Determination of ptaquiloside in ten species of Pteris in northwestern Argentina.

The toxic effect of ferns of the genus of Pteris in bovines is caused by ptaquiloside, the main carcinogenic toxin. In this study, ten species of Pteris fern in different phenologic stages and plant conditions were collected in northwest Argentina. The phytochemical analysis showed the presence of Pt in the recent collected samples (adults and young plants) but not in the herbarium specimens. The results show a great variation of Pt concentration that depends on the phenologic stage, plant condition, and collection site. Pt was measured in 6-4326 µg/g concentration, with a mean concentration of 644 µg/g. No Pt was detected in eight species of Pteris collected from herbarium samples; such results may be a false negative. It is important to notice that analysis of herbarium samples for Pt may not be a reliable method to determine its presence. It is important to further understand the potential toxicity caused by these ferns because of their effect on animals, public health, and the environment.

Occurrence and stability of ptesculentoside, caudatoside and ptaquiloside in surface waters.

The illudane glycosides ptesculentoside (PTE), caudatoside (CAU) and ptaquiloside (PTA) are found in bracken ferns (Pteridium sp.). PTA is known to contaminate water bodies adjacent to bracken ferns and hence contribute to water toxicity. This study for the first time reports the presence of PTE and CAU in surface waters with concentrations up to 5.3 µg L-1 and outlines their stability under semi-natural conditions using water of two diverse lakes at their natural pH or pH adjusted to 6.5, with temperature controlled at 5 or 15 °C, and in the presence or absence of microbial activity. Under the same set of tested conditions the three illudane glycosides degraded at similar rates: with half-lives of approximately two days at pH 7.4 and 15 °C, and approximately 12 days at pH 5.2-6.5 and 5 °C. The water origin had significant influence on the degradation rates, but only due to its difference in pH. In most cases, the degradation rates of all the three illudane glycosides could be predicted using the existing first-order model for PTA hydrolysis. As PTE and CAU exhibit the same leaching pattern and stability as PTA, previous predictions of bracken environmental impact are likely underestimated, as PTE and CAU have not been monitored and included in the risk assessment.

An Efficient Method for the Isolation of Toxins from Pteridium aquilinum and Evaluation of Ptaquiloside Against Cancer and Non-cancer Cells.

The common fern, bracken (Pteridium aquilinum), is well known for its toxic effects on livestock due principally to the carcinogenic constituent ptaquiloside ( 1: ), although other toxins are present including the cyanogenic glycoside, prunasin ( 2: ). Here, we report an improved and relatively "green" process for the isolation of 1: and 2: from fresh bracken fronds and the evaluation of 1: for cytotoxicity against several cancer cell lines. The results indicate that 1: displays selective toxicity against cancer cells relative to noncancer retinal epithelial cells, and the improved method for the isolation of 1: is expected to facilitate further exploration of its pharmacological properties.

Synthesis, biological activity, and four-dimensional quantitative structure-activity analysis of 2-arylidene indan-1,3-dione derivatives tested against Daphnia magna.

A series of 18 2-arylidene indan-1,3-dione derivatives was synthesized and tested against Daphnia magna to assess the environmental toxicity of these compounds. Aiming to investigate the toxicity mechanism for this series of compounds, a four-dimensional quantitative structure-activity analysis (4D-QSAR) was performed through the partial least square regression (PLS). The best PLS model was built with two factors and the selected field descriptors, of Coulomb (C) and Lennard-Jones (L) nature, describing 77.43% of variance and presenting the following statistics: r 2 = 0.89; SEC = 0.30; Q 2 = 0.81; SEV = 0.36. According to the literature, the bioactivity of α,ß-unsaturated ketones, a functionality present in the series of compounds under investigation, is related to the conjugated double bond with the carbonyl group. The presence of a positive Coulomb descriptor nearby the carbonyl moieties, obtained as a result of the regression model, indicates that these polar groups are also related to the toxicity on D. magna. From the PLS regression model, the toxicity EC50-48 h values increases with the positive Coulomb descriptor and diminishes with the negative Lennard-Jones descriptors. It could be concluded that the presence of small polar groups in the aromatic ring of the arylidene moiety tends to increase the toxicity, while bulkier apolar substituents lead to a decrease of the toxicity.

