Accumulation and depuration of the synthetic antioxidant ethoxyquin in the muscle of Atlantic salmon (Salmo salar L.).

The biological fate of the fish feed additive, ethoxyquin (EQ) was examined in the muscle of Atlantic salmon during 12 weeks of feeding followed by a 2 weeks depuration period. Parent EQ (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline), quinone imine (2,6-dihydro-2,2,4-trimethyl-6-quinolone), de-ethylated EQ (6-hydroxy-2,2,4-trimethyl-1,2-dihydroquinoline) and EQDM (EQ dimer or 1,8'-di(1,2-dihydro-6-ethoxy-2,2,4-trimethyl-quinoline) were found to be the ubiquitous metabolites of dietary EQ, with EQDM as a main metabolite. A rapid decrease in the level of EQ (2.4 days of half-life) was balanced by an increase in EQDM, giving an unchanged net sum following 2 weeks of depuration. The mandatory 14 days depuration period prior to slaughtering of farmed salmon in Norway was not sufficient for complete elimination of EQ-derived residuals. Post depuration, EQDM accounted for 99% of sum of the two compounds in all treatment groups; possible toxicological effects of EQDM are not known. The individual concentrations of EQ and EQDM and their sum are dependent on EQ level in the feed, consequently, their residual concentrations may be controlled. The theoretical amount of EQ and EQDM consumed in one meal of farmed salmon would be under the recommended ADI, provided that the fish were raised on feed with no more than 150 mg EQ/kg feed, which is the EU maximum limit for EQ in fish feed.     

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.     

Accumulation and elimination kinetics of dietary endosulfan in Atlantic salmon (Salmo salar)

The carry-over of dietary endosulfan to the fillet of farmed Atlantic salmon was studied. The uptake and elimination rate constants of the alpha and beta isoform of endosulfan were determined in seawater adapted Atlantic salmon (initial weigh 173+/-25 g) fed on endosulfan enriched diets (724 and 315 microg kg(-1) for alpha- and beta-endosulfan, respectively) for 92 days, followed by a 56 days depuration period with feeding on control diets (<0.3 microg kg(-1) endosulfan). The accumulation of the toxic metabolite endosulfan sulphate, which was not detected (<0.5 microg kg(-1)) in the experimental feeds, was also determined. Dietary beta-endosulfan was more persistent than alpha-endosulfan as demonstrated by a higher uptake (41+/-8% vs. 21+/-2%) and lower elimination (26+/-2 x 10(-3) day(-1) vs. 40+/-1 x 10(-3) day(-1)) rate constants, and a higher biomagnification factor (0.10+/-0.026 vs. 0.05+/-0.003, p<0.05). Based on the decrease in diastereometric factor over time, biotransformation was estimated to account for at least 50% of the endosulfan elimination. The formation of the metabolite endosulfan sulphate comprised a maximum 1.2% of the total accumulation of endosulfan. Using a simple one-compartmental model and the experimentally-determined rate constants, it was predicted that only dietary concentration higher than 7.5 mg kg(-1) would cause the estimated maximum residue level (MRL) in fillet of 0.47 mg kg(-1) to be exceeded. Model estimations also predict that current EU maximum permitted levels in fish feeds (0.005 mg kg(-1)) would give fillet concentrations that are under the detection limit (0.3 microg kg(-1)), which is in agreement with the general absence of endosulfan in monitored farmed Atlantic salmon.     

