Ethoxyquin, a Lipid Peroxidation Inhibitor, Has Protective Effects against White Matter Lesions in a Mouse Model of Chronic Cerebral Hypoperfusion.

White matter lesions induced by chronic cerebral hypoperfusion can cause vascular dementia; however, no appropriate treatments are currently available for these diseases. In this study, we investigated lipid peroxidation, which has recently been pointed out to be associated with cerebrovascular disease and vascular dementia, as a therapeutic target for chronic cerebral hypoperfusion. We used ethoxyquin, a lipid-soluble antioxidant, in a neuronal cell line and mouse model of the disease. The cytoprotective effect of ethoxyquin on glutamate-stimulated HT-22 cells, a mouse hippocampal cell line, was comparable to that of a ferroptosis inhibitor. In addition, the administration of ethoxyquin to bilateral common carotid artery stenosis model mice suppressed white matter lesions, blood-brain barrier disruption, and glial cell activation. Taken together, we propose that the inhibition of lipid peroxidation may be a useful therapeutic approach for chronic cerebrovascular disease and the resulting white matter lesions.

Ethoxyquin is a Competent Radical-Trapping Antioxidant for Preventing Ferroptosis in Doxorubicin Cardiotoxicity.

ABSTRACT: Doxorubicin (DOX) is an effective anti-cancer agent for various malignancies. Nevertheless, it has a side effect of cardiotoxicity, referred to as doxorubicin-induced cardiomyopathy (DIC), that is associated with a poorer prognosis. This cardiotoxicity limits the clinical use of DOX as a therapeutic agent for malignancies. Recently, ferroptosis, a form of regulated cell death induced by the accumulation of lipid peroxides, has been recognized as a major pathophysiology of DIC. Ethoxyquin is a lipophilic antioxidant widely used for food preservation and thus may be a potential therapeutic drug for preventing DIC. However, the efficacy of ethoxyquin against ferroptosis and DIC remains to be fully elucidated. Here, we investigated the inhibitory action of ethoxyquin against GPx4-deficient ferroptosis and its therapeutic efficacy against DOX-induced cell death in cultured cardiomyocytes and cardiotoxicity in a murine model of DIC. In cultured cardiomyocytes, ethoxyquin treatment effectively prevented GPx4-deficient ferroptosis. Ethoxyquin also prevented DOX-induced cell death, accompanied by the suppression of malondialdehyde (MDA) and mitochondrial lipid peroxides, which were induced by DOX. Furthermore, ethoxyquin significantly prevented DOX-induced cell death without any suppression of caspase cleavages representing apoptosis. In DIC mice, ethoxyquin treatment ameliorated cardiac impairments, such as contractile dysfunction and myocardial atrophy, and lung congestion. Ethoxyquin also suppressed serum lactate dehydrogenase and creatine kinase activities, decreased the levels of lipid peroxides such as MDA and acrolein, inhibited cardiac fibrosis, and reduced TUNEL-positive cells in the hearts of DIC mice. Collectively, ethoxyquin is a competent antioxidant for preventing ferroptosis in DIC and can be its prospective therapeutic drug.

Development of EQ-6, a Novel Analogue of Ethoxyquin to Prevent Chemotherapy-Induced Peripheral Neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a common and often dose-limiting side effect of many cancer drugs. Because the onset of neuronal injury is known, it is an ideal clinical target to develop neuroprotective strategies. Several years ago, we had identified ethoxyquin as a potent neuroprotective drug against CIPN through a phenotypic drug screening and demonstrated a novel mechanism of action, inhibition of chaperone domain of heat shock protein 90. To improve its drug-like properties we synthesized a novel analogue of ethoxyquin and named it EQ-6 (6-(5-amino)-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline hydrochloride). Here we show that EQ-6 prevents axon degeneration in primary dorsal root ganglion neurons in vitro, and this axon protection is associated with preserved levels of nicotinamide adenine dinucleotide, a key metabolite in programmed axon degeneration pathway. We also found that EQ-6 prevents loss of epidermal nerve fibers in a mouse model of CIPN induced by paclitaxel and that doses of EQ-6 that provide neuroprotection are associated with reduced tissue levels of SF3B2, a potential biomarker of target engagement. Furthermore, we show that EQ-6 is safe in vitro and in mice with daily administration for a month. We found that oral bioavailability is about 10%, partly due to rapid metabolism in liver, but EQ-6 appears to be concentrated in neural tissues. Given these findings, we propose EQ-6 as a first-in-class drug to prevent CIPN.

