The Reassessed Impact of Nicotine against Neurotoxicity in Mesencephalic Dopaminergic Cell Cultures and Neuroblastoma N18TG2 Cells

• Abstract
• Introduction
• Results
• Discussion
• Materials and Methods
• Materials
• Preparation of mesencephalic dopaminergic cell cultures
• Treatment of primary mesencephalic cells
• Treatment of neuroblastoma cell line (N18TG2)
• Resazurin reduction assay
• Identification of THir neurons
• Determination of neurite outgrowth in THir neurons
• JC-1 staining
• Measurement of total GSH
• PI uptake
• DAPI measurement
• Statistical analysis
• Contributorsʼ Statement
• References

Abstract

Neuroprotective effects of nicotine are still under debate, so further studies on its effectiveness against Parkinsonʼs disease are required. In our present study, we used primary dopaminergic cell cultures and N18TG2 neuroblastoma cells to investigate the effect of nicotine and its neuroprotective potential against rotenone toxicity. Nicotine protected dopaminergic (tyrosine hydroxylase immunoreactive) neurons against rotenone. This effect was not nAChR receptor-dependent. Moreover, the alkaloid at a concentration of 5 µM caused an increase in neurite length, and at a concentration of 500 µM, it caused an increase in neurite count in dopaminergic cells exposed to rotenone. Nicotine alone was not toxic in either cell culture model, while the highest tested concentration of nicotine (500 µM) caused growth inhibition of N18TG2 neuroblastoma cells. Nicotine alone increased the level of glutathione in both cell cultures and also in rotenone-treated neuroblastoma cells. The obtained results may be helpful to explain the potential neuroprotective action of nicotine on neural cell cultures.

Introduction

Nicotine is a commonly known alkaloid produced in some Solanaceae species, for instance in Nicotiana tabacum L., which is believed to contribute to smoking addiction, that leads to the premature death of over 8 million people each year, according to an estimation by the World Health Organization [1]. By contrast, it has been suggested and vividly discussed that cigarette smoking may protect from Parkinsonʼs disease (PD) since smokers have lower rates of PD [2], [3], [4], [5]. On one hand, the presumable therapeutic potential of nicotine has gained more attention. On the other hand, some results have revised nicotineʼs addictive potential [6], indicating that cigarette smoke, which contains more than 5600 compounds, is the main factor for smoking dependence [7]. Nonetheless, an increasing number of studies have shed new light on nicotine as a pharmacological agent, indicating that nicotine possesses therapeutic benefits [8], [9], [10].

Since nicotine is a probable candidate of an antiParkinson agent in tobacco [11], this study aims to elucidate its neuroprotective potential. PD is characterized by the formation of Lewi bodies and the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), which is manifested by rigidity, bradykinesia, tremor, and postural instability [12], [13]. The damage in SNpc is supposed to be due to the deleterious effect of oxidative stress on dopaminergic neurons [14]. Treatment of PD is based on overcoming the lack of neurotransmitters by delivery of L-DOPA, which is burdened with severe side effects, or dopamine agonists, which compensate for the loss of DA in the striatum [15]. Additionally, monoamine oxidase B inhibitors, antioxidants, catechol-O-methyltransferase inhibitors, and other drugs are used to optimize PD therapy, considering the progression of the disease and individual pathogenesis.

Rotenone is a pesticide compound isolated from Derris plants with lipophilic properties, which easily crosses membranes [16]. Rotenone is known to be cytotoxic to primary dopaminergic neurons [17]. Its cytotoxic mechanism of action is due to its function as a specific inhibitor of mitochondrial complex I with consequent formation of free radicals, especially superoxide radicals [17], [18], [19], [20]. These can be transformed into hydrogen peroxide via superoxide dismutases. Hydrogen peroxide can take part in the Fenton reaction, leading to the formation of the even more reactive hydroxyl radical. Betarbet et al. showed that, besides an increase of reactive oxygen species levels, the formation of Lewi bodies also can be induced by rotenone [16]. Since Betarbetʼs study, complex I inhibition by rotenone is a common model for PD. In murine mesencephalic cell cultures, rotenone and MPP⁺, another complex I inhibitor that only affects dopaminergic neurons due to its specific uptake mechanism, are used as animal replacement PD models [17], [21], [22], while in cell lines, usually only rotenone is used.

There are many theories concerning the probable neuroprotective effects of nicotine, including both receptor- and nonreceptor-mediated mechanisms of action. Nicotine, as an agonist of the nicotinic acetylcholine receptors (nAChRs), increases the levels of dopamine (DA) within the central nervous system (CNS), and therewith supports the L-DOPA treatment to attenuate the symptoms of the PD [10]. Nicotine can also cause up-regulation of anti-apoptotic proteins like BCL-2 and BCL-X, which seem to be mediated by nAChRs [23]. It is also known that nicotinic receptors containing a beta 2-subunit serve as a critical link between the release of acetylcholine and the dopaminergic reward control system [24].

Nicotine was shown to protect mammalian dopaminergic neurons from 6-hydroxydopamine, a dopaminergic neuron-specific neurotoxin, in an nAChR-dependent manner [25]. More recently, nicotine-induced neuroprotection has been suggested to involve a calcium-modulated, mitochondrial stress-activated PTEN-induced kinase 1 (PINK1)/Parkin-dependent pathway (PDR-1) [26]. It has been suggested also that nicotine may attenuate amyloid-β-mediated neurotoxicity and NMDA-induced excitotoxicity. Further, it prevents oxidative stress-induced injury through α7-nAChR involvement [27]. Moreover, nicotine was efficacious to limit the damage caused by glutamate-induced excitotoxicity, probably by the prevention of triggering intracellular cell death pathways [28]. In the above-mentioned MPP⁺ model of neurodegeneration, nicotine was reported to protect neurons by suppressing SIRT6, a member of the sirtuin family comprising NAD+-dependent enzymes, which can promote apoptosis in neurons [29].

This study was designed to investigate the effects of pure nicotine against rotenone, a PD model drug in primary mesencephalic cultures, as well as in neuroblastoma cell lines N18TG2 (NB). Since rotenone treatment leads to superoxide radical formation, and the involvement of mitochondria was proposed, we aimed to investigate whether nicotine can restore mitochondrial function and induce the increase of endogenous antioxidant glutathione (GSH) levels. Further, we wanted to know whether nicotine-induced neuroprotection in our primary cell culture model is nAChR-mediated.

