Freshwater pollution biomarker: response of brain acetylcholinesterase activity in two fish species

The effect of prolonged exposure at two sites along the Reconquista River (Argentina), a highly polluted peri-urban water body, on brain acetylcholinesterase (AChE, EC 3.1.1.7, acetylcholine acetylhydrolase) of two teleosts was examined. Caged Cyprinus carpio and field-captured Cnesterodon decemmaculatus were used as sentinel organisms. Eserine concentration inhibiting 50% of AChE activity (IC50) and inhibition kinetic parameters were also evaluated. Interspecies IC50 differences were found to agree with observed kinetic parameters (KA, ki and kc), indicating that carps were more sensitive to eserine. Data obtained disclosed spatial differences and demonstrated the high sensitivity of AChE activity as an exposure biomarker. Marked species-related differences were detected, showing that enzyme determination of C. decemmaculatus is more effective in highly polluted sites. Considering the river water physicochemical profile, observed changes in AChE activities can be partly attributed to long-lasting raised concentrations of dissolved heavy metals.     

Separation and detection of vitellogenin in fish plasma by capillary zone electrophoresis

A method for coupling capillary zone electrophoresis (CZE) with rapid membrane chromatography purification (RMCP) was established for the analysis of vitellogenin (VTG) in male fish plasma induced with 17ss-estrodiol. CZE analyses of purified VTG were performed in a buffer containing 25 mM sodium borate (pH 8.4). A 50 microm i.d. fused-silica capillary was used for separation and the detection was carried out by UV-diode array at 214 nm. Inter- and intra-assay variabilities of the proposed method were less than 10.06 and 1.95%, respectively. The method has good linear relationship over the scope of 15-2250 microg/ml with a correlation coefficient of R2 = 0.9965 and a detection limit of 7.0 microg/ml. The established CZE method was also applied to directly separate and identify VTG from fish plasma. The results indicated this method could minimize interferences from plasma proteins, allowing the detection of at least 62.5 microg/ml of VTG proteins in total proteins. This is a rapid and easy method to determine the quantity and purity of VTG compared to Bradford method and SDS-PAGE.     

Immunoassay of haemoglobin-acrylonitrile adduct in rat as a biomarker of exposure

Acrylonitrile (AN) is a rat carcinogen. Human exposure may come from chemical industries and smoking. A haemoglobin adduct of acrylonitrile (Hb-AN) has been used as a biomarker of exposure by means of gas chromatography-mass spectrometry (GC-MS) analysis. We have developed specific monoclonal antibodies (Mab) to human Hb-AN and wish to report evaluation of an immunoassay in rats using an Mab that cross-reacts with rat Hb-AN. A dose response study of LD 0, 10, 50, and 90 in Sprague-Dawley rats was undertaken, with each rat receiving [2,3-14C]AN at 50 Ci kg-1 sc, and Hb from an aliquot of blood was taken for covalent binding analysis by liquid scintillation spectrometry and fluorescence ELISA. The dose responses of rats at 0.25, 0.5, 1.0, and 2.0 h after AN doses of 20, 50, 80, 115 mg kg-1 were compared by both methods with Hb and globin samples. Regression analysis showed a linear relationship between immunoassay and 14C-AN binding. This indicates that an antigenic form of Hb-AN may be used as a surrogate of Hb-AN adduct. The sensitivity of ELISA was tested in rats exposed for 1 h to sub-toxic doses of AN (10-1.1 mg kg-1). Quantification of Hb-AN by immunoassay was achieved by calibration with a synthetic adduct HbAN4h, a reference adduct prepared by treating rat Hb with excess AN for 4 h. ELISA and GC-MS analysis of N-terminal valine-AN in the Hb-AN adduct were compared and similar detection levels were found. This rat study appears to have validated the new immunoassay method for biomonitoring of AN exposure.     

