Berberine alleviates rotenone-induced cytotoxicity by antioxidation and activation of PI3K/Akt signaling pathway in SH-SY5Y cells.

Berberine, an isoquinoline alkaloid isolated from traditional Chinese medicine, has been widely studied for its efficacy in the treatment of neurodegenerative diseases. However, berberine-mediated neuroprotection in the pathogenesis of Parkinson's disease is still uncertain. In this study, the effects of berberine on rotenone-induced neurotoxicity in SH-SY5Y cells were investigated. The results showed that berberine treatment significantly alleviated rotenone-induced decrease in the cell viability in SH-SY5Y cells. Further studies demonstrated that berberine suppressed the production of intracellular reactive oxygen species, restored the mitochondrial transmembrane potential, increased Bcl-2/Bax ratio, and decreased caspase-3 activation that induced by rotenone. Furthermore, berberine also restored the phosphorylation of Akt, which was downregulated by rotenone in SH-SY5Y cells. These results suggest that berberine protects rotenone-treated SH-SY5Y cells by antioxidation and activation of PI3K/Akt signaling pathway.

Docosahexaenoic acid protection in a rotenone induced Parkinson's model: Prevention of tubulin and synaptophysin loss, but no association with mitochondrial function.

Rotenone, a classic mitochondrial complex I inhibitor, leads to dopaminergic neuronal death resulting in a Parkinson's-like-disease. Docosahexaenoic acid (DHA) has shown neuroprotective effects in other experimental models of Parkinson's disease, but its effect on the rotenone-induced parkinsonism is still unknown. We tested whether DHA in vivo exerts a neuroprotective effect on rotenone-induced parkinsonism and explored the mechanisms involved, including mitochondrial function and ultrastructure as well as the expression of tubulin and synaptophysin. We pretreated eighty male Wistar rats with DHA (35 mg/kg/day) for seven days and then administered rotenone for eight days. We then measured rearing behavior, number of dopaminergic neurons, tyrosine hydroxylase content, tubulin and synaptophysin expression, mitochondrial complex I, respiratory control ratio, mitochondrial transmembrane potential, ATP production activity and mitochondrial ultrastructure. We found that in vivo DHA supply exerted a neuroprotective effect, evidenced by decreased dopaminergic neuron cell death. Although we detected rotenone induced mitochondrial ultrastructure alterations, these were not associated with mitochondrial dysfunction. Rotenone had no effect on mitochondrial complex I, respiratory control ratio, mitochondrial transmembrane potential or ATP production activity. DHA also prevented a rotenone-induced decrease in tubulin and synaptophysin expression. Our results support the neuroprotective effect of DHA on rotenone-induced parkinsonism, and a possible effect on early stage Parkinson's disease. This protective effect is not associated with mitochondrial function improvement, but rather with preventing loss of tubulin and synaptophysin, proteins relevant to synaptic transmission.

Neuroprotective Effects of Açaí (Euterpe oleracea Mart.) against Rotenone In Vitro Exposure.

Neuropsychiatric diseases, such as bipolar disorder (BD) and schizophrenia (SCZ), have a very complex pathophysiology. Several current studies describe an association between psychiatric illness and mitochondrial dysfunction and consequent cellular modifications, including lipid, protein, and DNA damage, caused by cellular oxidative stress. Euterpe oleracea (açaí) is a powerful antioxidant fruit. Açaí is an Amazonian palm fruit primarily found in the lowlands of the Amazonian rainforest, particularly in the floodplains of the Amazon River. Given this proposed association, this study analyzed the potential in vitro neuropharmacological effect of Euterpe oleracea (açaí) extract in the modulation of mitochondrial function and oxidative metabolism. SH-SY5Y cells were treated with rotenone to induce mitochondrial complex I dysfunction and before and after we exposed the cells to açaí extract at 5 µg/mL. Treated and untreated cells were then analyzed by spectrophotometric, fluorescent, immunological, and molecular assays. The results showed that açaí extract can potentially increase protein amount and enzyme activity of mitochondrial complex I, mainly through NDUFS7 and NDUFS8 overexpression. Açaí extract was also able to decrease cell reactive oxygen species levels and lipid peroxidation. We thus suggest açaí as a potential candidate for drug development and a possible alternative BD therapy.

MITOsym®: A Mechanistic, Mathematical Model of Hepatocellular Respiration and Bioenergetics.