Results

Due to the high vulnerability of dopaminergic neurons to oxidative stress, we stained these neurons immunocytochemically against tyrosine hydroxylase. Tyrosine hydroxylase converts tyrosine to L-DOPA, the precursor of dopamine. This allows the evaluation of dopaminergic cell (THir cell) survival and morphology. The mean of dopaminergic neurons per well was 545 ± 127, corresponding to around 0.21% of the whole cell population. Treatment of cultures with 80 nM of rotenone for 48 h significantly decreased the number of tyrosine hydroxylase immunoreactive (THir) neurons by 51.1%, compared to cultures treated with vehicle alone. Concomitant treatment with nicotine in concentrations of 0.5 and 5 µM significantly increased the number of surviving cells by 78.2% and 63.4%, respectively ([Fig. 1]). Moreover, a strong but not significant tendency for an increased number of THir neurons after treatment with nicotine alone in lower concentrations could be observed.

Rotenone treatment led to malformations and undirected outgrowth of neurites and shrunken cell bodies while nicotine had no effect. In cells treated with nicotine and rotenone, the neurite outgrowth recovered to a large extent. Nonetheless, the cell bodies seemed not to reach the size and shape of those in control cultures ([Fig. 2]).

While treatment with nicotine alone has no effect (data not shown), administration of rotenone significantly decreased the average number of primary neurites and the neurite lengths of THir neurons by 38.2% and 52.8%, respectively, when compared to controls. Compared to those values, nicotine could increase the average number of primary neurites dose-dependently, which was significant at a concentration of 500 µM (increase by 26.0% compared to rotenone controls) ([Fig. 3 a]). Likewise, the average length of the primary neurites was significantly higher in cultures additionally treated with nicotine compared to those treated with rotenone alone ([Fig. 3 b]). At a concentration of 5 µM, the beneficent effect of nicotine reached a maximum, with an increase to 80.9% from 47.2% in cells affected by rotenone. To investigate a proposed nACh receptor dependency, the nAChR antagonist mecamylamine was administered in cultures treated concomitantly with rotenone, but no changes of the count of tyrosine hydroxylase positive cells could be observed ([Fig. 4]).

Inducing oxidative stress by rotenone can be counteracted by the upregulation of endogenous antioxidative mechanisms, such as an increase of GSH levels. Treatment of cultures with rotenone decreased the total level of GSH nonsignificantly by about 9.6% compared to controls. Treatment of cultures with nicotine alone significantly increased the total level of GSH in cells by 23.7% at a concentration of 0.5 µM and 42.0% at 500 µM ([Fig. 5 a]). In cultures concomitantly treated with rotenone, this increase was slackened, so influencing GSH levels seem not to be the protective effect of nicotine. Nicotine is discussed to have antioxidant abilities, but its impact on mitochondrial activity and oxidative stress in primary cell cultures has not been reported before.

JC-1 localizes to the inner mitochondria membrane where it forms red fluorescent dimers in mitochondria with intact membrane potential. In damaged mitochondria, aggregation does not take place, and green fluorescence of JC-1 monomers predominate. Because of that, JC-1 staining is a reliable marker for the integrity of mitochondria. Treatment of cultures with rotenone (80 nM) on the 12th DIV for 48 h significantly decreased the red/green fluorescence ratio by 10.8% compared to controls ([Fig. 5 b]). Concomitant treatment with nicotine had no effect.

The above-described results were done in primary cultures that contain around 60% glial cells. To exclude the glial participation on the effect of nicotine, we performed further experiments on neuroblastoma cells.

Healthy, metabolic active cells can convert the nontoxic resazurin to the molecule resorufin, while the color of the reagent changes from blue to pink. The amount of resazurin reduction is proportional to the overall metabolism activity. In N18TG2 cells, resazurin reduction was diminished by 23.5% after rotenone administration in comparison to control. Nicotine had no significant influence on the resorufin production of NB cells in both control and rotenone cultures ([Fig. 6 a]).

Changes in the cultureʼs overall metabolism can be the consequence of a reduced metabolism of the cells but also a reduced cell number. Therefore, we used propidium iodide (PI) for detecting the dying and dead neuroblastoma cells, since it can enter only cells with disintegrated membranes. Nicotine was not toxic to the cells as shown in PI staining, but a minor dose-dependent decrease of total cell density by 9.5% could be observed, which was significant at a concentration of 500 µM, by using the nuclear fluorescence dye DAPI ([Fig. 6 b]). In rotenone cultures, a decrease of DAPI fluorescence by 31.8% was detected, which was further reduced by 27.8% in cultures administered with nicotine (500 µM) concomitantly.

Treatment of NB cells with rotenone significantly decreased the total level of GSH by 52.2%, compared to vehicle control, while treatment with nicotine alone in lower concentrations like 0.5 µM and 5 µM for 48 h significantly increased the total level of GSH by 10.2% and 5.7%, respectively, when compared to controls ([Fig. 7]). Simultaneous treatment of NB cells with nicotine and rotenone caused a significant increase in total GSH levels in cells treated with a concentration of 0.5, 5, and 50 µM by 20.2, 16.0, and 13.0% respectively, in comparison to control cells exposed only to the rotenone.

Discussion

Nicotine has gained interest in treatment for smoking addiction as an active compound of the smoking cessation therapy (nicotine replacement therapy, NRT). Marginal attention has been paid to the therapeutic potential of nicotine. It has been suggested that cigarette smoking correlates with a diminished prevalence of PD [2], [3], [4], [5]. The exact etiology of PD is still unknown, but oxidative stress, genetic predisposition, microbiome disturbance, protein misfolding, the involvement of certain pesticides, and exposure to heavy metals as triggers for the pathogenesis of this disease have been suggested [30]. In a case-control study, Ritz et al. [31] found a strong inverse association between smoking and PD. Not only those who smoked but also those using NRT were less likely to develop PD. According to other epidemiologic studies, there exists a dose-response relationship between smoking and the disease [32], [33]. These results are discussed controversially because of the study designs that create a survivor bias due to premature death. Nevertheless, the main candidate responsible for the protection seems to be nicotine [2]. Despite a large number of studies, the precise relationship between nicotine and neuroprotection remains to be elucidated.

Our study was designed to assess the effect of nicotine in rotenone-induced cell death in neuroblastoma and dopaminergic cells in primary mesencephalic cultures, which are used to study cellular and molecular aspects of PD [34] and neuroblastoma cells. According to Shimohama et al. [23], nicotine inhibited both motor deficits and DA neuronal cell loss in the substantia nigra of rotenone-treated mice.

