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Background: Recent studies have shown that harringtonine (HT) could specifically bind with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein and host cell transmembrane serine protease 2 (TMPRSS2) to block membrane fusion, which is an effective antagonist for SARS-CoV-2., Purpose: Our study focused on in-depth exploration of in vitro pharmacokinetic characteristics of HT in lung., Methods: HPLC-fluorescence detection method was used to detect changes of HT content. Incubation systems of lung microsomes for phase I metabolism and UGT incubation systems for phase II metabolism were performed to elucidate metabolites and metabolic mechanisms of HT, and then the metabolic enzyme phenotypes for HT were clarified by chemical inhibition method and recombinant enzyme method. Through metabolomics, we comprehensively evaluated the physiological dynamic changes in SD rat and human lung microsomes, and revealed the relationship between metabolomics and pharmacological activity of HT., Results: HPLC-fluorescence detection method showed strong specificity, high accuracy, and good stability for rapid quantification of HT. We confirmed that HT mainly underwent phase I metabolism, and the metabolites of HT in different species were all identified as 4'-demethyl HT, with metabolic pathway being hydrolysis reaction. CYP1A2 and CYP2E1 participated in HT metabolism, but as HT metabolism was not NADPH dependent, the esterase HCES1 in lung also played a role. The main KEGG pathways in SD rat and human lung microsomes were cortisol synthesis and secretion, steroid hormone biosynthesis and linoleic acid metabolism, respectively. The downregulated key biomarkers of 11-deoxycortisol, 21-deoxycortisol and 9(10)-EpOME suggested that HT could prevent immunosuppression and interfere with infection and replication of SARS-CoV-2., Conclusion: HT was mainly metabolized into 4'-demethyl HT through phase I reactions, which was mediated by CYP1A2, CYP2E1, and HCES1. The downregulation of 11-deoxycortisol, 21-deoxycortisol and 9(10)-EpOME were key ways of HT against SARS-CoV-2. Our study was of great significance for development and clinical application of HT in the treatment of COVID-19., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2024. Published by Elsevier GmbH.)

This study aimed to characterize the effects of arsenic exposure on the expression of microsomal epoxide hydrolase (mEH or EPHX1) and soluble epoxide hydrolase (sEH or EPHX2) in the liver and small intestine. C57BL/6 mice were exposed to sodium arsenite in drinking water at various doses for up to 28 days. Intestinal, but not hepatic, mEH mRNA and protein expression was induced by arsenic at 25 ppm, in both males and females, whereas hepatic mEH expression was induced by arsenic at 50 or 100 ppm. The induction of mEH was gene specific, as the arsenic exposure did not induce sEH expression in either tissue. Within the small intestine, mEH expression was induced only in the proximal, but not the distal segments. The induction of intestinal mEH was accompanied by increases in microsomal enzymatic activities toward a model mEH substrate, cis -stilbene oxide, and an epoxide-containing drug, oprozomib, in vitro, and by increases in the levels of PR-176, the main hydrolysis metabolite of oprozomib, in the proximal small intestine of oprozomib-treated mice. These findings suggest that intestinal mEH, playing a major role in converting xenobiotic epoxides to less reactive diols, but not sEH, preferring endogenous epoxides as substrates, is relevant to the adverse effects of arsenic exposure, and that further studies of the interactions between drinking water arsenic exposure and the disposition or possible adverse effects of epoxide-containing drugs and other xenobiotic compounds in the intestine are warranted. SIGNIFICANCE STATEMENT: Consumption of arsenic-contaminated water has been associated with increased risks of various adverse health effects, such as diabetes, in humans. The small intestinal epithelial cells are the main site of absorption of ingested arsenic, but they are not well characterized for arsenic exposure-related changes. This study identified gene expression changes in the small intestine that may be mechanistically linked to the adverse effects of arsenic exposure and possible interactions between arsenic ingestion and the pharmacokinetics of epoxide-containing drugs in vivo., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

Organotins have been widely used in various industrial applications. This study investigated the structure-activity relationship as inhibitors of human, pig, and rat gonadal 3β-hydroxysteroid dehydrogenases (3β-HSD). Human KGN cell, pig, and rat testis microsomes were utilized to assess the inhibitory effects of 18 organotins on the conversion of pregnenolone to progesterone. Among them, diphenyltin, triethyltin, and triphenyltin exhibited significant inhibitory activity against human 3β-HSD2 with IC 50 values of 114.79, 106.98, and 5.40 μM, respectively. For pig 3β-HSD, dipropyltin, diphenyltin, triethyltin, tributyltin, and triphenyltin demonstrated inhibitory effects with IC 50 values of 172.00, 100.19, 87.00, 5.75, and 1.65 μM, respectively. Similarly, for rat 3β-HSD1, dipropyltin, diphenyltin, triethyltin, tributyltin, and triphenyltin displayed inhibitory activity with IC 50 values of 81.35, 43.56, 55.55, 4.09, and 0.035 μM, respectively. They were mixed inhibitors of pig and rat 3β-HSD, while triphenyltin was identified as a competitive inhibitor of human 3β-HSD2. The mechanism underlying the inhibition of organotins on 3β-HSD was explored, revealing that they may disrupt the enzyme activity by binding to cysteine residues in the catalytic sites. This proposition was supported by the observation that the addition of dithiothreitol reversed the inhibition caused by all organotins except for triethyltin, which was partially reversed. In conclusion, this study provides valuable insights into the structure-activity relationship of organotins as inhibitors of human, pig, and rat gonadal 3β-HSD. The mechanistic investigation suggests that these compounds likely exert their inhibitory effects through binding to cysteine residues in the catalytic sites., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Castanopsis tribuloides belongs to the oak species (Fagaceae) and it is commonly distributed in evergreen forests of Bangladesh, India, Myanmar, Nepal, China, and Thailand. Our present study aimed at uncovering the antipyretic potential of methanol extract of C. tribuloides bark (CTB) in the mice models. Baker's yeast pyrexia model was employed to determine the antipyretic potentials of the extract. Besides, molecular docking and dynamics simulation of CTB phenolic compounds were explored to validate the experimental results and gain insight into the possible antipyretic mechanism of action that can lead to the design and discovery of novel drugs against mPGES-1. The results revealed that CTB (400 mg/kg) significantly inhibited (P < 0.001) the elevated body temperature of mice since 0.5 h, which is more prominent than the standard. At dose 200 mg/kg, the bark extract also produced significant (P < 0.05) antipyretic activity since 2 h. HPLC-DAD analysis identified and quantified nine polyphenolic compounds from the extract, including rutin hydrate, (-) epicatechin, caffeic acid, catechin hydrate, catechol, trans-ferulic acid, p-coumaric acid, vanillic acid, and rosmarinic acid. Molecular docking study suggested probable competition of these phenolic compounds with glutathione, an essential cofactor for microsomal prostaglandin E synthase-1 (mPGES-1) activity. Additionally, RMSF, RMSD, Rg, and hydrogen bonds performed during MD simulations revealed that rutin hydrate (rich in CTB) bound to the mPGES-1 active site in a stable manner and thus inactivating mPGES-1. Therefore, it can be concluded that rutin hydrate reduces pyrexia in mice via downregulating PGE2 synthesis by inhibiting mPGES-1 activity., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Prostaglandin E 2 (PGE 2 ) plays pivotal roles in controlling microglial activation with the EP 2 receptor, a PGE 2 receptor subtype. Activated microglia are often reported to increase cyclooxygenase (COX)-2 expression, followed by PGE 2 production, but it is unclear whether extracellular PGE 2 is involved in microglial PGE 2 synthesis. In the present study, we report that PGE 2 increases COX-2 protein in microglia. In a culture system, PGE 2 at 10 -6 M for 3 h increased COX-2 and microsomal PGE synthase (mPGES)-1 mRNA levels, and reduced mPGES-2, but did not affect COX-1 or cytosolic PGE synthase (cPGES) in microglia. PGE 2 at 10 -6 M for 3 h also increased the COX-2 protein level, but did not affect COX-1, mPGES-1, mPGES-2, or cPGES. An EP 2 agonist, ONO-AE1-259-01, also increased COX-2 and mPGES-1 mRNA levels, and reduced mPGES-2, but did not affect COX-1 or cPGES, whereas an EP 1 agonist, ONO-DI-004, an EP 3 agonist, ONO-AE-248, and an EP 4 agonist, ONO-AE1-329, had no effect. Similar to PGE 2 , ONO-AE1-259-01 increased the COX-2 protein level, but did not affect COX-1, mPGES-1, mPGES-2, or cPGES. In addition, the effects of PGE 2 were inhibited by an EP 2 antagonist, PF-04418948, but not by an EP 1 antagonist, ONO-8713, an EP 3 antagonist, ONO-AE3-240, or an EP 4 antagonist, ONO-AE3-208, at 10 -6 M. On the other hand, lipopolysaccharide (LPS) increased PGE 2 production, but the LPS-induced PGE 2 production was not affected by ONO-8713, PF-04418948, ONO-AE3-240, or ONO-AE3-208. These results indicate that PGE 2 increases COX-2 protein in microglia through the EP 2 receptor supporting the idea that extracellular PGE 2 has a triggering aspect for microglial activation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Aim: This study investigated our Enzymelinks, COX-2-10aa-mPGES-1 and COX-2-10aa-PGIS, as cellular cross-screening targets for quick identification of lead compounds to inhibit inflammatory PGE 2 biosynthesis while maintaining prostacyclin synthesis. Methods: We integrated virtual and wet cross-screening using Enzymelinks to rapidly identify lead compounds from a large compound library. Results: From 380,000 compounds virtually cross-screened with the Enzymelinks, 1576 compounds were identified and used for wet cross-screening using HEK293 cells that overexpressed individual Enzymelinks as targets. The top 15 lead compounds that inhibited mPGES-1 activity were identified. The top compound that specifically inhibited inflammatory PGE 2 biosynthesis alone without affecting COX-2 coupled to PGI 2 synthase (PGIS) for PGI 2 biosynthesis was obtained. Conclusion: Enzymelink technology could advance cyclooxygenase pathway-targeted drug discovery to a significant degree.

