Pharmacokinetics of α-amanitin in mice using liquid chromatography-high resolution mass spectrometry and in vitro drug-drug interaction potentials.

The aim of this study was to determine pharmacokinetics of α-amanitin, a toxic bicyclic octapeptide isolated from the poisonous mushrooms, following intravenous (iv) or oral (po) administration in mice using a newly developed and validated liquid chromatography-high resolution mass spectrometry. The iv injected α-amanitin disappeared rapidly from the plasma with high a clearance rate (26.9-30.4 ml/min/kg) at 0.1, 0.2, or 0.4 mg/kg doses, which was consistent with a rapid and a major excretion of α-amanitin via the renal route (32.6%). After the po administration of α-amanitin at doses of 2, 5, or 10 mg/kg to mice, the absolute bioavailability of α-amanitin was 3.5-4.8%. Due to this low bioavailability, 72.5% of the po administered α-amanitin was recovered from the feces. When α-amanitin is administered po, the tissue to plasma area under the curve ratio was higher in stomach > large intestine > small intestine > lung ~ kidneys > liver but not detected in brain, heart, and spleen. The high distribution of α-amanitin to intestine, kidneys, and liver is in agreement with the previously reported major intoxicated organs following acute α-amanitin exposure. In addition, α-amanitin weakly or negligibly inhibited cytochrome P450 and 5'-diphospho-glucuronosyltransferase enzymes activity in human liver microsomes as well as major drug transport functions in mammalian cells overexpressing transporters. Data suggested remote drug interaction potential may be associated with α-amanitin exposure.

Chronic exposure to arsenic in tap water reduces acetylcholine-induced relaxation in the aorta and increases oxidative stress in female rats.

The aim of this work is to determine whether consuming tap water containing arsenic (20 microg/L) alters oxidative stress levels in female rats and changes vascular response. Whereas nitric oxide produces complete relaxation, arsenic (7 months of exposure) impairs the acetylcholine-induced endothelial relaxation in the rat aorta compared with control rats. Arsenic exposure results in a marked elevation in reactive oxygen species in blood, and delta-aminolevulinic acid dehydratase activity, which is a sensitive biomarker for arsenic toxicity and oxidative stress, is significantly decreased in erythrocytes from 7-month-old rats. Diastolic blood pressure increases significantly in 7-month-old arsenic-treated versus control rats. The percentage of change in peripheral resistance increases. The results indicate that chronic environmental exposure to low levels of arsenic alters the release of vasoactive substances, causes changes in oxidative stress, and increases blood pressure in female rats.

In search of the Holy Grail: Poisons and extended release local anesthetics.

Regional anesthesia has been advocated as adjunct to a multimodal analgesia regimen. The limited duration of the action of available local anesthetics limits their application. Catheters, perineural or IV adjuvants, or repetition of blocks are modalities available to prolong the analgesic benefit of LRA. All of these approaches have their shortcomings. New extended release local anesthetic formulations may provide time-efficient and longer duration of analgesia with a single injection. Available data on liposomal bupivacaine are promising, and more recently, it has been FDA approved for use in interscalene brachial plexus block but not for other nerve blocks at this time. Several other new formulations and compounds, such as HTX-011, Neosaxitoxin, and SABER-Bupivacaine, are also being developed and tested for their safety and analgesic potential.

Evaluation of hepatic uptake clearance of tetrodotoxin in the puffer fish Takifugu rubripes.

