Ethoxyquin attenuates enteric oxidative stress and inflammation by promoting cytokine expressions and symbiotic microbiota in heat-stressed broilers.

Intestinal oxidative stress in broilers is produced by chronic heat stress (HS) and has a negative impact on poultry performance as it induces intestinal inflammation and promotes the invasion of gram-negative bacteria, such as bacterial lipopolysaccharide (LPS). Therefore, dietary inclusion of the antioxidant compound, ethoxyquin (EQ), could improve enteric antioxidant capacity, immune responses, and the epithelial barrier, and maintain the symbiotic gut microbiota community. To investigate the effects of EQ supplementation on alleviating enteric oxidative stress in heat-stressed broilers, 200 one-day-old male Ross 308 broilers were randomly assigned to 4 groups (n = 50 chicks/group; n = 10 chicks/replicate) and fed a basal diet supplemented with 0 (CT), 50 (EQ-50), 100 (EQ-100), and 200 (EQ-200) mg EQ/ kg-1 for 5 wk. The chicks were raised in floor pens inside the broiler farm at a temperature and humidity index (THI) of 29 from d 21 to d 35. Growth performance traits, relative organ index, hepatic antioxidant enzymes, serum immunity, total adenylate, and cytokine activities were improved in the EQ-50 group (linear or quadratic P < 0.05), promoting the relative mRNA expression of cytokine gene-related anti-inflammatory and growth factors. A distinct microbial community colonised the gut microbiota in the EQ-50 group, with a high relative abundance of Lactobacillus, Ligilactobacillus, Limosilactobacillus, Pediococcus, Blautia, and Faecalibacterium compared to the other groups. Dietary supplementation with 50 mg EQ/ kg-1 for 5 wk attenuates enteric oxidative stress and intestinal inflammation by enhancing serum immune and cytokine content (IgG, IL-6, and TGF-ß,) and symbiotic microbiota in heat-stressed broilers. EQ promotes the expression of Hsp70, SOD2, GPx 4, IL-6, and IGF-1 cytokine gene-related anti-inflammatory and growth factors in heat-stressed hepatic broilers. Collectively, EQ-50 could be a suitable feed supplement for attenuating enteric oxidative stress and intestinal inflammation, thereby promoting the productivity of heat-stressed broilers.

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.

Determination of ethoxyquin by ultra-high performance liquid chromatography with tandem mass spectrometry and a Singapore survey of ethoxyquin residues in eggs, egg products and poultry.

In this study, an advanced ultra-high performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method was developed for quantifying ethoxyquin (EQ). The approach employed a distinctive antioxidant added extraction step designed to prevent ethoxyquin decomposition and maintain analytical precision. This method effectively determines residue levels of EQ in eggs, processed egg products, poultry muscle, salmon, and liquid milk. The method was shown to have a limit of quantitation (LOQ) for eggs, milk, salmon, and chicken muscle of 1.5 µg/kg, 1.9 µg/kg, 2.1 µg/kg, and 1.2 µg/kg, respectively. The recoveries of EQ ranged from 79.2% to 107.6%, with a relative standard deviation (RSD) below 8.4%. A surveillance study for the presence of EQ in different types of eggs and poultry muscle available in Singapore was conducted and a total of 140 samples were tested. EQ residues in all samples were found to be below the U.S. Food and Drug Administration (FDA) MRLs of 500 µg/kg. Some samples of salted and preserved eggs from China were detected with higher concentration of EQ.

Monitoring the exposure to ethoxyquin between 2000 and 2021 in urine samples from the German Environmental Specimen Bank.

