Ferroptosis-Specific Inhibitor Ferrostatin-1 Relieves H2O2-Induced Redox Imbalance in Primary Cardiomyocytes through the Nrf2/ARE Pathway.

OBJECTIVE: Ischemic heart disease (IHD) has always been the focus of attention of many researchers in cardiovascular disease, and its pathogenesis is also very complicated. Ferroptosis may be involved in the occurrence and development of IHD. METHODS: First, primary cardiomyocytes were treated with H2O2 to simulate the IHD in vitro model. After pretreatment with different concentrations of ferrostatin-1, cell survival rate was detected by MTT method, cell apoptosis was detected by TUNEL staining and flow cytometry, and the expression of oxidative stress, ferroptosis, and related molecules of Nrf2/ARE pathway was detected by Western blotting (WB) and quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The mortality of primary cardiomyocytes in the H2O2 group was obviously increased. Ferrostatin-1 treatment can effectively inhibit cell death, improve antioxidant enzyme activity, inhibit the expression of ferroptosis-related molecules, and activate Nrf2/ARE pathway expression. CONCLUSION: Ferroptosis-specific inhibitor ferrostatin-1 relieves H2O2-induced redox imbalance in primary cardiomyocytes through the Nrf2/ARE pathway, inhibits ferroptosis, and thereby slows cardiomyocyte death.

Radical scavenging and anti-inflammatory activities of (hetero)arylethenesulfonyl fluorides: Synthesis and structure-activity relationship (SAR) and QSAR studies.

A series of (hetero)arylethenesulfonyl fluorides (1-58) were synthesized and screened for their in vitro antioxidant (DPPH, ABTS and DMPD methods) and anti-inflammatory activities. The results revealed that compounds 4, 15, 16, 24, 25, 26, 38, 39, 40, and 54 exhibited excellent antioxidant activity using all the three performed antioxidant methods, which were superior to the standard antioxidants ascorbic acid and gallic acid. Compounds 6-9, 11, 18, 19, 21, 22, 30, 39, 40, 44, 45, 48-50, 54, 55 and 57 displayed promising anti-inflammatory activity, which were better than the reference drug indomethacin. Preliminary structure-activity relationship (SAR) revealed that compounds containing electron donating (OH and OCH3) groups on the phenyl ring possessed excellent antioxidant properties while compounds containing electron-withdrawing (Cl, NO2, F and Br) groups on the phenyl ring were found to be most potent anti-inflammatory agents. The presence of SO2F group played a crucial role in increases both antioxidant and anti-inflammatory activities.

Curiouser and Curiouser: The Macrocyclic Lactone, Abamectin, Is also a Potent Inhibitor of Pyrantel/Tribendimidine Nicotinic Acetylcholine Receptors of Gastro-Intestinal Worms.

Nematode parasites may be controlled with drugs, but their regular application has given rise to concerns about the development of resistance. Drug combinations may be more effective than single drugs and delay the onset of resistance. A combination of the nicotinic antagonist, derquantel, and the macrocyclic lactone, abamectin, has been found to have synergistic anthelmintic effects against gastro-intestinal nematode parasites. We have observed in previous contraction and electrophysiological experiments that derquantel is a potent selective antagonist of nematode parasite muscle nicotinic receptors; and that abamectin is an inhibitor of the same nicotinic receptors. To explore these inhibitory effects further, we expressed muscle nicotinic receptors of the nodular worm, Oesophagostomum dentatum (Ode-UNC-29:Ode-UNC-63:Ode-UNC-38), in Xenopus oocytes under voltage-clamp and tested effects of abamectin on pyrantel and acetylcholine responses. The receptors were antagonized by 0.03 µM abamectin in a non-competitive manner (reduced Rmax, no change in EC50). This antagonism increased when abamectin was increased to 0.1 µM. However, when we increased the concentration of abamectin further to 0.3 µM, 1 µM or 10 µM, we found that the antagonism decreased and was less than with 0.1 µM abamectin. The bi-phasic effects of abamectin suggest that abamectin acts at two allosteric sites: one high affinity negative allosteric (NAM) site causing antagonism, and another lower affinity positive allosteric (PAM) site causing a reduction in antagonism. We also tested the effects of 0.1 µM derquantel alone and in combination with 0.3 µM abamectin. We found that derquantel on these receptors, like abamectin, acted as a non-competitive antagonist, and that the combination of derquantel and abamectin produced greater inhibition. These observations confirm the antagonistic effects of abamectin on nematode nicotinic receptors in addition to GluCl effects, and illustrate more complex effects of macrocyclic lactones that may be exploited in combinations with other anthelmintics.

