Assessment of pollution in sewage ponds using biomarker responses in wild African sharptooth catfish (Clarias gariepinus) in Tanzania.

The interactive effects of mixed pollutants in sewage wastewater on biomarker responses were investigated using wild male African sharptooth catfish (Clarias gariepinus) in Morogoro, Tanzania. A total of 58 fish were used, of which 21 were from Mindu dam (reference site) and 22, 9 and 10 from Mafisa, Mazimbu and Mzumbe sewage ponds, respectively. Liver somatic index (LSI) and gonadosomatic index (GSI) were significantly greater (two- to threefold) and (five- to sixfold), respectively, in fish from all sewage ponds. Haemoglobin concentration and gill filament 7-ethoxyresurufin O-deethylase (EROD) activities were significantly higher (1.2-fold and twofold, respectively) in fish from Mzumbe sewage ponds than in fish from Mindu dam, whereas liver EROD activity was significantly higher in fish from Mzumbe and Mafisa sewage ponds (5-fold). A HPLC method for determination of enzymatically formed p-nitrophenyl-glucuronide (PNPG) was developed and applied to measure UDP-glucuronosyl transferase (UGT) activities that was significantly higher in fish from all sewage ponds (2-2.5-fold) than in fish from Mindu dam. Kinetic characteristics and assay dependence of UGT were studied with microsomal preparations. Metallothionein (MT) content was significantly lower (three- to fourfold) in fish from sewage ponds than in fish from Mindu dam, and corresponded with cumulative levels of cadmium, lead and mercury. Condition factor, vitellogenin (Vtg), acetylcholinesterase (AChE) activities in plasma, eyes and brain, haematocrit, plasma protein and cytosolic glutathione S-transferase (GST) activities were comparable in fish from sewage ponds and Mindu dam. Although specific pollutants other than the metals were not identified by chemical analysis, application of a suite of biomarkers in C. gariepinus demonstrated that all sewage ponds were contaminated by pollutants of public health concern.

Vitellogenin in African sharptooth catfish (Clarias gariepinus): purification, characterization, and ELISA development.

Vitellogenin (Vtg) induction in African sharptooth catfish (Clarias gariepinus) was assessed in order to develop a method for monitoring estrogenic pollution in African freshwater systems. Clarias gariepinus Vtg (Cg-Vtg) was purified from serum obtained from 17alpha-ethynylestradiol (EE2)-exposed fish and polyclonal antibodies against Cg-Vtg were raised. An enzyme-linked immunosorbent assay (ELISA) was developed and the induction and kinetics of Vtg were assessed in male fish in three different exposure trials using both natural estrogen (17alpha-estradiol [E2]) and synthetic EE2. Concentrations of EE2 in water and levels of EE2 conjugates in bile were quantified by liquid chromatography-mass spectrometry (LC-MS). In addition, co-administration of E2 and benzo[a]pyrene (BaP) were studied. Vtg was induced in all exposure trials and the maximum induction was observed 1 wk after exposure. Exposure of male C. gariepinus to 1.4, 2.7, and 13.9 microg/ml EE2 induced Vtg synthesis at all concentrations. BaP did not influence the Vtg kinetics. However, an increased rate of biliary excretion of EE2 was observed when BaP was additionally administered. In conclusion, Vtg is induced in male C. gariepinus after exposure to both E2 and EE2, rendering it a suitable biomarker for endocrine-disrupting chemicals in African freshwater systems.

Estrogenic effects of selected hydroxy polychlorinated biphenyl congeners in primary culture of Atlantic Salmon (Salmo salar) hepatocytes.

Many persistent organic pollutants are known to have endocrine-disrupting effects in several aquatic and terrestrial species. In this regard, hydroxylated metabolites of polychlorinated biphenyls (OH-PCBs) represent serious health and environmental concern because they are shown to act agonistic or antagonistic at hormone receptors (HRs) or to cause hormone-receptor-mediated responses. In the present study, salmon primary hepatocytes were used to study alterations in an estrogen signaling pathway resulting from exposure to four hydroxylated (4OH-CB 107, 4OH-CB146, 4OH-CB187, and 3OH-CB138) metabolites of PCB at different concentrations using quantitative real-time polymerase chain reaction. The effects of the PCB metabolites were compared to the mRNA expression in 17alpha-ethynylestradiol (EE2)-treated cells. Concentration-specific increase of vitellogenin (Vtg) mRNA transcription after exposure to OH-PCBs was observed. Decreased mRNA transcription was observed for zona radiata protein (Zr-protein) and cytochrome P450 side-chain cleavage (P450scc) enzyme. For estrogen receptor beta (ERbeta), the mRNA expression pattern was OH-PCB-metabolite congener-specific. A novel aspect of this study is that OH-PCBs produced effects on hepatic steroidogenic pathways by targeting the StAR protein and P450scc genes. Given that endocrine toxicology research mainly has focused on estrogenicity involving direct ER-mediated effects and that steroidogenic enzyme and proteins are highly tissue- and cell-type-specific and controlled by different promoters and second-messenger pathways, the present findings provide potential new targets for interaction with xenobiotics such as hydroxylated congeners of certain chemicals. The quantitative expression patterns of hepatic and extrahepatic steroidogenic genes and proteins after exposure to environmental contaminants are the subject of systematic investigations in our laboratory.

