Ethoxyresorufin O-deethylase Activity Research Articles

Application of a series of biomarkers in Scallop Chlamys farreri to assess the toxic effects after exposure to a priority hazardous and noxious substance (HNS)—Acrylonitrile

The antioxidant enzymes and detoxification parameters responses of the scallop Chlamys farreri to different degree of acrylonitrile (AN) were investigated. Accordingly, the median lethal concentration (LC50) at 96 h was 98.5 mg/L AN. Results from chronic toxicity test demonstrated that superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were stimulated in the highest two doses of AN (2.0 and 5.0 mg/L), but significantly inhibited in the highest concentration (5.0 mg/L) at the end of the exposure. The levels of DNA strand breaks, lipid peroxidation (LPO) and protein carbonyl (PC) contents showed damage effects exposed AN at the highest two doses. Additionally, AN significantly induced the enzymatic activity of glutathione-s-transferase (GST), related mRNA expression levels of P-glycoprotein (P-gp) and GST-pi; and no significant changes were found on CYP1A1 mRNA expression and ethoxyresorufin O-deethylase (EROD) activity. Our results indicated that P-gp and GST-pi mRNA expression in digestive glands of the scallop C. farreri may potentially be used in ecological risk assessment of hazardous and noxious substances (HNS) contamination of marine.

Historical Pollution of an Urban Watershed Based in Geochemical, Geoacumulation, and EROD Activity in PLHC-1 Analyses in Sediment Cores.

In the present study, the environmental changes over the past 100years in the Cambé River watershed in Southern Brazil were investigated. For this, a sediment core was collected to examine the distribution of metals, polycyclic aromatic hydrocarbons (PAHs), and ecotoxicological markers. The core corresponds from 1914 to 2012, which was obtained by the decay of 210Pb. Changes in the study area also were examined by: Geoaccumulation index (Igeo) and enrichment factor (EF), sediment quality guidelines (SGQs), and ethoxyresorufin-O-deethylase (EROD) activity in Poeciliopsis lucida hepatocellular carcinoma (PLHC-1) cells line. The Σ16 PAHs ranged from 242.6 to 40,775ngg-1, with pyrogenic source at the beginning of the core, which likely corresponds to the burning of forests to establish the city and the later use of fossil fuels from 1960 to 2012. In the 1930s, most of metals presented a concentration below the background. After the 1930s, high concentrations can be associated with the intense use of fertilizers (Cd, Cu, Zn, Hg) and increases in urban traffic (Zn and Pb). Igeo showed that the distribution of Cu is considered moderately to strongly polluted, and the EF of Pb was considered as moderately polluted. Statistical analyses showed a strong relation between the presence of the ANP, FLU, PHE, BaP, IND, and Bghi compounds and the induction of EROD activity, and no correlation with heavy metals. A prediction model for the linear regression was obtained between the ANP and BaP concentrations and the EROD activity, with an accuracy of 99%.

Integration of multi-tissue PAH and PCB burdens with biomarker activity in three coastal shark species from the northwestern Gulf of Mexico

Tissue-based burdens of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) were integrated with ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST) enzyme activity in bull (Carcharhinus leucas), blacktip (Carcharhinus limbatus), and bonnethead (Sphyrna tiburo) sharks from Galveston Bay, TX. The potential toxicity of these burdens was evaluated by calculation of toxic equivalents (TEQs). Concentrations of total PAHs (∑PAHs) were significantly greater in blacktip and bonnethead sharks than bull sharks in liver, but did not exhibit differences in muscle among species. Hepatic concentrations of ∑PAHs in these sharks (range of means: 1560–2200 ng/g wet wt.) were greater than concentrations previously reported in oysters from Galveston Bay (range of means: 134–333 ng/g dry wt.), which suggests that trophic dilution of PAHs may not be reflected in sharks. Total PCBs (∑PCBs) were significantly greatest in bull sharks and lowest in bonnetheads, while blacktips were intermediate to these species. EROD activity was greater in bonnetheads than the other species, whereas GST activity was significantly higher in blacktips and bonnetheads than in bull sharks. Integration of hepatic burdens with biomarker activity via constrained multivariate analysis found correlations for only a small number of individual PAH/PCB congeners. Hepatic TEQ measurements suggest potential physiological effects of these burdens compared to established TEQ thresholds for other taxa, although the likelihood of similar effects in sharks requires further study and the inclusion of toxic endpoints. Our findings indicate that sharks may be prone to the accumulation of PAHs and PCBs, which may result in negative health outcomes for these cartilaginous fishes.

