Ethoxyresorufin O-deethylase Activity Research Articles

Identification of biomarkers responsive to chronic exposure to pharmaceuticals in target tissues of Carcinus maenas

A 28-day bioassay was performed with Carcinus maenas to evaluate chronic effects caused by exposure to caffeine and ibuprofen (0.1–50 μg L−1) in sea water. Lysosomal membrane stability (LMS) was evaluated in hemolymph applying the neutral red retention assay (NRRA); several biomarkers including ethoxyresorufin O-deethylase (EROD), dibenzylfluorescein dealkylase (DBF), glutathione S-transferase (GST), glutathione peroxidase (GPX), lipid peroxidation (LPO) and DNA damage were studied in gill, hepatopancreas, muscle and gonad tissues. In crabs exposed to environmental concentrations of the drugs, retention time was reduced by 50%. EROD and DBFOD activities were induced by caffeine in muscle and hepatopancreas tissues (p < 0.05); GST activity was activated by ibuprofen in gill, hepatopancreas and muscle at the highest concentrations tested (p < 0.05). All tissues showed GPX activity and LPO induction (p < 0.05). Crabs exposed to caffeine and ibuprofen showed evidence of DNA damage mainly in hepatopancreas tissues (p < 0.05). Environmental concentrations of pharmaceuticals induce LMS and the biochemical responses studied in this crab. This methodology is a suitable technique for assessing pharmaceutical toxicity in the marine environment.

Membrane Phospholipid Augments Cytochrome P4501a Enzymatic Activity by Modulating Structural Conformation during Detoxification of Xenobiotics

Cytochrome P450 is a superfamily of membrane-bound hemoprotein that gets involved with the degradation of xenobiotics and internal metabolites. Accumulated body of evidence indicates that phospholipids play a crucial role in determining the enzymatic activity of cytochrome P450 in the microenvironment by modulating its structure during detoxification; however, the structure-function relationship of cytochrome P4501A, a family of enzymes responsible for degrading lipophilic aromatic hydrocarbons, is still not well defined. Inducibility of cytochrome P4501A in cultured catfish hepatocytes in response to carbofuran, a widely used pesticide around the world, was studied earlier in our laboratory. In this present investigation, we observed that treating catfish with carbofuran augmented total phospholipid in the liver. We examined the role of phospholipid on the of cytochrome P4501A-marker enzyme which is known as ethoxyresorufin-O-deethylase (EROD) in the context of structure and function. We purified the carbofuran-induced cytochrome P4501A protein from catfish liver. Subsequently, we examined the enzymatic activity of purified P4501A protein in the presence of phospholipid, and studied how the structure of purified protein was influenced in the phospholipid environment. Membrane phospholipid appeared to accelerate the enzymatic activity of EROD by changing its structural conformation and thus controlling the detoxification of xenobiotics. Our study revealed the missing link of how the cytochrome P450 restores its enzymatic activity by changing its structural conformation in the phospholipid microenvironment.

Inhibitory Potentials of Five Phytoestrogens from Pueraria candollei var. mirifica on CYP1A1 and CYP1A2 Proteins in Mouse Liver Microsomes and in silico

Pueraria candollei var. mirifica (PM) is a Thai traditional medicinal plant for rejuvenation and estrogen replacement therapy in menopausal women. CYP1A1 and CYP1A2 proteins are the members of hepatic cytochrome P450 (CYP) enzymes to activate a procarcinogen, in which ethoxyresorufin O -deethylase (EROD) and methoxyresorufin O -demethylase (MROD) activities are the specific markers for CYP1A1 and CYP1A2, respectively. In the present study, the effects of five phytoestrogens isolated form the bark of PM tuberous roots namely miroestrol, deoxymiroestrol, khawkhurin, isomiroestrol, and methoxyisomiroestrol, on EROD and MROD activities were examined in mouse hepatic microsomes, compared to a typical CYP1A1/2 inducer and substrate beta-napthoflavone (BNF). The bindings of these five compounds to either CYP1A1 or CYP1A2 enzymes were analyzed using molecular docking with homology modeling technique. Rank of the median inhibitory concentration (IC 50 ) of these compounds on EROD activity corresponded to that of MROD, namely BNF > miroestrol > kwakhurin > deoxymiroestrol > methoxyisomiroestrol> isomiroestrol, respectively. Interestingly, the binding pose energy of these compounds to CYP1A1 and CYP1A2 proteins were consistent to those of inhibitory effects on EROD and MROD activities. The observations suggested for the first time that the active phytoestrogens from PM possessed inhibitory potentials on CYP1A1 and CYP1A2 via EROD and MROD activities, respectively. Furthermore, the binding energy of the compounds to CYP1A1 and CYP1A2 proteins might be a useful tool to predict the effects of a compound on these two CYP enzymes.

