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Pyrrolizidine alkaloids (PA) exert their toxic effects only after bioactivation. Although their toxicity has already been studied and metabolic pathways including important metabolites were described, the quantification of the latter revealed a large unknown portion of the metabolized PA. In this study, the qualitative and quantitative metabolite profiles of structurally different PAs in rat and human liver microsomes were investigated. Between five metabolites for europine and up to 48 metabolites for lasiocarpine were detected. Proposals for the chemical structure of each metabolite were derived based on fragmentation patterns using high-resolution mass spectrometry. The metabolite profiles of the diester PAs showed a relatively good agreement between both species. The metabolic reactions were summarized into three groups: dehydrogenation, oxygenation, and shortening of necic acid(s). While dehydrogenation of the necine base is considered as bioactivation, both other routes are considered as detoxification steps. The most abundant changes found for open chained diesters were dealkylations, while the major metabolic pathway for cyclic diesters was oxygenation especially at the nitrogen atom. In addition, all diester PAs formed several dehydrogenation products, via the insertion of a second double bond in the necine base, including the formation of glutathione conjugates. In rat liver microsomes, all investigated PAs formed dehydropyrrolizidine metabolites with the highest amount formed by lasiocarpine, whereas in human liver microsomes, these metabolites could only be detected for diesters. Our findings demonstrate that an extensive analysis of PA metabolism can provide the basis for a better understanding of PA toxicity and support future risk assessment.

Background Senecio jacobaea contains pyrrolizidine alkaloids that can induce severe hepatic intoxication in horses, either acute when ingested in high amounts or chronic when consumed over a long period. The aim of this study was to determine horses’ rejection behaviour towards the presence of Senecio jacobaea in hay when fed ad libitum. We hypothesized that adult horses can sort Senecio jacobaea out of the contaminated hay when hay is fed ad libitum. Six warmblood geldings with a mean (±SD) age of 15 ± 2 years were included. In a randomized study, Senecio jacobaea contaminated hay (5% or 10% contamination level) was provided at several timepoints over the day for 1 hour to six. Hay was provided ad libitum for the rest of the day. The horses’ rejection behaviour towards Senecio jacobaea was observed. If a horse ingested two Senecio jacobaea plants twice at different timepoints, then the horse was excluded from the experiment. Results Two out of six horses had to be excluded from the study after three out of 12 observation periods due to repeated Senecio jacobaea intake. Two other horses had to be excluded after nine and 11 out of 12 observation periods. Only two horses were able to sort out the various amounts (5 and 10% contamination level) of Senecio jacobaea during the whole experiment. Conclusions Horses’ intake of Senecio jacobaea cannot be avoided despite being fed with hay ad libitum. Due to the risk of chronic intoxication by pyrrolizidine alkaloids intake, feeding Senecio jacobaea contaminated hay must be avoided, and pastures with Senecio jacobaea growth are considered inappropriate for feed production.

Retrorsine is a hepatotoxic pyrrolizidine alkaloid (PA) found in herbal supplements and medicines, food and livestock feed. Dose-response studies enabling the derivation of a point of departure including a benchmark dose for risk assessment of retrorsine in humans and animals are not available. Addressing this need, a physiologically based toxicokinetic (PBTK) model of retrorsine was developed for mouse and rat. Comprehensive characterization of retrorsine toxicokinetics revealed: both the fraction absorbed from the intestine (78%) and the fraction unbound in plasma (60%) are high, hepatic membrane permeation is dominated by active uptake and not by passive diffusion, liver metabolic clearance is 4-fold higher in rat compared to mouse and renal excretion contributes to 20% of the total clearance. The PBTK model was calibrated with kinetic data from available mouse and rat studies using maximum likelihood estimation. PBTK model evaluation showed convincing goodness-of-fit for hepatic retrorsine and retrorsine-derived DNA adducts. Furthermore, the developed model allowed to translate in vitro liver toxicity data of retrorsine to in vivo dose-response data. Resulting benchmark dose confidence intervals (mg/kg bodyweight) are 24.1–88.5 in mice and 79.9–104 in rats for acute liver toxicity after oral retrorsine intake. As the PBTK model was built to enable extrapolation to different species and other PA congeners, this integrative framework constitutes a flexible tool to address gaps in the risk assessment of PA.

Pyrrolizidine alkaloids (PA) are secondary metabolites of certain flowering plants. The ingestion of PAs may result in acute and chronic effects in man and livestock with hepatotoxicity, mutagenicity, and carcinogenicity being identified as predominant effects. Several hundred PAs sharing the diol pyrrolizidine as a core structure are formed by plants. Although many congeners may cause adverse effects, differences in the toxic potency have been detected in animal tests. It is generally accepted that PAs themselves are biologically and toxicologically inactive and require metabolic activation. Consequently, a strong relationship between activating metabolism and toxicity can be expected. Concerning PA susceptibility, marked differences between species were reported with a comparatively high susceptibility in horses, while goat and sheep seem to be almost resistant. Therefore, we investigated the in vitro degradation rate of four frequently occurring PAs by liver enzymes present in S9 fractions from human, pig, cow, horse, rat, rabbit, goat, and sheep liver. Unexpectedly, almost no metabolic degradation of any PA was observed for susceptible species such as human, pig, horse, or cow. If the formation of toxic metabolites represents a crucial bioactivation step, the found inverse conversion rates of PAs compared to the known susceptibility require further investigation.

