Anxiolytic-like effect of natural product 2-hydroxy-3,4,6-trimethoxyacetophenone isolated from Croton anisodontus in adult zebrafish via serotonergic neuromodulation involvement of the 5-HT system.

Benzodiazepines are highly effective in combating anxiety; however, they have considerable adverse effects, so it is important to discover new safe anxiolytic agents. This study was designed to investigate the effect of the natural product 2-hydroxy-3,4,6-trimethoxyacetophenone (HTMCX) on anxiety and seizure behavior in adult zebrafish and its possible mechanisms of action. The acute toxicity of 96 h of HTMCX was analyzed, and the open and light/dark field tests (n = 6 animals/group) were used to assess the anxiety behavior of animals treated with HTMCX. In addition, the mechanisms of action were investigated with antagonists of the GABAA, 5-HT receptors, and molecular anchorage study. Pentylenetetrazole (PTZ) was used to induce seizure by immersion. As a result, acetophenone HTMCX (1, 3 and 10 mg/kg; v.o.) was non-toxic and affected locomotor activity. The higher doses (3 and 10 mg/kg; v.o.) produced signs of anxiolytic action in the light/dark test, and this effect was reversed by the pizotifen (antagonist 5HTR1 and 5HTR2A/2C), having the potential to form a complex with 5HTR1B. However, the anxiolytic effect of HTMCX has not been abolished by flumazenil (antagonist GABAA), cyproheptadine (antagonist 5HTR2A), and granisetron (antagonist 5HTR3A/3B). Therefore, HTMCX demonstrated an anxiolytic effect, suggesting that the 5HTR1 and 5HTR2C receptors may be involved in the pharmacological performance of this acetophenone in the central nervous system.

In Silico and In Vivo Toxicological Evaluation of Paeonol.

Paeonol is a phenolic compound reported for its various pharmacological activities such as antioxidant, anti-inflammatory and antidiabetic activity. There are no systematic scientific reports on the in vivo toxicity of paeonol. So, the present work was designed to study in silico and in vivo toxicity of paeonol. In silico toxicity predictions were carried out using pkCSM, ProTox-II, pre-ADMET server and OSIRIS property explorer. Acute oral toxicity study of paeonol was carried out in female Sprague Dawley rats at a single dose of 300 mg/kg, 2000 mg/kg and 5000 mg/kg. Animals were observed for toxicity signs and mortality for 14 days. Repeated dose oral toxicity study of paeonol was carried out in female and male Sprague Dawley rats at a dose of 50, 100 and 200 mg/kg body weight for 28 days. At the end of the study, hematological, biochemical and urine parameters were assessed. Histopathology of vital organs was also carried out. In silico toxicity study predicted that paeonol is non-toxic. The maximally tolerated dose of paeonol was found to be 5000 mg/kg in acute toxicity study in female rats. Paeonol was found to be safe at a dose of 50, 100 and 200 mg/kg in repeated dose toxicity study in male and female rats.

Cepharanthine and Curcumin inhibited mitochondrial apoptosis induced by PCV2.

BACKGROUND: Porcine circovirus type 2 (PCV2) is an immunosuppressive pathogen with high prevalence rate in pig farms. It has caused serious economic losses to the global pig industry. Due to the rapid mutation of PCV2 strain and co-infection of different genotypes, vaccination could not eradicate the infection of PCV2. It is necessary to screen and develop effective new compounds and explore their anti-apoptotic mechanism. The 13 natural compounds were purchased, with a clear plant origin, chemical structure and content and specific biological activities. RESULTS: The maximum no-cytotoxic concentration (MNTC) and 50% cytotoxic concentration (CC50) of 13 tested compounds were obtained by the cytopathologic effect (CPE) assay and (3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method in PK-15 cells. The results of qPCR and Western blot showed that, compared with the PCV2 infected group, the expression of Cap in Paeonol (0.4 mg/mL and 0.2 mg/mL), Cepharanthine (0.003 mg/mL, 0.0015 mg/mL and 0.00075 mg/mL) and Curcumin (0.02 mg/mL, 0.001 mg/mL and 0.005 mg/mL) treated groups were significantly lowered in a dose-dependent manner. The results of Annexin V-FITC/PI, JC-1, Western blot and ROS analysis showed that the expression of cleaved caspase-3 and Bax were up-regulated Bcl-2 was down-regulated in Cepharanthine or Curcumin treated groups, while ROS and MMP value were decreased at different degrees and the apoptosis rate was reduced. In this study, Ribavirin was used as a positive control. CONCLUSIONS: Paeonol, Cepharanthine and Curcumin have significant antiviral effect. And the PCV2-induced Mitochondrial apoptosis was mainly remitted by Cepharanthine and Curcumin.

