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Edmund Maser - One of the best experts on this subject based on the ideXlab platform.
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Construction of a biosensor mutant of Comamonas testosteroni for Testosterone determination by cloning the EGFP gene downstream to the regulatory region of the 3,17β-HSD gene.
Chemico-biological interactions, 2014Co-Authors: Guangming Xiong, Edmund MaserAbstract:Comamonas testosteroni (C. testosteroni) is able to catabolize a variety of steroids and polycyclic aromatic hydrocarbons. 3,17β-Hydroxysteroid dehydrogenase (3,17β-HSD) from C. testosteroni is a Testosterone-inducible protein and a key enzyme in steroid degradation. 3,17β-HSD is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. We found that a 2.4 kb regulatory DNA fragment upstream of the 3,17β-HSD gene (βhsd) responds to steroids and triggers βhsd gene induction. To exploit this cis-acting regulatory element for a steroid determination system, plasmids pK2.4-EGFP-4 and pBB2.4-EGFP-8 were constructed. Both plasmids contain the EGFP gene fused downstream to a 2.4 kb DNA fragment from C. testosteroni. However, whereas pK2.4-EGFP-4 could integrate into the chromosomal DNA of C. testosteroni and knock out the βhsd gene promoter, pBB2.4-EGFP-8 could replicate in C. testosteroni cells as a free plasmid DNA. After integration of pK2.4-EGFP-4 into the βhsd gene promoter, 3,17β-HSD expression could not be induced such that EGFP expression in the mutant cells was at low levels. In contrast, in C. testosteroni cells transformed with pBB2.4-EGFP-8 the expression of EGFP was induced with Testosterone. Our results showed that fluorescence counts (relative fluorescence units; RFU) increased in parallel with Testosterone concentrations. Of note, estradiol and cholesterol could not induce the EGFP reporter gene. In summary, this new biosensor system might be used for the specific determination of Testosterone.
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Identification and isolation of a regulator protein for 3,17β-HSD expressional regulation in Comamonas testosteroni.
Chemico-biological interactions, 2014Co-Authors: Pu Huang, Guangming Xiong, Edmund MaserAbstract:Comamonas testosteroni (C. testosteroni) is able to catabolize a variety of steroids and polycyclic aromatic hydrocarbons. 3,17β-Hydroxysteroid dehydrogenase (3,17β-HSD) from C. testosteroni is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. It is an inducible and key enzyme in steroid degradation. Elucidating the mechanism of 3,17β-HSD gene (βhsd) regulation may help us to generate prospective C. testosteroni mutants for bioremediation. The genome of C. testosteroni ATCC11996 was sequenced in our previous work. Upon examining the genome with bioinformatics tools, a gene (brp) coding for a regulator protein (BRP) for 3,17β-HSD expression was found upstream of the βhsd gene. A Blast search revealed high identities to a nucleotide binding protein with unknown function in other bacteria. Two potential promoters and two repeat sequences (RS, 16 bp), spaced to each other by 1661 bp, were also found upstream of the βhsd gene C. testosteroni. The brp gene was cloned into plasmid pK18 and pET-15b, expressed in Escherichia coli, and the recombinant BRP protein was purified on a Ni-column. In addition, a brp gene knock-out mutant of C. testosteroni was prepared. These knock-out mutants showed an enhanced expression of both the βhsd gene and the hsdA gene (the latter coding for 3α-HSD/CR) in the presence of Testosterone. To characterize the BRP functional DNA domain, different fragments of the βhsd upstream regulatory region were tested in a cotransformation system. Our data reveal that the βhsd gene undergoes complex regulation involving the two promoters, a loop structure via the two repeat sequences, and the steroid Testosterone. Furthermore, a proximal repressor gene for βhsd expression, phaR, had been identified in our previous investigations. The exact interplay between all these factors will be determined in future experiments.
