Phoneutria nigriventer spider toxin Tx2-6 induces priapism in mice even after cavernosal denervation

Accepted Manuscript Phoneutria nigriventer spider toxin Tx2-6 induces priapism in mice even after cavernosal denervation Katherine Garcia Ravelli, Adriana de Toledo Ramos, Luana Baracho Gonçalves, Fábio Carlos Magnoli, Lanfranco Ranieri Paolo Troncone PII:

S0041-0101(17)30072-7

DOI:

10.1016/j.toxicon.2017.02.026

Reference:

TOXCON 5582

To appear in:

Toxicon

Received Date: 12 December 2016 Revised Date:

27 January 2017

Accepted Date: 23 February 2017

Please cite this article as: Ravelli, K.G., Ramos, A.d.T., Gonçalves, L.B., Magnoli, F.C., Troncone, L.R.P., Phoneutria nigriventer spider toxin Tx2-6 induces priapism in mice even after cavernosal denervation, Toxicon (2017), doi: 10.1016/j.toxicon.2017.02.026. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Title: Phoneutria nigriventer spider toxin Tx2-6 induces priapism in mice even after cavernosal denervation.

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Katherine Garcia Ravelli (Ravelli, K. G.) Instituto Butantan – Lab. of Phamacology - São Paulo – Brasil – [email protected]

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Adriana de Toledo Ramos (Ramos, A. T.) Instituto Butantan – Lab. of Phamacology - São Paulo – Brasil [email protected]

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Luana Baracho Gonçalves (Goncalves, L. B.) Instituto Butantan – Lab. of Phamacology - São Paulo – Brasil [email protected] Fábio Carlos Magnoli (Magnoli, F. C.) Instituto Butantan – Lab. of Immunochemistry - São Paulo – Brasil [email protected] Lanfranco Ranieri Paolo Troncone (Troncone, L. R. P.)

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Authors:

Instituto Butantan – Lab. of Phamacology - São Paulo – Brasil [email protected]

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Corresponding author : Lanfranco R. P. Troncone

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e-mail:

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Address:

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[email protected] Instituto Butantan, Lab. Pharmacology

Av. Vital Brasil, 1500, São Paulo, SP, Brazil Postal Code: 05300-900

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Keywords: Phoneutria nigriventer, Tx2-6, spider toxin, priapism, penile erection, mouse, nitric oxide, in vivo, denervation, cavernous nerve, prostatectomy.

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The Phoneutria nigriventer spider toxin Tx2-6 causes priapism in humans and mice. This toxin produces a delay in Sodium channel inactivation, generalized vascular congestion and death by respiratory failure. NO-Synthase inhibitors seem to abolish toxin-induced priapism. The understanding of the ultimate molecular mechanism involved in toxin-induced priapism may shed light on aspects of erectile function/dysfunction. This study investigates if cavernosal denervation can abolish the toxin-induced priapism. Surgical cavernosal nerve excision/denervation was performed in mice and confirmed by infertility, histological assessment of fibrosis and immunohistochemical staining for synaptophysin. Denervated mice showed intense fibrosis of the cavernosal tissue as well as absence of synaptophysin IHC staining; surprisingly mice showed toxin-induced priapism when tested 15, 30 or 60 days after denervation. While sham-operated mice presented full priapism, denervated animals showed only partial priapism possibly due to the fibrosis. These results reveal that erection caused by Tx2-6 toxin may not depend on cavernosal nerves integrity. The effect of this toxin on sodium channels seem not directly involved in priapism as many toxins have identical effects but do not induce priapism. Discussion approaches the many different potential sites of intervention listed in the signaling cascades of NO/cGMP, RhoA/Rho-Kinase, as well as the emerging new gasotransmitter H2S. The pharmacological inhibition of Rho-kinase and toxin Tx2-6 have similar effects in vivo.

