Erythromycin N-demethylase Activity Research Articles

Metabolism of β-naphthoflavone by cytochrome P450 isoforms in three air breathing fish, Channa punctatus, Heteropneustes fossilis and Clarias batrachus

The crucial phase I xenobiotic metabolising enzyme cytochrome P450 (CYP 450) has a variety of isoforms and a wide range of substrate specificities. While the roles of mammalian CYP 450 are well established, those of non-mammalian species are much less defined. These roles are frequently extrapolated from mammalian data and assumed to be similar across vertebrate species. In the present study, three air breathing fish species, Channa punctatus, Heteropneustes fossilis and Clarias batrachus were injected intraperitoneally (IP) with 50 mg/kg β-naphthoflavone (β-NF) and its effects on hepatic CYP 450 system were evaluated. β-NF treatment resulted in significant increase (p<0.01) in CYP 450 content and CYP1A mediated EROD (Ethoxyresorufin O-deethylase) activity in all three treated fish groups compared to control. CYP2B mediated N,N-dimethylaniline demethylase activity and CYP2E1 mediated aniline hydroxylase activity showed no response suggesting insensitivity to β-NF treatment. All three fish species showed an increase in CYP3A4 mediated erythromycin N-demethylase activity, but only H. fossilis displayed a significant difference (p<0.05). Of all the activities investigated, EROD activity mediated by CYP1A responded towards β-NF treatment the best and could serve as a sensitive biomarker to gauge the effects of chemical pollutants in the aquatic environment.

The effect of co-administration of berberine, resveratrol, and glibenclamide on xenobiotic metabolizing enzyme activities in diabetic rat liver

It is possible to use plant-derived antioxidant molecules in the form of dietary supplements. However, dietary supplement–drug interaction pattern has not been well defined for most of these products. The aim of this study was to determine the effects of berberine, resveratrol, and glibenclamide on xenobiotic metabolizing enzyme activities in diabetic rats. Streptozotocin was administered to create experimental diabetes. Resveratrol (5 mg/kg) (R), glibenclamide (5 mg/kg) (G), and berberine (10 mg/kg) (B) were administered individually or in combinations in DMSO by intraperitoneal administration route to the diabetic rats. DMSO was also given to non-diabetic control (C) and diabetic control (D) groups. Livers of rats were taken under anesthesia at the end of the treatment period (12 days). Ethoxyresorufin O-deethylase (EROD), pentoxyresorufin O-depentylase (PROD), aniline 4-hydroxylase (A4H), erythromycin N-demethylase (ERND), glutathione S-transferase (GST), catalase (CAT), and glutathione reductase (GR) activities were measured in microsomes and cytosols. In addition, histomorphological studies were also performed in the liver tissues. EROD activity of D+R was significantly higher than C and D+R+B. PROD activity of D+R was significantly higher than C, D, D+R+G, D+R+B, and D+R+B+ G. PROD activity of D+B was significantly higher than C and D+R+B. ERND activity of D+R was significantly higher than D+R+G and D+R+B. GST activity of D+R was significantly higher than D+R+G. CAT activity of D+B was significantly lower than C. It is clear that co-administration of resveratrol, berberine, and glibenclamide modifies some of the important xenobiotic metabolizing enzyme activities. Resveratrol and berberine have the potential to cause dietary supplement–drug interaction.

In vitro interactions of abiraterone, erythromycin, and CYP3A4: implications for drug-drug interactions.

Potential drug-drug interactions of the antitumor drug abiraterone and the macrolide antibiotic erythromycin were studied at the stage of cytochrome P450 3A4 (CYP3A4) biotransformation. Using differential spectroscopy, we have shown that abiraterone is a type II ligand of CYP3A4. The dependence of CYP3A4 spectral changes on the concentration of abiraterone is sigmoidal, which indicates cooperative interactions of CYP3A4 with abiraterone; these interactions were confirmed by molecular docking. The dissociation constant (Kd ) and Hill coefficient (h) values for the CYP3A4-abiraterone complex were calculated as 3.8±0.1μM and 2.3±0.2, respectively. An electrochemical enzymatic system based on CYP3A4 immobilized on a screen-printed electrode was used to show that abiraterone acts as a competitive inhibitor toward erythromycin N-demethylase activity of CYP3A4 (apparent Ki =8.1±1.2μM), while erythromycin and its products of enzymatic metabolism do not affect abiraterone N-oxidation by CYP3A4. In conclusion, the inhibition properties of abiraterone towardCYP3A4-dependent N-demethylation oferythromycin and the biologically inert behavior of erythromycin toward abiraterone hydroxylation were demonstrated.

