Induction Of Ethoxyresorufin O-deethylase Activity Research Articles

Steroid Hormones Protect against Fluoranthene Ethoxyresorufin-O-Deethylase (EROD) Activity Inhibition

The physiological conditions of an organism may influence its ability to cope with environmental stressors, such as contaminants. Biotransformation and the endocrine system interact with each other to promote animal’s fitness. However, little is known regarding the interaction between hormones and response to pollutants such as polycyclic aromatic hydrocarbons (PAHs). In this in vitro study, we aimed to increase the knowledge regarding the effects of steroid hormones on ethoxyresorufin-O-deethylase (EROD) activity inhibited by contaminants. The effects on in vivo induced EROD activity of Anguilla anguilla were assessed by conducting single and combined exposures to fluoranthene (FL) and to physiological levels of two major steroid hormones (cortisol and 17ß-estradiol). Hepatic microsome exposure to the lowest concentrations of FL (0.1 and 0.3 µM), as well as to cortisol and 17ß-estradiol (E2), led to significant EROD activity induction. However, the highest tested concentrations of FL (0.9 and 2.7 µM) significantly inhibited this enzymatic activity. When microsomes were simultaneously exposed to 0.9 µM FL and one of the hormones, both cortisol and E2 were able to decrease the inhibitory effects, with the former completely reverting EROD activity inhibition. These findings support the idea that cortisol and E2 can help prevent the inhibitory effects of PAHs over biotransformation enzymes, highlighting the physiological relevance of these hormones.

Tris (2,3-Dibromopropyl) Isocyanurate (TDBP-TAZTO or TBC) Shows Different Toxicity Depending on the Degree of Differentiation of the Human Neuroblastoma (SH-SY5Y) Cell Line

Tris (2,3-dibromopropyl) isocyanurate (TDBP-TAZTO or TBC) is a heterocyclic hexabromated flame retardant. It is widely used during the production of many synthetic compounds. High concentrations of TDBP-TAZTO were found in river water, surface sediments, soil, earthworms, and carp tissues. Moreover, it has been shown that this compound can cross the blood–brain barrier and accumulate in the gut and brain of carp. The aryl hydrocarbon receptor (AhR) has been characterized as a multifunctional intracellular sensor and receptor. AhR is an activator of cytochrome P450 1A1 and 1A2, which metabolize various toxic compounds. The aim of the study was to explain how/whether TDBP-TAZTO increases the expression and/or activity of the CYP1A1 enzyme and the AhR and TUBB3 expression during SH-SY5Y cell differentiation. SH-SY5Y cells were differentiated for 7 and 14 days using retinoic acid. Cell viability, ethoxyresorufin-O-deethylase (EROD) activity, and mRNA expression of CYP1A1, AhR, and TUBB3 were assessed. Our experiment showed that, during the differentiation process, the ability of TDBP-TAZTO to induce EROD activity in SH-SY5Y cells subsequently decreased, which may have been an effect of cell differentiation into neurons. Moreover, the results suggest that TDBP-TAZTO can affect the differentiation process. Since no CYP2B6 mRNA expression was detected, the CAR receptor may not be involved in the TDBP-TAZTO mechanism of action. However, more research is needed in this field to elucidate this mechanism precisely.

Toxicological effects of micronized tire crumb rubber on mummichog (Fundulus heteroclitus) and fathead minnow (Pimephales promelas)

