Cytotoxicity Of Rotenone Research Articles

Traditionally used edible medicinal plants protect against rotenone induced toxicity in SH-SY5Y cells-a prospect for the development of herbal nutraceuticals

Plants are good sources of pharmacologically active compounds. The present study aimed to examine the neuroprotective potentials of the methanol extracts of Salix tetrasperma Roxb. leaf (STME) and Plantago asiatica L. (PAME), two edibles medicinal plants of Manipur, India against neurotoxicity induced by rotenone in SH-SY5Y cells. Free radical quenching activities were evaluated by ABTS and DPPH assays. The cytotoxicity of rotenone and the neuronal survival were assessed by MTT assay and MAP2 expression analysis. DCF-DA, Rhodamine 123 (Rh-123), and DAPI measured the intracellular reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP), and apoptotic nuclei, respectively. Superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities were also assessed. LC-QTOF-MS analysis was performed for the identification of the compounds present in STME and PAME. The study showed that both the plant extracts (STME and PAME) showed antioxidant and neuroprotective capabilities in rotenone-induced neurotoxicity by preventing oxidative stress through the reduction of intracellular ROS levels and reversing the activities of GPx, SOD, and CAT caused by rotenone. Further, both plants prevented apoptotic cell death by normalizing the steady state of MMP and protecting nuclear DNA condensation. LC-QTOF-MS analysis shows the presence of known neuroprotective compounds like uridine and gabapentin in STME and PAME respectively. The two plants might be an important source of natural antioxidants and nutraceuticals with neuroprotective abilities. This could be investigated further to formulate herbal nutraceuticals for the treatment of neurodegenerative disease like Parkinson's disease.

Neuroprotective potential of traditionally used medicinal plants of Manipur against rotenone-induced neurotoxicity in SH-SY5Y neuroblastoma cells

Ethnopharmacological relevanceAlternanthera sessilis (L.) R. Br. ex DC., Eryngium foetidum L., and Stephania japonica (Thunb.) Miers plants are traditionally used to treat various central nervous system disorders like paralysis, epilepsy, seizure, convulsion, chronic pain, headache, sleep disturbances, sprain, and mental disorders. However, their possible neuroprotective effects have not been evaluated experimentally so far. Aim of the studyThe study aims to examine the neuroprotective potential of the three plants against cytotoxicity induced by rotenone in SH-SY5Y neuroblastoma cells and assess its plausible mechanisms of neuroprotection. Materials and methodsThe antioxidant properties of the plant extracts were determined chemically by DPPH and ABTS assay methods. The cytotoxicity of rotenone and the cytoprotective activities of the extracts were evaluated using MTT assays. Microtubule-associated protein 2 (MAP2) expression studies in cells were performed to assess neuronal survival after rotenone and extract treatments. Mitochondrial membrane potential and intracellular levels of reactive oxygen species were evaluated using Rhodamine 123 and DCF-DA dye, respectively. Catalase, glutathione peroxidase, and superoxide dismutase activities were also measured. Apoptotic nuclei were examined using DAPI staining. Liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS) analysis of the plant extracts was also performed. ResultsThe methanol extracts of A. sessilis, S. japonica, and E. foetidum showed excellent free radical scavenging activities. MAP2 expression studies show that A. sessilis and S. japonica have higher neuroprotective effects against rotenone-induced neurotoxicity in SH-SY5Y cells than E. foetidum. Pre-treating cells with the plant extracts reverses the rotenone-induced increase in intracellular ROS. The plant extracts could also restore the reduced mitochondrial membrane potential induced by rotenone treatment and reinstate rotenone-induced increases in catalase, glutathione peroxidase, and superoxide dismutase activities. All the extracts inhibited rotenone-induced changes in nuclear morphology and DNA condensation, an early event of cellular apoptosis. LC-QTOF-MS analysis of the plant extracts shows the presence of neuroprotective compounds. ConclusionsThe plant extracts showed neuroprotective activities against rotenone-treated SH-SY5Y cells through antioxidant and anti-apoptotic mechanisms. These findings support the ethnopharmacological uses of these plants in treating neurological disorders. They probably are a good source of neuroprotective compounds that could be further explored to develop treatment strategies for neurodegenerative diseases like Parkinson's disease.

