Induction Of EROD Activity Research Articles

Assessment of the Bioavailability of PAHs in Rats Exposed to a Polluted Soil by Natural Routes: Induction of EROD Activity and DNA Adducts and PAH Burden in Both Liver and Lung

In order to assess bioavailability of polycyclic aromatic hydrocarbons (PAHs) present in soils, male laboratory rats were exposed to litters of control and polluted soils. After 88±2 h of exposure, several biomarkers were measured in both liver and lung. When rats were exposed to SIV soil, contaminated by a mixture of at least 13 PAHs, (1) only 2 or 3 PAH compounds were detected in liver and lung; (2) cytochrome P450-dependent monooxygenase activity, followed by 7-ethoxyresorufin O-deethylase (EROD) activity measurement, was highly induced in liver (13-fold-induction) and lung (up to 78-fold); and (3) DNA adducts were significantly increased. For what concerns soil artificially contaminated by only one PAH (phenanthrene or B[a]P), EROD activity was not or fewly induced, respectively. These results demonstrate the occurrence of a high bioavailability of PAHs to mammals in natural conditions of exposure. First results concerning DNA adducts must be profounded, but they already show that a short exposure of mammals to PAH-polluted soils can lead to potential genotoxic effects. EROD activity can be used as a sensitive biomarker in both liver and lung of rats maintained on litters of soils in the laboratory, and such a test can be used routinely to contribute to risk assessment.

Constitutive and Inducible Cytochromes P450 in Rat Lung Mitochondria: Xenobiotic Induction, Relative Abundance, and Catalytic Properties

The presence of xenobiotic-inducible CYP1A1, 2B1/2, and 3A1/2 in rat lung mitochondria was investigated using mitochondrial preparations of defined purity. The mitochondrial P450 content in uninduced lung was 1.5-fold higher compared to microsomes. Administration of BNF induced the P450 contents by twofold in both mitochondrial and microsomal membrane fractions. BNF treatment induced EROD activity to about 40-fold in the microsomal fraction and 25-fold in the mitochondrial fraction. The microsomal induction was observed at 4 days of BNF treatment, while the mitochondrial induction required 10 days of treatment. Consistent with the activity profile, Western blot analysis showed the presence of CYP1A1 antibody reactive protein only in lung mitochondria from BNF-treated rats. BNF administration also caused a 50 to 80% reduction in the CYP2B1/2-associated PROD and BROD activities and CYP3A1/2-associated ERND activity in both mitochondria and microsomes. There was also a parallel reduction in the antibody reactive CYP2B1/2 and 3A1/2 proteins in both of these membrane fractions. Administration of DEX for 4 days induced mitochondrial and microsomal ERND activity by 1.7- and 2.5-fold, respectively. Mitochondrial EROD activity was inhibited by antibodies to P450MT2, as well as Adx, but not by antibody against P450 reductase, indicating the mitochondrial localization of CYP1A1. Protease protection and alkaline extraction experiments indicated that CYP1A1 associated with lung mitochondria is localized inside the inner membrane and exists as a membrane extrinsic protein. In summary, this is probably the first report of inducible P450s in rat lung mitochondria, and our results suggest a possible functional role for these mitochondrial enzymes in xenobiotic metabolism.

Time- and Concentration-Dependent Induction of CYP1A1 and CYP1A2 in Precision-Cut Rat Liver Slices Incubated in Dynamic Organ Culture in the Presence of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin

