The Interaction of Heptakis (2,6-di-O-Methyl)-ß-cyclodextrin with Mianserin Hydrochloride and Its Influence on the Drug Toxicity.

One tetracyclic antidepressant, mianserin hydrochloride (MIA), has quite significant side effects on a patients' health. Cyclodextrins, which are most commonly used to reduce the undesirable features of contained drugs within their hydrophobic interior, also have the potential to alter the toxic behavior of the drug. The present paper contains investigations and the characteristics of interaction mechanisms for MIA and the heptakis (2,6-di-O-methyl)-ß-cyclodextrin (DM-ß-CD) system, and evaluated the effects of the complexation on MIA cytotoxicity. In order to assess whether there was an interaction between MIA and DM-ß-CD molecules, isothermal titration calorimetry (ITC) have been chosen. Electrospray ionization mass spectrometry (ESI-MS) helped to establish the complex stoichiometry, and circular dichroism spectroscopy was used to describe the process of complex formation. In order to make a wider interpretative perspective, the molecular docking results have been performed. The viability of Chinese hamster cells were investigated in the presence of DM-ß-CD and its complexes with MIA in order to estimate the cytotoxicity of the drug and the conjugate with the chosen cyclodextrin. The viability of B14 cells treated with MIA+DM-ß-CD is lower (the toxicity is higher) than with MIA alone, and no protective effects have been observed for complexes of MIA with DM-ß-CD in any ratio.

Effects of the antidepressant, mianserin, on early development of fish embryos at low environmentally relevant concentrations.

Pharmaceuticals have been considered as emerging organic contaminants in the environment that might pose huge risk to the non-target aquatic organisms. Mianserin, a tetracyclic antidepressant, is present at low detectable concentrations in the aquatic environment; however, limited attention has been devoted to its potential adverse effects on the aquatic animals. In the present study, we first performed an acute toxicity test for mianserin exposure using zebrafish (Danio rerio) embryos during 4-124h post fertilization (hpf). Time-dependent lethal concentrations of mianserin exposure on the zebrafish embryos were firstly determined at mg/L levels. Then, a series of sublethal concentrations of 0.01, 0.1, 1, 10, 100, and 1000µg/L of mianserin were prepared for the short-term exposure of zebrafish embryos for 120h. The results showed that mianserin exposure reduced the body length of zebrafish larvae, in addition to altering multiple physiological and biochemical parameters in the exposed embryos/larvae. A dose-dependent inhibition of the total antioxidant capacity and total cholinesterase activity was revealed in the exposed fish larvae upon increasing the concentrations of mianserin exposure. A U-shaped concentration-dependent response curve was observed for the adrenocorticotropic hormone; however, an inversed U-shaped response curve was obtained for the monoamine oxidase level in response to mianserin exposure. Activities of the total adenosine triphosphatase (T-ATPase), Na+/K+-ATPase, and Ca2+/Mg2+-ATPase were significantly increased in the fish larvae exposed to relatively high doses of mianserin; interestingly however, low dose of mianserin at 10ng/L inhibited their Na+/K+-ATPase and T-ATPase activities. Additionally, the coordinated regulation of cyclic adenosine monophosphate and protein kinase A was observed in the mianserin-exposed fish larvae, implying a reserved signaling pathway involved in the fish response to the antidepressant. Therefore, our study demonstrated that mianserin exposure significantly affected the early development of fish embryos at environmentally relevant concentrations, and suggested that the risk of pharmaceutical contamination of the aquatic environment, even at low doses, should receive more attention.

Global transcriptomic analysis of zebrafish in response to embryonic exposure to three antidepressants, amitriptyline, fluoxetine and mianserin.