Identification of a novel aromatic-turmerone analog that activates chaperone-mediated autophagy through the persistent activation of p38.

Introduction: Aromatic (Ar)-turmerone is a bioactive component of turmeric oil obtained from Curcuma longa. We recently identified a novel analog (A2) of ar-turmerone that protects dopaminergic neurons from toxic stimuli by activating nuclear factor erythroid 2-related factor 2 (Nrf2). D-cysteine increases Nrf2, leading to the activation of chaperone-mediated autophagy (CMA), a pathway in the autophagy-lysosome protein degradation system, in primary cultured cerebellar Purkinje cells. In this study, we attempted to identify novel analogs of ar-turmerone that activate Nrf2 more potently and investigated whether these analogs activate CMA. Methods: Four novel analogs (A4-A7) from A2 were synthesized. We investigated the effects of A2 and novel 4 analogs on Nrf2 expression via immunoblotting and CMA activity via fluorescence observation. Results: Although all analogs, including A2, increased Nrf2 expression, only A4 activated CMA in SH-SY5Y cells. Additionally, A4-mediated CMA activation was not reversed by Nrf2 inhibition, indicating that A4 activated CMA via mechanisms other than Nrf2 activation. We focused on p38, which participates in CMA activation. Inhibition of p38 significantly prevented A4-mediated activation of CMA. Although all novel analogs significantly increased the phosphorylation of p38 6h after drug treatment, only A4 significantly increased phosphorylation 24h after treatment. Finally, we revealed that A4 protected SH-SY5Y cells from the cytotoxicity of rotenone, and that this protection was reversed by inhibiting p38. Conclusion: These findings suggest that the novel ar-turmerone analog, A4, activates CMA and protects SH-SY5Y cells through the persistent activation of p38.

Codonopsis pilosula polysaccharide alleviates rotenone-induced murine brain organoids death through downregulation of gene body DNA methylation modification in the ZIC4/PGM5/CAMTA1 axis

Here, the protective mechanism of Codonopsis pilosula polysaccharide (CpP) against mouse brain organoids (mBO) damage was analyzed, and the rotenone affected the genomic epigenetic modifications and physiological activity of mouse brain organoids was examined. Pathological experiments have shown that rotenone significantly damaged the subcellular organelles of mouse brain organoids. According to RRBS-Seq, rotenone significantly promoted gene body hypermethylation modifications in mouse brain organoids. Molecular biology experiments have confirmed that rotenone significantly promoted the hypermethylation modification of Zic4, Pgm5, and Camta1 gene bodies in mouse brain organoids, and their expression levels were significantly lower than those of the control group. Bioinformatic analysis suggested that multiple binding motif of transcription factors ZIC4 (Zinc finger protein of the cerebellum 4) were present at the promoters of both the Pgm5 (Phosphoglucomutase 5) and Camta1 (Calmodulin binding transcription activator 1) genes. When the expression of Zic4 was silenced, the proliferation of mouse brain organoids was significantly reduced and the expression level of PGM5 was also significantly decreased. In addition, Codonopsis pilosula polysaccharide treatment of mouse brain organoids significantly reduced the cytotoxicity of rotenone, promoted cell cycle progression, increased intracellular glutathione activity, significantly induced the demethylation modification of the Zic4, Pgm5, and Camta1 gene bodies, and promoted the high expression of ZIC4 and PGM5. Therefore, the study confirmed that Codonopsis pilosula polysaccharide alleviated rotenone-induced mouse brain organoids death by downregulating DNA gene bodies methylation modification of the Zic4/Pgm5/Camta1 axis.

Growth Differentiation Factor 15 Protects SH-SY5Y Cells From Rotenone-Induced Toxicity by Suppressing Mitochondrial Apoptosis.

