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.

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.

Hepatic metabolism, phase I and II biotransformation enzymes in Atlantic salmon (Salmo Salar, L) during a 12 week feeding period with graded levels of the synthetic antioxidant, ethoxyquin.

The synthetic antioxidant ethoxyquin (EQ) is a widely used additive in animal feeds, including farmed fish feed. The use of EQ as food additive is prohibited and it is also undesirable in farmed meat and fish products. The possible negative aspects of EQ in fish feeds, such as modulation of hepatic detoxifying enzymes and possible effects through "carry-over" to edible parts of fish are not known. In addition, the subsequent consequences for human consumers have not been previously studied. In the present work, the alteration in gene and protein expression patterns, and catalytic activities of phase I and II hepatic biotransformation enzymes due to prolonged exposure to graded levels of dietary EQ in the range of 11-1800 mg EQ/kg feed were studied. The kinetics of parent EQ and its major metabolite, ethoxyquin dimer (EQDM) was also studied. In general two weeks seem to be the critical point in the entire toxicological response of salmon to dietary consumed EQ. Biotransformation of EQ to EQDM is shown to be a rapid process. However, the decrease in biotransformation rate results in the accumulation of EQ metabolites, high concentration of which was postulated to alter translation and post-translational modification of CYP3A, GST and UDPGT at feeding day 14 and 42, with subsequent decreases in the biotransformation of consumed EQ. Decrease in the biotransformation of consumed EQ produced the retention of un-metabolized EQ rather than metabolites in salmon liver. This may be considered as undesirable effect, since it could lead to the transport and accumulation in other organs and edible tissues. It may also cause a new wave of biotransformation with formation of metabolites inhibiting detoxifying enzymes. In general, these processes may prolong the excretion of dietary EQ from the fish body and produce EQ-derived residues in the ready-to-consume salmon or fish products. These EQ residues may have higher toxicological effects for human consumers than the parent compound and therefore need to be studied in more detail.

Hepatic biotransformation and metabolite profile during a 2-week depuration period in Atlantic salmon fed graded levels of the synthetic antioxidant, ethoxyquin.

The synthetic antioxidant ethoxyquin (EQ) is increasingly used in animal feeds and has been candidate for carcinogenicity testing. EQ has the potential for toxicological and adverse health effects for both fish and fish consumers through "carryover" processes. The toxicological aspects of EQ have not been systematically investigated. The present study was performed to investigate the hepatic metabolism, metabolite characterization, and toxicological aspects of EQ in salmon during a 2-week depuration after a 12-week feeding period with 18 mg (low), 107 mg (medium), and 1800 mg/kg feed (high). The alteration in gene expressions and catalytic activities of hepatic biotransformation enzymes were studied using real-time polymerase chain reaction with specific primer pairs and by kinetics of two identified hepatic metabolites. Analysis of EQ metabolism was performed using high performance liquid chromatography (HPLC) method and showed the detection of four compounds of which two were quantified, parent EQ and EQ dimer (EQDM). Two metabolites were identified as de-ethylated EQ (DEQ) and quinone imine, but these were not quantified. The concentration of the quantified EQ-related compounds in the liver at day 0 showed a positive linear relationship with measured dietary EQ (R2= 0.86 and 0.92 for parent EQ and EQDM, respectively). While the low-EQ-feeding group showed a time-specific increase of aryl hydrocarbon receptor (AhR) mRNA expression, the medium-dose group showed decreased AhR mRNA at depuration day 7. Expression of CYP1A1 was decreased during the depuration period. Consumption of dietary EQ produced the expression of CYP3A, glutathione S-transferase (GST), and uridine diphosphate glucuronosyl-transferase (UDPGT) mRNA during the depuration period. A similar pattern of effect was observed for both CYP3A and phase II genes and supports our previous postulation of common regulation of these enzymes by the same inducer, namely EQ metabolites. The increase of CYP3A, UDPGT, and GST gene expressions at day 7 was in accordance with the low concentration of DEQ. The low concentration of putative DEQ may induce the CYP3A with subsequent increase in the biotransformation of EQ into DEQ. The increase in UDPGT may seem to be a synchronizing mechanism required for the excretion of DEQ. The biotransformation of dietary EQ is proven by simultaneous induction of both phase I and II detoxification system in the liver of Atlantic salmon. Therefore, the apparent low concentration of putative DEQ may account for the induced phase I and II detoxifying enzymes at least during depuration. This speculated hypothesis is currently a subject for systematic investigation in our laboratory using in vitro and genomic approaches.

