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Multi-level biomarker responses (molecular/biochemical and histological/physiological levels) were studied to assess the sublethal toxicities of copper (Cu: 1.25, 5, and 20μg/L) and benzo[a]pyrene (BaP: 0.5, 5, and 50μg/L) induced in the freshwater pale chub Zacco platypus. Except for the kidney tissues when exposed to 20μg Cu/L, no significant differences were observed at the histological or physiological levels among the treatment groups. However, various molecular and biochemical responses were observed in Z. platypus, and these responses primarily depended on exposure time. Upon Cu exposure, both DNA single-strand breaks (COMET) and metallothionein (MT) concentration significantly increased after 4 days, whereas there were no significant changes after 14 days of exposure. Both 4 and 14 days of BaP exposure induced significant increases in COMET and 7-ethoxyresorufin-O-deethylase (EROD) activity, but there was no significant difference between them. Additionally, both Cu and BaP induced acetylcholinesterase (AChE) activity only after 14 days of exposure. The current findings demonstrate that the differences in the responses of MT and EROD are associated with each chemical's particular mode of action. Biomarker responses at the molecular and biochemical levels were quantized in terms of the integrated biomarker response (IBR) index to compare the toxicities of Cu and BaP. The IBR values were well correlated with the concentrations of Cu and BaP, and the correlations were enhanced at 4 days of exposure (r(2)=0.849 and 0.945, respectively) compared with 14 days (r(2)=0. 412 and 0.634, respectively). These results suggest that the IBR index may be a useful tool for the integrative quantification of the molecular and biochemical biomarker responses in a short-term exposure to Cu and BaP., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aryl hydrocarbon (Ah) receptor (Ah-agonist) effects of environmental samples containing polychlorinated aromatic hydrocarbons were evaluated using a 7-ethoxyresorufin-O-deethylase (EROD) assay of a primary hepatocyte culture from grass carp (Ctenopharyngodon idellus). The results were compared with those obtained from the assay using the rat hepatoma cell line H4IIE and chemical analysis using high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS). A dose-response relationship was observed between the EROD activities, either from primary hepatocyte culture assay or from H4IIE assay, and concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The results showed that the assay based on the H4IIE cell line (EC50=0.83 pg/mL) is more sensitive to TCDD than the assay based on primary hepatocyte culture (EC50=9.7 pg/mL). In tests of environmental samples, the results from the assay using primary hepatocyte culture were comparable to those from the assay using the H4IIE cell line and chemical analysis of concentrations of mixtures of polychlorinated dibenzo-p-dioxin and dibenzofuran (PCDD/PCDF). The lack of a change in the activities of glutathione-S-transferase (GST) and lactate dehydrogenase (LDH) in cell culture upon exposure to TCDD indirectly indicates that the compound is persistent to biodegradation in the cell culture system.

Anguilla anguilla L. were caged for 8 and 48 h in harbor water of Aveiro Lagoon, Portugal. Respiratory burst activity (RBA) of peritoneal, head kidney, and gill phagocytes was measured. Lipid peroxidation (LPO) was estimated in gill, kidney, and liver. Liver ethoxyresorufin-O-deethylase (EROD) activity, cytochrome P450 (Cyt P450) content, and bile metabolites were assayed. Various antioxidant enzymes, viz., glutathione peroxidase, catalase, and glutathione S-transferase and nonenzymatic antioxidant, viz., total reduced glutathione were also studied. Harbor water xenobiotics induced a significant RBA increase in gill after 8 h; whereas in peritoneum and head kidney it increased after 48 h exposure. These responses were adversely associated with tissue-specific peroxidative damage since significant LPO increase was observed in gill (8 and 48 h), kidney (48 h), and liver (48 h). The tissue most affected was gill. Moreover, liver EROD activity, Cyt P450 content and bile metabolites remain unaltered after 8 h; in contrast, 48 h exposure showed significant EROD activity decrease and pyrene-type bile metabolites increase. Decreased EROD activity may be associated with concomitant liver damage, as increased LPO was observed after 48 h. Furthermore, the tissue-specific damage corresponded to the differences in the antioxidant potentials of the tissues, since the initial exposure period caused a significant increase in liver antioxidant activities, whereas gill and kidney showed a significant decrease, demonstrating that liver is highly adaptive to oxidative damage. However, at 48 h exposure gill, kidney, and liver showed a suppressive antioxidant effect, probably due to PAHs, since a significant induction at PAH-type bile metabolites has been seen. Our results demonstrate that phagocyte activation and associated peroxidative damage are concomitantly corroborated with enzymatic and nonenzymatic antioxidant activity differences. In addition, hepatic antioxidant induction after short-term exposure may serve as a potent biomarker for water pollutants in fish.