Antidepressants are among the most commonly detected pharmaceuticals in aqueous systems, and, as emerging organic pollutants, may exert negative effects on non-target aquatic organisms. Previously, it has been revealed that antidepressant exposure significantly inhibits the growth and development of fish during their early developmental stages. Thus, in the present study, we aimed to identify and compare the underlying mechanisms of action of different antidepressants at the transcriptional level using zebrafish (Danio rerio) embryos. Through high-throughput RNA sequencing (RNA-Seq) data analysis, 32, 34, and 130 differentially expressed genes (DEGs) were obtained from zebrafish larvae after 120h of embryonic exposure to sublethal concentrations of amitriptyline, fluoxetine, and mianserin, respectively. The expression profiles of the identified DEGs showed similar trends in response to the three antidepressant treatments, suggesting consistent toxic effects of low concentrations of these three drugs on the regulation of gene expression in fish. Several metabolic and signaling pathways, including glycolysis/gluconeogenesis and the insulin pathway, were affected in the exposed fish larvae. The expression profiles of selected DEGs were then verified by the qRT-PCR method, which indicated significant positive correlations with the RNA-Seq results. Next, we determined the concentration-dependent expression patterns of 6 selected DEGs in fish larvae exposed to three antidepressants at a series of environmentally relevant concentrations. The results revealed a significant concentration-dependent reduction in the levels of dual-specificity phosphatase 5 (dusp5) mRNA, as well as a non-concentration-dependent gene expression inhibition of prostaglandin D2 synthase b (ptgdsb); the circadian rhythm-related genes, i.e. those encoding nuclear receptor subfamily 1, group D, member 1 (nr1d1) and period 2 (per2); and genes encoding early growth response factors (egr1 and egr4), in the antidepressant-treated fish larvae. In summary, to our knowledge, our findings demonstrate, for the first time, that the three different categories of antidepressants have common effects on the gene expression involved in multiple biological processes and signaling pathways during the early development of fish and thus provide information for characterizing the adverse outcome pathways and on the ecological risk assessment of these pharmaceutical pollutants in the aquatic environment.

Mirtazapine toxicity in cats: retrospective study of 84 cases (2006-2011).

Objectives Mirtazapine is commonly used in veterinary medicine at doses of 1.88 or 3.75 mg as an appetite stimulant. The objectives of this study were to determine the most common adverse effects reported and the dose associated with these signs. Methods Records of cats with mirtazapine exposure (2006-2011) were obtained from the American Society for the Prevention of Cruelty to Animals' Animal Poison Control Center. The following parameters were recorded: signalment, weight, outcome, agent ingested, amount ingested, route of exposure, clinical signs observed, intended of use, onset time of signs and duration of signs. Results The 10 most commonly observed adverse effects reported in 84 cats exposed to mirtazapine included vocalization (56.0% of cats; mean dose 2.56 mg/kg), agitation (31.0%; 2.57 mg/kg), vomiting (26.2%; 2.92 mg/kg), abnormal gait/ataxia (16.7%; 2.87 mg/kg), restlessness (14.3%; 3.55 mg/kg), tremors/trembling (14.3%; 2.43 mg/kg), hypersalivation (13.0%; 2.89 mg/kg), tachypnea (11.9%; 3.28 mg/kg), tachycardia (10.7%; 3.04 mg/kg) and lethargy (10.7%; 2.69 mg/kg). Fifty-nine (70.2%) cases were considered accidental ingestions and 25 (29.8%) cases were given mirtazapine as prescribed. The doses associated with signs of toxicity were 15.00 mg (40 cats), 3.75 mg (25 cats), 7.50 mg (four cats), 30.00 mg (one cat), 18.75 mg (one cat), 11.25 mg (one cat), 5.80 mg (one cat) and 1.88 mg (one cat). For cats with available information, the onset of clinical signs ranged from 15 mins to 3 h, and time to resolution of clinical signs ranged from 12-48 h. Conclusions and relevance The greater number of adverse effects at 3.75 mg rather than 1.88 mg suggests that the latter may be a more appropriate starting dose for stimulating appetite while limiting toxicity. The benefit of dispensing exact doses of mirtazapine is implied given the likelihood of accidental administration of a full tablet (15 mg) and the resulting toxicity.

Evaluation of direct and indirect photodegradation of mianserin with high-performance liquid chromatography and short-term bioassays.

