Analytical and pharmacological consequences of the in vivo deamidation of trastuzumab and pertuzumab.

A liquid chromatography-tandem mass spectrometry method is presented for the quantitative determination of the in vivo deamidation of the biopharmaceutical proteins trastuzumab and pertuzumab at an asparagine in their complementarity determining regions (CDRs). For each analyte, two surrogate peptides are quantified after tryptic digestion of the entire plasma protein content: one from a stable part of the molecule, representing the total concentration, and one containing the deamidation-sensitive asparagine, corresponding to the remaining non-deamidated concentration. Using a plasma volume of 10 µL and a 2-h digestion at pH 7, concentrations between 2 and 1000 µg/mL can be determined for the various protein forms with values for bias and CV below 15% and without unacceptable in vitro deamidation taking place. A considerable difference between the total and non-deamidated concentrations, and thus a substantial degree of deamidation, was observed in plasma for both trastuzumab and pertuzumab. After a 56-day forced deamidation test 40% of trastuzumab and 68% of pertuzumab was deamidated, while trastuzumab and pertuzumab showed up to 47% and 35% of deamidation, respectively, in samples collected from breast cancer patients during treatment with a combination of both drugs. A good correlation between the non-deamidated concentration results and those of a receptor binding assay indicate a loss of receptor binding for both trastuzumab and pertuzumab along with the deamidation in their CDRs. Deamidated trastuzumab also lost its capability to inhibit the growth of breast cancer cells in a cell-based viability assay, suggesting a relation between the degree of deamidation and pharmacological activity.

Comparative Study on the Performance of Three Detection Methods for the Quantification of Pacific Ciguatoxins in French Polynesian Strains of Gambierdiscus polynesiensis.

Gambierdiscus and Fukuyoa dinoflagellates produce a suite of secondary metabolites, including ciguatoxins (CTXs), which bioaccumulate and are further biotransformed in fish and marine invertebrates, causing ciguatera poisoning when consumed by humans. This study is the first to compare the performance of the fluorescent receptor binding assay (fRBA), neuroblastoma cell-based assay (CBA-N2a), and liquid chromatography tandem mass spectrometry (LC-MS/MS) for the quantitative estimation of CTX contents in 30 samples, obtained from four French Polynesian strains of Gambierdiscus polynesiensis. fRBA was applied to Gambierdiscus matrix for the first time, and several parameters of the fRBA protocol were refined. Following liquid/liquid partitioning to separate CTXs from other algal compounds, the variability of CTX contents was estimated using these three methods in three independent experiments. All three assays were significantly correlated with each other, with the highest correlation coefficient (r2 = 0.841) found between fRBA and LC-MS/MS. The CBA-N2a was more sensitive than LC-MS/MS and fRBA, with all assays showing good repeatability. The combined use of fRBA and/or CBA-N2a for screening purposes and LC-MS/MS for confirmation purposes allows for efficient CTX evaluation in Gambierdiscus. These findings, which support future collaborative studies for the inter-laboratory validation of CTX detection methods, will help improve ciguatera risk assessment and management.

Unlabeled lysophosphatidic acid receptor binding in free solution as determined by a compensated interferometric reader.

