Significant outcomes
This work shows that mitochondria isolated from the frontal cortex and hippocampus of vitamin A-treated rats are more sensitive to an in vitro exposition to amyloid-β (Aβ) peptides 1–40 or 1–42 (at 0.2 or 0.1 μM, respectively) regarding mitochondrial complex enzyme activity, mitochondrial 3-nitrotyrosine content and superoxide anion radical (O2−•) production. These data suggest that vitamin A supplementation at clinical doses (1000–9000 IU/kg/day) did affect both directly and indirectly the mitochondrial function, increasing the susceptibility of such organelle to a posterior deleterious stimulus.
Limitations
It is necessary to investigate whether other pro-oxidant agents also affect mitochondria isolated from vitamin A-treated rats.
Introduction
Vitamin A modulates several biological events from organogenesis to maintenance of mature tissues (Reference Lane and Bailey1). On the other hand, vitamin A-related toxicity has long been investigated, but the mechanisms by which it acts are not completely understood (Reference Snodgrass2). This vitamin has been characterised as a redox agent, which is able to interfere with redox homeostasis through either an antioxidant or a pro-oxidant action. We have shown that vitamin A supplementation at pharmacological doses induces a pro-oxidant effect in some rat brain regions and impaired rat performance in different behavioural tests (Reference De Oliveira and Moreira3–Reference De Oliveira, Oliveira, Behr and Moreira10). Recently, increased mortality rates among users of vitamin A supplementation even at low doses were shown (Reference Bjelakovic, Nikolova, Gluud, Simonetti and Gluud11). In addition, the combination of vitamin A supplementation and smoking was shown to induce tumour growth among patients with lung cancer (Reference Omenn, Goodman and Thornquist12,Reference Omenn, Goodman and Thornquist13). Nevertheless, vitamin A utilisation at moderate to high doses (values ranging from 30 000 to 300 000 IU/day) is still being applied in the treatment of some pathologies, as for instance dermatological disturbances, leukaemia and immunodeficiency (Reference Tsunati, Iwasaki, Kawai, Tanaka, Ueda, Uchida and Nakamura14–Reference Myhre, Carlsen, Bohn, Wold, Laake and Blomhoff17). More alarming is the fact that vitamin A supplementation at about 8500 IU/kg/day is recommended as safe during weight gain therapy for very-low-weight preterm infants (Reference Mactier and Weaver18). Vitamin A – or its derivatives, the retinoids – possesses the ability to impair the homeostasis of several mammalian brain regions and, consequently, to induce cognitive decline characterised by irritability, decreased capabilities to learning and memory, anxiety and depression, which affect directly patient's life quality and work production (Reference Myhre, Carlsen, Bohn, Wold, Laake and Blomhoff17,Reference Jick, Kremers and Vasilakis-Scaramozza19).
On the basis of the fact that vitamin A supplementation induces mitochondrial dysfunction in vivo and that mitochondrial impairment has been associated with neurodegenerative diseases, we tested here the susceptibility of mitochondria isolated from the frontal cortex and hippocampus of vitamin A-treated rats to fragment length of Aβ peptides 1–40 and 1–42 at low concentrations in vitro, as such molecules take a pivotal role during either ageing or Alzheimer's disease progression by inducing cognitive impairment among humans (Reference Halliweel20). The aim of this work was to analyse whether mitochondria isolated from vitamin A-treated rat brain areas are more sensitive than control mitochondria when a challenge with Aβ peptides was applied with respect to mitochondrial electron transfer chain (METC) enzyme activity, superoxide anion radical (O2−•) production and nitrosative stress.
Materials and methods
Animals
Adult male Wistar rats (280–300 g) were obtained from our own breeding colony. They were caged in groups of five with free access to food and water and were maintained on a 12-h light-dark cycle (7:00–19:00 h), at a temperature-controlled colony room (23 ± 1 °C). These conditions were maintained constant throughout the experiments. All experimental procedures were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH publication number 80–23, revised 1996) and the Brazilian Society for Neuroscience and Behavior recommendations for animal care. Our research protocol was approved by the Ethical Committee for animal experimentation of the Federal University of Rio Grande do Sul.
Drugs and reagents
Arovit® (retinol palmitate, a water-soluble form of vitamin A) was purchased from Roche, Sao Paulo, SP, Brazil. Polyclonal antibody to 3-nitrotyrosine was obtained from Calbiochem, San Diego, CA, USA. All other chemicals were purchased from Sigma, St. Louis, MO, USA. Vitamin A treatment was given daily and administered during night.
