Erythrocytic Nuclear Abnormalities Research Articles

Long-Term Toxicity Effects of Cadmium and Lead on Bufo raddei Tadpoles

Cadmium (Cd) and lead (Pb), entering aquatic systemsfrom industrial and consumer waste can cause contamina-tion of the aquatic environment. As a result of bioaccu-mulation when they enter the food chain, Cd and Pb cancause serious damage to physiological processes or tissuesin aquatic organisms even at concentrations far below thelethal level (He et al., 2005).Erythrocytic micronuclei (EMN) and erythrocytic nu-clear abnormalities (ENA) have been routinely applied toassess water quality and the genotoxicity of contaminantson organisms (Chandra and Khuda-Bukhsh, 2004; Zhuet al., 2004). Superoxide dismutase (SOD) is an enzymethat can scavenge superoxide anions and protect organismsfrom adverse effects. Adenosine triphosphatase (ATPase)is an enzyme that catalyzes adenosinetriphosphate hydro-lysis and provides chemical energy for organisms. Theactivities of SOD and ATPase have been used as biomar-kers for assessing water quality (Achuba, 2002; Brauneret al., 2003).Like fish, tadpoles are inevitably affected by heavymetals in aquatic systems. Perez-Coll and Herkovits (1990;1996) and Herkovits and Perez-Coll (1993) have docu-mented the accumulation of Cd or Pb in the tissues andorgans of embryos and tadpoles. Lefcort et al. (1998) re-ported that heavy metals could alter the survival, growth,metamorphosis and antipredatory behavior of Rana lutei-ventris tadpoles. Rosenberg et al. (1998) studied the sub-lethal effect of lead on erythrocyte osmotic fragility in Bufoarenarum. Naab et al. (2001) reported that Zn could inhibitthe pentose phosphate pathway in the ovaries of adultB. arenarum females, resulting in a decrease of the embryosurvival ratio. Carattino et al. (2004) showed that bothglucose 6-phosphate dehydrogenase and 6-phosphogluco-nate dehydrogenase activities could be used as biomarkersfor Cu but not for Cd toxicity. Fonovich de Schroeder(2005) proposed a possible role for glucose 6-phosphatedehydrogenase as a biomarker for Zn in B. arenarumovarian tissue. Bufo raddei is the most common species inNorth China and is highly adaptable to polluted environ-ments. Our previous work (Huang et al., 2004; Long et al.,2004) showed that Cd and Pb increased the mortality andmalformation ratio and prolonged the development andmetamorphosis period in tadpoles, but had little effect onthe hatching of B. raddei. Although various reports haveshown that heavy metals can affect tadpoles’ growth anddevelopment, little is known about the chronic toxicityeffects of Cd and Pb on the physiological processes of B.raddei. tadpoles. The purpose of this research is to explorethe effects of Cd and Pb on EMN and ENA frequencies,SOD and ATPase activities in target organs of B. raddeitadpoles with increasing concentrations of heavy metal andexposure times.Materials and MethodsBovine serum albumin (BSA) and Giemsa were obtainedfrom Sigma (USA). All other chemicals and reagents wereof analytical grade and were procured from local chemicalcompanies.

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.

Monitoring of nuclear abnormalities in peripheral erythrocytes of three fish species from the Goksu Delta (Turkey): genotoxic damage in relation to water pollution

Goksu Delta is a specially protected area in the Mediterranean region of Turkey. The delta is classified as a Wetland of International Importance according to the RAMSAR Convention on Wetlands of International Importance. Increases in population have recently taken place in this region due to heavy agricultural activities and discharges of anthropogenic wastes. In the present study, frequencies of erythrocytic nuclear abnormalities such as, micronuclei (MN) and nuclear buds (NB) were investigated in peripheral blood of three fish species; Clarias gariepinus (Catfish), Alburnus orontis (Bleak), and Mugil cephalus (Mullet) from Akgol (AG) and Paradeniz (PD) lagoons of Goksu Delta. Concentrations of heavy metals (Cu, Cd, Ni, Pb) were also measured in the water, sediment samples. MN and NA frequencies were elevated in fish from AG and PD lagoons in comparison with those from upstream regions. The results of this study indicate that the lagoons of Goksu Delta contaminated with genotoxic pollutants and that the genotoxicity is related to the agricultural activities and to the discharge of anthropogenic waste waters.

