7-ethoxycoumarin O-deethylase Research Articles

Optimization of assay conditions to quantify ECOD activity in vivo in individual Daphnia magna. Assay performance evaluation with model CYP 450 inducers/inhibitors

Screening the activity of the cytochrome P450 (CYP450) mixed function oxidase system in aquatic invertebrates received seldom applications in ecotoxicology due to low baseline enzymatic activities characteristic for these organisms. In this study, an existing in vivo spectrofluorometric assay method based on quantifying the cytochrome P450 mediated conversion of 7-ethocycoumarin (EtC) used as substrate to the product 7-hydroxycoumarin (HCm) called: ethoxycoumarin-O-deethylase (ECOD) activity, initially applicable on pooled samples of Daphnia magna, was optimized for use on individual organisms. Optimal assay conditions have been established for as small as 3- and 6 days old individuals, and the limits of spectrofluorometric detection of HCm excreted by daphnids in the incubation media were defined. The modified assay was tested by screening the modulation of ECOD activity in daphnids following 24 h exposure to β-naphthoflavone (β-NF, reference CYP450 inducer) and to prochloraz (PCZ), a potent CYP450 inhibitor. Maximal ECOD activity levels in daphnids were recorded following 2 hours of incubation to 200 nM EtC. The limit of spectrofluorometric detection of HCm in the incubation media was 6.25 nM, achieved by more than 80% of three days old daphnids and all six days old individuals. Exposure of daphnids to β-NF demonstrated a bell-shaped ECOD activity induction potential, while PCZ elicited partial (60%) inhibition of ECOD activity. This optimized in vivo ECOD activity assay may serve as a cost-effective tool to study the responsiveness of Phase-I metabolism in D. magna to toxic pressure and its applicability to other aquatic invertebrates is also worth for consideration.

Liver Antioxidant Capacity and Steatosis in Laying Hens Exposed to Various Quantities of Lupin (Lupinus angustifolius) Seeds in the Diet.

Despite the many beneficial properties of legume plants, their use in diets for poultry is limited by the presence of antinutritional factors. The aim of the study was to determine the activity of DT-diaphorase, ethoxycoumarin O-deethylase, and catalase, and the concentration of malondialdehyde in liver tissue, as well as the activity of SOD and CAT in the serum of Hy-line Brown hens fed a diet supplemented with various doses of Lupinus angustifolius seeds. The results indicate that the use of large amounts of lupin in the diet resulted in an increase in MDA concentration in the liver and the lipid vacuolization of hepatocytes. A significant increase in DTD activity was observed in chickens receiving 15% lupin. Regardless of lupin dose, no increase in SOD activity was observed in chicken serum after 33 days of the experiment. From the 66th day of the experiment, an increase in catalase activity in the serum of laying hens was observed, while low activity of this enzyme was found in the liver. It can be concluded that the short-term use of lupin in the diet of laying hens does not affect the activity of antioxidant enzymes and, therefore, does not affect the oxidative-antioxidant balance of their body.

Effects of chronic cirrhosis induced by intraperitoneal thioacetamide injection on the protein content and Michaelis-Menten kinetics of cytochrome P450 enzymes in the rat liver microsomes.

Chronic intraperitoneal injection of thioacetamide (TAA) in rats has been used as an animal model of human cirrhosis to study the effects of the disease on drug metabolism. However, TAA inhibits P450 enzymes directly and independently of cirrhosis. We investigated the effects of chronic cirrhosis in rats, induced by 10 weeks of intraperitoneal TAA, on the P450 enzymes after a 10-day washout period to eliminate TAA. Liver histology and serum biomarkers of hepatic function confirmed cirrhosis in all animals. Microsomal total P450 content, P450 reductase activity and ethoxycoumarin O-deethylase activity, a general marker of P450 activity, were significantly reduced by 30%-50% in cirrhotic animals. Additionally, the protein content and Michaelis-Menten kinetics of the activities of CYP2D, CYP2E1 and CYP3A were investigated. Whereas cirrhosis reduced the microsomal protein contents of CYP2D and CYP3A by 70% and 30%, respectively, the protein contents of CYP2E1 were not affected. However, the activities of all the tested isoenzymes were substantially lower in the cirrhotic livers. It is concluded that the TAA model of cirrhosis that incorporates a 10-day washout period after intraperitoneal injection of the chemical to rats produces isoenzyme-selective reductions in the P450 proteins or activities, which are independent of the direct inhibitory effects of TAA.