Treatment
The animals were treated once a day for 28 days. The treatments were carried out at night (i.e. when the animals are more active and take a greater amount of food) in order to ensure maximum vitamin A absorption, as this vitamin is better absorbed during or after a meal. The animals were treated once a day with vehicle (0.15 M saline; n = 8–10 animals), 1000 (n = 8–10), 2500 (n = 8–10), 4500 (n = 8–10) or 9000 IU/kg (n = 8–10) of retinol palmitate (vitamin A) orally via a metallic gastric tube (gavage) in a maximum volume of 0.6 ml. Adequate measures were taken to minimise pain or discomfort.
Mitochondrial isolation
Mitochondria from fresh rat frontal cortex and hippocampus were isolated as described elsewhere (Reference Klamt, De Oliveira and Moreira21). Briefly, frontal cortex and hippocampus of Wistar rats were suspended in ice-cold isolation buffer A [220 mM mannitol, 70 mM sucrose, 5 mM HEPES (pH 7.4), 1 mM EGTA and 0.5 mg/ml fatty-acid free bovine serum albumin] and gently homogenised with a glass homogeniser and centrifuged at 2000 ×g for 10 min at 4 °C. Approximately three quarters of the supernatant were further centrifuged at 10 000 ×g for 10 min at 4 °C in a new tube. The fluffy layer of the pellet was removed by gently shaking with buffer A and the firmly packed sediment was resuspended in the same buffer without EGTA and centrifuged at 10 000 ×g for 10 min at 4 °C. The mitochondrial pellet was resuspended in buffer B [210 mM mannitol, 70 mM sucrose, 10 mM HEPES-KOH (pH 7.4), 4.2 mM succinate, 0.5 mM KH2PO4 and 4 μg/ml rotenone]. This procedure, which was designed to isolate intact mitochondria rather than to recover all of that present in the frontal cortex and hippocampus, yielded about 7 mg of mitochondrial protein per gram of tissue.
Mitochondrial challenge with Aβ peptides 1–40 and 1–42
After obtaining mitochondria from rat frontal cortex and hippocampus, we incubated mitochondria with Aβ peptides 1–40 (0.2 μM) or 1–42 (0.1 μM) to verify whether the organelles isolated from vitamin A-treated rats are more sensitive to a low concentration of Aβ peptides, which would alter mitochondrial function only if it were previously affected through vitamin A supplementation. The challenge was carried out in buffer B at room temperature in open tubes for 10 min before the quantification of mitochondrial respiratory chain enzyme activity, superoxide anion radical (O2−•) production and 3-nitrotyrosine content analyses.
METC activity
Complex I–CoQ–III activity. It was determined by following the increase in absorbance due to the reduction of cytochrome c at 550 nm with 580 nm as the reference wavelength (∈ = 19.1 mM−1 cm−1). The reaction buffer contained 210 mM mannitol, 70 mM sucrose, 20 mM potassium phosphate, pH 8.0, 2.0 mM KCN, 10 μM EDTA, 50 μM cytochrome c and 20–45 μg supernatant protein. The reaction started by the addition of 25 μM NADH and was monitored at 30 °C for 3 min before the addition of 10 μM rotenone, after which the activity was monitored for an additional 3 min. Complex I–III activity was the rotenone-sensitive NADH:cytochrome c oxidoreductase activity (Reference Shapira, Mann and Cooper22).
Complex II–CoQ–III activity. It was measured by following the increase in absorbance due to the reduction of cytochrome c at 550 nm with 580 nm as the reference wavelength (∈ = 21 mM−1 cm−1). The reaction mixture consisting of 170 mM mannitol, 70 mM sucrose, 40 mM potassium phosphate, pH 7.4, 16 mM succinate was preincubated with 50–100 μg supernatant protein at 30 °C for 30 min. Subsequently, 4.0 mM sodium azide and 7.0 μM rotenone were added and the reaction started by the addition of 0.6 μg/ml cytochrome c and monitored for 5 min at 30 °C (Reference Fisher, Ruitenbeek and Berden23).
Complex IV activity. It was measured by following the decrease in absorbance due to the oxidation of previously reduced cytochrome c at 550 nm with 580 nm as the reference wavelength (€ = 19.15 mM−1 cm−1). The reaction buffer contained 220 mM mannitol, 70 mM sucrose, 10 mM potassium phosphate, pH 7.0, 0.6 mM n-dodecyl-β-d-maltoside, 2–4 μg supernatant protein and the reaction was started with the addition of 0.7 μg reduced cytochrome c. The activity of complex IV was measured at 25 °C for 10 min (Reference Rustin, Chretien, Bourgeron, Gérard, Rötig, Saudubray and Munnich24).
