Doses Of Rotenone Research Articles

Acute intranigral infusion of rotenone in rats causes progressive biochemical lesions in the striatum similar to Parkinson's disease

We examined in Sprague–Dawley rats whether intranigral administration of complex-I inhibitor, rotenone, produces biochemical lesions in the striatum similar to those observed in Parkinson's disease (PD). Unilateral stereotaxic infusion of rotenone (2–12 μg in 1 μl) into substantia nigra (SN) pars compacta caused significant inhibition of complex-I activity and increased production of hydroxyl radicals in vivo as measured employing spectrophotometric and HPLC-electrochemical procedures, respectively. It also caused a significant time- and dose-dependent reduction of dopamine level, but not serotonin, in the ipsilateral striatum when assayed using an HPLC electrochemical method. This effect was found to be progressive for 90 days. A dose-dependent decrease in nigral glutathione level, as measured fluorimetrically, was also observed to be progressive till 90th day. A significant decrease in tyrosine hydroxylase immunoreactivity in the striatum (73 ± 8.4% as assessed by densitometric studies) or in SN ipsilateral to the side of infusion suggested nigrostriatal neuronal degeneration. A dose of rotenone (6 μg in 1 μl) that caused 55% striatal dopamine depletion when infused into the SN failed to affect serotonin levels in the terminal regions when infused into the nucleus raphe dorsalis, indicating rotenone's specificity of action towards dopaminergic neurons. Our findings suggest that unilateral infusion of rotenone reproduces neurochemical and neuropathological features of hemiparkinsonism in rats and indicate an active involvement of oxidative stress in rotenone-induced nigrostriatal neurodegeneration. The present study also demonstrates more sensitivity of dopaminergic neurons towards rotenone and establishes mitochondrial complex-I damage as one of the major contributory components of neurodegeneration in PD. The progressive nature of pathology in this model closely mimics idiopathic PD, and absence of mortality warrants the use of this model in drug discovery programs.

Rotenone‐induced caspase 9/3‐independent and ‐dependent cell death in undifferentiated and differentiated human neural stem cells

We used human neural stem cells (hNSCs) and their differentiated cultures as a model system to evaluate the mechanism(s) involved in rotenone (RO)- and camptothecin (CA)-induced cytotoxicity. Results from ultrastructural damage and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining indicated that RO-induced cytotoxicity resembled CA-induced apoptosis more than H(2)O(2)-induced necrosis. However, unlike CA-induced, caspase 9/3-dependent apoptosis, there was no increased activity in caspase 9, caspase 3 or poly (ADP-ribose) polymerase (PARP) cleavage in RO-induced cytotoxicity, in spite of time-dependent release of cytochrome c and apoptosis-inducing factor (AIF) following mitochondrial membrane depolarization and a significant increase in reactive oxygen species generation. Equal doses of RO and CA used in hNSCs induced caspase 9/3-dependent apoptosis in differentiated cultures. Time-dependent ATP depletion occurred earlier and to a greater extent in RO-treated hNSCs than in CA-treated hNSCs, or differentiated cultures treated with RO or CA. In conclusion, these results represent a unique ultrastructural and molecular characterization of RO- and CA-induced cytotoxicity in hNSCs and their differentiated cultures. Intracellular ATP levels may play an important role in determining whether neural progenitors or their differentiated cells follow a caspase 9/3-dependent or -independent pathway in response to acute insults from neuronal toxicants.

Rotenone induces oxidative stress and dopaminergic neuron damage in organotypic substantia nigra cultures

