In Vitro and In Vivo Evaluation of Pellotine: A Hypnotic Lophophora Alkaloid.

Quality of life is often reduced in patients with sleep-wake disorders. Insomnia is commonly treated with benzodiazepines, despite their well-known side effects. Pellotine (1), a Lophophora alkaloid, has been reported to have short-acting sleep-inducing properties in humans. In this study, we set out to evaluate various in vitro and in vivo properties of 1. We demonstrate that 1 undergoes slow metabolism; e.g. in mouse liver microsomes 65% remained, and in human liver microsomes virtually no metabolism was observed after 4 h. In mouse liver microsomes, two phase I metabolites were identified: 7-desmethylpellotine and pellotine-N-oxide. In mice, the two diastereomers of pellotine-O-glucuronide were additionally identified as phase II metabolites. Furthermore, we demonstrated by DESI-MSI that 1 readily enters the central nervous system of rodents. Furthermore, radioligand-displacement assays showed that 1 is selective for the serotonergic system and in particular the serotonin (5-HT)1D, 5-HT6, and 5-HT7 receptors, where it binds with affinities in the nanomolar range (117, 170, and 394 nM, respectively). Additionally, 1 was functionally characterized at 5-HT6 and 5-HT7, where it was found to be an agonist at the former (EC50 = 94 nM, Emax = 32%) and an inverse agonist at the latter (EC50 = 291 nM, Emax = -98.6). Finally, we demonstrated that 1 dose-dependently decreases locomotion in mice, inhibits REM sleep, and promotes sleep fragmentation. Thus, we suggest that pellotine itself, and not an active metabolite, is responsible for the hypnotic effects and that these effects are possibly mediated through modulation of serotonergic receptors.

Towards a microfluidic H295R steroidogenesis assay-biocompatibility study and steroid detection on a thiol-ene-based chip.

The development of cell-based microfluidic assays offers exciting new opportunities in toxicity testing, allowing for integration of new functionalities, automation, and high throughput in comparison to traditional well-plate assays. As endocrine disruption caused by environmental chemicals and pharmaceuticals represents a growing global health burden, the purpose of the current study was to contribute towards the miniaturization of the H295R steroidogenesis assay, from the well-plate to the microfluidic format. Microfluidic chip fabrication with the established well-plate material polystyrene (PS) is expensive and complicated; PDMS and thiol-ene were therefore tested as potential chip materials for microfluidic H295R cell culture, and evaluated in terms of cell attachment, cell viability, and steroid synthesis in the absence and presence of collagen surface modification. Additionally, spike-recovery experiments were performed, to investigate potential steroid adsorption to chip materials. Cell aggregation with poor steroid recoveries was observed for PDMS, while cells formed monolayer cultures on the thiol-ene chip material, with cell viability and steroid synthesis comparable to cells grown on a PS surface. As thiol-ene overall displayed more favorable properties for H295R cell culture, a microfluidic chip design and corresponding cell seeding procedure were successfully developed, achieving repeatable and uniform cell distribution in microfluidic channels. Finally, H295R perfusion culture on thiol-ene chips was investigated at different flow rates (20, 10, and 2.5 µL/min), and 13 steroids were detected in eluting cell medium over 48 h at the lowest flow rate. The presented work and results pave the way for a time-resolved microfluidic H295R steroidogenesis assay.

Non-aqueous electrophoresis integrated with electrospray ionization mass spectrometry on a thiol-ene polymer-based microchip device.

