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PubMed1998

Comparison of the in vitro cytotoxicity of hydroxylamine metabolites of sulfamethoxazole and dapsone.

The differential incidence of adverse drug reactions (ADR) between trimethoprim-sulfamethoxazole and dapsone might be explained, in part, by differences in the inherent toxicity of the hydroxylamine metabolites of sulfamethoxazole and dapsone. To test this hypothesis, the in vitro cytotoxicities of sulfamethoxazole hydroxylamine, dapsone hydroxylamine, and monoacetyldapsone hydroxylamine were compared using peripheral blood mononuclear cells (PBMC) from healthy volunteers. After 3 hr of exposure to hydroxylamine metabolites, PBMC were washed thoroughly to remove residual hydroxylamine, and viability was assessed 16 hr later by determination of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) conversion. A concentration-dependent toxicity was observed with each hydroxylamine metabolite. While dapsone hydroxylamine and monoacetyldapsone hydroxylamine were not significantly different, both showed significantly greater cytotoxic potency than sulfamethoxazole hydroxylamine (P < 0.05). This differential potency was not a function of differential stability in aqueous medium and was maintained over time. The effects of red blood cells (RBC), impermeable RBC "ghosts," and RBC lysate on hydroxylamine-induced cytotoxicity were determined using a two-compartment dialysis system. Amelioration of hydroxylamine-dependent cytotoxicity occurred when RBC were included in PBMC incubations. This apparent detoxifying effect was markedly greater using RBC lysate in comparison with impermeable "ghosts" (P < 0.05). No difference in detoxification was observed between sulfamethoxazole hydroxylamine and monoacetyldapsone hydroxylamine. Differences in the inherent cytotoxicity of their hydroxylamine metabolites do not appear to explain the differential incidence of ADR between trimethoprim-sulfamethoxazole and dapsone.

Cell Survival↗
PubMed1993

Reduction of dapsone hydroxylamine to dapsone during methaemoglobin formation in human erythrocytes in vitro.

The fate of the toxic metabolite of dapsone, dapsone hydroxylamine, has been studied in the human red cell. Twice-washed red cells were incubated at 37 degrees with dapsone hydroxylamine: at 3 and 5 min, 27.0 +/- 2.2 and 33.2 +/- 2.7% of the haemoglobin had been converted to methaemoglobin, leading to a maximum at 45 min (45 +/- 1.8%). HPLC analysis revealed that parent amine was produced from dapsone hydroxylamine during methaemoglobin formation in the red cells. At 3 min, conversion of dapsone hydroxylamine to dapsone reached 7.0 +/- 3.9% leading to a maximum at 30 min (18.1 +/- 3.7%). There was a linear relationship between hydroxylamine-dependent methaemoglobin formation and conversion of hydroxylamine to dapsone (r = 0.97). At 4 degrees, methaemoglobin and dapsone formation was greatly retarded, and did not exceed 10%. Co-incubation of diethyl dithiocarbamate (DDC) with dapsone hydroxylamine and red cells led to a marked increase in methaemoglobin formation (61.4 +/- 3.4%) compared with hydroxylamine and red cells alone (45.0 +/- 1.8%, P < 0.001) at 45 min, and conversion of dapsone hydroxylamine to dapsone was almost doubled at 45 min (35.7 +/- 5.3%) compared with hydroxylamine and red cells (18.1 +/- 2.5%). A linear relationship between methaemoglobin formation and dapsone formation (r = 0.96) was also shown to occur in the presence of DDC. Incubation of red cells with DDC and dapsone hydroxylamine caused a significantly greater reduction in glutathione levels (98.3 +/- 1.6%) compared with red cells and dapsone hydroxylamine alone (84.8 +/- 2.7%) at 5 min (P < 0.001), although there was no significant difference between the groups at 15 min (96.9 +/- 2.6 vs 98.1 +/- 2.2%). Intra-erythrocytic glutathione was then depleted by 75 +/- 3.4%, by pretreatment with diethyl maleate (6 mM), and these cells in the presence of the hydroxylamine showed a significant fall in both methaemoglobin generation (29.7 +/- 1.2 vs 35.0 +/- 1.7%) and parent amine formation (11.1 +/- 0.2 vs 16.5 +/- 1.1%) compared with untreated red cells at 45 min. It is possible that a cycle exists between hepatic oxidation of dapsone to its hydroxylamine and reduction to the amine within the red cell, which may lead to re-oxidation by hepatic cytochrome P450. This process may contribute to the persistence of the drug in vivo.

