Adverse Events Due to Insomnia Drugs Reported in a Regulatory Database and Online Patient Reviews: Comparative Study.

BACKGROUND: Patient online drug reviews are a resource for other patients seeking information about the practical benefits and drawbacks of drug therapies. Patient reviews may also serve as a source of postmarketing safety data that are more user-friendly than regulatory databases. However, the reliability of online reviews has been questioned, because they do not undergo professional review and lack means of verification. OBJECTIVE: We evaluated online reviews of hypnotic medications, because they are commonly used and their therapeutic efficacy is particularly amenable to patient self-evaluation. Our primary objective was to compare the types and frequencies of adverse events reported to the Food and Drug Administration Adverse Event Reporting System (FAERS) with analogous information in patient reviews on the consumer health website Drugs.com. The secondary objectives were to describe patient reports of efficacy and adverse events and assess the influence of medication cost, effectiveness, and adverse events on user ratings of hypnotic medications. METHODS: Patient ratings and narratives were retrieved from 1407 reviews on Drugs.com between February 2007 and March 2018 for eszopiclone, ramelteon, suvorexant, zaleplon, and zolpidem. Reviews were coded to preferred terms in the Medical Dictionary for Regulatory Activities. These reviews were compared to 5916 cases in the FAERS database from January 2015 to September 2017. RESULTS: Similar adverse events were reported to both Drugs.com and FAERS. Both resources identified a lack of efficacy as a common complaint for all five drugs. Both resources revealed that amnesia commonly occurs with eszopiclone, zaleplon, and zolpidem, while nightmares commonly occur with suvorexant. Compared to FAERS, online reviews of zolpidem reported a much higher frequency of amnesia and partial sleep activities. User ratings were highest for zolpidem and lowest for suvorexant. Statistical analyses showed that patient ratings are influenced by considerations of efficacy and adverse events, while drug cost is unimportant. CONCLUSIONS: For hypnotic medications, online patient reviews and FAERS emphasized similar adverse events. Online reviewers rated drugs based on perception of efficacy and adverse events. We conclude that online patient reviews of hypnotics are a valid source that can supplement traditional adverse event reporting systems.

Using FluoZin-3 and fura-2 to monitor acute accumulation of free intracellular Cd2+ in a pancreatic beta cell line.

The understanding of cellular Cd2+ accumulation and toxicity is hampered by a lack of fluorescent indicators selective for intracellular free Cd2+ ([Cd2+]i). In this study, we used depolarized MIN6 mouse pancreatic beta cells as a model for evaluating [Cd2+]i detection with commercially available fluorescent probes, most of which have been traditionally used to visualize [Ca2+]i and [Zn2+]i. We trialed a panel of 12 probes including fura-2, FluoZin-3, Leadmium Green, Rhod-5N, indo-1, Fluo-5N, and others. We found that the [Zn2+]i probe FluoZin-3 and the traditional [Ca2+]i probe fura-2 responded most consistently and robustly to [Cd2+]i accumulation mediated by voltage-gated calcium channels. While selective detection of [Cd2+]i by fura-2 required the omission of Ca2+ from extracellular buffers, FluoZin-3 responded to [Cd2+]i similarly in the presence or absence of extracellular Ca2+. Furthermore, we showed that FluoZin-3 and fura-2 can be used together for simultaneous monitoring of [Ca2+]i and [Cd2+]i in the same cells. None of the other fluorophores tested were effective [Cd2+]i detectors in this model.

M3-subtype muscarinic receptor activation stimulates intracellular calcium oscillations and aldosterone production in human adrenocortical HAC15 cells.

A previous body of work in bovine and rodent models shows that cholinergic agonists modulate the secretion of steroid hormones from the adrenal cortex. In this study we used live-cell Ca2+ imaging to investigate cholinergic activity in the HAC15 human adrenocortical carcinoma cell line. The cholinergic agonists carbachol and acetylcholine triggered heterogeneous Ca2+ oscillations that were strongly inhibited by antagonists with high affinity for the M3 muscarinic receptor subtype, while preferential block of M1 or M2 receptors was less effective. Acute exposure to carbachol and acetylcholine modestly elevated aldosterone secretion in HAC15 cells, and this effect was also diminished by M3 inhibition. HAC15 cells expressed relatively high levels of mRNA for M3 and M2 receptors, while M1 and M5 mRNA were much lower. In conclusion, our data extend previous findings in non-human systems to implicate the M3 receptor as the dominant muscarinic receptor in the human adrenal cortex.

Fluorescence detection of intracellular cadmium with Leadmium Green.