Native interactions between lysophospholipids (LPs) and their cognate LP receptors are difficult to measure because of lipophilicity and/or the adhesive properties of lipids, which contribute to high levels of nonspecific binding in cell membrane preparations. Here, we report development of a free-solution assay (FSA) where label-free LPs bind to their cognate G protein-coupled receptors (GPCRs), combined with a recently reported compensated interferometric reader (CIR) to quantify native binding interactions between receptors and ligands. As a test case, the binding parameters between lysophosphatidic acid (LPA) receptor 1 (LPA1; one of six cognate LPA GPCRs) and LPA were determined. FSA-CIR detected specific binding through the simultaneous real-time comparison of bound versus unbound species by measuring the change in the solution dipole moment produced by binding-induced conformational and/or hydration changes. FSA-CIR identified KD values for chemically distinct LPA species binding to human LPA1 and required only a few nanograms of protein: 1-oleoyl (18:1; KD = 2.08 ± 1.32 nM), 1-linoleoyl (18:2; KD = 2.83 ± 1.64 nM), 1-arachidonoyl (20:4; KD = 2.59 ± 0.481 nM), and 1-palmitoyl (16:0; KD = 1.69 ± 0.1 nM) LPA. These KD values compared favorably to those obtained using the previous generation back-scattering interferometry system, a chip-based technique with low-throughput and temperature sensitivity. In conclusion, FSA-CIR offers a new increased-throughput approach to assess quantitatively label-free lipid ligand-receptor binding, including nonactivating antagonist binding, under near-native conditions.

Assay for advanced glycation end products generating intracellular oxidative stress through binding to its receptor.

Advanced glycation end products (AGEs) are a heterogenous group of glycation adducts on amino acids produced with sugars or dicarbonyls. Intracellular inflammation triggered by binding of AGEs to receptor for AGEs (RAGE) is linked to some chronic diseases. Here, we established a competitive assay format to comprehensively quantify AGEs which bound to RAGE. RAGE-binding activities of sugar- and dicarbonyl-derived AGEs were correlated with oxidative stress in cultured cells generated by the respective AGEs, suggesting that this would be a promising method for evaluating AGEs which could affect cellular functions despite limited information on individual glycation adducts.

Receptor-binding affinities of bisphenol A and its next-generation analogs for human nuclear receptors.

An endocrine-disrupting chemical Bisphenol A (BPA) binds specifically to a nuclear receptor (NR) named ERRγ. Although the importance of receptor-binding evaluation for human NRs is often stressed, the binding characteristics of so-called next-generation (NextGen) bisphenol compounds are still poorly understood. The ultimate objective of this investigation was to evaluate BPA and its NextGen analogs for their abilities to bind to 21 human NRs, the greatest members of NRs for which tritium-labeled specific ligands were available. After establishing the detailed assay conditions for each NR, the receptor binding affinities of total 11 bisphenols were evaluated in competitive binding assays. The results clearly revealed that BPA and the NextGen bisphenols of BPAF, BPAP, BPB, BPC, BPE, and BPZ were highly potent against one or more of NRs such as CAR, ERα, ERß, ERRγ, and GR, with IC50 values of 3.3-73 nM. These bisphenols were suggested strongly to be disruptive to these NRs. BPM and BPP also appeared to be disruptive, but less potently. BPF exhibited only weak effects and only against estrogen-related NRs. Surprisingly, most doubtful bisphenol BPS was supposed not to be disruptive. The NRs to which BPA and NextGen bisphenols did not bind were RARα, RARß, RARγ, and VDR. PPARγ, RORα, RORß, RORγ, RXRα, RXRß, and RXRγ, exhibited very weak interaction with these bisphenols. The ten remaining NRs, namely, ERRγ, ERß, ERα, CAR, GR, PXR, PR, AR, LXRß, and LXRα, showed distinctly strong binding to some bisphenols in this order, being likely to have consequential endocrine-disruption effects.

A plate-based histo-blood group antigen binding assay for evaluation of human norovirus receptor binding ability.

Human norovirus is a leading cause of gastroenteritis worldwide. Although two in vitro cultivation methods have been reported, they cannot provide mechanistic insights into viral inactivation. Receptor-binding assays supplement these assays and give insight into capsid integrity. We present a streamlined version of a receptor-binding assay with minimal time-to-result while maintaining accuracy and high throughput. We validate assay performance for physical and chemical inactivation treatments of a norovirus GII.4 capsid. The assay produces a high positive/negative ratio (25.3 ± 4.9) in <2.5 h and has a limit of detection of 0.1 µg/ml capsid. This method is a valuable additional tool for understanding human norovirus inactivation.