In this study, we investigated the hepatic uptake clearance (CL(uptake)) of tetrodotoxin (TTX) in the marine puffer fish Takifugu rubripes by integration plot analysis after a single bolus injection of 0.25mg TTX/kg body weight into the hepatic vein at 20 degrees C. The blood concentration of TTX decreased over time after the injection, from 1451+/-45 ng/mL at 10 min to 364+/-59 ng/mL at 60 min. TTX concentrations in the spleen and kidney decreased in parallel with the blood concentrations, whereas those in the muscle and skin remained almost the same throughout the experiment. In contrast, the TTX concentration in the liver gradually increased, reaching 1240+/-90 ng/g liver at 60 min after injection. The amount of TTX that had accumulated in the liver 60 min after injection accounted for 63+/-5% of the administered dose. Integration plot analysis indicated a CL(uptake) of 3.1 mL/min/kg body weight in the liver for TTX, a rate far below that of the hepatic portal vein blood flow rate (at most, 9%). This finding is consistent with negligible extraction of TTX by the liver. The results demonstrated conclusively that the liver-specific distribution of TTX in T. rubripes is achieved by removal from the systemic circulation, but not by the hepatic first-pass effect.

Arsenic accumulation, elimination, and interaction with copper, zinc and manganese in liver and kidney of rats.

The arsenic accumulation, distribution and influences on metallothionein-1 (MT-1) expression and other trace elements in various organs were examined in rats orally exposed to sodium arsenate (iAs(V)). Rats received a dose of 0, 1, 10 and 100ppm of iAs(V) in drinking water daily for 4- and 16-weeks. Arsenic seems to be distributed in all of the tissues, and was accumulated relatively higher in the spleen, lung and kidney compared to the liver, and much lower in skin and cerebrum. High dose of iAs(V)-exposure significantly increased the concentration of copper in the kidney, but did not influence other trace elements such as zinc and manganese in the liver. The mRNA expression of MT-1 was dose-dependently increased by iAs(V)-exposure in the liver whereas it was decreased in the kidney. These data indicate that arsenic is widely distributed and significantly accumulated in various organs and influences on other trace elements, and also modulates MT-1 expression in the liver and kidney.

[Cause of Death: 'Intoxication' - a Matter of the Concentration?] / Todesursache «Intoxikation¼ ­ alles eine Frage der Konzentration?

Cause of Death: 'Intoxication' - a Matter of the Concentration? Abstract. Elucidation of the cause of death is one of the main reasons for medico-legal investigations. In clinical toxicology, the severity of a given poisoning is typically assessed with the blood concentration of a pharmacologically or toxicologically active compound. Such an interpretation proves to be difficult or even impossible in postmortem toxicology. Numerous biochemical and biological processes beginning immediately after death may render the calculated drug concentration unreliable. Concentrations obtained from postmortem samples do not necessarily reflect the blood concentration at the time of death. A prediction if and to what extent such postmortem changes might have occurred is still impossible for individual cases. Interpretation therefore needs to be done with care, considering case circumstances and all available information.

Application of precolumn oxidation HPLC method with fluorescence detection to evaluate saxitoxin levels in discrete brain regions of rats.

Saxitoxin (STX) is one of several related toxins that cause paralytic shellfish poisoning (PSP). This toxin blocks neuronal transmission by binding to the voltage-gated Na+ channel and for this reason, it has been widely used in the study of Na+ channel. The aim of this study was to analyze STX distribution in different rat brain regions after its acute intraperitoneal (i.p.) administration. Male rats (150-200 g) were injected i.p. with STX (5 and 10 microg/kg of body weight). After three time intervals of 30, 60, and 120 min (for 5 microg/kg STX dose) and 30 min (for 10 microg/kg STX dose) animals were sacrificed by cervical dislocation. Brains were removed and dissected in seven regions. STX concentration was measured using a precolumn oxidation high-performance liquid chromatographic method with fluorescence detection (HPLC/FLD). STX was found in all the regions evaluated at ppm levels meaning that STX peripherical administered across the blood-brain barrier and is distributed along the whole brain.

[The importance and value of the toxicological and biochemical results in the management of the poisoned patient]. / Znaczenie i interpretacja wyników badan toksykologicznych i biochemicznych w postepowaniu z zatrutym pacjentem.

