Induction Of EROD Activity Research Articles

Comparative toxicity of beach mesoplastics from South Spain: An in vitro approach

Plastics, particularly mesoplastics, dominate beach debris and act as carriers of hazardous chemicals, either initially present in plastics or absorbed from the surrounding environment. In this study, mesoplastics were collected from five beaches in the southern region of Spain to investigate their potential impact on marine life. In vitro assays employing fish liver cells (PLHC-1) were conducted to evaluate the toxicity of methanolic extracts derived from intact mesoplastics and after simulated photodegradation. LC-MS analysis of the methanolic extracts revealed the presence of organophosphate esters, phthalates, and phthalate alternatives. The extracts from photodegraded plastics generally showed higher cytotoxicity, ability to generate reactive oxygen species (ROS), and genotoxicity (micronuclei formation) than those from intact mesoplastics. All the extracts induced EROD activity in PLHC-1 cells, indicating the presence of significant amounts of CYP1A inducers in beach mesoplastics. Thus, mesoplastics contain chemicals able to induce cytotoxicity and genotoxicity in PLHC-1 cells, and further photodegradation of mesoplastics facilitates the release of additional chemicals, increasing the overall toxicity. This work also highlights the usefulness of cell-based assays to better define the risks of plastic pollution.

Age matters: Comparing life-stage responses to diluted bitumen exposure in coho salmon (Oncorhynchus kisutch)

Millions of liters of diluted bitumen (dilbit), a crude oil product from Canada's oil sands region, is transported through critical Pacific salmon habitat each day. While the toxicity of the water-soluble fraction of dilbit (WSFd) to early life-stages of salmon is known, quantitative data on life-stage differences in sensitivity to WSFd is missing. To fill this knowledge gap, we exposed two juvenile life-stages of coho salmon (O. kisutch) in parallel to very low (parts per billion), environmentally-relevant concentrations of WSFd for acute (48 h) and sub-chronic (4 wk) durations. The relative sensitivities of the two life-stages (fry and parr) were assessed by comparing the timing and magnitude of biological responses using common organismal and molecular endpoints of crude oil exposure. A significant reduction in body condition occurred in both fry and parr after 4 wk exposure to WSFd. Both life-stages also experienced a concentration-dependent decrease in time-to-loss-of-equilibrium during a hypoxia challenge test at both 48 h and 4 wk of exposure. Although organismal responses were similar, molecular responses were distinct between life-stages. In general, unexposed fry had higher baseline values of hepatic phase I biotransformation indicators than unexposed parr, but induction of EROD activity and cyp1a mRNA expression in response to WSFd exposure was greater in parr than in fry. Neither gst nor hsp70 mRNA expression, markers of phase II biotransformation and cell stress, respectively, were reliably altered by WSFd exposure in either life-stage. Taken together, results of this study do not support differential sensitivities of coho fry and parr to WSFd. All the same, the potential for ontogenic differences in the expression and induction of phase I biotransformation need to be considered because age does matter for these endpoints if they are used as bioindicators of exposure in post-spill impact assessments.

The Cichlidae fish Geophagus brasiliensis (Quoy & Gaimard, 1824) has suitability as a sentinel species for changes caused by xenobiotics?