Immunoassay of haemoglobin-acrylonitrile adduct in rat as a biomarker of exposure

Acrylonitrile (AN) is a rat carcinogen. Human exposure may come from chemical industries and smoking. A haemoglobin adduct of acrylonitrile (Hb-AN) has been used as a biomarker of exposure by means of gas chromatography-mass spectrometry (GC-MS) analysis. We have developed specific monoclonal antibodies (Mab) to human Hb-AN and wish to report evaluation of an immunoassay in rats using an Mab that cross-reacts with rat Hb-AN. A dose response study of LD 0, 10, 50, and 90 in Sprague-Dawley rats was undertaken, with each rat receiving \[2,3-14C]AN at 50 Ci kg-1 sc, and Hb from an aliquot of blood was taken for covalent binding analysis by liquid scintillation spectrometry and fluorescence ELISA. The dose responses of rats at 0.25, 0.5, 1.0, and 2.0 h after AN doses of 20, 50, 80, 115 mg kg-1 were compared by both methods with Hb and globin samples. Regression analysis showed a linear relationship between immunoassay and 14C-AN binding. This indicates that an antigenic form of Hb-AN may be used as a surrogate of Hb-AN adduct. The sensitivity of ELISA was tested in rats exposed for 1 h to sub-toxic doses of AN (10-1.1 mg kg-1). Quantification of Hb-AN by immunoassay was achieved by calibration with a synthetic adduct HbAN4h, a reference adduct prepared by treating rat Hb with excess AN for 4 h. ELISA and GC-MS analysis of N-terminal valine-AN in the Hb-AN adduct were compared and similar detection levels were found. This rat study appears to have validated the new immunoassay method for biomonitoring of AN exposure.     

Decline in serum antibodies to methyltetrahydrophthalic anhydride after cessation of exposure.Implications for use as a biomarker

T he association between exposure intensity and serum levels of immunoglobulins E and G against low molecular weight compounds was evaluated. The decay of levels of specific IgE and IgG antibodies was studied after cessation of exposure in workers exposed to the inhalant allergen methyltetrahydrophthalic anhydride in a plant using epoxy resins. Sera have been collected in workers for 18-84 (mean value 54) months after cessation of exposure. Specific IgE and IgG was assessed by RAST and ELISA, respectively. The mean of individual half-times for IgE ( N = 10) and IgG ( N = 8) was 0 9 (range 0 1-1 8) and 0 4 (range 0 2-0 6) years, respectively, after total avoidance of exposure. Corresponding decreases of IgE and IgG were also observed after reduction, but not total elimination, of exposure. No correlation was seen between biologic halftimes of specific IgE and total IgE, atopy, smoking habits or gender. The results indicate that the levels of specific antibodies in sensitized individuals reflect long term exposure, and may persist for years after the end of exposure.     

Toenail mercury concentration as a biomarker of methylmercury exposure

The aim of the study was to assess the value of toenail mercury as an alternative biomarker of methylmercury exposure compared with blood and hair. Blood, hair and toenail total mercury concentrations were determined simultaneously in a southern urban sea n = 35 and an eastern rural lake n = 37 group and separately in a central Finnish rural lake n = 39 group. A questionnaire based index was used for estimating frequency of exposure taking into account high vs low mercury fish. Total mercury concentration was determined by cold vapour atomic absorption spectrophotometry. The mean Fish Consumption Frequency Index varied from 4.7 to 9.9 on a scale of 1-20. The blood, hair and toenail mercury mean concentrations ranged from 2.9 to 14.6 g l-1, 0.45 to 1.57 mg kg-1 and 0.20 to 0.54 mg kg-1, respectively. In the combined southern and eastern groups n = 72 sampled simultaneously, the correlations between blood and hair mercury were r = 0.92 and that of blood and toenails r = 0.78 p 0.0001 . In the central Finnish group the correlations were more moderate. In the three groups all three biomarkers correlated highly with the fish consumption index, r = 0.43-0.76 p 0.0001 . Men consumed high mercury fish more frequently than women, 8.6 vs 6.6 n.s. . The mean mercury levels of blood and hair were two fold, but toenail mercury levels were only 30 higher in men compared with those of the women. The relative sensitivity slope of the sources decreased in the order, blood hair toenails. In conclusion, toenails, an easily accessible tissue for the estimation of methylmercury exposure, have been shown to be closely correlated with the well established samples for biomarkers, viz. blood and hair mercury.     