Non-aqueous capillary electrophoresis (NACE) on microfluidic chips is still a comparatively little explored area, despite the inherent advantages of this technique and its application potential for, in particular, lipophilic compounds. A main reason is probably the fact that implementation of NACE on microchips largely precluded the use of polymeric substrate materials. Here, we report non-aqueous electrophoresis on a thiol-ene-based microfluidic chip coupled to mass spectrometry via an on-chip ESI interface. Microchips with an integrated ESI emitter were fabricated using a double-molding approach. The durability of thiol-ene, when exposed to different organic solvents, was investigated with respect to swelling and decomposition of the polymer. Thiol-ene exhibited good stability against organic solvents such as methanol, ethanol, N-methylformamide, and formamide, which allows for a wide range of background electrolyte compositions. The integrated ESI emitter provided a stable spray with RSD% of the ESI signal ≤8%. Separation efficiency of the developed microchip electrophoresis system in different non-aqueous buffer solutions was tested with a mixture of several drugs of abuse. Ethanol- and methanol-based buffers provided comparable high theoretical plate numbers (≈ 6.6 × 104-1.6 × 105 m-1) with ethanol exhibiting the best separation efficiency. Direct coupling of non-aqueous electrophoresis to mass spectrometry allowed for fast analysis of hydrophobic compounds in the range of 0.1-5 µg mL-1 and 0.2-10 µg mL-1 and very good sensitivities (LOD ≈ 0.06-0.28 µg mL-1; LOQ ≈ 0.20-0.90 µg mL-1). The novel combination of non-aqueous CE on a microfluidic thiol-ene device and ESI-MS provides a mass-producible and highly versatile system for the analysis of, in particular, lipophilic compounds in a wide range of organic solvents. This offers promising potential for future applications in forensic, clinical, and environmental analysis. Graphical abstract.

A quantitative method for the selective 5-HT2A agonist 25CN-NBOH in rat plasma and brain.

In recent years, agonists of the 5-HT2A receptor have gained increasing attention for their potential therapeutic use to treat psychological disorders such as anxiety and depression. Here, we report the development and validation of an LC-MSMS based analytical method for the quantification of the novel selective 5-HT2A agonist 25CN-NBOH in rat plasma and brain. As simple and efficient sample clean-up we applied the Phree Phospholipid Removal approach from Phenomenex, which is particularly novel for brain samples. In order to investigate the metabolic stability of 25CN-NBOH in vitro biotransformation studies with recombinant enzymes and human liver microsomes were conducted. Several biotransformation products and pathways could be identified. Based on the in vitro study one of the putative metabolites (2C-CN) was included in the analytical method development. To test the methods applicability 25CN-NBOH was quantified in plasma and brain samples from a pharmacokinetic in vivo study with Wildtype Long Evans rats. Both the in vitro metabolism data as well as the in vivo PK data suggest that 25CN-NBOH is susceptible to metabolism, but is degraded slower and is more stable compared to other NBOMe's investigated to date. The developed analytical method might serve as basis to include further 25CN-NBOH metabolites. It is expected to facilitate further preclinical and clinical investigations of 25CN-NBOH in biological matrices.

A thiol-ene microfluidic device enabling continuous enzymatic digestion and electrophoretic separation as front-end to mass spectrometric peptide analysis.

One of the most attractive aspects of microfluidic chips is their capability of integrating several functional units into one single platform. In particular, enzymatic digestion and chemical separation are important steps in processing samples for many biochemical assays. This study presents the development and application of a free-flow electrophoresis microfluidic chip, and its upstream combination with an enzyme microreactor with immobilized pepsin in the same miniaturized platform. The whole microfluidic chip was fabricated by making use of thiol-ene click chemistry. As a proof of concept, different fluorescent dyes and labeled amino acids were continuously separated in the 2D electrophoretic channel. The protease pepsin was immobilized using a covalent linkage with ascorbic acid onto a high-surface monolithic support, also made of thiol-ene. To show the potential of the microfluidic chip for continuous sample preparation and analysis, an oligopeptide was enzymatically digested, and the resulting fragments were separated and collected in a single step (prior to mass spectrometric detection), without the need of further time-consuming liquid handling steps.

The classic azole antifungal drugs are highly potent endocrine disruptors in vitro inhibiting steroidogenic CYP enzymes at concentrations lower than therapeutic Cmax.

