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Allopregnanolone-mediated protective effects of progesterone on tributyltin-induced neuronal injury in rat hippocampal slices
Journal of Steroid Biochemistry & Molecular Biology 135 (2013) 1–6
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Allopregnanolone-mediated protective effects of progesterone on tributyltin-induced neuronal injury in rat hippocampal slices Yasuhiro Ishihara, Tomohito Kawami, Atsuhiko Ishida, Takeshi Yamazaki ∗ Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan
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Article history: Received 30 August 2012 Received in revised form 20 December 2012 Accepted 20 December 2012 Keywords: Neuroprotection Progesterone Allopregnanolone Tributyltin Hippocampal slices GABAA receptor
a b s t r a c t Increasing evidence shows that progesterone, a neuroactive steroid, has protective actions in central nervous system, but there is little evidence to show the protective mechanism of progesterone on neurotoxicity induced by environmental chemicals. In this study, we examined the effects of progesterone on neuronal injury induced by tributyltin (TBT) in rat hippocampal slices. Treatment with progesterone dose-dependently suppressed hippocampal neuronal injury induced by TBT. The neuroprotective action of progesterone was completely canceled with pretreatment by finasteride, a 5␣-reductase inhibitor, but it was not affected by mifepristone, a progesterone receptor antagonist, or by SU-10603, a cytochrome P450 17␣ inhibitor. The content of allopregnanolone in the slices was significantly increased by treatment with progesterone, and this increment was greatly suppressed with a pretreatment of finasteride. Treatment with allopregnanolone attenuated neuronal injury induced by TBT in a dose-dependent manner. The neuroprotective effects not only of progesterone but also of allopregnanolone were canceled by bicuculline, a potent gamma-aminobutyric acid A (GABAA ) receptor antagonist. Pretreatment with muscimol, a GABAA receptor agonist, attenuated hippocampal neuronal injury elicited by TBT. Taken together, allopregnanolone converted from progesterone in hippocampal slices could protect neurons from TBTinduced neurotoxicity due to a GABAA receptor-dependent mechanism. One of the physiological roles of neuroactive steroids might be neuroprotection from environmental chemicals. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Steroid hormones synthesized in and secreted from peripheral endocrine glands pass through the blood–brain barrier and then perform functions in the central nervous system. In addition, it was observed that the brain possesses an inherent endocrine system and synthesizes some steroid hormones [1]. Recently, increasing evidence has shown that a neuroactive steroid, progesterone, protects neurons. Treatment with progesterone increased the expression of Bcl-2 and decreased the contents of active caspase-3 to suppress apoptosis [2,3]. Progesterone also seems to be beneficial in preventing mitochondrial dysfunction, which results in the loss of hippocampal cells after a controlled cortical contusion [4]. Furthermore, it has been reported that progesterone protects hippocampal slice cultures from cell death following oxygen-glucose
Abbreviations: BDNF, brain-derived neurotrophic factor; GABA, gammaaminobutyric acid; NGF, nerve growth factor; ROS, reactive oxygen species; PI, propidium iodide; TBT, tributyltin. ∗ Corresponding author at: Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, 1-7-1, Kagamiyama, Higashi-Hiroshima 739-8521, Hiroshima, Japan. Tel.: +81 82 424 6527; fax: +81 82 424 0759. E-mail address: [email protected] (T. Yamazaki). 0960-0760/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jsbmb.2012.12.013
deprivation, largely due to its conversion to allopregnanolone via a gamma-aminobutyric acid A (GABAA ) receptor-dependent mechanism [5]. Progesterone is readily metabolized, in the brain, to allopregnanolone by activities of 5␣-reductase and 3␣hydroxysteroid dehydrogenase [5]. Organotin compounds have long been used as thermal stabilizers, catalytic agents and biocidal compounds for preserving wood, textiles, cordage fibers and electronic equipment. Among them, tributyltin (TBT) has been most widely used in paint formulations to prevent marine fouling on ships, boats and fish-farming nets. Environmental surveying and monitoring of TBT are conducted to prevent the consumption of bioaccumulated TBT by humans. The average intake of TBT by humans from market-bought seafood has been estimated to vary worldwide between 0.18 and 2.6 g per day per person [6], and the presence of butyltin compounds, including TBT, reportedly exists at concentrations between 50 nM and 400 nM in human blood [7]. Therefore, the effects of TBT on the human brain are now of great concern. Because many environmental chemicals, including organotin compounds, are lipophilic and have high blood–brain barrier permeability, the central nervous system is exposed to exogenous chemicals. The administration of TBT elicited abnormal behavior and the reduction of brain weight within the cerebellum and decreased synaptogenesis in rats [8–10]. This is evidence
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suggesting that the central nervous system is a primary target of TBT. However, the central nervous system is equipped with some endogenous neuroprotectants against harmful chemicals. Some of the most well-known factors are neutrophins such as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). BDNF attenuated cellular injury of differentiated neural stem cells induced by trimetyltin by activating the pathways of mitogen-activated protein kinase and phosphoinositide-3kinase/Akt signals [11]. NGF was up-regulated in murine astrocytes by treatment with trimethyltin and plays an important role to protect neurons [12]. Recently, in addition to neutrophins, neuroactive steroids have been noted as endogenous neuroprotective molecules. However, there is no existing report to show the effects of neuroactive steroids on neurotoxicity induced by organotin compounds. In this study, we examined the protective effects of progesterone on neuronal injury induced by TBT using rat organotypic hippocampal slice cultures. TBT has been one of the most commonly used organotin compounds and thus accumulates significantly in the environment.