Phellodendron amurense Extract Protects Human Keratinocytes from PM2.5-Induced Inflammation via PAR-2 Signaling.

Dietary supplement and personal care products aiming to provide protection from air pollution have been of great interest for decades. Epidemiology demonstrated that PM10 and PM2.5 particulate matter (PM) are an actual threat to public health worldwide, but the detailed processes of how these particles attack the cells are not fully understood. Here, we report that the measurement of intracellular calcium concentration ([Ca2+]i) using human respiratory or skin cells can illustrate pollutant challenges by triggering Ca2+ influx in these cells. This signal was generated by proteinase-activated receptor-2 (PAR-2), confirmed by competition analyses, and Phellodendron amurense bark extract (PAE), a traditional medicine, was able to control the response and expression of PAR-2. Increase in proinflammatory cytokines and decrease in cell adhesion components could suggest a severe damage status by air pollutants and protection by PAE. Finally, we identified 4-O-feruloylquinic acid (FQA), an active compound of PAE, showing the same effects on Ca2+ influx and PAR-2 regulation. The results presented here should help understand the underlying mechanism of PM insults and the beneficial effect of standardized PAE as dietary supplement or cosmetical ingredient.

Ptaquiloside and Pterosin B Levels in Mature Green Fronds and Sprouts of Pteridium arachnoideum.

Pteridium arachnoideum, a fern of the Pteridium aquilinum species complex found in South America, is responsible for several different syndromes of poisoning. Cases of bovine enzootic hematuria and upper alimentary squamous cell carcinoma are both frequent occurrences in Brazil, whereas only bovine enzootic hematuria is noted with any frequency around the world. The reason for the high frequency of upper alimentary squamous cell carcinoma in Brazil is not currently known. One possible explanation may be the higher levels of ptaquiloside and pterosin B in Brazilian Pteridium than those present in the plant in other countries. However, these levels have not yet been determined in P. arachnoideum. Thus, the present study aimed to measure and compare ptaquiloside and pterosin B levels in mature green fronds and sprouts of P. arachnoideum collected from different locations in Brazil. Samples of P. arachnoideum were collected from the states of Minas Gerais and Rio Grande do Sul. A total of 28 mature leaf samples and 23 sprout samples were used. The mean concentrations of ptaquiloside and pterosin B present in the mature green fronds of P. arachnoideum ranged from 2.49 to 2.75 mg/g and 0.68 to 0.88 mg/g, respectively; in P. arachnoideum sprouts, mean concentrations of ptaquiloside and pterosin B ranged from 12.47 to 18.81 mg/g, and 4.03 to 10.42 mg/g for ptaquiloside and pterosin B, respectively. Thus, ptaquiloside and pterosin B levels in P. arachnoideum samples collected in Brazil were higher in sprouts than in mature green fronds, as observed in other countries. However, there was no variation in ptaquiloside levels among plants collected from different cities in Brazil. The high frequency of upper alimentary squamous cell carcinoma in Brazilian cattle may not be attributed to greater levels of ptaquiloside and pterosin B in P. arachnoideum than in other Pteridium species in other countries.

Pharmacodynamics of intrathecal and epidural fadolmidine, an α2-adrenoceptor agonist, after bolus and infusion in dogs-comparison with clonidine.

An α2-adrenoceptor agonist, clonidine, is extensively used in both anesthesia and intensive care medicine. However, clonidine may produce pronounced hemodynamic side effects such as hypotension and bradycardia which may limit its usefulness in certain conditions. Fadolmidine is a potent α2-adrenoceptor agonist with different physicochemical properties than clonidine. Here, the effects of fadolmidine and clonidine on analgesia (an increase in thermal skin twitch response latency), sedation, blood pressure, heart rate, respiratory rate, and body temperature were evaluated either up to 8 h after either intrathecal or epidural bolus injections or during a 24-h continuous intrathecal infusion at equipotent analgesic doses in non-anesthetized Beagle dogs. Fadolmidine and clonidine produced a dose-dependent and equipotent maximal antinociception after intrathecal bolus injection (ED50: 67 µg and 78 µg, respectively), but the duration of action of fadolmidine was more long-lasting. During the intrathecal infusion, fadolmidine achieved a good analgesic effect without evoking cardiovascular side effects, e.g., hypotension; these were evident during clonidine infusion. Epidurally, the antinociceptive potency of fadolmidine was weaker (ED50: 128 µg) than when intrathecally administered and weaker than that of epidural clonidine (ED50: 51 µg). At analgesic doses, fadolmidine injection induced moderate initial hypertension concomitantly with a decrease in heart rate whereas clonidine evoked hypotension and bradycardia. These results suggest that especially when non-opioid long-term pain relief is needed, an intrathecal infusion of fadolmidine can provide long-term antinociception with less of the known use-limiting adverse effects associated with clonidine.

