Dapsone-induced agranulocytosis-possible involvement of low-activity N-acetyltransferase 2.

Dapsone-induced agranulocytosis is a rare but potentially fatal adverse drug reaction (ADR). A 45-year-old male Caucasian patient developed agranulocytosis caused by dapsone (diamino-diphenyl sulfone), which he was prescribed for leukocytoclastic vasculitis. Patient's treatment consisted of termination of dapsone, antibiotic therapy, and granulocyte colony-stimulating factor leading to prompt improvement of symptoms and normalization of laboratory blood values. Diagnostic evaluation revealed methemoglobinemia and excluded glucose-6-phosphate dehydrogenase deficiency. Pharmacogenetics testing showed that he was a carrier of NAT2 *5/*6 genotype, predisposing to low activity of the N-acetyltransferase 2 enzyme. This was the first and only ADR to dapsone reported in Croatia. In total, there have been 73 ADR to dapsone recorded worldwide, including only four cases of agranulocytosis.

Preservation of basophils in dapsone-induced agranulocytosis suggests a possible pathogenetic role for leucocyte peroxidases.

We describe a hitherto unreported laboratory observation, namely the selective preservation of basophils, in a case of severe dapsone-induced agranulocytosis. Dapsone is known to be metabolised by peroxidases to nitroso derivatives in non-hemopoietic cells where these can in turn act as haptens. Our observation that basophils are selectively spared in this syndrome supports the hypothesis that leucocyte peroxidases function in a similar way to facilitate the pathogenesis of this form of agranulocytosis in susceptible individuals.

Impact of myeloperoxidase and NADPH-oxidase polymorphisms in drug-induced agranulocytosis.

Therapy of the atypical antipsychotic drug clozapine is limited by a comparatively high incidence of agranulocytosis in 0.8% of patients. This severe side effect is possibly based on the clozapine-mediated stimulation of cytokines and soluble cytokine receptors release, followed by induction of granulocyte proliferation and induction of myeloperoxidase (MPO) and NADPH-oxidase. Because NADPH-oxidase/MPO may oxidize clozapine to highly reactive nitrenium ions, we investigated the role of hereditary polymorphisms in the NADPH oxidase/myeloperoxidase system in agranulocytosis patients who received clozapine (n = 49), ticlopidine (n = 11), and other drugs prior to the event. The low active MPO -436A allelic variant frequency was 22.2% in cases and 19.9% in controls, but AA carriers were overrepresented among cases compared with the sum of AG and GG-carriers (odds ratio 4.16, 95% confidence limits 0.86-20.3, P = 0.056). Particularly in clozapine-induced agranulocytosis, this finding was most pronounced (P = 0.04). In the CYBA gene, encoding the p22phox subunit of the NADPH-oxidase, 2 polymorphisms were investigated. C242T (His72Tyr) had an allele frequency of 31.9% and 32.2% (P = NS) and A640G in the 3'-UTR was less frequent in cases (48.7%) than controls (60.0%), odds ratio 0.63 (0.39-1.02), P = 0.048. CYBA 640GG-carriers were marginally less frequent in cases compared with controls (28.2% vs. 38.7%, P = 0.062). Sequencing the entire coding region of the NADPH subunit CYBB (gpS1phase) disclosed that CYBB is a highly conserved gene, which does not represent a risk factor for clozapine-induced agranulocytosis. The impact of the polymorphic myeloperoxidase, however, needs further verification to predict a patient's risk to develop drug-induced agranulocytosis.

Pathogenesis of granulocytopenia and bone marrow atrophy during classical swine fever involves apoptosis and necrosis of uninfected cells.