2. Materials and methods 2.1. Materials Tributyltin chloride was obtained from Tokyo Chemical Industry (Tokyo, Japan). Progesterone was obtained from Sigma–Aldrich (St. Louis, MO, USA). Finasteride and (−)-bicuculline methiodide were purchased from Enzo Life Science International (Farmingdale, NY, USA). Allopregnanolone was obtained from Agrisera (Vannas, Sweden). Mifepristone and muscimol were purchased from Cayman Chemical (Ann Arbor, MI, USA). SU-10603 was a kind gift from Dr. C.R. Jefcoate, University of Wisconsin. All other chemicals were obtained from Nacalai Tesque (Kyoto, Japan) or Sigma–Aldrich and were of reagent grade.
2.4. Drug treatment Stock solutions of TBT (3 mM), progesterone (10 mM), allopregnanolone (10 mM) and finasteride (100 mM) were prepared with ethanol. Stock solutions of mifepristone (20 mM) and SU10603 (10 mM) were prepared with dimethyl sulfoxide. Bicuculline (10 mM) and muscimol (10 mM) were dissolved in phosphatebuffered saline. Final concentrations of these reagents for treatment of the slices were decided by preliminary experiments to fulfill the following criteria; (i) the reagents alone did not induce neuronal toxicity and (ii) the concentrations of the reagents were not largely different from those in previous studies. Progesterone or allopregnanolone was added to the serum-free culture medium 2 h before treatment with TBT. The pre-treatment period was decided by preliminary experiments. Mifepristone, finasteride, SU-10603 or bicuculline was added 20 min before treatment with progesterone or allopregnanolone. Muscimol was added 20 min before treatment with TBT. 2.5. Measurement of cell death To visualize neuronal cell death in three different regions of the slice (the CA1, CA3 and dentate gyrus (DG)), hippocampal slices were stained by adding propidium iodide (PI) into the culture medium at a concentration of 1 g/mL throughout the TBT or vehicle treatment, according to the method reported previously [13]. Slices were excited with a 540 ± 25 nm light, and the emitted fluorescence was acquired at 605 ± 55 nm on an inverted fluorescent microscope (BZ-9000, Keyence, Osaka, Japan) to determine the cellular PI uptake. All cells were killed after the experiment by keeping the cultures at 4 ◦ C for 48 h, and the value of the maximum PI uptake for each slice was obtained by microscopic observation and subsequent analysis. The cell injury was expressed as the percentage of cell death, which was calculated by the formula: (PI uptake/maximum PI uptake) × 100. Five to seven slices were used for each of three to five separate experiments. 2.6. Quantification of allopregnanolone
2.2. Animals All procedures performed on animals were in accordance with the Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, Japan and the Animal Care and Use Committee of Hiroshima University, Hiroshima, Japan. Postnatal 7day-old Wistar rats were obtained from CLEA Japan (Tokyo, Japan) and were maintained in a temperature-controlled animal facility with 12 h light–dark cycles.
Hippocampal slices were washed with ice-cold phosphatebuffered saline and then collected. The allopregnanolone concentration was determined by Asuka Pharmamedical Co. Ltd. (Kawasaki, Japan) using liquid chromatography–tandem mass spectrometry methods. 2.7. Statistical analyses All data are expressed as the mean ± standard error (S.E.). Data were statistically analyzed by one way analysis of variance (ANOVA) followed by Bonferroni post hoc analysis. Probability (P) values <0.05 were considered to be statistically significant.
2.3. Organotypic hippocampal slice cultures 3. Results Rat organotypic hippocampal slices were prepared according to our previous report [13]. Briefly, hippocampi were dissected from postnatal 9- to 11-day-old Wistar rats and then cut transversely into 300 m slices. Slice culture of hippocampus from older than 15-day rat was not succeeded in our experimental condition. Tissue sections were plated on Millicell or Omnipore membranes (Millipore, Bedford, MA) that were then inserted into culture plates filled with the culture medium. The slices were maintained in a humidified CO2 incubator at 37 ◦ C. The culture medium was changed every 2 days, and sections were cultured for 6 or 7 days. One day before any treatment, the slices were transferred to serum-free medium and then used in the experiments.
3.1. Suppressive effects of progesterone on neuronal cell death induced by TBT The treatment of rat hippocampal slices with various concentrations of TBT (0.1, 0.3, 1, 3 and 10 M) for 24 h increased the PI-derived fluorescence in a dose-dependent manner, indicating that dose-dependent cell death was elicited by TBT (Fig. 1), as reported previously [13]. Hippocampal neurons in CA1, CA3 and DG were similarly injured by treatment with TBT (Fig. 1). Because approximately 50% of the hippocampal cells were dyed by treatment with 3 M TBT, this condition was suitable for analyzing the mechanism of TBT-induced neurotoxicity.
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Fig. 1. Neuronal injury induced by TBT in rat hippocampal slices. Rat hippocampal slices were exposed to different concentrations of TBT (0.1, 0.3, 1, 3 and 10 M) for 24 h, and then cell death was assessed by PI uptake. The reported values are the mean ± S.E. of 5 separate experiments.