In a previous 24-h study, precision-cut rat liver slices were validated as a usefulin vitromodel for assessing the dose-related induction of CYP1A1 and CYP1A2 in rat liver following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Further assessment of the utility of this model was accomplished by initially exposing rat liver slices to medium containing TCDD (0.01 nM) for 24 h and incubating the slices up to an additional 72 h in TCDD-free medium. The slices remained viable throughout the incubation period with an intracellular potassium content varying from 45.2 ± 2.3 μmol/g at 48 h to 50.0 ± 1.6 μmol/g at 72 h. In TCDD-exposed slices, CYP1A1 protein and its respective enzymatic activity, theO-deethylation of ethoxyresorufin (EROD), significantly increased with time over the 96-h incubation period, with EROD activity increasing from 63.6 ± 14.2 at 24 h to 905 ± 291 pmol/mg/min at 96 h. Under identical incubation conditions, but in the absence of TCDD, the EROD activity for the control liver slices ranged from 14.3 ± 4.3 to 44.9 ± 11.9 pmol/min/mg. Conversely, the level of CYP1A2 protein and its respective activity (acetanilide hydroxylation) transiently decreased from 24 to 96 h with no significant differences observed between the control (0 nM TCDD) and treatment group (0.01 nM TCDD). The concentration–effect relationship at 96 h was characterized by incubating rat liver slices for the initial 24 h in medium containing TCDD at concentrations ranging from 0.1 pM to 10 nM. Induction of CYP1A1 protein and EROD activity was observed for all treatment groups with the 10 nM TCDD treatment group displaying greater than 100-fold induction compared to control (0 nM TCDD). Immunohistochemical localization of CYP1A1 protein within liver slices supported the time- and concentration-dependent induction of EROD activity by TCDD. The induction of CYP1A1 was initially observed to be centrilobular, with increased expression due to both elevated CYP1A1 within cells and the recruitment of additional cells expressing CYP1A1 throughout the entire liver slice. Additionally, the immunohistochemical analysis of the liver slices demonstrated the conservation of tissue architecture following up to 96 h of incubation in dynamic organ culture and provided further evidence for maintenance of tissue viability. In comparison to CYP1A1, the induction of CYP1A2 at 96 h was a less sensitive response, with significant induction of CYP1A2 protein and its respective activity occurring at a medium concentration of 0.1 nM TCDD (686 pg/g liver). In general, increasing the incubation period from 24 to 96 h markedly increased TCDD-induced expression of CYP1A1 and minimally enhanced CYP1A2 expression. Moreover, extending the incubation period to 96 h resulted inin vitroinduction profiles for CYP1A1 and CYP1A2 that were qualitatively and quantitatively similar to that previously observed followingin vivoexposure to TCDD (Drahushuket al., Toxicol. Appl. Pharmacol.140, 393–403, 1996).

Comparison of two bioassays, a fish liver cell line (PLHC-1) and a midge ( Chironomus riparius), in monitoring freshwater sediments

Two bioassays, a fish hepatoma cell line (PLHC-1) and a midge ( Chironomus riparius), were used to monitor surficial sediments from Lake Võrtsjärv and River Narva, Estonia. L. Võrtsjärv is polluted with mainly polycyclic aromatic hydrocarbons (PAHs) but R. Narva possesses complex contamination (PAHs, heavy metals, sulphates, chlorides). The PLHC-1 cells were exposed to the extracted lipid soluble compounds (PAH fraction) of the sediments, after which the cytotoxicity (total protein content) and cytochrome P4501A (CYP1A) inducibility (7-ethoxyresorufin O-deethylase activity (EROD) and CYP1A protein content) were measured. The midges were grown in whole sediments after which the midge growth (larval growth and survival as endpoints) or the emergence (larval survival and adult emergence) were tested. Contents of selected PAHs and heavy metals in the sediments were also evaluated. In the PLHC-1 screening experiments, most of the sediments from R. Narva were more toxic and caused higher EROD activities at lower doses than the sediments from L. Võrtsjärv. The most polluted sediment in R. Narva (total PAH content 744 ng g −1 dry weight sediment) gave 15 mg dry sediment ml −1 as the ED 50 for induction of EROD activity in the cells exposed for 3 days. In the midge growth test, larvae seemed to grow better in the sediments from L. Võrtsjärv than from R. Narva and the mortality was somewhat higher in two areas in R. Narva than in other study areas. Adult emergence did not show such clear trends between these two watersheds, though emergence was accelerated in some sediments. A sediment from a point source of pollution (accidental release of asphalt) from R. Narva was also studied and found to be the most toxic for the PLHC-1 cells and the most potent inducer of CYP1A (ED 50s 0.59 and 0.56 mg dry sediment ml −1). None of the midge larvae survived in this sediment. In a time-course study, the highest EROD activity in the PLHC-1 cells was reached at lower doses of PAH fraction after 24-h exposure than after 48 or 72 h, suggesting metabolism of PAHs in the cultures. Further, CYP1A induction was still seen as elevated amounts of CYP1A protein in cases where catalytic EROD activity was decreased at higher doses of PAH fraction. Overall, the PLHC-1 bioassays were shown to be sensitive methods for detecting PAH pollution. The midge bioassays reflected better the bioavailability and the in situ effects of the complex mixture of compounds in the sediments.