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a novel transcriptional repressor phar for the steroid inducible expression of the 3 17β hydroxysteroid dehydrogenase gene in comamonas testosteroni atcc11996
Chemico-Biological Interactions, 2013Co-Authors: Guangming Xiong, Edmund MaserAbstract:Comamonas testosteroni is able to catabolize a variety of steroids and polycyclic aromatic hydrocarbons and might be used in the bioremediation of contaminated environments. 3,17β-Hydroxysteroid dehydrogenase (3,17β-HSD) from C. testosteroni is a member of the short-chain dehydrogenase/reductase (SDR) superfamily and a key enzyme in steroid degradation. The genome of C. testosteroni ATCC11996 was sequenced in our previous work. In addition to the gene coding for 3,17β-HSD (βhsd), a novel transcriptional repressor phaR gene (phaR) which locates 2290 bp upstream of the βhsd gene was found. PhaR knock-out mutants of C. testosteroni were prepared and shown to grow better than wild-type C. testosteroni in the presence of 1 mM Testosterone, 0.5 mM estradiol or 0.5 mM cholesterol in both Standard 1 Nutrient (SIN) medium and 1:10 diluted SIN medium. After 1 mM Testosterone induction, 3,17β-HSD expression in the mutant was 2.5 times higher than in wild type C. testosteroni. Accordingly, PhaR is a repressor that controls 3,17β-HSD expression. Moreover, phaR knock-out mutants grow at higher rates and produce more protein in the presence of steroids as carbon source. However, ELISA results showed that 0.5 mM estradiol and cholesterol could not induce βhsd gene expression in both wild-type and mutant C. testosteroni. Probably, in addition to the βhsd gene, PhaR regulates some other genes that relate to steroid degradation. The genes coding for PhaR and 3,17β-HSD together with their promoter domains were cloned into plasmids pK18 and pUC19. Escherichia coli HB101 was co-transformed with these plasmids. The results suggest that PhaR is a repressor, which might bind on a special βhsd promoter domain (214 bp). A 2509 bp DNA fragment that contained a putative promoter for the βhsd gene (without the phaR gene) was cloned into plasmid pUC2.5-3. The plasmid was transformed into HB101 (E. coli) and induced with Testosterone. As a result, 3,17β-HSD expression was at a high level, but could not be further enhanced by Testosterone. Taken together, phaR knock-out mutants have better ability to degrade steroids than wild-type C. testosteroni ATCC11996 and might therefore be used in bioremediation.
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identification and characterization of the lysr type transcriptional regulator hsdr for steroid inducible expression of the 3α hydroxysteroid dehydrogenase carbonyl reductase gene in comamonas testosteroni
Applied and Environmental Microbiology, 2012Co-Authors: Wenjie Gong, Guangming Xiong, Edmund MaserAbstract:3 alpha-Hydroxysteroid dehydrogenase/carbonyl reductase (3 alpha-HSD/CR) from Comamonas testosteroni is a key enzyme in steroid degradation in soil and water. 3 alpha-HSD/CR gene (hsdA) expression can be induced by steroids like Testosterone and progester-one. Previously, we have shown that the induction of hsdA expression by steroids is a derepression where steroidal inducers bind to two repressors, RepA and RepB, thereby preventing the blocking of hsdA transcription and translation, respectively (G. Xiong and E. Maser, J. Biol. Chem. 276:9961-9970, 2001; G. Xiong, H. J. Martin, and E. Maser, J. Biol. Chem. 278:47400-47407, 2003). In the present study, a new LysR-type transcriptional factor, HsdR, for 3 alpha-HSD/CR expression in C. testosteroni has been identified. The hsdR gene is located 2.58 kb downstream from hsdA on the C. testosteroni ATCC 11996 chromosome with an orientation opposite that of hsdA. The hsdR gene was cloned and recombinant HsdR protein was produced, as was anti-HsdR polyclonal antibodies. While heterologous transformation systems revealed that HsdR activates the expression of the hsdA gene, electrophoresis mobility shift assays showed that HsdR specifically binds to the hsdA promoter region. Interestingly, the activity of HsdR is dependent on decreased repression by RepA. Furthermore, in vitro binding assays indicated that HsdR can come into contact with RNA polymerase. As expected, an hsdR knockout mutant expressed low levels of 3 alpha-HSD/CR compared to that of wild-type C. testosteroni after Testosterone induction. In conclusion, HsdR is a positive transcription factor for the hsdA gene and promotes the induction of 3 alpha-HSD/CR expression in C. testosteroni.