Azole antifungal drugs are used worldwide to treat a variety of fungal infections such as vulvovaginal candidiasis, particularly in pregnant women who are at increased risk. The aim of this study was to mechanistically investigate the endocrine disrupting potential of four commonly used azole antifungal drugs; clotrimazole, miconazole, ketoconazole and fluconazole in vitro using the H295R cell assay and two recombinant, CYP17A1 and CYP19A1 (aromatase), assays. Steroids were quantified using LC-MS/MS. In both recombinant assays, all four azoles inhibited the CYP enzymes investigated, at therapeutically relevant concentrations. However, responses were much more complex in the H295R cell line. Clotrimazole inhibited steroid production in a dose-dependent manner with IC50 values for CYP17A1 and CYP19A1 in the range 0.017-0.184 µM. Miconazole and ketoconazole increased all steroids on the hydroxylase axis (IC50 MIC: 0.042-0.082 µM, KET: 0.041-1.2 µM), leading to accumulation of progestagens and corticosteroids and suppression of androgens and estrogens, indicating inhibition of CYP17A1, in particular lyase activity. However, ketoconazole suppressed all steroids at higher concentrations, resulting in bell-shaped curves for all steroids on the hydroxylase axis. Fluconazole was found to inhibit CYP17A1-lyase activity, causing suppression of androgens (IC50 = 114-209 µM) and estrogens (IC50 = 28 µM). The results indicate that these four azole drugs are highly potent in vitro and, based on plasma Cmax values, may exert endocrine disrupting effects at therapeutically relevant concentrations. This raises concern for endocrine related effects in patients using azole antifungal drugs, particularly when taken during sensitive periods like pregnancy.

Enantioselective mixture toxicity of the azole fungicide imazalil with the insecticide α-cypermethrin in Chironomus riparius: Investigating the importance of toxicokinetics and enzyme interactions.

The fungicide imazalil is a chiral compound with one R- and one S-enantiomer. Enantiomers, while having the same chemical properties, can differ in their biological activity expressed as efficacy/toxicity as well as in their degradation kinetics and pathways. Azoles such as imazalil have been shown to synergize the effect of pyrethroid insecticides like α-cypermethrin through inhibition of cytochrome P450 monooxygenase responsible for pyrethroid detoxification. The aim of this study was to investigate, if the enantiomers of imazalil are selective in their synergistic potential in a mixture with a pyrethroid insecticide tested in Chironomus riparius. Potential enantioselectivity was studied on the level of uptake and elimination, inhibition of cytochrome P450 activity measured in vitro and in vivo and on synergistic potential of α-cypermethrin induced immobilization. Synergy was measured as an increase in α-cypermethrin toxicity after 144h applying a constant non-lethal imazalil concentration of 0.65 µmol/L. The R- and S-imazalil enantiomers increased α-cypermethrin toxicity from an EC50 of 1580 ±â€¯980 pmol/L to an EC50 of 83 ±â€¯10 pmol/L and 53 ±â€¯8 pmol/L, respectively. The relatively small potency difference between imazalil enantiomers could not be explained by the in vitro cytochrome P450 inhibition, as the IC50 values were similar (0.11 ±â€¯0.01 and 0.09 ±â€¯0.01 µmol/L for R- and S-imazalil). Measuring in vivo P450 inhibition and the toxicokinetic of imazalil did not show a clear trend of selectivity towards one or the other enantiomer. The study therefore suggests that cytochrome P450 enzymes involved in detoxification in C. riparius are not enantioselective for imazalil.

Can Toxicokinetic and Toxicodynamic Modeling Be Used to Understand and Predict Synergistic Interactions between Chemicals?