Antioxidant, mutagenic and antimutagenic activities of an aqueous extract of Limoniastrum guyonianum gall.

An aqueous extract of Limoniastrum guyonianum gall (G extract) was tested on Salmonella typhimurium to assess its mutagenic and antimutagenic effects. This extract showed no mutagenicity when tested with S. typhimurium strain TA104 either with or without exogenous metabolic activation mixture (S9), whereas our findings revealed that the aqueous gall extract induced a mutagenic effect in S. typhimurium TA1538 when tested in the presence, as well as in the absence, of S9 activation mixture at the concentration of 500 µg/mL. Thus, the same concentration produced a mutagenic effect, when incubated with S. typhimurium TA100 in the presence of metabolic activation mixture. In contrast, our results showed a weak antimutagenic potential of the same extract against sodium azide in the presence of S. typhimurium TA100 and S. typhimurium TA1538 without metabolic activation (S9), whereas, in the presence of S. typhimurium TA104, we obtained a significant inhibition percentage (76.39%) toward 3.25 µg/plate of methylmethanesulfonate. Antimutagenicity against aflatoxin B1, 4-nitro-o-phenylene-diamine and 2-aminoanthracène was significant, with an inhibition percentage of, respectively, 70.63, 99.3 and 63.37% in the presence of, respectively, S. typhimurium TA100, S. typhimurium TA1538 and S. typhimurium TA104 strains at a concentration of 250 µg/plate after metabolic activation (S9). Antioxidant capacity of the tested extract was evaluated using the enzymatic (xanthine/xanthine oxidase assay) and the nonenzymatic (2,2-diphenyl-1-picrylhydrazyl) system. G extract exhibited high antioxidant activity.

Functional effects of the KCNQ modulators retigabine and XE991 in the rat urinary bladder.

The anticonvulsant retigabine has previously been reported to inhibit bladder overactivity in rats in vivo but the mechanism and site of action are not known. In the present study we investigated the effect of retigabine in isolated rat bladder tissue. Bladders from Sprague-Dawley rats were cut transversally into rings and mounted on an isometric myograph. The average tension, the amplitude and frequency of bladder muscle twitches were measured. The bladder tissue was stimulated with carbachol, KCl (5, 10 and 60mM), and by electric field stimulation. Dose-response curves were obtained with increasing concentrations of the KCNQ((2-5)) selective positive modulator, retigabine or with the KCNQ((1-5)) negative modulator XE991. Retigabine experiments were repeated in the presence of 10 microM XE991. Retigabine reduced both the contractility and the overall tonus of bladder tissue independent of the mode of stimulation with EC(50) values ranging from 3.3 microM (20mM KCl) to 8.3 microM (0.2 microM carbachol). In support of a KCNQ-specific effect, retigabine had only weak effects after 60mM KCl pre treatment and all retigabine effects could be reversed by XE991. XE991 increased both the amplitude and mean tension of the bladder but was more potent at increasing the number rather than the size of the stimulated twitches. In conclusion, this study demonstrates an efficacious KCNQ dependent effect of retigabine and XE991 on rat bladder contractility.

Retigabine-induced population primary afferent hyperpolarisation in vitro.

Retigabine is a compound of potential interest in analgesia which acts as an M-channel opener to depress neuronal excitability. Here we study the effects of retigabine and its antagonist XE-991 on populations of primary afferents. Experiments were performed using a hemisected spinal cord preparation from rat pups maintained under in vitro conditions. Recording from dorsal roots were performed using tight fitting suction electrodes coupled to AC and DC amplifiers. The adjacent dorsal root was electrically stimulated at regular intervals. The effects of the modulators on basal potential, spontaneous potentials and dorsal root-dorsal root responses were studied. Superfusion of retigabine (10 microM) produced long lasting and robust hyperpolarisation of primary afferents which persisted during superfusion of picrotoxin (20 microM) and tetrodotoxin (0.5 microM). Other effects of retigabine were (1) increase in stimulation threshold; (2) increase in size of responses to suprathreshold stimuli; and (3) increase in amplitude and decrease in frequency of spontaneous dorsal root potentials. Superfusion of XE-991 had little effect on its own but blocked all the effects of retigabine. These results indicate the presence of functional M-currents in central terminals of primary afferents and in the interneurones that mediate dorsal root potentials. The depressant effects of retigabine on the excitability of these structures may contribute to its analgesic effects after pain-inducing treatments.

A notable antimutagenicity of two nonmutagenic novel oxadiazoles in Salmonella mutagenicity assay.