Leadmium Green is a commercially available, small molecule, fluorescent probe advertised as a detector of free intracellular cadmium (Cd(2+)) and lead (Pb(2+)). Leadmium Green has been used in various paradigms, such as tracking Cd(2+) sequestration in plant cells, heavy metal export in protozoa, and Pb(2+) absorption by vascular endothelial cells. However very little information is available regarding its affinity and selectivity for Cd(2+), Pb(2+), and other metals. We evaluated the in vitro selectivity of Leadmium Green using spectrofluorimetry. Consistent with manufacturer's claims, Leadmium Green was sensitive to Cd(2+) (KD ~600 nM) and also Pb(2+) (KD ~9.0 nM) in a concentration-dependent manner, and furthermore proved insensitive to Ca(2+), Co(2+), Mn(2+) and Ni(2+). Leadmium Green also responded to Zn(2+) with a KD of ~82 nM. Using fluorescence microscopy, we evaluated Leadmium Green in live mouse hippocampal HT22 cells. We demonstrated that Leadmium Green detected ionophore-mediated acute elevations of Cd(2+) or Zn(2+) in a concentration-dependent manner. However, the maximum fluorescence produced by ionophore-delivered Zn(2+) was much less than that produced by Cd(2+). When tested in a model of oxidant-induced liberation of endogenous Zn(2+), Leadmium Green responded weakly. We conclude that Leadmium Green is an effective probe for monitoring intracellular Cd(2+), particularly in models where Cd(2+) accumulates rapidly, and when concomitant fluctuations of intracellular Zn(2+) are minimal.

A comparison of Zn2+- and Ca2+-triggered depolarization of liver mitochondria reveals no evidence of Zn2+-induced permeability transition.

Intracellular Zn(2+) toxicity is associated with mitochondrial dysfunction. Zn(2+) depolarizes mitochondria in assays using isolated organelles as well as cultured cells. Some reports suggest that Zn(2+)-induced depolarization results from the opening of the mitochondrial permeability transition pore (mPTP). For a more detailed analysis of this relationship, we compared Zn(2+)-induced depolarization with the effects of Ca(2+) in single isolated rat liver mitochondria monitored with the potentiometric probe rhodamine 123. Consistent with previous work, we found that relatively low levels of Ca(2+) caused rapid, complete and irreversible loss of mitochondrial membrane potential, an effect that was diminished by classic inhibitors of mPT, including high Mg(2+), ADP and cyclosporine A. Zn(2+) also depolarized mitochondria, but only at relatively high concentrations. Furthermore Zn(2+)-induced depolarization was slower, partial and sometimes reversible, and was not affected by inhibitors of mPT. We also compared the effects of Ca(2+) and Zn(2+) in a calcein-retention assay. Consistent with the well-documented ability of Ca(2+) to induce mPT, we found that it caused rapid and substantial loss of matrix calcein. In contrast, calcein remained in Zn(2+)-treated mitochondria. Considered together, our results suggest that Ca(2+) and Zn(2+) depolarize mitochondria by considerably different mechanisms, that opening of the mPTP is not a direct consequence of Zn(2+)-induced depolarization, and that Zn(2+) is not a particularly potent mitochondrial inhibitor.

The interaction of biological and noxious transition metals with the zinc probes FluoZin-3 and Newport Green.

Zinc-sensitive fluorescent probes have become increasingly important in the investigation of the cellular roles of zinc. There is, however, little information on how the other transition metals in cells may influence the measurement of zinc. We have characterized in vitro the interaction of the nominal zinc indicators FluoZin-3 and Newport Green with all the cationic transition metals found within cells, Cr, Mn, Fe, Co, and Cu, as well as Ni and Cd, by measuring their dissociation constants. In addition, we have shown how FluoZin-3 can be used to quantify the concentration of copper in a cell-free assay and report that the fluorescence of Newport Green is boosted by both Cu(I) and Fe(II). Furthermore, we have introduced diagnostics for detecting the interference of metals other than zinc with its measurement within cells.

Glutamate mobilizes [Zn2+] through Ca2+ -dependent reactive oxygen species accumulation.