ObjectiveParkinson’s disease (PD) is the second most common neurodegenerative disorder worldwide. Mitochondrial dysfunction is suspected as one of the pathogenic mechanisms of PD. Growth/differentiation Factor-15 (GDF15) has been reported to affect mitochondrial function in PD. However, the relationship between mitochondrial function and GDF15 induction has not been explained well. Hence, we aimed to reveal the effect of GDF15 induction on SH-SY5Y cells with rotenone toxicity, a cell model of PD.MethodsSH-SY5Y cells were exposed to 1 μM rotenone as a PD model. Cells were transfected with a GDF15-overexpression plasmid and empty vector. We then analyzed the expression level of GDF15, BCL-2/BAX, P53, PGC1-α, α-syn, and TH in GDF15-overexpressing cells by western blotting, enzyme-linked immunosorbent assay, and quantitative real-time polymerase chain reaction. The cytotoxicity of rotenone was measured by CCK-8 assays. Cell apoptosis was detected by flow cytometric and TUNEL assays. The effect of GDF15 on oxidative stress and mitochondrial function was revealed using DCFH-DA, mito-SOX, and JC-10 assays and a Seahorse XF Cell Mito Stress Test.ResultsGDF15 protected rotenone-treated SH-SY5Y cells from toxicity by preserving mitochondrial function and decreasing apoptosis, during which GDF15 might function by influencing PGC1α through the regulation of p53. In addition, GDF15 overexpression could improve Akt and mTOR phosphorylation, leading to PI3K/Akt/mTOR pathway activation. However, these protective effects were eliminated when cells were treated with the PI3K/Akt specific inhibitor LY294002.ConclusionOur findings suggest that GDF15 can protect mitochondrial function and inhibit apoptosis in SH-SY5Y cells after exposure to rotenone by upregulating PGC1α via p53. These properties might comprise its anti-apoptotic effects, mediated by the PI3K/Akt/mTOR signaling pathway.

In Vitro Evaluation of Antioxidant and Anticancer Activity of Lemongrass (Cymbopogon citratus (D.C.) Stapf)

Cancer is considered a multifactorial disease and its development could be associated with several factors, for example, rotenone exposition. Unfortunately, many cancers are resistant to chemotherapy, as cervical cancer. Regarding this, lemongrass is a remarkable natural product that presents antioxidant and anticancer activities, which could show therapeutic action against rotenone and cervical cancer. Thus, this study aimed to investigate the antioxidant and anticancer action of lemongrass. An in vitro study was conducted using VERO (kidney cells) and SiHa cell lines (cervical cancer cells). VERO cells were exposed to rotenone and lemongrass extract for 24 and 72 h. While SiHa cells were exposed to lemongrass isolated and associated to chemotherapy, 5-fluorouracil, during 24 and 48 h. After, levels of viability, proliferation, and oxidative metabolism were determined. The results showed that lemongrass presents antioxidant activity on VERO cells by increasing cell viability and proliferation and decreasing oxidative stress caused by rotenone. Moreover, lemongrass showed anticancer activity by decreasing cell viability and increasing oxidative stress parameters on SiHa. Besides, lemongrass had no alteration in the chemotherapy activity. Therefore, this study revealed that lemongrass presents antioxidant and anticancer activity since it can protect against the cytotoxicity of rotenone and reduce the cell viability of cervical cancer.

Rotenone restrains colon cancer cell viability, motility and epithelial‑mesenchymal transition and tumorigenesis in nude mice via the PI3K/AKT pathway.

