The European eel (Anguilla anguilla) is a euryaline fish suitable for investigating exposure, acc... more The European eel (Anguilla anguilla) is a euryaline fish suitable for investigating exposure, accumulation patterns and biological effects of lipophilic pollutants such as polychlorinated biphenyls (PCBs). Eels were collected from the Orbetello Lagoon (Tyrrhenian coast of Italy) and injected with increasing doses of Aroclor 1254 to better characterise EROD activity as biomarker in eels from Mediterranean brackish environments. Fish muscle
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2006
We describe the acetylcholinesterase polymorphisms of two bivalve molluscs, Adamussium colbecki a... more We describe the acetylcholinesterase polymorphisms of two bivalve molluscs, Adamussium colbecki and Pecten jacobaeus. The research was aimed to point out differences in the expression of pesticide-resistant acetylcholinesterase forms in organisms living in different ecosystems such as the Ross Sea (Antarctica) and the Mediterranean Sea. In A. colbecki, distinct acetylcholinesterase molecular forms were purified and characterized from spontaneously soluble, low-salt-soluble and low-salt-Triton extracts from adductor muscle and gills. They consist of two non-amphiphilic acetylcholinesterases (G(2), G(4)) and an amphiphilic-phosphatidylinositol-membrane-anchored form (G(2)); a further amphiphilic-low-salt-soluble G(2) acetylcholinesterase was found only in adductor muscle. In the corresponding tissues of P. jacobaeus, we found a non-amphiphilic G(4) and an amphiphilic G(2) acetylcholinesterase; amphiphilic-low-salt-soluble acetylcholinesterases (G(2)) are completely lacking. Such results are related with differences in cell membrane lipid compositions. In both scallops, all non-amphiphilic AChEs are resistant to used pesticides. Differently, the adductor muscle amphiphilic forms are resistant to carbamate eserine and organophosphate diisopropylfluorophosphate, but sensitive to organophoshate azamethiphos. In the gills of P. jacobaeus, amphiphilic G(2) forms are sensitive to all three pesticides, while the corresponding forms of A. colbecki are sensitive to eserine and diisopropylfluorophosphate, but resistant to azamethiphos. Results indicate that organophosphate and/or carbamate resistant AChE forms are present in species living in far different and far away environments. The possibility that these AChE forms could have ensued from a common origin and have been spread globally by migration is discussed.
Marine environments are continuously being threatened by a large number of xenobiotics from anthr... more Marine environments are continuously being threatened by a large number of xenobiotics from anthropogenic sources. Even in sparsely populated and relatively pristine areas, such as Antarctica, hazardous chemicals can pose a serious environmental problem. The main aims of the present study were to (1) validate and optimize an analytical technique utilizing a microtitre-plate photometer to ascertain background levels of esterase activities in the Antarctic bivalve Adamussium colbecki, (2) carry out in situ monitoring of esterase activities to assess any potential environmental impacts of the Italian Scientific Antarctic Base "Terra Nova Bay" on the surrounding marine area. Results showed the presence of organophosphorous-sensitive cholinesterase (ChE) and carboxylesterase (CbE) activities in the gills of A. colbecki and optimal assay conditions were comparable with those found for bivalve species from temperate areas. A higher sensitivity of ChE versus acetylthiocholine activity in A. colbecki to chlorpyrifos compared to species from temperate areas may also be inferred. The in situ study indicated no differences in the environmental quality of the three study sites located around the Italian Base.
... Doyotte, A., Cossu, C., Jacquin, MC, Babut, M., and Vasseur (1997). Antioxidant enzymes, glut... more ... Doyotte, A., Cossu, C., Jacquin, MC, Babut, M., and Vasseur (1997). Antioxidant enzymes, glutathione and lipid peroxidation as relevant biomarkers of experimental or field exposure in the gills and the digestive gland of the freshwater bivalve Unio tumidus. ... Forshaw, PJ (1977). ...