Differences in ethoxyquin nephrotoxicity between male and female F344 rats.

Dose-response studies have shown a sharp threshold for the renal papillary toxic effect in male rats between 0.25% and 0.5% ethoxyquin (Eto) in the diet over 6 months. Although similar elevated urinary protein (albumin) levels resulted from dietary Eto (0.5%) in both males and females, papillary necrosis was male specific. Following [14C]Eto administration, radiolabel was associated with urinary albumin but not alpha 2 globulin (alpha(2mu)-g). Autoradiographic studies indicated that the sex differences in nephrotoxicity do not involve differences in distribution or retention of Eto. Faecal and urinary metabolic profiles were also similar in the two sexes. The sharp threshold of toxicity in the male rat could indicate a fine balance between toxifying/detoxifying metabolism of Eto.

Carbohydrate fermentation and nitrogen metabolism of a finishing beef diet by ruminal microbes in continuous cultures as affected by ethoxyquin and(or) supplementation of monensin and tylosin.

Long-term feedlot studies have shown positive effects (i.e., improved ADG and reduced morbidity and mortality) of dietary supplementation with ethoxyquin (AGRADO). This may be due to improving the antioxidant capacity at the ruminal, postruminal, or postabsorption levels. This study was designed to investigate the role of ethoxyquin at the rumen level. A finishing diet (12.5% CP; DM basis) was formulated to contain (on a DM basis) 77.5% flaked corn, 10% corn cobs, 10% protein/vitamin/mineral supplement, and 2.5% tallow. In a randomized complete block design experiment, the treatments were arranged as a 2 x 2 factorial. The main factors were two ethoxyquin treatments (without or with 150 ppm) and two monensin/tylosin treatments (without or with monensin and tylosin at 0.0028 and 0.0014% of dietary DM, respectively). Eight dual-flow, continuous culture fermenters were used in two experimental periods (blocks; 8 d each with 5 d for adjustment and 3 d for sample collection) to allow for four replications for each treatment. No interactions (P > 0.05) were detected for any of the measurements evaluated. Therefore, results of the main factors were summarized. Ethoxyquin supplementation improved (P < 0.05) true digestibility of OM (from 38.8 to 45.0%) but it did not alter (P > 0.05) concentrations of total VFA (averaging 131 mM) or acetate (averaging 58.8 mM). Ethoxyquin decreased (P < 0.05) propionate concentration from 51.1 to 42.4 mM and increased (P < 0.05) butyrate concentration from 18.4 to 22.9 mM. Digestion of total nonstructural carbohydrates was not altered (P > 0.05) by the treatments and averaged 86%. With the exception of increased (P < 0.05) concentration of propionate (from 42.0 to 51.5 mM) and decreased (P < 0.05) concentration of butyrate (from 25.9 to 16.3 mM), no effects (P > 0.05) were detected for monensin/tylosin. Ruminal N metabolism, including efficiency of bacterial protein synthesis (averaging 21.2 g N/kg OM truly digested), was not affected (P > 0.05) by the treatments. Results suggest positive effects of ethoxyquin on ruminal digestion of OM and unique changes in VFA production.

Induction of glutathione S-transferase activity and protein expression in brown bullhead (Ameiurus nebulosus) liver by ethoxyquin.