Two novel CYP3A isoforms in marine mussel Mytilus coruscus: Identification and response to cadmium and benzo[a]pyrene

CYP3A enzymes play a crucial role in metabolic clearance of a variety of xenobiotics. However, their genetic information and function remain unclear in molluscs. In the present study, two novel CYP3A genes i.e. McCYP3A-1 and McCYP3A-2 were identified and characterized from the thick shell mussel Mytilus coruscus, and their tissue distribution as well as the response to cadmium (Cd) and benzo[a]pyrene (B[α]P) exposure were addressed using real time quantitative RT-PCR (qRT-PCR) and erythromycin N-demethylase (ERND) assay. McCYP3A-1 and McCYP3A-2 possess typically domains of CYP family such as helix-C, helix-I, helix-K, PERF and the heme binding domain as well as the characteristic domains of CYP3s including six SRS motifs. McCYP3A-1 and McCYP3A-2 transcripts were constitutively expressed in all examined tissues with high expression level in digestive glands, hepatopancreas and gonads. Upon B[α]P exposure, McCYP3A-1 and McCYP3A-2 mRNA expression in digestive glands showed a pattern of up-regulation followed by down-regulation, while under Cd exposure, showed a time-dependent induction profile. In addition, ERND activity, generally used as an indicator of CYP3, increased in a time-dependent manner after exposure to Cd and B[α]P. These results collectively indicated that McCYP3A-1 and McCYP3A-2 are CYP3A family member and may play a potential role in metabolic clearance of xenobiotics. Meanwhile, the current results may provide some baseline data to support McCYP3A-1 and McCYP3A-2 as candidate biomarkers for monitoring of PAHs and heavy metal pollution.

Comparison of the florfenicol metabolic rate in Portunus trituberculatus individuals with different body weights

We analysed the metabolic rate of large individuals (LIs) and small individuals (SIs) (based on body weight) of 80-day-old specimens of Portunus trituberculatus injected with 40 mg/kg florfenicol (FLR). The results showed that FLR contents in the gills and hepatopancreas were higher in the SIs than in the LIs. The activities of erythromycin N-demethylase and 7-ethoxyresorufin-O-deethylase related to cytochrome oxidase P450 (CYP) were induced after FLR injection and showed a size-dependent pattern; however, no significant difference in aminopyrine N-demethylase activity was found between the LIs and SIs. The activity of the phase II enzyme glutathione S-transferase (GST) increased in the hepatopancreas after FLR injection, and it was higher in the SIs than the LIs. Moreover, induction of CYP2, CYP3, GST, ABCB and ABCG was observed in the first 24 hr after FLR injection. FLR caused more biomolecular damage in the SIs than the LIs, displaying an obvious body weight- and time-dependent pattern. In summary, the SIs exhibited faster FLR uptake and slower depuration in this species, and FLR caused more biomolecular damage to the SIs.

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.

Biotransformation and tissue distribution of protopine and allocryptopine and effects of Plume Poppy Total Alkaloid on liver drug-metabolizing enzymes

In this study, the biotransformation in the plasma, urine and feces of rats following oral administration of protopine (PRO) and allocryptopine (ALL)were explored using HPLC-QqTOF MS. An HPLC-MS/MS method for the determination of tissues was developed and applied to the tissue distribution study in rats following intragastric administration of Plume Poppy Total Alkaloid for 3 weeks. A total of ten PRO metabolites and ten ALL metabolites were characterized in rats in vivo. Among these metabolites, six PRO metabolites and five ALL metabolites were reported for the first time. The predicated metabolic pathways including ring cleavage, demethylation following ring cleavage, and glucuronidation were proposed. The low-concentration residue of PRO and ALL in various tissues was detected at 24 h and 48 h after dosing, which indicated that both compounds could be widely distributed in tissues and exist as low levels of residue. The activities of erythromycin N-demethylase, aminopyrine N-demethylase and NAD (P)H quinone oxidoreductase in female rats can be induced post-dose, but these activities were inhibited in male rats. The proposed biotransformation and residues of PRO and ALL and their effects on enzymes may provide a basis for clarifying the metabolism and interpreting pharmacokinetics.