Rotenone, a pesticide and complex I inhibitor, causes nigrostriatal degeneration similar to Parkinson disease pathology in a chronic, systemic, in vivo rodent model [M. Alam, W.J. Schmidt, Rotenone destroys dopaminergic neurons and induces parkinsonian symptoms in rats, Behav. Brain Res. 136 (2002) 317–324; R. Betarbet, T.B. Sherer, G. MacKenzie, M. Garcia-Osuna, A.V. Panov, J.T. Greenamyre, Chronic systemic pesticide exposure reproduces features of Parkinson's disease, Nat. Neurosci. 3 (2000) 1301–1306; S.M. Fleming, C. Zhu, P.O. Fernagut, A. Mehta, C.D. DiCarlo, R.L. Seaman, M.F. Chesselet, Behavioral and immunohistochemical effects of chronic intravenous and subcutaneous infusions of varying doses of rotenone, Exp. Neurol. 187 (2004) 418–429; T.B. Sherer, J.H. Kim, R. Betarbet, J.T. Greenamyre, Subcutaneous rotenone exposure causes highly selective dopaminergic degeneration and alpha-synuclein aggregation, Exp. Neurol. 179 (2003) 9–16.]. To better investigate the role of mitochondria and complex I inhibition in chronic, progressive neurodegenerative disease, we developed methods for long-term culture of rodent postnatal midbrain organotypic slices. Chronic complex I inhibition over weeks by low dose (10–50 nM) rotenone in this system lead to dose- and time-dependent destruction of substantia nigra pars compacta neuron processes, morphologic changes, some neuronal loss, and decreased tyrosine hydroxylase (TH) protein levels. Chronic complex I inhibition also caused oxidative damage to proteins, measured by protein carbonyl levels. This oxidative damage was blocked by the antioxidant α-tocopherol (vitamin E). At the same time, α-tocopherol also blocked rotenone-induced reductions in TH protein and TH immunohistochemical changes. Thus, oxidative damage is a primary mechanism of mitochondrial toxicity in intact dopaminergic neurons. The organotypic culture system allows close study of this and other interacting mechanisms over a prolonged time period in mature dopaminergic neurons with intact processes, surrounding glia, and synaptic connections.

Chronic Exposure to Rotenone Models Sporadic Parkinson's Disease inDrosophila melanogaster

Parkinson's disease (PD) is a movement disorder characterized by the selective degeneration of nigrostriatal dopaminergic neurons. Both familial and sporadic cases present tremor, rigidity, slowness of movement, and postural instability. Although major insights into the genes responsible for some rare hereditary cases have arisen, the etiology of sporadic cases remains unknown. Epidemiological studies have suggested an association with environmental toxins, mainly mitochondrial complex I inhibitors such as the widely used pesticide rotenone. In recent years, Drosophila melanogaster has been used as a model of several neurodegenerative diseases, including a genetic model of PD. Here, we studied the neurodegenerative and behavioral effects of a sublethal chronic exposure to rotenone in Drosophila. After several days, the treated flies presented characteristic locomotor impairments that increased with the dose of rotenone. Immunocytochemistry analysis demonstrated a dramatic and selective loss of dopaminergic neurons in all of the brain clusters. The addition of l-dopa (3,4-dihydroxy-L-phenylalanine) into the feeding medium rescued the behavioral deficits but not neuronal death, as is the case in human PD patients. In contrast, the antioxidant melatonin (N-acetyl-5-methoxytryptamine) alleviated both symptomatic impairment and neuronal loss, supporting the idea that this agent may be beneficial in the treatment of PD. Therefore, chronic exposure to pesticides recapitulates key aspects of PD in Drosophila and provides a new in vivo model for studying the mechanisms of dopaminergic neurodegeneration.

Behavioral and immunohistochemical effects of chronic intravenous and subcutaneous infusions of varying doses of rotenone

