Erythrocytic Nuclear Abnormalities Frequency Research Articles

Assessment of DNA damage in Ardea cinerea and Ciconia ciconia: A 5-year study in Portuguese birds retrieved for rehabilitation

Over the past decades, the presence of micronucleated blood cells has been used to detect genotoxic effects of xenobiotics in fish, amphibians and birds. This study assessed the frequency of micronuclei (MN) and other nuclear abnormalities in erythrocytes of individuals of Ardea cinerea and Ciconia ciconia retrieved for rehabilitation in order to evaluate the influence of age, temporal and spatial factors on the occurrence of DNA damage in Portuguese wild birds. Blood smears from 65 birds with different life-history backgrounds (e.g. geographic origin, age) were collected between 2007 and 2011 and the frequency of erythrocyte nuclear abnormalities (ENAs) was analysed. Differences in DNA damage between ages were observed to occur in C. ciconia, with chicks displaying significantly higher frequencies of ENAs (both when looking at total ENAs or only MN frequency) than juveniles and adults. Additionally, significant differences in ENAs frequencies were observed between different years and geographic origins, whereas MN frequency alone did not show significant alterations concerning spatial and temporal variations. These results suggest that the assessment of ENAs rather than MN frequency alone may be a useful and valuable tool to complement the evaluation of DNA damage in populations of birds, as prompted by individual life-history traits and environmental factors.

Acute exposure to a quinalphos containing insecticide (convoy) causes genetic damage and nuclear changes in peripheral erythrocytes of silver barb, Barbonymus gonionotus

The present study was aimed to assess the genotoxic effect in fish caused by convoy, an insecticide commercial formulation containing quinalphos, present in the aquatic waterbody. For this purpose a freshwater teleost, silver barb was exposed to sublethal concentrations (25% and 50% of LC50) of convoy and erythrocytic cellular abnormalities (ECA) and erythrocytic nuclear abnormalities (ENA) tests were performed in addition to the commonly used micronucleus (MN) assay using peripheral erythrocytes and DNA contents in the different tissues after 1, 24, 36, 48, and 72 h of exposures. The obtained results indicated that acute exposure of different sub lethal concentrations of convoy to the fish resulted in significant alterations of erythrocytes as well as significant reduction of DNA contents in blood and vital organs and tissues, such as the brain, liver, kidney and muscle. Compared to each treatment excluding control group, frequencies of ECA, ENA, and MN were found to be elevated with exposure time of the doses. From this study, we conclude that convoy is a hazardous chemical to silver barb. Bioassays can be used as a tool for screening aquatic pollution, especially for insecticides.

Impact of treated wastewater on organismic biosensors at various levels of biological organization

Relating the treated wastewater quality and its impact on organismic biosensors (Prussian carp, Carassius gibelio and earthworm, Eisenia fetida) was the main objective of the study. The impact on health status of fish living downstream, microbiological contamination and antimicrobial resistance, fish tissue structure, blood biochemistry, oxidative stress, genotoxic effects, as well as multixenobiotic resistance mechanism (MXR) was assessed. Treated wastewater discharged from the WWTP modified the environmental parameters and xenobiotic concentrations of the receiving surface waters. Potential bacterial pathogens from fish and respective waters were found in relatively low numbers, although they comprised aeromonads with a zoonotic potential. High resistance profiles were determined towards the tested antimicrobial compounds, mostly sulfamethoxazole and erythromycin. Histopathology primarily revealed gill lamellar fusion and reduction of interlamellar spaces of effluent fish. A significant increase in plasma values of urea, total proteins, albumins and triglycerides and a significant decrease in the activity of plasma superoxide dismutase were noted in carp from the effluent-receiving canal. Micronucleus test did not reveal significant differences between the examined groups, but a higher frequency of erythrocyte nuclear abnormalities was found in fish sampled from the effluent-receiving canal. Earthworms indicated to the presence of MXR inhibitors in water and sludge samples, thus proving as a sensitive sentinel organism for environmental pollutants. The integrative approach of this study could serve as a guiding principle in conducting evaluations of the aquatic habitat health in complex bio-monitoring studies.

Common toad Rhinella arenarum (Hensel, 1867) and its importance in assessing environmental health: test of micronuclei and nuclear abnormalities in erythrocytes.