Biochemical characterization of cholinesterase activity (ChE) was carried out on the Antarctic sc... more Biochemical characterization of cholinesterase activity (ChE) was carried out on the Antarctic scallop Adamussium colbecki collected in winter 2000 from Campo Icaro (Ross Sea, Antarctica) in order to increase its suitability as a sentinel organism for monitoring the Antarctic environment. The digestive gland, gills and adductor muscle were investigated for substrate specificity and inhibitors sensitivity using acetylthiocholine iodide (ASCh) and butyrylthiocholine iodide (BSCh) as substrates and tetra (monoisopropyl)pyrophosphor-tetramide (Iso-OMPA), 1,5-bis(4-allyldimethylammoniumphenyl)-penthan-3-one dibromide (BW284c51) and the insecticide chlorpyrifos as inhibitors. Effect of in vivo exposure to ZnCl2 was also investigated. All the tissues expressed ChE activity (gill > adductor muscle > digestive gland) and low substrates specificity throughout the hydrolysis of both ASCh and BSCh substrates. Partial (25–29%) and total inhibition (100%) of ChE activity in gills was demonstrated following in vitro incubation with Iso-OMPA and BW284c51 (3 mM), respectively. Concentration-dependent inhibition was also evident with chlorpyrifos in the range 10−4–10−10 M (IC50 10−6) while in vivo exposure to ZnCl2 did not seem to affect ChE activity in the scallop. The potential use of ChE in the A. colbecki as biomarker for monitoring water contamination in the marine Antarctic environment is discussed.
Comparative Biochemistry and Physiology B-biochemistry & Molecular Biology, 2006
We describe the acetylcholinesterase polymorphisms of two bivalve molluscs, Adamussium colbecki a... more We describe the acetylcholinesterase polymorphisms of two bivalve molluscs, Adamussium colbecki and Pecten jacobaeus. The research was aimed to point out differences in the expression of pesticide-resistant acetylcholinesterase forms in organisms living in different ecosystems such as the Ross Sea (Antarctica) and the Mediterranean Sea. In A. colbecki, distinct acetylcholinesterase molecular forms were purified and characterized from spontaneously soluble, low-salt-soluble and low-salt-Triton extracts from adductor muscle and gills. They consist of two non-amphiphilic acetylcholinesterases (G2, G4) and an amphiphilic-phosphatidylinositol-membrane-anchored form (G2); a further amphiphilic-low-salt-soluble G2 acetylcholinesterase was found only in adductor muscle. In the corresponding tissues of P. jacobaeus, we found a non-amphiphilic G4 and an amphiphilic G2 acetylcholinesterase; amphiphilic-low-salt-soluble acetylcholinesterases (G2) are completely lacking. Such results are related with differences in cell membrane lipid compositions. In both scallops, all non-amphiphilic AChEs are resistant to used pesticides. Differently, the adductor muscle amphiphilic forms are resistant to carbamate eserine and organophosphate diisopropylfluorophosphate, but sensitive to organophoshate azamethiphos. In the gills of P. jacobaeus, amphiphilic G2 forms are sensitive to all three pesticides, while the corresponding forms of A. colbecki are sensitive to eserine and diisopropylfluorophosphate, but resistant to azamethiphos. Results indicate that organophosphate and/or carbamate resistant AChE forms are present in species living in far different and far away environments. The possibility that these AChE forms could have ensued from a common origin and have been spread globally by migration is discussed.
In order to gain preliminary knowledge about the threat to marine ecosystems due to leakage of ch... more In order to gain preliminary knowledge about the threat to marine ecosystems due to leakage of chemical warfare agents (CWAs) and other pollutants from rusting bombshells on the seabed, a case study was conducted in a dumping area in the southern Adriatic Sea (depth 200–300 m). Following electroacoustic and magnetometric surveys of the CWA dumping area, an integrated ecotoxicological approach was used. This approach was based on analysis of CWA residues and their metabolites, including arsenic, in sediment and organisms, as well as multimarker methodology including the Health Assessment Index, histological lesion analysis and enzyme assays. Two sentinel species were selected, the blackbelly rosefish [Helicolenus dactylopterus (Delaroche, 1809)] and the European conger (Conger conger L., 1758). Sediment analysis revealed the presence of CWA degradation products, including 1-4-thioxane and 1-4-dithiane. Tissues of fish from the CWA dumping site showed higher levels of arsenic than those from the reference site. Neither CWAs nor their metabolites were detected in fish tissues. Arsenic levels recorded in blackbelly rosefish were well above those reported for other fish species from the southern Adriatic, much higher than the FDA limit for food (2.6 mg kg−1) and close to the LD50 calculated for mammals (20 mg kg−1 body weight). The presence of pollutants in the CWA dumping site was also confirmed by pathological lesions in both species and EROD activity, two to three times higher than in fish from the reference site (16.45±8.08 and 8.05±5.87 pmol min−1 mg protein−1 in blackbelly rosefish and 269±24.92 and 78.71 pmol min−1 mg protein−1 in European conger, respectively). Cholinesterase activity seemed unaffected in muscle of both species, whereas in brain they were one-third of those recorded in fish from the reference site (14.22±10.05 and 72.87 nmol min−1 mg protein−1, respectively). This suggests that acetylcholinesterase is sensitive to CWAs. In conclusion, the agreement of all the chemical and biological parameters investigated suggests that the integrated ecotoxicological approach used is appropriate to reveal the presence and biological effects of CWAs in the marine ecosystem.
