Effects of Unsaturated Fatty Acid Esters of Testosterone on Neuronal, Behavioral and Hormonal Parameters in Male Rats Subjected to the Formalin Test


Chronic diseases are often accompanied by inflammatory and degenerative processes. Estrogens have repeatedly been found to be involved in these processes. Testosterone (T) is the main precursor of estrogen in the brain and T replacement in chronic diseases has become important in recent years, prompting research on new T-conjugated molecules. We recently synthesized three new molecules including unsaturated fatty acid esters: T-linoleate (TL), T-oleate (TO) and T-eicosapentanoate (TEPA). These substances were s.c. administered for 7 days to intact male rats subjected to the formalin test (FT). Three other groups were included as comparisons: NAIVE, receiving no substance, OIL, treated with almond oil (vehicle), and TN, treated with T-undecanoate, a saturated fatty acid. Spontaneous behaviors and pain-induced responses were determined during the FT, hormones (T and dihydrotestosterone, DHT) were determined in blood, while estrogen receptors (ERα and β) were detected at the genomic and proteomic levels in the hippocampus, hypothalamus and spinal cord. In the hippocampus, ERα and ERβ mRNA levels were increased respectively by TN and TL treatments with respect to OIL, whereas the hypothalamus TO and TL caused a decrease of ERα mRNA levels. At the proteomic level, TO, TL and TEPA decreased the levels of ERα in the hypothalamus, whereas TEPA decreased ERβ in the spinal cord, hippocampus and hypothalamus. There was no effect of treatment on the spontaneous behaviors, while the TO and TL groups showed lower pain-induced behaviors (paw jerk frequency and licking duration) than the OIL group. TN increased paw jerk frequency and decreased licking duration with respect to OIL. The treatments had no effect on T and DHT plasma levels. These results clearly indicate the possibility of pain and ER modulation by T-esters.

Share and Cite:

Petroni, A. , Fiorenzani, P. , Tomei, V. , Garofalo, A. , Aiello, F. , Seta, D. , Giordano, A. , Vodo, S. , Ceccarelli, I. and Aloisi, A. (2014) Effects of Unsaturated Fatty Acid Esters of Testosterone on Neuronal, Behavioral and Hormonal Parameters in Male Rats Subjected to the Formalin Test. Open Journal of Endocrine and Metabolic Diseases, 4, 167-179. doi: 10.4236/ojemd.2014.46017.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Paul, S.M. and Purdy, R.H. (1992) Neuroactive Steroids. The FASEB Journal, 6, 2311-2322.
[2] Rupprecht, R. and Holsboer, F. (1999) Neuroactive Steroids: Mechanisms of Action and Neuropsychopharmacological Perspectives. Trends in Neurosciences, 22, 410-416.
[3] Rupprecht, R., di Michele, F., Hermann, B., Strohle, A., Lancel, M., Romeo, E., et al. (2001) Neuroactive Steroids: Molecular Mechanisms of Action and Implications for Neuropsychopharmacology. Brain Research Reviews, 37, 59-67. http://dx.doi.org/10.1016/S0165-0173(01)00123-0
[4] Fillingim, R.B. and Ness, T.J. (2000) Sex-Related Hormonal Influences on Pain and Analgesic Responses. Neuroscience & Biobehavioral Reviews, 24, 485-501. http://dx.doi.org/10.1016/S0149-7634(00)00017-8
[5] Negri-Cesi, P., Colciago, A., Celotti, F. and Motta, M. (2004) Sexual Differentiation of the Brain: Role of Testosterone and Its Active Metabolites. Journal of Endocrinological Investigation, 27, 120-127.
[6] Smith, S.S. and Woolley, C.S. (2004) Cellular and Molecular Effects of Steroid Hormones on CNS Excitability. Cleveland Clinic Journal of Medicine, 71, S4-10.
[7] Aloisi, A.M. and Bonifazi, M. (2006) Sex Hormones, Central Nervous System and Pain. Hormones and Behavior, 50, 1-7. http://dx.doi.org/10.1016/j.yhbeh.2005.12.002
[8] Vodo, S., Bechi, N., Petroni, A., Muscoli, C. and Aloisi, A.M. (2013) Testosterone-Induced Effects on Lipids and Inflammation. Mediators of Inflammation, 2013, Article ID: 183041.
