Lethal and sublethal toxicity assessment of Bacillus thuringiensis var. israelensis and Beauveria bassiana based bioinsecticides to the aquatic insect Chironomus riparius.

Despite being considered environmentally safe, a deeper environmental risk assessment is needed for microbial insecticides; special attention should be devoted to their sublethal toxicity to non-target species. This study evaluated effects of VectoBac® 12AS - VB (based on the bacterium Bacillus thurigiensis var. israelensis) and Naturalis®-L - NL (based on the fungus Beauveria bassiana) on the aquatic insect Chironomus riparius life-history and biochemical responses. Acute tests estimated a 48 h-LC50 (median lethal concentration) of 1.85 µg/L (VB) and 34.7 mg/L (NL). Under sublethal exposure, VB decreased adults' emergence (LOEC - lowest observed effect concentration of 80 ng/L) while NL impaired larval growth (LOEC of 0.32 mg/L) and delayed emergence (LOEC of 2 mg/L for males and 0.8 mg/L for females). Despite not being monotonic, phenoloxidase activity increased (LOEC of 20 ng/L (VB) and 2 mg/L (NL)), suggesting activation of the immune system. There were no indications of oxidative damage nor neurotoxicity. Catalase activity was stimulated with all VB treatments, possibly associated with detoxification of immune response products. Under NL exposure, glutathione-S-transferase activity increased but did not show a dose-dependent response and, total glutathione decreased in the highest concentration. Exposure to both formulations caused the increase in protein content, while carbohydrate and lipids were not altered. This study revealed the susceptibility of C. riparius to VB and NL at concentrations below the ones recommended for field application, with potential population-level effects. These results add important information for the risk assessment of these microbial insecticides in aquatic ecosystems, considering relevant sublethal endpoints and raising concern about the adverse effects on non-target aquatic organisms.

Influence of biochar particle size on biota responses.

Despite the increasing interest for biochar as a soil amendment, a knowledge gap remains on its impacts on non-target soil and aquatic species. We hypothesised that biochar particle size and application rate can play a role in the toxicity to biota. Pine woodchip biochar was incorporated in a clean soil at three particle size classes: small (<0.5 mm), medium (1-2 mm), and large (<4 mm), and at two concentrations: 1% and 6% w/w. A laboratory screening with earthworm Eisenia andrei avoidance behaviour bioassay was carried out to test the most adequate application rates, particle sizes and soil-biochar pre-incubation period. Thereafter, a 28-day greenhouse microcosm experiment was conducted as an ecologically more representative approach. Survival, vertical distribution and weight changes of E. andrei, and bait-lamina consumption were recorded. Soil leachates from the microcosms were collected to evaluate their impact on Daphnia magna immobilisation and Vibrio fischeri (Microtox®) bioluminescence. A feeding experiment with E. andrei was also performed to address earthworm weight changes and to conduct a screening of PAH-type metabolites in their tissue. The 6% <0.5 mm treatment pre-incubated for 96 h induced significant avoidance of the earthworms. Significantly lower bait-lamina consumption was observed in microcosms for the 6% <0.5 mm treatment. Moreover, particle size was a statistically significant factor regarding the loss of weight in the feeding experiment and higher concentration of naphthalene-type metabolites detected in E. andrei tissue, when exposed to <0.5 mm biochar particles. The leachates had no adverse effects on the aquatic species. The results suggest that particles <0.5 mm of pine woodchip biochar can pose sub-lethal effects on soil biota.

Involvement of the antioxidant system in differential sensitivity of Carcinus maenas to fenitrothion exposure.

Carcinus maenas is an invertebrate with worldwide distribution and high ability to adapt to different environments, which is frequently used in environmental monitoring. Despite this, it is not clear how historical exposure to moderate contamination may influence sensitivity to further chemical stress in this important decapod species. This study investigated differential responses to organophosphate fenitrothion of C. maenas from a moderately contaminated estuary and a low impacted one, using in vitro and in vivo biomarker assays. To clarify potential differences in sensitivity, a biochemical characterisation of muscle cholinesterases was first performed. The results indicated acetylcholinesterase (AChE) as the main form present in C. maenas muscle. Exposure assays revealed that crabs from the moderately contaminated site were less sensitive to fenitrothion showing lower AChE inhibition than those from the low impacted site. Other biomarker changes detected in these animals were: increased anaerobic metabolism (muscle lactate dehydrogenase), enhanced phase II biotransformation (glutathione S-transferases in the digestive gland) and antioxidant defences (i.e., activities of glutathione reductase, glutathione peroxidase and catalase, and levels of total glutathiones in the digestive gland). Altogether, the results pointed out a role for the glutathione redox system towards tolerance to fenitrothion exposure.