Biochemical markers and ovarian histology were investigated in prespawning females of grass goby ... more Biochemical markers and ovarian histology were investigated in prespawning females of grass goby (Zosterisessor ophiocephalus) and grey mullet (Mugil cephalus) collected, respectively, in late spring and summer 2000 in four sites of a highly eutrophic brackish ecosystem of central Italy, the Orbetello Lagoon. Exposure to chlorinated and aromatic hydrocarbons was evaluated in fish livers by the somatic liver index (SLI) and by measuring 7-ethoxyresorufin-O-deethylase (EROD) and benzo(a)pyrene monooxygenase (BaPMO) activities. Acetylcholinesterase (AChE) activity was measured in brain and gills to evaluate exposure to organophosphates (OPs) and carbamates (CBs). The gonad somatic index (GSI) was used to confirm ovarian maturation and ovarian histology was investigated as a potential biomarker for environmental effects. Samples from the Western Basin, near a sewage treatment plant (STP) off the town of Orbetello, showed higher SLI values and higher EROD and BaPMO activities than those collected from the Ansedonia Canal (AC) in the Eastern Basin (p<0.05) and respect to those from reference sites: the Albegna River (AR) Delta for grass goby and the Nassa Canal (NC), connected with the sea, for grey mullet both located in the Western Basin as well. Low brain AChE activity was observed in both species from the reference sites (AR and NC) in association with the presence of anomalies in developing oocytes: unexpectedly small in grass goby and irregular disintegrated cytoplasm in grey mullet. The results indicate that the Western Basin is more polluted than the Eastern Basin particularly in the Orbetello where the sewage treatment plant may be a source of aromatic and chlorinated compounds while the Albegna River and the Nassa Canal may be sources of OPs and CBs.
An integrate approach will be discussed for investigations on oxidative stress in xenobiotic toxi... more An integrate approach will be discussed for investigations on oxidative stress in xenobiotic toxicity. While the analysis of individual antioxidants is useful for their sensitivity and to understand the mode of action of a stressor, the integration with the analysis of the total antioxidant capacity provides a more holistic assessment of the overall biological significance of such variations. TOSC has a greater predictive value on the health condition of the organisms and allows to discriminate the different role of specific ROS in oxidative stress syndrome.
In this work, susceptibility to oxidative stress was analyzed under laboratory conditions in the ... more In this work, susceptibility to oxidative stress was analyzed under laboratory conditions in the European eel Anguilla anguilla. Eels were treated with increasing concentrations of benchmark environmental pollutants, namely, benzo[a]pyrene ([BaP], at 0, 0.1, 1, 10, and 50 mg/kg), beta-naphthoflavone ([BNF], at 0, 0.1, 1, 10, and 50 mg/kg), Arochlor 1254 (at 0, 0.1, 1, 10, and 50 mg/kg), and 2,3,7,8-tetrachlorodibenzo p-dioxin ([TCDD], at 0, 0.01, 0.1, 1, and 2 microg/kg). The integral relationships were analyzed between induction of ethoxyresorufin O-deethylase (EROD) activity, its involvement in perturbing oxyradical metabolism, and the role of cytochrome P450 and/or oxidative stress in mediating genotoxic effects. To reveal whether the oxidative status in exposed organisms was altered as a result of chemical exposure, measurements of the main endogenous antioxidant defenses were integrated with the measurement of total oxyradical scavenging capacity (TOSC) toward peroxyl radicals and hydroxyl radicals (*OH). This approach permits discriminating the resistance of a tissue toward different forms of oxyradicals, thereby indicating a differential role for specific reactive oxygen species (ROS) in perturbing the balance between prooxidant and antioxidant mechanisms. All the analyzed chemicals promoted EROD induction (reflective of CYP1A) and altered either the levels or the activities of the antioxidants studied, which might be anticipated to exert alterations in oxyradical metabolism. Analysis of TOSC suggested the prevalence of metabolic oxidative pathways leading to the more reactive *OH on exposure to the chemicals studied. Of these chemicals, enhanced EROD activity correlated with genotoxic damage only in the cases of the nonhalogenated hydrocarbons BaP and BNF. The highest degree of genotoxic damage was consistently observed in organisms in which the capacity to absorb or scavenge OH was lowest. These data suggest a general relationship between oxidative stress and loss of DNA integrity in juvenile eels exposed to the chemicals studied herein.