Indirect ELISA to 3-nitrotyrosine
To realise indirect enzyme-linked immunosorbent assay (ELISA), rat brain regions were rapidly homogenised (T < 1min) in lysis buffer containing 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton-X-100, 10% glycerol, 2 mM sodium orthovanadate and Complete™ protease inhibitor cocktail (Roche). Indirect ELISA was performed to analyse changes in the content of nitrotyrosine by utilising a polyclonal antibody to nitrotyrosine (Calbiochem) diluted 1:2000 in phosphate-buffered saline (PBS), pH 7.4, with 5% albumin. Briefly, microtitre plates (96-well flat-bottom) were coated for 24 h with the samples diluted 1:2 in PBS with 5% albumin. Plates were then washed four times with wash buffer (PBS with 0.05% Tween-20), and the specific antibodies were added to the plates for 2 h at room temperature. After washing (four times), a second incubation with peroxidase-conjugated anti-rabbit antibody (diluted 1:1000) for 1 h at room temperature was carried out. After addition of substrates (hydrogen peroxide and 3,3′,5,5′-tetramethylbenzidine, 1:1 – v:v), the samples were read at 450 nm in a plate spectrophotometer. Results are expressed as changes in percentage among the groups and the experiments were performed in triplicate.
Superoxide anion radical (O2−•) production
Briefly, to obtain submitochondrial particles (SMPs), frontal cortex and hippocampus were dissected and homogenised in 230 mM mannitol, 70 mM sucrose, 10 mM Tris-HCl and 1 mM EDTA (pH 7.4). The samples were centrifuged for 10 min at 600 ×g (4 °C). The supernatants were then centrifuged (8000 ×g for 10 min at 4 °C) two times to isolate mitochondria. Then, freezing and thawing (three times) the mitochondrial solution gave rise to superoxide dismutase-free SMP. The SMP solution was also washed (twice) with 140 mM KCl, 20 mM Tris-HCl (pH 7.4) to ensure mitochondrial superoxide dismutase (SOD) release from mitochondria (centrifugation to wash at 5400 ×g for 10 min at 4 °C). To quantify O2−• production, SMP was incubated in reaction medium consisting of 230 mM mannitol, 70 mM sucrose, 10 mM HEPES-KOH (pH 7.4), 4.2 mM succinate, 0.5 mM KH2PO4, 0.1 μM catalase and 1 mM epinephrine, and the increase in the absorbance (auto-oxidation of adrenaline to adrenochrome) was read at 480 nm in a spectrophotometer at 32 °C, as previously described (Reference De Oliveira and Moreira3,Reference Poderoso, Carreras, Lisdero, Riobo, Schopfer and Boveris25). The experiments were performed in triplicate.
Statistical analyses
Results are expressed as means ± SEM; pvalues were considered significant when p < 0.05. Differences in experimental groups were determined by one-way analysis of variance (ANOVA) followed by the post hoc Duncan's test whenever necessary.
Results
METC enzyme activity
As depicted in Fig. 1a, vitamin A supplementation at 4500 or 9000 IU/kg/day induced an increase in complex I–III enzyme activity in mitochondria incubated in the absence of Aβ peptides (p < 0.05). However, this increase was abolished in the presence of Aβ peptides 1–40 or 1–42, which decreased complex I–III enzyme activity (p < 0.05). Complex I–III enzyme activity of mitochondria isolated from saline-treated rats was not affected by the challenge with Aβ peptides.
Fig. 1. Vitamin A supplementation effects on METC enzyme activity in a challenge with Aβ peptides 1–40 or 1–42. (a) Frontal cortex complex I–III enzyme activity; (b) hippocampal complex I–III enzyme activity; (c) frontal cortex complex II–III enzyme activity; (d) hippocampal complex II–III enzyme activity; (e) frontal cortex complex IV enzyme activity and (f) hippocampal complex IV enzyme activity. Data are mean ± SEM (n = 8–10 per group). *p < 0.05 (vitamin A vs. control group), #p < 0.05 (vitamin A vs. control group in the presence of Aβ peptides 1–40 or 1–42), a,b,c,dp < 0.05 (vitamin A dose in the presence of Aβ peptides 1–40 or 1–42 vs. the same vitamin A dose in the absence of Aβ peptides 1–40 or 1–42; one-way ANOVA followed by the post hoc Duncan's test).