Ethnopharmacological Relevance: Shengmai San (SMS) has been commonly used as a traditional Chinese medicine for the treatment of cardiovascular disorders, of which drug interactions need to be assessed for the safety concern. There is little evidence for the alterations of hepatic and intestinal drug-metabolizing enzymes after repeated SMS treatments to assess drug interactions., Aim of the Study: The studies aim to illustrate the effects of repeated treatments with SMS on cytochrome P450s (CYPs), reduced nicotinamide adenine dinucleotide (phosphate)-quinone oxidoreductase (NQO), uridine diphosphate-glucuronosyltransferase (UGT), and glutathione S-transferase (GST) using in vivo rat model., Materials and Methods: The SMS was prepared using Schisandrae Fructus, Ginseng Radix, and Ophiopogonis Radix (OR) (1:2:2). Chromatographic analyses of decoctions were performed using ultra-performance liquid chromatography (UPLC) and LC-mass spectrometry. Sprague-Dawley rats were orally treated with the SMS and its component herbal decoctions for 2 or 3 weeks. Hepatic and intestinal enzyme activities were determined. CYP3A expression and the kinetics of intestinal nifedipine oxidation (NFO, a CYP3A marker reaction) were determined., Results: Schisandrol A, schisandrin B, ginsenoside Rb1 and ophiopogonin D were identified in SMS. SMS selectively suppressed intestinal, but not hepatic, NFO activity in a dose- and time-dependent manner. Hepatic and intestinal UGT, NQO and GST activities were not affected. A 3-week SMS treatment decreased the maximal velocity of intestinal NFO by 50%, while the CYP3A protein level remained unchanged. Among SMS component herbs, the decoction of OR decreased intestinal NFO activity., Conclusions: These findings demonstrate that 3-week treatment with SMS and OR suppress intestinal, but not hepatic CYP3A function. It suggested that the potential interactions of SMS with CYP 3A drug substrates should be noticed, especially the drugs whose bioavailability depends heavily on intestinal CYP3A., (Copyright © 2021 Elsevier B.V. All rights reserved.)

A new oleanane triterpenoid ( 2 ) was isolated from the roots of Rubia philippinensis . The structure of 2 was determined by analysis of HRMS and NMR data and identified as a rubiprasin analogue, 16 β -hydroxyrubiprasin B. Four related known compounds were also encountered which include rubiprasin B ( 1 ), maslinic acid ( 3 ), 4- epi -hederagenin ( 4 ) and oleanolic acid ( 5 ). The compounds 3 - 5 displayed moderate inhibitory activity against the synthesis of the eicosanoid 20-HETE.

Small molecules targeting the PF74 binding site of the HIV-1 capsid protein (CA) confer potent and mechanistically unique antiviral activities. Structural modifications of PF74 could further the understanding of ligand binding modes, diversify ligand chemical classes, and allow identification of new variants with balanced antiviral activity and metabolic stability. In the current work, we designed and synthesized three series of PF74-like analogs featuring conformational constraints at the aniline terminus or the phenylalanine carboxamide moiety, and characterized them using a biophysical thermal shift assay (TSA), cell-based antiviral and cytotoxicity assays, and in vitro metabolic stability assays in human and mouse liver microsomes. These studies showed that the two series with the phenylalanine carboxamide moiety replaced by a pyridine or imidazole ring can provide viable hits. Subsequent SAR identified an improved analog 15 which effectively inhibited HIV-1 (EC 50 = 0.31 μM), strongly stabilized CA hexamer (ΔTm = 8.7 °C), and exhibited substantially enhanced metabolic stability (t 1/2 = 27 min for 15 vs. 0.7 min for PF74). Metabolic profiles from the microsomal stability assay also indicate that blocking the C5 position of the indole ring could lead to increased resistance to oxidative metabolism.

Background: Simmondsia chinensis (jojoba) is the only plant known to store wax esters instead of triacylglycerols in its seeds. Wax esters are composed of very-long-chain monounsaturated fatty acids and fatty alcohols and constitute up to 60% of the jojoba seed weight. During jojoba germination, the first step of wax ester mobilization is catalyzed by lipases. To date, none of the jojoba lipase-encoding genes have been cloned and characterized. In this study, we monitored mobilization of storage reserves during germination of jojoba seeds and performed detailed characterization of the jojoba lipases using microsomal fractions isolated from germinating seeds., Results: During 26 days of germination, we observed a 60-70% decrease in wax ester content in the seeds, which was accompanied by the reduction of oleosin amounts and increase in glucose content. The activity of jojoba lipases in the seed microsomal fractions increased in the first 50 days of germination. The enzymes showed higher activity towards triacylglycerols than towards wax esters. The maximum lipase activity was observed at 60 °C and pH around 7 for triacylglycerols and 6.5-8 for wax esters. The enzyme efficiently hydrolyzed various wax esters containing saturated and unsaturated acyl and alcohol moieties. We also demonstrated that jojoba lipases possess wax ester-synthesizing activity when free fatty alcohols and different acyl donors, including triacylglycerols and free fatty acids, are used as substrates. For esterification reactions, the enzyme utilized both saturated and unsaturated fatty alcohols, with the preference towards long chain and very long chain compounds., Conclusions: In in vitro assays, jojoba lipases catalyzed hydrolysis of triacylglycerols and different wax esters in a broad range of temperatures. In addition, the enzymes had the ability to synthesize wax esters in the backward reaction. Our data suggest that jojoba lipases may be more similar to other plant lipases than previously assumed.

Several natural-based compounds and products are reported to possess anti-inflammatory and immunomodulatory activity both in vitro and in vivo. The primary target for these activities is the inhibition of eicosanoid-generating enzymes, including phospholipase A2, cyclooxygenases (COXs), and lipoxygenases, leading to reduced prostanoids and leukotrienes. Other mechanisms include modulation of protein kinases and activation of transcriptases. However, only a limited number of studies and reviews highlight the potential modulation of the coupling enzymatic pathway COX-2/mPGES-1 and Th17/Treg circulating cells. Here, we provide a brief overview of natural products/compounds, currently included in the Italian list of botanicals and the BELFRIT, in different fields of interest such as inflammation and immunity. In this context, we focus our opinion on novel therapeutic targets such as COX-2/mPGES-1 coupling enzymes and Th17/Treg circulating repertoire. This paper is dedicated to the scientific career of Professor Nicola Mascolo for his profound dedication to the study of natural compounds.

The Ames test has become one of the most commonly used tests to assess the mutagenic potential of medicinal plants since they have several biological activities and thus have been used in traditional medicine and in the pharmaceutical industry as a source of raw materials. Accordingly, this review aims to report previous use of the Ames test to evaluate the mutagenic potential of medicinal plants. A database was constructed by curating literature identified by a search on the electronic databases Medline (via Pubmed), Science Direct, Scopus, and Web of Science from 1975 to April 2020, using the following terms: "genotoxicity tests" OR "mutagenicity tests" OR "Ames test" AND "medicinal plants." From the research, 239 articles were selected, including studies of 478 species distributed across 111 botanical families, with Fabaceae, Asteraceae and Lamiaceae being the most frequent. It was identified that 388 species were non-mutagenic. Of these, 21% (83/388) showed antimutagenic potential, most notable in the Lamiaceae family. The results also indicate that 18% (90/478) of the species were mutagenic, of which 54% were mutagenic in the presence and absence of S9. Strains TA98 and TA100 showed a sensitivity of 93% in detecting plant extracts with mutagenic potential. However, the reliability of many reviewed studies regarding the botanical extracts may be questioned due to technical issues, such as testing being performed only in the presence or absence of S9, use of maximum doses below 5 mg/plate and lack of information on the cytotoxicity of tested doses. These methodological aspects additionally demonstrated that a discussion about the doses used in research on mixtures, such as the ones assessed with botanical extracts and the most sensitive strains employed to detect the mutagenic potential, should be included in a possible update of the guidelines designed by the regulatory agencies., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Published by Elsevier B.V.)

