Biochemical biomarkers in chronically metal-stressed daphnids

Biochemical biomarkers are a popular measure of toxic effects on organisms due to their assumed fast response, and are usually assessed after acute exposure of the organism to the stressor. However, increasing interest in the use of biochemical biomarkers in environmental pollution monitoring calls for more laboratory long-term studies of contaminants' effects on biochemical endpoints. In this study, four biochemical biomarkers (protein content, activity of cholinesterase (ChE), catalase (CAT) and glutathione S-transferase (GST), were correlated with standardised reproductive and survival endpoints of water fleas (Daphnia magna) after chronic exposure to Cr (VI) and Cd. No effect on the reproduction and survival was noticed up to the highest tested concentration of Cr (VI) (52.5 microg/L), while the protein content, and the ChE and CAT activity decreased, and GST activity increased. Cd affected reproduction of daphnids above 0.656 microg/L, but the protein content and ChE activity were changed at 0.328 microg/L and 0.082 microg/L of Cd, respectively. Biochemical biomarkers in some cases proved to be equally or more sensitive than reproduction and mortality. We recommend more frequent use of a battery of biochemical biomarkers in combination with other higher-level biomarkers also in chronic studies and not only in the acute ones.     

Oxidative DNA damage induced by iron chloride in the larvae of the lace coral Pocillopora damicornis

Biochemical and molecular biomarkers tools are utilized as early warning signatures of contaminant exposure to target and non-target organisms. The objective of this study was to investigate the sublethal effects of iron chloride to the larvae of the lace coral Pocillopora damicornis by measuring a suit of oxidative-stress biomarkers. The larvae were exposed to a range of sublethal concentrations of iron chloride (0.01, 0.1, 1, 10, and 100 ppm) for seven days. With reference to oxidative stress biomarkers, the no-observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) of iron chloride were observed to be 0.01 and 100 ppm respectively. At the end of the seventh day the antioxidant status of the larvae was evaluated by the levels of glutathione (GSH), glutathione peroxidase (GPX), glutathione reductase (GR), and glutathione-S-transferase (GST), in both experimental and control groups. For the quantification of cellular oxidative damage, lipid peroxidation (LPO) activity was determined in the same and the extent of DNA damage was assessed by the expression of DNA apurinic/apyrimidinic (AP) sites. Iron chloride exhibited a concentration-dependent inhibition of GSH and GPX and induction of GR, GST, LPO, and DNA-AP sites in the P. damicornis larvae when compared to the control group. The oxidative stress biomarkers of the larvae exposed to 0.1, 1, and 10 ppm of iron chloride did not show any significant overall differences when compared to the control group. However the activities of LPO, GSH, GPX, GR, GST and DNA-AP in the larval group exposed to 100 ppm of iron chloride exhibited statistically significant (P=0.002, 0.003, 0.002, 0.002, 0.005 and 0.007) differences when compared to the control group. The research results indicated that iron chloride in concentrations at the 100 ppm level caused oxidative stress in the P. damicornis larvae.     

Effects of copper and cadmium on cholinesterase and glutathione S-transferase activities of two marine gastropods (Monodonta lineata and Nucella lapillus)

With the view of using Nucella lapillus and Monodonta lineata as bioindicators in biomonitoring programs in the NW coast of Portugal, the sensitivity to copper and cadmium of these two common species in Atlantic coasts of Europe was investigated. Assays based on mortality and on the activity of the enzymes cholinesterases (ChE) and glutathione S-transferases (GST) were used, as these biomarkers have been used in biomonitoring studies in the area. ChEs present in foot muscle of both species were characterised and found to show properties of both typical acetylcholinesterase and pseudocholinesterase. Cadmium LC50s for N. lapillus and M. lineata were 2.64 and 2.44 mg/L, respectively, while the lowest observed effect concentration (LOEC) was 1.53 mg/L for both species. LOEC value for cooper was 0.075 mg/L for both. Cadmium in vivo exposure increased ChE activity of N. lapillus, but had no effects on M. lineata ChE. No in vitro effects of cadmium on ChE activity of any of the tested species was observed. Copper had no significant in vivo effects on ChE activity, although it inhibited the ChE of both species in vitro, the IC50s being 5.87 and 12.17 mg/L for N. lapillus and M. lineata, respectively. Cadmium had no significant effects on GST activity of either species, while copper caused a significant reduction of N. lapillus GST (LOEC=0.044 mg/L) but had no effect on M. lineata GST. Results indicate that (i) N. lapillus and M. lineata have a similar acute sensitivity to cadmium and copper; (ii) ChE and GST of these species are sensitive to cadmium (iii) ChEs of both species are inhibited by copper at concentrations in the mg/L range and therefore, its use is limited to heavily polluted sites; (iv) N. lapillus GST does not seem to be a suitable biomarker for copper, at least in the range of concentrations tested, since it was inhibited by copper, but no clear concentration-response relationship was observed.     