The inducibility of hepatic cytosolic glutathione S-transferases (GSTs) was examined in brown bullheads, a freshwater fish that is highly susceptible to hepatic neoplasia following exposure to carcinogen-contaminated sediments. Juvenile bullheads were fed a semi-purified antioxidant-free diet supplemented with ethoxyquin (0.5% w/w dissolved in 3% corn oil), a prototypical rodent GST-inducing agent, twice daily for 14 days. Control bullheads received the antioxidant-free diet supplemented with corn oil (3% w/w). A significant increase (1.6-fold, p < or = 0.01) in hepatic cytosolic GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB) was observed in the ethoxyquin-treated bullheads relative to control fish. A trend toward increased GST-NBC activity was observed in the ethoxyquin-treated fish (1.2-fold, p = 0.06), whereas no treatment-related effects were observed on GST activities toward ethacrynic acid (ECA). In contrast, GST activity toward (+/-)-anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE) was repressed in affinity-purified cytosolic fractions prepared from ethoxyquin-treated bullheads relative to control bullheads. Silver staining and densitometric analysis of isoelectric-focused, affinity-purified GST proteins revealed increased expression of two basic GST-like isoforms in ethoxyquin-treated fish. In summary, exposure to ethoxyquin increases brown bullhead GST-CDNB catalytic activity and hepatic cationic GST protein expression. However, the increase in overall GST-CDNB activity by ethoxyquin is associated with repression of GST-BPDE activity, suggesting differential effects on hepatic bullhead GST isoforms by ethoxyquin. The potential repression of bullhead GST isoforms that conjugate the carcinogenic metabolites of PAH metabolism under conditions of environmental chemical exposure could be a contributing factor in the sensitivity of bullheads to pollutant-associated neoplasia.

Comparative xenobiotic metabolism between Tg.AC and p53+/- genetically altered mice and their respective wild types.

The use of transgenic animals, such as v-Ha-ras activated (TG:AC) and p53+/- mice, offers great promise for a rapid and more sensitive assay for chemical carcinogenicity. Some carcinogens are metabolically activated; therefore, it is critical that the altered genome of either of these model systems does not compromise their capability and capacity for metabolism of xenobiotics. The present work tests the generally held assumption that xenobiotic metabolism in the TG:AC and p53+/- mouse is not inherently different from that of the respective wild type, the FVB/N and C57BL/6 mouse, by comparing each genotype's ability to metabolize benzene, ethoxyquin, or methacrylonitrile. Use of these representative substrates offers the opportunity to examine arene oxide formation, aromatic ring opening, hydroxylation, epoxidation, O-deethylation, and a number of conjugation reactions. Mice were treated by gavage with (14)C-labeled parent compound, excreta were collected, and elimination routes and rates, as well as (14)C-derived metabolite profiles in urine, were compared between relevant treatment groups. Results of this study indicated that metabolism of the 3 parent compounds was not appreciably altered between either FVB/N and TG:AC mice or C57BL/6 and p53+/- mice. Further, expression of CYP1A2, CYP2E1, CYP3A, and GST-alpha in liver of naive genetically altered mice was similar to that of corresponding wild-type mice. Thus, these results suggest that the inherent ability of TG:AC and p53+/- mice to metabolize xenobiotics is not compromised by their altered genomes and would not be a factor in data interpretation of toxicity studies using either transgenic mouse line.

Effects of dietary oltipraz and ethoxyquin on aflatoxin B1 biotransformation in non-human primates.

Following aflatoxin B1 (AFB) exposure, rats readily develop liver tumors. However, treatment of rats with a variety of compounds, including the synthetic dithiolthione oltipraz and the antioxidant ethoxyquin, protects these rodents from AFB-induced hepatocarcinogenesis. Several epidemiological studies strongly suggest that AFB is also a causative agent of liver cancer in humans. However, relatively little is known about the efficacy of cancer chemoprevention in human and non-human primates. To this end, we examined the effects of chemopreventive agents on AFB metabolism in non-human primates. Hepatic aflatoxin B1 metabolism profiles of macaque (Macaca nemestrina) and marmoset (Callithrix jacchus) monkeys were determined and compared to humans. Quantitatively, the oxidative metabolism of this mycotoxin was similar in the three primate species. In contrast to macaques, both humans and marmosets lacked AFB-glutathione conjugating activity. It was concluded that marmosets resembled human AFB metabolism more closely than the macaques, and therefore, marmoset monkeys were chosen for this study. Eleven adult male marmosets were randomly assigned to three groups. Animals received the synthetic dithiolthione oltipraz, the antioxidant ethoxyquin, or vehicle only. In addition, two single doses of AFB were also administered orally before and after animals were treated with aforementioned compounds. Both oltipraz and ethoxyquin induced aflatoxin B1-glutathione conjugating activity in the livers of some but not all marmosets. In addition, 10 microM oltipraz inhibited cytochrome P450-mediated activation of AFB to the ultimate carcinogenic metabolite, aflatoxin B1-8,9-epoxide, in vitro, up to 51%. Furthermore, animals treated in vivo with oltipraz, but not ethoxyquin, exhibited a significant reduction (53% average) in AFB-DNA adduct formation relative to the control animals (p < 0.05). Together, our data suggest that chemoprevention is also effective in primates; however, most likely to a lesser degree than in rodents.