Rotenone, a natural hydrophobic pesticide, has been reported to display anticancer activity in a variety of cancer cells. However, the mechanism of rotenone on colon cancer (CC) cell migration, invasion and metastasis is still unknown. In the present study, the cytotoxicity of rotenone on CC cells were detected by the Cell Counting Kit-8 assay and confirmed by clone formation assay. The effects of rotenone on CC cell invasion and migration activity were determined in vitro by Transwell invasion and wound healing assays, respectively. In addition, to reveal whether rotenone affected the epithelial-mesenchymal-transition (EMT) process, reverse transcription-quantitative PCR, western blotting and immunofluorescence assays were used to detect the expression of EMT markers. The expression levels of the key markers of the PI3K/AKT pathway after rotenone treatment alone or in combination with a PI3K/AKT signaling activator in CC were also detected by western blotting. Finally, the in vivo anti-tumor effects of rotenone were evaluated in a subcutaneous xenotransplant tumor model treated with an intraperitoneal injection of rotenone. The results of the present study demonstrated that rotenone treatment induced CC cell cytotoxicity and greater effects were observed with increasing concentrations and inhibited cell proliferation compared with untreated cells. In vitro cell function assays revealed that rotenone inhibited CC cell migration, invasion and EMT compared with untreated cells. Mechanically, the phosphorylation levels of AKT and mTOR were downregulated in rotenone-treated CC cells compared with untreated cells. Additionally, AKT and mTOR phosphorylation levels were increased by the PI3K/AKT signaling activator insulin-like growth factor 1 (IGF-1), which was reversed by rotenone treatment. The cell function assays confirmed that the IGF-1-activated cell proliferation, migration and invasion were decreased by rotenone treatment. These results indicated that rotenone affected CC cell proliferation and metastatic capabilities by inhibiting the PI3K/AKT/mTOR signaling pathway. In addition, rotenone inhibited tumor growth and metastatic capability of CC, which was confirmed in a xenograft mouse model. In conclusion, the present study revealed that rotenone inhibited CC cell viability, motility, EMT and metastasis in vitro and in vivo by inhibiting the PI3K/AKT/mTOR signaling pathway.

Activation of the immunoproteasome protects SH-SY5Y cells from the toxicity of rotenone

This study investigated the expression and role of immunoproteasome (i-proteasome) in a cell model of Parkinson’s disease (PD). The cytotoxicity of rotenone was measured by CCK-8 assay. The i-proteasome β1i subunit PSMB9 was suppressed by a specific shRNA or transfected with an overexpression plasmid in the SH-SY5Y cells. Under the exposure to rotenone or not, the expression of constitutive proteasome β subunits, i-proteasome βi subunits, antigen presentation related proteins, α-syn and TH were detected by Western blot in PSMB9-silenced or -overexpressed cells, and the proteasomal activities were detected by fluorogenic peptide substrates. The location of i-proteasome βi subunits and α-syn were detected by immunofluorescence staining. The levels of ROS, GSH and MDA were measured by commercial kits. Cell apoptosis was detected by flow cytometry. Besides impairing the constitutive proteasomes, rotenone induced the expression of βi subunits of i-proteasome and antigen presentation related proteins such as TAP1, TAP2 and MHC-I. Silencing or overexpressing PSMB9 had no obvious effect on the levels of other subunits, but could regulate the chymotrypsin-like activity of 20S proteasome and the expression of TAP1, TAP2 and MHC-I. Three βi subunits (PSMB9, PSMB10, PSMB8) of i-proteasome were all co-localized with α-syn. PSMB9 knockdown aggravated accumulation of α-syn, degradation of TH, release of ROS, increased level of MDA, decreased level of GSH and eventually promoted apoptosis in SH-SY5Y cells after rotenone treatment, while over-expression of PSMB9 could attenuate these toxic effects of rotenone. I-proteasome is activated in SH-SY5Y cells treated with rotenone and may play a neuroprotective role.

The novel mechanism of rotenone-induced α-synuclein phosphorylation via reduced protein phosphatase 2A activity

Rotenone has been shown to induce many parkinsonian features and has been widely used in chemical models of Parkinson’s disease (PD). Its use is closely associated with α-synuclein (α-syn) phosphorylation both in vivo and in vitro. However, the mechanisms whereby rotenone regulates α-syn phosphorylation remain unknown. Protein phosphatase 2A (PP2A) has been shown to play an important role in α-syn dephosphorylation. We therefore investigated if rotenone caused α-syn phosphorylation by down-regulation of PP2A activity in mice. Rotenone increased the phosphorylation of α-syn at Ser129, consistent with the inhibition of PP2A activity by increased phosphorylation of tyrosine 307 at the catalytic subunit of PP2A (pTyr307 PP2Ac). We further explored the interactions among rotenone, PP2A, and α-syn in SK-N-SH cells and primary rat cortical neurons. Rotenone inhibited PP2A activity via phosphorylation of PP2Ac at Tyr307. The reduction in PP2A activity and rotenone cytotoxicity were reversed by treatment with the PP2A agonist, C2 ceramide, and the Src kinase inhibitor, SKI606. Immunoprecipitation experiments showed that rotenone induced an increase in calmodulin–Src complex in SK-N-SH cells, thus activating Src kinase, which in turn phosphorylated PP2A at Tyr307 and inhibited its activity. C2 ceramide and SKI606 significantly reversed the rotenone-induced phosphorylation and aggregation of α-syn by increasing PP2A activity. These results demonstrate that rotenone-reduced PP2A activity via Src kinase is involved in the phosphorylation of α-syn. These findings clarify the novel mechanisms whereby rotenone can induce PD.