Allometric scaling of hepatic biotransformation in rainbow trout

Biotransformation may substantially impact the toxicity and accumulation of xenobiotic chemicals in fish. However, this activity can vary substantially within and among species. In this study, liver S9 fractions from rainbow trout (4–400 g) were used to evaluate relationships between fish body mass and the activities of phase I and phase II metabolic enzymes. An analysis of log-transformed data, expressed per gram of liver (g liver−1), showed that total cytochrome P450 (CYP) concentration, UDP-glucuronosyltransferase (UGT) activity, and glutathione S-transferase (GST) activity exhibited small but significant inverse relationships with fish body weight. In contrast, in vitro intrinsic clearance rates (CLIN VITRO,INT) for three polycyclic aromatic hydrocarbons (PAHs) increased with increasing body weight. Weight normalized liver mass also decreased inversely with body weight, suggesting a need to express hepatic metabolism data per gram of body weight (g BW−1) in order to reflect the metabolic capabilities of the whole animal. When the data were recalculated in this manner, negative allometric relationships for CYP concentration, UGT activity, and GST activity became more pronounced, while CLIN VITRO,INT rates for the three PAHs showed no significant differences across fish sizes. Ethoxyresorufin O-deethylase (EROD) activity normalized to tissue weight (g liver−1) or body weight (g BW−1) exhibited a non-monotonic pattern with respect to body weight. The results of this study may have important implications for chemical modeling efforts with fish.

Selection of biochemical and physiological parameters in the croaker Micropogonias furnieri as biomarkers of chemical contamination in estuaries using a generalized additive model (GAM)

Biochemical and physiological parameters in the croaker Micropogonias furnieri were evaluated as biomarkers of chemical contaminants in estuaries. Juvenile croakers (10–20 cm total body length) were collected in summer and winter (2011 and 2012), in two sites at the Lagoa dos Patos estuary (southern Brazil). Fish were also collected in summer (2011 and 2012) in one site at the Barra do Chui estuary (southern Brazil). Tissue (gills, muscle or liver) samples were dissected and analyzed for contaminants [metals, polycyclic aromatic hydrocarbons (PAHs) and organochlorine pesticides (OCPs)] and selected biochemical and physiological parameters [metallothionein-like proteins (MTLP), acetylcholinesterase (AChE) activity, ethoxyresorufin-O-deethylase (EROD) activity, and lipid peroxidation (LPO)]. Additionally, water samples were collected for water chemical analyses (salinity, pH, temperature, dissolved oxygen, total alkalinity, and sulfate concentration). Data obtained were integrated and analyzed, using Principal Component Analysis (PCA) and Generalized Additive Model (GAM) approaches. Results showed that changes in concentrations of chemical contaminants and responses of biochemical and physiological parameters did not show any pattern according to the site, season and year of fish collection. However, they were influenced by fish body length and water temperature and salinity. Liver LPO and EROD activity were not responsive to PAHs. However, liver LPO was responsive to HCB, p,p-DDD, p,p-DDT and endosulfan sulfate. In turn, gill MTLP concentration, muscle AChE activity and liver EROD activity were responsive to non-essential metals (Pb, Cd and Ag). Considering that the ecotoxicological modeling approach adopted (GAM) accounted for biological, spatial and temporal variability of data associated with fish body size and site, season and year of fish collection, gill MTLP concentration, muscle AChE activity, as well as liver LPO and EROD activity can be considered as reliable biomarkers of fish exposure to chemical contaminants in estuaries.