Functional characterization of a common CYP4F11 genetic variant and identification of functionally defective CYP4F11 variants in erythromycin metabolism and 20-HETE synthesis

CYP4F11, together with CYP4F2, plays an important role in the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) from arachidonic acid. We identified 21 variants by whole exome sequencing, including 4 non-synonymous variants in Korean subjects. The proteins of the wild-type CYP4F11 and the four coding variants (C276R, D315N, D374Y, and D446N) were expressed in Escherichia coli DH5α cells and purified to give cytochrome P450-specific carbon monoxide difference spectra. Wild-type CYP4F2 was also expressed and purified to compare its activity with the CYP4F11 wild-type. Wild-type CYP4F11 exhibited the highest maximal clearance for erythromycin N-demethylase activity followed by the variants D374Y, D446N, C276R, and D315N. In particular, the CYP4F11 D315N protein showed about 50% decrease in intrinsic clearance compared to the wild type. The ability of wild-type CYP4F11 and the variants to synthesize 20-HETE from arachidonic acid was similar; the CYP4F11 D315N variant, however, showed only 68% of wild-type activity. Furthermore, the ability of CYP4F2 to synthesize 20-HETE was 1.7-fold greater than that of CYP4F11. Overall, our results suggest that the metabolism of CYP4F11 substrates may be reduced in individuals carrying the CYP4F11 D315N genetic variant and individuals carrying the common D446N CYP4F11 variant likely exhibit comparable 20-HETE synthesis as individuals expressing wild-type CYP4F11.

Single nucleotide polymorphism of CYP3A4 intron 2 and its influence on CYP3A4 mRNA expression and liver enzymatic activity in human liver.

In adult liver, CYP3A4 plays an important role in the metabolism of a wide range of endogenous and exogenous compounds. To investigate whether there is a single nucleotide polymorphism (SNP) of CYP3A4 intron 2 in the liver and its effects on the mRNA expression and enzymatic activity of CYP3A4, genomic DNA was extracted from 96 liver tissue samples obtained from patients who had undergone liver surgery. An SNP of CYP3A4 intron 2 was identified by polymerase chain reaction (PCR)-single-strand confirmation polymorphism and DNA sequencing. The mRNA expression of CYP3A4 was determined by the fluorescence quantitative PCR technique. The enzymatic activity of CYP3A4 was measured using erythromycin and testosterone as probe substrates. Twelve patients were found to have the SNP/T4127G CYP3A4 within intron 2. The mRNA levels of CYP3A4 in wild-type and SNP/T4127G samples were 2.62±1.09 and 2.79±1.63, respectively (P>0.05). Erythromycin N-demethylase activity in wild-type and SNP/T4127G samples were 121.2±32.8 and 124.7±61.6 nmol·mg(-1)·min(-1), respectively (P>0.05). The activity of testosterone 6β-hydroxylase was significantly different between wild-type (648±173 pmol·mg(-1)·min(-1)) and SNP/T4127G samples (540±196 pmol·mg(-1)·min(-1); P<0.05). In conclusion, the SNP/T4127G of CYP3A4 intron 2 exists in the liver. This SNP does not affect the mRNA expression of CYP3A4 but significantly decreases the hepatic microsomal testosterone 6β-hydroxylase activity of CYP3A4. Furthermore, this study indicates that the appropriate selection of probe substrates is very important in studying the relationship between the genotype and phenotype of CYP3A4.