Accumulation and oxidative stress biomarkers in Japanese flounder larvae and juveniles under chronic cadmium exposure

This study investigated how Cd exposure affected oxidative biomarkers in Japanese flounder, Paralichthys olivaceus, at early life stages (ELS). Fish were exposed to waterborne Cd (0–48 µg L^− 1) from embryonic to juvenile stages for 80 days. Growth, Cd accumulation, activities of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), glutathione S-transferase (GST, EC 2.5.1.18), and levels of glutathione (GSH) and lipid peroxidation (LPO) were investigated at three developmental stages. Flounder growth decreased and Cd accumulation increased with increasing Cd concentration. In metamorphosing larvae, CAT and SOD activities were inhibited and GSH level was elevated, while LPO was enhanced by increasing Cd concentrations. CAT and GST activities of settling larvae were inhibited but GSH level was elevated at high Cd concentrations. In juveniles, SOD activity and LPO level were increased but GST activity was inhibited as Cd concentration increased. Antioxidants in flounder at ELS were able to develop ductile responses to defend against oxidative stress, but LPO fatally occurred due to Cd exposure. These biochemical parameters could be used as effective oxidative biomarkers for evaluating Cd contamination and toxicity in marine environments: CAT, SOD, GSH, and LPO for metamorphosing stage; CAT, GSH, and GST for settling stage; and SOD, GST, and LPO for juvenile stage.     

Oxidative stress biomarkers in the freshwater characid fish, Brycon cephalus, exposed to organophosphorus insecticide Folisuper 600 (methyl parathion)

Methyl parathion (MP) is an organophosphorus insecticide used worldwide in agriculture and aquaculture due to its high activity against a broad spectrum of insect pests. The effect of a single exposure to 2 mg L(- 1) of a commercial formulation of MP (MPc: Folisuper 600(R), MP 600 g L(- 1)) on catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione S-transferase (GST), reduced glutathione (GSH) and lipid peroxidation (LPO) of the liver, white muscle and gills of Brycon cephalus was evaluated after 96 h of treatment. MPc exposure resulted in a significant induction of SOD, CAT and GST activity in all tissues. However, the GPx activity decreased significantly in white muscle and gills, whereas no alterations were observed in hepatic GPx activity. MPc also induced a significant increase in LPO values in the white muscle and gills, while hepatic LPO levels did not show any significant alteration. The current data suggest that MPc has oxidative-stress-inducing potential in fish, and that gills and white muscle are the most sensitive organs of B. cephalus, with poor antioxidant potentials. The various parameters studied in this investigation can also be used as biomarkers of exposure to MPc.     

Effects of benzo(k)fluoranthene exposure on the biomarkers of scallop Chlamys farreri

Scallops (Chlamys ferrari) were cultured for 30 days in seawater containing benzo(k)fluoranthene (BkF) at 0.5, 1.0 and 10.0 microg/L. No effects were noted on 7-ethoxyresorufin O-deethylase (EROD) activity in digestive gland at low concentrations (0.5 and 1 microg/L) of BkF, but BkF increased the glutathione-S-transferase (GST) activity. At 10 microg/L BkF increased EROD activity significantly, and depressed GST activity. Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in digestive gland increased significantly in 0.5 and 1 microg/L BkF. In 10 microg/L concentrations of BkF, the activity of three antioxidant enzymes increased first and reached a peak after a few days, before tapering off towards the end of the 30 day exposure. In high concentrations of BkF, activity of three antioxidant enzymes in gill showed an early peak (12 h), before dropping off. Lipid peroxidation (LPO) levels increased along with sampling times, and there were time- and concentration-effects between LPO levels and BkF. The responses of the gills and the digestive gland were not always parallel which can be explained by differences in the bioavailability of the toxicant. The performance of each biomarker is assessed in the context of the role and advantages of selecting a battery of biomarkers for detecting contamination problems. The use of C. ferrari as a sentinel species for biomonitoring potential toxic effects in situ is discussed as well as mechanisms of BKF toxicity and alexipharmic strategies of C. ferrari.     