Structural Requirements for Potent Direct Inhibition of Human Cytochrome P450 1A1 by Cannabidiol: Role of Pentylresorcinol Moiety

Our recent work has shown that cannabidiol (CBD) exhibits the most potent direct inhibition of human cytochrome P450 1A1 (CYP1A1) among the CYP enzymes examined. However, the mechanism underlying this CBD inhibition remains to be clarified. Thus, to elucidate the structural requirements for the potent inhibition by CBD, the effects of CBD and its structurally related compounds on CYP1A1 activity were investigated with recombinant human CYP1A1. Olivetol, which corresponds to the pentylresorcinol moiety of CBD, inhibited the 7-ethoxyresorufin O-deethylase activity of CYP1A1; its inhibitory effect (IC50=13.8 µM) was less potent than that of CBD (IC50=0.355 µM). In contrast, d-limonene, which corresponds to the terpene moiety of CBD, only slightly inhibited CYP1A1 activity. CBD-2'-monomethyl ether (CBDM) and CBD-2',6'-dimethyl ether inhibited CYP1A1 activity with IC50 values of 4.07 and 23.0 µM, respectively, indicating that their inhibitory effects attenuated depending on the level of methylation on the free phenolic hydroxyl groups in the pentylresorcinol moiety of CBD. Cannabidivarin inhibited CYP1A1 activity, although its inhibitory potency (IC50=1.85 µM) was lower than that of CBD. The inhibitory effects of Δ(9)-tetrahydrocannabinol and cannabielsoin (IC50s ≈10 µM), which contain a free phenolic hydroxyl group and are structurally constrained, were less potent than that of CBDM, which contains a free phenolic hydroxyl group and is rotatable between pentylresorcinol and terpene moieties. These results suggest that the pentylresorcinol structure in CBD may have structurally important roles in direct CYP1A1 inhibition, although the whole structure of CBD is required for overall inhibition.

Contrasting retinoid and thyroid hormone status in differentially-contaminated northern fulmar colonies from the Canadian Arctic, Svalbard and the Faroe Islands

The northern fulmar (Fulmarus glacialis) has previously been shown to accumulate a wide range, and occasionally high concentrations of organochlorines (OCs) (e.g., PCBs, chlorobenzenes, DDT- and chlordane-related compounds, dioxins and furans). The present study aimed to investigate, using a meta-analysis approach, the variations in cytochrome P450 (CYP) 1A-like enzyme induction based on ethoxyresorufin O-deethylase activity (EROD) and selected physiological variables (retinoids and thyroid hormones) in northern fulmar breeding in three differentially OC-exposed populations: Nunavut (Canadian Arctic), Svalbard (Norwegian Arctic) and the Faroe Islands. Substantially higher (roughly two-fold) OC levels were uncovered in the liver of this long-lived fulmarine petrel breeding in the Faroe Islands relative to Svalbard and Nunavut. Liver levels of PCDDs, PCDFs and non-ortho PCBs in Faroe Islands fulmars were amongst the highest reported thus far in any seabirds from the northern regions. Positive correlations were depicted in combined fulmars (all three populations) between hepatic EROD activity and concentrations of OCs, in which strongest associations were found for dioxin-like compound (PCDFs and PCDDs) and TEQ concentrations. Moreover, moderate to strong positive correlations were found between liver OC concentrations and plasma total thyroxin (TT4) levels and TT4/total triiodothyronine (TT3) level ratios, as well as strong negative correlations between the same suite of OCs and plasma TT3 levels. Hepatic OC concentrations (PCBs, PCDDs, PCDFs, HCB, p,p'-DDE and oxychlordane) also were positively correlated with hepatic retinyl palmitate levels which, in turn, were associated with a significant decrease in plasma retinol levels and somewhat unchanged liver retinol levels. The present meta-analysis investigation on northern fulmar breeding in three geographically-distant sites illustrated that OC exposure (mainly PCBs and dioxins/furans) may be associated with modulation of the thyroid and retinoid homeostasis. However, the impact of confounding environmental factors (e.g., temperature and nutritional status) on current physiological variable variations could not be ruled out, and thus any cause-effect linkages between thyroid and retinoid system perturbation and OC exposure cannot be ascertained.