PURPOSE: MITOsym, a new mathematical model of hepatocellular respiration and bioenergetics, has been developed in partnership with the DILIsym® model with the purpose of translating in vitro compound screening data into predictions of drug induced liver injury (DILI) risk for patients. The combined efforts of these two models should increase the efficiency of evaluating compounds in drug development in addition to enhancing patient care. METHODS: MITOsym includes the basic, essential biochemical pathways associated with hepatocellular respiration and bioenergetics, including mitochondrial oxidative phosphorylation, electron transport chain activity, mitochondrial membrane potential, and glycolysis; also included are dynamic feedback signals based on perturbation of these pathways. The quantitative relationships included in MITOsym are based primarily on published data; additional new experiments were also performed in HepG2 cells to determine the effects on oxygen consumption rate as media glucose concentrations or oligomycin concentrations were varied. The effects of varying concentrations of FCCP on the mitochondrial proton gradient were also measured in HepG2 cells. RESULTS: MITOsym simulates and recapitulates the reported dynamic changes to hepatocellular oxygen consumption rates, extracellular acidification rates, the mitochondrial proton gradient, and ATP concentrations in the presence of classic mitochondrial toxins such as rotenone, FCCP, and oligomycin. CONCLUSIONS: MITOsym can be used to simulate hepatocellular respiration and bioenergetics and provide mechanistic hypotheses to facilitate the translation of in vitro data collection to predictions of in vivo human hepatotoxicity risk for novel compounds.

Mechanisms of neuroprotection by glucose in rat retinal cell cultures subjected to respiratory inhibition.

PURPOSE: Previous experiments have demonstrated that short-term hyperglycemia in rats renders the retina resistant to subsequent metabolic insults. The present study aimed to elucidate putative mechanisms involved in this protective response. METHODS: Retinal cultures comprising neurons and glia were treated with the mitochondrial complex I inhibitor, rotenone, at a range of concentrations, for up to 24 hours. In some cases, glucose or the alternative energy substrates, pyruvate or lactate, and/or inhibitors of glycolysis or the pentose phosphate pathway (PPP) were also applied. Cell viability was assessed using complementary techniques: immunocytochemistry, immunoblotting, cytotoxicity assay, and TUNEL. Cellular levels of ATP, reactive oxygen species (ROS), and nicotinamide adenine dinucleotide phosphate (NAD[P]H) were also assessed. RESULTS: Rotenone caused the preferential loss of neurons from retinal cultures in a concentration-dependent manner; glial cells were also affected, but only at a higher concentrations (10 µM). Cell loss was by apoptosis, and was preceded by a reduction of both cellular ATP and NAD(P)H levels and an increase in the production of ROS. Glucose counteracted the detrimental effects of rotenone. This involved a reduction in ROS levels and an increase in the cellular ATP/NAD(P)H ratio. The protective effect of glucose was partially reversed by either PPP or glycolysis inhibition. CONCLUSIONS: Glucose rescued cultured rat retinal cells from rotenone-induced toxicity. Glucose acted via both the PPP and the glycolytic pathway, maintaining cellular ATP and NAD(P)H levels and reducing ROS production. These data have implications for treatment of retinal diseases that involve metabolic compromise to neurons.

Glyceraldehyde-3-phosphate dehydrogenase: activity inhibition and protein overexpression in rotenone models for Parkinson's disease.

Rotenone, a widely used pesticide and an environmental risk factor for Parkinson's disease (PD), induces nigrostriatal injury, Lewy body-like inclusions, and Parkinsonian symptoms in rat models for PD. Our previous data indicated that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) overexpression and glycolytic inhibition were co-current in rotenone-induced PC12 (rat adrenal pheochromocytoma cells) cell death. However, whether GAPDH overexpression plays any role in dopaminergic neurodegeneration in vivo remains unknown. In this study, we have found that GAPDH overexpression and GAPDH-positive Lewy body-like aggregates in nigral dopaminergic neurons while nigral GAPDH glycolytic activity decreases in rotenone-based PD animal models. Furthermore, GAPDH knockdown reduces rotenone toxicity significantly in PC12. These in vitro and in vivo data suggest that GAPDH contributes to the pathogenesis of Parkinson's disease, possibly representing a new molecular target for neuroprotective strategies and alternative therapies for PD.

Protective effect of lycopene on oxidative stress and cognitive decline in rotenone induced model of Parkinson's disease.

