HPLC determination of a novel aroylhydrazone iron chelator (o-108) in rabbit plasma and its application to a pilot pharmacokinetic study

A high performance liquid chromatographic method for the determination of a biocompatible iron chelator, pyridoxal 2-chlorobenzoyl hydrazone (o-108), in rabbit plasma was developed and validated. The separation was achieved on a C18 column with the mobile phase composed of a mixture of 0.01 M phosphate buffer (pH 6) with the addition of EDTA (2 mM), methanol and acetonitrile (42:24:14; v/v/v). The method was validated with respect to selectivity, linearity (0.8-150 microg/mL), intra- and inter-day variability and stability. This method was successfully applied to the analysis of the samples obtained from a pilot pharmacokinetic experiment, in which the chelator was administered intravenously to rabbits.     

S-nitrosylation of IRP2 regulates its stability via the ubiquitin-proteasome pathway

Nitric oxide (NO) is an important signaling molecule that interacts with different targets depending on its redox state. NO can interact with thiol groups resulting in S-nitrosylation of proteins, but the functional implications of this modification are not yet fully understood. We have reported that treatment of RAW 264.7 cells with NO caused a decrease in levels of iron regulatory protein 2 (IRP2), which binds to iron-responsive elements present in untranslated regions of mRNAs for several proteins involved in iron metabolism. In this study, we show that NO causes S-nitrosylation of IRP2, both in vitro and in vivo, and this modification leads to IRP2 ubiquitination followed by its degradation in the proteasome. Moreover, mutation of one cysteine (C178S) prevents NO-mediated degradation of IRP2. Hence, S-nitrosylation is a novel signal for IRP2 degradation via the ubiquitin-proteasome pathway.     

Iron targeting to mitochondria in erythroid cells

Immature erythroid cells have an exceptionally high capacity to synthesize haem that is, at least in part, the result of the unique control of iron metabolism in these cells. In erythroid cells the vast majority of Fe released from endosomes must cross both the outer and the inner mitochondrial membranes to reach ferrochelatase, which inserts Fe into protoporphyrin IX. Based on the fact that Fe is specifically targeted into erythroid mitochondria, we have proposed that a transient mitochondria-endosome interaction is involved in Fe transfer to ferrochelatase [Ponka (1997) Blood 89, 1-25]. In this study, we examined whether the inhibition of endosome mobility within erythroid cells would decrease the rate of (59)Fe incorporation into haem. We found that, in reticulocytes, the myosin light-chain kinase inhibitor, wortmannin, and the calmodulin antagonist, W-7, caused significant inhibition of (59)Fe incorporation from (59)Fe-transferrin-labelled endosomes into haem. These results, together with confocal microscopy studies using transferrin and mitochondria labelled by distinct fluorescent markers, suggest that, in erythroid cells, endosome mobility, and perhaps their contact with mitochondria, plays an important role in a highly efficient utilization of iron for haem synthesis.     

DNA-adducts and atherosclerosis: a study of accidental and sudden death males in the Czech Republic

Atherosclerosis and carcinogenesis may share some common mechanisms of the genotoxic action of exogenous compounds, such as polycyclic aromatic hydrocarbons (PAHs). The main objective of this study was to test the hypothesis that "bulky" aromatic DNA-adducts in smooth muscle cells (SMCs) of thoracic aortas taken at autopsy from sudden and accidental death male subjects, aged between 30 and 60 years (N=133), are associated with the stage of atherosclerosis. The subjects with severe atherosclerotic damage were treated as "Cases" (N=66). The subjects meeting diagnostic criteria for slight and moderate total atherosclerotic body damage were treated as "Controls" (N=67). An additional objective of the study was to evaluate the effect of known atherogenic risk factors and possible modifiers of atherosclerotic changes, such as age, smoking, plasma lipid and antioxidant vitamin levels and some genetic susceptibility markers, e.g. polymorphisms of GSTM1, GSTT1, NAT2, CYP1A1 or apolipoprotein E (APO E) genes. We found significantly higher DNA-adduct levels in "Cases" as compared with "Controls" (2.11+/-1.07 adducts/10(8) nucleotides versus 1.49+/-0.55 adducts/10(8) nucleotides, P<0.001). "Cases" were significantly older and had elevated heart weight and plasma cholesterol levels and a higher frequency of overweight subjects as compared with "Controls". No significant differences in DNA-adduct levels between smokers and non-smokers within either group were detected. Multivariate logistic regression revealed that the "bulky" aromatic DNA-adducts, which are the most likely related to environmental exposure to genotoxic chemicals, remain a statistically significant predictor of the stage of atherosclerosis (OR=3.76, 95% CI=1.54-9.18, P=0.004) even after adjustment for age, smoking, obesity, heart weight and genetic susceptibility markers (GSTT1 and CYP1A1-MspI polymorphisms) that were also significant predictors. The fact that the "bulky" aromatic DNA-adduct levels predict the progression of atherosclerosis independently of smoking indicates that the formation of atherosclerotic plaques may also be initiated by environmental exposures other than tobacco smoke.     

