Toxicity of aromatic disulphides. III. In vivo haemolytic activity of aromatic disulphides

Diphenyl disulphide, 4,4'-diaminodiphenyl disulphide, 2,2'-diaminodiphenyl disulphide, 4,4'-dimethyldiphenyl disulphide and 4,4'-dinitrodiphenyl disulphide, when administered orally to rats, induced haematological and pathological changes indicative of erythrocyte destruction in vivo. No evidence of haemolysis was detected, however, in animals receiving diphenyl disulphide-2,2'-dicarboxylic acid or dibenzyl disulphide. The order of activity of the various aromatic disulphides in provoking in vivo haemolysis was similar to that previously recorded for 'active oxygen' generation and erythrocyte damage in vitro. The results of this investigation suggest that in vivo haemolysis may be anticipated from any disulphide or thiol which undergoes appreciable autoxidation at neutral pH. While aromatic or alpha beta-unsaturated thiols and disulphides would be expected to be the most active haemolytic agents, other thiols or disulphides may precipitate the destruction of erythrocytes whose defences against oxidative attack are deficient.     

Steric effects on the haemolytic activity of aromatic disulphides in rats

Certain derivatives of diphenyl disulphide are known to cause haemolytic anaemia in rats, by a mechanism possibly involving intra-erythrocytic redox cycling with concomitant generation of 'active oxygen' species. In ring-substituted diphenyl disulphide derivatives, electronic effects of substituents have been shown markedly to affect the rate of 'active oxygen' production in vitro and toxicity in vivo. In the present study, the influence of steric effects of substituents on these parameters has been investigated. The severity of the haemolysis induced in groups of seven rats by oral dosing with 4,4'-dimethoxydiphenyl disulphide and 4,4'-dimethyldiphenyl disulphide at doses of 500 mumol/kg/day for 6 days was greater than that of the 2,2' isomers and the haemolytic activity of a series of 2,2'-dialkyl derivatives decreased with increasing size of the alkyl group. In vitro, the haematin-catalysed oxidation rates and the rates of redox cycling of the corresponding thiols in the presence of glutathione were similarly influenced by steric hindrance. The structure-activity relationships identified in the present investigation, together with knowledge of the electronic effects of substituents, should permit accurate prediction of the toxicity of new or untested aromatic thiols and disulphides.     

二(2-吡啶基-N-氧化物)二硫化物对K562细胞增殖的抑制作用

目的研究二(2-吡啶基-N-氧化物)二硫化物(L)对K562细胞增殖的影响。方法应用MTT比色法检测L对K562细胞增殖的影响;应用透射电镜检测L对K562细胞凋亡形态学的影响;应用流式细胞仪检测L对K562细胞周期的影响。结果 L可抑制K562细胞的增殖,呈现浓度依赖性;L可使K562细胞呈现凋亡形态学特征;L可使K562细胞周期发生阻滞。结论 L在体外具有抑制K562细胞增殖的作用。     

Investigation of thiol-disulphide balance in patients with acute urticaria and chronic spontaneous urticaria

Background: Thiol–disulphide balance plays a major role in health and diseases. This balance may be disrupted by various diseases. We aimed to determine status of the effect of thiol–disulphide balance in urticaria.

Objectives: We aimed to investigate the thiol–disulphide balance in patients with acute urticaria (AUP) and chronic spontaneous urticaria (CSU).

Methods: Study included 53 AUP and 47 healthy controls plus 57 patients with chronic spontaneous urticaria (CSUP) and 57 healthy controls. Levels of native thiols, disulphides and total thiols were evaluated in plasma using a new and automated spectrophotometric method. Ratios of disulphides/total thiols, disulphides/native thiols and native thiols/total thiols were calculated.

Results: For AU, there was no statistical difference compared to control group in levels of native thiols, disulphides and total thiols. For CSU, however, there was an increase in levels of native thiols, disulphides and total thiols and the ratio of thiol/disulphide in favour of disulphide.

