Formaldehyde and Cancer Pain Figure 1. Formaldehyde concentration in the cultured cancer cell lines in vitro and in vivo. (A) MRMT-1 cancer cells. (B) Human H1299 lung cancer cells. (C) Human SY5Y cancer cells. (D) Ascites from peritoneal inoculation of Walker 256 mammary gland carcinoma cells. (E) Bone morrow of MRMT-1 breast cancer pain model rat in vivo. (F) Tumor tissues from lung and beast cancer patients. * p,0.05, ** p,0.01, compared with that of the first day. doi:10.1371/journal.pone.0010234.g001 CHO (TRPV1-CHO) cells. As expected, formaldehyde (.0.1 mM) induced an increase of cytosolic [Ca2+]i in a concentration- dependent manner (Fig. 4, G and H). As a control, in the untransfected CHO cells, formaldehyde at 100 mM elicited only slight Ca2+ influx (data not shown). Formaldehyde induced Ca2+ influx in the TRPV1-CHO cells was significantly inhibited by the TRPV1 antagonist capsazepine and melatonin (Fig. 4, I and J). Formaldehyde and pH 6.0 synergistically elicited currents in TRPV1-CHO cells in vitro Since nociceptive behaviors (pain) induced by formaldehyde at pH 6.0 were sensitive to the TRPV1 antagonist, formaldehyde at pH 6.0 may act directly on TRPV1 (Fig. 3B). We recorded the TRPV1 current induced by capsaicin and formaldehyde (with or without pH 6.0) using patch clamp recording in TRPV1-CHO cells. Capsaicin at 10 mM induced an inward current with voltage clamped at 260 mV. Capsazepine, a TRPV1 antagonist, strongly suppressed the capsaicin-induced current. Similarly, formaldehyde at 3 mM (concentration detected in human tumor tissues) induced an inward current in TRPV1-CHO cells in a concentration- dependent manner and 10 mM capsazepine blocked the formal- dehyde-induced current (Fig. 5A). As controls, neither 3 mM formaldehyde, nor 10 mM capsaicin, nor formaldehyde plus capsaicin induced any current in the untransfected CHO cells (data not shown). Although low pH of 6.0 in extracellular solution had little effect on TRPV1-CHO cells, currents induced by formaldehyde at 1,10 mM were significantly potentiated by pH 6.0 (Fig. 5, A and B). This result indicates that there is a synergistic effect between formaldehyde and an acidic environment. As a positive control, formaldehyde at 1 and 3 mM also markedly potentiated capsaicin (1 mM)-induced current in the TRPV1-CHO cells (Fig. 5, C and D). These data suggest that formaldehyde directly activates TRPV1 with more efficiency at low pH. Formaldehyde and pH 5.0 synergistically elicited C-fiber discharges via TRPV1 in vivo Of greater importance, we tried to determine whether formal- dehyde within the concentration range detected in the cancer tissues from patients is functional in exciting peripheral nociceptive nerve fibers. Peripheral C-fibers transduce nociceptive information to conduct nociceptive information. An increase in nociceptive C-fiber firings is a common characteristic of pain. Since formaldehyde at pH 5.0 (mimicking an extremely acidic tumor microenvironment) induced more severe pain behaviors than pH 5.0 alone (Fig. 3B), we tested whether formaldehyde can excite C-fibers. It was found that formaldehyde (1 mM, similar to the levels detected in human lung cancer tissues) at pH 5.0 can directly excite C-fibers. The number of action potentials increased significantly after formaldehyde (under pH 5.0) injection into the receptive field of the C-fibers. This C-fiber excitation could be blocked by the TRPV1 antagonist capsazepine (CPZ) (Fig. 5, E and F). Formaldehyde scavengers inhibited formaldehyde- induced neurotoxicity in cultured DRG neurons We further tested the neurotoxicity of formaldehyde within the above-measured concentration range (.0.1 mM) in the cultured DRG neurons. It was shown that formaldehyde was neurotoxic to DRG neurons in a dose-dependent manner (Fig. 6A). Resveratrol, an exogenous formaldehyde scavenger, and glutathione, an endogenous formaldehyde scavenger, showed spontaneous chem- ical interaction with formaldehyde in PBS solution within 40 minutes (Fig. 6B). These formaldehyde scavengers decreased formaldehyde-induced neurotoxicity with a concentration-depen- dent manner (Fig. 6, C and D). Formaldehyde scavengers and TRPV1 antagonists attenuated bone cancer pain behaviors in rats As shown in Fig. 7A, X-ray revealed that no radiological change (score = 0) was found in animals treated with heat-killed tumor cells or with PBS solution. However, 7 days after injection with MRMT-1 cells, the bone showed some loss of medullary bone and apparent erosion of the cortical bone. Further deterioration was detected on day 15 post-injection with additional full thickness unicortical bone loss. Formaldehyde scavengers (glutathione and resveratrol) and melatonin all significantly decreased bone destruction; however, capsazepine, a TRPV1 antagonist, did not protect bone structure from erosion on day 15 (Fig. 7, A and B). Pain behaviors including thermal hyperalgesia and mechanical allodynia were observed from 7 to 15 days after injection of MRMT-1 cancer cells. It was also found that the pain behaviors were attenuated by capsazepine, melatonin and formaldehyde scavengers (glutathione and resveratrol) on days 11 and 15 after MRMT-1 inoculation (Fig. 7, C and D). It was of interest to note that even though capsazepine and melatonin both attenuated bone cancer pain behaviors, the formaldehyde concentration in the spinal cord and blood of the cancer pain model rats on days 15 was still kept higher than that of the control rats. More importantly, resveratrol and glutathione (formaldehyde scavengers) inhibited bone cancer pain behaviors by decreasing excessive formaldehyde in the spinal cord (Fig. S1, A and B). Discussion Tumor tissues directly secrete endogenous formaldehyde The physiological formaldehyde level was reported to be approximately 0.1 mM in the blood or brain of human and non-human animal [21]. Surprisingly, clinical data showed that formaldehyde concentrations were significantly elevated (2,8 fold) in urine from patients with bladder cancer and prostate cancer [3], in the expiration of some patients suffering from breast cancer [4] and especially high in blood samples (8,10 folds) from certain patients with tumor [24]. Formaldehyde was also elevated in lymphocytes in chronic lymphocytic leukemia [7]. The expression and activity of formaldehyde generating enzymes, such as lysine- specific demethylase 1 (LSD1) [25,26], semicarbazide-sensitive amine oxidase (SSAO) [27,28] and cytochrome P-450 [29,30], formaldehyde degrading enzymes, such as aldehyde dehydroge- nase 2 (ALDH2) and class III alcohol dehydrogenase (ADH3) [31,32], are considered to have critical roles in the pathogenesis of breast cancer. Over-expression of ADH3 has been found in cancer tissues, which defenses formaldehyde [33]. This implies that tumor tissues can tolerate formaldehyde at abnormal levels. The present PLoS ONE | www.plosone.org 4 April 2010 | Volume 5 | Issue 4 | e10234