Lovro Stanovnik

Risk assessment of buckwheat flour contaminated by thorn-apple (Datura stramonium L.) alkaloids: a case study from Slovenia

In Slovenia, a mass poisoning incident involving 73 consumers with symptoms such as dry mouth, hot red skin, blurred vision, tachycardia, urinary retention, ataxia, speech disturbance, disorientation and visual hallucinations occurred in 2003. In all cases, consumers had eaten buckwheat flour food products within the last few hours. Investigations by responsible authorities identified the contamination of a range of buckwheat food products with thorn-apple (Datura stramonium L.) seeds containing toxic alkaloids, atropine and scopolamine. To ensure the safe consumption of buckwheat food products, we carried out risk characterisation and proposed provisional maximum residue levels (MRLs) of atropine and scopolamine mixture in buckwheat flour. In the absence of critical “no observed adverse effect levels” for atropine and scopolamine, we based our estimation of the acute reference doses on the lowest recommended therapeutic doses. Taking into account the additive effect of the two alkaloids, we calculated acute reference doses of the mixture, that is 0.05 µg/kg of body mass for atropine and 0.03 µg/kg of body mass for scopolamine. MRLs for atropine and scopolamine mixture in buckwheat flour were estimated in a worst-case scenario, that is consumption of 100 g of flour by a child weighing 10 kg and taking into account a range of atropine/scopolamine ratio in implicated food products, that is 0.85–3.3. We proposed the national MRLs for atropine/scopolamine mixture in buckwheat food products: 4.0 µg/kg (atropine) and 2.0 µg/kg(scopolamine). However, in view of the large variability in the alkaloid content, depending on the origin of the Datura, we propose that risk assessment should be carried out on a case-by-case basis, taking into account the ratio between atropine and scopolamine content in a particular sample.

The effect of compound 48/80 and of electrical field stimulation on mast cells in the isolated mouse stomach

It has been shown that compound 48/80 evokes acid secretion in the isolated mouse stomach by releasing histamine from mast cells. The aim of the present work was to study the distribution and type of mast cells in the isolated mouse stomach and to examine the effect of compound 48/80 and of electrical field stimulation (EFS) on these cells.Histological examination of the stomachs showed only the presence of connective tissue mast cells (CTMC) in all parts of the stomach except in the glandular mucosa where mucosal mast cells (MMC) predominated (MMC−71%, CTMC−29%). Compound 48/80 and EFS did not affect MMC, whereas CTMC showed marked degranulation in all parts of the stomach including the glandular mucosa. It can be concluded that CTMC, present in the gastric mucosa and being sensitive also to EFS, may be involved in the regulation of gastric secretion in the mouse.

TRICYCLIC ANTIDEPRESSANTS CHANGE PLASMA HISTAMINE KINETICS AFTER ITS SECRETION INDUCED BY COMPOUND 48/80 IN THE RAT

Tricyclic antidepressants (TCA) influence secretion in rat mast cells [1, 2]. In the rat, they reduce experimental inflammation [3–5]. It is possible that at least part of their anti-inflammatory action is due to the inhibition of histamine release and/or to changing the rate of its elimination from the site of inflammation. After administration of compound 48/80 in the rat there is an increase of blood histamine levels [6]. A lowering of such increase may serve as an indirect measure for the inhibition of histamine secretion from mast cells. Using the rat, we studied the effects of three TCA: amitriptyline (AM), doxepin (DO), imipramine (IM) on plasma histamine levels after compound 48/80 and also their effects on the rate of histamine disappearance from the circulation.