Sestrin2 Protects Dopaminergic Cells against Rotenone Toxicity through AMPK-Dependent Autophagy Activation.

Dysfunction of the autophagy-lysosomal pathway (ALP) and the ubiquitin-proteasome system (UPS) was thought to be an important pathogenic mechanism in synuclein pathology and Parkinson's disease (PD). In the present study, we investigated the role of sestrin2 in autophagic degradation of α-synuclein and preservation of cell viability in a rotenone-induced cellular model of PD. We speculated that AMP-activated protein kinase (AMPK) was involved in regulation of autophagy and protection of dopaminergic cells against rotenone toxicity by sestrin2. The results showed that both the mRNA and protein levels of sestrin2 were increased in a TP53-dependent manner in Mes 23.5 cells after treatment with rotenone. Genetic knockdown of sestrin2 compromised the autophagy induction in response to rotenone, while overexpression of sestrin2 increased the basal autophagy activity. Sestrin2 presumably enhanced autophagy in an AMPK-dependent fashion, as sestrin2 overexpression activated AMPK, and genetic knockdown of AMPK abrogated autophagy induction by rotenone. Restoration of AMPK activity by metformin after sestrin2 knockdown recovered the autophagy activity. Sestrin2 overexpression ameliorated α-synuclein accumulation, inhibited caspase 3 activation, and reduced the cytotoxicity of rotenone. These results suggest that sestrin2 upregulation attempts to maintain autophagy activity and suppress rotenone cytotoxicity through activation of AMPK, and that sestrin2 exerts a protective effect on dopaminergic cells.

Rotenone isolated from Pachyrhizus erosus displays cytotoxicity and genotoxicity in K562 cells

Pachyrhizus erosus (Fabaceae) is a herb commonly known as ‘yam bean’, which has been cultivated in México since pre-Columbian times for its edible tubers. The seeds are also known for their acaricidal and insecticidal properties due to rotenone and other isoflavonoid contents. Rotenone has exhibited cytotoxic activity against several human tumour cell lines; however, its mechanism of action is still not fully understood. In this study, we determined the cytotoxicity of rotenone isolated from P. erosus seeds on K562 human leukaemia cells. Rotenone exhibited significant cytotoxic activity (IC50 = 13.05 μM), as determined by the MTT assay. Three other isolated isoflavonoids were not cytotoxic. Rotenone genotoxicity was detected using the comet assay. Rotenone induced cell death, and caspase-3 activation as indicated by TUNEL assay, and immunocytofluorescence. Plasmid nicking assay indicated that rotenone does not interact directly with DNA.

Salubrinal protects against rotenone-induced SH-SY5Y cell death via ATF4-parkin pathway

Parkinson's disease (PD) is a progressive neurodegenerative disorder, for which there are no effective disease-modifying therapies. Growing evidence from studies in human PD brain, in addition to genetic and toxicological models, indicates that endoplasmic reticulum (ER) stress is a common feature of the disease and contributes to neurodegeneration. We examine whether salubrinal, a ER stress inhibitor, can protect the rotenone-induced SH-SY5Y cell death and explore the mechanisms underlying this protection. Our results demonstrated that rotenone induced a significant ER stress response and caused cell apoptosis, which was inhibited by salubrinal. Activating transcription factor 4 (ATF4), a member of the ATF/CREB family of basic leucine zipper transcription factors, has been implicated in the pathogenesis of neurodegeneration. We showed that salubrinal increased the up-regulation of ATF4 expression. An ATF4 siRNA significantly increased the rotenone cytotoxicity and decreased the salubrinal's protection. Further, we showed that ATF4 siRNA inhibited the expression of parkin, and parkin knockdown similarly aggravated the rotenone cytotoxicity and reduced the salubrinal's protection. Additionally, the protein level of parkin was declined after treatment with rotenone, whereas this reduction was rescued by salubrinal. These findings indicate ATF4-parkin pathway plays an important role in the salubrinal-mediated neuroprotection of rotenone-induced dopaminergic cell death.