Recent studies on the distribution of microplastics in the Charleston Harbor, SC, USA revealed that a large part of the microplastic particles that are found in the intertidal sediments are tire wear particles. These particles originate from the wear of tire treads on roadways, and wash into the estuary during rain events. The abundance of these particles has raised questions about potential toxicity to aquatic organisms that ingest these particles. The synthetic rubber in car tires consists of a large variety of chemicals, which can vary between manufacturers, but usually contains styrene-butadiene rubber, carbon black and zinc. To investigate the potential toxicity of tire wear particles, both mummichogs (Fundulus heteroclitus) and fathead minnow (Pimephales promelas) were exposed to different concentrations of crumb rubber particles (38-355 µm) in a 7-day static renewal exposure. Dissection of the fish revealed that crumb rubber was ingested and accumulated in the intestinal tract. At the highest concentration tested (6 g/L) partial mortality was observed in the fathead minnow, which came close to the assumed LC50. To investigate if polynuclear aromatic hydrocarbons (PAHs) were leaching from the particles, bile fluorescence was measured, together with potential induction of cytochrome P450-1A through the ethoxyresorufin-O-deethylase (EROD) assay. Elevated levels of 2-, 4-, and 5-, ring structures representative of PAHs were detected in the bile of exposed animals. Bile fluorescence indicated that 4-ring PAH compounds were the most bioavailable from the crumb rubber particles. Induction of EROD activity was observed in exposed animals at environmentally relevant concentrations of the crumb rubber particles (<1-2 g/L), and this elevated EROD activity indicated that PAH compounds from the crumb rubber particles were being metabolized in both mummichogs and fathead minnow.

Pueraria candollei var. mirifica-Induced CYP1A1 and CYP1A2 expression in human choriocarcinoma bewo cells

Background: The human placenta metabolizes many endogenous substances, drugs, and xenobiotics. Cytochrome P450 family 1 (CYP1) is expressed in both early- and full-term placenta. The Thai medicinal plant Pueraria candollei var. mirifica (PM) is traditionally consumed for rejuvenation and has neuroprotective, anti-osteoporotic, and antioxidant activities. Objectives: The objective of this study was to compare the effects of PM and the CYP1A inducer β-naphthoflavone (BNF) on the expression of CYP1, aryl hydrocarbon receptor (AHR), AHR nuclear translocator (ARNT), and the transporter ABCG2. Materials and Methods: Human choriocarcinoma BeWo cells were treated with BNF (10 μM), ethanolic extract of PM (PM-EtOH), or column chromatographic fractions of PM-EtOH (F2, F4, and F6) at 1, 10, and 100 μg/mL for 24 h. The mRNA expression of target genes was determined using real-time quantitative polymerase chain reaction. The activity of ethoxyresorufin-O-deethylase (EROD), a marker for CYP1, was measured at the RNA harvesting time point. Results and Discussion: PM-EtOH, F2, and F4 significantly induced EROD activity and expression of CYP1A1 and CYP1A2 while CYP1B1 and AHR were slightly suppressed and ARNT was unchanged. ABCG2 was slightly induced by F2. Therefore, the expression of CYP1 in BeWo cells appears to be independent of the AHR/ARNT regulatory pathway. Conclusion: The use of PM-containing products at high quantities or for long periods during pregnancy is of concern due to likely herb–drug interactions and toxicological risks through activation of CYP1A1 and CYP1A2 transcription.

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%.

Response of gene expression in zebrafish exposed to pharmaceutical mixtures: Implications for environmental risk

Complex mixtures of pharmaceutical chemicals in surface waters indicate potential for mixture effects in aquatic organisms. The objective of the present study was to evaluate whether effects on target gene expression and enzymatic activity of individual substances at environmentally relevant concentrations were additive when mixed. Expression of zebrafish cytochrome P4501A (cyp1a) and vitellogenin (vtg) genes as well as activity of ethoxyresorufin-O-deethylase (EROD) were analyzed after exposure (96h) to caffeine-Caf, ibuprofen-Ibu, and carbamazepine-Cbz (0.05 and 5µM), tamoxifen-Tmx (0.003 and 0.3µM), and after exposure to pharmaceutical mixtures (low mix: 0.05µM of Caf, Ibu, Cbz and 0.003µM of Tmx, and high mix: 5µM of Caf, Ibu, Cbz and 0.3µM of Tmx). Pharmaceuticals tested individually caused significant down regulation of both cyp1a and vtg, but EROD activity was not affected. Exposure to low mix did not cause a significant change in gene expression; however, the high mix caused significant up-regulation of cyp1a but did not affect vtg expression. Up-regulation of cyp1a was consistent with induction of EROD activity in larvae exposed to high mix. The complex mixture induced different responses than those observed by the individual substances. Additive toxicity was not supported, and results indicate the need to evaluate complex mixtures rather than models based on individual effects, since in environment drugs are not found in isolation and the effects of their mixtures is poorly understood.