Limitations of the Anticholinergic Activity Assay and Assay-Based Anticholinergic Drug Scales.

OBJECTIVE: The anticholinergic activity (AA) assay is a common method to determine a patient's anticholinergic load. Several limitations, however, are expected when applying the AA assay to patients or using drug scales to estimate anticholinergic burden based on AA levels. This study aims to demonstrate common pitfalls in an experimental setting and outline their clinical consequences. METHODS: The AA was analyzed for five drugs with reported interaction with muscarinic receptors. Concentration-response curves were constructed for furosemide (weak anticholinergic), diphenhydramine (moderate anticholinergic), the strong anticholinergic amitriptyline and its metabolite nortriptyline, and the cholinergic pilocarpine. The Combination Index (CI) was used to assess the interaction of three drug combinations with amitriptyline. RESULTS: All compounds displaced the radioactive tracer from its receptor binding site in a concentration-dependent manner, and full displacement was reached for all compounds except furosemide (Emax 16%). The CI indicated that amitriptyline and thioridazine have antagonistic effects (CI = 1.46) at low and synergistic effects (CI = 0.88) at higher concentrations (p < 0.0001), whereas synergistic effects (CI = 0.47-0.48) were observed for amitriptyline in any concentration combined with pilocarpine (p < 0.001). CONCLUSION: When the patient's anticholinergic load is estimated using AA levels, the actual exposure, combination of anticholinergic drugs, their active metabolites, and also drugs with an opposite pharmacologic action will contribute to AA levels, whereas weak anticholinergic drugs in therapeutic concentrations are rather negligible.

Plant membrane assays with cytokinin receptors underpin the unique role of free cytokinin bases as biologically active ligands.

Cytokinin receptors play a key role in cytokinin-dependent processes regulating plant growth, development, and adaptation; therefore, the functional properties of these receptors are of great importance. Previously the properties of cytokinin receptors were investigated in heterologous assay systems using unicellular microorganisms, mainly bacteria, expressing receptor proteins. However, within microorganisms receptors reside in an alien environment that might distort the receptor properties. Therefore, a new assay system has been developed allowing studies of individual receptors within plant membranes (i.e. closer to their natural environment). The main ligand-binding characteristics of receptors from Arabidopsis [AHK2, AHK3, and AHK4] and maize (ZmHK1) were refined in this new system, and the properties of full-length Arabidopsis receptor AHK2 were characterized for the first time. Ligand specificity profiles of receptors towards cytokinin bases were comparable with the profiles retrieved in bacterial assay systems. In contrast, cytokinin-9-ribosides displayed a strongly reduced affinity for receptors in the plant assay system, indicating that ribosides as the common transport form of cytokinins have no or very weak cytokinin activity. This underpins the central role of free bases as the sole biologically active cytokinin compounds. According to molecular modelling and docking studies, N (9)-ribosylation alters the bonding pattern in cytokinin-receptor interaction and prevents ß6-ß7 loop movement important for tight hormone binding. A common feature of all receptors was a greatly reduced ligand binding at low (5.0-5.5) pH. The particularly high sensitivity of ZmHK1 to pH changes leads to the suggestion that some cytokinin receptors may play an additional role as pH sensors in the lumen of the endoplasmic reticulum.

In vitro study on the agonistic and antagonistic activities of bisphenol-S and other bisphenol-A congeners and derivatives via nuclear receptors.

