Occupational methaemoglobinaemia. Mechanisms of production, features, diagnosis and management including the use of methylene blue

Methaemoglobin is formed by oxidation of ferrous (FeII) haem to the ferric (FeIII) state and the mechanisms by which this occurs are complex. Most cases are due to one of three processes. Firstly, direct oxidation of ferrohaemoglobin, which involves the transfer of electrons from ferrous haem to the oxidising compound. This mechanism proceeds most readily in the absence of oxygen. Secondly, indirect oxidation, a process of co-oxidation which requires haemoglobin-bound oxygen and is involved, for example, in nitrite-induced methaemoglobinaemia. Thirdly, biotransformation of a chemical to an active intermediate that initiates methaemoglobin formation by a variety of mechanisms. This is the means by which most aromatic compounds, such as amino- and nitro-derivatives of benzene, produce methaemoglobin. Methaemoglobinaemia is an uncommon occupational occurrence. Aromatic compounds are responsible for most cases, their lipophilic nature and volatility facilitating absorption during dermal and inhalational exposure, the principal routes implicated in the workplace. Methaemoglobinaemia presents clinically with symptoms and signs of tissue hypoxia. Concentrations around 80% are life-threatening. Features of toxicity may develop over hours or even days when exposure, whether by inhalation or repeated skin contact, is to relatively low concentrations of inducing chemical(s). Not all features observed in patients with methaemoglobinaemia are due to methaemoglobin formation. For example, the intravascular haemolysis caused by oxidising chemicals such as chlorates poses more risk to life than the methaemoglobinaemia that such chemicals induce. If an occupational history is taken, the diagnosis of methaemoglobinaemia should be relatively straightforward. In addition, two clinical observations may help: firstly, the victim is often less unwell than one would expect from the severity of 'cyanosis' and, secondly, the 'cyanosis' is unresponsive to oxygen therapy. Pulse oximetry is unreliable in the presence of methaemoglobinaemia. Arterial blood gas analysis is mandatory in severe poisoning and reveals normal partial pressures of oxygen (pO2) and carbon dioxide (pCO2,), a normal 'calculated' haemoglobin oxygen saturation, an increased methaemoglobin concentration and possibly a metabolic acidosis. Following decontamination, high-flow oxygen should be given to maximise oxygen carriage by remaining ferrous haem. No controlled trial of the efficacy of methylene blue has been performed but clinical experience suggests that methylene blue can increase the rate of methaemoglobin conversion to haemoglobin some 6-fold. Patients with features and/or methaemoglobin concentrations of 30-50%, should be administered methylene blue 1-2 mg/kg/bodyweight intravenously (the dose depending on the severity of the features), whereas those with methaemoglobin concentrations exceeding 50% should be given methylene blue 2 mg/kg intravenously. Symptomatic improvement usually occurs within 30 minutes and a second dose of methylene blue will be required in only very severe cases or if there is evidence of ongoing methaemoglobin formation. Methylene blue is less effective or ineffective in the presence of glucose-6-phosphate dehydrogenase deficiency since its antidotal action is dependent on nicotinamide-adenine dinucleotide phosphate (NADP+). In addition, methylene blue is most effective in intact erythrocytes; efficacy is reduced in the presence of haemolysis. Moreover, in the presence of haemolysis, high dose methylene blue (20-30 mg/kg) can itself initiate methaemoglobin formation. Supplemental antioxidants such as ascorbic acid (vitamin C), N-acetylcysteine and tocopherol (vitamin E) have been used as adjuvants or alternatives to methylene blue with no confirmed benefit. Exchange transfusion may have a role in the management of severe haemolysis or in G-6-P-D deficiency associated with life-threatening methaemoglobinaemia where methylene blue is relatively contraindicated.     