Bioactive indanes: Development and validation of an LC-MS/MS bioanalytical method for the determination of PH46A, a new potential anti-inflammatory agent, in dog and rat plasma and its application to a pharmacokinetic study in dog.

A new chemical entity, which is a chiral indane dimer, PH46A, has been developed by our research group. As a clinical candidate. PH46A has recently completed Phase I clinical studies in man. Previously, during its pre-clinical development, in in vivo pre-clinical studies PH46A showed potent anti-inflammatory properties, which can be targeted at a range of diseases, including inflammatory bowel disease (IBD). To support the pre-clinical development of this drug candidate, we developed a LCMS/MS method for determining PH46 (the acid form of PH46A salt) in both dog and rat plasma using Compound 1 as internal standard (IS). Those species were selected for safety pharmacology and toxicology, as well as pharmacokinetics studies. The method was validated over the range 10-10000 ng/mL for both matrices and the linearity, accuracy, precision and specificity over this range were demonstrated to be acceptable. No significant matrix effects or carryover were observed for both PH46 and IS and recovery was consistent. PH46 was found to be stable in both dog and rat plasma under the test conditions, such as at room temperature for >24 h, through 3 freeze/thaw cycles, and at -20 °C for >1 month. PH46 and IS in dog and rat plasma extracts were also found to be stable in the autosampler against fresh standard extracts on re-injection after 143.5 h and 243.5 h, respectively at 4 °C. 10- and 100-fold dilutions with control matrix were found not to affect the performance of the assay. This method was successfully applied to a pharmacokinetic study in the dog. With the exception of one dog, 003 M, oral administration of PH46A in gelatine capsules was well tolerated at a dose level of 100 mg/kg. The highest Cmax was observed in animal 003 M. The rapid absorption and high plasma concentration observed for animal 003 M compared to the data for animals 001 M and 002 M may account for the sickness observed in this animal; however, the reasons for this have not been investigated.

Brodifacoum Toxicity in American Kestrels (Falco sparverius) with Evidence of Increased Hazard on Subsequent Anticoagulant Rodenticide Exposure.

A seminal question in ecotoxicology is the extent to which contaminant exposure evokes prolonged effects on physiological function and fitness. A series of studies were undertaken with American kestrels ingesting environmentally realistic concentrations of the second-generation anticoagulant rodenticide (SGAR) brodifacoum. Kestrels fed brodifacoum at 0.3, 1.0, or 3.0 µg/g diet wet weight for 7 d exhibited dose-dependent hemorrhage, histopathological lesions, and coagulopathy (prolonged prothrombin and Russell's viper venom times). Following termination of a 7-d exposure to 0.5 µg brodifacoum/g diet, prolonged blood clotting time returned to baseline values within 1 wk, but brodifacoum residues in liver and kidney persisted during the 28-d recovery period (terminal half-life estimates >50 d). To examine the hazard of sequential anticoagulant rodenticide (AR) exposure, kestrels were exposed to either the first-generation AR chlorophacinone (1.5 µg/g diet) or the SGAR brodifacoum (0.5 µg/g diet) for 7 d and, following a recovery period, challenged with a low dose of chlorophacinone (0.75 µg/g diet) for 7 d. In brodifacoum-exposed kestrels, the challenge exposure clearly prolonged prothrombin time compared to naive controls and kestrels previously exposed to chlorophacinone. These data provide evidence that the SGAR brodifacoum may have prolonged effects that increase the toxicity of subsequent AR exposure. Because free-ranging predatory and scavenging wildlife are often repeatedly exposed to ARs, such protracted toxicological effects need to be considered in hazard and risk assessments. Environ Toxicol Chem 2020;39:468-481. © 2020 SETAC.