Effects of vegetables–fruit extracts and indole-3-carbinol on stearic acid-modulated intercellular communication and cytochrome P450-IA activity

Modulatory effects were investigated of extracts of a vegetables–fruit mixture and indole-3-carbinol (I3C) on stearic acid-modulated gap junctional intercellular communication (GJIC) and cytochrome P450-IA activity (EROD). In V79 cells, pure water and hexane extracts of a vegetables–fruit mixture and 25 μg/ml I3C significantly protected against decreased GJIC caused by 10 μM stearic acid. Furthermore, pure, 10× and 100× diluted vegetables–fruit extracts significantly maintained their capacity to induce EROD activity in Caco-2 cells, but only when these extracts were added to the cells in media already containing 500 μM stearic acid for 48 h. Stearic acid itself did not induce EROD activity. I3C (10, 25, and 50 μg/ml) clearly induced EROD activity in Caco-2 cells, irrespective of the order at which I3C and stearic acid were added to the cells. In conclusion, the present in vitro study showed that vegetables–fruit extracts and I3C modulate effects of stearic acid on intercellular communication and cytochrome P450-IA activity.

In vitro induction of ethoxyresorufin‐O‐deethylase and porphyrins by halogenated aromatic hydrocarbons in avian primary hepatocytes

AbstractEthoxyresorufin‐O‐deethylase (EROD) and porphyrin induction responses of primary hepatocytes to halogenated aromatic hydrocarbons (HAHs) were examined in newly hatched domestic chickens, herring gulls, ring‐billed gulls, double‐crested cormorants, and Forster's terns. Concentration–response relationships were determined for both biochemical responses in hepatocyte preparations derived from individual avian livers (except for the tern). The choice of vehicle used to dose chicken hepatocytes greatly affected the potencies and efficacies of HAHs. Dimethyl sulfoxide resulted in median effective concentration (EC50) values for EROD induction that were between 10 and 15 times less than isooctane (isooctane was used throughout the study). Neither vehicle induced EROD activity by itself. Concentration‐dependent increases in EROD activity were observed with several HAHs, and their potencies (EC50 values) were compared to that of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) within each hepatocyte preparation to determine relative potency factors (RPFs). Differences in sensitivity to these responses were observed among individuals within each of the species and among species. Median EC50 values (nM) for EROD induction by TCDD were 0.72, 13, 20, 25, and 150 for the chicken, cormorant, ring‐billed gull, herring gull, and tern hatchling, respectively. Relative potency factors for several HAHs were different, in both ranking and potency, from those generally derived in mammalian hepatocytes. Porphyrin accumulation was observed occasionally with the most potent aryl hydrocarbon receptor agonists, but most HAHs were not tested at concentrations sufficiently high to observe a consistent response. This study provides information on interindividual and interspecies differences in responsiveness to TCDD‐like compounds and provides species‐specific RPFs that may prove useful for the purpose of hazard and risk assessment for fish‐eating birds.