Neurotoxicity of TBT was elicited quickly, within three hours after the addition of the toxin [13]. So, we pre-added progesterone before administration of TBT to evaluate the protective effect. When rat hippocampal slices were pretreated with various concentrations of progesterone for 2 h and then cultured with 3 M TBT for 24 h, neuronal cell death induced by TBT was attenuated in a dose-dependent manner (Fig. 2). Statistical analysis showed that the protective effects of progesterone, above the concentration of 0.1 M, were significant (Fig. 2). These results indicate the protective action of progesterone on TBT-induced neuronal injury in rat hippocampal slices. 3.2. Suppressive effects of allopregnanolone, which is converted from progesterone, on neuronal cell death induced by TBT Progesterone is known to bind to the progesterone receptor to protect cells via genomic or non-genomic pathways [14]. In addition, 17-estradiol and allopregnanolone, which are metabolites of progesterone, were also reported to provide neuroprotective action [15]. Due to this previous knowledge, we then examined
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the protective mechanism of progesterone against TBT-induced neurotoxicity. Mifepristone is a progesterone receptor antagonist. Finasteride is a 5␣-reductase inhibitor that blocks the conversion of progesterone to allopregnanolone (Fig. 3A). SU-10603 is an inhibitor of cytochrome P450 17␣ and thus suppresses the synthesis of 17-estradiol via testosterone (Fig. 3A). Mifepristone (10 M), finasteride (100 M) or SU-10603 (20 M) alone causes no toxicity against hippocampal neurons. Treatment with 1 M progesterone significantly attenuated cell death induced by TBT (Fig. 3B), as presented in Fig. 2. While pretreatment with mifepristone or SU-10603 showed no effect on the protective actions of progesterone on TBT-induced neuronal injury, finasteride completely canceled the protective effects of progesterone (Fig. 3B). The allopregnanolone content in untreated rat hippocampal slices was close to the detection limit (Fig. 3C). Treatment of the slices with progesterone greatly increased the allopregnanolone content (Fig. 3C). Importantly, pretreatment with finasteride greatly suppressed the increment of allopregnanolone induced by progesterone (Fig. 3C). Furthermore, pretreatment with allopregnanolone clearly suppressed neuronal injury induced by TBT in a dosedependent manner (Fig. 4). Statistical analysis revealed that the protective effects of allopregnanolone above a concentration of 0.1 M were significant (Fig. 4). These results indicate that allopregnanolone converted from progesterone protects hippocampal neurons from TBT toxicity. 3.3. Involvement of the GABAA receptor in the suppression of TBT-induced neurotoxicity by progesterone Increasing evidence shows that allopregnanolone acts on GABAA receptors as an agonist [16]. Therefore, we next investigated the contribution of GABAA receptors on neuroprotection by progesterone. Treatment with 1 M progesterone or 1 M allopregnanolone significantly attenuated neuronal cell injury induced by TBT (Fig. 5), as presented in Figs. 2 and 4. Note that pretreatment with bicuculline, a potent GABAA receptor antagonist, significantly abrogated the neuroprotective actions of progesterone or allopregnanolone (Fig. 5). Treatment with bicuculline alone showed no toxicity in the hippocampal slices (Fig. 5). Furthermore, pretreatment with muscimol, a potent GABAA receptor agonist, partially but significantly suppressed neuronal cell death induced by TBT (Fig. 6). These data suggest that the GABAA receptor is involved in the protective effects of progesterone on neuronal injury induced by TBT. 4. Discussion
Fig. 2. Protective effects of progesterone on neuronal injury induced by TBT. Rat hippocampal slices were pretreated with various concentrations of progesterone (Prog, 0.01, 0.1, 1 and 10 M). After a 2-h incubation, the slices were exposed to 3 M TBT for 24 h. Cell death was evaluated by PI uptake. The reported values are the mean ± S.E. of 5 separate experiments. *P < 0.05, **P < 0.01 vs. 3 M TBT-treated group.
In this study, we examined the protective effects of progesterone on hippocampal neuronal injury induced by TBT. Progesterone dose-dependently attenuated neuronal cell death induced by TBT. In addition, judging from the following 3 points, the protective effects of progesterone from TBT-induced neurotoxicity were mediated by allopregnanolone, which was converted from progesterone in the hippocampal slices: (1) the neuroprotective action of progesterone was suppressed by treatment with finasteride, which inhibits 5␣-reductase to abrogate the conversion from progesterone to allopregnanolone; (2) allopregnanolone alone showed neuroprotective effects; and (3) treatment of the slices with progesterone increased the contents of allopregnanolone, whereas pretreatment with finasteride attenuated the increment of allopregnanolone evoked by treatment with progesterone. Allopregnanolone is an active metabolite of progesterone. The anesthetic and anxiolytic effects of allopregnanolone, caused by directly interacting with GABAA receptors to potentiate currents, are well demonstrated [16,17]. Recently, allopregnanolone was
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Fig. 3. Abrogation of the protective action of progesterone by finasteride. (A) Pathway of progesterone-related metabolism in the rat hippocampus. StAR, steroidogenic acute regulatory protein (Star); TSPO, translocator protein (Tspo); 3-HSD, 3-hydroxysteroid dehydrogenase/5-4 isomerase (Hsd3b1/2); 3␣-HSD, 3␣-hydroxysteroid dehydrogenase (Akr1c14); 17-HSD3, 17-hydroxysteroid dehydrogenase type-3 (Hsd17b3). (B) Rat hippocampal slices were pretreated with a progesterone receptor antagonist, mifepristone (Mife, 10 M), a 5␣-reductase inhibitor, finasteride (Fina, 100 M) and a cytochrome P450 17␣ inhibitor, SU-10603 (SU, 20 M); then, progesterone (1 M) was added to the culture. After a 2-h incubation, the slices were exposed to 3 M TBT for 24 h. Cell death was evaluated by PI uptake. The reported values are the mean ± S.E. of 5 separate experiments. **P < 0.01 vs. 3 M TBT-treated group. ## P < 0.01 vs. 3 M TBT and 1 M progesterone-treated group. (C) Rat hippocampal slices were pretreated with finasteride (100 M), and then progesterone (1 M) was added to the culture. After a 2-h incubation, the slices were collected, and the concentration of allopregnanolone was measured by liquid chromatography–tandem mass spectrometry methods. The reported values are the mean ± S.E. of 3 separate experiments. **P < 0.01 vs. untreated group. ## P < 0.01 vs. 1 M progesterone-treated group.
