Missed chances? Sequestration and non-sequestration of alkaloids by moths (Lepidoptera).

Some butterflies and moths sequester and retain noxious phytochemicals for defence against predators. In the present study, three moth species, the garden tiger moth, Arctia caja, the death hawk moth, Acherontia atropos, and the oleander hawk moth, Daphnis nerii, were tested whether they sequester alkaloids from their host plants. Whereas A. caja consistently sequestered atropine from Atropa belladonna, also when atropine sulfate was added to the alkaloid-free diet of the larvae, A. atropos and D. nerii were found to be unable to sequester alkaloids, neither atropine nor eburnamenine from Vinca major, respectively. Instead of acquiring toxicity as chemical defence, nocturnal lifestyle and cryptic attitudes may improve their chances of survival.

Assay of ethanol and congener alcohols in serum and beverages by headspace gas chromatography/mass spectrometry.

The analysis of ethanol and of its congeners in blood plays an important role in forensic cases, especially when allegations are made that alcohol has been consumed after an accident. In alcoholic beverages, congener alcohols are by-products and are generated during fermentation. The assay of these compounds in serum samples and beverages has been previously performed using headspace-gas chromatography-flame ionization detection methods (HS-GC-FID). As an alternative, a robust headspace-gas chromatography-mass spectrometry (HS-GC-MS) procedure was developed and validated, which has the following advantages:•Simultaneous determination of ethanol, congener alcohols and other endogenous substances.•Reduction of matrix interference by increasing selectivity and specificity.•Clear separation of the positional isomers 3-methyl-1-butanol and 2-methyl-1-butanol.

l-Thyroxin and the Nonclassical Thyroid Hormone TETRAC Are Potent Activators of PPARγ.

Thyroid hormones (THs) operate numerous physiological processes through modulation of the nuclear thyroid hormone receptors and several other proteins. We report direct activation of the nuclear peroxisome proliferator-activated receptor gamma (PPARγ) and retinoid X receptor (RXR) by classical and nonclassical THs as another molecular activity of THs. The T4 metabolite TETRAC was the most active TH on PPARγ with nanomolar potency and binding affinity. We demonstrate that TETRAC promotes PPARγ/RXR signaling in cell-free, cellular, and in vivo settings. Simultaneous activation of the heterodimer partners PPARγ and RXR resulted in high dimer activation efficacy. Compared to fatty acids as known natural ligands of PPARγ and RXR, TETRAC differs markedly in its molecular structure and the PPARγ-TETRAC complex revealed a distinctive binding mode of the TH. Our observations suggest a potential connection of TH and PPAR signaling through overlapping ligand recognition and may hold implications for TH and PPAR pharmacology.

Excretion of 4-fluoroamphetamine and three metabolites in urine after controlled oral ingestion.

Each year, synthetic drugs are occurring in high numbers in the illicit drug market. But data on their pharmacology and toxicology are scarcely available. Therefore, a pilot study was performed to evaluate excretion of 4-fluoroamphetamine (4FA) in humans and identify metabolites in urine. Twelve subjects ingested 100 mg and five 150 mg 4-FA in a bitter lemon drink. Urine samples were scheduled at baseline and 4 times during the following 12 h and analyzed by liquid chromatography-mass spectrometry (LC-MSMS). Concentrations of 4-FA were in the range of 0.7-38 mg/l which is in accordance with the data in previously reported cases. A marked decrease of creatinine excretion in the first two samples was noted. The creatinine normalized concentrations show a maximum 4 h after ingestion in accordance with serum pharmacokinetics. Three products of two metabolic pathways were identified in very low concentrations, two diastereomers of 4-fluorophenylpropanolamine and one ring hydroxylated 4-FA that was conjugated to a large extent. The concentration-time courses paralleled those of 4-FA. The study results show the range of 4-FA concentrations to be expected in urine after oral ingestion of typical dosages and show two pathways of 4-FA metabolism.