Granulocytopenia, a hematological hallmark of classical swine fever, is partially responsible for the suppression of innate immune defenses during classical swine fever. The present report demonstrates that this depletion was apparent as early as 3 days postinfection (p.i.). Both mature peripheral and bone marrow neutrophils were affected, whereas immature neutrophils increased absolutely in the periphery and coincidentally immature myeloid progenitors in the bone marrow. These data suggest that a pathogenic relationship exists between these compartments. The central event was not the arrest of hematopoietic cell proliferation or of the mobilization process, but instead apoptosis and possibly also necrosis were shown to play a role. This increase in apoptotic and dead cells was detected as early as 1-3 days p.i. In contrast, viral RNA in bone marrow hematopoietic cells (BMHC) was first detected 5 days p.i., and significant amounts of infected BMHC were detected only 7 days p.i., with the major target being the myeloid compartment. The increased caspase-3 activity observed supported a role for apoptotic cell death. Furthermore, the elevated caspase-9 activity indicated the involvement of the mitochondrial apoptotic pathway. Taken together, the results demonstrate that granulocytopenia and bone marrow atrophy are mediated by hematopoietic cell death and that indirect virus-host-mediated mechanisms are likely to be responsible.

Polymorphisms of NAT2 in relation to sulphasalazine-induced agranulocytosis.

Agranulocytosis is a rare, but serious adverse reaction to sulphasalazine. The polymorphic enzyme N-acetyltransferase 2 (NAT2) plays an important role in the metabolism of sulphasalazine. This study was conducted to analyse whether the risk of sulphasalazine-induced agranulocytosis is increased in slow acetylators. Patients were treated for inflammatory disease, mostly joint disease, with a mean dose of 2 g sulphasalazine daily. Thirty-nine patients reacted with agranulocytosis, while 75 patients had been treated for a minimum of 3 months without haematological side-effects. A population-based control panel of 448 individuals was used for comparison. All subjects were genotyped for NAT2 by polymerase chain reaction followed by restriction enzyme digestion. The six most common allelic variants were analysed: NAT2*4, NAT2*5A, NAT2*5B, NAT2*5C, NAT2*6 and NAT2*7. The proportion of slow acetylators was significantly higher in patients with sulphasalazine-induced agranulocytosis (69%) and population-based controls (64%) compared to patients who tolerated sulphasalazine (45%); odds ratio 2.71 [95% confidence interval (CI) 1.20; 6.15], P = 0.015, and odds ratio 2.17 (95% CI 1.32; 3.56), P = 0.002, respectively. Patients who developed agranulocytosis did not differ from population-based control subjects in the frequency of slow acetylators; odds ratio 1.25 (95% CI 0.62; 2.53), P = 0.535. The risk of agranulocytosis did not appear to be increased in slow acetylators, provided that the difference compared with sulphasalazine-treated control subjects was not due to a predominance of fast acetylators among patients with inflammatory joint disease. Instead, selection bias was suspected since more slow acetylators may have discontinued sulphasalazine therapy because of drug-intolerance.

Activation of CGS 12094 (prinomide metabolite) to 1,4-benzoquinone by myeloperoxidase: implications for human idiosyncratic agranulocytosis.

Many marketed pharmaceuticals are known to cause idiosyncratic agranulocytosis in humans. Similarly prinomide, an antiinflammatory drug, was associated with a low incidence of agranulocytosis (<0.3%) in clinical trials, even though chronic toxicity studies in rodents and primates showed no evidence of agranulocytosis with either prinomide or its parahydroxy metabolite, CGS 12094. To investigate mechanisms for this human specific toxicity, experiments were conducted to study the metabolism of prinomide and CGS 12094 by myeloperoxidase (MPO), a major enzyme of neutrophils and leukocyte progenitor cells. Although prinomide was not metabolized by human MPO, CGS 12094 was rapidly metabolized (>90%; 2 min); this reaction was dependent on H2O2 and MPO and was inhibited by azide. During the MPO-catalyzed metabolism of CGS 12094, reactive intermediates that irreversibly bound to protein and cysteine were generated. One of the reactive metabolites generated was identified by mass spectroscopy and trapping with cysteine as 1,4-benzoquinone, a compound implicated in the myelotoxicity associated with benzene. Thus during conditions which lead to elevated levels of H2O2 (e.g., active inflammation), CGS 12094 is rapidly metabolized by MPO to reactive intermediates that may be related to prinomide-induced agranulocytosis.

