Dopamine Receptor Subtypes,IP3, cAMP and cGMP Functional Regulation in Parkinsonism Induced by Unilateral Infusion of Rotenone in Rats: Recovery with Bone Marrow Cells,Serotonin and GABA Supplementation

In the present study, the effects of 5-HT, GABA and Bone Marrow Cells infused intranigrally to substantia nigra individually and in combinations on unilateral rotenone infused Parkinsonism induced rats. Scatchard analysis of DA, DA D1 and D2 receptors in the corpus striatum, cerebral cortex, cerebellum, brain stem and hippocampus showed a significant increase in the Brain regions of rotenone infused rat compared to control. Real Time PCR amplification of DA D1, D2, Bax and ubiquitin carboxy-terminal hydrolase were up regulated in the brain regions of rotenone infused rats compared to control. Gene expression studies of -Synuclien, cGMP and Cyclic AMP response element-binding protein showed a significant down regulation in Rotenone infused rats compared to control. Behavioural studies were carried out to confirm the biochemical and molecular studies.Our study demonstrated that BMC administration alone cannot reverse the above said molecular changes occurring in PD rat. 5-HT and GABA acting through their specific receptors in combination with bone marrow cells play a crucial role in the functional recovery of PD rats. 5-HT, GABA and Bone marrow cells treated PD rats showed significant reversal to control in DA receptor binding and gene expression. 5-HT and GABA have co-mitogenic property. Proliferation and differentiation of cells re-establishing the connections in Parkinson's disease facilitates the functional recovery. Thus, it is evident that 5-HT and GABA along with BMC to rotenone infused rats renders protection against oxidative, related motor and cognitive deficits which makes them clinically significant for cellbased therapy. The BMC transformed to neurons when co-transplanted with 5-HT and GABA which was confirmed with PKH2GL and nestin. These newly formed neurons have functional significance in the therapeutic recovery of Parkinson’s disease.

Rotenone induces degeneration of photoreceptors and impairs the dopaminergic system in the rat retina

Rotenone is a widely used pesticide and a potent inhibitor of mitochondrial complex I (NADH-quinone reductase) that elicits the degeneration of dopaminergic neurons and thereby the appearance of a parkinsonian syndrome. Here we have addressed the alterations induced by rotenone at the functional, morphological and molecular levels in the retina, including those involving both dopaminergic and non-dopaminergic retinal neurons. Rotenone-treated rats showed abnormalities in equilibrium, postural instability and involuntary movements. In their outer retina we observed a loss of photoreceptors, and a reduced synaptic connectivity between those remaining and their postsynaptic neurons. A dramatic loss of mitochondria was observed in the inner segments, as well as in the axon terminals of photoreceptors. In the inner retina we observed a decrease in the expression of dopaminergic cell molecular markers, including loss of tyrosine hydroxylase immunoreactivity, associated with a reduction of the dopaminergic plexus and cell bodies. An increase in immunoreactivity of AII amacrine cells for parvalbumin, a Ca2+-scavenging protein, was also detected. These abnormalities were accompanied by a decrease in the amplitude of scotopic and photopic a- and b-waves and an increase in the b-wave implicit time, as well as by a lower amplitude and greater latency in oscillatory potentials. These results indicate that rotenone induces loss of vision by promoting photoreceptor cell death and impairment of the dopaminergic retinal system.

A preliminary study of the influence of Velsicol AR-60 upon the insecticidal effectiveness of dusts containing rotenone or DDT.

"Literature cited": leaf 84.

The synthesis of novel nitroxides and their application as small-molecule antioxidants and profluorescent probes for oxidative stress