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

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

Bioaccessibility of Hg, Cd and As in cooked black scabbard fish and edible crab

Regular consumption of seafood has been widely recommended by authorities. Yet, some species accumulate high levels of contaminants like Hg, Cd and As. In addition, the risks associated to the consumption of such seafood may increase if consumers use cooking practices that enhance the concentration of contaminants and their bioaccessibility. In this study, the bioaccessibility of Hg, Cd and As was assessed with in vitro human digestion of raw and cooked black scabbard fish (Hg; steamed, fried and grilled) and edible crab (Cd and As; steamed and boiled) tissues. Additionally, the toxicological hazards associated with the consumption of these products were also discussed. Generally, Hg, Cd and As bioacessibility increased throughout the digestion process. Cadmium and As revealed high bioaccessibility rates in raw and cooked samples (up to 100%), whereas lower bioaccessible fractions of Hg was observed (up to 40%). Furthermore, this study pointed out the importance of food matrix, elemental chemical properties and cooking practices in the bioaccessibility of Hg, Cd and As. The toxicological hazards revealed that edible crab brown meat (Cd) and grilled black scabbard fish (MeHg) consumption in children should be moderated. In contrast, edible crab muscle (Cd) and fried or steamed black scabbard fish (MeHg) should be consumed to minimize exposure. The use of bioaccessible contaminant data strongly reduced the toxicological risks of MeHg, whereas less risk reduction occurred with Cd and inorganic As.     

Review on the toxicity, occurrence, metabolism, detoxification, regulations and intake of zearalenone: an oestrogenic mycotoxin.

Zearalenone (ZEA) is a mycotoxin produced mainly by fungi belonging to the genus Fusarium in foods and feeds. It is frequently implicated in reproductive disorders of farm animals and occasionally in hyperoestrogenic syndromes in humans. There is evidence that ZEA and its metabolites possess oestrogenic activity in pigs, cattle and sheep. However, ZEA is of a relatively low acute toxicity after oral or interperitoneal administration in mice, rat and pig. The biotransformation for ZEA in animals involves the formation of two metabolites alpha-zearalenol (alpha-ZEA) and beta-zearalenol (beta-ZEA) which are subsequently conjugated with glucuronic acid. Moreover, ZEA has also been shown to be hepatotoxic, haematotoxic, immunotoxic and genotoxic. The exact mechanism of ZEA toxicity is not completely established. This paper gives an overview about the acute, subacute and chronic toxicity, reproductive and developmental toxicity, carcinogenicity, genotoxicity and immunotoxicity of ZEA and its metabolites. ZEA is commonly found on several foods and feeds in the temperate regions of Europe, Africa, Asia, America and Oceania. Recent data about the worldwide contamination of foods and feeds by ZEA are considered in this review. Due to economic losses engendered by ZEA and its impact on human and animal health, several strategies for detoxifying contaminated foods and feeds have been described in the literature including physical, chemical and biological process. Dietary intakes of ZEA were reported from few countries from the world. The mean dietary intakes for ZEA have been estimated at 20 ng/kgb.w./day for Canada, Denmark and Norway and at 30 ng/kgb.w./day for the USA. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) established a provisional maximum tolerable daily intake (PMTDI) for ZEA of 0.5 microg/kg of body weight.     

Risk and benefits from consuming salmon and trout: a Canadian perspective.

Recent reports on the presence of persistent organic chemicals in wild and farmed salmon have left consumers and health professionals confused regarding the safety of regular fish consumption. The objectives of this study were (1) to compare concentrations of key contaminants and the essential nutrients omega-3 fatty acids between farmed and wild salmon and trout, and (2) to balance risks and benefits from regularly consuming these species. Concentrations of mercury, polychlorinated biphenyls, dioxins and furans as well as omega-3 fatty acids were determined in fillets from farmed salmon and trout bought in various markets located in Quebec, Canada, as well as in fillets from wild salmonids obtained from fishermen and various Canadian agencies. While differences were observed between market (farmed) and wild fish with regard to the concentrations of mercury and polychlorinated biphenyls, overall the concentrations of contaminants were low, such that the regular consumption of these fish would not cause tolerable daily intakes to be exceeded. Our results indicate that salmon and trout sold in Quebec markets, which as in markets located elsewhere in North America originate for the most part from Chilean farms, can be consumed regularly to achieve optimal nutritional benefits from omega-3 fatty acids, without incurring significant contaminant related health risks.     