Time-dependent transcriptomic and biochemical responses of 6-formylindolo[3,2-b]carbazole (FICZ) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are explained by AHR activation time

6-Formylindolo[3,2-b]carbazole (FICZ) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are ligands of the aryl hydrocarbon receptor (AHR) and bind to the AHR with high affinity. Until recently, TCDD was considered to be the most potent AHR agonist, but several recent studies indicate that FICZ binds with greater affinity to the AHR than TCDD. To advance our understanding of the similarities and differences of the effects of FICZ and TCDD exposure in chicken embryo hepatocyte (CEH) cultures, we compared relative expression changes of 27 dioxin-responsive genes by the use of a chicken PCR array, porphyrin accumulation and ethoxyresorufin-O-deethylase (EROD) activity at different time points. In addition, an egg injection study was performed to assess the effects of FICZ on the developing chicken embryo. The results of the current study showed: (1) mean EROD-derived relative potency values for FICZ compared to TCDD changed as a function of time (i.e. 9, 0.004, 0.0008 and 0.00008 at 3, 8, 24, and 48h, respectively) in CEH cultures; (2) FICZ exposure did not result in porphyrin accumulation in CEH cultures; (3) concordance between gene expression profiles for FICZ and TCDD was time- and concentration-dependent, and (4) no mortality or morphological abnormalities were observed in chicken embryos injected with 0.87ng FICZ/g egg into the air cell. The results presented herein suggest that while FICZ and TCDD share similar molecular targets, transient versus sustained AHR activation by FICZ and TCDD result in differential transcriptomic responses. Moreover, rapid metabolism of FICZ in hepatocytes resulted in a significant decrease in the induction of EROD activity.

Potency of polycyclic aromatic hydrocarbons (PAHs) for induction of ethoxyresorufin-O-deethylase (EROD) activity in hepatocyte cultures from chicken, Pekin duck, and greater scaup.

The potency of tetrachlorodibenzo-p-dioxin (TCDD) and 18 polycyclic aromatic hydrocarbons (PAHs) for induction of ethoxyresorufin-O-deethylase (EROD) activity was assessed in primary hepatocyte cultures prepared from chicken (Gallus domesticus), Pekin duck (Anas platyrhynchos domesticus), and greater scaup (Aythya marila). TCDD and 8 of the PAHs induced EROD activity in a concentration-dependent manner. Seven of these were previously shown to be acutely toxic to avian embryos, while the 10 congeners that did not produce an EROD response caused limited mortality. The rank order potency of the EROD-active congeners in all three species was as follows: TCDD>dibenz[ah]anthracene>benzo[k]fluoranthene>indeno[1,2,3-cd]pyrene>benzo[a]pyrene>chrysene≈benz[a]anthracene≈benz[ghi]perylene>benzo[b]naphtho[2,3-d]thiophene. Chicken hepatoctyes were more sensitive than duck hepatocytes to EROD induction by all test compounds, but the gap in species sensitivity was 100-fold for TCDD, and generally ≤10-fold for PAHs. This study is the first to use in vitro methods to rank the AHR-mediated potency of PAHs in birds. These data may be useful for assessing risks associated with exposure to PAHs in the environment.

Detection of effects caused by very low levels of contaminants in riverine sediments through a combination of chemical analysis, in vitro bioassays, and farmed fish as sentinel.