Evidence from clinical and experimental studies indicate that oxidative stress is involved in pathogenesis of Parkinson's disease. The present study was designed to investigate the neuroprotective potential of lycopene on oxidative stress and neurobehavioral abnormalities in rotenone induced PD. Rats were treated with rotenone (3 mg/kg body weight, intraperitoneally) for 30 days. NADH dehydrogenase a marker of rotenone action was observed to be significantly inhibited (35%) in striatum of treated animals. However, lycopene administration (10 mg/kg, orally) to the rotenone treated animals for 30 days increased the activity by 39% when compared to rotenone treated animals. Rotenone administration increased the MDA levels (75.15%) in striatum, whereas, lycopene administration to rotenone treated animals decreased the levels by 24.33%. Along with this, significant decrease in GSH levels (42.69%) was observed in rotenone treated animals. Lycopene supplementation on the other hand, increased the levels of GSH by 75.35% when compared with rotenone treated group. The activity of SOD was inhibited by 69% in rotenone treated animals and on lycopene supplementation; the activity increased by 12% when compared to controls. This was accompanied by cognitive and motor deficits in rotenone administered animals, which were reversed on lycopene treatment. Lycopene treatment also prevented release of cytochrome c from mitochondria. Collectively, these observations suggest that lycopene supplementation along with rotenone for 30 days prevented rotenone-induced alterations in antioxidants along with the prevention of rotenone induced oxidative stress and neurobehavioral deficits. The results provide an evidence for beneficial effect of lycopene supplementation in rotenone-induced PD and suggest therapeutic potential in neurodegenerative diseases involving accentuated oxidative stress.

Reversal of rotenone-induced dysfunction of astrocytic connexin43 by opening mitochondrial ATP-sensitive potassium channels.

Growing evidence suggests that the astrocytic gap junctions (GJs), mainly formed by connexin 43 (Cx43), play an important role in physiological maintenance and various central nervous system (CNS) pathological conditions. However, little is known about the role of Cx43 in Parkinson's disease (PD). In this article, we report that rotenone, a classic neurotoxin for PD, could inhibit expression of astrocytic Cx43 and gap junction permeability. ATP-sensitive potassium (K(ATP)) channel openers, iptakalim (IPT) and diazoxide (DZ), exerted protective effect on rotenone-induced dysfunction of Cx43 and astrocyte apoptosis, which was reversed by selective mitochondrial K(ATP) (mitoK(ATP)) channel blocker 5-hydroxydecanoate (5-HD). Taken together, our findings reveal that rotenone-induced dysfunction of astrocytic Cx43 may be involved in the pathology of PD. Moreover, opening mitoK(ATP) channels in astrocytes can reverse rotenone-induced dysfunction of astrocytic Cx43 and therefore protect against toxicity of rotenone on astrocytes.

Attenuation of rotenone-induced mitochondrial oxidative damage and neurotoxicty in Drosophila melanogaster supplemented with creatine.

Creatine (Cr), an ergogenic nutritional supplement is demonstrated to possess bioenergetic, antiexcitotoxic and antioxidant properties. This study investigated the neuroprotective effects of Cr against rotenone induced oxidative stress, mortality and neurotoxicty in Drosophila melanogaster. We found significant diminution in the endogenous levels of oxidative markers in whole body homogenates of flies exposed to Cr (2-10 mM). Cr supplementation resulted in reduced mortality in flies exposed to rotenone (500 microM) and better performance in a negative geotaxis assay. Further Cr (10 mM) markedly offset rotenone induced mitochondrial oxidative stress, completely restored the GSH levels, nitric oxide levels, activity of Mn-SOD and dopamine depletion. In an oxidative stress bioassay, flies given Cr prophylaxis exhibited marked resistance to paraquat exposure. These data allow us to hypothesize that the neuroprotective action of Cr in Drosophila may be related to its direct antioxidant activity and ability to abrogate rotenone induced mitochondrial oxidative stress.

Comparative effects of lantadene A and its reduced metabolite on mitochondrial bioenergetics.