Biochemical characterization of cholinesterases in Enchytraeus albidus and assessment of in vivo and in vitro effects of different soil properties, copper and phenmedipham.

Enchytraeus albidus are important organisms of the soil biocenosis, used as standard test species in environmental risk assessment. The inhibition of cholinesterases (ChE) activity of several species has been widely used to assess the exposure and effects of anti-cholinesterase environmental contaminants. Several studies have shown the association between ChE activity inhibition and adverse effects on behaviour and survival. Extensive studies addressing survival and behavioural endpoints, as well as other biomarkers, have been done in E. albidus with different types of soil contaminants. The main objectives of this study were: (1) to characterize biochemically the ChE present in the soluble post-mitochondrial fraction of E. albidus whole body homogenates, using different substrates and selective inhibitors; (2) to assess the in vivo effects of copper, phenmedipham and different soil properties (pH, organic matter, clay) on the ChE activity; (3) to assess the in vitro effects of copper and phenmedipham on the ChE activity. The results suggest the presence of one ChE in the soluble post-mitochondrial fraction of E. albidus whole body homogenates, which displays properties of both acetylcholinesterase and pseudocholinesterase considering the typical mammalian enzymes. It is also shown that ChE activity is not inhibited by exposure to different soil properties and that copper and phenmedipham inhibited ChE activity both in in vivo and in in vitro conditions and therefore ChE inhibition seems to be a robust biomarker for this herbicide and this heavy metal. This study showed that ChE activity in E. albidus might be correlated to previously determined higher level effects like survival and reproduction, as well as avoidance behaviour.

Biochemical effects and polycyclic aromatic hydrocarbons (PAHs) in senegal sole (Solea senegalensis) from a Huelva estuary (SW Spain).

Relations between several stress oxidative biomarkers and aromatic polycyclic hydrocarbon (PAH) concentrations have been studied in wild sole, Solea senegalensis collected in the vicinity of a petrochemical industry. Antioxidant enzyme activities in eco-toxicological studies constitute excellent markers for exposure to a large variety of pollutants. The 16 PAHs in sediment as well as oxidative damage (LPO), activity of catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), glutathione reductase (GR) and PAHs type metabolites in sole liver were analysed. Significant correlations (p<0.05) were established between some biomarkers as GST, GPx and CAT and PAHs metabolites in liver (naphthalene, pyrene and phenanthrene) and PAHs concentrations in sediments (fluoranthene, acenaphthene, anthracene and chrysene). PAHs accumulated in the sediment and organisms are inducers of antioxidant defences. GST, GPx and CAT were robust biomarkers showing correlations with both PAHs in sediments and liver PAH metabolites showing different responses to low and high molecular weight PAHs.

Acute effects of copper and mercury on the estuarine fish Pomatoschistus microps: linking biomarkers to behaviour.