First, we wanted to know whether nicotine can counteract rotenone toxicity. While dopaminergic neurons can be detected distinctively by immunocytochemistry and possess a high sensitivity to oxidative stress, we counted THir neurons and measured their neurite outgrowth. Concomitant treatment with rotenone and nicotine in concentrations of 0.5 and 5 µM significantly increased the number of surviving cells. Also, a strong (but not significant) tendency for an increased number of THir neurons after treatment with nicotine alone was observed. The obtained results were reflected by the morphology of the cells and were in accordance with results obtained for the neurite outgrowth. Rotenone administration leads not only to increased oxidative stress that induces cell degenerating cascades but also results in a decreased formation of ATP [35]. Neurite outgrowth is an energy-consuming process. Therefore, the numbers and lengths of primary neurites were detected to quantify the effect of nicotine under mitochondrial impairment and to validate the impression given by THir microscopy.

Thus, our data give evidence that nicotine provided significant protection regarding the morphology of neurons against rotenone. Our results may suggest that nicotine improves the energy supply for the cells.

Our results remain partially consistent with results from Takeuchi et al. [11], where nicotine prevented a decrease of dopaminergic neurons in SNpc caused by rotenone as well as a decrease of TH-positive neurons in the striatum. The protection of primary mesencephalic cultures was supposed to be mediated via nAChRs and through activation of the PI3K-Akt/PKB pathway.

Receptor-mediated action of nicotine was also stated in a study by Quik et al. [36], where activation of nicotinic receptors by nicotine-protected nigral dopaminergic neurons from 1-methyl-4-phenyl-1,2,3,6-tetrahydropteridine (MPTP) toxicity in vitro. It was also pointed out that the protection seems to be particularly through α7- and α4β2-nicotinic receptors. MPTP is converted to the above-mentioned complex I inhibitor MPP⁺, so the main effect seems to be consistent with our findings. Interestingly, the survival rate of dopaminergic neurons is higher in the rotenone than in the MPP⁺ model, though in our cultures even the astrocytes suffered from complex I inhibition. This was not expected, since rotenone and MPP⁺ are different models for PD: While MPP⁺ enters only dopaminergic cells through dopamine transporters, rotenone affects all cells. Therewith, this finding may point to an involvement of astrocytes in neuroprotection, since it is known that astrocytes at least in the hippocampus have functional α7 nAChRs [37].

Moreover, it has been also suggested, that nicotine acts not only on cell surface nicotinergic receptors, but mitochondrial nAChR and respiratory complex I also are suggested targets of direct nicotine action in mitochondria with different effects on the reactive oxygen species (ROS) production. The effects can be both detrimental and beneficial, but they vary between studies depending on how the nicotine is administered and mitochondria are isolated [38].

On the contrary, treating cells with an nAChR antagonist in our study did not show any significant change in observed results concerning the viability of dopaminergic neurons, indicating that the effects we observed were not receptor-mediated.

Since rotenone administration leads to an increase of superoxide radicals, we investigated whether nicotine can increase the endogenous antioxidative capacity of the cells. Treatment of cultures with nicotine alone significantly increased the total level of GSH in cells, but in cultures concomitantly treated with rotenone, this increase was slackened, so influencing GSH levels seemed not to be the protective effect of nicotine. Sherer et al. [30] have shown that a short-term rotenone exposure has an insignificant trend toward increased cellular GSH levels, but just chronic rotenone treatment (3 – 4 weeks) caused a significant reduction in cellular GSH levels. So, we assume that modification of total GSH levels can be triggered by rotenone in a time-dependent manner. After 48 h in our culture system, we detected only a tendency of decreased total GSH levels in rotenone-treated cultures, and the effect of nicotine in rotenone-treated primary cultures was significant at a concentration of 0.5 and 500 µM but comparably low.

So, we do not expect the induction of endogenous antioxidation via GSH to be a main protective mechanism of nicotine. Nevertheless, this effect should not be neglected, taking into account that the GSH levels are decreased in PD [39]. The effects of nicotine on GSH levels are cell type- and experimental model-specific. For instance, it is known that nicotine may inhibit the activity of glutathione reductase in the cell culture of mesenchymal stem cells. [40]. The influence of astrocytes in primary cell culture has also to be taken into consideration, as GSH released from astrocytes plays an important role in the whole supply of GSH in neural culture [41]. This could also be the case in reference to our primary culture. So far, it is also known that activation of astroglial α7-nicotinic receptors may play a role in neuroprotection by decreasing inflammation and oxidative stress [42].

Nicotine is discussed to have antioxidant abilities, but its impact on mitochondrial activity and oxidative stress in primary cell cultures has not been reported before. Rotenone as an inhibitor of complex I of the respiratory chain causes mitochondrial impairment. To exclude that nicotine directly interacts with rotenone and to investigate whether nicotine interferes with mitochondria, we measured the red/green fluorescent ratio of JC-1, an indicator for impaired mitochondrial function. Nicotine is known to compete on complex I of the mitochondrial electron transfer chain and to decrease oxygen consumption [43]. Due to these characteristics, it may protect the CNS. Treatment of cultures with rotenone significantly decreased mitochondrial integrity. Concomitant treatment with nicotine had no effect, pointing to neither a molecular interaction with rotenone nor recovery of mitochondrial function.

JC-1 data in primary cells showed that mitochondrial function is not recovered in rotenone-treated mesencephalic cultures by nicotine, while cells are still able to continue ATP-consuming neuronal outgrowth. While treatment of cells with nicotine showed an increasing tendency (not significant) to preserve the mitochondrial membrane potential, this finding does not explain the protective effect against rotenone.

Combined with the low effect of nicotine on GSH levels, it could also be possible that nicotine may enable the cells to switch the cell metabolism to another source of ATP than the mitochondrial respiratory chain, since in our cultures, nicotine treatment in rotenone cultures lead to a significant increase of neurite outgrowth and a partial restoration of dopaminergic cell morphology. It has to be elucidated whether nicotine can increase ATP formation in cells (e.g., from other sources than mitochondria, like glycolysis).

In a study by Linert et al. [44], the possible antioxidant effects of nicotine in vitro were presented. This might result from its characteristic binding of Fe²⁺ and the reduction of transferrin-mediated iron uptake as well as the reduction of Fenton activity in the presence of nicotine and dopamine. Additionally, in the study of Bridge et al. [45], which was examining the electrochemical behavior of an Fe(II)/Fe(III) redox couple in the presence of nicotine, the ability of nicotine to complex with free iron and to reduce its reactivity was stated. Moreover, according to Williams and Linert [10], it may be also possible that the metabolites of nicotine may have more enhanced chelating abilities than nicotine itself.