Industrial wastewater and agricultural practices are among the main activities discharging organic pollutants, such as Polychlorinated biphenyl (PCB) congeners and organochlorine insecticides (e.g. DDT - dichloro diphenyl trichloroethane), to the environment. In this study, we used the native Cichlidae fish species Geophagus brasiliensis as a sentinel to evaluate the hepatic 7-ethoxyresorufin-O-deethylase EROD activity, a biomarker of exposure to CYP1A-inducing pollutants, to assess the bioavailability of xenobiotics in two reaches of a large lotic system in Southeast Brazil: a less disturbed area (site 1) in the upper stretch, and an area in the middle stretch, which receives various industrial and agricultural effluents from upstream cities (site 2). In addition, G. brasiliensis were exposed to a single dose of 50 mg kg-1 beta-naphthoflavone (BNF) or of 50 mg kg-1 dimethylbenzoanthracene (DMBA) to test the effects on, respectively, the hepatic EROD activity in S9 supernatant fraction, and the frequency of micronucleated erythrocytes three days after the i.p. treatments, and compared to an unexposed group, to test its potential as a sentinel for biomonitoring studies. The EROD activity was approximately two-fold higher in fish from the impacted stretches than in fish from the less disturbed stretches (p < 0.05). Micronuclei (MN) frequency was also significantly different (p < 0.01) in DMBA-treated fish. The induced EROD activity in the impacted site suggests that organochlorinated pollutants are reaching the biota of the Paraíba do Sul River, confirming the suitability of Geophagus brasiliensis as a useful sentinel species to detect changes caused by xenobiotics

Assessing the effects of textile leachates in fish using multiple testing methods: From gene expression to behavior

The textile industry, while of major importance in the world economy, is a toxic industry utilizing and emitting thousands of chemical substances into the aquatic environment. The aim of this project was to study the potentially harmful effects associated with the leaching of chemical residues from three different types of textiles: sportswear, children’s bath towels, and denim using different fish models (cell lines, fish larvae and juvenile fish). A combination of in vitro and in vivo test systems was used. Numerous biomarkers, ranging from gene expression, cytotoxicity and biochemical analysis to behavior, were measured to detect effects of leached chemicals. Principle findings indicate that leachates from all three types of textiles induced cytotoxicity on fish cell lines (RTgill-W1). Leachates from sportswear and towels induced mortality in zebrafish embryos, and chemical residues from sportswear reduced locomotion responses in developing larval fish. Sportswear leachate increased Cyp1a mRNA expression and EROD activity in liver of exposed brown trout. Leachates from towels induced EROD activity and VTG in rainbow trout, and these effects were mitigated by the temperature of the extraction process. All indicators of toxicity tested showed that exposure to textile leachate can cause adverse reactions in fish. These findings suggested that chemical leaching from textiles from domestic households could pose an ecotoxicological threat to the health of the aquatic environment.

An active biomonitoring approach using three-spined stickleback (Gasterosteus aculeatus, L.) to assess the efficiency of a constructed wetland as tertiary treatment of wastewater

The present work aimed to assess the efficiency of a constructed wetland as tertiary treatment on urban wastewater with a multibiomarker approach using caged three-spined sticklebacks (Gasterosteus aculeatus L.). Fish were caged on three sites: at the entrance of the Constructed Wetland (CW), directly inside the wastewater effluent, at the exit of the CW, and in a weakly impacted site considered as a reference. After 21 days of caging, sticklebacks state of health was assessed using several biomarkers representing some biological functions such as innate immune and antioxidant systems, biotransformation enzymes, reproduction parameters and synaptic transmission. A strong inhibition of the innate immune system, an induction of EROD activity and an alteration of the hepatosomatic index were observed in fish caged at the entrance of the CW compared to those caged in the reference site. In addition, wastewater effluent induced a decrease of antioxidant system without induced oxidative damage on cell membranes. No improvement of these biomarkers was observed for antioxidant parameters at the exit of the CW. However, in fish caged at the exit of the CW, the EROD induction observed at the entrance was reduced and the innate immune system presented the same level compared to fish caged in the reference site, underlying the beneficial effect of the CW for these parameters. Integrated Biomarker response (IBR) was equal to 25.2 at the entrance of the CW and 17.4 at the exit of the CW which highlighted the global positive effect of the CW on water quality based on fish biomarker measurement.

Nanoplastics Decrease the Toxicity of a Complex PAH Mixture but Impair Mitochondrial Energy Production in Developing Zebrafish.