Mitochondrial toxins such as the complex 1 inhibitor rotenone are widely used as pesticides and may be present in military environments. Administration of rotenone can induce biochemical and histological alterations similar to those of Parkinson's disease in rats. However, only a subset of animals show these effects and it is unclear whether more subtle alterations are caused by chronic administration of rotenone in those animals that appear resistant to its toxic effects on dopaminergic nerve terminals. To address this question, vehicle or rotenone (2.0, 2.5, or 3.5 mg/kg/day) was administered intravenously or subcutaneously for 21 days to adult rats, and rotenone effects on survival, motor behavior, and striatal tyrosine hydroxylase immunoreactivity (TH-IR) were examined. Both intravenous and subcutaneous rotenone induced a dose-dependent decrease in survival rates. Surviving animals showed a decrease in spontaneous rearing. Locomotor activity and movement initiation time were also altered in some of the experimental groups. Confirming previous results, TH-IR in the striatum was markedly decreased in rats that fell ill early in the study and in a few of the surviving rats with high rotenone doses. However, none of the surviving rats receiving 2.0 mg/kg/day showed TH-IR loss reminiscent of Parkinson's disease, and loss of striatal TH-IR across doses was not correlated with motor behavior in individual rats. Thus, chronic administration of low doses of rotenone induces motor anomalies even in animals that do not develop histological signs of Parkinson's disease, indicating a pervasive neurological effect of moderate mitochondrial dysfunction in vivo.

1-Methyl-1,2,3,4-tetrahydroisoquinoline protects against rotenone-induced mortality and biochemical changes in rat brain

The effect of single and multiple administration of the neurotoxic pesticide, rotenone, and the potentially neuroprotective compound, 1-methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ), on the concentration of dopamine and its metabolites (homovanillic acid-HVA, 3,4-dihydroxyphenylacetic acid-DOPAC, and 3-methoxytyramine-3-MT)) in three brain areas was studied by high-performance liquid chromatography (HPLC) with electrochemical detection in Wistar rats. The rate of dopamine catabolism in the striatum along the N-oxidative and O-methylation pathways was assessed by calculation of the ratio of dopamine metabolites to dopamine. In addition, the effect of rotenone on mortality and general behavior of rats was investigated. We have found that the neurotoxic pesticide, rotenone, administered in a single dose (12 mg/kg s.c.) did not produce evident behavioral or biochemical effects. In contrast, repeated administration of rotenone in doses (12–15 mg/kg) causing abnormalities in general behavior, produced considerable mortality and dramatic increases in dopamine metabolism, which may be ascribed to an increase in the oxidative pathway. Interestingly, it depressed the concentration of the extracellular dopamine metabolite, 3-MT. These behavioral and biochemical changes were effectively counteracted by administration of 1MeTIQ before each dose of rotenone. In summary, we demonstrated that multiple systemic rotenone injections are strongly toxic, and induce alterations of cerebral dopamine metabolism, and that 1MeTIQ may be considered as a potential protective agent against environmental factors affecting the function of the dopaminergic system.

Subcutaneous Rotenone Exposure Causes Highly Selective Dopaminergic Degeneration and α-Synuclein Aggregation

Previous studies demonstrated that chronic systemic exposure to the pesticide and mitochondrial toxin rotenone through jugular vein cannulation reproduced many features of Parkinson's disease (PD) in rats, including nigrostriatal dopaminergic degeneration and formation of α-synuclein-positive cytoplasmic inclusions in nigral neurons (R. Betarbet et al., 2000, Nat. Neurosci. 3, 1301–1306). Although novel and conceptually important, the rotenone model of PD suffered from being extremely labor-intensive. The current paper demonstrates that these same features of PD can be reproduced by chronic, systemic exposure to rotenone following implantation of subcutaneous osmotic pumps. Chronic subcutaneous exposure to low doses of rotenone (2.0–3.0 mg/kg/day) caused highly selective nigrostriatal dopaminergic lesions. Striatal neurons containing DARPP-32 (dopamine and cAMP-regulated phosphoprotein) remained intact with normal morphology, and NeuN staining revealed normal neuronal nuclear morphology. Neurons of the globus pallidus and subthalamic nucleus were spared. Subcutaneous rotenone exposure caused α-synuclein-positive cytoplasmic aggregates in nigral neurons. This new protocol for chronic rotenone administration is a substantial improvement in terms of simplicity and throughput.