Mouhape et. al. [46] stated that nicotine protects against rotenone in primary mesencephalic cultures by acting on the iron turnover in cells. Nicotine was suggested to reduce the level of the cellular labile iron pool and the redox-active iron, which was a receptor-mediated action. This remains in agreement with the findings of Bridge et al. [45], although it sheds new light on this by stating that it is a receptor-mediated effect. Moreover, in the study by Mouhape et al. [46], nicotine protected cells against rotenone in both in vitro and in vivo models of PD. Our data of GSH levels and mitochondrial integrity (JC-1) show that nicotine has no significant effect in rotenone-treated cultures. This supports the suggestion that nicotine might act by the reduction of the Fenton reaction rather than to interact with mitochondrial function to reduce superoxide radical levels.

To exclude glial participation on the effect of nicotine, further experiments were performed in neuroblastoma cells. The first step was to assess the general toxicity of nicotine alone in N18TG2 neuroblastoma cells. For this purpose, we measured the influence of nicotine in the resazurin reduction assay and PI uptake in cells. We could observe no toxicity of nicotine. Taking into account that nicotine has, for many years, been generally perceived as a toxin and just a short time ago its toxicity was reassessed, our results seem to be consistent with the opinion that nicotine is not as harmful as it was thought to be previously [47]. Additionally, cell count (DAPI) analysis showed that nicotine in the highest tested concentration may moderately inhibit cell proliferation in neuroblastoma cells since no increase of cell death (PI uptake) could be observed. Since the cell number was not influenced by nicotine, the resazurin reduction data point to unchanged overall cell metabolism.

The mentioned effects were accompanied by a modest increase in the total GSH levels in control and rotenone-affected cells, which corresponds to the finding in mesencephalic cultures. Nonetheless, no increase in cell survival in rotenone-cotreated cultures was found, pointing to a substantial role of astrocytes in nicotine-mediated neuroprotection.

In our study, nicotine can mitigate the effect of rotenone, but this effect is less pronounced when high concentrations were administered. It is known that nicotine itself can be toxic. For example, it was shown that nicotine can increase oxidative stress in cells after exposition to higher concentrations. The overproduction of ROS may cause damages to basic cellular components resulting in dysfunction or leading to cell death [48]. Observations about the effects caused by low nicotine concentrations may also be in agreement with those described in our previous research on human melanocytes [49], where lower concentrations of nicotine caused induction of specific biochemical processes in cells. Also, neurotoxic effects of nicotine were described [50]. We suppose that protecting and damaging characteristics of nicotine counteract, but in our study, we found no cell toxicity induced by nicotine but rather a weakening of cell protection against rotenone by higher concentrations of nicotine.

To conclude, our study shows that neuroprotection of nicotine against complex I inhibition does not target mitochondria. We propose an astrocyte-dependent, compensative mechanism to increase ATP levels since the mitochondrial integrity was not recovered by nicotine in rotenone-affected cultures. Our developing understanding of the molecular effect of nicotine in cell cultures, together with continuous progress in the development of pharmaceutical formulations containing nicotine may one day lead to finding new concepts in the therapy of neurodegenerative diseases.

Materials and Methods

Materials

Pregnant OF1/SPF mice were purchased from the Institute for Laboratory Zoology and Veterinary Genetics, Himberg (Austria), the N18TG2 cells from the German Collection of Microorganisms and Cell Cultures GMBH. Poly-D-lysine hydrobromide, colorless DMEM, HEPES buffer, FBS (heat-inactivated), horse-serum, L-glutamine, nicotine (purity ≥ 99%), DAPI, resazurin sodium salt, paraformaldehyde, PI, rotenone, sodium pyruvate, and 3,3′-diaminobenzidine tetrahydrochloride-hydrate (DAB) were purchased from Sigma-Aldrich. Dulbeccoʼs PBS saline 1 × (DPBS), Hankʼs balanced salt solution 1 × (HBSS), B27 supplement minus AO, trypsin-EDTA, and trypsin inhibitor were obtained from Invitrogen. ABC-Kit Vectastain and peroxidase mouse IgG were from Vector Laboratories. Hydrogen peroxide, D(+)-glucose monohydrate, DMSO, and Kaiserʼs glycerol gelatine were obtained from Merck. Penicillin/streptomycin, DNase I, and Triton X-100 were purchased from Roche. The mouse anti tyrosine hydroxylase antibody was from Szabo, Austria, and the glutathione assay kit and mecamylamine (hydrochloride) were from Cayman Chemical Company. JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolyl-carbocyanine iodide) was purchased from Thermo Fisher Scientific.

Preparation of mesencephalic dopaminergic cell cultures

Experiments were discussed and approved by the institutional ethics and animal welfare committee under GSP guidelines and national legislation (Sep 28, 2015). Pregnant OF1/SPF mice were cared for and handled following the guidelines of the European Union Council (2010/63/EU) for the use of laboratory animals. Pregnant mice were sacrificed on gestation day 14 before the embryos were carefully removed and transferred to a Petri dish containing PBS saline (pH 7.4) for dissection. Under a stereoscope (Nikon SMZ-1B), the ventral mesencephala were excised and primary cultures prepared according to Koutsilieri et al. [51]. Briefly, after removal of the meninges, tissues were mechanically cut into small pieces in DPBS (pH 7.4) and subsequently triturated and dissociated with a fire-polished Pasteur pipette in DMEM supplemented with HEPES buffer (25 mM), glucose (30 mM), GSH (2 mM), penicillin-streptomycin (10 U/mL and 10 µg/mL, respectively) and heat-inactivated fetal calf serum (FCS, 10%). The cell suspension was placed into 48-well plates (Greiner, Bio One, Inc.) precoated with poly-D-lysine (50 µg/mL; density 750 000 cells per mL, 340 µL cell suspension per well). Cultures were grown at 37 °C in an atmosphere of 5% CO₂ air. On the 1st and 3rd day in vitro, the medium was changed, and on the 5th day, half of the medium was replaced by B27 (1.4%) containing medium. From the 6th day on, B27 supplemented medium was used.

