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Mitochondrial complex I inhibition depletes plasma testosterone in the rotenone model of Parkinson's disease
Physiology & Behavior 83 (2004) 395 – 400
Mitochondrial complex I inhibition depletes plasma testosterone in the rotenone model of Parkinson’s disease M. Alam, W.J Schmidt* Zoological Institute, Department of Neuropharmacology, Morgenstelle 28E, University of Tuebingen, 72076 Tuebingen, Germany Received 5 April 2004; received in revised form 29 July 2004; accepted 11 August 2004
Abstract Age-related depletion of testosterone may increase the brain’s vulnerability to parkinsonian- or Alzheimer’s-like neurodegenerative disorders. In rats, rotenone, a mitochondrial complex I inhibitor, causes specific nigral dopaminergic neurodegeneration producing parkinsonian symptoms. In this study, rotenone was administered on a daily basis (2 mg/kg i.p.) to two groups of rats, over a period of 30 and 60 days, respectively. In order to contribute towards the validation of the rotenone rat model, the changing level of the peripheral sex steroid hormone, testosterone, which would also mimic those found in Parkinson’s disease (PD) patients, was evaluated. Parallel to this, prolactin, luteinizing hormone (LH), the nonsexual steroid thyroid-stimulating hormone, and the corticosterone hormone levels in the peripheral blood plasma were measured to show whether other hormones have also been affected by complex I inhibition. The rotenone treatment caused a decrease of testosterone level in the peripheral blood plasma. There were no differences in the thyroid hormone and prolactin but increases in leutinizing hormone and corticosterone were observed. Data from this study indicate that rotenone depleted the sex steroid hormone which is preferentially produced in the periphery, e.g., adrenal gland and testis. In conclusion, because a decrease in testosterone levels is also one of the comorbidities which are found in male PD patients, our results indicate that the rotenone model mimics PD symptoms not only on a neuronal and behavioral level, but also on the testosterone levels. D 2004 Elsevier Inc. All rights reserved. Keywords: Androgen; Testosterone; Corticosterone; Prolactin; Complex I; Dopamine
1. Introduction NADH-ubiquinone oxidoreductase deficiency of mitochondrial complex I can be present in a wide variety of biochemical and symptomatic phenotypes. Complex I is a huge multienzyme complex of 43 separately encoded gene products derived from both nuclear and mitochondrial genomes [19,29]. Rotenone inhibits mitochondrial complex I (NADH: ubiquinone oxidoreductase), which is a proximal component of the mitochondrial electron transport chain (ETC), and it produces the energy necessary for maintenance of all neuronal or cell functions.
* Corresponding author. Tel.: +49 7071 2974571; fax: +49 7071 295144. E-mail address: [email protected] (W.J. Schmidt). 0031-9384/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2004.08.010
The involvement of mitochondrial inhibition in neurodegeneration has received particular attention. Mitochondrial abnormalities have been identified in more common sporadic neurological disorders, including Alzheimer’s disease (AD) and Parkinson’s disease (PD), and they also occur as part of normal aging [25]. The chronic exposure of rotenone induces dopaminergic neuronal degeneration and formation of alpha-synuclein-positive cytoplasmic inclusions in nigral neurons [5,36]. PD is a movement disorder and mostly it is related to the disturbance of the nigrostriatal dopaminegric system. In most cases of PD, the mesolimbic dopaminergic system is also disturbed, which plays an important role as a modulator of the complex reward process that organizes motivated behaviors, such as drinking, feeding and sex, by the elevation of salient environmental stimuli. The mesolimbic dopaminergic system is known to play an
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M. Alam, W.J. Schmidt / Physiology & Behavior 83 (2004) 395–400
