Claire E. Gavin

Mn2+ sequestration by mitochondria and inhibition of oxidative phosphorylation

Manganese is known to accumulate in mitochondria and in mitochondria-rich tissues in vivo. Although Ca2+ enhances mitochondrial Mn2+ uptake, ATP-bound Mn2+ is not sequestered by suspended rat brain mitochondria, and ATP binds Mn2+ even more tightly than it binds Mg2+. Physiological levels of the polyamine spermine enhanced 54 Mn2+ uptake at the low [Ca2+]s characteristic of unstimulated cells (approximately 100 nM). With succinate as substrate, Mn2+ inhibited oxygen consumption by suspensions of rat liver mitochondria after the addition of ADP but not after the addition of uncoupler. With glutamate/malate as substrate, Mn2+ inhibited ADP-stimulated respiration and also slightly inhibited uncoupler-stimulated respiration. State 4 (resting) respiration was unchanged in all cases, indicating that the inner membrane retained its impermeability to protons. These results suggest that Mn2+ was not oxidized and that it can interfere directly with oxidative phosphorylation, most likely by binding to the F1 ATPase. Mn2+ may also bind to the NADH dehydrogenase complex, but not strongly enough to affect electron transport in vivo. It is suggested that accumulation of manganese within the mitochondria of globus pallidus may help explain the distinctive pathology of manganism.

Manganese neurotoxicity and the role of reactive oxygen species

Manganese (Mn) is an essential dietary nutrient, but an excess or accumulation can be toxic. Disease states, such as manganism, are associated with overexposure or accumulation of Mn and are due to the production of reactive oxygen species, free radicals, and toxic metabolites; alteration of mitochondrial function and ATP production; and depletion of cellular antioxidant defense mechanisms. This review focuses on all of the preceding mechanisms and the scientific studies that support them as well as providing an overview of the absorption, distribution, and excretion of Mn and the stability and transport of Mn compounds in the body.

Determination of the oxidation states of manganese in brain, liver, and heart mitochondria

Excess brain manganese can produce toxicity with symptoms that resemble those of Parkinsonism and causes that remain elusive. Manganese accumulates in mitochondria, a major source of superoxide, which can oxidize Mn2+ to the powerful oxidizing agent Mn3+. Oxidation of important cell components by Mn3+ has been suggested as a cause of the toxic effects of manganese. Determining the oxidation states of intramitochondrial manganese could help to identify the dominant mechanism of manganese toxicity. Using X‐ray absorbance near edge structure (XANES) spectroscopy, we have characterized the oxidation state of manganese in mitochondria isolated from brain, liver, and heart over concentrations ranging from physiological to pathological. Results showed that (i) spectra from different model manganese complexes of the same oxidation state were similar to each other and different from those of other oxidation states and that the position of the absorption edge increases with oxidation state; (ii) spectra from intramitochondrial manganese in isolated brain, heart and liver mitochondria were virtually identical; and (iii) under these conditions intramitochondrial manganese exists primarily as a combination of Mn2+ complexes. No evidence for Mn3+ was detected in samples containing more than endogenous manganese levels, even after incubation under conditions promoting reactive oxygen species (ROS) production. While the presence of Mn3+ complexes cannot be proven in the spectrum of endogenous mitochondrial manganese, the shape of this spectrum could suggest the presence of Mn3+ near the limit of detection, probably as MnSOD.

An analysis of the effects of Mn2+ on oxidative phosphorylation in liver, brain, and heart mitochondria using state 3 oxidation rate assays

Manganese (Mn) toxicity is partially mediated by reduced ATP production. We have used oxidation rate assays--a measure of ATP production--under rapid phosphorylation conditions to explore sites of Mn(2+) inhibition of ATP production in isolated liver, brain, and heart mitochondria. This approach has several advantages. First, the target tissue for Mn toxicity in the basal ganglia is energetically active and should be studied under rapid phosphorylation conditions. Second, Mn may inhibit metabolic steps which do not affect ATP production rate. This approach allows identification of inhibitions that decrease this rate. Third, mitochondria from different tissues contain different amounts of the components of the metabolic pathways potentially resulting in different patterns of ATP inhibition. Our results indicate that Mn(2+) inhibits ATP production with very different patterns in liver, brain, and heart mitochondria. The primary Mn(2+) inhibition site in liver and heart mitochondria, but not in brain mitochondria, is the F₁F₀ ATP synthase. In mitochondria fueled by either succinate or glutamate+malate, ATP production is much more strongly inhibited in brain than in liver or heart mitochondria; moreover, Mn(2+) inhibits two independent sites in brain mitochondria. The primary site of Mn-induced inhibition of ATP production in brain mitochondria when succinate is substrate is either fumarase or complex II, while the likely site of the primary inhibition when glutamate plus malate are the substrates is either the glutamate/aspartate exchanger or aspartate aminotransferase.