Plastics are recognized as a worldwide threat to the environment, possibly affecting human health and wildlife. Small forms of plastics such as micro- and nanoplastics can interact with other organic contaminants, potentially acting as chemical carriers and modulating their toxicity. In this study, we investigated the toxicity of polystyrene nanoparticles (Nano-PS) and a real-world environmental PAH mixture (Elizabeth River Sediment Extract, ERSE, comprised of 36 detected PAHs) to zebrafish embryos and larvae. Embryos were exposed to Nano-PS (0.1-10 ppm) or ERSE (0.1-5% v/v, equivalent to ΣPAH 5.07-25.36 ppb) or coexposed to a combination of both. Larvae exposed to Nano-PS did not exhibit developmental defects, while larvae exposed to ERSE (2-5%) showed classic signs of PAH toxicity such as heart malformation and deformities in the jaw, fin, and tail. ERSE (5%) also impaired vascular development in the brain. When coexposed, Nano-PS decreased the developmental deformities and impaired vascular development caused by ERSE. This was strongly correlated to the lower PAH bioaccumulation detected in the coexposed animals (whole larvae, as well as the yolk sac, brain, and heart). Our data suggest that PAHs are sorbing to the surface of the Nano-PS, decreasing the concentration, uptake, and toxicity of free PAHs during the exposure. Such sorption of PAHs increases the agglomeration rate of Nano-PS during the exposure time, potentially decreasing the uptake of Nano-PS and associated PAHs. Despite that, similar induction of EROD activity was detected in animals exposed to ERSE in the presence or not of Nano-PS, suggesting that enough PAHs were accumulated in the organisms to induce cellular defense mechanisms. Nano-PS exposure (single or combined with ERSE) decreased the mitochondrial coupling efficiency and increased NADH production, suggesting an impairment on ATP production accompanied by a compensatory mechanism. Our data indicate that nanoplastics can sorb contaminants and potentially decrease their uptake due to particle agglomeration. Nanoplastics also target and disrupt mitochondrial energy production and act as vectors for the mitochondrial uptake of sorbed contaminants during embryonic and larval stages. Such negative effects of nanoplastics on energy metabolism and efficiency could be detrimental under multiple-stressors exposures and energy-demanding scenarios, which remains to be validated.

Toxicity assessment of pollutants sorbed on environmental microplastics collected on beaches: Part II-adverse effects on Japanese medaka early life stages

While microplastics are present in great abundance across all seas and oceans, little is known about their effects on marine life. In the aquatic environment, they can accumulate a variety of chemicals and can be ingested by many marine organisms including fish, with chronic physical and chemical effects. The purpose of this paper is to evaluate the toxic effects of pollutants sorbed at the surface of environmental microplastics (MPs), collected on various beaches from three islands of the Pacific Ocean. Developmental toxicity of virgin MPs or artificially coated with B[a]P and environmental MPs from Easter Island, Guam and Hawaii was evaluated on embryos and prolarvae of Japanese medaka. Mortality, hatching success, biometry, malformations, EROD activity and DNA damage were analyzed after exposure to DMSO extracts. No toxicity was observed for extracts of virgin MPs whatever the endpoint considered. Extracts of virgin MPs coated with 250 µg.g−1 of B(a)P induced lethal effects with high embryo mortality (+81%) and low hatching rate (−28%) and sublethal effects including biometry and swimming behavior changes, increase of EROD activity (+94%) and DNA damage (+60%). Environmental MPs collected on the three selected islands exhibited different polymer, pollutant and toxicity patterns. The highest toxicity was detected for MPs extract from Hawaï with head/body length and swimming speed decreases and induction of EROD activity and DNA stand breaks. This study reports the possible sublethal toxicity of organic pollutants sorbed on MPs to fish early life stages.