Comparative metabolism and disposition of ethoxyquin in rat and mouse. I. Disposition.

1. The biological fate of the antioxidant [3-14C]ethoxyquin (EQ) was investigated in the male F344 rat and the B6C3F1 mouse following either p.o. or i.v. administration. 2. The disposition of single doses up to 25 mg/kg was similar in the rat and mouse. About 90% of a total dose was excreted in urine and faeces within 24 h post-dosing. In contrast, no more than 60% of a higher dose of 250 mg/kg was excreted within 24 h following p.o. administration. 3. Metabolism of EQ was rapid in both the rat and mouse following either p.o. or i.v. administration. Little or no parent compound was detected in cumulative 24-h excreta. 5. EQ-derived radioactivity bioaccumulated in some tissues following repeated exposure to rat of either 25 or 250 mg/kg by gavage. However, the fold-increases in concentrations of EQ-derived radioactivity in tissues following repeated administration of the higher dose were generally less than those observed following repeated administration of the lower dose. Repeated high dose administration may overcome delayed gastric emptying (observed following single dose administration of 250 mg/kg) and/or lead to auto-induction of EQ metabolism.

Comparative metabolism and disposition of ethoxyquin in rat and mouse. II. Metabolism.

1. The major pathways of ethoxyquin (EQ) metabolism in both the rat and mouse are O-deethylation and conjugation to endogenous substrates. 2. The two major EQ-derived metabolites excreted in rat urine were in the form of sulphate conjugates, 1,2-dihydro-6-hydroxy-2,2,4-trimethylquinoline sulphate, and 1,2,3,4-tetrahydro-3,6-dihydroxy-4-methylene-2,2-dimethylquinoline sulphate. The latter apparently arises from an intramolecular rearrangement of the 3,4-epoxide of ethoxyquin. 3. Mouse urine contained one major glucuronide, 1,2-dihydro-6-hydroxy-2,2,4-trimethylquinoline glucuronide as well as one major sulphate conjugate, 1,2-dihydro-6-hydroxy-2,2,4-trimethylquinoline sulphate. 4. EQ-derived radioactivity was excreted in rat bile, mainly as GSH conjugates, with little unchanged EQ present. Two of the biliary metabolites are glutathione conjugates of ethoxyquin 3,4-epoxide; the third appears to be a conjugate of either ethoxyquin 7,8-epoxide or 2,2,4-trimethylquinol-6-one.

The influence of dietary ethoxyquin on the vitamin E status in broilers.

In a feeding experiment with 48 male broiler chickens was the effect of dietary ethoxyquin and dl-alpha-tocopherol acetate on vitamin E status of chickens investigated. The experimental diets were offered the chickens from the first day of age until the end of the experiment at 42 days. Chickens were fed on diets containing 0, 75, or 150 mg ethoxyquin/kg feed, with (30 mg/kg feed) or without added dl-alpha-tocopherol acetate. The concentration of alpha-tocopherol in plasma of chickens on dl-alpha-tocopherol acetate supplemented diets were significantly higher than those of chickens fed on the nonsupplemented diets. Supplementation with alpha-tocopherol acetate reduced the haemolysis in vitro. Ethoxyquin seemed to have an increasing effect on the concentration of alpha-tocopherol in plasma, but not in the liver. Addition of ethoxyquin to diets without dl-tocopherol acetate reduced the concentration of gamma-tocopherol in plasma. Significant interactions between the addition of ethoxyquin and the addition of dl-alpha-tocopherol acetate to the feed were observed on the haemolysis in vitro and the activity of glutathione peroxidase in plasma. The results indicate that incorporation of ethoxyquin in the diets of chickens exert an improving effect on the vitamin E status of the organism.

Ovine urinary metabolites of ethoxyquin.