The mutagenic activity of two newly synthesized oxadiazoles: 1,3-bis(5-benzylthio-1,3,4-oxadiazol-2-yl) benzene (M1) and 1,4-bis(5-benzylthio-1,3,4-oxadiazol-2-yl) benzene (M2) was studied in Salmonella typhimurium strains TA97, TA100, TA102 and TA1537 in the presence and absence of S9mix. The antimutagenicity of M1 and M2 against H2O2, sodium azide (SA) and 4-nitro-o-phenylene diamine (NPD) using the tester strains TA102, TA100 and TA97, respectively, was also investigated. The two compounds were found to be nonmutagenic using the four tester strains. However, they showed high mutagenic repression activity against hydrogen peroxide (95% and 97% for M1 and M2, respectively, at a concentration of 335 micrograms/plate). Moderate mutagenic repression against NPD (58% and 55% for M1 and M2, respectively, at a concentration of 167.5 micrograms/plate) and low mutagenic repression against SA (21% and 33% for M1 and M2 respectively, at a concentration of 335 micrograms/plate) was detected. The obtained results are very encouraging to test the above mentioned compounds as anticarcinogens.

Antimutagenic activities of acetone and methanol fractions of Terminalia arjuna.

The antimutagenic effect of benzene, chloroform, acetone and methanol fractions from Terminalia arjuna, a well-known medicinal plant, was determined against Acid Black dye, 2-aminofluorene (2AF) and 4-nitro-o-phenylenediamine (NPD) in TA98 Frameshift mutagen tester strain of Salmonella typhimurium. Among the different fractions, the antimutagenic effect of acetone and methanol fractions was more than that observed with other fractions. Co-incubation and pre-incubation modes of experimentation did not show much difference in the antimutagenic activity of the extracts. Moreover, these fractions inhibited the S9-dependent mutagens, 2AF and Acid Black dye more effectively than the direct-acting mutagens. Studies are under way to isolate and elucidate the nature of the antimutagenic factor in acetone and methanol fractions.

Melanoidins exert a weak antiradical activity in watery fluids.

The Maillard reaction has a dramatic impact on the overall acceptance and nutritional quality of most of the foods consumed daily in European countries. Melanoidins are polymeric structures formed in the last stage of the Maillard reaction with nearly unknown effect on the human health. The antiradical activity of several melanoidins isolated from model systems and coffee has been studied. A novel antiradical efficiency concept has been applied to describe the antiradical activity in an aqueous medium by bleaching the radical cation N,N-dimethyl-p-phenylenediamine (DMPD(*)(+)). Melanoidins exerted a significantly lower antiradical activity than classical antioxidant compounds (tannic acid, ferrulic acid, caffeic acid, gallic acid, and Trolox) in an aqueous medium. Significant differences have been observed according to the type of amino acid used as reactant during the formation of the melanoidin structure and the antiradical efficiency exerted.

Anti-mutagenic activity of Phyllanthus amarus Schum & Thonn in vitro as well as in vivo.

Methanolic extract of Phyllanthus amarus was tested for its anti-mutagenic activity in Salmonella typhimurium strains TA1535, TA100, and TA102 (Ames test). P. amarus extract was able to inhibit the activation and mutagenicity of 2-acetaminofluorene (2-AAF) and aflatoxinB(1) at concentrations of 0.25-2 mg/plate. It was also found to inhibit mutagenicity induced by direct acting mutagens sodium azide (NaN(3)), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), and 4-nitro-0-phenylenediamine (NPD), at concentrations of 1 mg to 0.25 mg/plate. Urinary mutagenicity produced in rats by benzo[a] pyrene was found to be significantly inhibited by the oral administration of Phyllanthus extract. These results indicate significant anti-mutagenicity of the extract in vitro as well as in vivo.

Inhibition of irritation and contact hypersensitivity by phenoxyacetic acid methyl ester in mice.

New anti-irritant treatments are required to prevent irritation and sensitization reactions to consumer medicines and dermatological drugs. We report here that phenoxyacetic acid methyl ester (PAME) is an effective agent to prevent and treat irritant and allergic contact dermatitis. Balb/c mice skin-treated with 1% PAME do not lose weight relative to vehicle-treated mice, nor is it irritating to mouse skin. Topical PAME prevents skin irritation to a wide variety of irritants including: arachidonic acid, capsaicin, sodium lauryl sulfate (SLS), disodium laureth sulfosuccinate and tetradecanoylphorbol-13-acetate. Histological studies showed that 1% PAME greatly diminished dermal neutrophilic infiltration and dermal capillary vessel dilation, and prevented epidermal hyperproliferation and hyperkeratosis that accompanies detergent (SLS)-induced skin irritation. Topical PAME inhibited ear swelling following ear challenge during the elicitation phase of contact hypersensitivity in mice sensitized with 1-chloro-2, 4-dinitrochlorobenzene (DNCB), oxazolone and the hair coloring dye rho-phenylenediamine (PPD). Finally, topical administration of 1% PAME prior to PPD or DNCB sensitization prevented the induction phase of contact hypersensitivity. These results indicate that PAME represents a potential new category of potent topical anti-inflammatory agents.