The main objective of the present study was to investigate possible links between biomarkers and swimming performance in the estuarine fish Pomatoschistus microps acutely exposed to metals (copper and mercury). In independent bioassays, P. microps juveniles were individually exposed for 96 h to sub-lethal concentrations of copper or mercury. At the end of the assays, swimming performance of fish was measured using a device specially developed for epibenthic fish (SPEDE). Furthermore, the following biomarkers were measured: lipid peroxidation (LPO) and the activity of the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH), glutathione S-transferases (GST), 7-ethoxyresorufin-O-deethylase (EROD), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx). LC(50)s of copper and mercury (dissolved throughout metal concentrations) at 96h were 568 microg L(-1), and 62 microg L(-1), respectively. Significant and concentration-dependent effects of both metals on swimming resistance and covered distance against water flow were found at concentrations equal or higher than 50 microg L(-1) for copper and 3 microg L(-1) for mercury (dissolved throughout metal concentrations). These results indicate that SPEDE was efficacious to quantify behavioural alterations in the epibenthic fish P. microps at ecologically relevant concentrations. Significant alterations by both metals on biomarkers were found including: inhibition of AChE and EROD activities, induction of LDH, GST and anti-oxidant enzymes, and increased LPO levels, with LOEC values ranging from 25 to 200 microg L(-1), for copper and from 3 to 25 microg L(-1) for mercury (dissolved throughout metal concentrations). Furthermore, significant and positive correlations were found between some biomarkers (AChE and EROD) and behavioural parameters, while negative correlations were found for others (LPO, anti-oxidant enzymes and LDH) suggesting that disruption of cholinergic function through AChE inhibition, decreased detoxification capability due to EROD inhibition, additional energetic demands to face chemical stress, and oxidative stress and damage may contribute to decrease the swimming performance of fish. Since a reduced swimming capability of fish may reduce their ability to capture preys, avoid predators, and interfere with social and reproductive behaviour, the exposure of P. microps to copper and/or mercury concentrations similar to those tested here may decrease the fitness of wild populations of this species.

Effects of natural and chemical stressors on Enchytraeus albidus: can oxidative stress parameters be used as fast screening tools for the assessment of different stress impacts in soils?

Enchytraeids are important organisms of the soil biocenosis. They improve the soil pore structure and the degradation of organic matter. These organisms are used in standardized testing, using survival and reproduction (6 weeks) as endpoints. The use of biomarkers, linked to ecologically relevant alterations at higher levels of biological organization, is a promising tool for Environmental Risk Assessment. Here, enchytraeids were exposed for different time periods (two days and three weeks) to different soils (OECD artificial soil, different compositions in its organic matter, clay or pH value, and LUFA 2.2 natural soil) and different chemicals (Phenmedipham and copper). The main question addressed in the present study was if the effects of chemicals and different soil properties are preceded by alterations at the sub-cellular level, and if these endpoints may be used reliantly as faster screening tools for the assessment of different stress conditions in soils. The parameters measured in E. albidus whole body were: lipid peroxidation (LPO), total glutathione (TG), as well as the enzymatic activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione S-transferase (GST). The results showed that biomarker responses in E. albidus were significantly affected by the soil type (GST, CAT, GPx, GR and LPO) and the duration of exposure in OECD artificial soil (GST, GPx, GR, CAT and LPO) but not in LUFA 2.2 natural soil. For the abiotic factors studied, after 2 days, low pH decreased significantly the TG levels and the activities of CAT and GR,and low OM also significantly decreased CAT and GR activities. After 3 weeks, differences in soil properties caused a decrease in GR and GPx activities, whereas increased GST activity was observed due to low organic matter and pH. Copper significantly increased the activities of CAT, GPx and GR, and decreased the activity of GST after 2 days as well as inscreasing LPO levels after 3 weeks. Phenmedipham increased LPO levels, associated with increased levels of TG as well as increased activities of CAT and GPx and decreased GST activity after 3 weeks exposure. This study shows that both abiotic and chemical stresses could be followed through biomarker analysis and that some of these determinations are potential endpoints in a quick soil contamination assessment procedure.

Oxidative stress, liver biotransformation and genotoxic effects induced by copper in Anguilla anguilla L.--the influence of pre-exposure to beta-naphthoflavone.