The role of bracken fern illudanes in bracken fern-induced toxicities.

Bracken fern is carcinogenic when fed to domestic and laboratory animals inducing bladder and ileal tumours and is currently classified as a possible human carcinogen by IARC. The carcinogenic illudane, ptaquiloside (PTQ) was isolated from bracken fern and is widely assumed to be the major bracken carcinogen. However, several other structurally similar illudanes are found in bracken fern, in some cases at higher levels than PTQ and so may contribute to the overall toxicity and carcinogenicity of bracken fern. In this review, we critically evaluate the role of illudanes in bracken fern induced toxicity and carcinogenicity, the mechanistic basis of these effects including the role of DNA damage, and the potential for human exposure in order to highlight deficiencies in the current literature. Critical gaps remain in our understanding of bracken fern induced carcinogenesis, a better understanding of these processes is essential to establish whether bracken fern is also a human carcinogen.

Cytotoxicity of new psychoactive substances and other drugs of abuse studied in human HepG2 cells using an adopted high content screening assay.

New psychoactive substances (NPS) are still an emerging issue in clinical and forensic toxicology. Information about their cytotoxic potential is limited or even unavailable before distribution and thus their intake can be of high risk for consumers. The aim of the presented study was to develop a strategy to identify cytotoxic potential of NPS based on a high content screening assay (HCSA) using HepG2 cell line and four fluorescent dyes, namely Hoechst33342, TMRM, CAL-520, and TOTO-3. The HCSA was optimized to work without an automated analyzer by using the model compounds fluvastatin, paracetamol, propranolol, and simvastatin. The following parameters were monitored: stained nuclei as a measure for cell count as well as nuclear size and nuclear intensity (all Hoechst33342), mitochondrial membrane potential (TMRM), cytosolic calcium level (CAL-520), and plasma membrane integrity (TOTO-3). The present study showed strong cytotoxic potential for the NPS 5F-PB-22 and MDAI, moderate effects for MDMA, MDPV, methylone, cathinone, 4-MEC, and mephedrone, and no toxic effects for methamphetamine. To assess the metabolic suitability of HepG2 cells under the chosen conditions, cell culture supernatants were analyzed by liquid chromatography-high resolution-tandem mass spectrometry. Metabolites were merely detected for lipophilic drugs such as 5F-PB-22 and MDPV and in addition with a much lower abundance in comparison to the parent compound but the study only allowed a qualitative look for metabolites and the used liver cell line might not ideal when considering metabolism.

Rational Design and Multibiological Profiling of Novel Donepezil-Trolox Hybrids against Alzheimer's Disease, with Cholinergic, Antioxidant, Neuroprotective, and Cognition Enhancing Properties.

A novel series of donepezil-trolox hybrids were designed, synthesized, and evaluated as multifunctional ligands against Alzheimer's disease (AD). Biological assays showed that these derivatives possessed moderate to good inhibitory activities against acetylcholinesterase (AChE) and monoamine oxidase B (MAO-B) as well as remarkable antioxidant effects. The optimal compound 6d exhibited balanced functions with good inhibition against hAChE (IC50 = 0.54 µM) and hMAO-B (IC50 = 4.3 µM), significant antioxidant activity (41.33 µM IC50 by DPPH method, 1.72 and 1.79 trolox equivalent by ABTS and ORAC methods), excellent copper chelation, and Aß1-42 aggregation inhibition effect. Furthermore, cellular tests indicated that 6d has very low toxicity and is capable of combating oxidative toxin (H2O2, rotenone, and oligomycin-A) induced neurotoxicity. Most importantly, oral administration of 6d demonstrated notable improvements on cognition and spatial memory against scopolamine-induced acute memory deficit as well as d-galactose (d-gal) and AlCl3 induced chronic oxidative stress in a mouse model without acute toxicity and hepatotoxicity. In summary, both in vitro and in vivo results suggested that 6d is a valuable candidate for the development of a safe and effective anti-Alzheimer's drug.