The detection and potential treatment of oxidative stress in biological systems has been explored using isoindoline-based nitroxide radicals. A novel tetraethyl-fluorescein nitroxide was synthesised for its use as a profluorescent probe for redox processes in biological systems. This tetraethyl system, as well as a tetramethyl-fluorescein nitroxide, were shown to be sensitive and selective probes for superoxide in vitro. The redox environment of cellular systems was also explored using the tetramethylfluorescein species based on its reduction to the hydroxylamine. Flow cytometry was employed to assess the extent of nitroxide reduction, reflecting the overall cellular redox environment. Treatment of normal fibroblasts with rotenone and 2-deoxyglucose resulted in an oxidising cellular environment as shown by the lack of reduction of the fluorescein-nitroxide system. Assessment of the tetraethyl-fluorescein nitroxide system in the same way demonstrated its enhanced resistance to reduction and offers the potential to detect and image biologically relevant reactive oxygen species directly. Importantly, these profluorescent nitroxide compounds were shown to be more effective than the more widely used and commercially available probes for reactive oxygen species such as 2’,7’-dichlorodihydrofluorescein diacetate. Fluorescence imaging of the tetramethyl-fluorescein nitroxide and a number of other rhodamine-nitroxide derivatives was undertaken, revealing the differential cellular localisation of these systems and thus their potential for the detection of redox changes in specific cellular compartments. As well as developing novel methods for the detection of oxidative stress, a number of novel isoindoline nitroxides were synthesised for their potential application as small-molecule antioxidants. These compounds incorporated known pharmacophores into the isoindoline-nitroxide structure in an attempt to increase their efficacy in biological systems. A primary and a secondary amine nitroxide were synthesised which incorporated the phenethylamine backbone of the sympathomimetic amine class of drugs. Initial assessment of the novel primary amine derivative indicated a protective effect comparable to that of 5-carboxy-1,1,3,3- tetramethylisoindolin-2-yloxyl. Methoxy-substituted nitroxides were also synthesised as potential antioxidants for their structural similarity to some amphetamine type stimulants. A copper-catalysed methodology provided access to both the mono- and di-substituted methoxy-nitroxides. Deprotection of the ethers in these compounds using boron tribromide successfully produced a phenolnitroxide, however the catechol moiety in the disubstituted derivative appeared to undergo reaction with the nitroxide to produce quinone-like degradation products. A novel fluoran-nitroxide was also synthesised from the methoxy-substituted nitroxide, providing a pH-sensitive spin probe. An amino-acid precursor containing a nitroxide moiety was also synthesised for its application as a dual-action antioxidant. N-Acetyl protection of the nitroxide radical was necessary prior to the Erlenmeyer reaction with N-acetyl glycine. Hydrolysis and reduction of the azlactone intermediate produced a novel amino acid precursor with significant potential as an effective antioxidant.

Serine protease inhibition and mitochondrial dysfunction associated with cisplatin resistance in human tumor cell lines : targets for therapy

Indicators of mitochondrial function were studied in two different cell culture models of cis-diamminedichloroplatinum-II (CDDP) resistance: the intrinsically resistant human ovarian cancer cell line CI-80-13S, and resistant clones (HeLa-S1a and HeLa-S1b) generated by stable expression of the serine protease inhibitor—plasminogen activator inhibitor type-2 (PAI-2), in the human cervical cancer cell line HeLa. In both models, CDDP resistance was associated with sensitivity to killing by adriamycin, etoposide, auranofin, bis[1,2-bis(diphenylphosphino)ethane]gold(I) chloride {[Au(DPPE)2]Cl}, CdCl2 and the mitochondrial inhibitors rhodamine-123 (Rhl23), dequalinium chloride (DeCH), tetraphenylphosphonium (TPP), and ethidium bromide (EtBr) and with lower constitutive levels of ATP. Unlike the HeLa clones, CI-80-13S cells were additionally sensitive to chloramphenicol, 1-methyl-4-phenylpyridinium ion (MPP+), rotenone, thenoyltrifluoroacetone (TTFA), and antimycin A, and showed poor reduction of 1-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), suggesting a deficiency in NADH dehydrogenase and/or succinate dehydrogenase activities. Total platinum uptake and DNA-bound platinum were slightly lower in CI-80-13S than in sensitive cells. The HeLa-S1a and HeLa-S1b clones, on the other hand, showed poor reduction of triphenyltetrazolium chloride (TTC), indicative of low cytochrome c oxidase activity. Total platinum uptake by HeLa-S1a was similar to HeLa, but DNA-bound platinum was much lower than for the parent cell line. The mitochondria of CI-80-13S and HeLa-S1a showed altered morphology and were fewer in number than those of JAM and HeLa. In both models, CDDP resistance was associated with less platinum accumulation and with mitochondrial and membrane defects, brought about one case with expression of a protease inhibitor which is implicated in tumor progression. Such markers may identify tumors suitable for treatment with gold phosphine complexes or other mitochondrial inhibitors.