Simple validated LC-MS/MS method for the determination of atropine and scopolamine in plasma for clinical and forensic toxicological purposes.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of atropine and scopolamine in 100μL human plasma was developed and validated. Sample pretreatment consisted of protein precipitation with acetonitrile followed by a concentration step. Analytes and levobupivacaine (internal standard) were separated on a Zorbax XDB-CN column (75mm×4.6mm i.d., 3.5μm) with gradient elution (purified water, acetonitrile, formic acid). The triple quadrupole MS was operated in ESI positive mode. Matrix effect was estimated for deproteinised plasma samples. Selected reaction monitoring (SRM) was used for quantification in the range of 0.10-50.00ng/mL. Interday precision for both tropanes and intraday precision for atropine was <10%, intraday precision for scopolamine was <14% and <18% at lower limit of quantification (LLOQ). Mean interday and intraday accuracies for atropine were within ±7% and for scopolamine within ±11%. The method can be used for determination of therapeutic and toxic levels of both compounds and has been successfully applied to a study of pharmacodynamic and pharmacokinetic properties of tropanes, where plasma samples of volunteers were collected at fixed time intervals after ingestion of a buckwheat meal, spiked with five low doses of tropanes.

Acute effects of a low‐dose atropine/scopolamine mixture as a food contaminant in human volunteers

To verify the assumptions in our previous risk assessment of an atropine/scopolamine mixture in buckwheat flour, we performed a randomized, double‐blind, placebo‐controlled cross‐over study in 20 healthy, adult volunteers. The volunteers ingested a traditional Slovenian buckwheat meal, made of boiled buckwheat flour to which alkaloids were added. In addition to the placebo they ingested 0.12/0.10, 0.37/0.29, 1.22/0.95, 3.58/2.81 and 12.10/9.50 µg kg–1 body mass (BM) of the atropine/scopolamine mixture. The changes in body temperature, heart rate, salivary and sweat secretion, pupil size, near‐point vision and subjective symptoms were recorded regularly for 4 h after the ingestion. Decreased salivary and sweat secretion, increased heart rate and pupil size and reduced near‐point vision accompanied by characteristic subjective symptoms were observed at 12.10/9.50 µg kg–1 BM. At doses of 0.37/0.29 and 1.22/0.95 µg kg–1 BM, a significant decrease in the heart rate was noted, which we consider to be a critical effect of a low‐dose exposure to the atropine/scopolamine mixture. Although this did not have any clinical relevance in our subjects, it may have serious implications if it occurred in people with pre‐existent cardiac conditions or those on medications that may cause bradycardia. No significant changes in the observed end points were noted at 0.12/0.10 µg kg–1 BM. We estimate that the NOAEL (No Observed Adverse Effect Level) for the atropine/scopolamine mixture lies between the lower two administered doses. Applying the uncertainty factor of 10, we propose a new provisional Acute Reference Doses (ARfDs) of the mixture, i.e. 0.01 µg kg–1 BM for each alkaloid, and a further refinement using higher‐tier approaches. Copyright

The effect of compound 48/80, substance P and pentagastrin on the isolated guinea pig atrium.

It has been shown that histamine is present in guinea pig hearts. In the present work, the effect of some substances, known to liberate mast cell histamine, on the isolated guinea pig atria was studied. Compound 48/80 (100 μg/ml), pentagastrin (10−6 M) and substance P (10−5 M) were added 2–3 times to the isolated organs and the frequency of contractions was measured. At the end of experiments, the atria were examined histologically for mast cell degranulation.Compound 48/80 and pentagastrin increased the frequency of contractions of isolated atria. Substance P provoked a dose-dependent decrease of contraction frequency; this effect was diminished by atropine (10−5 M). All three substances provoked pronounced degranulation of mast cells present in the atrium, the effect of substance P being significantly greater than the effects of the other two substances. It can be concluded that mast cells, present in guinea pig atrium, are sensitive to the histamine liberators used; histamine is released in quantities high enough to produce an effect.

The role of tissue mast cells in exocrine secretion: studies in the submandibular gland of the cat.

Histamine was found to be stored in the submandibular gland of the cat mainly in the mast cells. The amine is released from mast cells by compound 48/80 and pilocarpine. This was demonstrated in thein vivo andin vitro experiments and by histological examination. During the physiological stimulation of the gland, via the electrical stimulation of the chorda tympani nerve, significant changes of histamine content were not found. This could be explained by the increased synthesis of histamine during physiological stimulation. The physiological role of histamine in salivary secretion was demonstrated. The similarity between the roles of histamine, mast cells and chromaffine-like cells in salivary and gastric secretion is discussed.

Mechanisms of changes in coronary arterial tone induced by bee venom toxins.