Deletion of microsomal prostaglandin E 2 synthase-1 (mPGES-1) inhibits inflammation and protects against atherosclerotic vascular diseases but displayed variable influence on pathologic cardiac remodeling. Overactivation of β -adrenergic receptors ( β -ARs) causes heart dysfunction and cardiac remodeling, whereas the role of mPGES-1 in β -AR-induced cardiac remodeling is unknown. Here we addressed this question using mPGES-1 knockout mice, subjecting them to isoproterenol, a synthetic nonselective agonist for β -ARs, at 5 or 15 mg/kg per day to induce different degrees of cardiac remodeling in vivo. Cardiac structure and function were assessed by echocardiography 24 hours after the last of seven consecutive daily injections of isoproterenol, and cardiac fibrosis was examined by Masson trichrome stain in morphology and by real-time polymerase chain reaction for the expression of fibrosis-related genes. The results showed that deletion of mPGES-1 had no significant effect on isoproterenol-induced cardiac dysfunction or hypertrophy. However, the cardiac fibrosis was dramatically attenuated in the mPGES-1 knockout mice after either low-dose or high-dose isoproterenol exposure. Furthermore, in vitro study revealed that overexpression of mPGES-1 in cultured cardiac fibroblasts increased isoproterenol-induced fibrosis, whereas knocking down mPGES-1 in cardiac myocytes decreased the fibrogenesis of fibroblasts. In conclusion, mPGES-1 deletion protects against isoproterenol-induced cardiac fibrosis in mice, and targeting mPGES-1 may represent a novel strategy to attenuate pathologic cardiac fibrosis, induced by β -AR agonists. SIGNIFICANCE STATEMENT: Inhibitors of microsomal prostaglandin E 2 synthase-1 (mPGES-1) are being developed as alternative analgesics that are less likely to elicit cardiovascular hazards than cyclooxygenase-2 selective nonsteroidal anti-inflammatory drugs. We have demonstrated that deletion of mPGES-1 protects inflammatory vascular diseases and promotes post-myocardial infarction survival. The role of mPGES-1 in β-adrenergic receptor-induced cardiomyopathy is unknown. Here we illustrated that deletion of mPGES-1 alleviated isoproterenol-induced cardiac fibrosis without deteriorating cardiac dysfunction. These results illustrated that targeting mPGES-1 may represent an efficacious approach to the treatment of inflammatory cardiovascular diseases., (Copyright © 2020 by The Author(s).)

Nicotine is the primary psychoactive chemical in both traditional and electronic cigarettes (e-cigarettes). Nicotine levels in both traditional cigarettes and e-cigarettes are an important concern for public health. Nicotine exposure due to e-cigarette use is of importance primarily due to the addictive potential of nicotine, but there is also concern for nicotine poisoning in e-cigarette users. Nicotine concentrations in e-liquids vary widely. Additionally, there is significant genetic variability in the rate of metabolism of nicotine due to polymorphisms of CYP2A6, the enzyme responsible for the metabolism of approximately 80% of nicotine. Recent studies have shown CYP2A6 activity is also reduced by aromatic aldehydes such as those added to e-liquids as flavoring agents, which may increase nicotine serum concentrations. However, the impacts of flavored e-liquids on CYP2A6 activity are unknown. In this study, we investigated the impact of three flavored e-liquids on microsomal recombinant CYP2A6. Microsomal recombinant CYP2A6 was challenged at e-liquid concentrations ranging up to 0.125% (v/v) and monitored for metabolic activity using a probe molecule approach. Two e-liquids exhibited dose-dependent inhibition of CYP2A6 activity. Mass spectrometry was conducted to identify flavoring agents in flavored e-liquids that inhibited CYP2A6. Microsomal recombinant CYP2A6 was subsequently exposed to flavoring agents at concentrations ranging from 0.03 μM to 500 μM. Cinnamaldehyde and benzaldehyde were found to be the most potent inhibitors of microsomal CYP2A6 of the flavoring agents tested, with identified IC 50 values of 1.1 μM and 3.0 μM, respectively. These data indicate certain aromatic aldehyde flavoring agents are potent inhibitors of CYP2A6, which may reduce nicotine metabolism in vivo. These findings indicate an urgent need to evaluate the effects of flavoring agents in e-cigarette liquids on the pharmacokinetics of nicotine in vivo.

In this study we performed the comprehensive pharmacological analysis of two stereoisomers of 4-chloro-meta-cresol (4CMC), a popular ryanodine receptor (RyR) agonist used in muscle research. Experiments investigating the Ca 2+ -releasing action of the isomers demonstrated that the most potent isomer was 4-chloro-orto-cresol (4COC) (EC 50 = 55 ± 14 μM), although 3-chloro-para-cresol (3CPC) was more effective, as it was able to induce higher magnitude of Ca 2+ flux from isolated terminal cisterna vesicles. Nevertheless, 3CPC stimulated the hydrolytic activity of the sarcoplasmic reticulum ATP-ase (SERCA) with an EC 50 of 91 ± 17 μM, while 4COC affected SERCA only in the millimolar range (IC 50 = 1370 ± 88 μM). IC 50 of 4CMC for SERCA pump was 167 ± 8 μM, indicating that 4CMC is not a specific RyR agonist either, as it activated RyR in a similar concentration (EC 50 = 121 ± 20 μM). Our data suggest that the use of 4COC might be more beneficial than 4CMC in experiments, when Ca 2+ release should be triggered through RyRs without influencing SERCA activity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing interest., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Synthetic cannabinoid receptor agonists (SCRAs) possess high abuse liability and complex toxicological profiles, making them serious threats to public health. EG-018 is a SCRA that has been detected in both illicit products and human samples, but it has received little attention to date. The current studies investigated EG-018 at human CB 1 and CB 2 receptors expressed in HEK293 cells in [ 3 H]CP55,940 competition binding, [ 35 S]GTPγS binding and forskolin-stimulated cAMP production. EG-018 was also tested in vivo for its ability to produce cannabimimetic and abuse-related effects in the cannabinoid tetrad and THC drug discrimination, respectively. EG-018 exhibited high affinity at CB 1 (21 nM) and at CB 2 (7 nM), but in contrast to typical SCRAs, behaved as a weak partial agonist in [ 35 S]GTPγS binding, exhibiting lower efficacy but greater potency, than that of THC at CB 1 and similar potency and efficacy at CB 2 . EG-018 inhibited forskolin-stimulated cAMP with similar efficacy but lower potency, compared to THC, which was likely due to high receptor density facilitating saturation of this signaling pathway. In mice, EG-018 (100 mg/kg, 30 min) administered intraperitoneally (i.p.) did not produce effects in the tetrad or drug discrimination nor did it shift THC's ED 50 value in drug discrimination when administered before THC, suggesting EG-018 has negligible occupancy of brain CB 1 receptors following i.p. administration. Following intravenous (i.v.) administration, EG-018 (56 mg/kg) produced hypomotility, catalepsy, and hypothermia, but only catalepsy was blocked by the selective CB 1 antagonist rimonabant (3 mg/kg, i.v.). Additional studies of EG-018 and its structural analogues could provide further insight into how cannabinoids exert efficacy through the cannabinoid receptors., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Recently, we identified a novel fatty acid amide hydrolase (FAAH) inhibitor, PKM-833 [(R)-N-(pyridazin-3-yl)-4-(7-(trifluoromethyl)chroman-4-yl)piperazine-1-carboxamide]. The aim of the present study is to characterize the pharmacological profile of PKM-833 in vitro and in vivo. PKM-833 showed potent inhibitory activities against human and rat FAAH with IC 50 values of 8.8 and 10 nmol/L, respectively, 200-fold more selectivity against other 137 molecular targets, and irreversible mode of action. In pharmacokinetic and pharmacodynamic studies, PKM-833 showed excellent brain penetration and good oral bioavailability, and elevated anandamide (AEA) concentrations in the rat brain. These data indicate that PKM-833 is a potent, selective, orally active, and brain-penetrable FAAH inhibitor. In behavioral studies using rat models, PKM-833 significantly attenuated formalin-induced pain responses (3 mg/kg) and improved mechanical allodynia in complete freund's adjuvant (CFA)-induced inflammatory pain (0.3-3 mg/kg). On the other hand, PKM-833 did not show the analgesic effects against mechanical allodynia in chronic constriction injury (CCI)-induced neuropathic pain up to 30 mg/kg. Regarding side effects, PKM-833 had no significant effects on catalepsy and motor coordination up to 30 mg/kg. These results indicate that PKM-833 is a useful pharmacological agent that can be used to investigate the role of FAAH and may have therapeutic potential for the treatment of inflammatory pain without undesirable side effects., (© 2020 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