Pharmacokinetic properties of 4-fluoroamphetamine in serum and oral fluid after oral ingestion.

INTRODUCTION: Each year, synthetic drugs occur in high numbers on the illicit drug market. But data on their pharmacology and toxicology are scarce. Therefore, a controlled study was performed to evaluate pharmacokinetic parameters of 4-fluoroamphetamine (4-FA) in humans and to compare it with effects. METHODS: Twelve subjects ingested 100 mg and five subjects also received 150 mg 4-FA in a bitter lemon drink. Blood and oral fluid samples were taken during the following 12 hours and analyzed for 4-FA and traces of amphetamine as impurity by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: For 12 hours after ingestion, the concentration-time course of 4-FA was similar to that of amphetamine with maximal concentrations appearing in serum after about 2 hours (in median 195 ng/mL after the 100 mg dose, range 155-316 ng/mL). The elimination half-life was approximately 8-9 hours and shorter than that of amphetamine but it exhibited a marked variation (5.5-16.8 hours). In oral fluid, 4-FA could also be detected for 12 hours and concentrations were higher than in serum. During the first 3 hours after ingestion concentrations were higher, most probably due to oral contamination. Serum concentrations in forensic cases were in the range of those observed in the present study suggesting dosages in recreational use in the range of those tested here. CONCLUSIONS: The pharmacokinetic properties of 4-FA are similar to that of amphetamine including a marked variation in elimination. However, recreational dosages may already exhibit prominent adverse effects and may even have life-threatening consequences.

Lack of alkaloids and tetrodotoxin in the neotropical frogs Allobates spp. (Aromobatidae) and Silverstoneia flotator (Dendrobatidae).

Frogs of the family Dendrobatidae are known to contain toxic alkaloids in their skin secretion, but Allobates species of the closely related Aromobatidae family are considered to lack toxic secretions. However, contradictory results have been reported. Analyses of alcohol extracts from three different Allobates species from South-America (Guiana Shield), Central America (Costa Rica), and from the dendrobatid frog Silverstoneia flotator confirm the absence of alkaloids and tetrodotoxin in aromobatids and in a dendrobatid of the subfamily Colostethinae.

Loss of skin alkaloids in poison toads, Melanophryniscus klappenbachi (Anura: Bufonidae) when fed alkaloid-free diet.

Toads of the genus Melanophryniscus contain toxic alkaloids sequestered from a diet of mainly ants and mites. Wild-caught specimens of M. klappenbachi were fed an alkaloid-free diet and their alkaloid profile was analyzed during 38 months in captivity. Individual alkaloid patterns varied considerably. Over time, the concentration of two alkaloids, pumiliotoxin 251D and 3,5-disubstituted indolizidine 195B, significantly declined, suggesting that the toads depend on continuous access to alkaloid-containing prey to maintain natural levels of their chemical defense.

Excretion of metabolites of the synthetic cannabinoid JWH-018 in urine after controlled inhalation.

Each year, synthetic cannabinoids are occurring in high numbers on the illicit drug market but data obtained after controlled application are rare. The present study on pharmacokinetics in urine is part of a pilot study on adverse effects of JWH-018, which is one of the oldest and best known synthetic cannabinoids. Six subjects inhaled smoke from 2 and 3mg JWH-018. The drug and ten potential metabolites were analyzed in urine samples collected during 12h after inhalation by liquid chromatography-mass spectrometry (LC-MS/MS) without and with conjugate cleavage. The parent compound was not detectable, but 13 of its metabolites, all of which were conjugated. Concentrations of the predominant metabolite, JWH-018 pentanoic acid, were less than 5ng/ml, but in two subjects it was still detected up to 4 weeks after ingestion. Other major metabolites were 5- and 4-HOpentyl-JWH-018, JWH-073 butanoic acid and a hypothetically dihydroxylated and dehydrogenated metabolite of JWH-018. Occasionally, further hydroxylated metabolites were found. Generally, hydroxylated metabolites were detected in concentrations lower than 1ng/ml already 10h after inhalation. All concentrations were much lower than reported for urine samples of authentic JWH-018 users. The formation of the metabolite JWH-018 pentanoic acid was found to be slightly delayed, but its rather high concentrations and detection over several weeks after single dosing makes it a useful target for urine analysis. The different excretion of carboxylic acid and hydroxylated metabolites may aid in evaluation of time of use.