Effects of hydroxy-polychlorinated biphenyl (OH-PCB) congeners on the xenobiotic biotransformation gene expression patterns in primary culture of Atlantic salmon (Salmo salar) hepatocytes.

Hydroxylated metabolites of PCBs [OH-PCBs] represent new health and environmental concern because they have been shown to have agonist or antagonist interactions with hormone receptors (HRs) or hormone-receptor mediated responses. The present study was designed to investigate the estrogenic potency based on anti-AhR signalling effect of three 4-OH substituted PCB congeners (#107, #146 and #187), one 3-OH substituted congener (#138), and the pharmaceutical synthetic estrogen, ethynylestradiol (EE2) in fish in vitro system using primary culture of Atlantic salmon hepatocytes. The effects were studied by quantifying changes in transcripts with gene-sequence primer pairs for a suite of gene responses (AhRalpha, ARNT, CYP1A1, CYP3A, UGT and GST) belonging to the xenobiotic biotransformation system. Our data show that OH-PCB congeners and EE2, decreased AhRalpha and ARNT transcript levels, and CYP1A1, UGT and GST gene expressions, together with CYP3A gene expression. The decreased expression of transcripts for xenobiotic biotransformation system is related to the concentration of individual OH-PCB congener and these responses are typical of reported estrogenic and estrogen-like effects on the CYP system. Modulation of biotransformation pathways by OH-PCBs may alter xenobiotic metabolism leading to the production of toxic reactive molecules, altering pharmacokinetics and diminishing the clearance rate of individual chemicals from the organism.

Influence of 17alpha-ethynylestradiol on CYP1A, GST and biliary FACs responses in male African sharptooth catfish (Clarias gariepinus) exposed to waterborne Benzo[a]Pyrene.

Dose dependent effects of Benzo[a]pyrene (BaP) on cytochrome P4501A (CYP1A), glutathione-S-transferase (GST) and fluorescent aromatic compounds (FACs) metabolites biomarker responses were studied in African sharptooth catfish (Clarias gariepinus) following 24 h of waterborne exposures. Based on biomass of C. gariepinus in different tanks, BaP concentrations of 1.60, 3.44, and 18.21 microg/L that corresponded to 0.5, 1.0, and 5.0 mg/kg body weight were used. Significant induction of EROD activities in gill filaments was observed at all doses and the accumulation of FACs metabolites in bile was significantly different between groups. Accumulation of FACs metabolites in bile strongly correlated (r (2) = 0.99) with BaP doses. Hepatic EROD activities were undetectable and no effect on GST activities was observed. The highest dose of BaP from the dose dependent study was further studied to assess the interactive and temporal responses of C. gariepinus on CYP1A, GST, and FACs metabolites biomarkers following exposure to either BaP alone, 17alpha-ethynylestradiol (EE(2)) alone or a combination of both compounds at concentrations of 54.17 microg/L for BaP, 51.38 microg/L for EE(2) and 54.44 microg/L for each of both compounds. Based on biomass in each tank, these concentrations corresponded to 5 mg/kg body weight. While a group of six fish was sacrificed on day 0 from the control tank only, other groups of six fish were sacrificed after 1, 3, and 6 days of exposure from the control and exposed groups. Maximum induction of gill filament and hepatic EROD activities was observed after 1 day of exposure. Both EROD activities in gill filaments and liver were significantly induced by exposure to BaP alone or co-administration with EE(2). Gill filament EROD induction was significantly inhibited (50%) by co-administration of BaP and EE(2) compared to administration of BaP alone. Levels of FACs in bile for BaP and BaP + EE(2) exposed groups were significantly different from the control at all doses and time points. A significant induction of GST activities was observed in fish exposed to BaP and BaP + EE(2) after 3 days. Exposure to EE(2) alone caused significant induction of this enzyme after day 6. This study reports for the first time the significant antagonistic influence of EE(2) on BaP in gills of fish following waterborne exposures. The results also indicate that chemical mixtures may affect biomarker responses differently from compounds administered alone and that the sensitivity of CYP1A to interactive chemicals is different in gills and liver.

Evaluation of the gill filament-based EROD assay in African sharptooth catfish (Clarias gariepinus) as a monitoring tool for waterborne PAH-type contaminants.