Relative potency of PCB126 to TCDD for sublethal embryotoxicity in the mummichog (Fundulus heteroclitus)

The relative potency (ReP) of 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for sublethal responses was assessed in Fundulus heteroclitus embryos. Eggs were treated with intravitelline injections of graded sublethal doses of PCB126 (312–5000pgg−1 wet weight, ww) or TCDD (5–1280pgg−1 ww). At 16 days post-fertilization (DPF), craniofacial deformities were observed in larvae hatched from eggs treated with the two highest doses of PCB126 (2500–5000pgg−1 ww). Both compounds caused a dose-responsive reduction of larval growth and prey capture ability (at ≥1250pgg−1 ww), and induction of ethoxyresorufin-O-deethylase (EROD) activity (at ≥80pgg−1 ww). The dose–response relationships for EROD activity for PCB126 and TCDD had similar slopes and the ReP of PCB126 to TCDD for EROD activity was estimated at 0.71. This is 140-fold higher than the World Health Organization (WHO) TCDD equivalency factor (TEF) of PCB126 for fish (0.005), which is based on rainbow trout (Oncorhynchus mykiss) embryolethality data. The slope of the dose–response relationship for prey capture ability for PCB126 was steeper than for TCDD, suggesting different mechanisms of action. Expression levels of several genes were also studied by quantitative real-time polymerase chain reaction (qPCR) following exposure to single doses of TCDD or PCB126 (1280 and 1250pgg−1 ww, respectively) causing similar EROD induction. A different pattern of responses was observed between PCB126 and TCDD: PCB126 appeared to induce antioxidant responses by inducing sod2 expression, while TCDD did not. These results suggest that relative potencies are species-specific and that the current ReP for PCB126 underestimates its toxicity for some fish species. It is recommended to develop species-specific RePs for a variety of sublethal endpoints and at environmentally relevant doses.

Effect of in situ exposure history on the molecular responses of freshwater bivalve Anodonta anatina (Unionidae) to trace metals

The goal of the study was to assess the adequacy of molecular responses in mollusks in relation to their in situ exposure history. Freshwater male bivalve mollusks Anadonta anatina (Unionidae) from polluted (A) and unpolluted (F) sites were subjected to 14 days of exposure to copper (Cu2+, 10μgL−1), zinc (Zn2+, 130μgL−1) or cadmium (Cd2+, 15μgL−1). The comparison of two control groups showed that the specimens from site A had higher levels of Cu, Zn and Cd and metallothionein (measured both through metal (MT-Me), and protein (MT-SH) levels) in the tissues. Cytotoxicity (low lysosomal membrane stability), low glutathione level, high antioxidant and apoptotic enzymes activities, lipid and protein oxidative injury, depletion of ethoxyresorufin-O-deethylase (EROD) in digestive gland, high vitellogenin-like protein (Vtg-LP) concentration in gonads confirmed the effect of toxic environment on this group.Exposures provoked increased number of hemocytes with micronuclei (by 100–500%) and nuclear abnormalities (by 50–400%) (genotoxicity), elevation of caspase-3 (in 1.5–10 times) and/or Vtg-LP (by 70–310%) levels in all groups. However, the responses were strongly dependent on the origin of mussels. Exposed mussels from site F demonstrated typical for the effect of toxic metals elevation of MT-SH (by 100–380%) and MT-Me (up to seven times) levels and accumulation of metals (with a few exceptions) in the tissues. Conversely, in the mussels inhabiting site A, exposures caused the decrease of metal (by 37% for Cu, by 62% for Zn, by 50% for Cd), MT-SH (by 68% in ZnA group) and MT-Me (by 50–68%) levels. That was accompanied with increase of cytotoxicity and EROD activity (by 144–240%). High level of protein carbonyls was the distinguished feature of all groups from site A. Hence, despite high efficiency of metal detoxification and oxidative stress responses in the mussels, in the specimens from spontaneously polluted site they were impaired.