Sepsis-induced liver dysfunction was ameliorated by propofol via suppressing hepatic lipid peroxidation, inflammation, and drug interactions

AimsOur previous study showed that propofol can protect against sepsis-induced insults through suppressing liver nitrosation and inflammation. This study further evaluated the mechanisms of propofol-caused protection from sepsis-induced liver dysfunction. Main methodsMale Wistar rats were subjected to cecal ligation and puncture (CLP) and then exposed to propofol. Levels of hepatic oxidative stress and lipid peroxidation were consecutively measured. Expressions of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-4 messenger (m)RNA or proteins were quantified. Effects of propofol on microsomal pentoxyresorufin O-dealkelase (PROD) and ethoxycoumarin O-deethylase (ECOD) activities were determined. Key findingsAdministration of propofol to CLP-treated rats significantly attenuated sepsis-induced insults. CLP caused augmented serum aspartate aminotransferase and alanine aminotransferase activities and concurrently triggered liver damage. In contrast, treatment with propofol protected against CLP-induced liver dysfunction. As to the mechanisms, the CLP-induced increases in oxidative stress and lipid peroxidation levels and TNF-α and IL-1β mRNA and protein expressions were subsequently attenuated by propofol. Furthermore, administration of CLP-treated rats with propofol augmented levels of IL-4 in the liver. Phenobarbital treatment of liver microsomes in CLP-treated rats produced less amplification of PROD and ECOD activities, and a smaller amount of 4-hydroxypropofol was metabolized from propofol by liver microsomes. In contrast, more drug interactions occurred with propofol, which decreased PROD and ECOD activities in liver microsomes of CLP-treated rats. SignificanceTaken together, the present study showed that propofol can protect against sepsis-induced liver dysfunction through suppressing hepatic oxidative stress, lipid peroxidation, inflammation, and drug biotransformation and interactions in the liver.

Tramadol-induced hepato- and nephrotoxicity in rats: Role of Curcumin and Gallic acid as antioxidants.

Tramadol is an analgesic used to treat moderate to severe pain caused by cancer, osteoarthritis, and other musculoskeletal diseases. Cytochrome P450 system metabolizes tramadol and induces oxidative stress in different organs. Therefore, the present study aims at investigating the changes in the activities and the protein expressions of CYPs isozymes (2E1, 3A4, 2B1/2), antioxidants status, free radicals levels after pretreatment of rats with Curcumin and/or Gallic as single- and/or repeated-doses before administration of tramadol. In repeated-dose treatments of rats with tramadol, the activities of cytochrome P450, cytochrome b5, and NADPH-cytochrome-c-reductase, and the antioxidant enzymes including glutathione reductase, glutathione peroxidase, glutathione S-transferase, catalase, superoxide dismutase, and levels of glutathione were inhibited in the liver and the kidney of rats. Interestingly, such changes caused by tramadol restored to their normal levels after pretreatment of rats with either Curcumin and/or Gallic acid. On the other hand, repeated-dose treatment of rats with tramadol increased the activities of both dimethylnitrosamine N-demethylase I (DMN-dI), and aryl hydrocarbon hydroxylase (AHH) compared to the control group. However, pretreatment of rats with Curcumin and/or Gallic acid prior to administration of tramadol restored the inhibited DMN-dI activity and its protein expression (CYP 2E1) to their normal levels. On the other hand, tramadol inhibited the activity of ethoxycoumarin O-deethylase (ECOD) and suppressed its protein marker expression (CYP2B1/2), whereas Curcumin, Gallic acid and/or their mixture restored such changes to their normal levels. In conclusion, Curcumin and/or Gallic acid alleviated the adverse effects caused by tramadol. In addition, patients should be advice to take Curcumin and/or Gallic acid prior to tramadol treatment to alleviate the hepatic and renal toxicities caused by tramadol.