Aquatic organisms are often exposed to mixtures of low levels of pollutants whose presence and effects can pass easily unnoticed if only traditional monitoring strategies are employed. The main aim of this work was to assess the presence and effects of trace levels of pollutants in a scarcely affected area through the combination of chemical and biological approaches. Sediments were collected along a river with little anthropogenic pressure and assayed for cytochrome P450 (Cyp1a)-dependent ethoxyresorufin-O-deethylase (EROD) activity with the rainbow trout gonadal cell line RTG-2. Chemical analyses were performed in these sediments using two-dimensional gas chromatography-time-of-flight mass spectrometry. Sediment samples induced EROD activity, and chemical analyses evidenced the presence of a wide variety of contaminants in the range of nanograms per gram of dry weight. Correlation analysis between EROD induction and chemical analyses data showed an r value of 0.840 (p < 0.05). In addition, fish from a fish farm located downstream of the sampling points exhibited high hepatic EROD levels as well as an induced expression of cyp1a and cyp3a. In conclusion, only an appropriate combination of biological and chemical techniques allowed the detection of the presence of trace levels of contaminants in a theoretically nonaffected river.

SIN3A, Generally Regarded as a Transcriptional Repressor, Is Required for Induction of Gene Transcription by the Aryl Hydrocarbon Receptor

CYP1A1 bioactivates several procarcinogens and detoxifies several xenobiotic compounds. Transcription of CYP1A1 is highly induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via the aryl hydrocarbon receptor. We recently described an RNAi high throughput screening performed in the Hepa-1 mouse hepatoma cell line, which revealed that SIN3A is necessary for the induction of CYP1A1-dependent ethoxyresorufin-o-deethylase (EROD) enzymatic activity by TCDD. In the current studies, we sought to provide insight into the role of SIN3A in this process, particularly because studies on SIN3A have usually focused on its repressive activity on transcription. We report that ectopic expression of human SIN3A in Hepa-1 cells enhanced EROD induction by TCDD and efficiently rescued TCDD induction of EROD activity in cells treated with an siRNA to mouse SIN3A, thus validating a role for SIN3A in CYP1A1 induction. We demonstrate that SIN3A is required for TCDD induction of the CYP1A1 protein in Hepa-1 cells but not for expression of the aryl hydrocarbon receptor protein. In addition, siRNAs for SIN3A decreased TCDD-mediated induction of CYP1A1 mRNA and EROD activity in human hepatoma cell line Hep3B. We establish that TCDD treatment of Hepa-1 cells rapidly increases the degree of SIN3A binding to both the proximal promoter and enhancer of the Cyp1a1 gene and demonstrate that increased binding to the promoter also occurs in human Hep3B, HepG2, and MCF-7 cells. These studies establish that SIN3A physically interacts with the CYP1A1 gene and extends the transcriptional role of SIN3A to a gene that is very rapidly and dramatically induced.

Applicability of the TCDD-TEQ approach to predict sublethal embryotoxicity in Fundulus heteroclitus

The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxic equivalent quantity (TCDD-TEQ) approach was used successfully to predict lethal embryotoxicity in salmonids, but its applicability to sublethal effects of mixtures of organohalogenated compounds in other fish species is poorly known. The sublethal toxicity of two dioxin-like compounds (DLCs), 3,3′,4,4′-tetrachlorobiphenyl (PCB77) and 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PnCDF), two non-dioxin-like (NDL) polychlorinated biphenyls (PCBs), 2,2′,5,5′-tetrachlorobiphenyl (PCB52) and 2,3,3′,4′,6-pentachlorobiphenyl (PCB110), and of Aroclor 1254, a complex commercial mixture of PCBs, was assessed in Fundulus heteroclitus embryos exposed by intravitelline injection. At 16 days post-fertilization, the two DLCs and Aroclor 1254 altered prey capture ability in addition to inducing classical aryl hydrocarbon receptor-mediated responses: ethoxyresorufin-O-deethylase (EROD) induction, craniofacial deformities and reduction in body length. None of these responses was induced by the two NDL PCBs, at doses up to 5400ngg−1wet weight. Dose–response curves for prey capture ability for the 2 DLCs tested were not parallel to that of TCDD, violating a fundamental assumption for relative potency (ReP) estimation. Dose–response curves for EROD induction were parallel for 2,3,4,7,8-PnCDF and TCDD, but the ReP of 2,3,4,7,8-PnCDF for F. heteroclitus was 5-fold higher than the World Health Organization (WHO) fish toxic equivalent factor (TEF) based on embryolethality in salmonids. The chemically derived TCDD-TEQs of Aroclor 1254, calculated using 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) concentrations and it ReP for F. heteroclitus, overestimated its potency to induce EROD activity possibly due to antagonistic interactions among PCBs. This study highlights the limitations of using TEFs based on salmonid toxicity data alone for risk assessment to other fish species. There is a need to assess the variability of RePs of DLCs in different species for a variety of endpoints and to better understand interactions between DLCs and other toxic chemicals.