[9] Mhyre, A.J. and Dorsa, D.M. (2006) Estrogen Activates Rapid Signaling in the Brain: Role of Estrogen Receptor Alpha and Estrogen Receptor Beta in Neurons and Glia. Neuroscience, 138, 851-858.
[10] Pike, C.J., Nguyen, T.V., Ramsden, M., Yao, M., Murphy, M.P. and Rosario, E.R. (2008) Androgen Cell Signaling Pathways Involved in Neuroprotective Actions. Hormones and Behavior, 53, 693-705.
[11] Craft, R.M., Mogil, J.S. and Aloisi, A.M. (2004) Sex Differences in Pain and Analgesia: The Role of Gonadal Hormones. European Journal of Pain, 8, 397-411.
[12] Fillingim, R.B., King, C.D., Ribeiro-Dasilva, M.C., Rahim-Williams, B. and Riley 3rd, J.L. (2009) Sex, Gender, and Pain: A Review of Recent Clinical and Experimental Findings. The Journal of Pain, 10, 447-485. http://dx.doi.org/10.1016/j.jpain.2008.12.001
[13] Kaergaard, A., Hansen, A.M., Rasmussen, K. and Andersen, J.H. (2000) Association between Plasma Testosterone and Work-Related Neck and Shoulder Disorders among Female Workers. Scandinavian Journal of Work, Environment & Health, 26, 292-298. http://dx.doi.org/10.5271/sjweh.545
[14] Aloisi, A.M., Ceccarelli, I., Carlucci, M., Suman, A., Sindaco, G., Mameli, S., Paci, V., Ravaioli, L., Passavanti, G., Bachiocco, V. and Pari, G. (2011) Hormone Replacement Therapy in Morphine-Induced Hypogonadic Male Chronic Pain Patients. Reproductive Biology and Endocrinology, 9, 26.
[15] Ceccarelli, I., Scaramuzzino, A., Massafra, C. and Aloisi, A.M. (2003) The Behavioral and Neuronal Effects Induced by Repetitive Nociceptive Stimulation Are Affected by Gonadal Hormones in Male Rats. Pain, 104, 35-47. http://dx.doi.org/10.1016/S0304-3959(02)00460-8
[16] Gaumond, I., Arsenault, P. and Marchand, S. (2005) Specificity of Female and Male Sex Hormones on Excitatory and Inhibitory Phases of Formalin-Induced Nociceptive Responses. Brain Research, 1052, 105-111. http://dx.doi.org/10.1016/j.brainres.2005.06.011
[17] Vincent, K., Warnaby, C., Stagg, C.J., Moore, J., Kennedy, S. and Tracey, I. (2013) Brain Imaging Reveals that Engagement of Descending Inhibitory Pain Pathways in Healthy Women in a Low Endogenous Estradiol State Varies with Testosterone. Pain, 154, 515-524.
[18] Aloisi, A.M. (2003) Gonadal Hormones and Sex Differences in Pain Reactivity. Clinical Journal of Pain, 19, 168-174. http://dx.doi.org/10.1097/00002508-200305000-00004
[19] Ceccarelli, I., Casamenti, F., Massafra, C., Pepeu, G., Scali, C. and Aloisi, A.M. (1999) Effects of Novelty and Pain on Behavior and Hippocampal Extracellular ACh Levels in Male and Female Rats. Brain Research, 815, 169-176. http://dx.doi.org/10.1016/S0006-8993(98)01171-8
[20] Zimmerman, M.E., Pan, J.W., Hetherington, H.P., Lipton, M.L., Baigi, K. and Lipton, R.B. (2009) Hippocampal Correlates of Pain in Healthy Elderly Adults: A Pilot Study. Neurology, 73, 1567-1570.
[21] Mutso, A.A., Radzicki, D., Baliki, M.N., Huang, L., Banisadr, G., Centeno, M.V., Radulovic, J., Martina, M., Miller, R.J. and Apkarian, A.V. (2012) Abnormalities in Hippocampal Functioning with Persistent Pain. Journal of Neuroscience, 32, 5747-5756. http://dx.doi.org/10.1523/JNEUROSCI.0587-12.2012
[22] Gondo, M., Moriguchi, Y., Kodama, N., Sato, N., Sudo, N., Kubo, C. and Komaki, G. (2012) Daily Physical Complaints and Hippocampal Function: An fMRI Study of Pain Modulation by Anxiety. NeuroImage, 63, 1011-1019. http://dx.doi.org/10.1016/j.neuroimage.2012.07.025
[23] Korol, D.L. (2004) Role of Estrogen in Balancing Contributions from Multiple Memory Systems. Neurobiology of Learning and Memory, 82, 309-323. http://dx.doi.org/10.1016/j.nlm.2004.07.006
[24] Hajszan, T. and MacLusky, N.J. (2006) Neurologic Links between Epilepsy and Depression in Women—Is Hippocampal Neuroplasticity the Key? Neurology, 66, S13-S22.