Effect of Yajieshaba, a preparation of Dai indigenous medicine, on enhanced liver detoxification

ObjectiveTo explore the mechanistic effects of Yajieshaba (YJSB) on enhanced liver detoxification. MethodsThe effects of YJSB on alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were assayed in five acute chemical liver injury models [carbon tetrachloride (CCl4), D-galactosamine (D-Glan), 4-acetamidophenol (AAP), thioacetamide (TAA) and 1-naphthyl isothiocyanate (ANIT)]. Sleep latency and sleep time of pentobarbital sodium were tested in control mice and CCl4 model miceafter oral YJSB administration. The effects of YJSB on drug metabolism enzymes of liver microsomes were tested in control rats and CCl4 model rats. The levels of cytochrome P450 (CYP450) and Cyt b5 in liver microsomes were assayed using the method by Omura and Sato, and activities of erythromycin N-demethylase (ERD) and aminopyrine N-demethyl (ADM) were evaluated by Nash colorimetry. Probe substrate-based high performance liquid chromatography (HPLC) methods were established for CYP3A4 and CYP1A2. ResultsThe level of serum ALT was reduced by YJSB at 3.51 g/kg in the five models as follows: CCl4 > D-Glan, AAP, ANIT > TAA. YJSB treatment did not reduce the level of serum AST. YJSB at 3.51 g/kg prolonged the sleep latency in control mice and shortened the sleep time of control mice and CCl4 model mice. For control rats, YJSB at 2.43 g/kg increased the levels of CYP450 and Cyt b5 and induced the activities of ERD and ADM; for liver injuries induced by CCl4 in rats, YJSB at 2.43 g/kg increasedthe levels of CYP450 and Cyt b5. These results suggest that YJSB at 2.43 g/kg induces CYP3A4 and CYP1A2. ConclusionThese results suggest that YJSB enhanced liver detoxification and the mechanisms may be partially related to CYP3A4 and CYP1A2 induction.

Effects of baicalin on oral pharmacokinetics of caffeine in rats.

Scutellaria baicalensis is one of the most widely used herbal medicines in East Asia. Because baicalein and baicalin are major components of this herb, it is important to understand the effects of these compounds on drug metabolizing enzymes, such as cytochrome P450 (CYP), for evaluating herb-drug interaction. The effects of baicalin and baicalein on activities of ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), benzyloxyresorufin O-debenzylase (BROD), p-nitrophenol hydroxylase and erythromycin N-demethylase were assessed in rat liver microsomes in the present study. In addition, the pharmacokinetics of caffeine and its three metabolites (i.e., paraxanthine, theobromine and theophylline) in baicalin-treated rats were compared with untreated control. As results, EROD, MROD and BROD activities were inhibited by both baicalin and baicalein. However, there were no significant differences in the pharmacokinetic parameters of oral caffeine and its three metabolites between control and baicalin-treated rats. When the plasma concentration of baicalin was determined, the maximum concentration of baicalin was below the estimated IC50 values observed in vitro. In conclusion, baicalin had no effects on the pharmacokinetics of caffeine and its metabolites in vivo, following single oral administration in rats.

Effects of Nonylphenol on the Enzyme Activity of ERND in Gastropods (&lt;i&gt;Cipangopaludina chinensis&lt;/i&gt;)

Cipangopaludina chinensis were exposed to different concentrations of Nonylphenol (NP) in experimental groups. The effects of NP on the enzyme activity of erythromycin N-demethylase (ERND) in stomach were studies. The result showed that different concentrations of NP in settlings had different influence on the enzyme activity of ERND. The overall trend was that the activity of enzyme ERND showed decrease tendency at the beginning, then increased. Later, the enzyme activity of ERND in the stomach in the median concentration and high concentration was higher than Group Ⅲ (Environment group). Consolidating half-lethal concentration measurement results NP was shown that it has toxicity, so its impact on the environment can not be ignored.

Effect of Roxithromycin on the Activity of Enzyme ERND in the Kidney of Mollusk (&lt;i&gt;Cipangopaludina&lt;/i&gt;&lt;i&gt; chinensis&lt;/i&gt;)

Cipangopaludina chinensis which is a species of benthic macroinvertebrates was exposed to roxithromycin, and tested the erythromycin N-demethylase (ERND) of kidney, then explored the effect of roxithromycin on the biochemical indicator of C.chinensis. The results showed that roxithromycin had effect on the activity of ERND in the kidney of C.chinensis and the overall trend was promoting at the beginning then suppressing at the end, but the differences of the influence caused by different concentrations of roxithromycin was not obvious. In addition, it appeared from the survival condition of control group and experimental group, roxithromycin which is a kind of antibiotic, had no lethal effect on C.chinensis. But the concentration of roxithromycin which is more than 250 times, 500 times and 750 times of the environment used in this experiment had significant influence on the enzyme activity of the ERND in the kidney of C.chinensis.