reported to suppress neuronal injury occurring in various animal models such as cerebral ischemia [18], Alzheimer’s disease [19] and excitotoxicity by kainate [20]. Furthermore, neuroprotection by allopregnanolone was revealed to be dependent on the activity of GABAA receptors [21]. In this study, neuroprotection by allopregnanolone as well as progesterone was canceled by pretreatment with bicuculline, a potent GABAA receptor antagonist. Therefore, we considered that allopregnanolone protects hippocampal neurons from TBT via a GABAA receptor-dependent mechanism. This mechanism is supported by the data indicating that a GABAA receptor agonist, muscimol, attenuated TBT-induced neuronal injury. Taken together, our data indicate that allopregnanolone, which is converted from progesterone in the hippocampal slices, attenuates TBT-induced neuronal injury by the stimulation of GABAA receptors. The brain has been considered to be a target of the peripheral endocrine system, but it was recently discovered that the
original endocrine system is present in the brain and synthesizes steroid hormones. We revealed that 17-estradiol was synthesized in rat hippocampal slices, and the content of 17-estradiol in 3-day-old cultured slices was approximately 50 fmol/mg protein [25]. Although progesterone content in the hippocampus has never been reported, the amount of progesterone in the mouse brain and in the rat cerebellum was reportedly 6.0 fmol/mg tissue [26] and 15 fmol/mg tissue [27], respectively. This study showed that allopregnanolone content in untreated hippocampal slices was below 1 fmol/mg tissue. Therefore, the intracerebral concentrations of progesterone and allopregnanolone are considered to be much lower than those of 17-estradiol in the brain. However, interestingly, because the amount of allopregnanolone increased greatly with the addition of progesterone, allopregnanolone-synthesizing enzymes from progesterone (5␣-reductase and 3␣-hydroxy-5steroid dehydrogenase) are suggested to maintain high activity. Higashi et al. demonstrated that high levels of allopregnanolone
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Fig. 4. Protective effects of allopregnanolone on neuronal injury induced by TBT. Rat hippocampal slices were pretreated with various concentrations of allopregnanolone (Allo, 0.01, 0.1, 1 and 10 M). After a 2-h incubation, the slices were exposed to 3 M TBT for 24 h. Cell death was evaluated by PI uptake. The reported values are the mean ± S.E. of 5 separate experiments. **P < 0.01 vs. 3 M TBT-treated group.
were detected in the brains of stressed rats but not in the brains of normal rats [28]. This leads us to believe that the increase in allopregnanolone is a defensive response to acute stress. In the stressed condition, increases in progesterone supply either by de novo synthesis as a neurosteroid or from peripheral tissues are considered to cause the increase in allopregnanolone in the brain to protect neuronal cells. We previously reported that ROS were produced by treatment with TBT in rat hippocampal slices [13]. Some steroid hormones are known to directly scavenge ROS [22]. However, in the present study, the neuroprotective effects of progesterone and allopregnanolone were completely canceled by pretreatment with finasteride or bicuculline. Therefore, the ROS-eliminating activity of progesterone and allopregnanolone was considered not to be involved in the mechanism of neuroprotection. Nakatsu et al. showed that the potentiation of glutamate release occurred upstream of ROS generation when rat cortical neurons were stimulated by TBT [23]. Furthermore, a GABAA receptor agonist,
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Fig. 6. Protective action of muscimol on neuronal injury induced by TBT. Rat hippocampal slices were pretreated with 10 M muscimol (Mus) for 20 min, and then 3 M TBT was added to the dish. After a 24-h incubation, cell death was evaluated by PI uptake. The reported values are the mean ± S.E. of 5 separate experiments. **P < 0.01 vs. 3 M TBT-treated group.
muscimol, was demonstrated to attenuate glutamate release and subsequent ROS production to suppress cell death induced by amyloid  proteins (25–35) in rat cortical neurons [24]. Therefore, in our experimental system, the attenuation of excitotoxicity and subsequent oxidative injury induced by TBT might be involved in the neuroprotective effects of progesterone due to the activation of GABAA receptors. The protective effect of progesterone and allopregnanolone from TBT stimuli was, however, only partial in the hippocampal neurons, indicating the diverse mechanisms of cell death induced by TBT. Further study will be needed to explore this point. In conclusion, progesterone protected hippocampal neurons from TBT-induced cell death. Progesterone was readily converted to allopregnanolone, and the neuroprotective activity could be controlled with modulation of GABAA receptor activity by allopregnanolone. This is the first study to reveal the protective effects of a neuroactive steroid, in this case progesterone, on neurotoxicity by TBT, which is an environmental toxin largely accumulated in the worldwide hydrosphere. Acknowledgements We thank Dr. Eiji Munetsuna, Fujita Health University for technical contributions to the hippocampal slice cultures. This work was supported by Grants-in Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. References
Fig. 5. Attenuation of protective effects of progesterone and allopregnanolone by bicuculline. Rat hippocampal slices were pretreated with 100 M bicuculline (Bicuc) for 20 min progesterone (1 M) or allopregnanolone (1 M) was added and cultured for 2 h. The slices were exposed to 3 M TBT for 24 h, and then cell death was evaluated by PI uptake. The reported values are the mean ± S.E. of 5 separate experiments. **P < 0.01 vs. 3 M TBT-treated group. ## P < 0.01 vs. 3 M TBT and 1 M progesterone or 1 M allopregnanolone treated group.