Treatment of primary mesencephalic cells

To investigate the neuroprotective potential of nicotine against rotenone toxicity, cultures were concomitantly treated with rotenone (80 nM in DMSO; the final DMSO concentration was 0.0008%), and nicotine (0.5, 5, 50, and 500 µM) in the presence or absence of 5 µM of mecamylamine hydrochloride (in DMSO; the final DMSO concentration was 0.025%) as a nicotine antagonist on the 12th day in vitro for 48 h. For primary cells and neuroblastoma cells, different parameters were determined. Only total GSH levels were measured in either model. The concentration of rotenone was nearly the LD50 of this compound in our cell culture system and chosen according to Radad et al. [17].

Treatment of neuroblastoma cell line (N18TG2)

The murine neuroblastoma cell line was cultivated according to Rupprecht et al. [52]. For measurement of resazurin reduction, GSH level, and PI uptake, cells were seeded with a final concentration of 300 000 cells/mL in poly-D-lysine pre-coated 96-well plates (150 µL per well; Greiner, Bio One, Inc.) and grown in control media (DMEM with high glucose [24 mM], supplemented with 2 mM pyruvate, 3.9 mM GSH, and B27 serum-free supplement), or in media containing nicotine (0.5, 5, 50, and 500 µM) and rotenone (80 nM) for 48 h. For each independent experiment, another passage of N18TG2 cells was used.

Resazurin reduction assay

To determine the overall metabolic activity of the N18TG2 cell line, a resazurin reduction assay was used. The measurement was performed 48 h after treatment of cells by adding 30 µL of resazurin to each well of a 96-well plate and measuring the spectrometric absorbance of these wells (the final concentration of resazurin was 50 µM). Absorbance was determined by using a plate reader (Spark multimode reader, Tecan) at 570/600 nm after 0 and 4 h of resazurin administration. The percentage of resazurin reduction of the control group was considered as 100% of metabolic activity.

Identification of THir neurons

At the end of the experiments, primary mesencephalic cell cultures were fixed in 4% paraformaldehyde for 45 min at 4 – 8 °C. Cultures were rinsed with DPBS (pH 7.4) between each step of the staining. After washing, cells were permeabilized with 0.4% Triton X-100 for 30 min and incubated with 5% horse serum (Vectastain ABC Kit) for 90 min to block nonspecific binding sites at room temperature. Then, cells were incubated with anti-TH antibody overnight at 4 °C. Biotinylated secondary antibody and avidin-biotin-horseradish peroxidase complex were consecutively added for 90 min each at room temperature. The reaction product was developed in a solution of diaminobenzidine (DAB, 1.4 mM) in DPBS (pH 7.4) containing 3.3 mM H₂O₂. The total THir cells were counted in 10 randomly selected fields (0.01 mm²/field) at 100 × magnification with a Nikon inverted microscope by using a tally counter.

Determination of neurite outgrowth in THir neurons

After anti-TH staining, neurite lengths and numbers of dopaminergic cells were measured. For this reason, 6 photographs per condition were taken under an inverted microscope at the same magnification (200 ×). Since the DAB oxidation product is a dark brown precipitate, no image processing was done. The pictures were edited with Adobe Photoshop CS3, and the neurite lengths were calculated by pixel evaluation. A calibration standard was used for reference.

JC-1 staining

The JC-1 aggregate/monomer ratio has been used as a tool to estimate changes in the mitochondrial membrane potential. Primary cell cultures were treated with nicotine and rotenone for 48 h. At the end of the incubation period, the culture medium was removed, and the cells were loaded with JC-1 for 15 min at 37 °C. A 10 µg/mL stock solution of JC-1 was prepared in DMSO and added to DMEM medium (final concentration of JC-1 was 0.1 µg/mL). After the loading period, the cells were rinsed twice with DPBS (pH 7.4) and photographed with a Nikon inverted microscope with epifluorescence equipment using a rhodamine filter set (510 DM/520 BA, B-2A) and a ProgRes Speed XT camera (Jenoptik). Semiquantitation of red/green fluorescence intensity was carried out by measuring the overall fluorescence intensity in 6 randomly selected areas for each well with aid of Adobe Photoshop software in the histogram mode. Quantitation was expressed as the mean ± standard error of the mean (SEM) of red/green fluorescence ratios, as % of control.

Measurement of total GSH

To investigate the effect of nicotine on total GSH levels in cultured N18TG2 and mesencephalic primary cells, cultures were treated with nicotine (0.5, 5, 50, 500 µM) and rotenone (80 nM) for 48 h. GSH concentrations were determined using a commercially available GSH detection kit (Cayman Chemical Company) following the protocol of the manufacturer.

In brief, 50 µL of the supernatant of the lysed and centrifuged cells (12 000 g for 5 min) was transferred into a 96-well plate. An assay cocktail was prepared containing glutathione reductase, NADP^+, and 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB). The amount of GSH is directly proportional to the yellow-colored reduction product of the DTNB, 5-thio-2-nitrobenzoic acid. The formation of the product was kinetically detected using a plate reader for measurement at 410 nm. Since glutathione reductase was used in the assay, both GSH and GSSG were measured, and the results reflect total GSH.

PI uptake

PI staining was performed after 2 days of incubation for detecting degenerating neuroblastoma cells. The cell culture medium was replaced by 100 µL of a PI solution (670 ng/mL in pre-warmed DMEM) and incubated at 37 °C for 10 min. After incubation with the dye, cultured cells were washed with colorless DMEM, and 10 photos per well were taken by a digital camera (at 1000 magnification; Nikon) attached to an inverted microscope (Nikon) with epifluorescence equipment using the TRITC filter (G-2A). Color intensities for each photo were analyzed by Adobe Photoshop software. The averaged color intensity was measured with the histogram modus.

DAPI measurement

Blue fluorescent DAPI nucleic acid stain was used for counting the whole cell population of neuroblastoma cells. It passes through intact membranes of cells where it binds strongly to DNA. DAPI solution (in pre-warmed DMEM, 2 µM final concentration) was added to the cultures at 37 °C for 10 min and analyzed after washing with colorless DMEM. Ten photos per wells were taken randomly by a digital camera (1000 magnification; Nikon) using the UV-filter on the inverted microscope (Nikon, Japan) from each well and then analyzed by Adobe Photoshop software. The averaged color intensity was measured with the histogram modus.

Statistical analysis

Data were expressed as mean ± SEM. After a Kolmogorov-Smirnov test showing that data do not follow a normal distribution, statistical differences were determined using the Kruskal-Wallis (H)-test followed by the χ2 test. Statistical analysis was done using StatView 5.0, SAS Institute Inc., and Microsoft Excel 2016 software. Differences of *p < 0.05 were regarded as statistically significant. To determine statistical significance between 2 independent sample groups (control vs. rotenone), the nonparametric Mann-Whitney U-test was used. Differences with ^#p < 0.05 were regarded as statistically significant.

