Bioconcentration and behavioral effects of four benzodiazepines and their environmentally relevant mixture in wild fish.

We studied the adverse effects of four benzodiazepines frequently measured in European surface waters. We evaluated bioaccumulation potential of oxazepam, bromazepam, temazepam, and clobazam in freshwater fish species - perch (Perca fluviatilis) and we conducted a series of behavioral trials to assess their potential to alter boldness, activity, and social behavior. All selected endpoints were studied individually for each target benzodiazepine and as a mixture of all tested compounds to assess possible combinatory effects. We used a three-dimensional automated tracking system to quantify the fish behavior. The four compounds bioconcentrated differently in fish muscle (temazepam > clobazam > oxazepam > bromazepam) at high exposure (9.1, 6.9, 5.7, 8.1 µg L-1, respectively) and low exposure (0.5, 0.5, 0.3, 0.4 µg L-1, respectively) concentrations. A significant amount of oxazepam was also measured in fish exposed to temazepam, most likely because of the metabolic transformation of temazepam within the fish. Bromazepam, temazepam, and clobazam significantly affected fish behavior at high concentration, while no statistically significant changes were registered for oxazepam. The studied benzodiazepines affected behavior in combination, because the mixture treatment significantly changed several important behavioral traits even at low concentration, while no single compound exposure had such an effect at that dose. Based on our results, we conclude that effects of pharmaceuticals on aquatic environments could be underestimated if risk assessments only rely on the evaluation of single compounds. More studies focused on the combinatory effects of environmentally relevant mixtures of pharmaceuticals are necessary to fill the gaps in this knowledge.

Behaviour and cardiac response to stress in signal crayfish exposed to environmental concentrations of tramadol.

Evidence of the ecological and biological impact of pharmaceuticals in surface waters on aquatic organisms is increasing. Tramadol is a synthetic opioid analgesic used to treat chronic and acute pain. To investigate its long-term effects at environmentally relevant levels, we evaluated heart rate (HR) and locomotion of signal crayfish Pacifastacus leniusculus during a 21-day exposure to 1 µg L-1 tramadol followed by 14 days depuration. Locomotion and HR were recorded over a period 30 min before and 30 min after exposure to physiological fluids of an injured conspecific, a natural stressor, four times during the tramadol exposure and four times during depuration. A significant increase in HR following stress induction was found in the majority of tramadol-exposed and control crayfish, as well as significant group-specific HR changes between both groups. Locomotor activity during tramadol treatment differed from that during depuration, in general showing less time spent in locomotion and lower distance moved. The tramadol exposed crayfish exhibited higher velocity during depuration than during the exposure period. Results may suggest a potential shift in prey-predator relationships.

Use of chiral liquid chromatography for the evaluation of stereospecificity in the carbonyl reduction of potential benzo[c]fluorene antineoplastics benfluron and dimefluron in various species.

Benfluron (B) [5-(2-dimethylaminoethoxy)-7H-benzo[c]fluorene-7-one hydrochloride] is a potential antineoplastic agent. In the organism, B undergoes a rapid phase I biotransformation through oxidative and reductive metabolic pathways. The carbonyl reduction of B leads to reduced benfluron, red-B, this is one of the principal pathways for the deactivation of this compound. The structure of B was modified to suppress its rapid deactivation via the carbonyl reduction on C7. Dimefluron, D (3,9-dimethoxy-benfluron) is one of the derivatives of B, in which an alternative metabolic pathway (O-desmethylation) prevails over the carbonyl reduction. The goal of this study was to develop HPLC methods enabling chiral separations of the red-B and -D enantiomers. The separation of red-B enantiomers was successful done on a Chiralcel OD-R column (250 mm x 4.6 mm ID, 5 microm) using a mobile phase acetonitrile-1 M NaClO4 (40:60, v/v). Another mobile phase, methanol-1 M NaClO4 (75:25, v/v), had to be employed for the sufficient resolution of red-D enantiomers. Flow rate was 0.5 ml min(-1) in both cases. Red-B was detected at 340 nm, red-D at 370 nm. The above chiral HPLC methods were used for the study of the biotransformation of B and D in the microsomal fractions of liver homogenates prepared from various species (rat, rabbit, pig, guinea pig, goat and human). The enantiospecificity of the respective carbonyl reductases was evaluated and discussed for both prochiral compounds, B and D.

Disposition study of a new potential antineoplastic agent dimefluron in rats using high-performance liquid chromatography with ultraviolet and mass spectrometric detection.

The disposition of a new potential antineoplastic drug dimefluron after an oral administration to rats was investigated. Dimefluron, 3,9-dimethoxy-5-(2-dimethylaminoethoxy)-7H-benzo[c]fluoren-7-one hydrochloride, was administered in a single oral dose (250 mg kg(-1) of body weight) in the form of an aqueous solution via a gastric probe. Dimefluron metabolites were being searched for in rat faeces. Synthetic standards of the expected phase I metabolites (the products of O- and N-desmethylation, N-oxidation and carbonyl reduction of dimefluron) were prepared and used together with dimefluron and internal standard in the development of two HPLC bioanalytical methods based on different separation principles. The first separation of dimefluron and the phase I metabolites was tested on a 250 mm x 4 mm chromatographic column with LiChrospher 60 RP-selectB 5 microm (Merck) using an isocratic mobile phase containing 0.01 M nonylamine buffer (pH 7.4) and acetonitrile in the 1:2 ratio (v/v). The second separation was performed on a 250 mm x 4 mm chromatographic column Discovery HS F5, 5 microm (Supelco) using a linear gradient mode with the mobile phase containing acetonitrile and phosphate buffer (0.05 M KH2PO4, pH 3). The flow rate was 1 ml min(-1) in both cases. UV detection was performed in the dual wavelength mode, with 317 nm having been used for dimefluron and all 7H-benzo[c]fluoren-7-one metabolites, 367 nm for 7H-benzo[c]fluoren-7-ol metabolites. A higher homologue of dimefluron served as an internal standard. The identity of the dimefluron metabolites in biological samples was confirmed using HPLC-MS experiments. The elimination study showed that the concentration maximum for dimefluron and its metabolites in rat faeces was reached 48 h after the administration of the parent drug. O-Desmethylated derivatives of dimefluron prevailed among the phase I metabolites.