The objective of this study was to evaluate the effects of bleached/unbleached kraft mill effluents (B/UKME) on the reproductive parameters of free-ranging Florida largemouth bass (Micropterus salmoides floridanus). The reproductive parameters measured included gonadosomatic index (GSI), histological evaluation of gonads, and plasma concentrations of vitellogenin (VTG), 17beta-estradiol, and 11-ketotestosterone (11-KT). Hepatic ethoxyresorufin-O-deethylase (EROD) activity was measured as a marker of exposure to cytochrome P450-inducing agents in these effluents. Endpoints were compared among adult bass sampled from tributary and mainstream effluent-contaminated and reference sites. Females sampled from the site closest to the mill outfall had a significant five-fold increase in EROD activity compared to bass sampled from reference streams. Although sex hormones were significantly reduced in bass from exposed sites, there were no differences in VTG and GSI across sites. The absence of organism-level responses was probably not related to a lack of sensitivity, as previous studies in our laboratory have shown that bass exposed to these effluents exhibit changes in GSI and in other measures associated with reproductive success. In females, inverse relationships were observed between VTG and GSI and EROD activity. These relationship, however, were not consistent within all of the sites studied. Collectively, our findings indicate that hepatic EROD induction is an effective marker of B/UKME exposure in largemouth bass and that it might be associated with antiestrogenic effects in this species.

In the present study, the estrogenic response in the reproductive cycle of female carp, Cyprinus carpio, in relation to a sewage treatment works (STWs) was measured as: alterations in plasmatic and hepatic vitellogenin (VTG) content, plasma levels of testosterone (T) and estradiol (E2), and effects on key enzymes of phases I and II biotransformation, ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST). Low plasma VTG content in females collected 4 km downstream of an effluent discharge suggested possible estrogenic disturbances; however, hepatic VTG content and sex hormone levels did not mirror this. In fact, E2 and T largely fluctuated among fish, with individual variations even greater than among sampling stations or periods. This suggests that at moderately polluted sites, biological variations of female carp and genetic particularities are prevalent. Hepatic biotransformation enzymes, such as EROD, increased with increasing water temperature while, in contrast, GST was maximal at the lowest water temperature.

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

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

The effects of naphthalene (NAP) and beta-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 microM) 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 2h 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 8h 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.

Chronic changes were measured in fish downstream from a pulp and paper mill located in the Humber Arm, Newfoundland, Canada. Cunner (Tautogolabrus adspersus) downstream of the mill were longer and heavier at an earlier age than reference fish. Condition factor was similar among sites. Hepatosomatic indices and ethoxyresorufin-O-deethylase activity were higher in fish near the mill. Gondal condition suggested that gametogenesis might have been delayed in these same fish. Although splenosomatic indices were greater in exposed fish, splenic hemosiderin was not significantly different from reference values. Gill and liver changes in exposed cunner were significant. Gill parasites showed no site difference; however, the prevalence and abundance of gastrointestinal acanthocephalans were significantly greater in fish from impact sites. These results suggest that effluent from this mill may be responsible for observed biological effects; however, further study is needed to determine whether gradients in nutrient loading and untreated sewage discharge may also contribute to fish health in the Humber Arm.

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% (E25) and 50% (E50)]; 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 E25, as well as at 16, 24, and 72 h exposure to E50. S exposure induced liver EROD activity only at 24h. 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 24h exposure to E25. 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 E50 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 E25, as well as at 8, 16, and 24 h exposure to E50. 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 24h exposure to E25 and E50, and 72 h to E50. A. anguilla L. increased ENA frequency was detected in T at 16, 24, and 72 h, whereas in E25 and S it was observed at 8, 16, and 24 h. Furthermore, E50 ENA frequency increased at 24 h exposure.

