Pak Lai Tang

Effects by doxorubicin on the myocardium are mediated by oxygen free radicals

We hypothesized that doxorubicin (DOX) induces cardiotoxicity of myocardium via oxygen radicals. The present study is aimed at examining the membrane alterations by oxygen radicals generated by DOX in adult rats and cultured neonatal myocytes. Our results showed that DOX 1) decreased beta-adrenoceptor (BAR) density in the cell membrane, 2) increased the membrane permeability of cultured neonatal rat myocytes and 3) altered the ultrastructure of myofibrils and subplasmalemmal actin networks. These effects were reproducible by exogenous hydrogen peroxide. The antioxidant melatonin (MLT) inhibited enzyme leakage and peroxidation in a concentration-dependent manner. It is concluded that DOX induces cardiotoxicity through lipid peroxidation and melatonin is an effective antioxidant against the reactive oxygen intermediates generated by DOX.

MECHANISMS OF IRON UPTAKE BY MAMMALIAN CELLS

On the basis of the discussion in this paper, the process of transferrin and iron (transferrin-bound iron and non transferrin-bound iron) uptake and transferrin release by reticulocytes are summarized diagrammatically. Although we were able to outline the pathways shown in the figure, there is still a long way to go before we achieve total understanding of the mechanisms of iron uptake. In addition, many important questions need to be answered. For example: what is the nature and properties of the iron carrier on the membrane? What is the relationship between the iron carrier and transferrin receptor? Is the iron carrier system in membranes of cells from different tissues similar or different? And how does iron cross the membrane of the endosomes after it is released from transferrin? All of these questions merit further investigation.

Fluctuation of Blood Melatonin Concentrations With Age: Result of Changes in Pineal Melatonin Secretion, Body Growth, and Aging

Melatonin in the systemic circulation of rats fluctuates with age, and the causes for such changes were investigated. Male rats (aged 7 days, 16 days, 18 days, 20 days, 30 days, 48 days, 60 days, and > 17 months) were adapted under a lighting regime of 12L: 12D for at least 7 days. Pineals and blood samples from the trunk or confluens sinuum were collected in the dark period. Melatonin in tissues was extracted, identified, and determined by gas chromatography‐mass spectrometry (GC‐MS) and/or radioimmunoassay. Tissue melatonin levels obtained by radioimmunoassay correlated closely with those quantified by GC‐MS. Thus, the melatonin radioimmunoassay used is a reliable assay method for melatonin in the plasma and pineal of the rat.

2-[125l]lodomelatonin Binding Sites in the Lung and Heart: A Link between the Photoperiodic Signal, Melatonin, and the Cardiopulmonary System

The pineal gland plays an important role in seasonal adaptation including variation in energy requirement. Animals exhibiting seasonal changes in their energy expenditure would be benefited if their cardiac and pulmonary systems respond to the pineal photoperiodic signal, melatonin, appropriately. Thus, we would like to hypothesize that melatonin receptors are present in the heart and lung. Using a specific labeled melatonin agonist, 2-[125I]iodomelatonin, binding sites were demonstrated in the lung and heart of birds and other animals. In the chicken lung, there were high affinity (equilibrium dissociation constant, Kd = 9.11 +/- 0.73 pmol/l) and low density (maximum number of binding sites, Bmax = 1.29 +/- 0.16 fmol/mg protein) 2-[125I]iodomelatonin binding sites that were highly specific to melatonin. Similar binding with lower density was demonstrated in the quail and frog lungs. In the duck heart, specific 2-[125I]iodomelatonin binding sites with a Kd of 30.5 +/- 3.5 pmol/l and a Bmax of 0.46 +/- 0.1 fmol/mg protein (n = 4) were demonstrated. Competitive studies suggested that these binding sites were specific to melatonin. Thus, saturable and reversible 2-[125I]iodomelatonin binding was present in the lung and heart membrane preparations of birds and possibly other animals. The picomolar affinity, femtomolar density and highly specific pharmacological profile of these binding sites suggest that they can be classified as ML-1 melatonin receptors. The 2-[125I]iodomelatonin binding sites described in the lung and heart as well as those binding sites demonstrated in other peripheral tissues suggest the ubiquitous direct action of melatonin on peripheral tissues.

Polysaccharide peptide (PSP) restores immunosuppression induced by cyclophosphamide in rats.

Polysaccharide peptide (PSP) is a protein-bound polysaccharide extracted from an edible mushroom, Coriolus versicolor. Effects of PSP (2g/kg/day) on cyclophosphamide (CPA, 40 mg/kg/2 days)-induced immunosuppression were investigated by determining lymphocyte proliferation, Natural killer (NK) cell formation, IgG and IL-2 concentration, WBC count and the weight of organs after rats were treated with or without CPA in the presence or absence of PSP. The results demonstrated that PSP possessed immunopotentiating effect, being effective in restoring CPA-induced immunosuppression such as depressed lymphocyte proliferation, Natural Killer cell function, production on white blood cell and the growth of spleen and thymus in rats as well as in increasing both IgG and IL-2 production on which CPA did not have significant effects under the conditions of our experiments. PSP can partly restore CPA-induced immunosuppression. Based on our findings and the data accumulated so far, it was suggested that PSP should be considered as an useful adjuvant especially combined with CPA or other chemotherapy in clinical treatment of cancer patients. The mechanism by which PSP restores the immunosuppression induced by CPA is unclear.