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The first reports of priapism in humans after Phoneutria nigriventer spider bites triggered investigations to identify the toxins and mechanisms involved in this effect. This had been previously reported in 1966 by Schenberg and PereiraLima (Schenberg and Lima, 1966), who described fasciculation, hypersalivation, tremors, piloerection and death after the animals were injected with crude venom. In human envenomation events, the main complaint is local pain, which demands local anesthetics and also priapism in children (Bucaretchi et al., 2000). In 1998 we reported for the first time that a peptide toxin isolated from this venom was responsible for the priapism observed (Troncone et al., 1998). This toxin was previously described to delay Sodium channel inactivation (Araujo et al., 1993; Rezende Junior et al., 1991) and was then called Tx2-6. An isoform toxin, called Tx2-5, differs by roughly 5 amino-acids over 48. Later, we reported that toxin-induced priapism was strongly dependent on NO, as LNAME and 7-NI (two nitric-oxide synthase inhibitors) prevented the toxic effects (Yonamine et al., 2004). Using micro-array techniques, we investigated the changes in gene expression in mouse penile tissue after priapism induced by toxin in vivo. Of the several genes that had their expression levels altered, endothelin B receptor was the most noteworthy with a 3.5-fold increase in expression. After signaling pathways analysis, we suggested the involvement of this signaling molecule in the toxin-induced priapism (Villanova et al., 2009). Given the multiple signs of intoxication that ultimately lead to death, an investigation on the possible cause of death was performed using a histopathological approach. We observed a remarkable amount of vascular congestion in many organs, particularly the lungs, heart and kidney, suggesting that the cause of death is related to respiratory collapse (Leite et al., 2012). In another study we showed that iodinated toxin can penetrate the blood-brain barrier (Yonamine et al., 2005). Therefore, a c-fos gene transcription brainmapping in mice was undertaken to determine whether a central effect could be related to the pro-erectile action of the toxin. Our results demonstrated that only stress-related brain structures were clearly activated and, in addition, intracerebral injections of tolerable doses of toxin were not capable of inducing priapism (Troncone et al., 2011).

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All together, these evidences argue in favor of a peripheral site of action related to the toxin-induced priapism. Following this rationale, in the present study we tested whether cavernosal nerve ablation can avoid priapism. Denervation was confirmed by infertility, synaptophysin IHC and Masson’s Trichrome histology for denervation-induced fibrosis of the cavernosal tissues. Surprisingly, our results showed that Tx2-6 continued to induced priapism even after total cavernosal nerve ablation. The molecular mechanisms involved in toxin-induced priapism may represent a new investigative avenue to establish novel treatments for erectile dysfunction after cavernosal nerve injury.

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Spider venom purification as described earlier (Troncone et al., 1995; Yonamine et al., 2004). Peptide identification was based on in vivo testing for priapism and mass spectrometry performed in an AB SCIEX Q-TOF triple-Quadrupole. The typical molecular mass of 5287 Da was characteristic of Tx2-6.

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2.2- Toxin

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We used Swiss male mice weighting 25-28g and male C57Bl6 mice. The animals were kept in our animal facility with light, temperature and ventilation all controlled and having water and standard pelleted food ad libitum. All procedures were previously submitted and approved by the Committee for Animal Use in Research of Instituto Butantan and found in accordance to the National rules (CEUAIB No. 453/08).

2.3- Surgical cavernosal denervation

Thirty-six male Swiss mice weighing 26-35g and 10 C57Bl6 mice weighing 2430g underwent either surgical bilateral cavernosal denervation, or were subjected to the identical procedure but nerves were left untouched (shamoperated). Surgery was carried out under Isoflurane anesthesia using a Matrx VIP3000 vaporizer at 2-3% in medicinal Oxygen. After a longitudinal incision on the skin and muscular layer, the prostate was recognized, allowing identification and isolation of both cavernosal nerves. A section of 1-2mm of each cavernosal nerve was excised. The muscular layer and skin were sutured with Vicryl 4-0 suture. One week later, every animal was paired with a female mouse for 30 days to check whether the male was able to mate, so to ascertain impotence.

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2.1- Animals

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2.- Methods

2.4- Masson’s trichrome histology Two months after cavernosal denervation (or sham-denervation) six mice of each group underwent euthanasia; penises were dissected and immersed in 10% formaldehyde overnight. They were then dehydrated, and submitted to paraffin embedding for sectioning in a microtome, 6µm thick, mounted in slides and submitted to standard Masson`s Trichrome staining to evidence fibrosis.

