Effects of acute exposure to exogenous ammonia on cerebral monoaminergic neurotransmitters in juvenile Solea senegalensis

The present study explored the potential role of brain catecholaminergic and serotoninergic systems as neuronal targets for the toxicological effects of acute ammonia exposure (0.28 mg l⁻¹ of un-ionized ammonia for 12 and 24 h) in juvenile sole (Solea senegalensis). In addition, plasma cortisol levels were measured. The results showed significant increases in their concentrations that were similar after 12 and 24 h of exposure. These data indicate that acute exposure (12 and 24 h) to ammonia initiates a typical stress response in the Senegalese sole, with stimulation of the hypothalamus–pituitary–interrenal axis. The concentrations of dopamine (DA), serotonin (5-hydroxytryptamine; 5-HT) and noradrenaline (NA), and their metabolites, 3, 4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxy-3-indoleacetic acid (5HIAA), were measured in the hypothalamus, telencephalon and optic tectum. The main changes induced by acute exposure to ammonia were decreases in the concentrations of 5-HT and DA, which were significant in most of the brain regions studied. The ratios of 5-HIAA/5-HT and DOPAC/DA increased in all regions and at all times studied, although in the case of the DOPAC/DA ratio, the increases were only significant in the hypothalamus (24 h exposure) and in the optic tectum (12 and 24 h exposure). These changes indicated that exposure to ammonia elicited time-dependent increases in serotoninergic and dopaminergic activity in the hypothalamus, telencephalon and optic tectum.     

Multi-biomarker responses to estuarine habitat contamination in three fish species: Dicentrarchus labrax, Solea senegalensis and Pomatoschistus microps

Several biomarker responses were determined in three fish species, Dicentrarchus labrax, Solea senegalensis and Pomatoschistus microps, from two estuaries of the Portuguese coast, Ria de Aveiro and Tejo. Both estuaries have significant anthropogenic influences from multiple sources (industrial, agricultural and shipping activities), which was evident from sediment chemical characterization concerning metal (copper, zinc, nickel, lead and chromium) and polycyclic aromatic hydrocarbon (PAH) concentrations. Spatial variability in fish responses was observed across species for most biomarkers of exposure [the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST), and metallothionein concentrations (MT)] and effect biomarkers [lipid peroxidation (LPO), RNA to DNA ratio (R:D), protein and lipid content]. In general, the interspecific differences in biomarker responses were greater than the spatial differences, due to differences in the behavior and habitat use of the species. Nevertheless, similarities were also observed considering both chemical load and biomarker responses. In highly polluted sites fish showed in general a significant antioxidant enzyme induction, associated with decreased R:D values, while fish from the least impacted site had little enzyme induction and better condition indices (high R:D and low LPO values). EROD activity was also higher for all species in the Tejo than Ria de Aveiro estuary, despite the generally higher total PAH measured in Ria de Aveiro, most likely due to a higher proportion of 4 and 6-ring PAHs, considered more toxic than low molecular weight PAHs, in the Tejo. In conclusion, this multi-biomarker approach considering multiple species provided improved understanding of the diverse responses and effects of exposure to contaminants and the effective risk it poses for different fish species.     

The effect of CYP1A induction on amiodarone disposition in the rat

In the treatment of cardiac arrhythmias, amiodarone (AM) has emerged as a primary therapeutic agent. In addition to other cytochrome P450 (CYP), 1A1 and 1A2 facilitate the biotransformation of AM to the pharmacologically and toxicologically active metabolite, desethylamiodarone (DEA). The exposure to polycyclic aromatic hydrocarbons can induce these isoforms. This study was aimed at investigating the effect of CYP1A induction on the disposition of AM, after single and multiple intravenous doses, using β-naphthoflavone (BNF) treated rats to model induction of CYP1A. After a single dose (25 mg/kg), the plasma AUC of DEA were significantly induced (～3-fold). With multiple doses, AM AUC_{0–24 h} was significantly reduced in the BNF plasma (30%), lung (35%), liver (48%), kidney (52%), heart (34%), and intestine (43%). In contrast the DEA AUC_{0–24 h} was increased significantly in the BNF plasma (36%), lung (56%), liver (101%), kidney (65%), and heart (73%). The DEA/AM ratios of the AUC_{0–24 h} were increased in the BNF plasma, lung, liver, kidney, and heart by 1.9-, 2.5-, 3.8-, 3.4-, and 2.7-fold, respectively. In both groups of rats, the highest concentrations of AM and DEA were in the lung. Exposure to BNF was shown to increase DEA concentrations in the rat. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:539–548, 2010     

