Showing papers by "Karl A. Nath published in 1993"

Endothelial-cell heme uptake from heme proteins: induction of sensitization and desensitization to oxidant damage.

Abstract: Iron-derived reactive oxygen species are implicated in the pathogenesis of various vascular disorders including atherosclerosis, vasculitis, and reperfusion injury. The present studies examine whether heme, when liganded to physiologically relevant proteins as in hemoglobin, can provide potentially damaging iron to intact endothelium. We demonstrate that reduced ferrohemoglobin, while relatively innocuous to cultured endothelial cells, when oxidized to ferrihemoglobin (methemoglobin), greatly amplifies oxidant (H2O2)-mediated endothelial-cell injury. Drawing upon our previous observation that free heme similarly primes endothelium for oxidant damage, we posited that methemoglobin, but not ferrohemoglobin, releases its hemes that can then be incorporated into endothelial cells. In support, cultured endothelial cells exposed to methemoglobin--in contrast to exposure to ferrohemoglobin, cytochrome c, or metmyoglobin--rapidly increased their heme oxygenase mRNA and enzyme activity, thereby supporting heme uptake; ferritin production was also markedly increased after such exposure, thus attesting to eventual incorporation of Fe. These cellular methemoglobin effects were inhibited by the heme-scavenging protein hemopexin and by haptoglobin or cyanide, agents that strengthen the liganding between heme and globin. If the endothelium is exposed to methemoglobin for a more prolonged period (16 hr), it accumulates large amounts of ferritin; concomitantly, and presumably associated with iron sequestration by this protein, the endothelium converts from hypersusceptible to hyperresistant to oxidative damage. We conclude that when oxidation of hemoglobin facilitates release of its heme groups, catalytically active iron is provided to neighboring tissue environments. The effect of this relinquished heme on the vasculature is determined both by extracellular factors--i.e., plasma proteins, such as haptoglobin and hemopexin--as well as intracellular factors, including heme oxygenase and ferritin. Acutely, if both extra- and intracellular defenses are overwhelmed, cellular toxicity arises; chronically, when ferritin is induced, resistance to oxidative injury may supervene.

Tumor cell heme uptake induces ferritin synthesis resulting in altered oxidant sensitivity: possible role in chemotherapy efficacy.

Abstract: Neovascularization and hemorrhage are common features of malignant tumors. We wondered whether hemoglobin derived from extravasated RBC deposits heme-derived iron into the tumor, which could modulate the sensitivity of cancer cells to oxidant-mediated injury. A brief exposure (1 h) of 51 Cr-radiolabeled breast cancer cells (BT-20) but not colon cancer cells (Caco-2) to hemin (10 µm) or FeSO 4 (10 µm) significantly enhances cytotoxicity mediated by 0.5 mm hydrogen peroxide (H 2 O 2 ). Associated with Caco-2 resistance, these cells were found to be enriched in the endogenous iron chelator, ferritin. If cellular ferritin is even further increased through 1 h incubation (24 h prior to H 2 O 2 exposure) of both cell types with hemin, FeSO 4 , or exogenous spleen apoferritin itself (24 h), marked resistance to H 2 O 2 -mediated cytotoxicity is manifest. Under several conditions, the sensitivity of tumor cells to oxidant-mediated lysis is inversely proportional to their ferritin content. Pretreatment of BT-20 and Caco-2 cells with hemin or FeSO 4 rapidly increases H-ferritin mRNA but only slightly increases L-ferritin mRNA; nevertheless, large increases in overall ferritin content of iron-exposed cells result. Data analogous to those with H 2 O 2 -mediated cytotoxicity were obtained in studies of bleomycin-engendered DNA strand breakage and cell damage, i.e. , brief treatment of BT-20 cells with both hemin or FeSO 4 significantly increases their sensitivity to bleomycin (100 µg/ml), whereas treatment followed by 24 h incubation with media alone significantly protects against bleomycin toxicity. We speculate that acute exposure of tumors to iron ( e.g. , derived from heme-proteins in hemorrhagic cancerous lesions) may increase sensitivity of some cancer cells, particularly those relatively low in endogenous ferritin, to oxidant-mediated lysis. In contrast, repeated, more chronic, exposure may result in resistance of various tumors to oxidant-producing immune effector cells or chemotherapeutic agents, an effect derived from their increased synthesis and accumulation of the intracellular iron scavenger, ferritin.

Autoxidation of cysteine generates hydrogen peroxide: Cytotoxicity and attenuation by pyruvate

Abstract: The reactivity of cysteine presents a paradox: although regarded as an antioxidant, cysteine interacts with oxygen in a metal-catalyzed reaction to produce reactive species. Because ischemia provokes the appearance of millimolar amounts of cysteine and increased amounts of transition metals, we studied whether cysteine, in the presence of transition metals, consumes oxygen, generates hydrogen peroxide, and is toxic. Using fluorescence cytometry, we provide direct evidence that hydrogen peroxide is copiously generated during cysteine autoxidation. Pyruvate attenuates such generation of hydrogen peroxide and cytotoxicity. Cysteine oxidation is stimulated by an EDTA-chelatable diethyl-dithiocarbamate-chelatable constituent of kidney extract; this suggests that copper is the catalytically active metal. The toxicity resulting from cysteine oxidation is less than that induced by amounts of reagent hydrogen peroxide that produce comparable fluorescence. Cysteine also prevents hydrogen peroxide-induced toxicity. Thus, although cysteine generates hydrogen peroxide, it can guard against hydrogen peroxide toxicity, possibly by binding metals on which the toxicity of hydrogen peroxide is dependent. Thus the behavior of cysteine can be salutary or pernicious; the net effect of cysteine, within this wide ambit of actions, is decisively influenced by the conditions to which cysteine is exposed.

Effect of Proteinuria on Renal Interstitium: Effect of Products of Nitrogen Metabolism

Abstract: Tubulointerstitial disease is an invariant finding in proteinuric renal disease regardless of the underlying disease or the compartment in which the disease originates. Such histologic changes are functionally significant in that scores of such injury rather than glomerular histologic injury correlate with decrements in GFR. Proteinuria, the consequence of a loss of glomerular permselectivity incurred by glomerular diseases, also provides an index of renal functional decline. This review provides evidence that proteins leaked into the urinary space may directly or indirectly provoke tubulointerstitial injury, a linkage that may underlie the functional significance of proteinuria and tubulointerstitial disease. This review also highlights two products of nitrogen metabolism, ammonia and nitric oxide, in the pathogenesis of tubulointerstitial disease.