Mitochondrial electron transport chain complex I

The way that Rotenone affects ENS signaling involves the intimate involvement of mitogen-activated polypeptide (MAPK), Toll-like receptors, Wnt, and Ras [2].

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Inhibition of mitochondrial respiratory chain complex I by Rotenone had been found to induce cell death in a variety of cells. However, the mechanism is still elusive. Because reactive oxygen species (ROS) play an important role in apoptosis and inhibition of mitochondrial respiratory chain complex I by Rotenone was thought to be able to elevate mitochondrial ROS production, we investigated the relationship between rotenone-induced apoptosis and mitochondrial reactive oxygen species. Rotenone was able to induce mitochondrial complex I substrate-supported mitochondrial ROS production both in isolated mitochondria from HL-60 cells as well as in cultured cells. Rotenone-induced apoptosis was confirmed by DNA fragmentation, cytochrome c release, and caspase 3 activity. A quantitative correlation between rotenone-induced apoptosis and rotenone-induced mitochondrial ROS production was identified. Rotenone-induced apoptosis was inhibited by treatment with antioxidants (glutathione, N-acetylcysteine, and vitamin C). The role of rotenone-induced mitochondrial ROS in apoptosis was also confirmed by the finding that HT1080 cells overexpressing magnesium superoxide dismutase were more resistant to rotenone-induced apoptosis than control cells. These results suggest that rotenone is able to induce apoptosis via enhancing the amount of mitochondrial reactive oxygen species production.[5]

Rotenone can be utilized in animal modeling to develop Parkinson's regulation models. Rotenone induces a considerable rise in excitatory neurotransmitters; significant decreases in glutamate and aspartate, as well as excitatory, GABA, glycine, and taurine, are detected in the cerebellum of PD animals [1]. Rotenone (1.5, 2, or 2.5 mg/kg) resulted in increased alpha-synuclein dose needs in the substantia nigra. Furthermore, at dosages of 2 and 2.5 mg/kg, rotenone induced significant decreases in the number of tyrosine kinase-immunoreactive neurons in the substantia nigra and dopamine in the striatum [4].

Respiration Measurement[5]
Oxygen consumption was measured with a Clark oxygen electrode as described before. Briefly, 1 × 107 HL-60 cells were treated with various concentrations of Rotenone for 30 min. Cells were then collected and resuspended in a medium containing 0.3 m mannitol, 10 mmpotassium HEPES (pH 7.4), 5 mm potassium phosphate (pH 7.4), and 1 mm MgCl2. The cells were injected into a respiration chamber that was then sealed. The total volume of the respiration chamber was 1.6 ml. Respiration was then measured and calculated as the rate of change in the oxygen concentration, assuming the initial oxygen concentration to be 6.8 mg/liter. Cell respiration was converted to percentage of control.

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ATP Determination[5]
For ATP measurement, a commercially available luciferin-luciferase assay kit was used. Briefly, HL-60 cells were treated with various concentrations of Rotenone for 24 h and then collected in 1-ml Eppendorf tubes. After a single wash with ice-cold PBS, cells were lysed with the somatic cell ATP-releasing reagent provided by the kit. Luciferin substrate and luciferase enzyme were added and bioluminescence was assessed on a Perkin Elmer 3B spectroflurometer. Whole-cell ATP content was determined by running an internal standard. The cellular ATP level was converted to percentage of untreated cells (control).

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Measurement of Cellular Superoxide Production by Flow Cytometry[5]
Measurement of cellular superoxide production was performed as described previously, with some modifications. For HL-60 cells, cells were treated with various concentrations of Rotenone for 30 min. Cells were then collected and washed in Hank's balanced salt solution (HBSS) at 250 × g. Cells were resuspended in HBSS containing 10 μm hydroethidine (HE) and incubated at 37 °C for 10 min. For HT1080 fibrosarcoma cells, cells were treated with Rotenone for 30 min, the medium was then removed, and cells were washed once with HBSS. Cells were then incubated HBSS containing 0.25% trypsin for 5 min. Cells were then resuspended in HBSS containing 10 μm hydroethidine (HE), and incubated at 37 °C for 10 min. All cell suspensions were placed into 12 × 75-mm tubes for assay. Flow cytometry studies were carried out on a Beckman-Coulter XL flow cytometer. Ethidium fluorescence was collected using a 610-nm long-pass filter.