Contributorsʼ Statement

Data collection: M. Delijewski, C. Krewenka, B. Kranner, R. Moldzio; design of the study: M. Delijewski, R. Moldzio; statistical analysis: M. Delijewski, R. Moldzio; analysis and interpretation of the data: M. Delijewski, K. Radad, C. Krewenka, B. Kranner, R. Moldzio; drafting the manuscript: M. Delijewski, R. Moldzio; critical revision of the manuscript: K. Radad, R. Moldzio.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgements

The authors acknowledge Alexander Unterberger for his esteemed assistance. The study was supported by the University of Veterinary Medicine, Vienna. In the present study, cell culture is used as an animal experiment replacement model. It is the utmost concern of the authors to help reduce animal experiments. Experiments were discussed and approved by the institutional ethics and animal welfare committee following GSP guidelines and national legislation. Pregnant OF1/SPF mice were cared for and handled following the guidelines of the European Union Council (2010/63/EU) for the use of laboratory animals.

• References

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• 23 Shimohama S. Nicotinic receptor-mediated neuroprotection in neurodegenerative disease models. Biol Pharm Bull 2009; 32: 332-336
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• 30 Sherer TB, Betarbet R, Stout AK, Lund S, Baptista M, Panov AV, Cookson MR, Greenamyre JT. An in vitro model of Parkinsonʼs disease: linking mitochondrial impairment to altered alpha-synuclein metabolism and oxidative damage. J Neurosci 2002; 22: 7006-7015
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  CrossrefPubMedSearch in Google Scholar
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• 40 Aspera-Werz RH, Ehnert S, Heid D, Zhu S, Chen T, Braun B, Sreekumar V, Arnscheidt C, Nussler AK. Nicotine and cotinine inhibit catalase and glutathione reductase activity contributing to the impaired osteogenesis of SCP-1 cells exposed to cigarette smoke. Oxid Med Cell Longev 2018; e2018: 3172480
  PubMedSearch in Google Scholar
• 41 Dringen R, Pfeiffer B, Hamprecht B. Synthesis of the antioxidant glutathione in neurons: supply by astrocytes of CysGly as precursor for neuronal glutathione. J Neurosci 1999; 19: 562-569
  CrossrefPubMedSearch in Google Scholar
• 42 Patel H, McIntire J, Ryan S, Dunah A, Loring R. Anti-inflammatory effects of astroglial alpha7 nicotinic acetylcholine receptors are mediated by inhibition of the NF-kappaB pathway and activation of the Nrf2 pathway. J Neuroinflammation 2017; 14: 192
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• 43 Cormier A, Morin C, Zini R, Tillement JP, Lagrue G. In vitro effects of nicotine on mitochondrial respiration and superoxide anion generation. Brain Res 2001; 900: 72-79
  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 45 Bridge MH, Williams E, Lyons ME, Tipton KF, Linert W. Electrochemical investigation into the redox activity of Fe(II)/Fe(III) in the presence of nicotine and possible relations to neurodegenerative diseases. Biochim Biophys Acta 2004; 1690: 77-84
  CrossrefPubMedSearch in Google Scholar
• 46 Mouhape C, Costa G, Ferreira M, Abin-Carriquiry JA, Dajas F, Prunell G. Nicotine-induced neuroprotection in rotenone in vivo and in vitro models of Parkinsonʼs disease: evidences for the involvement of the labile iron pool level as the underlying mechanism. Neurotox Res 2019; 35: 71-82
  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 48 Mari M, Colell A, Morales A, Montfort C, Garcia-Ruiz C, Fernandez-Checa JC. Redox control of liver function in health and disease. Antioxid Redox Signal 2010; 12: 1295-1331
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Correspondence