Comparison of the metabolism of 10 cosmetics-relevant chemicals in EpiSkin™ S9 subcellular fractions and in vitro human skin explants.

An understanding of the bioavailability of topically applied cosmetics ingredients is key to predicting their local skin and systemic toxicity and making a safety assessment. We investigated whether short-term incubations with S9 from the reconstructed epidermal skin model, EpiSkin™, would give an indication of the rate of chemical metabolism and produce similar metabolites to those formed in incubations with human skin explants. Both have advantages: EpiSkin™ S9 is a higher-throughput assay, while the human skin explant model represents a longer incubation duration (24 hours) model integrating cutaneous distribution with metabolite formation. Here, we compared the metabolism of 10 chemicals (caffeine, vanillin, cinnamyl alcohol, propylparaben, 4-amino-3-nitrophenol, resorcinol, 4-chloroaniline, 2-amino-3-methyl-3H-imidazo[4,5-F]quinoline and 2-acetyl aminofluorene) in both models. Both models were shown to have functional Phase 1 and 2 enzymes, including cytochrome P450 activities. There was a good concordance between the models with respect to the level of metabolism (stable vs. slowly vs. extensively metabolized chemicals) and major early metabolites produced for eight chemicals. Discordant results for two chemicals were attributed to a lack of the appropriate cofactor (NADP+ ) in S9 incubations (cinnamyl alcohol) and protein binding influencing chemical uptake in skin explants (4-chloroaniline). These data support the use of EpiSkin™ S9 as a screening assay to provide an initial indication of the metabolic stability of a chemical applied topically. If required, chemicals that are not metabolized by EpiSkin™ S9 can be tested in longer-term incubations with in vitro human explant skin to determine whether it is slowly metabolized or not metabolized at all.

End-product inhibition of skatole-metabolising enzymes CYP1A, CYP2A19 and CYP2E1 in porcine and piscine hepatic microsomes.

The hepatic cytochrome p450 enzymes 1 A, 2A19 and 2E1 is very important for the elimination of skatole from the body of pigs. Impaired skatole metabolism, results in skatole accumulation, which give rise to off flavor of the meat. Several metabolites of skatole has been identified, however the role of these metabolites in the inhibition of the skatole metabolizing enzymes are not documented. Using microsomes from pigs and fish, we determined the ability of several skatole metabolites to inhibit CYP1 A, CYP2A19 and CYP2E1 dependent activity. Our results show that 2-aminoacetophenone is an inhibitor of porcine CYP2A19 and CYP2E1 activity, but not the piscine orthologues. In conclusion, there is species specific differences in the inhibition of CYP1 A and CYP2A19 dependent metabolism of probe substrates. This is relevant to the evaluation of different model systems and to the reduction of off flavor of meat.

Paeonol exerts potential activities to inhibit the growth, migration and invasion of human gastric cancer BGC823 cells via downregulating MMP­2 and MMP­9.

Paeonol (Pae) is an herbal extract that has attracted extensive attention for its anti­cancer effects demonstrated by a number of studies, which have predominantly demonstrated inhibition of cell proliferation and induction of apoptosis. The influence of Pae on cancer cell metastasis has been less widely reported. The present study aimed to investigate the under­reported effects of Pae on the growth, invasion and migration of poorly differentiated BGC823 gastric cancer cells with strong invasive and metastatic abilities. The anti­proliferative and pro­apoptotic effects of Pae on BGC823 cells were verified by Cell Counting kit­8 and Annexin V­fluorescein isothiocyanate/propidium iodide assays. Cell scratch­wound healing and Transwell methods were applied, and it was demonstrated that Pae could exert inhibitory activities on the invasion and migration of BGC823 cells. Furthermore, it was indicated by western blot analysis that Pae could downregulate the protein expression levels of matrix metalloproteinase (MMP)­2 and ­9 in a concentration­dependent manner, which may support a novel potential mechanism accounting for its anti­cancer effects on gastric cancer.

Physiological effects of autotoxicity due to DHAP stress on Picea schrenkiana regeneration.