Methaemoglobinaemia associated with the use of cocaine and volatile nitrites as recreational drugs: a review

Methaemoglobinaemia can cause significant tissue hypoxia, leading to severe, potentially life-threatening clinical features and/or death. Over recent years there have been increasing reports of methaemoglobinaemia related to recreational drug use. There have been 25 articles describing methaemoglobinaemia related to recreational use of volatile nitrites (poppers) and more recently, four reports of methaemoglobinaemia in association with recreational cocaine use. In this article we discuss the mechanisms by which methaemoglobinaemia occurs in relation to the use of both volatile nitrites and cocaine, and summarize the published cases of recreational drug-related methaemoglobinaemia. The volatile nitrites can cause methaemoglobinaemia directly through their activity as oxidizing agents. However, with cocaine, methaemoglobinaemia is related to adulterants such as local anaesthetics or phenacetin, rather than to the cocaine itself. Clinicians managing patients with acute recreational drug toxicity should be aware of the potential for methaemoglobinaemia in these patients, particularly in patients with cyanosis or unexplained low oxygen saturations on pulse oximetry, and ensure that appropriate and timely management is provided, including, where appropriate, the use of methylthioninium chloride (methylene blue).     

Poisoning due to urea herbicides

Urea herbicides, which act by inhibiting photosynthesis, were introduced in 1952 and are now used as pre- and post-emergence herbicides for general weed control in agricultural and non-agricultural practices. Urea herbicides are generally of low acute toxicity and severe poisoning is only likely following ingestion when nausea, vomiting, diarrhoea and abdominal pain may occur. As urea herbicides are metabolised to aniline derivatives, which are potent oxidants of haemoglobin, methaemoglobinaemia (18-80%) has been documented, as well as haemolysis. Treatment is supportive and symptomatic. Methylthioninium chloride (methylene blue) 1-2mg (the dose depending on the severity of features) should be administered intravenously over 5-10 minutes if there are symptoms consistent with methaemoglobinaemia and/or a methaemoglobin concentration >30%.     

Erythrocyte membrane alterations as the basis of chlorate toxicity

The effects of sodium chlorate and of sodium nitrite on human erythrocytes were studied in vitro. Nitrite rapidly oxidised haemoglobin and glutathione; reduction of methaemoglobin (Hbi) by methylene blue was complete during 3 h of incubation with nitrite. With chlorate, a concentration-dependent lag phase was seen before Hbi was formed. After prolonged incubation, Hbi could no longer be reduced with methylene blue. Several other effects were observed that explain the clinical picture of chlorate poisoning which involves haemolysis followed by disseminated intravascular coagulation and renal failure: increased permeability to cations, increased resistance to hypotonic haemolysis and prolonged filtration time through polycarbonate membranes with cylindrical pores of 5 micron diameter. This suggests an increased membrane rigidity due to membrane protein polymerisation, as demonstrated by SDS polyacrylamide gel electrophoresis. Simultaneously, erythrocyte enzymes were inactivated, primarily glucose-6-phosphate dehydrogenase which is necessary for the therapeutic effect of methylene blue. This explains the inefficacy of methylene blue in the treatment of a case of chlorate poisoning that we observed (Arch. Toxicol., 48 (1981) 281).     

Use of a metabolic inhibitor to reduce dapsone-dependent haematological toxicity

Aside from its established use as an antileprotic and anti-inflammatory drug, dapsone is also effective in the therapy of Pneurnocystis carinii pneumonia. Unfortunately, its use is often limited by its dose-dependent toxicity, such as methaemoglobinaemia and haemolysis; the latter condition occurs most frequently in gIucose-6-dehydrogenase deficient individuals. It is also responsible for occasional life-threatening disorders such as agranulocytosis. Dapsone may undergo acetylation, but its toxicity is due to the product of its oxidative metabolism, dapsone hydroxylamine. This is generated in man by the constitutive hepatic cytochrome P450 enzyme IIIA4. Studies in the rat revealed that dapsone-dependent methaemoglobinaemia could be greatly diminished by the co-administration of metabolic inhibitors. In the isolated perfused rat liver, dapsone hydroxylamine levels and hence methaemoglobin formation fell significantly in the presence of cimetidine. In addition, the clearance of the parent drug was retarded, and perfusate concentrations of monoacetyl dapsone increased. The protective effect of cimetidine also reduced methaemoglobin formation in the whole rat during the chronic administration of dapsone. Incubation of dapsone in a two-compartment in vitro system using human tissues in the presence of cimetidine or ketoconazole resulted in a decrease in methaemoglobin formation in all the human livers tested. Although cimetidine was only effective if incubated with microsomes and NADPH prior to the addition of dapsone. Administration of cimetidine (3 × 400 mg daily) to volunteers 3 days prior to and 4 days post administration of a single dose of 100 mg dapsone caused drug concentrations to increase by almost 30%. There was a marked fall in peak methaemoglobin levels and the percentage of the dose excreted in urine as dapsone hydroxylamine N-glucuronide was reduced by almost one third. During high dose dapsone therapy it may be possible that the co-administration of cimetidine might reduce toxicity while maintaining drug efficacy.     