Identification of human cytochrome P450 enzymes involved in the hepatic and intestinal biotransformation of 20(S)‐protopanaxadiol

20(S)‐Protopanaxadiol (aPPD), a ginseng sapogenin, has been shown to be a promising anti‐cancer compound and anti‐depressant agent. Although the bacterial biotransformation of ginsenosides has been studied thoroughly, few have reported on the cytochrome P450 (P450) mediated metabolism of aPPD. Taken orally, aPPD must first undergo absorption and metabolism in the intestine before further metabolism in the liver. The present study investigated the comparative biotransformation profile of aPPD in human intestinal microsomes (HIM) and human liver microsomes (HLM) and characterized the human P450 enzymes involved in aPPD metabolism. Three major monooxygenated metabolites and five minor dioxygenated metabolites were identified as the predominant products in aPPD incubations with HIM and HLM using liquid chromatography–mass spectrometry. Reaction phenotyping studies were performed with a panel of specific P450 chemical inhibitors, antibody inhibition and human recombinant P450 enzymes. Ketoconazole, a CYP3A inhibitor, blocked the formation of oxygenated metabolites of aPPD in both HIM and HLM in a concentration dependent manner. Among the human recombinant P450 enzymes assayed, CYP3A4 exhibited the highest activity towards aPPD oxidative metabolite formation, followed by CYP3A5. In summary, the results have shown that aPPD is extensively metabolized by HIM and the metabolite profile following in vitro incubations is similar in HIM and HLM. CYP3A4 and CYP3A5 isoforms are the predominant enzymes responsible for oxygenation of aPPD in HIM and HLM. The characterization of aPPD as a CYP3A substrate may facilitate better prediction of drug–herb interactions when aPPD is taken concomitantly with other therapeutic agents. Copyright © 2013 John Wiley & Sons, Ltd.     

Benzo(a)pyrene induces hepatic AKR1A1 mRNA expression in tilapia fish (Oreochromis niloticus)

AKR1A1 or aldehyde reductase is a member of the aldo-keto reductases superfamily that is evolutionarily conserved among species. AKR1A1 is one of the five AKRs (AKR1A1 and 1C1-1C4) implicated in the metabolic benzo(a)pyrene (BaP) activation to reactive BaP 7,8-dione. BaP is a polycyclic aromatic hydrocarbon (PAH) widely distributed in aquatic ecosystems and its metabolic activation is necessary to produce its toxic effects. Although the presence of AKR1A1 in fish has been reported, its tissue distribution in tilapia (Oreochromis niloticus) and AKR1A1 inducibility by BaP are not known yet. Moreover, cytochrome P4501A (CYP1A) mRNA expression in fish has been used as a PAH biomarker of effect. Therefore, BaP effects on AKR1A1 and CYP1A gene expressions in tilapia, a species of commercial interest, were investigated by real-time RT-PCR. A partial AKR1A1 cDNA was identified, sequenced and compared with AKR1A1 reported sequences in the GenBank DNA database. Constitutive AKR1A1 mRNA expression was detected mainly in liver, similarly to that of CYP1A. BaP exposure resulted in statistically significant AKR1A1 and CYP1A mRNA induction in liver (20- and 120-fold, respectively) at 24 h. On the other hand, ethoxyquin (EQ) was used as control inducer for AKR1A1 mRNA. Interestingly, EQ also induced CYP1A mRNA levels in tilapia liver. Our results suggest that teleost AKR1A1, in addition to CYP1A, are inducible by BaP. The mechanism of AKR1A1 induction by BaP and its role in fish susceptibility to BaP toxic effects remains to be elucidated.     

Characterization of in vitro metabolites of cudratricusxanthone A in human liver microsomes