Biological responses and contaminant levels in biological tissues were investigated in fish speci... more Biological responses and contaminant levels in biological tissues were investigated in fish specimens of grass goby (Zosterisessor ophiocephalus) collected in spring 2000 in a moderately polluted brackish ecosystem, the Orbetello lagoon (southern coast of Tuscany, Italy). The complexity of harmful effects that contaminants may cause in fish over time was assessed. General condition indices (somatic liver index, and gonadal somatic index), cytochrome P450 enzymes, acetylcholinesterase activity and ovarian morphology were integrated with the content of organochlorines (OCs—hexachlorobenzene, DDTs and polychlorinated biphenyls) in liver and ovaries and p-nonylphenol (NP) and lower ethoxylate (NPE1–2) content in muscle and ovaries as well. Samples collected from the western basin of the lagoon near the Orbetello sewage treatment plant had somatic liver index values and two P450 activities significantly higher than those collected from the same basin at the Albegna river delta and in the Ansedonia canal in the eastern basin (P < 0.05). On the other hand, while muscle content of p-NP and NPE1–2 was significantly higher in samples from the Orbetello and Albegna river sites than in samples from the Ansedonia canal site, OC levels in liver (DDTs > PCBs > HCB) were particularly lower in samples from STP compared to levels detected in sites AC and AR. Light microscopy of ovaries revealed that although the gonads were sexually mature, the diameter of the oocytes and eggs was smaller than that commonly reported. High levels of OCs and NPs were, in fact, observed in grass goby ovaries, and the differences observed between acetylcholinesterase activities among investigated sites suggest exposure to insecticides as well. The integration of biological responses and contaminant tissue content reveals that certain areas of the lagoon are selectively impacted by various human activities and helps in distinguishing relationships between biological responses and contaminant load in the lagoon environment.Les réponses biologiques et les niveaux de contamination des tissus ont été recherchés dans des spécimens de gobies d'herbiers (Zosterisessor ophiocephalus) récoltés au printemps 2000 dans un écosystème saumâtre modérément pollué, la lagune d'Orbetello (côte sud de la Toscane). La complexité des effets nuisibles des contaminants sur les poissons est suivie au cours du temps. Des indices globaux (indice somatique hépatique, indice somatique gonadique), les enzymes du cytochrome P450, l'activité acétylcholinestérase et la morphologie de l'ovaire sont utilisés ainsi que le contenu organochloré (hexachlorobenzène, DDTs et biphényles polychlorinatés) du foie et des ovaires, les teneurs en p-nonylphénol et en éthoxylates inférieurs du muscle et des ovaires. Des échantillons provenant du bassin ouest de la lagune près de la station d'épuration d'Orbetello présentent, pour l'indice somatique hépatique et pour les deux activités P450, des valeurs nettement supérieures à celles qui sont relevées dans le même bassin dans le delta de la rivière Albegna et dans le canal Ansedonia dans le bassin est (P < 0,05). D'autre part, alors que le contenu des muscles en p-nonylphénol et en éthoxylates inférieurs apparaît nettement supérieur dans les échantillons provenant des sites « station d'épuration d'Orbetello » et « rivière Albegna » comparé aux échantillons du site « canal Ansedonia », les niveaux d'organochlorés dans le foie (DDTs > PCBs > HCB) sont particulièrement faibles au site « station d'épuration d'Orbetello » comparé à ceux des sites « canal Ansedonia » et « rivière Albegna ». L'examen microscopique des ovaires révèle que, même si les gonades sont sexuellement mûres, le diamètre des oocytes et des œufs est plus petit qu'habituellement. De hautes concentrations d'hexachlorobenzènes et de p-nonylphenols sont en fait observées dans les ovaires des gobies des herbiers et les différences entre les activités acetylcholinestérase des différents sites suggèrent une exposition aux insecticides. L'intégration des réponses biologiques et du contenu tissulaire en contaminants indique que certaines aires de la lagune subissent un impact sélectif des activités humaines. Ceci met en évidence les relations entre les réponses biologiques et la charge en contaminants dans l'environnement lagunaire.