Effects of tebuconazole on cytochrome P450 enzymes, oxidative stress, and endocrine disruption in male rats.

The major objective of the present study was to determine the ability of a triazole fungicide tebuconazole to induce cytochrome P450-dependent monooxygenases, oxidative stress, and endocrine-disrupting activity using male rats treated with tebuconazole at 10, 25, and 50 mg/kg p.o. once daily for 28 days. In liver, tebuconazole dose-dependently increased microsomal contents of cytochrome P450 and cytochrome b5 and the activities of NADPH-cytochrome P450 reductase, 7-ethoxyresorufin O-deethylase, methoxyresorufin O-demethylase, pentoxyresorufin O-dealkylase, 7-ethoxycoumarin O-deethylase, aniline hydroxylase, and erythromycin N-demethylase. In kidney, tebuconazole increased 7-ethoxycoumarin O-deethylase activity without affecting other monooxygenase activities. In marked contrast to liver and kidney, tebuconazole decreased testicular 7-ethoxyresorufin O-deethylase, methoxyresorufin O-demethylase, 7-ethoxycoumarin O-deethylase, aniline hydroxylase, and erythromycin N-demethylase activities. The results of immunoblot analysis of liver microsomes of controls and tebuconazole-treated rats revealed that tebuconazole induced CYP1A1/2, CYP2B1/2, CYP2E1, and CYP3A proteins in liver. Additions of tebuconazole to liver microsomes inhibited microsomal 7-ethoxycoumarin O-deethylase activity in vitro (IC50 = 1.50-1.69 µM). Treatment of rats with tebuconazole decreased glutathione content and increased glutathione S-transferase, superoxide dismutase, catalase, and glutathione peroxidase activities in liver; increased superoxide dismutase activities in kidney and testis; but decreased glutathione S-transferase activity in testis. Treatments with tebuconazole decreased serum testosterone concentration and cauda epididymal sperm count. The present study demonstrates that tebuconazole induces a multiplicity of CYPs and oxidative stress in liver; inhibits testicular P450 and glutathione S-transferase activities; and produces anti-androgenic effects in male rats.

Monitoring and biochemical characterization of beta-cypermethrin resistance in Spodoptera exigua (Lepidoptera: Noctuidae) in Sichuan Province, China

The beet armyworm Spodoptera exigua, a major pest affecting numerous cultivated crops in China, has developed a serious resistance to many traditional chemical insecticides. The resistance levels of the field-collected populations from different districts in Sichuan Province, China, to nine insecticides were detected with a diet-incorporation bioassay. Compared to the Lab-ZN strain, five (in 2014) and three (in 2016) field populations displayed either high or extremely high levels of resistance to beta-cypermethrin. All the field populations collected in 2014 were susceptible to emamectin benzoate, hexaflumuron, methoxyfenozide, chlorantraniliprole, cyantraniliprole and indoxacarb but exhibited low or moderate levels of resistance to abamectin. The resistances of field populations collected in 2016 were significantly higher than two years earlier, especial for chlorantraniliprole and cyantraniliprole with RRs rising from 173.4- to 582.6-fold and 175.3- to 287.6-fold, respectively, even though the field populations had retained moderate or low levels of resistance to chlorpyrifos and hexaflumuron. The synergism experiment revealed that the resistance of the LS16 population to beta-cypermethrin may be mainly related to cytochrome P450 monooxygenases (P450s), which was responsible for the highest increase ratio of 37.97-fold, for piperonyl butoxide, rather than either carboxylesterase (CarE) or glutathione S-transferase (GST). The cytochrome P450 ethoxycoumarin O-deethylase activity of the LS16 population was also the strongest among the treatments (P < 0.05). Non-denaturing polyacrylamide gel electrophoresis (native PAGE) indicated that enhanced E11, E13 and E15-E16 bands in the LS16 population likely contribute to the development of resistance to beta-cypermethrin.