Fish are exposed in the aquatic ecosystems to different classes of pollutants. Polycyclic aromatic hydrocarbons (PAHs) and heavy metals represent two important classes of aquatic contaminants. Thus, one lot of European eels (Anguilla anguilla L.) was pre-exposed during 24 h to 2.7 microM beta-naphthoflavone (BNF; a PAH-like compound), and subsequently exposed during 24 h to 0, 1 and 2.5 microM copper (Cu). Additionally, another lot not pre-exposed to BNF was exposed to the same Cu concentrations. BNF pre-exposure promoted a significant increase in liver ethoxyresorufin O-deethylase (EROD) activity, but did not change the other responses investigated in eels. On the other hand, both Cu concentrations did not modify the liver EROD activity either in eels pre-exposed to BNF or not. Liver total cytochrome P450 was increased in eels exposed to Cu 2.5 microM, being significantly only in eels not pre-exposed to BNF. Free sulfhydryl group content was decreased by 1 and 2.5 microM in eels pre-exposed to BNF or not pre-exposed, being significant at 2.5 microM Cu in eels not pre-exposed compared to its control. Liver total glutathione (TG), reduced glutathione (GSH) and GSH/oxidized glutathione (GSSG) levels were slightly decreased by 1 and 2.5 microM Cu in eels pre-exposed to BNF, whereas a slight tendency to increase was observed in eels not pre-exposed. Thus, liver TG and GSH significantly decreased in 2.5 microM Cu BNF pre-exposed eels compared to eels not pre-exposed to BNF. Liver glutathione reductase and catalase activities were significantly inhibited by 1 and 2.5 microM Cu in eels pre-exposed to BNF, concomitantly with a slight liver glutathione peroxidase tendency to decrease. Lipid peroxidation was significantly increased by 1 microM Cu in eels either pre-exposed or not pre-exposed to BNF. Liver H(2)O(2) was significantly increased by 1 microM Cu in eels pre-exposed to BNF. Liver DNA integrity was significantly decreased by 1 and 2.5 microM Cu in eels pre-exposed to BNF. The oxidative stress and genotoxic effects induced by Cu in eels pre-exposed to BNF revealed that the metal effects are potentiated by previous exposure to BNF.

Oxidative stress and genotoxic responses to resin acids in Mediterranean mussels.

This study represents the first attempt to investigate the genotoxic effects and oxidative stress of resin acids in Mediterranean mussels (Mytilus galloprovincialis Lmk). Mussels were exposed to 2.7 microM abietic acid (AA) and dehydroabietic acid (DHAA) for 6, 12, 18, and 24h. Gill and hepatopancreas conjugation activity, antioxidant defense system, lipid peroxidation (LPO), and DNA damage were determined as reduced glutathione (GSH), glutathione S-transferase (GST) activity, glutathione peroxidase (GPx) activity, catalase (CAT) activity, LPO, and DNA strand breaks. AA caused significant GST inhibition in mussel gills at 12, 18, and 24h. Activity of the antioxidant enzymes, namely, GPx and CAT, was inhibited at 24 and 18 h, respectively, in mussel gills. A significant increase in gill LPO was observed at 24h. The DNA integrity of mussel hepatopancreas significantly decreased after 12 and 24 h exposure to AA. A significant increase in LPO was observed after 6h exposure to DHAA, in either mussel gills or hepatopancreas. DNA integrity was significantly decreased in mussel hepatopancreas after 12 and 24 h exposure to DHAA. AA induced oxidative damage and genotoxicity in mussels, because it promoted increases in LPO in gills and DNA strand breaks in hepatopancreas. DHAA promoted oxidative damage and genotoxicity in mussels, as significant increases were observed in LPO in gills and hepatopancreas and in DNA strand breaks in hepatopancreas.

Juvenile sea bass biotransformation, genotoxic and endocrine responses to beta-naphthoflavone, 4-nonylphenol and 17 beta-estradiol individual and combined exposures.

Juvenile sea bass, Dicentrarchus labrax L., were exposed during 2, 4, 8, and 24 h to 0.9 microM beta-naphthoflavone (BNF), 131 nM 17 beta-estradiol (E(2)), 4.05 microM 4-nonylphenol (NP), as well as to BNF combined either to E(2) or NP (maintaining the previous concentrations). Liver cytochrome P450 content (P450), ethoxyresorufin-O-deethylase (EROD), and glutathione S-transferase (GST) activities were measured in order to evaluate biotransformation responses. Genotoxicity was assessed as erythrocytic nuclear abnormalities (ENA) frequency. The effects on endocrine function were evaluated as plasma cortisol and glucose. Cortisol was not affected by xeno/estrogens tested, either in single exposure or mixed with BNF. Nevertheless, the intermediary metabolism was affected since glucose concentration increased after 4 h exposure to E(2), and after all BNF+NP exposure lengths. Moreover, a synergism between BNF and NP was thoroughly demonstrated, whereas a sporadic antagonistic interaction was found at 4 h BNF + E(2) exposure. Liver EROD and GST activities were not significantly altered by single E(2) or NP exposure. However, both compounds were able to induce EROD activity in the presence of BNF. NP single exposure was able to significantly increase liver P450 content, while its mixture with BNF displayed an antagonistic interference. Considering the xeno/estrogens single exposures, only NP induced an ENA increase; however, both mixtures (BNF + E(2) and BNF + NP) displayed genotoxic effects. Fish responses to mixtures of xenobiotics are complex and the type of interaction (synergism/potentiation or antagonism) in a particular mixture can vary with the evaluated biological response.