Gene expression patterns and related enzymatic activities of detoxification and oxidative stress systems in zebrafish larvae exposed to the 2,4-dichlorophenoxyacetic acid herbicide

The present study aims to assess the effects of 2,4-D herbicide on biotransformation and oxidative stress status of zebrafish larvae. Animals were exposed to a range of sublethal concentrations (0.02–0.8 mg/L) and biomarkers at transcriptomic level and biochemical level were assessed. Chemical analysis with showed that the bioaccumulation of 2,4-D in 96 hpf zebrafish larvae were increased in a concentration-dependent manner. This herbicide induced significant effects at both gene expression and enzymatic activities levels after at 96 hpf. Results of mRNA expression showed a differential transcription regulation with all target genes depending on the tested concentrations. The mRNA level of gsr and cyp1a were up regulated at the highest dose of herbicide (0.8 mg/L). The gene expression of gstp1 showed an up regulation at lower dose (0.02 mg/L) and a down regulation at the highest dose (0.8 mg/L) of 2,4-D. A significant induction of EROD activity and inhibition of GST activity were noted in groups exposed to 0.8 mg/L of 2,4-D. Considering the antioxidant defenses, the activity of CAT was increased in larvae exposed to 0.8 mg/L of herbicide and GPx activity was induced at lower doses of 2,4-D (0.02 and 0.051 mg/L). Moreover, peroxidative damage, assessed as MDA content, was markedly increased in larvae exposed to high 2,4-D concentration. Overall, the present study data indicate that bioaccumulation of 2,4-D in 96 hpf zebrafish larvae and alterations in detoxification and oxidative stress related parameters, likely associated with ROS production, which may endanger the embryo-larval stages development of fish.

Comparative biomarker responses in Japanese medaka (Oryzias latipes) exposed to benzo[a]pyrene and challenged with betanodavirus at three different life stages

It is now well documented that several contaminants can modulate the fish immune system, leading to disrupted host resistance against pathogens and increased incidence of disease. Since fish are usually co-exposed to chemicals and pathogens in the natural environment, analysis of the immunotoxic effects of pollutants is particularly relevant. The authorities in the European Union have recommended the development of toxicity assays on cell cultures and embryos, as an alternative to testing in vertebrates. This is why in our study, a fish immune challenge assay was developed for the early life stages of Japanese medaka to evaluate and compare the relevance of new biomarkers. Fish were exposed to benzo[a]pyrene (BaP), a model pollutant, for 8days at the embryonic stage, or for 48h at the larvae and juvenile stages, and fish were infected with betanodavirus by bath-challenge of 106TCID50/mL. Biometric changes and induction of malformations were observed after embryonic exposure. DNA damage and induction of EROD activity were recorded at the end of all chemical exposures. Viral infection increased the mortality rate significantly and disturbed the behavior of fish after light stimulation. While BaP exposure increased swimming speed, betanodavirus infection slowed swimming activity. In larvae co-exposed to BaP and the virus, the viral titer in the whole body was higher than in fish infected only with the virus. This study highlighted the sensitivity and usefulness of the immune challenge assay on the early life stages of Japanese medaka to evaluate the toxic effects of pollutants.

Butyrate alters expression of cytochrome P450 1A1 and metabolism of benzo[a]pyrene via its histone deacetylase activity in colon epithelial cell models.

Butyrate, a short-chain fatty acid produced by fermentation of dietary fiber, is an important regulator of colonic epithelium homeostasis. In this study, we investigated the impact of this histone deacetylase (HDAC) inhibitor on expression/activity of cytochrome P450 family 1 (CYP1) and on metabolism of carcinogenic polycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP), in colon epithelial cells. Sodium butyrate (NaBt) strongly potentiated the BaP-induced expression of CYP1A1 in human colon carcinoma HCT116 cells. It also co-stimulated the 7-ethoxyresorufin-O-deethylase (EROD) activity induced by the 2,3,7,8-tetrachlorodibenzo-p-dioxin, a prototypical ligand of the aryl hydrocarbon receptor. Up-regulation of CYP1A1 expression/activity corresponded with an enhanced metabolism of BaP and formation of covalent DNA adducts. NaBt significantly potentiated CYP1A1 induction and/or metabolic activation of BaP also in other human colon cell models, colon adenoma AA/C1 cells, colon carcinoma HT-29 cells, or in NCM460D cell line derived from normal colon mucosa. Our results suggest that the effects of NaBt were due to its impact on histone acetylation, because additional HDAC inhibitors (trichostatin A and suberanilohydroxamic acid) likewise increased both the induction of EROD activity and formation of covalent DNA adducts. NaBt-induced acetylation of histone H3 (at Lys14) and histone H4 (at Lys16), two histone modifications modulated during activation of CYP1A1 transcription, and it reduced binding of HDAC1 to the enhancer region of CYP1A1 gene. This in vitro study suggests that butyrate, through modulation of histone acetylation, may potentiate induction of CYP1A1 expression, which might in turn alter the metabolism of BaP within colon epithelial cells.

