Alina Błaszczyk

Ethoxyquin: An Antioxidant Used in Animal Feed

Ethoxyquin (EQ, 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline) is widely used in animal feed in order to protect it against lipid peroxidation. EQ cannot be used in any food for human consumption (except spices, e.g., chili), but it can pass from feed to farmed fish, poultry, and eggs, so human beings can be exposed to this antioxidant. The manufacturer Monsanto Company (USA) performed a series of tests on ethoxyquin which showed its safety. Nevertheless, some harmful effects in animals and people occupationally exposed to it were observed in 1980s which resulted in the new studies undertaken to reevaluate its toxicity. Here, we present the characteristics of the compound and results of the research, concerning, for example, products of its metabolism and oxidation or searching for new antioxidants on the EQ backbone.

Comparative analysis of cytotoxic, genotoxic and antioxidant effects of 2,2,4,7-tetramethyl-1,2,3,4-tetrahydroquinoline and ethoxyquin on human lymphocytes.

2,2,4,7-Tetramethyl-1,2,3,4-tetrahydroquinoline (THQ) is a new synthetic compound with potential antioxidant activity. In this study, cytotoxic, genotoxic and antioxidant activities of THQ were studied on human lymphocytes with the use of the trypan blue exclusion assay, the TUNEL method, the comet assay and the micronucleus test. The activities of THQ were compared with those of a structurally similar compound-ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline, EQ), which is used in animal feeds as a preservative. Cytotoxic effects of THQ were observed after 1-h treatment at the concentration of 500 microM and after 24-h treatments at the concentrations of 250-500 microM. Although the micronucleus test did not reveal a genotoxic effect of THQ, in the comet assay the statistically significant increase in DNA damage was observed as compared with the control. On the other hand, the protection of human lymphocytes against DNA damage induced by hydrogen peroxide suggests an antioxidant activity of THQ. The comparative analysis of THQ and EQ activities performed in these studies revealed that THQ was less cytotoxic and less genotoxic than EQ. Slightly lower antioxidant activity of THQ was also shown in the comet assay when it was used at the lower studied doses (1-5 microM), but for the highest one (10 microM) its efficiency was similar to that of EQ. In the micronucleus assay THQ was more effective than EQ in protecting the cultured lymphocytes from clastogenicity of H2O2. We believe that THQ is worthy of further detailed studies on its antioxidant properties to confirm its usefulness as a preservative.

Modulation of ethoxyquin genotoxicity by free radical scavengers and DNA damage repair in human lymphocytes

N-tert-butyl-alpha-phenylnitrone (PBN) and its new derivative N-(Pyridine-4-ylmethylidene)-2-carboxy-tert-butylamine N-oxide (PBNC) were synthesized and used to modulate ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline, EQ) genotoxicity. Ethoxyquin, an antioxidant used mainly as a preservative in animal feeds, was shown to cause DNA breaks in human lymphocytes. The aim of the study was to evaluate the involvement of free radicals in the genotoxicity of EQ and its modulation by cellular repair systems. Human lymphocytes treated with EQ (10-50 microM) and nitrone free radical scavengers (100 microM) were tested with the comet assay. It was shown that both PBN and PBNC reduced the level of EQ-induced DNA damage, but PBN was slightly more effective. The modulation of the level of DNA damage was also observed as a result of DNA repair by cellular repair systems. Moreover, induction of oxidized bases by ethoxyquin was showed; lymphocytes exposed to ethoxyquin and treated with endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (FpG), enzymes recognizing oxidized bases, displayed greater extent of DNA damage than those not treated with the enzymes.

Cytotoxicity of nitroxyl (HNO/NO−) against normal and cancer human cells

Nitroxyl (HNO/NO(-)) is a molecule which results from one-electron reduction of nitric oxide (NO). It is considered to be a pharmacologically important particle because of its cardiovascular effects and potential anticancer activity. Due to molecule instability studies on nitroxyl biological activity require the use of donor compounds. In the present study Angelis salt (Na2N2O3) was used as the nitroxyl donor and cytotoxicity of HNO/NO(-) against human lymphocytes and A549 and HT29 cancer cells was examined. The studies were performed under different pH conditions (pH 6.2 and 7.4) because it was suggested that formation of HNO and its toxicological effects may be stronger in tissues subjected to acidosis such as cancerous ones. It was shown that nitroxyl exerted cytotoxic effect against all three types of cells. Nitroxyl induced both apoptotic and necrotic cell death. The cytotoxic effect analyzed directly after cell treatment was stronger than that observed 24h later. Differences in cell sensitivity to nitroxyl were observed--proliferating lymphocytes were the most sensitive cells. It was also shown that pro-oxidant activity of nitroxyl was stronger under slightly acidic conditions as compared to physiological pH but this difference was not always reflected in the observed cytotoxic effect, especially when the effect was measured 24h after cell treatment. Thus, relatively high toxicity of nitroxyl against normal cells and its ability to induce not only apoptotic but also necrotic cell death make use of this molecule in cancer therapy questionable.

Cytotoxicity and Genotoxicity of Ethoxyquin Used As an Antioxidant

Ethoxyquin (EQ; 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline) is widely used as an antioxidant in animal feed and may be carried over to food products of animal origin. It is believed that there are no adverse effects to human health resulting from exposure to EQ as long as it is used at the permitted concentrations. Nevertheless, some harmful effects in animals were observed in the 1980s and some studies were undertaken to evaluate EQ toxicity. This paper reviews EQ’s potential pro-oxidant activity and the ability to cause DNA damage and chromosome aberrations, which may have negative biological consequences. Food safety aspects are also discussed.

Evaluation of the genotoxic and antioxidant effects of two novel feed additives (ethoxyquin complexes with flavonoids) by the comet assay and micronucleus test

The complexes of antioxidant ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline; EQ) with rutin or quercetin (EQ-R and EQ-Q, respectively) were studied in human lymphocytes for genotoxic and antioxidant activities with the use of the comet assay and micronucleus test. The study was undertaken to search for new potential antioxidants, and was motivated by reports of unfavourable side-effects observed in animals fed with feeds containing EQ, which is allowed up to 150 mg kg−1 (0.015%) in complete animal feed. It was shown that EQ-R induced DNA damage in human lymphocytes when used at all the concentrations studied (1–25 µM), while after EQ-Q treatment, the genotoxic effect was observed mainly after higher doses (10 and 25 µM). An increase in the number of micronuclei was observed only for EQ-Q after a dose of 50 µM. The studied compounds decreased the degree of DNA damage induced by hydrogen peroxide (10 µM) in the comet assay. The results obtained in both tests showed that the antioxidant activity of EQ-Q was comparable with that of EQ, so further detailed studies are necessary to estimate its possible usefulness as a feed preservative.

Synthesis and cytotoxicity of (R)- and (S)-ricinoleic acid amides and their acetates

An environment-friendly, free of solvent, process for the synthesis of (R)- and (S)-ricinoleic acid amides has been developed. Starting from methyl ricinoleates and pyrrolidine or ethanolamine, the corresponding amides were obtained with yields ranging from 83-88%. Among 12 synthesized derivatives of ricinoleic acid, including the starting methyl esters, amides, and their acetates, nine compounds were obtained and tested for the first time. Studies on ricinoleic acid derivatives cytotoxicity showed that methyl esters were the least cytotoxic compounds and modification of their structure resulted in increasing cytotoxicity of the obtained products against both cancer cells and normal lymphocytes. Both enantiomers of the ethanolamine-derived amides showed the most promising anticancer potential.