Anthropogenic activities may generate significant changes in the integrity of aquatic ecosystems, so long-term monitoring of populations that inhabit them is crucial. Counting micronucleated erythrocytes (MN) and erythrocytic nuclear abnormalities (ENA) in peripheral blood is a widely used method for detecting chromosomal damage due to chemical agents in the water. We analyzed MN and ENA frequency in blood obtained from the common toad Rhinella arenarum populations in sites with different degrees of environmental degradation. The results of this study indicate that there is an association between the frequency of micronuclei and nuclear abnormalities and the degree of environmental alteration recorded for the sites studied.

Fresh water fish, Channa punctatus, as a model for pendimethalin genotoxicity testing: A new approach toward aquatic environmental contaminants.

Pendimethalin (PND) is one of the common herbicides used worldwide. Fresh water fish, Channa punctatus, was exposed to PND in aquaria wherein its LC50 value was recorded to be 3.6 mg/L. Three sublethal (SL) concentrations, namely, 0.9, 1.8, and 2.7 mg/L were selected for the evaluation of genotoxicity and oxidative stress generated in the fish. In vivo comet assay was carried out in the blood, liver, and gill cells after exposing the fish to aforesaid SL concentrations of PND for 24, 48, 72, and 96 h. The results of the comet assay demonstrated the genotoxicity of PND in all the three tissues. Induction of oxidative stress in the gill cells was affirmed by the increased lipid peroxidation (LPO) and decreased levels of reduced glutathione, superoxide dismutase, and catalase. Frequencies of erythrocytic nuclear abnormalities (ENA) and micronuclei (MN) were also used to assess the genotoxic potential of PND on C. punctatus. MN frequency did not show any enhancement after PND exposure, but the frequency of ENA such as kidney-shaped nuclei, segmented nuclei and lobed nuclei, showed a significant increase after 24-96 h. Thus, ENA seems to be a better biomarker than MN for PND induced genotoxicity. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1520-1529, 2016.

Erythrocytic abnormalities in three Antarctic penguin species along the Antarctic Peninsula: biomonitoring of genomic damage

Pollutants and toxic contaminants produced in all parts of the world are transported to remote regions including Antarctica. Tourism, research, and fishing activities on this continent are another source of contamination. Toxic substances affect Antarctic species, and some produced genomic damage to the fauna. The genetic damage can be detected by microscopic observation of erythrocytic nuclear abnormalities (ENAs). We counted the number of ENAs in seven populations of three Pygoscelid penguin species, Adélie (Pygoscelis adeliae), Chinstrap (Pygoscelis antarctica), and Gentoo (Pygoscelis papua), and found important differences among species exposed to the same conditions. ENAs were more frequent in Adélie penguins than in the other two species. Inter-population comparisons within species showed remarkable differences in Adélie and Chinstrap penguins but not in Gentoo penguin. Frequency of ENAs in Adélie penguins was the highest in Yalour Island population, intermediate in King George Island population, and the lowest in Torgersen Island and Avian Island populations. In Chinstrap penguins, the highest number of ENAs was found on Deception Island, and significant differences were found only between Deception Island and King George Island populations. This information will provide baseline data to be used for assessing the evolution of genomic damage of penguins along the Antarctic Peninsula in the future.

Assessment of chromosomal damage induced by a deltamethrin-based insecticide in fish (Anguilla anguilla L.) – A follow-up study upon exposure and post-exposure periods

The pyrethroid insecticide Decis®, containing deltamethrin as active ingredient, is among the most popular broad-spectrum biocides, with wide application in agriculture and home pest control. The occurrence of deltamethrin in the aquatic environment is well-established, but the possible genotoxic effects of Decis® in non-target organisms, namely fish, remain unknown. Hence, this work aimed to evaluate the cytogenetic damaging potential of Decis® in European eel (Anguilla anguilla L.), adopting the erythrocytic nuclear abnormalities (ENAs) assay. In addition, it was intended to investigate the damage progression in the post-exposure period. The frequency of immature erythrocytes (IE) was also determined to provide indirect information on the erythrocyte catabolism and erythropoiesis rate. Fish were exposed to 17.5 and 35μgL−1 of Decis® (equivalent to 0.05 and 0.1μgL−1 of deltamethrin, respectively) during 1 and 3days. Thereafter, fish were transferred to clean water and kept for 1, 7 and 14days. The results demonstrated a clear potential to induce chromosomal damage following 3days exposure, depicted in an ENA frequency increase for both Decis® concentrations. The transient nature of this cytogenetic damage was also demonstrated, as ENA frequency returned to the control level 1 and 7days after cessation of the exposure, respectively for the higher and the lower Decis® concentration. Moreover, this response pattern suggested a rapid metabolization and elimination of the formulation constituents by A. anguilla, combined with an increased erythrocyte turnover in fish exposed to the higher Decis® concentration, as pointed out by the IE frequency rise. Overall, the demonstrated genotoxic properties of Decis® pointed out increased risk factors to fish exposed to this insecticide.

