ToxSci Advance Access originally published online on June 21, 2006
Toxicological Sciences 2006 93(1):11-21; doi:10.1093/toxsci/kfl044
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Hepatic Biotransformation and Metabolite Profile during a 2-Week Depuration Period in Atlantic Salmon Fed Graded Levels of the Synthetic Antioxidant, Ethoxyquin
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* National Institute for Nutrition and Seafood Research (NIFES), PO Box 2029, Nordnes, N-5817 Bergen, Norway; and Department of Biology, Norwegian University of Science and Technology (NTNU), Høgskoleringen 5, N-7491 Trondheim, Norway
1 To whom correspondence should be addressed. Fax: +47 73 591309. E-mail: arukwe{at}bio.ntnu.no.
Received April 24, 2006; accepted June 12, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
Key Words: synthetic antioxidant; ethoxyquin; salmon; biotransformation; metabolites.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The synthetic antioxidant ethoxyquin (EQ) is an aromatic amine additive (Fig. 1) widely used to preserve feed stuffs and fish feed from spontaneous oxidation (Hayes et al., 1998
; Manson et al., 1997). The function of EQ is based on its ability to scavenge free radicals formed during lipid oxidation (Rossing et al., 1985; Thorisson et al., 1992a). The by-products of lipid oxidation are responsible for the rancidity of feeds that causes weight reduction, oxidative stress (Cominacini et al., 1997; Saxena et al., 2000), and vitamin E and C shortage (Hamre et al., 1997) among other documented toxicological effects when fed to farmed fish. In this respect, the addition of synthetic antioxidants to feeds should be beneficial for overall fish health (Christiansen et al., 1995; Thorisson et al., 1992a). We have recently shown that substantial amount of dietary EQ is incorporated in salmon muscle with extensive partitioning in other tissues with equal concentration in blood, kidney, heart, brain, gut, muscle, spleen, and gills after 12 weeks of feeding (Bohne et al., 2006a). High concentration of parent EQ was found in adipose tissue and liver, de-ethylated EQ (DEQ) was found in liver, and EQ-dimer (EQDM) was found in adipose tissue of salmon fed graded levels of EQ (Bohne et al., 2006b).
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Studies performed on EQ metabolism in mammalian systems showed that EQ is extensively metabolized (Burka et al., 1996
; Kim et al., 1992; Sanders et al., 1996; Skaare and Solheim, 1979). The major pathways of EQ metabolism in rodents are three cytochrome P450 (CYP)-mediated O-deethylation, hydroxylation, and epoxydation followed by conjugation with endogenous products that are subsequently excreted through urine, bile, or faeces (Burka et al., 1996). Eight urinary EQ metabolites have been identified and four of these were structurally determined, representing two sulphated and two glucuronidated products (Burka et al., 1996). EQ was recently shown to cause a number of negative side effects in animals fed diets containing this compound, in addition to adverse effects in people exposed occupationally (Blaszczyk and Skolimowski, 2005).
Recently, we have shown in salmon that 80–98% of dietary EQ was accumulated in the muscle. The steady state in the muscle was not achieved during 12 weeks of feeding exposure with EQ ranging from 11 to 107 mg/kg feed. As a consequence, the concentration of parent EQ in the muscle increased consistently during the feeding period (Bohne et al., 2006a). Enzymes participating in the metabolic transformation of chemotherapeutics, steroids, and xenobiotics are classified into phase I and II biotransformation pathway. The phase I enzymes are generally involved in the functionalization of exogenous compounds, thereby creating a more polar and water-soluble compound (Van den Berg et al., 1994). The CYP1A1 and CYP3A enzymes participate in the phase I biotransformation process (Bucheli and Fent, 1995; Förlin et al., 1994). Phase II enzymes, such as uridine diphosphate glucuronosyl-transferase (UDPGT) and glutathione S-transferase (GST), use the products of phase I reactions to form large endogenous molecules (George, 1994).
The production of farmed Atlantic salmon requires a mandatory 2-week depuration period to ensure the elimination of veterinary medicaments and other undesirable components. The effect of starvation on the toxicological effects of EQ and its metabolites retained in salmon body has not been previously evaluated. Salmonids store energy in form of lipids predominantly found in adipose (visceral) and muscular tissues (Zhou et al., 1995, 1996). During starvation, fats stored in adipose tissues are utilized before the muscular fat, hepatic glycogen, and protein sources (Czesny et al., 2003). Therefore, the high release of parent EQ into circulating system under starvation could affect the hepatic metabolism as well as biotransformation gene and protein expression patterns. The aims of this study were (1) to study the modulation of gene and enzyme expressions for phase I (CYP1A1, CYP3A, and aryl hydrocarbon receptor [AhR]) and phase II (GST and UDPGT) biotransformation systems, (2) to study the relationship between expression of phase I and phase II systems based on the concept that the balance between activation and inactivation reactions are decisive for the accumulation of reactive and potentially damaging metabolites within cells, and (3) to study the kinetics of parent EQ and its metabolites in liver.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
Ethoxyquin PESTANAL of over 97.5% purity was purchased from Sigma (Oslo, Norway). EQ metabolites, quinone imine (QI), EQDM, and DEQ of over 90% purity, were synthesized according to known protocols (He and Ackman, 2000a
; Thorisson et al., 1992b) by Synthetica AS (Oslo, Norway). All other reagents used were of analytical grade and obtained from general laboratory suppliers.