Using activity guided purification, four known compounds, sesquiterpene atractylenolide III (1), and the polyacetylenes 14-acetoxy-12-senecioyloxytetradeca-2E,8E,10E-trien-4,6-diyn-1-ol (2), 14-acetoxy-12-α-methylbutyl-2E,8E,10E-trien-4,6-diyn-1-ol (3), and 14-acetoxy-12-β -methylbutyl-2E,8E,10E-trien-4,6-diyn-1-ol (4), were isolated from a traditional herbal medicine, Atractylodes rhizome. Structurally similar 3 and 4 (3/4 mixture) were obtained as a mixture. In intact Chinese hamster ovary (CHO) K1 cell assays, 1, 2, and a 3/4 mixture selectively inhibited cholesterol [ 14 C]oleate synthesis from [ 14 C]oleate with IC 50 values of 73.5 µM, 35.4 µM, and 10.2 µM, respectively, without any effects on cytotoxicity. As a potential target of these inhibitors involved in cholesteryl ester (CE) synthesis, effects on sterol O-acyltransferase (SOAT) activity were investigated using microsomes prepared from CHO-K1 cells as an enzyme source. Hence, these compounds inhibit SOAT activity with IC 50 values (211 µM for 1, 29.0 µM for 2, and 11.8 µM for 3/4 mixture) that correlate well with those measured from intact cell assays. Our results strongly suggest that these compounds inhibit CE synthesis by blocking SOAT activity in CHO-K1 cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Novel purine and purine isosteres containing a ferrocene motif and 4,1-disubstituted ( 11a - 11c , 12a - 12c , 13a - 13c , 14a - 14c , 15a - 15c , 16a , 23a - 23c , 24a - 24c , 25a - 25c ) and 1,4-disubstituted ( 34a - 34c and 35a - 35c ) 1,2,3-triazole rings were synthesized. The most potent cytotoxic effect on colorectal adenocarcinoma (SW620) was exerted by the 6-chloro-7-deazapurine 11c (IC 50 = 9.07 µM), 6-chloropurine 13a (IC 50 = 14.38 µM) and 15b (IC 50 = 15.50 µM) ferrocenylalkyl derivatives. The N -9 isomer of 6-chloropurine 13a containing ferrocenylmethylene unit showed a favourable in vitro physicochemical and ADME properties including high solubility, moderate permeability and good metabolic stability in human liver microsomes.

The side effects of nonsteroidal anti-inflammatory drugs (NSAIDs) in the cardiovascular system mainly result from its inhibitory effect on cyclooxygenase-2 (COX-2). Since NSAIDs are one of the most commonly used anti-inflammatory drugs in the clinic, it is necessary to identify new anti-inflammatory drugs that are safer than NSAIDs. Nardosinanone N (NAN), a compound isolated from the roots and rhizomes of Nardostachys chinensis, was evaluated for its anti-inflammatory effects using the lipopolysaccharide (LPS)-stimulated RAW264.7 cell line and rat peritoneal macrophage models. First, we found that NAN down regulated the levels of nitric oxide (NO), inducible nitric oxide synthase (iNOS) and prostaglandin E 2 (PGE 2 ), but not cyclooxygenase-2 (COX-2). Additionally, NAN reduced the M1 macrophage phenotype and increased the M2 macrophage phenotype. Furthermore, mechanistic studies showed that NAN activated the nuclear factor-erythroid 2 -related factor 2 (Nrf2) signaling pathway, which, in turn, increased the expression of antioxidant protein heme oxygenase-1 (HO-1) to achieve its anti-inflammatory effect. Finally, Nrf2 siRNA and the HO-1 inhibitor significantly attenuated the anti-inflammatory effect of NAN. More interestingly, we found that NAN did not affect COX-2 expression and activity but reduced the PGE 2 concentration by selective inhibition of microsomal prostaglandin E synthase-1 (mPGES-1). In conclusion, NAN may be a new anti-inflammatory drug that has fewer side effects than NSAIDs and can be a new potential Nrf2 activator and mPGES-1 inhibitor., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Effect-based analyses are being recognized as excellent tools to a comprehensive and reliable water quality evaluation to complement physical and chemical parameters. The Salmonella/microsome mutagenicity test was introduced in the São Paulo State water quality-monitoring program in 1999 and waters from 104 sites used to the production of drinking water were analyzed. Samples were tested after organic extraction, using the microsuspension version of the Salmonella/microsome assay with strains TA98 and TA100 with and without S9-mammalian metabolic system. Of the 1720 water samples analyzed in 20 years, 20% were positive; TA98 was the most sensitive strain, detecting alone 99%. Results were presented in hazard categories to facilitate water managers' understanding and general public communication. Hot spots of mutagenicity were identified, and pollution sources investigated. A flow scheme with instructions of how to proceed in case of mutagenic samples was developed and implemented in the monitoring program. Enforcement actions were taken to reduce exposure of humans and aquatic biota to mutagenic compounds. The results presented provide scientific basis for the incorporation of the Salmonella/microsome assay in a regulatory framework, and to guide water-quality managers. The inclusion of a mutagenicity assay using standardized conditions proved to be an opportunity to improve the quality of water, and the strategy presented here could be applied by any environmental agency around the world. Environ. Mol. Mutagen. 61:200-211, 2020. © 2019 Wiley Periodicals, Inc., (© 2019 Wiley Periodicals, Inc.)

Norovirus (NV), is the most common cause of acute gastroenteritis worldwide. To date, there is no specific anti-NV drug or vaccine to treat NV infections. In this study, we evaluated the inhibitory effect of different stilbene-based analogs on RNA genome replication of human NV (HNV) using a virus replicon-bearing cell line (HG23). Initial screening of our in-house chemical library against NV led to the identification of a hit containing stilbene scaffold 5 which on initial optimization gave us a vinyl stilbene compound 16c (EC 50  = 4.4 μM). Herein we report our structure-activity relationship study of the novel series of vinyl stilbene analogs that inhibits viral RNA genome replication in a human NV-specific manner. Among these newly synthesized compounds, several amide derivatives of vinyl stilbenes exhibited potent anti-NV activity with EC 50 values ranging from 1 to 2 μM. A trans-vinyl stilbenoid with an appended substituted piperazine amide (18k), exhibited potent anti-NV activity and also displayed favorable metabolic stability. Compound 18k demonstrated an excellent safety profile, the highest suppressive effect, and was selective for HNV replication via a viral RNA polymerase-independent manner. Its potential host-targeting antiviral mechanism was further supported by specific activation of heat shock factor 1-dependent stress-inducible pathway by 18k. These results suggest that 18k might be a promising lead compound for developing novel NV inhibitors with the novel antiviral mechanism., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Atmospheric particulate matter (PM) organic fractions from urban centers are frequently mutagenic for the Salmonella/microsome assay. This mutagenicity is related to both primary and secondary pollutants, and meteorological conditions have great influence on the secondary pollutant's formation. Our objective was to compare the mutagenicity of atmospheric total suspended particulates (TSP) from three cities with marked different meteorological conditions and TSP concentrations: Limeira (Brazil) with 99.0 μg/m 3 , Stockholm (Sweden) with 6.2 μg/m 3 , and Kyoto (Japan) with 28.0 μg/m 3 . For comparison, we used the same batch of filters, sample extraction method, and Salmonella/microsome testing protocol with 11 strains of Salmonella with and without metabolic activation. Samples were collected during winter and pooled into one single extract representing each city. All samples were mutagenic for all tested strains, except for TA102. Based on the strain's selectivity, nitroarenes, polycyclic aromatic hydrocarbons, and aromatic amines play a predominant role in the mutagenicity of these samples. The mutagenic potencies expressed by mass of extracted organic material (EOM; revertants/μg EOM) were similar (~twofold difference) among the cities, despite differences in meteorological conditions and pollution sources. In contrast, the mutagenic potencies expressed by air volume (rev/m 3 ) varied ~20-fold, with Limeira > Kyoto ≈ Stockholm. These results are the first systematic assessment of air mutagenicity from cities on three continents using the same protocols. The results confirm that the mutagenic potency expressed by EOM mass is similar regardless of continent of origin, whereas the mutagenic potency expressed by air volume can vary by orders of magnitude. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc., (© 2019 Wiley Periodicals, Inc.)

Staphylococcus aureus is one of the most common pathogens causing hospital-acquired and community-acquired infections. Methicillin-resistant S. aureus (MRSA)-formed biofilms in wounds are difficult to treat with conventional antibiotics. By targeting FabB/FabF of bacterial fatty acid synthases, platensimycin (PTM) was discovered to act as a promising natural antibiotic against MRSA infections. In this study, PTM and its previously synthesized sulfur-Michael derivative PTM-2t could reduce over 95% biofilm formation by S. aureus ATCC 29213 when used at 2 μg/mL in vitro. Topical application of ointments containing PTM or PTM-2t (2 × 4 mg/day/mouse) was successfully used to treat MRSA infections in a BABL/c mouse burn wound model. As a potential prodrug lead, PTM-2t showed improved in vivo efficacy in a mouse peritonitis model compared with PTM. Our study suggests that PTM and its analogue may be used topically or locally to treat bacterial infections. In addition, the use of prodrug strategies might be instrumental to improve the poor pharmacokinetic properties of PTM.