Serine protease inhibition and mitochondrial dysfunction associated with cisplatin resistance in human tumor cell lines: targets for therapy

Indicators of mitochondrial function were studied in two different cell culture models of cis-diamminedichloroplatinum-II (CDDP) resistance: the intrinsically resistant human ovarian cancer cell line CI-80-13S, and resistant clones (HeLa-S1a and HeLa-S1b) generated by stable expression of the serine protease inhibitor—plasminogen activator inhibitor type-2 (PAI-2), in the human cervical cancer cell line HeLa. In both models, CDDP resistance was associated with sensitivity to killing by adriamycin, etoposide, auranofin, bis[1,2-bis(diphenylphosphino)ethane]gold(I) chloride {[Au(DPPE)2]Cl}, CdCl2 and the mitochondrial inhibitors rhodamine-123 (Rhl23), dequalinium chloride (DeCH), tetraphenylphosphonium (TPP), and ethidium bromide (EtBr) and with lower constitutive levels of ATP. Unlike the HeLa clones, CI-80-13S cells were additionally sensitive to chloramphenicol, 1-methyl-4-phenylpyridinium ion (MPP+), rotenone, thenoyltrifluoroacetone (TTFA), and antimycin A, and showed poor reduction of 1-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), suggesting a deficiency in NADH dehydrogenase and/or succinate dehydrogenase activities. Total platinum uptake and DNA-bound platinum were slightly lower in CI-80-13S than in sensitive cells. The HeLa-S1a and HeLa-S1b clones, on the other hand, showed poor reduction of triphenyltetrazolium chloride (TTC), indicative of low cytochrome c oxidase activity. Total platinum uptake by HeLa-S1a was similar to HeLa, but DNA-bound platinum was much lower than for the parent cell line. The mitochondria of CI-80-13S and HeLa-S1a showed altered morphology and were fewer in number than those of JAM and HeLa. In both models, CDDP resistance was associated with less platinum accumulation and with mitochondrial and membrane defects, brought about one case with expression of a protease inhibitor which is implicated in tumor progression. Such markers may identify tumors suitable for treatment with gold phosphine complexes or other mitochondrial inhibitors.

Oxidative activities in mitochondria-like particles from Setaria digitata, a filarial parasite

The oxidative metabolic potential of Setaria digitata, a filarial parasite found in the intraperitoneal cavity of cattle, was investigated. These worms showed active wriggling movements which were not affected by respiratory poisons such as cyanide, rotenone and malonate. They also possessed cyanide-insensitive and glucose-independent oxygen consumption pathways. By differential centrifugation of sucrose homogenates, a fraction containing mitochondria-like particles was obtained in which the activity of the marker enzyme, succinate dehydrogenase, was recovered. This fraction catalysed succinate- and NADH-dependent reduction of both cytochrome c and dyes. Oxygen uptake found with succinate, NADH and ascorbate as substrates was not sensitive to cyanide. Cytochromes could not be detected in either this fraction or homogenates of the worms. H2O2 generation with a number of substrates and lipid peroxidation by measuring malondialdehyde formed as well as by accompanying oxygen uptake were demonstrated in the mitochondria-like particles. A lipid quinone, possibly with a short side chain and related to ubiquinone, was detected in the worms. The results suggested the existence of two cyanide-insensitive oxygen-consuming reactions in Setaria: one respiratory substrate-independent lipid peroxidation, and a second substrate-dependent reaction that requires an auto-oxidizable quinone but not a cytochrome system.

Introduction of high-content screening method for studying drug-induced mitochondrial dysfunction in hepatocytes

Drug induced liver injury is one of the frequent reasons for the drug removal from the market. During the recent years there has been a pressure to develop more cost efficient, faster and easier ways to investigate drug-induced toxicity in order to recognize hepatotoxic drugs in the earlier phases of drug development. High Content Screening (HCS) instrument is an automated microscope equipped with image analysis software. It makes the image analysis faster and decreases the risk for an error caused by a person by analyzing the images always in the same way. Because the amount of drug and time needed in the analysis are smaller and multiple parameters can be analyzed from the same cells, the method should be more sensitive, effective and cheaper than the conventional assays in cytotoxicity testing. Liver cells are rich in mitochondria and many drugs target their toxicity to hepatocyte mitochondria. Mitochondria produce the majority of the ATP in the cell through oxidative phosphorylation. They maintain biochemical homeostasis in the cell and participate in cell death. Mitochondria is divided into two compartments by inner and outer mitochondrial membranes. The oxidative phosphorylation happens in the inner mitochondrial membrane. A part of the respiratory chain, a protein called cytochrome c, activates caspase cascades when released. This leads to apoptosis. The aim of this study was to implement, optimize and compare mitochondrial toxicity HCS assays in live cells and fixed cells in two cellular models: human HepG2 hepatoma cell line and rat primary hepatocytes. Three different hepato- and mitochondriatoxic drugs (staurosporine, rotenone and tolcapone) were used. Cells were treated with the drugs, incubated with the fluorescent probes and then the images were analyzed using Cellomics ArrayScan VTI reader. Finally the results obtained after optimizing methods were compared to each other and to the results of the conventional cytotoxicity assays, ATP and LDH measurements. After optimization the live cell method and rat primary hepatocytes were selected to be used in the experiments. Staurosporine was the most toxic of the three drugs and caused most damage to the cells most quickly. Rotenone was not that toxic, but the results were more reproducible and thus it would serve as a good positive control in the screening. Tolcapone was the least toxic. So far the conventional analysis of cytotoxicity worked better than the HCS methods. More optimization needs to be done to get the HCS method more sensitive. This was not possible in this study due to time limit.