Effects of flumequine on cytochrome P450 enzymes in allogynogenetic silver crucian carp, Carassius auratus gibelio

The effects of flumequine on activities of several microsomal P450 monooxygenases of allogynogenetic silver crucian carp (Carassius auratus gibelio) were determined and the induction of fluemquine on CYP1A was further explored at the level of protein expression and mRNA transcription.After 24 h following a single intraperitoneal injection at a dose of 35 mg/kg,the ethoxyresorufin O-deethylase (EROD) activity in the liver of the Crucian carp was 54.33 pmol/(mg·min),which was significantly higher than that of the control carps [34.00 pmol/(mg·min)](P0.01).It was revealed that flumequine could significantly induce EROD activity(P0.01).However,the other P450 monooxygenases,such as erythromycin N-demethylase (ERND) [177.98 pmol/(mg·min)],aminopyrine N-demethylase [934.40 pmol/(mg·min)] and ethoxycoumarin O-deethylase (ECOD) [9.84pmol/(mg·min)]were not significantly affected by flumequine,comparing with these activities in control [140.90 pmol/(mg·min),850.71 pmol/(mg·min) and 8.93 pmol/(mg·min)],respectively.Except that the ERND activity in kidney tissue was higher than that in liver,the activities of APD,EROD and ECOD were the highest in liver tissue.An immunoblot analysis with rabbit anti-rat P4501A polyclonal antibody showed that CYP 1A protein expression in flumequine-treated carps was markedly higher than that in control carps,which was consistent with the trend in EROD activities.These findings suggest that P450 1A isoform,constitutively expressed in crucian carp,was particularly susceptible to activation by flumequine.Semiquantitative RT-PCR indicated that CYP1A mRNA was not affected by flumequine.In vitro assay,microsomes incubating with various concentrations of flumequine demonstrated that this fluoroquinolone antibiotic had no induction or inhibition of CYP1A.On the basis of the above,liver CYP1A induction of flumequine on carp occurs at the post-translation level,which may enhance protein stability.

Assessment of the toxicity of organochlorine pesticide endosulfan in clams Ruditapes philippinarum

This study is aimed at evaluating the effects of endosulfan in clams (Ruditapes philippinarum). For this purpose, a study was performed on clams exposed to 0.005, 0.05 and 0.5μg/L endosulfan for 15 days. S ubsequently, the level of ethoxyresorufin-O-deethylase (EROD) activity, glutathione-S-transferase (GST) activity, glutathione (GSH) content, superoxide dismutase (SOD) activity, lipid peroxidation (LPO) and DNA strand break was determined in gills and digestive glands. Among the parameters, endosulfan caused significant changes in induction of EROD activity and oxidative stress in clams R. philippinarum. The exposure to endosulfan increased the concentration of EROD, GST, GSH, MDA and decreased the concentration of SOD. Moreover, according to the correlation analysis results, the EROD activity and GSH content in digestive gland as well as GST activity, LPO and DNA damage in both tissues had excellent correlation with endosulfan concentration. These results provided information on potential biomarkers that could be effectively applied to the biomonitoring of aquatic ecosystem in areas susceptible to persistent organochlorine compounds contamination, and also information on toxic effects.

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.

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.