Diets, fish, rearing conditions, and feeding experiment.
The diets were produced in one batch as 4-mm pellets at Fiskeriforskning (Bergen, Norway), according to established recipes. The feed requirement was calculated with a start weight of 0.2 kg of each fish, predicted daily growth rate of 1%, and the feed conversion factor of 1 (dry weight feed/fresh weight fish). Three diets with increasing levels of EQ (18, 107, and 1800 mg/kg feed) were fed in triplicate tanks to Atlantic salmon (Salmo salar). The levels of EQ in the diets will be further referred to as low (18 mg/kg), medium (107 mg/kg), and high (1800 mg/kg) dose. After 12 weeks, the feeding was terminated, and fish underwent a starving period of 2 weeks. Five fish from each of nine tanks (triplicate per feeding group) were sacrificed on the first day of feeding trial (control) and first day of the feeding termination (day 0) and thereafter at 3, 7, and 14 days of starving (depuration period). The feeding experiment was conducted on totally 900 individuals of salmon produced at AquaGen (Kyrkaeteroyra, Norway).
The experimental fish were half-year smolt with mean weight of 200 g at the start of acclimatization period and 400 g at the start of the feeding. To ensure the equal toxicological status of individuals at the beginning of the exposure, fish were fed experimental diet containing the lowest achieved amount of EQ for 1 month before the experiment started. Fish were stocked in the 1.5 x 1.5 x 0.5–m glass fibre tanks. The salinity and temperature during experiment were monitored and kept at 27 ± 2
and 9.5 ± 0.8°C, respectively. The daily light and dark cycle was artificially adjusted to follow the natural photoperiod at Matre (near Bergen Norway, 60°N), and feeding was performed automatically every 5 min from 8:00 A.M. to 4:00 P.M. in winter month and from 8:00 A.M. to 8:00 P.M. from April to July. Unconsumed feed pellets were collected and weighed, and the daily feed and EQ intake were calculated. During sampling five fish from each tank were randomly selected and anesthetized in a bath of benzocain (ethyl amino benzoat), prepared from 5 ml of stock solution of 0.1% benzocaine in 96% ethanol in 10 l seawater. The bile bladder was carefully removed, and bile was collected. The liver was dissected, rinsed in physiological solution, and immediately frozen on dry nitrogen. The samples were kept at –80°C in air- and light-tight containers until analysis. The average fish weight in a tank was measured three times during the experiment and was used in the determination of the growth rate. The individual fish and liver weights were recorded during the sampling and were used for the calculation of liver somatic index (%).
HPLC analysis of liver samples and feed nutrient analysis.
EQ and its metabolites were extracted with hexane from liver homogenates or bile dissolved in ethanol/NaOH and protected from air- and light-mediated oxidation by addition of saturated EDTA, ascorbic acid, and pyrogallol as described in Bohne et al. (2006b). The method was based on reversed-phase HPLC combined with fluorescence detection. Briefly, the successful separation of EQ-derived compounds was achieved on tandem coupled phenyl-hexyl and C18 columns by elution with two different mobile phases. Mobile phase 1 was automatically blended by a delivery system from 20% buffer that contains 0.6% acetic acid with 1% diethyl amine and 80% of 0.1% solid ascorbic acid solution in acetonitrile (ACN). Mobile phase 2 contained a solution of 0.1% solid ascorbic acid in ACN. Liver extracts (10 µl) were run in a cyclic 23.5-min sequence: 6.5 min with 20% of mobile phase 1 and 80% of phase 2 at 0.35 ml/min; 2.5 min gradient change to 100% phase 2 at 0.35 ml/min; 7 min with 100% mobile phase 2 at 0.35 ml/min; 1.0 min gradient change to 20% of mobile phase 1 and 80% mobile phase 2 at 0.40 ml/min; 5.5 min with 20% of mobile phase 1 and 80% of mobile phase 2 at 0.40 ml/min and 1.0 min with 20% of mobile phase 1 and 80% of mobile phase 2 at 0.35 ml/min. The detection limit (at 358/433 nm) of matrix-spiked EQ compounds was 0.02 µg/l for EQ and 0.06 µg/l for EQDM. The quantification of EQ-derived compounds was performed using a calibration curve with internal standard chemicals as described in Bohne et al. (2006b). EQ and metabolites were extracted from fish feed by a one-step procedure with ACN as adopted by the Association of Official Analytical Chemists International (Schreier and Greene, 1997). All analyses of salmon muscle and feed were performed in duplicates. 