How flood events affect rainbow trout: Evidence of a biomarker cascade in rainbow trout after exposure to PAH contaminated sediment suspensions

Increasing frequency and intensity of flood events are major concerns in the context of climate change. In addition to the direct hydrological implications of such events, potential ecotoxicological impacts are of increasing interest. It is vital to understand mechanisms of contaminant uptake from suspended particulate matter (SPM) and related effects in aquatic biota under realistic conditions. However, little is known about these processes. Due to recent changes in climate, during summer temperatures of German rivers frequently exceed 25°C. Effects of re-suspension of sediments on biota under elevated temperature regimes are likely to differ from those under lower temperature regimes. To elucidate this differential response of aquatic vertebrates, rainbow trout were exposed to suspensions of sediment from the Rhine River that was spiked with a mixture of polycyclic aromatic hydrocarbons (PAH). The experiments were conducted under two different temperature regimes (24°C or 12°C). Physicochemical parameters, including concentration of PAHs in SPM, and biomarkers in fish (biliary PAH metabolites, 7-ethoxyresorufin O-deethylase activity, lipid peroxidation (LPO), mRNA expression of some genes and micronuclei) were measured over the course of a 12d study. Concentrations of pyrene and phenanthrene decreased over time, while no decrease was observed for chrysene and benzo[a]pyrene. The biomarker cascades, more specifically the temporal dynamics of biomarker reactions, did not only show quantitative differences (i.e. different induction intensity or rate of biomarker responses) at the two temperatures but also qualitative differences, i.e. different biomarker responses were observed. A slight significant increase of biliary metabolites in fish was observed in un-spiked sediment at 24°C. In bile of fish exposed to PAH spiked sediment concentrations of 1-hydroxypyrene and 1-hydroxyphenanthrene increased significantly during the first two days, and then decreased. At 12°C uptake of PAHs was slower and maximum metabolite concentrations in bile were less than in fish exposed at 24°C. Following a latency of two days, concentrations of PAH metabolites in bile of fish exposed at 24°C were followed by a peak in LPO. PAHs spiked into sediments under laboratory conditions were significantly more bioavailable than the PAHs that were already present in un-spiked field-collected sediments.

A simple and reliable in vivo EROD activity measurement in single Fundulus heteroclitus embryo and larva

Dioxin-like compounds (DLC) induce toxic responses in early life stages of fish through activation of the aryl hydrocarbon receptor (AhR) which is frequently assessed by ethoxyresorufin-O-deethylase (EROD) activity. A novel spectrofluorimetric method was developed to quantitatively assess EROD activity in individual living embryos and prolarvae of a marine model fish species, the mummichog Fundulus heteroclitus. This in vivo method is based on the measurement of the production of resorufin by single live embryos or prolarvae after 5 h incubation with ethoxyresorufin. Freshly fertilized eggs were treated topically from 2.5 to 50 pg egg−1, with 3,3′,4,4′,5-pentachlorobiphenyl (PCB126), a prototypical DLC. EROD activity was assessed in embryos (7 days post-fertilization) and prolarvae (16 days post-fertilization). Resorufin was measured both in the culture medium (25‰ seawater) and in whole fish homogenates, to assess the percentage retained in the body. Approximately 95% and 17% of the resorufin produced in vivo was retained in embryos and prolarvae respectively. EROD activity in homogenates of embryos and in the culture medium of prolarvae increased linearly with dose. EROD activity measured by the in vivo method was highly correlated to that measured by a traditional in vitro technique using S9 fractions for both embryos and prolarvae. Both in vivo and in vitro EROD activity were higher in prolarvae than in embryos pretreated with PCB126. EROD induction measured in prolarvae by the in vivo and in vitro methods were similar whereas higher induction was measured in vivo than in vitro in embryos. The in vivo method was more sensitive and as reliable as the in vitro technique, and required a lower number of fish (4 compared to 3 pools of 5). This in vivo method is useful to link EROD induction in individual embryos or prolarvae to other organism-level responses. Further studies with other categories of xenobiotics should be performed to assess potential toxic effects on resorufin absorption/excretion processes which could affect in vivo measurements.