Effect of troglitazone on CYP1A1 induction

Several peroxisome proliferators enhance CYP1A1 activity, but the mechanisms involved in this enhancement remain unknown. In this study, we examined the effect of troglitazone, a peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist, on CYP1A1 gene expression and explored the mechanisms involved in these effects. Troglitazone increased gene expression of CYP1A1 mRNA and also increased CYP1A1-specific 7-ethoxyresorufin-O-deethylase (EROD) activity in a dose-dependent manner. Moreover, concomitant treatment with troglitazone and GW9662, a PPAR antagonist, markedly reduced the troglitazone-inducible EROD activity. Luciferase reporter assays using Hepa-1c1c7 cells showed a significant transactivation by troglitazone with a reporter plasmid containing a region from -1395 to +7 of the CYP1A1 gene. We found that a putative peroxisome proliferator-response element (PPRE) between -521 and -500 is located in the CYP1A1 gene promoter. Their inactivation by deletion mutagenesis suppressed the inductive effect of troglitazone on CYP1A1 promoter activation. Electrophoretic mobility shift assay revealed that troglitazone induced the activation of the PPAR-gamma to a form capable of binding specifically to the PPRE sequence of the CYP1A1 gene promoter. Furthermore, troglitazone increased the formation of the benzo[a]pyrene (BaP)-DNA adduct. Overall, our results suggest that troglitazone induces CYP1A1 enzyme activity and gene expression through PPAR-gamma activation, and may be involved in carcinogenesis.     

Biomonitoring toxicity of natural sediments using juvenile Hyalella curvispina (Amphipoda) as test species: evaluation of early effect endpoints

The utility of early effect endpoints as biomarkers of ecotoxicity of natural sediments in water–sediment static system was investigated. The particular goal was to evaluate the ecotoxicity of the sediment samples from La Choza stream, located in upper basin of the Reconquista river, the second most polluted river of Argentina. Native juveniles Hyalella curvispina were used as test organisms evaluating survival, growth, oxidative stress parameters (SOD; CAT, TBARS) and the electron transport system (ETS) activity as early toxic effect. This study used methodologies and techniques that allow the assessment of sediment pollution with a native species as test organism and provided data to discuss the viability of sublethal endpoints as tools for freshwater sediment assessment. In spring and in summer two ten-day series of whole-sediment assays were conducted simultaneously: (a) standard assays and (b) biomarkers assays. A control sediment was ran simultaneously in which no––effect on survival was measured. In summer there was a significant increase in length and biomass in both exposed and control groups. In spring an inhibitory effect on growth and an increase in oxidative damage with a concomitant rise in antioxidant defenses, was observed in animals exposed to La Choza sediment. ETS measurement indicated a significant depression of metabolic activity of amphipods exposed to contaminated sediments. The measured biomarkers represent the first record for juvenile H. curvispina exposed to polluted natural sediments under standardized laboratory conditions. The used bioanalytical tools demonstrated higher sensitivity and a more accurate assessment of the effects than those obtained by the standard tests of survival and growth. We propose their adoption in biomonitoring of freshwater sediment toxicity.     

Preferential induction of CYP1A1 and CYP1B1 in CCSP-positive cells.

Both benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are potent ligands of aryl hydrocarbon receptors (AhR). Although animal studies indicate that both compounds induce pathological changes in the peripheral lung, the specific cell type involved remains unclear. Clara cells, expressing Clara cell specific protein (CCSP) and abundant in cytochrome P450, are nonciliated bronchiolar epithelial cells in the peripheral lung. Here we explore the hypothesis that CCSP-positive Clara cells are highly responsive to AhR ligands and are the primary cell type involved in BaP- and TCDD-induced toxicities. The responsiveness to AhR ligands was evaluated by measuring the respective mRNA and protein levels of cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1) using real-time RT-PCR and immunocytochemistry assays. Two in vitro models were used: primary cultures of human small airway epithelial (SAE) cells and rat lung slice cultures. In the presence of calcium, human SAE cells differentiated into CCSP-positive cells. BaP- and TCDD-induced mRNA and protein levels of CYP1A1 and CYP1B1 levels were significantly elevated in CCSP-positive cell cultures. Similarly, AhR mRNA and protein levels were increased in CCSP-positive cell cultures, as determined by real-time RT-PCR and Western blot analysis. When rat lung slice cultures were treated with BaP or TCDD for 24 h, CYP1A1 and CYP1B1 proteins were strongly induced in Clara cells. These results indicate that, in the peripheral lung of both rats and humans, CCSP-positive cells (Clara cells) may be more sensitive to AhR ligands than other cell types.     