Toxicological evaluation of 5-methoxy-2-aminoindane (MEAI): Binge mitigating agent in development.

5-Methoxy-2-aminoindane (MEAI) is a psychoactive compound of the aminoindane class, which in recent years has been recreationally used by many people, who reported of a mild euphoric, alcohol-like tipsy experience and reduced desire to consume alcoholic beverages. In the light of these observations it was decided to progress MEAI through a preliminary drug development route and evaluate the acute and subacute toxicity of MEAI administrated orally to Sprague Dawley rats, as well as to determine potential in-vitro cytotoxic and mutagenic effects using state-of-the-art protocols. Furthermore, the interaction of MEAI at the highest non-toxic concentration (100mg/L) with ethanol at cytotoxic levels of 6% and 7.5% was explored, in order to identify possible additive or synergistic effects. MEAI showed a good safety profile in rats at 10 and 30mg/kg body weight, corresponding to the human doses of 1.6mg/kg and 4.8mg/kg body weight, respectively. Cytotoxic effect was demonstrated using concentrations of 500 and 1000mg/L with calculated IC50 value of 368.2mg/L for rat brain striatum primary neurons and 403.1mg/L for human primary healthy hepatocytes. The combination of 6% or 7.5% ethanol with 100mg/L MEAI revealed no statistically significant increase of cytotoxic effect. Further studies, especially long term chronic and addictive behavior studies, are required in-order to assess MEAI safety profile.

Synthesis and biological activity of the novel indanedione anticoagulant rodenticides containing fluorine.

Here, 3 fluorinated intermediates of drug were synthesized: (M1), (M2), (M3). Three new anticoagulant rodenticides were designed which were based on 4-hydroxycoumarin or 1,3-indandione, added acute toxicity groups containing fluorine. The structures of synthesized compounds were analyzed and proved by FT-IR spectroscopy and 1H nuclear magnetic resonance (1H-NMR). The compounds were also evaluated for their anticoagulant and acute biologic activity. In addition, both the acute orally toxicity and the feeding indexes of R1 and R2 were tested. The result of the experiment proved that the new synthesis of 1, 3 - indan diketone for maternal new anticoagulant rodenticide can replace the current 4 - hydroxyl coumarin as the mother of the second generation anticoagulant rodenticide and 1, 3 - indan diketone for maternal new anticoagulant rodenticides will have a good development prospect.

The proof-of-concept of ASS234: Peripherally administered ASS234 enters the central nervous system and reduces pathology in a male mouse model of Alzheimer disease.

BACKGROUND: The heterogeneity of Alzheimer disease requires the development of multitarget drugs for treating the symptoms of the disease and its progression. Both cholinergic and monoamine oxidase dysfunctions are involved in the pathological process. Thus, we hypothesized that the development of therapies focused on these targets might be effective. We have developed and assessed a new product, coded ASS234, a multipotent acetyl and butyrylcholinesterase/monoamine oxidase A-B inhibitor with a potent inhibitory effect on amyloid-ß aggregation as well as antioxidant and antiapoptotic properties. But there is a need to reliably correlate in vitro and in vivo drug release data. METHODS: We examined the effect of ASS234 on cognition in healthy adult C57BL/6J mice in a model of scopolamine-induced cognitive impairment that often accompanies normal and pathological aging. Also, in a characterized transgenic APPswe/PS1ΔE9 mouse model of Alzheimer disease, we examined the effects of short-term ASS234 treatment on plaque deposition and gliosis using immunohistochemistry. Toxicology of ASS234 was assessed using a quantitative high-throughput in vitro cytotoxicity screening assay following the MTT assay method in HepG2 liver cells. RESULTS: In vivo, ASS234 significantly decreased scopolamine-induced learning deficits in C57BL/6J mice. Also, reduction of amyloid plaque burden and gliosis in the cortex and hippocampus was assessed. In vitro, ASS234 exhibited lesser toxicity than donepezil and tacrine. LIMITATIONS: The study was conducted in male mice only. Although the Alzheimer disease model does not recapitulate all features of the human disease, it exhibits progressive monoaminergic neurodegeneration. CONCLUSION: ASS234 is a promising alternative drug of choice to treat the cognitive decline and neurodegeneration underlying Alzheimer disease.

