Heavy-metal and microbial depuration of the clam Ruditapes decussatus and its effect on bivalve behavior and physiology

The bivalve Ruditapes decussatus was evaluated as a possible biomonitor of heavy-metal contamination. Concentrations of copper (Cu), cobalt (Co), iron (Fe), nickel (Ni), and manganese (Mn) were measured in R. decussatus. Water and sediment samples were collected at two stations of Timsah Lake in Ismailia, Egypt, from October to November 2002, using atomic absorption spectrophotometry (AAS). Results from the heavy-metal and microbial analyses indicated that site II was less contaminated than site I. The bivalve showed accumulation of metals, with a bioaccumulation factor (BAF) greater than 4. The bioaccumulation of metals varied strongly according to the sampling site. After 48 h of depuration, Fe, Ni, Co, Cu, and Mn were reduced significantly, to 46.8% and 47.7%, 19.9% and 20.3%, 27.3% and 27.9%, 35.9% and 36.6%, and 18.2% and 26.6%, compared with the initial concentrations, in clam tissue at the two stations. In bivalves from site II the counts of total bacteria, fecal coliforms, and bacterial pathogens were reduced by more than 90%, whereas phage counts were only reduced by 56% after 4 days of depuration. The depuration of bivalves collected from the heavily polluted site (I) was not effective, as coliforms were reduced only by 85% after 4 days, whereas counts of pathogens and pathogenic indicators such as Vibrio, fecal Streptococcus, and coliphage decreased to less than 50% of the initial concentration. The time necessary to decrease contamination to 10%, 50%, and 90% for clams at both stations was consistently shorter for heavy metals than for microorganisms. Investigation of the effects of heavy-metal and microbiological depuration on valve movement and physiological rates (oxygen consumption and ammonia excretion) was carried out on R. decussatus to test the utility of physiological stress indices in assessing the health of depurated animals. Clams in the experimental tanks exhibited various states of activity, which were rated by identifying and scoring (0-4) the different parameters, including shell gap, siphon extension, and foot protrusion. Moreover, an increase in ammonia excretion was usually associated with an increase in respiration rate. The oxygen-to-nitrogen ratio provided a sensitive indicator of bivalve health. It can be concluded that shellfish monitoring and depuration data depended on the initial concentration of the pollutants. There were differences in the physiological responses of clams from the two sites during the periods of pre- and postdepuration of the contaminants. There was a significant correlation between reduction of metal concentration in clam tissue and enhancement of valve movement, as well as activity and increasing respiration rate.     

Oxidative stress in the clam Ruditapes decussatus (Linnaeus, 1758) in relation to polycyclic aromatic hydrocarbon body burden

Seasonal variation of antioxidant enzymes activities (superoxide dismutase, catalase, and glutathione peroxidases) and lipid peroxidation (LPO) were studied in the clam Ruditapes decussatus in relation to body burdens of polycyclic aromatic hydrocarbons (PAHs). Clams were sampled in eight sites from the Ria Formosa lagoon. PAH concentrations were seasonally rather than spatially dependent, being higher in summer (August). Antioxidant enzymes activities and LPO levels in the clam digestive gland were also seasonally dependent. Antioxidant enzymes presented distinct seasonal variations: Mit SOD (superoxide dismutase activity measured in the mitochondrial fraction) was induced in the summer and down-regulated in winter and spring, while Cyt SOD activity (measured in the cytosolic fraction) was highest in autumn and lower in the summer. Neither Mit nor Cyt SOD were related to the clam PAH body burden, suggesting that cells are using other antioxidant systems to eliminate oxyradicals. Catalase (CAT), however, was induced in spring and down-regulated in summer, the inverse of the PAH concentrations in clam tissues. CAT induction in spring appears to be related to the excess of oxyradicals arising from the metabolic activity associated with the reproductive cycle. Conversely, the decrease in CAT activity in the summer may be related to the high water temperatures reached in the Ria Formosa (up to 30 degrees C). Glutathione peroxidases (total fraction - T-GPx and dependent on selenium - Se-GPx) presented a similar seasonal pattern, and were negatively related to PAH concentrations, which may indicate a precarious state of the clams, associated with PAH toxicity. Similarly, LPO was also inversely correlated to the PAH concentrations indicating that increases in PAH concentrations were not causing membrane oxidative damage in R. decussatus digestive gland. The results suggest that antioxidant enzymes in R. decussatus digestive gland are strongly affected by seasonal factors stressing the need of other experiments to clarify the PAHs effect on this clam species.     