Toenail mercury concentration as a biomarker of methylmercury exposure

The aim of the study was to assess the value of toenail mercury as an alternative biomarker of methylmercury exposure compared with blood and hair. Blood, hair and toenail total mercury concentrations were determined simultaneously in a southern urban sea n = 35 and an eastern rural lake n = 37 group and separately in a central Finnish rural lake n = 39 group. A questionnaire based index was used for estimating frequency of exposure taking into account high vs low mercury fish. Total mercury concentration was determined by cold vapour atomic absorption spectrophotometry. The mean Fish Consumption Frequency Index varied from 4.7 to 9.9 on a scale of 1-20. The blood, hair and toenail mercury mean concentrations ranged from 2.9 to 14.6 g l-1, 0.45 to 1.57 mg kg-1 and 0.20 to 0.54 mg kg-1, respectively. In the combined southern and eastern groups n = 72 sampled simultaneously, the correlations between blood and hair mercury were r = 0.92 and that of blood and toenails r = 0.78 p 0.0001 . In the central Finnish group the correlations were more moderate. In the three groups all three biomarkers correlated highly with the fish consumption index, r = 0.43-0.76 p 0.0001 . Men consumed high mercury fish more frequently than women, 8.6 vs 6.6 n.s. The mean mercury levels of blood and hair were two fold, but toenail mercury levels were only 30 higher in men compared with those of the women. The relative sensitivity slope of the sources decreased in the order, blood hair toenails. In conclusion, toenails, an easily accessible tissue for the estimation of methylmercury exposure, have been shown to be closely correlated with the well established samples for biomarkers, viz. blood and hair mercury.     

Induction of vitellogenin production in male tilapia (Oreochromis mossambicus) by commercial fish diets

Mozambique tilapia, (Oreochromis mossambicus), are a euryhaline teleost and an important biological model species. Captive male tilapia frequently have high levels of the estrogen-induced yolk precursor protein vitellogenin (Vg), a common indicator of exposure to estrogenic compounds. Sex steroids are found in commercial fish diets, but relatively few studies have examined the relationship between commercial diets and Vg production. In a fasting experiment to ascertain a dietary role in male Vg production, plasma Vg was reduced to negligible levels after 2 weeks of fasting, while no change in estrogen receptor (ER) expression was seen. When male tilapia were fed a squid-based diet that replaced the commercial trout diet, plasma Vg was reduced to undetectable levels over 40 days, concomitant with significant reductions in hepatic expression of Vgs A, B, and C, and ERβ, compared with control fish fed commercial trout diet. Female tilapia fed the squid-based for 20 days had no change in these parameters. When male tilapia were fed a defined, soy-based diet, plasma Vg reduced to 20% of levels in fish given either commercial trout diet or a defined, fishmeal-based diet. Overall, results from these studies suggest that estrogens in a commercial trout diet induce vitellogenin production by increasing expression of Vg, but not ER genes in male tilapia.     

Development and application of a monoclonal antibody-based sandwich ELISA for quantification of Japanese medaka (Oryzias latipes) vitellogenin

Vitellogenin (Vtg) was purified from ascitic fluid of a 17beta-estradiol (E2)-treated female Japanese medaka by anion-exchange chromatography. The molecular mass of medaka Vtg by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), corresponding to the Vtg monomer, was 200 kDa. BALB/c mice were immunized with purified-Vtg and two hybridoma clones producing specific antibodies against medaka Vtg were selected. The specificity of these monoclonal antibodies (mAbs) was evaluated by Western blot analysis of the plasma proteins separated on SDS-PAGE, and no cross-reactivity was observed with plasma proteins from control males. A sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of medaka Vtg was developed using these mAbs. The assay range was between 1 and 100 ng/ml, and the intra- and inter-assay variations determined from plasma samples were within 7.7 and 8.5%, respectively. Recovery of medaka Vtg added to plasma was 92-111%. In a plasma dilution test, plots of Vtg concentration gave a straight line. After exposure of male medaka to E2 (10 ng/l), Vtg appeared in liver and plasma on the first day and reached a maximum on the 3rd to 5th day. The sandwich ELISA could be useful for the detection of estrogenic properties, and the medaka Vtg bioassay could be a very sensitive and good tool for screening of endocrine disrupting compounds.     