Conclusion: Thiol–disulphide balance was not affected by AU but shifted towards to disulphide in CSU indicating the presence of oxidative stress (OS).     

The crude venom from the sea anemone Stichodactyla helianthus induces haemolysis and slight peroxidative damage in rat and human erythrocytes.

The haemolytic and peroxidative effects of crude venom of the sea anemone Stichodactyla helianthus were evaluated in rat and human erythrocytes. Venom extract caused a significant concentration-dependent effect on haemolysis (release of haemoglobin). Human erythrocytes were more sensitive (0.094 mg protein/ml) than those of the rats (0.3787 mg protein/ml). In contrast, a light effect on lipid peroxidation (LP, an index of oxidative damage to membrane lipids) was recorded. The concentrations needed to produce a significant effect on LP in rat and human erythrocytes were, respectively, 2-fold and 7-fold higher than those required to produce significant haemolysis. The differential effect of S. helianthus venom on haemolysis and oxidation of membrane lipids is not common for venoms of other sea anemones, which usually show a tightly related effect on LP and haemolytic damage.     

Toxicity of aromatic disulphides. II. Intraerythrocytic hydrogen peroxide formation and oxidative damage by aromatic disulphides

Diphenyl disulphide has been shown to generate hydrogen peroxide in erythrocytes in vitro. It also induces oxidative damage (reversible and irreversible haemoglobin oxidation, depletion of non-protein and protein-bound thiols) in these cells. Such changes were also recorded in erythrocytes exposed to 4,4'-diaminodiphenyl disulphide, 2,2'-diaminodiphenyl disulphide, 4,4'-dimethyldiphenyl disulphide, 4,4'-dinitrodiphenyl disulphide, diphenyl disulphide-2,2'-dicarboxylic acid and dibenzyl disulphide. The relative potency of these compounds in causing erythrocyte damage is correlated with their ability to generate 'active oxygen' species in vitro.     

Bradykinin-potentiating activity of captopril disulphide dimer (SQ 14,551)

The ability of captopril and one of its metabolites, the disulphide dimer have been studied for their ability to affect angiotensin I and bradykinin responses. Captopril disulphide dimer (i.v.) potentiated the vasodilatory effects of bradykinin in urethane-anaesthetized rats, at doses of 0.1 and 0.3 mg/kg but did not inhibit the angiotensin I-mediated pressor response at these same concentrations. This activity of captopril disulphide dimer in vivo was not seen with bradykinin responses in isolated guinea pig ileum except at bath concentrations much higher than for captopril (10(-5) M). However captopril and captopril disulphide dimer at oral doses of 10 mg/kg both lowered systolic and diastolic blood pressures in spontaneously hypertensive rats. These studies are consistent with an in vivo bradykinin-potentiating activity of captopril disulphide dimer and suggest a possible antihypertensive activity of disulphide metabolites of captopril.     

Dynamic thiol/disulphide homeostasis in patients with basal cell carcinoma

Background: The aim of this study is to measure and compare the dynamic thiol/disulphide homeostasis of patients with basal cell carcinoma and healthy subjects with a newly developed and original method.

Objective: Thirty four patients attending our outpatient clinic and clinically and histopathologically diagnosed as nodular basal cell carcinoma, and age and gender matched 30 healthy individuals have been involved in the study. Thiol/disulphide homeostasis tests have been measured with a novel automatic spectrophotometric method developed and the results have been compared statistically.

Results: Serum native thiol and disulphide levels in the patient and control group show a considerable variance statistically (p?=?0.028, 0.039, respectively). Total thiol levels do not reveal a considerable variation (p?=?0.094). Disulphide/native thiol ratios and native thiol/total thiol ratios also show a considerable variance statistically (p?=?0.012, 0.013, 0.010, respectively).

