Hydrazine compounds inhibit glycation of low-density lipoproteins and prevent the in vitro formation of model foam cells from glycolaldehyde-modified low-density lipoproteins.

AIMS/HYPOTHESIS: Previous studies have shown that glycation of LDL by methylglyoxal and glycolaldehyde, in the absence of significant oxidation, results in lipid accumulation in macrophage cells. Such 'foam cells' are a hallmark of atherosclerosis. In this study we examined whether LDL glycation by methylglyoxal or glycolaldehyde, and subsequent lipid loading of cells, can be inhibited by agents that scavenge reactive carbonyls. Such compounds may have therapeutic potential in diabetes-associated atherosclerosis. MATERIALS AND METHODS: LDL was glycated with methylglyoxal or glycolaldehyde in the absence or presence of metformin, aminoguanidine, Girard's reagents P and T, or hydralazine. LDL modification was characterised by changes in mobility (agarose gel electrophoresis), cross-linking (SDS-PAGE) and loss of amino acid residues (HPLC). Accumulation of cholesterol and cholesteryl esters in murine macrophages was assessed by HPLC. RESULTS: Inhibition of LDL glycation was detected with equimolar or greater concentrations of the scavengers over the reactive carbonyl. This inhibition was structure-dependent and accompanied by a modulation of cholesterol and cholesteryl ester accumulation. With aminoguanidine, Girard's reagent P and hydralazine, cellular sterol levels returned to control levels despite incomplete inhibition of LDL modification. CONCLUSIONS/INTERPRETATION: Inhibition of LDL glycation by interception of the reactive aldehydes that induce LDL modification prevents lipid loading and model foam cell formation in murine macrophage cells. Carbonyl-scavenging reagents, such as hydrazines, may therefore help inhibit LDL glycation in vivo and prevent diabetes-induced atherosclerosis.

The intracellular oxidation of 2',7'-dichlorofluorescin in murine T lymphocytes.

Intracellular reactive oxygen species (ROS) production by activated murine T lymphocytes was investigated by analyzing intracellular dichlorofluorescin (DCFH(2)) oxidation in lymph node cells (LNC). An increase in DCFH(2) oxidation in LNC induced by phorbol myristate acetate (PMA) was detected by flow cytometry. It was confirmed that this increase was present in Thy1(+) LNC. We examined the contribution to intracellular DCFH(2) oxidation of ROS released by leukocytes other than T cells present in the LNC suspension. Superoxide dismutase, catalase, and glutathione/glutathione peroxidase inhibited the PMA-induced increase in intracellular DCFH(2) oxidation. Furthermore, PMA failed to elicit DCFH(2) oxidation in LNC isolated from mice lacking a functional NADPH oxidase (gp91(phox) gene knockout mice), but this response could be restored in these cells by the addition of T cell-depleted LNC from wild-type litter mates. This study highlights the necessity for caution in using the DCFH(2) assay to demonstrate specific intracellular ROS production in heterogeneous cell populations. It also suggests that cells other than T cells in lymph node populations may, through production of ROS, influence the intracellular redox state of T lymphocytes.

The macrophage in atherosclerosis: modulation of cell function by sterols.

Lipid-laden macrophage foam cells are an early and persistent component of atherosclerotic lesions. As such they are likely to play a key role in disease progression, both as scavengers of lipid and as inflammatory mediators. The sterol content of macrophage foam cells is largely native cholesterol together with a small but significant proportion of oxidized cholesterol (oxysterols). Few in vitro investigations of the influence of sterol accumulation on macrophage function have used cells that contain physiologically or even pathologically representative amounts of cholesterol or, more particularly, oxysterols. However, recent studies, using macrophages with a sterol content much closer to that of authentic foam cells, show that the presence of oxysterols causes an impairment in macrophage cholesterol export, suggesting a key role for oxysterols in the maintenance of the foam cell phenotype. The implications of physiologically relevant levels of oxysterols on a wider range of macrophage function remain to be investigated.

Prooxidant and antioxidant activities of macrophages in metal-mediated LDL oxidation: the importance of metal sequestration.