Inhibitory action of Epilobium hirsutum extract and its constituent ellagic acid on drug-metabolizing enzymes.

Epilobium hirsutum (EH) is a medicinal plant for treating various diseases. Despite its wide usage, there is no available information about its potential influences on drug metabolism. The present study was undertaken to determine the in vivo effects of EH on hepatic CYP2B, CYP2C, CYP2D, and CYP3A enzymes that are primarily involved in drug metabolism. Male Wistar rats were injected intraperitoneally with EH water extract (EHWE) and ellagic acid (EA) at a daily dose of 37.5 and 20mg/kg, respectively, for 9days and hepatic drug-metabolizing enzymes were assessed at activity, protein and mRNA levels. Erythromycin N-demethylase activity was inhibited by 53 and 21% in EHWE- and EA-treated rats, respectively. Benzphetamine N-demethylase and 7-benzyloxyresorufin-O-debenzylase activities were decreased by 53 and 43%, and 57 and 57% in EHWE-and EA-treated rats, respectively. Moreover, protein levels of CYP2B1, CYP2C6, CYP2D2, and CYP3A1 also decreased by 55, 15, 33, and 82% as a result of EHWE treatment of rats, respectively. Similarly, CYP2B1, CYP2C6, CYP2D2, and CYP3A1 protein levels decreased by 62, 63, 49, and 37% with EA treatment, respectively. qRT-PCR analyses also showed that mRNA levels of these enzymes were significantly inhibited with bothEHWE and EA treatments. In conclusion, inhibition of drug clearances leading to drug toxicity because of the lowered activity and expression of drug-metabolizing enzymes might be observed in the people who used EH as complementary herbal remedy that might be contributed by its EA content.

Ergovaline in tall fescue and its effect on health, milk quality, biochemical parameters, oxidative status, and drug metabolizing enzymes of lactating ewes1

Ergovaline (EV) produced by symbiotic association of Epichloë coenophiala with tall fescue (Lolium arundinaceum) causes toxicoses in livestock. In this study, 16 lactating ewes (BW 76.0 ± 0.6 kg) were used to determine the effects of feeding endophyte-infected (FE+) or endophyte free (FE-) tall fescue hay on animal health and performances and to investigate the putative mechanisms of action of EV. The mean EV concentrations in FE+ and FE- diets were 497 ± 52 and <5 µg/kg DM, respectively. Decreased hay consumption and BW were observed in the FE+ group. Prolactin (PRL) concentrations decreased (P < 0.02) in the FE+ group from d 3 to 28 of the study compared to the FE- group, but no consequences were observed on milk quantity or quality. Skin temperature and the thermocirculation index were lower (P < 0.05) in the FE+ than in the FE- group from d 3 to 7, but this effect disappeared from d 14 to 28. Hematocrit, mineral and biochemical, and enzymatic analyses of plasma revealed no differences between the 2 groups. Measurement of oxidative damage and antioxidant enzyme activities revealed a decrease in the activities of plasma catalase (P < 0.05), kidney glutathione reductase and peroxidase and in kidney total glutathione and malondialdehyde contents (P < 0.02) in ewes fed FE+. Hepatic flavin monooxygenase enzyme activities decreased (P < 0.01) in ewes fed FE+, except for a marked increase in the demethylation of erythromycin. This activity is linked to cytochrome P4503A content and is known to be involved in ergot alkaloid metabolism. Glutathione S-transferase activity in the kidneys decreased (P < 0.02) in the FE+ group, whereas no difference was observed in uridine diphosphate-glucuronosyltransferase activity in the liver or kidneys. The reversibility of the effect of FE+ hay on skin temperature and the increase in erythromycin N-demethylase activity may contribute to the relative resistance of ewes to EV toxicity.