The most important tasks of modern analytic toxicology can be summarized as follows: identification and quantification of toxicants potentially responsible for the intoxication of an emergency patient; confirmation or exclusion of poisoning diagnoses; grading and prognosis of an intoxication; monitoring of elimination therapies; testing for drugs of abuse; exclusion of the presence of central depressants before organ explanation or discontinuation of life support; checking the compliance of patients with prescribed drug therapy; therapeutic drug monitoring. The interpretation of toxicologic results is one of the most difficult tasks in toxicology. It is not always possible to relate symptoms and blood concentrations of drugs and poisons. In many cases, it is necessary to monitor biochemical parameters in the management of poisoned patient.

Apiaceous vegetable constituents inhibit human cytochrome P-450 1A2 (hCYP1A2) activity and hCYP1A2-mediated mutagenicity of aflatoxin B1.

In humans, apiaceous vegetables (carrots, parsnips, celery, parsley, etc.) inhibit cytochrome P-450 1A2, a biotransformation enzyme known to activate several procarcinogens, including aflatoxin B1 (AFB). We evaluated eight phytochemicals from apiaceous vegetables for effects on human cytochrome P-450 1A2 (hCYP1A2) activity using a methoxyresorufin O-demethylase (MROD) assay and a trp-recombination assay. Saccharomyces cerevisiae was used for heterologous CYP1A2 expression and this yeast strain is also diploid and auxotrophic for tryptophan due to mutations in the trp5 alleles. When these two alleles undergo AFB-induced mitotic recombination, gene conversion occurs, allowing yeast to grow in the absence of tryptophan. The apiaceous constituents psoralen, 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP), and apigenin were potent inhibitors of hCYP1A2-mediated MROD activity in yeast microsomes, whereas quercetin was a modest hCYP1A2 inhibitor. Naringenin, caffeic acid, and chlorogenic acid did not inhibit hCYP1A2-mediated MROD activity. The 2-h pretreatment of intact yeast cells with psoralen, 5-MOP, and 8-MOP significantly improved cell survival after subsequent 4-h AFB treatment and reduced hCYP1A2-mediated mutagenicity of AFB. Apigenin also significantly decreased mutagenicity. These results suggest that in vivo CYP1A2 inhibition by apiaceous vegetables may be due to the phytochemicals present and imply that apiaceous vegetable intake may be chemopreventive by inhibiting CYP1A2-mediated carcinogen activation.

Strain-dependent disposition of inorganic arsenic in the mouse.

Recent studies have suggested that polymorphisms in the methylation of inorganic arsenic (iAs) exist in animals and humans. Methylation of iAs is an important step in the elimination of arsenic. The objective of this study was to examine whether there are differences in iAs disposition, and hence methylation, between three strains of mice. Ninety-day-old female mice (strains: C3H/HeNCrlBR, C57BL/6NCrlBR, and B6C3F1/CrlBR) were administered [73As]arsenate or [73As]arsenite orally at dose levels of 0.5 or 5.0 mg As/kg. Another group of mice were administered [73As]arsenate (5.0 mg As/kg) intraperitoneally (i.p.). Disposition of [73As] was assessed by whole-body counting, and analysis of urine, feces and tissues for radioactivity. Urine was analyzed by chromatography for arsenic metabolites. Several strain- and dose-related effects in the disposition of [73As] were observed with both arsenicals. After oral administration, the clearance of [73As]arsenate, measured by whole-body counting, was dependent on the strain. However, because there was no strain dependence on clearance of [73As]arsenate administered i.p., the effect after oral administration may be due to a difference in absorption of arsenate between the strains. With increased oral dose of arsenate and arsenite, the clearance of [73As] was slower and there was higher tissue retention of [73As]. The percentage of metabolites excreted in urine also was affected by the administered dose. With increased dose, the percentage of arsenite and monomethylarsonic acid were significantly increased, and dimethylarsinic acid decreased. However, our results suggest there is no overall difference between these strains of mice with respect to disposition of iAs. A better understanding of the role of phenotype in the disposition and toxicity of iAs would reduce the uncertainty in arsenic risk assessment.

