Derek A. Drechsel

Mitochondria Are a Major Source of Paraquat-induced Reactive Oxygen Species Production in the Brain

Paraquat (PQ2+) is a prototypic toxin known to exert injurious effects through oxidative stress and bears a structural similarity to the Parkinson disease toxicant, 1-methyl-4-pheynlpyridinium. The cellular sources of PQ2+-induced reactive oxygen species (ROS) production, specifically in neuronal tissue, remain to be identified. The goal of this study was to determine the involvement of brain mitochondria in PQ2+-induced ROS production. Highly purified rat brain mitochondria were obtained using a Percoll density gradient method. PQ2+-induced hydrogen peroxide (H2O2) production was measured by fluorometric and polarographic methods. The production of H2O2 was evaluated in the presence of inhibitors and modulators of the mitochondrial respiratory chain. The results presented here suggest that in the rat brain, (a) mitochondria are a principal cellular site of PQ2+-induced H2O2 production, (b) PQ2+-induced H2O2 production requires the presence of respiratory substrates, (c) complex III of the electron transport chain is centrally involved in H2O2 production by PQ2+, and (d) the mechanism by which PQ2+ generates H2O2 depends on the mitochondrial inner transmembrane potential. These observations were further confirmed by measuring PQ2+-induced H2O2 production in primary neuronal cells derived from the midbrain. These findings shed light on the mechanism through which mitochondria may contribute to ROS production by other environmental and endogenous redox cycling agents implicated in Parkinsons disease.

Role of reactive oxygen species in the neurotoxicity of environmental agents implicated in Parkinson's disease

Among age-related neurodegenerative diseases, Parkinsons disease (PD) represents the best example for which oxidative stress has been strongly implicated. The etiology of PD remains unknown, yet recent epidemiological studies have linked exposure to environmental agents, including pesticides, with an increased risk of developing the disease. As a result, the environmental hypothesis of PD has developed, which speculates that chemical agents in the environment are capable of producing selective dopaminergic cell death, thus contributing to disease development. The use of environmental agents such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, rotenone, paraquat, dieldrin, and maneb in toxicant-based models of PD has become increasingly popular and provided valuable insight into the neurodegenerative process. Understanding the unique and shared mechanisms by which these environmental agents act as selective dopaminergic toxicants is critical in identifying pathways involved in PD pathogenesis. In this review, we discuss the neurotoxic properties of these compounds with specific focus on the induction of oxidative stress. We highlight landmark studies along with recent advances that support the role of reactive oxygen and reactive nitrogen species from a variety of cellular sources as potent contributors to the neurotoxicity of these environmental agents. Finally, human risk and the implications of these studies in our understanding of PD-related neurodegeneration are discussed.

Respiration-dependent H2O2 Removal in Brain Mitochondria via the Thioredoxin/Peroxiredoxin System

Mitochondrial reactive oxygen species (ROS) play an important role in both physiological cell signaling processes and numerous pathological states, including neurodegenerative disorders such as Parkinson disease. While mitochondria are considered the major cellular source of ROS, their role in ROS removal remains largely unknown. Using polarographic methods for real-time detection of steady-state H2O2 levels, we were able to quantitatively measure the contributions of potential systems toward H2O2 removal by brain mitochondria. Isolated rat brain mitochondria showed significant rates of exogenous H2O2 removal (9–12 nmol/min/mg of protein) in the presence of substrates, indicating a respiration-dependent process. Glutathione systems showed only minimal contributions: 25% decrease with glutathione reductase inhibition and no effect by glutathione peroxidase inhibition. In contrast, inhibitors of thioredoxin reductase, including auranofin and 1-chloro-2,4-dinitrobenzene, attenuated H2O2 removal rates in mitochondria by 80%. Furthermore, a 50% decrease in H2O2 removal was observed following oxidation of peroxiredoxin. Differential oxidation of glutathione or thioredoxin proteins by copper (II) or arsenite, respectively, provided further support for the thioredoxin/peroxiredoxin system as the major contributor to mitochondrial H2O2 removal. Inhibition of the thioredoxin system exacerbated mitochondrial H2O2 production by the redox cycling agent, paraquat. Additionally, decreases in H2O2 removal were observed in intact dopaminergic neurons with thioredoxin reductase inhibition, implicating this mechanism in whole cell systems. Therefore, in addition to their recognized role in ROS production, mitochondria also remove ROS. These findings implicate respiration- and thioredoxin-dependent ROS removal as a potentially important mitochondrial function that may contribute to physiological and pathological processes in the brain.