Cudratricusxanthone A (CTXA), isolated from the roots of Cudrania tricuspidata, exhibits several biological activities; however, metabolic biotransformation was not investigated. Therefore, metabolites of CTXA were investigated and the major metabolic enzymes engaged in human liver microsomes (HLMs) were characterized using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). CTXA was incubated with HLMs or human recombinant CYPs and UGTs, and analysed by an LC‐MS/MS equipped electrospray ionization (ESI) to qualify and quantify its metabolites. In total, eight metabolites were identified: M1–M4 were identified as mono‐hydroxylated metabolites during Phase I, and M5–M8 were identified as O‐glucuronidated metabolites during Phase II in HLMs. Moreover, these metabolite structures and a metabolic pathway were identified by elucidation of MSⁿ fragments and formation by human recombinant enzymes. M1 was formed by CYP2D6, and M2–M4 were generated by CYP1A2 and CYP3A4. M5–M8 were mainly formed by UGT1A1, respectively. While investigating the biotransformation of CTXA, eight metabolites of CTXA were identified by CYPs and UGTs; these data will be valuable for understanding the in vivo metabolism of CTXA. Copyright © 2015 John Wiley & Sons, Ltd.     

Synthesis and studies on antioxidants: ethoxyquin (EQ) and its derivatives

In our study ethoxyquin (EQ) and its two complexes with flavonoids were obtained from ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline, EQ) and quercetin (EQ-Q, 1:1) or rutin (EQ-R, 1:1). Cytotoxicity of the tested compounds was studied using the trypan blue exclusion method and the properties of the studied compounds were also analyzed with the TUNEL method evaluating their ability to induce apoptosis. It was shown that EQ induced apoptosis in cultured human lymphocytes, especially at 0.25 and 0.5 mM concentrations. The same effects were also observed after the incubation of lymphocytes with EQ-Q and EQ-R, but the numbers of apoptotic cells observed were lower than for EQ.     

New compounds, 6-hydroxykarahanaenone and 10-hydroxykarahanaenone,from biotransformation of karahanaenone by CYP2A6

The in vitro biotransformation of karahanaenone was examined in cytochrome P450 (CYP) 2A6. The biotransformation of karahanaenone by CYP2A6 was investigated by gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS). Karahanaenone was found to be oxidized to two metabolites by CYP2A6. In order to produce large quantity of metabolites by CYP2A6, the biotransformation of karahanaenone by Salmonella typhimurium OY1002/2A6 was investigated. Similarly, two metabolites were confirmed by GC and GC–MS. The structure of metabolites was determined by 1D NMR, 2D NMR, and infrared, as a result there were new compounds, (6R)-hydroxykarahanaenone and 10-hydroxykarahanaenone.     

Probing Mechanisms of CYP3A Time-Dependent Inhibition Using a Truncated Model System

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Pharmacokinetics of thiamine in female Baltic salmon (Salmo salar L.) broodfish

A thiamine deficiency disease termed M74 syndrome affects the yolk-sac fry of wild and feral Baltic salmon. However, very little is known about the pharmacokinetics of thiamine in salmon. This study examined the pharmacokinetics of thiamine hydrochloride injected intraperitoneally into feral and farmed Baltic salmon females preparing for spawning. The total thiamine concentrations in the eggs and white muscle of feral females were only half of those in the farmed fish after injection. The relative inefficiency of the incorporation of thiamine into the eggs in feral Baltic salmon may be a predisposition factor for M74 syndrome. The relative bioavailability of intraperitoneally-injected thiamine hydrochloride was shown to be ca. 94% of that administered intra-aortically. A far lower intraperitoneal dose (ca. 20mgkg(-1) fish) than currently used was shown to elevate the total thiamine concentration in the eggs above the critical threshold of M74 syndrome.     

CYP1-mediated antiproliferative activity of dietary flavonoids in MDA-MB-468 breast cancer cells

