Erik Noaksson

Altered steroid metabolism in several teleost species exposed to endocrine disrupting substances in refuse dump leachate

Endocrine disruption associated with reproductive failure has been reported previously in female perch (Perca fluviatilis) and roach (Rutilus rutilus) from Lake Molnbyggen in Sweden and in female brook trout (Salvelinus fontinalis) from Vadbäcken, a stream emptying into Molnbyggen. Both Molnbyggen and Vadbäcken have been contaminated by toxic leachate from a municipal refuse dump. In this study, female perch were caught in Molnbyggen and the reference lake, Lake Djursjön, to further investigate the endocrine mechanism behind the significant numbers of sexually immature (SIM) female perch in Molnbyggen. Blood plasma analysis of progesterone (P), 17alpha-hydroxyprogesterone (17alpha-OHP), testosterone (T), and 17beta-oestradiol (E2), as well as analysis of brain aromatase activity (P450arom), were carried out. The exceptional high numbers of SIM female perch in Molnbyggen was confirmed in February 1999. In July 1999, at an early stage of oogenesis, perch from Molnbyggen showed significantly decreased gonadosomatic index (GSI) and aromatase activity. The presence of aromatase inhibiting substances in lake sediments were therefore tested in vitro. The aromatase activity was dose-dependently inhibited by clotrimazole, reaching 50% inhibition at a concentration of 0.9 microM. Aromatase inhibiting substances were found both in Molnbyggen and reference sediment extracts, indicating that they were naturally occurring substances and not of anthrophogenic origin. The similar decrease in levels of circulating steroids (P, 17alpha-OHP, T, and E2), aromatase, and GSI therefore suggest that the low aromatase activity is due to down-regulation rather than inhibition. To further investigate the steroidogenesis prior to T, P, and 17alpha-OHP were analysed in perch caught in 1997 and 1998 in Lakes Molnbyggen, Kvarntjärn (downstream), Yxen (upstream), and Djursjön, in female roach caught in Molnbyggen and Djursjön in 1997, and in brook trout caught in Vadbäcken and the reference stream Björntjärnsbäcken in 1998. The absence of differences in P and 17alpha-OHP levels, combined with a significantly lower T level in female perch and roach from Molnbyggen in 1997, could be the result of either increased metabolism and excretion of T, or a disruption downstream of 17alpha-OHP formation. The unaffected P levels and significantly lower 17alpha-OHP levels, together with significantly decreased T and E2 levels, found in adult (>45g) female brook trout from Vadbäcken, further indicated that an altered steroidogenesis downstream of P is one possible mechanism underlying the low T levels and thus the high number of SIM female fish, since too low T levels might be insufficient to activate the brain-pituitary-gonadal axis.

Endocrine disruption in brook trout (Salvelinus fontinalis) exposed to leachate from a public refuse dump

Lake Molnbyggen was previously found to harbour a large number of sexually immature female perch (Perca fluviatilis) suffering from endocrine disruption. In an attempt to pin-point the source of the endocrine-disrupting substance(s) (EDSs), brook trout (Salvelinus fontinalis) from Vadbäcken, a stream contaminated by leachate from a public refuse dump and which empties into Lake Molnbyggen, were investigated. In addition, female perch from Lakes Yxen and Kvarntjärn, located up-stream and down-stream of Lake Molnbyggen, were investigated. Only 16.7% of the adult female brook trout in Vadbäcken were sexually mature, associated with decreased gonadosomatic index, lower brain aromatase activity, and lower circulating levels of testosterone and 17beta-oestradiol, in comparison to female brook trout from the reference stream Björntjärnsbäcken. Male brook trout showed decreased gonadosomatic index, in addition to bile duct hyperplasia in the liver, which was also found in female brook trout livers from Vadbäcken. In Lake Molnbyggen, 57.6% of the female perch were found to be sexually immature with high frequencies of skin lesions, such as sores and fin erosion, significantly decreased gonadosomatic index, lower aromatase activity, and lower levels of testosterone and 17beta-oestradiol. No signs of reproductive disorders or endocrine disruption were seen in female perch from Lakes Yxen and Kvarntjärn compared to female perch from the reference lake, Lake Djursjön. Since brook trout of both sexes from Vadbäcken displayed the same kind of serious adverse impairment of gonad development and endocrine disruption as perch from Lake Molnbyggen, very strong evidence are provided that the refuse dump is the source for the responsible EDS(s), since both Vadbäcken and Lake Molnbyggen are known to be contaminated by leachate from that dump. The low levels of PAHs and PCBs in the surface sediments of Lake Molnbyggen suggest that these pollutants are not the responsible EDS(s).