[25] Acaz-Fonseca, E., Sanchez-Gonzalez, R., Azcoitia, I., Arevalo, M.A. and Garcia-Segura, L.M. (2014) Role of Astrocytes in the Neuroprotective Actions of 17β-Estradiol and Selective Estrogen Receptor Modulators. Molecular and Cellular Endocrinology, 389, 48-57.
[26] De Maddalena, C., Bellini, M., Berra, M., Meriggiola, M.C. and Aloisi, A.M. (2012) Opioid-Induced Hypogonadism: Why and How to Treat It. Pain Physician, 15, ES111-ES118.
[27] Tajar, A., McBeth, J., Lee, D.M., Macfarlane, G.J., Huhtaniemi, I.T., Finn, J.D., Bartfai, G., Boonen, S., Casanueva, F.F., Forti, G., Giwercman, A., Han, T.S., Kula, K., Labrie, F., Lean, M.E., Pendleton, N., Punab, M., Silman, A.J., Vanderschueren, D., O’Neill, T.W. and Wu, F.C. (2011) Elevated Levels of Gonadotrophins but Not Sex Steroids Are Associated with Musculoskeletal Pain in Middle-Aged and Older European Men. Pain, 152, 1495-1501. http://dx.doi.org/10.1016/j.pain.2011.01.048
[28] Saad, F., Aversa, A., Isidori, A.M. and Gooren, L.J. (2012) Testosterone as Potential Effective Therapy in Treatment of Obesity in Men with Testosterone Deficiency: A Review. Current Diabetes Reviews, 8, 131-143. http://dx.doi.org/10.2174/157339912799424573
[29] Siri-Tarino, P.W., Sun, Q., Hu, F.B. and Krauss, R.M. (2010) Saturated Fat, Carbohydrate, and Cardiovascular Disease. American Journal of Clinical Nutrition, 91, 502-509.
[30] Petroni, A. (2007) Medicament Based on a Monoester of Steroids with Long Chain Fatty Acids. Pub. No. WO/2007/ 068434.
[31] Phillips, G., Pinkernell, B. and Jing, T. (1994) The Association of Hypotestosteronemia with Coronary Artery Disease in Men. Arteriosclerosis, Thrombosis, and Vascular Biology, 14, 701-706.
[32] Ceccarelli, I., Rossi, A., Maddalena, M., Weber, E. and Aloisi, A.M. (2009) Effects of Morphine on Testosterone Levels in Rat C6 Glioma Cells: Modulation by Anastrozole. Journal of Cellular Physiology, 221, 1-4. http://dx.doi.org/10.1002/jcp.21830
[33] Aiello, F., Garofalo, A., Aloisi, A.M., Lamponi, S., Magnani, A. and Petroni, A. (2013) Synthesis of Esters of Androgens with Unsaturated Fatty Acids for Androgen Requiring Therapy. Journal of Endocrinological Investigation, 36, 390-395.
[34] Zimmermann, M. (1983) Ethical Guidelines for Investigations of Experimental Pain in Conscious Animals. Pain, 16, 109-110. http://dx.doi.org/10.1016/0304-3959(83)90201-4
[35] Dominguez, R. and Mitchevych, P. (2010) Estradiol Rapidly Regulates Membrane Estrogen Receptor α Levels in Hypothalamic Neurons. Journal of Neuroscience, 30, 12589-12596.
[36] Shughrue, P.J. and Merchenthalerm, I. (2001) Distribution of Estrogen Receptor β Immunoreactivity in the Rat Central Nervous System. Journal of Comparative Neurology, 436, 64-81.
[37] Perez, C., Falero, A., Llanes, N., Hung, B.R., Herve, M.E., Palmero, A. and Martii, E. (2003) Resistance to Androstanes as an Approach for Androstandienedione Yield Enhancement in Industrial Mycobacteria. Journal of Industrial Microbiology and Biotechnology, 30, 623-626.