Among the different mechanisms proposed to explain the cancer-protecting effect of dietary flavonoids, substrate-like interactions with cytochrome P450 CYP1 enzymes have recently been explored. In the present study, the metabolism of the flavonoids chrysin, baicalein, scutellarein, sinensetin and genkwanin by recombinant CYP1A1, CYP1B1 and CYP1A2 enzymes, as well as their antiproliferative activity in MDA-MB-468 human breast adenocarcinoma and MCF-10A normal breast cell lines, were investigated. Baicalein and 6-hydroxyluteolin were the only conversion products of chrysin and scutellarein metabolism by CYP1 family enzymes, respectively, while baicalein itself was not metabolized further. Sinensetin and genkwanin produced a greater number of metabolites and were shown to inhibit strongly in vitro proliferation of MDA-MB-468 cells at submicromolar and micromolar concentrations, respectively, without essentially affecting the viability of MCF-10A cells. Cotreatment of the CYP1 family inhibitor acacetin reversed the antiproliferative activity noticed for the two flavones in MDA-MB-468 cells to 13 and 14 μM respectively. In contrast chrysin, baicalein and scutellarein inhibited proliferation of MDA-MB-468 cells to a lesser extent than sinensetin and genkwanin. The metabolism of genkwanin to apigenin and of chrysin to baicalein was favored by CYP1B1 and CYP1A1, respectively. Taken together the data suggests that CYP1 family enzymes enhance the antiproliferative activity of dietary flavonoids in breast cancer cells, through bioconversion to more active products.     

CYP2C8 and CYP3A4 are the principal enzymes involved in the human in vitro biotransformation of the insulin secretagogue repaglinide

AIMS: To identify the principal human cytochrome P450 (CYP) enzyme(s) responsible for the human in vitro biotransformation of repaglinide. Previous experiments have identified CYP3A4 as being mainly responsible for the in vitro metabolism of repaglinide, but the results of clinical investigations have suggested that more than one enzyme may be involved in repaglinide biotransformation. METHODS: [14C]-Repaglinide was incubated with recombinant CYP and with human liver microsomes (HLM) from individual donors in the presence of inhibitory antibodies specific for individual CYP enzymes. Metabolites, measured by high-performance liquid chromatography (HPLC) with on-line radiochemical detection, were identified by liquid chromatography-mass spectrophotometry (LC-MS) and LC-MS coupled on-line to a nuclear magnetic resonance spectrometer (LC-MS-NMR). RESULTS: CYP3A4 and CYP2C8 were found to be responsible for the conversion of repaglinide into its two primary metabolites, M4 (resulting from hydroxylation on the piperidine ring system) and M1 (an aromatic amine). Specific inhibitory monoclonal antibodies against CYP3A4 and CYP2C8 significantly inhibited (> 71%) formation of M4 and M1 in HLM. In a panel of HLM from 12 individual donors formation of M4 and M1 varied from approximately 160-880 pmol min-1 mg-1 protein and from 100-1110 pmol min-1 mg-1 protein, respectively. The major metabolite generated by CYP2C8 was found to be M4. The rate of formation of this metabolite in HLM correlated significantly with paclitaxel 6alpha-hydroxylation (rs = 0.80; P = 0.0029). Two other minor metabolites were also detected. One of them was M1 and the other was repaglinide hydroxylated on the isopropyl moiety (M0-OH). The rate of formation of M4 in CYP2C8 Supersomes was 2.5 pmol min-1 pmol-1 CYP enzyme and only about 0.1 pmol min-1 pmol-1 CYP enzyme in CYP3A4 Supersomes. The major metabolite generated by CYP3A4 was M1. The rate of formation of this metabolite in HLM correlated significantly with testosterone 6beta-hydroxylation (rs = 0.90; P = 0.0002). Three other metabolites were identified, namely, M0-OH, M2 (a dicarboxylic acid formed by oxidative opening of the piperidine ring) and M5. The rate of M1 formation in CYP3A4 Supersomes was 1.6 pmol min-1 pmol-1 CYP enzyme but in CYP2C8 Supersomes it was only approximately 0.4 pmol min-1 pmol-1 CYP enzyme. CONCLUSIONS: The results confirm an important role for both CYP3A4 and CYP2C8 in the human in vitro biotransformation of repaglinide. This dual CYP biotransformation may have consequences for the clinical pharmacokinetics and drug-drug interactions involving repaglinide if one CYP pathway has sufficient capacity to compensate if the other is inhibited.     