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2.5- IHC for Synaptophysin

Seven sham-operated controls and seven denervated Swiss mice were anesthetized as described above and transcardially perfused with PBS 0.1M, pH=7.4 followed by fixative solution of 4% paraformaldehyde in the same PBS. After perfusion, penises were dissected and kept in the fixative for 4 hours. Penises were then cut in a cryostat into 22µm longitudinal slices, mounted on slides and submitted to IHC procedure as follows: three 10 minute washes in 4

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PBS, overnight incubation at room temperature in phosphate buffer plus Triton X-100 0,3%, normal goat serum and monoclonal primary antibody antisynaptophysin diluted 1:1000. Then followed three 10 minute washes in PB, incubation for two hours with biotinylated secondary antibody diluted 1:200 in PB plus 3% Triton-X100 at room temperature. After that, three 10 minute washes in PB and incubation with avidin-peroxidase for 1.5 hours at room temperature. After completing 3 washes of 10 minutes each in PB, the complex location was revealed by diaminobenzidine in PB for 3 minutes, which was followed by solution of 0,3% H2O2. After chromogen reaction, slides were washed 3 times in PB, 10 minutes each, and allowed to dry on a heated plate (37oC) overnight. Slides were then immersed in water for 5 minutes and submerged in 0,05% OsO4 for 15 seconds for color intensification. Slides were then washed 3 times in PB, dehydrated and mounted with Permount.

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2.6- Toxin treatment and priapism evaluation

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One group of saline injected mice was handled in the same way in order to assess whether this manipulation could stimulate penile erection, however no sign of erection was observed. Groups of 5 to 7 denervated mice were tested after 15, 30 and 60 days of surgery. Groups of sham operated mice were used as controls in the same time schedule.

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The priapism-inducing toxin Tx2-6 was injected intra-peritonealy into mice at the dose of 0.6µg/kg, in saline. This dose was standardized in previous studies and was demonstrated to induce priapism with minimal deaths (Leite et al., 2012). Priapism was evaluated using a rating scale with four instances: One cross attributed when a darkening bluish color is observed at the base of the penis but no exposure is obtained after a mild pressure to the surrounding tissues, two crosses when a small exposure can be obtained, three crosses when a clear erection is observed and four crosses when the penis assumes a conical shape (shown below). From 40 to 120 minutes after toxin injection the animals were examined for priapism signs at 5 minute intervals, each time measuring the maximal degree of erection.

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Chi-square analysis was applied to the priapism data obtained after 60 days of surgery (Table I). Data was grouped in a table with three conditions: score below 3 crosses, three crosses, and four crosses. Immuno-histochemical data and Masson’s trichromic histochemistry data were considered of qualitative nature.

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3.- RESULTS

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3.1- Denervation, fibrosis assessment and synaptophysin IHC

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Synaptophysin IHC showed an almost complete absence of immune-reactivity for synaptophysin, as can be seen in Figure 1. It was visible that little remained of the nerve fibers after we scanned a great number of slices from seven different mice.

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Denervated cavernosal tissue showed intense blue staining in Masson`s Trichrome histology representing predominant collagen-rich structures, in clear contrast to the dominant red-colored tissue represented by smooth muscle of sham-operated mice, as can be seen in Figure 2. These tissues were obtained from mice after 2 months of the surgical procedure.

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3.2- Toxin-induced priapism after cavernosal denervation

The typical signs of Tx2-6 intoxication, piloerection, salivation and respiratory distress were present in control and denervated animals. As can be seen in Table 1, Tx2-6 toxin continued to produce priapism even after total cavernous denervation. Fifteen days after denervation four animals out of five, showed some degree of priapism, three of them showed complete erection and one of them failed. Three of the control mice failed to show priapism while the remaining two were positive. Thirty days after denervation, three out of the seven denervated animals showed incomplete priapism (while the other four failed), compared with the three out of five controls that showed incomplete priapism at that time. In addition to the groups of seven mice (sham and denervated) tested after 60 days of surgery, a group of five denervated and five sham-operated animals (injected with the toxin as described) was tested to confirm and document these results, but this time using C57Bl6 mice. All twelve animals tested 60 days after denervation showed partial (three crosses) priapism while ten of the sham-operated mice showed four crosses priapism, one three crosses and one failed to show priapism. Statistical analisys showed a significant difference up to p≤0,01 (DF=2 and the value of X2 obtained was 12.19). Illustrations of the complete priapism and the partial three-cross degree are presented in Figure 3, with control and denervated animals.

