Isobavachalcone induces hepatotoxicity in zebrafish embryos and HepG2 cells via the System Xc--GSH-GPX4 signaling pathway in ferroptosis response.

Isobavachalcone (IBC) is a flavonoid component of the traditional Chinese medicine Psoraleae Fructus, with a range of pharmacological properties. However, IBC causes some hepatotoxicity, and the mechanism of toxicity is unclear. The purpose of this paper was to investigate the possible mechanism of toxicity of IBC on HepG2 cells and zebrafish embryos. The results showed that exposure to IBC increased zebrafish embryo mortality and decreased hatchability. Meanwhile, IBC induced liver injury and increased expression of ALT and AST activity. Further studies showed that IBC caused the increase of ROS and MDA the decrease of CAT, GSH, and GSH-Px; the increase of Fe2+ content; and the changes of ferroptosis related genes (acsl4, gpx4, and xct) and iron storage related genes (tf, fth, and fpn) in zebrafish embryos. Through in vitro verification, it was found that IBC also caused oxidative stress and increased Fe2+ content in HepG2 cells. IBC caused depolarization of mitochondrial membrane potential (MMP) and reduction of mitochondrial ATP, as well as altered expression of ACSl4, SLC7A11, GPX4, and FTH1 proteins. Treatment of HepG2 cells with ferrostatin-1 could reverse the effect of IBC. Targeting the System Xc--GSH-GPX4 pathway of ferroptosis and preventing oxidative stress damage might offer a theoretical foundation for practical therapy and prevention of IBC-induced hepatotoxicity.

Isoliquiritigenin induces neurodevelopmental-toxicity and anxiety-like behavior in zebrafish larvae.

Isoliquiritigenin, a flavonoid compound, exhibits a variety of pharmacological properties, including anti-inflammatory, anti-oxidative, anti-microbial, anti-viral, and anti-tumor effects. In the past few years, the consumption of isoliquiritigenin-containing dietary supplements has increased due to their health benefits. Although the neuroprotective effects of isoliquiritigenin have been well-investigated, these studies were performed in cells and adult animals. The potential effects of isoliquiritigenin on the development, especially the neurodevelopment, of certain populations, such as zebrafish larvae, have not been investigated. In this study, zebrafish larvae were employed as a model to investigate the effects of isoliquiritigenin on development and neurodevelopment. Zebrafish embryos treated with high concentrations of isoliquiritigenin (10 and 15 µM) exhibited high rates of mortality, hatching, and malformation, indicating that isoliquiritigenin can affect zebrafish development. In addition, isoliquiritigenin impeded the development of central nervous system regions and the length of dopaminergic neurons located in midbrains and thalami of transgenic zebrafish larvae. The locomotor ability of zebrafish larvae exposed to high concentrations of isoliquiritigenin was negatively affected. The total distance and the average velocity significantly decreased, and anxiety-related behaviors were observed under light-dark challenge. Furthermore, the levels of gap43, tuba1b, mbp, hcrt, vmat2, and pomc, which mediate neurodevelopment, neurotoxicity, and anxiety were significantly decreased in zebrafish larvae exposed to isoliquiritigenin. These results indicate that isoliquiritigenin can disrupt the development of dopaminergic neurons and the function of the central nervous system in zebrafish, causing anxiety-like symptoms.

Novel sulfonamide-chalcone hybrid stimulates inflammation, angiogenesis and upregulates vascular endothelial growth factor (VEGF) in vivo.

Chalcones and sulfonamides are well-known chemical groups associated with several biological activities such as antibiotic, anti-inflammatory, and antitumor activities. Over the past few decades, a series of sulfonamide-chalcone hybrids have been synthesized and assessed to develop compounds with interesting biological properties for application in disease therapy. In the present study, a new sulfonamide-chalcone hybrid µ - (2,5-dichloro-N-{4-[(3E)-4-(3-nitrophenyl) buta-1,3-dien-2-yl] phenyl} benzene sulfonamide), or simply CL185, was synthesized, and its angiogenic activity was assessed using the chick embryo chorioallantoic membrane (CAM) assay at different concentrations (12.5, 25, and 50 µg/µL). To further investigate the role of CL185 in the angiogenic process, we evaluated the levels of vascular endothelial growth factor (VEGF) in all treated CAMs. The results showed that all concentrations of CL185 significantly increased tissue vascularization (p < 0.05) as well as the parameters associated with angiogenesis, in which inflammation was the most marked phenomenon observed. In all CAMs treated with CL185, VEGF levels were significantly higher than those in the negative control (p < 0.05), and at the highest concentration, VEGF levels were even higher than in the positive control (p < 0.05). The pronounced angiogenic activity displayed by CL185 may be related to the increase in VEGF levels that were stimulated by inflammatory processes observed in our study. Therefore, CL185 presents a favorable profile for the development of drugs that can be used in pro-angiogenic and tissue repair therapies.