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DNA Fragmentation[5]
The DNA fragmentation assay was performed according to the method described previously with some modifications. HL-60 cells were treated with various concentrations of Rotenone in the presence or absence of antioxidants. Cells (2 × 107) were washed once with PBS (4 °C, pH 7.4) and collected by centrifugation at 250 × g for 5 min. The pellet was then treated with 0.5 ml of lysis buffer (10 mmTris-HCL, pH 7.4, 10 mm EDTA, 0.5% sodium dodecyl sulfate) for 10 min on ice. After treatment with RNase A (final concentration, 100 μg/ml) for 1 h at 37 °C, the cells were incubated at 50 °C for 4 h in the presence of 100 μg/ml proteinase K. DNA was precipitated by addition of 50 μl of 3 m sodium acetate (pH 5.2) and 1 ml of cold (4 °C) 100% ethanol to the solution. DNA was then collected and dissolved in TE buffer (10 mm Tris pH 8.0, EDTA 1 mm). For analysis, 10–20 μl of DNA was loaded on a 1.2% agarose gel containing 10 μg/ml ethidium bromide. Electrophoresis was performed in 0.5 × Tris borate-EDTA buffer (18 mm Tris-base (pH 8.0), 18 mm boric acid, and 1 mm EDTA) at 70 V for 2 h. DNA was visualized under ultraviolet light and photographed.

Cell Counting Kit-8[2]
The effect of rotenone on the viability of ENS cells was determined using a Cell Counting Kit-8 (CCK8) assay. A total of 2000 cells were plated into each well of a 96-well plate, and cultured for 24 h. The culture medium was then removed, and the ENS cells were exposed to rotenone at concentrations of 0.01, 0.02, 0.07, 0.21, 0.62, 1.85, 5.56, 16.67, and 50 μM for 48 h. Cells without any rotenone treatment were used as controls. Next, 10 µL of CCK-8 was added to each well and incubated for 3 h, and a microplate reader was used to detect the absorbance at 450 nm.

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Total RNA Preparation[2]
Total RNA was prepared according to the TRIZOL method from cells treated with/without 0.3 μM rotenone for 48 h. And DNA contaminated in total RNA was eliminated using DNaseI. RNA concentration was detected using NanoDrop 1000 and 1 μg of RNA from each sample was used to analyze the RNA integrity by 1% agarose gel electrophoresis, the remaining RNA was stored at −80 °C for RNA-seq and real time-polymerase chain reaction (RT-PCR).

The aim of the present work was to investigate the neurochemical changes induced in the cerebellum of rat model of Parkinson's disease (PD). Rats were divided into two groups; control and rat model of PD induced by the intrastriatal injection of Rotenone. As compared to control, a significant increase in the excitatory amino acid neurotransmitters; glutamate and aspartate together with a significant decrease in the inhibitory amino acids, GABA, glycine and taurine were observed in the cerebellum of rat model of PD. This was associated with a significant increase in lipid peroxidation, nitric oxide and tumor necrosis factor-α and a significant decrease in reduced glutathione. A significant decrease in acetylcholinesterase and a significant increase in Na+,K+-ATPase were recorded in the cerebellum of rat model of PD. In addition the cerebellar sections from rat model of PD showed marked necrosis of Purkinje cells, irregular damaged cells, cytoplasmic shrinkage, necrosis and perineuronal vacuolation. The present results indicate that the disturbance in the balance between the excitatory and inhibitory amino acids may have a role in the pathogenesis of PD. According to the present neurochemical and histopathological changes, the cerebellum should be taken into consideration during the treatment of PD.[1]

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Parkinson disease (PD) is a neurodegenerative disorder affecting mainly the motor system, as a result of death of dopaminergic neurons in the substantia nigra pars compacta. The present scenario of research in PD is directed to identify novel molecules that can be administered individually or co-administered with L-Dopa to prevent the L-Dopa-Induced Dyskinesia (LID) like states that arise during chronic L-Dopa administration. Hence, in this study, we investigated whether Morinda citrifolia has therapeutic effects in Rotenone-induced Parkinson's disease (PD) with special reference to mitochondrial dysfunction mediated intrinsic apoptosis. Methods: Male Sprague-Dawley rats were stereotaxically infused with Rotenone(3 µg in both SNPc and VTA) and co-treated with the ethyl acetate extract of Morinda citrifolia and levodopa.[3]