important facilitory role in copulation [26]. But it is clear that in PD, the tubero-infundibular dopaminergic neurons are spared [1]. There is substantial evidence indicating that steroid hormones modify vulnerability to neuronal insults. Prolonged elevation of corticosterone was shown to increase the vulnerability of nerve cells to the neurodegenerative processes in aging, cerebral ischemia and possibly other neurodegenerative diseases [35]. An increase in levels of glucocorticoids, like corticosterone, may exert many of their toxic effects via an interaction with dopamine (DA) and glutamate. It has been shown that this stress hormone also inhibits the uptake of DA [9]. Likewise, glucocorticoids can block the uptake of glutamate [14] which can further exacerbate the neurotoxic effect through excitotoxicity. Gonadal steroids, like testosterone in the male, play a role in the nervous system development. A decrease in circulating testosterone contributes to an age-related increase in glial fibrillary acidic protein (GFAP) which could render the brain more susceptible to neurodegeneration during aging [8]. Low serum testosterone has been associated with poor cognitive performance in elderly men and AD [20,28]. A number of in vivo studies have shown that androgens can rescue specific populations of motoneurons from both ontogenic- and axotomy-induced death. In in vitro studies, it has been shown that glucose-deprived hippocampal neurons are rescued by testosterone and testosterone itself also acts as an antioxidant via androgen-receptor-mediated mechanism [2,15]. Like other steroid hormones, the cellular effects of testosterone can be broadly grouped into genomic and nongenomic categories. The neuroprotective role of testosterone is mostly known through the genomic pathway via an androgen-receptor-dependent manner. The polyglutamine (CAG) polymorphism of androgen receptors has been associated with the risk of AD in men and with other brain disorders [24]. In the neonatal period, the presence of circulating testosterone is essential for the development of the substantia nigra region which exerts proconvulsant effects throughout the rat’s life, a unique feature of the male substantia nigra. The final maturation of the substantia nigra occurs in the prepubertal period, and is, in part, regulated by testosterone as well [38]. The influence of testosterone in neurodegenerative diseases, such as PD and AD, can be translated into new cures of the disorders with distinct sex-specific and more effective therapies. The level of circulating testosterone plays a key role in the development organization of the substantia nigra pars reticulata and its GABAA-ergic circuit that modify motor behavior in a precise, age-dependent fashion [38]. In contrast, gonad steroids increase the number and size of tyrosine hydroxylase (TH) immunoreactive neurons in some dopaminergic neurons’ population in both male and female rats [34,40]. Therefore, it seems that androgen levels are capable of influencing dopaminergic function via modulation of TH mRNA expression. The sex hormone testosterone has a
general and more prominent influence on the brain than reproduction and sexuality. Although it is well known that most pesticides have a pronounced effect on sex steroids, most of them are however not complex I inhibitors. Therefore, the present study explored whether a specific complex I inhibitor has a specific effect on the male sex steroid and other hormones.
2. Experimental procedures 2.1. Animals Forty adult male Sprague–Dawley rats (Charles River, Sulzfeld, Germany) aged 7 weeks were chosen for the experiment. At the beginning of the experiment, the rats weighed 230–240 g. Animals were housed in cages under a 12:12-h light/dark cycle in a room maintained at a constant temperature of 22 8C and 50–60% humidity with food and water available ad libitum. To minimize the stress for the rats during the injections, they were weighed and handled for 5 to 10 min the day before the injection. All treatments used in this experiment adhere to the international ethical standards and the German AnimalProtection Law and have been approved by local animal care committee (Tierschutzkommission, Regierungspra¨sidium Tu¨bingen, ZP 2/03). 2.2. Chemicals Rotenone was purchased from Sigma (St. Louis, MO) and was dissolved in sterile natural oil [middle chain triglycerides (MCT); Miglyol 812, clinical pharmacy, University Tuebingen]. Luciferin–luciferase enzymes were obtained from Sigma and protein assay kit from Bio-Rad (Richmond, CA, USA). 2.3. Treatment Rotenone emulsified in natural oil at 2.0 mg/ml was given intraperitoneally once a day at 1 ml/kg during 60 days. Oil was injected as vehicle to control rats (1 ml/kg). For the determination of the serum hormone assay and tissue adenosine triphosphate (ATP) from the adrenal gland and testis, two groups of rats were used, namely, vehicletreated (n=10) and rotenone-treated (n=10) groups. Control groups received oil for 30 and 60 days and rats were treated with rotenone for 30 and 60 days. 2.4. Blood sampling Rats were killed by decapitation with the use of a guillotine after 30 and 60 days and the trunk blood was collected after an overnight fast from both groups in a blood sample tube containing lithium-heparin (15 IU heparin/ml).