Biomarker responses in fish exposed to polycyclic aromatic hydrocarbons (PAHs): Systematic review and meta-analysis

Biomarkers of antioxidant and biotransformation systems are commonly used to assess polycyclic aromatic hydrocarbons (PAHs) pollution in fish. Despite their extensive application of biomarkers, contradictory results are vastly reported in the literature, even for the same species in similar contamination scenarios. This study aims to verify response patterns of biomarkers in fish exposed to PAHs. Through systematic reviews and meta-analyses, we were able to evaluate: (i) overall magnitude of PAHs effects on biotransformation and oxidative stress biomarkers; (ii) patterns of response among experimental approaches (laboratory, field and active biomonitoring), environment (marine and freshwater) and fish habitat (pelagic, demersal, etc.); (iii) effects of exposure route, time and concentration of PAHs; and (iv) which biomarkers respond best to PAHs exposure. Overall, biomarker responses were significantly affected by PAHs exposure. The activities of ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST), superoxide dismutase (SOD), glutathione peroxidase (GPx) and levels of oxidized glutathione (GSSG) and lipid peroxide (LPO) significantly increased in fish exposed to PAHs, whereas catalase (CAT) and glutathione reductase (GR) activities and levels of reduced glutathione (GSH) were not affected. Amongst responsive biomarkers, EROD and GST activities significantly differed among approaches and between marine and freshwater environments, but were not affected by fish habitat. GSSG levels were higher in fish from laboratory bioassays compared to the field, but did not differ between environments nor habitats. Exposure route played a major role only for GST and GPx responses. Finally, increasing PAHs concentration and exposure time had a significant effect on all assessed biomarkers, except for CAT. We conclude that EROD and GST are robust biomarkers to assess PAHs effects in fish. Contrarily, CAT is an inadequate biomarker of PAHs exposure since no significant response was observed. Our study also highlighted some research gaps in PAHs contamination studies, such as a clear lack of active biomonitoring experiments.

Joint toxicity on hepatic detoxication enzymes in goldfish (Carassius auratus) exposed to binary mixtures of lead and paraquat

Compared to single exposure, chemical mixtures might induce joint toxicity including additive, synergistic and antagonistic effects on both organisms and environment. Owing to the specific toxicity of oxidative stress and binding to proteins, lead (Pb) is generally recognized a non-essential and threatening heavy metal to animals and human. Paraquat (PQ) is a widely used herbicide in agriculture and can trigger oxidative stress as well as Pb. Little information was available about joint effects of the two chemicals on toxicological responses in organisms, especially in fish. In our present study, goldfish (Carassius auratus) were randomly exposed to single and combined experiments with different concentrations of Pb and PQ for 28 days. Activities of four enzyme biomarkers in liver, ethoxyresorufin-O-deethylase (EROD), 7-benzyloxy-4-trifluoromethyl-coumarin-O-debenzyloxylase (BFCOD), glutathione-S-transferase (GST) and UDP-glucuronosyltransferase (UGT) were evaluated in each experimental group on day 14 and 28. The results showed four enzyme levels were markedly reduced with the increase of concentrations in mixtures and prolonged exposure. The inhibitory EROD and BFCOD activities were not significantly changed in goldfish following PQ-treated groups with or without 0.5 mg/L Pb, which indicated PQ has more inhibitory toxicity on CYP450 enzymes than Pb in co-exposure groups. However, the reduced values of GST were observed only in the combinations containing high doses of Pb or PQ during experimental periods. Although the responses of UGT activity were similar to GST on 14th day, all combinations of Pb and PQ generated stronger inhibitions on UGT activities compared to individual Pb and PQ-treated group. These results suggested that combined exposure of Pb and PQ have more inhibitory toxicity on phase I enzymes than phase II enzymes.

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.