Pharmacokinetic properties of the synthetic cannabinoid JWH-018 in oral fluid after inhalation.

Each year, synthetic cannabinoids occur in high numbers on the illicit drug market, but data on their detectability are rarely available. A pilot study was performed to assess adverse effects of JWH-018, which is one of the oldest and best known synthetic cannabinoids. Oral fluid has been evaluated as a specimen for drug monitoring. Six subjects inhaled smoke derived from 2 and 3 mg JWH-018. The drug and 10 of its metabolites were analyzed in oral fluid samples collected during the following 12 hours using the Quantisal collection device by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Maximum concentrations of JWH-018 reached 2.2-2036 (median 25.7) ng/mL after inhalation and decreased during the next hour to only 0.08-8.42 (median 0.89) ng/mL. Metabolites were not found. During the elimination phase (median half-life 1.69 hours), detection of the drug over 6-12 hours (median 8 hours) after inhalation was achieved (0.024 ng/mL limit of quantification). Oral fluid/serum ratios varied considerably intra- and inter-individually in a range of 0.05-555 (median 1.38). The detection of JWH-018 in oral fluid requires high analytical sensitivity even 1 hour after inhalation. The pharmacokinetic properties of inhaled JWH-018 are similar to those of THC. Times for detection are typically less than 12 hours. High variability of the oral fluid/serum ratio precludes extrapolation of oral fluid concentrations to blood.

Influence of bleaching and coloring on ethyl glucuronide content in human hair.

Ethyl glucuronide (EtG) is increasingly used in forensic toxicology as a marker for alcohol use in analyses of hair samples, especially in abstinence control. Some cosmetic treatments are considered to markedly reduce the EtG content. In view of especially many women with coloured hair the present study was performed to further investigate the effect of a variety of colouring procedures (bleaching, tinting, permanent and semi-permanent dyeing, henna) on the EtG content. Untreated hair samples (n = 12, EtG 13.9-64.7 pg/mg) were re-analyzed (gas chromatography- negative chemical ionization mass spectrometry, 0.8 pg/mg quantification limit) after different treatment procedures. A decrease of the EtG content of at least 10% occurred in every case. The reduction in comparison to the untreated hair was expectedly high for permanent dyeing and bleaching with 18.1% of the initial content (median, range 0.0-50.9%) and 18.4% (0.0-46.7%), respectively. For henna this was 38.3% (0.0-83.0%), for tinting 70.4% (29.0-90.8%), for semi-permanent dyeing 41.9% (0.0-77.4%). With permanent hair dye the EtG content was decreased to below 7 pg/mg in 10 of 12 cases, in 3 cases even below the LOD (0.2 pg/mg). Surprisingly henna treatment without oxidative component had a marked influence, EtG was below 2 pg/mg in 2 of 12 samples. The study showed that all tested coloration procedures markedly affected the deposited EtG content. Even temporary or henna coloration may have a marked effect. The present data support the recommendation to exclude hair samples with colour manipulations for analysis on the EtG content as a precaution in alcohol abstinence programs. Copyright © 2017 John Wiley & Sons, Ltd.

Estimating the age of the adult stages of the blow flies Lucilia sericata and Calliphora vicina (Diptera: Calliphoridae) by means of the cuticular hydrocarbon n-pentacosane.