Human Biomonitoring (HBM) of emerging chemicals gained increasing attention within the EU in recent years. After evaluating the metabolism, we established a new HBM method for ethoxyquin (EQ), a feed additive, which was banned in 2017 due to concerns regarding the possible exposure of the general population to it and its highly toxic precursor p-phenetidine. The method was applied to 250 urine samples from the Environmental Specimen Bank collected between 2000 and 2021. The major metabolite EQI was quantified in the majority of the study samples illustrating the ubiquitous exposure of the non-occupationally exposed population. A rather constant exposure was observed until 2016 with a significant decline from 2016 to 2021. This drop falls within the EU wide ban of the chemical as a feed additive from June 2017 which led to a gradual removal until its complete suspension in June 2020. The daily intake (DI) was evaluated with respect to the reported derived no-effect level (DNEL) to estimate the potential health risks from EQ exposure. The median DI of 0.0181 µg/kg bw/d corresponds to only 0.01 % of the DNEL. Even the observed maxima up to 13.1 µg/kg bw/d only accounted for 10 % of the DNEL. Nevertheless, the values suggest a general exposure with the risk of higher burden in a low fraction of the population. In regard to the EQ associated intake of the carcinogen and suspected mutagen p-phenetidine, this level of exposure cannot be evaluated as safe. The recent decrease and the broad exposure substantiate the need for future HBM campaigns in population representative studies to further investigate the observed reductions, potentially find highly exposed subgroups and clarify the impact of the ban as feed additive on EQ exposure.

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.

Use of (Q)SAR genotoxicity predictions and fuzzy multicriteria decision-making for priority ranking of ethoxyquin transformation products.

Ethoxyquin (EQ; 6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline) has been used as an antioxidant in feed for pets and food-producing animals, including farmed fish such as Atlantic salmon. In Europe, the authorization for use of EQ as a feed additive was suspended, due to knowledge gaps concerning the presence and toxicity of EQ transformation products (TPs). Recent analytical studies focusing on the detection of EQ TPs in farmed Atlantic salmon feed and fillets reported the detection of a total of 27 EQ TPs, comprising both known and previously not described EQ TPs. We devised and applied an in silico workflow to rank these EQ TPs according to their genotoxic potential and their occurrence data in Atlantic salmon feed and fillet. Ames genotoxicity predictions were obtained applying a suite of five (quantitative) structure-activity relationship ((Q)SAR) tools, namely VEGA, TEST, LAZAR, Derek Nexus and Sarah Nexus. (Q)SAR Ames genotoxicity predictions were aggregated using fuzzy analytic hierarchy process (fAHP) multicriteria decision-making (MCDM). A priority ranking of EQ TPs was performed based on combining both fAHP ranked (Q)SAR predictions and analytical occurrence data. The applied workflow prioritized four newly identified EQ TPs for further investigation of genotoxicity. The fAHP-based prioritization strategy described here, can easily be applied to other toxicity endpoints and groups of chemicals for priority ranking of compounds of most concern for subsequent experimental and mechanistic toxicology analyses.

Microfluidic Electrochemistry Meets Trapped Ion Mobility Spectrometry and High-Resolution Mass Spectrometry-In Situ Generation, Separation, and Detection of Isomeric Conjugates of Paracetamol and Ethoxyquin.

Over the last 3 decades, electrochemistry (EC) has been successfully applied in phase I and phase II metabolism simulation studies. The electrochemically generated phase I metabolite-like oxidation products can react with selected reagents to form phase II conjugates. During conjugate formation, the generation of isomeric compounds is possible. Such isomeric conjugates are often separated by high-performance liquid chromatography (HPLC). Here, we demonstrate a powerful approach that combines EC with ion mobility spectrometry to separate possible isomeric conjugates. In detail, we present the hyphenation of a microfluidic electrochemical chip with an integrated mixer coupled online to trapped ion mobility spectrometry (TIMS) and time-of-flight high-resolution mass spectrometry (ToF-HRMS), briefly chipEC-TIMS-ToF-HRMS. This novel method achieves results in several minutes, which is much faster than traditional separation approaches like HPLC, and was applied to the drug paracetamol and the controversial feed preservative ethoxyquin. The analytes were oxidized in situ in the electrochemical microfluidic chip under formation of reactive intermediates and mixed with different thiol-containing reagents to form conjugates. These were analyzed by TIMS-ToF-HRMS to identify possible isomers. It was observed that the oxidation products of both paracetamol and ethoxyquin form two isomeric conjugates, which are characterized by different ion mobilities, with each reagent. Therefore, using this hyphenated technique, it is possible to not only form reactive oxidation products and their conjugates in situ but also separate and detect these isomeric conjugates within only a few minutes.