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Testosterone inducible regulator is a kinase that drives steroid sensing and metabolism in comamonas testosteroni
Journal of Biological Chemistry, 2008Co-Authors: Andre Gohler, Guangming Xiong, Simone Paulsen, Gabriele Trentmann, Edmund MaserAbstract:The mechanism of gene regulation by steroids in bacteria is still a mystery. We use steroid-inducible 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) as a reporter system to study steroid signaling in Comamonas testosteroni. In previous investigations we cloned and characterized the 3alpha-HSD/CR-encoding gene, hsdA. In addition, we identified two negative regulator genes (repA and repB) in the vicinity of hsdA, the protein products which repress hsdA expression on the level of transcription and translation, respectively. Recently, a positive regulator of hsdA expression, TeiR (Testosterone-inducible regulator), was found by transposon mutagenesis, but the mode of its action remained obscure. In the present work we produced a TeiR-green fluorescent fusion protein and showed that TeiR is a membrane protein with asymmetrical localization at one of the cell poles of C. testosteroni. Knock-out mutants of the teiR gene revealed that TeiR provides swimming and twitching motility of C. testosteroni to the steroid substrate source. TeiR also mediated an induced expression of 3alpha-HSD/CR which was paralleled by an enhanced catabolism of Testosterone. We also found that TeiR responds to a variety of different steroids other than Testosterone. Biochemical analysis with several deletion mutants of the teiR gene revealed TeiR to consist of three different functional domains, an N-terminal domain important for membrane association, a central steroid binding site, and a C-terminal part mediating TeiR function. Finally, we could demonstrate that TeiR works as a kinase in the steroid signaling chain in C. testosteroni. Overall, we provide evidence that TeiR mediates steroid sensing and metabolism in C. testosteroni via its steroid binding and kinase activity.
Guangming Xiong - One of the best experts on this subject based on the ideXlab platform.
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Construction of a biosensor mutant of Comamonas testosteroni for Testosterone determination by cloning the EGFP gene downstream to the regulatory region of the 3,17β-HSD gene.
Chemico-biological interactions, 2014Co-Authors: Guangming Xiong, Edmund MaserAbstract:Comamonas testosteroni (C. testosteroni) is able to catabolize a variety of steroids and polycyclic aromatic hydrocarbons. 3,17β-Hydroxysteroid dehydrogenase (3,17β-HSD) from C. testosteroni is a Testosterone-inducible protein and a key enzyme in steroid degradation. 3,17β-HSD is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. We found that a 2.4 kb regulatory DNA fragment upstream of the 3,17β-HSD gene (βhsd) responds to steroids and triggers βhsd gene induction. To exploit this cis-acting regulatory element for a steroid determination system, plasmids pK2.4-EGFP-4 and pBB2.4-EGFP-8 were constructed. Both plasmids contain the EGFP gene fused downstream to a 2.4 kb DNA fragment from C. testosteroni. However, whereas pK2.4-EGFP-4 could integrate into the chromosomal DNA of C. testosteroni and knock out the βhsd gene promoter, pBB2.4-EGFP-8 could replicate in C. testosteroni cells as a free plasmid DNA. After integration of pK2.4-EGFP-4 into the βhsd gene promoter, 3,17β-HSD expression could not be induced such that EGFP expression in the mutant cells was at low levels. In contrast, in C. testosteroni cells transformed with pBB2.4-EGFP-8 the expression of EGFP was induced with Testosterone. Our results showed that fluorescence counts (relative fluorescence units; RFU) increased in parallel with Testosterone concentrations. Of note, estradiol and cholesterol could not induce the EGFP reporter gene. In summary, this new biosensor system might be used for the specific determination of Testosterone.
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Identification and isolation of a regulator protein for 3,17β-HSD expressional regulation in Comamonas testosteroni.