Dapsone↗
PubMed2000

A study on the interaction between hydroxylamine analogues and oxyhemoglobin in intact erythrocytes.

The oxidative potency of hydroxylamine (HYAM) and its O-derivatives (O-methyl- and O-ethyl hydroxylamine) is generally larger than the effects of the N-derivatives (N-methyl-, N-dimethyl-, and N,O-dimethyl hydroxylamine). The effects of the two groups of hydroxylamines also differ in a qualitative sense. To elucidate this difference in toxicity profiles we investigated the hemoglobin dependence of the toxicity, the occurrence of cell-damaging products like superoxide and H(2)O(2), and the cellular kinetics of the hydroxylamine analogues. All hydroxylamines were found to depend on the presence and accessibility of oxyhemoglobin to exert their toxicity. This did not provide an explanation for the different toxicity profiles. The interaction of some hydroxylamines with oxyhemoglobin is known to lead to the formation of radical intermediates. Differences in the stability of these radical products are known to occur, and in some cases secondary products are formed. This can contribute to the differences in toxicity. In this respect, production of superoxide radicals was demonstrated for all hydroxylamines in the reaction with oxyhemoglobin. Evidence for H(2)O(2) generation during the reaction of HYAM, O-methyl, O-ethyl-, and N-dimethyl hydroxylamine with oxyhemoglobin was also found. Next to variations in the products formed, differences in cellular kinetics are likely to be among the most important factors that explain the different toxicity patterns seen for the hydroxylamines in erythrocytes. Indeed, differences were found to exist for the kinetics of methemoglobin formation in erythrocytes. Not only was the final level of methemoglobin formed much lower for the N-derivatives, but also the reaction rate with oxyhemoglobin was slower than with HYAM and its O-derivatives. Except for N,O-dimethyl hydroxylamine (NODMH), the same pattern was seen in hemolysates. NODMH tripled its effect on hemoglobin in hemolysate compared with incubations in erythrocytes. This implies that cellular uptake is a limiting factor for NODMH. Since formation of H(2)O(2) is most likely a result of an interaction with hemoglobin, differences in kinetics of methemoglobin formation can be an explanation for the fact that NMH and NODMH did not produce H(2)O(2) to a detectable level. These results indicate that (a) the toxicity of all hydroxylamines depends on an interaction with oxyhemoglobin; (b) the interaction with hemoglobin produces radical intermediates and concomitantly superoxide radicals and H(2)O(2); and (c) differences in uptake, reaction rate with hemoglobin, and stability of the intermediates formed do exist for the different hydroxylamines and contribute to their differences in toxicity.

Ditiocarb↗
PubMed1998

Two mechanisms for toxic effects of hydroxylamines in human erythrocytes: involvement of free radicals and risk of potentiation.

The toxic potency of three industrially used hydroxylamines was studied in human blood cells in vitro. The parent compound hydroxylamine and the O-ethyl derivative gave very similar results. Both compounds induced a high degree of methemoglobin formation and glutathione depletion. Cytotoxicity was visible as Heinz body formation and hemolysis. High levels of lipid peroxidation occurred, in this respect O-ethyl hydroxylamine was more active than hydroxylamine. In contrast H2O2 induced lipid peroxidation was lowered after O-ethyl hydroxylamine or hydroxylamine treatment, this is explained by the ferrohemoglobin dependence of H2O2 induced radical species formation. Glutathione S-transferase (GST) and NADPH methemoglobin reductase (NADPH-HbR) activities were also impaired, probably as a result of the radical stress occurring. The riboflavin availability was decreased. Other enzyme activities glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G6PDH), glucose phosphate isomerase and NADH methemoglobin reductase, were not or only slightly impaired by hydroxylamine or O-ethyl hydroxylamine treatment. A different scheme of reactivity was found for N,O-dimethyl hydroxylamine. This compound gave much less methemoglobin formation and no hemolysis or Heinz body formation at concentrations up to and including 7 mM. Lipid peroxidase induction was not detectable, but could be induced by subsequent H2O2 treatment. GST and NADPH-HbR activities and riboflavin availability were not decreased. On the other hand GR and G6PDH activities were inhibited. These results combined with literature data indicate the existence of two different routes of hematotoxicity induced by hydroxylamines. Hydroxylamine as well as O-alkylated derivatives primarily induce methemoglobin, a process involving radical formation. The radical stress occurring is probably responsible for most other effects. N-alkylated species like N,O-dimethyl hydroxylamine primarily lead to inhibition of the protective enzymes G6PDH and GR. Since these enzymes play a key role in the protection of erythrocytes against oxidative stress a risk of potentiation during mixed exposure does exist.