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.

Ethoxyquin is neuroprotective and partially prevents somatic and autonomic neuropathy in db/db mouse model of type 2 diabetes.

Ethoxyquin (EQ), a quinolone-based antioxidant, has demonstrated neuroprotective properties against several neurotoxic drugs in a phenotypic screening and is shown to protect axons in animal models of chemotherapy-induced peripheral neuropathy. We assessed the effects of EQ on peripheral nerve function in the db/db mouse model of type II diabetes. After a 7 week treatment period, 12-week-old db/db-vehicle, db/+ -vehicle and db/db-EQ treated animals were evaluated by nerve conduction, paw withdrawal against a hotplate, and fiber density in hindlimb footpads. We found that the EQ group had shorter paw withdrawal latency compared to vehicle db/db group. The EQ group scored higher in nerve conduction studies, compared to vehicle-treated db/db group. Morphology studies yielded similar results. To investigate the potential role of mitochondrial DNA (mtDNA) deletions in the observed effects of EQ, we measured total mtDNA deletion burden in the distal sciatic nerve. We observed an increase in total mtDNA deletion burden in vehicle-treated db/db mice compared to db/+ mice that was partially prevented in db/db-EQ treated animals. These results suggest that EQ treatment may exert a neuroprotective effect in diabetic neuropathy. The prevention of diabetes-induced mtDNA deletions may be a potential mechanism of the neuroprotective effects of EQ in diabetic neuropathy.

Ethoxyquin and Butylated Hydroxy Toluene Distrub the Hematological Parameters and Induce Structural and Functional Alterations in Liver of Rats.

The current experiment aimed to assess the effect of the synthetic antioxidants ethoxyquin (EQ) and/or butylated hydroxytoluene (BHT) on the liver function tests, hematological parameters, and liver histoarchitecture in rats. A total of 50 male Sprague-Dawley rats were divided into five groups of 10 animals per group. The first group served as the control and did not receive any treatments, and the second group served as the vehicle control and was orally administrated 1 ml of corn oil day after day for consecutive 45 and 90 days. The third group (EQ) was orally administered 1 ml of EQ dissolved in corn oil day after day for consecutive 45 and 90 days in a dose of 1/5 LD50, and the fourth group (BHT) was orally received 1 ml of BHT dissolved in corn oil day after day for consecutive 45 and 90 days in a dose of 1/5 LD50. The fifth group (combination group) was orally administered both EQ and BHT at the same doses and durations described above. The present results showed that the final body weight was significantly decreased in the EQ- or BHT-treated group particularly at 90 days of exposure to both compounds. Furthermore, the liver weight was significantly elevated in EQ, BHT, and co-exposed groups at 45 and 90 days of exposure, compared to the control group. Moreover, EQ, BHT, and their co-exposure caused a significant elevation in the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes, as well as total bilirubin at 45 and 90 days of exposure. On the other hand, there was no significant change in the total albumin. Hemoglobin value, red blood cells, white blood cells, platelets, and differential leucocyte count at 45 and 90 days of exposure were significantly decreased. Histopathological significant findings in the liver were observed as vascular congestions, vacuolations, hydropic degenerations, lipidosis, and swelling, particularly in the co-exposed group for 90 days. These findings confirmed the hepatotoxic potential of EQ and BHT; therefore, it is recommended to control and limit the utilization of such chemicals.

Development of a human biomonitoring method for assessing the exposure to ethoxyquin in the general population.

