Journal of Hepatology
Volume 40, Issue 4 , Pages 696-698, April 2004

Rusty notions of cell injury

Department of Cell and Developmental Biology, School of Medicine, University of North Carolina, CB 7090, 236 Taylor Hall, Chapel Hill, NC 27599-7090, USA

See Article, pages 607–615

Article Outline

 

In this issue of the Journal Rauen and co-workers from the University Hospital in Essen characterize the cellular mechanisms underlying iron toxicity to cultured rat hepatocytes [1]. Although an essential nutrient, iron is an old and well characterized human toxicant causing acute hepatocellular necrosis after accidental overdose of dietary iron supplements and chronic hepatic injury in hereditary hemochromatosis, a genetic disease of excess iron uptake. The article by Rauen et al. implicates a broader role for iron in pathophysiology.

Free or ‘chelatable’ intracellular iron concentrations in biological tissues and fluids are generally believed to be extremely small, in part because the transition metal is such an active partner in redox reactions generating toxic free radicals. Previously, the Essen group developed evidence that an increase of intracellular chelatable iron was a key factor leading to oxidative stress and death of sinusoidal endothelial cells and hepatocytes after cold ischemia and reperfusion [2]. Indeed, iron has been implicated in a variety of diseases, including diabetes, cancer, cardiovascular disease and alcoholic liver disease [3], but the mechanistic basis of iron-mediated toxicity has never been well characterized. Here, Rauen et al. examined directly the cytotoxicity of intracellular chelatable iron to rat hepatocytes.

Possibly because of its toxicity, hepatocytes take up free iron poorly. Accordingly, ferric iron (Fe3+) was loaded as a membrane permeable complex with 8-hydroxyquinoline. The consequence was rapid cell killing that increased from about 10% over control after 2 h to about 60% after 4 h. Although apoptosis is more fashionable to study, the character of killing after iron treatment was predominantly oncosis (oncotic necrosis). Features of oncotic necrosis included cell swelling, formation of large clear blebs that rarely contained organelles and ATP depletion. Typical features of apoptosis were largely absent, including cell shrinkage, nuclear fragmentation, and internucleosomal DNA cleavage. Evidence presented that neither fructose nor glycine protected against iron-induced cell killing might argue against oncotic necrosis. However, fructose as an ATP-generating glycolytic substrate may not protect if mitochondrial injury causes uncoupling, as likely occurs after iron treatment [4], [5], [6]. Moreover, iron caused oxidative stress, and glycine is reported not to protect against oxidative stress-induced cytotoxicity to hepatocytes [7]. Nonetheless, some apoptosis occurred as evidenced by a few cells with characteristic condensed, lobulated and fragmented nuclei. Such observations illustrate how shared pathways initiate both necrosis and apoptosis, a phenomenon that has been called ‘necrapoptosis’ [8]. Cell killing with features of both necrosis and apoptosis is especially prominent after toxicant and hypoxia-induced injuries.

The novel finding by Rauen et al. is that intracellular loading with chelatable iron quickly induces onset of the mitochondrial permeability transition (MPT). Opening of high conductance permeability transition (PT) pores causes the MPT [9]. PT pores non-specifically conduct solutes of molecular mass less than about 2000Da, and PT pore opening quickly causes mitochondrial depolarization, uncoupling of oxidative phosphorylation and large amplitude colloid osmotic swelling of the inner membrane-matrix compartment. Uncoupling of oxidative phosphorylation leads to cellular ATP depletion and a pattern of necrotic cell death, whereas mitochondrial swelling causes outer membrane release and release of cytochrome c and other proapoptotic factors. If ATP depletion-dependent necrosis can be prevented by an alternative glycolytic source of ATP, then cytochrome c release promotes caspase-dependent apoptosis instead [10], [11]. Both necrosis and apoptosis under such conditions are blocked by MPT inhibitors like cyclosporin A and trifluoperazine.

Using confocal fluorescence microscopy, Rauen et al. documented directly intracellular onset of the MPT by the redistribution of normally impermeant calcein into the mitochondrial matrix space and the simultaneous release of the potential-indicating fluorophore, TMRM. Moreover, trifluoperazine and cyclosporin A decreased iron-induced cell killing and blocked mitochondrial depolarization. Thus, induction by iron of the MPT was a key mechanism in the killing of hepatocytes by chelatable iron.