The widespread use of pharmaceuticals has lead to their detection in surface and ground waters. In the last year antidepressants in particular have shown very high growth dynamics of consumption and numerous research shows that these pharmaceuticals are detected in the environment and even in drinking water. Drugs and their metabolites can be subject to two types of photoreaction, direct and indirect photodegradation. These pharmaceuticals even at low concentration can have adverse effects on aquatic life, and the resulting photoproducts can be more toxic than parents compounds. The aim of this study was to evaluate the direct and indirect photodegradation of mianserin. The kinetics of the process and the identification of photoproducts were investigated by HPLC-PDA and HPLC-MS/MS, respectively. Ecotoxicity of mianserin before and after irradiation was assessed with a battery of assays with bacteria, protozoa and crustacea. The results show that mianserin was not toxic to Vibrio fischeri (Microtox), but its toxicity to protozoan Spirostomum ambiguum (Spirotox) and crustacean Thamnocephalus platyurus (Thamnotoxkit F(™)) was comparable to other antidepressants. On the basis of the results of the toxicity and HPLC before and after irradiation it can be seen that the decrease toxicity of mianserin was related only to a decrease of its concentration. The photoproducts had no impact to toxicity. The direct photodegradation of mianserin was more effective in UV/vis light than vis light. However the presence of humic acid in the indirect photodegradation increases the rate of degradation without regard to the kind of used light.

The in vitro genotoxic and cytotoxic effects of remeron on human peripheral blood lymphocytes.

Remeron (Mirtazapine) is an antidepressant drug which exerts its action by blocking presynaptic α-2-adrenergic receptors and postsynaptic serotonin types 2 and 3 receptors. In this in vitro analysis, human peripheral blood lymphocytes was treated by remeron (10, 25, 40 and 55 µg/mL) for 24 hours and 48 hours periods, then it was attempted to study of genotoxic and cytotoxic effects of the substance on human peripheral blood lymphocytes by some tests such as sister chromatid exchange (SCE), chromosomal abnormalities (CA) and micronucleus (MN) tests. Also proliferating effect of the substance was investigated. Remeron didn't significantly cause chromosomal abnormalities and sister chromatid exchange while caused micronucleus at 40 µg/mL concentration and 24 h periodic time and increased proliferation index of the both 24 and 48 hours treated cells was decreased in a concentration manner. Also, exposing to the remeron for 24 and 48 hours leaded to a decrease in mitotic index and nucleus division index in the cells by concentration dependent manner. These findings showed that remeron did not have significantly genotoxic effects on human peripheral blood lymphocytes while it showed cytotoxic effects on the cells, which is the first report on genotoxic and cytotoxic properties of remeron.

Photodegradation kinetics, cytotoxicity assay and determination by stability-indicating HPLC method of mianserin hydrochloride.

A stability-indicating HPLC method for the determination of mianserin hydrochloride in coated tablets was developed and validated. Also, drug photodegradation kinetics and cytotoxicity were determined. Chromatographic analyses were performed in an Ace RP-18 octadecyl silane column (250 mm x 4.6 mm i.d., particle size 5 microm) maintained at ambient temperature (25 degrees C). The mobile phase was composed of methanol, 50 mM monobasic potassium phosphate buffer and 0.3% triethylamine solution adjusted to pH 7.0 with phosphoric acid 10% (85:15, v/v) in isocratic mode at a flow rate of 1.0 mL x min(-1). The performed degradation conditions were: acid and basic media with HCl 1.0 M and NaOH 1.0 M, respectively, oxidation with H2O2 3% and the exposure to UV-C light. No interference in the mianserin hydrochloride elution was verified by degradation products formed. Linearity was assessed and ANOVA showed non-significant linearity deviation (p > 0.05). Adequate results were obtained for repeatability, intermediate precision, accuracy and robustness. The photodegradation kinetics of mianserin hydrochloride was evaluated in methanol. The degradation of mianserin could be better described as zero order kinetic (r = 0.9982). The UV-C degraded samples of mianserin hydrochloride were also studied in order to determine the preliminary cytotoxicity in vitro against mononuclear cells.

Inhibition of cardiac hERG potassium channels by tetracyclic antidepressant mianserin.

