Restoration of sensitivity to oxazaphosphorines by inhibitors of aldehyde dehydrogenase activity in cultured oxazaphosphorine-resistant L1210 and cross-linking agent-resistant P388 cell lines. (57/62)

The sensitivity of cultured L1210 and P388 cells sensitive (L1210/0, P388/0) and resistant (L1210/OAP, P388/CLA) to oxazaphosphorines, to 4-hydroperoxycyclophosphamide, ASTA Z-7557, phosphoramide mustard, and acrolein was determined in the absence and presence of known (disulfiram, diethyldithiocarbamate, cyanamide) or suspected [ethylphenyl(2-formylethyl)phosphinate] inhibitors of aldehyde dehydrogenase activity. The L1210/OAP cell line is resistant specifically to the oxazaphosphorines; P388/CLA cells are partially cross-resistant to other cross-linking agents. All four inhibitors of aldehyde dehydrogenase activity potentiated the cytotoxic action of the oxazaphosphorines, 4-hydroperoxycyclophosphamide and ASTA Z-7557, against L1210/OAP and P388/CLA cells; in the presence of a sufficient amount of inhibitor, sensitivity was essentially fully restored in both cases. The inhibitors did not potentiate the cytotoxic action of the nonoxazaphosphorines, phosphoramide mustard and acrolein, against these cell lines. The cytotoxic action of the oxazaphosphorines and nonoxazaphosphorines against L1210/0 and P388/0 cells was not potentiated by any of the aldehyde dehydrogenase inhibitors. Inhibitors of xanthine oxidase or aldehyde oxidase activities did not potentiate the cytotoxic action of the oxazaphosphorines against L1210/OAP cells. These observations strongly suggest that (a) aldehyde dehydrogenase activity is an important determinant with regard to the sensitivity of a cell population to the oxazaphosphorines; (b) L1210/0 and P388/0 cells lack the relevant aldehyde dehydrogenase activity; (c) the phenotypic basis for the resistance to oxazaphosphorines by L1210/OAP cells is aldehyde dehydrogenase activity; and (d) the major reason that P388/CLA cells are resistant to oxazaphosphorines is aldehyde dehydrogenase activity.  (+info)

Hydrogen transfer between ethanol molecules during oxidoreduction in vivo. (58/62)

Rates of exchange catalysed by alcohol dehydrogenase were determined in vivo in order to find rate-limiting steps in ethanol metabolism. Mixtures of [1,1-2H2]- and [2,2,2-2H3]ethanol were injected in rats with bile fistulas. The concentrations in bile of ethanols having different numbers of 2H atoms were determined by g.l.c.-m.s. after the addition of [2H6]ethanol as internal standard and formation of the 3,5-dinitrobenzoates. Extensive formation of [2H4]ethanol indicated that acetaldehyde formed from [2,2,2-2H3]ethanol was reduced to ethanol and that NADH used in this reduction was partly derived from oxidation of [1,1-2H2]ethanol. The rate of acetaldehyde reduction, the degree of labelling of bound NADH and the isotope effect on ethanol oxidation were calculated by fitting models to the found concentrations of ethanols labelled with 1-42H atoms. Control experiments with only [2,2,2-2H3]ethanol showed that there was no loss of the C-2 hydrogens by exchange. The isotope effect on ethanol oxidation appeared to be about 3. Experiments with (1S)-[1-2H]- and [2,2,2-2H3]ethanol indicated that the isotope effect on acetaldehyde oxidation was much smaller. The results indicated that both the rate of reduction of acetaldehyde and the rate of association of NADH with alcohol dehydrogenase were nearly as high as or higher than the net ethanol oxidation. Thus, the rate of ethanol oxidation in vivo is determined by the rates of acetaldehyde oxidation, the rate of dissociation of NADH from alcohol dehydrogenase, and by the rate of reoxidation of cytosolic NADH. In cyanamide-treated rats, the elimination of ethanol was slow but the rates in the oxidoreduction were high, indicating more complete rate-limitation by the oxidation of acetaldehyde.  (+info)

Incorporation of the 1-pro-R and 1-pro-S hydrogen atoms of ethanol in the reduction of acids in the liver of intact rats and in isolated hepatocytes. (59/62)