Similarly, increased complex I–III enzyme activity was observed in mitochondria isolated from hippocampus of the rats that were administered vitamin A supplementation at 4500 or 9000 IU/kg/day (p < 0.05; Fig. 1b). Aβ peptides challenge induced a decrease in complex I–III enzyme activity of mitochondria isolated from vitamin A-treated rats at any dose (p < 0.05; Fig. 1b).
According to Fig. 1c, complex II–III enzyme activity did not change in the mitochondria obtained from the frontal cortex of vitamin A-treated rats and incubated in the absence of Aβ peptides. On the contrary, it was observed that Aβ peptides challenge decreased complex II–III enzyme activity only in the mitochondria obtained from vitamin A-treated rats (Fig. 1c; p < 0.05). A similar effect was observed in mitochondria isolated from the hippocampus of the rats that received vitamin A supplementation at any dose tested (Fig. 1d; p < 0.05).
Complex IV enzyme activity of the mitochondria incubated in the absence of Aβ peptides did not change in the rats that were treated with vitamin A supplementation (Fig. 1e). However, it was observed that there was decreased complex IV enzyme activity in mitochondria isolated from the frontal cortex of vitamin A-treated rats when Aβ peptides were added to the incubation media (p < 0.05; Fig. 1e). Decreased complex IV enzyme activity was observed in the mitochondria isolated from the hippocampus of the rats that were administered vitamin A supplementation at 4500 or 9000 IU/kg/day and whose mitochondria were incubated in the absence of Aβ peptides (p < 0.05; Fig. 1f). When Aβ peptides were present in the incubation media, it was observed that there was decreased complex IV enzyme activity in the mitochondria obtained from the hippocampus of the rats that were treated with vitamin A supplementation at any dose tested (p < 0.05; Fig. 1f).
Superoxide anion radical (O2−•) production
As shown in Fig. 2a, mitochondria isolated from the frontal cortex of the rats that were administered vitamin A supplementation at 2500, 4500 or 9000 IU/kg/day produced more O2−• than control mitochondria even in the absence of Aβ peptides (p < 0.05). However, an increment in O2−• production in the presence of Aβ peptides was observed (p < 0.05; Fig. 2a). Mitochondria obtained from the hippocampus of the rats that received vitamin A supplementation at 2500, 4500 or 9000 IU/kg/day produced significantly more O2−• than mitochondria isolated from control rats in the absence of Aβ peptides (p < 0.05; Fig. 2b). The incubation with Aβ peptides increased O2−• production in hippocampal mitochondria obtained from vitamin A-treated rats (p < 0.05; Fig. 2b).
Fig. 2. Vitamin A supplementation effects on superoxide anion radical (O2−•) production in (a) frontal cortex and (b) hippocampal mitochondrial incubations with or without Aβ peptides 1–40 or 1–42. The effects of vitamin A supplementation on 3-nitrotyrosine content in mitochondrial membranes during incubations with or without Aβ peptides 1–40 or 1–42 are shown in (c) and (d). *p < 0.05 (vitamin A vs. control group), #p < 0.05 (vitamin A vs. control group in the presence of Aβ peptides 1–40 or 1–42), a,b,c,dp < 0.05 (vitamin A dose in the presence of Aβ peptides 1–40 or 1–42 vs. the same vitamin A dose in the absence of Aβ peptides 1–40 or 1–42; one-way ANOVA followed by the post hoc Duncan's test).
Mitochondrial membrane 3-nitrotyrosine content
Mitochondrial membranes obtained from the frontal cortex of vitamin A-treated rats show increased 3-nitrotyrosine content even in the absence of Aβ peptides in the incubation media (p < 0.05; Fig. 2c). Interestingly, although increased 3-nitrotyrosine content in mitochondrial membranes of frontal cortex was observed, it was not changed by the challenge with Aβ peptide 1–40 (Fig. 2c). On the other hand, the challenge with Aβ peptide 1–42 did increase 3-nitrotyrosine content in membranes of mitochondria that were obtained from the frontal cortex of vitamin A-treated rats (p < 0.05; Fig. 2c).
We observed increased 3-nitrotyrosine content in mitochondrial membranes obtained from the hippocampus of vitamin A-treated rats in the absence of Aβ peptides challenge (p < 0.05; Fig. 2d). However, the challenge with either Aβ peptides 1–40 or 1–42 did increase 3-nitrotyrosine content in hippocampal mitochondrial membranes obtained from vitamin A-treated rats (p < 0.05; Fig. 2d).