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4.- DISCUSSION

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The complete loss of physiological erection after bilateral ablation of the cavernosal nerves was confirmed by different parameters: denervated mice failed to mate and reproduce, synaptophysin immune staining was absent and cavernosal fibrosis was demonstrated histologically by the Masson’s trichromic histology, showing the extension of the Wallerian degeneration and its consequences.

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ACCEPTED MANUSCRIPT To our surprise, we observed the persistence of toxin-induced priapism after total cavernosal denervation. This unexpected result represents an important clue about the ultimate molecular mechanism of action of Tx2-6 related to priapism. The nerve ablation experiment was repeated using two mice lines, Swiss and C57Bl6, with identical results. The replication of this experiment with a different mouse line followed no specific rationale except to confirm the initial results. It is noteworthy that priapism observed after toxin treatment in all denervated mice was unequivocal but incomplete, i.e. the penises did not expand in a conical shape since the extensive fibrosis prevented the full expansion of erectile tissues. The effect of this toxin on sodium channels (Matavel et al., 2002; Rizzi et al., 2007) seem not directly involved in priapism as many toxins have similar effects but do not induce priapism (Rizzi et al., 2007). The same considerations have been raised by other authors in a recent review on arthropod toxins involved in priapism (Nunes et al., 2013).

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As penile erection is a complex neurovascular process involving both sympathetic, parasympathetic and superior CNS components, its physiology is far from being completely understood. The current literature proposes that penile erection involves at least two components: one initial nerve-triggered smooth muscle relaxation involving neuronal nitric oxide synthase, allowing for blood to fill the sinusoidal spaces, and a second phase dependent on vascular endothelial distension and endothelial NO production that sustains a persistent erection (Burnett, 2004). The role of endothelium in sustaining erection is extensively documented (Andersson, 2011; Hurt et al., 2002). The eNOS is a calcium-calmodulin dependent enzyme that can be activated by sheer-stress in endothelial cells lining the cavernosal sinusoids after the initial distension. Our results now demonstrate that the priapism observed in mice injected with Tx2-6 toxin is independent of nerve activity. Even then, NO seems to play an important role in toxin effects as L-NAME and 7-NI inhibitors blocked toxicity (Yonamine et al., 2004). Seems likely that toxin-induced priapism depends exclusively on eNOS. Interestingly, human cavernosal tissue retains endothelial function after radical prostatectomy and consequent cavernosal denervation (Martinez-Salamanca et al., 2015). It has been demonstrated that cavernosal nerve ablation in rats eliminates nNOS from penile tissues while eNOS is not affected (Podlasek et al., 2001).

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In a comprehensive review on the mechanisms governing penile erection, the roles of NO and several chemical mediators were approached (Andersson, 2011). According to this review, the relative contribution of nNOS and eNOS to the erectile process was not completely determined. As discussed by Mizusawa and col.(Mizusawa et al., 2001), the NADPH-Diaphorase-positive (NOS) nerve labelling in rat cavernosal tissue seems restricted to the penile arteries (Schirar et al., 1994; Vanhatalo et al., 1996) though not exclusively (Keast, 1992). However, according to his own results in mice, vesicular ACh transporter- and

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ACCEPTED MANUSCRIPT NOS-immunoreactivity overlap in cavernosal smooth muscle bundles, suggesting an important role for NOS in the mouse cavernous tissue. Yet, functional in vitro experiments further argue in favor of a smaller importance of endothelial NOS for erection in the rat compared to mouse tissue (Hedlund et al., 1999). Therefore, the role of endothelium-derived NO in mice resemble more that of humans (Hedlund et al., 1999; Mizusawa et al., 2001). In a different approach, eNOS appears up regulated in transgenic nNOS-/- mouse (Burnett et al., 1996). The existence of splice variants of nNOS (penile nNOS) further complicates the interpretation of experiments with transgenic animals lacking nNOS and/or eNOS (Andersson, 2011; Hurt et al., 2006).