-Tocopherol was analyzed by HPLC as described in Lie et al. (1994). Astaxanthin was analyzed as described by Nordgarden et al. (2003), and thiobarbituric acid–reactive substances (TBARs) were analyzed according to Hamre et al. (2001). Vitamin A was measured in feed according to procedure described by Nöll (1996) and modified by Ørnsrud et al. (2002).
Preparation of cytosol and microsomal fractions.
Cytosolic and microsomal fractions were prepared by differential ultracentrifugation as described by Pesonen and Andersson (1987). The liver samples were thawed on ice, and thereafter 2 g was sliced with a pair of scissors and homogenized in 1:4 ratio of liver weight and volume of 0.1M phosphate buffer (pH. 7.4), respectively, with four to six up and down strokes using a Potter-Elvehjem–type teflon-glass homogenizer. The homogenate was first centrifuged for 20 min x 12,000 x g at 4°C. The pellets were discarded leaving the postmitochondrial or S12 supernatant. The S12 fraction was further centrifuged at 60 min x 100,000 x g at 4°C. The resulting supernatant was collected, and microsomal pellet was resuspended in 1:1 (weight/volume per original liver weight) of 0.1M phosphate buffer (pH 7.6) with 20% glycerol. Both the cytosol and microsomes were stored in aliquots at – 80°C until analyzed.
Protein and enzyme assays.
Total amounts of cytosolic and microsomal proteins were determined with the method of Bradford (1976), with bovine serum albumin as standard. Measurements were simplified using a Synergy HT microplate reader from Bio-Tek Instruments Inc. (Winnoski, VT) for absorbance reading. UDPGT activity toward p-nitrophenol was measured in hepatic microsomal samples as described by Andersson et al. (1985), and GST activity in hepatic cytosolic samples was measured using 1-chloro-2,4-dinitrobenzene as substrate as described previously (Habig et al., 1974). Both UDPGT and GST assays were adapted to 96-well plate reader and measured spectrophotometrically using the Synergy HT for absorbance reading. The reaction was run in duplicates, and enzyme activity values were normalized to protein content in the samples. As a quality control, two known samples were assayed in parallel with all assay series to assure the consistency of the results obtained with unknown samples. All enzyme activities were assayed at room temperature.
RNA isolation and cDNA synthesis.
Total RNA was isolated from liver tissues homogenized in Trizol reagent according to manufacturer's protocol, and RNA concentrations were determined using NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE). Total cDNA for the real-time polymerase chain reactions (PCRs) was generated from 1 µg DNase-treated total RNA from all samples using poly-T primers from iScript cDNA Synthesis Kit as described by the manufacturer (Bio-Rad, Hercules, CA).
Primer optimization and quantitative (real time) PCR.
PCR primers for amplification of 76- to 146-bp PCR products were designed from conserved regions of the studied genes using the primer design software (http://www.biodirectory.com/directory/Bioinformatics/PCR_180.html). The primer sequences, their amplicon size, and the optimal annealing temperatures are shown in Table 1. Prior to all real-time PCRs, all primer pairs were used in titration reactions in order to determine optimal primer pair concentrations and their optimal annealing temperatures, and real-time PCRs were run using reverse transcriptase reactions without enzyme. All chosen primer pair concentrations used at the selected annealing temperatures gave a single-band pattern for the expected amplicon size in all reactions. The studied genes were cloned and used for the generation of real-time PCR standard curves (see below).
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Quantitative (real time) PCR was used for evaluating gene expression profiles. For each treatment, the expression of individual target genes was analyzed using the Mx3000P REAL-TIME PCR SYSTEM (Stratagene, La Jolla, CA) as described by Arukwe (2005)
and Mortensen et al. (2006) with some modifications. Controls lacking cDNA template were included to determine the specificity of target cDNA amplification. Cycle threshold (Ct) values obtained were converted into mRNA copy number using standard plots of Ct versus log copy number. The criterion for using the standard curve is based on equal amplification efficiency with unknown samples, and this is usually checked prior to extrapolating unknown samples to the standard curve. The standard plots were generated for each target sequence using known amounts of plasmid containing the amplicon of interest. Data obtained from triplicate runs for target cDNA amplification were averaged and expressed as nanogram per milligram of initial total RNA used for reverse transcriptase (cDNA) reaction. It should be noted that our real-time PCR approach has been validated and gives consistent results. We do not use the so-called housekeeping genes in normalizing our real-time PCRs since we are yet to find a toxicologically neutral gene (Arukwe, 2005) for normalization purposes.