Chemico-biological interactions, 2014Co-Authors: Pu Huang, Guangming Xiong, Edmund MaserAbstract:Comamonas testosteroni (C. testosteroni) is able to catabolize a variety of steroids and polycyclic aromatic hydrocarbons. 3,17β-Hydroxysteroid dehydrogenase (3,17β-HSD) from C. testosteroni is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. It is an inducible and key enzyme in steroid degradation. Elucidating the mechanism of 3,17β-HSD gene (βhsd) regulation may help us to generate prospective C. testosteroni mutants for bioremediation. The genome of C. testosteroni ATCC11996 was sequenced in our previous work. Upon examining the genome with bioinformatics tools, a gene (brp) coding for a regulator protein (BRP) for 3,17β-HSD expression was found upstream of the βhsd gene. A Blast search revealed high identities to a nucleotide binding protein with unknown function in other bacteria. Two potential promoters and two repeat sequences (RS, 16 bp), spaced to each other by 1661 bp, were also found upstream of the βhsd gene C. testosteroni. The brp gene was cloned into plasmid pK18 and pET-15b, expressed in Escherichia coli, and the recombinant BRP protein was purified on a Ni-column. In addition, a brp gene knock-out mutant of C. testosteroni was prepared. These knock-out mutants showed an enhanced expression of both the βhsd gene and the hsdA gene (the latter coding for 3α-HSD/CR) in the presence of Testosterone. To characterize the BRP functional DNA domain, different fragments of the βhsd upstream regulatory region were tested in a cotransformation system. Our data reveal that the βhsd gene undergoes complex regulation involving the two promoters, a loop structure via the two repeat sequences, and the steroid Testosterone. Furthermore, a proximal repressor gene for βhsd expression, phaR, had been identified in our previous investigations. The exact interplay between all these factors will be determined in future experiments.
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a novel transcriptional repressor phar for the steroid inducible expression of the 3 17β hydroxysteroid dehydrogenase gene in comamonas testosteroni atcc11996
Chemico-Biological Interactions, 2013Co-Authors: Guangming Xiong, Edmund MaserAbstract:Comamonas testosteroni is able to catabolize a variety of steroids and polycyclic aromatic hydrocarbons and might be used in the bioremediation of contaminated environments. 3,17β-Hydroxysteroid dehydrogenase (3,17β-HSD) from C. testosteroni is a member of the short-chain dehydrogenase/reductase (SDR) superfamily and a key enzyme in steroid degradation. The genome of C. testosteroni ATCC11996 was sequenced in our previous work. In addition to the gene coding for 3,17β-HSD (βhsd), a novel transcriptional repressor phaR gene (phaR) which locates 2290 bp upstream of the βhsd gene was found. PhaR knock-out mutants of C. testosteroni were prepared and shown to grow better than wild-type C. testosteroni in the presence of 1 mM Testosterone, 0.5 mM estradiol or 0.5 mM cholesterol in both Standard 1 Nutrient (SIN) medium and 1:10 diluted SIN medium. After 1 mM Testosterone induction, 3,17β-HSD expression in the mutant was 2.5 times higher than in wild type C. testosteroni. Accordingly, PhaR is a repressor that controls 3,17β-HSD expression. Moreover, phaR knock-out mutants grow at higher rates and produce more protein in the presence of steroids as carbon source. However, ELISA results showed that 0.5 mM estradiol and cholesterol could not induce βhsd gene expression in both wild-type and mutant C. testosteroni. Probably, in addition to the βhsd gene, PhaR regulates some other genes that relate to steroid degradation. The genes coding for PhaR and 3,17β-HSD together with their promoter domains were cloned into plasmids pK18 and pUC19. Escherichia coli HB101 was co-transformed with these plasmids. The results suggest that PhaR is a repressor, which might bind on a special βhsd promoter domain (214 bp). A 2509 bp DNA fragment that contained a putative promoter for the βhsd gene (without the phaR gene) was cloned into plasmid pUC2.5-3. The plasmid was transformed into HB101 (E. coli) and induced with Testosterone. As a result, 3,17β-HSD expression was at a high level, but could not be further enhanced by Testosterone. Taken together, phaR knock-out mutants have better ability to degrade steroids than wild-type C. testosteroni ATCC11996 and might therefore be used in bioremediation.