Effects of dihydrolipoic acid (DHLA), α-lipoic acid. N-acetyl cysteine and ascorbate on xenobiotic-mediated methaemoglobin formation in human erythrocytes in vitro

α-Lipoic acid, dihydrolipoic acid (DHLA), N-acetyl cysteine and ascorbate were compared with methylene blue for their ability to attenuate and/or reduce methaemoglobin formation induced by sodium nitrite, 4-aminophenol and dapsone hydroxylamine in human erythrocytes. Neither α-lipoic acid, DHLA, N-acetyl cysteine nor ascorbate had any significant effects on methaemoglobin formed by nitrite, either from pre-treatment, simultaneous addition or post 30 min addition of the agents up to the 60 min time point, although N-acetyl cysteine did reduce methaemoglobin formation at 120 min (P<0.05). In all three treatment groups at 30, 60 and 120 min, there were no significant effects mediated by DHLA or N-acetyl cysteine on 4-aminophenol (1 mM)-mediated haemoglobin oxidation. Ascorbate caused marked significant reductions in 4-aminophenol methaemoglobin in all treatment groups at 30–120 min except at 30 min in the simultaneous addition group (P<0.0001). Neither α-lipoic acid, nor N-acetyl cysteine showed any effects on hydroxylamine-mediated methaemoglobin formation at 30 and 60 in all treatment groups. In contrast, DHLA significantly reduced hydroxylamine-mediated methaemoglobin formation at all three time points after pre-incubation and simultaneous addition (P<0.001), while ascorbate was ineffective. Compared with methylene blue, which was effective in reducing methaemoglobin formation by all three toxins (P<0.01), ascorbate was only highly effective against 4-aminophenol mediated methaemoglobin, whilst the DHLA-mediated attenuation of dapsone hydroxylamine-mediated methaemoglobin formation indicates a possible clinical application in high-dose dapsone therapy.     

Severe chlorate poisoning: Report of a case

A case of severe sodium chlorate poisoning was observed within 5 h after suicidal ingestion of 150–200 g of the herbicide. Methaemoglobinaemia was the early symptom of the intoxication. Treatment with methylene blue and ascorbic acid could not prevent a massive haemolysis with disseminated intravascular coagulation. Hypercoagulation and hyperfibrinolysis could be treated successfully with exchange transfusions, heparin and fresh plasma. During the first hours, 70 mmol chlorate were excreted before complete renal failure occurred which required haemodialysis for several weeks. Clinical observations and in vitro experiments provide evidence that methylene blue is effective only in the very early stages of chlorate poisoning. Consequently, the following treatment is suggested: gastric lavage, exchange transfusion, bicarbonate infusion, haemodialysis, anticoagulation with heparin and substitution of clotting factors if necessary.Dedicated to Prof. Gustav Adolf Martini on occasion of his sixty-fifth birthdayA preliminary report has been given at the Spring Meeting of the German Pharmacological Society, Mainz, March 1981     

A Dapsone-induced Blood Dyscrasia in the Mouse: Evidence for the Role of an Active Metabolite

In the female mouse, dapsone (50–500 mg kg^?1, p.o.) caused a dose-related methaemoglobinaemia which peaked at 0m?5-1 h with recovery to baseline values occurring by 4h. Cimetidine (100 mg kg^?1, p.o.), a known inhibitor of several hepatic P450 isozymes administered 1 h before dapsone, prevented the methaemoglobinaemia. In-vitro, dapsone required activation by mouse hepatic microsomes to cause methaemoglobin formation in mouse erythrocytes and cytotoxicity to human mononuclear leucocytes. In both instances, the toxic effects were markedly reduced by cimetidine. Daily dosing of mice with dapsone (50 mg kg^?1, p.o.) for 3 weeks induced a blood dyscrasia, characterized by a fall of platelet and white blood cell counts, which was inhibited by cimetidine (100 mg kg^?1, p.o. daily). It is concluded that an active metabolite of dapsone arising from a P450-dependent pathway is involved in the genesis not only of the methaemoglobinaemia but also the blood dyscrasia arising from repeated administration of the drug in this species.     