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2 vol (g/ml)], homogenized at 250–260 rpm for 3 min in the ice bath, and centrifuged at 960g at 4 8C for 15 min and followed by recentrifuging at 8000g for 10 min at 4 8C to give a supernatant. The supernatant was used to measure activities of ATP. 2.6. Hormone and tissue protein assay The in vitro testosterone test was carried out by enzyme sandwich immunoassay (Vitro ECI, Ortho Diagnostics, Rochester, NY, USA) in immunodiagnostics system. Intra- and interassay variations were b9.4%. The tissue protein assay was carried out using a Bio-Rad protein assay kit. 2.7. Evaluation of ATP content Tissue supernatants were incubated with a luciferin– luciferase mixture and the resulting luminescence was measured with a luminometer (LUMAT LB 9507; Berthhold Technologies, Bad Wildbad, Germany) according to the method [39] and the ratio ATP per unit protein was calculated. The analysis was done twice per animal. The mean values of relative intensityFS.D. was taken to plot the graph. 2.8. Statistical analysis Data are expressed as meansFS.E.M. Statistical analysis was carried out using the GB-Stat V5.4 software (Dynamic Microsystem, Silver Spring, MD). The data for peripheral hormone analysis were evaluated using two-way ANOVA followed by Fisher’s LSD comparison which was used for group differences. *Pb0.05 and **Pb0.01 were taken as levels of statistical significance.
3. Results Fig. 1. (a, b and c) Plasma concentrations of the (a) testosterone; (b) luteinizing hormone; and (c) thyroid-stimulating hormone in (2 mg/kg) rotenone-treated animals (n=10) in white bars and (1 ml/kg) oil-treated control animals (n=10) in hatched bars on day 30 and day 60. Values are shown as meanFS.E.M. P represents statistical analysis: *significant at Pb0.05 and **significant at Pb0.01.
The blood was centrifuged at 1500g for 15 min at 4 8C. The plasma was frozen at 20 8C until analyzed.
The depletion of serum concentration of total testosterone in rotenone-treated rats were 62.33% and 85.5% compared to oil-treated animals on days 30 and 60 (Fig. 1a). A significant effect of treatment was found in both groups. The levels of testosterone between groups were
Table 1 Levels of corticosterone and prolactin in control and rotenone-treated animals
2.5. Adrenal gland and testis sample preparation Adrenal glands were rapidly removed, adhering fat was cleaned, and after weighing, capped tightly and stored at 70 8C until the measurement of ATP. The left testis were removed from each animal’s body, weighed immediately, minced in cold phosphate buffer [10 mM, pH 7.0,
Day 30
Corticosterone (nmol/l) Prolactin (mIU/l)
Day 60
Cont
Rotenone
Cont
Rotenone
27.93F1.81
50.5F2.23
33.26F1.95
51.56F2.80
7.16F1.14
7.31F2.34
8.96F1.05
7.6F1.49
Values are shown as meanFS.E.M.
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F(1,18)=19,90 Pb0.003. The levels of sex hormonebinding globulin (SHBG) was the same in both groups of animals (data are not shown). The levels of luteinizing hormone (LH) and corticosterone in rotenone-treated animals were significantly higher than control animals (Fig. 1b and Table 1). On days 30 and 60, the levels of LH between the groups were F(1,18)=13.91, Pb0.001 and the level of corticosterone were F(1,18)=71.05, Pb0.0001, respectively. There were no differences in serum thyroidstimulating hormone (TSH) level and prolactin as compared to control on days 30 and 60 (Fig. 1c and Table 1). The levels of ATP in the adrenal gland and testis homogenates were significantly decreased within 30 and 60 days in a time-dependent manner compared to control which correlates with highly significant depletion of serum testosterone in peripheral circulation (Fig. 2a and b). The levels of ATP in adrenal glands and testis were F(1,12)=37.64, Pb0.0001 and F(1,12)=200.18, Pb0.0001, respectively.
Fig. 2. (a and b) The depletion of ATP in percentage on day 30 and day 60 in peripherally rotenone-treated animals group (2 mg/kg) has been shown by white bars and oil-treated animals (1 mg/kg) as a control group by hatched bars. Values are shown as meanFS.E.M. P represents statistical analysis: *significant at Pb0.05 and **significant at Pb0.01.