Benzo(a)pyrene-induced metabolic responses in Manila clam Ruditapes philippinarum by proton nuclear magnetic resonance ((1)H NMR) based metabolomics

Benzo(a)pyrene is an important polycyclic aromatic hydrocarbon (PAH) which causes carcinogenic, teratogenic and mutagenic effects in various species and the level of contamination of this toxic agent in the marine environment is of great concern. In this study, metabolic responses induced by two doses (0.02 and 0.2 μM) of BaP were characterized in the gill tissues of Manila clam Ruditapes philippinarum after exposure for 24, 48 and 96 h. The high dose (0.2 μM) of BaP induced the disturbances in energy metabolism and osmotic regulation based on the metabolic biomarkers such as succinate, alanine, glucose, glycogen, branched chain amino acids, betaine, taurine, homarine, and dimethylamine in clam gills after 24 h of exposure. In addition, hormesis induced by BaP was found in clams exposed to both doses of BaP. Overall, our results demonstrated the applicability of metabolomics for the elucidation of toxicological effects of marine environmental contaminants in a selected bioindicator species such as the Manila clam.     

Inhibitory effect of stiripentol on carbamazepine and saquinavir metabolism in human

AIMS: To characterize the in vitro and in vivo inhibitory effect of stiripentol, a new anticonvulsant, on the metabolism of carbamazepine and saquinavir, which are substrates of CYP3A4. METHODS: Human liver microsomes and cDNA-expressed CYP enzymes were used for the in vitro experiments. Pharmacokinetic data from epileptic children and healthy adults were used for the carbamazepine and saquinavir in vivo studies, respectively. RESULTS: Carbamazepine biotransformation to its 10,11-epoxide by human liver microsomes (Vmax = 10.3 nmol min(-1) nmol(-1) P450, apparent Km = 362 microm), cDNA-expressed CYP3A4 (Vmax = 1.17 nmol min(-1) nmol(-1) P450, apparent Km = 119 microm) and CYP2C8 (Vmax = 0.669 nmol min(-1) nmol(-1) P450, apparent Km = 757 microm) was inhibited by stiripentol (IC50 14, 5.1, 37 microM and apparent Ki 3.7, 2.5, 35 microm, respectively). Saquinavir biotransformation to its major metabolite M7 by human liver microsomes (Vmax = 5.7 nmol min(-1) nmol(-1) P450, apparent Km = 0.79 microm) was inhibited by stiripentol (IC50 163 microM, apparent Ki 86 microm). In epileptic children treated with carbamazepine and stiripentol, the plasma concentration ratio of carbamazepine epoxide/carbamazepine was decreased by 65%. The in vivo apparent Ki for stiripentol ranged from 10.5 to 41.4 microm. The pharmacokinetics of saquinavir was not modified by stiripentol in healthy adults. The 95% confidence intervals for the difference for Cmax and AUC of saquinavir between the placebo and stiripentol phase were (-39.8, 39.8) and (-33.2, 112), respectively. CONCLUSIONS: These results showed that stiripentol was a weak inhibitor of saquinavir metabolism both in vitro and in vivo. In contrast, stiripentol is a potent inhibitor of carbamazepine 10,11-epoxide formation in vitro and in vivo in epileptic patients.     