The European eel (Anguilla anguilla) is a euryaline fish suitable for investigating exposure, acc... more The European eel (Anguilla anguilla) is a euryaline fish suitable for investigating exposure, accumulation patterns and biological effects of lipophilic pollutants such as polychlorinated biphenyls (PCBs). Eels were collected from the Orbetello Lagoon (Tyrrhenian coast of Italy) and injected with increasing doses of Aroclor 1254 to better characterise EROD activity as biomarker in eels from Mediterranean brackish environments. Fish muscle
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2006
We describe the acetylcholinesterase polymorphisms of two bivalve molluscs, Adamussium colbecki a... more We describe the acetylcholinesterase polymorphisms of two bivalve molluscs, Adamussium colbecki and Pecten jacobaeus. The research was aimed to point out differences in the expression of pesticide-resistant acetylcholinesterase forms in organisms living in different ecosystems such as the Ross Sea (Antarctica) and the Mediterranean Sea. In A. colbecki, distinct acetylcholinesterase molecular forms were purified and characterized from spontaneously soluble, low-salt-soluble and low-salt-Triton extracts from adductor muscle and gills. They consist of two non-amphiphilic acetylcholinesterases (G(2), G(4)) and an amphiphilic-phosphatidylinositol-membrane-anchored form (G(2)); a further amphiphilic-low-salt-soluble G(2) acetylcholinesterase was found only in adductor muscle. In the corresponding tissues of P. jacobaeus, we found a non-amphiphilic G(4) and an amphiphilic G(2) acetylcholinesterase; amphiphilic-low-salt-soluble acetylcholinesterases (G(2)) are completely lacking. Such results are related with differences in cell membrane lipid compositions. In both scallops, all non-amphiphilic AChEs are resistant to used pesticides. Differently, the adductor muscle amphiphilic forms are resistant to carbamate eserine and organophosphate diisopropylfluorophosphate, but sensitive to organophoshate azamethiphos. In the gills of P. jacobaeus, amphiphilic G(2) forms are sensitive to all three pesticides, while the corresponding forms of A. colbecki are sensitive to eserine and diisopropylfluorophosphate, but resistant to azamethiphos. Results indicate that organophosphate and/or carbamate resistant AChE forms are present in species living in far different and far away environments. The possibility that these AChE forms could have ensued from a common origin and have been spread globally by migration is discussed.
Marine environments are continuously being threatened by a large number of xenobiotics from anthr... more Marine environments are continuously being threatened by a large number of xenobiotics from anthropogenic sources. Even in sparsely populated and relatively pristine areas, such as Antarctica, hazardous chemicals can pose a serious environmental problem. The main aims of the present study were to (1) validate and optimize an analytical technique utilizing a microtitre-plate photometer to ascertain background levels of esterase activities in the Antarctic bivalve Adamussium colbecki, (2) carry out in situ monitoring of esterase activities to assess any potential environmental impacts of the Italian Scientific Antarctic Base &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;Terra Nova Bay&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; on the surrounding marine area. Results showed the presence of organophosphorous-sensitive cholinesterase (ChE) and carboxylesterase (CbE) activities in the gills of A. colbecki and optimal assay conditions were comparable with those found for bivalve species from temperate areas. A higher sensitivity of ChE versus acetylthiocholine activity in A. colbecki to chlorpyrifos compared to species from temperate areas may also be inferred. The in situ study indicated no differences in the environmental quality of the three study sites located around the Italian Base.
... Doyotte, A., Cossu, C., Jacquin, MC, Babut, M., and Vasseur (1997). Antioxidant enzymes, glut... more ... Doyotte, A., Cossu, C., Jacquin, MC, Babut, M., and Vasseur (1997). Antioxidant enzymes, glutathione and lipid peroxidation as relevant biomarkers of experimental or field exposure in the gills and the digestive gland of the freshwater bivalve Unio tumidus. ... Forshaw, PJ (1977). ...