Our study elucidates some mechanisms of contractions or relaxations of isolated porcine left anterior descending coronary artery (LAD) induced by two peptides from the honeybee venom, melittin and apamin. Contractions or relaxations were measured on relaxed or precontracted arteries, respectively. Melittin at lower concentrations (0.1-10 microg/ml) induced transient relaxation, and contraction at higher concentrations (>or=7 microg/ml). The removing of the endothelium diminished the melittin-induced relaxation but did not affect the maximal contraction. The inhibition of prostaglandin and nitric oxide (NO) synthesis (by indomethacin and by N-omega-Nitro-l-arginine, respectively) and the use of K(+) channel inhibitors (apamin and charybdotoxin) showed that melittin evoked relaxation via an endothelium-dependent mechanism (NO production), and by activation of charybdotoxin-sensitive K(+) channels of smooth muscle. Apamin alone did not affect contraction or relaxation, but the inhibition of NO and prostanoid production revealed the involvement of apamin-sensitive K(+) channels of smooth muscle in melittin-induced relaxation. Our data show that melittin and apamin could affect contractility of porcine LAD at concentrations similar to those encountered in multiple honeybee stings in humans. Melittin could directly affect contractility of porcine LAD, whereas apamin acts as a modulator of the relaxant response to melittin.

Stereoselective and endothelium-independent action of nicardipine on the isolated porcine coronary artery

The qualitative and quantitative effects of the (+)-S and (-)-R enantiomers and of the racemic mixture of the Ca2+ channel antagonist, nicardipine, were compared on the isolated porcine coronary artery with intact and removed endothelium. All three forms of nicardipine inhibited the contractions induced by KCl (5-90 mM) in both vessel preparations. The potency (IC50) of the (+)-S and (-)-R enantiomers and of the racemic mixture was 6.6, 31.8 and 10.9 nM in the vessel with endothelium and 6.4, 41.9 and 9.8 nM in the vessel without endothelium. The parameters of the concentration-response curves for each form of nicardipine at a submaximal KCl (60 mM) concentration and the potency ratios between the two enantiomers ((+)-S/(-)-R) were not statistically significantly different (P>0.05) in the two vessel preparations. In conclusion, qualitatively, all three forms of nicardipine showed only Ca2+ channel antagonistic effects in both vessel preparations. Quantitatively, the inhibition of contraction was stereoselective, the (+)-S enantiomer being the most potent, and was endothelium-independent.

Direct thrombin inhibitors built on the azaphenylalanine scaffold provoke degranulation of mast cells

The main structural feature of direct thrombin inhibitor LK-732 responsible for the appropriate interaction at the thrombin active site is a strong basic group. A possibility that a strong basic group of LK-732 might contribute to the mast cell degranulation effect and consequent reduction of tracheal air flow (TAF) and fall of mean arterial blood pressure (MAP) in rats was investigated in the present study. At doses up to 5 mg/kg (i.v.), LK-732 did not cause significant changes of TAF and MAP. At 7 mg/kg (i.v.), a sudden reduction of TAF and a fall of MAP was observed within 5 min after LK-732 administration (75% mortality, p = 0.007). A less basic direct thrombin inhibitor LK-658 (21 mg/kg, i.v.) did not significantly disturb TAF and MAP. A reduction of TAF and a fall of MAP caused by LK-732 (7 mg/kg, i.v.) was almost completely abolished in rats with degranulated mast cells (0% mortality, p = 0.008). LK-732 concentration-dependently degranulated rat peritoneal mast cells in vitro (pEC(50) = 1.92 +/- 0.05 muM). A structure-activity relationship (SAR) study revealed that the terminal basic groups attached to the aromatic ring are responsible for the mast cell degranulation effect. A good correlation was observed between mast cell degranulation and pK(b) of analogues of LK-732 (R(2) = 0.49), but not between mast cell degranulation and thrombin K(i) (R(2) = 0.23). LK-732-induced reduction of TAF, the fall of MAP and high mortality originate from LK-732-induced mast cell degranulation. As judged by the SAR study, this effect could be overcome by reducing the basicity of LK-732.