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The role of penile smooth muscle cholinergic innervation should also be considered. Penile tissue is rich in cholinergic terminals (Hedlund et al., 1999), and acetylcholine stimulates muscarinic receptors on cavernosal smooth muscle (M2-type) and endothelial cells (M3-type) (Traish et al., 1995). However, more recently the existence of nicotinic receptors in endothelial cells have been described in a number of different blood vessels (Li and Wang, 2006), but no specific search for these receptors have been undertaken in cavernosal tissue, leaving open the possibility of having a nicotinic effect on penile functions in addition to the reported muscarinic. In this sense, apparently, α4β2, α3β4 and α7 nicotinic receptors are possibly involved in cavernosal smooth muscle relaxation through NO release (Ozturk Fincan et al., 2010). Endothelial cell nicotinic α7 receptors have been implicated also in angiogenesis (Cooke and Ghebremariam, 2008; Heeschen et al., 2002; Li and Wang, 2006) and, notably, endothelial cells have the complete machinery to handle acetylcholine (Cooke and Ghebremariam, 2008). The α7 subtype of nicotinic receptors is of particular interest as it has a large conductance to calcium (for a review (Dajas-Bailador and Wonnacott, 2004)). Upon stimulation, α7 receptors could lead to a rise in intracellular (endothelial) calcium levels, triggering the calmodulin-eNOS complex, so inducing NO synthesis and release, which would potentially interfere with penile erection. As denervation does not eliminate eNOS in cavernosal tissue (Martinez-Salamanca et al., 2015), this enzyme could be involved in the persistence of priapism under Tx2-6 intoxication as a denervation-induced compensatory up-regulation of cholinergic receptors could take place. On a different signaling cascade, at least in nerve cells, α7 nicotinic receptors coupled to heterotrimeric G-protein regulates RhoA-GTPase and neurite growth (King and Kabbani, 2016), which rises the interest to investigate whether α7 receptor activation in cavernosal smooth muscle cells could be involved in the erectile process. The RhoA/Rho-kinase cascade is well documented in penile smooth muscle and endothelial cells (Chitaley et al., 2001; Jin and Burnett, 2006; Sopko et al., 2014). According to this molecular cascade the RhoA/Rho-kinase complex is basically active in penile smooth muscle and responsible for the contracted/flaccid state of the penis. By the interactions of different signaling pathways involving GPCR, RhoA activates

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The closest effect we could find using a pharmacological tool was the in vivo effect of RhoA/Rho-Kinase inhibitor Y-27632 (Chitaley et al., 2001). Would Tx26 share a similar effect? In fact, Uvin and coworkers have recently published results on human cavernosal tissue supporting that Y-27632 and PDE5 inhibitors have additive effects on relaxation and possibly, on erection (Uvin et al., 2017). Further strengthening this hypothesis, Lasker and colleagues have tested another RhoK inhibitor, azaindole-1 injected intra-cavernosaly on cavernosal pressure in the rat and observed a long lasting increase in cavernosal pressure, resembling priapism (Lasker et al., 2013).

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Finally, a recent line of investigation has demonstrated that hydrogen sulfide (H2S), a gaseous transmitter just like NO, may have a role in endotheliumdependent vasorelaxation, as well as angiogenesis (Beltowski and JamrozWisniewska, 2014; Coletta et al., 2012). H2S and NO may also have a compensatory mechanism in play when one of them is lacking (Yetik-Anacak et al., 2016). Further investigation in this field may lead to new explanations for a number of medical issues involving erectile function and dysfunction, as well as a possible involvement in the effects of the toxin Tx2-6.