Ethoxyquin (EQ) is commonly used as an antioxidant in animal feeds. Although EQ is not permitted for usage in food products for humans within the EU, residues of EQ and its transformation products could be determined in food of animal origin. Despite its widespread use and concerns on its toxicological profile, no information about the systemic exposure to EQ in the general population is available. Hence, we developed a human biomonitoring (HBM) method for EQ. Our approach included a metabolism study with five subjects, who were administered an oral dose of 0.005 mg EQ/kg body weight. Unchanged EQ and the major metabolite 2,2,4-trimethyl-6(2H)-quinolinone (EQI) were identified as urinary excretion products of EQ. While small amounts of EQ could be determined in high concentrated samples from the metabolism study only, 28.5% of the orally applied EQ dose could be recovered as EQI. Toxicokinetic parameters were determined for EQI, the potential biomarker of exposure. In addition, an analytical method for EQI (LOQ = 0.03 µg/L) in urine based on UHPLC-MS/MS comprising enzymatic glucuronide hydrolysis and salt-assisted liquid-liquid extraction was developed, validated and applied to 53 urine samples from the general population. EQI could be quantified in 11 (21%) of the samples in levels up to 1.7 µg/L urine, proving the suitability of the developed method for the intended purpose.

Analysis of ethoxyquin residues in animal feed using QuEChERS and gas chromatography tandem mass spectrometry and its results from Catalonian production 2018-2019.

Ethoxyquin (EQ) is a quinolone commonly used as an antioxidant additive and a fungicide. However, Regulation (EU) 2017/962 suspended its authorisation as a feed additive for all animal species and categories. The aim of this study is thus to ensure compliance with this regulation by developing a method of analysing EQ in animal feed. For analysis, EQ was extracted from animal feed by using a modified QuEChERS protocol that used an ascorbic acid buffer to minimise its degradation. The extracts were analysed by gas chromatography coupled to triple quadrupole mass spectrometry (GC-QQQ), obtaining two chromatographic peaks corresponding to EQ and its transformation products. A study of these peaks was subsequently carried out using different standards, evincing that EQ had indeed been converted into several different transformation products. Quantification required the sum of the areas of the different peaks. The method was validated according to European Commission guidelines, namely SANTE/12682/2019. The obtainment of pesticide-free samples for carrying out the validation process was a critical achievement, as EQ residues were detected in 90% of the analysed samples. This was made possible by manufacturing 20 different feed samples from a mixture of several cereals used in animal feed in the laboratory. Method validation yielded excellent results in terms of accuracy (recoveries 70-120%), precision (RSD < 20%) and linearity (r2≥ 0.99) at the studied levels, as well as excellent sensitivity and selectivity (retention time ±0.1 min; ratio < 30%) to the LoQ. Over the course of 2018 and 2019, 70 samples of various feed matrices from agricultural production in Catalonia were analysed, garnering positive results 43% of the time.

CGSEA: A Flexible Tool for Evaluating the Associations of Chemicals with Complex Diseases.

The etiology of many human complex diseases or traits involves interactions between chemicals and genes that regulate important physiological processes. It has been well documented that chemicals can contribute to disease development through affecting gene expression in vivo In this study, we developed a flexible tool CGSEA for scanning the candidate chemicals associated with complex diseases or traits. CGSEA only need genome-wide summary level data, such as transcriptome-wide association studies (TWAS) and mRNA expression profiles. CGSEA was applied to the GWAS summaries of attention deficiency/hyperactive disorder, (ADHD), autism spectrum disorder (ASD) and cervical cancer. CGSEA identified several significant chemicals, which have been demonstrated to be involved in the development or treatment of ADHD, ASD and cervical cancer. The CGSEA program and user manual are available at https://github.com/ChengSQXJTU/CGSEA.

The food preservative ethoxyquin impairs zebrafish development, behavior and alters gene expression profile.