盐酸小檗碱对大鼠体内卡马西平及其代谢产物药动学的影响

目的:研究盐酸小檗碱(BBR)与卡马西平(CBZ)合用对大鼠体内CBZ及其代谢产物10,11-环氧卡马西平(ECBZ)药动学的影响。方法:大鼠随机分为CBZ单用组和BBR低、中、高剂量合用组,经灌胃2周BBR后,HPLC法测定大鼠口服CBZ后CBZ及ECBZ的血药浓度,3P97药动学软件计算药动学参数。结果:与CBZ单用组相比,BBR合用组的CBZ与ECBZ的药动学参数差异无显著性(P>0.05)。结论:体内研究表明合用BBR对大鼠口服CBZ的药动学无显著影响。     

Single and combined effects of carbamazepine and copper on nervous and antioxidant systems of zebrafish (Danio rerio)

Various pollutants co‐exist in the aquatic environment such as carbamazepine (CBZ) and copper (Cu), which can cause complex effects on inhabiting organisms. The toxic impacts of the single substance have been studied extensively. However, the studies about their combined adverse impacts are not enough. In the present study, zebrafish were exposed to environmental relevant concentrations of CBZ (1, 10, and 100 μg/L), Cu (0.5, 5, and 10 μg/L) and the mixtures (1 μg/L CBZ + 0.5 μg/L Cu, 10 μg/L CBZ + 5 μg/L Cu, 100 μg/L CBZ + 10 μg/L Cu) for 45 days, the effects on nervous and antioxidant systems of zebrafish were investigated. The results demonstrated that, in comparison with single exposure group, the combined presence of CBZ and Cu exacerbated the effect of antioxidant system (the ability of inhibition of hydroxyl radicals (IHR), superoxide dismutase (SOD), catalase (CAT) and glutathione S‐transferase (GST)) but not nervous system (Acetylcholinesterase [AChE]). The qPCR results supported the changes of corresponding enzymes activities. Hepatic histopathological analysis verified the results of biomarkers. Our work illustrated that the toxicity of mixed pollutants is very complicated, which cannot simply be inferred from the toxicity of single pollutant, and calls for more co‐exposure experiments to better understanding of the co‐effects of pollutants on aquatic organisms.     

Uptake,distribution, metabolism and clearance of chlordecone by channel catfish (Ictalurus punctatus)

Channel catfish (Ictalurus punctatus) were exposed to a [¹⁴C]chlordecone-contaminated diet for a period of 90 days, followed by a chlordecone-free diet for 30 days. The uptake and clearance rate constants were 0.237 day^?1 and 0.080 day^?1, respectively. The half-life of chlordecone in these fish was 8.7 days. The dietary accumulation factor (DAF), defined as the equilibrium concentration of pesticide in fish/daily dietary dosage level was calculated to be 3.0.Highest concentrations of chlordecone in channel catfish were found in the blood and brain. White catfish exposed to the [¹⁴C]chlordecone-contaminated diet contained highest concentrations in the brain. In both species lowest concentrations were measured in the mesenteric adipose and carcass.Chlordecone may be eliminated with bile via the intestinal contents. There is evidence for an extrabiliary source of fecal chlordecone in channel catfish. Chlordecone may be excreted across the gills and sloughed off with epidermal mucus. Urinary excretion is negligible.Channel catfish are capable of reducing chlordecone to chlordecone alcohol as shown by the presence of this metabolite in the bile. Enzymatic digestion of bile with β-glucuronidase did not significantly affect the recovery of chlordecone or chlordecone alcohol.     

Preclinical absorption,distribution, metabolism and excretion (ADME) characterization of ICAM1988, an LFA-1/ICAM antagonist,and its prodrug

1. Intercellular adhesion molecule (ICAM)-1988 is a small molecule lymphocyte function-associated antigen-1 (LFA-1) antagonist being considered for its anti-inflammatory properties. Following intravenous administration of ICAM1988, clearances in mice, rats, dogs, and monkeys were 17.8, 3.31, 15.4, and 6.85 ml min^?1 kg^?1, respectively.