Muscular and brain cholinesterase sensitivities to azinphos methyl and carbaryl in the juvenile rainbow trout Oncorhynchus mykiss

The organophosphate azinphos methyl (AzMe) and the carbamate carbaryl are the insecticides mostly used in the irrigated valley of Río Negro and Neuquén, Patagonia, Argentina. Juvenile rainbow trout were exposed to AzMe and carbaryl and the sensitivity of skeletal muscular cholinesterase (ChE) and the time course of inhibition and recovery were evaluated. EC50 values demonstrated that AzMe was a stronger in vivo inhibitor of muscular ChE (1.05+/-0.23 microg/L) than carbaryl (270+/-62.23 microg/L). Muscular ChE was significantly less sensitive to both insecticides than brain ChE. EC50 values obtained for muscular ChE were closer than those for brain ChE to the respective pesticide lethal concentrations, pointing out the relevance of the muscular enzyme in determining acute toxicity. The recovery process of ChE activity after carbaryl exposure (500 microg/L) was fast, whereas no significant recovery was observed with AzMe (1 microg/L) after 21 days in uncontaminated media. Brain and muscular ChE were inhibited and showed a significant but not complete recovery after three consecutive 48-h exposures to AzMe (1 microg/L) followed by a recovery period of 7 days. This scheme mimics the periodical application of the insecticides in the region and suggests a certain probability of a sustained ChE inhibition under field conditions, affecting fish development and survival.     

Evaluation of biomarkers in oyster larvae in natural and polluted conditions

Crassostrea gigas D-shaped larvae were subjected to different conditions of temperature and salinity for 24 h and four biomarkers (acetylcholinesterase (AChE) activity, thiobarbituric acid reactive substances (TBARS) levels, glutathione S-transferase (GST) and catalase (CAT) activities) were measured. AChE activity decreased when salinity increased from 25 to 30 and 35 psu at 20 and 25 degrees C. Temperature did not seem to have an influence on AChE activity. TBARS levels increased as a function of salinity when the temperature was maintained at 20 degrees C, whereas at 25 degrees C no effect of salinity could be observed. Variations in GST and CAT activities were not significant with salinity and temperature except that catalase activity was higher at 25 degrees C than at 20 degrees C. Exposure experiments were conducted at 23 degrees C and 30 psu with carbofuran (100 and 1000 microg/l) and malathion (100 and 300 microg/l). There was an inhibition of AChE activity with carbofuran, and a toxic effect shown by an increase in TBARS levels counteracted by increases in GST and CAT activities which protected the larvae. When two pairs of adults producing larvae were taken into consideration, significant differences in biomarker levels were noted between the larval offspring of each pair. Malathion induced a decrease in AChE activity and an increase in CAT activity.     

Effects of azinphos methyl and carbaryl on Rhinella arenarum larvae esterases and antioxidant enzymes

Organophosphate (OP) and carbamate pesticides are anticholinesterasic agents also able to alter antioxidant defenses in different organisms. Amphibian larvae are naturally exposed to these pesticides in their aquatic environments located within agricultural areas. We studied the effect of the carbamate carbaryl (CB) and the OP azinphos methyl (AM), compounds extensively used in Northern Patagonian agricultural areas, on reduced glutathione (GSH) levels and the activities of esterases and antioxidant enzymes of the toad Rhinella arenarum larvae. Larvae were exposed 48 h to AM 3 and 6 mg/L or CB 10 and 20 mg/L. Cholinesterase and carboxylesterases were strongly inhibited by CB and AM. In insecticide-exposed larvae, carboxylesterases may serve as alternative targets protecting cholinesterase from inhibition. GSH-S-transferase (GST) activity was significantly increased by CB and AM. Superoxide dismutase activity increased in tadpoles exposed to 6 mg/L AM. Conversely, catalase (CAT) was significantly inhibited by both pesticides. GSH levels, GSH reductase and GSH peroxidase activities were not significantly affected by pesticide exposure. GST increase constitutes an important adaptive response to CB and AM exposure, as this enzyme has been related to pesticide tolerance in amphibian larvae. Besides, the ability to sustain GSH levels in spite of CAT inhibition indicates quite a good antioxidant response. In R. arenarum larvae, CAT and GST activities together with esterases could be used as biomarkers of CB and AM exposure.     