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identification and characterization of the lysr type transcriptional regulator hsdr for steroid inducible expression of the 3α hydroxysteroid dehydrogenase carbonyl reductase gene in comamonas testosteroni
Applied and Environmental Microbiology, 2012Co-Authors: Wenjie Gong, Guangming Xiong, Edmund MaserAbstract:3 alpha-Hydroxysteroid dehydrogenase/carbonyl reductase (3 alpha-HSD/CR) from Comamonas testosteroni is a key enzyme in steroid degradation in soil and water. 3 alpha-HSD/CR gene (hsdA) expression can be induced by steroids like Testosterone and progester-one. Previously, we have shown that the induction of hsdA expression by steroids is a derepression where steroidal inducers bind to two repressors, RepA and RepB, thereby preventing the blocking of hsdA transcription and translation, respectively (G. Xiong and E. Maser, J. Biol. Chem. 276:9961-9970, 2001; G. Xiong, H. J. Martin, and E. Maser, J. Biol. Chem. 278:47400-47407, 2003). In the present study, a new LysR-type transcriptional factor, HsdR, for 3 alpha-HSD/CR expression in C. testosteroni has been identified. The hsdR gene is located 2.58 kb downstream from hsdA on the C. testosteroni ATCC 11996 chromosome with an orientation opposite that of hsdA. The hsdR gene was cloned and recombinant HsdR protein was produced, as was anti-HsdR polyclonal antibodies. While heterologous transformation systems revealed that HsdR activates the expression of the hsdA gene, electrophoresis mobility shift assays showed that HsdR specifically binds to the hsdA promoter region. Interestingly, the activity of HsdR is dependent on decreased repression by RepA. Furthermore, in vitro binding assays indicated that HsdR can come into contact with RNA polymerase. As expected, an hsdR knockout mutant expressed low levels of 3 alpha-HSD/CR compared to that of wild-type C. testosteroni after Testosterone induction. In conclusion, HsdR is a positive transcription factor for the hsdA gene and promotes the induction of 3 alpha-HSD/CR expression in C. testosteroni.
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Testosterone inducible regulator is a kinase that drives steroid sensing and metabolism in comamonas testosteroni
Journal of Biological Chemistry, 2008Co-Authors: Andre Gohler, Guangming Xiong, Simone Paulsen, Gabriele Trentmann, Edmund MaserAbstract:The mechanism of gene regulation by steroids in bacteria is still a mystery. We use steroid-inducible 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) as a reporter system to study steroid signaling in Comamonas testosteroni. In previous investigations we cloned and characterized the 3alpha-HSD/CR-encoding gene, hsdA. In addition, we identified two negative regulator genes (repA and repB) in the vicinity of hsdA, the protein products which repress hsdA expression on the level of transcription and translation, respectively. Recently, a positive regulator of hsdA expression, TeiR (Testosterone-inducible regulator), was found by transposon mutagenesis, but the mode of its action remained obscure. In the present work we produced a TeiR-green fluorescent fusion protein and showed that TeiR is a membrane protein with asymmetrical localization at one of the cell poles of C. testosteroni. Knock-out mutants of the teiR gene revealed that TeiR provides swimming and twitching motility of C. testosteroni to the steroid substrate source. TeiR also mediated an induced expression of 3alpha-HSD/CR which was paralleled by an enhanced catabolism of Testosterone. We also found that TeiR responds to a variety of different steroids other than Testosterone. Biochemical analysis with several deletion mutants of the teiR gene revealed TeiR to consist of three different functional domains, an N-terminal domain important for membrane association, a central steroid binding site, and a C-terminal part mediating TeiR function. Finally, we could demonstrate that TeiR works as a kinase in the steroid signaling chain in C. testosteroni. Overall, we provide evidence that TeiR mediates steroid sensing and metabolism in C. testosteroni via its steroid binding and kinase activity.
Robert M Sapolsky - One of the best experts on this subject based on the ideXlab platform.
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rapid stimulatory effects of Testosterone upon myotubule metabolism and sugar transport as assessed by silicon microphysiometry
Aggressive Behavior, 1996Co-Authors: Larry W Tsai, Robert M SapolskyAbstract:A considerable number of studies bave revealed bebavioral circumstances tbat give rise to small or transient differences in circulating Testosterone concentrations; however, carefut consideration of androgen physiology leads to tbe disquieting conclusion that these differences are often untikely to have much physiotogic or behavioral significance. In the present report, we observe that small transients of Testosterone secretion could have very rapid anabolic effects on a cultured muscle-derived cell line. Specifically, we have examined the effects of Testosterone on metabolism in cultured C2CI2 myotubules, using a silicon microphysiometer. The instrument monitors cellular extrusion of protons and acidic metabolites, and such extrusion is directly linked to ATP hydrolysis, thus providing a real-time measure of cellular metabolism. Te.stosterone caused a small but significant increase in metabolism. Tbe most striking feature of this effect wa.s its rapidity, in that it occurred within 3 br. This rapid enhancement of metabolism suggested that Testosterone might be enhancing substrate uptake even more rapidly. Indeed., we found that Testosterone increased 2-deoxyglucose uptake within I min. The rapidity of this effect seemed to preclude mediation by classical Intracellular steroid receptors. In support of this, we were unahle to detect specific intracellular binding of Testosterone. These findings sbow that Testosterone can exert rapid anabolic effects on substrate transport and metabolism in myotuhules. Should this finding general to muscle in vivo, it suggests that relatively small individual differences in Testosterone profiles, in response to various social interactions, may have very real consequence for subsequent muscle physiology. © 19% Wilej-Liss, Inc.