Individual and Mixture Toxicity of Commercial Formulations Containing Glyphosate, Metsulfuron-Methyl, Bispyribac-Sodium, and Picloram on Rhinella arenarum Tadpoles

We investigated the effects of four commercial formulations of herbicides (glyphosate [GLY], metsulfuron-methyl [MET], bispyribac-sodium [BIS], and picloram [PIC]) individually, and in three 50:50 mixtures (GLY–MET, GLY–BIS, GLY–PIC) on the common toad Rhinella arenarum (Anura: Bufonidae) tadpoles. Enzymatic parameters such as, glutathione S-transferase (GST), butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) activities, as well as erythrocyte nuclear abnormalities (ENA) were studied. Interactions between herbicides in mixtures were evaluated and classified as additive, synergistic, or antagonistic. Toxicity results (48-h LC50) showed that PIC was the most toxic herbicide, followed by BIS, GLY, and MET, while GLY–PIC was the most toxic mixture, followed by GLY–BIS, and GLY–MET. All commercial herbicide formulations and their mixtures significantly inhibited BChE activity in exposed tadpoles. The AChE activity was also inhibited by all herbicides and their mixtures, except by GLY–BIS. The inhibition of GST activity was only significant for GLY, MET, PIC, and GLY–MET. A significant increase in the frequency of ENA was found for tadpoles exposed either to commercial herbicide formulations or to mixtures, except for GLY. All the mixtures showed synergism for BChE activity while for AChE only the GLY–MET and GLY–PIC mixtures acted synergistically. GLY–MET showed synergism for GST, whereas for ENA, the mixture GLY–BIS was antagonistic. This study with R. arenarum tadpoles demonstrates that the interactions between three of the most intensively used herbicides in soybean crops results in synergistic effects on mortality and neurotoxicity and synergistic or additive effects in genotoxicity.

Mercury-Induced Chromosomal Damage in Wild Fish (Dicentrarchus labrax L.) Reflecting Aquatic Contamination in Contrasting Seasons

Ria de Aveiro (mainly Laranjo basin, Portugal) has been subjected to mercury contamination from a chlor-alkali plant, currently presenting a well-described mercury gradient. This study aimed to assess mercury genotoxicity in this area by measuring the frequency of erythrocytic nuclear abnormalities (ENA) in the European sea bass (Dicentrarchus labrax), addressing the relation with total mercury concentration in the blood and the modulatory role of seasonal variables. Fish were collected, in warm and cold periods, at three locations differing in their distances to the main mercury source: reference (R), moderately (M), and highly (H) contaminated sites. Genotoxicity was detected in both degrees of contamination (M and H) and in both periods of the year (warm and cold), which is in line with the greater levels of mercury measured in fish blood. No significant seasonal variations were observed for mercury bioaccumulation or ENA frequency. The apparent low imperviousness of ENA frequency to seasonal factors reinforced its consistency as a genotoxicity biomarker, thus enabling a clearer identification of cause-and-effect relationships. Overall, the results reflected a serious environmental risk to native ichthyofauna at Laranjo basin due to mercury contamination, showing a potential of mercury to induce genetic damage in fish blood cells through clastogenic and/or aneugenic actions.

Golden grey mullet and sea bass oxidative DNA damage and clastogenic/aneugenic responses in a contaminated coastal lagoon

Several xenobiotics or their metabolites have redox-cycling properties and potential to induce oxidative stress and DNA damage. The current work aimed to study, under environmental conditions, oxidative DNA damage (8-hydroxy-2′-deoxyguanosine (8-OHdG)) and its association with chromosomal damage measured as erythrocytic nuclear abnormalities (ENAs), in Liza aurata and Dicentrarchus labrax, caught at a costal lagoon (Ria de Aveiro, Portugal) having sites with different contamination profiles. The quantified parameters were also used to assess the lagoon’s environmental status. Five critical sites were assessed comparing to a reference site. L. aurata displayed higher 8-OHdG levels and ENAs frequency, respectively, at Laranjo and Vagos. D. labrax 8-OHdG levels were higher at the sites where quantification was possible whereas no differences were found in terms of ENAs. No correlation was found in both species between 8-OHdG and ENAs. Despite no direct linkage between the two biomarkers was found, this study demonstrates species and site dependent genotoxic responses.