Bisphenols are a group of chemicals structurally similar to bisphenol-A (BPA) in current use as the primary raw material in the production of polycarbonate and epoxy resins. Some bisphenols are intended to replace BPA in several industrial applications. This is the case of bisphenol-S (BPS), which has an excellent stability at high temperature and resistance to sunlight. Studies on the endocrine properties of BPS have focused on its interaction with human estrogen receptor alpha (hERα), but information on its interaction with other nuclear receptors is scarce. The aim of this study was to investigate interactions of BPS, BPF, BPA and its halogenated derivatives, tetrachlorobisphenol A (TCBPA), and tetrabromobisphenol A (TBBPA), with human estrogen receptors (hERα and hERß), androgen receptor (hAR), and pregnane X receptor (hPXR), using a panel of in vitro bioassays based on competitive binding to nuclear receptors (NRs), reporter gene expression, and cell proliferation assessment. BPS, BPF, and BPA efficiently activated both ERs, while TCBPA behaved as weak hERα agonist. Unlike BPF and BPA, BPS was more active in the hERß versus hERα assay. BPF and BPA were full hAR antagonists (BPA>BPF), whereas BPA and BPS were weak hAR agonists. Only BPA, TCBPA, and TBBPA, were hPXR agonists (TCBPA>TBBPA>BPA). These findings provide evidence that BPA congeners and derivatives disrupt multiple NRs and may therefore interfere with the endocrine system. Hence, further research is needed to evaluate the potential endocrine-disrupting activity of putative BPA substitutes.

Emerging organophosphate ester resorcinol bis(diphenyl phosphate) exerts estrogenic effects via estrogen receptor pathways.

Environmental occurrence and human exposure of emerging organophosphate esters (eOPEs) have increased significantly in recent years. Resorcinol bis(diphenyl) phosphate (RDP) is one of the major eOPEs detected in indoor dust, but the knowledge on its toxicities and health risks is rather limited. In this study, we investigated the in vitro estrogenic effects and underlying mechanism of RDP in comparison with a legacy OPE triphenyl phosphate (TPHP). Our results showed that RDP promoted MCF-7 cell proliferation with the lowest effect concentration of 2.5 µM, and the maximum enhancement of 1.6 folds is greater than that of TPHP (1.3 folds). The effect was inhibited completely by an estrogen receptor (ER) antagonist, suggesting that ER activation was responsible for the enhancement. In luciferase reporter gene assays both RDP and TPHP activated ER transcriptional activity at 2.5 µM, but RDP activity was higher than TPHP. Competitive fluorescence binding assays showed that RDP bound to ER with an IC10 of 0.26 µM, which is 20 folds lower than TPHP (5.6 µM). Molecular docking simulation revealed that both RDP and TPHP interacted with ER at the binding pocket of estradiol, although the hydrogen bonds were different. Taken together, RDP exerted stronger estrogenic effects than TPHP through ER-mediated pathways and may pose more health risks.

Assay of CB1 Receptor Binding.

Type-1 cannabinoid receptor (CB1), one of the main targets of endocannabinoids, plays a key role in several pathophysiological conditions that affect both the central nervous system and peripheral tissues. Today, its biochemical identification and pharmacological characterization, as well as the screening of thousands of novel ligands that might be useful for developing CB1-based therapies, are the subject of intense research. Among available techniques that allow the analysis of CB1 binding activity, radioligand-based assays represent one of the best, fast, and reliable methods.Here, we describe radioligand binding methods standardized in our laboratory to assess CB1 binding in both tissues and cultured cells. We also report a high-throughput radioligand binding assay that allows to evaluate efficacy and potency of different compounds, which might represent the basis for the development of new drugs that target CB1-dependent human diseases.

Evaluation of relative potency of calibrated ciguatoxin congeners by near-infrared fluorescent receptor binding and neuroblastoma cell-based assays.