Genetic factors and risk of agranulocytosis from metamizol.

The role of genetic factors in the pathogenesis of agranulocytosis was investigated in agranulocytosis patients by phenotyping for N-acetyltransferase and glucose-6-phosphate polymorphism; by typing for gene products of the major histocompatability complex, ABO- and RH-blood groups, and haemoglobins; and by performing cytogenetic analysis of chromosome aberrations. Nine persons were identified as agranulocytosis cases in the period from 1982 to 1987 among the population of Sofia. They were contacted again 10 years after recovery from the disease. Five of them were associated with metamizol (dipyrone) use. The results obtained revealed significant differences between the agranulocytosis patients and the healthy population in the human lymphocyte antigen (HLA) allele frequencies, and in the degree and the frequency of chromosome aberrations. A higher frequency of the HLA24 antigen (relative risk 13.60, p = 0.05) and a lower frequency of the DQA1*0501 allele were evident for the ex-agranulocytosis patients as compared to the controls (11% versus 57% respectively, p = 0.05). In the patients exposed to metamizol, an A24-B7 haplotype was found with a frequency higher than that in the non-exposed patients and the reference group (p < 0.05). The HLA-DQwl antigen and metamizol-related agranulocytosis were evidently associated in all cases (5/5;100%) in contrast to the patients not exposed to metamizol and the controls. The HL-A2 antigen was absent in four of the five metamizol-associated agranulocytosis cases (20%), while in the control group it was present in 56%. The degree of structural rearrangements (0.62 +/- 0.2%) and the frequency of chromosome breakages (7.75 +/- 0.68%) in agranulocytosis patients were higher than those in the healthy population (0.3 +/- 0.12%, p < 0.05 and 1.42 +/- 0.27%, p < 0.01, respectively). The abnormalities affected predominantly chromosomes 1(1p13), 2(2p12) and 5(5p12). No differences were found between the agranulocytosis patients and the healthy population when considering the haemoglobin subtypes, ABO-and RH-blood groups, glucose-6-phosphate dehydrogenase activity and the rates of slow and rapid acetylators.

Free radical scavenging enzyme activity and related trace metals in clozapine-induced agranulocytosis: a pilot study.

We hypothesized that patients who had experienced clozapine-induced agranulocytosis would have abnormalities in their free radical scavenging enzyme activity (FRESA) and levels of related trace metals. We therefore measured FRESA profiles and related trace metals in four groups: post-clozapine agranulocytosis (POST CLOZ AGRAN) (N = 9); clozapine no agranulocytosis (CLOZ NO AGRAN) (N = 12); West Coast controls (WC CONTROLS) (N = 14); and Long Island Jewish Medical Center controls (LIJ CONTROLS) (N = 12). Glutathione peroxidase (GSH-Px, P1) levels in plasma were slowest in the POST CLOZ AGRAN group (34.3 +/- 6.9 IU/dl [standard deviation; SD]; p < 0.002); red blood cell glutathione peroxidase (GSH-Px, RBC) was highest in the WC CONTROLS (38.7 +/- 4.7 IU/g hemoglobin [Hgb]; p < 0.008); and selenium (SE) levels in plasma were lower in both the POST CLOZ AGRAN group (111.6 +/- 14.7 ng/ml) and the CLOZ NO AGRAN group(115.0 +/- 17.8) than in the WC CONTROLS (142.5 +/- 18.3; p < 0.0006). SE was also lower in the POST CLOZ AGRAN group than in the LIJ CONTROLS (129.1 +/- 21.6; p < 0.04). The presence of at least one of the following: (1) GSH-Px, P1 < 37.6 IU/dl; (2) GSH-Px, RBC < 31.0 IU/g Hgb; or (3) SE < 112.4 ng/ml, distinguished POST CLOZ AGRAN subjects from the WC CONTROLS, but not from the LIJ CONTROLS. Data from this cross-sectional pilot study suggest that abnormalities in the body's antioxidant defense system may be involved in the pathogenesis of clozapine-associated agranulocytosis. If confirmed in large-scale, prospective studies, these preliminary findings have potential clinical application in the screening and prophylaxis of clozapine agranulocytosis.