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Allopregnanolone-mediated protective effects of progesterone on tributyltin-induced neuronal injury in rat hippocampal slices Yasuhiro Ishihara, Tomohito Kawami, Atsuhiko Ishida, Takeshi Yamazaki ∗ Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan
a r t i c l e
i n f o
Article history: Received 30 August 2012 Received in revised form 20 December 2012 Accepted 20 December 2012 Keywords: Neuroprotection Progesterone Allopregnanolone Tributyltin Hippocampal slices GABAA receptor
a b s t r a c t Increasing evidence shows that progesterone, a neuroactive steroid, has protective actions in central nervous system, but there is little evidence to show the protective mechanism of progesterone on neurotoxicity induced by environmental chemicals. In this study, we examined the effects of progesterone on neuronal injury induced by tributyltin (TBT) in rat hippocampal slices. Treatment with progesterone dose-dependently suppressed hippocampal neuronal injury induced by TBT. The neuroprotective action of progesterone was completely canceled with pretreatment by finasteride, a 5␣-reductase inhibitor, but it was not affected by mifepristone, a progesterone receptor antagonist, or by SU-10603, a cytochrome P450 17␣ inhibitor. The content of allopregnanolone in the slices was significantly increased by treatment with progesterone, and this increment was greatly suppressed with a pretreatment of finasteride. Treatment with allopregnanolone attenuated neuronal injury induced by TBT in a dose-dependent manner. The neuroprotective effects not only of progesterone but also of allopregnanolone were canceled by bicuculline, a potent gamma-aminobutyric acid A (GABAA ) receptor antagonist. Pretreatment with muscimol, a GABAA receptor agonist, attenuated hippocampal neuronal injury elicited by TBT. Taken together, allopregnanolone converted from progesterone in hippocampal slices could protect neurons from TBTinduced neurotoxicity due to a GABAA receptor-dependent mechanism. One of the physiological roles of neuroactive steroids might be neuroprotection from environmental chemicals. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Steroid hormones synthesized in and secreted from peripheral endocrine glands pass through the blood–brain barrier and then perform functions in the central nervous system. In addition, it was observed that the brain possesses an inherent endocrine system and synthesizes some steroid hormones [1]. Recently, increasing evidence has shown that a neuroactive steroid, progesterone, protects neurons. Treatment with progesterone increased the expression of Bcl-2 and decreased the contents of active caspase-3 to suppress apoptosis [2,3]. Progesterone also seems to be beneficial in preventing mitochondrial dysfunction, which results in the loss of hippocampal cells after a controlled cortical contusion [4]. Furthermore, it has been reported that progesterone protects hippocampal slice cultures from cell death following oxygen-glucose
Abbreviations: BDNF, brain-derived neurotrophic factor; GABA, gammaaminobutyric acid; NGF, nerve growth factor; ROS, reactive oxygen species; PI, propidium iodide; TBT, tributyltin. ∗ Corresponding author at: Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, 1-7-1, Kagamiyama, Higashi-Hiroshima 739-8521, Hiroshima, Japan. Tel.: +81 82 424 6527; fax: +81 82 424 0759. E-mail address: [email protected] (T. Yamazaki). 0960-0760/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jsbmb.2012.12.013
deprivation, largely due to its conversion to allopregnanolone via a gamma-aminobutyric acid A (GABAA ) receptor-dependent mechanism [5]. Progesterone is readily metabolized, in the brain, to allopregnanolone by activities of 5␣-reductase and 3␣hydroxysteroid dehydrogenase [5]. Organotin compounds have long been used as thermal stabilizers, catalytic agents and biocidal compounds for preserving wood, textiles, cordage fibers and electronic equipment. Among them, tributyltin (TBT) has been most widely used in paint formulations to prevent marine fouling on ships, boats and fish-farming nets. Environmental surveying and monitoring of TBT are conducted to prevent the consumption of bioaccumulated TBT by humans. The average intake of TBT by humans from market-bought seafood has been estimated to vary worldwide between 0.18 and 2.6 g per day per person [6], and the presence of butyltin compounds, including TBT, reportedly exists at concentrations between 50 nM and 400 nM in human blood [7]. Therefore, the effects of TBT on the human brain are now of great concern. Because many environmental chemicals, including organotin compounds, are lipophilic and have high blood–brain barrier permeability, the central nervous system is exposed to exogenous chemicals. The administration of TBT elicited abnormal behavior and the reduction of brain weight within the cerebellum and decreased synaptogenesis in rats [8–10]. This is evidence
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suggesting that the central nervous system is a primary target of TBT. However, the central nervous system is equipped with some endogenous neuroprotectants against harmful chemicals. Some of the most well-known factors are neutrophins such as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). BDNF attenuated cellular injury of differentiated neural stem cells induced by trimetyltin by activating the pathways of mitogen-activated protein kinase and phosphoinositide-3kinase/Akt signals [11]. NGF was up-regulated in murine astrocytes by treatment with trimethyltin and plays an important role to protect neurons [12]. Recently, in addition to neutrophins, neuroactive steroids have been noted as endogenous neuroprotective molecules. However, there is no existing report to show the effects of neuroactive steroids on neurotoxicity induced by organotin compounds. In this study, we examined the protective effects of progesterone on neuronal injury induced by TBT using rat organotypic hippocampal slice cultures. TBT has been one of the most commonly used organotin compounds and thus accumulates significantly in the environment.