Induction of cytochrome P450 1A1 in rat liver slices by 7-ethoxycoumarin and 4-methyl-7-ethoxycoumarin

7-Ethoxycoumarin (EC) is widely used as a model substrate for monooxygenase function, its O-deethylation representing cytochrome P450 (P450) activity mainly of 1A but also of 2B isoforms. Reports on investigations of its own capacity to induce or suppress P450 activities, however, have not been found in biomedical literature. To avoid the influence of in vivo pharmacokinetics, studies can well be undertaken with liver slice incubation. Therefore in the present investigation precision-cut rat liver slices from male 43-63-day-old male HAN:Wistar outbred rats were incubated at 30 degrees C in carbogen saturated William's Medium E for 24 h. EC was added previously to final concentrations of 10, 25, 50, 75 or 100 microM. After incubation, homogenate was prepared from slices and used for model reactions (7-ethoxyresorufin O-deethylation [EROD] and 7-pentoxyresorufin O-depentylation [PROD]). EROD, indicating activities of 1A isoforms, was enhanced by incubation with EC at 25 and 50 microM to about doublefold but showed control or lower values at 75 and 100 microM. Incubation with beta-naphthoflavone in comparison led to variable increases (3-5-fold of controls). For PROD as an indicator of the phenobarbital inducible P450 isoforms 2B1 and 2B2 no enhancement was found, but a decrease by incubation with 75 and 100 microM EC. To further investigate the correlation between enzyme activity and gene expression after slice incubation, P450 1A1 mRNA content was measured by RT-PCR. Induced gene expression for 1A1 was seen with different EC concentrations to a variable extent, though not as strong as with BNF. Similar incubation with 4-methyl-7-ethoxycoumarin revealed an even stronger induction of EROD activity with maxima at about 10-32 microM, reaching BNF values. In contrast incubation with 7-benzyloxycoumarin had no evident inducing or suppressing effect, neither on EROD nor on PROD activity.

Induction of erod activity in HEPA‐1 mouse hepatoma cells and estrogenicity in mcf‐7 human breast cancer cells by extracts of pulp mill effluents, sludge, and sediment exposed to effluents

AbstractExtracts of effluents and sludges from the primary and secondary clarifiers of an activated sludge treatment plant at a Finnish bleached kraft pulp and paper mill were analyzed in two cell bioassays. Total dioxin‐like activities were determined by measuring the induction of ethoxyresorufin‐O‐deethylase (EROD) activity in Hepa‐1 mouse hepatoma cells. Estrogenicity was studied by measuring luciferase activity in MCF‐7 ERE‐luc, which are MCF‐7 human breast cancer cells stably transfected with an estrogen‐responsive element linked to a luciferase promoter. Sediments collected near the pulp mill and from other sites in Lake Saimaa as well as fillets of whitefish exposed to effluents were examined and EROD activity was determined for complex mixtures of compounds extractable with dichloromethane (DCM) from the pulp mill effluent or compounds collected by semipermeable membrane devices (SPMDs) from the same effluent. Extracts of effluents, particulates, SPMDs, and sludges all caused measurable EROD induction. Because the induction potencies of the total DCM extract and the fraction of neutral compounds were similar, it was concluded that most of the EROD induction in pulp mill effluents was due to neutral compounds. Sediment collected from the vicinity of the pulp mill had greater EROD‐inducing potency compared to that of the control sites from the same lake. Induction of EROD potencies of muscle extracts of whitefish exposed to diluted effluents were not significantly greater than those of control fish exposed to dilution water only. All extracts contained some estrogen agonist activity when tested in the MCF‐7 bioassay.