Calculations and statistics.
Specific growth rate (SGR), as % body weight per day:
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Feeding rate, as % of body weight (at start) eaten per day:
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Statistical differences between sampling points within a same diet were calculated using nested ANOVA, followed by Tukey's HSD test (Statistica, version 6.1; StatSoft, Tulsa, OK). Differences among dietary treatments at the same time points were assessed by one-way ANOVA, followed by Tukey's HSD test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Physiological Parameters
The formulation and proximate analyses of the experimental feeds were performed, and there were no significant differences in the lipid, astaxantin, tTBARs, and natural antioxidants content of the different diets (data not shown). There were no fish mortalities during the experimental period. No significant differences among treatments were observed for feeding rate, specific growth rate, and biological FCR (Table 2). After 2 weeks depuration period, there were no significant effects of EQ levels on growth or liver weight (Table 2), although minor weight losses were observed.
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Hepatic Metabolism of Ethoxyquin
Analysis of EQ metabolism showed the detection of more than 10 metabolites of dietary EQ in hepatic tissue (not shown) with no differences between liver patterns of observed metabolites among treatment groups. Four EQ-related compounds were identified (Fig. 1) and two were quantified, namely parent EQ and EQDM. Third and fourth metabolites were identified as DEQ and QI. All named metabolites were identified by spiking experiments with synthesized chemical standards, identities of which were confirmed by hydrogen (proton) nuclear magnetic resonance (1H-NMR), mass spectrometry (MS), and HPLC. The concentration of QI in most samples was below the quantification limit. The recovery of spiked standards of EQ and EQDM was in the range of 82–105% and 94–100%, respectively. Moreover, the within-assay reproducibility of quantification in organs and body fluids other than muscle was 3–10% for EQ in the range 0.4–91 µg/kg and 0.4–16% for EQDM in the range 0.4–4493 µg/kg (Bohne et al., 2006b
). Due to poor recovery (55%), the quantification of DEQ is not reported in this paper. The concentration of each metabolite in the liver at day 0 (that is at the end of the feeding) showed a positive linear relationship with measured EQ in the diet with R2 = 0.86 and 0.92 for EQ and EQDM, respectively. The metabolites' kinetic differences within and between treatment groups during the 2-week depuration period are shown in Figure 2. In the low (18 mg EQ/kg feed)-dose group, the levels of EQ metabolite (EQDM) showed a time-specific significant increase with decreasing parent EQ during the depuration period (Fig. 2A). The contrast is true for the intermediate (107 mg EQ/kg feed)- and high (1800 mg EQ/kg feed)-dose groups where the EQ metabolite, EQDM, first increased with increasing parent EQ during the feeding period and thereafter decreased with decreasing parent EQ levels during the 2-week depuration period (Fig. 2B and 2C, respectively). In general, EQDM increased more than DEQ (not shown) in the low-dose groups, while EQDM decreased more than DEQ in the intermediate- and high-dose groups.
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Expression of AhR, CYP1A1, and CYP3A Genes
It should be noted that the control and depuration day 0 represent start and end (week 12) of feeding experiment, respectively, and during this period AhR mRNA levels decreased in the low-dose, increased in the medium-dose, and remained unchanged in the high-dose group (Fig. 3A). During the depuration period, AhR mRNA was highly expressed in all feeding groups with variable EQ effects at different time intervals (Fig. 3A). At depuration day 0, the expression of AhR mRNA levels was apparently EQ dose specific (Fig. 3A) with the medium (107 mg EQ/kg feed)-dose group showing the highest mRNA level. While no differences were observed at day 3, AhR mRNA showed significant reduction in the medium (107 mg EQ/kg feed)- and high (1800 mg EQ/kg feed)-dose feeding groups compared to the low (18 mg EQ/kg feed)-dose group (Fig. 3A). In general, while the low–EQ-feeding group showed a time-specific increase of AhR mRNA expression, the medium-dose group did not show any changes, except at depuration day 7, when AhR mRNA expression decreased. In contrast, AhR mRNA expression in the high-dose groups first increased at depuration day 3, and thereafter, time-specific significant decreases were observed (Fig. 3A).