The antidepressant mianserin exhibits a tetracyclic structure that is different from typical tricyclic antidepressants (TCA) and that of selective serotonin reuptake inhibitors. In comparison to the older TCA, mianserin has been shown to have a superior risk profile regarding proarrhythmic effects, both in vitro and in vivo. However, the underlying molecular electrophysiological basis has not been elucidated to date. Therefore, we studied the effects of mianserin on cardiac hERG potassium channels, the predominant target of drug-induced proarrhythmia. HERG channels were expressed in the Xenopus oocyte expression system and in human embryonic kidney (HEK) cells and currents were measured with two-microelectrode voltage-clamp and whole-cell patch-clamp, respectively. Mianserin inhibited hERG currents in a dose-dependent manner with an IC(50) of 3.2 micromol/l in HEK cells. Onset of blockade was slow and the inhibitory effect was not reversible upon wash-out of the drug. In hERG channel mutants, Y652A and F656A, lacking aromatic residues in the S6 domain, the effect of mianserin was significantly reduced in comparison to the wild type. Mianserin inhibited hERG currents in the open and inactivated state, but not in the closed states. HERG inactivation kinetics were significantly altered by mianserin without marked effects on channel activation kinetics. The inhibitory effect was not frequency dependent. In conclusion, mianserin is a low-affinity hERG-blocking agent. However, taken together with the lack of APD-prolongation shown in other studies, mianserin seems to have a good safety profile. Lack of consistent QT prolonging effects of mianserin in previous studies may therefore be linked to additional effects such as inhibition of other cardiac ion channels. However, as demonstrated by clinical case reports, mianserin can induce proarrhythmic effects in susceptible patients. Therefore, in patients with complex co-medication (i.e., additional hERG-blocking agents) and in patients with risk factors for acquired long QT syndrome as well as in cases of overdose, adequate monitoring should be recommended.

Neuropharmaceuticals in the environment: mianserin-induced neuroendocrine disruption in zebrafish (Danio rerio) using cDNA microarrays.

Because of their environmental occurrence and high biological activity, human pharmaceuticals have received increasing attention from environmental and health agencies. A major bottleneck in their risk assessment is the lack of relevant and specific effect data. We developed an approach using gene expression analysis in quantifying adverse effects of neuroendocrine pharmaceuticals in the environment. We studied effects of mianserin on zebrafish (Danio rerio) gene expression using a brain-specific, custom microarray, with real-time polymerase chain reaction as confirmation. After exposure (0, 25, and 250 microg/L) for 2, 4, and 14 d, RNA was extracted from brain tissue and used for microarray hybridization. In parallel, we investigated the impact of exposure on egg production, fertilization, and hatching. After 2 d of exposure, microarray analysis showed a clear effect of mianserin on important neuroendocrine-related genes (e.g., aromatase and estrogen receptor), indicating that antidepressants can modulate neuroendocrine processes. This initial neuroendocrine effect was followed by a "late gene expression effect" on neuronal plasticity, supporting the current concept regarding the mode of action for antidepressants in mammals. Clear adverse effects on egg viability were seen after 14 d of exposure at the highest concentration tested. Based on the specific molecular impact and the effects on reproduction, we conclude that further investigation of the adverse effects on the brain-liver-gonad axis is needed for a correct ecological risk assessment of antidepressants.

Predicting drug-induced agranulocytosis: characterizing neutrophil-generated metabolites of a model compound, DMP 406, and assessing the relevance of an in vitro apoptosis assay for identifying drugs that may cause agranulocytosis.