The case for manganese interaction with mitochondria

In “Manganese accumulates primarily in nuclei of cultured brain cells”, Kalia et al (Kalia et al., 2008) report that upon fractionation of neuronal cells, labeled Mn was found primarily in nuclei, with virtually none in the mitochondrial fraction and therefore, that mitochondria may play an insignificant role in subcellular Mn distribution. They also say that “there has been no direct evidence --- on subcellular distribution of Mn,” and that the recent report by Morello et al (Morello et al., 2008) concluded that nuclear components may represent the “preferential targets for Mn accumulation and toxicity” (Kalia et al., 2008). In fact, a number of other studies (Ayotte and Plaa, 1985; Lai et al., 1999; Liccione and Maines, 1988; Maynard and Cotzias, 1955; Miller et al., 1975) have determined the subcellular distribution of Mn after tissue fractionation following treatment with Mn in vivo, and all demonstrated treatment-related increases in intramitochondrial Mn. In addition, other workers have found that Mn decreases energy metabolism in vivo and in vitro, including decreases in the activities of mitochondrial enzymes, in membrane potential, and ATP production (Brouillet et al., 1993; Du et al., 1997; Galvani et al., 1995; Gavin et al., 1992; Malecki, 2001; Malthankar et al., 2004; Roth et al., 2000; Roth et al., 2002; Wolters et al., 1989; Zwingmann et al., 2003). Using electron energy-loss spectroscopy with electron microscopy, Morello et al (Morello et al., 2008) reported that although nuclei contained more Mn than mitochondria, treatment-related increases were greater in the mitochondria. They concluded that “the relevant distribution of Mn is not limited to the mitochondria.” Consideration of why the results of Kalia et al differ from those of other distribution studies requires an understanding of mitochondrial ion transport and its response to fractionation. Mitochondrial sequestration of Ca2+ or Mn2+ does not represent simple binding but weak binding within a steady state controlled by both influx and efflux of these ions (Gunter and Pfeiffer, 1990). Mn2+ is sequestered by the mitochondrial Ca2+ uniporter, primarily energized by the internally negative membrane potential (ΔΨ), and effluxed by the Na+-independent mechanism, primarily energized by the pH gradient (Gavin et al., 1990; Gunter and Pfeiffer, 1990; Gunter and Sheu, 2008). Both are maintained by energy-dependent proton pumping across an intact inner membrane. If ΔΨ falls, uptake velocity decreases precipitously – by over 83% as ΔΨ falls from 180 to 160 mV, for example (Gunter and Sheu, 2008). If ΔΨ falls near zero, the weakly bound ions rush out by reverse uniport (Gunter et al., 1975; Gunter et al., 1978) The “isolated mitochondria” produced by fractionation, whether by mechanical action or by detergents, represent resealed fragments of the original cellular mitochondrial network (Gunter and Sheu, 2008; Chan, 2006). In these resealed mitochondria, ΔΨ is dissipated during fractionation, then rebuilt by proton pumping energized by endogenous substrate -- e.g., pyruvate, a product of glycolysis that in the intact cell is transported continually into mitochondria for use in the TCA cycle. However, the fractionation procedure greatly dilutes glycolytic enzymes and substrates, and the amount of endogenous substrate within isolated, resealed mitochondria is greatly reduced and no longer replenished. It has been well known since the 1960’s that appreciable Ca2+ or Mn2+ uptake by these resealed mitochondria requires addition of mitochondrial substrate; however, none was added in the fractionation studies cited above. Why, then, did all except Kalia et al find Mn in the mitochondria? Maynard and Cotzias (1955) stressed that they treated animals with less Mn than that present in the food. Other ex vivo Mn distribution studies (Lai et al., 1999; Liccione and Maines, 1988) examined Mn concentrations in brain fractions; since brain Mn uptake is limited both by the blood-brain barrier and by rigorous homeostatic control of absorption and excretion, the amounts reaching mitochondria were probably not large. In contrast, Kalia et al exposed their cells for 24 hours to 100 μM Mn2+. Based on the uptake that we measured in PC12 cells at 100 μM for 24 hours (11.7 nmoles/mg cell protein) (Gunter et al., 2005), we estimate that the mitochondria of Kalia et al were exposed to [Mn2+]s over 100 times higher than those of Maynard and Cotzias and much higher than in the other fractionation studies. Following fractionation, the resealed mitochondria would begin to resequester and cycle the surrounding Mn2+, using energy from endogenous substrate. However, in the presence of large amounts of Mn2+, as in the experiments of Kalia et al, Mn cycling would quickly dissipate the endogenous substrate, ΔΨ would fall, and the Mn2+ would be released again from the mitochondria to bind to available sites such as nuclei. (For example, Mn2+ binds to DNA with an affinity of about 33 μM (Kennedy and Bryant, 1986)). In the presence of small amounts of Mn2+, as with the other fractionation studies, ion cycling would dissipate the available energy much more slowly, and the mitochondria would likely retain measurable amounts of Mn. While conducting earlier experiments (Gunter et al., 2004), we determined the medium [Mn2+] to which isolated mitochondria would pump varying amounts of added Mn2+ (Fig. 1). Notice that if 2μM Mn2+ is added, the mitochondria will pump the external [Mn2+] to around 80 nM, while if 142 μM Mn2+ is added, the mitochondria will only pump the external [Mn2+] to around 3.4 μM. This is because the more extensive Mn2+ cycling in the latter case lowers ΔΨ, shifting the steady state toward less uptake. These concentrations represent the levels at which energized mitochondria compete with other cellular binding sites, such as those in the nucleus. These observations suggest that mitochondrial substrates should be added in fractionation studies to minimize redistribution of Mn2+. Figure 1 Concentration of free Mn2+ in the medium in the presence of several preparations of energized mitochondria (4 mg/ml) as a function of the total concentration of Mn2+ added. 160 μM Ca2+ was also added to the data indicated by the filled square ...

