HPLC Instrument (Hewlett-Packard, USA) with a fluorodetector was used. The method was as described in our previous report [72]. Cytosolic Ca2+ concentration in TRPV1-transfected CHO cells and DRG neurons For transfection of CHO cells with TRPV1 plasmids, CHO cells were harvested after brief trypsin digestion and seeded onto confocal plate chambered cover-glasses (LabTek, Nunc) precoated with 20 mg/ml poly-L-lysine. Actively growing cells were trans- fected with a pcDNA3.1-TRPV1 plasmid by lipofectamine plus (Life Technologies, Rockville, MD, USA). Control cells were transfected with pcDNA3.1 plasmid only. After 16,24 h culture, cells were challenged with formaldehyde or capsaicin. DRG neurons were freshly isolated with trypsin and collegenase digestion as in our previous report [73]. TRPV1-CHO cells and DRG neurons were loaded with 50 mM Fluo-3/AM (Molecular Probes) and incubated for 30 min at 37uC. Fluo-3/AM was excited at 488 nm, and its emitted fluorescence was collected at 515 nm. The control bath solution (pH 7.4) was used as described previously [69]. Changes in cytosolic [Ca2+]i concentration in the TRPV1-CHO cells and in the DRG neurons were measured with a confocal laser scanning microscope (Leica Company Ltd., Germany). Whole cell patch-clamp recording of TRPV1 current in vitro Recordings in TRPV1-CHO cells were made with standard whole-cell patch-clamp method as described in our previous report [73]. Experiments were carried out at room temperature (22,24uC). The extracellular solution consisted of 130 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 30 mM glucose and 25 mM HEPES-NaOH and the pH was adjusted to 7.3. Patch pipettes (resistance 2,5 MV) were filled with 140 mM CsCl, 4 mM MgCl2, 10 mM EGTA and 10 mM HEPES-CsOH (pH 7.3). Reagents were applied with an automated perfusion device. Extracellular electrophysiological recording of C-fiber firing in vivo Male Sprague-Dawley rats weighing 300,350 g were anesthe- tized with urethane (1.5 g/kg, i.p.). Animal preparation and the teased fiber recording method were carried out as previously reported [73]. Briefly, the L5 dorsal root was exposed by lumbar laminectomy and covered with warmed (36uC) paraffin oil. Fine axon bundles (microfilaments), cut centrally but in continuity with the DRG distally, were separated from the dorsal root near its point of entry into the spinal cord. C-fiber was recorded for 2 min to detect its receptive field and to confirm its conduction velocity (less than 2.0 m/s). C-fiber firing within the following 5 min was References 1. Ghilardi JR, Ro¨hrich H, Lindsay TH, Sevcik MA, Schwei MJ, et al. (2005) Selective blockade of the capsaicin receptor TRPV1 attenuates bone cancer pain. J Neurosci 25: 3126–3131. 2. Mantyh PW, Clohisy DR, Koltzenburg M, Hunt SP (2002) Molecular mechanisms of cancer pain. Nat Rev Cancer 2: 201–209. 3. Spanel P, Smith D, Holl TA, Al Singary W, Elder JB (1999) Analysis of formaldehyde in the headspace of urine from bladder and prostate cancer patients using selected ion flow tube mass spectrometry. Rapid Commun Mass Spectrom 13: 1354–1359. 4. Ebeler SE, Clifford AJ, Shibamoto T (1997) Quantitative analysis by gas chromatography of volatile carbonyl compounds in expired air from mice and human. J Chromatogr B Biomed Sci Appl 702: 211–215. 5. Ghilardi JR, Ro¨hrich H, Lindsay TH, Sevcik MA, Schwei MJ, et al. (2005) Selective blockade of the capsaicin receptor TRPV1 attenuates bone cancer pain. J Neurosci 25: 3126–3131. 