Direct and metabolism-dependent toxicity of sulphasalazine and its principal metabolites towards human erythrocytes and leucocytes.

1. The role of metabolites in sulphasalazine-mediated toxicity has been investigated in vitro by the use of human red blood cells and mononuclear leucocytes as target cells, with methaemoglobin formation and cytotoxicity respectively, being the defined toxic end-points. 2. Of the metabolites of sulphasalazine investigated, only sulphapyridine was bioactivated by human liver microsomes in the presence of NADPH to a metabolite which caused marked methaemoglobinaemia and a small, but statistically significant degree of mononuclear leucocyte cell death. 3. Methaemoglobinaemia was inhibited by ketoconazole but not by ascorbic acid (100 microM), glutathione (500 microM) and N-acetylcysteine (50 microM). In contrast, ascorbic acid and the thiols afforded complete protection for mononuclear leucocytes. 4. Sulphapyridine (100 microM) was converted in vitro to a metabolite (metabolite conversion 6.8 +/- 0.3%), the retention time of which on h.p.l.c. corresponded to synthetic sulphapyridine hydroxylamine. The half-life of sulphapyridine hydroxylamine in phosphate buffer (pH 7.4) was found to be 8.1 min. 5. In the absence of microsomes and NADPH, sulphapyridine hydroxylamine caused a concentration-dependent (10-500 microM) increase in methaemoglobinaemia (2.9%-24.4%) and cytotoxicity (5.4%-51.4%), whereas sulphasalazine, sulphapyridine, 5-hydroxy sulphapyridine and 5-aminosalicylic acid had no effect.     

An investigation of the role of metabolism in dapsone-induced methaemoglobinaemia using a two compartment in vitro test system.

1. We have utilized a two compartment system in which two teflon chambers are separated by a semi-permeable membrane in order to investigate the role of metabolism in dapsone-induced methaemoglobinaemia. Compartment A contained a drug metabolizing system (microsomes prepared from human liver +/- NADPH), whilst compartment B contained target cells (human red cells). 2. Incubation of dapsone (1-100 microM) with human liver microsomes (2 mg protein) and NADPH (1 mM) in compartment A (final volume 500 microliters) led to a concentration-dependent increase in the methaemoglobinaemia (15.4-18.9% at 100 microM) compared with control (2.3 +/- 0.4%) detected in the red cells within compartment B. In the absence of NADPH dapsone had no effect. 3. Of the putative dapsone metabolites investigated, only dapsone-hydroxylamine caused methaemoglobin formation in the absence of NADPH (40.6 +/- 6.3% with 100 microM). However, methaemoglobin was also detected when monoacetyl-dapsone, 4-amino-4'-nitro-diphenylsulphone and 4-aminoacetyl-4'-nitro-diphenylsulphone were incubated with human liver microsomes in the presence of NADPH. 4 Dapsone-dependent methaemoglobin formation was inhibited by addition of ketoconazole (1-1000 microM) to compartment A, with IC50 values of 285 and 806 microM for the two liver microsomal samples studied. In contrast, methaemoglobin formation was not inhibited by cimetidine or a number of drugs pharmacologically-related to dapsone. The presence of glutathione or ascorbate (500 microM) did not alter the level of methaemoglobin observed.     

Begrenzende Faktoren der Methämoglobinbildung durch Phenylhydroxylamin in Hämolysaten verschiedener Tierarten

Summary 1. Addition of ATP, G-6-P, G-6-PDH and NADP to haemolysates of erythroyctes from pigs, cows, and sheep increased the formation of methaemoglobin through phenylhydroxylamine by various degrees, according to the animal source used. The results revealed limiting factors for the formation of methaemoglobin in haemolysates. These factors are compared with the limiting factors found in intact erythrocytes.2. Phosphorylation of glucose limits NADP reduction and, consequently, the methaemoglobin formation in haemolysates of erythrocytes from the three animals. The individual limiting factors are as follows: in sheep erythrocytes, the activity of hexokinase; in pig erythrocytes, the glucose utilisation; in cow erythrocytes, the ATP concentration.3. When the hexokinase reaction is circumvented by the addition of G-6-P, the activity of NADPH-diaphorase limits the formation of methaemoglobin in the haemolysates of cow erythrocytes. However, the formation of methaemoglobin in haemolysates of pigs and cows can be increased by the addition of NADP before the activity of diaphorase becomes the limiting factor.4. After adding a complete NADPH regenerating system, the velocities of methaemoglobin formation in haemolysates from the three animals were found to correlate with the corresponding disphorase activities.