Larry W Tsai - One of the best experts on this subject based on the ideXlab platform.
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rapid stimulatory effects of Testosterone upon myotubule metabolism and sugar transport as assessed by silicon microphysiometry
Aggressive Behavior, 1996Co-Authors: Larry W Tsai, Robert M SapolskyAbstract:A considerable number of studies bave revealed bebavioral circumstances tbat give rise to small or transient differences in circulating Testosterone concentrations; however, carefut consideration of androgen physiology leads to tbe disquieting conclusion that these differences are often untikely to have much physiotogic or behavioral significance. In the present report, we observe that small transients of Testosterone secretion could have very rapid anabolic effects on a cultured muscle-derived cell line. Specifically, we have examined the effects of Testosterone on metabolism in cultured C2CI2 myotubules, using a silicon microphysiometer. The instrument monitors cellular extrusion of protons and acidic metabolites, and such extrusion is directly linked to ATP hydrolysis, thus providing a real-time measure of cellular metabolism. Te.stosterone caused a small but significant increase in metabolism. Tbe most striking feature of this effect wa.s its rapidity, in that it occurred within 3 br. This rapid enhancement of metabolism suggested that Testosterone might be enhancing substrate uptake even more rapidly. Indeed., we found that Testosterone increased 2-deoxyglucose uptake within I min. The rapidity of this effect seemed to preclude mediation by classical Intracellular steroid receptors. In support of this, we were unahle to detect specific intracellular binding of Testosterone. These findings sbow that Testosterone can exert rapid anabolic effects on substrate transport and metabolism in myotuhules. Should this finding general to muscle in vivo, it suggests that relatively small individual differences in Testosterone profiles, in response to various social interactions, may have very real consequence for subsequent muscle physiology. © 19% Wilej-Liss, Inc.
Fanton, Letícia Esmanhoto - One of the best experts on this subject based on the ideXlab platform.
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Mechanisms underlying Testosterone protective effect on temporomandibular joint nociception development in rats
[s.n.], 2018Co-Authors: Fanton, Letícia EsmanhotoAbstract:Orientadores: Cláudia Herrera Tambeli, Luana FischerDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: As disfunções temporomandibulares são mais prevalentes em mulheres que em homens, sendo o papel dos hormônios sexuais um dos fatores apontados como causa dessa diferença. Nesse contexto, a testosterona apresenta um efeito protetor ao diminuir o risco de ratos desenvolverem dor na articulação temporomandibular (ATM), já que a injeção de 0,5% de formalina na ATM não é capaz de induzir nocicepção em machos intactos, mas induz em machos gonadectomizados (Gx) e fêmeas. O objetivo deste trabalho foi investigar se o papel protetor da testosterona no desenvolvimento da dor da ATM em ratos: (a) depende da ação organizacional da testosterona durante o período de diferenciação sexual do sistema nervoso central; (b) é mediado diretamente pela ação andrógena ou pela ação do estrógeno sintetizado a partir da testosterona; (c) é mediado pela ativação do sistema opioide endógeno central; e (d) resulta de um menor risco de machos desenvolverem edema na ATM. A formalina foi utilizada como estímulo nociceptivo e inflamatório na ATM. Para testar se o efeito protetor da testosterona depende da sua ação organizacional no período de diferenciação sexual, ratos orquidectomizados com um dia de vida ou na fase adulta e fêmeas intactas receberam testosterona antes do teste da formalina na ATM. Para testar a ação direta da testosterona ou do estrógeno sintetizado a partir da testosterona, ratos machos receberam injeção subaracnoide, na região do núcleo sensorial trigeminal, de flutamide (antagonista de receptores androgênicos) ou de ICI 182 780 (antagonista de receptores estrogênicos), antes do teste da formalina na ATM. Para testar o envolvimento de um mecanismo neural central dependente da ativação do sistema opioide, ratos machos receberam injeção de naloxona (antagonista de receptores opioides), na região do núcleo sensorial trigeminal, antes do teste da formalina na ATM. Para verificar