Lithocholic acid is a cytotoxic bile acid oxidized at the C-3 position by human cytochrome P450 3A (CYP3A) to form 3-ketocholanoic acid, but it is not known whether this metabolite is cytotoxic. Tocotrienols, in their various isomeric forms, are vitamin E analogues. In the present study, the hypothesis to be tested is that tocotrienols inhibit CYP3A-catalyzed lithocholic acid 3-oxidation, thereby influencing lithocholic acid cytotoxicity. Our enzyme catalysis experiments indicated that human recombinant CYP3A5 in addition to CYP3A4, liver microsomes, and intestinal microsomes catalyzed lithocholic acid 3-oxidation to form 3-ketocholanoic acid. Liver microsomes with the CYP3A5*1/*3 and CYP3A5*3/*3 genotypes were associated with decreased lithocholic acid 3-oxidation. α-Tocotrienol, γ-tocotrienol, δ-tocotrienol, and a tocotrienol-rich vitamin E mixture, but not α-tocopherol (a vitamin E analogue), differentially inhibited lithocholic acid 3-oxidation catalyzed by liver and intestinal microsomes and recombinant CYP3A4 and CYP3A5. Compared to lithocholic acid 3-oxidation, CYP3A-catalyzed testosterone 6β-hydroxylation was inhibited to a lesser extent by α-tocotrienol, γ-tocotrienol, δ-tocotrienol, and a tocotrienol-rich vitamin E mixture. δ-Tocotrienol inhibited lithocholic acid 3-oxidation by a mixed mode. Like lithocholic acid, 3-ketocholanoic acid was also cytotoxic in human intestinal and liver cell models. δ-Tocotrienol decreased the extent of lithocholic acid 3-oxidation and this inhibition was associated with enhanced cytotoxicity in LS180 cells treated with δ-tocotrienol and lithocholic acid. Overall, vitamin E analogues inhibited in vitro lithocholic acid 3-oxidation in an isomer-dependent manner, with inhibition occurring with tocotrienols, but not α-tocopherol. The enhanced lithocholic acid toxicity by δ-tocotrienol in a human intestinal cell model warrants future investigations in vivo., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The endoplasmic reticulum (ER) contains both α-glucosidases and α-mannosidases which process the N-linked oligosaccharides of newly synthesized glycoproteins and thereby facilitate polypeptide folding and glycoprotein quality control. By acting as structural mimetics, iminosugars can selectively inhibit these ER localized α-glycosidases, preventing N-glycan trimming and providing a molecular basis for their therapeutic applications. In this study, we investigate the effects of a panel of nine iminosugars on the actions of ER luminal α-glucosidase I and α-glucosidase II. Using ER microsomes to recapitulate authentic protein N-glycosylation and oligosaccharide processing, we identify five iminosugars that selectively inhibit N-glycan trimming. Comparison of their inhibitory activities in ER microsomes against their effects on purified ER α-glucosidase II, suggests that 3,7a-diepi-alexine acts as a selective inhibitor of ER α-glucosidase I. The other active iminosugars all inhibit α-glucosidase II and, having identified 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) as the most effective of these compounds, we use in silico modeling to understand the molecular basis for this enhanced activity. Taken together, our work identifies the C-3 substituted pyrrolizidines casuarine and 3,7a-diepi-alexine as promising "second-generation" iminosugar inhibitors., (© The Author(s) 2019. Published by Oxford University Press.)

New pyrazole derivatives 2-5 were synthesized and evaluated for their COX-1 and COX-2 inhibitory activity in vitro. All compounds showed good inhibitory activity at a nanomolar level and most compounds exhibited selectivity towards COX-2 inhibition. Compounds 2a, 3b, 4a, 5b and 5e exhibited IC 50 towards COX-2 enzyme of 19.87, 39.43, 61.24, 38.73 and 39.14 nM, respectively. Furthermore, compounds 3b, 4a, 5b and 5e exhibited a selectivity index of 22.21, 14.35, 17.47 and 13.10, respectively. The most active compounds were further subjected to in vivo anti-inflammatory assay. The tested compounds showed better or comparable activity to celecoxib as positive control. In order to explore their binding mode and selectivity behaviour, molecular docking in the active site of COX-2 was carried out for these derivatives. Analysis of the docked poses of the compounds showed that they adopt similar conformations to the highly selective COX-2 inhibitor, SC-558. The docking pose of compound 3b was confirmed by molecular dynamics. All the tested compounds exhibited potent inhibitory effect on the production of PGE 2 , in addition to their inhibition of COX-2 enzyme., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Tuberculosis (TB) remains a significant global health problem for which new therapeutic options are sorely needed. The ability of the causative agent, Mycobacterium tuberculosis, to reside within host macrophages and form biofilm-like communities contributes to the persistent and drug-tolerant nature of the disease. Compounds that can prevent or reverse the biofilm-like phenotype have the potential to serve alongside TB antibiotics to overcome this tolerance, and decrease treatment duration. Using Mycobacterium smegmatis as a surrogate organism, we report the identification of two new 2-aminoimidazole compounds that inhibit and disperse mycobacterial biofilms, work synergistically with isoniazid and rifampicin to eradicate preformed M. smegmatis biofilms in vitro, are nontoxic toward Galleria mellonella, and exhibit stability in mouse plasma., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Human dihydroorotate dehydrogenase (hDHODH) is a flavin-dependent enzyme essential to pyrimidine de novo biosynthesis, which serves as an attractive therapeutic target for the treatment of autoimmune disorders. A novel series of hDHODH inhibitors was developed based on a lead which was obtained by a medicinal chemistry exploration. Most compounds showed moderate to significant potency against hDHODH, compounds 5d, 5e, and 6a effectively inhibited the activities of hDHODH with IC 50 values from 0.9 to 2.8 μM. Further studies showed that compound 5e also effectively suppressed proliferation of the activated PBMCs (IC 50  = 20.35 μM). Surprisingly, compound 5e also showed anti-pulmonary fibrotic activity similar to that of pirfenidone in vitro assay. Therefore, compound 5e might have potential to be developed as a novel hDHODH inhibitors for autoimmune diseases therapy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

This Virtual Special Issue of Mutation Research is dedicated to Professor Bruce N. Ames in recognition of his 90 th birthday in December 2018. His pioneering work in the field of chemical mutagenesis resulted in the well-known Ames Salmonella/mammalian-microsome mutagenicity assay that has played a pivotal role since the 1970s in the field of genetic toxicology. The assay is usually referred to as the Ames test and was gradually developed by improving the sensitivity of the test based on available scientific discoveries. When a chemical is determined to be a mutagen in the Ames test it has the potential of also being a carcinogen based on the somatic mutation theory of carcinogenesis. For nearly 20 years, I was responsible for running the Ames mutagenicity testing laboratory at SRI International on a contractual basis with commercial and government funding. Now I feel privileged having been given the opportunity to provide a historical overview of how the Ames test was developed., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Ayahuasca is a beverage used in religious rituals of indigenous and nonindigenous groups, and its therapeutic potential has been investigated. Ayahuasca is obtained by decoction of the Banisteriopsis caapi that contains β-carbolines (harmine, harmaline, and tetrahydroharmine) plus Psychotria viridis that contains N,N-dimethyltryptamine. Although plants used in folk medicine are recognized as safe, many of them have genotoxic potential. The Salmonella/microsome assay is usually the first line of the mutagenicity evaluation of products intended for therapeutic use. Our objective was to evaluate the mutagenicity of ayahuasca beverage and their constituents using the Salmonella/microsome assay with TA98 and TA100. We analyzed two ayahuasca samples, and also beverage samples prepared each individual plant P. viridis and B. caapi. Harmine and harmaline were also tested. All beverage samples were chemically characterized and both ayahuasca samples could be considered representative of the beverages consumed in religious rituals. Both ayahuasca samples were mutagenic for TA98 and TA100 with and without S9, with similar potencies. The beverage obtained from P. viridis was not mutagenic, and beverage obtained from B. caapi was mutagenic for TA98 with and without S9. Harmine was nonmutagenic and harmaline was mutagenic only for TA98 without S9. Harmaline fully explain the mutagenicity observed with TA98 without S9 of both ayahuasca samples and the B. caapi beverage samples. We conclude that the ayahuasca samples are mutagenic and this effect is partially explained by harmaline, one of the β-carbolines present in the beverage. Other mutagenic compounds seem to be present and need to be further investigated. Environ. Mol. Mutagen. 60:269-276, 2019. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

In the presence of ciprofloxacin (CPFX), free radical adduct formation was demonstrated in rat cerebral microsomes using a spin trap α-(4-pyridyl-1-oxide)-N- tert -butyl-nitrone by electron spin resonance spectroscopy. Active microsomes, dihydronicotinamide-adenine dinucleotide phosphate, and ciprofloxacin were necessary for the formation of a spin trap/radical adduct. Adduct formation increased dose-dependently at 0.5-1 mM CPFX concentration for 180 min, and 0.3-1 mM concentration level for 240 min. The addition of SKF 525A, ZnCl 2 or desferrioxamine to the incubation system caused complete inhibition of the radical formation. However, pretreatment of microsomal system with superoxide dismutase (SOD) did not induce any protective effect. Induction of lipid peroxidation, and depletion of thiol levels by CPFX were also shown in the system. These results strongly suggested that CPFX produces free radical(s) in the cerebral microsomes of rats.