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Stipendiat der Alexander von Humboldt-Stiftung 1965.     

Chronic Copper Poisoning in Sheep. I. The Relationship of Methaemoglobinemia to Heinz Body Formation and Haemolysis during the Terminal Crisis

Abstract The values of haematocrit, total haemoglobin in plasma, methaemo-globin percentages in erythrocytes and plasma, the osmotic fragility of the erythrocytes, and the occurrence of Heinz bodies were investigated during the terminal crisis in 4 cases of experimental chronic copper poisoning in sheep. At the beginning of the crisis, which lasted for well over one day, 10–20 per cent methaemoglobin was detected in the erythrocytes, before any haemolysis occurred. Later on severe haemolysis developed, and maximum levels of haemoglobin in the plasma were close to 2.5 g/100 ml. During the haemolytic stage both methaemoglobin and haemoglobin were detected in the plasma at approximately the same proportions as in the erythrocytes. No changes were observed in the osmotic fragility of the red cells until the onset of the haemolysis. It is concluded that the methaemoglobin formation is mainly an intra-corpuscular process and that most of the methaemoglobin detected in plasma in chronic copper poisoning in sheep, comes from the erythrocytes.     

Cytotoxicity of myeloperoxidase-activated catechols: oxidative injury to the red blood cell.

The effects of two catechols (1,2-benzenediol and nordihydroguaiaretic acid) on the myeloperoxidase-Cl(-)-H2O2 antimicrobial/cytotoxic system of the human neutrophil were investigated. To determine the cytotoxicity of myeloperoxidase-generated oxygen metabolites (mainly chlorinated oxidants such as hypochlorite) and catechol oxidation products, the well characterized erythrocyte was used as a target. At relatively low concentrations (less than 10 microM), the catechols acted as redox catalysts by stimulating the generation of chlorinated oxidants. This is visualized as a promotion of haemolysis which reached a maximum and then decreased again with increasing concentrations of the catechol. In this respect, the dicatechol, nordihydroguaiaretic acid, was more potent. At higher concentrations, the catechols competed more effectively with Cl- as electron donors and the generation of chlorinated oxidants decreased with a consequent decrease in haemolysis. Above 200 microM nordihydroguaiaretic acid, complete haemolysis occurred which might be due to high membrane concentrations of the catechol due to its high lipid solubility. In contrast, high 1,2-benzenediol concentrations did not induce haemolysis. The catechols stimulated methaemoglobin formation in a concentration-dependent fashion with 1,2-benzenediol more potent than nordihydroguaiaretic acid. There was some correlation between membrane microviscosity and haemolysis which in turn did not correlate with haemoglobin oxidation. No direct correlation existed between intracellular methaemoglobin formation and the precipitation of haemoglobin oxidation products on the membrane. Disulphide crosslinks were not involved in the covalent polymerization of haemoglobin subunits.     

Hyperbaric Oxygenation in the Treatment of Life-Threatening Isobutyl Nitrite-Induced Methemoglobinemia—A Case Report

Methemoglobinemia usually results from exposure to oxidizing substances such as nitrates or nitrites. Iron within hemoglobin is oxidized from the ferrous (Fe²⁺) state to the ferric (Fe³⁺) state, resulting in the inability to transport oxygen and carbon dioxide. Clinically, this condition causes functional cyanosis. As methemoglobin levels increase, patients show evidence of cellular hypoxia in all tissues. Death usually occurs when methemoglobin fractions approach 70% of total hemoglobin. We describe the case of a 35-year-old female patient with severe life-threatening isobutyl nitrite-induced methemoglobinemia of 75% of total hemoglobin. Toluidine-blue was administered as first-line antidotal therapy immediately, followed by hyperbaric oxygenation. The patient recovered uneventfully and could be discharged 3 days later.     