Increased striatal dopamine turnover following acute administration of rotenone to mice

Because of the potential role of mitochondrial dysfunction in nigrostriatal degeneration in Parkinson’s disease, the effects of rotenone (an inhibitor of mitochondrial NADH dehydrogenase and a naturally occurring toxicant) on the levels of striatal dopamine (DA) and DA metabolites were evaluated after acute and subchronic administration to mice. Systemic acute treatment with relatively high doses of rotenone did not affect DA concentration, but caused a significant increase in both DA metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). DOPAC and HVA changes were measured at 1 day and were reversed within 1 week, paralleling the time course of rotenone-induced increase in striatal lactate levels. Subchronic administration with a relatively mild dose of rotenone did not significantly alter the striatal levels of DA and DOPAC, while it slightly reduced HVA concentration. No neurochemical signs of dopaminergic damage were seen when mice were co-exposed to rotenone and diethyldithiocarbamate, a compound known to enhance nigrostriatal injury caused by the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Also, rotenone did not cause additional injury to animals previously lesioned by MPTP. Taken together, data indicate that rotenone is not capable of causing overt dopaminergic toxicity under the testing paradigms used in this study. Rather, an increase in DA turnover, as indicated by a higher (DOPAC+HVA)/DA ratio, seems to be associated to rotenone-induced striatal energy impairment.

Oral Toxicity of Rotenone for Common Carp

Abstract Rotenone was administered in a food bolus by gavage to common carp Cyprinus carpio. The experimental formulations also contained a constant level of a surfactant, polysorbate 80. The lethal dose of rotenone to 50% of the test animals (LD50) was 8.1 mg/kg of fish with a 95% confidence interval of 7.7–8.5 mg/kg. The extrapolated lethal dose to 99% of the test animals was 11.6 mg/kg. Behavior of the fish receiving an oral dose of rotenone was similar to that of fish exposed to rotenone in water, and all deaths occurred within 16 h of gavage. Rotenone was made available for absorption via the intestine by including a surfactant in the formulation.

Inhibition of ovulation in PMSG/hCG-treated immature rats by rotenone, a specific inhibitor of mitochondrial oxidation.

Immature Wistar rats were induced to ovulate by treatment with PMSG and hCG. Control animals ovulated 43.5 +/- 0.36 ova/rat. Intraperitoneal injection of rotenone doses of 0.125, 0.25 and 0.50 mg/kg reduced the ovulation rate to 24.0 +/- 3.08, 8.0 +/- 0.88 and 1.5 +/- 0.44 ova/rat, respectively. The rotenone significantly reduced ovarian cytochrome oxidase activity and progesterone production, but not production of oestradiol or testosterone. Thyroxine treatment at a dose of 5 mg/kg s.c. reversed the rotenone inhibition of ovulation. The results suggest that an increase in mitochondrial respiration is an essential feature of the ovulation process in mammals.

Effect of injected rotenone on the production and composition of urine from the rainbow trout (Salmo Gairdneri)

Renal function was evaluated in adult rainbow trout ( Salmo gairdneri) dosed i.a. with rotenone at 225 and 275 μg/kg. The chemical composition of urine samples and urine flow rates collected over a 5-h pretreatment period were compared with hourly urine samples collected over a 5-h posttreatment period. Significant increases in osmolality and in concentrations of sodium, potassium, chloride, glucose, and total protein were observed in the urine of treated fish. Urine solute concentrations reached maximum values within 1 to 3 h after treatment and decreased thereafter, indicating that the effects were reversible. Concentrations of sodium and chloride were highly correlated in 2-h posttreatment urine samples at the low ( r = 0.922) and high ( r = 0.981) rotenone treatments. Urine flow rates were reduced in trout at each dose of rotenone but the decrease in volume of urine voided was not dose-dependent. In a separate study, [ 14C]polyethylene glycol was used as a filtration marker to determine the effect of rotenone treatment (225 μg/kg) on urine flow rate, glomerular filtration rate, and renal water reabsorption. We showed that posttreatment urine flow rates were reduced partly by reduced glomerular filtration and partly by increased water reabsorption. Transient increases in plasma osmolality and hematocrit also were observed 0.5 h after rotenone treatment.