钩虾胆碱酯酶（ChE）和谷胱甘肽转硫酶（GST）的敏感性和特异性比较研究

研究了有机磷农药甲地基嘧啶流磷,有机氯农药林丹,菊酯类农药氯菊酯,表面活性剂直链苯磺酸钠和重金属Zn对钩虾（Gammarus pulex L.)胆碱酯酶（ChE）和谷胱甘肽转硫酶（GST）活性变化以及毒性影响,结果表明,在暴露24h和48h后,仅有机磷农药甲基嘧硫磷显著抑制胆碱酯的酶的活性,在暴露48h后,有机氯农药林丹和菊酯类农药氯菊酯能显著提高谷胱甘肽转硫酶活性,在暴露24h后,仅梵在导致谷胱甘肽转硫酶明显升高,作为生物标志物,胆碱酯酶比谷胱甘肽转硫酶具有更高的特异性,这两种生物标志物较毒性试验方法具有更高的敏感性。     

可食用文蛤中氧化性胁迫的生物标记物与甲基毒死蜱和草甘膦的生物富集

近几十年期间,在种群和个体受干扰后,对作为早期指示剂的生物标记物的研究受到越来越多的关注。我们用对水生生态系统污染敏感的生物标记物双壳类软体动物(文蛤)来评估两种有机磷杀虫剂(甲基毒死蜱、草甘膦)的影响。文蛤是水生生态系统污染的一种敏感的指示物种。在不同时间段测定文蛤中不同组织的非酶的(谷胱苷肽)和酶的(过氧化氢酶)抗氧化剂,作为文蛤中生物标记物的反应。在实验室条件下,测定了脂质过氧化作用、蛋白羰基含量、总蛋白含量、总脂质含量以及胆碱酯酶的活性。对不同的生物标记与杀虫剂的生物体内积累的相互关系进行了研究。甲基毒死蜱在文蛤组织中具有最大的诱导氧化胁迫的潜能,导致脂质过氧化反应增加并抑制抗氧化剂。而且,鳃是对该反应最敏感的器官。文蛤是一种极好的甲基毒死蜱的积聚者,因为暴露60天后,可以测定到其组织中浓度为824.0 mg/kg w.w的甲基毒死蜱。随着在草甘膦中暴露时间的增加,与背景水平相比,组织中草甘膦的浓度增加大约8×103mg/kg w.w。可以得出这样的结论:在一种生物中测定几种生物标记物是有用的。在双壳类中,蛋白质的羰基诱导可用于双壳类中化学污染物诱导的氧化胁迫的生物指示剂。抗氧化剂的防御成分是敏感的参数,是评估污染的水生生态系统的有用的生物标记物。辅以蛤组织的化学分析,生物标记参数能够提供一种有力的监测工具。     

Biomarkers as a tool to assess effects of chromium (VI): Comparison of responses in zebrafish early life stages and adults

The present work aims to compare the sensitivity of embryos and adult zebrafish to chromium (VI) (as potassium dichromate) focusing on biomarkers (cholinesterase, glutathione S-transferase and lactate dehydrogenase) as endpoints. Zebrafish eggs showed less sensitivity to Cr (VI) (96 h-LC₅₀ = 145.7 mg/L) than adults (96 h-LC₅₀ = 39.4 mg/L) probably due to the protective action of the chorion. However, biomarkers were much more responsive in larvae than in adults and gave clear indications about Cr (VI) mode of action: it seems to be neurotoxic (inhibited cholinesterase), to inhibit glutathione S-transferase activity and to interfere with cellular metabolic activity (changes in lactate dehydrogenase activity) in larvae. In adults, only glutathione S-transferase was responsive, showing a clear inhibition. The responsiveness of the analyzed biomarkers in larvae reinforces the idea of the usefulness of early life stage assays in the assessment of chemicals effects. Moreover, early life stage assays also contributed with relevant information regarding anomalies in larvae development and behavior. Further research should focus on the use of biomarkers to assess long term effects which are ecologically more relevant.     