Ciguatera fish poisoning (CFP) is a foodborne illness affecting > 50,000 people worldwide annually. It is caused by eating marine invertebrates and fish that have accumulated ciguatoxins (CTXs). Recently, the risk of CFP to human health, the local economy, and fishery resources have increased; therefore, detection methods are urgently needed. Functional assays for detecting ciguatoxins in fish include receptor binding (RBA) and neuroblastoma cell-based assay (N2a assay), which can detect all CTX congeners. In this study, we made these assays easier to use. For RBA, an assay was developed using a novel near-infrared fluorescent ligand, PREX710-BTX, to save valuable CTXs. In the N2a assay, a 1-day assay was developed with the same detection performance as the conventional 2-day assay. Additionally, in these assays, we used calibrated CTX standards from the Pacific determined by quantitative NMR for the first time to compare the relative potency of congeners, which differed significantly among previous studies. In the RBA, there was almost no difference in the binding affinity among congeners, showing that the differences in side chains, stereochemistry, and backbone structure of CTXs did not affect the binding affinity. However, this result did not correlate with the toxic equivalency factors (TEFs) based on acute toxicity in mice. In contrast, the N2a assay showed a good correlation with TEFs based on acute toxicity in mice, except for CTX3C. These findings, obtained with calibrated toxin standards, provide important insights into evaluating the total toxicity of CTXs using functional assays.

Recent advances in estrogen receptor-targeted probes conjugated to BODIPY dyes.

Estrogens, is a class of steroid hormones associated with the occurrence and development of breast cancer, that bind to estrogen receptors (ER). The development of BODIPY-based fluorescent ligands for the ER has continued to gain tremendous attention over the past 20 years. This review focuses on the synthesis methods, optical properties, and biological activity of BODIPY fluorescent probes conjugated to ER ligands. These will provide new strategy for designing fluorescent probes for targeting estrogen receptors.

Characterization of Influenza Virus Binding to Receptors on Isolated Cell Membranes.

An interplay between receptor-binding properties of influenza viruses (IVs) and spectrum of sialic acid-containing receptors on target cells in birds and mammals determine viral host range, tissue tropism, and pathogenicity. Here, we describe method that allows to characterize binding of IVs to biologically relevant cellular receptors using a conventional solid-phase enzyme-linked assay. In this method, we isolate plasma membranes from respiratory and intestinal epithelial cells of animal origin (Subheading 3.2). We adsorb the membranes in the wells of 96-well ELISA plates, incubate the membrane-coated wells with serially diluted IVs, and determine amounts of IVs attached to the membranes using viral ability to bind peroxidase-labeled sialoglycoprotein fetuin. Based on the concentration dependence of IV binding to the membrane, we estimate binding avidity and number of binding sites. We describe two variants of the assay in Subheadings 3.6 and 3.7 and provide examples.

Effect of Modified Yukmijihwang-Tang on Sleep Quality in the Rat.

Many plants have been used in Korean medicine for treating insomnia. However, scientific evidence for their sedative activity has not been fully investigated. Thus, this study was carried out to investigate the sedative effects of the extracts of medicinal plants, including Yukmijihwang-tang and its various modified forms through the 5-HT2c receptor binding assay, and to further confirm its sleep-promoting effects and the underlying neural mechanism in rats utilizing electroencephalography (EEG) analysis. Enzyme-linked immunosorbent assay (ELISA) was used to measure serotonin (5-HT) in the brain. The water extracts of modified Yukmijihwang-tang (YmP) displayed binding affinity to the 5-HT2C receptor (IC50 value of 199.9 µg/mL). YmP (50 mg/kg) administration decreased wake time and increased REM and NREM sleep based on EEG data in rats. Additionally, treatment with YmP significantly increased the 5-HT level in the hypothalamus. In conclusion, the sedative effect of YmP can be attributed to the activation of the central serotonergic systems, as evidenced by the high affinity of binding of the 5-HT2C receptor and increased 5-HT levels in the brain of the rat. This study suggests that YmP can be a new material as a sleep inducer in natural products.

Optimization binding studies of opioid receptors, saturation and competition, using [3H]-DAMGO.