Polycyclic aromatic hydrocarbon exposure impairs pre-migratory fuelling in captively-dosed Sanderling (Calidris alba)

Efficient fuelling is essential for migratory birds because fuel loads and fuelling rates affect individual fitness and survival during migration. Many migrant shorebirds are exposed to oil pollution and its toxic constituents, polycyclic aromatic hydrocarbons (PAHs), at migratory staging sites, which has the potential to interfere with avian refuelling physiology. In this study, we orally dosed shorebirds with environmentally-relevant PAH mixtures to simulate dietary exposure during staging. Forty-nine wild-caught Sanderling (Calidris alba) were exposed to 0 (control), 12.6 (low), 126 (medium), or 1260 (high) μg total PAH/kg body weight/day. Birds were dosed during a 21-day period of autumn pre-migratory fuelling to mimic the typical staging duration of Sanderling. We measured daily changes in mass and fat loads, as well as ethoxyresorufin-O-deethylase (EROD) activity, serum biochemical profiles, and liver mass and lipid content following dosing. All dose groups gained fat and increased in mass (size-corrected) during the study period, with females having a higher average body mass than males. However, mass gain was 3.9, 5.4, and 3.8 times lower in the low, medium, and high dose groups, respectively, relative to controls, and body mass in the medium and high dose groups significantly declined near the end of the experiment. EROD activity showed a dose-dependent increase and was significantly elevated in the high dose group relative to controls. Higher individual EROD activity was associated with reduced serum bile acid and elevated serum creatine kinase concentrations in both sexes, and with elevated serum lipase concentrations in females. These results suggest that PAH exposure in Sanderling can interfere with mechanisms of lipid transport and metabolism, can cause muscle damage, and can lead to reduced overall fat loads that are critical to staging duration, departure decisions, migratory speed, and flight range. Given that many shorebirds migrate thousands of kilometers between the breeding and wintering grounds and frequently aggregate at key staging sites that are subject to contamination, PAH exposure likely represents a significant threat to shorebird migratory success.

In vitro effects of brominated flame retardants, selected metals and their mixtures on ethoxyresorufin-O-deethylase activity in Mossambica tilapia liver

The in vitro effects of individual brominated flame retardants (BFRs), selected metals, and their binary mixtures on ethoxyresorufin-O-deethylase (EROD) activity were evaluated using a plate-reader method. The BFRs, 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47), decabromodiphenyl oxide (BDE-209), hexabromocyclododecane (HBCD), and tetrabromobisphenol A (TBBPA), were tested at doses ranging from 0.1 ng/L to 100 µg/L. Selected metals (Cu2+, Cd2+, Hg2+, and Zn2+) were screened at doses of 0.1 mg/L to 50 mg/L. The activity of EROD was significantly induced by TBBPA, BDE-209, and Zn2+, while HBCD, Cu2+, Cd2+, and Hg2+ decreased EROD activity. Moreover, following exposure to binary mixtures of metals and BFRs, the EROD activity dose-response curves were similar to those of the metals alone, indicating that EROD activity was governed by the metals.

Biomarkers in fish as a measure of the state of marine environment of Kuwait.

The health of a marine ecosystem can effectively be monitored by studying the levels of biomarkers in a representative species. A change in background level of a biomarker indicates exposure to a specific type of pollutants. It also identifies bioavailability and the organism response to the causative agent among the compounds present in the surrounding water body. Yellowfin seabream (Acanthopagrus latus), a local variety of fish, was examined for parent PAHs in the liver, its metabolites in bile by the GC-MS method as exposure biomarkers and cytochrome P4501A1 by assay of ethoxyresorufin-o-deethylase (EROD) in the liver as an effect biomarker. A comparison was made between fish collected in 2015 with the fish collected in 2005-2006 and stored at - 80°C in the fish bank. The objective was to examine the extent of changes in the environmental quality of the Kuwait marine area and the status of fish health concerning oil-related pollutants since Arabian Gulf is surrounded by oil-producing countries. Interestingly, insignificant differences between the liver PAH content and EROD activity were observed in fish over the sampling periods. The fish efficiently metabolized PAHs and excreted hydroxy-metabolites in bile. The study suggested that environmental quality of the Kuwait marine area was not deteriorated to any serious extent in the last decade and biomarkers can be used effectively in assessing the thrust of sub-optimal levels of various contaminants present in the marine area on the resident biota.