Cytochrome P450 activity in individual Chironomus riparius larvae was measured using a microtiter plate adaptation of the ethoxyresorufin-O-deethylase (EROD) assay. The sensitivity of this biomarker was tested by exposing larvae to phenobarbital (0.5 and 1.0 mM) and permethrin (1 and 10 microg/g). Both chemicals induced EROD activity in C. riparius larvae by up to 1.58-fold with PB and 2.47-fold with permethrin. EROD induction was more pronounced after 48 h. The initially high EROD activity in the controls suggested that P450s are induced by stress. Feeding levels prior to exposure also had a significant effect on EROD activity. EROD activity compared to the control was highest when larvae were fed double the normal ration. These results indicate that EROD activity in individual C. riparius may be a useful biomarker to add to a suite of biomarkers for the detection of freshwater pollution., (Copyright 2002 Elsevier Science (USA))

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 microM) 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 microM) 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 microM 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.

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

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 microM 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 microM BaP, whereas 0.3 microM BaP exposure induced a significant liver EROD increase from 2 up to 144 h. Liver cytochrome P-450 content was significantly increased at 8 h to 2.7 microM 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 microM 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 microM BaP, respectively. The A. anguilla L. genotoxic response to BaP, measured as ENA induction, was significantly increased at 144 h exposure to 0.3 microM BaP. The intermediate BaP concentration tested (0.9 microM) induced a significant three fold ENA increase at 48 and 72 h exposure compared to their controls. The highest BaP concentration (2.7 microM) induced a significant increase in ENA frequency at 72, 144 and 216 h exposure.

Is Anguilla anguilla L. (eel) liver ethoxyresorufin O-deethylase (EROD) induction absolutely necessary in order to convert promutagens as benzo[a]pyrene (BaP) into a mutagenic compound? Eels were exposed for 8 h to clean (control) and 0.3 microM beta-naphthoflavone (BNF)-contaminated water. The 8-h exposure to 0.3 microM BNF brought about a very high EROD induction (10 pmol/min/mg protein) compared to control animals (1 pmol/min/mg protein). The Ames test (Maron and Ames, 1983) was carried out with Salmonella typhimurium TA 98 strain (TA98 His-) and eel isolated S9 liver fraction was used as a metabolic BaP activator. The BaP and BNF dose range concentrations tested were 0 (blank), 0.015, 0.08, 0.15, 0.38, 0.75, 1.5, 3.8, and 7.5 microM/plate and 0 (blank), 0.412, 1.235, 3.704, 11.1, 33.0, and 100 nM BNF, respectively. A dose-response relationship between BaP concentration and mutagenic activity was observed in the presence of S9 fractions in control and 0.3 microM BNF-exposed eels. Significant positive results, as TA98 His+ revertants, were observed at 0.38, 0.75, 1.5, 3.8, and 7.5 microM BaP/plate induced by BNF S9 fractions. Significant BaP mutagenic activation by liver control S9 was detected only at 1.5, 3.8, and 7.5 microM/plate. The BaP 1.5, 3.8, and 7.5 microM/plate mutagenic activation by BNF S9 and control S9 were not significantly different. Relative to BNF activation, it was only possible to detect His+ reversion at 11.1 nM BNF concentration with 0.3 microM BNF-induced S9. The above results demonstrate that the eel S9 liver fraction has the capacity to biotransform high BaP concentrations and convert it into a mutagenic compound with or without previous liver BNF biotransformation induction. The same does not apply to low BaP concentrations, where liver S9 induction by BNF is necessary to promote mutagenesis.

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

Potential masculinization, detoxification, and oxidative stress responses were assessed in domesticated female guppies (Poecilia reticulata) exposed for 42 days to diluted effluent from a modern Swedish kraft pulp mill or a model androgen. Methyltestosterone induced male-like coloration and transformation of the anal fin into a gonopodium-like structure. The effluent did not induce any apparent changes of the anal fin morphology; however, the exposed guppies became more colored than control fish, which could be an androgenic response. A better understanding of the physiological mechanisms involved in these responses would be required for a full evaluation. Both primary effluent and effluent which had undergone activated sludge treatment caused a moderate but significant induction of hepatic ethoxyresorufin-O-deethylase (EROD) activity. However, the general toxicity of both effluents was low, as mortality was negligible even at 25% dilutions. There was a continuous production of offspring in all groups (47-62% female fry), except by methyltestosterone-treated females, which did not reproduce. There were no indications that either effluent caused oxidative stress since hepatic glutathione reductase, glutathione S-transferase and DT-diaphorase activities remained unchanged compared with controls.

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

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