Formaldehyde and Cancer Pain Figure 4. Formaldehyde-induced increase in cytosolic [Ca2+]i in cultured DRG neurons and in TRPV1-CHO cells. (A) Formaldehyde- induced dose-dependent increase of cytosolic [Ca2+]i in DRG neurons. (B) Statistical analysis of formaldehyde-induced [Ca2+]i influx in DRG neurons. (C and D) Inhibition of capsazepin (CPZ, 100 mM) and melatonin (MT, 200 mM) on formaldehyde-induced [Ca2+]i influx in DRG neurons. (E and F) Inhibition of MT and CPZ on capsaicin (CAP, 10 mM)-induced Ca2+ influx in DRG neurons. (G) Formaldehyde-induced dose-dependent increase of cytosolic [Ca2+]i in TRPV1-CHO cells. (H) Statistical analysis of formaldehyde-induced Ca2+ influx in TRPV1-CHO cells. (I and J) Inhibition of MT and CPZ on formaldehyde-induced Ca2+ influx in TRPV1-CHO cells. ** p,0.01, compared with controls. n = 5,10. doi:10.1371/journal.pone.0010234.g004 paw in the MRMT-1 breast cancer pain model rats (Fig. 7, B and C); this result is similar to that observed in a previous report [43]. The MRMT-1 bone cancer pain model is widely used in breast cancer bone pain research [44]. TRPV1 antagonists attenuated endogenous formaldehyde-induced bone cancer pain behaviors (Fig. 7, B and C). The selective TRPV1 antagonists, such as iodo- resiniferatoxin [45] and capsazepine, and the non-selective antagonist ruthenium red [46], inhibited formalin-induced pain behaviors. These findings suggest that TRPV1 may participate in formaldehyde-evoked pain. In our behavior tests, we found that formaldehyde at low pathological (3 mM, based on concentration detected in human cancer tissues) in an acidic environment induced rat pain responses via TRPV1 in vivo (Fig. 3B). Capsazepine (a TRPV1 antagonist) attenuated capsaicin- or formaldehyde- (pH 6.0) induced pain responses in rats (Fig. 3, B–D). A recent study also showed that TRPV1 participates in nociception especially under extremely acidic conditions [16]. Recent researches have shown that both TRPA1 and TRPV1 are possible targets of endogenous formaldehyde in vitro and in vivo [9]. In the report of Macpherson et al, formaldehyde-evoked calcium responses in DRG neurons and nocifensive behaviors were almost abolished in TRPA12/2 mice. At the same time, formaldehyde could still evoke pain responses in the TRPA12/2 mice. This suggests that formaldehyde does not merely activate TRPA1. In our present study, formaldehyde (.0.1 mM) was found to activate TRPV1 (Fig. 1A), especially in the acidic environment. We think that TRPV1 or TRPA1 are all under the mechanisms of pain. AP-18 (a TRPA1 antagonist) partially decreased formaldehyde-induced pain (pH 5.0,6.0) and did not attenuated capsaicin-induced pain behaviors (Fig. 3B). This implies that under an acidic microenvironment of cancer tissues, TRPV1 may play a more critical role than TRPA1. Whether TRPA1 also participates in bone cancer pain is unknown, but will be investigated in our further research. Formaldehyde under acidic environment induces pain responses via TRPV1 With patch clamp recording, it was found that formaldehyde (.3 mM) activated TRPV1 directly in TRPV1-transfected CHO cells. While neither an acidic environment alone (pH 6.0), nor formaldehyde at low concentration alone (,3 mM) elicited currents, formaldehyde at the same low concentration under an acidic environment (pH 6.0) dose-dependently induced currents via TRPV1 (Fig. 5, A and B). In fact, formaldehyde (1,10 mM) did not elicit currents in ASIC1a-transfected CHO cells (data not shown). These data indicate that TRPV1 (and not ASIC1a) is the direct target of formaldehyde, especially formaldehyde in an acidic environment. Formaldehyde also enhanced capsaicin-induced currents in vitro (Fig. 5, C and D). Formaldehyde level was elevated to about 0.6 mM in the bone morrow of this model in vivo (Fig. 1E), and formaldehyde (1 mM) under an acidic environment (pH 5.0) elicited C-fiber discharges in vivo (Fig. 5, E and F). Formaldehyde-induced pain responses in rat were obviously enhanced under an acidic environment (pH 5.0) in vivo (Fig. 3, A and B). It has been reported that microenvironment of tumor tissues has pH values of 4,5 [12], and that pain behaviors could be induced at a pH as low as 5.0 through activation of ASICs and/ or TRPV1 [15]. These data suggests that accumulated formalde- hyde and acidic environment in tumor tissues synergistically induce pain responses by activating TRPV1 in afferent C-fiber of bone marrow or skin. Moreover, formaldehyde up-regulated NGF expression in mast cells in vitro, and NGF secreted by mast cells and macrophages could up-regulate TRPV1 [2]. This implies that formaldehyde secreted by tumor tissues possibly up-regulates TRPV1 expression. Interesting- ly, over-expression of TRPV1 has been found in bone morrow, DRG neurons and afferent C-fibers in bone cancer pain models [1,47]. Therefore, we hypothesize that the proliferating cancer cells secrete excessive endogenous formaldehyde in the initial stages, and then formaldehyde up-regulates TRPV1 expression in the afferent nerves. Consequently, over-expression of TRPV1 increases me- chanical sensitization by decreasing pain thresholds of patients with cancer. Then, as the tumor progresses, acceleration of acidification and chronic accumulation of formaldehyde lead to mechanical allodynia or severe pain via ASICs and/or TRPV1 in skin or bone marrow of the cancer patients (Fig. S2). Although, blockade of TRPV1 has been suggested as a possible therapeutic target to relieve pain [1], recent research has shown that the chronic blockade of this receptor may increase risk of cancer development [48]. In our study, we found that although capsazepine and melatonin all attenuated bone cancer pain responses, they did not decrease local formaldehyde levels in spinal cord and blood (Fig. S1, A and B). More importantly, formaldehyde can promote proliferation of cells [49], and it is a risk factor for cancer development [7]. This hints that formalde- hyde may be a critical factor of the glial over-proliferation in the spinal cord of this bone cancer pain model [44]. Interestingly, melatonin has been used clinically for breast cancer [50]. We found that it inhibited acute formaldehyde- and capsaicin-induced pain behaviors (Fig. 3, A and C), as shown previously [22,51–52]; and it blocked formaldehyde or capsaicin-elicited Ca2+ influx in DRG neurons and TRPV1-transfected CHO cells (Fig. 4). But, the melatonin receptor is not expressed in DRG neurons and CHO cells [53,54]. Melatonin may act by antagonizing TRPV1. Potential side effects of chronic blockade of TRPV1 require further investigation. Formaldehyde scavengers decrease pain responses by decreasing formaldehyde level In the classical formalin test, resveratrol (exogenous formalde- hyde scavenger) [55,56] and glutathione (endogenous formalde- hyde scavenger) [57–59] inhibited formalin-induced pain respons- es (Fig. 3, C and D). To test whether resveratrol and glutathione are formaldehyde scavengers, at the molecular level, we found that resveratrol and glutathione brought about chemical deactivation of formaldehyde in vitro (Fig. 6B). At the cellular level, they also inhibited formaldehyde-induced neurotoxicity (Fig. 6, C and D); at the tissues level, resveratrol and glutathione attenuated MRMT-1 bone cancer pain responses in rats by decreasing endogenous formaldehyde levels in the spinal cord in vivo (Fig. S1A). These data further confirm that they are formaldehyde scavengers. Resveratrol inhibits proliferation of cancer cell by scavenging PLoS ONE | www.plosone.org 8 April 2010 | Volume 5 | Issue 4 | e10234