Endocrine and metabolic responses of Anguilla anguilla L. caged in a freshwater–wetland (Pateira de Fermentelos—Portugal)

The present short-term in situ study was carried out in a freshwater-wetland--Pateira de Fermentelos--considered an enlargement of Cértima River, in the centre of Portugal. This ecosystem is an important fishing and recreation place, receiving effluents from different origins namely, electroplating industrial effluents containing heavy metals, domestic wastes, as well as pesticides and fertilizers resulting from agriculture activities. The aim of the present research work was to monitor the effects induced by the contaminants present in Pateira de Fermentelos water, using Anguilla anguilla L. (European eel) as a bioindicator. The eels were caged for 48 h at four Pateira de Fermentelos sites, differing in their distances to the main known pollution source (Cértima River): A (close to the lagoon entrance), B, C and D (the farthest from the Cértima River). A reference site was selected near the Cértima river spring. The following parameters were measured: liver ethoxyresorufin-O-deethylase (EROD) and alanine transaminase (ALT) activities, plasma levels of cortisol, 17beta-estradiol (E2), thyroid-stimulating-hormone (TSH), free thyroxine (T4), free triiodothyronine (T3), as well as glucose and lactate. The erythrocytic nuclear abnormalities (ENA) frequency was also scored as a genotoxicity indicator. The results revealed increased plasma cortisol and glucose concentrations at all exposure sites, displaying a similar response pattern. Plasma T3 showed a significant decrease only at site A when compared to reference site, whereas plasma E2 increased at sites B and D when compared to all the other sites, including reference site. The present results indicate the Pateira de Fermentelos water contamination, demonstrating the usefulness of the adopted strategy.

Cytochrome P4501A, genotoxic and stress responses in golden grey mullet ( Liza aurata) following short-term exposure to phenanthrene

This study represents a first approach to short-term effects of phenanthrene (Phe) in fish. The teleost Liza aurata was exposed to 0.1–2.7 μM Phe during 16 h. CYP1A induction was assessed as liver ethoxyresorufin- O-deethylase (EROD) activity. Genotoxicity was evaluated in gill and liver as DNA integrity (by alkaline unwinding), whereas in blood the erythrocytic nuclear abnormalities (ENA) frequency was determined. Stress responses were determined as cortisol, glucose and lactate plasma levels. Liver EROD activity was significantly increased by Phe 0.3–2.7 μM. Phe genotoxicity in gill was not found, whereas liver DNA integrity significantly decreased after exposure to Phe 0.1 and 0.9 μM demonstrating its genotoxicity which did not correlate with liver CYP1A induction. Phe genotoxicity in blood was demonstrated by a significant ENA increase from 0.1 up to 2.7 μM. In terms of stress responses, plasma cortisol was significantly increased by Phe 0.3–2.7 μM, though plasma glucose was only significantly increased by Phe 0.9 and 2.7 μM. The Phe observed effects on L. aurata detected at different levels demonstrate a physiological unbalance and a probable ecological risk to ichthyofauna.

Biomonitoring of the genotoxic potential (micronucleus and erythrocyte nuclear abnormalities assay) of the Admiralty Bay water surrounding the Brazilian Antarctic Research Station “Comandante Ferraz,” King George Island

The micronucleus (Mn) and other kinds of erythrocyte nuclear abnormality (ENA) assays were used to assess the genotoxic potential of the seawater in front of the Brazilian Antarctic Research Station “Comandante Ferraz” (King George Island). In two consecutive summers, the fish Trematomus newnesi was exposed to the seawater in front of the fuel storage tanks and the sewage discharge outlet of the Station in laboratory bioassays and in situ caging assays. After the exposure, frequencies of ENA and Mn were scored on blood smears. Significantly higher Mn and ENA frequencies in relation to those of the controls were observed in fish exposed to both types of water, in both types of experiment. Seawater in front of the fuel tanks and sewage discharge outlet at the Station was found to induce the formation of micronuclei and other ENAs, indicators of genotoxicity. The need for further investigation and the suitability of the methods for screening mutagenic pollution in polar waters are discussed.