Experimental vermeersiekte (Geigeria ornativa O. Hoffm. poisoning) in sheep. I: An evaluation of diagnostic aids and an assessment of the preventive effect of ethoxyquin.

Dried milled Geigeria ornativa, which had been stored at room temperature for 4 years, was fed in rations to sheep. The usefulness of contrast radiography of the oesophagus, biopsies of oesophageal and skeletal muscles, electrocardiography, and clinical pathological parameters for diagnosing vermeersiekte were evaluated. Ethoxyquin was evaluated as a preventive agent. All the sheep developed signs of vermeersiekte but regurgitation was observed in only one animal. Contrast radiography and the examination of tissue biopsies were of diagnostic value in 8 out of 13 and 6 out of 6 sheep respectively. Electrocardiography did not aid in antemortal diagnosis of the condition and selected chemical parameters were non-specific. The inclusion of ethoxyquin in the diet of experimental animals (n = 3) prior to and during feeding of G. ornativa did not prevent the development of vermeersiekte. This study confirmed that dried stored G. ornativa retains its toxicity and that regurgitation is not a consistent clinical sign in sheep with vermeersiekte. The diagnostic value of contrast radiography of the oesophagus and examination of oesophageal and muscle biopsies is reported for the first time.

Antioxidants delay the onset of thyroiditis in obese strain chickens.

Dietary iodine has been shown to be important in the induction of thyroiditis in susceptible chicken strains although the underlying mechanism remains unknown. Iodine may exert its effects through the formation of reactive oxidative radicals which would cause thyroidal injury and initiate infiltration. We have tested this hypothesis by examining the ability of butylated hydroxyanisole (BHA), ethoxyquin, and other antioxidants to prevent thyroiditis in Obese strain (OS) chickens, a strain that develops severe disease by 4 weeks of age. BHA, when administered from hatching until death at 5 weeks of age, reduced thyroidal infiltration and serum levels of antibodies binding thyroglobulin, T3, T4. Similar effects were observed with the antioxidant ethoxyquin. Weaker antioxidants such as vitamins C and E and beta-carotene had only slight or negligible effects on these parameters. BHA reduced thyroiditis in OS chicks killed at 3 and 5 weeks of age, but not at 8 weeks. When BHA treatment was initiated after the development of severe disease, it did not reduce thyroglobulin antibody levels. To determine the mechanism by which BHA reduces thyroiditis, studies were performed to assess the effect of BHA on thyroid function and on the immune responses to exogenous antigens. BHA had no effect on thyroid function in normal strain chickens since thyroidal radioiodine uptake and organification and serum T3 and T4 levels were unaffected. BHA did not alter immune responses to exogenous antigens such as sheep red blood cells or Brucella abortus in OS chickens. In summary, potent antioxidant drugs delayed the onset of thyroiditis when treatment was initiated before the onset of disease, suggesting that reactive oxygen intermediates are involved in the early stages of pathogenesis. However, the site of action remains unknown since they had no detectable effects on thyroid function or general immune responses.

Effect of vitamin E and synthetic antioxidants on the survival rate of mercury-poisoned Japanese quail.