Differential Contribution of the Mitochondrial Respiratory Chain Complexes to Reactive Oxygen Species Production by Redox Cycling Agents Implicated in Parkinsonism

Exposure to environmental pesticides can cause significant brain damage and has been linked with an increased risk of developing neurodegenerative disorders, including Parkinsons disease. Bipyridyl herbicides, such as paraquat (PQ), diquat (DQ), and benzyl viologen (BV), are redox cycling agents known to exert cellular damage through the production of reactive oxygen species (ROS). We examined the involvement of the mitochondrial respiratory chain in ROS production by bipyridyl herbicides. In isolated rat brain mitochondria, H2O2 production occurred with the following order of potency: BV > DQ > PQ in accordance with their measured ability to redox cycle. H2O2 production was significantly attenuated in all cases by antimycin A, an inhibitor of complex III. Interestingly, at micromolar (< or = 300 microM) concentrations, PQ-induced H2O2 production was unaffected by complex I inhibition via rotenone, whereas DQ-induced H2O2 production was equally attenuated by inhibition of complex I or III. Moreover, complex I inhibition decreased BV-induced H2O2 production to a greater extent than with PQ or DQ. These data suggest that multiple sites within the respiratory chain contribute to H2O2 production by redox cycling bipyridyl herbicides. In primary midbrain cultures, H2O2 differed slightly with the following order of potency: DQ > BV > PQ. In this model, inhibition of complex III resulted in roughly equivalent inhibition of H2O2 production with all three compounds. These data identify a novel role for complex III dependence of mitochondrial ROS production by redox cycling herbicides, while emphasizing the importance of identifying mitochondrial mechanisms by which environmental agents generate oxidative stress contributing to parkinsonism.

Inhibition of Mitochondrial Hydrogen Peroxide Production by Lipophilic Metalloporphyrins

Many studies have established a role for oxidative stress and mitochondrial dysfunction as an important mechanism in the pathogenesis of neuronal disorders. Metalloporphyrins are a class of catalytic antioxidants that are capable of detoxifying a wide range of reactive oxygen species. The AEOL112 series of glyoxylate metalloporphyrins were designed with increased lipid solubility for better oral bioavailability and penetration of the blood-brain barrier. The goal of this study was to develop an in vitro assay using rat brain mitochondria to reliably detect endogenously released hydrogen peroxide (H2O2) and identify glyoxylate metalloporphyrins based on rank order of potency for removal of physiologically relevant H2O2. A polarographic method was established for the sensitive, accurate, and reproducible detection of low levels of H2O2. The assay identified several potent glyoxylate metalloporphyrins with H2O2 scavenging potencies (IC50) in the nanomolar range. These results provide a simplified in vitro model system to detect physiologically generated mitochondrial H2O2 as a screening tool to predict the biological efficacy of potential therapeutic entities.

Chapter 21 Paraquat-induced production of reactive oxygen species in brain mitochondria.

Paraquat (PQ) is a prototypical redox cycling agent commonly used experimentally to generate reactive oxygen species and oxidative stress. Recently, PQ has also come under investigation as a potential environmental neurotoxin associated with increased risk for neurodegenerative disease developing after chronic exposure. The interactions of PQ with mitochondria remain an important aspect of its toxicity, particularly in the brain, although the underlying mechanisms are relatively uncharacterized. Here, we describe the basic measurement of PQ-induced hydrogen peroxide (H(2)O(2)) production in isolated brain mitochondria by use of two independent methods, polarography and fluorometry. The advantages of the use of these two independent methods include the capability to validate results and overcoming limitations in the use of either method exclusively. These simplified in vitro techniques for measurement of mitochondrial-generated H(2)O(2) can be easily applied to other tissues and models.

Brain Mitochondrial Drug Delivery: Influence of Drug Physicochemical Properties

ABSTRACTPurposeTo determine the influence of drug physicochemical properties on brain mitochondrial delivery of 20 drugs at physiological pH.MethodsThe delivery of 8 cationic drugs (beta-blockers), 6 neutral drugs (corticosteroids), and 6 anionic drugs (non-steroidal anti-inflammatory drugs, NSAIDs) to isolated rat brain mitochondria was determined with and without membrane depolarization. Multiple linear regression was used to determine whether lipophilicity (Log D), charge, polarizability, polar surface area (PSA), and molecular weight influence mitochondrial delivery.ResultsThe Log D for beta-blockers, corticosteroids, and NSAIDs was in the range of −1.41 to 1.37, 0.72 to 2.97, and −0.98 to 2, respectively. The % mitochondrial uptake increased exponentially with an increase in Log D for each class of drugs, with the uptake at a given lipophilicity obeying the rank order cationic>anionic>neutral. Valinomycin reduced membrane potential and the delivery of positively charged propranolol and betaxolol. The best equation for the combined data set was Log % Uptake = 0.333 Log D + 0.157 Charge – 0.887 Log PSA + 2.032 (R2 = 0.738).ConclusionsDrug lipopohilicity, charge, and polar surface area and membrane potential influence mitochondrial drug delivery, with the uptake of positively charged, lipophilic molecules being the most efficient.

Transmission of mutant phenotypes from ES cells to adult mice

Genetic manipulation of embryonic stem (ES) cells has been used to produce genetically engineered mice modeling human disorders. Here we describe a novel, additional application: selection for a phenotype of interest and subsequent transmission of that phenotype to a living mouse. We show, for the first time, that a cellular phenotype induced by ENU mutagenesis in ES cells can be transmitted and recapitulated in adult mice derived from these cells. We selected for paraquat-resistant (PQR) ES clones. Subsequent injection of these cells into blastocysts resulted in the production of germline chimeras, from which tail skin fibroblasts exhibited enhanced PQR. This trait was also recovered in progeny of the chimera. We avoided PQ toxicity, which blocks the ability to involve the germline, by developing a sib-selection method, one that could be widely applied wherever the selection itself might diminish the pluripotency of the ES cells. Thus, phenotype-driven screens in ES cells are both feasible and efficient in producing intact mouse models for in vivo studies.