THF-diols (9,12-oxy-10,13-dihydroxyoctadecanoic and 10,13-oxy-9,12-dihydroxyoctadecanoic acids) are endocrine disrupters in rats and mitogens in breast cancer cells. sites and blocked sexual behavior in male and female rats [4] at a very low dose (~0.30 mg/kg body weight/day). The second mitogenic HPLC peak of mitogenic activity was subsequently identified as 9,10-dihydroxy-12-octadecenoic acid (leukotoxin diol; LTX-diol), a well-known leukotoxin [5]. A synthetic mixture of LTX-diol, and its 12,13-dihydroxy-9-octadecenoic acid isomer (iso-leukotoxin diol; i-LTXdiol), were separated by HPLC. Both LTX-diol isomers blocked sexual behavior in female rats at a low dose (~0.80 mg/kg body weight/day). However, unlike the THF-diols, the LTX-diol isomers did not disrupt male sexual behavior in adult male rats. Recent studies show that THF-diols and LTX-diols act additively to disrupt endocrine function at concentrations (0.5 to 1 1.0 ppm) [6], far lower than those of classical phytoestrogens [7]. While the corncob mitogens are powerful endocrine disruptors, the mechanism through which these mitogens act has not been elucidated. Recently, our laboratory demonstrated that THF-diol stimulates phospholipase A2 (PLA2), lipoxygenases (LOX-5 and LOX-12) and cyclooxygenases (COX-1 and COX-2) in MCF-7 cells [8]. The products of these enzymes (prostaglandins, hydroxyeicosatetraenoic acids (HETEs) and hydroxyoctadecenioc acids (HODEs)) are well known regulators of cell growth [9-11]. The data presented in this manuscript suggests that the THF-diols may function by activating the production of nitric oxide. Materials and Methods MCF-7 Cell Growth Conditions Stock cultures of MCF-7 human breast cancer cells were grown in T-150 flasks as previously described [12]. For microarray studies, the cells were seeded into T-75 flasks containing 10 mls of phenol buy 59870-68-7 red free DMEM Acvrl1 media containing 5% charcoal-stripped fetal calf serum (FCS, Gibco, Carlsbad, CA, USA) and 1% penicillin-streptomycin and allowed to attach for 24 hours. After a media change, the cells grown for 24 hours in media containing 2 l of ethanol (controls) or 8 g/ml of THF-diol (synthesized in our lab as previously described [3]) in 2 l of ethanol. After incubation, 5 million cells from triplicate flasks for each treatment group were harvested for RNA isolation. Viable, attached cell numbers were monitored by hemocytometer counts based on trypan blue dye exclusion [13]. RNA and Protein Preparation Cells from EtOH controls or THF-diol-treated flasks were washed buy 59870-68-7 with PBS and collected with 0.25% trypsin-EDTA (4 mls). Following 5 minute incubation, the trypsin was inactivated by the addition of 10 mls of media containing 10% FCS. Approximately 5.0 106 cells from each flask were centrifuged (2000 rpm 5 minutes) in RNAse/DNAse free tubes, resuspended in 1 ml of PBS and 4 mls of RNAlater (Qiagen, Valencia, CA, USA) and stored at ?20oC. The frozen cells were thawed on ice, collected by centrifugation and lysed by resuspension in 0.6 mls of RTL buffer (Qiagen) containing -mercaptoethanol. The lysed cells were homogenized by centrifugation through a Qiashredder (18,000 g 2 minutes). The pass through from the Qiashredder containing nucleic acids was diluted with an equal amount of 70% EtOH and loaded onto a RNeasy spin column. The column was washed with RW1 followed by RNase-free DNase digestion to remove residual DNA and further washed with RPE buffers according to the manufacturers instructions. Purified total RNA was eluted with 50 l of RNAse-free water following buy 59870-68-7 5 minute RT incubation. RNA integrity was verified on an Agilent 2100 Bioanalyzer in the Baylor College of Medicine Microarray Core Laboratory. Microarray Analyses RNA from ethanol controls or THF-diol treated MCF-7 cells was subjected to oligo(deoxythymidine)-Reverse Transcription to generate cDNA, followed by transcription and biotin-labeling of to generate cRNA (Enzo Biochem, Farmingdale, NY, USA). The fragmented, biotin-labeled cRNA was hybridized to Human Genome U133 Plus 2.0 oligonucleotide arrays (Affymetrix, Santa Clara, CA, USA) containing approximately 54,000 probe sets (38,500 genes). Each transcript is represented on the chip as 11 probe pairs and each pair contained a perfect match and mismatch. Microarrays were stained with strepavidin antibody and streptavidin-phycoerythrin in an Affymetrix Fluidics station. Arrays were scanned at 3 M with a GeneArray scanner (Affymetrix). All experiments were performed in triplicate with independent pools of cRNA from EtOH controls and THF-diol-treated MCF-7 cells using six separate microarray chips. Following low-level quantification of the scanned data using GeneChip Operating System (GCOS, version 1.4, Affymetrix), data were further analyzed with dChip 2006 (Harvard University, Cambridge, MA, USA) to adjust the arrays to.