Insecticide resistance and enhanced cytochrome P450 monooxygenase activity in field populations of Spodoptera litura from Sichuan, China

Spodoptera litura (Fabricius) is a widely distributed worldwide insect pest, particularly of vegetable crops. To determine the sensitivity of S. litura to insecticides, the toxicities of beta-cypermethrin, chlorpyrifos, emamectin benzoate, methoxyfenozide, and indoxacarb were tested from 2014 to 2016 using a diet incorporation assay on populations of S. litura collected from different districts in Sichuan Province and a susceptible strain (Lab-HN). Compared to the Lab-HN strain, field populations showed high levels of resistance to beta-cypermethrin (267.3- to 1789.2-fold). Field populations exhibited low or moderate resistance to chlorpyrifos in 2014 (8.9- to 24.4-fold) and high levels in 2016 (440.5- to 950.5-fold). All populations collected in 2014 and 2016 were susceptible or had a low or moderate level of resistance to indoxacarb (2.2- to 31.0-fold). No field populations exhibited resistance to emamectin benzoate or methoxyfenozide (0.7- to 1.5-fold and 0.9- to 1.4- fold) in 2014, whereas the resistance in 2016 increased to 29.8- to 55.2-fold and 38.1- to 59.0- fold, respectively. A synergist experiment indicated that the Lab-HN strain and field-collected population from Shuangliu in 2016 (SL16 population) to beta-cypermethrin may be related to cytochrome P450 monooxygenases (P450s), with the highest synergism ratios (SR) of piperonyl butoxide (PBO) of 34.4- and 73.3-fold, but not to carboxylesterase (CarE) or glutathione-S- transferase (GST). We detected the activities of detoxification enzymes and found that the ethoxycoumarin O-deethylase activities were also the strongest in the Lab-HN strain and the SL16 population with the highest SR values of 2.6- and 4.1-fold, respectively. Non-denaturing polyacrylamide gel electrophoresis (native PAGE) indicated that the inhibition of in vivo P450 activity caused by PBO was due to the binding of the synergist to the E5 or E6 isozyme, which were absent in the Lab-HN strain.

Acute toxicity and sublethal effects of myclobutanil on respiration, flight and detoxification enzymes in Apis cerana cerana

Myclobutanil is currently used on the flowering plants. Little is known about how Apis cerana cerana respond to myclobutanil exposure. Hence, the acute toxicity of myclobutanil and its sublethal effects on respiration, flight and detoxification enzymes [7-ethoxycoumarin O-deethylase (ECOD) and glutathione S-transferases (GSTs)] in A. cerana cerana were investigated. The results indicated that formulation grade myclobutanil showed moderate toxicity to A. cerana cerana either contact (LD50=4.697μg/bee) or oral (LD50=2.154μg/bee) exposure. Sublethal dose of myclobutanil significantly reduced the respiration rate of workers at 24h and 48h regardless of the exposure method. However, myclobutanil didn't significantly affect the take-off flight. After nurse bees exposure to the dose (LD5) of formulation-grade myclobutanil, ECOD activity was significantly induced when compared with control, but GST activity didn't change. In the forager bees, no enzyme markers response was obtained in this test. From the present study we can infer that myclobutanil disturb respiration and P450-mediated detoxification of the individual bees of A. cerana cerana. Thus, myclobutanil may has risk for A. cerana cerana, it should be cautiously used.

In vitro interactions of malachite green and leucomalachite green with hepatic drug-metabolizing enzyme systems in the rainbow trout (Onchorhyncus mykiss)