Radiosensitization by Inhibiting Complex I activity in Human Hepatoma HepG2 cells to X-ray Radiation

The purpose of this study is to investigate the influence of mitochondrial respiratory chain complex I inhibition on the radiosensitivity of HepG2 cells. The complex I inhibitor rotenone was used to inhibit complex I activity on HepG2 cells before X-ray irradiation. The cytotoxicity of rotenone was analyzed by MTT assay at various doses. Rotenone induced dissipation of mitochondrial membrane potential and increase of intracellular ROS production were observed. Intracellular ATP production level was determined using luciferin-luciferase assay kit. We further analyzed cell survival and cell cycle distribution of a combined treatment which HepG2 cells underwent 0.5 µM rotenone pretreatment firstly and irradiated with different doses of X-ray radiation afterwards. Our results suggest rotenone pretreatment prior to X-ray irradiation could induce a sensitizing effect on HepG2 cells by enhancing X-ray radiation induced proliferation inhibition and cell apoptosis.

GSK3β and endoplasmic reticulum stress mediate rotenone-induced death of SK-N-MC neuroblastoma cells

Rotenone, an environmental toxin that inhibits mitochondrial complex I, has been used to induce experimental Parkinsonism in animals and cell cultures. We investigated the mechanism underlying rotenone-induced death of SK-N-MC neuroblastoma cells. Rotenone-induced cell death preceded intracellular accumulation of reactive oxygen species, and antioxidants failed to protect cells, indicating that oxidative stress was minimally involved in rotenone-induced death of SK-N-MC cells. Glycogen synthase kinase 3β (GSK3β), a multifunctional serine/threonine kinase, has been implicated in the pathogenesis of neurodegeneration. We showed that rotenone activated GSK3β by enhancing its phosphorylation at tyrosine 216 while inhibiting phosphorylation at serine 9. Inhibitors of GSK3β and dominant negative (kinase deficient) GSK3β partially protected SK-N-MC cells against rotenone cytotoxicity. Rotenone also induced endoplasmic reticulum (ER) stress which was evident by an increase in phosphorylation of PERK, PKR, and eIF2α as well as the expression of GRP78. Rotenone had a modest effect on the expression of CHOP. An eIF2α siRNA significantly reduced rotenone cytotoxicity. ER stress was experimentally induced by tunicamycin and thapsigargin, but tunicamycin/thapsigargin did not activate GSK3β in SK-N-MC cells. Down-regulation of eIF2α also offered partial protection against rotenone cytotoxicity. Combined treatment of GSK3β inhibitors and eIF2α siRNA provided much greater protection than either treatment alone. Taken together, the results suggest that GSK3β activation and ER stress contribute separately to rotenone cytotoxicity.

Epigallocatechin gallate (EGCG) potentiates the cytotoxicity of rotenone in neuroblastoma SH-SY5Y cells

Exposure to rotenone, a widely used pesticide, has been suggested to increase the risk of developing Parkinson's disease. Studies indicate that the neurotoxicity of rotenone may be related to its ability to generate reactive oxygen species. The present work was conducted to determine to what extent (−)-epigallocatechin-3-gallate (EGCG), a widely used dietary supplement, modulates the cytotoxicity of rotenone in human neuroblastoma SH-SY5Y cells. Our results indicate that EGCG shows concentration-dependent effects on ROS production and cytotoxicity in SH-SY5Y cells. Treatment of these dopaminergic cells with rotenone (1–50 μM) alone or EGCG (25 or 50 μM) alone caused a significant decrease in cell viability. Pretreatment of SH-SY5Y cells with 25 or 50 μM EGCG potentiated the cytotoxicity of rotenone. The exacerbating effect of EGCG on rotenone toxicity may involve an apoptotic mechanism as shown by the enhancement of caspase-3 activity and activation of other caspases in rotenone-treated SH-SY5Y cells. The potentiating effect of EGCG on rotenone toxicity may be attributed to the enhanced production of intracellular superoxide in SH-SY5Y cells. The enhanced intracellular production of ROS by rotenone-EGCG combination may also account for the increased formation of protein carbonyls in 10,000× g fraction of SH-SY5Y cells detected by anti-HNE antibody. For instance, core histones and nuclear ribonuclear proteins were identified as major putative in vivo targets of HNE. Our present findings indicate that more detailed mechanistic studies are necessary to fully understand the chemistry of EGCG and to justify its use as potentially health-promoting dietary supplement, e.g. in the prevention of neurodegenerative diseases associated with oxidative stress.