Microbial Conjugation Studies of Licochalcones and Xanthohumol.

Microbial conjugation studies of licochalcones (1-4) and xanthohumol (5) were performed by using the fungi Mucor hiemalis and Absidia coerulea. As a result, one new glucosylated metabolite was produced by M. hiemalis whereas four new and three known sulfated metabolites were obtained by transformation with A. coerulea. Chemical structures of all the metabolites were elucidated on the basis of 1D-, 2D-NMR and mass spectroscopic data analyses. These results could contribute to a better understanding of the metabolic fates of licochalcones and xanthohumol in mammalian systems. Although licochalcone A 4'-sulfate (7) showed less cytotoxic activity against human cancer cell lines compared to its substrate licochalcone A, its activity was fairly retained with the IC50 values in the range of 27.35-43.07 µM.

Hydroxylated Chalcones as Aryl Hydrocarbon Receptor Agonists: Structure-Activity Effects.

Hydroxylated chalcones are phytochemicals which are biosynthetic precursors of flavonoids and their 1,3-diaryl-prop-2-en-1-one structure is used as a scaffold for drug development. In this study, the structure-dependent activation of aryl hydrocarbon receptor (AhR)-responsive CYP1A1, CYP1B1, and UGT1A1 genes was investigated in Caco2 colon cancer cells and in non-transformed young adult mouse colonocytes (YAMC) cells. The effects of a series of di- and trihydroxychalcones as AhR agonists was structure dependent with maximal induction of CYP1A1, CYP1B1, and UGT1A1 in Caco2 cells observed for compounds containing 2,2'-dihydroxy substituents and this included 2,2'-dihydroxy-, 2,2',4'-trihydroxy-, and 2,2',5'-trihydroxychalcones. In contrast, 2',4,5'-, 2'3',4'-, 2',4,4'-trihydroxy, and 2',3-, 2',4-, 2',4'-, and 2',5-dihydroxychalcones exhibited low to non-detectable AhR activity in Caco2 cells. In addition, all of the hydroxychalcones exhibited minimal to non-detectable activity in YAMC cells, whereas 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced CYP1A1, CYP1B1, and UGT1A1 in Caco2 and YAMC cells. The activity of AhR-active chalcones was confirmed by determining their effects in AhR-deficient Caco2 cells. In addition, 2,2'-dihydroxychalcone induced CYP1A1 protein and formation of an AhR-DNA complex in an in vitro assay. Simulation and modeling studies of hydroxylated chalcones confirmed their interactions with the AhR ligand-binding domain and were consistent with their structure-dependent activity as AhR ligands. Thus, this study identifies hydroxylated chalcones as AhR agonists with potential for these phytochemicals to impact AhR-mediated colonic pathways.

Isoliquiritigenin triggers developmental toxicity and oxidative stress-mediated apoptosis in zebrafish embryos/larvae via Nrf2-HO1/JNK-ERK/mitochondrion pathway.

Isoliquiritigenin (ISL) is an emerging natural flavonoid found in the roots of licorice, exhibits antioxidant, anti-cancer, anti-inflammatory, anti-allergic, cardioprotective, hepatoprotective and neuroprotective properties. However, the effect of ISL in embryonic development is yet to be elucidated, and the mechanisms underlying its target-organ toxicity and harmful side effects are still unclear. In the present study, we employed zebrafish embryos to study the developmental toxicity effect of ISL and its underlying mechanisms. Zebrafish embryos upon treatment with either vehicle control (0.1% DMSO) or ISL solutions for 4-96 h post fertilization (hpf) showed that ISL exposure instigated severe developmental toxicity in heart, liver, and nervous system. Mortality and morphological abnormalities were also observed. High concentrations of ISL exposure resulted in abnormal phenotypes and embryonic malformations including pericardial edema, swim bladder defects, yolk retention, curved body shape and shortening of body length. Moreover, ISL exposure led to significant loss of dopaminergic neurons accompanied by reduced locomotor behaviour. Apoptotic cells were predominantly located in the heart area of 96 hpf embryo. Additionally, ISL significantly increased the levels of reactive oxygen species, lipid peroxidation content and decreased antioxidant enzyme activities. The expressions pattern of apoptosis-related genes Bad, Cyto c, Caspase-9, Caspase-3 and Bax/Bcl-2 indicated that the oxidative stress-induced apoptosis triggered by ISL suggest involvement of Nrf2-HO1/JNK-ERK/mitochondrion pathways. In conclusion, here we provide first evidence that demonstrate ISL-induced dose-dependent developmental toxicity in zebrafish embryos. Furthermore, gene expression patterns in the embryos correlate the above and reveal potential genetic mechanisms of developmental toxicity.