Haematological and biochemical alterations in leprosy patients already treated with dapsone and MDT

Dapsone (DDS) is useful in the treatment of a number of inflammatory conditions which are characterized by neutrophil infiltration. It is the drug of choice for the treatment of leprosy and prophylaxis of malaria. Haematological side effects of methaemoglobinaemia and haemolysis have been long recognized. However, the frequency and severity of these side effects in patients already treated with DDS as a single drug or as part of a multidrug therapy (MDT) have not been well documented. We report herein an investigation of the effect of dapsone long-term treatment on the haematological and biochemical alterations in leprosy patients undergoing dapsone as a single drug (DDS group) or as part of a multidrug therapy in combination with rifampin and clofazimine (MDT group). Methaemoglobinaemia and haemolytic anaemia were the principal side effects observed. Reticulocytes were found to be elevated (> 1.5%) in 90% of the patients. Heinz bodies were also detected (6.6% of the patients). The osmotic fragility test showed a reduction in cell resistance and in the evaluation of white cells a severe eosinophilia was found. Hepatic, pancreatic and renal evaluation by the determination of biochemical parameters showed rare and occasional changes of no apparent clinical significance. We conclude that haematological side effects of dapsone are significant even at doses currently used to treat leprosy (100 mg/day) and that rifampin and clofazimine do not increase the incidence of these effects during long-term treatment.     

An investigation into the haematological toxicity of structural analogues of dapsone in-vivo and in-vitro.

With microsomes prepared from a single human liver, 4,4'-diaminodiphenyl sulphone (DDS), 4-acetyl-4-aminodiphenyl sulphone (MADDS), 4-acetyl-4-aminodiphenyl thioether (MADDT) and 4,4'-diacetyldiphenyl thioether (DADDT) caused significantly greater methaemoglobin formation compared with control. In-vitro in the rat, the pattern of toxicity was slightly different:DADDT was not haemotoxic, whilst 3,4'-diaminodiphenyl sulphone (3,4'DDS) and 3,3'-diaminodiphenyl sulphone (3,3'DDS) as well as DDS, MADDS and MADDT were significantly greater than control. 4,4' Acetyl diphenyl sulphone (DADDS), 4,4' diaminodiphenyl thioether (DDT), 4,4'-diaminodiphenyl ether (DDE) and 4,4' diaminooctofluorodiphenyl sulphone (F8DDS) did not cause significant methaemoglobinaemia in either human or rat liver microsomes. DDS, MADDS, and MADDT were not significantly different in haemotoxicity generation in-vitro in the presence of human microsomes. In the rat in-vitro, DDS, MADDS, and 3,4'DDS did not differ significantly in red cell toxicity, and were the most potent methaemoglobin formers. The 3,3'DDS and MADDT derivatives were both significantly less toxic compared with DDS. None of the compounds tested caused haemoglobin oxidation in the absence of NADPH in-vitro. In the whole rat, DDS, MADDS and MADDT caused significantly higher levels of methaemoglobin compared with control. None of the remaining compounds caused methaemoglobin formation which was significantly greater than control. DDS and MADDS were the most potent methaemoglobin formers tested, in-vivo and in-vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitrite intoxication: Protection with methylene blue and oxygen

The lethal effects of sodium nitrite can be antagonized by the administration of various combinations of methylene blue, oxygen, and hyperbaric oxygen. The potency ratios were compared in groups of mice pretreated with methylene blue (2 or 20 mg/kg) and/or oxygen (1 ATA or 2 ATA). Hyperbaric oxygen (2 ATA) was found to be more effective than oxygen (1 ATA) or air with or without methylene blue at 2 mg/kg. When the dose of methylene blue was increased to 20 mg/kg, oxygen (1 ATA) was more effective than air, but hyperbaric oxygen was no more efficacious than oxygen (1 ATA).     

The use of cimetidine as a selective inhibitor of dapsone N-hydroxylation in man.