In vivo and in vitro effects of tunnel wash water and traffic related contaminants on aquatic organisms

In order to maintain the construction and safety of road tunnels, they are routinely washed. The wash water appears to be highly polluted with a plethora of contaminants in elevated concentrations. In addition, new and emerging compounds are likely to occur. The discharge water has shown acute toxic and sub-lethal effects in several organisms. In this study, ecotoxicity tests with algae (Pseudokirchneriella subcapitata) and in vitro tests with primary rainbow trout (Oncorhynchus mykiss) hepatocytes were used to characterize the effect of TWW from three different tunnels. In addition, selected N- and Cl-PAHs were tested for cytotoxicity, EROD activity and CYP1A protein production. TWW samples and/or extracts from two tunnels reduced the algal growth and induced cytotoxicity, EROD activity and CYP1A protein production in vitro. Four of the eight tested Cl- and N-substituted PAHs induced EROD activity and CYP1A protein production at micro-molar concentrations. N-PAHs were detected in samples from the tunnel wash, highlighting substituted PAHs as potentially important traffic-related contaminants.

Assessment of biological effects of environmental pollution in Mersin Bay (Turkey, northeastern Mediterranean Sea) using Mullus barbatus and Liza ramada as target organisms

The increasing emphasis on the assessment and monitoring of marine ecosystems has revealed the need to use appropriate biological indicators for these areas. Enzyme activities and histopathology are increasingly being used as indicators of environmental stress since they provide a definite biological end-point of pollutant exposure. As part of an ecotoxicological assessment of Mersin Bay, EROD enzyme activity and histopathological response in selected organs and tissues of two species of fish, Mullus barbatus (red mullet) and Liza ramada (thinlip grey mullet), captured from area were examined. Pollutant (Organochlorines (OC), alkylphenols (APs) and BPA) levels and biomarker responses in tissue samples were evaluated together for their potential to alter the metabolism and cellular aspects in liver and gonad. Elevated induction of EROD activity and histopathological alterations in contaminated samples from Mersin Bay was observed compared to reference site indicating the exposure to potential pollutants.

Temporal variations in embryotoxicity of Lake Ontario American eel (Anguilla rostrata) extracts to developing Fundulus heteroclitus

The recruitment of American eel (Anguilla rostrata) juveniles to Lake Ontario (LO), Canada has declined significantly since the 1980s. To investigate the possible contribution of maternally-transferred persistent organic pollutants (POPs) to this decline, this study measured temporal variations in the toxicity of complex organic mixtures extracted from LO American eels captured in 1988, 1998 and 2008 to developing Fundulus heteroclitus exposed by intravitelline (IVi) injection. The 1988 and 1998 eel extracts were most toxic, causing a pattern of sublethal embryotoxic responses similar to those previously reported in F. heteroclitus embryos exposed to single dioxin-like compounds (DLCs): stunted growth, craniofacial deformities, EROD activity induction, and reduced predatory capacities. The potency of extracts declined over time; the only significant effect of the 2008 eel extracts was EROD induction. The chemically-derived TCDD-TEQs of eel extracts, calculated using measured concentrations of some DLCs and their relative potencies for F. heteroclitus, overestimated their potency to induce EROD activity possibly due to interactions among POPs. Other POPs measured in eel extracts (non-dioxin-like PCBs, PBDEs and organochlorinated pesticides) did not appear to be important agonistic contributors to the observed toxicity. The toxicity of the complex mixtures of POPs measured in LO eels may have been underestimated as a result of several factors, including the loss of POPs during extracts preparation and a focus only on short-term effects. Based on the model species examined, our results support the hypothesis that contamination of LO with DLCs may have represented a threat to the American eel population through ecologically-relevant effects such as altered larval prey capture ability. These results prioritize the need to assess early life stage (ELS) toxicity of DLCs in Anguilla species, to investigate long-term effects of complex eel extracts to ELS of fish, and to develop biomarkers for potential effects in eel ELS sampled in the field.