Induction of EROD activity in gill epithelial cells from rainbow trout

Induction of EROD activity in gill epithelial cells from rainbow trout

Accumulation and effects of benzo(a)pyrene on cytochrome P450 1A in waterborne exposed and intraperitoneal injected juvenile turbot ( Scophthalmus maximus)

Juvenile turbot ( Scophthalmus maximus) were exposed to benzo(a)pyrene (BaP) for 14 d using a glass bead generator flow-through system. Exposure was followed by a recovery period of 16 d. The highest BaP concentration in the edible portion of the fish, 16.5 ± 4.3 μg BaP/kg, was observed on the first day. Then concentrations dropped following first-order kinetics. BaP was below detection level at the end of the experiment. A statistically significant increase in bile fluorescence was observed from day 9 until the end of the experiment, suggesting the elimination of BaP metabolites by this route. No significant differences between control and exposed fish in EROD activity and CYP1A concentration, measured by immunodetection method, were observed. Intraperitoneal injection of 2.5 mg BaP/kg in juvenile turbot induced EROD activity. Under waterborne experimental conditions, bile fluorescence was observed to be a more sensitive biomarker of BaP exposure than EROD activity and CYP1A measurement.

A fish hepatoma cell line (PLHC-1) as a tool to study cytotoxicity and CYP1A induction properties of cellulose and wood chip extracts

Cytotoxicity and CYP1A induction properties of celluloses and wood chips were studied with a teleost liver cell line, PLHC-1. Cells were exposed to acetone extracts of celluloses produced using new bleaching techniques (elemental chlorine free, ECF; totally chlorine free, TCF) in two sulphate mills or without any bleaching (unbleached, UB) in a sulphite mill. In another set of exposures, celluloses (ECF and TCF bleached) and wood chips (from pine and birch) were collected from a sulphate mill, extracted with acetone, and the extracts used to treat the cells. After exposure, O-deethylation of 7-ethoxyresorufin (EROD, a measure of cytochrome P4501A (CYPIA) catalytic activity), and total protein content, a measure of cytotoxicity, were assayed. The presence of the CYP1A protein in the exposed cells was assessed by immunoblotting. The cellulose and wood chip extracts were able to cause both cytotoxicity and EROD induction in the PLHC-1 cells. In the exposures conducted with the material from three different mills, the celluloses made of birch were more cytotoxic and more potent inducers of EROD activity than were the celluloses of pine. Further, UB celluloses increased EROD activity and caused cytotoxicity at lower doses than material bleached with modem bleaching techniques. In the exposures made with material from one single mill, there were no clear trends between the celluloses made of pine or birch. Wood chips of pine, however, were more cytotoxic than wood chips of birch. Especially with pine wood chips, cytotoxicity interfered with the induction of EROD activity, thus complicating the evaluation of CYPIA induction. CYPlA protein content was not detected in cells exposed to extracts of celluloses or wood chips, possibly due to low amounts of protein available for the assay. Wood and pulp processing, like bleaching, may change the chemical composition of the raw material in a way that reduces the potency for biological effects of the final product, cellulose. This could explain why both UB celluloses and wood chips were more potent in the cells than ECF or TCF bleached celluloses. In this study the PLHC-1 cell line showed its potential for use in evaluating the biological activity existing in pulp and paper mill products and raw materials. The identity and source of the compounds that were able to affect the PLHC-1 cell line remain to be determined.

Cytochromes P450 in liver of the turtle Chrysemys picta picta and the induction and partial purification of CYP1A-like proteins