Pyrethroid insecticide lambda-cyhalothrin induces hepatic cytochrome P450 enzymes, oxidative stress and apoptosis in rats

This study aimed to examine in rats the effects of the Type II pyrethroid lambda-cyhalothrin on hepatic microsomal cytochrome P450 (CYP) isoform activities, oxidative stress markers, gene expression of proinflammatory, oxidative stress and apoptosis mediators, and CYP isoform gene expression and metabolism phase I enzyme PCR array analysis. Lambda-cyhalothrin, at oral doses of 1, 2, 4 and 8mg/kg bw for 6days, increased, in a dose-dependent manner, hepatic activities of ethoxyresorufin O-deethylase (CYP1A1), methoxyresorufin O-demethylase (CYP1A2), pentoxyresorufin O-depentylase (CYP2B1/2), testosterone 7α- (CYP2A1), 16β- (CYP2B1), and 6β-hydroxylase (CYP3A1/2), and lauric acid 11- and 12-hydroxylase (CYP4A1/2). Similarly, lambda-cyhalothrin (4 and 8mg/kg bw, for 6days), in a dose-dependent manner, increased significantly hepatic CYP1A1, 1A2, 2A1, 2B1, 2B2, 2E1, 3A1, 3A2 and 4A1 mRNA levels and IL-1β, NFκB, Nrf2, p53, caspase-3 and Bax gene expressions. PCR array analysis showed from 84 genes examined (P<0.05; fold change>1.5), changes in mRNA levels in 18 genes: 13 up-regulated and 5 down-regulated. A greater fold change reversion than 3-fold was observed on the up-regulated ALDH1A1, CYP2B2, CYP2C80 and CYP2D4 genes. Ingenuity Pathway Analysis (IPA) groups the expressed genes into biological mechanisms that are mainly related to drug metabolism. In the top canonical pathways, Oxidative ethanol degradation III together with Fatty Acid α-oxidation may be significant pathways for lambda-cyhalothrin. Our results may provide further understanding of molecular aspects involved in lambda-cyhalothrin-induced liver injury.

Markers of oil exposure in cold-water benthic environments: Insights and challenges from a study with echinoderms

In spite of increasing naval activities and petroleum exploration in cold environments, there is currently a paucity of tools available to monitor oil contamination in boreal marine life, especially in sedentary (non-fish) species that dominate benthic communities. This research aimed to identify biotic sources of variation in biomarkers using subarctic echinoderms, and to identify suitable biomarkers of their exposure to hydrocarbons. The focal species included the sea star Asterias rubens, the brittle star Ophiopholis aculeata, the sea urchin Strongylocentrotus droebachiensis, and the sea cucumber Cucumaria frondosa, which are among the most abundant echinoderms in the North Atlantic and Arctic Oceans. The latter two species are also commercially exploited. A series of 96-h acute exposures of the water-accommodating fraction (WAF) of used lubricating oil (ULO) were performed in different seasons (i.e. distinct reproductive stages). Digestive and reproductive tissues were analyzed for baseline and response levels of glutathione peroxidase (GPx) and ethoxyresorufin-O-deethylase (EROD). GPx activity was detected in the pyloric caecum, stomach, and gonad of sea stars, the intestine and gonad of sea cucumbers, and the gonad of brittle stars and sea urchins. No seasonal variation in baseline GPx activity occurred. Upon exposure to the ULO WAF, sex-based differences were elicited in the GPx activity of sea star stomachs (lower in females than males). EROD activity was present in the pyloric caeca of sea stars, and the gonads of brittle stars and sea urchins. An interaction between season and sex on baseline EROD activity was measured in the gonads of sea urchins. Ovaries exhibited significant seasonal variation in EROD activity and had greater activity than testes during the spawning and post-spawning seasons. Seasonal variation in EROD activity also occurred in sea star pyloric caeca and brittle star gonads. Furthermore, testes of sea urchins exposed to the ULO WAF exhibited suppressed EROD activity compared to baseline levels. The nearly universal presence of GPx activity highlights its potential as a useful biomarker, while EROD activity was much more limited. Findings suggest a complex relationship between temporal and biotic factors on both the baseline and response levels of enzymatic activity, emphasizing the need to consider sex and sampling season in studies of biomarkers of hydrocarbon exposure in boreal indicator species that display annual reproductive cycles.