In the present study, we investigated the detrimental effects of ethoxyquin (EQ) on zebrafish embryonic development using different endpoints including lethality, malformations, locomotion and gene expression. EQ is primarily used as a preservative in animal feed and it has been shown to have negative impacts on different laboratory animals. However, studies on the adverse effects of EQ in aquatic animals are still limited. In this study, zebrafish eggs were exposed to different concentrations of EQ ranging from 1 to 100 µM for six days. In the 100 µM treated groups 95 and 100% mortality was observed at 24 and 48 h, respectively. Delayed development, decreased pigmentation and pericardial edema were observed in larvae. Behavioral analysis of larvae demonstrated a distinct locomotive pattern in response to EQ both in light and dark indicating a possible developmental neurotoxicity and deficits in locomotion. The expression levels of genes involved in several physiological pathways including stress response, cell cycle and DNA damage were altered by EQ. Our results demonstrate that EQ could cause developmental and physiological toxicity to aquatic organisms. Hence, its toxic effect should be further analyzed and its use and levels in the environment must be monitored carefully.

Modelling of the feed-to-fillet transfer of ethoxyquin and one of its main metabolites, ethoxyquin dimer, to the fillet of farmed Atlantic salmon (Salmon salar L.).

Ethoxyquin (EQ) is an antioxidant supplemented to feed ingredients, mainly fish meal, which is currently under re-evaluation for use in the food production chain. EQ is partly metabolized into several metabolites of which the ethoxyquin dimer (EQDM) accumulates most in the farmed fish fillet. In this study, the feed-to-fillet transfer of dietary EQ and EQDM in Atlantic salmon fillet was investigated, and a physiologically based pharmacokinetic (PBPK-) two-compartmental model was developed, based on experimental determined EQ and EQDM uptake, metabolism, and elimination kinetics. The model was verified with an external data-set and used to simulate the long term (>1.5 years) EQ and EQDM feed-to fillet transfer in Atlantic salmon under realistic farming conditions such as the seasonal fluctuations in feed intake, growth, and fillet fat deposition. The model predictions showed that initial EQDM levels in juvenile fish are the driving factor in final levels found in food-producing animals, while for EQ the levels in feed, and seasonal variations were the driving factor for food EQ levels.

Determination of the Synthetic Antioxidant Ethoxyquin and Its Metabolites in Fish and Fishery Products Using Liquid Chromatography-Fluorescence Detection and Stable-Isotope Dilution Analysis-Liquid Chromatography-Tandem Mass Spectrometry.

The use of the synthetic antioxidant ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline, EQ) as a flame retardant in fish meal transported by sea is required by international authorities to prevent self-ignition. Because of extensive carry-over within the food chain, selective and sensitive analytical methods are required for investigations on human exposure and the safety of EQ and its metabolites. Therefore, a simple, fast, and rugged liquid-chromatography (LC) method was developed for the detection of EQ and its metabolites in fish and fishery products after liquid-liquid extraction using QuEChERS. For screening purposes, a fluorescence detector was used (LC-FLD) with the EQ-analogue methoxyquin serving as an internal standard. For stable-isotope dilution analysis by liquid chromatography-tandem mass spectrometry (SIDA-LC-MS/MS), deuterated analogues of EQ and its metabolites were synthesized for the first time and allowed for sensitive quantification and thus confirmation of screening results. Both methods were validated and successfully applied to commercially available fish samples.

Identification of ethoxyquin and its transformation products in salmon after controlled dietary exposure via fish feed.

Ethoxyquin (EQ) is an additive present in fish feed and its fate in fish should be carefully characterized due to food safety concerns regarding this compound. Therefore, the objective of this work was to identify the transformation products (TPs) of EQ in Atlantic salmon. Salmon in independent tanks were given feed containing ethoxyquin concentrations of 0.5 mg/kg, 119 mg/kg or 1173 mg/kg for 90 days. After the feeding trial, salmon fillets were extracted in acetonitrile and analyzed by liquid chromatography with traveling-wave ion mobility spectrometry coupled to high resolution mass spectrometry (UHPLC-TWIMS-QTOFMS). EQ was transferred from the feed to salmon fillets and 23 TPs were characterized, resulting from dimerization, oxygenation, cleavage, cleavage combined with oxygenation, cleavage combined with conjugation, and other uncategorized alterations. Moreover, EQ and some TPs were also detected in commercial salmon randomly sampled from different Norwegian fish farms. This study confirmed that the dimer 1,8'-EQDM was the main TP of EQ and, together with previous research, brought the overall number of characterized TPs to a total of 47.