Age estimation of insects like blow flies plays an important role in forensic entomology and can answer questions in regard to time of death. So far the focus is on the immature stages of these insects, but recently the adult fly became a target of interest. It has been established that the profile of specific cuticular hydrocarbons (CHCs) changes in a consistent pattern as adult insects age; thus, their analysis could be a promising tool for the age estimation of adult insects. We investigated the CHC n-pentacosane (nC25) on the legs of the adult blow flies Lucilia sericata and Calliphora vicina with gas chromatography-mass spectrometry. The flies were kept at room temperature (17°C±2°C) and 12:12 L:D from Day 1 to Day 20 post-emergence. For each of five flies per species, the amount of nC25 on all legs was determined daily. The amounts of nC25 on C. vicina increased linearly (R2=0.949). No significant difference between sexes could be detected. While L. sericata showed the same linear increase in general, we found significant (p<0.001) differences in the amount of nC25 between males and females. Although the amounts of nC25 increased linearly for both sexes (males: R2=0.948; females: R2=0.920), female L. sericata produced more nC25 than males. An equation for the prediction of fly age is constructed from these data. Although the influence of various environmental factors, e.g., fluctuating temperatures, still needs to be tested, nC25 seems to be a promising tool for the age estimation of adult flies.

Pharmacokinetic properties of the synthetic cannabinoid JWH-018 and of its metabolites in serum after inhalation.

Each year, synthetic cannabinoids are occurring in high numbers in the illicit drug market, but data on their pharmacology and toxicology are scarcely available. Therefore, a pilot study was performed to assess adverse effects of JWH-018, which is one of the oldest and best known synthetic cannabinoids. Six subjects inhaled smoke from 2 and 3mg JWH-018. The drug and nine of its metabolites were analyzed in their blood samples taken during the following 12h by liquid chromatography-mass spectrometry (LC-MSMS). The maximum concentration of JWH-018 reached 2.9-9.9ng/ml after inhalation and markedly decreased during the next 1.5h, followed by a multiexponential decline (t1/2 in median 1.3h and 5.7h). The concentration of the pentanoic acid metabolite was slightly higher than that of the 3-, 4- and 5-hydroxypentyl metabolites and of the 6-hydroxyindol metabolite. The data also suggest a multiexponential decline and slow terminal elimination of JWH-018 and all metabolites. The detection of JWH-018 and of its metabolites in serum requires high analytical sensitivity. The pharmacokinetic properties of inhaled JWH-018 are similar to that of THC. A slow terminal elimination of drug and metabolites may lead to accumulation in chronic users.

Feeding on toxic prey. The praying mantis (Mantodea) as predator of poisonous butterfly and moth (Lepidoptera) caterpillars.

Caterpillars of the monarch butterfly, Danaus plexippus, feed on milkweed plants, Asclepias spp. (Apocynaceae), and sequester their toxic cardenolides aimed at deterring predators. Nevertheless, Chinese praying mantids, Tenodera sinensis, consume these caterpillars after removing the midgut ("gutting") including its plant content. In the present study, monarch caterpillars raised on A. curassavica, and those of the death's-head hawkmoth, Acherontia atropos, raised on Atropa belladonna containing atropine, were fed to mantids, Hierodula membranacea, which removed the gut of both species discarding about 59% of cardenolides and more than 90% of atropine, respectively. The ingestion of these compounds produced no apparent ill effects in the mantids and both were excreted with faeces. On the other hand, when mantids were fed with larvae of two moth species, Amata mogadorensis and Brahmaea certia, raised on non-poisonous host plants, the mantids showed the same gutting behaviour, thereby discarding indigestible plant material. As polar compounds, e.g. cardenolides and atropine, are not absorbed from the mantids midgut and do not pass the gut membrane, this enables the mantids to feed on toxic prey.

Death of a South American fur seal (Arctocephalus australis) after the ingestion of toads--evaluation of toad poisoning by toxicological analysis.