Cytochrome P4501A induction in avian hepatocyte cultures exposed to polychlorinated biphenyls: Comparisons with AHR1-mediated reporter gene activity and in ovo toxicity

Avian-specific toxic equivalency factors (TEFs) were developed by the World Health Organization to simplify environmental risk assessments of dioxin-like compounds (DLCs), but TEFs do not account for differences in the toxic and biochemical potencies of DLCs among species of birds. Such variability may be due to differences in species sensitivity to individual DLCs. The sensitivity of avian species to DLCs was recently associated with the identity of amino acids 324 and 380 in the aryl hydrocarbon receptor 1 (AHR1) ligand binding domain. A luciferase reporter gene (LRG) assay, measuring AHR1-mediated induction of a cytochrome P450 1A5 (CYP1A5) reporter gene, in combination with a species' AHR1 ligand binding domain sequence, were also shown to predict avian species sensitivity to polychlorinated biphenyls (PCBs) and PCB relative potency in a given species. The goals of the present study were to (1) characterize the concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and PCBs 126, 77, 105 and 118 on induction of ethoxyresorufin O-deethylase (EROD) activity and CYP1A4/5 mRNA in chicken, ring-necked pheasant and Japanese quail embryo hepatocytes and (2) compare these in vitro results to those previously generated by the LRG assay and in ovo toxicity studies. EROD activity and CYP1A4/5 mRNA expression data support and complement the findings of the LRG assay. CYP1A enzyme activity and mRNA expression were significantly correlated both with luciferase activity and in ovo toxicity induced by PCBs. Relative potency values were generally similar between the LRG and EROD assays and indicate that the relative potency of some PCBs may differ among species.

Assessment of toxic effects of triclosan on the swordtail fish (Xiphophorus helleri) by a multi-biomarker approach

The toxic effects of triclosan (TCS) on the swordtail fish (Xiphophorus helleri) were assessed based on various biomarkers including enzymatic activities of ethoxyresorufin O-deethylase (EROD), erythromycin N-demethylase (ERND) and glutathione-s-transferase (GST) and mRNA expression levels of CYP1A, CYP3A, glutathione S-transferase (GST) and P-glycoprotein (P-gp). The acute toxicity test showed the LC50 value of 1.47mgL−1 for TCS. The mRNA expressions of CYP1A, CYP3A, GST and P-gp showed dose–effect relationships in female swordtail fish when exposed to TCS, These mRNA expression levels were found more sensitive to TCS exposure than the enzymatic activities of EROD, ERND and GST do. In addition, the male fish displayed higher gene expression levels and more dramatic changes in enzyme activities than the females did. Our data further demonstrated that TCS was a typical inducer to Phase I and Phase II metabolism enzymes and genes, suggesting it is a potential ecotoxicological risk to aquatic ecosystems.

Effluent from drug manufacturing affects cytochrome P450 1 regulation and function in fish

We have previously reported very high concentrations of pharmaceuticals in the effluent from a treatment plant receiving wastewater from about 90 bulk drug manufacturers near Hyderabad, India. The main objective of the present study was to examine how high dilutions of this effluent affect mRNA expression of cytochrome P450 (CYP) 1 family genes and ethoxyresorufin O-deethylase (EROD) activity in exposed wildlife, using the three-spined stickleback (Gasterosteus aculeatus) as a model. In gill filaments exposed to diluted effluent ex vivo, EROD activity was strongly inhibited in a concentration-dependent manner. In a subsequent in vivo study, groups of fish were exposed (24h) to three concentrations of effluent, 0.8%, 1.6% or 3.2%. In this experiment, EROD in gills was induced 27-, 52- or 60-fold, respectively. Accordingly, CYP1A mRNA was markedly up-regulated in gill, liver and brain of fish exposed to all three effluent concentrations. Expression of mRNA for CYP1B1 and CYP1C1 was induced in gills at all concentrations while effects on these genes in liver and brain were weak or absent. The results of a time course study suggested that most CYP1-inducing substances in the effluent were readily metabolised or excreted, because the induced EROD activity and mRNA expression decreased when the fish were transferred to clean water. Considering that CYP1 enzymes play important roles in biotransformation of endogenous and foreign compounds, the observed dual effect of the effluent on CYP1 catalytic activity and mRNA expression suggests that multiple physiological functions could be affected in exposed wildlife.