se a testosterona reduz o risco de desenvolvimento de edema na ATM, foi avaliado o extravasamento plasmático em ratos machos Gx ou sham Gx depois da injeção de formalina na ATM. A orquidectomia com um dia de vida ou na fase adulta, seguida de reposição hormonal antes do experimento comportamental, não afetou o comportamento nociceptivo dos ratos. A administração de testosterona em fêmeas intactas diminuiu o comportamento nociceptivo induzido pela injeção de formalina na ATM. Em machos sham Gx, a administração subaracnoide de flutamide (120 µg) ou de naloxona (15 µg) aumentou o comportamento nociceptivo induzido pela injeção de formalina na ATM. Já a administração subaracnoide de ICI 182 780 não alterou o comportamento nociceptivo de ratos sham Gx. O extravasamento plasmático na região da ATM foi similar em machos sham Gx e em machos Gx que receberam injeção de formalina na ATM. Dessa forma, podemos concluir que o efeito protetor da testosterona no desenvolvimento da dor da ATM em ratos: (a) depende do efeito ativacional da testosterona, que medeia um mecanismo neural central dependente da ativação de receptores andrógenos e da liberação de peptídeos opioides e (b) não se deve a uma redução do risco de desenvolvimento de edema na região da ATM de ratos machos.Abstract: Temporomandibular disorders are known to be around two times more prevalent in women than in men, and one of the factors pointed out as a possible cause for such a difference is the role of sexual hormones. In this context, Testosterone presents a protective effect by diminishing the risk of rats developing temporomandibular joint (TMJ) pain, since 0.5% formalin injection does not induce nociception in naive males but it does in gonadectomized (Gx) male and female rats. The aim of this study was to investigate whether the protective role of Testosterone in the development of TMJ pain in rats: (a) depends on the organizational action of Testosterone during the sexual differentiation period of the central nervous system; (b) is mediated directly by androgens action or by the action of estrogens synthesized from Testosterone; (c) is mediated by the activation of the endogenous opioid system; (d) results from a reduced risk of males developing TMJ edema. The nociceptive and inflammatory agent formalin was injected into the TMJ region of rats. To test whether the protective role of Testosterone depends on its organizational effect at the critical period of sexual differentiation, one-day-old or six-week-old orchidectomized rats and naive females received Testosterone before the formalin TMJ test. To test whether the effect of Testosterone was direct or indirect via estrogen derived from its aromatization, male rats received a subarachnoid injection in the vicinities of the spinal trigeminal nucleus, respectively of flutamide (androgenic receptor antagonist) or of ICI 182 780 (estrogenic receptor antagonist), before the formalin TMJ test. To test the involvement of a central activation of the opioid system, male rats received a subarachnoid injection in the vicinities of the spinal trigeminal nucleus of naloxone (opioid receptor antagonist) before the formalin TMJ test. To test whether Testosterone induces a lower risk of males developing TMJ edema, we evaluated plasma protein extravasation in sham Gx and Gx male rats, after the TMJ injection of formalin. Orchidectomy with one day of life or in adulthood followed by Testosterone administration prior to the behavioral assay, did not affect nociceptive behavior in rats. Testosterone administration in naive females diminished the TMJ formalin-induced nociceptive behavior. In sham Gx male rats, subarachnoid administration of flutamide (120 µg) or naloxone (15 µg) augmented the nociceptive behavior induced by TMJ formalin injection. On the other hand, subarachnoid injection of ICI 182 780 did not affect significantly the nociceptive behavior of sham Gx male rats. Plasma protein extravasation was similar in sham Gx and Gx male rats that received TMJ formalin injection. Therefore, we can conclude that the protective effect of Testosterone on the development of TMJ pain in rats: (a) depends on Testosterone activational effects which mediates a neural central mechanism dependent of the activation of androgen receptors and subsequent release of opioid peptides and (b) it is not due to a lower risk of male rats developing TMJ edema.MestradoFisiologia OralMestre em Odontologi