The present study examined cellular effects of the atypical antipsychotic drug clozapine on blood cells of treated patients with and without clozapine-induced agranulocytosis (CA). Blood from one patient who commenced clozapine treatment was examined at weekly intervals for 128 days. Olanzapine-treated (n = 5) and polymedicated (n = 14) schizophrenic patients, as well as healthy subjects (n = 19) and septic shock patients (n = 8), were studied for comparison. We observed dramatically increased numbers of native neutrophils stained for superoxide anion production (P < or = 0.005, n = 10) and significantly elevated expression levels of the proapoptotic genes p53 (P < or = 0.020), bax alpha (P < or = 0.001), and bik (P < or = 0.002) in all tested non-CA patients (n = 19) and CA patients (n = 4). In non-CA patients, the expression of these genes did not correlate to the percentage of apoptotic neutrophils (2.0% +/- 1.3%), but in CA patients about 37% of the neutrophils show morphologic signs of apoptosis (P < or = 0.001). Under G-CSF therapy of CA, the number of apoptotic neutrophils and the expression of the proapoptotic genes decreased significantly. In conclusion, high production of reactive oxygen species in neutrophils of clozapine-treated patients, together with increased expression of proapoptotic genes, suggests that neutrophils are predisposed to apoptosis in schizophrenic patients under clozapine therapy. The correlation between drug and proapoptotic markers was highest for clozapine and bax alpha as well as superoxide anion radicals. This indicates oxidative mitochondrial stress in neutrophils of clozapine-treated patients which probably contributes to the induction of apoptosis and sudden loss of neutrophils and their precursors in CA patients.

The atypical antipsychotic, clozapine, exerts superior efficacy in therapy-resistant schizophrenia, but unfortunately induces agranulocytosis with an incidence of 0.8 - 1 %. In this study, we investigated the cellular uptake of clozapine into human promyelocytic leukaemia HL-60 cells using HPLC with electrochemical detection. On incubation with 1.25 to 40 microM clozapine for 30 min, a saturable, energy- and temperature-dependent uptake process takes place (K m = 18.8 microM, k cat = 1.36 nmol/5 min/mg protein at 37 degrees C). This suggests membrane passage of clozapine by a carrier mechanism. 10 microM Indatraline, an inhibitor of dopamine, noradrenaline and serotonin (5-HT) reuptake, but not the selective 5-HT reuptake inhibitor fluvoxamine, markedly reduced the transport of clozapine by 62 %, whereas addition of 10 mM glucose to the incubation medium increased intracellular clozapine concentrations by 28 %. Since cyclosporine A, vinblastine or verapamil up to a final concentration of 10 microM did not alter the intracellular accumulation of clozapine, an involvement of P-glycoprotein seems to be unlikely. In summary, clozapine uptake into HL-60 cells meets criteria of an active unidirectional transport. Its molecular correlates remain to be established.

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By differential hybridization we have identified cDNA clones that are derived from iron-regulated genes in Saccharomyces cerevisiae. Sequencing of seven cDNA clones revealed that five clones correspond to TP/1 encoding FRG triosephosphate isomerase (Tpil p) and one corresponds to TDH3 encoding glyceraldehyde-3-phosphate dehydrogenase (Tdh3p). During iron-limited growth mRNA levels for Tpilp and Tdh3p were at least 3-fold lower than during iron-saturated growth; as shown with a hem7 mutant strain this regulation does not require haem synthesis. mRNA half-lives of TP/7 (TDH3) were 11-5 min (18 min) in low-iron medium and 30 min (32-5 min) in high-iron medium, indicating iron-regulation of transcript half-lives; the stabilities of the ACT7 and PDCl transcripts were not influenced by iron. Increased glycerol production during growth in low-iron, as compared to high-iron medium, is consistent with a modification of the glycolytic flux during iron-limited growth in 5. cerevisiae.

Using qualitative and microquantitative histo-chemical techniques, alcohol dehydrogenase and aldehyde dehydrogenase activity was studied in the gastric mucosa of male and female rats. Alcohol dehydrogenase was demonstrated by staining reactions with maximum activity in surface and neck cells and with clearly weaker activity also in parietal cells. Aldehyde dehydrogenase could be detected in surface and neck cells, and also to a comparable degree in the parietal cells. Quantitative analyses of microdissected samples yielded high values for alcohol dehydrogenase activity exclusively in the superficial part of the gastric mucosa, whereas low-Km aldehyde dehydrogenase activity showed a decreasing gradient from the surface to the deeper parts of the mucosa. Sex differences could not be confirmed.