Nuclear abnormalities in erythrocytes of parrots (Aratinga canicularis) related to genotoxic damage

Nuclear abnormalities in erythrocytes, as micronuclei and nuclear buds (BE), are considered potential biomarkers of genotoxic exposure. We described previously the frequency of spontaneous micronucleated erythrocytes (MNE) in the species Aratinga canicularis. Here, we have used this species to evaluate the induction of MNE and BE by mitomycin-C. Animals were given a single intracoelomic injection of 0, 2, 3 or 4 mg/kg mitomycin-C on two consecutive days. A drop of blood was obtained after 0, 24, 48 and 72 h, and stained smears were used to count micronucleated polychromatic erythrocytes (MNPCE) and polychromatic erythrocytes with buds (BPCE)/1000 polychromatic erythrocytes. The number of MNE and BE in 10 000 total erythrocytes was also counted. MNPCE and BPCE frequencies were elevated at 24, 48, and 72 h after the administration of the lower dose (P<0.03). At a 3 mg/kg dose, the frequency of MNPCE increased at 48 and 72 h (P<0.04) whereas the number of BPCE increased, but not significantly. Administration of 4 mg/kg mitomycin-C increased the number of MNE observed at 72 h (P<0.03), the number of MNPCE at 48 h (P<0.01) and 72 h (P<0.006), the BE frequency at 72 h (P<0.05), and the frequency of BPCE at 48 and 72 h (P<0.001). While mitomycin-C appears to produce a parallel increase in MNPCE and BPCE frequencies, the MNE seemed to be a more sensitive indicator of genotoxicity than the BE. This suggests that evaluating BE and MNE in routine haematological analysis should be considered to evaluate environmental genotoxic exposure.

Biotransformation, stress and genotoxic effects of 17β-estradiol in juvenile sea bass (Dicentrarchus labrax L.)

The effects of 17β-estradiol (E2) on fish became a matter of concern, since significant levels of this hormone were detected in the aquatic environment released mainly by domestic sewage treatment plants. In this perspective, the current study was focused on E2 effects upon biotransformation, stress and genotoxic responses of juvenile Dicentrarchus labrax L. (sea bass). Fish were exposed to E2 during 10 days in two different ways: water diluted (200 ng/L or 2000 ng/L) and i.p. injected (0.5 mg/kg or 5 mg/kg). A battery of biological responses was evaluated: liver ethoxyresorufin-O-deethylase (EROD) and alanine transaminase (ALT) activities, liver somatic index (LSI), plasma cortisol, glucose and lactate concentrations, as well as erythrocytic nuclear abnormalities (ENA).All the exposure conditions induced endocrine disruption, measured as plasma cortisol decrease, and genotoxicity, measured as ENA increase. Thus, no differences were detected either between different exposure routes or tested concentrations. Concerning liver EROD and ALT activities, as well as plasma glucose and lactate concentrations no differences were found between treated and control groups. LSI was the only parameter to respond differently in the two exposure routes, as only E2 water diluted induced a significant increase in this hepatic indicator.

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.

Anguilla anguilla L. liver EROD induction and genotoxic responses after retene exposure

Anguilla anguilla L. (European eel) were exposed for 8, 16, 24, and 72 h to 0 (control), 0.1, 0.3, 0.9, and 2.7 μM retene. A. anguilla L. liver ethoxyresorufin- O-deethylase (EROD) activity significantly increased during the whole exposure period to all retene concentrations, when compared to their controls. The liver cytochrome P450 content only increased after exposure to high retene concentrations (0.9 and 2.7 μM) from 8 to 24 and 72 h, respectively. Generally, liver DNA integrity decreased with increased retene concentrations. Thus, a low retene concentration (0.1 μM) was only effective at 16 h, 0.3 and 0.9 μM had an early and prolonged effect up to 24 h, and 2.7 μM decreased liver DNA integrity during the whole exposure period. However, blood DNA integrity decrease was observed in eels after 24 h exposure to 0.1 μM retene, and at 16 h to 0.3 and 0.9 μM retene, despite an early blood DNA integrity decrease at 8 and 16 h exposure to 2.7 μM retene. An early genotoxic response to retene was also observed as erythrocyte nuclear abnormalities plus Notched (ENA+ Not) frequency increase at 8, 16, and 24 h exposure to 0.1 and 0.3 μM retene as well as at 8, 16, 24, and 72 h to 0.9 μM retene. Though, the highest retene concentration (2.7 μM) only induced ENA+ Not and erythrocyte nuclear abnormalities minus Notched (ENA− Not) at 16 h exposure. The eel ENA+ Not increase was more sensitive than the ENA− Not increase as a measure of retene genotoxicity. Eel liver alanine amino transferase (ALT) increased activity reveals its enhanced transamination capacity after short-term exposure to retene. The A. anguilla L. ratio between hemoglobin concentration and red blood cells count (Hb/RBC) increased at 8 h exposure to 0.1, 0.3, and 0.9 μM retene, suggesting an initial homeostatic process.