2. In mass balance studies using [¹⁴C]-ICAM1988 in rats dosed intravenously, unchanged ICAM1988 contributed to 25.1% of the dose.

3. In rats, the systemic bioavailability of ICAM1988 was improved to 0.28 when the drug was administered orally as its isobutyl ester, ICAM2660. In rats, this was consistent with the complete in vitro conversion of ICAM2660 to ICAM1988 in plasma, and liver and intestinal S9.

4. In dogs and monkeys, ICAM2660 did not improve the bioavailability of ICAM1988. This is consistent with limited in vitro conversion of ICAM2660 to ICAM1988 in plasma and liver S9.

5. In human in vitro studies, ICAM2660 conversion to ICAM1988 in liver was similar to rats while no conversion in plasma and intestinal S9 fractions were observed. Based on the in vitro metabolism similarities of human and rat, it would be anticipated that in human oral administration of ICAM2660 would improve the systemic exposure of ICAM1988.

     

Quantitation of the Relative Amounts of Anhydrous Carbamazepine (C15H12N2O) and Carbamazepine Dihydrate (C15H12N2O · 2H2O) in a Mixture by Solid-State Nuclear Magnetic Resonance (NMR)

The application of solid state nuclear magnetic resonance (NMR) for the quantitation of the relative amounts of carbamazepine anhydrate (I) and Carbamazepine dihydrate (II) in a mixture is presented. The techniques of cross polarization, dipolar decoupling, and magic angle spinning have been used to obtain high-resolution NMR spectra of the samples in the solid state. Although the chemical shifts of I and II were similar, the proton spin lattice relaxation time of II was much shorter than that of I. A delay time of 10 sec between pulses resulted in saturation of the signal from I and in a spectrum arising solely from II. The dependence of the observed signal intensity on the contact time was evaluated for II and glycine, the internal standard, to allow theoretical estimation of the peak area ratios. Various molar ratios of I and II were then mixed with glycine, and the resulting peak area ratios of II to the area of the alpha and the carbonyl carbons of glycine was linearly related to the relative proportion of II in the mixture.     

Analysis of carbamazepine and its 10,11-epoxide in serum by direct sample injection using surfactant containing eluents and column switching

Column switching is used in conjunction with surfactant containing mobile phases and traditional reverse phase LC columns to provide a highly reproducible and accurate analytical procedure for carbamazepine and its 10,11-epoxide in serum. This approach eliminates the tedious sample preparation steps commonly used in the analysis of drugs by HPLC, while providing a high degree of protein removal prior to the final analysis. Various pre-columns were investigated and a pellicular reverse phase column was found suitable for optimum concentration of drugs and removal of serum proteins. A variety of standard reverse phase columns could be used for the analytical separation. The separation of the drugs could be accomplished with a high degree of reproducibility. Tandem pre-column operation was demonstrated to give a sample throughput of 10 h-1.     

Toxic effects and depuration after the dietary lead(II) exposure on the bioaccumulation and hematological parameters in starry flounder (Platichthys stellatus)

Platichthys stellatus (mean length 20 ± 2 cm, mean weight 160.15 ± 15 g) were exposed to the different levels of dietary lead(II) at the concentrations of 0, 30, 60, 120, 240 mg/kg for 4 weeks. Depuration was conducted for 2 weeks after exposure. The lead exposure over 60 mg Pb/kg induced the significant bioaccumulation in tissues of P. stellatus (5–30 μg/g tissue), except for brain and muscle where the exposure to 240 mg Pb/kg caused the bioaccumulation (2–4 μg/g tissue). The hematological parameters such as red blood cell (RBC) counts, hematocrit (Ht) value and hemoglobin (Hb) concentration were substantially decreased over 60 mg Pb/kg, and lasted even after the depuration period. For plasma components, calcium and magnesium levels in plasma were generally decreased over 60 mg Pb/kg, and glucose level was also mainly increased over 60 mg Pb/kg. Total protein was significantly decreased over 120 mg Pb/kg after 4 weeks exposure. Glucose and total protein showed the restoration after the depuration period in groups of fish exposed previously to over 60 and 120 mg Pb/kg, respectively. However, other parameters that changed during the exposure over 60 mg Pb/kg did not recovered. For enzymatic components in plasma, glutamic oxalate transminase (GOT), glutamic pyruvate transminase (GPT) and alkaline phosphatase (ALP) were significantly increased over 120 mg Pb/kg, and there was only restoration observed after the depuration for ALP over 120 mg Pb/kg.     