Induction of cytochrome P4501A by highly purified hexachlorobenzene in primary cultures of ring-necked pheasant and Japanese quail embryo hepatocytes

Primary cultures of ring-necked pheasant (Phasianus colchicus) and Japanese quail (Coturnix japonica) embryo hepatocytes were used to compare the potencies of highly purified hexachlorobenzne (HCB-P), reagent-grade HCB (RG-HCB) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as inducers of ethoxyresorufin O-deethylase (EROD) activity, cytochrome P4501A (CYP1A4) messenger ribonucleic acid (mRNA) and CYP1A5 mRNA. HCB-P, RG-HCB and TCDD all induced EROD activity and up-regulated CYP1A4 and CYP1A5 mRNA. Induction was not caused by contamination of HCB with polychlorinated dibenzo-p-dioxins, dibenzofurans or biphenyls. Based upon a comparison of the EC50 and ECthreshold values for EROD and CYP1A4/5 concentration–response curves, the potency of HCB relative to TCDD was 0.001 in ring-necked pheasant and 0.01 in Japanese quail embryo hepatocytes. Differences in species sensitivity to HCB were found to be mainly dictated by differences in species sensitivity to TCDD rather than differences in the absolute potency of HCB. Consequently, ring-necked pheasant and Japanese quail embryo hepatocytes were found to be equally sensitive to HCB exposure. Species sensitivity comparisons were also made with chicken (Gallus gallus domesticus) and revealed that chicken embryo hepatocytes were less responsive to EROD induction (lower maximal response) by HCB compared to the embryo hepatocytes of pheasant and quail.

Highly purified hexachlorobenzene induces cytochrome P4501A in primary cultures of chicken embryo hepatocytes

Some uncertainty exists regarding the purity of hexachlorobenzene (HCB) used in past toxicity studies. It has been suggested that reported toxic and biochemical effects initially attributed to HCB exposure may have actually been elicited by contamination of HCB by polychlorinated dibenzo- p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). Herein, primary cultures of chicken embryo hepatocytes (CEH) were used to compare the potencies of two lots of reagent-grade hexachlorobenzene (HCB-old [HCB-O] and HCB-new [HCB-N]), highly purified HCB (HCB-P) and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) as inducers of ethoxyresorufin O-deethylase (EROD) activity, cytochrome P4501A4 (CYP1A4) messenger ribonucleic acid (mRNA) and CYP1A5 mRNA. The study also compared the EROD- and CYP1A4/5 mRNA-inducing potencies of HCB to the potencies of two mono- ortho substituted polychlorinated biphenyls (PCBs), 2,3,3′,4,4′-pentachlorobiphenyl (PCB 105) and 2,3′4,4′,5-pentachlorobiphenyl (PCB 118). HCB-O, HCB-N and HCB-P all induced EROD activity and up-regulated CYP1A4 and CYP1A5 mRNAs. Induction was not caused by contamination of HCB with PCDDs or PCDFs. Based upon a comparison of the EC 50 and EC threshold values for EROD and CYP1A4/5 mRNA concentration–response curves, the potency of HCB relative to the potency of TCDD was 0.0001, and was similar to that of PCB 105 and PCB 118. The maximal EROD activity and CYP1A4/5 mRNA expression differed greatly between HCB and TCDD, and may contribute to an overestimation of the ReP value calculated for highly purified HCB.

Impact of selenate treatment on broccoli glucosinolate profile and bioactivity

Selenium (Se)‐enriched broccoli has potent anticarcinogenic effects, which are suggested to be the result of changes in detoxification enzyme activity, induction of apoptosis, and inhibition of histone deacetylation (HDAC) and angiogenesis. We previously showed that feeding high‐Se broccoli powder (H‐SeB; 4.28 ppm) to male Copenhagen rats induced hepatic ethoxyresorufin‐O‐deethylase (EROD) and NAD(P)H:quinone oxidoreductase (NQO1) activity 1.6 and 1.9‐fold over low‐Se broccoli (L‐SeB; 0.25 ppm), respectively. To elucidate the relative role of Se in induction of these enzymes, mouse hepatoma Hepa‐1c1c7 cells were exposed to aqueous extracts of H‐SeB or L‐SeB. Extracts of H‐SeB increased EROD and NQO1 activity 2.7 and 1.3‐fold, respectively. Adding Se as selenite or Se‐methylselenocysteine (SeMSC) to L‐SeB extracts had no impact on EROD or QR activity. Likewise, Lee et al. reported that SeMSC up to 2.5 mM had no direct effect on HDAC in prostate cancer cells. Selenium‐enrichment of broccoli increased levels of the glucosinolate neoglucobrassicin, the hydrolysis product of which is known to induce EROD activity. We conclude that the increase in detoxification enzyme activity is not a direct effect of Se, but the result of a (plant) metabolite of Se and/or changes in the glucosinolate profile. Supported by grants from USDA/AFRI and the Illinois Council on Food and Agricultural Research.