Cytochromes P450 (CYP) in hepatic microsomes from the turtle Chrysemys picta picta and their response to inducers were examined. Freshly caught turtles had one protein (59 kDa) detected in western blot with monoclonal antibody 1-12-3 to scup CYP1A. That same band and a second band were detected with polyclonal anti-mouse Cyp1a1. Polyclonal anti-scup P450B (putative CYP2B) recognized three bands and anti-scup P450A (putative CYP3A), one band. TCB (3,3′,4,4′-tetrachlorobiphenyl) at 5 mg kg −1 injected once induced EROD activity 3-fold. Repeated high-dose injections of TCB, 2,3,3′,4,4′-pentachlorobiphenyl, Aroclor 1254 or β-naphthoflavone induced CYP1A 20-fold and P450B-related proteins 2–3-fold. Rates of ethoxy- (EROD) methoxy- (MROD) and pentoxyresorufin O-dealkylases and benzo[a]pyrene (B[a]P) hydroxylase (AHH) were induced by these treatments, and were correlated with putative CYP1A content. Phenobarbital slightly elevated only MROD activity. Ethoxycoumarin (EC) O-deethylase rates were high, 1.6–2.2 nmol min −1 mg −1 in control and treated turtles, suggesting that EC is not a turtle CYP1A substrate. Highly induced EROD rates were 0.06 nmol min −1 mg −1, while AHH rates exceeded 4 nmol min −1 mg −1, suggesting that C. picta picta CYP1A may prefer PAH substrates. Induction of AHH was reflected in the formation of metabolites 3-OH-, 9-OH- and 7-OH-BP and BP-7,8-dihydrodiol (DHD). BP-4,5-DHD was not detected. Chromatographic procedures resolved the 59 kDa putative CYP1A from the second protein recognized by anti-Cyp1a1. The 59-kDa protein was also specifically and highly immunopurified by Mab 1-12-3. Thus, several CYP including two CYP1A-related proteins are expressed in turtle liver. Multiple CYP1A genes in reptiles may provide an insight into the origin of divergence in the CYP1A subfamily. Induction of a CYP1A may be a useful indicator of exposure to Ah receptor agonists in turtles.

Laboratory and Field-Caging Studies on Hepatic Enzymatic Activities in European Eel and Rainbow Trout

Caging field experiments were conducted on young European eel (Anguilla anguilla) and on rainbow trout (Onchorynchus mykiss) for the determination of a biomonitoring protocol of freshwater streams. Hepatic monooxygenase and conjugation enzyme activities were measured as biomarkers of exposure to some organic pollutants. Laboratory studies were performed to compare the responsiveness of these two species concerning their enzymatic activities. The induction of monooxygenase activity, measured as the deethylation of 7-ethoxyresorufin (EROD activity) was examined byβ-naphtoflavone (β-NF) treatment. Dose–response study revealed that EROD activity increased significantly from 1 mgβ-NF/kg bw and was on a plateau at 50 mgβ-NF/kg bw for both species. No significant effect on glutathione S-transferase (GST) activity was observed. During a time-course study, EROD activity increased, starting from the second day for both species. In European eel, this enzymatic activity decreased significantly from the seventh day, whereas no significant decrease was observed for rainbow trout after 14 days. During a 2-week field study, EROD and GST activities were measured in fish held in cages upstream and downstream of a polluted area. For both species, the induction of EROD activity was observed in the polluted site after 14 days of caging; GST activity was not significantly affected. Monooxygenase activity appeared to be a sensitive tool for biomonitoring freshwater streams for both species.

Cytochrome P450 1A1 induction in rat lymphoid tissues following in vivo and in vitro exposure to 2,3,7,8-tetrachlorodibenzo- p-dioxin requires protein kinase C

The induction of cytochrome P450 1A1 (CYP1A1) is one of the most sensitive responses associated with exposure to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and related compounds. The mechanisms that underlie this response are not completely understood, particularly in lymphoid tissues that may be used in biomarker studies in humans. CYP1A1 mRNA expression and enzyme activity (ethoxyresorufin- o-deethylase, EROD) were investigated in rat thymus and spleen and isolated thymocytes and splenocytes in culture. Thymus- or spleen-derived microsomes from rats treated in vivo with TCDD showed induced EROD activity after as little as 12 h following a single exposure to TCDD (5 μg/kg body weight). Resting rat thymocytes in culture had detectable levels of EROD activity and CYP1A1 mRNA expression which increased following in vitro exposure to ≥0.1 nM TCDD for 24 or 48 h. Interestingly, concomitant in vitro exposure of rat thymocytes to TCDD and the mitogen concanavalin A (Con A) inhibited the induction of EROD activity, which is in contrast to the response of cultured human peripheral blood lymphocytes (Landi et al., 1994; Pharmacogenetics 4, 242–246). Resting rat splenocytes in culture had no detectable EROD activity and CYP1A1 activity could not be induced by in vitro TCDD exposure, in the presence or absence of Con A. These results suggest that the relative maturation state of the cells is important in regulating the expression of CYP1A1, since splenocytes represent a more mature population of B and T lymphocytes. TCDD-induced CYP1A1 expression in cultured rat thymocytes was inhibited by the addition of calphostin C, a specific protein kinase C (PKC) inhibitor, suggesting a role for PKC as a second messenger in the CYP1A1 induction pathway. In vivo co-exposure with phorbol-myristate-acetate (PMA) and TCDD also inhibited CYP1A1 induction. Again, suggesting a role for PKC in CYP1A1 induction. Together, these results indicate that relative lymphocyte maturation state and the PKC pathway are important factors in regulating the expression of CYP1A1.