The effect of vitamin E and the synthetic antioxidants, 6-ethyoxy,1,2-dihydro 2,2,4-trimethylquinoline (ethoxyquin), 2,6 bis(1,1 dimethyethyl)-4-methylphenol (BHT), N,N-diphenyl-p-phenylene diamine (DPPD), bis-(diethyl thiocarbamoyl) disulfide (Antabuse), and 2 tertiary-butyl-4-methoxyphenol (BHA) on organic mercury-induced mortality was investigated in Japanese quail. When the synthetic antioxidants, ethoxyquin, BHT, and Antabuse were fed at 1% of the diet, they induced mortality. Ethoxyquin was less toxic in combination with mercury (Hg) than when it or mercury was given alone. Of the antioxidants tested at .5% of the diet, only Antabuse was toxic as shown by increased mortality. At .5% of the diet, both ethoxyquin and DPPD reduced mortality associated with organic Hg poisoning. Neither BHA nor BHT had any effect in reducing Hg toxicity. In fact, mortality from organic Hg was greater when organic Hg was given in combination with .5% BHT than when given alone. Vitamin E was equal or superior to all synthetic antioxidants tested in alleviating the toxicity of organic Hg poisoning. The cause of observed antioxidant protection during organic Hg stress is not known but the protection may result from the ability to scavenge free radicals generated by induction of in vivo peroxidation by the Hg compound.

Ability of ethoxyquin and butylated hydroxytoluene to counteract deleterious effects of dietary aflatoxin in chicks.

The antioxidants ethoxyquin and butylated hydroxytoluene (BHT) were added to diets of chicks in concentrations three and eight times above that usually found in poultry feed beginning 15 days after hatch; the chicks had been placed on feed containing 1000 or 3000 ppb aflatoxin on the day of hatch. These diets were continued until chicks were 6 weeks of age. At that time, deleterious effects of aflatoxin on weight gain, feed efficiency, and organ weights (spleen, bursa) were evident. BHT alleviated these effects, but ethoxyquin did not. Pretreatment with ethoxyquin did not protect chicks either. Ethoxyquin was not able to induce the activities of chick liver enzymes that detoxify aflatoxin and other foreign compounds. Lack of effect of ethoxyquin on these enzymes may hinder ability of this antioxidant to protect chicks from aflatoxin.

Tissue thiol induction in mice and protective effect against pyrrolizidine alkaloids by dietary ethoxyquin and methionine hydroxy analog.

Dietary administration of ethoxyquin (EQ) and methionine hydroxy analog (MHA) induced the thiol concentrations in liver, kidney, small intestine and stomach of mice accompanied with hepatic hypertrophy. The hepatic thiol levels among EQ, EQ-MHA, or EQ-methionine treated mice were significantly higher (p less than 0.01) than that of the controls. Hepatic hypertrophy was apparent with 0.031% EQ-0.063% MHA in the diet. EQ feeding, as 0.5% w/w of feed with or without another additive, also resulted in reduced weight gain. The toxicity of pyrrolizidine alkaloids (PAs) was reduced by feeding diets containing 0.063% EQ-0.125% MHA or 0.125% EQ-0.25% MHA. The intraperitoneal LD50 values of PAs in these two EQ-MHA feeding groups were 92.8 and 94.0 mg/kg, respectively, compared to that of 71.3 mg/kg for the controls.

Elimination of adverse effects of ethoxyquin (EQ) by methionine hydroxy analog (MHA). Protective effects of EQ and MHA for bitterweed poisoning in sheep.

Dietary ethoxyquin (EQ) and methionine hydroxy analog (MHA) protected 6-8-month-old wethers from toxic doses of bitterweed (Hymenoxys odorata DC.). The EQ-MHA group received sweet feed (corn, oats, dehydrated alfalfa pellets, cane molasses and minerals), 500 g/day/sheep, supplemented with EQ and MHA (0.5% and 1.0% of feed, respectively) for 9 days prior to the poisoning with bitterweed while the MHA group received the same feed without EQ and controls received the same amount of feed with no additives. Two of 6 MHA-treated and 3 of 7 controls died whereas all 7 EQ-MHA-treated sheep survived after receiving 5 doses of bitterweed (5 X 5.5 g/kg) in 6 days. Coadministration of MHA and EQ eliminated the adverse effect of EQ; dietary EQ lowered the serum albumin, calcium, and alkaline phosphatase content while protecting the animals from bitterweed poisoning. EQ is the most promising protective agent tested for bitterweed poisoning in sheep.