Liberation of zinc from intracellular stores contributes to oxidant-induced neuronal injury. However, little is known regarding how endogenous oxidant systems regulate intracellular free zinc ([Zn(2+)](i)). Here we simultaneously imaged [Ca(2+)](i) and [Zn(2+)](i) to study acute [Zn(2+)](i) changes in cultured rat forebrain neurons after glutamate receptor activation. Neurons were loaded with fura-2FF and FluoZin-3 to follow [Ca(2+)](i) and [Zn(2+)](i), respectively. Neurons treated with glutamate (100 microM) for 10 min gave large Ca(2+) responses that did not recover after termination of the glutamate stimulus. Glutamate also increased [Zn(2+)](i), however glutamate-induced [Zn(2+)](i) changes were completely dependent on Ca(2+) entry, appeared to arise entirely from internal stores, and were substantially reduced by co-application of the membrane-permeant chelator TPEN during the glutamate treatment. Pharmacological maneuvers revealed that a number of endogenous oxidant producing systems, including nitric oxide synthase, phospholipase A(2), and mitochondria all contributed to glutamate-induced [Zn(2+)](i) changes. We found no evidence that mitochondria buffered [Zn(2+)](i) during acute glutamate receptor activation. We conclude that glutamate-induced [Zn(2+)](i) transients are caused in part by [Ca(2+)](i)-induced reactive oxygen species that arises from both cytosolic and mitochondrial sources.

Zinc causes loss of membrane potential and elevates reactive oxygen species in rat brain mitochondria.

Emerging evidence suggests that Zn2+ may impair neuronal metabolism. We examined how Zn2+ affects the activity of isolated brain mitochondria fueled with glutamate + malate, succinate or glycerol 3-phosphate. Submicromolar levels of Zn2+ dissipated membrane potential and inhibited oxygen utilization in all three substrate conditions. Zn(2+)-induced depolarization was reversed by the membrane-impermeant metal chelator, EGTA, and was inhibited by uniporter blockade. Cyclosporin A did not block Zn(2+)-induced depolarization. Added Zn2+ increased accumulation of reactive oxygen species (ROS) in glutamate + malate or glycerol 3-phosphate conditions, but inhibited succinate-supported ROS accumulation. These results show that Zn2+ blocks mitochondrial function in all physiologically relevant substrate conditions.

Direct visualization of mitochondrial zinc accumulation reveals uniporter-dependent and -independent transport mechanisms.

Current evidence suggests that zinc kills neurons by disrupting energy production, specifically by inhibiting mitochondrial function. However it is unclear if the inhibitory effect requires zinc accumulation, and if so, precisely how zinc enters mitochondria. Here, using fluorescence microscopy to visualize individual rat brain mitochondria, we detected matrix zinc uptake using the fluorophore FluoZin-3. Fluorescence increased rapidly in mitochondria treated with micromolar free zinc, and was quickly returned to baseline by membrane permeant chelation. Zinc uptake occurred through the calcium uniporter, because depolarization or uniporter blockade reduced fluorescence changes. However, increased fluorescence under these conditions suggests that zinc can enter through a uniporter-independent pathway. Fluorescence steadily declined over time and was unaffected by acidification or phosphate depletion, suggesting that zinc precipitation is not a mechanism for reducing matrix zinc. Uniporter blockade with ruthenium red also did not change the rate of zinc loss. Instead, zinc appears to exit the matrix through a novel efflux pathway not yet identified. Interestingly, dye-loaded mitochondria showed no fluorescence increase after treatment with strong oxidants, arguing against oxidant-labile intra-mitochondrial zinc pools. This study is the first to directly demonstrate zinc accumulation in individual mitochondria and provides insight about mechanisms mediating mitochondrial zinc uptake and efflux.

Simultaneous detection of intracellular free calcium and zinc using fura-2FF and FluoZin-3.

Elevation of intracellular free zinc ([Zn2+]i) probably contributes to cell death in injury paradigms involving calcium deregulation and oxidative stress such as glutamate excitotoxicity. However, it is difficult to monitor both ions simultaneously in live cells. Here we present a new method using fluorescence microscopy and the ion sensitive indicators fura-2FF and FluoZin-3 to monitor both [Ca2+]i and [Zn2+]i in primary cortical neurons. We show that the new single wavelength dye FluoZin-3 responds robustly to small zinc loads, is insensitive to high Ca2+ or Mg2+, and is relatively unaffected by low pH or oxidants. The ratiometric indicator fura-2FF is sensitive to both Ca2+ and Zn2+. However, in conditions analogous to excitotoxic glutamate exposure where [Ca2+]i is high relative to [Zn2+]i, we found that fura-2FF responds mostly to [Ca2+]i but is relatively unaffected by low [Zn2+]i. Moreover, fura-2FF ratio changes caused by high [Ca2+]i or high [Zn2+]i could be distinguished because each ion produces a different spectral response. Finally, dual dye experiments showed that FluoZin-3 and fura-2FF respond robustly to [Zn2+]i and [Ca2+]j, respectively, in the same neurons during intense glutamate exposure. These studies provide a novel method for the simultaneous detection of both calcium and zinc in cells.

Divergent consequences arise from metallothionein overexpression in astrocytes: zinc buffering and oxidant-induced zinc release.