Picea Schrenkiana as one of the most important zonal vegetation was an endemic species in Middle Asia. Natural regeneration of P. Schrenkiana is a long existing problem troubling scientists. The autotoxicity of 3,4-dihydroxy-acetophenone (DHAP) was found to be a causative factor causing the failure of P. Schrenkiana natural regeneration. The effects of concentrations of DHAP treatment on the viability of root cell, activities of antioxidant enzymes and levels of P. Schrenkiana phytohormones were performed to disclose the physiological mechanism of DHAP autotoxicity. It was observed that high concentration of DHAP could inhibit the seed germination and seedling growth, but had a hormesis at low concentrations. Analyses showed that the root cells significantly lost their viability treated with high DHAP. The enzymes activities of seedlings were significantly stimulated by the treatment of 0.5 mM DHAP to give a transient increase and then decrease as DHAP concentration increased to 1.0 mM except for GR (glutathione reductase) in which DHAP treatment had little effect on its activity. Comparing with the control, an increase in the levels of phytohormones ZT (zeatin), GA3 (gibberellic acid) and IAA (indole acetic acid) was induced by the treatment of DHAP at low concentrations (0.1-0.25 mM), but the significant deficiency was found treated by high concentrations (0.5-1.0 mM). In addition, the ABA (abscisic acid) level increased in all experimental observations. These results suggested that DHAP significantly affected indices of growth and physiology, and provided some new information about different effect in P. Schrenkiana treated with DHAP.

Relationship between auxiliary gamma subunits and mallotoxin on BK channel modulation.

The large-conductance, calcium- and voltage-activated K+(BK) channel consists of the pore-forming α subunits (BKα) and auxiliary subunits. The auxiliary γ1-3 subunits potently modulate the BK channel by shifting its voltage-dependence of channel activation toward the hyperpolarizing direction by approximately 145 mV (γ1), 100 mV (γ2), and 50 mV (γ3). Mallotoxin is a potent small-molecule BK channel activator. We analyzed the relationship between mallotoxin and the γ subunits in their BK channel-activating effects in membrane patches excised from HEK-293 cells. We found that mallotoxin, when applied extracellularly, shifted the half-activation voltage (V1/2) of BKα channels by -72 mV. The channel-activating effect of mallotoxin was greatly attenuated in the presence of the γ1, γ2, or γ3 subunit, with resultant ΔV1/2 (+/- mallotoxin) values of -9, -28, or -15 mV, respectively. Most examined γ1 mutant subunits antagonized mallotoxin's channel-activating effect in a manner that was largely dependent on its own modulatory function. However, mallotoxin caused an irreversible functional and structural disengagement of the γ1-F273S mutant from BK channels. We infer that the auxiliary γ subunit effectively interferes with mallotoxin on BK channel modulation via either a direct steric competition or an indirect allosteric influence on mallotoxin's binding and action on BKα.

Evaluation of the toxicity of α-(phenylselanyl) acetophenone in mice.

Selenium is an essential micronutrient with several biological roles in the human body, but supra nutritional consumption can cause toxic effects. The potential deleterious effects of organoselenium compounds are controversial. The compound α-(phenylselanyl) acetophenone (PSAP) exhibits antioxidant, antidepressant-like and glutathione peroxidase-like activity, which makes important the elucidation of any toxic effects. Hence, the present study aims to investigate the in vitro toxicity of PSAP in Chinese Hamster ovary cells (through MTT assay) and analyse its genotoxicity using the comet assay in mice leukocytes after acute or chronic treatments, alongside with biochemical analyses. Our results demonstrate that the oral administration of PSAP in acute (1, 5, 10, 50, 200 mg/kg) and chronic (1, 10, 50, 200 mg/kg) doses did not cause genotoxicity. The compound presented cytotoxic effect in the MTT assay just at 500 µM after 24 h of administration and at 250 and 500 µM after 48 and 72 h of administration. According to biochemical analysis, PSAP presented a minor toxic effect by altering δ-ALA-D activity in liver and catalase activity in kidney at the highest tested concentration. Taking together, these data indicate that PSAP has low toxic effects after chronic administration in mice.

Paeonol Inhibits the Proliferation, Invasion, and Inflammatory Reaction Induced by TNF-α in Vascular Smooth Muscle Cells.