Dimethylamines↗
PubMed1977

Activation of guanylate cyclase in cerebral cortex of rat by hydroxylamine.

Hydroxylamine actived guanylate cyclase in particulate fraction of cerebral cortex of rat. Activation was most remarkable in crude mitochondrial fraction. When the crude mitochondrial fraction was subjected to osmotic shock and fractionated, guanylate cyclase activity recovered in the subfractions as assayed with hydroxylamine was only one-third of the starting material. Recombination of the soluble and the particulate fractions, however, restored guanylate cyclase activity to the same level as that of the starting material. When varying quantities of the particulate and soluble fractions were combined, enzyme activity was proportional to the quantity of the soluble fraction. Heating of the soluble or particulate fraction at 55 degrees for 5 min inactivated guanylate cyclase. The heated particulate fraction markedly activated guanylate cyclase activity in the native soluble fraction, while the heated soluble fraction did not stimulate enzyme activity in the particulate. The particulate fraction preincubated with hydroxylamine at 37 degrees for 5 min followed by washing activated guanylate cyclase activity in the soluble fraction in the absence of hydroxylamine. Further fractionation of the crude mitochondrial fraction revealed that the factor(s) needed for the activation by hydroxylamine is associated with the mitochondria. The mitochondrial fraction of cerebral cortex activated guanylate cyclase in supernatant of brain, liver, or kidney in the presence of hydroxylamine. The mitochondrial fraction prepared from liver or kidney, in turn, activated soluble guanylate cyclase in brain. Activation of guanylate cyclase by hydroxylamine was compared with that of sodium azide. Azide activated guanylate cyclase in the synaptosomal soluble fraction, while hydroxylamine inhibited it. The particulate fraction preincubated with azide followed by washing did not stimulate guanylate cyclase activity in the absence of azide. The activation of guanylate cyclase by hydroxylamine is not due to a change in the concentration of the substrate GTP, Addition of hydroxylamine did not alter the apparent Km value of guanylate cyclase for GTP. Guanylate cyclase became less dependent on manganese in the presence of hydroxylamine. Thus the activation of guanylate cyclase by hydroxylamine is due to the change in the Vmax of the reaction.

Animals↗
PubMed1987

Reactions of hydroxylamine with the electron-donor side of photosystem II.

The reaction of hydroxylamine with the O2-evolving center of photosystem II (PSII) in the S1 state delays the advance of the H2O-oxidation cycle by two charge separations. In this paper, we compare and contrast the reactions of hydroxylamine and N-methyl-substituted analogues with the electron-donor side of PSII in both O2-evolving and inactivated [tris(hydroxymethyl)aminomethane- (Tris-) washed] spinach PSII membrane preparations. We have employed low-temperature electron paramagnetic resonance (EPR) spectroscopy in order to follow the oxidation state of the Mn complex in the O2-evolving center and to detect radical oxidation products of hydroxylamine. When the reaction of hydroxylamine with the S1 state in O2-evolving membranes is allowed to proceed to completion, the S2-state multiline EPR signal is suppressed until after three charge separations have occurred. Chemical removal of hydroxylamine from treated PSII membrane samples prior to illumination fails to reverse the effects of the dark reaction, which argues against an equilibrium coordination of hydroxylamine to a site in the O2-evolving center. Instead, the results indicate that the Mn complex is reduced by two electrons by hydroxylamine, forming the S-1 state. An additional two-electron reduction of the Mn complex to a labile "S-3" state probably occurs by a similar mechanism, accounting for the release of Mn(II) ions upon prolonged dark incubation of O2-evolving membranes with high concentrations of hydroxylamine. In N,N-dimethylhydroxylamine-treated, Tris-washed PSII membranes, which lack O2 evolution activity owing to loss of the Mn complex, a large yield of dimethyl nitroxide radical is produced immediately upon illumination at temperatures above 0 degrees C. The dimethyl nitroxide radical is not observed upon illumination under similar conditions in O2-evolving PSII membranes, suggesting that one-electron photooxidations of hydroxylamine do not occur in centers that retain a functional Mn complex. We suggest that the flash-induced N2 evolution observed in hydroxylamine-treated spinach thylakoid membrane preparations arises from recombination of hydroxylamine radicals formed in inactivated O2-evolving centers.