Lantana (Lantana camara Linn.) is a noxious weed to which certain medicinal properties have been attributed, but its ingestion has been reported to be highly toxic to animals and humans, especially in the liver. The main hepatotoxin in lantana leaves is believed to be the pentacyclic triterpenoid lantadene A (LA), but the precise mechanism by which it induces hepatotoxicity has not yet been established. This work addressed the action of LA and its reduced derivative (RLA) on mitochondrial bioenergetics. At the concentration range tested (5-25 microM), RLA stimulated state-4 respiration, inhibited state-3 respiration, circumvented oligomycin-inhibited state-3 respiration, dissipated membrane potential and depleted ATP in a concentration-dependent manner. However, LA did not stimulate state-4 respiration, nor did it affect the other mitochondrial parameters to the extent of its reduced derivative. The lantadenes didn't inhibit the CCCP-uncoupled respiration but increased the ATPase activity of intact coupled mitochondria. The ATPase activity of intact uncoupled or disrupted mitochondria was not affected by the compounds. We propose, therefore, that RLA acts as a mitochondrial uncoupler of oxidative phosphorylation, a property that arises from the biotransformation (reduction) of LA, and LA acts in other mitochondrial membrane components rather than the ATP synthase affecting the mitochondrial bioenergetics. Such effects may account for the well-documented hepatoxicity of lantana.

Pretreatment with near-infrared light via light-emitting diode provides added benefit against rotenone- and MPP+-induced neurotoxicity.

Parkinson's disease (PD) is a movement disorder caused by the loss of dopaminergic neurons in the substantia nigra pars compacta, leading to nigrostriatal degeneration. The inhibition of mitochondrial respiratory chain complex I and oxidative stress-induced damage have been implicated in the pathogenesis of PD. The present study used these specific mitochondrial complex I inhibitors (rotenone and 1-methyl-4-phenylpyridinium or MPP(+)) on striatal and cortical neurons in culture. The goal was to test our hypothesis that pretreatment with near-infrared light (NIR) via light-emitting diode (LED) had a greater beneficial effect on primary neurons grown in media with rotenone or MPP(+) than those with or without LED treatment during exposure to poisons. Striatal and visual cortical neurons from newborn rats were cultured in a media with or without 200 nM of rotenone or 250 microM of MPP(+) for 48 h. They were treated with NIR-LED twice a day before, during, and both before and during the exposure to the poison. Results indicate that pretreatment with NIR-LED significantly suppressed rotenone- or MPP(+)-induced apoptosis in both striatal and cortical neurons (P<0.001), and that pretreatment plus LED treatment during neurotoxin exposure was significantly better than LED treatment alone during exposure to neurotoxins. In addition, MPP(+) induced a decrease in neuronal ATP levels (to 48% of control level) that was reversed significantly to 70% of control by NIR-LED pretreatment. These data suggest that LED pretreatment is an effective adjunct preventative therapy in rescuing neurons from neurotoxins linked to PD.

The role of dopamine transporter in selective toxicity of manganese and rotenone.

The dopamine transporter has been shown to be the most relevant target site for the specificity of 1-methyl-4-phenylpyridinium ion (MPP+), a neurotoxin for dopaminergic neurons. In contrast, the mechanisms underlying the selective toxicity of manganese and rotenone, potentially toxic agents implicated in dopaminergic neuronal cell death, remain unknown. The aim of this study was to determine the cellular mechanisms of manganese or rotenone uptake in dopaminergic cells via the dopamine transporter. PC12 cells overexpressing the dopamine transporter, which were exposed to 10microM MPP+, showed extensive DNA fragmentation, a biochemical hallmark of apoptosis, whereas wild-type PC12 cells or vector-transfected PC12 cells, which were exposed to 5mM MPP+, did not show DNA fragmentation. In contrast, manganese and rotenone induced DNA fragmentation at slightly lower concentrations in PC12 cells overexpressing the dopamine transporter compared to control cells. Dopamine transporter inhibitors, such as mazindol, nomifensine, or GBR12909, inhibited MPP+-induced DNA fragmentation but did not affect manganese- and rotenone-induced DNA fragmentation in PC12 cells overexpressing the dopamine transporter. Finally, manganese accumulated to similar levels in PC12 cells overexpressing the dopamine transporter and control PC12 cells following incubation with manganese chloride. These results suggested that the dopamine transporter dose not confer cytotoxicity to manganese and rotenone.

Evaluation of olive oil mill wastewaters acute toxicity: a study on the mitochondrial bioenergetics.

Acute toxicity of olive mill wastewaters (OMW), collected from a continuous olive mill, was evaluated in rat liver mitochondrial bioenergetics. Inhibition of respiratory activities in state 4, state 3, and uncoupled respiration are essentially mediated through partial inhibitions of mitochondrial complexes II and III. ATPase activity was considerably less depressed by OMW than ATP synthase activity (a difference of 42%). The inhibition observed on ATP synthase is mostly the result of an inhibition on the redox complexes. Ultimately, the OMW-induced loss of phosphorylation capacity was not only the result of a direct effect of OMW on the enzymatic complex (F(0)-F(1) ATPase), but also the result of a deleterious effect on the integrity of the mitochondrial membrane, which can promote an inhibition of the respiratory complexes and an increase of the proton permeability of the inner membrane.