2. Materials and methods 2.1. Materials Tributyltin chloride was obtained from Tokyo Chemical Industry (Tokyo, Japan). Progesterone was obtained from Sigma–Aldrich (St. Louis, MO, USA). Finasteride and (−)-bicuculline methiodide were purchased from Enzo Life Science International (Farmingdale, NY, USA). Allopregnanolone was obtained from Agrisera (Vannas, Sweden). Mifepristone and muscimol were purchased from Cayman Chemical (Ann Arbor, MI, USA). SU-10603 was a kind gift from Dr. C.R. Jefcoate, University of Wisconsin. All other chemicals were obtained from Nacalai Tesque (Kyoto, Japan) or Sigma–Aldrich and were of reagent grade.
2.4. Drug treatment Stock solutions of TBT (3 mM), progesterone (10 mM), allopregnanolone (10 mM) and finasteride (100 mM) were prepared with ethanol. Stock solutions of mifepristone (20 mM) and SU10603 (10 mM) were prepared with dimethyl sulfoxide. Bicuculline (10 mM) and muscimol (10 mM) were dissolved in phosphatebuffered saline. Final concentrations of these reagents for treatment of the slices were decided by preliminary experiments to fulfill the following criteria; (i) the reagents alone did not induce neuronal toxicity and (ii) the concentrations of the reagents were not largely different from those in previous studies. Progesterone or allopregnanolone was added to the serum-free culture medium 2 h before treatment with TBT. The pre-treatment period was decided by preliminary experiments. Mifepristone, finasteride, SU-10603 or bicuculline was added 20 min before treatment with progesterone or allopregnanolone. Muscimol was added 20 min before treatment with TBT. 2.5. Measurement of cell death To visualize neuronal cell death in three different regions of the slice (the CA1, CA3 and dentate gyrus (DG)), hippocampal slices were stained by adding propidium iodide (PI) into the culture medium at a concentration of 1 g/mL throughout the TBT or vehicle treatment, according to the method reported previously [13]. Slices were excited with a 540 ± 25 nm light, and the emitted fluorescence was acquired at 605 ± 55 nm on an inverted fluorescent microscope (BZ-9000, Keyence, Osaka, Japan) to determine the cellular PI uptake. All cells were killed after the experiment by keeping the cultures at 4 ◦ C for 48 h, and the value of the maximum PI uptake for each slice was obtained by microscopic observation and subsequent analysis. The cell injury was expressed as the percentage of cell death, which was calculated by the formula: (PI uptake/maximum PI uptake) × 100. Five to seven slices were used for each of three to five separate experiments. 2.6. Quantification of allopregnanolone
2.2. Animals All procedures performed on animals were in accordance with the Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, Japan and the Animal Care and Use Committee of Hiroshima University, Hiroshima, Japan. Postnatal 7day-old Wistar rats were obtained from CLEA Japan (Tokyo, Japan) and were maintained in a temperature-controlled animal facility with 12 h light–dark cycles.
Hippocampal slices were washed with ice-cold phosphatebuffered saline and then collected. The allopregnanolone concentration was determined by Asuka Pharmamedical Co. Ltd. (Kawasaki, Japan) using liquid chromatography–tandem mass spectrometry methods. 2.7. Statistical analyses All data are expressed as the mean ± standard error (S.E.). Data were statistically analyzed by one way analysis of variance (ANOVA) followed by Bonferroni post hoc analysis. Probability (P) values <0.05 were considered to be statistically significant.
2.3. Organotypic hippocampal slice cultures 3. Results Rat organotypic hippocampal slices were prepared according to our previous report [13]. Briefly, hippocampi were dissected from postnatal 9- to 11-day-old Wistar rats and then cut transversely into 300 m slices. Slice culture of hippocampus from older than 15-day rat was not succeeded in our experimental condition. Tissue sections were plated on Millicell or Omnipore membranes (Millipore, Bedford, MA) that were then inserted into culture plates filled with the culture medium. The slices were maintained in a humidified CO2 incubator at 37 ◦ C. The culture medium was changed every 2 days, and sections were cultured for 6 or 7 days. One day before any treatment, the slices were transferred to serum-free medium and then used in the experiments.
3.1. Suppressive effects of progesterone on neuronal cell death induced by TBT The treatment of rat hippocampal slices with various concentrations of TBT (0.1, 0.3, 1, 3 and 10 M) for 24 h increased the PI-derived fluorescence in a dose-dependent manner, indicating that dose-dependent cell death was elicited by TBT (Fig. 1), as reported previously [13]. Hippocampal neurons in CA1, CA3 and DG were similarly injured by treatment with TBT (Fig. 1). Because approximately 50% of the hippocampal cells were dyed by treatment with 3 M TBT, this condition was suitable for analyzing the mechanism of TBT-induced neurotoxicity.
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Fig. 1. Neuronal injury induced by TBT in rat hippocampal slices. Rat hippocampal slices were exposed to different concentrations of TBT (0.1, 0.3, 1, 3 and 10 M) for 24 h, and then cell death was assessed by PI uptake. The reported values are the mean ± S.E. of 5 separate experiments.