Oxidative stress is a well established inducer of the MPT, and oxidative stress appeared to be the principal cause of the iron-dependent MPT in hepatocytes. Iron caused lipid peroxidation, and both cell killing and lipid peroxidation increased with increasing oxygen. Iron and other transition metals, such as copper, catalyze the so-called Fenton chemistry to generate the highly reactive hydroxyl radical from hydrogen peroxide and superoxide:

(1)
(2)
(3)
where O2·− is superoxide, O2 is oxygen, H2O2 is hydrogen peroxide, OH· is hydroxyl radical and Fe2+ and Fe3+ are ferrous and ferric iron [12]. Consistent with a role of iron in oxygen radical generation, Rauen et al. found that killing was blocked by the novel cell-permeant catalase mimetic, TAA-1/Fe, but not by exogenous extracellular catalase. Somewhat surprisingly, however, the authors found that the cell permeable superoxide dismutase mimetic, MnTBAP which degrades O2·− to oxygen and hydrogen peroxide, did not protect despite the fact that MnTBAP also has catalase activity [13]. These findings suggest a more complex chemistry involving iron–oxygen species. Alternatively, since mitochondria accumulate iron ions, direct reduction of Fe3+ to Fe2+ by mitochondrial oxidoreductases may bypass Reaction (1) in the Fenton chemistry [14]. In other systems, H2O2 potentiates iron toxicity, consistent with an important role of Reaction (2) in iron-dependent oxygen radical formation [15].

The work presented in today's issue is an extension of elegant earlier studies applying fluorescent probes like phen green SK to visualize changes of intracellular chelatable iron [16], [17]. Unlike many other ion-indicating fluorophores, iron binding to phen green SK causes fluorescence to decrease. Iron also quenches the fluorescence of calcein used to mark the intracellular space and to identify changes of mitochondrial membrane permeability. In previous studies of calcein-loaded hepatocytes, calcein fluorescence decreased in models of oxidative stress, hypoxia, ischemia/reperfusion and drug toxicity, often in a relatively abrupt fashion [18], [19]. Loss of fluorescence was attributed to dye leakage, but loss of fluorescence occurs even when the dye is present in similar concentration in the extracellular space (K. Kon, J.-S. Kim, J.J. Lemasters, unpublished observations). The intriguing possibility to explain these observations is that chelatable or free iron increases late in these injuries and contributes to the MPT that subsequently occurs.

Changes of free iron may also contribute to normal physiological processes. In Kupffer cells, chelatable iron increases transiently after LPS stimulation, and iron chelators block NFκB activation leading to cytokine formation [20], [21], [22]. Iron chelation also produces hypoxia inducible factor-1α transactivation and expression of transferrin receptors [23], [24]. By such a process, chelatable iron levels for heme and iron-containing protein synthesis may be homostatically regulated.

If free or chelatable iron increases in response to various stimuli and stresses, then where does the iron come from? Clearly, proteolysis in lysosomes, proteosomes and elsewhere acts to recycle iron for biosynthetic reactions. Similarly, heme oxygenase releases iron for recycling. Excess iron is stored in a non-reactive, highly chelated form as ferritin and hemosiderin. However, these stores of iron may not be amenable to rapid mobilization as may occur in toxic stress and physiological signaling. Mitochondria also accumulate iron, and release of mitochondrial iron after the MPT may help perpetuate the MPT throughout a cell.

The acidic lysosomal/endosomal compartment may represent the most important source of rapidly mobilized iron. In addition to being a major site of proteolysis, the endosomal/lysosomal compartment is continuously receiving iron as a result of transferrin receptor-mediated endocytosis [25]. Transferrin is an iron-binding plasma protein. With two iron binding sites, 30% iron occupancy and a plasma concentration of 5–10 μM, transferrin accounts in large part for the non-heme iron content of plasma. Via endocytosis, transferrin is constantly delivered into the endosomal/lysosomal compartment as the major site of iron uptake. An iron transporter, nRAMP2, then mediates release of iron into the cytosol from the endosomal/lysosomal lumen [26]. Thus, lysosomes and endosomes are potential rust buckets primed to release iron in response to pathophysiological stresses and physiological stimuli. In pathological processes during oxidative stress and hypoxia/ischemia, lysosomes rupture [18], [27], [28], [29]. Such rupture appears to be a source of iron release and consequent pro-oxidant cell damage [30], [31]. Similarly, in LPS-stimulated cytokine formation by macrophages, a lysosome-dependent step seems involved, which may involve lysosomal release of divalent cation [32]. It is tempting to speculate therefore that lysosomes are the source of the transient increase of chelatable iron during macrophage activation.

In conclusion, free or chelatable iron is an emerging dynamic regulator of cellular function and important mediator of cell injury. Like the much better characterized calcium ion, iron in its free ferrous and ferric states may represent both a signaling agent regulating normal cellular responses and an intracellular mediator of toxicity when normal iron homeostasis becomes compromised. Because increased iron intake and storage are also increasingly linked to chronic human diseases, including diabetes, cancer, and cardiovascular and liver disease, the study of chelatable iron in cellular toxicity using the approaches pioneered by Rauen and co-workers should only increase.

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This work was supported, in part, by grants from the National Institutes of Health.

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References 

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PII: S0168-8278(04)00073-X

doi:10.1016/j.jhep.2004.02.015

Journal of Hepatology
Volume 40, Issue 4 , Pages 696-698, April 2004

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