Malachite green (MG) has been widely used in aquaculture to treat a number of microbial and parasitic diseases. It is currently banned in the EU because of the high cytotoxicity and carcinogenic activity, which is also shared by leucomalachite green (LMG), a reduced MG metabolite that can persist in fish tissues for months. There is scant information about the ability of either compound to interact with drug metabolizing enzymes in fish. Therefore we evaluated the in vitro effects of MG and LMG (25, 50 and 100μM) on some DMEs and glutathione (GSH) content in rainbow trout liver subfractions. LMG did not affect any of the examined parameters. In contrast, MG proved to deplete GSH and to depress to a various extent the activities of NAD(P)H cytochrome c reductase, 7-ethoxycoumarin O-deethylase, 1-naphthol uridindiphosphoglucuronyl-transferase and maximally those of 7-ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) accepting 1-chloro2,4-dinitrobenzene (CDNB) as substrate. The inhibition mechanisms of EROD and GST were investigated by means of non-linear Michaelis-Menten kinetics and Lineweaver-Burk plots using 0.175–8μM MG. The calculated IC50 for EROD was 7.1μM, and the inhibition appeared to be competitive (Ki 2.78±0.24μM). In the case of GST, the calculated IC50 was 0.53μM. The inhibition was best described as competitive toward GSH (Ki 0.39±0.02μM) and of mixed-type toward CDNB (Ki 0.64±0.06μM). Our findings indicate that, contrary to LMG, MG behaves as a relatively strong inhibitor of certain liver DMEs and can reversibly bind GSH.

Development of in vivo biotransformation enzyme assays for ecotoxicity screening: In vivo measurement of phases I and II enzyme activities in freshwater planarians

The development of a high-throughput tool is required for screening of environmental pollutants and assessing their impacts on aquatic animals. Freshwater planarians can be used in rapid and sensitive toxicity bioassays. Planarians are known for their remarkable regeneration ability but much less known for their metabolic and xenobiotic biotransformation abilities. In this study, the activities of different phase I and II enzymes were determined in vivo by directly measuring fluorescent enzyme substrate disappearance or fluorescent enzyme metabolite production in planarian culture media. For phase I enzyme activity, O-deethylation activities with alkoxyresorufin could not be detected in planarian culture media. By contrast, O-deethylation activities with alkoxycoumarin were detected in planarian culture media. Increases in 7-ethoxycoumarin O-deethylase (ECOD) activities was only observed in planarians exposed to 1μM, but not 10μM, β-naphthoflavone for 24h. ECOD activity was inhibited in planarians exposed to 10 and 100μM rifampicin or carbamazepine for 24h. For phase II enzyme activity, DT-diaphorase, arylsulfatases, uridine 5′-diphospho (UDP)-glucuronosyltransferase or catechol-O-methyltransferase activity was determined in culture media containing planarians. The results of this study indicate that freshwater planarians are a promising model organism to monitor exposure to environmental pollutants or assess their impacts through the in vivo measurement of phase I and II enzyme activities.

A point mutation (L1015F) of the voltage-sensitive sodium channel gene associated with lambda-cyhalothrin resistance in Apolygus lucorum (Meyer–Dür) population from the transgenic Bt cotton field of China

In China, the green mirid bug, Apolygus lucorum (Meyer–Dür), has caused severe economic damage to many kinds of crops, especially the cotton and jujubes. Pyrethroid insecticides have been widely used for controlling this pest in the transgenic Bt cotton field. Five populations of A. lucorum collected from cotton crops at different locations in China were evaluated for lambda-cyhalothrin resistance. The results showed that only the population collected from Shandong Province exhibited 30-fold of resistance to lambda-cyhalothrin. Neither PBO nor DEF had obvious synergism when compared the synergistic ratio between SS and RR strain which was originated from the Shandong population. Besides, there were no statistically significant differences (p>0.05) in the carboxylesterase, glutathione S-transferase, or 7-ethoxycoumarin O-deethylase activities between the Shandong population and the laboratory susceptible strain (SS). The full-length sodium channel gene named AlVSSC encoding 2028 amino acids was obtained by RT-PCR and rapid amplification of cDNA ends (RACE). One single point mutation L1015F in the AlVSSC was detected only in the Shandong population. Our results revealed that the L1015F mutation associated with pyrethroid resistance was identified in A. lucorum populations in China. These results will be useful for the rational chemical control of A. lucorum in the transgenic Bt cotton field.