Neurotoxicity of TBT was elicited quickly, within three hours after the addition of the toxin [13]. So, we pre-added progesterone before administration of TBT to evaluate the protective effect. When rat hippocampal slices were pretreated with various concentrations of progesterone for 2 h and then cultured with 3 M TBT for 24 h, neuronal cell death induced by TBT was attenuated in a dose-dependent manner (Fig. 2). Statistical analysis showed that the protective effects of progesterone, above the concentration of 0.1 M, were significant (Fig. 2). These results indicate the protective action of progesterone on TBT-induced neuronal injury in rat hippocampal slices. 3.2. Suppressive effects of allopregnanolone, which is converted from progesterone, on neuronal cell death induced by TBT Progesterone is known to bind to the progesterone receptor to protect cells via genomic or non-genomic pathways [14]. In addition, 17-estradiol and allopregnanolone, which are metabolites of progesterone, were also reported to provide neuroprotective action [15]. Due to this previous knowledge, we then examined
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the protective mechanism of progesterone against TBT-induced neurotoxicity. Mifepristone is a progesterone receptor antagonist. Finasteride is a 5␣-reductase inhibitor that blocks the conversion of progesterone to allopregnanolone (Fig. 3A). SU-10603 is an inhibitor of cytochrome P450 17␣ and thus suppresses the synthesis of 17-estradiol via testosterone (Fig. 3A). Mifepristone (10 M), finasteride (100 M) or SU-10603 (20 M) alone causes no toxicity against hippocampal neurons. Treatment with 1 M progesterone significantly attenuated cell death induced by TBT (Fig. 3B), as presented in Fig. 2. While pretreatment with mifepristone or SU-10603 showed no effect on the protective actions of progesterone on TBT-induced neuronal injury, finasteride completely canceled the protective effects of progesterone (Fig. 3B). The allopregnanolone content in untreated rat hippocampal slices was close to the detection limit (Fig. 3C). Treatment of the slices with progesterone greatly increased the allopregnanolone content (Fig. 3C). Importantly, pretreatment with finasteride greatly suppressed the increment of allopregnanolone induced by progesterone (Fig. 3C). Furthermore, pretreatment with allopregnanolone clearly suppressed neuronal injury induced by TBT in a dosedependent manner (Fig. 4). Statistical analysis revealed that the protective effects of allopregnanolone above a concentration of 0.1 M were significant (Fig. 4). These results indicate that allopregnanolone converted from progesterone protects hippocampal neurons from TBT toxicity. 3.3. Involvement of the GABAA receptor in the suppression of TBT-induced neurotoxicity by progesterone Increasing evidence shows that allopregnanolone acts on GABAA receptors as an agonist [16]. Therefore, we next investigated the contribution of GABAA receptors on neuroprotection by progesterone. Treatment with 1 M progesterone or 1 M allopregnanolone significantly attenuated neuronal cell injury induced by TBT (Fig. 5), as presented in Figs. 2 and 4. Note that pretreatment with bicuculline, a potent GABAA receptor antagonist, significantly abrogated the neuroprotective actions of progesterone or allopregnanolone (Fig. 5). Treatment with bicuculline alone showed no toxicity in the hippocampal slices (Fig. 5). Furthermore, pretreatment with muscimol, a potent GABAA receptor agonist, partially but significantly suppressed neuronal cell death induced by TBT (Fig. 6). These data suggest that the GABAA receptor is involved in the protective effects of progesterone on neuronal injury induced by TBT. 4. Discussion
Fig. 2. Protective effects of progesterone on neuronal injury induced by TBT. Rat hippocampal slices were pretreated with various concentrations of progesterone (Prog, 0.01, 0.1, 1 and 10 M). After a 2-h incubation, the slices were exposed to 3 M TBT for 24 h. Cell death was evaluated by PI uptake. The reported values are the mean ± S.E. of 5 separate experiments. *P < 0.05, **P < 0.01 vs. 3 M TBT-treated group.
In this study, we examined the protective effects of progesterone on hippocampal neuronal injury induced by TBT. Progesterone dose-dependently attenuated neuronal cell death induced by TBT. In addition, judging from the following 3 points, the protective effects of progesterone from TBT-induced neurotoxicity were mediated by allopregnanolone, which was converted from progesterone in the hippocampal slices: (1) the neuroprotective action of progesterone was suppressed by treatment with finasteride, which inhibits 5␣-reductase to abrogate the conversion from progesterone to allopregnanolone; (2) allopregnanolone alone showed neuroprotective effects; and (3) treatment of the slices with progesterone increased the contents of allopregnanolone, whereas pretreatment with finasteride attenuated the increment of allopregnanolone evoked by treatment with progesterone. Allopregnanolone is an active metabolite of progesterone. The anesthetic and anxiolytic effects of allopregnanolone, caused by directly interacting with GABAA receptors to potentiate currents, are well demonstrated [16,17]. Recently, allopregnanolone was
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Fig. 3. Abrogation of the protective action of progesterone by finasteride. (A) Pathway of progesterone-related metabolism in the rat hippocampus. StAR, steroidogenic acute regulatory protein (Star); TSPO, translocator protein (Tspo); 3-HSD, 3-hydroxysteroid dehydrogenase/5-4 isomerase (Hsd3b1/2); 3␣-HSD, 3␣-hydroxysteroid dehydrogenase (Akr1c14); 17-HSD3, 17-hydroxysteroid dehydrogenase type-3 (Hsd17b3). (B) Rat hippocampal slices were pretreated with a progesterone receptor antagonist, mifepristone (Mife, 10 M), a 5␣-reductase inhibitor, finasteride (Fina, 100 M) and a cytochrome P450 17␣ inhibitor, SU-10603 (SU, 20 M); then, progesterone (1 M) was added to the culture. After a 2-h incubation, the slices were exposed to 3 M TBT for 24 h. Cell death was evaluated by PI uptake. The reported values are the mean ± S.E. of 5 separate experiments. **P < 0.01 vs. 3 M TBT-treated group. ## P < 0.01 vs. 3 M TBT and 1 M progesterone-treated group. (C) Rat hippocampal slices were pretreated with finasteride (100 M), and then progesterone (1 M) was added to the culture. After a 2-h incubation, the slices were collected, and the concentration of allopregnanolone was measured by liquid chromatography–tandem mass spectrometry methods. The reported values are the mean ± S.E. of 3 separate experiments. **P < 0.01 vs. untreated group. ## P < 0.01 vs. 1 M progesterone-treated group.