A potential CYP4B1 suicide gene application in engineered T-cell treatment of blood cancers has revived interest in the use of 4-ipomeanol (IPO) in gene-directed enzyme prodrug therapy, in which disposition of the administered compound may be critical. IPO contains one chiral center at the carbon bearing a secondary alcohol group; it was of interest to determine the effect of stereochemistry on 1) CYP4B1-mediated bioactivation and 2) (UGT)-mediated glucuronidation. First, ( R )-IPO and ( S )-IPO were synthesized and used to assess cytotoxicity in HepG2 cells expressing rabbit CYP4B1 and re-engineered human CYP4B1, where the enantiomers were found to be equipotent. Next, a sensitive UPLC-MS/MS assay was developed to measure the IPO-glucuronide diastereomers and product stereoselectivity in human tissue microsomes. Human liver and kidney microsomes generated ( R ) - and ( S )-IPO-glucuronide diastereomers in ratios of 57:43 and 79:21, respectively. In a panel of 13 recombinantly expressed UGTs, UGT1A9 and UGT2B7 were the major isoforms responsible for IPO glucuronidation. ( R )-IPO-glucuronide diastereoselectivity was apparent with each recombinant UGT, except UGT2B15 and UGT2B17, which favored the formation of ( S )-IPO-glucuronide. Incubations with IPO and the UGT1A9-specific chemical inhibitor niflumic acid significantly decreased glucuronidation in human kidney, but only marginally in human liver microsomes, consistent with known tissue expression patterns of UGTs. We conclude that IPO glucuronidation in human kidney is mediated by UGT1A9 and UGT2B7. In human liver, it is mediated primarily by UGT2B7 and, to a lesser extent, UGT1A9 and UGT2B15. Overall, the lack of pronounced stereoselectivity for IPO's bioactivation in CYP4B1-transfected HepG2 cells, or for hepatic glucuronidation, suggests the racemate is an appropriate choice for use in suicide gene therapies., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

The use of chemical substances for the management of fish farming activities may compromise the quality of the tank water itself and of water bodies that receive the effluents. As studies that assess the environmental effect caused by pisciculture are scarce, the present study aimed at evaluating the water quality in two fish farms in the region of Grande Dourados, Brazil, from the site of water collection to the site of water disposal. The tools used for this purpose were the analysis of land use and cover and the determination of physical, chemical, and biological parameters of water samples. Maps of land use and cover were created, and water samples were collected at four sampling sites in two fish farms. The Allium cepa test, assays with Astyanax lacustris, and the Salmonella/microsome assay were performed. In addition, physical and chemical parameters were measured and metal and emerging contaminants in the water samples were investigated. The A. lacustris demonstrated the genotoxicity and the Salmonella/microsome assay suggested the mutagenic potential of water samples from the fish farms and indicated higher genotoxicity in the disposal tanks than in the collection tanks of the Brilhante fish farm. However, all the samples at the Dourados fish farm were genotoxic, and mutagenicity was shown to start at the water collection site. With regard to the A. cepa test, there was no statistical difference between the collection sites in both fish farms. Moreover, the observed genetic damage may be associated with the presence of metals and emerging contaminants in the water samples, which suggests that these chemicals have potential genotoxic and mutagenic effects that are related to the type of land use and cover in the area of the region studied. Considering that contaminated waters can potentially disturb the structure and functioning of natural ecosystems, the present study demonstrated the importance of treating fish farm effluent to minimize the negative effect of this activity on water bodies.

We provide a kinetic characterization of (Na + , K + )-ATPase activity in a posterior gill microsomal fraction from a hololimnetic population of the diadromous Amazon River shrimp Macrobrachium amazonicum. Sucrose density gradient centrifugation reveals two distinct membrane fractions showing considerable (Na + , K + )ATP-ase activity, but also containing other microsomal ATPases. Only a single immune-reactive (Na + , K + )-ATPase with M r of ≈110 kDa is present that hydrolyzes ATP with V M  = 130.3 ± 4.8 nmol Pi min -1 mg protein -1 and K 0.5  = 0.065 ± 0.00162 mmol L -1 , exhibiting site-site interactions. Stimulation by Na + (V M  = 127.5 ± 5.3 nmol Pi min -1 mg protein -1 , K 0.5  = 5.3 ± 0.42 mmol L -1 ), Mg 2+ (V M  = 130.6 ± 6.8 nmol Pi min -1 mg protein -1 , K 0.5  = 0.33 ± 0.042 mmol L -1 ), K + (V M  = 126.7 ± 7.7 nmol Pi min -1 mg protein -1 , K 0.5  = 0.65 ± 0.0079 mmol L -1 ) and NH 4 + (V M  = 134.5 ± 8.6 nmol Pi min -1 mg protein -1 , K 0.5  = 1.28 ± 0.44 mmol L -1 ) also obeys cooperative kinetics. Ouabain (K I  = 0.18 ± 0.058 mmol L -1 ) inhibits total ATPase activity by ≈70%. This study reveals considerable differences in the kinetic characteristics of the gill (Na + , K + )-ATPase in a hololimnetic population that appear to result from the adaptation of diadromous Macrobrachium amazonicum populations to different limnic habitats., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Dysregulation of neuronal intracellular Ca 2+ homeostasis can play a crucial role in many neurotoxic effects, including impaired brain development and behavioral dysfunctions. This study examined 40 suspected neurotoxicants from different chemical classes for their capacity to alter Ca 2+ release and uptake from rat cortical microsomes. First, ten suspected neurotoxicants have been tested using a well-established cuvette-based Ca 2+ flux assay. Five out of ten compounds (TOCP, endosulfan, PCB-95, chlorpyrifos and BDE-49) showed a significant, concentration-dependent alteration of Ca 2+ release and uptake in adult rat cortical microsomes. The original cuvette assay was downscaled and customized to a fast, higher throughput microplate method and the 40 suspected neurotoxicants were screened for their effects on intracellular Ca 2+ homeostasis. In decreasing order of potency, the 15 test compounds that showed the strongest alteration of Ca 2+ levels in adult rat microsomes were TOCP, endosulfan, BDE-49, 6-OH-BDE-47, PCB-95, permethrin, alpha-cypermethrin, chlorpyrifos, bioallethrin, cypermethrin, RDP, DEHP, DBP, BDE-47, and PFOS. Results from co-exposure experiments with selective inhibitors suggested that for some compounds Ca 2+ releasing effects could be attributed to RyR activation (PFOS, DBP, and DEHP) or to SERCA inhibition (a potential novel mechanism of action for all four tested pyrethroid insecticides). The effects of the two most potent compounds, endosulfan and TOCP, were not blocked by any of the inhibitors tested, indicating other possible mechanism of action. For all other potent test compounds, a combined effect on RyR, IP 3 R, and/or SERCA has been observed. PFOS and 6-OH-BDE-47 caused increased Ca 2+ release from adult but not from neonatal rat brain microsomes, indicating age-dependent difference in susceptibility to these test compounds. The current study suggests that the neurotoxic potential of compounds belonging to different chemical classes could partly be attributed to the effects on intracellular Ca 2+ release and uptake. Although further validation is required, the downscaled method developed in this study presents technical advance that could be used for the future screening of suspected intracellular Ca 2+ disruptors., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Leucas zeylanica (L.) W.T. Aiton is a popular, multi-purpose medicinal plant in Sri Lanka but the pharmacological potential and the chemical profile have not been systematically investigated to understand and rationalize the reported ethnobotanical significance., Aim of the Study: The present study was undertaken to scientifically validate the traditional usage of this plant for the treatment of inflammatory conditions, gout and microbial infections. Inhibition of 5-lipoxygenase (5-LO), microsomal prostaglandin E 2 synthase (mPGES)-1 and xanthine oxidase (XO) by different extracts of L. zeylanica was investigated to determine the anti-inflammatory and anti-gout activity, respectively. The antibacterial and antifungal activities were also studied and the relevant constituents in the bioactive extracts were tentatively identified., Materials and Methods: Cell-free and/or cell-based assays were employed in order to investigate the effects of the extracts against the activity of human 5-LO, mPGES-1 and XO as well as to assess antioxidant properties. The antibacterial activity of the extracts was determined by the broth micro-dilution method against Gram positive and Gram negative bacteria including methicillin-resistant Staphylococcus aureus while the agar dilution method was employed to determine the anti-Candida activity. Gas chromatography coupled to mass spectrometric (GC-MS) analysis enabled the characterization of secondary metabolites in the extracts., Results: The dichloromethane extract of L. zeylanica efficiently inhibited 5-LO activity in stimulated human neutrophils (IC 50 = 5.5 µg/mL) and isolated human 5-LO and mPGES-1 (IC 50 = 2.2 and 0.4 µg/mL). Potent inhibition of XO was observed by the same extract (IC 50 = 47.5 μg/mL), which is the first report of XO-inhibitory activity of a Sri Lankan medicinal plant. Interestingly, significant radical scavenging activity was not observed by this extract. Only the n-hexane extract exhibited antibacterial activity against Staphylococcus aureus and Staphylococcus saprophyticus with a MIC of 250 µg/mL while the anti-Candida activity was moderate. GC-MS analysis revealed the presence of phytosterols, fatty acids, sesquiterpenes, diterpenes and several other types of secondary metabolites., Conclusions: Potent inhibition of 5-LO, mPGES-1 and XO rationalizes the ethnopharmacological use of L. zeylanica as anti-inflammatory and anti-gout remedy. Interestingly, the antimicrobial activities were not prominent, despite its wide utility as an antimicrobial medication., (Copyright © 2018 Elsevier B.V. All rights reserved.)