Publication History

Received: 22 December 2020

Accepted after revision: 05 June 2021

Article published online:
06 July 2021

© 2021. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

• References

• 1 World Health Organization. Tobacco Fact sheet N°339, updated May 2020, World Health Organization, Geneva. Accessed December 4, 2020 at: http://www.who.int/mediacentre/factsheets/fs339/en/
• 2 Morens DM, Grandinetti A, Reed D, White LR, Ross GW. Cigarette smoking and protection from Parkinsonʼs disease: false association or etiologic clue?. Neurology 1995; 45: 1041-1051
  CrossrefPubMedSearch in Google Scholar
• 3 Fratiglioni L, Wang HX. Smoking and Parkinsonʼs and Alzheimerʼs disease: review of the epidemiological studies. Behav Brain Res 2000; 113: 117-120
  CrossrefPubMedSearch in Google Scholar
• 4 Checkoway H, Powers K, Smith-Weller T, Franklin GM, Longstreth WT, Swanson PD. Parkinsonʼs disease risks associated with cigarette smoking, alcohol consumption, and caffeine intake. Am J Epidemiol 2002; 155: 732-738
  CrossrefPubMedSearch in Google Scholar
• 5 De Reuck J, De Weweire M, Van Maele G, Santens P. Comparison of the age of onset and development of motor complications between smokers and non-smokers in Parkinsonʼs disease. J Neurol Sci 2005; 231: 35-39
  CrossrefPubMedSearch in Google Scholar
• 6 Talhout R, Opperhuizen A, van Amsterdam JG. Role of acetaldehyde in tobacco smoke addiction. Eur Neuropsychopharmacol 2007; 17: 627-636
  CrossrefPubMedSearch in Google Scholar
• 7 Perfetti TA, Rodgman A. The complexity of tobacco and tobacco smoke. Beitr Tabakforsch Int 2011; 24: 215-232
  PubMedSearch in Google Scholar
• 8 Wang D, Gao T, Zhao Y, Mao Y, Sheng Z, Lan Q. Nicotine exerts neuroprotective effects by attenuating local inflammatory cytokine production following crush injury to rat sciatic nerves. Eur Cytokine Netw 2019; 30: 59-66
  PubMedSearch in Google Scholar
• 9 Gandelman JA, Newhouse P, Taylor WD. Nicotine and networks: potential for enhancement of mood and cognition in late-life depression. Neurosci Biobehav Rev 2018; 84: 289-298
  CrossrefPubMedSearch in Google Scholar
• 10 Williams E, Linert W. In vitro evidence supporting the therapeutic role of nicotine against neurodegeneration. In Vivo 2004; 18: 391-399
  PubMedSearch in Google Scholar
• 11 Takeuchi H, Yanagida T, Inden M, Takata K, Kitamura Y, Yamakawa K, Sawada H, Izumi Y, Yamamoto N, Kihara T, Uemura K, Inoue H, Taniguchi T, Akaike A, Takahashi R, Shimohama S. Nicotinic receptor stimulation protects nigral dopaminergic neurons in rotenone-induced Parkinsonʼs disease models. J Neurosci Res 2009; 87: 576-585
  CrossrefPubMedSearch in Google Scholar
• 12 Dunnett SB, Bjorklund A. Prospects for new restorative and neuroprotective treatments in Parkinsonʼs disease. Nature 1999; 399: 32-39
  CrossrefPubMedSearch in Google Scholar
• 13 Shimohama S, Sawada H, Kitamura Y, Taniguchi T. Disease model: Parkinsonʼs disease. Trends Mol Med 2003; 9: 360-365
  CrossrefPubMedSearch in Google Scholar
• 14 Sherer TB, Betarbet R, Greenamyre JT. Environment, mitochondria, and Parkinsonʼs disease. Neuroscientist 2002; 8: 192-197
  PubMedSearch in Google Scholar
• 15 Olanow CW, Agid Y, Mizuno Y, Albanese A, Bonuccelli U, Damier P, De Yebenes J, Gershanik O, Guttman M, Grandas F, Hallett M, Hornykiewicz O, Jenner P, Katzenschlager R, Langston WJ, LeWitt P, Melamed E, Mena MA, Michel PP, Mytilineou C, Obeso JA, Poewe W, Quinn N, Raisman-Vozari R, Rajput AH, Rascol O, Sampaio C, Stocchi F. Levodopa in the treatment of Parkinsonʼs disease: current controversies. Mov Disord 2004; 19: 997-1005
  CrossrefPubMedSearch in Google Scholar
• 16 Betarbet R, Sherer TB, MacKenzie G, Garcia-Osuna M, Panov AV, Greenamyre JT. Chronic systemic pesticide exposure reproduces features of Parkinsonʼs disease. Nat Neurosci 2000; 3: 1301-1306
  CrossrefPubMedSearch in Google Scholar
• 17 Radad K, Rausch WD, Gille G. Rotenone induces cell death in primary dopaminergic culture by increasing ROS production and inhibiting mitochondrial respiration. Neurochem Int 2006; 49: 379-386
  CrossrefPubMedSearch in Google Scholar
• 18 Ferrante RJ, Schulz JB, Kowall NW, Beal MF. Systemic administration of rotenone produces selective damage in the striatum and globus pallidus, but not in the substantia nigra. Brain Res 1997; 753: 157-162
  CrossrefPubMedSearch in Google Scholar
• 19 Alam M, Schmidt WJ. Rotenone destroys dopaminergic neurons and induces parkinsonian symptoms in rats. Behav Brain Res 2002; 136: 317-324
  CrossrefPubMedSearch in Google Scholar
• 20 Moldzio R, Piskernik C, Radad K, Rausch WD. Rotenone damages striatal organotypic slice culture. Ann N Y Acad Sci 2008; 1148: 530-535
  CrossrefPubMedSearch in Google Scholar
• 21 Moldzio R, Pacher T, Krewenka C, Kranner B, Novak J, Duvigneau JC, Rausch WD. Effects of cannabinoids Δ(9)-tetrahydrocannabinol, Δ(9)-tetrahydrocannabinolic acid and cannabidiol in MPP⁺ affected murine mesencephalic cultures. Phytomedicine 2012; 19: 819-824
  CrossrefPubMedSearch in Google Scholar
• 22 Gille G, Rausch WD, Hung ST, Moldzio R, Janetzky B, Hundemer HP, Kolter T, Reichmann H. Pergolide protects dopaminergic neurons in primary culture under stress conditions. J Neural Transm 2002; 109: 633-643
  CrossrefPubMedSearch in Google Scholar
• 23 Shimohama S. Nicotinic receptor-mediated neuroprotection in neurodegenerative disease models. Biol Pharm Bull 2009; 32: 332-336
  CrossrefPubMedSearch in Google Scholar
• 24 Changeux JP. Nicotinic receptors and nicotine addiction. C R Biol 2009; 332: 421-425
  CrossrefPubMedSearch in Google Scholar
• 25 Ryan RE, Ross SA, Drago J, Loiacono RE. Dose-related neuroprotective effects of chronic nicotine in 6-hydroxydopamine treated rats, and loss of neuroprotection in alpha4 nicotinic receptor subunit knockout mice. Br J Pharmacol 2001; 132: 1650-1656
  CrossrefPubMedSearch in Google Scholar
• 26 Nourse jr. JB, Harshefi G, Marom A, Karmi A, Cohen Ben-Ami H, Caldwell KA, Caldwell GA, Treinin M. Conserved nicotine-activated neuroprotective pathways involve mitochondrial stress. iScience 2021; 24: 102140
  CrossrefPubMedSearch in Google Scholar
• 27 Dong Y, Bi W, Zheng K, Zhu E, Wang S, Xiong Y, Chang J, Jiang J, Liu B, Lu Z, Cheng Y. Nicotine prevents oxidative stress-induced hippocampal neuronal injury through α7-nAChR/Erk1/2 signaling pathway. Front Mol Neurosci 2020; 13: 557647