Concurrent agranulocytosis and hepatitis secondary to clomipramine therapy.

A 67-year-old man developed concurrent severe agranulocytosis and elevation of hepatic transaminases as a result of treatment with clomipramine. Although such adverse drug reactions can be considered rare events, the potentially serious nature of these reactions vindicate the routine monitoring of blood picture, and liver function tests, after initiation of treatment with tricyclic antidepressants.

Infantile agranulocytosis with survival into adolescence: periodontal manifestations and laboratory findings. A case report.

A case of infantile agranulocytosis with survival into adolescence is presented. The polymorphonuclear leukocyte is considered an important source of lysosomal enzymes in gingival crevicular fluid, and evaluation of connective tissue-degrading enzymes in the fluid was performed. The activity of beta-glucuronidase, a ground substance-degrading enzyme that may serve as a marker for polymorphonuclear leukocytes, was markedly reduced in the fluid compared to samples from systemically healthy adults with periodontitis. The activities of the ground substance-degrading enzyme arylsulfatase, and collagenase, were in the low-normal range. The plaque microbiology, as characterized by dark-field microscopy and selective culturing, was consistent with advanced periodontitis. A review of the medical history revealed a series of bacterial infections since infancy. Improvement in the systemic health of the patient occurred at about the age of 15, and the intake of antibiotics to control infections was correspondingly reduced after this time. An exacerbation of the patient's periodontal disease, as evaluated by loss of alveolar bone on radiographs, occurred 1 to 2 years later. The progression of periodontal disease observed in this patient was apparently associated with the withdrawal of antibiotics administered for control of systemic (nonoral) infections.

Angiotensin converting enzyme concentrations in the lung lavage of normal rabbits and rabbits treated with nitrogen mustard exposed to hyperoxia.

Increased concentrations of angiotensin converting enzyme (ACE) were found in lung lavages from rabbits exposed to hyperoxia for 72 h and the concentrations of ACE were correlated with ratios of extravascular lung water to body weight (r = 0.69, p less than 0.05) and albumin concentrations in lung lavages (r = 0.89, p less than 0.01). In parallel studies, rabbits treated with nitrogen mustard in which granulocytopenia was maintained throughout the 72-h hyperoxic exposure period had less evidence of edematous lung injury and lower concentrations of ACE in their lung lavages than similarly treated rabbits in which granulocytopenia was not maintained. The results suggested that granulocytes contribute to acute edematous lung injury from hyperoxia and that ACE concentrations in lung lavages reflect this process.

Serum myeloperoxidase and lactoferrin in neutropenia.

Radioimmunosorbent assays for determination of serum content of the neutrophil proteins myeloperoxidase and lactoferrin are described. Serial studies were performed in patients with neutropenia. In 2 cases of cyclic neutropenia the myeloperoxidase level showed slight variations within the normal range during the cycle while lactoferrin displayed a clear correlation with neutrophil counts. In 1 case with persistent agranulocytosis myeloperoxidase was normal but lactoferrin was extremely low. During the regeneration phase of drug-induced neutropenia neutrophil counts and serum lactoferrin increased in a parallel fashion. Since serum myeloperoxidase was normal during profounded neutropenia it is suggested to derive primarily from myeloperoxidase-rich granulopoietic precursor cells of the marrow. Serum lactoferrin on the other hand seems to derive from leakage of more granulopoietic cells of blood and marrow. Studies of neutrophil proteins of serum may aid in evaluation of neutropenic patients.