The ability of African sharptooth catfish (Clarias gariepinus) in inducing cytochrome P-450 class 1A (CYP1A) and glutathione S-transferase (GST) biomarkers was determined in liver and gill filaments after 4 days of waterborne exposure to the polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P). Male (n=6) and female (n=6) fish were exposed to B[a]P added to the water (30 microg/l) corresponding to 5 mg/kg total body weight. Five female and six male fish, exposed to acetone alone added to the water served as controls. The 7-ethoxyresurufin-O-deethylase (EROD) activity was measured in hepatic microsomes and gill filaments. In addition, GST activity was determined in the hepatic cytosolic fraction and fluorescent aromatic compounds (FACs) in bile and biliverdin. Benzo[a]pyrene strongly induced EROD activities in gill filaments of both sex. Levels of FACs per ml of bile were 17-fold higher in exposed fish compared to the controls. Correlations between induction of EROD activities in gill filaments and liver and between induction of EROD activities in gill filaments and levels of biliary FACs metabolites were strong. GST activities in the hepatic cytosolic fraction were similar amongst the treated and control groups. This is the first report on studies determining EROD activities in gill filaments and hepatic tissue, FACs in bile and GST in hepatic tissues of C. gariepinus after waterborne exposure to B[a]P. The findings suggest that the gill filament-based EROD assay in C. gariepinus can be used to monitor the pollution of AhR agonists in aquatic ecosystems in eastern and southern African countries.

Thyroid hormone status in gray seal (Halichoerus grypus) pups from the Baltic Sea and the Atlantic Ocean in relation to organochlorine pollutants.

Marine apex predators, such as the gray seal (Halichoerus grypus), are exposed to high concentrations of organochlorine (OC) compounds via biomagnification in the food web. These lipophilic pollutants are transferred from the female to her offspring during lactation, and there is particular concern for endocrine disruption in the offspring. Herein we examined associations between the blubber concentrations of polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethane (DDT) and metabolites, chlordanes, hexachlorocyclohexanes (HCHs) and hexachlorobenzene (HCB), and plasma concentrations of thyroid hormones (thyroxine [T4] and tri-iodothyronine [T3]) in free-ranging, newly weaned gray seal pups from the Baltic Sea and the Norwegian waters of the Atlantic Ocean. Concentrations of total T3 (TT3) and free T3 (FT3) were significantly lower in the polluted seals from the Baltic Sea. Blubber concentrations of the sum of the analyzed PCB congeners (sigmaPCBs) and the sum of the DDT compounds (sigmaDDTs) were correlated negatively with plasma TT3 and FT3 concentrations. The results from this study indicate that OCs may affect levels of thyroid hormones in free-ranging gray seal pups, and that more focus should be put on T3 when addressing the possible effects of OCs on thyroid hormone function and status in nonrodent species.

Relationships between PCBs and thyroid hormones and retinol in female and male polar bears.

We studied the relationships between polychlorinated biphenyls (PCBs) and thyroid hormones (THs) and retinol within two groups of female polar bears (Ursus maritimus), females with cubs of the year (FWCOY) and females without cubs of the year (FWOCOY), and within a group of males. Concentrations of five of the six quantified PCB congeners, i.e., PCB-99, PCB-153, PCB-156, PCB-180, PCB-194 (sigma PCB5), correlated with each other, whereas the concentrations of PCB-118 did not correlate with the other congeners. sigma PCB5 and PCB-118 did not differ between the three different groups of polar bears, and the plasma levels ranged from 16.7 to 203.2 ng/g wet weight (ww) for sigma PCB5 and from 0.09 to 0.93 ng/g ww for PCB-118. PCBs did not affect the retinol status in any of the three groups. In FWCOY, we found negative correlations between sigma PCB5 and the three TH variables free thyroxin (FT4) (r2 = 0.35), free triiodothyronine (FT3) (r2 = 0.30), and the total T4:total T3 ratio (TT4:TT3) (r2 = 0.92). In FWOCOY, sigma PCB5 was negatively correlated to TT4 (r2 = 0.14) and positively correlated to TT3:FT3 (r2 = 0.31), whereas PCB-118 was positively correlated to FT3 (r2 = 0.21) and negatively correlated to TT3:FT3 (r2 = 0.26). In males, sigma PCB5 was negatively correlated to FT3 (r2 = 0.56) and positively correlated to FT4:FT3 (r2 = 0.78), whereas PCB-118 was negatively correlated to FT4:FT3 (r2 = 0.53). Thus, PCBs affected five TH variables in the female polar bears (TT4, FT4, FT3, TT3:FT3, TT4:TT3), but PCBs affected only two TH variables in males (FT3, FT4:FT3). Female polar bears could be more susceptible to TH-related effects of PCBs than are males. PCBs also affected T3 to a larger degree than T4.