Discovery of chalcone-modified estradiol analogs as antitumour agents that Inhibit tumour angiogenesis and epithelial to mesenchymal transition.

Angiogenesis plays an essential role in tumourigenesis and tumour progression, and anti-angiogenesis therapies have shown promising antitumour effects in solid tumours. 2-Methoxyestradiol (2ME2), an endogenous metabolite of estradiol, has been regarded as a potential antitumour agent mainly targeting angiogenesis. Here we synthesized a novel series of chalcones based on 2-methoxyestradiol and evaluated their potential activities against tumours. Compound 11e was demonstrated to have potent antiangiogenic activity. Further studies showed that 11e suppressed tumour growth in human breast cancer (MCF-7) xenograft models without obvious side effects. Evaluation of the mechanism revealed that 11e targeted the epithelial to mesenchymal transition (EMT) process in MCF-7 cells and inhibited HUVEC migration and then contributed to hindrance of angiogenesis. Thus, 11e may be a promising antitumour agent with excellent efficacy and low toxicity.

New chalcone-sulfonamide hybrids exhibiting anticancer and antituberculosis activity.

New sulfonamides 5/6 derived from 4-methoxyacetophenone 1 were synthesized by N-sulfonation reaction of ammonia (3) and aminopyrimidinone (4) with its sulfonyl chloride derivative 2. Sulfonamides 5 and 6 were used as precursors of two new series of chalcones 8a-f and 9a-f, which were obtained through Claisen-Schmidt condensation with aromatic aldehydes 7a-f. Compounds 5/6, 8a-d, 8f, 9a-d, and 9f were screened by the US National Cancer Institute (NCI) at 10 µM against sixty different human cancer cell lines (one-dose trial). Chalcones 8b and 9b satisfied the pre-determined threshold inhibition criteria and were selected for screening at five different concentrations (100, 10, 1.0, 0.1, and 0.01 µM). Compound 8b exhibited remarkable GI50 values ranging from 0.57 to 12.4 µM, with cytotoxic effects being observed in almost all cases, especially against the cell lines K-562 of Leukemia and LOX IMVI of Melanoma with GI50 = 0.57 and 1.28 µM, respectively. Moreover, all compounds were screened against Mycobacterium tuberculosis H37Rv, chalcones 8a-c and 9a-c were the most active showing MIC values between 14 and 42 µM, and interestingly they were devoid of antibacterial activity against Mycobacterium smegmatis and Staphylococcus aureus. These antituberculosis hits showed however low selectivity, being equally inhibitory to M. tuberculosis and mammalian T3T cells. The chalcone-sulfonamide hybrids 8a-f and 9a-f resulted to be appealing cytotoxic agents with significant antituberculosis activity.

Design, synthesis and QSAR study of 2'-hydroxy-4'-alkoxy chalcone derivatives that exert cytotoxic activity by the mitochondrial apoptotic pathway.

Eleven 4'-alkoxy chalcones were synthesized and biologically evaluated for their antiproliferative activity against four human tumor cell lines (PC-3, MCF-7, HF-6, and CaSki). Compounds 3a-3d and 3f were selective against PC-3, with IC50 values ranging from 8.08 to 13.75 µM. In addition, chalcones 3a-3c did not affect the normal fibroblasts BJ cells. The most active and selective compounds were further evaluated for their effect on the progression of cell cycle in PC-3 cells, and chalcones 3a and 3c induced a G2/M phase arrest. Furthermore, it was found that these three chalcones induced the mitochondrial apoptotic pathway by regulating Bax and Bcl-2 transcripts and by increasing caspase 3/7 activation. Otherwise, the QSAR model indicates that the double bond of the α,ß-unsaturated carbonyl, as well as the planar structure geometry, are important to the biological activity of the synthetized chalcones. Based on these studies, it was concluded that withdrawing substituents in ring A, decrease the antiproliferative activity. This is related to the possible mechanism of action of these compounds, where a Michael addition needs to take place in order to be a potent anticancer agent.