Hypoxia and the pharmaceutical diclofenac influence the circadian responses of three-spined stickleback

Pollution with low concentrations of pharmaceuticals, especially when combined with low-oxygen conditions (hypoxia), is a threat to aquatic ecosystems worldwide. The non-steroidal anti-inflammatory drug diclofenac is commonly detected in wastewater effluents, and has potential to accumulate in the bile of fish. Diclofenac has been shown to activate aryl hydrocarbon receptor (AHR), which induces transcription in the metabolic enzyme cytochrome P450 1a (cyp1a). Previously, crosstalk has been shown to occur between AHR and hypoxia inducible factor 1 (HIF-1). In addition, both of these transcription factors interact with the proteins regulating circadian (24-h) rhythms in vertebrates. Yet little is known about the significance of these interactions during simultaneous exposure to chemicals and hypoxia in fish in vivo. We exposed wild-caught three-spined sticklebacks (Gasterosteus aculeatus) to diclofenac (1μg/L, 14 days), hypoxia (2.0mg/L, up to 24h) and the combination of both. We then analyzed markers of chemical biotransformation (EROD activity, cyp1a and ahr mRNA levels), glycolysis (lactate dehydrogenase (LDH) enzyme activity, ldh and enolase 1a mRNA levels), and the transcription of core circadian clock genes clock and period 1 in liver tissue. Samples were taken at three time points during the light period in order to address disturbances in the circadian variation of metabolic processes. The results show that mRNA levels and LDH activity tended to be lowest before the dark period, but this pattern was disturbed by hypoxia and diclofenac. Diclofenac and hypoxia co-exposure induced EROD activity more strongly than diclofenac exposure alone, while cyp1a mRNA level was increased also by hypoxia and diclofenac alone. LDH activity and mRNA expression showed a clear time-dependent response during hypoxia, which is consistent with the previously suggested decreased accumulation of HIF-1 during the dark period. Furthermore, LDH activity and transcription was disturbed by diclofenac, indicating important effects of environmental pollutants in disturbing natural acclimation. This study demonstrates the need for more studies to understand the potential disturbances in endogenous rhythms caused by environmental pollution in natural populations.

Polar polycyclic aromatic compounds from different coal types show varying mutagenic potential, EROD induction and bioavailability depending on coal rank

Investigations of the bioavailability and toxicity of polycyclic aromatic compounds (PAC) have rarely considered the heterogeneity of coals and the impact of more polar PAC besides polycyclic aromatic hydrocarbons (PAH).Earlier, we investigated the toxicity of eight heterogeneous coals and their extracts. In the present study, the hazard potential with respect to mechanism-specific toxicity of polar fractions of dichloromethane extracts from coals was studied. Polar extract fractions of all coal types except for anthracite induced EROD activity (determined in RTL-W1 cells), independent of coal type (Bio-TEQs between 23±16 and 52±22ng/g). The polar fractions of all bituminous coal extracts revealed mutagenic activity (determined using the Ames Fluctuation test). No significant mutation induction was detected for the polar extract fractions from the lignite, sub-bituminous coal and anthracite samples, which indicates a higher dependency on coal type for polar PAC here.Additionally, information on bioavailability was derived from a bioaccumulation test using the deposit-feeding oligochaete Lumbriculus variegatus which was exposed for 28days to ground coal samples. Despite the high toxic potential of most coal extracts and a reduced biomass of Lumbriculus in bituminous coal samples, bioaccumulation of PAH and mortality after 28days were found to be low. Limited bioaccumulation of PAH (up to 3.6±3.8mg/kg EPA-PAH) and polar PAC were observed for all coal samples. A significant reduction of Lumbriculus biomass was observed in the treatments containing bituminous coals (from 0.019±0.004g to 0.046±0.011g compared to 0.080±0.025g per replicate in control treatments).We conclude that bioavailability of native PAC from coals including polar PAC is low for all investigated coal types. In comparison to lignite, sub-bituminous coals and anthracite, the bioavailability of PAC from bituminous coals is slightly increased.

