA peaks: 5,11,14–20:3 and 5,11,14,17–20:4, respectively (Figure 2). These two LCPUFA are analogues to arachidonic acid (5,8,11,14–20;4) and eicosa- pentaenoic acid (5,8,11,14,17–20:5), respectively. Our data demonstrate that in the substantial absence of D8-desaturase activity (FADS2), the D5-desaturase (FADS1) operates on 20:2n26 and 20:3n23. D6-desaturation (FADS2) is normally about 10-fold more active than D5-desaturation (FADS1), thus it is plausible that during cell transformation FADS1 activity would persist when FADS2 is inactive. However, the preferred substrates for FADS1 are not essential fatty acids (18:2n26 and 18:3n23), but their elongation products 20:2n26 and 20:3n23. FADS1 acting on these substrates generates unusual butylene-interrupted carbon products and our results show that these rare fatty acids may be produced in cancer cells. This is the first molecular evidence showing novel FADS1 substrate specificity for 20:2n26 Loss of FADS2 Causes Unusual Fatty Acid Synthesis and 20:3n23 fatty acids. Moreover, FADS2-transfected cells demonstrate conclusively that the FADS2 gene product D8- desaturates 20:2n26 and 20:3n23 to 20:3n26 (DGLA) and 20:4n23 (ETA) respectively, equivalent to our results in heterologously transformed yeast [14]. Moreover, while it is well known that both n23 and n26 PUFA substrates compete for same enzymes, our current data show FADS1 and FADS2 competing for the same substrates (Figure 2). The net consequence is the substitution of the inactive 5,11,14– 20:3 and 5,11,14,17–20:4 for ARA (5,8,11,14–20:4) and EPA (5,8,11,14,17–20;5), respectively, with unpredictable consequences for eicosanoid-mediated cell-cell paracrine signaling. Dysregula- tion of eicosanoid signaling is linked to tumor development, angiogenesis and metastasis in animal models [15]. A double bond at position 8 is required for cyclooxygenase (COX), lipoxygenase (LOX) and thromboxane biosynthesis, and thus the absence of the double bond at position 8 renders 5,11,14–20:3 and 5,11,14,17– 20:4 inactive as substrates for biosynthesis of most eicosanoids [16,17,18]. Moreover, the possible action of the butylene- interrupted PUFA as competitive inhibitors of eicosanoid biosynthetic enzymes is unknown, as is the activity of other key eicosanoid synthetic enzymes (e.g. cytochrome P450) whose actions on normal parts of the unusual fatty acids would result in products with unknown activit(ies). FADS genes, encoding enzymes required for PUFA biosynthesis arose evolutionarily by gene duplication events. Despite key importance of FADS2 and the loss of its function in several cancer cells, the molecular details and consequences of FADS2 loss has not been described or investigated. FADS2 is known to have activity towards at least seven substrates (18:2n26, 18:3n23, 20:2n26, 20:3n23, 24:4n26, 24:5n23, 16:0). The loss of FADS2-encoded activities in cancer cells completely shuts down the classical and alternative PUFA pathways, eliminating eicosa- noid and docosanoid precursor biosynthesis from the plant-based PUFA linoleic and linolenic acids and thus limiting cell-cell signaling (Figure 3). Many studies have found strong associations between single nucleotide polymorphisms within the FADS gene cluster and complex phenotypes related to chronic disease, as well Figure 1. GC results of transfected MCF7 cells with18:2n-6 and18:3n-3 fatty acids. A: No product is seen in FADS1-transfected cells. B: FADS2 transfected cells D6-desaturate 18:2n26R18:3n26 (9,12–18:2 R 6,9,12–18:2) and 18:3n–3R18:4n23 (9,12,15–18:3 R 6,9,12,15–18:4). doi:10.1371/journal.pone.0028186.g001 PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e28186