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During the feeding and depuration periods, CYP1A1 showed steady time-specific decreases (Fig. 3B). CYP1A1 mRNA level showed a dose-specific increase at depuration day 0 (Fig. 3B) in the high-dose group. At depuration day 7, CYP1A1 mRNA showed a significant increase in the low-dose group, compared to medium- and high-dose groups (Fig. 3B). In general there were no significant time-related changes in CYP1A1 mRNA levels observed in the low (18 mg EQ/kg feed)-EQ group (Fig. 3B). In contrast, the high-EQ group (1800 mg EQ/kg feed) showed time-specific (not significant) reductions in CYP1A1 mRNA expression (Fig. 3B). The observed day 7 reduction in medium-dose group was restored at the end of the depuration period (Fig. 3B). Significant dose-related increases were observed at days 0 and 7, caused by the highest and lowest doses of EQ, respectively.
For CYP3A, transcription rate during the depuration period followed the same pattern as seen with AhR and CYP1A1 expression (Fig. 3C). When compared to the start of feeding (control), CYP3A mRNA levels increased in all feeding groups at the end of feeding (depuration day 0, Fig. 3C). There is no dose dependency in the expression of CYP3A mRNA at depuration day 0. The high-dose group showed elevated CYP3A mRNA at day 3 and so did the low (18 mg EQ/kg feed)-dose group at depuration day 7 (Fig. 3C). In general, while the low–EQ-feeding group showed apparent time-specific changes of CYP3A mRNA expression during the feeding and depuration period, the medium (107 mg EQ/kg feed)-dose group did not show any changes (despite the initial increase during feeding), except at depuration day 7 when CYP3A mRNA expression was decreased. In contrast, CYP3A mRNA expression in the high-dose groups first increased between control (start of feeding) and depuration days 0 and 3, decreasing thereafter from day 7 to 14 (Fig. 3C).
Expression of GST and UDPGT Genes
The UDPGT and GST genes shared very similar, with few exceptions, pattern of expression that is comparable to phase 1 gene expression (Figs. 4 and 5, respectively). These unique patterns are characterized as follows for UDPGT; respective reduction and increase in the high-dose (1800 mg EQ/kg feed) group at depuration days 0 and 3, compared to control, low (18 mg EQ/kg feed)-dose, and medium (107 mg EQ/kg feed)-dose groups (Fig. 4A). At depuration day 7, UDPGT expression showed a reduction in the high-dose group compared to the low- and medium-dose groups (Fig. 4A). In general, the low–, medium–, and high–EQ-feeding groups showed slight but time-specific increases of UDPGT mRNA expression during the feeding and depuration periods (Fig. 4A). UDPGT mRNA expression in the high-dose group first increased between feeding and depuration days 0 and 3, decreasing thereafter from day 7 to14 (Fig. 4A).
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The GST gene expression followed almost a similar pattern as described for UDPGT, increasing in all dose groups between feeding and depuration days 0 and 3 (Fig. 4B). At day 3, the high-dose (1800 mg EQ/kg feed) group showed a significant increase of GST mRNA expression, compared to low (18 mg EQ/kg feed)- and medium (107 mg EQ/kg feed)-dose groups (Fig. 4B). At depuration day 7 (also at day 14 for the high-dose group), GST expression showed significant reduction in the high- and medium-dose groups compared to the low-dose group (Fig. 4B). In general, while the low–EQ-feeding group showed slight increases of GST mRNA expression during the feeding and depuration periods, the medium-dose group showed only minor changes, except at depuration day 7, when GST mRNA expression was decreased (Fig. 4B). In contrast, GST mRNA expression in the high-dose group first increased between feeding and depuration days 0 and 3, decreasing thereafter from day 7 to14 (Fig. 4B).
UDPGT and GST Enzyme Activities
Due to the interesting pattern of gene expressions observed in all feeding groups during the depuration period, feeding dose 107 mg EQ/kg feed was selected for evaluation of catalytic patterns. Specific activity of phase II enzymes was measured in liver samples from salmon fed 107 mg EQ/kg feed. Similar to gene expression patterns, time-dependent alterations in specific UDPGT and GST activities were documented to be similar for both enzymes' activities (Fig. 5A and 5B, respectively). The UDPGT enzyme activity was decreased at depuration day 14 in the medium (107 mg EQ/kg feed)-dose group (Fig. 5A). The alterations in GST activity showed decreased levels at depuration days 7 and 14 (Fig. 5B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In Norwegian aquaculture industry, the marked requirements of more fatty fish flesh have led to the increase of lipid content in fish feed and consequently resulted in the increased use of antioxidants to prevent auto-oxidation of lipids. EQ is increasingly used in animal feeds and has been candidate for carcinogenicity testing (Bammler et al., 2000
). EQ has the potential for toxicological and adverse health effects for both fish and fish consumers through carryover processes, and these toxicological aspects 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. To our knowledge, this type of investigation has not been performed using fish as a model.