Conclusions: Thiol disulphide homeostasis in patients with basal cell carcinoma alters in the way that disulphide gets lower and thiols get higher. Thiol/disulphide level is likely to have a role in basal cell carcinoma pathogenesis.     

Interaction of cysteamine with the thiol and disulphide groups in deoxyribonucleoproteins

The thiol content of deoxyribonucleoproteins (DNP), isolated from Ehrlich ascites tumor cells by 1 M NaCl extraction, was measured by amperometric titration with AgNO₃ before and after reduction of the disulphide bonds with NaBH₄. The amount of -SH and SS groups was 59 μmoles and 19 $\text{μmoles}\text{g}$ protein, respectively. About 20 μmoles -$\text{SH}\text{g}$ protein of the total thiol content of DNP could be extracted with a weak acid after reduction of the disulphide bonds. In addition to proteins, the acid soluble fraction contained also glutathione and unidentified -SH containing polypeptides with molecular weights larger than glutathione. The radioprotective compound cysteamine was found to form mixed disulphides with the -SH groups of DNP, and to reduce existing disulphide bonds and thereby release DNP-bound peptides. The possible importance of -SH and disulphide groups in DNP for radiation protection is discussed.     

D-penicillamine metabolism: the role of transformation in blood plasma

The transformation of D-penicillamine (D-pen) was studied in orally- and intravenously-dosed rats and in human plasma in vitro. In each case, low molecular weight (LMW) metabolites (previously identified as disulphides) and a mixed disulphide between D-pen and albumin (D-pen-protein) formed. The rates of D-pen elimination, other than through protein conjugation, were comparable in the rat groups to the rate of oxidation to LMW metabolites in vitro. The rates of transformation to D-pen-protein were also comparable in the in vitro preparations and in orally-treated rats. These qualitative and quantitative similarities suggest blood plasma may be an important site of transformation in vivo. Extracellular oxidation of D-pen may be linked to its antirheumatic action, either through reduction of oxygen species or through formation of D-pen-protein disulphides at surfaces of mononuclear leukocytes.     

Haemolytic activity and nephrotoxicity of 2-hydroxy-1,4-naphthoquinone in rats

The short-term toxicity of 2-hydroxy-1,4-naphthoquinone (lawsone) and 2-methyl-1,4-naphthoquinone (menadione) has been compared in rats. 2-Methyl-1,4-naphthoquinone has been shown previously to cause haemolytic anaemia in animals, and this was confirmed in the present experiment. 2-Hydroxyl-1,4-naphthoquinone was found also to cause haemolysis, in a dose-dependent manner, as reflected by decreased blood packed cell volumes and haemoglobin levels and by histopathological changes in spleen, liver and kidney. With both naphthoquinones, the haemolysis was of the oxidative type, characterized by the presence of Heinz bodies within erythrocytes. Haemolysis was the only toxic change identified in rats dosed with 2-methyl-1,4-naphthoquinone. In contrast, 2-hydroxyl-1,4-naphthoquinone was not only a haemolytic agent but also a nephrotoxin, causing renal enlargement, elevated plasma levels of urea and creatinine and histologically-identified tubular necrosis, largely confined to the distal segment of the proximal convoluted tubules. The relationship between the in vivo toxic effects of these naphthoquinones and previously-reported data on their in vitro cytotoxic action is discussed.     

Relevance of NADPH depletion and mixed disulphide formation in rat lung to the mechanism of cell damage following paraquat administration