[38] Toran-Allerand, C.D., Singh, M. and Setalo Jr., G. (1999) Novel Mechanisms of Estrogen Action in the Brain: New Players in an Old Story. Frontiers in Neuroendocrinology, 20, 97-121.
[39] Purves-Tyson, T.D. and Keast, J.R. (2004) Rapid Actions of Estradiol on Cyclic Amp Response-Element Binding Protein Phosphorylation in Dorsal Root Ganglion Neurons. Neuroscience, 129, 629-637. http://dx.doi.org/10.1016/j.neuroscience.2004.08.019
[40] Sohrabji, F., Miranda, R.C. and Toran-Allerand, C.D. (1994) Estrogen Differentially Regulates Estrogen and Nerve Growth Factor Receptor mRNAs in Adult Sensory Neurons. Journal of Neuroscience, 14, 459-471.
[41] Allen, A.L. and McCarson, K.E. (2005) Estrogen Increases Nociception-Evoked Brain-Derived Neurotrophic Factor Gene Expression in the Female Rat. Neuroendocrinology, 81, 193-199.
[42] Ji, Y., Tang, B. and Traub, R.J. (2011) Spinal Estrogen Receptor Alpha Mediates Estradiol-Induced Pronociception in a Visceral Pain Model in the Rat. Pain, 152, 1182-1191.
[43] Spooner, M.F., Robichaud, P., Carrier, J.C. and Marchand, S. (2007) Endogenous Pain Modulation during the Formalin Test in Estrogen Receptor Beta Knockout Mice. Neuroscience, 150, 675-680.
[44] ?sterlund, M.K., Gustafsson, J.A., Keller, E. and Hurd, Y.L. (2000) Estrogen Receptor β (ERβ) Messenger Ribonucleic Acid (mRNA) Expression within the Human Forebrain: Distinct Distribution Pattern to ERα mRNA. Journal of Clinical Endocrinology & Metabolism, 85, 3840-3846.
[45] Schreihofer, D.A., Stoler, M.H. and Shupnik, M.A. (2000) Differential Expression and Regulation of Estrogen Receptors (ERs) in Rat Pituitary and Cell Lines: Estrogen Decreases ERα Protein and Estrogen Responsiveness. Endocrinology, 141, 2174-2184.
[46] Tena-Sempere, M., Gonzalez, L.C., Pinilla, L., Huhtaniemi, I. and Aguilar, E. (2001) Neonatal Imprinting and Regulation of Estrogen Receptor Alpha and Beta mRNA Expression by Estrogen in the Pituitary and Hypothalamus of the Male Rat. Neuroendocrinology, 73, 12-25.
[47] Alarid, E.T., Bakopoulos, N. and Solodin, N. (1999) Proteasome-Mediated Proteolysis of Estrogen Receptor: A Novel Component in Autologous Down-Regulation. Molecular Endocrinology, 13, 1522-1534. http://dx.doi.org/10.1210/mend.13.9.0337
[48] Patisaul, H.B., Whitten, P.L. and Young, L.J. (1999) Regulation of Estrogen Receptor Beta mRNA in the Brain: Opposite Effects of 17β-Estradiol and the Phytoestrogen, Coumestrol. Molecular Brain Research, 67, 165-171. http://dx.doi.org/10.1016/S0169-328X(99)00058-3
[49] Prange-Kiel, J., Wehrenberg, U., Jarry, H. and Rune, G.M. (2003) Para/Autocrine Regulation of Estrogen Receptors in Hippocampal Neurons. Hippocampus, 13, 226-234.
[50] Aloisi, A.M., Ceccarelli, I., Fiorenzani, P., De Padova, A.M. and Massafra, C. (2004) Testosterone Affects Formalin-Induced Responses Differently in Male and Female Rats. Neuroscience Letters, 361, 262-264. http://dx.doi.org/10.1016/j.neulet.2003.12.023
[51] Rossmeisl, M., Medrikova, D., van Schothorst, E.M., Pavlisova, J., Kuda, O., Hensler, M., Bardova, K., Flachs, P., Stankova, B., Vecka, M., Tvrzicka, E., Zak, A., Keijer, J. and Kopecky, J. (2013) Omega-3 Phospholipids from Fish Suppress Hepatic Steatosis by Integrated Inhibition of Biosynthetic Pathways in Dietary Obese Mice. Biochimica et Biophysica Acta, 1841, 267-278.

Copyright © 2023 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.