Vehicle development, pharmacokinetics and toxicity of the anti-invasive agent 4-fluoro-3',4',5'-trimethoxychalcone in rodents.

Effective inhibitors of invasion and metastasis represent a serious unmet clinical need. We have recently identified 4-fluoro-3',4',5'-trimethoxychalcone or C16 as a potent anti-invasive molecule. In this paper, we report on the development of an optimized vehicle for oral administration of C16. We also explore its pharmacokinetic and toxicity profile in rodents as a prelude to a broad-scope evaluation as a pharmacological tool in animal models of disease. C16 showed suboptimal pharmacokinetics with limited oral bioavailability and whole blood stability. Rapid metabolism with elimination via glutathione conjugation was observed. An oral dosing routine using medicated gels was developed to overcome bioavailability issues and yielded sustained whole blood levels above the half maximal effective concentration (EC50) in a 7-day study. The compound proved well-tolerated in acute and chronic experiments at 300 mg/kg PO dosing. The medicated gel formulation is highly suitable for evaluation of C16 in animal models of disease.

Monoamine oxidase inhibitory activity of methoxy-substituted chalcones.

The MAO-B inhibitory activity of chalcone (1, 3- diphenyl-2-propen-1-one) based compounds arise from its structural similarity with 1, 4-diphenyl-2-butene, a known MAO-B inhibitor. Based on our previous report, the methoxy-substituted with fluorine containing chalcones are promising reversible MAO-B inhibitors, while in the present study, a series of methoxylated chalcones (C1-C9) bearing substitution on the para position of ring B was synthesized and evaluated for their human monoamine oxidase inhibitory activity. With the exception of (2E)-1-(4-methoxyphenyl)-3-(4-nitrophenyl) prop-2-en-1-one (C7), which is a nonselective inhibitor, the chalcones exhibited competitive, selective, and reversible inhibition of hMAO-B. The most potent compound, (2E)-3-[4-(dimethylamino) phenyl]-1-(4-methoxyphenyl) prop-2-en-1-one (C5), showed the best inhibitory activity towards hMAO-B (IC50=0.29±0.011µM;Ki=0.14±0.001µM). The reversibility of MAO-B inhibition by compound C5 was demonstrated by the recovery of enzyme activity after dialysis of mixtures containing enzyme and inhibitor. The reversiblity of C5 was 25.38±1.40 and 92.00±3.87% before and after dialysis, respectively. PAMPA was carried out to evaluate the blood-brain barrier effects of the designated compounds. Moreover, the most potent MAO-B inhibitor, C5, was found to be nontoxic towards cultured hepatic cells at 5 and 25µM, with 97 and 90% viability. Molecular docking study was performed against hMAO-B to observe the binding site interactions of the lead compound.

Methoxylated 2'-hydroxychalcones as antiparasitic hit compounds.

Chalcones display a broad spectrum of pharmacological activities. Herein, a series of 2'-hydroxy methoxylated chalcones was synthesized and evaluated towards Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum. Among the synthesized library, compounds 1, 3, 4, 7 and 8 were the most potent and selective anti-T. brucei compounds (EC50 = 1.3-4.2 µM, selectivity index >10-fold). Compound 4 showed the best early-tox and antiparasitic profile. The pharmacokinetic studies of compound 4 in BALB/c mice using hydroxypropil-ß-cyclodextrins formulation showed a 7.5 times increase in oral bioavailability.

Cardamonin (2',4'-dihydroxy-6'-methoxychalcone) isolated from Boesenbergia rotunda (L.) Mansf. inhibits CFA-induced rheumatoid arthritis in rats.

Boesenbergia rotunda (L.) Mansf. had been traditionally used as herbs to treat pain and rheumatism. Cardamonin (2',4'-dihydroxy-6'-methoxychalcone) is a compound isolated from Boesenbergia rotunda (L.) Mansf.. Previous study had shown the potential of cardamonin in inhibiting the release of pro-inflammatory cytokines such as tumour necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) in vitro. Thus, the possible therapeutic effect of cardamonin in the rheumatoid arthritis (RA) joints is postulated. This study was performed to investigate the anti-arthritic properties of cardamonin in rat model of induced RA, particularly on the inflammatory and pain response of RA. Rheumatoid arthritis paw inflammation was induced by intraplantar (i.pl.) injection of complete Freund's adjuvant (CFA) in Sprague Dawley rats. Using four doses of cardamonin (0.625, 1.25, 2.5, and 5.0mg/kg), anti-arthritic activity was evaluated through the paw edema, mechanical allodynia and thermal hyperalgesia responses. Enzyme-linked immunosorbent assay (ELISA) was carried out to evaluate the plasma level of TNF-α, IL-1ß, and IL-6. Histological slides were prepared from the harvested rat paws to observe the arthritic changes in the joints. Behavioral, biochemical, and histological studies showed that cardamonin demonstrated significant inhibition on RA-induced inflammatory and pain responses as well as progression of joint destruction in rats. ELISA results showed that there was significant inhibition in TNF-α, IL-1ß, and IL-6 levels in plasma of the cardamonin-treated RA rats. Overall, cardamonin possesses potential anti-arthritic properties in CFA-induced RA rat model.