reported to suppress neuronal injury occurring in various animal models such as cerebral ischemia [18], Alzheimer’s disease [19] and excitotoxicity by kainate [20]. Furthermore, neuroprotection by allopregnanolone was revealed to be dependent on the activity of GABAA receptors [21]. In this study, neuroprotection by allopregnanolone as well as progesterone was canceled by pretreatment with bicuculline, a potent GABAA receptor antagonist. Therefore, we considered that allopregnanolone protects hippocampal neurons from TBT via a GABAA receptor-dependent mechanism. This mechanism is supported by the data indicating that a GABAA receptor agonist, muscimol, attenuated TBT-induced neuronal injury. Taken together, our data indicate that allopregnanolone, which is converted from progesterone in the hippocampal slices, attenuates TBT-induced neuronal injury by the stimulation of GABAA receptors. The brain has been considered to be a target of the peripheral endocrine system, but it was recently discovered that the
original endocrine system is present in the brain and synthesizes steroid hormones. We revealed that 17-estradiol was synthesized in rat hippocampal slices, and the content of 17-estradiol in 3-day-old cultured slices was approximately 50 fmol/mg protein [25]. Although progesterone content in the hippocampus has never been reported, the amount of progesterone in the mouse brain and in the rat cerebellum was reportedly 6.0 fmol/mg tissue [26] and 15 fmol/mg tissue [27], respectively. This study showed that allopregnanolone content in untreated hippocampal slices was below 1 fmol/mg tissue. Therefore, the intracerebral concentrations of progesterone and allopregnanolone are considered to be much lower than those of 17-estradiol in the brain. However, interestingly, because the amount of allopregnanolone increased greatly with the addition of progesterone, allopregnanolone-synthesizing enzymes from progesterone (5␣-reductase and 3␣-hydroxy-5steroid dehydrogenase) are suggested to maintain high activity. Higashi et al. demonstrated that high levels of allopregnanolone
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Fig. 4. Protective effects of allopregnanolone on neuronal injury induced by TBT. Rat hippocampal slices were pretreated with various concentrations of allopregnanolone (Allo, 0.01, 0.1, 1 and 10 M). After a 2-h incubation, the slices were exposed to 3 M TBT for 24 h. Cell death was evaluated by PI uptake. The reported values are the mean ± S.E. of 5 separate experiments. **P < 0.01 vs. 3 M TBT-treated group.
were detected in the brains of stressed rats but not in the brains of normal rats [28]. This leads us to believe that the increase in allopregnanolone is a defensive response to acute stress. In the stressed condition, increases in progesterone supply either by de novo synthesis as a neurosteroid or from peripheral tissues are considered to cause the increase in allopregnanolone in the brain to protect neuronal cells. We previously reported that ROS were produced by treatment with TBT in rat hippocampal slices [13]. Some steroid hormones are known to directly scavenge ROS [22]. However, in the present study, the neuroprotective effects of progesterone and allopregnanolone were completely canceled by pretreatment with finasteride or bicuculline. Therefore, the ROS-eliminating activity of progesterone and allopregnanolone was considered not to be involved in the mechanism of neuroprotection. Nakatsu et al. showed that the potentiation of glutamate release occurred upstream of ROS generation when rat cortical neurons were stimulated by TBT [23]. Furthermore, a GABAA receptor agonist,
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Fig. 6. Protective action of muscimol on neuronal injury induced by TBT. Rat hippocampal slices were pretreated with 10 M muscimol (Mus) for 20 min, and then 3 M TBT was added to the dish. After a 24-h incubation, cell death was evaluated by PI uptake. The reported values are the mean ± S.E. of 5 separate experiments. **P < 0.01 vs. 3 M TBT-treated group.
muscimol, was demonstrated to attenuate glutamate release and subsequent ROS production to suppress cell death induced by amyloid  proteins (25–35) in rat cortical neurons [24]. Therefore, in our experimental system, the attenuation of excitotoxicity and subsequent oxidative injury induced by TBT might be involved in the neuroprotective effects of progesterone due to the activation of GABAA receptors. The protective effect of progesterone and allopregnanolone from TBT stimuli was, however, only partial in the hippocampal neurons, indicating the diverse mechanisms of cell death induced by TBT. Further study will be needed to explore this point. In conclusion, progesterone protected hippocampal neurons from TBT-induced cell death. Progesterone was readily converted to allopregnanolone, and the neuroprotective activity could be controlled with modulation of GABAA receptor activity by allopregnanolone. This is the first study to reveal the protective effects of a neuroactive steroid, in this case progesterone, on neurotoxicity by TBT, which is an environmental toxin largely accumulated in the worldwide hydrosphere. Acknowledgements We thank Dr. Eiji Munetsuna, Fujita Health University for technical contributions to the hippocampal slice cultures. This work was supported by Grants-in Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. References
Fig. 5. Attenuation of protective effects of progesterone and allopregnanolone by bicuculline. Rat hippocampal slices were pretreated with 100 M bicuculline (Bicuc) for 20 min progesterone (1 M) or allopregnanolone (1 M) was added and cultured for 2 h. The slices were exposed to 3 M TBT for 24 h, and then cell death was evaluated by PI uptake. The reported values are the mean ± S.E. of 5 separate experiments. **P < 0.01 vs. 3 M TBT-treated group. ## P < 0.01 vs. 3 M TBT and 1 M progesterone or 1 M allopregnanolone treated group.
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