The suitability of gastropods as biomarkers

The measurement and induction of mixed function oxyigenases (MFOs) of limnetic gastropods were investigated to estimate their suitability as biomarkers. A determination of MFO activities was performed through the measurement of ethoxyresorufin-O-dealkylase (EROD), pentoxy-(PROD) and benzoxyresorufin-O-dealkylase-activity (BROD). Optimal measuring conditions of these activities were investigated in preparations of the digestive gland of 4 species of limnetic gastropods. Results indicate that MFO-activity inhibiting substances are accumulating in the microsomal pellet, the fraction commonly used for the measurement of MFO-activities. Therefore, the fraction used for induction studies was the postmitochondrial supernatant (PMS). EROD, PROD, and BROD activity of Planorbis planorbis and Planorbis carinatus were measured after a treatment with Aroclor 1254 for 1-17 days. Maximal induction of EROD and PROD were 6 and 10 times the value in the control group of P. carinatus, respectively induction in P. planorbis was lower. BROD-activity could not been measured in P. planorbis. In P. carinatus, BROD-activity increased to a maximum of 2 times after treatment with Aroclor 1254. The level of induction of EROD-activity is comparable to results described in analog studies with fish.

Induction of cytochrome P4501A1 by photooxidized tryptophan in Hepa lclc7 cells

Mouse hepatoma Hepa-lclc? (Hepa-1) cells were cultivated in the presence of UV-irradiated amino acids. The results demonstrated that all of the amino acids tested, UV-oxidized tryptophan caused the highest induction of 7-ethoxyresorufin O-deethylase (EROD) activity compared with the controls (P < 0.01). The induction of EROD activity by oxidized tryptophan was dose dependent, and maximal induction was obtained at 12 hr after administration. Studies with various Hepa-1 mutants, which are defective in either the aryl hydrocarbon (Ah) receptor or Ah receptor nuclear translocator protein, indicated that the induction of EROD activity by oxidized tryptophan occurs through the Ah receptor. Gel mobility shift assays using nuclear extracts of Hepa-1 cells revealed that oxidized products of tryptophan can induce both Ah receptor transformation and binding of the liganded Ah receptor complex to its specific DNA recognition site. CYP1A1 mRNA, quantified by reverse transcription-polymerase chain reaction, and CYP1A1 protein were induced markedly in the oxidized tryptophan group compared with the controls. Injection of isolated oxidized tryptophan products into adult male rats caused significant induction of EROD activity in the pulmonary and hepatic microsomes compared with the controls (P < 0.01). These results demonstrated that oxidized tryptophan induces Ah receptor activation and binding of the liganded Ah receptor complex to its specific DNA recognition site, thereby initiating transcription and translation of the CYP1A1 gene with concomitant increase of EROD activity in Hepa-1 cells. Induction of EROD activity in the liver and lungs after injection of isolated oxidized tryptophan products into rats suggests that a similar mechanism may be operative in vivo.