DMP 406 is a clozapine analogue developed by Dupont-Pharma for the treatment of schizophrenia. Unfortunately it caused agranulocytosis in dogs during preclinical studies. Clozapine also causes agranulocytosis and this is believed to be due to a reactive nitrenium ion metabolite produced by neutrophils. We studied the oxidation of DMP 406 by activated neutrophils and found that the major reactive species that is produced is not a nitrenium ion but rather an imine. This metabolite is similar to the reactive metabolite that has been proposed to be responsible for mianserin-induced agranulocytosis. Therefore we also studied the oxidation of mianserin by activated neutrophils and found that, although the major species is an iminium ion, it also bears a lactam moiety in the piperazine ring resulting from further oxidation. We usually find that HOCl is a good model system for the production of reactive metabolites of drugs that are formed by activated neutrophils, but in the case of both DMP 406 and mianserin, the products produced were significantly different than those formed by activated neutrophils. In contrast, the combination of horseradish peroxidase and hydrogen peroxide (HRP/H(2)O(2)) formed a very similar pattern of products, and this system was used to produce sufficient quantities of metabolites to allow for identification. The reactive metabolites of both DMP 406 and mianserin reacted with a range of nucleophiles, but in many cases the reaction was reversible. The best nucleophile for trapping these reactive metabolites was cyanide. It has been demonstrated that the products of clozapine oxidation by HRP/H(2)O(2), presumably the nitrenium ion, induced apoptosis in neutrophils at therapeutic concentrations of clozapine. It has been suggested that this process is involved in the mechanism of clozapine-induced agranulocytosis. We tested DMP 406 and mianserin in this system to see if the ability of a reactive metabolite of a drug to cause apoptosis could predict the ability of that drug to cause agranulocytosis. We used clozapine as a positive control and we also tested olanzapine, a drug that forms a reactive metabolite similar to that of clozapine but is given at a lower dose and does not cause agranulocytosis. We found that DMP 406 did not increase apoptosis at concentrations below 50 microM, and although mianserin did increase apoptosis at 10 microM this is above the therapeutic concentration. Olanzapine caused an increase in apoptosis at the same concentration as clozapine (1 microM), but because its therapeutic concentration is lower, this concentration was above the pharmacological range. There was no increase in apoptosis with any drug in the absence of HRP/H(2)O(2). These results indicate that this assay is unable to reliably predict the ability of different types of drugs to cause agranulocytosis. This is not a surprising result given that different drugs may induce agranulocytosis by different mechanisms.

Imipramine- and mianserin-induced acute cell injury in primary cultured rat hepatocytes: implication of different cytochrome P450 enzymes.

The antidepressants, imipramine and mianserin, have been reported to cause liver damage. We investigated a role of cytochrome P450 (CYP)-mediated formation of a reactive metabolite in antidepressant-induced acute cell injury using hepatocytes isolated from male and female Wistar rats, and male Dark Agouti rats, which have different relative abundance of CYP enzymes. Culture of the hepatocytes with imipramine and mianserin caused a marked decrease in glutathione followed by protein thiol, which preceded lactate dehydrogenase leakage. The decreases in glutathione and protein thiol contents by imipramine were significantly slower in hepatocytes from male Dark Agouti rats than those from male Wistar rats, whereas no significant sex difference in Wistar rats was observed. The decrease in thiol by mianserin was significantly slower in hepatocytes from female Wistar than those from male Wistar rats, whereas no significant differences were found between Wistar and Dark Agouti males. Results consistent with alteration of the thiols were obtained for lactate dehydrogenase leakage induced by imipramine and mianserin. These findings indicated that CYP-mediated metabolic activation was involved in acute cell injury induced by the antidepressants; namely a CYP2D enzyme(s), which is deficient in Dark Agouti rats, and a male specific CYP enzyme(s) were suggested to be responsible for the cytotoxicity of imipramine and mianserin, respectively.

Acute lung failure induced by tricyclic antidepressants.

Overdosing of several drugs, such as tricyclic antidepressants, salicylates, and opiates, is known to induce effects like those seen in patients with adult respiratory distress syndrome. By exposing isolated perfused and ventilated rat lungs via the perfusate to six different tricyclic antidepressants (amitriptyline, nortriptyline, imipramine, desipramine, mianserine, and maprotiline), we investigated possible effects on ventilation (conductance and dynamic compliance), lung perfusion flow, and edema formation. The effects of these substances were pronounced and appeared within 15 min after exposure. Amitriptyline was studied in greater detail and caused a dose-related (0.01-1.0 mM) reduction in ventilation and perfusion flow. At the highest drug concentration pronounced lung edema was observed. Morphological studies were conducted with a transmission electron microscope. The microscopic preparations showed dose-related edema (amitriptyline 0.1 and 1.0 mM). The effects noted in our experimental studies are similar to those described in patients who have taken an overdose of tricyclic antidepressants. This emphasizes the possibility of a noncardiogenic edema component in these patients.

Amiodarone induces two different types of disorders in mouse alveolar macrophages.