Some chemicals are known to enhance the effect of other chemicals beyond what can be predicted with standard mixture models, such as concentration addition and independent action. These chemicals are called synergists. Up until now, no models exist that can predict the joint effect of mixtures including synergists. The aim of the present study is to develop a mechanistic toxicokinetic (TK) and toxicodynamic (TD) model for the synergistic mixture of the azole fungicide, propiconazole (the synergist), and the insecticide, α-cypermethrin, on the mortality of the crustacean Daphnia magna. The study tests the hypothesis that the mechanism of synergy is the azole decreasing the biotransformation rate of α-cypermethrin and validates the predictive ability of the model on another azole with a different potency: prochloraz. The study showed that the synergistic potential of azoles could be explained by their effect on the biotransformation rate but that this effect could only partly be explained by the effect of the two azoles on cytochrome P450 activity, measured on D. magna in vivo. TKTD models of interacting mixtures seem to be a promising tool to test mechanisms of interactions between chemicals. Their predictive ability is, however, still uncertain.

Determining lower threshold concentrations for synergistic effects.

Though only occurring rarely, synergistic interactions between chemicals in mixtures have long been a point of focus. Most studies analyzing synergistic interactions used unrealistically high chemical concentrations. The aim of the present study is to determine the threshold concentration below which proven synergists cease to act as synergists towards the aquatic crustacean Daphnia magna. To do this, we compared several approaches and test-setups to evaluate which approach gives the most conservative estimate for the lower threshold for synergy for three known azole synergists. We focus on synergistic interactions between the pyrethroid insecticide, alpha-cypermethrin, and one of the three azole fungicides prochloraz, propiconazole or epoxiconazole measured on Daphnia magna immobilization. Three different experimental setups were applied: A standard 48h acute toxicity test, an adapted 48h test using passive dosing for constant chemical exposure concentrations, and a 14-day test. Synergy was defined as occuring in mixtures where either EC50 values decreased more than two-fold below what was predicted by concentration addition (horizontal assessment) or as mixtures where the fraction of immobile organisms increased more than two-fold above what was predicted by independent action (vertical assessment). All three tests confirmed the hypothesis of the existence of a lower azole threshold concentration below which no synergistic interaction was observed. The lower threshold concentration, however, decreased with increasing test duration from 0.026±0.013µM (9.794±4.897µgL-1), 0.425±0.089µM (145.435±30.46µgL-1) and 0.757±0.253µM (249.659±83.44µgL-1) for prochloraz, propiconazole and epoxiconazole in standard 48h toxicity tests to 0.015±0.004µM (5.651±1.507µgL-1), 0.145±0.025µM (49.619±8.555µgL-1) and 0.122±0.0417µM (40.236±13.75µgL-1), respectively, in the 14-days tests. Testing synergy in relation to concentration addition provided the most conservative values. The threshold values for the vertical assessments in tests where the two could be compared were in general 1.2 to 4.7 fold higher than the horizontal assessments. Using passive dosing rather than dilution series or spiking did not lower the threshold significantly. Below the threshold for synergy, slight antagony could often be observed. This is most likely due to induction of enzymes active in metabolization of alpha-cypermethrin. The results emphasize the importance of test duration when assessing synergy, but also show that azole concentrations within the typically monitored range of up to 0.5µgL-1 are not likely to cause severe synergy concerning Daphnia magna immobilization.

Separation, isolation and stereochemical assignment of imazalil enantiomers and their quantitation in an in vitro toxicity test.

A simple method for the separation of the enantiomers of the fungicide imazalil was developed. Racemic imazalil was separated into its enantiomers with an enantiomeric purity of 99% using HPLC-UV with an enantioselective column (permethylated cyclodextrin) operated in reversed phase mode (water with 0.2% trimethylamine and 0.08% acetic acid and methanol). The absolute configuration of the separated enantiomers was assigned and unequivocally confirmed by optical rotation as well as by vibrational circular dichroism (VCD) and electronic circular dichroism (ECD) combined with ab-initio calculations. The same enantioselective column was also used to develop an HPLC-MS/MS method for the quantification of imazalil enantiomers. The HPLC-MS/MS method reached limits of quantification (LOQs) of 0.025mg/mL with 5µL injections. This method was used to verify imazalil concentrations and enantiomeric fractions in samples from an in vitro test on effects on human steroidogenesis (H295R steroidogenesis assay). The quantification verified the stability of the enantiomers of imazalil during the in vitro tests.