Genotoxic Effects of Aluminum on the Neotropical Fish Prochilodus lineatus

Applying an integrated approach using the Comet, micronucleus (MN), and random amplified polymorphic DNA (RAPD) assays, occurrence of erythrocytic nuclear abnormalities (ENAs) and the liver activity of antioxidants enzymes (catalase and glutathione-S-transferase (GST)) was carried out to evaluate the effects of acute (6, 24, and 96 h) and subchronic (15 days) exposures to aluminum on fish Prochilodus lineatus. The Comet assay showed that fish erythrocytes exhibited significantly higher DNA damage after 6 and 96 h of Al exposure. MN frequencies were very low and did not increase significantly after Al exposures, while ENAs frequency increased significantly after all exposure periods. RAPD profiles obtained with DNA from fish fins collected before the toxicity tests were compared to the profiles with DNA from gills and liver of the same fish sampled after Al exposures. Alterations in RAPD profiles, including appearance and disappearance of bands, after 6 h, 24 h, and 15 days of Al exposure were detected. Fish exposed to Al for 6 and 24 h also showed significant increases in GST and catalase activities. These results indicated that Al exposure was genotoxic to P. lineatus, inducing DNA damage in peripheral erythrocytes. The induction of antioxidant enzymes might be an indication that Al causes oxidative damage to DNA, while the very low frequency of MN suggests that Al does not produce clastogenic or aneugenic effects. Genotoxic effects after 15 days of Al exposure was revealed only by RAPD, showing that this assay represents a sensitive method to detect genotoxic damage, occasionally not detected by other genotoxic tests used in toxicological genetics studies.

Erythrocytic nuclear abnormalities in wild and caged fish ( Liza aurata) along an environmental mercury contamination gradient

Laranjo basin (Aveiro, Portugal) has been subjected to mercury contamination from a chlor-alkali plant, presenting a well-described mercury gradient. This study aims the assessment of mercury genotoxicity in this area by measuring erythrocytic nuclear abnormalities (ENA) frequency in the mullet Liza aurata, and its relation with total mercury concentration (Hg t) in blood. Wild fish were seasonally analysed, and, complementarily, fish were caged for 3 days at three locations differing on their distances to the mercury source. The results from Laranjo were compared with those from a reference area (S. Jacinto). Wild fish from Laranjo showed elevated ENA frequency in summer and autumn in concomitance with increased blood Hg t. Surprisingly, no ENA induction was found in winter, despite the highest blood Hg t, which may be explained by haematological dynamics alterations, as supported by a decreased immature erythrocytes frequency. Caged fish displayed ENA induction only at the closest site to the contamination source, also showing a correlation with blood Hg t.

Genotoxic effects of binary mixtures of xenoandrogens (tributyltin, triphenyltin) and a xenoestrogen (ethinylestradiol) in a partial life-cycle test with Zebrafish (Danio rerio)

A partial life-cycle test with the model fish Danio rerio was performed in order to evaluate the genotoxic potential of binary mixtures of xenoandrogenic (tributyltin — TBT; triphenyltin — TPT) and an estrogenic compound (ethinylestradiol — EE2). Five days post-fertilisation larvae were diet-exposed to environmental relevant concentrations of TBT and TPT (25 ng/g–100 ng/g), and water-exposed to ethinylestradiol (3.5 ng/L) for a four-month period; binary mixtures of TBT plus EE2 and TPT plus EE2 were run in parallel. The erythrocytic nuclear abnormalities (ENA) assay in circulating erythrocytes was used to evaluate genotoxicity in the end of the four-month exposure period. A significant increase (p<0.05, Kruskall–Wallis non-parametric ANOVA) in ENA frequency, in comparison with control animals, was observed in those animals exposed to TBT and TPT (the highest doses only), and to EE2 and the binary mixtures, although neither synergistic nor additive effects of the tested compounds were evident. Overall, the results clearly indicate that chronic exposure to low levels of TBT, TPT, EE2 and binary mixtures of TBT plus EE2 and TPT plus EE2 are genotoxic to zebrafish, which may suggest that wild fish populations may be under increased DNA damage in areas contaminated by these endocrine disrupting chemicals.