Lamotrigine inhibition of rotenone- or 1-methyl-4-phenylpyridinium-induced mitochondrial damage and cell death

Defects in mitochondrial function have been shown to participate in the induction of neuronal cell injury. The aim of the present study was to assess the effect of antiepileptic lamotrigine against the cytotoxicity of mitochondrial respiratory complex I inhibitors rotenone and 1-methyl-4-phenylpyridinium (MPP +) in relation to the mitochondria-mediated cell death process and oxidative stress. Both rotenone and MPP + induced the nuclear damage, the changes in the mitochondrial membrane permeability, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species and the depletion of GSH in differentiated PC12 cells. Lamotrigine significantly attenuated the rotenone- or MPP +-induced mitochondrial damage leading to caspase-3 activation, increased oxidative stress and cell death. The preventive effect of lamotrigine against the toxicity of rotenone was greater than its effect on that of MPP +. The results show that lamotrigine seems to reduce the cytotoxicity of rotenone and MPP + by suppressing the mitochondrial permeability transition formation, leading to cytochrome c release and subsequent activation of caspase-3. The preventive effect may be ascribed to its inhibitory action on the formation of reactive oxygen species and depletion of GSH. Lamotrigine seems to exert a protective effect against the neuronal cell injury due to the mitochondrial respiratory complex I inhibition.

Effect of fraxetin and myricetin on rotenone-induced cytotoxicity in SH-SY5Y cells: comparison with N-acetylcysteine

The purpose of this study was to investigate the potential neuroprotective effects of myricetin (flavonoid) and fraxetin (coumarin) on rotenone-induced apoptosis in SH-SY5Y cells, and the possible signal pathway involved in a neuronal cell model of Parkinson's disease. These two compounds were compared to N-acetylcysteine. The viability of cells was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and cytotoxicity was assayed by lactate dehydrogenase (LDH) released into the culture medium. Parameters related to apoptosis, such as caspase-3 activity, the cleavage of poly(ADP-ribose) polymerase and the levels of reactive oxygen species were also determined. Rotenone caused a time- and dose-dependent decrease in cell viability and the degree of LDH release was proportionally to the effects on cell viability. Cells were pretreated with fraxetin, myricetin and N-acetylcysteine at different concentrations for 30 min before exposure to rotenone. Cytotoxicity of rotenone (5 μM) for 16 h was significantly diminished as well as the release of LDH into the medium, by the effect of fraxetin, myricetin and N-acetylcysteine, with fraxetin (100 μM) and N-acetylcysteine (100 μM) being more effective than myricetin (50 μM). Rotenone-induced apoptosis in SH-SY5Y cells was detected by an increase in caspase-3 activity and in the cleavage of poly(ADP-ribose) polymerase. After exposing these cells to rotenone, a significant increase in reactive oxygen species preceded apoptotic events. Fraxetin (100 μM) and N-acetylcysteine (100 μM) not only reduced rotenone-induced reactive oxygen species formation, but also attenuated caspase-3 activity and poly(ADP-ribose) polymerase cleavage at 16 h against rotenone-induced apoptosis. The effect of fraxetin in both experiments was similar to that of N-acetylcysteine. These results demonstrated the protective action of fraxetin and suggest that it can reduce apoptosis, possibly by decreasing free radical generation in SH-SY5Y cells. Myricetin at 100 μM was without any preventive effect.