Protective effects of antioxidants on bitterweed (Hymenoxys odorata DC) toxicity in sheep.

Antidotal effects of the 2 antioxidants butylated hydroxyanisole (BHA) and ethoxyquin (EQ) were evaluated in bitterweed (Hymenoxys odorata DC) toxicosis in sheep. Bitteerweed contains a toxic sesquiterpene lactone, hymenoxon, the toxicity of which is reduced by cysteine. Both BHA and EQ are known to induce hepatic glutathione production in rodents. Treatment of sheep with EQ (2.5 g/sheep/day for 9 days before poisoning) gave significant protection from toxic doses of bitterweed, but the protective effect of BHA was insignificant. Of 6 sheep given EQ in the feed, 5 survived 7 doses of bitterweed (4 g/kg/day or higher for 7 days), whereas 5 of 7 controls and 4 of 7 sheep given feed with added BHA died. The added EQ in the feed decreased the serum alkaline phosphatase activity and total protein, albumin, and calcium concentrations. Seemingly, EQ is the first protective agent with field application potential for bitterweed toxicity.

[Biological activity of vitamin E in feed mixtures with a varying santoquin content]. / Prouchvaniia vurkhu biologichnata aktivnost na vitamin E vuv furazhni smesi s razlichno sudurzhanie na santokvin.

Studied was the content of vitamins A and E as well as that of fats, aldehydes, and peroxides and the biologic activity of vitamin E with regard to the amount of santoquin in combined forage mixtures for birds. The experiments were carried out with a finisher forage mixture for broilers to which santoquin was added in amounts of 0.010, 0.016, 0.020, and 0.025 per cent. It was found that at an antioxidant concentration of 0.020 per cent a best protection of the biologic vitamin E activity was provided. The rapid inactivation of vitamin E at a level of santoquin lower than 0.016 per cent was associated with the intense deposition of products of the final oxidation when fats turned rancid. Taking into consideration the importance of fats as a source of energy a 0.020 per cent level of santoquin in the forage mixtures for broilers is suggested.

Preventive effect of selenium, methionine and antioxidants against encephalomalacia of chicks induced by dilauryl succinate.

The protective effect of supplemental selenium, methionine, ascorbic acid, menaquinone and five antioxidants against encephalomalacia of chicks fed a diet containing dilauryl succinate was examined. Diauryl succinate induces vitamin E deficiency signs such as fragility of the erythrocytes and encephalomalacia. Supplementation of selenium and methionine with or without simultaneous supplementation of a low level of dl-alpha-tocopheryl acetate had little effect on preventing encephalomalacia. The preventive effect of ascorbic acid, methylene blue, ethoyquine, 2,6-ditertiary-butyl-p-cresol and butylated hydroxyanisole was roughly in proportion to their dietary level, and a high level of any of them could almost completely protect the chicks from encephalomalacia, while diphenyl-p-phenylenediamine was not as effective and the effect was not proportional to the dose. Menaquinone had little effect. No difference was observed in the plasma tocopherol levels and peroxide levels in the adipose tissueof the chick fed eith er dilauryl succinate or cornstarch. The effect of dilauryl succinate appears to be independent of peroxides generated in the chick.

Free radical theory of aging: inhibition of amyloidosis in mice by antioxidants; possible mechanism.

The antioxidants, alpha-tocopherol acetate and a quinolone derivative (Santoquin), inhibited the development of amyloidosis when added to the diet of casein-injected C3HeB/FeJ male mice. Santoquin, and to a lesser extent butylated hydroxytoluene (BHT), also depressed the appearance of a plasma protein fraction in these mice; the effect of alpha-tocopherol was not determined. Consideration of the current knowledge of amyloid, in the light of these antioxidant studies, prompted the following hypothesis. Amyloidosis is largely the result of an enhanced rate of oxidative degradation of a connective-tissue glycoprotein(s) coupled with oxidative/enzymatic changes in the plasma, both of the tissue-derived substances and of immunoglobulins, to form the amyloid fibril protein subunits (AL and AA) which subsequently aggreagate to form the amyloid fibrils.