Dicentrarchus labrax biotransformation and genotoxicity responses after exposure to a secondary treated industrial/urban effluent.

The present research work was designed to study Dicentrarchus labrax (sea bass) biotransformation and genotoxicity responses to the soluble fraction of a secondary treated industrial/urban effluent (SF-STIUE) discharged through a submarine pipe outlet into the Aveiro coastal area. Sea bass was exposed for 4, 8, 16, 24, 48, and 96 h to 0%, 0.1%, and 1% SF-STIUE and the following biological responses were measured: (1) liver cytochrome P450 (P450) content and ethoxyresorufin-O-deethylase (EROD) activity, as phase I biotransformation parameters; (2) liver gluthathione S-transferase (GST) activity as a phase II conjugation enzyme; (3) biliary and liver cytosol naphthalene (Naph)- and benzo(a)pyrene (B(a)P)-type metabolites, by fixed wavelength fluorescence detection (FF); (4) liver DNA strand breaks, erythrocytic micronuclei (EMN), and erythrocytic nuclear abnormalities (ENA) as genotoxicity parameters. Both SF-STIUE dilutions (0.1% and 1%) failed to significantly increase liver EROD activity, despite a significant increase of liver P450 at 16 and 48 h exposure to 0.1%. Liver GST activity increased significantly at 4h of sea bass exposure to 1% SF-STIUE, being inhibited at 96 h of exposure to this SF-STIUE dilution. Naph- and B(a)P-type metabolite contents were not significantly increased in bile. However, Naph-type metabolite contents increased significantly in liver cytosol at 4h exposure to 1% SF-STIUE, and at 24h exposure to 0.1% and 1% SF-STIUE. Furthermore, B(a)P-type metabolites increased significantly in liver cytosol at 4h exposure to 1% SF-STIUE, and 16 h exposure to 0.1% and 1% SF-STIUE. EMN and ENA frequencies increased significantly at 4, 8, 16, 24, 48, and 96 h exposure to 0.1% and 1% SF-STIUE. Liver DNA integrity decreased significantly at 96 h of sea bass exposure to 1% SF-STIUE. The STIUE discharged into Aveiro coastal area is of great ecotoxicological concern due to its genotoxic potential.

Genotoxicity biomarkers' association with B(a)P biotransformation in Dicentrarchus labrax L.

Dicentrarchus labrax (sea bass) were exposed during 0, 4, 8, 16, 24, 48, and 96 h to 0 and 0.1 microM benzo(a)pyrene (B(a)P), an environmental pollutant, and the following biomarkers were measured: (1) liver cytochrome P450 (P450) content and ethoxyresorufin-O-deethylase (EROD) activity as phase I biotransformation parameters, (2) liver gluthathione-S-transferase (GST) activity as a phase II biotransformation conjugation enzyme, (3) biliary and liver cytosolic B(a)P-type metabolites by fixed wavelength fluorescence detection (FF), and (4) erythrocytic micronuclei (EMN) and erythrocytic nuclear abnormalities (ENA) as genotoxicity biomarkers. Liver EROD activity (4 h), P450 content (24 h), GST activity (4, 8, and 96 h), bile (4-96 h), and liver cytosolic (4-24 h) B(a)P-type metabolites increased significantly in sea bass exposed to B(a)P as well as EMN (8-96 h) and ENA (4-96 h) frequencies. B(a)P genotoxicity is associated with increase in B(a)P-type metabolites in liver cytosol due to an impaired phase II conjugation. This increase seems to be responsible for the decrease in liver EROD and GST activities.

Juvenile sea bass liver biotransformation and erythrocytic genotoxic responses to pulp mill contaminants.