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Subcutaneous administration of Rotenone has recently attracted attention because of its convenience, simplicity and efficacy in replicating features of Parkinson's disease (PD) in animal models. However, the wide range of doses reported in the literature makes it difficult to evaluate the effectiveness of this technique objectively. The aim of the present study was to identify the optimum dose of subcutaneous Rotenone for establishing a model of PD. We injected male Wistar rats subcutaneously with one of three doses of Rotenone (1.5, 2, or 2.5mg/kg) daily for 5 weeks. Rotenone caused a dose-dependent increase in α-synuclein in the substantia nigra. Furthermore, at 2 and 2.5mg/kg, Rotenone caused a significant decrease in the number of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra, and dopamine in the striatum. However, mortality at 2.5mg/kg was 46.7%, compared with just 6.7% at 2mg/kg; the high mortality observed at 2.5mg/kg would limit its application. The 2mg/kg dose showed no detrimental effect on body weight after 5 weeks of daily injections. Furthermore, rats in the 2mg/kg group showed a longer latency to descend from a horizontal bar and a grid wall, decreased rearing, and shorter latency to fall from a rotarod than rats that received vehicle or saline. Mitochondrial damage, observed by transmission electron microscopy, was also evident at this dose. Together, our data indicate that daily subcutaneous injection of 2mg/kg Rotenone in rats facilitates the formation of α-synuclein and reproduces the typical features of PD, while maintaining low mortality.[4]

Absorption, Distribution and Excretion
Gastrointestinal absorption is low and incomplete. In animals, rotenone is hundreds of times more toxic intravenously than orally. Fats and oils increase absorption.
Male and female Sprague Dawley rats were dosed with purified rotenone (99.23% pure). ... Preliminary excretion balance, excretion balance, pharmacokinetic, and enterohepatic circulation studies were conducted in both sexes using oral and intravenous (iv) doses of (14C)-rotenone at 0.01 to 5.0 mg/kg. Urinary and fecal metabolites were analyzed by thin layer chromatography. ... Feces were the major route of excretion for iv and oral exposures with small amounts of (14C) being recovered in the urine. Excretion was nearly complete in 48 hours in both sexes after iv doses and in males after oral doses. Female excretion was nearly complete 72 hours after oral dosing. Tissue retention of (14C) was low. The pharmacokinetics data were described by a two-compartment model. Data were consistent with enterohepatic recycling. ...
Early experiments with rabbits and dogs fed with rotenone indicated retention and/or metabolism of the toxicant. No intact material was obtained from the urine, but the feces contained rotenone for at least 8 days after administration. A somewhat different picture emerged for mice 48 hr after treatment with (14)C-labeled rotenone, 20% of the (14)C was in the urine, 0.3% was expired, 5% remained in the body, and the rest was in the feces.
When the fate and distribution of (14)C-labeled rotenone in different organs were followed in mice, 21.6% of the radioactivity was found in the small intestine, 19.5% in the urine, and 4.4% in the liver.
Exhalation of (14)CO2 within 50 hr after oral or ip dosing of 5'beta-[3-methoxy-(14)C]rotenone to mice and rats respectively was 27 and 12.5%. /5'beta-[3-methoxy-(14)C]Rotenone/

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Metabolism / Metabolites
Male and female Sprague Dawley rats were dosed with purified rotenone (99.23% pure). ... Nine metabolites were detected in urine and feces. Four of these cochromatographed with (14)C-labeled impurities in the standard. No parent compound could be detected in feces or urine. The major metabolite (S0) was very polar and in feces accounted for 40.82 to 72.99% of the excreted (14)C by males and 33.48% to 65.76% by females. A similar polar metabolite in male urine accounted for 69.67% to 93.37% of the excreted (14)C and in females 43.51 to 94.88%. Only one metabolite (S8) from one rat comigrated with a known standard (rotenolone). It represented about 0.23% of the dose (8.2% of the urinary (14)C X 2.79% of the dose that was excreted in the urine).
The principal metabolites formed both in vivo and in vitro from rotenone for these species are basically the same and are, in decreasing order of mammalian toxicity, as follows: 8'-hydroxyrotenone, 6a beta,12a beta-rotenolone, 6a beta,12a alpha-rotenolone, and 6',7'-dihydro-6',7'-dihydroxyrotenone. The hydroxylated metabolites also have reduced inhibitory activity to insect and rat liver mitochondria. The formation of water-soluble conjugates, which were more abundant in mammalian than in insect tissues, was also noted in these studies. A phenolic metabolite resulting from 3-O-demethylation was also identified.
In vitro, rotenone is subject to a variety of cytochrome P-450-catalyzed oxidations in vertebrate and insect tissues. Oxidation occurs (e.g., at both the methyl group and the double bond of the isopropenyl group, and at the carbon atom adjacent to the keto group between the B and C rings to yield a pair of enantiomers, rotenolones I and II).
Rotenone can also be oxidized completely to carbon dioxide (CO2) such that up to 13% and 27% of the compound administered to mice and rats appeared as exhaled. Some 20% of the ingested rotenone can be accounted for as urinary metabolites in rats and mice.
For more Metabolism/Metabolites (Complete) data for ROTENONE (9 total), please visit the HSDB record page.