Enantioselective endocrine disrupting effects of omeprazole studied in the H295R cell assay and by molecular modeling.

Enantiomers possess different pharmacokinetic and pharmacodynamic properties and this may not only influence the therapeutic effect of a drug but also its toxicological effects. In the present work we investigated the potential enantioselective endocrine disrupting effects of omeprazole (OME) and its two enantiomers on the human steroidogenesis using the H295R cell line. Differences in production of 16 steroid hormones were analyzed using LC-MS/MS. Additionally, to evaluate the differences in binding modes of these enantiomers, docking and molecular dynamics (MD) simulations of S-omeprazole (S-OME) and R-omeprazole (R-OME) in CYP17A1, CYP19A1 and CYP21A2 were carried out. Exposing H295R cells to OME and its enantiomers resulted in an increase of progesterone (PRO) and 17α-hydroxy-progesterone (OH-PRO) levels. At the same time, a decrease in the corticosteroid and androgen synthesis was observed, indicating inhibition of CYP21A2 and CYP17A1. In both cases, the effect of R-OME was smaller compared to that of the S-OME and a certain degree of enantioselectivity of CYP17A1 and CYP21A2 was suggested. Docking indicated that the N-containing rings of OME possibly could interact with the iron atom of the heme for S-OME in CYP17A1 and S- and R-OME in CYP21A2. However, density functional theory calculations suggest that the direct N-Fe interaction is weak. The study demonstrates enantioselective differences in the endocrine disrupting potential of chiral drugs such as omeprazole. These findings may have potential implications for drug safety and drug design.

Suspended particles only marginally reduce pyrethroid toxicity to the freshwater invertebrate Gammarus pulex (L.) during pulse exposure.

Current ecotoxicological research on particle-associated pyrethroids in freshwater systems focuses almost exclusively on sediment-exposure scenarios and sediment-dwelling macroinvertebrates. We studied how suspended particles influence acute effects of lambda-cyhalothrin and bifenthrin on the epibenthic freshwater amphipod Gammarus pulex (L.) using brief pulse exposures followed by a 144 h post exposure recovery phase. Humic acid (HA) and the clay mineral montmorillonite (MM) were used as model sorbents in environmentally realistic concentrations (5, 25 and 125 mg L(-1)). Mortality of G. pulex was recorded during the post exposure recovery phase and locomotor behavior was measured during exposure to lambda-cyhalothrin. We found that HA in concentrations ≥25 mg L(-1) adsorbed the majority of pyrethroids but only reduced mortality of G. pulex up to a factor of four compared to pyrethroid-only treatments. MM suspensions adsorbed a variable fraction of pyrethroids (10% for bifenthrin and 70% for lambda-cyhalothrin) but did not significantly change the concentration-response relationship compared to pure pyrethroid treatments. Behavioral responses and immobilisation rate of G. pulex were reduced in the presence of HA, whereas behavioral responses and immobilisation rate were increased in the presence of MM. This indicates that G. pulex was capable of sensing the bioavailable fraction of lambda-cyhalothrin. Our results imply that suspended particles reduce to only a limited extent the toxicity of pyrethroids to G. pulex and that passive uptake of pyrethroids can be significant even when pyrethroids are adsorbed to suspended particles.

What causes the difference in synergistic potentials of propiconazole and prochloraz toward pyrethroids in Daphnia magna?