Chlorophyll↗
PubMed1981

The steady-state kinetics of the NADH-dependent nitrite reductase from Escherichia coli K 12. Nitrite and hydroxylamine reduction.

The reduction of both NO2- and hydroxylamine by the NADH-dependent nitrite reductase of Escherichia coli K 12 (EC 1.6.6.4) appears to follow Michaelis-Menten kinetics over a wide range of NADH concentrations. Substrate inhibition can, however, be detected at low concentrations of the product NAD+. In addition, NAD+ displays mixed product inhibition with respect to NADH and mixed or uncompetitive inhibition with respect to hydroxylamine. These inhibition characteristics are consistent with a mechanism in which hydroxylamine binds during catalysis to a different enzyme form from that generated when NAD+ is released. The apparent maximum velocity with NADH as varied substrate increases as the NAD+ concentration increases from 0.05 to 0.7 mM with 1 mM-NO2- or 100 mM-hydroxylamine as oxidized substrate. This increase is more marked for hydroxylamine reduction than for NO2- reduction. Models incorporating only one binding site for NAD can account for the variation in the Michaelis-Menten parameters for both NADH and hydroxylamine with [NAD+] for hydroxylamine reduction. According to these models, activation of the reaction occurs by reversal of an over-reduction of the enzyme by NADH. If the observed activation of the enzyme by NAD+ derives both from activation of the generation of the enzyme-hydroxylamine complex from the enzyme-NO2- complex during NO2- reduction and from activation of the reduction of the enzyme-hydroxylamine complex to form NH4+, then the variation of Vapp. for NO2- or hydroxylamine with [NAD+] is consistent with the occurrence of the same enzyme-hydroxylamine complex as an intermediate in both reactions.

Escherichia coli↗
PubMed1993

Reduction of dapsone hydroxylamine to dapsone during methaemoglobin formation in human erythrocytes in vitro--II. Movement of dapsone across a semipermeable membrane into erythrocytes and plasma.

We have used an in vitro two-compartment model, to investigate the ability of dapsone, formed by erythrocyte-mediated detoxification of its hydroxylamine metabolite, to escape the cells and cross a semi-permeable membrane into both plasma and other erythrocytes. Both diethyl dithiocarbamate (DDC) treated and untreated erythrocytes were incubated with dapsone hydroxylamine and dialysed against either fresh cells or plasma. Methaemoglobin was predominantly detectable in compartment A although the presence of low levels of methaemoglobin in compartment B indicated that the hydroxylamine itself had crossed the membrane. In contrast to methaemoglobin disposition, recovery of dapsone was higher (P < 0.05) in compartment B compared with A for all three treatment groups at 30 and 60 min, but not at the remaining time points. Regression analysis of the cumulative recovery of dapsone over 150 min in all three treatment groups for both compartments A and B showed correlation coefficients close to unity. In compartment A, analysis of the mean slopes of the regression lines indicated that, overall, significantly more dapsone was recovered from group 1 (erythrocytes, hydroxylamine and DDC dialysed against untreated red cells) compared with group 3 (erythrocytes and hydroxylamine dialysed against plasma) (0.22 +/- 0.05 vs 0.09 +/- 0.005; P < 0.025). Also in compartment A, significantly more dapsone was recovered from group 2 (erythrocytes and hydroxylamine dialysed against untreated red cells) compared with group 3 (erythrocytes and hydroxylamine dialysed against plasma: 0.16 +/- 0.02 vs 0.09 +/- 0.005). In compartment B, dapsone recovery was significantly greater in group 1 (erythrocytes, hydroxylamine and DDC dialysed against untreated red cells; slope of regression line: 0.59 +/- 0.05) compared with group 2 (erythrocytes and hydroxylamine dialysed against untreated red cells; slope of line: 0.28 +/- 0.02, P < 0.005). In addition, dapsone recovery was significantly greater in group 1 (0.59 +/- 0.05) compared with group 3 (erythrocytes and hydroxylamine dialysed against plasma; 0.21 +/- 0.02, P < 0.005). Dialysis of erythrocytes with dapsone itself over 120 min caused no detectable methaemoglobin formation. The process of erythrocyte-mediated dapsone formation from its hydroxylamine may feasibly occur in vivo and contribute to the systemic persistence and therapeutic effect of dapsone.

Cell Membrane Permeability↗