This research represents the first study concerning liver phase I biotransformation induction, measured as cytochrome P-450 (P450) and ethoxyresorufin-O-deethylase (EROD), and genotoxic responses, measured as erythrocytic micronuclei (EMN) and nuclear abnormalities (ENA), in Dicentrarchus labrax L. (sea bass) exposed to pulp and paper mill contaminants. Juvenile sea bass were exposed for 6 h to 0 (control) and 0.0125 microM concentrations of the resin acids (RAs) abietic acid (AA) and dehydroabietic acid (DHAA) or 7-isopropyl-1-methylphenanthrene (retene) (Experiment 1). Sea bass were exposed for 6 h to 0 (control), 0.78, 1.56, 3.12, 6.25, and 12.5% bleached kraft pulp mill environmental contaminated water (BKPMECW) collected at the old sewage outlet of a pulp and paper industry (Experiment 2). The time-dependent response was studied in sea bass at 0, 8, 16, 24, 48, and 72 h exposure to 0.78 and 12.5% BKPMECW (Experiment 3). The experimental results demonstrated the presence of genotoxic compounds in BKPMECW. AA, DHAA, and retene may be the constituents responsible for high BKPMECW genotoxicity, since they induced similar sea bass EMN and ENA frequency increases. The BKPMECW, the RAs, and retene failed to significantly increase liver EROD activity and P450 content at 6 h. Furthermore, 3.12% BKPMECW, 0.0125 microM AA, and 0.0125 microM retene significantly decreased liver EROD activity. However, P450 was significantly increased from 8 up to 72 h exposure to BKPMECW. Therefore, the low or inhibited EROD levels could be a consequence of a general membrane disturbance by BKPMECW, RAs, and retene. However, liver ALT results indicate significant liver damage or enzyme inhibition only at 8, 16, 48, and 72 h exposure to BKPMECW.

Juvenile sea bass liver biotransformation induction and erythrocytic genotoxic responses to resin acids.

Juvenile Dicentrarchus labrax L. (sea bass) were exposed for 0, 2, 4, 6, and 8 h to abietic acid (AA) and dehydroabietic acid (DHAA) in concentration ranges 0, 0.0125, 0.025, 0.05, 0.1, 0.3, 0.9, and 2.7 microM. Liver cytochrome P-450 content (P450) and liver ethoxyresorufin-O-deethylase activity (EROD) were determined as phase I biotransformation biomarkers. Genotoxicity was measured as erythrocytic micronuclei (EMN) and nuclear abnormalities (ENA). Liver damage was assessed as liver alanine aminotransferase activity (ALT) and liver somatic index (LSI) was used as a general health indicator. AA inhibited EROD (at 2 h exposure to 0.05 microM) or failed to induce it, whereas a significant P450 increase was observed at 2 h exposure to 0.05 (2.3-fold) and 2.7 microM (6.3-fold). A significant P450 increase was also observed at 6 and 8 h exposure, respectively, to 0.0125 (3.4-fold) and 0.025 microM (4.9-fold) AA. EMN and ENA frequency were significantly increased at 2 h exposure to 0.9 microM AA. A significant EMN and ENA time-related increase was observed with an increased exposure length up to 8 h. Therefore, all the AA concentrations tested promoted an EMN and ENA increase at 8 h exposure. DHAA induced a significant EROD increase (3.2-fold) at 2 h exposure to the lowest concentration tested. Liver P450 was significantly increased at 2 h exposure to 0.025 (1.8-fold) and 0.3 microM (2.5-fold), at 4 h exposure to 0.1 microM (3.6-fold), and at 6 h exposure to 0.1 (3.2-fold) and 0.3 microM (2.8-fold), whereas it was significantly decreased (30% of control value) at 4 h exposure to 0.9 microM. EMN and ENA frequency were significantly increased from 0 to 2 h exposure to all DHAA concentrations tested and remained high from 2 to 8 h. EMN frequency was increased 20 times over control at 2 h exposure to 0.0125 microM DHAA, whereas it increased 9-fold at 2 h exposure to 0.9 microM AA. Furthermore, ENA frequency was increased 3 times over control at 2 h exposure to 0.0125 microM DHAA, and 2-fold after exposure to 0.9 microM AA. Maximal EMN and ENA induction was observed at 2 h exposure to 0.0125 microM DHAA and only at 4 h exposure to 2.7 microM AA. Therefore, the comparative analysis of the two resin acids genotoxic effects, measured as EMN and ENA, indicated that DHAA is more genotoxic than AA.