We have examined the possibility that the mechanism of paraquat toxicity in the lung involves both the formation of mixed disulphides (the amount of NPSH or GSH involved in protein disulphide formation) and the prolonged oxidation of NADPH, leading to NADPH depletion. We have compared the oxidation-reduction status of the lung, 2, 8 and 24 hr after dosing rats subcutaneously with 20 mg paraquat/kg (a dose which causes extensive lung damage 24 hr after dosing) or 20 mg diquat/kg (a chemically related bipyridyl which only causes very minimal lung damage 24 hr after dosing). Lung NADP⁺ levels were not affected 2, 8 or 24 hr after dosing with either bipyridyl. However, although NADPH levels were unchanged 2 hr after dosing with paraquat, and 2, 8 and 24 hr after dosing with diquat, there was a significant decrease in NADPH levels by 8 and 24 hr after dosing with paraquat. The changes in NADPH levels were coincident with the lung damage (characterized previously) caused by these treatments. In contrast with these effects on NADPH levels, there was an increase in NPSH and GSH levels in the lung by 8 and 24 hr after dosing with paraquat or diquat. Thus, there was no simple relationship between lung NADPH levels and lung sulphydryl levels.Lung mixed disulphide levels (the amounts of NPSH or GSH involved in disulphide formation) were increased 2, 8 and 24 hr after dosing with paraquat or diquat, although oxidized glutathione levels remained normal. Thus, an early and persistent biochemical effect of paraquat and diquat in the lung involves an increase in mixed disulphide levels, which is probably a consequence of the lungs' response to an increase in the oxidation of NADPH and GSH. As suggested previously, the increase in mixed disulphide levels appears to be a mechanism for regulating the normal redox state of the lung. However, despite this regulatory mechanism, NADPH depletion occurs 8 and 24 hr after dosing with paraquat, but not diquat, coincident with the development of lung damage.In conclusion, we suggest that mixed disulphide formation is not only a regulatory mechanism, but in some circumstances also may cause changes in essential biosynthetic and regulatory functions of the lung. This may ultimately lead to the drop in NADPH levels, which we propose is a critical biochemical event in the development of alveolar epithelial cell damage following the administration of paraquat.     

Increase in hepatic mixed disulphide and glutathione disulphide levels elicited by paraquat

Paraquat (1 mM), when added to isolated haemoglobin-free perfused rat liver, leads to an increase of intracellular mixed disulphides from 1.3 μmole GSH equivalents per g wet weight in the controls to 2.5 μmole/g. This raises the proportion of mixed disulphides to total glutathione equivalents from about 0.2 at the beginning of the perfusion to about 0.4. The mixed disulphides are predominantly protein-bound, with low molecular weight compounds being quantitatively negligible.The content of intracellular glutathione disulphide (GSSG) is increased from 17 nmole/g in the controls to 38 nmole/g in the presence of paraquat. In addition, there is an increased rate of release of GSSG into the extracellular (biliary) space, reported previously.It is suggested that, in a reaction catalysed by thioltransferase(s), the rise in GSSG is correlated with the rise in mited disulphides (reaction 1). Occupancy of potential cellular mixed disulphide sites is about $\text{1}\text{2}$ in the controls, and rises to about $\text{2}\text{3}$ in the presence of paraquat.The ratio of cellular contents, NADPH/NADP⁺, is decreased from 5.1 in the controls to 2.3 in the presence of paraquat, while the sum of NADPH plus NADP⁺ remains unaltered.The perturbation in the glutathione status may be related to metabolic effects such as the stimulation of the pentose-phosphate pathway activity, and possibly also to the expression of toxic effects.     

The determination of D-penicillamine and its disulphide in plasma by reversed-phase ion-pair high-performance liquid chromatography

Reversed-phase ion-pair conditions are used for the determination of D-penicillamine and penicillamine disulphide. Two chromatographic systems were employed, one for penicillamine and the other for penicillamine disulphide. The procedures permit the determination of total penicillamine (protein-bound, free and as disulphides) in whole plasma, and total penicillamine (free and as disulphides) in plasma ultrafiltrate, using an incubation step in the presence of dithiothreitol. Free penicillamine and penicillamine disulphide may be determined independently by direct injection of plasma ultrafiltrate. Both solutes may be measured at an on-column sensitivity of 10 ng, utilizing an electrochemical detector based on a glassy carbon electrode.     