Regulation of UDP-glucuronosyltransferase (UGT) 1A1 by progesterone and its impact on labetalol elimination

The authors recently reported the increased oral clearance of labetalol in pregnant women. To elucidate the mechanism of the elevated oral clearance, it was hypothesized that female hormones, at the high concentrations attainable during pregnancy, enhance hepatic metabolism of labetalol. Labetalol glucuronidation, which is the major elimination pathway of labetalol, was characterized by screening six recombinant human UGTs (UGT1A1, 1A4, 1A6, 1A9, 2B4, and 2B7) for their capacity to catalyse labetalol glucuronidation. The effect of female hormones (progesterone, oestradiol, oestriol, or oestrone) on the promoter activities of relevant UDP glucuronosyltransferases (UGT) was investigated using a luciferase reporter assay in HepG2 cells. The involvement of oestrogen receptor α (ERα) and pregnane X receptor (PXR) was examined by co-transfecting ERα- or PXR-constructs. UGT1A1 and UGT2B7 were identified as the major UGT enzymes producing labetalol glucuronides (trace amount of glucuronide conjugate was formed by UGT1A9). The activities of the UGT1A1 promoter containing PXR response elements were enhanced by progesterone, but not by oestrogens, indicating PXR-mediated induction of UGT1A1 promoter activity by progesterone. Results from semi-quantitative real-time polymerase chain reaction (PCR) assays are consistent with the above findings. This effect of progesterone on UGT1A1 promoter activities was concentration dependent. Promoter activities of UGT2B7 were not affected by either oestrogens or progesterone. The results suggest a potential role for progesterone in regulating labetalol elimination by modulating the expression of UGT1A1, leading to enhanced drug metabolism during pregnancy.     

Pharmacokinetics and metabolism of (R,R)-methoxyfenoterol in rat

1. (R,R)-fenoterol (Fen), a β₂-adrenoceptor agonist, is under clinical investigation in the treatment of congestive heart disease. The pharmacokinetics and metabolism of the 4-methoxyphenyl derivative of (R,R)-Fen, (R,R)-MFen, have been determined following intravenous and oral administration to the rat and compared with corresponding results obtained with (R,R)-Fen. Results from the study suggest that (R,R)-MFen can offer pharmacokinetic and metabolic advantages in comparison to an earlier (R,R)-Fen.

2. The oral administration revealed that the net exposure of (R,R)-MFen was about three-fold higher than that of (R,R)-Fen (7.2 versus 2.3?min × nmol ml^?1), while intravenous administration proved that the clearance was significantly reduced, 48 versus 146?ml min^?1 kg^?1, the T_1/2 was significantly longer, 152.9 versus 108.9?min, and the area under the curve (AUC) was significantly increased, 300 versus 119?min × nmol ml^?1.

3. (R,R)-MFen was primarily cleared by glucuronidation associated with significant presystemic glucuronidation of the compound. After intravenous and oral administration of (R,R)-MFen, (R,R)-Fen and (R,R)-Fen-G were detected in the urine samples indicating that (R,R)-MFen was O-demethylated and subsequently conjugated to (R,R)-Fen-G. The total (R,R)-Fen and (R,R)-Fen-G as a percentage of the dose after intravenous administration was 3.6%, while after oral administration was 0.3%, indicating that only a small fraction of the drug escaped presystemic glucuronidation and was available for O-demethylation.