Liver phase I and phase II enzymatic induction and genotoxic responses of beta-naphthoflavone water-exposed sea bass.

Sea bass were exposed to 0 (control), 0.1, 0.3, 0.9, and 2.7 microM beta-naphthoflavone (BNF) for 0, 2, 8, and 16 h in order to assess the chronological and concentration relationships between BNF phase I and II biotransformation responses, such as liver cytochrome P450 (P450) content, ethoxyresorufin-O-deethylase (EROD), uridine diphosphate-glucuronosyl transferase (UDP-GT), and the genotoxic effects, measured either by erythrocytic micronuclei (EMN) or erythrocytic nuclear abnormalities (ENA) tests. Liver alanine aminotransferase (ALT) activity and liver somatic index (LSI) were also measured. A significant liver EROD activity was found at 8 h exposure, respectively, to 0.1, 0.3, 0.9, and 2.7 microM BNF. Maximal liver EROD activity increase was observed at 16 h exposure to 0.9 microM BNF, whereas the highest liver P450 was reached at 8 h exposure to 2.7 microM BNF. Liver UDP-GT activity was significantly increased at 2 h exposure to 0.1 and 0.3 microM BNF and at 8 h exposure to 0.1, 0.3, and 0.9 microM BNF, decreasing at 16 h, for every exposure concentration. Significant ENA increase was observed at 2h exposure, respectively, to 0.3, 0.9, and 2.7 microM BNF. Maximal ENA increase was observed at 16 h exposure to 0.9 microM BNF. The MN was significantly increased at 8 and 16 h exposure, respectively, to 2.7 and 0.9 microM BNF. Liver ALT activity significantly increases at 8 h exposure to 0.1 and 0.3 microM BNF, whereas liver somatic index was significantly increased from 2 to 16 h exposure for every BNF concentration. A slight liver EROD activity increase with a concomitant lack of liver UDP-GT activity is able to induce significant erythrocytic genotoxic effects. Liver UDP-GT high levels are important in sea bass BNF detoxification. However, high liver UDP-GT activity is not enough to prevent the BNF metabolite genotoxic effects on sea bass erythrocytes when liver EROD activity is induced at 2 and 8 h exposure to 0.3 and 0.9 microM BNF. The genotoxic effects measured as EMN and ENA suggest that the balance between the rates of liver BNF reactive and conjugated metabolites seems to be critical.

Beta-naphthoflavone liver EROD and erythrocytic nuclear abnormality induction in juvenile Dicentrarchus labrax L.

Juvenile Dicentrarchus labrax L. (sea bass) was exposed to five different beta-naphthoflavone (BNF) concentrations-0, 0.1, 0.3, 0.9 and 2.7 microM-for 0, 2, 4, 6, 8, 16, 24, 48, 72, 144, and 216 h. A battery of biomarkers was investigated, such as liver ethoxyresorufin-O-deethylase (EROD), liver cytochrome P450 (P450 content), liver aminotransferase (ALT activity), liver somatic index (LSI), micronuclei (MN), and erythrocytic nuclear abnormality (ENA) frequencies. Juvenile D. labrax L. liver EROD induction started at 2 h exposure to 2.7 microM BNF and 6 h exposure to 0.1, 0.3, and 0.9 microM BNF, respectively. A significant liver EROD decrease was observed between 8 and 16 h for all BNF concentrations, followed by a slight increase after 48 h exposure to 0.9 and 2.7 microM BNF and a definitive decrease from 72 h exposure onward. Liver P450 content significantly increased at 2, 6, and 8 h exposure, respectively, to 2.7 microM, 0.9, 0.3, and 0.1 microM BNF. However, liver P450 content remained significantly higher than that of the control from 72 to 216 h in the sea bass exposed to 2.7 microM BNF. Sea bass ENA induction started at 4h exposure to 0.9 and 2.7 microM BNF, and significantly increased to 16 and 24 h exposure, whereas for 0.3 microM BNF a significant increase started after 8 h exposure. A significant ENA frequency increase was still observed at 144 and 216 h exposure to 0.9 and 2.7 microM BNF. The micronuclei induction was observed at 4, 6, and 8 h, respectively, after 2.7, 0.9, and 0.3 microM BNF exposure. However, there was a micronuclei frequency decrease for 0.3, 0.9, and 2.7 microM BNF exposure concentrations between 8 and 16 h, followed by a slight increase after 48, 72, and 144 h exposure, respectively, to 2.7, 0.9, and 0.3 microM BNF. Liver somatic index significantly increased after 216 h, whereas ALT activity significantly decreased at 144 and 216 h 2.7 microM BNF exposure.