CYP1A induction and blue sac disease in early developmental stages of rainbow trout (Oncorhynchus Mykiss) exposed to retene

Early life stages of rainbow trout were exposed to different regimes of water-borne retene (7-isopropyl-1-methylphenanthrene) to determine if there is an ontogenic stage particularly sensitive to retene toxicity, and if cytochrome P-4501A (CYP1A) induction is a forerunner to blue sac disease (BSD), the syndrome of toxicity. CYP1A protein concentrations, measured by immunohistochemistry, were first detected during organogenesis, when organ and enzyme systems are first being developed, and steadily increased until swim-up. The prevalence of signs of BSD rose 1 wk following a marked increase in CYP1A activity after hatch, suggesting that CYP1A induction is related to BSD. The larval stage was the most sensitive to retene toxicity, based on CYP1A induction and a high prevalence of BSD. The most common signs of BSD were hemorrhaging, yolk-sac edema, and mortality, but hemorrhaging was the first and most frequently observed response. Tissue concentrations of retene were elevated just after fertilization, but decreased steadily as fish developed to the swim-up stage, most likely due to the establishment of more efficient metabolic and excretory systems in later stages of development.     

Disposition of 2,3,7,8-tetrabromodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat: biliary excretion and induction of cytochromes CYP1A1 and CYP1A2

The biologic activity and pharmacokinetic properties of 2,3,7,8-tetrabromodibenzo-p-dioxin (TBDD) are similar to those of the chlorinated congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Metabolism of both compounds appears to be rate-limiting for excretion, which is primarily via the feces. Therefore, the biliary elimination of TBDD and TCDD was examined as an indirect assessment of metabolism. Male F344 rats were anesthetized with pentobarbital, and 1 nmol/kg [3H]TBDD or [3H]TCDD was administered iv. Bile was collected for up to 8 hr while rats were maintained under anesthesia. The rate of biliary excretion of radioactivity was slightly greater for TCDD than TBDD (10% vs 7% in 5 hr). All biliary radioactivity was attributable to metabolites. High pressure liquid chromatographic (HPLC) profiles of biliary radioactivity were similar for [3H]TBDD and [3H]TCDD. To determine if pretreatment altered elimination kinetics, a single dose of 100 nmol/kg TBDD or TCDD was administered to rats by oral gavage 3 days prior to iv injection of 1 nmol/kg [3H]TBDD or [3H]TCDD, respectively. Biliary excretion of the radiolabeled dose was quantitatively and qualitatively unaffected by pretreatment despite a twofold increase in hepatic levels of radiolabel in the pretreated animals. Therefore, under these experimental conditions, autoinduction of TCDD and TBDD metabolism did not occur in the rat in vivo at doses which elicited enhanced hepatic uptake. In a second set of studies, the dose-response profiles for induction of cytochromes CYP1A1 and CYP1A2 by TBDD were characterized. The ED50 value for CYP1A1 induction (measured by ethoxyresorufin O-deethylase activity and radioimmunoassay (RIA) was estimated to be 0.8-1.0 nmol/kg, similar to what has been reported for TCDD. Induction of CYP1A2 (RIA) by TBDD appeared to be a more sensitive response over the dose range studied. Finally, comparison of hepatic CYP1A2 induction vs hepatic concentrations of TBDD 3 days following treatment with 10 vs 1 nmol/kg TBDD suggested that induction of CYP1A2 alone may not account for nonlinearities in dioxin disposition exemplified by dose-related increases in the ratio of dioxin concentrations in liver and adipose tissue.     