In tandem mass spectrometry the multiple reaction monitoring (MRM) mode is normally used for targeted analysis but this mode also has the potential to screen for structural similarities of analytes. On the basis of the fact that in general similar molecular structures result in similar fragments or losses of neutrals, this approach was used for pyrrolizidine alkaloid (PA) screening but could also be easily adapted to screen for other compound classes. PA are plant toxins of which several hundred individual compounds have been identified. Our MRM screening approach uses the structural relation and similar core structure of all PA which results in a common and thus predictable mass spectrometric fragmentation behaviour. On this basis a method was developed which screens for PA structures by MRM transitions and allows the detection of each individual PA down to a low microgram per kilogram concentration range. The approach was applied to investigate plants from the families of Asteraceae (several species of Senecio and Eupatorium ), Boraginaceae ( Echium , Cynoglossum , Borago and Anchusa officinalis as well as Heliotropium europaeum ) and Fabaceae ( Crotalaria incana ) for a complete qualitative and quantitative PA characterisation. All analytes that were detected as possible PA by MRM screening were further investigated by recording product ion spectra. Analytes which exhibited a typical PA fragmentation pattern were either confirmed as PA or otherwise deleted as false positive signals (false positive rate was below 10 %). Sum formulas of confirmed PA were determined by additional measurements applying high resolution mass spectrometry. In that way 121 unknown PA were identified and for the first time complete PA profiles of different PA plants were delivered.

The ingestion of seafood contaminated with the marine biotoxin okadaic acid (OA) can lead to diarrhetic shellfish poisoning with symptoms like nausea, vomiting and abdominal cramps. Both rat and the human hepatic cytochrome P450 monooxygenases (CYP) metabolize OA. However, liver cell toxicity of metabolized OA is mainly unclear. The aim of our study was to detect the cellular effects in HepG2 cells exposed to OA in the presence of recombinant CYP enzymes of both rat and human for the investigation of species differences. The results should be set in correlation with a CYP-specific metabolite pattern. Comparative metabolite profiles of OA after incubation in rat and human recombinant CYP enzymes were established by using LC-MS/MS technique. Results demonstrated that metabolism of OA to oxygenated metabolites correlates with detoxification which was mainly catalyzed by human CYP3A4 and CYP3A5. Detoxification by rat Cyp3a1 was lower compared to human CYP3A enzymes and activation of OA by Cyp3a2 was observed, coincident with minor overall conversion capacity of OA. By contrast human and rat CYP1A2 seem to activate OA into cytotoxic intermediates. In conclusion, different mechanisms of OA metabolism may occur in the liver. At low OA doses, the human liver is likely well protected against cytotoxic OA, but for high shellfish consumers a potential risk cannot be excluded.

Background, Aim and Scope One of the first occurrences of pharmaceutically active compounds in groundwater was reported from the sewage irrigation farms south of Berlin. At these sewage irrigation farms treated sewage effluent passed the soil and unsaturated zone before reaching the aquifer. Clofibric acid was detected in pore water from soils of those sewage irrigation farms in concentrations between 65 ng/L and 1430 ng/L. The aim of this study was to investigate the transport behavior of regularly detected clofibric acid, diclofenac, ibuprofen, and propyphenazone under conditions comparable to those at the sewage irrigation farms in a multiple compound sand column laboratory experiment. Materials and Methods Sediment column experiments were conducted to study the transport of pharmaceuticals in the unsaturated zone. The migration was measured in fine to medium grained sand and leaching solution containing 1 mg/L of pharmaceutically active compounds and 61 mg/L of the tracer lithium chloride (LiCl). For the analysis of the pharmaceutical compounds the water samples were adjusted to a pH value of 2 and then extracted by solid-phase extraction (SPE). Before extraction, the samples were spiked with a surrogate standard for analytical quality control. The sample extracts were analyzed by capillary gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring (SIM). Depending on the sample volume (100 to 200 mL) and the matrix, the limits of detection were between 1 and 10 ng/L, and the limits of quantitation were between 5 and 25 ng/L. Analysis for calcium, magnesium and lithium were carried out using a 'Trace Scan' ICP-AES from Thermo Jarrel Ash. Sodium, potassium, iron and manganese were analyzed using a Philips PU 9400 flame AAS. Analysis of anions was performed on a Dionex ion chromatograph DX 120. Results At the sewage irrigation farms the average concentrations of clofibric acid in the unsaturated zone declined from higher values near ground surface (480 ng/L) to lower values near the groundwater table (65 ng/L). From the pharmaceuticals analyzed only clofibric acid, primidone and propyphenazone could be analyzed in the first (upper) aquifer at the sewage irrigation farms. All other pharmaceuticals could neither be detected in the first aquifer nor in the deeper aquifers. Breakthrough curves from soil column experiments revealed no transformation and no retardation for clofibric acid, whereas transformation of diclofenac was so high (79%) that no retardation factor could be calculated. Ibuprofen was significantly transformed (37%), transformation of propyphenazone (17%) was quite low and retardation (Rf = 2.05) was in the range of previously conducted column experiments. Discussion The results confirm previously conducted experiments with clofibric acid where this compound was identified as highly mobile and persistent. The results that diclofenac and ibuprofen are significantly transformed where unexpected as other studies exhibited much lower transformation under saturated conditions at least for diclofenac. However, lower pH values and higher oxygen contents in the unsaturated zone compared to the aquifer may explain this observed high transformation of these compounds at the column experiments. Conclusions We conclude that irrigation with sewage effluent containing the compounds used in our experiments will lead to an input into groundwater of clofibric acid, whereas diclofenac and ibuprofen will most likely be transformed during the passage. Propyphenazone will be retarded but will most likely occur in groundwater. These results from the column experiments coincide very well with the occurrence of the pharmaceuticals clofibric acid, primidone, and propyphenazone in the first aquifer. Recommendations and Perspective : The results underline the need to study the sorption of pharmaceuticals on various materials. e.g. organic matter, surfaces at pH values occurring in the unsaturated zone. Future field studies will also include the investigation of desorption behavior in the unsaturated zone.