High Pressure Liquid Chromatographic Determination of Rotenone and Degradation Products in Animal Chow and Tissues

An analytical procedure is described for determining residues of rotenone, rotenolone, dehydrotenone, and rotenone in admixture in animal chow and tissues. The methanol or ethyl ether extracts from samples of chow and tissues, respectively, are subjected to a liquid-liquid partitioning cleanup with hexane-acetonitrile, further cleanup on a column of silica gel, and subsequent analysis by high pressure liquid chromatography using an ultraviolet absorption detector set at 295 nm. Animal chow, mouse fetuses, and gastrointestinal tracts spiked with 0.5 ppm of each compound in admixture yielded average recoveries of 92, 51, and 79%, respectively; minimum quantities of the 4 compounds detectable in the 3 substrates averaged 0.12, 0.04, ajd 0.14 ppm, respectively. Stability studies indicate that rotenone reacts with animal chow with a half-life of 7--8 days and is photodegraded in incandescent light with a half-life of 0.65 day. No transplacental transfer of rotenone or its products was observed in fetuses from mice receiving 7 consecutive daily doses of rotenone at levels up to 25 mg/kg.

Role of ATP on the initial rate of amino acid uptake in ehrlich ascites cells

Incubation with graded doses of rotenone can bring about a graded lowering of the cellular ATP level. Using cells with varying ATP levels, it can be shown that the initial uptake of [ 14C]glycine, before the cellular concentration exceeds that of the medium, is decreased as ATP decreases. Alterations in cellular cations cannot account for the difference in glycine influx. Prolonged exposure of cells to a lowered ATP content increases the exodus of cellular glycine. Valinomycin reduces the steady-state level of glycine uptake, but its effects can to a major extent be overcome by the addition of glucose. At high extracellular K + (70 mM) neither the sum of the Na ++K + gradients, nor the electrochemical potential of Na + provides sufficient energy to account for glycine and 2-aminoisobutyrate accumulation if a 1:1 coupling between Na + and the amino acid occurs.

Inhibition of the mitochondrial respiratory chain in isolated atria—A comparision of rotenone and amytal

The functional and metabolic effects of amytal and rotenone, two inhibitors of the oxidation of reduced nicotinamide dinucleotide (NADH) by the mitochondrial respiratory chain, have been studied in the isolated atrium of the guinea pig. Rotenone in concentrations of 1–8 μM produces a negative inotropic and chronotropic response which develops gradually over 60 min. At the highest dose, contractile tension is depressed about 50 per cent while the frequency is decreased by about 60 per cent after 60 min of exposure to rotenone. A further, 20–30 per cent of the total atrial ubiquinone (UQ) was found in the oxidized form in the rotenone-treated atria at all dose levels, reflecting inhibition of NADH oxidation. The amounts of lactate were elevated to 200 per cent of control levels in atria exposed to 1 and 4 μM rotenone. This is a further indication of the blockade of NADH oxidation. Energy balance was adversely affected as shown by 20 and 30 per cent decreases in the creatine phosphate level and in the ratio of ATP to ADP. In contrast, amytal produced no demonstrable effects on UQ redox status, lactate and pyruvate levels, or energy balance after 60 min at a concentration of 0.1 mM, but did depress contractility by about 20 per cent. The depression developed more rapidly with amytal than with rotenone. Frequency was not affected. At 2.5 mM amytal, contractility was completely abolished within 10 min and the UQ redox status was shifted by about 23 per cent to the oxidized side, a shift comparable to that seen with all doses of rotenone. The ratio of lactate to pyruvate was increased 350 per cent above control levels. Energy balance was severely affected even in the absence of any contractile activity, as indicated by a 70 per cent decrease in creatine phosphate levels and a 54 per cent decrease in the ATP/ADP quotient. It is concluded that amytal, at concentrations below 0.5 mM, depresses atrial contractility independent of any effect on the mitochondrial respiratory chain. At concentrations between 0.5 and 2.5 mM, the blockade of NADH oxidation in the atrial mitochondria can be demonstrated and may play a role in the functional depression. At the highest dose, additional inhibition of mitochondrial energy transfer reactions contributes to the negative energy balance over and above what might be expected on the basis of inhibition of electron transport alone.