DNA adducts as a biomarker of polycyclic aromatic hydrocarbon exposure in aquatic organisms: relationship to carcinogenicity

The use of DNA adduct measurement as a biomarker of exposure to polycyclic aromatic hydrocarbons (PAHs) is now well established in ecotoxicology. In particular, DNA adduct levels in aquatic organisms has been found to produce a better correlation with PAH exposure than PAH concentrations in organisms. DNA adducts levels are most commonly determined using the ³²P-postlabelling assay which measures total aromatic adducts. The relationship between relative DNA adduct formation and carcinogenicity has been investigated for a number of carcinogenic and non-carcinogenic PAHs using an in vitro system. Our results demonstrate that relatively high levels of DNA adducts can be produced by some non-carcinogenic PAHs, while other non-carcinogenic compounds do not produce detectable adducts. In addition, it has been shown that all carcinogenic PAHs investigated produce DNAadducts and that a relationship exists between relative adduct formation and carcinogenic potency. An investigation of adduct levels in fish liver and crustacean hepatopancreas in Oxley Ck, Brisbane has shown that higher than expected DNA adduct levels were correlated with the presence of carcinogenic and non-carcinogenic PAHs with high relative adduct forming potential.     

DNA adducts as a biomarker of polycyclic aromatic hydrocarbon exposure in aquatic organisms: relationship to carcinogenicity

The use of DNA adduct measurement as a biomarker of exposure to polycyclic aromatic hydrocarbons (PAHs) is now well established in ecotoxicology. In particular, DNA adduct levels in aquatic organisms has been found to produce a better correlation with PAH exposure than PAH concentrations in organisms. DNA adducts levels are most commonly determined using the ³²P-postlabelling assay which measures total aromatic adducts. The relationship between relative DNA adduct formation and carcinogenicity has been investigated for a number of carcinogenic and non-carcinogenic PAHs using an in vitro system. Our results demonstrate that relatively high levels of DNA adducts can be produced by some non-carcinogenic PAHs, while other non-carcinogenic compounds do not produce detectable adducts. In addition, it has been shown that all carcinogenic PAHs investigated produce DNAadducts and that a relationship exists between relative adduct formation and carcinogenic potency. An investigation of adduct levels in fish liver and crustacean hepatopancreas in Oxley Ck, Brisbane has shown that higher than expected DNA adduct levels were correlated with the presence of carcinogenic and non-carcinogenic PAHs with high relative adduct forming potential.     

Effects of exposure to 17alpha-ethinylestradiol during early development on sexual differentiation and induction of vitellogenin in zebrafish (Danio rerio)

The effect of acute stress on plasma beta-corticosterone (B), testosterone (T) and estradiol-17beta (E2) concentrations in juvenile alligators collected from sites with varying sediment contaminants was examined in this study. Dramatic increases in plasma B concentrations were observed in alligators from all of the sites after 2 h of capture although females from the intermediate contaminant site exhibited a significantly lower percentage increase in B than females from the other two sites. Males from the site with the highest contaminant levels exhibited elevated initial B concentrations relative to the other sites. This pattern was not observed after 2 h of restraint. Females from the highest contaminant site exhibited depressed initial T when compared to the other sites although this pattern was not observed after 2 h of restraint. Neither E2 nor T decreased after 2 h in females, whereas T concentrations decreased in all males over the same time period. The variance associated with these endpoints was also examined to determine whether it could serve as a more sensitive marker for perturbations of the endocrine system and stress response. Females from the higher and intermediate contaminant sites exhibited the lowest and highest standard errors (respectively) associated with 2 h plasma B concentrations with no differences among mean concentrations suggesting a perturbation of the stress response in these animals that was not detected by examining the means. We concluded that the environmental contaminants could be acting as stressors, leading to the observed differences.     