Metabolites of Dietary EQ
Biotransformation of dietary EQ in Atlantic salmon during the 2-week depuration resulted in at least four major compounds, namely parent EQ, DEQ, QI, and EQDM. Identified EQ metabolites were the same in the liver and bile of all feeding groups. Parent EQ was the main compound observed and represented 73–99% and 55–75% of total biliary (Bohne et al., 2006b) or liver EQ and EQDM at depuration day 0 (that is after 84 days of the feeding), respectively. This observation is in accordance with the ratio of parent EQ among biliary metabolites reported in rats (Skaare and Solheim, 1979). DEQ was identified as the major urine metabolite in rat (Skaare and Solheim, 1979) and was postulated as the intermediate biliary metabolite of EQ in rodents (Burka et al., 1996), but the metabolite was not identified by Sanders et al. (1996). In the present study, DEQ and QI were detected in salmon liver samples but were not quantified. This finding is in contrast to the study by He and Ackman (2000b) where DEQ was not identified in fish liver samples. The different HPLC separation methods used by these studies (Bohne et al., 2006b; He and Ackman, 2000b) may account for the different finding. At the same time, it should be noted that DEQ formation represents 10–20% of EQ metabolites in salmon liver and bile. This low recovery of DEQ might represent a weakness in the elution procedure used in the present study or a specie-specific pattern of EQ metabolism in fish that needs to be studied in more detail. Another fish-specific product of EQ, metabolite EQDM, not observed in mammalian system (Burka et al., 1996; Skaare and Solheim, 1979), was found to be a minor compound of both bile and liver of salmon fed EQ. Concentration of EQDM in liver at the termination of feeding was in the range of 0.06–0.8 mg/kg tissue. This value is comparable with the reported 0.25 mg/kg in the liver of steelhead salmon treated with EQ (He and Ackman, 2000b).
Effects on AhR, CYP1A1, and CYP3A Genes
In fish, the AhR is essential for the initiation of the CYP isozyme gene transcriptions. While significant increase in the expression of AhR and CYP3A mRNA were observed at depuration day 3, CYP1A1 mRNA was not significantly altered during the depuration period in any of the tested groups, although a trend toward down-regulation was observed. In mammals, studies of phase I drug metabolism has revealed that administration of synthetic antioxidants to rodents caused modest changes in the levels of CYP1A1 enzyme activities (Kahl, 1984). The time course fluctuation in CYP1A1 mRNA concentration during 14 days of depuration was not significant and will henceforth only be described as a trend. CYP1A1 mRNA level in fish receiving low EQ dose increased during the first week of depuration and decreased to day 0 level during the following week. The opposite was observed in fish receiving the medium and high EQ doses, where a decrease was observed during first week and increased during the next week of depuration. The levels of CYP1A1 mRNA in all treatment groups were in general lower than levels at day 0 of feeding period, suggesting a possible decrease of gene expression during the depuration period. Induction of CYP1A1 gene expression and enzyme activity have been described as biomarker of exposure to endogenous and exogenous compounds (Bucheli and Fent, 1995; Goksøyr and Förlin, 1992).
Given that AhR mRNA showed a nonparallel expression with CYP1A1 whose mRNA levels decreased from experimental feeding start to end, we speculate that the CYP1A1 might have suffered some inhibitory effect. There are several hypotheses that may account for possible CYP1A1 down-regulation at feeding and depuration periods. The inhibitory action of EQ could be mediated, at least in part, through the hepatic antioxidant response elements (ARE) where EQ-ARE complex can interfere with CYP1A1 gene directly or alternatively may interact with AhR and indirectly regulate CYP1A1 gene expression through binding with promoters and/or enhancers (Buetler et al., 1995; Hayes et al., 2000). Furthermore, EQ may modulate CYP1A1 through AhR repressor (AhRR), as has been shown in a recent study (Maradonna et al., 2004). It is also possible that the basic-helix-loop-helix-PAS (Per-AhR/ARNT-Sim homology sequence) of transcription factor usually associated with each other to form heterodimers, AhR/ARNT or AhRR/ARNT, and bind the ARE sequences in the promoter regions of target genes to regulate their expression (Buetler et al., 1995). The absence of CYP1A1 gene increase might also be a pitfall in the sampling procedure, such as delay in CYP1A1 synthesis with time and by other biological factors such as sex and/or season. Seasonal changes in CYP1A1 protein and activity levels, including reproductive period, have been documented in fish species (Arukwe and Goksøyr, 1997). In our experiment, both parameters were estimated simultaneously and fish were not separated by sex.