Induction of Fish Biomarkers by Synthetic-Based Drilling Muds

The study investigated the effects of chronic exposure of pink snapper (Pagrus auratus Forster), to synthetic based drilling muds (SBMs). Fish were exposed to three mud systems comprised of three different types of synthetic based fluids (SBFs): an ester (E), an isomerized olefin (IO) and linear alpha olefin (LAO). Condition factor (CF), liver somatic index (LSI), hepatic detoxification (EROD activity), biliary metabolites, DNA damage and stress proteins (HSP-70) were determined. Exposure to E caused biologically significant effects by increasing CF and LSI, and triggered biliary metabolite accumulation. While ester-based SBFs have a rapid biodegradation rate in the environment, they caused the most pronounced effects on fish health. IO induced EROD activity and biliary metabolites and LAO induced EROD activity and stress protein levels. The results demonstrate that while acute toxicity of SBMs is generally low, chronic exposure to weathering cutting piles has the potential to affect fish health. The study illustrates the advantages of the Western Australian government case-by-case approach to drilling fluid management, and highlights the importance of considering the receiving environment in the selection of SBMs.

EROD activity induction in peripheral blood lymphocytes, liver and brain tissues of rats orally exposed to polycyclic aromatic hydrocarbons

Little is known in terms of multi-matrix cytochrome P450 activity induction under repeated oral exposure to planar halogenated and polycyclic aromatic hydrocarbons (PHH, PAH). In the present study, 60 rats were daily exposed, during 28 days, to oral ingestion of a mixture consisting of phenanthrene, pyrene and benzo(a)pyrene at 0, 6 or 600 μg/day. EROD activity, reflecting almost exclusively CYP1A1 and CYP1B1 activities, was measured in brain and liver microsomes as well as in peripheral blood lymphocytes (PBLs). All induction kinetics could be appropriately fitted using logistic-like models. After 28 days of exposure to a 6 μg/day dose, EROD activity was found to be 91, 152 and 94-fold increased in lymphocytes, liver and brain, respectively, compared to day 0. Plateau activities could be appropriately fitted versus ingested doses using Hill or Michaelis-Menten models. Correlations between matrices made it possible to conclude that EROD activity in PBL should be considered as a sensitive, convenient and non-destructive approach for (i) evaluating EROD activity in liver, which was found to represent 98% of the observed EROD activities in the three tested matrices and (ii) evaluating oral exposure of homogeneous groups of farm animals (race, diet) to CYP inducing PAH and PHH.

Biomarker responses in persian sturgeon (Acipenser persicus) exposed to benzo-a-pyrene and beta-naphthoflavone