Biochemical characterization of cholinesterase activity (ChE) was carried out on the Antarctic sc... more Biochemical characterization of cholinesterase activity (ChE) was carried out on the Antarctic scallop Adamussium colbecki collected in winter 2000 from Campo Icaro (Ross Sea, Antarctica) in order to increase its suitability as a sentinel organism for monitoring the Antarctic environment. The digestive gland, gills and adductor muscle were investigated for substrate specificity and inhibitors sensitivity using acetylthiocholine iodide (ASCh) and butyrylthiocholine iodide (BSCh) as substrates and tetra (monoisopropyl)pyrophosphor-tetramide (Iso-OMPA), 1,5-bis(4-allyldimethylammoniumphenyl)-penthan-3-one dibromide (BW284c51) and the insecticide chlorpyrifos as inhibitors. Effect of in vivo exposure to ZnCl2 was also investigated. All the tissues expressed ChE activity (gill > adductor muscle > digestive gland) and low substrates specificity throughout the hydrolysis of both ASCh and BSCh substrates. Partial (25–29%) and total inhibition (100%) of ChE activity in gills was demonstrated following in vitro incubation with Iso-OMPA and BW284c51 (3 mM), respectively. Concentration-dependent inhibition was also evident with chlorpyrifos in the range 10−4–10−10 M (IC50 10−6) while in vivo exposure to ZnCl2 did not seem to affect ChE activity in the scallop. The potential use of ChE in the A. colbecki as biomarker for monitoring water contamination in the marine Antarctic environment is discussed.
Comparative Biochemistry and Physiology B-biochemistry & Molecular Biology, 2006
We describe the acetylcholinesterase polymorphisms of two bivalve molluscs, Adamussium colbecki a... more We describe the acetylcholinesterase polymorphisms of two bivalve molluscs, Adamussium colbecki and Pecten jacobaeus. The research was aimed to point out differences in the expression of pesticide-resistant acetylcholinesterase forms in organisms living in different ecosystems such as the Ross Sea (Antarctica) and the Mediterranean Sea. In A. colbecki, distinct acetylcholinesterase molecular forms were purified and characterized from spontaneously soluble, low-salt-soluble and low-salt-Triton extracts from adductor muscle and gills. They consist of two non-amphiphilic acetylcholinesterases (G2, G4) and an amphiphilic-phosphatidylinositol-membrane-anchored form (G2); a further amphiphilic-low-salt-soluble G2 acetylcholinesterase was found only in adductor muscle. In the corresponding tissues of P. jacobaeus, we found a non-amphiphilic G4 and an amphiphilic G2 acetylcholinesterase; amphiphilic-low-salt-soluble acetylcholinesterases (G2) are completely lacking. Such results are related with differences in cell membrane lipid compositions. In both scallops, all non-amphiphilic AChEs are resistant to used pesticides. Differently, the adductor muscle amphiphilic forms are resistant to carbamate eserine and organophosphate diisopropylfluorophosphate, but sensitive to organophoshate azamethiphos. In the gills of P. jacobaeus, amphiphilic G2 forms are sensitive to all three pesticides, while the corresponding forms of A. colbecki are sensitive to eserine and diisopropylfluorophosphate, but resistant to azamethiphos. Results indicate that organophosphate and/or carbamate resistant AChE forms are present in species living in far different and far away environments. The possibility that these AChE forms could have ensued from a common origin and have been spread globally by migration is discussed.
In order to gain preliminary knowledge about the threat to marine ecosystems due to leakage of ch... more In order to gain preliminary knowledge about the threat to marine ecosystems due to leakage of chemical warfare agents (CWAs) and other pollutants from rusting bombshells on the seabed, a case study was conducted in a dumping area in the southern Adriatic Sea (depth 200–300 m). Following electroacoustic and magnetometric surveys of the CWA dumping area, an integrated ecotoxicological approach was used. This approach was based on analysis of CWA residues and their metabolites, including arsenic, in sediment and organisms, as well as multimarker methodology including the Health Assessment Index, histological lesion analysis and enzyme assays. Two sentinel species were selected, the blackbelly rosefish [Helicolenus dactylopterus (Delaroche, 1809)] and the European conger (Conger conger L., 1758). Sediment analysis revealed the presence of CWA degradation products, including 1-4-thioxane and 1-4-dithiane. Tissues of fish from the CWA dumping site showed higher levels of arsenic than those from the reference site. Neither CWAs nor their metabolites were detected in fish tissues. Arsenic levels recorded in blackbelly rosefish were well above those reported for other fish species from the southern Adriatic, much higher than the FDA limit for food (2.6 mg kg−1) and close to the LD50 calculated for mammals (20 mg kg−1 body weight). The presence of pollutants in the CWA dumping site was also confirmed by pathological lesions in both species and EROD activity, two to three times higher than in fish from the reference site (16.45±8.08 and 8.05±5.87 pmol min−1 mg protein−1 in blackbelly rosefish and 269±24.92 and 78.71 pmol min−1 mg protein−1 in European conger, respectively). Cholinesterase activity seemed unaffected in muscle of both species, whereas in brain they were one-third of those recorded in fish from the reference site (14.22±10.05 and 72.87 nmol min−1 mg protein−1, respectively). This suggests that acetylcholinesterase is sensitive to CWAs. In conclusion, the agreement of all the chemical and biological parameters investigated suggests that the integrated ecotoxicological approach used is appropriate to reveal the presence and biological effects of CWAs in the marine ecosystem.