SEGMENTAL ARTERIAL MEDIOLYSIS: A COMMON MISDIAGNOSIS AND UNDER-RECOGNIZED PHENOMENON

Indole-3-carbinol (I3C) is a secondary plant metabolite produced in vegetables of the Brassica genus, including cabbage, cauliflower, and brussels sprouts. I3C is both an anti-initiator and a promoter of carcinogenesis. Consumption of I3C by humans and rodents can lead to marked increases in activities of cytochrome P-450-dependent monooxygenases and in a variety of phase II drug-metabolizing enzymes. We have reported previously that the enzyme-inducing activity of I3C is mediated through a mechanism requiring exposure of the compound to the low-pH environment of the stomach. We report here the aromatic hydrocarbon responsiveness-receptor Kd values (22 nM-90 nM), determined with C57BL/6J mouse liver cytosol and the in vitro- and in vivo-molar yields (0.1-6%) of the major acid condensation products of I3C. We also show that indolo[3,2-b]carbazole (ICZ) is produced from I3C in yields on the order of 0.01% in vitro and, after oral intubation, in vivo. ICZ has a Kd of 190 pM for aromatic hydrocarbon responsiveness-receptor binding and an EC50 of 269 nM for induction of cytochrome P4501A1, as measured by ethoxyresorufin O-deethylase activity in murine hepatoma Hepa 1c1c7 cells. The binding affinity of ICZ is only a factor of 3.7 x 10(-2) lower than that of the highly toxic environmental contaminant and cancer promoter 2,3,7,8-tetrachlorodibenzo-p-dioxin. ICZ and related condensation products appear responsible for the enzyme-inducing effects of dietary I3C.

Chromium accumulation and biomarker responses in the Neotropical fish Prochilodus lineatus caged in a river under the influence of tannery activities

In order to assess the effects of tannery effluents (TE) in organism health, juveniles of Prochilodus lineatus were submitted to in situ tests at four different river locations: site A – upstream of the tannery; site B – next to the tannery; and sites C and D – downstream of the tannery. After 96 h exposure in the river, samples of fish tissue, river water and sediment were collected in order to quantify chromium (Cr) concentrations. Tissue samples were used to assess the activity of ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST), the content of glutathione (GSH) and metallothionein (MT) and the occurrence of lipid peroxidation (LPO) and DNA damage. Higher Cr concentrations were detected in the water and sediments from site B and in the liver of fish confined at site B, compared to the other sites. Fish caged at site B demonstrated higher levels of liver MT and hepatic EROD activity in relation to fish caged at the other sites. Moreover, fish from site B presented increased liver and branchial GST activities, as well as more GSH in the liver, than fish from site A. There were no significant variations in the occurrence of LPO and DNA damage among fish caged at the different sites. Thus, TE increased Cr levels in the water, sediments, and fish livers and stimulated the synthesis of MT and GSH and the activities of EROD and GST. In conclusion, TE affect the quality of the river and promote changes in biochemical biomarkers and Cr accumulation in P. lineatus.