Azole fungicides (imidazoles and triazoles) are known to function synergistically with several compounds, especially with pyrethroid insecticides, most likely by inhibiting cytochrome P450. Different azole fungicides have been shown to differ in their synergistic potentials usually with the imidazoles being stronger synergists than the triazoles. This study investigated whether the toxicokinetic and toxicodynamic (TKTD) properties of the imidazole prochloraz and triazole propiconazole can explain their different synergistic potential toward the freshwater macroinvertebrate Daphnia magna. Pulse exposure to external concentrations of propiconazole (1.4µM) and prochloraz (1.7µM) for 18h resulted in internal concentrations of 22.7 and 53.5µmolkg(-1)w.w. for propiconazole and prochloraz, respectively. This 2-fold difference in bioaccumulation corresponded very well with the observed 2.7-fold lower external EC50-estimate (7 days) for prochloraz compared to propiconazole. The estimated IC50 for the in vivo inhibition of cytochrome P450 (ECOD) activity, however, measured as transformation of 7-ethoxycoumarin into 7-hydroxycoumarin, was almost 500-fold higher for prochloraz (IC50: 0.011±0.002µM) compared to propiconazole (IC50: 4.9±0.06µM). When indirectly measuring the binding strength of the two azoles, daphnids exposed to propiconazole recovered roughly 80% of their ECOD activity compared to the control shortly after being moved to azole-free medium, indicating that propiconazole causes reversible inhibition of cytochrome P450. In contrast, the ECOD-activity remained inhibited in the prochloraz-exposed daphnids for 12h following transfer to azole-free medium, which correlated with elimination of the measured internal prochloraz concentration (DT95≈13h). These results indicate that lethal toxicity of the azole fungicides is mainly driven by toxicokinetics through their hydrophobicities resulting in different internal concentrations. Their synergistic potential toward pyrethroid toxicity, on the other hand, is mainly governed by their toxicodynamic effects measured as the differences in IC50-values toward in vivo cytochrome P450 (ECOD) activity together with the proposed binding strength measured indirectly through the recovery of ECOD activity as a function of internal azole concentrations.

Measuring internal azole and pyrethroid pesticide concentrations in Daphnia magna using QuEChERS and GC-ECD--method development with a focus on matrix effects.

Pyrethroids are highly toxic towards aquatic macroinvertebrates such as Daphnia magna and can be synergized when co-occurring with azole fungicides. A sensitive analytical method for the measurement of azole-pyrethroid mixtures in aquatic macroinvertebrates is not available at present. We developed and validated an extraction, cleanup, and quantification procedure for four pyrethroid insecticides and four azole fungicides at the picograms per milligram wet weight level in D. magna using a QuEChERS approach and GC-ECD analysis. Short- and long-term matrix effects were analyzed by injection of a series of extracts from D. magna, and the best surrogate standards were identified through correlation analysis of analyte responses. The presence of matrix clearly stabilized the analyte responses (≤6% relative standard deviation of peak area compared to up to 22% when injected without matrix). The sensitivity was high with detection limits and limits of quantification between 58-168 and 119-571 pg mg(wet weight)(-1) for the azoles and 5.8-27 and 12-84 pg mg(wet weight)(-1) for the pyrethroids, respectively. Accuracy (% recovery) was between 95 and 111% and the precision (repeatability) below 10% relative standard deviation for all analytes. In the case of prochloraz, α-cypermethrin, and deltamethrin, normalization to surrogate standards led to a clear improvement of accuracy and precision by up to 8 and 4%, respectively. The method was successfully applied to the measurement of internal α-cypermethrin concentrations in D. magna under environmentally relevant exposure conditions (exposure to a pulse in the micrograms per liter range) with and without co-exposure to propiconazole.

Measuring cytochrome P450 activity in aquatic invertebrates: a critical evaluation of in vitro and in vivo methods.