Metallothionein and cortisol receptor expression in gills of Atlantic salmon, Salmo salar, exposed to dietary cadmium

Commercial fish feeds may contain significant levels of cadmium (Cd). However, little is known about the effects of dietary cadmium on fish organs, especially gills, the key osmoregulatory organ. We therefore studied the effects of dietary cadmium on metallothionein (MT) and cortisol receptor (GR) immunoreactivity in the branchial epithelium of the Atlantic salmon (Salmo salar). Cadmium was daily administered via food at 0.2mg (control), 5mg (low dose) and 125 mg (high dose) Cd per kilogram dry pellet weight. Fish were sampled after four and eight weeks. After both four and eight weeks, plasma cadmium concentration had increased significantly only in fish fed the high cadmium dose. Plasma calcium, sodium, chloride and cortisol levels were not affected. In the controls, most MT was colocated with the chloride cell marker, Na(+)/K(+)-ATPase, but some MT was present in pavement and respiratory cells. GR expression was found in chloride, pavement, respiratory and undifferentiated cells in all fish groups, but cadmium accumulation and a marked stimulation of MT expression were seen only in the chloride cells in the gills of fish fed the high cadmium dose. Cadmium treatment did not alter GR expression. When the double staining technique for MT and GR was applied, a marked heterogeneity became apparent in the chloride, pavement and respiratory cells of both groups of cadmium-treated fish and in the control fish. Some fish showed double staining, others stained only for one of the antibodies, whereas other cells were negative for both. We conclude that cadmium entering the gut also enters the gills, where it accumulates in chloride cells and stimulates MT expression.     

Assessing the sensitivity of Atlantic salmon (Salmo salar) to dietary endosulfan exposure using tissue biochemistry and histology

The incorporation of plant-based ingredients, and the possible carry-over of pesticides such as endosulfan, in fish feeds may present new toxicological challenges to aquacultural species. Biological responses of Atlantic salmon (Salmo salar) to a 35-day dietary endosulfan exposure at levels ranging from 4 to 710 microgkg(-1) were assessed using tissue histology and biochemistry. Liver 7-ethoxyresorufin-O-deacetylase (EROD) activity was significantly elevated in the highest exposure group (710 microgkg(-1)) by day 35. Other hepatic indicators of stress impacts and responses (glutathione-S-transferase and glutathione peroxidase activities and hepatic alpha-tocopherol content) remained unchanged. Branchial Na(+), K(+)-ATPase activity was significantly reduced at day 14 in the highest exposure group, but returned to control levels by day 35. Conversely, intestinal Na(+), K(+)-ATPase activity was significantly inhibited at day 35, but again only at the highest exposure level. In contrast to the biochemical results, hepatic and intestinal histology revealed effects of exposure even at the lowest dose tested (4 microgkg(-1)). In the posterior intestine, pathology was characterised by vacuolation and fusion of villi, and in the most severe cases, loss of epithelial integrity in villi tips. In the liver the primary effects were glycogen depletion and lipidosis. These changes were typical of a generalised stress response. While histology endpoints may prove to be the most sensitive indicators of dietary endosulfan exposure, the organismal relevance of these structural changes must be considered in the absence of effects in other biomarkers at dietary levels less than 710 microgkg(-1).     

Investigation of the metabolism of 14C/13C-practolol in rat using directly coupled radio-HPLC-NMR-MS

1. The metabolic fate of ¹⁴C ¹³C-practolol was investigated using on-line HPLCNMR-MS following oral administration to rat. The major route of elimination for the radiolabel was via the urine with the principal biotransformation products confirmed as the 2-hydroxy- and 2-hydroxyglucronide metabolites. 2. In addition, futile deacetylation, determined by the replacement of ¹³C-labelled acetyl groups with endogenous ¹²C-acetyls accounted for ~7-10% of the urinary metabolites, corresponding to ~5% of the dose undergoing N-deacetylation. 3. Evidence for chiral metabolism was sought via NMR of isolated metabolites using beta cyclodextrin as a chiral shift agent. Practolol was excreted as a racemate. However, some enantioselective metabolism excretion had occurred as the hydroxy- and hydroxyglucuronide were not excreted as racemic mixtures. 4. Directly coupled radio-HPLC-NMR-MS is extremely effective for the identification of the metabolites of radiolabelled xenobiotics in urine samples.