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To the best of our knowledge Tx2-6 spider toxin is the only substance capable of inducing priapism in normal (and now denervated) mice and may constitute a model for the study of this medical emergency. Other priapism models involve transgenic mutations, such as the incorporation of sickle cell hemoglobin (Paszty, 1997), expression of opiorphin (Tong et al., 2008), or the deletion of adenosine deaminase (Mi et al., 2008). There is a model employing mechanical constriction of the penis after vacuum-induced engorgement in order to retain blood, however this does not constitute a real world pathological condition but rather mimics ischemic priapism (Sanli et al., 2004).

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In conclusion, Tx2-6 may cause priapism by positively interfering in smooth muscle-relaxing steps - NO/cGMP accumulation, H2S synthesis/accumulation, cytoplasmic calcium decrease - or negatively interfering in smooth musclecontracting steps - receptor-triggered IP3 accumulation, RhoA/Rho-Kinase activation. Further investigations will offer a definitive answer to this question.

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ACCEPTED MANUSCRIPT The authors thankfully acknowledge the research grants 94/1214-6 and 02/04545-7 to LRPT and Post-Doctoral fellowship 15/08098-5 to ATR, all from FAPESP. CNPq Masters degree fellowship to KGR. FUNDAP Professional improvement fellowship to LBG. We also thank Mr. Wilson de B. D’Avila for technical assistance.

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Figure 1 – Immunohistochemical staining for Synaptophysin: Left panel shows denervated cavernosal tissue with remains of nerve fibers two months after denervation. Right panel shows control tissue with abundant nerve fibers (black arrows). Objective 40x.

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Figure 2 – Masson`s Trichromic staining of cavernosal tissue from 12 mice. Denervated tissue represented on the left side and sham-operated mice tissue on the right. Notice the abundant blue collagen staining of denervated mice representing fibrosis. Samples taken two months after denervation or shamsurgery. Objective 10x.

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Figure 3 - Typical Tx2-6 toxin induced priapism in control sham-operated mouse; notice the conical shape of the penis representing a complete erection (right). Toxin induced priapism in cavernous nerve-ablated mouse; notice the evident erection with limited expansion of the penis, given to fibrotic atrophy (left). Toxin injected intraperitoneally.

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Table I – Tx2-6 toxin-induced priapism as observed in different groups challenged at days 15, 30 and 60 after denervation or control sham operation. Crosses attributed according to ratings described in text. Groups composed of 5