BACKGROUND: Opioid analgesics are prescribed for the moderate to severe pain in the clinic. New analogs of µ-opioid receptors are introduced because they may have less adverse effects and better efficacy. However, these new analogs have to be screened for their receptor affinity before entering clinical trial phases. A common method to do such screening is using radioligand-binding-assay, which is a fast and precise screening technique if the assays are done at an optimum condition. One of the main challenges in this type of screening is to separate free/unbound radioligands from bound radioligands. In this study, we applied a centrifugation method instead of a filtration method to separate free radioligands from bound radioligands, and also optimized the conditions for radioligand receptor binding studies of µ-opioid receptors, saturation, and the competition. METHODS: We used the midbrain and brainstem of naltrexone-treated rats as a source of µ-opioid receptors, and [3H]-DAMGO as the radioligand. Naloxone was also used to determine non-specific binding. A given amount of membrane protein was incubated with an increasing amount of radioligand at 37 °C to saturate the receptors at equilibrium and the amount of radioligand saturated in the receptors were used in competition studies. RESULTS: 160 µg membrane protein saturated with 20 nM [3H]-DAMGO at 37 °C for 35 min with Kd (15.06 nM, 95% CI 8.117-22.00) and Bmax (0.4750 pmol/mg, 95% CI 0.3839-0.5660). CONCLUSION: Applying the centrifugation method instead of the filtration to separate free from bound radioligand produced repeatable and reliable results. The optimum conditions for radioligand binding were used in competition studies which resulted in the expected outcomes.

Melatonin binds with high affinity and specificity to beta-amyloid: LC-MS provides insight into Alzheimer's disease treatment.

To study the potential relationship between melatonin and beta-amyloid (Abeta), we established a liquid chromatography-mass spectrometry (LC-MS) method to quantitatively analyze melatonin, deuterated isotopes (melatonin-D4), and internal standard 6-iodo-2-(4'-dimethylamino-) phenyl-imidazo(1,2) pyridine (IMPY) under positive (+) mode. The gradient elution was set to 6 min, and the corresponding peak time of melatonin and its isotope melatonin-D4 was 3.14 min, while the peak time for the internal standard IMPY was 3.24 min. Next, we established and optimized the molecule receptor saturation binding assay based on LC-MS to determine the melatonin affinity for beta-amyloid (Abeta). Melatonin showed a high and specific binding for Abeta. The corresponding equilibrium dissociation constant (Kd) of melatonin with Abeta 1-40 and Abeta 1-42 was 814.37 ± 36.62 and 628.33 ± 13.57 nmol·L-1 ; besides, the Kd of melatonin with mixed plaques (1-40 and 1-42) was 461.13 ± 45.37 nmol·L-1 . The results may suggest the potential mechanism of action of MT on Abeta and provide a theoretical basis for the improvement of MT treatment of Alzheimer's disease.

Investigating (anti)estrogenic activities within South African wastewater and receiving surface waters: Implication for reliable monitoring.

Natural and synthetic steroid hormones and many persistent organic pollutants are of concern for their endocrine-disrupting activities observed in receiving surface waters. Apart from the demonstrated presence of estrogen- and estrogen-mimicking compounds in surface waters, antagonistic (anti-estrogenic) responses originating from wastewater effluent have been reported but are less known. Estrogenicity and anti-estrogenicity were assessed using recombinant yeast estrogen receptor binding assays (YES/YAES) at ten South African wastewater treatment works (WWTWs) and receiving rivers in two separate sampling campaigns during the summer- and winter periods in the area. Four WWTWs were then further investigated to show daily variation in estrogenic endocrine-disrupting activities during the treatment process. Although estrogenicity was notably reduced at most of the WWTWs, some treated effluent and river water samples were shown to be above effect-based trigger values posing an endocrine-disrupting risk for aquatic life and potential health risks for humans. Furthermore, estrogenicity recorded in samples collected upstream from some WWTW discharge points also exceeded some calculated risk trigger values, which highlights the impact of alternative pollution sources contributing towards endocrine disrupting contaminants (EDCs) in the environment. The YAES further showed variable anti-estrogenic activities in treated wastewater. The current study highlights a variety of factors that may affect bioassay outcomes and conclusions drawn from the results for risk decision-making. For example, mismatches were found between estrogenic and anti-estrogenic activity, which suggests a potential masking effect in WWTW effluents and highlights the complexity of environmental samples containing chemical mixtures having variable endocrine-disrupting modes of action. Although the recombinant yeast assay is not without its limitations to show endocrine-disrupting modulation in test water systems, it serves as a cost-effective tier-1 scoping assay for further risk characterisation and intervention.