1. The N-hydroxylation of dapsone is thought to be responsible for the methaemoglobinaemia and haemolysis associated with this drug. We wished to investigate the effect of concurrent administration of cimetidine (400 mg three times per day) on the disposition of a single dose (100 mg) of dapsone in seven healthy volunteers in order to inhibit selectively N-hydroxylation. 2. The AUC of dapsone (31.0 +/- 7.2 micrograms ml-1 h) was significantly increased (P less than 0.001) in the presence of cimetidine (43.3 +/- 8.8 micrograms ml-1 h). 3. Peak methaemoglobin levels observed after dapsone administration (2.5 +/- 0.6%) were significantly (P less than 0.05) reduced in the presence of cimetidine (0.98 +/- 0.35%). 4. The percentage of the dose excreted in urine as the glucuronide of dapsone hydroxylamine was significantly (P less than 0.05) reduced in the presence of cimetidine (34.2 +/- 9.3 vs 23.1 +/- 4.2%). 5. Concurrent cimetidine therapy might reduce some of the haematological side-effects of dapsone.     

Dapsone toxicity: Some current perspectives

• 1.1. Dapsone is a potent anti-inflammatory and anti-parasitic compound, which is metabolised by cytochrome P-450 to hydroxylamines, which in turn cause methaemoglobinaemia and haemolysis. However, during the process of methaemoglobin formation, erythrocytes are capable of detoxifying the hydroxylamine to the parent drug, which may either reach the tissues to exert a therapeutic effect or return to the liver and be re-oxidised in a form of systemic cycling. This glutathione-dependent effect, combined with the un-ionised state of the drug at physiological pH, may contribute to its efficacy.
• 2.2. Paradoxically, other aspects of the glutathione-dependent cycling of the hydroxylamine metabolite may contribute to the major adverse reaction of the drug, agranulocytosis. Erythrocytes exposed to the metabolite and repeatedly washed may still release the hydroxylamine in sufficient concentration to kill mononuclear leucocytes in vitro. Thus, erythrocytes may be a conduit for the hydroxylamine to reach the bone marrow to covalently bind to granulocyte precursors, which may trigger an immune response in certain individuals and may lead to the potentially fatal eradication of granulocytes from the circulation.
• 3.3. Attempts to increase patient tolerance to dapsone have been most successful using a metabolic inhibitor to reduce hepatic oxidation of the drug to the hydroxylamine. Methaemoglobin formation in the presence of cimetidine was maintained at 30% below control levels for almost 3 mo, and patients' reported side effects such as headache and lethargy were significantly reduced.
• 4.4. As clinical application of new and safer dapsone analogues is years away, the use of cimetidine provides an immediate route to increasing patient compliance during dapsone therapy, especially in those maintained on dapsone dosages in excess of 200 mg/day.

     

Silymarin protection against major reactive oxygen species released by environmental toxins: exogenous H2O2 exposure in erythrocytes

Silymarin is a polyphenolic plant flavonoid (a mixture of flavonoid isomers such as silibinin, isosilibinin, silidianin and silichristin) derived from Silymarin marianum that has anti-inflammatory, hepatoprotective and anticarcinogenic effects. Our earlier studies have shown that silymarin plays a protective role against the oxidative damage induced by environmental contaminants like benzo(a)pyrene in erythrocyte haemolysates. During the detoxification of these environmental contaminants, the major reactive oxygen species generated is hydrogen peroxide (H(2)O(2)). Because H(2)O(2 )can easily penetrate into the cell and cause damage to biomolecules, the protective role of silymarin was further assessed against this cytotoxic agent in vitro in erythrocyte haemolysates. The protective effect was monitored by assessing the levels of the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione-s-transferase, glutathione peroxidase and malondialdehyde (LPO) in three groups: vehicle control, H(2)O(2)-exposed groups and drug co-incubation group (H(2)O(2) + silymarin). The protective effect of silymarin on the non-enzymic antioxidant glutathione and haemolysis, methaemoglobin content and protein carbonyl content were also assessed. It was observed that the activities of antioxidant enzymes and glutathione were reduced and the malondialdehyde levels were elevated after H(2)O(2 )exposure. There were also alterations in haemolysis, methaemoglobin content and protein carbonyl content, whereas after the administration of silymarin, the antioxidant enzyme activities reversed to near normal with reduced malondialdehyde content and normalized haemolysis, methaemoglobin content and protein carbonyl content. The results suggest that silymarin possesses substantial protective effect and free radical scavenging mechanism against exogenous H(2)O(2)-induced oxidative stress damages, hence, can be used as a protective drug against toxicity induced by environmental contaminants.