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Andersson, K.E., 2011. Mechanisms of penile erection and basis for pharmacological treatment of erectile dysfunction. Pharmacol. Rev. 63, 811859. Araujo, D.A., Cordeiro, M.N., Diniz, C.R., Beirao, P.S., 1993. Effects of a toxic fraction, PhTx2, from the spider Phoneutria nigriventer on the sodium current. N-S. Arch. Pharmacol 347, 205-208. Beltowski, J., Jamroz-Wisniewska, A., 2014. Hydrogen sulfide and endotheliumdependent vasorelaxation. Molecules 19, 21183-21199. Bucaretchi, F., Deus Reinaldo, C.R., Hyslop, S., Madureira, P.R., De Capitani, E.M., Vieira, R.J., 2000. A clinico-epidemiological study of bites by spiders of the genus Phoneutria. Rev.Inst.Med.Trop.Sao Paulo 42, 17-21. Burnett, A.L., 2004. Novel nitric oxide signaling mechanisms regulate the erectile response. Int.J.Impot.Res. 16 Suppl 1, S15-S19. Burnett, A.L., Nelson, R.J., Calvin, D.C., Liu, J.X., Demas, G.E., Klein, S.L., Kriegsfeld, L.J., Dawson, V.L., Dawson, T.M., Snyder, S.H., 1996. Nitric oxidedependent penile erection in mice lacking neuronal nitric oxide synthase. Mol. Med. 2, 288-296. Chitaley, K., Wingard, C.J., Clinton Webb, R., Branam, H., Stopper, V.S., Lewis, R.W., Mills, T.M., 2001. Antagonism of Rho-kinase stimulates rat penile erection via a nitric oxide-independent pathway. Nat. Med. 7, 119-122. Coletta, C., Papapetropoulos, A., Erdelyi, K., Olah, G., Modis, K., Panopoulos, P., Asimakopoulou, A., Gero, D., Sharina, I., Martin, E., Szabo, C., 2012. Hydrogen sulfide and nitric oxide are mutually dependent in the regulation of angiogenesis and endothelium-dependent vasorelaxation. Proc. Natl. Acad. Sci. U. S. A. 109, 9161-9166. Cooke, J.P., Ghebremariam, Y.T., 2008. Endothelial nicotinic acetylcholine receptors and angiogenesis. Trends Cardiovasc. Med. 18, 247-253. Dajas-Bailador, F., Wonnacott, S., 2004. Nicotinic acetylcholine receptors and the regulation of neuronal signalling. Trends Pharmacol.Sci. 25, 317-324. Hedlund, P., Alm, P., Andersson, K.E., 1999. NO synthase in cholinergic nerves and NO-induced relaxation in the rat isolated corpus cavernosum. Br. J. Pharmacol. 127, 349-360. Heeschen, C., Weis, M., Aicher, A., Dimmeler, S., Cooke, J.P., 2002. A novel angiogenic pathway mediated by non-neuronal nicotinic acetylcholine receptors. J. Clin. Invest. 110, 527-536. Hurt, K.J., Musicki, B., Palese, M.A., Crone, J.K., Becker, R.E., Moriarity, J.L., Snyder, S.H., Burnett, A.L., 2002. Akt-dependent phosphorylation of endothelial nitric-oxide synthase mediates penile erection. Proc. Natl. Acad. Sci. U. S. A. 99, 4061-4066. Hurt, K.J., Sezen, S.F., Champion, H.C., Crone, J.K., Palese, M.A., Huang, P.L., Sawa, A., Luo, X., Musicki, B., Snyder, S.H., Burnett, A.L., 2006. Alternatively spliced neuronal nitric oxide synthase mediates penile erection. Proc.Natl.Acad.Sci.U.S.A 103, 3440-3443. Jin, L., Burnett, A.L., 2006. RhoA/Rho-kinase in erectile tissue: mechanisms of disease and therapeutic insights. Clin Sci (Lond) 110, 153-165. Keast, J.R., 1992. A possible neural source of nitric oxide in the rat penis. Neurosci. Lett. 143, 69-73.

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mice: a study using radiotracer technique. J. Venom. Anim. Tox. incl. Trop. Dis. 11, 39-50. Yonamine, C.M., Troncone, L.R., Camillo, M.A., 2004. Blockade of neuronal nitric oxide synthase abolishes the toxic effects of Tx2-5, a lethal Phoneutria nigriventer spider toxin. Toxicon : official journal of the International Society on Toxinology 44, 169-172.

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15 days

30 days

60 days Denervated

Saline

Denervated

Sham

Denervated

Sham

*

Sham

1 2 3 4 5 6 7 8(C57) 9(C57) 10(C57) 11(C57) 12(C57)

0 0 0 0 0

0 ++++ ++++ ++ ++++

0 ++++ +++ 0 0

+++ +++ +++ 0 0 0 0

+++ +++ ++++ 0 0

+++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++

++++ ++++ ++++ ++++ ++++ +++ 0 ++++ ++++ ++++ ++++ ++++

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Figure 1

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Figure 2

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ACCEPTED MANUSCRIPT Highlights: Phoneutria nigriventer spider toxin Tx2-6 causes priapism; Toxin effect are reverted by NOS inhibitors; Penile erection depends on cavernosal nerve integrity; Tx2-6 effects were evaluated after cavernosal nerve ablation; Tx2-6 induced priapism even after cavernosal nerve ablation;

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INSTITUTO BUTANTAN

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LABORATÓRIO DE FARMACOLOGIA AV. VITAL BRASIL, 1500 CEP 05503-900 - SÃO PAULO – SP

São Paulo, December 09, 2016.

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Ethical Statement

We, the authors of the manuscript entitled: “Phoneutria nigriventer spider toxin

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Tx2-6 induces priapism in mice even after cavernosal denervation” declare that this study was not published and is not currently under consideration by other journals.

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The corresponding author, on behalf of all the authors,

Dr. Lanfranco R. P. Troncone

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Lab. Pharmacology – Instituto Butantan Av. Vital Brasil 1500 São Paulo – SP - BRAZIL