Hypoxia-Mediated Impairment of the Mitochondrial Respiratory Chain Inhibits the Bactericidal Activity of Macrophages

In infected tissues oxygen tensions are low. As innate immune cells have to operate under these conditions, we analyzed the ability of macrophages (M phi) to kill Escherichia coli or Staphylococcus aureus in a hypoxic microenvironment. Oxygen restriction did not promote intracellular bacterial growth but did impair the bactericidal activity of the host cells against both pathogens. This correlated with a decreased production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates. Experiments with phagocyte NADPH oxidase (PHOX) and inducible NO synthase (NOS2) double-deficient M phi revealed that in E. coli- or S. aureus-infected cells the reduced antibacterial activity during hypoxia was either entirely or partially independent of the diminished PHOX and NOS2 activity. Hypoxia impaired the mitochondrial activity of infected M phi. Inhibition of the mitochondrial respiratory chain activity during normoxia (using rotenone or antimycin A) completely or partially mimicked the defective antibacterial activity observed in hypoxic E. coli-or S. aureus-infected wild-type M phi, respectively. Accordingly, inhibition of the respiratory chain of S. aureus-infected, normoxic PHOX-/- NOS2(-/-) M phi further raised the bacterial burden of the cells, which reached the level measured in hypoxic PHOX-/- NOS2(-/-) M phi cultures. Our data demonstrate that the reduced killing of S. aureus or E. coli during hypoxia is not simply due to a lack of PHOX and NOS2 activity but partially or completely results from an impaired mitochondrial antibacterial effector function. Since pharmacological inhibition of the respiratory chain raised the generation of ROI but nevertheless phenocopied the effect of hypoxia, ROI can be excluded as the mechanism underlying the antimicrobial activity of mitochondria.

Length-weight relationship of some marine fish species in Reunion Island, Indian Ocean

The length-weight relationship of 29 marine fish species form Reunion Island (SW Indian Ocean) belonging to 14 families were computed. Data from 5,340 individuals were used for this purpose. Fish were sampled using different techniques, mainly with rotenone poisoning on coral reef flats, beach seine and handlines on shallow coastal bays, and longline fishing in the nearby open sea.

Length-weight relationship of fishes from coral reefs and lagoons of New Caledonia: an update

Length-weight relationships of 316 reef and lagoon fish from New Caledonia (SW Pacific Ocean) belonging to 68 families are computed. A total of 43,750 individuals was used for this purpose. Fish were sampled by different techniques such as rotenone poisoning, handline and bottom longline fishing, gill and trammel nets, and trawling in various isotopes (coral reefs, lagoon bottoms and mangroves).

Regional Fisheries Advisory Committee Meeting 30th January, 1984

This is the report from the Regional Fisheries Advisory Committee meeting, which was held on the 30th January, 1984. The report contains information on the use of fyke nets and an application to use Rotenone fish Toxicant. Also included is information on Dunsop Trout Farm and the establishment of migratory fish rearing facilities, the decline of salmon and sea trout catches in South Cumbria, and the decline of salmon stocks in the River Eden. The Fisheries Advisory Committee was part of the Regional Water Authorities, in this case the North West Water Authority. This preceded the Environment Agency which came into existence in 1996.

Regional Fisheries Advisory Committee Meeting 18th June, 1984

This is the report from the Regional Fisheries Advisory Committee meeting, which was held on the 18th June, 1984. The report contains information on fishing licence duties, the Flimby Arrangement and the Disease of Fish Acts 1937 and 1983. Also covered is the use of Rotenone in the Lancaster Canal at Stainton, and the Salmon and Freshwater Fisheries Act, 1975. The Fisheries Advisory Committee was part of the Regional Water Authorities, in this case the North West Water Authority. This preceded the Environment Agency which came into existence in 1996.