Effect of metals on polycyclic aromatic hydrocarbon induction of CYP1A1 and CYP1A2 in human hepatocyte cultures

Environmental cocontamination by polycyclic aromatic hydrocarbons (PAHs) and metals could affect the carcinogenic consequences of PAH exposure by modifying PAH induction of PAH-bioactivating CYP1A. The effect of As, Pb, Hg, or Cd (ranked as the most hazardous environmental metals by EPA and ATSDR) on CYP1A1 and 1A2 induction by benzo[a]pyrene (BaP), benzo[b]fluoranthene (BbF), dibenzo[a,h]anthracene (DBahA), benzo[a]anthracene (BaA), and benzo[k]fluoranthene (BkF) has thus been investigated in fresh human hepatocyte cultures. Induction was probed by ethoxyresorufin-O-deethylase activity, by immunoblots, and by RT-PCR. Uptake of PAHs into the hepatocytes varied according to PAH and liver donor: 84% of 5 microM BaA and 25-40% of 5 microM DBahA was taken up in 24 h. Hepatocytes retained viability up to 1 microM Cd and 5 microM Pb, Hg, or As and 5 microM PAHs. PAH induction of CYP1A in hepatocytes was variable, some cultures expressed CYP1A1 and others CYP1A1 and 1A2, and to variable extents. Induction efficiency (relative to DMSO controls) at 2.5 microM PAH concentration was in the order BkF (7.6-fold) > DBahA (6.1 fold) > BaP (5.7 fold) > BbF (3.9-fold) > BaA (2.5-fold). All four metals (1-5 microM) decreased CYP1A1/1A2 induction by some of the PAHs with dose-, metal-, and PAH-dependency. Arsenic (5 microM) decreased induction by 47% for BaP, 68% for BaA, 45% for BbF, 79% for BkF, and 53% for DBahA. Induced CYP1A2 protein was much more extensively decreased than 1A1 protein, and CYP1A2 mRNA and, to variable extents, CYP1A1 mRNA were decreased by As. Thus the metals in PAH/metal mixtures could diminish PAH carcinogenicity by decreasing induction of their bioactivation by CYP1A1/1A2.     

Growth retardation of fetal rats exposed to nicotine in utero: possible involvement of CYP1A1, CYP2E1, and P-glycoprotein

To elucidate the possible metabolic mechanism of intrauterine growth retardation induced by nicotine, this study determines the effects of prenatal nicotine exposure on fetal development and cytochrome P4501A1 (CYP1A1), CYP2E1, and P-glycoprotein (Pgp) expression in maternal liver and placenta. Pregnant rats were given 1.0 mg/kg nicotine subcutaneously twice a day from gestational day (GD) 8 to GD 15, 18, or 21. In nicotine-treated groups, fetal developmental parameters including body weight were significantly lower. The activities of CYP1A1 and CYP2E1 in maternal liver microsomes in nicotine-treated groups increased significantly with progressing gestation when compared with the corresponding control, but returned to the level similar to the control in late pregnancy. Nicotine-treated groups induced pathological changes and increased malondialdehyde (MDA) content in the placenta when compared with the control. The gene expressions of CYP1A1 and CYP2E1 in the placenta increased significantly in nicotine-treated groups on GD 15 and GD 18, but returned to the level similar to the corresponding control on GD 21. In nicotine group, there was a decrease of mdr1a expression on GD 15, GD 18, and GD 21, with the most significant decrease on GD 15. In contrast, no significant difference was found in mdr1b mRNA expression between the nicotine-treated animals and the corresponding control. In comparison with the corresponding control, the placental Pgp protein significantly decreased on GD 15 and GD 18. Our results showed that prenatal nicotine exposure resulted in inhibition of fetal growth significantly. The induction of CYP2E1 and CYP1A1 gene expression by nicotine in the maternal liver and placenta may be involved with the observed increase in oxidative stress and lipid peroxidation. The inhibition of the placental Pgp expression by nicotine may also contribute to an increased susceptibility of the fetus to environmental toxins.     

The effect of ozone on the expression of metallothionein in tissues of rats chronically exposed to cadmium

Our aims were to evaluate the expression of metallothionein (MT) in an experimental rat model which experienced chronic exposure to cadmium (Cd) and to measure its expression after ozone therapy (OT) or oxygen (Ox) in the same model, as compared to the control group, which was exposed to neither cadmium nor ozone.Forty male Wistar rats were divided into 5 groups: control, Cd, Cd and Ox, Cd and Oz, and Oz. During our research, Cd concentration (ASA) and MT concentration (ELISA) were determined in supernatants of the kidneys, liver and pancreas. SDS-PAGE analyses and immunohistochemical localization were used to evaluate the level of MT expression in the tissue. In rats intoxicated with Cd, the highest concentration of both Cd and MT was observed in the kidneys and liver, with a significantly lower concentration measured in the pancreas. Ozone therapy reduces the accumulation of cadmium in the liver and kidneys, resulting in a reduced expression of metallothionein in those tissues.     