Target identification of drug induced mitochondrial toxicity using immunocapture based OXPHOS activity assays.

Mitochondrial dysfunction has been shown to be a pharmacotoxicological response to a variety of currently-marketed drugs. In order to reduce attrition due to mitochondrial toxicity, high throughput-applicable screens are needed for early stage drug discovery. We describe, here, a set of immunocapture based assays to identify compounds that directly inhibit four of the oxidative phosphorylation (OXPHOS) complexes: I, II, IV, and V. Intra- and inter-assay variation were determined and specificity tested by using classical mitochondrial inhibitors. Twenty drugs, some with known mitochondrial toxicity and others with no known mitochondrial liability, were studied. Direct inhibition of one or more of the OXPHOS complexes was identified for many of the drugs. Novel information was obtained for several drugs including ones with previously unknown effects on oxidative phosphorylation. A major advantage of the immunocapture approach is that it can be used throughout drug screening from early compound evaluation to clinical trials.

Caenorhabditis elegans MPP+ model of Parkinson's disease for high-throughput drug screenings.

The neurotoxin MPTP and its active metabolite MPP+ cause Parkinson's disease (PD)-like symptoms in vertebrates by selectively destroying dopaminergic neurons in the substantia nigra. MPTP/MPP+ models have been established in rodents to screen for pharmacologically active compounds. In addition to being costly and time consuming, these animal models are not suitable for large scale testings using compound libraries. We present a novel MPP+-based model for high-throughput screenings using the nematode Caenorhabditis elegans. Incubation of C. elegans with MPTP or its active metabolite MPP+ resulted in strong symptomatic defects including reduced mobility and increased lethality, and is correlated with a specific degeneration of the dopaminergic neurons. The phenotypic consequences of MPTP/MPP+ treatments were recorded using automated hardware and software for quantification. Incubation of C. elegans with a variety of pharmacologically active components used in PD treatment reduced the MPP+-induced defects. Our data suggest that the C. elegans MPTP/MPP+ model can be used for the quantitative evaluation of anti-PD drugs.

Dysfunction of mitochondrial complex I and the proteasome: interactions between two biochemical deficits in a cellular model of Parkinson's disease.

Two biochemical deficits have been described in the substantia nigra in Parkinson's disease, decreased activity of mitochondrial complex I and reduced proteasomal activity. We analysed interactions between these deficits in primary mesencephalic cultures. Proteasome inhibitors (epoxomicin, MG132) exacerbated the toxicity of complex I inhibitors [rotenone, 1-methyl-4-phenylpyridinium (MPP+)] and of the toxic dopamine analogue 6-hydroxydopamine, but not of inhibitors of mitochondrial complex II-V or excitotoxins [N-methyl-d-aspartate (NMDA), kainate]. Rotenone and MPP+ increased free radicals and reduced proteasomal activity via adenosine triphosphate (ATP) depletion. 6-hydroxydopamine also increased free radicals, but did not affect ATP levels and increased proteasomal activity, presumably in response to oxidative damage. Proteasome inhibition potentiated the toxicity of rotenone, MPP+ and 6-hydroxydopamine at concentrations at which they increased free radical levels >/= 40% above baseline, exceeding the cellular capacity to detoxify oxidized proteins reduced by proteasome inhibition, and also exacerbated ATP depletion caused by complex I inhibition. Consistently, both free radical scavenging and stimulation of ATP production by glucose supplementation protected against the synergistic toxicity. In summary, proteasome inhibition increases neuronal vulnerability to normally subtoxic levels of free radicals and amplifies energy depletion following complex I inhibition.

Mitochondrial complex inhibitors preferentially damage substantia nigra dopamine neurons in rat brain slices.