Murine macrophages incubated in metal-supplemented RPMI could block or promote oxidation of low-density lipoprotein (LDL) depending on the degree of metal supplementation. Only at high concentrations of Cu (1 micromol/L) and Fe (30 micromol/L) were cells prooxidant, leading to an accelerated rate of LDL oxidation over that measured in comparable cell-free media. At lower concentrations of Cu and Fe in RPMI, LDL oxidation in the presence of macrophages was inhibited relative to the cell-free condition. This appeared to be dependent on a stable modification of the culture medium, because preconditioning of media by incubation with macrophages could also decrease their capacity to sustain subsequent cell-free LDL oxidation. This was due, in part, to a removal of metal from the media during preconditioning. However, resupplementation of media with metals did not fully restore oxidative capacity, indicating that other cell-dependent antioxidant modifications occurred. This did not involve significant alterations to the thiol content of the media. This study highlights the complexity of the role that cells such as macrophages have with regards to LDL oxidation in vitro and demonstrate that there are both antioxidative and prooxidative components.

The iron-selective chelator desferal can reduce chelated copper.

It was shown that the iron-selective chelator desferal (desferrioxamine mesylate:DFO) can reduce Cu(II) as judged by measuring the formation of the complex between Cu(I) and a specific chelator for this species, neocuproine (NC), in phosphate buffer. It was found that under optimal conditions, 3 moles of Cu(II) could be reduced per mole of DFO. Studies of the kinetics of Cu(II) reduction by DFO revealed that the rate of Cu(II) reduction by DFO was considerably slower than that by ascorbate. In the case of both reductants, even in the absence of NC, Cu(I) complexes remained in aqueous solutions for at least 30 min. DFO could also reduce Cu complexed to histidine. The results presented highlight the interpretive dangers which can arise in studies involving multiple transition metals, especially in the presence of multiple chelators. Specifically, when desferal is used, it is important to be aware that any copper present may become reduced, and that any Cu(I) formed might participate in ongoing redox reactions.

Batch-to-batch variation of Chelex-100 confounds metal-catalysed oxidation. Leaching of inhibitory compounds from a batch of Chelex-100 and their removal by a pre-washing procedure.

Removal of adventitious redox-active metals from buffers by treatment with Chelex resin is a widely used procedure in free radical research. Use of a new batch of Chelex-100 resin in our laboratory coincided with a sudden inability to oxidise low-density lipoprotein with copper. We found that copper-mediated oxidation of ascorbate in water treated with the same batch of Chelex was inhibited when compared with untreated water and water treated with a different batch of the resin. Washing the Chelex removed the inhibitory effect suggesting that material was leaching from the resin. The washing procedure for Chelex-100 described is simple and can be scaled up. Oxidation of ascorbate with low concentrations of copper can be used to test the quality of batches of the resin.

In vitro effects of three iron chelators on mitogen-activated lymphocytes: identification of differences in their mechanisms of action.

The effects of three iron chelators (ADR-529/ICRF-187; omadine/pyrithione; and a newly synthesized pyridoxal-based iron chelator, SAG-15) on cultured BALB/c murine lymph node cells stimulated with phorbol myristate acetate and ionomycin have been investigated. All three agents were found to inhibit [3H]-thymidine incorporation after 66-72 h incubation. Pretreatment of ADR-529 and omadine with Fe(III) or Fe(II) ions did not prevent their inhibitory effects. However, pretreatment of SAG-15 with Fe(II) or Fe(III) ions led to a significant increase in the ID50. Time-course studies of cell viability and thymidine incorporation demonstrated that the inhibitory effect of omadine was attributable to cell killing while for ADR-529 and SAG-15 there were both cytostatic and cytotoxic effects. Cell cycle analysis showed that treatment of cells with ADR-529 led to arrest in G2/M while treatment with SAG-15 led to a G0/G1 arrest. Iron has an obligatory role in T-lymphocyte activation that may be related to the formation of reactive oxygen species. SAG-15 is a new iron chelator that will help in the elucidation of the precise role of iron in lymphoproliferation. Since SAG-15 is an extremely effective iron chelator in vivo it has potential as an immunosuppressive agent.