Can melatonin delay oxidative damage of human erythrocytes during prolonged incubation?

PURPOSE: Melatonin (MEL) is an effective antioxidant in numerous experimental models, both in vitro and in vivo. However, it should be stressed that there are also papers reporting limited antioxidative activity of MEL or even giving evidence for its pro-oxidative properties. In the present paper we investigated the influence of MEL on the oxidative damage of human erythrocytes during prolonged incubation. MATERIAL/METHODS: Human erythrocytes suspended in phosphate-buffered saline (PBS), pH 7.4 were incubated at 37ºC either in absence or presence of melatonin at concentration range 0.02 mM-3 mM for up to 96 hrs. The influence of MEL on erythrocyte damage was assessed on the basis of the intensity of intracellular oxidation processes (the oxidation of HbO2, GSH, fluorescent label DCFH2) as well as damage to the plasma membrane (lipid peroxidation, the potassium leakage) and the kinetics of hemolysis. RESULTS: The prolonged incubation of erythrocytes induced a progressive destruction of erythrocytes. Melatonin prevented lipid peroxidation and hemolysis whereas the oxidation of HbO2 and DCFH2 was enhanced by melatonin at concentrations higher than 0.6 mM. In the case of erythrocytes incubated with 3 mM of MEL, the hemolysis rate constant (0.0498±0.0039 H%•h⁻¹) was 50% lower than that of the control while the HbO2 oxidation rate constants were about 1.4 and 1.5 times higher for 1.5 and 3 mM of MEL, respectively. Melatonin had no influence on the oxidation of GSH and the potassium leakage. CONCLUSIONS: Probably, MEL can stabilize the erythrocyte membrane due to interaction with lipids, thus prolonging the existence of cells. On the contrary, in the presence of MEL the accelerated oxidation of HbO2 and generally, increased oxidative stress was observed in erythrocytes. Pro- and antioxidative properties of melatonin depend on the type of cells, redox state, as well as experimental conditions.

Comparison of the effect of phenol and its derivatives on protein and free radical formation in human erythrocytes (in vitro).

The effect of phenolic compounds: phenol, 2,4-dichlorophenol (2,4-DCP), 2,4-dimethylphenol (2,4-DMP) and catechol on human erythrocytes was studied. The level of fluorescent label - 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA) oxidation by phenolic compounds in erythrocytes as well as the carbonyl group content and hemoglobin denaturation were monitored. H(2)DCFDA has been utilized extensively as a marker for studies of oxidative stress at the cellular level. We noted that 2,4-DCP, 2,4-DMP and catechol induced an increase in the concentration- and time-dependent H(2)DCFDA oxidation. We also observed an increase in carbonyl group content and the changes in parameter T (denaturation of hemoglobin) in erythrocytes incubated with 2,4-DCP, catechol and 2,4-DMP. The highest level of H(2)DCFDA oxidation was provoked by 2,4-DCP. The biggest changes of proteins in erythrocytes measured as the carbonyl group content were induced by 2,4-DMP, but measured as parameter T they were induced by catechol. It was observed that phenol did not oxidize H(2)DCFDA up to the concentration of 2.5 mM after 3 h of incubation. Phenol did not affect the carbonyl group content but decreased parameter T (induced denaturation of hemoglobin). To sum up, the kind of the substituent in a phenolic ring determines the molecular mechanism of action of the individual compound and the capacity of reactive oxygen species generation and thus damages the specified structures in human erythrocytes.

Effect of dose-rate and dose fractionation on radiation-induced hemolysis of human erythrocytes.

Human erythrocytes suspended in an isotonic Na-phosphate buffer, pH 7.4 (hematocrit 2%) were exposed under air to gamma radiation at a dose rates of 2.2 kGy.h-1 and 4.2 kGy.h-1. The dose-response curves for hemolysis of erythrocytes indicated that the process of hemolysis is inversely related to the dose-rate. At both dose-rates we observed a reduced level of hemolysis, when erythrocytes were irradiated with a split dose (0.4 kGy + 2.3 kGy with an interval time between the subsequent exposures from 1 to 4 h) in comparison with the same single dose (2.7 kGy). The maximal effect of fractionation was observed when the interfraction time was equal to 3.5 h. The influence of the interfraction temperature on this effect was observed. The results obtained indicate that enucleated human erythrocytes under suitable radiation conditions are capable of repairing radiation damage which leads to hemolysis.