Cytochrome P450 induction in Ictalurus punctatus following exposure to wastewater treatment plant effluent

Induction of the cytochrome P450 enzymes in fish, measured as ethoxyresorufin-O-deethylase (EROD) activity, has been extensively used as a biomarker in assessing exposures and responses of aquatic organisms to contaminants. This study focused on whether exposure to Troy (Alabama) wastewater treatment plant (TWWTP) effluent at the Walnut Creek mixing zone, induced transcription of mRNA for cytochrome P4501A1 enzyme production and increased EROD activity in channel catfish (Ictalurus punctatus). Water from Big Creek (Barbour County, AL), regarded as least impacted by pollutants, was used as a reference source for administration of a positive control chemical (PCB – Aroclor 1254). All water samples were transported from field sites to the Troy University laboratory for conducting the study. Reverse transcriptase-polymerase chain reaction (RT-PCR) indicated the presence of compounds capable of inducing transcription of CYP1A1 mRNA in catfish liver. Increased transcription of mRNA for cytochrome P4501A1 enzyme production, confirmed by a 3-fold induction of EROD activity, was found in catfish exposed to water from the TWWTP mixing zone on Walnut Creek compared to catfish exposed to Walnut Creek Upstream. Catfish exposed to water from Big Creek treated with PCBs were found to have only slightly higher enzyme activities than those exposed to water from Big Creek Control, but a 2.7-fold EROD level was found in catfish exposed to Big Creek Control compared to Walnut Creek Upstream. Determining the induction of cytochrome P450 and subsequent enzymatic activity in catfish and other fish species common to this region could be useful early molecular warnings of possible pollution effects, beyond those methods typically used to characterize water quality.

Oil dispersion increases the apparent bioavailability and toxicity of diesel to rainbow trout (Oncorhynchus mykiss)

Diesel is a complex mixture containing polycyclic aromatic hydrocarbons, which persist after a spill, pass readily from water into tissues, and are toxic to early life stages of fish. The bioavailability and chronic toxicity of hydrocarbons dissolved into water from floating diesel (water-accommodated fraction) and chemically dispersed diesel (chemically enhanced water-accommodated fraction) were measured by the extent of ethoxyresorufin-O-deethylase (EROD) induction in juvenile rainbow trout (Oncorhynchus mykiss) and by the severity of blue sac disease in embryos. The water-accommodated fraction of floating diesel was virtually nontoxic to embryos at nominal concentrations up to 1,000 mg/L, causing only small weight changes. Liver EROD induction in juvenile trout was only observed at the highest nominal water-accommodated fraction concentration (10,000 mg/L). Chemical dispersion increased the bioavailability and toxicity of diesel to trout by 100-fold. Diesel chemically enhanced water-accommodated fraction induced EROD activity, caused blue sac disease, and impaired development and growth of embryonic trout at nominal concentrations as low as 10 mg/L; 88% mortality occurred at 100 mg/L. However, when total hydrocarbon concentrations were measured, differences between dispersed and undispersed diesel disappeared, with a median lethal concentration of 8 mg/L of total hydrocarbons and sublethal median effective concentrations ranging from 1.3 to 6.1 mg/L. Dispersion of diesel by high-energy mechanical mixing was recently reported to cause acute lethality to juvenile trout between 40 and 200 mg/L. Therefore, dispersion of oil by any means increases the bioavailability and apparent toxicity of diesel to fish embryos without changing the toxicity of its components. Nevertheless, in an actual spill, dispersion of diesel increases the effects of oil on fish populations.