Immunotoxicity and hepatotoxicity of PFOS and PFOA in tilapia (Oreochromis niloticus)

Perfluorinated organic compounds (PFOCs) are emerging persistent organic pollutants (POPs) widely distributed in the environment, wildlife and human. We studied the toxicology of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) on immunotoxicity and hepatotoxicity in primarily cultured hepatocytes of freshwater tilapia (Oreochromis niloticus). Cultured hepatocytes were exposed to PFOS or PFOA (0, 10, 20 and 30 mg/L) for 24, 48, 72, 96, 120 and 144 hours, respectively. Oral doses of these compounds that induce significantly detectable immunotoxicity and hepatotoxicity were employed in the study. In response to PFOS, the leukocytes, B cells, granulocytes, and macrophages among the isolation of intrahepatic immune cells (IHIC) from PFOS-treated tilapia produced significant levels of immune cells compared with that of the control group. The numbers of leukocytes, B cells, granulocytes, and macrophages for PFOS-treated tilapia increased with the incremental exposure concentration. Moreover, similar to the findings in PFOA toxicity effects, the erythropoietin levels in tilapia increased with the increase of the PFOS and PFOA concentrations. The lowest doses (10 mg/L) of PFOS exposure led to a marked inhibition in the hepatocyte viability in tilapia. Similarly, tilapia exposed to PFOA demonstrated a similar pattern, and a dose-dependent decrease in the hepatocyte viability was observed in the following treatment of PFOA. In the 72 h exposures, ethoxy-resorufin-O-deethylase (EROD) activity was significantly induced with the increase concentrations in tilapia liver (p<0.05). Tilapia showed a strong EROD induction in livers, and significantly difference in EROD activity was observed between control, PFOS and PFOA-exposed tilapias. The liver glycogen content showed that PFOS and PFOA exposure caused significant changes in the liver glycogen content, which depended on the duration of exposure. And it appeared that the decrease in blood glucose level during the acclimation was followed by significant increase in liver glycogen content in tilapia.

Induction of detoxification processes in Oncorhynchus mykiss by trace levels of contaminants

In order to study the variability of detoxification activities associated with the cytochromes P450 1A (CYP1A) and 3A (CYP3A), several continental fish farms of Oncorhynchus mykiss were routinely sampled. In fish, CYP1A plays a key role in the biotransformation of awide variety of pollutants. This cytochrome is induced after ligand-activation of the aryl hydrocarbon receptor. CYP3A is also implicated in themetabolismof a wide variety of xenobiotics, including pharmaceuticals however its activity is mediated by the pregnane-X-receptor. CYP1A and CYP3A inductions have been widely used as biomarkers of environmental pollution in aquatic systems, in particular when measured at the enzymatic level through ethoxyresorufin-O-deethylase (EROD) and benzyloxy-4-[trifluoromethyl]-coumarin-O-debenzyloxylase (BFCOD) activities, respectively. In one of the monitored fish farms, EROD activity, which initially showed basal levels, exhibited a sharp increase difficult to explain by seasonal variability. In order to confirm the increased induction of the cytochrome activities, 32 animals were transferred to a control farm and maintained for one month. Periodic samplings of eight animals at days 0, 7, 15 and 30 were performed and both EROD and BFCOD activities in the livers measured. Under these controlled conditions, enzymatic activities associated with detoxification processes gradually decreased to basal levels indicating thepresence of some factor in the fish farm inducing the cytochromes. In order to investigate possible causes of such induction both water and sediment samples, along the river feeding the fish farm, were taken and analysed byaGCxGC-TOFMS system. This systemprovedveryuseful and allowed us to identify non-target compounds in some samples, among them personal care, pharmaceutical products, and industrial chemicals (i.e. PAHs, phthalates, alkylphenolic compounds) that couldbe related to the observed induction.

Impact of Oxidative Stress Indicated by Thiobarbituric Acid Reactive Substances (TBARS) and Protein Carbonyl Levels (PC) on Ethoxyresorufin-O-deethylase (EROD) Induction in Common Carp (Cyprinus carpio)