Attenuation of carbon tetrachloride toxicity in acute hepatosteatosis induced by intake of a high carbohydrate diet after food deprivation (959.1)

Hepatic fat accumulation constitutes a major component in the pathogenesis of liver injury. We determined the impact of lipid deposition in liver on cytochrome P‐450 (CYP) system and CCl4‐induced hepatotoxicity. Rats were fasted for 48 h, and then provided with a high‐carbohydrate/fat‐free diet (FH) or a normal diet (FN) for 48 h. Hepatic triglyceride level was not different between the normal control and the FN group, but 4 times greater in the FH group, which was consistent with the histopathological observation. FN intake resulted in a small increase in microsomal p‐nitrophenol hydroxylase, p‐nitroanisole O‐demethylase, and erythromycin N‐demethylase activities, but aminopyrine N‐demethylase was decreased. The microsomal enzyme activities determined were all inhibited markedly by FH intake. Immunoblotting analysis showed that proteins of CYP2E1, 1A, 3A, and 2B1/2 were decreased in the rats fed FH. Expression of CYP mRNAs was also down‐regulated. A dose of CCl4 was administered to the rats fed the different diets. The resulting liver injury was significantly lower in the FH group than in the FN group as determined by elevation of serum enzyme activities and histopathological examination. The results indicate that acute hepatosteatosis can precipitate erratic responses to various endogenous and exogenous substances via alterations in hepatic CYP‐mediated metabolizing capacity.

Toxic effects of Triclosan on the detoxification system and breeding of Daphnia magna

The toxic effects of different concentrations of Triclosan (TCS) (1-128 μg/L) on Daphnia magna (D. magna) were investigated by acute (48 h) and chronic (21-day) toxicity tests. The response of antioxidase system and Phase I metabolism process of D. magna exposed to TCS were investigated by measuring a series of biomarkers including glutathione S-transferase (GST), catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), 7-ethoxyresorufin O-deethylase (EROD), Erythromycin N-demethylase (ERND) and Aminopyrine N-demethylase (APND). The 48 h LC50 of TCS was 330 μg/L for D. magna. In the chronic test, total number of neonates per female, body length and the intrinsic rate of natural increase (r) of D. magna increased at the low exposure concentrations (1-16 μg/L) and decreased at the high concentrations (64-128 μg/L), while the total number of molting per adult decreased continually. The GST and CAT activities showed no significant increase in all treatments, and SOD activities were induced after 24-h exposure and inhibited after 48-h exposure at 4-128 μg/L of concentrations. The MDA content increased after 6-h exposure but decreased after 48-h exposure at 4-128 μg/L. EROD activities initially increased after 6-h exposure, but decreased after 24 and 48-h exposure, ERND and APND activities showed a similar temporal pattern among different treatments groups. SOD, MDA and APND were sensitive to TCS, thus they are suitable as potential biomarkers for the exposure to TCS.

Induction of CYP3A gene and enzyme activity expression in kidney cell of grass carp (Ctenopharyngodon idellus)

Cytochrome P450 3A(CYP3A) is the major cytochrome P450 responsible for the metabolism of en-dogenous and xenobiotic substrates.Unexpected drug–drug interactions in fish are generally associated with the induction of CYP3A activity,and can lead to the formation of drug residues,which threaten the safety of fishery products.We determined the in vitro effects of rifampicin(RIF) on CYP3A mRNA expression and enzyme activity in a freshwater teleost,the grass carp(Ctenopharyngodon idellus).Recently,a number of CYP3A genes have been isolated from several teleost species.Although CYP3A genes in fish contain multiple paralogs differing in gene expression pattern and tissue distribution,CYP3A enzymes exhibit similar catalytic properties because of their structural similarities.The CYP3A gene and its full-length cDNA of 1 849 bp were cloned from grass carp by RT-PCR.The CYP3A gene has an open reading frame(ORF) of 1 542 bp,a stop codon,a 3'-untranslated regions(3' UTR),and a polyA signal is present in the 3'UTR region.The ORF encodes a 513 amino acid(aa) protein,which has a signal peptide(29aa),two transmembrane helixes(23aa and 23aa),a heme binding domain(HBD,21aa),and six substrate recognition sites(SRS1-6).Multiple alignments showed that the CYP3A amino acid se-quence from grass carp is 60% to 92% similar to other vertebrate CYP3As.CYP3A gene expression after induc-tion by RIF in grass carp kidney cells(CIK) was assayed by quantitative real-time PCR.CYP3A-dependent erythromycin N-demethylase(ERND) activity was determined by spectrophotometry,and CYP3A activity was assessed by measuring the formation of formaldehyde.In the induced group,CYP3A mRNA expression reached a plateau at 8h,while the highest level of CYP3A activity occurred at 10h.The results indicated that CYP3A mRNA expression and enzyme activity were significantly higher than in the control group(P0.05).The highest level of CYP3A mRNA expression appeared 2 hours before the maximum of enzyme activity.In addition,the transcription of level CYP3A was apparently higher than the enzyme activity,implying that induction by RIF affected enzyme activity by transcriptional regulation of the CYP3A gene.