Qualité des milieux aquatiques continentaux : intérêts de l'utilisation d'un marqueur biochimique chez le poisson

Biological assessment is useful in addition to conventional chemical mesures in order to monitor the water quality. Biochemical markers are early warning systems of the exposition of organisms to pollutants. Among them, the induction of the fish EROD activity enables the detection of specific compounds PAHs, PCBs. dioxines.EROD induction was evaluated for three cyprinids species in two French river sites : Durance and Moselle. In the river Durance, the closer to a chemical plant the sampling site was, the higher the measured EROD induction. In the river Moselle, the fish showed a significant induced EROD activity after industrial effluent discharges. In both cases the measure was species dependent.Although the EROD activity seems a fitted tool to assess the water quality, complementary studies are necessary to understand the effects of biotic and abiotic variables, on the results.

Biotransformation enzymes and their induction by β-naphthoflavone during embryolarval development in salmonid species

Biotransformation enzymes were studied in vitro in subcellular fractions from early life stages of Arctic charr ( Salvelinus alpinus), whitefish ( Coregonus lavaretus) and grayling ( Thymallus thymallus). Each species showed an increase in 7-ethoxyresorufin O-deethylase (EROD), NADPH-cytochrome c reductase and glutathione S-transferase (GST) throughout embryolarval development. Significant increase in enzymatic activities seemed to occur around hatching period in the three species, especially for EROD activity. Interspecies comparison led to different results depending on how activities were expressed (specific, per individual, or normalized with respect to the size of individual). Arctic charr embryos and eleuteroembryos exposed for 72 h to 0.12 ppm β-naphthoflavone showed a sixfold induction of EROD activity whereas NADPH-cytochrome c reductase and GST activities were not modified.

Differences in cypia response between flounder ( Platichthys flesus) and plaice ( Pleuronectes platessa) after long-term exposure to harbour dredged spoil in a mesocosm study

Flounder (Platichthys flesus) and plaice (Pleuronectes platessa) were exposed to three different levels of pollution involving contaminants from harbour dredged spoil in a large-scale mesocosm study lasting 30 months. Exposure of fish to PCBs and PAHs was estimated from liver concentrations of CB-153 and biliary levels of 1-OH pyrens in flounder. Both marker compounds were significantly elevated in fish from the polluted mesocosms in comparison to the reference mesocosm. Of the three parameters used to characterise CYPIA induction in flounder, only CYPIA-mRNA in female fish was significantly induced by pollution. However, significant induction of EROD activity and immunochemically measured CYPlA protein content was observed in plaice. To inivestigate in more detail the apparent lack of CYP1A induction, a parallel experiment was run in which flounder were injected with a single i.p. injection of benzo[a]pyrene (BaP) and sacrificed after 48 hours. Since this approach did result in significant induction of all the parameters used to characterise CYP1A induction (EROD, CYPIA, CYPIA mRNA), it appears that external factors may have prevented induction in the mesocosm experiments. Down-regulating effects of the possible presence of a compound with oestrogenic activity, or altered membrane-CYPIA or membrane-receptor interaction, could have contributed to this apparent lack of response, despite the intrinsic ability of flounder to increase CYPIA levels after exposure to a model inducer (BaP).

Hepatic enzymatic activities of the european eel Anguilla anguilla as a tool for biomonitoring freshwater streams: laboratory and field caging studies

Hepatic monooxygenase activity and conjugation enzyme activity were studied in young european eel (Anguilla anguilla). Laboratories studies were performed by treatment with β-naphtoflavone (β-NF). Two sources of eels, wild and reared eels were compared during a time-course study. MO activities measured as 7-ethoxyresorufin O-deethylase (EROD) were induced by β-NF, peak values were reached 2 and 4 days after treatment. The time-course induction of EROD activity was similar for wild and reared eels. In contrast, the conjugation reaction enzymes measured as glutathione-S-transferase (GST) didn't respond to this treatment. During field studies, EROD and GST activities were studied in wild and reared eels held in cages upstream and downstream of a PAH polluted area. Similar induction of EROD activity was observed in the polluted site after 14 days of caging. The period of caging was studied and EROD activities were significantly induced in wild eels caged in the polluted site after 7 and 14 days. GST activities were not affected by the caging in the polluted site. The results indicate the usefulness of EROD activity as a biomarker of PAH contamination in the model of wild eels caging in the field.