CYP3A mRNA levels were already elevated during feeding period and were further enhanced during the depuration period. Generally, mRNA levels were 10- and 5-fold higher during depuration period than at the beginning and the end of the feeding period, respectively. Thus, CYP3A gene expression was induced during the depuration period, in contrast to CYP1A gene, whose expression was inhibited. CYP3A is the enzyme of phase I detoxifying reactions (Celander and Stegeman, 1997), induction of which is correlated to active metabolism of endogenous and exogenous compounds. The increase in CYP3A expression was correlated to high level of immunoreactive protein during the whole depuration period (not shown). The conformity in gene and protein expression confirmed CYP3A increase during the depuration period, which may be needed for the biotransformation of dietary EQ during the depuration period.
The induction of CYP3A expression is mediated by the pregnane X receptor (PXR), at least in mammals (Wang and LeCluyse, 2003; Willson and Kliewer, 2002). The remarkable chemical and structural diversity of known PXR activators has fueled discussions about their physiological functions (Wang and LeCluyse, 2003; Willson and Kliewer, 2002). PXR has been shown to be activated by various xenobiotics (e.g., rifampicin, clotrimazole, the bisphosphonate ester SR12813, hyperforin), natural and synthetic steroids (e.g., 5ß-pregnane-3, 20-dione, pregnelenone 16-carbonitrile, dexamethasone), and bile acids (e.g., lithocholic acid and 6-keto lithocholic acid) (Kliewer et al., 1998; Lehmann et al., 1998; Moore et al., 2000; Staudinger et al., 2001). There is mounting evidence that PXRs evolved to serve as promiscuous xenoreceptors for detecting potentially harmful compounds of both endogenous and exogenous origin (Moore et al., 2002). Based on the above-named studies, we speculate that some EQ intermediate may regulate CYP3A gene expression by feedback regulation through the PXR and/or other receptors-coactivators/repressors. Whether EQ or its metabolites modulated CYP3A levels between start of feeding and depuration day 7 in all dose groups are mediated via the PXR should be investigated in a differently designed study, such as nuclear run-on experiments, and the mechanisms involved should be studied by full-length cloning of the PXR cDNA and promoter characterization.
Effects on UDPGT and GST Genes and Enzyme Activities
The UDPGT transcript levels increased fivefold during depuration period compared to control and day 0. Significant time course increase of gene expression was observed on days 3 and 7 of depuration for low and high doses, respectively. Time course alterations in mRNA levels in medium-dose group were, in general, moderate. The decreased levels of UDPGT between days 3 and 7 were restored to basal level at day 14. Specific activity of UDPGT protein measured in medium-dose group did not reflect these alterations and maintained the same trend during the whole depuration period. In general, the same pattern of UDPGT increase was also observed for GST in the lower and highest dose groups. The decrease in mRNA levels was observed in medium-dose group during the first week of depuration followed by increases in transcript levels during the next week. The levels decreased between days 3 and 7 and were restored to original levels at day 14. Specific activity of UDPGT protein measured in the medium-dose group did not reflect these alterations and maintained the same trend during the whole depuration period. The same pattern of increase for GST was observed as for UDPGT in the low- and high-dose groups. In general, it is electrophilic compounds that are conjugated with glutathione by GST, and this makes them more water soluble and suitable for elimination via the bile, urine, or gills. Thus, low specific activity of GST during the last week of depuration could be due to inhibition/inactivation of protein by competitors in the form of EQ intermediates, in which high concentrations in heptaocytes is due to inhibition or reduction (at day 7, restored however at day 14) of gene transcription of CYP1A1 and CYP3A, respectively.