Biochemical markers and ovarian histology were investigated in prespawning females of grass goby ... more Biochemical markers and ovarian histology were investigated in prespawning females of grass goby (Zosterisessor ophiocephalus) and grey mullet (Mugil cephalus) collected, respectively, in late spring and summer 2000 in four sites of a highly eutrophic brackish ecosystem of central Italy, the Orbetello Lagoon. Exposure to chlorinated and aromatic hydrocarbons was evaluated in fish livers by the somatic liver index (SLI) and by measuring 7-ethoxyresorufin-O-deethylase (EROD) and benzo(a)pyrene monooxygenase (BaPMO) activities. Acetylcholinesterase (AChE) activity was measured in brain and gills to evaluate exposure to organophosphates (OPs) and carbamates (CBs). The gonad somatic index (GSI) was used to confirm ovarian maturation and ovarian histology was investigated as a potential biomarker for environmental effects. Samples from the Western Basin, near a sewage treatment plant (STP) off the town of Orbetello, showed higher SLI values and higher EROD and BaPMO activities than those collected from the Ansedonia Canal (AC) in the Eastern Basin (p<0.05) and respect to those from reference sites: the Albegna River (AR) Delta for grass goby and the Nassa Canal (NC), connected with the sea, for grey mullet both located in the Western Basin as well. Low brain AChE activity was observed in both species from the reference sites (AR and NC) in association with the presence of anomalies in developing oocytes: unexpectedly small in grass goby and irregular disintegrated cytoplasm in grey mullet. The results indicate that the Western Basin is more polluted than the Eastern Basin particularly in the Orbetello where the sewage treatment plant may be a source of aromatic and chlorinated compounds while the Albegna River and the Nassa Canal may be sources of OPs and CBs.
An integrate approach will be discussed for investigations on oxidative stress in xenobiotic toxi... more An integrate approach will be discussed for investigations on oxidative stress in xenobiotic toxicity. While the analysis of individual antioxidants is useful for their sensitivity and to understand the mode of action of a stressor, the integration with the analysis of the total antioxidant capacity provides a more holistic assessment of the overall biological significance of such variations. TOSC has a greater predictive value on the health condition of the organisms and allows to discriminate the different role of specific ROS in oxidative stress syndrome.
In this work, susceptibility to oxidative stress was analyzed under laboratory conditions in the ... more In this work, susceptibility to oxidative stress was analyzed under laboratory conditions in the European eel Anguilla anguilla. Eels were treated with increasing concentrations of benchmark environmental pollutants, namely, benzo[a]pyrene ([BaP], at 0, 0.1, 1, 10, and 50 mg/kg), beta-naphthoflavone ([BNF], at 0, 0.1, 1, 10, and 50 mg/kg), Arochlor 1254 (at 0, 0.1, 1, 10, and 50 mg/kg), and 2,3,7,8-tetrachlorodibenzo p-dioxin ([TCDD], at 0, 0.01, 0.1, 1, and 2 microg/kg). The integral relationships were analyzed between induction of ethoxyresorufin O-deethylase (EROD) activity, its involvement in perturbing oxyradical metabolism, and the role of cytochrome P450 and/or oxidative stress in mediating genotoxic effects. To reveal whether the oxidative status in exposed organisms was altered as a result of chemical exposure, measurements of the main endogenous antioxidant defenses were integrated with the measurement of total oxyradical scavenging capacity (TOSC) toward peroxyl radicals and hydroxyl radicals (*OH). This approach permits discriminating the resistance of a tissue toward different forms of oxyradicals, thereby indicating a differential role for specific reactive oxygen species (ROS) in perturbing the balance between prooxidant and antioxidant mechanisms. All the analyzed chemicals promoted EROD induction (reflective of CYP1A) and altered either the levels or the activities of the antioxidants studied, which might be anticipated to exert alterations in oxyradical metabolism. Analysis of TOSC suggested the prevalence of metabolic oxidative pathways leading to the more reactive *OH on exposure to the chemicals studied. Of these chemicals, enhanced EROD activity correlated with genotoxic damage only in the cases of the nonhalogenated hydrocarbons BaP and BNF. The highest degree of genotoxic damage was consistently observed in organisms in which the capacity to absorb or scavenge OH was lowest. These data suggest a general relationship between oxidative stress and loss of DNA integrity in juvenile eels exposed to the chemicals studied herein.