Effect of Dimethyl Sulfoxide and Melatonin on the Isolation of Human Primary Hepatocytes

The availability of fully functional human hepatocytes is critical for progress in human hepatocyte transplantation and the development of bioartificial livers and in vitro liver systems. However, the cell isolation process impairs the hepatocyte status and determines the number of viable cells that can be obtained. This study aimed to evaluate the effects of using dimethyl sulfoxide (DMSO) and melatonin in the human hepatocyte isolation protocol. Human hepatocytes were isolated from liver pieces resected from 10 patients undergoing partial hepatectomy. Each piece was dissected into 2 equally sized pieces and randomized, in 5 of 10 isolations, to perfusion with 1% DMSO-containing perfusion buffer or buffer also containing 5 m<smlcap>M</smlcap> melatonin using the 2-step collagenase perfusion technique (experiment 1), and in the other 5 isolations to standard perfusion or perfusion including 1% DMSO (experiment 2). Tissues perfused with DMSO yielded 70.6% more viable hepatocytes per gram of tissue (p = 0.076), with a 26.1% greater albumin production (p < 0.05) than those perfused with control buffer. Melatonin did not significantly affect (p > 0.05) any of the studied parameters, but cell viability, dehydrogenase activity, albumin production, urea secretion, and 7-ethoxycoumarin O-deethylase activity were slightly higher in cells isolated with melatonin-containing perfusion buffer compared to those isolated with DMSO. In conclusion, addition of 1% DMSO to the hepatocyte isolation protocol could improve the availability and functionality of hepatocytes for transplantation, but further studies are needed to clarify the mechanisms involved.

Acute toxicity and sublethal effects of fipronil on detoxification enzymes in juvenile zebrafish (Danio rerio)

The acute toxicity of fipronil and its sublethal effects on detoxification enzymes (carboxylesterases (CarEs), glutathione S-transferases (GSTs), and 7-ethoxycoumarin O-deethylase (ECOD)) in zebrafish (Danio rerio) were investigated. The results indicated that the 24-h LC50 of fipronil for zebrafish was 220.4μg/L (95% CI: 173.7–272.4μg/L). Sublethal concentrations of fipronil did not cause significant changes in CarEs activities. In the liver and muscle tissues, GST activities at the tested concentrations did not significantly differ from those in the control. In the brain and gill tissues, GST activities at a concentration of 4μg/L were significantly lower than those at a concentration of 2μg/L. The results suggest that CarEs and GSTs were not suitable biomarkers for fipronil effects in D. rerio. A significant induction in the ECOD activities in the brain, gill, liver, and muscle tissues was observed compared with the control. Moreover, the dose-dependent responses of the ECOD activity were observed after treatment with sublethal concentrations of fipronil in the range of 2–20μg/L. The results suggested that ECOD could be a suitable biomarker of fipronil effects in D. rerio.

Seasonal patterns of polycyclic aromatic hydrocarbons in digestive gland and arm of octopus (Octopus vulgaris) from the Northwest Atlantic

Among organic pollutants existing in coastal areas, polycyclic aromatic hydrocarbons (PAHs) are of great concern due to their ubiquity and carcinogenic potential. The aim of this study was to evaluate the seasonal patterns of PAHs in the digestive gland and arm of the common octopus (Octopus vulgaris) from the Northwest Atlantic Portuguese coast. In the different seasons, 18 PAHs were determined and the detoxification capacity of the species was evaluated. Ethoxyresorufin O-deethylase (EROD) and ethoxycoumarin O-deethylase (ECOD) activities were measured to assess phase I biotransformation capacity. Individual PAH ratios were used for major source (pyrolytic/petrogenic) analysis. Risks for human consumption were determined by the total toxicity equivalence approach. Generally, low levels of PAHs were detected in the digestive gland and in the arm of octopus, with a predominance of low molecular over high molecular weight compounds. PAHs exhibited seasonality in the concentrations detected and in their main emission sources. In the digestive gland, the highest total PAH levels were observed in autumn possibly related to fat availability in the ecosystem and food intake. The lack of PAH elimination observed in the digestive gland after captivity could be possibly associated to a low biotransformation capacity, consistent with the negligible/undetected levels of EROD and ECOD activity in the different seasons. The emission sources of PAHs found in the digestive gland varied from a petrogenic profile observed in winter to a pyrolytic pattern in spring. In the arm, the highest PAH contents were observed in June; nevertheless, levels were always below the regulatory limits established for food consumption. The carcinogenic potential calculated for all the sampling periods in the arm were markedly lower than the ones found in various aquatic species from different marine environments. The results presented in this study give relevant baseline data for environmental monitoring of organic pollution in coastal areas.