6. Sabino MA, Mantyh PW (2005) Pathophysiology of bone cancer pain. J Support Oncol 3: 15–24. Formaldehyde and Cancer Pain taken as the basal firing. After injection of 1 mM formaldehyde with pH 5.0 (mimicking an extremely acidic tumor microenvi- ronment) or vehicle only (normal saline, pH 5.0) into the receptive field of a C-fiber, firing was recorded for another 5 min. The frequency of the C-fiber firing before and after formaldehyde injection was compared. Data analysis Data are expressed as mean 6 S.E.M. Data from in vitro experiments were analyzed using the Student’s t test for comparison of independent means. In vivo data were analyzed by analysis of variance (ANOVA) followed by Dunnett’s post hoc test repeated measures of ANOVA, and post hoc tests including Student’s t test (ex vivo test). p values less than 0.05 were considered statistically significant. Supporting Information Figure S1 Formaldehyde concentration in bone cancer pain model rats in (A) spinal cord and (B) blood. In MRMT-1 pain model of rats, formaldehyde (FA) concentration increased. TRPV1 antagonists capsazepine (CPZ 0.1 mg/ml) and melatonin (MT, 5 mg/ml) had no obvious influence on formaldehyde concentra- tion, but formaldehyde scavengers resveratrol (Res, 0.4 mg/ml) and glutathione (GSH, 25 mg/ml) decreased FA concentration. * p,0.05, ** p,0.01; # p,0.05, ## p,0.01, all compared with PBS groups. n = 10. Found at: doi:10.1371/journal.pone.0010234.s001 (0.33 MB TIF) Figure S2 A putative scheme that excessive formaldehyde secreted by tumor tissues and its induction on bone cancer pain under an acidic microenvironment. Found at: doi:10.1371/journal.pone.0010234.s002 (4.24 MB TIF) Acknowledgments We thank Dr. David Julius in University of California at San Francisco for the TRPV1 plasmid; Novartis Oncology Research, Basel for MRMT-1 rat mammary gland carcinoma cells; Dr. Fenglin Han (Peking University Third Hospital) for evaluation of X-rays; Xinhong Wang in our institute for technical support. The authors would like to thank Dr. Sonya G. Lehto in CA, USA for her kind help in the preparation of this manuscript. Author Contributions Conceived and designed the experiments: ZT YW. Performed the experiments: ZT WL YW FY YH HL HL BD QM. Analyzed the data: ZT TLX JW HZ. Contributed reagents/materials/analysis tools: HT JW HZ. Wrote the paper: ZT. 7. Thorndike J, Beck WS (1977) Production of formaldehyde from N5- methyltetrahydrofolate by normal and leukemic leukocytes. Cancer Res 37: 1125–1132. 8. Tian LJ, Du YR, Xiao Y, Lv ZM, Yu YQ, et al. (2009) Mediating roles of the vanilloid receptor TRPV1 in activation of rat primary afferent nociceptive neurons by formaldehyde. Sheng Li Xue Bao 61: 404–416. 9. Macpherson LJ, Xiao B, Kwan KY, Petrus MJ, Dubin AE, et al. (2007) An ion channel essential for sensing chemical damage. J Neurosci 27: 11412–11415. 10. McNamara CR, Mandel-Brehm J, Bautista DM, Siemens J, Deranian KL, et al. (2007) TRPA1 mediates formalin-induced pain. Proc Natl Acad Sci 104: 13525–13530. 11. Fujita F, Uchida K, Moriyama T, Shima A, Shibasaki K, et al. (2008) Intracellular alkalization causes pain sensation through activation of TRPA1 in mice. J Clin Invest 118: 4049–4057. 12. Delaisse JM, Vaes G (1992) Mechanism of mineral solubilization and matrix degradation in osteoclastic bone resorption. In: Rifkin BR, Gay CV, eds. Biology and Physiology of the Osteoclast. CRC Press, Boca Raton. pp 289–314. PLoS ONE | www.plosone.org 13 April 2010 | Volume 5 | Issue 4 | e10234