CYP1A induction and blue sac disease in early life stages of white suckers (Catostomus commersoni) exposed to oil sands

The objectives of this study were to evaluate the influence of natural oil sands on the early developmental stages of white sucker (Catostomus commersoni) and to determine whether biochemical responses in this species were similar to native fish caught in the Athabasca Oil Sands area. Early life stage (ELS) sediment toxicity tests were conducted using controls, reference sediments, natural oil sands, and industrially contaminated (wastewater pond) sediments collected from sites along the Athabasca River, Alberta (Canada). Eggs and larvae were observed for mortality, hatching, deformities, growth, and cytochrome P-4501A (CYP1A) activity using immunohistochemistry. E-Nat-, S-Nat-, and wastewater pond sediment-exposed groups showed significant premature hatching, reduced growth, and exposure-dependent increases in ELS mortality and larval malformations relative to controls. The most common larval deformities included edemas (pericardial, yolk sac, and subepidermal), hemorrhages, and spinal defects. Juveniles exposed to oil sands and wastewater pond sediments (96 h) demonstrated significantly increased 7-ethoxyresorufin-O-deethylase (EROD) activity (30- to 50-fold) as compared to controls. Reference sediment-exposed groups and water controls demonstrated reliable embryo and larval survival, minimal malformations, and negligible CYP1A staining. These observed signs of blue sac disease (ELS mortality, malformations, growth reductions, CYP1A activity induction) may produce deleterious reproductive effects in natural fish populations exposed to oil sands mixtures.     

Development of in vitro methods to predict induction of CYP1A2 and CYP3A4 in humans

The important role of cytochrome P450 (CYP) drug-metabolizing enzymes has been studied for many years, and the potential liabilities of inducing these enzymes are well understood. Though several mechanisms of induction have been studied, a growing consensus is developing that the aryl hydrocarbon receptor (AHR) and the pregnane X receptor (PXR) have evolved as the primary mechanisms responsible for clinically relevant drug-drug interactions caused by induction of drug-metabolizing factors. AHR and PXR have been identified as inducers of a variety of Phase I and Phase II drug-metabolizing enzymes, drug transporters, and other factors involved in drug metabolism. Though many genes are induced through these regulating factors, CYP1A2 and CYP3A4 have been the most reliable biomarkers to identify compounds with potential induction liabilities through AHR and PXR, respectively. Here are presented several in vitro methods to detect AHR- and PXR-mediated induction of CYP1A2 and CYP3A4 in fresh and cryopreserved primary human hepatocytes, stable transfectants, and transiently transfected immortalized cells.     

The relationships among CYP1A induction, toxicity, and eye pathology in early life stages of fish exposed to oil sands

Exposure of the early life stages of fish to oil sands constituents is associated with mortality and larval malformations such as edemas, hemorrhages, and skeletal, craniofacial, and eye defects. In fathead minnow (Pimephales promelas) and white sucker (Catostomus commersoni) larvae, indices of total eye pathology increased significantly following oil sands exposure. Structural, cytoplasmic, inflammatory, and degenerative eye alterations included poor retinal differentiation, microphthalmia, optic fissures, dysphasic retinas and lenses, inflammatory infiltrates, retinal epithelial lifting, and necrotic foci. Cytochrome P-4501A (CYP1A) was expressed in ocular (retina, lens) and kidney endothelial tissues, as indicated by immunohistochemistry. Although the kinetics of exposure-response curves for mortality and CYP1A expression were similar in both species, species differences in the magnitude and sensitivity of the responses were observed. Oil sands were twofold more toxic to fathead minnows (TPAH LC50 = 47-330 microg/g) than to white sucker (TPAH LC50 = 95-860 microg/g) larvae. For both species, larval mortality was significantly related to CYP1A protein concentrations in kidneys, and severity of these effects rose with oil sands exposure. The relationships among eye damage, mortality, and CYP1A indices warrants further investigation, and may lead to the use of CYP1A induction as an indicator of adverse effects rather than just contaminant exposure.