4. Discussion In the present study, we reveal an interesting relationship between complex I inhibition and a decrease of peripheral testosterone in male rats; in addition, a time-dependent decrease of testosterone and an increase of corticosterone and LH in the plasma has been observed. The present experiment also shows that the hypothalamic–pituitary axis was spared because the increase in LH in peripheral blood was due to a decrease of serum testosterone in periphery circulation, which means that the feedback mechanism was not disrupted; however, the TSH and prolactin secretion was unaffected. Testosterone is physiologically secreted by the adrenal glands and testis; its serum level is higher in men and normally decreases during life. This hormone is transported by the SHBG and albumin. Normally, SHBG is inversely related to circulating testosterone. In aging, the level of testosterone decreases and the levels of SHBG increase. The specific mechanism in the depletion of testosterone in different types of neurodegeneration are varied; for example, in Kwashiorkor and anorexia nervosa, the SHBG is elevated. In amyotrophic lateral sclerosis (ALS), the reduced level of testosterone might also be related to an altered binding to either SHBG or albumin [21]. However, in our experiment, we did not find any changes in SHBG in both the control and rotenone-treated groups. Glucocorticoids (cortisol in primates, corticosterone in rat) are secreted from the adrenal cortex upon activation of the hypothalamic–pituitary–adrenal axis as a response to stressful environmental stimuli. It has become evident that these hormones can modulate the neurodegenerative process that occurs in nerotoxic trauma, aging and AD [12,13]. Stress hormones, like corticosterone, have been found to impair learning and memory function (within several tens of minutes) rapidly via unknown neuronal mechanisms [27]. In primary hippocampal cultures, corticostrone decreases glutathione peroxidase, an antioxidant enzyme, and nicotinamide dinucleotide phosphate, which is an important substrate for the regeneration of glutathione [33]. Corticosterone resulted in extreme prolongation of the time duration of NMDA-receptor-mediated calcium signals, resulting in calcium overload neurotoxicity. High levels of corticosterone induce a rapid and nongenomic prolongation of NMDA-receptor-mediated calcium elevation, resulting in calcium-induced neurotoxicity [37]. In our experiment, we found that the level of corticosterone was higher compared to control. ATP is a source of energy and almost all cellular energy depends on mitochondrial pathways. Thus, the disruption of any pathway of mitochondria could be a cause of different types of diseases. The type of disease depends on which parts of tissues or organs are mostly or primarily affected. In our experiment, peripheral administration of rotenone decreased ATP to 34% and 73% on days 30 and 60 in adrenal glands. In the testis tissues on day 30, ATP levels
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were decreased by 34% and on day 60, ATP levels were decreased by 63%, which might be a further cause of depletion of testosterone as an energy-dependent manner. A variety of studies in animals and clinical observations have demonstrated that certain autonomic reflexes are altered under conditions of androgen deprivation. There are numerous possible mechanisms by which testosterone deprivation could influence reflex function. Growing evidence suggests a putative neuroprotective role for testosterone in the CNS, and in particular in motorneurons [6,18]. Testosterone reduces neuronal secretion of Alzheimer’s h-amyloid peptides [16]. However, evidence has linked testosterone deficiency to PD, AD and other neurodegenerative disorders [30,31], and it has also been shown that some nonmotor symptoms of PD may respond to testosterone replacement therapy [32]. Recently, it has been shown that specific complex I inhibitors are a cause of decreased tyrosine hydroxylase degeneration of dopaminergic neurons [3,23]. In contrast, gonadoectomized rats and also in some amphibians, e.g., in frogs, there are significantly fewer tyrosine hydroxylase immunoreactive neurons in number and size in different areas of dopaminergic neurons in the brain compared to testosterone-treated males [7]. Another study also showed that the potentially important sites of testosterone in the development of the motor pathway are predominantly present in the pelvic autonomic ganglion cells [22] and have a relation with autonomic reflex function. It is clear that in Parkinson’s disease, the autonomic reflex is also delayed or disturbed. Testosterone also works via androgen receptors and mostly the androgen receptors are located in noradrenergic and cholinergic neurons [10,11,17]. Kainic acid resulted in significant neuronal loss in the hilus of castrated male rats compared to animals treated with vehicles. Testosterone administration prevented the neurodegenerative effect of kainic acid [4]. In this study, we would like to hypothesize that complex I inhibitors, which are responsible for reproducing symptoms of PD in rats, also deplete serum testosterone and decrease levels of ATP in the peripheral organs, such as the adrenal glands and testis. The depletion of energy levels and excess oxidative stress due to complex I inhibition also could be a cause for a decrease of testosterone and an increase of corticosterone in peripheral plasma, without disrupting the thyroid and prolactin hormone levels.