4. The glucuronidation pattern was confirmed by the results from in vitro studies where incubation of (R,R)-MFen with rat hepatocytes produced (R,R)-MFen-G, (R,R)-Fen and (R,R)-Fen-G, while incubation with rat intestinal microsomes only resulted in the formation of (R,R)-MFen-G.

     

Toxicokinetics of tetrabromoethylcyclohexane (TBECH) in juvenile brown trout (Salmo trutta) and effects on plasma sex hormones

Technical 1,2-dibromo-4-(1,2 dibromoethyl)cyclohexane or tetrabromoethylcyclohexane (TBECH) used primarily as an additive flame retardant in polystyrene foams, contains two diastereoisomers, α- and β- present in equimolar amounts. At temperatures in excess of 125 °C, isomerization to two other isoforms, δ- and γ- is possible. The recent detection of TBECH in the environment and studies suggesting that isomers are androgenic prompted us to examine the toxicokinetics and biochemical effects of one of the isomers, β-, in a controlled laboratory environment. Juvenile brown trout (Salmo trutta) were exposed to three different amounts of the β-isomer (low, medium and high) via the food followed by a period in which they were exposed to unfortified food. A fourth group of fish was exposed to unfortified food for the duration of the experiment. On days 0, 7, 14, 21, 35, 49, 56, 63, 77, 91, 105, and 133, eight fish from each treatment group were euthanized and liver, plasma, lower jaw (i.e., thyroid tissue) and gonad were collected and the remaining tissue (‘whole-fish’) was retained. β-Isomer content was measured in whole-fish and in liver while estradiol (E2), 11-ketotestosterone (11-KT) and testosterone (T) were measured in plasma. Based on liver and gonad somatic indices, no apparent effects on liver or gonad development in fish from any of the treatment groups were observed. The bioaccumulation of β-isomer was similar in fish from all treatment groups with steady-state occurring before the end of the uptake phase. Depuration of the β-isomer from fish obeyed first order kinetics and there were no statistically significant differences in the depuration half life (t_1/2) among the treatment groups: 22.5 ± 10.4 (low), 13.5 ± 5.9 (med) and 13.8 ± 2.2 (high) days. Steady-state biomagnification factors were much smaller than 1 for fish in all treatment groups. Debrominated metabolites were not detected in composite liver or whole-fish extracts and there was no evidence of isomerization of the β-isomer to other isoforms in vivo. While there were occasional differences among treatment groups in circulating plasma E2, T and 11-KT levels there was no clear, temporal trend or dose-response.     

Characterization of increased drug metabolism activity in dimethyl sulfoxide (DMSO)-treated Huh7 hepatoma cells

1. The objective of this study was to characterize Huh7 cells’ baseline capacity to metabolize drugs and to investigate whether the drug metabolism was enhanced upon treatment with dimethyl sulfoxide (DMSO).

2. The messenger RNA (mRNA) levels of major Phase I and Phase II enzymes were determined by quantitative real-time-polymerase chain reaction (RT-PCR), and activities of major drug-metabolizing enzymes were examined using probe drugs by analysing relevant metabolite production rates.

3. The expression levels of drug-metabolizing enzymes in control Huh7 cells were generally very low, but DMSO treatment dramatically increased the mRNA levels of most drug-metabolizing enzymes as well as other liver-specific proteins. Importantly, functionality assays confirmed concomitant increases in drug-metabolizing enzyme activity. Additionally, treatment of the Huh7 cells with 3-methylcholanthrene induced cytochrome P450 (CYP) 1A1 expression.

4. The results indicate that DMSO treatment of Huh7 cells profoundly enhances their differentiation state, thus improving the usefulness of this common cell line as an in vitro hepatocyte model.