Design, synthesis, biological evaluation, and molecular modeling studies of chalcone-rivastigmine hybrids as cholinesterase inhibitors.

A series of novel chalcone-rivastigmine hybrids were designed, synthesized, and tested in vitro for their ability to inhibit human acetylcholinesterase and butyrylcholinesterase. Most of the target compounds showed hBChE selective activity in the micro- and submicromolar ranges. The most potent compound 3 exhibited comparable IC50 to the commercially available drug (rivastigmine). To better understand their structure activity relationships (SAR) and mechanisms of enzyme-inhibitor interactions, kinetic and molecular modeling studies including molecular docking and molecular dynamics (MD) simulations were carried out. Furthermore, compound 3 blocks the formation of reactive oxygen species (ROS) in SH-SY5Y cells and shows the required druggability and low cytotoxicity, suggesting this hybrid is a promising multifunctional drug candidate for Alzheimer's disease (AD) treatment.

Effects of isoliquiritigenin on ovarian antral follicle growth and steroidogenesis.

Isoliquiritigenin is a botanical estrogen used as a dietary supplement. Previous studies show that other botanical estrogens affect ovarian estradiol synthesis, but isoliquiritigenin's effects on the ovary are unknown. Thus, this study tested the hypothesis that isoliquiritigenin inhibits ovarian antral follicle growth and steroidogenesis. Antral follicles from CD-1 mice were cultured with vehicle control (dimethyl sulfoxide; DMSO) or isoliquiritigenin (0.6µM, 6 µM, 36 µM, and 100 µM) for 48-96h. During culture, follicle diameters were measured daily to assess follicle growth. After culture, media were collected for hormone assays and follicles were collected for gene expression analysis of steroidogenic enzymes. Isoliquiritigenin inhibited antral follicle growth and altered estradiol, testosterone, and progesterone levels. Additionally, isoliquiritigenin altered the mRNA levels of cytochrome P450 steroid 17-α-hydroxylase 1, aromatase, 17ß-hydroxysteroid dehydrogenase 1, and steroidogenic acute regulatory protein. These data indicate that exposure to isoliquiritigenin inhibits growth and disrupts steroid production in antral follicles.

Influence of S-Oxidation on Cytotoxic Activity of Oxathiole-Fused Chalcones.

Synthesis, in vitro cytotoxic activity, and interaction with tubulin of oxidized, isomeric 1-(5-alkoxybenzo[d][1,3]oxathiol-6-yl)-3-phenylprop-2-en-1-ones and 1-(6-alkoxybenzo[d][1,3]oxathiol-5-yl)-3-phenylprop-2-en-1-ones are described. Most of the compounds demonstrated cytotoxic activity at submicromolar concentrations. It was found that oxidation of sulfur atom of the oxathiole-fused chalcones strongly influenced activity of the parent compounds, and that depending on relative position of the sulfur atom in the molecule, the activity was either increased or diminished. For isomers with sulfur atom para to the chalcone carbonyl group, oxidation led to increase in activity, while for isomers with sulfur atom meta to the carbonyl the activity dropped down. It was demonstrated that the compounds interact with tubulin at the colchicine binding site, and the interaction was evaluated using molecular modeling. It was concluded that the observed profound influence of oxidation of the sulfur atom on cytotoxic activity cannot be solely related to interaction of the compounds with tubulin.

Peptide reactivity associated with skin sensitization: The QSAR Toolbox and TIMES compared to the DPRA.