This study examined the impact of oxidative stress indicated by thiobarbituric acid reactive substances (TBARS) and protein carbonyl (PC), induced by intensive exercise and cadmium chloride (CdCl2) on ethoxyresorufin-O-deethylase (EROD) activity in juvenile carp (Cyprinus carpio). In the first experiment, fish were divided into three groups: (1) control, (2) carp exposed to intensive exercise, and (3) carp that was not exercised but previously, as well as carp in group 2, received single dose of 3-methylcholantrene (3-MC). The third and sixth day fish were sacrificed and the measurements were conducted. In the second experiment, fish were divided into (1) control, (2) carp in water containing CdCl2, and (3) carp in dechlorinated tap water (2 and 3 received single dose of 3-MC on the seventh day after exposure to CdCl2). The carp were killed 6 days later and livers were excised for biochemical analyses. In the first experiment, on the sixth day after treatment with 3-MC, results show statistically significant increase in EROD activity in non-exercised carp, while that increase in carp exposed to intensive exercise was significantly lower. Three days after exposure to 3-MC, statistically significant increase in TBARS was observed in both exercised and non-exercised carp. Six days after exposure to 3-MC, PC levels were significantly higher in exercised carp. Pretreatment with CdCl2, in the second experiment, caused oxidative stress and reduction of EROD activity. Results show linkage between expression of EROD activity and oxidative stress biomarkers and possible influence of oxidative stress on the cell membrane structures and consequently on EROD activity.

Structural influence of isothiocyanates on expression of cytochrome P450, phase II enzymes, and activation of Nrf2 in primary rat hepatocytes

Primary cultures of rat hepatocytes were used to investigate whether and how eight isothiocynates (ITCs) with different chemical structures (the aromatic benzyl, 4-hydroxybenzyl, phenethyl isothiocyanates and the aliphatic allyl, napin, iberin, raphasatin isothiocyanates and sulforaphane) derived from hydrolyzed glucosinolates, were able to modulate cytochrome P450 (CYP) and antioxidant/detoxifying enzymes and to activate the Nrf2 transcription factor. The aromatic ITCs at 40μM markedly increased the transcription of CYP1A1 and 1A2 mRNA and increased the associated ethoxyresorufin O-deethylase (EROD) activity after 24h of treatment. By contrast, the aliphatic ITCs (40μM) decreased CYP1A1 and 1A2 transcription, together with the corresponding EROD activity. The same treatment also caused a striking and similar transcriptional repression of CYP3A2, and the corresponding benzyloxyquinoline debenzylase activity in response to all the ITCs tested. In the same culture conditions, most of the antioxidant/detoxifying enzymes were significantly up-regulated by 40μM ITCs. In particular, NAD(P)H:quinone oxidoreductase and heme oxygenase-1 were induced, although to different levels, at transcriptional, protein and/or activity levels by all the ITCs. However, glutathione S-transferase activity was not induced by the allyl, benzyl, and 4-hydroxybenzyl ITCs, glutathione reductase activity was not induced by benzyl, and 4-hydroxybenzyl ITCs and catalase activity was not induced by allyl ITC. As for the Nrf2 transcription factor, a partial translocation of its protein from the cytosol to the nucleus was revealed by immunoblotting after 1h of treatment for all the ITCs tested. The ability of ITCs to induce the antioxidant and phase II enzymes did not appear to be affected by their hydrophilicity or other structural factors. Taken together, these results show that these ITCs are effective inducers of ARE/Nrf2-regulated antioxidant/detoxifying genes and have the potential to inhibit, at least in rat liver, the bioactivation of carcinogens dependent on CYP3A2 catalysis.

Differences in the induction of cyp1A and related genes in cultured rainbow trout Oncorhynchus mykiss. Additional considerations for the use of EROD activity as a biomarker

Two rainbow trout Oncorhynchus mykiss fish farms were repeatedly sampled in order to observe the variability of ethoxyresorufin-O-deethylase (EROD) activity and of related genes in the liver. Fish coming from fish farm A exhibited EROD levels that could be considered as basal according to the scientific literature, however, EROD activity in fish coming from fish farm B was significantly increased. This was accompanied by augmented aryl hydrocarbon receptor (ahr) and cytochrome P4501A (cyp1A) messenger RNA expression and reduced oestrogen receptor (er) and vitellogenin (vtg) transcription. Only sediment extracts from the entry channel of fish farm B induced EROD activity in O. mykiss cultured cells, however, this induction could not be explained by the levels of polyaromatic hydrocarbons (PAH) and polychlorinated biphenyls (PCB) measured in the sediments. The results of this study point out that O. mykiss cultured in fish farms could be used as sentinels for indication of pollution. In this particular work, however, no conclusive evidence has been found for a relationship between the presence of PAHs and PCBs and the observed EROD induction.