Discussion
Neurodegenerative pathologies affect dramatically life quality of patients suffering from such diseases. It has been shown that there was decreased life expectancy and production at work. Moreover, the governmental costs for treatment of neuropathological conditions increase annually in both developed and developing countries (Reference Ferri, Prince and Brayne26–Reference Hawton and Van Heeringen28). Vitamin A has been shown to induce neurotoxicity and also impaired mammalian behaviour [as reviewed in (Reference Allen and Haskell16,Reference Myhre, Carlsen, Bohn, Wold, Laake and Blomhoff17)]. Decreased metabolic rates were observed in the cerebral cortex of patients under retinoid treatment against acne (Reference Bremner, Fani and Ashraf29). Hallucinations, confusion, anxiety, depression and suicide ideation may also be listed among the effects associated with vitamin A supplementation use (Reference O'Reilly, Bailey and Lane30). Nonetheless, it still remains on debate by which mechanisms such vitamin elicits neurotoxicity.
We have shown that mitochondria are a target of vitamin A-related toxicity in vitro and in vivo. Retinol at concentrations similar to that observed physiologically induced mitochondrial swelling and facilitated cytochrome c release from the organelle in vitro(Reference Klamt, De Oliveira and Moreira21). Recently, we showed increased levels of oxidative stress markers in SMPs isolated from some brain regions of rats that were subjected to long-term vitamin A supplementation. This effect was accompanied by increased rates of O2−• production (Reference De Oliveira and Moreira3,Reference De Oliveira and Moreira6–Reference De Oliveira, Oliveira, Behr and Moreira10). In addition, vitamin A supplementation is able to impair METC enzyme activity in vivo, which may give rise to free radical production (Reference De Oliveira and Moreira3–Reference De Oliveira, Oliveira, Behr and Moreira10).
In this work, we show, for the first time, that mitochondria isolated from the frontal cortex and hippocampus of vitamin A-treated rats are more sensitive to a challenge with Aβ peptides at low doses regarding METC enzyme activity, O2−• production and nitrosative stress. Aβ peptides take a central role in Alzheimer's disease and their increased levels have been mentioned as a cause of this pathology (Reference Berg, Youdim and Riederer31). Aβ peptides interact with mitochondria in vivo physiologically, but such interaction becomes harmful depending on other circumstances, as for instance the redox and bioenergetics environments (Reference Manczak, Anekonda, Henson, Park, Quinn and Reddy32–Reference Pavlov, Petersen, Glaser and Ankarcrona34). Moreover, Aβ intramitochondrial accumulation occurs before extracellular Aβ deposition during Alzheimer's disease (Reference Du, Guo and Fang35). It was recently shown that mitochondrial bioenergetic impairment preceded Alzheimer's disease pathology in an animal model of this pathology, and it was positively correlated with increased mitochondrial Aβ levels (Reference Yao, Irwin, Zhao, Nilsen, Hamilton and Brinton36). Then, based on the data obtained in our work, we suggest that vitamin A supplementation may induce a predisposition to mitochondrial deficit elicited by the presence of Aβ peptides, favouring a loss of energetic homeostasis.
It is noteworthy that Aβ peptides decreased cortical and hippocampal METC enzyme activities of the rats that were administered vitamin A at any dose tested. In some cases, vitamin A supplementation alone induced an increase in METC enzyme activities, which was decreased by the combination of previous vitamin A supplementation and Aβ peptides. On the other hand, when vitamin A supplementation was not effective in altering METC activity, it rendered mitochondria to be more sensitive to Aβ peptides challenge (Fig. 1).
The mechanism by which Aβ peptides interfere with the function of mitochondria (complex I–III, complex II–III and complex IV enzyme activity) isolated from vitamin A-treated rats still remains to be clarified, but by decreasing mitochondrial complex enzyme activity, Aβ peptides may increase O2−• production due to electron leakage and consequent O2 partial reduction at complex IV, as previously reviewed (Reference Halliweel20). Then, based on the data presented here, we propose that vitamin A supplementation in vivo facilitates mitochondrial dysfunction in future events that may interfere with mitochondrial dynamics.
The data presented here may be useful, at least in part, to show that the use of vitamin A supplements as an antioxidant during the treatment (or prevention) of neurodegenerative diseases should be revised. The faddism around utilisation of vitamin supplements in an attempt to prevent ageing progress, for example, may otherwise trigger negative, and perhaps irreversible, consequences.
Acknowledgements
This work was supported by grants from CNPq, CAPES and Rede Instituto Brasileiro de Neurociência (IBN-Net) # 01.06.0842-00. The authors declare that they have no conflicts of interest.