Animals in zoological gardens are at risk of severe and even lethal poisoning when they accidentally ingest toads. Here we report the case of an eleven month old male South American fur seal (Arctocephalus australis) which was found dead in its outdoor enclosure in the zoo of Dortmund, Germany. Autopsy revealed the presence of two adult, partly digested common toads (Bufo bufo) in the stomach. Toxicological analysis of the stomach content using high performance liquid chromatography coupled to time-of-flight mass spectrometry (LC-TOF MS) proved the presence of bufadienolides, the major cardiotoxic components of toad poisons. Using electrochemical luminescens immunoassay (ECLIA) compounds equivalent to digitoxin were detected in the blood sample confirming the absorption of toad poison components from the intestines into the circulation potentially leading to cardiac failure. In zoological gardens special precautions are necessary to protect non-native animals from encountering toads and the risk of poisoning, particularly in early spring, the spawning period of the toads.

Pitfall in cannabinoid analysis--detection of a previously unrecognized interfering compound in human serum.

In clinical and forensic toxicology, high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS) is increasingly used since it allows the development of sensitive and fast drug analysis procedures. During development of a LC-MS/MS method for determination of the psychoactive cannabinoid Δ(9)-tetrahydrocannabinol (THC) and of its two metabolites 11-hydroxy-THC (THCOH) and 11-nor-9-carboxy-THC (THCCOOH) in serum, a previously unrecognized interfering compound was detected. Extending the fast gradient elution program by an isocratic phase leads to sufficient separation of the interfering compound, initially co-eluting with THCCOOH and exhibiting the same fragments. For characterization, product ion scans and precursor ion scans were performed. Samples from cannabis users were analyzed to estimate the abundance of the interfering compound. The mass spectrometric experiments showed that the interfering compound exhibited the same molecular mass as THCCOOH and a similar fragmentation pattern except for relative fragment intensities. This compound was exclusively detectable in authentic samples. Concentrations were in the range of 4.5 to 51 % (median 14.6 %, n = 73) of those of THCCOOH. After further optimization of the gradient, the method was sufficiently selective and sensitive and validation parameters were within acceptance limits. A new compound related to cannabis use was detected in human serum, and data suggest an isomeric structure to THCCOOH. Considering the rather high amounts observed, it was surprising that this compound had not been detected previously. Further studies on its structure and origin are necessary.

Retrospective analysis of synthetic cannabinoids in serum samples--epidemiology and consumption patterns.

Herbal mixtures contain synthetic cannabinoids, which can cause severe intoxications. Due to the great variety and the changing spectrum of substances on the drug market, prevalence data are limited, and data on prevalence rates of synthetic cannabinoids in forensic cases are not available. The present study was performed to survey the prevalence of synthetic cannabinoids in cases of traffic and criminal offences in the German state Hesse in 2010. The applied analytical method covered all synthetic cannabinoids on the drug market at that time, and with 20% of the blood samples (422 out of 2201) a representative number was reanalyzed. In twelve samples synthetic cannabinoids were identified and a prevalence of 2.8% was estimated. Consumption patterns showed predominantly cases of multi-drug consumption (10 cases); the combination with cannabis or alcohol was frequent (four cases each). The observed deficits were moderate with the exception of aggravation of paranoia in one case. The symptoms were either compatible with the effects of cannabinoid agonists or attributable to alcohol or other drugs found in the blood samples. Our current analytical strategy is to perform such analyses only in cases where use is suspected or where symptoms are not explained by routine toxicological analyses. Hence, the positive rate is rather low highlighting the need to keep up with the developments on the drug market and to establish sensitive screening methods covering a broad range of substances that can be updated fast, e.g., relying on collections of mass spectrometric reference data.

Poor alkaloid sequestration by arrow poison frogs of the genus Phyllobates from Costa Rica.