Biotransformation and Genotoxic Biomarkers in Mullet Species (LIZA SP.) From a Contaminated Coastal Lagoon (Ria De Aveiro, Portugal)

The aim of the present study was to assess liver ethoxyresorufin-O-deethylase (EROD) activity and erythrocytic nuclear abnormalities (ENA) frequency in three mullet species, as biotransformation and genotoxicity biomarkers, respectively, for monitoring aquatic ecosystems. Specimens of Liza aurata, Liza ramada and Liza saliens were caught bimonthly, between May 2000 and March 2001, at nine stations in the Ria de Aveiro (Portugal), a polluted coastal lagoon. The survey revealed two critical areas (Vagos and Carregal) where it was observed elevated EROD activity and ENA frequency, comparing to the reference sites. The results often reflect a contamination by a mixture of pollutants, from point and non-point sources. Nevertheless, the responses observed at Vagos are probably attributable to high polycyclic aromatic hydrocarbon (PAH) water levels measured at that station. All species were able to detect inter-site differences; however L. aurata seems to be the most appropriate bio-indicator due to its larger geographical distribution and abundance during the whole year. The adopted biomarkers, applied to mullet species, can be successfully employed for biomonitoring anthropogenic contamination in both Atlantic and Mediterranean European coastal waters.

Physiological and genetic responses of European eel (Anguilla anguilla L.) to short-term chromium or copper exposure?Influence of preexposure to a PAH-like compound

Anguilla anguilla L. (European eel) was exposed for 24 h to chromium (Cr-100 microM and 1 mM) or copper (Cu-1 and 2.5 microM), with or without a 24-h preexposure to beta-naphthoflavone (BNF-2.7 microM), a polycyclic aromatic hydrocarbon (PAH)-like compound, simulating sequential exposure to PAHs and heavy metals. Plasma cortisol, thyroid-stimulating hormone (TSH), free triiodothyronine (T3), and free thyroxine (T4) were determined in order to assess the effects on endocrine function. Plasma glucose and lactate also were measured. The frequency of erythrocytic nuclear abnormalities (ENA) was scored as a genotoxicity indicator. Plasma T4 decreased in eels when exposed to Cr only. The interference of BNF preexposure on Cr effects was observed as a significant plasma glucose increase. Single exposures to Cu elevated plasma cortisol and glucose (2.5 microM), as well as plasma lactate (1 microM), whereas a T4 decrease was found for both concentrations. BNF preexposure prevented plasma cortisol and lactate increases; however, a greater T4 decrease was observed in eels exposed to 2.5 microM Cu. Moreover, this pretreatment was crucial for genotoxicity expression because only BNF+2.5 microM Cu-exposed fish exhibited significant ENA induction. In general, plasma T4 was the most affected hormone, as it responded to all Cr and Cu exposure conditions.

Anguilla anguilla L. liver ethoxyresorufin O-deethylation, glutathione S-tranferase, erythrocytic nuclear abnormalities, and endocrine responses to naphthalene and β-naphthoflavone