  CrossrefPubMedSearch in Google Scholar
• 28 Kaur J, Rauti R, Nistri A. Nicotine-mediated neuroprotection of rat spinal networks against excitotoxicity. Eur J Neurosci 2018; 47: 1353-1374
  CrossrefPubMedSearch in Google Scholar
• 29 Nicholatos JW, Francisco AB, Bender CA, Yeh T, Lugay FJ, Salazar JE, Glorioso C, Libert S. Nicotine promotes neuron survival and partially protects from Parkinsonʼs disease by suppressing SIRT6. Acta Neuropathol Commun 2018; 6: 120
  CrossrefPubMedSearch in Google Scholar
• 30 Sherer TB, Betarbet R, Stout AK, Lund S, Baptista M, Panov AV, Cookson MR, Greenamyre JT. An in vitro model of Parkinsonʼs disease: linking mitochondrial impairment to altered alpha-synuclein metabolism and oxidative damage. J Neurosci 2002; 22: 7006-7015
  CrossrefPubMedSearch in Google Scholar
• 31 Ritz B, Lee PC, Lassen CF, Arah OA. Parkinson disease and smoking revisited: ease of quitting is an early sign of the disease. Neurology 2014; 83: 1396-1402
  CrossrefPubMedSearch in Google Scholar
• 32 Grandinetti A, Morens DM, Reed D, MacEachern D. Prospective study of cigarette smoking and the risk of developing idiopathic Parkinsonʼs disease. Am J Epidemiol 1994; 139: 1129-1138
  CrossrefPubMedSearch in Google Scholar
• 33 Gorell JM, Rybicki BA, Cole Johnson C, Peterson EL. Occupational metal exposures and the risk of Parkinsonʼs disease. Neuroepidemiology 1999; 18: 303-308
  CrossrefPubMedSearch in Google Scholar
• 34 Shastry P, Basu A, Rajadhyaksha MS. Neuroblastoma cell lines–a versatile in vitro model in neurobiology. Int J Neurosci 2001; 108: 109-126
  CrossrefPubMedSearch in Google Scholar
• 35 JanssenDuijghuijsen LM, Grefte S, de Boer VCJ, Zeper L, van Dartel DAM, van der Stelt I, Bekkenkamp-Grovenstein M, van Norren K, Wichers HJ, Keijer J. Mitochondrial ATP depletion disrupts Caco-2 monolayer integrity and internalizes claudin 7. Front Physiol 2007; 8: 794
  CrossrefPubMedSearch in Google Scholar
• 36 Quik M, Jeyarasasingam G. Nicotinic receptors and Parkinsonʼs disease. Eur J Pharmacol 2000; 393: 223-230
  CrossrefPubMedSearch in Google Scholar
• 37 Shen JX, Yakel JL. Functional alpha7 nicotinic ACh receptors on astrocytes in rat hippocampal CA1 slices. J Mol Neurosci 2012; 48: 14-21
  CrossrefPubMedSearch in Google Scholar
• 38 Malińska D, Więckowski M, Michalska B, Drabik K, Prill M, Patalas-Krawczyk P, Walczak J, Szymański J, Mathis C, Van der Toorn M, Luettich K, Hoeng J, Peitsch M, Duszyński J, Szczepanowska J. Mitochondria as a possible target for nicotine action. J Bioenerg Biomembr 2019; 51: 259-276
  CrossrefPubMedSearch in Google Scholar
• 39 Smeyne M, Smeyne RJ. Glutathione metabolism and Parkinsonʼs disease. Free Radic Biol Med 2013; 62: 13-25
  CrossrefPubMedSearch in Google Scholar
• 40 Aspera-Werz RH, Ehnert S, Heid D, Zhu S, Chen T, Braun B, Sreekumar V, Arnscheidt C, Nussler AK. Nicotine and cotinine inhibit catalase and glutathione reductase activity contributing to the impaired osteogenesis of SCP-1 cells exposed to cigarette smoke. Oxid Med Cell Longev 2018; e2018: 3172480
  PubMedSearch in Google Scholar
• 41 Dringen R, Pfeiffer B, Hamprecht B. Synthesis of the antioxidant glutathione in neurons: supply by astrocytes of CysGly as precursor for neuronal glutathione. J Neurosci 1999; 19: 562-569
  CrossrefPubMedSearch in Google Scholar
• 42 Patel H, McIntire J, Ryan S, Dunah A, Loring R. Anti-inflammatory effects of astroglial alpha7 nicotinic acetylcholine receptors are mediated by inhibition of the NF-kappaB pathway and activation of the Nrf2 pathway. J Neuroinflammation 2017; 14: 192
  CrossrefPubMedSearch in Google Scholar
• 43 Cormier A, Morin C, Zini R, Tillement JP, Lagrue G. In vitro effects of nicotine on mitochondrial respiration and superoxide anion generation. Brain Res 2001; 900: 72-79
  CrossrefPubMedSearch in Google Scholar
• 44 Linert W, Bridge MH, Huber M, Bjugstad KB, Grossman S, Arendash GW. In vitro and in vivo studies investigating possible antioxidant actions of nicotine: relevance to Parkinsonʼs and Alzheimerʼs diseases. Biochim Biophys Acta 1999; 1454: 143-152
  CrossrefPubMedSearch in Google Scholar
• 45 Bridge MH, Williams E, Lyons ME, Tipton KF, Linert W. Electrochemical investigation into the redox activity of Fe(II)/Fe(III) in the presence of nicotine and possible relations to neurodegenerative diseases. Biochim Biophys Acta 2004; 1690: 77-84
  CrossrefPubMedSearch in Google Scholar
• 46 Mouhape C, Costa G, Ferreira M, Abin-Carriquiry JA, Dajas F, Prunell G. Nicotine-induced neuroprotection in rotenone in vivo and in vitro models of Parkinsonʼs disease: evidences for the involvement of the labile iron pool level as the underlying mechanism. Neurotox Res 2019; 35: 71-82
  CrossrefPubMedSearch in Google Scholar
• 47 Mayer B. How much nicotine kills a human? Tracing back the generally accepted lethal dose to dubious self-experiments in the nineteenth century. Arch Toxicol 2014; 88: 5-7
  CrossrefPubMedSearch in Google Scholar
• 48 Mari M, Colell A, Morales A, Montfort C, Garcia-Ruiz C, Fernandez-Checa JC. Redox control of liver function in health and disease. Antioxid Redox Signal 2010; 12: 1295-1331
  CrossrefPubMedSearch in Google Scholar
• 49 Delijewski M, Wrześniok D, Otręba M, Beberok A, Rok J, Buszman E. Nicotine impact on melanogenesis and antioxidant defense system in HEMn-DP melanocytes. Mol Cell Biochem 2014; 395: 109-116
  CrossrefPubMedSearch in Google Scholar
• 50 Ferrea S, Winterer G. Neuroprotective and neurotoxic effects of nicotine. Pharmacopsychiatry 2009; 42: 255-265
  Thieme ConnectPubMedSearch in Google Scholar
• 51 Koutsilieri E, Chen TS, Kruzik P, Rausch WD. A morphometric analysis of bipolar and multipolar TH-IR neurons treated with the neurotoxin MPP⁺ in co-cultures from mesencephalon and striatum of embryonic C57BL/6 mice. J Neurosci Res 1995; 41: 197-205
  CrossrefPubMedSearch in Google Scholar
• 52 Rupprecht A, Sittner D, Smorodchenko A, Hilse KE, Goyn J, Moldzio R, Seiler AE, Brauer AU, Pohl EE. Uncoupling protein 2 and 4 expression pattern during stem cell differentiation provides new insight into their putative function. PLoS One 2014; 9: e88474
  CrossrefPubMedSearch in Google Scholar