Ethanol oxidation causes redox effects. The coupling of this oxidation via NADH to intermediary metabolism was studied in order to reveal the underlying mechanisms. Isolated rat hepatocytes were incubated with [1,1-2H2]-, (1R)-[1-2H]- and (1S)-[1-2H]-ethanol and the 2H incorporation was measured in lactate, beta-hydroxybutyrate, fumarate, malate, succinate, alpha-oxoglutarate and citrate. The results differed in the following ways from results obtained in intact rats. Lactate became labelled to an increasing extent, and in more than one position, indicating labelling of pyruvate. A small and constant fraction of malate and fumarate was formed without access to [2H]coenzyme. Addition of aspartate increased this fraction, which was concluded to be formed in the mitochondria. Citrate was essentially unlabelled. The 2H from (1R)-[1-2H]ethanol contributed to malate to a larger extent and to beta-hydroxybutyrate to a smaller extent, and 2H from (1S)-[1-2H]ethanol contributed to lactate to a smaller extent. These results indicate that the exchange via shuttle system was less efficient in isolated hepatocytes than in intact rats. The 2H incorporation was independent of concentration of [1,1-2H2]ethanol when this was above 5mM. Additions known to increase ethanol elimination, and cyanamide, which decreases it, had no marked effect on the 2H incorporation. This indicates equilibration of the NADH bound to alcohol dehydrogenase with free NADH. Disulfiram and cyanamide caused a decrease in the relative incorporation from (1S)-[1-2H]ethanol into malate in liver of intact rats. Addition of cyanamide to incubations with hepatocytes resulted in a decrease of the contribution of 2H from (1S)-[1-2H]ethanol in lactate, beta-hydroxybutyrate and malate. This indicates that acetaldehyde was only oxidized in the mitochondrial compartment.  (+info)

Selective suppression of schedule-induced ethanol drinking by antialcoholic drugs in rats. (60/62)

Effects of disulfiram and calcium cyanamide, antialcoholic drugs, on schedule-induced ethanol drinking as well as on schedule-controlled response (lever-pressing) under a fixed interval 1 min schedule of food reinforcement were investigated in Wistar strain rats. When ethanol solution was available, the schedule-induced ethanol drinking decreased depending on the ethanol concentration (2-8%). However, the dose of ethanol intake during the 1 hr experimental session was at maximum (2.8 g/kg) when 4% ethanol solution was available. Thereafter, 4% ethanol solution was used in the experiment for studying the effects of disulfiram and calcium cyanamide on the schedule-induced ethanol drinking. Disulfiram (100-200 mg/kg, p.o.), pretreated at 1 hr before the start of the experiment, tended to suppress schedule-induced water drinking. However, the same treatment of calcium cyanamide (5-10 mg/kg, p.o.) did not produce a marked change in it. In contrast, disulfiram (100 and 200 mg/kg) and calcium cyanamide (5 and 10 mg/kg) markedly suppressed schedule-induced ethanol drinking without eliciting a marked change in schedule-controlled response. The present results suggest that both disulfiram and calcium cyanamide selectively suppress ethanol drinking in rats.  (+info)

Interaction of calcium and lead in human erythrocytes. (61/62)

The interactions of calcium and lead on the human erythrocytes have been studied in vitro using 45Ca and 203Pb as tracers. The chemical groups binding calcium and lead on the erythrocytes were also investigated. Calcium ions in the plasma were shown to be capable of replacing the 203Pb on the red cells. More than 85% of the 203Pb in the erythrocyte was associated with the cytoplasmic components, and the rest was bound to the stromal membrane. About 90% of 45Ca was attached to erythrocyte membrane. Extraction of 45Ca and 203Pb-labelled erythrocyte membranes with chloroform/methanol mixture showed that the distribution patterns of these two nuclides are similar, with over 88% protein bound, less than 10% lipid bound, and traces in the aqueous phase. Chemical modification of erythrocyte membrane proteins with carbodi-imide, p-chloromercuribenzoate (PCMB), and maleic anhydride suggested that the carboxyl groups are responsible for binding lead and calcium to the red cell membrane. The SH groups may have a minor role in the binding for both cations. Amino groups did not appear to affect the binding of these cations. Gel chromatography of 45Ca-labelled erythrocyte membrane indicated that Ca++ bound to the same fraction of membrane proteins as 203Pb, corresponding to a molecular weight of about 130 000 to 230 000. A possible implication of these findings is that lead and calcium may compete for the same binding site(s) on the erythrocyte.  (+info)

Cyanamide-induced granulocytopenia. (62/62)

We report a 64-year-old male with granulocytopenia and dermatitis due to cyanamide treatment. We administered cyanamide for alcoholism. After about one month he suffered from scaly erythema over his whole body and granulocytopenia (granulocyte; 140/microliter) with maturation arrest in bone marrow. After cessation of cyanamide and the start of granulocyte colony-stimulating factor administration, the skin eruption ameliorated gradually, and the peripheral blood granulocyte counts increased. Cyanamide showed positive results in the drug lymphocyte stimulation test (198%) and the patch test led to the diagnosis of granulocytopenia and dermatitis induced by cyanamide. After restarting glibenclamide and diazepam administration, his granulocytopenia did not reoccur. To our knowledge, this is the first report of a case with granulocytopenia induced by cyanamide.  (+info)