Synergistic neuroprotection by bis(7)-tacrine via concurrent blockade of N-methyl-D-aspartate receptors and neuronal nitric-oxide synthase

The excessive activation of the N-methyl-D-aspartate receptor (NMDAR)/nitric oxide (NO) pathway has been proposed to be involved in the neuropathology of various neurodegenerative disorders. In this study, NO was found to mediate glutamate-induced excitotoxicity in primary cultured neurons. Compared with the NO synthase (NOS) inhibitor, N(G)-monomethyl-L-arginine (L-NMMA), and the NMDAR antagonist memantine, bis(7)-tacrine was found to be more potent in reducing NO-mediated excitotoxicity and the release of NO caused by glutamate. Moreover, like L-NMMA but not like 5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) and memantine, bis(7)-tacrine showed greater neuroprotection and inhibition on NO release when neurons were pretreated for a prolonged time between 0 and 24 h and remained quite potent even when neurons were post-treated 1 h after the glutamate challenge. Bis(7)-tacrine was additionally found to be as moderately potent as memantine in competing with [(3)H]MK-801, inhibiting NMDA-evoked currents and reducing glutamate-triggered calcium influx, which eventually reduced neuronal NOS activity. More importantly, at neuroprotective concentrations, bis(7)-tacrine substantially reversed the overactivation of neuronal NOS caused by glutamate without interfering with the basal activity of NOS. Furthermore, in vitro pattern analysis demonstrated that bis(7)-tacrine competitively inhibited both purified neuronal and inducible NOS with IC(50) values at 2.9 and 9.3 microM but not endothelial NOS. This result was further supported by molecular docking simulations that showed hydrophobic interactions between bis(7)-tacrine and three NOS isozymes. Taken together, these results strongly suggest that the substantial neuroprotection against glutamate by bis(7)-tacrine might be mediated synergistically through the moderate blockade of NMDAR and selective inhibition of neuronal NOS.     

Evidence for redox cycling of lawsone (2-hydroxy-1,4-naphthoquinone) in the presence of the hypoxanthine/xanthine oxidase system

This study reports that lawsone (2-hydroxy-1,4-naphthoquinone) undergoes redox cycling in the presence of the hypoxanthine/xanthine oxidase system. The rate of cytochrome c reduction obtained in the presence of 80 microM lawsone was almost three times the rate of cytochrome c reduction measured in its absence. This increase in the rate of cytochrome c reduction was partially inhibited by superoxide dismutase, suggesting the involvement of O(2)(.-) in this process. It is remarkable to note that, even though lawsone is considered to be a non-redox-cycling quinone in vitro, this quinone was shown to be more toxic in vivo in rats than menadione, causing haemolytic anemia of an oxidative nature and renal damage. The view that this quinone is a non-redox-cycling quinone was based on the inability of one-electron-transferring flavoenzymes such as NADPH-cytochrome c reductase to reduce this naphthoquinone. Our finding that lawsone, like menadione, undergoes redox cycling in the presence of the hypoxanthine/xanthine oxidase system could explain the observed oxidative damage of tissues inflicted by this quinone in rats in vivo. Such an observation therefore reconciles the in vivo toxicity results of this naphthoquinone with those of in vitro experiments.     

The role of quinine in haemolysis

Quinine produces haemolysis of rabbit and human red blood cells in concentrations up to 1 in 700. In a concentration of 1 in 10,000 it increases the degree of haemolysis produced by various haemolytic agents like saponin, bile salts and digitonin in vitro. Quinine when injected intravenously in 35 mg./kg. dose in rabbits increases the susceptibility of red blood cells to the haemolytic action of saponin. Intravascular haemolysis following the administration of quinine is seen in cases of blackwater fever, and on the basis of experimental work described in the paper it is suggested that quinine precipitates these haemolytic episodes by rendering the red blood cells more susceptible to the action of tissue lytic factors.     