Juvenile sea bass liver P450, EROD induction, and erythrocytic genotoxic responses to PAH and PAH-like compounds.

Sea bass were exposed, for 0, 2, 4, 6 and 8 h, to 0 or 2.7 microM beta-naphthoflavone (BNF), or to 0, 0.1, 0.3, 0.9, or 2.7 microM benzo[a]pyrene (B(a)P) and naphthalene (NAPH). Liver cytochrome P-450 content (P450) and liver ethoxyresorufin-O-deethylase activity (EROD) induction were determined as phase I biotransformation responses, whereas erythrocytic micronuclei (EMN) and erythrocytic nuclear abnormalities (ENA) tests were performed to assess genotoxic effects. Liver alanine aminotransferase activity and liver somatic index were determined, respectively, as liver damage and common health indicators. A significant increase in sea bass EROD activity began, respectively, at 2 and 4 h exposure to 2.7 microM B(a)P and 2.7 microM BNF, whereas NAPH failed to induce EROD activity. Maximal EROD activity was observed after 6 h exposure to 2.7 microM BNF (9-fold increase), 2.7 microM B(a)P (4-fold increase), and 2.7 microM NAPH (2-fold increase), indicating BNF as the most potent EROD inducer (BNF>B(a)P>NAPH). A significant increase in liver P450 content was observed at 6 h exposure to 2.7 microM BNF (6.5-fold increase), indicating BNF as the most potent P450 inducer. A significant P450 increase was observed at 8 h exposure only to 0.1 microM B(a)P (2-fold increase), whereas it slightly increased at 2 h exposure to 2.7 microM NAPH, within a wide variable range. The BNF, NAPH, and B(a)P genotoxic potential was demonstrated as sea bass EMN and ENA. B(a)P promoted at 2 h exposure a significant EMN (24-fold) and ENA (2.2-fold) increase, whereas NAPH exhibited similar results only at 8 h exposure. BNF also increased significantly sea bass EMN (8-fold) and ENA (1.5-fold) after 8 h. The results indicated B(a)P as the most genotoxic compound, followed by NAPH and BNF (B(a)P>NAPH>BNF). Despite the low liver P450 content and EROD activity induction by NAPH and B(a)P, their genotoxic potential was higher than that of BNF.

Ca2+-H+ antiport activity in synaptic vesicles isolated from sheep brain cortex.

Synaptic vesicles isolated from sheep brain cortex exhibit an ATP-dependent Ca2+ accumulation that is inhibited by the protonophore uncoupler carbonyl cyanide m-chorophenylhydrazone (CCCP) and completely released by the Ca2+ ionophore ionomycin. This transport activity was sensitive to the V-type ATPase inhibitor, bafilomycin, but not to the P-type ATPase inhibitor, vanadate. We also observed that the proton gradient, established across the synaptic vesicle membranes in the presence of ATP, is partially dissipated by the addition of Ca2+ (100-860 microM) in correlation to an increase of ATP hydrolysis by the H+-pumping ATPase. In contrast, the activity of the H+-ATPase, measured under uncoupling conditions (presence of CCCP), appears to be unaltered by the calcium ion. The Ca2+-induced H+ release visualized by fluorescence quenching of acridine orange correlates well with the Ca2+ uptake determined isotopically. These results indicate that synaptic vesicles accumulate Ca2+, via a low affinity Ca2+-H+ antiport system energized by the protonmotive force originated from the H+-pumping ATPase activity.