XANES spectroscopy: a promising tool for toxicology: a tutorial.

X-ray absorption near edge structure (XANES) spectroscopy can provide information on the oxidation state of metal ions within a biological sample and also the complexes in which it is found. This type of information could be of great use to toxicologists in understanding the mechanism of action of many toxic agents. The prospect of using a sophisticated physical technique such as XANES may be somewhat intimidating for those without a strong physical background. Here, we explain the concepts necessary to understand XANES spectroscopy at a level that can be easily understood by biological scientists without a strong physics background and describe useful sample preparation and data analysis techniques which can be adapted for a variety of applications. Examples are taken from an ongoing study of manganese in brain mitochondria and neuron-like cells.

Human trophoblast cultures: Models for implantation and peri-implantation toxicology

Implantation is the process that leads from blastocyst attachment to its embedding in the uterine wall. It is widely believed that failure of implantation is a common cause of pregnancy loss. Toxic agents can interfere directly with the process of implantation and therefore may account for unexplained implantation failures. Our knowledge of human implantation remains limited, mainly due to the lack of adequate experimental models. Studies of mechanisms underlying implantation in humans are by nature and for ethical reasons restricted to in vitro models. The aim of this review is to provide a critical evaluation of various in vitro models of implantation in humans, as well as essential background knowledge required for application of these models to the assessment of peri-implantation toxicity. Particular attention has been devoted to cell-cell and cell-matrix interactions as possible endpoints in the screening of toxic agents.

Mn2+ transport across biological membranes may be monitored spectroscopically using the Ca2+ indicator dye antipyrylazo III

The metallochromic indicator antipyrylazo III can be used for the rapid and convenient monitoring of Mn2+ transport in biological systems. The apparent KD of the Mn-antipyrylazo III complex in buffered 150 mM KCl (pH 7.2 at 20 degrees C) is approximately 2.5 x 10(-5) M. The sensitivity of antipyrylazo III to Mn2+ is comparable to that of arsenazo III to Ca2+. Mn2+ can be measured without interference from Ca2+, by using dual-wavelength spectrophotometry at the wavelength pair 510-590 nm, or 530-565 nm in cell or mitochondrial suspensions. Ca2+ can be monitored at the wavelength pair 720-790 nm without interference from Mn2+. This paper represents the first application of this technique, here used to characterize mitochondrial efflux kinetics of Mn2+. We report that Mn2+ is transported out of liver mitochondria with a Vmax of 1-2 nmol/(mg.min) and a Km of about 12 nmol/mg. These results are in close agreement with results of measurements using 54Mn.

The Role of Mitochondrial Oxidative Stress and ATP Depletion in the Pathology of Manganese Toxicity

The mitochondrial electron transport chain (ETC) creates most of the reactive oxygen species (ROS) produced by the average eukaryotic cell. These ROS, primarily superoxide radical, hydrogen peroxide, and hydroxyl radical, can damage the proteins, phospholipids, and nucleic acids of the mitochondria and the entire cell, though they also play important roles in cell signaling. Addition of ions such as Ca2+ or Mn2+ to mitochondria is known to increase ROS production. Excessive manganese (Mn) uptake by the brain, particularly by the globus pallidus and striatum, can cause signs and symptoms somewhat similar to those of Parkinson’s disease (PD); however, unlike PD, manganese toxicity, or manganism, is characterized pathologically by apoptotic cell death in the globus pallidus (GP). Initial effects of excessive brain Mn2+ include inhibition of ATP production and increased generation of mitochondrial ROS. We do not know the exact pathways that lead from these initial insults to the death of GP cells. However, ATP inhibition and increased ROS have been shown to contribute to the activation of apoptotic processes. ROS increase the probability of induction of the mitochondrial permeability transition (MPT), which causes rapid swelling of the mitochondrial matrix, tearing or leakage of the mitochondrial outer membrane, and release of factors from the mitochondrial intermembrane space which activate apoptosis. It is likely that treatments which ameliorate mitochondrial ROS damage will prove beneficial in minimizing the signs and symptoms of Mn toxicity.