David P. Cox

Hydroxyl radical is produced via the Fenton reaction in submitochondrial particles under oxidative stress: implications for diseases associated with iron accumulation

Abstract Mitochondrial dysfunction and reactive oxygen species (ROS) are often implicated in diseases involving oxidative stress and elevated iron. As mitochondria produce ATP by oxidative phosphorylation, ROS by-products are generated from the electron transport chain. Although superoxide and hydrogen peroxide have been thoroughly investigated, little evidence documents hydroxyl radical (HO•) production in mitochondria. In order to determine whether HO• is generated under oxidative stress conditions by a Fenton-type mechanism, bovine heart submitochondrial particles were examined for HO• in the presence and absence of iron ligands, antioxidant enzymes and HO• scavengers. HO• was measured as 2,3- and 2,5-dihydroxybenzoic acid (DHBA), using HPLC with electrochemical detection. The iron ligand desferrioxamine significantly decreased DHBAs, indicating that HO• generation required iron redox-cycling. In addition, results from exogenous SOD and catalase, exogenous hydrogen peroxide, and HO•-scavenger studies support a Fenton-type reaction mechanism. The results indicate that increased HO• levels occur in mitochondria under oxidative stress and that the HO• levels can be modulated with antioxidant enzymes and iron ligands. Our findings together with reports on iron accumulation in degenerative diseases highlight the importance of developing mitochondrial-targeted antioxidants for the therapeutic intervention of diseases associated with mitochondrial dysfunction and oxidative stress.

Distinct Nrf1/2-independent mechanisms mediate As3+-induced glutamate-cysteine ligase subunit gene expression in murine hepatocytes

Trivalent arsenite (As(3+)) is a known human carcinogen that is also capable of inducing apoptotic cell death. Increased production of reactive oxygen species is thought to contribute to both the carcinogenic and the cytotoxic effects of As(3+). Glutathione (GSH) constitutes a vital cellular defense mechanism against oxidative stress. The rate-limiting enzyme in GSH biosynthesis is glutamate-cysteine ligase (GCL), a heterodimeric holoenzyme composed of a catalytic (GCLC) and a modifier (GCLM) subunit. In this study, we demonstrate that As(3+) coordinately upregulates Gclc and Gclm mRNA levels in a murine hepatocyte cell line resulting in increased GCL subunit protein expression, holoenzyme formation, and activity. As(3+) increased the rate of transcription of both the Gclm and the Gclc genes and induced the posttranscriptional stabilization of Gclm mRNA. The antioxidant N-acetylcysteine abolished As(3+)-induced Gclc expression and attenuated induction of Gclm. As(3+) induction of Gclc and Gclm was also differentially regulated by the MAPK signaling pathways and occurred independent of the Nrf1/2 transcription factors. These findings demonstrate that distinct transcriptional and posttranscriptional mechanisms mediate the coordinate induction of the Gclc and Gclm subunits of GCL in response to As(3+) and highlight the potential importance of the GSH antioxidant defense system in regulating As(3+)-induced responses in hepatocytes.

Gain and Loss of Function for Glutathione Synthesis: Impact on Advanced Atherosclerosis in Apolipoprotein E–Deficient Mice

Objective—Glutamate-cysteine ligase (GCL) is the rate-limiting step in glutathione synthesis. The enzyme is a heterodimer composed of a catalytic subunit, GCLC, and a modifier subunit, GCLM. We generated apolipoprotein E (apoE)−/− mice deficient in GCLM (apoE−/−/Gclm−/−) and transgenic mice that overexpress GCLC specifically in macrophages (apoE−/−/Gclc-Tg) to test the hypothesis that significantly altering the availability of glutathione has a measurable impact on both the initiation and progression of atherosclerosis. Methods and Results—Atherosclerotic plaque size and composition were measured in the innominate artery in chow-fed male and female mice at 20, 30, 40, and 50 weeks of age and in the aortic sinus at 40 and 50 weeks of age. The apoE−/−/Gclm−/− mice more rapidly developed complex lesions, whereas the apoE−/−/Gclc-Tg mice had reduced lesion development compared with the littermate apoE−/− control mice. Transplantation of bone marrow from the apoE−/−/Gclm−/− and apoE−/−/Gclc-Tg mice into apoE−/− mice with established lesions also stimulated or inhibited further lesion development at 30 weeks posttransplant. Conclusion—Gain and loss of function in the capacity to synthesize glutathione especially in macrophages has reciprocal effects on the initiation and progression of atherosclerosis at multiple sites in apoE−/− mice.