The first step in xenobiotic detoxification in aquatic invertebrates is mainly governed by the cytochrome P450 mixed function oxidase system. The ability to measure cytochrome P450 activity provides an important tool to understand macroinvertebrates' responses to chemical stressors. However, measurements of P450 activity in small aquatic invertebrates have had variable success and a well characterized assay is not yet available. The general lack of success has been scarcely investigated and it is therefore the focus of the present work. In particular, the suitability of the substrate selected for the assay, the sensitivity of the assay and the possible inhibition/attenuation of enzymatic activity caused by endogenous substances were investigated. 7-ethoxycoumarin-O-dealkylation activity of Daphnia magna, Chironomus riparius larvae and Hyalella azteca was assessed in vivo and in vitro and possible inhibition of enzymatic activity by macroinvertebrates homogenate was investigated. Activities of D. magna and C. riparius larvae measured in vivo were 1.37 ± 0.08 and 2.2 ± 0.2 pmol h(-1) organism(-1), respectively, while activity of H. azteca could not be detected. In vitro activity could be measured in C. riparius larvae only (500-1000 pmol h(-1) mg microsomal protein(-1)). The optimization of the in vitro assay has been especially long and resource consuming and particularly for D. magna, substances that inhibited cytochrome P450 activity seemed to be released during tissue homogenization preventing activity measurements in vitro. We therefore recommend testing the P450 inhibition potential of homogenate preparations prior to any investigation of P450 activity in vitro in macroinvertebrates.

The synergistic potential of the azole fungicides prochloraz and propiconazole toward a short α-cypermethrin pulse increases over time in Daphnia magna.

Pyrethroid insecticides are highly toxic to non-target aquatic invertebrates. Their high toxicity is synergized when co-occurring with azole fungicides in the aquatic environment. Little is known about the importance of synergy, when pyrethroids only occur during a short pulse of a few hours, as it is likely to happen in the environment, nor about the persistence of synergy over time. This study analyzed the synergistic potential of the fungicides propiconazole and prochloraz toward Daphnia magna, when exposed to a pulse (7.2 h) of α-cypermethrin at different concentrations (average pulse concentrations 0.07-11 nM). Immobilization was monitored during exposure and a subsequent recovery period (87.5h) with and without continuous co-exposure to the azoles (1.4 and 1.7 µM, respectively). EC50 values for immobilization decreased exponentially over time with a higher rate in the presence of the azoles. EC50 values for α-cypermethrin determined at the end of the experiment were 3.3±0.5 nM in the absence of azoles and 0.26±0.04, and 0.08±0.01 nM in the presence of propiconazole and prochloraz, respectively. The synergistic potential of the azoles was strongly dependent on time: no synergism could be detected during the pulse, but with azole co-exposure EC50 values decreased during the recovery period by a factor of up to 13 (propiconazole) and 61 (prochloraz) compared to values without azole exposure. Such high synergistic ratios have not been reported for pesticide mixtures in literature before. Our findings highlight that a pulse of the pyrethroid α-cypermethrin is synergized far beyond the actual pulse and beyond standardized test durations. Long post-exposure times are therefore mandatory in order to capture full synergism.

Characterization of acetylcholinesterase inhibition and energy allocation in Daphnia magna exposed to carbaryl.

The inhibition of acetylcholinesterase (AChE) activity and energy allocation in the freshwater organism Daphnia magna exposed to carbaryl and potential recovery from the effects was examined. The binding of carbaryl-AChE was characterized through in vitro assays. To evaluate the recovery from inhibition and the alteration in energy budget, in vivo exposure and recovery regime tests were conducted. In comparison to diazoxon, the active metabolite of the insecticide diazinon, the stability of enzyme-carbaryl complex was fifteen times lower and the reactivity toward the active site was two times lower, resulting in approximately 30 times lower overall inhibition rate than for diazoxon. The in vitro reactivation rate constant of the inhibited enzyme and the in vivo recovery rate constant of AChE activity were 1.9 h⁻¹ and 0.12 h⁻¹ for carbaryl, respectively, which are much higher than the corresponding rate constants for diazoxon. The lower AChE inhibition and greater reactivation/recovery rates are in accordance with the lower toxicity of carbaryl compared to diazinon. Carbaryl exposure also altered the profile of the energy reserve: the decrease in lipid and glycogen and the increase in protein content resulted in the reduction of the total energy budget by about 45 mJ/g(ww). This corresponds to 26 percent of the available energy, which might allocate for external stressors. The mechanistic model of AChE inhibition is helpful to get an insight into (eco-)toxicological effects of AChE inhibitors on freshwater crustaceans under environmentally realistic conditions.