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Mitochondrial complex I inhibition depletes plasma testosterone in the rotenone model of Parkinson’s disease M. Alam, W.J Schmidt* Zoological Institute, Department of Neuropharmacology, Morgenstelle 28E, University of Tuebingen, 72076 Tuebingen, Germany Received 5 April 2004; received in revised form 29 July 2004; accepted 11 August 2004
Abstract Age-related depletion of testosterone may increase the brain’s vulnerability to parkinsonian- or Alzheimer’s-like neurodegenerative disorders. In rats, rotenone, a mitochondrial complex I inhibitor, causes specific nigral dopaminergic neurodegeneration producing parkinsonian symptoms. In this study, rotenone was administered on a daily basis (2 mg/kg i.p.) to two groups of rats, over a period of 30 and 60 days, respectively. In order to contribute towards the validation of the rotenone rat model, the changing level of the peripheral sex steroid hormone, testosterone, which would also mimic those found in Parkinson’s disease (PD) patients, was evaluated. Parallel to this, prolactin, luteinizing hormone (LH), the nonsexual steroid thyroid-stimulating hormone, and the corticosterone hormone levels in the peripheral blood plasma were measured to show whether other hormones have also been affected by complex I inhibition. The rotenone treatment caused a decrease of testosterone level in the peripheral blood plasma. There were no differences in the thyroid hormone and prolactin but increases in leutinizing hormone and corticosterone were observed. Data from this study indicate that rotenone depleted the sex steroid hormone which is preferentially produced in the periphery, e.g., adrenal gland and testis. In conclusion, because a decrease in testosterone levels is also one of the comorbidities which are found in male PD patients, our results indicate that the rotenone model mimics PD symptoms not only on a neuronal and behavioral level, but also on the testosterone levels. D 2004 Elsevier Inc. All rights reserved. Keywords: Androgen; Testosterone; Corticosterone; Prolactin; Complex I; Dopamine
1. Introduction NADH-ubiquinone oxidoreductase deficiency of mitochondrial complex I can be present in a wide variety of biochemical and symptomatic phenotypes. Complex I is a huge multienzyme complex of 43 separately encoded gene products derived from both nuclear and mitochondrial genomes [19,29]. Rotenone inhibits mitochondrial complex I (NADH: ubiquinone oxidoreductase), which is a proximal component of the mitochondrial electron transport chain (ETC), and it produces the energy necessary for maintenance of all neuronal or cell functions.
* Corresponding author. Tel.: +49 7071 2974571; fax: +49 7071 295144. E-mail address: [email protected] (W.J. Schmidt). 0031-9384/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2004.08.010
The involvement of mitochondrial inhibition in neurodegeneration has received particular attention. Mitochondrial abnormalities have been identified in more common sporadic neurological disorders, including Alzheimer’s disease (AD) and Parkinson’s disease (PD), and they also occur as part of normal aging [25]. The chronic exposure of rotenone induces dopaminergic neuronal degeneration and formation of alpha-synuclein-positive cytoplasmic inclusions in nigral neurons [5,36]. PD is a movement disorder and mostly it is related to the disturbance of the nigrostriatal dopaminegric system. In most cases of PD, the mesolimbic dopaminergic system is also disturbed, which plays an important role as a modulator of the complex reward process that organizes motivated behaviors, such as drinking, feeding and sex, by the elevation of salient environmental stimuli. The mesolimbic dopaminergic system is known to play an
396
M. Alam, W.J. Schmidt / Physiology & Behavior 83 (2004) 395–400
important facilitory role in copulation [26]. But it is clear that in PD, the tubero-infundibular dopaminergic neurons are spared [1]. There is substantial evidence indicating that steroid hormones modify vulnerability to neuronal insults. Prolonged elevation of corticosterone was shown to increase the vulnerability of nerve cells to the neurodegenerative processes in aging, cerebral ischemia and possibly other neurodegenerative diseases [35]. An increase in levels of glucocorticoids, like corticosterone, may exert many of their toxic effects via an interaction with dopamine (DA) and glutamate. It has been shown that this stress hormone also inhibits the uptake of DA [9]. Likewise, glucocorticoids can block the uptake of glutamate [14] which can further exacerbate the neurotoxic effect through excitotoxicity. Gonadal steroids, like testosterone in the male, play a role in the nervous system development. A decrease in circulating testosterone contributes to an age-related increase in glial fibrillary acidic protein (GFAP) which could render the brain more susceptible to neurodegeneration during aging [8]. Low serum testosterone has been associated with poor cognitive performance in elderly men and AD [20,28]. A number of in vivo studies have shown that androgens can rescue specific populations of motoneurons from both ontogenic- and axotomy-induced death. In in vitro studies, it has been shown that glucose-deprived hippocampal neurons are rescued by testosterone and testosterone itself also acts as an antioxidant via androgen-receptor-mediated mechanism [2,15]. Like other steroid hormones, the cellular effects of testosterone can be broadly grouped into genomic and nongenomic categories. The neuroprotective role of testosterone is mostly known through the genomic pathway via an androgen-receptor-dependent manner. The polyglutamine (CAG) polymorphism of androgen receptors has been associated with the risk of AD in men and with other brain disorders [24]. In the neonatal period, the presence of circulating testosterone is essential for the development of the substantia nigra region which exerts proconvulsant effects throughout the rat’s life, a unique feature of the male substantia nigra. The final maturation of the substantia nigra occurs in the prepubertal period, and is, in part, regulated by testosterone as well [38]. The influence of testosterone in neurodegenerative diseases, such as PD and AD, can be translated into new cures of the disorders with distinct sex-specific and more effective therapies. The level of circulating testosterone plays a key role in the development organization of the substantia nigra pars reticulata and its GABAA-ergic circuit that modify motor behavior in a precise, age-dependent fashion [38]. In contrast, gonad steroids increase the number and size of tyrosine hydroxylase (TH) immunoreactive neurons in some dopaminergic neurons’ population in both male and female rats [34,40]. Therefore, it seems that androgen levels are capable of influencing dopaminergic function via modulation of TH mRNA expression. The sex hormone testosterone has a
general and more prominent influence on the brain than reproduction and sexuality. Although it is well known that most pesticides have a pronounced effect on sex steroids, most of them are however not complex I inhibitors. Therefore, the present study explored whether a specific complex I inhibitor has a specific effect on the male sex steroid and other hormones.
2. Experimental procedures 2.1. Animals Forty adult male Sprague–Dawley rats (Charles River, Sulzfeld, Germany) aged 7 weeks were chosen for the experiment. At the beginning of the experiment, the rats weighed 230–240 g. Animals were housed in cages under a 12:12-h light/dark cycle in a room maintained at a constant temperature of 22 8C and 50–60% humidity with food and water available ad libitum. To minimize the stress for the rats during the injections, they were weighed and handled for 5 to 10 min the day before the injection. All treatments used in this experiment adhere to the international ethical standards and the German AnimalProtection Law and have been approved by local animal care committee (Tierschutzkommission, Regierungspra¨sidium Tu¨bingen, ZP 2/03). 2.2. Chemicals Rotenone was purchased from Sigma (St. Louis, MO) and was dissolved in sterile natural oil [middle chain triglycerides (MCT); Miglyol 812, clinical pharmacy, University Tuebingen]. Luciferin–luciferase enzymes were obtained from Sigma and protein assay kit from Bio-Rad (Richmond, CA, USA). 2.3. Treatment Rotenone emulsified in natural oil at 2.0 mg/ml was given intraperitoneally once a day at 1 ml/kg during 60 days. Oil was injected as vehicle to control rats (1 ml/kg). For the determination of the serum hormone assay and tissue adenosine triphosphate (ATP) from the adrenal gland and testis, two groups of rats were used, namely, vehicletreated (n=10) and rotenone-treated (n=10) groups. Control groups received oil for 30 and 60 days and rats were treated with rotenone for 30 and 60 days. 2.4. Blood sampling Rats were killed by decapitation with the use of a guillotine after 30 and 60 days and the trunk blood was collected after an overnight fast from both groups in a blood sample tube containing lithium-heparin (15 IU heparin/ml).