Mechanism of bis(7)-tacrine inhibition of GABA-activated current in cultured rat hippocampal neurons

Bis(7)-tacrine is a novel dimeric acetylcholinesterase inhibitor derived from tacrine that shows promise for the treatment of Alzheimer's disease. We have previously reported that bis(7)-tacrine inhibits GABA_A receptors. In the present study we investigated the mechanism of bis(7)-tacrine inhibition of GABA_A receptor function using whole-cell patch-clamp recording in cultured rat hippocampal neurons. Bis(7)-tacrine produced a gradual decline of GABA-activated current to a steady-state, but this was not an indication of use-dependence, as the gradually declining component could be eliminated by exposure to bis(7)-tacrine prior to GABA application. In addition, bis(7)-tacrine inhibition did not require the presence of agonist, and GABA-activated current recovered completely from inhibition by bis(7)-tacrine in the absence of agonist. The slow onset of inhibition by bis(7)-tacrine was not apparently due to an action at an intracellular site, as inclusion of 25 μM bis(7)-tacrine in the recording pipette did not alter inhibition by bis(7)-tacrine applied externally. Bis(7)-tacrine shifted the GABA concentration-response curve to the right in a parallel manner and the pA₂ value estimated from a Schild plot was 5.7. Bis(7)-tacrine increased the time constant of activation of GABA-gated ion channels without affecting the time constants of deactivation or desensitization. These results suggest that bis(7)-tacrine is a competitive GABA_A receptor antagonist with slow onset and offset kinetics. The competitive inhibition of GABA receptors by bis(7)-tacrine could contribute to its ability to enhance memory.     

Antifungal evaluation of bis Mannich bases derived from acetophenones and their corresponding piperidinols and stability studies

The development of resistance to current antifungal therapeutics drives the search for effective new agents. The fact that some acetophenone-derived Mannich bases had shown antifungal activities in our previous studies led us to design and synthesize acetophenone-derived bis Mannich bases, B1-B5, bis(beta-aroylethyl)methylamine hydrochlorides, to evaluate their antifungal activity. These bis Mannich bases were then converted to the corresponding piperidinols, C1-C5, which are structural isomers of bis derivatives, 3-aroyl-4-aryl-1-methyl-4-piperidinol hydrochlorides, to see alterations in biological activity. A stability study of B1 and Cl was also carried out to estimate whether they alkylate the thiols. All compounds studied have shown antifungal activity, especially against dermatophytes (Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, and Microsporum canis), in the concentration range studied (2-128 microng/ml). The activity was especially apparent against T. tonsurans. All compounds had at least equal antifungal activity compared with the reference compound amphotericin-B against T. tonsurans. Bis Mannich bases were generally found to be more potent compounds than their structural isomer piperidinols. The results of our stability studies suggest that thiol alkylation may contribute to the antifungal activity of the Mannich bases synthesized. Even though all compounds showed antifungal activity against dermatophytes, bis Mannich bases B1, B2, B4, and B5 appear to have potential for developing novel antifungal agents against dermatophytes.     

Lack of antagonism between thioglycerol and an oxytocin analogue not containing a disulphide bond

1. The antagonistic effect of thioglycerol against oxytocin and an analogue of oxytocin not containing a disulphide bridge (desamino-1-carba-oxytocin) has been compared in the rat isolated depolarized uterus.2. Thioglycerol clearly differentiated between the two compounds, 40 mM producing a dose ratio of 12 with oxytocin but only 1.5 with the carba compound.3. It was confirmed that thioglycerol produces no appreciable destruction of oxytocin in vitro.4. The mode of action of thiols in antagonizing specifically S-S polypeptides is discussed. It is concluded that they probably do not produce their effect either by inactivating receptors or by inactivating S-S polypeptides in solution. A possible mechanism is that thiols potentiate the destruction of S-S polypeptides at the receptor site.