1. Quercetin is a dietary flavonoid has extremely low water solubility and found to possess CYP3A inhibitory activity. The purpose of the present study was to evaluate the effect of quercetin and quercetin nanoparticles (NQC) on the pharmacokinetics of bromocriptine (BRO) in rats. 2. NQC prepared by antisolvent precipitation method and characterized by SEM and dissolution test. The following methods were used in this study i.e. in vitro liver and intestinal CYP3A microsomal activity and in vitro non-everted sac method. To confirm these findings, an in vivo pharmacokinetic study was also performed. 3. The results indicate that quercetin significantly (p < 0.05) inhibited the CYP3A activity in liver and intestinal microsomes. In non-everted sac study, the intestinal transport and P app of BRO were significantly increased in NQC and quercetin groups. Furthermore, in vivo study revealed that the increased levels of C max and AUC were comparatively high in NQC pretreated group than quercetin group. In addition, pretreatment with quercetin and NQC significantly (p < 0.05) decreased the mean CL/F and Vd/F of BRO. 4. NQC pretreatment might be result in higher plasma levels of quercetin that could inhibit the CYP3A enzyme and enhanced the bioavailability of BRO.

Daidzein, one of the major soy isoflavones, has a number of beneficial bioactivities for human health. It is mainly metabolized into 7- and/or 4'-glucuronides by UDP-glucuronosyltransferase (UGT) enzymes in mammals, including humans. The present study was conducted to examine the regioselective glucuronidation of daidzein at the 7- and 4'-hydroxyl groups in the liver and intestinal microsomes of humans, monkeys, rats, and mice. Daidzein glucuronidation activities at substrate concentrations of 1.0-200 µM were assessed, and Eadie-Hofstee plots were constructed. The kinetics for 7- and 4'-glucuronidation in the liver microsomes fit the Michaelis-Menten model, except for an atypical model for 7-glucuronidation in rats and a biphasic model for 4'-glucuronidation in monkeys. These kinetics in the intestinal microsomes followed the Michaelis-Menten model, except for a biphasic model for 7-glucuronidation in mice. The CL int values for 7-glucuronidation were in the order of monkeys (49) ≫ rats (5.3) > humans (1.0) > mice (0.7) for liver microsomes, and rats (2.4) ≥ monkeys (2.2) > humans (1.0) ≥ mice (0.8) for intestinal microsomes. On the other hand, the CL int values for 4'-glucuronidation were in the order of monkeys (4.0) > mice (1.0) ≈ humans (1.0) > rats (0.4) for liver microsomes, and humans (1.0) ≫ monkeys (0.08) ≥ mice (0.07) > rats (0.05) for intestinal microsomes. These results demonstrated that the metabolic abilities of UGT enzymes toward daidzein in the liver and intestines markedly differed among humans, monkeys, rats, and mice, and suggest that species and regioselective differences are closely associated with the bioactivities of soy isoflavones.

Soil contamination enters aquatic ecosystems affecting sediment quality. The region studied is the Taquari River, Brazil, close to a site contaminated by wood preservatives, with a runoff route into the river. The first stage of the remediation process (In this article, the terms intervention and remediation have been used with slightly different meanings. We consider intervention to be the first phase of the remediation process, which aims to remove active sources) was an intervention to remove the main active sources. The Salmonella/microsome assay and polycyclic aromatic hydrocarbons (PAHs) were used to assess sediment quality in organic extracts during different intervention phases. The strains used were TA98, TA97a, and TA100 with and without S9mix (±S9). The results indicated the presence of pro-mutagens at site Ta010 (closest to the contaminated site) in all samplings, and the highest result occurred before intervention for TA100 + S9 (1,672 ± 215.9 rev/g). These values decreased during (83 ± 23.6 rev/g) and after this process (403 ± 105.9 rev/g), although the PAHs concentrations increased. Samples from this site presented PAHs with a carcinogenic potential during the assessed periods. After intervention, Ta006 (4 km downstream from Ta010) showed the most significant mutagenesis for TA100 + S9 (764 ± 230.2 rev/g) and, although the total PAHs values were lower, the species considered carcinogenic had higher concentrations. Mutagenesis predicted values of PAHs confirmed that carcinogenic species were predominantly detected by TA100, and the other PAHs by TA97a strains. Marked contaminant release to the river was observed, mainly in Ta010 at different periods. Mutagenicity and PAHs values in an internal stream, upstream from Ta010, showed a dispersion route of these agents. Thus, contamination in Ta010 and possible contribution to Ta006, after intervention, provides a warning regarding environmental quality in the region. Environ. Mol. Mutagen. 59:625-638, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most abundant and carcinogenic tobacco-specific nitrosamine in tobacco and tobacco smoke. The major metabolic pathway for NNK is carbonyl reduction to form the (R) and (S) enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) which, like NNK, is a potent lung carcinogen. The goal of this study was to characterize NNAL enantiomer formation in human lung and identify the enzymes responsible for this activity. While (S)-NNAL was the major enantiomer of NNAL formed in incubations with NNK in lung cytosolic fractions, (R)-NNAL comprised ~60 and ~95% of the total NNAL formed in lung whole cell lysates and microsomes, respectively. In studies examining the role of individual recombinant cytosolic reductase enzymes in lung NNAL enantiomer formation, AKR1C1, AKR1C2, AKR1C3, AKR1C4 and CBR1 all exhibited (S)-NNAL-formation activity. To identify the microsomal enzymes responsible for (R)-NNAL formation, 28 microsomal reductase enzymes were screened for expression by real-time PCR in normal human lung. HSD17β6, HSD17β12, KDSR, NSDHL, RDH10, RDH11 and SDR16C5 were all expressed at levels ≥HSD11β1, the only previously reported microsomal reductase enzyme with NNK-reducing activity, with HSD17β12 the most highly expressed. Of these lung-expressing enzymes, only HSD17β12 exhibited activity against NNK, forming primarily (>95%) (R)-NNAL, a pattern consistent with that observed in lung microsomes. siRNA knock-down of HSD17β12 resulted in significant decreases in (R)-NNAL-formation activity in HEK293 cells. These data suggest that both cytosolic and microsomal enzymes are active against NNK and that HSD17β12 is the major active microsomal reductase that contributes to (R)-NNAL formation in human lung., (© The Author(s) 2018. Published by Oxford University Press.)

Opioid peptides are key regulators in cellular and intercellular physiological responses, and could be therapeutically useful for modulating several pathological conditions. Unfortunately, the use of peptide-based agonists to target centrally located opioid receptors is limited by poor physicochemical (PC), distribution, metabolic, and pharmacokinetic (DMPK) properties that restrict penetration across the blood-brain barrier via passive diffusion. To address these problems, the present paper exploits fluorinated peptidomimetics to simultaneously modify PC and DMPK properties, thus facilitating entry into the central nervous system. As an initial example, the present paper exploited the Tyr 1 -ψ[( Z)CF═CH]-Gly 2 peptidomimetic to improve PC druglike characteristics (computational), plasma and microsomal degradation, and systemic and CNS distribution of Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu). Thus, the fluoroalkene replacement transformed an instable in vitro tool compound into a stable and centrally distributed in vivo probe. In contrast, the Tyr 1 -ψ[CF 3 CH 2 -NH]-Gly 2 peptidomimetic decreased stability by accelerating proteolysis at the Gly 3 -Phe 4 position.

Starting from a weak screening hit, potent and selective inhibitors of the MALT1 protease function were elaborated. Advanced compounds displayed high potency in biochemical and cellular assays. Compounds showed activity in a mechanistic Jurkat T cell activation assay as well as in the B-cell lymphoma line OCI-Ly3, which suggests potential use of MALT1 inhibitors in the treatment of autoimmune diseases as well as B-cell lymphomas with a dysregulated NF-κB pathway. Initially, rat pharmacokinetic properties of this compound series were dominated by very high clearance which could be linked to amide cleavage. Using a rat hepatocyte assay a good in vitro-in vivo correlation could be established which led to the identification of compounds with improved PK properties., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The Salmonella/microsome assay (Ames test) is the most widely used mutagenicity test for the evaluation of pure chemicals and environmental samples. There are several versions of protocols available in the literature, including those that reduce the amount of sample needed for testing with liquid and agar media. The microsuspension version of the Salmonella/microsome assay is more sensitive than the standard protocol. It is performed using 5-times concentrated bacteria and less sample and S9 mixture, but still uses conventional Petri dishes (90 × 15 mm). It has been extensively used for environmental sample testing, including in effect-directed analysis (EDA). The objective of this study was to miniaturize the microsuspension assay using 12-well microplates instead of the conventional plates. For validation of this miniaturization, thirteen mutagenic compounds were tested using three Salmonella strains that were selected based on their different spontaneous reversion frequencies (low, medium, and high). The conditions of the miniaturized procedure were made as similar as possible to the microsuspension protocol, using the same testing design, metabolic activation, and data interpretation, and the tests were conducted in parallel. The miniaturized plate assay (MPA) and microsuspension procedures provided similar sensitivities although MPA is less laborious and require less sample and reagents, thereby reducing overall costs. We conclude that the MPA is a promising tool and can be particularly suitable for environmental studies such as EDA or monitoring programs. Environ. Mol. Mutagen. 59:488-501, 2018. © 2018 Wiley Periodicals, Inc., (Copyright © 2018 Wiley Periodicals, Inc.)