The applicability of acetylcholinesterase and glutathione S-transferase in Daphnia magna toxicity test

The most commonly used toxicity test worldwide is the acute Daphnia magna test. The relevance of acetylcholinesterase (AChE) and glutathione S-transferase (GST) activity in D. magna exposed to chromium, cadmium, and diazinon was evaluated in connection with this standard test. We found no link between enzyme activities and immobility. Concentrations of Cr(6+) up to 280 microg/L had no effect on AChE and GST activities, while 20% immobility was observed. At concentrations of 20-25 microg/L of Cd(2+) AChE activity was increased by about 50%. The effect of diazinon on both enzymes was insignificant up to concentrations that caused 27% immobility. Consequently, while the use of AChE and GST activities is recommended when the mode of action of chemicals is studied, the value of these biomarkers in routine acute toxicity tests is limited because the relationship between enzyme activities and immobility of D. magna exposed to different chemicals is unclear.     

The effects of selenium on oxidative stress biomarkers in the freshwater characid fish matrinxã, Brycon cephalus (Günther, 1869) exposed to organophosphate insecticide Folisuper 600 BR (methyl parathion)

Methyl parathion (MP), an organophosphate widely applied in agriculture and aquaculture, induces oxidative stress due to free radical generation and changes in the antioxidant defense system. The antioxidant roles of selenium (Se) were evaluated in Brycon cephalus exposed to 2 mg L(-1) of Folisuper 600 BR (MP commercial formulation - MPc, 600 g L(-1)) for 96 h. Catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione S-transferase (GST), reduced glutathione (GSH) and lipid peroxidation (LPO) levels in the gills, white muscle and liver were evaluated in fish fed on diets containing 0 or 1.5 mg Se kg(-1) for 8 weeks. In fish treated with a Se-free diet, the MPc exposure increased SOD and CAT activities in all tissues. However, the GPx activity decreased in white muscle and gills whereas no alterations were observed in the liver. MPc also increased GST activity in all tissues with a concurrent decrease in GSH levels. LPO values increased in white muscle and gills and did not change in liver after MPc exposure. A Se-supplemented diet reversed these findings, preventing increases in LPO levels and concurrent decreases in GPx activity in gills and white muscle. Similarly, GSH levels were maintained in all tissue after MPc exposure. These results suggest that dietary Se supplementation protects cells against MPc-induced oxidative stress.     

Toxicity of the aromatase inhibitor letrozole to Japanese medaka (Oryzias latipes) eggs, larvae and breeding adults

Letrozole is a synthetic aromatase inhibitor and interferes in the committed step in the synthesis of endogenous estrogens from androgens. To evaluate potential effects on the early life stages of Japanese medaka, larvae and fertilized eggs were exposed to letrozole for 96 h and 14 days, respectively. No larvae died and no adverse effects were found on embryonic development at concentrations up to 3125 microg/L. Reproductive effects were assessed by exposing adults to 1, 5, 25, 125 and 625 microg/L letrozole for 21 days. A dose-dependent decrease in fecundity (>25 microg/L) and fertility (>5 microg/L) accompanied by histological changes suggested the inhibition of oocyte growth and possibly maturation. At 625 microg/L, the fish ceased spawning during the last week of exposure. Letrozole (>5 microg/L) reduced plasma vitellogenin levels in females in a dose-dependent manner. Transgenerational effects were evaluated by removing freshly-laid F1 eggs from letrozole-contaminated water and raising them to 15 days post-hatching in control water. Hatchability and time to hatching were detrimentally affected (>5 microg/L), but no morphological deformities were observed. Furthermore, a dose-dependent increase in the proportion of genotypic F1 males was found (>5 microg/L).     

Reproductive effects of prenatal exposure to nonylphenol on zebrafish (Danio rerio)

Mature female and male zebrafish were separated and exposed to nonylphenol (NP) at 0.1, 1, 10, 50, 100 and 500 microg/L, respectively, for 3 weeks. Gonadosomatic index (GSI) in both sexes and vitellogenin (VTG) induction in males was measured as the bioindicators for the impairment to the parents. The results indicated that 50 microg/L of NP was the non-observed effect concentration (NOEC) for GSI and VTG induction. Afterwards, the 50 microg/L NP exposed females and males, and the control females and males were cross-wise pair-bred in the control water for one week to examine the reproductive effects. The embryonic cathepsin D (CAT D) activity, eggshell thickness, fecundity, hatching rate and malformation (vertebral column flexure) rate of offspring were determined in the four pair-bred groups. While endpoints remained unchanged in the groups with exposed males, prenatal exposure of females to 50 microg/L of NP resulted in the impairment of reproduction in groups with exposed females including inhibition of CAT D activity (P < 0.05), decrease of eggshell thickness (by 23.6%) and elevation of malformation rate (P < 0.001). These results suggested NP could induce reproductive damage to zebrafish at NOEC for parents. The results also imply that alterations of CAT D activity and eggshell thickness may be more sensitive biomarkers to indicate the reproductive effects caused by endocrine disrupting chemicals.     