DNA adduct formation and persistence in liver and extrahepatic tissues of northern pike (Esox lucius) following oral exposure to benzo[a]pyrene, benzo[k]fluoranthene and 7H-dibenzo[c, g]carbazole

The formation and persistence of DNA adducts in liver, intestinal mucosa, gills and brain of juvenile northern pike (Esox lucius) following oral exposure to benzo[a]pyrene (BaP), benzo[k]fluoranthene (BkF) and 7H-dibenzo[c,g]carbazol (DBC) were analysed by 32P-postlabelling. The dosage was 25 micromol/kg body weight of each substance, administered on 5 occasions with an interval of 12-14 days. Sampling was carried out 9 days after the second treatment, and 9, 16, 33 and 78 days after the fifth treatment. Pikes were also fed with the substances singly for comparison of adduct patterns. A complex pattern of adducts was detected in all examined tissues from fish treated with the mixture. Total adduct levels were highest in intestine (347+/-17.4 nmol adducts/mol nucleotides, mean+/-SE), followed by liver (110+/-9.3), gills (69+/-6) and brain (14+/-4.2). In pike treated with BaP alone, one major adduct was detected in all examined tissues. This BaP-adduct made up approximately 50% of the total amount of adducts in the brain. Corresponding values in liver, intestine and gills were 23, 31 and 34%, respectively. One relatively weak BkF-adduct and at least 10 different DBC-adducts were detected in all analysed tissues. Total adduct level in the intestine declined to 29.4% of the maximum value 78 days after the last exposure, while there was no significant decline in adduct levels in liver, gills or brain. The results suggest that intestine is more susceptible to adduct formation than liver after oral exposure, and that adduct levels in the intestine represent ongoing or relatively recent exposure. DNA adducts in the other investigated tissues were much more persistent and may therefore accumulate during long-term exposure.

Biochemical biomarkers in adult female perch (Perca fluviatilis) in a chronically polluted gradient in the Stockholm recipient (Sweden)

A battery of biochemical biomarkers and the SigmaPCB concentration in adult female perch (Perca fluviatilis) verified an aquatic pollution gradient with the city of Stockholm (Sweden) as a point source of anthropogenic substances. The investigation included both an upstream gradient, 46 km westwards through Lake Mälaren, and a downstream gradient, 84 km eastwards through the Stockholm archipelago. Besides the main gradient from Stockholm, there were strong indications of pollution coming from the Baltic Sea. The results indicated a severe pollution situation in central Stockholm, with poor health status of the perch, characterised by increased specific EROD activity in the liver, increased liver EROD somatic index, decreased AChE activity in the muscle, increased amount of DNA adducts in the liver, and a high concentration of biliary 1-pyrenol. In addition, laboratory exposure to common EROD inducers elicited an abnormal response, suggestive of chronic intoxication.

Studies with oxythiamine to mimic reproduction disorders among fish early life stages

Abstract Newly fertilised eggs of sea trout (Salmo trutta) were injected with the thiamine antagonist oxythiamine or with oxythiamine in combination with 3,3′,4,4′-tetrachlorobiphenyl (PCB #77). Rainbow trout (Oncorhynchus mykiss) eggs were injected with PCB #77. A decrease in liver transketolase activity was used as a biomarker to indicate a physiological deficiency in thiamine. Exposure to oxythiamine caused deformities in the newly hatched larvae, as well as high mortality during the larval stages. Exposure to oxythiamine in combination with PCB #77 caused a synergistic higher frequency of disorders. Analysis of liver transketolase activity in the larvae injected with PCB #77 indicated that PCB #77 did not decrease the half-life of thiamine in the offspring. The data obtained indicate that oxythiamine increased the sensitivity to PCB #77 in the offspring, however through mechanisms other than those causing thiamine deficiency in feral fish.

