Oxidative effects in human erythrocytes caused by some oximes and hydroxylamine.

Both oximes and hydroxylamine (HYAM) are compounds with known oxidative capacity. We tested in vitro whether acetaldoxime (AAO), cyclohexanone oxime (CHO), methyl ethyl ketoxime (MEKO) or HYAM affect haemoglobin oxidation (into HbFe3+), formation of thiobarbituric acid reactive substances (TBARS), and glutathione (GT) depletion in human haemolysate, erythrocytes or blood. All these parameters are known to be related to oxidative stress. Glutathione S-transferase (GST) activity was measured as it may be affected by oxygen radicals. All three oximes caused a low degree of HbFe3+ accumulation in erythrocytes. This was higher in haemolysates indicating that membrane transport may be limiting or that protective mechanisms within erythrocytes are more effective. HbFe3+ accumulation was lower for the oximes than for HYAM. AAO and HYAM caused TBARS formation in blood. For HYAM this was expected as free radicals are known to be generated during HbFe3+ formation. Free radical generation by AAO and HYAM in erythrocytes was confirmed by the inhibition of GST. For the other two oximes (CHO and MEKO) some special effects were found. CHO did inhibit erythrocyte GST while it did not cause TBARS formation. MEKO was the least potent oxime as it caused no TBARS formation, little HbFe3+ accumulation and little GST inhibition in erythrocytes. However, GT depletion was more pronounced for MEKO than for the other oximes, indicating that glutathione conjugation occurs. TBARS formation, GT depletion and GST modulation caused by the oximes and HYAM were also tested in rat hepatocytes. However, no effects were found in hepatocytes. This suggests that a factor present in erythrocytes is necessary for free radical formation. Studies with proposed metabolites of the oximes (i.e. cyclohexanone, acetaldehyde or methylethyl ketone) and addition of rat liver preparations to the erythrocyte incubations with oximes, suggest that metabolism is not a limiting factor in erythrocyte toxicity.

Glutathione depletion in human erythrocytes and rat liver: a study on the interplay between bioactivation and inactivation functions of liver and blood.

The interplay between bioactivation and inactivation functions of human erythrocytes and rat liver was studied. Glutathione depletion was used as a measure of the amount of reduced glutathione (GSH)-reactive compound. Iodoacetamide (IAcA), N-ethylmaleimide (NEM) and diethyl maleate (DEM), which are electrophiles that need no metabolic activation, were able to deplete GSH in incubations with either aqueous GSH solution or erythrocytes. These results indicate that these compounds can pass the erythrocyte membrane. Cyclophosphamide (CP), 3-hydroxyacetanilide (3-HAA) and 2-methylfurane (2-MF) needed metabolic activation by rat liver microsomes to deplete glutathione in incubations with aqueous GSH solution or erythrocytes. By measuring the sum of both reduced and oxidized glutathione [ = total glutathione (GT)] it became clear that GSH-reactive metabolites are generated out of CP, 3-HAA and 2-MF by the action of microsomes and that these metabolites can pass through the erythrocyte membrane. As GT depletion was higher when microsomes of phenobarbital-pretreated rats were used, the metabolites were (are expected to be) generated by phenobarbital-inducible enzymes. GT was also depleted in incubations with haemolysate and 3-HAA or 2-MF but not in incubations with aqueous GSH solution, which indicates that erythrocyte cytosol can metabolize 3-HAA and 2-MF into GSH-reactive compounds. The pesticides monuron and monulinuron did not affect GT concentrations when aqueous GSH solution, haemolysate or erythrocytes with or without microsomal activating system were tested. When hepatocytes were incubated with 3-HAA or CP (2 mm), about 2 mm of internal GT concentration was depleted. The hepatocytes excreted GSH-reactive metabolites generated from 3-HAA and CP (about 20% of the metabolites formed for 3-HAA). Erythrocyte GT was not depleted in co-incubations of hepatocytes and erythrocytes with 3-HAA. This can be explained by the amounts of GSH-reactive metabolites excreted by the hepatocytes, which would require very effective uptake by the erythrocytes in order to be detectable.

Glutathione depletion in human erythrocytes as an indicator for microsomal activation of cyclophosphamide and 3-hydroxyacetanilide.