Multiple biomarker response in rainbow trout during exposure to hexavalent chromium

This research investigated hexavalent chromium toxicity in rainbow trout using a panel of biomarkers at different levels of biological organization. A time-course experiment in which rainbow trout were exposed in hard water (63.5 mg/L CaCO3) to a sublethal concentration of hexavalent chromium (10 mg/L) for a period of 28 days was conducted. The responses of multiple biomarkers were measured in gill and liver tissues at varying time points. Significant differences in metallothionein induction, superoxide dismutase activity, lipid peroxidation, cellular morphology, and growth were observed. Results indicated that gill tissues were more sensitive than hepatic tissues to chromium toxicity, yet hepatic tissues appeared to play a larger role in the organism's adaptive response to chromium compared to gill tissues. This study highlights the importance of using a set of integrated biomarkers to assess contaminant exposure and effects.     

Mercapturic acids as biomarkers of exposure to electrophilic chemicals:applications to environmental and industrial chemicals

The use of mercapturic acids (N-acetyl-L-cysteine S-conjugates, MAs) in the biological monitoring of human exposure to environmental and industrial chemicals is receiving more and more attention. Mercapturic acids (MAs) are formed from glutathione (GSH) S-conjugates via the MA-pathway. Although this pathway can lead to different end-products, the formation of MAs is the predominant route in most species, including man. Two GSH S-transferases (GSTs) show genetic polymorphisms in humans and this can have major consequences for individual susceptibility to toxic effects and for MA formation. In occupational toxicology, adducts to biomacromolecules are also used as biomarkers. DNA adducts are a measure for the effective dose, while protein adducts are related to the dose at critical site. Both type of adducts are normally determined in blood, while MAs are determined in urine. Most MAs are excreted with relatively short half-lifes, allowing a direct evaluation of the occupational circumstances. For many compounds similar (linear) dose-dependency was found for MA excretion, formation of macromolecular adducts, and for various biomarkers of toxic effects. These relations together with fact that MAs relate to the electrophilic character of compounds, allows for the conclusion that MAs are biomarkers of toxicologically relevant internal doses of chemicals or their metabolites. An overview will be given here of the use of MAs in the assessment of internal human exposure to electrophilic environmental and industrial chemicals. Additionally, the formation of GSH S-conjugates, their catabolism to MAs and several of the frequently used analytical approaches are discussed. When appropriate, the influence of genetic polymorphisms on formation of MAs and on susceptibility to toxicity will be discussed for different chemicals as well.     

Occupational exposure to HDI: Progress and challenges in biomarker analysis

1,6-Hexamethylene diisocyanate (HDI) is extensively used in the automotive repair industry and is a commonly reported cause of occupational asthma in industrialized populations. However, the exact pathological mechanism remains uncertain. Characterization and quantification of biomarkers resulting from HDI exposure can fill important knowledge gaps between exposure, susceptibility, and the rise of immunological reactions and sensitization leading to asthma. Here, we discuss existing challenges in HDI biomarker analysis including the quantification of N-acetyl-1,6-hexamethylene diamine (monoacetyl-HDA) and N,N′-diacetyl-1,6-hexamethylene diamine (diacetyl-HDA) in urine samples based on previously established methods for HDA analysis. In addition, we describe the optimization of reaction conditions for the synthesis of monoacetyl-HDA and diacetyl-HDA, and utilize these standards for the quantification of these metabolites in the urine of three occupationally exposed workers. Diacetyl-HDA was present in untreated urine at 0.015–0.060 μg/l. Using base hydrolysis, the concentration range of monoacetyl-HDA in urine was 0.19–2.2 μg/l, 60-fold higher than in the untreated samples on average. HDA was detected only in one sample after base hydrolysis (0.026 μg/l). In contrast, acid hydrolysis yielded HDA concentrations ranging from 0.36 to 10.1 μg/l in these three samples. These findings demonstrate HDI metabolism via N-acetylation metabolic pathway and protein adduct formation resulting from occupational exposure to HDI.     