Hydroxybenzoate paralytic shellfish toxins induce transient GST activity depletion and chromosomal damage in white seabream (Diplodus sargus)

Fish are routinely exposed to harmful algal blooms that produce noxious compounds and impact the marine food web. This study investigates the role of phase I and II detoxification enzymes on metabolism of the novel paralytic shellfish toxins (PSTs), the hydroxybenzoate analogues recently discovered in Gymnodinium catenatum strains, in the liver of white seabream, assessing ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST) activities. Additionally, the genotoxic potential of hydroxybenzoate PSTs was examined through the erythrocytic nuclear abnormality (ENA) assay. Fish were injected with hydroxybenzoate PSTs into the coelomic cavity and sacrificed 2 and 6 days later for biochemical and cytogenetic analyses. While the activity of EROD was unresponsive to toxins, a significant GST activity decrease was observed at 2 days after injection indicating an impairment of this line of the detoxification system. The genotoxic potential of PSTs was demonstrated by the induction of clastogenic/aneugenic effects at 2 days, as measured by the ENA assay. Overall, this study contributes to better understand the impact of toxins produced by G. catenatum blooms in fish, revealing effects that, even transitory, point out a risk associated to hydroxybenzoate analogues.

Abstract 4427: Role of cytochrome P450 (CYP)1A2 in the sustained induction of CYP1A1 by the carcinogen, 3-methylcholanthrene (MC) in mouse heptoma cells (hepa-1)

Abstract Humans are constantly exposed to environmental carcinogenic polycyclic aromatic hydrocarbons (PAHs) through cigarette smoke, diesel exhausts, charcoal-broiled meats, etc. Cytochrome P4501A (CYP1A) enzymes play important roles in the activation of PAHs such as 3-methylcholanthrene (MC) to carcinogenic DNA-binding metabolites. We reported earlier that MC causes persistent induction of hepatic and pulmonary CYP1A1 in mice for several weeks after MC ithdrawal, and that the phenomenon of sustained hepatic CYP1A1 induction is lost in Cyp1a2-null mice. In this study, we tested the hypothesis that MC elicits induction of persistent CYP1A1 nduction in hepa-1 cells, and that CYP1A2 contributes mechanistically to this henomenon. Hepa-1 cells were treated ith the MC (2.5 µM), or dimethylsulfoxide (DMSO) as control, and at selected ime points, CYP1A1 promoter activity, CYP1A1 enzyme activities, contents, and CYP1A1 mRNA levels were studied. We fund that MC markedly and persistently induced CYP1A1 promoter activity, transcription, apoprotein expression, and the CYP1A1 associated ethoxyresorufin O-deethylase (EROD) activities for up to 5 days. Transfection of CYP1A2 siRNA resulted in knockdown of CYP1A2 mRNA by 70%, but a statistically significant increase of basal CYP1A1 mRNA by 35-40%. The induction of CYP1A1 promoter ctivity, CYP1A1 mRNA, CYP1A1 protein, and EROD activity by MC were not affected by CYP1A2 siRNA at the 24 h time point, but was significantly ttenuated by Cyp1a2 siRNA on day 5. These results suggest that CYP1A2, possibly via a metabolite, contributes to the sustained induction of CYP1A1 by MC in hepa-I cells. Further investigations into the mechanisms of persistent induction of CYP1A1 by MC could lead to novel preventative/therapeutic strategies against PAH-mediated carcinogenesis in humans. (Supported in part by 2R01 ES009132.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4427. doi:1538-7445.AM2012-4427

Design and One‐Pot and Microwave‐Assisted Synthesis of 2‐Amino/5‐Aryl‐1,3,4‐oxadiazoles Bearing a Benzimidazole Moiety as Antioxidants

In this study, two new series of 2-amino-1,3,4-oxadiazoles and 5-aryl-1,3,4-oxadiazoles carrying a benzimidazole moiety were synthesized. The antioxidant properties of these compounds were investigated in vitro by the determination of the microsomal NADPH-dependent inhibition of lipid peroxidation levels (LP), the microsomal ethoxyresorufin O-deethylase activity (EROD), and DPPH radical scavenger effects. Among the tested compounds, 2-[(2-(4-chlorophenyl)-1H-benzo[d]imidazole-1-yl)methyl]-5-(4-fluorophenyl)-1,3,4-oxadiazole (9) was found to be the most active compound in all three in vitro systems.