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2 vol (g/ml)], homogenized at 250–260 rpm for 3 min in the ice bath, and centrifuged at 960g at 4 8C for 15 min and followed by recentrifuging at 8000g for 10 min at 4 8C to give a supernatant. The supernatant was used to measure activities of ATP. 2.6. Hormone and tissue protein assay The in vitro testosterone test was carried out by enzyme sandwich immunoassay (Vitro ECI, Ortho Diagnostics, Rochester, NY, USA) in immunodiagnostics system. Intra- and interassay variations were b9.4%. The tissue protein assay was carried out using a Bio-Rad protein assay kit. 2.7. Evaluation of ATP content Tissue supernatants were incubated with a luciferin– luciferase mixture and the resulting luminescence was measured with a luminometer (LUMAT LB 9507; Berthhold Technologies, Bad Wildbad, Germany) according to the method [39] and the ratio ATP per unit protein was calculated. The analysis was done twice per animal. The mean values of relative intensityFS.D. was taken to plot the graph. 2.8. Statistical analysis Data are expressed as meansFS.E.M. Statistical analysis was carried out using the GB-Stat V5.4 software (Dynamic Microsystem, Silver Spring, MD). The data for peripheral hormone analysis were evaluated using two-way ANOVA followed by Fisher’s LSD comparison which was used for group differences. *Pb0.05 and **Pb0.01 were taken as levels of statistical significance.
3. Results Fig. 1. (a, b and c) Plasma concentrations of the (a) testosterone; (b) luteinizing hormone; and (c) thyroid-stimulating hormone in (2 mg/kg) rotenone-treated animals (n=10) in white bars and (1 ml/kg) oil-treated control animals (n=10) in hatched bars on day 30 and day 60. Values are shown as meanFS.E.M. P represents statistical analysis: *significant at Pb0.05 and **significant at Pb0.01.
The blood was centrifuged at 1500g for 15 min at 4 8C. The plasma was frozen at 20 8C until analyzed.
The depletion of serum concentration of total testosterone in rotenone-treated rats were 62.33% and 85.5% compared to oil-treated animals on days 30 and 60 (Fig. 1a). A significant effect of treatment was found in both groups. The levels of testosterone between groups were
Table 1 Levels of corticosterone and prolactin in control and rotenone-treated animals
2.5. Adrenal gland and testis sample preparation Adrenal glands were rapidly removed, adhering fat was cleaned, and after weighing, capped tightly and stored at 70 8C until the measurement of ATP. The left testis were removed from each animal’s body, weighed immediately, minced in cold phosphate buffer [10 mM, pH 7.0,
Day 30
Corticosterone (nmol/l) Prolactin (mIU/l)
Day 60
Cont
Rotenone
Cont
Rotenone
27.93F1.81
50.5F2.23
33.26F1.95
51.56F2.80
7.16F1.14
7.31F2.34
8.96F1.05
7.6F1.49
Values are shown as meanFS.E.M.
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F(1,18)=19,90 Pb0.003. The levels of sex hormonebinding globulin (SHBG) was the same in both groups of animals (data are not shown). The levels of luteinizing hormone (LH) and corticosterone in rotenone-treated animals were significantly higher than control animals (Fig. 1b and Table 1). On days 30 and 60, the levels of LH between the groups were F(1,18)=13.91, Pb0.001 and the level of corticosterone were F(1,18)=71.05, Pb0.0001, respectively. There were no differences in serum thyroidstimulating hormone (TSH) level and prolactin as compared to control on days 30 and 60 (Fig. 1c and Table 1). The levels of ATP in the adrenal gland and testis homogenates were significantly decreased within 30 and 60 days in a time-dependent manner compared to control which correlates with highly significant depletion of serum testosterone in peripheral circulation (Fig. 2a and b). The levels of ATP in adrenal glands and testis were F(1,12)=37.64, Pb0.0001 and F(1,12)=200.18, Pb0.0001, respectively.
Fig. 2. (a and b) The depletion of ATP in percentage on day 30 and day 60 in peripherally rotenone-treated animals group (2 mg/kg) has been shown by white bars and oil-treated animals (1 mg/kg) as a control group by hatched bars. Values are shown as meanFS.E.M. P represents statistical analysis: *significant at Pb0.05 and **significant at Pb0.01.