Metabolites of ethoxyquin (EQ, 1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) in the urine of sheep and rats were separated and identified by gas chromatography-mass spectrometry (GC-MS). Sheep were given diets containing EQ or EQ.HCl (0.5% of total diet) and urine samples were collected for the first 24 h and for another 24-h period after 12 d of feeding. Rats were given EQ/corn oil (0.08 g EQ/d/rat) orally for 7 d and urine samples were collected at ambient temperature for a 24-h period following 6 d of dosing. The urine samples were extracted with ethyl acetate at pH 5, and the concentrated extracts were analyzed by GC-MS. Ethoxyquin was identified in all sheep urine samples collected during the first 24 h of feeding, and EQ and hydroxylated EQ were identified in all urine samples collected after 12 d of feeding. In contrast, EQ, hydroxylated EQ, and dihydroxylated EQ were identified in urine collected from rats fed EQ for 7 d.

Effects of high concentrations of dietary vitamin E and ethoxyquin on the performance of laying hens.

1. Three experiments were carried out with light strain laying hens to evaluate the effects of relatively high doses of dietary vitamin E (125 mg/kg food) or ethoxyquin (EQ) (250 mg/kg food) on their laying performance. The control diet contained 5 and 125 mg/kg vitamin E and EQ, respectively. The experimental diets were fed either from one or 32 weeks until 88 or 89 weeks of age. 2. The two antioxidants did not affect the growth of the pullets, age at first egg, final body weight, average egg weight or relative abdominal fat pad size and liver weight at the termination of the experiments. In two out of three experiments, vitamin E and EQ did not affect egg production, food efficiency or mortality; in the third experiment vitamin E significantly (P less than 0.05) improved egg production and food efficiency after an outbreak of Newcastle disease which occurred at 34 weeks of age. EQ significantly reduced mortality during the course of this experiment, but did affect the variables of performance. In two experiments vitamin E consistently improved shell density, although a significant effect was observed in only one of the eight determinations carried out. EQ did not affect this variable. 3. The uterine muscle was more susceptible to oxidation than the drumstick meat, as evaluated by TBA values. In both tissues, vitamin E significantly and consistently decreased TBA values and restricted their increase during incubation, while EQ was less effective, particularly in the drumstick meat. 4. It is concluded that increasing vitamin E and EQ concentrations in diets of laying hens have no effect on the decrease in egg production due to aging. However, vitamin E may minimize the decline in egg production and food efficiency following the outbreaks of some diseases and slightly improve--under certain yet undefined conditions--shell density.

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.

Dietary restriction alone and in combination with oral ethoxyquin/2-mercaptoethylamine in mice.

To investigate effects of dietary caloric restriction (DR) combined with antioxidant feeding, long-lived hybrid mice were divided into four dietary groups at weaning, and followed until natural death. Groups "C" and "R" received control (97 kcal/wk) and restricted (56 kcal/wk) diets respectively. Groups "C+ alpha ox" and "R+ alpha ox" received C or R diets supplemented with an antioxidant mixture (2-mercaptoethylamine plus ethoxyquin). R mice (mean life span 41 months) significantly outlived the other three groups (mean life span 30-34 months). Hepatic degeneration and increased hepatoma in the R+ alpha ox group suggested unusual hepatotoxicity of this regimen. Antioxidants had little effect on splenic cell mitogen response in similarly fed mice sacrificed at 12-15 months. Gompertz analysis suggests that the beneficial effect of DR may be due to reductions in initial vulnerability or rate-of-aging parameters, or both, and that the relative influence of each factor may vary with animal strain and DR protocol used.

Experimental prevention of bitterweed (Hymenoxys odorata) poisoning of sheep.