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Biological Half-Life
... The half-life of rotenone in the head, viscera, and carcass of bluegills was about 22, 11, and 28 days, respectively, and the major metabolites identified were rotenolone and 6',7'-dihydro-6',7'-dihydroxyrotenone.

Toxicity Summary
Rotenone works by interfering with the electron transport chain in mitochondria. Specifically, it inhibits the transfer of electrons from iron-sulfur centers in complex I to ubiquinone. This prevents NADH from being converted into usable cellular energy (ATP). (L1274)

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Non-Human Toxicity Values
LD50 Rabbit oral 1500 mg/kg
LD50 Mouse oral 350 mg/kg
LD50 Rat oral 25 mg/kg /oil solution/
LD50 Rat oral 60 mg/kg
For more Non-Human Toxicity Values (Complete) data for ROTENONE (9 total), please visit the HSDB record page.

[1]. Cerebellar neurochemical and histopathological changes in rat model of Parkinson's disease induced by intrastriatal injection of rotenone. Gen Physiol Biophys. 2016 Nov 30.

[2]. RNA-Seq Expression Analysis of Enteric Neuron Cells with Rotenone Treatment and Prediction of Regulated Pathways. Neurochem Res. 2016 Nov 30.

[3]. Morinda citrifolia mitigates rotenone-induced striatal neuronal loss in male Sprague-Dawley rats by preventing mitochondrial pathway of intrinsic apoptosis. Redox Rep. 2016 Nov 24:1-12.

[4]. Subcutaneous rotenone rat model of Parkinson's disease: dose exploration study. Brain Res. 2016 Nov 19. pii: S0006-8993(16)30776-4.

[5]. Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production. J Biol Chem. 2003 Mar 7;278(10):8516-25.

Therapeutic Uses
Vet: ectoparasiticide
Rotenone is also used to treat scabies and head lice on humans as well as various ectoparasites on livestock and pet animals. /Former use/
MEDICATION (VET): In veterinary medicine, rotenone is used in the power form to control parasitic mites on chickens and other fowl, and for lice and ticks on dogs, cats, and horses.

C23H22O6

394.42

394.141

C, 70.04; H, 5.62; O, 24.34

83-79-4

83-79-4

6758

White to light yellow solid powder

1.3±0.1 g/cm3

559.8±50.0 °C at 760 mmHg

159-164 °C(lit.)

244.6±30.2 °C

0.0±1.5 mmHg at 25°C

1.591

4.65

0

6

3

29

664

3

O=C1[C@H]2C3C=C(OC)C(OC)=CC=3OC[C@H]2OC2C3C[C@H](C(=C)C)OC=3C=CC1=2

JUVIOZPCNVVQFO-HBGVWJBISA-N

InChI=1S/C23H22O6/c1-11(2)16-8-14-15(28-16)6-5-12-22(24)21-13-7-18(25-3)19(26-4)9-17(13)27-10-20(21)29-23(12)14/h5-7,9,16,20-21H,1,8,10H2,2-4H3/t16-,20-,21+/m1/s1

(1S,6R,13S)-16,17-dimethoxy-6-prop-1-en-2-yl-2,7,20-trioxapentacyclo[11.8.0.03,11.04,8.014,19]henicosa-3(11),4(8),9,14,16,18-hexaen-12-one

Ro-KO; Rotenone; Cube root; 83-79-4; Dactinol; Paraderil; Barbasco; Tubatoxin; (-)-Rotenone; Derris; Ronone; HSDB 1762; bCube-Pulver

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~50 mg/mL (~126.8 mM)
H2O: < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.34 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (6.34 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.34 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2.5 mg/mL (6.34 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)