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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., (Copyright © 2016. Published by Elsevier Inc.)

During the past decade the Swan-Canning estuary, Western Australia, has shown signs of stress which has been attributed to high nutrient inputs. There is little information on the effect of nonnutrient contaminants on biota inhabiting the estuary. A suite of biomarkers was measured on black bream (Acanthopagrus butcheri) to determine whether annual variations in fish biomarkers exist in the wet (winter) and dry (summer) seasons. Serum sorbitol dehydrogenase showed no significant differences between years, indicating that measured mixed-function oxygenase (MFO) enzyme activities were not affected by annual variations in hepatic tissue damage. Ethoxyresorufin-O-deethylase activity was lower in female black bream than in male fish while ethoxycoumarin-O-deethylase activity was not influenced by gender. Biomarker levels measured at various sites confirm that major roads and drains are significant contributors of MFO-inducing chemicals into the Swan-Canning estuary. No consistent upstream or downstream gradient in biomarker response was identified. The ratio of naphthalene-type to benzo(a)pyrene-type biliary metabolites was linked to runoff from urban areas into the estuary. There was high annual variability in all biomarkers in both seasons, suggesting that biannual monitoring is required to evaluate the effect of contaminants on the biota in the estuary.

Several investigations have demonstrated that the increase in chemicals in the environment may have caused effects on aquatic life and wildlife. The impact from oil production activities on the benthic community structure and shrimp (Farfantepenaeus duorarum and Litopenaeus setiferus) biochemical markers were studied in the Gulf of Mexico, which is heavily contaminated by polycyclic aromatic hydrocarbons and heavy metals. Shrimp were collected from a control area and from an area close to oil production platforms during October and November 2002. There was no spatial difference in exposure and response probably because shrimp migrate, as results did not show significant differences in cytochrome P4501A (as measured by ethoxycoumarin O-deethylase (ECOD) metabolism) between the two sites. In October, shrimp ECOD activities were higher and statistically different from those measured in the samples taken in November. As for ECOD activities, with the concentration of vitellogenin as another biomarker, there were no differences between the shrimp collected from the control area and the shrimp collected on the oil production platforms. In this case the concentrations were higher in shrimp collected in November vs. shrimp collected in October. However, there are significant correlations between contaminants and responses (biomarkers), indicating an effect of pollution. One of the most important considerations brought up by this kind of study is that, although the majority of groups studying the effects of endocrine disruption have focused almost exclusively on human health or vertebrate wildlife-related issues, it is necessary to increase research focused on understanding the function of hormones in invertebrate species exposed in the field to different kinds of pollutants.

Polychlorinated biphenyls (PCBs) are organochlorinated micropollutants ubiquitously distributed in the environment. They are known to be strong inducers of hepatic monooxygenases in fish. This can adversely affect reproduction by increasing steroid metabolism. In this work, adult barbels were contaminated with food containing Aroclor 1260, a commercial PCB mixture from Monsanto, at environmentally relevant concentrations. A significant increase in cytochrome P450 was observed, and two particularly sensitive enzymes, ethoxyresorufin o-deethylase (EROD) and ethoxycoumarin o-deethylase (ECOD), were strongly induced. Electron microscopy revealed alterations in liver ultrastructure in contaminated fish, principally an increase in the number of cisternae of the rough endoplasmic reticulum, drastic glycogen depletion, dissolution of mitochondrial contents, and appearance of myelin figures. Contamination was also studied in relation to reproductive success in a hatchery. Contaminated males displayed no alteration in milt quality, but PCBs did alter female reproductive parameters. Total mortality of eggs and larvae increased significantly with the level of PCBs in the eggs. The most highly contaminated fish did not even spawn. All the adverse effects recorded here tended to be reversible when the intoxication ended, sometimes after only a 1-year detoxication period., (Copyright 1999 Academic Press.)