Accumulation and effects of 90‐day oral exposure to Dechlorane Plus in quail (Coturnix coturnix)

While a number of studies have addressed bioaccumulation of the flame retardant Dechlorane Plus (DP), little information is available regarding the adverse effects of DP on animals, especially on bird species. In the present study, male common quails (Coturnix coturnix) were consecutively exposed to commercial DP-25 by gavage for 90 d at 1-mg/kg/d, 10-mg/kg/d, and 100-mg/kg/d dosages. Concentrations of DP isomers in liver, muscle, and serum were determined after exposure. Liver enzyme activity involved in xenobiotic biotransformation processes and oxidative stress was measured, as well as glutathione and maleic dialdehyde content. The results showed that DP was more prone to accumulate in the liver than in muscle and serum in all exposed groups. In tested tissues, syn-DP dominated in the high-exposure groups (10 and 100 mg/kg/d), whereas anti-DP tended to accumulate in the low-exposure group (1 mg/kg/d). The concentration ratios of anti-DP to total DP (fanti values) in the tissues examined were close to commercial DP in the low-exposure group; however, the fanti values were significantly decreased in the high-exposure groups. Enzyme activity of 7-pentoxyresorufin-O-demethylase (PROD) decreased significantly in all exposed groups compared with the control group, whereas activity of erythromycin N-demethylase (ERND) and the antioxidant enzyme catalase significantly increased in high-exposure groups. The results implied that DP exposure levels influenced isomeric compositions in organs and that DP exposure altered hepatic alkoxyresorufin O-dealkylase (AROD) activity and contributed to the biological effects of DP.

Effect of atrazine and chlorpyrifos exposure on cytochrome P450 contents and enzyme activities in common carp gills

Chlorpyrifos (CPF) and atrazine (ATR) are the most widely used organophosphate insecticides and triazine herbicides, respectively, worldwide. This study aimed at investigating the effects of ATR, CPF and mixture on common carp gills following 40-d exposure and 40-d recovery experiments. Cytochrome P450 content, activities of aminopyrine N-demethylase (APND) and erythromycin N-demethylase (ERND) and the mRNA levels of the CYP1 family (CYP1A, CYP1B, and CYP1C) were determined. In total, 220 common carps were divided into eleven groups, and each group was treated with a specific concentration of ATR (4.28, 42.8 and 428μg/L), CPF (1.16, 11.6 and 116μg/L) or ATR–CPF mixture (1.13, 11.3 and 113μg/L). The results showed that P450 content and activities of APND and ERND in fish exposed to ATR and mixture were significantly higher than those in the control group. After the 40-d recovery treatment (i.e., depuration), the P450 content and the activities of APND and ERND in fish decreased to the background levels. A similar tendency was also found in the mRNA levels of the CYP1 family (CYP1A, CYP1B, and CYP1C) in common carp gills. The CPF-treated fish showed no significant difference from the control groups, except for a significant CYP1C induction. These results indicated that CYP enzyme levels are induced by ATR but were only slightly affected by CPF in common carp gills. In addition, the ATR and CPF exposure showed an antagonistic effect on P450 enzymes in common carp gills.