The aim of this study was to evaluate the effect of paeonol on the proliferation, migration, and inflammation induced by tumor necrosis factor (TNF-α) of rat vascular smooth muscle cells (VSMCs). Primary rat VSMCs were identified by immunofluorescence assay. The inhibition of VSMCs proliferation induced by TNF-α was observed after paeonol treatment in a dose-dependent manner. Treatment with 100 µM paeonol significantly reduced the expression of proliferating cell nuclear antigen (PCNA). On the other hand, transwell assay showed that treatment with paeonol suppressed the invasion of TNF-α-induced VSMCs and the production of inflammation factors stimulated by TNF-α. For apoptosis induced by paeonol, Western blot analysis showed that cleaved caspase-3 and -9 were detected, and pro-apoptotic protein Bax was up-regulated, whereas anti-apoptotic protein Bcl-2 was down-regulated by paeonol in TNF-α-stimulated VSMCs. ELISA analysis data showed that both levels of IL-1ß and IL-6 produced by the stimulation of TNF-α were decreased by paeonol in a dose-dependent manner in VSMCs. These results suggest that paeonol can effectively inhibit the proliferation through apoptotic induction through caspase pathway in VSMCs induced by TNF-α. Also, paeonol significantly reduced the invasion and the inflammation stimulated by TNF-α in VSMCs.

Co-exposure with fullerene may strengthen health effects of organic industrial chemicals.

In vitro toxicological studies together with atomistic molecular dynamics simulations show that occupational co-exposure with C60 fullerene may strengthen the health effects of organic industrial chemicals. The chemicals studied are acetophenone, benzaldehyde, benzyl alcohol, m-cresol, and toluene which can be used with fullerene as reagents or solvents in industrial processes. Potential co-exposure scenarios include a fullerene dust and organic chemical vapor, or a fullerene solution aerosolized in workplace air. Unfiltered and filtered mixtures of C60 and organic chemicals represent different co-exposure scenarios in in vitro studies where acute cytotoxicity and immunotoxicity of C60 and organic chemicals are tested together and alone by using human THP-1-derived macrophages. Statistically significant co-effects are observed for an unfiltered mixture of benzaldehyde and C60 that is more cytotoxic than benzaldehyde alone, and for a filtered mixture of m-cresol and C60 that is slightly less cytotoxic than m-cresol. Hydrophobicity of chemicals correlates with co-effects when secretion of pro-inflammatory cytokines IL-1ß and TNF-α is considered. Complementary atomistic molecular dynamics simulations reveal that C60 co-aggregates with all chemicals in aqueous environment. Stable aggregates have a fullerene-rich core and a chemical-rich surface layer, and while essentially all C60 molecules aggregate together, a portion of organic molecules remains in water.

Olfactory mucosal necrosis in rats following acute intraperitoneal administration of 1,2-diethylbenzene, 1,2-diacetylbenzene and 2,5-hexanedione.

1,2-Diethylbenzene (1,2-DEB) is used in the manufacture of some plastics. Exposure to 1,2-DEB has been shown to induce peripheral neuropathy in rats. This neurotoxicity is thought to be caused by a metabolite, 1,2-diacetylbenzene (1,2-DAB), a γ-diketone-like compound. 1,2-DEB was previously shown to be extensively and rapidly taken up by the nasal mucosa in male rats. In the present study, the nasal mucosa in rats exposed to 1,2-DEB and 1,2-DAB were examined histologically. Results were compared to sections from rats exposed to two other DEB isomers - 1,3-diethylbenzene (1,3-DEB) and 1,4-diethylbenzene (1,4-DEB) - and to two other neurotoxic compounds - n-hexane and its γ-diketone metabolite, 2,5-hexanedione (2,5-HD). A single intraperitoneal dose of 1,2-DEB (200mg/kg) induced time-dependent necrosis in the olfactory epithelium and Bowman's glands, with lesions appearing from the earliest observation time (4h) in the dorsomedial olfactory mucosa. Lesions spread through the lateral and ventral parts of the ethmoturbinates over the following days. The dorsal and medial zones of the nasal cavity started to regenerate from 72h after treatment, with the new epithelium showing metaplasia. One month after treatment, most of the olfactory epithelium had returned to normal. 1,2-DAB (40mg/kg) caused the same lesions as those observed after treatment with 1,2-DEB. Treatment with 2,5-HD (1g/kg) also caused lesions of the olfactory epithelium, mainly at level IV. However, these were comparatively less severe than those observed after exposure to 1,2-DEB. In contrast, intraperitoneal injection of 1,3-DEB (800mg/kg), 1,4-DEB (800mg/kg) and n-hexane (2g/kg) did not affect the nasal mucosa. Pretreatment of rats with 5-phenyl-1-pentyne, an inhibitor of CYP2F2 and CYP2E1 completely inhibited the olfactory toxicity caused by 1,2-DEB. These results suggest that metabolic activation of 1,2-DEB may be responsible for the toxicity observed.