Effects of flumequine on cytochrome P450 enzymes in allogynogenetic silver crucian carp, Carassius auratus gibelio

The effects of flumequine on activities of several microsomal P450 monooxygenases of allogynogenetic silver crucian carp (Carassius auratus gibelio) were determined and the induction of fluemquine on CYP1A was further explored at the level of protein expression and mRNA transcription.After 24 h following a single intraperitoneal injection at a dose of 35 mg/kg,the ethoxyresorufin O-deethylase (EROD) activity in the liver of the Crucian carp was 54.33 pmol/(mg·min),which was significantly higher than that of the control carps [34.00 pmol/(mg·min)](P0.01).It was revealed that flumequine could significantly induce EROD activity(P0.01).However,the other P450 monooxygenases,such as erythromycin N-demethylase (ERND) [177.98 pmol/(mg·min)],aminopyrine N-demethylase [934.40 pmol/(mg·min)] and ethoxycoumarin O-deethylase (ECOD) [9.84pmol/(mg·min)]were not significantly affected by flumequine,comparing with these activities in control [140.90 pmol/(mg·min),850.71 pmol/(mg·min) and 8.93 pmol/(mg·min)],respectively.Except that the ERND activity in kidney tissue was higher than that in liver,the activities of APD,EROD and ECOD were the highest in liver tissue.An immunoblot analysis with rabbit anti-rat P4501A polyclonal antibody showed that CYP 1A protein expression in flumequine-treated carps was markedly higher than that in control carps,which was consistent with the trend in EROD activities.These findings suggest that P450 1A isoform,constitutively expressed in crucian carp,was particularly susceptible to activation by flumequine.Semiquantitative RT-PCR indicated that CYP1A mRNA was not affected by flumequine.In vitro assay,microsomes incubating with various concentrations of flumequine demonstrated that this fluoroquinolone antibiotic had no induction or inhibition of CYP1A.On the basis of the above,liver CYP1A induction of flumequine on carp occurs at the post-translation level,which may enhance protein stability.

Effects of norfloxacin on the drug metabolism enzymes of two sturgeon species (Acipenser schrenckiandAcipenser ruthenus)

Summary The enzyme activities of 7-ethoxycoumarin O-deethylase (ECOD) and glutathione S-transferase (GST), and on glutathione (GSH) content, involved in metabolism of the antibiotic Norfloxacin (NFLX), were investigated in Acipenser schrencki and Acipenser ruthenus. Sturgeons weighing 45–55 g were kept in an aquarium (0.5 m × 0.5 m × 0.9 m) for two weeks under controlled conditions (fish density 88 individuals per m3, 18°C) before the experiment. The two species of sturgeon were divided into five groups each (n = 15 in each group), with each group subdivided into three replicates of five fish per tank. A control group in which distilled water was administered orally was also tested. NFLX was forced into the stomachs of the fish at a concentration of 20, 40, 60, 80 and 100 mg kg−1 body weight, respectively. ECOD activities in liver microsomes, and GST activity and GSH content in liver microsomes and blood plasma, were measured and compared. Results indicate that ECOD activity is progressively inhibited with increasing NFLX concentrations. ECOD activity varied from 0.12 nmol mg−1 min−1 to 0.07 nmol mg−1 min−1, demonstrating an inhibition rate of 60.83% in A. schrencki and 65.14% in A. ruthenus. In both species tested, GST and GSH levels exhibited a trend of first increasing, and then decreasing with increasing NFLX levels, reaching a peak value at 40 mg per kg−1 body weight. Thus, the presented results indicate that NFLX can induce a change in the activity of some drug metabolism related enzymes such as ECOD and GST in vivo.