The induction of GST by EQ is a well-documented mechanism of EQ-induced chemoprotection against aflatoxin B1 in rodents (Hayes et al., 1998; Henson et al., 2001; Kahl and Kahl, 1983) and nonhuman primates (Bammler et al., 2000). This is in accordance with the previous speculation that the principal beneficial effect of phenolic antioxidants appears to involve induction of phase II drug-metabolizing enzymes (Hayes et al., 2000). For example, studies of GST induction in rodents indicate that antioxidant compounds such as EQ and butylated hydroxyanisole are more effective inducers of GST expression than are AhR agonists (Buetler et al., 1995). The mechanism by which cells sense the presence of inducing agents in order to initiate transcriptional activation through the ARE is unknown. Because metabolites of phenolic antioxidants are thiol-active agents, it has been postulated that these compounds are required to react with certain protein cysteine residues in order to mediate transcriptional activation of phase II drug-metabolizing genes (Hayes and Pulford, 1995). While monofunctional inducers are defined as electrophile compounds that can react with glutathione and selectively induce phase II biotransformation enzymes (Talalay et al., 1988), bifunctional inducers are capable of both phase I and II enzymes. The observed effects and differences on phase I and II biotransformation system are in agreement with reported bifunctional nature of the EQ in vivo (Buetler et al., 1995) and is in contrast to monofunctional nature documented in vitro (Miao et al., 2004)
Metabolic Profile of EQ and Biotransformation Profile
UDPGT reacts with compounds that contain hydroxyl (OH) group, which are present on parent compound or a product of the phase I biotransformation. DEQ is the only identified EQ metabolite in the present study with an OH group and therefore a potential substrate for glucuronidation. In rodents, DEQ was transformed to epoxide and further excreted as glutathione conjugate (Burka et al., 1996). Phase I and II metabolizing enzymes were postulated to share the same receptor-mediated and non–receptor-mediated regulation pathways (Rushmore and Kong, 2002). Thus, expression pattern of CYP1A1, CYP3A, and GST may be compared to UDPGT and to the ratio of EQ metabolites measured in the liver. In this study, a common feature in gene expression levels was the dose-specific increase at day 7 where the low EQ dose (18 mg/kg) enhanced increase of CYP1A1 and CYP3A gene expression at day 7 compared to medium- and high-dose feeding groups. Noting that dietary EQ in salmon liver is found to be parent EQ, DEQ, and EQDM, the observed increase of gene expression may be explained by the relative ratio between these compounds. Thus, the observed increase of gene expression in fish fed 18 mg EQ may possibly reflect the relatively low levels of DEQ compared to the measured level in the other two dose groups. Therefore, we speculate that DEQ may be, at least in part, responsible for the induced phase I and II detoxifying enzymes during the depuration period. This speculated hypothesis is currently a subject of systematic investigation in our laboratory using in vitro and genomic approaches.
Food Safety Aspect
The biological fate of graded levels of dietary EQ was also evaluated in salmon muscle during the 12-week feeding and 2-week depuration periods. Note that the production of farmed Atlantic salmon in Norway requires a mandatory 2-week depuration period to ensure the elimination of veterinary medicaments and other undesirable components. Parent EQ, QI, DEQ, and EQDM were identified in all muscle samples and were among a total of 14 compounds observed on the chromatograms. Among the unidentified metabolites, one major compound was termed UMEQ (unknown metabolite of EQ) whose concentration in the muscle (expressed in arbitrary units) correlated with administered dietary EQ doses. The same was true for the parent EQ, DEQ, and EQDM whose concentrations in the muscle correlated with administered dietary EQ doses (Bohne, Arukwe and Hamre, in preparation). It should be noted that QI was not quantified in the present study due to low muscle levels. Since concentration of QI in salmon muscle was under the quantification limit for the applied method, it was not possible to document the potential dose dependence on dietary EQ and subsequent metabolic origin of QI in the muscle. Discussed in Bohne et al. (2006b), the decrease in biotransformation enzymes at day 14 of feeding with 107 mg EQ/kg feed produced the accumulation of parent EQ in both hepatic and muscle tissues of salmon. In contrary, during 2 weeks depuration period, the concentration of parent EQ in muscle decreased by 98%, with calculated half-life of 2.4 days. At the same time the concentration of EQDM significantly increased by 41%. Thus, the net concentration of the sum of parent EQ and its dimer remained unchanged during the depuration period. We speculate that two mechanisms or combination of these could explain the observed results (1) biotransformation of parent EQ into EQDM and (2) transport of EQDM from other organs, such as between liver and adipose tissue, which was the main depot of EQDM (Bohne, Arukwe and Hamre, unpublished). Thus, the edible part of the ready-to-consume salmon, namely the muscle, contains high concentration of EQDM whose toxicological effect on humans has not been previously investigated. The daily intake of EQ and EQDM by a 60-kg person through the consumption of one portion (200 g) of salmon fillet had been estimated to be 3377 ng/kg body weight. This is below the established acceptable daily intake for EQ alone, which is 5000 ng/kg body weight (WHO). Nevertheless, several compounds with the possibility of having EQ-derived metabolic origin were observed in this study, and we are performing studies to identify and characterize these compounds.
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ACKNOWLEDGMENTS |
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This work was supported by Norwegian Research Council under the project no. 143314/130 SYNTOX. The help from Harald Mundheim at Fiskeriforskning (Norway, Bergen) in the formulation and production of the experimental diets is highly appreciated. The authors are grateful to the technical staff at Matre Research Station for excellent stalling of the fish and technical staff at FettVit laboratory at NIFES for the help in the sampling. We also thank Anne Mortensen for highly professional technical assistance.
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REFERENCES |
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