The effects of naphthalene (NAP) and β-naphthoflavone (BNF) on phase I biotransformation and genotoxicity in Anguilla anguilla L. were evaluated. Phase II biotransformation and cortisol levels were also assessed in NAP-treated fish. Two groups of eels were exposed to either a NAP or a BNF concentration range (0.1–2.7 μM) for different exposure periods (2–72 h). An early significant ethoxyresorufin O-deethylation (EROD) activity inhibition was observed, especially for the highest NAP concentrations at 2–6 h exposure and for BNF at 2 h exposure. However, a significant EROD activity increase was detected from 16 to 72 h exposure for NAP and from 4 to 72 h exposure for BNF. The cytochrome P450 (P450) content was not dose related. However, with regard to BNF exposure, P450 was the first biomarker to respond. Liver alanine transaminase (ALT) activity was measured as an indicator of hepatic health condition. ALT results demonstrated that the EROD activity decrease, previously described for NAP, was not related to tissue damage. Nevertheless, the highest BNF concentrations were demonstrated to induce liver damage and to impair the EROD activity response. An increased genotoxic response, measured as erythrocytic nuclear abnormalities (ENA), was observed during the first 8 h NAP exposure. However, for exposures longer than 8 h, ENA frequency returned to the control levels. This response profile may reflect a considerable DNA repair capacity and/or a metabolic adaptation providing an efficient NAP biotransformation and consequent detoxification. BNF revealed no ENA alterations for all concentrations and exposure lengths. In the NAP experiment a causal relationship between immature erythrocytes (IE) and ENA frequency disappearance was not found. BNF results with regard to IE frequency revealed an ability to alter the balance between erythropoiesis and removal of erythrocytes. Liver glutathione S-transferase activity was significantly induced after 2 and 48 h NAP exposure. A cortisol-impaired response seems to occur from 4 to 24 h NAP exposure, demonstrating an endocrine disruption. However, an adaptation process seems to occur after 48 h, since the plasma cortisol had a tendency to increase. The present findings confirm the usefulness of the adopted biomarkers. The ecological risk associated with aquatic contamination by NAP was also confirmed by the present data.

Genotoxic and biochemical responses in caged eel ( Anguilla anguilla L.) after short-term exposure to harbour waters

European eel ( Anguilla anguilla L.) were caged and exposed in situ for 8 and 48 h to the Aveiro offward fishing harbour water (HW) and to clean seawater under laboratory conditions (Control). Eel liver biotransformation (Phase I) was measured as ethoxyresorufin- O-deethylase (EROD) activity, cytochrome P450 ( P450) and glutathione S-transferase (GST) activity (Phase II). Genotoxic responses were determined as blood, liver and kidney DNA strand breaks as well as erythrocytic nuclear abnormalities (ENAs). HW failed to significant increase liver EROD, GST activities and ENA frequency. Nevertheless, P450 content was significantly increased after 8 and 48 h exposure. Genotoxicity measured as DNA integrity decrease was found in blood after 8 and 48 h exposure to HW, whereas in liver and kidney, it was observed after 48 h exposure to HW. Blood, kidney and liver genotoxicity may be due to the presence of polycyclic aromatic hydrocarbons (PAHs) which are genotoxic compounds and the main HW organic contaminants.

Genotoxic and hepatic biotransformation responses induced by the overflow of pulp mill and secondary-treated effluents on Anguilla anguilla L.

Pulp and paper mill effluent compounds pollute the aquatic environment and are responsible for increased biochemical alterations and genotoxicity in aquatic organisms such as fish. Adult eels ( Anguilla anguilla L) were exposed during 8, 16, 24, and 72 h to the following conditions: (1) aerated, filtered, and dechlorinated tap water ( C); (2) 2.5% (v/v) sewage water previously treated with activated sludge ( T); (3) bleached kraft pulp and paper mill effluent collected at the river Vouga, close to an ancient sewage outlet (Portucel), diluted in tap water [25% ( E 25 ) and 50% ( E 50 )]; and (4) bleached kraft pulp and paper mill effluent sediment [water-soluble fraction ( S)]. Liver biotransformation induced by the above conditions was measured as ethoxyresorufin- O-deethylase (EROD), cytochrome P450 (P450) (Phase I), and glutathione- S-transferase (GST) (Phase II). Genotoxicity was also determined as blood/liver DNA strand breaks and erythrocytic nuclear abnormalities (ENA) induced on European eel ( A. anguilla L). Liver EROD activity was significantly increased in eels at 8 and 16 h exposure to E 25 , as well as at 16, 24, and 72 h exposure to E 50 . S exposure induced liver EROD activity only at 24 h. A significant decrease in liver P450 was observed at 72 h exposure to T, whereas a significant P450 increase at 16 h was followed by a significant decrease at 24 h exposure to E 25 . Another P450 significant increase was noticed at 72 h exposure to S. Liver GST activity (Phase II) demonstrated a significant increase at 72 h exposure to E 50 and to S. A significant decrease in blood DNA integrity was observed at 72 h exposure to T and at 24 and 72 h to S. Blood DNA integrity significantly decreased at 16 and 24 h exposure to E 25 , as well as at 8, 16, and 24 h exposure to E 50 . Liver DNA integrity significantly decreased at 72 h exposure to T and at 16 h exposure to S. Moreover , liver DNA integrity was significantly decreased at 24 h exposure to E 25 and E 50 , and 72 h to E 50 . A. anguilla L. increased ENA frequency was detected in T at 16, 24, and 72 h, whereas in E 25 and S it was observed at 8, 16, and 24 h. Furthermore, E 50 ENA frequency increased at 24 h exposure.