4. Discussion In the present study, we reveal an interesting relationship between complex I inhibition and a decrease of peripheral testosterone in male rats; in addition, a time-dependent decrease of testosterone and an increase of corticosterone and LH in the plasma has been observed. The present experiment also shows that the hypothalamic–pituitary axis was spared because the increase in LH in peripheral blood was due to a decrease of serum testosterone in periphery circulation, which means that the feedback mechanism was not disrupted; however, the TSH and prolactin secretion was unaffected. Testosterone is physiologically secreted by the adrenal glands and testis; its serum level is higher in men and normally decreases during life. This hormone is transported by the SHBG and albumin. Normally, SHBG is inversely related to circulating testosterone. In aging, the level of testosterone decreases and the levels of SHBG increase. The specific mechanism in the depletion of testosterone in different types of neurodegeneration are varied; for example, in Kwashiorkor and anorexia nervosa, the SHBG is elevated. In amyotrophic lateral sclerosis (ALS), the reduced level of testosterone might also be related to an altered binding to either SHBG or albumin [21]. However, in our experiment, we did not find any changes in SHBG in both the control and rotenone-treated groups. Glucocorticoids (cortisol in primates, corticosterone in rat) are secreted from the adrenal cortex upon activation of the hypothalamic–pituitary–adrenal axis as a response to stressful environmental stimuli. It has become evident that these hormones can modulate the neurodegenerative process that occurs in nerotoxic trauma, aging and AD [12,13]. Stress hormones, like corticosterone, have been found to impair learning and memory function (within several tens of minutes) rapidly via unknown neuronal mechanisms [27]. In primary hippocampal cultures, corticostrone decreases glutathione peroxidase, an antioxidant enzyme, and nicotinamide dinucleotide phosphate, which is an important substrate for the regeneration of glutathione [33]. Corticosterone resulted in extreme prolongation of the time duration of NMDA-receptor-mediated calcium signals, resulting in calcium overload neurotoxicity. High levels of corticosterone induce a rapid and nongenomic prolongation of NMDA-receptor-mediated calcium elevation, resulting in calcium-induced neurotoxicity [37]. In our experiment, we found that the level of corticosterone was higher compared to control. ATP is a source of energy and almost all cellular energy depends on mitochondrial pathways. Thus, the disruption of any pathway of mitochondria could be a cause of different types of diseases. The type of disease depends on which parts of tissues or organs are mostly or primarily affected. In our experiment, peripheral administration of rotenone decreased ATP to 34% and 73% on days 30 and 60 in adrenal glands. In the testis tissues on day 30, ATP levels
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were decreased by 34% and on day 60, ATP levels were decreased by 63%, which might be a further cause of depletion of testosterone as an energy-dependent manner. A variety of studies in animals and clinical observations have demonstrated that certain autonomic reflexes are altered under conditions of androgen deprivation. There are numerous possible mechanisms by which testosterone deprivation could influence reflex function. Growing evidence suggests a putative neuroprotective role for testosterone in the CNS, and in particular in motorneurons [6,18]. Testosterone reduces neuronal secretion of Alzheimer’s h-amyloid peptides [16]. However, evidence has linked testosterone deficiency to PD, AD and other neurodegenerative disorders [30,31], and it has also been shown that some nonmotor symptoms of PD may respond to testosterone replacement therapy [32]. Recently, it has been shown that specific complex I inhibitors are a cause of decreased tyrosine hydroxylase degeneration of dopaminergic neurons [3,23]. In contrast, gonadoectomized rats and also in some amphibians, e.g., in frogs, there are significantly fewer tyrosine hydroxylase immunoreactive neurons in number and size in different areas of dopaminergic neurons in the brain compared to testosterone-treated males [7]. Another study also showed that the potentially important sites of testosterone in the development of the motor pathway are predominantly present in the pelvic autonomic ganglion cells [22] and have a relation with autonomic reflex function. It is clear that in Parkinson’s disease, the autonomic reflex is also delayed or disturbed. Testosterone also works via androgen receptors and mostly the androgen receptors are located in noradrenergic and cholinergic neurons [10,11,17]. Kainic acid resulted in significant neuronal loss in the hilus of castrated male rats compared to animals treated with vehicles. Testosterone administration prevented the neurodegenerative effect of kainic acid [4]. In this study, we would like to hypothesize that complex I inhibitors, which are responsible for reproducing symptoms of PD in rats, also deplete serum testosterone and decrease levels of ATP in the peripheral organs, such as the adrenal glands and testis. The depletion of energy levels and excess oxidative stress due to complex I inhibition also could be a cause for a decrease of testosterone and an increase of corticosterone in peripheral plasma, without disrupting the thyroid and prolactin hormone levels.
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