Excessive accumulation of the heavy metal zinc is cytotoxic. As a consequence, cellular vulnerability to zinc-induced injury may be regulated by the abundance of proteins that maintain intracellular free zinc concentrations ([Zn2+]i). In this study, we overexpressed the zinc-binding protein metallothionein-II (MT) in astrocytes to assess its impact as (1) an acute zinc buffering mechanism, and (2) an oxidant-releasable zinc pool. Overexpression of MT in primary astrocyte cultures was accomplished using an adenoviral vector. Using the zinc-sensitive fluorescent indicator mag-fura-2, we monitored [Zn2+]i after stimulating zinc influx or oxidant treatment. With MT overexpression, we observed an acute buffering effect manifested as a dampening of stimulus-induced increases in [Zn2+]i. In contrast, we also saw enhanced zinc release with application of the sulfhydryl oxidizing agent 2,2'-dithiodipyridine. These results indicate that overexpression of a zinc-binding protein can quickly diminish [Zn2+]i following zinc influx, but elevate [Zn2+]i under conditions of oxidative stress, providing protective yet potentially endangering effects.

Zinc inhibition of cellular energy production: implications for mitochondria and neurodegeneration.

An increasing body of evidence suggests that high intracellular free zinc promotes neuronal death by inhibiting cellular energy production. A number of targets have been postulated, including complexes of the mitochondrial electron transport chain, components of the tricarboxylic acid cycle, and enzymes of glycolysis. Consequences of cellular zinc overload may include increased cellular reactive oxygen species (ROS) production, loss of mitochondrial membrane potential, and reduced cellular ATP levels. Additionally, zinc toxicity might involve zinc uptake by mitochondria and zinc induction of mitochondrial permeability transition. The present review discusses these processes with special emphasis on their potential involvement in brain injury.

A reevaluation of neuronal zinc measurements: artifacts associated with high intracellular dye concentration.

The emergence of zinc as a potent neurotoxin has prompted the development of techniques suitable for the measurement of intracellular free zinc ([Zn(2+)](i)) in cultured cells. Accordingly, a new family of Zn(2+)-sensitive fluorophores has become available. Using ionophore-induced elevations of [Zn(2+)](i) in cultured neurons, we measured [Zn(2+)](i)-induced changes in the novel dyes FuraZin-1 and FluoZin-2 and compared them with the established [Zn(2+)](i)-sensitive fluorophores mag-fura-2 and Newport Green. All of these dyes effectively detected [Zn(2+)](i), and FuraZin-1, FluoZin-2, and Newport Green showed selectivity for [Zn(2+)](i) over [Ca(2+)](i) and [Mg(2+)](i). However, the dyes showed little difference in their apparent sensitivity to [Zn(2+)](i), even though their in vitro affinities for Zn(2+) varied from 20 nM to 3 microM. We show herein that this is a consequence of the relatively high concentrations of intracellular dye used in experiments of this nature. Thus, for the measurement of [Zn(2+)](i), the sensitivity of the reporting system is dominated by the intracellular dye concentration, whereas dye affinity is unimportant. We extend these findings to show that calibration of dye signal to ion concentration is critically dependent on precise measurement of intracellular dye concentration.

The relationship between intracellular free iron and cell injury in cultured neurons, astrocytes, and oligodendrocytes.

Iron is an essential element for cells but may also be an important cytotoxin. However, very little is known about iron transport, redox status, or toxicity specifically inside cells. In this study, we exploited the sensitivity of fura-2 to quenching by ferrous iron (Fe(2+)) to detect intracellular free iron ([Fe(2+)](i)) in neurons, astrocytes, and oligodendrocytes in primary culture. All cell types exposed to Fe(2+) in the presence of the ionophore pyrithione rapidly accumulated Fe(2+) to a similar extent. The heavy-metal chelators bipyridyl and N,N,N',N'-tetrakis(2-pyridalmethyl)ethyl-enediamine rapidly reversed the increase in [Fe(2+)](i), whereas desferrioxamine had little effect. Interestingly, the Fe(2+)-mediated quenching of fura-2 fluorescence was reversed in a concentration-dependent manner by hydrogen peroxide. This was likely caused by the oxidation of Fe(2+) to Fe(3+) inside the cell. Acute exposure of cells to Fe(2+) was only toxic when the metal was applied together with pyrithione, showing that Fe(2+) is only toxic when elevated inside cells. Interestingly, only neurons and oligodendrocytes were injured by this elevation in [Fe(2+)](i), whereas astrocytes were unaffected, although [Fe(2+)](i) was elevated to the same degree in each cell type. These studies provide a novel approach for detecting [Fe(2+)](i) in a manner sensitive to the redox state of the metal. These studies also provide a model system for the study of the toxic consequences of elevated [Fe(2+)](i) in neural cells.