limited. Whether excessive endogenous formaldehyde induces cancer pain still remains to be determined. A recent report has indicated that formaldehyde (.0.013 mM) can elicit currents via transient receptor potential vanilloid receptor 1 (TRPV1) and this current could be blocked by the specific TRPV1 antagonist capsazepine in dorsal root ganglion (DRG) neurons [8]. Furthermore, formaldehyde (.0.02 mM) can induce Ca2+ influx via TRPV1 and transient receptor potential ankyrin 1 (TRPA1) in transfected-CHO cells. TRPA1 is more sensitive to formaldehyde than TRPV1 [9,10], however, TRPA1 is sensitive to an intracellular alkalization, not an acidic microenvi- ronment (pH,6.0) [11]. It is well known that tumor tissues typically exist in an acidic microenvironment in the range of pH 4,5 [12]. TRPV1 is a cation channel activated by capsaicin, noxious heat, low pH (pH,5.5) and endogenous vanilloids [13,14]. More importantly, TRPV1-mediated currents induced by capsaicin, endogenous vanilloids and ethanol can be enhanced by low pH [15]. TRPV1 participates in nociception especially under acidic conditions [16] and is considered to play an important role in cancer pain [17]. Clinical investigation found TRPV1 over-expression in patients with pancreatic carcinoma [18], bladder cancer [19] and breast cancer [20], and such an over-expression is positively correlated with the intensity of pain [18]. Therefore, in the present study, we postulated that excessive cancer tissue-derived endogenous formaldehyde induces bone cancer pain via TRPV1 especially under an acidic tumor microenvironment. Results Formaldehyde concentration increased in cultured cancer cell lines and tumor tissues from cancer patients We first investigated whether formaldehyde concentration was elevated in cultured tumor cell lines in vitro. Formaldehyde concentrations in rat breast cancer cell line MRMT-1 cells were significantly higher on day 2 than those of controls on the first day after cell density reached 105 cells/ml and 86105 cells/ml respectively, but decreased on day 3 when the cell density decreased (n = 6) (Fig. 1A). Formaldehyde concentration was also significantly increased in human lung cancer cell line H1299 cells and SY5Y cells (Fig. 1, B and C). Syngeneic Walker 256 mammary gland carcinoma cells were cultured by seeding into the abdominal cavity. A significant increase in the formaldehyde concentration was found in 0.5 ml ascitic fluid (26107 cells/ml) 6 d and 12 d after inoculation. Formaldehyde concentration was elevated two fold on day 6 after inoculation (from 0.04 mM to 0.08 mM) and decreased on day 12 when tumor cells grew into terminal phase. The highest concentration was 0.10 mM (Fig. 1D). Formaldehyde concentration was significantly elevated in bone marrows of MRMT-1 cancer pain model as compared with that in normal bone marrow (Fig. 1E). These results indicate that the formaldehyde concentration was elevated in all tested tumor cell lines. Pain visual analogue scores (VAS) were used in groups of patients with (n = 19) and without (n = 6) breast pain. Nineteen patients had significantly suffered from cancer pain. Only 1 patient reported thermal pain descriptors (burning, hot), while most reported ache and tenderness. VAS scores in these patients and controls were 3.860.3 and 0.360.2 respectively. Formaldehyde concentration was examined in tumor tissues from patients (Fig. 1F). In preparations from lung cancer patients, the average formaldehyde concentration was 0.7260.06 mM (n = 10) with the highest concentration 1.01 mM. This was significantly higher than that in the normal tissues adjacent to the cancer (0.1960.06 mM). Formaldehyde and Cancer Pain In breast cancer tissues from patients, the formaldehyde concentration was 0.7560.12 mM with the highest concentration 2.35 mM. Although the breast tumor adjacent tissues (as controls) were not gained, levels of formaldehyde in human tissues were approximately 0.1,0.2 mM as previously reported [21]. These levels are similar to the average level (0.1960.06 mM) found in human lung cancer adjacent tissues in the present experiment. Taken together, these data show that the tumor-derived formaldehyde concentration is elevated in cancer tissues, strongly suggesting that tumor tissues secrete formaldehyde. Formaldehyde scavengers and TRPV1 antagonists attenuated formalin-induced pain behaviors The formalin test (5% formalin, i.e. 1662 mM formaldehyde) is a classic pain model commonly used to evaluate analgesic medicines. We found that formaldehyde scavengers glutathione (GSH) and resveratrol (Res), and TRPV1 antagonists capsazepine (CPZ) and melatonin (MT) significantly decreased the number of flinchings in a dose-dependent manner in both acute and tonic phases (Fig. 2, A–D), similar to that in previous reports [1,22]. The solvent used for these regents, DMSO (final concentration ,10%) by itself did not show significant effect (Fig. 2A). Low concentration formaldehyde-induced pain behaviors via TRPV1 was enhanced by low pH Formalin 5% functioning as a chemical irritant can induce nociceptive behavioral responses (pain). This in turn raises an interesting question of whether formaldehyde at pathologically low concentrations (1,3 mM, based on the concentrations of formaldehyde detected in human tumor tissues) can induce pain responses, and whether TRPV1 or TRPA1 is involved in the pain responses. Intraplantar injection of formaldehyde (0.1 mM to 100 mM) to the right hind paw evoked dose-dependent, short- lasting (5 min) pain responses of the injected paw in normal rats. Capsazepine, melatonin and AP-18 (a TRPA1 antagonist) all attenuated the low concentration formaldehyde (5 mM)-induced pain responses (Fig. 3A). These results indicate that formaldehyde at low pathological concentration can induce pain behavioral responses, possibly through activation of TRPV1 and TRPA1. In addition, formaldehyde (3 mM) with a low pH of 5.0 or 6.0 (mimicking the acidic cancer microenvironment) induced more severe pain responses than formaldehyde in a neutral environment (pH 7.4). These responses were partially inhibited by AP-18, but almost completely inhibited by capsazepine (a TRPV1 antagonist) (Fig. 3B). Moreover, capsazepine and melatonin attenuated capsaicin-induced pain responses (Fig. 3C), but AP-18 did not (Fig. 3D). This result is similar to a previous report [23]. These data suggest that TRPV1, but not TRPA1, plays a key role in low concentration formaldehyde-induced pain behaviors under acidic environment. Formaldehyde induced Ca2+ influx in dorsal root ganglion (DRG) neurons and TRPV1-CHO cells in vitro We further used calcium imaging to test whether low concentration formaldehyde can directly excite DRG neurons via TRPV1. As expected, formaldehyde at concentrations of 1 mM to 100 mM induced a concentration-dependent increase of [Ca2+]i in freshly isolated rat DRG neurons (Fig. 4, A and B). TRPV1 antagonists capsazepine and melatonin inhibited the [Ca2+]i increase evoked by formaldehyde (Fig. 4, C and D) or by capsaicin (Fig. 4, E and F). To verify whether formaldehyde directly activates TRPV1, we next examined the effect of formaldehyde on TRPV1-transfected PLoS ONE | www.plosone.org 2 April 2010 | Volume 5 | Issue 4 | e10234