Uptake of DOPA and 5-HTP in pancreatic islets and parafollicular cells studied by combined autoradiographic and fluorescence microscopic techniques

Summary White albino mice were simultaneously injected with ³H-5-HTP and unlabelled DOPA or ³H-DOPA and unlabelled 5-HTP. The distribution patterns of the formed indol- and catecholamines in a) the pancreatic islets and b) the parafollicular cells were then compared by use of a combined autoradiographic-fluorescence microscopic technique.Localization in the same sections of radioactivity and fluorescence representing the respective monoamines corresponded very well for all the time intervals studied both in the pancreatic islets and in the parafollicular cells.In the pancreatic islets the monoamines were localized in all cells shortly after the injection of the precursors. The monoamines then first disappeared from the -cells leaving the -cells with the highest concentration of activity and fluorescence. In the thyroid the activity and fluorescence, localized in the same parafollicular cells, persisted for a longer time than in the islets.The authors wish to express their gratitude to Professor Sven Ullberg, Head of the Department of Toxicology, University of Uppsala, for much valuable advice during this work. The investigation was supported by grants from the Swedish Medical Research Council (No. 12x-713-01), Knut and Alice Wallenbergs Stiftelse and Ragnar and Torsten Söderbergs Stiftelse.     

Transferrin and iron uptake by human lymphoblastoid and K-562 cells

Two human lymphoblastoid cell lines and K-562 cells were found to take up radioiodinated transferrin and transferrin-bound iron in amounts comparable to reticulocytes. These cell lines were also shown to possess transferrin receptors whose numbers and affinity for transferrin were similar to those of reticulocytes. However, unlike reticulocytes, in which at least 90% of the iron taken up is incorporated into heme, in the lymphoblastoid and K-562 cells only around 10% of the incorporated iron is found in heme. In addition, in contrast to the hemoglobin synthesizing cells, excess heme does not inhibit the removal of iron from transferrin by the lymphoblastoid and K-562 cells, suggesting that only during erythroid differentiation do cells acquire a specific mechanism for removing iron from transferrin which is subject to feedback inhibition by heme.     

Transferrin-receptor-independent but iron-dependent proliferation of variant Chinese hamster ovary cells

The purpose of this study is to clarify the role of iron, transferrin, an iron-binding protein in vertebrate plasma, and transferrin receptors in cell proliferation. Transferrin, which is indispensable for most cells growing in tissue culture, is frequently referred to as a "growth factor". Proliferating cells express high numbers of transferrin receptors, and the binding of transferrin to their receptors that is needed for cells to initiate and maintain their DNA synthesis is sometimes regarded as analogous to other growth factor-receptor interactions. Although numerous previous experiments strongly indicate that the only function of transferrin in supporting cell proliferation is supplying cells with iron, they did not completely rule out some direct or signaling role transferrin receptors could play in cell proliferation. To address this issue, we exploited transferrin-receptor-deficient mutant Chinese hamster ovary (CHO) cells (McGraw, T. E., Greenfield, L., and Maxfield, F. R., 1987, J. Cell. Biol. 105, 207-214) in which various aspects of iron and transferrin metabolism in relation to their capacity to proliferate were investigated. Variant cells neither specifically bind transferrin nor do their extracts contain any detectable functional transferrin receptors, yet they proliferate and synthesize DNA with rates comparable to those observed with parent CHO cells. Desferrioxamine, an iron chelating agent, inhibits growth and DNA synthesis of both variant and control CHO cells. This inhibition can be fully alleviated, in both cell types, by ferric pyridoxal isonicotinoyl hydrazone, which can supply cells with a utilizable form of iron by a pathway not requiring transferrin and their receptors. Studies of 59Fe uptake and 125I-transferrin binding revealed that parent cells can take up iron by at least three mechanisms: from transferrin by receptor-dependent and -independent (nonspecific, nonsaturable, not requiring acidification) pathways and from inorganic iron salts (initially present in the medium as FeSO4). Although variant CHO cells are unable to acquire transferrin iron via the receptor pathway, two remaining mechanisms provide these cells with sufficient amounts of iron for DNA synthesis and cell proliferation. In conclusion, although transferrin receptors are dispensable in terms of their absolute requirement for proliferating cells, a supply of iron is still needed for their DNA synthesis. Transferrin-receptor-deficient CHO cells may be a useful model for investigating receptor-independent iron uptake from transferrin and nontransferrin iron sources.