Hepatic Biotransformation and Metabolite Profile during a 2-Week Depuration Period in Atlantic Salmon Fed Graded Levels of the Synthetic Antioxidant, Ethoxyquin

Abstract

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.

Antitrypanosomal Activity of 1,2-Dihydroquinolin-6-ols and Their Ester Derivatives

“…We propose that compounds esterified at the 6-oxygen atom are hydrolyzed rapidly by trypanosomal esterases, followed by two-electron oxidation to a quinone imine species. 28,45 This quinone imine could then undergo a single electron reduction to form a semiquinone that may enhance the rate of formation of reactive oxygen species such as superoxide and/or other free radicals. 46,47 The added strain on the parasite's antioxidant machinery could lead to lethal consequences for the organism (see Scheme 6).…”

Hepatic Retention and Toxicological Responses during Feeding and Depuration Periods in Atlantic Salmon (Salmo salar) Fed Graded Levels of the Synthetic Antioxidant, Butylated Hydroxytoluene

“…PCR primers for amplification of 76-146 bp gene-specific PCR-products were designed from conserved regions of the studied genes. The primer sequences, their amplicon size, and the optimal annealing temperatures are shown in Table 3 and were performed according to established methods in our laboratory (17,24). Sequences were confirmed using NCBI nucleotide BLAST software (http://www.ncbi.nlm.nih.gov/BLAST).…”

Ethoxyquin: An Antioxidant Used in Animal Feed

“…It was in agreement with the results obtained earlier by Skaare and Roald [ 27 ]. EQ is considered as a model inducer of phase II enzymes involved in the metabolism of xenobiotics, but influence of EQ on phase I enzyme gene transcript levels was also observed [ 28 , 29 ]. The key role in mediating phase I reactions (e.g., oxidation or reduction) producing more hydrophilic compounds is played by the CYP (cytochrome P450) enzyme family.…”

“…The key role in mediating phase I reactions (e.g., oxidation or reduction) producing more hydrophilic compounds is played by the CYP (cytochrome P450) enzyme family. Bohne et al [ 28 , 29 ] observed the alteration of CYP3A gene expression; an increase in the amount of CYP3A transcripts was detected in salmon after feeding them with the diet containing EQ at the highest dose used (1800 mg kg −1 ). The authors speculate that EQ may regulate CYP3 gene expression by interaction, for example, with pregnane X nuclear receptor (PXR) whose function is to sense the presence of toxic xenobiotics and in response enhance the expression of proteins involved in their detoxification.…”