Toxicokinetic and toxicodynamic model for diazinon toxicity--mechanistic explanation of differences in the sensitivity of Daphnia magna and Gammarus pulex.

A mechanistic toxicokinetic and toxicodynamic model for acute toxic effects (immobilization, mortality) of the organothiophosphate insecticide diazinon in Daphnia magna is presented. The model was parameterized using measured external and internal (whole-body) concentrations of diazinon, its toxic metabolite diazoxon, and the inactive metabolite 2-isopropyl-6-methyl-4-pyrimidinol, plus acetylcholinesterase (AChE) activity measured during exposure to diazinon in vivo. The toxicokinetic and toxicodynamic model provides a coherent picture from exposure to the resulting toxic effect on an organism level through internally formed metabolites and the effect on a molecular scale. A very fast reaction of diazoxon with AChE (pseudo first-order inhibition rate constant k(i) = 3.3 h(-1)) compared with a slow formation of diazoxon (activation rate constant k(act) = 0.014 h(-1)) was responsible for the high sensitivity of D. magna toward diazinon. Recovery of AChE activity from inhibition was slow and rate-determining (99% recovery within 16 d), compared with a fast elimination of diazinon (99% elimination within 17 h). The obtained model parameters were compared with toxicokinetic and toxicodynamic parameters of Gammarus pulex exposed to diazinon from previous work. This comparison revealed that G. pulex is less sensitive because of a six times faster detoxification of diazinon and diazoxon and an approximately 400 times lower rate for damage accrual. These differences overcompensate the two times faster activation of diazinon to diazoxon in G. pulex compared to D. magna. The present study substantiates theoretical considerations that mechanistically based effect models are helpful to explain sensitivity differences among different aquatic invertebrates.

Toxicokinetic-toxicodynamic modeling of quantal and graded sublethal endpoints: a brief discussion of concepts.

We report on the advantages and problems of using toxicokinetic-toxicodynamic (TKTD) models for the analysis, understanding, and simulation of sublethal effects. Only a few toxicodynamic approaches for sublethal effects are available. These differ in their effect mechanism and emphasis on linkages between endpoints. We discuss how the distinction between quantal and graded endpoints and the type of linkage between endpoints can guide model design and selection. Strengths and limitations of two main approaches and possible ways forward are outlined.

Mechanistic toxicodynamic model for receptor-mediated toxicity of diazoxon, the active metabolite of diazinon, in Daphnia magna.

The organothiophosphate diazinon inhibits the target site acetylcholinesterase only after activation to its metabolite diazoxon. Commonly, the toxicity of xenobiotics toward aquatic organisms is expressed as a function of the external concentration and the resulting effect on the individual level after fixed exposure times. This approach does not account for the time dependency of internal processes such as uptake, metabolism, and interaction of the toxicant with the target site. Here, we develop a mechanistic toxicodynamic model for Daphnia magna and diazoxon, which accounts for the inhibition of the internal target site acetylcholinesterase and its link to the observable effect, immobilization, and mortality. The model was parametrized by experiments performed in vitro with the active metabolite diazoxon on enzyme extracts and in vivo with the parent compound diazinon. The mechanism of acetylcholinesterase inhibition was shown to occur irreversibly in two steps via formation of a reversible enzyme-inhibitor complex. The corresponding kinetic parameters revealed a very high sensitivity of acetylcholinesterase from D. magna toward diazoxon, which corresponds well with the high toxicity of diazinon toward this species. Recovery of enzyme activity but no recovery from immobilization was observed after in vivo exposure to diazinon. The toxicodynamic model combining all in vitro and in vivo parameters was successfully applied to describe the time course of immobilization in dependence of acetylcholinesterase activity during exposure to diazinon. The threshold value for enzyme activity below which immobilization set in amounted to 40% of the control activity. Furthermore, the model enabled the prediction of the time-dependent diazoxon concentration directly present at the target site.