Effect of lipid peroxidation on transferrin-free iron uptake by rabbit reticulocytes

The relationship between lipid peroxidation and uptake of transferrin- free iron, Fe(II), by reticulocytes in an experimental system for studying membrane transport of Fe(II) was investigated by using free radical scavengers: BHA (butylated hydroxyanisole), BHT (butylated hydroxytoluene), superoxide dismutase, alpha-tocopherol, propyl gallate and DPPD (N,N-diphenyl-1,4-phenylenediamine), and producers: t-butyl hydroperoxide, cumene hydroperoxide, H2O2 and aluminium carbonate. Measurements were made of MDA (malondialdehyde) and the rate of Fe(II) uptake from a sucrose solution buffered at pH 6.5 by Pipes. Most scavengers and producers used could increase or decrease only slightly the rate of Fe(II) uptake and some of them had no effect on Fe(II) uptake and MDA could not be detected at iron concentration of lower than 10 microM and incubation time of 20 min. At iron concentration of higher than 100 microM and incubation time of 4 h, there was the production of MDA which increased with the increment of iron concentration of incubation medium and BHT could inhibit the production of MDA. In addition, no difference was found in the rates of Fe(II) uptake in three experimental groups whose incubation medium was buffered by Pipes, Mops and Mes respectively. The results suggested that iron could induce free radical reaction under experimental conditions, especially at high concentration of iron and longer incubation time; however, at low concentration of iron (<10 microM) and the usual incubation time (20 min) free radical reaction was very slight and the extent of the reaction was not enough to damage the integrity and function of the membrane of reticulocytes, and that Fe(II) uptake by reticulocytes was not the result of free radical reaction and lipid peroxidation. It was therefore concluded that iron could not initiate its own membrane transport in rabbit reticulocytes by free radical reaction and lipid peroxidation and that the experimental system we used for studying membrane transport of Fe(II) is valid.

Differential behaviour of cell membranes towards iron-induced oxidative damage and the effects of melatonin.

The ability of melatonin to protect iron-induced lipid peroxidation was studied in various rat cell membranes. The concentration of cellular membrane malondialdehyde (MDA) was used as an index of induced oxidative membrane damage. Cell membranes from the brain, heart, kidney and liver of the male Sprague-Dawley rat were incubated with ferric ammonium citrate (20 microg/ml iron) alone for 3 h and concomitant with varying concentrations of melatonin ranging from 125 to 2,000 microM. The basal MDA levels of all the cell membranes were 25.0+/-1.4 (brain), 21.2+/-0.2 (heart), 10.0+/-0.9 (kidney) and 20.7+/-0.4 (liver) microM/g membrane protein, and the highest lipid peroxidation after exposure to iron occurred in the kidney (314.4%), followed by the heart (151.3%), the liver (130.4%) and the brain (121.7%). This peroxidative effect was completely (ED50 846.7 microM for the heart) and partially suppressed by melatonin (ED50 462.1 microM for the brain, 178.3 microM for the kidney and 886.6 microM for the liver). This inhibition effect on MDA production by these cell membranes was also found - except for the liver - if melatonin was used alone. These results show that the direct effect of lipid peroxidation on cellular membrane following iron exposure is markedly reduced by melatonin.

INHIBITORY MECHANISM OF LEAD ON TRANSFERRIN-BOUND IRON UPTAKE BY RABBIT RETICULOCYTES : A FRACTAL ANALYSIS

Experimental data of transferrin and transferrin-bound iron uptake byrabbit reticulocytes in the presence or absence of extracellular lead isanalyzed by means of a fractal model. A highly significant correlation offractal dimension (Df) of intracellular transferrin or transferrin-boundiron uptake with varying extracellular concentrations of lead (0 ~ 25umol/L) was observed (Transferrin: r = 0.897, p = 0.015; transferrin-boundiron: r = 0.947, p = 0.004). The Df of membrane-bound transferrin (r =-0.618, p = 0.191) or transferrin-bound iron (r = 0.144, p = 0.786) did notappear to be markedly altered by lead. Further analysis shows thatinhibitory degree of lead on intracellular iron uptake is higher than thaton intracellular transferrin uptake. These results suggest that theinhibitory effect of lead on the iron uptake may occur in intracellularprocess rather than in membrane binding step, probably inhibitingtranslocation of iron across the endosomal membrane.

Transferrin-bound iron uptake by the cultured cerebellar granule cells

Excessive brain iron has been found in several neurodegenerative diseases. However, little information is available about mechanism of iron uptake by different types of brain cells including neurons. In this study, transferrin-bound iron (Tf-Fe) accumulation in the cultured cerebellar granule cell was investigated in vitro. After 5 days of culture, the cells were incubated with 1 microM of double-labelled transferrin (1251-Tf-59Fe) at 37 degrees C for 60 min. The cellular Tf-Fe and transferrin (Tf) uptake was analysed. The result showed (1) Tf uptake by the cells increased rapidly at the first 5 min, reaching its maximum after about 20 min of incubation; (2) Tf-Fe uptake kept increasing in a linear manner during the whole period of incubation; (3) the addition of either NH4Cl or CH3NH2, the blockers of Tf-Fe uptake via inhibiting iron release from Tf within endosomes, decreased the cellular Tf-Fe uptake but had no significant effect on Tf uptake; (4) trypsin and unlabelled Tf-Fe inhibited the uptake rate of Tf-Fe as well as Tf. The results suggested that Tf-Fe transport across the membrane of this type of neuron, much like other mammalian cells, was mediated by Tf-TfR endocytosis. Dysfunction of Tf or TfR would possibly lead to iron irregulation in the brain and consequently cause damage to neuronal functions.

Differential inhibitory effects of melatonin analogs and three naphthalenic ligands on 2‐[125I]iodomelatonin binding to chicken tissues

Pang CS, Tang PL, Song Y, Pang SF, Ng KW, Guardiola‐Lemaitre B, Delagrange P, Brown G.M. Differential inhibitory effects of melatonin analogs and three naphthalenic ligands on 2‐[l25I]iodomelatonin binding to chicken tissues. J. Peneal Res. 1997; 23:148–155.