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.

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

Juvenile sea bass, Dicentrarchus labrax L., were exposed during 2, 4, 8, and 24 h to 0.9 μM β-naphthoflavone (BNF), 131 nM 17β-estradiol (E 2), 4.05 μM 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.

Anguilla anguilla L. genotoxic and liver biotransformation responses to abietic acid exposure

Adult eels ( Anguilla anguilla L.) were exposed for 8, 16, 24, and 72 h to 0, 0.1, 0.3, 0.9, and 2.7 μM abietic acid (AA). Genotoxicity was measured as erythrocytic nuclear abnormalities (ENA), as well as DNA strand breaks in blood and liver. Liver cytochrome P450 (P450) content, liver ethoxyresorufin O-deethylase (EROD), and glutathione S-transferase (GST) activities were determined as biotransformation biomarkers. Liver alanine transaminase (ALT) activity was also measured as an indication of tissue damage. Low AA concentrations, such as 0.1 and 0.3 μM, result in a delayed induction of A. anguilla L. liver EROD activity, whereas the higher AA concentration (2.7 μM AA) also has a delayed effect probably as a consequence of liver tissue high inhibitory concentration. The current eel liver GST activity results demonstrate that only low AA concentrations promote liver increases in GST, whereas high AA concentrations, such as 0.9 and 2.7 μM, do not alter it. The results concerning eel liver ALT activity indicate that significant liver damage is induced by high AA concentrations, such as 2.7 and 0.9 μM. The eel ENA result analysis reveals that AA is a weak ENA inducer in A. anguilla L. Blood DNA integrity results suggest that low AA concentrations promote late decreases in blood DNA integrity; nevertheless, high AA concentrations are early blood genotoxic inducers compared with low AA doses. According to the present research results with respect to eel liver DNA damage, all of the AA exposure concentrations decreased liver DNA integrity.

Micronuclei, nuclear lesions and interphase silver-stained nucleolar organizer regions (AgNORs) as cyto-genotoxicity indicators in Oreochromis niloticus exposed to textile mill effluent

In this study, cyto-genotoxic effects of a textile mill effluent on fish Oreochromis niloticus were investigated using the micronucleus (MN) test and methods to analyze interphase silver-stained nucleolar organizer regions (AgNORs). Fishes were exposed to three different concentrations of textile mill effluent (5, 10 and 20% (v/v)) for 3, 6 and 9 days. Cyclophosphamide (2 mg/l) was used as a positive control. Micronucleus frequencies were examined in peripheral blood erythrocytes and gill cells. Nuclear abnormalities (NA) other than micronuclei such as binuclei, lobed nuclei, blebbed nuclei and notched nuclei were also evaluated in peripheral erythrocytes. Interphase AgNOR parameters were examined in epithelial cells obtained from the edge of caudal fins after 90 and 180 min of exposure. As a result, dose-dependent increases in the frequencies of micronuclei and other NA in erythrocytes were observed. MN frequencies in gill cells also significantly increased, while the interphase AgNOR parameters in fin cells decreased, as a result of textile effluent and cylophosphamide treatments.

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.

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 4 h 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 4 h exposure to 1% SF-STIUE, and at 24 h exposure to 0.1% and 1% SF-STIUE. Furthermore, B( a)P-type metabolites increased significantly in liver cytosol at 4 h 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.

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.

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 μM 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.

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.