Ptaquiloside from bracken (Pteridium spp.) inhibits tumour-infiltrating CD8+ T cells in HPV-16 transgenic mice.

Bracken is a fern with worldwide distribution. Exposure to bracken toxins such as ptaquiloside is hypothesized to increase the risk of papillomavirus-related cancers of the upper digestive tract. Ptaquiloside is thought to be an immunosupressor, thus allowing for the development of viral lesions. We have used a human papillomavirus type 16-transgenic (K14-HPV16) mouse model to study the effects of ptaquiloside on tumour-infiltrating CD8+ T lymphocytes, which are critical players in anti-tumour immunity. HPV16+/- mice received ptaquiloside (0.5 mg/mouse/week) for 10 weeks. These were then euthanized at 30 weeks of age, along with age-matched untreated controls. Skin samples were enzymatically digested and CD8+ T cells analysed for CD107a and CD44 surface expression. Ptaquiloside-exposed HPV16+/- mice showed a significantly decreased percentage (P < 0.05) of CD8+CD107a+ and CD8+CD44 + T cells when compared with untreated HPV16+/- animals. Histologically, 100% of ptaquilosidetreated mice showed diffuse epidermal dysplasia, compared with 50% of the untreated mice. These findings suggest that ptaquiloside exerts an immunosuppressive role by decreasing CD8+ T cell activation and degranulation in HPV-induced lesions. Given the key role of CD8+ T lymphocytes against HPV-induced lesions, this effect is likely to contribute for viral persistence, tumour progression and increased aggressiveness in patients with HPV-related malignancies.

Anticancer activity of gallic acid template-based benzylidene indanone derivative as microtubule destabilizer.

Benzylidene indanones have been designed and synthesized from gallic acid, a plant phenolic acid as possible anticancer agent. The best analogue of the series, that is, 3-(3',4',5'-trimethoxyphenyl)-4,5,6-trimethoxy-2-(4˝-nitrobenzylidene)-indan-1-one (8) exhibited potent cytotoxicity (IC50 =3-10 µm) against several human cancer cell lines through microtubule destabilization (IC50 =1.54 µm) after occupying colchicine-binding site of ß-tubulin. In cell cycle analysis, compound 8 exerted G2/M phase arrest in both MCF-7 and MDA-MB-231 cells and induced apoptosis. It reduced 34.8% solid tumor in in vivo Ehrlich ascite carcinoma in Swiss albino mice at 30 mg/kg dose. In acute oral toxicity experiment, it was tolerable up to 300 mg/kg doses in Swiss albino mice. The lead compound 8 needs to be optimized for better activity.

Synthesis and evaluation of multi-target-directed ligands for the treatment of Alzheimer's disease based on the fusion of donepezil and melatonin.

A novel series of compounds obtained by fusing the acetylcholinesterase (AChE) inhibitor donepezil and the antioxidant melatonin were designed as multi-target-directed ligands for the treatment of Alzheimer's disease (AD). In vitro assay indicated that most of the target compounds exhibited a significant ability to inhibit acetylcholinesterase (eeAChE and hAChE), butyrylcholinesterase (eqBuChE and hBuChE), and ß-amyloid (Aß) aggregation, and to act as potential antioxidants and biometal chelators. Especially, 4u displayed a good inhibition of AChE (IC50 value of 193nM for eeAChE and 273nM for hAChE), strong inhibition of BuChE (IC50 value of 73nM for eqBuChE and 56nM for hBuChE), moderate inhibition of Aß aggregation (56.3% at 20µM) and good antioxidant activity (3.28trolox equivalent by ORAC assay). Molecular modeling studies in combination with kinetic analysis revealed that 4u was a mixed-type inhibitor, binding simultaneously to catalytic anionic site (CAS) and the peripheral anionic site (PAS) of AChE. In addition, 4u could chelate metal ions, reduce PC12 cells death induced by oxidative stress and penetrate the blood-brain barrier (BBB). Taken together, these results strongly indicated the hybridization approach is an efficient strategy to identify novel scaffolds with desired bioactivities, and further optimization of 4u may be helpful to develop more potent lead compound for AD treatment.