Effect of microcystin on ion regulation and antioxidant system in gills of the estuarine crab Chasmagnathus granulatus (Decapoda,Grapsidae)

The objective of this work was to evaluate mechanisms of microcystin toxicity on crustacean species. Adult male crabs of Chasmagnathus granulatus (13.97+/-0.35 g) acclimated to low salinity (2 per thousand ) were injected with saline (control) or Microcystis aeruginosa aqueous extract (39.2 microg/l) at 24 h intervals for 48 h. After the exposure period, the anterior and posterior gills were dissected, measuring Na(+),K(+)-ATPase and glutathione-S-transferase (GST) activity. Total oxyradical scavenging capacity (TOSC) and lipid peroxides (LPO) content were also determined. Na(+),K(+)-ATPase activity in anterior gills was significantly lower in crabs injected with toxin than in control crabs, while no significant difference in the enzyme activity was detected in posterior gills. Both sodium and chloride concentration in the hemolymph were not affected by toxin exposure. Significant changes in GST activity were detected in posterior gills, with higher values being observed in the toxin-injected crabs. Crabs exposed to microcystin also showed a significant increase in the TOSC value against peroxyl radicals, for both anterior and posterior gills. Lipid peroxides level did not change in both gill types after exposure to the toxin. The increased levels of TOSC suggest the occurrence of a crab response against oxidative stress induced by toxin injection, which prevents lipid peroxidation.     

Oxidative stress and genotoxic effects in gill and kidney of Anguilla anguilla L. exposed to chromium with or without pre-exposure to beta-naphthoflavone

Fish in the aquatic environment can be subjected to a multipollution state and the occurrence of sequential exposures is an important aspect of eco-toxicological research. In this context, a preceding exposure can affect a toxic response to a subsequent exposure. Therefore, the current study was based on sequential exposures, viz. to a PAH-like compound (beta-naphthoflavone, BNF) followed by a heavy metal (chromium, Cr), focusing on the assessment of oxidative stress responses and their role in induction of genotoxicity. Oxidative stress responses in gill and kidney were investigated in European eel (Anguilla anguilla L.), and measured as lipid peroxidation (LPO), glutathione peroxidase (GPX), catalase (CAT) and glutathione S-transferase (GST) activity, and reduced glutathione (GSH) concentration, whereas genotoxicity was measured as DNA strand breakage. Fish were exposed for 24 h to two Cr concentrations (100 microM, 1 mM), with or without pre-exposure to BNF (2.7 microM, 24 h). In gill, a GSH decrease was observed along with loss of DNA integrity at all exposure conditions except at the lowest Cr concentration, showing a crucial role of GSH over genotoxicity. Moreover, sporadic induction of antioxidant enzymes was not effective in the protection against genotoxicity. However, a different mechanism seems to occur in kidney, since the loss of DNA integrity detected for all exposed groups was not accompanied by alterations in antioxidant levels. With regards to peroxidative damage, both organs showed an LPO increase after sequential exposure to BNF and 100 microM Cr. However, no association between LPO induction and antioxidant responses could be established, showing that LPO is not predictable solely on the basis of antioxidant depletion. The interference of BNF pre-exposure with the response of organs to Cr showed a marked dependence on the Cr concentration. Gill showed synergistic effects on LPO and GPX increase, as well as on CAT and GSH decrease for the lowest Cr concentration. However, for the highest concentration an additive effect on decrease of DNA integrity and an antagonistic effect on the increase of GPX were observed. In kidney, synergistic effects were evident on LPO increase and GSH decrease for the lowest Cr concentration, as well as on CAT and GST decrease for the highest concentration. In contrast, an antagonistic action was observed on DNA integrity loss for both Cr concentrations. The current results are relevant in assessing the interactions of PAHs and metals and contribute to a better knowledge about oxidative stress and mechanisms of genotoxicity in fish.