In the present study, the antigenotoxic activity of poly(d,l-lactic- co-glycolic acid) (PLGA) nanoparticles (NPs) loaded with caffeic acid phenethyl ester (CAPE) was investigated in comparison to free CAPE using the Ames Salmonella/microsome assay. Additionally, to elucidate the impacts of the type of solvent effect on antigenotoxic activity, the following systems were tested: CAPE in water (poor solvent), ethyl alcohol (good solvent), and PLGA NPs (unknown). The effect of the NP system on solubility was investigated for the first time by assessing the antigenotoxic potential. In this study, the CAPE/PLGA NPs were synthesized using an oil-in-water (o/w) single-emulsion solvent evaporation method with an average size of 206.2 ± 1.2 nm, ζ potential of -19.8 ± 2.5 mV, encapsulation efficiency of 87.2 ± 2.5%, and drug loading of 53.3 ± 1.8%. According to the results of the antigenotoxic activity, the highest antimutagenic activity in both applied strains was found for CAPE in ethanol, and the lowest activity was detected for CAPE in water. Our study has shown that NP systems exhibit high antigenotoxic activity, which is similar to the results of CAPE dissolved in ethanol. These results have shown that NP systems increase biological activity of hydrophobic substances by increasing their solubility and that the use of PLGA instead of organic solvents in drug production may provide an increase in their medical utility.

Incubation of B10.MLM cells, a cell line of alveolar macrophages, with lycopene, a carotenoid, leads to an increase of lycopene content in their microsomal fraction. That increase was higher and developed faster when the cells were incubated with immune complexes formed by lycopene and mAb 6B9 (L-6B9 mAb), a monoclonal hapten-specific antibody raised against lycopene, as compared with dimethyl sulfoxide (DMSO)-dissolved lycopene (DMSO-L). Moreover, incubation of B10.MLM cells with L-6B9 mAb complexes was accompanied by more efficient accumulation of lipid droplets in the cultured cells and more significant inhibition of mRNA for 3-hydroxy-3-methylglutaryl-coenzyme (HMG-CoA) reductase, a rate-limiting enzyme of cholesterol biosynthesis known to be targeted by lycopene. Additionally, there was a better inhibition of Chlamydia trachomatis infection in B10.MLM cells infected with the pathogen and incubated thereafter with L-6B9 mAb complexes as compared with DMSO-L. Altogether, the results suggest that association with monoclonal antibody promotes intracellular delivery of lycopene in cultured cells possibly through Fc-receptor mediated uptake.

Despite recent progress in diagnosis and treatment, survival for children with high-risk metastatic neuroblastoma is still poor. Prostaglandin E 2 (PGE 2 )-driven inflammation promotes tumor growth, immune suppression, angiogenesis and resistance to established cancer therapies. In neuroblastoma, cancer-associated fibroblasts (CAFs) residing in the tumor microenvironment are the primary source of PGE 2 . However, clinical targeting of PGE 2 with current non-steroidal anti-inflammatory drugs or cyclooxygenase inhibitors has been limited due to risk of adverse side effects. By specifically targeting microsomal prostaglandin E synthase-1 (mPGES-1) activity with a small molecule inhibitor we could block CAF-derived PGE 2 production leading to reduced tumor growth, impaired angiogenesis, inhibited CAF migration and infiltration, reduced tumor cell proliferation and a favorable shift in the M1/M2 macrophage ratio. In this study, we provide proof-of-principle of the benefits of targeting mPGES-1 in neuroblastoma, applicable to a wide variety of tumors. This non-toxic single drug treatment targeting infiltrating stromal cells opens up for combination treatment options with established cancer therapies., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Cytochrome P450 2J2 (CYP2J2) is involved in the metabolism of drugs, including albendazole, astemizole, ebastine, and endogenous substrates. In a previous study, we used recombinant CYP2J2 and determined whether danazol, hydroxyebastine, telmisartan, and terfenadone inhibited CYP2J2 by using four representative CYP2J2 substrates, namely albendazole, astemizole, ebastine, and terfenadine. In this study, we evaluated the inhibitory potential of these four chemicals on human liver and intestinal microsomes, which are commonly used in a reaction phenotyping study. Among the four CYP2J2 inhibitors tested, terfenadone was strongest inhibitor of CYP2J2-mediated metabolism of albendazole, astemizole, and terfenadine with IC 50 values of 0.31, 0.15, and 2.11 μM, respectively, in human liver microsomes (HLMs). In addition, terfenadone had strong inhibitory effect on the metabolism of the abovementioned drugs in human intestinal microsomes (HIMs), with IC 50 values of 0.43, 0.08 and 1.07 μM, respectively. Danazol, weakly inhibited CYP2J2-mediated metabolism of albendazole and astemizole with IC 50 values of 13.8 and 18.3 μM, respectively in HLMs, whereas it strongly inhibited the CYP2J2-mediated ebastine hydroxylase activity in HLMs and HIMs (IC 50  = 1.93-1.95 μM). Our data suggest that terfenadone may be used as a general CYP2J2 inhibitor in reaction phenotyping study using HLMs and HIMs regardless of the substrate used., (Copyright © 2018 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Green tea ( Camellia sinensis ) is a popular beverage worldwide, raising concern for adverse interactions when co-consumed with conventional drugs. Like many botanical natural products, green tea contains numerous polyphenolic constituents that undergo extensive glucuronidation. As such, the UDP-glucuronosyltransferases (UGTs), particularly intestinal UGTs, represent potential first-pass targets for green tea-drug interactions. Candidate intestinal UGT inhibitors were identified using a biochemometrics approach, which combines bioassay and chemometric data. Extracts and fractions prepared from four widely consumed teas were screened (20-180 μ g/ml) as inhibitors of UGT activity (4-methylumbelliferone glucuronidation) in human intestinal microsomes; all demonstrated concentration-dependent inhibition. A biochemometrics-identified fraction rich in UGT inhibitors from a representative tea was purified further and subjected to second-stage biochemometric analysis. Five catechins were identified as major constituents in the bioactive subfractions and prioritized for further evaluation. Of these catechins, (-)-epicatechin gallate and (-)-epigallocatechin gallate showed concentration-dependent inhibition, with IC 50 values (105 and 59 μ M, respectively) near or below concentrations measured in a cup (240 ml) of tea (66 and 240 μ M, respectively). Using the clinical intestinal UGT substrate raloxifene, the K i values were ∼1.0 and 2.0 μ M, respectively. Using estimated intestinal lumen and enterocyte inhibitor concentrations, a mechanistic static model predicted green tea to increase the raloxifene plasma area under the curve up to 6.1- and 1.3-fold, respectively. Application of this novel approach, which combines biochemometrics with in vitro-in vivo extrapolation, to other natural product-drug combinations will refine these procedures, informing the need for further evaluation via dynamic modeling and clinical testing., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

The use of fish cell cultures has proven to be an effective tool in the study of environmental and aquatic toxicology. Valuable information can be obtained from comparisons between cell lines from different species and organs. In the present study, specific chemicals were used and biomarkers (e.g. 7-Ethoxyresorufin-O-deethylase (EROD) activity and reactive oxygen species (ROS)) were measured to assess the metabolic capabilities and cytotoxicity of the fish hepatic cell lines Hepa-E1 and RTH-149, and the fish gill cell lines RTgill-W1 and G1B. These cell lines were exposed to β-naphthoflavone (BNF) and benzo[a]pyrene (BaP), the pharmaceutical tamoxifen (TMX), and the organic peroxide tert-butylhydroperoxide (tBHP). Cytotoxicity in gill cell lines was significantly higher than in hepatic cells, with BNF and TMX being the most toxic compounds. CYP1-like associated activity, measured through EROD activity, was only detected in hepatic cells; Hepa-E1 cells showed the highest activity after exposure to both BNF and BaP. Significantly higher levels of CYP3A-like activity were also observed in Hepa-E1 cells exposed to TMX, while gill cell lines presented the lowest levels. Measurements of ROS and antioxidant enzymes indicated that peroxide levels were higher in gill cell lines in general. However, levels of superoxide were significantly higher in RTH-149 cells, where no distinctive increase of superoxide-related antioxidants was observed. The present study demonstrates the importance of selecting adequate cell lines in measuring specific metabolic parameters and provides strong evidence for the fish hepatocarcinoma Hepa-E1 cells to be an excellent alternative in assessing metabolism of xenobiotics, and in expanding the applicability of fish cell lines for in vitro studies., (Copyright © 2018 Elsevier Inc. All rights reserved.)