It has been reported that amiodarone induces disorders of alveolar macrophages and pulmonary fibrosis, but the mechanism is not well-understood. This study was performed to elucidate the toxic mechanism from the standpoint of cellular function. Using alveolar macrophages obtained from a male Slc:ICR mouse, several injuries caused by amiodarone were compared to those caused by amantadine and mianserin as cationic amphiphilic drugs (CADs). As parameters for the drug effects, H(+)-ATPase and acid sphingomylinase activities, cellular pH, cytokine and prostaglandin releases, phagocytosis and neutral red uptake were measured. Amiodarone decreased H(+)-ATPase activity initially and subsequently increased cellular pH and decreased acid sphingomyelinase activity. These changes, which were also observed with amantadine and mianserin, were considered to be CAD-related. Amiodarone increased cytokine and prostaglandin releases and suppressed neutral red uptake and phagocytosis. These changes, being not induced by amantadine and mianserin, were considered to be specific for amiodarone. The above data suggest that amiodarone has two types of toxic effects on alveolar macrophages.

Toxicity of amiodarone on mouse pulmonary endothelial cells cultured with or without alveolar macrophages.

This study was designed to specify the toxicity of amiodarone toward mouse pulmonary endothelial cells in comparison with that of another cationic amphiphilic drug, i.e., mianserin. These examinations were performed in the absence and presence of mouse alveolar macrophages under transmembrane co-culture or in direct contact with the endothelial cells to assess the contribution of macrophages to the toxicities toward the endothelial cells. As a result of 24-hr treatment, amiodarone caused a decrease in cell viability, in H(+)-ATPase, acid sphingomyelinase, and acid phospholipase A2 activities, and in neutral red uptake, and an increase in permeability of the endothelial cells. Because the magnitude of changes in the endothelial cells was the greatest under direct contact with macrophages, and was the mildest without macrophages, macrophages were considered to enhance the toxicity of amiodarone toward the endothelial cells. Additionally, the toxic effect of amiodarone on the cells was depressed by pretreatment of them with docosahexaenoic acid (DHA) or alpha-tocopherol for 2 days and co-treatment with these agents for 1 day, but not with prednisolone or indomethacin co-treatment. DHA and alpha-tocopherol protected endothelial cells from the toxicity of amiodarone. The effect was more potent for DHA than alpha-tocopherol.

Elucidation of the structural requirements for the bioactivation of mianserin in-vitro.

The aim of this study was to investigate structure-activity relationships among a series of compounds related to the antidepressant drug, mianserin, with respect to their ability to produce cytotoxic metabolites. Human peripheral lymphocytes were used as target cells and these were exposed to the individual compounds, in the presence or absence of a drug metabolizing system derived from human liver. The individual enantiomers of mianserin showed differences in their cytotoxicity profiles; the R-(-) isomer giving NADPH-dependent cytotoxicity while the S-(+) isomer showed direct cytotoxicity at high concentrations. Cytotoxicity was reduced by removal from mianserin of the nitrogen atom at the 5 position and by substitution of a methyl group for a hydrogen atom at position 14b. In contrast, insertion of an oxygen atom at position 10 of the drug molecule, precluding the formation of a carbonium ion, had little effect on cytotoxic metabolite formation. The data are consistent with the proposal that one or more iminium ions derived from mianserin are responsible for the cytotoxicity observed in this in-vitro system and that appropriate chemical modification may preclude bioactivation of mianserin by P450 enzymes.

Effect of mianserin on blood glucose level in rabbits.

Blood glucose level was estimated in 18 h fasted albino rabbits following acute feeding of graded doses of mianserin. Mianserin (6.0 mg/kg) produced a gradually increasing hyperglycemic effect which became significant (P < 0.01) at 10 h and onwards. This appears to be due to increased turnover and release of noradrenaline by the drug. The same dose of mianserin also produced glucose intolerance during early hours probably by interfering with gastrin functions.

Structure-activity relationships of tricyclic antidepressants and related compounds in the wing somatic mutation and recombination test of Drosophila melanogaster.