Biotransformation, genotoxic, and histopathological effects of environmental contaminants in European eel ( Anguilla anguilla L.)

A prolonged toxicity study was carried out in young European eel ( Anguilla anguilla L.) to evaluate the effects of environmental contaminants, namely, two individual standard compounds, benzo[ a]pyrene (BaP) and dehydroabietic acid (DHAA), and a complex mixture, bleached kraft pulp mill effluent (BKPME). Fish were exposed to BaP (0.22, 0.45, and 0.9 μM) and BKPME (3.12%, 6.25%, and 12.5% (v/v)) for 3, 7, and 30 days and to DHAA (0.07, 0.15, and 0.30 μM) for 3, 7, 30, 90, and 180 days. The biomarkers include biotransformation and genotoxicity indicators, such as total ethoxyresorufin O-deethylase (EROD) activity and frequency of erythrocytic nuclear abnormalities (ENAs), respectively. Hematological dynamics was assessed as frequency of immature erythrocytes (IEs). Histopathological examinations were carried out for the highest concentrations and for 30 days and longer exposures. Total EROD increases significantly only after 180 days of DHAA exposure. However, significant ENA induction was generally observed during exposure to all contaminants tested. Nevertheless, some of the ENA results suggest an altered genotoxic response, which may arise either from short-term exposures to the highest contaminant levels or long-term exposures to the lowest contaminant levels. IE frequency decreased significantly after 30 days of exposure to 0.45 μM BaP and 180 days of exposure to the entire DHAA concentration range. Increased density of pigmented macrophage aggregates in 30-day BaP- and BKPME-exposed fish as well as in 90- and 180-day DHAA-exposed fish confirmed histopathological liver alterations. Bile accumulation in hepatocytes after BaP treatment, cytoplasmic vacuolization and cell atrophy following DHAA exposure, as well as liver loss of parenchymal cells in BKPME-exposed fish, were also detected. Dispersed necrosis and focal inflammation were observed in the livers of all treated groups. Fish exposed to DHAA and BKPME showed skin and gill disruption as well as kidney Malpighian corpuscle alterations. All 30-day-treated groups revealed intense spleen hemosiderosis, indicating increased erythrophagia. This splenic effect may be strongly correlated with the observed disappearance of ENAs. Neoplastic lesions were not found. A multibiomarker strategy, which includes EROD, ENA, and IE assays as well as histopathological studies, contributed to a better understanding of the global toxic process.

Anguilla anguilla L. Biochemical and Genotoxic Responses to Benzo[ a]pyrene

Eels ( Anguilla anguilla L.) were exposed for 2, 4, 6, 8, 16, 24, 48, 72, 144, and 216 h to 0 (control), 0.3, 0.9, and 2.7 μM benzo[ a]pyrene (BaP). The biotransformation induced by BaP was measured as liver ethoxyresorufin O-deethylase (EROD) activity and cytochrome P450 content, and compared with the genotoxic effects, such as erythrocytic nuclear abnormalities (ENA), and blood and liver DNA strand breaks. The liver exhibited a highly significant EROD activity increase from 2 up to 216 h exposure to 0.9 and 2.7 μM BaP, whereas 0.3μM BaP exposure induced a significant liver EROD increase from 2 up to 144h. Liver cytochrome P-450 content was significantly increased at 8 h to 2.7 μM BaP exposure. Liver DNA integrity was decreased at 16 h, from 8 up to 144 h and 8 up to 72 h exposure to 0.3, 0.9, and 2.7 μM BaP, respectively. A significant decrease in blood DNA integrity was observed at 48, 72, 144 h, from 8 up to 72, and from 6 up to 72 h exposure to 0.3, 0.9, and 2.7 μM BaP, respectively. The A. anguilla L. genotoxic response to BaP, measured as ENA induction, was significantly increased at 144 h exposure to 0.3 μM BaP. The intermediate BaP concentration tested (0.9 μM) induced a significant three fold ENA increase at 48 and 72 h exposure compared to their controls. The highest BaP concentration (2.7μM) induced a significant increase in ENA frequency at 72, 144 and 216 h exposure.