Response of Cytochrome P450 Expression to Maize Volatiles in Helicoverpa armigera (Hübner)

The 7-ethoxycoumarin O-deethylase (ECOD) activities of cytochrome P450s and differential expression of six cytochrome P450 genes induced by the volatiles from both damaged and undamaged maize plants were investigated in the cotton bollworm, Helicoverpa armigera (Hübner). The ECOD activity changed with time of exposure to maize volatiles. At 36 h after cotton bollworm larvae exposure to maize volatiles, the ECOD activities in cotton bollworm damaged and artificially damaged groups were 2.36 and 4.53 times higher than the control group respectively. The relative expression levels of CYP4S1, CYP6B2 and CYP6B7 in the cotton bollworm were significantly increased in artificially damaged plant group, which was 2.93, 5.09 and 10.66 times higher than that in the control group, respectively. The expression levels of CYP6B2, CYP6B6, CYP9A12, and CYP9A14 were much lower in the larvae exposure to volatiles from both healthy and pest damaged maize seedlings than in the control group at 12 h after larvae exposure to maize volatiles. For the cotton bollworm damaged maize group, the expression of CYP4S1 and CYP9A14 increased.

Metabolism of 7‐ethoxycoumarin, safrole, flavanone and hydroxyflavanone by cytochrome P450 2A6 variants

CYP 2A6 is a human enzyme that metabolizes many xenobiotics including coumarin, indole, nicotine and carcinogenic nitrosamines. The gene for CYP2A6 is polymorphic. There are few data available to clarify the relationship between P450 genetic variants and the metabolism of materials in food. The CYP 2A6 wild-type protein and 13 mutants (CYP2A6.1, CYP2A6.2, CYP2A6.5, CYP2A6.6, CYP2A6.7, CYP2A6.8, CYP2A6.11, CYP2A6.15, CYP2A6.16, CYP2A6.17, CYP2A6.18, CYP2A6.21, CYP2A6.23 and CYP2A6.25) were co-expressed with NADPH-cytochrome P450 reductase in E. coli. The hydroxylase activities toward 7-ethoxycoumarin, coumarin, safrole, flavanone and hydroxyflavanone were examined. Ten types of CYP2A6 variants except for CYP2A6.2, CYP2A6.5 and CYP2A6.6 showed Soret peaks (450 nm) typical of P450 in the reduced CO-difference spectra and had 7-ethoxycoumarin O-deethylase activities. CYP2A6.15 and CYP2A6.18 showed higher activities for safrole 1'-hydroxylation than CYP2A6.1. CYP2A6.25 and CYP2A6.7 had lower safrole 1'-hydroxylase activities. CYP2A6.7 had lower flavanone 6- and 2'-hydroxylase activities, whereas CYP2A6.25 had higher 6-hydroxylase activity and lower 2'-hydroxylase activity. Hydroxyflavanone was metabolized by CYP2A6.25, but was not metabolized by wild-type CYP2A6.1. These results indicate that CYP2A6.25 possessed new substrate specificity toward flavonoids.

Field studies using fish biomarkers – How many fish are enough?

Significant efforts are invested in field studies using fish, and it is important to optimize the number of organisms collected to evaluate the possible impacts of contamination. This paper provides ecotoxicologists with the approximate numbers of fish needed to identify statistically significant differences among samples using physiological indices and biochemical markers of fish health. The numbers of fish to collect are reported for ethoxyresorufin-o-deethylase (EROD); ethoxycoumarin-o-deethylase (ECOD), serum sorbitol dehydrogenase (sSDH), stress proteins, gonadosomatic index, liver somatic index, condition factor, and biliary metabolites of polycyclic aromatic hydrocarbons. The number of fish to collect was as few as four for ECOD activity (with a power of 80%), but as high as 106 for CF (with a power of 95%). Achieving statistical significance between sites does not help in the interpretation of the biological significance of a parameter, but well-planned field samplings will maximize the chances of correctly identifying areas of concern.