Advances in Detecting Ciguatoxins in Fish.

Ciguatera fish poisoning (CFP) is currently the most common marine biotoxin food poisoning worldwide, associated with human consumption of circumtropical fish and marine invertebrates that are contaminated with ciguatoxins. Ciguatoxins are very potent sodium-channel activator neurotoxins, that pose risks to human health at very low concentrations (>0.01 ng per g of fish flesh in the case of the most potent Pacific ciguatoxin). Symptoms of CFP are nonspecific and intoxication in humans is often misdiagnosed. Presently, there is no medically approved treatment of ciguatera. Therefore, to mitigate the risks of CFP, reliable detection of ciguatoxins prior to consumption of fish tissue is acutely needed, which requires application of highly sensitive and quantitative analytical tests. During the last century a number of methods have been developed to identify and quantify the concentration of ciguatoxins, including in vivo animal assays, cell-based assays, receptor binding assays, antibody-based immunoassays, electrochemical methods, and analytical techniques based on coupling of liquid chromatography with mass spectrometry. Development of these methods, their various advantages and limitations, as well as future challenges are discussed in this review.

Cyclic imine toxins survey in coastal european shellfish samples: Bioaccumulation and mode of action of 28-O-palmitoyl ester of pinnatoxin-G. first report of portimine-A bioaccumulation.

Cyclic imine toxins exhibit fast acting neurotoxicity and lethality by respiratory arrest in mice explained by their potent antagonistic activity against muscular nicotinic acetylcholine receptors. We performed a survey of gymnodimine-A, 13-desmethyl spirolide-C, 13,19-didesmethyl spirolide-C, 20-methyl spirolide-G, pinnatoxin-A, pinnatoxin-G, portimine-A and 28-O-palmitoyl ester of pinnatoxin-G in 36 shellfish samples collected in coastal areas of 8 European countries using a microplate receptor binding assay and UPLC-MS/MS for toxin identification and quantification. The major toxins found in these samples were pinnatoxin-G, 20-methyl spirolide-G, 13-desmethyl spirolide-C, gymnodimine-A and portimine-A. Traces of 13,19-didesmethyl spirolide-C, pinnatoxin-A and 28-O-palmitoyl ester of pinnatoxin-G were also detected. The rapid death of mice was correlated with higher pinnatoxin-G concentrations in mussel digestive gland extracts injected intraperitoneally. Our survey included nontoxic control samples that were found to contain moderate to trace amounts of several cyclic imine toxins. Shellfish may bioaccumulate not only cyclic imine toxins but also a large number of acyl derivatives as a product of metabolic transformation of these neurotoxins. This is the first report in which portimine-A and 28-O-palmitoyl ester of pinnatoxin-G were detected in shellfish extracts from digestive glands of mussels collected in Ingril lagoon. The bioaccumulation of portimine-A is particularly of concern because it is cytotoxic and is able to induce apotosis. The mode of action of 28-O-palmitoyl ester of pinnatoxin-G was studied by receptor binding-assay and by two-electrode voltage clamp electrophysiology. The antagonistic behavior of the acylated pinnatoxin-G towards nicotinic acetylcholine receptor of muscle type is shown here for the first time. Since cyclic imine toxins are not regulated further monitoring of these emerging toxins is needed to improve evidence gathering of their occurrence in shellfish commercialized for human consumption in Europe given their potent antagonism against muscle and neuronal nicotinic acetylcholine receptors.