Tolerance and dose-response assessment of subchronic dietary ethoxyquin exposure in Atlantic salmon (Salmo salar L.).

Ethoxyquin (EQ; 6-Ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline) has been used as an antioxidant in feed components for pets, livestock and aquaculture. However, possible risks of EQ used in aquafeed for fish health have not yet been characterized. The present study investigated the toxicity and dose-response of subchronic dietary EQ exposure at doses ranging from 41 to 9666 mg EQ/kg feed in Atlantic salmon (Salmo salar L.). Feed at concentrations higher than 1173 mg EQ/kg were rejected by the fish, resulting in reduced feed intake and growth performance. No mortality was observed in fish exposed to any of the doses. A multi-omic screening of metabolome and proteome in salmon liver indicated an effect of dietary EQ on bioenergetics pathways and hepatic redox homeostasis in fish fed concentrations above 119 mg EQ/kg feed. Increased energy expenditure associated with an upregulation of hepatic fatty acid ß-oxidation and induction and carbohydrate catabolic pathways resulted in a dose-dependent depletion of intracytoplasmic lipid vacuoles in liver histological sections, decreasing whole body lipid levels and altered purine/pyrimidine metabolism. Increased GSH and TBARS in the liver indicated a state of oxidative stress, which was associated with activation of the NRF2-mediated oxidative stress response and glutathione-mediated detoxification processes. However, no oxidative DNA damage was observed. As manifestation of altered energy metabolism, the depletion of liver intracytoplasmic lipid vacuoles was considered the critical endpoint for benchmark dose assessment, and a BMDL10 of 243 mg EQ/kg feed was derived as a safe upper limit of EQ exposure in Atlantic salmon.

The effects of ethoxyquin and Angelica sinensis extracts on lipid oxidation in fish feeds and growth, digestive and absorptive capacities and antioxidant status in juvenile red carp (Cyprinus carpio var. xingguonensis): a comparative study.

Firstly, a linoleic and linolenic acid emulsion and fish feeds were incubated with graded levels of ethoxyquin (EQ) and petroleum ether extract, ethyl acetate extract (EAE), ethanol extract and aqueous extract of Angelica sinensis. The results showed that EQ and extracts of Angelica sinensis (EAs) inhibited lipid oxidation in material above. Of all of the examined EAs, EAE showed the strongest protective effects against the lipid oxidation. Moreover, EAE at high concentrations showed a stronger inhibitory effect on lipid oxidation than that of EQ. Next, 7 experimental diets that respectively supplemented 0.0, 0.2, 0.8 and 3.2 g kg-1 of EQ and EAE were fed to 280 juvenile red carp (Cyprinus carpio var. xingguonensis) with seven treatment groups for 30 days. The results indicated that dietary EAE improved growth performance in carp. Moreover, dietary EAE increased the activities of trypsin, lipase, alpha-amylase, alkaline phosphatase, glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase (GPT) and decreased plasma ammonia content in carp. Meanwhile, dietary EAE reduced the levels of malondialdehyde and raised the activities of anti-superoxide anion, anti-hydroxyl radical, superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase and the content of reduced glutathione in the hepatopancreas and intestine of carp. However, with the exception of GPT, dietary EQ got the opposite results to dietary EAE in carp. These results revealed that dietary EAE improved the digestive, absorptive and antioxidant capacities in fish. However, dietary EQ inhibited the digestive, absorptive and antioxidant capacities in fish. So, EAE could be used as a natural antioxidant for replacing EQ in fish feeds.