Biological responses and contaminant levels in biological tissues were investigated in fish speci... more Biological responses and contaminant levels in biological tissues were investigated in fish specimens of grass goby (Zosterisessor ophiocephalus) collected in spring 2000 in a moderately polluted brackish ecosystem, the Orbetello lagoon (southern coast of Tuscany, Italy). The complexity of harmful effects that contaminants may cause in fish over time was assessed. General condition indices (somatic liver index, and gonadal somatic index), cytochrome P450 enzymes, acetylcholinesterase activity and ovarian morphology were integrated with the content of organochlorines (OCs—hexachlorobenzene, DDTs and polychlorinated biphenyls) in liver and ovaries and p-nonylphenol (NP) and lower ethoxylate (NPE1–2) content in muscle and ovaries as well. Samples collected from the western basin of the lagoon near the Orbetello sewage treatment plant had somatic liver index values and two P450 activities significantly higher than those collected from the same basin at the Albegna river delta and in the Ansedonia canal in the eastern basin (P < 0.05). On the other hand, while muscle content of p-NP and NPE1–2 was significantly higher in samples from the Orbetello and Albegna river sites than in samples from the Ansedonia canal site, OC levels in liver (DDTs > PCBs > HCB) were particularly lower in samples from STP compared to levels detected in sites AC and AR. Light microscopy of ovaries revealed that although the gonads were sexually mature, the diameter of the oocytes and eggs was smaller than that commonly reported. High levels of OCs and NPs were, in fact, observed in grass goby ovaries, and the differences observed between acetylcholinesterase activities among investigated sites suggest exposure to insecticides as well. The integration of biological responses and contaminant tissue content reveals that certain areas of the lagoon are selectively impacted by various human activities and helps in distinguishing relationships between biological responses and contaminant load in the lagoon environment.Les réponses biologiques et les niveaux de contamination des tissus ont été recherchés dans des spécimens de gobies d'herbiers (Zosterisessor ophiocephalus) récoltés au printemps 2000 dans un écosystème saumâtre modérément pollué, la lagune d'Orbetello (côte sud de la Toscane). La complexité des effets nuisibles des contaminants sur les poissons est suivie au cours du temps. Des indices globaux (indice somatique hépatique, indice somatique gonadique), les enzymes du cytochrome P450, l'activité acétylcholinestérase et la morphologie de l'ovaire sont utilisés ainsi que le contenu organochloré (hexachlorobenzène, DDTs et biphényles polychlorinatés) du foie et des ovaires, les teneurs en p-nonylphénol et en éthoxylates inférieurs du muscle et des ovaires. Des échantillons provenant du bassin ouest de la lagune près de la station d'épuration d'Orbetello présentent, pour l'indice somatique hépatique et pour les deux activités P450, des valeurs nettement supérieures à celles qui sont relevées dans le même bassin dans le delta de la rivière Albegna et dans le canal Ansedonia dans le bassin est (P < 0,05). D'autre part, alors que le contenu des muscles en p-nonylphénol et en éthoxylates inférieurs apparaît nettement supérieur dans les échantillons provenant des sites « station d'épuration d'Orbetello » et « rivière Albegna » comparé aux échantillons du site « canal Ansedonia », les niveaux d'organochlorés dans le foie (DDTs > PCBs > HCB) sont particulièrement faibles au site « station d'épuration d'Orbetello » comparé à ceux des sites « canal Ansedonia » et « rivière Albegna ». L'examen microscopique des ovaires révèle que, même si les gonades sont sexuellement mûres, le diamètre des oocytes et des œufs est plus petit qu'habituellement. De hautes concentrations d'hexachlorobenzènes et de p-nonylphenols sont en fait observées dans les ovaires des gobies des herbiers et les différences entre les activités acetylcholinestérase des différents sites suggèrent une exposition aux insecticides. L'intégration des réponses biologiques et du contenu tissulaire en contaminants indique que certaines aires de la lagune subissent un impact sélectif des activités humaines. Ceci met en évidence les relations entre les réponses biologiques et la charge en contaminants dans l'environnement lagunaire.
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