Biotransformation enzymes of xenobiotics (ethoxyresorufin-O-deethylase, cytochrome P4501A1 content and glutathione-S-transferase) were investigated in the liver of Persian Sturgeon (Acipenser persicus) after a 96-hour exposure to polycyclic aromatic hydrocarbons (PAHs), premutagenic benzo[a]pyrene (BaP) and beta-naphthoflavone (BNF). The fish were injected 10 mg/kg wet-body weight in corn oil for 96 hours every days. Ethoxyresorufin-O-deethylase activity (EROD) and glutathione s-transferase activity (GST) were measured in the fish liver. Cytochrome P4501A1 (CYP1A1) content was estimated by indirect enzyme-linked immunosorbent assay (ELISA). The response appeared as early as 12 hours post exposure. A time-dependent response was observed in the EROD activity, being significantly higher at 48 hours post exposure to 10 mg/kg of BaP. The greatest induction occurred in the fish treated with 10 mg/kg BaP, in which a 32.1- fold increase in EROD activity was observed. Results showed that EROD activity in A. persicus is significantly increased by BaP and BNF treatments. Both chemicals showed higher values of EROD activity compared to the liver CYP1A content. There was a rise in glutathione-S-transferase activity in fish exposed to BNF, but no increase was observed in fish treated with BaP. The results showed that hepatic CYP1A expression in terms of induction of EROD activity might be suited as a biomarker of organic contamination in aquatic environments and led to lower sensitivity of the second phase in the detoxification enzyme.

Effects of individual and a mixture of pharmaceuticals and personal‐care products on cytotoxicity, EROD activity and ROS production in a rainbow trout gonadal cell line (RTG‐2)

The presence of pharmaceuticals and personal-care products (PPCPs) in aquatic environments is of concern. Although measured concentrations of individual substances are low, little consideration has been given to the likely chronic nature of the exposures or to the potential for mixture effects. The purpose of the present study was to use the RTG-2 rainbow trout cell line to analyse sub-lethal and cytotoxic effects of PPCPs present in a wastewater-treatment-plant (WWTP) effluents and their mixtures. Interactions with cytochrome P450 1A enzyme, oxidative stress, cellular senescence and cell viability were assessed using 7-ethoxyresorufin-o-deethylase (EROD), reactive oxygen species (ROS), ß-galactosidase (ß-gal) and neutral red (NR) uptake assays, respectively. Not all of the compounds that were tested exhibited significant effects. The lowest-observed-effect concentrations and half maximal effective concentrations (EC50 ) were within the range 0.15 to 784.47 µg l(-1) . Clear dose-response curves were found for cells exposed to different mixtures of PPCPs. The lowest-observed-effect concentrations and concentrations causing EC50 were within the range 0.05 to 54.61 µg l(-1) . Four out the seven tested mixtures induced EROD activity. ROS production was detected in two mixtures. The ß-gal inhibition response was observed in six out the seven tested mixtures and occurred at a higher concentration than was observed for EROD induction activity or ROS generation. The present study clearly shows that the stress response through which cells mount a homeostatic response to toxicants can be potentially used for an initial, rapid and cost-effective assessment of the complex mixtures of PPCP that present in WWTP effluents are difficult and expensive to analyse chemically.

Effects of 2,4-dinitrotoluene exposure on enzyme activity, energy reserves and condition factors in common carp ( Cyprinus carpio)

In this study relative condition factor (RCF) and hepatosomatic index (HSI) as well as the available energy reserves of common carp ( Cyprinus carpio) by 2,4-DNT semi-static bioassay were determined and linked to effects of enzymes in liver tissues. Fish were exposed at sublethal concentrations of 2,4-DNT (0.13 μg/L, 0.1, 0.5, 1.0 mg/L) for 7 and 15 d. Based on the results, there was no significant change in all parameters measured in fish exposed to 2,4-DNT at environmental related concentration, but 2,4-DNT stress in fish exposed to higher concentrations reflected the significant changes of physiological and biochemical responses. 2,4-DNT stress resulted in EROD activity induction in the liver, and the levels of EROD activity ranged from 0.39- to 1.83-fold higher than control. For GK, Na +/K +-ATPase, and GST, these enzyme activity continued to decline after exposure to 0.1, 0.5 and 1.0 mg/L 2,4-DNT, whereas the trend on GK and Na +/K +-ATPase was more obvious than GST. Through principal component analysis, effects by 2,4-DNT-stress in each test group were distinguished. Additionally, indications of a trade-off between metabolic cost of toxicant exposure and processes vital to the survival of the organism were seen at the enzyme activity level as well as on higher levels of biological organization.