A small volatile bacterial molecule triggers mitochondrial dysfunction in murine skeletal muscle.

Mitochondria integrate distinct signals that reflect specific threats to the host, including infection, tissue damage, and metabolic dysfunction; and play a key role in insulin resistance. We have found that the Pseudomonas aeruginosa quorum sensing infochemical, 2-amino acetophenone (2-AA), produced during acute and chronic infection in human tissues, including in the lungs of cystic fibrosis (CF) patients, acts as an interkingdom immunomodulatory signal that facilitates pathogen persistence, and host tolerance to infection. Transcriptome results have led to the hypothesis that 2-AA causes further harm to the host by triggering mitochondrial dysfunction in skeletal muscle. As normal skeletal muscle function is essential to survival, and is compromised in many chronic illnesses, including infections and CF-associated muscle wasting, we here determine the global effects of 2-AA on skeletal muscle using high-resolution magic-angle-spinning (HRMAS), proton ((1)H) nuclear magnetic resonance (NMR) metabolomics, in vivo (31)P NMR, whole-genome expression analysis and functional studies. Our results show that 2-AA when injected into mice, induced a biological signature of insulin resistance as determined by (1)H NMR analysis-, and dramatically altered insulin signaling, glucose transport, and mitochondrial function. Genes including Glut4, IRS1, PPAR-γ, PGC1 and Sirt1 were downregulated, whereas uncoupling protein UCP3 was up-regulated, in accordance with mitochondrial dysfunction. Although 2-AA did not alter high-energy phosphates or pH by in vivo (31)P NMR analysis, it significantly reduced the rate of ATP synthesis. This affect was corroborated by results demonstrating down-regulation of the expression of genes involved in energy production and muscle function, and was further validated by muscle function studies. Together, these results further demonstrate that 2-AA, acts as a mediator of interkingdom modulation, and likely effects insulin resistance associated with a molecular signature of mitochondrial dysfunction in skeletal muscle. Reduced energy production and mitochondrial dysfunctional may further favor infection, and be an important step in the establishment of chronic and persistent infections.

Acaricidal potentials of active properties isolated from Cynanchum paniculatum and acaricidal changes by introducing functional radicals.

This study evaluated the acaricidal activities of acetophenone and its derivatives for their potentials as natural acaricides using an impregnated fumigant bioassay against Dermatophagoides spp. and Tyrophagus putrescentiae . On the basis of the LD50 values against D. farinae, 3'-methoxyacetophenone (0.41 µg/cm(2)) was 89.9 times more toxic than DEET (36.87 µg/cm(2)), followed by 4'-methoxyacetophenone (0.52 µg/cm(2)), 2'-methoxyacetophenone (0.75 µg/cm(2)), 2'-hydroxy-5'-methoxyacetophenone (1.03 µg/cm(2)), 2'-hydroxy-4'-methoxyacetophenone (1.29 µg/cm(2)), acetophenone (1.48 µg/cm(2)), 2'-hydroxyacetophenone (1.74 µg/cm(2)), 2',5'-dimethoxyacetophenone (1.87 µg/cm(2)), 2',4'-dimethoxyacetophenone (2.10 µg/cm(2)), and benzyl benzoate (9.92 µg/cm(2)). In regard to structure-activity relationships between acaricidal activity and functional radicals (hydroxyl and methoxy groups) on the acetophenone skeleton, a monomethoxy group (2'-, 3'-, and 4'-methoxyacetone) on the acetophenone skeleton was more toxic than were the other groups (2',4'- and 2',5'-dimethoxyacetophenone, 2'- and 4'-hydroxyacetophenone, 2'-hydroxy-4'-methoxyacetophenone, 2'-hydroxy-5'-methoxyacetophenone, and 4'-hydroxy-3'-methoxyacetophenone). These results indicated that acaricidal activity against three mite species changed with the introduction of functional radicals (hydroxyl and methoxy groups) onto the acetophenone skeleton.