Heme oxygenase expression as a biomarker of exposure to amphiphilic polymer-coated CdSe/ZnS quantum dots.

Abstract Because of their unique optical properties, quantum dots (QDs) have become a preferred system for ultrasensitive detection and imaging. However, since QDs commonly contain Cd and other heavy metals, concerns have been raised regarding their toxicity. QDs are thus commonly synthesised with a ZnS cap structure and/or coated with polymeric stabilisers. We recently synthesised amphiphilic polymer-coated tri-n-octylphosphine oxide - poly(maleic anhydride-alt-1-tetradecene (TOPO-PMAT) QDs, which are highly stable in aqueous environments. The effects of these QDs on viability and stress response in five cell lines of mouse and human origins are reported here. Human and mouse macrophages and human kidney cells readily internalised these QDs, resulting in modest toxicity. TOPO-PMAT QD exposure was highly correlated with the induction of the stress response protein heme oxygenase-1 (HMOX1). Other stress biomarkers (glutamate cysteine ligase modifier subunit, NAD(P)H, necrosis) were only moderately affected. HMOX1 may thus be a useful biomarker of TOPO-QDOT QD exposure across cell types and species.

Glutathione (GSH) and the GSH synthesis gene Gclm modulate plasma redox and vascular responses to acute diesel exhaust inhalation in mice

Abstract Context: Inhalation of fine particulate matter (PM2.5) is associated with acute pulmonary inflammation and impairments in cardiovascular function. In many regions, PM2.5 is largely derived from diesel exhaust (DE), and these pathophysiological effects may be due in part to oxidative stress resulting from DE inhalation. The antioxidant glutathione (GSH) is important in limiting oxidative stress-induced vascular dysfunction. The rate-limiting enzyme in GSH synthesis is glutamate cysteine ligase and polymorphisms in its catalytic and modifier subunits (GCLC and GCLM) have been shown to influence vascular function and risk of myocardial infarction in humans. Objective: We hypothesized that compromised de novo synthesis of GSH in Gclm−/+ mice would result in increased sensitivity to DE-induced lung inflammation and vascular effects. Materials and methods: WT and Gclm−/+ mice were exposed to DE via inhalation (300 μg/m3) for 6 h. Neutrophil influx into the lungs, plasma GSH redox potential, vascular reactivity of aortic rings and aortic nitric oxide (NO•) were measured. Results: DE inhalation resulted in mild bronchoalveolar neutrophil influx in both genotypes. DE-induced effects on plasma GSH oxidation and acetylcholine (ACh)-relaxation of aortic rings were only observed in Gclm−/+ mice. Contrary to our hypothesis, DE exposure enhanced ACh-induced relaxation of aortic rings in Gclm−/+ mice. Discussion and conclusion: These data support the hypothesis that genetic determinants of antioxidant capacity influence the biological effects of acute inhalation of DE. However, the acute effects of DE on the vasculature may be dependent on the location and types of vessels involved. Polymorphisms in GSH synthesis genes are common in humans and further investigations into these potential gene-environment interactions are warranted.

Fluorescent assessment of intracellular calcium in individual cells.

Calcium is an important intracellular ion involved in numerous cell processes. There are multiple factors that contribute to the release of Ca²⁺. Some factors induce release as part of intracellular signaling cascades, while others result in unwanted changes to both basal and inducible Ca²⁺ levels. The accurate measurement of intracellular Ca²⁺ is, therefore, an important tool in neurotoxicology for assessing compounds/substances that disrupt Ca²⁺ homeostasis. Fluorescent, Ca²⁺-sensitive probes Indo-1 and Fluo-3 allow for the quantification of intracellular Ca²⁺ in individual cells to determine what effects neurotoxins have on both basal and stimulus-dependent Ca²⁺ concentrations.