Hepatic phase I and II biotransformation responses and contaminant exposure in long-finned pilot whales from the Northeastern Atlantic

Faroe Island pilot whales have been documented to have high body burdens of organohalogen contaminants (OHCs), including polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs), but low burdens of their respective hydroxylated metabolites (OH-PCBs and OH-PBDEs). The present study investigated the hepatic expression and/or catalytic activities of phase I and II biotransformation enzymes in relation to hepatic concentrations of target OHCs, including OH-PCBs and OH-PBDEs, in long-finned pilot whales (Globicephala melas) from the Northeastern Atlantic. CYP1A, 2B, 2E and 3A protein expressions were identified in juveniles and adult males, but not in adult females. Ethoxyresorufin-O-deethylase (EROD) activity was significantly lower in adult females than in juveniles and adult males. Using multivariate analyses to investigate relationships between biological responses and OHC concentrations, a positive relationship was identified between EROD and OHCs. The activity levels of phase II conjugating enzymes (uridine 5′-diphospho-glucuronosyltransferase [UDPGT], and glutathione S-transferase [GST]) were low. The analyses of mRNA expression did not show correlative relationships with OHC concentrations, but cyp1a and ahr transcripts were positively correlated with EROD activity. We suggest that the low concentrations of OH-PCBs and OH-PBDEs reported in pilot whales is probably due to the identified low phase I biotransformation activities in the species.

Comparative live-imaging of in vivo EROD (ethoxyresorufin-O-deethylase) induction in zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) embryos after exposure to PAHs and river sediment extracts

The measurement of EROD (ethoxyresorufin-O-deethylase) activity to determine the induction of CYP1A after exposure to dioxin-like substances is a well-established biomarker in fish. For reasons of animal welfare and implementations of new chemicals regulations (REACh), in vivo methods using zebrafish (Danio rerio) and medaka (Oryzias latipes) embryos have recently been developed to quantify CYP1A induction, which is visualized as mean intensity of the autofluorescent resorufin formed in living anaesthetized embryos.In the present study, concentration ranges of three PAHs (benzo[a]pyrene, β-naphthoflavone, benzo[k]fluoranthene) as examples of known CYP1A inducers as well as extracts of two well-characterized sediment samples of the lower Neckar river (Southern Germany) were used to determine the suitability of the fathead minnow (Pimephales promelas) embryo for the in vivo EROD assay. Data for zebrafish embryos were generated for comparison. Fathead minnow embryos were principally suitable to show in vivo EROD induction via live-imaging. Since in fathead minnow embryos both signal area and fluorescence intensities are lower than in zebrafish embryos, the induction potentials of the three model PAHs and the environmental samples proved to be species-dependent.Among the three PAHs tested, benzo[k]fluoranthene lead to the strongest EROD signal followed by β-naphthoflavone and benzo[a]pyrene in comparison to the positive control. Whereas benzo[k]fluoranthene and β-naphthoflavone showed a dose-response relationship for the EROD induction, benzo[a]pyrene failed to induce a significant signal in fathead minnow embryos. If compared to the model PAHs, the extracts of both sediments from the lower Neckar River induced stronger EROD signals in both fathead minnow and zebrafish embryos. Observations thus documented fathead minnow embryos to be as suitable for biomonitoring purposes as are zebrafish embryos.

Methylated polycyclic aromatic hydrocarbons and/or their metabolites are important contributors to the overall estrogenic activity of polycyclic aromatic hydrocarbon-contaminated soils.

In the present study 42 polycyclic aromatic compounds (PACs) were investigated for their estrogenic potential using the VM7Luc4E2 transactivation assay. Relative potencies were determined for mass-balance analysis. In addition, compounds were tested in combination with the estrogen receptor (ER) antagonist ICI182,780 (ICI) and the aryl hydrocarbon receptor antagonist/CYP1A1 inhibitor α-naphthoflavone. Luciferase induction and CYP1A1-dependent ethoxyresorufin-O-deethylase (EROD) activity were measured to assess whether the estrogenic activity was elicited by the compound itself and/or by its metabolites. Relative potencies ranged between 10-7 and 10-4 . The ability of ICI to decrease luciferase activity stimulated by all compounds indicated that the induction responses were ER-dependent. The aryl hydrocarbon receptor antagonist/CYP1A1 inhibitor α-naphthoflavone decreased luciferase induction and EROD activity by several compounds, including the methylated chrysenes, suggesting that metabolites of these chemicals contributed to ER activation. Several PACs, such as acridine and its derivatives, appear to directly activate the ER. Furthermore, extracts of soils from industrial areas were examined using this bioassay, and estrogenic activity was detected in all soil samples. Mass-balance analysis using a combination of relative potencies and chemical analysis of the samples suggested that polycyclic aromatic hydrocarbons (PAHs) and alkylated PAHs, such as 1- and 3-methylchrysene, are important contributors to the overall estrogenic activity. However, these results revealed that a considerable proportion of the estrogenic activity in the soil remained unexplained, indicating the presence of other significant estrogenic compounds. Environ Toxicol Chem 2018;37:385-397. © 2017 SETAC.