Frogs of the genus Phyllobates from Colombia are known to contain the highly toxic alkaloid batrachotoxin, but species from Central America exhibit only very low levels or are entirely free of this toxin. In the present study alcohol extracts from 101 specimens of Phyllobates lugubris and Phyllobates vittatus and 21 of three sympatric species (Dendrobates pumilio, Dendrobates auratus, Dendrobates granuliferus) from Costa Rica were analyzed by gas chromatography-mass spectrometry. Whereas the extracts of the Dendrobates species exhibited typical profiles of toxic alkaloids, those of the two Phyllobates species contained low levels of few alkaloids only, batrachotoxin was not detected. Although the feeding pattern of the Dendrobates and Phyllobates species are similar as revealed by examination of their stomach content (mainly ants and mites), the Phyllobates species are poorly sequestering alkaloids from their food source in contrast to the Dendrobates frogs.

Cantharidin and demethylcantharidin (palasonin) content of blister beetles (Coleoptera: Meloidae) from southern Africa.

In two species of meloid beetles, Hycleus oculatus and Hycleus tinctus, from southern Africa, cantharidin and demethylcantharidin (palasonin) were assayed quantitatively. For cantharidin the mean value per specimen was about 1 mg for H. oculatus and 0.2 mg for H. tinctus, the mean palasonin concentration was 20 (H. oculatus) and 12 times (H. tinctus) lower, respectively. However, considerable individual variation in the cantharidin concentration was observed and values of more than 6 mg of this compound per beetle were measured pointing to the high risk of severe and even fatal poisoning when ingesting these insects.

Criminal poisoning of commuters in Bangladesh: prospective and retrospective study.

Travel-related poisoning is an emerging social and public health emergency in Bangladesh but its cause and significance have not been determined. To investigate this syndrome we performed a prospective clinical study and retrospective analysis of hospital records in a general medicine unit of a public tertiary care teaching hospital in Dhaka, Bangladesh, using toxicological analysis by fluorescence polarization immunoassay (FPIA) and liquid chromatography coupled to time-of-flight mass spectrometry (LC-TOF MS). The participants of the prospective study were 130 consecutive patients aged 16-80 years who were admitted with central nervous system depression (Glasgow Coma Score 3-14) after using public transportation, in the absence of other abnormalities, from January through June 2004, and a convenience sample of 15 such patients admitted during 3 days in May 2006. In 2004-2006, travel-related poisoning increased from 6.1 to 9.5% of all admissions (210-309 of 3266-3843 per year), representing 46.6-55.7% of all admitted poisoning cases. Incidents were associated with bus (76%), taxi, train, and air travel, or local markets; 98% of patients remembered buying or accepting food or drinks before losing consciousness. Direct financial damage (missing property) was diverse and frequently existential. Among 94 urine samples analyzed by FPIA, 74% tested positive for benzodiazepines. Among 15 urine samples analyzed by LC-TOF MS, lorazepam was detected in all; five also contained diazepam or metabolites; nitrazepam was present in three. FPIA results obtained for these 15 samples were below the recommended cut-off in eight (53%; lorazepam only). Our findings show that the massive medicosocial emergency of travel-related poisoning in Bangladesh is the result of drug-facilitated organized crime and that benzodiazepine drugs are used to commit these crimes, suggesting modifications to the local emergency management of the victims of this type of poisoning. They also highlight the need for more research in the neglected field of acute poisoning in Bangladesh, and for criminal investigations of the use of benzodiazepine drugs in this country.

Further studies on pumiliotoxin 251D and hydroquinone content of the skin secretion of Melanophryniscus species (Anura, Bufonidae) from Uruguay.

In whole animal ethanolic extracts from adult specimens of Melanophryniscus atroluteus (27 specimens) and M. devincenzii (16 specimens) as well as of two egg clutches and four tadpole samples from the latter species, the major alkaloid pumiliotoxin (PTX) 251D and hydroquinone were assayed quantitatively by gas chromatography/mass spectrometry. All toad extracts contained high concentrations of PTX 251D and hydroquinone and exhibited considerable variation in the content of these compounds among individual specimens. The extracts of the eggs and tadpoles were entirely free of alkaloids as well as hydroquinone, pointing to a dietary origin of these compounds.