A model system for the detection of reactive metabolites, using glutathione depletion after microsomal activation, has been described previously. We developed a battery of complementary test systems using rat liver microsomes for metabolism and aqueous glutathione solutions, human erythrocytes or hemolysate derived therefrom, as target. Reactive metabolite formation and the ability of metabolites to pass the erythrocyte membrane were tested using 3-hydroxyacetanilide (3-HAA) and cyclophosphamide (CP) as substrates. Neither unchanged 3-HAA nor CP depleted glutathione in erythrocytes or in aqueous reduced glutathione solutions (GSH solutions). Addition of fortified normal or liver microsomes from rats pretreated with phenobarbital (PB microsomes) induced a 3-HAA/CP concentration-dependent glutathione depletion in both systems. With PB microsomes, higher depletions were found. While unchanged 3-HAA did not deplete aqueous GSH solutions or glutathione in erythrocytes, a significant depletion in hemolysate was found. The results indicate that both CP and 3-HAA metabolites are able to pass through the erythrocyte membrane. While both substances can metabolically be activated by rat liver microsomes, only 3-HAA can be activated by soluble factors in erythrocytes. However, unchanged 3-HAA has no effect on GSH in erythrocytes. This might be caused by an inability of unchanged 3-HAA to enter the erythrocyte. More generally, an adequate combination of the test systems described can be used to detect (a) the reactivity of unchanged substances and their metabolites, and (b) the ability of unchanged substances and their reactive metabolites to pass through the erythrocyte membrane.

Toxicokinetics of dimethylacetamide (DMAc) in rat isolated perfused liver.

Dimethylacetamide (DMAc) is a skin-penetrating solvent able to induce hepatic damage after chronic exposure. Previous research has indicated that metabolism may be saturated at its present TLV/TWA (10 ppm). Biological monitoring of monomethylacetamide (MMAc), the primary metabolite of DMAc, might therefore underestimate exposure to DMAc and related health hazards. We used the recirculating perfusion technique in isolated rat liver to evaluate DMAc metabolism. Medium concentrations starting at about 30, 50, 100 and 275 microM, respectively, were tested. Perfusate samples were taken regularly and analysed for DMAc; pharmacokinetic parameters (extraction ratio and clearance) were calculated for each perfusion. Inlet DMAc concentrations were calculated and concentration groups divided in 16, 36, 70, 160, 225 microM. The extraction ratio of the 16 microM group differed significantly from the other concentration groups tested. DMAc metabolism was saturated at a DMAc concentration of 36 microM. Extraction ratios were unaffected when cimetidine, an inhibitor of cytochrome P450 activity, was added to the perfusion medium or when cimetidine-pretreated animals were used. DMAc clearance was 2.20 ml min-1 at a medium concentration of about 36 microM. Extrapolation of the observed (rat) liver clearance to man showed that airborne concentrations of 18 ppm would, under the presumptions used, lead to saturated metabolism of DMAc; however, saturation at even lower concentrations could not be excluded.

Changes in blood glutathione concentrations, and in erythrocyte glutathione reductase and glutathione S-transferase activity after running training and after participation in contests.

Previously sedentary men (n = 23) and women (n = 18) were trained to run a half marathon contest after 40 weeks. Total blood glutathione had increased by 20 weeks of training and had returned to normal after 40 weeks. Erythrocyte glutathione reductase activity had increased by 20 weeks and remained elevated after 40 weeks. This effect was accompanied by decreases in glutathione reductase coefficients, which indicated that increases in the presence of riboflavin may have been responsible for the changes in reductase activity. Erythrocyte glutathione S-transferase activity had increased slightly after 20 weeks of training and a much more marked increase was found after 40 weeks. This may have been indicative of the occurrence of lipid peroxidation in this phase of training. The participants ran a 15-km race after the first 20 weeks of training and a half marathon after 40 weeks. Blood glutathione tended to decrease after the 15-km race and increased after the half marathon. In both cases it had returned to normal values 5 days after the race. Erythrocyte glutathione reductase was elevated 1 day after the races, and had returned to normal after 5 days. This could also have been explained from concurrent changes in the riboflavin content of the erythrocytes. Erythrocyte glutathione S-transferase activity decreased after both races, but was restored 5 days after the half marathon while such was not the case after the 15-km race.

Influence of oxygen supply on liver condition and elimination of dimethylacetamide in the isolated perfused rat liver.

The effects were studied of improved oxygen supply on the integrity and metabolic activity towards dimethylacetamide of the isolated perfused rat liver. Improvement of oxygen supply by increased medium oxygenation or addition of chemical oxygen carriers (perfluortributylamine) or erythrocytes led to increased bile secretion. Leakage of lactate dehydrogenase and aspartate aminotransferase could be prevented during a 1-hr perfusion when either chemical oxygen carriers or erythrocytes were added. Improved medium oxygenation alone was not sufficient to prevent high enzyme leakage during the second half of the perfusion period. Histological evaluation confirmed the conclusion that less damage occurred when erythrocytes or perfluortributylamine were added to the perfusion medium. The metabolic clearance of dimethylacetamide by the perfused rat liver was not significantly improved when erythrocytes were added to the medium. The results show that addition of perfluortributylamine, or erythrocytes at a level of 4 g haemoglobin/litre, is necessary to maintain liver integrity for at least 1 hr in the liver perfusion system used in this study.