Activity measurements of enzymes catalyzing (i) the oxidation of xenobiotics (phase I) and (ii) the conjugation of metabolites produced in phase I (phase II) were carried out in ruffe to test its suitability for biological monitoring. Ruffe typically lives in the lower regions of rivers and in estuaries where monitoring is of particular interest for estimating the amount of pollutants introduced into the sea. The flounder, already examined for this purpose, is used as reference organism. In ruffe, the enzymatic activity (7-ethoxycoumarin-O-deethylase (ECOD) and 7-ethoxyresorufin-O-deethylase (EROD)) increased with the contamination of the river toward Hamburg whereas in flounder it did not. The absence of a correlation between induction of the enzymatic activity in flounder and environmental contamination is attributed to the higher readiness of the flounder to migrate. Therefore, mixed function oxygenase activity found in flounders may not represent the inducing capacity of the local concentrations of xenobiotics. Since ECOD activity in ruffe was 5 to 20 times higher compared to the flounder, histopathological findings of investigations of the liver tissue were considered. Destruction or pathological changes of the endoplasmatic reticulum (where ECOD and EROD are located) is often reported in flounder but not in the ruffe, which may influence the expression of some enzymatic activities. The enzymatic activity of the phase II enzyme glutatione-S-transferase, which is considered to have a protective function in the cell against damages, caused by reactive metabolites, was more induced in ruffe than in flounder. Thus the ruffe may be less subjected to cell injury.

Hepatocytes isolated from fetal quail livers (Coturnix coturnix japonica) were cultured in vitro. Their capacity to metabolize drugs and xenobiotics was explored with typical cytochrome P450 substrates: ethoxycoumarin (known to be metabolized by several P450s), ethoxyresorufin (essentially dealkylated by P450IA1), and testosterone (specifically hydroxylated at several positions by several P450s). The cells could be kept metabolically active in culture for at least 4 days. Their drug-metabolizing activities were inducible by the usual P450 inducers, like phenobarbital and benzanthracene, but also by Aroclor 1254, a PCB mixture. The results obtained indicate that this experimental model could certainly be very helpful in ecotoxicological studies.

The xenobiotic-metabolizing enzyme system of hatchery golden ide [Leuciscus idus (L.)] were tested for applicability in biological monitoring in rivers. The golden ide, cyprinid fish used for toxicity testing, were caged for 1 to 3 weeks in flowthrough systems at various locations of the river Elbe. The activities of 7-ethoxycoumarin-O-deethylase (ECOD) and the cytochrome P450 (P450) concentrations in the livers were analyzed. Exposure in the river Elbe increased the ECOD activity and the P450 concentration. In a flowthrough basin located in the harbor of Hamburg the ECOD activity increased up to 3.5 times, and the P450 concentration increased slightly by a factor of 1.5. During exposure of the golden ide at two Elbe stations upstream and downstream the city of Hamburg, levels of P450 in the livers did not change significantly when compared with laboratory controls. There was, however, a significant increase of ECOD activity between stations Bunthaus (upstream) and Blankenese (downstream). The ECOD activity in the livers of golden ide caged downstream were significantly higher compared to those at the upstream station. The results presented in this study suggest that golden ide can be employed as experimentals for routine measurements in limnic habitats using biochemical assays.

The newt (Pleurodeles waltl) is an amphibian species used in a mutagenicity test (micronucleus). This study was carried out to establish if the inducibility of hepatic cytochromes P450 of this species is similar to that of the rat. Our results showed that the newt is characterized by a lower level of hepatic cytochrome P450-dependent activities than the rat. Variations of enzymatic activities according to sex and season were observed. Specific activities in newt were characterized by an almost complete insensitivity to induction by phenobarbital pretreatment. On the other hand, pretreatment by 3-methylcholanthrene resulted in an increase in the metabolism of several hydroxycoumarin and resorufin derivatives, similar to the effects observed in rat liver. Occurrence of specific forms of cytochromes P450 was assessed by specific antibodies.

The PLHC-1 fish hepatoma cell line (Poeciliopsis lucida) was used in the neutral red assay to evaluate the acute cytotoxicities of direct-acting (alkylbenzenes, phthalate diesters, and pesticides) and metabolism-mediated (benzo[a]pyrene) toxicants. The sequence of cytotoxic potencies for the alkylbenzenes and phthalate diesters appeared to be a direct function of their hydrophobicity (as described by logarithmic octanol/water partition coefficients). The organochlorine pesticides (alachlor and p,p'-methoxychlor) were more cytotoxic than the organophosphorus pesticides (EPN, diazinon, and malathion). The PLHC-1 cell line apparently maintained sufficient xenobiotic-metabolizing capacity, as the hepatoma cells were able to metabolize benzo[a]pyrene to cytotoxic intermediates. Xenobiotic-metabolizing capacity was temperature dependent, with enzymatic activity increasing as the temperature was increased from 28 to 34 to 37 degrees C, was inducible by Aroclor 1254 (a chemical inducer of cytochrome P450-dependent monooxygenase activity), and was reduced by EPN (an inhibitor of P450 activity).