Using cell apoptosis, micronuclei and immune alternations as biomarkers of phenanthrene exposure in yellowfin seabream (Acanthopagrus latus)

In the present study, the apoptosis and tissue changes in the spleen, as well as humoral immune-related parameters, micronuclei (MN) induction in blood cells and Ethoxyresorufin-O-deethylase (EROD) activity were investigated in yellowfin seabream (Acanthopagrus latus) after short-term exposure to phenanthrene (Phe). The fish were intraperitoneally injected with different concentrations (2, 20 and 40 mg kg−1) of Phe and tissue and blood samples were collected 1, 4, 7 and 14 days after injection. The concentrations of Phe in the fish liver increased 4 days after the experiment. EROD activity showed a pattern consistent with Phe concentration in the liver. Apoptotic index in the spleen increased dose dependently in Phe-exposed fish. Exposure to Phe caused significant decrease in the plasma level of immunoglobulin M, phagocytic and respiratory burst activity after 4 days of exposure. The frequency of MN in the erythrocytes of the treated fish was significantly higher than control. The main pathological alterations in the spleen included the increase in melanomacrophage centers (MMCs), destroyed red blood cell and hemorrhage. The degree of tissue changes in the spleen of the exposed fish ranged from slight to moderate damage. The size and number of MMCs in the spleen were significantly higher in Phe-treated fish compared to the control. Our results showed that Phe could suppress immune responses in fish, induce cell apoptosis, histological changes in the spleen and MN formation. This may suggest those parameters consider as useful biomarkers for monitoring of the health status of fish during exposure to Phe.

Responses of immune organs after single-dose exposure to phenanthrene in yellowfin seabream (Acanthopagrus latus): CYP1A induction and oxidative stress

The effect of phenanthrene (Phe) on induction of ethoxyresorufinO-deethylase (EROD) activity and oxidative stress was examined in immune organs of yellowfin seabream Acanthopagrus latus. Fish were treated with a single intraperitoneal injection at 2, 20, or 40 mg kg−1. The Phe concentration in spleen, EROD activity, superoxide dismutase (SOD) and catalase (CAT) activity, ascorbic acid (AsA), total glutathione (GSH), lipid peroxidation (LPO), and protein carbonylation (PC) levels in spleen and head kidney were assessed at one, four, seven, and 14 days post-injection. Dose response relationship was explored for data on day four. Phe concentration reached the highest observed level on day four, showed decline on day seven, and was undetectable at the end of trial. EROD activity in both organs followed the pattern of Phe level in all treated groups. Catalase and SOD activity at low Phe concentrations was significantly higher than controls, while LPO and PC level showed no differences from controls. In contrast, at 20 and 40 mg kg−1, CAT and SOD activity, an indicator of oxidative stress, was significantly lower than in untreated controls, while AsA, GSH, LPO, and PC levels were higher on days 4 and 7. No parameter of any treatment group in either organ tissue showed difference from control values at the end of the experiment. The SOD and CAT activity in both organs exhibited a biphasic (initial stimulatory effect) effect, whereas other parameters showed a monophasic effect in terms of dose-response. Results suggest that Phe induced CYP1A and antioxidant responses in immune organs.