Isolation, characterization and electron microscopic single particle analysis of the NADH : ubiquinone oxidoreductase (complex I) from the hyperthermophilic eubacterium Aquifex aeolicus

The proton-translocating NADH:ubiquinone oxidoreductase (complex I) has been purified from Aquifex aeolicus, a hyperthermophilic eubacterium of known genome sequence. The purified detergent solubilized enzyme is highly active above 50 degreesC. The specific activity for electron transfer from NADH to decylubiquinone is 29 U/mg at 80 degreesC. The A. aeolicus complex I is completely sensitive to rotenone and 2-n-decyl-quinazoline-4-yl-amine. SDS polyacrylamide gel electrophoresis shows that it may contain up to 14 subunits. N-terminal amino acid sequencing of the bands indicates the presence of a stable subcomplex, which is composed of subunits E, F, and G. The isolated complex is highly stable and active in a temperature range from 50 to 90 degreesC, with a half-life of about 10 h at 80 degreesC. The activity shows a linear Arrhenius plot at 50-85 degreesC with an activation energy at 31.92 J/mol K. Single particle electron microscopy shows that the A. aeolicus complex I has the typical L-shape. However, visual inspection of averaged images reveals many more details in the external arm of the complex than has been observed for complex I from other sources. In addition, the angle (90degrees) between the cytoplasmic peripheral arm and the membrane intrinsic arm of the complex appears to be invariant.

Later Life Consequences of Developmental Mitochondrial DNA Damage in C. elegans

Mitochondria are responsible for producing the vast majority of cellular ATP, and are therefore critical to organismal health [1]. They contain thir own genomes (mtDNA) which encode 13 proteins that are all subunits of the mitochondrial respiratory chain (MRC) and are essential for oxidative phosphorylation [2]. mtDNA is present in multiple copies per cell, usually between 103 and 104 , though this number is reduced during certain developmental stages [3, 4]. The health of the mitochondrial genome is also important to the health of the organism, as mutations in mtDNA lead to human diseases that collectively affect approximately 1 in 4000 people [5, 6]. mtDNA is more susceptible than nuclear DNA (nucDNA) to damage by many environmental pollutants, for reasons including the absence of Nucleotide Excision Repair (NER) in the mitochondria [7]. NER is a highly functionally conserved DNA repair pathway that removes bulky, helix distorting lesions such as those caused by ultraviolet C (UVC) radiation and also many environmental toxicants, including benzo[a]pyrene (BaP) [8]. While these lesions cannot be repaired, they are slowly removed through a process that involves mitochondrial dynamics and autophagy [9, 10]. However, when present during development in C. elegans, this damage reduces mtDNA copy number and ATP levels [11]. We hypothesize that this damage, when present during development, will result in mitochondrial dysfunction and increase the potential for adverse outcomes later in life.

To test this hypothesis, 1st larval stage (L1) C. elegans are exposed to 3 doses of 7.5J/m2 ultraviolet C radiation 24 hours apart, leading to the accumulation of mtDNA damage [9, 11]. After exposure, many mitochondrial endpoints are assessed at multiple time points later in life. mtDNA and nucDNA damage levels and genome copy numbers are measured via QPCR and real-time PCR , respectively, every 2 day for 10 days. Steady state ATP levels are measured via luciferase expressing reporter strains and traditional ATP extraction methods. Oxygen consumption is measured using a Seahorse XFe24 extra cellular flux analyzer. Gene expression changes are measured via real time PCR and targeted metabolomics via LC-MS are used to investigate changes in organic acid, amino acid and acyl-carnitine levels. Lastly, nematode developmental delay is assessed as growth, and measured via imaging and COPAS biosort.

I have found that despite being removed, UVC induced mtDNA damage during development leads to persistent deficits in energy production later in life. mtDNA copy number is permanently reduced, as are ATP levels, though oxygen consumption is increased, indicating inefficient or uncoupled respiration. Metabolomic data and mutant sensitivity indicate a role for NADPH and oxidative stress in these results, and exposed nematodes are more sensitive to the mitochondrial poison rotenone later in life. These results fit with the developmental origin of health and disease hypothesis, and show the potential for environmental exposures to have lasting effects on mitochondrial function.

Lastly, we are currently working to investigate the potential for irreparable mtDNA lesions to drive mutagenesis in mtDNA. Mutations in mtDNA lead to a wide range of diseases, yet we currently do not understand the environmental component of what causes them. In vitro evidence suggests that UVC induced thymine dimers can be mutagenic [12]. We are using duplex sequencing of C. elegans mtDNA to determine mutation rates in nematodes exposed to our serial UVC protocol. Furthermore, by including mutant strains deficient in mitochondrial fission and mitophagy, we hope to determine if deficiencies in these processes will further increase mtDNA mutation rates, as they are implicated in human diseases.