The molecular initiating event (MIE) of skin sensitization is the binding of a hapten to dermal proteins. This can be assessed using the in chemico direct peptide reactivity assay (DPRA) or in silico tools such as the QSAR Toolbox and TIMES SS. In this study, the suitability of these methods was analyzed by comparing their results to in vivo sensitization data of LLNA and human studies. Compared to human data, 84% of non-sensitizers and sensitizers yielded consistent results in the DPRA. In silico tools resulted in 'no alert' for 83%-100% of the non-sensitizers, but alerted only 55%-61% of the sensitizers. The inclusion of biotic and abiotic transformation simulations yielded more alerts for sensitizers, but simultaneously dropped the number of non-alerted non-sensitizers. In contrast to the DPRA, in silico tools were more consistent with results of the LLNA than human data. Interestingly, the new "DPRA profilers" (QSAR Toolbox) provided unsatisfactory results. Additionally, the results were combined in the '2 out of 3' prediction model with in vitro data derived from LuSens and h-CLAT. Using DPRA results, the model identified 90% of human sensitizers and non-sensitizers; using in silico results (including abiotic and biotic activations) instead of DPRA results led to a comparable high predictivity.

In vitro and in silico toxicity evaluation of bioactive 4'-aminochalcone derivatives.

The 4'-aminochalcones compounds are open-chain flavonoids structures which have shown a known array of pharmacological activities, such as antibacterial, antifungal, anti-inflammatory and antitumor effects. There is little toxicological information available about these compounds in the literature. Therefore, the investigation of toxic effects of three 4'-aminochalcone derivatives was performed using in silico and in vitro assays. In silico provided results that indicated the occurrence of mutagenic and genotoxic effects. In vitro tests, using Cellular Proliferation and Viability, Micronucleus, and DNA damage by Comet assay, showed that the compounds studied also present mutagenic and genotoxic effects, which confirm the result determined by the in silico analysis. The use of experimental and computational models is complementary to each other and the results determined for 4'-aminochalones suggest that the chalcones should also be carefully considered since they show some risks to cause toxic effects to human cells.

Licochalcone A Induced Suicidal Death of Human Erythrocytes.

BACKGROUND: The anti-inflammatory, immunomodulatory, and antimicrobial Glycyrrhiza inflata extract component licochalcone A triggers apoptosis of tumor cells and is thus considered for the treatment of malignancy. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the triggering of eryptosis includes Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i), and ceramide. The present study explored, whether and how licochalcone A induces eryptosis. METHODS: Human erythrocytes drawn from healthy individuals were exposed for 24 hours to 1-10 µg/ml licochalcone A. Flow cytometry was subsequently employed to estimate phosphatidylserine exposure at the cell surface from annexin V binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, and ceramide utilizing specific antibodies. In addition, hemolysis was quantified from hemoglobin release. RESULTS: Licochalcone A significantly increased the percentage of annexin-V-binding cells (≥ 5 µg/ml), significantly decreased forward scatter (2.5 - 5 µg/ml), significantly increased Fluo3-fluorescence (≥ 7.5 µg/ml), and significantly increased ceramide abundance (10 µg/ml). The effect of licochalcone on annexin-V-binding was not significantly modified, but hemolysis significantly enhanced by removal of extracellular Ca2+. CONCLUSIONS: Licochalcone triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect independent from Ca2+ entry and presumably in part due to ceramide.

Toxicological Evaluation of Anti-Scrapie Trimethoxychalcones and Oxadiazoles.

An altered form of the cellular prion protein, the PrPScor PrPRes, is implicated in the occurrence of the still untreatable transmissible spongiform encephalopathies. We have previously synthesized and characterized aromatic compounds that inhibit protease-resistant prion protein (PrPRes) accumulation in scrapie-infected cells. These compounds belong to different chemical classes, including acylhydrazones, chalcones and oxadiazoles. Some of the active compounds were non-toxic to neuroblastoma cells in culture and seem to possess drugable properties, since they are in agreement with the Lipinski´s rule of 5 and present desirable pharmacokinetic profiles as predicted in silico. Before the evaluation of the in vivo efficacy of the aromatic compounds in scrapie-infected mice, safety assessment in healthy mice is needed. Here we used Swiss mice to evaluate the acute toxicity profile of the six most promising anti-prionic compounds, the 2,4,5-trimethoxychalcones (J1, J8, J20 and J35) and the 1,3,4-oxadiazoles (Y13 and Y17). One single oral administration (300 mg/kg) of J1, J8, J20, J35, Y13 and Y17 or repeated intraperitoneal administration (10 mg/kg, 3 times a week, for 4 weeks) of J1, J8 and J35, did not elicit toxicity in mice. We strongly believe that the investigated trimethoxychalcones and oxadiazoles are interesting compounds to be further analyzed in vivo against prion diseases.