Biological effects on viviparous blenny exposed to chrysene and held in synthetic as well as in natural brackish water

Abstract Viviparous blenny ( Zoarces viviparous ) were exposed to chrysene and held in natural brackish water as well as in two different synthetic brackish waters, all with a salinity of 6.8%. After 10 weeks of exposure a number of different biomarkers was analysed. The result indicated increased ethoxyresorufin- O -deethylase activity and the formation of two different DNA adduct spots in the liver tissue, as a result of the chrysene exposure. Very similar results of the chrysene exposure were obtained in blenny held in the two synthetic brackish waters. No influence of the synthetically made waters compared to the natural brackish water could be observed on the biomarkers investigated in the study.

Reproductive disorders in fish from Lake Molnbyggen and an adjacent stream contaminated by leakage water from a refuse dump

Abstract Fish were examined in Lake Molnbyggen, a remote Swedish lake with a refuse dump in the vicinity, and an adjacent stream which drains the refuse dump area. The results showed significantly decreased gonadosomatic index (GSI) in females of four teleost species: perch ( Perca fluviatilis ), northern pike ( Esox lucius ), roach ( Rutilus rutilus ) and brook trout ( Salvelinus fontinalis ). Results show that female perch and female brook trout with decreased GSI have a low brain aromatase activity (CYP 19), close to the activity found in juvenile fish. The testosterone levels in blood plasma were also significantly lower in female perch with decreased GSI values. A frequent occurrence of fin deformations and open wounds were also found on perch, pike and roach. The biomarkers ethoxyresorufin O -deethylase, glutathione reductase and hydrophobic DNA adducts indicated a low general exposure to anthropogenic substances, although glutathione- S -transferase and catalase were induced in female perch. In conclusion, these results suggest serious adverse impairment of endocrine functions caused by specific organic pollutants in leakage water from the refuse dump.

DNA adducts in northern pike (Esox lucius) exposed to benzo[a]pyrene, benzo[k]flouranthene and 7H-dibenzo[c,g]carbazole

Abstract Formation and persistence of DNA adducts in liver, intestine, gills and brain of juvenile northern pike (Esox lucius) were analysed by 32P-postlabelling, following repeated oral exposure to benzo[a]pyrene (BaP), benzo[k]fluoranthene and 7H-Dibenzo[c,g]carbazole, singly or in a mixture. A complex pattern of adducts was detected in all examined tissues from fish treated with the mixture. Total adduct levels were highest in intestine, followed by liver, gills and brain. A major BaP-adduct made up approximately 50% of the total amount of adducts in the brain. Corresponding values in liver, intestine and gills were 23, 31 and 34%, respectively. Total adduct level in the intestine had declined to 29.4% of the maximum value 78 days after the last exposure, while there was no significant decline in adduct levels in liver, gills or brain. The results suggest that intestine is more susceptible to adduct formation than liver after oral exposure, and that adduct levels in the intestine represent ongoing or relative recent exposure.

Simultaneous determination of several natural steroids in blood plasma from perch (Perca fluviatilis) by GC-HRMS

A gas chromatography-high resolution mass spectrometry (GC-HRMS) procedure for the simultaneous determination of 18 endogenous steroid hormones in blood plasma from teleost fish has been developed. Proteins were removed by precipitation in methanol and lipids were removed by a liquid–liquid extraction. The protein and lipid free extract was further purified by using two successive solid phase extraction (SPE) methods (C18 and NH2). The isolated steroid hormones were silylated with a mixture of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA): iodotrimethylsilane (TMSI):dithioerythritol (DTE) prior to determination with GC-HRMS working in selective ion recording mode. A volume of 200 µL blood plasma was sufficient for accurate determination of the steroid hormone levels, which enabled determination in individual fish. The detection limits ranged from 0.0003 to 0.2 ng g−1 blood plasma from perch (Perca fluviatilis), approximately 10 to 100 times lower then previously reported in this field. The recoveries for the entire procedure were in the range 58 to 150% with a variation, expressed as standard deviation (SD), below 10% with some exceptions. Despite the multi-step clean-up procedure, the intra-assay coefficient of variation, i.e. the within-day variation, for most steroid hormones was well below 14%. Finally, the procedure has been successfully applied to the determination of steroid hormones in blood plasma from female perch caught in two Swedish lakes.