Systematic toxicological analysis: computer-assisted identification of poisons in biological materials.

A new software was developed to improve the chances for identification of a "general unknown" in complex biological materials. To achieve this goal, the total ion current chromatogram was simplified by filtering the acquired mass spectra via an automated subtraction procedure, which removed mass spectra originating from the sample matrix, as well as interfering substances from the extraction procedure. It could be shown that this tool emphasizes mass spectra of exceptional compounds, and therefore provides the forensic toxicologist with further evidence-even in cases where mass spectral data of the unknown compound are not available in "standard" spectral libraries.

Do surface-active lipids in food increase the intestinal permeability to toxic substances and allergenic agents?

The incidence of many common diseases has increased during the last decades. High fat intake is a risk factor for many diseases. We propose that some of the negative effects of fat are caused by lipid-induced damage of the gastrointestinal epithelium, thus compromising the epithelial function as a barrier for passage of toxic substances and allergenic agents to the circulatory system. Monoglycerides (MGs), phospholipids and fatty acids (FAs) are surface-active molecules that in pharmaceutical studies act as permeability enhancers for hydrophilic drugs with low absorption. Three possible mechanisms were proposed: (a) lipid-induced alterations in intracellular events may cause destabilization of tight junctions between the GI epithelial cells, (b) lipids may destabilize cell membranes, (c) lipids cause intestinal cell damage, which increase the permeability of the GI epithelium. These "side effects" of lipids may partly explain the association between fat intake and disease observed in epidemiological studies.

Fatal strychnine poisoning: application of gas chromatography and tandem mass spectrometry.

The history and toxicological findings in a case of suicidal fatal strychnine poisoning are presented along with a description of the analytical methods. Detection and quantitation of strychnine in body fluids and tissues was performed by gas chromatography (GC) with nitrogen-phosphorus detection, using organic extraction and calibration by a standard addition method. Strychnine concentrations in subclavian blood (1.82 mg/mL), inferior vena cava blood (3.32 mg/mL), urine (3.35 mg/mL), bile (11.4 mg/mL), liver (98.6 mg/kg), lung (12.3 mg/kg), spleen (11.8 mg/kg), brain (2.42 mg/kg), and skeletal muscle (2.32 mg/kg) were determined. Confirmation of strychnine in blood and tissue was performed by GC with detection by tandem ion-trap mass spectrometry (MS). GC-MS-MS analysis, employing electron ionization followed by unit mass resolution and collision-induced dissociation of strychnine, resulted in confirmatory ions with mass-to-charge ratios of 334 (parent ion), 319, 306, 277, 261, 246, 233, and 220. Additional confirmation was provided by GC-MS-MS-MS analysis of each confirmatory ion, revealing an ion fragmentation pathway consistent with the molecular structure of strychnine. The case demonstrates body tissue and fluid distribution of strychnine in a fatal poisoning and the application of tandem MS in medical examiner casework.

General unknown screening in postmortem tissue and blood samples: a semi-automatic solid-phase extraction using polystyrene resins followed by liquid-liquid extraction.

The identification of general unknown poisons in complex biological materials like postmortem blood and tissue is a great challenge for the forensic toxicologist. Therefore, a screening procedure utilizing a semi-automatic work-up with an ASPEC system was developed. A broad range of different compounds can be isolated by using non-selective and generally applicable organic polymeric sorbents such as OASIS HLB or Isolute 101. Because colloidal solutions were applied to these sorbents, the denaturation of proteins, which can result in an irreversible loss of significant compounds by adsorption and occlusion, could be avoided. Because of the process of micellar chromatography followed by liquid-liquid extraction of the crude extract, very clean fractions were obtained from such complex matrices as postmortem blood, liver, and brain samples. High recoveries (72-100%) and good day-to-day relative standard deviations (1-17%) could be achieved with both polymeric sorbents. The procedure paves the way for the identification of general unknown poisons in target organs and is therefore a useful tool in the field of forensic toxicology.