Using a rat brain slice preparation, we investigated the role of energy impairment on the selective loss of dopamine neurons in the substantia nigra (SN). Brain slices (400 microm) were incubated at 35 degrees C for 2 h in the presence or absence of mitochondrial complex inhibitors, rotenone, MPP+, 3-nitropropionic acid, and antimycin A. Slices were also incubated in rotenone with excitatory amino acid (EAA) receptor antagonists, MK-801 and CNQX, to determine whether rotenone-induced damage was mediated by EAAs. The slices were then fixed, recut into 30-microm sections, and immunolabeled for tyrosine hydroxylase (TH) to identify catecholamine neurons and to quantify loss of TH-labeled dendrites after treatment. Quantitative comparison was made between SN dopamine neurons, in which rotenone-induced dendrite loss was severe, and hypothalamic A11 dopamine neurons, which were spared. Adjacent sections that were immunolabeled for calbindin or stained with cresyl violet also revealed a striking dendritic degeneration of SN neurons in rotenone-exposed slices, whereas noncatecholamine neurons, such as those in the perifornical nucleus (PeF), were more resistant. Preferential damage to SN dopamine neurons was also evident with other mitochondrial complex inhibitors, MPP+ and antimycin A. EAA receptor antagonists provided partial protection to SN neurons in slices incubated with rotenone (3 microM). The particular vulnerability of SN dopamine neurons in the slice is consistent with the vulnerability of SN in Parkinson's disease. The selective effect of mitochondrial complex inhibition in SN dopamine neurons implies a fundamental deficit in the capacity of these neurons to defend against toxic insult.

Bioenergetic consequences of opening the ATP-sensitive K(+) channel of heart mitochondria.

There is an emerging consensus that pharmacological opening of the mitochondrial ATP-sensitive K(+) (K(ATP)) channel protects the heart against ischemia-reperfusion damage; however, there are widely divergent views on the effects of openers on isolated heart mitochondria. We have examined the effects of diazoxide and pinacidil on the bioenergetic properties of rat heart mitochondria. As expected of hydrophobic compounds, these drugs have toxic, as well as pharmacological, effects on mitochondria. Both drugs inhibit respiration and increase membrane proton permeability as a function of concentration, causing a decrease in mitochondrial membrane potential and a consequent decrease in Ca(2+) uptake, but these effects are not caused by opening mitochondrial K(ATP) channels. In pharmacological doses (<50 microM), both drugs open mitochondrial K(ATP) channels, and resulting changes in membrane potential and respiration are minimal. The increased K(+) influx associated with mitochondrial K(ATP) channel opening is approximately 30 nmol. min(-1). mg(-1), a very low rate that will depolarize by only 1-2 mV. However, this increase in K(+) influx causes a significant increase in matrix volume. The volume increase is sufficient to reverse matrix contraction caused by oxidative phosphorylation and can be observed even when respiration is inhibited and the membrane potential is supported by ATP hydrolysis, conditions expected during ischemia. Thus opening mitochondrial K(ATP) channels has little direct effect on respiration, membrane potential, or Ca(2+) uptake but has important effects on matrix and intermembrane space volumes.

Kinetic analysis of 75selenium uptake by mitochondria of germinating Vigna radiata of different selenium status.

Earlier studies in our laboratory demonstrated the beneficial role of Se in Vigna radiata, a Se-deficient legume, during germination, as reflected in growth-related parameters and specific uptake of 75Se. Uptake of Na2(75)SeO3, added in vitro by mitochondria isolated from seedlings germinated in control (without Se), and Se-supplemented groups (0.5, 1.0, and 2.0 ppm Se) indicated a proportional increase in the uptake with added Na2(75)SeO3, in concentrations up to 25 microM. The uptake of 75Se, increased linearly with time up to 15 min and a definite efflux followed at 30 min. The results were indicative of cooperative effects during Se transport. Kinetic analyses of the uptake of 75Se during time intervals of 15 and 30 min were carried out both in the whole mitochondria and the mitochondrial protein fractions. Graphical analyses using Lineweaver-Burk plot, Hill plot, log [v] vs log [A] and Scatchard plot confirmed the existence of negative cooperativity during 75Se uptake. Hill coefficient (nH) values were estimated to be around 0.7-0.8. Scatchard plots for 75Se uptake were biphasic, suggesting the probable presence of two classes of binding sites. The number of high and low affinity binding sites were estimated to be around 4-7 and 26-30 nmol/mg protein, respectively. Studies with mitochondrial respiratory inhibitors indicated about 10-20% of the total 75Se uptake to be energy dependent. Inhibition of 75Se uptake by about 60-70% by sulfate and sulfite (5-25 microM) implies the involvement of dicarboxylate port in Se transport. A decrease in the uptake of 75Se by 40-60% effected by CdCl2, HgCl2, mersalyl, and NEM confirmed the interaction of thiols in the process. Evidence for the regulatory nature of 75Se uptake by mitochondria of V. radiata emerges from the present study.