     

Utility of spatially-resolved atmospheric pressure surface sampling and ionization techniques as alternatives to mass spectrometric imaging (MSI) in drug metabolism

1. Tissue distribution studies of drug molecules play an essential role in the pharmaceutical industry and are commonly undertaken using quantitative whole body autoradiography (QWBA) methods.

2. The growing need for complementary methods to address some scientific gaps around radiography methods has led to increased use of mass spectrometric imaging (MSI) technology over the last 5 to 10 years. More recently, the development of novel mass spectrometric techniques for ambient surface sampling has redefined what can be regarded as “fit-for-purpose” for MSI in a drug metabolism and disposition arena.

3. Together with a review of these novel alternatives, this paper details the use of two liquid microjunction (LMJ)-based mass spectrometric surface sampling technologies. These approaches are used to provide qualitative determination of parent drug in rat liver tissue slices using liquid extraction surface analysis (LESA) and to assess the performance of a LMJ surface sampling probe (LMJ-SSP) interface for quantitative assessment of parent drug in brain, liver and muscle tissue slices.

4. An assessment of the utility of these spatially-resolved sampling methods is given, showing interdependence between mass spectrometric and QWBA methods, in particular there emerges a reason to question typical MSI workflows for drug metabolism; suggesting the expedient use of profile or region analysis may be more appropriate, rather than generating time-intensive molecular images of the entire tissue section.

     

Uptake,biotransformation, and elimination of rotenone by bluegills (Lepomis macrochirus)

Yearling bluegills (Lepomis macrochirus) were exposed to sublethal concentrations of [¹⁴C]rotenone (5.2 μg/l) for 30 days in a continuous flow exposure system and then transferred to clean, flowing water for an additional 21-day depuration period. Rates of uptake and elimination and profile of the rotenoid metabolites in head, viscera, and carcass components were evaluated by ¹⁴C counting and by high performance liquid chromatography. Total [¹⁴C]rotenone derived activity was relatively uniform in all body components within 3 days after initial exposure and remained constant during the ensuing 27 days of exposure. Initial uptake rate coefficients were highest in viscera (K_u = 80 · h^?1) and were nearly identical for head (K_u = 14 · h^?1) and carcass (K_u = 10 · h^?1). Analyses of tissue extracts by high performance liquid chromatography confirmed the presence of at least six biotransformation products of rotenone. More than 60% of the activity extracted from viscera was present as a single peak which represented a compound that was extremely soluble in water. Rotenone composed only 0.3% of the extractable activity in viscera taken from fish exposed to rotenone for 30 days; however, rotenone accounted for 15.4% of extractable activity in the head and 20.1% in the carcass components. Rotenolone and 6′,7′-dihydro-6′,7′-dihydroxyrotenolone were tentatively identified as oxidation products in all tissue extracts.Elimination of ¹⁴C activity from all body components was biphasic; both phases followed first-order kinetics. The rate of elimination was nearly equal for all body components during the initial phase but was most rapid from viscera during the second phase of elimination. Bioconcentration factors for the head, viscera, and carcass were 165, 3,550, and 125, respectively, when calculated on the basis of total ¹⁴C activity but only 25.4, 11, and 26 when calculated as the concentration of parent material.     

Distribution of butyltins (TBT, DBT, MBT) in sediments of Gulf of Cádiz (Spain) and its bioaccumulation in the clam Ruditapes philippinarum

Surface sediment samples were analyzed for organotins namely tributyltin (TBT), dibutyltin and monobutyltin from six areas located in the Gulf of Cádiz (14 stations), Spain. The total butyltin ranged between undetected and 1,580 ng Sn g⁻¹. TBT generally prevailed in most of the samples, suggesting fresh inputs of butyltin compounds and/or less degradation of TBT. The observed levels of butyltins at several sites are much higher than that required to induce toxic effect on marine organisms, suggesting that these sediments are polluted with butyltin compounds. The clam Ruditapes philippinarum was used for studying bioaccumulation of butyltins by exposing them to contaminated sediments from the Gulf of Cádiz over a period of 28 days under laboratory conditions. Biota-sediment accumulation factor (BSAF) ranged from 0.44 to 3.99.     