Excreted thiocyanate detects live reef fishes illegally collected using cyanide--a non-invasive and non-destructive testing approach.

Cyanide fishing is a method employed to capture marine fish alive on coral reefs. They are shipped to markets for human consumption in Southeast Asia, as well as to supply the marine aquarium trade worldwide. Although several techniques can be used to detect cyanide in reef fish, there is still no testing method that can be used to survey the whole supply chain. Most methods for cyanide detection are time-consuming and require the sacrifice of the sampled fish. Thiocyanate anion (SCN(-)) is a metabolite produced by the main metabolic pathway for cyanide anion (CN(-)) detoxification. Our study employed an optical fiber (OF) methodology (analytical time <6 min) to detect SCN(-) in a non-invasive and non-destructive manner. Our OF methodology is able to detect trace levels (>3.16 µg L(-1)) of SCN(-) in seawater. Given that marine fish exposed to cyanide excrete SCN(-) in the urine, elevated levels of SCN(-) present in the seawater holding live reef fish indicate that the surveyed specimens were likely exposed to cyanide. In our study, captive-bred clownfish (Amphiprion clarkii) pulse exposed for 60 s to either 12.5 or 25 mg L(-1) of CN(-) excreted up to 6.96±0.03 and 9.84±0.03 µg L(-1) of SCN(-), respectively, during the 28 days following exposure. No detectable levels of SCN(-) were recorded in the water holding control organisms not exposed to CN(-), or in synthetic seawater lacking fish. While further research is necessary, our methodology can allow a rapid detection of SCN(-) in the holding water and can be used as a screening tool to indicate if live reef fish were collected with cyanide.

[What is a "poison"? Proposal of definition]. / ¿Qué es un «tóxico¼? Una propuesta de definición.

We discuss different interpretations of the term poison as well as the need of bringing up to date the changes in this matter according to the science progress. A clear and exact definition is proposed after analysing the factors that affect the relativity of the concept and its boundaries. The proposal for a definition is presented taking into account the most broadly extended concepts concerning its significance. That is to say: "a poison is, for human beings and their non-pathogenic and non-harmful biological environment, an electromagnetic or corpuscular radiation, or a non-infectious chemical agent, structured no larger in size than a small particle or fibre that, after being generated internally or after contact, penetration and/or absorption by a live organism, in sufficiently high dose, can produce or produces a direct or indirect adverse effect unrelated to its temperature or measurable electrical potential difference". The scientific knowledge needs accurate definitions to avoid ambiguities.

First report about saxitoxins in freshwater fish Hoplias malabaricus through trophic exposure.

Cyanobacterial waterblooms, such as the saxitoxin (STX) producer Cylindrospermopsis raciborskii, have been a worldwide concern in environmental health. However, the bioaccumulation of this neurotoxin in the trophic chain is not completely known. The aim of the present work was to evaluate STX bioaccumulation through chemical analyses and the toxic and trophic effects using biomarkers in the tropical freshwater fish Hoplias malabaricus. They were fed once every five days with Astyanax sp. before being subjected to intraperitoneal inoculation with STX extract (0.08 µg/100 g) obtained by lysis of toxic C. raciborskii strain (T3). After 20 days the brain was collected for acetylcholinesterase (AChE), superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), glutathione peroxidase (GPx), glutathione (GSH), lipoperoxidation (LPO), protein carbonylation (PCO), and comet assay analysis. The muscle was collected for STX chemical analysis. The activities of SOD and concentrations of PCO and LPO increased. The CAT, GST, and GPx activities decreased. Genotoxicity was observed in the experimental group. STX was not detected in muscle samples. Thus, an oxidative stress was observed in the brain, leading to the damage of lipids, proteins, and DNA. The mechanism of action of the neurotoxin in this subchronic exposure suggests an apoptotic cellular process.