Effects of ethynylestradiol on vitellogenin synthesis and sex differentiation in juvenile grey mullet (Mugil cephalus) persist after long-term exposure to a clean environment

We investigated the continuing effects of exposure to ethynylestradiol (EE₂) in juvenile grey mullet after transfer to a clean environment. Eleven-month-old juvenile fish containing immature phenotype gonad were fed dry diets; the low and high EE₂-treated groups were fed diets with 0.04 and 4 μg EE₂/g body weight for 4 weeks, respectively. After treatment, they were transferred to clean seawater, and reared with an EE₂ free diet for 350 days. Vitellogenin (VTG) was not detected in the serum of the control group throughout the experimental period. However, in both treatment groups, abnormal values of serum VTG were detected until approximately 100 days after transfer to a clean environment. In the control group, sex differentiation was not confirmed until 206 days after transfer to a clean environment. However, some of the fish in the 0.04 μg EE₂-treated group had ovarian cavity and oocytes at 26 days. In most of the fish in the 4 μg EE₂-treated group, the ovarian cavity had already appeared at the end of EE₂ treatment (0 day), and oocytes were observed at 26 days, suggesting that EE₂ accelerates ovarian differentiation. These results suggest that previous exposure to EE₂ has long-term effects on VTG synthesis and gonadal development.     

Feeding of an Iberian stream cyprinid assemblage: seasonality of resource use in a highly variable environment

Food resource use by seven cyprinids from an Iberian stream was analysed over 9 months. Differences in food resource use were found both between species and within species between seasons. Plant material was a more important food for carp, nase, goldfish and barbel than for gudgeon, roach and chub, irrespective of the season. Chironomid larvae were the staple animal food for the former five species throughout the year. Roach and chub, especially the latter, displayed large seasonal variations in prey use, with chironomid larvae only being important during autumn. Ephemerellid nymphs and ephemeropteran imagos dominated the animal diet of chub during spring and summer, respectively. Dipteran adults and Formicidae were the most important prey for roach during spring and summer, with other common prey being ephemerellid nymphs and hydropsychid larvae. Food resource overlap among the three dominant species (roach, barbel and chub) displayed a large seasonal variation. High overlaps were observed during autumn when these species used the same resources. During summer overlaps were much lower with each species specialising on different prey. The remaining less abundant species had large diet overlaps amongst themselves and with barbel, over all seasons. It is suggested that morphological constraints, habitat partitioning and temporal changes in food resource limitation may be involved in producing these patterns of food resource use.     

Validation of urinary sphingolipid metabolites as biomarker of effect for fumonisins exposure in Kenyan children

Context: Fumonisins (FNs), a group of mycotoxins produced mainly by Fusarium species, are ubiquitous food contaminants, especially for maize. Fumonisin B₁ (FB₁) caused severe toxicities in farm animals, induced kidney and liver tumours in rodents and is associated with many human adverse health effects, including oesophageal cancer. International Agency for Research on Cancer (IARC) categorizes FB₁ as a possible human carcinogen (Group 2B). Inhibition of ceramide synthesis and disruption of sphingolipids metabolism are well studied as the major mechanisms of FB₁-induced toxicity. Increases in sphinganine (Sa) and decrease in sphingosine (So) levels and their ratio are validated biomarkers of FB₁ effects.

Methods: In this study, we measured urinary levels of Sa, So and Sa/So in 284 children aged 1–14?years who consume maize as a staple diet. Exfoliated cells from urine were processed and sphingolipids quantified by High Pressure Liquid Chromatography.

Results and conclusions: Sa and So were detectable in 95.07% and 98.94% of samples, respectively. Creatinine adjusted mean levels and standard deviation of Sa, So and Sa/So ratio were 1.23?±?2.18, 4.99?±?8.3 and 0.296?±?0.587?nM. These results further confirmed the findings in studies with human adults, i.e. urinary Sa, So levels and Sa/So ratio are good biomarkers to assess FNs exposure in children.