The different metabolism of morusin in various species and its potent inhibition against UDP-glucuronosyltransferase (UGT) and cytochrome p450 (CYP450) enzymes

1.?The aim of this study was to investigate the inhibitory effect of morusin on Glucuronosyltransferase (UGT) isoforms and cytochrome P450 enzymes (CYP450s). We also investigated the metabolism of morusin in human, rat, dog, monkey, and minipig liver microsomes.

2.?100?μM of morusin exhibited strong inhibition on all UGTs and CYP450s. The half inhibition concentration (IC₅₀) values for CYP3A4, CYP1A2, CYP2C9, CYP2E1, UGT1A6, UGT1A7, and UGT1A8 were 2.13, 1.27, 3.18, 9.28, 4.23, 0.98, and 3.00?μM, and the inhibition kinetic parameters (K_i) were 1.34, 1.16, 2.98, 6.23, 4.09, 0.62, and 2.11?μM, respectively.

3.?Metabolism of morusin exhibited significant species differences. The quantities of M₁ from minipig, monkey, dog, and rat were 7.8, 11.9, 2.0, and 6.3-fold of human levels. The K_m values in HLMs, RLMs, MLMs, DLMs, and PLMs were 7.84, 22.77, 14.32, 9.13, and 22.83?μM, and V_max for these species were 0.09, 1.23, 1.43, 0.15, and 0.75?nmol/min/mg, respectively. CL_int (intrinsic clearance) values (V_max/K_m) for morusin obeyed the following order: monkey?>?rat?>?minipig?>?dog?>?human. CL_H (hepatic clearance) values for humans, dogs, and rats were calculated to be 8.28, 17.38, and 35.12?mL/min/kg body weight, respectively.

4.?This study provided vital information to understand the inhibitory potential and metabolic behavior of morusin among various species.     

Uptake and metabolism of technical nonylphenol and its brominated analogues in the roach (Rutilus rutilus)

Alkylphenols have been implicated as one of the causative agents of oestrogenic contamination in fish. This study reports the fate of a technical mixture of secondary and tertiary isomers of the environmental contaminant 4-nonylphenol (NP) in mature female roach. Fish were exposed to a concentration of 4.9microg/l radio-labelled technical NP over a 4-day period in a flow-through aquarium. NP residues were extracted from all tissues and analysed by reverse phase radio HPLC. The concentration of NP residues was highest in bile and liver, with apparent bioconcentration factors (apparent BCFs) of 34,121 and 605, respectively. In other tissues, apparent BCF values were recorded between 13 and 250. NP was the only residue detected in brain, muscle and blood cells whereas an additional NP derivative was detected in other soft tissues, including gonads and liver, which was identified as the brominated derivative of technical NP, dibromo-4-nonylphenol. Traces of the dibrominated NP were also detected in aquarium water but only in the presence of NP, which suggested that bromination of the NP parent compound occurred in situ in the water due to the presence of trace amounts of oxidised bromine species in municipal water following disinfection. There was no evidence of further metabolism of the brominated derivative in roach, but a single major metabolite of NP was present in liver and bile, which was identified as the glucuronide conjugate of 4-(hydroxy-nonyl)-phenol. This detection of only one NP metabolite suggests that, in contrast to other fish species, the variety of metabolic pathways available for the deactivation of 4-alkylphenols in roach is limited. This study indicates that brominated alkylphenols bioconcentrate in a range of tissues and, as they have been detected in the aquatic environment, they could contribute to the body burden of alkylphenolics in fish. The formation of these brominated derivatives in aquarium water suggest that they could confound toxicity tests to assess the effects of alkyphenols to aquatic organisms.