Four antidepressants and one neuroleptic drug were tested for genotoxicity using the somatic mutation and recombination test (SMART) in wing cells of Drosophila melanogaster. Three-day-old larvae trans-heterozygous for two linked recessive wing hair mutations (multiple wing hairs and flare) were fed the test compounds in water or solvents mixed with a standard dry food for 48 h. Wings of the emerging adult flies were scored for the presence of spots of mutant cells which can result from either somatic mutation or mitotic recombination. The tricyclic antidepressant clomipramine, which is closely related to imipramine (previously shown to be genotoxic in somatic cells of Drosophila), was clearly genotoxic at concentrations above 10 mM. The structurally related antidepressants lofepramine and mianserin were positive only at 100 mM which is the maximum tolerated dose. The antidepressant maprotiline and the antipsychotic chlorpromazine, which are distinguished from the other compounds by a 6-membered central ring instead of a 7-membered one, were not genotoxic in the same dose range. These results lend further support for the hypothesis that an N atom in the heterocyclic 7-membered ring of the tricyclic molecule is responsible for the genotoxic property of the compounds in Drosophila.

A stereochemical investigation of the cytotoxicity of mianserin metabolites in vitro.

1. The metabolism of the enantiomers of mianserin to stable, chemically reactive and cytotoxic metabolites by human liver microsomes has been investigated in vitro. 2. Both enantiomers were metabolised to three major oxidation products: 8-hydroxymianserin, desmethylmianserin and mianserin 2-oxide. Hydroxylation occurred more readily with the S-enantiomer, whereas desmethylmianserin was always the major metabolite of the R-enantiomer. 3. The generation of chemically reactive metabolites exhibited a marginal degree of stereoselectivity, as assessed by irreversible binding of drug to microsomal protein (S greater than or equal to R; P less than or equal to 0.05). 4. The formation of metabolites which were cytotoxic towards human mononuclear leucocytes was greater (P less than or equal to 0.001] for R(-)-mianserin than for S(+)-mianserin and showed a significant correlation with N-demethylation (r = 0.84, P less than or equal to 0.01).

An in vitro study of the microsomal metabolism and cellular toxicity of phenytoin, sorbinil and mianserin.

1. The cytotoxicity of metabolites generated from phenytoin, sorbinil and mianserin by human and mouse liver microsomes was assessed by co-incubation with human mononuclear leucocytes as target cells. Cytotoxicity was determined by trypan blue dye exclusion. 2. Phenytoin and sorbinil were metabolised by NADPH-dependent murine microsomal enzymes to cytotoxic metabolites. Cytotoxicity produced by both drugs was significantly enhanced by the epoxide hydrolase inhibitor trichloropropane oxide (TCPO). No significant cytotoxicity was observed in the presence of human liver microsomes. 3. Mianserin was metabolised by both human and mouse liver microsomes to a cytotoxin. Cytotoxicity was greater in the presence of human liver microsomes (13.7 +/- 2.2%; mean +/- s.d. for four livers, compared with 6.0 +/- 2.4%, mean +/- s.d., n = 4, with mouse liver microsomes), and was unaffected by pretreatment with TCPO. 4. Stable metabolites were quantified by radiometric high performance liquid chromatography. Phenytoin and sorbinil were metabolised to 5-(p-hydroxyphenyl)-5-phenyl-hydantoin (0.3-0.5% of incubated radioactivity) and 2-hydroxysorbinil (0.4-2.7% of incubated radioactivity), respectively, by both human and mouse liver microsomes. 5. Mianserin was metabolised to 8-hydroxymianserin and desmethylmianserin by both human and mouse liver microsomes. Desmethylmianserin was the major product in incubations with human liver microsomes (32.3 +/- 12%, mean +/- s.d. for four livers), whereas 8-hydroxymianserin was the predominant metabolite generated by mouse liver microsomes (25.9 +/- 1.5%, mean +/- s.d., n = 4). 6. Generation of electrophilic metabolites was assessed by determination of the amount of radiolabelled material which became irreversibly bound to protein. Only mouse liver microsomes activated phenytoin to a chemically reactive metabolite, whereas both mouse and human liver microsomes generated reactive metabolites from sorbinil and mianserin. 7. These studies show that drug cytotoxicity can be mediated by low concentrations (circa microM) of metabolites generated by NADPH-dependent hepatic microsomal enzymes; however demonstration of cytotoxicity in vitro has not been established as a means of predicting in vivo toxicity.