To examine the effects on bitterweed toxicity of dietary factors known to increase thiol concentrations in the body, 36 lambs were fed one of the following diets (12 lambs/diet) for a minimum of 9 days prior to bitterweed administration: diet 1, 10% crude protein; diet 2, 20% crude protein, 0.5% methionine, 0.5% sodium sulfate, and 1,102 IU of vitamin E/kg; and diet 3, diet 2 with 0.5% ethoxyquin hydrochloride added. Four lambs fed each diet were euthanatized prior to bitterweed administration (initial euthanasia group). Four lambs fed each diet were administered bitterweed (0.68% hymenoxon, air-dried basis) at a rate of 0.25% of live weight for 5 consecutive days. The remaining four lambs on each diet served as unchallenged controls. In the initial euthanasia group, diet 2 increased extracellular blood thiol concentrations (1.12 vs 0.94 mg of SH/d1, P less than 0.10), rumen fluid thiol concentrations (4.46 vs 1.88 mg of SH/d1, P less than 0.05), and liver thiol concentrations (263.6 vs 109.3 micrograms SH/g of wet wt, P less than 0.05), compared with diet 1. Ethoxyquin hydrochloride (diet 3) reduced blood thiol concentrations (0.94 vs 1.12 mg of SH/dl, P less than 0.10) and liver thiol concentrations (151.6 vs 263.6 micrograms of SH/g of wet wt, P less than 0.05), compared with diet 2. Kidney thiols were unaffected by treatments.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparation and dietary effects of ethoxyquin hydrochloride.

Ethoxyquin (EQ, 1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) was purified and converted to a crystalline, stable ethoxyquin hydrochloride (EQ-HCl). The readily available (technical grade) oily EQ reacted with concentrated hydrochloric acid (HCl) and precipitated as a crystalline salt (EQ-HCl) in acetone, leaving most of the impurities in solution. The regenerated free base (EQ) from the EQ-HCl was further purified by silicagel column chromatography to remove several minor contaminants, and the pure unstable EQ was immediately converted into a pure stable salt (EQ-HCl). The dietary administration of EQ-HCl, 0.25 or 0.5% in the feed, induced hepatic and intestinal thiols in mice and provided protection against toxic doses of pyrrolizidine alkaloids. The LD50 values of the 0.125 and 0.25% EQ-HCl-pretreated mice were 94.0 and 98.5 mg/kg, respectively, compared to that of controls, 71.3 mg/kg. The EQ-HCl-supplemented feed appeared to be more palatable, but other effects, such as the hepatic hypertrophy, the tissue thiol induction, and the protective effects, were comparable to those of unpurified EQ.

Antimutagenic effects on male germ cells of mice.

The alkylating agent cyclophosphamide (CPA) and the antioxidant ethoxyquin (EQ) were administered perorally to NMRI mice. The strong clastogenic action of CPA on spermatogonia was diminished by simultaneous doses of EQ. Higher doses of the antioxidant produced greater anticlastogenic action. Furthermore, the action of the mutagen and the antioxidant on the late spermatids and the spermatozoa was observed using the dominant lethal test. The antioxidant had only a weak influence on these postmeiotic stages.

Effect of oxidized fish oil, DL-alpha-tocopheryl acetate and ethoxyquin supplementation on the vitamin E nutrition of rainbow trout (Salmo gairdneri) fed practical diets.

A factorial experiment was conducted using two degrees of oxidation of the 7.5% supplemental fish oil (peroxide values of 5 and 120 meq/kg oil), two levels of supplemental DL-apha-tocopheryl acetate (0 and 33 mg/kg diet) and two levels of ethoxyquin (0 and 125 mg/kg diet) supplementation. Dietary thiobarbituric acid number, weight percentage of polyunsaturated fatty acids and omega-three fatty acids in the total fatty acids were significantly (P less than 0.05) different between diets with fresh and highly oxidized oil. Dietary RRR-alpha-tocopherol was significantly (P less than 0.05) reduced by the addition of highly oxidized oil after 24 weeks storage of the feed while supplemental DL-alpha-tocopheryl acetate level was not changed. Fish fed the various diets showed no differences in growth, feed:gain ratio, carcass composition or plasma glutathione peroxidase activity. The mortality, percent red cells hemolyzed by hydrogen peroxide, plasma and liver RRR-alpha-tocopherol concentrations were significantly (P less than 0.05) affected by the addition of highly oxidized oil or DL-alpha-tocopheryl acetate but not by ethoxyquin except that mortality was reduced by supplementary ethoxyquin. The results of this study suggested that no vitamin E or ethoxyquin supplementation was needed to prevent a deficiency of vitamin E in rainbow trout fed a practical diet containing 7.5% of a good quality herring oil for 24 weeks.