Diaminotoluenes induce intrachromosomal recombination and free radicals in Saccharomyces cerevisiae.

The carcinogenicity of aniline-based aromatic amines is poorly reflected by their activity in short-term mutagenicity assays such as the Salmonella typhimurium reverse mutation (Ames) assay. More information about the mechanism of action of such carcinogens is needed. Here we report the effects on DEL recombination in Saccharomyces cerevisiae of the carcinogen 2,4-diaminotoluene and its structural isomer 2,6-diaminotoluene, which is reported to be non-carcinogenic. Both compounds are detected as equally mutagenic in the Salmonella assay. In the absence of any external metabolizing system both compounds were recombinagenic in the DEL assay with the carcinogen being a more potent inducer of deletions than the non-carcinogen. In the presence of Aroclor-induced rat liver S9, however, the carcinogen 2,4-diaminotoluene became a 2-fold more potent inducer of deletions, and the non-carcinogen 2,6-diaminotoluene was rendered less toxic and no induced recombination was observed. 2,4-Diaminotoluene is distinguished from its non-carcinogen analog in the DEL assay, therefore, on the basis of a preferential activation of the carcinogen in the presence of a rat liver microsomal metabolizing system. Free radical species are produced by several carcinogens and have been implicated in carcinogenesis. We further investigated whether exposure of yeast to either 2,4-diaminotoluene or 2,6-diaminotoluene resulted in a rise in intracellular free radical species. The effects of the free radical scavenger N-acetylcysteine on toxicity and recombination induced by the two compounds and intracellular oxidation of the free radical-sensitive reporter compound dichlorofluorescin diacetate were studied. Both 2,4- and 2,6-diaminotoluene produced tree radical species in yeast, indicating that the reason for the differential activity of the compounds for induced deletions is not reflected in any difference in the production of free radical species.

Antimutagenic effects of ajoene, an organosulfur compound derived from garlic.

The antimutagenic effects of ajoene, which is an organosulfur compound derived from garlic, were investigated by the Ames test. Ajoene inhibited mutagenesis induced by both benzo[a]pyrene (B[a]P) and 4-nitro-1,2-phenylenediamine (NPD) in a dose-dependent manner. In particular, NPD-induced mutagenesis was more effectively suppressed by ajoene than the B[a]P-induced type. Furthermore, the inhibition of mutagenesis by ajoene was more effective for transition-type mutations than for the frame shift type. HPLC analysis of B[a]P metabolism in the presence of the rat liver microsomal fraction (S-9) showed that ajoene dose-dependently inhibited the metabolic activation of B[a]P. This suggests that ajoene affected the metabolic enzymes in the S-9 fraction.

[Suppression of phosphofructokinase (PFK) by sera from cancer patients, and mechanism of the antagonistic effect of PSK].

Body fluids from cancer patients (sera, pleural effusions and ascites) tended to inhibit phosphofructokinase (ATP: D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) (PFK), the rate-limiting key enzyme in the glycolysis pathway, when analysed using aqueous ATP solution separately from the main reaction mixture. A protein-bound polysaccharide from Coliolus versicolor QUEL (Krestine, PSK) antagonistically elevated the activity of the enzyme. It was found that PSK stabilized PFK in a similar way to certain enzyme stabilizers such as proteins and polysaccharides. Furthermore, it was clarified that PSK worked as an ion radical scavenger that could capture 1O2, O-2, and OH X radicals released from lipoperoxides. In other words PSK protects PFK from hyperoxidation by lipoperoxides.

2,4-diaminoanisole-induced thyroid pigmentation in rats inhibited by m-phenylenediamine.

The effect of the carcinogenic hair dye component 2,4-diaminoanisole (2,4-DAA) on thyroid and pituitary morphology was studied. Heavy pigmentation of the hypertrophied thyroid epithelium was present when the animals were fed with 2,4-DAA for 6 weeks. Another hair dye component m-phenylenediamine (m-PDA), which lacks the methoxy group, and which is not carcinogenic, prevented the dark pigmentation of the thyroid epithelium caused by 2,4-DAA, but had only a slight effect on the hypertrophy of the gland. In the pituitary gland of 2,4-DAA-fed animals only a few aldehyde fuchsin positive thyrotrope cells were present. When 2,4-DAA and m-PDA were fed simultaneously, the number of hypertrophied chromophobic cells was greatly increased and the aldehyde fuchsin-positive cells were practically absent.